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Effects of the Herbicide Atrazine on Ambystoma tigrinum Metamorphosis: Duration, Larval Growth, and Hormonal Response


These results indicate that not only is tiger salamander metamorphic climax responsive to sublethal concentrations of atrazine in the environment, but that the response is substantively different at different concentrations of the herbicide. At the lower concentration, larvae reached stage 4 later, but at a size and weight comparable to the control group. By contrast, at the higher concentration, larvae progressed to stage 4 at the same time, but at a smaller size and lower weight than control larvae.

Figure 2.
Figure 2. Days to stage (a), snout-vent length (b), and weight (c) for larvae at stage 2 (open bars) or stage 4 (filled bars) exposed to 0, 75, and 250 µg/L atrazine. Shown are least squares means + 1 standard error of the mean. Larvae in the 250 µg/L treatment took longer to reach stage 2 than the controls, but reached stage 4 more quickly, and at a smaller size and lower weight than the controls. Larvae in the 75 µg/L treatment reached stage 4 later, but at a size comparable to larvae in the control group.

The hormonal underpinnings of these life-history observations appear to revolve around circulating concentration of corticosterone relative to thyroxine. Larvae exposed to either concentration of atrazine responded with elevated thyroxine, essentially a physiological decision to curtail growth and speed metamorphic progress. However, suppression of corticosterone presumably moderated the effect of elevated thyroxine in larvae in the lower-atrazine concentration by slowing conversion of thyroxine to triiodothyronine, thereby slowing metamorphic progress and allowing additional larval growth between stages. The advantage to such moderation is transformation at a larger size, with attendant higher survival and fecundity prospects (Rose and Armentrout 1976).

Previous studies on amphibian larvae have demonstrated that the speed of metamorphic progress is responsive to environmental factors such as temperature (Smith-Gill and Berven 1979), food availability (Alford and Harris 1988; Tejedo and Reques 1994), and food quality (Kupferberg 1997). Here, we have shown that age and size at metamorphosis also can depend on the presence of a contaminant in the environment. The mechanism by which larvae discern the presence of these environmental factors is the subject of some debate, however. Wilbur and Collins (1973) hypothesized that changes in growth rate, coupled with overall larval size, dictate age and size at metamorphosis: rapidly growing larvae should begin metamorphosis later and at a larger size than larvae growing slowly. Alford and Harris (1988) and Tejedo and Reques (1994) found experimental support for this hypothesis, but Smith-Gill and Berven (1979) did not. Crump (1981) proposed that energy accumulation, as opposed to growth per se, could account for changes in timing of metamorphosis.

Our study was not designed to address the question of larval growth history effects on metamorphic progress; we did not measure growth of individual larvae. However, we can examine the effect of growth rate on larval size at stages 2 and 4 and on days-to-stage by calculating the expected growth rate based on the regression equation describing the growth of the control larvae and determining the ratio of observed to expected growth rate in atrazine-exposed larvae. Because weight can decline as metamorphosis progresses, we used snout-vent length as the measure of growth. Control larvae growth rate can be expressed as:

L = 53.37 + 0.30 D ,
with r2 = 0.77; L = snout-vent length (mm) and D = days-to-stage. Using this equation, we calculated the expected snout-vent lengths of each of the atrazine-exposed larvae and determined the ratio of the observed-to-expected size. The ratios varied significantly among treatments (F = 16.37; df = 2, 202; P = 0.0001); larvae in 75 or 250 µg/L atrazine grew at a slower rate than control larvae. This allowed us to partition variance in days-to-stage into that accounted for by the observed-to-expected growth ratio, by dose, by stage, and by the interaction of growth ratio and dose, using analysis of covariance. Of course, stage accounted for the majority of the variation in days-to-stage, but we found that growth ratio accounted for more variance in days-to-stage than did atrazine concentration (Table 3). This suggests that metamorphic progress may be mediated by the effect of atrazine on growth rate (or energy assimilation), rather than a direct effect of atrazine concentration on the endocrine system.

Table 3. Results of ANOVA to partition variance in days-to-stage

Factor df F P
Observed: expected growth rate 1,198 6.3 .0126
Atrazine concentration 2,198 1.4 .2548
Stage 1,198 278.6 .0001
Growth rate x atrazine concentration 2,198 1.4 .2547

The implications of this study are twofold. First, the effect of atrazine on tiger salamander metamorphosis seems not to be mediated through the interrenal axis in the way we had originally hypothesized. Corticosterone concentrations did not rise in response to the presumed stress of atrazine contamination, at least when measured at the onset of metamorphic climax, nor was are suppressed uniformly by prolonged contact of larvae with the contaminant. Instead, we propose that corticosterone acts as an integrator of information on growth rate relative to environmental condition, such that differentiation is slowed to maximize growth if environmental conditions limit energy assimilation, but larvae are still growing at some (unknown) minimum rate (Fig. 3). If energy assimilation is too low to allow this minimum growth rate, corticosterone is not suppressed and the rate of differentiation is maximized. Elevated thyroxine concentrations under poor environmental conditions provide the raw material to allow accelerated metamorphosis but require the addition of corticosterone to maximize the rate of differentiation. This model represents a mechanistic extension of the model put forward by Wilbur and Collins (1973), in which the risk of remaining in an aquatic environment is balanced against the risk of metamorphosis. The model also suggests an indirect form of endocrine disruption (as defined by Crisp et al. 1998), in that the contaminant does not appear to be mimicking either corticosterone or thyroxine but instead is modifying an environmental cue that triggers production of the hormones.

The second implication of this study is that agricultural chemicals have the potential to affect amphibian life history. Larvae subjected to low levels of contamination, as in the lower-atrazine concentration in this study, will spend more time in the wetland prior to transformation. In the northern Great Plains, this means that larvae may fail to transform before cold temperatures set in, thus making them vulnerable to anoxia in ice-covered wetlands. In more highly contaminated environments, as in the higher-atrazine concentration, larvae will likely complete transformation at a smaller size than they would in the absence of the contaminant, and smaller size is associated with lower survival and fecundity. Unlike the laboratory environment, however, larvae under natural conditions will be subjected not to the contaminant in isolation, but in combination with other stressors associated with agricultural practices, as well as influences of weather and resource availability. Field studies will be necessary to determine interactions among all these factors and their collective effect on tiger salamander life history.

Figure 3.
Figure 3. Model for regulation of differentiation by corticosterone and thyroxine in response to growth rate. To the left of L, larvae are too small and circulating levels of thyroxine are too low to initiate metamorphosis. Between L and a, differentiation is maximal to hasten escape from a poor environment where growth is slow, regardless of the consequences for adult fitness in terrestrial habitat; neither corticosterone nor thyroxine limit differentiation, although nutrition may limit thyroxine production (Kupferberg 1997). Between a and b, differentiation is slowed to allow continued growth before transformation and, thus, increase adult fitness; corticosterone limits differentiation by lack of synergism with thyroxine. To the right of b, larvae transform at a larger size to optimize adult fitness; corticosterone concentrations rise to promote metamorphosis, but low thyroxine concentrations limit differentiation. To the right of H, we propose that thyroxine declines below the threshold necessary for metamorphosis; larvae mature as gilled (paedomorphic) adults.

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