Environmental context determines nitrate toxicity in Southern toad (Bufo terrestris) tadpoles

Aquatic nitrate contamination has escalated over the past 50 years, primarily due to intensified fertilizer application and sewage production worldwide. Nitrate's role in the decline of amphibian populations remains unclear, although studies suggest that nitrate exposure affects larval development. We exposed Bufo terrestris tadpoles to environmentally relevant nitrate concentrations from Gosner stage 25 through forelimb emergence. Tadpoles were exposed to fluctuating (0-30 mg/L NO(3)-N, alternated three times per week) or constant nitrate concentrations (0, 5, 15, or 30 mg/L NO(3)-N), and effects were compared in two water types: natural spring water and reverse-osmosis filtered water, fortified with electrolytes (RO(e)). We measured growth and developmental rates, survival, time to metamorphosis, metamorph body size, hepatosomatic index, and whole-body thyroxine (T(4)) concentrations at forelimb emergence. Based on our observations, we reached three main conclusions: (1) in constant nitrate, tadpoles in RO(e) water grew faster, and were generally larger with higher or similar T(4) at metamorphosis than tadpoles raised in spring water, irrespective of nitrate concentration, (2) in fluctuating nitrate (flux), there were no differences in time to or size at metamorphosis in either water type relative to controls; however, mean T(4) concentrations in the flux treatment showed a reversed pattern in the two water types (SP>RO(e)) compared to the general pattern observed with constant nitrate exposure (RO(e)>SP), and (3) the effect of nitrate on growth and development depended on water type. In RO(e) water with high nitrate (RO(e)-30 mg/L NO(3)-N), tadpoles metamorphosed an average of 5 days (13%) earlier than control animals, but were similar in size to controls. However, spring water tadpoles reared in high nitrate (30 mg/L) delayed metamorphosis by 7 days (18%) compared to animals reared in SP-0, and 11 days (32%) compared to tadpoles raised in RO(e)-30. This delayed development allowed SP-30 tadpoles to reach a larger size that was more similar to metamorphs raised in RO(e) water. Based on information from other studies, we conclude that, in RO(e) water, tadpoles exhibited an expected stress response to nitrate (e.g. metamorphosed earlier as nitrate concentration increased). However, we suggest that, in spring water, tadpoles were exposed to additional stressors that decreased growth rate and thyroxine concentrations, and that this effect was modified by nitrate.