SUMMARY OF THE REMEDIATION TECHNOLOGIES DEVELOPMENT FORUM
PHYTOREMEDIATION OF ORGANICS ACTION TEAM
TPH IN SOIL SUBGROUP
CONFERENCE CALL

March 18, 1998
12:00 p.m. to 2:00 p.m.

On March 16, 1998, members of the Remediation Technologies Development Forum (RTDF), Phytoremediation of Organics Action Team, Total Petroleum Hydrocarbons (TPH) in Soil Subgroup, met via a conference call. The following members participated:

Lucinda Jackson, Chevron Corporation (RTDF Action Team Co-Chair and Subgroup Co-Chair)
Phil Sayre, U.S. Environmental Protection Agency (EPA) (Subgroup Co-Chair)
Steve Rock, EPA (RTDF Action Team Co-Chair)
Leslie Karr, Naval Facilities Engineering Service Center (NFESC)
Dawn Carroll, EPA
Evelyn Drake, Exxon Research and Engineering
John Finn, RETEC, Inc.
Wayne Fishs, Phillips Petroleum Company
Greg Harvey, U.S. Air Force
Joe Kreitinger, RETEC, Inc.
Peter Kulakow, Kansas State University
Ernest Lory, NFESC
Ryan Miya, University of California-Berkeley
Terry McIntyre, Environment Canada
C. M. (Mike) Reynolds, U.S. Army Cold Regions
Bruce Pivetz, ManTech Environmental Research Services Corporation
Lauren Trasell, Elf Aquitane, Inc. [Note: Spelling of name and affiliation not confirmed.]
David Tsao, Amoco Research Center
Duane Wolf, University of Arkansas

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

INTRODUCTIONS

Lucinda Jackson and Phil Sayre, the two co-chairs for the TPH in Soil Subgroup, welcomed participants to the Subgroup's first official conference call. Sayre said that the Subgroup has attracted a great deal of enthusiasm, with about 75 people expressing interest. To eliminate excessive confusion, the co-chairs invited only about 25 people to participate in the conference call. Sayre plans to distribute a summary of the meeting proceedings to those who were unable to participate in the call.

An agenda was distributed before the conference call. Sayre noted that the agenda was full, but hoped the Subgroup would be able to accomplish the following during the conference call:

• Identify people who are seriously interested in promoting phytoremediation as a technology used to remediate TPH in soil. Sayre hoped to identify a half dozen people who want to develop a standardized protocol and go forth with field testing.
• Talk about generating a database that will compile all of the information that has already been collected for phytoremediation of petroleum hydrocarbons in soil. (Sayre wants the database to include information that is not available through literature searches.)

Sayre asked people to introduce themselves and to briefly describe their involvement with phytoremedial technologies. (Note: The list does not include all of the participants because some people joined the call late.) The following people introduced themselves:

• Steve Rock, an environmental engineer with EPA, serves as a co-chair for the Phytoremediation of Organics Action Team and as a co-chair for the Alternative Covers Subgroup. The Alternative Covers Subgroup is setting up a network of test sites across the United States—a direction that Rock suspects the TPH in Soil Subgroup will also follow.
• Ryan Miya, a graduate student at the University of California-Berkeley, is working with Mary Firestone in the University's Department of Environmental Science, Policy, and Management. Miya is studying mechanisms that are involved with enhanced microbial degradation of organic compounds in rhizosphere soil.
• Terry McIntyre is manager of the biotechnology program at Environment Canada. His organization supports an active phytoremediation program and is currently conducting research on hearty oat and wheat species in their cold laboratory research facility. McIntyre is interested in collaborating with the Subgroup.
• Ernest Lory, a representative from NFESC, is involved with two phytoremedial projects at Port Hueneme in California. One of the projects involves methyl-tertiary-butyl ether (MTBE) cleanup. The other project—led by Purdue University's Katherine Banks and Paul Schwab—is using grasses and clover to remediate soil contaminated by waste oil. (At this site, TPH concentration falls between 2,000 and 4,000 parts per million [ppm].)
• C. M. (Mike) Reynolds, a research soil scientist with the U.S. Army Cold Regions, is working on several phytoremedial projects in Alaska. For the last 3 years, Reynold's group has been planting annual rye grass crops and collecting data at a small site near Fairbanks. The group will initiate field work in Barrow, Galena, and near Ketchikan in the summer of 1998. (The project in Barrow is a field demonstration of rhizosphere-enhanced remediation of TPH-contaminated soil.) Reynolds is eager to work closely with the TPH in Soil Subgroup on methodology issues to make sure that he stays "synchronized" with what others are doing. Specifically, he wants feedback on what parameters should be measured and how they should be measured.
• Leslie Karr, a representative of the NFESC, has been working with both Lory and Reynolds.
• Peter Kulakow, with the Agronomy Department at Kansas State University, has worked with Jackson, Paul Schwab, and Katherine Banks. Kulakow is mainly interested in identifying which plants are best suited for phytoremediation.
• David Tsao is with Amoco Research Center. Amoco is considering phytoremediation as a remedial option at a variety of sites. Amoco is investigating whether plants can be used to 1) remediate hydrocarbons and metallic salts, and 2) establish hydraulic control.
• Bruce Pivetz, a project scientist with ManTech Environmental Research Services Corporation, serves as a contractor to EPA. He provides technical assistance to different regions, including information about phytoremediation. Pivetz is particularly interested in the possibility of using phytoremedial technologies at refinery sites and wood preservative sites.
• Duane Wolf, from the University of Arkansas, is trying to establish field test sites in southern Arkansas. Wolf also works with Reynolds.
• Dawn Carroll, a representative from EPA's Technology Innovation Office (TIO), is involved with all three of the RTDF Phytoremediation of Organics Action Team's Subgroups.
• Joe Kreitinger is associated with RETEC, Inc., and Cornell University. He is currently working with the Gas Research Institute (GRI) and the Niagra Mohawk Power Corporation to evaluate hydrocarbon degradation.
• Wayne Fishs is a representative with the Phillips Petroleum Company. His company is interested in using plants to remediate petroleum hydrocarbons for two different scenarios, namely, salt-impacted soils and soils that are not salt-impacted.
• Greg Harvey, a representative of the U.S. Air Force, is the co-chair for the Trichloroethylene (TCE) in Ground Water Subgroup.
• Lauren Trasell, a representative with Elf Aquitane, Inc., is interested in learning more about the possibility of the RTDF Subgroup joining efforts with the Petroleum Environmental Research Forum (PERF). [Note: Spelling of participant's name and affiliation not confirmed.]

MECHANISMS FOR PHYTOREMEDIATION OF TPH

As noted by Jackson, investigators are not sure how plant systems degrade petroleum hydrocarbons. She recently talked with Miya and thought the Subgroup would benefit from hearing about his research.

Miya opened his discussion by describing some of the advantages and disadvantages associated with using phytoremediation to clean TPH in soil. The major advantage for using phytoremediation is cost savings. Compared to other types of remedial technology, phytoremediation is relatively inexpensive. Plants can also establish hydraulic control over an area and limit the potential for contaminant migration to occur. Limitations associated with the technology include:

• The technology only cleans soils that are located in the root zone.
• Some plants may be adversely impacted by contaminants.
• Some plants are unable to grow in certain climatic and soil conditions.
• The efficacy of plant remediation may be location-specific (i.e., some plants can only remediate specific contaminants, and only when the contaminant is located in a particular type of soil).

Miya noted that the degradation of petroleum hydrocarbons is enhanced within the rhizosphere. Although many researchers have documented this phenomenon, little experimental work has been conducted to determine why this occurs. One hypothesis is that plants remove contaminants from the soil by absorbing them (i.e., plant uptake) through their roots. Miya does not think that the "plant uptake mechanism" plays a large role in causing hydrocarbons to disappear from the rhizosphere, however. Given the large molecular weight and the hydrophobic properties of petroleum hydrocarbons, Miya thinks that plants would have difficulty absorbing contaminants. Miya thinks microbial activity is the primary force driving contaminant degradation. He listed several proposed mechanisms that could help explain why microbes are able to degrade contaminants more rapidly in the rhizosphere than in other soil zones:

• Larger microbial populations. Compared to other soil zones, more microorganisms are present in the rhizosphere. The greater the number of microorganisms present, the greater the likelihood that microbes will contact a contaminant.
• Increased microbial activity. It is possible that microorganisms in the rhizosphere "work harder" (or have increased metabolic activity) than other microorganisms. Increased activity could be fostered if the plant roots provide microorganisms with a source of nitrogen or a vitamin.
• Rhizosphere conditions allow microorganisms to retain their ability to degrade contaminants over a longer period of time. David Creole's work suggests that microorganisms living in the rhizosphere are able to preserve their genetic stability for longer periods of time. [Note: Spelling of researcher's name not confirmed.]
• Selective enrichment of degrading-type microorganisms. It is possible that something in the rhizosphere environment may help sustain degrading-type microbial populations.

Aside from being important in the degradation process, microbes may help plants become established in contaminated environments. Some researchers suspect that rhizosphere microbes protect plants from harmful toxicants.

In an effort to better understand what mechanisms are actually operating, Miya is conducting three studies:

• Assessing changes in the quality and quantity of degraders in rhizosphere soil versus non-rhizosphere soil. For this study, Miya is exposing oil-degrading microbes to phenanthrene and hexa-decane. He is using an overlay technique to isolate the degraders.
• Analyzing how contaminant degradation is affected by various components of the rhizosphere. For this study, Miya will measure contaminant degradation rates in an environment that does not have roots. He will then add different rhizosphere components (e.g., root exudates and root debris) to see how each component impacts the degradation rate. This experiment will help determine which components of the rhizosphere are most important for promoting petroleum hydrocarbon degradation.
• Ultimate fate analysis. Miya will use radio-labeled hydrocarbons to determine whether hydrocarbons are taken up by plants. Sayre asked Miya if this study will show whether the polar metabolic byproducts of microbial activity are being absorbed by plants. (Sayre noted that a French team [Chaineau, C.H., et al. 1997. Phytotoxicity and plant uptake of fuel hydrocarbons. J. Environ. Qual. Vol. 26] recently concluded that plants do not take up hydrocarbons, but they may be able to take up polar metabolic byproducts that result from microbial activity.) Miya's experiments will not address this issue. Miya said the only way to determine whether plants are taking up byproducts involves setting up an abiotic (i.e., no microbes) environment. Sayre thinks it would be interesting to find out if the byproducts are being absorbed. If they are, it would be interesting to find out if the byproducts could pose a health hazard if a person ate the plants.

The plant species that Miya is using is a wild oat grass species (i.e., Avena barbata). He selected this grass because it has been shown to grow well in TPH-contaminated soils.

UPDATE ON THE PERF ACTIVITIES

Evelyn Drake provided a summary of PERF's activities. In 1994, 10 oil companies and GRI initiated laboratory experiments analyzing whether plants can remediate hydrocarbons in 1) crude oil contaminated soil, and 2) gas pit sludge contaminated soil. When the laboratory experiment was initiated, five to six plant species were planted, but only two to three of the species were predominant by the end of the studies. Significant biodegradation of hydrocarbons was reported in both types of soil. Due to the success documented in the laboratory, PERF is eager to initiate field studies.

For the field studies, PERF plans to set up demonstrations at one crude oil contaminated and at one gas pit sludge contaminated site. PERF has drafted an affordable field test design that involves:

• Identifying potential sites. Candidates for the crude oil contaminated site have been identified in the Gulf and in the western part of the country. For the gas pit sludge contaminated site, ARCO has a site in California that is being considered as a potential candidate.
• Characterizing potential sites. PERF will ask the site owner to help with site characterization.
• Making a final selection
• Planting appropriate perennial plant species. PERF plans to initiate field activities in the spring of 1998. Drake suspects that PERF will choose a grass species. To date, the PERF team has not had the "luxury" of testing different grasses to determine which ones exhibit the metabolic characteristics that lead to the most effective hydrocarbon degradation. Rather than being based on performance, the choice of which plants to use will be decided by climatic suitability, ease of establishment, and its ability to tolerate contaminated soils. One participant asked if PERF's final site selection will hinge on the ease with which a plant species could be established at the site. Drake said that this will be one of the factors considered in the decision-making process.
• Monitoring for hydrocarbon removal for three years. Drake said that PERF plans to monitor for hydrocarbon removal at the beginning and end of the season for three consecutive years. Kulakow and Reynolds noted that a three-year monitoring period may not be sufficient. They thought a longer monitoring period would be preferable since plant species take a long time to establish and mature. Drake took their considerations seriously and plans to ask PERF decision-makers to consider increasing the monitoring period to 5 years.
• Determining whether hydrocarbons are being transferred to plant material. PERF will probably analyze plant tissues to determine whether hydrocarbons are being transferred to plant tissues.

PERF has identified three partners (i.e., Chevron, Exxon, and Elf Aquitane, Inc.) who are committed to donating money to help promote the field phase of PERF's project. PERF is hoping to identify other participants. Ideally, they are looking for partners who can contribute $25,000.

POSSIBILITY OF MERGING THE PERF AND RTDF EFFORTS

Many discussions have been held regarding the possibility of merging the PERF and RTDF efforts. Drake hopes that the two groups can work together. She thinks working with the RTDF TPH in Soil Subgroup will help:

• Strengthen PERF's field test design. Drake noted that the PERF design is very simplistic. She hopes that the RTDF Subgroup can help make the plan more "robust."
• Provide guidance on which plant species to use. As noted above, PERF has not performed studies to determine which plants are the most effective in remediating petroleum hydrocarbons. Because plants are the "bread and butter" of this technology, Drake is eager for someone to perform work in this area. She is hoping that the RTDF Subgroup will look at this issue.
• Provide regulatory guidance. Drake noted the importance of having regulatory guidance throughout field testing so that investigators can ensure that field tests address regulatory concerns. Before setting up demonstrations, Drake thinks the following questions need to be answered:

• What is the cleanup target for the site?

• What is the likelihood that the cleanup level will be achieved within a three year period?

• To what depth do soils need to be remediated to satisfy regulators: 0 to 6 inches, 0 to 2 feet, or 0 to 4 feet? Drake asked the participants whether regulatory expectations vary widely between different individuals and different states. Rock suspects expectations differ from state to state. Drake noted that New Jersey regulators used to want the first 2 feet of soil remediated, but more recently they have started to focus more attention on the first 6 inches.

  Phytoremedial technologies will not be overly successful if they are expected to clean deep soils. Nobody knows exactly how deep roots will go. As noted by Drake, work done in the laboratory cannot always be used to predict what will happen in the field. She noted a site where roots did not grow as deep as expected. (After 1 to 2 years, only fine root hairs extended below 2 feet.) Reynolds experienced the opposite; at his site in Fairbanks, Alaska, roots grew deeper than expected.

Sayre said that Paula Estornell—a representative of EPA's Office of Solid Waste and Emergency Response—plans to be a part of future Subgroup conference calls.

If PERF and the RTDF Subgroup do join efforts, they will work within a cooperative research and development agreement (CRADA). Drake asked whether project information is available to people who are not contributing funds. Rock assured her that CRADAs are set up to protect information (e.g., site names) that companies don't want to spread widely. Only those people who contribute money or in-kind (e.g., laboratory time, labor, or characterization studies) services can get access to information before it is published. Rock stressed that everything will eventually need to be published since EPA research is in the public domain. Sayre knows that some PERF participants have encountered the problem of being steered heavily by people who are not contributing much to a project. To avoid this situation, Sayre thinks the PERF-RTDF relationship could be setup so that people have to "pay to play"—a situation where those who contribute the most can provide the most input in decision-making. Rock told Drake to call him with any additional questions regarding CRADAs. Sayre recommended contacting EPA's CRADA expert, Larry Fradkin (513-569-7960), for additional information.

The discussion on CRADAs prompted a discussion about the legalities involved with transferring information between EPA and Environment Canada. Environment Canada has a Memorandum of Understanding (MOU) with the EPA that allows transfer of information between the two agencies. Sayre noted that other international agencies have participated in other RTDF groups. McIntyre and Sayre agreed that the issue will need to be explored further to determine how Environment Canada can participate within a CRADA.

Drake has asked representatives from Chevron, Exxon, and Elf Aquitane, Inc., to consult with their managers to see if they would be willing to work within the context of the RTDF and within a CRADA. She expects that the participants will have answers at the next conference call (April 6, 1998).

REGULATIONS THAT THE SUBGROUP NEED TO CONSIDER

McIntyre noted that researchers may face some regulatory opposition when trying to work with certain plants. In Canada, he had difficulty gaining approval to use a U.S. Department of Agriculture (USDA) plant because the plant represented a whole new product line for the Canadian Department of Agriculture.

In the United States, Sayre thinks some naturally occurring plants are subject to USDA oversite under the Plant Pesticide Act. Other than that, he is not aware of any federal regulations that would restrict plants from crossing state or country lines. Rock said that some states do have regulations preventing certain plants from entering.

John Finn reminded participants that federal quarantine regulations apply if soil is attached to plants. This is not an issue when seeds are used.

Sayre noted that the group may have to deal with additional regulations in the future if genetically-engineered plants become a viable option. Laws for transgenic plants include the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and Toxic Substances Control Act (TSCA).

MISSION STATEMENT

Sayre proposed the following as a mission statement:

Combine expertise, technologies, and resources to conduct phytoremediation field tests for degradation of petroleum hydrocarbons in order to develop guidelines for future applications and to gain public and regulatory acceptance.

None of the participants objected to the mission statement.

SUBGROUP'S GOALS

Selection of Most Efficacious Plants

Jackson noted that most investigators are using grasses as a remedial plant. Miya noted several advantages that grasses offer over other types of plant species, including:

• Grasses are genetically diverse.
• Grasses have low biological concentration factors.
• Grasses have an extensive rooting system that allows them to 1) establish an extensive rhizosphere zone, and 2) reduce water infiltration, thereby limiting the potential for migration to occur.

Several participants agreed that grasses and native plants are the best options to pursue. Jackson asked the participants if they wanted to limit their investigations only to grasses. Kreitinger told the group that GRI and the Niagara Mohawk Power Company have set up pilot scale projects to determine whether willow groves can remediate polyaromatic hydrocarbons (PAHs). (Some of these test sites are located in New York.) The interest to use willow was prompted by a DOE-sponsored program aimed at identifying alternative energy sources.

The participants discussed ways to evaluate which plants are the most effective at remediating petroleum hydrocarbons. Three methods were suggested:

• Conduct extensive field testing. Drake suggested setting up 10 different plots (each with a different plant species) at one test site. Reynolds thinks this is a great idea and has considered pursuing this avenue in the past. He warned the group that two obstacles standing in the way are 1) determining how to monitor performance, and 2) cost.

• Analyze plant metabolic properties. Drake summarized work that Laura Carreira is doing to analyze immunoassay systems for enzymes that are excreted from plants. While with the University of Georgia, Carreira investigated nitrogenases. She is now a partner in her own company, PhytoWorks, and she has expanded her scope to include a wide variety of enzymes, including dehydrogenases—enzymes that degrade PAHs. Drake thinks metabolic methodologies could provide investigators with a screening technique that will help determine which plant species are the best at breaking down hydrocarbons. Rock agreed that Carreira's work is interesting and has sent a couple of people to see her. He does not think the RTDF Subgroup could enlist her help, however, because she is charging a high price for her services and she is making her clients sign a disclosure agreement.
• Analyze the ways that plants modify the physical environment. Kreitinger said that Cornell is trying to develop screening techniques to evaluate changes in bioavailability of hydrocarbons in rhizosphere soil. This group is evaluating desorption, kinetics, and changes in soil pH. Kreitinger doesn't know if this approach will prove to be cheaper than Carreira's approach.

Selection of Key Hydrocarbon Mixtures

Jackson asked participants to list the types of hydrocarbon mixtures that they would like the Subgroup to pursue. The participants listed crude oil, refinery waste, and gas pit sludge. By the end of the conversation, the Subgroup decided that it would be better to identify "trigger" contaminants— compounds that cause the biggest health problems and attract the most regulatory interest. Jackson thinks the group should focus on trigger PAHs, regardless of their initial source.

During this part of the conversation, Lory asked the Subgroup to consider fuel additives. At some sites that he is working on, fuel additives are starting to "rear their ugly heads." He noted MTBE as an agent that causes potential concern. One participant noted that MTBE travels quickly to groundwater, spending limited time in soils. As a result, the participants thought that Lory's concerns about MTBE would be better assessed by a subcommittee within the TPH in Soil Subgroup or by the Vegetative Cap Subgroup. (Trees could be used to achieve hydraulic control and to prevent contaminants from leaching downward.)

PROPOSED PROJECT

Jackson and Sayre attached a description of a proposed project to the conference call agenda. The project involves setting up a series of test sites across a variety of climatic regions. The same plant species and the same data collection protocol would be conducted at each of the test sites. (Ideally, multiple plant species could be tested at each site, against a control plot.) Such an effort would provide insight about which plant species are most efficient under different climatic and edaphic conditions. At a minimum, each site would be monitored to determine the extent of contamination reduction in soil. Other analyses might include plant tissue contaminant analysis and microbial monitoring.

In order for the Subgroup to successfully conduct the proposed project, it will need to identify several potential test sites and participants who are truly dedicated to promoting the technology.

Field Sites

Sayre and Pivetz described a "high desert" site in Lovell, Wyoming. Participants were unsure exactly who is involved with remedial activities. Sayre thinks that Johanna Miller (EPA Region VIII) is in charge of remedial decisions at the site. The Coast Guard was involved, but is now trying to pull out. This 26-acre site has about 52,000 cubic yards of refinery sludge. Soils are contaminated with weathered TPH (average concentration of 30,000 ppm). The site is saturated with water immediately below the surface. Alfalfa is going to be planted to dry the surface soils, and was chosen as an option after "discing" efforts failed to work. Bioremediation pilot studies are scheduled for this site. Originally, the Subgroup identified it as a potential site for phytoremedial activities. While the group may be able to set up a test demonstration at this site, Sayre doubts that the Subgroup would be able to design a test that would align with what they hope to do at other sites.

Jackson said that Chevron has potential sites located in Cincinnati, Ohio, and Richmond, California.

McIntyre said that Environment Canada may be able to identify as many as 700 potential sites, the vast majority of which are refinery sites. He hopes to have more detail about these sites by the next conference call.

Wolf knows of three potential test sites, and Tsao said that Amoco has candidate sites. Lory added that the Navy has an aboveground tank farm that may serve as a candidate site.

People Who Are Interested in Promoting the Subgroup's Activities

By the time that Jackson asked who was interested in promoting the Subgroup's activities, some participants had already gotten off the phone. People who did respond with enthusiasm included:

• Kreitinger, who thinks GRI and the Niagara Mohawk Power Company will be interested in collaborating with the Subgroup's field program.
• Reynolds
• Tsao

Lory would like to be involved, but he is not sure to what extent.

CREATING A STANDARDIZED PROTOCOL

Participants think there is a great need to develop a standardized protocol. Drake cautioned, however, that the protocol will have to be flexible. Reynolds thought it would be helpful to draft a protocol that prioritizes activities since budget constraints often prevent groups from sticking to the ideal protocol.

Jackson will take the lead in developing a standardized protocol. She plans to use the PERF protocol as a springboard, and will work closely with Drake, Sayre, and Rock to generate a draft. It will be completed by March 30, 1998, and circulated to participants so that the protocol can be discussed during the next conference call.

CLOSING REMARKS AND DATE FOR NEXT CONFERENCE CALL

Sayre thanked the participants for their enthusiasm and support. He noted that the one of the goals for the conference call—talking about a database—was not discussed. He said that this topic will be dealt with on another occasion.

The participants talked briefly about the IBC conference that will be held in Houston, Texas. Rock said a room is reserved between 6:30 p.m. and 10:00 p.m.. The Subgroup can use this time slot to chat among themselves and to let others know about their activities.

The next conference call is scheduled for April 7, 1998. Before that time, Sayre and Rock will to try to encourage regulatory involvement.