wEPA
            United States
            Environmental Protection
            Agency
              Office of Solid Waste and
              Emergency Response
              Washington DC 20460
EPA/540/R-93/519a
August 1993
        for Conducting
Treatability Studies
Under CERCLA
            Biodegradation
            Remedy Selection

            Interim Guidance
SUPERFUND INNOVATIVE
TECHNOLOGY EVALUATION
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                                                             EPA/540/R-93/519a
                                                             August 1993
                          GUIDE FOR CONDUCTING
                 TREATABILITY STUDIES UNDER CERCLA:
                  BIODEGRADATION REMEDY SELECTION

                              INTERIM GUIDANCE
                             U.S. Environmental Protection Agency
                             Risk Reduction Engineering Laboratory
                             Office of Research and Development
                                 Cincinnati, Ohio 45268

                                       and

                           Office of Emergency and Remedial Response
                          Office of Solid Waste and Emergency Response
                                 Washington, D.C. 20460
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                                         DISCLAIMER
                       The information in this document has been funded wholly or in
                       part by the U.S. Environmental Protection Agency (EPA) under
                       ContractNo. 68-C8-0062, Work Assignment 3-43 and Contract
                       No.  68-CO-0048,  Work  Assignment 0-38,  to  Science
                       Applications International  Corporation (SAIC).  It has  been
                       subjected to the Agency's peer and administrative reviews and it
                       has been approved for publication as an EPA document. Mention
                       of trade names or commercial products does not constitute
                       endorsement or recommendation for use.
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                                                   FOREWORD
                                Today's rapidly developing and changing technologies and
                                industrial products and practices frequently carry with them the
                                increased generation of materials that, if improperly dealt with,
                                can threaten both public health and the environment. The U. S.
                                Environmental Protection Agency (EPA) is charged by
                                Congress with protecting the nation's land, air, and water
                                resources. Under a mandate of national environmental laws, the
                                agency strives to formulate and implement actions leading to a
                                compatible balance between human activities and the ability of
                                natural systems to support and nurture life. These laws direct
                                EPA to perform research to define our environmental problems,
                                measure the impacts, and search for solutions.

                                The EPA Risk Reduction Engineering Laboratory is responsible
                                for planning, implementing, and managing research,
                                development, and demonstration programs to provide an
                                authoritative, defensible engineering basis in support of the
                                policies, programs, and regulations of the EPA with respect to
                                drinking water, wastewater, pesticides, toxic substances, solid
                                and hazardous wastes, and Superfund-related activities. This
                                publication is one of the products of that research and provides
                                a vital communications link between the researcher and the user
                                community.

                                The primary purpose of this guide is to provide standard
                                guidance for designing and implementing a biodegradation
                                treatability study in support of remedy selection testing.
                                Additionally, it describes a three-tiered approach that consists
                                of 1) remedy screening testing, 2) remedy selection testing, and
                                3) remedial design/remedial action testing. It also presents a
                                guide for conducting treatability studies in a systematic and
                                stepwise fashion for determination of the effectiveness of
                                biodegradation in remediating a site regulated under the
                                Comprehensive Environmental Response, Compensation, and
                                Liability Act. The intended audience for this guide includes
                                Remedial Project Managers, On-Scene Coordinators, Potentially
                                Responsible Parties, consultants, contractors, and technology
                                vendors.
                                                               E. Timothy Oppelt, Director
                                                    Risk Reduction Engineering Laboratory
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                                               ABSTRACT
                           Systematically conducted, well-documented treatability studies are
                           an important component of the remedial investigation/feasibility
                           study  process and the  remedial design/remedial  action process
                           under the Comprehensive Environmental Response, Compensation,
                           and Liability Act  (CERCLA).  These  studies  provide valuable
                           site-specific  data  necessary  to  aid  in  the  selection and
                           implementation  of  the  remedy.  This  manual  focuses  on
                           biodegradation treatability studies conducted in support of remedy
                           selection testing  prior to developing the Record of Decision (ROD).

                           This  manual  presents   a  standard  guide   for  designing and
                           implementing a biodegradation remedy selection treatability study.
                           The manual  describes  and  discusses  the  applicability and
                           limitations  of  biodegradation  technologies,  and  defines  the
                           prescreening and field measurement data needed to determine if
                           treatability testing is required. It also presents an overview of the
                           process of conducting treatability tests and the applicability of tiered
                           treatability testing for evaluating biodegradation technologies. The
                           specific goals for each tier of testing are defined and performance
                           levels are presented, which should be met at the remedy selection
                           testing level in support of the ROD.  The elements of a treatability
                           study work plan  are also defined and detailed discussions  on the
                           design and execution of the remedy selection treatability studies are
                           provided.

                           The manual  is  not  intended  to  serve as  a  substitute  for
                           communication with experts or regulators or as the sole basis for the
                           selection of biodegradation as a particular remediation technology.
                           This manual is designed to be used in conjunction with the Guide for
                           Conducting Treatability Studies  Under CERCLA  (Final)'52' and the
                           Guide  for Conducting Treatability Studies Under CERCLA: Aerobic
                           Biodegradation  Remedy Screening  (Interim  Guidance).(53) The
                           intended  audience for this  guide  includes  Remedial  Project
                           Managers, On-Scene Coordinators, Potentially Responsible Parties,
                           consultants, contractors, and technology vendors.
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                                        TABLE OF CONTENTS
     Section                                                                                                 Page
          DISCLAIMER  	ii
          FORWARD  	111
          ABSTRACT  	iv
          FIGURES  	  vii
          TABLES 	viii
          ABBREVIATIONS, ACRONYMS, AND SYMBOLS 	 ix
          ACKNOWLEDGMENTS	 xi
     1.    Introduction  	1
          1.1   Background  	1
          1.2   Purpose and Scope  	1
          1.3   Intended Audience  	  2
          1.4   Use of This Guide	2
     2.    Technology Description and Preliminary Screening 	3
          2.1   Technology Description 	3
               2.1.1   In Situ Biological Technologies	4
               2.1.2   Ex Situ Biological Technologies	7
               2.1.3   Anaerobic Bioremediation Applications  	10
          2.2   Preliminary Screening and Technology Limitations 	11
               2.2.1   Literature/Database Review  	13
               2.2.2   Technical Assistance  	14
               2.2.3   Prescreening Characteristics  	15
               2.2.4   Technology Limitations  	16
     3.    The Use of Treatability Studies in Remedy Evaluation 	21
          3.1   Process of Treatability Testing in Selecting a Remedy  	21
          3.2   Application of Treatability  Tests	21
          3.3   Biodegradation Treatability Tests 	26
               3.3.1   Remedy  Screening  	26
               3.3.2   Remedy  Selection	29
               3.3.3   Remedial Design/Remedial Action 	29
     4.    Treatability Study Work Plan	33
          4.1   Test Goals 	33
               4.1.1   Remedy  Selection Treatability Study  Goals 	34
          4.2   Experimental Design and Procedures  	35
               4.2.1   Remedy  Selection Experimental Design 	36
               4.2.2   pH 	36
               4.2.3   Soil Characteristics	36
               4.2.4   Temperature  	37
               4.2.5   Moisture 	37
               4.2.6   Nutrients 	37
               4.2.7   Electron Acceptors  	37
               4.2.8   Microorganisms 	38
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                                     TABLE OF CONTENTS (continued)


     Section                                                                                                   Page

               4.2.9   Test Duration  	38
               4.2.10  Chemical Inhibition  	38
               4.2.11  Nonbiological Removal Processes  	39
               4.2.12  Toxicity Testing  	39
               4.2.13  Bioavailability  	39
               4.2.14  Experimental Design of In Situ Systems  	40
               4.2.15  Experimental Design of Ex Situ Systems	41
               4.2.16  Anaerobic Studies  	43
          4.3  Equipment and Materials  	44
          4.4  Sampling and Analysis  	45
               4.4.1   Field Sampling	45
               4.4.2   Media Analysis During the Treatability Study  	47
               4.4.3   Monitoring and Process Control Measurements  	47
               4.4.4   Treatment Product Sampling and Analysis  	48
          4.5  Data Analysis and Interpretation  	49
          4.6  Reports	50
          4.7  Schedule  	50
          4.8  Management and Staffing	51
          4.9  Budget 	51
     5.    Sampling and Analysis Plan	55
          5.1  Field Sampling Plan  	55
          5.2  Quality Assurance Project Plan 	55
               5.2.1   Project Description	56
               5.2.2   Quality Assurance Objectives 	56
               5.2.3   Sampling Procedures 	56
               5.2.4   Analytical Procedures and Calibration 	56
               5.2.5   Data Reduction, Validation and Reporting	57
               5.2.6   Quality Control Reports  	57
     6.    Treatability Data Interpretation  	59
          6.1  Technology  Evaluation 	59
               6.1.1   Remedy Screening Phase  	59
               6.1.2   Remedy Selection Phase  	59
               6.1.3   Remedial Design/Remedial Action Phase	62
          6.2  Estimation of Costs  	62
     7.    References  	63
     8.    Appendix A 	67
                                                           VI
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                                                     FIGURES

     Number                                                                                                  Page

     2-1    Superfund Remedial Actions: Summary of Alternative Treatment Technologies Through FY91 	4
     2-2    In Situ Bioremediation of Saturated Soils and Groundwater  	6
     2-3    Bioventing 	7
     2-4    Solid-Phase Bioremediation	8
     2-5    Soil Heap Bioremediation  	8
     2-6    Open Windrow Composting	9
     2-7    Above-Ground Slurry-Phase Bioremediation  	10
     2-8    Slurry-Phase Bioremediation in Existing Lagoon  	11
     2-9    Earth Biofilter Treatment 	11
     2-10   Biofilter/Biotower Treatment  	12
     2-11   A Graphic Representation of the Contaminant Removal Asymtote  	19
     3-1    Flow Diagram of the Tiered Approach to Conducting Treatability Studies 	22
     3-2    The Role of Treatability Studies in the RI/FS and RD/RA Process  	23
     4-1    Sample Treatability Testing Schedule for Remedy Selection Evaluation of
           In Situ Bioremediation 	51
     4-2    Sample Project Schedule for Laboratory Remedy Selection Evaluation of
           Solid- and Slurry-Phase Bioremediation 	52
     4-3    Sample Organization Chart  	52
                                                           VII
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                                                       TABLES
     Number                                                                                                    Page

     2-1    Comparison of Biological Remediation Technologies  	5
     2-2    Site and Soil Characteristics Identified as Important in Biological Treatment  	16
     3-1    Ability of Remedy Selection Treatability Studies to Address RI/FS Criteria  	27
     3-2    Biodegradation Criteria For Each Treatability Study Tier  	28
     4-1    Suggested Organization of Biodegradation Treatability Study Work Plan  	33
     4-2    Remedy Selection Treatability Study Characteristics	40
     4-3    Characteristics of Anaerobes Classified According to Physiological Nature  	45
     4-4    Equipment and Materials  	46
     4-5    Equipment for Field Collection of Soil Samples	47
     4-6    Commonly Used Analytical Chemistry Methods  	48
     4-7    Guidance for an Operational Monitoring Program 	49
     4-8    Major Cost Elements Associated with Biological Remedy Selection Treatability Studies	53
     5-1    Suggested Organization of the Sampling and Analysis Plan  	56
                                                           vm
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                                ABBREVIATIONS, ACRONYMS,
                                           AND SYMBOLS
                    ANOVA      analysis of variance
                    ARAR       applicable or relevant and appropriate requirement
                    ASTM       American Society for Testing and Materials
                    ATP         adenosine triphosphate
                    ATTIC       Alternative Treatment Technology Information Center
                    BBS         bulletin board system
                    BDAT       Best Demonstrated Available Technology
                    BOD         biological oxygen demand
                    BTEX        benzene, toluene, ethylbenzene, and xylene
                    CAMU       Corrective Action Management Unit
                    CERCLA     Comprehensive Environmental Response,
                                 Compensation, and Liability Act
                    CFR         Code of Federal Regulations
                    CLU-IN      Cleanup Information Database
                    CMP         chemical manufacture production
                    COC         contaminant of concern
                    COD         chemcial oxygen demand
                    COLIS       Computerized On-Line Information System
                    CPAH       carcinogenic polynuclear aromatic hydrocarbon
                    DNAPL      dense non-aqueous phase liquid
                    DO          dissolved oxygen
                    EPA         U.S. Environmental Protection Agency
                    ERT         Emergency Response Team
                    ETSC        Engineering Technical Support Center
                    FSP         Field Sampling Plan
                    FY          fiscal year
                    GC          gas chromatography
                    GC/MS       gas chromatography/mass spectroscopy
                    HOPE        high density polyethylene
                    HPLC        high-performance liquid chromatography
                    HWSFD      Hazardous Waste Superfund Collection Database
                    IR           infrared spectrometry
                    LAN         local area network
                    LDR         Land Disposal Restrictions
                    LNAPL       light non-aqueous phase liquid
                    MPN         most probable number
                    NAPL        non-aqueous phase liquid
                    NPDES       National Pollutant Discharge Elimination System
                    NPL         National Priorities List
                    NTIS         National Technical Information Service
                    O&G         oil and grease
                    OERR        Office of Emergency and Remedial Response
                    ORD         Office of Research and Development
                    OSC         On-Scene Coordinator
                    OSWER      Office of Solid Waste and Emergency Response
                                                      IX
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                                    ABBREVIATIONS (continued)
                     PAH          polynuclear aromatic hydrocarbon
                     PCB          polychlorinated biphenyl
                     PCP          pentachlorophenol
                     PMN         premanufacturer notification
                     POTW        publicy-owned treatment works
                     ppb          parts per billion
                     PPE          personal protection equipment
                     ppm          parts per million
                     ppmv         parts per million volume
                     PRP          Potentially Responsible Party
                     QA          quality assurance
                     QAPP        Quality Assurance Project Plan
                     QC           quality control
                     RCRA        Resource Conservation and Recovery Act
                     RD/RA        remedial design/remedial action
                     RI/FS         remedial investigation/ feasibility study
                     RITZ         Regulatory and Investigative Treatment Zone
                     ROD          Record of Decision
                     RPD          relative percent difference
                     RPM         Remedial Project Manager
                     RREL         Risk Reduction Engineering Laboratory
                     RSKERL      Robert S. Kerr Environmental Research Laboratory
                     SAIC         Science Applications International Corporation
                     SAP          Sampling and Analysis Plan
                     SITE          Superfund Innovative Technology Evaluation
                     SMOS        Subsurface Modeling Support
                     SRT          Subsurface Remediation Technology
                     STF          Soil Transport and Fate
                     SVOC        semivolatile organic compound
                     SW-846       Test Methods for Evaluating Solid Waste, Third Ed., SW-846
                     TQ           time zero
                     TCE          trichloroethylene
                     TIO          Technology Innovation Office
                     TOC          total organic carbon
                     TSC          Technical Support Center
                     TSCA        Toxic  Substance Control Act
                     TSDF         treatment, storage, and disposal facility
                     TSP          Technical Support Project
                     UST          underground storage tank
                     VIP          Vadose Zone Interactive Processes
                     VISITT        Vendor Information System for Innovative Treatment Technologies
                     VOC          volatile organic compound
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                                         ACKNOWLEDGMENTS

                      This guide was prepared for the U.S. Environmental Protection Agency (EPA), Officeof
                      Research and Development (ORE)), Risk Reduction Engineering Laboratory (RREL),
                      Cincinnati,  Ohio, by Science Applications International Corporation (SAIC) under
                      Contract No. 68-C8-0062 and Contract No. 68-CO-0048. Mr. Edward Opatken served as
                      the EPA Technical Project Monitor. Mr. Jim Rawe was SAICs Work Assignment
                      Manager. Mr. Derek Ross of Environmental Resources Management, Inc. served as a
                      technical expert. The primary authors for this guide were Mr. Jim Rawe, Ms.  Sharon
                      Krietemeyer, and Ms. Evelyn Meagher-Hartzell of SAIC. The project team included Mr.
                      Kurt Whitford of SAIC and Mr. Clyde Dial, SAIC's Senior Reviewer.

                      The authors want to give special thanks to Joe Healy, EPA Region IX; Steve Safferman,
                      EPA, RREL; Leo Lehmicke, ECO VA Corporation; and Keith Piontek, CH2 M Hill, for then-
                      continued involvement in the development of this document. The following other
                      Agency and  contractor personnel have contributed their time and comments by
                      participating in the technical workshop and/or peer reviews of the draft document:

                                   Harry Allen     U. S. EPA - ERT
                                   Ralph Baker     ENSR Consultants and Engineers
                                   Rick Bartha     Rutgers University
                                   Ed Bates        U. S. EPA - RREL
                                   DickBleam      Bioscience Management
                                   Frank Castaldi   Radian Corp.
                                   Chuck Coyle    U.S. Army Corps of Engineers
                                   James P. Earley  University of Notre Dame
                                   John Finn       Hart Crowser, Inc.
                                   Robert Finn     Cornell University
                                   Paul Flathman   O.H. Materials Corp.
                                   John Glaser     U. S. EPA - RREL
                                   Jones Grubbs   Solmar Corp.
                                   Patrick Haas    U. S. Air Force
                                   Eugene Harris   U. S. EPA - RREL
                                   Suxuan Huang   Keystone Environmental Resources
                                   James Hyzy     Wastestream Technology
                                   Robert Irvine    University of Notre Dame
                                   Fran Kremer     U. S. EPA - RREL
                                   Kim Kneton     U. S. EPA - RREL
                                   Ron Lewis      U. S. EPA - RREL
                                   Carol Litchfield  Environmental Technology Application
                                   John Mathews   U. S. EPA - RSKERL
                                   Lisa Nichols    U. S. EPA - Region III
                                   Ross Miller     U. S. Air Force
                                   Dave Smith     U. S. EPA - Region VIII
                                   Jim Spain       U.S. Air Force
                                   Todd Stevens   Pacific Northwest Lab
                                   Timothy Vogel   University of Michigan
                                   Paul S. Yocum   University of Notre Dame
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                                                  SECTION  1
                                             INTRODUCTION
     1.1  BACKGROUND

     Section  121(b)  of  the   Comprehensive  Environmental
     Response,  Compensation,  and Liability Act (CERCLA)
     mandates the U.S. Environmental Protection Agency (EPA) to
     select remedies to restore hazardous waste sites that "utilize
     permanent solutions and alternative treatment technologies or
     resource recovery  technologies to the maximum  extent
     practicable" and to prefer remedial actions in which treatment
     that "permanently  and significantly reduces the volume,
     toxicity, or mobility of hazardous substances, pollutants, and
     contaminants  is a principal element."  Treatability studies
     provide data to support treatment technology selection and
     remedy implementation. If treatability studies are used, they
     should be performed as soon as it is evident that insufficient
     information is available to select and implement a technology.
     Conducting  treatability  studies   early in  the   remedial
     investigation/feasibility study  (RI/FS) process  reduces
     uncertainties associated with selecting the remedy based on
     limited information and provides a sound basis for the Record
     of Decision (ROD). EPA regional planning should factor in the
     time and resources required for these studies.

     Treatability studies conducted during the RI/F S phase indicate
     whether the technology can meet the cleanup goals for the
     site,  whereas  treatability  studies conducted during the
     remedial design/remedial action (RD/RA) phase  establish
     design  and  operating parameters  for  optimization of
     technology performance. Although the purpose and scope of
     these studies  differ, they complement one another since
     information  obtained in support  of the remedy  selection
     process may also be used to support RD/ RA.(75)

     This document refers to three levels, or tiers, of treatability
     studies: remedy screening, remedy selection, and RD/RA
     testing. Three tiers of treatability studies are also defined in
     the Guide for Conducting Treatability Studies Under CERCLA,
     Final, hereinafter referred to as the "generic guide."(52) The
     generic guide refers to the three treatability study tiers, based
     largely on the scale of test equipment, as laboratory screening,
     bench-scale testing,  and  pilot-scale  testing.  Laboratory
     screening is typically used to screen potential  remedial
     technologies  and  is  equivalent to  remedy  screening:
     Bench-scale testing is typically used for remedy selection
     testing; however, it may  fall  short of providing enough
     information for remedy selection. Bench-scale studies can, in
     some cases, provide enough information for full-scale design.
Pilot-scale studies are normally used for RD/RA, but may be
required for remedy selection testing in some cases. Because
of the overlap of these tiers, and because of differences in the
applicability  of each tier  to  different  technologies, the
functional descriptions  of the treatability study tiers (i.e.,
remedy screening, remedy selection, and RD/RA testing) are
used in this document.

Some or all of the treatability study levels may be needed on
a case-by-case basis. The time and cost necessary to perform
the  studies are balanced against the improved confidence in
the  selection of treatment alternatives. These decisions are
based on the quantity and quality of data available and on
other factors (e.g., State and community acceptance  of the
remedy,  additional site  data,  and experience  with the
technology). The need for each level of treatability testing is
a management decision. Section 3 discusses in greater detail
howtreatability studies are used in remedy evaluation. Section
6 provides guidance on  interpreting treatability study results
and generating cost estimates.

1.2 PURPOSE AND SCOPE

This guide helps ensure a reliable and consistent approach to
conducting remedy selection studies. Although there has been
increased interest  in  using  microbes  to  treat   media
contaminated with inorganics and metals, this document is
limited to providing guidance on performing remedy selection
studies  that  evaluate  treatment  alternatives  for  media
contaminated with  organic  contaminants.  The remedy
screening level of treatability testing is discussed in the Guide
for Conducting Treatability Studies Under CERCLA: Aerobic
Biodegradation  Remedy  Screening   (Interim   Guidance),
hereinafter referred to  as  the  "biodegradation  screening
guide. "(53) Remedy screening  studies provide  quick  and
relatively inexpensive indications of whether biodegradation
is a potentially viable remedial technology. Remedy selection
treatability testing provides data to help  determine if  a
technology can be used singly or in combination with another
technology to reduce contaminant concentrations to  levels
that comply with site cleanup goals. Remedy selection studies
also provide preliminary estimates of the cost and performance
data necessary to design either an RD/RA study or afull-scale
remediation system.

In general, RD/RA studies will be  required to optimize
full-scale system design. Presumably, before RD/RA studies
are conducted, remedy selection testing has al-
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     ready been ready been used to determine that biodegradation
     is an economically and technically viable treatment alternative.
     RD/RA testing will be site-specific and will utilize equipment
     employed  during full-scale  treatment.  Consequently,  an
     in-depth discussion of RD/RA testing is beyond the scope of
     this guidance document.

     1.3 INTENDED AUDIENCE

     This document  is  intended for use by  Remedial Project
     Managers (RPMs),On-SceneCoordinators(OSCs),Potentially
     Responsible  Parties (PRPs), consultants, contractors, and
     technology vendors, Each has a different role in conducting
     treatability studies under CERCL A. Specific responsibilities for
     each can be found in the generic guide. (52)

     1.4 USE OF THIS GUIDE

     This guide is organized into seven sections and reflects the
     basic information required to  perform  treatability studies
     during the RI/FS process. Section 1  is an introduction that
     defines the role of the guide, describes the purpose and scope
     of the guide, and outlines its intended audience.  Section 2
     describes  different  biodegradation  processes  currently
     available and discusses how to conduct preliminary screening
     to determine if  biological treatment  is a potentially viable
     solution.  Section 2 also identifies factors that may limit the
     feasibility of biodegradation. Section  3 provides an overview
     of the different levels of treatability testing and discusses how
     to determine the need for treatability studies.   Section  4
     provides an overview of remedy selection treatability studies,
     describes the contents of a typical Work Plan, and discusses
     the major issues to consider when conducting a treatability
     study. Section 5 discusses the Sampling  and Analysis Plan
     (SAP), including the Field SamplingPlan (FSP) and the Quality
     Assurance Project Plan (QAPP).  Section 6  explains how to
     interpret the data produced from treatability studies and how
     to determine if further RD/RA testing is justified.  Section 7
     lists the references. Although each section has been written to
     address a specific topic, they have been designed to provide
     enough  background  information  to  allow the reader to
     understand the topic being addressed without needing to refer
     to another section  of  the  document  for  clarification of
secondary issues. Although some repetition exists within this
document, every effort was taken to minimize redundancy.

This guide is one of a series of guidance documents being
developed by EPA. It is a companion document to the generic
guide (52:i and the biodegradation screening  guide. (53) In an
effort to avoid redundancy, supporting information in these
and  other  readily  available  guidance  documents  is  not
repeated in this document.

Treatability studies for biodegradation are in their infancy.
Procedures for conducting mathematical modeling and for
performing field-  and  larger-scale  tests have  not been
standardized. There are numerous site-specific conditions that
can  impact biodegradation; many  of  these  cannot  be
accounted for or controlled during testing and/ or remediation,
and controversy exists concerning the usefulness of these
tools. The lack of consensus stems, in part, from uncertainties
associated with the use of in situ technologies.  In order to
thoroughly  address the  various design  considerations
associated with biological treatability studies, this document
provides guidance on the available alternatives,  including a
discussion of their relative advantages and disadvantages.
Hopefully, this information will provide a  sound basis for
approaching the treatability study process. This document is
not intended to serve as a substitute for communication with
regulators or experts in the field of biodegradation. This
document should never be the sole basis for the selection of
biodegradation as a remedial alternative or the exclusion of
biodegradation from consideration.

As treatability study experience is gained, EPA anticipates
furthercomments and possible revisions to this document. For
this reason, EPA encourages constructive  comments  from
outside sources. Direct written comments to:

        Mr. Edward Opatken
        U.S. Environmental Protection Agency
        Office of Research and Development
        Risk Reduction Engineering Laboratory
        26 W. Martin Luther King Drive
        Cincinnati, Ohio 45268
        (513) 569-7855
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                                                SECTION  2
                          TECHNOLOGY  DESCRIPTION  AND
                                  PRELIMINARY SCREENING
     This section presents a brief description of various full-scale
     biological treatment technologies and a discussion of the
     information necessary for prescreening the technology before
     committing to a treatability test program. Subsection 2.1
     describes  several types of full-scale remediation systems.
     Subsection 2.1 is divided into three additional subsections:
     the first  two subsections  address  in  situ and  ex situ
     technologies separately; the thirdsecti on discusses anaerobic
     applications. The distinction between in situ and ex situ is
     made in other sections throughout the document and reflects
     the significant differences that exist between in situ and ex
     situ treatment. Subsection 2.2 discusses available literature,
     databases, and technical assistance, and reviews field data
     necessary to prescreen  these  technologies.  Technology
     limitations are also reviewed in this subsection.

     2.1 TECHNOLOGY DESCRIPTION

     Bioremediation generally refers to the breakdown of organic
     compounds (contaminants) by microorganisms. Solid-phase,
     slurry-phase, soil heap bioremediation, bioventing in situ
     bioremediation, and composting technologies can be used to
     remediate  contaminated  soils biologically. (3o:i(74)  In situ
     technologies  encourage contaminant biodegradation by
     promoting biological activity (e.g.,  nutrient  and  oxygen
     availability)   without  relocating  the  impacted   media.
     Disadvantages associated with in situ treatment include a
     limited ability to control the sites-specific variables affecting
     biodegradation and  the potential for offsite contaminant
     migration. In contrast, ex situ techniques physically isolate
     the contaminants from the environment prior to or during
     treatment, thereby limiting the potential for contaminant
     migration  during treatment, while increasing the ability to
     control conditions that regulate biological degradation. These
     advantages  must be balanced against the high  costs
     associated with materials handling, space  requirements, and
     an increased potential for fugitive emissions  during media
     excavation and  transport. As  the  number  of variables
     requiring  control increases,  the more complicated (i.e.,
     problematic) implementation becomes. For example, it would
     be less problematic to implement a remedial design, which
     modified only one parameter during treatment (i.e., oxygen
     concentrations),  than an  application  that  required  the
     modification  of  multiple  factors  (e.g.,  pH,  oxygen
     concentrations, nutrients, microbes,  or buffering  agents).
     Biodegradation can be used as the sole treatment technology
     at a site  or in conjunction with other  technologies in a
treatment train. The technology shows promise for degrading
or transforming  a large  number of organic compounds
commonly   found  at   contaminated   sites  to
environmentally-acceptable  or  less  mobile  compounds.
Recently, biological mechanisms have been used to reduce the
toxicity of metals as well  as to increase metals recoveries.
Bioremediation has also been used to treat water contaminated
with nitrate, phosphate, and other inorganic compounds. These
applications, however, are not discussed  extensively in this
guide.

As of October 1992, approximately 149 CERCLA, Resource
Conservation and Recovery Act (RCRA), underground storage
tank (UST), and  other governmentally regulated sites have
been identified by EPA Regions and States as considering
(e.g., performing  treatability studies), planning, operating
full-scale,  or having  used  biological  treatment  systems.
Approximately 62 percent of the sites are CERCLA, 14 percent
are RCRA, and 10 percent are UST sites. The remaining 14
percent represent Toxic Substance Control Act (TSCA) and
other Federal and State efforts/41' Of the  149 sites discussed
above, approximately 27 percent are presently operational at
the full-scale level, and 14 percent have been completed.

At the end of EPA fiscal  year 1991 (FY91), there were 45
Superfund sites where bioremediation had been  selected.
These sites represent 9  percent of the total number of
Superfund sites (Figure 2-1).(59) Historically, bioremediation has
been  primarily  applied  at  sites  containing  petroleum
hydrocarbons, creosote, pesticides, herbicides, and solvents.
Bioremediation is presently being investigated at a number of
sites contaminated  with  explosives  and polychlorinated
biphenyls (PCBs). Although full-scale applications have yet to
occur, bioremediation has been selected in a number of RODs
as a potential technology for treating media contaminated with
explosives and PCBs. Full-scale applications are scheduled to
begin at these and other non-CERCLA sites in the near future.

Brief discussions of in situ and ex situ technologies follow. The
majority of the text in these  subsections was  adapted from
material presented in the biodegradation  screening guide.(53)
Except for the addition of two new subsections describing
bioventing and  biofilters,  and  a table (Table  2.1)  that
synopsizes  the   advantages,  disadvantages,  and  the
appropriate applications of  the different technologies, the
majority of the text in Subsections 2.1.1  (In Situ Biological
Technologies) and 2.1.2 (Ex situ
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                                                  Thermal Desorption (28) 6%
                                  Chemical Treatment (1) <1%
                               In situ Vitrification (3) <1%
                               Dechlorination (8) 2%,
              Soil Vapor Extraction (84) 17%
         In situ Flushing (16) 3%'
      Other Innovative * (3) <1%

               Offsite Incineration (85) 17%
        In situ Bioremediation ** (20) 4%
             Ex situ Bioremediation (25) 5%
                    Solvent Extraction (6) 1%
                           Soil Washing (16) 3%
                                                                                    Onsite Incineration (65) 13%
   Solidification/Stabilization (128) 26%
                                               Other Established* (10) 2%
                INNOVATIVE TECHNOLOGIES
                          (210)42%
        ESTABLISHED TECHNOLOGIES
                   (288)  58%
                             Total  Number of Technologies - 498
                                                                             ***
          NOTES:

          () Number of times this technology was selected or used.
          *  "Other" established technologies are soil aeration, in situ flaming, and chemical neutralization.  "Other" innovative
                technologies are air sparging and contained recovery of oily wastes.
          ** Includes nine in situ groundwater treatment remedies.
          *** Data are derived from 1982-1991 Records of Decision (RODs) and anticipated design and construction activities as of
                February 1992. More than one technology per site may be used.


            Figure 2-1. Superfund remedial actions: summary of alternative treatment technologies through FY91.
    Biological Technologies) underwent minor technical and
    editorial changes. Significant modifications, however, were
    made to Subsection 2.2.1 through 2.2.4. In Subsections 2.2.1
    (Literature/Database   Review)  and  2.2.2   (Technical
    Assistance), additional literature and database sources, as
    well as organizations to contact for technical assistance,
    were  recommended.  Subsections  2.2.3  (Prescreening
    Characteristics) and 2.2.4 (Technology Limitations) were
    completely rewritten, with significant changes made to the
    technical scope of these discussions.

    A series of engineering bulletins is being prepared by EPA's
    RiskReduction Engineering Laboratory (RREL) in Cincinnati,
    Ohio/45^46' Readers interested in more detailed discussions
    of certain biodegradation technologies are encouraged to
    utilize these documents. These bulletins provide additional
    information  on  certain  biodegradation technologies
    including  the applicability  of the technology, the most
    current performance data, the status of the technology, and
    sources for further information.
2.1.1  In Situ Biological Technologies

In situ biological technologies treat contaminants in place,
eliminating the need for soil excavation and limiting volatile
releases into the atmosphere. As a result, many of the risks and
costs associated  with materials handling are reduced  or
eliminated. Under some circumstances, these technologies can
be used to clean up soil contamination responsible for impaired
groundwater quality; they have been most frequently employed
to treat  soils with moderate to high permeabilities. In situ
biological technologies may enhance traditional pump and treat
technologies by reducing the time needed to achieve aquifer
cleanup standards.

In Situ Bioremediation

During in situ bioremediation,  contaminant biodegradation
within the subsurface soil  and water is enhanced
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                            Table 2-1. Comparison of Biological Remediation Technologies*.
      Technology
       Advantages
      Disadvantages
   Typical application
      In situ
      Bioventing
      Solid-phase
      Soil heaping
                                Inexpensive
                                low exposure risks
                                excavation not required
excavation not required
faster degradation than other in
situ technologies
simple procedure
inexpensive
currently accepted method

inexpensive
low degradation rates
control of operating parameters
is difficult
hydrological characteristics can
affect treatment

hydrological characteristics can
affect treatment
contaminant volatilization can
occur during treatment

some exposure risks**
some exposure risks**
                                                                saturated soils
                                                                aerobic or anaerobic
                                                                permeable soils
permeable soils
unsaturated soils
surface contamination
aerobic


surface contamination
aerobic
      Composting
inexpensive
self-heating
needs bulking agents
some exposure risks**
residual contamination
surface contamination
aerobic
      Slurry bioreactors
      Biofilters
good operational control
good microbe/compound
contact
enhanced desorption of
compound from soil
high degradation rates
can be operated cyclically
without loss in performance
can treat a heterogeneous mixes
of contaminants
high degradation rates
high capital outlay
limited by reactor size
some exposure risks**
prone to clogging
odors may result
filter media must be installed
by hand
air loading rates are low
surface contamination
recalcitrant compounds
soils that bind compound
tightly
aerobic or anaerobic

gaseous contamination
light aliphatic compounds
chlorinated aliphatic and
aromatic compounds
      *      Adapted from reference number 28.
      **     Fugitive emissions may occur during excavation.

     without using excavation. The technology usually involves
     enhancing natural biodegradation  processes by adding
     nutrients, oxygen (if the process is  aerobic),  and in  some
     cases,  microorganisms to  stimulate the biodegradation of
     contaminants. Moisture control may also be required to
     enhance biodegradation in unsaturated soils. If oxygen is the
     rate-limiting parameter, oxygen sources such as air, oxygen, or
     hydrogen peroxide (H2O2) may be used. If the percolation of
     aqueous amendments is being considered, rough calculations
     should be made to estimate  the amount of oxygenated water
     that will be reguired to mineralize the contaminants at the site
     (see  Subsection 4.2.7 for an expanded discussion of this
     analysis). This concept is equivalent to estimating biological
     or chemical oxygen demand (BOD or COD) and can be used to
     verify that sufficient oxygen (i.e., electron acceptor) will be
     present. Laboratory and field studies have indicated that the
     addition of methane or other primary substrates may aid in the
     co-metabolic   biodegradation  of  low  molecular weight
     chlorinated organics. Recent evidence suggests that anaerobic
     processes that use nitrate as  a terminal electron acceptor may
     be effective for the in situ treatment of benzene, toluene,
     xylenes,1-14-"-13-1 and some polynuclear aromatic hydrocarbons
     (PAHs).(17)

     In situ bioremediation is often used  in conjunction with a
                                  groundwater-pumping and soil-flushing system to circulate
                                  nutrients and oxygen through a contaminated aquifer and
                                  associated soils. The process usually involves introducing
                                  aerated, nutrient-enriched water into the contaminated zone
                                  through a series of injection wells or infiltration trenches and
                                  recovering the  water downgradient. Highly water-soluble
                                  contaminants are usually flushed  out  of a permeable soil
                                  before significant biodegradation  can occur; less soluble
                                  contaminants usually  remain  in   the  soil  and  may  be
                                  biodegraded.  The recovered water can then be treated,  if
                                  necessary, and reintroduced to the soil on site or discharged
                                  to the surface (Figure 2-2). Whether amendments can stimulate
                                  in situ biodegradation  depends in part  on contaminant
                                  accessibility. Water table fluctuations within the treatment
                                  zone can impact in situ  bioremediation by affecting critical
                                  factors  such  as: nutrient  and oxygen  concentrations,  air
                                  permeability, contaminant distribution, moisture content, and
                                  microbial composition. A low permeability soil (low hydraulic
                                  conductivity) can hinder the movement of water, nutrients,
                                  and, to a  lesser extent oxygen, through the  contamination
                                  zone. The soil' s hydraulic conductivity must be low enough to
                                  allow the  microbes sufficient time to incorporate  needed
                                  nutrients as amendmentladen water percolates through the
                                  soil.  Hydraulic conductivity  also  affects  delivery  of
                                  aqueous-phase electron
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      acceptors (e.g., hydrogen peroxide and nitrate). Variable
      hydraulic  conductivities  in  different soil strata within a
      contaminated area can complicate the design of flow  control.
      The ability to reinject  or  discharge  water to the surface is
      dependent upon local  regulations. Recovered groundwater
      may  require  pretreatment followed  by discharge  to  a
      publicly-owned treatment works (POTW).

      Except for bioventing, in situ technologies have primarily been
      used to  treat saturated  soils. Generally unsaturated soil
      treatment  has  been limited to fairly shallow regions over
      groundwaterthat is already contaminated. The bioremediation
      of contaminants present in unsaturated soils has been limited,
      in  part  due to difficulties associated with ensuring that
      sufficient time is available for microbes to utilize amendments
      present within the percolating water. Attempting to overcome
      these difficulties by increasing the flow of amendment-laden
      water into the soil can lead to a decrease in the soil's air
      permeability. This decrease in permeability is associated with
      increased soil  saturation and inhibited  gaseous oxygen (air)
      delivery. Futhermore, since the solubility of oxy gen in water is
      limited (i.e.,  less than 8 mg/L  at 20°C), in most  situations
      oxygenated water will be unable to meet oxygen requirements.
      Thus, in order to operate effectively, percolation techniques
      used to introduce amendment-laden water to the soil should be
      combined with air injection or vacuum extraction techniques
      used to  oxygenate the unsaturated soil.  Alternate electron
      acceptors may be utilized as an option. It should be noted, in
 situations where underlying groundwater is not contaminated,
the risk of contaminating the groundwater by infiltration from
the overlying treatment zone often limits the application of
bioremediation to the unsaturated zone.

Bioventing

In situ bioventing uses relatively low-flow soil aeration techniques to
enhance the bioremediation of soil contaminated  with organic
contaminants. Aeration systems  similar to those employed during
soil vapor extraction are used to supply oxygen to the soil. Typically
a vacuum extraction, air injection, or combination vacuum extraction
and air injection system is employed(77) An air pump, one or more air
injection or vacuum extraction probes, and emissions monitors at the
ground surface  (Figure 2-3)  are commonly used.  Although no
peer-reviewed data have been released on the use of vapor-phase
nutrients (e.g., ammonia and phosphorus) at least one vendor has
developed  a system designed to provide these nutrients to  the
subsurface.(77) However, in most field applications to date, nutrient
additions have been found to provide no additional benefits.*78'

In general, low air pressures and airflow rates are used to maximize
biodegradation while minimizing contaminant volatilization. Some
systems, however, utilize higher air flow rates, thereby combining
bioventing with soil vapor  extraction. (36>(39X41>(74> Although  the
technology is predominantly used to treat reasonably permeable
unsaturated soils,  research  is  being  performed  regarding  its
applicability to less permeable soils, saturated soils, and ground-
                                               Nutrients
                                                 Aeration
                                                     Microorganisms
                                                                          Treatment
                                                                        (If Necessary)
                                      ;::5^3£^
                                      •';...:•;; Groundwater Flow Direction ,:
                                      •"^^^^^"•^^""^^"™—™^H"^™T^-"™^-^^™T^^™^p"™™*l^™ii'^T^^^^y^
                                                     ...         ..
                                                    l Low Permeability Bedrock
                              Figure 2-2. In situ bioremediation of saturated soils and groundwater.
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                                                      Low rate
                                                     air injection
                   Surface monitoring
                         to ensure
                       no emissions
                Monitoring of soil
              gas to assess vapor
                 biodegradation
                 Biodegradation of
                 contaminated soils
              Biodegradation of vapors
                                                       Figure 2-3. Bioventing.
     water (using air sparging techniques).*22' A Test Plan and Technical
     Protocol for a Field Treatability Test for  Bioventing has  been
     developed  by the U.S. Air Force.(35) This document has  been
     reviewed and is supported by EPA.

     2.1.2  Ex Situ  Biological Technologies

     Solid-Phase Bioremediation

     Solid-phase bioremediation (sometimes referred to as land treatment
     or landfarming) is a process that treats soil in above-ground treatment
     systems using conventional soil management practices to enhance the
     microbial degradation of contaminants. Solid-phase bioremediation,
     in many instances, can be performed without triggering land disposal
     restrictions  (LDRs).  Subsection  3.2   further  discusses  the
     applicability of LDRs to bioremediation projects.

     Solid-phase bioremediation  at  CERCLA sites  usually  involves
     placing excavated soil in an above-grade soil treatment area (Figure
     24). If required, nutrients and microorganisms are added to the soil,
     which is tilled at regular intervals to improve aeration and contact
     between the microorganisms and the contaminants. During the
     operation of a solid-phase bioremediation system, pH, temperature,
     nutrient concentrations, and moisture content are maintained within
     ranges conducive to microbial  activity (optimal  ranges for  these
     parameters are discussed in Subsection 4.2). If necessary, highly
     contaminated soil can be mixed with less contaminated soil from the
     same site to reduce the contaminant concentrations to levels that do
     not inhibit microbial activity.  Depending  on the nature of the
     contaminant, the type of soil, and a number of other site-specific
     factors, mixing may not reduce toxicity at a micro-environment level.
     Addition-
ally, regulatory approval may be required before a less contaminated
soil may be mixed with a more highly contaminated soil. Solid-phase
treatment systems can be modified  to contain and to treat soil
leachate by adding underdrain and liquid treatment systems. Volatile
organic compounds (VOCs) can be contained by adding an optional
cover. Conventional VOC treatment can be added as part of a
treatment train.

A variety of processes in addition to bioremediation influence the fate
of contaminants during solid-phase treatment. These include physical
and chemical processes such as leaching, adsorption, desorption,
photodecomposition, oxidation, volatilization, and hydrolysis. The
physical and chemical properties of the contaminants interact with
site-specific variables (i.e., soil properties) to influence the fate of the
contaminants. The contaminants may  be degraded or transformed to
environmentally-acceptable or less mobile compounds.(19) While
most of these reactions occur in the top 6 to 12 inches of the
treatment zone, some contaminant decomposition and immobilization
occurs within underlying layers.

Soil Heap Bioremediation

Soil heap bioremediation, which is very  similar in  nature  to
solid-phase  bioremediation, involves piling contaminated  soil  in
heaps up to several meters high (Figure 2-5). If required, aeration is
usually provided by pulling a vacuum through the heap.  Simple
irrigation techniques are generally used to maintain moisture content,
pH, and nutrient concentrations within ranges conducive to the
biodegradation of contaminants. The system can be designed to
control the release of VOCs by passing the exhaust from the vacuum
through activated carbon  or biofilters. Moisture control and flow
rates can be varied to favor biodegradation rather than volatilization.
Simi-
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                             Excavation
                            So/7 Screening
            Microorganisms
            Nutrients
            Aeration  I

                 i   I
        (Optional)
 Plastic Film Greenhouses
                  Leachate Collection System
                                     Figure 24. Solid-phase bioremediation.
                       Visqueen Cover
                               \
                  Asphalt
                                                   L.
                         Soil        Nutrients
                                    Aeration
                           N       Microorganisms
7
                                           Side View        Plastic Piping
                                                             (compatible with contaminants)
                                                                    S.  •!"«*
                                           Top V/ew
                                      Figure 2-5. Soil heap bioremediation.
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      lar modifications are employed during bioventing (Subsection 2.1.1).

      Composting

      Like soil heap bioremediation, composting is similar to solid-phase
      bioremediation. In contrast, composting technologies typically employ
      a bulking agent and encourage the thermophilic degradation of the
      contaminants of interest.

      Composting involves the storage of biodegradable waste with a bulking
      agent (e.g., chopped hay or wood chips). The structurally-firm bulking
      agent is usually biodegradable. Typically, two parts bulking agent are
      mixed with  one  part  contaminated  soil  to  improve  the  soil
      permeability. Adequate aeration; optimum temperature, moisture, and
      nutrient concentrations; and the presence of an appropriate microbial
      population are necessary to enhance the decomposition of organic
      compounds. The biodegradation process may be thermophilic. If so,
      microorganisms that occur naturally in the decaying organic matter
      may biodegrade the contaminants of concern. However, the elevated
      temperatures associated with thermophilic biodegradation may limit
      the activity of nonthermophilic indigenous and exogenous organisms.
      Althoughbiodegradation is usually the  mechanism through which
      contaminant reduction is sought, some contaminants (e.g., nitroaroma
      tics) or their degrada tion products may be strongly adsorbed on humic
      materials with covalent bonds, limiting their environmental mobility
      and thus reducing the potential for exposure(20)(29)(34)

      The three basic types of composting are open windrow systems, static
      windrow systems, and in-vessel (reactor)  systems. In the open
      windrow system, the compost is stacked into elongated piles (Figure
      2-6). Aeration is
accomplished by tearing down and rebuilding the piles. In the static
windrow system, piles of compost can be aerated by a forced air
system (the piles are built on top of a grid of perforated pipes). The
in-vessel system involves placing the compost into a closed reactor.
Aeration is accomplished by tumbling, stirrin& and forced aeration.
Like soil heap bioremediation and solid-phase bioremediation, pH,
microbial, and nutrient supplementation, as well as fugitive emission
control, may be needed depending on the types and concentrations
of contaminants present in the soil.

Slurry-Phase Bioremediation (Liquid/
Solids Treatment)
In slurry-phase bioremediation, excavated contaminated soil is
typically combined  with water  and then placed in an onsite,
stirred-tank reactor(s) where the soil is combined with water to
form a slurry. The solids content of the slurry depends on the type
of soil, the type of mixing and aeration equipment available, and the
rates  of  contaminant removal  that  need  to   be achieved.
Contaminated  surface or groundwater may  be used as makeup
water, enabling slurry-phase units  to alleviate water and  soil
contamination  problems  simultaneously.  Depending  on  the
characteristics  of the soil, it may be directly fed into the slurry
system, pretreated  to remove  contaminants not amenable to
biodegradation, or pretreated using soil washing to achieve  a
significant reduction in the volume of material requiring treatment.
If required,   nutrients,   pH  amendments,  and/or  microbial
supplements may be added to the slurry. The  slurry is then aerated
and/or agitated to facilitate the  aerobic  biodegradation  of the
contaminants.   This  encourages  efficient  biodegradation  by
promoting  contact between contaminants,  microbes, nutrients,
carbon sources, water, and electron acceptors.
                       Windrow
                                               Figure 2-6. Open windrow composting.
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      The process can be operated in either a batch or a continuous mode
      (Figure 2-7).

      As with solid-phase bioremediation, the process can be designed to
      contain and treat VOCs. Additionally, slurry-phase bioremediation
      systems may be used to  treat sludges and sediments in existing
      lagoons and impoundments, thus eliminating the need for excavation
      (Figure 28). In such systems, an impermeable layer should be present
      under the slurry-phase system to prevent contaminant migration.

      Biofilters

      Microorganisms can  also be  used  to treat  organic  vapors  by
      employing biofilters.  Biofilters  operate in a manner similar  to
      processes used to biologically treat wastewater(e.g., trickling filters).
      As with theseprocesses, biofilters provide bacteria withasurface on
      which to  grow. Oxygen concentrations,  temperature,  nutrient
      concentrations, moisture levels, pH, and carbon levels are adjusted to
      optimize  contaminant degradation, resulting in  significant vapor
      phase contaminant reductions. The primary components of biofilters
      are: an air blower, an air distribution system, a moisturizing system,
      filter media, and a drainage system (See Figures 2-9 and 2-10). The
      technology is considered very effective in removing light aliphatic
      compounds (e.g., propane and isobutane) with removal efficiencies
      in the range of 95 to 99 percent. Chlorinated aliphatic and aromatic
      compounds can also be removed using biofilters, however somewhat
      lower removal rates have been reported.'16'
                          2.1.3  Anaerobic Biomediation
                                  Applications

                          The in situ and ex  situ technologies described in the previous
                          subsections normally function under aerobic conditions. However,
                          anaerobic biological processes can be applied to either in situ or ex
                          situ technologies. The application of nutrients and moisture and the
                          control of pH are common elements of anaerobic and aerobic
                          systems. Anaerobic systems use chemical oxygen sources as electron
                          acceptors. Oxygen is normally  limited for in situ systems eitherby'
                          natural conditions orby artificial means (surface flooding or other
                          surface barriers). If oxygen penetration is limited and a readily
                          degradable  substrate  is present,  indigenous  microorganisms will
                          rapidly  deplete the available  oxygen. The effectiveness of such
                          oxygen barriers will be limited until the oxygen content of the soil or
                          groundwater  is depleted.  Limiting oxygen  levels is easier  to
                          accomplish in ex situ (e.g., slurry reactors) than in situ applications.
                          Please note, however, that some processes may be micro-aerophilic
                          and will not work under strict anaerobic conditions. A number of
                          papers are available describing anaerobic slurry phase processes.(10)(28)

                         Anaerobic organisms can be facultative (organisms which can grow
                          either in the presence or the absence  of oxygen) or obligate
                          (organisms  which  grow  only  under  anaerobic conditions).
                         Denitrifying bacteria are typically aerobic organisms which utilize
                         nitrate  as  an electron acceptor  in the  absence of  oxygen.
                          Sulfate-reducing bacteria are strict anaerobes which utilize as electron
                         acceptors either eletion
                                Excavation
                                                  Soil Screening
                                    Water Recycle
                  Dewatered
                    Solids
                             L
J
                                                          Nutrients
                                                          pH Adjustment
                                                          Aeration
                                                          Microorganisms
                                                                                                      IIH
                                                         I
                               Dewatering
                                 Slurry Bioreactors
                                       Figure 2-7. Above-ground slurry-phase bioremediation.
                                                                 10
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Nutrients
   pH Adjustment
       Aeration
          Microorganisms

                Mixer
                                                         Mixer
                  Mixer
                                             Water
                                                ttWviv&iS-;'
                                                udge
                                                                                                Impermeable
                                                                                                Liner
                             Figure 2-8. Slurry-phase bioremediation in existing lagoon.
    mental sulfur or sulfur compounds. Methanogenic bacteria are
    obligate anaerobes which utilize carbon sources and produce
    methane gas. The following paragraphs discuss each of these
    groups of anaerobic organisms.

    Facultative anaerobic microorganisms have the ability to grow
    in the presence or absence of oxygen. In the presence of
    oxygen, the organisms are able to use oxygen as the terminal
    electron acceptor. In the absence of oxygen,  an alternative
    electron acceptor is utilized. Growth rates, biomass production,
    and metabolic rates are lower under anaerobic conditions.
    Alternative  electron acceptors may  be organic acids or
    inorganic  molecules such as  nitrate (in the case  of the
    denitrifying  bacteria).   Generally,   these  organisms  are
    heterotrophic in nature, and able to utilize a wide variety of
    carbon sources under aerobic or under anaerobic conditions.
    However, the  pathways used and metabolic  intermediates
    produced may  differ under aerobic and anaerobic conditions.

    Denitrifying bacteria utilize nitrate as an electron acceptor in
    the absence  of oxygen.(62) The majority of these organisms are
    classified as aerobic bacteria, since they are primarily found in
    oxygen-containing  environments.   The   ability  of  the
    denitrifying  bacteria to grow  under  essentially anaerobic
    conditions allows the use of an additional pool of metabolic
    activities for bioremediation. These microorganisms express
    alternative pathways, in many instances, for the degradation
    of organic compounds under  denitrifying conditions. For
    example, under either aerobic or denitrifying conditions,  a
    species  of  Pseudomonas  was  able  to  utilize  o-,  m-,
    p-phthalates;  benzoate;  cyclohex-1-ene carboxylate;  and
    cyclohex-3-carboxylate. However, m-hydroxybenzoate  and
    phydroxybenzoate could onlybe utilized under denitrifying
    conditions. This allows the consideration of reactors that use
    both aerobic and denitrifying strategies to expand the range of
    compounds  that  are  degradable  by  a  given microbial
    consortium.
Sulfate-reducing bacteria utilize sulfate, elemental sulfur, or
reduced sulfur compounds as electron acceptors. The product
of these energy reactions is hydrogen  sulfide (H2S). The
typical environments are mud and  sediments, which are
anaerobic, as well as the internal tracts of humans.  These
organisms utilize a variety of carbon sources, but many are not
degraded to  CO2; that is, very few are  mineralized. The
potential value of these organisms may be in their ability to
attack sulfur-containing compounds or in the treatment of
sulfate-  or sulfur-containing wastes.

The methanogenic bacteria are obligate anaerobic bacteria.
They have been utilized by the waste treatment industry for a
number of years. One group of these organisms is capable of
using hydrogen and CO2 for the production of methane.
Another group can ut Jize acetate for the formation of methane.
Generally, the methanogenic bacteria are found as part of a
consortium   composed  of  heterotrophic  organisms,
hydrogen-producingorganisms, and the methanogens. The
heterotrophic anaerobes degrade  available organic carbon
sources  to CO2  or acetate through a  series of reactions
involving a number of bacteria. This includes organisms that
will reduce organic acids to CO2 and acetate.  The hydrogen-
producing bacteria are  essential to, and generally occur in
close association with, the methanogens. The activity of the
methanogdns has been closely studied over the years because
of the value as a fuel source of the methane produced.

2.2     PRELIMINARY SCREENING AND
         TECHNOLOGY LIMITATIONS

The determination of the need for and the appropriate level of
treatability studies is dependent on available literature, expert
technical judgment, and site-specific factors. The first two
elements, the literature search and expert consultation, are
critical  to determining if adequate data are available or if a
treatability study is needed for decision-making.
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                                                                    Treated Vapors
                  Air
                Blower
Water
Pump
A  A           Sprinkler

7  ( ^---^£^~:." --.--.   )  t  t
   y:r/,f  ,r=^gr-*T-s  \    \ <  <   \
                                                  Biofiiter Media
                      Waste Air
                                     onnnnnnnnnnnnnnnnnoo on  /   Gravel Bed
                  In    o   o
                               n
                                                                                   Liner
                                            Air
                                        Distribution
                                           Ports
                                    Drainage
                                     Pipes
                                   Figure 2-9.  Earth biofilter treatment
         Biological
         Filter
         Section
         Distribution
         Section
                                              Excess Water Drainage
                                                                                 =0-
                                                          Blower
                                                         Exhaust to
                                                        Atmosphere
                                 Figure 2-10. Biofilter/Biotower treatment.
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     2.2.1 Literature/Database Review

     The following reports  and electronic  databases can  be
     consulted  when  planning  and conducting  treatability
     studiesand when prescreening bioremediation  for use at  a
     specific site. Existing reports include:

     •    Guide  for  Conducting  Treatability  Studies  Under
         CERCLA, Final. U.S. Environmental Protection Agency.
         EPA/540/R-92/071a, October 1992.(52)

     •    Guidance for Conducting Remedial  Investigations and
         Feasibility Studies Under CERCLA,  Interim Final. U.S.
         Environmental Protection Agency. EPA/540/G-89/004,
         October 1988.(51)

     •    Guide  for  Conducting  Treatability  Studies  Under
         CERCLA: Aerobic Biodegradation Remedy Screening,
         Interim Guidance. U.S. Environmental Protection Agency.
         EPA/540/2-91/013A, July 1991.(53)

     •    Superfund Treatability Clearinghouse Abstracts. U.S.
         Environmental Protection Agency. EPA/540/2-89/ 001,
         March 1989.(72)

     •    The Superfund  Innovative  Technology  Evaluation
         Program:  Technology  Profiles  Fifth  Edition.  U.S.
         Environmental Protection Agency. EPA/540/R-92/ 077,
         December 1992.(74)

     •    Summary  of TreatmentTechnology  Effectiveness  for
         Contaminated   Soil.  U.S.  Environmental  Protection
         Agency. EPA/540/8-89/053, June 1990.(71)

     •    Inventory of Treatability Study Vendors, Volumes I and II.
         U.S.  Environmental  Protection   Agency.   EPA/
         540/2-90/003a and b, March 1990.(61)

     •    Bioremediation in the Field. U. S. Environmental Protection
         Agency. EPA/540/N-92/004. (Published Quarterly)(41)

     •    Bioremediation of Contaminated Surface  Soils. U.S.
         Environmental  Protection Agency. EPA/600/2-89/073,
         August 1989.(42)

     •    Innovative Treatment Technologies: Semi-Annual Status
         Report.  U.S.  Environmental   Protection  Agency.
         EPA/542/R-92/011, October 1992.(59)

     •    User's Guide for Land Treatment-Compound Property
         Processor and Air Emissions Estimator (LAND7). U.S.
         Environmental  Protection Agency. EPA/540/3-87/026,
         November 1989.(76)

     •    Hazardous Waste Treatment,  Storage,  and Disposal
         Facilities  (TSDF)  -  Air   Emission  Models.  U.S.
         Environmental  Protection Agency. EPA/450/3-87/026,
         November 1989.(56)

     •    Innovative Hazardous Waste Treatment Technologies: A
         Developer's Guide to Support Services, Second Edition.
         U.S.   Environmental   Protection   Agency.
         EPA/540/2-91/0-12, June 1992.(57)

     •    Federal Remediation Technologies Roundtable. Federal
         Publications on Alternative and InnovativeTreatment
         Technologies for Corrective Action and Site Remediation,
    Second Edition. U.S. Environmental Protection Agency.
    EPA/542/B-92/001, August 1992.(49)

•   Innovative Treatment Technologies: Overview and Guide
    to Information Sources. U.S. Environmental Protection
    Agency. EPA/540/9-91/002, October 1991.(58)

Currently, RREL in Cincinnati, Ohio is expanding the RREL
Treatability Data Base. This expanded database contains data
from soil treatability studies. In addition, a repository for the
treatability study reports will be maintained at the Water and
Hazardous Waste Research Division of RREL in Cincinnati.
Contact Glenn Shaul in the Toxics Control Branch of RREL at
(513) 569-7408 regarding this database.

Robert S. Kerr Environmental Research Laboratory (RSKERL)
in Ada, Oklahoma is presently developing the Subsurface
Remediation Technology (SRT) Database, which will provide
site-specific information concerning subsurface contamination
and  remediation  activities currently  being proposed  or
conducted at hazardous waste sites throughout the United
States. The SRT Database will be available in early 1993 by
way of an electronic bulletin board system (BBS) operated by
RSKERL  or via the local area networks (LANs) a,t the EPA
Regional Offices. RSKERL has also developed a Soil Transport
and  Fate (STF)  Database  which  presents  information
concerning the behavior of organic and inorganic chemicals in
soil environments.  This database is packaged with a Model
Management System, which consists of the Vadose Zone
Interactive Processes (VIP) Model and the Regulatory and
Investigative  Treatment Zone  (RITZ)  Model. Additional
information on the  SRT and STF Databases as well as the
Model Management System can beobtained by calling Dr.
David  Burden at (405) 456-8500. His office is located in
RSKERL'sCenterfor Subsurface Modeling Support (CSMoS)
of the Application and Assistance Branch.

The Office of Solid Waste and Emergency Response (OS WER)
maintains the  Cleanup  Information (CLU-IN)  BBS  for
communicating ideas and disseminating information and to
serve as  a gateway to other OSWER electronic databases.
Currently, the CLU-IN BBS has eight different components,
including  news  and mail services  and conferences and
publications on specific technical areas. The contact is Dan
Powell, (703) 308-8827, of OSWER's Technology Innovation
Office (TIO).

TIO has also developed the Vendor Information System for
Innovative Treatment Technologies (VISITT) Database. This
database contains information provided to TIO by technology
developers, manufacturers, and suppliers regarding innovative
technologies for hazardous waste site remediation. To obtain
technical assistance or a copy of VISITT, call the VISITT
Hotline at (800) 245-4505 or (703) 883-8448.

The Office of Research and Development (ORD) headquarters
maintains the Alternative Treatment Technology Information
Center (ATTIC), which is a compendium of information from
many  available  databases. Data relevant to  the  use  of
treatment technologies in Superfund actions are collected and
stored in ATTIC. ATTIC searches other information systems
and databases and integrates the information into responses.
It also includes a  pointer system that refers the user  to
individual experts in EPA. The system currently encompasses
technical summaries for the Superfund Innovative Tech-
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     nology Evaluation (SITE) Program,  treatment technology
     demonstration  projects,  industrial  project  results,  and
     international program data.  Contact the ATTIC  System
     Operator at (301) 670-6294  or access the database with  a
     modem by calling (301) 670-3808.

     Several other databases also provide information that may be
     useful during bioremediation remedy selection treatability
     studies.  The Hazardous Waste Superfund Collection Data
     Base  (HWSFD)  contains  bibliographic references  and
     abstracts pertaining to the documents in the Hazardous Waste
     Superfund Collection at the EPA Headquarters Library. User
     support  for this database  can be  obtained by calling (800)
     334-2405. The National Technical Information Service (NTIS)
     Bibliographic Data Base is the largest single source for public
     access to federally-produced information. This database is
     available  to the public through  a number of  commercial
     vendors including the following: BRS, (800) 345-4277; CISTI,
     in  Canada, (613) 993-1210; DATA-STAR, (800) 221-7754;
     DIALOG, (800) 334-2564; ORBIT, in Virginia, (703) 4420900, and
     in the rest of the U.S., (800) 456-7248; and STN International,
     (800) 848-6533. The Records of Decision System (RODS) is an
     online database containing the full text of the Superfund RODs
     for National Priorities List  (NPQ sites nationwide. Contact the
     RODS Help Line (202) 260-3770 for assistance.(48)

     Finally, the RREL  Technical Support Branch is supporting a
     variety of treatability-related activities, including development
     of this  guide  and  other  technology-specific  guidance
     documents, preparation of engineering bulletins, compilation
     of a list of vendors who  perform treatability studies, and
     performance of treatability studies for EPA Regions.

     2.2.2 Technical Assistance

     Technical assistance can be obtained from the Technical
     Support Project (TSP) Team, which is made up of a number of
     Technical Support Centers (TSCs). It is a joint service of
     OSWER, ORD,  and EPA Regions. The  TSP offers direct,
     site-specific technical assistance  to OSCs and RPMs and
     develops technology workshops, issue papers, and other
     information for EPA Region staff. The TSP:

     •   Reviews contractor  work plans, evaluates  remedial
        alternatives,  reviews  RI/FS  reports,  and assists in
        selection and design of a final remedy

     •   Offers modeling assistance,  data analysis,  and data
        interpretation  Assists in developing  and evaluating
        sampling plans

     •   Conducts field  studies  (soil gas, hydrogeology, site
        characterization)

     •   Develops technical workshops and training, issue papers
        on groundwater topics, and generic protocols

     •   Assists in performance of treatability studies

     For further information on the TSP, contact:
             Technology Innovation Office
                Contact: Richard Steimle
                     (703) 308-8846

The following support centers provide technical information
and advice related to biodegradation and treatability studies:

1.       Ground-Water  Fate   and Transport Technical
        Support Center
        Roberts. Kerr Environmental Research Laboratory
        Ada, OK
        Contact: Don Draper
                (405) 332-8800

    RSKERL in Ada, Oklahoma, is EPA's center for fate and
    transport research, focusing its efforts on transport and
    fate of contaminants in the vadose and saturated zones of
    the subsurface, methodologies relevant to protection and
    restoration  of  groundwater quality, and evaluation of
    subsurface  processes for the treatment of hazardous
    waste. RSKERL provides technical assistance such as
    evaluating  remedial alternatives,  reviewing RI/FS and
    RD/RA Work Plans, and providing technical information
    and advice.

2.       Engineering Technical Support Center (ETSC)
        Risk Reduction Engineering Laboratory
        Cincinnati, OH
        Contact: BenBlaney or Joan Colson
                (513) 569-7406 or (513) 569-7501

    ETSC provides technical information and advice related to
    treatability  studies. The ETSC is sponsored by OSWER
    but operated by RREL; it handles site-specific remediation
    engineering problems. Access to thissupport center is
    available through the EPA site Project Managers.

    RREL offers expertise in contaminant  source control
    structures;  materials  handling  and  decontamination;
    treatment of soils, sludges, and sediments; and treatment
    of aqueous and organic liquids.  The  following are
    examples of the technical assistance that can be obtained
    through the ETSC:

        •   Review of the treatability aspects of the RI/F S

        •   Review of RI/FS treatability study Work Plans
            and final reports

        •   Oversight of RI/FS treatability studies

        •   Identification of alternative remedies

        •   Assistance   with   studies   of  innovative
            technologies

        •   Assistance in full-scale design and startup

3.   Emergency Response Team (ERT)
    Technical Support Center (TSC)
    Office of Emergency and Remedial Response
    (OERR) Branch
    Edison, NJ  Contact: Joseph Lafornara
                    (908) 321-6740
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         The ERT  TSC  is located at the OERR Environmental
         Response Branch in Edison, New Jersey. ERT provides
         technical  expertise  for  the  development  and
         implementation  of innovative  treatment technologies
         throughits Alternative Technology Section. The following
         are examples of the types of technical assistance that can
         be obtained through ERT:

         •    Consultation on  water and  air  quality criteria,
             ecologicalrisk assessment, andtreatability study test
             objectives

         •    Development and  implementation of sitespecific
             health and safety programs

         •    Performance of in-house  remedy screening and
             remedy selection treatability  studies of chemical,
             physical,  and biological treatment technologies

         •    Sampling and analysis of air, water, and soil

         •    Provision of onsite analytical support

         •    Oversight of treatability study performance

         •    Interpretation and evaluation of treatability study
             data

     In  addition to the TSCs,  the  Gulf Breeze  Environmental
     Research Laboratory in Gulf Breeze, Florida provides technical
     assistance  to  EPA  Regions.  Research  interests include
     biodegradation and bioremediationof pesticides,  petroleum
     hydrocarbons, PAHs, and chlorinated solvents. Contact Rick
     Cnpe at (904) 934-9261 for further information.

     2.2.3   Prescreening Characteristics

     Before a treatability study is conducted, a literature search
     should be performed to confirm whether the compounds of
     interest are known to be amenable to biological  treatment.
     Evidence of biodegradation under dissimilar conditions,  as
     well as data relating to compounds of similar structure,  should
     be  considered.  If  preliminary  research  indicates that
     bioremediation is  a poor candidate for selection,  further
     research may be warranted. Expert recommendations regarding
     the technology's  potential should be  obtained  before
     eliminating bioremediation from further consideration. Caution
     should  also  be   employed  when  reviewing studies
     demonstrating the degradation of pure chemicals. Chemical
     interactions or inhibitory effects of contaminants can alter the
     biodegradability  of  chemicals  in   the  complex  mixtures
     frequently  found  at  Superfund sites. Particular  attention
     should also be paid to degradation products, since they may
     be as toxic or more toxic than the parent compound. Studies
     reporting the disappearance of a specific compound as a
     measure  of biodegradation can be misleading since in some
     instances  disappearance  may occur concomitantly  with
     transformation to a more toxic compound. An example is the
     conversion  of   the  relatively   non-toxic  herbicide
     2,4-dichlorophenoxyacetic  acid  (2,4-D)  to the mutagenic
     compound 2,4,-dichlorophenol by a genetically engineered soil
     organism.1-31'

     Several documents and review articles that present detailed
information on contaminant biodegradability are listed in
Section 7, References.  However, discretion  should  be
exercised  when  using  these  reference  materials,  since
conditions that  allow the biodegradation of compounds
traditionally  considered nonbiodegradable  are continually
being discovered through ongoing research and development
efforts. Examples of classes of compounds thatareamenable to
bioremediation include?11) (MJPIJPSJPTXIH)

•   Petroleum hydrocarbons (e.g., gasoline and diesel fuel)

•   Nonchlorinated solvents (e.g., acetone,  ketones,  and
    alcohols)

•   Wood-treating   wastes   (e.g.,   creosote   and
    pentachlorophenol)

•   Aromatic compounds (e.g., benzene, toluene, xylenes, and
    phenols)

•   Some  chlorinated  aromatic    compounds   (e.g.,
    chlorobenzenes, biphenyl with fewer than  five chlorines
    per molecule)
The literature search should also investigate the chemical and
physical  properties  of  the  contaminants,  particularly
contaminant volatility,  solubility, and biological availability
(i.e., how strongly the contaminant is sorbed to the soil) in
order to  assess their  impacts on contaminant  removal.
Information  regarding  site conditions  and soil properties
should  be  compiled,   A  partial  list  of  site   and  soil
characteristics that can impact bioremediation are presented in
Table 2-2.(42:i The physical/chemical parameters  of the media
should  also  be  determined; these  include, salinity, total
organic carbon (TOC), oxygen availability, moisture content,
temperature, available electron acceptors, and  the presence
and chemical state of metals, especiallyiron. When possible,
data  should  be gathered from previous site characterization
efforts. If the quality of these data is questionable, it may be
necessary to perform preliminary testing. The  utility  of the
data  must be balanced  against the testing costs and time
considerations.

Furthermore, since biodegradation may not be able to reduce
contamination to target  levels within practical  time frames,
alternative technologies may be required to  supplement
biological treatment as part of a treatment train.

There is no steadfast rule that specifies when to  proceed with
remedy  screening,  when to eliminate biodegradation as  a
treatment technology, or when to proceed to remedy selection
testing based on a preliminary screening analysis. An analysis
of the existing literature coupled with the site characterization
will provide  the  information required to make  an educated
decision. However, when in doubt, a remedy screening study
is recommended. Several guidance documents are available to
aid   in   determining  the  keycontaminant   and  matrix
characteristics  that  are  needed  to  prescreen  various
technologies.^7-"-52-"-54-1 Example 1 is  a hypothetical literature
search provided to illustrate some of the complexities of this
analysis.
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                      Table 2-2. Site and Soil Characteristics Identified as Imoortant in Biological Treatment
                                                                                    In situ
                                           Ex situ
      Soil type

      Extent of contamination

      Soil profile properties

          Boundary characteristics
          Depth of contamination
          Texture*
          Structure
          Bulk density*
          Clay content
          Type of clay
          Cation exchange
          Organic matter content*
          pH*
          Redox potential*

      Hydraulic properties and conditions

          Soil water characteristic curve
          Field capacity/permanent wilting point
          Water holding capacity*
          Permeability*  (under saturated and 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

      Meterological and climatological data

          Wind velocity  and direction
          Temperature
          Precipitation
          Water budget
                     X

                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
                     X
X

X
X

X
X
X
X
X
X
X
X
X
X
     : Factors that may be managed to enhance soil treatment.
     2.2.4 Technology Limitations

     Many factors impact the feasibility of biodegradation. These
     factors should  be  addressed prior to the  selection  of
     biodegradation and prior to the investment of time and funds
     in further testing. Some of these factors are discussed in this
     section. A detailed discussion of these factors is beyond the
     scope of this document. The reader should consult references
     37, 42, 45, and 46,  and others, for more information.

     The physical form in which the contaminants are distributed
     within the media, as well as the amount, location,
and extent of the contamination, can have a profound impact
on the viability of bioremediation. In general, contaminants
may be dissolved in the groundwater, adsorbed onto thesoil,
absorbed into thesoil, or, depending on contaminant solubility
and density, distributed as "free product" or non-aqueous
phase liquid (NAPL). NAPLs can occur either on the top of the
watertable [e.g., light non-aqueous phaseliquids (LNAPLs) or
"floaters"] or at the bottom of the aquifer, against the bedrock
or some  other  impervious geologic structure [e.g., dense
non-aqueous phase liquids (DNAPLs) or "sinkers"). The
distribution of  contaminant into these different  phases is
ultimately a function of their physical and chemical prop-
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                                                       Example 1

        Soil and groundwater at a chemical manufacture production plant are contaminated with trichloroethylene (TCE)
        beneath buildings  and roadways at depths of 25 to 50 feet. The TCE plume is 600 yds in length, and  TCE
        concentrations are between 100 and 6,000 parts per billion (ppb). The drinking water standard is 5 ppb. A literature
        review was performed to determine, whether biological treatment can reduce TCE to these levels.

        Numerous papers in the academic literature show that TCE can be degraded in the presence of various cometabolites;
        aerobically in the presence of aromatic compounds like phenol or gaseous alkanes like methane and propane, and
        anaerobically in the presence of various simple organic compounds like acetate or benzoate. In the papers, which
        appear to have adequate QA/QC, biological treatment has accounted for losses ranging from 30 to 99.5  percent in 2-
        to 60-day tests.

        Although no full scale cleanups are on record, two well documented in situ pilot tests were found, one by a major
        university in conjunction with EPA and the other by a large environmental engineering firm. Both indicate positive results
        and recommend full-scale treatment as a viable option for those sites.  For these reasons, the RPM decided that a
        remedy screening study to assess the feasibility of using biological treatment at this site was warranted. The  RPM
        contacted several of the people involved in the first pilot test (the EPA oversight officer and the professor at the university)
        to seek suggestions on how to  proceed.
     erties   and  the  hydrogeological   and  geochernical
     characteristics of the formation..

     Variabilities in waste composition can cause inconsistent
     bacterial activity and, ultimately, inconsistent degradation.
     Heterogeneities such as debris, fill material, and geological
     anomalies (e.g., large clay lenses, rocks, and cavities) will
     influence air, water, contaminant movement, and excavation
     requirements. These formations can significantly  impede in
     situ bioremediation activities by obstructing the transport of
     nutrients or oxygen to the contaminated media.  Groundwater
     levels, contaminant depth, and the soil bearing capacity (as
     related to the soil's ability to support equipment) can also
     impact biological treatment. In combination, these parameters
     can determine  whether the media requiring treatment are
     amenable to either in situ or ex situ bioremediation.

     Soil  characteristics,  such  as  nonuniform particle  size
     distribution,  soil  type,  moisture  content,  hydraulic
     conductivity, and permeability, can also significantly affect
     biodegradation. Since organic contaminants tend to adsorb to
     fine particles such as silts and clays, variations in media
     composition and contaminant  concentrations can lead to
     variations in biological activity and inconsistent degradation
     rates. The presence of significant quantities of organic matter
     (humus, peat, nonregulated anthropomorphic compounds,
     etc.) may also cause high oxygen uptake rates, resulting in
     depleted oxygen supplies duringin situ applications. Low soil
     permeability can hinder the movement of water, nutrients, and
     oxygen through the contamination zone. Low percolation rates
     may cause amendments to be assimilated by soils immediately
     surrounding application points, preventing them fromreaching
     areas that are more remote either vertically or horizontally.
     Often only exsitu remedial technologies are applicable to sites
     that contain low-permeability  soils. This is true for both
     biological and nonbiological applications. Monitoring can be
     used  to  determine   amendment   fate.  Amendment
     concentrations and application frequencies can be adjusted to
     compensate for physical/ chemical depletion and/or high
     microbial demand. If these modifications fail to compensate for
microbial demand, remediation may occurby a sequential
deepening and widening of the active treatment layer (i.e., as
the contaminant is degraded  in areas near the amendment
addition points, and microbial activity decreases due to the
reduced substrate, the amendments move farther, increasing
microbial activity in those areas).

Even in relatively permeable soils, ion exchange and filtration
mechanisms can limit  the movement and  therefore  the
effectiveness of microbial and nutrient amendments.  It may be
necessary during treatment to improve the transport of water,
electron  acceptors, mineral  nutrients, co-substrates,  and
microorganisms by controlled pumping or by other means.
Care must be taken when performing the concomitant addition
of electron acceptors and donors through injection wells.
Excessive microbial growth or high concentrations of iron or
manganese may cause clogging in the well screen or the soil
pores  in the immediate vicinity of the well screen.1-60-1 Soils
prone  to  oxygen  transport  limitations may  be  most
appropriately treated using above-ground land treatment or
reactor approaches. Although  the above-soil characteristics
significantly impact in situ treatment, they canalso influence
the viability of ex situ treatment, specifically materials handling
and mixing requirements.

The presence of either an indigenous  or introduced microbial
population capable of degrading the contaminants of concern
is essential to the success of biological processes. Although
acclimated microbes have been known to tolerate very high
concentrations of metals  given long-term exposure, elevated
levels  of heavy metals, pesticides, highly chlorinated organics,
and some inorganic salts may inhibit microbial activity. Other
parameters  such  as  contaminant concentration,  pH,  and
temperature also affect microbial activity. In some instances,
these  characteristics  can be controlled or modified through
engineering practices. Metals maybe leached or complexed to
reduce microbial toxicity and improve  the  potential  for
treatment. Toxic effects may  be addressed by dilution, pH
control, metals control, (e.g.,
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     immobilization,  volatilization,  chelation,  and  washing),
     sequential treatment,  or  by employing microbial strains
     resistant to the toxicants. Physicochemical factors limiting
     biodegradation  such  as  temperature,  pH,  wateractivity,
     electron acceptors, nutrients, andtoxicity, must be addressed
     by either ameliorating the problem or by employing appropriate
     strains resistant to adverse conditions.

     In general, the effectiveness of these engineering practices
     must be assessed on a site-by-site basis. Generally, system
     operation can be easier to control and sampling simpler to
     perform during ex situ applications. Particular attention should
     be paid to any negative side-effects that may occur. Examples
     of problems that may be encountered include the following:

     •    Surface   active   agents   may  be  added   during
         bioremediation to increase the bioavailability of poorly
         water-soluble  or  sorbed  organic  pollutants. If the
         soil-water partition coefficient of the target contaminant
         is less than 10, modifying the soil's capacity to retain
         water may cause soluble compounds to leach into the
         groundwater.

     •    Excessive nitrate formation, which  may leach into the
         soil-water, may result from nitrogen addition.

     •    Some  nitrogen fertilizers tend to  change  soil  pH,
         necessitating further pH adjustment.

     •    By adding a carbon source to encourage the cometabolic
         degradation  of  a  specific  compound,  preferential
         degradation  of the added substrate may inhibit the
         degrada tion of the compounds of interest.

     Please note  that  each  contaminant   has   a  range  of
     concentrations atwhich the potential for biodegradation is
     maximized. Below this range microbial activity may not occur
     without the addition of a co-substrate.  Above  this range
     microbial  activity  may  be  inhibited   and,  once toxic
     concentrations are  reached, eventually arrested.  During
     inhibition, contaminant degradation  generallyoccurs ata
     reduced rate. In contrast, at toxic concentrations, con-
taminarit degradation does not occur. The concentrations at
which microbial growth  is either supported, inhibited,  or
arrested vary with the contaminant,  medium, and microbial
species.

Contaminant volatility is particularly  important, especially in
stirred and lor aerated reactors where the contaminants can
volatilize before being degraded. Example 2 illustrates how
contaminant  volatility  impacts  treatability  testing  and
potentially limits the application of a biological technology.

Contaminant  solubility should  also  be  determined,  since
highly soluble compounds can leach from the soil before being
degraded.  Attention should be paid to contaminant mixtures
that will behave differently from pure compounds. Interactions
between the contaminants and the soil may affect the reported
solubility, volatility,  and  partition coefficients  of the pure
compounds. Contaminant weathering may lead to binding in
soil pores, which can limit availability even of reportedly
soluble compounds.

Although preliminary data may indicate that the technology is
capable of reducing contamination levels to acceptable limits,
researchers are cautioned against stopping a study before site
cleanup goals are met. Although the initial rate of removal after
a potential lag period is generally rapid,  with time this rate
decreases  to a nearzero value. As shown  in Figure 2-11, the
concentration (theasymptote)  at  which the contaminant
removal rate is essentially zero represents, for all intents and
purposes, the lowest cleanup concentration that be achieved
during a remedial action. While additional contaminant removal
may occur over a very long period of time, this typically
non-zero concentration is the bioremediation end point from a
practical perspective. Typically, this asymptote is a function
of the folio wing:

•   Soil type - asymptote concentrations are higher in fine
    grained soils.

•   Initial contaminant concentration - the higher the initial
    concentration, the higher the end point tends to be.
                                                       Example 2

        A site contains soil contaminated with a mixture of VOCs and semivolatile organic compounds (SVOCs). A remedy
        screening shake flaskstudy measured greaterthan 90 percent biodegradation of the VOCs and SVOCs. Solid-phase
        bioremediation was being considered for full-scale application at the site. However, concerns were raised regarding
        organic carbon volatilization during solid-phase treatment.

        A remedy selection study was performed to determine the relative contribution of volatilization and biodegradation to
        the removal of the organic compounds. The study demonstrated that volatilization was the predominant mechanism
        forthe removal of the VOCs and the low molecular weight SVOCs. Air stripping removed 99 percent of VOCs within 21
        days. Biodegradation was the  major process for destruction  of the  high molecular weight organic compounds and
        removed  88 percent of SVOCS within 100 days.

        Based on the results of the  remedy selection study, an RD/RA study of a slurry-phase process was scheduled.
        Biotreatmentwas selected to maximize biodegradation of both the VOCs and SVOCs using a slurry-phase process that
        included off-gas collection and  recycling.
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     •    The "age" of the contaminated soil - the longer the soil
         has  been contaminated,  the more "irreversible" the
         contaminant partitioning, the lower the contaminant
         bioavailability, and the higher the endpoint.

     Since the asymptote is difficult to predict and is some-times
     greater than cleanup  criteria, treatability testing must be
     continued until either the removal goals have been met, the
     asymptote has been identified, or the allowable treatment time
     has been exceeded.
                                                              £300-1
                                                              H200-
                                                             FIGURE 2-11. A graphic representation of the
                                                                            contaminant removal asymptote.
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                                                  SECTION  3
                            THE USE  OF TREATABILITY STUDIES
                                       IN REMEDY EVALUATION
     This section presents an overview of the use of treatability
     studies in confirming the selection of biodegradation as the
     technology remedy under CERCLA. It also provides a decision
     tree that defines the tiered approach to the overall treatability
     study program with examples of the application of treatability
     studies to the RI/FS  and remedy selection testing processes.
     Subsection 3.1 presents an overview of the general process
     of,conducting treatability tests. Subsection 3.2 defines the
     applicability of each  tier of testing, based on the information
     obtained, to assess, evaluate, and confirm biodegradation as
     the selected remedy. Subsection 3.3 provides an expanded
     description  of the   tiered approach  to  biodegradation
     treatability testing.

     3.1      PROCESS OF TREATABILITY
             TESTING IN SELECTING A REMEDY

     Treatability  studies  should  be performed  in a  systematic
     fashion to ensure that the  data generated  can support the
     remedy evaluation process. This section describes a general
     approach that should be followed by RPMs,  potentially
     responsible parties (PRPs), and contractors during all levels of
     treatability testing. This approach may include some or all of
     the following:

     •   Selecting a contracting mechanism*
     •   Issuing the Work Assignment *
     •   Establishing data quality objectives
     •   Preparing the Work Plan
        Preparing the SAP
     •   Preparing the Health and Safety Plan
     •   Conducting community relations activities
     •   Complying with regulatory requirements
     •   Executing the study
     •   Analyzing and interpreting the data
     •   Reporting the results
     *   Tasks not performed by contractors.

     These elements are described in detail in the generic guide,
     which provides  information applicable to all  treatability
     studies. It also presents information specific to
remedy screening, remedy selection, and RD/RA testing.1-52-1

Treatability studies for a particular site will often entail multiple
tiers of  testing. Duplication of effort can be  avoided by
recognizing this possibility in the early planning phases of the
project.  The  Work  Assignment, Work  Plan,  and other
supporting documents should include all anticipated activities.

There are three levels or tiers of treatability studies: remedy
screening, remedy selection, and RD/RA testing.  Some or all
of the levels  may be needed on a caseby-case basis. By
balancing the  time and cost necessary to perform the testing
with the risks inherent in the decision (i.e., selection of an
inappropriate  treatment alternative), the level of treatability
testing required can be determined. These decisions arebased
on the quantity and  quality of data available and on other
decision factors (e.g., State and community acceptance of the
remedy and new site data). The flow diagram for the tiered
approach, Figure 3-1, traces the data review process and the
decision points and factors to be considered step by step.

Technologies  are generally evaluated first  at  the remedy
screening level and progress through remedy selection testing
to the RD/RA tier.  A technology may enter the process,
however, at whatever level is appropriate based on available
data on the technology and site-specific factors. For example,
if the technology  under study has been successfully applied
at a similar site, a remedy screening study may not be needed
to demonstrate potential applicability. Rather, treatability
studies may progress directly to remedy selection testing to
verify that performance standards can be met. It should be
noted, however, that treatability studies, at some level, will
normally be needed to ensure that the site target cleanup goals
can be achieved. Figure 3 -2 shows the relationship of the three
levels of treatability study to each other and to the RI/FS
process.

3.2     APPLICATION  OF TREATABILITY
        TESTS

Before conducting treatability studies, the objectives of each
tier of testing must be established. Biodegradation treatability
study objectives  are based upon the specific needs of the
RI/FS. There  are nine  evaluation criteria specified in the
document, Guidance for Conducting
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Site
Characterization

1
Technology
Screening
                                                                                                            MANAGEMENT DECISION FACTORS:

                                                                                                             • State and Community Acceptance
                                                                                                             • Schedule Constraints
                                                                                                             • Additional Data
Technology
 Potentially
  Viable?
                              Treatability
                               Studies
                               Needed?
                      Management
                     Decision Factors
                              Technology
                            Demonstrated for
                              Contaminant
                                MaJrix?
                       Remedy
                       Screening
                       Studies
Technology
 Feasible?
                                                                         Remedy
                                                                         Selection
                                                                         Studies
                                                                    Meet
                                                                 Performance
                                                                    Goals
                                                                                                                             Meet
                                                                                                                          Performance
                                                                                                                            Goals?
                                                               Detailed Analysis
                                                                of Alternatives
                                                                                                                            RD/RA
                                                                                                                           Studies
                                                Figure 3-1.  Row diagram of the tiered approach to conducting treatability studies.
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                               Remedial Investigation/
                               Feasibility Study (RI/FS)
                                                 Identification
                                                 of Alternatives
                                           Record of
                                           Decision -
                                            (ROD)

                                           Remedy
                                           Selection
                            Remedial Design/
                           - Remedial Action—
                                (RD/RA)
               Scoping
              - the  -
                RI/FS
     Site
Characterization
and Technology
   Screening
                                     REMEDY
                                   SCREENING
                                    to Determine
                                Technology Feasibility
  Evaluation
of Alternatives
                                                      REMEDY SELECTION
                                                       to Develop Performance
                                                           and Cost Data
 Implementation
~"  of Remedy
                                                                                             RD/RA
                                                                                        to Develop Scale-Up,
                                                                                        Design, and Detailed
                                                                                             Cost Data
                         Figure 3-2. The role of treatability studies in the RUFS and RD/RA processes.
    Remedial  Investigations and  Feasibility  Studies  Under
    CERCLA (Interim Final).(51)  A detailed analysis of different
    remedial alternatives using the nine CERCLA criteria is
    essential. Treatability studies provide data for as  many as
    seven of these criteria. These seven criteria are:

    •   Overall protection of human health and the environment
    •   Compliance with applicable or relevant and appropriate
        requirements (ARARs)
    •   Long-term effectiveness and permanence
    •   Reduction  of toxicity,  mobility, or volume through
        treatment
    •   Short-term effectiveness
    «   Implementability
    •   Cost

    The two remaining CERCLA criteria, State  and community
    acceptance, are based in part on the preferences and concerns
    of the State and community regarding alternative technologies.
    A viable remedia tion  technology may be eliminated from
    consideration if the State or community objects to its use.
    Although these criteria cannot be targeted for assessment
    during the  treatability study, process data may be produced
    that address State and community concerns.  Potentially this
    data may be used to change  the State's or community's
    perception of the  technology under study. The remainder of
    this subsection discusses the seven applicable criteria.

    The first criterion, overall  protection of human  healthand
                           theenvironment, is used to evaluate how a technology, as a
                           whole, can be used to  protect human health  and the
                           environment. In previous years, cleanup goals often reflected
                           background site conditions. Attaining background cleanup
                           levels through treatment has proved impractical in many
                           situations.  The present trend is toward the development of
                           site-specific cleanup levels that are risk-based rather than
                           background-based. In situations where unique cleanup criteria
                           have been designated  as  part  of a  site-specific  risk
                           assessment, the evaluation of a technology's ability to provide
                           for "overall protection of human health and the environment"
                           may be limited to assessing the technology's ability to attain
                           targeted  contaminant reductions.  Often,  however,  the
                           evaluation  of this criterion  draws  on the technology's
                           compliance with the other evaluation criteria,  specifically
                           long-term  effectiveness  and   permanence;   short-term
                           effectiveness; compliance with ARARs; and reduction of
                           toxicity, mobility, and volume through treatment. To assess
                           whether the technology is capable of protecting human health
                           and  the environment, treatability study  data from remedy
                           selection testing must be obtained to help to answer the
                           following questions:

                           «    What  will  be the  maximum  remaining  contaminant
                                concentrations?

                           •    Will the residual contaminant levels be sufficiently low to
                                meet   the  established  ARARs  or  the  risk-based
                                contaminant cleanup levels?
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     •    What are the contaminant concentrations and physical
         and chemical differences between the untreated and the
         treated fractions (e.g., have contaminant toxicity, mobility,
         and volume been reduced)?

     The second criterion, compliance with ARARs, ensures that
     the selected technology meets  all of the relevant Federal and
     State ARARs (as defined in CERCLA Section 121) that have
     been identified in  previous stages  of the RI/FS  process.
     ARARs may be categorized as chemical-specific requirements
     that define acceptable  exposure levels and thus preliminary
     remediation goals (i.e., maximum contaminant levels); as
     location-specific requirements that set restrictions on activities
     within specific locations,  such as floodplains, wetlands, or
     historic sites; and as action-specific requirements that set
     controls or restrictions for particular treatment and disposal
     activities related to the management of hazardous wastes (i.e.,
     RCRA minimum technology standards).1-51-1 Subsequent  text
     addresses in detail  limitation that two of the most common
     ARARs, LDRs and TSCA rulings have on the application and
     testing of biological technologies.

     The LDRs for Newly Listed Wastes and Contaminated Debris
     Rule,  promulgated  on August  18,  1992, may  limit the
     applicability of certain bioremediation technologies to certain
     sites. LDRs apply to hazardous wastes regulated by RCRA
     that are intended for  land disposal. For each category of
     hazardous waste, the LDRs establish treatment standards that
     are either concentration-based (hazardous constituents must
     be reduced to a set concentration before the material is eligible
     forland disposal) or technology-based (material containing the
     listed hazardous waste must be treated by the designated Best
     Demonstrated Available  Technology,  or BOAT). LDRs
     generally become applicable as soon as hazardous waste is
     excavated.  For  RCRA corrective  actions and  CERCLA
     remediations, however, LDRs would not apply if the hazardous
     waste is treated in its original Corrective Action Management
     Unit (CAMU) or in a temporary  unit (such as a bioreactor),
     which  will  be  removed  from  the  site  following
     treatment.1-40-"-41-"-47-"-51-1 In situ treatment (i.e., non-excavated) of
     hazardous waste also does not  trigger LDRs.

     The LDRs for Newly Listed Wastes and Contaminated Debris
     Rule was Phase 1 of a three-part regulation. Phase 2, LDRs for
     Newly Listed Waste and Contaminated Soil, is scheduled for
     issue in late summer 1993, while Phase 3 is scheduled for issue
     in March 1994. It is possible that bioremediation will be the
     BOAT standard forsoils contaminated with certain chemicals,
     but  until  Phase  2 is promulgated, all treatment must either
     comply with the existing LDRs or seek compliance alternatives.
     The available compliance alternatives include the following:(41)

     •    Treatability variances (for wastes that are considered
         more difficult  to  treat  than the waste on  which the
         standard was based)

     •    No-migration petitions (which require demonstrating that
         the waste cannot migrate from the disposal location for as
         long as it will remain hazardous)

     •    BOAT   exemption  for  groundwater  reinjection
         (groundwater can  be exempt from the LDRs if it is just
         pumped to the surface, amended, and reinjected)

     Additional information can  be obtained from the following
sources:1 '

    OSWERs TIO
    Contact: Michael Forlini
    703-308-8825

    RCRA/Superfund Hotline
    800-424-9346 or
    703-920-9810 (from Washington, D.C.)

TSCA   may   also   apply  to   certain  applications   of
bioremediation.  Genetically-modified  microorganisms  are
currently regulated under TSCA Section 5(1986), which is part
of  an  interagency  Coordinated  Framework   for
Biotechnology.(9)  Microorganisms   are  subject   to
premanufacture notification (PMN) reporting under TSCA
Section 5 when  they are  intended for TSCA uses, which
include bioremediation and other commercial applications.(40)

There   are  numerous   circumstances   under   which
microorganisms are exempt from  regulation  under TSCA
Section 5.  PMN reporting is required  only for  new
microorganisms and does not apply to naturally-occurring
microorganisms.   In   the   1986  policy  statement,  new
microorganisms were defined as microorganisms that contain
genetic material from  organisms of different genera/40'

New draft  TSCA biotechnology rules  entered EPA's Red
Border review process on December 27, 1991. The draft rules
propose that the definition of new microorganisms be changed
to  include  only  those  microorganisms  that   possess
deliberately-modified hereditary traits and are likely to exhibit
new behaviors. The draft rules also propose exemptions for
new microorganisms that fall into one or more of the following
categories:(4o:)

•   Test marketing.

•   Common microorganisms that have a history  of safe use.

•   Microorganisms that are listed in the regulations and have
    met specific criteria regarding introduced genetic material
    and containment practices. This category includes Tier I
    exemptions (one-time certification of compliance must be
    obtained before the first use of the microorganism) and
    Tier II exemptions (request must be filed 45 days before
    the microorganism is manufactured or imported).

•   Research and development  activities  in  which  the
    microorganisms are contained in a structure such as a
    greenhouse, a bioreactor, etc.

These new rules are still in draft form and are not likely to take
effect before 1995.(40)

Future   risks  to  human health and  the environment  are
evaluated when determining the third criterion, the long-term
effectiveness of  a remedial  action. The magnitude  of any
residual risk and the adequacy and reliability of controls must
be evaluated. Residual risk, as applied to  biodegradation,
assesses the risks associated with the residual contaminants
and  metabolites  or  byproducts in  the treated soil and
groundwater at the conclusion of all remedial activities. When
relatively toxic compounds or compounds with potential to be
transformed into toxic
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     byproducts require treatment, a mass balance to assess
     mineralization  using a  radiolabeled  compound may  be
     appropriate.  The volume,  toxicity,  and  mobility  of the
     residuals, as well as their propensity to bioaccumulate, should
     be determined during testing.

     Since mineralization studies can provide evidence indicating
     that  a  biological process is  capable of transforming the
     contaminants into benign endproducts, logically speaking,
     toxicity testing should not  be  considered unless  the
     mineralization data demonstrate that the biological process is
     incapable of actually mineralizing the target compounds. The
     potential for long-term release of adsorbed contaminants from
     the  treated soil matrix should also be  addressed during
     biological treatability testing. If controls are needed to manage
     the residuals, data should be compiled during testing to help
     determine both the type and degree of long-term management
     to be employed. Long-term  operation, maintenance,  and
     monitoring requirements, as well as difficulties and risks
     associated with long-term application of a control, will need to
     be obtained in order to assess whether a control is suitable for
     long-term  application. Attention should be paid to future site
     access   restrictions and monitoring  requirements.  Such
     assessments  are usually beyond the  scope of a remedy
     selection treatability study, but may be marginally  addressed
     based on remedy selection testing results.

     During the assessment of the fourth criterion, reduction of
     toxicity, mobility,  or  volume through treatment,  specific
     numerical  data requirements are targeted, including  (where
     applicable):

     •    What mass/volume of media was treated during the test?

     •    What were the contaminant removals experienced during
         treatment?  What  percentage  can be  attributed  to
         biological removal  mechanisms? How do these data
         compare  to background   levels  for  biological  and
         nonbioloical removal mechanisms?

     •    Have  the mass and  mobility of the toxic contaminants
         been reduced, and if so, by  how much? How do the
         mobility and toxicity of the leachate from the treated soil
         compare to the leachate from the untreated soil?

     •    Has the volume of toxic material been reduced, and if so,
         by how much?

     •    What residual contaminants and/or byproducts are left in
         the soil following treatment? What are the quantities and
         characteristics  of these residuals? What are the risks
         associated with these contaminants?

     Toxicity studies may need to be conducted on the treated and
     untreated media to determine toxicity reduction. Since toxicity
     studies measure a substance's effect on living organisms, they
     can also provide information regarding two other CERCLA
     criteria:  overall protection  of  human  health  and  the
     environment and long-term effectiveness and permanence.
     Toxicity studies are typically separated into two categories:
     environmental  effects  testing(77)(82:i  and  health  effects
     testing.(80) Environmental effects testing measures toxicity to
     certain plants and animals, while the health effects testing is
     used to estimate toxicity to humans based on existing data and
     tests conducted on single and multicellular organisms. Several
     specific toxicity tests are briefly described in the
compendium of tools presented in Appendix A. The design
and interpretation of toxicity studies require consultation with
trained professionals  because of the inability to  measure
human toxicity directly and because a substance that is toxic
to one organism might be more or less toxic to another.

The fifth criterion, short-term effectiveness, addresses the
effects of the treatment technology from remedy design and
construction through implementation and  completion  of
response objectives.  An  estimated  cleanup date  may be
projected from data obtained regarding residual contaminant
concentrations in the soil. Risks faced by the community,
workers, and the environment during the remedial action (e.g.,
uncontrolled  contaminant  volatilization   during  slurry
bioreactor treatment)  must be identified and appropriate
controls evaluated.

The sixth criterion, implementability, evaluates the  technical
and administrative feasibility of an alternative. This relates to
the availability of required goods and services as well as the
technical feasibility of biodegradation at the site. Determining
whether the contaminated soil is chemically  and physically
amenable to biological treatment under site-specific conditions
is essential. The following questions  must be answered  in
order to  address the implementability of a  bioremediation
technology:

•   What are the oxygen sources (i.e., electron acceptors) and
    nutrient availabilities of the site soils? Is supplementation
    possible? What are the costs and benefits associated with
    supplementation?

•   Is in situ treatment practical,  in  view of site and soil
    characteristics, or do heterogeneities exist that would
    inhibit  in  situ  biodegradation? If so, can the media be
    safely excavated for ex situ biodegradation?

•   What is the water infiltration rate?  Soil permeability? Ion
    exchange capacity? Is contaminant migration (through the
    air or groundwater) likely? To what depth does the
    vadose zone  extend?  Can  issues  regarding  these
    parameters be resolved or addressed?

•   What   are  the  characteristics   and  quantities   of
    contaminants that will remain after biodegradation? Is this
    concentration  within  project goals? Will  an additional
    treatment mechanism need to be employed to meet project
    goals?

•   What is the administrative feasibility associated with
    using this technology? Has it been used before within the
    Region? How quickly can it be approved for use? Will the
    State and local governments approve its use? Can existing
    time constraints be met?

Additionally, the implementability criterion evaluates whether
vendors and process equipment are available to perform the
remediation, if adequate space exists  to perform treatment
operations, and what materials handling problems might be
encountered if soil must be excavated.

The final EPA evaluation criterion that can  specifically be
addressed during a treatability study is cost. RD/RA
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     treatability studies provide data  to estimate the following
     important cost factors:

     •    The initial design of the full-scale unit

     •    The estimated capital, operating, and maintenance costs

     •    Initial estimate of the time required to  achieve  target
         cleanup levels, as reflected by operation and maintenance
         costs.

     In  some  cases, remedy selection  treatability studies  can
     provide preliminary estimates of the same cost and schedule
     factors. However, in order to evaluate this criterion adequately,
     a conceptual design of the bioremediation system is needed
     and tradeoffs between capital and operating costs must be
     made. Additional treatment and disposal  costs must also be
     considered.  A  properly  designed  biological  treatment
     technology should produce either  CO2 and water or other
     relatively innocuous degradation products, thus reducing the
     possibility that process residuals  will  require  additional
     treatment and disposal as hazardous or  regulated wastes.
     However,  certain  technologies,   particularly  ex   situ
     technologies, can be expected to generate residuals requiring
     some level of treatment and disposal. For example, aqueous
     and slurry-phase  technologies frequently generate  excess
     sludge (e.g., biomass), which requires treatment, dewatering,
     and disposal.

     In general, most smaller-scale remedy selection studies only
     show that biodegradation can  meet the required  target
     concentrations under experimental conditions. The results of
     successful smaller-scale laboratory selection studies must be
     combined with soil characterization data and performance data
     from similar sites to  evaluate the implementability of the
     technology at a specific site. Even after these steps are taken,
     there may be a high degree of uncertainty as to the ability of
     the technology to  reach the contaminant  target levels under
     field conditions in a reasonable time. As a result, larger-scale
     field studies are often recommended, particularly during the
     evaluation of an in situ bioremediation technology.

     Table 3-1 shows how remedy selection treatability studies
     addre ss seven of the nine criteria. The experimental parameters
     monitored during the  study are chosen to provide data on the
     ability of the test to meet the study goals.  Remedy selection
     treatability study  goals  and  experimental parameters  are
     discussed in Subsections 4.1 and 4.2, respectively.

     3.3 BIODEGRADATION TREATABILITY
         TESTS

     The following subsections describe the  tiered approach to
     biodegradation treatability testing. Basic elements of each tier
     of testing are provided. A detailed discussion  of remedy
     selection testing may be  found in Section 4.  Since this
     document is intended as guidance for remedy selection studies
     only, amore thorough description of the remedy screening and
     RD/RA studies is beyond the scope of this document.

     It is important to note, that as more information  is gathered
     regarding the application of a specific technology to
certain types  of contaminants,  testing requirements will
decrease.

3.3.1   Remedy Screening

Remedy screening is the first level of testing. It is used to
determine whether biodegradation  is  possible  with the
site-specific  waste material in question. These studies are
generally low  in cost (e.g., $10,000 to $50,000) and usually
require 1 weekto several months to complete. Additional time
must be  allowed for project planning,  chemical analyses,
interpretation of test data, and report writing.  Only limited
quality control (QC) is required. Remedy screening studies
yield  data indicating  a technology's  potential to  meet
performance goals. They generate little, if any, design or cost
data and should not be used as the sole basis for selection of
a remedy.

Typically, aerobic biological remedy screening studies are
performed  in test  reactors  containing  saturated  soil,
unsaturated soil, soil slurries, and aqueous solutions. Studies
employing simple shake flasks, soil pans, or slurry reactors are
usually employed/21^81' Normally pH, contaminant loading
rates, and oxygen and  nutrient availability are adjusted to
increase the chances of success. These reactors may be small
sacrificialbatchreactors (approximately 40mL to 1 L in size) or
larger microcosms (1 to 10 L) that are subsampled. (Only  a
portion of the contents are removed at each sampling time to
monitor the progress  of biodegradation.)  The microbial
population can  be either indigenous (e.g., acclimated or
nonacclimated) to the site, selectively cultured,  a proprietary
mixture provided by a  vendor, or any combination of the
preceding. Inhibited controls are employed to account for
abiotic removal during treatment. As an alternative, abiotic
losses can be monitored directly. The goal of a screening level
study is to determine whether biodegradation can occur. Since
the ability of a technology to meet treatment goals is not the
issue, it is usually not necessary to establish complete removal
of  the  contaminant  of interest. Thus,  a  reduction  in
contaminant concentration over a 3- to 6-week period of 20
percent  (minimum) to  50  or 60 percent (corrected  for
nonbiological  losses  through  photodecomposition,
volatilization, adsorption, etc.) would indicate that biological
treatment may  be feasible.

Contaminant reductions and other criteria used to evaluate
treatability study tiers are listed in Table 3-2. The information
required to determine the success of each level of treatability
study is  also  presented. While the  criteria listed are not
all-inclusive, they provide readers with  a  "yardstick" with
which they can  compare proposed treatability studies and
verify that the  appropriate tier is being investigated.

Example 3 illustrates the type of information that might result
froma remedy screening study and the conclusions that might
be drawn from that information. For more  detailed information
on  remedy  screening, please consult the biodegradation
screening guide  and  EPA's  Center Hill  facility  staff  in
Cincinnati, Ohio. RREL has recently developed a protocol for
performing biological remedy screening studies at this facility.
Information  regarding these  treatability  studies may  be
obtained from Eugene Harris at (513) 569-7862.(69)
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                        Table 3-1.  Ability of Remedy Selection Treatability Studies To Address RI/FS Criteria
       Study goals
        Experimental parameters
             RI/FS criteria*
       Compare performance, cost, etc.,
       of different treatment systems at a
       specific site
       Measure the initial and final
       contaminant concentrations, and
       calculate the percentage of
       contaminant removal from the soil,
       sludge, or water through
       biodegradation
       Estimate the type and concentration
       of residual contaminants and /or
       byproducts left in the soil after
       treatment
       Develop estimates for reductions  in
       contaminant toxicity, volume, or
       mobility
       Identify contaminant fate and the
       relative removals due to biological
       and nonbiological removal
       mechanisms
       Produce design information
       required for next level of testing
       Develop preliminary cost and time
       estimates for full-scale remediation
       Evaluate need for pretreatment and
       requirements for long-term
       operation, maintenance, and
       monitoring

       Evaluate need for additional steps
       within treatment train
       Assess ability of bioremediation to
       meet site-specific cleanup levels
       Determine optimal conditions for
       biodegradation and evaluate steps
       needed to stimulate biodegradation
Dependent on type of treatment systems
compared
Contaminant concentration
Contaminant/byproduct concentration
Contaminant concentration, toxicity
testing

Contaminant concentrations present in
solid, liquid, and gaseous phases taken
from test and control reactors, oxygen
uptake/CO2 evolution
Temperature, pH, moisture, nutrient
concentrations and delivery,
concentration and delivery of electron
donors and acceptors, microbial
composition, soil characteristics, test
duration, nonbiological removal
processes
Treatability study cost (i.e., material and
energy inputs, residuals quality and
production, O&M costs, where
appropriate), test duration, time requires
to meet performance goals
Soil characteristics, contaminant
concentration/toxicity
Soil characteristics, contaminant
concentration, nonbiological removal
processes, residual quality (relative to
further treatment and/or disposal
requirements)
Contaminant concentration
Temperature, pH, nutrient concentrations
and delivery, concentration and delivery
of electron donors and acceptors,
microbial composition, soil
characteristics, test duration,
contaminant concentration
Overall protection of human health and the
environment
Compliance with ARARs
Long-term effectiveness and permanence
Reduction of toxicity, mobility, and volume
through treatment
Short-term effectiveness
Implementability
Cost
Overall protection of human health and the
environment
Compliance with ARARs
Long-term effectiveness and permanence
Reduction of toxicity, mobility, and volume
through treatment

Overall protection of human health and the
environment
Compliance with ARARs
Long-term effectiveness and permanence

Reduction of toxicity, mobility, and volume
through treatment

Overall protection of human health and the
environment
Long-term effectiveness and permanence
Reduction of toxicity, mobility, and volume
through treatment
Short-term effectiveness

Implementability
Cost
Short-term effectiveness
Implementability
Cost
Compliance with ARARs
Long-term effectiveness and permanence
Short-term effectiveness
Implementability
Cost

Overall protection of human health and the
environment
Long-term effectiveness and permanence
Implementability
Cost

Overall protection of human health and the
environment
Compliance with ARARs
Long-term effectiveness and permanence
Reduction of toxicity, mobility, and volume
through treatment
Short-term effectiveness
Implementability
Cost
          Depending on specific components of the remedy selection treatability study, additional criteria may be applicable.
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                             Table 3-2. Biodegradation Criteria for Each Treatability Study Tier
      Criteria
Remedy screening
Remedy selection
Remedy design
      Biodegradation of most-     >20% net removal
      resistant contaminants of    compared to removal in
      concern(COCs)            inhibited control
      Initial contaminant
      concentration
      Environmental conditions



      Extent of biodegradation

      Biodegradation rate

      Estimate time to reach
      cleanup standards

      Mass balance


      Toxic byproducts

      Process control and
      reliability

      Microbial activity

      Process optimization

      Cost estimate for full-
      scale

      Bid specifications

      Experimental scale
Optimal for technology



Optimal for technology
(include site conditions if
possible)

Estimate*

Crude estimate*

NA


Crude*


Detect*

NA


Crude measure*

NA

NA


NA

Usually bench-scale
                            Meets cleanup standard
                            undertest conditions
Maximum concentration
expected during
remediation

Simulate expected site
treatment conditions
Quantify

Defensible estimate

Estimate


Closure or defensible
explanation

Test for if appropriate*

Assess potential


Verify/quantify*

Estimate*

Rough,-30%, +50%


NA

Either bench-or pilot-
scale
                           Meets cleanup standards
                           under site conditions
Actual range of
concentrations expected
during remediation

Actual site treatment
conditions for the specific
technology

Quantify

Quantify

Refined estimate


Closure or defensible
explanation

Test for if appropriate

Demonstrate


Quantify/monitor*

Refined estimate

Detailed/refined


Nearly complete

Usually pilot- or full-scale
         Not required, although sometimes possible to address significantly.
                                                      Example 3

          A former agricultural distributorship contained approximately 12,000 cubic yards of pesticide-contaminated soil,
          having combined concentrations of less than 200 parts per million (ppm) for2,4-dichlorophenoxyacetic acid (2,4-D)
          and 4-chloro-2-methylphenoxyacetic acid (MCPA). The average combined concentration of 2,4-D and MCPA in the
          soil was 86 ppm. Regulatory cleanup requirements for the site were 10 ppm. A remedy  screening study was
          performed to determine whether significant biodegradation could be achieved with a solid-phase bioremediation
          process.  Soil microcosms, designed to simulate a  full-scale  solid-phase  bioremediation  system,  were
          established to evaluate biodegradation. Initial and final contaminant concentrations, as well as microbial plate
          counts, were analyzed to assess performance.

          The soil  microcosm studies demonstrated  that the naturally-occurring microorganisms  in the soil could
          biodegrade the 2,4-D and MCPA, provided nutrient concentrations and moisture content were maintained within
          ranges conducive to biodegradation. The  average combined 2,4-D and MCPA concentrations decreased from 86
          to 5 ppm  in 12 weeks, a decrease of 94 percent. The concentration of  pesticides in the inhibited controls was
          reduced by 10 percent, indicating that biodegradation was the predominant removal mechanism. Based on the
          positive results from the remedy screening studies, a remedy-selection field-scale test was designed to determine
          whether bioremediation was capable of achieving the  site cleanup levels under field conditions.
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     Please note that  the biodegradation screening  guide does not
     address anaerobic treatability testing.  To accomplish this the
     reader must possess a basic understanding of anaerobic processes.
     (Note: anaerobic processes occur in an environment lacking in free
     oxygen  but possessing  alternative  electron  acceptors such as
     nitrate, carbonate, or sulfate.(43) Anaerobic conditions may either
     occur naturally or be established by modifying  site or media
     characteristics. Further information on these processes can be
     obtained from various sources.)(68) Some important characteristics
     of anaerobic applications and testing are mentioned in Subsection
     2.1.3 and  Section 4 of this document. Experimental designs for
     anaerobic  remedy screening studies can be developed by applying
     these considerations to the experimental designs described in the
     biodegradation screening guide. Other references may be consulted
     that provide additional information on anaerobic  treatment and
     treatability studies.(38)(43)(68)

     3.3.2    Remedy Selection

     Remedy selection testing  is the second level of testing. To the
     maximum extent practical, remedy selection tests should simulate
     site conditions during treatment, allowing researchers to identify
     the technology's  performance on a waste-specific basis for an
     operable unit. These studies  are generally of moderate cost (e.g.,
     $50,000 to $300,000) and may require several weeks to 2 years to
     complete.  They yield data that identify whether the technology is
     likely to meet expected cleanup goals and can provide information
     in support of the detailed analysis of the alternative (i.e., seven of
     the  nine   evaluation criteria).  Toxicity testing  of  residual
     contaminants and intermediate degradation  products  may be
     necessary. Slurry-phase reactors, soil pans,  or contained soil
     treatment   systems  are generally  used  to  evaluate ex situ
     bioremediation technologies, while  soil plots and soil columns
     (both within the laboratory and field) may be used to evaluate in
     situ technologies.

     Throughout this document, the phrase "contained soil treatment"
     is used to describe treatability studies conducted on excavated soil
     in  a  treatment cell.  These  studies are typically  larger-scale
     representations of compost  piles, soil heaps, or land treatment
     systems. Contained soil treatment systems are constructed on a
     larger scale than soil pans but may be similar to soil pans in other
     respects. The other testing methods discussed in this document are
     considered self-explanatory. Further information on the basic
     characteristics of contained soil treatment and the other test
     methods is provided in Table 4-2.

     Smaller-scale treatability  studies using  soil pans,  small soil
     columns, and small slurry-phase reactors, are generally performed
     in the laboratory and may last from  1 week to  6  months,
     depending on the type of study employed. The media (i.e., soil,
     sediments) treated during these studies should be taken from the
     contaminated site. Due to the relatively small amounts of media
     tested during these  treatability  studies  (refer to  Table 4-2),
     operating  parameters are relatively  easy  to control.  While this
     makes it easier for researchers to approximate ideal operating
     conditions, it unfortunately makes it less likely that these studies
     will simulate actual site conditions during full-scale treatment,
     especially  when  evaluating an in situ technology.  Studies
     performed to evaluate slurry reactors are the exception. These
     smaller-scale studies
should be designed to achieve mass balance closure. In reality,
results providing at least a semi-quantitative mass balance are
usually acceptable for remedy selection. In general, they are less
expensive than larger-scale field studies and typically cost from
$50,000 to $150,000.

Larger-scale treatability studies using soil plots and contained soil
systems are generally  performed in the field and  last from 2
months to 2 years. These studies typically  cost $100,000 to
$300,000 and are particularly appropriate for complex sites where
in situ biodegradation is being considered. Generally, these studies
are conducted onsite, preferably on a small portion of the area
requiring remediation. Large soil column studies, on the other hand,
are often performed in the laboratory. However, techniques to
assess biodegradation using soil columns within the field are being
developed. These buried columns will be able to examine microbial
activity at isolated depths using remote sensing instrumentation.(66)
Steps are  often taken to isolate the media physically from the
environment, thereby preventing possible contaminant migration.
Although  the  design of  treatability  studies  depends  on the
characteristics  of the specific  technology  under  analysis,  these
studies typically use techniques and equipment that are similar or
identical to those used during full-scale remediation, enabling these
studies to approximate full-scale treatment closely. These studies
often provide detailed information that may be used to supplement
RD/RA studies and can be used in the design of the full-scale
treatment system.

Table 3-2 lists the type  of information needed to determine the
success of a  remedy selection treatability study.  Example 4
describes the type of information collected during a hypothetical
remedy selection treatability study as well as the conclusions and
interpretations made from that information.

3.3.3    Remedial  Design/Remedial  Action

RD/RA testing is the third level of testing. By operating a field
unit  under conditions similar to those expected during full-scale
remediation, RD/RA testing can be used to:

•    Provide the  data required for final full-scale design

•    Develop more accurate cost and time estimates for full-scale
     remediation

•    Confirm biodegradation rates and cleanup levels determined
     during remedy selection

•    Optimize unit operating parameters

These studies are of moderate to high cost (e.g., $100,000 to
$500,000) and may require several months or more to complete.
They should be performed during the remedy implementation
phase of a site cleanup.

RD/RA tests usually consist of bringing a mobile treatment unit
onto the site or constructing a small-scale unit  for nonmobile
technologies. The size and scope of the RD/RA test may be
determined by several factors including the complexity of the
process and the availability of equipment, test material, funds, and
time. It is also critical that the RD/RA test equipment be sized so
that realistic scale-up factors can be used for the transition to
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                                                        Example 4

          An abandoned  refinery NPL site contains numerous pits holding approximately 60,000 cubic yards of waste
          contaminated with styrene tar and other organic materials. The site contains rubble and debris in the pits, posing
          significant materials handling problems. The contaminant of particular concern, phenanthrene, was detected at
          500 ppm, significantly above the acceptable limit of less than 1 ppm. VOCs were detected at 300 ppm. Average
          initial contaminant concentrations in the soil treated during the treatability study were 36.3 ppm and 26.0 ppm for
          phenanthrene and  VOCs, respectively. Although styrene tar is traditionally remediated by incineration, public
          resistance  prompted  an investigation into  biological alternatives.  Following  a laboratory  screening  study
          demonstrating  phenanthrene biodegradability,  a remedy selection field demonstration was initiated.  Final
          concentrations of less than 260 ppb and 5,800 ppb for VOCs and phenanthrene, respectively, were targeted. Site
          cleanup goals for phenanthrene were set at less than 1 ppm.

          A pilot-scale, solid-phase air stripping and biological treatment facility was constructed to demonstrate the
          feasibility of bioremediating  contaminated soils and organic residues. The  treatment facility consisted  of an
          enclosed, lined  treatment bed containing 200 cubic yards of contaminated soil from a backfilled storage lagoon
          at the former refinery bite. The liner was an 80-mil high density polyethylene (HOPE) synthetic membrane with
          heat-welded seams. A sand drainage layer was placed on top of the liner and a 6-inch layerof contaminated soil
          was placed on top  of the  sand. Nutrients were applied to  the treatment bed through an overhead spray system.
          The treatment bed was tilled daily to increase soil surface  area and provide aeration. Volatile emissions from the
          treatment bed were contained by  a plastic-film greenhouse and routed to  carbon adsorption  units. Aerobic
          heterotrophic and phenanthrene degrading microorganisms were periodically assessed to determine microbial
          activity.

          Sampling after 21 days of operation indicated that greater than 99 percent of the VOCs had been removed  by air
          stripping. Samples  collected after 94 days of operation demonstrated that an average of 89 percent of the SVOCs
          had been biodegraded. Phenanthrene concentrations were reduced an average of 84 percent. Phenanthrene had
          a half-life of 33 days, corresponding to approximately 130 days to reach the concentration approaching the
          analytical detection limit for phenanthrene (using EPA-approved procedures). This was a significant improvement
          in degradation rate overthe 69 and 298 day half-lives reported in two previous studies, which were identified during
          the literature search. (These studies were performed at two different sites.) The data indicated that approximately
          131 days would be required for the phenanthrene concentration to reach the  analytical detection limit using the
          EPA-approved procedures. The study demonstrated that soils could be remediated using a combination  of air
          stripping and bioremediation. Based on performance during testing, additional testing was recommended.
     full-scale operation. If possible, the RD/RA equipment should be
     designed so  that it can be readily converted to  the full-scale
     remediation  system. In some cases,  RD/RA tests may  be a
     continuation of remedy selection tests using the same apparatus. A
     complete mass balance, including all nonbiological pathways, should
     be performed at this level of testing. Typical testing periods are from
     2 to 6 months. For more complex sites, for example sites with
     different types of contaminants in different areas or with different
     geological structures in different
areas, longer testing periods may be required.

Given the limited availability of peer-reviewed published data on
full-scale applications using innovative technologies, RD/RA testing
will generally be necessary. Table 3-2 lists the type of information
needed  to assess the success of an RD/RA treatability study.
Example 5 illustrates the type of information that might be collected
during a hypothetical RD/RA study as well as the conclusions and
interpretations made from that information.
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                                                      Example 5

          The manufacture and handling of explosives at U.S. Army industrial facilities has resulted in significant soil
          contamination. Previous remedy selection testing demonstrated the feasibility of using composting to remediate
          soils  and  sediments  that  had   been  contaminated  with  explosives   [2,4,6-trinitrotoluene  (TNT);
          hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX); and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX)] over a
          period of years. RD/RA testing was performed  on soils from an Army depot site with 1,500 cubic yards of
          explosives-contaminated soil. Initial contaminant concentrations within the soil ranged from 200 to 3,700 ppm for
          TNT, RDX, and HMX combined. The average combined concentration in the soil/sediment treated during RD/RA
          testing was 1,700 ppm. Asite cleanup goal of 100 ppmfortotal explosives was targeted. The study was designed
          to determine the maximum soil/sediment loading level, optimal amendments and process parameters, and the
          feasibility of using mechanically-agitated compost pile technologies. Individual laboratory studies were conducted
          for amendment selection and sample homogenization.

          Four pilot-scale compost piles consisting of soiled livestock bedding material, livestock feed, hay, fertilizers, and
          explosives-contaminated sediments were constructed onsite. A process control/monitoring system was designed
          to control and record temperature, provide oxygen, and sample and analyze exhaust gas from each reactor for
          moisture and oxygen. Periodic sampling to determine explosives concentrations was also performed. Data were
          fed to a computer located in the site trailer. Two amendment selection tests and two soil loading tests  were
          conducted. Different soil loadings were employed within each pile. Relatively small amounts of material  were
          treated.

          Results to date indicate extensive removal of TNT,  HMX, and RDX at soil loading  levels high enough to justify
          full-scale implementation.   During  treatability  testing,  bioassays were  also  conducted in addition  to
          compound-specific analyses. These assays indicated that the toxicity reductions generally parallel TNT and RDX
          reductions. It is known that intermediates are formed in the degradation of explosives but the bioassays indicate
          that the intermediates  are significantly less toxic than the parent compounds.

          A mixture of 10 percent contaminated soil and 90 percent amendments proved optimal; a combined explosives
          concentration of 75 ppm was obtained, reflecting a removal of 96 percent. However, effective composting was
          achieved at soil loading rates up to 40 percent by volume.  Materials handling requirements, operation and
          maintenance costs, material costs, and overall analytical requirements were evaluated. At full scale, it is estimated
          that treatment costs would be $250 perton ofcontaminated soil. Materials handling requirements, operation and
          maintenance costs, materials costs, and overall analytical requirements were analyzed.
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                                                   SECTION 4
                              TREATABILITY STUDY WORK PLAN
     Section 4 of this document is written assuming that an RPM is
     requesting  treatability   studies  through  a  Work
     Assignment/Work Plan mechanism. Although the discussion
     focuses on this mechanism, it can also apply  to situations
     where other contracting mechanisms are used.

     This section focuses on specific elements of the Work Plan for
     bioremediation treatability studies. These include test goals,
     experimental design and procedures, equipment and materials,
     sampling  and analysis, data analysis  and interpretation,
     reports, schedule, management and staffing,  and budget.
     These elements are described in Subsections 4.1 through 4.9.
     Complementing these subsections are Section 5, Sampling and
     Analysis Plan, and Section 6, Treatability Data Interpretation,
     which address the sampling and data analysis elements of the
     Work Plan in greater detail. Table 4-1 lists all of the Work Plan
     elements.

     Carefully planned treatability studies are necessary to ensure
     that the data generated are useful for evaluating the validity or
     performance of a technology. The Work Plan, prepared by the
     contractor when the Work Assignment is in place, sets forth
     the contractor's proposed technical approach for completing
     the tasks outlined  in the  Work Assignment. It assigns
     responsibilities and establishes the project schedule. It may
     also establish costs, although vendor costs may be considered
     confidential. The Work Plan must  be approved by the RPM
     before initiating subsequent tasks.  For more information on
     each of these  sections, refer to the generic guide.(52)

     4.1   TEST GOALS

     Setting goals for the treatability study is critical to the ultimate
     utility of the data generated. Goals appropriate to the tier of
     study must be defined before starting the treatability study. It
     is essential to consider how the different tiers of testing relate
     to and build upon each other when defining the study goals.
     Typically, remedy screening tests  are used to  determine if
     bioremediation is feasible with the site-specific waste material
     in question. The ability of a technology to  meet treatment
     goals is not the issue during remedy screening. Since it is not
     usually necessary to establish complete removal of the
     contaminant of interest, data compiled at this level of testing
     are normally  used to  assess contaminant biodegradability.
     Remedy selection tests, on the other hand, are used to answer
     the questions, "Will biodegradation reduce con-
Table 4-1.  Suggested Organization of Biodegradation
            Treatability Study Work Plan
 No   Work plan elements
Subsection
  1.    Project technology description

  2.    Remedial technology description

  3.    Test goals

  4.    Experimental design and
       procedures
 5.    Equipment and materials

 6.    Sampling and analysis

 7.    Data management

 8.    Data analysis and interpretation

 9.    Health and safety

 10.   Residuals management

 11.   Community relations

 12.   Reports

 13.   Schedule

 14.   Management and staffing

 15.   Budget
    4.1

    4.2


    4.3

    4.4


    4.5
    4.6

    4.7

    4.8

    4.9
taminant concentrations to meet cleanup goals?" and "Can the
contaminant be treated in a cost-effective manner?" Asa result,
remedy selection test goals are typically site-specific and may
be based on cleanup levels, risk assessments, or other criteria.
RD/RA testing is used to develop detailed design and cost
data and to confirm the applicability of full-scale performance.
Test goals at this tier emphasize process optimization, cost
minimization, and the collection of specific design data.

Brief descriptions  of remedy screening and RD/RA study
goals  were presented in   Subsections   3.3.1   and  3.3.3,
respectively. For an in-depth discussion of remedy screening
goals, consult the biodegradation screening guide. (53)
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     4.1.1   Remedy Selection Treatability
             Study Goals

     Remedy selection treatability study goals are based on current
     site contaminant levels and cleanup goals for soils, sludges,
     and water at the site.  The ideal goals for a remedy selection
     treatability test are the cleanup criteria for the site. In previous
     years,  cleanup  goals often  reflected  background  site
     conditions. Attaining background cleanup levels through
     treatment has proved impractical in  many situations.  The
     present trend is toward  the  development of  site-specific
     cleanup   levels  that  are  risk-based  rather  than
     background-based.

     For several reasons,  such as ongoing waste analysis  and
     ARARs  determination, cleanup criteria  are sometimes not
     finalized until the ROD is signed, long after treatability studies
     must be initiated. Nevertheless, treatability study goals need
     to  be established before the study has begun in order to
     assess the study's success. In many instances, this may entail
     an "educated guess" as to what the final cleanup levels will be.
     In  the absence of set  cleanup levels, the RPM  can estimate
     goals for the treatability studies based on the first four criteria
     listed at the beginning of Subsection 3.2. Previous treatability
     study results may provide the basis for an estimate of the
     treatability study goals when site cleanup goals have not been
     set. Cleanup goals can be based on regulatory requirements
     that do not account for the risk present at the specific site.
     Meeting standards can be expensive and  time consuming.
     Studies can help project the  time required to achieve the
     various target levels being considered.

     Cleanup criteria directly relate to the final management of the
     material. They  may  dictate the  need for complementary
     treatment processes to remediate the entire waste stream. For
     example, while biodegradation may be used to treat organics,
     a follow-on or pretreatment technology may be needed to treat
     metals and inorganics. Such combinations must be considered
     when planning the treatability studies and during the overall
     remedy evaluation phase. The development of graduated goals
     for contaminant reduction may fully address these complex
     needs.  For  example,  if biodegradation  can  reduce  soil
     contaminant levels to 100 ppm, no further treatment may be
     necessary. If, however, biodegradation can only reduce the
     contaminant level to 1,000 ppm, treatment  with another
     technology may be required.

     Data obtained during remedy selection testing should be used
     to assess whether a technology can meet site-specific cleanup
     levels. Consequently, testing should last until the contaminant
     concentration  falls  below the  study  cleanup  goal or
     contaminant removal  has leveled off  and  contaminant
     reductions  cease to  occur at a reasonable rate  (i.e.,  the
     "asymptote"). To accomplish this, it may be necessary to
     extend the length of the treatability study. Often the removal
     asymptote associated with a specific matrix and technology is
     a function of the starting concentration.  Therefore, in most
     cases  a  soil   sample containing  the  highest  level of
     contamination expected at  the site in question should be
     employed during remedy selection testing. It is important that
     the contaminant concentration not be so high that microbial
     activity is inhibited. In the event the maximum concentration
     is representative of only a minor portion of the media being
     treated,  treatability  studies  using  soils  with  "average"
concentrations may be more appropriate. Treatability studies
using average concentration soils are also appropriate if the
soil will be diluted during treatment (i.e., slurry treatment).

Ideally, a preliminary full-scale design and cost analysis will be
conducted prior to the remedy selection treatability study.
This preliminary analysis  will indicate the parameters of
particularimportance in the optimization and evaluation of the
technology. The degree to which the study "mimics" the
proposed  technology, the quality and reliability of the data
and its interpretation will be significantly impacted. Thus,
studies that closely simulate field conditions will provide the
most reliable information about a technology. Specific goals of
the remedy selection tier of testing are:

•   Measure the initial and final contaminant concentrations
    in the media and calculate the percentage of contaminant
    removal from  the  soil, sludge, or water attributed to
    biodegradation

•   Determine  the type and concentrations of  residual
    contaminants and/or byproducts  left  in the soil after
    treatment

•   Estimate reductions in  contaminant toxicity, volume, or
    mobility

•   Identify contaminant fate and the relative removals due to
    biological and nonbiological removal mechanisms

•   Produce the design information required for the next level
    of testing, in the event RD/RA studies are warranted

•   Develop preliminary cost and time estimates for full-scale
    remediation

•   Evaluate the need for  pretreatment prior to biological
    treatment (e.g., add bulking agents prior to composting or
    remove oversize particles prior to slurry-phase treatment),
    as  well as  long-term  operation,  maintenance,  and
    monitoring requirements

•   Evaluate the need for additional steps within the treatment
    train  (e.g.,  soil washing to  remove metals, soil vapor
    extraction to remove VOCs prior to ex situ bioremediation)

•   Assess the ability of bioremediation to meet the cleanup
    levels for a specific site

•   Determine  optimal conditions for biodegradation and
    evaluate the steps needed to stimulate biodegradation
    (e.g.,  nutrient addition,  surfactant addition,  cultured
    microbial populations)

•   Compare the performance,  cost, feasibility, timeliness,
    permitting  requirements, etc.,  of different treatment
    systems at a specific site

Toxicity reduction  may also be an important goal  in some
remedy selection,  treatability  studies,  especially  if this
parameter has been identified as a cleanup criterion for the site.
Toxicity reduction can be  demonstrated by performing of
toxicity tests on the treated and untreated media.  Toxicity
testing may also be used to establish test goals. Information
on specific toxicity tests is provided in Appendix A.
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     Example 6 is provided to demonstrate typical goals for a
     remedy selection study as well as the type of decision that can
     be made when these goals are achieved.

     4.2   EXPERIMENTAL  DESIGN AND
           PROCEDURES

     Careful planning during the design of a treatability study  is
     required to ensure  that appropriate data are  obtained. The
     experimental design must identify the critical parameters and
     determine  the required number of  replicate  tests.  This
     subsection discusses the different elements remedy selection
     treatability  study  design.  A brief description of remedy
     screening and RD/RA studies, addressing goals, design, and
     purpose,  can be  found  in  Subsections  3.3.1  and  3.3.3,
     respectively.

     The information presented in this subsection is  intended
     merely  as  a guideline or  starting point.  Because remedy
     selection treatability studies are site- and contaminant specific,
     this information should be modified, as necessary, for a given
     site.  Subsection 4.2.1  presents  an  overview of remedy
     selection experimental design. It is beyond the scope of this
     document to go into great detail on statistical experimental
     design, but useful texts on the subject are available. ^2Jr>

     A number of factors commonly influence the basic design and
     operation of biological studies. These factors have a profound
     impact  on  both treatability study  operation  and utility.
     Important  factors  to be  considered  when designing  a
     biological treatability study include the following:

     •    Moisture
     •    Nutrients
     •   Electron acceptors (e.g., oxygen, nitrate, sulfate)
     •   Microorganisms
     •   Duration of test
•   Inhibitory compounds and their control

•   Impact  of  nonbiological  removal   processes  (e.g.,
    volatilization, sorption, photodecomposition, leaching)

•   Toxicity testing

•   Bioavailability

Readers are referred to Subsection 4.1 for a discussion of
treatability study objectives and specific removal goals. Brief
discussions of other factors important for the design and
operation of biological studies are included in Subsections
4.2.2 through 4.2.13. Within these subsections references are
made to optimizing study  parameters in order to maximize
performance. It is important to stress that the intent is to
maximize performance under achievable field conditions in a
cost-effective manner in order to achieve intended results.
Subsections 4.2.14 and 4.2.15 discuss design and operational
parameters unique to treatability studies for in situ and ex situ
technologies,  respectively.  Although  each method  is
mentioned singly, using a combination of different testing
methods at the laboratory  and/or field scale may provide a
more accurate, cost-effective assessment of the technology's
capabilities at the  remedy  selection  level. For  example,
although large-scale field applications reliably mimic full-scale
applications, it may be easier and more cost-effective to use
laboratory-scale  testing to determine the effects mixing
patterns,  treatment   coverage,  transport processes,
temperature,  and   pH  have   on  biodegradation  rates.
Furthermore, depending on the technology  and site under
study, one study alone may not be able to  provide sufficient
information to select a technology reliably.

The  guidance provided in the  referenced  subsections  is
primarily designed for aerobic treatability studies;  however,
with some modifications, this guidance can also be applied to
anaerobic  treatability studies. Subsection  2.1.3 provides an
overview  of  common types   of anaerobic  organisms
encountered.
                                                         Example 6

          A remedy-selection laboratory study was performed to determine whether biodegradation could be used to remediate
          PCP-contaminated soil from a wood treatment facility (i.e., a pole yard). Since PCP is known to be amenable to
          biotreatment at concentrations less than 500 ppm, the RPM was able to bypass remedy screening testing. The object
          of the remedy selection study was to determine the rate and extent of PCP biodegradation achievable using solid- and
          slurry-phase treatment processes. Small soil pan and slurry phase studies designed to simulate full-scale processes
          were established, as were inhibited controls to measure the effect of abiotic processes on PCP removal. The average
          PCP concentration in the soil was 100 ppm, which was representative of site conditions.

          The studies demonstrated that  both solid- and slurry-phase processes could be used to biodegrade the PCP
          effectively. However, the rate and extent of biodegradation achievable was greater with slurry- rather than solid-phase
          processes. Ninety percent of the  PCP was removed within 4 weeks with the slurry-phase processes. Sixty percent of
          the PCP was removed within 12 weeks with the solid-phase process. An additional 8 weeks was needed to remove
          90 percent of the PCP during the solid-phase study. Abiotic processes did not contribute significantly to the removal
          of PCP.

          Based on the results of the remedy selection study and the need for rapid cleanup, RD/RA slurry-phase testing was
          performed to provide the data required to design and implement a full-scale slurry-phase remediation process.
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     4.2.1   Remedy Selection Experimental
             Design

     In formulating an experimental design, the total number of
     samples  taken  depends  on  the desired  difference  in
     concentrations that the experimenter wishes to detect, the
     measurement variability (the analytical coefficient of variation),
     and  Type  I  and  II error probabilities.  The  probability
     associated with a Type I error reflects the chance that the
     experiment will indicate that there is a statistically significant
     treatment effect when, in reality, none exists (false positive).
     Conversely, the Type II error probability is the chance of not
     detecting a significant treatment effect when in reality, the
     treatment  is  effective  (false  negative).  Traditionally,
     experimental designs have been constructed so that these
     errorprobabilities are on the order of 5 percent (e.g., 95 percent
     confidence levels).

     Replicate systems or replicate subsamples (at least duplicate
     and preferably triplicate) are recommended  for all remedy
     selection treatability studies to ensure reliable data. Replicate
     samples are used to measure overall analytical precision and
     should be performed for approximately  10  percent of the
     samples analyzed. Matrix  spikes are  used to assess the
     accuracy (the agreement between the analytical result and the
     actual compound concentration) of analytical  data. Matrix
     spikes are known concentrations of target analy tes added to
     a sample of soil, water, sediment, or air (the sample matrix) prior
     to sample preparation and analysis. Matrix spikes are used to
     evaluate sample bias (the effect the matrix has on the ability to
     detect  the  target  analytes accurately).  Surrogate spikes
     (compounds  similar to  the  target  analytes  in  chemical
     composition and behavior, but not normally found in the
     environmental samples) are also used to measure accuracy
     during organic compound analyses.

     Equipment rinsate, trip, and method blanks are used to assess
     the potential for sample contamination from equipment during
     sample collection/preparation, during sample handling and
     shipping, and arising from sample processing during analytical
     testing,respectively. Further information on quality assurance
     can be found in Test Methods for Evaluating Solid Waste(73)
     and  Data  Quality  Objectives  for  Remedial Response
     Actions/44' In general, the analytical variability associated with
     soil and sludge sampling and analysis can be quite high (on
     the order of 20 to 50 percent). Therefore, a sufficient number of
     samples must be taken for statistically significant effects to be
     observed. Additional information on sample size selection is
     available in many statistics textbooks.(6)(18)(24)

     Remedy  selection  treatability  studies range  from  small
     laboratory studies employing soil pans, slurry-phase reactors,
     or soil columns, to relatively large field applications utilizing
     small plots  of land (field plots) or contained soil systems.
     Generally, slurry-phase reactors, soil pans, and contained soil
     treatment systems are used to evaluate ex situ bioremediation
     technologies, while soil columns and field plots are more
     commonly used to evaluate  in situ technologies. Ultimately,
     remedy  selection  studies  should strive  to  simulate the
     conditions encountered during full-scale applications of the
     technology under study.

     The size of equipment used in remedy selection testing is
influenced by a number of factors, including the following

•   The amount of time and money available for testing

•   The uncertainty associated with the technology

•   The number of technologies being tested (as related to
    space, cost, and time restrictions)

The test system used during remedy  selection testing can
consist of a single large reactor or multiple small reactors.
Studies that employ large reactors include field studies, large
flask  studies,  and  soil pan studies.  Multiple reactors
consisting of serum bottles, small slurry reactors, and small
soil reactors may be set up in place of a single large system. It
is typically expensive and time-consuming to use field-scale
equipment to conduct remedy selection testing, particularly if
numerous technologies are  being considered. It may also be
easier to  examine the effects of mixing patterns, transport
processes,  temperature, pH,  and  nutrient  addition  in
laboratory-scale equipment. Field studies, however, usually
provide the best approximation  of full-scale performance.
These studies can also estimate the environmental impact and
cost with  a  higher  level  of certainty.  All  of  these
considerations will influence the size and scale of the system
selected for a remedy selection study.


4.2.2   pH

Most microorganisms thrive  within  a neutral  range (pH
between 6.5 and 8.5). However, many acidic or alkaline soils
support a viable microbial population capable of degrading the
contaminants  of interest. The indigenous microbes within
these soils may have evolved to the point where they cannot
survive or are  inhibited at a  different pH. If pH adjustment is
required to optimize a particular microbial population, additives
such as hydrochloric acid, potassium hydroxide, lime, or buffer
solutions may be used during treatability testing. The amount
of acid or base added to a soil sample during testing varies
with the buffering capacity of the soil.  Care must be taken to
ensure that the addition of amendments does not inhibit
biological activity. Furthermore, the pH has a profound effect
on abiotic contaminant reactions within the soil. Depending on
the specific characteristics of the  soil, changes may cause
materials (i.e., metals) within the soil to precipitate and may
increase the mobility of hazardous contaminants present in the
soil. Alternatively, a change in pH may cause the contaminant
to become  strongly  sorbed  to the  soil, thus inhibiting
degradation.  Consequently,  although a  neutral pH will
generally enhance microbial activity, pH  adjustment should
not be employed unless  an associated increase  in the
biodegradation rate is first demonstrated, and only if the pH
control is deemed feasible during remediation. In situations
where biodegradation is limited by  an  extreme pH (i.e., less
than 2), additives may be used to adjust the medium's pH.


4.2.3   Soil Characteristics

Soil and contaminant heterogeneity can significantly impact
the quality of the data generated and therefore must
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     be considered when designing a study. In general, as long as
     the  test results are not compromised, the media may be
     homogenized to  address  heterogeneous  characteristics.
     However, it may not be appropriate to use homogenized media
     when obtaining specific types of data pertaining to in situ
     biodegradation. Alternatively, the number of replicate samples
     taken may be increased to account for soil heterogeneity. For
     small reactors, where the entire contents are sacrificed at a
     sampling time, more replicate reactors should be prepared. For
     large reactors, where only a portion  of the contents are
     removed at each sampling time, multiple samples from the
     reactor  should  be taken. Large  reactors  must be  sized
     accordingly, so  that removal of multiple  samples does not
     adversely affect the processes taking place in the reactor.

     4.2.4  Temperature

     The temperature of the medium should be routinely monitored
     during  testing in  order  to  assess  its impact  on system
     performance (e.g., removal rates). Depending on the type of
     study  being  performed  and   the   technology  under
     consideration, temperature control may be required in order to
     optimize  biodegradation.  The  optimum  temperature  for
     biodegradation depends on the microorganisms present but is
     usually between 15° and 30° C for aerobic processes and 25 °
     to 35° C for anaerobic processes. Temperature control may be
     difficult in large scare treatability studies, particularly those
     utilizing in  situ   systems.  Although  groundwater  and
     subsurface  soil temperatures do not significantly change
     throughout the year, some  in situ studies  performed on
     contaminated media above the frost zone may experience
     marked decreases in removal rates during the colder seasons.
     Temperature  control  techniques  utilized  during in situ
     treatability  studies include  covering  the treatment  area,
     blowing heated air through tunnels in the treatment area,
     Installing in-ground heaters, and percolating heated water
     through the media. Vegetation can provide a cover to prevent
     the  surface soil from heating in the summer and to act as
     insulation to reduce heat loss in the winter.1-54-1

     4.2.5  Moisture

     Moisture levels are also routinely monitored and modified
     during  treatability testing in order to  assess the impact
     moisture content has on system performance. It is generally
     desirable to maintain the soil moisture level between 40 to 80
     percent of field capacity for solid-phase aerobic treatability
     studies; however, the actual range employed during testing
     depends on the nature of the medium under treatment and the
     operational characteristics of the technology under study.
     During solid-phase anaerobic treatability studies, the treatment
     area may be flooded to help to maintain anaerobic conditions.
     Moisture availability is not a  concern for  slurry-phase
     treatment, since surplus water is available.

     4.2.6  Nutrients

     Nutrient availability is  frequently a limiting factor during
     biological treatment. As a result, nutrient amendments are
     commonly employed during bioremediation  and biological
     treatability studies. The nutrients most frequently
added are nitrogen (e.g., ammonia nitrogen) and phosphorus
(e.g., phosphate). Organic nitrogen may be required by some
organisms. Protein supplementation has also been shown to
increase the degradation of heavy oils. Nitrogen must be
added cautiously in order to avoid changing the soil pH and
to prevent groundwater contamination due to excessive nitrate
formation. Supplemental carbon sources (glucose, acetate,
citrate, and corn starch solutions), inorganics (micronutrients,
mineral salt, and ammonia salt solutions, etc.), and/or vitamins
may also be provided. Agricultural fertilizers  and products,
such as alfalfa, blood meal, wild rice hulls, and manure, are also
common. Carbon to nitrogen ratios may range from 100:0.5 to
100:7.0, while carbon to phosphorus ratios may range from
100:0.1 to 100:1.0. Depending on the site and technology under
consideration, nutrient ratios may be  determined based on
initial TOC as an indication of carbon content. (Note: accurate
carbon mass determinations are difficult to obtain with highly
heterogeneous  soils.) These ranges are merely guidelines;
optimum nutrient conditions  are  site-specific. In general,
nutrient concentrations should be monitored and maintained
at some reasonably moderate but steady state concentration
determined experimentally.  Biodegradation in one or more
systems with nutrient addition can  be  compared  to the
biodegradation in  one or more systems  without nutrient
addition.

Soil water can be monitored for ammonia (NH3) phosphate
(PO4), nitrate (NO3), and nitrite (NO2) in order to determine
whether additional augmentation is required. Alternatively,
amendments can be added when biological activity slows
down. During soil plot studies, it may be beneficial to monitor
nutrient concentrations in groundwater obtained from both
up-gradient and down-gradient locations.

4.2.7  Electron Acceptors

Oxygen is the most common terminal electron acceptor for
aerobic microorganisms. Oxygen availability is also a common
limiting  factor  for  biological  treatment.  Oxygen addition
methods vary widely, particularly between different types of
treatability studies. During small-scale slurry-phase studies,
oxygen is typically transferred from the headspace into the
slurry by  shaking  or mixing.  Oxygen  addition in  larger
slurry-phase systems typically utilizes diffusers or aerating
mixers.Ex situ solid-phase systems (soil pans or contained soil
treatment  systems) typically obtain oxygen from mixing or
tilling. In situ systems are typically provided with oxygen
through the injection of liquids such as  water  with  high
dissolved oxygen (DO) levels or hydrogen peroxide or through
forced aeration systems such as bioventing.  Air, oxygen,
hydrogen peroxide, and nitrate amendments may be employed.
Gas injection or infiltration of water containing these oxygen
sources may be further enhanced by introducing microscopic
bubbles of gas (gas aphrons) into the soil at levels greater
than their solubility limits. Treatability study data demonstrate
that the soil retains the gas aphrons longer than air or other
gases directly  injected  into  the  soil;  however,  studies
pertaining to the full-scale application of aphrons have not
been identified. Gas aphrons are best suited for sandy soils.1-54-1

Oxygen requirements cannot be calculated from the con-
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     taminant concentrations because naturally-occurring organics
     and inorganics will  also be  degraded and will therefore
     contribute to the oxygen demand. Oxygen uptake rates and the
     oxygen content within soil pore water (i.e., DO) should be
     monitored  to  assess  oxygen  requirements.  Oxygen
     consumption data collected during remedy selection testing
     will be used to design the oxygen supply system. Generally,
     oxygen consumption is easier to monitor in closed reactor
     systems.

     When evaluating whether to employ percolation techniques to
     introduce aqueous amendments to the vadose zone during the
     aerobic  biodegradation  of  contaminated  surface  water,
     groundwater, or soil, it is important to estimate the amount of
     oxygenated water that will be  required to mineralize the
     contaminants  (and other carbon sources) at the site. Rough
     calculations can be made by  remembering the following
     relationships: 1) the maximum solubility of oxygen in water is
     approximately   8  mg/L  at  20 °C;  and 2) the complete
     mineralization of one pound of hydrocarbon (e.g., hexane)
     stoichiometrically requires approximately 3 pounds of oxygen.
     The resulting estimate can be used to verify whether sufficient
     oxygen will be present, similar to estimates of BOD or COD.

     Non-oxygen electron acceptors, such as nitrate, sulfate, or
     carbonate, can be used singly or in combination to enhance
     anaerobic  biodegradation.  The type of electron acceptor
     employed depends on the class of anaerobe responsible for
     contaminant   degradation   (facultative   anaerobic,
     sulfate-reducing, methanogenic, and denitrifying bacteria).
     Subsection 4.2.16 lists anumber of common electron acceptors
     according to types of anaerobes that utilize them.
     4.2.8  Microorganisms

     Nutrient addition, temperature control, PH control, etc., are
     generally performed in order to encourage the growth of either
     an indigenous or introduced microbial population capable of
     biologically degrading the contaminants of concern. Usually
     an indigenous population exists in the medium, which has
     already developed the ability to utilize the contaminants of
     concern. The purpose of biological testing and remediation is
     to modify any conditions that have impeded the growth of
     these microbes  and maximize  their ability to  degrade the
     contaminants of concern. The metabolic  diversity  of the
     naturally-occurring  microbial  community   should  be
     determined. Microbes capable of using a wide range of organic
     substrates, as well as specific substrate degraders capable of
     degrading certain compounds of interest, should be evaluated.
     Bioassays using target species may need to be performed.
     Parallel testing  to  evaluate the degradation attributed  to
     introduced  and  indigenous bacteria should be performed.
     Bacteria should  be enumerated at the beginning and end of
     each experiment at a minimum. Intermediate analyses may be
     appropriate since biological activity can be measured relative
     to oxygen uptake rates and microbial plate counts.

     If a microbiological characterization of the medium indicates
     that the naturally-occurring microbial activity is insufficient to
     achieve  the required  rates of biodegradation, even after
     environmental conditions have been
enhanced,  inoculation  can be  evaluated.  Commercially
available cultures reported to biodegrade the contaminants of
concern or microorganisms enriched from site samples may be
used. Researchers are cautioned against employing microbial
supplements without first assessing the relative advantages
associated with their use, as well as potential competition that
may occur between the indigenous and introduced organisms.
Generally,this evaluation may be accomplished by inoculating
one of two groups of identical test cells. Care must be taken
during testing to ensure that samples are not contaminated
with airborne  microbes.  During the evaluation  of in situ
technologies, the impact of site conditions such as  climate,
precipitation, soil properties, and carbon levels,  should be
evaluated in  order to  assess their impact on microbial
movement from the injection point to  the  contamination
location. Potential competition with other microorganisms, the
ability of the microbes to survive in a foreign and possibly
hostile (i.e., toxic) environment, as well as the microbes' ability
to metabolize a wide range of substrates should be evaluated.
Additionally,  when  choosing  a  commercially-marketed
microbial supplement, the RPM should ensure that there are
independent, peer-reviewed data supporting its applicability.
4.2.9  Test Duration

The duration of the treatability study must be considered in
order to allocate personnel and funding properly, as well as
plan for appropriate monitoring efforts over time. In general, at
least three or four time periods should be studied, including
the time-zero (T0) analysis. However, if the study goals are met
prior to the completion of all time periods, it is not necessary
to continue sampling at additional time periods.

Researchers are cautioned against stopping a study before the
site cleanup  goals are met, since initially high removal rates
can decrease to near zero values at concentrations above the
site  cleanup goals (see Subsection  2.2.4  for expanded
description of this phenomena). For all practical purposes this
asymptotic behavior defines the bioremediation end point.
4.2.10 Chemical Inhibition

Although acclimated microbes have been known to tolerate
very high concentrations of contaminants and metals given
long-term exposure,  elevated  concentrations may  inhibit
microbial activity. Studies may be performed to determine
whether biological activity is inhibited by a given chemical or
combination of chemicals present in the soil. These tests
should  determine  contaminant concentrations at which
microbial growth is supported, inhibited, or arrested. Inhibitory
concentrations may be estimated by monitoring reductions in
the number of actively degrading microorganisms present as
contaminant concentrations increase. Toxic effects may be
addressed by dilution, pH control, metals  control (e.g.,
immobilization,  volatilization,  chelation,  and washing)
sequential treatment, or by  employing  microbial  strains
resistant to toxicants. Inhibition is typically studied in  soil
pans or small slurry-phase reactors rather than larger-scale
systems.
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     4.2.11  Nonbiological Removal
             Processes

     Remedy selection  treatability tests  should  also  include
     controls to measure the impact of nonbiological processes,
     such  as volatilization,  sorption, chemical  degradation,
     migration, and photodecomposition. Inhibited controls can be
     established by adding formaldehyde, mercuric chloride (during
     non-EPA studies), sulfuric acid (added to lower the pH to 2 or
     below), or sodium azide to retard microbial activity. The media
     may also be autoclaved in order to inhibit microbial  activity.
     (Note: considerable difficulty has been reported using some
     chemicals to inhibit microbial processes in soils.) Contaminant
     concentrations are measured in both the test reactors and the
     control reactors at the beginning  of the study  (T0),  at
     Intermediate times, and at the end of the study. The  mean
     contaminant concentrations in  both the control and test
     reactors at the end of the test can be compared to their initial
     concentrations to see if a statistically significant change in
     concentration has occurred. The decrease in the  control
     reactors may be attributed to abiotic mechanisms, while the
     decrease in the test reactors would be a result of abiotic and
     biotic  processes.  The  difference in  mean  contaminant
     concentrations between the test reactors and  the inhibited
     control reactors at each time interval sampled will show
     whether there is a  statistically  significant  reduction  in
     contaminant concentration due to microbial activity. Care
     should be taken to assess the effects  that the different
     sterilizing agents can have on the chemical behavior of the
     contaminant system. For example,  formaldehyde  has the
     potential to act as an electron  donor, while  sulfuric acid
     addition will  impact pH.  Sodium azide  can, under certain
     circumstances, promote spontaneous  explosive  reactions,
     while  mercuric  chloride  may complex  certain  petroleum
     hydrocarbons,   leading  to   artificially  low  hydrocarbon
     concentrations. Placing the media in an autoclave may  result
     in the desorption of volatile contaminants. Finally, sterilization
     agents may modify soil structure.

     Complete sterilization of soils can be difficult to accomplish.
     Incomplete mixing of sterilization agents with soils can  result
     in pockets of surviving microbes in soil pores. In some cases,
     microbial populations can  transform and detoxify sterilizing
     agents. Additional sterilizing agents can be provided during
     the  test  to maintain  reduced  biological  activity.  The
     effectiveness  of sterilizing agents can  be measured by
     techniques such as microbial enumeration, respirometry, and
     enzyme analysis. Unless these or similar techniques show no
     microbial activity,  it may not be possible to distinguish
     between removal of contaminants by abiotic and biological
     processes  in  the   control  reactors.  However,  complete
     sterilization of the control is not necessary provided biological
     activity  is  inhibited sufficiently  so that a  statistically
     significant difference between the test and control means can
     be determined.  If sterilization is not complete, substantial
     degradation  in  the control can mask the occurrence  of
     biodegradation in the test reactor. Both during and at the end
     of the study, plate cultures  can be performed to determine
     whether controls were adequately sterilized.

     In addition to employing controls, a number of methods exist
that can be used to assess system performance.

Oxygen uptake and/or  carbon dioxide evolution can  be
monitored to assess the biological activity in a closed system.
(2i)(si) Oxygen uptake measurements are useful indicators of
biological activity in both the  test and control reactors.
Volatilization may  also be estimated by establishing a closed
system   and  monitoring  off  gases  for  VOCs  and
SVOCS.(21)(32:ii:8i:i For smaller-scale studies, organic traps and
collection systems formedia analysis may be used to evaluate
more  precisely  both  biological   and  abiotic removal
mechanisms. Alternatively,  an independent vapor extraction
simulation may be used to  assess the maximum amount of
VOCs in the matrix. This will provide  an estimate of the
maximum amount of abiotic loss due to VOCs. If significant
VOC losses are experienced (i.e., greater than 25 percent),
VOCs should be quantitated directly.

Ideally, performance should be assessed using a mass balance
approach capable  of   accounting  for  mineralization,
transformation, volatilization,  and  residual concentrations.
Samples of the  solid, liquid, and gaseous phases should  be
analyzed when   appropriate.  The  concentrations  of
contaminants, as well as any added substrates, metabolites,
electron   acceptors,  radio   labeled  compounds,  and
nondegradable tracers generated  by or introduced to the test
media should be determined. Radio labeling may be employed
to help to evaluate the fate of the contaminants and to perform
a mass balance calculation. Due to the relatively high cost
associated with purchasing radio labeled compounds, this
technique should be used only when a less expensive method
for calculating mass balance is unavailable. In general, the cost
of the labeled  compounds is  usually proportional to the
complexity of the compound. Mineralization studies using 14C
labeling may be particularly appropriate for  studies involving
either relatively toxic compounds  or compounds with the
potential to be transformed into toxic byproducts.

4.2.12  Toxicity Testing

Toxicity testing that examines environmental  and health effects
can be used to determine  whether the  risk  posed  by the
mediumunder study is adequately reduced by bioremediation.
Examples of common toxicity testing techniques can be found
in Appendix A, "Compendium  of Tools." Toxicity tests may
also be conducted for one or more of the time periods studied
and may be used to determine whether treatment is complete.

4.2.13  Bioavailability

In order for biodegradation to occur, the  microorganisms
responsible for contaminant degradation must have access to
the  contaminants  requiring  treatment.  The   biological
availability, or bioavailability, of a contaminant is a function of
the contaminant's solubility in water and its tendency  to
adsorb on the  surface of the soil.  Adsorption is the major
mechanism affecting the fate and transport of most organic
and inorganic compounds in soils. The tendency of organic
molecules to adsorb on the soils determined by both the
contaminant's and soil's physical and chemical characteristics.
Important contaminant  properties  that affect  adsorption
include: chemical struc-
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     ture; contaminant acidity or basicity (pKa or pKb); water
     solubility; permanent charge; polarity; and molecule size. In
     general, the leaching potential of a chemical is proportional to
     the magnitude of its adsorption (partitioning) coefficient in the
     soil. The  bioavailability of poorly-water soluble or sorbed
     organic pollutants may be improved by using surface active
     agents or surfactants.

     4.2.14  Experimental Design of In Situ
             Systems

     The  following  subsections  contain  experimental design
     information specific to soil column and field plot treatability
     studies. These studies are traditionally used to evaluate in situ
     technologies.  Table  4-2  outlines  some  of the  basic
     characteristics of the different testing methods employed and
     should be referred to when reading these subsections.

     Soil Column Treatability Studies

     Soil columns may be composed of soil, sediment, sand, or
     stone and can vary in size from 0.01 to 3,200 cubic feet. As
     outlined in Table 4-2, these studies last from  1 week to  6
     months and may be performed in both the laboratory and field.

     EPA's RREL is currently performing studies using in situ
     columns that are 9 inches in diameter and approximately  8
     inches  in length. These columns are isolated from the
     surrounding medium by a cylinder that is gently driven into
     the  soil,  sediment, or sand.  The columns are  open  at the
     bottom and have a top through which temperature and carbon
     dioxide measurements can be taken. They can be installed at
     any excavatable depth and covered with the  excavated soil,
     providing data on subsurface biodegradation. Future research
     will include the addition of amendments to the in situ soil
     columns.(1)(12)(6(i)

     Alternatively, the column of contaminated medium may
                                        be relocated to a laboratory for the treatability study. In order
                                        to simulate in situ conditions more closely, the soil is often
                                        disturbed as little as possible. Degradation rates determined
                                        using soil columns filled with homogenized soil may, however,
                                        be more representative  of an entire site than those using
                                        undisturbed soil cores.  There  are other advantages and
                                        disadvantages associated  with  soil columns filled with
                                        homogenized soil: they can be sampled without disrupting the
                                        integrity of the system but they  do not provide an accurate
                                        representation of the hydraulic  conductivity or nutrient
                                        transport of the undisturbed soil. To maximize the applicability
                                        of the  tests to in situ treatment, soil columns filled with
                                        homogenized soil can be used to determine degradation rates
                                        and undisturbed soil cores  can be used to estimate hydraulic
                                        conductivity and other parameters that do not require soil
                                        sampling.^-1 When soil columns filled with homogenized soil
                                        are used, the representativeness of the study can often be
                                        improved by compacting  the soil  until its transport properties
                                        are similarto those of the  undisturbed soil. Depending on the
                                        size of the columns and the desired number of sampling points,
                                        replicate soil columns or  replicate  samples  from a  single
                                        column may be used.

                                        In addition to providing  information on nutrient adsorption,
                                        hydrogen  peroxide  decomposition  (aerobic  systems), and
                                        "plugging" potential within the soil, soil column treatability
                                        studies  can provide information relative to the degree  of
                                        biodegradation that can be expected at various depths. These
                                        studies may also be designed to assess vertical movement of
                                        bacteria within contaminated soil and the utility of alternative
                                        oxygen sources.  It  should be noted, however, that  other
                                        factors  influencing the effectiveness of bioremediation are not
                                        examined in undisturbed  soil column studies. These factors
                                        include lateral infiltration of air, water, and contaminants, and
                                        the effects of groundwater pumping on soil characteristics.

                                        As with most other treatability studies, pH, moisture, nutrient
                                        addition, oxygen availability, and temperature
                            Table 4-2.  Remedy Selection Treatability Study Characteristics
Type of study
Field plots
Soil columns
Applicability
In situ bioremediation
In situ bioremediation
Scale
Field-scale
Lab- and
field-scale
Size
1 to 1,111 yd 2 plot of land*
0.01 - 3,200 ft3 of soil,
sand, sediment, or stone
Duration
2 months to 2 years
1 week to 6 months
     Soil pans

     Slurry-phase
     reactors
Solid-phase treatment

Slurry-phase and
 solid-phase (occasionally)
 treatment
Lab-scale        2 to 100 Ibs of soil            1 to 6 months

Field-scale       Greater than 20 gallons of    2 to 3 months
                 slurried media

Lab-scale        1 fluid oz to 20 gallons        1 to 8 weeks
Contained soil
systems
Composting, soil heap
bioremediation, and
solid-phase treatment
Lab- and
field-scale
7ft3to3,900yds3of soil
10 days to
10 months
         Field plot sizes are given as areas rather than volumes because treatment depths are frequently undefined.

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     are often monitored and modified. Moisture monitoring (daily
     or weekly) and nutrient addition are typical. Sprinkler systems
     and upflow percolation systems are commonly used. In order
     to encourage biodegradation beyond the initial layer of soil,
     oxygen is almost always supplied, frequently by injecting a
     liquid oxygen source (i.e., hydrogen peroxide or aerated water)
     directly into the column or by inducing airflow through the
     unsaturated soil. If volatilization is a concern, an airtight soil
     column equipped for offgas monitoring using organic traps
     may be needed.

     Field Plot Treatability Studies

     Field plots may provide the closest approximation to fullscale
     in situ treatment. These treatability studies, which last from 2
     months to 2 years, are typically conducted on plots ranging in
     size from 1 to 1,111  square yards (i.e., one-fourth of an acre).
     These plots are usually located within  a portion of the area
     requiring remediation.  (Note: plot sizes are  given as areas
     rather than volumes  because treatment depths are frequently
     undefined.) Because field plots are relatively  large, field plot
     treatability studies typically use replicate sampling.

     Field plots often use techniques and equipment that are similar
     or identical  to those used in full-scale remediation. These
     studies can  closely  approximate many  aspects of full-scale
     treatment. The data obtained from these studies can often be
     used to:

     •    Develop the design for full-scale treatment

     «    Optimize specific operating parameters (e.g., nutrient and
         oxygen addition rates)

     •    Develop cost and schedule estimates for the full-scale
         system

     Field plot treatability studies frequently employ pH monitoring
     and adjustment (using lime or phosphoric  acid).  The soil
     moisture is also frequently monitored and adjusted duringfield
     plot treatability studies. Infiltration and irrigation systems are
     commonly used to add water to a field plot.

     The nutrient addition methods chosen for treatability studies
     that utilize field plots are similar to those chosen for full-scale
     treatment. Nutrient addition alternatives include the following:

     «    Addition of chemical nutrients to the water being applied
         to the soil

     •    Application of agricultural fertilizer

     Regular nutrient monitoring is also recommended to ensure
     that nutrient addition rates are sufficient but not excessive.
     One logical scheme consists of groundwater monitoring both
     up-gradient and down-gradient of the nutrient injection points.
     Typical analytes include nitrate (NO3"') nitrite (NO2"'), kjeldahl
     nitrogen, ammonia(NH3), and phosphate  (PO4"3); less common
     analytes inclule sulfate (SO4"2) and iron.

     The oxygen addition techniques chosen for treatability studies
     that utilize field plots are similar to those chosen for full-scale
     treatment. Oxygen addition alternatives  typically used at this
scale  include  forced aeration/bioventing  and  hydrogen
peroxide injection. Oxygen availability should be monitored
routinely to ensure that it is adequate.

Temperature should be considered in the design of treatability
studies utilizing field plots since the ground temperature above
the frost line naturally varies with the season and climate and
can significantly  impact  biodegradation rates. Temperature
control (typically  a heating system) may be helpful for some
studies.  However, studies are generally timed to occur during
those  seasons with  the  most favorable  weather  and
temperature conditions.  During those  studies  in  which
temperature variations are expected to impact biodegradation
processes, temperature monitoring should be employed to
assess its impact on the biodegradation rate. In some studies,
it may be helpful to monitor the temperature of both the soil
and the groundwater.

Instead  of using  inhibited controls, soil plot studies have
traditionally used control plots that are monitored and sampled
in an identical fashion to normal test plots, but do not receive
enhancement. The data obtained from test and control plots
are compared to determine whether any amendments (e.g.,
nutrients, oxygen) added to the test plots actually enhanced
biological activity.

Specific concerns regarding contaminant volatilization  or
migration  may require the  application of different types of
controls. If volatilization is a concern, the  plots may be
enclosed in airtight covers and the air monitored for volatile
contaminants. If migration is a concern, the test plots  and all
but one  control plot should be isolated from the surrounding
soil. The results will indicate whether it will be  necessary to
take steps to limit  volatilization or migration during full-scale
treatment. A leachate collection system may be required to
obtain a mass balance closure and to prevent contamination of
surrounding areas. Leachate and  underbedding material may
be sampled to assess the potential for contaminant migration.
Specialized volatilization sampling devices may be employed
to measure contaminants emitted to the atmosphere.

4.2.15  Experimental Design of Ex Situ
        Systems

Three ex situ  experimental designs are covered in  this
subsection: soil pans, contained soil treatment studies, and
slurry-phase tests. These studies are  generally shorter  in
duration than in  situ studies  and place less emphasis on
evaluatin g and accounting for specific site characteristics (e.g.,
soil permeability). Table 4-2 outlines  some  of  the basic
characteristics of the different testing methods employed and
should be referred to when reading these subsections.

Soil Pan Treatability Studies

As outlined in Table 4-2,  soil  pan studies  are  generally
short-term studies (1 to 6 months in duration) performed in the
laboratory within  shallow pans capable of holding between 2
and 100 pounds of soil.  The medium (i.e., soil, sediments)
treated during these studies will usually be taken from the site
and   should  possess contamination  levels  which  are
representative of the site. Because soil
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     pans are typically small, operating parameters (e.g., nutrient
     availability, pH,  moisture,  oxygen,  and temperature)  are
     relatively easy to control and study costs are relatively low
     (referto Table 4-2). Generally oxygen addition can be provided
     by tilling or mixing the soil one to three times per week, while
     moisture is monitored and amended either daily or weekly.
     Since conditions are usually so easy to control, these studies
     are more likely to reflect ideal operating conditions rather than
     the less-than-perfect conditions typically experienced during
     field applications.

     Because soil pan studies are typically small, replicate systems
     are recommended. These additional systems eliminate many of
     the data quality problems associated with collecting replicated
     samples from a single soil pan without generating substantial
     cost. When designing a soil pan treatability study, sampling
     requirements must be considered. If an entire soil pan is to be
     sacrificed at each sampling time, substantially more replicates
     need to be prepared. The volume of material in each pan,
     however, can be  significantly  smaller. If  subsampling  is
     employed, fewer replicates are generally required. The volume
     of soil in each pan,  however, must  be sufficient to allow
     removal of sample aliquots without adversely affecting the
     continued use of the pan for the study. During each sampling
     effort, a minimum of three samples (pans or aliquots) from the
     test group and two
samples from the control group are recommended.

An abiotic control can be prepared for soil pan treatability
studies in order to assess contaminant reduction due to
nonbiological mechanisms. Depending on previous testing
initiatives, inhibition testing may also need to be included
during the remedy selection treatability study. During other
types of  treatability studies,  inhibition  tests may not be
necessary. If volatilization is a concern, the soil pans may be
tested in  a closed (i.e.,  airtight) system  and monitored for
volatile contaminants. Alternatively, organic traps may be
employed to assess volatilization in closed systems.

Example 7 describes a simple experimental design for a remedy
selection treatability study utilizing a soil pan.

Contained Soil Treatment Experiments

Contained soil treatability  studies are frequently used to
assess the effectiveness of composting, soil heaping,  and
other solid-phase biotreatment technologies. Although they
can be performed within a laboratory setting, the majority of
these studies take place in the field using larger-scale systems.
As outlined in Table 4-2, these studies typically last from 10
days to 10 months and handle moderate to very large volumes
of soil (7 ft3 to 3,900 yd3).
                                                          Example 7

        Twenty thousand cubic yards of soil were contaminated with creosote during the life of a railroad tie treating plant. Approximately 4
        percent of the soil was com posed of com pounds that were extractable using benzene (i.e., benzene extractables). Average total PAH
        concentrations were 900 mg/kg. Total PAHs in the soil ranged from 100 to 2,000 ppm, and benzene extractables ranged from 2 to 10
        percent by weight. A soil pan study was performed to determine whether cleanup criteria (i. e., 100 ppm for target PAH compounds
        and 1 percent for benzene extractables) could be achieved using solid-phase biological land treatment.

        Testing was conducted using stainless-steel pans (6.0x10.0 x 2.5 inches). Each pan contained approximately 2 pounds of material.
        At the beginning of the study, water was added to obtain a 20 to 25 percent moisture content, a range conducive to microbial activity.
        The pans were incubated for 8 weeks at ambient temperature. The soil was tilled daily with a hand trowel to optimize aeration and
        contact between the microorganisms and the contaminants. Pans were covered with polyethylene film to minimize moisture loss during
        the incubation period without preventing oxygen transfer. Water was added to the pans to maintain the moisture content at 20 to 25
        percent. The pH of the pans was monitored at regular intervals to ensure that it remained within the range considered conducive to
        microbial  activity (7.5 to 8.5). Microbial activity was assessed by enumerating the numbers of microorganisms in the pans at regular
        intervals. The numbers of aerobic heterotrophic microorganisms were determined by standard enumerative techniques with a 1 -gram
        sample removed randomly from each pan initially and after 2, 4, 6, and 8 weeks of incubation  (Appendix A - Compendium of Tools).
        At each sampling point, nine random samples from the entire depth of the pan were removed and composited to provide the  sample
        for chemical analysis. Sampling points were sampled initially, and at 2,4, 6, and 8 weeks and analyzed for benzene extractables (which
        was  considered a inexpensive indicator of trend). Gas chromatography/mass spectrometry (GC/MS) techniques were used to measure
        the concentrations of VOCs and SVOCs (PAHs) initially and at 8 weeks. All experiments were performed in triplicate to ensure reliable
        data.

        Analytical data demonstrated  that the benzene  extractable and total  PAH contamination dropped  to  1.0 percent and 80 ppm,
        respectively, during the 8-week study. Based on these removals, as well as other operational data evaluated during parallel testing,
        researchers estimated that it will take 2 years to achieve the treatment goal of 100 ppm total PAHs and 1 percent benzene extractables
        at a cost of approximately $40 per cubic yard.
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     Although the design of contained soil treatment experiments
     depends on the characteristics  of the specific technology
     under analysis, these studies generally  provide detailed
     information regarding onsite applications of the technology
     that may be used to supplement RD/RA studies. Polyethylene
     liners, leachate collection systems, forced aeration systems,
     soil infiltration  systems,  mixing equipment, and humidity
     recorders are among the auxiliary  equipment that may be
     employed during these experiments.

     During both aerobic and anaerobic studies, pH control and
     regular (weekly or biweekly) pH monitoring are recommended.
     Supplements may  be added as needed. Bulking agents may
     also be required. If inhibition testing reveals that contaminant
     concentrations  are  inhibiting  microbial  activity,  the
     contaminant concentrations  may require  dilution by the
     addition of less-contaminated soil to maximize treatment.

     Moisture content,  rainfall, and pan evaporation rates may be
     monitored daily or weekly to help to evaluate watering needs.
     Readings should be taken at several depths to ensure the
     bottom of the treatment area is not saturated and becoming
     anaerobic.  The soil can  be maintained near field moisture
     capacity by using infiltration systems, water sprays, and
     irrigation systems.

     Nutrient augmentation  is often limited to nitrogen  or
     phosphorus addition, but may include potassium and carbon
     addition. The optimum C:N:P:K ratio is dependent on the
     amount  and type  of  waste  requiring treatment and the
     microorganisms  to be optimized.  Commercial fertilizers and
     manure are two of the more common supplements applied
     during confined solidphase treatability studies. Regular (daily
     or  weekly)  sampling   for  nutrient  concentrations   is
     recommended, as nutrients are usually added at the beginning
     of the treatability  study and whenever testing indicates that
     concentrations  are  below the  optimum  operating  range.
     Aeration is frequently accomplished using mechanicalmixing
     or forced aeration. Routine (daily or weekly) monitoring  is
     recommended to ensure that adequate oxygen is available.

     System temperature should be monitored daily. The optimum
     temperature range for most aerobic contained soil treatment
     test plots is similar to the range recommended for other aerobic
     treatment  methods (15° to  30°  C).  However,  certain
     microorganisms such as  white rot fungus achieve optimal
     degradation at significantly higher temperatures (i.e., 39° C for
     white rot fungus). The actual operating temperatures are often
     lower, however, since only a limited number of land treatment
     studies  are performed  within  a controlled  environment.
     Composting studies generally operate at higher temperatures
     (approximately 55° to 70°  C).

     Contaminant  reductions  associated  with  volatilization,
     adsorption, or chemical incorporation (covalent bonding) into
     the  compost  matrix,  or  chemical  degradation are  rarely
     evaluated during contained soil studies. Like soil plot studies,
     emphasis is placed on determining the relative increase  in
     biodegradation caused by enhancing conditions conducive to
     biodegradation. Thus researchers attempt to determine the net
     increase in biological degradation experienced by comparing
     removals in enhanced systems with removals in biologically
     active, nonenhanced systems.
Slurry-Phase Tests

During  slurry-phase   studies,  contaminated  media  are
suspended within an aqueous solution that is generally 60 to
90 percent liquid.  Continuous or  intermittent mixing to
encourage both aeration and contaminant/nutrient availability
is frequently employed. As shown in Table 4-2, the scale of
remedy selection slurry-phase experiments may range from 1
fluid ounce vials to sludge ponds with operating volumes of
up to 70,000 gallons. More common, intermediate sizes include
0.1 to 0.3 gallon flasks, 5 to 20 gallon reactors, and 0.3 to 130
gallon sequencing batch reactors. Large-scale field studies
(greater than 20 gallons) generally provide better information
relative to the  onsite application of the technology and are
often used to supplement RD/RA requirements. Please note,
however,  that  it is extremely  difficult to  subsample large
reactors efficiently over a period of a study, i.e., to remove the
same  solids-to-liquid ratio at each sampling point. Feed tanks,
carbon adsorbers, vapor absorbers, and digesters may be
included in the treatment trains used during large-scale field
studies.

Large-scale field applications treating volumes of 20 gallons or
greater last an average of 2 to 3 months.  Studies  using
sequencing batch  reactors are typically much faster, with
hydraulic  residence times  of 1 to  10 days.  Small-scale
laboratory experiments typically last between 1 and 8 weeks.

Temperature should be monitored daily to assess possible
impacts on biodegradation rates. Monitoring instrumentation
can range from a thermometer in a shaker-water bath,  to a
series of thermosensors within the batch reactors. Temperature
controls,  such as  covers or immersion heaters, may be
necessary. Laboratory testing is likely to take place at ambient
temperatures, while the temperatures in field-scale studies tend
to vary with the season and climate.

Since most aerobic  slurry-phase  treatability  tests  are
continually mixed, the application of chemical oxygen sources
is unnecessary. During large-scale testing, floating aerators,
downdraft mixer/surf ace aerator combinations, ordiffusersmay
be employed to provide oxygen. Oxygen uptake or DO content
may be measured to determine the degree of biological activity.
If inhibition testing reveals that contaminant concentrations
are excessive,  the samples may require dilution to maximize
testing results  and treatment.  If volatilization is a concern, the
slurry reactors may be  sealed  with airtight covers  and
monitored for  volatile contaminants. Alternatively, organic
traps may be employed to assess volatilization.

Example 8 describes a simple experimental design for a remedy
selection treatability study utilizing a slurry-phase  system.
4.2.16  Anaerobic Studies

During anaerobic treatability studies oxygen availability must
be reduced  or eliminated. This can  be accomplished  by
consuming the DO in the media (supplying excess electron
donors to the microbial population)  and by  limiting the
diffusion of more oxygen into the system (e. g., by flooding the
soil or establishing an oxygen-free
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     gaseous phase  above the surface  of the medium).  An
     oxygen-free gaseous  phase may be  established by:  1)
     evacuating the headspace with a suction pump and refilling
     the headspace with a non-oxygen containing gas (hydrogen,
     helium, ornitrogen), or 2)placingthe test system (i.e., soil pan)
     in a gloveboxwith an oxygen-free atmosphere. Alternatively,
     a gas pack generator can be used to produce an anaerobic
     atmosphere.  When  attempting   to  establish  anaerobic
     conditions using a hydrogen atmosphere, palladium-coated
     pellets of aluminum may be employed to promote the chemical
     binding between hydrogen and the last traces of oxygen. Some
     anaerobic microbes require CO2 which is usually readily
     available in soil systems. In such cases a blend of N2 and CO2
     can be used. Trace oxygen can be scrubbed from this medium
     by passing it over hot copper.

     Since facultative anaerobic, sulfate-reducing, methanogenic,
     and denitrifying bacteria typically employ different electron
     acceptors (nitrate, carbonate, or sulfate), as well as produce
     dissimilar byproducts  and metabolic intermediates,  test
     designs employed during anaerobic testing depend largely on
     the type of microorganisms used to perform biodegradation.^62)
     Table 4-3 outlines some of the different electron acceptors
     used  and byproducts produced by the different types of
     microorganisms.

     Many anaerobes fail to grow unless the medium has been
prereduced (i. e., poised) to a level at or below a particular redox
potential or Eh (usually -150 mV to -350 mV at pH 7). Therefore,
poising agents such as cy steine hydrochloride, ascorbic acid,
thioglycollate, and starch may need to be added during
testing. The precise medium-specific Eh that will support the
growth of a given anaerobe depends  on the size of the
inoculum (ongoing growth tends to lower the Eh of the
surrounding medium), the identity of the poising agent, and
the specific electron acceptor that is supplied.   ' The redox
indicator resazurin may be used to demonstrate that anaerobic
conditions are maintained throughout the study.1-7' Since the
Eh of the media will determine which groups of microorganism
are active, the particular physiological group of anaerobes to
be stimulated  should be identified during remedy screening
testing.

Two techniques commonly used during anaerobic testing, the
Mclntosh andFildes' anaerobic jar and the roll tube technique
are listed in Appendix A,  Compendium of Tools. Further
information on anaerobic processes can be obtained from
various sources.
              (68)
4.3  EQUIPMENT AND MATERIALS

Standard laboratory equipment such as mixing flasks and
sample collection bottles should be available for all treatability
studies. Additional equipment and material re-
                                                       Example 8

        An refinery impoundment was used for 40 years as a settling pond for oily waste streams. Following refinery shutdown,
        a total volume of 25,000 cubic yards of oily sludge was identified. A characterization of the sludge revealed that the
        material was 15 percent oil and grease (O&G) and 50 percent solids. Average PAH and carcinogenic PAH (CPAH)
        concentrations  of 1,180 and 98 ppm were also identified. A laboratory slurry-phase study was performed to evaluate
        the feasibility of using slurry bioremediation technology to remediate the oily sludge.  Data were also sought regarding
        the impact that  pH, surfactant addition, O&G concentrations, and total solid loadings can  have on treatment efficiency.
        Site cleanup goals of 2 percent for O&G, 100 ppm for PAHs, and  10 ppm for CPAHs were targeted.

        The slurry-phase study was conducted in 4 L stainless-steel tanks with spargers located on the bottom for aeration.
        The slurry was continually mixed with a rotating impeller located in the middle of the reactor. Sludge was combined with
        deionized water and nutrients as required. Dried sludge was obtained by air-drying at room temperature prior to
        make-up of the  slurry. The bioreactors were incubated at ambienttemperaturefor4 weeks. Tankvolume was monitored
        daily; pH and DO were monitored daily. Triplicate samples were taken on days 0, 1, 3, 5, 7, 14, 21, and 28 to determine
        O&G concentrations, PAH concentrations, and microbial activity. O&G concentrations were used to measure the rate
        of biodegradation of the contaminants in the soil  sludge. Microbial activity was  assessed  by both microscopic
        examination using a phase contrast microscope and standard  enumeration techniques. The numbers of aerobic
        heterotrophic microorganisms and phenanthrene-degrading microorganisms were determined (refer to Appendix A,
        Compendium of Tools).  Increases in microbial populations  in conjunction with losses in  contaminant indicated
        enhanced  biodegradation.  Triplicate samples were also  removed  initially  and at the end of the experiment for
        determination of VOC and SVOC concentrations by GC/MS. All experiments were  performed in triplicate  to ensure
        reliable data. Past experience with oily wastes ruled out the need for toxicity testing.

        Based on mass balance data obtained from the study, O&G, PAH, and CPAH contamination were reduced by 89, 93,
        and 95 percent, respectively. Corresponding final contamination levels within the sludge residuals from the reactors
        were 1.7 percent for O&G, 87 ppm for PAHs, and 5 ppm for CPAHs. Total solids was reduced by 15 percent. Preliminary
        estimates place treatment costs for the site at approximately $125 per cubic yard. Based on these data, a large-scale
        tank study was  proposed to evaluate the technology further.
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                     Table 4-3. Characteristics of Anaerobes Classified According to Physiological Nature
      Bacteria type
Electron acceptors
Byproducts
      Denitrifying


      Facultative anaerobic


      Sulfate-reducing


      Methanogenic
Nitrate and organic nitrogen, in the
presence or absence of oxygen

Organic acids or inorganic molecules, in
the absence of oxygen

Sulfate, elemental sulfur, reduced sulfur
compounds

Hydrogen and CO2, acetate
Excluding excess nitrate, unanticipated
and undesirable byproducts are unlikely

Metabolic intermediates differ under
aerobic and anaerobic conditions

Hydrogen sulfide
Methane
     quirements specific to the  type  of study employed (e.g.,
     slurry-phase, soil pan, etc.) are listed in Table 4-4.

     4.4   SAMPLING AND ANALYSIS

     The Work Plan should address the test's needs for sampling
     and analysis work, as well as quality assurance QA) support.
     The SAP, which will be prepared after Work Plan approval,
     helps to ensure that the samples are representative and that
     the quality of the analytical data is generally known. The SAP
     addresses field sampling, contaminant characterization,  and
     the  sampling and  analysis during treatability testing. It
     consists of two parts: the FSP and the  QAPP. Further
     discussion of the FSP and QAPP and specific sampling and
     analytical tests and protocols are presented in Section 5  and
     in the generic guide.
     4.4.1  Field Sampling

     Field samples  are taken to provide baseline  contaminant
     concentrations and  contaminated material for treatability
     studies. A sampling plan should be developed that directs the
     collection of representative samples from the site for the
     treatability test. The sampling plan should be site-specific and
     describe the number, location, and volume of samples to be
     collected. The objective  of the sampling  plan must  be
     consistent with treatability test objectives. For example, it may
     be more appropriate to perform testing on  a relatively
     "undisturbed" or intact soil sample when evaluating an in situ
     technology.  This  approach  is  particularly important for
     determining  baseline   information,  such  as  hydraulic
     conductivity  and  porosity.  When  consistency between
     samples is important, as in determining optimized nutrient
     addition rates, homogenized and subdivided soil samples may
     be preferred.  This approach minimizes  initial differences
     between  test  samples, increasing  the  confidence that
     differences in results are caused by the manipulated parameter.
     Homogenizations and composite  sampling are also preferred
     if an ex situ technology  is being considered, since the
     characteristics  of an intact soil sample (e.g., relative to its
     ability  to  mimic  permeability,  nutrient and  contaminant
     dispersal) are less relevant.  The EPA document,
                              Methods for Evaluating the Attainment of Cleanup Standards,
                              provides information on sampling plan design/64'

                              Generally, samples representative of conditions typical of the
                              entire site  or a defined area (e.g., hot spots) within the site
                              should be  collected.  The selection of soil sampling locations
                              should be  based on knowledge of the site. Information from
                              previous   soil samples,  soil   gas  analysis  using  field
                              instrumentation, and obvious odors or residues are parameters
                              that can be used to specify sample locations. Alternatively, a
                              random, stratified, or systematic sampling  plan  could be
                              implemented to allow results to be more easily expressed in
                              statistical  terms.1-64-1  This approach, which does not use the
                              sampler's knowledge of the site, may increase the likelihood of
                              missing hot spots bioremediation may not be capable of
                              effectively treating.  The EPA document, Test Methods for
                              Evaluating Solid Waste, provides a  discussion of random,
                              stratified and systematic sampling as well  as sample  size
                              requirements/73-1

                              Composited samples representative of the media requiring
                              remediation are ideal samples for treatability studies that do
                              not require intact undisturbed media. Compositing reduces the
                              variability  in contaminant concentration and provides more
                              accurate data on soil concentrations before and after testing.
                              Compositing  is usually appropriate for soils containing
                              nonvolatile constituents; however, if the target contaminants
                              are volatile, care  should be taken to minimize losses during
                              compositing. Compositing samples on ice is a good method of
                              reducing volatile compound losses, as long as the samples are
                              not allowed to freeze. The EPA document, Groundwater Issue:
                              Soil Sampling and Analysis for Volatile Organic Compounds,
                              provides additional information on this topic.(50)

                              When  obtaining media  samples to use during biological
                              treatability studies,  emphasis should also  be placed on
                              maintaining the biological integrity of the samples. Improper
                              handling of soil samples can reduce microbial populations
                              and/or inactivate  extracellular enzymes which are functional
                              under normal field conditions. Although changes to the soil
                              are inevitable during handling, it is important to minimize these
                              changes and their  impacts on  microbial studies. Drastic
                              changes in soil moisture, temperature, etc. should be avoided.
                              To the
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Table 4-4. Equipment and
Soil columns
Test systems ! Lab/field cylinders
Field plots
! In-ground barriers
! Above-ground
beams
Materials
Slurry reactors
! Lab reactors
! Small tanks
(lab/field)

Contained soil system
! Lined/beamed area in the
field
! Soil pans (lab)
      Contaminant
      sampling
Small coring
device
Split-spoon
Shelby tube
   Bailer
   Sample port
Split spoon
Shelby tube
      Moisture control
Sprinkler
Upflow
percolation
Water can
Sprinkler
Subsurface
irrigation
NA
Sprinkler
Watering can
      Temperature
      measurement
Temperature
probe
Soil thermometer
Temperature
probes
   Thermometer
   Temperature
   probe
Temperature probe
Soil thermometer
      Nutrient addition
      (Agricultural
      chemicals or
      other
      chemicals)
Pumps/sprinklers
for dissolved
nutrients
Shovel/rake/etc.
Tractor
Spreader or sprayer
Sprinkler/irrigation
system for
dissolved nutrients
   Metering
   pump
   Mix tank
Trowel/shovel/rake/etc.
Tractor
Sprinkler/irrigation
      Oxygen addition
      (for aerobic
      studies)
Aerator
Oxygenated water
injection system
H2O2 injection
system
H202
Tractor and disc
garden tiller
Bioventing/forced
aeration
   Floating
   aerators
   Diffusers
Trowel, hand tool, etc.
      pH control
pH probe for soil
dissolved in water
Acid
Base
pH probe for soil
dissolved in water
Lime
Phosphoric acid
   pH probe
   Acid
   Base
pH probe for soil
dissolved in water
Acid
Base
     extent possible, samples should be collected using procedures
     that minimize the addition or transfer of microbes between
     samples (e.g., steam cleaning sampling equipment between
     samples) and the  introduction of foreign material (i.e., by
     sampling devices or drilling residues/4-1 samples either should
     be used promptly or placed in thin-walled polyethylene bags
     or glass containers and stored at 5 to 10° C. The polyethylene
     bag will reduce moisture losses, while permitting  some gas
     exchange. Since the biological activity o fa sample decreases
     with time, samples held for greater than 48 hours are generally
     unsuitable forbiodegradation studies. Storage at sub-freezing
     temperatures should not be used to lengthen the acceptable
     storage period as it alters the characteristics of the microbial
     community.  Furthermore,  samples  which  are allowed  to
     completely air dry will most likely experience an anomalous
     burst of respiratory  activity  upon remoisturizing  and  a
     selection for the fungal components  of the
                                    microbial population.

                                    The method of sample collection is site-specific. For example,
                                    drill rigs or hand augers can be used to collect samples,
                                    depending on the depth of the sample required and the soil
                                    characteristics.  Soil  cores,  which  preserve  the media's
                                    structure, are ideal for determining air permeability, as well as
                                    for providing data regarding  the  impact  of geological
                                    formations,  contaminant/depth  relationships,  and  other
                                    site-specific media characteristics. Equipment for obtaining soil
                                    samples from upper layer soils can be found in Table 4-5.(68)

                                    Regardless of the technique used to collect the sample, an
                                    adequate volume of soil sample should be collected from each
                                    sampling location to account for replicate treatability tests and
                                    analytical  QA/QC requirements. Guidelines for statistical
                                    sampling procedures are  give in the documents Hazardous
                                    Waste Land Treatment and Test Meth-
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     ods for Evaluating Solid Waste (SW-846).
                          Table 4-5. Equipment for Field Collection of Soil Samples
     4.4.2   Media Analysis During the
             Treatability Study

     Contaminant concentrations  should  be determined at the
     beginning of the study and at the sample times chosen in the
     experimental design. Consult  SW-846(73) for the appropriate
     methods. GC or GC/MS techniques can be used to evaluate the
     biodegradation of a wide range of components and confirm
     that  the  bioremediation  process is treating all of the
     compounds of concern, and  not only a limited  set of the
     compounds. When determining VOCs and SVOCs, it may be
     possible to minimize costs  by substituting GC or other
     appropriate  methods  (e.g.,  high-performance  liquid
     chromatography (HPLC) for GC/ MS methods. However, this
     is not advised for heavily-contaminated soils that contain a
     significant  amount  of other   "non-priority   pollutant"
     compounds and degradation intermediates. All sampling and
     analysis should be  performed in accordance with the  SAP
     (Section 5). In order to obtain a statistically relevant measure
     of background contamination levels, it is necessary to take a
     significant number of replicate samples that are representative
     of the area being sampled.

     The concentrations of some important matrix parameters are
     determined by using standard analytical chemistry methods
     (Table 4-6). These parameters  are important for the design of
     remedy selection testing and RD/RA studies and should be
     determined  before  the treatability  study  begins. These
     methods  should not be  used  as an  indication of the
     inappropriateness of the technology.

     Direct microscopy  (e.g., fluorescent staining, buried-slide
     technique),  adenosine triphosphate (ATP) analysis, enzyme
     activity  analysis, and culture  counts  (e.g., plate counts,
     dilution counts) may  be used to monitor microbial activity
     during testing.1-42-1
                                                                Hand-driven equipment     Power-driven equipment
     4.4.3   Monitoring   and
             Measurements
Process   Control
     A monitoring program is  an  essential component of any
     remedy selection treatability study. Monitoring data can be
     used to assess degradation rates and to determine if system
     design  or operational changes are needed. During remedy
     selection testing biodegradation may be assessed by removing
     samples from the testing system (e.g., reactor, treatment bed),
     or in the case of smaller-scale, laboratory tests, by  sacrificing
     the entire contents of smaller test systems at predetermined
     time  intervals.  Contaminant  concentrations  should  be
     determined at the beginning, end, and one or more intermediate
     time points. Toxicity studies may also be conducted if toxicity
     reduction is included in the test goals. The length of the study
     will be determined by the biodegradability of the contaminants
     and the time required to achieve parallel test goals. Measures
     of microbial activity (CO2 evolution, oxygen uptake, etc.) may
     also be  used to identify appropriate sampling times.

     Process control measurements are also essential.  Nutrients,
     water, and pH are among the most common media parameters
     measured. Measurement of ambient and soil
                            Screw-type auger


                            Post-hole auger

                            Barrel auger

                            Dutch auger

                            Split-spoon sampler

                            Tube-type sampler

                            Auger/dry-tube corer
                           Continuous flight power
                           auger (hollow-stemmed)

                           Core sampler

                           Split-spoon sampler

                           Bucket auger

                           Cable-tool drill rig

                           Rotary drill rig
temperatures is also customary; weather conditions may also
be recorded/68' The effects different operating parameters have
on removal efficiency should be determined. Typically, tests
are run in triplicate.

In addition to monitoring contaminant disappearance and
process control parameters, it may be necessary to monitor
media  outside  the treatment zone  to  assess possible
contaminant migration. Depending on the scale of study,
groundwater, soil, runoff water, and/or air monitoring may be
required. By successfully combining these monitoring efforts,
an accurate picture of contaminant fate can be achieved.
Generally, as the degree of control associated with keeping the
media under study separated from the environment decreases,
the potential for contaminant migration increases, therefore the
need for additional levels of monitoring decreases.

During field studies, particularly large,  in situ and contained
soil (e.g., landfarming) studies, soil cores and soil-pore liquid
monitoring should be  used to determine  if  hazardous
constituents are migrating out of the testing area. Soil core
samples generally provide information regarding the movement
of the slower moving hazardous constituents, while soil-pore
liquid samples evaluate the movement  of the faster moving
contaminants. The number, location, and depth of soil core
and soil-pore liquid samples will provide  an accurate indication
of conditions below the testing area.  When determining
vertical contaminant migration, contaminant concentration
trends below the testing zone need to be monitored. Increasing
concentrations are indicative of migration from  the testing
zones.   Steady  or  decreasing  concentrations  without
indications of increased biological activity (e.g., no increase in
microbial counts) are indicative of minimal vertical migration.
However,  an increase in microbial  activity  suggests that a
previously  limiting  factor,  such as  substrate  (i.e., the
contaminants)  availability, has been removed.  Ultimately,
contaminant migration out of the testing zone cannot be
conclusively demonstrated by citing a decrease in contaminant
concentrate along with increased microbial activity.

The frequency and timing of sampling must be based on the
frequency,  timing,  and  rate  of  amendment  application,
groundwater proximity, soil  permeability, and rainfall. The
mobility of the contaminant and the impact treatment has on
contaminant mobility must be account -
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                                 Table 4-6. Commonly Used Analytical Chemistry Methods
      Analysis
Liquid
Soil/sludge
      Moisture

      Nitrate

      Total organic carbon

      Total kjeldahl nitrogen

      Soluble orthophosphate

      Soluble ammonia

      PH

      TPH by GC

      TPH by IR*


      Base, neutral, and acid extractable
      compounds

      VOCs

      VOCs by GC

      Total O&G
      (IR Method)*

      Total O&G
      (Gravimetric method)
SW-846 Method 9200

SW-846 Method 9060

U.S. EPA Method 351.2

U.S. EPA Method 365.1

U.S. EPA Method 350.1

SW-846 Method 9040

SW-846 Method 8015

U.S. EPA Method 418.1


SW-846 Method 8270


SW-846 Method 8240

SW-846 Method 8010/8020

U.S. EPA Method 413.2


U.S. EPA Method 413.2
ASTM2216



SW-846 Method 9060

ASTM E 778
SW-846 Method 9045

SW-846 Method 8015

SW-846 Method 9071
U.S. EPA Method 418.1

SW-846 Method 8270


SW-846 Method 8240

SW-846 Method 8010/8020

SW-846 Method 9071
U.S. EPA Method 413.2

SW-846 Method 9071
U.S. EPA Method 413.2
     *  infrared spectrometry


     ed for.  In addition  to  providing  data on  the  vertical
     displacement of the contaminant, soil core samples may also
     be used to provide data on treatment progress in the testing
     zone.(73) Lysimeters may also be used to evaluate migration
     potential during in  situ field studies.  Table 4-7 provides
     guidance for developing a monitoring program during large
     field treatability studies/73'

     When necessary,  groundwater should  be monitored  to
     determine whether contaminants are migrating out of the
     testing zone. Pressure vacuum lysimeters, trench lysimeters,
     and vacuum extractors may be used to  monitor soil-pore
     liquids and/or leachates. Generally, groundwater monitoring
     supplements the unsaturated zone monitoring system.  If
     runoff water analyses are needed a monitoring program should
     be instituted. The sampling and monitoring approach will vary,
     depending on whether the water is released as a continuous
     discharge or as a  batch discharge following treatment.
     Depending  on  the   technology  under study and the
     characteristics and volume of water produced, a National
     Pollutant Discharge Elimination System (NPDES) permit may
     be required.

     Due  to  the volatile  nature  of many  contaminants,  air
                   monitoring is a essential element of many site monitoring
                   plans. Besides providing data on  potential  contaminant
                   releases, air monitoring provides a means for evaluating the
                   effectiveness of vapor suppression techniques. Depending on
                   the  scale of the study, personal  monitoring equipment,
                   perimeter sampling,  and upwind/downwind sampling may be
                   needed to ensure the safety of residents and workers. High
                   efficiency particle filter samplers and gas/vapor samplers may
                   be used. Solid sorbent traps may be used to sample volatile
                   organic air pollutants. Continuous air monitoring may also be
                   advisable.(68) If significant emissions are anticipated during
                   treatability testing, RPMs should check with the appropriate
                   regulatory offices to identify potential monitoring, reporting,
                   and permitting requirements. Depending on the technology
                   under study, air monitoring data may help define contaminant
                   fate, particularly during mass balance calculations.

                   4.4.4   Treatment Product Sampling
                           and Analysis

                   Biodegradation,  especially  ex situ bioremediation, is  not
                   always a stand-alone process.  The treated solids, liquids, and
                   each of the other various waste streams (biological sludges)
                   should be analyzed for the contaminants identi-
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                                 Table 4-7. Guidance for an Operational Monitoring Program
      Medium to be monitored
Purpose
Parameters to be analyzed
      Soil cores
       (unsaturated zone)
      Soil-pore liquid
       (unsaturated zone)
      Groundwater

      Vegetation (if grown for food
       chain use)

      Runoff water

      Soil in the
       treatment zone

      Air
Determine slow moving hazardous
constituents
Determine highly mobile
constituents
Determine mobile constituents

Phototoxic and bioaccumulating
hazardous constituents (food chain
hazards)
Soluble or suspended constituents

Determine degradation, pH,
nutrients, and rate- and capacity-
limiting constituents
Personnel and population health
hazards
All hazardous constituents in the waste or the
principal hazardous constituents, metabolites
of hazardous constituents, and nonhazardous
constituents of concern

All hazardous constituents in the waste or the
principal hazardous constituents, mobile
metabolites of hazardous constituents, and
important mobile nonhazardous constituents
Hazardous constituents and metabolites or
select indicators
Hazardous metals and organics  and their
metabolites

Discharge permit and background parameters
plus hazardous organics
Hazardous constituents,  metabolites, pH, N,P,
K, moisture,  and microbial population  and
activity
Particulates (adsorbed hazardous
constituents) and hazardous volatiles
     fied in the original soil analyses and their known degradation
     products to see if additional treatment is needed.  In many
     cases, indicator contaminants, which are representative of a
     larger group of contaminants, can be analyzed in place of a full
     scan.  Caution  must  be  exercised  in  using   indicator
     contaminants since biodegradation efficiencies can vary from
     one contaminant to another. The process efficiency may be
     either understated or overstated when analyzing for indicator
     compounds.

     4.5   DATA ANALYSIS AND
           INTERPRETATION

     The Work Plan should discuss the techniques to be used in
     analyzing and interpreting  the data. The objective of data
     analysis and interpretation is to provide sufficient information
     to  the RPM,  OSC, and EPA management  to assess the
     feasibility of biodegradation as a remedial technology. After
     remedy selection testing is complete, the decision must be
     made whether to proceed to the RD/RA testing tier, to perform
     a full-scale bioremediation, or to rule out bioremediation as an
     alternative. The data analysis and interpretation are a critical
     part of the remedy selection testing process.

     The primary goal of the remedy selection biodegradation
     treatability study is to determine how well  the treatment
     method removes the contaminants. System performance
                          is affected by a variety of process design variables, including
                          contaminant concentration, nutrientand oxygen availability,
                          abiotic losses, pH, microbial acclimation, and temperature.
                          Often one or more of these variables must be adjusted to
                          enhance the remediation process suitably. In order to properly
                          evaluate the impact the various process variables have on
                          testing results, the following data should be reported for each
                          treatability test:

                          •  Concentration of chemicals  in  samples at the time of
                             sampling (field concentration) and before the samples are
                             added to the reactors (T0 reactor concentration)

                          •  Amount of soil used in the reactors and a description of all
                             modifications to the reactors

                          •  Quantity of residual chemicals in each of the reactors at
                             each sampling time

                          •  Quantity  of residual chemicals  lost  due to  abiotic
                             processes

                          •  Temperature profile over the entire experiment recorded in
                             a written log indicating type, extent, and time of any action

                          •  Any other additions, removals, changes, manipulations, or
                             mishaps that occur during the course of the
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        experiment should be recorded in a written log indicating
        type, extent, and time of any action

     •   All  cited  analytical  and  microbiological  procedures
        (recorded ina written log)

     •   All  QC  data (e.g.,  recovery  percentage  of spikes,
        contaminant concentrations, if any, in experimental and
        analytical blanks)

     Additional information on the interpretation of treatability
     study data is presented in Section 6 of this document.

     Assessing whether the bioremediation method under study
     can achieve site cleanup levels within reasonable time limits
     and under practical engineering conditions is the primary goal
     of the remedy selection treatability study. Adjustments should
     be made for the impact of the different design variables (e.g.,
     pH  or oxygen  availability).  Statistical  analysis of  data that
     follow a normal  distribution  can be performed using the
     analysis  of variance (ANO VA) techniques and other statistical
     methods. In some  instances, the  use  of  nonparametric
     evaluations may be more appropriate. For details on parametric
     evaluations, refer to  the documents  entitled Statistical
     Analysis of Groundwater Data at RCRA Facilities (Interim
     Final),(70) and Experimental Design and Analysis.(18) Models
     (conceptual, mathematical, and physical) may be used as a
     focus for data integration. These models should be capable of
     bridging laboratory and field applications.  A realistic scale-up
     to full-scale applications is essential.  Both stochastic and
     deterministic models should  be  used to identify limiting
     mechanisms  and  critical parameters. Best-  and worst-case
     scenarios should be used to define the operational parameters.
     Data obtained from a large field-scale study should be used to
     validate the model.

     4.6  REPORTS

     The last step of the treatability  study is interpreting and
     reporting the  results.  The Work Plan may  discuss the
     organization and content of interim and final reports. Complete,
     objective, and accurate reporting is critical, because decisions
     about implementability will be mostly based upon the outcome
     of the study. The RPM or OSC may not require formal reports
     at each  treatability  study tier. Interim  reports should be
     prepared after each tier. Project briefings should be made to
     interested parties to determine the need for and scope of the
     next tier of testing. To facilitate the reporting of results and
     comparisonsbetween treatment alternatives,  a suggested
     tableof contents is presented in the generic guide/52' At the
     completion of the study, a formal report is always required.

     OERR requires that a copy of all treatability study reports be
     submitted to the Agency's Superfund Treatability Database
     repo sitory. One copy of each treatability study report must be
     sent to:

          U.S. Environmental Protection Agency
          Superfund  Treatability Database (MS-445)
          ORD/RREL
          26 West Martin Luther King Dr.
          Cincinnati, Ohio 45268
4.7  SCHEDULE

The Work Plan includes a schedule  for completing the
treatability study. The schedule gives the anticipated starting
date and ending date for each of the tasks described in the
Work Plan and shows how the various tasks interface. Listed
below are some of the specific tasks that should always be
considered when scheduling:

•  Data review/literature search

•  Work Plan preparation, review, and revision

•  SAP preparation

•  Sample collection and disposal

•  Field sample analysis

•  Treatability test (including analyses)

•  Disposal of waste material generated during the test

•  Data validation

•  Report preparation, review, and revision

•  Meetings

The treatability test has the  greatest potential  for  time
variance. The schedule for this test can vary tremendously
depending on whether a small- or large-scale study is being
performed. Small laboratory-scale studies typically take from
3 to 6 months, whereas large fieldscale studies usually take
from 6 to 9 months. Contaminant types and concentrations
involved also can impact the test schedule. For example, a
laboratory-scale remedy selection treatability test for  soils
contaminated with benzene, toluene, ethylbenzene, andxylene
(BTEX) may be conducted within a 1 or 2 weeks, whereas tests
involving PAHs may take several months because of the
relative   biodegradability of  these   classes  of  organic
compounds. Sufficient time must be built into the schedule to
reach specified cleanup concentrations. The treatability study
must  continue  until either the removal goals have  been
achieved or the contaminant removal has reached a distinct
concentrationat which contaminant reductions cease to occur
at a reasonable rate.

The time span for each task accounts for the time required to
obtain the Work Plan,  subcontractor,  and other approvals
(e.g., disposal approval from a permitted commercial treatment,
storage, and disposal facility);  sample procurement time, if
necessary; analytical  turnaround time;  data  validation
intervals; and review and comment periods for reports and
other project deliverables. Some contingency should be built
into the schedule to accommodate unexpected delays (e.g.,
bad weather, equipment downtime) without affecting the
project completion date. Example schedules for in situ and ex
situ remedy selection studies are presented in Figures 4-1 and
4-2, respectively. If the study involves multiple tiers of testing,
all tiers should be shown on one schedule. Careful planning
before the start of tests is essential. Depending on the review
and approval process, planning can take up to several months.
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     Setup  of the  laboratory  and  procurements  of neces-
     sary   equipment    and   laboratory    supplies    for
     treatability studies may take a month or more. Depending on
     how rapidly laboratory results can be provided, analytical
     results can be available in less than 30 days. Shorter analytical
     turnaround time can be requested, but quick turnarounds will
     normally increase the costs. Turnaround times should be less
     than the  time between sampling points. Results from one
     sampling point are needed before the next sample is  taken
     becausethe sampling schedule may be extended if degradation
     is occurring at a slower rate than anticipated. This is especially
     important when sacrificial reactors are used for timepoints and
     a limited number of these reactors were set up at the beginning
     of the study. For this reason, inexpensive analyses with quick
     turnaround times are recommended formonitoring treatability
     studies even if  confirmatory analyses (GC/MS) need  to be
     performed at certain points.

     The schedules in Figures 4-1 and 4-2 are based on a 30-day
     analytical turnaround time. In the event 90-day turnarounds
     are experienced, the schedules in Figures 4-1 and 4-2 would
     increase to 26 months and  17 months, respectively, reflecting
     net  increases of 2 months.  These schedules do not reflect
     " standard" and/or" average" time-lines for treatability testing.
     The variability inherent in treatability testing would make any
     attempt at simulating these conditions meaningless.

     Interpretation of the results and final report writing usually
     requires 1 to 2 months, but this is highly dependent on the
     review process.  It is not unusual  for the remedy selection
     phase to take 3 to 9 months before treatability testing and final
     reporting can be completed.
                             4.8  MANAGEMENT AND STAFFING

                             The Work Plan discusses the management and staffing of the
                             remedy selection treatability study and specifically identifies
                             the personnel responsible for executing the treatability study
                             by name and qualifications. Generally, the following expertise
                             is  needed for the successful completion of the treatability
                             study:

                             •  Project Manager (Work Assignment Manager)

                             •  QA Manager

                             •  Chemist

                             •  Microbiologist,  Environmental  Scientist/Engineer,  or
                                Bioengineer

                             •  Lab Technician.

                             Responsibility for various aspects of the project is typically
                             shown in an organization chart such as the example shown in
                             Figure 4-3.

                             4.9  BUDGET

                             The Work Plan should discuss the budget for completion of
                             the remedy selection testing tier unless  this  information is
                             judged to be business-confidential by  EPA.  The cost of
                             remedy selection testing varies tremendously and is directly
                             related to the type of test (laboratory or field-scale), the
                             technology under study,  the method of sample
            Task Description
Month:   1   2  345  6  7  8 9  10  11 12 1314 15  16  17 18 19 20 21 22 23 24
              Work/Sampling Plan Preparation
             Review and Approval of Work Plan
                           System Design
               Mobilization/System Installation
                    System Testing/Start-up
                         System Operation
                           Demobilization
                        Laboratory Analysis
                           Data Validation
                             Draft Report
                             EPA Review
                             Final Report
                   Monthly Progress Reports
                                                                              Legend: Task Duration  ••  Report Due X
            Figure 4-1.  Sample treatability testing schedule for remedy selection evaluation of in situ bioremediation.
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Task Description Month: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Work/Sampling Plan Preparation
Review and Approval of Work Plan
Site Sampling
Baseline Chemical Characterization
Baseline Microbiological
Characterization
Bench-Scale Evaluation of
Solid-Phase Treatment
Bench-Scale Evaluation of
Slurry-Phase Treatment
Laboratory Analysis
Data Validation
Draft Report
EPA Review
Final Report
Monthly Progress Reports
—







—








•M








••H
•
•






mum
•


X





-



X










X










X









X









X








X





•••


X




«•


X





••1

X






u*m
X
                                                                       Legend: Task Duration
                               Report Due X
                    Figure 4-2. Sample project schedule for laboratory remedy selection evaluation of
                                      solid-and slurry-phase bioremediation.
                     CONTRACTOR or
             WORK ASSIGNMENT  MANAGER
           •  Report to EPA Remedial Project Manager
           •  Supervise Overall Project
          TREATABILITY STUDY PROJECT MANAGER

           •  Oversee Treatability Study execution
           •  Prepare applicable sections of Report
              and Work Plan
           •  Oversee sample collection and analysis
                      LAB TECHNICIAN

               Perform Treatability Study
               Sample collection and analysis
  QUALITY ASSURANCE MANAGER

Oversee Quality Assurance
Prepare applicable sections of Report
and Work Plan
                                      Figure 4-3. Sample organization chart.
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     collection, the number of samples collected, the type of and
     number of chemical analyses performed, and the number of
     replicate tests performed. The factor that most influences the
     cost of the remedy selection testing phase is whether the test
     is performed at the laboratory-scale or field-scale level. Larger
     field-scale  studies   are  more  expensive   than  small
     laboratory-scale  studies   because  they  require  field
     mobilization/demobilization, field crews to run the test, more
     analytical data,  and  are usually of longer duration than
     small-scale tests. The type and number of chemical analyses
     performed also have a significant impact on the cost of remedy
     selection testing. Laboratory setup costs also may be inflated
     due to government requirements. One method to minimize
     costs  is to use  an  inexpensive analysis as  an  indicator
     parameter and to perform a limited number of analyses for the
     more expensive volatile and semivolatile priority pollutants.
     Use of GC rather than  GC/MS methods, if applicable, should
     also help to minimize costs. Table 4-8 summarizes the major
     cost elements associated with remedy selection treatability
     tests.

     Sampling costs will be influenced by the contaminant types
     and depth of contamination found in the soil, sludge, or
     sediment. Depending on the depth of contamination and the
     regulatory requirements, field sampling can cost hundreds of
     thousands of dollars. The health and safety considerations
     during sampling activities are more extensive when certain
     contaminants, (e.g.,  VOCs) are present. Level B  personal
     protective equipment (PPE) rather than Level D(79)  PPE can
     increase the cost  component by an order of magnitude. In
     general,  most laboratory and field-based remedy  selection
     studies will require Level D PPE.  Sampling equipment for
     surface samples is much less complicated than the equipment
     needed for deep  samples.  Depending on the number of
     samples and tests specified, test residuals (e.g., contaminated
     solvent  and water)  will  require  proper treatment and/or
     disposal.  Since effluents and residual materials produced
     during testing often are treated  as a hazardous waste,
     regardless of whether the contaminant has been degraded,
     high disposal costs may have to be assumed.

     Other factors to consider include report preparation and the
     availability of essential equipment and laboratory  supplies.
     Generally, an initial draft of the report under-goes internal
     review prior to the final draft. Depending on the process, final
     report preparation can be time-
    Table 4-8.  Major Cost Elements Associated with
    Biological  Remedy Selection Treatability Studies
 Cost element
Approximate cost
range (thousands
    of dollars)
 Work plan preparation

 SAP preparation

 Health and safety plan
 preparation

 Field sample collection

 Field sample chemical analysis

 Laboratory setup/materials

 Treatability test operation

 Treatability test chemical
 analysis

 Data presentation/report/
 remediation cost estimate

 Total cost range
      2-5

      2-5

      1-5


      5-10

     5-100

      5-10

      5-15

     5-100


     20-50


    50 - 300
consuming as well as  costly.  Procurement of specialized
testing equipment  (e.g., reagents and  glassware) will  also
increase the costs.

The typical costs for the remedy selection testing phase are
estimated to range from $50,000 to $300,000. These estimates
are highly dependent on the factors discussed previously. Not
included  in  these costs are the  cost  of  government
procurement  procedures,  including  soliciting for  bids,
awarding contracts, etc.

To minimize costs, opportunities for cost savings should be
sought actively. For example, during bioventing  studies,
boreholes used to characterize  the site may be converted to
bioventing wells.
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                                                  SECTION 5
                                SAMPLING AND ANALYSIS PLAN
     The SAP consists of two parts: the Field Sampling Plan and
     the Quality Assurance Project Plan.  The purpose  of this
     section is to identify the contents of and aid in the preparation
     of these plans. The RI/FS requires a SAP for all field activities.
     The SAP ensures that samples obtained for characterization
     and testing are representative, and that the quality of the
     analytical data generated is known and appropriate. The SAP
     addresses field sampling, waste characterization, and sampling
     and analysis of the treated wastes  and  residuals from the
     testing apparatus or treatment unit.  The  SAP is  usually
     prepared after Work Plan approval.

     5.1   FIELD SAMPLING PLAN

     The FSP component of the SAP describes the sampling
     objectives; the type, location, and number of samples to be
     collected; the sample numbering system; the equipment and
     procedures  for collecting the   samples;   the   sample
     chain-of-custody procedures; and the required packaging,
     labeling, and shipping procedures.

     Field samples  are  taken to provide baseline contaminant
     concentrations and contaminated material  for treatability
     studies. The sampling objectives must be consistent with the
     treatability test objectives.

     The primary objectives of remedy selection treatability studies
     are to evaluate the extent to which specific chemicals are
     removed from  soil, sediment, sludge, or water. The primary
     objectives  for collecting samples  to be used in  remedy
     selection treatability testing include to following:

     •   Acquisition  of samples representative of  conditions
        typical of the entire site or defined areas within the site.
        Because a limited mass balance may be required, field
         sampling plans may be required. However, professional
        judgment  regarding the sampling  locations may be
        exercised to select sampling sites that are typical of the
         area (pit, lagoon, etc.) or appear to have above-average
        concentrations  of contaminants in the  area being
        considered for the treatability test. This may be difficult
        because reliable site characterization data may not be
         available early in the remedial investigation.

     •   Acquisition of sufficient sample volumes necessary for
        testing, analysis,  and  QA/QC.  The  biodegradation
         screening  guide recommends using about 5 kg of the
        contaminated medium. During remedy selec-
    tion testing, the amount of sample will depend on the size
    of the test and the number of test samples.

From these two primary objectives, more specific objectives
are developed. When developing the more detailed obj ectives,
consider the following types of questions:

•   Should  samples be  composited  to  provide  better
    reproducibility for the treatability test? This question is
    addressed in Subsection 4.4.1.

•   Are there adequate data to determine sampling locations
    indicative of the more contaminated areas of the site?
    Have soil gas surveys been conducted? Contaminants
    may be widespread or isolated in small areas (hot spots).
    Contaminants may be mixed with other contaminants in
    one location  and appear alone in others. Concentration
    profiles may vary significantly with depth.

•   Are  the  soils  and   contaminants  homogeneous  or
    heterogeneous? Soil types can vary across a site and will
    vary with depth. Depending on professional judgement,
    contaminated samples for various soil types may have to
    be taken to conduct treatability tests. Variations in soil
    composition  can   affect   the  effectiveness   of
    biodegradation as well as the accuracy of the analyses
    employed.

•   Are contaminants present in the sediment, sludge, or
    water? Different sampling methods must be used for each
    of these media. Will media exchange contaminants during
    treatment? Mass balances may be necessary.

•   Is  sampling  of a "worst-case" scenario  warranted?
    Assessment  of this question must  be made on a site-
    by-site basis. Hot spots and contaminants in different
    media may be difficult to treat. These should be factored
    into the test plan if they represent a significant portion of
    the waste site.

After identifying the sampling objectives, an  appropriate
sampling strategy is described. Specific items that should be
discussed briefly and included are listed in Table 5-1.

5.2   QUALITY ASSURANCE
      PROJECT PLAN

The QAPP consists of 11 sections. Since many of these
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         Table 5-1.  Suggested Organization of the
              Sampling and Analysis Plan

      FSP	

      1.       Site background
      2.       Sampling objectives
      3.       Sample location and frequency
              Selection
              Media type
              Sampling  strategy
              Location map

      4.       Sample designation
              Recording procedures

      5.       Sample equipment and procedures
              Equipment
              Calibration
              Sampling  procedures
      6.       Sample handling and analysis
              Preservation and holding times
              Chain-of-custody
     	Transportation	
      QAPP	

      1.       Project description
              Test goals
              Critical variables
              Test matrix
              Project  organization and responsibilities

      2.       QA objectives
              Precision, accuracy, completeness
              Representativeness and comparability
              Method detection limits
      3.       Sampling procedures and sample custody
      4.       Analytical procedures and calibration
      5.       Data reduction, validation, and reporting
      6.       Internal QC checks
      7.       Performance and system audits
      8.       Calculation of data quality indicators
      9.       Corrective action
      10.      QC reports to management
      11.      References
     sections are generic and applicable to any QAPP and are
     covered in available documents,1-44-"-67' this guide will discuss
     only those aspects of the QAPP that are affected by the
     treatability testing of biodegradation.

     5.2.1   Project Description

     Section 1 of the QAPP must include an experimental project
     description that clearly defines the experimental design, the
     experimental  sequence  of  events,  each  type of critical
     measurement to be made, each type of matrix (experimental
     setup) to be  sampled, and  each type of system to  be
monitored. This section may reference Section 4 of the Work
Plan. All details of the experimental design not finalized in the
Work Plan should be defined in this section.

Items  in this section  include, but are not limited to, the
following:

•   Number of samples (areas or locations) to be studied

•   Identification of treatment conditions  (variables) to be
    studied for each sample

•   Target compounds for each sample

•   Number of replicates per treatment condition

•   Criteria for technology retention or rej ection for each type
    of remedy selection test.

The Project Description clearly defines and distinguishes the
critical measurements from other  observations and system
conditions  (e.g., process  controls, operating parameters)
routinely  monitored.   Critical  measurements   are  those
measurements or data-generating activities that directly impact
the technical objectives of a project. At  a minimum, the
determination of the target compound (identified previously)
in the initial and treated samples will be critical measurements
for remedy  selection  tests.  Concentrations   of   target
compounds  in all  fractions  and the oxygen and nutrient
availability will be among the critical measurements for RD/RA
tests.

5.2.2   Quality  Assurance Objectives

Section 2 lists the QA objectives for each critical measurement
and sample matrix defined in Section 1. These objectives are
presented in terms of the six data quality indicators: precision,
accuracy, completeness, representativeness, comparability,
and, where applicable, method detection limit.

5.2.3   Sampling Procedures

The procedures used to obtain field samples for the treatability
study are described in the FSP. They need not be repeated in
this section, but should be incorporated by reference.

Section 3 of the QAPP contains a description of a credible plan
forsubsampling the material delivered to the laboratory for the
treatability study. The methods for aliquoting the material for
determination of chemical and physical characteristics, such as
bulk density or specific gravity, moisture content, contaminant
concentration, etc., must be described.

5.2.4   Analytical Procedures and
        Calibration

Section  4 describes  or references  appropriate  analytical
methods and standard operating procedures to be used for
each critical measurement. In addition, the calibration
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     procedures and frequency  of calibration are discussed or
     referenced for each analytical system, instrument, device, or
     technique used for each critical measurement.

     The methods for analyzing the treatability study samples are
     the same as those  for  chemical  characterization  of field
     samples.  Table 4-6  presents suitable analytical  methods.
     Preference is given to methods in SW-846.(73). Otherstandard
     methods may be used as appropriate.1-^1-3-"-64-1 Methods other
     than GC/MS techniques are recommended to reduce costs,
     when possible.

     5.2.5   Data Reduction, Validation, and
             Reporting

     Section 5 includes, for each critical measurement and each
     sample matrix,  specific presentation of the requirements for
     data reduction, validation,  and reporting. Aspects of these
     requirements are covered in Subsections 4.5 and 4.6.
5.2.6  Quality Control Reports

Section 10 describes the  QA/QC information  that will be
included in the final project report. At a minimum, reports
include:

    Changes to the QAPP

•   Limitations or constraints on the applicability of the data

•   The status of QA/QC programs, accomplishments, and
    corrective actions

•   Results of technical systems and performance evaluation
    QC audits

•   Assessments of data quality in terms of precision,
    accuracy,  completeness,  method  detection  limits,
    representativeness, and comparability.

The final report contains all the QA/QC information to support
the credibility of the data and the validity of the  conclusions.
This information may be presented in an appendix to the
report. Additional information on data quality objectives1-44-1
and preparation of QAPPs '-67-1 is  available in EPA  guidance
documents.
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                                                   SECTION  6
                           TREATABILITY  DATA INTERPRETATION
     This  section is designed to help the  site RPM, OSC, or
     contractor interpret treatability data. The test results and goals
     for each  tier  must be evaluated properly to assess the
     bioremediation potential.  Testing results are interpreted in
     relation to seven of the nine RI/FS  evaluation criteria, as
     appropriate. Subsection 3.2 describes the nine criteria and how
     they should be addressed for bioremediation.

     The remedy screening tier establishes the general applicability
     of  the technology.  The  remedy  selection  testing  tier
     demonstrates the applicability of the technology to a specific
     site. The RD/RA tier provides information in support of the
     evaluation criteria.  The  test objectives  are  based  on
     established cleanup  goals or  other  performance-based
     specifications (such as removal efficiency).

     Subsection 4.6 of this guide  discusses the need for the
     preparation of interim and final reports and refers  to a
     suggested format. In addition to the raw and summary data for
     the treatability study and associated QC, the treatability report
     should describe the meaning of the results and how to use
     these results in the feasibility study for both the screening and
     selection  of alternatives.  The report  must evaluate the
     performance of the technology and give an estimate of the
     costs and schedule for final remediation using the technology.

     6.1   TECHNOLOGY EVALUATION


     6.1.1   Remedy Screening Phase

     Remedy  screening treatability  studies typically  consist of
     simple  laboratory  reactor  tests. Normally,  contaminant
     concentrations in the matrix are  measured before and after
     treatment. A threshold of greater than 20 percent reduction in
     contaminant concentration,  compared to the  abiotic control,
     indicates that additional treatability studies may be warranted.
     Before- and after- treatment concentrations normally can be
     based on duplicate samples at  each time period. The mean
     values are compared to assess the success of the study. A
     number of statistical texts are available if more information is
     needed (5X15X18X7°)

     When sufficient  information  is available regarding the
     contaminant's degradability in the selected  media, remedy
     screening tests may be omitted. This information should be
     media- and contaminant-specific and may  or may not be
     applicable to other sites.
When the results of a screening study demonstrate that a
specific  contaminant is biodegradable under  laboratory
conditions, it should not be assumed that the contaminant will
be  degraded  in  a specific  soil/site  system.  Full-scale
application,particularly of in situ technologies, requires further
site-specific investigation as  part of a remedy selection
treatability study process.

6.1.2   Remedy Selection Phase

Remedy selection studies should be performed if the results of
either the literature review or the  remedy screening test
indicate that bioremediation is a potential cleanup option.
Remedy  selection  studies  are used to  identify  the
technology' s performance on a site- and contaminant-specific
basis. Costs for these studies generally range from $50,000 to
$300,000. Data from remedy selection studies may be used to
determine if the technology can meet expected cleanup goals
in a reasonable  time  frame  under practical  engineering
conditions. Data should be used to support  the detailed
analysis of the alternative with respect to seven of the nine
RI/FS evaluation  criteria  presented  in  Subsection  3.2.
Treatability data analysis during a remedy selection study is
demonstrated in Example 9.

When interpreting data relating to contaminant disappearance,
RPMs are cautioned against making claims based solely on
substrate  removal.  To  accurately  assess  risk reduction,
changes in toxicity, mobility, or  volume, and the long-term
implications of treatment, RPMs must first determine the extent
to  which the  contaminant  has  mineralized  and  the
concentration  and  characteristics  of  any  intermediate
byproducts remaining  in the media. Ideally,  this may be
assessed using amass balance approach. The concentrations
of contaminants as well as any added substrate, metabolites,
electron   acceptors,   radiolabeled  compounds,   and
nondegradable tracers generated  or introduced to the media
should  be determined.  Data pertaining to initial (baseline),
intermediate,  and  final  contaminant  and   byproduct
concentrations should be analyzed.  Nonbiological removal
mechanisms   also  must  be   considered  during  data
interpretation. Section 4.2.11 provides information on the use
of biologically inhibited controls  to determine the impact of
nonbiological removal.  During  data interpretation,  the
contaminant  concentrations in the  test and control cells
should  be compared. The difference in mean contaminant
concentrations between the test and control cells will indicate
whether a statistically significant amount of bio-
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                                                      Example 9
      A remedy selection treatability study was performed to evaluate a slurry-phase technology's ability to remediate an
      impoundmentcontaminated with petroleum refinery sludges. Surfactants and nutrients were added. Reactor performance
      was  monitored  by measuring the  oxygen uptake rate and O&G removal. Based on extensive experience with O&G
      biodegradation,  toxicity testing was not performed.
      The average initial O&G concentration in the sediment was 41,000 ppm, the maximum concentration expected in the full-
      scale (70,000 gallon), slurry bioreactor. A cleanup goal of 20,000 ppm O&G was targeted during the study. After 4 weeks
      the average O&G concentration in  the inhibited control was reduce to 39,000 ppm, a reduction of nearly 5 percent. The
      average O&G concentration in the biologically active system was reduced to 14,000 ppm a 66 percent reduction in the same
      time period. The leveling out of O&G concentrations at the end of the  experiment indicates that the maximum extent of
      biodegradation achievable under the test conditions had been reached.
                                                                        O&G
              Sample                           T0           T,            T2
              Bioreactor
              Replicate 1
              Replicate 2
              Replicate 3
              Mean Value

              Inhibited Control
              Replicate 1
              Replicate 2
              Replicate 3
              Mean Value
39,000
41,000
43,000
41,000
39,000
41,000
43,000
41,000
32,000
34,000
39,000
35,000
36,000
39,000
42,000
39,000
21,000
24,000
24,000
23,000
37,000
40,000
40,000
39,000
13,000
15,000
17,000
15,000
37,000
41,000
39,000
39,000
14,000
16,000
12,000
14,000
42,000
36,000
39,000
39,000
      The average contaminant concentration in the slurry-phase bioreactor, at each time-point,  is compared to the average
      contaminant concentration in the inhibited control, at the same time-point, to measure the biodegradation at that time-point.
      The inhibited  control accounts for contaminant losses due to volatilization,  adsorption to  soil particles, and chemical
      reactions.  Some contaminant loss in the control due to biodegradation may  occur since total sterilization  is difficult to
      accomplish. However, an O&G analysis of the extract generated from the slurry-phase reactor indicated that abiotic losses
      were due mainly to adsorption. Since a statistically significant difference between the test and control means exists, O&G
      reductions in the test bioreactor were attributed to biodegradation.
     degradation is occurring. As discussed in Section 4.2.11, the
     effectiveness and possible side-effects of the sterilizing agents
     added to the control cells must also be considered during data
     interpretation.
     When the final contaminant concentration is below detection
     limit, it must be reported as such. For example, if the detection
     limit is 100 mg/kg and the contaminant was not detected, the
     final concentration must be reported as "less than 100 kg/mg."
     If the initial  concentration was 200  mg/ kg, the removal
     efficiency must be reported as " greater than 50 percent," even
     though the actual removal efficiency may be significantly
     higher than 50 percent. In
                some cases, it may be possible to avoid this situation by
                selecting an analytical method with a lower detection limit.
                For remedy selection treatability testing, however, the ability
                of the technology to meet cleanup  goals is much more
                important than the removal efficiency. To provide a decisive
                evaluation of a technology's ability to reduce a contaminant
                concentration below the cleanup goal, the final concentration
                of that contaminant should  be  analyzed using a method
                detection limit that is less than or equal to the cleanup goal. If
                this is not done, a meaningful evaluation of the technology's
                ability to remediate the site cannot be performed.
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     In  addition to contaminant concentrations, data showing
     increased microbial counts, oxygen consumption, and carbon
     dioxide  evolution  often   are  considered  indicative  of
     contaminant biodegradation. While these data do indicate
     biological activity, accurate data interpretation must consider
     the possibility that the bacteria are consuming background
     carbon rather than the contaminants.

     RPMs also are cautioned against attributing improvements in
     performance to  specific  characteristics of the treatment
     process (e.g., microbial supplementation) without first:

     •    Verifying whether similar removals are experienced in a
         control cell in which this specific characteristic is varied

     •    Determining whether other mechanisms, not related to the
         technique under discussion, were actually responsible for
         the removal.

     Data should be analyzed to determine the impact operating
     parameters  (such  as pH, temperature, nutrient and oxygen
     concentrations, etc.) have on performance (i.e., contaminant
     and byproduct concentrations,  microbial activity, oxygen
     uptake rates, CO2 evolution). The resulting information then
     can be used to refine both time and cost estimates and to
     identify  specific operating parameters for the next level of
     testing.   Potential   pretreatment   and  post-treatment
     requirements may also be identified.

     When evaluating the technology, a rational scale-up from the
     remedy selection study to full-scale application must be made.
     Realistic but conservative estimates should be sought for
     actual treatment efficiencies, times, and schedules. Less than
     ideal (i.e., laboratory-based) conditions in the field must be
     identified and compensated for when scaling  up from a
     laboratory-based study to a field study. Best and worst case
     scenarios should be used to define operational parameters.

     A  sufficient number of data points and replicates must be
     obtained in order to perform a valid statistical analysis of the
     technology. The data must comply with established criteria for
     precision, accuracy, completeness, method detection  limits,
     representativeness and comparability. An established relative
     percent difference (RPD) between either the matrix duplicates
     or between the matrix spike/matrix spike duplicates should be
     defined  in order to assess precision. If QA objectives for
     precision and  accuracy are not met, the precision  and/or
     accuracy of the derived removal efficiency are decreased.
     Similarly, if completeness objectives are not met (i.e., the ratio
     of  the number of valid measurements to the total number of
     measurements planned), then the confidence limits associated
     with the results will  be decreased. Strict adherence  to the
     analytical methods and  defined calibration procedures is
     critical to the validity of the generated data. Results generated
     by  an unauthorized method, an unapproved deviation from the
     standard protocol, or during the operation of uncalibrated or
     malfunctioning equipment  should be rejected.  Data lying
     outside of specified acceptance limits established about the
     arithmetic mean of the project's entire data set should be
     identified but not used when determining  overall project
     results.

     As mentioned in Subsection 4.5, data following a  normal
     distribution can be analyzed using ANOVA techniques and
other  statistical  methods.1-18-"-70'   In  some   instances
nonparametric evaluations may be more appropriate. Models
(conceptual, mathematical, and physical) also may be used as
a focus for data integration. Both stochastic and deterministic
models may be used  to  identify limiting mechanisms and
critical parameters. Zero- and first-order reaction rate models
are  commonly  used to  describe the rate of contaminant
degradation as a  function of contaminant concentration.
Zero-order reaction rates are unaffected by the changes in
constituent concentration. In contrast, the rate of contaminant
transformation during  first-order reactions is proportional to
the  constituent concentration. Generally, the first-order rate
model is more widely  used because of the model's apparent
effectiveness in describing observed results.

Mathematical modeling also can be used to predict the fate
and behavior of organic constituents in a contaminated soil
system. Modeling results can help identify the potential for air,
leachate, or subsoil contamination. The RITZ and VIP models
commonly  are  used.  Both models  simulate vadose  zone
processes,  including  volatilization,  degradation,  sorption
/desorption, advection, and dispersion; however, the  VIP
model  also accounts for  the  dynamic behavior of organic
constituents in unsaturated soil systems under conditions of
variable precipitation, temperature, and waste loading. Data
regarding physical abiotic loss mechanisms and constituent
partitioning within the soil should be developed to ensure that
modeling results account  for contaminant losses due to both
biological and abiotic mechanisms.

Mathematical modeling  should not, however, be used to
proj ect cleanup levels below those attained during treatability
testing. Reaction rates can be used to interpolate data (i.e., to
project the time required to reach a contaminant concentration
between the initial and final concentrations measured during
testing), but should not be used to  extrapolate data beyond
the   final  concentration achieved  during  testing.  This
recommendation should  be  strictly  observed because, as
discussed in Section 2.2.4, biodegradation is an asymptotic
process. The concentration at which the contaminant removal
rate  is very close to zero  represents, from a practical
perspective, the lowest concentration that can be achieved by
the bioremediation technology being tested.

If required, several bioremediation processes can be evaluated
simultaneously to determine which process or combination of
processes is most appropriate for the cleanup of a given site.
For example, if the contaminated materials at a site can be
effectively  remediated with either  a  solid-phase or  a
slurry-phase biological treatment process,  both of these
processes   may   be  evaluated   simultaneously.   The
biodegradation rates measured during the solid-phase and
slurry-phase remedy selection evaluations can then be used to
estimate the treatment time, equipment, and land area required
by   each  treatment   process.   This  procedure  permits
determination of which process or combination of processes
can most cost-effectively  achieve the required cleanup levels
in the required period of time. If sufficient design and cost
information are acquired during the remedy selection tests to
permit full-scale system design, further RD/RA testing may be
unnecessary.
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     6.1.3   Remedial Design/Remedial
             Action Phase

     RD/RA testing is the third level of testing in the RI/FS process.
     The cost of these studies generally ranges from $100,000 to
     $500,000. As discussed in the preceding paragraph, RD/RA
     studies are not always required. When RD/RA  tests  are
     performed, they are typically post-ROD. Therefore, if RD/RA
     testing is conducted, it should produce the data required for
     final full-scale RD/RA and costing.  The RD/RA testing
     program is usually conducted on site and should test all
     equipment and processes so that accurate specifications can
     be made for the full-scale system.

     Example  10 demonstrates the decision process from remedy
     screening, through remedy selection testing, to the RD/RA._
     This example is a continuation of Example 6 in Subsection 4.1
     of this guide.

     The size and scope of the RD/RA testing programs may be
     decided by several factors, including the quantity of material
     available for testing, the complexity of the process, the cost,
     the available time, and the equipment availability.  When an
     RD/RA test is being setup, it is important that the equipment
     be sized so that realistic scale-up factors can be used for
     designing a full-scale operation.

     In conclusion, technologies generally are evaluated first at the
     remedy screening level and progress through remedy selection
     testing to the RD/RA tier. A technology may enter, however,
     at whatever tier or level is appropriate based on available data
     about the technology and site-specific factors. Forexample, a
     technology that has been studied extensively may not warrant
     remedy screening testing to determine whether  it has  the
     potential to work.  Rather, it may  go directly to remedy
     selection testing to determine if the performance standards can
     be met.
6.2  ESTIMATION OF COSTS

Before considering technologies for RD/RA testing, complete
and accurate cost estimates are required. Consequently, when
making preliminary cost estimates forfull-scale bioremediation,
achievable cleanup levels, degradation rates, the concentration
and application frequency of various degradation-enhancing
supplements  (e.g.,  nutrients,  lime,  water),  contaminant
migration controls,  and monitoring  requirements must be
considered.  The impact these  parameters have on labor,
analytical, material and energy costs, as well as the unit's
design and possible pre- and post-treatment requirements, also
must be considered.

Generally, large-scale field tests can be designed to simulate
full-scale performance and costs more accurately than smaller
laboratory studies. However,  estimating full-scale cost from
treatability study data still can be difficult. Given the variability
and interaction of factors such as soil temperature, moisture,
heterogeneous  contaminant  concentrations, and optimal
amendment concentrations, empirical results may not always
depict the range of  reasonable  bioremediation results. One
approach to examining the variability and interaction ofthese
factors is simulation modeling. Simulation models (e.g., Monte
Carlo Models) attempt to quantify the probability of a certain
set of events or values occurring, based on available empirical
data. Using probabilistic simulation methods can produce time
and cost estimates for a particular confidence interval and a
specific level of certainty (i.e.,  the researchers can state with 90
percent certainty that the cost of he proj ect will be within +40
percent   of  the  estimate.)  Additional  information  on
probabilistic simulations is available in most statistical text-
books/33'
                                                      Example 10

      Despite the reduction in PCP concentration during the remedy selection testing tier of treatability testing, the
      percentage of degradation, as compared to the control, indicated that the process may have been inhibiting
      microbial activity. The RPM decided to investigate mixing less-contaminated soil with the highly contaminated
      soil to lower PCP concentrations and stimulate biodegradation. Remedy selection testing, using the design
      modification suggested  by the remedy screening studies, resulted in an average removal of 93 percent of the
      PCP. RD/RA testing was performed to provide design information for a full-scale system, which was used to
      remediate the site successfully.
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                                                      SECTION  7
                                                    REFERENCES
     1.   Acton, D., D. Major, and E. Cox. Evaluating the Enhanced
         Aerobic Biodegradation of Trichloroethylene Using In Situ Test
         Columns. Presented at The Second International Symposium on
         In Situ and On-Site Bioreclamation, San Diego, California, April
         58,1993.

     2.   American Society for Testing and Materials. Annual Book of
         ASTM Standards. Philadelphia, Pennsylvania, 1987.

     3.   American Society of Agronomy, Inc. Methods of Soil Analysis,
         Part  1, Physical and Mineralogical Methods; Second Ed.,
         Madison, Wisconsin, 1986.

     4.   Barrano, F.T., J.L. Kocornik, I.D. MacFarlane, N.D. Durant,
         andL.P. Wilson. Subsurface Sampling Techniques Used For a
         Microbial Investigation. Presented at The Second International
         Symposium on In Situ and On-site Bioreclamation, San Diego,
         California, April 5-8, 1993.

     5.   Bevington, P.R. Data Reduction and Error Analysis for  the
         Physical Sciences. McGraw-Hill, Inc., New York, New York,
         1969. 336pp.

     6.   Box, G.E.P., W.G. Hunter, and J.S. Hunter.  Statistics  for
         Experimenters. John  Wiley & Sons, New York, New York,
         1978.

     7.   Davis, J.W. andS.S. Madsen. The Biodegradation of Methylene
         Chloride  in Soils. Environmental Toxicology and Chemistry,
         Vol.  10, 1991, pp. 463-474.

     8.   Domenico, P.A. and  F.W. Schwartz. Physical and Chemical
         Hydrology. John Wiley & Sons, New York, New York, 1990.

     9.   Federal Register. Coordinated Framework for the Regulation of
         Biotechnology. 51 FR 23302. Office of the Federal Register,
         Washington, D.C., June 26,1986.

     10.  Funk, S.B., D.L Crawford, D.J. Roberts, and R.L. Crawford.
         Initial  Phase  Optimization  for  the  Bioremediation  of
         Munitions-Contaminated Soils. Applied and Environmental
         Microbiology, July 1993.

     11.   Gibson, D.T. Microbial Degradation of Organic Compounds.
         Microbiology Series. Marcel Dekker, Inc., New York, New
         York, 1984.

     12.  Gillham, R.W, R.C. Starr, and D.J. Miller. A Device for In Situ
         Determination of Geochemical Transport
    Parameters, 2. Biochemical Reaction. Groundwater, 28:858-862.

13.  Hutchins, S.R., et al. Biodegradation of Aromatic Hydrocarbons
    by Aquifer Microorganisms Under Denitrifying Conditions.
    Environmental  Science and Technology, 25:68-75, 1991.

14.  Hutchins, S.R.. Biodegradation of Monoaromatic Hydrocarbons
    by Aquifer Microorganisms Using Oxygen, Nitrate or Nitrous
    Oxide  as  the  Terminal Electron Acceptor.  Applied and
    Environmental Microbiology, 57:2403-2407,1991.

15.  Kleinbaum  D.G.  and  L.L.  Kupper.  Applied Regression
    Analysis and Other Multivariable Methods. Duxbury Press,
    North Scituate, Massachusetts, 1978.556 pp.

16.  Kosky, K.F. and C.R. Neff. Innovative Biological Degradation
    Systems   for  Hydrocarbons  Treatment.   Presented  at
    NWWA/API Petroleum Hydrocarbons and Organic Chemicals
    in Ground Water  Conference, November 1988. Paper was
    revised in 1989 and included with vendor information.

17.  Kukor, J.J. andR. H. Olsen. Diversity of Toluene Degradation
    Following  LongTerm   Exposure  to  BTEX  In   Situ.  In:
    Biotechnology and Biodegradation. D. Kanely, A. Chakrabarty,
    and G. Omenn, eds. Gulf Publishing Co., Houston, Texas, 1989.
    pp. 405-421.

18.  Lentner, M.  and T. D. Bishop. Experimental Design and
    Analysis. Valley Book Company, Blacksburg, Virginia, 1986.

19.  Loehr,  R.C. Land Treatment as  a  Waste  Management
    Technology: An Overview.  Land Treatment: A Hazardous
    Waste Management Alternative. R.C. Loehr, et al., eds. Center
    for Research in Water Resources, The University of Texas at
    Austin, Austin, Texas,  1986. pp. 7-17.

20.  Macalady, D.L., P.G.  Tratnyek,  and  T.J. Grundl. Abiotic
    Reduction  Reactions of Anthropogenic Organic Chemicals in
    Anaerobic  Systems: A  Critical Review. J. Contam. Hydro!.,
    1:1-28,1986.

21.  Marinucci, A.C. and R. Bartha. Apparatus for Monitoring the
    Mineralization of Volatile 14C-Labeled Compounds. Applied
    and Environmental Microbiology, 38(5): 1020-1022, 1979.
                                                               63
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     22.  Marley, M.C., et al. The Application of the In Situ Air
          Sparging   as  an   Innovative  Soils  and  Groundwater
         Remediation Technology. Groundwater Monitoring Review,
          pp. 137-144, Spring 1992.

     23.  Munnecke, D.M., L.M. Johnson, H.W. Talbot, and S. Barik.
         Microbial Metabolism and Enzymology of Selected Pesticides.
         In:  Biodegradation  and Detoxification  of  Environmental
         Pollutants.  A.M. Chakrabarty, ed. CRC Press, Boca Raton,
         Florida, 1982.

     24.  Odeh R.E.  and M. Fox. Sample Size Choice. Marcel Dekker,
         Inc., New York, New York, 1975.

     25.  Fitter  P.   and  J.  Chudoba.  Biodegradability  of  Organic
         Substances  in the  Aquatic Environment. CRC Press, Boca
         Raton, Florida, 1990.

     26.  Pramer, D.  and R. Bartha. Preparation and Processing of Soil
         Samples for Biodegradation Studies. Environmental Letters,
         2(4):217-224,1972.

     27.  Reineke, W. and H. J. Knackmuss. Microbial Degradationof
         Haloaromatics. Ann. Rev. Microbial., 42:263-287, 1988.

     28.  Roberts, D.J., R.H.  Kaake, S.B. Funk, D.L Crawford, andR.L.
         Crawford.  Anaerobic  Remediation  of  Dinoseb  from
         Contaminated Soil. Applied Bio~hemistry and Biotechnology,
         Vol. 39/40,1993.

     29.  Roberts, D. J., S. Funk, D. L. Crawford, and R. L. Crawford.
         Anaerobic  Biotransformations  of   Munitions  Wastes.
         Symposium of Bloremediation of Hazardous Wastes: Research,
         Development and Field Evaluations. EPA/600/R-93/054, U.S.,
         Environmental Protection Agency, 1993.

     30.  Ross, D. Application of Biological Processes to the Clean Up
         of Hazardous Wastes. Presented at  The 17th Environmental
         Symposium: Environmental Compliance and Enforcement at
         DOD Installations in the  1990s, Atlanta, Georgia, 1990.

     31.  Semprini,   et al.  In Situ Biotransformation  of Carbon
         Tetrachloride, Freon-113, Freon-11, and 1,1,1-TCA Under
         Anoxic Conditions. In: In SituBioreclamation. Applications and
         Investigations  for  Hydrocarbon  and  Contaminated   Site
         Remediation, R.E.   Hinchee  and  R.F.   Olfenbuttel,  eds.
         Butterworth-Heinemann, Stoneham, Massachusetts, 1991.

     32.  Sims R. C.  Treatment Potential for 56 EPA Listed Hazardous
         Chemicals  In Soil.  EPA/600/6-88/001, U.S.  Environmental
         Protection Agency, 1988.

     33.  Snedecor, G.W. and W.G. Cochran. Statistical Methods. Iowa
         State University Press, Ames, Iowa,  1989.

     34.  Tiedje, J.M., A.J. Sexstone, T.B. Parkin, N.P. Revsbech  and
         D.R.  Shelton.  Anaerobic  Processes  in  Soil.  Plant  Soil,
         76:197-212. 1984.

     35.  U.S. Air Force. Test Plan and Technical Protocol for a Field
         Treatability TestforBioventing. Environmental Services Office,
         Air Force Center for Environmental Excellence, May 1992.
36.  U.S. Environmental Protection Agency. A Citizen's Guide to
    Bioventing. EPA/542/F-92/008, March 1992.

37.  U.S. Environmental  Protection  Agency. ACompendium of
    Technologies  Used in the Treatment of Hazardous Wastes.
    EPA/625/8-87/014, 1987.

38.  U.S. Environmental Protection Agency and Air and Waste
    Management  Association.  In:  Proceedings  of  the  1989
    A&WMA/EPA International Symposium on Hazardous Waste
    Treatment: Biosystems For Pollution Control, February 1989.

39.  U.S. Environmental Protection Agency. Bioremediation in the
    Field. EPA/540/2-91/018, August 1991.

40.  U.S. Environmental Protection Agency. Bioremediation in the
    Field. EPA/540/N-92/001, March 1992.

41.  U.S. Environmental Protection Agency. Bioremediation in the
    Field. EPA/540/N-92/004, October 1992.

42.  U.S. Environmental Protection  Agency.  Bioremediation  of
    Contaminated Surface Soils. EPA/600/289/073, August 1989.

43.  U.S. Environmental  Protection  Agency. Bioremediation of
    Hazardous Wastes.  EPA/600/R-92/126, August 1992.

44.  U.S. Environmental Protection Agency. Data Quality Objectives
    for Remedial Response Activities. EPA/540/G-87/003, March
    1987.

45.  U.S. Environmental Protection  Agency. Draft Engineering
    Bulletin: In Situ Biodegradation Treatment. Unpublished.

46.  U.S. Environmental Protection Agency. Engineering Bulletin:
    Slurry Biodegradation. EPA/540/2-90/ 016, September 1990.

47.  U.S. Environmental Protection Agency. Environmental Fact
    Sheet:   EPA  Issue  Final  Rules  for   Corrective  Action
    Management  Units and Temporary Units.

48.  U.S. Environmental Protection Agency, Federal Remediation
    Technologies  Roundtable. Accessing Federal Data Bases for
    Contaminated Site Clean-Up Technologies. EPA/542/B-92/002,
    August, 1992.

49.  U.S. Environmental Protection Agency. Federal Remediation
    Technologies  Roundtable. Federal Publications on Alternative
    and Innovative Treatment Technologies for Corrective Action
    and  Site Remediation, Second  Edition.  EPA/542/B-92/001,
    August  1992.

50.  U.S. Environmental Protection Agency. Ground-Water Issue:
    Soil  Sampling and Analysis for Volatile Organic Compounds.
    EPA/540/4-91 /001, February 1991.

51.  U.   S.  Environmental Protection  Agency.  Guidance  for
    Conducting Remedial  Investigations and Feasibility Studies
    Under CERCLA, Interim Final.  EPA/540/G-89/004, October
    1988.U.S.  Environmental Protection  Agency.  Guide  for
    Conducting  Treatability  Studies Under CERCLA, Final.
    EPA/540/R-92/071a, October 1992.
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     53.  U.S. Environmental Protection Agency. Guide for Conducting
         Treatability Studies Under CERCLA: Aerobic Biodegradation
         Remedy Screening, Interim Guidance. EPA/540/2-91/013 A, July
         1991.

     54.  U.S. Environmental Protection Agency. Handbook on In Situ
         Treatment  of  Hazardous  Waste  -  Contaminated Soils.
         EPA/540/2-90/002, January 1990.

     55.  U.S. Environmental Protection Agency. Hazardous Waste Land
         Treatment. SW-874,1983.

     56.  U.S. Environmental Protection Agency. Hazardous Waste
         Treatment,  Storage, and Disposal Facilities (TSDF) - Air
         Emission Models. EPA/450/3-87/026, 71. November 1989.

     57.  U.S. Environmental Protection Agency. Innovative Hazardous
         Waste Treatment  Technologies:  A Developer's  Guide to
         Support  Services,  Second  Edition.  EPA/540/2-91/012, June
         1992.

     58.  U.S.  Environmental  Protection  Agency.  Innovative 73.
         Treatment Technologies: Overview and Guide to Information
         Sources. EPA/540/9-91/002, October 1991.

     59.  U.S. Environmental Protection Agency. Innovative Treatment
         Technologies: Semi-Annual Status Report. EPA/542/R-92/011,
         October 1992.

     60.  U.S. Environmental Protection Agency. International Evaluation
         of  In-Situ   Biorestoration  of  Contaminated  Soil   and
         Groundwater. EPA/540/2-90/012, September 1990.

     61.  U.S.  Environmental   Protection  Agency.   Inventory of
         Treatability   Study  Vendors,   Volumes I  and  II. EPA/
         540/2-90/003a and b, March 1990.

     62.  U.S. Environmental Protection Agency. Laboratory  77. and
         Field Studies on BTEX Biodegradation in a Fuel- Contaminated
         Aquifer Under Denitrifying Conditions.  EPA/600/D-91/256,
         1991.

     63.  U.S. Environmental Protection Agency. Methods forChemical
         Analysis of Water and Wastes. EPA/600/4-79/020, March 1979.

     64.  U.S. Environmental Protection Agency. Methods for Evaluating
         the Attainment of Cleanup Standards, Volume I: Soils and Solid
         Media. EPA/230/02-89/042, February 1989.

     65.  U.S.  Environmental   Protection  Agency. Microbiological
         Decomposition  of  Chlorinated Aromatic Compounds.  EPA
         600/2-86/090,1986.

     66.  U.S.   Environmental  Protection  Agency.  Microcosms,
         Laboratory   Macrocosms,  and In  Situ Macrocosms. In:
         Nineteenth   Annual  RREL   Hazardous  Waste  Research
         Symposium Abstract Proceedings. EPA/600/R-93/040, April
         1993.
67.  U.S. Environmental Protection Agency. Preparation Aids for the
    Development of Category IV Quality Assurance Project Plans.
    EPA/600/8-91/006, February 1991.

68.  U.S. Environmental  Protection Agency. Review of In-Place
    Treatment  Techniques for  Contaminated  Surface  Soils.
    EPA/540/2-84/003b,  November 1984.

69.  U.S. Environmental Protection Agency. Screening Protocol for
    Evaluating  Biotreatability  Potential of Contaminated Soil.
    March 17,1992. Unpublished.

70.  U.S. Environmental Protection Agency. Statistical Analysis of
    Groundwater Monitoring Data at RCRA Facilities, Interim
    Final. EPA/530/SW-89/026, April 1989.

71.  U.S. Environmental Protection Agency. Summary of Treatment
    Technology   Effectiveness   for  Contaminated   Soil.
    EPA/540/8-89/053, June 1990.

72.  U.S. Environmental Protection Agency. Superfund Treatability
    Clearinghouse Abstracts. EPA/540/289/001, March 1989.

73.  U.S. Environmental Protection Agency.  Test Methods for
    Evaluating Solid Waste. Third Ed., SW-846, December 1987.

74.  U.S.  Environmental  Protection  Agency.  The Superfund
    Innovative  Technology  Evaluation  Program:  Technology
    Profiles, Fifth Edition. EPA/540/R-92/077, December 1992.

75.  U.S. Environmental  Protection Agency. Treatability Studies
    Under  CERCLA:  An   Overview.  OSWER Directive
    9380.3-02FS, December 1989.

76.  U.S. Environmental Protection Agency. Users Guide  for Land
    Treatment-Compound  Property Processor and Air Emissions
    Estimator (LAND 7). EPA/540/3-87/026, November  1989.

77.  U.S. Environmental  Protection Agency. Vendor Information
    System for  Innovative Treatment Technologies (VISITT).
    EPA/540/2-91/011, June 1991.

78.  Vogel, C., R. Hinchee, R. Miller, and  G.  Sayles. Bioventing
    Hydrocarbon-Contaminated Soil in a Sub-Arctic Environment.
    Presented at The Second International  Symposium on In Situ
    and On-Site Bioreclamation, San Diego, California, April 5-8,
    1993.

79  CFR   1910.120(a)  -  Hazardous  Waste   Operations and
    Emergency Response - Appendix B.

80.  40 CFR, Part 798. Subpart  F. Genetic Toxicity. Office of the
    Federal Register, Washington, D.C., July 1991.

81.   40 CFR, Section 796.3400. Inherent Biodegradability in Soil.
    Office of the Federal Register, Washington, D.C., July 1991.

82.  40 CFR, Section 797.2750. Seed Germination/Root Elongation
    Toxicity Test. Office of Federal Register, Washington, D.C.,
    July 1991.
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                         APPENDIX A
                  COMPENDIUM OF TOOLS
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            There are a number of tools available that can be useful during bioremediation remedy selection treatability studies.
     Specific tools are briefly described in Tables A-l through A-6. Additional information on bioremediation testing tools can be
     found in various references, including the ASTM Standards on Materials and Environmental Microbiology1-^. Other references
     that may provide further information are listed in Subsection 2.2.1 of this guide. Definitions for unfamiliar technical terms may be
     found in the Dictionary of Microbiology  (b) or the Dictionary of Biotechnology1-0'
                                     Table A-1. Tools Used for Toxicity Testing
Tool
Microtox
Description/Application
This automated test measures
toxicity to bacteria and can be
Advantages
Unit is easy to
operate.
Disadvantages
Correlation to human and
animal toxicity is not clear.
      Genotoxicity (40
      CFR 798.5100
      through
      798.5955)(d)
      Seed
      germination/root
      elongation (40
      CFR 797.2750)


      Earthworms
      Cerio daphnia
      Fathead
      minnows
      Genotoxicity
      (plants)
used to determine whether
treatment is reducing the
environmental toxicity of leachate
from treated soil.

These toxicity tests measure
genetic damage to bacteria and     human and animal
other organisms and can be used   toxicity is projected.
to determine whether treatment
is reducing toxicity to human
health.
A good correlation to   An automated testing unit is
                      not yet commercially
                      available.
This toxicity test can be used to
determine whether treatment is
reducing environmental toxicity.
This toxicity test measures
impact on earthworm deaths and
can be used to determine
whether
treatment is reducing
environmental toxicity.

This toxicity test measures
impact on cerio dephnia and can
be used to determine whether
treatment is reducing
environmental toxicity.

This toxicity test measures
impact on fathead minnows and
can be used to determine
whether treatment is reducing
environmental toxicity.

This toxicity test measures
genetic damage to plants and
can be used to determine
whether treatment is reducing
toxicity to the environment.
Test is very sensitive
to PAHs.
Applicable for
determining soil
toxicity.
Applicable for
determining the
toxicity of aqueous
media including
leachates.

Applicable for
determining the
toxicity of aqueous
media including
leachates.
Sensitivities vary for seed from
different plants. Also, this test
is not applicable for
contaminants that are not
water-soluble.

Not appropriate for
determining aquatic toxicity.
Cannot be used to directly
measure soil toxicity.
Cannot be used to directly
measure soil toxicity.
Extremely sensitive.    Requires lenghty training.
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                         Table A.2. Tool Used to Measure or Describe Biological Activity
      Tool
Description/Application
Advantages
Disadvantages
      Respirometry
      Fluorocene
      diacetate

      Resazurin (f)
      Microbial
      assay/enumeratio
      n (plate counts,
      etc.)

      Epifluorescence
      microscopy (f)
      Most probable
      number (MPN)
      methods (f)
      Arrhenius equation
      Reaction kinetics
Used to determine
biodegradability and reaction
kinetics from oxygen
consumption or carbon dioxide
evolution.

Enzyme-based used to
determine biological activity.

A redox indicator (can be used to
indicate whether anaerobic or
aerobic conditions are present).

Determining the type and under
of bacteria present in a sample to
determine biological activity.


Determining the total number of
active bacteria present in a
sample.
Estimating the number of
microorganisms in a sample that
are capable of degrading the
contaminants of interest.

Equation used to describe the
temperature dependence of a
reaction rate (such as a
biodegradation rate).

Equations used describe the rate
of degradation (or production) of
chemical compounds.
Can be used to test for
microbial inhibition; rapid
easy to operate.
Easy to use.
Standard technique.
Specific to groups of
microorganisms with
special degradation
abilities.

Allow reaction rate data
collected at one
temperature to be applied
at other temperatures.

Can be used  to estimate
cleanup times.
Oxygen consumption or
carbon dioxide evolution
due to chemical
degradation can yield a
false positive.

Not readily available
May not count the
microorganisms of
interest.
Measures total bacteria
and cannot be used
quantify a certain type of
bacteria.(g)

Labor intensive.
Not applicable for all
reactions at all
temperatures.


Because reaction may be
governed by multiple
mechanisms or rate-
limiting factors,  the
kinetics may change at
low contamination
concentrations.
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                                 Table A-3. Tools Used to Inhibit Biological Activity
      Tool
Description/Application
Advantages
Disadvantages
      Formaldehyde
      Mercuric chloride
      Sodium azide
      Low pH
Use to inhibit biological
activity in control cells.
Used to inhibit biological
activity in control cells.
Used to inhibit biological
activity in control cells (by
inhibiting respirometric
activity).

Used to inhibit biological
activity in control cells.
Less hazardous than
some inhibitors.
Effective.
Usually effective for
aerobic bacteria.
Effective.
Not always effective; can be
degraded by some
organisms.

Use of mercury compounds
may be restricted in some
laboratories. Not always
effective because it can
reduce measured petroleum
hydrocarbons
concentration.

Potentially explosive. Not
effective for bacteria that are
capable of anaerobic
degradation.
                                  Table A-4. Tools Used to Develop Mass Balances
Tool
Radiolabeling
Description/Application
Biodegradation studies can
Advantages
Can be used determine
Disadvantages
Cost can be high.
                             be studied using14 C
                             compounds.
                             degradation products. Removal
                             mechanisms, and mass balances.
      Liquid scintillation
      counter
Used to detect radiolabeled
compounds
Can be used to determine
degradation products, removal
mechanisms, and mass balances.
      Roll tube test
                                      Table A-5. Tools Used for Anaerobic Testing
Tool
Mclntosh and
Fildes' jar
Description/Application
Closed, flask-like reactor, which
employs a gas-pack generator H2 and
Advantages
Easy to use.
Disadvantages
Subsampling may not be
possible.
  CO2)and a pallidium catalyst to establish
  an O2-free system in which anaerobic
  testing can be assesses. Can be used
  to develop an anaerobic culture.

  Test tubes, containing an agar-like
  medium and N2, used to develop an
  anaerobic culture or to directly assess
  biodgradation.
            Easy to use.
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                                Table A-6. Tools Used for Physical Character of Soils
      Tool                                                   Description/Application

      Hydraulic conductivity                                   A soil property that determines the maxiumum flow
                                                             rate of water through the soil.

      Soil moisture retention01'                                 Once determined, the soil moisture retention can be
                                                             increased or decreased if necessary.

      Soil density                                            Measures soil density01
       (ASTM Methods D1556-82 and D2937-83)ffl

      Particle size analysis of soils                            Quantitive determination of the distribution of particle
       (ASTM Method D422-63)(i)                              size in soil.

      Soil water content                                       Measures water content of soil.
       (ANSI/ASTM D2216-80 and ASTM D3017-88)ffl

      Specific gravity of soils                                  Measures specific gravity of soil.
       (ASTM D854-83)ffl
    References

    a   ASTM Standards on Materials and Environmental Microbiology. American Society for Testing and Materials,
        Philadelphia, PA. 1987.

    b   Dictionary of Microbiology. Singleton. John Wiley and Sons. 1978.

    c   Dictionary of Biotechnology. Coombs. 1986.

    d   40 CFR, Section798 SubpartF. Genetic Toxicity. July 1, 1991.

    e   40 CFR, Section 797.2750. Seed germination/root elongation toxicity test. July 1, 1991.

    f   Davis, J. W. and S.  S. Madsen. The Biodegradation of Methylene Chloride in Soils. Environmental Toxicity and
        Chemistry, Vol. 10pp. 463-474,1991.

    g   Roberts, P. V., G. D. Hopkins, D. M. Mackay, and L. Semprini. A Field Evaluation of In-Situ Biodegradation of Chlorinated
        Ethenes: Part 1, Methodology  and Field Site Characterization. Groundwater, Vol. 28, No. 4, July- August 1990.

    h   Klute, A. Editor. Methods of Soil Analysis, Part 1: Physical And Mineralogical Methods. Second Edition. American
        Society of Agronomy, Inc. and Soil Science Society of America, Inc. Madison,  WI. 1986.

    i   Environmental Protection Agency. Handbook on In Situ Treatment of Hazardous Waste - Contaminated Soils.
        EPA/540/2-90/002. January 1990.

    j   Annual Book of ASTM Standards, American Society for Testing and Materials, Philadelphia, PA.
                                                          71

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          EPA
    PERMIT No. G-35
Word-searchable version — Not a true copy

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