Fate and Effects of Emulsions Product after Oil Spills in Estuaries

Richard F. Lee, Principal Investigator

Abstract
Stable water-in-oil emulsions, often formed after oil spills, contribute to the difficulties of cleanup due to their persistence and high viscosity. The primary objective of our studies was to determine the fate and effects of such emulsions after they enter estuaries. To achieve this objective, a stable emulsion formed from Kuwait crude oil, as well as nonemulsified oil, was added to estuarine mesocosms, followed by exposure of the estuarine grass shrimp (Palaemonetes pugio) to treated sediments. The mesocosm studies showed that emulsion treated sediments had polycyclic aromatic hydrocarbon concentrations (PAH) of 284, 198, 89, 49 and 7 µg/g sediment on after Days1, 14, 28, 42 and 56, respectively, while non-emulsified oiled sediments had concentrations of 271, 14, 3, 0.3 and 0.2 ug/g sediments on these same days, respectively. Female grass shrimp exposed to Day 14 emulsified oiled sediments did not produce embryos and exposure to Day 36 sediments resulted in reduced embryo production. In contrast, grass shrimp exposed to non-emulsified oiled sediments were not affected with respect to embryo production compared to controls. A second assay exposed grass shrimp embryos to sediment pore water followed by determination of embryo hatching rates and DNA strand breaks. Comparable results were found between the two assays (embryo hatching/DNA strand breaks versus embryo production) with sediments from Days 14 and 36. The persistence of PAHs in emulsified oiled sediments, relative to nonemulsified oiled sediments, is the likely explanation of the prolonged toxicity of the emulsified oiled sediments. Sediments after oil spills may have higher PAH concentrations (6000 to 100,000 µg/g sediment) than were present in our mesocosm studies. If these PAH decreases occur at the same rate observed in our studies, then emulsified oiled sediments would show toxicity for several months if emulsified oil entered an estuary during a spill. The results of the present project provide expected rates of PAH decrease in estuarine sediments. They suggest that if emulsified oil is not prevented from entering an estuary, water-in-oil emulsion can result in prolonged toxicity to grass shrimp. Grass shrimp can comprise up to 56% of the biomass of pelagic macrofauna in estuarine tidal creeks and are important in the diet of many estuarine fish. Methods to prevent formation of emulsions or emulsified oil from entering an estuary should be considered during an oil spill. The project also developed a pore water assay to rapidly assess the genotoxicity of sediment in large coastal areas and assess the presence of biological “hot spots” which require more attention. The sediment pore water assay can also be used to determine the recovery rate of grass shrimp in heavily oiled sites.

Keywords: Oil, Emulsions, Toxicity, Sediment, Comet Assay

Acknowledgements
Funding for this work was provided by the NOAA/UNH Coastal Response Research Center(Grant number 03-688). Karrie Brinkley, Ullrich Wartinger and Keith Maruya from Skidaway Institute of Oceanography were involved in various aspects of the project.

Table of Contents



1.0    Introduction
One of the consequences of oil spills in coastal waters can be the formation of stable water-in-oil emulsions with mixing energy provided by wind and tides (Daling et al., 2003; Fingas et al., 2001; Lee, 1999; Lunel et al., 1996). Stable water-in-oil emulsions are characterized by their persistence (5 days or longer), high water content (50 to 90%), high viscosity, small water droplets and higher density than the original oil (Brandvik and Daling, 1991; Fingas et al., 1994). The high viscosity results in a semi-solid or gel which contributes to the persistence of the emulsified oil and makes cleanup more difficult.

A variety of compounds and mixtures have been shown to promote and stabilize these emulsions, including sea water particulates, and fractions or compounds found in crude oil (Payne and Phillips, 1985; Lee, 1999 and references cited therein). It appears that compounds with higher solubility in the oil phase than in the aqueous phase (e.g., nickel porphyrins) are the emulsifying agents that can promote stable water-in-oil emulsions. Crude oils that form very unstable emulsions (e.g., Gullfaks crude from the North Sea) require weathering before stable emulsions are formed. The weathering may cause the formation of colloidal asphaltene particles and highly polar compounds that contribute to emulsion stabilization.

Payne et al. (1983) added Prudhoe Bay crude oil to a large wave tank and found that emulsification did not occur until a significant amount of evaporation had taken place to remove lower molecular weight compounds. Payne and Phillips (1985) found that most oils that formed emulsions in real spill events also formed stable emulsions in the laboratory. Spills involving oils with relatively high asphaltene concentrations (e.g.Torry Canyon, Tanker Arrow and IXTOC-1) produced stable water-in-oil emulsions (Payne and Phillips, 1985). In contrast, Ekofisk crude, which is low in asphaltenes (0.03%), formed unstable emulsions (Audunson, 1978). The work reported here supports the earlier studies of Payne and Phillips(1985) concerning the importance of asphaltenes in the oil, particularly porphyrins, in producing stable emulsions. We further showed that for Prudhoe crude oil that particles in the surrounding ocean water are also important in producing emulsions

Stranded oil is toxic to a variety of marine invertebrates including crustaceans, polychaetes and mollusks(Glemarec and Hussenot, 1982; Jackson et al., 1989; Lee et al., 1981; McGuiness, 1990; National Research Council, 1985; Peterson, 2000; Sanders et al., 1980). While many of the spilled oils formed emulsions, it is not clear from these field studies if exposure to emulsified oil produced different effects than exposure to non-emulsified oils.

The studies covered in this report include determination of the stability and properties of water-in-oil emulsions formed by five crude oils, photo-oxidation products in emulsions after solar exposure, and fate and effects of an emulsified and non-emulsified oil added to estuarine mesocosms.

Since emulsions often form after oil spills, they present major problems for effective cleanup after the spill, due to their persistence and physical properties. The results of this project provide information on the effects and fate of emulsified oil entering which can be used when decisions are made concerning cleanup and use of dispersants after an oil spill has occurred.

2.0    Objectives
The primary objective of this project was to determine the fate and effects of water-in-oil emulsions formed after oil spills in estuaries. Specific objectives to achieve this overall objective included determination of the: 1) stability and properties of water-in-oil emulsions formed from different crude oils; (2) changes over time of the concentrations of polycyclic aromatic hydrocarbons (PAHs) in estuarine sediments after the addition of emulsified/non-emulsified oils; and (3) changes over time in the toxicity (reproduction/embryo development/ DNA damage assays) of estuarine sediments to grass shrimp after the addition of emulsified/non-emulsified oils.

3.0 Methods
Preparation of water-in-oil emulsions
Water-in-oil emulsions were prepared by rotating 500 ml cylindrical separatory funnels containing 300 ml of estuarine water and 30 ml of Kuwait crude oil. The rotating apparatus run at 30 rpm for up to 24 hrs was similar to the one described by Hokstad et al. (1993). Water contents of the emulsions were noted at 0.5, 1, 2, 4, 6, 8, 12 and 24 hours and the data used to calculate an uptake t½ [defined by Hokstad et al. (1993) as the time needed to pick up half the maximum water content]. Water droplet sizes in the emulsion were determined with an Olympus microscope (Model 1X50) equipped with an image analyzer system and software (Image Pro Plus; Silver Springs,MD). Both white and blue fluorescence light were used in determining water droplet diameters.

Addition of emulsified and non-emulsified oil to mesocosms
The mesocosms used were 1m deep with a volume of 300 m3 and each contained approximately 1000 kg of estuarine sediment (Skidaway River estuary). A water-in-oil emulsion (360 ml of emulsion) made from Kuwait crude oil as described above was added to a 30 cm x 30 cm section in the center of the mesocosm. To a second mesocosm, non-emulsified Kuwait crude oil (360 ml) was added. A third mesocosm, where no oil was added, served as a control.

Analysis of PAHs in sediment
Sediment cores were taken from the control mesocosm and the oil treated mesocosms at Days 1, 14, 28, 42, 56, 70 and 84. Cores were sectioned at 0-3 cm and 3-6 cm. Procedures for the analysis of sediment sediment cores for PAHs were similar to those described earlier (Maruya et al., 1997). Sediment samples (10g) from each core were freeze dried, extracted with methylene chloride and extracts passed through a silica gel column. The PAH fractions from the silica gel column were analyzed with a Hewlet Packard 6890 Series Plus gas chromatograph coupled to a 5973 mass spectrometer. Twenty three individual PAH analytes were quantified with a detection limit of 10 ng/g for each analyte. Except for naphthalene, recovery of spiked PAH standards ranged from 89 to 95%, while naphthalene recovery ranged from 55 to 67%. Procedural blanks, NIST sediments, spiked matrices and replicate samples were analyzed using the same procedures outlined above. For PAH spiked sediments, recoveries were between 90-100%.

Grass shrimp reproduction assay
For this assay, approximately 1 kg of sediment taken from each mesocosm (control, treatment with emulsified Kuwait crude oil addition and treatment with non-emulsified Kuwait crude) was subdivided into three parts and each part added to one of three aquaria, followed by addition of estuarine water (40 L to each aquarium) and grass shrimp (20 shrimp to each aquarium) collected from the nearby estuary (Skidaway Island, GA). Sediments for testing were taken on Days 14, 36 and 58. The salinity of the water was 25-27 ppt and the temperature of the aquaria was maintained at 27oC with heaters. The grass shrimp were fed frozen Artemia sp. and kept under a l2 h light/12 h dark regime. Every 5 days the following parameters were determined in each aquarium: (a) mortality; (b) number of females with mature ovaries; and (c) number of females with attached embryos. Grass shrimp were exposed to sediments for 58 days with no water change. An egg bearing female from each of the three aquaria were removed and 48 embryos (stage 8) from each female were transferred to 48 well polystyrene plates with each well containing 1.1 ml of estuarine water and 1 embryo. These culture plates were kept in the dark at 27oC and the number of embryos hatched determined by daily examination of the plates under a dissecting microscope. Hatching was generally completed within 48 h after transfer to culture plates. Data is reported as mean ± standard deviation (n=3; 3 aquaria for each test sediment)

Pore water assay
Pore water was collected from oil treated and reference mesocosm sediments (Days 1, 7, 14, 28, 49 and 87) by centrifugation at 1500 x g. Approximately 3 ml of pore water was collected from 40 g of sediment. Forty eight stage 7 (undergoing organogenesis) embryos [Winston et al. (2004) described grass shrimp embryo stages] were used for the pore water assay. Grass shrimp embryos were removed with forceps from egg bearing females. One grass shrimp embryo (24 embryos for each time period) was placed in each well of a polystyrene tissue culture plate with each well containing 1.1 ml of diluted sediment pore water [1:10 diluted with filtered (0.25 µm filter) estuarine water]. Plates were kept at 27oC in an incubator in the dark. Hatching rates [percentage of embryos which hatched into the zoea stage (stage 12)] were determined by daily examination of embryos in the plates under a dissecting microscope.

Comet assay for pore water assay
Twenty embryos (Stage 7) from the same female used for the hatching test described above were added to a beaker containing 20 ml of diluted pore water (1:10 diluted with estuarine water) from each mesocosm and incubated at 27oC for 24 hours. The embryos were removed from the beaker after 24 hours and used for the comet assay. The procedures for the comet assay were a modification of those described by Singh et al. (1998) and Steinert et al. (1998). Embryos were placed in a microcentrifuge tube on ice, followed by addition of 1 ml of 4oC filtered estuarine water to each tube. Embryos were homogenized in a 1 ml ground glass tissue homogenizer and left to settle at 4oC for 5 min. During this settling, individual cells remained in suspension while intact tissue pieces and fragments sank to the bottom. A portion of the supernatant (800 µl) was transferred to a second microcentrifuge tube, followed by centrifugation at 14,000 x g for 2 min at 4oC. After removal of the supernatant, the pelleted cells were resuspended in 70 µl of 0.65% low-melting point agarose in Kenny’s salt solution (0.4 M NaCl, 9 mM KCl, 0.7 mM K2HPO4, 2 mM NaHCO3), cooled to ~25oC after melting. This suspension was pipetted onto a slide coated with 1% agarose and a coverslip applied. The agarose was allowed to harden on ice-cold trays for 3 min. Slides were transferred to lysis buffer (2.5 M NaCl, 0.1 M EDTA, 0.01 M TRIS-HCl, 10% dimethyl sulfoxide, 1% Triton X100, pH 10). After incubation overnight at 4oC, slides were rinsed with water (4oC). After this water rinse, slides were placed in unwinding buffer (0.2 M NaOH, 1 mM EDTA, pH>13.5, 4oC) for 15 min. Electrophoresis of slides in the unwinding buffer was carried out at 300mA and 25V for 20 min. At the end of the electrophoresis run slides were neutralized by rinsing them in 0.4 M TRIS-HCl (pH 7.5) and gels were fixed by rinsing in 100% ethanol (4oC) for 5 min. Once dry, cells were stained with 20 µl of ethidium bromide (20 µg/ml). DNA strand breaks were determined by quantitation of fluorescence (excitation at 540 nm) in the head and tail of 50 randomly chosen cells from each replicate slide using an inverted fluorescent microscope (Nikon Eclipse E400), high sensitivity charged coupled device (CCD) camera and an image analysis system (Komet 4.0 - Kinetic Imaging Ltd.; Nottingham, UK). Data is reported as the mean (percent of DNA in the tail) ± SEM (n=5) where 50 cells/slide were counted. Datasets were compared using 1-way ANOVA followed by Tukey’s post-test. Statistically significant differences were expressed as P < 0.05 or P < 0.01.



4.0 Results
Emulsion formation
Emulsions were made from a number of crude oils (Monterey, Prudhoe Bay, Kuwait, south Louisiana, fuel oil No.5 from Venezuela, Tia Juana) and their properties determined, including water content, time to reach half maximum water content, water droplet size and stability (Table 1). Fingas et al. (1994) defined stable emulsions as those where the water persists for at least 5 days. Stable emulsions are also characterized by a high water content (50 to 90%), high viscosity, small water droplets (1 to 10 µm) and higher density than the original oil (Brandvik and Daling, 1991; Fingas et al., 1994). Based on these parameters, Monterey, Kuwait, Prudhoe Bay and Fuel oil no. 5 formed stable water-in-oil emulsions.

Prudhoe Bay (North Slope) crude formed stable emulsions with estuarine water, but not with filtered estuarine water, indicating the importance of water particulates in the formation of stable emulsion with this crude (Figs.1 and 2). Unstable water-in-oil emulsions were characterized by large water droplet size (=20 µm) and maximum water uptake of less than 40%. For example, South Louisiana crude formed unstable emulsions with average water droplet size of 52 µm and water content of 22% 12 hours after mixing. A very stable emulsion was formed from Kuwait crude with a mean water droplet size of 3.1 ± 1.5 µm and water content of 95% (Figs. 3-5). Payne and Phillips (1985) found that most oils that formed emulsions in real spill events also formed stable emulsions in the laboratory.

Addition of Oil to Mesocosms
Four days after addition to estuarine sediment the emulsified Kuwait oil is highly viscous and remained firmly adsorbed to the sediment while the nonemulsified oil was mostly washed away by the tide and only an oily stain remained on the sediment (Fig.6).

a. Initial Toxicity Studies
Initial laboratory toxicity work showed high mortality within 48 hours when sediments treated with 200 ul of emulsified Kuwait oil were added to 100 g of sediment, but no acute toxicity, i.e no mortality, when 100 µl or 50 µl of emulsified oil were added. To avoid acute toxicity, based on these initial toxicity experiments, we added 360 ml of Kuwait emulsion to one mesocosm (~1000 kg of sediment), 360 ml of nonemulsified Kuwait oil to a second mesocosm and a third mesocosm that received no oil served as a control.

b. Changes in PAH Concentrations
The changes in total and individual PAH concentrations in the oil treated mesocosms are given in Fig. 7 and 8. The data shown are only for the upper core sections (1-3 cm), since elevated PAH concentrations were not observed in oiled sediments for the 3-6 cm sections. Total PAH concentrations decreased with time in both the emusified and non-emulsified oiled sediments, but the decrease was much more pronounced in the non-emulsified oiled sediment (Fig. 7). Oiled sediments showed a typical petrogenic PAH profile with a high concentrations of lower weight alkalated PAHs (e.g. alkylated naphthalenes and phenanthrenes) (Fig 8). The unoiled control sediment showed a low background concentration of total PAHs (0.1 µg/g sediment) composed primarily of high molecular weight pyrogenic type PAHs.

Total sediment PAH concentrations at Day 1 in the control, emulsified oil and non-emulsified oil treatments were 0.1, 271 and 284 µg/ g sediment, respectively, while on Day 14, PAH concentrations were 0.1, 14 and 198 µg/g sediment, respectively (Fig. 7). By Day 28, the non-emulsified oiled sediment had low total PAHs (3 µg/g sediment) with little evidence of petrogenic PAHs, while the the emulsified oiled sediment still had relatively high total PAH concentrations (89 µg/g sediment) including high concentrations of petrogenic PAHs. While total PAHs had decreased to 2 µg/g sediment by Day 84 in the emulsified oiled sediment there was still evidence of petrogenic type PAHs in the sediment.

A number of oxidized PAHs were detected in the oiled sediments at Days 14 and 28, including phenanthrene quinone, dibenzothiophene-S-oxide, 9-fluorenone,anthraquinone, benzanthrone, benz(a)anthraquinone, hydroxyfluoranthene (Fig. 9; Lee, 2003). The presence of these oxidized PAHs indicates that a variety of oxidation processes, e.g. photo-oxidation, was acting on the stranded oil.

c. Reproduction and Embryo Development Production
The effects of exposure to oiled and non-oiled sediment on grass shrimp reproduction, embryo production and embryo hatching are given in Table 2. A photograph of a female grass shrimp with attached embryos is shown in Fig. 10. Exposure of female shrimp to Day 14 emulsified oiled sediments resulted in no embryo production. Embryo production was reduced after exposure to Day 36 sediment compared with controls and there were no effects on reproduction or embryo production after exposure to Day 58 sediment. Quite contrasting results were observed when female shrimp were exposed to Days 14, 36 and 58 nonemulsified oiled sediments. There were no effects on reproduction, embryo production or hatching.

d. Pore water tests
The effects of pore water from oiled and nonoiled sediments on embryo DNA (strand breaks - comet assay) and embryo development (hatching) are reported in Table 3. The comet assay detects DNA strand breaks and alkali labile sites by measuring the migration of DNA from immobilized nuclear DNA (Singh et al., 1988; Fig. 11). Sediment pore water from Day 14, 36 and 58 emulsified oiled sediments caused more DNA strand breaks and reduced embryo hatching than pore water from either control or nonemulsified oiled sediments. DNA strand breaks and embryo hatching were not significantly different on Day 80 in control, emulsified and nonemulsified oiled sediments.



5.0 Discussion
Major findings of the project included a determination that emulsified oil persisted in estuarine sediments and that grass shrimp embryo production was reduced by exposure of females to emulsified oiled sediment. PAHs, which remained at high concentrations in emulsified oiled sediment, were assumed to be the cause of effects on shrimp embryo reproduction. Embryo production was not affected in grass shrimp exposed to nonemulsified oiled sediments. While PAH concentrations of emulsified oiled sediments were higher than nonemulsified oiled sediments (198 and 14 µg of total PAHs/g sediment on Day 14 in nonemulsified and emulsified sediments, respectively), even the highest concentrations were relatively low compared with the very high PAH concentrations found after oil spills. Concentrations of 6000 to 100,000 µg PAHs/g sediment have been reported in oiled intertidal sediments after oil spills (Lee and Page, 1997). If the initial concentration of stranded emulsified oil was 10,000 µg PAHs/g sediment, then after 70 days the sediment PAH concentration would be 140 µg/g sediment, assuming the same first order kinetics as observed in our experiments and other PAH degradation studies (e.g. Lee and Ryan, 1983). A PAH concentration of 140 µg/g sediment would be expected to affect both grass shrimp reproduction and embryo development (See data in Fig 7 and Table 2). Sediment PAH concentration in the case of sediment receiving non-emulsified oil would be less than 14 µg PAHs/g sediment after 70 days, a concentration not expected to affect grass shrimp reproduction. In addition, during an actual spill, the emulsified oil would not enter the estuary at a single time point but would likely enter repeatedly enter at different times. Thus, it seems likely that stranded emulsified oil, where initial PAH concentrations are very high, would show toxicity for several months after the spill.

A very rapid (1-2 days) and inexpensive assay, the sediment pore water assay, was used to assess genotoxicity and embryotoxicity in oiled and unoiled sediments (Table 3). This assay determines DNA strand breaks and embryo hatching after exposure of grass shrimp embryos to sediment pore water. The results with the pore water assay were quite comparable to the results obtained with reproduction/embryo production assay (compare results in Tables 2 and 3). Thus, it appears that the inexpensive and rapid pore water assay could be used to rapidly assess the genotoxicity of sediments over a broad area after an oil spill. This would allow a search for “hot spots” in a coastal area where a spill has occurred and also allow rapid monitoring for recovery, e.g decreased toxicity after an oil spill.

Possible use of project results for oil spill responses
There are several potential applications of the project’s results for oil spill response. One of the models (ADIOS) used after spills was developed by the HAZMAT /NOAA group in Seattle. ADIOS is used to provide answers as to the path of an oil spill based on winds, currents and the oil’s properties, including an emulsification constant. ADIOS may predict that an oil spill will enter a particular estuary, however, it does not provide information as to what happens to oil once it enters the estuary. Thus, our data show the rate of disappearance of both emulsified and non-emulsified oil that enters estuaries. One application of our project is to provide support for the prevention of the entrance of oil into estuaries (e.g.,use of dispersants, floating booms), particularly estuaries with endangered species and/or commercial shellfish. If emulsified oil enters estuaries, the oiled sediments can be toxic for many months after the spill. A second application of our project’s results is the use of the sediment pore water assay to rapidly assess the genotoxicity of sediment in a large coastal area to assess sites within the area posing the greatest threat to the biota. Grass shrimp can comprise up to 56% of the biomass of pelagic macrofauna in estuarine tidal creeks and are important in the diet of many estuarine fish (Reinsel et al., 2001; Scott et al., 1992). A third application, also using the sediment pore water assay, would be the determination of the recovery rate of heavily oiled sites.

6.0 Technology Transfer
The results of the work on the persistence and prolonged toxicity of emulsified oil have been submitted to Environmental Chemistry and Toxicology. They will also be submitted to the next annual meeting of the Society of Environmental Chemistry and Toxicology. A summary of the results with emulsified oil and its fate and effects were presented in a talks/workshops at the Hazardous Materials Response Division (HAZMAT) of the Office of Response & Restoration of NOAA in 2004 and 2005. This group developed the ADIOS software which allows a prediction of the path and fate of an oil spill, based on properties of the oil as well as currents and wind. In the directions for using this software, it is noted that many oil weathering processes depend on the extent of emulsification. In the instruction to ADIOS users it is noted that little information is available to calculate the emulsification constant. Our work described procedures to determine emulsification parameters for a particular oil (water content parameters, water droplet size and emulsion stability) and we list these parameters for a number of widely transported oils and whether they form stable emulsions. The results of this oil emulsification work should be of use of the next version of the ADIOS software. A talk on our emulsification work, the results of which are included in this final report, was given at HAZMAT in Seattle, WA. In addition, a summary of the work given at the HAZMAT workshop, on the oxidation of PAHs in crude oil, was published (Lee, 2003).


7.0 Achievement and Dissemination
Manuscripts
1. Lee RF. 2003. Photo-oxidation and photo-toxicity of crude and refined oils. Spill Sci. Technol. 8:157-162.
2. Lee RF, Maruya K, Warttinger U, Brinkley K. Prolonged toxicity of emulsified oil in estuarine sediments. Environ. Toxicol. Chem. (to be submitted)

Workshops
Workshop on Oil Emulsification. Hazardous Materials Response Division (HAZMAT) of the Office of Response & Restoration of NOAA(Seattle, WA; April, 2004, 2005). 8 participants

Graduate Students
Ulrich Warttinger, Fate of Emulsified Oil, Master’s Degress at Mannheim Fachhochschule (Germany) 2005, presently Ph.D. student in environmental science at University of South Carolina.

References
Audunson T. 1978. The fate and weathering of surface oil from the Bravo blowout. In: Proceedings of the Conference on Assessment of Ecological Impacts of Oil Spills.American Institute of Biological Sciences, Washington, DC, pp. 445-475.

Brandvik PJ, Daling PS. 1991. W/O-emulsion formation and w/o-emulsion stability testing – an extended study with eight oil types. DIWO Report No. 10, IKU Sintef Group, Trondheim, Norway

Daling, P.S., M. Ø. Moldestad, Ø. Johansen, Al. Lewis and J. Rødak, 2003. Norwegian testing of emulsion properties at sea – The importance of oil type and release conditions. Spill Science & Technology Bulletin 8: 123-136.

Fingas MF, Fieldhouse B, Mullin J. 1994. Studies of water-in-oil emulsions and techniques to measure emulsion treating agents. In: Proceedings of the Arctic Marine Oilspill Program Technical Seminar. Environment Canada, Ottawa, Ontario, pp. 213-244.

Fingas, M.F., B. Fieldhouse, J. Lane,and J.V. Mullin. 2001. What causes the formation of water-in-oil emulsions. In: Proceedings of the 2001 International Oil Spill Conference. American Petroleum Institute, Washington, DC, pp. 109-114.

Glemarec M, Hussenot E. 1982. A three-year ecological survey in Benoit and Wrac’h Abers following the Amoco Cadiz oil spill. Netherlands J Sea Res 16:483-490.

Hokstad JN, Daling PS, Lewis AS, Shrom-Kristiansen T. 1993. Metholodology for testing water-in-oil emulsions and demulsifers. Description of laboratory procedures. In Walker AH, Ducey DL, Gould JR, Orvik SB, eds, Formation and Breaking of Water-In-Oil Emulsions: Workshop Proceedings. MSRC Technical Report Series 93-018. Marine Spill Response Corp., Washington, DC, pp 239-353.

Jackson JBC, Cubit JD, Keller BD, Batista V, Burns K, Caffey HM, Caldwell RL, Garrity SD, Getter CD, Gonzalea C, Guzman HM, Kaufman KW, Knap AH, Levings, SC, Marshall MJ, Steger R, Thompson RC, Weil E. 1989. Ecological effects of a major oil spill on panamanian coastal marine communities. Science 243:37-44.

Lee RF. 1999. Agents which promote and stabilize water-in-oil emulsions. Spill Sci. Technol. Bull. 5: 117-126.
Lee RF. 2003. Photo-oxidation and photo-toxicity of crude and refined oils. Spill Sci. Technol. Bull. 8:157-162.

Lee RF, Page DS. 1997. Petroleum hydrocarbons and their effects in subtidal regions after major oil spills. Mar Pollut Bull 34:928-940.

Lee RF, Ryan C. 1983. Microbial and photochemical degradation of polycyclic aromatic hydrocarbons in estuarine waters and sediments. Can. J. Fish. Aquatic Sci. 40(Suppl. 2): 86-94.

Lee RF, Dornseif F, Gonsulin F, Tenore K, Hanson R. 1981. Fate and effects of a heavy fuel oil spill on a Georgia salt marsh. Mar Environ. Res 5:125-143.

Lunel, T., J. Revin, N. Bailey, C. Halliwell and L. Davis. 1996. A successful at sea response to the Sea Empress spill. In: Proceedings of the 19th AMOP seminar, June12-14, 1996 Canada, pp. 1499-1520.

Maruya KA, Loganathan BG, Kannan K, McCumber S, Lee R. 1997. Organic and organometallic compounds in estuarine sediments in the Gulf of Mexico (1993-1994). Estuaries 20:700-709.

McGuinness KA. 1990. Effects of oil spills on macro-invertebrates of saltmarshes and marine forests in Botany Bay, New South Wales, Australia. J Exp Mar Biol Ecol 142:121-135

National Research Council. 1985. Oil in the Sea: Inputs, Fates, and Effects. National Research Council, National Academy Press, Washington, DC.

Payne, JR, Phillips CR 1985. Petroleum spills in the marine environment. In: The Chemistry and Formation of Water-in-Oil Emulsions and Tar Balls. Lewis Publishers, Chelsea, Michigan, p. 148.

Payne JR, Kirsten BE, McNabb GD, Lamback JL, De Oliveira L, Jordan RED, Hom W. 1983. Multivariate analysis of petroleum hydrocarbon weathering in the subarctic marine environment. In: Proceedings of the 1983 Oil Spill Conference. American Petroleum Institute, Washington, DC, pp 423-434.

Peterson CH. 2000. The Exxon Valdez oil spill in Alaska: acute, indirect and chronic effects on the cocystem. Adv Mar Biol 39:3-84.

Reinsel KA, Glas PS, Rayburn JR, Pritchard MK, Fisher WS 2001. Effects of food availability on survival, growth ands reproduction of the grass shrimp Palaemonetes pugio: a laboratory study. Mar Ecol Prog Ser 220:231-239.

Sanders HL, Grassle JP, Hampson GR, Morse LS, Garner-Price S, Jones CC. 1980. Anatomy of an oil spill: long-term effects from the grounding of the barge Florida off West Falmouth, Massachusetts. J Mar Res 38:265-380.

Scott GI, Fulton MH, Crosby MC, Key PB, Daugomah JW. 1982. Agriculture insecticide runoff effects on estuarine organsisms: correlating laboratory and field toxicity tests, ecophysiology bioassays and ecotoxicological biomonitoring. US Environmental Proection Agency, Gulf Breeze Environmental Research Laboratory, Gulf Breeze, FL.

Singh NP, McCoy MT, Rice RR, Schneider A. 1988. A simple technique for the quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184-191.

Steinert SA, Streib-Montee R, Leather JM, Chadwick DB. 1198. Dna damage in mussels at sites in San Diego Bay. Mutat Res 399:65-85.

Winston GW, Lemaire DGE, Lee RF 2004. Antioxidants and total oxyradical scavenging capacity during grass shrimp, Palaemonetes pugio, embryogenesis. Comp Biochem Physiol 139C:281-288.