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

May 19, 1998
11:00 p.m.­12:30 p.m.

On May 19, 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
Pam Davis, Exxon Product Research (EPR)
Evelyn Drake, Exxon Research and Engineering
Steve Geiger, RETEC, Inc.
Peter Kulakow, Kansas State University (KSU)
Charlene Owens, EPR
C.M. (Mike) Reynolds, U.S. Army Cold Regions
David Tsao, Amoco Research Center
Duane Wolf, University of Arkansas

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

EVELYN DRAKE'S OUTLINE

During the May 1, 1998, conference call, Evelyn Drake agreed to compile an outline of the TPH in Soil Subgroup's planned activities. She completed the outline and distributed it to conference call participants prior to the May 19, 1998, conference call. She also distributed the outline to Arthur D. Little (ADL) and Batelle so that these two laboratories could use the outline to provide cost estimates. Drake provided a brief description of the outline and asked conference call participants for feedback.

Phytoremediation Field Test Design

Drake's outline states that each test site will consist of 12 plots. She arrived at this number by assuming that four replicate plots will be established for three different kinds of treatments (i.e., unvegetated control and two different kinds of vegetated treatments).

Soil Sampling Plan

Preliminary Site Characterization

Drake's outline states that 20 composite samples will be collected as part of the site characterization sampling at each site. Using this approach, 20 potential test plots would be established and then the 12 plots that are most similar in hydrocarbon type and concentration would be chosen for the field study. Drake argued that using this approach significantly reduces site variability.

Drake's outline recommends collecting the site characterization samples at a depth of 0 to 6 inches. David Tsao noted that he is required to clean the soil to a depth of 18 inches at an Amoco site in Texas City. Drake told Tsao that she would recommend collecting site characterization samples from a depth of 0 to 18 inches at this site.

Peter Kulakow thought Drake's approach was interesting and worthy of further consideration. He noted, however, that her plan differs from the one discussed during previous conference calls. In previous discussions, the Subgroup had agreed to use a randomized complete block design, where 20 random grab samples would be collected from test sites. Data from these samples, Kulakow explained, would then be channeled to a statistician, who would be able to assess the site's variability and determine how many replicates would be required to show X percent difference between treatment plots. Drake questioned this approach, asking whether it is valid to collect one type of sample (i.e., grab) during site characterization and another type (i.e., composite) during field testing. Both Kulakow and Drake agreed that the Subgroup should consult a statistician to get input on the best way to perform the initial characterization.

Mike Reynolds noted that the site characterization data will play a vital role in determining how many samples must be collected, how to lay out field plots, and how many replications are required. This means, he said, that investigators may need to leave ample time between collecting site characterization samples and establishing field test plots. To minimize the lag, Drake said, she has asked laboratories to complete analysis of the site characterization samples within 7 days. (Drake said she also asked the laboratories for a 7-day turnaround for the initial [T₀] field test sampling.)

Field Test Sampling

Drake estimated that 92 samples will be collected at each test site. According to her outline, 68 of these samples will be collected at 0 to 6 inches and 24 samples will be collected at a depth of 6 to 18 inches. For both the shallow and the deep samples, Drake's outline specifies that 12 samples (one composite sample per plot) should be collected per sampling event. Drake's outline recommends collecting shallow samples at T₀, after 6 months (T₁), after 18 months (T₂), and after 30 months (T₃). For the deep samples, Drake's outline recommends collecting samples at T₀ and T₃ and archiving the samples at -20°C.

The conference call participants talked about Drake's recommended sampling depths. As noted above, Tsao is required to clean the first 18 inches of soil at his site. Drake said that he could collect composite samples from 0 to 18 inches during field test sampling, but noted that using this approach would not allow the Subgroup to track important events in the 0 to 6 inch range. Drake strongly encouraged Tsao to collect 0 to 6 inch and 6 to 18 inch samples if his budget allows this. Steve Rock said he must clean the first 60 inches of soil at a Chevron site in Cincinnati. Rock said he plans to collect samples from 0 to 6 inches and 6 to 60 inches.

In her outline, Drake assumed that six sites will be involved in the RTDF Subgroup's program. According to Drake's projections, about 552 samples (6 sites x 92 samples) will be generated by the RTDF Subgroup. Rock agreed that six was a good estimate to start with. Participant noted, however, that as many as 10 to 12 sites may become involved. Potential test sites include:

• The Chevron site in Cincinnati (led by Rock)
• The Amoco site in Texas City (led by Tsao)
• Other Amoco sites. Tsao noted that other Amoco sites are likely candidates for phytoremediation. He was unsure, however, when these projects would be initiated.
• Three sites in Alaska (led by Reynolds)
• A site in Arkansas (led by Duane Wolf)
• Two Petroleum Environmental Research Forum (PERF) sites. Drake noted that the PERF is hoping to test a crude oil site and a gas pit sludge site.
• A site in Richmond, California. Phil Sayre said he thinks Lucinda Jackson is considering a site in this area.
• An ADL site. Sayre noted that ADL has a site that might prove to be a candidate.

Hydrocarbon Analyses

Drake noted that there are three kinds of hydrocarbon analyses that are required:

• TPH. Drake noted that there are several ways to obtain TPH measurements. In previous work, Drake subjected her samples to a methylene chloride extraction, applied a silica treatment (to remove polars), and used GC/FID to separate her TPHs into an aliphatic TPH fraction (F1) and an aromatic TPH fraction (F2). Drake realized that all of the RTDF Subgroup participants might not require such precise detail. She noted that some investigators may prefer to measure the total TPH (F1 + F2) using GC/FID or a gravimetric approach. Drake has asked ADL and Batelle to provide price quotes for the various analytical techniques.

  Sayre asked Drake how investigators choose which analytical methods to use for TPH analysis. Drake said that the decision is often influenced by regulatory requirements. For example, in Drake's state, at one time regulators preferred using infrared (IR) for TPH analysis, but now they approve of the GC/FID methodology. Rock said that regulators in Ohio do not want him to use IR analysis. Participants thought that most regulators would be satisfied if a GC/FID analysis was used. Drake noted that she has heard that the gravimetric methodology is difficult to use. Reynolds said that his group plans to perform the TPH analysis in-house (using GC/FID) rather than sending the samples to an outside laboratory.

• Biomarkers. Drake noted that the biomarker analysis requires using a single ion monitoring scan with GC-MS. Sayre said that ADL sent him some information about their biomarker analysis protocol. ADL sent two articles electronically in zip files, but Sayre has been unable to open these files. (ERG was helping Sayre but could not successfully open the files.) ADL's protocol is also described in the Federal Register. Sayre provided participants a reference: Federal Register. September 15, 1994. Volume 59, No. 178. Page 47384.

  Drake recommended performing biomarker analyses during the preliminary site characterization. Doing so, she argued, would allow investigators to evaluate the ratio between TPH (degrading components) and non-degrading components (biomarkers). She said that evaluating ratios rather than absolute TPH values will help reduce the variability at a site. Drake noted that the "ratio method" is useful in situations where only one type of hydrocarbon contaminant is present at a site. (If multiple types are present, the biomarkers vary across the site and provide meaningless ratios.) Drake said she is scheduled to give a talk on biomarker analysis at the June 1998 IBC conference in Houston, TX. To prepare for the talk, Drake intends to draft a one-page summary describing why it is beneficial to use ratios rather than absolute measurements. Drake said that she would distribute the summary to the conference call participants.

• Priority polyaromatic hydrocarbons (PAHs). Drake noted that a GC-MS scan is required to analyze for PAHs. As noted above, Reynolds said that he plans to conduct TPH analyses in-house. He is unsure, however, whether his group can match ADL's or Batelle's expertise for PAH analysis. Reynolds plans to send a sample to one of these laboratories for PAH analysis. Drake asked Reynolds if he plans to split his sample. Reynolds said he may extract the sample first and then split the extract. Drake thought this was a good idea.

Drake said she plans to have pricing information from ADL and Batelle by the next conference call. She noted that ADL and Batelle are very similar in their analytical capabilities. She suspects, therefore, that some members of the RTDF Subgroup will choose to use ADL and others will use Batelle.

STATISTICAL REVIEW OF PROTOCOL

Sayre asked Rock whether he had identified a statistician who could help develop the team's protocol. Rock has found a statistician who can work on the Chevron site in Cincinnati. Rock said he hopes to have this statistician evaluate site characterization data from all of the RTDF Subgroup's test sites. At this point, however, he is only authorized to use the statistician for the Cincinnati site.

SHIPPING PROTOCOL AND SAMPLE STORAGE

As noted during the May 8, 1998, conference call, ADL failed to send Sayre useful information on shipping and storage. Sayre has asked Joe Kreitinger (from RETEC, Inc.) to provide an SOP that will explain how to collect, handle, store, and ship samples. Kreitinger told Sayre that he would submit the SOP by May 22, 1998. Sayre agreed to distribute the SOP to conference call participants.

Rock asked Reynolds whether he has an SOP for shipping and storing samples. Reynolds said he does but it is not very detailed. Reynolds was looking forward to seeing what Kreitinger produces.

SEED MIX AND PLANTING DENSITY

Pam Davis noted that the RTDF Subgroup established percentages for the "specified mix" during the previous conference call. (As noted by Rock, participants had agreed to use 10 to 15 percent rye grass, 20 to 25 percent legume, and 60 to 70 percent tall fescue.) Drake asked whether the group was considering using perennial rye, annual rye, or both. Sayre said the RTDF Subgroup will use one or the other, but not both.

Kulakow warned that the percentages should not be determined by seed weight. He noted that different plants have different seeding rates. For example, while 1 pound of legume seed might be needed to plant a 1,000 square foot area, 10 pounds of grass might be required to cover the same area. Kulakow agreed to prepare an example describing seeding considerations and to distribute it among conference call participants.

Davis asked whether the group had decided how much seed should be planted at test sites. Reynolds and Kulakow said they have found that petroleum-contaminated sites require heavier seeding than uncontaminated pasture areas. At his sites in Fairbanks, Alaska, Reynolds had to use multiples of what was originally recommended by a university forage specialist. (Reynolds was using annual rye and a mixture of annual rye/red fescue.)

Kulakow recommended preparing enough seed for two complete seed batches. For contaminated sites, Kulakow recommended overseeding the site during the initial seeding and, if necessary, following with a second seeding six weeks later. Kulakow used this approach at Port Hueneme and was able to produce plots with excellent stance. Drake agreed that preparing twice as much seed is an excellent plan.

Conference call participants discussed some of the reasons why plant germination and growth are curtailed in contaminated areas. Reynolds said that studies indicate that a plant's reaction to TPH differs dramatically across plant species and hydrocarbon type and concentrations. Reynolds offered to send June Muniz, James Walworth, and Nancy Moore's paper to Sayre. In this paper, they describe conditions in which grasses can be grown in soils with diesel concentrations reaching 64,000 parts per million (ppm).

Reynolds said his group has found lighter-end hydrocarbons to be more toxic to plants than heavier-end hydrocarbons. Drake agreed with Reynold's finding. She has a site where 50,000 ppm of diesel-type material has been shown to inhibit plant growth. According to Drake, Larry Davis (from KSU) suspects that ethyl benzene might be responsible and could be destroying the root membrane's integrity.

Wolf's group has found a connection between temperature and a plant's ability to survive in a contaminated environment. Using diesel-contaminated soils in the range of 34,000 ppm, Wolf's team found that plants were able to germinate and grow at 15°C. When temperatures reached 25 to 27°C, however, the plants died.

For highly contaminated sites, Kulakow recommended performing a germination trial prior to establish seeded test plots. Drake agreed that this was a prudent initial step. Kulakow said a germination trial could be conducted within a two-week period and could be performed in a greenhouse, growth chamber, or in the field. Sayre said that the RTDF Subgroup's protocol will be revised to incorporate Kulakow's recommendation.

MICROBIAL ANALYSIS PROTOCOL

EPR's Protocol

Davis distributed EPR's microbial analysis protocol to conference call participants prior to the May 19, 1998, conference call. Davis briefly summarized the protocol. She said the procedure involves subjecting soil samples to a series of dilutions. The Bushnell Haas (BH) solution, a sterile, low-nutrient solution, is used in the protocol. Each time the soil is mixed in the BH solution, the sample is vortexed.

As noted by Davis and Sayre, the EPR protocol differs from the RTDF Subgroup's current protocol in two ways. First, the EPR protocol recommends injecting dilutions and oil into sterile vials that are prefilled with BH solution. (Davis noted that using completely sealed systems reduces the opportunity for evaporation and sample contamination.) Second, the EPR protocol does not recommend using a Redox die. (Davis noted that EPR's test offers a strictly visual verification of hydrocarbon degraders. With this test, investigators determine whether microbial activity is occurring based on the cloudiness of the broth and whether its sheen breaks up.)

Choosing an Oil for the Microbial Analysis

Sayre said the RTDF Subgroup must decide what kind of reference oil to use in the microbial analysis. He indicated that there are two options:

• Standard oil. Sayre contacted the American Petroleum Institute (API) to find out if standardized oils are available. Initially, he was told that no standards exist. Recently, he was told that API has three types of standard oils that have been used for toxicity tests. API told Sayre that 5 gallons are available for all three reference oils. According to Sayre, API is willing to give the RTDF Subgroup all but 1 liter of each of the three reference oils. Sayre was told, however, that the reference oils have not been well characterized.
• Site-specific oil. During the previous conference call, Jim Brown noted that site-specific oil samples could be obtained by extracting oil directly from the soil. Since that call, Brown has distributed information on the extraction methodology and associated cost. Using the extraction technique he describes, 5 milliliters of site-specific oil could be obtained for $200. Sayre and Davis thought the cost was high.

Sayre asked the group whether they want to use a standard oil or a site-specific oil. Initially, Drake said she preferred using a standard oil. Charlene Owens and Davis argued that it is important to use an oil that reflects site conditions. Davis said that hydrocarbon degrader counts are influenced by the type of reference oil that is used in the analysis. After hearing Owens and Davis' arguments, the conference call participants agreed that using site-specific oil is preferable to using a standardized oil. Conference call participants did not clarify exactly when the extract would be obtained. Sayre said he thought the extraction would be performed once at the initial start up of the project. Wolf noted that the site-specific composition of a hydrocarbon changes over time. Owens said the Subgroup could consider performing a second extraction three years after starting activities.

Sayre asked whether the participants approved of the extraction methodology that Brown distributed. Reynolds asked for additional information on how extensively the protocol has been used and whether the extraction pulls out a significant amount of carbon that is not related to petroleum hydrocarbons. Sayre agreed to call Brown to get answers to these questions.

MISCELLANEOUS

Rock asked the conference call participants if they were planning to attend the June 1998 IBC conference in Houston, TX. Sayre, Rock, Davis, Drake, Reynolds, Tsao, and Owens said they plan to attend. Kulakow and Wolf were unsure whether they could obtain funding to travel to the conference.

Kulakow asked whether Walter Kovalick has decided to issue Technology Innovation Office funds to the Subgroup. Sayre said that the matter has not been decided, but hopes to have an answer by the end of May.

ACTION ITEMS

• Sayre asked participants to gather relevant materials before May 27, 1998. He recommended sending these materials to ERG so they can be distributed to all conference call participants. The materials Sayre hopes to distribute included:
  
  

  —	SOP for soil handling and shipping. Sayre has asked Joe Kreitinger (from RETEC, Inc.) to provide an SOP that will explain how to collect, handle, store, and ship samples.
  —	Example of plant mixture. Kulakow agreed to prepare an example describing seeding considerations.
  —	Description of biomarker analysis. Drake agreed to draft a one-page summary describing why it is beneficial to use ratios rather than absolute measurements.

• Sayre agreed to contact Jim Brown for additional information on an oil extraction methodology.
• Drake said she would follow up with ADL and Batelle for cost estimates.
• Rock agreed to revise the Subgroup's protocol.
• Rock said he would try to get a statistician to offer some input on the protocol before the next conference call.

NEXT CONFERENCE CALL

The next conference call is scheduled for May 29, 1998, between 10:30 a.m. and 12:00 p.m. (EST).

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:	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. Native grass mix or other species, including trees. 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: pH and salinity (times 0 and 3) 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.) Petroleum (EPA method for saturated and aromatic hydrocarbons analyzed with GC, TPH measured, individual PAHs measured) (times 0, 1, 2, and 3) Biomarkers (times 0, 1, 2, and 3) Microbial analysis (times 0 and 3) 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 Banks' lab at KSU and by Exxon. 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 # of positive responses @ 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. Exxon uses the Bushnell Haas broth and multiwell plates. Each well contains 10 µl 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 would (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.