In Situ Bioremediation of Oiled Shoreline Environments

Kenneth Lee

INTRODUCTION

Microbial degradation is a principal process in the elimination of petroleum pollutants from the environment (Cerniglia 1993; Zobell 1964). In consideration of this fact, numerous strategies have been proposed and developed over the last 20 years to accelerate natural oil biodegradation rates. With the reported success of bioremediation operations on the beaches of Alaska after the Exxon Valdez oil spill (Atlas and Bartha 1992; Bragg and others 1994; Prince 1993; Pritchard and Costa 1991), and that of other controlled field trials (Lee and others 1997b; Swannell and others 1996; Venosa and others 1996), this technology is now considered one of the most promising oil spill countermeasures (Hoff 1993; Swannell and Head 1994).

BIOREMEDIATION STRATEGIES

There are two main approaches to oil spill bioremediation: 1) Bioaugmentation involves the addition of oil-degrading bacteria to supplement the existing microbial population; and 2) biostimulation involves the addition of nutrients or growth-enhancing cosubstrates and/or improvements in habitat quality to stimulate the growth of indigenous oil degraders.

Environmental Sciences Division, Maurice Lamontagne Institute, Fisheries and Oceans Canada, Mont-Joli, Quebec, Canada



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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP In Situ Bioremediation of Oiled Shoreline Environments Kenneth Lee INTRODUCTION Microbial degradation is a principal process in the elimination of petroleum pollutants from the environment (Cerniglia 1993; Zobell 1964). In consideration of this fact, numerous strategies have been proposed and developed over the last 20 years to accelerate natural oil biodegradation rates. With the reported success of bioremediation operations on the beaches of Alaska after the Exxon Valdez oil spill (Atlas and Bartha 1992; Bragg and others 1994; Prince 1993; Pritchard and Costa 1991), and that of other controlled field trials (Lee and others 1997b; Swannell and others 1996; Venosa and others 1996), this technology is now considered one of the most promising oil spill countermeasures (Hoff 1993; Swannell and Head 1994). BIOREMEDIATION STRATEGIES There are two main approaches to oil spill bioremediation: 1) Bioaugmentation involves the addition of oil-degrading bacteria to supplement the existing microbial population; and 2) biostimulation involves the addition of nutrients or growth-enhancing cosubstrates and/or improvements in habitat quality to stimulate the growth of indigenous oil degraders. Environmental Sciences Division, Maurice Lamontagne Institute, Fisheries and Oceans Canada, Mont-Joli, Quebec, Canada

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP Bioaugmentation As a result of extensive media coverage, there is a perception that marine oil spills may be effectively treated by the addition of oil degrading bacteria (“super bugs”). In reality, there is little or no need to add microorganisms to oil-contaminated ecosystems. Microbial ecologists have conclusively demonstrated that oil-degrading bacteria within sediments (Button and others 1992; Lee and Levy 1987; Prince 1993; Venosa and others 1997), open waters (Atlas 1993; Pierce and others 1975), and sea ice (Delille and others 1997) naturally increase in numbers after exposure to oil. Furthermore, field trials have shown that the addition of commercial mixtures (Lee and Levy 1987) or enriched cultures of indigenous oil-degrading bacteria (Fayad and others 1992; Venosa and others 1996) did not significantly enhance the rates of oil biodegradation over that achieved by nutrient enrichment alone. The concept of developing a genetically engineered super bug to degrade crude oil single-handedly is seriously flawed (Lethbridge and others 1994). Vast metabolic potential is required to deal with the diverse array of chemicals in crude oil. Even if it were technically feasible to incorporate all the necessary genetic information into recombinant microorganisms, the burden of maintaining all of these genes is likely to be so great as to make the recombinant strains noncompetitive in the natural environment. In summary, allochthonous microorganisms are generally unable to compete with the natural microflora (Lee and Levy 1987; Venosa and others 1996) in the open environment. Successful enhancement of oil degradation with allochthonous microbial cultures has been achieved only when chemostats or fermentors were used to control conditions and reduce competition from indigenous microflora (Wong and Goldsmith 1988). Although commercial seed cultures may be useful in the treatment of specific compounds within crude oil that are relatively resistant to degradation and isolated spills in confined areas (Lee and Levy 1989a), they appear to be of little benefit for the treatment of the bulk of petroleum contaminants in the open environment. Oil biodegradation within the marine environment is not limited to microbial inocula; therefore, further development of bioremediation agents that contain oil-degrading bacteria as the only active ingredient is difficult to justify. Biostimulation Addition of Nutrients Although the potential capability of indigenous microflora to degrade oil is a function of the physical and chemical properties of the seawater and oil, the environmental conditions, and the biota themselves, it is gen-

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP erally accepted that nutrient availability is the most common limiting factor (Atlas and Bartha 1973; Lee and Levy 1987). Fertilization with nitrogen and phosphorus offers great promise as a countermeasure against marine spills (Atlas and Bartha 1972, 1992; Prince 1993; Swannell and Head 1994; Walker and others 1976) and the ratios of carbon, nitrogen, and phosphorus to support optimal oil degradation rates have been defined (Bragg and others 1994; Reisfeld and others 1972; Venosa and others 1996). To optimize nutrient delivery, oleophilic nutrient formulations that retain optimal nutrient concentrations at the oil-water interface where biodegradation occurs have been developed (Atlas and Bartha 1973; Tramier and Sirvins 1983). An example is Inipol EAP22 (Elf Aquitaine, France), a microemulsion mixture composed of urea in brine encapsulated in oleic acid as the external phase with lauryl-ether-phosphate as a surfactant (Croft and others 1995; Ladousse and Tramier 1991). Its efficacy has been demonstrated on cobble beaches contaminated by the Exxon Valdez spill in Alaska (Prince 1993). However, additional research on the factors controlling the mechanisms of action is required, as it has not been proven to be effective under all conditions. Failure of bioremediation treatments has been attributed to the rapid loss of nutrients and/or acute toxic responses by the natural microflora to the oil (Lee and Levy 1987; Safferman 1991). Controlled studies suggest that optimum rates of degradation could be sustained by retaining high, nontoxic, renewable concentrations of nutrients within the interstitial pore water (Lee and others 1997; Venosa and others 1996). The feasibility of adding inorganic nutrients on a periodic basis has been demonstrated in field trials as a means of sustaining elevated nutrient concentrations within the sediments for effective bioremediation (Lee and Levy 1989b, 1991; Venosa and others 1996). The advantages of inorganic agricultural fertilizers as bioremediation agents include low cost, availability, and ease of application. Field and laboratory beach microcosm studies now suggest that concentrations of nitrate-N for optimal biostimulation should be between 1.0 and 2.5 mg 1−1 (Bragg and others 1994; Du and others 1999). Although these elevated nutrient concentrations within the interstitial waters in shorelines can be maintained by periodic additions of nutrients, it is not the most practical operational strategy. Nutrient delivery systems must be developed. In this regard, the development of slow-release fertilizer formulations and considerations of beach hydrodynamics in the dispersion of nutrients might decrease cost and effort (Boufadel and others 1999; Lee and others 1993). There is also renewed interest in having an organic carbon source mingled with bioremediation agents to promote rapid bacterial growth (Ladousse and Tramier 1991). This has led to the recent

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP development and testing of organic fertilizers composed of fish meal, animal meal, or fish bone meal (Basseres and others 1993; Lee and others 1995c). Theoretically, optimal nutrient concentrations can be maintained within oiled sediments for prolonged periods by internal nutrient regeneration processes coupled with the degradation of these products, which might also provide essential trace elements and other growth factors. Addition of Oxygen and Alternate Electron Acceptors Microbial oil degradation rates within sediments are very slow under anoxic conditions (Atlas and Bartha 1992; Lee and Levy 1991). Sediment tilling and raking have been shown to improve the bioremediation efficacy by increasing the penetration depth of oxygen and nutrient supplements (Sendstad and others 1984; Sergy and others 1998). Although commercial forms of chemical oxidants such as hydrogen, calcium, and magnesium peroxides have been used successfully in terrestrial environments for groundwater remediation, their application in the marine environment warrants further study. Although carbon transformations by aerobic microorganisms are inhibited in many fine-sediment/wetland environments, facultative and obligate anaerobes become active in anoxic environments and will degrade organic compounds (Patrick and others 1985). Carbon transfer processes in anoxic environments include fermentation, nitrate reduction, denitrification, and sulfate reduction (Valiela 1984). Except for fermentation in which the organic compound itself acts as the terminal electron acceptor, these processes require an inorganic oxidant (e.g., NO3− and SO42−). Feasibility of bioremediation strategies based on the addition of alternate electron acceptors should be evaluated. Phytoremediation Salt marshes are among the most sensitive of ecosystems and the most difficult to clean. Application of traditional oil spill cleanup techniques within this habitat may cause more damage than the oil itself. Foot and mechanical traffic will damage vegetation and drive the hydrocarbons into the anaerobic layer of the sediments where petroleum hydrocarbons may persist for decades (Baker and others 1993). Consideration is now being given to the inherent capacity of wetland plant species to aerate the rhizosphere as a means to stimulate aerobic oil biodegradation. Plants also may take up oil and release exudates and enzymes that stimulate microbial activity. Vegetative transplantation has been used in terrestrial environments for the cleanup of hazardous wastes (Schnoor and others 1995), including polycyclic aromatic hydrocarbons (Banks and

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP Schwab 1993). Although this process described as phytoremediation has not been used as a marine oil spill countermeasure, recent greenhouse studies with wetland plants (Spartina sp.) showed that the oil degradation rate in sediments was significantly enhanced by the application of fertilizer in conjunction with the presence of transplants (Lin and Mendelssohn 1998). Enhanced Dispersion (Chemical Dispersants, Biosurfactants, Oil-Mineral Fine interctions) Microbial attack of oil spilled in the marine environment occurs principally at the oil-water interface. Thus, facilitating an increase in the oil-water interface may enhance the rate and extent of biodegradation as the oil becomes more accessible to nutrients, oxygen, and bacteria. Increases in microbial activity and oil biodegradation have been correlated with the addition of chemical dispersants (Lee and others 1985; Swannell and Daniel 1999), surface agents such as powdered peat (Lee and others 1999), and fertilizers supplemented with biosurfactants for use as bioremediation agents. Research studies after the Exxon Valdez oil spill demonstrated the significance of clay-oil flocculation processes on the natural cleansing of oil residues from impacted shoreline sediment (Bragg and Owens 1994). Physical/chemical interactions with mineral fines reduce the adhesion of the residual oil to sediments by promoting the formation of stable micro-sized oil-fine aggregates (flocs) that are subsequently dispersed into the water column (Bragg and Owens 1994; Lee and others 1997a, 1998). An increase in the oil-water interface facilitated by such oil-mineral fine aggregate formation stimulates both the extent and rate of oil degradation (Lee and others 1997a; Weise and others 1999). Research during “Spills-of-Opportunity” In terms of a spill incident case study, the most rigorous study of bioremediation was conducted by Exxon and the US Environmental Protection Agency after the 1989 Exxon Valdez spill in Alaska. Preliminary laboratory experiments demonstrated the potential of nutrient enrichment as a bioremediation treatment (Pritchard and Costa 1991; Pritchard and others 1992). A large-scale (120 km of shoreline in 1989 using 23 tons of nitrogen) field operation was initiated after laboratory and field experiments that confirmed the effectiveness of bioremediation agents that included an oleophilic fertilizer (Bragg and others 1994; Button and others 1992; Glaser and others 1991) dissolved water-soluble (Glaser and others 1991; Pritchard and Costa 1991) and slow-release inorganic fertilizer formulations (Bragg and others 1994; Pritchard and Costa 1991; Safferman

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP 1991), and microbial inocula (Venosa and others 1992). Nutrient treatment was focused on the application of an oleophilic nutrient (Inipol EAP22) for the oil film on surface beach material, and the granular slow-release agricultural fertilizer (Customblen) for subsurface oil. By measuring changes over time in the oil composition relative to hopane, a conserved biomarker, the rate and extent of oil biodegradation was quantified with a high level of statistical confidence. Monitoring hydrocarbon losses relative to this conserved biomarker provided benchmark confirmation of oil biodegradation. Fertilizer additions were reported to accelerate the rate of oil removal by a factor of two to five. Furthermore, it was proven that the rate of oil biodegradation was a function of the nitrogen concentration maintained in the pore water of the intertidal sediment (Bragg and others 1994). These results suggested that the effectiveness of bioremediation can be improved by making real-time measurements of nutrients in sediments to ensure that adequate, but safe, levels of nutrients are maintained during treatment. In 1996, the Sea Empress grounded at the entrance of Milford Haven, United Kingdom, spilling approximately 65,000 tons of Forties Blend crude oil. Cleanup operations at Amroth Beach after this spill incident provided an opportunity to test the application of surf-washing operations as a means to accelerate the dispersion of oil within the beach sediments into the sea, where it was effectively biodegraded (Lee and others 1997a; Lunel and others 1995) at an enhanced rate. A RESEARCH NEED FOR OPERATIONAL GUIDELINES The decision to use bioremediation requires the demonstration of efficacy, reliability, and predictability. Despite successful field demonstrations of its efficacy (Bragg and others 1994; Lee and others 1997b; Prince 1993; Swannell and others 1997; Venosa and others 1996), bioremediation is still a controversial oil spill countermeasure. Part of the problem is that the guidelines for the proper use of the various bioremediation strategies in the marine environment are limited (Swannell and others 1996; Thomas and others 1995). To make informed decisions on the applicability and usage of bioremediation, additional information is required on (1) the testing and selection of bioremediation agents; (2) toxicity and other environmental impacts; (3) the influence of oil chemistry and environmental factors; and (4) the monitoring of efficacy and operational endpoints. Testing and Selection of Bioremediation Agents To assist response personnel in the selection and use of spill biore-

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP mediation agents, it is useful to have some simple, standard methods for screening performance and toxicity of available bioremediation products (Blenkinsopp and others 1995; Thomas and others 1995). There is no doubt about the utility of laboratory shaker flask studies to identify the potential impacts and rank the efficacy of various commercial bioremediation agents (Blenkinsopp and others 1995; Pritchard and others 1992; Venosa and others 1997; Wrenn and others 1994). However, laboratory flask studies cannot fully simulate the natural environment where conditions are in a constant state of flux due to tidal cycle inundation and washout, temperature variation, climatic changes, and fresh and saltwater interactions. For example, although ammonium has been used successfully as a nitrogen supplement in field trials (Lee and others 1997b), in small-scale laboratory systems with limited buffering capacity oil biodegradation can be suppressed by acid production associated with ammonia metabolism (Wrenn and others 1994). Indeed, the limitations of both shaker flask and mesocosm tests were recently demonstrated (Lee and others 1997b) as laboratory results could not be reproduced in the field due to physicochemistry changes that altered the interaction between residual oil and sediments. The need for controlled-release field experiments is evident. Advantages include statistically valid, replicated, randomized block designs with various treatments under conditions that address site heterogeneity and mechanisms of loss. Different methods have been used to test the efficacy of bioremediation agents in the field. There is now a need for a standard protocol that will allow interlaboratory comparison of results of experiments conducted in different environments (Lee and others 1995a; Merlin 1995). A coordinated effort by the scientific community will accelerate the development of an operational guideline based on a consolidated database of environmentally diverse data. Toxicity and Other Environmental Impacts The public has responded favorably to bioremediation strategies based on nutrient enrichment because the implicit goal is that of reducing toxic effects by converting organic molecules to benign cell biomass and “environmentally friendly” products like carbon dioxide and water (Atlas and Cerniglia 1995). Some environmentalists have expressed concern about the net benefit of bioremediation strategies because of the potential production of toxic metabolic by-products, possible toxic components in the formulation of bioremediation agents, and the ineffective degradation of the most toxic components of residual oils (Hoff 1991; OTA 1991). To date, detrimental effects from nutrient enrichment have not been observed

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP after actual field operations (Mearns and others 1997; Prince 1993), although the possibility of a future incident still exists. As an example, oxygen depletion and production of ammonia from excessive applications of a fish-bone meal fertilizer during one field experiment caused detrimental effects that included toxicity and the suppression of oil degradation rates (Lee and others 1995b). For safety assurance, future operational guidelines should include ecotoxicological-monitoring protocols. DNA analysis may be used to determine population shifts within functional microbial groups as a means to assess stress effects or changes in oil biodegradation potential after bioremediation treatment (Grossman and others 2000). Stable carbon (δ13C) and nitrogen (δ15N) isotopes have been used to monitor changes in trophic interactions after the application of bioremediation agents in the cleanup of oil residues from the Exxon Valdez spill (Coffin and others 1997). Evidence for the transfer of oil-carbon or fertilizer-nitrogen assimilated by bacteria to higher trophic levels has not been found. Assuming bioremediation was effective, additional bacterial biomass arising from oil degradation was either not transferred efficiently to higher trophic levels or not tidally transported from the beach to coastal waters. Influence of Oil Chemistry and Environmental Factors A fraction of the components in crude oils spilled within the marine environment are easily degraded; others are slowly or only partially degraded. Some compounds are totally nonbiodegradable (recalcitrant). As a guideline, the greater the complexity (number of alkyl-branched substituents or condensed aromatic rings) of the hydrocarbon structure, the slower the degradation and the greater the likelihood of accumulating partially oxidised intermediary metabolites. These and other factors such as volatility set the practical operational limits of bioremediation strategies. For instance, there is no advantage to bioremediate a surface spill of gasoline because it would evaporate rapidly. A detailed 7-month study on the bioremediation of a waxy crude oil in sand beach and salt marsh environments has demonstrated the influence of environmental factors on the outcome of a bioremediation treatment strategy (Lee and Levy 1991). Study results clearly demonstrated that the success of bioremediation depends on the nature of the contaminated shoreline. On a sandy beach contaminated with low concentrations of Terra Nova crude oil, toxicity to the oil-degrading bacteria was not a factor, and ambient concentrations of nitrogen and phosphorus were sufficient to result in rapid oil biodegradation. Under these conditions, nutrient enrichment provided little or no benefit and nature can be left to take its course (a nonaction strategy). However, higher oil levels provided a

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP carbon-enriched environment and the microbial community within the beach became nutrient-limited, and bioremediation treatment could effectively enhance the rate of oil removal. In the salt marsh environment treated with similar oil concentrations, oil penetrated into the anoxic layers of the sediment and the fertilization strategy was ineffective. In this particular case, the addition of oxygen may be required as a part of the bioremediation strategy. The intricacy of interactions influencing the success of bioremediation in this study is not unique. The ability of indigenous microbes of Prince William Sound, Alaska (Sugai and others 1997), to mineralize hexadecane, phenanthrene, and naphthalene has been shown to be influenced by the intensity of physical mixing, the method of bioremediation agent application, and the availability of alternative carbon sources. The efficacy of specific bioremediation formulations may be influenced by environmental conditions. For example, at temperate conditions greater than 15°C, slow-release (sulphur-coated urea) fertilizer formulations appear to be more effective in retaining elevated nutrient concentrations within the sediments than inorganic nitrogen (ammonium nitrate) fertilizers (Lee and others 1993). Lower temperatures are thought to reduce the permeability of the coating on the slow-release fertilizer, effectively suppressing nutrient release rates. For optimal effectiveness, the selection of bioremediation agents should take into account the environmental conditions, the type of contaminated shoreline, and the methods of application (Lee and others 1993; Prince 1993; Swannell and others 1995, 1996). Studies in the intertidal region of sandy beaches with lithium as a conservative tracer (Wrenn and others 1997) have demonstrated that dissolved nutrient transport is driven by tide-influenced hydraulic gradients and wave activity. Nutrient retention in the bioremediation zone of sand beach could be predicted from data on the extent of water coverage, and a suitable application schedule could be devised from the modeling of hydrodynamic data. In north-temperate environments, although winter temperatures do not affect the apparent number of heterotrophic bacteria in oiled sediments, the number of oil-degraders declines (Lee and Levy 1989b, 1991; Prince 1993; Swannell and others 1997). Further study is warranted to identify whether these observations are attributed to a physiological response or to physiochemical changes in the oil that alters its availability to the bacteria. It is now also apparent that the most important influence on the carrying capacity for hydrocarbon degraders in the marine environment may be the removal of biomass by physical processes such as scouring by breaking waves. If this is the case, the optimal level of oil degradation capacity can be provided by indigenous bacteria provided that

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP sufficient nutrients are present. The addition of exogenous hydrocarbon degraders (i.e., bioaugmentation) will not increase population density (Venosa and others 1996). Rapid biodegradation of crude oil stranded within intertidal environments can occur under temperate conditions. On the Delaware coast, natural nitrogen concentrations were found to be high enough to sustain rapid intrinsic rates of biodegradation without human intervention (Venosa and others 1996). Although nutrient addition at this site significantly accelerated the rate of hydrocarbon biodegradation, the incremental increase (slightly greater than 200% for the alkanes and 50% for the polynuclear aromatic hydrocarbon levels) is not high enough to warrant a major, perhaps costly, bioremediation effort in the event of a large crude oil spill in that area. A similar conclusion was also reached in a field trial to evaluate the influence of a slow-release fertilizer on the biodegradation rate of crude oil spilled on intertidal sediments of an estuary (Oudot and others 1998). Due to adaptation of marine bacteria to hydrocarbons along the coast of Brittany (Atlas and Cerniglia 1995) and high background levels of N and P at the study site, no significant difference in biodegradation rates was detected after nutrient addition. It was proposed that bioremediation by nutrient enrichment would be of limited use if background interstitial porewater levels of N exceed 100 µmoles 1-1. A strong correlation between the available concentrations of ammonia and phosphorus and the degradation rates of petroleum has been demonstrated in a recent study in Texas that monitored the relatively rapid recovery of an oil-impacted coastal wetland environment by intrinsic biodegradation (Harris and others 1999). In light of these results, it is suggested that interstitial nutrient levels be determined before any decision is made to apply bioremediation agents. Monitoring Remediation Effectiveness and Identification of Operational Endpoints Wide acceptance and use of bioremediation strategies by the oil spill response community has been limited by the lack of defined performance standards. For proper application of the technology, there is a need for monitoring programs to quantify intrinsic rates of oil loss and degradation, demonstrate treatment efficacy, and identify operational endpoints. A major obstacle is heterogeneity within the natural environment. Absolute levels of contamination can vary widely over a site and simple estimates of biodegradation based on sequential samples can be confounded by this heterogeneity, unless large numbers of samples are taken. This problem can be resolved by the normalization of data to conserved markers such as hopanes and chrysenes found within the oil (Lee and

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP others 1997b; Oudot and others 1998; Prince and others 1993; Venosa and others 1996). Though costly and time-consuming, these analyses by gas chromatography/mass spectrometry are necessary to demonstrate effectiveness at a level of precision and accuracy demanded by the scientific community. However, from an operational perspective, considering the numerous samples needed to characterize a spill site, other more rapid and less costly performance measures must be developed to satisfy regulators and managers. In situ measurement of microbial CO2 production by respirometry or radiotracer methods can be used to quantify oil mineralisation rates to estimate bioremediation success (Swannell and others 1994, 1997). Enumeration of potential oil-degrading bacteria by their isolation on specific media has become a benchmark in many bioremediation studies, although many bacteria within the natural environment are dormant or unculturable on the media used. Therefore, it is essential to show, by combined chemical and microbiological methods, that the oil-degrading bacteria are truly active. Recent studies have shown changes in the distribution of hydrocarbon-degrading genes in response to the hydrocarbon composition to which the bacterial population is exposed (Sotsky and others 1990). Future use of DNA and RNA gene probes for pollutant catabolic pathways may provide practical and evolutionary insights into how and why biodegradation activity is expressed (Greer and others 1993; Sayler and Layton 1990). As discussed, future operational guidelines will incorporate reliable microbial response and ecotoxicological monitoring protocols to verify efficacy for toxicity reduction over that of no treatment. In addition to direct chemical evidence of oil degradation, microscale biotests may provide an operational endpoint indicator for bioremediation on the basis of toxicity reduction; i.e., the site is acceptable as there is no detectable toxic effects, or the treatment is detrimental in that a toxic response is induced (Lee and others 1995b; Mearns and others 1995). CONCLUSIONS With the recent demonstrations of its efficacy in the field, bioremediation has been touted as the emerging oil spill countermeasure of the 21st century. An advantage of this environmentally friendly technology is its relatively low cost, as it does not require large numbers of personnel or highly specialized equipment for its application. However, its wide acceptance as an operational oil spill countermeasure has been limited by the lack of data showing its effectiveness relative to current technologies and operational guidelines for its application.

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OPPORTUNITIES FOR ENVIRONMENTAL APPLICATIONS OF MARINE BIOTECHNOLOGY: PROCEEDINGS OF THE OCTOBER 5-6, 1999, WORKSHOP Operational limitations exist for all oil spill countermeasures. In the context of shoreline cleanup, bioremediation should be considered a useful addition to the toolbox of oil spill treatment strategies, including the option of “no treatment.” Improvements in bioremediation technologies will result from basic research in microbial ecology, which will identify the factors controlling optimal rates of oil degradation. Future applied research is also needed to construct a database for decision making that includes information on the type of oil, application methodologies available (form and type of bioremediation agent, type and frequency of application), environmental conditions (availability of nutrients, bacteria, oxygen, temperature, and wave or tidal immersion), and defining treatment endpoints. REFERENCES Atlas RM. 1993 Bacteria and bioremediation of oil spills. Oceanus 36:71-73. Atlas RM, Bartha R. 1992 Hydrocarbon biodegradation and oil spill bioremediation. Adv Microb Ecol 12:287-338. Atlas RM, Bartha R. 1972 Degradation and mineralization of petroleum in seawater: Limitations by nitrogen and phosphorous. Biotechnol Bioeng 14:309-317. Atlas RM, Bartha R. 1973 Stimulated biodegradation of oil slicks using oleophilic fertilizers Environ Sci Technol 7:538-541. Atlas RM, Cerniglia CE. 1995 Bioremediation of petroleum pollutants. Bioscience 45:332-338. Baker JM, Guzman LM, Bartlett PD, Little DI, Wilson CM. 1993 Long-term fate and effects of untreated thick oil deposits on salt marshes. In: Proceedings of the International Oil Spill Conference. Washington, DC: American Petroleum Institute. p 395-399. Banks KM, Schwab AP. 1993 Dissipation of polycyclic aromatic hydrocarbons in the rhizosphere In: Symposium on Bioremediation of Hazardous Wastes: Research, Development and Field Evaluations, Washington, DC: Environmental Protection Agency, EPA/600/ R-93/054, p 246. Basseres A, Eyraud P, Ladousse A, Tramier B. 1993 Enhancement of spilled oil biodegradation by nutrients of natural origin. In: Proceedings of the International Oil Spill Conference. Washington, DC: American Petroleum Institute. p 495-501. Blenkinsopp S, Sergy G, Wang Z, Fingas MF, Foght J, and Westlake DWS. 1995 Oil spill bioremediation agents—Canadian efficacy test protocols 1995. In: Proceedings of the 1995 International Oil Spill Conference. Washington, DC: American Petroleum Institute. p 91-96. Boufadel MC, Suidan MT, Rauch CH, Ahn C-H, Venosa AD. 1999 Nutrient transport in beaches subjected to freshwater input and tides In: Proceedings of the International Oil Spill Conference. Washington, DC: American Petroleum Institute. (Publication 4686A, Paper 170).

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