Fort DJ et al. (2023), Importance of diet in amphibian metamorphosis-b...

XB-ART-59305

J Appl Toxicol 2023 Mar 01;433:360-372. doi: 10.1002/jat.4387.

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

Fort DJ , Leopold MA , Wolf JC , Todhunter KJ , Weterings PJJM .

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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- )-deficient FETAX solution supplemented with <0.025, 0.17, 0.52, 1.58, and 4.80 μg I- /L (measured concentrations 0.061, 0.220, 0.614, 1.65, and 4.73 μg I- /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- , respectively. Developmental rates and hind limb length of the 1.65 and 4.73 μg/L I- 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.

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Species referenced: Xenopus laevis
Genes referenced: smn1
GO keywords: thyroid hormone generation [+]

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	Figure 2. Representative Xenopus laevis larvae at study Day 32: from top to bottom, a tadpole from the group fed Sera Micron Nature diet in dechlorinated tap water per test guidance (OECD, 2009), followed by Frog Brittle fed tadpoles exposed to 0.061, 0.220, 0.614, 1.65, and 4.73 μg/L I− treatments.
	Figure 3. (A) Effect of iodide concentration on hind limb length in Xenopus laevis tadpoles exposed for 32 days from NF Stage 47. Error bars represent SEM. Solid bar represents Sera Micron Nature diet benchmark. Dashed line indicates significantly decreasing relative to Sera Micron Nature diet benchmark (Jonckheere–Terpstra test, p = 0.0000, 0.0000, 0.0029, and 0.0089 for the 0.061, 0.220, 0.614, and 1.65 μg/L I− treatments, respectively). Solid line indicates decreasing relative to 4.73 μg/L I− treatment (Jonckheere–Terpstra test, p = 0.0000, 0.0001, 0.035, and 0.0146, respectively; for the 0.061, 0.220, 0.614, 1.65, and 4.73 μg/l I− treatments). (B) Effect of iodide concentration on normalized hind limb length in X. laevis tadpoles exposed for 32 days from NF Stage 47. Error bars represent SEM. Solid bar represents Sera Micron Nature diet benchmark. Dashed line indicates significantly different relative to Sera Micron Nature diet benchmark (Dunnett's test, p = 0.0003, 0.0002, 0.0000, 0.0000, and 0.0000 for the 0.061, 0.220, 0.614, 1.65, and 4.73 μg/L I− treatments, respectively). Solid line indicates significantly decreasing relative to 4.73 μg/L I− supplementation treatment (Jonckheere–Terpstra test, p = 0.0000, 0.0000, 0.002, and 0.0144, respectively; for the 0.061, 0.220, 0.614, 1.65, and 4.73 μg/l I− treatments). (C) Effect of iodide concentration on snout–vent length in X. laevis tadpoles exposed for 32 days from NF Stage 47. Error bars represent SEM. Solid bar represents Sera Micron Nature diet benchmark. Dashed line indicates significantly decreasing relative to Sera Micron Nature diet benchmark (Jonckheere–Terpstra test, p = 0.0023, 0.001, 0.0017, 0.0022, and 0.0144 for the 0.061, 0.220, 0.614, 1.65, and 4.73 μg/L I− treatments, respectively). Not significantly decreasing relative to 4.73 μg/l I− treatment (Jonckheere–Terpstra test, p = 0.1561 [decreasing trend] and 0.8496 [increasing trend]). (D) Effect of iodide concentration on body weight in X. laevis tadpoles exposed for 32 days from NF Stage 47. Error bars represent SEM. Solid bar represents Sera Micron Nature diet benchmark. Dashed line indicates significantly decreasing relative to Sera Micron Nature diet benchmark (Jonckheere–Terpstra test, p = 0.0036, 0.0089, 0.0105, 0.0213, and 0.0141, respectively; for the 0.061, 0.220, 0.614, 1.65, and 4.73 μg/L I− treatments). Not significantly decreasing relative to 4.73 μg/L I− treatment (Jonckheere–Terpstra test, p = 0.2066 [decreasing trend] and 0.8102 [increasing trend]).

	Figure 5. (A) Guaranteed analysis of Sera Micron Nature diet, (https://www.Sera.de/en/) and Sera Micron Nature product specification. (B) Guaranteed analysis of Frog Brittle diet (https://www.enasco.com/xenopus-food-and-care). (C) Composition of Sera Micron Nature diet (https://www.Sera.de/en/, and Sera Micron Nature product specification. Note the significant contribution of the algal component of the diet. (D) Composition of the Frog Brittle diet (https://www.enasco.com/xenopus-food-and-care). Note the absence of the algal component of the diet. (E) Vitamin and mineral composition of Sera Micron Nature diet (https://www.Sera.de/en/) and Sera Micron Nature product specification. (F) Vitamin and mineral composition of the Frog Brittle diet (https://www.enasco.com/xenopus-food-and-care).