Opportunities for Environmental Applications of Marine Biotechnology: Proceedings of the October 5-6, 1999, Workshop (2000)

Ralph J. Portier

There is an estimated 3.2 million tons annual (mta) input of petroleum hydrocarbons into the world's oceans (NRC 1985). The majority is in small amounts from chronic sources, 0.7 mta from tanker operations, and 0.7 mta from municipal wastes. Accidental spills account for 0.42 mta, just 13% of the world's total input of petroleum hydrocarbons. The chronic, small amounts of oil are rapidly removed from the marine environment by a variety of processes—evaporation, dissolution, biodegradation, emulsification, and sedimentation—in a matter of days in normal conditions. When there is an accidental spill from oil production or transport leading to a large lens of visible brown/black oil, the environment 's natural capacity for self-purification is overwhelmed. The oil may persist for months if not decades. Serious acute and chronic ecological damage can occur, and economies and community health can be affected (Atlas and Bartha 1973; Kelso and Kendziorek 1991; Overton and others 1994). Because of the danger to health, ecology, and public relations represented by large oil spills that overwhelm natural capacity for purification, new marine biotechnology approaches are needed to move the “technology” forward for cleaning up impacted coastal and marsh environments.

The fate of petroleum hydrocarbons in the marine environment has been documented by Bartha (1986). A small oil spill will spread out until

Aquatic/Industrial Toxicology Laboratory, Institute for Environmental Studies, Louisiana State University, Baton Rouge, LA

it is just a sheen on the water surface; 1 g will cover 1-10 m². This thin film will be evaporated, emulsified, metabolized, or dissolved. Depending on temperature, mixing conditions and composition of the oil, 10-55% will be lost through evaporation and photo-oxidation (Baker and others 1993; Walker and others 1993). The more polar fractions of the oil, carbon lengths 12 and less (≤ C-12), will dissolve, ultimately to be metabolized by naturally occurring bacteria (Overton and others 1994). Natural processes will emulsify the remaining oil or it will have an impact on the sea bottom or marsh environment. If the oil undergoes emulsification and natural dispersion, then within 2 months, the bioavailable hydrocarbons will be metabolized, leaving behind a highly condensed, recalcitrant residue of complex hydrocarbons called asphaltenes and resins (Bartha 1986; Stewart and others 1993).

If conditions are poor for emulsification and dispersion of the oil, typical for marsh environments, it may emulsify only partly, forming a mousse, which is an oil-in-water emulsion (up to 80% water, depending on the oil) that is highly resistant to degradation. Mousse has been known to persist in sediments for decades (Atlas 1981; Baker and others 1993; Bartha 1986; NRC 1985).

Oil spills affect ecosystems in three ways: smothering plants and animals, massive input of organic carbon upsetting nutrient cycling, and toxicity (NRC 1985).

• Smothering. Smothering of plants and animals comes about due to oil's physical characteristics—its stickiness, buoyancy, and oleophilicity.

• Disruption of nutrient cycling and microbial diversity. The normal nutrient cycle will be disrupted by the massive influx of hydrocarbon. This will exert a selective pressure on the microbial biota for petroleum hydrocarbon degradation (Bartha 1986). This selection pressure will change the natural biodiversity, perhaps changing the flow of energy through the marine food web and ultimately changing what food sources are available to higher organisms.

• Toxicity. Oil exerts its toxic effects primarily through its water-soluble fractions. Hydrophobic fractions will exert toxic effects only if swallowed or adhered to the skin where hydrophobic compounds can dissolve into lipophilic tissues. The water-soluble fractions are more toxic because they dissolve in the water, thus coming into contact with marine biota not near the oil spill. As the more complex and less soluble compounds are oxidized in metabolism and photo-oxidation, they become water soluble and begin to affect the biota. Effects seen with toxic hydro-

carbon and hydrocarbon residues are changes in respiration, growth, reproduction, behavior, calcification, molting, ion transport, and enzyme activity (NRC 1985).

Oil spill response aims to prevent damaging effects by removing the oil from the endangered environment. A variety of spill-response methods exist and are generally broken down into two classes:

• Mechanical response. Mechanical response at sea is the use of booms and other physical devices to contain and aid in physical recovery of the oil. This method has rarely been used to its full theoretical capability due to bad weather, sea state, or logistical problems related to the volume of oil spilled in a catastrophic accident.

• Chemical response. Chemical response to oil spills at sea consists of applying dispersants to disperse the oil as tiny droplets into the water. This was used to great effect in the spill from the Sea Empress off the coast of Wales in February 1966 (Lunel and others 1997). Some success has also been achieved with surfactant beach cleaners that are designed to lift oil from beaches without dispersing it (Prince and others 1999).

However, there was and continues to be concern over the combined effect of oil and dispersants (George-Ares and others 1999; Wolfe and others 1998). Although dispersants are no longer more toxic than the oil they are supposed to remediate, they will increase the toxic effect of the oil. As stated above, it is primarily the water-soluble fraction of the oil that is toxic because of its transport through water to the organism. For the normal oil slick on the marsh surface, only the organisms near the air/ water interface of the oil will encounter high concentrations of toxins. When the oil has been dissolved into the water column, as happens with dispersants, deep water biota not normally affected by oil spills will encounter oil. The current thinking on spill response to coastal marine environments is summarized in Table 1.

The development of commercial inocula for industrial wastewater biotreatment is a mature industry. Microbial products are used daily by coastal zone industries to treat elevated wastewater discharges into littoral environments. Most of these products are adapted microflora packaged on a pasteurized wheat bran base. Minimal toxicological testing of these products has been conducted to date. Similar products proposed for use in oil spill response in these coastal environments have undergone a comprehensive series of tiered tests under federal guidelines (Portier 1991). Few products have been approved to date for US Coast Guard use in impacted marsh environments. Biologicals include the aforementioned whole cell products, enzyme preparations, co-oxidizing substrates, modifying agents, and nutrient amendments. There is a need to further expand the type, efficacy, and total number of such products available for marsh restoration. Critical needs for additional research are summarized in Table 2.

With the development of a more efficacious battery of biologicals, engineered systems that deliver the novel biotech product with precision and minimal impact are also needed. Current protocols for delivering biologicals are rather primitive. Mechanical sprayers are the current state of the art. Engineered systems are needed for preinvasive response to oiling and post-oil ablation. A robust screening protocol to test candidate engineered systems must be developed for the unique marsh habitat. Engineered systems approved for a neritic/pelagic environment may not be appropriate for the littoral environment. Positioning equipment that delivers biological and/or combination products with minimal marsh impact are still needed. Finally, spill response companies must be weaned off expensive, lucrative, but hopelessly ineffective booming and chemical treatment strategies (Portier and Ahmed 1988).

If a better biological coupled to an acceptable engineered system can be realized from marine biotechnology research, the question one must then pose is “How can we assess the efficacy of treatment? ” There has always been a linkage between spill response and analytical instrumentation. Traditional gas chromatography/mass spectroscopy protocols have been developed for assessing and fingerprinting oil, yet the instrumentation is bulky and not relatively mobile. A prototype portable device is under final field testing and will be available in 2000 (Overton and others 1994). However, this device is really the first of a new generation of handheld sophisticated tools for assessing impact from a spill. A summary of analytical instrumentation development linked to marine biotechnology research programs appears in Table 3.

Finally, there still is a need to predict risk and relative impact. Assuming logistics and intervention approaches have become more sophisticated through the years, there continues to be the problem of developing the environmental management tools to determine when and if a marine biotechnology delivery system will minimize and/or facilitate postspill remediation (Portier and Ahmed 1988; Smith and Portier 1997). Biological assays are effective tools in assessing impact from point-source wastewater discharges or from impacted soils. Few assays are available for assessing acute and chronic toxicity of benthic and marsh habitat. A battery of sophisticated, possibly genome-based, assays need to be developed for marsh grasses, marsh mammalian populations, microorganisms, and crustacea (Lee and Portier 1999; Lin and others 1999).

Marine biotechnology approaches can play a pivotal role in developing strategies for prevention and/or postevent restoration of marsh habitats. The focus for the past few decades has been on crude oil and refined petroleum products. Domestic sewage and small volume-generated point

sources pose greater threats to the coastal marsh environment annually. Thus, new tools will be needed to assess, model, prevent, and restore spills in our nation's coastal zone. To summarize, the following actions should be considered for fundamental research in marsh restoration:

• Establish linkages to existing National Science Foundation centers to further develop novel biologicals for spill response.

• Establish a program review on biotechnology products/engineered systems assessment and approval for field applications.

• Continue to look for low-tech or “no” tech approaches based on risk assessment strategies.

Atlas R. 1981 Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiolog Rev 45:180-209.

Atlas R, Bartha R. 1973 Stimulated biodegradation of oil slicks using oleophilic fertilizers Environ Sci Technol 7:538-541.

Baker J, Little D, Owens E. 1993 A review of experimental oil spills. In: Proceedings of 1993 Oil Spill Conference, American Petroleum Institute, Washington, DC. p 583-590.

Bartha R. 1986 Biotechnology of petroleum pollutant biodegradation. Microb Ecol 12:155-172.

George-Ares A, Clark JR, Biddiner GR, Hinman ML. 1999 Comparison of test methods and early toxicity characterization for five dispersants. Ecotoxicol Environ Safety 42:138-142.

Kelso D, Kendziorek M. 1991 Alaska's response to the Exxon Valdez oil spill. Environ Sci Technol 25:16-23.

Lee DJ, Portier RJ. 1999 In situ bioremediation of amines and glycol-contaminated soils using low intervention methods. Remediation 9:117-132.

Lin Q, Mendelssohn IA, Henry CB, Roberts PO, Walsh MM, Overton EB, Portier RJ. 1999 Effects of bioremediation agents on oil degradation in mineral and sandy salt marsh sediments. Environ Technol 200:825-837.

Lunel T, Rusin J, Halliwell C, Davis L. 1997 The net environmental benefit of a successful dispersant operation at the Seam Empress incident. In: Proceedings of the 1997 International Oil Spill Conference, American Petroleum Institute, Washington, DC. p 185-194.

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

Overton E, Sharp W, Roberts P. 1994 Toxicity of petroleum. In: Cockerham L, Shane B, eds. Basic Environmental Toxicology. Boca Raton, FL: CRC Press. p 133-156.

Portier RJ. 1991 Applications of adapted microorganisms for site remediation of contaminated soil and water. In: Martin AM, ed. Biological Degradation of Wastes. New York: Elsevier. p 247-259.

Portier RJ, Ahmed SI. 1988 A marine biotechnical approach for coastal and estuarine site remediation and pollution control. Mar Technol Soc J 22:34-42.

Prince RC, Varadaraj R, Fiocco RJ, Lessard RR 1999 Bioremediation as an oil spill response tool. Environ: 20:891-896.

Pritchard PH, Costa C. 1991 EPA's Alaska oil spill bioremediation project. Environ Sci Technol 25:372-379.

Smith TG, Portier RJ. 1997 A risk assessment of chlorinated aliphatics in bioremediated soils Remediation 7:107-132.

Stewart P, Tedaldi D, Lewis A, Goldman E. 1993 Biodegradation rates of crude oil in seawater. Water Environ Res 65:845-848.

Walker M, McDonagh M, Albone D, Grigson S, Wilkinson A, Baron G. 1993 Comparison of observed and predicted changes to oil after spills. In: Proceedings of 1993 Oil Spill Conference. Washington, DC: American Petroleum Institute. p 389-392.

Wolfe MF, Schwartz GBJ, Singaram S, Mielbrecht EE, Tjeerdema RS, Sowby ML. 1998 Influence of dispersants on the bioavailability of naphthalene from the water-accommodated fraction crude oil to the golden-brown algae Isochrysis galbana. Arch Environ Contam Toxicol 35:274-280.