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Atrazine Exposure and the Occurrence of Reproductive Abnormalities in Field Caught Bufo marinus From South Florida

By Timothy S. Gross1, Marisol Sepulveda1, Carr JA2, Giesy JP3, Hosmer AJ4, Kendall RJ2, Solomon K5, Smith EE2, and G. Van Der Kraak5
1U.S. Geological Survey Florida Integrated Science Center and University of Florida, Gainesville, 2Texas Tech University, Lubbock, 3Michigan State University, East Lansing, 4Syngenta, Greensboro, SC, 5University of Guelph, Guelph

Many chemicals in our environment are suspected or known to influence endocrine system function. Endocrine disrupting chemicals (EDCs) have been broadly defined as exogenous agents that interfere with the production, release, transport, metabolism, binding, action, or elimination of natural hormones responsible for maintenance of homeostasis and regulation of developmental processes. Since hormones are important modulators of tissue differentiation, developing organisms are thought to be especially sensitive to EDCs. Indeed, many agricultural contaminants such as herbicides, fungicides, and insecticides have been shown to induce developmental toxicity through alterations in hormonal activity. For example, the insecticide DDT and its metabolite p,p’DDE are known to affect development through an anti-androgenic mechanism.

Atrazine is the most commonly used herbicide in the United States. Recent reports have suggested that environmentally relevant levels of atrazine can alter sexual development in laboratory exposed African Clawed frogs (Xenopus laevis), however, similar experiments have been unable to replicate these findings. The goal of the current study was to document whether similar reproductive system anomalies were common in anurans from Florida sugarcane agricultural areas, which have the highest per acre atrazine use in the U.S. The giant toad or cane toad (Bufo marinus) was chosen as the focal species because they posses a nonfunctional rudimentary ovary (Bidder’s organ), which potentially makes them sensitive to EDCs that influence gonad development. They are an invasive species, so destructive sampling is not expected to cause negative impacts on local diversity, and they are found in large numbers in sugar cane fields were atrazine is extensively applied. The goal of the current study was to document whether exposure of frogs to sugarcane agricultural areas in south Florida would result in a higher incidence of intersex and/or developmental anomalies. Sugarcane agriculture has the highest per acre atrazine use in the U.S., which could represent the highest potential risk for exposure of native anuran species to atrazine.

graphs of atrazine concentrations in water for both sugarcane agricultural sites
Figure 1. Atrazine concentrations in water for both sugarcane agricultural sites. Line indicates mean concentrations across each site. [larger image]
To determine the distribution and concentration of atrazine at south Florida sites, multiple water samples were colleted from several canals/ditches at each of two agricultural sites every two weeks from February through June, 2002 (fig. 1). Adult toads were collected from two sugarcane agricultural areas Canal Point (CP), and Belle Glade (BG) as well as from a University of Miami pond/canal (reference site with little to no atrazine use or agricultural input) during April-June 2002. Adult Bufo marinus were collected from these three sites: Canal Point (N=55), Belle Glade (N=50), and University of Miami (N=24). Body weight, length, and coloration were recorded, blood was collected, and gonads were removed and weighed. This species is sexually dimorphic, with females having a mottled appearance and males having a solid coloration. Sex was identified as follows: the presence of ovarian tissue and absence of testicular tissue = female; presence of testes and absence of developing eggs, oviduct, and ovarian tissue = normal male; and presence of testes with developing eggs or oviduct or ovarian tissue = intersex . Macroscopic identification of additional testicular anomalies included: segmented testes, abnormal shaped testis, twisted or curled testes, and multiple testes. Gonads from each individual that had testicular tissue were both macroscopically and histologically examined. Blood plasma was analyzed for phospho-lipoprotein (an indirect measure of vitellogenin) and estradiol and testosterone concentrations were analyzed using RIA procedures.

Atrazine levels were highest at Canal Point during March, but were highest at Belle Glade in February. B. marinus tadpoles were potentially exposed to atrazine concentrations as high as 20ppb during development at Canal Point and 26ppb at Belle Glade during 2002. Toads collected from the nonagricultural /reference, University of Miami, site exhibited the characteristic gender-specific pattern which correlated to subsequent gonadal morphology and histology. However, all toads collected from both agricultural sites, Belle Glade and Canal Point, exhibited the distinctive female pattern, although subsequent gonadal morphology and histology demonstrated male, intersexed, and female toads. The frequency of males exhibiting “testis abnormalities” was not significantly different among sites. The frequency of intersexed animals was significantly different among sites: 39 percent and 29 percent of the individuals at the agricultural sites, Canal Point and Belle Glade. No individuals from the non-agricultural/reference site were intersexed. The types of abnormal female tissue found in association with testicular tissue varied between CP and BG. Plasma sex steroids did not differ between intersexed and normal males. However, plamsa phospholipoprotein (an indirect indicator of vitellogenin was increased in intersexed males to levels which were similar to those for vitellogenic females.

The purpose of this preliminary study was to determine if animals found in sugarcane exhibit reproductive abnormalities similar to those seen in African Clawed Frogs exposed to atrazine in the laboratory. The incidence of testicular anomalies, other than intersex were similar across sites. However, the incidence of intersex was increased for both agricultural sites as compared to the non-agricultural/reference site. Nonetheless, Bufo marinus adults were active and breeding at all sites. Data suggests that agricultural exposure, including exposure to atrazine, may explain the differences in the percent of intersexed individuals and length of oocytes between Canal Point and Belle Glade sites. However, we can not conclude that atrazine is responsible for these abnormalities, since other agricultural chemcials are likely present at both sites. In addition, water quality analyses were not conducted for the non-agricultural/reference site (University of Miami) and exposure to atrazine at this site is unknown. The University of Miami site is expected to have low levels of atrazine, but is probably not atrazine free. Further research should be conducted to determine whether atrazine is capable of causing the effects we have documented in B. marinus under controlled laboratory conditions as well as expanded field studies of these and other sites. Nonetheless, these results indicate an increased incidence of intersex in toads exposed to agricultural contaminants. The implications of these data to future and ongoing restoration is unknown, however, a redistribution of water resources in the greater everglades ecosystem could result in additional exposures for amphibian populations in this sensitive ecosystem.

Contact: Timothy S. Gross, USGS-FISC, 7920 NW 71st St., Gainesville, FL 32653., Phone: 352-378-8181 Ext 323, FAX: 352-378-4956,

(This abstract was taken from the Greater Everglades Ecosystem Restoration (GEER) Open File Report 03-54)

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