July 1999

PURPOSE: Determine efficacy of agricultural and non-crop plants for degradation of aged petroleum hydrocarbons in soil at multiple locations and under varied climatic conditions.
LOCATIONS: 6 - 12 sites in North America.
PLOT SIZE: 20' x 20' minimum

STATISTICAL DESIGN: Randomized complete block (place plots based on presence of TPHs in site characterization. For soil and plant samples, take 8 random sample cores per plot and make a composite sample.


  1. Local optimized treatment: grass, mix of species, including trees

  2. Unplanted and unfertilized control (kept weed-free; in descending order of preference use 1) Glyphosate or equivalent post-emergent, 2) hand-picking of weeds, 3) tilling, 4) pre-emergent herbicide).

  3. Standard Mixture of
    rye 10-15% (annual or perennial),
    legume 20-25% (alfalfa, clover, birds-foot trefoil), and
    fescue 60-70% (varieties chosen for local conditions) (see Attachment 1: Seeding Mixture)

FERTILIZATION: Over the course of the trial, vegetated plots should be fertilized with nitrogen and phosphorus at the rate corresponding to the carbon to nitrogen to phosphorus ratio of 50:2:1. Additional fertilizer can be added to account for plant uptake requirements. All vegetated plots should be fertilized at the same rates. The rate of fertilization should be divided into several applications to minimize adverse effects on plant growth due to excess nitrogen. Accurate records should be maintained on rates of fertilization and how it is applied. After considerable discussion it was decide that the unvegetated treatment should not be fertilized so that the vegetation treatments are compared with a control that represents no treatment. This trial will not attempt to separate the effects of plants from the effect of fertilizer. Participants are encouraged to include an unvegetated and fertilized control treatment if space and resources permit.

SOIL SAMPLING: See Attachment 2: Soil Sample Collection. Eight random sub-samples per plot composited make one sample. Take soil samples as described at the following times and soil depths sampling location at the following times:

T=I: Initial site characterization (after tilling):
samples taken in grid over whole site
20 - 0 to 6"
  0 - 6 to 12"
T=0: Before planting, after seed bed prep: 12 - 0 to 6"
12 - 6 to 18"
T=1: 6 months after planting, or end of first growing season: 12 - 0 to 6"
12 - 6 to18"*
T=2: 18 months after planting, or end of second growing season 12 - 0 to 6"
12 - 6 to18"*
T=3: 30 months after planting, or end of third growing season: 12 - 0 to 6"
12 - 6 to 18"

*samples may be analyzed or archived for possible later analysis

PLANT SAMPLING: Take plant shoot and root samples at time 3 (30 months) for hydrocarbon analysis. Take eight random sub-samples per plot to make a composite sample. (Attachment 3 : Plant Assessments: Plant hydrocarbon uptake analysis - to be developed).

SAMPLE ANALYSIS: Send composite soil samples for analysis of:

  1. Agronomic conditions: pH, salinity, available nutrients, soil analysis (texture, organic matter, EC, CEC, soil type.) The analyses should be tailored to the region. See Attachment 4: EPA Support Items.

  2. Contaminant concentrations: PAHs using EPA method with GC, TPH using DCM solvent

  3. Biomarkers (times 0, 1, 2, and 3)

  4. TPH fractions using TPHCWG method (times 0 and 3) See Attachment 6.

  5. Microbial analysis (times 0 and 3 for plate counting and MPN, times 0, 1, 2, and 3 for PLFA and DGGE analysis) See Attachment 5: Microbial Analysis.

  6. Alkylated PAHs and BTEX

Items 1, 2, 3, and 4 are required parts of the protocol. Items 5 and 6 are optional.

PLANT ASSESSMENTS: Evaluate plant growth and species coverage at 6 months, 18 months, and 30 months. Attachment 3: Plant Assessments. Small, one-species strip plots near test site are recommended for plant identification during evaluation.

DATA MANAGEMENT: Provided by cooperating CRADA Partners. See Attachment 4.

GROWING CONDITIONS and PLOT MANAGEMENT: Determine seed bed preparation, planting technique, planting rate, and irrigation in site-specific manner to establish good stand growth. Record procedures. Add soil amendments based on need from initial soil sampling. Record rainfall, irrigation, and average daily temperature (available from local airport) throughout growing season. Vegetation should be cut only during the dormant period to remove excess plant litter unless there is excessive growth that threatens to damage the stand.

Summary of analyses planned for each RTDF sampling event.

Sampling Event Matrix Analysis Purpose
T=i soil PAH, TPH, agro, biomarkers, TPHCWG determine variability, agronomic amendments
0 soil PAH, TPH,microbial, biomarkers, TPHCWG establish baseline
1 soil PAH, TPH, agro, biomarkers, plant assessment assess first season results
2 soil PAH, TPH, agro, biomarkers, plant assessment assess second season results
3 soil, plant PAH, TPH, agro, microbial, biomarkers,
TPHCWG, plant assessment, plant uptake
assess third season results, to assess plant uptake, if any

Attachment 1
Attachment 2
Attachment 3
Attachment 4
Attachment 5
Attachment 6

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