No threshold dose for estradiol-induced sex reversal of turtle embryos: How little is too much?

Risk assessments for nongenotoxic chemicals assume a threshold below which no adverse outcomes are seen. However, when an endogenous chemical, such as 17 beta-estradiol (E-2), occurs at a concentration sufficient to cause an effect, the threshold is already exceeded. Under these circumstances, exogenous estradiol is not expected to provide a threshold dose. This principle is demonstrated for E-2 in the red-eared slider, a turtle with temperature-dependent sex determination. In this species, gonadal sex is determined by egg incubation temperature; female development requires endogenous estrogen produced by elevated temperature. While normal production of females by endogenous estrogens is not an adverse effect, exogenous estrogens can sex reverse presumptive males, which can be an adverse effect. A large dose-response study was conducted using seven doses and a vehicle control (starting n = 300/group); a single E-2 dose was applied to the eggshell of recently laid eggs. Animals were sexed after hatching. The incubation temperature chosen, 28.6 degrees C, generates a minority of females. Thus, the criteria for testing the threshold hypothesis were met, i.e., there is evidence that there is endogenous estrogen and that it generates an irreversible response. The lowest E-2 dose tested, 400 pg/egg (40 ng/kg), sex reversed 14.4% of the animals, demonstrating very low dose sensitivity. The data were fit with a modified Michaelis-Menten equation, which provided an estimate of 1.7 ng/egg for endogenous estradiol, The median effective dose (ED50) was 5.0 +/- 2.0 ng/egg (35% confidence limits), of which 1.7 ng/egg was endogenous estradiol and 3.3 ng/egg came from the applied estradiol. There was no apparent threshold dose for E-2. A smaller replication confirmed these results. These results provide a simple biologically based dose-response model and suggest that chemicals which act mechanistically like E-2 may also show no threshold dose. If so, even low environmental concentrations of such chemicals may carry risk for sex reversal.