The Red-Eared Slider Turtle: An Animal Model for the Study of Low Doses and Mixtures1

SYNOPSIS. Current risk assessment techniques for almost all chemicals involve establishing a threshold dose, or the dose below which no adverse effects are seen. But threshold doses may not apply in situations where the chemical mimics the actions of an endogenous compound—such as a steroid hormone—that is important to development. In such cases, any exposure may exceed the threshold. Recent studies with the red-eared slider turtle have shown that exogenous estradiol—even when applied to eggshell in doses as low 0.4 ng—will affect sex development during embryogenesis. Considering that only 0.2% of the estradiol applied to the eggshell ends up in the embryo, it becomes apparent that even very low dosages of steroid hormones or their mimics can have profound biological effects. We tested this idea using eight compounds identified in the yolk of alligator eggs from Lake Apopka, Fla. Five of the compounds—the PCB mixture Aroclor 1242, trans-Nonachlor, cisNonachlor, p,p9-DDE, and chlordane—altered sex ratio outcomes when applied to eggshells during development. Aroclor 1242 produced the most powerful effects, shifting the ratio of females almost twofold, while chlordane had the greatest effect when combined with estradiol. Administration of all eight compounds together also increased the ratio of females to males. However, comparing the single-compound exposures at the same dosages indicate that these compounds behave differently in combination than they do singly, emphasizing the need for further studies using chemical mixtures reflecting proportions found in nature. The effect of chlordane and Aroclor 1242 on aromatase activity levels during embryogenesis in the brain and adrenal-kidney-gonad (AKG) complex was also examined. Chlordane, a suspected anti-androgen in this species, did not affect aromatase activity in either the brain or the AKG. However, Aroclor 1242 significantly altered aromatase activity levels in the red-eared slider turtle brain—but not in the AKG—during a crucial developmental period. After this crucial period, Aroclor 1242 caused an increase in aromatase activity in the AKG of embryos just prior to hatch. Additionally, hatchling males treated during embryogenesis with Aroclor 1242 and chlordane exhibited significantly lower testosterone levels than controls in response to folliclestimulating hormone administration, while chlordane-treated females had significantly lower progesterone, testosterone, and 5a-dihydrotestosterone levels relative to controls. These results are similar to those found in juvenile alligators from Lake Apopka. Males treated with Aroclor 1242 and trans-Nonachlor displayed an elevated estradiol response to FSH administration vs. control males. Taken together, these results suggest that EDCs exert effects during embryonic development that extend beyond birth. They also suggest that the alterations in sex steroid hormone levels observed in animals from contaminated areas may result from EDC-induced alterations in the neuroendocrine axis controlling gonadal sex steroid hormone production.