Environ. Sci. Technol. 2005, 39, 98-102
Physiological Basis for Large Differences in Resistance to Nitrite among Freshwater and Freshwater-Acclimated Euryhaline Fishes JOSEPH R. TOMASSO, JR.* Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634 MARTIN GROSELL The Rosenstiel School of Marine & Atmospheric Sciences and the Marine & Freshwater Biomedical Sciences Center, University of Miami, Miami, Florida 33149
Uptake of environmental NO2- by most freshwater fishes occurs at the gills where NO2- is actively transported into the blood by the Cl- uptake pathway. Some freshwater fishes do not concentrate NO2- in their plasma, regardless of environmental NO2- exposure and exhibit a high degree of resistance to NO2-. Recent studies indicate that freshwater-adapted killifish (Fundulidae: Fundulus heteroclitus) and European eel (Anguillidae: Anguilla anguilla) have no or minimal Cl- uptake activity at the gills relative to most freshwater fishes; rather, Cl- requirements are met in other ways (probably dietary). We hypothesized that different rates of Cl- uptake by the gill may explain the observed differences in NO2- uptake and consequent toxicity among freshwater fishes. Cl- influx rates of channel catfish (Ictaluridae: Ictalurus punctatus), a species that concentrates NO2- in the plasma and is sensitive to NO2-, and bluegill (Centrarchidae: Lepomis macrochirus), a species that does not concentrate NO2- in the plasma and is resistant to NO2-, were determined over a range of environmental Cl- concentrations. Channel catfish actively transported chloride into the plasma (Km ) 155.6 ( 101.2 µmol/L Cl-; Jmax ) 414.9 ( 51.4 nmol/g/h; ( SEM). In contrast, bluegill exhibited no observable Cl- uptake. We placed our results and previously reported results in a phylogenetic context and concluded that differences in Cl- uptake mechanisms among groups of freshwater fishes may explain, in large part, the wide range of sensitivity to environmental NO2-. NO2- uptake determinations may also prove to be an easy screening method when studying the phylogenetic distribution and nature of Cl- uptake mechanisms in the gills of fishes.
Introduction NO2- is an intermediate product of the oxidation of ammonia to nitrate. Due to imbalances of nitrifying bacteria in aquatic systems receiving high inputs of ammonia, NO2- concentrations may reach toxic concentrations. This has been par* Corresponding author phone: (864)656-2809; fax: (864)656-0435; e-mail:
[email protected]. 98
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 1, 2005
ticularly evident worldwide in the rapidly-expanding aquaculture industry (1) where fish and invertebrates are typically reared at high densities and ammonia is introduced to the system by deamination of amino acids from high-protein feeds (2). Early studies (3-8) established that environmental NO2is actively transported by the Cl- uptake pathway of the gill into the plasma where it exceeds environmental concentrations and that NO2- uptake could be competitively excluded by increasing the environmental Cl-:NO2- ratio. Once in the fish, NO2- oxidizes the iron in hemoglobin yielding methemoglobin, which is not capable of reversible oxygen binding. Fish with high levels of methemoglobin suffer from a functional hypoxia, which may lead to death. While methemoglobinemia and the ensuing hypoxia are generally considered the primary mechanism of toxicity, many other effects of NO2- have been reported (9-12). The idea that NO2- enters freshwater fishes by way of the Cl- uptake pathway of the gill became complicated with reports that members of the family Centrarchidae and Moronidae do not concentrate NO2- in the plasma and that manipulating environmental Cl- levels has no effect on either plasma NO2-concentrations or toxicity (13-15). The investigators speculated that the Cl- uptake pathway in members of these families may be more discriminatory than in fishes that concentrate NO2-. Other investigators (16) suggested that low Cl- uptake rates at the gills could explain the insensitivity of some fishes to NO2-. It is generally accepted that Cl- transport across freshwater-fish gills occurs via apical anion exchange with HCO3providing cellular substrate. The anion exchanger is sensitive to stilbenes, and the availability of HCO3- is dependent on carbonic anhydrase activity, which catalyzes CO2 hydration (17, 18). Cl- presumably moves through basolateral Clchannels down its electrochemical gradient to the extracellular fluids. The energy required for the active uptake of Clis probably provided by the apical proton pump since inhibition of the H+ ATPase with bafilomycin potently inhibits Cl- uptake (18, 19). The proton pump and active Cl- uptake appear to be linked by the proton extrusion, which prevents the reversal of the carbonic anhydrase mediated reaction, allowing cellular HCO3- concentrations to build to sufficient levels to drive Cl- exchange. An additional functional consideration is the titration of the boundary layer HCO3by the extruded protons to shift the HCO3- gradient in favor of the anion-exchange process (20). In contrast to freshwater fishes, marine fishes utilize an intestinal chloride cotransport pathway rather than apical anion-exchange pathway at the gills. This cotransport system is critically important in maintaining water balance in marine fishes (21) and has also been implicated in NO2- uptake (22, 23). Recent studies on Cl- uptake pathways in freshwater fishes prompted a reconsideration of why some fishes concentrate NO2- and some do not. Freshwater-acclimated killifish (Fundulidae: Fundulus heteroclitus) and eels (Anguillidae: Anguilla anguilla and Anguilla rostrata) were found to have minimal chloride uptake activity at the gills relative to most freshwater fishes (24-29); rather, Cl- requirements are met in other ways (probably dietary). We hypothesized that different rates of Cl- uptake by the gill may explain the observed differences in NO2- uptake and consequent toxicity (1000-fold, based on median-lethal concentrations; 30) among freshwater fishes. In an attempt to test this hypothesis, we determined Cl- influx rates of channel catfish (Ictaluridae: Ictalurus punctatus), a species that concentrates NO2in the plasma and is sensitive to nitrite, and bluegill 10.1021/es048900t CCC: $30.25
2005 American Chemical Society Published on Web 11/17/2004
(Centrarchidae: Lepomis macrochirus), a species that does not concentrate nitrite in the plasma and is resistant to NO2(14). In addition, we also determined the effect of environmental NO2- on Cl- uptake rates in these species.
Materials and Methods Experimental Animals. Juvenile bluegill L. macrochirus (0.6 ( 0.26 g; mean ( SEM) and channel catfish I. punctatus (6.7 ( 0.26 g) were obtained from (Southland Fish Farms; Hopkins, SC). Fish were maintained in 100-L, aerated, glass aquaria supplied with 1 L min-1 of dechlorinated Virginia Key tap water at 22 °C and were fed flake feed (Tetra; Blacksburg, VA; protein g 46% as shrimp and soy meal) daily until 48 h prior to experimentation. Chloride Uptake Kinetics. Individual channel catfish were placed in flux chambers, which consisted of 100 mL polypropylene bottles filled with 80 mL of water (see below) and placed horizontally. Each bottle contained a port for obtaining water samples and was aerated through polyethylene tubing (PE50) to ensure oxygenation and mixing. Flux chambers contained a Cl--free medium of pH 7.7 consisting of 0.5 mM CaSO4, 0.15 mM MgSO4, 0.5 mM NaHCO3, and 0.05 mM KHCO3. Measurements of Cl- uptake kinetics were initiated 30 min after fish were placed in chambers by adding 0.5 µCi (18.5 kBq) 36Cl- to each flux chamber. After a 5 min equilibration period, two initial water samples (each of 1 mL) were obtained for measurements of Cl- concentration and 36Cl- radioactivity. At the end of the flux period, a second set of samples were obtained for measurements of Clconcentrations and radioactivity. The Cl- concentration in the flux medium was then adjusted by addition of NaCl from a concentrated stock solution. After a 5 min equilibration period, the above procedure was repeated. Measurements of Cl- uptake were performed at three Cl- concentrations on each individual fish. Flux periods ranged from 30 to 180 min with times being longer in the higher chloride concentrations. Two groups, each consisting of 10 fish, were exposed to a different set of three Cl- concentrations yielding a total of six Cl- concentrations with n ) 10 for each concentration. The corresponding experiments with bluegill were conducted in the same manner except, due to the smaller size, fish were fluxed in 10 mL of solution (same composition as above) in polypropylene beakers using 0.2 µCi (7.4 kBq) per beaker. Due to the smaller volume of flux medium, only 300 µL samples were obtained for measurements of Cl- concentration and 36Cl- radioactivity at the beginning and end of each flux period. Cl- Uptake in the Presence and Absence of NO2-. As predicted, the above experiments revealed that bluegill do not rely on active uptake of Cl- from the water, at least not at rates that can be determined by the above protocol. To determine these low rates of Cl- uptake in the bluegill and to assess effects of NO2- on Cl- uptake in both bluegill and channel catfish, additional experiments were performed. The measurements were based on appearance of isotope in the fish rather than on disappearance from the water since this technique is much more sensitive. Four groups of 5 channel catfish were placed in 4-L Nalgene beakers each containing 0.5 L of the above flux medium adjusted to contain 200 µM Cl- (as NaCl). Two beakers served as duplicate control treatments while the other two received nominal 1.0 mM NO2- (as NaNO2). Measured NO2- concentrations were 0.89 mM in the catfish experiment and 1.08 mM in the bluegill experiment. Flux measurements were initiated by addition of 2.2 µCi (81.4 kBq) L-1 of 36Cl-. Water samples for determination of Cl- and NO2- concentration as well as 36Cl- radioactivity were obtained 5 min after isotope addition and immediately before the end of the flux period. Uptake of Cl- in bluegill was assessed in the same manner in beakers containing 300 mL. In agreement
with a protocol previously employed to measure Cl- uptake in fish with high and very low Cl- uptake rates (rainbow trout and European eel, respectively; 27) different exposure periods were employed for the two species used in the present study. For the channel catfish, a 4 h flux period was used while a longer flux period of 24 h was employed for the bluegill. At the end of the flux period, fish were subjected to a “cold displacement rinse” in 100 mM NaCl for 1 min, after which they were killed by an overdose of buffered MS-222 (0.3 g/L). Subsequently, fish were transferred to individual vials to which was added 5 times the volume of 10% HNO3. Capped vials were digested overnight at 80 °C, vortexed thoroughly, and centrifuged to obtain a clear supernatant. An aliquot of the supernatant was prepared for scintillation counting of 36Cl- radioactivity as described below. Calculations and Analytical Techniques. Concentrations of Cl- and NO2- in all water samples were determined by anion chromatography (Dionex DX120; Dionex Corporation, Sunnyvale, CA). Detection limit for both anions was 5 µM. The 36Cl- radioactivity in water samples and digests were determined by liquid scintillation counting (TmAnalytic Beta Tract 6895). To test for quenching arising from the acidity and coloration of the fish digests, spike recovery tests were performed on five randomly selected samples. Recovery of 93% of spiked radioactivity was observed, and appropriate corrections were performed. Calculation of Cl- uptake (JCl-) in the Cl- kinetics experiments was based on disappearance of 36Cl- radioactivity and the specific activity of 36Cl- in the flux medium:
JCl- ) {(36Cl-tCPMinitial - 36Cl-tCPMfinal)/
([(36Cl-CPM/Cl-)initial + (36Cl-CPM/Cl-)final])/2}/(t/m)
where 36Cl-tCPMinitial and 36Cl-tCPMfinal are the total amount of radioactivity (counts per minute; CPM) present in the flux chamber at the beginning and at the end of a flux period, respectively; and ([(36Cl-CPM/Cl-)initial + (36Cl-CPM/Cl-)final])/ 2 is the mean specific activity (CPM nmol-1) of a given flux period, respectively. Finally, t and m represents flux time (h) and fish mass (g), respectively. Correction for isotope “back flux” from the fish to the flux medium was unnecessary as estimates of internal specific activity were less than 5% of the external specific activity (31). Cl- uptake rates (JCl-) in the presence and absence of NO2- in both channel catfish and bluegill were determined by appearance of 36Cl- radioactivity in the fish:
JCl- ) {(CPMdigest/[(36Cl-CPM/Cl-)initial +
(36Cl-CPM/Cl-)final]/2)}/(t/m)
where CPMdigest denotes the total amount of radioactivity present in the fish determined from an aliquot of the digest, [(36Cl-CPM/Cl-)initial + (36Cl-CPM/Cl-)final]/2 is the mean specific activity in the water, and t and m have the same meanings as above. Data Presentation and Statistical Evaluation. Transport kinetics constants for Cl- uptake in channel catfish were determined using the nonlinear regression Wizard in SigmaPlot 8.0 (Systat Software, Incorporated, Point Richmond, CA). Comparison of means from different experimental groups were performed by unpaired, two tailed students t-tests, and values were considered significantly different at P < 0.05.
Results and Discussion Channel catfish exhibited active Cl- uptake (Figure 1) that increased with increasing environmental Cl- concentrations until a maximum uptake rate was reached (Km ) 155.6 ( 101.2 µmol/L Cl-; Jmax ) 414.9 ( 51.4 nmol/g/h). A similar VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
99
As predicted, bluegill did not exhibit observable uptake of Cl- (Figure 1). What appears to be an increase in Cl- efflux at the two highest environmental Cl- concentrations is probably a result of surface bound 36Cl- being displaced by environmental 35Cl- due to the decreased ambient specific activity at higher Cl- concentrations (see Materials and Methods for details). Such displacement due to reduced specific activity at higher Cl- concentrations presumably would also have occurred in the experiment with catfish (although differences in binding rates between scaled and scaleless fishes could reasonably be expected). If such displacement occurred, the Jmax for channel catfish Cl- uptake would be underestimated. Regardess, the absence or very low rate of Cl- uptake activity observed in the bluegill is similar to that observed in European and American eel (2428). FIGURE 1. Rates of chloride influx as a function of environmental chloride concentrations in chanel catfish (Ictalurus punctatus) and bluegill (Lepomis macrochirus). Uptake kinetics (mean ( SEM) were not calculated for bluegill because no uptake was apparent. See text for details of exposure duration.
FIGURE 2. Effect of environmental nitrite on influx (mean ( SEM) of environmental chloride in channel catfish (Ictalurus punctatus) and bluegill (Lepomis macrochirus). See text for details of exposure conditions. uptake pattern has been observed in a number of other teleosts (18, 32-36).
Influx of Cl- was significantly inhibited in channel catfish when NO2- was added to the environment (Figure 2). Our observations are consistent with that of Williams and Eddy (16), who observed decreased Cl- influx after exposing rainbow trout (Oncorhyncus mykiss) and perch (Perca fluviatilis) to combinations of environmental chloride and NO2-. Presumably, NO2- is replacing Cl- in the Cl- uptake pathway. This assertion is supported by the recent observation that plasma NO2- concentrations increase and plasma Clconcentrations decrease in grass carp (Ctenopharyngodon idella) when exposed to NO2- (37). NO2- inhibition of the very-low or absent Cl- uptake rates in the bluegill was not observed (Figure 2). When the differences in Cl- uptake rates (Figure 1) and the effects of environmental NO2- on Cl- uptake rates (Figure 2) are considered together, it is apparent that the large differences in toxicity to NO2- (96-h median-lethal concentrations of 0.51 mmol for channel catfish and 5.70 mmol for bluegill; 14) are due to the differential uptake rates of both Cl- and NO2-, which are reflected in large differences in accumulation of NO2- in the plasma (14). Based on available information, freshwater and freshwater-acclimated euryhaline fishes can be divided into two groupssfishes that
FIGURE 3. Phylogeny, based on Nelson (41), of families of fishes for which information on nitrite uptake and/or chloride uptake at the gill are available. Within uptake columns, Y ) yes for uptake, N ) no uptake (no for chloride refers to uptake rates similar to bluegill, eel, and Fundulus; no for nitrite indicates that nitrite does not concentrate in plasma relative to environmental concentrations) based on superscripted references (8, 13-16, 18, 19, 26-28, 35-40). Chloride uptake information for Centrarchidae and Ictaluridae are presented in Figure 1. Nitrite uptake information for Fundulidae is unpublished (Tomasso and Grosell). Nitrite classifications for Anguillidae and Percidae are estimates based on published median-lethal concentrations. 100
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 1, 2005
concentrate environmental NO2- in the plasma and fishes that do not (Figure 3). For fishes where both Cl- and NO2uptake information is available, species that exhibit little or no Cl- uptake do not concentrate NO2- and species with higher Cl- uptake rates do concentrate NO2- (Figure 3). If our assertion is correct that NO2- uptake is a function of Cl- uptake rates on the gills of fishes, then NO2- uptake may provide a convenient screening method in Cl- uptake studies and may provide some insight into the evolution of ionoregulation in freshwater fishes. As summarized in Figure 3, representatives of five of seven orders of Superorder Neopterygii/Division Teleostei investigated by us or others did not concentrate NO2- in the plasma. Of these, representatives from three orders were studied and found to demonstrate little or no Cl- uptake across the gills. Within one order (Perciformes), representatives of three families concentrated NO2- and two families did not. Given that the uptake of Cl- (either from the diet or environment) is critical for freshwater fishes, the phylogenetically widespread and inconsistent absence of chloride uptake at the gill described here provides support for multiple parallel origins of freshwater osmoregulatory strategies and pathways as suggested by Marshall (20). An alternative explanation is that the minimal branchial chloride uptake rates exhibited by some groups of fishes are an adaptation to cope with some aspect of past or present environments. Our findings also point out the need to have an understanding of the physiological basis for nitrite uptake and toxicity, at least to the family level, when setting acceptable levels of nitrite for aquaculture operations or establishing site-specific criteria for public waters. Such an understanding can be related to the culture species or fish assemblage to ensure adequate protection.
(14) (15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
Acknowledgments This is technical contribution 5043 of the South Carolina Agricultural Experiment Station.
(26)
Literature Cited (1) Tomasso, J. R. Global aquaculture production with an emphasis on the United States. In Aquaculture and the Environment in the United States; Tomasso, J. R., Ed.; U.S. Aquaculture Society/ World Aquaculture Society: Baton Rouge, LA, 2002; pp 1-8. (2) Tomasso, J. R. Toxicity of nitrogenous wastes to aquaculture animals. Rev. Fish. Sci. 1994, 2, 291-314. (3) Crawford, R. E.; Allen, G. H. Seawater inhibition of nitrite toxicity to Chinook salmon. Trans. Am. Fish. Soc. 1977, 106, 105-109. (4) Perrone, S. J.; Meade, T. L. Protetive effect of chloride on nitrite toxicity to coho salmon (Oncorhynchus kisutch). J. Fish. Res. Board. Can. 1977, 34, 486-492. (5) Wedemeyer, G. A.; Yasutake, W. T. Prevention and treatment of nitrite toxicity in juvenile steelhead trout (Salmo gairdneri). J. Fish. Res. Board Can. 1978, 35, 822-827. (6) Tomasso, J. R.; Simco, B. A.; Davis, K. B. Chloride inhibition of nitrite-induced methemoglobinemia in channel catfish (Ictalurus punctatus). J. Fish. Res. Board Can. 1979, 36, 1141-1144. (7) Eddy, F. B.; Kunzlik, P. A.; Bath, R. N. Uptake and loss of nitrite from the blood of rainbow trout Salmo gairdneri Richardson, and the Atlantic salmon Salmo salar L. in fresh water and dilute sea water. J. Fish Biol. 1983, 23, 105-116. (8) Bath, R. N.; Eddy, F. B. Transport of nitrite across fish gills. J. Exp. Zool. 1980, 214, 119-121. (9) Mensi, P.; Arillo, A.; Margiocco, C.; Schenone, G. Lysosomal damage under nitrite intoxication in rainbow trout (Salmo gairdneri Rich.). Comp. Biochem. Physiol. 1982, 73C, 161-165. (10) Margiocco, C.; Arillo, A.; Mensi, P.; Schenone, G. Nitrite bioaccumulation in Salmo gairdneri Rich. and hematological consequences. Aquat. Toxicol. 1983, 3, 261-270. (11) Scarano, G.; Saroglia, M. G. Recovery of fish fro functional and hemolytic anemia after brief exposure to a lethal concentration of nitrite. Aquaculture 1984, 43, 421-426. (12) Jensen, F. B. Nitrite disrupts multiple physiological functions in aquatic animals. Comp. Biochem. Physiol. 2003, 135A, 9-24. (13) Palachek, R. M.; Tomasso, J. R. Toxicity of nitrite to channel catfish (Ictalurus punctatus), tilapia (Tilapia aurea), and large-
(27)
(28)
(29) (30)
(31)
(32)
(33)
(34)
(35)
(36)
mouth bass (Micropterus salmoides): evidence for a nitrite exclusion mechanism. Can. J. Fish. Aquat. Sci. 1984, 41, 17391744. Tomasso, J. R. Comparative toxicity of nitrite to freshwater fishes. Aquat. Toxicol. 1986, 8, 129-137. Mazik, P. M.; Hinman, M. L.; Winklemann, D. A.; Klaine, S. J.; Simco, B. A. Influence of nitrite and chloride concentrations on survival and hematological profiles of striped bass. Trans. Am. Fish. Soc. 1991, 120, 247-254. Williams, E. M.; Eddy, F. B. Chloride uptake in freshwater teleosts and its relationship to nitrite uptake and toxicity. J. Comp. Physiol. 1986, 156B, 867-872. Perry, S. F.; Payan, P.; Girard, J. P. The effects of perfusate HCO3and PCO2 on chloride uptake in perfused gills of rainbow trout. Can. J. Fish Aquat. Sci. 1984, 41, 1768-1773. Boisen, A. M. Z.; Amstrup, J.; Novak, I.; Grosell, M. Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio). Biochim. Biophys. Acta 2003, 1618, 207218. Fenwick, J. C.; Wendelaar Bonga, S. E. W.; Potts, W. T. W. In vivobafilomycin-sensitive Na+ uptake in young freshwater fish J. Exp. Biol. 1999, 202, 3659-3666. Marshall, W. S. Na+, Cl-, Ca2+ and Zn2+ transport by fish gills: retrospective review and prospective synthesis. J. Exp. Zool. 2002, 293, 264-283. Karnaky, K. J. Osmotic and ionic regulation. In The Physiology of Fishes, 2nd ed.; Evans, E. D., Ed; CRC Press: Boca Raton, FL, 1998; pp 157-176. Grosell, M.; Jensen, F. B. NO2- uptake and HCO3- excretion in the intestine of the European flounder (Platichthys flesus). J. Exp. Biol. 1999, 202, 2103-2110. Grosell, M.; Jensen, F. B. NO2- uptake and HCO3- excretion in the intestine of the European flounder (Platichthys flesus). Aquat. Toxicol. 2000, 50, 97-107. Kirsch, R. The kinetics of peripheral exchanges of water and electrolytes inthe silver eel (Anguilla anguilla L.) in fresh water and seawater. J. Exp. Biol. 1972, 57, 489-512. Bornancin, M.; DeRenzis, S.; Maetz, J. Branchial Cl- transport, anion stimulated ATPase, and acid-base balance in Anguilla anguilla adapted to freshwater: effects of hyperoxia. Comp. Physiol. 1977, 117, 313-322. Goss, G. G.; Perry, S. F. Different mechanisms of acid-base regulation inrainbow trout (Oncorhynchus mykiss) and American eel (Anguilla rostrata) during NaHCO3 infusion. Phys. Zool. 1994, 67, 381-406. Patrick, M. L.; Pa¨rt, P.; Marshall, W. S.; Wood, C. M. Characteristics of ionand acid-base transport in the freshwater adapted mummichog (Fundulus heteroclitus). J. Exp. Zool. 1997, 279, 208-219. Grosell, M.; Hogstrand, C.; Wood, C. M.; Hansen, H. J. M. A nose-to-nosecomparison of the physiological effects of exposure to ionic silver versus silver chloride in the European eel (Anguilla anguilla) and the rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 2000, 48, 327-342. Wood, C. M.; Laurent, P. Biochim. Biophys. Acta 2003, 1618, 106-119. Russo, R. C.; Thurston, R. V. Toxicity of ammonia, nitrite and nitrate to fishes. In Aquaculture and Water Quality; Brune, D. E., Tomasso, J. R., Eds; World Aquaculture Society: Baton Rouge, LA, 1991; pp 58-89. Maetz, J. Les e´changes de sodium chez le poisson Carassius auratus L. Action d’un inhibiteur de l’anhydrase carbonique. J. Physiol., Paris 1956, 48, 1085-1099. Krogh, A. Osmotic regulation in fresh water fishes by active absorption ofchloride ions. Z. Vergl. Physiol. 1937, 24, 565666. Maetz, J.; Romeu, F. G. The mechanism of sodium and chloride uptake bythe gills of a freshwater fish, Carrasius auratus. II. Evidence for NH4+/Na+ and HCO3-/Cl- exchangers. J. Gen. Physiol. 1964, 47, 1209-1227. Kerstetter, T. H.; Kirschner, L. B. Active chloride transport by the gills ofrainbow trout (Salmo gairdneri). J. Exp. Biol. 1972, 56, 263-272. De Renzis, G.; Maetz, J. Studies on the mechanism of chloride absorptionby the goldfish gill. Relation with acid-base regulation. J. Exp. Biol. 1973, 59, 339-358. De Renzis, G. The brachial chloride pump in the goldfish Carrasius auratus: relationship between Cl-/HCO3- and Cl-/ Cl- exchanges and the effects of thiocyanate. J. Exp. Biol. 1975, 63, 587-602.
VOL. 39, NO. 1, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
101
(37) Alcaraz, G.; Rangel, L. Early signs of nitrite toxicity in juvenile grass carp Ctenopharyngodon idella. J. World Aquacult. Soc. 2004, 35, 94-99. (38) Atwood, H. L.; Tomasso, J. R.; Smith, T. I. J. Nitrite toxicity to southernflounder Paralichthys lethostigma. J. World Aquacult. Soc. 2001, 32, 348-351. (39) Wise, D. J.; Tomasso, J. R. Acute toxicity of nitrite to red drum Sciaenops ocellatus: effect of salinity. J. World Aquat. Soc. 1989, 20, 193-198.
102
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 1, 2005
(40) Fontenot, Q. C.; Isely, J. J.; Tomasso, J. R. Characterization and inhibitionof nitrite uptake in shortnose sturgeon fingerlings. J. Aquat. Animal Health 1999, 11, 76-80. (41) Nelson, J. S. Fishes of the World, 3rd ed.; John Wiley & Sons: New York, 1994.
Received for review July 15, 2004. Revised manuscript received September 27, 2004. Accepted October 1, 2004. ES048900T
