United States
Environmental Protection
Agency
Municipal Environmental Research
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S2-81-208 Oct. 1981
Project Summary
Restoring Hazardous Spill-
Damaged Areas: Technique
Identification/Assessment
R. S. Wentsel, R. H. Foutch, W. E. Harward, III, W. E. Jones, 111, and J. F.
Kitchens
This study identifies and assesses
methods for accelerating the restor-
ation of lands damaged by spills of
hazardous materials. The first phase of
the study involved a literature review
to determine what response methods
had been used in the past to clean up
spills on land and to identify other
techniques that could be developed
for detoxification of hazardous-spill-
damaged lands.
In the second phase of the study,
four primarily biological techniques
for accelerating the restoration of
spill-damaged lands were evaluated in
the laboratory:
Technique
Chemical
Enhancement of Chlorobenzene,
microbial degra- Ethion
dation by indige-
nous organisms
2. Addition of
mixed microor-
ganisms from
primary sewage
effluent
Formaldehyde,
aniline
3. Addition of Dinitrophenol,
adapted/mutant chlordane
microbial cultures
4. Selective ab-
sorption by
harvestable
plants
Lead nitrate,
cadmium nitrate
The accelerated removal of one or
both chemicals was observed in
techniques 2. 3, and 4. The effects of
the spilled chemical on the soil
chemistry and microorganisms were
also monitored.
During the third phase, a plan for
field testing of techniques 2 and 4 was
designed. Recommended land resto-
ration methods for spills of the 271
hazardous chemicals listed in the
Federal Register (1978) were compiled
during the fourth phase.
This Project Summary was devel-
oped by EPA's Municipal Environmen-
tal Research Laboratory, Cincinnati,
OH, to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
This study focused on the identifica-
tion and development of biological
techniques that have the potential for
restoration of spill-damaged landsanda
laboratory assessment of the feasibility
of these techniques. A literature search
was conducted to determine the current
biological, chemical, and physical
techniques used to treat spill-damaged
land. Viable biological techniques were
then identified, and four were selected
for laboratory evaluation.
The first technique examined was the
enhancement of microbial degradation
by indigenous organisms. This tech-
nique was tested for several reasons.
First, the microorganisms that have
-------
survived the initial shock from the
spilled chemical are relatively resistant
to the chemical. If the surviving microbial
population can be increased rapidly
with the addition of nutrients, they may
then be able to degrade the chemical.
Also, the organisms used are from the
spill area and do not have to be specially
cultured.
The second technique (T-1) involved
the use of microorganisms in primary
sewage effluent to degrade a spilled
chemical. Primary sewage effluent
contains a variety of microorganisms, is
usually high in nutrients, is cheap, and
is readily available. Primary sewage
effluent can be used to replenish the
microbial population in the spill area. If
nutrients are supplied, the microorgan-
isms from the primary sewage effluent
can degrade the spilled chemical.
The third technique (T-2) was the
addition of adapted/mutant microbial
cultures. Soil or primary sewage effluent
microorganisms were cultured in nu-
trient medium containing the chemical
to be degraded. The chemical was the
sole carbon source for the organisms.
After several subcultures, a culture was
developed that could utilize the chemical
of concern. This culture, with nutrients,
could then be applied to the soil to
degrade the spilled chemical.
The fourth technique evaluated was
the uptake of heavy metals by harvest-
able plants. Plants were selected for use
based on data from the literature search
and preliminary tests. For the heavy-
metal-contaminated soil, conditions for
uptake by the plants were optimized by
adjusting the pH and adding disodium
ethylenediaminetetra acetic acid, a
chelating agent.
A plan for field testing of the two most
promising techniques frorri the labora-
tory tests was then developed. The plan
included the chemicals to be spilled,
restoration treatments to be applied,
and sampling techniques.
Finally, recommended land restora-
tion methods were developed for the
hazardous chemicals under considera-
tion. The chemicals were environment-
ally classified, and restoration techni-
ques were proposed for treatment.
Experimental Methodology
Two 2.7- x 5.8-m greenhouses were
used in the study. Each greenhouse
contained nine enclosed environmental
chambers that were constructed in
groups of three, with each group
supported by a wood frame. A diagram
Coolant Water
Condenser
Carbon Filter
S— Drains
Figure 1. Set of three environmental chambers.
£
of three environmental chambers is
shown in Figure 1.
A steady flow of fresh air through the
environmental chambers was induced
by connecting the air exit line from the
chamber to a vacuum system. Fresh
outside air was admitted into each
environmental chamber, recirculated
within the chambers by means of a
small fan, and then passed through a
water-cooled condenser, a carbon filter,
a sodium hydroxide bubbler, and a
vacuum pump to the outside. The air
system permitted all airborne chemicals
to be collected for analysis.
A liquid drainage hole was drilled in
the glass bottom of each environmental
chamber. Any liquid collected in the
bottom of the chambers could be
drained and analyzed.
In the bottom of each chamber were
placed 2.5 cm of sand, then 5 cm of
gravel, and another 2 cm of sand.
Approximately 30 to 45 cm of soil were
then placed in the chambers (high-
organic soil in three chambers, sandy-
loam in three chambers, and clay in the
remaining three chambers). Once the
chambers were filled with soil and
packed down, the soils were seeded
with perennial rye grass and allowed to
equilibrate for 3 weeks.
Two liters of the liquid organic
chemical under study were evenly
sprinkled over each of the nine chambers.
2,4-Dinitrophenol, a solid at room
temperature, was applied at a density of
500 g/chamber. After application of the
chemical, the chambers were closed,
and the air circulation system was put
into operation. Core samples were
taken 24 and 48 hours after chemical
addition and approximately every week
thereafter.
One set of three chambers (organic,
sandy, and clay soils) was used as a
control and received no treatment. The
other two sets received experimental
treatments falling within the definition
of the three techniques outlined above.
Planters 45 cm in diameter and 45 cm
high were used to study the uptake of
heavy metals by harvestable plants. A
diagram of the planters and the experi-
mental treatments are presented in
Figure 2. Twelve planters were used for
each metal. Three soils were used in the
test—organic, sandy-loam, and clay—
and four planters were used for each
soil. For each soil, one planter acted as a
control and the other three received the
heavy metal contamination. Three of
the four planters for each soil type were
sprinkled with 250 ml of 0.025 molar Cd
-------
Chamber A
Chamber B
Chamber C Chamber D
Chamber Treatments
A (the control): no metal, pH adjusted.
B and D: metal added. pH adjusted.
C: metal added, pH adjusted, chelating agent added.
Figure 2. Experimental design for plant uptake studies.
MC-labeled molecules). The heavy
metal tests also included an analysis of
the soil and of sampled plant tissue for
Cd or Pb.
Results and Discussion
Monochlorobenzene
Monochlorobenzene was somewhat
toxic to soil bacteria and was highly
z, and another set of planters was
doused with 250 ml of 0.025 molar Pb
(N03)2.
The soil samples from each chamber
that had been doused with organic
compounds were analyzed for soil pH,
percent moisture, percent organic
matter, nitrate, soil bacteria, soil fungi,
and soil 14C activity (except for 2,4-
dinitrophenol, which did not contain
toxic to grass. Twenty-four hours after
the spill, the grass had completely
turned brown. Monochlorobenzene was
rapidly lost from the soil, as evidenced
by the high concentration of it found in
the carbon fitters and traps.
No evidence of microbial degradation
of monochlorobenzene was observed
during the tests. No additional com-
pounds were seen in the GC traces, and
no 14C02 was found in the sodium
hydroxide bubblers.
The monochlorobenzene provided a
good example of the movement in soils
of a volatile compound that is low in
polarity and low in water solubility. The
monochlorobenzene evaporated from
the soils tested within 28 days after the
spills. The treatment methods applied to
the soils were relatively ineffective
because they involved wetting the soil
surface. The wet soil reduced the
evaporation rate of the monochloro-
benzene in the treated chambers. The
control chambers had uninhibited
evaporation of the monochlorobenzene
from the soil and a faster recovery of
normal soil fauna.
Ethion
Ethion did not have an immediate
effect on the grass, which remained
healthy in all chambers for about 1
month after the spill and then slowly
wilted and turned brown. During the
first 5 months after the spill, the Ethion*
levels in the soils gradually decreased.
The initial treatments, with nutrients(T-
1 chambers) or nutrients with aeration
(T-2 chambers) were not effective in
significantly increasing the degradation
rate of Ethion by indigenous micro-
organisms. Adjustment of soil pH with
lime addition on day 44, 46, and 53 did
not improve the Ethion degradation
rate.
The microbial populations were
sufficiently high in the treated cham-
bers, and the microbes appeared to be
capable of living in the presence of
Ethion. But, either the organisms were
incapable of degrading the Ethion, or
the Ethion was unavailable to the
organisms. Two methods were tried to
improve the water solubility of Ethion
and thus its availability to the microbial
populations: (a) the addition of a
surfactant (Tween 80), and (b) the
addition of ethanol. These treatments
were applied to the T-1 and T-2
"Mention of trade names or commercial products
does not constitute endorsement or recommenda-
tion for use.
-------
chambers on day 95 and day 122,
respectively, but they did not improve
the Ethion degradation rate by microor-
ganisms.
On day 210, day 219, and day 220, the
T-2 chambers were each treated with a
total of 10 liters of a 50:50 ethanol-
water solution containing 6.8% sodium
hydroxide to determine if the Ethion
could be hydrolyzed in the soil. The pH of
the top layer of soil was raised to 10 to
12 by this treatment. Because of the
high pH, microbial levels dropped
drastically. In the organic soil, the
hydrolysis treatment reduced the Ethion
levels from 55,000 ppm to less than
4,000 ppm. Ethion levels in the top layer
of the sandy soil decreased from
approximately 60,000 ppm to less than
10,000 ppm. The Ethion levels in the
clay soil were not substantially affected
by the treatment.
Following the ethanol/sodium hy-
droxide treatment, a number of tenta-
tive degradation products were observed
by gas chromatography/mass spectros-
copy. None of the observed peaks could
be matched to entries in the NIH/EPA
Mass Spectral Search System (MSSS).
Tentative molecular formulae and
possible structural assignments for
several of the peaks are presented.
Confirmation of identity without isola-
tion of sufficient material for nuclear
magnetic resonance (NMR) and infrared
spectra is not possible.
The 14C radiation levels in all soils
were variable but indicated a loss of
activity with time. After the ethanol/
sodium hydroxide treatment, the radia-
tion levels did not decrease significantly,
indicating that the S-CH2-S segment of
the Ethion molecule (the 14C-labeled
site) was still present in the soil.
Assuming a first-order decay of
Ethion in the three soil types, the half-
lives of Ethion were calculated from the
14C measurements and from chemical
analysis (Table 1).
Formaldehyde
The spill of formaldehyde was toxic to
both the soil microorganisms and the
plant life. The grass was dead by the end
of the second day after the spill. On days
2 and 25 after the formaldehyde spill,
one set of soil chambers was treated
with primary sewage effluent (T-1) and
another set was treated with primary
sewage effluent cultured with 5000
ppm formaldehyde (T-2). In the organic
soils, formaldehyde concentrations in
the treated and control chambers were
Table 1. Ethion Half-Lives
Time (days)
Soil Type
Organic
Sandy
Clay
Chemical analysis
157
200
123
">C
162
117
159
Table 2.
Rate of Formaldehyde Loss
Chemical Analysis (ppm/day)
4C Activity (cpm/dayj
Chambers Organic Sandy
Clay
Organic Sandy
Clay
Control
T-1
T-2
145
110
122
82
90
51
48
64
72
2.3
4.5
1.8
1.3
1.1
0.5
0.7
1.0
1.0
essentially the same for the first 25
days. The second microbial treatment
applied on day 25 was again ineffective
in accelerating the removal of formalde-
hyde from the soil. The radiometric data
generally confirm the formaldehyde
analyses.
The microbial populations in both the
control and treated chambers gained
viability when formaldehyde levels
decreased to approximately 1000 ppm.
Once this occurred, the formaldehyde
levels in the soil decreased rapidly.
When the formaldehyde is reduced to
tolerable levels, the organic soil provides
adequate nutrients for microbial
metabolism.
The sandy soil upper layers exhibited
only slight differences in formaldehyde
levels between the treated and control
chambers over the initial 25 days. After
the second treatment was applied on
the 25th day, however, both T-1 and T-2
chamber soils had lower formaldehyde
levels than the control. The T-1 chambers
had the most rapid loss of formaldehyde.
The middle sandy soil layers of the three
chambers had approximately the same
formaldehyde concentrations for the
first 20 days, but they continued to
decline from day 20 to 66 in the T-1 and
T-2 chambers and the concentration in
the control chamber remained effectively
stable. The treated and control chambers
had no significant differences in formal-
dehyde concentrations at the lower
level. The concentrations gradually
declined throughout the study.
The formaldehyde levels in the upper
layer of all the clay soil chambers were
approximately the same during the first
25 days after the spill. The second
treatment of microorganisms (day 25)
increased the rate of depletion of the
formaldehyde in the treated chambers.
The formaldehyde concentration dis-
appeared from the T-2 chambers at
faster rate than from the T-1 chamber
In the control chamber, however, th
rate of disappearance of formaldehyo
remained constant. By the 47th da'
both the T-1 and T-2 chambers ha
upper layer formaldehyde concentre
tions of less tha n 100 ppm, whereas th
control chamber contained more tha
1000 ppm of formaldehyde in the soil
The middle soil layer in the cla
chambers had initial formaldehyd
concentrations between 500 and 10O
ppm. The T-2 chamber middle laye
reached 0 ppm of formaldehyde on da
52. The lower clay soil layers showed n
significant trends in formaldehyd
concentration that could be attributed t
the treatments used. All lower cla
layers had formaldehyde concentration
of less than 30 ppm on the 46th day.
The rate of formaldehyde loss deter
mined from regression lines fitted ti
formaldehyde concentration versu:
time measurements in the upper soi
layers and to the 14C counts per minuti
(cpm) data are given in Table 2.
The higher rate of formaldehyd(
removal from the sandy T-1 and clay T-i
chambers correlates with the loss of 14(
activity in the chambers.
Aniline
The aniline spill was toxic to soi
microbes and plants. The grass in thi
chamber was dead by the end of th<
fourth day after the spill.
The initial treatments with priman
sewage effluent (T-1) on days 2 and 4i
or with 15% hydrogen peroxide solutior
and primary sewage effluent (T-2) or
days 2 and 12 were relatively ineffective
in reducing aniline levels in the organic
soil. On day 96, however, the T-1
chambers were treated with a mixe<
-------
culture of microorganisms and a nutrient
salt solution that resulted in aniline
levels significantly lower than in the
control chamber by day 113. The
addition of nutrient salt and yeast
extract solutions to theT-1 chambers on
days 126 and 154 continued to reduce
the aniline levels. By day 160, aniline
concentration in theT-1 chambers was
less than 100 ppm. The treatments
applied to the T-2 chambers were not
effective in reducing the aniline concen-
trations in the soil.
The hydrogen peroxide treatments
reduced the aniline concentrations in
the T-2 sandy chamber by 20% to 40%.
This T-2 chamber had the lowest aniline
concentrations for the remainder of the
experiment. The initial addition of
primary sewage effluent to the sandy
soil T-1 chamber was ineffective; but
after the addition of the mixed microbial
culture and the nutrient salts on day 96,
the aniline level decreased by 6000 to
8000 ppm. Another decrease in the soil
aniline concentration in this T-1 cham-
ber was observed after the nutrient salts
and yeast extract were added on day
154.
None of the treatments were effective
in accelerating the removal of aniline
from the clay soil. Aniline levels in all
three chambers decreased at a similar
rate.
The half-life data for aniline in the
organic and sandy soils are presented in
Table 3. The half-life was calculated
assuming a first-order decay. For the
organic soil, the aniline concentration in
the control chamber on day 12 was used
as the initial value for the calculations.
The half-life value of aniline in the T-1
chamber was about half those obtained
in the control and T-2 chambers.
The half-lives for the sandy soil
chambers were separated in parts. In
the first part of the experiment (days 7 to
91), the T-2 chamber (which received
hydrogen peroxide) had the shortest
half-life. In the second part of the
experiment (days 91-166), the T-1
chamber had the shortest half-life. The
T-1 chamber received the adapted
mixed microbial culture and nutrient
salts during this period.
Chlordane
The chlordane spill was not signifi-
cantly toxic to soil microbes, but the
grass was completely dead within 48
hours after the spill. On days 2, 8, and
28, adapted microbial culture and
nutrient salts were added to both sets of
chambers (T-1 and T-2), and the T-2
chambers were also treated with lime.
In general, there was no loss of
chlordane from the clay or the organic
soils. The organic T-1 soil showed a
slight decline in chlordane; however,
the data are too scattered to draw any
definite conclusions. In all sandy soil
upper layers, chlordane concentrations
decreased. The control and treated
chambers appeared to lose chlordane at
a similar rate. The 14C radiation levels in
the sandy soils also show a gradual
decrease in activity. No chlordane was
found in the traps or carbon filters or in
any of the chambers. The UC levels in
the bubblers from the sandy chambers
were below the detection limits.
From the data, it appears that chlor-
dane is persistent in the soils with the
possible exception of the sandy soils.
Whether the apparent decrease in
chlordane levels in sandy soil resulted
from degradation or volatilization is not
known. No degradation products were
found in the GC traces, but column
conditions, or extraction conditions, or
both may not have been favorable for
their detection.
2,4-Dinitrophenol
This chemical killed the grass cover in
the chambers within 5 days after the
spill and depressed the soil microbial
populations. On days 2 and 11, adapted
microbial culture, nutrient salts, and
yeast extract were added to one set of
chambers. In addition, these chambers
were treated with lime on day 2.
The 2,4-dinitrophenol concentrations
in the upper layer of the treated and
control sandy soil chambers were
similar throughout the experiment. The
concentration was observed to decrease
from near 4000 ppm at day 1 to 1200 to
1400 ppm at day 62. The treatments
were not effective in increasing the 2,4-
Table 3.
Chamber
Half-Life of Aniline in Organic and Sandy Soils
Organic soil Sandy soil
Days 12 to 166
Days 7 to 91
Days 91 to 166
Control
T-1
T-2
44
19
58
39
63
28
45
24
49
dinitrophenol removal rate from the
sandy soil.
The treated clay soil chamber ap-
peared to have an initial 50% loss of 2,4-
dinitrophenol concentration in the
upper layer during the first 12 days.
After day 12, the upper layer 2,4-
dinitrophenol concentrations were
relatively stable between 500 and 1000
ppm. The 2,4-dinitrophenol concentra-
tion in the upper layer clay soil control
chamber was near 2000 ppm on day 12,
and it gradually decreased to 1000 ppm
by day 72. After the initial treatment of
the microorganisms, the 2,4-dinitro-
phenol concentrations in the treated
clay soil chamber were reduced by at
least 1000 ppm. But further reductions
in the 2,4-dinitrophenol levels did not
occur.
Heavy Metal Bioaccumulation
The experimental design for the
heavy metal tests is presented in Figure
2. The plants grown in the chelated soils
had higher metal contents. Chelating
agents increased the plant uptake of
metals by solubilizing them and increas-
ing their diffusion to root surfaces. The
disodium EDTA was added to the soil
only once in the early part of the
experiment. This chemical is biodegrad-
able and was probably removed from
the soil during the course of the
experiments. Repeated applications of
EDTA or other chelating agents would
assuredly increase the removal rate of
the metals from the soil. For cadmium,
EDTA significantly increased the uptake
of the metal by the vegetation. The
results were not as dramatic for lead.
The reduction of cadmium concentra-
tions in the chelated soils during the
experiment was due to the uptake of
cadmium by the plants and the leaching
of the metal from the soil. The cadmium
concentrations in the chelated sandy
soil chamber decreased by about 50%.
The grasses sampled from the chamber
during the first 30 days accounted for
1% to 2% of the metal loss. If the grass
had been planted in the entire chamber
and complete harvesting had been
conducted, the amount of metal removed
would have been greater. But at least
several harvests would need to be
conducted before significant losses of
cadmium from the soil would occur.
The full report was submitted in
partial fulfillment (Task 11) of Contract
No. 68-03-2648 by Rockwell Inter-
national, Newbury, California 91320,
under the sponsorship of the U.S.
Environmental Protection Agency.
-------
/?. S. Wentsel, Ft. H. Foutch, W, E. Harward, III. W. £. Jones, HI, andJ. F. Kitchens
are with the Atlantic Research Corporation, Alexandria, VA 22314.
John Brugger is the EPA Project Officer (see below).
The complete report, entitled "Restoring Hazardous Spill-Damaged Areas:
Technique Identification/Assessment," (Order No. PB 82-103 870; Cost:
$26.00, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Oil and Hazardous Materials Spills Branch
Municipal Environmental Research Laboratory—Cincinnati
U.S. Environmental Protection Agency
Edison, NJ 08837
U.S. GOVERNMENT PRINTING OFFICE:1981--559-092/3318
-------
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
RETURN POSTAGE GUARANTEED
CSS ?JSlK2P«OTECTlON AGENCY
HC£T;-,£ S L16KHKY
2i6~o ugArfrtOKN STHEEl
CHICAGO IL oOt»04
------- |