Hayes TB , Stuart AA , Mendoza M , Collins A , Noriega N , Vonk A , Johnston G , Liu R , Kpodzo D .

	Figure 1. Proposed mechanism of atrazine action on amphibians. The induction of aromatase results in a decrease in androgens (aromatase substrate) and a subsequent increase in estrogens (aromatase product). Atrazine thus demasculinizes (chemically castrates) and feminizes amphibians. Molecular and endocrine support for the proposed mechanism has been demonstrated in amphibians as well as all other vertebrate classes examined (see “Discussion”).
	Figure 2. . Experimental design for testing the effects of partial E2 exposure on sex differentiation in X. laevis larvae. The larval period from hatching [NF stage (St.) 48] to the completion of metamorphosis (complete tail reabsorption, NF stage 66) lasts 65 days on average under standard conditions in our laboratory. Sex differentiation occurs between NF stages 52 and 54 (~ 25 days posthatching). Animals were treated with E2 (100 μg/L) for 7 days (beginning at NF stage 50, day 19 posthatching, and ending at NF stage 53, day 26), for 14 days (beginning at NF stage 50, day 19 posthatching, and ending at NF stage 55, day 33), or 49 days (until complete tail reabsorption, NF stage 66). Treatment periods are shown by the solid green horizontal arrows, and termination of treatments, by the vertical dashed lines. In the final experimental group, treatment ended for each individual as they metamorphosed. Black arrows indicate that the animals were reared without E2 exposure after the treatments were completed. Larval pictures adapted from Niewkwoop and Faber (1994).
	Figure 3. Gonads of a control postmetamorphic (NF stage 66) male (A,C) and female (B,D) African clawed frog (X. laevis). Abbreviations: OV, ovarian vesicle; K, kidney. (A,B) The entire kidney–interrenal–gonadal complex. The yellow color is the result of Bouin’s fixative. Arrowheads show the rostral and caudal ends of the animal’s right gonad. (C,D) Transverse cross-sections (8 μm) through the geometric center of each animal’s right gonad. Sections were stained with Mallory’s trichrome stain. Arrow indicates melanophore in the ovary. Scale bar: (A,B) 0.1 mm; (C,D) 10 μm. Figure adapted from Hayes et al. (2002a).
	Figure 4. Gonads of an NF stage 66 male X. laevis showing SSP. The individual shown was exposed to 0.1 ppb atrazine. (A) The entire dissected Bouin’s-fixed kidney–interrenal–gonadal complex. Each of the six testes is numbered. (B,C) Sagittal sections (stained in Mallory’s trichrome stain) through the animal’s right and left gonad, respectively. The six testes are numbered corresponding to numbers in A. Each is a distinct organ based on review of the entire series of sagittal sections. Scale bar: (A,B) 0.1 mm; (C) 40 μm.
	Figure 5. A mixed hermaphrodite at NF stage 66, treated with 0.1 ppb atrazine. Abbreviations: FB, fatbody; K, kidney; O, ovary; T, testis. (A) The entire dissected, Bouin’s-fixed kidney–interrenal–gonadal complex. (B–E) Transverse cross-sections (8 μm) stained in Mallory’s trichrome stain. Sections were taken through areas indicated by the black lines. Note the absence of pigment in the ovaries, a conditional typical in hermaphrodites. Scale bar: (A) 0.1 mm; (B–E) 25 μm.
	Figure 6. A lateral hermaphrodite at NF stage 66, treated with 0.1 ppb atrazine. Abbreviations: O, ovary, T, testis. (A) The entire dissected, Bouin’s-fixed kidney–interrenal–gonadal complex. (B) A transverse cross-section (8 μm) stained in Mallory’s trichrome stain. The section was taken from the center of the gonad indicated by the arrow in A. Scale bar: (A) 0.1 mm; B) 25 μm.
	Figure 7. A rostral-caudal hermaphrodite at NF stage 66, treated with 0.1 ppb atrazine. Abbreviations: K, kidney, O, ovary, T, testis. (A) The entire dissected, Bouin’s-fixed kidney–interrenal–gonadal complex. (B,C) Transverse cross-sections (8 μm) of the animal’s right gonad stained in Mallory’s trichrome stain. The sections were taken from the areas shown by the white arrowheads: right rostral testis in cross-section (B) and right caudal ovary (C). (D–G) Sagittal sections (8 μm) taken from the animal’s left gonad from the areas indicated by the white arrows: sagittal sections of the left rostral testis (D,E) and through the caudal ovary ( F,G). Note absence of pigment in the ovary. Scale bar: (A) 0.1 mm; (B–C) 10 μm; (D–G) 50 μm.
	Figure 8. An animal with nonpigmented ovaries. The individual shown was exposed to 0.1 ppb atrazine. Abbreviations: K, kidney; O, ovary; UO, unpigmented ovaries. (A) The entire dissected, Bouin’s-fixed kidney–interrenal–gonadal complex. Although the individual shown has distinct lobes, some animals with this morphology had only very shallow indications of lobes. (B) A transverse cross-section (8 μm) through the geometric center of the animal’s kidney and gonads. The section is stained in Mallory’s trichrome stain. This histology revealed that the animals were in fact females, even though their gonads lacked pigment, typical of normal ovaries. Scale bar: (A) 0.1 mm; (B) 40 μm.
	Figure 9. (A) Frequency of males, females, and specimens with gonadal malformations in controls and atrazine-treated animals (0.1–25 ppb). Numbers above bars are samples sizes and represent the number surviving to metamorphosis (of 90). Dashed line indicates 50%. (B) Frequency of gonadal malformations only. X-axis is categorical.
	Figure 10. Unpigmented ovaries induced by CPA exposure. Abbreviations: FB, fat body; K, kidneys; UO, unpigmented ovary. Scale bar = 0.1 mm.
	Figure 11. Frequency of males, females, and animals with nonpigmented ovaries in animals exposed to CPA compared with solvent (methanol)-treated controls. Numbers above bars are samples sizes and represent the number surviving to metamorphosis (of 90). Dashed line indicates 50%.
	Figure 12. SSP (A) and hermaphroditism (B) in animals treated for 7 days (NF stage 50–53) with 100 μg/L E2. Six testes (three on each side) are numbered in A. Abbreviations: O, ovary; T, testis. Scale bar = 0.1 mm for A and B.
	Figure 13. Frequency of males, females, animals with SSP, and hermaphrodites in animals treated for s7, 14, or 49 days with 100 μg/L E2. X-axis is categorical. Control bar shows the control sex ratio. NF stages below the bars indicate the stage at which E2 exposure was terminated. All E2 exposures began at NF stage 50 (Figure 2). Numbers above bars are samples sizes and represent the number surviving to metamorphosis (of 90). Dashed line indicates 50%.

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