SUMMARY OF THE REMEDIATION TECHNOLOGIES DEVELOPMENT FORUM
PHYTOREMEDIATION ACTION TEAM
TOTAL PETROLEUM HYDROCARBON IN SOIL SUBGROUP
CONFERENCE CALL

April 5, 1999
12:00 p.m.-1:00 p.m.

On April 5, 1999, the following members of the Phytoremediation Action Team, Total Petroleum Hydrocarbon (TPH) in Soil Subgroup, met in a conference call:

Phil Sayre, U.S. Environmental Protection Agency (EPA) (Subgroup Co-Chair)
Steve Geiger, RETEC, Inc.
Peter Kulakow, Kansas State University (KSU)
C. M. (Mike) Reynolds, U.S. Army Cold Regions
Steve Rock, EPA
Ross Smart, Chevron Corporation
David Tsao, Amoco Research Center

Also present was Christine Hartnett of Eastern Research Group, Inc. (ERG)..

UPDATE ON CONTRACTUAL AGREEMENTS AND POTENTIAL SITES

The TPH in Soil Subgroup has created a field study program to evaluate how effectively plants degrade petroleum hydrocarbons across a range of test sites. Several site owners have expressed interest in participating in the Subgroup's field program and some have already initiated activities. Conference call participants gave reports on the following sites:

• Chevron's California site. Ross Smart noted that field activities were initiated at Chevron's California site in late 1998. He said that plant growth is strong at this site, with some plants reaching 12 inches in height. (Smart said that the robust growth is due to a recent fertilizer application and three rainfalls.) Smart said that weed growth on the control plot is being controlled by RoundUp. Site characterization and time zero (T₀) samples have been collected, Smart said, but the results are not yet available for the latter. In April, Smart continued, Peter Kulakow will visit the site and collect plant biomass samples. T₁ samples will be collected in June 1999.
• Chevron's Ohio site. Steve Rock said that Chevron and EPA are working together on a site in Ohio. Rock said that site characterization samples will be collected on April 6, 1999. During the week of April 19, 1999, Rock continued, T₀ samples will be collected and grasses and trees will be planted.
• Petroleum Environmental Research Forum (PERF) sites. Smart noted that PERF, an organization of petroleum companies, plans to participate in the TPH Subgroup's field study program. During the last conference call, he said, Evelyn Drake explained that the PERF contract had been drafted based on the following assumptions: (1) at least four petroleum companies will participate in PERF, (2) each company will contribute $20,000, and (3) PERF will be collectively responsible for field studies at two sites. Smart said that it is still unclear whether these assumptions can be met. To date, he said, only three companies (Chevron, Elf Aquitane, and Phillips Petroleum Company) have committed to the project and only one PERF site has been identified. He said that he is trying to determine whether Chevron's Ohio site (listed above) can serve as the second PERF site. He warned, however, that PERF will not be able to fund two sites unless a fourth company commits to the project. He said that Exxon is still considering whether to participate.
• Amoco's sites. David Tsao said that he recently identified a new site, located in Missouri, that might serve as a good candidate for field study. Funding for this site, however, will not be made available until next year. Tsao said that it is unclear whether Amoco will be participating in the Subgroup's field program at all. Assuming that it does, Tsao said, the work will be conducted under a Cooperative Research and Development Agreement (CRADA). He said that Amoco's lawyers had some questions about the CRADA and that these were forwarded to EPA's Larry Fradkin. Tsao said he will wait for Fradkin's response before proceeding.
• New York site. Steve Geiger said that he plans to conduct a demonstration project at a site in New York. Investigators have evaluated the site, he said, and chosen areas for site characterization sampling. Geiger said that the site's owners are in the process of evaluating a CRADA.
• Kansas site. Kulakow said that KSU plans to conduct a phytoremediation study at a military site in Kansas. He and Rock agreed to talk offline about establishing a research agreement between EPA and KSU.
• Alaska sites. Mike Reynolds said that field studies are underway at three Alaskan sites, located in Barrow, Galena, and Ketchikan. Seeds have already been planted, he noted, at the Galena and Ketchikan sites and field characterization samples have been collected at all three sites. The results indicate that (1) contaminant concentrations are most variable at the Ketchikan site, (2) biomarkers (i.e., hopane) are present at all three, and (3) microbial numbers are significantly higher at the Galena site. Expanding on the latter point, Reynolds said that he was not surprised that Galena had higher microbial counts because the site consists of a well-aerated and well-fertilized portion of a biopile. (Reynolds said that his team collected microbial information on bacteria, fungi, and actinomycetes.) Reynolds agreed to investigate the possibility of establishing a Memorandum of Understanding between EPA and his team.

SAMPLE ANALYSIS

Conference call participants discussed the following topics regarding sample analysis:

• Quantities used for agronomic analyses. Kulakow recommended collecting two to four samples for agronomic analyses at each site. He did stress, however, that the optimal number is site-specific and that investigators should use their best judgement as to how many samples will be needed to characterize a site. Kulakow said that KSU must have about 1 kilogram of soil per sample to run the agronomic analyses. Rock agreed to send samples to KSU from Chevron's Ohio site by April 9, 1999.
• Laboratory fees. Smart said that he contacted the Arthur D.. Little (ADL) and Battelle laboratories to confirm their prices. He said that both have agreed to charge the same fee for their services.
• Using standard samples. Smart said that laboratories can test their accuracy by analyzing a standard sample of known contaminant composition. He said that a standard sample has been collected and will be homogenized and ready for distribution within the next couple of weeks.

MICROBIAL ANALYSIS PROTOCOL

Kulakow said that Reynolds recently sent him a microbial analysis protocol. This will be incorporated, Kulakow noted, into the Subgroup's protocol. Conference call participants talked briefly about the microbial analysis protocol and discussed the following points:

• Collecting rhizosphere soils. Phil Sayre noted that Reynolds' protocol does not offer clear instruction on how to collect rhizosphere soils. Reynolds said that his team uses the word "rhizosphere" loosely, and usually assumes that the bulk soil in a planted area represents the rhizosphere. He acknowledged that there are more precise ways to collect rhizosphere soil (e.g., extract roots, shake them, and bang them against a glass plate), but said that these methods are very tedious.
• Using automated methodologies to collect most probable number (MPN). Sayre and Rock said that they are glad that Reynolds' protocol included more automated methodologies for collecting MPN. Compared to using tubes, Reynolds said, using a microplate method allows investigators to use less media and time. He did note, however, that tubes may support growth for longer durations because they are better at preventing drying. (Reynolds said that investigators are not sure whether evaluating longer-growing samples could be advantageous in some way.)
• Carbon sources. Rock noted that Reynolds' protocol recommends using some unconventional carbon sources. Reynolds said that vegetable oil is recommended for alkanes. Rock recommended putting a specific brand name in the protocol so that everyone is using the same growth medium.
• Evaluating three taxonomic groups. Sayre noted that Reynolds' protocol recommends collecting information on bacteria, fungi, and actinomycetes). He asked whether all three need to be monitored. Reynolds said that collecting information on all three will provide information on how each affects the other; he would particularly like to know whether different groups peak in activity when others are dormant. Information of this sort, he said, will let investigators know whether different groups play more predominant roles during certain times of the year. To date, Reynolds said, most of the data collected have been on bacteria because these are the easiest organisms to study. Reynolds' said that species from the other taxonomic groups can be enumerated on plates impregnated with Martin's medium. Reynolds said that this medium contains substances (e.g., streptomycin) that inhibit bacterial growth. It also slows fungal growth to the point that colonies, rather than mycelia, form. The former are easier to count.
• FAME. Reynolds said that tubes and microplates are useful for obtaining MPN, but that the FAME technique might be able to accomplish the same in a less work-intensive fashion. Reynolds said his group is trying to determine how FAME's results correlate with those of other methods. He said that Duane Wolf is also planning to use FAME.

Reynolds said that he wants to encourage other Subgroup participants to collect samples for microbial analyses at the same time that they collect samples for chemical analyses. Sayre recommended calling Bob Menzer to discuss initiating an extensive microbial analysis program. Sayre said that Menzer might have funding to support such a project.

APRIL 1999 TPH IN SOIL SUBGROUP MEETING

Sayre noted that a Battelle conference is scheduled in San Diego, California, on April 19-22. He said that the TPH in Soil Subgroup will meet on the afternoon of the 21st, during a lull in Battelle's sessions. The meeting will take place at the San Diego Sheraton Hotel and Marina's Harbor Island #1 room and will start around 2:00PM. Sayre stressed that it will be an informal gathering; conference call participants agreed that including too many people dilutes discussions on core issues.

Sayre said that he drafted a preliminary agenda in March. He encouraged conference call participants to provide comments on the agenda so that he can revise it and redistribute it by mid-April. Conference call participants talked about the following items on the meeting's agenda:

• Updates on field test projects, problems encountered, and data gathered to date. Sayre asked conference call participants if they wanted to give site updates and status reports at the meeting. Tsao said that he did not. Reynolds said he could give an update on his Alaskan sites and provide a summary of the T₀ and biomarker results. Smart agreed to provide an update on Chevron's California site; he said that he would gather site characterization and biomarker data for the presentation.
• Protocol review. Conference call participants expressed interest in having the Subgroup's protocol reviewed at the conference. Sayre said that he has asked some regulators from California to attend the meeting and is hoping that they will provide comments. He said that he also hopes to get feedback from Al Venosa because he has worked on several hydrocarbon cleanup projects in marine and near-shore environments. Tsao said that he sent the protocol to regulators from Rhode Island, Illinois, and New Jersey and that he would be willing to present their comments and concerns if they offer any feedback. (Tsao said that Bob Mueller, an active member of the Interstate Technology Regulatory Cooperation [ITRC], was one of the regulators who received the TPH in Soil Subgroup protocol. Tsao said that ITRC has already released a document about using phytoremediation for metals and plans to release one on organics in the future.)
• TPH Criteria Working Group (TPHCWG) methodology. Geiger agreed to give a presentation on the TPHCWG methodology.
• Biomarkers. Smart said that ADL's Greg Douglas has agreed to give a presentation on this topic. Reynolds said that Battelle's Kevin McCarthy has also expressed interest. Reynolds agreed to call Douglas and McCarthy to make sure their presentations do not overlap in content. After talking to them, Reynolds said, he will e-mail Rock, Sayre, and Lucinda Jackson to let them know what topics Douglas and McCarthy have agreed on.

ACTION ITEMS

• Kulakow and Rock agreed to talk offline about establishing a research agreement between EPA and KSU.
• Reynolds agreed to investigate the possibility of establishing a Memorandum of Understanding between EPA and his team.
• Rock agreed to send samples for agronomic analyses to KSU by April 9, 1999.
• Kulakow agreed to incorporate Reynolds' microbial protocol into the Subgroup's protocol.
• Sayre asked Reynolds to call Bob Menzer to discuss initiating an extensive microbial analysis program.
• Sayre agreed to revise and redistribute the April 1999 TPH in Soil meeting agenda.
• Smart agreed to provide an update on Chevron's California site at the April 1999 meeting. He said that he would gather site characterization and biomarker data for the presentation. Reynolds also agreed to prepare a presentation.
• Reynolds agreed to call Douglas and McCarthy to make sure their presentations at the April 1999 meeting will not overlap. After talking to them, Reynolds said, he will e-mail Rock, Sayre, and Jackson to let them know what topics Douglas and McCarthy have agreed on.