National Academy of Sciences reports on atrazine as
the cause of many frog mutations
TB, A Collins, M Lee, M Mendoza, N Noriega, AA Stuart,
and A Vonk. 2002. Hermaphroditic,
demasculinized frogs after exposure to the herbicide,
atrazine, at low ecologically relevant doses.
Proceedings of the National Academy of Sciences (US) 99:5476-5480.
et al. demonstrate that at exposure levels far
beneath those found in the lakes, rivers, streams,
drinking water and even rainwater, atrazine causes
frogs to mature with multiple, mixed gonads and to become
demasculinized. These effects occurred at exposure levels
10,000 - 30,000 times beneath levels previously identified
as non-toxic to frogs.
impact on frogs appears to be caused by this herbicide's
ability to promote the conversion of testosterone to estrogen
via activity of the enzyme aromatase. This mechanism is
found not just in frogs, but other vertebrates as well,
including mammals. Their study raises important, and as
yet unanswered, questions about the possible role of atrazine
in world-wide frog population declines, and about the
potential for atrazine to affect human health via the
same enzymatic mechanism.
is one of the most widely and heavily used agricultural
chemicals in the world. Each year, American farmers alone
apply 60 million pounds to U.S. farmland to prevent growth
of weeds competing with corn and other crops. The US EPA
considers short-term exposure to 200 ppb acceptable for
people and allows 3 ppb atrazine in drinking water. In
rainwater in regions where atrazine is applied, atrazine
contamination has been measured up to 40 ppb. It has even
been found at 1 ppb in rainwater in regions where
atrazine is not used. Hence it is virtually certain
that frogs (and people) living in the real world are regularly
exposed to atrazine at levels many times greater than
what Hayes et al. report is sufficient to harm
did they do? Hayes et al. used two different
experimental designs to study the effect of atrazine on
the laboratory rat of the amphibian world, the African
Clawed Toad Xenopus laevus. In both designs, Hayes
took great care to make sure that the scientists measuring
the exposed animals were unaware of which treatment they
had received. Only after all the measurements were taken
did a coding system reveal what treatment each animal
sample sizes used in the experiments were quite large
(90 animals per treatment); these experiments were repeated
four times; and experiments with atrazine at these low
levels were replicated 51 times in Hayes' lab with similar
1: expose developing tadpoles to different levels of atrazine
and examine individuals for morphological effects after
metamorphisis. The tadpoles were exposed to concentrations
of atrazine from 0.01 ppb to 25 ppb.
2: expose adults directly to 25 ppb atrazine and measure
testosterone and estrogen levels. Adults were used because
they found it not possible to obtain enough serum from
tadpoles to do the hormonal assays.
did they find?
Experiment 1: When exposed as tadpoles to as little as
0.1 part per billion atrazine, individuals mature with
hermaphroditic deformities in the reproductive tract.
Normal adult frogs have two gonads, either two testes
if male or two ovaries if female. Between 16% and 20%
of animals treated with atrazine at 0.1 ppb and above
had either two many gonads and/or mixtures of ovaries
and testes. This is not normal. Hayes et al. comment
that they had never seen these effects in 6 years of study
of Xenopus involving over 10,000 animals.
figures below, from Hayes et al. shows the deformed reproductive
tract of one hermaphroditic frog. This particular individual
has three testes and three ovaries. The microscopic photograph
on the left shows the entire complex of the animals gonads
and kidneys. The microphotograph on the right shows cross-sections
through gonadal material at places specified by the arrows
on the left. T= testis; O= ovary.
tadpoles were exposed to atrazine at 1 ppb and above,
Hayes et al. noted a demasculinization of the adults
secondary sexual characteristics, specifically a reduction
in the size of the males' larynges (vocal chords). Normally
male larynges are larger than female. The atrazine-treated
males had larynges that were intermediate in size between
normal male and normal female larynges. There was no effect
on the female larynges size.
2: Adult males exposed to 25 ppb atrazine showed
a 10-fold decrease in testosterone compared to controls.
This effect is highly significant statistically.
Treated male testosterone levels were indistinguishable
from control females.
Hayes et al.
does it mean?
This research eliminates any doubt about atrazine as an
endocrine disruptor at extraordinarily low levels of exposure.
It puts endocrine disruption high onto the list of plausible
factors contributing to frog population declines. And
because of the apparent mechanism of action, via enhancement
of aromatase conversion of testosterone to estrogen, it
raises important concerns about atrazine disrupting the
hormonal control of development in other organisms, including
Hayes et al. summarize in their paper, atrazine
contamination is extremely widespread , including, via
atmospheric transport, in rainwater in regions where atrazine
is not used for agricultural crops. The level of atrazine
sufficient to cause reproductive abnormalities in Xenopushermaphroditismis
1/30th of the level allowed by the US EPA in drinking
recommended application level of atrazine ranges from
2,500,000 29,300,000 ppb, the allowable contaminant
level for atrazine in drinking water is 3 ppb, and
short-term exposures of 200 ppb are not considered
a health risk. Atrazine can be as high as 21 ppb in
ground water, 42 ppb in surface waters, 102 ppb in
river basins in agricultural areas, up to 224 ppb
in Midwestern streams, and up to 2,300 ppb in tailwater
pits in Midwestern agricultural areas. Atrazine can
be found in excess of 1 ppb in precipitation in localities
where it is not used and up to 40 ppb in rainfall
in Midwestern agricultural areas."
in many places in the world are undoubtedly exposed to
atrazine at these levels. It remains to be determined
whether other frog species are as sensitive to atrazine
as is Xenopus, and whether the effects are similar.
Hayes' work should reinvigorate efforts to understand
the contribution of chemical exposure to frog declines.
has strongly implicated infection by a chytrid fungus
as an important factor driving frog extinctions, other
causes, including contaminants and the introduction of
exotic species, remain plausible contributors to what
is most likely a multi-factoral process. And given the
ability of some contaminants to reduce
disease resistance, it is not implausible to hypothesize
that contaminants are involved in the rapid pace at which
amphibians have succumbed to the fungal infection. No
research has assessed the impact of low-level atrazine
exposure on the development of frog immune competency.
research by Hayes et al. also points out the crucial
need to carry out experiments assessing toxicological
impacts at environmentally-relevant levels. Prior work
had seemingly dismissed atrazine as an endocrine disruptor
affecting frogs, but the research used exposures that
were 10,000 to 30,000 times higher than the dosage found
by Hayes et al. to produce developmental disruption:
teratogenesis,growth inhibition, and mortality in amphibians
in response to atrazine were not considered environmental
of the high doses required to produce these effects. Effects
in the current study, however, occurred at levels 10,000
times lower than the dose required to produce effects
in amphibians in these previous studies. Allran
and Karasov (2001) reached the conclusion that atrazine
was probably not a significant factor in amphibian declines
based on their studies of toxicity, deformities, and effects
on feeding and ventilation in leopard frogs that did not
produce noticeable effects below 3 ppm. The current data
show that negative effects on sex differentiation occur
at doses 30,000 times lower than effective doses reported
by Allran and Karasov."