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

May 8, 1998
11:00 p.m.­12:30 p.m.


On May 8, 1998, the following members of the Phytoremediation of Organics 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 Rock, EPA
Kathy Banks, Purdue University
Paul Schwab, Purdue University
Jim Brown, Roy F. Weston
Pam Davis, EPR
Peter Kulakow, Kansas State University (KSU)
C.M. (Mike) Reynolds, U.S. Army Cold Regions
David Tsao, Amoco Research Center

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


SAMPLE ANALYSIS--CHOOSING AN ANALYTICAL LABORATORY

During the May 1, 1998, conference call, participants agreed to research the analytical capabilities and cost rates of three laboratories: Arthur D. Little (ADL), Batelle, and KSU/Purdue.

    ADL

As noted by Steve Rock, the RTDF Subgroup is well aware of ADL's excellent reputation for polyaromatic hydrocarbon (PAH) and biomarker analyses. Rock said that everyone he has talked to within EPA has given ADL excellent reviews. Phil Sayre recently received three papers describing ADL's biomarker analysis protocol. Sayre agreed to distribute these papers to the conference call participants.

Rock said he was unable to obtain a cost estimate from ADL in time for the conference call. ADL has agreed to prepare a quote once it receives more information about the RTDF Subgroup's needs. Evelyn Drake had told ADL that she would fax the laboratory a sample outline during the first week of May, but she was unable to do so.

    Batelle

Rock noted that he contacted the Batelle laboratory in Columbus and learned that it can perform agronomic analyses. Mike Reynolds said that he will visit the Batelle laboratory in Duxbury, Massachusetts, on May 11, 1998. He agreed to present the sampling outline to Batelle if Drake could provide it in time. Sayre told Reynolds to contact Drake directly for the outline.

    KSU/Purdue

Paul Schwab and Kathy Banks discussed KSU/Purdue's capabilities. They were familiar with the RTDF Subgroup's draft protocol and recognized that the Subgroup requires the following analyses:

Schwab said that he and Banks had discussed cost but did not have a price quote at the time of the conference call. Rock explained that the Subgroup has incentives to use a Hazardous Substances Research Center (HSRC) laboratory, which are that (1) EPA has established contracts with HSRC laboratories and (2) Technology Innovation Office (TIO)'s Walter Kovalick may be willing to contribute money for sample analysis if the Subgroup chooses an HSRC laboratory. (Sayre said that he and Kovalick planned to talk further about this possibility on May 8, 1998.)

Banks said that KSU/Purdue can offer cheaper analyses than ADL but that KSU/Purdue cannot process samples as quickly as ADL. Banks asked the RTDF Subgroup the turnaround time it requires. For the T-1 sampling (i.e., the site characterization sampling), Rock said the team will need results within 2 weeks. (About 20 samples will be collected per site for the T-1 sampling.) Banks thought this could be problematic given the number of proposed sites. Rock assured Banks that the samples would not all arrive at once. He estimated that activities would be initiated at four or five sites this spring. He also said that the T-1 samples would require only the PAH and biomarker analyses rather than the full suite of analyses. Sayre said that bacterial counts might be required at this stage as well, depending on how the final microbial analysis protocol is drafted.

Bank asked Rock how soon the RTDF Subgroup needs to start having samples analyzed. Rock said the group needs results very soon. Schwab explained that the Purdue laboratory is still being set up and that its instrumentation is just starting to go on line. He doubted that Purdue would be able to process any samples for at least another 6 weeks. Rock said this could be problematic. Ideally, Rock said, the Subgroup wants to use the same laboratory throughout the program rather than switch mid-program.


STATISTICAL REVIEW OF PROTOCOL

Sayre noted that it is imperative for the group to identify a statistician who can help develop the team's protocol. Once a statistician is chosen, Sayre recommended having him or her review Banks' work. (Banks' report indicates that investigators should aim to detect a 9 to 19 percent difference between their seeded plots and their unseeded plots.)

Jim Brown said that he had contacted Charlie Kufs, a statistician formerly employed by Roy F. Weston and that Kufs is very interested in helping the RTDF Subgroup. Brown asked Rock whether he had spoken with Kufs. Rock said he had left a message with Kufs but had not heard from him. Brown agreed to recontact Kufs and to encourage him to call Rock.


SHIPPING PROTOCOL

During the May 1, 1998, conference call, ADL agreed to send Sayre a copy of its shipping protocol. Sayre said he had received a fax from ADL but that the fax merely stated that ADL will send appropriate shipping containers and will provide guidelines on collection and holding techniques once activities begin.


MICROBIAL ANALYSIS PROTOCOL

Sayre asked Banks whether she could provide a description of the microbial analysis protocol she uses. Banks said that she is publishing the protocol but needs another month to finalize the publication. Banks noted that the RTDF Subgroup's draft microbial analysis protocol is like the one she uses. Differences between the two include the amount of soil and the amount of phosphate buffer used.

Banks said that she and Pam Davis use similar methods and recommended asking Davis for a written protocol. Davis agreed that she and Banks have used similar approaches but noted that Sara McMillen had changed the EPR protocol. According to Davis, McMillen suggested using a syringe to inject oil and dilutions into prefilled and sealed viles directly. This approach, Davis noted, reduced the potential for evaporation. Banks said that McMillen's protocol sounded "good" and asked Davis whether it had been published. Davis was unsure but said she would find out. Sayre asked Davis to send him a copy of the protocol. Davis said that she must first ensure she is allowed to distribute the protocol.


BACTERIAL ENUMERATION TECHNIQUES

During the April 24, 1998, conference call, participants discussed whether the RTDF team should use a standard weathered crude for the bacterial enumeration procedures. Since then, Sayre and David Tsao have talked to API staff and have learned that a standardized weathered crude is not available.

Brown said that representatives from Micro-Innotech, a laboratory in St. Louis, told him that it is important to use a reference oil that is characteristic of the site. Based on this information, Brown recommended that the Subgroup choose two or three types of reference oils (e.g., a weathered crude and a diesel oil) rather than just one. Rock noted that the potential test sites generally have weathered or refined oil.

While Brown believed two or three different reference oils would suffice, he noted that site-specific oils would be ideal. In New Jersey, he noted, a senior scientist had derived a protocol for extracting oils from contaminated soil. While Brown said the extraction methodology is fairly straightforward, he also said he realized that performing an extraction at each site might be too labor intensive and costly. Brown agreed to distribute information about the extraction procedure's methodology and cost.


WEED CONTROL

Rock said the RTDF Subgroup must choose a weed-control option. Rock said there are four options:

Rock said that some scientists are reluctant to use chemical control because they are unsure how herbicides impact microbial populations. Uncertainty about potential impacts prompted some of the conference call participants to recommend hand picking or tilling. Other participants noted, however, that these two options are too labor intensive at some sites. If chemical controls are used, Rock recommended a post-emergent herbicide (e.g., RoundUp) rather than a pre-emergent one. (Rock said that he does not like using pre-emergents because they require spraying herbicide over large areas.) Peter Kulakow, Banks, and Schwab said that they have used RoundUp at their sites because they did not have the labor for hand picking or tillage. Kulakow said RoundUp is a good choice because its effects are short lived. Reynolds said that he has talked with Duane Wolf and other soil microbiologists and they agree that RoundUp is a reasonable compromise.

Because budgets and labor availability differ so dramatically from site to site, Kulakow did not think the RTDF Subgroup's protocol should favor one form of weed control. He thought it best to make the decision site specific. Rock agreed with this approach and said that the protocol will simply require that investigators specify their approach.

Rock asked participants which methods they plan to use at their sites. Reynolds said he was leaning toward a hand-picking approach because he thought he would have ample local labor for his projects. Tsao was unsure which weed control method he would use but had narrowed it to hand picking or post-emergent chemical control. Tsao said that he plans to investigate the economics of each approach before making a final decision. Rock said that he was leaning toward RoundUp at his site because he was unsure of his labor availability.


SEED MIX

Rock noted that the group had decided to use a variety of plant groups in its control mix. He asked the group for guidance in establishing percentages for the mix. Kulakow and Rock together generated the following percentages:

None of the conference call participants objected to these recommendations. Rock said that he would write this recommendation into the protocol. He also said that he would not object to people altering these percentages at sites. Kulakow stressed that the most important step is that investigators document their actions. Kulakow said he would be happy to talk to people about using plant species in different climates.


MISCELLANEOUS

Tsao said that he had collected and frozen samples from one of his sites and plans to initiate sampling activities at a few other sites within the month.

Rock said that he hopes to collect samples in mid-May.

According to Rock, Lucinda Jackson plans to initiate activities within the year at a California site.

Rock asked Brown whether Royal Nadeau is considering testing phytoremediation at a Wyoming site. Brown said he could not speak for Nadeau.

Reynolds noted that his group is performing a full analysis on some samples and that he is investigating whether he can perform these analyses at other sites. Reynolds said he hoped these efforts would help form a database. Rock thought this was a "fantastic" idea.


ACTION ITEMS


NEXT CONFERENCE CALL

The next conference call is scheduled for May 19, 1998, between 11:00 a.m. and 12:30 p.m. (EST).


Attachment A

Field Study Protocol


REMEDIATION TECHNOLOGIES DEVELOPMENT FORUM
PHYTOREMEDIATION ACTION TEAM
FIELD STUDY PROTOCOL
1 May 1998 Version

TITLE: PHYTOREMEDIATION OF PETROLEUM HYDROCARBONS IN SOIL
PURPOSE: Determine efficacy of vegetating with agricultural and native plants for degradation of petroleum hydrocarbons in soil at multiple locations and under varied climatic conditions.
TREATMENTS:
1. Mixture of (a) rye (annual or perennial), (b) a legume (alfalfa, clover, birds-foot trefoil), and (c) fescue, with varieties chosen to optimize growth under local conditions.
2. Native grass mix or other species, including trees.
3. Unplanted control (kept weed-free; use RoundUp or hand-picking of weeds).

LOCATIONS:
Approximately 12 sites in North America.
SOIL SAMPLING: Take soil samples in each sampling location at the following times:
Initial site characterization (samples taken 0 to 6" after tilling)
0:       Before planting, after seed bed prep (samples taken at 0 to 6" and 1 to 2')
1:       6 months after planting (samples taken at 0 to 6" only)
2:       18 months after planting (samples taken at 0 to 6" only)
3:       30 months after planting (samples taken at 0 to 6" and 1 to 2')

Use 1" diameter GEOPROBE "large bore" sampler is suggested. Place each sample in a clean plastic sample liner; use hand sampling devices.
PLANT SAMPLING: Take plant shoot samples at time 3 (30 months) for hydrocarbon analysis. Method will be provided.
SAMPLE STORAGE: Sample storage and shipping protocol will be provided.
SAMPLE ANALYSIS: Send composite soil samples to Arthur D. Little, Inc. (ADL), Environmental Monitoring and Analysis Unit, for archiving and analysis of:

1. pH and salinity (times 0 and 3)
2. Available nutrients (times 0 and 3). (The analyses should be tailored to the soils in that region of the country and to the plant species used in tests.)
3. Petroleum (EPA method for saturated and aromatic hydrocarbons analyzed with GC, TPH measured, individual PAHs measured) (times 0, 1, 2, and 3)
4. Biomarkers (times 0, 1, 2, and 3)
5. Microbial analysis (times 0 and 3)
6. Soil analysis (texture, organic matter, EC, CEC, soil type, etc.) (Time 0)


PLOT SIZE:
20' x 20' minimum
REPLICATIONS: 4
STATISTICAL DESIGN: Randomized complete block (place plots based on presence of TPHs in site characterization. For soil and plant samples, take 4 random sample cores per plot and make a composite sample.
GROWING CONDITIONS: Conduct seed bed preparation, planting technique, planting rate, and irrigation in site-specific manner to establish good stand growth. Record procedures.

Fertilization is based on need from initial soil sampling.
PLANT EVALUATIONS: Evaluate plant characteristics at 6 months, 18 months, and 30 months:
  • Percent cover

  • Shoot height

  • Rooting characteristics (root depth and density)

WEATHER CONDITIONS:
Record rainfall and average daily temperature (available from local airport) throughout growing season.
SITE HISTORY: Source of contamination
Past site uses
Previous cleanup attempts
Climate

Protocols for Bacterial Analyses

Bacteria to monitor: Total hydrocarbon degraders, based on growth on a standard weathered crude oil. Note that this protocol accounts only for bacteria and therefore does not lead to enumeration of other hydrocarbon-degrading taxa, like fungi.

Soil sample size needed and treatment of sample before analysis: Sample size not yet specified. Sample handling should minimize freeze-thaw cycles on samples.

Hydrocarbon mixture to test with: A standard crude hydrocarbon that can be weathered in a standardized way. [Options: an Exxon or ?Amoco standard, an American Petroleum Institute Standard Oil; Weathering protocol not yet discussed]

Protocols for bacterial counts: [exact protocol to use and costs not settled]

Two apparently similar MPN techniques have been developed by Kathy Bank's lab at KSU and by Exxon. The Banks' technique uses an MPN approach, which relies on dilution of soil in a Bushnell Haas broth (mineral salts broth lacking carbon source). Soil is diluted 10-9 with 5 replicates of each dilution. A tetrazolium (redox) dye is added to each suspension and vortexed. The petroleum substrate was added to each suspension, then capped with an aerated cap. Controls of the dye and hydrocarbon were included as precautions against contamination. Changes in dye color indicate microbial respiration and were recorded weekly until activity ceased. The number of positive responses at each dilution level were used for the final calculations. MPN tables were used to determine the number of degraders in each soil sample. Exxon used a technique similar to that of Kathy Banks. He uses the Bushnell Haas broth, and multiwell plates. Each well contains 10 ul of weathered crude oil on top of broth, and growth is assessed visually (sheen broken, growth noted, etc. "sheen-screen" method?). Four replications are used for each dilution. Heterotrophs are also enumerated in a rich medium, with cloudiness indicating growth, and no dyes used.

Microbe Innotec Labs uses an automated MPN technique that employs software developed by Al Venosa (EPA). This software automates the process for determining the number of heterotrophs and TPH degraders. Paul Flaffman of OHM is also familiar with this technique. Costs are estimated to be $68/sample for heterotroph analysis and $195/sample for 48-hour TPH degrader analysis.

The 1980 APHA/AWWA/WPCF reference book Standard Methods for the Analysis of Water and Wastewater, 15th edition, has a Method 907 that uses the same Bushnell and Haas agar and a hydrocarbon supplement to enumerate hydrocarbon degraders in soil. Some later editions of the text do not have the protocol, however.

Timing of Sampling for TPH Degraders, Identifying Number of Degraders Needed for Efficient Degradation

Hydrocarbon degraders often make up 10 percent of heterotrophs at a contaminated site. Usually, there are enough hydrocarbon degraders to affect remediation if time 0 counts are on the order of 104 TPH degraders per gram of soil.

Protocol currently calls for microbial sampling at time 0 (before fertilization) and time 3 (30 months). The time 0 sampling is intended to determine if sufficient degraders are present. The time 3 sampling is intended to see if degraders have increased in number due to rhizosphere effect (and, to a lesser degree, nutrient amendments) in phytoremediated plots. Further sampling between times 0 and 3 (e.g., at the end of each growing season) would help determine if bacterial numbers have changed due to plant presence. Because TPH degraders tend to increase in numbers in the first few weeks after fertilization, samples should probably be taken 2 months after the last fertilization. All fertilization dates/application rates should be specified.