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Journal of Veterinary Advances

Toxicological Impact of Crude Oil Contaminated Water on the Serum Biochemistry of Adult Rabbits in the Humid Tropics

Nyeche V. N., Ajuogu P. K. and Nodu M. B.

J Vet Adv 2015, 5(4): 685-692

DOI: 10.5455/jva.20150421011656

Online version is available on:

Online version is available on:

ISSN: 2251-7685


Original Article

Toxicological Impact of Crude Oil Contaminated Water on the Serum Biochemistry of Adult Rabbits in the Humid Tropics

1 Nyeche V. N., 2 Ajuogu P. K. and 3 Nodu M. B.

1 Department of Animal Science, Faculty of Agriculture, Rivers State University of Science and Technology, Port Harcourt, Rivers State, Nigeria. 2 Department of Animal Science and Fisheries, Faculty of Agriculture, University of Port Harcourt, Choba East west Road Rivers State, Nigeria. 3 Department of Animal Science and Fisheries, Faculty of Agriculture, Niger Delta University, Amasoma Bayelsa State, Nigeria.


This work was carried out to evaluate the toxicological responses of Rabbits serum biochemistry exposed to graded levels of crude oil contaminated water. Thirty two (32) adult New Zealand white breed of rabbits were randomly assigned into four treatment groups designated A, B, C, and D respectively according to the graded levels of crude oil contaminated water as follows: Treatment A (control) 0.00% contaminated water, treatment B 0.01%, treatment C 0.02% and treatment D 0.03% crude oil in a completely randomized experimental design (CRD). From the results, all the serum biochemical parameters of the rabbits (Cholesterol, glucose, protein, urea, creatinine, total bilirubin and conjugated bilirubin) exposed to the treatment were significantly (P < 0.05) affected by the treatment amongst the treatment groups except albumin that was not impacted significantly (P > 0.05). In conclusion crude oil contaminated water was adjudged to have adverse influence on the blood chemistry and the general physiology of the rabbits.

Keywords: Crude oil, contamination, water, serum, biochemistry, rabbits, tropics.

Corresponding author: Department of Animal Science and Fisheries, Faculty of Agriculture, University of Port Harcourt, Choba East west Road Rivers State, Nigeria. Received on: 21 Mar 2014 Revised on: 30 Apr 2014 Accepted on: 21 Apr 2015 Online Published on: 30 Apr 2015

685 J. Vet. Adv., 2015, 5(4): 685-692



Farm animals like cattle, sheep, rabbits and poultry including game animals are the major sources of animal protein in Nigeria. The lives of these animals have however been jeopardized especially in the Delta region of Nigeria where oil exploratory/exploitation activities have resulted in spillages and environmental degradation. Rabbits constitute one of the most recently well-recognized source of rapid animal protein supply in Nigeria (Ekpenyong, 1986; Aduku and Olukosi, 1990; Berepubo, 1994; Nodu, 2000). This has made rabbit farming a fast growing sub-sector of the animal industry. Some of the unique features that have endeared rabbits to Nigeria livestock farmers and researchers include their efficient conversion of waste food and surplus garden produce as well as other unconventional “feeds” (not useful to man) into edible meat (Owen et al., 2008). Other prospects are the fact that rabbit meat (“lagum”) is less prone to religious bias nationwide and the rabbit has on average a short gestation period of 31 days. Some of the oil exploration installations are located on the most productive farmlands and valuable ecosystems in Rivers State of Nigeria and other operational areas in the Niger Delta region. Amadi et al., (1993) established that all oil installation, exploration, production and transportation activities carry risk of oil spillage through human error, equipment failure, blowouts, operational discharge, accidents, outright sabotage, natural and even unknown causes. As Cairns and Pratt (1993) puts it, the impairment and progressive deterioration of the environment exposed to different levels of oil pollution in the region was clearly noted from early times but the control measures were taken only after some serious incidents involving human victims occurred. These complaints are further worsened by the frequent oil spillage occurring from burst oil pipes crisscrossing the area. The toxicology of crude oil which is an area that studies or examines the effects of dosages of given compounds (material or chemical) on the


lives of animals or plants exposed to it has continued to receive wide attention (Yahaya 2001; Ovuru et al., 2004; Ngodigha, 2009, George 2010). Crude oil has been noted to contain several poisonous compounds, which accumulate in the body and induce toxic symptoms that sometimes result in death (Heintz et al., 1999). Crude oil, is broadly characterized as paraffinic/naphtanic or aromatic (Chapelle, 1993), contains smaller proportions of non-hydrocarbon compounds, such as oxygen, thiols, heterocyclic nitrogen and sulphur compounds, as well as metalloporphyrins (Anoliefo 1991, Traven 1992, Chapelle, 1993). Baker (1981) noted that within each series of hydrocarbons, the small molecules are more toxic than the larger molecules. The Effects of poison brought about by the components of crude oil on life of animals cannot be accurately evaluated. This is because of the complicated nature of crude oil pollution since toxic chemicals seldom occur singly. Gibson (1991). Several of these chemicals or components of the oil act together to produce an effect on the subject (animal or plant) giving a synergistic effect. Sometimes this effect results in weakening of the immune system so that the animal may fall victim much more easily to various parasitic and other disease organisms (Yahaya, 2001). Some are identified as carcinogens, mutagens, and teratogens (CDC, 1999). Exposure of humans and animals to these chemicals is increasing in terms of the environmental level and the different usage of crude oil (Patrick-Iwuanyanwu et al., 2011). Difference in exposure or contact will occur based on location, work, personal activities, age, diet use of protective equipment and other factors. The toxic effect can be acute lethal, sub-lethal or both, depending on the level of exposure, organism exposed and the dosage it is exposed to (Rothman et al., 1996). In several organs, mainly heart and liver, cell damage is followed by increased activities of a number of cytoplasmic enzymes in the blood, a phenomenon that provides the basis for clinical diagnosis of diseases e.g. liver enzymes are usually raised in acute hepatotoxicity but tend to decrease

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with prolonged intoxication due to damage to the liver cells. A number of investigations have been conducted on the direct and indirect effects of crude oil on poultry (Nwokolo et al., 1984), goat (Ngodigha et al., 1999) and rabbits (Monsi et al., 1991; Berepubo et al., 1994; Ovuru et al., 2004). Owu et al., (2005) reported a significant reduction in red blood cell and haematocrit value in Guinea pigs which were exposed to crude oil. Also, Olawale and Owuora (2007), observed that crude oil contaminated diet could pose serious effect on the hormonal system, which may consequently affect the reproduction process in organisms found in a crude oil polluted environment, and change in the endocrine system which may cause changes in reproductive development, growth or behaviour that can affect the animals or human or their offspring (Naz et al., 1999). According to Monosson et al., (1999), anti-androgens in adult animals increase the serum levels of androgens, luteinizing hormone and estrogen. This may imply that the crude oil is acting as anti-androgenic compounds, and thereby inducing spontaneous abortions, still birth and reproductive malfunction (Alvarez et al., 2007). Cytotoxic and biochemical derangement are associated with ingestion of marine animals in polluted area (Nwankwoala and George, 2000). Brain damage such as cerebral cortex malfunctions was also been observed in fetuses of pregnant rats exposed to bonny light crude oil, (Fischer et al., 2005). The advantageous features of the rabbit have encouraged its choice for this study whose broad aim is to determine the possible consequences of crude oil spillages on the physiological performance of livestock and/or game in crude petroleum-bearing ecosystem or environment. The objective of this work is to assess the impact of crude oil contamination on the serum chemistry of rabbits in the Niger Delta.

Materials and Methods

The study was carried out at the rabbitry section of Rivers State University of Science and Technology Teaching and Research Farm, Nkpolu-Oroworukwo, Port Harcourt, a city

687 J. Vet. Adv., 2015, 5(4): 685-692

located in the south-south region (Niger Delta) of Nigeria. Thirty two (32) post pubertal adult (7-8 months) New Zealand breed of rabbits were used for this study. They were housed in conventional standard single tie hutches of two compartments, and were randomly assigned into four treatment groups of eight animals, designated A, B, C and D respectively. In a complete randomized experimental design (CRD), each treatment was further sub-divided into four replicates of two (2) per replicate. The treatment groups were allocated to four graded levels of crude oil contaminated water as follows:

Treatment A (control) 0.00mls crude oil contaminated water. Treatment B.O.O1% crude oil contaminated water. Treatment C.O.02% crude oil contaminated water. Treatment D.O.03% crude oil contaminated water.

Experimental Technique The crude petroleum oil used in this study was Bonny light grade, obtained from Nigeria Agip oil Company (NAOC) at Obirikom flow station in Ogba-Egbema Ndoni Local Government Area of Rivers State. They were exposed for 24 hours in a sheltered pan to allow evaporation of light fraction in order to ensure a stable product (White 1992). This is to simulation the natural occurring condition following oil spillages in the oil-bearing communities and was diluted with borehole water to obtain concentrations of 0.01%, 0.02 %, and 0.03%, assigned to treatments B, C, and D respectively. Treatment A (0.00%), represent the control. On the arrival, the animals were pre- conditioned for two weeks to get them acclimatized to the new environment, during which feeds (growers mash and forages) and clean water were given ad libitum. After the pre-conditioning period, the rabbits were exposed to treated water containing graded levels of crude oil contaminated water as thus, Treatment (A) (control) 0.0% crude oil,


(B) = 0.01% crude oil, (C) (0.02%) crude oil and (D) O. 03% crude oil respectively. Other management practices were strictly observed, for instance regular washing and disinfections of the feeders and drinkers, regular deworming with ivomec, intermittent administration of anti-biotics, prophylactic administration of coccidiostart etc.

Blood Collection and Analysis After the twelve weeks experimental periods, 10ml blood samples were collected from all the experimental rabbits via the ear vein and decanted into a sterile well labeled heparinized sample bottles, and stored in an ice pack, which was immediately taken to the laboratory for serum biochemical analysis. The blood samples were allowed to coagulate at room temperature and centrifuged for 10mins at 3000 rmp. The total Protein concentration was assessed using Biuret method. 5.0ml of Biuret reagent was pipetted into tubes labeled blank, standard, test, and control. 0.1ml of distilled water, standard, sample and control were pipetted into their respective tubes, mixed and incubated for 30minutes at 25ºC. The absorbance’s were measured against the reagent blank at wavelength of 546nm. The concentration of total protein was calculated by dividing the absorbance of sample against absorbance of standard multiplied by concentration of standard (Henry et al., 1974). Bromocresol green (BCG) method by Doumas et al., (1971) was used for albumin estimation. 3ml of Bromocresol green reagent was pipetted into tubes labeled blank, standard, sample and control. 0.01ml of distilled water, standard, sample and control was pipetted into their respective tubes, mixed and incubated at 25 ºC for 5minutes. The absorbance were measured at 578nm against the reagent blank. The concentration of Albumin was determined by dividing the absorbance of sample against absorbance of standard multiplied by concentration of standard. The liver to body weight ratio was determined by taking the weight of the whole liver and comparing it with the final body weight as described by Sunmonu and Oloyede (2007). urea, creatinine and cholesterol were carried out following standard procedures of (Meyer et al.,


1992), using Quimica Clinica Aplicada (QCA) test kits (Quimica Clinica Aplicada, Spain) and a Spectrum lab 21A Spectrophotometer (Spectrum lab, England). Total Bilirubin is determined in the presence of caffeine, which releases albumin bound Bilirubin by the reaction with diazotized sulphanlic acid (Meyer et al., 1992). The glucose level was determined in the presence of glucose oxidase, formed when hydrogen peroxide reacts with phenol and 1- aminophenazone to give red violet colour as indicator (Barham et al., 1972 and Teuscher et al.,


Total Protein















treatments between the treatment groups. The



values are 50.50 g/l, 66.50 g/l, 70.80 g/l and



increment was











contamination. The






Albumin There was significant difference (p < 0.05) between the treatment groups. Treatment D 49.00 g/l is significantly higher than C (48.00 g/l), B (46.00 g/l) and A (44.55 g/l).

Creatinine There was significant difference (p < 0.05) on the creatinine values between the treatment groups according to the increased levels of crude oil contaminated water. Treatment D (4.35 mmol/L) had higher creatinine status than treatments C (3.15 mmol/L) and B (2.50 mmol/L). The least was recorded in treatment group a (1.55 mmol/L).

Urea There was a progressive significant (p < 0.05) increase on the urea level as the treatment increases. Treatment D (8.42 mmol/L) was significantly higher than groups C (5.35 mmol/L), B (5.65 m mol/L) andA (4.75 m mol/L).

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Glucose The influence of crude oil contaminated water on glucose did not differ significantly (p > 0.05) defer between the treatment groups. But there was observable numerical difference between the groups. Treatment D (6.35 m mol/L) was numerically higher than A, (5.85 m mol/L), B (6.30 m mol/L) and D (6.30 m mol/L).

Total Bilirubin Statistical analysis of the total Bilirubin was significantly (p < 0.05) impacted amongst the treatment groups. There was a progressive significant increment in total bilirubin status as the treatments increases, their values are 3.74 g/L, 5.80 g/L, 6.25 g/L and 7.40 g/L for treatments A, B, C and D respectively. The

highest and the least values were recorded in treatments D (7.40 g/L) and A (3.74 g/L).

Conjugated Bilirubin Treatment effect on conjugated bilirubin was significantly different (p < 0.05) between the treatment groups. Treatments D (3.85 m mol/U) are significantly (p < 0.05) higher than treatments B (2.35 m mol/U), C (2.98 m mol/U) and A (2.01 m mol/U) respectively.

Cholesterol Influence of the treatment on the cholesterol status was significantly different (P < 0.05) between the treatment groups. Treatments A (1.13 m mol/L) and B (1.58 m mol/L) are significantly (p < 0.05) lower than treatment C (2.55 m mol/L) and D (2.60 m mol/L).

Table 1: Effect of crude oil contaminated water on Serum Biochemical parameters.



Serum Biochemical parameters






Total Protein (g/l)

50.50 d 4.64 44.55 a 2.05 1.55 d 0.50 4.752 c 0.02

66.50 c 4.55

70.80 b 2.42

75.00 a 2.82 49.00 a 0.50


Albumin (g/l)

46.00 a 2.05

48.00 a



Creatinine (m mol/L)

2.50 c 5.65 b


3.15 b


4.35 a



Urea (m mol/L)


5.35 b


8.42 a 6.35 a 7.40 a 3.85 a 2.60 a



Glucose (m mol/L)

5.85 a


6.30 a 5.80 c


6.30 a




Total Bilirubin (g/L)

2.74 d



6.25 b




Conjugated bilirubin (m mol/U)

2.01 d


2.35 c


2.98 b




Cholesterol (m mol/L)

1.13 c


1.58 b


2.55 a



Mean SEM on the some row with different superscript differs significantly (p < 0.05).


Serum biochemistry investigated in this study showed an elevated increase according to the increased levels of crude oil contaminated water amongst the treated groups. Increase in urea and creatinine have been used as important indices for the evaluation of the effects of chemicals on the kidney (Davis and Berndt, 1994). Urea and creatinine are important metabolites that are usually excreted by the kidney. Their presence in the blood suggests kidneys inability to excrete them; this also implies a decrease in glomerular filtration rate, which may have been induced by the hydrocarbon fractions present in the diet. The

689 J. Vet. Adv., 2015, 5(4): 685-692

significant increase in serum urea and creatinine levels observed in this study, is in line with Henry (2001), who reported that serum and urinary concentrations of urea and creatinine are indices of renal function, and low urinary clearance of urea and/or creatinine evident by their low urinary concentrations and consequent rise in their serum levels are pointers to impaired renal function. Also Counts et al., (1995) reported that chemically induced nephrotoxicity by halogenated hydrocarbons, injure the proximal tubule monolayer, resulting in gaps in the epithelial lining leading to back leak of filtrate and diminished glomerular filtration rate. Similarly, George (2010) established increased urea and creatinine status of rabbits exposed to


crude oil contaminated food. The significant increase in the bilirubin levels (both conjugated and total bilirubin) amongst the tested groups may suggest the haemolytic process or toxicity to the liver caused by crude oil intoxication (Saneky et al., 1993). Another associated reason could be a metabolic disturbance in the liver involving defective conjugation and/or excretion of bilirubin. Its rate of elimination is an important contributing source to the excretion of the xenobiotic, but is of primary importance for the excretion of the animal metabolites (Ovuru et al., 2004). ). Bilirubin is a by-product of the breakdown of heamoglobin from red blood cells in the liver. Its level is a good indication of the liver function. Its abnormal elevation suggests liver damage or disease. Mononucleosis, hemolytic anaemia, low levels of exposure to the sun, and toxic effects of some products (Udem, et al., 2011), http//wwwcarbonbasedcom/ebother; htm\\liver function). Since the liver encounters environmental toxicants and waste products, within this frame work, it extracts the environmental toxicants and waste products to prevent their circulation to other parts of the body. A linear progressive increase in cholesterol concentration according to the increasing levels of crude oil contaminated water may be related to the general increase in lipid mobilization. Cholesterol is a key intermediate in the biosynthesis of related sterols such as bile acids, adenocortical hormones (stress indicator), androgens and estrogens (Rahmani, et al., 1988). Ovuru et al., (2004) also reported that increased cholesterol level in the serum, is an index of stress in animals. In this study, increase in surum concentration of cholesterol with increasing concentration of crude oil contaminated water was observed. Serum cholesterol rises when there is renal retention damage resulting in diminished removal of lipoprotein from plasma, thus causing the concentration to increase markedly. This supports previous workers kato et al., (1982), Quazi et al., (1983) who reported an increase in serum cholesterol of rats fed triglycerides and liver lipids fed PCB. These findings including


the results of this research supports the fact that increased cholesterol level is an index of stress in animals of which crude oil caused (stressor). Also the retention of protein in the blood is an indicator of major functional changes in kidney and liver (Agrawal and John, 1990). Protein has nutritive transporting protective, buffering and energy function (Cheesborough, 1992). In this study, the increased protein in the serum with increasing levels of crude oil contamination, may suggest a haemo concentration, presence of abnormal globulins or some form of liver and kidney dysfunction (Ganong, 2005). Serum glucose in this study increased with increasing crude oil contamination which may also indicate stress due to increased glucose concentration in the blood as a result of hypocalcaemia and the interference with the secretion of insulin from the pancreas (Ganong, 2005). It could also imply reduced functional ability of the pancreas in its insulin production due to crude oil toxicity. Sahal et al., (1994) reported increased blood glucose concentration leading to diabetes mellitus. Udem et al., (2011) reported that mononucleosis, haemolytic anaemia, toxins leads to increase in serum bilirubin due to liver damage. Ovuru et al., (2004) in their work, revealed that impairment of organs like liver and kidney functions leads to accumulation of metabolites and Xenobiotics.


In conclusion, this study shows that rabbits’ exposure to petroleum hydrocarbon is a risk factor for impairment of renal and liver function as indicated by serum metabolites assessed in this study. Its’ devastating consequences on the general physiology of the test animals were revealed which ultimately will affect productivity and food security (Summonu and Oloyede, 2009; Ngodigha 2009; Ovuru et al., 2004). Also, the present study, substantiated already existing, reports on the crude oil intoxication of both aquatic and Teretial life (Summonu and Oloyede 2009; Berepubo et al., 2001) using rabbits as the test animal.

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