Field fluorometers for assessing oil dispersion at sea.

Oil dispersion by the application of chemical dispersants is an important tool in oil spill response, but it is difficult to quantify in the field in a timely fashion that is useful for coordinators and decision-makers. One option is the use of rugged portable field fluorometers that can deliver essentially instantaneous results if access is attainable. The United States Coast Guard has suggested, in their Special Monitoring of Applied Response Technologies (SMART) protocols, that successful oil dispersion can be identified by a five-fold increase in oil fluorescence. Here we test three commercial fluorometers with different excitation/emission windows (SeaOWL, Cyclops 7FO, and Cyclops 7F-G) that might prove useful for such applications. Results show that they have significantly different dynamic ranges for detecting oil and that using them (or similar instruments) in combination is probably the best option for successfully assessing the effectiveness of oil dispersion operations. Nevertheless, the rapid dilution of dispersed oil means that measurements must be made within an hour or two of dispersion, suggesting that one feasible scenario would be monitoring ship-applied dispersants by vessels following close behind the dispersant application vessel. Alternatively, autonomous submersibles might be pre-deployed to monitor aerial dispersant application, although the logistical challenges in a real spill would be substantial.

Temporal chemical composition changes in water below a crude oil slick irradiated with natural sunlight.

Photooxidation can alter the environmental fate and effects of spilled oil. To better understand this process, oil slicks were generated on seawater mesocosms and exposed to sunlight for 8 days. The molecular composition of seawater under irradiated and non-irradiated oil slicks was characterized using ion mobility spectrometry-mass spectrometry and polyaromatic hydrocarbons analyses. Biomimetic extraction was performed to quantify neutral and ionized constituents. Results show that seawater underneath irradiated oil showed significantly higher amounts of hydrocarbons with oxygen- and sulfur-containing by-products peaking by day 4-6; however, concentrations of dissolved organic carbon were similar. Biomimetic extraction indicated toxic units in irradiated mesocosms increased, mainly due to ionized components, but remained <1, suggesting limited potential for ecotoxicity. Because the experimental design mimicked important aspects of natural conditions (freshly collected seawater, natural sunlight, and relevant oil thickness and concentrations), this study improves our understanding of the effects of photooxidation during a marine oil spill.

Bioremediation of Petroleum Hydrocarbons in the Upper Parts of Sandy Beaches.

The biodegradation of dispersed crude oil in the ocean is relatively rapid (a half-life of a few weeks). However, it is often much slower on shorelines, usually attributed to low moisture content, nutrient limitation, and higher oil concentrations in beaches than in dispersed plumes. Another factor may be the increased salinity of the upper intertidal and supratidal zones because these parts of the beach are potentially subject to prolonged evaporation and only intermittent inundation. We have investigated whether such an increase in salinity has inhibitory effects on oil biodegradation in seashores. Lightly weathered Hibernia crude oil was added to beach sand at 1 or 10 mL/kg, and fresh seawater, at salinities of 30, 90, and 160 g/L, was added to 20% saturation. The biodegradation of oil was slower at higher salinities, where the half-life increased from 40 days at 30 g/L salts to 58 and 76 days at 90 and 160 g/L salts, respectively, and adding fertilizers somewhat enhanced oil biodegradation. Increased oil concentration in the sand, from 1 to 10 mL/kg, slowed the half-life by about 10-fold. Consequently, occasional irrigation with fertilization could be a suitable bioremediation strategy for the upper parts of contaminated beaches. However, dispersing oil at sea is probably the most suitable option for the optimal removal of spilled crude oil from the marine environment.

Oil Irradiation Experiments Document Changes in Oil Properties, Molecular Composition, and Dispersant Effectiveness Associated with Oil Photo-Oxidation.

While chemical dispersants are a powerful tool for treating spilled oil, their effectiveness can be limited by oil weathering processes such as evaporation and emulsification. It has been suggested that oil photo-oxidation could exacerbate these challenges. To address the role of oil photo-oxidation in dispersant effectiveness, outdoor mesocosm experiments with crude oil on seawater were performed. Changes in bulk oil properties and molecular composition were quantified to characterize oil photo-oxidation over 11 days. To test relative dispersant effectiveness, oil residues were evaluated using the Baffled Flask Test. The results show that oil irradiation led to oxygen incorporation, formation of oxygenated hydrocarbons, and higher oil viscosities. Oil irradiation was associated with decreased dispersant efficacy, with effectiveness falling from 80 to <50% in the Baffled Flask Test after more than 3 days of irradiation. Increasing photo-oxidation-induced viscosity seems to drive the decreasing dispersant effectiveness. Comparing the Baffled Flask Test results with field data from the Deepwater Horizon oil spill showed that laboratory dispersant tests underestimate the dispersion of photo-oxidized oil in the field. Overall, the results suggest that prompt dispersant application (within 2-4 days), as recommended by current oil spill response guidelines, is necessary for effective dispersion of spilled oil.

Hypersaline Pore Water in Gulf of Mexico Beaches Prevented Efficient Biodegradation of Deepwater Horizon Beached Oil.

The 2010 Deepwater Horizon (DWH) blowout released 3.19 million barrels (435 000 tons) of crude oil into the Gulf of Mexico. Driven by currents and wind, an estimated 22 000 tons of spilled oil were deposited onto the northeastern Gulf shorelines, adversely impacting the ecosystems and economies of the Gulf coast regions. In this work we present field work conducted at the Gulf beaches in three U.S. States during 2010-2011: Louisiana, Alabama, and Florida, to explore endogenous mechanisms that control persistence and biodegradation of the MC252-oil deposited within beach sediments as deep as 50 cm. The work involved over 1500 measurements incorporating oil chemistry, hydrocarbon-degrading microbial populations, nutrient and DO concentrations, and intrinsic beach properties. We found that intrinsic beach capillarity along with groundwater depth provides primary controls on aeration and infiltration of near-surface sediments, thereby modulating moisture and redox conditions within the oil-contaminated zone. In addition, atmosphere-ocean-groundwater interactions created hypersaline sediment environments near the beach surface at all the studied sites. The fact that the oil-contaminated sediments retained near or above 20% moisture content and were also eutrophic and aerobic suggests that the limiting factor for oil biodegradation is the hypersaline environment due to evaporation, a fact not reported in prior studies. These results highlight the importance of beach porewater hydrodynamics in generating unique hypersaline sediment environments that inhibited oil decomposition along the Gulf shorelines following DWH.

Crude oil biodegradation in upper and supratidal seashores.

The salinity of the upper parts of seashores can become higher than seawater due to evaporation between tidal inundations. Such hypersaline ecosystems, where the salinity can reach up to eight-fold higher than that of seawater (30-35 g/L), can be contaminated by oil spills. Here we investigate whether such an increase has inhibitory effects on oil biodegradation. Seawater was evaporated to a concentrated brine and added to fresh seawater to generate high salinity microcosms. Artificially weathered Hibernia crude oil was added, and biodegradation was followed for 76 days. First-order rate constants (k) for the biodegradation of GC-detectable hydrocarbons showed that the hydrocarbonoclastic activity was substantially inhibited at high salt - k decreased by ~75% at 90 g/L salts and ~90% at 160 g/L salts. This inhibition was greatest for the alkanes, although it extended to all classes of compounds measured, with the smallest effect on four-ring aromatics (e.g., chrysenes). Genera of well-known aerobic hydrocarbonoclastic bacteria were only identified at 30 g/L salts in the presence of oil, and only a few halophilic Archaea showed a slight enrichment at higher salt concentrations. These results indicate that biodegradation of spilled oil will likely be slowed in supratidal ecosystems and suggest that occasional irrigation of oiled supratidal zones could be a useful supporting strategy to remediation processes.

Occurrence and biodegradation of hydrocarbons at high salinities.

Hypersaline environments are found around the world, above and below ground, and many are exposed to hydrocarbons on a continuous or a frequent basis. Some surface hypersaline environments are exposed to hydrocarbons because they have active petroleum seeps while others are exposed because of oil exploration and production, or nearby human activities. Many oil reservoirs overlie highly saline connate water, and some national oil reserves are stored in salt caverns. Surface hypersaline ecosystems contain consortia of halophilic and halotolerant microorganisms that decompose organic compounds including hydrocarbons, and subterranean ones are likely to contain the same. However, the rates and extents of hydrocarbon biodegradation are poorly understood in such ecosystems. Here we describe hypersaline environments potentially or likely to become contaminated with hydrocarbons, including perennial and transient environments above and below ground, and discuss what is known about the microbes degrading hydrocarbons and the extent of their activities. We also discuss what limits the microbial hydrocarbon degradation in hypersaline environments and whether there are opportunities for inhibiting (oil storage) or stimulating (oil spills) such biodegradation as the situation requires.

Integrating Dispersants in Oil Spill Response in Arctic and Other Icy Environments.

Future oil exploration and marine navigation may well extend into the Arctic Ocean, and government agencies and responders need to plan for accidental oil spills. We argue that dispersants should play an important role in these plans, since they have substantial logistical benefits, work effectively under Arctic conditions, and stimulate the rapid biodegradation of spilled oil. They also minimize the risk of surface slicks to birds and mammals, the stranding of oil on fragile shorelines and minimize the need for large work crews to be exposed to Arctic conditions.

The Rate of Crude Oil Biodegradation in the Sea.

Various groups have studied the rate of oil biodegradation in the sea over many years, but with no consensus on results. This can be attributed to many factors, but we show here that the principal confounding influence is the concentration of oil used in different experiments. Because of dilution, measured concentrations of dispersed oil in the sea are sub-parts-per-million within a day of dispersal, and at such concentrations the rate of biodegradation of detectable oil hydrocarbons has an apparent half-life of 7-14 days. This can be contrasted with the rate of degradation at the higher concentrations found in oil slicks or when stranded on a shoreline; there the apparent half-life varies from many months to many years.

The biodegradation of crude oil in the deep ocean.

Oil biodegradation at a simulated depth of 1500m was studied in a high-pressure apparatus at 5°C, using natural seawater with its indigenous microbes, and 3ppm of an oil with dispersant added at a dispersant:oil ratio of 1:15. Biodegradation of the detectable hydrocarbons was prompt and extensive (>70% in 35days), although slower by about a third than under otherwise identical conditions equivalent to the surface. The apparent half-life of biodegradation of the total detectable hydrocarbons at 15MPa was 16days (compared to 13days at atmospheric pressure), although some compounds, such as the four-ring aromatic chrysene, were degraded rather more slowly.

Marine Oil Biodegradation.

Crude oil has been part of the marine environment for millions of years, and microbes that use its rich source of energy and carbon are found in seawater, sediments, and shorelines from the tropics to the polar regions. Catastrophic oil spills stimulate these organisms to "bloom" in a reproducible fashion, and although oil does not provide bioavailable nitrogen, phosphorus or iron, there are enough of these nutrients in the sea that when dispersed oil droplets dilute to low concentrations these low levels are adequate for microbial growth. Most of the hydrocarbons in dispersed oil are degraded in aerobic marine waters with a half-life of days to months. In contrast, oil that reaches shorelines is likely to be too concentrated, have lower levels of nutrients, and have a far longer residence time in the environment. Oil that becomes entrained in anaerobic sediments is also likely to have a long residence time, although it too will eventually be biodegraded. Thus, data that encompass everything from the ecosystem to the molecular level are needed for understanding the complicated process of petroleum biodegradation in marine environments.

Oil spill dispersants: boon or bane?

Dispersants provide a reliable large-scale response to catastrophic oil spills that can be used when the preferable option of recapturing the oil cannot be achieved. By allowing even mild wave action to disperse floating oil into tiny droplets (<70 µm) in the water column, seabirds, reptiles, and mammals are protected from lethal oiling at the surface, and microbial biodegradation is dramatically increased. Recent work has clarified how dramatic this increase is likely to be: beached oil has an environmental residence of years, whereas dispersed oil has a half-life of weeks. Oil spill response operations endorse the concept of net environmental benefit, that any environmental costs imposed by a response technique must be outweighed by the likely benefits. This critical review discusses the potential environmental debits and credits from dispersant use and concludes that, in most cases, the potential environmental costs of adding these chemicals to a polluted area are likely outweighed by the much shorter residence time, and hence integrated environmental impact, of the spilled oil in the environment.

Biodegradation of dispersed oil in Arctic seawater at -1°C.

As offshore oil and gas exploration expands in the Arctic, it is important to expand the scientific understanding of arctic ecology and environmental impact to mitigate operational risks. Understanding the fate of oil in arctic seawater is a key factor for consideration. Here we report the chemical loss due to the biodegradation of Alaska North Slope (ANS) crude oil that would occur in the water column following the successful dispersion of a surface oil slick. Primary biodegradation and mineralization were measured in mesocosms containing Arctic seawater collected from the Chukchi Sea, Alaska, incubated at -1°C. Indigenous microorganisms degraded both fresh and weathered oil, in both the presence and absence of Corexit 9500, with oil losses ranging from 46-61% and up to 11% mineralization over 60 days. When tested alone, 14% of 50 ppm Corexit 9500 was mineralized within 60 days. Our study reveals that microorganisms indigenous to Arctic seawater are capable of performing extensive biodegradation of chemically and physically dispersed oil at an environmentally relevant temperature (-1°C) without any additional nutrients.

A protocol for assessing the effectiveness of oil spill dispersants in stimulating the biodegradation of oil.

Dispersants are important tools in oil spill response. Taking advantage of the energy in even small waves, they disperse floating oil slicks into tiny droplets (<70 µm) that entrain in the water column and drift apart so that they do not re-agglomerate to re-form a floating slick. The dramatically increased surface area allows microbial access to much more of the oil, and diffusion and dilution lead to oil concentrations where natural background levels of biologically available oxygen, nitrogen, and phosphorus are sufficient for microbial growth and oil consumption. Dispersants are only used on substantial spills in relatively deep water (usually >10 m), conditions that are impossible to replicate in the laboratory. To date, laboratory experiments aimed at following the biodegradation of dispersed oil usually show only minimal stimulation of the rate of biodegradation, but principally because the oil in these experiments disperses fairly effectively without dispersant. What is needed is a test protocol that allows comparison between an untreated slick that remains on the water surface during the entire biodegradation study and dispersant-treated oil that remains in the water column as small dispersed oil droplets. We show here that when this is accomplished, the rate of biodegradation is dramatically stimulated by an effective dispersant, Corexit 9500. Further development of this approach might result in a useful tool for comparing the full benefits of different dispersants.

Lab tests on the biodegradation of chemically dispersed oil should consider the rapid dilution that occurs at sea.

Most crude oils spread on open water to an average thickness as low as 0.1 mm. The application of dispersants enhances the transport of oil as small droplets into the water column, and when combined with the turbulence of 1 m waves will quickly entrain oil into the top 1 m of the water column, where it rapidly dilutes to concentrations less than 100 ppm. In less than 24 h, the dispersed oil is expected to mix into the top 10 m of the water column and be diluted to concentrations well below 10 ppm, with dilution continuing as time proceeds. Over the multiple weeks that biodegradation takes place, dispersed oil concentrations are expected to be below 1 ppm. Measurements from spills and wave basin studies support these calculations. Published laboratory studies focused on the quantification of contaminant biodegradation rates have used concentrations orders of magnitude greater than this, as it was necessary to ensure the concentrations of hydrocarbons and other chemicals were higher than the detection limits of chemical analysis. However, current analytical methods can quantify individual alkanes and PAHs (and their alkyl homologues) at ppb and ppm levels. To simulate marine biodegradation of dispersed oil at dilute concentrations commonly encountered in the field, laboratory studies should be conducted at similarly low hydrocarbon concentrations.

The primary biodegradation of dispersed crude oil in the sea.

Dispersants are important tools for stimulating the biodegradation of large oil spills. They are essentially a bioremediation tool - aiming to stimulate the natural process of aerobic oil biodegradation by dispersing oil into micron-sized droplets that become so dilute in the water column that the natural levels of biologically available nitrogen, phosphorus and oxygen are sufficient for microbial growth. Many studies demonstrate the efficacy of dispersants in getting oil off the water surface. Here we show that biodegradation of dispersed oil is prompt and extensive when oil is present at the ppm levels expected from a successful application of dispersants - more than 80% of the hydrocarbons of lightly weathered Alaska North Slope crude oil were degraded in 60 d at 8 °C in unamended New Jersey (USA) seawater when the oil was present at 2.5 ppm by volume. The apparent halftime of the biodegradation of the hydrocarbons was 13.8 d in the absence of dispersant, and 11 d in the presence of Corexit 9500 - similar to rates extrapolated from the field in the Deepwater Horizon response.

Localizing the chemical forms of sulfur in vivo using X-ray fluorescence spectroscopic imaging: application to onion (Allium cepa) tissues.

Sulfur has a particularly rich biochemistry and fills a number of important roles in biology. In situ information on sulfur biochemistry is generally difficult to obtain because of a lack of biophysical techniques that have sufficient sensitivity to molecular form. We have recently reported that sulfur K-edge X-ray absorption spectroscopy can be used as a direct probe of the sulfur biochemistry of living mammalian cells [Gnida, M., et al. (2007) Biochemistry 46, 14735-14741]. Here we report an extension of this work and develop sulfur K-edge X-ray fluorescence spectroscopic imaging as an in vivo probe of sulfur metabolism in living cells. For this work, we have chosen onion (Allium cepa) as a tractable model system with well-developed sulfur biochemistry and present evidence of the localization of a number of different chemical forms. X-ray absorption spectroscopy of onion sections showed increased levels of lachrymatory factor (LF) and thiosulfinate and decreased levels of sulfoxide (LF precursor) following cell breakage. In intact cells, X-ray fluorescence spectroscopic imaging showed elevated levels of sulfoxides in the cytosol and elevated levels of reduced sulfur in the central transport vessels and bundle sheath cells.

Characterization of a modified nitrogenase Fe protein from Klebsiella pneumoniae in which the 4Fe4S cluster has been replaced by a 4Fe4Se cluster.

The Azotobacter vinelandii nifS gene product has been used with selenocysteine to reconstitute Klebsiella pneumoniae nitrogenase Fe protein. Chemical analysis and extended X-ray absorption fine structure (EXAFS) spectroscopy show that the 4Fe4S cluster present in the native protein is replaced by a 4Fe4Se cluster. As well, EXAFS spectroscopy shows that the bond lengths to the cysteine thiolate ligands shrink by 0.05 A (from 2.28 to 2.23 A) upon reduction, whereas the Fe-Fe distance is essentially unchanged. Thus, the core of the 4Fe4Se cluster remains essentially static on reduction, whilst the external cysteine thiolate ligands are pulled in towards the cluster. Compared with native (S)-Fe protein, the (Se)-Fe protein has a 20-fold increased rate of MgATP-induced Fe chelation, a sixfold decreased specific activity for acetylene reduction, a fivefold decreased rate of MgATP-dependent electron transfer from (Se)-Fe protein to MoFe protein, and a fourfold increase in the ATP to 2e (-) ratio. The high ATP to 2e (-) ratio and decreased specific activity are consistent with a lower rate of dissociation of oxidized (Se)-Fe protein from reduced MoFe protein. Thus, the relatively small adjustments in the Fe protein structure necessary to accommodate the 4Fe4Se cluster are transmitted both to adjacent residues that dock at the surface of the MoFe protein and to the ATP hydrolysis sites located approximately 19 A away.

Field metabolomics and laboratory assessments of anaerobic intrinsic bioremediation of hydrocarbons at a petroleum-contaminated site.

Field metabolomics and laboratory assays were used to assess the in situ anaerobic attenuation of hydrocarbons in a contaminated aquifer underlying a former refinery. Benzene, ethylbenzene, 2-methylnaphthalene, 1,2,4- and 1,3,5-trimethylbenzene were targeted as contaminants of greatest regulatory concern (COC) whose intrinsic remediation has been previously reported. Metabolite profiles associated with anaerobic hydrocarbon decay revealed the microbial utilization of alkylbenzenes, including the trimethylbenzene COC, PAHs and several n-alkanes in the contaminated portions of the aquifer. Anaerobic biodegradation experiments designed to mimic in situ conditions showed no loss of exogenously amended COC; however, a substantive rate of endogenous electron acceptor reduction was measured (55 ± 8 µM SO(4) day(-1)). An assessment of hydrocarbon loss in laboratory experiments relative to a conserved internal marker revealed that non-COC hydrocarbons were being metabolized. Purge and trap analysis of laboratory assays showed a substantial loss of toluene, m- and o-xylene, as well as several alkanes (C(6)-C(12)). Multiple lines of evidence suggest that benzene is persistent under the prevailing site anaerobic conditions. We could find no in situ benzene intermediates (phenol or benzoate), the parent molecule proved recalcitrant in laboratory assays and low copy numbers of Desulfobacterium were found, a genus previously implicated in anaerobic benzene biodegradation. This study also showed that there was a reasonable correlation between field and laboratory findings, although with notable exception. Thus, while the intrinsic anaerobic bioremediation was clearly evident at the site, non-COC hydrocarbons were preferentially metabolized, even though there was ample literature precedence for the biodegradation of the target molecules.