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

Results

Water Quality and Toxicity Testing

Water quality characteristics of ASTM very hard water were measured prior to use in toxicity tests (Table 1) and were within acceptable guidelines (ASTM 1989). Throughout the experiment, dissolved oxygen concentrations were maintained between 20% - 60% saturation before renewal and 50% - 70% saturation after renewal.

Measured concentrations of atrazine in the three exposure treatments were 0.23 ± 0.21 µg/L in the 0 µg/L treatment, 81.83 + 3.77 µg/L in the 75 µg/L treatment, and 299.71 + 7.32 µg/L in the 250 µg/L treatment. There were no exposure-related mortalities.

Table 1: Summary of water-quality characteristics of blended water used
for exposure of tiger salamander larvae to atrazine (n = 6 grab samples).

Parameter Mean ± SE
pH 8.10 ± .1
Conductivity at 25°C (µmhos/cm) 1,045 ± 11
Hardness (mg/L as CaCO3) 333 ± 7
Alkalinity (mg/L as CaCO3) 231 ± 2
Calcium (mg/L) 55 ± 2
Magnesium (mg/L) 48 ± 0
Chloride (mg/L) 8 ± 0
Sulfate (mg/L) 330 ± 14

Effects of Atrazine on Corticosterone and Thyroxine

The overall ANOVA indicated significant differences in corticosterone concentration, with significant main effect of stage (F = 19.27; df = 1, 95; P = 0.0001) and significant treatment-by-stage interaction (F = 3.22; df = 2, 95; P = 0.04). Average plasma corticosterone concentrations increased significantly between stages 2 and 4 in larvae subjected to either no atrazine (P = 0.0089) or 250 µg/L atrazine (P = 0.001); larvae in 75 µg/L atrazine did not increase plasma corticosterone between stages 2 and 4 (P = 0.49; Fig. 1a). Corticosterone concentration was significantly lower at stage 4 in larvae in the 75 µg/L treatment than in larvae in the 250 µg/L treatment (P = 0.005 for preplanned contrast); the control group did not differ from those in the 250 µg/L (P = 0.26) or the 75 µg/L (P = 0.077) treatment at stage 4.

The overall ANOVA for plasma thyroxine concentration indicated a highly significant main effect of stage (F = 42.16; df = 1, 95; P = 0.0001) and a nearly significant main effect of atrazine treatment (F = 2.85; df = 2, 95; P = 0.06). Plasma thyroxine concentrations increased significantly between stages 2 and 4 in each of the three treatment groups (P = 0.015, 0.0002, and 0.0001 for 0, 75, and 250 µg/L, respectively). Thyroxine concentration at stage 4 was significantly elevated in the two atrazine treatments compared with the control (P = 0.0155 and 0.0359 for 0 vs. 75 µg/L and 0 vs. 250 µg/L, respectively; Figure 1b).

Figure 1.
Figure 1. Plasma corticosterone (a) and plasma thyroxine (b) concentrations in stage 2 (open bars) and stage 4 (filled bars) larvae exposed to 0, 75, and 250 µg/L atrazine. Shown are least squares means + 1 SE of the mean. We found a significant treatment-by-stage interaction in which corticosterone concentrations increased between stages 2 and 4 in both the control larvae and the larvae exposed to 250 µg/L atrazine but not in those exposed to 75 µg/L atrazine. Plasma thyroxine concentration increased significantly between stages 2 and 4 in all treatments and was significantly higher at stage 4 in the two atrazine treatments compared with the control.

Growth and Timing

For each aspect of metamorphic timing and growth (days-to-stage, weight, and snout-vent length), we found significant differences with respect to stage and a significant stage-by-atrazine treatment interaction (Table 2). Preplanned contrasts indicated that larvae in the 250 µg/L treatment took longer to reach stage 2 (P = 0.04), and were smaller (P = 0.0007) and weighed less (P = 0.006; Fig. 2) at stage 4 than larvae in the control treatment. Larvae in the 75 µg/L treatment were marginally smaller at stage 2 (P = 0.056) and reached stage 4 later (P = 0.05; Figure 2) than larvae in the control group. Larvae in the 250 µg/L treatment reached stage 4 sooner (P = 0.01), but were smaller (P = 0.01) and weighed less (P = 0.004; Fig. 2) at stage 4 than larvae in the 75 µg/L treatment.

Table 2: Results of ANOVA for differentiation and growth
of tiger salamander larvae exposed to 0, 75, and 250 µg/L atrazine

Factor
df
F
P
Days-to-stage:
    Treatment 2,199 .8 .4425
    Stage 1,199 279.1 .0001
    Treatment x stage 2,199 5.7 .0039
Weight:
    Treatment 2,199 3.5 .0332
    Stage 1,199 232.7 .0001
    Treatment x stage 2,199 3.4 .0344
Snout-vent length:
    Treatment 2,199 5.40 .0052
    Stage 1,199 191.06 .0001
    Treatment x stage 2,199 3.02 .0509

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