Stenohaline freshwater stingrays (Potamotrygon spp.) are endemic to the very dilute (Na+, Cl-, Ca2+ ≤ 30 μmol L-1), often acidic blackwaters of the Rio Negro despite gill Na+ and Cl- transport characteristics that appear unfavorable (high Km, low Jmax). We evaluated the possible protective role of blackwater itself, which is rich in dissolved organic carbon (DOC), as well as the importance of Ca2+ in allowing this tolerance of dilute, acidic conditions. Responses of stingrays in natural blackwater (DOC = 8.4 mg L-1) were compared with those in a natural reference water with similar ion levels but low DOC (0.6 mg L-1). Comparing these two water types, we found that differences in Na+ and Cl- unidirectional fluxes (JinX, JoutX; measured with radiotracers) and net fluxes (JnetX), influx and outflux kinetic relationships, and net ammonia excretion (JAmm) were generally small at pH 6.3, though the balance points where Jin = Jout shifted from >300 μmol L-1 in reference water (low DOC) to about 100 μmol L-1 in blackwater (high DOC). In reference water, both JinNa and JinCl were inhibited >90%, both JoutNa and JoutCl more than doubled, and JAmm did not change at pH 4.0. In blackwater, the inhibition of influxes was attenuated, the increases in outflux did not occur, and JAmm increased by 60% at pH 4.0. Addition of 100 μmol L-1 Ca2+ to reference water prevented the increases in JoutNa and JoutCl and allowed JAmm to increase at pH 4.0, which demonstrates that the gills are sensitive to Ca2+. However, addition of Ca2+ to blackwater had no effect on the responses to pH 4.0. Addition of commercial humic acid to reference water did not duplicate the effects of natural Rio Negro blackwater at the same DOC level; instead, it greatly exacerbated the increases in JoutNa and JoutCl at low pH and prevented any protective influence of added Ca2+. Thus, blackwater DOC appears to be very different from commercial humic acid. Biogeochemical modeling indicated that blackwater DOC prevents Ca2+ binding, but not H+ binding, to the gills and that the protective effects of blackwater cannot be attributed to its higher buffer capacity or its elevated Al or Fe levels. Natural DOC may act directly at the gills at low pH to exert a protective effect and, when doing so, may override any protective action of Ca2+ that might otherwise occur.
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