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Sublethal Effects of Sodium Nitrate on Developmental Rate and Body Length in Southern Toad (Bufo terrestris) Tadpoles


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SUBLETHAL EFFECTS OF SODIUM NITRATE ON DEVELOPMENTAL RATE AND BODY LENGTH IN SOUTHERN TOAD (Bufo terrestris ) TADPOLES By ESTHER MARIE LANGAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003

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ii ACKNOWLEDGMENTS I wish to thank my advisor, Dr. Max Nickerson, and my committee, Dr. Peter Frederick and Dr. Louis Guillette, Jr., for thei r invaluable guidance, advice, and support in this project. Dr. Frank Nordlie, Dr. La uren Chapman, and Dr. Gary Butcher provided essential equipment during the course of this study. I also wish to thank all of my friends, es pecially Daniel Janes, Patricia Gomez and Thea Edwards for their assistance during the course of this project and for providing adequate motivation when necessary. My gratitude is furt her extended to my undergraduate volunteers, without whom this project would not have been completed. I am indebted to my parents for their undyi ng support and encourag ement when morale was lacking.

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iii TABLE OF CONTENTS page ACKNOWLEDGMENTS..................................................................................................ii LIST OF FIGURES...........................................................................................................iv ABSTRACT....................................................................................................................... vi CHAPTER 1 AMPHIBIAN DECLINES AND NITRATES.............................................................1 Global Amphibian Declines.........................................................................................1 Nitrate Fertilizers..........................................................................................................2 Effects of Nitrates on Amphibians...............................................................................3 Summary.......................................................................................................................5 2 SOUTHERN TOAD (Bufo terrestris ) AND TADPOLE DEVELOPMENT AND GROWTH.....................................................................................................................7 Natural history..............................................................................................................7 Tadpole metamorphosis: growth and development......................................................8 3 SUBLETHAL EFFECTS OF SODIUM NITRATE ON DEVELOPMENTAL RATE AND BODY LENGTH IN SOUTHERN TOAD TADPOLES (Bufo terrestris ).....................................................................................................................11 Introduction.................................................................................................................11 Materials and Methods...............................................................................................13 Animals................................................................................................................13 Materials..............................................................................................................14 Experimental design............................................................................................15 Statistical Analysis..............................................................................................17 Results........................................................................................................................ .17 Discussion...................................................................................................................20 Future Research..........................................................................................................25 LIST OF REFERENCES...................................................................................................50 BIOGRAPHICAL SKETCH.............................................................................................55

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iv LIST OF FIGURES Figure page 3-1 Mean tadpole body length by treatment over time...................................................28 3-2 Mean body length by clutch in 0 mg/L NaNO3 treatment.......................................29 3-3 Mean body length by clutch in 5 mg/L NaNO3 treatment.......................................30 3-4 Mean body length by clutch in 15 mg/L NaNO3 treatment.....................................31 3-5 Mean body length by clutch in 15 mg/L NaNO3 treatment, excluding outlier........32 3-6 Mean body length by clutch in 30 mg/L NaNO3 treatment....................................33 3-7 Average time to metamorphosis per treatment ........................................................34 3-8 Mean tadpole Gosner stage by treatment over time.................................................35 3-9 Mean Gosner stage by clutch in 0 mg/L NaNO3 treatment......................................36 3-10 Mean Gosner stage by clutch in 5 mg/L NaNO3 treatment......................................37 3-11 Mean Gosner stage by clutch in 15 mg/L NaNO3 treatment....................................38 3-12 Mean Gosner stage by clutch in 15 mg/L NaNO3 treatment excluding outlier .......39 3-13 Mean Gosner stage by clutch in 30 mg/L NaNO3 treatment....................................40 3-14 Maximum and minimum temperatures for 0 mg/L NaNO3 treatment.....................41 3-15 Maximum and minimum temperatures for 5 mg/L NaNO3 treatment.....................42 3-16 Maximum and minimum temperatures for 5 mg/L NaNO3 treatment.....................42 3-17 Overall mortality ......................................................................................................43 3-18 Total number of dead tapdoles per replicate ............................................................43 3-19 Total number of dead tadpoles per clutch. ...............................................................44

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v 3-20 Proportion of deceased individuals, metamorphs, and tadpoles for 0 mg/L NaNO3 treatment......................................................................................................45 3-21 Proportion of deceased individuals, metamorphs, and tadpoles for 5 mg/L NaNO3 treatment......................................................................................................46 3-22 Proportion of deceased individuals, metamorphs, and tadpoles for 15 mg/L NaNO3 treatment......................................................................................................47 3-23 Proportion of deceased individuals, metamorphs, and tadpoles for 30 mg/L NaNO3 treatment......................................................................................................48 3-24 Proportion of new metamorphs................................................................................49

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vi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SUBLETHAL EFFECTS OF SODIUM NITRATE ON DEVELOPMENTAL RATE AND BODY LENGTH IN SOUTHERN TOAD (Bufo terrestris ) TADPOLES By Esther M. Langan August 2003 Chair: Max A. Nickerson Department: Wildlife Ecology and Conservation Agricultural industries are the major c ontributors of nonpoint nitrate pollution in the environment. Runoff from agricultural ar eas ultimately resides in aquatic habitats where amphibians breed and develop in Nort h America. Many studies have documented adverse effects of nitrates on amphibians in cluding mortality, edema, reduced feeding and swimming activity, spinal curvature, and reduced growth and developmental rates. My study used low levels of sodium nitrat e (0 mg/L, 5 mg/L, 15 mg/L, and 30 mg/L) on Southern toad tadpoles (Bufo terrestris ) to determine if there was an effect on body length and developmental rate. Levels used in this experiment reflect nitrate concentrations in areas where this species develops. Tadpol es were obtained from one population and three separate clutches. I staged and meas ured them every other day until all tadpoles had either metamorphosed (Gosner stage 45) or died. All tadpoles were at the same Gosner stage at the beginni ng of the experiment. Water temperature and pH were

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vii sampled frequently. Dissolved oxygen was measured randomly throughout the experiment. There was no relationship among water temperature, pH, dissolved oxygen and tadpole body length. Time to metamorphosis wa s significantly differe nt between 0 mg/L and 30 mg/L treatments, 5 mg/L and 30 mg /L treatments, and 15 mg/L and 30 mg/L treatments. Time to metamorphosis was not significantly differe nt among 0 mg/L, 5 mg/L, and 15 mg/L sodium nitrate treatment s. Water parameters were relatively consistent among tadpole tanks throughout the experiment. Nitrate treatment had no effect on tadpole body length (m m). It is possible that the population from which I obtained tadpoles contained animals tolerant to nitrate contamination (because of past selection favoring individuals more tolerant to nitrate). Individuals from this population may have higher phenotypic plasticity with resp ect to nitrate tolerance. It is also plausible that the levels used in the expe riment were too low to produce effects on body length (since published studies found effects in many species at much higher levels). Nitrate treatment accelerated developmenta l rate. Tadpoles may have accelerated development in order to escape an unfavorable habitat. Alternativel y, sodium nitrate may have stimulated developmental rate by in teracting with horm ones responsible for metamorphosis. Clutch had an effect on tadpole length and stag e, with one clutch developing more slowly than the other two clutches and exhibiting smaller body lengths throughout most of the experiment. This re sult could be due to genetic factors or nongenetic factors from maternal influences such as yolk content and egg size.

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1 CHAPTER 1 AMPHIBIAN DECLINES AND NITRATES Global Amphibian Declines Global decline in amphibian populations is a relatively recent concern (Barinaga 1990; Phillips 1990; Dunson et al. 1992; Howe et al. 1998). Although many extinctions have occurred, amphibian declines alarm sc ientists because they are occurring at unnaturally high rates and because anthr opogenic factors are most likely involved (Barinaga 1990; Hecnar 1995; Schuytema and Nebeker 1999a). Increased exposure to ultraviolet radiation due to ozone depletion has been studied inte nsively as a potential cause of decline, especially in amphibian populations that occur at higher elevations where ultraviolet radiat ion may have greater effects (Bla ustein et al. 1994; Hays et al 1996; Langhelle et al. 1999). Fungal and bacterial epidem ics, iridoviruses, droughts, temperature changes, habitat destruction and fragmentation, exotic species, heavy metals, acid rain, pesticides and fertilizers have al so been proposed as agents of amphibian decline (Barinaga 1990; Phillip s 1990; Dunson et al. 1992; Howe et al. 1998; Adams 1999; Hayes et al. 2002). Species-specific differences in response to (and tolerance of) these different agents further complicate research on amphibian declin es (Rouse et al. 1999; Hayes et al. 2002). In addition, most studies focus on individual ag ents and their effects on amphibians rather than including multiple agents in the study. Th is is important since agents most likely work synergistically to advers ely affect amphibian populations (Rouse et al. 1999). It is necessary to study thes e effects since amphibians are im portant predators and prey in

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2 their ecosystems. Such studies are also n ecessary to effectively conserve and monitor amphibian populations (Hecnar 1995). Nitrate Fertilizers Although nitrogen enters ecosystems from sewa ge effluents, industrial waste, urban runoff, atmospheric deposition, and livestock feed lots, agricultur al application of nitrogen-based fertilizers remains the ma jor nonpoint source of nitrates in the environment (Camargo & Ward 1995; Carp enter et al. 1998; Rouse et al. 1999). Nitrogen fertilizers are widely used in the United States and Canada. In less than 50 years, the use of nitrogen fertilizer in the United States has increased from 0.45 million metric tons to more than 9.98 million tons (Lanyon 1995). Nitrogen becomes present at high levels in aquatic systems as ammonia, am monium ion, nitrite, and nitrate. The most common form of nitrogen found in fe rtilizers is ammoni um nitrate, NH4NO3 (Oldham et al. 1997). Fertilizers are applied to agricultural crops to provide them with nitrogen, phosphate, and potassium needed for plant grow th. Agricultural ope rations tend to apply more fertilizer than necessary to the landscape, and the crops ar e not able to absorb all of the nutrients in the fertilizer (Hecnar 1995). This excess ferti lizer is either washed (via runoff) into aquatic habitats where embryos and larvae develop; or pe netrates the soil and ultimately contaminates groundwater (Hecnar 1995). Safe levels of nitrate in groundwater for humans have been designa ted at 10 mg/L nitrate (Camargo & Ward 1995). Exceeding this level may result in methemoglobinemia (blue-baby syndrome), cancer in humans, or neural tube defects in infants (Hecnar 1995; Rouse et al. 1999; Croen et al. 2001). Methemoglobinemia occurs when nitrate is converted to nitrite by symbiotic intestinal fauna, resulting in th e oxidation of hemoglobin to methemoglobin (a

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3 molecule that is unable to bind with oxygen). In aquatic ha bitats, nitrate levels below 10 mg/L produce mortality in salmonid eggs and fr y (Kincheloe et al. 1979), in two species of insects (Camargo and Ward 1995), and in amphibians (Rouse et al 1999). Therefore, when agricultural practices overapply fertilizers, the excess ultimately ends up in aquatic habitats where amphibians breed and lay their eggs; and where embryos and tadpoles develop (Meyer-Aurich et al. 1998). Effects of Nitrates on Amphibians Most research on the effects of nitrogen-b ased fertilizers has focused on lethal and sublethal effects of nitrates on embryonic and larval stages since they are considered to be more susceptible than adults to aquatic cont aminants. This is due to the restriction of larvae to aquatic habitats where fertilizer s can be concentrated during development; the permeability of the egg membrane; the presence of gills; and the highly cutaneous nature of their skin (Hecnar 1995). Sublethal effects of nitrat e-based fertilizers include deformities, smaller body size, decreased move ment, and decreased feeding activity that would make larvae more vulnerable to pr edators (Hecnar 1995; Watt and Oldham 1995; Xu and Oldham 1997; Laposta and Dunson 1998; Marco et al. 1999; Schuytema and Nebeker 1999a, 1999b; Rouse et al. 1999). Other indirect po pulation effects could result if chronically exposed individua ls survive to become adults and reproduce. Smaller body sizes can affect the ability of male amphibian s to attract mates and smaller females have been shown to lay fewer and smaller eggs than larger females (Watt & Oldham 1995). In general, the application of agricultural fertilizers occurs primarily in the spring when most amphibians breed and when embryos and larvae develop (Xu & Oldham 1997). Adult amphibians may, therefore, migr ate over landscapes where fertilizers were

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4 recently applied (Oldham et al. 1997). Howeve r, fertilizers’ effects on adult amphibians have not been studied intensively. The amount of fertilizer required to produce 50% mortality in test subjects varies among species and type of nitrogen-based fertilizer (ammonium, ammonium nitrate, sodium nitrate, or ammonium sulfate) (R ouse et al. 1999; Schuytema & Nebeker 1999a). Depending on the amount of fertilizer use d, sublethal effects can occur such as embryonic or larval deformities, cardiac or abdominal edema, disequilibrium, small body size at metamorphosis, and decreased sw imming and feeding activity (Hecnar 1995; Marco et al. 1999; Rouse et al. 1999; Schuytema & Nebeker 1999a). The larval deformity, curling defect, refers to the curvature of the larva’s tail. Curling defects occur when the vitelline membrane surrounding the embryo fails to expand during embryonic development; and forc es the growing spine to curl. The underlying cause of curling is not well unde rstood although one hypothesis suggests that pH affects osmotic potential within the vitell ine membrane. Anothe r hypothesis suggests that pH affects inhibition of a hatching enzyme that digests the vite lline membrane (Freda 1991; Laposata & Dunson 1998). Schuytema and Nebeker (1999a) found that Xenopus laevis (Anura: Pipidae) tadpoles exposed to 50.9 mg/L ammonium nitrate all exhibited curling defects. Since ammoni um nitrate is commonly used in agricultural landscapes, a lower pH may be present in aquatic sy stems where amphibians congregate, thus potentially producing curling def ects in larvae. Such deform ities likely affect foraging and swimming ability in tadpoles, which could increase the likelihood of mortality due to nutritional deficiencies, predation, and higher incidence of infection resulting from poor nutritional status (Andrn et al 1988). Population studies rela ting to this sublethal effect

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5 of low pH have not yet been conducted, but are necessary to determine long-term survivability of species. Other deformities of low pH such as tail retention and limb malformations in metamorphs would most lik ely increase predation risk because they would decrease mobility (Xu & Oldham 1997). Slow growth rates have been found in ma ny amphibian species exposed to varying levels of nitrate (Hecnar 1995; Xu & Oldham 1997; Schuytema & Nebeker 1999a). Hecnar (1995) proposed that a small size at metamorphosis was related to decreased foraging as a direct result of meth emoglobinemia in amphibian larvae. Methemoglobinemia would cause decreased oxyge n transport in larvae and would reduce activity levels, including foraging. Small si ze at metamorphosis may be avoided if the larva undergoes a longer metamo rphic period (Larson et al. 1998). However, both small size at metamorphosis and a long metamorphi c period carry with them the costs of increased susceptibility to predation and desiccation (Alf ord and Harris 1988). Smaller individuals may not reproduce as early as la rger individuals, thereby reducing fecundity (Alford and Harris 1988) and may have higher pr edation risks, higher rates of water loss, and a smaller range of viable prey items, a ll serving to decrease chances of survival (Crump 1980). A long metamorphic period ma y expose larvae to de siccation if larvae develop in ephemeral habitats and do not metamorphose before the habitat dries up (Hecnar 1995). Summary Researchers are investigating many po ssible causes of amphibian declines worldwide, particularly anthropogenic factors. Nitrate fertilizers are heavily used in agricultural practices. Water runoff of these fertilizers into amphibian habitats may be negatively affecting populations. Laboratory studies on several amphibian species using

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6 different nitrate fertilizers have resulted in tadpoles with slower growth, smaller size at metamorphosis, reduced feeding and swi mming activity, edema, curling defects and mortality. Studies need to focus on how nitrate contamination may be affecting amphibians in the wild. Mesocosm studi es are needed that mimic temporary pond communities. Such studies may elucidate di rect and indirect effects of nitrates on amphibians. In addition, since there is am ple evidence that nitrates produce adverse effects on amphibians, research should concentrate on determining the underlying mechanisms producing these effects. It is also essential to compare amphibian populations and species existing in high-usage areas of nitr ate fertilizer with those existing in areas with little or no nitrate fertil izer usage. Such studies may reveal possible adaptive tolerance of species exposed to constant levels of nitrate.

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7 CHAPTER 2 SOUTHERN TOAD (Bufo terrestris ) AND TADPOLE DEVELOPMENT AND GROWTH Natural History The southern toad Bufo terrestris is one of six species be longing to the americanus toad complex, the most broadly distributed group of the Bufo genus (Anura: Bufonidae) in North America. It is a common species that exists in the Coastal Plain of the United States from Louisiana south to Florida and north to the extrem e southeast of Virginia. An isolated population is also found in Sout h Carolina (Blair 1972, Conant and Collins 1998). Bufo terrestris is found throughout mainland Florid a and the lower Florida Keys. Toads inhabiting the Keys have a noticea bly smaller body size than those on the mainland. The typical snout vent length of mainland toads is 80 mm. In Florida, the southern toad is found in mesophytic hammo cks, scrubs, wet prairies, pinelands and urban and agricultural areas (Duellman and Schw artz 1958). It is nocturnal, spending the daytime hiding in burrows and the nighttime foraging (Conant and Collins 1998). Its diet consists mainly of invertebrates, particular ly beetles (Duellman a nd Schwartz 1958). In Florida, B terrestris breeds from March through August in any lentic body of water. Females are capable of laying thousands of eggs contained in two gela tinous strings. The strings are typically attached to submerged vegetation and multiple females may lay their eggs in the same area (Martof et al. 1980). Eggs hatch after a few days; and tadpoles school together in groups of between 100 and 1000 individuals (McDiarmid and Altig 1999). Bufo terrestris tadpoles prefer warmer temperat ures (> 30C) in their aquatic

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8 habitats (Noland and Ultsch 1981). Tadpoles metamorphose into 7 to 21 mm toadlets in approximately 1 to 2 months (Martof et al. 1980). Tadpole Metamorphosis: Growth and Development Metamorphosis is the change of an aquatic tadpole into a terrestrial adult. During this process, radical external and internal morphologica l and physiological changes occur primarily due to changes in several ho rmone levels including thyroid hormone (T3 and T4), thyroid-stimulating hormone (TSH), prolactin (PRL), corticosterone (B), aldosterone, deoxycorticosterone, estradiol (E2), and testosterone (T). Many other hormones (e.g. melatonin, somatostatin, and gonadotropin-releasi ng hormone) may also influence amphibian metamorphosis but conclusi ve scientific evidence is lacking (Shi 2000). Factors such as temperature (Hayes et al. 1993), water amount light, and density (Shi 2000) may also influe nce hormone levels. Tadpoles typically exhibit a period of maximum growth af ter hatching from the egg (Gosner Stages 25-41) that is accompanied by little development. During metamorphosis (Gosner Stages 42-46), however, significant de velopment occurs while little growth is obtained (McDiarmid and Altig 1999). Many factors affect developmental and growth rates including photoperiod, temperature, food abundance and quality, tadpole density, pH, and oxygen concentration. Temperature pr oduces the greatest effect on development and growth rates. Lower temperatures typically produce slower growth and developmental rates in anuran larvae with developmental rate being affected more than growth rate (Harkey and Semlitsch 1988; He rreid and Kinney 1967). Tadpoles raised at lower temperatures, however, are usually larger than those raised at higher temperatures since developmental rate is more sensitive to temperature than growth rate (Harkey and Semlitsch 1988; McDiarmid and Altig 1999; Smith-Gill and Berven 1979). Bufo

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9 terrestris tadpoles prefer the warmest temperatures in their aquatic environment; and often exhibit body temperatures greater than 30C. This confers faster developmental times to reach metamorphosis before pond desicc ation since this species typically breeds in ephemeral waters (Crump 1989; Noland and Ultsch 1981). Temperature also affects thyroid hormone, a major hormone produci ng metamorphosis in anurans. Lower temperatures slow down metamorphosis i nduced by thyroid hormone by requiring much higher levels of thyroid hormone in orde r for metamorphosis to occur (Harkey and Semlitsch 1988; Shi 2000). Therefore, variations in temperature can have significant effects on growth and developmental rates in tadpoles (Smith-Gill and Berven 1979). Tadpole density affects growth and developmen tal rates in that individuals raised in low-density conditions with high food availability tend to maximize growth rates and decrease developmental rate to achieve a maximum body size at metamorphosis. However, individuals raised in high-dens ity conditions where food abundance is low tend to exhibit lower growth rate s and higher developmental rates. These individuals generally achieve a smaller body size at metamo rphosis. Higher densities tend to affect growth rate more than developmental rate (Pough and Kamel 1984; Smith-Gill and Berven 1979; Wilbur and Collins 1973). In high food availability conditions more resources are allocated to growth. In low f ood conditions more resources are allocated to development to escape unfavorable conditions (Alford and Harris 1988). Individuals from high food conditions will metamorphos e later at larger body sizes while the majority of individuals from low food condi tions will metamorphose earlier at a smaller body size (McDiarmid and Altig 1999).

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10 As with temperature and density, pH can affect developmental and growth rates, thus affecting size at metamorphosis. Low pH levels produce slower growth (Beebee 1986; Cummins 1986; Freda and Dunson 1986; Grant and Licht 1993; Warner et al. 1991) and developmental rates (Cummins 1986; Grant and Licht 1993) in tadpoles of several anuran species. Metamorphs exposed to low pH have exhibite d tail retention and malformed limbs (Xu and Oldham 1997). A dditionally, Beebee (1986) determined that small tadpoles were more susceptible to adve rse effects of low pH than large tadpoles. Low pH also affects fertilization in adult Rana pipiens by decreasing sperm motility (Schlichter 1981).

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11 CHAPTER 3 SUBLETHAL EFFECTS OF SODIUM NITRATE ON DEVELOPMENTAL RATE AND BODY LENGTH IN SOUTHERN TOAD (Bufo terrestris ) TADPOLES Introduction Sublethal effects of various contaminants such as deformities, smaller body sizes at metamorphosis, and slower gr owth and developmental rates have been documented in many larval amphibians (Hecnar 1995; Xu & Oldham 1997). Sublethal effects have resulted specifically from sodium nitrate, a common type of agricultural fertilizer that appears in aquatic habitats due to runoff. Schuytema and Nebeker (1999a) found that 578 mg/L nitrate-nitrogen from sodium n itrate produced 50% mortality in Pseudacris regilla embryos (Anura: Hylidae). Mo rtality was attributed to the nitrate ion rather than the sodium ion since P. regilla embryos required much less ammonium nitrate (25 mg/L NH4-N) to produce 50% mortality. Additionally, 9% of the su rviving embryos from the sodium nitrate treatment regime exhibited edem a and curling defects. Smaller lengths in P regilla and Xenopus laevis (Anura: Pipidae) were seen at 111 mg/L nitrate-nitrogen from sodium nitrate (Schuytema and Nebeker 1999a). Mortality occurred in all Rana aurora (Anura: Ranidae) embyros at 918 mg/L n itrate from sodium nitrate. Embryo body length in this species was adversely affected at <29.1 mg/L nitrate-nitrogen (Schuytema and Nebeker 1999b). Sodium nitrate negatively affected growth rate and survival of Bufo bufo tadpoles at concentrations of 40 mg/L and 100 mg/L of the nitrate ion. Tadpole size increased with lower concentrations of the nitrate i on (Baker and Waights 1993). A concentration

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12 of 16.45 mg /L nitrate-nitrogen (from potassium nitrate) produced 50% mortality in Rana pretiosa Other effects included reduced feed ing, lethargy, disequilibrium, edema and curling defects at 16.45 mg/L or higher. Th e 10 mg/L nitrate maximum in U.S. drinking water was deemed toxic to Rana pretiosa (Marco et al. 1999). Hecnar (1995) found that 13.6 and 39.3 mg/L nitrate (from ammonium n itrate) was sufficient to produce 50% mortality in two differe nt populations of Bufo americanus tadpoles. At 2.5 mg/L nitrate, all tadpoles of three anuran species displayed less feed ing and swimming activity. Anuran species are affected differently by contaminant concentrations (Hecnar 1995; Marco et al. 1999). In this study, I exposed Bufo terrestris tadpoles to low levels (0, 5, 15, and 30 mg/L ) of sodium nitrate to determine if these leve ls produced adverse effects on body length and developmental ra te. These concentrations are 0, 0.824, 2.472, and 4.944 mg/L nitrate-nitroge n, respectively, based on molecular weight. These levels were chosen because they repres ent nitrate levels found in Bufo terrestris breeding habitats in Florida. I chose sodium nitrate as a contaminant since it is used frequently in agricultural practices. From available water data, pond nitrate-nitrogen amounts in Florida typically range from 0.1 to 1 mg/L alth ough values of 3 mg/L or higher have been reported (United States Geological Su rvey. NWISWeb Data for the Nation. http://waterdata.usgs.gov/nwis. Accessed Apr il 2004). Furthermore, since very few studies have used such low nitrate-nitrogen levels, this study will he lp determine a nitrate toxicity range for this species. I predicted that adverse effects would be mo re dramatic in the higher sodium nitrate treatment levels, and that adve rse effects would be manifested in tadpoles as shorter body lengths and retarded development. Most of the tadpoles in previous experiments have

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13 developmental times longer than 60 days. It is known that speci es are influenced differently by varying types and concentrations of sodium nitrate. Perhaps larval period length also influences how greatly a ta dpole is affected by a contaminant since individuals with faster growth di splay higher metabolic rates. Materials and Methods Animals I obtained seven pairs of amplexing Bufo terrestris adults at approximately 0200 hours on the morning of 1 June 2002. Pairs we re collected from a retention pond in a commercial area in Gainesville, Florida. Ampl ecting pairs were placed in separate plastic shoe boxes with approximately 2.54-3.81 cm of standing water from their native pond. Each of the seven pairs of Bufo terrestris laid eggs in the hours among time of capture and 1100 hours on 1 June 2002. Adults were re moved from the plas tic containers and released at their site of capture. One clut ch of eggs perished due to a possible fungal infection. At 2030 hours on the same day, the remaining six clutches of eggs were separately placed into 55-liter containers. Before egg introduction, the containers were filled with approximately 47 quarts of chlorine -free tap water. Each container was also furnished with two air pumps to provide ad equate oxygen flow in the water. Embryos remained in these containers until they reached a feeding and free-swimming tadpole stage, Stage 25 of the Gosner (1960) staging system, at whic h time I selected tadpoles for the experiment. Using a lottery, I randomly selected three of the six clutches to be used in the experiment and released the remaining three cl utches of tadpoles to their native pond. I used a total of 180 tadpoles in the experiment (60 tadpoles from each clutch). Tadpoles were selected for the experiment by dipping small fish nets in different areas in the clutch

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14 containers. I placed five tadpoles from each cl utch into each of the 12 tanks, providing a total of 15 tadpoles per tank. Materials I used 0 mg/L, 5 mg/L, 15 mg/L a nd 30 mg/L sodium nitrate (NaNO3) as my treatment levels. These levels as nitr ate-nitrogen are 0 mg/L, 0.824 mg/L, 2.472 mg/L, and 4.944 mg/L, respectively, calculated by mo lecular weight of nitrogen present in NaNO3. Levels chosen for use in this experi ment represent the low range of nitrate concentrations in Florida waters. Stock solutions of sodium nitrate were created immediately before the experiment so that each time water was to be changed, each tank would receive only 2 mL of stock solution from its respective treatment level. Another scientist labeled the solution bottles either E, F, H, or J so that I was blind to the concentrations. On 3 June 2002, I obtained twelve 10-g allon glass aquaria to house Bufo terrestris tadpoles. Each treatment had three replicates, producing a to tal of twelve aquaria. I washed and scrubbed the tanks with a dilute d bleach solution (~10% bleach) and then thoroughly rinsed them with fresh tap water. The next day, the tanks were again rinsed with fresh tap water and allowed to dry. On 5 June 2002, I set up the twelve 10-gallon tanks in an outdoor, open-air, covered setting. This sett ing provided tadpoles with ambient temperatures and some direct sun light throughout the expe riment but prevented the addition of rainwater to the tanks. I set up three 1-ga llon, cloth painting sieves in each tank Each sieve separated tadpole clutches within the tank. Sieves were attached to the top of the tanks using offi ce clips and the tanks were cove red with aluminum mesh to minimize the introduction of detritus and to prevent possible predation by insects. A 151-liter glass tank that was filled with tap water and constantly had air running through

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15 it using two air pumps served as a water rese rvoir that would be used to replenish tank water. Tadpoles were fed rabbit pellets ad libitum throughout the experiment. Experimental Design Using a lottery system, tanks were randomly assigned to one of four treatments: H, E, F, or J, representing 0 mg of sodium nitrate per liter, 5 mg/L, 15 mg/L, or 30 mg/L, respectively. Clutch order w ithin each tank was also randomi zed using a lottery so that clutch order and clutch neighbors within each tank would vary among tanks. I labeled each tank with its designated treatment letter and clutch order (e.g., 1 2 3 or 1 3 2). I assigned tanks a position in the lab setting and randomly moved them every other day throughout the experiment using a lottery system Clutch order within each tank did not change during the course of the experiment. Seven liters of water were added to each tank followed by two mL of sodium nitrate stock solution (either E, F, J, or H, depending on each tank’s previous random assignment). Tanks were agitated to thoroughly mix the solutions in the water. On 6 June 2002 (5 days after egg collect ion), all tadpoles were measured and staged. Body length (mm) was measured from th e tip of the snout to the posterior part of the body where the tail’s lateral line joined the body. Develo pmental staging of tadpoles was performed using a dissecting microscope and the Gosner (1960) staging system. I performed body length measurements and stagin g every other day. In order to stage and measure tadpoles, I removed each tadpole from its treatment tank and placed it on a water-saturated cloth. Body le ngths were determined using calipers and staging required that the tadpoles be placed under a bright light for microscope use. Tadpoles were typically out of water for one minute to obtain body length and stage data.

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16 Tank water was changed every other day, on the days when tadpoles were not staged or measured. Before changing wate r for each tank, tadpoles in each sieve were transferred to a small, plastic weighing boat by gently reversing the sieve. The tanks were rinsed with high-pressure water and sieves were also ri nsed out to remove leftover food. Again, seven liters of water were added per tank, followed by two mL of the appropriate nitrate solution. Wa ter that was added to the tanks was obtained from tap water that had been sitting in the 151-li ter tank for approximately 48 hours with air pumps aiding in chlorine removal. Tadpoles were added to each sieve and each sieve was placed in its corresponding position in its tank according to the previously arranged clutch order. One pellet of rabbit food was a dded to each clutch sieve and the tanks were again covered with aluminum mesh. Water, pH, and temperature per tank we re measured frequently throughout the experiment. On days when the water was changed, pH and temperature were measured twice: once before changing the water a nd once after adding new water and nitrate concentrations. This provided a range fo r these parameters during the experiment. Furthermore, the use of maximum/minimum temperature thermometers provided daily temperature ranges for one tank in each tr eatment. Every day the maximum/minimum thermometers were placed in a new tank within the same treatment level. Dissolved oxygen was measured randomly during the experiment. I checked tanks daily for tadpole mortality and abnormalities such as edema, erratic swimming, and body malformations. I recorded deceased or deformed tadpoles, noting treatment level (E, F, H, or J) and clutch number. Deceased tadpoles were removed from the experiment and a final body length and st age, if possible, we re recorded. Any

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17 tadpoles showing abnormalities were noted yet remained in the experiment. Towards the end of the experiment, each tank was also checked daily for new metamorphs (Gosner Stage 42, forelimb emergence). The presence of metamorphs was recorded, but they were only removed from the experiment when they reached Gosner Stage 45, when Bufo terrestris tadpoles have fully functioning lungs (Wassersug and Seibert 1975). The experiment lasted a total of 33 days, until a ll of the tadpoles had either reached Gosner Stage 45 (four limbs present, tail stub present, terrestrial stage) or died. Statistical Analysis I calculated average body le ngths per clutch in each tank. A square root transformation was performed to linearize the data. Comparisons of body length among clutch and treatment were performed over time using a mixed linear model in SAS. This determined whether clutch, treatment, or ta nk influenced body length in tadpoles. Water, pH, and temperature were also compared among tanks in the mixed linear model to determine any possible relationship with ta dpole body length. Time to metamorphosis (days) was analyzed per tadpole using a mixe d linear model as well. Stage 42 (forelimb emergence) was considered metamorphosis since this stage is easily recognizable. Results One clutch of eggs perished, presumably due to a fungal inf ection, as noted by a white, opaque covering on the eggs. There wa s no significant difference in body length (F = 0.79, p = 0.5063) among treatments (Fig. 3-1) or among tanks (p = 0.28). There was a clutch effect on body length (p = 0.0006) over time (Figs. 3-2 3-6). Figure 3-5 excludes an outlier tadpole in the 15 mg/L treatment. This tadpole remained at Stage 25 throughout the entire experiment. Elimina tion of this tadpole produced much lower standard errors. Towards the end of the e xperiment this individual’s body began swelling

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18 and became transparent. On Day 28, body le ngth (BL) of this tadpole was 10.29 mm while its body width (BW) was 8.56 mm. On Day 31, its BL was the longest at 11.91 mm with the widest BW of 12.00 mm. On Day 33, it had a BL of 10.86 mm and a BW of 10.30 mm. Ultimately, its body swelling produced a width practically the same dimension as its length, an abnormal feature for a typical tapdole in this species. Body lengths of Clutch 2 were typically smaller than Clutches 1 and 3. However, after the elapse of three-quarters of th e experimental time, Clutch 2 in each treatment typically attained an equal or greater si ze than either or both Clut ches 1 and 3. This typically occurred when Clutches 1 and 3 were in metamorphic climax. Time to metamorphosis was significan tly different among control and 30 mg/L tadpoles (p = 0.0346), 5 mg/L and 30 mg/L ta dpoles (p = 0.0334), and 15 mg/L and 30 mg/L tadpoles (p = 0.0499). Time to meta morphosis was not significantly different among control and 5 mg/L tadpoles (p = 0.9787), control and 15 mg/L tadpoles (p = 0.6814), and 5 mg/L and 15 mg/L tadpoles (p = 0.6954). Average time to metamorphosis per treatment is depicted in figure 3-7 Figure 3-8 depicts the average Gosner stage per treatment per day. A clutch effect on tim e to metamorphosis was not present (p = 0.2190). Figures 3-9 3-13 depict mean Gosner Stage of tadpoles by clutch over time. Figure 3-12 exludes the outlier tadpole from Clutch 1 which maintained Gosner Stage 25 throughout the entire experiment. Dissolved oxygen appeared constant among tanks as well (Table 3-1). Temperature and pH did not vary significantl y during the course of the experiment and exhibited no apparent relationship with tadpole body length (p = 0.2810, p = 0.3628).

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19 These environmental factors are known to influe nce life history traits such as size and developmental rate. Days 13 and 26 had the highest temperatures at about 31C and 30C (Figs. 3-14 3-16). Differences in maximum and minimum treatment temperatures can be attributed to variation in thermometers and tank placem ent. Some tanks may have received more sunlight on a given day than other tanks Possible confounding e ffects of the latter variability were minimized in the experi ment by randomly moving tank position every other day. Mortality was unequal among treatments. Mortality was 42.2%, 17.8%, 15.6%, and 28.9% in the sodium nitrate treatments from lowest to highest c oncentrations. Clutch 1 had the highest mortality and Clutch 3 ha d the lowest mortality overall (Fig. 3-17). Mortality was also present in each replicate (tank) with no apparent pattern (Fig. 3-18). No replicate exhibited mortality of all 15 tadpoles. Clutch 1 tadpoles had higher mortality than Clutch 2 or 3 tadpoles, rega rdless of treatment regime, and Clutch 2 tadpoles had higher mortality than Clutch 3 ta dpoles in all treatmen ts with the exception of the 5 mg/L sodium nitrate treatment (Fig. 319). In this instance, Clutch 2 and 3 tadpoles had equal mortality (one tadpole each). Higher proportions of mortality in the control and 30 mg/L treatments are reflected in lower proportions of total metamorphs throughout the experiment (Figs. 3-20 – 3-23) Proportion of metamorphs was higher for 5 mg/L and 15 mg/L treatment levels due to decreased amount of mortality. Metamorphs began appearing by Day 16 of the experi ment. Clutch 1 tadpoles typically metamorphosed before Clutches 2 and 3 (Fig. 3-24). The majority of Clutch 1 tadpoles metamorphosed among Days 16 and 18 with th e greatest proportion occurring on Day 16.

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20 The majority of Clutch 2 tadpoles metamorphosed among Days 18 and 20 with the greatest proportion occurring on Day 20. The majority of Clutch 3 tadpoles metamorphosed among Days 18 and 20 also. Un like Clutch 2, however, the greatest proportion of metamorphs for Clutch 3 occurred on Day 18. Discussion Concentrations of 5 mg/L, 15 mg/L a nd 30 mg/L sodium nitrate (0.824 mg/L, 2.472 mg/L, and 4.944 mg/L nitrate-nitrogen) produ ced no observable effects on body length in Bufo terrestris tadpoles. Tadpoles in di fferent tanks had statistically similar body lengths, suggesting that variation as a result of tank set up was minimal, if presen t at all. Tadpoles of different treatment regimes exhibited st atistically similar bo dy lengths throughout the course of the experiment. Higher concentra tions of nitrate can produce negative effects on body length in this species as seen in other literature (Hec nar 1995; Schuytema and Nebeker 1999a and 1999b). However, exposing earlier stages (egg and embryo) of Bufo terrestris to the experimental concentrations may produce the proposed lethal and sublethal effects since earlier stages may differ in their susceptibility to nitrate contamination (Xu and Oldham 1997). Temp erature and pH, factors known to greatly influence body length and developmental rate did not vary greatly among tanks during the course of the experiment. Time to metamorphosis differed among the 30 mg/L sodium nitrate treatment and all other treatments. Tadpoles exposed to 30 mg/L sodium nitrate obtained Gosner Stage 42 in the shortest amount of time. Tadpoles exposed to 15 mg/L sodi um nitrate achieved forelimb emergence before 5 mg/L and c ontrol tadpoles. Control tadpoles reached Gosner Stage 42 before 5 mg/L tadpoles. Thes e data suggest that higher concentrations of sodium nitrate (30 mg/L and 15 mg/L) accelerated developmental rate in Bufo

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21 terrestris tadpoles. It is possible that the ta dpoles in the 30 mg/L treatment increased developmental rate to escape the unfavorable habitat created by sodi um nitrate. Another possibility is that a hormetic response occurred such th at these low levels of sodium nitrate stimulated developmental rate by interacting with hormones responsible for metamorphosis. Hormesis refers to stimul atory effects of contaminants at low doses followed by inhibitory effects at higher c ontaminant levels (Ba iler et al. 2003). Contaminants such as cadmium, polycyclic aromatic hydrocarbons, and dioxin have been shown to produce hormetic effects in some species (Calabrese and Baldwin 2003). Boone et al. (2001) found that the insecticid e, carbaryl, accelerated development in Rana clamitans tadpoles. Although a faster developmental rate may be beneficial in instances where tadpoles are in a habitat that will quickly dry up, it may produce smaller body sizes at metamorphosis that could have negative e ffects on fecundity and survival (Alford and Harris 1988; Wilbur and Collins 1973). All tadpoles used in the experiment originated from a single population in the wild. Hecnar (1995) report ed that 13.6 mg/L of NH4-N was sufficient to produce 50% mortality in one population of Bufo americanus tadpoles while another population required 39.3 mg/L of NH4-N to produce 50% mortality. The latter population originated from an area where intensive agricultur e occurs, suggesting that this population developed resistance to ammonium nitrate. Laurila et al. (20 02) observed significant differences in body size at metamorphos is and larval period length in Rana temporaria tadpoles. In their study, tadpoles from a northern population did not vary these life history traits with respect to water quantity while tadpol es from a southern population exhibited faster developmental times and smaller body sizes when exposed to quickly

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22 decreasing water volume. In nature, the s outhern population inhabits ponds which dry out frequently and erratically. The northern population, however, inhabits ponds that are not prone to complete drying. Since populational differences exist, determining lethal and sublethal effects of contaminants based on one or a few populations may not apply to all populations within a speci es, resulting in conservati on efforts that may not be adequate to preserve the whole species. In a survey of 26 Florida springs, nitrate (NO3-N) ranged from 1.10 to 38.00 mg/L (Katz et al. 1999). Total nitrogen in four lakes (Wauberg, Watermelon Pond, Orange and Newnan) in Alachua County, Florida ranged from a minimum of 0.273 to a maximum of 11.500 mg/L (Lakewatch 2001). Nitrate amounts (as NO3) in Florida ponds typically range from 0.1 to 1 mg/L, although some ponds have nitrate amounts at the 3 mg/L range or higher (USGS 2003). One pond in Gainesvi lle, Florida (Alachua County) where B terrestris bred in 2003 had 0.0048 mg/L nitratenitrogen. The pond where I obtained breeding adults for use in this experiment was also located in Gainesville, Florida. This pond was not situated near agricultural sites a nd likely exhibited nitr ate levels similar to other Florida ponds (< 1 mg/L). The levels used in this experiment were ecologically relevant at 0 mg/L, 0.824 mg/L, 2.472 mg/L, and 4.944 mg/L nitrate-nitrogen. Although Bufo terrestris tadpole body length was not adve rsely affected by the nitrate concentrations in this study, higher con centrations may produce smaller body lengths since studies that used higher levels found such an effect. Clutch affected tadpole body length during the course of this experiment. This could be due to genetic variation or nongeneti c variation from matern al influences such as egg size and nutrient input in egg yolk (Travis 1980) Travis (1980) observed a

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23 genetic effect on growth rates in Hyla gratiosa by cross-breeding a limited number of males and females and observing the growth rate s in offspring. Semlitsch et al. (2000) performed a cross-breeding experiment with Hyla versicolor and concluded that genetic variation in tadpoles, rather than maternal influences, largely affected tadpol e tolerance to the insecticide carbaryl. However, a separa te, crossbreeding experi ment would have to be conducted to conclusively state whether clutch differences in body length and Gosner stage of Bufo terrestris are due to genetic effects. Tadpoles from Clutch 2 typically had smaller body lengths than tadpoles from either Clutch 1 or 3 until a bout Day 24 in the control and 5 mg/L treatment, Days 18-22 and Day 28 for 15 mg/L, and Day 22 for 30 mg/L A potential explanation for Clutch 2 tadpoles having longer body lengths later in the experiment c ould be the decrease in body length during metamorphic climax, Gosner Stages 42-46 (Emerson 1986). Since tadpoles in Clutches 1 and 3 were ahead in devel opmental stages they also had smaller body lengths when in Gosner Stages 42-46 (met amorphic climax). Tadpoles from Clutch 2 typically had slightly slower development than tadpoles from Clutches 1 and 3, although clutch did not have a statistically significant effect on time to metamorphosis. Since density and competition were not fact ors, longer larval periods did not appear to translate into smaller body sizes at metamorphosis. At the beginning of the experiment, all tadpoles from Clutch 2 were sm aller than tadpoles from Clutches 1 and 3. At the end of the experiment, Clutch 2 ta dpoles in the control group achieved larger body lengths than Clutch 1 or 3 ta dpoles (Fig. 3-2). In the 5 mg/L and 30 mg/L treatments, Clutch 2 tadpoles were still smaller than Clutch 1 or 3 tadpole s at the end of the experiment, although the differe nce was not as large as it was at the onset of the

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24 experiment (Figs. 3-3 and 3-6). At the end of the experiment, Clutch 2 tadpoles in the 15 mg/L treatment level were larger than Clut ch 3 tadpoles. However, they were still smaller than Clutch 1 tadpoles although the size difference was not as great as at the onset of the experiment (Fig. 3-5). Clutch 2 tadpoles also had s lightly longer larval periods although this was not st atistically significant. Indi viduals with longer larval periods spend more time in a stable environment and maximize growth at the risk of increased mortality from predation and desi ccation thereby achieving a larger body size at metamorphosis (Alford and Harris 1988; Hecnar 1995). Although most Clutch 2 tadpoles were not larger than Clutch 1 or 3 tadpoles, th eir body lengths did increase during the course of the experiment such that their final lengths showed much less disparity among Clutches 1 and 3 as compared to the beginning of the experiment. One tadpole from the 15 mg/L treatment ( 2.472 mg nitrate/L) had a grossly swollen and transparent body. Hecnar (1995) noted ma ny tadpoles with a similar condition when he exposed them to chronic levels (2.5, 5, a nd 10 mg/L) of nitrate-n itrogen and suggested that nitrate produced the defo rmity by affecting tadpole osmo regulation. It is possible that nitrate produced the sa me condition in my experiment. Although only one tadpole exhibited the swelling, it may have been more susceptible to nitrate since individuals vary in their response to contaminants. Howe ver, since only one tadpole exhibited this condition in my experiment, it is uncertain what the causitive agent may have been. The fact that the control a nd 30 mg/L treatments had the highest mortality appear to be a stochastic event unrelat ed to nitrate concentration. All water parameters among treatments were similar, there was no vari ability in body length, and all tadpoles were handled equally by the inves tigators. Length of larval period cannot account for

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25 mortality since control tadpoles required appr oximately the same amount of time to reach metamorphosis as tadpoles in the 5 and 15 mg/L treatments. Furthermore, since tadpoles in these groups reached metamorphosis at a pproximately the same time, handling stress from body measurements and staging among these groups would have been fairly consistent and cannot account for mortality differences. A scientific explanation for Clutch 1 displaying the greatest mortality in each treatment is also lacking and may be stochastic as well. Similar studies which st aged tadpoles typically did so with much less frequency. Semlitsch and Schmiedehausen (1994) used only one stage measurement (Gosner Stage 42, forelimb emergence) for a measure of developmental rate. Performing fewer tadpole measurements would certai nly decrease stress to the animals and, therefore, possibly reduce mortality. Future Research Determining effects of varying nitrate le vels on all life stages of amphibians inhabiting areas where such contamination occurs is essential to prevent possible population and species declines Since many amphibian species breed in temporary bodies of water, future experiments should compare effects of nitrates among several populations that inhabit a range of nitrate-contaminated and uncontaminated areas, using levels consistent with those found in the fiel d. Such levels are likely to be much lower than those in current literatu re. Data on nitrate concentra tions in temporary bodies of water are relatively scant, therefore this type of study would add to that body of literature as well by providing such data. Additionally, scant information exists on nitrate effects on developmental rate in amphibians. If n itrate levels decrease developmental rates, amphibians inhabiting temporary ponds may be in danger of dessiccation since metamorphosis may not be completed by the time their pond dries completely. If nitrates

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26 increase developmental rates, it may lead to individuals with smaller body sizes at metamorphosis since a longer la rval period typically produces larger metamorphs (Alford and Harris 1988; McDiarmid and Altig 1999). This could reduce fitness if smaller females produce fewer and smaller eggs (Wa tt and Oldham 1995), ultimately resulting in reduced amphibian populations. Comparing effects of differen t types of nitrate would be useful since they produce varying effects on amphibians. Ammonium n itrate, for example, is more toxic to amphibians than sodium nitrate, owing to th e ammonium ion and is used extensively in agricultural settings (Sc huytema and Nebeker 1999b). Effects on fecundity and fertilization are also lacking in the litera ture with only one st udy linking low pH to decreased sperm motility (Schli cther 1981). Decreased sperm motility could negatively affect population growth in habitats heavily contaminated with ammonium nitrate. Ultimately, many factors work synergistically in the environment to create adverse effects. Without the addition of contaminants, larval mortality is relatively high so that very few individuals reach reproductive ages. With the added stress of contaminants, mortality likely will increase and reduce populations in the futu re (Semlitsch et al. 2000). Although contaminants may select for resist ant individuals in the population, allowing the population to persist and grow, loss of ge netic diversity could reduce adaptiveness of the population to future selective pressures. Pesticides, fertilizers, competition, food abundance and water quality all likely have complex roles in developmental and growth rates of larval amphibians. It is necessary to understand the impact of each factor independently and interdependently to predic t the future of amphibian populations and create realistic conservation goals.

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27 More detailed studies comparing contamin ant effects on species with different life history characteristics need to be undertaken as well. Bufo terrestris exhibits synchronous hatching and metamorphosis. Studies have shown that high density and low food abundance can affect development and gr owth in other species by producing smaller metamorphs (Smith-Gill and Berven 1979; Wil bur and Collins 1973). Nitrate has been shown to affect body length, larval period lengt h, and feeding activity in other species as well (Hecnar 1995; Marco et al. 1997; Oldham et al. 1997; Rouse et al. 1999). It may be reasonable, therefore, to conc lude that nitrate contamina tion may differentially affect species with varying life hist ory characteristics such as population density and time to metamorphosis, producing more drastic effect s in species that breed synchronously in ephemeral bodies of water compared to spec ies with asychronous br eeding in permanent bodies of water. Since temper ature can greatly affect the le ngth of the larval period, a laboratory experiment using tadpoles of the same species with varying nitrate concentrations and three temperature regimes may be useful in determining how larval period may interact with contaminants. Mesocosms are experimental tank commun ities that mimic natural temporary pond conditions but provide more control, manipulation, and accurate censusing data than natural ponds (Morin 1983). Mesocosm studi es would be particularly useful in determining indirect nitrate effects on ta dpoles resulting from algal effects, a major tadpole food source. Mesocosm experiments may more accurately reflect contaminant interactions in nature and provide more insight into co mplex ecological interactions of contaminants in nature.

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28Figure 3-1. Mean tadpole body length by treatm ent over time. Data were pooled among cl utches for each treatment. Data points represent repeated measures of body le ngth throughout the experiment. Day re presents time since the start of the experiment when tadpoles were first expos ed to nitrate treatments. As tadpoles reached metamorphosis (Gosner Stage 45) in the experiment, their final body length before release was used in calculating the averages for the duration of the experiment. All standard errors (+ SE) per day were less than 0.20. 3 4 5 6 7 8 9 10 11 2468101214161820222426283133 DayMean body length (m m 0 mg/L 5 mg/L 15 mg/L 30 mg/L

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29Figure 3-2. Mean body length of tadpoles by clut ch in 0 mg/L sodium nitrate treatment. Each clutch represents a separate grou p of siblings. Day refers to time since the st art of the experiment. Error bars represen t standard error. Standard error ranged from 0.065 to 0.89. 3 4 5 6 7 8 9 10 11 12 2468101214161820222426283133 DayMean body length (m m Clutch 1 Clutch 2 Clutch 3

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30Figure 3-3. Mean body length of tadpoles by clut ch in 5 mg/L sodium nitrate treatment. Each clutch represents a separate grou p of siblings. Day refers to time since the start of the experiment. Error bars repr esent standard error. 3 4 5 6 7 8 9 10 11 12 2468101214161820222426283133 DayBody length (m m Clutch 1 Clutch 2 Clutch 3

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31Figure 3-4. Mean body length of tadpoles by clut ch in 15 mg/L sodium nitrate treatment. Each clutch represents a separate gro up of siblings. Day refers to time since the start of th e experiment. Error bars represent standard error. 3 4 5 6 7 8 9 10 11 12 2468101214161820222426283133 DayBody length (m m Clutch 1 Clutch 2 Clutch 3

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32Figure 3-5. Mean tadpole body length of tadpoles by clutch in 15 mg/L NaNO3 treatment, excluding outlier fr om Clutch 1. Data were pooled among tadpoles for each clutch and each clutch represents a separate group of siblings. Day refers to time since the start of the experiment Error bars represent + SE. Elimination of the outlier tadpole produced slightly larger body lengths for Clutch 1 tadpoles on some days. 3 4 5 6 7 8 9 10 11 12 2468101214161820222426283133 DayBody length (m m Clutch 1 Clutch 2 Clutch 3

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33Figure 3-6. Mean body length of tadpoles by clut ch in 30 mg/L sodium nitrate treatment. Each clutch represents a separate gro up of siblings. Day refers to time since the start of th e experiment. Error bars represent standard error. 3 4 5 6 7 8 9 10 11 12 246810121416182022242628 DayBody length (m m Clutch 1 Clutch 2 Clutch 3

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34 Figure 3-7. Average time to metamorphosis (days) per treatment. Metamorphosis was defined as Gosner Stage 42 (forelimb emergence). Error bars represent standard error (+SE). 17 17.5 18 18.5 19 19.5 20 TreatmentTime to metamorphosis (day 0 mg/L 5 mg/L 15 mg/L 30 mg/L

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35 Figure 3-8. Mean tadpole Gosner stage by tr eatment over time. Data were pooled among cl utches for each treatment. Data points represent repeated measures of Gosner stage throughout the experiment. Day re presents time since the start of the experiment when tadpoles were first expos ed to nitrate treatments. As tadpoles reached metamorphosis (Gosner Stage 45) in the experiment, their final Gosner st age before release was used in calculating the averag es for the duration of the experiment. All standard errors (+ SE) per day were less than 0.55. 26 28 30 32 34 36 38 40 42 44 46 2468101214161820222426283133 DayMean Gosner stag e 0 mg/L 5 mg/L 15 mg/L 30 mg/L

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36Figure 3-9. Mean Gosner stage of tadpoles by clutch in 0 mg/L s odium nitrate treatment. Each clut ch represents a separate grou p of siblings. Day refers to time since the start of th e experiment. Error bars represent standard error. 24 26 28 30 32 34 36 38 40 42 44 46 2468101214161820222426283133 DayMean Gosner stag e Clutch 1 Clutch 2 Clutch 3

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37Figure 3-10. Mean Gosner stage of tadpoles by clutch in 5 mg/L sodium n itrate treatment. Each clutch represents a separate gro up of siblings. Day refers to time since the start of th e experiment. Error bars represent standard error. 24 26 28 30 32 34 36 38 40 42 44 46 2468101214161820222426283133 DayMean Gosner stag e Clutch 1 Clutch 2 Clutch 3

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38Figure 3-11. Mean Gosner stage of tadpoles by clutch in 15 mg/L sodium nitrate treatment. Each clut ch represents a separate gr oup of siblings. Day refers to time since the start of th e experiment. Error bars represent standard error. 24 26 28 30 32 34 36 38 40 42 44 46 2468101214161820222426283133 DayMean Gosner stag e Clutch 1 Clutch 2 Clutch 3

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39Figure 3-12. Mean Gosner stage of tadpoles by clutch in 15 mg/L sodium nitrate trea tment excluding one individual from Clutch 1 that did not develop and remained at Stage 25 throughout the experiment. Elim ination of this individual produced a smaller standard error for Clutch 1 than represented in figure 9. Each clutch represents a se parate group of siblings. Day refers to time since the start of the experime nt. Error bars represent standard error. 24 26 28 30 32 34 36 38 40 42 44 46 2468101214161820222426283133 DayMean Gosner stag e Clutch 1 Clutch 2 Clutch 3

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40Figure 3-13. Mean Gosner stage of tadpoles by clutch in 30 mg/L sodium n itrate treatment. Each clut ch represents a separate g roup of siblings. Day refers to time since the start of th e experiment. Error bars represent standard error. 24 26 28 30 32 34 36 38 40 42 44 46 24681012141618202224262831 DayMean Gosner stag e Clutch 1 Clutch 2 Clutch 3

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41 Table 3-1. Dissolved oxygen samples from ra ndom days per treatment and tank within treatment. Signify tanks that have completed experime ntation and are therefore empty. Values are amount of dissolved oxygen in ppm (mg/L). Each value represents one measurement per tank. Figure 3-14. Maximum and minimum temperatures ( C) for 0 mg/L sodium nitrate treatment. 0 mg/L5 mg/L15 mg/L30 mg/L TankTankTankTank Day123123123123 158.838.818.868.808.808.808.818.808.208.798.788.77 168.528.488.508.528.538.548.518.528.548.518.518.51 218.428.398.408.408.408.408.438.418.418.328.378.33 28**9.808.90*9.30*9.20***9.20 31**8.108.20*8.20*8.50**** 33**8.60**8.40*8.40**** Treatment 20 22 24 26 28 30 32 34 356791315161718192021232425262728293133 DayTemperature (C) Tmax Tmin

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42 Figure 3-15. Maximum and minimum temperatures ( C) for 5 mg/L sodium nitrate treatment. Figure 3-16. Maximum and minimum temperatures ( C) for 15 mg/L sodium nitrate treatment. 20 22 24 26 28 30 32 34 356791315161718192021232425262728293133 DayTemperature (C) Tmax Tmin 20 22 24 26 28 30 32 34 356791315161718192021232425262728293133 DayTemperature (C) Tmax Tmin

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43 Figure 3-17. Overall mortality in each treatment per clutch. Figure 3-18. Total number of dead tapdoles pe r replicate (single ta nk) within treatment 0 1 2 3 4 5 6 7 8 051530 Sodium Nitrate Treatment (mg/L)Mortalit y Clutch 1 Clutch 2 Clutch 3 0 1 2 3 4 5 6 7 8 9 123 Replicate (Tank Number)Total number dea d 0 mg/L 5 mg/L 15 mg/L 30 mg/L

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44 Figure 3-19. Total number of dead ta dpoles per clutch within treatment. 0 2 4 6 8 10 12 123 ClutchTotal number of dea d 0 mg/L 5 mg/L 15 mg/L 30 mg/L

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45 Figure 3-20. Proportion of deceased indi viduals, metamorphs, and tadpoles for 0 mg/L sodium nitrate treatment. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 12345678910111213141516171819202122232425262728 DayProportio n Deceased Metamorphs Tadpoles

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46 Figure 3-21. Proportion of deceased indi viduals, metamorphs, and tadpoles for 5 mg/L sodium nitrate treatment. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 12345678910111213141516171819202122232425262728293031 DayProportion Deceased Metamorphs Tadpoles

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47 Figure 3-22. Proportion of deceased indi viduals, metamorphs, and tadpoles for 15 mg/L sodium nitrate treatment. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 12345678910111213141516171819202122232425262728293031323334 DayProportion Deceased Metamorphs Tadpoles

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48 Figure 3-23. Proportion of deceased indi viduals, metamorphs, and tadpoles for 30 mg/L sodium nitrate treatment. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 12345678910111213141516171819202122232425262728 DayProportio n Deceased Metamorphs Tadpoles

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49 0.0 0.1 0.2 0.3 0.4 0.5 1516171819202122232425262728 DayProportion of new metamorphs Clutch 1 Clutch 2 Clutch 3 Figure 3-24. Proportion of ne w metamorphs (Gosner Stage 42, forelimb emergence) each day per clutch. Proportions are nu mber of new metamorphs per day for each clutch divided by total number of metamorphs each clutch ultimately produced.

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52 Hays, J. B., A. R. Blaustein, J. M. Kieseck er, D. P. Chivers, and R. G. Anthony. 1996. Developmental response of amphibians to solar and artificial UVB sources: a comparative study. Photochemistry and Photobiology 64(3): 449-456. Hayes, T., R. Chan, and P. Licht. 1993. Inter actions of temperature and steroids on larval growth, development, and metamorphosis in a toad (Bufo boreas ). Journal of Experimental Zoology 266:206-215. Hayes, T., K. Haston, M. Tsui, A. Hoang, and A. V. Haeffele. 2002. Feminization of male frogs in the wild. Nature 419:895-896. Hecnar, S. J. 1995. Acute and chronic toxi city of ammonium n itrate fertilizer to amphibians from southern Ontario. Environmental Toxicology and Chemistry 14(12):2131-2137. Herreid II, C. F. and S. Kinney. 1967. Temp erature and development of the wood frog, Rana sylvatica in Alaska. Ecology 48(4):579-590. Howe, G. E., R. Gillis, and R. C. Mowbray. 1998. Effect of chemical synergy and larval stage on the toxicity of at razine and alachlor to amphi bian larvae. Environmental Toxicology and Chemistry 17(3):519-525. Katz, B. G., H. D. Hornsby, J. F. B ohlke, and M. F. Mokray. 1999. Sources and chronology of nitrate contamination in spring waters, Suwannee River Basin, Florida. U. S. Geological Survey Wate r-Resources Investigations Report 99-4252. Kincheloe, J. W., G. A. Wedemyer, and D. L. Koch. 1979. Tolerance of developing salmonid eggs and fry to nitrate e xposure. Bulletin of Environmental Contamination and Toxicology 23:575-578. Langhelle, A., J. Lindell-Mans, and P. Nystro m. 1999. Effects of u ltraviolet radiation on amphibian embryonic and larval developm ent. Journal of Herpetology 33(3): 449456. Lanyon, L. E. 1995. Does nitrogen cycle?: Ch anges in the spatial dynamics of nitrogen following the industrialization of nitr ogen fixation. Journal of Productivity Analysis 8:70-78. Laposata, M. M., and W. A. Dunson. 1998. Effects of boron and nitrate on hatching success of amphibian eggs. Archives of Environmental Contamination and Toxicology 35:615-619. Larson, D. L., S. McDonald, A. J. Fivizzani, W. E. Newton, and S. J. Hamilton. 1998. Effects of the herbicide atrazine on Ambystoma tigrinum metamorphosis: duration, larval growth, and hormonal response. Physiological Zoology 71(6):671-679.

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53 Laurila, A., S. Karttunen, and J. Meri l. 2002. Adaptive phenotypic plasticity and genetics of larval lif e histories in two Rana temporaria populations. Evolution 56(3):617-627. Marco, A., C. Quilchano, and A. R. Blaustein. 1999. Sensitivity to nitrate and nitrite in pond-breeding amphibians from the Pacifi c Northwest, USA. Environmental Toxicology and Chemistry 18(12):2836-2839. Martof, B. S., W. M. Palmer, J. R. Bailey, and J. R. Harrison III. 1980. Amphibians and reptiles of the Carolinas and Virginia. The University of North Carolina Press, Chapel Hill. McDiarmid, R. W. and R. Altig. 1999. Ta dpoles: the Biology of Anuran Larvae. The University of Chicago Press, Chicago. Meyer-Aurich, A., P. Zander., A. Werner, and R. Roth. 1998. Developing agricultural land use strategies appropriate to natu re conservation goals and environmental protection. Landscape and Ur ban Planning 41:119-127. Morin, P. J. 1983. Predation, competition, a nd the composition of larval anuran guilds. Ecological Monographs 53(2):119-138. Noland, R. and G. R. Ultsch. 1981. The roles of temperature and dissolved oxygen in microhabitat selection by the tadpoles of a frog (Rana pipiens ) and a toad (Bufo terrestris ). Copeia 1981:645-652. Oldham, R. S., D. M. Latham, D. Hilton-Brow n, M. Towns, A. S. Cooke, and A. Burn. 1997. The effect of ammonium nitrate fertiliser on frog (Rana temporaria ) survival. Agriculture, Ecosystems and Environment 61:69-74. Phillips, K. 1990. Where have all the frogs and toads gone? Biosci ence 40(6):422-424. Pough, F. H. and S. Kamel. 1984. Post-metamorphic change in activity metabolism of anurans in relation to life history. Oecologia 65:138-144. Rouse, J. D., C. A. Bishop, and J. Struger. 1999. Nitrogen Pollution: An assessment of its threat to amphibian surviv al. Environmental Health Perspectives 107(10): 799-803. Schlichter, L. C. 1981. Low pH affects th e fertilization and development of Rana pipiens eggs. Canadian Journal of Zoology 59:1693-1699. Schuytema, G. S., and A. V. Nebeker. 1999a Comparative effects of ammonium and nitrate compounds on Pacific treefrog and Af rican clawed frog embryos. Archives of Environmental Contamination and Toxicology 36:200-206. Schuytema, G. S., and A. V. Nebeker. 1999b. Effects of ammonium nitrate, sodium nitrate, and urea on red-legged frogs, Paci fic treefrogs, and African clawed frogs. Bulletin of Environmental Contam ination and Toxicology 63:357-364.

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54 Semlitsch, R. D., C. M. Bridges, and A. M. Welch. 2000. Genetic variation and a fitness tradeoff in the tolerance of gray treefrog (Hyla versicolor ) tadpoles to the insecticide carbaryl. Oecologia 125:179-185. Semlitsch, R. D. and S. Schmiedehausen. 1994. Parental contributions to variation in hatchling size and its relati onship to growth and metamorphosis in tapdoles of Rana lessonae and Rana esculenta Copeia 1994(2):406-412. Shi, Y. 2000. Amphibian Metamorphosis: Fr om Morphology to Molecular Biology. John Wiley and Sons, Inc., New York. Smith-Gill, S. J. and K. A. Berven. 1979. Predicting amphibian metamorphosis. The American Naturalis t 113(4):563-585. Travis, J. 1980. Genetic variation for larval specific growth rate in the frog Hyla gratiosa Growth 44:167-181. Warner, S.C., W. A. Dunson, a nd J. Travis. 1991. Interaction of pH, density, and priority effects on the surviviorship a nd growth of two species of hylid tadpoles. Oecologia 88:331-339. Wassersug, R. J. and E. A. Seibert. 1975. Behavioral responses of amphibian larvae to variation in dissolved ox ygen. Copiea 1975(1):86-103. Watt, P. J., and R. S. Oldham, R. S. The e ffect of ammonium nitr ate on the feeding and development of larvae of the smooth newt, Triturus vulgaris (L.), and on the behaviour of its food source, Daphnia Freshwater Biology 33:319-324. Wilbur, H. M. and J. P. Collins. 1973. Ecol ogocial aspects of amphibian metamorphosis. Science 182:1305-1314. Xu, Q. and R. S. Oldham. 1997. Lethal a nd sublethal effects of nitrogen fertilizer ammonium nitrate on common toad (Bufo bufo ) tadpoles. Archives of Environmental Contamination and Toxicology 32(3): 298-303.

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55 BIOGRAPHICAL SKETCH Esther M. Langan was born in Melbourne, Florida, on 25 February 1975. An Air Force upbringing moved her to Germany, Virg inia, North Dakota, and finally Tampa, Florida where she has lived most of her life She received her Bachelor’s of Science degree from the Department of Wildlife Ec ology and Conservation at the University of Florida in 1997.


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Copyright Date: 2008

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SUBLETHAL EFFECTS OF SODIUM NITRATE ON DEVELOPMENTAL RATE
AND BODY LENGTH IN SOUTHERN TOAD (Bufo terrestris) TADPOLES
















By

ESTHER MARIE LANGAN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2003















ACKNOWLEDGMENTS

I wish to thank my advisor, Dr. Max Nickerson, and my committee, Dr. Peter

Frederick and Dr. Louis Guillette, Jr., for their invaluable guidance, advice, and support

in this project. Dr. Frank Nordlie, Dr. Lauren Chapman, and Dr. Gary Butcher provided

essential equipment during the course of this study.

I also wish to thank all of my friends, especially Daniel Janes, Patricia Gomez and

Thea Edwards for their assistance during the course of this project and for providing

adequate motivation when necessary. My gratitude is further extended to my

undergraduate volunteers, without whom this project would not have been completed. I

am indebted to my parents for their undying support and encouragement when morale

was lacking.
















TABLE OF CONTENTS
page

A C K N O W L E D G M E N T S .................................................................................................. ii

LIST OF FIGURES ......... ............................... ........ ............ iv

ABSTRACT .............. ......................................... vi

CHAPTER

1 AMPHIBIAN DECLINES AND NITRATES ................................... ...............1

G lob al A m phibian D declines .............................................................. ..................... 1
Nitrate Fertilizers ................ ........ ....... .......... 2
Effects of N itrates on A m phibians ........................................ ............................... 3
S u m m ary ................................................................................................... . 5

2 SOUTHERN TOAD (Bufo terrestris) AND TADPOLE DEVELOPMENT AND
G R O W T H .................................................................................... . 7

Natural history .......................... .... ............ ...............7
Tadpole metamorphosis: growth and development........................................................8

3 SUBLETHAL EFFECTS OF SODIUM NITRATE ON DEVELOPMENTAL
RATE AND BODY LENGTH IN SOUTHERN TOAD TADPOLES (Bufo
te rre stri s) ............................................................................................................... 1 1

In tro d u ctio n ............................................................................................. 1 1
M materials and M methods ....................................................................... .................. 13
A n im als....................................................13
M a te ria ls .....................................................................................................1 4
Experimental design .................. ............................. ........... .... .......... 15
Statistical A naly sis .......................................... ............ ...... ........17
R e su lts ...........................................................................................1 7
D isc u ssio n ............................................................................................................. 2 0
F utu re R research ................................................................2 5

LIST OF REFERENCES ................................................................. ...........50

B IO G R A PH IC A L SK E T C H ....................................................................................... 55
















LIST OF FIGURES


Figure page

3-1 Mean tadpole body length by treatment over time............................................28

3-2 Mean body length by clutch in 0 mg/L NaNO3 treatment ....................................29

3-3 Mean body length by clutch in 5 mg/L NaNO3 treatment ....................................30

3-4 Mean body length by clutch in 15 mg/L NaNO3 treatment ...................................31

3-5 Mean body length by clutch in 15 mg/L NaNO3 treatment, excluding outlier ........32

3-6 Mean body length by clutch in 30 mg/L NaNO3 treatment ..................................33

3-7 Average time to metamorphosis per treatment ................................ ............... 34

3-8 Mean tadpole Gosner stage by treatment over time............... .....................35

3-9 Mean Gosner stage by clutch in 0 mg/L NaNO3 treatment.............................. 36

3-10 Mean Gosner stage by clutch in 5 mg/L NaNO3 treatment.............................. 37

3-11 Mean Gosner stage by clutch in 15 mg/L NaNO3 treatment.............................. 38

3-12 Mean Gosner stage by clutch in 15 mg/L NaNO3 treatment excluding outlier .......39

3-13 Mean Gosner stage by clutch in 30 mg/L NaNO3 treatment.............................. 40

3-14 Maximum and minimum temperatures for 0 mg/L NaNO3 treatment ...................41

3-15 Maximum and minimum temperatures for 5 mg/L NaNO3 treatment ...................42

3-16 Maximum and minimum temperatures for 5 mg/L NaNO3 treatment ...................42

3-17 O overall m mortality ...................... ...................... .. .. ......... ......... 43

3-18 Total number of dead tapdoles per replicate .................................... .................43

3-19 Total number of dead tadpoles per clutch. ..................................... ............... 44









3-20 Proportion of deceased individuals, metamorphs, and tadpoles for 0 mg/L
N aN O 3 treatm ent. ................................................ .. .... ........ ......... 45

3-21 Proportion of deceased individuals, metamorphs, and tadpoles for 5 mg/L
N aN O 3 treatm ent ......................... ...................... .. .... ...... .. ........... 46

3-22 Proportion of deceased individuals, metamorphs, and tadpoles for 15 mg/L
N aN O 3 treatm ent. ................................................ .. .... ........ ......... 47

3-23 Proportion of deceased individuals, metamorphs, and tadpoles for 30 mg/L
N aN O 3 treatm ent ......................... .... .................. .. .... ..... .. ........... 48

3-24 Proportion of new metamorphs ................... ................................ 49















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

SUBLETHAL EFFECTS OF SODIUM NITRATE ON DEVELOPMENTAL RATE
AND BODY LENGTH IN SOUTHERN TOAD (Bufo terrestris) TADPOLES

By

Esther M. Langan

August 2003

Chair: Max A. Nickerson
Department: Wildlife Ecology and Conservation

Agricultural industries are the major contributors of nonpoint nitrate pollution in

the environment. Runoff from agricultural areas ultimately resides in aquatic habitats

where amphibians breed and develop in North America. Many studies have documented

adverse effects of nitrates on amphibians including mortality, edema, reduced feeding

and swimming activity, spinal curvature, and reduced growth and developmental rates.

My study used low levels of sodium nitrate (0 mg/L, 5 mg/L, 15 mg/L, and 30 mg/L) on

Southern toad tadpoles (Bufo terrestris) to determine if there was an effect on body length

and developmental rate. Levels used in this experiment reflect nitrate concentrations in

areas where this species develops. Tadpoles were obtained from one population and

three separate clutches. I staged and measured them every other day until all tadpoles

had either metamorphosed (Gosner stage 45) or died. All tadpoles were at the same

Gosner stage at the beginning of the experiment. Water temperature and pH were









sampled frequently. Dissolved oxygen was measured randomly throughout the

experiment.

There was no relationship among water temperature, pH, dissolved oxygen and

tadpole body length. Time to metamorphosis was significantly different between 0 mg/L

and 30 mg/L treatments, 5 mg/L and 30 mg/L treatments, and 15 mg/L and 30 mg/L

treatments. Time to metamorphosis was not significantly different among 0 mg/L, 5

mg/L, and 15 mg/L sodium nitrate treatments. Water parameters were relatively

consistent among tadpole tanks throughout the experiment. Nitrate treatment had no

effect on tadpole body length (mm). It is possible that the population from which I

obtained tadpoles contained animals tolerant to nitrate contamination (because of past

selection favoring individuals more tolerant to nitrate). Individuals from this population

may have higher phenotypic plasticity with respect to nitrate tolerance. It is also

plausible that the levels used in the experiment were too low to produce effects on body

length (since published studies found effects in many species at much higher levels).

Nitrate treatment accelerated developmental rate. Tadpoles may have accelerated

development in order to escape an unfavorable habitat. Alternatively, sodium nitrate may

have stimulated developmental rate by interacting with hormones responsible for

metamorphosis. Clutch had an effect on tadpole length and stage, with one clutch

developing more slowly than the other two clutches and exhibiting smaller body lengths

throughout most of the experiment. This result could be due to genetic factors or

nongenetic factors from maternal influences such as yolk content and egg size.














CHAPTER 1
AMPHIBIAN DECLINES AND NITRATES

Global Amphibian Declines

Global decline in amphibian populations is a relatively recent concern (Barinaga

1990; Phillips 1990; Dunson et al. 1992; Howe et al. 1998). Although many extinctions

have occurred, amphibian declines alarm scientists because they are occurring at

unnaturally high rates and because anthropogenic factors are most likely involved

(Barinaga 1990; Hecnar 1995; Schuytema and Nebeker 1999a). Increased exposure to

ultraviolet radiation due to ozone depletion has been studied intensively as a potential

cause of decline, especially in amphibian populations that occur at higher elevations

where ultraviolet radiation may have greater effects (Blaustein et al. 1994; Hays et al.

1996; Langhelle et al. 1999). Fungal and bacterial epidemics, iridoviruses, droughts,

temperature changes, habitat destruction and fragmentation, exotic species, heavy metals,

acid rain, pesticides and fertilizers have also been proposed as agents of amphibian

decline (Barinaga 1990; Phillips 1990; Dunson et al. 1992; Howe et al. 1998; Adams

1999; Hayes et al. 2002).

Species-specific differences in response to (and tolerance of) these different agents

further complicate research on amphibian declines (Rouse et al. 1999; Hayes et al. 2002).

In addition, most studies focus on individual agents and their effects on amphibians rather

than including multiple agents in the study. This is important since agents most likely

work synergistically to adversely affect amphibian populations (Rouse et al. 1999). It is

necessary to study these effects since amphibians are important predators and prey in









their ecosystems. Such studies are also necessary to effectively conserve and monitor

amphibian populations (Hecnar 1995).

Nitrate Fertilizers

Although nitrogen enters ecosystems from sewage effluents, industrial waste, urban

runoff, atmospheric deposition, and livestock feed lots, agricultural application of

nitrogen-based fertilizers remains the major nonpoint source of nitrates in the

environment (Camargo & Ward 1995; Carpenter et al. 1998; Rouse et al. 1999).

Nitrogen fertilizers are widely used in the United States and Canada. In less than 50

years, the use of nitrogen fertilizer in the United States has increased from 0.45 million

metric tons to more than 9.98 million tons (Lanyon 1995). Nitrogen becomes present at

high levels in aquatic systems as ammonia, ammonium ion, nitrite, and nitrate. The most

common form of nitrogen found in fertilizers is ammonium nitrate, NH4N03 (Oldham et

al. 1997).

Fertilizers are applied to agricultural crops to provide them with nitrogen,

phosphate, and potassium needed for plant growth. Agricultural operations tend to apply

more fertilizer than necessary to the landscape, and the crops are not able to absorb all of

the nutrients in the fertilizer (Hecnar 1995). This excess fertilizer is either washed (via

runoff) into aquatic habitats where embryos and larvae develop; or penetrates the soil and

ultimately contaminates groundwater (Hecnar 1995). Safe levels of nitrate in

groundwater for humans have been designated at 10 mg/L nitrate (Camargo & Ward

1995). Exceeding this level may result in methemoglobinemia (blue-baby syndrome),

cancer in humans, or neural tube defects in infants (Hecnar 1995; Rouse et al. 1999;

Croen et al. 2001). Methemoglobinemia occurs when nitrate is converted to nitrite by

symbiotic intestinal fauna, resulting in the oxidation of hemoglobin to methemoglobin (a









molecule that is unable to bind with oxygen). In aquatic habitats, nitrate levels below 10

mg/L produce mortality in salmonid eggs and fry (Kincheloe et al. 1979), in two species

of insects (Camargo and Ward 1995), and in amphibians (Rouse et al. 1999). Therefore,

when agricultural practices over-apply fertilizers, the excess ultimately ends up in aquatic

habitats where amphibians breed and lay their eggs; and where embryos and tadpoles

develop (Meyer-Aurich et al. 1998).

Effects of Nitrates on Amphibians

Most research on the effects of nitrogen-based fertilizers has focused on lethal and

sublethal effects of nitrates on embryonic and larval stages since they are considered to

be more susceptible than adults to aquatic contaminants. This is due to the restriction of

larvae to aquatic habitats where fertilizers can be concentrated during development; the

permeability of the egg membrane; the presence of gills; and the highly cutaneous nature

of their skin (Hecnar 1995). Sublethal effects of nitrate-based fertilizers include

deformities, smaller body size, decreased movement, and decreased feeding activity that

would make larvae more vulnerable to predators (Hecnar 1995; Watt and Oldham 1995;

Xu and Oldham 1997; Laposta and Dunson 1998; Marco et al. 1999; Schuytema and

Nebeker 1999a, 1999b; Rouse et al. 1999). Other indirect population effects could result

if chronically exposed individuals survive to become adults and reproduce. Smaller body

sizes can affect the ability of male amphibians to attract mates and smaller females have

been shown to lay fewer and smaller eggs than larger females (Watt & Oldham 1995).

In general, the application of agricultural fertilizers occurs primarily in the spring

when most amphibians breed and when embryos and larvae develop (Xu & Oldham

1997). Adult amphibians may, therefore, migrate over landscapes where fertilizers were









recently applied (Oldham et al. 1997). However, fertilizers' effects on adult amphibians

have not been studied intensively.

The amount of fertilizer required to produce 50% mortality in test subjects varies

among species and type of nitrogen-based fertilizer (ammonium, ammonium nitrate,

sodium nitrate, or ammonium sulfate) (Rouse et al. 1999; Schuytema & Nebeker 1999a).

Depending on the amount of fertilizer used, sublethal effects can occur such as

embryonic or larval deformities, cardiac or abdominal edema, disequilibrium, small body

size at metamorphosis, and decreased swimming and feeding activity (Hecnar 1995;

Marco et al. 1999; Rouse et al. 1999; Schuytema & Nebeker 1999a).

The larval deformity, curling defect, refers to the curvature of the larva's tail.

Curling defects occur when the vitelline membrane surrounding the embryo fails to

expand during embryonic development; and forces the growing spine to curl. The

underlying cause of curling is not well understood although one hypothesis suggests that

pH affects osmotic potential within the vitelline membrane. Another hypothesis suggests

that pH affects inhibition of a hatching enzyme that digests the vitelline membrane (Freda

1991; Laposata & Dunson 1998). Schuytema and Nebeker (1999a) found that Xenopus

laevis (Anura: Pipidae) tadpoles exposed to 50.9 mg/L ammonium nitrate all exhibited

curling defects. Since ammonium nitrate is commonly used in agricultural landscapes, a

lower pH may be present in aquatic systems where amphibians congregate, thus

potentially producing curling defects in larvae. Such deformities likely affect foraging

and swimming ability in tadpoles, which could increase the likelihood of mortality due to

nutritional deficiencies, predation, and higher incidence of infection resulting from poor

nutritional status (Andren et al. 1988). Population studies relating to this sublethal effect









of low pH have not yet been conducted, but are necessary to determine long-term

survivability of species. Other deformities of low pH such as tail retention and limb

malformations in metamorphs would most likely increase predation risk because they

would decrease mobility (Xu & Oldham 1997).

Slow growth rates have been found in many amphibian species exposed to varying

levels of nitrate (Hecnar 1995; Xu & Oldham 1997; Schuytema & Nebeker 1999a).

Hecnar (1995) proposed that a small size at metamorphosis was related to decreased

foraging as a direct result of methemoglobinemia in amphibian larvae.

Methemoglobinemia would cause decreased oxygen transport in larvae and would reduce

activity levels, including foraging. Small size at metamorphosis may be avoided if the

larva undergoes a longer metamorphic period (Larson et al. 1998). However, both small

size at metamorphosis and a long metamorphic period carry with them the costs of

increased susceptibility to predation and desiccation (Alford and Harris 1988). Smaller

individuals may not reproduce as early as larger individuals, thereby reducing fecundity

(Alford and Harris 1988) and may have higher predation risks, higher rates of water loss,

and a smaller range of viable prey items, all serving to decrease chances of survival

(Crump 1980). A long metamorphic period may expose larvae to desiccation if larvae

develop in ephemeral habitats and do not metamorphose before the habitat dries up

(Hecnar 1995).

Summary

Researchers are investigating many possible causes of amphibian declines

worldwide, particularly anthropogenic factors. Nitrate fertilizers are heavily used in

agricultural practices. Water runoff of these fertilizers into amphibian habitats may be

negatively affecting populations. Laboratory studies on several amphibian species using









different nitrate fertilizers have resulted in tadpoles with slower growth, smaller size at

metamorphosis, reduced feeding and swimming activity, edema, curling defects and

mortality. Studies need to focus on how nitrate contamination may be affecting

amphibians in the wild. Mesocosm studies are needed that mimic temporary pond

communities. Such studies may elucidate direct and indirect effects of nitrates on

amphibians. In addition, since there is ample evidence that nitrates produce adverse

effects on amphibians, research should concentrate on determining the underlying

mechanisms producing these effects. It is also essential to compare amphibian

populations and species existing in high-usage areas of nitrate fertilizer with those

existing in areas with little or no nitrate fertilizer usage. Such studies may reveal possible

adaptive tolerance of species exposed to constant levels of nitrate.














CHAPTER 2
SOUTHERN TOAD (Bufo terrestris) AND TADPOLE DEVELOPMENT AND
GROWTH

Natural History

The southern toad Bufo terrestris is one of six species belonging to the americanus

toad complex, the most broadly distributed group of the Bufo genus (Anura: Bufonidae)

in North America. It is a common species that exists in the Coastal Plain of the United

States from Louisiana south to Florida and north to the extreme southeast of Virginia. An

isolated population is also found in South Carolina (Blair 1972, Conant and Collins

1998). Bufo terrestris is found throughout mainland Florida and the lower Florida Keys.

Toads inhabiting the Keys have a noticeably smaller body size than those on the

mainland. The typical snout vent length of mainland toads is 80 mm. In Florida, the

southern toad is found in mesophytic hammocks, scrubs, wet prairies, pinelands and

urban and agricultural areas (Duellman and Schwartz 1958). It is nocturnal, spending the

daytime hiding in burrows and the nighttime foraging (Conant and Collins 1998). Its diet

consists mainly of invertebrates, particularly beetles (Duellman and Schwartz 1958). In

Florida, B. terrestris breeds from March through August in any lentic body of water.

Females are capable of laying thousands of eggs contained in two gelatinous strings. The

strings are typically attached to submerged vegetation and multiple females may lay their

eggs in the same area (Martof et al. 1980). Eggs hatch after a few days; and tadpoles

school together in groups of between 100 and 1000 individuals (McDiarmid and Altig

1999). Bufo terrestris tadpoles prefer warmer temperatures (> 30C) in their aquatic









habitats (Noland and Ultsch 1981). Tadpoles metamorphose into 7 to 21 mm toadlets in

approximately 1 to 2 months (Martof et al. 1980).

Tadpole Metamorphosis: Growth and Development

Metamorphosis is the change of an aquatic tadpole into a terrestrial adult. During

this process, radical external and internal morphological and physiological changes occur

primarily due to changes in several hormone levels including thyroid hormone (T3 and

T4), thyroid-stimulating hormone (TSH), prolactin (PRL), corticosterone (B),

aldosterone, deoxycorticosterone, estradiol (E2), and testosterone (T). Many other

hormones (e.g. melatonin, somatostatin, and gonadotropin-releasing hormone) may also

influence amphibian metamorphosis but conclusive scientific evidence is lacking (Shi

2000). Factors such as temperature (Hayes et al. 1993), water amount, light, and density

(Shi 2000) may also influence hormone levels.

Tadpoles typically exhibit a period of maximum growth after hatching from the egg

(Gosner Stages 25-41) that is accompanied by little development. During metamorphosis

(Gosner Stages 42-46), however, significant development occurs while little growth is

obtained (McDiarmid and Altig 1999). Many factors affect developmental and growth

rates including photoperiod, temperature, food abundance and quality, tadpole density,

pH, and oxygen concentration. Temperature produces the greatest effect on development

and growth rates. Lower temperatures typically produce slower growth and

developmental rates in anuran larvae with developmental rate being affected more than

growth rate (Harkey and Semlitsch 1988; Herreid and Kinney 1967). Tadpoles raised at

lower temperatures, however, are usually larger than those raised at higher temperatures

since developmental rate is more sensitive to temperature than growth rate (Harkey and

Semlitsch 1988; McDiarmid and Altig 1999; Smith-Gill and Berven 1979). Bufo









terrestris tadpoles prefer the warmest temperatures in their aquatic environment; and

often exhibit body temperatures greater than 30C. This confers faster developmental

times to reach metamorphosis before pond desiccation since this species typically breeds

in ephemeral waters (Crump 1989; Noland and Ultsch 1981). Temperature also affects

thyroid hormone, a major hormone producing metamorphosis in anurans. Lower

temperatures slow down metamorphosis induced by thyroid hormone by requiring much

higher levels of thyroid hormone in order for metamorphosis to occur (Harkey and

Semlitsch 1988; Shi 2000). Therefore, variations in temperature can have significant

effects on growth and developmental rates in tadpoles (Smith-Gill and Berven 1979).

Tadpole density affects growth and developmental rates in that individuals raised in

low-density conditions with high food availability tend to maximize growth rates and

decrease developmental rate to achieve a maximum body size at metamorphosis.

However, individuals raised in high-density conditions where food abundance is low tend

to exhibit lower growth rates and higher developmental rates. These individuals

generally achieve a smaller body size at metamorphosis. Higher densities tend to affect

growth rate more than developmental rate (Pough and Kamel 1984; Smith-Gill and

Berven 1979; Wilbur and Collins 1973). In high food availability conditions more

resources are allocated to growth. In low food conditions more resources are allocated to

development to escape unfavorable conditions (Alford and Harris 1988). Individuals

from high food conditions will metamorphose later at larger body sizes while the

majority of individuals from low food conditions will metamorphose earlier at a smaller

body size (McDiarmid and Altig 1999).









As with temperature and density, pH can affect developmental and growth rates,

thus affecting size at metamorphosis. Low pH levels produce slower growth (Beebee

1986; Cummins 1986; Freda and Dunson 1986; Grant and Licht 1993; Warner et al.

1991) and developmental rates (Cummins 1986; Grant and Licht 1993) in tadpoles of

several anuran species. Metamorphs exposed to low pH have exhibited tail retention and

malformed limbs (Xu and Oldham 1997). Additionally, Beebee (1986) determined that

small tadpoles were more susceptible to adverse effects of low pH than large tadpoles.

Low pH also affects fertilization in adult Rana pipiens by decreasing sperm motility

(Schlichter 1981).














CHAPTER 3
SUBLETHAL EFFECTS OF SODIUM NITRATE ON DEVELOPMENTAL RATE
AND BODY LENGTH IN SOUTHERN TOAD (Bufo terrestris) TADPOLES

Introduction

Sublethal effects of various contaminants such as deformities, smaller body sizes at

metamorphosis, and slower growth and developmental rates have been documented in

many larval amphibians (Hecnar 1995; Xu & Oldham 1997). Sublethal effects have

resulted specifically from sodium nitrate, a common type of agricultural fertilizer that

appears in aquatic habitats due to runoff. Schuytema and Nebeker (1999a) found that

578 mg/L nitrate-nitrogen from sodium nitrate produced 50% mortality in Pseudacris

regilla embryos (Anura: Hylidae). Mortality was attributed to the nitrate ion rather than

the sodium ion since P. regilla embryos required much less ammonium nitrate (25 mg/L

NH4-N) to produce 50% mortality. Additionally, 9% of the surviving embryos from the

sodium nitrate treatment regime exhibited edema and curling defects. Smaller lengths in

P. regilla and Xenopus laevis (Anura: Pipidae) were seen at 111 mg/L nitrate-nitrogen

from sodium nitrate (Schuytema and Nebeker 1999a). Mortality occurred in all Rana

aurora (Anura: Ranidae) embyros at 918 mg/L nitrate from sodium nitrate. Embryo body

length in this species was adversely affected at <29.1 mg/L nitrate-nitrogen (Schuytema

and Nebeker 1999b).

Sodium nitrate negatively affected growth rate and survival of Bufo bufo tadpoles

at concentrations of 40 mg/L and 100 mg/L of the nitrate ion. Tadpole size increased

with lower concentrations of the nitrate ion (Baker and Waights 1993). A concentration









of 16.45 mg /L nitrate-nitrogen (from potassium nitrate) produced 50% mortality in Rana

pretiosa. Other effects included reduced feeding, lethargy, disequilibrium, edema and

curling defects at 16.45 mg/L or higher. The 10 mg/L nitrate maximum in U.S. drinking

water was deemed toxic to Rana pretiosa (Marco et al. 1999). Hecnar (1995) found that

13.6 and 39.3 mg/L nitrate (from ammonium nitrate) was sufficient to produce 50%

mortality in two different populations ofBufo americanus tadpoles. At 2.5 mg/L nitrate,

all tadpoles of three anuran species displayed less feeding and swimming activity.

Anuran species are affected differently by contaminant concentrations (Hecnar

1995; Marco et al. 1999). In this study, I exposed Bufo terrestris tadpoles to low levels

(0, 5, 15, and 30 mg/L ) of sodium nitrate to determine if these levels produced adverse

effects on body length and developmental rate. These concentrations are 0, 0.824, 2.472,

and 4.944 mg/L nitrate-nitrogen, respectively, based on molecular weight. These levels

were chosen because they represent nitrate levels found in Bufo terrestris breeding

habitats in Florida. I chose sodium nitrate as a contaminant since it is used frequently in

agricultural practices. From available water data, pond nitrate-nitrogen amounts in

Florida typically range from 0.1 to 1 mg/L although values of 3 mg/L or higher have been

reported (United States Geological Survey. NWISWeb Data for the Nation.

http://waterdata.usgs.gov/nwis. Accessed April 2004). Furthermore, since very few

studies have used such low nitrate-nitrogen levels, this study will help determine a nitrate

toxicity range for this species.

I predicted that adverse effects would be more dramatic in the higher sodium nitrate

treatment levels, and that adverse effects would be manifested in tadpoles as shorter body

lengths and retarded development. Most of the tadpoles in previous experiments have









developmental times longer than 60 days. It is known that species are influenced

differently by varying types and concentrations of sodium nitrate. Perhaps larval period

length also influences how greatly a tadpole is affected by a contaminant since

individuals with faster growth display higher metabolic rates.

Materials and Methods

Animals

I obtained seven pairs of amplexing Bufo terrestris adults at approximately 0200

hours on the morning of 1 June 2002. Pairs were collected from a retention pond in a

commercial area in Gainesville, Florida. Amplecting pairs were placed in separate plastic

shoe boxes with approximately 2.54-3.81 cm of standing water from their native pond.

Each of the seven pairs of Bufo terrestris laid eggs in the hours among time of capture

and 1100 hours on 1 June 2002. Adults were removed from the plastic containers and

released at their site of capture. One clutch of eggs perished due to a possible fungal

infection. At 2030 hours on the same day, the remaining six clutches of eggs were

separately placed into 55-liter containers. Before egg introduction, the containers were

filled with approximately 47 quarts of chlorine-free tap water. Each container was also

furnished with two air pumps to provide adequate oxygen flow in the water. Embryos

remained in these containers until they reached a feeding and free-swimming tadpole

stage, Stage 25 of the Gosner (1960) staging system, at which time I selected tadpoles for

the experiment.

Using a lottery, I randomly selected three of the six clutches to be used in the

experiment and released the remaining three clutches of tadpoles to their native pond. I

used a total of 180 tadpoles in the experiment (60 tadpoles from each clutch). Tadpoles

were selected for the experiment by dipping small fish nets in different areas in the clutch









containers. I placed five tadpoles from each clutch into each of the 12 tanks, providing a

total of 15 tadpoles per tank.

Materials

I used 0 mg/L, 5 mg/L, 15 mg/L and 30 mg/L sodium nitrate (NaNO3) as my

treatment levels. These levels as nitrate-nitrogen are 0 mg/L, 0.824 mg/L, 2.472 mg/L,

and 4.944 mg/L, respectively, calculated by molecular weight of nitrogen present in

NaNO3. Levels chosen for use in this experiment represent the low range of nitrate

concentrations in Florida waters. Stock solutions of sodium nitrate were created

immediately before the experiment so that each time water was to be changed, each tank

would receive only 2 mL of stock solution from its respective treatment level. Another

scientist labeled the solution bottles either E, F, H, or J so that I was blind to the

concentrations.

On 3 June 2002, I obtained twelve 10-gallon glass aquaria to house Bufo terrestris

tadpoles. Each treatment had three replicates, producing a total of twelve aquaria. I

washed and scrubbed the tanks with a diluted bleach solution (-10% bleach) and then

thoroughly rinsed them with fresh tap water. The next day, the tanks were again rinsed

with fresh tap water and allowed to dry. On 5 June 2002, I set up the twelve 10-gallon

tanks in an outdoor, open-air, covered setting. This setting provided tadpoles with

ambient temperatures and some direct sunlight throughout the experiment but prevented

the addition of rainwater to the tanks. I set up three 1-gallon, cloth painting sieves in

each tank Each sieve separated tadpole clutches within the tank. Sieves were attached to

the top of the tanks using office clips and the tanks were covered with aluminum mesh to

minimize the introduction of detritus and to prevent possible predation by insects. A

151-liter glass tank that was filled with tap water and constantly had air running through









it using two air pumps served as a water reservoir that would be used to replenish tank

water. Tadpoles were fed rabbit pellets ad libitum throughout the experiment.

Experimental Design

Using a lottery system, tanks were randomly assigned to one of four treatments: H,

E, F, or J, representing 0 mg of sodium nitrate per liter, 5 mg/L, 15 mg/L, or 30 mg/L,

respectively. Clutch order within each tank was also randomized using a lottery so that

clutch order and clutch neighbors within each tank would vary among tanks. I labeled

each tank with its designated treatment letter and clutch order (e.g., 1 2 3 or 1 3 2). I

assigned tanks a position in the lab setting and randomly moved them every other day

throughout the experiment using a lottery system. Clutch order within each tank did not

change during the course of the experiment. Seven liters of water were added to each

tank followed by two mL of sodium nitrate stock solution (either E, F, J, or H, depending

on each tank's previous random assignment). Tanks were agitated to thoroughly mix the

solutions in the water.

On 6 June 2002 (5 days after egg collection), all tadpoles were measured and

staged. Body length (mm) was measured from the tip of the snout to the posterior part of

the body where the tail's lateral line joined the body. Developmental staging of tadpoles

was performed using a dissecting microscope and the Gosner (1960) staging system. I

performed body length measurements and staging every other day. In order to stage and

measure tadpoles, I removed each tadpole from its treatment tank and placed it on a

water-saturated cloth. Body lengths were determined using calipers and staging required

that the tadpoles be placed under a bright light for microscope use. Tadpoles were

typically out of water for one minute to obtain body length and stage data.









Tank water was changed every other day, on the days when tadpoles were not

staged or measured. Before changing water for each tank, tadpoles in each sieve were

transferred to a small, plastic weighing boat by gently reversing the sieve. The tanks

were rinsed with high-pressure water and sieves were also rinsed out to remove leftover

food. Again, seven liters of water were added per tank, followed by two mL of the

appropriate nitrate solution. Water that was added to the tanks was obtained from tap

water that had been sitting in the 151-liter tank for approximately 48 hours with air

pumps aiding in chlorine removal. Tadpoles were added to each sieve and each sieve

was placed in its corresponding position in its tank according to the previously arranged

clutch order. One pellet of rabbit food was added to each clutch sieve and the tanks were

again covered with aluminum mesh.

Water, pH, and temperature per tank were measured frequently throughout the

experiment. On days when the water was changed, pH and temperature were measured

twice: once before changing the water and once after adding new water and nitrate

concentrations. This provided a range for these parameters during the experiment.

Furthermore, the use of maximum/minimum temperature thermometers provided daily

temperature ranges for one tank in each treatment. Every day the maximum/minimum

thermometers were placed in a new tank within the same treatment level. Dissolved

oxygen was measured randomly during the experiment.

I checked tanks daily for tadpole mortality and abnormalities such as edema, erratic

swimming, and body malformations. I recorded deceased or deformed tadpoles, noting

treatment level (E, F, H, or J) and clutch number. Deceased tadpoles were removed from

the experiment and a final body length and stage, if possible, were recorded. Any









tadpoles showing abnormalities were noted yet remained in the experiment. Towards the

end of the experiment, each tank was also checked daily for new metamorphs (Gosner

Stage 42, forelimb emergence). The presence of metamorphs was recorded, but they

were only removed from the experiment when they reached Gosner Stage 45, when Bufo

terrestris tadpoles have fully functioning lungs (Wassersug and Seibert 1975). The

experiment lasted a total of 33 days, until all of the tadpoles had either reached Gosner

Stage 45 (four limbs present, tail stub present, terrestrial stage) or died.

Statistical Analysis

I calculated average body lengths per clutch in each tank. A square root

transformation was performed to linearize the data. Comparisons of body length among

clutch and treatment were performed over time using a mixed linear model in SAS. This

determined whether clutch, treatment, or tank influenced body length in tadpoles. Water,

pH, and temperature were also compared among tanks in the mixed linear model to

determine any possible relationship with tadpole body length. Time to metamorphosis

(days) was analyzed per tadpole using a mixed linear model as well. Stage 42 (forelimb

emergence) was considered metamorphosis since this stage is easily recognizable.

Results

One clutch of eggs perished, presumably due to a fungal infection, as noted by a

white, opaque covering on the eggs. There was no significant difference in body length

(F = 0.79, p = 0.5063) among treatments (Fig. 3-1) or among tanks (p = 0.28). There was

a clutch effect on body length (p = 0.0006) over time (Figs. 3-2 3-6). Figure 3-5

excludes an outlier tadpole in the 15 mg/L treatment. This tadpole remained at Stage 25

throughout the entire experiment. Elimination of this tadpole produced much lower

standard errors. Towards the end of the experiment this individual's body began swelling









and became transparent. On Day 28, body length (BL) of this tadpole was 10.29 mm

while its body width (BW) was 8.56 mm. On Day 31, its BL was the longest at 11.91

mm with the widest BW of 12.00 mm. On Day 33, it had a BL of 10.86 mm and a BW of

10.30 mm. Ultimately, its body swelling produced a width practically the same

dimension as its length, an abnormal feature for a typical tapdole in this species. Body

lengths of Clutch 2 were typically smaller than Clutches 1 and 3. However, after the

elapse of three-quarters of the experimental time, Clutch 2 in each treatment typically

attained an equal or greater size than either or both Clutches 1 and 3. This typically

occurred when Clutches 1 and 3 were in metamorphic climax.

Time to metamorphosis was significantly different among control and 30 mg/L

tadpoles (p = 0.0346), 5 mg/L and 30 mg/L tadpoles (p = 0.0334), and 15 mg/L and 30

mg/L tadpoles (p = 0.0499). Time to metamorphosis was not significantly different

among control and 5 mg/L tadpoles (p = 0.9787), control and 15 mg/L tadpoles (p =

0.6814), and 5 mg/L and 15 mg/L tadpoles (p = 0.6954). Average time to metamorphosis

per treatment is depicted in figure 3-7. Figure 3-8 depicts the average Gosner stage per

treatment per day. A clutch effect on time to metamorphosis was not present (p =

0.2190). Figures 3-9 3-13 depict mean Gosner Stage of tadpoles by clutch over time.

Figure 3-12 excludes the outlier tadpole from Clutch 1 which maintained Gosner Stage 25

throughout the entire experiment.

Dissolved oxygen appeared constant among tanks as well (Table 3-1).

Temperature and pH did not vary significantly during the course of the experiment and

exhibited no apparent relationship with tadpole body length (p = 0.2810, p = 0.3628).









These environmental factors are known to influence life history traits such as size and

developmental rate.

Days 13 and 26 had the highest temperatures at about 310C and 300C (Figs. 3-14 -

3-16). Differences in maximum and minimum treatment temperatures can be attributed

to variation in thermometers and tank placement. Some tanks may have received more

sunlight on a given day than other tanks. Possible confounding effects of the latter

variability were minimized in the experiment by randomly moving tank position every

other day.

Mortality was unequal among treatments. Mortality was 42.2%, 17.8%, 15.6%,

and 28.9% in the sodium nitrate treatments from lowest to highest concentrations. Clutch

1 had the highest mortality and Clutch 3 had the lowest mortality overall (Fig. 3-17).

Mortality was also present in each replicate (tank) with no apparent pattern (Fig. 3-18).

No replicate exhibited mortality of all 15 tadpoles. Clutch 1 tadpoles had higher

mortality than Clutch 2 or 3 tadpoles, regardless of treatment regime, and Clutch 2

tadpoles had higher mortality than Clutch 3 tadpoles in all treatments with the exception

of the 5 mg/L sodium nitrate treatment (Fig. 3-19). In this instance, Clutch 2 and 3

tadpoles had equal mortality (one tadpole each). Higher proportions of mortality in the

control and 30 mg/L treatments are reflected in lower proportions of total metamorphs

throughout the experiment (Figs. 3-20 3-23). Proportion of metamorphs was higher for

5 mg/L and 15 mg/L treatment levels due to decreased amount of mortality. Metamorphs

began appearing by Day 16 of the experiment. Clutch 1 tadpoles typically

metamorphosed before Clutches 2 and 3 (Fig. 3-24). The majority of Clutch 1 tadpoles

metamorphosed among Days 16 and 18 with the greatest proportion occurring on Day 16.









The majority of Clutch 2 tadpoles metamorphosed among Days 18 and 20 with the

greatest proportion occurring on Day 20. The majority of Clutch 3 tadpoles

metamorphosed among Days 18 and 20 also. Unlike Clutch 2, however, the greatest

proportion of metamorphs for Clutch 3 occurred on Day 18.

Discussion

Concentrations of 5 mg/L, 15 mg/L and 30 mg/L sodium nitrate (0.824 mg/L, 2.472

mg/L, and 4.944 mg/L nitrate-nitrogen) produced no observable effects on body length in

Bufo terrestris tadpoles. Tadpoles in different tanks had statistically similar body lengths,

suggesting that variation as a result of tank setup was minimal, if present at all. Tadpoles

of different treatment regimes exhibited statistically similar body lengths throughout the

course of the experiment. Higher concentrations of nitrate can produce negative effects

on body length in this species as seen in other literature (Hecnar 1995; Schuytema and

Nebeker 1999a and 1999b). However, exposing earlier stages (egg and embryo) of Bufo

terrestris to the experimental concentrations may produce the proposed lethal and

sublethal effects since earlier stages may differ in their susceptibility to nitrate

contamination (Xu and Oldham 1997). Temperature and pH, factors known to greatly

influence body length and developmental rate, did not vary greatly among tanks during

the course of the experiment.

Time to metamorphosis differed among the 30 mg/L sodium nitrate treatment and

all other treatments. Tadpoles exposed to 30 mg/L sodium nitrate obtained Gosner Stage

42 in the shortest amount of time. Tadpoles exposed to 15 mg/L sodium nitrate achieved

forelimb emergence before 5 mg/L and control tadpoles. Control tadpoles reached

Gosner Stage 42 before 5 mg/L tadpoles. These data suggest that higher concentrations

of sodium nitrate (30 mg/L and 15 mg/L) accelerated developmental rate in Bufo









terrestris tadpoles. It is possible that the tadpoles in the 30 mg/L treatment increased

developmental rate to escape the unfavorable habitat created by sodium nitrate. Another

possibility is that a hormetic response occurred such that these low levels of sodium

nitrate stimulated developmental rate by interacting with hormones responsible for

metamorphosis. Hormesis refers to stimulatory effects of contaminants at low doses

followed by inhibitory effects at higher contaminant levels (Bailer et al. 2003).

Contaminants such as cadmium, polycyclic aromatic hydrocarbons, and dioxin have been

shown to produce hormetic effects in some species (Calabrese and Baldwin 2003).

Boone et al. (2001) found that the insecticide, carbaryl, accelerated development in Rana

clamitans tadpoles. Although a faster developmental rate may be beneficial in instances

where tadpoles are in a habitat that will quickly dry up, it may produce smaller body sizes

at metamorphosis that could have negative effects on fecundity and survival (Alford and

Harris 1988; Wilbur and Collins 1973).

All tadpoles used in the experiment originated from a single population in the

wild. Hecnar (1995) reported that 13.6 mg/L of NH4-N was sufficient to produce 50%

mortality in one population of Bufo americanus tadpoles while another population

required 39.3 mg/L of NH4-N to produce 50% mortality. The latter population originated

from an area where intensive agriculture occurs, suggesting that this population

developed resistance to ammonium nitrate. Laurila et al. (2002) observed significant

differences in body size at metamorphosis and larval period length in Rana temporaria

tadpoles. In their study, tadpoles from a northern population did not vary these life

history traits with respect to water quantity while tadpoles from a southern population

exhibited faster developmental times and smaller body sizes when exposed to quickly









decreasing water volume. In nature, the southern population inhabits ponds which dry

out frequently and erratically. The northern population, however, inhabits ponds that are

not prone to complete drying. Since populational differences exist, determining lethal

and sublethal effects of contaminants based on one or a few populations may not apply to

all populations within a species, resulting in conservation efforts that may not be

adequate to preserve the whole species.

In a survey of 26 Florida springs, nitrate (NO3-N) ranged from 1.10 to 38.00 mg/L

(Katz et al. 1999). Total nitrogen in four lakes (Wauberg, Watermelon Pond, Orange and

Newnan) in Alachua County, Florida ranged from a minimum of 0.273 to a maximum of

11.500 mg/L (Lakewatch 2001). Nitrate amounts (as NO3) in Florida ponds typically

range from 0.1 to 1 mg/L, although some ponds have nitrate amounts at the 3 mg/L range

or higher (USGS 2003). One pond in Gainesville, Florida (Alachua County) where B.

terrestris bred in 2003 had 0.0048 mg/L nitrate-nitrogen. The pond where I obtained

breeding adults for use in this experiment was also located in Gainesville, Florida. This

pond was not situated near agricultural sites and likely exhibited nitrate levels similar to

other Florida ponds (< 1 mg/L). The levels used in this experiment were ecologically

relevant at 0 mg/L, 0.824 mg/L, 2.472 mg/L, and 4.944 mg/L nitrate-nitrogen. Although

Bufo terrestris tadpole body length was not adversely affected by the nitrate

concentrations in this study, higher concentrations may produce smaller body lengths

since studies that used higher levels found such an effect.

Clutch affected tadpole body length during the course of this experiment. This

could be due to genetic variation or nongenetic variation from maternal influences such

as egg size and nutrient input in egg yolk (Travis 1980) Travis (1980) observed a









genetic effect on growth rates in Hyla gratiosa by cross-breeding a limited number of

males and females and observing the growth rates in offspring. Semlitsch et al. (2000)

performed a cross-breeding experiment with Hyla versicolor and concluded that genetic

variation in tadpoles, rather than maternal influences, largely affected tadpole tolerance to

the insecticide carbaryl. However, a separate, crossbreeding experiment would have to

be conducted to conclusively state whether clutch differences in body length and Gosner

stage of Bufo terrestris are due to genetic effects.

Tadpoles from Clutch 2 typically had smaller body lengths than tadpoles from

either Clutch 1 or 3 until about Day 24 in the control and 5 mg/L treatment, Days 18-22

and Day 28 for 15 mg/L, and Day 22 for 30 mg/L. A potential explanation for Clutch 2

tadpoles having longer body lengths later in the experiment could be the decrease in body

length during metamorphic climax, Gosner Stages 42-46 (Emerson 1986). Since tadpoles

in Clutches 1 and 3 were ahead in developmental stages they also had smaller body

lengths when in Gosner Stages 42-46 (metamorphic climax). Tadpoles from Clutch 2

typically had slightly slower development than tadpoles from Clutches 1 and 3, although

clutch did not have a statistically significant effect on time to metamorphosis.

Since density and competition were not factors, longer larval periods did not appear

to translate into smaller body sizes at metamorphosis. At the beginning of the

experiment, all tadpoles from Clutch 2 were smaller than tadpoles from Clutches 1 and 3.

At the end of the experiment, Clutch 2 tadpoles in the control group achieved larger body

lengths than Clutch 1 or 3 tadpoles (Fig. 3-2). In the 5 mg/L and 30 mg/L treatments,

Clutch 2 tadpoles were still smaller than Clutch 1 or 3 tadpoles at the end of the

experiment, although the difference was not as large as it was at the onset of the









experiment (Figs. 3-3 and 3-6). At the end of the experiment, Clutch 2 tadpoles in the 15

mg/L treatment level were larger than Clutch 3 tadpoles. However, they were still

smaller than Clutch 1 tadpoles although the size difference was not as great as at the

onset of the experiment (Fig. 3-5). Clutch 2 tadpoles also had slightly longer larval

periods although this was not statistically significant. Individuals with longer larval

periods spend more time in a stable environment and maximize growth at the risk of

increased mortality from predation and desiccation thereby achieving a larger body size

at metamorphosis (Alford and Harris 1988; Hecnar 1995). Although most Clutch 2

tadpoles were not larger than Clutch 1 or 3 tadpoles, their body lengths did increase

during the course of the experiment such that their final lengths showed much less

disparity among Clutches 1 and 3 as compared to the beginning of the experiment.

One tadpole from the 15 mg/L treatment (2.472 mg nitrate/L) had a grossly swollen

and transparent body. Hecnar (1995) noted many tadpoles with a similar condition when

he exposed them to chronic levels (2.5, 5, and 10 mg/L) of nitrate-nitrogen and suggested

that nitrate produced the deformity by affecting tadpole osmoregulation. It is possible

that nitrate produced the same condition in my experiment. Although only one tadpole

exhibited the swelling, it may have been more susceptible to nitrate since individuals vary

in their response to contaminants. However, since only one tadpole exhibited this

condition in my experiment, it is uncertain what the causitive agent may have been.

The fact that the control and 30 mg/L treatments had the highest mortality appear to

be a stochastic event unrelated to nitrate concentration. All water parameters among

treatments were similar, there was no variability in body length, and all tadpoles were

handled equally by the investigators. Length of larval period cannot account for









mortality since control tadpoles required approximately the same amount of time to reach

metamorphosis as tadpoles in the 5 and 15 mg/L treatments. Furthermore, since tadpoles

in these groups reached metamorphosis at approximately the same time, handling stress

from body measurements and staging among these groups would have been fairly

consistent and cannot account for mortality differences. A scientific explanation for

Clutch 1 displaying the greatest mortality in each treatment is also lacking and may be

stochastic as well. Similar studies which staged tadpoles typically did so with much less

frequency. Semlitsch and Schmiedehausen (1994) used only one stage measurement

(Gosner Stage 42, forelimb emergence) for a measure of developmental rate. Performing

fewer tadpole measurements would certainly decrease stress to the animals and,

therefore, possibly reduce mortality.

Future Research

Determining effects of varying nitrate levels on all life stages of amphibians

inhabiting areas where such contamination occurs is essential to prevent possible

population and species declines. Since many amphibian species breed in temporary

bodies of water, future experiments should compare effects of nitrates among several

populations that inhabit a range of nitrate-contaminated and uncontaminated areas, using

levels consistent with those found in the field. Such levels are likely to be much lower

than those in current literature. Data on nitrate concentrations in temporary bodies of

water are relatively scant, therefore this type of study would add to that body of literature

as well by providing such data. Additionally, scant information exists on nitrate effects

on developmental rate in amphibians. If nitrate levels decrease developmental rates,

amphibians inhabiting temporary ponds may be in danger of dessiccation since

metamorphosis may not be completed by the time their pond dries completely. If nitrates









increase developmental rates, it may lead to individuals with smaller body sizes at

metamorphosis since a longer larval period typically produces larger metamorphs (Alford

and Harris 1988; McDiarmid and Altig 1999). This could reduce fitness if smaller

females produce fewer and smaller eggs (Watt and Oldham 1995), ultimately resulting in

reduced amphibian populations.

Comparing effects of different types of nitrate would be useful since they produce

varying effects on amphibians. Ammonium nitrate, for example, is more toxic to

amphibians than sodium nitrate, owing to the ammonium ion and is used extensively in

agricultural settings (Schuytema and Nebeker 1999b). Effects on fecundity and

fertilization are also lacking in the literature with only one study linking low pH to

decreased sperm motility (Schlicther 1981). Decreased sperm motility could negatively

affect population growth in habitats heavily contaminated with ammonium nitrate.

Ultimately, many factors work synergistically in the environment to create adverse

effects. Without the addition of contaminants, larval mortality is relatively high so that

very few individuals reach reproductive ages. With the added stress of contaminants,

mortality likely will increase and reduce populations in the future (Semlitsch et al. 2000).

Although contaminants may select for resistant individuals in the population, allowing

the population to persist and grow, loss of genetic diversity could reduce adaptiveness of

the population to future selective pressures. Pesticides, fertilizers, competition, food

abundance and water quality all likely have complex roles in developmental and growth

rates of larval amphibians. It is necessary to understand the impact of each factor

independently and interdependently to predict the future of amphibian populations and

create realistic conservation goals.









More detailed studies comparing contaminant effects on species with different life

history characteristics need to be undertaken as well. Bufo terrestris exhibits

synchronous hatching and metamorphosis. Studies have shown that high density and low

food abundance can affect development and growth in other species by producing smaller

metamorphs (Smith-Gill and Berven 1979; Wilbur and Collins 1973). Nitrate has been

shown to affect body length, larval period length, and feeding activity in other species as

well (Hecnar 1995; Marco et al. 1997; Oldham et al. 1997; Rouse et al. 1999). It may be

reasonable, therefore, to conclude that nitrate contamination may differentially affect

species with varying life history characteristics such as population density and time to

metamorphosis, producing more drastic effects in species that breed synchronously in

ephemeral bodies of water compared to species with asychronous breeding in permanent

bodies of water. Since temperature can greatly affect the length of the larval period, a

laboratory experiment using tadpoles of the same species with varying nitrate

concentrations and three temperature regimes may be useful in determining how larval

period may interact with contaminants.

Mesocosms are experimental tank communities that mimic natural temporary pond

conditions but provide more control, manipulation, and accurate censusing data than

natural ponds (Morin 1983). Mesocosm studies would be particularly useful in

determining indirect nitrate effects on tadpoles resulting from algal effects, a major

tadpole food source. Mesocosm experiments may more accurately reflect contaminant

interactions in nature and provide more insight into complex ecological interactions of

contaminants in nature.





















* 0 mg/L
-- 5 mg/L
--15 mg/L
-- 30 mg/L


2 4 6 8 10 12 14 16 18 20 22 24 26 28 31 33

Day


Figure 3-1. Mean tadpole body length by treatment over time. Data were pooled among clutches for each treatment. Data points
represent repeated measures of body length throughout the experiment. Day represents time since the start of the
experiment when tadpoles were first exposed to nitrate treatments. As tadpoles reached metamorphosis (Gosner Stage 45)
in the experiment, their final body length before release was used in calculating the averages for the duration of the
experiment. All standard errors (+SE) per day were less than 0.20.












12

11

10

9

8 O Clutch 1
0 Clutch 2
7 7
2 *Clutch 3

6 -

5

4 -



2 4 6 8 10 12 14 16 18 20 22 24 26 28 31 33

Day

Figure 3-2. Mean body length of tadpoles by clutch in 0 mg/L sodium nitrate treatment. Each clutch represents a separate group of
siblings. Day refers to time since the start of the experiment. Error bars represent standard error. Standard error ranged
from 0.065 to 0.89.












12

11

10

f 9

8 Clutch 1
0E Clutch 2
7 Clutch 3

6

5-

4 -



2 4 6 8 10 12 14 16 18 20 22 24 26 28 31 33

Day

Figure 3-3. Mean body length of tadpoles by clutch in 5 mg/L sodium nitrate treatment. Each clutch represents a separate group of
siblings. Day refers to time since the start of the experiment. Error bars represent standard error.











12

11

10

S9

S8 Clutch 1
SEl Clutch 2
S7 Clutch 3
6

5 -

4 _


2 4 6 8 10 12 14 16 18 20 22 24 26 28 31 33
Day

Figure 3-4. Mean body length of tadpoles by clutch in 15 mg/L sodium nitrate treatment. Each clutch represents a separate group of
siblings. Day refers to time since the start of the experiment. Error bars represent standard error.






















8 E Clutch 1
O Clutch 2
7 Clutch 3

6 -

5 -

4



2 4 6 8 10 12 14 16 18 20 22 24 26 28 31 33

Day

Figure 3-5. Mean tadpole body length of tadpoles by clutch in 15 mg/L NaNO3 treatment, excluding outlier from Clutch 1. Data were
pooled among tadpoles for each clutch and each clutch represents a separate group of siblings. Day refers to time since
the start of the experiment. Error bars represent + SE. Elimination of the outlier tadpole produced slightly larger body
lengths for Clutch 1 tadpoles on some days.














11

10

E 9

8 E- 1 Clutch 1
[E Clutch 2
7-
e 7 Clutch 3
0
6

5-

4



2 4 6 8 10 12 14 16 18 20 22 24 26 28

Day

Figure 3-6. Mean body length of tadpoles by clutch in 30 mg/L sodium nitrate treatment. Each clutch represents a separate group of
siblings. Day refers to time since the start of the experiment. Error bars represent standard error.












19.5

19

18.5

18

17.5


S0 mg/L
S5 mg/L
S15 mg/L
M 30 mg/L


Treatment

Figure 3-7. Average time to metamorphosis (days) per treatment. Metamorphosis was
defined as Gosner Stage 42 (forelimb emergence). Error bars represent
standard error (+SE).


_L























0 mg/L
- 5 mg/L
--15 mg/L
-- 30 mg/L


2 4 6 8 10 12 14


16 18 20 22 24


26 28 31 33


Day


Figure 3-8. Mean tadpole Gosner stage by treatment over time. Data were pooled among clutches for each treatment. Data points
represent repeated measures of Gosner stage throughout the experiment. Day represents time since the start of the
experiment when tadpoles were first exposed to nitrate treatments. As tadpoles reached metamorphosis (Gosner Stage 45)
in the experiment, their final Gosner stage before release was used in calculating the averages for the duration of the
experiment. All standard errors (+SE) per day were less than 0.55.















44-

42

40

c 38
3 3O Clutch 1
S36 -
o 30 Clutch 2
34 -
U Clutch 3
S32-

30

28 -

26 -

24
2 4 6 8 10 12 14 16 18 20 22 24 26 28 31 33

Day

Figure 3-9. Mean Gosner stage of tadpoles by clutch in 0 mg/L sodium nitrate treatment. Each clutch represents a separate group of
siblings. Day refers to time since the start of the experiment. Error bars represent standard error.






















El Clutch 1
36 -
[]3 Clutch 2
34 Clutch 3

S32

30

28 -

26 -

24
2 4 6 8 10 12 14 16 18 20 22 24 26 28 31 33
Day

Figure 3-10. Mean Gosner stage of tadpoles by clutch in 5 mg/L sodium nitrate treatment. Each clutch represents a separate group of
siblings. Day refers to time since the start of the experiment. Error bars represent standard error.














44

42

40

c 38
E Clutch 1
S36 -
S-O Clutch 2
34 Clutch 3

| 32

30

28

26 -

24
2 4 6 8 10 12 14 16 18 20 22 24 26 28 31 33
Day

Figure 3-11. Mean Gosner stage of tadpoles by clutch in 15 mg/L sodium nitrate treatment. Each clutch represents a separate group of
siblings. Day refers to time since the start of the experiment. Error bars represent standard error.














44

42

40

C 3 38
~,E Clutch 1
S36 -
SO Clutch 2
S34 Clutch 3

S32

30

28 -

26 -

24 ----
2 4 6 8 10 12 14 16 18 20 22 24 26 28 31 33
Day
Figure 3-12. Mean Gosner stage of tadpoles by clutch in 15 mg/L sodium nitrate treatment excluding one individual from Clutch 1
that did not develop and remained at Stage 25 throughout the experiment. Elimination of this individual produced a
smaller standard error for Clutch 1 than represented in figure 9. Each clutch represents a separate group of siblings. Day
refers to time since the start of the experiment. Error bars represent standard error.














44

42

40

Cj 38
O Clutch 1
8 36 -
0 Clutch 2
34 a 5 Clutch 3

C 32

30

28 -

26 -

24
2 4 6 8 10 12 14 16 18 20 22 24 26 28 31
Day

Figure 3-13. Mean Gosner stage of tadpoles by clutch in 30 mg/L sodium nitrate treatment. Each clutch represents a separate group
of siblings. Day refers to time since the start of the experiment. Error bars represent standard error.










Table 3-1. Dissolved oxygen samples from random days per treatment and tank within
treatment.

Treatment
0 mg/L 5 mg/L 15 mg/L 30 mg/L
Tank Tank Tank Tank
Day 1 2 3 1 2 3 1 2 3 1 2 3
15 8.83 8.81 8.86 8.80 8.80 8.80 8.81 8.80 8.20 8.79 8.78 8.77
16 8.52 8.48 8.50 8.52 8.53 8.54 8.51 8.52 8.54 8.51 8.51 8.51
21 8.42 8.39 8.40 8.40 8.40 8.40 8.43 8.41 8.41 8.32 8.37 8.33
28 9.80 8.90 9.30 9.20 9.20
31 8.10 8.20 8.20 8.50 *
33 8.60 8.40 8.40 *
* Signify tanks that have completed experimentation and are therefore empty. Values are
amount of dissolved oxygen in ppm (mg/L). Each value represents one measurement per
tank.


32

30

28

S26
E
24

22

20


-- Tmax
------ Tmin


-- n .




3 5 6 7 9 13 15 16 17 18 19 20 21 23 24 25 26 27 28 29 31 33

Day


Figure 3-14. Maximum and minimum temperatures (C) for 0 mg/L sodium nitrate
treatment.




















S28


i 26


24


22


-- Tmax
----.- Tmin


R --. -. L.
,-- t. i..m" ."" I -
"


3 5 6 7 9 13 15 16 17 18 19 20 21 23 24 25 26 27 28 29 31 33

Day

Figure 3-15. Maximum and minimum temperatures (C) for 5 mg/L sodium nitrate
treatment.





34


- Tmax
------ Tmin


(U
28


S26


24

22


, ... -. m -i.
\ --.1 "1 if "


3 5 6 7 9 13 15 16 17 18 19 20 21 23 24 25 26 27 28 29 31 33

Day


Figure 3-16. Maximum and minimum temperatures (C) for 15 mg/L sodium nitrate
treatment.






43


8
7
6
5 Clutch 1
t 4 O Clutch 2
3 Clutch 3

2-



0 5 15 30

Sodium Nitrate Treatment (mg/L)

Figure 3-17. Overall mortality in each treatment per clutch.




9
8- -
-7 -
6 0 mg/L
5 O 5 mg/L
4 I 15 mg/L
3 I- 30 mg/L




1 2 3

Replicate (Tank Number)

Figure 3-18. Total number of dead tapdoles per replicate (single tank) within treatment









12

10-

o 8 0 mg/L
6O 5 mg/L
6
|* 15 mg/L
S 4 30 mg/L

2


1 2 3

Clutch

Figure 3-19. Total number of dead tadpoles per clutch within treatment.
























* Deceased
E Metamorphs
E Tadpoles


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Day


Figure 3-20. Proportion of deceased individuals, metamorphs, and tadpoles for 0 mg/L sodium nitrate treatment.


0.9

0.8

0.7

= 0.6

a 0.5

0.4

0.3

0.2

0.1

0.0


























SDeceased
] Metamorphs
* Tadpoles


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Day


Figure 3-21. Proportion of deceased individuals, metamorphs, and tadpoles for 5 mg/L sodium nitrate treatment.


























SDeceased
] Metamorphs
I Tadpoles


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Day


Figure 3-22. Proportion of deceased individuals, metamorphs, and tadpoles for 15 mg/L sodium nitrate treatment.





















* Deceased
E Metamorphs
E Tadpoles


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28


Day


Figure 3-23. Proportion of deceased individuals, metamorphs, and tadpoles for 30 mg/L sodium nitrate treatment.

























In


15 16 17 18 19 20


[I [M


n F


21 22 23 24 25 26 27 28
Day


Figure 3-24. Proportion of new metamorphs (Gosner Stage 42, forelimb emergence)
each day per clutch. Proportions are number of new metamorphs per day for
each clutch divided by total number of metamorphs each clutch ultimately
produced.


] Clutch 1
* Clutch 2
* Clutch 3
















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BIOGRAPHICAL SKETCH

Esther M. Langan was born in Melbourne, Florida, on 25 February 1975. An Air

Force upbringing moved her to Germany, Virginia, North Dakota, and finally Tampa,

Florida where she has lived most of her life. She received her Bachelor's of Science

degree from the Department of Wildlife Ecology and Conservation at the University of

Florida in 1997.