12 Protection by Natural Blackwater against Disturbances in Ion Fluxes Caused by Low pH Exposure in Freshwater Stingrays Endemic to the Rio Negro Chris M. Wood 1,2, * Aline Y. O. Matsuo 1,2 Rod W. Wilson 1,3 R. J. Gonzalez 1,4 Marjorie L. Patrick 1,5 Richard C. Playle 6 Adalberto Luis Val 1 1 Laboratory of Ecophysiology and Molecular Evolution, National Institute for Amazon Research, Alameda Cosme Ferreira, 1756, 69 083-000 Manaus, Amazonas, Brazil; 2 Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada; 3 School of Biological Sciences, Hatherly Laboratories, University of Exeter, Exeter EX4 4PS, United Kingdom; 4 Department of Biology, University of San Diego, 5998 Alcala Park, San Diego, California 92110; 5 Department of Cell Biology and Neuroscience, University of California, Riverside, California 92521; 6 Department of Biology, Wilfrid Laurier University, Waterloo, Ontario N2L 3C5, Canada Accepted 11/19/02 ABSTRACT Stenohaline freshwater stingrays (Potamotrygon spp.) are en- demic to the very dilute (Na + , Cl - , mmol L -1 ), often 2+ Ca ≤ 30 acidic blackwaters of the Rio Negro despite gill Na + and Cl - transport characteristics that appear unfavorable (high K m , low J max ). We evaluated the possible protective role of blackwater itself, which is rich in dissolved organic carbon (DOC), as well as the importance of Ca 2+ in allowing this tolerance of dilute, acidic conditions. Responses of stingrays in natural blackwater ( mg L -1 ) were compared with those in a natural DOC p 8.4 reference water with similar ion levels but low DOC (0.6 mg L -1 ). Comparing these two water types, we found that differ- ences in Na + and Cl - unidirectional fluxes ( , ; measured X X J J in out with radiotracers) and net fluxes ( ), influx and outflux ki- X J net *Corresponding author. Address for correspondence: Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Can- ada; e-mail: woodcm@mcmail.cis.mcmaster.ca. Physiological and Biochemical Zoology 76(1):12–27. 2003.  2003 by The University of Chicago. All rights reserved. 1522-2152/2003/7601-2101$15.00 netic relationships, and net ammonia excretion (J Amm ) were generally small at pH 6.3, though the balance points where shifted from 1300 mmol L -1 in reference water (low J p J in out DOC) to about 100 mmol L -1 in blackwater (high DOC). In reference water, both and were inhibited 190%, both Na Cl J J in in and more than doubled, and J Amm did not change at pH Na Cl J J out out 4.0. In blackwater, the inhibition of influxes was attenuated, the increases in outflux did not occur, and J Amm increased by 60% at pH 4.0. Addition of 100 mmol L -1 Ca 2+ to reference water prevented the increases in and and allowed J Amm Na Cl J J out out to increase at pH 4.0, which demonstrates that the gills are sensitive to Ca 2+ . However, addition of Ca 2+ 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 and at low pH Na Cl J J out out and prevented any protective influence of added Ca 2+ . Thus, blackwater DOC appears to be very different from commercial humic acid. Biogeochemical modeling indicated that blackwater DOC prevents Ca 2+ binding, but not H + binding, to the gills and that the protective effects of blackwater cannot be attrib- uted 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 pro- tective action of Ca 2+ that might otherwise occur. Introduction The ion-poor acidic blackwaters of the Rio Negro and its trib- utaries, characterized as “slightly contaminated distilled water” by Sioli (1968, p. 271), support a surprising diversity of fish life that is thought to represent about 8% of the world’s ich- thyofauna (reviewed by Val and Almeida-Val 1995). These blackwaters are so called because of their dark tea color, which is caused by a high content of dissolved organic carbon (DOC) comprising humic, fulvic, and other organic acids derived from the breakdown of jungle vegetation (Leenheer 1980; Sioli 1984; Ertel et al. 1986; Kuchler et al. 1994). In addition to the many teleost families with representatives endemic to blackwaters (Gery 1969; Fink and Fink 1979; Val and Almeida-Val 1995), one family of elasmobranchs, the Potamotrygonidae, has suc- cessfully colonized this extreme habitat. These freshwater sting-