Chemical Contaminants

(September 23, 2003)

There is a growing body of evidence that chemical contaminants are in some way responsible for amphibian declines (Blaustein et al. 2003). The consequences of chemical stressors, such as pesticides, heavy metals, acidification and nitrogen based fertilizers, on amphibians are lethal, sublethal, direct and indirect. The sublethal affects of contaminants on amphibians include hampered growth, development and behavior, which could lead to developmental and behavioral abnormalities (Bridges 1997, Bridges 2000). These developmental and behavioral abnormalities may alter susceptibility to predation (Bridges 1999a) and competition and decrease reproductive success (Bridges 1999b, Relyea and Mills 2001, Boone and Semlitsch 2002). Chemical contaminants also weaken the immune system making amphibians more susceptible to parasites, disease and UV radiation (Blaustein et al. 2003, Christin et al. 2003, Daszak et al. 2003, Gendron et al. 2003). Certain pesticides can disrupt the endocrine system, resulting in sexual malformations, such as hermaphroditism (Hayes et al. 2002b, Hayes et al. 2003). Other contaminates indirectly affect amphibians by altering food web dynamics (Boone and Bridges 2003). This page briefly summarizes resent research on the effects of four main types of chemical stressors on amphibians: pesticides, heavy metals, acidification and nitrogen pollution.

PESTICIDES

Approximately 19,000 to 20,000 pesticides, which broadly include insecticides, herbicides and fungicides, are currently approved for release by the United States, Environmental Protection Agency (EPA) (Boone and Bridges 2003). The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), the first major pesticide law to be signed in the United States in 1942, mandates that a safe level for human and non-target wildlife be determined before a pesticide is approved for use. The most common study preformed for licensing a pesticide is an acute toxicity (short-term poisoning potential) test, such as an LC50. The LC stands for "Lethal Concentration". The LC50 concentration represents the concentration that causes the death of 50% of a group of test animals, where the group of test animals is exposed to the contaminant all at once (hence an acute toxicity test).

The standard test animals used for aquatic environments are usually bluegill sunfish, fathead minnows and rainbow trout. Initially, researchers thought amphibians would be more sensitive to contaminants then the standard vertebrates used in LC50 tests, due to their unshelled eggs and permeable skin; but, research by Bridges et al. (2002) suggests that for some amphibian species, pesticide concentrations necessary to induce mortality may, in fact, be comparable to and in some cases higher than concentrations that induced mortality in some fish species. Researchers are finding that there is a wide variation in tolerance levels among amphibians even between closely related species (Bridges et al. 2002). Therefore, conclusions drawn from studies on only a few species cannot reveal the full effects of potentially harmful chemicals to amphibians in general (McDiarmid and Mitchell 2000). The table below illustrates the range of 96 hour LC50 values for three amphibians and how those values compare to commonly tested vertebrates (modified with permission from Bridges et al. 2002).

Chemical	Southern Leopard frog tadpoles	Boreal toad tadpoles	Bluegill Sunfish	Fathead minnow	Rainbow trout
4nonylphenol (ìg/L)	0.34 (0.31-0.37)	0.12 (0.09-0.15)	N/A	0.27	0.19
carbaryl (mg/L)	8.4 (7.4-9.6)	12.31 (10.3-14.7)	6.2	5.21	1.88
copper (mg/L)	0.23 (0.21-0.25)	0.12 (0.07-0.18)	7.3	0.47	0.88
PCP (mg/L)	0.14 (0.12-0.17)	0.37 (0.25-0.42)	0.192	0.25	0.016
Permethrin (ìg/L)	18.2	>10	6.2	9.38	3.31

“New generation” pesticides

The fastest growing group of pesticides are called the “new generation” pesticides (post World War II), which are predominantly insecticides and herbicides. Common “new generation” insecticides, such as organophosphates, carbamates, and pyrenthoids, are neurotoxins that function by inhibiting nervous system acetylcholinesterase (AChE), causing the constant firing of nerve impulses (Boone and Bridges 2003). Boone and others have been studying the affects of carbaryl, a carbamate insecticide, since 1995. For carbaryl, higher concentrations than those found in the environment are needed to induce mortality in larval amphibians (Bridges 1999a). However, chronic exposure, as a posed to acute exposure, to carbaryl concentrations an order of magnitude lower than environmental concentrations resulted in an increase in larval mortality and extremely high deformity rates (Bridges 2000). Because, lethal concentrations from LC50 data are high, there is a greater chance that sublethal concentrations are affecting amphibian populations. Bridges (1999, 1999 and 1997) found that sublethal concentrations of carbaryl alters tadpole behavior, making them more vulnerable to predation, and decrease feeding rates resulting in a smaller size at metamorphosis. Interestingly, carbaryl had an indirect positive affect on Woodhouse’s toad, Bufo woodhousii, by altering food web dynamics (Boone and Bridges 2003). Zooplankton (which feed on algae) is very sensitive to carbaryl, and the reduction or elimination of them frequently results in algal blooms (tadpole food source), thus having a positive effect on tadpole growth and mass at metamorphosis. However, carbaryl is likely to have a negative indirect affect on salamanders via starvation because their invertebrate food supply is eliminated.

Herbicides are generally considered to have little effect on fish and wildlife because they primarily function to disrupt the photosynthetic pathways of plants. However, recent findings have revealed that Atrazine, the most commonly used herbicide in the United States, causes hermaphroditism in the African clawed frog (Xenopus laevis) and the northern leopard frog (Rana pipiens) in laboratory studies (Hayes et al. 2002a, Hayes et al. 2002b). In addition to laboratory studies, Hayes et al. (2003) surveyed wild populations of R. Pipiens from different regions of the United States. To Hayes’s surprise, he found hermaphroditic frogs at every location sampled where Atrazine levels were equal to or greater than 0.1 parts per billion (Hayes et al. 2003).

Contaminant	Species	Effect	Reference
Atrazine	Xenopus laevis	Disrupts steroidogenesis resulting in demasculanization and hermaphroditism	Hayes et al. 2002
Atrazine	Rana pipiens	Disrupts steroidogenesis resulting in demasculanization and hermaphroditism	Hayes et al. 2003
Chlorinated hydrocarbons	Necturus maculosus	Changes in secretion of corticosterone, which could hinder reproductive performance	Gendron et al 1997
Endosulfan (a cyclodiene organochlorine insecticide)	Notopthalamus viridescencs	Altered morphology of pheromonal glands in females and interfered with hormonal signaliing and mating success	Parker et al. 2001
carybaryl	Hyla versicolor	killed 60-98% of tadpoles when predatory cues were also present	Reylea and Mills 2001

HEAVY METALS AND ACIDIFICATION

The intense agricultural and industrial production from mines has increased the prevalence of heavy metals in surface waters. Heavy metals, such as, aluminum (Al), lead (Pb), zinc (Zn), cadmium (Cd), mercury (Hg), silver (Ag), copper (cu), arsenic (As), manganese (Mn), molybdenum (Mo) and antimony (SB) may adversely affect amphibian populations (Blaustein et al. 2003). For example, Rowe et al. (1996, 1998) found that coal ash increased the incidence of oral deformities, increased metabolic rate and lowered larval survival of the American bullfrog, Rana catesbeiana, larvae. In the Southern toad, Bufo terrestris, coal ash increased corticosterone and testosterone levels and lowered larval survival (Rowe et al. 2001).

Acidification can also have adverse effects on amphibian growth and development ultimately contributing to population declines. Extremely low pH can arrest embryo development (Freda et al. 1990). At low but slightly higher pH levels, embryo development proceeds but the enzymes that induce hatching are inhibited, thus, trapping the fully developed embryo inside the egg capsule (Clark and Lazerte 1987).

Acidification and heavy metal contamination often work synergistically because the solubility of heavy metals in water increases as pH drops. As a result, heavy metals leach more quickly from contaminated soils in contact with acidic water (Blaustein et al. 2003). Furthermore, studies have found that inorganic monomeric aluminum acts synergistically with pH to cause embryo mortality (Clark and Hall 1985, Clark and Lazerte 1985, Freda and McDonald 1990).

Contaminant	Species	Effect	Reference
Coal ash	Rana catesbeiana	Increased incidence of oral deformities, higher metabolic rates and low larval survival	Rowe et al. 1996, Rowe et al. 1998a, Rowe et al. 1998b
Coal ash	Bufo terrestris	Lower larval survival. When transplanted into a polluted site all larvae died before metamorphosis.	Rowe et al. 2001
Coal ash	B. terrestris	Increased corticosterone and testosterone levels.	Hopkins et al 1997
Acidification	Ambystoma tigrinum	Potential cause of population declines in Colorado.	Hoffman 1989
Acidification	B. calamita	Seems to have played a role in population declines in Britain	Beebee et al 1990
Aluminum and acidification	B. americanus and R. sylvatica	Reduced hatching success	Clark and LaZerte 1987

NITROGEN POLLUTION

Nitrogen pollution is becoming a severe problem world wide with unknown consequences on amphibian populations. A recent review by Rouse et al. (1999), found that several watersheds in North America have high enough nitrate concentrations to cause death and developmental anomalies in amphibians (Rouse et al. 1999). Nitrogen pollution from anthropogenic sources enters aquatic ecosystems via agricultural runoff, livestock, precipitation and effluents from industrial and human waste. Nitrogen is found in the aquatic environment in four forms: ammonium ion, ammonia, nitrite and nitrate. Ammonia is the most toxic, followed by nitrite and nitrate; but ammonia and nitrite are rarely found in excess in the environment because they are quickly oxidized to nitrate by bacteria and algae (Rouse et al. 1999). In the table below, we summarize the effects of nitrogen pollution on different amphibian species.

Contaminant	Species	Effect	Reference
Nitrite	Rana pretiosa, Rana aurora, Bufo boreas, Hyla regilla and Ambystoma gracile	Reduced feeding activity, swim less vigorously, display disequilibrium, develop malformations of the body and death	Marco et al. 1999
Nitrate	Rana pretiosa, Rana aurora, Bufo boreas, Pseudacris regilla and Ambystoma gracile	Reduced feeding activity, swim less vigorously, display disequilibrium, develop malformations of the body and die	Marco et al. 1999 and De Solla et al. 2002
Urea fertilizers	R. cascadae and B. boreas	Recently metamorphosed frogs altered their feeding behavior	Hatch et al 2001
Ammonium Perchlorate	X. leavis	Inhibited forelimb emergence, skewed sex ratio, disrupted thyroid function and reduced hatching success	Goleman et al. 2002a,b
Ammonium	B. americanus, Pseudachris triseriata, R. pipiens and R. clamitans	Decreased larval survivorship to metamorphosis, decreased activity and rapid weight loss	Hecnar 1995

Literature Cited

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Boone, M. D., and C. M. Bridges. 2003. Effects of pesticides on amphibian populations. Pages 152-167 in R. D. Semlitsch, editor. Amphibian Conservation. Smithsonian Institution, Washington.

Boone, M. D., and R. D. Semlitsch. 2002. Interactions of an insecticide with competition and pond drying in amphibian communities. Ecological applications 12:307-316.

Bridges, C. M. 1997. Tadpole swimming performance and activity affected by acute exposure to sublethal levels of carbaryl. Environmental Toxicology & Chemistry 16:1935-1939.

Bridges, C. M. 1999a. Effects of a pesticide on tadpole activity and predator avoidance behavior. Journal of Herpetology 33:303-306.

Bridges, C. M. 1999b. Predator-prey interactions between two amphibian species: Effects of insecticide exposure. Aquatic Ecology 33:205-211.

Bridges, C. M. 2000. Long-term effects of pesticide exposure at various life stages of the southern leopard frog (Rana sphenocephala). Archives of Environmental Contamination and Toxicology 39:91-96.

Bridges, C. M., F. J. Dwyer, D. K. Hardesty, and D. W. Whites. 2002. Comparative contaminant toxicity: Are amphibian larvae more sensitive than fish? Bulletin of Environmental Contamination & Toxicology [print] 69:562-569.

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Clark, K. L., and B. D. Lazerte. 1985. A Laboratory Study of the Effects of Aluminum and Ph on Amphibian Eggs and Tadpoles. Canadian Journal of Fisheries & Aquatic Sciences 42:1544-1551.

Clark, K. L., and B. D. Lazerte. 1987. Intraspecific Variation in Hydrogen Ion and Aluminum Toxicity in Bufo-Americanus and Ambystoma-Maculatum. Canadian Journal of Fisheries & Aquatic Sciences 44:1622-1628.

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Freda, J., and D. G. McDonald. 1990. Effects of Aluminum on the Leopard Frog Rana-Pipiens Life Stage Comparisons and Aluminum Uptake. Canadian Journal of Fisheries & Aquatic Sciences 47:210-216.

Gendron, A. D., D. J. Marcogliese, S. Barbeau, M. S. Christin, P. Brousseau, S. Ruby, D. Cyr, and M. Fournier. 2003. Exposure of leopard frogs to a pesticide mixture affects life history characteristics of the lungworm Rhabdias ranae. Oecologia 135:469-476.

Hayes, T., K. Haston, M. Tsui, A. Hoang, C. Haeffele, and A. Vonk. 2002a. Herbicides: Feminization of male frogs in the wild. Nature 419:895-896.

Hayes, T., K. Haston, M. Tsui, A. Hoang, C. Haeffele, and A. Vonk. 2003. Atrazine-induced hermaphroditism at 0.1 ppb in American Leopard frogs (Rana pipiens): Laboratory and field evidence. Environmental Health Perspectives [print] 111:568-575.

Hayes, T. B., A. Collins, M. Lee, M. Mendoza, N. Noriega, A. A. Stuart, and A. Vonk. 2002b. Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proceedings of the National Academy of Sciences of the United States of America 99:5476-5480.

Relyea, R. A., and N. Mills. 2001. Predator-induced stress makes the pesticide carbaryl more deadly to gray treefrog tadpoles (Hyla versicolor). Proceedings of the National Academy of Sciences of the United States of America [print] 98:2491-2496.

Rouse, J. D., C. A. Bishop, and J. Struger. 1999. Nitrogen pollution: An assessment of its threat to amphibian survival. Environmental Health Perspectives 107:799-803.

Rowe, C. L., W. A. Hopkins, and V. R. Coffman. 2001. Failed recruitment of southern toads (Bufo terrestris) in a trace element-contaminated breeding habitat: Direct and indirect effects that may lead to a local population sink. Archives of Environmental Contamination & Toxicology [print] 40:399-405.

Rowe, C. L., O. M. Kinney, and J. D. Congdon. 1998. Oral deformities in tadpoles of the bullfrog (Rana catesbeiana) caused by conditions in a polluted habitat. Copeia 1998:244-246.

Rowe, C. L., O. M. Kinney, A. P. Fiori, and J. D. Congdon. 1996. Oral deformities in tadpoles (Rana catesbeiana) associated with coal ash deposition: Effects on grazing ability and growth. Freshwater Biology 36:723-730.