Effects of sublethal concentrations of potassium permanganate on African catfish

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International Journal of Integrative Biology

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ISSN 0973-8363

Effects of sublethal concentrations of potassium permanganate on

nitrogenous waste products of African catfish:
Clarias gariepinus (Burchell, 1822)
Kori-Siakpere Ovie *
Department of Animal and Environmental Biology, Delta State University, Abraka, Nigeria
Submitted: 8 Aug. 2008; Revised: 27 Oct. 2008; Accepted: 27 Nov. 2008

Abstract
Changes in the nitrogenous waste product of the fish Clarias gariepinus (Burchell, 1822) subjected to sublethal
concentrations (2.0; 6.0and 10.0 mg/L) of potassium permanganate over a period of 192 hours were studied in a
semistatic (renewal) system. The nitrogenous waste metabolism is reflected in the changes in total plasma
bilirubin, plasma uric acid, plasma creatinine and plasma urea. Slight rise in plasma level of total bilirubin
suggest a metabolic disturbance in liver involving defective conjugation and/or excretion of bilirubin; increased
creatinine and uric acid concentration in the blood suggest the inability of the kidney to excrete these products, a
manifestation of nephritic damage; decreased plasma urea may be attributable to the depleted plasma protein
levels.
Keywords: Potassium permanganate, nitrogenous waste, plasma bilirubin, plasma uric acid, plasma creatinine.

INTRODUCTION
Biochemical and physiological biomarkers are
frequently used for detecting or diagnosing sublethal
effects in fish exposed to different toxic substances (De
La Torre et al., 1998). Sublethal effects are biochemical
in origin as most toxicants exert their effects at basic
level of the organism by reacting with enzymes or
metabolites and other functional components of the cell.
Such effects might lead to irreversible and detrimental
disturbances of integrated functions such as behaviour,
growth, reproduction and survival (EIFAC, 1975;
Waldichuk, 1979).
Many biochemical techniques have been developed
during last few years to allow the detection of the
effects of various pollutants on aquatic biota. Some of
these are based on clinical chemical approaches
originally developed to assess human health; others
derived from basic studies of the mechanism of action
of specific toxicants (usually in mammalian systems)
(Addison, 1988).

*
Corresponding author:
Kori-Siakpere Ovie, Ph.D.
Department of Animal and Environmental Biology,
Delta State University, P, M. B. 1,,
Abraka, Nigeria
Email: oviekori@yahoo.com

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Changes in energetic metabolites and other biomarkers

have been studied as possible tools for aquatic
toxicological research (Moore and Simpson, 1992;
Arellano et al., 2000; Abou El-Naga et al., 2001).
Nitrogenous waste products associated with fish
include urea, uric acid, creatinine and bilirubin.
Literature on the effects of toxicants on nitrogenous
waste products of fish is scanty. Apart from the work of
Adamu and Kori-Siakpere (2008) who reported on the
effect of Portland cement on the nitrogenous waste
products of Clarias gariepinus. Klyszejko and
Lyezywek (1999) and Grosell et al. (2004) reported the
effect of deltametrin and copper on nitrogenous waste
products of fish. Most other literature data available are
on the effect of toxicants on rats. For example, Khleifat
et al. (2002), Kamis et al. (2003), Idowu et al. (2006)
and Ashour et al. (2007) reported the effect of toxicants
on nitrogenous waste products of rats.
The research work is conducted with the general
objective of providing more information that will
enhance the biological potential of Clarias gariepinus
as culturable fish and the findings will give us some
information about internal disturbances before the fish
shows apparent external signs of poisoning, and
increase our knowledge/understanding of the mode of
action of potassium permanganate.
The specific
objective of the research was the determination of the
effect of sublethal concentration of KMnO4 exposure

IJIB, 2008, Vol. 4, No. 1, 40

Effects of sublethal concentrations of potassium permanganate on African catfish

on the nitrogenous waste products: total plasma

bilirubin, creatinine, uric acid and urea of advanced
juveniles of C. gariepinus.

MATERIALS AND METHODS

Apparently healthy live specimens of Clarias
gariepinus (advanced juveniles: mean weight,
165.15+3.45g; mean length 29.42+6.56cm) were
purchased from Tomab Fish Farms, Obiaruku, Delta
State, Nigeria; at different times, as the need arose,
transported to the Animal and Environmental Biology
Research Laboratory, Delta State University, Abraka
and kept in large plastic drums supplied with clean
borehole water.
Three replicates of the same experimental design were
conducted. In each replicate, four tanks were stocked
with 20 randomly selected fish and the fish were
acclimatized to the experimental conditions for two
weeks. At the end of the acclimatization period, each
tank was randomly assigned to one of four treatments.
Three tanks were dosed with either one, three or five
times (x1, x3 or x5) the therapeutic dose/concentration
(2mg/L) of potassium permanganate (KMnO4): (i.e.
2mg/L, 6mg/L or 10mg/L)
and one received no
KMnO4 (0mg/L; control).
The experimental tanks consisted of large plastic
containers of 150litres capacity. Experimental fish were
fed daily with commercial fish pellets at 3% of their
body weights. Mortality during the period of
acclimatization was less than 2%. The fish were not fed
24hours prior to and during the experimental period
which lasted 192 hours. The water quality parameters
of the experimental set up with KMnO4 toxicant and
control bioassay were measured at every sampling time
by the procedures according to APHA (1998)
procedures. The water quality parameters measured
included pH 6.48+0.32, temperature 28.4+1.2oC,
dissolved oxygen 7.36+1.12mg/L, free carbon dioxide
4.85+0.06 mg/L and total alkalinity 34.6+1.54 mg/L.
The test was performed by the semistatic (renewal)
method in which the exposure medium was exchanged
every sampling time to maintain toxicant strength and
level of dissolved oxygen as well as minimizing the
level of ammonia excretion during this experiment.
After the acclimatization period, solutions of different
concentrations of KMnO4 were added to all tanks
except the control tank to achieve the predetermined
concentrations.
Two fish were randomly caught individually using a
small hand net from each experimental tank at each
sampling time. The experiments were conducted three
times, yielding a total of six fish for each treatment at
each sampling time. The sampling was done before the
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initial addition of KMnO4 (0hr = start) and then at 12,

24, 48, 96 and 192 hours. The selected fish was placed
belly upwards and blood samples obtained from the
caudal circulation with the aid of a heparinised 2cm3
disposable plastic syringe and a 21 gauge disposable
hypodermic needle. Blood was taken under gentle
aspiration until about 1cm3 has been obtained; then the
needle was withdrawn and the blood gently transferred
into heparinized plastic containers. The samples were
then mixed gently but thoroughly.
Plasma was obtained from blood samples by
centrifugation and then drawn into a 1cm3 plastic
syringe and transferred into a universal bottle, diluted
1:20 with deionised water. The diluted plasma was then
stored in a refrigerator and later used for analysis for
plasma nitrogenous waste products: total plasma
bilirubin, creatinine, uric acid and urea. All
determinations were carried out in duplicates for each
sample. Plasma nitrogenous waste products of total
plasma bilirubin, creatinine, uric acid and urea were all
determined
colorimetrically
using
commercial
diagnostic kits (Quimica Clinica Aplicada S. A., Spain
and Randox Ltd, UK) following the manufacturers
instruction; with the aid of a spectrophotometer
(Spectrumlab 21A, Lenjguang Tech, China).
The total plasma bilirubin was measured by using
Jedrassik-Grof method which is based on the reaction
of total bilirubin with diazotized sulfanilic acid, in the
presence of caffeine, with the final production of an
azopigment whose colour is proportional to the
concentration of bilirubin present in the sample. The
measurements were done using a commercial kit
(Quimica Clinica Aplicada S. A., Spain). The plasma
creatinine was measured by using the standard Biuret
method which is based on the reaction between the
creatinine and picrate to form a coloured complex in
alkaline solution. The measurements were done using a
commercial kit (Randox Laboratories Limited, U.K.).
The plasma uric acid was measured by using the
enzymatic colorimetric method which is based on the
reaction in which uric acid is converted by uricase to
allantoin and hydrogen peroxide, which under the
catalytic influence of peroxidase, oxides 3,5-Dichloro2-hydroxybenzenesulfonic acid and 4-aminophenazone
to form a red-violet quinoneimine compound. The
measurements were done using a commercial kit
(Randox Laboratories Limited, U.K.).
The plasma urea was measured by using the UreaseBerthelot colorimetric method which is based on the
fact that urea in plasma is hydrolysed to ammonia in
the presence of urease. The ammonia is then measured
photometrically by Berthelots reaction. The
measurements were done using a commercial kit
(Randox Laboratories Limited, U.K.).

IJIB, 2008, Vol. 4, No. 1, 41

Effects of sublethal concentrations of potassium permanganate on African catfish

RESULTS
The changes obtained in the nitrogenous waste products
following sublethal exposure of the African catfish:
Clarias gariepinus to the various concentrations of
potassium permanganate (KMnO4) over a period of
192 hours are presented herein.
Total Pla sma Bilirub in (mg/dL)

17
Sta rt

the toxicant was significantly different from that of the

zero time at 12 and 96 hours exposure and fish exposed
to 10mg/L toxicant concentration at 24 hours exposure
period. The highest percentage elevation in the values
of the total plasma bilirubin of 5.25 and 5.52 were
recorded in C. gariepinus exposed to 2mg/L KMnO4 at
12 and 96 hours respectively.
15
Pla sma Crea tinine (mg /d L)

Results obtained for the replicates from all three

experiments were combined and subjected to statistical
analysis using two-way analysis of variance (ANOVA)
to test for level of significance between the various
levels of sublethal concentrations of KMnO4 and the
exposure periods. Multiple comparisons of the means
were analyzed by the Bonferroni tests. All analyses
were performed using the software programme
(GraphPads Prism Software version 5.0, San Diego,
CA). Results were considered significant at the 95%
confidence level (P< 0.05).

Sta rt
12Hours
24Hours

12

48Hours
96Hours
192Hours

6
0

10

Conc entra tion (mg /L KMnO4)

Figure 2: Mean values of plasma creatinine of C. gariepinus

following exposure to the various concentrations of potassium
permanganate over a period of 192 hours. Each column represents the
mean value and vertical bars indicate the standard error of the mean.

12Hours
24Hours

16

48Hours
96Hours
192Hours

15

14

10

Conc entra tion (mg /L KMnO4)

Figure 1: Mean values of total plasma bilirubin of C. gariepinus

following exposure to the various concentrations of potassium
permanganate over a period of 192 hours. Each column represents the
mean value and vertical bars indicate the standard error of the
mean.cid, plasma creatinine, plasma urea, Clarias gariepinus, Nigeria

The mean levels of total plasma bilirubin in C.

gariepinus exposed to various concentrations of
potassium permanganate under different exposure
period is graphically shown in Fig. 1, while the
percentage variations of the total plasma bilirubin is
presented in Table 1 [Supplementary data]. The control
values were within a narrow range of 15.64 mg/dL and
15.95 mg/dL.
The mean levels of total plasma
bilirubin in C. gariepinus did not vary much with
increase in concentration levels of the toxicant. It
fluctuated between 15.95mg/dL in the control at zero
time to a mean level of 16.62mg/dL recorded in C.
gariepinus exposed to 2mg/L KMnO4 following 96
hours exposure. Analysis of variance (ANOVA) result
showed that there was no significant difference in mean
level of total plasma bilirubin in C. gariepinus (P<0.05)
with increase in concentration. However, multiple
comparisons using Bonferroni test showed that the
control (0.00) was similar to the mean levels of total
plasma bilirubin observed for C. gariepinus exposed to
6mg/L of the toxicant at all exposure time. The mean
levels of total plasma bilirubin in exposed to 2mg/L of

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Plasma creatinine levels in C. gariepinus exposed to the

various concentrations of potassium permanganate
under different exposure period is given in Fig. 2; while
the percentage variations of the plasma creatinine is
presented in Table 2 [Supplementary data]. The control
values were within a narrow range of 10.64mg/dL and
10.95mg/dL. The mean levels of plasma creatinine did
not vary tremendously. There was a minimal increase
from the control to 2mg/L. The mean level increased to
11.15mg/dL in the 6mg/L concentration and further
increased in the 10mg/L concentration. Analysis of
variance (ANOVA) test reveal that the mean levels of
plasma creatinine levels in C. gariepinus increased with
increase in concentration (P<0.05). Further analysis
using a post hoc test (Bonferroni) showed that the zero
time values were similar with the mean levels
represented in C. gariepinus exposed to all toxicant
concentrations at 12 hours. On the other hand, all
treatment following 48 through 192 hours exposure in
the three exposure concentrations showed a significant
increase (P<0.05) in plasma creatinine levels as
compared with the fish at zero time.
Plasma creatinine levels in the treated fish showed a
seemingly dose- and time-dependent trend with the
maximum elevation percentage (40.16) being recorded
in 10mg/L KMnO4 exposed fish at 192hours. Plasma
uric acid levels in C. gariepinus exposed to various
concentration of potassium permanganate under
different exposure period is shown graphically in Fig. 3;
while the percentage variation with respect to the
control values are listed in Table 3 [Supplementary data].
The mean control levels of the plasma uric acid of the
treated C. gariepinus were within a narrow range
between 8.18mg/dL and 8.78mg/dL.

IJIB, 2008, Vol. 4, No. 1, 42

Effects of sublethal concentrations of potassium permanganate on African catfish

Plasma Uric acid (mg/dL)

30

Start
12Hours

25

24Hours

20

48Hours
15

96Hours
192Hours

10
5
0
0

2
6
Concentration (mg/L KMnO4)

10

Figure 3: Mean values of plasma uric acid of C. gariepinus following

exposure to the various concentrations of potassium permanganate
over a period of 192 hours. Each column represents the mean value
and vertical bars indicate the standard error of the mean.
600

Sta rt

Pla sma Urea (mg /d L)

550

12Hours

500

24Hours

450

48Hours

400

96Hours

350

192Hours

300
250
200
150
2

10

10

Concentra tion (mg /L KMnO4)

Figure 4: Mean values of plasma urea of C. gariepinus following

exposure to the various concentrations of potassium permanganate
over a period of 192 hours. Each column represents the mean value
and vertical bars indicate the standard error of the mean.

There was a gradual increase in the mean levels of

plasma uric acid with increase in concentration. For
example, the mean levels increase steadily from
8.33mg/dL in the control to 11.09, 15.96 and
13.14mg/L in 2mg/L, 6mg/L and 10mg/L KMnO4
respectively. Similarly, the mean plasma uric acid
levels in C. gariepinus were elevated with increase in
exposure period. In the 10mg/L KMnO4 exposed fish,
the plasma uric acid levels were 60.64, 111.52, 140.24,
218.88 and 201.76% above the zero time levels
following 12, 24, 48, 96 and 192 hours exposure
respectively. Analysis of variance (ANOVA) result
showed that there was significant difference (P<0.05)
in the mean levels of plasma uric acid in C. gariepinus
with increase in concentration of the toxicant and
increase in the exposure period. Multiple comparison
using Bonferroni showed that the means of the zero
time values were not significantly (P<0.05) different
from the mean levels of plasma uric acid in C.
gariepinus exposed to 2mg/L and 10mg/L KMnO4 at
12 hours exposure. However, the mean levels of the
parameter in 6mg/L KMnO4 at 12 hours were
significantly different. Analysis of variance (ANOVA)
result clearly shows that there was significant
difference in the mean levels of plasma uric acid in C.
gariepinus with increase in the exposure period. Using
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post hoc test (Bonferroni) showed that the mean at 0hrs

was significantly different from the mean at 24, 48, 96
and 192 hours exposure periods.
The mean levels of plasma urea for the test organisms
exposed to various concentrations of potassium
permanganate at different exposure times in given in
Fig. 4; while the percentage variations of the plasma
urea in the treated groups are indicated in Table 4
[Supplementary data] . The control values of the plasma
urea varied between 501.87mg/dL and 520.52mg/dL.
From an initial mean of 509.65mg/dL in the control to
502.39, 495.50 and 419.34mg/dL in the test fish
exposed to 2mg/L, 6mg/L and 10mg/L KMnO4 at 12
hours exposure mean of 509.65mg/dL in the control to
502.39, 495.50 and 419.34mg/dL in the test fish
exposed to 2mg/L, 6mg/L and 10mg/L KMnO4 at 12
hours exposure time.
Thereafter there was a
progressive decrease with the mean levels of plasma
urea in the fish with increase in the concentration level.
For example, the mean levels in the 10mg/L KMnO4
exposed fish were 419.34, 390.98, 303.97 244.65 and
215.41mg/dL at 12, 24, 48, 96 and 192hours
respectively. Analysis of variance result (ANOVA)
showed that there was significant difference (P<0.05)
in the mean levels of the test organisms with increase in
the concentration of the toxicant. Multiple comparison
of means using Bonferroni tests revealed that the mean
levels of plasma urea for the zero time were not
significantly different from the test fish exposed to
2mg/L KMnO4 at 12, 24, 48 and 96hours; and 6mg/L
KMnO4 at 12, 24 and 48hours of exposure. However,
there were significant (P<0.05) different between the
zero time values and the mean levels of plasma urea in
the test fish exposed to 2mg/L KMnO4 at 192hours, the
test fish exposed to 6mg/L KMnO4 at 96 and 192hours;
and 10mg/L KMnO4 at all the exposure periods. There
was thus a dose and time-dependent decrease in the
mean values of the plasma urea. The maximum
percentage reduction of -58.62 was recorded in the
10mg/L KMnO4 exposed fish.

DISCUSSION
Nitrogenous waste products associated with fishes
include urea, uric acid creatinine and bilirubin. Urea
occurs in natures as the major nitrogen containing end
product of protein metabolism by vertebrates, which
excrete urea in urine. Uric acid is a purine, which is
produced from the breakdown of body cells and the
consumed food. Creatinine is a nitrogenous waste
product, which is synthesized in the body at a fairly
constant rate from creatine; while bilirubin is a
metabolic waste product which formed from the
breakdown of erythrocytes. Klyszejko and Lyezywek,
(1999) and Grosell, et al., (2004) have reported the
effect of deltametrin and copper on nitrogenous waste
product of fish. While, Khleifat, et al., (2002), Kamis,
IJIB, 2008, Vol. 4, No. 1, 43

Effects of sublethal concentrations of potassium permanganate on African catfish

et al., (2003), Idowu, et al., (2006) and Ashour, et al.,

(2007) have all reported the effect of toxicant on
nitrogenous waste products of rats.

Adamu KM and Kori-Siakpere O (2008) Effect of Sublethal

Concentrations of Portland Cement Powder in Solution on
Nitrogenous Waste Products of the African catfish: Clarias
gariepinus. (Burchell 1822). Acta Zoologica Lituanica 18(1): 55 - 60

This study has demonstrated that total bilirubin in the

potassium permanganate exposed fish were not
significantly affected. The slight rise in plasma level of
total and conjugated bilirubin suggest liver cell damage
in which, usually, there is an increase in both
metabolites (Cheesborough, 1992). Another possible
reason may be a metabolic disturbance in liver
involving defective conjugation and/or excretion of
bilirubin. The bilirubin route of elimination is perhaps
most important contributing source to the excretion of
xenobiotics, but is of primary importance for the
excretion of the animal's metabolites. Since the liver
encounters nutrients, environmental toxicants and
waste products, within this framework, it extracts the
environmental toxicants and waste products to prevent
their circulation to other parts of the body.

Addison RF (1988) Biochemical effects of a pollutant gradient

introduction. Marine Ecology Progress Series 46: 31.

The effect of potassium permanganate on the levels of

creatinine and uric acid were elevated while urea was
depleted in this investigation. These compounds are the
most abundant nonprotein nitrogen constituents in the
body and their determination are the most commonly
ordered tests of the kidneys ability to excrete
metabolic wastes (Tresseler, 1988). The data show
significant increases in the levels of creatinine and uric
acid in most of the treatments. Since increases in these
values are used as indicators of renal failure, it can be
postulated that exposure of the experimental fish is
related to the impairment of renal function. The
presence of increasing creatinine and uric acid
concentration in the blood suggest the inability of the
kidney to excrete these products, which further suggest
a decrease in glomerular filtration rate (GFR). A
common manifestation of nephritic damage is acute
renal failure characterised by decline in glomerular
filtration rate, which may have been induced by the
potassium permanganate present in the water. This
assertion is supported by Counts et al. (1995) who
reported that chemically induced nephrotoxicity by
halogentated hydrocarbons, injure the proximal tubule
monolayer, resulting in gaps in the epithelial lining,
leading to back leak of filtrate and diminished
glomerular filtration rate.

APHA (1998) Standard methods for examination of water and

wastewater. 20th Edn. American Public Health Association
Washington D C. 1976p
Arellano JM, et al. (2000) Accumulation and histopathological
effects of copper in gills and liver of Senegales sole, Solea
senegalensis and Toadfish, Halobatrachus didactylus. Ecotoxicology
and Environmental Restoration. 3(1): 22 28.
Ashour AA, et al. (2007) Blood, serum glucose and renal parameters
in lead- loaded Albino rats and treatment with some chelating agents
and Natural oils. Turkish Journal of Biology 31: 25 - 34
Cheesborough M (1992) Medical Laboratory Manual for Tropical
Countries. Butterworth-Heinemann Ltd., Hakkey Court, Jordan Hill.
Vol. I pp.472-505.
Counts RS, et al. (1995) Nephrotoxicant inhibition of renal proximal
tubule cell regeneration. American Journal of Physiology 169F: 274
- 281.
De La Torre FR, et al. (1998) Enzyme activities as biomarkers of
fresh water pollution: Response of fish branchial (Na-K) ATPase and
liver transaminases. Environmental Toxicology 14: 313 319.
EIFAC (1975) Report on fish toxicity testing procedures. Prepared by
European Inland Fisheries. Technical Paper 24.
Grosell M, et al. (2004) Effects of prolonged copper exposure in the
marine gulf toad fish Opsanus beta. I. Hydromineral balance and
plasma nitrogenous waste products. Aquatic Toxicology 68: 249
262
Idowu AB, et al. (2006) Effect of Hyptis suaveolens ethanolic extract
on the physiology of albino rats. The Zoologist 1(4): 75-79.
Kamis AB, et al. (2003) Preliminary biochemical and haematological
effects of aqueous suspension of pulp of Hyphaena thebeica (L) mart
in rats. Biokemistri 13: 1-7
Khaleifat K, et al. (2002) The chronic effects of Teucrium pollum on
some Blood parameters and histopathology of liver and kidney in the
rat. Turkish Journal of Biology 26: 65 71
Klyszejko B and Lyezywek G (1999) Effects of a sublethal
concentration of Deltametrin on biochemical parameters of the blood
serum of Carp Cyprinus carpio L; Acta Ichthyologica et Piscatoria 2:
109 115
Moore MN and Simpson MG (1992) Molecular and cellular
pathology in environmental impact assessment. Aquatic Toxicology
22: 313 322.

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Tresseler KM (1988) Clinical laboratory and diagnostic tests. 2nd Ed.

Prentice Hall Inc., Englewood Cliffs, NY.

Abou El-Naga, et al. (2001) Effect of cadmium and copper on the

digestive gland enzymes of the Limpet (Patella sp, Mollusca,
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Waldichuk M (1979) Review of the problem. In: The assessment of

the sublethal effect of pollutants in the sea. Ed. H. A. Cole, p 399
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