Polycyclic Aromatic Hydrocarbons ( PAH ) in fingerlings of Clarias gariepinus ( Burchell , 1822 ) to Petroleum

One hundred and twenty (120) fingerlings of Clarias gariepinus (mean weight: 0.96 ± 0.1g) were randomly exposed to 4 experimental treatments of petroleum, based on LC50 values (6.4mg/L of crude oil, 8.7mg/L of petrol, 8.0mg/L of kerosene and 7.8mg/L of diesel oil) and replicated thrice, to determine polycyclic aromatic hydrocarbons (PAH) in exposed fish for 96 h. There was no significant difference (P > 0.05) in total (PAHs) between crude oil (97.1 ng/uL) and diesel (97.2 ng/uL) exposed fish and also between petrol (53.2 ng/uL) and kerosene (49.6 ng/uL) exposed fish, but there was a significant difference (P < 0.05) in PAH levels of the crude oil/diesel exposed -groups of fish compared to petrol/kerosene exposed -groups of fish (97.1/97.2 and 53.2/49.6 ng/uL). Naphthalene correlated positively to benzo a anthracene (r=0.672, (P < 0.05), benzo b fluoranthene (r=0.681, P < 0.05) and chrysene (r=0.615, P < 0.05) but did not correlate to fluorene. Benzo a anthracene correlated positively to benzo a pyrene (r=0.578, P < 0.05), phenathrene (r=0.685, P < 0.05) but did not correlate to acenaphthene. Fluorene correlated positively to benzo a pyrene (r=0.695, P < 0.05) but did not correlate to chrysene. Chrysene correlated positively to dibenzo a,h, pyrene (r=0.658, P < 0.05) to phenathrene and benzo b fluoranthene (r=0.659, P< 0.05). Indeno 123 cdpyrene and fluranthene however did not correlate to other PAHs except naphthanene, acenaphthene and acenaphthylene. The level of PAH in fish may translate to the toxicity effect since crude oil and diesel with lower LC50 (6.4 and 7.8 mg/L) deposited greater PAH than kerosene and petrol with higher LC50 (8.7 and 8.0 mg/L) in fingerlings of C. gariepinus. High risk to cancer disorders may occur in exposed fish to petroleum with high incidence of fluorene , anthracene, pyrene and benz a anthracene which correlated positively to benzo a pyrene which provide some basis for predicting impact of oil spills on fingerling population.


Introduction
Polycyclic Aromatic Hydrocarbons (PAHs) are group of compounds consisting of two or more fused aromatic rings which are formed during incomplete combustion of organic materials such as wood and fossil fuels, petroleum products and coal (Li et al., 2001).They are ubiquitous pollutants frequently found in a variety of environments such as freshater marine sediments, atmosphere and ice [3].Many PAHs and their expoxides are highly toxic to microorganisms, fishes and man [15].In recent times, PAHs have received much attention due to their potential cause of cancer, mutagenic diorders and birth defects [15,11].Adverse effects of PAHs have also been observed in marine organisms and they include growth reduction, endocrine alteration, and malformation of embryo and larvae and DNA damage.Ingestion of contaminated food and diffusion from water across their gills and skin are major routes of PAHs exposure to fish.Due to the lipophilic nature and high chemical stability of PAHs, they accumulate in the fatty tissues of fish following their uptake.Fishes are therefore good indicators of pollution in inland and coastal waters [5].Two broad groups exist based on their physical and biological properties including, high molecular weight (HMW) and low molecular weight (LMW) PAHs.The HMW PAHs consists of 4-6 aromatic rings and are less readily bio-degraded by indigenous microorganisms, hence can persist in the aqueous environment by bio-accumulating in aquatic organisms like fish and mussels and are more carcinogenic.The LMW PAHs consists of 2-3 aromatic rings and although less carcinogenic, also pose toxic effect to many aquatic organisms [2].The concentrations of petroleum products toxic to aquatic organisms depend on the type and hydrocarbon constituents, as well as the species involved.[10] Estimated concentrations of petroleum toxic to fish eggs and fingerlings to be 0.5-10 mg/L.Benzo a pyrene binds to DNA to cause cancer and is frequently used as marker for carcinogenic disorders and may provide basis for predicting impact of exposures of PAH to C.gariepinus fingerlings [14].Although fishes have oxidative enzymes for metabolic detoxification of xenobiotics including aromatic petroleum hydrocarbons [14], little is known about the PAH level of petroleum in exposed fish.The uptake and translocation of crude oil and other petroleum products /or compounds in fish may be the gills, guts or the intestinal wall [17].The parent compounds readily solublize in cell membranes and are probably carried via the erythrocytes to the general circulation of the blood.Some of the compounds may be carried by lipoproteins and leukocytes in the blood to the liver.The major route of excretion of petroleum metabolites is through the bile; into the intestine and out with faeces.Some are excreted through the gills and kidney [7].The African catfish of genus Clarias are esteemed group of fish with high market value in Tropical Africa [13].Their hardy nature and possession of accessory air-breathing organs enable them to tolerate adverse aquatic conditions [4].Nonetheless, Clarias gariepinus fingerlings are very delicate and sensitive to aquatic pollutants including crude oil and other petroleum products [16].This study was undertaken to determine the comparative toxic level of PAH of various petroleum products on C. gariepinus fingerlings

Results and Discussion
No significant difference (P > 0.05) in total (PAHs) between crude oil (97.1 ng/uL, Table 1) and diesel (97.2 ng/uL) exposed fish and between petrol (53.2 ng/uL) and kerosene (49.6 ng/uL) exposed fish gave an indication that kerosene and petrol PAH in fish exposed for 96h are similar as well as PAH in fish to crude oil and diesel for the same period of time.A significant difference (P < 0.05) in PAH levels of the crude oil/diesel exposed -groups of fish compared to petrol/kerosene exposed -groups of fish (97.1/97.2 and 53.2/49.6 ng/uL, Table 1) shows that heavier petroleum deposit more PAH in fish than light ones after short exposure duration for 4 days [6].Naphthalene correlated positively to benz a anthracene (r=0.672,(P < 0.05, Table 2), benzo b fluoranthene (r=0.681,P < 0.05) and chrysene (r=0.615,P < 0.05) but did not correlate to fluorene.Benz a anthracene correlated positively to benzo a pyrene (r=0.578,P < 0.05), phenathrene (r=0.685,P < 0.05) but did not correlate to acenaphthene.Fluorene correlated positively to benzo a pyrene (r=0.695,P < 0.05) but did not correlate to chrysene.Chrysene correlated positively to dibenz a,h, pyrene (r=0.658,P < 0.05) to phenathrene and benzo b fluoranthene (r=0.659,P< 0.05).Indeno 123-cd pyrene and fluoranthene however did not correlate to other PAHs except naphthanene, acenaphthene and acenaphthylene.The lighter PAH LMW were more predominant in kerosene and petrol (naphthalene, acenaphthene and acenaphthylene) in exposed fish, and correlated more than heavier PAH HMW (indeno 123 cd pyrene and fluoranthene).The level of PAH in fish may translate to toxicity effect since crude oil and diesel with lower LC 50 (6.4and 7.8 mg/L) and therefore more toxic [6], deposited greater PAH than kerosene and petrol with higher LC 50 (8.7 and 8.0 mg/L) and therefore less toxic to fingerlings of C. gariepinus.Greater PAH in HMW than LMW agrees with the findings of [2] and may provide basis for predicting the impact of oil spills on fingerlings population.High risk to cancer disorders may occur in exposed fish to petroleum with elevated levels of fluorene , anthracene, pyrene and benzo a anthracene which correlated positively to benzo a pyrene (Figure 1) frequently used as carcinogenic marker [14].

Acute toxicity test
Toxicity of petroleum to C. gariepinus was carried out according to the OECD guideline for testing of chemicals No. 203 in a semi-static renewal system by using 200L capacity glass aquaria.Thirty (30) fish were randomly exposed to 4 experimental treatments of petroleum based on LC 50 values (6.4 mg/L of crude oil, 8.7 mg/L of petrol, 8.0mg/L of kerosene and 7.8mg/L of diesel oil) [10,13] and replicated thrice, to determine polycyclic aromatic hydrocarbons (PAH) in exposed fish for 96 h.The exposure pollutant was renewed each day and was also analysed using LC-MS/MS to ensure the agreement between nominal and actual concentrations of the petroleum in the aquaria [9].The experiment was conducted under the natural photoperiod of 12:12 light-dark cycle.The physicochemical parameters of the test water were analysed daily, using standard methods [1] and were recorded (dissolved oxygen 7.50 ± 0.45 mg L −1 , temperature 27.75 ± 0.5 0 C, pH 7.8 ± 0.13 and free carbon dioxide 4.28 ± 0.6 mg L −1 ) [13].The test fish were sampled in each replicate to determine level of PAH in exposed fish.A portion of each sample was taken for extraction and analysis of PAH [12].2.3 Statistical Analysis Data obtained were expressed as standard mean ± standard error of mean and analyzed using the statistical package SPSS 20.0 computer program (SPSS Inc.Chicago Illinois, USA).Differences in the test between low molecular weight LMW and high molecular weight HMW PAHs were subjected to one way analysis of variance (ANOVA) followed by Turkey's multiple range test to determine level of difference at 95% probability level.Pearson correlation was used to determine relationship between individual PAHs.

Conclusion
The level of PAH in fish may translate to toxicity effect since crude oil and diesel with lower LC 50 (6.4and 7.8 mg/L) deposited greater PAH than kerosene and petrol with higher LC 50 (8.7 and 8.0 mg/L) in fingerlings of Clarias gariepinus.The quality and quantity of PAH in exposed fish to petroleum may provide some basis for predicting the impact of oil spills on fingerling population.High risk to cancer disorders may occur in exposed fish to petroleum with incidence of fluorene , anthracene, pyrene and benz a anthracene which correlated to benzo a pyrene.

Figure 1 :
Figure 1: Positive correlation of benzo a pyrene with pyrene, anthracene, benz a anthracene and pyrene 2.1 Experimental fish and petroleum A total of one hundred and twenty (120) juveniles of African catfish (mean weight 0.96 ± 0.1g) were obtained from local outskirts in Enugu Nigeria and transported to Fisheries Wet Laboratory of the Department of Animal/Fisheries Science and Management, Enugu State University of Science and Technology ESUT, Enugu Nigeria.They were held in four fiber reinforced plastic (FRP) tanks, containing 320 L of de-chlorinated tap water.Aeration was provided to all tanks round the clock in order to maintain dissolved oxygen contents.Before the commencement of the study, the fish were acclimatized for two weeks and were fed with commercial fish diet composed of 40% crude protein.The faecal matter and other waste materials were siphoned off daily to reduce ammonia content in water.Petroleum was obtained from Nigerian National Petroleum Cooperation Enugu.Ethical clearance from the Enugu State University of Science and Technology Committee on Experimental Animal Care was obtained and followed.

Table 1 :
PAH in fish exposed to Petroleum