Human activities in agriculture, industry, power generation, urbanization and
transportation result in the emission of several wastes products in solid, liquid
or gaseous forms into the aquatic environment (Ayoola, 2008).
In many developing countries such as Nigeria, the activities of informal mechanical
workshops, usually results in uncontrolled discharge of spent engine oil into
drainage channels and canals and this eventually end up in the aquatic bodies.
The indiscriminate discharge of spent engine oil into the environment and its
negative effects on the environment demands the development of various control
strategies. Mechanical workshops now use dispersants, emulsifiers, degreasers
or detergents in washing off spent engine oil thus ensuring easy dispersal.
Dispersants are imported into the Nigeria from Europe, North America, Asia and
other temperate countries. In spite of legislation limiting the disposal of
toxic chemicals, pollution of aquatic environments still occurs (Fleeger
et al., 2003). Aquatic species are not equally susceptible to toxic
substances and much information on the toxicities of spent engine was based
on species not native to Nigeria and in environmental conditions different from
those of Nigeria. The Department of Petroleum Resources (DPR), the apex body
in Nigeria regulating activities of the petroleum industries has since 1980,
demanded 96 h LC50 toxicity tests of drilling mud systems, base oil,
oil-based muds, chemical lubricant and degreaser on local species under Nigerian
conditions to determine the safety of their use before these chemicals are approved
to be used in the petroleum industry in Nigeria (Odiete,
Spent engine oil which is chemically referred to as used mineral-based crankcase
oil is simply described as the brown to black, oily liquid removed from the
engine of a motor vehicle when the oil is changed. It is consists of branched
alkanes; cycloalkanes; Polyaromatic Hydrocarbons (PAHS); linear alkanes; decomposition
products; additives and contaminants of worn engine parts which include heavy
metals such as chromium, iron, lead, nickel, silicon, tin, aluminum, copper,
magnesium. Spent engine is a common and toxic environmental contaminant that
is not naturally found in the environment. However, disposal of spent engine
oil into gutters, water drains, open vacant plots and farms is commonly practice
in Nigeria, especially by motor mechanics that change oil from motor vehicles
and generators (Fleeger et al., 2003).
Many environmental contaminants exert their effects via genotoxic and metabolically
toxic mechanisms simultaneously causing carcinogenesis, embryo toxicity and
implicit a long term alteration in organisms by active through several generations
(Ali et al., 2008). Environmental contaminants
has developed different methods for double and single strand breaks of DNA,
DNA- adducts, micronuclei formation and chromosome aberrations. One of the most
popular and promising is the micronucleus test (MN). It is a marker of cytogenetic
damage usually caused by clastogenic or aneugenic compounds. The assessment
of cytogenetic damage has been presented assay in identification of pollution
hazards in marine environment.
In fish, the kidney is responsible for erythropoiesis as well as filtration.
Upon fish exposure to toxins, defective erythrocytes undergo passage from the
kidney into the peripheral blood, from where they are removed by the hemocatheresis
organs (Palhares and Grisolia, 2002). Chromosomal aberration
studies with preponderant native fish species represent an important effort
in delineating the extent of a particular chromosome damage and change such
as micronuclei formations and likely agents inducing the visible aberration
in the fish genome.
According to Odiete (2003), test organism to be used
for toxicity test must be ecological and economically important, occupy trophic
positions leading to humans or other important species, have adequate background
biology, be widely distributed, be genetically stable, have its early stages
(larvae, fry, juveniles) available throughout the year and be sensitive. The
African catfish Clarias gariepinus is an ecologically important and commercially
valued fish in Nigeria (Ayoola, 2008). These mudfish
are frequently and widely cultured in ponds and they also occur freely in Nigerian
natural freshwaters. Clarias gariepinus has been chosen because it is
sensitive being in its early life stage when compared to the adult (Odiete,
2003). This study intend to investigate the acute toxicity of spent engine
oil using Clarias gariepinus and evaluate the genotoxic effects of spent
engine oil using micronucleus assay in Clarias gariepinus.
MATERIALS AND METHODS
Clarias gariepinus of 8.0±0.2 cm in length and with an average weight of 7.8±0.2 cm were bought from a private fish farm in Jakande estate area of Lagos state. They were placed in a plastic container which was properly aerated and transported to the marine sciences laboratory at the University of Lagos.
The fish were kept in a glass tank (50x30x30 cm) for two weeks in properly aerated water. The stocking water was changed every day and the juveniles were fed twice daily with commercially prepared feed obtained from the fish farm until, feeding was stopped a day before the bioassay test.
Test compound: spent engine oil: Spent engine oil was collected in gallons from different mechanic workshops and fuel stations located in the Osodi-Isolo metropolis for the bioassays on Clarias gariepinus juveniles. The spent oil collected from different mechanic workshops were mixed uniformly by adding equal volumes of oil from each source into the same container and this served as the test compound.
Bioassay procedure: The preliminary tests were carried out at first to determine suitable range of concentration on the Clarias gariepinus juveniles for 96 h that four days. The concentration ranges for the range finding test were 100, 200, 400, 60, 70 and 900 mL-1. these concentrations were carefully measured out with a measuring cylinder to make out the correct measurements in triplicate of six plastic containers. While clear undiluted water served as control. In each of these bowls, 10 juveniles of 8.0±0.2 in length with mean weight of 7.8±0.2 g were introduced.
Assessment of quantal response (mortality): Mortality assessment was
carried out every 24 h over a 96 h range finding experimental period. Fish was
assumed to be dead when there was no body or operculum movement, even when prodded
with a glass rod. The results gotten from the range findings test are used in
calculating the definitive test as described by Ayoola (2008).
Definitive test was also carried out every 24 h over a four day period (96 h).
Ten active animals were introduced into the test medium containing spent engine
oil. Each treatment was set in triplicates, giving a total of 30 fishes per
test medium, including untreated media (control). The test animals were exposed
to the following concentrations of the spent lubricant oil: Spent lubricant
oil: 400, 500, 600, 700, 800 and 0 mL L-1 (control) v/v. These values
were generated from the range finding experiment.
Cytogenetic analysis of Clarias gariepinus exposed to sublethal concentration of spent lubricant oil: In this analysis, Clarias gariepinus juveniles were exposed to sub lethal concentration of spent lubricant oil as follows: Spent lubricant oil: 100, 200, 300, 400 and 0 mL L-1 (control). These values were generated from the definitive test experiment.
Each treatment was set in triplicates, giving a total of 30 fishes per treatment including untreated media (control). A total of 150 test animals were exposed per sub lethal concentrations including control. The sub-lethal toxicity tests carried out for 14 days was to investigate the rate at which Clarias gariepinus accumulates spent engine oil. The semi-static renewal bioassays procedure was adopted. To avoid drastic changes in concentration of test media via evaporation and reduction in dissolved oxygen levels, the test media were removed from their respective containers once every twenty-four (24 h) and replaced into freshly prepared test media over a fourteen day (14 day) period of sub lethal experimentation.
At determined time intervals,(day 7 and 14), five (5) live Clarias gariepinus
were randomly selected from each concentration of a triplicate including control,
blood samples were collected from the selected fish and smeared on microscope
slides, they were then fixed, stained with Giemsa (sigma) solution, they were
then rinsed with ethanol and then left to air dry over night before examining
the slides with Microscope using oil-immersion (x1500). For the scoring of micronuclei,
the following criteria were adopted from Campana et al.
(2003); the diameter of the micronucleus marginally overlap with main nucleus
as long as there is clears identification of the nuclear boundary and MN should
have similar staining as the main nucleus.
Micronuclei analysis: Blood samples were obtained by caudal vein puncture using a heparinized syringe and directly smeared on microscopic slides. The microscope slides were air dried for 24 h fixed in methanol for 20 min and left to air dry for twenty-four hours and stained in 10% Giemsa (sigma) solution for 20 min and left to air dry for twenty four h The stained slides were analyzed under a light microscope at a magnification of 1000x. Micronucleus was identified according to the following criteria:
Spherical or ovoid-shaped extra nuclear bodies in the cytoplasm, diameter of 1/3-1/20 of the main nucleus on-refractory bodies, color texture and optical features resembling those of the nucleus and the bodies are completely separated from the main nucleus.
Physicochemical parameters of the test media: The pH, temperature and dissolved oxygen content of the control and test media in the experimental set-up were determined. Temperature measurement was determined in situ by means of a simple mercury-in-glass thermometer. A highly sensitive pH meter (Philips pH meter model 9405) with glass electrode was used for the determination of pH sample. The Dissolved Oxygen (DO) was measured using appropriate digital instruments (Horiber U-10).
Acute toxicity test: The probit analysis was generated through a computer generated programme designed and implemented by Ge-le Pattaurriel Imperial College, London run by an IBM computer. The values against log-dose value for LC5, LC50 and LC95 were obtained and tabulated graphs of probit values against log dose values were obtained and tabulated. Graphs of probit values against log values were plotted using the line of best fit for a straight line curve. The following indices of toxicity and their 95% confidence limits were derived:
||LC95 value (Lethal concentration that will cause
mortality of 95% of the exposed population of test organisms)
||LC50 value (Lethal concentration that will cause mortality
of 50% of the exposed population of test organisms)
||LC5 value (Lethal concentration that will cause mortality of
5% of the exposed population of test organisms)
Statistical analysis: The data from micronucleus were analyzed using graphical representation, ANOVA and Duncan multiple Range test (DMRT) to test for significant difference (5% level) in the mean frequency of micronucleus induction in Clarias gariepinus exposed to different sub lethal concentration of spent engine oil.
Percentage mortality of Clarias gariepinus is presented in Table 1, probit of mortality against log concentration of spent engine oil against Clarias gariepinus at 96 h LC50 is presented in Fig. 1. The analysis of concentration-mortality data of spent engine oil when tested against Clarias gariepinus revealed that the derived toxicity indices (LC50) is 2.75 (562 mL-1). On the basis of computed Toxicity Factor (TF), using 96 h LC50, spent lubricant oil was found to be very toxic to the Clarias gariepinus juveniles (Fig. 1).
||Probit of mortality against log concentration of spent engine
oil against Clarias gariepinus at 96 h LC50
|| Percentage mortality of Clarias gariepinus fingerlings
exposed to spent engine oil
|Mean values followed by same superscript in each row are not
significantly different at p<0.05
||Mean frequencies of micronucleus induction in erythrocytes
of Clarias gariepinus exposes to sub lethal dose of spent lubricant
|Mean values followed by same superscript in each row are not
significant different at p<0.05
Analysis of variance (ANOVA), showed that there was significant difference (p<0.05) in the quantal response (mortality) of Clarias gariepinus to different concentrations of spent engine oil at 24, 48, 72 and 96 h of exposure (Table 1).
Micronucleus analysis : Results reveal that the Clarias gariepinus specie shows various degrees of sensitivity in monitoring genetic damage (especially clastogenic effect). This is indicated by variations in averages of the micro nucleated cells among species at various test solutions. The obtained results are summarized in Table 2. The chromosomal aberrations represented by the formation of micronucleus showed marked increase in the following level of occurrences; 100, 200, 300 and 400 mL L-1. Test solution of concentration 400 mL-1 was observed to possess fish with higher level of micronucleus frequencies.
The mean frequencies of micronucleus in Clarias gariepinus exposed to
different concentrations of spent engine oil ranged from (10.33±230-71.54±20.01).
||Micronucleated erythrocyte (arrow) of Clarias gariepinus
treated with 200 mL L-1 concentration of engine oil
||Binucleated erythrocyte (arrow) of Clarias gariepinus
treated with 400 mL L-1 concentration of engine oil
The lowest value was 10.33±2.30 and was recorded at day 7 in organisms
exposed to control (0.00 mL L-1) experiment and highest value was
71.54±20.01 and was recorded in organisms exposed to 400 mL L-1
test solutions. There was no significant difference in the frequencies of observed
micronucleus in Clarias gariepinus exposed to different concentrations
in day 7 and day 14 exposures. There was a significant difference in the micronucleus
frequency observed between control solution and the 400 mL L-1 test
Figure 2 shows micro nucleated cell in 200 mL L-1
concentration, Fig. 3 shows binucleated cell in 400 mL L-1
concentration, Fig. 4 shows normal cell in control, Fig.
5 shows micro nucleated cell in 100 mL L-1 and Fig.
6 shows binucleated cell in 300 mL L-1 concentration in different
fish Clarias gariepinus used for this study. Cytological examination
after treatment with different doses of spent engine oil revealed that binucleated
cells, deformed nuclei in addition to the main type of aberration (micronucleus)
||Micronucleated cell (arrow) of Clarias gariepinus treated
with 100 mL L-1 concentration of engine oil
||Binucleated cell (arrow) of Clarias gariepinus treated
with 300 mL L-1 concentration of engine oil
It is obvious that peripheral erythrocytes are sensitive for the damage induced
by the aquatic contaminants (approximately 150% compared with kidneys erythrocytes).
The high percentage of micronuclei (MN) in peripheral blood of Clarias gariepinus
may represent evidence that its genome well tolerates such type of cytogenetic
damage without apoptosis. Peripheral blood cells were shown to be sensitive
in Clarias gariepinus species for monitoring mutagenic and/or clastogenic
effect induced by the aquatic environment.
Exposure to genotoxic agents may result in mutation, metabolic disorder, damage
embryos and reduced fertility. The use of genotoxicity testing is essential
for the assessment of potential livestock toxicity so that hazard can be controlled.
Regulations in many countries are beginning to limit point source discharges
of toxic chemicals into water sources; however, historical and current industrial
and urban discharges are still responsible for high concentrations of toxic
substances in aquatic environments (Rodriguez-Cea et
al., 2003). The results obtained from this study showed that spent engine
oil was toxic to Clarias gariepinus with toxicity increasing with time,
exposure and concentration. The 96 h Lethal Concentration (LC50)
obtained was similar to results obtained from Olaifa et
al. (2004) on the lethal and sub lethal effects of copper on the African
catfish (Clarias gariepinus). Pollution from spent engine oil is one
of the growing environmental problems in Nigeria. It is pertinent to point out;
however, that pollution by spent engine oil in Nigeria occurs mostly in sub
lethal concentrations because the present level of spent oil in contaminated
waters is not high enough to cause acute toxicity effects on aquatic pelagic
and benthic organisms. This is in agreement with Russo et
al. (2004) on contaminants present in the aquatic environment that endanger
the survival and physiology of the organisms and also induces genetic alterations.
In this study, there was significant difference (p<0.05) between the frequencies
of micronuclei obtained from the peripheral blood smears collected on day seven
(7) and day fourteen (14). (Ali et al., 2008)
observed that Clarias gariepinus from freshwaters of Egypt have higher
incidence of the chromosomal aberrations of micronuclei in its genome than the
Clarias gariepinus juveniles employed in this study, maintaining the
species to be highly tolerant of that particular genetic damage without triggering
the genetically programmed event that allows cells to die. Hence, the statistical
results of significant difference between the species of catfish from the waters
of Egypt and Clarias gariepinus in this study might be as a result of
physiological variations and responses to the local agents inducing the chromosomal
damage, the micronuclei frequencies may vary according to the kind of pollution
involved and the species of fish. This study indicates that nuclear abnormalities
are induced in response to exposure to genotoxic agents which is similar with
the observation of Malla and Ganesh (2009). Cell attachments
are rarer than micro nuclei in normal cells, but can be found in up to 20% of
cells treated with genotoxic substances such as spent engine oil. These attachments
may result from problems in segregating tangled chromosomes. In a stress situation,
erythrocyte count is one of the first parameters that are affected. In this
study, it was found to increase almost immediately after the fish was transferred
from control to the spent engine oil test solutions. An organism such as fish
in polluted water is under stress and the extent of the stress depends on the
type of pollutant and of course the amount or concentration of pollutants in
the water. Most fishes have a way of detecting polluted areas but in the case
where the whole water is contaminated, the fish is faced with the problem of
using other means of respiration apart from the gills. In this study, low concentrations
of spent engine oil do not show much increase in micronuclei or nuclear abnormalities
except in high concentrations of spent engine oil. During this study, it was
obvious that Clarias gariepinus tend to increase its atmospheric air
breathing activities as the concentration of spent engine oil increased. The
results of this study suggest that higher frequencies of micronuclei and nuclear
abnormalities determined in the cells of Clarias gariepinus from the
test solution may be indicative of damage caused by the pollutant in the test
solution. The positive relationship between micronuclei and nuclear abnormality
induction suggests that nuclear abnormalities may be useful complementary assay
for genotoxicity analyses when fish are used as experimental animals. In conclusion,
the findings in this study related to toxic response and sensitivity towards
spent engine oil underline the need for further caution in order to ensure reduction
in the risk of damage caused by multiple pollutants as they occur in ecosystems.