Comparison of two field systems for determination of crude oil biodegradation in cold seawater.

Marine oil spills have devastating environmental impacts and extrapolation of experimental fate and impact data from the lab to the field remains challenging due to the lack of comparable field data. In this work we compared two field systems used to study in situ oil depletion with emphasis on biodegradation and associated microbial communities. The systems were based on (i) oil impregnated clay beads and (ii) hydrophobic Fluortex adsorbents coated with thin oil films. The bacterial communities associated with the two systems displayed similar compositions of dominant bacterial taxa. Initial abundances of Oceanospirillales were observed in both systems with later emergences of Flavobacteriales, Alteromonadales and Rhodobacterales. Depletion of oil compounds was significantly faster in the Fluortex system and most likely related to the greater bioavailability of oil compounds as compared to the clay bead system.

Comprehensive Two-Dimensional Gas Chromatography with Peak Tracking for Screening of Constituent Biodegradation in Petroleum UVCB Substances.

Petroleum substances, as archetypical UVCBs (substances of unknown or variable composition, complex reaction products, or biological substances), pose a challenge for chemical risk assessment as they contain hundreds to thousands of individual constituents. It is particularly challenging to determine the biodegradability of petroleum substances since each constituent behaves differently. Testing the whole substance provides an average biodegradation, but it would be effectively impossible to obtain all constituents and test them individually. To overcome this challenge, comprehensive two-dimensional gas chromatography (GC × GC) in combination with advanced data-handling algorithms was applied to track and calculate degradation half-times (DT50s) of individual constituents in two dispersed middle distillate gas oils in seawater. By tracking >1000 peaks (representing ∼53-54% of the total mass across the entire chromatographic area), known biodegradation patterns of oil constituents were confirmed and extended to include many hundreds not currently investigated by traditional one-dimensional GC methods. Approximately 95% of the total tracked peak mass biodegraded after 64 days. By tracking the microbial community evolution, a correlation between the presence of functional microbial communities and the observed progression of DT50s between chemical classes was demonstrated. This approach could be used to screen the persistence of GC × GC-amenable constituents of petroleum substance UVCBs.

Ultimate and Primary Biodegradation of a Range of Nonpolymeric and Polymeric Surfactants in Seawater.

Surfactants are chemicals commonly used in a wide range of domestic and industrial products. In the present study, ultimate biodegradation of 18 surfactants representing different classes (including several polymeric alcohol ethoxylates [AEs]) was determined in seawater at 20 °C by a Closed Bottle test method. After 28 days of incubation, 12 surfactants reached 60% biodegradation and were considered to be readily biodegradable in seawater. The results for the six additional surfactants indicated that the 60% pass level may be reached by extended incubation time, or that reduced biodegradation could be associated with toxicity of the chemicals. All these six surfactants were biodegraded >20% after 28 days, indicative of primary biodegradation in seawater. Polymeric ethoxylates with high numbers of ethylene oxide (EO) groups (40-50 EO groups) were more slowly biodegraded than polyethoxylates with 4 to 23 EO groups. Biodegradation experiments of the AE C12 EO9 (3 to 18 EO groups) in a carousel system at 20 °C with natural seawater and a surfactant concentration of 500 µg/L showed rapid primary biodegradation by targeted analyses of the AE, with >99% primary biodegradation after 2 days of incubation. The surfactant depletion coincided with temporary formation of polyethylene glycols, suggesting that central fission is an important degradation step in seawater. A primary biodegradation experiment in the carousel system with C12 EO9 was conducted in the presence of suspended particulate materials (SPMs; marine phytoplankton and clay particles), showing that the presence of SPMs did not hamper the primary biodegradation of the surfactant. Separation of fractions in 20-µm steel filters indicated some particle association of the surfactant. Environ Toxicol Chem 2023;42:1472-1484. © 2023 SETAC.

The fate of conventional and potentially degradable gillnets in a seawater-sediment system.

Abandoned gillnets in the marine environment represent a global environmental risk due to the ghost fishing caused by the nets. Degradation of conventional nylon gillnets was compared to that of nets made of polybutylene succinate co-adipate-co-terephthalate (PBSAT) that are designed to degrade more readily in the environment. Gillnet filaments were incubated in microcosms of natural seawater (SW) and marine sediments at 20 °C over a period of 36 months. Tensile strength tests and scanning electron microscopy analyses showed weakening and degradation of the PBSAT filaments over time, while nylon filaments remained unchanged. Pyrolysis-gas chromatography/mass spectrometry revealed potential PBSAT degradation products associated with the filament surfaces, while nylon degradation products were not detected by these analyses. Microbial communities differed significantly between the biofilms on the nylon and PBSAT filaments. The slow deterioration of the PBSAT gillnet filaments shown here may be beneficial and reduce the ghost fishing periods of these gillnets.

A critical review of marine snow in the context of oil spills and oil spill dispersant treatment with focus on the Deepwater Horizon oil spill.

Natural marine snow (NMS) is defined as the "shower" of particle aggregates formed by processes that occur in the world's oceans, consisting of macroscopic aggregates of detritus, living organisms and inorganic matter. Recent studies from the Deepwater Horizon oil spill suggest that marine snow is also formed in association with oil spills and was an important factor for the transport of oil to the seabed. This review summarizes the research and literature on MS, mainly from the DWH oil spill, with a focus on the relation between the use of oil spill dispersants and the formation and fate of oil-related marine snow (ORMS). Studies are still required to determine ORMS processes at oil concentrations as relevant as possible for chemically dispersed oil.

Petroleum hydrocarbon and microbial community structure successions in marine oil-related aggregates associated with diatoms relevant for Arctic conditions.

Oil-related aggregates (ORAs) may contribute to the fate of oil spilled offshore. However, our understanding about the impact of diatoms and associated bacteria involved in the formation of ORAs and the fate of oil compounds in these aggregates is still limited. We investigated these processes in microcosm experiments with defined oil dispersions in seawater at 5 °C, employing the Arctic diatom Fragilariopsis cylindrus and its associated bacterial assemblage to promote ORA formation. Accumulation of oil compounds, as well as biodegradation of naphthalenes in ORAs and corresponding water phases, was enhanced in the presence of diatoms. Interestingly, the genus Nonlabens was predominating the bacterial communities in diatom-supplemented microcosms, while this genus was not abundant in other samples. This work elucidates the relevance of diatom biomass for the formation of ORAs, microbial community structures and biodegradation processes in chemically dispersed oil at low temperatures relevant for Arctic conditions.

Biodegradation-mediated alterations in acute toxicity of water-accommodated fraction and single crude oil components in cold seawater.

Hydrocarbon biodegradation may be slower in cold Arctic than in temperate seawater, and this will affect the toxicity time window of the hydrocarbons. In this study, the acute toxicities of water-soluble phases of 1,3-dimethylnaphthalene, phenanthrene, fluoranthene, and low energy water-accommodated fractions (LE-WAFs) of an evaporated (200 °C+) crude oil, were screened by a Microtox bioassay during biodegradation in cold seawater (4-5 °C). The water-solubility of fluoranthene was too low to provoke a toxic response at any time, whereas the toxicity of 1,3-dimethylnaphthalene and phenanthrene decreased over time in relation to biotransformation of these compounds. In LE-WAFs, the Microtox EC50 was associated with biodegradation of the predominant hydrocarbons (naphthalenes, 2- to 3-ring PAH), as well as with phenol degradation products. The acute toxicities of single hydrocarbons and LE-WAFs persisted for a longer period in the cold seawater than previously shown at higher seawater temperatures. These results suggest implications for fate and effects assessment of hydrocarbons after oil spills in cold environments, like the Arctic. However, further biodegradation studies using Arctic seawater and relevant species for toxicity testing are needed for confirmation.

Dispersibility and biotransformation of oils with different properties in seawater.

Dispersants are used to remove oils slicks from sea surfaces and to generate small oil-droplet dispersions, which may result in enhanced biodegradation of the oil. In this study, dispersibility and biodegradation of chemically dispersed oils with different physical-chemical properties (paraffinic, naphthenic and asphaltenic oils) were compared in natural temperate SW at 13 °C. All selected oils were chemically dispersible when well-known commercial dispersants were used. However, interfacial tension (IFT) studies of the dispersed oils showed different IFT properties of the oils at 13 °C, and also different leaching of the dispersants from oil droplet surfaces. Biodegradation studies of the chemically dispersed oils were performed in a carousel system, with initial median droplet sizes <30 µm and oil concentrations of 2.5-2.8 mg/L. During biodegradation, oil droplet concentrations were rapidly reduced, in association with the emergence of macroscopic 'flocs'. Biotransformation results showed that half-lives of semivolatile total extractable organic carbon (TEOC), single target 2- to 4-ring PAH, and 22 oil compound groups used as input data in the oil spill contingency model OSCAR, did not differ significantly between the oils (P > 0.05), while n-alkanes half-lives differed significantly (P < 0.05). Biotransformation was associated with rapid microbial growth in all oil dispersions, in association with n-alkane and PAH biotransformation. These results have implications for the predictions of biodegradation of oil slicks treated with dispersants in temperate SW.

Biodegradation of dispersed oil in seawater is not inhibited by a commercial oil spill dispersant.

Chemical dispersants are well-established as oil spill response tools. Several studies have emphasized their positive effects on oil biodegradation, but recent studies have claimed that dispersants may actually inhibit the oil biodegradation process. In this study, biodegradation of oil dispersions in natural seawater at low temperature (5°C) was compared, using oil without dispersant, and oil premixed with different concentrations of Slickgone NS, a widely used oil spill dispersant in Europe. Saturates (nC10-nC36 alkanes), naphthalenes and 2- to 5-ring polycyclic aromatic hydrocarbons (PAH) were biotransformed at comparable rates in all dispersions, both with and without dispersant. Microbial communities differed primarily between samples with or without oil, and they were not significantly affected by increasing dispersant concentrations. Our data therefore showed that a common oil spill dispersant did not inhibit biodegradation of oil at dispersant concentrations relevant for response operations.

Microbial communities related to biodegradation of dispersed Macondo oil at low seawater temperature with Norwegian coastal seawater.

The Deepwater Horizon (DWH) accident in 2010 created a deepwater plume of small oil droplets from a deepwater well in the Mississippi Canyon lease block 252 ('Macondo oil'). A novel laboratory system was used in the current study to investigate biodegradation of Macondo oil dispersions (10 µm or 30 µm median droplet sizes) at low oil concentrations (2 mg l(-1)) in coastal Norwegian seawater at a temperature of 4-5°C. Whole metagenome analyses showed that oil biodegradation was associated with the successive increased abundances of Gammaproteobacteria, while Alphaproteobacteria (Pelagibacter) became dominant at the end of the experiment. Colwellia and Oceanospirillales were related to n-alkane biodegradation, while particularly Cycloclasticus and Marinobacter were associated with degradation of aromatic hydrocarbons (HCs). The larger oil droplet dispersions resulted in delayed sequential changes of Oceanospirillales and Cycloclasticus, related with slower degradation of alkanes and aromatic HCs. The bacterial successions associated with oil biodegradation showed both similarities and differences when compared with the results from DWH field samples and laboratory studies performed with deepwater from the Gulf of Mexico.

Biodegradation of dispersed Macondo oil in seawater at low temperature and different oil droplet sizes.

During the Deepwater Horizon (DWH) accident in 2010 a dispersant (Corexit 9500) was applied at the wellhead to disperse the Macondo oil and reduce the formation of surface slicks. A subsurface plume of small oil droplets was generated near the leaking well at 900-1300 m depth. A novel laboratory system was established to investigate biodegradation of small droplet oil dispersions (10 µm or 30 µm droplet sizes) of the Macondo oil premixed with Corexit 9500, using coastal Norwegian seawater at a temperature similar to the DWH plume (4-5°C). Biotransformation of volatile and semivolatile hydrocarbons and oil compound groups was generally faster in the 10 µm than in the 30 µm dispersions, showing the importance of oil droplet size for biodegradation. These data therefore indicated that dispersant treatment to reduce the oil droplet size may increase the biodegradation rates of oil compounds in the deepwater oil droplets.

Depletion and biodegradation of hydrocarbons in dispersions and emulsions of the Macondo 252 oil generated in an oil-on-seawater mesocosm flume basin.

Physically and chemically (Corexit 9500) generated Macondo 252 oil dispersions, or emulsions (no Corexit), were prepared in an oil-on-seawater mesocosm flume basin at 30-32 °C, and studies of oil compound depletion performed for up to 15 days. The use of Corexit 9500 resulted in smaller median droplet size than in a physically generated dispersion. Rapid evaporation of low boiling point oil compounds (C⩽15) appeared in all the experiments. Biodegradation appeared to be an important depletion process for compounds with higher boiling points in the dispersions, but was negligible in the surface emulsions. While n-alkane biodegradation was faster in chemically than in physically dispersed oil no such differences were determined for 3- and 4-ring PAH compounds. In the oil dispersions prepared by Corexit 9500, increased cell concentrations, reduction in bacterial diversity, and a temporary abundance of bacteria containing an alkB gene were associated with oil biodegradation.

Chemical and toxicological characterization of an unresolved complex mixture-rich biodegraded crude oil.

Chemical and toxicological characterization of unresolved complex mixtures in the water-soluble fraction of an artificially weathered Norwegian Sea crude oil was determined by a combination of chemical analysis and toxicity testing in fish in vitro bioassays. The water-soluble fraction of the crude oil was separated into 14 increasingly polar fractions by preparative high-pressure liquid chromatography. The in vitro toxicity (7-ethoxyresorufin O-deethylase activity, estrogenicity, and metabolic inhibition) of these fractions was characterized in a primary culture of liver cells (hepatocytes) from rainbow trout (Oncorhynchus mykiss). The main contributor to toxicity was one of the most polar fractions, accounting gravimetrically for more than 70% of the organic material in the water-soluble fraction and dominated by an unresolved complex mixture. Chemical analysis by gas chromatography-mass spectrometry and comprehensive two-dimensional gas chromatography-time of flight-mass spectrometry identified a large number of cyclic and aromatic sulfoxide compounds and low amounts of benzothiophenes (<0.1% of total mass) in this fraction. Commonly monitored toxic components of crude oil (e.g., naphthalenes, polycyclic aromatic hydrocarbons, and alkylated phenols) eluted in less polar fractions, characterized by somewhat lower toxicity. Normalization of in vitro responses to the mass in each fraction demonstrated a more even distribution of toxicity, indicating that toxicity in the individual fractions was related to the amount of material present. Although polar and nonpolar compounds contribute additively to crude oil toxicity, the water-soluble fraction was dominated by polar compounds because of their high aqueous solubility and the high oil-water loading. Under these conditions, the polar unresolved complex mixture-rich fraction might account for a large portion of crude oil toxicity because of its high abundance in the water-soluble fraction.

Biodegradation of petroleum hydrocarbons in seawater at low temperatures (0-5 degrees C) and bacterial communities associated with degradation.

In this study biodegradation of hydrocarbons in thin oil films was investigated in seawater at low temperatures, 0 and 5 degrees C. Heterotrophic (HM) or oil-degrading (ODM) microorganisms enriched at the two temperatures showed 16S rRNA sequence similarities to several bacteria of Arctic or Antarctic origin. Biodegradation experiments were conducted with a crude mineral oil immobilized as thin films on hydrophobic Fluortex adsorbents in nutrient-enriched or sterile seawater. Chemical and respirometric analysis of hydrocarbon depletion showed that naphthalene and other small aromatic hydrocarbons (HCs) were primarily biodegraded after dissolution to the water phase, while biodegradation of larger polyaromatic hydrocarbons (PAH) and C(10)-C(36) n-alkanes, including n-hexadecane, was associated primarily with the oil films. Biodegradation of PAH and n-alkanes was significant at both 0 and 5 degrees C, but was decreased for several compounds at the lower temperature. n-Hexadecane biodegradation at the two temperatures was comparable at the end of the experiments, but was delayed at 0 degree C. Investigations of bacterial communities in seawater and on adsorbents by PCR amplification of 16S rRNA gene fragments and DGGE analysis indicated that predominant bacteria in the seawater gradually adhered to the oil-coated adsorbents during biodegradation at both temperatures. Sequence analysis of most DGGE bands aligned to members of the phyla Proteobacteria (Gammaproteobacteria) or Bacteroidetes. Most sequences from experiments at 0 degree C revealed affiliations to members of Arctic or Antarctic consortia, while no such homology was detected for sequences from degradation experiment run at 5 degrees C. In conclusion, marine microbial communities from cold seawater have potentials for oil film HC degradation at temperatures < or =5 degrees C, and psychrotrophic or psychrophilic bacteria may play an important role during oil HC biodegradation in seawater close to freezing point.

Biotransformation and dissolution of petroleum hydrocarbons in natural flowing seawater at low temperature.

The objective of this study was to establish methods for controlled studies of hydrocarbon depletion from thin oil films in cold natural seawater, and to determine biotransformation in relation to other essential depletion processes. Mineral oil was immobilized on the surface of hydrophobic Fluortex fabrics and used for studies of microbial biodegradation in an experimental seawater flow-through system at low temperatures (5.9-7.4 degrees C) during a test period of 42 days. The seawater was collected from a depth of 90 m, and microbial characterization by epifluorescence microscopy, fluorescence in situ hybridization, and most-probable number analysis showed relatively larger fractions of archaea and oil-degrading microbes than in the corresponding surface water. Chemical analysis of hydrocarbons attached to the fabrics during the test period showed that n-alkanes (C10-C36) were decreased by 98% after 21 days, while naphthalenes were depleted by 99-100% during the same period. At the end of the period 4-5 ring polyaromatic hydrocarbon (PAH) compounds were removed by 82% from the fabrics. Analysis of the recalcitrant pentacyclic triterpane C30 17alpha(H),21beta(H)-hopane showed that the oil remained adsorbed to the fabrics during the test period. Comparison of depletion analysis with calculation of hydrocarbon dissolution in a flow-through system indicated that naphthalenes and smaller PAH compounds (alkylated 2-ring and 3-ring compounds) were removed from the fabrics by dissolution. The data further implied that depletion of n-alkanes and 4-5 ring PAH hydrocarbons were the result of biotransformation processes. PCR amplification of bacterial 16S rRNA genes from microbes adhering on the immobilized oil surfaces showed the dominance of a few bands when analysed in denaturing gradient gel electrophoresis (DGGE). Sequence analysis of DGGE bands revealed phylogenetic affiliation to the alpha- and gamma-subdivisions of proteobacteria and to the Chloroflexus-Flavobacterium-Bacteroides group.