Comparative Toxicity of Crude oil, Dispersant and Crude Oil-Plus-Dispersant to Tilapia guineensis

The acute toxicity of Nigeria crude oil (Bonny Light), reference compound (Sodium Dodecyl Sulphate (SDS)), dispersant (Nalco-D4106) and dispersant-plus-crude oil to Tilapia guineensis were studied. Tests were conducted over a 96 h period after acclimatization of individual in the laboratory. There were initial ranges finding test to determine the concentrations of the toxicants to be administered on the test organisms in the definitive tests. The tests were semi-static bioassays in which the exposure media were replaced every 24 h, at which the T. guineensis were also examined for mortality. Tilapia guineensis were exposed to he following concentrations of crude oil (0, 40, 80, 160, 240 and 320 mg L^-1), SDS (0, 5, 25, 50, 75 and 100 mg L^-1), dispersant (0, 1500, 2000, 4000, 7500 and 10000 mg L^-1) and dispersant-plus-crude oil (0, 540, 1080, 2160, 3240 and 4320 mg L^-1). The 96 h LC₅₀ of Bonny Light crude oil, SDS, dispersant and dispersant-plus-crude oil were 125.89, 25.12, 3162.28 and 1995.26 mg L^-1, respectively. Sodium dodecyl sulphate was the most toxic of the toxicants and the dispersant (Nalco-D4106) reduced the toxicity of Bonny Light crude oil by 16 folds.

INTRODUCTION

Oil spillage as a result of petroleum industry activities and pipe-line vandalization by saboteurs is a frequent occurrence in Nigeria. There have been over three thousand eight hundred and fifty four (3,854) reported cases of oil spillage in Nigeria from 1986 to 2000 (Adeyemi, 2004). These incidences of oil spillage have had profound negative effects on the aquatic flora and fauna of the oil-producing areas. Some aquatic species have become endangered while others have gone into extinction. Apart from this, the socio-economic lives of the indigenes of the oil-producing areas have been adversely affected, which have resulted in militancy in Nigeria in the last decade.

One of the methods used in oil spill clean-up is the application of dispersants. Dispersants are group of chemicals designed to be sprayed onto oil slicks to accelerate the process of natural dispersion. Chemical dispersants are widely used on waters in Nigeria during routine cleaning of oil spillage to break-up the oil into emulsion, which are rapidly diluted by water bodies thereby preventing recombination of the oil to form a slick (Odiete, 1999). Spraying dispersants may be the only means of removing oil from the sea surface particularly when mechanical recovery is not possible.

The use of dispersants to combat oil spills is a controversial decision because it introduces a new pollutant into the aquatic environment and does not remove the oil but only disperses it (Lin and Mendelssohn, 1998). Other opinions are in favour of its use because they remove the oil from the surface and facilitate degradation and dilution thus, preventing the oil from coming a shore (Riepsaite and Stankevicius, 2005).

Dispersant toxicity to aquatic organisms has been well documented since they were first developed as a spill response technology (Hall et al., 1989). Dispersants are intended to enhance oil mobilization into water column, thus, potentially resulting in increased biological stresses in affected areas because of elevated oil concentrations (Fuller et al., 2004). Supporting laboratory studies have shown that surfactants can increase crude oil concentrations in the water column, thereby increasing the observed toxicity compared to no-surfactant controls (Kanga et al., 1997). However, a lot of studies have also shown that application of dispersant reduces the toxicity of crude oil (Singer et al., 1991; Fuller et al., 1999, 2004; Page et al., 2000, 2002; Harris et al., 2002; Mueller et al., 2003).

The explicit consideration of toxicological effects resulting from dispersant use is not a straightforward task. In fact, it can be quite confusing and confounding to decision-making process. The bottom line of toxicity to an ecosystem or a specific living resource is a function of at least five components: the dispersant, the oil being dispersed, the nature of the exposure, the organism in question and life stage of the organism in question. The combination of these factors, as well as others that may be relevant in specific situations, will determine the ultimate impact on resources (Singer et al., 1999).

The objectives of this study was to investigate the toxicity of Bonny Light crude oil, dispersant (Nalco-D4106) and a mixture of Bonny Light crude oil-plus-dispersant (Nalco-D4106) on Tilapia sdguineensis by the determination of median lethal concentration (96 h LC₅₀), median lethal time (LT₅₀), toxicity factor of dispersant and the synergistic or joint action factor of oil-plus-dispersant among other variables.

MATERIALS AND METHODS

Experimental Set-up and Fish Management
Acute toxicity tests were conducted to compare the toxicities of a crude oil (Bonny Light), an oil dispersant (Nalco-D4106), a reference compound (Sodium dodecyl sulphate) and dispersant-plus-oil mixture on Tilapia guineensis. The bioassay was carried out in the Department of Wildlife and Fisheries Management, University of Ibadan, Nigeria in 2007. The method of bioassay employed was the one outlined by APHA (1998).

Acclimatization of Test Organisms
The fish (Tilapia guineensis) were collected from Lagos Lagoon, Lagos, Nigeria in thick transparent polyethylene bags with artificial oxygen to sustain the fish during transportation to the laboratory. The fish were held in glass tanks (60x30x45 cm³) for 14 days prior to the start of the experiment. Each acclimatization tank had the habitat water from where the fish were collected. The habitat water in the tanks was replaced every 2 days. During acclimatization, the temperature was maintained at 29+2°C while aeration was continued throughout the period with aquarium pumps. The photo-period was 12 h light and 12 h darkness. The fish were fed with commercial pelleted fish feed (40% protein) at 3% body weight ad libitum (Odiete, 1999).

Preparation of Test Medium and Application of Test Chemicals
Experiment 1
A preliminary (range-finding) test as described by Solbe (1995) and Rahman et al. (2002) was conducted to determine the main experimental concentrations for the crude oil (Bonny Light), dispersant (Nalco-D4106), reference compound (Sodium dodecyl sulphate) and dispersant-plus-crude oil. The main experimental concentrations for the toxicants (crude oil, dispersant, reference compound and dispersant-plus-crude oil) above were determined based on 0-100% mortality of Tilapia guineensis in 24 h.

Definitive Test
Experiment 2
The second stage of the experiment gives details of the main experimental concentrations for the toxicants as described by Solbe (1995) and Daka and Ekweozor (2004).

Crude Oil (Bonny Light) against T. guineensis
The followings were set up: 0.0, 0.1, 0.2, 0.4, 0.6 and 0.8 mL crude oil (Specific gravity: 0.7942 g mL^-1) in 2 L of Lagos Lagoon water corresponding to 0.0, 40.0, 80.0, 160.0, 240.0 and 320.0 mg crude oil, respectively per liter of water.

Dispersant (Nalco-D4106) against T. guineensis
The following were set up: 0.0, 3.0, 4.0, 8.0, 15.0 and 20.0 mL of dispersant in 2 L of Lagos Lagoon water corresponding to 0, 1500, 2000, 4000, 7500 and 10,000 mg of dispersant, respectively per liter of water.

Sodium Dodecyl Sulphate (SDS) against T. guineensis
The followings were set up: 0.0, 10.0, 50.0, 100.0, 150.0 and 200.0 mg SDS in 2 L of Lagos Lagoon water corresponding to 0.0, 5.0, 25.0, 50.0, 75.0 and 100.0 mg SDS, respectively per liter of water.

Crude Oil (Bonny Light)-Plus-Dispersant (Nalco-D4106) against T. guineensis
The followings were set up: 0.0, 0.1, 0.2, 0.4, 0.6 and 0.8 mL Bonny Light crude oil and 0.0, 1.0, 2.0, 4.0, 6.0 and 8.0 mL dispersant in 2 L of Lagos Lagoon water corresponding to a combined concentration of 0.0, 540.0, 1080.0, 2160.0, 3240.0 and 4320.0 mg crude oil-plus- dispersant, respectively per liter of water.

Selection of Organisms (T. guineensis) for the Bioassay
Crude Oil (Bonny Light)
One hundred and twenty (120) fingerlings of T. guineensis of mean length 7.50±1.30 cm and mean weight 12.17±1.55 g were randomly assigned in equal number (20) into six test tanks (60x30x45 cm³) separately, containing the following concentration of the crude oil 0.0 Mg L ^-1 (control), 40.0, 80.0, 160.0, 240.0 and 320.0 mg L^-1. Each of these experimental units were replicated thrice to give a total of 18 experimental units (test tanks) containing 360 fingerlings of T. guineensis.

The control (0.0 mg L^-1) contained only 20 fingerlings of T. guineensis from Lagos Lagoon without the test toxicant (Bonny Light crude oil). During the bioassay, the test solution in each tank was renewed every 24 h.

Dispersant (Nalco-D4106), Sodium Dodecyl Sulphate and Crude Oil-Plus-Dispersant
A similar experiment as the one described above was set up for dispersant, sodium dodecyl sulphate and crude oil-plus-dispersant using the concentrations determined after the preliminary tests (Experiment 1). Six experimental units were each set up for the toxicants (dispersant, sodium dodecyl sulphate and crude oil-plus-dispersant). These were the concentration of the toxicants: 0 mg L^-1 (Control) and 1500, 2000, 4000, 7500 and 10000 mg L^-1 for dispersant; 0.0 mg L^-1 (control), 5, 25, 50, 75 and 100 mg L^-1 for sodium dodecyl sulphate and 0 mg L^-1 (control), 540, 1080, 2160, 3240 and 4320 mg L^-1 for the crude oil-plus-dispersant. Each of these experimental units was replicated thrice to give a total of 54 experimental units containing 1,080 fingerlings of T. guineensis. The controls (0 mg L^-1) contained 20 fingerlings of T. guineensis without the toxicant (dispersant, sodium dodecyl sulphate or crude oil-plus-dispersant). In all, there was 72 experimental units containing 1440 fingerlings of T. guineensis.

Monofilament nettings were used to cover the tanks to prevent the specimen from jumping out of test solutions. The behavior of specimens was observed and death was recorded for the 24, 48, 72 and 96 h test periods. Death was defined as complete immobility with no flexion of the abdomen upon forced extensions (Bryan, 1976).

Statistical Analysis
Each test concentration was converted into a logarithm and the corresponding percentage (%) mortality was transformed into probit (Sprague, 1969). The median lethal toxicity (LC₅₀), median lethal time (LT₅₀), minimum lethal concentration and minimum lethal time were determined according to the method described by Finney (1971). Analysis of Variance (ANOAV) was used to test for significant differences in the number of survivors in the concentrations of the test toxicants (crude oil, dispersant, sodium dodecyl sulphate and dispersant-plus-crude oil).

The toxicity factor for dispersant and synergistic or joint action factor was determined using the formula described by Odiete (1999).

RESULTS

The results of the 96 h median lethal concentrations (96 h LC₅₀) obtained for the toxicants (Dispersant (Nalco-D4106), Reference compound (sodium dodecyl sulphate), crude oil (Bonny Light) and a mixture of Dispersant (Nalco-D4106)-plus-crude oil (Bonny Light) is shown in Fig. 1-4 while, the LC₅₀ values for the toxicants is shown in Table 1. There were very strong and positive correlations between log concentration of crude oil and probit mortality as shown by the values of regression analysis in Fig. 1 (r² = 0.80, N = 5, α = 0.05), Fig. 2 (r² = 0.86, N = 5, α = 0.05), Fig. 3 (r² = 0.98, N = 5, α = 0.05) and Fig. 4 (r² = 0.88, N = 5, α = 0.05). Coefficient of determination (r²) varied from the value (r² = 0.80, N = 5, α = 0.05) to (r² = 0.98 N = 5 α = 0.05) which shows that 80-98% of the association is dependent on the variable (Log concentration and probit mortality) for Tilapia guineensis.

Table 2 shows the median lethal time (LT₅₀) of the toxicants to T. guineensis within a 96 h period. The values were obtained by plotting probit mortality against log time for each of the concentration (Odiete, 1999). The highest (61 h) LT₅₀ for the dispersant (Nalco-D4106) was obtained at a concentration of 2000 mg L^-1 while the lowest (24 h) occurred at 10000 mg L^-1.

Fig. 1:	The 96 h LC50 of dispersant (Nalco-d4106) to Tilapia guineensis

Fig. 2:	The 96 h LC50 of reference compound (sodium dodecyl sulphate) to Tilapia guineensis

Fig. 3:	The 96 h LC50 of crude oil (Bonny Light) to Tilapia guineensis

Fig. 4:	The 96 h LC50 of dispersant (Nalco-d4106)-plus-crude oil (Bonny Light) to Tilapia guineensis

Table 1:	96 h LC50 Toxicants to Tilipia guineensis

Table 2:	Median Lethal Time (LT50) of toxicants to Tilapia guineensis

Table 3:	Minimum concentration and minimum time of toxicants to Tilapia guineensis

Table 4:	Survivors of fish (T. guineensis) exposed to different concentration (mg L-1) of toxicants

Ninety six hours was the highest LT₅₀ value obtained for the reference compound (sodium dodecy sulphate) at a concentration of 50 mg L^-1 while, 30 h was the lowest and it occurred at a concentration of 100 mg L^-1. The highest LT₅₀ values for Bonny Light crude oil (90 h) and a mixture of dispersant (Nalco-D4106) and Bonny Light crude oil (90 h) occurred at concentrations of 160 and 3240 mg L^-1, respectively, while, the lowest values were 49 h and 78 h and they occurred at 320 and 4320 mg L^-1, respectively.

Table 3 shows the minimum concentration of each toxicant that can cause death of T. guineensis and the minimum time that these concentrations can cause death. These values were obtained by plotting median lethal time (LT₅₀) values of each toxicant against the corresponding log of concentration (Table 2) (Odiete, 1999). The values obtained showed that the toxicant that can cause death of T. guineensis within the shortest time among the toxicants investigated is the dispersant (Nalco-D4106) and this occurred in 24 h while Bonny Light crude oil had the highest minimal time (90 h) to cause death of T. guineensis. The reference compound (sodium dodecyl sulphate) had the least minimal concentration (50.12 mg L^-1) that could cause the death of T. guineensis while the mixture of dispersant (Nalco-D4106) and Bonny Light crude oil had the highest minimal concentration (3235.93 mg L^-1) that can cause death of T. guineensis.

The results of the survivors of T. guineensis exposed to different concentrations of the toxicants are shown in Table 4. The number of survivors in each concentration of the dispersant (Nalco-D4106) is significantly (p<0.05) different from the others. A similar result was obtained for the other toxicants; sodium dodecyl sulphate, Bonny Light crude oil and dispersant (Nalco-D4106) (Table 4).

The toxicity factor of the dispersant (Nalco-D4106) to T. guineensis was 125.89 while the synergistic or joint action factor of the mixture of the dispersant (Nalco-D4106) and Bonny Light crude oil to T. guineensis was 15.85.

DISCUSSION

The 96 h median lethal concentration (LC₅₀) of the investigated toxicants to T. guineensis revealed that the most toxic of the toxicants was the reference compound (sodium dodecyl sulphate (SDS)} with a 96 h LC₅₀ value of 25.12 mg L^-1. This value agrees with the 96 h LC₅₀ value (22.5 mg L^-1) obtained in young Pimephales promelas by Newsome (1982). However, the above value (that is, 96 h LC₅₀ of SDS on T. guineensis) is lower than the 96 h LC₅₀ of Bonny Light crude oil (125. 89 mg L^-1) on T. guineensis recorded in this study (Table 1).

The results of the 96 h LC₅₀ of Bonny Light crude oil (125.89 mg L^-1) recorded in this study is similar to the values obtained in previous study by Daka and Ekweozor (2004). The study was on the effect of size on the acute toxicity of Nigerian crude oil (Egbogoro Liner II) to the mangrove oyster (Crassostrea gasar) using semi-static renewal bioassay (Reish and Oshida, 1987). The LC₅₀ value obtained was 135 mg L^-1 for small size (10-30 mm) oyster. However, Akbari et al. (2004) use a static condition to test the acute toxicity of Malaysian crude oil on seabass fry under tropical conditions. The results showed that the 96 h LC₅₀ of Water-Soluble Fractions (WSF) of crude oil to seabass was 23.1 mg L^-1. The difference in median lethal concentrations obtained in these studies might be due to the method of bioassay employed and the type of crude oil administered (Vanderhorst et al., 1976; Fuller et al., 2004).

The results of median lethal concentration (96 h LC₅₀) of the Bonny Light crude oil (125.89 mg L^-1) and the dispersant (Nalco-D4106) (3162.28 mg L^-1) show that the Bonny Light crude oil is more toxic to T. guineensis than the dispersant (Nalco-D4106). This is not in agreement with the study of Otitoloju and Popoola (2009) which reported that Biosolve (a dispersant) was about 27, 284 times more toxic than crude oil while another dispersant (OSD 9460) was just about 4 times more toxic than crude oil when acting alone against Clarias gariepinus. The reason for the variation in the toxicity of the dispersant might be due to their chemical nature.

The mixture of the dispersant (Nalco-D4106) and crude oil (Bonny Light) was not as toxic as the reference compound (SDS) and the crude oil (Bonny Light) only but was more toxic than the dispersant only. The median lethal concentrations of the dispersant-plus-crude oil and crude oil only were 1995.26 and 3162.28 mg L^-1, respectively. This result is in agreement with the study carried out by Fuller et al. (2004). In this study, the toxicity of Arabian medium crude oil, dispersant and oil-plus- dispersant to four organisms was carried out. The organisms were two fish species (Cyprinodon variegatus and Menidia beryllina), one shrimp species (Americamysis bahia) and a luminescent bacteria (Vibrio fisheri). The results indicated that the 96 h LC₅₀ of the dispersant only was higher than the mixture of dispersant-plus-oil for the four organisms indicating that the mixture of Arabian medium crude oil-plus-dispersant was more toxic than the dispersant only. However, the study carried out by Kanga et al. (1997) showed that dispersants increases the toxicity of crude oil to aquatic organisms.

The number of organisms that survived in each concentration of the dispersant (Nalco-D4106) differ significantly (p<0.05). A similar result was also obtained for the reference compound (SDS), crude oil (Bonny Light) and the mixture of dispersant-plus-crude oil.

The toxicity factor of the dispersant which is a measure of how much more or less toxic a dispersant is to the test organism than the reference compound was 125.89. It Indicates that the reference compound (SDS) is about 126 times were toxic than the dispersant (Nalco-D4106). The synergistic or joint action factor of the mixture of dispersant-plus-crude oil which indicates how much more (potentiation) or less (reduction) toxic the dispersant-plus- crude oil is to the test organism than the crude oil was 15.85. This shows that crude oil only is about 16 times more toxic than the dispersant-plus-crude oil mixture. The implication is that the dispersant (Nalco-D4106) was able to reduce the toxicity of crude oil to T. guineensis by 16 folds.

CONCLUSION

Although, the use of dispersants in oil spill clean up have been discouraged because of their adverse effects on aquatic organisms. However, the dispersant (Nalco-D4106) investigated in this study appears to reduce the toxicity of crude oil to T. guineensis. While scientists are seeking biological remedies to oil spillage, relatively low toxic dispersants like Nalco-D4106 might still be used if research shows that they do not have serious and profound effects on that aquatic ecosystem. Ultimately, the use of dispersants will be discontinued when biological remedial technique like bioremediation and phytoremediation are fully adaptive in real case scenario.