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Body mass, developmental stage, and interspecific differences in acid tolerance of larval anurans.

Abstract. -- The relationships of body mass and developmental stage to acid tolerance were examined in tadpoles of three species of anurans from central Texas: Bufo woodhousei, Bufo valliceps, and Gastrophryne olivacea. Significant differences in survival time at low pH occurred among all three species (survival time of Bufo valliceps > Bufo woodhousei > Gastrophryne olivacea). Body mass and developmental stage were significantly correlated, and their relationship to survival time was complex and species specific. Multiple regression analysis indicated that for Bufo woodhousei, mass was positively associated with survival time; there was no effect of developmental stage independent of its correlation with mass. For Bufo valliceps, mass was positively associated with survival time, whereas developmental stage was negatively associated with survival time. Neither mass nor developmental stage were significantly associated with survival time in Gastrophryne olivacea.


Reports of widespread amphibian declines (Barinaga 1990; Wake 1991) and concerns about the deleterious influence of acid precipitation on amphibian communities have stimulated an increase in recent research on the effects of acidity on amphibians (Freda 1986; Pierce 1993). Although there is little current evidence that acid deposition is responsible for amphibian declines (Dunson et al. 1992; Pierce 1993), this research has provided important information as to how amphibians respond to variation in pH. In general, amphibian larvae are more tolerant of acidity than embryos, and acid tolerance of amphibian larvae increases as larvae grow and develop (Pierce et al. 1984; Freda & Dunson 1985; Freda & MacDonald 1989).

In addition to this ontogenetic variation, there are interspecific (Freda & Dunson 1985; Warner et al. 1991), interpopulation (Pierce & Harvey 1987), and intrapopulation (Pierce & Sikand 1985; Pierce & Wooten 1992) differences in acid tolerance of tadpoles. However, the extent to which these differences in acid tolerance are related to differences in body mass remains unclear. For example, body mass and developmental stage are usually correlated within species, and the increased acid tolerance reported for more developmentally advanced larvae might be simply a consequence of increased body mass. Also, if body mass is important in determining acid tolerance among larvae, the effect of mass should be removed before assessing inter- and intraspecific differences. This investigation examines these issues by studying the associations of body mass and developmental stage to acid tolerance in larvae of three species of anurans: Bufo woodhousei, Bufo valliceps, and Gastrophryne olivacea.

Materials and Methods

Tadpoles of Bufo woodhousei, Bufo valliceps, and Gastrophryne olivacea were collected from a small ephemeral pond located in McLennan County, Texas on 20 May 1991. Adults of all three species had reproduced two weeks earlier during rainfall that filled the pond; thus all larval specimens were approximately the same age. Following collection, the tadpoles were transported to the laboratory, separated by species, and placed in neutral (pH 7.2-7.7) reconstituted soft water (RSW), which consisted of 48 mg NaHC[O.sub.3], 30 mg MgS[O.sub.4], 30 mg CaS[O.sub.4] * 2[H.sub.2]O, and 2 mg KC1 dissolved in one liter of deionized [H.sub.2]O (Stephen 1975). Specimens were kept in an incubator at 25 C on a 12 hr light: 12 hr dark photoperiod for 24 hours prior to testing and were not fed during this period.

Immediately prior to testing acid toxicity, each specimen was weighed with the following procedure. A small capped vial containing neutral RSW was weighed on an analytical balance to the nearest 0.0001 g. The specimen was carefully removed from its container with a small mesh net, briefly placed on a wet sponge to remove excess water adhering to the body, and transferred to the weighed vial with a stainless steel spatula. The vial was then capped and reweighed to the nearest 0.0001 g. Wet body mass of the tadpole was then obtained by subtraction. After weighing, each specimen was placed in an individually marked glass beaker containing neutral RSW. The weighing procedure causes no injury or obvious stress to tadpoles (Cummins 1986).

After all specimens were weighed, they were transferred to the test solutions, which consisted of unaltered, neutral RSW (pH 7.2-7.7) or RSW acidified to pH 3.5 with dilute sulfuric acid. A pH 3.5 test solution was used because preliminary tests indicated that this pH produced mortality in all three species during the 72-hour test period. Each specimen was tested individually in 300 ml of solution contained within a 400 ml glass beaker covered with a watch glass. Prior to testing, all beakers and watch glasses were washed with soap and water, rinsed once with tap water, rinsed twice with deionized water, rinsed once with 0.1 M HN[O.sub.3], and rinsed twice more with deionized water. Fifteen specimens of each species were tested in the neutral solution, and 30 specimens of each species were tested in the acidic solution, for a total of 135 test subjects. Specimens were randomly assigned to the treatment groups.

Mortality was assessed by lightly touching each specimen with a small, flat, stainless steel spatula; specimens that failed to respond to this stimulus by exhibiting movement were considered dead. All surviving tadpoles were checked for mortality at 1, 2, 4, 8, 12, 24, 36, 48, 60, and 72 hours after the start of the experiment. Throughout the test period, each beaker was maintained at 25 C on a 12 hr light: 12 hr dark photoperiod. (The dark phase was interrupted briefly during mortality checks.) The positions of beakers within the incubator were randomized at the beginning of the experiment, and they were rerandomized every 24 hours. To minimize pH drift, test solutions were replaced with fresh solution every 24 hours. Before changing solutions, pH was measured in five randomly selected beakers of acidic solution and five randomly selected beakers of neutral solution. Specimens were not fed during the test period.

All specimens that expired during the experiment were immediately preserved in 10% formalin. At the end of the experiment, five surviving specimens of each species from the neutral solution were randomly selected, anesthetized in MS-222, and then preserved in 10% formalin. All other surviving specimens were returned to their native pond. Developmental stage (Gosner 1960) was determined on preserved specimens by examination using a dissecting microscope. For statistical testing, survival time was log transformed ([log.sub.10] of [time + 1]). Analysis of variance and regression were used to examine the relationship of mass, developmental stage, and species to survival time. The experiment-wise error rate was considered to be 0.05.


The pH of the test solutions drifted slightly during the 24 hours between solution changes. For the pH 3.5 solution, the average pH at the end of 24 hours between solution changes was 3.61 (range = 3.54-3.68); for the pH 7.2-7.6 solution, the average pH at the end of 24 hours was 7.56 (range = 7.36-7.7).

The tadpoles of the three species tested differed in developmental stage (F = 53.83; d.f. = 2, 96; P < 0.001). Bufo valliceps tadpoles were the most advanced, with an average Gosner stage of 29 (Table 1). The Scheffe-F test for a posteriori comparisons indicated that the larvae of Bufo valliceps were more advanced than those of Bufo woodhousei and Gastrophryne olivacea, but the latter two species did not differ significantly from one another. Significant differences also occurred in average tadpole mass of the three species at the beginning of the experiment (F = 62.17; d.f. = 2, 132; P < 0.001). Bufo valliceps and Gastrophryne olivacea did not differ significantly in wet mass (both averaged 0.047 g), but both were significantly heavier than Bufo woodhousei tadpoles (average mass = 0.025 g, Table 1). Developmental stage and wet mass were positively correlated within each species (for Bufo woodhousei r = 0.60, d.f. = 31, P < 0.001; for Bufo valliceps r = 0.78, d.f. = 33, P < 0.001); for Gastrophryne olivacea r = 0.70, d.f. = 32, P < 0.001). The slopes of the regressions of mass and stage were similar in all three species.

No specimen in the neutral pH treatment group expired during the 72-hour test; therefore, our analysis is restricted to survival in the acidic solution (pH 3.5). In the acidic solution, all tadpoles died within 60 hours, and there were significant differences in survival time among species (F = 80.06; d.f. = 2, 87; P = 0.0001). A posteriori comparisons (Scheffe-F test) indicated that each species differed significantly from the other two species in survival time. Bufo valliceps tadpoles lived much longer on the average than did tadpoles of the other two species (Table 1). Large numbers of Gastrophryne olivacea and Bufo woodhousei larvae died early in the experiment (100% mortality at 4 and 12 hours, respectively), whereas mortality of Bufo valliceps larvae began later and occurred over a broader range of time (4 hours to 60 hours). Although both Gastrophryne olivacea and Bufo woodhousei tadpoles died relatively early compared to Bufo valliceps, Bufo woodhousei tadpoles survived significantly longer than those of Gastrophryne olivacea.

Mass and survival time at low pH were significantly correlated in Bufo woodhousei tadpoles (r = 0.65, d.f. = 29, P = 0.03), but there was no significant relationship between mass and survival time in Bufo valliceps (r = 0.24, d.f. = 29, P = 0.21) nor in Gastrophryne olivacea (r = 0.27, d.f. = 29, P = 0.27). Similarly, developmental stage and survival time were significantly correlated in Bufo woodhousei (r = 0.42, d.f. = 26, P = 0.03), but not in Gastrophryne olivacea (r = 0.34, d.f. = 27, P = 0.08) nor in Bufo valliceps (r = -0.061, d.f. = 28, P = 0.75).

Because mass and developmental stage are correlated and both are correlated with survival time in Bufo woodhousei, we used multiple regression to examine the independent effects of mass and stage on survival time (Table 2). For Bufo woodhousei, the overall regression was significant. Mass was positively associated with survival time (standardized beta = 0.704), but there was no significant association of stage independent of its correlation with mass. For Bufo valliceps, the overall regression was significant, and both stage and mass had independent effects on survival time. Mass was positively correlated with survival time (standardized beta = 0.81), whereas developmental stage exhibited a negative correlation (standardized beta = -0.71) once the effect of mass on survival time was removed. The overall regression in Gastrophryne olivacea was not significant.


Previous studies found that acid tolerance of amphibian larvae increases with development (Pierce et al. 1984; Freda & Dunson 1985; Freda & MacDonald 1989) and that interspecific and intraspecific differences occur in tadpole acid tolerance (Freda & Dunson 1985; Pierce & Sikand 1985; Pierce & Harvey 1987). However, the extent to which differences in body mass were responsible for these developmental and individual differences was not examined. In tadpoles, surface-to-volume ratio, buffering capacity, and general hardiness (all of which likely affect the sensitivity of amphibian larvae to low pH conditions) are correlated with body mass. Thus, there is reason to suspect that increases in body mass associated with growth and development in tadpoles are likely to result in increased acid tolerance, but the precise relationship between body mass, developmental stage, and acid tolerance has not been studied previously in these animals.

The tadpoles used in this experiment were collected from a pond with relatively high pH (pH 7-8), so the test subjects would not have been previously exposed to acidic conditions. However, the ranges of all three species extend into areas with poorly buffered soils and acidic habitats (in east Texas). Some ponds and streams in east Texas have pHs regularly below 4 and as low as 3.1; we have collected tadpoles from one pond where the pH was frequently below 4.0 (unpublished observations). Thus, tadpoles may be exposed to low pHs under natural conditions.

In interpreting the results of our experiments, it is important to keep in mind that this study examined acid tolerance over a relatively restricted range of body mass and development stage. Larval specimens were purposely chosen that were approximately the same age, so that it could be determined whether species differences are explained by differences in growth and developmental rates. Different results might have been obtained had one used larvae with larger differences in body mass, or larvae that were at the extremes of the developmental sequence.

Results obtained during this study point to several important conclusions. First, it is clear that different amphibian species exhibit different relationships among body mass, developmental stage, and survival time in low pH conditions. Body mass and stage were associated with survival time in Bufo tadpoles (though their interactions were complex), but there was no evidence for an effect of either mass or stage on acid tolerance in Gastrophryne olivacea. Second, body mass displayed a positive relationship to survival time; this was evident from both the univariate analysis and the multiple regression, although the correlation was not significant in all three species. The significant effect of body mass in the multiple regression (for Bufo woodhousei and Bufo valliceps) indicates that mass has an association with survival time that is independent of developmental stage. This association makes sense because increasing body mass increases buffering capacity and reduces the surface-to-volume ratio of the animal, both of which reduce sensitivity to acidity. These findings suggest that the effects of body mass should be assessed and statistically removed in future studies that examine ontogenetic, interspecific, or intraspecific differences in acid tolerance of larvae.

The relationship between developmental stage and survival time was more complex. In the univariate analysis, stage exhibited a positive correlation with survival time, although the correlation was only significant for Bufo woodhousei. However, this correlation appears to be largely the result of the positive correlation between mass and developmental stage. When multiple regression was applied and the effect of body mass removed, developmental stage actually had a negative relationship to survival time (significant only in Bufo valliceps). This finding indicates that among tadpoles of similar mass, those that are more developmentally advanced may actually be less tolerant. Thus, the increase in acid tolerance previously observed with development (Pierce et al. 1984; Freda & Dunson 1985; Freda & MacDonald 1989) may be largely the result of increases in body mass. However, it is important to note that this study examined acid tolerance over a narrow range of tadpole development, and developmental variation may play a more important role in acid tolerance among tadpoles from a wider range of stages.

An important question is whether species differences in acid tolerance are independent of variation in body mass and developmental stage. Normally, this question might be addressed with covariance, using body mass and stage as covariates in an analysis of mean differences in survival time among the species. However, analysis of covariance requires that the slopes of each covariate (body mass of developmental stage) with the dependent variable (survival time) be similar among groups. This is clearly not the case in this study, as the relationships were significant in some species and not significant in others; thus the assumptions of covariate analysis cannot be met. Nevertheless, simple inspection of the data suggests that body mass and developmental stage do not account for all the differences which were observed in survival time among the three species. For example, Bufo valliceps and Gastrophryne olivacea had the same mean body mass, yet Bufo valliceps exhibited much longer survival time than Gastrophryne olivacea. Also, Bufo woodhousei and Gastrophryne olivacea did not differ in developmental stage, but their survival times were significantly different. These observations indicate that there are fundamental differences in acid tolerance between species that are not a simple function of differences in body mass and developmental stage.
Table 1. Mean ([+ or -] standard error) of mass, developmental stage
(Gosner 1960), and survival time at pH 3.5 of Bufo woodhousei, Bufo
valliceps, and Gastrophryne olivacea tadpoles.

Species Mass (g) Stage Survival
 Time (hr)

Bufo woodhousei 0.025 [+ or -] 0.001 26.4 [+ or -] 0.14 4.8 [+ or -]
Bufo valliceps 0.047 [+ or -] 0.002 28.9 [+ or -] 0.20 22.5 [+ or -]
Gastrophryne 0.047 [+ or -] 0.002 27.0 [+ or -] 0.19 2.9 [+ or -]
olivacea 0.20

Table 2. Multiple regression of mass and stage on survival time in three
species of tadpoles. d.f. = degrees of freedom for correlation, r =
correlation coefficient, ssmass = standardized beta coefficient for
mass, tmass = t value for mass, ssstage = standardized beta coefficient
for stage, and tstage = t value for stage in multiple regression.
* Designates P < 0.05; ** designates P < 0.01.

Species F d.f. r ssmass tmass ssstage tstage

Bufo woodhousei 9.7 26 0.67** 0.70 3.45** -0.05 0.27
Bufo valliceps 4.25 28 0.50* 0.81 2.89** -0.71 2.52*
Gastrophryne 1.81 27 0.36 0.14 0.54 0.25 0.97


We thank Dr. Kevin Gutzwiller for commenting on an earlier draft of the paper. This work was completed as a part of an undergraduate honors thesis at Baylor University.

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Neeta Verma* and Benjamin A. Pierce

Department of Biology, Baylor University, Waco, Texas 76798-7388

* Present address: Texas A & M College of Medicine, College Station, Texas 77843
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Author:Verma, Neeta; Pierce, Benjamin A.
Publication:The Texas Journal of Science
Geographic Code:1U7TX
Date:Nov 1, 1994
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