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.

References [+] :

Ankley, Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment. 2010, Pubmed

Ankley, Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment. 2010, Pubmed
Ballew, Fitness Consequences of Boldness in Juvenile and Adult Largemouth Bass. 2017, Pubmed
Barinaga, Where have all the froggies gone? 1990, Pubmed
Bonifacio, Integrated ecotoxicological assessment of the complex interactions between chlorpyrifos and glyphosate on a non-target species Cnesterodon decemmaculatus (Jenyns, 1842). 2020, Pubmed
Bosisio, Developmental toxicity, uptake and distribution of sodium chromate assayed by frog embryo teratogenesis assay-Xenopus(FETAX). 2009, Pubmed , Xenbase
Bredeweg, The integrative effects of behavior and morphology on amphibian movement. 2019, Pubmed
Burke, Ethoxyresorufin: direct fluorimetric assay of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. 1974, Pubmed
Collins, Amphibian decline and extinction: what we know and what we need to learn. 2010, Pubmed
Colombo, Exposure to the organophosphorus pesticide chlorpyrifos inhibits acetylcholinesterase activity and affects muscular integrity in Xenopus laevis larvae. 2005, Pubmed , Xenbase
Derbalah, Temporal trends in organophosphorus pesticides use and concentrations in river water in Japan, and risk assessment. 2019, Pubmed
ELLMAN, A new and rapid colorimetric determination of acetylcholinesterase activity. 1961, Pubmed
Edginton, Comparative effects of pH and Vision herbicide on two life stages of four anuran amphibian species. 2004, Pubmed , Xenbase
Ford, The Role of Behavioral Ecotoxicology in Environmental Protection. 2021, Pubmed
Güngördü, Evaluation of in vitro and in vivo toxic effects of newly synthesized benzimidazole-based organophosphorus compounds. 2013, Pubmed , Xenbase
Habig, The identity of glutathione S-transferase B with ligandin, a major binding protein of liver. 1974, Pubmed
Hutchinson, Acute and chronic effects of carrier solvents in aquatic organisms: a critical review. 2006, Pubmed
Laetz, The synergistic toxicity of pesticide mixtures: implications for risk assessment and the conservation of endangered Pacific salmon. 2009, Pubmed
Ockleford, Scientific Opinion on the state of the science on pesticide risk assessment for amphibians and reptiles. 2018, Pubmed
Peltzer, Biotoxicity of diclofenac on two larval amphibians: Assessment of development, growth, cardiac function and rhythm, behavior and antioxidant system. 2019, Pubmed
Richards, Biochemical effects of chlorpyrifos on two developmental stages of Xenopus laevis. 2002, Pubmed , Xenbase
Richards, Physical effects of chlorpyrifos on two stages of xenopus laevis. 2003, Pubmed , Xenbase
Sandin, Spatial and temporal patterns of pesticide concentrations in streamflow, drainage and runoff in a small Swedish agricultural catchment. 2018, Pubmed
Sneddon, Considering aspects of the 3Rs principles within experimental animal biology. 2017, Pubmed
Ubaid Ur Rahman, A comprehensive review on chlorpyrifos toxicity with special reference to endocrine disruption: Evidence of mechanisms, exposures and mitigation strategies. 2021, Pubmed
Van Buskirk, Influence of tail shape on tadpole swimming performance. 2000, Pubmed
Venturino, Biomarkers of effect in toads and frogs. 2003, Pubmed
Wang, Increasing the reliability and reproducibility of aquatic ecotoxicology: Learn lessons from aquaculture research. 2018, Pubmed
Wijesinghe, Chlorpyrifos-induced toxicity in Duttaphrynus melanostictus (Schneider 1799) larvae. 2011, Pubmed
Xuereb, Acetylcholinesterase activity in Gammarus fossarum (Crustacea Amphipoda): linking AChE inhibition and behavioural alteration. 2009, Pubmed
Yu, Lethal and sublethal effects of three insecticides on two developmental stages of Xenopus laevis and comparison with other amphibians. 2013, Pubmed , Xenbase
Yu, Effects of chlorothalonil on development and growth of amphibian embryos and larvae. 2013, Pubmed , Xenbase
Ávila-Díaz, Chlorpyrifos and Dimethoate in Water and Sediments of Agricultural Drainage Ditches in Northern Sinaloa, Mexico. 2021, Pubmed

	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.