Importance of diet in amphibian metamorphosis‐based studies designed to assess the risk of thyroid active substances

Abstract

The present study evaluated the hypothesis that dietary quality used in historical studies may impact the effects of chemical stressors on premetamorphic development and metamorphosis due to suboptimal nutritional quality. A modified Amphibian Metamorphosis Assay (AMA) was performed in which Nieuwkoop and Faber (NF) Stage 47 tadpoles of Xenopus laevis were exposed for 32 days to iodide (I À )deficient FETAX solution supplemented with <0.025, 0.17, 0.52, 1.58, and 4.80 μg I À /L (measured concentrations 0.061, 0.220, 0.614, 1.65, and 4.73 μg I À /L) and fed a pureed Frog Brittle (FB) diet. An AMA guideline benchmark group (four replicates) exposed to dechlorinated tap water and fed standard Sera Micron Nature ® (SMN) diet was evaluated concurrently. Developmental delay, observed as changes in stage distribution or median developmental stage, occurred in FB treatments with 0.061, 0.220, and 0.614 μg/L I À , respectively. Developmental rates and hind limb length of the 1.65 and 4.73 μg/L I À groups were similar to each other, but both treatments fell short of the developmental rate achieved by the SMN benchmark. Iodide supplementation also had no impact on nonthyroidal growth endpoints, which were markedly reduced in FB-fed frogs compared with their SMN-fed counterparts. All larvae that received the FB diet had mildly to severely hypoplastic/atrophic thyroids, a condition for which iodine supplementation had little if any ameliorative effect. Collectively, these results suggested that nutritional deficiencies in the FB diet negatively affected both growth and metamorphic development, the latter of which was only compensated to a limited extent by iodine supplementation.

Inefficacy of dietary test substance administration in Amphibian Metamorphosis Assay (AMA) studies

“…The influence of diet type, dietary regime, and dietary quality on standardized developmental toxicity studies using amphibians, such as the AMA and LAGDA, was evaluated recently by Fort et al (2022) and Marini et al (2023). Fort et al (2022) (Leloup & Buscagalia, 1977;Shi, 2000), which is generally consistent with the findings of the present study.…”

“…The influence of diet type, dietary regime, and dietary quality on standardized developmental toxicity studies using amphibians, such as the AMA and LAGDA, was evaluated recently by Fort et al (2022) and Marini et al (2023). Fort et al (2022) (Leloup & Buscagalia, 1977;Shi, 2000), which is generally consistent with the findings of the present study. However, in the Fort et al ( 2022) study, non-thyroidal growth endpoints including SVL and body weight were markedly reduced in FB-fed frogs compared to their SMN-fed counterparts.…”

“…The food provided to the tadpoles is a dry diet (Sera ® Micron Nature [SMN], Sera, Heinsberg, Germany) that is liquified and fed as a concentrated suspension. Although SMN is used almost exclusively in traditional AMA studies based on recommendations provided by OECD (2009) and USEPA (2015) and is considered the gold standard (Fort, Leopold, Wolf, Todhunter, & Weterings, 2022), other diets could be used that provide equivalent results. Test solutions flow in and out of the aquaria at a constant rate; generally at 25 ml/min.…”

An Amphibian Metamorphosis Assay Dietary Restriction Study: Lessons for Data Interpretation

“…It should be recognized that the pattern of endpoint effects in the present study, consisting of inhibited thyroid development combined with reductions in other growth and development metrics, is not consistent with any well-recognized mechanism of chemical-induced thyroid endocrine disruption. Furthermore, it is likely no coincidence that similar effect patterns were observed in X. laevis tadpoles that received a suboptimal dietary formulation (Fort, Leopold, et al, 2022) and in those exposed to a systemically toxic levels of copper, as evidenced by multi-organ cytotoxicity . Thyroid gland development may be dependent on the nutritional status of the tadpole.…”

Influence of systemic copper toxicity on early development and metamorphosis in Xenopus laevis

“…Included in the former category are differences from control involving HLL (often normalized by SVL to normalize for growth‐related differences), developmental stage, and thyroid histopathology, while the latter category includes SVL and mean body weight (OECD, 2009; USEPA, 2009). However, recent evidence suggests that reductions in developmental stage may, in some instances, be caused by non‐thyroidal factors such as reduced dietary energy intake, alterations in iodine availability, and/or systemic toxicity (Fort et al, in press). In the current study, evidence that Cu caused developmental arrest via a non‐thyroid mechanism is provided by the fact that divergence in developmental stage began prior to NF stage 54 during premetamorphosis (Figure 3).…”