Boualit L et al. (2022), The Amphibian Short-Term Assay: Evaluation of a...

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Environ Toxicol Chem 2022 Nov 01;4111:2688-2699. doi: 10.1002/etc.5436.

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The Amphibian Short-Term Assay: Evaluation of a New Ecotoxicological Method for Amphibians Using Two Organophosphate Pesticides Commonly Found in Nature-Assessment of Biochemical, Morphological, and Life-History Traits.

Boualit L , Cayuela H , Cattin L , Chèvre N .

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Amphibia is the most threatened class among vertebrates, with >40% of the species threatened with extinction. Pollution is thought to alter amphibian population dynamics. With the growing interest in behavioral ecotoxicology, the neurotoxic organophosphate pesticides are of special concern. Understanding how exposure to neurotoxics leads to behavioral alterations is of crucial importance, and mechanistic endpoints should be included in ecotoxicological methods. In the present study, we tested an 8-day assay to evaluate the toxicity of two organophosphates, diazinon and chlorpyrifos, on Xenopus laevis, that is, on biochemical, morphological, and life-history traits related to locomotion capacities. The method involves measuring biomarkers such as glutathione-S-transferase (GST) and ethoxyresorufin-O-deethylase (EROD; two indicators of the detoxifying system) in the 8-day-old larvae as well as acetylcholinesterase (AChE) activity (involved in the nervous system) in 4-day-old embryos and 8-day-old larvae. Snout-to-vent length and snout-to-tail length of 4-day-old embryos and 8-day larvae were recorded as well as the corresponding growth rate. Fin and tail muscle widths were measured as well for testing changes in tail shape. Both tests showed effects of both organophosphates on AChE activity; however, no changes were observed in GST and EROD. Furthermore, exposure to chlorpyrifos demonstrated impacts on morphological and life-history traits, presaging alteration of locomotor traits. In addition, the results suggest a lower sensitivity to chlorpyrifos of 4-day-old embryos compared to 8-day-old larvae. Tests on other organophosphates are needed to test the validity of this method for the whole organophosphate group. Environ Toxicol Chem 2022;41:2688-2699. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

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Species referenced: Xenopus laevis
Genes referenced: ache
GO keywords: acetylcholinesterase activity [+]

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	Figure 1 Morphological endpoint measurements on embryos (top) and larvae (bottom). (A) Snout-to-vent length, (B) snout-to-tail length, (C) fin width, (D) muscle width.
	Figure 2 Acetylcholinesterase (A), ethoxyresorufin-O-deethylase (B), and glutathione-S-transferase (C) activities measured on 8-day-old larvae continuously exposed to six concentrations of diazinon (p = 0.01–0.05; p = 0.001–0.01; **p < 0.001). p values are adjusted with the Bonferroni-Holm method. The central bar of the boxplot is the group median. Upper and lower hinges correspond to the 25th and 75th quantiles, respectively. Upper and lower whiskers extend from the closest hinge, respectively, to the largest and the smallest values at most 1.5 times the interquartile range. Violins represent the smoothed histograms of the data distribution. Extreme outliers are not displayed in the graphs. AchE = acetylcholinesterase; DZN = diazinon; EROD = ethoxyresorufin-O-deethylase; GST = glutathione-S-transferase.
	Figure 3 Acetylcholinesterase measured on 4-day-old embryos (A) and 8-day-old larvae (B) and ethoxyresorufin-O-deethylase and glutathione-S-transferase activities measured on 8-day-old larvae (C,D) continuously exposed to six concentrations of chlorpyrifos (p = 0.01–0.05; p = 0.001–0.01; **p < 0.001). p values are adjusted with the Bonferroni-Holm method. Plates of origin are set as a random effect in statistical models. The central bar of the boxplot is the group median. Upper and lower hinges correspond to the 25th and 75th quantiles, respectively. Upper and lower whiskers extend from the closest hinge, respectively, to the largest and the smallest values at most 1.5 times the interquartile range. Violins represent the smoothed histograms of the data distribution. Extreme outliers are not displayed in the graphs. AchE = acetylcholinesterase; DMSO = dimethyl sulfoxide; CPF = chlorpyrifos; EROD = ethoxyresorufin-O-deethylase; GST = glutathione-S-transferase.
	Figure 4 Snout-to-tail to snout-to-vent length ratio measured on 4-day-old embryos (A) and 8-day-old larvae (B) and fin width to muscle width ratio (C) measured on 4-day-old embryos continuously exposed to six concentrations of chlorpyrifos (p = 0.01–0.05; p = 0.001–0.01; **p < 0.001). p values are adjusted with the Bonferroni-Holm method. Plates of origin are set as a random effect in statistical models. The central bar of the boxplot is the group median. Upper and lower hinges correspond to the 25th and 75th quantiles, respectively. Upper and lower whiskers extend from the closest hinge, respectively, to the largest and the smallest values at most 1.5 times the interquartile range. Violins represent the smoothed histogram of the data distribution. Extreme outliers are not displayed in the graphs. STL = snout-to-tail length; SVL = snout-to-vent length; DMSO = dimethyl sulfoxide; CPF = chlorpyrifos; FW = fin width; MW = muscle width.
	Figure 5 Snout-to-vent length measured on 4-day-old embryos (A) and 8-day-old larvae (B) continuously exposed to six concentrations of chlorpyrifos and the corresponding growth rate (C) (p = 0.01–0.05; p = 0.001–0.01; **p < 0.001). p values are adjusted with the Bonferroni- Holm method. Plates of origin are set as a random effect in statistical models. The central bar of the boxplot is the group median. Upper and lower hinges correspond to the 25th and 75th quantiles, respectively. Upper and lower whiskers extend from the closest hinge, respectively, to the largest and the smallest values at most 1.5 times the interquartile range. Violins represent the smoothed histogram of the data distribution. Extreme outliers are not displayed in the graphs. DMSO = dimethyl sulfoxide; CPF = chlorpyrifos.

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	Figure 1 Morphological endpoint measurements on embryos (top) and larvae (bottom). (A) Snout-to-vent length, (B) snout-to-tail length, (C) fin width, (D) muscle width.
	Figure 2 Acetylcholinesterase (A), ethoxyresorufin-O-deethylase (B), and glutathione-S-transferase (C) activities measured on 8-day-old larvae continuously exposed to six concentrations of diazinon (p = 0.01–0.05; p = 0.001–0.01; **p < 0.001). p values are adjusted with the Bonferroni-Holm method. The central bar of the boxplot is the group median. Upper and lower hinges correspond to the 25th and 75th quantiles, respectively. Upper and lower whiskers extend from the closest hinge, respectively, to the largest and the smallest values at most 1.5 times the interquartile range. Violins represent the smoothed histograms of the data distribution. Extreme outliers are not displayed in the graphs. AchE = acetylcholinesterase; DZN = diazinon; EROD = ethoxyresorufin-O-deethylase; GST = glutathione-S-transferase.
	Figure 3 Acetylcholinesterase measured on 4-day-old embryos (A) and 8-day-old larvae (B) and ethoxyresorufin-O-deethylase and glutathione-S-transferase activities measured on 8-day-old larvae (C,D) continuously exposed to six concentrations of chlorpyrifos (p = 0.01–0.05; p = 0.001–0.01; **p < 0.001). p values are adjusted with the Bonferroni-Holm method. Plates of origin are set as a random effect in statistical models. The central bar of the boxplot is the group median. Upper and lower hinges correspond to the 25th and 75th quantiles, respectively. Upper and lower whiskers extend from the closest hinge, respectively, to the largest and the smallest values at most 1.5 times the interquartile range. Violins represent the smoothed histograms of the data distribution. Extreme outliers are not displayed in the graphs. AchE = acetylcholinesterase; DMSO = dimethyl sulfoxide; CPF = chlorpyrifos; EROD = ethoxyresorufin-O-deethylase; GST = glutathione-S-transferase.
	Figure 4 Snout-to-tail to snout-to-vent length ratio measured on 4-day-old embryos (A) and 8-day-old larvae (B) and fin width to muscle width ratio (C) measured on 4-day-old embryos continuously exposed to six concentrations of chlorpyrifos (p = 0.01–0.05; p = 0.001–0.01; **p < 0.001). p values are adjusted with the Bonferroni-Holm method. Plates of origin are set as a random effect in statistical models. The central bar of the boxplot is the group median. Upper and lower hinges correspond to the 25th and 75th quantiles, respectively. Upper and lower whiskers extend from the closest hinge, respectively, to the largest and the smallest values at most 1.5 times the interquartile range. Violins represent the smoothed histogram of the data distribution. Extreme outliers are not displayed in the graphs. STL = snout-to-tail length; SVL = snout-to-vent length; DMSO = dimethyl sulfoxide; CPF = chlorpyrifos; FW = fin width; MW = muscle width.
	Figure 5 Snout-to-vent length measured on 4-day-old embryos (A) and 8-day-old larvae (B) continuously exposed to six concentrations of chlorpyrifos and the corresponding growth rate (C) (p = 0.01–0.05; p = 0.001–0.01; **p < 0.001). p values are adjusted with the Bonferroni- Holm method. Plates of origin are set as a random effect in statistical models. The central bar of the boxplot is the group median. Upper and lower hinges correspond to the 25th and 75th quantiles, respectively. Upper and lower whiskers extend from the closest hinge, respectively, to the largest and the smallest values at most 1.5 times the interquartile range. Violins represent the smoothed histogram of the data distribution. Extreme outliers are not displayed in the graphs. DMSO = dimethyl sulfoxide; CPF = chlorpyrifos.