The toxic effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) to fathead minnow (Pimephales promelas), channel catfish (Ictalurus punctatus), lake herring (Coregonus artedii), medaka (Oryzias latipes), white sucker (Catastomus commersoni), northern pike (Esox lucius), and zebrafish (Danio danio) were observed during early life‐stage development after waterborne exposure of fertilized eggs. Species sensitivity based on TCDD‐Cegg (TCDD concentration in eggs) was determined by effects observed over a 32‐d period for all species except lake herring in which a 100‐d period was used. Signs of TCDD toxicity, including edema, hemorrhaging, and craniofacial malformations were essentially identical to those observed in salmonids following TCDD egg exposure and preceded or accompanied mortality most often during the period from hatch through swim‐up. The no‐observed‐effect concentrations and lowest‐observed‐effect concentrations, based on significant decreases in survival and growth as compared to the controls, ranged from 175 and 270 pg/g for lake herring to 424 and 2,000 pg/g for zebrafish, respectively. Shapes of concentration–response curves, expressed as TCDD‐Cegg versus percent mortality, were similar for all species and were consistently steep suggesting that the mechanism of action of TCDD is the same among these species. The LCegg50s (concentrations in eggs causing 50% lethality to fish at test termination) ranged from 539 pg/g for the fathead minnow to 2,610 pg/g for zebrafish. Comparisons of LCegg50s indicate that the tested species were approximately 8 to 38 times less sensitive to TCDD than lake trout, the most sensitive species evaluated to date. When LCegg50s are normalized to the fraction lipid in eggs (LCegg,l50s), the risk to early life stage survival for the species tested ranges from 16‐ to 180‐fold less than for lake trout.

Abstract-The toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to fathead minnow (Pimephales promelas), channel catfish (Ictalurus punctatus), lake herring (Coregonus artedii), medaka (Oryzias latipes), white sucker (Catastomus commersoni), northern pike (Esox lucius), and zebrafish (Danio danio) were observed during early life-stage development after waterborne exposure of fertilized eggs. Species sensitivity based on TCDD-C egg (TCDD concentration in eggs) was determined by effects observed over a 32-d period for all species except lake herring in which a 100-d period was used. Signs of TCDD toxicity, including edema, hemorrhaging, and craniofacial malformations were essentially identical to those observed in salmonids following TCDD egg exposure and preceded or accompanied mortality most often during the period from hatch through swim-up. The no-observedeffect concentrations and lowest-observed-effect concentrations, based on significant decreases in survival and growth as compared to the controls, ranged from 175 and 270 pg/g for lake herring to 424 and 2,000 pg/g for zebrafish, respectively. Shapes of concentration-response curves, expressed as TCDD-C egg versus percent mortality, were similar for all species and were consistently steep suggesting that the mechanism of action of TCDD is the same among these species. The LC egg 50s (concentrations in eggs causing 50% lethality to fish at test termination) ranged from 539 pg/g for the fathead minnow to 2,610 pg/g for zebrafish.Comparisons of LC egg 50s indicate that the tested species were approximately 8 to 38 times less sensitive to TCDD than lake trout, the most sensitive species evaluated to date. When LC egg 50s are normalized to the fraction lipid in eggs (LC egg,ᐉ 50s), the risk to early life stage survival for the species tested ranges from 16-to 180-fold less than for lake trout.

In response to the initial Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommendations, research was conducted on the development of a Xenopus laevis based tail resorption assay for evaluating thyroid axis disruption. This research highlighted key limitations associated with relying on tail resorption as a measure of anti/thyroid activity. The most critical limitation being that tail tissues of tadpoles at metamorphic climax are insensitive to perturbation by thyroid axis agonists/antagonists. To improve upon the initial proposal, we have conducted experiments comparing the sensitivity of pre-metamorphic (stage 51) and pro-metamorphic (stage 54) larvae to the model thyroid axis disruptors methimazole (control, 6.25, 12.5, 25, 50, 100 mg/l), 6-propylthiouracil (PTU) (control, 1.25, 2.5, 5, 10, and 20 mg/l), and thyroxine (T4) (0.25, 0.5, 1, 2, 4 microg/l). Exposures were conducted using two different experimental designs. For experimental design 1, tadpoles were exposed to methimazole or PTU starting at either NF stage 51 or NF 54 for 14 days. For experimental design 2, tadpoles were exposed to PTU or T4 starting at NF stage 51 or NF 54 for 14 and 21 days, respectively. Methimazole and PTU, which are thyroid hormone synthesis inhibitors, both caused a concentration dependent delay in larval development. As determined from this endpoint, there were only minor differences in sensitivity observed among the two stages examined. Further, both compounds caused concentration dependent changes in thyroid gland morphology. These changes were characterized as reduced colloid, glandular hypertrophy, and cellular hyperplasia and hypertrophy. Treatment failed to negatively affect growth, even in tadpoles that experienced significant metamorphic inhibition. T4 treatment resulted in a concentration dependent increase in developmental rate, as would be expected. Similar to studies with methimazole, there were no differences in sensitivity among the two developmental stages examined. These results indicate that tadpoles in the early stages of metamorphosis are sensitive to thyroid axis disruption and that development of a short-term, diagnostic amphibian-based thyroid screening assay shows considerable promise.

Abstract-Recently a suite of relatively specific hindlimb deformities have been observed in several anuran species in North America. These deformities include ectopic and supernumerary limbs and missing limbs, limb segments, or digits. The objective of this study was to assess two stressors hypothesized as responsible for limb malformations in amphibians: methoprene, an insect growth regulator that, through interaction with the retinoic acid signaling system, could possibly cause limb deformities, and ultraviolet (UV) light. Northern leopard frogs (Rana pipiens) were exposed to several different concentrations of methoprene both in the absence and presence of UV light designed to mimic the UV wavelength spectrum present in sunlight. Exposures were initiated at early embryonic stages (newly fertilized eggs) and continued through emergence of the forelimbs of the frogs. At the highest methoprene concentration tested, both in the absence and presence of UV light, severe developmental effects were observed, with all organisms dying within 12 to 16 d of test initiation. However, exposure to the pesticide did not cause limb malformations. Irrespective of methoprene treatment, a very high percentage (ϳ50%) of animals held under the UV light for 24 d developed hindlimb malformations. These malformations usually were bilateral and sometimes completely symmetrical, and consisted of missing limb segments and missing or reduced digits. A complete proximal to distal representation of the deficiencies occurred, ranging from missing or malformed femurs to the absence of single digits or digit segments. The developmental period of greatest sensitivity to UV light occurred during very early limb bud development, corresponding with formation of the apical ectodermal ridge. The significance of these findings in terms of deformed frogs in the field is uncertain. Although the deformity types observed (i.e., missing limb segments and digits) were similar to those seen in some field specimens, the UV light treatment did not cause the full range of malformations observed in animals from the field (e.g., supernumerary limbs, nonbilateral deformities). Furthermore, although the artificial light spectrum utilized mimicked the relative UV spectrum present in sunlight, it did not match full sunlight intensity, and did not accurately mimic visible wavelengths. Finally, the relationship of the UV light dose used in the laboratory to that actually experienced by amphibians in the field is uncertain. Despite these questions, our findings suggest that UV light should be further considered as a plausible factor contributing to amphibian malformations in field settings.

The perchlorate anion inhibits thyroid hormone (TH) synthesis via inhibition of the sodium-iodide symporter. It is, therefore, a good model chemical to aid in the development of a bioassay to screen chemicals for affects on thyroid function. Xenopus laevis larvae were exposed to sodium perchlorate during metamorphosis, a period of TH-dependent development, in two experiments. In the first experiment, stage 51 and 54 larvae were exposed for 14 d to 16, 63, 250, 1,000, and 4,000 microg perchlorate/ L. In the second experiment, stage 51 larvae were exposed throughout metamorphosis to 8, 16, 32, 63, and 125 microg perchlorate/L. Metamorphic development and thyroid histology were the primary endpoints examined. Metamorphosis was retarded significantly in the first study at concentrations of 250 microg/L and higher, but histological effects were observed at 16 microg/L. In the second study, metamorphosis was delayed by 125 microg/L and thyroid size was increased significantly at 63 microg/L. These studies demonstrate that inhibition of metamorphosis readily can be detected using an abbreviated protocol. However, thyroid gland effects occur at concentrations below those required to elicit developmental delay, demonstrating the sensitivity of this endpoint and suggesting that thyroidal compensation is sufficient to promote normal development until perchlorate reaches critical concentrations.