1.0 INTRODUCTION:

Crude oil(a natural occurring flammable liquid) was discovered in Nigeria in 1956 at Oloibiri in Bayelsa State of the Niger Delta Region by the Shell Petroleum Development Company (SPDC). Following the discovery, pioneer production began in 1958 from the Company’s oil field at Oloibiri (Aghalino, 1999).

Crude oil,a fossil fuel, is the remains of plants and animals that were buried in sediments for several millions of years where they decomposed to form a colloidal mixture of hydrocarbons from which various petroleum products such as gasoline, kerosine, fuel oil, lubricating oil, wax and asphalt are derived (Pitts, 2013). Several toxic components of crude oil such as polycyclic aromatic hydrocarbons (PAHs) and water soluble fractions (W.S.F) have been documented. (Edema, 2010).

Crude oil and its refined petroleum products are mainly hydrocarbons with some inorganic elements such as oxygen, nitrogen, iron, vanadium, nickel, chromium and sulphur. (Edema, 2006., Pitts, 2013) Nigeria’s crude oil is classified mostly as ‘light’ and ‘sweet’ as the oil is largely free of sulphur (NRC, 1985). Nigeria is the highest producer of ‘sweet oil’ in the Organization of Petroleum Exporting Countries (OPEC). This ‘light’ and ‘sweet’ oil is often referred to as ‘Bonny light’ (Zabbey et al, 2007).

Petroleum production and export play a dominant role in Nigerian economy and account for about 90% of her gross earnings. (Ndukuba, 2007., Sunmonu and Oloyede, 2007) In spite of the stupendous wealth which Nigeria has generated from the production and sale of oil, many problems have been created on the environment. Various oil spills have been reported in Nigeria. From 1976 to 1997, for instance, there were 5,334 cases of oil spillage releasing about 2.8 million barrels of oil into land, swamp, estuaries and coastal waters (Dublin-Green et al,1998) Crude oil has also polluted the Nigerian environment through refinery effluents, natural seepages as well as sabotage (Zabbey et al,2007).

Major causes of oil spillage in Nigeria include:- corrosion of pipelines and tankers (which accounts for 50% of all spills) sabotage (accounts for 28% of all spills) other oil production operations (account for 21% of all oil spills) and finally inadequate or non-functional production equipments which account for just 1% of all oil spills. (Awobayo, 1981;Ekedo, 2012).

Corrosion accounts for such a high percentage of all spills in the Niger Delta Region because due to the small size of the oil field, there is extensive network of pipelines which are poorly maintained and have outlived their estimated life span. (Ekedo, 2012) Sabotage, on the other hand, is performed through what is popularly called ‘bunkering’ whereby the saboteur taps a pipeline and in the process of extraction, sometimes the pipeline is damaged. Oil tapped from these pipelines is sold illegally. (Awobayo, 1981).

Thus oil spillage has over the years led to the pollution of many aquatic and terrestrial ecosystems (Afolabi et al, 1985., Akpofure, et al , 2000) Immense tracts of Mangrove forests in the Niger Delta Region have virtually been destroyed due to petroleum exploitation (Diop, 1993). It has been reported that crude oil caused the mortality of a huge number of marine organisms like crustaceans and fishes (Anderson, et al 1974). A short exposure to crude oil killed several marine invertebrate populations (Suchanek, 1993) Similarly, a short exposure of crude oil contaminated feed to weaner rabbits caused significant retardation of growth of the rabbits. (Berepubo et al, 1994) A brief exposure to ‘Bonny light’ crude oil caused significant mortality of Heterobranchus bidorsalis fingerlings. (Nwamba et al, 2001). Ovuru and Ekweozor (2004) observed that crude oil contaminated diets caused detrimental changes in blood parameters of semi adult rabbits. Crude oil exposure on the African catfish, Clarias gariepinus caused detrimental changes in the activities of liver enzymes of the catfish. (Sunmonu and Oloyede, 2006).

Thus, there is a lot of literature on the lethal effects of crude oil on several freshwater and marine animals. However, there is very little information on sublethal concentration of crude oil by which some Nigerian fishes could tolerate and so this study was undertaken. In the Niger Delta region of Nigeria where petroleum exploration is carried out, capture fisheries activities are rapidly declining while more attention is paid to aquaculture. A knowledge of crude oil tolerance limits of fishes is environmentally significant in assessing their distribution as well as the effects of oil spillage on fishes found in Nigerian inland and coastal waters, especially in the Niger Delta Region where there is constant oil spillage. (Akpofure et al, 2000). The findings would therefore guide us in culturing P.annectens as well as other indigenous fishes, especially in areas where there is much of crude oil exploration (extraction, refinery, transportation or spillage) The findings would also serve as baseline information which regulatory agencies could refer to, whilst setting up national standards for water quality management, especially in the Niger Delta region of Nigeria.

2.0 MATERIALS AND METHODS:

2.1 Procurement of test organisms, P.annectens.

A hundred and fifty specimens of P.annectens procured from Oguta Lake in Imo State, Nigeria were, within 3 hours from the moment of procurement, brought alive into Physiology laboratory of Animal and Environmental Biology Department of Abia State University, Uturu- Nigeria.

2.2 Acclimation and maintenance of P.annectens in the laboratory.

The standard length and weight of all procured specimens of P.annectens were quickly determined to the nearest 0.1cm and 0.1g respectively. They were then introduced into fourteen plastic tanks (0.54x 0.38 x 0.30 m) each of which contained 5 liters of dechlorinated water that were neither aerated with air pumps nor covered, where they remained for 14 days. During this acclimation period, the lungfish were kept at room temperature of 26.5 ± 1.4 ^OC and fed on boiled rice and beans as well as on fish feed obtained from the Regional Aquaculture Centre, Aluu in Rivers State, Nigeria ad libitum until used for the experiment. The water in all tanks was renewed daily to prevent accumulation of excess or uneaten food, waste materials and the fish’s mucous secretions.

2.3 Procurement of crude oil.

‘Bonny light’ crude oil used for this study was procured from the Shell Petroleum Development Company Port Harcourt, with authority got from the Department of Petroleum Resources, Nigerian National Petroleum Corporation (NNPC) Enugu.

2.4 Preparation of various concentrations of crude oil.

Five litres of each of the following percentages of crude oil were prepared:- 0%, 0.4% 0.8%, 1.2%, 1.6%, 2.0%, 2.4%, 2.8%, 3.2%, 3.6%, 4.0%, 4.4%, 4.8% and 5.2%. This was carried out by mixing the crude oil with the appropriate volume of dechlorinated tap water (Table 1). The prepared concentrations of crude oil were transferred into fourteen empty tanks (0.54 x 0.38 x 0.30 m) each tank containing a particular concentration of crude oil.

2.5 Selection of fishes and determination of crude oil tolerance limits of P.annectens.

Fifty six specimens of the lungfish, P.annectens were selected from amongst the survivors of acclimation and randomly introduced into the fourteen tanks that contained various concentrations of crude oil at a stocking density of four fish per tank. This was meant to mimick the effect of natural dilution of crude oil, as the oil used to float along the water surface whenever there was oil spillage. However, only healthy looking and active lungfish without any external signs of physical injuries or disease were selected. All fish were fed and all tanks were neither aerated with air pumps nor covered throughout the twenty one day period of immersion in dilute crude oil. The room temperature was maintained at 26.5 ± 1.4 ^oC The tolerance limits of P.annectens in various concentrations of crude oil as well as the changes in body weights were noted.

2.6 Gradual acclimation of P.annectens to crude oil.

A second set of experiments was later carried out so as to monitor the gradual acclimation of P.annectens to crude oil. Fifteen specimens of fingerling P.annectens (mean length 12.6 ±.2.1cm, mean weight, 55.6 ± 6.2g) were placed in 0.5% concentration of crude oil.

After one week, they were transferred to 1.0% concentration of crude oil. After another week, they were introduced in 1.5% concentration of crude oil and still after every one week, were introduced into 2.0% and 2.1% concentration of crude oil respectively.

This was repeated using juveniles (mean length, 29.8 ± 3.3cm, mean weight 168.8 ± 9.4g) and adult specimens (mean length 38.6 ± 4.5cm, mean weight 480.2 ± 21.7g) respectively. The tolerance of every maturity stage (fingerling, juvenile, adult) of P. annectens in each of these crude oil concentrations was observed.

2.7 Histological studies of the gills of fingerling and adult P.annectens.

Histological studies of the gills of fingerling P.annectens (standard length, 11.1cm; body weight 49.6g) and adult P.annectens (standard length, 38.7cm., body weight, 494.8g) were made so as to ascertain the anatomical basis which could be useful in the interpretation of some results. The gills were fixed in alcoholic Bouins fluid (picric acid, 1.0g; 40% formaldehyde, 60ml; glacial acetic acid, 15ml; and 80% Ethanol, 150ml) for 2 hours. It was then dehydrated through ascending percentages of ethanol, cleared in xylene and embedded in molten paraffin wax. Thin sections of between 5 and 6 µm thick were stained in Eosin – haematoxylin after the method of Onwuchekwa and Okafor (2001) and modified by Okafor (2011).

3.0 RESULTS:

3.1 Toxicological analysis

All maturity stages of P.annectens (fingerlings, juveniles, adults) survived for over twenty one days of the experiment in crude oil concentration of 2.0% downwards Many even survived for over three months in 2.0% crude oil concentration downwards. Mortality occurred before 24 hours in crude oil concentration of 2.1% and above. When P.annectens was introduced into 2.1% concentration of crude oil, the fish made violent movements in the tank as if it wanted to escape, then after a period of between one to four hours, the fish went into a state of “crude oil coma”. Then before the next 20 hours, it died, after having lost an average of 28.8% of its initial body weight.

3.2 Histological studies of the gills.

The epithelial layers of the gills of both fingerlings and adult, P.annectens contained some special “hydrocarbon emission cells” (Plate 1a, b)

Plate 1. a. adult b. fingerling

Histological section of the gills of P. annectens which reveals

the Hydrocarbon emission cells

Key 1= hydrocarbon emitting cell ; 2= cartilage

4.0 DISCUSSION:

Since all maturity stages of the lungfish (fingerlings, juveniles, adults) P.annectens were able to survive for three months in crude oil concentration of 2.0% and below, indicates that P.annectens is capable of thriving in water bodies mildly polluted by crude oil. In any case, if the crude oil concentration of that polluted water exceeds 2.0%, the lungfish would die or might escape into an unpolluted area. The relatively high mortality recorded for P.annectens exposed to crude oil concentration of 2.1% and above could be attributed to low dissolved oxygen content of the water. According to Escaravage (1990) certain chemicals or pollutants in water (like crude oil) reduce the dissolved oxygen content of that water body thereby making it toxic to fishes and other organisms in that water.

Table 1. The preparation of various concentrations of crude oil , each with a volume of 5 litres.

Concentration of Crude oil. (%)	Volume of dechlorinated tap water. (litres)	Volume of Bonny light, crude oil.(litres)
5.2	4.74	0.26
4.8	4.76	0.24
4.4	4.78	0.22
4.0	4.80	0.20
3.6	4.82	0.18
3.2	4.84	0.16
2.8	4.86	0.14
2.4	4.88	0.12
2.0	4.90	0.10
1.6	4.92	0.08
1.2	4.94	0.06
0.8	4.96	0.04
0.4	4.98	0.02
0.0	5.00	0.00

The result conforms to those of some other workers who reported the lethal and sublethal effects of crude oil on several aquatic animals. (Nwamba et al, 2001, 2006.,Ndukuba, 2007, Ugwu et al, 2008, 2010) For instance, Lee (1975) observed that a short exposure to crude oil concentration of 0.01m1/ litre caused heavy mortality of marine zooplankton. Similarly a short exposure to crude oil of 1.25 m1/litre concentration led to significant mortality of Heterobranchus bidorsalis fingerlings and also caused increased leucocyte counts of H bidorsalis juveniles (Nwamba et al (2001, Nwamba, 2010). Ugwu et al (2011) observed that exposure of Clarias gariepinus to 1.5 m1/ liter of crude oil caused high mortality of the fingerlings. However, in this study, all maturity stages (fingerlings, juveniles, adults) of P. annectens were able to survive up to 2.0m1/ litre of crude oil for over three months.

Roubal et al (1977) was of the opinion that the uptake of crude oil in fish might be either via the gills, the gut or the intestinal walls.

Death of aquatic organisms due to toxic effects of crude oil, especially the more sensitive fingerlings and juvenile forms could be through coating of the gills with oil, resulting in asphyxiation (Anderson et al, 1974; Stagemen and Sabo, 1976). In addition, the incorporation of finely dispersed particles of oil and oil products into organisms can negatively affect organs and systems either directly or due to bioaccumulation processes.

Death could also occur as the crude oil caused blockage of atmospheric oxygen from dissolving in the ambient water thereby limiting the supply of oxygen to the fish (Ugwu et al, 2011).

Crude oil readily passes the cell membranes into the blood from where it is carried by lipoproteins to the liver. (Stagemen and Sabo, 1976). In the liver, crude oil is detoxified and excreted into bile from where it enters the duodenum and leaves the body along with faeces. (Stagemen and Sabo, 1976) Crude oil might also be excreted via kidney and gills (Stagemen and Sabo, 1976). However, in this study, it is suggested that crude oil is excreted mainly via the gills. The relative high tolerance to crude oil exhibited by P.annectens is actually attributed to the possession of hydrocarbon secreting cells along the epithelial layers of the gills which are involved in the active transportation of hydrocarbons from the blood into the ambient water (Plate 1).

In this study, crude oil was considered to be the cause of mortality of P.annectens. However, it is possible that there might be other factors which could play vital roles in ameliorating or exacerbating crude oil tolerance. Some examples of such factors include water quality (inappropriate dissolved oxygen content, unsuitable pH, temperature, salinity, CO₂, NO₃, NO_2,H₂S, and organic matter levels) as well as some biological variables like genetic difference, previous exposure to crude oil or any of its refined products etc.

The responses of P.annectens in this study tend to provide some basis for predicting the overall impact of Nigerian crude oil spills on fish population.

Continuous oil spills in the Niger Delta Region of Nigeria due to oil exploitation have not only caused degradation to the environment and destroyed the traditional livelihood of the region, but have caused environmental pollution that has affected weather conditions, soil fertility, water ways aquatic habitats and wild life. (Akpofure, et al, 2000) This inhuman situation continues to attract the interest of environmental observers and calls for regular evaluation of the exploitation activities by the Nigerian Petroleum industry. (Ekedo,2012).

The study therefore provides useful information in the culturing of P.annectens or other culturable fishes found around the Niger Delta Region. It can also serve as guideline to capture fisheries activities in the Niger Delta Region of Nigeria.

ACKNOWLEDGEMENT:

The authors are grateful to Ekedo, M.C., Nwaogu, C and Dike, M for the assistance they provided during the course of the study.

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Received on 21.11.2013 Modified on 10.12.2013

Asian J. Research Chem 7(1): January 2014; Page 76-80