ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 60 (2005) 198202

Crude oil plus dispersant: always a boon or bane?

Adebayo Akeem Otitoloju
Ecotoxicology Laboratory, Department of Zoology, Faculty Of Science, University of Lagos, Akoka, Lagos, Nigeria Received 10 August 2003; received in revised form 2 December 2003; accepted 12 December 2003

Abstract The toxicities of a Nigerian brand of crude oil (Forcados Light), a newly approved dispersant for use in Nigerian ecosystems (Biosolve), and their mixtures, based on ratios 9:1, 6:1 and 4:1 (v/v), were evaluated against the juvenile stage of prawn, Macrobrachium vollenhovenii, in laboratory bioassays. On the basis of the derived toxicity indices, crude oil with 96-h LC50 value of 0.28 ml/L was found to be about six times more toxic than the dispersant (96-h LC50 1.9 ml/L) when acting alone against M. vollenhovenii. Toxicity evaluations of the mixtures of crude oil and dispersant meant to simulate the environmental control settings of crude oil spillages in aquatic ecosystems revealed that effects of the crude oil/dispersant mixtures varied, depending largely upon the proportion of addition of the mixture components. The interactions between mixture of crude oil and dispersant at the test ratios of 9:1 and 4:1 were found to conform with the model of synergism (RTU=1.2 and 2.1, respectively), while the interactions between the mixture prepared based on ratio 6:1 conformed with the model of antagonism (RTU=0.16), based on the concentrationaddition model. Furthermore, the mixtures prepared based on ratios 9:1 and 6:1 were found to be less toxic than crude oil when acting singly against M. vollenhovenii while the mixture prepared based on ratio 4:1 was found to have similar toxicity with crude oil when acting singly, based on the derived synergistic ratio values. The importance of results obtained from the joint-action tests in setting effective and environmentally safe dispersal ratios is discussed. r 2004 Elsevier Inc. All rights reserved.
Keywords: Crude oil; Dispersant; Joint action; Mixture toxicity; Spill control

1. Introduction Almost all activities involved in the exploration and exploitation of crude oil results in the discharge of this compound into the environment. In recent times, the volume of crude oil being spilled into the environment has increased signicantly, especially now that oil seems to have taken the center stage as the major source of energy to mankind. Spilled oil on the surface of water bodies limits gaseous exchange, entangles and kills surface organisms, and coats the gills of sh (Wells et al., 1995; Spies et al., 1996). It also depresses phytoplankton photosynthesis, respiration and growth, kills or causes developmental abnormalities in zooplankton and the young stages of many aquatic organisms, and causes tainting of sh, shellsh etc. (Afolabi et al., 1985; National Research Council (NRC), 1985; Otitoloju and Adeoye, 2003; Powell et al., 1985). The spillage of crude oil into the environment and its far-reaching negative effects on the environment
E-mail address: bayotitoloju@yahoo.com. 0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2003.12.021

demands the development of various control strategies such as containment and recovery using booms, skimmers or pumps, sinkers, burning or dispersants (Westermeyer, 1991). The volume of oil spilled, the sensitivity of the receiving environment, the topography of the area, etc. will always dictate the type of control strategies deployed. Dispersants are usually deployed when there is need to urgently eliminate the oating mass of oil slick, especially when the spillage is close to shorelines (NRC, 1989). The dispersants act by emulsifying or breaking up the oil slick into smaller pieces, thus ensuring easy dispersal and or mixing in aquatic ecosystems. It is, however, important to note that most of the dispersants that have been initially introduced into the market were found either to be very toxic on their own against organisms inhabiting the receiving ecosystems or, to in some cases, to enhance, the toxicity of the spilled crude oil to the exposed organisms when deployed or utilized to control oil spills (Oyewo, 1986). Recent information on joint-action toxicity of mixtures of compounds has shown that the types of interactions exhibited by components of mixtures are

ARTICLE IN PRESS
A.A. Otitoloju / Ecotoxicology and Environmental Safety 60 (2005) 198202 199

largely dependent on the proportion of their occurrence in the mixture (Otitoloju, 2002, 2003). Therefore, it is not impossible that those dispersants that in the past were found to have enhanced (synergism) the toxic effect of crude oil to exposed organisms could actually have caused a reduction (antagonism) in the toxic effect of crude oil or the mixture at other dispersal ratios. Indeed, it is a fact that the recommended mixture or dispersing ratio of previously introduced dispersants took into consideration mainly the optimum dispersal ratio that could achieve the greatest emulsifying capabilities with minimal consideration for the type of joint action that may be exhibited by the mixtures at the proposed mixing ratio (Kingham, 1981). It is therefore important to carry out extensive toxicity evaluation of dispersants and crude oil in various mixing ratios before permits are given to allow the use of such chemicals in managing oil spills in aquatic resources.

Dispersant. Biosolve, a water-based biodegradable hydrocarbon mitigation agent newly approved for oil spill control in Nigeria, was obtained from a representative of the marketers. 2.3. Preparation of test media including application of toxicants A given volume of lagoon water (16% salinity) was measured using a measuring cylinder into clean, dry bioassay containers, and a predetermined volume of crude oil or dispersant or the mixture (9:1, 6:1, and 4:1; crude oil:dispersant) was added into the water to make it up to 1000 mL (total volume of test media) to achieve the desired test concentration. The joint-action tests were carried out based on ratios 9:1, 6:1, and 4:1 (crude oil:dispersant); ratio 9:1 represents the prescribed mixing ratios for oil spill control in aquatic ecosystems by the regulatory body in Nigeria while ratios 6:1 and 4:1 are the recommended ratios by the manufacturers of the dispersant. 2.4. Assessment of quantal response (mortality)

2. Materials and methods 2.1. Test animal Macrobrachium vollenhovenii (Arthropoda, Crustacea, Decapoda) of similar sizes ranging from 6 to 7 cm and body weights of 230 to 250 mg were utilized for the bioassays in this study. This animal was selected for this study because it is one of the test organisms approved for toxicity testings involving dispersants in Nigeria by the department of petroleum resources (DPR). The animals were collected from the Lagos lagoon, Nigeria with the aid of set nets and cast nets. Efforts were made to obtain the animals from the same location over the experimental period to reduce variability in biotypes. The prawns, M. vollenhovenii, were kept in a glass holding tank (75 30 32 cm) which was quarter lled with water from the lagoon and left for a minimum of 7 days to allow them to adapt to experimental conditions (temp., 3072 C; R.H. 7972; salinity, 16%). During the period of acclimatization, the animals were fed on the prepared sh meal (20 g per 100 animals). The lagoon water in the glass holding tank was aerated with a Dyna free 1000 air pump to dechlorinate it and the water was changed once every 3 days to prevent the accumulation of waste metabolites and decaying food materials. 2.2. Test chemicals Crude oil. Forcados light was obtained from the Shell (SPDC) production platform in Forcados, Port Harcourt, Nigeria. Some of the physico-chemical characteristics of the Forcados light brand of crude oil include sulfur content 0.2%, API gravity 60/60F, rapid vapor pressure 2.5 psi, and pour point 25. Prawns, M. vollenhovenii, were assumed to be dead if there was no movement of the appendages, opercula, or mouth even when prodded with a blunt glass rod. 2.5. Bioassays Single-action toxicity of crude oil tested against the prawns, M. vollenhovenii. Twenty active prawns of similar sizes in three replicates were introduced randomly into the test media in bioassay containers. A total of 60 prawns were exposed per treatment including untreated control (lagoon water). The prawns were exposed to varying concentration of crude oil as follows: 0.4, 1.2, 1.6, 1.8, 2.0, 4.0, 5.0, 7.0 mL/L and untreated control. Mortality assessments were carried out once every 24 h for 4 days. Joint-action toxicity of mixtures of crude oil and dispersant against prawns, M. vollenhovenii. A procedure similar to that just described for single action tests was carried out but in this instance the test medium contained both crude oil and dispersants in the ratios 9:1, 6:1 or 4:1. The prawns were exposed to varying concentrations of the mixtures of crude oil and dispersant as follows: 0.2, 4.0, 0.6, 0.8, 1.0, 1.4, 1.8, 2.2, 2.6, 3.0, 3.4 mL/L and untreated control. 2.6. Statistics Concentrationresponse data analysis. Toxicological data involving quantal response (mortality) for both single- and joint-action studies were analyzed by probit analysis (Finney, 1971). The indices of toxicity

ARTICLE IN PRESS
200 A.A. Otitoloju / Ecotoxicology and Environmental Safety 60 (2005) 198202

measurement derived from these analyses were LC50 (median lethal concentration that causes 50% response (mortality) of exposed organisms), LC95 (lethal concentration that causes 95% response (mortality) of exposed organisms), and T.F. (toxicity factor for relative potency measurements, e.g., ratio of 96-h LC50 of a compound to LC50 values at equivalent time intervals). Analysis of data and measurement of joint-action toxicity of multiple mixtures of test compounds. For the joint-action toxicity of heavy metal mixtures, the two models employed for the classications are the concentration-addition model by Anderson and Weber (1975) with slight modication (relative toxic units (RTU) estimations; Otitoloju, (2001)) and the synergistic ratios (SR) model after Hewlett and Plackett (1969). The concentration-addition model assumes that, when similarly acting toxicants are mixed in any proportion, they will add together to give the observed response. In evaluating the joint action, a predicted response value(s) (e.g., LC50) is derived by summing up the LC50 values of the separate toxicants according to the proportion of their contribution in the mixture. The predicted LC50 value(s) is then compared to the observed LC50 value of the mixture so as to classify the type of interaction among the components of the mixture as: (i) additive if the observed LC50 value of the mixture is equal to the predicted LC50 value, (ii) synergistic if the observed LC50 value of the mixture is less than the predicted LC50 value, or (iii) antagonistic if the observed LC50 value of the mixture is greater than the predicted LC50 value. The relationship of observed LC50 values to predicted LC50 (RTU) is estimated as RTU predicted LC50 value=observed LC50 value; where RTU=1 describes additive action, RTUo1 describes antagonism, and RTU41 describes synergism. The synergistic ratios model is SR LC50 of a chemical acting alone ; LC50 of chemical additive mixture

where SR=1 describes additive action, SRo1 describes antagonism and SR41 describes synergism.

3. Results 3.1. Acute toxicity of crude oil and dispersant tested against prawns, M. vollenhovenii The analysis of concentration-mortality data of crude oil when tested against the prawns revealed that the derived toxicity indices (LC50) ranged from 0.28 (96-h LC50) to 1.18 mL/L (24-h LC50), while for the dispersant, the derived toxicity indices ranged from 1.90 (96-h LC50) to 2.63 mL/L (24-h LC50) (Table 1). On the basis of the computed toxicity factor (96-h LC50) ratios, the crude oil, Forcados light, was found to be about six times more toxic against M. vollenhovenii than the dispersant, Biosolve (Table 1). 3.2. Joint-action toxicity of mixtures (9:1, 6:1, and 4:1 vol/vol) of crude oil and dispersant against prawns, M. vollenhovenii The analysis of doseresponse data for the mixture of crude oil and dispersant (9:1, 6:1, and 4:1) revealed that the observed 96-h LC50 values of the mixtures prepared based on ratios 9:1, 6:1, and 4:1 were 0.38, 2.4, and 0.28 mL/L, respectively (Table 2); therefore the mixture prepared based on ratio 4:1 was the most toxic followed by ratios 9:1 and 6:1 (the least toxic). Comparison of the observed 96-h LC50 value of the mixtures to the predicted 96-h LC50 based on the concentration-addition model shows that the interaction between the crude oil and the dispersant when prepared based on ratios 9:1 and 4:1 is in conformity with the model of synergism (RTU41) while interaction between the components when prepared based on ratio 6:1 conforms with the model of antagonism (RTUo1) (Table 2).

Table 1 Relative acute toxicity of crude oil, Forcados light and dispersant, Biosolve against the prawns, Macrobrachium vollenhovenii Chemicals Crude oil (mL/L) 24 h 48 h 72 h 96 h Dispersant (mL/L) 24 h 48 h 72 h 96 h LC50 (95% C.L) 1.2 (0.91.8) 3.5 (2.35.3) 0.3 (0.20.5) 0.28 (0.20.4) LC95 (95% C.L) 14.5 (12.817.5) 11.7 (10.513.2) 3.3 (2.73.8) 3.1 (2.43.5) Slope7SE 0.870.5 3.171.6 1.670.9 1.370.8 Probit line equation Y Y Y Y 0:1 0:8X 1:7 3:1X 0:8 1:6X 0:7 1:3X Toxicity factor

6.8

2.6 3.2 2.2 1.9

(1.93.2) (2.25.1) (1.22.5) (1.42.3)

5.5 5.2 4.7 3.4

(4.86.2) (4.35.9) (4.15.6) (2.73.9)

5.172.7 3.471.9 4.972.8 6.473.5

Y Y Y Y

2:1 5:1X 1:7 3:4X 1:6 4:9X 1:8 6:4X

C.L, 95% condence limit; SE, standard error; toxicity factor, 96-h LC50 of dispersant/96-h LC50 of crude oil.

ARTICLE IN PRESS
A.A. Otitoloju / Ecotoxicology and Environmental Safety 60 (2005) 198202 201 Table 2 Analysis (based on concentration-addition and synergistic ratio models) of the 96-h LC50 values of crude oil and dispersant when acting jointly or singly against the prawns, Macrobrachium vollenhovenii Mixtures of crude oil:dispersant 9:1 6:1 4:1 Crude oil only Observed 96-h LC50 (95% C.L) ml/L 0.38 2.40 0.28 0.28 (0.200.56) (2.103.30) (0.180.33) (0.20.4) Predicted 96-h LC50 (95% C.L) ml/L 0.44 (0.320.59) 0.39 (0.370.67) 0.60 (0.440.78) 3.1 (2.43.5) Probit line equation Y Y Y Y 1:69 4:03X 3:85 6:58X 1:98 3:59X 0:7 1:3X RTU 1.16 0.16 2.14 Synergistic ratio (SR) 0.74 0.12 1.0

C.L, 95% condence limit; predicted 96-h LC50=sum total of the single-action 96-h LC50 values of constituent toxicants according to proportion of contribution in the test mixture; RTU=predicted 96-h LC50/observed 96-h LC50; RTU=1 indicates additive action; RTU41 indicates synergism; RTUo1 indicates antagonism; synergistic ratio=96-h LC50 of crude oil acting singly/96-h LC50 of the mixture.

Further analysis based on the synergistic ratios model shows that mixtures prepared based on ratios 9:1 and 6:1 were found to be less toxic (SRo1) (antagonism) than the crude oil when acting singly while the mixture prepared based on ratio 4:1 was found to have similar toxicity level with crude oil when acting alone (SR=1) (additive action) (Table 2).

4. Discussion In this study, the acute toxicity level based on the 96-h LC50 value of Forcados light brand of crude oil was found to be 0.28 mL/L when tested against prawns, M. vollenhovenii. This result is in agreement with the ndings of Imevbore et al. (1984), who carried out studies on the toxicity of three brands of Nigerian fresh crude oils, namely, Forcados, Bonny Light, and Bonny Medium, against some brackish water organisms, Tilapia guineensis, Aphocheilchythys spilauchena, and Tympanotonus fuscatus, and two freshwater organisms, Sarotherodon niloticus and Paniocephacus sp. The authors further reported that Bonny Light was the most toxic of the three brands of crude oil studied. The acute toxicity level based on the 96-h doseresponse data for the dispersant, Biosolve against M. vollenhovenii was found to be 1.90 mL/L. Although in this study the dispersant was not as toxic as the crude oil tested, it nevertheless was found to be toxic against the test animal at relatively low concentration. This observation is in agreement with the ndings of Oyewo (1986), who reported that some dispersants used in Nigeria were relatively highly toxic to some brackish water species. Comparison of the toxicity of crude oil and the dispersant, Biosolve on the prawns, M. vollenhovenii, however, showed that the crude oil Forcados light was about six times more toxic than the dispersant, Biosolve. The studies on the joint-action toxicity of crude oil and the dispersant Biosolve when mixed in ratios 9:1, 6:1, and 4:1 and tested against M. vollenhovenii revealed that the interactions between the crude oil and the

dispersant Biosolve when tested jointly varied depending on the proportion of addition of the mixture components. The analysis of the joint-action results based on the concentration-addition model showed that the toxicity of the mixtures of crude oil and dispersant at the test ratios of 9:1 and 4:1 was found to conform largely with the model of synergism (RTU=1.2 and 2.1, respectively), indicating that the toxicity of the mixture of crude oil and dispersant was enhanced at these test ratios. However, the toxicity of the mixture prepared based on ratio 6:1 was found to conform with the model of antagonism (RTU=0.16), indicating that the toxicity of the mixture of crude oil and dispersant was reduced by over six times at this test ratio. The implication of these observations is that the mixtures of crude oil and the dispersant in ratios 9:1 and 4:1 would be expected to cause more damage to the exposed animals when utilized for oil spill control at these mixture ratios, since the type of interaction exhibited by the components of the mixture is synergistic. However, with regard to mixture ratio prepared based on ratio 6:1, the interaction between the mixture components resulted in a reduction in their toxicity by over six times, thus indicating that this mixture ratio (6:1) would be an environmentally safer mixture ratio if Biosolve were to be deployed for oil spill control. Furthermore, comparison of the toxicity of the mixtures at the different test ratios (9:1, 6:1, and 4:1) to that of crude oil when acting singly showed that mixtures prepared according to ratios 9:1 and 6:1 were less toxic (SR=0.74 and 0.12, respectively) than crude oil when acting singly against the test animal M. vollenhovenii. Hence the deployment of the dispersant for oil spill control using these mixture ratios could reduce the toxic effects of crude oil, if acting singly, to the exposed test organism. The mixture prepared based on ratio 4:1 was, however, found to have similar toxicity prole as the crude oil when acting singly (SR=1.0). The results obtained in this study therefore imply that the deployment of new-generation dispersants for oil spill control in aquatic environments may indeed minimize or reduce overall biological damage if the

ARTICLE IN PRESS
202 A.A. Otitoloju / Ecotoxicology and Environmental Safety 60 (2005) 198202 Eco-development Consultancy Group, Institute of Ecology, University of Ife, Nigeria, 272pp. Kingham, J.D., 1981. Oil spill chemicals: environmental implication and use policy. Proceedings of the international seminar on the petroleum industry and the Nigerian Environment. FMW&H/ NNPC PTI Effurun, pp. 179185. National Research Council, 1985. Oil in the seaInputs, fates and effects. National Research Council Marine Board, National Academy Press, Washington, DC, 601pp. National Research Council, 1989. Using oil spill dispersants on the sea. National Research Council Marine Board, National Academy Press, Washington, DC, 335pp. Otitoloju, A.A., 2001. Joint action toxicity of heavy metals and their bioaccumulation by benthic animals of the Lagos lagoon. Ph.D. Thesis, University of Lagos, 234pp. Otitoloju, A.A., 2002. Evaluation of the joint action toxicity of binary mixtures of heavy metals against the mangrove periwinkle Tympanotonus fuscatus var radula (L.). Ecotoxicol. Environ. Saf. 53 (3), 404415. Otitoloju, A.A., 2003. Relevance of joint action toxicity evaluations in setting realistic environmental safe limits of heavy metals. J. Environ. Manage. 67 (2), 121128. Otitoloju, A.A., Adeoye, O.A., 2003. Tainting and weight changes in Tilapia guineensis exposed to sublethal doses of crude oil. Biosci. Res. Commun. 15 (1), 9199. Oyewo, E.O., 1986. The acute toxicity of three dispersant. Environ. Pollut. 41, 2331. Powell, C.B., Baranowska-Dutiewicz, B., Isoun, M., Ibiebele, D.D., Ofoegbi, F.U., Whyte, S.A., 1985. Oshika oil spill environmental impact: effect on aquatic environment. Proceedings of the International Seminar on the Petroleum Industry and the Nigerian Environment. FMW&H/NNPC, Kaduna, pp. 181201. Spies, R.B., Rice, S.D., Wolfe, D.A., Wright, B.A., 1996. The effects of the Exxon Valdez oil spill on the Alaskan coastal environment. In: Rice, S.D., Spies, R.B., Wolfe, D.A., Wright, B.A. (Eds.), Proceedings of the Exxon Valdez oil Spill Symposium. American Fisheries Society, Bethesda, MD, pp. 116. Wells, P.G., Butler, J.N., Hughes, J.S., 1995. Exxon Valdez Oil Spill: Fate and Effects in Alaskan Waters. American Society for Testing and Materials, Philadelphia, pp. 338. Westermeyer, W.E., 1991. Oil spill response capabilities in the United States. Environ. Sci. Technol. 25 (2), 196200.

joint-action effects of the crude oil and dispersant at varying mixing ratios are taken into consideration. Extreme caution must therefore be exercised to balance efcacy of proposed mixture ratios to emulsify spilled crude oil with the resultant impact on the toxicity of the mixture component before recommending mixture ratios of crude oil to dispersant for use in oil spill controls.

Acknowledgments The author is grateful to members of the staff of the Shell Petroleum Development Co., Port Harcourt, Nigeria for assistance rendered in obtaining crude oil and to Professor K.N. Don-Pedro for providing some materials used for this study.

References
Afolabi, O.N., Adeyemi, S.A., Imevbore, A.M.A., 1985. Studies of the toxicity of some Nigerian crude oils to some aquatic organisms. In: Proceedings of the International Seminar on the Petroleum Industry and the Nigerian Environment. FMW&HH/NNPC, pp. 269273. Anderson, P.D., Weber, L.J., 1975. The toxicity to aquatic population of mixtures containing certain heavy metals. In: Proceedings of the International Conference on Heavy Metal in the Environment. Inst. of Environmental Studies, Univ. of Toronto, pp. 933953. Finney, D.J., 1971. Probit Analysis. Cambridge University Press, London. Hewlett, P.S., Plackett, R.L., 1969. A Unied theory for quantal responses to mixtures of drugs: non-interactive action. Biometrics 15, 591610. Imevbore, A.M.A., Adeyemi, S.A., Afolabi, O.A., 1984. Studies on the toxicity of Nigerian crude oils: A technical report submitted to the Nigerian National Petroleum Corporation (NNPC) by