Atrazine-Induced Hermaphroditism
By: Michelle Lin
What is Atrazine?
What is Atrazine?

Systematic herbicide that blocks photosynthesis
What is Atrazine?


Systematic herbicide that blocks photosynthesis
Metabolized to four hydroxyatrazine compounds & to
three chlorinated atrazine compounds.
What is Atrazine?


Systematic herbicide that blocks photosynthesis
Metabolized to four hydroxyatrazine compounds & to
three chlorinated atrazine compounds.
 Predominate metabolites found in plants
What is Atrazine?


Systematic herbicide that blocks photosynthesis
Metabolized to four hydroxyatrazine compounds & to
three chlorinated atrazine compounds.
 Predominate metabolites found in plants
 Desethylated atrazine, desisopropyl atrazine, &
diaminochlorotriazine (DACT) predominate in animal
tissues, & in soils and water
What is Atrazine?



Systematic herbicide that blocks photosynthesis
Metabolized to four hydroxyatrazine compounds & to
three chlorinated atrazine compounds.
 Predominate metabolites found in plants
 Desethylated atrazine, desisopropyl atrazine, &
diaminochlorotriazine (DACT) predominate in animal
tissues, & in soils and water
commonly detected pesticide in ground and surface
water
What is Atrazine?




Systematic herbicide that blocks photosynthesis
Metabolized to four hydroxyatrazine compounds & to
three chlorinated atrazine compounds.
 Predominate metabolites found in plants
 Desethylated atrazine, desisopropyl atrazine, &
diaminochlorotriazine (DACT) predominate in animal
tissues, & in soils and water
commonly detected pesticide in ground and surface
water
detected in streams, rivers, groundwater, and
reservoirs is related directly to both its volume of
usage, and its tendency to persist in soils and move
with water.
It’s a pesticide, so what?
It’s a pesticide, so what?

Most widely used herbicide in the world
It’s a pesticide, so what?


Most widely used herbicide in the world
Most common contaminant in ground and surface
waters
It’s a pesticide, so what?



Most widely used herbicide in the world
Most common contaminant in ground and surface
waters
About 64 to 75 million lbs of active ingredient are
applied per year
It’s a pesticide, so what?




Most widely used herbicide in the world
Most common contaminant in ground and surface
waters
About 64 to 75 million lbs of active ingredient are
applied per year
Three-fourths of all field corn & sorghum are treated
with atrazine annually for weed control
It’s a pesticide, so what?





Most widely used herbicide in the world
Most common contaminant in ground and surface
waters
About 64 to 75 million lbs of active ingredient are
applied per year
Three-fourths of all field corn & sorghum are treated
with atrazine annually for weed control
Also used on sugarcane, wheat, guava, macadamia nuts
orchard grass and hay
What does Atrazine cause?
What does Atrazine cause?

Hermaphroditism in American Leopard Frogs (Rana
pipiens)
What does Atrazine cause?

Hermaphroditism in American Leopard Frogs (Rana
pipiens)
 21 ppb atrazine exposure in 48 hrs can results in
severe gonadal dygenesis in African clawed frogs
(Xenopus laevis)
What does Atrazine cause?

Hermaphroditism in American Leopard Frogs (Rana
pipiens)
 21 ppb atrazine exposure in 48 hrs can results in
severe gonadal dygenesis in African clawed frogs
(Xenopus laevis)
 at concentrations of 0.1 ppb, atrazine induces
hermaphroditism
The experiment
The experiment

Leopard frogs obtained from Sensiba March, Brown
County, Wisconsin then shipped to the University of
California Berkeley
The experiment


Leopard frogs obtained from Sensiba March, Brown
County, Wisconsin then shipped to the University of
California Berkeley
Larvae were treated by immersion with nominal
concentrations of 0, 0.1, or 25 ppb atrazine (98% pure)
The experiment



Leopard frogs obtained from Sensiba March, Brown
County, Wisconsin then shipped to the University of
California Berkeley
Larvae were treated by immersion with nominal
concentrations of 0, 0.1, or 25 ppb atrazine (98% pure)
Atrazine was predissolved in ethanol, and all
treatments contained 0.0036% ethanol
The experiment




Leopard frogs obtained from Sensiba March, Brown
County, Wisconsin then shipped to the University of
California Berkeley
Larvae were treated by immersion with nominal
concentrations of 0, 0.1, or 25 ppb atrazine (98% pure)
Atrazine was predissolved in ethanol, and all
treatments contained 0.0036% ethanol
Exposed throughout larval period from 2 days
posthatching until complete tail reabsorbtion.
The experiment





Leopard frogs obtained from Sensiba March, Brown
County, Wisconsin then shipped to the University of
California Berkeley
Larvae were treated by immersion with nominal
concentrations of 0, 0.1, or 25 ppb atrazine (98% pure)
Atrazine was predissolved in ethanol, and all
treatments contained 0.0036% ethanol
Exposed throughout larval period from 2 days
posthatching until complete tail reabsorbtion.
All dosing and analyses were conducted blindly with
color-coded tanks and treatments.
The experiment continued
The experiment continued

At metamorphosis, each animal was weighed and
measured then euthanized in 0.2% benzocaine, fixed in
Bouin’s fixative, and preserved in 70% ethanol until
further analysis.
The experiment continued


At metamorphosis, each animal was weighed and
measured then euthanized in 0.2% benzocaine, fixed in
Bouin’s fixative, and preserved in 70% ethanol until
further analysis.
Sex of all individuals were determined based on gross
gonadal morphology using a Nikon SMZ 10A
The experiment continued



At metamorphosis, each animal was weighed and
measured then euthanized in 0.2% benzocaine, fixed in
Bouin’s fixative, and preserved in 70% ethanol until
further analysis.
Sex of all individuals were determined based on gross
gonadal morphology using a Nikon SMZ 10A
Tissues dissected and dehydrated in graded alcohols
followed by infiltration with Histoclear and paraffin
The experiment continued




At metamorphosis, each animal was weighed and
measured then euthanized in 0.2% benzocaine, fixed in
Bouin’s fixative, and preserved in 70% ethanol until
further analysis.
Sex of all individuals were determined based on gross
gonadal morphology using a Nikon SMZ 10A
Tissues dissected and dehydrated in graded alcohols
followed by infiltration with Histoclear and paraffin
Serial histological sections cut at 8µm through entire
gonad
The experiment continued





At metamorphosis, each animal was weighed and
measured then euthanized in 0.2% benzocaine, fixed in
Bouin’s fixative, and preserved in 70% ethanol until
further analysis.
Sex of all individuals were determined based on gross
gonadal morphology using a Nikon SMZ 10A
Tissues dissected and dehydrated in graded alcohols
followed by infiltration with Histoclear and paraffin
Serial histological sections cut at 8µm through entire
gonad
Slides were stained in Mallory’s trichrome stain and
images of gonads were recorded
The collection sites
The collection sites
Localities based on atrazine use determined by sales
The collection sites
Localities based on atrazine use determined by sales
Countries with <0.4kg/km² atrazine use were chosen
as potential control sites
The collection sites
Localities based on atrazine use determined by sales
Countries with <0.4kg/km² atrazine use were chosen
as potential control sites
>9.3 kg/km² were chosen as potential atrazineexposed sites
The collection sites
Localities based on atrazine use determined by sales
Countries with <0.4kg/km² atrazine use were chosen
as potential control sites
>9.3 kg/km² were chosen as potential atrazineexposed sites
Collection sites continued
Collection sites continued

Sampled in Utah on 15 July 2001 and moved eastward
Collection sites continued


Sampled in Utah on 15 July 2001 and moved eastward
Collected 100 animals in 8 sites for a total of 800
animals
Collection sites continued



Sampled in Utah on 15 July 2001 and moved eastward
Collected 100 animals in 8 sites for a total of 800
animals
Selected small individuals in an attempt to sample
newly metamorphosed animals
Collection sites continued




Sampled in Utah on 15 July 2001 and moved eastward
Collected 100 animals in 8 sites for a total of 800
animals
Selected small individuals in an attempt to sample
newly metamorphosed animals
Animals were euthanized in benzocaine, fixed in
Bouin’s fixative for 48 hours and preserved in 70%
ethanol
Collection sites continued





Sampled in Utah on 15 July 2001 and moved eastward
Collected 100 animals in 8 sites for a total of 800
animals
Selected small individuals in an attempt to sample
newly metamorphosed animals
Animals were euthanized in benzocaine, fixed in
Bouin’s fixative for 48 hours and preserved in 70%
ethanol
They were measured and sex was determined and
histological analysis was conducted on the gonads of 20
males from each site and a subset of females from each
site
Chemical Analysis
Chemical Analysis

At each site, 100mL of water was collected
Chemical Analysis

At each site, 100mL of water was collected
 Frozen on dry ice
Chemical Analysis

At each site, 100mL of water was collected
 Frozen on dry ice
 Atrazine levels were determined
Chemical Analysis

At each site, 100mL of water was collected
 Frozen on dry ice
 Atrazine levels were determined
 Water sample extracted in organic solvent followed
by aqueous/organic extraction
Chemical Analysis


At each site, 100mL of water was collected
 Frozen on dry ice
 Atrazine levels were determined
 Water sample extracted in organic solvent followed
by aqueous/organic extraction
Analyzed by liquid chromatography/mass
spectrophotometry using daughter ion
Chemical Analysis



At each site, 100mL of water was collected
 Frozen on dry ice
 Atrazine levels were determined
 Water sample extracted in organic solvent followed
by aqueous/organic extraction
Analyzed by liquid chromatography/mass
spectrophotometry using daughter ion
Positive controls contained mixtures of pesticides at
both 0.1 and 10.0 ppb
Chemical Analysis




At each site, 100mL of water was collected
 Frozen on dry ice
 Atrazine levels were determined
 Water sample extracted in organic solvent followed
by aqueous/organic extraction
Analyzed by liquid chromatography/mass
spectrophotometry using daughter ion
Positive controls contained mixtures of pesticides at
both 0.1 and 10.0 ppb
detection limits at 0.1 ppb
Gonadal analysis on lab-animals
Gonadal analysis on lab-animals

Control animals sexually
differentiated at
metamorphosis
Gonadal analysis on lab-animals

Control animals sexually
differentiated at
metamorphosis
 earliest males to
metamorphose had solid
testicular lobules
Gonadal analysis on lab-animals

Control animals sexually
differentiated at
metamorphosis
 earliest males to
metamorphose had solid
testicular lobules
 testes were more
differentiated (contained
more distinct tubules and
germ cells) anteriorly
than posteriorly
Gonadal analysis on lab-animals

Control animals sexually
differentiated at
metamorphosis
 earliest males to
metamorphose had solid
testicular lobules
 testes were more
differentiated (contained
more distinct tubules and
germ cells) anteriorly
than posteriorly
Gonadal analysis continued
Gonadal analysis continued

Animals that metamorphosed
later had open lobules
Gonadal analysis continued

Animals that metamorphosed
later had open lobules
 Contained both primary
spermatogonia and
spermatids
Gonadal analysis continued

Animals that metamorphosed
later had open lobules
 Contained both primary
spermatogonia and
spermatids
 Females had numerous
oocytes in their gonads
and a central ovarian
cavity
Gonadal analysis continued

Animals that metamorphosed
later had open lobules
 Contained both primary
spermatogonia and
spermatids
 Females had numerous
oocytes in their gonads
and a central ovarian
cavity
The Results
The Results

Atrazine-treated males (0.1 and
25 ppb) were sexually
differentiated at metamorphosis
The Results

Atrazine-treated males (0.1 and
25 ppb) were sexually
differentiated at metamorphosis
 36 and 12% of males treated
with 0.1 and 25 ppb atrazine
suffered gonadal dysgenesis
The Results

Atrazine-treated males (0.1 and
25 ppb) were sexually
differentiated at metamorphosis
 36 and 12% of males treated
with 0.1 and 25 ppb atrazine
suffered gonadal dysgenesis
 Gonadal dysgenesisunderdeveloped testes with
poorly structured, closed
lobules or no lobules at all
and low to absent germ
cells
The Results

Atrazine-treated males (0.1 and
25 ppb) were sexually
differentiated at metamorphosis
 36 and 12% of males treated
with 0.1 and 25 ppb atrazine
suffered gonadal dysgenesis
 Gonadal dysgenesisunderdeveloped testes with
poorly structured, closed
lobules or no lobules at all
and low to absent germ
cells
The Results Continued
The Results Continued

29% of the 0.1 ppb-treated animals and 8% that were
treated with 25 ppb displayed varying degrees of sex
reversal
The Results Continued


29% of the 0.1 ppb-treated animals and 8% that were
treated with 25 ppb displayed varying degrees of sex
reversal
Testicular lobules of sex-reversed males contained
oocytes
The Results Continued



29% of the 0.1 ppb-treated animals and 8% that were
treated with 25 ppb displayed varying degrees of sex
reversal
Testicular lobules of sex-reversed males contained
oocytes
Males that metamorphosed later contained large
number of oocytes
The Results Continued




29% of the 0.1 ppb-treated animals and 8% that were
treated with 25 ppb displayed varying degrees of sex
reversal
Testicular lobules of sex-reversed males contained
oocytes
Males that metamorphosed later contained large
number of oocytes
Males that underwent complete sex reversal had gonads
almost completely filled with oocytes and only a
limited number of lobules remained.
The Results Continued





29% of the 0.1 ppb-treated animals and 8% that were
treated with 25 ppb displayed varying degrees of sex
reversal
Testicular lobules of sex-reversed males contained
oocytes
Males that metamorphosed later contained large
number of oocytes
Males that underwent complete sex reversal had gonads
almost completely filled with oocytes and only a
limited number of lobules remained.
No observable effects in atrazine-treated females
Observations in the Wild
Observations in the Wild

Chemical analysis of water revealed none of the sites
were atrazine free
Observations in the Wild

Chemical analysis of water revealed none of the sites
were atrazine free
 Only one site (Juab County, Utah) contained <0.2
ppb atrazine levels
Observations in the Wild

Chemical analysis of water revealed none of the sites
were atrazine free
 Only one site (Juab County, Utah) contained <0.2
ppb atrazine levels
 Sites in Utah and Wyoming did not have detectable
levels of atrazine metabolites
Observations in the Wild


Chemical analysis of water revealed none of the sites
were atrazine free
 Only one site (Juab County, Utah) contained <0.2
ppb atrazine levels
 Sites in Utah and Wyoming did not have detectable
levels of atrazine metabolites
All sites with atrazine sales exceeding 0.4 kg/km² and
contaminant levels that exceeded 0.2 ppb had males
that displayed sex reversal similar to those that
resulted from laboratory
Observations in the Wild Continued
Observations in the Wild Continued

Site 3, North Platte River
in Wyoming had most
advanced cases of
hermaphroditism
Observations in the Wild Continued

Site 3, North Platte River
in Wyoming had most
advanced cases of
hermaphroditism
 92% testicular oocytes
Observations in the Wild Continued

Site 3, North Platte River
in Wyoming had most
advanced cases of
hermaphroditism
 92% testicular oocytes
 Advanced stages of
complete sex reversal
Observations in the Wild Continued


Site 3, North Platte River
in Wyoming had most
advanced cases of
hermaphroditism
 92% testicular oocytes
 Advanced stages of
complete sex reversal
All other sites varied in
frequency and severity of
gonadal abnormalities
Observations in the Wild Continued


Site 3, North Platte River
in Wyoming had most
advanced cases of
hermaphroditism
 92% testicular oocytes
 Advanced stages of
complete sex reversal
All other sites varied in
frequency and severity of
gonadal abnormalities
Contaminant levels
Contaminant levels
The Collection Sites
The Collection Sites
Contaminant levels continued
Contaminant levels continued
What does all this mean?
What does all this mean?

Atrazine exposure disrupts gonadal development in
exposed larvae
What does all this mean?


Atrazine exposure disrupts gonadal development in
exposed larvae
Widespread atrazine contamination was accompanied
by observation of hermaphroditic animals in the field
What does all this mean?



Atrazine exposure disrupts gonadal development in
exposed larvae
Widespread atrazine contamination was accompanied
by observation of hermaphroditic animals in the field
Studies suggest that atrazine impacts amphibians in the
wild
What does all this mean?



Atrazine exposure disrupts gonadal development in
exposed larvae
Widespread atrazine contamination was accompanied
by observation of hermaphroditic animals in the field
Studies suggest that atrazine impacts amphibians in the
wild
 Atrazine contamination of water coincides with
amphibian breeding activity
What does this mean to us?
What does this mean to us?

Not so much that amphibian population declines
(decline is minimal)
What does this mean to us?


Not so much that amphibian population declines
(decline is minimal)
We drink the same water
What does this mean to us?



Not so much that amphibian population declines
(decline is minimal)
We drink the same water
Atrazine effects on amphibians vs. atrazine effects on
humans
What does this mean to us?




Not so much that amphibian population declines
(decline is minimal)
We drink the same water
Atrazine effects on amphibians vs. atrazine effects on
humans
May cause reproduction problems if long term exposure
to atrazine
What does this mean to us?





Not so much that amphibian population declines
(decline is minimal)
We drink the same water
Atrazine effects on amphibians vs. atrazine effects on
humans
May cause reproduction problems if long term exposure
to atrazine
“Adverse effects on hypothalamic-pituitary function in
humans”--Scientific Advisory Panel convened in June
2000
Risk and Exposure
Risk and Exposure
The Bigger Picture
The Bigger Picture

Why should we care?
The Bigger Picture

Why should we care?
 Atrazine contamination may affect us also
The Bigger Picture

Why should we care?
 Atrazine contamination may affect us also
 May not be same effects exhibited in amphibians
The Bigger Picture

Why should we care?
 Atrazine contamination may affect us also
 May not be same effects exhibited in amphibians
 More aware of other pesticides
The Bigger Picture


Why should we care?
 Atrazine contamination may affect us also
 May not be same effects exhibited in amphibians
 More aware of other pesticides
How do we solve Atrazine Contamination?
The Bigger Picture


Why should we care?
 Atrazine contamination may affect us also
 May not be same effects exhibited in amphibians
 More aware of other pesticides
How do we solve Atrazine Contamination?
 Prohibit atrazine distribution
The Bigger Picture


Why should we care?
 Atrazine contamination may affect us also
 May not be same effects exhibited in amphibians
 More aware of other pesticides
How do we solve Atrazine Contamination?
 Prohibit atrazine distribution
 Come up with new pesticide
Sources



UNITED STATES ENVIRONMENTAL PROTECTION
AGENCY WASHINGTON, D.C. 20460
http://www.epa.gov/oppsrrd1/reregistration/atrazine/
hed_redchap_16apr02.PDF
Atrazine-Induced Hermaphroditism at 0.1 ppb in
American Leopard Frogs (Rana pipiens): Laboratory and
Field Evidence
http://ehp.niehs.nih.gov/members/2003/5932/5932.p
df
Feminized Frogs: Herbicide disrupts sexual growth
http://www.sciencenews.org/articles/20020420/fob1.a
sp