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: huertas@porthos.bio.ub.es (M. Huertas).

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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
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