SHORTER COMMUNICATIONS Journal of Herpetology, Vol. 44, No. 2, pp. 297–300, 2010 Copyright 2010 Society for the Study of Amphibians and Reptiles

Water from Urban Streams Slows Growth and Speeds Metamorphosis in Fowler’s Toad (Bufo fowleri) Larvae K. BARRETT,1 C. GUYER,

AND

D. WATSON

Department of Biological Sciences, Auburn University, Auburn, Alabama 36849 USA ABSTRACT.—We evaluated the effect of a potentially stressful urban aquatic environment on growth and development of Fowler’s Toad (Bufo fowleri) larvae. We reared larvae to metamorphosis in water from urban and forested streams in a laboratory setting. We found no evidence of oral disc anomalies associated with urban environments, but we did find that tadpoles in these environments were smaller at 26 days of age (but not at metamorphosis) and metamorphosed faster than tadpoles reared in water from forested streams. The observed results were partially consistent with the predictions of H. M. Wilbur and J. P. Collins, who suggested in 1973 that stressful aquatic environments should result in an earlier date of metamorphosis for larvae attempting to escape that environment. We suggest further work to pinpoint factor(s) responsible for the results we observed, and we relate our findings to previous findings of declines in amphibian species richness in the study area. Several reviews have cataloged both the abiotic and biotic consequences of urbanization for stream ecosystems (Paul and Meyer, 2001; Allan, 2004; Walsh et al., 2005). Consistently, urbanization has been shown to result in streams with increased spate frequency, altered water chemistry (i.e., higher nitrates and conductivity), wider and deeper stream beds, and decreased nutrient uptake within the stream ecosystem (Walsh et al., 2005). These factors translate to a decrease in density and diversity of many stream organisms (Paul and Meyer, 2001; Walsh et al., 2005). Despite the growing number of studies that examine the response of stream invertebrates and fishes to urbanization (Paul and Meyer, 2001; Walsh et al., 2005), relatively few studies have investigated amphibian response to urban development (but see Gibbs et al., 2005; Parris, 2006; Price et al., 2006; Pehek and Mazor, 2008). For example, amphibians are not even addressed in three major reviews of urbanization’s impact on stream systems (Paul and Meyer, 2001; Allan, 2004; Walsh et al., 2005). We believe that responses of amphibians to changes in water quality as lands become urbanized are likely to be worthy of investigation given the permeability of amphibian skin and (in many species) the presence of aquatic eggs and larvae (Wilbur, 1980; Blaustein and Johnson, 2003). All of these features are likely to make amphibians sensitive to polluted waters. Although few studies specifically address responses of amphibians to urban development, several address how amphibians respond to a variety of potential pollutants (Boone et al., 2001; Chen et al., 2006; Edwards et al., 2006). In these cases, typical experimental designs involve exposure of amphibian(s) to a pollutant by application of moderate to large doses of a suspected toxin to water in a laboratory setting. Here, we take a slightly different approach and investigate effects of water and substrate from urban streams on tadpole growth in a controlled laboratory setting, without directly applying any given toxin. 1 Corresponding Author. Present address: Warnell School of Forestry and Natural Resources, University of Georgia, Athens, Georgia 30602 USA; E-mail: [email protected]

Our interest is in determining whether cumulative effects of all accumulated pollutants generate differential growth and metamorphosis in larvae of a species with rapid development (Bufo fowleri). Peterson et al. (2008) have successfully used this approach. In the urban system we are evaluating (Columbus, Georgia), two past studies suggest water quality may impact stream biota. First, Helms et al. (2005) described an increased incidence of fish tumors and lesions in urban streams within the Columbus area relative to nearby forested streams. Second, Barrett and Guyer (2008) documented amphibian species richness to be nearly 75% lower in urban stream habitats relative to their forested counterparts. Based on previous studies (Rowe et al., 1998; Burger and Snodgrass, 2000), we predicted tadpoles reared in water from urban streams would grow more slowly and show oral disc abnormalities relative to tadpoles reared in water from forested streams because of toxins that accumulate in urban environments. Additionally, based on Wilbur and Collins (1973), we predicted that larvae would undergo metamorphosis more quickly and at smaller sizes in urban stream water because accumulated toxins act as stressors; thus, an early exit from the environment could be advantageous to the larvae. MATERIALS AND METHODS Urban streams used in this experiment were located within Columbus, Georgia, and forested streams were located north of Columbus (within 20 km of the urban streams). Urban streams were defined as those draining a watershed with at least 30% of land cover as impervious surface, and forested streams were defined as those draining a watershed with at least 75% forested land and less than 3% impervious surface. The categorical grouping of urban and forested streams is further defined and supported in Barrett and Guyer (2008). Chemical analysis of the water, which was conducted prior to this study (Schoonover et al., 2005), indicated a clear and consistent difference in the chemical makeup of the two stream categories (Table 1). We collected individuals from two clutches of B. fowleri tadpoles on 8 June 2008 from a forested stream

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TABLE 1. Water chemistry data for the six streams from which we collected water to rear tadpoles. Data are from Schoonover et al. (2005) and J. E. Schoonover and B. S. Helms (unpubl. data). Stream

NO{ 3 (mg L21)

NHz Conductivity 4 (mg L21) (mS/cm)

pH

Reference mean BLN MO MU3

0.22 0.27 0.15 0.24

0.00 0.00 0.00 0.00

45.6 29.1 35.5 72.1

6.63 6.82 6.61 6.45

Urban mean BU1 BU2 RB

1.68 1.83 1.52 1.69

0.15 0.10 0.19 0.15

105.0 99.0 107.7 108.3

6.72 6.93 6.77 6.47

that is separated from the watersheds used as sources of water and substrate in this study. These clutches were presumed to have hatched seven days prior to collection because the site was first visited on 1 June 2008, when some members of both clutches still were retained within each egg mass and others were in close proximity to the egg masses. We combined the two sets of tadpoles to maximize genetic diversity and, on 9 June 2008, established the following design. Treatments consisted of water (0.75 L) and substrate (135 g) collected from three urban streams and three forested streams and placed into separate 1.2-L plastic containers. We changed water and substrate every 8– 14 days, using samples collected from the field within the previous 24 h. The interval for water changes was the same for all treatments. We placed eight-day-old tadpoles into individual, 1.2-L containers (diameter 5 14.5 cm, height 5 7.0 cm) to avoid any effect of competition on growth or development. Containers were held in the laboratory, and temperatures were maintained at 23–25uC. The laboratory space contained windows, which allowed the tadpoles to be exposed to sunlight and natural photoperiods throughout the experiment. Fifteen replicates were established for each of the six streams, for a total of 90 tadpoles (45 exposed to water and substrate from an urban stream and 45 exposed to water and substrate from a forested stream). Tadpoles were fed 0.04 g of aquatic turtle pellets (Rep-Cal Aquatic Turtle Food) every 3–4 days (ad libitum). All individuals were fed on the same day throughout the experiment. Six tadpoles from each stream were sacrificed 26 days after hatching using 0.04% unbuffered MS 222 solution; thus, they could be evaluated for oral disc abnormalities. We also recorded wet mass and length measurements on these individuals. The sacrificed individuals were Stage 37 (Gosner, 1960), which was chosen because the oral disc is known to atrophy during later stages of development (Peterson et al., 2008). To assess oral disc development, we first looked for and recorded any severe malformations to the disc. Subsequently, we counted the number of teeth in each tooth row and used this as a response variable indicative of oral disc quality. The remaining tadpoles from each stream were allowed to develop through metamorphosis, which

we defined as emergence of forelimbs and resorption of at least one-half of the tail. When tadpoles reached this stage, we recorded wet mass, snout–vent length (SVL), and elapsed time (days since hatching). Finally, we noted all mortality within each treatment group. We used a directional, mixed-model, nested analysis of variance (ANOVA) with land use category as a main effect and stream as a random factor nested within land use to model our response variables (i.e., size or tooth number of 26-day tadpoles). The ANOVAs were directional tests based on our a priori predictions that both size and tooth number would be greater in tadpoles reared in water from forested streams, because directional tests are more powerful than conventional tests when the response of the treatment group (urban water) is hypothesized to differ from the control in a given direction (Rice and Gaines, 1994). Because we measured several aspects of body size (total length, body length, tail height, and mass), we conducted a principal components analysis on these measurements to account for intercorrelation of the variables. The first two axes in this analysis explained 91% of the variance in the size data. PC1 was equally defined by mass, body length, and total length. PC2 was predominately defined by tail height. We conducted two separate nested ANOVA with each principal component as a response variable. PC1 data were ln(10 + x), where x 5 PC1 value, transformed to meet the assumptions of normality. We also used a directional, mixed-model, nested ANOVA with the same predictor variables outlined above for the data on individuals recently completing metamorphosis. For these data, we used mass and SVL as response variables, both of which we predicted to be greater in tadpoles reared in forested water. Finally, we conducted a directional ANOVA with the above predictor variables using the residuals of the regression of body mass against SVL (body condition index). All analyses were conducted in Minitab Version 13. Alpha was set at 0.05. RESULTS The 26-day old tadpoles reared in water from urban streams were significantly smaller (PC1) than those reared in water from forested streams (main effect of land use category, F1,30 5 6.58, P 5 0.02; Fig. 1A). There was no significant difference between the two treatments for tail height as represented by PC2 (F1,30 5 2.66, P 5 0.11). We found no consistent difference in numbers of teeth for tadpoles grown in water from urban versus forested streams (F1,30 5 5.39, P 5 0.97). In all tests for an effect of land use on tadpole size or development, the F-test for a stream effect was not statistically significant (P . 0.05), and the variance component of the stream variable was 4% or less. Four tadpoles raised in water and substrate from forested streams died before transforming, as did five raised in water and substrate from urban streams. Mean size at metamorphosis for individuals raised in water and substrate from forested streams (10.89 mm SVL; 0.15 g) was not significantly different from individuals raised in water and substrate from urban streams (10.62 mm SVL; 0.14 g). This was true when size was assessed as SVL (F1,39 5 0.82, P 5 0.37) or mass (F1,39 5 0.76, P 5 0.39). Mean body condition index at metamorphosis for tadpoles reared in water and

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FIG. 1. Effects of water and substrate from forested (black bars) and urban (gray bars) streams on growth of Bufo fowleri tadpoles. (A) Tadpole total length at day 26 of development (N 5 6 for all streams). Total length was highly correlated with body length and mass; hence, analyses were run on the scores of the first principal component for these variables. (B) Number of days following hatching required for tadpoles to reach metamorphosis when grown in water and substrate from forested and urban streams. Sample sizes listed in bars. Mean (6 SE) values for urban streams 5 37.1 6 0.78 days; forested streams 5 39.1 6 0.74 days. substrate from forested streams was positive (i.e., mean value of residuals above the line of best fit for regression of body mass vs. SVL). Mean body condition index at metamorphosis was negative for those tadpoles reared in water and substrate from urban streams (i.e., mean value of residuals below the line of best fit for regression of body mass vs. SVL). The difference in mean body condition index between treatments was not statistically significant (F1,39 5 0.23, P 5 0.63). Tadpoles reared in urban stream water and substrate took significantly fewer days on average to achieve metamorphosis relative to tadpoles reared in water from forested streams (F1,39 5 5.82, P 5 0.02; Fig. 1B). In all tests for an effect of land use on size or time to metamorphosis, the F-test for a stream effect was not statistically significant (P . 0.05), and the variance component of the stream variable was 9% or less. DISCUSSION Our results were mixed with respect to the predictions of Wilbur and Collins (1973), who stated that amphibian larvae exposed to a stressful aquatic

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environment will undergo metamorphosis at an earlier date and a smaller size to escape the stressor. Although tadpoles exposed to water and substrate from urban streams did complete metamorphosis two days sooner than tadpoles reared in water and substrate from forested streams, our data did not match the prediction of Wilbur and Collins (1973) for size at metamorphosis. The statistically significant size difference we observed between urban and forested treatments in 26-day-old tadpoles did not yield smaller individuals at metamorphosis for tadpoles grown in water and substrate from urban streams. Instead, tadpoles experiencing urban water and substrate grew faster after day 26, achieving the same final size as tadpoles experiencing water from forested streams. Because we fed all tadpoles a diet that was nutrient rich, our methods might have facilitated levels of growth in the lab that are not attainable in field settings. Nevertheless, our data suggest that water quality alone can alter tadpole growth. Several studies have evaluated the impact of competitors or predators on tadpoles. Resource availability and predation pressure were the focus of Wilbur and Collins (1973) as the external stressors that could regulate timing of metamorphosis. More recently, some researchers have evaluated the ability of tadpoles to adjust date of and size at metamorphosis in response to polluted water (Edwards et al., 2006). Although no physiological pathway has been mapped conclusively that would demonstrate tadpoles can detect and respond to toxins by reducing age at metamorphosis, such an adaptive response has been judged to be likely (Rose, 2005). Our data suggest effects on growth trajectories in polluted waters might be complex, while still yielding the expected reduction in age at metamorphosis. Although we did not measure potential toxins directly as part of this study, other monitoring efforts in the same streams from which we drew water, coupled with previous research at other sites, provide some specific variables that should be evaluated in future studies such as ours. Specifically, past studies have measured elevated levels of nitrates and conductivity (Schoonover et al., 2005; J. E. Schoonover and B. S. Helms, unpubl. data) from our urban water and substrate sources. Laboratory and mesocosm studies have provided evidence that nitrates do (Smith et al., 2005) and do not (Allran and Karasov, 2000; Meredith and Whiteman, 2008) lead to significant developmental changes in amphibians. Elevated levels of nitrates and conductivity (a composite measure of several pollutants entering the stream from runoff), in conjunction with various herbicides or pesticides applied to residences, are the most likely contributors to our observed results. Additionally, an emerging body of literature suggests synergistic effects from multiple pollutants are the most likely reasons for observed developmental anomalies and population declines in frogs (Blaustein and Kiesecker, 2002; Pahkala et al., 2002). Studies such as ours offer another way to assess such synergisms, because they provide size data from tadpoles developing in water containing a variety of pollutants. Lab studies manipulating pollutants singly, or in more complex multivariate designs, can use growth patterns obtained from our design as a reference point for environmentally relevant growth responses.

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Bufo fowleri appears to be a hearty species, because we consistently observed adults at both urban and forested field sites. Mechanisms underlying the absences of other, potentially more sensitive, species from urban watersheds may be explained from further investigations using B. fowleri. Certainly additional investigation is warranted because data from this study, evidence of declines in amphibian species richness in urban areas (Barrett and Guyer, 2008), and data on increased incidence of fish lesions in urban waters (Helms et al., 2005) all demonstrate the strong negative effect urban environments can exert on the local biota. Acknowledgments.—We would like to thank S. T. Samoray for helping to maintain the experiment and D. J. McMoran for finding the tadpoles. This research was approved by the Auburn University Institutional Animal Care and Use Committee (PRN 2007-1207). LITERATURE CITED ALLAN, J. D. 2004. Landscapes and riverscapes: the influence of land use on stream ecosystems. Annual Review of Ecology, Evolution, and Systematics 35:257–284. ALLRAN, J. W., AND W. H. KARASOV. 2000. Effects of atrazine and nitrate on Northern Leopard Frog (Rana pipiens) larvae exposed in the laboratory from posthatch through metamorphosis. Environmental Toxicology and Chemistry 19:2850–2855. BARRETT, K., AND C. GUYER. 2008. Differential responses of amphibians and reptiles in riparian and stream habitats to land use disturbances in western Georgia, USA. Biological Conservation 141:2290–2300. BLAUSTEIN, A. R., AND P. T. J. JOHNSON. 2003. The complexity of deformed amphibians. Frontiers in Ecology and the Environment 1:87–94. BLAUSTEIN, A. R., AND J. M. KIESECKER. 2002. Complexity in conservation: lessons from the global decline of amphibian populations. Ecology Letters 5:597–608. BOONE, M. D., C. M. BRIDGES, AND B. B. ROTHERMEL. 2001. Growth and development of larval Green Frogs (Rana clamitans) exposed to multiple doses of an insecticide. Oecologia 129:518–524. BURGER, J., AND J. W. SNODGRASS. 2000. Oral deformities in several species of frogs from the Savannah River Site, USA. Environmental Toxicology and Chemistry 19:2519–2524. CHEN, T. H., J. A. GROSS, AND W. H. KARASOV. 2006. Sublethal effects of lead on Northern Leopard Frog (Rana pipiens) tadpoles. Environmental Toxicology and Chemistry 25:1383–1389. EDWARDS, T. M., K. A. MCCOY, T. BARBEAU, M. W. MCCOY, J. M. THRO, AND L. J. GUILLETTE, JR. 2006. Environmental context determines nitrate toxicity in Southern Toad (Bufo terrestris) tadpoles. Aquatic Toxicology 78:50–58. GIBBS, J. P., K. K. WHITELEATHER, AND F. W. SCHUELER. 2005. Changes in frog and toad populations over 30 years in New York State. Ecological Applications 15:1148–1157. GOSNER, K. L. 1960. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16:183–190.

HELMS, B. S., J. W. FEMINELLA, AND S. PAN. 2005. Detection of biotic responses to urbanization using fish assemblages from small streams of western Georgia, USA. Urban Ecosystems 8:39–57. MEREDITH, C. S., AND H. H. WHITEMAN. 2008. Effects of nitrate on embryos of three amphibian species. Bulletin of Environmental Contamination and Toxicology 80:529–533. PAHKALA, M., K. RA¨SA¨NEN, A. LAURILA, U. JOHANSON, L. O. BJO¨RN, AND J. MERILA¨. 2002. Lethal and sublethal effects of UV-B/pH synergism on common frog embryos. Conservation Biology 16:1063–1073. PARRIS, K. M. 2006. Urban amphibian assemblages as metacommunities. Journal of Animal Ecology 75:757–764. PAUL, M. J., AND J. L. MEYER. 2001. Streams in the urban landscape. Annual Review of Ecology and Systematics 32:333–365. PEHEK, E., AND R. MAZOR. 2008. Effects of a stormwater impoundment on streamside salamander populations on Staten Island, New York. In J. C. Mitchell, R. E. J. Brown, and B. Bartholomew (eds.), Urban Herpetology, pp. 85–94. Society for the Study of Amphibians and Reptiles, Salt Lake City, UT. PETERSON, J. D., V. A. PETERSON, AND M. T. MENDONC¸A. 2008. Growth and development effects of coal combustion residues on Southern Leopard Frog (Rana sphenocephala) tadpoles exposed throughout metamorphosis. Copeia 2008:499–503. PRICE, S. J., M. E. DORCAS, A. L. GALLANT, R. W. KLAVER, AND J. D. WILLSON. 2006. Three decades of urbanization: estimating the impact of land-cover change on stream salamander populations. Biological Conservation 133:436–441. RICE, W. R., AND S. D. GAINES. 1994. ‘‘Heads I win, tails you lose’’: testing directional alternative hypotheses in ecological and evolutionary research. Trends in Ecology and Evolution 9:235–237. ROSE, C. S. 2005. Integrating ecology and developmental biology to explain the timing of frog metamorphosis. Trends in Ecology and Evolution 20:129–135. ROWE, C. L., O. M. KINNEY, AND J. D. CONGDON. 1998. Oral deformities in tadpoles of the bullfrog (Rana catesbeiana) caused by conditions in a polluted habitat. Copeia 1998:244–246. SCHOONOVER, J. E., B. G. LOCKABY, AND S. PAN. 2005. Changes in chemical and physical properties of stream water across an urban-rural gradient in western Georgia. Urban Ecosystems 8:107–124. SMITH, G. R., K. G. TEMPLE, H. A. DINGFELDER, AND D. A. VAALA. 2005. Effects of nitrate on the interactions of the tadpoles of two ranids (Rana clamitans and R. catesbeiana). Aquatic Ecology 40:125–130. WALSH, C. J., A. H. ROY, J. W. FEMINELLA, P. D. COTTINGHAM, P. M. GROFFMAN, AND R. P. MORGAN. 2005. The urban stream syndrome: current knowledge and the search for a cure. Journal of the North American Benthological Society 24:706–723. WILBER, H. M. 1980. Complex life cycles. Annual Review of Ecology and Systematics 11:67–93. WILBUR, H. M., AND J. P. COLLINS. 1973. Ecological aspects of amphibian metamorphosis. Science 182:1305–1314. Accepted: 26 June 2009.