Received date: April 29, 2016; Accepted date: June 08, 2016; Published date: June 15, 2016
Citation: Dussauze M, Pichavant-Rafini K, Theron M, Floch SL, Milinkovitch T, et al. (2016) An Experimental System to Assess Potential Biological Impact of Operational Response During an Oil Spill. J Pet Environ Biotechnol 7: 282. doi:10.4172/2157-7463.1000282
Copyright: © 2016 Dussauze M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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In order to evaluate the potential toxicity of dispersant application in an oil slick in near-shore areas, this study presents an experimental system designed to perform toxicity tests on fish. Three possible oil spill scenario issues during an oil spill were tested on juvenile sea bass (Dicentrarchus labrax); the Water Soluble Fraction of oil; the Mechanical Dispersion of oil; the Chemical Dispersion of oil. Preliminary toxicity assays suggest that the experimental system is appropriate to assess the three experimental conditions during a period of 24 hours. This experimental system allowed obtaining “quick” and relevant results of acute toxicity in an emergency context such an oil spill.
Dispersed oil; Acute toxicity test; Water soluble fraction of oil; Oil droplets; Dicentrarchus labrax
- A communication for the validation of an experimental system.
- A system designed to perform toxicity tests on small water organisms
- A system devised to simulate the behaviour and the toxicity of the petroleum following dispersant use.
Experimentations were done on juvenile European seabass, Dicentrarchus labrax. Seabass is a commercially important demersal species present in near-shore areas. Sea bass (4.6 ± 0.2 g) were provided by an aquaculture facility (E.M.G., Gravelines, France) and acclimated for 1 month in a 300-L flow-through tank (35%, 19.0 ± 0.1°C, with 12 hours light:12 hours dark photoperiod) prior to bioassays. During acclimation, they were fed daily with fish food (Neosupra AL2 from Le Gouessant Aquaculture). The diet composition was 58% proteins, 13% lipids, 0.5% cellulose, 10% ash, 10% moisture.
The petroleum used in the study was a crude Arabian light (CAL). The CAL is oil used in other eco-toxicological studies [14-17]. This oil is composed of 54% saturated hydrocarbons, 10% polar compounds and 36% aromatic hydrocarbons. To recreate the most realistic conditions of an oil slick drifting at sea for a few days, the oil was evaporated in a 1 m3 tank for 24 hours. This weathering caused initial evaporation of the lighter compounds inducing change in the oil composition. The weathered CAL contained 54% saturated hydrocarbons, 12% polar compounds and 34% aromatic hydrocarbons. The total evaporation of oil was approximately 7%. Details of CAL are presented in supplementary data. The viscosity of the oil was under 5000 cst, allowing the application of dispersants . Two formulations of thirdgeneration dispersant (1 and 2), manufactured by Total Fluides and Innospec (Gamlen) were selected.
The experimental system was adapted from Blackman et al. . It is composed of twelve experimental tanks (units). Each tank is a 20 L cylinder fitted with a removable central column 77 mm in diameter that houses a stainless steel shaft and 3 bladed propellers. The central cylinder has two sets of two apertures situated at the top and the bottom. The apertures are covered with a metallic mesh screen to exclude test animals from the propeller housing. The propeller rotates (at 1000 rpm) to produce a small vortex within the central cylinder, thus drawing the exposure solutions in through the upper apertures and expelling them through the lower ones. This homogenization allowed maintaining oil droplets throughout the water column. The system is a static water system, e.g. without water supply, maintained in a temperature controlled room (19°C) and equipped of an aeration supply. The experimental device complies with the French AFNOR standard  to determine the acute toxicity of a substance.The experimental system was adapted from Blackman et al. . It is composed of twelve experimental tanks (units). Each tank is a 20 L cylinder fitted with a removable central column 77 mm in diameter that houses a stainless steel shaft and 3 bladed propellers. The central cylinder has two sets of two apertures situated at the top and the bottom. The apertures are covered with a metallic mesh screen to exclude test animals from the propeller housing. The propeller rotates (at 1000 rpm) to produce a small vortex within the central cylinder, thus drawing the exposure solutions in through the upper apertures and expelling them through the lower ones. This homogenization allowed maintaining oil droplets throughout the water column. The system is a static water system, e.g. without water supply, maintained in a temperature controlled room (19°C) and equipped of an aeration supply. The experimental device complies with the French AFNOR standard  to determine the acute toxicity of a substance.
All the exposure conditions were prepared in 22 L glass exposure tanks. The four basic exposure conditions to be used were prepared separately; they included Water Soluble Fraction (WSF), Mechanically Dispersed oil (MD) and Chemically Dispersed oil using the two dispersant formulations (CD1 and CD2). The WSF was prepared with 95 g of weathered CAL in 20 L of seawater following the lower energy method of Singer et al. . Only the liquid phase was used as the exposure environment. Mechanically dispersed oil (MD) was prepared using 20 L of sea water and 95 g of weathered CAL; the mixture was agitated using a propeller mixer (RW 16 Basic IKA) fitted with the same 3 bladed-propeller and using the same rotor speed (1000 rpm) as used in the experimental system (described in 2.3). Chemically dispersed oil solutions, using dispersants 1 and 2 (CD1 and CD2), were prepared using 20 L of sea water, 95 g of weathered CAL, and 5 g of dispersants 1 or 2 respectively (following the manufacturer’s recommended application ratio of 20:1), and with the same mixing procedure as for the mechanically dispersed oil solution. Once the exposure media had been prepared, they were diluted in sea water at six concentrations (0%, 2.4%, 12%, 18%, 24% and 40%) and distributed in the experimental system.
Groups of 10 fish were exposed to one dilution of each exposure condition medium for 24 hours in an experimental tank. Physicochemical parameters (pH, dissolved oxygen, water temperature, salinity) were monitored. Two exposure conditions were tested simultaneously: chronologically CD1 and WSF, then CD2 and MD. At the end of the 24 hours exposure period, the fish in each tank were gently transferred to clean sea water for a 24 hour period, as recommended by Blackman et al. . For this purpose, 22 L glass flow-through tanks were used. After 24 hours, each tank was inspected and dead fish were counted. Fish were considered dead when no gill movement and no response to a caudal pinch were observed.
Measurements of total petroleum hydrocarbon (TPH) seawaterconcentrations: The total petroleum hydrocarbon (TPH) concentration in each dilution of each exposure medium was assessed at the beginning (T0) and at the end of the exposure period (T1),using the mean of three replicated measurements for each time point. TPH concentrations were quantified by spectrophotometry (UV-Vis spectrophotometer, Unicam at 390 nm) of dichloromethane extracted samples, as described by Fusey and Oudot .
Measurement of the droplet size of dispersed oil: The oil droplet size distribution (diameter in microns) of CD1, CD2 and MD conditions were analyzed 6 hours after the beginning of fish exposure at a nominal concentration of 1250 mg/L. The measurements were performed by laser granulometry (Malvern Mastersizer 2000) based on the principle of Fraunhofer according to the intensity of diffracted radiation, whereby the diffraction angle dependent on the particle size. A water sample flow rate of 1200 mL/min and an obscuration of 10% were the conditions used during the measurements.
The LC50 values (the TPH concentration of the exposure media that caused the death of 50% of a group of test animals) were calculated using the trimmed Spearman-Karber method and a US EPA probit program, and expressed as values (lower 95% confidence interval - upper 95% confidence interval). The difference between MD, CD1 and CD2, concerning TPH concentration, was evaluated following the Quade test procedure: exposure media (MD, CD1 and CD2) were considered as treatment and the % of stock solutions (0%, 2.4%, 12%, 18%, 24%, 40%) were considered as blocks. Thus, the values obtained for each exposure environment at several dilutions were considered as repeated measurements. The statistical analysis was carried out using Systat 12 software and the significance of the results was ascertained at α=0.05.
The goal of this study was to simulate a possible scenario of dispersion of a drafting oil spill. For fish exposed to 0% of stock solution, no mortality was found. Moreover all physicochemical parameters, temperature (19.1 ± 0.2°C), pH (8.04 ± 0.03), dissolved oxygen (97.5 ± 0.9% of O2 satuation) and salinity (35.2 ± 0.0 PSU), remained constant throughout the experimental periodfor all exposure conditions. The possibility to maintain viable juvenile of Dicentrarchus labrax suggests that the experimental system makes possible to use early life stage in oil and dispersant toxicity assessment.
When measurements of total petroleum hydrocarbon concentration for mechanical (MD) and chemical (CD1 and CD2) dispersion are compared, it appears that the dispersant application increases significantly the concentration of TPH (mean over 24 hours) in the water column (Table 1). This result is in accordance with previous observations obtained in field operations and in situ experimentation [22,23]. Taken together and very logically, these results show that the transfer of petroleum from the surface to the water column is increased when dispersant is applied.
|% of stock solution||[TPH] (mg/L)||Fish mortality (%)||[TPH] (mg/L)||Fish mortality (%)||[TPH] (mg/L)||Fish mortality (%)|
|2.4||45 (65-25)||0||107 (118-96)||0||102 (130-74)||0|
|12||214 (235-196)||0||554 (659-449)||0||585 (647-523)||0|
|18||213 (293-133)||0||971 (1037-905)||50||744 (881-607)||0|
|24||306 (405-207)||0||1116 (1269-963)||100||964 (1050-878)||30|
|40||373 (443-302)||0||1547 (1542-1553)||100||1879 (1948-1810)||100|
|LC50 (mg/L)||n.c.||873 (782-976)||1227 (1091-1379)|
|The results are expressed as mean concentrations over 24 hours (concentration at T0 - concentration at T1). Respecting Quade test procedures, values obtained for each exposure condition at several % of stock solution are considered as repeated measure and *indicates significant differences (P < 0.05) of TPH concentrations between exposure conditions. LC50 values are expressed as values (lower 95% CI-upper 95% CI). n.d. = not detected. n.c. = not calculable.|
Table 1: Fish mortality (%). % of stock solution and total petroleum hydrocarbon concentration (mg/L) in sea water for mechanical dispersion (MD) and chemical dispersion (CD1 and CD2) during the 24 hour exposures.
Analysis of oil droplet size distribution showed a significant difference between the two chemically dispersed conditions and the mechanically dispersed condition (Table 2). This result can explain a part of the higher TPH concentration of CD1 and CD2 conditions compared to those observed in MD condition. Indeed, application of surfactant increases the bioavailability of oil . The two commercial formulations of dispersant used in this study had different surfactant concentrations. This difference could conduct to difference in oil bioavailability for the two chemical dispersion conditions Consequently, oil bioavailability could be one of the reasons of differences observed between MD, CD1 and CD2 and CD2 conditions in their fish mortalities.
|d (0.1)||d (0.5)||d (0.9)|
|CD1||2.0 ± 0.0 a||5.2 ± 0.0 a||12.5 ± 0.0 a|
|CD2||1.8 ± 0.0 a||4.1 ± 0.0 a||11.8 ± 0.2 a|
|MD||107.6 ± 0.9 b||227.9 ± 3.3 b||437.3 ± 11.4 b|
In this table, d (0.5), d (0.1) and d (0.9) correspond respectively to the median and the two deciles of a Normal distribution. (n = 7). Differences in letters indicate statistical differences between groups (p < 0.05).
Table 2: Size (µm) of oil droplets in CD1, CD2 and MD obtained with laser granulometry.
Without dispersant application a part of the oil slick could solubilised in the water column. The water soluble fraction (WSF) of oil is commonly used in dispersed crude oil toxicity tests. Thus, we exposed fish to this treatment in order to compare our results with those obtained in the literature. Preliminary results of this study suggest that chemically dispersed oil was more toxic than WSF of oil since no mortality was found for WSF whatever the percentage of stock solution tested (0, 2.4, 12, 18, 24 and 40% of stock solution in seawater for WSF). In our results, no TPH were detected for each % of stock solution tested. These results are in accordance with other studies using different experimental approaches [8,10,25- 27]. It can therefore, be concluded that our experimental system is suitable for assessing the toxicity of dispersant application.
A rapid decrease in TPH concentration is commonly observed in offshore field operations . At the opposite, since near-shore areas have a lower dilution potential and important natural mixing processes (e.g. wave action), natural dispersion of the oil slick can be maintained and the oil slick can even be displaced from the surface to the water column (as described by Lunel  during the Braer oil spill). For example, during Sea Empress and Braer oil spills, high concentration of oil was observed over more than one week (respectively  and . In this study, the experimental system was under static condition (e.g. without dilution due to water supply), consequently the evolution of TPH concentration depends on the turbulent mixing energy. This allows reproducing different scenarios making possible to simulate a natural dispersion in near-shore areas.
The device presented here makes possible to measure the evolution of the total petroleum hydrocarbon concentration during this period and our results show no important reduction of TPH concentration over the 24 hours of contamination (Table 1). Thus, the experimental approach succeeds in simulating dispersant application in near-shore areas.
Bioassays must be considered with caution due to the complexity of work with living material . Indeed, numerous settings could influence the results of the experimentation if they cannot be controlled. However, during an emergency context such an oil spill, bioassay could be relevant to evaluate the potential biological impact of operational response.
Our concern was to find an efficient and reliable method for rapid toxicity assessment of a dispersed oil spill even in near-shore areas. This study presents an experimental system designed to perform reproducible toxicity tests on small marine organisms and to simulate the increasing transfer of petroleum from the surface to the water column when dispersant is applied (comparing MD and CD1, CD2). Moreover comparisons of preliminary toxicity assays performed on juvenile fish with published literature suggest that the experimental system is suitable for assessing the toxicity of dispersant application. Nevertheless, in this system attention must be paid to the fact that a part of the evolution of TPH concentrations and size of oil droplets are dependent on the given turbulent mixing energy. This mixing energy can be changed to respond to different oil spill scenario.
Thus, the present experimental approach seems of interest in order to establish a comprehensive framework in an emergency context and especially to dispersant use during an oil spill. However, the natural environment is complex and full of interaction between biotic and abiotic parameters. These laboratory results can differ with in situ observations. Consequently, for a better comprehension of these interactions, these acute toxicity studies can be coupled with studies focused on the sub-lethal effects of oil-dispersant mixtures.
This study was supported by a PhD grant from the Conseil Général of the Charente-Maritime. The Agence Nationale de la Recherche is acknowledged for financial support for the project ‘DISCOBIOL’. The authors also acknowledge Total Fluides and Innospech for providing chemicals.
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