Survival and Growth Responses of Lithobates Pipiens Tadpoles to an Herbicide and an Algaecide Used to Control Aquatic Invasive Plants

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SURVIVAL AND GROWTH RESPONSES OF LITHOBATES PIPIENS TADPOLES TO AN HERBICIDE AND AN ALGAECIDE USED TO CONTROL AQUATIC INVASIVE PLANTS

Caitlin B. Thomas

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

August 2015

Committee:

M. Gabriela Bidart-Bouzat, Advisor

Juan L. Bouzat

Daniel D. Wiegmann

Caitlin B. Thomas

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ABSTRACT

M. Gabriela Bidart-Bouzat, Advisor

Chemical herbicides are currently one of the most common methods for managing aquatic invasive plants. Despite their widespread use, little is known about the potential negative effects that several of the most commonly used herbicides may have on vertebrates. Amphibians may be particularly at risk because they can easily absorb toxic substances through their skin. In a controlled chamber experiment, Northern Leopard frogs (Lithobates pipiens) were exposed to environmentally relevant concentrations of copper-sulfate and diquat-dibromide, two herbicides commonly used to control invasive aquatic plants. The effect of these herbicides, either alone or combined, were evaluated on survival and growth-related traits of tadpoles over the course of the experiment. Tadpole survival was significantly decreased by copper-sulfate applications and the combination of this herbicide with diquat-dibromide. The copper-sulfate treatment also negatively affected the growth of tadpoles throughout the experiment, as evidenced by lower measures of body weight and body length. Conversely, diquat-dibromide caused a significant increase in tadpole growth, although it was only a transient effect. This study provides relevant information regarding the potential effect of two herbicides (commonly applied simultaneously) on an amphibian species that is considered to be an indicator of the quality of natural wetlands.

Therefore, it has important implications for the management of aquatic environments that provide habitats for a multitude of non-target species.

ACKNOWLEDGMENTS

To my advisor, Dr. Gabriela Bidart-Bouzat, a very big thank you for her help, guidance, advice, and support in overcoming all the minor setbacks I had in completing my project. Thank you to my committee members, Dr. Juan Bouzat and Dr. Daniel Wiegmann for their advice when

I first proposed my thesis. A special thanks to Dr. Eileen Underwood for her invaluable advice on rearing tadpoles in the lab. Also, thank you to my lab mate, Amanda Curtis, for her suggestions on carrying out tadpole measurements.

I would also like to thank Susan Schooner, who helped make sure my shipment of frog eggs survived when they arrived in the stockroom, or delivered them to the lab before the stockroom closed so I could get them. Additionally, I am grateful to Carolina Biological for shipping me a new clutch of eggs when the first one froze during delivery in the middle of the winter. Many thanks to Jen Baranski and the Animal Facility for allowing me to house my tadpoles when room in Dr. Bidart-Bouzat’s lab was limited.

Thank you to Rob Brown, one of the managers at Lake La Su An wildlife area, and to the

Ohio Division of Wildlife for providing me with information about wildlife areas near Bowling

Green, and for granting me permission to look for frog egg masses (even if I could not find any).

Thanks to Don Schooner as well for allowing me to look on his property at Schooner Farms.

Finally, a tremendous thanks to my family, who think I am a crazy “tree-hugging” biologist but love and support me just the same.

TABLE OF CONTENTS

Page

FITNESS RELATED RESPONSES OF LITHOBATES PIPIENS TADPOLES TO TWO

HERBICIDES COMMONLY USED TO CONTROL AQUATIC INVASIVE PLANTS... 1

I INTRODUCTION...... 1

M MATERIALS AND METHODS...... 4

S Study Organisms and Animal Husbandry...... 4

C Chemical Treatments...... 6

E Experimental Design...... 8

S Statistical Analyses...... 9

R RESULTS...... 9

D DISCUSSION...... 11

L LITERATURE CITED...... 17

APPENDIX A: 2013 INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE

APPROVAL………………………………………………………………………………...... 28

APPENDIX B: 2014 INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE

APPROVAL………………………………………………………………………………...... 29

LIST OF TABLES

Table Page

1 Mixed-model repeated measures ANOVA evaluating effects of treatment and time on

tadpole body weight and length……………………………………………….…… 25

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LIST OF FIGURES

Figures Page

1 Effects of herbicide treatments on tadpole survival. Asterisks indicate significant

differences in tadpole survival between each treatment and the control group from

Fisher’s exact tests. * P<0.0001…………………………………………….……… 26

2 Number of individuals that survived in each treatment and at each date. Asterisks indicate

significant differences in tadpole survival between each treatment and the control group

from Fisher’s exact tests. * P<0.0001………………..……………………………. 26

3 Effects of the herbicide diquat-dibromide, the algaecide copper-sulfate, and the

combination of both chemicals on tadpole weight (a), length (b), and tail length (c)

throughout the experiment. Asterisks indicate significant differences between treatment

means and those from the control group. *P<0.05…………………………………. 27

FITNESS-RELATED RESPONSES OF LITHOBATES PIPIENS TADPOLES TO TWO HERBICIDES COMMONLY USED TO CONTROL AQUATIC INVASIVE PLANTS

INTRODUCTION

The control of invasive species is quickly becoming a priority for the conservation of native species in many wildlife areas throughout the U.S. Naturalists are therefore trying to findways to protect natural aquatic and riparian habitats where herbicide use represents a large component of all aquatic weed control (Timmons 2005). However, chemical management of submerged aquatic plants is complicated by water movement, which can limit the amount of chemicals that are actually taken up by plants (Anderson 2003; Masser et al. 2013). This can lead to increased chemical applications that could be toxic to non-target organisms, such as fishes and amphibians (Pimental and Edwards 1982). In fact, previous studies have found negative effects of herbicides and pesticides on several aquatic organisms, including amphibians

(Wilson and Bond 1969; May et al. 1973; Fleeger et al. 2003; Relyea 2004; Relyea 2005;

Coutellec et al. 2008). Amphibians are considered particularly sensitive to habitat changes because they respire through their skin and can easily absorb toxic substances (Wake and

Vredenburg 2008). Therefore, the potential toxicity of chemicals used for the management of invasive plants on amphibians should be of major concern in conservation programs.

Frogs typically spend the first part of their biphasic life cycle in bodies of water as tadpoles. As adults, many frog species live near water or moist habitats to avoid desiccation (Vitt et al. 1990). All these aspects of the amphibian life cycle suggest that the physical boundary between frogs and their environment can be crossed quite easily; and thus, they are likely to be highly affected by any toxic chemical present in their habitat (Souder 2000). The sensitivity of amphibians to chemical pollutants is one of the main reasons why they have often been considered environmental indicators; that is, species that provide an indirect measure of the

2 environmental degradation of a particular ecosystem (Roy 2002; Blaustein and Johnson 2003).

For example, after an outbreak of frog deformities in the midwest United States in the late

1990’s, researchers believed this was indicative of a much larger environmental problem, and chemical pollutants were hypothesized to be the main cause of the deformities (Souder 2000). A range of negative side effects from herbicides and pesticides have been observed in a number of frog species, such as induced morphological changes in tadpoles (Relyea 2012), reduced size and weight of adults (Christin et al. 2012), increased tadpole mortality (Jones et al. 2009), absence of limbs or the presence of extra limbs (Ouellet et al. 1997), and altered expression of genes involved in development (Navarro-Martín et al. 2014). Clearly, herbicides can affect frog populations, but a few studies have also observed little to no negative effects of exposure in some frog species (Edge et al. 2011; Edge et al. 2014), which suggests species-specific responses

(Relyea 2009) or population-level variation in toxicity (Edge and Gahl et al. 2014).

Multiple herbicides are applied to bodies of water worldwide, including atrazine, glyphosate (marketed as Round-up®), diquat-dibromide and copper-based chemicals, such as copper sulfate. Diquat-dibromide is frequently used to treat aquatic weeds in the United States, despite being restricted or banned in other countries (Birch et al. 2011; Nault et al. 2011). This herbicide is toxic to both aquatic plants and animals because it forms reactive oxygen compounds that can attack plant chloroplasts and mitochondria (Gorzerino et al. 2009). One study showed that exposure of freshwater snails to commonly used concentrations of diquat- dibromide in the wild reduced their hatching rates and embryonic survival, as well as increased their juvenile mortality and food consumption (Ducrot et al. 2010; Coutellec et al., 2008). This herbicide has also been demonstrated to be up to five times more toxic to grass shrimp larvae than atrazine (Chung et al. 2008). Another chemical that is commonly used as an algaecide in

3 ponds and lakes is a copper sulfate solution marketed as Crystal Plex®. Because many algal species are potential food sources for tadpoles, the use of this algaecide can eliminate an important source of nutrients for amphibians and other aquatic herbivores (Kupferberg 1997;

Flecker et al. 1999). In addition, many organisms can be negatively affected by copper- containing chemicals. Previous studies have observed higher copper sensitivity in frog species compared to common fish species (Bridges et al. 2002), along with reduced survivability, increased deformity rates, decreased time to metamorphosis, and poor swimming performance in copper-containing environments (Chen, Gross, and Karasov 2007). Thus, incidental exposure of amphibians to this herbicide may be a concern.

Mixtures of herbicides and other contaminants may have synergistic effects that can severely affect amphibians in ways conservationists are only beginning to understand. Indeed, it has been observed that mixtures of insecticides and herbicides applied to an artificial habitat resulted in 99% mortality in Northern leopard frogs, Lithobates pipiens, even while individual chemicals themselves did not have negative impacts (Relyea 2008). Diquat-dibromide and copper-sulfate are two chemicals that are usually applied simultaneously, because their effectiveness to control invasive aquatic plants appears to increase when they are mixed together than when applied individually (Madsen 2000; Kammerer and Ledson 2001). Diquat-dibromide has been shown to increase the uptake of copper in aquatic plants, while, at the same time, copper-sulfate improves plant uptake of diquat-dibromide (Sutton et al. 1970, 1972; Pennington et al. 2001). If diquat-dibromide enhances copper uptake in aquatic plants, then it may also increase copper uptake and toxicity in aquatic animals. The decision to apply this herbicide combination in wildlife areas seems to be a double-edged sword for conservation land managers.

On the one hand, if invasive aquatic plants are left unchecked, they may outcompete and replace

4 native plant communities and alter the local ecosystem (Charudattan 2001; Rodriguez 2006;

Chilton et al. 2009). On the other hand, using herbicides to eliminate invasive plants may have unforeseen effects on non-target native species.

The main goal of this study is to evaluate the individual and interactive effects of the aquatic herbicide diquat-dibromide (marketed as Reward®) and a copper sulfate-based algaecide

(marketed as Crystal Plex®) on the survival and growth of Northern Leopard frog (Lithobates pipiens) tadpoles. Among other chemicals, these herbicides are frequently applied in managed wildlife areas to treat invasive aquatic plant species. Little is known about potential negative effects of diquat-bromide on amphibians; however, there is evidence that copper can affect growth rate, survival, development, and time to metamorphosis in Lithobates pipiens (Chen et al.

2007; García-Muñoz et al. 2010). Therefore, a copper-based herbicide such as Crystal Plex® is expected to be toxic to this frog species. Furthermore, the simultaneous application of diquat- dibromide and copper sulfate could produce an additive effect and result in a more pronounced negative effect on growth and mortality of Lithobates pipiens tadpoles than when either herbicide is used alone. Results from this study will be invaluable for wildlife managers using chemical mixtures to control invasive aquatic plant species.

MATERIALS AND METHODS

Study Organism and Animal Husbandry

The Northern Leopard frog (Lithobates pipiens) is one of the most widespread anuran species in the U.S. (Cook 1984). However, populations have been on the decline since the mid-1960’s throughout most of their range (Stebbins and Cohen 1995). Individuals are most often recognized by the distinctive large dark spots on their back and legs (Souder 2000). This frog species

5 occupies multiple habitats throughout the year to meet their life cycle requirements, including ponds in the spring for breeding and tadpole rearing, damp grassy areas in the summer for feeding, and larger lakes and streams for overwintering (Merrill 1977; Pope et al. 2000; Souder

2000). The relative abundance of L. pipiens in a variety of habitats, as well as their commercial availability, makes it an ideal study organism for assessing the potential effects of herbicides on amphibian species.

Two Lithobates pipiens egg clutches were ordered from Carolina Biological Supply

Company (Burlington, NC). Eggs were produced from artificially inseminated adult females.

The egg clutches arrived on March 26, 2014 in clear sealed bags containing highly oxygenated water. The bags remained sealed and were set on a bench in an open lab at Bowling Green State

University at room temperature (ranging from 23oC to 25oC), where they developed undisturbed.

On March 28, 2014 the eggs began hatching. Per instructions by Carolina Biological, the eggs and hatchlings were removed from their oxygenated bags and placed in containers with clean aged tap water (Frog Eggs, A Carolina Caresheet). All aged tap water used in the experiment was allowed to sit uncovered in large plastic containers for at least 4 days so that any chlorine present could evaporate out. By March 29, 2014, most tadpoles from both egg clutches had hatched and were free swimming. On March 31, 2014, randomly selected tadpoles were placed individually in containers set-up for the experiment. They were given 5 days to acclimate to their new environment before any chemicals were added. Experimental containers consisted of 1.7 L clear

PETE plastic containers (Superior Shipping Supplies, Van Nuys, CA) with 500 mL of aged tap water and a 10 cm artificial plant substrate to provide cover. Prior to the experiment, the artificial plants were soaked in tap water for one week to leach out any potential harmful chemicals. Each container was sealed with a lid to decrease water loss due to evaporation. All containers were

6 pre-punched with twelve 4 mm diameter holes at the top to allow for air flow. Each tadpole was fed 30 mg of Sera Micron tadpole food every 1-2 days. Tadpoles were maintained in controlled growth chambers in the lab at a temperature of 25°C and a light:dark 12:12 hours photoperiod.

Containers were rotated among shelves inside chambers and among chambers after every other feeding to avoid any potential position and chamber effects.

Chemical Treatments

Reward® (Syngenta Crop Protection, Inc. Greensborough, NC) is a diquat-dibromide chemical- based herbicide that can be used to treat both terrestrial and aquatic invasive plants. Tadpoles assigned to the diquat-dibromide treatment group (D) were exposed to a concentration of 0.015 mg/L of this chemical. This concentration was chosen because according to the Reward® label, it was considered to be within an “environmentally safe” range.

The second chemical chosen for this study, Crystal Plex (Sanco Industries INC, Ft.

Wayne, IN), is a registered aquatic algaecide containing 19.8% copper sulfate pentahydrate. It is typically added to bodies of water to control a broad range of algae. For the first 20 days of the experiment, which began on April 4th, 2014, tadpoles were exposed to weekly applications of 0.1 mg/L of copper-sulfate (CS). This was originally chosen because it closely approximated the amount recommended by the Crystal Plex® herbicide label to treat algae in the environment.

However, due to a high initial mortality rate of tadpoles in this group during the first 20 days of treatment applications (e.g., over fifty percent of tadpoles died), the concentration was reduced by half to 0.05 mg/L. This amount fell within the range considered appropriate for the same species in a previous study (Chen et al, 2007).

The treatment group consisting of simultaneous applications of diquat-dibromide and copper sulfate herbicides (D+CS) was exposed to the same concentrations of both chemicals that were added to the individual treatment groups. Copper-sulfate concentration in the D+CS group followed the same profile as the individual treatment. That is, the initial concentration of this chemical was reduced by half after the first 20 days of the treatment.

Stock solutions were prepared according to instructions in the chemical labels. To reach the desired copper-sulfate and diquat-dibromide concentration of 0.1 mg/L and 0.015 mg/L, 125

µL and 75 µL of the copper-sulfate and diquat-dibromide stock solutions, respectively, were added to 500 mL of aged tap water in each container in each of the respective treatment groups

(D, CS, and D+CS). Both of these concentrations were also added to the combined treatment group. A control group was housed similarly in containers that held only aged tap water. To reduce disturbance to tadpoles, only half of the water in each container was replaced during weekly water changes; that is, 250 mL of water were siphoned out of each container and replaced with 250 mL of aged tap water. For the control group, nothing further was added after water changes. In the chemical treatment groups, half of the original amount of diquat-dibromide

(37.5 µL) and copper-sulfate (67.5 µL) stock solutions, respectively, was added. On day 20 of the experiment, half water changes were carried out but copper-sulfate was not renewed during this week due to the observed high mortality rates in this treatment. Diquat-dibromide was also not applied during this week to maintain the same schedule of applications for both herbicides.

Herbicide applications were resumed the following week using the reduced copper-sulfate concentration of 0.05 mg/L and the original concentration of diquat-dibromide.

Experimental Design

After hatching, 120 tadpoles were randomly selected and arbitrarily assigned to either a control or an herbicide treatment group, with another 15 individuals set aside to serve as replacements should any of the selected experimental tadpoles die during the acclimation period. These were raised in conditions identical to those in the control group. Tadpoles were given five days to acclimate to the experimental environment before any chemicals were added. After this acclimation period, on April 4, 2014 (7 days post-hatch), the water in each container was replaced completely with clean aged tap water and herbicides were added to their respective treatment groups. Herbicide treatments were applied weekly, except, as previously mentioned, during week 3. Each treatment and the control group were replicated 30 times for a total of 120 tadpoles. Tadpoles were checked daily and any dead individuals were removed without replacement. Half water changes were carried out weekly.

On days 1, 10, 20, 31, 41, and 52 of the experiment, all tadpoles were briefly removed from their containers and measured for total body mass (to the nearest 0.0001 mg) using a digital balance. They were also measured with digital calipers (to the nearest 0.01 mm) for total body length (snout tip to tip of tail), and tail length (beginning of dorsal fin to tip of tail). Body width

(measured just behind the eyes) was only measured on days 1 and 52. Tail length was not measured on day 10. The experiment was terminated after the final measurement was taken on day 52. To corroborate the final diquat-dibromide and copper-sulfate concentrations in the treatment containers, water samples were randomly selected from one container of the diquat- dibromide and copper-sulfate treatment groups and sent for analysis to an independent lab

(AquaKnow water testing lab, Ewing, NJ). All tadpoles were euthanized in an MS-222 solution

(Western Chemical, INC, Ferndale, WA), as requested by the IACUC animal protocol.

Statistical Analysis

Statistical analyses were performed using SAS 9.3 software (SAS Institute, INC, Cary, NC).

Analyses of variance (ANOVAs) were performed to evaluate the effect of the different treatments (control, D, CS, and D+CS) on tadpole growth measures (body length, width, weight and tail length). Mixed model repeated measures ANOVAs (rmANOVA) were also performed to analyze how tadpole growth measures were affected by the treatments throughout the duration of the study. These analyses examined the effects of treatment, time, treatment × time, time2, and time2 × treatment on tadpole body weight and total body length. Post-hoc multiple comparisons among treatment means were performed using Fisher’s least significant difference t-tests only when the ANOVA model was significant (protected LSD). Measured variables were transformed when necessary to satisfy assumptions of normality and homocedasticity of ANOVA models

(i.e., square root, inverse or log transformations). Tadpole survival across the different treatments was analyzed using Fisher’s exact test.

RESULTS

Results from a Fisher’s exact test showed that survivability in tadpoles was affected by herbicide exposure (P< 0.0001). When compared to the control group, survival was significantly lower in the CS treatment and the D+CS treatment (both P=0.0033; Fig 1). Specifically, 12 out of 30 individuals survived in both of these groups (Fig. 2). There was no significant effect of the diquat-dibromide treatment on survival. Survival was the same in the D group as in the control.

Eighty percent of individuals survived (Fig. 1), or 24 out of 30 survived (Fig. 2)

A mixed model repeated measures ANOVA revealed significant effects of treatment, time, and time2 on both tadpole body weight, total body length and tail length (P<0.0001; Table

1). In other words, these 3 variables were influenced by both linear and non-linear components of time. However, the effects of time × treatment, and time2 × treatment were not significant, denoting that responses to treatments were similar through time (Table 1).

Treatment-induced differences in tadpole total body weight were detected as early as during the second measurement on day 10. On this date, body weight was found to be significantly lower in tadpoles exposed to copper-sulfate compared to those in the control group and D group. This trend was seen throughout the rest of the experiment (Fig. 3a). From the measurement made on day 20 until the end of the experiment (total of 4 measurements), tadpoles in the CS treatment weighed significantly less than those in the control group (Fig. 3a). On the other hand, less significant effects were found on body weight in tadpoles exposed to the diquat- dibromide herbicide. Tadpoles in the D treatment group weighed significantly more than the control group, but only on day 20 (Fig. 3a). There were no observed significant differences in body weight between tadpoles in the D+CS treatment group compared to the control group, or even the CS group (Fig. 3a).

As with body weight, there were also significant differences observed in tadpole body length between the treatment groups, especially for tadpoles exposed to the copper-sulfate herbicide (Fig. 3b). Tadpoles in the CS group were significantly shorter in total body length (Fig.

3b) when compared to the control, from day 20 until the end of the experiment and when compared to the D+CS group on days 31 and 41. No significant differences between tadpole body length in the D group, or D+CS group, and the control were observed besides on day 20

(Fig. 3b). Individuals in the D group were significantly longer in comparison to the control, and tadpoles in the D+CS group were significantly shorter. Additionally, on days 20, 31, and 52 the

D group had a significantly longer mean body length than the D+CS group.

A similar pattern was observed for tadpole tail length (Fig. 3c). Significant differences in tail lengths were observed between treatment groups on day 20. Tadpoles exposed to copper- sulfate and D+CS had tail lengths that were significantly shorter than those in the control group

(Fig. 3c). Tail lengths of tadpoles in the CS group remained significantly shorter than those in the control from day 20 until the end of the experiment (Fig. 3c). Tadpoles in the CS group was also significantly shorter in tail length when compared to the D+CS group on days 41 and 52.

Conversely, tadpoles in the D treatment had a significantly longer mean tail length compared to those in the control group; but again, this trend was found only on day 20 (Fig. 3c).

In addition, there was a significant effect of treatment on tadpole body width, which was measured only on days 1 and 52 (F3,68 = 3.21; P=0.0283). The only observed effect was related to a significantly narrower body width in tadpoles exposed to copper sulfate. No significant differences in tadpole body width between the D or D+CS groups and the control were found.

DISCUSSION

Results from this study showed that individual and combined applications of different herbicides can influence growth and survival of amphibians, such as Lithobates pipiens’ tadpoles. In particular, the herbicide copper sulfate had a consistent negative effect on growth- related traits of tadpoles, such as weight, tail and total body length, and body width. Survival of tadpoles exposed to copper sulfate was highly affected, as only 40% of the individuals assigned to this herbicide treatment survived until the end of the experiment. These results were consistent with those of a previous study showing that environmentally relevant levels of copper negatively affected the development, survival and behavior of L. pipiens tadpoles (Chen et al. 2007).

Copper-sulfate herbicides have also been found to be toxic to other amphibian species, causing a

12 delay in larval development (García-Muñoz 2008), lower body weight (Pritchard et al. 1973), reduced growth rates and swimming ability (Gürkan and Hayretdağ 2012), as well as abnormal limb development (Fort and Storer 1997; Chen et al. 2007). Given the high copper sensitivity previously observed in amphibians (Horne and Dunson 1995; Bridges et al. 2002; Redick and La

Point 2004), including that reported in the current study, treating wildlife areas with copper- sulfate should be a concern, as it may be contributing to the current decline in amphibian populations (Blaustein et al. 2003).

The decreased growth observed in copper-exposed tadpoles may have severe fitness and ecological consequences, because smaller tadpoles are generally more susceptible to predation

(Semlitsch and Gibbons 1988). Neuromuscular function in tadpoles can also be affected by copper-exposure (Chen et al. 2006 and 2007), causing decreased swimming speeds, and making it less likely that tadpoles can successfully evade predators (Raimondo et al. 1998). Copper- exposed amphibians may also be at a competitive disadvantage compared to larger amphibians by having an increased time to metamorphosis, and even decreased feeding times (Lefcort et al.

1998; Chen et al. 2007). Because of a potentially higher risk of predation, smaller tadpoles often adapt by limiting their activity, including time spent foraging for food (Peacor and Werner

2000). In this way, exposure to copper could indirectly further limit growth. Amphibian sensitivity to copper can also increase when it interacts with other factors, like UV-B (Baud and

Beck 2005), or potentially with other chemicals present in the environment, which was the main focus of the present study. Indeed, a previous study showed that that mixtures of insecticides combined with herbicides in an artificial habitat resulted in 99% mortality of Northern leopard frogs (Relyea 2008). In this study, even though the combined application of the herbicides diquat-dibromide and copper-sulfate (D+CS) did not appear to be as detrimental to tadpoles as

13 the exposure to copper-sulfate alone, tadpole growth was negatively affected by the D+CS treatment earlier in the development. In addition, tadpole mortality in the D+CS treatment was as high as in the copper-sulfate group alone. However, this effect could have been caused by the effect of copper-sulfate per se, since individual applications of diquat-dibromide did not affect tadpole survival.

Simultaneous applications of copper-sulfate and diquat-dibromide appear to enhance the uptake of these chemicals by plants (Sutton et al. 1970, 1972; Kammerer and Ledson 2001).

Therefore, the combined application of these chemicals is common practice, as suggested by the treatment label of the diquat-based herbicide marketed as Reward®. Sutton et al. (1970) found higher copper concentrations in aquatic hydrilla plant tissue when copper-sulfate was applied in combination with diquat-dibromide than when copper-sulfate was used alone. Plant weight was also decreased with the same chemical combination compared with diquat-dibromide itself

(Sutton et al. 1972). Because this dual exposure appears to increase chemical uptake in plants, one could expect that diquat could also increase the uptake of copper-sulfate in non-target organisms like larval amphibians. Because there are already documented negative effects of copper-sulfate on amphibians (Chen et al. 2007; García-Muñoz et al. 2008;

García-Muñoz et al. 2010; Gürkan and Hayretdağ 2012), these effects could potentially be enhanced by simultaneous applications of copper-sulfate and diquat-dibromide. However, in contrast to this prediction, results from this study showed that tadpoles exposed to this herbicide combination were less negatively affected than by copper-sulfate itself. A similar study performed in southern Florida investigated the effects of copper-sulfate and diquat-dibromide combination to treat hydrilla within a wildlife refuge, and found no negative effects on growth or survival of apple snails either under field or lab conditions (Winger et al. 1984). In any case,

14 previous studies have shown negative effects of diquat-dibromide on fish and tadpoles (Dial and

Dial 1987; Salah El-Deen and Rogers 1993); therefore, more information is needed on field applications of these two herbicides and other chemical blends, which may have unexpected negative effects on non-target plants and animals.

Diquat-dibromide is a nonselective herbicide that desiccates both terrestrial and aquatic plants when it comes into contact with them (Environmental Protection Agency, 1995; California

EPA, 2000). Diquat-dibromide specifically targets photosynthetic tissues in plants via electron interception during photosynthetic light reactions (Yocum and San Pietro 1969; Clayton 1996;

Koschnick and Haller 2006). Because amphibians lack these chloroplasts and photosynthetic tissues, one could hypothesize that diquat-dibromide may not be very toxic to these organisms.

However, prior studies have recorded numerous negative effects of diquat-dibromide on fish, such as lethargy, gill hemorrhages, avoidance behaviors, and a decrease in plasma and muscle protein (Leung et al. 1983; Salah El-Deen and Rogers 1993; Berry 1984). In contrast, other previous studies have failed to find negative effects of diquat-dibromide on eel and other fish species, as well as on amphibians, such as Lithobates tempolithobates and Bufo bufo (Mitra

1959; Mackenzie and Hall 1967; Blackburn and Weldon 1970; Cooke 1977; Yeo and Dechoretz

1976; Campbell et al. 2000; Tremblay 2004). Even though this study did not find any negative effects of diquat-dibromide on growth and survival of Lithobates pipiens tadpoles, Dial and Dial

(1987) found that a similar herbicide named paraquat dichloride caused lethargy and convulsive tremors to tadpoles of this species.

The lack of negative effects of diquat-dibromide on growth-related traits of L. pipiens in this study could have resulted from increased feeding by tadpoles to compensate for oxidative stress potentially induced by this herbicide (Dodge and Harris 1970; Abdollahi et al. 2004).

However, this is only a prediction that should be tested in future studies. Oxidative stress caused by this herbicide may affect lungs and respiratory systems (Gage 1968; Koschnick and Haller

2006). Tadpoles often compensate for this type of stress by increasing the rate of flow in their respiratory stream, and thereby, their food intake (Savage 1952; Kenny 1969; Cooke 1977; Feder et al. 1984; Jorgensen 2000). In the present experiment, it is possible that tadpoles exposed to diquat-dibromide could have altered their respiratory function, causing higher feeding rates and thus resulted in temporarily increased growth rates. During weekly water changes, there appeared to be more excrement at the bottom of containers housing tadpoles exposed to the diquat-dibromide treatment than in containers from other treatment groups (personal observation). This suggests that these tadpoles might have been consuming more food. However, no certainty exists that this was the case, as this prediction was not formally tested and intestinal contents of each tadpole were not weighed and examined in this experiment.

Because simultaneous application of copper-sulfate and diquat-dibromide to wildlife areas is a current common practice (Yeo and Dechoretz 1976; Timmons 2005; Murray-Gulde et al. 2008; Siemering et al. 2008; Gianou et al. 2012; Hasenbein et al. 2013), knowing if there are risks to non-target species is critical because there could be inadvertent chemical exposure.

Therefore, it is important to better understand what can make the simultaneous application of these two chemicals more potent in killing plants and algae, and determine if it can potentially affect sensitive amphibian species in a similar manner. This study represents a step toward understanding both the individual and combined effects of these two herbicides on tadpole growth and survival, and thus, it has important implications for the management of aquatic environments that provide habitats for a multitude of non-target species. Similar studies in the future should focus on longer-term effects of diquat-dibromide and copper-sulfate on amphibians

16 as well as the impact of other chemical blends over a wider range of environmentally relevant concentrations.

LITERATURE CITED

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Table 1. Mixed-model repeated measures ANOVA evaluating effects of treatment and time on tadpole body weight, body length, and tail length.

Source of Variation Body Weight Body Length Tail Length DF F DF F DF F

Treatment 3, 116 18.01* 3, 116 16.55* 3, 116 19.21*

Time 1, 385 122.52* 1, 385 201.54* 1, 282 286.29*

Time2 1, 385 394.00* 1, 385 342.84* 3, 282 0.17

Time × Treatment 3, 385 0.88 3, 385 0.49 1, 282 71.04*

Time2 × Treatment 3, 385 0.10 3, 385 0.23 3, 282 0.42

* P<0.0001

Figure 1. Effects of herbicide treatments on tadpole survival. Asterisks indicate significant differences in tadpole survival between each treatment and the control group from Fisher’s exact tests. * P<0.0001

Figure 2. Number of individuals that survived in each treatment group and measurement date. Asterisks indicate significant differences in tadpole survival between each treatment and the control group from Fisher’s exact tests. * P<0.0001

Figure 3. Effects of the herbicide diquat-dibromide, the algaecide copper-sulfate, and the combination of both chemicals on tadpole weight (a), length (b), and tail length (c) throughout the experiment. Asterisks indicate significant differences between treatment means and those from the control group. * P<0.05

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APPENDIX B: 2014 INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE APPROVAL