Evaluation of Activities of Transferases and Phosphatase in Plasma and
Organs of Clarias gariepinus Exposed
to Fluazifop-p-Butyl
Iniobong Reuben Inyang, Seiyefa Thomas and
Sylvester Chibueze Izah*
Journal of
Environmental Treatment Techniques, Vol. 4, No. 3, pp. 94-97, September 2016
Environmental Toxicity Research Unit, Department of
Biological Sciences, Niger Delta University, Wilberforce Island Bayelsa State
Received: 30/08/2016 Accepted: 18/09/2016 Published: 30/09/2016
Abstract
The aim of this study was to evaluate the
activities of transferases and phosphotase in plasma,
liver and kidney of Clarias gariepinus. This
study was carried out in the department of fisheries and animal science, Niger
Delta University, Wilberforce Island, Bayelsa State,
between September, 2014 and April 2015. Adult Clarias gariepinus were exposed in four
replicates to varying concentrations of fluazifop-p-butyl
(ranging from 0.01 -0.03ppm) in a 30 day semi static bioassay. Samples were
obtained from the liver, kidney and plasma. A statistically significant
increase (p<0.05) was recorded in the plasma. Liver alkaline amino
transferase (ALT) and Alkaline phosphatase (ALP) unveiled a significance
decrease, while liver aspartate amino transferase (AST) showed a clear
progressive increase compared to the control. Kidney enzyme values were
significant. A progressive decrease in value were
recorded (not in a dose dependent pattern). This toxicant could be toxic at
high concentration. These parameters could serve as useful biomarkers of sublethal effect of fluazifop-p-butyl
in non-target organism in the aquatic environment.
Keywords: Fluazifop-p-butyl,
Clarias gariepinus, Plasma
enzymes, Fish bioassay.
1 Introduction
Pollution via pesticides is one of the major problems
in the aquatic environment. Unlike the early 1970’s, pesticide production is
fast increasing because of its exigency in agriculture and anthropogenic
purposes. However, pesticides and related chemicals that
destroy the delicate balance between species that characterizes a functioning
ecosystem [1]. Pesticides produce many physiological and biochemical
changes in aquatic organisms by influencing the activities of several enzymes
[1].
These pesticides, even when applied in restricted
areas are washed and carried away by rains and flood to large water bodies like
ponds and rivers. The offshoot will lead to alteration of physiological
properties of water [2]. Pesticides are toxic, not only to fishes but also to
other organisms which form food of the fishes [3]. Fishes are very sensitive to
a wide variety of toxicants in water, various species of fish show uptake and
accumulation of many contaminatants or toxicants such
as pesticides [4].
Fluaxifop-p-butyl
is a post emergence phenoxy herbicide,
it is absorbed rapidly via leaf surfaces and quickly hydrolysis to fluazifop acid. The acid is transported primarily in the
phloem and accumulates in the meristems where it disrupts the synthesis of lipids
in susceptible species [5,6]. Fluazifop-p-butyl
inhibit acetyl CoA Carboxylase, an enzyme that catalyses
an early step in fatty acid synthesis in organisms. This xenobiotics can pass
readily into fish tissue, and is highly toxic to fish and other aquatic species
including invertebrates [7].
The effect of xenobiotic contamination in an
ecosystem can be estimated through analysis of biochemical changes in organism
inhabiting that polluted environment [8, 9, 10]. The
biochemical response of aquatic organism to pollution is given by changes in
several key enzymes, especially those of biotransformation systems. The value
of tissue enzyme activities in the diagnosis of the effect of pollutant is an
emerging area in aquatic toxicology and remediation programmes
[11].
In this study, the native Nigerian fish species Clarias gariepinus was
chosen as an assay organism due to its hardy nature and being the most cultured
fish species in Nigeria [12]. The goal was to access an effect of fluazifop-p-butyl on enzymes in plasma, liver and kidney of
Clarias gariepinus (a
widely cultured fish in Nigeria). This parameter was chosen because of its role
in fish metabolism and general physiology.
2 Materials and Methods
2.1 Experimental stock
Fish samples for this study were obtained from
biotechnology resource centre, Odi,
Bayelsa State, Nigeria. They were transported to the
wet laboratory of the department of fisheries and animal science, Niger Delta
University, Wilberforce Island, Bayelsa State,
Nigeria, where the assays were conducted. Thirty (30) healthy adult Clarias gariepinus with
mean length 16.04±0.23cm and mean weight 94.04±0.6g were acclimatized
individually in a rectangular aquaria for seven days.
Fish were fed once a day at 10.00 – 11.00hrs with 35% crude protein.
2.2
Experimental design
Completely randomized design (CRD) was used for
the experiment. There were four treatment levels with three replicates. A range
finding test (trial test) was carried out using the toxicant fluazifop-p-butyl. Four (4) concentrations of the toxicant
were prepared from the original (150g/l).
2.3
Test chemical
Sublethal concentrations of fluazifop-p-butyl
for the assay (0.01, 0.02 and 0.03ppm was determined based on the range finding
test [13]. These were prepared by transferring 0.02, 0.04, 0.06mls respectively
from the original concentration (150g/l) of the toxicant and making it up with
borehole water in the test aquaria, 30L of the diluents was used as control.
2.4
General bioassay
Four replications of each treatment level (concentration)
and control were set up by introducing fishes individually into each aquarium.
The exposed media were renewed every 48hours, the physiochemical
characterization of the water used for the bioassay was carried out using
standard methods of APHA [14] and the following values were obtained:
Temperature 24.05 – 24.20oC, pH 6.15 – 6.24., Conductivity 97.43 –
135.02µ/cm, Alkalinity, 11.31 -17.13mg/l, Dissolved Oxygen, 5.21 – 7.13mg/l,
and Turbidity, 103 – 146NTU. The acitivities of
aspartate amino tranferase (AST) and alanine amio transferase (ALT) in plasma and organs were assayed
using the colorimetric method of Reitman and Frankel [15] while Kind and King
[16] method was used for analysis of alkaline phosphatase (ALP).
2.5
Statistical analysis
Data were expressed as mean ± standard error.
The data were subjected to analysis of variance (ANOVA) and Duncan multiple
range test (DMRT) were used to test for pair wise significant differences
(p<0.05) between treatments.
3 Results
3.1 Plasma enzymes
Table 1 unveiled the result of enzyme
parameters of Clarias gariepinus exposed
to fluazifop-p-butyl. ALT values were statistically
significant (p<0.05). a sudden rise in value At
0.01ppm was recorded and subsequent drop in values, albeit higher than the
control value. AST values recorded was akin to ALT (fluctuation in values),
while ALP values recorded unveiled a significant drop in values and significant
rise in values at 0.02 and 0.03ppm (Table 1).
3.2
Liver enzymes
A statistically significant (p<0.05) values were
obtained in liver ALT (Table 2). Values decreased as the concentration of the
toxicant increases in a dose dependent pattern while AST values increased as
the concentration of the toxicant increased except at 0.03ppm (albeit, still
higher than the control value). Liver
ALP shows a clear elevation of values at 0.01ppm compared to the control and
then a sudden drop in values as the concentration of the toxicant increases
(Table 2).
3.3
Kidney enzymes
An obvious
fluctuation in values were recorded in kidney ALT, albeit experimental values
were slightly lower than the control while AST values decreases as the
concentration of the toxicant increases in a dose dependent pattern. ALP values
were statistically significant at 0.01 and 0.02ppm. The second test
concentration (0.02ppm) recorded the highest value (930.00±10.13) compared to
the control that had 556.00±8.41.
4 Discussion
Fish species are
sensitive to enzymatic and hormone disruptors. Chronic exposure to low levels
of pesticides may have a more significant effect on fish population than acute
poisoning [17]. Doses of pesticides that are not high enough to kill fish are
associated with subtle changes in behaviour and
physiology that impair both survival and reproduction [18]. Biochemical changes
induced by pesticidal stress usually lead to
metabolic disturbances, inhibition of important enzymes, retardation of growth
and reduction in the fecundity and longevity of the organism [19].
4.1 Fish plasma enzymes
Activities of enzymes in fish are essential metabolic processes [20]. We observed an increased in transferases (ALT and AST) in the plasma except AST at 0.03ppm. ALT and AST are found in heart, liver, skeletal muscle, kidney, pancreas, spleen, gill, red cells and brain tissue [21]. When disease or injury affect these tissues and the cells are destroyed, especially liver ALT and AST are released into the blood stream. The amount of ALT or AST is directly related to the number of cells affected by the disease or injury [22]. Increase in activities of these enzymes in plasma may be due to liver damage, which results in the liberation of these intracellular enzymes, hence the elevation of plasma ALT and AST level.
Experiment with
common carp, Cyprinus carpio exposed
to diazinon showed a clear elevation of values in the
plasma [22]. Again exposure of Nile tilapia (Oreochromis niloticus) to cypermethrin
also unveiled a progressive increase in plasma values [23].
A statistically
significant (p<0.05) levels was also recorded in ALP in the plasma. This
elevation in values is caused by the toxicant, flauzifop-p-butyl.
The significant elevation in the activity of ALP in the plasma is an indication
of tissue damage to organs that produces the enzyme such as the liver and
kidney. According to Nagat [24], this will eventually
lead to leakage of lysosomal enzyme into the cytoplasm and renal necrosis.
Table 1: Activities of ALT, AST and ALP
in plasma of Clarias gariepinus exposed
to fluazifop-p-butyl
for 30 days
|
Conc of Fluazifop (ppm) |
ALT (µ/l) |
AST (µ/l) |
AST (µ/l) |
|
0.00 |
66.00±0.12bc |
267.00±10.01c |
19.50±1.02c |
|
0.01 |
102.00±0.68a |
423.00±10.08a |
17.00±0.09cd |
|
0.02 |
68.50±1.12bc |
331.50±7.11b |
20.50±0.08b |
|
0.03 |
70.50±1.08b |
240.00±4.56d |
25.30±0.08a |
Means with the same superscript within
column are not significantly different (p>0.05)
Table
2: Activities of ALT, AST and ALP in liver of Clarias gariepinus exposed to fluazifop-p-butyl
for 30 days
|
Conc of Fluazifop (ppm) |
ALT (µ/l) |
AST (µ/l) |
AST (µ/l) |
|
0.00 |
137.00±11.10a |
81.10±0.99c |
109.00±4.00b |
|
0.01 |
71.00±3.92b |
191.00±8.43b |
211.50±3.05a |
|
0.02 |
60.10±0.07bc |
376.50±7.01a |
101.00±0.98c |
|
0.03 |
55.30±0.06c |
300.00±10.10a |
93.00±6.41c |
Means with the same superscript within
column are not significantly different (p>0.05)
Table
3: Activities of ALT, AST and ALP in kidney of Clarias gariepinus exposed to fluazifop-p-butyl
for 30 days
|
Conc of Fluazifop (ppm) |
ALT (µ/l) |
AST (µ/l) |
AST (µ/l) |
|
0.00 |
13.50±1.23a |
248.50±6.83a |
556.00±8.41b |
|
0.01 |
11.00±0.82b |
182.50±3.93b |
304.00±5.12c |
|
0.02 |
6.50±0.04cd |
69.00±0.05c |
930.00±10.13a |
|
0.03 |
9.50±0.03c |
68.50±0.46c |
536.00±3.75b |
Means with the same superscript within
column are not significantly different (p>0.05)
4.2
Liver and kidney enzymes
The present
research revealed a decrease in ALT and ALP values while AST recorded a steady increase
except at 0.03ppm (the highest concentration). These enzymes are present in
large amount in the liver and kidney of fishes, hence
any injury or damage in these organs will eventually lead to a decrease in
production. A decrease may also arise a result of
leakage of these enzymes into the blood circulation as a result of injury
sustained by the probe. Evidently, plasma enzymes in this present study
recorded a steady increase, confirming this fact (Table 1). According to Kamen [25], the reduction of these enzymes in the organs
may be attributed to interference of the toxicant (fluazifop-p-butyl)
with protein metabolism in the hepatic cells or inhibition of the enzymes.
ALP activity is a
reflection of changes in endoplasmic reticulum mass. It is also known to occur
in the cell membrane and may be involved in metabolite transport [26]. Thus the
decrease may denote a decrease in membrane transport of the probe organism.
5 Conclusion
This study
confirmed that exposure to pesticide (fluazifop-p-butyl)
can result in a significant changes in enzymes of Clarias gariepinus. This
results indicate that this toxicant could be toxic at high
concentration, therefore further studies are required to evaluate the potential
environmental risk of fluazifop-p-butyl, one of the
most prevalent herbicide in the Niger Delta, Nigeria.
Acknowledgement
This publication
is based on part of Master of Science project work of the second author
(Thomas, S) supervised by the lead author (Dr Inyang, IR).
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