Edith Holder

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure.

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The methods accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.

Determining the dispersibility of South Louisiana crude oil by eight oil dispersant products listed on the NCP Product Schedule.

We recently conducted a laboratory study to measure the dispersion effectiveness of eight dispersants currently listed on the National Contingency Plan Product Schedule. Results are useful in determining how many commercial dispersant products would have been effective for use on South Louisiana crude oil in the Deepwater Horizon oil spill. The test used was a modification of the Baffled Flask Test (BFT), which is being proposed to replace the current Swirling Flask Test (SFT). The modifications of the BFT in this study included use of one oil rather than two, increasing replication from 4 runs to 6, and testing at two temperatures, 5 °C and 25 °C. Results indicated that temperature was not as critical a variable as the literature suggested, likely because of the low viscosity and light weight of the SLC. Of the eight dispersants tested, only three gave satisfactory results in the laboratory flasks at both temperatures.

Laboratory evaluation of oil spill bioremediation products in salt and freshwater systems

Ten oil spill bioremediation products were tested in the laboratory for their ability to enhance biodegradation of weathered Alaskan North Slope crude oil in both freshwater and saltwater media. The products included nutrients to stimulate inoculated microorganisms, nutrients plus an oil-degrading inoculum, nutrients plus compounds intended to stimulate oil-degrading activity, or other compounds intended to enhance microbial activity. The product tests were undertaken to evaluate significant modifications in the existing official United States Environmental Protection Agency (EPA) protocol used for qualifying commercial bioremediation agents for use in oil spills. The EPA protocol was modified to include defined formulas for the exposure waters (freshwater, saltwater), a positive control using a known inoculum and nutrients, two negative controls (one sterile, the other inoculated but nutrient-limited), and simplified oil chemical analysis. Three analysts conducted the product test independently in each type of exposure water in round-robin fashion. Statistical tests were performed on analyst variability, reproducibility, and repeatability, and the performance of the various products was quantified in both exposure media. Analysis of variance showed that the analyst error at each time-point was highly significant (P values ranged from 0.0001 to 0.008, depending on water type and oil fraction). In the saltwater tests, six products demonstrated various degrees of biodegradative activity against the alkane fraction of the crude oil and three degraded the aromatic hydrocarbons by >10%. In the freshwater tests, eight products caused >20% loss of alkane hydrocarbons, of which five degraded the alkanes by >50%. Only four products were able to degrade polycyclic aromatic hydrocarbons (PAHs) by >20%, one of which caused 88% removal. However, when the variability of the analysts was taken into consideration, only one of the ten products was found to yield significant percent removals of the PAH fraction and only in freshwater. Viable microorganism population analysis (most-probable-number method) was also performed on every sample by each operator to measure the changes in aromatic and alkane hydrocarbon-degrading organism numbers. In general, little evidence of significant growth of either alkane- or PAH-degraders occurred among any of the ten products in either the saltwater or freshwater testing.

Protocol for laboratory testing of crude-oil bioremediation products in freshwater conditions

In 1993, the Environmental Protection Agency, National Risk Management Research Laboratory (EPA, NRMRL), with the National Environmental Technology Application Center (NETAC), developed a protocol for evaluation of bioremediation products in marine environments [18]. The marine protocol was adapted for application in freshwater environments by using a chemically defined medium and an oil-degrading consortium as a positive control. Four products were tested using the modified protocol: two with nutrients and an oleophilic component; one with nutrients, sorbent, and organisms; and one microbial stimulant. A separate experiment evaluated the use of HEPES and MOPSO buffers as replacements for phosphate buffer. The oleophilic nutrient products yielded oil degradation similar to the positive control, with an average alkane removal of 97.1±2.3% and an aromatic hydrocarbon removal of 64.8±1.2%. The positive control, which received inoculum plus nutrients, demonstrated alkane degradation of 98.9±0.1% and aromatic degradation of 52.9±0.1%. The sorbent-based product with inoculum failed to demonstrate oil degradation, while the microbial stimulant showed less oil degradation than the positive control. Replacement of phosphate buffer with other buffers had no significant effect on one products performance. Differences in product performance were easily distinguishable using the protocol, and performance targets for alkane and aromatic hydrocarbon degradation are suggested.

Influence of tide and waves on washout of dissolved nutrients from the bioremediation zone of a coarse-sand beach: Application in oil-spill bioremediation

Abstract Successful bioremediation of oil-contaminated beaches requires maintenance of a sufficient quantity of growth-limiting nutrients in contact with the oiled beach material. A conservative tracer study was conducted on a moderate-energy, sandy beach on Delaware Bay to estimate the washout rate for dissolved nutrients from the bioremediation zone at different stages during the lunar tidal cycle. When an aqueous solution of the conservative tracer (LiNO3) was applied to the beach surface in the upper intertidal zone at the full moon spring tide, it was completely removed within one day. When it was applied at neap tide, however, the tracer persisted in the bioremediation zone for several days. The amount of nutrient remaining in the bioremediation zone was highly correlated with the maximum extent to which the treated area had previously been submerged by water at high tide: submergence resulted in nearly complete removal of dissolved compounds from the bioremediation zone. This high rate of nutrient washout was confirmed by daily monitoring of nutrient concentrations in the bioremediation zone during an oil-spill bioremdiation field study that was conducted on a nearby beach.

Characterization of emissions and residues from simulations of the Deepwater Horizon surface oil burns

The surface oil burns conducted by the U.S. Coast Guard from April to July 2010 during the Deepwater Horizon disaster in the Gulf of Mexico were simulated by small scale burns to characterize the pollutants, determine emission factors, and gather particulate matter for subsequent toxicity testing. A representative crude oil was burned in ocean-salinity seawater, and emissions were collected from the plume by means of a crane-suspended sampling platform. Emissions included particulate matter, aromatic hydrocarbons, polychlorinated dibenzodioxins/dibenzofurans, elements, and others, the sum of which accounted for over 92% by mass of the combustion products. The unburned oil mass was 29% of the original crude oil mass, significantly higher than typically reported. Analysis of alkanes, elements, and PAHs in the floating residual oil and water accounted for over 51% of the gathered mass. These emission factors, along with toxicity data, will be important toward examining impacts of future spill burning operations.

Development of a Rational Oil Spill Dispersant Effectiveness Protocol

ABSTRACT Chemical dispersants are used in oil spill response operations to enhance the dispersion of oil slicks at sea as small oil droplets in the water column. To be considered for use, the dispersants must be listed in the National Contingency Plan (NCP) Product Schedule. Since 1994, dispersants were required to pass an effectiveness test known as the Swirling Flask Test (SFT), which is described in Appendix C of 40 CFR 300. Listing of a dispersant on the NCP Product Schedule is contingent on the dispersant being at least 45% effective in dispersing South Louisiana crude (SLC) and Prudhoe Bay crude (PBC) oils as measured and calculated by the test. Shortly after adopting the SFT, the U.S. Environmental Protection Agency (EPA) began to receive complaints that the test was too rigorous and few dispersants that were previously listed on the NCP Product Schedule could achieve the 45% effectiveness criterion. Additionally, the SFT has been found to give widely varying results in the hands of different testi...

Laboratory Assessment of Bioremediation Products Under Freshwater Conditions

ABSTRACT The recovery and transportation of petroleum and petroleum products creates the potential for spillage of these materials into the environment. Bioremediation is a recently applied technology for clean up after a petroleum spill, and a protocol for evaluating bioremediation products in marine systems has been instituted. The U.S. Environmental Protection Agency (EPA) undertook this research to extend the protocol to freshwater systems, and to validate the protocol by examining commercial products for effectiveness in the laboratory. EPAs shake flask protocol developed for marine systems was adapted for this work. Commercial products were applied per manufacturers recommended dosage. All flasks received weathered Alaska North Slope oil at 5 g/L. The test flasks contained the commercial products with an inoculum developed from freshwater sources unless the product contained microbes. The control was an oil degrading inoculum in Bushneil Haas (Difco) medium. Triplicate flasks for each treatment we...

Overview of Aquatic Toxicity Testing under the U.S. EPA Oil Research Program

2017-063 ABSTRACT The U.S. EPA Office of Research and Development is developing baseline data on the ecotoxicity of selected petroleum products, chemical dispersants, and other spill mitigating sub...

Biodegradability of Orimulsion in Saltwater and Freshwater Environments1

ABSTRACT Shake flask and respirometer experiments were executed to test the biodegradability of Orimulsion in freshwater and saltwater. For each experimental setup, two concentrations of the Orimulsion and the appropriate bacterial inoculum were added to artificial seawater and freshwater solutions, and the concentrations of Orimulsion hydrocarbons were monitored with time. Respirometers were used to monitor oxygen (O2) uptake and carbon dioxide (CO2) evolution to determine when to sample the shake flasks. Sampling of shake flasks occurred periodically by sacrificing triplicate flasks for each treatment. Residual hydrocarbons were extracted with dichloromethane and quantified by gas chromatography/mass spectrometry (GC/MS). The respirometry flasks were sacrificed on the last shake-flask sampling event and similarly evaluated for residual hydrocarbon content and for Microtox toxicity. Data reported confirm literature citations that Orimulsion is indeed biodegradable, at least to some extent. In the 10 g/L ...