Differential bioconcentration and depuration of diphenhydramine in Xenopus laevis tadpoles and frogs

Though bioconcentration of ionizable pharmaceuticals has received increased attention in fish and invertebrates, an understanding of bioconcentration of these contaminants of emerging concern by amphibians is lacking. Here, we investigated the bioconcentration, elimination and metabolism of a model weak base, the antihistamine diphenhydramine (DPH), at different developmental stages in the African clawed frog (Xenopus laevis), a well-established amphibian model. These developmental stages of X. laevis included tadpoles at pre-metamorphic stages (NF 49-52), tadpoles at pro-metamorphic and climax stages (NF 56-60) and newly metamorphosed frogs (NF 66). During metamorphosis from tadpoles to frogs, significant structural and functional changes occur in X. laevis, affecting respiration (gill resorption and lung development), feeding, digestion and skin structure. Animals were exposed to 10 μg/L DPH for 96 h, a concentration that did not affect mortality, development, growth, or behaviour, and then transferred to DPH-free water for 72 h. Relatively low steady-state bioconcentration factors (BCF_SS) of DPH were determined, ranging from 21 to 36; kinetic BCF (BCF_K) values ranged from 26 to 161. The depuration half-life (t_1/2) and 95 % loss in the depuration phase (t₉₅) of DPH were 19 and 20 h, and 83 and 85 h in pre-metamorphic and pro-metamorphic tadpoles, respectively. DPH had an approximately three times longer t_1/2 (66 h) and a t₉₅ (283 h) in juvenile frogs, suggesting differences in excretion and/or metabolism with age. Using non-targeted screening, DPH-N-oxide and DPH-N-desmethyl were identified at Schymanski level 2 as the exclusive DPH metabolites in all stages of X. laevis examined, indicating a major difference in the metabolism of DPH in frogs compared to other animals and humans. To our knowledge, this study provides initial observations for bioconcentration, elimination, and metabolism of an ionizable pharmaceutical in amphibians.