The fungicide chlorothalonil is nonlinearly associated with corticosterone levels, immunity, and mortality in amphibians

Abstract

Background: Contaminants have been implicated in declines of amphibians, a taxon with vital systems similar to those of humans. However, many chemicals have not been thoroughly tested on amphibians or do not directly kill them.

Objective: Our goal in this study was to quantify amphibian responses to chlorothalonil, the most commonly used synthetic fungicide in the United States.

Methods: We reared Rana sphenocephala (southern leopard frog) and Osteopilus septentrionalis (Cuban treefrog) in outdoor mesocosms with or without 1 time (1×) and 2 times (2×) the expected environmental concentration (EEC) of chlorothalonil (~ 164 μg/L). We also conducted two dose-response experiments on O. septentrionalis, Hyla squirella (squirrel treefrog), Hyla cinerea (green treefrog), and R. sphenocephala and evaluated the effects of chlorothalonil on the stress hormone corticosterone.

Results: For both species in the mesocosm experiment, the 1× and 2× EEC treatments were associated with > 87% and 100% mortality, respectively. In the laboratory experiments, the approximate EEC caused 100% mortality of all species within 24 hr; 82 μg/L killed 100% of R. sphenocephala, and 0.0164 μg/L caused significant tadpole mortality of R. sphenocephala and H. cinerea. Three species showed a nonmonotonic dose response, with low and high concentrations causing significantly greater mortality than did intermediate concentrations or control treatments. For O. septentrionalis, corticosterone exhibited a similar nonmonotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities (< 16.4 μg/L).

Conclusions: Chlorothalonil killed nearly every amphibian at the approximate EEC; at concentrations to which humans are commonly exposed, it increased mortality and was associated with elevated corticosterone levels and changes in immune cells. Future studies should directly quantify the effects of chlorothalonil on amphibian populations and human health.

Figure 1

Survival of tadpoles in the mesocosm experiment shown by the number of O. septentrionalis and R. sphenocephala tadpoles surviving after exposure to measured concentrations of chlorothalonil (1× EEC, ~ 164 μg/L; 2× EEC, ~ 328 μg/L; single pulse) relative to controls (water and solvent combined). Both species had 0% survival at 328 μg/L. For O. septentrionalis , n = 25/treatment; for R. sphenocephala, n = 10/treatment.

Figure 2

Survival of tadpoles in laboratory experiments I and II. Survival (A) and time to death (B) of O. septentrionalis (15 tadpoles/tank) and H. squirella (5 tadpoles/tank) exposed to several concentrations of chlorothalonil (0.164, 1.64, 16.4, 164, and 1,640 μg/L) and controls (water and solvent combined) for laboratory experiment I (n = 3 for all chlorothalonil concentrations; n = 6 for controls). (C) Survival of O. septentrionalis, H. cinerea, and R. sphenocephala exposed to several concentrations of chlorothalonil (0.0164, 0.164, 1.64, 16.4, 82.0, and 164 μg/L) and control treatments (water and solvent combined) for laboratory experiment II (n = 6 for all chlorothalonil concentrations: n = 12 for controls). Values shown are mean ± SE. Different lowercase letters indicate that responses for a given species were significantly different (p < 0.05) among treatment levels according to Fisher’s LSD multiple comparison tests.

Figure 3

Effect of chlorothalonil on tadpole liver health and immunity. Density of liver tissue (A) and number of melanomacrophages and granulocytes in the liver (B) of O. septentrionalis tadpoles exposed to several concentrations of chlorothalonil [0.0164 (n = 9), 0.164 (n = 4), 1.64 (n = 6), 16.4 (n = 5), 82.0 (n = 5), and 164 μg/L (n = 3) and controls (water and solvent combined (n = 6)]. Values shown are mean ± SE and best-fit lines.

Figure 4

Effects of chlorothalonil on corticosterone per gram of O. septentrionalis tissue shown as least squares means ± 1 SE. Means were averaged across the three chlorothalonil exposure durations (4, 28, and 100 hr), except for the 164 μg/L concentration, where only the 4 hr duration mean is shown because longer exposure killed the tadpoles. Also shown is the significant third-order polynomial function (y = 1.886571 + 0.035582x – 0.000668x² + 0.000003x³) for the relationship between chlorothalonil concentration and log corticosterone, adjusted for the effect of exposure duration. The corticosterone level for the 164 μg/L concentration is underestimated because it is the only mean based on 4 hr, rather than an average of 44 hr, of chlorothalonil exposure, and corticosterone increased significantly and log-linearly with the duration of chlorothalonil exposure (coefficient for log exposure duration = 0.269). Concentrations with different lowercase letters are significantly different from one another by Fisher’s LSD multiple comparison test (n = 13, 5, 7, 6, and 2 for 0, 0.164, 16.4, 82.0, and 164 μg/L, respectively).