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 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

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                                                                                      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

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 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

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 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

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                    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

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      (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

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                            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

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 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

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 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

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 •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

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                                    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
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 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)
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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

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                                     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

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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*
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 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].
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    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

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 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

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                                    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

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 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

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                                     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*

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                                      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

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 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

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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

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 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

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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

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       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

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      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

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 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

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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

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      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

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