Soft-tissue mineralization of bullfrog larvae (Rana catesbeiana) at a wastewater treatment facility.

Bullfrog tadpoles (Rana catesbeiana) from a wastewater treatment facility were identified with severe lesions consisting of large, up to 1-cm in diameter, mineralized nodules protruding from the tail or gular region. Sectioning of formalin-fixed specimens revealed more extensive mineralization involving the vertebrae or muscles of the head and tail. Nodules examined microscopically were not associated with parasitic or infectious agents. Large nodules consisted of mineralized aggregates surrounded by a margin of granulomatous inflammation. Individual connective-tissue fibers and muscle cells were mineralized at some foci. The nodules consisted entirely of calcium phosphate, and the lesions appeared to be novel. Total serum-calcium concentrations of tadpoles and calcium concentrations in water samples did not differ significantly with increasing distance from the discharge site. Affected tadpoles had elevated cholecalciferol (25-OH-vitamin D3) levels. Effluent from this wastewater treatment facility is divided into 3 streams, each passing through a separate series of wetlands allowing for replicated evaluation of tadpoles with increasing distance from the proximate inputs of treated wastewater. The prevalence of lesions was correlated with proximity of cells to the initial wastewater discharge site, and 28.5% of bullfrog larvae in the first cells had lesions. None were affected in the fifth cells. Southern leopard frog larvae (Rana sphenocephala), the only other species affected, had a much lower prevalence of lesions (<1%) than bullfrog tadpoles and were only affected in the first cells. To date, the primary cause of elevated cholecalciferol is undetermined, but it appears to be remediated by passage of water through the wetlands.