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Research Article
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Comparative Effects of Petrol and Diesel on Enzyme Activity in Tympanotonus fuscatus after Sublethal Exposure |
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O.S. Edori,
C. Festus
and
E.S. Edori
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ABSTRACT
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Pollution of the aquatic environment by petroleum and its
products is common the world over. This study is aimed at examining sublethal
effects of petrol and diesel on enzymes in Tympanotonus fuscatus namely:
Aspartate Transaminase (AST) (E.C. 2.6.1.1), Alanine Transaminase (ALT) (E.C.
2.6.2.2) and Alkaline Phosphatase (ALP) (E.C. 3.1.3.1) activity after exposure.
The periwinkles were exposed to 10.40, 15.60, 21.00, 26.00 ml L-1
and a control. The organs were removed on the sixth day and were prepared for
enzymatic analysis. Enzyme activities were compared to the control value and
between the toxicants. The effects of the toxicants on AST activity in the muscle
and viscera were significantly different (p>0.05) from the control value
(137.50±15.10 IU L-1). AST activity were raised more in petrol
concentrations than the diesel concentrations in the muscle. The reverse was
the case in the viscera at 15.60 ml L-1 (227.50±24.75 IU L-1).
ALT activity in the muscle were not significant (p>0.05) between the toxicant
media. In the viscera, significant differences (p>0.05) were observed in
some of the concentrations with petrol showing higher activity. ALP activity
in the muscle were not significant (p>0.05) in both media, but were more
elicited in the diesel concentrations. In the viscera, the activities of ALP
were more pronounced in the petrol concentrations and were significant (p>0.05)
at the higher diesel concentrations. The exposure of Tympanotonus fuscatus
to petrol and diesel concentrations caused changes in the enzymatic activities
in the organism with those of petrol more pronounced than those of the diesel.
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Received: March 30, 2013;
Accepted: May 25, 2013;
Published: November 26, 2013
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INTRODUCTION
Petroleum or crude oil is the major source of hydrocarbons in the world all
over. The various components of the crude oil are collected as distillate over
wide range of temperatures during fractional distillation. These distillates
are transported to different parts of the country (Nigeria) for different uses
or purposes. Nigeria, which has a wide range of pipeline network and deports
for the transportation and distribution of crude oil and its fractions (Renner
et al., 2008) has lost a lot due to vandalisation and bunkering activities.
Apart from vandalisation, most of these pipelines are old and poorly maintained
which results in oil spillages and pollution of the aquatic environment (Brume,
2004) resulting in stress of aquatic organism.
Crude oil and its refined products which are found to contain polycyclic aromatic
hydrocarbons and other toxic substances are constantly discharged into the aquatic
environment through different anthropogenic activities (Afolabi
et al., 1985). Although, the components of petroleum share similar
physical and chemical properties, yet the toxicological properties of the components
can be quite different (Anderson et al., 1974;
Chukwu and Okhumale, 2009) based on specific individual
physical and chemical properties of the component under investigation. It is
also established that in the event of oil spill, that man, animals, vegetation,
soil and the entire environment are affected through the injection of toxic
substances into the environment (Ngodigha et al.,
1999; Amakiri et al., 2009). Crude oil and
its fraction are found to cause mortality in organisms (Moles
and Norcoss, 1998; Renner et al., 2008)
and changes in species composition, low abundance, loss of species and tainting
(Widdows et al., 1982).
To effectively manage the environment, the effect of crude oil and its components
on aquatic species should not only be examined in the macro level but also in
the micro level where examination of their effects should be on the internal
changes within the organism. Such internal changes may be alteration in the
biochemistry and physiology of the organism, which may not be immediately pronounced
but can be noticed after long time of exposure in the event of chronic or sublethal
concentrations.
This study therefore was conducted to examine the effects of petrol and diesel
on the enzyme activity of a very important commercial brackish gastropod, periwinkle
(T. fuscatus).
MATERIALS AND METHODS
Source of test animals/acclimation of animals: The test animals (periwinkle)
of length 4.5-5.5 cm were handpicked from the Eagle Cement area of the New Calabar
River near the Ignatius Ajuru University of Education Rumuolumeni Port Harcourt,
Rivers State, Nigeria at low tide and were transported in a plastic container
to the Chemistry Department Laboratory of the University. Four hundred periwinkles
were acclimated to laboratory conditions for four days in plastic tanks of dimension
30x30x10 cm half filled with brackish fetched from same source.
Preparation of substrate: Sediments were collected from same source.
The sediments were air dried to constant weight and macerated in a mortar with
pestle and sieved with 2 mm mesh to separate stones. Two hundred and fifty grams
of the finely prepared sediments were measured into each of the plastic tanks
to serve as substrate.
Experimental design: The Completely Randomized Design (CRD) was used
for the experiment. The experiment was divided into four treatment levels and
a control with three replicates.
Preparation of toxicant/exposure of periwinkle to the toxicant: The
toxicants (petrol and diesel) were prepared in the following concentrations
(10.40, 15.60, 21.00 and 26.00 ml L-1) and a control. These concentrations
were chosen based on the 96 hr LC50 (104.68 ml L-1) observed
by Renner et al. (2008) on periwinkle exposed
to petrol. Ten periwinkles were exposed to each of the replicates and allowed
in the solution for three days before fresh solution or toxicant concentrations
were prepared and left of the next three days, all totaling six days of exposure.
Collection and preparation of samples: On the sixth day, the periwinkles
were brought out of the toxicant media and the shells were broken with a small
steel rod to separate the tissues from the shell. The tissue was then separated
into the muscle and the viscera. About 0.5 g of each of the organs were macerated
or homogenized in a ceramic mortar. The homogenized organs were mixed with physiological
saline (normal saline solution) and centrifuged at 3000 rpm for ten min. The
supernatant was collected with a dropper or teat pipette and transferred into
labeled 5 mL plain bottles for enzyme analysis.
Sample analysis: The samples were analysed for Aspartate Transaminase
(AST), Alanine Transaminase (ALT) and Alkaline Phosphatase (ALP). The colorimetric
end point technique by Reitman and Frankel (1957) was
used to determine the activities of AST and ALT in the organs. ALP activity
was analysed by the method of Bessey et al. (1946),
which take into consideration the effect of colour change of a buffered phenolphthalein
substrate.
Statistical analysis: The data obtained were subjected to Analysis of
Variance (ANOVA) to determine if there is any significant difference between
the exposures. Where differences existed, Duncans multiple range test
(DMRT) was used to compare the means (Zar, 1984).
RESULTS
The activity of AST in the muscle in petrol concentrations increased significantly
(p>0.05) when compared to the control value which was 137.50±15.10
IU L-1. The increase were higher in the lower concentrations, 10.40
and 15.60 ml L-1 which were 357.50±31.82 and 295.00±0.00,
respectively than the higher concentration 21.00 and 26.00 ml L-1
which were 232.00±38.89 and 240.00±48.49 IU L-1. In
the test concentrations of diesel, the activity of AST were either higher or
lower than the value of the control. The higher values were observed in the
middle concentrations, 15.60 and 21.00 ml L-1 which were 157.00±16.82
and 260.00±0.00. In the viscera, the activity of the enzyme were significantly
(p>0.05) higher than that of the control value (57.50±3.82) in both
the petrol and the diesel test concentrations. The highest activity was recorded
in the diesel concentration at 15.60 ml L-1 (227.50±24.75)
which was followed by the value observed for petrol at 10.40 ml L-1
(220.00±18.57) as compared to the value of l57.50±3.82 for the
control (Table 1).
The activity of ALT in the muscle in petrol concentrations decreased significantly
(p>0.05) below the control value (227.50±15.32 IU L-1)
except at 15.60 ml L-1 where the same value as that of the control
was observed. In diesel concentrations, there was a significant (p>0.05)
dose response decrease in the activity of ALT. The least activity observed was
92.50±15.32 IU L-1 at 26.00 ml L-1.
Table 1: |
Aspartate transaminase, AST ( IU L-1) in the muscle
and viscera of Tympanotonus fuscatus after exposure to petrol and
diesel for six days (Mean±SD) |
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Values with the same superscript in the same in the same row
are not significantly different (p>0.05) |
Table 2: |
Alanine transaminase, ALT ( IU L-1) in the muscle
and viscera of Tympanotonus fuscatus after exposure to petrol and
diesel for six days (Mean±SD) |
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Values with the same superscript in the same in the same row
are not significantly different (p>0.05) |
Table 3: |
Alkaline phosphatase, ALP ( IU L-1) in the muscle
and viscera of Tympanotonus fuscatus after exposure to petrol and
diesel for six days (Mean±SD) |
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Values with the same superscript in the same in the same row
are not significantly different (p>0.05) |
In the viscera, the activity of ALT were significantly (p>0.05) raised above
the control value (40.00±5.82 IU L-1) in both the petrol and
the diesel test concentrations. However, ALT activity observed at 10.40 ml L-1
was 127.60±20.32 IU L-1 in the petrol test medium, while the
values observed at 15.60 ml L-1 for both petrol and diesel which
were 125.50±22.00 and 125.50±0.00 IU L-1, respectively
(Table 2).
The activity of ALP in the muscle in petrol concentrations decreased significantly
(p>0.05) below the control value (147.50±8.75 IU L-1).
The most noticeable decrease was observed at 10.40 ml L-1 which was
97.50±10.61 IU L-1. In the diesel concentrations, the activity
of ALP were all below the control value except at 21.00 ml L-1 which
was equal to the control value. In the viscera, the activity of ALP in both
toxicant concentrations were significantly (p>0.05) higher than the value
of the control (215.00±16.49 IU L-1) except at 10.40 ml L-1
where decrease in activity was observed in both toxicant media (Table
3).
DISCUSSION
The behaviour of organism is a neurotropically controlled phenomena which is
mediated by neurotransmitter substances which alters the internal biochemistry
of organisms (Sambasiva Rao, 1999). Enzymes are fragile
substances which are denatured or deactivated in unsuitable conditions. These
unsuitable conditions mainly results from mans interference with the natural
environment. The changes observed in the activities of the enzymes in this study
is similar to those observed in other studies with toxicants (Greenway
and Storey, 2001; Humtsoe et al., 2007;
Mousa et al., 2008; Sreekala
and Zutshi, 2010). In stress induced reactions organisms need energy to
detoxify, biotransform and excrete the toxicant inorder to reduce the effect
of the toxicant. This can be achieved by the use of the immediate and principal
source of energy which is carbohydrate (Umminger, 1977).
Generally, there was an increase in AST activities in the muscle and the viscera
of the periwinkle in both petrol and diesel toxicant media while decrease in
ALT activity was only in the muscle in both toxicant media but increased in
the viscera in both media. In all cases, petrol was found to have induced or
elicited more enzymatic activity than the diesel. In the event of environmental
assault, AST and ALT may either be stepped up or down so that transamination
process can favour the organism. The associated increase in the activities of
AST and ALT in the organs in the toxicant media infers active/effective transamination
(Gabriel, et al., 2011). During stress conditions
the transaminases are raised to gain more energy so as to nullify the effect
of higher demand of carbohydrate and its precursors to maintain the glycolytic
pathway and TCA cycles at sustained levels (Tiwari and Singh,
2004) so that the organism can cope with the new environmental conditions.
Increase in the transferase indicates stress augmentation resulting from the
toxicants which in this case is petrol and diesel. Therefore the observed increase
in these enzymes was to fulfill the organisms need through amino acid pool (Tiwari
and Singh, 2004). Changes in these enzymes results from alteration in enzymatic
activities which was more in the petrol than the diesel. Enzyme changes depicts
disturbance in the structure and integrity of cell organelles (Roy,
2002; Karatas and Kalay, 2002). According to Roy
(2002), variation in enzyme activity is due to either increased or decreased
permeability of cell as well as the toxicant. The higher values of the activities
of the enzymes in the organs in petrol medium to that of diesel showed that
petrol may have induced the sites responsible for enzymatic reaction than the
diesel and that it may have penetrated more into the tissues than the diesel
to have caused more interaction with the tissue biochemistry of the organism.
However, decrease was observed in the ALT activity in the muscle in both petrol
and diesel media. ALT is more indicative of cell injury than AST (Gabriel
et al., 2011) and this implies that there was tissue damage. Since
AST activity did not decline in any of the tissues, it follows that the synthesis/production
of the amino acids and its precursors are generated through the aspartate pathway
(Tiwari and Singh, 2004), a reaction which shifts towards
anaerobic respiration of the organism which can lead to death after long exposure.
The biochemical control of ATP production can be enhanced by increased AST and
ALT activity which promotes the production pathway for protein synthesis (Greenway
and Storey, 2001).
Alkaline phoshatase (ALP) is present in all tissues of organism. It is a hydrolytic
enzyme that is mainly concerned with phosphate group transfer and plays an important
role in the general energetics of an organism (Sreekala
and Zutshi, 2010). ALP along with acid phosphatase (ACP) is associated with
the metabolic transport of carbohydrates, nucleotides, phosphoproteins and phospholipids
and also active in protein synthesis (Srivastava et
al., 1995).
There were marked increase in ALP activity in the viscera of the periwinkle
with a corresponding decrease in the muscle of the organism in both petrol and
diesel media. In the viscera, petrol was found to have elicited more ALP activity
than the diesel while the reverse was the case in the muscle. Increase in ALP
in the viscera may have resulted from phosphate absorption and ingestion (Durrieu
and Tran-Minh, 2002). Its increase plays the role of general energetics
of the organism by converting high energy compounds such as NADP to NAD (Sreekala
and Zutshi, 2010). Increase in ALP also promotes the synthesis of glycogen
by deactivating phosphorylase enzymes (Parthasarthi and
Karuppasamy, 1998), therefore its variation can cause changes in glycogen
content which may not be healthy for the organism.
However, decrease in ALP activity was also observed in the muscle in the two
toxicant media (petrol and diesel). Decrease in the activity of ALP increases
the rate of degradation of protein synthesis in anoxic conditions (Greenway
and Storey, 2001) which can cause reduction in the activities of several
other enzymes such as AST and ALT. Decrease in ALT suggests uncoupling of phoshorylation
which results from toxicity on the organism. According to Goldfischer
et al. (1964), ALP splits various phosphate esters at alkaline pH
and mediates membrane transport and therefore its decrease production can cause
altered transport and inhibitory effect on cell growth and multiplication. Another
possible cause of reduction in ALP can be due to acidosis (Shaikila
et al., 1993) which may be a mechanistic pathway for adaptive changes
by the periwinkle to meet the required energy during anaerobic breakdown of
glycogen. Inhibition of this enzyme (ALP) can also be from the interaction of
the toxicants with co-factors and regulators (Shaikila
et al., 1993; Ramesh et al., 1994).
Generally, all the enzymes followed the same pattern of activity in both petrol
and diesel media. Also the alteration in the activities of AST, ALT and ALP
in the organs/tissues of the periwinkle in the toxicant media (petrol and diesel)
is an indication of disturbance in the structure and integrity of cell organelles,
endoplasmic reticulum and membrane transport system (Nchumbeni
et al., 2007) of the organism. Again, it was observed that petrol
induced more activity in all the enzymes than the diesel which is an indication
that petrol was more toxic to the organism than diesel. Since petrol is more
volatile than diesel, it may have been easier for the diesel to penetrate into
the tissues of the organism (periwinkle) to cause biochemical alterations than
the diesel.
CONCLUSION
Petrol and diesel caused changes in the enzymatic responses of T. fuscatus.
This implies that they are toxic to T. fuscatus and the environment in
general even at very low concentrations. Generally, the effects produced by
toxicants were more pronounced in petrol than diesel thus signifying that petrol
is more toxic to T. fuscatus than diesel. If the presence of these substances
in the environment goes unchecked it can result in death of many aquatic organisms
after long exposure.
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