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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

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what is atrazine3
What is Atrazine?
  • Systematic herbicide that blocks photosynthesis
what is atrazine4
What is Atrazine?
  • Systematic herbicide that blocks photosynthesis
  • Metabolized to four hydroxyatrazine compounds & to three chlorinated atrazine compounds.
what is atrazine5
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 atrazine6
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 atrazine7
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 atrazine8
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 isrelated directly to both its volume of usage, and its tendency to persist in soils and move with water.
it s a pesticide so what10
It’s a pesticide, so what?
  • Most widely used herbicide in the world
it s a pesticide so what11
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 what12
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 what13
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 what14
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 cause16
What does Atrazine cause?
  • Hermaphroditism in American Leopard Frogs (Rana pipiens)
what does atrazine cause17
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 cause18
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 experiment20
The experiment
  • Leopard frogs obtained from Sensiba March, Brown County, Wisconsin then shipped to the University of California Berkeley
the experiment21
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 experiment22
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 experiment23
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 experiment24
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 continued26
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 continued27
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 continued28
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 continued29
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 continued30
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 sites32
The collection sites
  • Localities based on atrazine use determined by sales
the collection sites33
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 sites34
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 atrazine-exposed sites
the collection sites35
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 atrazine-exposed sites
collection sites continued37
Collection sites continued
  • Sampled in Utah on 15 July 2001 and moved eastward
collection sites continued38
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 continued39
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 continued40
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 continued41
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 analysis43
Chemical Analysis
  • At each site, 100mL of water was collected
chemical analysis44
Chemical Analysis
  • At each site, 100mL of water was collected
    • Frozen on dry ice
chemical analysis45
Chemical Analysis
  • At each site, 100mL of water was collected
    • Frozen on dry ice
    • Atrazine levels were determined
chemical analysis46
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 analysis47
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 analysis48
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 analysis49
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 animals51
Gonadal analysis on lab-animals
  • Control animals sexually differentiated at metamorphosis
gonadal analysis on lab animals52
Gonadal analysis on lab-animals
  • Control animals sexually differentiated at metamorphosis
    • earliest males to metamorphose had solid testicular lobules
gonadal analysis on lab animals53
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 animals54
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 continued56
Gonadal analysis continued
  • Animals that metamorphosed later had open lobules
gonadal analysis continued57
Gonadal analysis continued
  • Animals that metamorphosed later had open lobules
    • Contained both primary spermatogonia and spermatids
gonadal analysis continued58
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 continued59
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 results61
The Results
  • Atrazine-treated males (0.1 and 25 ppb) were sexually differentiated at metamorphosis
the results62
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 results63
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 dysgenesis- underdeveloped testes with poorly structured, closed lobules or no lobules at all and low to absent germ cells
the results64
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 dysgenesis- underdeveloped testes with poorly structured, closed lobules or no lobules at all and low to absent germ cells
the results continued66
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 continued67
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 continued68
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 continued69
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 continued70
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 wild72
Observations in the Wild
  • Chemical analysis of water revealed none of the sites were atrazine free
observations in the wild73
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 wild74
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 wild75
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 continued77
Observations in the Wild Continued
  • Site 3, North Platte River in Wyoming had most advanced cases of hermaphroditism
observations in the wild continued78
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 continued79
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 continued80
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 continued81
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
what does all this mean89
What does all this mean?
  • Atrazine exposure disrupts gonadal development in exposed larvae
what does all this mean90
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 mean91
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 mean92
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 us94
What does this mean to us?
  • Not so much that amphibian population declines (decline is minimal)
what does this mean to us95
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 us96
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 us97
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 us98
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
the bigger picture102
The Bigger Picture
  • Why should we care?
the bigger picture103
The Bigger Picture
  • Why should we care?
    • Atrazine contamination may affect us also
the bigger picture104
The Bigger Picture
  • Why should we care?
    • Atrazine contamination may affect us also
    • May not be same effects exhibited in amphibians
the bigger picture105
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 picture106
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 picture107
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 picture108
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
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.pdf
  • Feminized Frogs: Herbicide disrupts sexual growth http://www.sciencenews.org/articles/20020420/fob1.asp