1. Atrazine-Induced Hermaphroditism
By: Michelle Lin

2. What is Atrazine?

3. What is Atrazine?
Systematic herbicide that blocks photosynthesis

4. What is Atrazine? Systematic herbicide that blocks photosynthesis
Metabolized to four hydroxyatrazine compounds & to three chlorinated atrazine compounds.

5. What is Atrazine? Systematic herbicide that blocks photosynthesis
Metabolized to four hydroxyatrazine compounds & to three chlorinated atrazine compounds.
Predominate metabolites found in plants

6. 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

7. 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

8. 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.

9. It’s a pesticide, so what?

10. It’s a pesticide, so what?
Most widely used herbicide in the world

11. It’s a pesticide, so what?
Most widely used herbicide in the world
Most common contaminant in ground and surface waters

12. 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

13. 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

14. 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

15. What does Atrazine cause?

16. What does Atrazine cause?
Hermaphroditism in American Leopard Frogs (Rana pipiens)

17. 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)

18. 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

19. The experiment

20. The experiment
Leopard frogs obtained from Sensiba March, Brown County, Wisconsin then shipped to the University of California Berkeley

21. 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)

22. 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 % ethanol

23. 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 % ethanol
Exposed throughout larval period from 2 days posthatching until complete tail reabsorbtion.

24. 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 % 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.

25. The experiment continued

26. 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.

27. 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

28. 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

29. 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

30. 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

31. The collection sites

32. The collection sites
Localities based on atrazine use determined by sales

33. The collection sites
Localities based on atrazine use determined by sales
Countries with <0.4kg/km² atrazine use were chosen as potential control sites

34. 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

35. 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

36. Collection sites continued

37. Collection sites continued
Sampled in Utah on 15 July 2001 and moved eastward

38. 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

39. 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

40. 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

41. 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

42. Chemical Analysis

43. Chemical Analysis
At each site, 100mL of water was collected

44. Chemical Analysis At each site, 100mL of water was collected
Frozen on dry ice

45. Chemical Analysis At each site, 100mL of water was collected
Frozen on dry ice
Atrazine levels were determined

46. 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

47. 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

48. 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

49. 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

50. Gonadal analysis on lab-animals

51. Gonadal analysis on lab-animals
Control animals sexually differentiated at metamorphosis

52. Gonadal analysis on lab-animals
Control animals sexually differentiated at metamorphosis
earliest males to metamorphose had solid testicular lobules

53. 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

54. 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

55. Gonadal analysis continued

56. Gonadal analysis continued
Animals that metamorphosed later had open lobules

57. Gonadal analysis continued
Animals that metamorphosed later had open lobules
Contained both primary spermatogonia and spermatids

58. 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

59. 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

60. The Results

61. The Results
Atrazine-treated males (0.1 and 25 ppb) were sexually differentiated at metamorphosis

62. 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

63. 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

64. 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

65. The Results Continued

66. 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

67. 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

68. 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

69. 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.

70. 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

71. Observations in the Wild

72. Observations in the Wild
Chemical analysis of water revealed none of the sites were atrazine free

73. 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

74. 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

75. 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

76. Observations in the Wild Continued

77. Observations in the Wild Continued
Site 3, North Platte River in Wyoming had most advanced cases of hermaphroditism

78. Observations in the Wild Continued
Site 3, North Platte River in Wyoming had most advanced cases of hermaphroditism
92% testicular oocytes

79. 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

80. 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

81. 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

82. Contaminant levels

83. Contaminant levels

84. The Collection Sites

85. The Collection Sites

86. Contaminant levels continued

87. Contaminant levels continued

88. What does all this mean?

89. What does all this mean?
Atrazine exposure disrupts gonadal development in exposed larvae

90. 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

91. 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

92. 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

93. What does this mean to us?

94. What does this mean to us?
Not so much that amphibian population declines (decline is minimal)

95. What does this mean to us?
Not so much that amphibian population declines (decline is minimal)
We drink the same water

96. 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

97. 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

98. 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

99. Risk and Exposure

100. Risk and Exposure

101. The Bigger Picture

102. The Bigger Picture
Why should we care?

103. The Bigger Picture Why should we care?
Atrazine contamination may affect us also

104. The Bigger Picture Why should we care?
Atrazine contamination may affect us also
May not be same effects exhibited in amphibians

105. 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

106. 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?

107. 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

108. 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

109. Sources
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C
Atrazine-Induced Hermaphroditism at 0.1 ppb in American Leopard Frogs (Rana pipiens): Laboratory and Field Evidence
Feminized Frogs: Herbicide disrupts sexual growth