Acute exposure of Siberian sturgeon (Acipenser baeri, Brandt) yearlings to nitrite: median-lethal concentration (LC50) determination ...
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Aquatic Toxicology 57 (2002) 257– 266 www.elsevier.com/locate/aquatox Acute exposure of Siberian sturgeon (Acipenser baeri, Brandt) yearlings to nitrite: median-lethal concentration (LC50) determination, haematological changes and nitrite accumulation in selected tissues M. Huertas a,*, E. Gisbert a, A. Rodrı́guez a, L. Cardona b, P. Williot c, F. Castelló-Orvay a a Laboratory Acuicultura, Department of Biologia Animal, Fac. Biologia, Uni6ersitat de Barcelona, A6. Diagonal, 645, E-08028 Barcelona, Spain b Department of Biology, I.U.S.C., C/. Fontanella, 19, E-08010 Barcelona, Spain c Cemagref, Group. Bordeaux, Unité de Ressources Aquatiques Continentales, 50 a6. de Verdum, 33611 Gazinet Cedex, France Received 9 November 2000; received in revised form 10 April 2001; accepted 17 May 2001 Abstract Exposure of Siberian sturgeon (Acipenser baeri ) yearlings (172.0 918 g; mean 9 S.D.) to several NO− 2 –N concentrations (0, 25, 130, 180 and 275 mg/l) was studied for 72 h in static tests. At 72 h, the median-lethal concentration of NO− 2 –N was 130 mg/l in water with high chloride content (130.5 mg/l). Nitrite exposure produced high levels of methaemoglobin (MetHb) but did not seem to cause mortality, as surviving fish showed higher levels (82.7 95.6%) than torpid specimens (60.8 94.5%). Levels of MetHb were unrelated to environmental and plasmatic nitrite concentrations, as both torpid and surviving fish exposed to the highest nitrite levels (275 mg/l of NO− 2 –N) presented similar concentrations of MetHb to those exposed to 25 mg/l of NO− 2 – N, thus indicating the ability of Siberian sturgeon yearlings to regulate plasmatic nitrite levels and maintain them lower than the environmental concentration of the toxicant. Nitrite exposure caused changes in the plasmatic electrolyte balance, which is characterised by extracellular hyperkalemia, high plasmatic chloride levels and low plasmatic sodium concentration. Differences between the nitrite concentration in the liver of torpid (46.3 9 9.0 mg/l) and surviving specimens (19.1 9 13.1 mg/l) exposed to several concentrations of NO− 2 – N suggest a significant contribution of the liver in nitrite detoxification pathways, and would thus explain a possible nitrite tolerance of Siberian sturgeon yearlings. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Nitrite acute toxicity; Methaemoglobinemia; Siberian sturgeon * Corresponding author. Tel.: + 34-934-021-447; fax: + 34-934-035-740. E-mail address: email@example.com (M. Huertas). 0166-445X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 0 1 ) 0 0 2 0 7 - 7
258 M. Huertas et al. / Aquatic Toxicology 57 (2002) 257–266 1. Introduction several haematological and tissular parameters in Siberian sturgeon (Acipenser baeri, Brandt), one Nitrite can reach high concentrations in recircu- of the most farmed acipenserid species in Western lating aquaculture systems in which high densities Europe (Williot et al., 1993). of fish are kept and bacteria transform ammonia, the main nitrogenous waste product of fishes, to nitrite (Palachek and Tomasso, 1984). Nitrite con- 2. Material and methods centrations can also be high in streams which receiving effluents from wastewater treatment 2.1. Fish acclimation plants because of imbalances among species of nitrifying bacteria (Scarano et al., 1984). In both Siberian sturgeon juveniles were obtained from cases, nitrite levels can become toxic and even low the CREA (Centre de Recherche Aquacole, concentrations of this compound are toxic for Cemagref, France) and transferred to the Labora- many fish species (Russo and Thurston, 1977; tory of Aquaculture (Facultat de Biologia, Uni- Tomasso, 1994). versitat de Barcelona, Spain) where they were Nitrite is actively taken up across the gills in acclimatised for 30 days in rectangular 500 l con- competition with chloride (Bath, 1980; Eddy and crete tanks (filled with 440 l) with dechlorinated Williams, 1987). Nitrite accumulation in blood tap water. During acclimation, fish were fed, ac- plasma causes haemoglobin (Hb) oxidation to cording to Williot et al. (1988), a dry commercial methaemoglobin (MetHb) and ensuing hypoxia diet containing 45% protein, 12% lipid, 10% ash (Tomasso, 1986), as MetHb does not bind and and 3.5% carbohydrate (Marina 3, ProAqua S.A.) transport molecular oxygen (Scarano et al., 1984). until 2 days before the beginning of the experi- However, nitrite-induced methaemoglobinemia ment. At the end of the acclimation period, fish does not seem to be the primary cause of death, body weight was 172.09 18 g (mean9 S.D.). since several studies have revealed that some spe- Water quality was as follows: [Cl−]= 130.5 mg/ cies can survive for long periods with low concen- l, [Ca2 + ]= 52 mg/l, [Na+]= 37 mg/l, [NH+ 4 ]= trations of functional Hb (Bath and Eddy, 1980; 0.4 mg/l, [HCO− 2− 3 ]= 141 mg/l, [SO4 ]= 117 mg/l, Colt et al., 1981; Palachek and Tomasso, 1984; [NO− 3 ]= 10 mg/l, pH=7.3. Water temperature Doblander and Lackner, 1997). was maintained at 18 °C and dissolved oxygen Recent studies have shown that the primary was 6.29 0.4 mg/l. Fish were exposed to a 12-h cause of nitrite toxicity may vary among fish light–dark photoperiod using overhead fluores- species (Doblander and Lackner, 1997). This is cent lights. not only due to a sole mechanism but to a combi- nation of several toxic mechanisms, such as a 2.2. Experimental procedure reduction in the functional Hb concentration in blood and the subsequent functional anaemia Siberian sturgeon yearlings were exposed to (Tomasso, 1986), hepatocyte degeneration as a four nitrite concentrations (0-control group, 25, consequence of liver hypoxia (Arillo et al., 1984a), 130, 180 and 275 mg/l of NO− 2 –N) for 72 h, and damage of lysosomal and microsomal membranes three replicate trials at each level were performed. in the liver (Mensi et al., 1982; Arillo et al., 1984b; One day before each trial, ten specimens were Doblander and Lackner, 1996), the formation of introduced into each rectangular tank (440 l), in DNA-damaging nitroso derivates (De Flora and which initial fish density was 1.8–2.2 kg/m3. At Arillo, 1983) and changes in plasma homeostasis, the beginning of each test series, water was no such as extracellular hyperkalemia (Doblander longer renewed and was aerated by compressed and Lackner, 1997; Knudsen and Jensen, 1997). air and mixed using an external pump. Nitrite The present study aimed to determine median- concentrations were obtained by adding NaNO2 lethal concentrations (LC50) of environmental ni- to dechlorinated tap water. Fish were not fed trite and assess the effects of acute exposure on during the experiment in order to reduce nitrogen
M. Huertas et al. / Aquatic Toxicology 57 (2002) 257–266 259 excretion and maintain water quality, especially Plasma and tissue nitrite levels were determined nitrite concentration. At the beginning and end according to Shechter et al. (1972). As nitrite is of each trial, nitrite levels were measured. In all not stable in an acidic environment, an alkaline cases, levels remained constant and did not dif- extraction in six parts of a zinc sulphate solution fer from the nominal value. Although no specific (4.31%) and five parts NaOH (0.8%) was neces- measurements were performed to evaluate NH+ 4 sary. The resultant solution was maintained at –N and NH3 –N levels in experimental tanks, 0 °C for 60 min and later centrifuged for 15 the excretion rates observed by Salin and Williot min at 1000 rpm. An aliquot of the resulting (1991a) suggest that NH+ supernatant was used for nitrite determination 4 – N concentration at the end of the experimental period should be by adding of sulphanilic acid and Cleve’s acid. lower than 0.42 mg/l and that of NH3 – N lower This produce a final red– violet product, which than 0.005 mg/l. These values are very much was compared spectrophotometrically at 520 nm using standard nitrite solutions, obtained by lower than the LC50 reported by Salin and adding NaNO2 to distilled water. Williot (1991b) and consequently, collateral am- monia toxicity effects on experimental fish are not expected. 2.4. Statistical analysis During the experiment, samples of blood and tissue were taken from two kinds of specimens: At 72 h LC50, the median-lethal concentration of NO− 2 –N for Siberian sturgeon juveniles was torpid fish, and those that survived for 72 h. calculated by means of sigmoidal regression (Sig- Torpid specimens were recognised by a dramatic maStat 2.0) using the following equation reduction in their ventilatory frequency and op- ercular movements, a loss of equilibrium, over- y= a/[1+ e − (x − x0/b)]. turning and dull colour. Previous assays (data here not shown) showed that torpid fish did not Data from distinct treatments were subjected survive after 72 h; therefore, this stage was con- to analysis of variance (Zar, 1988). When a sig- sidered the precursor of fish death. nificant difference was detected (PB 0.05), the Heparinised syringes were used to obtain ANOVA was followed by a Scheffe multiple- blood samples from the caudal vein (3 ml). range test to identify which treatments were sig- These samples were then immediately analysed. nificantly different. Data obtained from surviving and torpid fish exposed to the same The following haematological parameters were nitrite concentration were compared by means of measured: Hb, MetHb, haematocrit (Hc), nitrite the Student’s t-test (Zar, 1988). plasma concentration and Na+, Cl−, K+ and Ca2 + concentrations. Nitrite concentration in se- lected tissues (muscle, liver and gills) was also determined; tissue samples were kept at − 80 °C 3. Results until analysis to prevent nitrite oxidation. 3.1. Median-lethal concentration (LC50) 2.3. Chemical analysis At 72 h, the median-lethal concentration of NO− 2 –N for Siberian sturgeon juveniles was cal- The concentration of Hb, and percentage of culated using sigmoidal regression [x0 = 139.4 MetHb were determined with a Hemoximeter (31.3 S.D.); a= 99.5 (21.7 S.D.); b= 43.7 (27.7 OSM3. Hc was measured by centrifugation (2 S.D.); r= 0.99; PB0.01] was 130 mg/l (Fig. 1). min at 12 000 revs/min) in glass capillaries. Almost all the fish survived the 72-h exposure to Plasma Na+, Cl−, K+ and Ca2 + concentrations 0 and 25 mg/l NO− 2 –N, while 66 and 10% of were measured by atomic absorption spec- fish survived the 72-h exposure to 180 and 275 trophotometry (Radiometer mod. EML-100). mg/l NO− 2 –N, respectively.
260 M. Huertas et al. / Aquatic Toxicology 57 (2002) 257–266 between fish exposed to various NO− 2 –N concen- trations and those in control tanks (ANOVA; P\ 0.05), nor between torpid and surviving speci- mens exposed to the same NO− 2 –N concentration (Student’s t-test; P\ 0.05). Statistically significant differences were ob- served (ANOVA; PB 0.05) in the plasmatic Cl−, Na+ and K+ concentrations of fish exposed to distinct levels of NO− − 2 –N and those in NO2 –N free water (Fig. 3). Controls and torpid fish ex- posed to 25 mg/l of NO− 2 –N (ANOVA; P\0.05) Fig. 1. Mortality (%; mean 9 S.D.) of Siberian sturgeon year- showed no significant differences in Cl− concen- lings after 72 h exposure to several NO− 2 –N concentrations. tration, while higher NO− 2 –N concentrations, be- tween 130 and 275 mg/l of NO− 2 –N, caused a 3.2. Haematological parameters statistically significant median increase of 13.5% in plasmatic Cl− concentration (ANOVA; PB The percentage of MetHb increased signifi- 0.05). Statistically significant differences in the cantly (ANOVA; PB 0.05) in all fish exposed to Cl− concentration between controls and surviving different NO− 2 –N concentrations compared with fish exposed to different levels of NO− 2 –N controls, and reached concentrations of MetHb (ANOVA; PB 0.05) were detected. In all treat- comprised between 56.0 and 87.0% of the total ments, the plasmatic Cl− concentration of surviv- Hb (Fig. 2). After 72-h exposure, the percentage ing fish was higher than in torpid specimens of MetHb in surviving fish (82.7 9 5.6) was signifi- (11.0%) (Student’s t-test; PB 0.05). Na+ plas- cantly higher than in torpid specimens (60.89 4.5) matic levels showed a statistically significant de- (Student’s t-test; PB 0.05 in all treatments). crease of 11.5% in torpid and surviving fish No statistically significant differences were de- exposed to 25, 130, 180 and 275 mg/l of NO− 2 –N tected in Hc (%) and Hb concentration (mg/l) (ANOVA; PB0.05). No statistically significant Fig. 2. Hb concentration (mg/l), and percentage of Hc and MetHb (mean 9S.D.) of Siberian sturgeon yearlings exposed to several NO− 2 – N concentrations. Means not sharing a common letter are significantly different (ANOVA; PB 0.05). The asterisk denotes statistically significant differences between torpid and surviving fish exposed to the same concentration of NO− 2 – N (Student’s t-test, P B 0.05).
M. Huertas et al. / Aquatic Toxicology 57 (2002) 257–266 261 Fig. 3. Plasma ion concentration (mmol/l; mean 9 S.D.) in Siberian sturgeon yearlings exposed to several NO− 2 – N concentrations. Means not sharing a common letter are significantly different (ANOVA; PB0.05). The asterisk denotes statistically significant differences between torpid and surviving fish exposed to the same concentration of NO− 2 – N (Student’s t-test, PB 0.05). differences were detected between Na+ plasmatic exposed to 25, 130 and 180 mg/l of NO− 2 –N levels in torpid and surviving specimens (Student’s (54.09 15.3 mg/l) (ANOVA; PB 0.05). No sig- t-test; P\0.05). K+ plasmatic concentration sig- nificant differences were detected between plas- nificantly increased in torpid fish exposed to in- matic NO− 2 –N levels in torpid and surviving fish creasing NO− 2 –N concentrations (ANOVA; exposed to various levels of this toxic compound P B 0.05), while no significant differences were (Student’s t-test; P\ 0.05). detected between controls and those that survived Gills of fish exposed to 130, 180 and 275 mg/l at the end of the trial (ANOVA; P \0.05). of NO− 2 –N accumulated it at higher levels than No significant differences (ANOVA; P\0.05) specimens exposed to 25 mg/l of NO− 2 –N (76.09 in Ca2 + concentration were found amongst ni- 14.8 vs. 53.8913.9 mg/l in torpid fish and 70.79 trite-exposed fish and those kept in NO− 2 – N free 18.0 vs. 34.59 3.5 mg/l in surviving ones) water, nor between torpid and surviving fish ex- (ANOVA; PB 0.05). No significant differences posed to the same treatment (Student’s t-test; were found between gill NO− 2 –N concentrations P\ 0.05). in torpid and surviving specimens (Student’s t- test; P\ 0.05). 3.3. NO− 2 –N concentration in selected tissues Similarly, muscle accumulated NO− 2 –N, but to a lesser extent (26.199.5 mg/l). No statistical Siberian sturgeon yearlings exposed to NO− 2 –N significant differences were detected between fish accumulated this toxic substance in the four tis- exposed to several concentrations of NO− 2 –N sues studied: liver, gills, muscle and blood plasma (ANOVA; P\ 0.05), or between torpid and sur- (ANOVA; PB 0.05, Fig. 4). viving specimens (Student’s t-test; P\ 0.05). In fish exposed to 275 mg/l of NO− 2 – N, plas- In torpid and surviving fish, the hepatic NO−2 – matic concentrations of the toxicant were higher N concentrations of animals exposed to 25, 130, (195.2948.8 mg/l) than those recorded in fish 180 and 275 mg/l of NO− 2 –N were virtually iden-
262 M. Huertas et al. / Aquatic Toxicology 57 (2002) 257–266 tical (ANOVA; P\0.05), and averaged 43.39 (Tomasso, 1994). However, our results cannot be 13.4 and 16.2910.5 mg/l in torpid and surviving directly compared with those of these studies, specimens, respectively. When the NO− 2 – N con- because of differences in the experimental design centration in the liver of surviving specimens was and water quality used. Especially relevant is the compared with that of torpid fish, Student’s t-test concentration of chloride, since this compound showed that hepatic NO− 2 – N levels were statisti- has been described as an effective protector cally significant lower (37.4%) in surviving than in against nitrite toxicity due to the competitive inhi- torpid fish (P B 0.05). bition of chloride toward nitrite uptake through the gills (Scarano et al., 1984; Williams and Eddy, 1986; Tomasso, 1994). The present trial was con- 4. Discussion ducted using water with a high chloride concen- tration (130.5 mg/l), whereas most of mentioned At 72 h, the median-lethal concentration of studies used water with low chloride levels (B 1– NO− 20 mg/l). The inverse relationship between envi- 2 –N for Siberian sturgeon fingerlings was 130 mg/l, a value similar to that reported for Mi- ronmental chloride and nitrite toxicity suggests cropterus salmoides (Palachek and Tomasso, 1984) that under low-chloride water conditions, the tol- but much higher than values reported for other erance level of Siberian sturgeon juveniles to NO− 2 freshwater species, such as A. bre6irostrum –N is lower, although further studies are needed (Fontenot and Isely, 1998), Oncorhynchus mykiss to correlate both variables in this species. (Margiocco et al., 1983; Lewis and Morris, 1986), One of the major effects of nitrite exposure is Ictalurus punctatus, Tilapia aurea (Palachek and the oxidation of haem groups of Hb. The result- Tomasso, 1984), Cyprinus carpio (Lewis and Mor- ing MetHb cannot bind and transport oxygen; ris, 1986; Jensen, 1990) and Anguilla anguilla hence specimens with high levels of Fig. 4. NO−2 – N concentration (mg/l; mean 9S.D.) of blood plasma, gills, muscle and liver of Siberian sturgeon yearlings exposed to several NO− 2 – N concentrations. Means not sharing a common letter are significantly different (ANOVA; P B0.05). The asterisk denotes statistically significant differences between torpid and surviving fish exposed to the same concentration of NO− 2 –N (Student’s t-test, P B 0.05).
M. Huertas et al. / Aquatic Toxicology 57 (2002) 257–266 263 methaemoglobinemia suffer functional anaemia Tomasso, 1984), plasmatic nitrite levels were owing to a reduction of the oxygen-carrying ca- never higher than those in the environment, which pacity of the blood. This, in turn, results in tissue seems to indicate the existence of a mechanism of hypoxia (Eddy and Williams, 1987; Jensen, 1990; nitrite exclusion from plasma. Tomasso, 1994). As expected, the exposure of According to differences between the NO− 2 –N Siberian sturgeon yearlings to NO− 2 – N in the concentration in the liver of torpid and surviving range of 25–275 mg/l caused an increase in specimens exposed to different concentrations of MetHb levels. However, several studies (Smith the toxicant, it seems that the hepatocytes of and Williams, 1974; Brown and Mc Leay, 1975; Siberian sturgeon have a nitrite detoxification Tucker and Schwerder, 1983) have revealed that mechanism which removes this toxic compound nitrite-induced methaemoglobinemia and the sub- from the blood. This mechanism is probably simi- sequent functional anaemia is not the primary lar to that previously described by Doblander and cause of death. This is also true for Siberian Lackner (1996) in O. mykiss. These authors re- sturgeon yearlings, as surviving specimens showed ported that fish hepatocytes efficiently oxidise ni- higher levels of methaemoglobinemia than torpid trite to nitrate by two possible mechanisms, fish, which was mainly due to a longer exposure. namely the catalase and cytochrome oxidase In several teleost species such as I. punctatus, T. pathways. aurea, M. salmoides (Palachek and Tomasso, Another mechanism of nitrite detoxification has 1984), Lepomis cyanellus (Tomasso, 1986), O. been reported in D. labrax (Scarano et al., 1984) mykiss (Hofer and Gatumu, 1994) and Dicen- and O. mykiss (Cameron, 1971; Stormer et al., trarchus labrax (Scarano et al., 1984), it has been 1996; Doblander and Lackner, 1997), since these reported that the percentage of MetHb is closely species oxidise nitrite to nitrate by means of the correlated to the concentration of nitrite in water MetHb reductase system, which has been reported and blood. In contrast, our results show that in I. punctatus (Huey et al., 1982) and in mam- levels of MetHb are not related to environmental mals (Huennekens et al., 1968). According to or plasmatic nitrite concentrations, as torpid and Scarano et al. (1984), such a mechanism reduces surviving fish exposed to the highest nitrite level the half-life of the red blood cell, resulting in its (275 mg/l of NO− 2 – N) presented MetHb values destruction by the spleen and kidney similar to those of animals exposed to 25 mg/l of macrophages, thus causing haemolytic anaemia. NO− 2 –N (66.998.6% in torpid and 84.89 7.1% In Siberian sturgeon, exposure to environmental in surviving fish vs. 56.09 8.6% in torpid and nitrite did not lead a decrease in Hc and Hb 86.7911.6% in surviving fish). levels. This observation suggests that the adapta- Under present experimental conditions, the tive mechanism of nitrite detoxification did not NO− 2 –N concentration in the gills and plasma of take place, and would thus confirm the key role of fish exposed to 130, 180 and 275 mg/l of NO− 2 –N hepatic detoxification pathways in this primitive was lower than the concentration of the toxicant species. in the environment. These results suggest that Environmental nitrite also caused changes in Siberian sturgeon yearlings can regulate plasmatic the plasmatic electrolyte balance of Siberian stur- nitrite levels and keep them lower than those in geon juveniles. In agreement with other studies the environment. Indeed, in most of the species (Jensen, 1987, 1990, 1992; Stormer et al., 1996), studied to date, such as O. mykiss (Bath and extracellular hyperkalemia was one of the most Eddy, 1980; Margiocco et al., 1983; Vedel et al., prominent nitrite-induced effects detected. Ac- 1998), O. kisutch (Meade and Perrone, 1980), I. cording to Stormer et al. (1996), Knudsen and punctatus and T. aurea (Palachek and Tomasso, Jensen (1997), the large increase in plasmatic K+ 1984), nitrite is actively concentrated in blood and levels in O. mykiss and C. carpio resulted from a reaches higher concentrations than those regis- loss of K+ from both erythrocytes and skeletal tered in the environment. However, in A. baeri muscle, the latter being quantitatively greater due (present study) and M. salmoides (Palachek and to the larger total mass. Our results suggest that
264 M. Huertas et al. / Aquatic Toxicology 57 (2002) 257–266 in torpid specimens, exposure to nitrite also in- mental nitrite concentrations ranging from 130 to duced a K+ efflux from skeletal musculature and 275 mg/l. Nitrite-induced hypertrophy of other possible intracellular compartments rather branchial chloride cells and their subsequent hy- than from erythrocytes, since a K+ efflux from perfunctionality, as described in O. mykiss (Mar- erythrocytes would have resulted in a reduction of giocco et al., 1983; Gaino et al., 1984), together red blood cell volume and Hc (Jensen, 1992). The with the high-chloride water concentrations in the mechanism underlying the nitrite-induced K+ present study, would also explain such an increase efflux from skeletal muscle tissue is not known in plasmatic Cl− levels. Such hypertrophy and/or (Knudsen and Jensen, 1997). In our experimental a differential selectivity of chloride cells to nitrite conditions, the dramatic imbalance of extracellu- (Palachek and Tomasso, 1984) would increase the lar K+, which was only detected in torpid fish, physiological levels of chloride, despite NO− 2 indicates that surviving specimens were able to competition or the decrease in HCO− 3 production. control extracellular hyperkalemia by K+ re- However, further histological evidence is needed moval, probably across the gills and through renal to verify this hypothesis. excretion, thereby allowing the net efflux K+ In conclusion, methaemoglobinemia is unre- from the animal to keep pace with the efflux of lated to fish mortality in nitrite acute exposure K+ from intracellular to external compartments, tests. Although the present study was conducted as described in O. mykiss (Stormer et al., 1996). under high chloride water conditions and high We also observed a parallel decrease in plasmatic chloride levels may contribute to lower plasma Na+ levels in nitrite-exposed Siberian sturgeon; a nitrite levels than those observed in the environ- possible explanation could be a NO− 2 -induced ment, differences between torpid and surviving interference (e.g. inhibition) with the Na+ – K+ fish suggest that Siberian sturgeon yearlings have ATPase, resulting in tissue K+ efflux and Na+ a nitrite detoxification mechanism in the liver, uptake, as previously reported in C. carpio which removes this toxic compound from the (Jensen, 1987; Knudsen and Jensen, 1997). blood and maintains plasmatic nitrite levels lower In nitrite-exposed O. mykiss (Stormer et al., than those in the environment. Nitrite exposure 1996) and C. carpio (Jensen, 1987; Knudsen and also leads to ion imbalance, which is probably Jensen, 1997), nitrite uptake was associated with a related to tissue damage, the probable final cause severe decrease in plasmatic Cl− levels. The un- of death. derlying mechanisms for such changes in the chlo- ride levels rely on branchial ion exchange processes. In freshwater fish, active chloride up- Acknowledgements take is assumed to be primarily associated with the lamellar epithelium and involves coupled Cl−/ The authors thank M. Pelard, D. Mercier and HCO− T. Rouault (Cemagref) for rearing fish. This re- 3 exchange. The competitive inhibition of this mechanism by nitrite may explain nitrite ac- search was sponsored by an ABM/acs/ACI-98 cumulation in exposed specimens and the decrease grant from the Direcció General de Recerca (Gen- in plasmatic levels of Cl− (Jensen, 1987). In con- eralitat de Catalunya). trast to O. mykiss, a large increase in plasmatic chloride levels was detected in Siberian sturgeon. References According to Knudsen and Jensen (1997), in fish exposed to nitrite there is a concomitant loss of Arillo, A., Gaino, E., Margiocco, C., Mensi, P., Schenone, G., K+ and Cl− from skeletal musculature associated 1984a. Biochemical and ultrastructural effects of nitrite in with a K+/Cl− co-transport mechanism or a stim- rainbow trout: liver hypoxia as the root of the acute toxicity mechanism. Environ. Res. 34, 135 – 154. ulation of separate conductive K+ and Cl− chan- Arillo, A., Mensi, P., Pirozzi, G., 1984b. Nitrite binding to nels. This mechanism would partially explain the cytochrome P-450 from liver microsomes of trout (Salmo dramatic increase in plasmatic Cl− levels in gairdneri Rich.) and effects on two microsomal enzymes. Siberian sturgeon juveniles exposed to environ- Toxicol. Lett. 21, 369 – 374.
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