Developmental toxicity of dibutyl phthalate and citrate ester plasticizers in Xenopus laevis embryos

Citrate esters have been considered as alternatives to phthalate plasticizers. Being considered to have low toxicity in mammals, their toxicological information for aquatic animals remains poorly understood. We examined the developmental toxicity of citrate esters including tributyl O-acetylcitrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylcitrate (ATHC) together with dibutyl phthalate (DBP) based on the frog embryo teratogenesis assay-Xenopus (FETAX). ATBC has the lowest 96 h LC50 and 96 h EC50 values. In RT-qPCR, the ratio of bax and bcl-2 mRNA was significantly increased by DBP, but not by ATBC, ATEC and ATHC. DNA fragmentation was obvious in DBP-treated tadpoles, but not in those treated with ATBC and ATEC, whereas ATHC caused necrotic DNA degradation. Lipid hydroperoxide levels in tadpoles were significantly increased by DBP and ATHC, but not by ATBC and ATEC, suggesting that induction of oxidative stress by DBP and ATHC in embryos. In tadpoles with head abnormalities, basihyal bone, ceratohyal bone and Meckel's cartilage were frequently missed together with reduction in branchial gill bones. Col2a1 mRNA in the head of tadpoles was significantly decreased by low concentration of DBP, ATHC, and high concentration of ATEC. In stage 25 embryos FoxN3 mRNA, a master regulator for differentiation of neural crest cells to chondrocytes in head, was significantly decreased by DBP and ATHC, but not by ATBC and ATEC. In conclusion, ATEC was recommended as the alternative to phthalate plasticizer having the lowest developmental toxicity in amphibian embryos.