Abstract
Amphibians face a variety of anthropogenic environmental perturbations that could act alone or in combination to influence population size. We investigated interactive effects of warming conditions, a moderate pulse of nitrogen pollution, and conspecific density on larvae of the common frog, Rana temporaria. The 16-day experiment had a 2 × 2 × 2 factorial design implemented in 80-l outdoor mesocosms. High density and warm temperature both resulted in reduced activity and visibility; tadpoles grew and developed more quickly at low density and high temperature. The high-nitrogen treatment did not influence behavior, growth, or development rate. We attribute this to several realistic features of our study, including a pulsed treatment application and natural denitrification within the mesocosms. There was only a single interaction among the three factors: higher temperature exacerbated density-dependence in growth rate. These results illustrate that climate warming may benefit temperate amphibians, although the benefits may be counteracted by enhanced larval crowding.
Introduction
Organisms are exposed to environmental stress when conditions fall outside the range within which individual fitness is high (Hoffman and Parsons, 1991; Ghalambor et al., 2007). This definition of stress emphasizes that environmental change is not inherently stressful, but becomes so only after conditions move beyond a certain range. For example, amphibian larvae often respond to a slight increase in water temperature with improved growth and development, whereas further warming causes physiological stress and eventually mortality (Newman, 1998; Duarte et al., 2012). Responses to stress are interesting for many reasons: they reflect the physiological capabilities of organisms, reveal the nature of trade-offs among fitness traits, and can influence the distribution of species (Hoffman and Parsons, 1991).
Humans are changing the environment in ways that, when sufficiently severe, can be detrimental to many organisms. Studies of amphibian responses to nitrogen pollution and pesticides have figured prominently in the literature (Mann et al., 2009; Marco and Ortiz-Santaliestra, 2009; Sparling et al., 2010; Egea-Serrano et al., 2012; Baker et al., 2013). Curiously, results from different studies suggest that pollution may have either positive or negative impacts on amphibian life history traits and survival (reviewed in Camargo et al., 2005; Egea-Serrano et al., 2012). Some of this variation is probably caused by real differences among species in their reaction to pollution (e.g., Bridges and Semlitsch, 2000), but there may also be methodological explanations for divergent results. For example, experiments have been conducted in a variety of venues, which can exhibit sharply different pollution dynamics and impacts. In the meta-analysis of Egea-Serrano et al. (2012), treatment with nitrogenous compounds caused a 4.4% decline in mass and 14.4% decline in survival in laboratory studies, but a 49.2% increase in mass and 5.6% increase in survival in outdoor mesocosms. The positive effects of pollution in more natural settings may result from microbial break-down of nitrogen pollution (Kemp and Dodds, 2002) combined with a fertilizing effect of nitrogen on the food of amphibian larvae (Boone et al., 2007). Second, treatment levels vary tremendously among these experiments (e.g., Johansson, Räsänen and Merila, 2001; Ortiz-Santaliestra et al., 2006; Egea-Serrano, Tejedo and Torralva, 2009, 2011; Oromi, Sanuy and Vilches, 2009). Finally, there is evidence that effects of anthropogenic chemicals are context-dependent: experiments demonstrating interactions among multiple factors are frequent (e.g., Boone and Bridges, 1999; Boone and Semlitsch, 2001; Hatch and Blaustein, 2003; Egea-Serrano, Tejedo and Torralva, 2009; Ortiz-Santaliestra et al., 2010; Ortiz-Santaliestra, Fernández-Benéitez and Marco, 2012). Although competitors and predators generally reduce amphibian survival, in polluted environments the negative effects of species interactions may be ameliorated by other changes in the aquatic community (Mills and Semlitsch, 2004; Distel and Boone, 2010). Taken together, these considerations suggest that laboratory studies may present a biased account of the impact of nitrogen pollution on amphibian larvae because of their low environmental realism (e.g., they do not simulate community-level processes well). This has been recently discussed in the context of herbicides (Lanctot et al., 2013; Edge et al., 2014). Here, we describe an experiment manipulating three potential sources of stress in semi-natural outdoor mesocosms while applying treatment levels that are representative of human-impacted landscapes.
Our aim was to test for interactive effects of conspecific density, elevated temperature and nitrogen pollution on larval anurans. For all three factors, treatment levels represent realistic variation observed in freshwater wetlands. We recorded effects on traits related to performance (survival, growth, and development) and on potential early indicators of sub-lethal impacts (activity and refuge use). Much previous work suggests that modest warming should improve the growth and development rates of ectothermic organisms such as tadpoles (e.g., Newman, 1998; Laugen et al., 2003; Castano et al., 2010). Resource availability should be enhanced by moderately elevated temperature and nitrogen concentration because these agents will fertilize and increase growth of the algae and small animals consumed by tadpoles. Temperature and nitrogen may be especially important at high density when resources are otherwise scarce (Wilbur, 1977a; Van Buskirk and Yurewicz, 1998).
Materials and methods
The study organism was Rana temporaria, a widely-distributed Eurasian frog occurring in natural, agricultural, and residential landscapes. The aquatic larvae develop in wetlands ranging from small puddles to large lakes. This broad habitat distribution means that R. temporaria tadpoles can be naturally exposed to agricultural runoff and to a range of temperatures and densities.
We manipulated water temperature, nitrogen level, and density of larval R. temporaria in a 2 × 2 × 2 complete factorial design, with six replicates of each treatment. Experimental units were 48 mesocosms (plastic tubs with 80 l water, 0.28 m2), arranged outdoors in six spatial blocks in a field at the University of Zurich. Mesocosms were filled with water on 6 March 2011, inoculated soon after with water and zooplankton from a natural pond, provided with 40 g of dried leaf litter (mostly Fagus sylvatica) and 2 g of rabbit food (JR Farm, Holzheim, Germany), and covered with lids constructed of 43% shade cloth.
Water temperature was either ambient (unmanipulated) or artificially increased. Warm temperature was achieved by placing a 100 W aquarium heater on the bottom of the mesocosm, set to operate at 24°C and turned on between 08:00 h and 17:00 h each day. Surface temperature in the warm treatment averaged 3.2°C higher than in the ambient treatment in late afternoon and 2.5°C higher in early morning (see online Supplementary Appendix A).
Nitrogen treatments included an unmanipulated control and a nitrogen-addition treatment. Each mesocosm in the high-nitrogen treatment received a single aliquot of 41.6 ml from a stock solution (76.8 g NH4NO3/l) diluted with 958.4 ml of dechlorinated tap water, mimicking a single pulse of pollution. Low-nitrogen mesocosms received 1 l of dechlorinated tap water. This was done on 6 April, one day after the tadpoles were introduced. The nominal concentration in the high-nitrogen treatment was 39.9 mg/l NH4NO3, corresponding to 9 mg/l NH4-NH4NO3 and 31 mg/l NO3-NH4NO3. Our treatment level was higher than 151 of the 185 nitrate values (81.6%) reported in published surveys of natural ponds (Secondi et al., 2009; Sahuquillo et al., 2012; Wexler, Hiscock and Dennis, 2012; Merchán et al., 2014). This treatment may therefore represent exposure to levels of pollution occurring only occasionally, perhaps after run-off events. Nitrate could not be manipulated independently from ammonium because we used NH4NO3 as a nitrogen source and, consequently, the nominal ammonium concentration was higher than values found in natural ponds (Griffis-Kyle and Ritchie, 2007; Sahuquillo et al., 2012). Nevertheless, this is realistic because NH4NO3 is among the most commonly used fertilizers throughout the world (FMA, MAFF and SOAFD, 1993). In natural systems, pulses of nitrogen rapidly decline due to nutrient uptake and/or microbial denitrification (Kemp and Dodds, 2002; Camargo and Alonso, 2006; Smith et al., 2009). We confirmed that this occurred in our mesocosms as well. Nitrate concentration in a randomly-chosen sample of eight low-nitrogen and eight high-nitrogen mesocosms was measured on 10 April and 19 April (see online Supplementary Appendix B). Nitrate levels were much higher during the first week of the experiment in the high-nitrogen treatment (supplementary table S2, supplementary fig. S2), approximately equal to the nominal concentration (30.2 mg/l NO3− in high-nitrogen; 1.69 mg/l NO3− in low-nitrogen). By the second week of the experiment, the nitrate level in the high-nitrogen treatment had declined to only slightly greater than that in the low-nitrogen treatment. Details are available in Appendix B. Ammonium was not assessed, but the fact that nitrate concentration remained high for the first four days supports the hypothesis that ammonium was, at least partially, transformed to nitrite and eventually to nitrate over time. Nitrogen addition also caused higher salinity in the high-nitrogen treatment, as observed previously by Egea-Serrano, Tejedo and Torralva (2009) (see online Supplementary Appendix C). However, salinity did not reach levels at which tadpole survival or behavior might be affected (ca. 500 mg/l; Winkler and Forte, 2011).
Density was manipulated by adding to each mesocosm either 8 or 32 tadpoles (29/m2 or 114/m2). These two densities are lower than 26% and 15%, respectively, of 182 estimates of R. temporaria density made in natural populations part-way through the larval stage (Van Buskirk, 2005). Eight egg clutches contributed equally to each mesocosm. At the beginning of the experiment (5 April), tadpoles were 7 days old, and were at Gosner (1960) stage 25; the mean mass of a randomly selected sample of 80 larvae not included in the experiment was 30 ± 0.8 mg (mean ± 1 SE). The eggs originated from a permanent pond in a mature deciduous forest (coordinates 47.389N, 8.562E; elevation 645 m). The pond is within a city park of Zurich, Switzerland, and there is no known history of agriculture or urban development within its watershed.
We observed the behavior of tadpoles on two occasions (10-11 April and 18-19 April). Lids were removed from the mesocosms 30 minutes before observations began, and then mesocosms were visited four times on each day between 10:00 and 16:00. On each visit we recorded the number of tadpoles that were active (swimming or feeding; Egea-Serrano et al., 2011), the total number of individuals that were visible, and the number that were within 1 cm of the surface of the water. The three behaviors were recorded during three separate 30-second observations of the mesocosm so that each visit lasted 90 seconds in total. The proportion active, the proportion visible, and the proportion at the surface were calculated after estimating the number alive in the mesocosm assuming a constant daily risk of mortality. According to this calculation, activity reflects the proportion active if hiding individuals are primarily inactive. We chose these behaviors because they are related to foraging effort and can exhibit responses to environmental contamination (Smith and Van Buskirk, 1995; Marco and Blaustein, 1999; Egea-Serrano et al., 2011; Denoël et al., 2012).
The experiment was terminated after 16 days (21 April), at which point we recorded survival and selected five larvae randomly from each mesocosm to be weighed (±0.001 g) and photographed in lateral aspect. Developmental stage was determined from the photographs.
Statistical analysis
Survival was analyzed using a generalized linear mixed model, with logit link and binomial error. Temperature, nitrogen, density, and their interactions were fixed factors. Block and mesocosm (nested within block) were random factors.
All other analyses were conducted on mesocosm means. Growth, calculated as the daily proportional increase in body mass, and log-transformed developmental stage were centered and then analyzed simultaneously with a mixed-effects multivariate analysis of variance. The three behavioral responses were arcsin square root transformed, centered, and then analyzed with a mixed-effects multivariate repeated measures analysis of variance. Random factors were the same as in the analysis of survival, and the fixed effects included temperature, nitrogen, density, trait (growth/development or activity/surface/visible), and their interactions. Analysis of behavior included a fixed effect of date (10-11 April or 18-19 April). Multivariate analyses were followed by univariate mixed-effects ANOVAs for each response, including only block in the random part.
These models were implemented in R 3.1.0 with lmer and lmerTest, using Type III SS and Satterthwaite’s approximation to estimate denominator degrees of freedom (Baayen, Davidson and Bates, 2008).
Summary of multivariate and univariate analyses of variance on growth rate and developmental stage of Rana temporaria tadpoles exposed to manipulated temperature, nitrogen, and density. Stage was log-transformed, and then both response variables were centered before analysis. Boldface highlights effects that were significant at . Random effects were never important (all P-values ⩾ 0.9).
Life history responses of R. temporaria tadpoles after exposure for 16 days to variation in nitrogen concentration, temperature, and density in outdoor mesocosms. Symbols are treatment means ± 1 SE (). Black lines and circles: ambient temperature; gray lines and triangles: warm. Open symbols and dashed lines: low-nitrogen, filled symbols and solid lines: high-nitrogen.
Citation: Amphibia-Reptilia 37, 1 (2016) ; 10.1163/15685381-00003029
Results
Survival was high and unaffected by any of the treatments or interactions ( in all cases; see online Supplementary Appendix D). MANOVA on growth and development revealed significant effects of temperature and density (table 1). Tadpoles grew and developed more slowly under ambient temperature than under artificially increased temperature (fig. 1; table 1). For growth rate, high density also had a negative effect. The temperature-by-density interaction in univariate analysis of growth was caused by a greater impact of increasing density at warm temperature (fig. 1A; table 1).
Tadpole behavior changed through time and was affected by temperature and density (table 2). Univariate analyses revealed that the proportions active and at the water surface decreased at high density and warm temperature (fig. 2; table 2). For tadpoles at the surface, the negative effect of density was reduced under warm conditions. The proportion of larvae visible decreased in the high density treatment (fig. 2, table 2). Activity, proportion at the surface, and visibility declined significantly with tadpole age. Multi-way interactions involving time were mostly weak, indicating that treatment effects on behavior were largely consistent across the two observation dates. Nitrogen and its interactions did not influence behavior (table 2).
Multivariate and univariate mixed-effects repeated measures analyses on three measures of behavior in Rana temporaria tadpoles. “Activity” is the proportion of tadpoles moving, “surface” is the proportion within 1 cm of the surface, and “visible” is the proportion of tadpoles present that were not hiding above the leaf litter. The three response variables were arcsin-sqrt transformed and then centered before analysis. Boldface highlights effects that were significant at . Random effects were never important (all P-values ⩾ 0.9).
Discussion
Warm water had a positive effect on the growth and developmental rate of R. temporaria tadpoles, and crowding had negative effects. These impacts on performance have been studied extensively in amphibian larvae. The beneficial effect of increasing temperature is attributable to a combination of physiological and ecosystem mechanisms. Enzymatic activity and metabolism of ectotherms is enhanced in warm environments (Angilletta, 2009, ch. 3), animals may eat more food at warm temperature (Warkentin, 1992; Bernardo et al., 2011), and digestive conversion efficiency tends to increase with temperature (Lindgren and Laurila, 2005; Stoks, Swillen and De Block, 2012). In the laboratory, these are the presumed causes of temperature effects on amphibian growth and development (Newman, 1998; Laugen et al., 2003). Under natural conditions, ecosystem changes triggered by warm temperature include enhanced growth rate of primary producers (Goldman and Carpenter, 1974; DeNicola, 1996), which can contribute to a growth response of herbivores (O’Regan, Palen and Anderson, 2014). This process may have contributed to the temperature treatment effect on tadpole growth in our semi-natural outdoor mesocosms. The density effect is primarily attributable to reduced per capita availability of food (Wilbur, 1977b; Steinwascher, 1978). These impacts of temperature and larval density may have consequences at the population level or contribute to population regulation, because declines in larval performance sometimes translate into reduced recruitment at reproduction (Smith, 1987; Berven, 1990).
Behavioral responses of R. temporaria tadpoles to variation in nitrogen concentration, temperature, and density. Data are averaged across the two observation occasions because interactions involving date were generally unimportant (table 2). Symbols are means ± 1 SE (). Black lines and circles: ambient temperature; gray lines and triangles: warm. Open symbols and dashed lines: low-nitrogen, filled symbols and solid lines: high-nitrogen.
Citation: Amphibia-Reptilia 37, 1 (2016) ; 10.1163/15685381-00003029
High levels of nitrogen pollution are known to harm larval amphibians (e.g., Egea-Serrano et al., 2012; Baker et al., 2013). In our experiment, however, the behavioral data and performance measures indicated that nitrogen was not stressful. This is presumably related to realistic features of the experimental venue and the modest treatment levels that we deployed. As described in Methods, our nominal concentration of 39.9 mg/l NH4NO3 is fairly high in comparison with published environmental data, but is much lower than concentrations used in many previous experiments (reviewed in Camargo et al., 2005; Mann et al., 2009). In addition, the outdoor mesocosms that we used may generate less severe impacts of nitrogen pollution, for three reasons (Egea-Serrano et al., 2012). First, experiments carried out in mesocosms commonly simulate a single pollution event, whereas treatment levels in laboratory studies are typically designed to remain constant. Microbial denitrification in mesocosms causes sharply reduced concentration of nitrate within a few days or weeks, just as it does in natural wetlands and field experiments (Kemp and Dodds, 2002; Camargo and Alonso, 2006; Griffis-Kyle and Ritchie, 2007; Massal et al., 2007). This leads to shorter exposure to nitrogen pollution than in laboratory settings. Second, conditions in outdoor mesocosms can be less crowded than in the lab, which may produce less stress, allowing tadpoles to invest more heavily in detoxification (Egea-Serrano et al., 2012). Finally, at the realistic concentrations that we used, nitrate acts to fertilize periphyton, and this may counter-balance any direct negative effects (Boone et al., 2007; Egea-Serrano and Tejedo, 2014). This process cannot occur in the lab, where resources are controlled and water changes are frequent.
Taken together, our data indicate that relevant concentrations of nitrogen pollution may be less stressful in natural conditions than suggested by laboratory results. The factors that reduce impacts in mesocosms, described in the preceding paragraph, are likely to be generally fulfilled in natural wetlands, and therefore we suspect that toxicology experiments overestimate the real-world impacts of exposure to nitrogen. The meta-analysis results of Egea-Serrano et al. (2012) described in the Introduction are consistent with this conclusion, because they show that negative impacts of nitrogen fertilizers in the lab become neutral or positive when assessed in mesocosms. Others have reached similar conclusions – for similar reasons – regarding the impacts of herbicides on aquatic organisms in natural wetlands (Lanctot et al., 2013; Baker et al., 2014; Edge et al., 2014). At this stage, however, comparison of the effects of pollution between lab and natural conditions is difficult because so few experiments have been performed in the field (Peltzer et al., 2006; Griffis-Kyle and Ritchie, 2007; Karraker et al., 2008; Egea-Serrano and Tejedo, 2014).
Our results suggest that direct effects of habitat warming on R. temporaria will be positive, caused by enhanced growth and development rates during the larval stage. This is based on treatment levels that were realistic in the sense that the temperature increase was close to that expected in global surface air temperature by the year 2100 (Meehl et al., 2007). Indeed, positive effects of climate warming are expected in many organisms that currently occur below their thermal optima (Deutsch et al., 2008; Duarte et al., 2012; Thomas et al., 2012). This appears to be the case for R. temporaria in central and northern Europe (Duarte et al., 2012), and for many other temperate amphibians (Alvarez and Nicieza, 2002; Sanuy, Oromí and Galofré, 2008; Orizaola and Laurila, 2009; Castano et al., 2010). However, the population-level consequences of these effects are uncertain because warming is likely to have additional effects on amphibian larval habitats, such as shortening the hydroperiod (Carey and Alexander, 2003) and increasing tadpole density and the concentration of nitrogen and salts as the pond desiccates (Tejedo and Reques, 1994; Tan, 2002; Fernández-Aláez and Fernández-Aláez, 2010). Positive effects of warming may compensate for an abbreviated hydroperiod because enhanced development allows animals to escape drying ponds (O’Regan, Palen and Anderson, 2014), but it is not known whether the same is true for changes in salinity or pollution.
Several studies of amphibian larvae have reported interactions between density and temperature or pollutants (Boone and Bridges, 1999; Boone and Semlitsch, 2001; Mills and Semlitsch, 2004; Govindarajulu and Anholt, 2006; Distel and Boone, 2009; Ortiz-Santaliestra, Fernández-Benéitez and Marco, 2012). Although we observed relatively few interactions, those that were detected may be important. For example, individual growth rate was lower in the high density treatment, but the decrease was strongest when temperature was warm. Crowded conditions therefore reduced the positive effect of warming on tadpole performance, probably because resource depletion at high density prevented animals from exploiting the more favorable growth conditions represented by warm temperature. O’Regan, Palen and Anderson (2014) illustrate how a warmer climate compensates for the stress imposed on larval amphibians by pond drying, but our data suggest the opposite message: warmer conditions can also exacerbate the negative effects of some natural stressors.
This study suggests that the direct effects of anthropogenic factors generally held to be stressful for amphibian larvae may in fact be neutral (in the case of nitrogen pollution) or even positive (habitat warming). Further data on these and other changes in the environment will be valuable because of the context-dependence of many sources of stress (e.g. the effect of temperature may depend on density), and because embryonic and adult stages may respond differently (Galloy and Denöel, 2010).
Acknowledgements
We are grateful to Christopher Robinson for help with nitrate assessment. Funding came from the Swiss National Science Foundation and the Conselho Nacional de Desenvolvimento Científico y Tecnológico – CNPq of Brazil (ref.370592/2013-1). We thank three anonymous reviewers for comments on the manuscript. Ethics permits were provided by the Veterinary Office of Canton Zurich and collection permits by the nature conservation office of Canton Zurich. A.E.S. designed and performed the experiment, collected and analyzed data, and wrote the manuscript. J.V.B. obtained funding and permits, designed the experiment, analyzed the data, and wrote the manuscript.
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Footnotes
Associate Editor: Sebastian Steinfartz.
