The effects of nitrate on three species of male tree frogs Hyla regilla, H. cadaverina, and H. chrysoscelis Megan Tracey, Becky Talyn & Erik Melchiorre, California State University, San Bernardino Abstract Amphibians are good bioindicator species for aquatic environments. Nitrate is a known contaminant, but based on previous fieldwork does not affect frog distribution. However, high levels of nitrate have been shown to negatively affect tadpoles. In this experiment we observed tree frogs’ behavioral response to nitrate contamination under controlled laboratory conditions to better understand the implications of nitrate contamination on amphibian populations. We tested the effects of nitrate on three species of tree frogs (Fig. 1): Hyla regilla (N=7), H. chrysoscelis (N=6) and H. cadaverina (N=8). We used 7, 10, 20, and 50 ppm, and control (0 ppm) treatments in blind trials. A small habitat cage was placed upon wooden supports, with a mirror for observation underneath. A Kim wipe was placed randomly on the right or left side of the habitat cage, with 5ml of one of the test solutions. Frogs were observed for up to 15 minutes or until they left the treatment. We noted whether or not they were absorbing the treatment, and how much time they spent squatting. Results show no relationship between time spent on treatment, absorbing or squatting and the level of nitrate used. Thus none of the three species responded to the presence or absence of nitrate in the lab, corroborating earlier field results. As studies have shown that exposure to nitrate puts tadpoles at risk for decreased growth rate and likelihood of survival, this lack of behavioral response to nitrate contamination may contribute to population decline, particularly in heavily farmed areas. Introduction This behavioral experiment was done to determine the implications of sub-lethal nitrate contamination on amphibian populations. The experiment follows on a pond study in which water samples were collected to determine whether frogs choose which ponds to use for reproduction based on chemical contamination of the water. Nitrate is a known local contaminant, especially in agricultural areas, but based on previous fieldwork does not affect frog distribution (Talyn et al. 2003). However, high levels of nitrate have been shown to negatively affect tadpoles, and their development (Rouse et al. 1999). This experiment was designed to observe tree frogs’ behavioral response to nitrate contamination under controlled laboratory conditions to better understand the implications of nitrate contamination on amphibian populations. Specifically, we assessed the behavioral response of adult male tree frogs to nitrate in water. Methods The experiment was performed using the male tree frogs Hyla regilla (Fig. 1 top; N=6) and H. cadaverina (Fig. 1 bottom right; N=7) found locally in areas surrounding California State University San Bernardino, and H. chrysoscelis (Fig. 1 bottom left; N=5) from Indiana. Frogs were exposed to 5 ml of nitrate-contaminated isosmotic solutions at 0, 7, 10, 20, and 50 ppm. Each trial was conducted in a half gallon aquarium placed on wooden supports above with a mirror. Each frog was carefully voided using gentle abdominal pressure, and weighed to 0.1 grams. We then placed the frog onto the solution with a small weigh boat for restraint. Approximately 20 seconds later the weigh boat was lifted off and observations started. Frogs were observed continuously for up to 15 minutes, recording absorption and squatting behavior. Absorbing occurred when the whole abdomen of the frog was fully pressed onto the solution. Squatting behavior accompanied increased breathing rate with just the pelvic region actively absorbing the treatment. If the frog was squatting he was absorbing, but the reverse is not true. When a frog left the treatment the trial was stopped. Results were analyzed separately for each behavior (length of trial, time absorbing, time squatting) using a two way ANOVA with species and nitrate concentration as factors. Figure 2: Nitrate concentration does not alter behavior of treefrogs. Figure 3: Three species of treefrogs do not respond differently to nitrate contamination. A) Time spent on solution, B) Time squatting. A B Results Our data show no relationship between time spent on treatment, absorbing or squatting and the level of nitrate used (Fig. 2; MANOVA: overall F=1.304, P=0.219; on treatment F=0.767, P=0.550; absorbing F=0.943, P=0.444; squatting F=1.391, P=0.246). While the three species differed in some of the behaviors (MANOVA: Species: overall F=6.424, P<0.0005; time on treatment F=12.972, P<0.0005; squatting F=0.574, P=0.566; absorbing F=12.134, P<0.0005), none of them responded to the presence or absence of nitrate in the lab (MANOVA: Species * nitrate concentration: overall F=0.842, P=0.968; time on treatment F=0.095, P=0.999, Fig. 3a; squatting F=0.439, P=0.894, Fig. 3b; absorbing F=0.090, P=0.999), corroborating earlier field results. Discussion and Conclusions  Amphibians are a god bioindicator species of aquatic environments.  v Tree frogs do not respond to the presence of nitrate contamination.  v Exposure to nitrate puts tadpoles at risk for decreased growth rate and likelihood of survival (Johnsson et al, 2001)  v Lack of behavioral response to nitrate contamination may contribute to population decline, particularly in heavily farmed areas. Acknowledgments We would like to thank Cindy Chrisler for assistance with frog maintenance, and Ivan Phillipsen for suggesting appropriate field sites. H. cadaverina photo from USGS Western Ecology Research Center web page. References 1.Boyer R, Grue C. The need for water quality criteria for frogs. Environmental Health Perspectives 1995; 103: Johnsson M, Rasanen K, Merila J. Comparison of nitrate tolerance between different populations of the common frog. Ranna temporaria. Aquatic Toxicology 2001; 54: Rouse J, Bishop C, Struger J. Nitrogen pollution: An assessment of its threat to amphibian survival. Environmental Health Perspectives 1999; 107: Talyn B, Hancock S, Melchiorre E. Land use effects on Water Chemistry and Gray Tree frog Habitat choices in Rural Ohio: Involving the local community. Figure 1: Top – Hyla regilla. Bottom left – H. chrysoscelis. Bottom right H. cadaverina.