Full and Partial Lifecycle Amphibian Toxicity Assay

We are currently developing and validating a standard protocol of evaluating reproductive toxicity in Xenopus . The current protocol entails a 30-45 day exposure or dosing period following super-ovulation, followed by a full reproductive work-up which includes mating, embryo counts, fertilization and necrosis rates, viability assessment, and short-term (4-d) teratological examination. An evaluation of ovary pathology, oocyte count, stage, and viability, testis pathology, sperm count, and dysmorphology assessment is also performed as warranted. Crossover experiments in which exposed females are mated with unexposed males and are also commonly performed with the assay. Dosing/exposure may be accomplished by parenteral routes, oral (intubation or feed), or by ambient exposure in the culture water or natural site waters/sediments. These protocols have also been adapted for use with native species, including the leopard frog Rana pipiens . Chronic exposure and lifecycle exposure study protocols (60-d or 180 d, respectively) are currently being developed and validated using Xenopus tropicalis . Results are encouraging.

Amphibian Metamorphosis Assay

The amphibian metamorphosis assays involve a partial lifecycle test format using prometamorphic Xenopus larvae to detect EDCs that alter thyroid activity. The tests involve exposing either NF stage 51 X. laevis larvae to test various materials for 21 d, or NF stage 54 X. laevis for 14 d, respectively.  At the conclusion of exposure, measurements of development stage, weight, and thyroid gland histology are made.  Thyroid histology has proven to be a highly sensitive and valuable endpoint.

Testing is performed under either flow-through or static renewal conditions depending on the characteristics of the test material. Observations and feeding are performed daily with dead organisms removed and recorded at this time using a digital or digital-video camera coupled with digitizing software. Statistical analyses and development of concentration-response curves are then performed to ascertain quantitative endpoints from the test. In addition, complimentary molecular assays (gene expression and arrays) are currently being developed. These assays will be used in conjunction with new analytical methods for thyroid hormone analysis (LC/GC-MS).