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Aquaculture 277 (2008) 192 – 196
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Ammonia and pH effects on some metabolic parameters and gill histology of
silver catfish, Rhamdia quelen (Heptapteridae)
Denise dos S. Miron a , Bibiana Moraes a , Alexssandro G. Becker b , Márcia Crestani a ,
Rosélia Spanevello a , Vania L. Loro a , Bernardo Baldisserotto b,⁎
b
a
Departamento de Química, Universidade Federal de Santa Maria, RS, Brazil
Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, 97105.900-Santa Maria, RS, Brazil
Received 5 September 2007; received in revised form 13 February 2008; accepted 15 February 2008
Abstract
The aim of the present study was to assess the effect of ammonia exposure at different pH on survivorship and metabolic parameters in the
liver, muscle and gill histology of silver catfish (Rhamdia quelen). The 96 h-LC50 of un-ionized ammonia (mg L− 1) at pH 6.0, 7.5 and 8.2 were:
0.44 (C.I. 0.38–0.49), 1.45 (C.I. 1.25–1.65) and 2.09 (C.I. 1.85–2.36), respectively. Survival of juveniles exposed to different ammonia levels was
altered by pH, and fish exposed to all ammonia levels and different pH showed muscle glucose, muscle and liver glycogen reduction. Liver
glucose and muscle and liver lactate levels increased in all fish exposed to ammonia as compared to the control. Exposure to waterborne ammonia
increased total ammonia levels in both tissues and also induced gill epithelium damages such as lamellar fusion and edema as compared with
controls at different pH. Silver catfish exposed to pH 6.0 and different NH3 levels presented significantly higher hepatic glucose and protein levels
when compared to those maintained at low NH3 levels. Juveniles exposed to NH3 levels at pH 7.5 and 8.2 showed lower hepatic protein levels
compared to those maintained at low NH3 levels. These parameters are indicative of pH dependence on ammonia toxicity in silver catfish. The
metabolic parameters and gill histology may be used as early indicators of ammonia toxicity in silver catfish.
© 2008 Elsevier B.V. All rights reserved.
Keywords: Silver catfish; Ammonia; pH; Metabolism; Gill histology; LC50
1. Introduction
Ammonia is a common aquatic pollutant and is toxic to fish. It
enters in natural aquatic systems through industrial and
agricultural wastes, and is also a natural product of nitrogenous
organic matter breakdown (Thurston and Russo, 1983). The
main nitrogenous compound excreted by fish is also ammonia,
which may reach toxic concentrations in high-density fish
culture, reducing growth and productivity (Tomasso et al., 1980;
Frances et al., 2000). The main source of endogenous ammonia
is direct deamination of amino acids that provide precursors for
both gluconeogenesis and lipogenesis in the fish liver (Le-Ruyet
et al., 1997). The toxicity of ammonia to aquatic organisms has
been attributed mainly to the un-ionized form (NH3), while the
⁎ Corresponding author. Tel.: +55 55 3220 9382; fax: +55 55 3220 8241.
E-mail address: bernardo@smail.ufsm.br (B. Baldisserotto).
0044-8486/$ - see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2008.02.023
ionized form (NH4+) is considered less toxic (Thurston et al.,
1984, Wood, 2001).
The pH plays an important role in homeostasis of aquatic
animals. Increase or decrease of pH is reported to cause
disturbances in acid–base balance, ion regulation and ammonia
excretion (Wood, 2001). Fish exposed to alkaline waters showed
increased plasma ammonia because a significant proportion of
excreted ammonia remains as NH3 in the water. In alkaline waters
the amount of H+ available to react with NH3 and produce NH4+ is
low, and in this condition the NH3 plasma–water gradient is
reduced, decreasing NH3 excretion and consequently accumulating in the plasma and tissues (Wilkie and Wood, 1996).
Studies have shown that ammonia exposure might lead to
histopathologic changes in gill and liver (Smith and Piper, 1975;
Thurston et al., 1984), and elevated ammonia and low pH induced
gill damage in juvenile brook trout (Salvelinus fontinalis)
(Mueller et al., 1991). However, some authors claim that acute
D.S. Miron et al. / Aquaculture 277 (2008) 192–196
193
0.300 mg L− 1 NH3) and 8.2 ± 0.1 (0.010 ± 0.0002, 0.8 ± 0.073, 2.0 ± 0.146, 2.3 ±
0.190, 2.4 ± 0.187 and 3.8 ± 0.263 mg L− 1 NH3). All experimental tests
including controls for each pH were made in triplicate. The pH was adjusted
to acidic or alkaline pH with 10% sulfuric acid or 10% sodium hydroxide,
respectively. NH3 levels were reached by adding concentrated NH4Cl solution.
All feces and residues were removed daily by suction, and consequently
approximately 20% of the water in the boxes was replaced by water with
previously adjusted pH and NH3 concentration. Mortality was determined at 12,
24, 48, 72, and 96 h after exposure to experimental conditions, and juveniles were
removed when immobile and respiratory movements ceased. The 96 h-LC50 for
NH3 were calculated by the methods of probits (Finney, 1971).
Fig. 1. 96 h-LC50 of un-ionized (NH3), ionized (NH+4 ) and total (NH3 + NH+4 )
ammonia for silver catfish juveniles exposed to different pH.
exposure to ammonia did not cause serious morphologic damage
to the gills (Wood, 2001). In addition, some freshwater fishes
exposed to high NH3 levels (1–4 mg L− 1) presented several
alterations on metabolic parameters (Shaffi, 1980).
The silver catfish, Rhamdia quelen (Quoy and Gaimard,
1824, Heptapteridae), occurs from Southern Mexico to Central
Argentina, and its husbandry is spreading in Southern Brazil
(Baldisserotto, 2004). Silver catfish was chosen for this toxicity
test due to its regional ecological and economic importance.
Several studies were done to determine best pH (Zaions and
Baldisserotto, 2000; Lopes et al., 2001; Copatti et al., 2005) and
water hardness (Townsend and Baldisserotto, 2001; Silva et al.,
2003, 2005; Townsend et al., 2003) for survival and growth, but
no studies regarding ammonia toxicity have been performed for
this species. Thus, the aim of this study was to verify the effects
of different ammonia and pH levels on some metabolic
parameters and gill histology, as possible indicators of toxicity
and to determine ammonia lethal concentration (96 h-LC50) for
silver catfish.
2. Materials and Methods
2.1. Fish
Silver catfish juveniles (11.04 ± 0.18 g and 11.07 ± 0.07 cm) were obtained
from a commercial fish farm near Santa Maria (Rio Grande do Sul, Brazil). Fish
were placed in continuously aerated 250 L tanks (100 juveniles per tank).
Temperature was maintained at 23–25 °C, pH 7.4, dissolved oxygen 7.2 mg L− 1,
water hardness 20 mg L− 1 CaCO3, maximum un-ionized ammonia level
0.007 mg L− 1, and maximum nitrite level 0.04 mg L− 1. Photoperiod was 12 h
light–12 h darkness. Fish were fed a total of 5% total biomass daily with
commercial feed (Purina, 45% crude protein) divided between two feedings
(08:30 and 17:30) for 7 days. Feeding was discontinued 24 h prior the beginning
of 96 h-LC50 test.
2.2. 96 h-LC50 tests
After acclimation, juveniles were randomly redistributed in continuously
aerated 40 L freshwater polyethylene boxes (10 fish per box). Silver catfish were
exposed to different NH3 levels at three different pH: 6.0 ± 0.1 (0.007 ± 0.0001,
0.25 ± 0.017, 0.4 ± 0.021, 0.5 ± 0.045, and 1.0 ± 0.071 mg L− 1 NH3), 7.5 ± 0.1
(0.008 ± 0.0001, 0.8 ± 0.074, 1.6 ± 0.180, 1.8 ± 0.231, 2.6 ± 0.312, and 3.2 ±
Fig. 2. Tissue glycogen and lactate of silver catfish exposed to different NH3
levels at (A) pH 6.0, (B) pH 7.5 and (C) pH 8.2. ⁎Indicate a significant
difference from control (0 mg L− 1 NH3) by one-way ANOVA and Tukey test
( p b 0.05) (n = 10).
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D.S. Miron et al. / Aquaculture 277 (2008) 192–196
Table 1
Metabolic parameters in liver and muscle tissue of silver catfish exposed to
water pH 6.0, 7.5 or 8.2 at different waterborne NH3 concentrations
pH 6.0
NH3 (mg L− 1)
0
Muscle
Glucose
Protein
Ammonia
Liver
Glucose
Protein
Ammonia
0.25
0.5
1.0
7.55 ± 0.74
97.0 ± 10.4
7.80 ± 2.75
4.30 ± 0.08⁎
107.0 ± 10.8⁎
8.40 ± 1.39⁎
4.40 ± 0.09⁎
127.0 ± 7.8⁎
9.54 ± 0.53⁎
4.27 ± 0.05⁎
133.0 ± 11.0⁎
14.70 ± 0.46⁎
25.67 ± 1.78
96.5 ± 8.6
28.50 ± 7.99
30.36 ± 2.14⁎
113.0 ± 6.3⁎
28.20 ± 3.70
32.50 ± 2.43⁎
112.3 ± 5.3⁎
36.00 ± 1.84⁎
34.60 ± 0.80⁎
111.0 ± 6.5⁎
44.00 ± 7.20
0
0.8
1.8
2.6
7.60 ± 0.72
103.5 ± 10.0
11.90 ± 1.27
4.42 ± 0.20⁎
131.7 ± 15.2⁎
11.31 ± 0.58
4.40 ± 0.07⁎
137.6 ± 15.3⁎
12.70 ± 0.84
4.37 ± 0.03⁎
142.0 ± 13.44⁎
27.70 ± 1.12⁎
15.80 ± 1.08
128.2 ± 14.2
20.00 ± 4.74
17.86 ± 4.50⁎
89.0 ± 10⁎
29.00 ± 1.30⁎
26.00 ± 4.40⁎
93.6 ± 9.4⁎
30.10 ± 2.15⁎
40.00 ± 4.28⁎
95.0 ± 8.0
30.10 ± 4.77⁎
0
2.0
2.3
2.4
pH 7.5
homogenized with 10% trichloroacetic acid using a motor-driven teflon pestle
and centrifuged at 1000 ×g for 10 min. Deproteinated supernatant was used for
the determination of lactate (Harrower and Brown, 1972), soluble sugar (Park
and Johnson, 1949) and ammonia (Boyd and Tucker, 1992).
The gills were removed and fixed in Bouin's fluid for 24 h, washed with
70% ethanol and dehydrated through a graded series of ethanol. They were
embedded in paraffin, sectioned at 5 μm thickness using a rotary microtome.
Sections were rehydrated in distilled water and stained with hematoxylin and
eosin. Morphological techniques were performed according to Karan et al.
(1998).
2.5. Statistical analysis
Homogeneity of the variances among the groups was verified with the
Levene test. Data of metabolic parameters exhibited homogeneous variances, so
comparisons among different NH3 treatments in the same pH were made by oneway ANOVA, followed by Tukey test (n = 10). All the tests were made with the
NH3 (mg L− 1)
Muscle
Glucose
Protein
Ammonia
Liver
Glucose
Protein
Ammonia
pH 8.2
NH3 (mg L− 1)
Muscle
Glucose
Protein
Ammonia
Liver
Glucose
Protein
Ammonia
8.50 ± 1.59
98.3 ± 3.0
8.20 ± 3.73
4.10 ± 0.04⁎
77.5 ± 7.02⁎
8.60 ± 1.20
4.28 ± 0.06⁎
68.7 ± 7.8⁎
9.28 ± 2.09⁎
4.10 ± 0.05⁎
70.3 ± 4.8⁎
11.20 ± 0.69⁎
27.86 ± 1.10
125.4 ± 5.4
12.68 ± 2.83
33.68 ± 1.46⁎
78.5 ± 3.7⁎
19.80 ± 3.67⁎
35.08 ± 0.91⁎
72.0 ± 7.6⁎
29.70 ± 1.24⁎
39.08 ± 1.80⁎
60.0 ± 9.9⁎
29.40 ± 2.93⁎
Glucose and ammonia expressed as μmol g tissue− 1 and protein as mg g
tissue− 1.
⁎Indicate a significant difference from 0 mg L− 1 by one-way ANOVA and
Tukey test ( p b 0.05) (n = 10).
2.3. Water sampling and analysis
Water quality was analyzed daily. Temperature (25.0± 0.5 °C) and dissolved
oxygen (7.35± 0.06 mg L− 1) were measured with an oxygen meter (YSI Y5512,
YSI Inc., Yellow Springs, Ohio, USA). Water hardness (20.0± 2.6 mg L− 1 CaCO3)
was analyzed by the EDTA titrimetric method, alkalinity (5.0± 2.3 mg L− 1 CaCO3
in the treatment at pH 6.0, and 60.0 ± 1.6 mg L− 1 CaCO3 at pH 7.5 and 8.2) and
nitrite (maximum level 0.04 ± 0.15 mg L− 1) according to Boyd and Tucker (1992).
The pH was measured with a DMPH-2 pH meter (Digimed, São Paulo, Brazil).
Total ammonia levels (NH3 + NH+4) were determined according to Boyd and Tucker
(1992) and NH3 was calculated as described by Piper et al. (1982). NH+4 levels were
calculated from the difference between total ammonia and NH3.
2.4. Biochemical and histological analysis
At the end of the exposure period (96 h), juveniles were sampled and tissues
(liver and muscle) were removed, frozen in liquid nitrogen and then stored at
− 20 °C. Glycogen was determined according to Bidinotto et al. (1997) and
tissue protein according to Lowry et al. (1951). Tissue samples were
Fig. 3. Gills of silver catfish exposed to (A) control (0 mg L− 1 NH3), (B) pH 6.0
and 0.5 mg L− 1 NH3, (C) pH 8.2 and 2.3 mg L− 1 NH3. HE, 400×.
D.S. Miron et al. / Aquaculture 277 (2008) 192–196
software Statistica (version 5.1). The minimum significance level used was 95%
( p b 0.05).
3. Results
NH3 96 h-LC50 at water pH 6.0, 7.5 and 8.2 were 0.44 (C.I. 0.38–
0.49 mg L− 1), 1.45 (C.I. 1.25–1.65 mg L− 1) and 2.09 mg L− 1 (C.I.
1.85–2.36 mg L− 1), respectively. Consequently, there was an increase
of NH3 96 h-LC50 with the increase of water pH. On the other hand,
NH+4 and NH3 + NH+4 96 h-LC50 of juveniles exposed to pH 7.5 and 8.2
were much lower than of those exposed to pH 6.0 (Fig. 1).
Survival and metabolic parameters in the liver and muscle of control
silver catfish were not changed by pH, but the effect of exposure to
different NH3 levels was altered by pH (Fig. 2 and Table 1). Fish exposed
to all NH3 levels showed significantly lower muscle glycogen and
glucose and hepatic glycogen, but higher hepatic glucose than control fish
maintained at the same pH (Fig. 2 and Table 1). Significantly higher
lactate levels were observed in the liver of juveniles exposed to all NH3
levels compared to control juveniles at the same pH. However, lactate
levels in the muscle were significantly higher only in those exposed to
0.25 mg L− 1 NH3 and pH 6.0, 0.8 and 2.6 mg L− 1 NH3 and pH 7.5 and all
NH3 levels at pH 8.2 compared to their respective controls at the same pH
(Fig. 2). Protein levels in the muscle were significantly higher in fish
exposed to all NH3 levels and pH 6.0 and 7.5, but significantly lower in
those maintained at pH 8.2 and exposed to all NH3 levels compared to
their respective controls at the same pH. Juveniles exposed to all NH3
levels presented significantly higher protein levels at pH 6.0, but lower at
pH 7.5 and 8.2 compared to their respective controls (Table 1). Exposure
to almost all NH3 levels significantly increased ammonia levels in both
tissues compared to their respective controls at the same pH (Table 1).
No significant changes on gill histology were observed among
controls at pH 6.0, 7.5 and 8.2 (Fig. 3A). Occurrence and severity of
gill tissue alterations were directly related to NH3 levels linked with
pH, i.e, these histological gill alterations were more evident at NH3
levels similar or higher than 96 h-LC50 at each pH. Fish maintained at
pH 6.0 and 0.5 mg L− 1 NH3 (or higher NH3 levels) showed hyperplasia
of gill lamellae resulting in fusion and necrosis (Fig. 3B). Fish exposed
to pH 7.5 and 1.8 mg L− 1 NH3 (or higher NH3 levels) (data not shown)
and to pH 8.2 and 2.3 mg L− 1 NH3 (or higher NH3 levels) presented a
40–60% increase of gill lamellae thickness (Fig. 3C).
4. Discussion
Ammonia toxicity and pH were directly related in this study.
The increase of pH led to an increase of NH3 96 h-LC50 and
decrease of NH4+ and NH3 + NH4+ 96 h-LC50 for silver catfish.
These results are in agreement with those obtained for rainbow
trout and fathead minnows (Thurston et al., 1981) but in channel
catfish (Ictalurus punctatus) only NH3 + NH4+ 96 h-LC50
decreased progressively with the increase of water pH (NH3
96 h-LC50 at pH 9.0 was higher than at pH 8.0) (Tomasso et al.,
1980). Values of NH3 96 h-LC50 for silver catfish at water pH
7.5 and 8.2 were similar to those determined by Tomasso et al.
(1980) for channel catfish at water pH 7.0 and 8.0 and water
hardness of 40 mg L− 1 CaCO3 (1.39 ± 0.06 and 1.82 ± 0.06,
respectively). However, NH3 96 h-LC50 of fathead minnows,
rainbow trout (Thurston et al., 1981) and cutthroat trout (Salmo
clarki) (Thurston et al., 1978) were lower in spite of using
higher water hardness (180–200 mg L− 1 CaCO3) in these
experiments. Consequently, silver catfish juveniles are rela-
195
tively resistant to acute waterborne ammonia exposure. Some
authors claim that safe NH3 levels for growth of a given species
are around 10% NH3 96 h-LC50 (see Tomasso, 1994), and
consequently safe NH3 levels for growth of silver catfish would
be 0.044, 0.14 and 0.21 mg L− 1 NH3 for pH 6.0, 7.5 and 8.2,
respectively. However, a review of the effect of NH3 on fish
growth revealed that the safe NH3 level for growth may be 3–
12% according to the species (Tomasso, 1994). Therefore, our
study indicates the NH3 levels to perform additional studies
regarding NH3 effect on silver catfish growth, but not the
precise safe level. In addition, as a slight pH reduction may be
used to reduce the fraction of NH3 and decrease water flow
requirements in flow-through systems (Eshchar et al., 2006), the
present study also demonstrated that would be interesting to
investigate the use of a slightly acidic pH to reduce NH3 effects
on silver catfish growth.
The reduction of hepatic and muscular glycogen in silver catfish
exposed to high NH3 levels at different pH may indicate that stress
generated by ammonia toxicity is accompanied by a rapid
degradation of tissue glycogen. In fact, carbohydrates stored in
liver and muscle are the first nutrients used in response to stress
conditions (Vijayavel et al., 2006). Lower glycogen levels in liver
and muscle were also observed in other freshwater fishes exposed
to high NH3 levels (1–4 mg L− 1) (Shaffi, 1980). The muscle lactate
elevation also indicated metabolic disorders and a clear response
against energy depletion generated by ammonia and pH exposure.
Fish white muscles constitute more than 50% of the whole
body mass, and anaerobic processes require glycogen for their
action (Knox et al., 1980). Probably in the exposure to elevated
NH3 levels a hypoxic condition was generated and fish developed
a preference for anaerobic glycogen breakdown. Elevated
concentrations of liver and muscle lactate suggest that this
metabolite can enhance rates of muscle glycolysis and in liver it
can be used as a substrate for gluconeogenesis. In accordance to
this hypothesis, higher levels of serum glucose and lactate were
observed in fish exposed to 1–4 mg L− 1 NH3 compared to
controls (Shaffi, 1980). Results concerning protein at pH 8.2 may
indicate a compensatory response to NH3 exposure: the high
energy demand caused by NH3 toxicity might have led to an
increase of protein catabolism following an increase of ammonia
levels in muscle and liver of silver catfish.
In fish the gill tissue is recognized as the major site of toxic
impact for many toxicants. In the present study, the major changes
in the gills of silver catfish exposed to ammonia levels above 96 hLC50 (mainly in those exposed to pH 6.0) were edema and fusion
of the secondary lamellae. All these lesions may reduce gill
functional surface for gaseous exchange, impairing respiratory
function. Gill damage was also observed after exposure of
rainbow trout to 0.01–0.07 mg L− 1 NH3 for months (Thurston
et al., 1984), cutthroat trout to 0.34 mg L− 1 NH3 for 29 days
(Thurston et al., 1978) and silver perch (Bidyanus bidyanus) to
0.36 mg L− 1 NH3 for 39 days (Frances et al., 2000).
It can be concluded that acute NH3 toxicity for silver catfish
is pH-dependent. In addition, exposure to high NH3 levels at
different pH indicated a preference for the anaerobic pathway to
energy production in muscle tissue. Ammonia toxicity induced
at different pH was confirmed by gill histology analysis.
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D.S. Miron et al. / Aquaculture 277 (2008) 192–196
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