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.
11
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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.
12
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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-
13
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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
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22. Marley, M.C., et al. The Application of the In Situ Air
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30. Ross, D. Application of Biological Processes to the Clean Up
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33. Snedecor, G.W. and W.G. Cochran. Statistical Methods. Iowa
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34. Tiedje, J.M., A.J. Sexstone, T.B. Parkin, N.P. Revsbech and
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38. U.S. Environmental Protection Agency and Air and Waste
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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.
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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
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Contaminated Site Clean-Up Technologies. EPA/542/B-92/002,
August, 1992.
49. U.S. Environmental Protection Agency. Federal Remediation
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and Innovative Treatment Technologies for Corrective Action
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53. U.S. Environmental Protection Agency. Guide for Conducting
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81. 40 CFR, Section 796.3400. Inherent Biodegradability in Soil.
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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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