A common fungicide kills frog tadpoles, may contribute to amphibian declines.

What did they do?

Researchers at the University of South Florida for the first time assessed the effects of chlorothalonil on tadpoles from four frog species, including Cuban, green and squirrel treefrogs and southern leopard frogs. They evaluated survival, levels of the stress hormone corticosterone, and immune system markers.

To mimic environmental exposures, the researchers created a natural frog habitat – a mesocosm – with water, leaf litter, phytoplankton, zooplankton crayfish and insects. Ten southern leopard frog tadpoles and 25 Cuban treefrog tadpoles were placed into the mesocosm.

The researchers used a computer program created by the EPA to estimate levels of chlorothalonil expected to occur in water after fungicide application. They estimated the environmentally relevant level at 164 micrograms per liter (μg/L).

The tadpoles were exposed to either the predicted amount, twice as much – 328 (μg/L) – or none. The researchers compared the number of deaths and the rate of development with and without the fungicide to determine effects on the tadpoles.

The researchers also exposed two species – five squirrel treefrog tadpoles and 15 Cuban treefrog tadpoles – to different concentrations of chlorothalonil. Concentrations ranged from 10 times the predicted environmental level of the fungicide to 1,000 times less.

Frog mortality was also measured in jars containing three tadpoles each of Cuban treefrog, southern leopard frog or green treefrog. These species were individually exposed to eight different levels of chlorothalonil ranging from the environmentally predicted level to 10,000 times less.

At the end of the experiment, surviving Cuban treefrog tadpoles were tested for liver and immune system toxicity.

Finally, researchers exposed Cuban treefrog tadpoles to different amounts of the fungicide for 4, 28 or 100 hours. After the exposure period ended, they measured levels of corticosterone in the surviving tadpoles. Corticosterone is a steroid hormone that is elevated in response to natural or chemical stressors like fungicides.

What did they find?

When the researchers created a natural environment to house the animals, they found that only tadpoles exposed to the fungicide died. They also noted that the fungicide caused death quickly: most animals died within the first 72 hours of the experiment.

When squirrel and Cuban treefrog tadpoles were housed together in jars, researchers found that the chemical killed Cuban treefrog tadpoles at levels far below what was predicted to be present in the environment.

Interestingly, high or low concentrations of the fungicide – but not intermediate levels – killed the tadpoles.

In the same experiment, they were unable to assess mortality in squirrel treefrog tadpoles because most of the unexposed tadpoles died in the experiment. The researchers suggest that the Cuban treefrogs out-competed the squirrel treefrogs for available food and so, they died.

In a critical experiment, researchers found that 100 percent of Cuban treefrog, southern leopard frog and green treefrog tadpools died after 10 hours of exposure to the environmentally estimated level of the fungicide.

In the same experiment, tadpoles were exposed to lower levels of chlorothalonil – up to 10,000 times less than the environmentally estimated level or 0.0164 µg/L. Sensitivity to the fungicide varied. For example, southern leopard treefrogs were the most sensitive species – 86 percent of the tadpoles died when exposed to 1,000 times less fungicide than the environmentally predicted level. The least sensitive species tested was the Cuban treefrog.

Researchers found that high or low concentrations of chlorothalonil – but not intermediate levels – also killed the southern leopard and green treefrog tadpoles. 

Cuban treefrog tadpoles that survived fungicide exposure had smaller livers, fewer liver immune cells and higher corticosterone levels. This experiment raises concerns about the health and fitness of tadpoles that survive chlorothalonil exposure. 

What does it mean?

Second to habitat loss, chemicals in the environment pose the largest threat to declining amphibian populations. This study is important because it is one of a handful of examples where high mortality was seen in frogs exposed to environmentally realistic levels of fungicide.

Additionally, this study reports that exposure to chlorothalonil kills frog tadpoles at levels far below those currently found in the environment. And, researchers found that tadpoles that survived exposure to the fungicide may have weakened or stressed immune systems. This raises critical concerns about the general health and fitness of surviving frogs and their ability to fight off infections.

Together, these findings suggest that chlorothalonil exposure can impact amphibian populations in two different ways. One, the fungicide can directly kill tadpoles. Two, it can indirectly kill frogs by weakening their immune systems and rendering them more susceptible to infectious disease.

The study also reported that low or high levels, but not intermediate levels, of the fungicide increase tadpole death and stress hormone levels. This is at odds with the conventional belief that higher concentrations of a chemical always cause greater harm. Instead, the study supports the growing concept that a wide variety of agrochemicals have adverse effects at very low, environmentally relevent levels.

In the current study, environmentally relevant levels of chlorothalonil were estimated using a computer tool created by the EPA. Future work is needed to more accurately understand real-world exposure levels of chlorothalonil.