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Atrazine-Induced Hermaphroditism

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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Atrazine-Induced Hermaphroditism

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  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 isrelated 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 0.0036% 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 0.0036% 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 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.

  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

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