OCR error (C:\Conversion\JobRoot\00000CEF\tiff\20013N69.tif): Unspecified error
-------
Online Library System (OLS) Libraries | US EPA Page 1 of 3
http://cave.epa.gov/cgi/nph-bwcgis/BASIS/ncat/pub/ncat/DDW?W%3DCALLNUM+PH+IS+%
90203%27+ORDER+BY+YR/Descen^^
jwrj JitJ |ftil3 IHB4 L uispiay Hecoros as bipiiograpny j [ item statusj
RECORD NUMBER: i OF 1
Main Title Approach to bioremediation of contaminated soil
Author Sims, Judith L.; Matthews., J. E.
CORP Author Utah State Univ., Logan.;Robert S. Kerr Environmental Research Lab.,
Ada, OK.
Publisher ! Mary Ann Liebert, Inc.,
Year Published ! 1990
Report EPA/600/J-90/203
Number
Stock Number ] PB9i-n6i52
OCLC Number ! 26995497
Subjects ; Organic compounds; Soils; Ground water; Water pollution; Soil
I microorganisms; Fertilizing; Cultivation; pH; Moisture control; Sites;
: Design; Utah; Reprints; Bioremediation; Vadose zone
Subject Added Soil pollution
Ent
Collation p. 117-149 ; 26 cm.
Holdings LIBRARY CALL NUMBER LOCATION
EMAD EPA/600/J-90/203 Region 6
Library/Dallas,TX
NTIS PB91-116152 Most EPA NTIS
libraries have a
fiche copy filed
under the call
number
shown. Check
with individual
http://cave.epa.gov/cgi/nph-bwcgis/BASIS/ncat/pub/ncat/DDW?W%3DCALLNUM+PH... 10/19/2006
-------
PB91-116152
i to MM* IM Mt^m* MM,. EPA/600/J-90/203
HAZARDOUS WASTE • HAZARDOUS MATERIALS
Vota»e7,N«mter2,l»90
Msiy ABB Ucfcat. lac.. PnUfebcn
Approach to Bioremediation of
Contaminated Soil
JUDITH L. SIMS
(/MA Water Research Laboratory
Utah State University
Logon. UT 94322-8200
RONALD C. SIMS
1 s \'
Department of Civil and Environmental Engineering
Utah State University
Logan. UT94322-4I10
SX '
JOHN E. MATTHEWS
Robert S. Ken Environmental Research Laboratory
, \ US. Environmental Protection Agency
P.O. Box 1198
Ada, OK 74*20
ABSTRACT
/\ Biological proeeases. including aicrobial degradation, have been identified as
•< ' critical mechani sms for attenuating organic contaminants during transit through the
•\ vadose zone to the groundwater. On-site soil remedial measures using biological
, ^ processes can reduce or eliminate groundwater contamination, thus reducing the need
,'' for extensive groundwater monitoring and treataent requirements. On-site remedial
systems that utilize the soil aa the treataent system accomplish treataent by using
fj naturally occurring microorganisms to treat the contaminants. Treataent often may
^ be enhanced by a variety of physical/chemical methods, such as fertilisation,
,\J tilling, soil Ph adjustment, moisture control, etc. The development of a biorcmedi-
'*^ ation program for a apecific contaminated soil system includes! (1) a thorough
"- site/soil/waste characterization; (2) testability studies; and (3) design and
implementation of the bioremediation plan.
Biological remediation of soils contaminated with organic chemicals has been
demonstrated to be an alternative treatment technology that can often aeet the goal
of achieving a permanent clean-up remedy at hazardous waste sites, as encouraged by
the U.S. Environmental Protection Agency (U.S. EPA) for implementation of the Super-
fund Amendments and Reauthorization Ace (SARA) of 1986. Biorcmedistion is especial-
ly promising if it is incorporated in a remediation plan that uses an integrated
approach to the cleanup of the complete site. i.e.. a plan that involves the concept
of a 'treatment train* of physical, chemical, and/or biological processes to address
remediation of all source* of contaminants at the site.
INTRODUCTION
' Biological remediation of soils contaminated with organic chemicals is an
alternative treatment technology that can often aeet the goal of achieving a pcrma-
•\ nent clean-up remedy at hazardous waate sites, as encouraged by the U.S. Environ-
< mental Protection Agency (U.S. EPA) for implementation of The Superfund Amendments
and Reauthorization Act (SARA) of 1986. Bioremediation is consistent with the
philosophical thrust of SARA, for it involves the use of naturally occurring micro-
organisms to degrade and/or detoxify hazardous constituents in the soil at a con-
taminated site to protect public health and the environment. Bioremediation of con-
117
-------
taminated soils, ingi«"»*qt application* and limitation*, hat been addretaed at
several recent scientific meetings and conference* (1. 2. 9, 4]. With regard speci-
fically to weed praaenring contaminated sites McGinn!* et al. (5) have stated that
reliable, safe, economical bioremedlatlon techniques using soil systems are attrac-
tive and warrant thorough study and evaluation. The uae of bioremedlatlon tech-
niques in conjunction with *>>i«««*e*i and physical treatment processes, i.e.. the use
of « 'treatment train. • Is an effective mean* for comprehensive site-specific reme-
diation [6]. An example of-a treatment train Is the use of soil vacuum extraction
to remove free product, followed by bioremedlatlon to remove residual contaminants
In the aoil. The length of time required for bioremedlatlon to achieve clean-up
goals will be dependent upon the specific hazardous constituent* of concern and the
aite characteristic* that influence the rate and extent of degradation.
Wilson [7] identified biological proceaaea, including microbial degradation.
a* a mechanism for attenuating contaminant* during tranait through the vados* zone
to the groundwater (the vadoa* zone 1* the region extending from the ground curface
of the earth to the upper surface of the principal water-bearing formation [•]).
On-*it* aoil remedial measures tuing biological proceaae* can reduce or eliminate
groundwater contamination, thu* reducing the need for extenilve groundwater moni-
toring and treatment requirements [7, 9, 10). Lehr (11] alao emphasized that moni-
toring for attenuation of contaminants occurring la the vadose zone provides Infor-
mation for understanding their movement In and through the vadose zone and in the
groundwater.
te-site bioremediation of contaminated aolls generally is accomplished by using
one of three types of systemst
(1) In situ;
(2) Prepared bed; or
(3) Bioreactor (e.g., slurry reactors) systems.
This discussion focuses on In situ and prepared bed systems, which utilize the soil
as the treatment medium, a* contracted to bioreactor syctem*, In which contaminated
aoil i* treated in an aqueou* medium.
An in *itu system consists of treating contaminated soil in place. Contami-
nated soil Is not moved from the alte. In general, naturally occurring microorga-
nism* are allowed to treat the contaminants. Treatment often may be enhanced by a
variety of phyaical/chemical methoda. cuch aa fertilization, tilling. *oil pH
adjustment, molature control, etc. In some instances, addition of supplemental
populations of adapted organism* may aerve to enhance treatment.
In a prepared bed aystem. the contaminated aoil may be either (1) physically
moved from its original aite to a newly prepared area, which baa been designed to
enhance bioremediation and/or to prevent transport of contaminants from the aite;
or (2) removed from the aite to a atorage area while the original location i* pre-
pared for uae, then returned to the bed. where the treatment i* accomplished.
Preparation of the bed may consist of such activities as placement of a clay or
plastic liner to retard transport of contaminants from the aite. or addition of
uncontaminated soil to provide additional treatment medium. Treatment may alao be
enhanced with phyaical/chemical methods, as with in situ systems.
Overview of Soil Biodegradatioii and Other Soil Processes
Bioremediation of a soil contaminated with organic chemicals is accomplished
by degradation of specific organic constituents, i.e.. the 'parent' compounds. The
term degradation may refer to complete mineralisation of the constituents to carbon
dioxide, water, inorganic compounds, and cell protein. The ultimate products of
aerobic metabolism are carbon dioxide and water. Under anaerobic conditions (i.e..
in the absence of oxygen), metabolic activities also result in the formation of
incompletely oxidized simple organic substances such aa organic acids as well aa
other products such as methane or hydrogen gas. However, blodegradatlon of a com-
pound is frequently a atepwiae process involving many enzymea and many tpeciea of
organisms. Therefore, in the natural environment, a constituent may not be com-
pletely degraded, but only transformed to intermediate product(a) that may be lea*.
lit
-------
equally, or more hazardous than the parent compound. as well as more or less mobile
ia the environment.
The goal of on-site bioremediation i* degradation that results in detoxifica-
tion of a parent compound to a product or product(*) that are no longer harardoui
to human health and/or the environment. Information on degradation and detoxifica-
tion of a parent compound may be obtained using chemical and bioassay analyses [12.
13. 14). Chemical analysis and identification of intermediate products m*y yield
information about biochemical degradation pathways and products. but are often time-
consuming and expensive. Bioassays may be used to demonstrate detoxification of
parent compounds and are usually less expensive and time consuming. Before bioreme-
diation is implemented at a contaminated site, degradation pathways for specific
constituents present and /or detoxification demonstrations require investigation so
that environmental end health protection can be achieved.
Degradation of most organic compounds in soil systems may be described by moni-
toring their disappearance in a soil through time. Disappearance, or rate of degra-
dation, is often expressed as a function of the concentration of one or more of the
constituents being degraded. This is termed the order ef the reaction and is the
value of the exponential used to describe the reaction [15]. Either zero or first
order power rate models are often used in environmental studies. A useful ten to
describe the reaction kinetics is the half-life. tj/,. which is the time required to
transform 50Z of the initial constituent.
A xero rate order model is one in which the rate of transformation of an
organic constituent is unaffected by changes in the constituent concentration
because the reaction rate is determined by some other factor than the constituent
concentration. The first order rate model is widely used because of its effective-
ness in describing observed results as well as its inherent simplicity. Zts use
also allows comparison of results obtained from different studies. In a first order
rate reaction, the rate of transformation of a constituent is proportional to the
constituent concentration. First order kinetics generally apply when the concentra-
tion of the compound being degraded is low relative to the biological activity in
the soil. However, very low concentrations may be insufficient to initiate enzyme
induction or support maintenance requirements necessary for microbial growth, even
if the compound can be used as an energy source (16).
Another model used to describe degradation in soils is the hyperbolic rate
model, which is similar to Michaelis-Menten enzyme kinetics. The model contains a
constant that represents the msTlmnm rate of degradation that is approached as the
concentration increases. This model simulates'a catalytic process in which degra-
dation may be catalyzed by microorganisms.
Often an organic compound that cannot be used as a sole carbon and energy
source for microorganisms is degraded. Biodegradation of the compound does not lead
to energy production or cell growth. This biodegredation process is referred to as
cometabolism [17], or co-oxidation if the transformation involves an oxidation reac-
tion [18]. Cometabolism occurs when an enzymt produced by an organism to degrade
one substance that supports growth also degrades another nongrowth substrate that
is neither essential for, nor sufficient to, support microbial growth. The non-
growth substrate is only incompletely oxidized, or otherwise transformed, by the
microorganism involved, although other microorganism* may utilize by-products of the
cometabolic process. Cometabolism may be a prerequisite for the mineralization of
many recalcitrant substances found in the environment, such as polynuclear aromatic
hydrocarbons [19J. .. • ' :
Measurement of physical abiotic loss mechanisms and partitioning of organic
constituents in a soil should be used ia conjunction with conventional degradation
studies to ensure that information generated frojTmodeling degradation represents
only biological degradation of parent compounds, and not other possible disappear-
ance mechanisms of .-the constituents 'in the -soil system.
The soil is a complex system, consisting of four phases (Figure l)t (1) soil
gases; (2) soil water; (3) .inorganic -solids; and (4) organic solids. Cases and
water, which are found in the pore spaces of a soil, together comprise about SOZ (by
volume) of a typical soil if An organic constituent, depending upon its solubility
and its tendency to volatilize, may be found ia varying proportions in these two
119
-------
Gas
15-35%
Water
15-35%
Inorganic
3845%
Typical Volu
PZCOU 1
tric Compositi
of Soil
phases. Port sizes and continuity and relative proportions of water and air in the
pom art examples of factors that affect the Mobility of contaaiaants (both upward
oat of the soil and downward to the saturated cone) in a specific soil. Depending
upon tits-specific soil characteristics and constituent-specific chemical and physi-
cal properties, constituents in these two phases may be relatively mobile or
immobile.
Soil solids are comprised of organic and inorganic components. The inorganic
components are comprised of sparingly soluble chemicals known as minerals, which are
primarily sand. silt, and clay particles in most coils. The solids may contain
highly reactive charged surfaces that play an important role in immobilizing organic
constituents in a specific soil. Certain types of clays are especially high in
negative charges, thus exhibiting what is termed as a high cation exchange capacity.
Clays may also contain positively charged surfaces and act as anion exchange media
for negatively charged constituents.
SoU organic matter also has many highly reactive charged surfaces and may aid
in retaining organic constituents in a soil system. The term humus refers to the
relatively stable portion of soil organic matter that remains in soil after the
chemicals comprising plant and animal residues have decomposed. Bydrophobic organic
constituents may partition from soil water into soil organic matter and thus become
less mobile in the soil system. Immobilization of constituents may result in
additional time for biodegradation to occur. However, immobilisation also could
result in less Unavailability to microorganisms. Research is required to discover
whether such immobilization constitutes adequate treatment if the constituent is so
tightly and irreversibly bound that it poses no harm to human health and the
environment.
Soil solidyorganie chemical interactions may be quite complex. The structure
of an organic constituent, as it affects such properties as molecular volume, water
solubility, octaaol-water partition coefficients, and vapor pressure, determines the
magnitude of eorption onto the surfaces of a specific soil. Specific aspects of
chemical structure that affect sorption onto soil surfaces have been summarized by
Dragun [20] i
(1) In general, the larger the molecule, the greater its tendency to exist in
the adsorbed state. This is attributed to multiple Van der Veal's forces
arising from many points of contact between the soil surface and the
adsorbed molecule;
130
-------
(2) Bydrophobicity refer* to the preferential migration to and accumulation
of an organic chemical is hydrophobic solvent* or oa hydrophobic surfaces
such as aoil organic matter, in preference to aqocou* solvent* or hydro-
philic surfaces. In general, molecular groups comprised of carbon,
hydrogen. bromine, chlorine, and iodine are hydrophobic groups, while
molecular groups containing nitrogen, sulfur, oxygen, and phosphorus are
primarily hydrophilic groups. The net hydrophobicity of a molecule is
determined by the combined effects of hydrophobic and hydrophilic groups
that comprise the molecule;
(3) Some organic "*>-p<«-«i« contain functional groups with permanent positive
negative or positive charges. These compounds will interact with charged
soil solids and adsorb onto soil surfaces. Soils typically possess a
significantly greater number of negative surfaces than positive ones,
thus negatively charged organic aaions may be repelled by soil surfaces.
Some organic chemicals contain functional groups that may or may not
possess a positive or negative charge, depending upon the acidity of the
soil/water system. The pi, of a chemical is a mathematical description
of the effect of acidity on the charge of the chemical. The relative
ratio of charged to uncharged molecules at a pfl level in a soil/water
system may be estimated and used in identification of the effect of a
molecular charge on the extent of adsorption. For chemicals that possess
both types of functional groups, i.e.. ones that can acquire a positive
charge and ones that can acquire a negative charge, the isoelectric point
(ZP) may be used to predict the effect of pH on the adsorption of these
chemicals. The IP is the pB at which the organic chemical has sero
charge. Above the ZP, the organic chemical has a aet negative charge;
below the ZP, the organic chemical has a net positive charge. The ZP
represents a general summation of the effects of the pK.s of each
functional group in the molecule;
(4) Hydrogen bonding occurs when a hydrogen atom serves as a bridge between
two electronegative atoms. The hydrogen atom is linked to one electro-
negative atOB by a covalent bond and to the other by an electrostatic
bond;
(5) Adsorption potential of a chemical Is affected by intramolecular reac-
tions of adjacent molecular groups or fragments or interference with a
particular adsorption mechanism caused by the presence of one or more
functional groups or molecular fragments; and
(6) Coordination is the formation of a weak bond between an organic molecule
that is capable of donating electrons and adsorbed cations that are
capable of accepting electrons. The net result is a partial overlap of
orbitals and a partial exchange of electron density. Coordination can
occur between organic chemicals and cations in the water phase of a soil
system as well as with soil particle surfaces and with adsorbed cations.
Other abiotic loss mechanisms in addition to surface sorption/desorption reactions
that may account for loss of parent compounds include:
(1) hydrolysis- a chemical reaction in which an organic chemical reacts with
water or a hydroxide ion;
(2) substitution and elimination- reactions where other chemicals in the soil
react with an organic chemical;
(3) oxidation- the reaction resulting in the removal of electrons from a
chemical. This removal generally occurs by two different pathwaysi (a)
heterolytic or polar reactions, (an electrophilic agent attacks an
organic molecule and removes an electron pair leading to the formation of
an oxidized product): or (b) homolytic or free-radical reaction (an agent
removes only one electron to form a radical that undergoes further
reaction); and
(4) reduction- a reaction that results in a net gain of electrons [20].
Successful bioremediation depends on a thorough characterisation and evaluation
of the pathways of movement and potential removal mechanisms of organic constituents
at a specific site, as illustrated in Figure 2. To assess the potential for use of
121
-------
t
1
u
SOLNIEIMCnONS I
PMASSS: HUD UOUD OAS I
FIGURE 2
Fate of Hazardous Contamii
ats in Soil
bioremediation, the transport rate of tht constituents may ba compared to the degra-
dation rate to determine if it is significant.
A Mans of predicting rate of transport of a constituent through a soil system
is to describe its mobility (or relative immobility) by predicting its retardation.
Ketardation is a factor that describes the relative velocity of the constituent corn-
pared to the rate of movement of water through the soil, i.e.:
* •
(1)
where t » retardation factor; V. - average water velocity: and Ve - average consti-
tuent velocity. A retardation factor greater than one indicates that a constituent
is moving more slowly than water through a soil. A factor developed from a trans-
port model combined with a description of sorption processes , as defined by a linear
Freundlich isotherm (21. 22], can be calculated from the following equation:
I - I +
(2)
where f • soil bulk density; K« • soil water partition coefficient, which describes
the partitioning between the soil solid phase and soil water; and I * volumetric
moisture content. This information can be used to manage a contaminated soil system
(i.e., through control of soil moisture, changes in bulk density, or addition of
amendments to the soil that affect the soil water partition coefficient) so that
constituents can be 'captured' or contained within the system, thus allowing time
for implementation and performance of bioremediation treatment techniques.
Vaste and Soil
Interfacing 'son-based behavioral characteristics' of specific orgaaics with
specific site and soil properties allows a determination of potential for bioreaedi-
122
-------
ation of • cite and potential for contamination of other media, i.e., the ground
water under the contaminated area, the atmosphere over the site or at the site boun-
daries, surface waters, etc.
Specific characteristics important for describing and assessing the environ-
mental behavior and fate for organic constituents in soil are listed in Table 1.
For each chemical, or checi : -la:5. the information required can be sumoarized as<
(1) characteristics relu, 10 potential leaching, e.g.. water solubility,
octanol/water partition coefficient, solid sorption coefficient; (2) characteristics
related to potential volatilisation, e.g., vapor pressure, relative volatilisation
index; (3) characteristics related to potential hiodegradation, e.g., half-life,
degradation rate, biodegradability index; and (*) characteristics related to chemi-
cal reactivity, e.g.. hydrolysis half-life, soil redox.potential [21].
TABLE I
Soil-Based Waste Characterisation [21]
Chemical Class
Acid
Base
Polar neutral
Nonpolar neutral
Inorganic
Chemical Properties
Molecular weight
Melting point
Specific gravity
Structure
Water solubility
Chemical Reactivity
Oxidation
Reduction
Hydrolysis
Precipitation
Polymerization
Soil Sorption Parameters
Freundlich sorption constants (I.U)
Sorption based on organic carbon content
Octanol water partition coefficient (K^,)
Soil Degradation Parameters
Half-life (tl/2)
Rate constant (first order)
Relative biodegradability
Soil Volatilization Parameters
Air:water partition coefficient (X*)
Vapor pressure
Henry's lav constant
Sorption based on organic carbon content
Water solubility
Soil Contamination Parameters
Concentration in soil
Depth of contamination
Date of contamination
An adequate site characterisation, including surface soil characteristics, sub-
surface hydrogeology. and microbiological characteristics, is the basis for the
123
-------
rational design of a bioreoediation system. Sit* constraints may limit rat* and
or/extent of treatment of the contaminated vadose con*; therefor*, a thorough ait*
characteritation is necessary to determine both the three-dimensional extent of
contamination aa well as engineering constraints and opportunities.
Important soil hydraulic. physical, aad chemical properties that affect the
behavior of organic cosstitucnts in the vadose zone are presented in Table 2. In
this con*, water prima: _iy coexists with air. though saturated regions may occur.
Perched water tables may develop at interfaces of layers with differing textures.
Prolonged infiltration may also result in saturated conditions.
The vadose zone usually consists of topsoils, typically three to six feet deep.
which arc weathered geological materials, arranged in more or leas well developed
profiles. Vater movement in the vadose con* is usually unsatitrated, with soil water
at lets than atmospheric pressure. Weathered topsoil materials gradually merge with
underlying earth materials, which may include residual or transported claya or
TABLE 2
Sit* and Soil Characteristics Identified as Important for In Situ Treatment [21]
Site location/topography and slope
Soil type, and extent
Soil profile properties
boundary characteristics
depth
texture*
amount and type of coarse fragments
structure*
color
degree of mottling
bulk density*
clay content f
type of clay '
cation exchange capacity*
organic matter content*
pH*
Eh*
aeration status*
Hydraulic properties and conditions
soil water characteristic curve
field capacity/permanent wilting point
water holding capacity*
permeability* (under saturated aad a range of unsaturated conditions)
infiltration rates*
depth to impermeable layer or bedrock
depth to groundwater.* including seasonal variations
flooding frequency
runoff potential*
Geological and hydrogeological factors
subsurface geological features
groundwater flow patterns and characteristics
Meteorological and climatological data
wind velocity and direction
temperature
precipitation
water budget
•Factors that may be managed to enhance soil treatment
124
-------
•and*. The topsoil differ* from the soil material below in that it is more
weathered, contains organic matter, and it the rone of plant root growth. In •one
regime, the vadose zone nay be hundred* of feet thick and the travel tine of con-
stituents to ground water hundreds or thousands of years. Other regions nay be
underlain by shallow potable aquifers especially susceptible to contamination due
to short transport tines and reduced potential of soil materials and processes for
pollutant attenuation.
Microbiological characterisation of a contaminated site should be conducted to
ensure that the site has a viable cemounity of microorganisms to accomplish biode-
gradation of the organic constituents present at the site. Approaches for esti-
mating the kinds, numbers, and metabolic activities.of coil organisms includet (1)
determination of the form, arrangement, and biomass of microorganisms in the soil;
(2) isolation and characterization of subgroups and species; and (3) detection and
measurement of metabolic processes [15). Examples of techniques to accomplish these
activities include direct microscopy of soil (e.g.. fluorescent staining, buried-
slide technique), biomass measurement by chemical techniques (e.g.. measurement of
ATP), measurement of enzyme activity, and cultural counts of microorganisms (e.g..
plate counts, dilution counts, isolation of specific organisms). Biotransformation
studies that measure the disappearance of contaminants or mineralization studies
that indicate complete destruction of contaminants to carbon dioxide and water may
be used to confirm the potential for biodegradation of specific organic chemicals.
Specific techniques include batch culture and electrolytic respirometer studies.
Controls to detect abiotic transformation of the contaminants and tests to detect
toxic effects of contaminants on microbial activity should be included in the
studies.
Information from waste and soil/site characterization studies of a specific site
and from laboratory evaluations of biodegradation and immobilization potential of
specific constituents at the site may be integrated by the use of predictive mathe-
matical model*. The resulting mathematical description may be used to: (1) evalu-
ate the effectiveness of use of on-site bioremediation for treatment of the contami-
nated soil; (2) develop appropriate containment structures to prevent unacceptable
waste transport from the treatment zone; and (3) design performance monitoring
strategies.
Mierobial Factors Affecting Biodegradation
The upper layers of soil contain large numbers and diversity of microorganisms.
Biodegradation of organic constituents is accomplished by enzymes produced by the
microorganisms. Since many enzymes are not released by microbial cells, substances
to be degraded must contact or be transported into the cells. Enzymes are generally
specific in the substances they affect, so many types may be required to complete
biodegradation of organic constituents. The production of enzymes is genetically
controlled, thus mutations and adaptations of the native soil microbial populations
can improve their ability to degrade organic substances (23]. Microbial ecologists
have identified ranges of critical environmental conditions that affect the activity
of soil microorganisms (Table 3). Many of these conditions are controllable and can
be changed to enhance biodegradation of organic constituents.
Vater is necessary for microbial life, and the soil water matric potential
against which microorganism* must extract water from the soil regulates their
activity (the coil matric potential is the energy required to extract water from the
soil pores to overcome capillary and adsorptive forces). Soil water also serves a*
the transport medium through which many nutrients and organic constituents diffuse
to the microbial cell, and through which metabolic waste products are removed. Soil
water also affects soil aeration status, nature and amount of soluble materials.
soil water osmotic pressure, and the pB of the soil solution [15].
Microbial respiration, plant root respiration, and respiration of other orga-
nisms remove oxygen from the soil atmosphere and enrich it with carbon dioxide.
Cases diffuse into the soil from the air above it, and gases in the soil atmosphere
diffuse into the air. However, oxygen concentration in a soil may be much less than
in air while carbon dioxide concentrations may be many times that of air. Even so.
12S
-------
TABLE 3
Critical Environmental factor* for Microbial Activity
[15. 21. 24J
Environmental Factor Optimum Level*
Available soil water 25 • 8S1 of water holding capacity;
-0.01 MPa
Oxygen Aerobic metabolism: Greater than
0.2 ag/1 dissolved oxygen, mini-
mum air-filled pore space of 10Z
by volume;
Anaerobic metabolism: Oj concen-
trations less than 1Z by volume
Redox potential Aerobes and facultative anaerobest
greater than 50 millivolts;
Anaerobess less than SO millivolts
pB 5.5 - 6.5
Nutrient* Sufficient nitrogen, phosphorus.
and other nutrients so not
limiting to Bicrobial growth
(Suggested C
-------
ponding increase in electron density, resulting is • progressively increased nega-
tive potential. Kedox potential is measured as I*, expressed in millivolts, or as
?s. which is equal to -log [«-] where {»'] is the concentration of negatively charged
electrons.
Oxygen levels in a coil system can be maintained by»
(1) prevention of saturation with water;
(2) presence of sandy and loamy soil materials (excessive clay contents are
undesirable);
(3) moderate tilling:
(4) avoidance of compaction of soil; and
(5) limited addition of additional carbonaceous materials (23].
Soil pB also affects the activity of soil microorganisms. Fungi are generally
more tolerant of acidic soil conditions {below pB 5) than are bacteria. The solu-
bility of phosphorus, an important nutrient in biological systems, is maximized at
a pa value of 6.5. A specific contaminated soil system may require management of
soil pH to achieve levels that maximize microbial activity. Control of pB to enhance
microbial activity may also aid in the immobilisation of hazardous metals in a soil
system (a pB level greater than 6 is recoonended to minimize metal transport).
Microbial metabolism and growth is dependent upon adequate supplies of essential
macro- and micronutrients. Required nutrients must be present and available to
microorganisms in: (1) a usable form; (2) appropriate concentrations; and (3)
proper ratios [20]. If the wastes present at the site are high in carbonaceous
materials and low in nitrogen (N) and phosphorus (P). the soils may become depleted
of available H and t required for biodegradation of the organic constituents.
Fertilization may be required at some contaminated tites. as a management technique
to enhance microbial degradation.
Biodegradation of organic constituents declines with lowering of soil tempera-
ture due to reduced microbial growth and metabolic activity. Biodegradation has
been shown to essentially stop at a temperature of 0* C. Soils exhibit a variation
in the temperature of the surface layers, both diurnally and seasonally. Diurnal
changes of temperature decrease with depth of the soil profile. Due to the high
specific heat of water, wet soils are less subject to large diurnal changes than dry
soils [15]. Factors that affect soil temperature include soil aspect (direction of
slope), steepness of slope, degree of shading, soil color, and surface cover.
The environmental factors presented in Table 3. as well as soil and waste
characteristics, interact to affect microbial activity at a specific contaminated
site. Computer modeling techniques are useful to attempt to describe the inter-
actions and their effects on treatment of organic constituents in a specific
situation.
Treatability Studies for Determination of Bioreaediation Potential
Treatability studies for sites contaminated with organic wastes are used to pro-
vide specific information concerning the potential rate and extent of bioremediation
of surficial soil and deeper vadose zone soils by providing information on fate and
behavior of organic constituents at a specific contaminated aite. Treatability
studies can be conducted in laboratory microcosms, at pilot scale facilities, or in
the field. To determine whether a specific site is suitable for bioremediation,
information from treatability studies is combined with information concerning aite
and waste characteristics in order to determine potential applications and limita-
tion* of the technology. Ultimate limitations to the use of bioremediation at a
specific site are usually related to: (1) time required for cleanup. (2) level of
clean-up attainable, and (3) cost of clean-up using bioremediation.
Information from treatability studies alao is used to prepare an approach to the
engineering design and implementation of a bioremediation system at • specific aite.
An engineering design to accomplish bioremediation at the aite is generally based
upon information from simulations (e.g.. mathematical modeling) or estimates of
pathways of migration of chemicals. These simulations or estimates are generated
from treatability data and site/soil characterization data tot (1) determine
containment requirements to prevent contamination of off-site receiver systems; (2)
127
-------
develop techniques to «f «•!•-••»• mass transfer of chemicals affecting microorganism
activity (addition of mineral natri«xt>. oxygen, additional energy sources. pB con-
trol products, ate.; removal of toxic products) in order to enhance bioremediation;
and (3) design a cost-effactive and efficient monitoring program to evaluate effec-
tiveness of treatment.
During the performance of a traatability study, biodegradation, detoxification.
and partitioning (immobilisation) processes are evaluated as tbey affect the fate
and behavior of organic constituents in the soil.
To assess the potential for biological degradation at.a specific contaminated
site, the use of treatability stadias incorporating materials balance and minerali-
zation approaches to determine the environmental fate and behavior of the consti-
tuent! in the specific soil is recommended (Table 4). Rate of degradation is calcu-
lated by measuring the loss of parent compound and the production of carbon divide
with, time of treatment. Degradation rate is often reported as half-life, which
represents the time required for SO percent of the compound to disappear based upon
a first-order kinetic model.
Calculation of the rate of decrease of parent compound, however, by itself does
not provide complete information concerning mechanisms and pathways by which organic
constituents are interacting with the soil environment [25]. Further information
is necessary to understand whether a constituent is simply transferred from one
TABLE 4
Materials Balances and Mineralization Approaches
to Biodegradation Assessment (24)
Biodegradation Approach Process Examined
Materials balances Recovery of parent compound in the
air. soil water, soil solids
(extractable)
Recovery of transformation products
in the air. soil water, and soil
solids (extractable)
Mineralization Production of carbon dioxide, and/
or methane from the parent com-
Balease of substituent groups,
e.g., chloride or bromide ions
phase (e.g.. solid phase) to another (e.g.. air phase) through a process of inter-
phase transfer, or is chemically altered eo that the properties of the parent com-
pound are destroyed. Evaluation of the fate of a constituent in a soil also
requires identification and measurement of the distribution of the constituent among
the physical phases that comprise the system as well as differentiation of the
mechanisms by which the constituent may be chemically altered in a soil system.
tadiolabslled (primarily l*C) compounds are often used to aid in the assessment of
biodegradation and interphase transfer potential, especially at environmentally
realistic, low concentrations (24).
A laboratory flask apparatus that can be need as a microcosm to measure inter-
phase transfer and bioJegradation potential in a laboratory treatability study is
illustrated in Figure S. The contaminated soil material is placed in a flask, which
is than closed and incubated under controlled conditions for a period of time.
During the incubation period, air is drawn through the flask and then through a sor-
121
-------
Enuent Pings Gas
Figure 3
Liberator? Flask Apparatus Bead for Mass Balance Measure
bent material. Volatilised materials «r« collected by the sorbnt and arc Manured
to provide an estimate of volatilization lota of the constituents of interest. At
the end of the incubation period, a portion of the contaminated eoil it treated with
an extracting aolution to determine the extent of lota of the constituents in the
toil matrix. Ihic lo«« can be attributed to biodegradation and poatible imaobiliza-
tion in the aoil materials.
Selection of an appropriate extracting solution ia necessary to maximize consti-
tuent recovery from the aoil. Another portion of the aoil it leached with water to
determine leaching potential of remaining constituents. Abiotic processes involved
in removal of the parent compound are also evaluated by comparing Biennially active
soil/waste mixtures with mixtures that have been treated with a microbial poison.
e.g., mercuric chloride or propylene oxide. The use of several poisons ia recom-
mended to aateat the effects of the poisons on chemical and physical properties of
the toil and waate constituents. A procedure incorporating features illustrated by
the uss of this microcosm it crucial to obtain a materials balance of waste consti-
tuents in the toil system.
Examples of such protocols may be found in (14. 25, 26, 27]. A certain amount of
material is added to the aoil, and tracking the fate of the material as it moves
through the multiple phases of the aoil system provides a materials balance.
Transformation refers to the partial alteration of hazardous constituents into
intermediate products. Intermediate products may be lett toxic or more toxic than
the parent compound, and therefore the rate and extent of detoxification of the con-
taminated material ahould be evaluated. Samples generated from the different phases
of the toil system in the microcosm studies can be analyzed for intermediate degra-
dation products and used in bioaatay ttudies to provide information concerning
transformation and detoxification proceates.
Bioassayt to quantify toxicity measure the effect of a chemical on a test
epecies under specified tett conditions [14). The toxicity of a chemical is propor-
tional to the severity of the chemical on the monitored response of the teat orga-
nism(s). Toxicity aasays utilize teat species that include ratt, fiah, inverte-
brates, microorganisms, and aeeda. The ataays may utilize tingle or multiple
epecies of test organisms. The use of a tingle bioaaaay procedure does not provide
129
-------
a comprehensive •valuation of the toxicity of a chemical in the toil/organic
chemical-impacted system. Often a battery of bloat cays it utilized that may include
measurements of effect! on general microbial activity (e.g.. retpiration. dehydro-
genate activity) at well at attayt relating to activity of subgroupt of the micro-
bial community (e.g., nitrification, nitrogen fixation, celluloae decomposition).
Bioattayt utilizing organisms from different ecological trophic levels may alto
be used to determine toxicological effects. However, use of a single assay as a
screening test to identify relative tozicity reduction in the environment is a
common procedure employed in treatability studies. Assays using microorganisms are
often used due to their speed, simplicity, ease in handling, coat effectiveness, and
use of a statistically significant number of test organisms that is required to
detect the effects of potentially toxic materials in the environment [28, 29].
Two microbial bioassays that have been used to evaluate toxicity of wastes.
including parent compounds and transformation products, in soil systems are the Ames
Salmonella cjrpaiaurium mammalian microsome assay and the Microtox™ test system.
The Ames assay is a measure of the mutagenic potential of hazardous compounds [30,
31] and bat been widely used to evaluate environmental samples [32. 33. 34. 35. 36].*
A high correlation has bean shown between carcinogenicity and mutagenicity. where
about 90Z of known carcinogens tested mutagenic in the Ames assay [37]. Special
strains of Saloonell* typbimariiat that require histidine to grow are uted to test
for mutagenicity. When plated on a histidine-free medium, the only bacteria able
to form colonies are thote that have reverted to the 'wild* state and are able to
produce their own histidine. Without the addition of test chemicals, this back
mutation occurs at a rate specific to each strain type (spontaneous reversion rate).
The addition of chemicals that are mutagenic increases the reversion rate. Mutagen-
icity is measured as a ratio of the number of colonies that grew in the presence of
a test sample (e.g., chemical, mixture of chemicals, or extract of an environmental
sample) to the number of colonies in the absence of the test sample. Since growth
occurs in proportion to mutagenic potential, growth will be greater in the presence
of a mutagen and will increase as the dose of mutagen is increased. The increase
in growth in response to dose is depicted graphically in dose-response curves. The
minimum mutagenic ratio (ratio of number of colonies that form in the pretence of
a test sample to the number of colonies on a control growth plate) is 2.0.
Therefore, a sample exhibiting a mutagenic ratio greater than 2.0 is considered to
possess mntagenic properties.
Some mutagens act directly en the bacterial cells while others require activa-
tion by mammsllsTi microsomes. These microtomes are generally obtained from liver
extracts of Aroclor 1254-induced rats (i.e.. rats injected with the polychlorinated
biphenyl (PCB). Aroclor 1254). The extract, referred to as the S-9 fraction, con-
tains enzymes that mstabolically convert certain chemicals to active mutagens, simu-
lating the activity that occurs in living mamas lien systems. Several etrains of
Salmonella typtdmaxiu* have bean developed in order to detect different types of
mutagens. The recommended strains for general mutagenicity testing include TA97,
TA98, TA100. TA102. TA97 and TA98 detect frameshift mutagens. TA100 detects muts-
gens causing bate-pair substitutions, while TA102 detects a variety of mutagens not
detected by the other strains.
The Microtox™ assay is a at end a nil red, instrumental-based general toxicity
assay that measures the reduction in light output produced by a suspension of marine
luminescent bacteria (Aoeoaaeeeriom abosnaoreom) in response to an environmental
sample [38]. The assay is used to measure acute toxicity of aqueous solutions or
water soluble fraction extracts. The bioassay organisms are handled like chemical
reagents. Suspensions of about one million bacteria are •challenged' with additions
of serial dilutions ef an aqueous sample or extract. Light output from each bac-
terial suspension is measured before and after each addition of sample. Results are
presented as ZCSO values, which are defined as sample concentrations resulting in
a SOZ decrease of light produced by the luminescent bacteria. High EC30 values
indicate lower toxicity than low values. Biolamineseence of the test organism
depends on a complex chain ef biochemical reactions. Chemical inhibition of any of
the biochemical reactions causes a reduction in bacterial luminescence. Therefore.
the Microtox™ test considers the physiological effect of a toxicant and not just
mortality. Matthews and Bulich [39] have described a method of using the Microtox1*
130
-------
assay to predict the land treatability of hazardous organic wastes. Matthew* and
Baiting* [40] described a Method uiing the Microtek assay to determine an appro-
priate range of waste application loading for soil-based treatment systems. Symons
and Sims [41] utilized the assay to assess the detoxification of a complex petroleum
waste in a soil environment. The assay was also included as a recommended bioassay
in the U.S. ZPA Permit Guidance Manual on Hazardous Vaste Land Treatment Demon-
strations [26].
Immobilization refers to extent of retardation of the downward transport
(leaching potential) and upward transport (volatilization potential) of waste con-
stituent*. Interphase transfer potential for waste constituents among soil oil
(waste), water, air. and solid (organic and inorganic) phases is affected by the
relative affinity of the waste constituents for each phase, and may be quantified
through calculation of partition coefficients [26]. Partition coefficients are cal-
culated as the ratio of the concentration of a chemical in the soil. oil. or air
phase to the concentration of a chemical in the water phase, and are expressed as
K. (oil/water). t± (air/water), and S« (solid/water). Calculation of retardation
factors (Equations 1 and 2) also may be used to predict immobilization of constitu-
ents in a soil system [22. 42].
Either laboratory microcosm, pilot scale reactors, or field plots may be used
to generate treatability data. The set of experimental conditions, e.g.. tempera-
ture, moisture, waste concentration, etc., under which the studies were conducted
should be presented along with experimental results.
Treatability study results provide information relating to rates and extent of
treatment of hazardous organic constituents when mass transfer rates of potential
limiting substances are not limiting the treatment. Treatability studies usually
represent optimum conditions with respect to mixing, contact of soil solid materials
with waste constituents and with microorganisms, and homogeneous conditions through-
out the microcosm. Therefore, treatability studies provide information concerning
potential levels of treatment achievable at a specific site. Under field condi-
tions, the rate and extent of bioremediation is generally limited by accessibility
and rate of mass transfer of chemical substances (oxygen, nutrients, etc.) to the
contaminated soil as well as by mass transfer of the contaminants to the microbial
population and removal of microbial degradation products.
Integration of Information from Site Characterization and TreatabilitT Studies
Information from the performance of aite characterization and treatability
atudies may be integrated with the uae of comprehensive mathematical modeling. In
general, models are used to analyze the behavior of an environmental system aider
both current (or past) conditions and anticipated (or future) conditions [43]. A
mathematical model provides a tool for integrating degradation and partitioning pro-
ceases with site/soil- and waste-specific characterization for simulating the
behavior of organic constituents in a contaminated soil and for predicting the path-
ways of migration through the contaminated area, and therefore pathways of exposure
to humans and to the environment. Models may also be used to approximate and esti-
mate the rates and extent of treatment that may be expected at the field acale under
varying conditions. DiCiulio and Suffet [44] have presented guidance on the selec-
tion of appropriate vadose zone models for site-specific applications, focusing on
recognition of limitations of process descriptions of models and difficulties in
obtaining input parameters required by these process descriptions.
The Regulatory and Investigative Treatment Zone Model (RITZ Model, developed at
the U.S. EPA Robert S. Kerr Environmental Research Laboratory by Short [45]} is an
example of a model that has been used to describe the potential fate and behavior
of organic constituents in a contaminated soil system [46]. The Ritz Model is based
on an approach by Jury [47]. An expanded version of RITZ, the Vadose Zone Inter-
active Processes (VIP) model incorporates predictive capabilities for the dynamic
behavior of organic constituents in unsaturated soil systems under conditions of
variable precipitation, temperature, and waste loadings [26, 48, 49, 50. SI]. Both
models simulate vadose zone processes, including volatilization, degradation, aorp-
tion/desorption, advection. and dispersion [52].
131
-------
One* interphase transfer potential and pathways of escape have been identified
by treatability studies aad simulation modeling, containment requirements for the
constituents of interest at the site can ba determined.. If the major pathway of
transport ia volatilisation, containment with respect to volatilization control is
required. An inflatable plastic dome erected over a contaminated site is a contain-
ment method that has been used to control escape of volatile constituents. Vola-
tile* ere drawn from the dome through a conduit and treated in an above ground
treatment system. If leaching ha* been identified as important, control of soil
water movement should be implemented. For example, if contaminated materials are
expected to leach downward from the site, the contaminated materials can be tempor-
arily removed from the site, and a plastic or clay liner placed under the site. When
downward as well as upward migration are significant, both volatilization and
leaching containment systems can be installed. Some hydrophobic chemicals do not
tend to volatilize or to leach but are persistent within the soil solid phase;
therefore containment efforts may not be required.
A critical and cost-effective use of modeling is in the analysis of proposed or
alternative future conditions, i.e.. the model it used a* a management or decision-
making tool to help answer 'what if' type questions [43]. Attempting to answer such
questions through data collection programs would be expensive and practically
impossible in many situations. For example, information can be generated to evalu-
ate the effects of using different approaches for enhancing microbial activity and
for accelerating biodegradation and detoxification of the contaminated area by
altering environmental conditions that affect microbial activity.
Results of modeling alao can aid in the identification of constituents that will
require treatment in the air (volatile) phase, in the leachate phase, and in the
solid (soil) phase. Monitoring efforts therefore can be concentrated on monitoring
the appropriate environmental phase to evaluate treatment effectiveness. Xf a com-
prehensive and thorough evaluation of a specific contaminated system has been con-
ducted, not all enemiesIs need to be monitored in each phase.
Potential AnBli.CAti.ont MsA Lini.tAtl.ont o£ Ri.orenftfiiAti.an T«
Existing information for constituents of interest at a specific site/soil con-
taminated system should be collected as a first step in the investigation of the
application of bioremediation as a potential treatment technology. Many organic
constituents from a wide range of chemical classes have been shown to be amenable
to biodegradation in laboratory studies, using both single strains of microbial
species or consortia of microbial populations. Biodegradation has also been demon-
strated in both aqueous cultures or soil microcosm studies. A summary of biodegra-
dation and disappearance rates for almost 300 chemicsls has been prepared by Dragun
[20]. Examples of specific chemical classes shown to be biodegradable includei
amines and alcohols [14]; polycyclic aromatic hydrocarbon* (PAHs) [5. 12, 13. S3.
54]; chlorinated and non-chlorinated phenols 114]; chlorinated aromatic hydrocarbons
[23]; polychlorinated biphenyls (PCB«) [35], halogenated aliphatic compounds (51.
57]; pesticides [13, 4B, 58. 59. 60. 61]; and various hazardous substances [13. 62].
Industrial wastee from petroleum refining, wood preserving, leather >-Mtifig. coal
gasification/liquefaction, food processing, pulp end paper manufacturing, organic
chemical production, animal production, munitions production, textile manufacturing.
pesticide manufacturing, and pharmaceutical manufacturing, aa well as municipal
wastewaters, sludges, and septage from septic tanks, have all been successfully
treated in land treatment systems [14. 20].
RSXERL. as part of its responsibilities to manage research programs to determine
the fate, transport, and transformation rates of pollutants in the soil, the
unsaturated and the saturated zones of the subsurface environment, initiated a
research program to develop comprehensive screening data on the treatability in soil
of specific listed hazardous organic chemicals and specific listed hazardous wastes.
Research results have been presented by Sims et al. [13], Loahr [14]. and McCinnis
et al. [5J. A Soil Transport and Fate (STF) Data Base was also developed for RSKERL
[63]. The Data Base contains quantitative and qualitative information on degrada-
tion, transformation, partitioning among the soil phases, and toxicity of hazardous
132
-------
organic constituents in toil systems. It may be used as • tool for
•ite assessment and remediation activities. The Database provides. input data
concerning degradation rates, partition coefficients, and chemical property data for
mathematical models simulating the behavior and.fate of chemical constituents in
contaminated surface and subsurface soils. The information is also useful for
providing assistance in determining treatment potential at contaminated sites using
in situ techniques. Chemicals maybe evaluated with respect to the importance of
natural processes in controlling persistence and transport potential, and. therefore
the susceptibility to degradation or retardation within a subsurface environment.
A report was prepared for the U.S. EPA evaluating the effectiveness of soil
treatment practices at Superfund sites. ZPA Office of ftesearch and Development
tests. Department of Defense and Department of Energy studies, state remediation
efforts, private party studies, and vendor demonstrations [64. 65]. Bioremediation
was shown to successfully treat many non-halogenated compounds, but was less sue-
cessful with halogenated compounds. Removal efficiencies for non-halogenated aro-
matics. heterocyclics and other polar compounds were greater than 952. Halogenated
aliphatic compounds were also successfully treated, with removal efficiencies
averaging 98Z; however, volatilization may have contributed to observed losses.
More complex halogenated and nitrated compounds exhibited lower removal
efficiencies, ranging from 50 to 6SZ.
Even though a specific organic constituent has been shown to biodegrade under
laboratory conditions, whether or not it will degrade in a specific soil/site system
is dependent on many factors [23]. Potential degradability requires investigation
in site-specific treatability studies. Available oxygen may be limiting in some
cases, while other compounds may require the presence of anaerobic conditions.
Other environmental conditions that may place restrictions on biological activity
include pB. temperature, and moisture. Open exposure to the soil environment, the
constituent may be biologically or chemically Altered so as to be rendered
persistent and/or toxic in the environment.
The system may lack other nutrients required for microbial activity. Other
chemicals present may serve as preferred substrates, or act to repress required
enzyme activities. High concentrations of metal salts may be inhibitory or toxic
to many microorganisms.
Most chemicals require the presence of a consortia of microbial species for
mineralization, some of which may not be present at the specific site. Also, most
organisms require a period of acclimation to the constituent before metabolism
occurs. During this period, the level of constituent must be high enough to promote
acclimation without being toxic or inhibitory. Prior exposure to the constituent
or similar constituents may help to shorten the acclimation period.
Example of Bioremediation Potential for Polvevelic Aromatic Hydrocarbons (PAHsl in
a Soil System
To demonstrate the potential effectiveness of bioremediation, results are pre-
sented for the semi-volatile chemical class of compounds known as the polycyclic
aromatic hydrocarbons (PAHs). These compounds are of environmental significance
because of their recalcitrance to biological degradation, their chronic toxic
effects on humans, and their widespread occurrence at contaminated waste sites.
Specifically* PAH compounds are associated with oily wastes, such as wastes from
petroleum refining operations and wastes from the wood preserving industry. The
higher molecular weight PAH compounds are of special concern, because they exhibit
mutagenic. carcinogenic, and teratogenic potential.
The degradation of PAB compounds in soils has been demonstrated in laboratory
treatability studies [66]. The results presented in Table 5 for PAH compounds
present in a complex oily waste show that the half-lives for four of the five com-
pounds ranged from only 15 to 139 days. However, the half-life for benxo(g.h.i)pery-
alene. a higher molecular weight PAB compound, was still quite long (1661 days).
McGinnis et al. [5] in a laboratory soil treatability study of PAH compounds present
in creosote waste sludges also found that degradation of PAB was dependent .on
molecular weight and number of aromatic rings. PABs with two rings generally
133
-------
TABLE 5
Degradation of PAHs Present in a Complex Oily Watte,
Applied at 21 Oil and Grease in Clay Loam Soil [66]
9SZ Confidence
Interval (t1/2) (days)
Compound
days
Lower
tipper
Fluoranthene 351
Pyrene 283
Beazo(a)anthracene 86
Benzo (g,h.i)perylene 8
Indenopyrene 5
IS
32
139
1661
69
0.966
0.884
0.397
0.006
0.559
13
26
87
13«
43
18
41
347
HD
139
*Co • Initial Concentration
*tj/z - Half-life (first order kinetics)
TABLE 6
Effect of Manure and pH Amendments on PAH Degradation in a
Complex Waste Incorporated into Soil [67]
Half-Life in Waste/Soil Mixture (days)
PAH Compound
Acenaphthylene
Acenaphthene
Flttorene
Pheaanthrene
Anthracene
Fluoranthene
Pyrene
Benz ( a ) anthracene
Chrysene
Benzo ( b } f luoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
benzo ( ghi ) perylene
DibenzU ,h)anthracene
Indeno( 1 . 2 . 3-cd)pyrene
Without Amendaents
78
96
64
69
28
104
73
123
70
85
143
91
74
179
57
With Amendments
14
45
39
23
17
29
27
52
42
65
74
69
42
70
42
134
-------
exhibited h»lf-lives less than ten days, while three ring compound* IB most case*
exhibited longer half-live*, which were usually lees than one hundred day*. Most
of the four or five ring PAHs exhibited half livec of one hundred toys or nore. The
results of these two studies suggest that Man* of enhancing biological degradation
of more recalcitrant FAB compounds should be investigated.
When additional carbon and energy sources were provided and soil pH was adjusted
from 6.1 to 7.5. the half-lives of PAH waste constituents present in a complex
fossil fuel waste added to a soil were decreased, as shown in Table 6 167]. In this
laboratory study using first order kinetic modeling of degradation, the use of
manure as an amendment and control of soil pH significantly decreased the t,/2 of
the PAH constituents studied. For example, the half-life of phenanthrene decreased
from 69 to 23 days. benz(a)anthracene from 123 to 52 days, and benz(a)pyrene from
91 to 69 days.
The control of soil moisture also resulted in enhanced biodegradation of PAHs,
as shown in Table 7 (67). Soil moisture in this study was described in term of
percent of field capacity. Field capacity is defined as the percentage of soil
moisture remaining in a soil after having been saturated and after free drainage has
practically ceased. Therefore, soils with moisture levels of 60 to 802 of field
capacity are wetter than soils with levels of 20 to 402 of field capacity. At
higher levels of soil moisture, the half-life of the PAH constituents studied
decreased. For example, for f luoranthene, the half-life decreased from SS9 days to
231 days. At a specific site where containment has been achieved, the addition and
control of soil moisture may be a tool to accomplish faster degradation of the
constituents.
An increase in soil temperature can also decrease the time required to accom-
plish degradation, especially the loss of lower molecular weight PAHs [68]. In a
laboratory study, the half-life of fluorene decreased from 60 days to 47 days to 32
days at 10*. 20*. and 30* C, respectively (Table 8). At a field site, toil tempera-
ture may be difficult to control. However, if a cover is used at the site to
control the release of volatile materials. an increase in soil temperature may also
occur. Seasonal climatic changes will affect the rate of degradation of organic
constituents, as well as geographical location of a specific contaminated cite.
If a soil has been exposed previously to similar or the same type of contamina-
tion, the soil microbial population may have become acclimated to the waste, and
waste degradation may occur at a faster rate. la a laboratory study investigating
the. acclimation of a soil to a fossil fuel waste, a greater reduction in concentra-
tion of all the waste FAB compounds studied was achieved in 22 days in an acclimated
soil, compared to the reduction seen in 40 days in an unacclimated toil (Table »)
[67]. These results show that at a site that has been contaminated for a period of
time, the indigenous microbial population may become acclimated to the presence of
wastes, and techniques to stimulate microbial activity may produce significant
degradatioa. Mixing of a small amount of a contaminated soil that has developed an
acclimated population with the contaminated soil to be treated may also result in
TABLE 7
Effect of Soil Moisture on PAH Degradation [67]
Half-life in Vaste/Soil Mixture (days)
Moisture Anthracene Phenanthrene Fluoranthene
20-40Z field capacity 43 61 559
60-80Z field capacity 37 54 231
135
-------
TABLE 8
Percentages of PAH leeiaining at the End of the 240 Day Study Period
•ad Estimated Apparent Los* Balf Litres [68]
Percent of PAH
Remaining
Estiaated Half Life (day)'
10"C 20»C 30«C
10»C
20»C
30»C
Acenaphthene
Flourene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Bens(a)anthracene
Chrysene
5 0 0
832
36 19 2
83 SI 58
94 71 IS
93 89 43
82 71 SO
85 88 86
Benxo(b)fluoranthene 77 75 62
Beaso(k)fluoranthene 93 93 89
Benzo(a)pyrene
73 54 S3
Dibenz(a.h)anthracene 88 87 83
Benzo(f.h.i)perylene 81 76 75
lndenoU.2,3-c.d)pyren« 80 77 70
<60
60 47 32
(+11/-10) (*«/-S) (+S/-3)
200
(+40/-40)
<60
<60
460 260 200
(+310/-UO) (+160/-70) (+90/-30)
+ 440 140
(+560/-160) (**0/-20)
+ 1900 210
(+6200/-800) (+160/-60)
680 430 240
(+300/.160) (+110/-70) (+40/-40)
980 1000 730
(+320S-270) (+900y-250) (+370/-180)
S80 610 360
(*520/.180) (+590/-200) (+150/-BO)
910 1400 910
(+690/-270)(+3300/-560)(+4400/-410)
530 290 220
(+1700/-230)(+570/-120) (+160/-60)
820 750 940
(+1100/-300)(+850/-260)(+12000/-4SO)
650 600 590
(+650/-230) (+570/-190) (+1800/-250)
600 730 630
(+310/-150)(+1100/-270)(+2SOO/-280)
* ti/2 OS percent confidence interval)
* Least squares elope (for calculation of tu,) - tero with 95 percent confidence.
13*
-------
TABLE 9
Acclimation of Soil to Complex Fo««il Fuel Waste [67]
Dnaccliaated Soil Acclimated Soil
Initial Soil Reduction in Soil Reduction in
FAB Compound Concentration 40 days (Z) Concentration* 22 days (I)
(mg/kg-dry wt)
Naphthalene
Pbenanthrene
Anthracene
Fluoranthene
Pyrene
Benz(a)anthracene
Chrysene
Benx(a)pyrene)
38
30
3B
154
177
30
27
10
90
70
58
SI
47
42
25
40
98
30
38
159
180
40
33
12
100
83
99
82
86
70
61
50
* After first rempplication of waste (after 168 days incubation at initial level)
(mg/kg-dry wt)
faster cleanup of a site. Amendment of the soil with exogenous microorganisms
developed in laboratory batch cultures would not be required.
A method to assess detoxification of waste constituents in a soil system
involves use of the Microtox1" assay [S9J. which was described previously. In a
clay loam soil, a petroleum refinery waste was added to soil at standard industry
application rates of 22, 42. and 82 by weight of oil and grease [41]. The results
of the study are shown in Figure 4. Time of incubation is plotted on the x-axis.
and ECSO values, as determined by the Microtox1" assay .on the y-axis. Detoxifi-
cation of a contaminated soil system is indicated by increased ECSO values approach-
ing 100Z. A value of 1002 is considered as non-toxic. At the 22 loading rate, the
waste material was detoxified to an K50 value of 100 in a period of about 100 days
(Figure 4a). At the highest level of contamination (82 loading rate), the materials
remained toxic, even after 180 days. In addition to providing evidence of detoxifi-
cation of waste constituents, this study also showed the potential for enhancement
of biodegradation by mixing oncontaainated soil with contaminated aoil to produce
a treatment medium with waste contents at levels not toxic to microbial populations.
In a sandy loan soil amended with the same contaminated material, a longer
period (about 170 days) was required to detoxify the 22 contamination level to an
ECSO value of 1002 (Figure 4b). Results of these studies show that mixing of con-
taminated soils with uncontaminated soils can result in detoxification. However.
since the rate of detoxification may be a function of soil type, these results also
Illustrate the site specificity of bioremediation efforts and underscore the need
to perform site-specific characterization of the contaminated area.
137
-------
Nunn clay loam
e
0 30 60 90 120 150 180
• 2% Oil & Grease
• 4% OB* Grease
a 8% Oi& Grease
Kidman sandy loam
100
T
E
•2.
• 2%Oil Grease
• 4% Oi A Grease
• 8%OS* Grease
20
4b
30. CO 90 120 ISO 180
•HUE (days)
Figure *
ZCSO as a function of time for two soils and three waste loading rates (41]
A study to evaluate detoxification of mutagenic potential of a complex fossil
fuel waste containing PAH compounds treated in a soil system was conducted utilizing
the ASMS assay (67]. Mutagenic ratios for 5. traUmazium strain TA98 (a test strain
used to detect frameahift autageaa such as PABs) with metabolic activation (to siau-
late aaamalian Metabolism by the addition of a mammalian liver extract (referred to
as the S9 fraction)), and without metabolic activation (without the addition of the
89 fraction) were determined immediately after waste incorporation and after 42 days
of incubation of the waste in the soil, lesults as shown in dose response curves
showed that the mutagenic ratios decreased from about 4.5 and 7.0 at the highest
dose levels tested immediately after waste incorporation (Figure 5) to borderline
matagenic levels (i.e., mutagenic ratios of about 2) after 42 days of treatment
(Figure 6). For a different 5. erpUaurium strain, (TA100. a test strain used to
detect mutagens causing base-pair substitutions), no dose-response effects or muta-
genic activity were measured during the study.
Results of a pilot scale field study have also demonstrated that bioremdiation
of PAH contaminated soils is a technology that can result in significant cleanup of
contaminated soils [69]. A coal gasification waste was thoroughly mixed into a soil
at a one-half acre site. Sampling of soil cores was performed at 10 feet intervals
138
-------
Dotting/pale)
Figure 5
Anec assay results for waste; soil mixture immediately
after waste incorporation into soil
across 100 feet rows. Data presented in Table 10 are conposite values from the
stapling efforts. In all cases, concentrations of the PAH coapounds in the soil
were greatly reduced after 91 days. Data quality was poorer at the 91 day sampling
period, as Measured by the coefficient of variation (CV), which is the Man value
measured (AVC) divided by the standard deviation (SD). The poorer data quality was
attributed to increased analytical difficulties when levels of constituents are
•easured near detection limits.
The fate and environmental impact of transformation products is an ares of bio-
remediation that needs more consideration. In a laboratory study, the transfor-
mation of a l*C radio-labelled PAH compound, 7,12- dimethylbenz(a)anthraccne, in a
GOO 2000 3000 4000 9000 WOO TOGO (GOO
Figure 6
Ames assay results for waste; soil mixture after 42 days incubation [67]
139
-------
TABLE 10
Field Results for Soil Treatment of FAHs
in Coal Gasification Wastes [69]
C,*(Mg/K> C After 91 days (jtg/g)
i^MMH^Mltlli
naphthalene
Acenaphthene
Phenanthrene
Benz ( a ) anthracene
Dibenz(a ,h)anthracene
AV?
1S6
729
78
86
32
SO
68
276
28
42
36
CV(X)
37
38
36
49
69
AVC
3
1
2.6
2
ND
SD
1.8
1.8
0.6
0.8
—
CV(I)
61
157
23
38
—
*CQ • Initial Soil Concentration
sandy loaa soil was investigated for a 28 day incubation period (70]. At time 0.
62Z of the applied parent compound was recovered from the soil, which represents the
extraction efficiency of the teat (Table 11). After 28 days of incubation, only 20Z
of the parent compound was recovered. Since recoveries in control reactors poisoned
with nercuric chloride were not significantly different over the 28 day incubation
TABU 11
Transformations of (WC) DMBA
by McLaurin Sandy Loan Soil* [70]
UC Appearing in Each Fraction. Percent
Soil Extract
Time. DMBA.
days parent compound Metabolites Soil Residue C02 Total
0 62(69)
14 26
28 20(60)
4(6)
43
53(11)
12(13)
16
17(16)
0(0)
0
0(0)
78(88)
85
90(87)
'Poisoned (control) data in parentheses.
140
-------
period, biological treatment was the proposed mechanism of compound removal free the
•oil system. Table 11 also shows that the decrease in parent "C was accompanied
by an increase in the metabolite "C fraction. The appearance of transformation pro-
ducts increased from 4Z of the total "C applied at time 0 to 5SZ after 28 days.
None of the radiolabelled carbon appeared as COj in thic study, bat 12 to 172 of the
radiolabelled material was associated with the solid phase of the soil during the
incubation period. The mass balance for the study ranged from 78 to 901 recovery of
the applied radiolabelled carbon. Therefore, the appearance, toxicity, fate, and
behavior of a metabolite fraction miy need to be evaluated on a site-specific basis.
The environmental significance and fate and behavior of many transformation pro-
ducts of PAH constituents, as veil as transformation .products from many other
organic constituents, are not yet known. Therefore, incorporating detoxification
assessment into a bioremediation plan is recommended to evaluate these concerns.
ion at Sitet
ed vith Organi
c Wastes
A recent survey conducted for the U.S. EPA concerning the use of bioremediation
at sites with soils contaminated with wood preserving wastes identified ten sites
that currently plan to use bioremediation techniques to clean-up contaminated soils
and sediments (Table 12) [71]. Sims observed a wide range of variability in target
clean-up levels. A wide variability also was observed in criteria for selecting
target levels (maximal contaminant levels (MCLs) based on drinking water standards
vs. negotiated levels vs. risk assessment-based levels) and in selection of soil
phases that must meet target levels (solid phase, leachate phase, and/or air phase).
Target levels were determined on a site-specific basis. .
An example of a bioremediation plan for a facility identified in the survey was
presented by Lynch and Genes [72]. Da-site treatment of creosote-contaminated soils
from a shallow, unlined surface impoundment was demonstrated at a disposal facility
for a wood-preserving operation in Minnesota. The contaminated soils contained
creosote constituents consisting primarily of PABs at concentrations ranging from
TABLE 12
Vood Preserving Sites Where Bioremediation has
been Proposed for Soil or Lagoon Sediments [71]
Site Hame
State (U.S. EPA Region)
Proposed Remediation
L.A. Clark and Son
Brown Wood Preserving
Burlington Northern (Brainard)
Horth Cavalcade Street
Baited Creosoting Company
Baxter /Union Pacific
Burlington Northern (Somers)
Libby (Champion International)
VA (XII)
TL (IV)
MH (V)
TX (VI)
TX (VI)
WY (VI11)
KT (VIII)
MT (VIII)
Bioremediation
Bioremediation
Landfarm
Bioremediation
In Situ Remediation
Bioremediation
Landfarm
InSituBioremedia
tion
Koppers. Co.. J. B. Baxter
CA (IX)
and Landfarm
Bioremediation
141
-------
1.000 to 10.000 ppm. Prior to implementation of the full scale treatment operation.
bench-scale and pilot-scale studies simulating proposed full-scale conditions were
conducted to define operation and design parameters. Over a four-month period. 62Z
to 80Z removal of total PABs were achieved in all test plots and laboratory reac-
tors. Two-ring PAB compounds were reduced by 80-90Z. 3-ring PABs by 82-93Z. and 4+-
ring PABs by 21-60Z.
The full-scale system -involved preparation of a treatment area within the con-
fines of the existing impoundment. A lined waste pile for temporary storage of the
sludge and contaminated soil from the impoundment was constructed. All standing
water from the impoundment was removed, and the sludges were excavated and segre-
gated for subsequent free oil recovery. Three to five feet of 'visibly' contami-
nated eoil was excavated and stored in the lined waste pile. The bottom of the
impoundment was stabilized as a base for the treatment area. The treatment area was
constructed by installation of a polyethylene liner, a leaehate collection system.
four feet of clean backfill, and addition of manure to achieve a carbontnitrogen
ratio of 50il. A sump for collection of stormwster and leaehate and a center pivot
irrigation system were also installed. The lined treatment area was required
because the natural soils at the site were highly permeable. A cap was also needed
for residual contaminants left in place below the liner. Contaminated soil was
periodically applied to the treatment facility and roto-tilled into the treatment
soil. Soil moisture was maintained near field capacity with the irrigation system.
During the first year of operation, greater than 95Z reductions in concentration
were obtained for 2- and 3-ring PABs. Greater than 70Z of 4- and 5-ring PAB com-
pounds were degraded during the first year. Comparison of half-lives of PABs in the
full-scale facility were in the low end of the range of half-lives reported for the
test plot units. Only two PAB compounds were detected in drain tile water samples.
at concentrations near analytical detection limits.
Bioremediation of a Texas oil field site with storage pit backfill soils con-
taminated with styrene, still bottom tars, and chlorinated hydrocarbon solvents was
demonstrated en a pilot scale [73]. The remediation efforts also included chemical
and physical treatment strategies. The pilot scale, solid-phase biological treat-
ment facility consisted of a plastic film greenhouse enclosure, a lined soil treat-
ment bed with an underdrain. an overhead spray system for distributing water.
nutrients, and inocula. an organic vapor control system consisting of activated car-
bon absorbers, and a fermentation vessel fer preparing microbial inoculum or
treating contaminated leaehate from the backfill soils. Soils were excavated from
the contaminated area and transferred to the treatment facility. Average concentra-
tions of volatile organic compounds (VOCs) were reduced by more than 99Z during the
94 day period of operation of the facility; most of the removal was attributed to
air stripping. Biodegradatien of semivolatile compounds reduced average concentra-
tions by 89Z during the treatment period.
A solid-phase treatment system to remediate petroleum contaminated soil at *
hazardous waste site in California was described by toss at al. [ 6 ]. The treatment
process involved stimulating the existing microbial population in the soil to
degrade petroleum hydrocarbon contaminants. A biotreatability evaluation prior to
full-scale operation demonstrated that the existing microorganisms in the soil could
degrade the petroleum hydrocarbons, but that the nutrient levels in the soil were
not sufficient to maintain growth and support complete degradation of the hydro-
carbon contaminants. Vith adequate nutrients, hydrocarbons decreased from 3500 ppm
to lese than 100 ppm in 4 weeks in bench scale studies. The degradation process
exhibited biphasic kinetics, likely due to the fact the petroleum hydrocarbons were
a mixture of a lighter diesel fuel and more recalcitrant waste motor oils. An
initial rapid rate of hydrocarbon removal was predominately due to degradation of
the lighter diesel fuel, while a second, slower rate reflected removal of the waste
oils. The full scale facility, which began operation in 1988, consists of a four
acre treatment site that has had 30 inches of contaminated soil applied to the sur-
face. Bioremediation of -.he top 15 inches was proceeding by the addition of nutri-
ents, daily tilling and maintenance of adequate eoil moisture levels. When the
first 15 inches of contaminated soil have been remediated to the target cleanup
level of 100 ppm. it will be removed and the second IS inches will be treated.
During the first four weeks of operation, the average concentration of petroleum
hydrocarbons was reduced from 2,800 to 280 mg/kg. The rate of hydrocarbon biodegra-
142
-------
dation measured in the field was consistent with the rate measured in the
laboratory.
A solid phase treatment system to clean up pesticides in soil contaminated as
a result of a fire at a chemical storage facility was also described by Ross et si.
[6]. Hater used to extinguish the fire carried large amounts of insecticides and
herbicides into the soil beneath the warehouse facility. Laboratory biotreatability
studies showed that moderately contaminated soils (90 rag/kg of 2,4-D) could be
treated in a soil treatment system to meet regulatory criteria (total MCPA and 2.4-D
• 10 mg/kg). while highly contaminated soils (2,4-D concentrations greater than 200
mg/kg) required treatment in a soil/water slurry bioreactor. A five acre soil
treatment area was constructed with an engineered clay liner 12 inches thick and a
drainage system to control water movement. Ten thousand cubic yards of soil contami-
nated with a complex mixture of herbicides and insecticides, including 2.4-D.
alachlor, trifluralin. carbofuran. and MCPA. were spread on the treatment bed to an
average depth of IS inches. During operation, soil conditions were optimized for
biological activity by daily tilling and by maintenance of soil moisture content
between 81 and 152 by weight. During three months of operation, the combined 2.4-D
and MCPA concentrations decreased from 86 ppm to 5 ppm.
Brubaker and Zxner [74] reported on two case histories that involved microbial
degradation of chemical contaminants to remediate chemical spills. Both sites also
involved other remediation tools in addition to microbial remediation, emphasizing
the need to examine complementary and synergistic remediation techniques. At the
first site, residual contamination from a formaldehyde spill vas treated using
chemical oxidation with hydrogen peroxide, followed by microbial •polishing* to com-
plete the remediation. A commercial inoculum of microorganisms acclimated for for-
maldehyde degradation and a nutrient solution were mixed in an aeration tank and
then sprayed on the site. Water was collected in a sump and recycled through the
aeration tank. Treatment effectiveness was measured by reduction of concentration
of formaldehyde in the aeration tank. After 25 days, concentrations had dropped
from over 700 mg/1 to less than 1 mg/1. At the second site, a gasoline leak from
an underground storage tank was remediated with enhanced bioreclamation techniques.
which consisted of addition of nutrients and hydrogen peroxide as an oxygen source.
A series of injection and recovery wells were used to recycle water through the
site. Soil samples showed a decrease in volatile fuel hydrocarbons from an average
of 245 ppm at the initiation of the bioreclamation process to 0.8 ppm after 200
days.
Bioremediation of'a site contaminated with PCBs, which have generally been con-
sidered resistant to biodegradation in the environment, has been demonstrated at a
drag-racing track in Mew York (55). Laboratory testability studies using contami-
nated soils from the sites inoculated with pure resting cell cultures of PCB-
degrading organisms that had been isolated from environmental samples showed sub-
stantial PCB biodegradation. up to 511 of the PCBs present in three days. Follow-up
laboratory studies were conducted using only 3-42 of the number of cells used in the
earlier studies, lower moisture content, lower temperatures, and no shaking or
aeration of the reaction mixtures. PCB degradation was not observed until 30 days
after the initiation of the study. In an undisturbed soil sample inoculated three
times weekly with the PCB-degrading microorganisms, 502 of the PCBs in the top 1 cm
of soil was degraded in IS weeks. Only 102 degradation was seen at depths below l
cm. When a duplicate of the undisturbed soil experiment was mixed at three months
with continued inoculation, the redistributed soil again exhibited the highest
degradation rate at the surface. In experiments where soils were inoculated three
times weekly and mixed after each application. 352 of the PCBs were degraded after
23 weeks at all depths. This degree of degradation represents a greater amount of
PCB destruction since the PCBs were degraded throughout the whole sample and not
just at the surface. Thus mixing was identified as an important site management
variable. Preliminary results at a field scale test site at the drag-racing track
indicated significant PCB degradation after eight to ten weeks.
CONCLUSIONS
Consideration of bioremediation for remediation of a site contaminated with
143
-------
organic constituents requires a detailed site, soil, and waste characterisation that
mat be conducted in order to evaluate the potential application of the technology
at the cite and to demonstrate the feasibility of the approach. A sound and
thorough engineering remediation plan developed at the on-set of the project will
allow cost-effective and efficient use of resources for implementation of site
clean-up. The use of treatability studies and sianilation modeling are also
necessary components of the bioremediation plan so that necessary data to evaluate
potential use and to identify pathways of migration are collected in a cost-
effective manner. Bioremediation of sites contaminated with organic chemicals is
a promising technology, especially if it is incorporated in a remediation plan that
uses an integrated approach to the cleanup of the complete site, i.e., a plan that
involves the concept of a 'treatment train* of physical, chemical, aad/or biological
processes to address remediation of all sources of contaminants at the site.
REFERENCES
1. Omenn. C.S. (ed.)> 1988. Environmental Biotechnology - Reducing Risks from
Environmental Chemicals through Biotechnology. Plenum Press. Mew York, MY.
505 pp.
2. Engineering Foundation. 1988. Proceedings, Conference on Biotechnology
Applications in Hazardous Waste Treatment. Engineering Foundation
Conferences. Longboat Key, Florida, October "31- November 4.
3. AWMA/EPA. 1989. Proceedings of the Internatl. Symposium on Hazardous Waste
Treatment: Biosystems for Pollution Control. Air and Waste Management
Association and tl.S. Environmental Protection Agency, Cincir,. ti, Ohio,
February 20-23.
4. U.S. EPA. 1989. Bioremediation of Hazardous Waste Sites Workshop. CERI-89-11.
U.S. Environmental Protection Agency, Cincinnati. OB.
5. McCinnis. 6.0., B. Borazjani, L.K. McParland. D.F. Pope, and D.A. Strobel.
1989. Characterization and Laboratory Soil Treatability Studies for Creosote
and Pentachlorophenol Sludges and Contaminated Soil. EPA/600/2-88/055. Robert
S. terr Environmental Research Laboratory, U.S. Environmental Protection
Agency, Ada. OK.
6. Ross. 0.. T. P. Marxian, and A.L. Bourquin. 1988. Bioremediation of
hazardous waste sites in the OSAt Case histories, pp. 395-397. in: Superfund
•88. Proe. 9th Hatl. Conf., Hazardous Materials Control Research Institute.
Silver Spring. MD.
7. Wilson. L.C. 1983. Monitoring in the vadose zone: Part III. Ground Water
Monitoring Review (Winter)t155-166.
8. Everett. L.6.. E.W. Hoylman. L.6. McMillion, and L.C. Wilson. 1982. Vadose
zone monitoring concepts at landfills, impoundments, and land treatment
disposal anas. Xai Management of Uncontrolled Hazardous Waste Sites.
Hazardous Materials Control Research Institute. Silver Spring. MD.
9. Wilson. L.C. 1981. Monitoring in the vadose cone: Part I. Storage changes.
Ground Water Monitoring Review (Fall):32-41.
10. Wilson. L.G. 1982. Monitoring in the vadose zonei Part II. Ground Water
Monitoring Review (Spring):31-42.
11. Lehr. J.B. 1988. The misunderstood world of unsaturated flow. Ground Water
Monitoring Review (Spring)t4-6.
12. Sims. R.C.. J.L. Sims, D.L. Sorensen. W.J. Doucctte. and L.L. Bastings. 1986.
144
-------
Waste/Soil Treatability Studies for Four Complex Industrial Wastes:
Methodologies and Results. Vol. 1 and 2. EPA/600/6-86/003a and b. Robert S.
Karr Environmental Research Laboratory. U.S. Environmental Protection Agency.
Ada. OK.
13. Sim*. R.C.. V.J. Ooucette. J.E. McLean. W.J. Crenney, and R.R. Dupont. 1988.
Treatment Potential for 56 ZPA Listed Hazardous Chemicals in Soil. EPA/600/6-
86-001. Robert S. Kerr Environmental Research Laboratory. U.S. Environmental
Protection Agency. Ada, OK. ^
1*. Loehr. R. 1989. Treatability Potential for ZPA Listed Harardous Wastes in
Soil. EPA/600/2-89/Oil. Robert S. Kerr Environmental Research Laboratory.
U.S. Environmental Protection Agency. Ada, OK.
15. Paul, Z.A.. and f. E. Clark. 1989. Soil Microbiology sad Biochemistry.
Academic Press. Inc.. San Diego. CA.
16. Rittmann. B.E., and P.L. McCarty. 1980. Model of steady-state biofilm
kinetics. Biotech. Bioeng. 22i 2343.
17. Borvath. R.S. 1972. Microbial co-metabolism and the degradation of organic
compounds in nature. Bacteriol. Rev. 36:146-155.
18. Perry, J.J. 1979. Microbial cooxidation involving hydrocarbons. Microbiol.
Rev. 43:59-72.
19. Keck, J.. R.C. Sims, M. Coover. K. Park, and B. Symons. 1989. Evidence for
cooxidation of polynuclear aromatic hydrocarbons in soil. Water Res. 23:1467-
1476.
20. Dragon. J. 1988. The Soil Chemistry of Hazardous Materials. Hazardous
Materials Control Research Institute, Silver Spring. MD.
21. Sims, R.C.. D.L. Sorensen, J.L. Sims. J.I. McLean, R. Mahmood, and R.R.
Dupont. 1984. Reviev of In Place Treatment Techniques for Contaminated
Surface Soils. Volume 2t Background Information for Xa Situ Treatment.
ZPA/540/2-84-003*. Municipal Environmental Research Laboratory. U.S.
Environmental Protection Agency, Cincinnati, OB.
22. Mahmood. R.J.. and R.C. Sims. 1986. Mobility of organics in land treatment
systems. J. Environ. Eng., Am. Soc. Civil Bog. 112:236-245.
23. Rochkind. M.L.. J.W. Blackburn and C.S. Sayler. 1986. Microbial Decomposition
of Chlorinated Aromatic Compounds. EPA/600/2-86/090. Basardous Waste
Engineering Research Laboratory, U.S. Environmental Protection Agency.
Cincinnati, OB.
24. Buddleston. -R.L.. C.A. Bleckmann. and J.R. Wolfe. 1986. Land treatment
biological degradation processes, pp. 41-61. In» R.C. Loehr and J.F. Malina.
Jr. (eds.) Land Treatment! A Harardous Waste Management Alternative. Water
Resources Symposium No. 13. Center for Research in Water Resources. The
University of Texas at Austin, Austin. TX.
25. Park. K.S.. R.C. Sims. R.R. Dupont. W.J. Ooucette. and J. E. Matthews. 1989.
Pate of PAB compounds in two soil types: Influence of volatilisation. abiotic
loss, and biological activity. Environ. Toxicol. Chem. (In press).
26. U.S. EPA. 1986. Permit Guidance Manual on Bacardons Waste Land Treatment
Demonstration*. EPA-530/SW.66-032. Office of Solid Waste and Emergency
Response. U.S. Environmental Protection Agency. Washington, DC.
27. U.S. EPA. 1988. Interim Protocol for Determining the Aerobic Degradation
145
-------
Potential of Hazardous Organic Constituents in Soil. 9.S. EPA Scientific
Starring Committee, Bioeystems Technology Development Program, and Soil
Treatment Processes Committee. Robart S. Karr Environmental Research
Laboratory. U.S. Environmental Protection Agency. Ada, OK.
28. Liu, D., and B.J. Dutka (ads.)- 1984. Toxicity Tatting Procedures using
Bacterial Systems. Marcel Dakker. Inc., Hew York. Inc.
29. Dutka. B.J.. aad 6. Bitten. 1986. Toxicity Testing using Microorganisms. CRC
Prats, Inc.. Boca Raton. PL.
30. Ames. B.B.. J. McCaaa, and E. Yamaaaki. E. 1975. Methods for detecting
carcinogens and mutagana with the SalmanalZa/mamnalian-microsome mutagenicity
teat. Mutation Res. 31:347-36*.
31. Mar on. D.M.. and B.H. Anas. 1983. Revised methods for the Salmonella
•utagenicity test. Mutation Res. 113:173-215.
32. Sims. R.C.. J.L. Sims, and R.R. Dupont. 1984. Human health affects assays. J.
Water Pollut. Control Fed. 56: 791-800.
33. Sims. R.C.. J.L. Sims, and R.R. Dupont. 1985. Human health effects assays. J.
Vatar Pollut. Control Fed. 57: 728-742.
34. Sims. R.C.. J.L. Sims, and R.R. Dupont. 1986. Human health effects assays. J.
Vatar Pollut. Control Fed. 58: 703-717.
35. Sims, R.C.. J.L. Sims, and R.R. Dupont. 1987. Human health effects asaays. J.
Water Pollut. Control Fed. 59: 601-614.
36. Sims, R.C., J.L. Sims, aad R.R. Dupont. 1988. Human health effects asssys. J.
Water Pollut. Control Fed. 60: 1093-1196.
37. McCaaa, J.R.. R. Choi. E. Yamasaki. and B.H. Ames. 1975. Detection of
carcinogens as mutagens in the Salmoaclla/microsoaa test: Aasay of 300
chemicals. Free. Matl. Acad. Sci. 72:5135-5139.
38. Bulich. A.A. 1979. Use of luminescent bacteria for determining toxicity in
aquatic environments, p. 98-106. lat L.L. Markings aad R.A. Rimer la, ads.
Aquatic toxicology. ASTM 667. Amer. Soc. for Testing aad Materials.
Philadelphia. FA.
39. Matthews, J.E. aad A.A. Bulich. 1984. A toxicity reduction teat system to
assist predicting land treatability of hazardous vaates. pp. 176-191. In:
J.I. Patroa, Jr.. V.J. Lacy, and R.A. Convay. eds.. Hazardous aad Industrial
Solid Waata Tasting: Fourth Symposium, STP-B86. American Society of Testing
aad Materials, Philadelphia, FA,
40. Matthews. J.E. sad L. Hastings. 1987. Evaluation of toricity teat procedure
for screening treatability potential -f waste in soil. Toxicity Assessment:
An Zntenatl. Quarterly 2: 265-281.
41. Symons, B.D. aad R.C. Sims. 1988. Assessing detoxification of a complex
hazardous waste, using the Microtox™ bioasaay. Arch. Environ. Contamination
ToxiCOl.l7t 497-505.
42. Bordea. R.C.. aad F.B. Bedient. 1987. In situ measurement of adsorption and
biotraasfozmation at a hazardous waste site. pp. 629-636. lat M. A. Marino
(ed.) Subsurface Flow aad Contamination Methods of Analysis aad Parameter
Uncertainty. AWRA Monograph Series Ho. 8, Am. Vater Resources Assoc.,
Bethesda. MD.
146
-------
43. Donagian, A.S.. Jr.. and P.S.C. Rao. 1986. Overview of terrestrial process**
•ad modeling, pp. 1-1-32. la: S.C. Ben and S.M. Melancen (eds.) Guidelines
for Field Testing Soil Fate and Transport Models. Final Report. EFA/600/4-
•6/020, Environmental Monitoring Systems Laboratory* U.S. Environmental
Protection Agency. Las Vegas. KV.
44. Digiulio, B.C.. and I.E. Suffet. 1988. Effects of physical, chemical, and
biological variability in modeling organic contaminant migration through
soil. pp. 152-137. In: Superfond '88. Proc. 9th Natl. Conf.. Hazardous
Materials Control Research Institute. Silver Spring. MD.
45. Short. T.E. 1986. Modeling processes in the unsaturated zone. pp. 211-240.
IB< E.G. Loehr and J.F. Malina. Jr. (eds.) Land Treatment: A Hazardous Vaste
Management Alternative. Vater Resources Symposium No. 13. Center for Research
in Vater Resources. The University of Texas at Austin. Austin, TZ.
46. U.S. EPA. 1988. Interactive Simulation of the Fate of Hazardous Chemicals
during Land Treatment of Oily Vastest RITZ User's Guide. EPA/600/8-88-001.
Robert S. Kcrr Environmental Research Laboratory, U.S. Environmental
Protection Agency. Ada, OK.
47. Jury. V.A., V.F. Spencer, and V.J. Farmer. 1983. Behavior assessment model
for trace organics in soil: Model description. J. Environ. Qual. 12: 558-564.
48. McLean. J.E.. R.C. Sims. V.J. Doucette. C.R. Canpp. and V.J. Crenney. 1988.
Evaluation of mobility of pesticides in soil using U.S. EPA methodology. J.
Environ. Eng.. Am. Soc. Civil Eng. 114> 689-703.
49. Stevens. O.K., V.J. Crenney. and Z. Yan. 1988. User's Manual: Vadose Zone
Interactive Processes Model. Dept. of Civil and Environ. Eng.. Utah State
Univ.. Logan, UT.
SO. Stevens, D.K.. V.J. Crenney. Z. Yan, and R.C. Sims. 1989. Sensitive
Parameter Evaluation for a Vadose Zone Fate and Transport Model. EPA/600/2-
B9/039. Robert S. Kerr Environmental Research Laboratory, U.S. Environmental
Protection Agency, Ada, OK.
51. Symons, B.D.. R.C. Sims, and V.J. Crenney. 1988. Fate and transport of
organics in soil: Model predictions and experimental results. J. Vater
Pollut. Control Fed. CO: 1684.1693.
52. Crenney, V.J.. C.L. Caupp. R.C. Sims, and T.E. Short. 1987. A mathematical
model for the fate of hazardous substances in soil: Model description and
experimental results. Hazardous Vastes I Hazardous Materials 4:223-239.
S3. Sims. R.C. and Overcash, M.R. 1983. Fate of polvnuclear aromatic compounds
(PHAs) in soil-plant systems. Residue Reviews 88: 1.68.
54. Bulman. T.. S. Lesage. P.J. A. Fowlie. and M.D. Vebber. 1985. The persistence
of polynuclear aromatic hydrocarbons in soil. PACE Report Ho. 85-2. Petroleum
Association for Conservation of the q*nfditn Environment, Ottawa, Canada.
55. Unterman. R.. D.L. Bedard. M.J. Brennan. L.B. Bopp. F.J. Mondello. R.E.
Brooks. D.P. Mobley, J. B. McDermott,.C. C. Schwartz, and D.K. Dietrich.
1988. Biological approaches for polychlorinated biphenyl degradation, pp.
253-269. In: C.S. Omenn (ed.), Environmental Biotechnology - Reducing Risks
from Environmental Chemicals through Biotechnology. Plenum Press. New York.
NY.
56. Vogel, T.M., C.S. Griddle, and P.L. McCarty. 1987. Transformations of
halogenated aliphatic compounds. Environ. Sci. Technol. 21:722-736.
147
-------
57. McCarty F.L. 1988. Bioengineering issues related to in situ remediation of
contaminated soils and groundwater. pp. 143-162. Int G.S. Omenn (ed.).
Environmental Biotechnology - Reducing Risks from Environmental Chemicals
through Biotechnology, Plenum Press, Mev York. MY.
58. Cuenzi. V.D. (ed.). 1974. Pesticides in Soil and Vater. Monograph. Soil Scl.
Soc. am.. Madison. VX.
59. Coring. C.A.X., and J.V. Hamaker (eds.). 1972. Organic Chemicals in the Soil
Environment. Marcel Dekker, Inc. Mev York, RY.
60. Goring. C.A.X.. D.A. Laskovski, J.V. Hamaker, R.V. Miekle. 1975. Principles
of pesticide degradation in soil. Int R. Haque and V.H. Freed (eds.)
Environmental Dynamics of Pesticides. Plenum Press, Mew York. MY.
61. Rao, P.S.C., and'J.M. Davidson. 1982. Estimation of pesticide retention and
transformation parameters required in nonpoint source pollution models. Xnt
M.R. Overcash and J.M. Davidson (eds.). Environmental Impact of Monpoint
Source Pollution. Ann Arbor Science, Ann Arbor. MX.
62. Overcash. M.R.. and D. Pal. 1979. Design of Land Treatment Systems for
Industrial Vastest Theory and Practice. Ann Arbor Science. Ann Arbor, MX.
63. U.S. EPA. 1988. Soil Transport and Fate Database and User's Manual (Draft).
Cooperative Agreement Mo. 813211. Robert S. Kerr Environmental Research
Laboratory. U.S. Environmental Protection Agency. Ada. OK.
64. Offutt. C.K., J.O. Xnapp. E. Cord-Duthinh. D.A. Bissex. A.V. Oravetz. Jr..
G.D. Lacy, P.J. Kenney. E.L. Green, and D. Bhinge. 1988. Analysis of
contaminated soil treatment effectiveness, pp. 429-434. Xnt Superfund *88.
Proc. 9th Matl. Conf.. Hazardous Materials Control Research Institute, Silver
Spring, MD.
65. COM Federal Programs Corporation. 1988. Summary of Treatment Technology
Effectiveness for Contaminated Soil. Office of Emergency and Remedial
Response. U.S. Environmental Protection Agency. Washington. DC.
66. Ryan. J.. R. Loehr. and R. Sims. 1987. The Land Treatability of Appendix VIII
Constituents Present in Petroleum Refinery Wastes t Laboratory and Modelling
Studies. American Petroleum Institute, Land Treatment Committee. 1220 L.
Street. Washington. D.C. (8 volumes).
67. Sims. R.C. 1986. Loading rates and frequencies for land treatment systems.
pp. 151-170. Int R.C. Loehr and J.F. Malina. Jr. (eds.) Land Treatmenti A
Hazardous Waste Management Alternative. Water Resources Symposium Mo. 13.
Center for Research in Water Resources. The University of Texas at Austin.
Austin. TJC.
68. Coover. M.P. and R.C. Sims. 1987. The effect of temperature on polycyclic
aromatic hydrocarbon persistence in an unacclimated agricultural soil.
Hazardous Waste & Hazardous Materials 4t 69-82.
69. Siau. R.C. 1986. Soil Treatability Study Results-Coal Gasification Process
Vater Fond Residuals. Utah Vater Research Laboratory, Utah State University,
Logan, UT.
70. Park, K.S., R.C. Sims, V.J. Doucette. and J.E. Matthews. 1988. Biological
transformation and detoxification of 7.l2-dimethylbenz(a)anthracene in soil
systems. J. Vater Pollut. Control Fed. 60: 1822-1825.
71. Sims. R.C. 1989. Overview of bioremediation in soil and ground water«
148
-------
theoretical and practical considerations, pp.11-38. Xnt Free.. Forum on
Bioremediation of Wood Treating Vaste. Mississippi Forest Products
Utilisation Laboratory. Mississippi State University. March 14-15 (In press).
72. Lynch. J.. and B.R. Genes. 1989. Land treatment of hydrocarbon contaminated
soils. Ch. 14. pp. 163-174. In: P.T. Kostecfci and E. J. Calabrese (eds.).
Petroleum Contaminated Soils. Vol It Remediation Techniques. Environmental
Fate, and Risk Assessment. Lewis Publishers, Chelsea, MX.
73. St. John. V.D. and D.J. Sikes. 1988. Complex industrial waste sites, pp. 237-
252. Xn< 6.S. Omenn (ed.). Environmental Biotechnology • Reducing Risks from
Environmental Chemicals through Biotechnology, Plenum Press. Mew York, MY.
74. Brubaker. C.R.. and J.B. Esner. 1988. Bioremediation of chemical spills, pp.
163-171. Zns C.S. Omenn (ed.), Environmental Biotechnology - Reducing Risks
from Environmental Chemicals through Biotechnology. Plenum Press. Mew York.
•Y.
Address Reprint Requests To:
Judith L. Simt
Utah Water Research Laboratory
Utah State University
Logan. OT 84322-8200
-------
-------
•0
IH >
o fi 3 oJ
£ v v
2*S a
T3
CX3
O
£ pj
------- |