Journal of Trace Elements in Medicine and Biology 27 (2013) 70–75
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Journal of Trace Elements in Medicine and Biology
journal homepage: www.elsevier.de/jtemb
Toxicology
Short-term mercury exposure on Na+ /K+ -ATPase activity and ionoregulation in
gill and brain of an Indian major carp, Cirrhinus mrigala
Rama Krishnan Poopal a , Mathan Ramesh a,∗ , Bheeman Dinesh b
a
b
Unit of Toxicology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore 641046, Tamil Nadu, India
Department of Neuroscience, UNM School of Medicine, University of New Mexico, USA
a r t i c l e
i n f o
Article history:
Received 13 August 2011
Accepted 17 June 2012
Keywords:
Cirrhinus mrigala
Organs
Na+ /K+ -ATPase
Ionoregulation
Mercuric chloride
a b s t r a c t
Recently mercury pollution has been increased considerably in aquatic resources throughout the world
and it is a growing global concern. In this study, the 96 h LC50 value of waterborne mercuric chloride for
Cirrhinus mrigala was found to be 0.34 mg/L (with 95% confidence limits). Fingerlings of C. mrigala were
exposed to 0.068 and 0.034 mg/L of mercuric chloride for 96 h to assess the Na+ /K+ -ATPase activity and
ionoregulation (Na+ , K+ and Cl− ) in gill and brain. Results showed that Na+ /K+ -ATPase activity and ionic
levels (Na+ , K+ and Cl− ) in gill and brain of fish exposed to different concentrations of mercuric chloride
were found to be significantly (p < 0.05) decreased throughout the study period. Mercury inactivates many
enzymes by attaching to sulfur atoms in which the enzyme Na+ /K+ -ATPase is highly sensitive to mercury.
The inhibition of gill and brain Na+ /K+ -ATPase activity might have resulted from the physicochemical
alteration of the membrane due to mercury toxicity. Moreover, inhibition of Na+ /K+ -ATPase may affect the
ion transport and osmoregulatory function by blocking the transport of substances across the membrane
by active transport. The present study indicates that the alterations in these parameters can be used in
environmental biomonitoring of mercury contamination in aquatic ecosystem.
© 2012 Elsevier GmbH. All rights reserved.
Introduction
Metals are naturally occurring components that are ubiquitous
in the earth’s crust [1,2]; unfortunately it is considered to be a major
aquatic problem [3]. Aquatic contamination by heavy metals is a
major ecological and health concerns worldwide [4]. Among the
heavy metals, mercury (Hg) is one of the nonessential toxic heavy
metal and found everywhere in the earth [5,6]. Recently mercury
pollution has increased considerably and it is a growing global concern [7]. Mercury exists in the environment in several physical and
chemical forms like vapor elemental Hg (Hg◦ ), inorganic Hg (Hg+2 ),
and organic Hg (CH3 Hg) which are non-biodegradable in nature
[8,9]. Mercury enters the body of living organism in the form of
inorganic salts or organic or elemental mercury that has diverse
toxicological profiles at the cell, organism, and ecosystem level [10].
The atmospheric mercury can be transported from one place to
another place and may convert to methylmercury (CH3 Hg+ ) and
accumulated in the food chain causing a serious threat to human
health which results in neurological disorder and death [11–15].
Asian countries become the main contributor of atmospheric mercury (Hg), accounting half of the global emission [12].
∗ Corresponding author. Tel.: +91 422 2428493; fax: +91 422 2422387.
E-mail address: [email protected] (M. Ramesh).
0946-672X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.jtemb.2012.06.002
In aquatic environment, mercury is present in many physical
and chemical forms with a range of properties, consequently determining complex distribution, bioavailability and toxicity patterns
[16,17]. Normally, in freshwater environment, inorganic form of
mercury is present in large quantities [8,18]. Due to its high toxicity
and its widespread occurrence in the environment, its monitoring
has attracted special attention [17]. Hence the understanding of
toxicant uptake, behavior and responses in fish may perhaps, have
a high ecological significance. Fish is the top most organism of the
aquatic food web and are susceptible to waterborne mercury toxicity and most of the studies are assessed on central nervous system
(CNS) and olfactory organs [19]. To assess the possible disturbances
in the physiology of fish, suitable biomarkers are used to monitor
the environmental contamination of xenobiotics.
Enzymes are sensitive biochemical marker for metal contamination in aquatic ecosystem. In aquatic organisms particularly in fish
gills, the enzyme Na+ /K+ -ATPase is play a major role in the maintenance of ion balance [20] and its activity (increase or decrease)
prove to be an vital index for tolerable levels of environmental contaminants and also as a potential indicators of toxic stress [21–23].
Further, Na+ /K+ -ATPase activity can be used as an early warning
of pollutant, because the inhibition of this enzyme occur before
gross osmoregulatory dysfunction [24]. Brain is more vulnerable for
mercury poisoning, particularly for inorganic form, which disrupts
Na+ /K+ -ATPase system, ionoregulatory activities and neurological
R.K. Poopal et al. / Journal of Trace Elements in Medicine and Biology 27 (2013) 70–75
function [19,25,26]. The changes in the tissue or an organ or group
of organism are measured using various biomarkers, in which electrolytes are considered as a sensitive biomarker, because even a
low concentration of waterborne metals may disrupt ionoregulation [18,27–29]. The changes in the ionoregulation activity lead to
cardiovascular collapse and then ultimately death [30].
In India, approximately 200 t of mercury and its compounds are
released into the environment annually as effluents from various
industrial sectors [1,31]. Groundwater, fish and sediment samples
from different states like Punjab, Haryana, Mumbai, Maharashtra,
Tamilnadu, etc., showed surprisingly high levels of Hg [32–34]. To
our knowledge study on neuro-toxicological effects of mercury in
Indian major carps are very limited. Hence the present study has
been carried out to assess the acute toxicity of mercuric chloride
on gill and brain Na+ /K+ -ATPase activity and ionoregulation of an
Indian major carp Cirrhinus mrigala. The carp is endemic to IndoGangetic riverine systems, cultivated widely in Southeast Asian
countries. The carp was taken as a test species due to its commercial importance, taste and also a suitable indicator for monitoring
of environmental pollution.
Materials and methods
Fish and maintenance in the laboratory
Specimens of C. mrigala, was selected as an experimental animal model. Fish with an average weight of 8.0 ± 0.5 g and length of
6.0 ± 0.5 cm were purchased from Aliyar Fish Farm, Aliyar, Tamilnadu and India. Fish were safely brought to the laboratory in
well-packed aerated polythene bags. After arriving to laboratory,
fish were stocked in a large cement tank (1000 L capacity) for a
minimum period of 25 days. During acclimation period fish were
fed ad libitum with rice bran and ground nut oil cake in dough
form once in day before replacement of water. Three forth of the
water was changed daily to remove excess feed and fecal materials. Dechlorinated tap water was used throughout the study period,
with the following hydrological features such as; temperature
26.2 ± 1.5 ◦ C, pH 7.1 ± 0.05, salinity 0.27 ± 0.7 ppt, dissolved oxygen 6.6 ± 0.04 mg/L and total hardness 17.1 ± 0.8 mg/L. Before the
commencement of the experiment, healthy fingerlings of C. mrigala
were transferred to clean glass aquarium tanks (200 L capacity) and
that served as the stock for the experimental schedule.
Toxicity assessment of 96 h LC50 value
Preliminary toxicity tests were carried out to determine the
median lethal tolerance limit of fish C. mrigala to mercuric chloride for 96 h. Separate circular plastic water tubs (50 L) were taken
and different concentrations of mercuric chloride such as 0.05, 0.1,
0.2, 0.3, 0.4, 0.5 mg/L were added. Then 10 healthy fish from the
stock were randomly collected and introduced into each tub, which
were starved for a period of 48 h prior to the experiment. To each
concentration three replicates were maintained. Simultaneously, a
control group (toxicant free) was also maintained in three different
aquaria under identical conditions. The mortality/survival of fish in
control and mercuric chloride tubs were recorded after 96 h. The
median lethal concentration for 96 h was found to be 0.34 mg/L,
which was calculated by probit analysis method of Finney [35] and
homogenicity of the population was tested using chi-square test of
Busvine [36]. The dead fish in the tank were removed immediately.
Short term toxicity studies
For acute toxicity study, six tubs with 50 L of capacity were taken
and divided into two groups with three tubs in each. One group of
71
tubs were received 1/5th (0.068 mg/L) value of 96 h LC50 of mercuric chloride (Treatment I) and the other group were received
1/10th (0.034 mg/L) value of 96 h LC50 of mercuric chloride (Treatment II). To each tub 15 fish from the stock were introduced. A
control was also maintained with similar setup. After 96 h, fish
from the Control, Treatment I and Treatment II were randomly collected and organs (gill and brain) were removed for the estimation
of Na+ /K+ -ATPase activity and ionoregulation (Na+ , K+ and Cl− ).
Sample preparation
Fish were thoroughly washed with double distilled water and
dehydrated with absorbent paper. 100 mg of gill and brain were
removed from the Control, Treatment I and Treatment II groups and
homogenized with 1.0 mL of 0.1 M Tris–HCl buffer (pH 7.5) in icecold condition. The homogenate was centrifuged at 1000 rpm for
15 min at −4 ◦ C, then, the supernatant was used for the estimation
of Na+ /K+ -ATPase activity and ionoregulation (Na+ , K+ and Cl− ).
Assessment of Na+ /K+ -ATPase activity
To determine the Na+ /K+ -ATPase activity, 100 mg of gill and
brain tissue from Control, Treatments I and II were collected and
homogenized with 1.0 mL of 0.1 M Tris–HCl buffer (pH 7.4) in icecold condition using a Teflon homogenizer and the contents were
centrifuged at 1000 rpm at 4 ◦ C for 15 min. The supernatant was
used for the estimation of Na+ /K+ -ATPase activity [37] and the values were expressed as ␮g/h/g.
Estimation of ionoregulation
Estimation of sodium and chloride. Sodium and chloride level in gill
and brain was estimated following the method of Maruna [38].
To determine the sodium level 0.01 mL of sample from Control,
Treatments I and II was taken in a test tube and to this 1.0 mL of
precipitating reagent was added. To the tube marked as standard,
0.01 mL of standard reagent was added. All the test tubes were
mixed well and allowed to stand at room temperature for 5 min.
Then the contents were centrifuged at 2000–3000 rpm for 2 min to
obtain a clear supernatant. To 0.02 mL of the supernatant 1.00 mL of
color reagent was added mixed well and allowed to stand at room
temperature for 5 min. A standard tube was also used with 0.01 mL
of standard reagent. The optical density of the Control, Standard,
Treatments I and II were measured against distilled water using
UV Spectrophotometer at 530 nm within 10 min and readings were
expressed as mmol/L.
To determine the chloride level, 10 mL of supernatant from Control, Treatments I and II were taken in a test tube and to this
1000 ␮L of thiocyanate reagent was added. Similarly, 10 mL of distilled water was taken in a test tube and marked as blank. For
standard, 10 mL of standard chloride reagent was added to the tube
marked as standard. Then the contents of the tubes were mixed well
and kept for 10 min at room temperature and the optical density
of Control, Standard, Treatments I and II were measured against
‘Blank’ using UV Spectrophotometer at 505 nm and the readings
were expressed as mmol/L.
Estimation of potassium. Potassium level in gill and brain were estimated the following the method of Young et al. [39] and Tietz
[40]. 1.0 mL of boron reagent was taken in a test rube and to this
0.05 mL of supernatant from Control, Standard, Treatments I and II
were added. To the test tube marked as standard 0.05 mL of potassium standard was added. Then all the tubes were mixed well and
allowed to stand for 10 min at room temperature. After 10 min the
absorbance of samples were measured against distilled water using
UV Spectrophotometer at 620 nm and the readings were expressed
as mmol/L.
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R.K. Poopal et al. / Journal of Trace Elements in Medicine and Biology 27 (2013) 70–75
Na+ /K+ -ATPase activity was also decreased significantly (p < 0.05),
in both the treatments when compare to control groups (Fig. 2).
A maximum percent decrease of 61.61% and 52.49% was noted in
Treatment I and II at the end of 96 h, respectively. In both the treatments a maximum percent decrease in Na+ /K+ -ATPase activity was
noted in gill when compared to brain.
Effect of mercuric chloride on ionic levels in gill and brain
Sodium, potassium and chloride levels in gill and brain of fish
exposed to Treatment I (0.068 mg/L) and Treatment II (0.034 mg/L)
for 96 h were significantly (p < 0.05) lower in relation to the respective control groups (Fig.3–8). A maximum percent decrease was
noted at the end of 96 h in both the treatments. Among the tissue
studied a maximum percent decrease in sodium, potassium and
chloride levels were noted in gill.
Discussion
Figs. 1–2. Inhibition of Na+ /K+ -ATPase activity in C. mrigala exposed to different concentrations of mercury for 96 h. Treatment I (0.068 mg/L) and Treatment
II (0.034 mg/L). (1) Gill Na+ /K+ -ATPase level (␮g/h/g), (2) brain of Na+ /K+ -ATPase
level (␮g/h/g). Values are mean ± S.E. of five individual observations. *Significant at
p < 0.05 (based on t-test).
Statistical analysis
The significance at p < 0.05 level between the value of Control,
Treatment I and Treatment II were analyzed by Student’s t-test and
the 96 h LC50 value of mercuric chloride with 95% confidence was
calculated by probit analysis method.
Results
Toxicity and preliminary changes in fish
During the acclimation period there was no mortality or visible
disease in experimental and control fish. When the fish C. mrigala
exposed to different concentrations of mercuric chloride showed
behavioral changes, such as, erratic swimming, restlessness, mucus
secretion, convulsions, and mislaid balance. The observed behavioral changes were found to be dose dependent showing maximum
changes in higher concentration of mercuric chloride. The 96 h
LC50 value of mercuric chloride was found as 0.34 mg/L. For the
present study, we selected 1/5th of 96 h LC50 value (0.068 mg/L) as
Treatment I and 1/10th value (0.034 mg/L) as Treatment II. Fish population used in the present examination was found as homogenous
based on Chi-square test.
Effect of mercuric chloride on gill and brain Na+ /K+ -ATPase
activity
Fig. 1 provides the Na+ /K+ -ATPase activity in gill of fish C.
mrigala exposed to mercuric chloride (0.068 mg/L – Treatment
I and 0.034 mg/L – Treatment II) for a period of 96 h. In both
the treatments there was a significant (p < 0.05) inhibition of gill
Na+ /K+ -ATPase activity as compared to the control groups. A maximum percent inhibition of 84.86% and 65.06% was noted in
Treatment I and II at the end of 96 h, respectively. Similarly, brain
Among the mercury fractions, inorganic mercury can be easily
transported in the environment and has harmful effects on aquatic
organisms [41]. In toxicological study, the LC50 (ppm or mg/L−1 )
concentration is used to evaluate the toxicity of the chemical; the
chemical is considering to be highly toxic at a concentration lesser
than 1 mg/L−1 . In the present study, the median lethal concentration of mercuric chloride to the fish C. mrigala for 96 h was found to
be 0.34 mg/L, indicating that mercuric chloride is highly toxic. The
observed LC 50 value is more or less equal to previously reported
LC 50 value of mercury toxicity to many fish species; 0.314 mg/L−1
in Channa marulius [42], 0.3 mg/L−1 in Heteropneustes fossilis [43]
and 0.35 mg/L−1 Channa punctatus [44]. The variation in LC50 value
may be depends on many factors such as species, age, sex, size of
fish, and water chemistry [45]. Heavy metals which are in dissolved
form in water are easily entered into the body of fish via active or
passive processes and may retain in the body [46]. We conclude
that the high toxicity of mercuric chloride to the fish C. mrigala
might have resulted from the uptake of dissolved mercuric chloride and its accumulation in the body may interact or bind with the
SH groups.
The binding of mercury with SH groups or S S bridges is mostly
associated with the availability of molecular form of mercury, location and their physico chemical properties [47]. The toxicity of the
mercury also depends on the molecular interactions that occur at
critical nucleophilic sites in and around target cells; the interaction that occurs between mercuric ions and thiol group of proteins,
peptides and amino acids [9] and this may cause cellular oxidative stress. Further the used concentration of mercuric chloride
might have damaged the structure of the gill or gill process which
may be responsible for the mortality of fish during acute treatment. In the present investigation, during 96 h acute treatment of
mercuric chloride, the fish C. mrigala showed behavioral changes
such as erratic movement, sluggish and settlement at the bottom.
According to Scott and Sloman [48] metabolic, neurological, sensorial interruption of the pollutants could change the behavior of the
fish.
Recently, to assess the polluted water bodies and the health
of aquatic organism’s biomarkers are widely used as early warning signals or diagnostic tools [49,50]. Cell organelles and enzymes
are sensitive to metal toxicity [51]. ATPase is a group of enzymes
that are highly sensitive to heavy metal toxicity and has an
important role in intracellular functions [52]. Na+ /K+ -ATPase is a
membrane bound enzyme located on the basolateral membranes
of the ion-transporting cells of the branchial epithelium and play
an important role in the active transport of ions [53]. In the present
study, Na+ /K+ -ATPase activity in gill and brain was inhibited in
R.K. Poopal et al. / Journal of Trace Elements in Medicine and Biology 27 (2013) 70–75
73
Figs. 3–8. Alterations in ionic levels (Na+ , K+ and Cl− ) of C. mrigala exposed to different concentrations of mercury for 96 h. Treatment I (0.068 mg/L) and Treatment II
(0.034 mg/L), (3) gill sodium level (mmol/L), (4) brain sodium level (mmol/L), (5) gill potassium level (mmol/L), (6) brain potassium level (mmol/L), (7) gill chloride level
(mmol/L), (8) brain chloride level (mmol/L). Values are mean ± S.E. of five individual observations. *Significant at p < 0.05 (based on t-test).
both the treatments. Similar result was also noted in the brain of
C. punctatus [54] and Notopterus notopterus [52]. In teleost fish,
gill plays an important role on ion regulation, osmoregulation,
gas exchange, acid–base balance and nitrogenous waste excretion and also likely to be a site action of heavy metals [28,46].
Many authors have reported severe gill damage of fish exposed
to water-borne organic and inorganic mercury [55–57]. Stagg et al.
[24] reported that branchial Na+ /K+ -ATPase activity was lower in
flounder Platichtys flesus collected from contaminated areas when
compared to less contaminated areas and noted a strong negative
correlation between mercury concentration and Na+ /K+ -ATPase
activity.
In the present study the inhibition of gill Na+ /K+ -ATPase activity might have resulted from the direct toxicity of inorganic
mercury on ATPase function. Mercury inhibits Na+ /K+ -ATPase
activity, which acts as a key enzyme for neurological function
in fish [44,52,58]. In the present study the inhibition of Na+ /K+ ATPase activity in brain might have resulted from binding of
mercuric chloride to sulfhydryl groups with similar affinity [59].
Moreover, alteration of the physicochemical properties of the
membrane due to mercuric toxicity may form another reason for
the observed decrease of Na+ /K+ -ATPase activity in brain. Inhibition
of Na+ /K+ -ATPase activity might have resulted from an increase in
neurotransmitter due to depolarization of nerve cells caused by
ionic imbalance [60]. The alteration of the enzyme activity or function due to metal exposure might have resulted from the binding of
the metal with sulfydryl groups which may cause conformational
changes and prevent substrate binding [46]. Xenobiotics can alter
the activity of the enzyme Na+ /K+ -ATPase through the disruption of
energy producing metallic pathways or direct interaction with the
enzyme [61]. However, the degree of inhibition of Na+ /K+ -ATPase
activity may vary from one species to another species indicating
that the exposure duration, dose, ecological conditions and the
toxicant play an important role in the inhibition of this enzyme.
Freshwater fish are hyper osmotic to their medium and maintain
their regular physiological process and body fluid homeostasis with
the help of ion/osmoregulatory processes [62]. Inorganic ions, Na+ ,
K+ , and Cl− are distributed throughout the body fluids and utilized
for the normal functioning of tissue, neuromuscular irritability,
coenzyme, acid base balance constancy of cell volume and osmotic
74
R.K. Poopal et al. / Journal of Trace Elements in Medicine and Biology 27 (2013) 70–75
pressure [63]. In the present investigation the levels of electrolytes
(Na+ , K+ , and Cl− ) are found to be declined in fish throughout the
exposure period. Aquatic pollutants may alter the permeability of
gill membrane resulting alterations in iono regulation or osmoregulatory dysfunction [29]. The waterborne metals are well known for
their ionic inhibition [18,53,64]. Fish exposed to both Hg (II) and
MeHg caused an increase in gill permeability to water and results a
change in the gill ion permeability; this may lead to lower plasma
ion levels by increasing diffusive ion losses and water uptake by
osmosis [65]. Moreover, Hg accumulation in gills also disrupts ion
regulation in fish and other organisms [24,66,67]. In the present
investigation the decreased level of sodium, potassium and chloride in gill tissue might have resulted from accumulation and toxic
effect of mercuric chloride in the gill surface leading to lesser intake
of these ions into the body or efflux of the same to the exterior. Generally, Na+ /K+ -ATPase was very sensitive to heavy metals. In vivo
and in vitro studies of mercurial compounds have significantly
inhibited the Na+ /K+ -ATPase activity in brain tissues [68]. Acute
effects of inorganic Hg toxicity results in decreasing of Na+ and
K+ levels in the organ, due to the derangement in respiration system and suppressed activities of prominent energy linked enzyme
(Na+ /K+ -ATPase) [18,55]. Further toxicant induced renal dysfunction may also leads to a significant decrease in ionic levels [63]. In
the present investigation, the decrease in ionic levels in brain of
C. mrigala exposed to acute concentration of mercury might have
resulted from suppressed activities of Na+ /K+ -ATPase. Further, mercuric chloride may disturb the ionic regulation and osmoregulation
leading to a decrease in ionic levels in brain.
Conclusion
In the present study mercuric chloride at acute concentrations
caused significant inhibition of Na+ /K+ -ATPase in gill and brain of
fish C. mrigala which in results alterations in ionic levels in these
organs/tissue. The changes in Na+ /K+ -ATPase activity and alterations in ionoregulation may be used as a biomarker for metal
pollution in aquatic environment. Since, fish is a chief and major
source of protein to people of India, the discharge of mercurial compounds into nearby aquatic bodies to be controlled and remediation
techniques should be introduced to remove mercury pollution from
freshwater ecosystem.
Conflict of interest statement
There is no conflict of interest.
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