Toxicological Effects Of Fluoride On The Growth And Development Of Chinese Giant Salamander And Chinese Wood Frog

Fluorine is a necessary trace element for animals. Fluoride at suitable dose can promote the development of animals, while excessive fluoride intake may result in toxic effects to animals, such as dental and skeleton fluorosis, damages of liver, kidney, thyroid structure and function. Excessive fluoride can inhibit enzyme activity, ultimately interrupting normal physiological processes. Currently, fluoride concentration in aquatic ecosystems is rising as a consequence of anthropogenic activities, including industrial discharges, release of sewage, pesticides use. High fluoride concentration in polluted waters was measured at 49 mg/L, in extreme cases even up to 96.8 mg/L.In spite of the fact that fluoride must be considered as a serious pollutant, relatively little is known about fluoride toxicity to amphibian. Bufo gargarizan and Rana chensinensis are wide amphibian species distributed in China, the embryos and larvae are particularly susceptible to toxins. Therefore, these two species has been used as model animals for exploring the toxic effects of fluoride in amphibian growth and development.In our study, B. gargarizans larvae were exposed to 0.68,4.63,21.42,43.16, and 61.26 mg/L F" from G26 (developing hind-limb buds) to G46:(completed metamorphosis). R. chensinensis embryos at G7 were also randomly assigned to different F- concentrations. The toxic effects of fluoride on the B. gargarizan and R. chensinensis were investigated using statistical, morphological, histopathological and molecular biology methods. Our main research results are as follows:1. For B. gargarizan, after a long-term fluoride exposure, mortalities of groups exposed to controls,0.68,4.63,21.42, and 43.16 mg/L F" was 2.50%,2.50%,3.33%, 11.67% or 34.17%, respectively. And exposure to 61.26 mg/L F- even caused 100% mortality within 21 days. In addition, the mortality decreased sharply, at the earlier exposure stage then declined following and reached relatively steady after 30 days exposure. Since young larvae are more susceptible to fluoride than older larvae and adults. The rapid increase of mortality might reflect the initial shock stage of fluoride acclimation, whereas, the relatively steady state of mortality rate could respond to the increased tolerance of larvae after a long-term fluoride exposure.2. On 20 days, exposure to 43.16 mg/L F- significantly decreased the body mass, snout-vent length and total body length of B. gargarizans larvae compared with controls. This decrease persisted on 40 days at 43.16 mg/L F-. Exposure to 43.16 mg/L F- also significantly inhibited the development of B. gargarizans larvae. All above results indicated that high fluoride exposure affected the growth and development of B. gargarizans larvae.3. B. gargarizans larvae exposure to 43.16 mg/L F- failed to reach G46 at the same time node. Double-stained method was used to investigate the effects of fluoride on skeleton development of larvae at G46. Results showed that skeleton development was enhanced in 4.63 mg/L F", while skeleton development was inhibited in 21.42 mg/L F".4. For B. gargarizan at G42, metamorphosis percent of larvae was 50% in controls, while only 17.78% could be observed in 43.16 mg/L F-. Meanwhile, metamorphosis percent of larvae exposed to 0.68,4.63, and 21.42 mg/L F- was 42.22%,41.11% and 26.67%, respectively. The results demonstrated that high dose of fluoride may retard the metamorphosis of B. gargarizan larvae.5. For B. gargarizan at stage G42:histological analysis was conducted to access the thyroid gland lesions. Morphological alterations of thyroid were observed in 43.16 mg/L F-, including distinct loosely-arranged irregular follicular epithelial cells and partial colloid depletion in follicles. Moreover, RT-qPCR was utilized to evaluate the endocrine-disrupting effects caused by fluoride. Results showed that exposure to 21.42 and 43.16 mg/L F" significantly down-regulated thyroid endocrine relevant genes (Dio2, Dio3, TRa and TRβ) expression among liver, tail and hind-limb of B. gargarizan larvae.6. For R. chensinensis:embryos malformation occurred in all fluoride treatments sucha as axial flexures, edemas, ruffled dorsal and ventral fin. Additionally,19.62 and 42.41 mg/L F- could significantly inhibit embryos growth and development.7. For R. chensinensis:on 15 days, total body length and body mass of larvae were significantly decreased in 19.62 and 42.41 mg/L F-; on 30 days significant reductions in total body length and body mass were observed only in 42.41 mg/L F-. Moreover, significant metamorphic delay was found in larvae exposed to 42.41 mg/L F".In conclusion, high dose of fluoride can increase mortality and incidence deformities, delay growth and development, cause thyroid lesions, and down-regulate relevant genes expression of thyroid endocrine system in B. gargarizan and R. chensinensis, respectively. We suggested that the toxicity of fluoride was correlated with thyroid endocrine-disrupting effects in amphibian. Our results provided certain references for further research.