In the present work, for the first time, a rapid and sensitive liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry (LC/APPI-MS/MS) method has been developed and validated for the simultaneous quantitation of testosterone, estradiol, ethinyl estradiol, and 11-ketotestosterone in fathead minnow fish plasma using no more than 10 microL of plasma. Compounds present in plasma were directly derivatized with dansyl chloride and 25 microL of the derivatized mixture was injected into the LC/APPI-MS/MS system. The gradient chromatographic elution was achieved on an Agilent Zorbax SB-C18 analytical column (2.1 mm x 50 mm, 1.8 microm particle size) with mobile phases consisting of acetonitrile, water and acetic acid. The flow rate was 0.5 to 0.7 mL/min and the total run time was 11.5 min. The lower limits of quantitation for testosterone, estradiol, ethinyl estradiol, and 11-ketotestosterone and were 1, 1, 1, and 2.5 ng/mL, respectively. Intra-batch precision was less than 19.4% and inter-batch precision was less than 11.7% for all four analytes. Accuracy was within 83.5-115.4% of nominal concentrations. This method is used for quantitation of sex steroid levels in fathead minnow tested in endocrine disruptor screening experiments.

The U.S. Environmental Protection Agency (U.S. EPA) has included an amphibian metamorphosis assay (AMA) to detect thyroid active chemicals in Tier 1 testing of their endocrine screening program. To understand the variability, specificity, and reliability of the key endpoints of this assay, two exposure studies with Xenopus laevis tadpoles were conducted with two known thyroid-active compounds, namely, methimazole or L-thyroxine, for a total of 21 d. In addition, various increased-flow-rate treatments were included in the exposures to evaluate the effects of physical stress on metamorphic development. The endpoints examined in the exposures were wet weight, snout-vent length, hind-limb length, developmental stage, and thyroid and gonadal histopathology. As expected, the results indicated that both methimazole and L-thyroxine were thyroid active in the AMA, hind-limb length and thyroid histopathology being the most sensitive endpoints of thyroid activity. Tadpoles that were exposed to the various physical stressors in these experiments showed no signs of altered metamorphic development, and exposure to the thyroid-active compounds had no effect on the developing gonad of X. laevis. Taken together, these results support the use of the AMA as a Tier 1 endocrine screen for detection of potential thyroid pathway activity; however, the lack of a true negative response (no-effect) during the validation process prevents a full evaluation of this assay's specificity at this time.

The amphibian metamorphosis assay (AMA) and the fish short-term reproduction assay (FSTRA) are screening assays designed to detect potential endocrine activity of a test substance. These assays are included in a battery of assays in Tier 1 of U.S. Environmental Protection Agency's Endocrine Disruptor Screening Program. Based on our laboratory's experience with these two assays, we have noted several challenges in the conduct and interpretation of the AMA and FSTRA, including, but not limited to, diseased/parasitized test organisms, failure to meet some guideline performance criteria, and issues selecting and maintaining test concentrations. Various approaches are described for addressing the challenges associated with both the conduct and interpretation of these assays. Historical control data for both the AMA and FSTRA are presented to further understand background occurrences of histopathological phenomena and variability associated with the measured endpoints in these assays. In the historical control database for the AMA, wet weight on day 7 was the most variable endpoint (coefficient of variation = 26%), while developmental stage on day 21 was least variable (coefficient of variation = 0.47%). In the FSTRA, vitellogenin concentrations were the most variable endpoint (coefficient of variation = 47-84%), while fertility was the least variable endpoint (coefficient of variation = 1.5%) among historical controls.

2,4-Dichlorophenoxyacetic acid (2,4-D) was evaluated in both the Amphibian Metamorphosis Assay (AMA) and the Fish Short Term Reproduction Assay (FSTRA). In the AMA, tadpoles were exposed to mean measured 2,4-D concentrations of 0 (water control), 0.273, 3.24, 38.0 and 113 mg acid equivalents (ae)/L for either seven or 21 days. In the FSTRA, fathead minnows were exposed to mean measured 2,4-D concentrations of 0 (water control), 0.245, 3.14, 34.0, and 96.5 mg ae/L for 21 days. The respective concentrations of 2,4-D were not overtly toxic to either Xenopus laevis tadpoles or fathead minnows (Pimephales promelas). In the AMA, there were no signs of either advanced or delayed development, asynchronous development, or significant histopathological effects of the thyroid gland among 2,4-D exposed tadpoles evaluated on either day seven or day 21 of the exposure. Therefore, following the AMA decision logic, 2,4-D is considered "likely thyroid inactive" in the AMA with a No Observable Effect Concentration (NOEC) of 113 mg ae 2,4-D/L. In the FSTRA, there were no significant differences between control and 2,4-D exposed fish in regard to fertility, wet weight, length, gonado-somatic indices, tubercle scores, or blood plasma concentrations of vitellogenin. Furthermore, there were no treatment-related histopathologic changes in the testes or ovaries in any 2,4-D exposed group. The only significant effect was a decrease in fecundity among fish exposed to 96.5 mg ae 2,4-D/L. The cause of the reduced fecundity at the highest concentration of 2,4-D tested in the assay was most likely due to a generalized stress response in the fish, and not due to a specific endocrine mode of action of 2,4-D. Based on fish reproduction, the NOEC in the FSTRA was 34.0 mg ae 2,4-D/L.