Atrazine Exposure and the Occurrence of Reproductive Abnormalities in Field
Caught Bufo marinus From South Florida
Timothy S. Gross1, Krista A. McCoy1,*, Marisol Sepulveda1,
Carr JA2, Giesy JP3, Hosmer AJ4, Kendall RJ2, Solomon K5, Smith EE2, Van
Der Kraak G5.
1
USGS-FISC & University of Florida, Gainesville, 2Texas Tech University,
Lubbock, 3Michigan State University, East Lansing, 4Syngenta, Greensboro, SC,
5
University 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.
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 (Figure 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). Adult Bufo marinus were
collected from these three sites: Canal Point (N=55) Belle Glade (N=50)
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% and 29% of the individuals at the agricultural sites,
Canal Point and Belle Glade. No individuals from the non-gricultural/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 nonagricultural/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.
Timothy S. Gross, USGS-FISC, 7920 NW 71st St., Gainesville, FL 32653.
Phone: 352-378-8181 Ext 323, FAX: 352-378-4956, Tim_s_gross@usgs.gov