PNL-4601
EPA-600/3-83-%3
EPA GUIDE FOR IDENTIFYING
CLEANUP ALTERNATIVES AT
HAZARDOUS-WASTE SITES AND SPILLS:
BIOLOGICAL TREATMENT
Prepared for
Office of Emergency and Remedial Response
U.S. Environmental Protection Agency
Prepared by
Pacific Northwest Laboratory
Operated for the U.S. Department of Energy
by Battelle Memorial Institute
Project Management by
Corvallis Environmental Research Laboratory
U.S. Environmental Protection Agency
Corvallis, Oregon
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DISCLAIMER
This manual describes the scientifically relevant and important functional activities
that concern regional, state, and local authorities during the response to hazardous
substance releases It is intended to convey technical recommendations only and not
to constitute agency policy The manual should not supersede specific procedures
and docu mentation for quality assurance, chain-of-custody, and other requirements
addressed by Environmental Protection Agency regulations and policy guidance
documents currently in effect The research described in this report has been funded
by the United States Environmental Protection Agency through interagency agree-
ment no AD-89-F-2A115 to the United States Department of Energy. This report has
been subjected to the Agency’s required peer and policy review
This report was prepared as an account of work sponsored by an agency of the
United States Government Neither the United States Government nor any agency
thereof, nor any of their employees, makes any warranty, express or implied, or
assumes any legal liability or responsibility for the accuracy, completeness, or use-
fulness of any information, apparatus, product, or process disclosed, or represents
that its use would not infringe privately owned rights Reference herein to any
specific commercial product, process, or service by trade name, trademark, manu-
facturer, or otherwise, does not necessarily constitute or imply its endorsement,
recommendation, or favoring by the United States Government or any agency
thereof Theviews and opinions of authors expressed herein do not necessarilystate
or reflect those of the United States Government or any agency thereof
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PNL-4601
EPA-600/3-83-063
EPA GUIDE FOR IDENTIFYING
CLEANUP ALTERNATIVES AT
HAZARDOUS-WASTE SITES AND SPILLS:
BIOLOGICAL TREATMENT
Prepared for
Office of Emergency and Remedial Response
U S Environmental Protection Agency
Prepared by
Pacific Northwest Laboratory
Operated for the U S Department of Energy
by Battelle Memorial Institute
Project Officer- L. C. Raniere
Corvallis Environmental Research Laboratory
U S Environmental Protection Agency
Corvallis, Oregon
Interagency Agreement No. AD-89-F-2A115
Publication No EPA-600/3-83-063
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PREFACE
The Comprehensive Environmental Response, Compensation, and Liability Act of 1980
(CERCIA) grants the President the authority to respond to releases of hazardous chemical
substances that imminently and substantially threaten public health or welfare, or the
environment The Act, which establishes a $1 6-billion Superfund to finance response
actions, and which charges the Environmental Protection Agency (EPA) with administering
critical portions of the response program, was designed to build on the existing environ-
mental response authority given to EPA under Section 311 of the Clean Water Act
In accordance with this mandate, the EPA is preparing a series of documents to assist state
and regional officials who are responsible for instituting cleanup actions at specific
hazardous-waste sites This guide deals exclusively with biological processes as tools for
cleaning up a site
This guide supplies the information and sources that an official needs to decide how to
proceed at a contaminated site This document also can be used by management to assist
in and overview staff cleanup activities. Additionally, it is a training tool for persons who
are new to the business of cleaning up hazardous-waste sites Finally, it is a resource for
anyone who wishes to know more about the cleanup of hazardous-waste sites
iii
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ACKNOWLEDGMENTS
Pacific Northwest Laboratory (PNL), operated by Hattelle Memorial institute for the U.S.
Department of Energy, prepared this document The work was performed under inter-
agency agreement no AD-89-F-2A115 with the U S Environmental Protection Agency
(EPA) Office of Research and Development, Corvallis Environmental Research Laboratory,
Corvallis . Oregon The work was performed at the request of the EPA Office of Emergency
and Remedial Response Dr Lawrence C Raniere was the EPA Project Officer and
Dr Richard A Craig was the PNL Project Manager Lead author for this work was
Dr. C Dale Becker Other PNL authors were C E. Cushing, D A Neitzel, C M Novich,
E Rogers and D J Silviera, with assistance from R. L. Aaberg, j S Burlison, S W Li, and
I L Posakony Technical review and guidance were provided by Harry Allen, John Bruger,
Karen Z Burgan, Phil Cook, Barbara Elkus, William E Fallon, A Galli, A F Gasperino,
john E Mathews, Leon H Meyers, Royal Nadeau, Spencer A Peterson, John Sainsberg,
Don Sanning and Richard Stanford
In addition, technical peer reviews were provided by Mark Harwell of Cornell University,
Anthony Dvorak of Argonne National Laboratory, and Naomi Barkeley of EPA ’s Municipal
Environmental Research Laboratory, Cincinnati
V
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EXECUTIVE SUMMARY
Under the provisions of Superfund (CERCLA of 1980), the Environmental Protection
Agency (EPA) has been charged with responsibility for cleaning up hazardous-waste dis-
posal sites This guide provides direction to on-scene personnel who must assess hazard-
ous wastes in the environment and may want to implement biological cleanup methods
Treatment For hazardous wastes are broadly grouped into physical, chemical, and biolog-
ical methods This document emphasizes biological treatment, but all three methods can
be integrated, as appropriate, to achieve optimal elfectiveness Physical or chemical pre-
treatment may be necessary prior to biotreatment if a waste is toxic to microorganisms in
its current form or concentration Almost all organic compounds and some inorganic
compounds can be degraded microbiologically, given suitable physical and chemical con-
ditions and sufficient time to do so
Pre-treatment site assessment is critical to provide the background data necessary to
evaluate and select treatment methods Information is needed about the wastes and their
concentrations, the danger of the wastes to workers, the features of the site environment,
and the biotransformation capacity of microorganisms Post-treatment site monitoring is
recommended to establish effectiveness of the methods applied
Biological methods can be applied to hazardous wastes either onsite or offsite Biological
methods include a no-treatment option (natural biodegradation), which relies on detoxifi-
cation of wastes by natural processes Biological treatment of hazardous wastes on soil is
called land treatment and includes both direct application and compositing Biological
methods can also be applied with special facilities developed for wastewater treatment
These facilities, called bioreactors, include the activated-sludge, trickling filter, rotating
disc, and aerobic and anaerobic lagoon or pond systems Hazardous wastes may also be
cleaned from a site by bioaccumulation and removal techniques.
Biotreatment of haLardous waste may be enhanced by controlling environmental condi-
tions Biotransformation of hazardous materials can be aided by dilution and by chemical
and physical pre-treatment Environmental conditions in land treatment and bioreactor
systems can also be enhanced to favor growth and enzymatic activities of microorganisms
Enhancement of the waste environs includes adlustment of pH, temperature, nutrient
levels, and aeration In some cases, specialized microorganisms can be developed and
introduced to aid microbial transformation Examples of such organisms are generic
microorganisms that are indigenous to the waste, the site, or the system, enriched
microorganisms thai are cultured to degrade a specific waste, or adapted microorganisms
that are cultured for site-specific conditions
The advantages and disadvantages of various biological treatment methods are discussed
in the text. Diagrams are provided to help personnel decide which treatment to select
The relative effectiveness of different biotreatment procedures at locations with hazardous
wastes is largely site-specific Relative effectiveness can be expected to vary from site to
site because environmental parameters will vary, as will types, quantities, and conditions
of hazardous-waste materials Thus, different methods may be favored at different
locations.
vii
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CONTENTS
PREFACE . . i i i
ACKNOWLEDGMENTS v
EXECUTIVE SUMMARY ... . v i i
1 0 INTRODUCTION . .. . . . 11
2 0 BIOTREATMENT OPTIONS . 2 1
3 0 SELECTION OF POTENTIAL BIOTREATMENT OPTIONS . 3.1
3 1 IS BIOTREATMENT VIABLE AT THE SITE? 3 1
3 2 CAN THE WASTE AT THE SITE BE TREATED BIOLOGICALLY? 3 3
3,3 WHAT QUESTIONS MUST BE ANSWERED TO EVALUATE
BIOTREATMENT OPTIONS? 3 5
4 0 SITE-SPECIFIC CONSIDERATIONS AT HAZARDOUS-WASTE SITES OR SPILLS .. 4 1
41 WHAT INFORMATION SHOULD BE COLLECTED BEFORE
TREATMENT? 41
4.2 WHAT INFORMATION SHOULD BE COLLECTED AFTER
TREATMENT? . ... . 415
4.3 CHAPTER REFERENCES ... . 4.24
5.0 DESCRIPTION OF BIOTREATPVIENT OPTIONS .. .. 5 1
51 NATURAL BIODEGRADATION ... . .... 5 1
52 LANDTREATMENT 51
5 3 BIOREACTOR SYSTEMS 5 7
54 BIOACCUMULATIONANDREMOVAL 515
5 5 ENHANCEMENT OF BIOTREATMENT PROCESSES 5 17
5.6 CHAPTER REFERENCES . 5 27
6 0 BIOTRANSFORMATION CONCEPTS 6.1
61 ROLE OF MICROORGANISMS IN B iOTRANSFORMATION . . . 6 1
6.2 BIOTRANSFORMATION PROCESSES . 6 6
63 RECALCITRANT COMPOUNDS 614
6.4 BIOACCUMULATION 618
65 ACTIVATION 620
6.6 METALS—SPECIAL CASES 6 21
6 7 ENV iRONMENTAL INFLUENCES . . 6.23
6 8 CHAPTER REFERENCES 6.25
ix
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FIGURES
11 Summary of Guide Chapters as They Relate to Specific User Needs .... . ... 11
1.2 Steps in Remedial Action Process for Responding to an Uncontrolled
Hazardous-Waste Site or Spill . 1 2
2 1 Concept of Onsite and Offsite Treatment Options Used for Biotransforminj
Hazardous Wastes 2 1
3 1 Selection of Biotreatment as a Cleanup Option .... 3 1
3 2 Flow Chart for Determining Whether or Not Chemicals Can Be
Treated Biologically 3.4
4 1 A Flow Chart for Categorizing Priority Pollutants 4 11
4 2 Decision Matrix for Planning Post-Treatment Monitoring 4.18
5.1 Matrix Comparison of Advantages and Disadvantages of Biotreatment Options for
Hazardous Wastes .... 5 4
5 2 Decision Process for Evaluating Land Application (Including Composting) for
Biotreatment of Hazardous Wastes 5 6
5 3 Decision Process for Evaluating Activated Sludge Biotreatment ... 5 10
5 4 Decision Process for Evaluating Trickling Filter Biotreatment . . . 5 11
5 5 Decision Process for Evaluating Rotating Disc Biotreatment .... .. . .. 5 13
5 6 Decision Process for Evaluating Lagoon and/or Pond Treatment 5 14
5 7 Decision Process for Evaluating Bioaccumulation by Algae and
Higher Plants (Autotrophs) .. . .. 5 17
5 8 Decision Process for Determining if Enhancement is Required
for Biotreatment .. 5.20
6 1 Hypothetical Model for Population Changes and Metabolism of a Chemcal
Exposed to Mineralizing and Co-metabolizing Populations . 6.8
x
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TABLES
4 1 EPA List of Hazardous Chemical Compounds 4.6
4 2 Known or Suspect Carcinogenic Materials . . . 4 7
4 3 Compounds, Category Rank and Environmental Compartments of EPA
Priority Pollutants . . . 4 8
6.1 A Summary of Microorganisms Involved in Biotransformation of Chemical
Compounds and Their Selective Characteristics . .. 6.3
6 2 Information on the Biotransformation of Chemical Compounds,
Including Reaction Type, Microorganisms Involved, Condition,
and Transformation Products 6 9
6.3 Relative Persistence of Selected Pesticides in Soil Under
Natural Conditions 615
6 4 Some Organic Compounds that Persist in Soils and Water for
Relatively Long Periods of Time .. . 6.17
6.5 Some Synthetic Polymers Resistant to Transformation by Microorganisms 6 17
6 6 Examples of Bioaccumulation of Synthetic Compounds by
Different Microorganisms . 6 18
xi
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1.0 INTRODUCTION
WHY THIS GUIDE?
This document provides guidance to help decide whether or not biological
treatment is an appropriate component of the remedial action process at a
hazardous-waste site or spi 1 i The guide describes biological treatment
methods, conditions and factors that limit the use of biotreatment, and types
of data needed to evaluate and select biotreatment methods Flow charts are
included to help users determine the usefulness of each biotreatment method
at specific hazardous-waste sites and spills
WHO CAN USE THIS GUIDE?
This guide is intended for use primarily by people who have limited expe-
rience in participating in or coordinating cleanup activities at uncontrolled
hazardous-waste sites In addition to being a training tool, an educational
resource, and a source of assistance during remedial-action analysis, this guide
may be used by managers to maintain a broad overview of cleanup programs
This guide contains material designed to serve many different users (Fig-
ure 1 1) As a decision-making tool, the document supplies information and
sources of information needed for users to make decisions about cleanup
activities
DECISION-MAKING
MANAGEMENT
EDUCATION
CHAPTER
FIGURE 1.1 Summary of Guide Chapters as They Relate to Specific User Needs
11
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introduction
As a management tool, the document contains flow charts that will help the
user select cleanup alternatives U also lists advantages and disadvantages of
various treatment methods to assist in and overview staff cleanup activities
As a training tool, the document reviews biotransformation techniques, dis-
cusses information needs related to selection of cleanup alternatives, and out-
lines assessment activities required at most cleanup sites
As an educational tool, the document reviews basic information that a user
needs to understand biological cleanup methods Obviously, all the biological,
chemical, and physical information necessary to understand biotransformation
of hazardous waste cannot be discussed in a guide More complete informa-
tion can be obtained by reading the references cited.
WHERE DOES THIS GUIDE FIT INTO THE REMEDIAL ACTION
PROCESS?
This guide is intended for use during the time when chemical, physical and
biological cleanup alternatives are being identified, that is, after the waste and
site have been characterized and health risks have been assessed, and before
the cleanup alternatives are analyzed in detail (see shaded area, Figure 1 2)
The guide is not intended as a design guide nor as an encyclopedic guide for
analyzing remedial-action alternatives Rather, when used, it ensures that
biological methods are given proper consideration during the development of
cleanup alternatives
I DISCOVERY 1
I STABILIZATION I
PRELIMINARY I
SURVEY
I ____________________________________________________________
DECISION FOR I PLANNED
FURTHER RESPONSE RESPONSE
SITE
CHARACTERIZATION
ANALYSIS OF LI SELECTION OF
ALTERNATIVESJJREMEDIAL ACTION
[ ASSESSMENT
FIGURE 1.2 Steps in Remedial Action Process for Responding to an uncontrolled
Hazardous-Waste Site or Spill
iDENTiFiCATiON OF
ALTERNATIVES
DESIGN OF
REMEDIAL ACTION
IMPLEMENTATION
OF
REMEDIAL ACTION
12
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Introducison
WHAT IS BIOLOGICAL TREATMENT?
Biological treatment uses the activity of microorganisms to transform hazard-
ous chemical compounds into innocuous materials. Almost all organic com-
pounds, and some inorganic compounds, can be degraded biologically if
given the proper physical and chemical conditions and sufficient time Biologi-
cal processes are particularly useful for detoxifying aqueous solutions that con-
tain dilute concentrations of hazardous materials
Microorganisms normally metabolize organic matter to produce energy by
pathways involving a variety of enzyme-catalyzed reactions Simple organic
compounds that occur naturally are readily incorporated into the cells of
microorganisms and oxidized The larger, more complex molecules are
degraded at a much slower rate Although microorganisms may be adapted
and grown on many types of organic materials, some complex organic com-
pounds are not degraded or are degraded very slowly, these compounds are
called “recalcitrant” organic compounds (refractory compounds) Other
organic compounds are toxic to microorganisms or inhibit their activity.
Special methods may be necessary to enhance biotransformation of refractory,
toxic, or inhibitory organic compounds
Inorganic materials (such as metals) and some organic compounds may be par-
tially removed from the environment by concentration, through the mecha-
nisms of adsorption or transport, in the microorganism during biological
treatment (bioaccumulation) However, inorganic materials are rarely
destroyed by biological treatment and may inhibit biodegradation at higher
concentrations
The behavior of chemical substances in the environment and during biological
treatment is influenced by their degradability and toxicity On the basis of
these two characteristics, chemical compounds can be divided into four
groups
• degradable and non-toxic
• degradable and toxic
• non-degradable and non-toxic
• non-degradable and toxic
Compounds in the first group are not oblectionable over the long term Com-
pounds in the second group, after being sufficiently diluted to prevent toxic
effects, can eventually be transformed by natural biological processes Com-
pounds of the third group may be aesthetically or otherwise objectionable
Compounds of the fourth group must be prevented from entering the envi-
ronment The degradable, toxic materials (second category are the focus of
this guide
13
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2.0 BIOTREATMENT OPTIONS
Treatment of hazardous materials can be conducted onsite or offsite (Figure
2.1) Onsite treatment may be prevented by local restrictions, state regulations,
population density, or other reasons The hazardous waste and contaminated
soil or water must then be taken elsewhere for treatment. For both onsite and
offsite treatment, the facility or degradation site must be secured to prevent
unwanted human or animal activity Boih onsite and offsite treatments include
three options
FIGURE 2.1 Concept of Onsute and Offsite Treatment Options Used for
Biotransformung Hazardous Wastes
ATMENT OPTION
OPTION
.I_
I ENHANCEME
IMPROVE PHYSICAL
CHEMICAL CONDITIONS
ADD MICROORGANISMS
• GENERIC
• ADAPTED
• ENRICHED
SURFACE
APPLICATION
SOLL
INCORPORATION
COMPOSTING
IBIOREACTO
SYSTEMS
ACTIVATED SLUDGE
TRICKLING FILTER
ROTATING DISCS
LAGOONS AND PONDS
(AEROBIC & ANAEROBIC (
OTHER
21
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B,otreaiment Options
a Destroy the waste by physical or chemical methods
b Allow the waste to degrade naturally
c. Degrade the waste at a biotreatment facility (which might apply chemical
and physical methods as well as biological methods)
When a waste site or spill is secured or the hazardous material is placed in an
authorized disposal site, wastes may degrade naturally (3) Most hazardous
organic wastes in the environment, given sufficient time, will eventually be
transformed or degraded Thus, sites where hazardous materials occur will
eventually return to their natural state.
The rate of waste degradation can be controlled with biotreatment methods
The type of biotreatment (4) at onsite and offsite locations will depend on the
feasibility of land treatment methods, the availability of special facilities such as
bioreactor systems, and the opportunity for enhancing site-specific biological
processes.
Land treatment (5) can be used to degrade solid, semi-solid, and liquid wastes.
Application of the method requires consideration of waste loading rates) com-
posting, and other methods such as surface tilling. Land treatment is econom-
ical but may require the commitment of large land areas for long periods of
time
Bioreactor systems (6) are used primarily for liquid wastes. Bioreactors are
derived from established systems that routinely treat municipal and industrial
waste streams. Degradation of hazardous materials will usually take place at
higher rates in bioreactor systems than in field situations because operating
conditions can be controlled Bioreactor systems are usually costly to con-
struct, operate, and maintain but are often the most effective cleanup alterna-
tive Currently, few waste-treatment systems are operating for the sole
purpose of detoxifying hazardous wastes
Some wastes may be accumulated in the tissue of plants (7) The plants can be
subsequently harvested and removed from the waste site or spill area Bio-
accumulation is usually a final step in the cleanup process The suitability of
bloaccumulation techniques depends on the toxicity of the waste, the time
available to clean the site or spill, the ability to dispose of removed material,
and safety and environmental factors.
Site enhancement (8) includes modifying site-specific physical and chemical
conditions to improve the effectiveness of biotreatment. In some situations,
degradation of wastes can be enhanced biologically by adding micro-
organisms Enhancement methods can be applied to land treatment, bio-
reactor systems, and bioaccumulation techniques
22
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3.0 SELECTION OF POTENTIAL BIOTREATMENT OPTIONS
Biological, physical, and chemical alternatives must be considered when
selecting a cleanup strategy This chapter addresses questions used to qualify
biological treatment as a viable method to treat hazardous waste
3.1 IS BIOTREATMENT VIABLE AT THE SITE?
Questions for determining whether or not biotreatrnent is a viable cleanup
alternative are outlined in Figure 3 1 Requirements for making decisions in
Steps 1 through 5 of Figure 3 1 are discussed below The items listed below
each question are the types of information and the sources of information
needed to answer the questions. More detailed discussion of specific consid-
erations and questions are included in subsequent chapters of this guide.
IS THE SITE ENVIRONMENT NO
COMPATIBLE WITH
BIOTREATMENT?
YES 1
CAN THE WASTE BE [ NO
TREATED BIOLOGICALLY? [
YES
IS BIOTREATMENT 1 YES CONSIDER NON -
PREVENTED BY BIOTREATMENT
REGULATION? I ALTERNATIVES
__________J® ______
NO
WILL SAFETY OR 1
ENVIRONMENTAL YES
CONSIDERATIONS PRECLUDE I
BIOLOGICAL TREATMENT?__I®
NO
WILL PUBLIC HEALTH AND
WELFARE CONSIDERATIONS YES
PREVENT TIMELY
BIOLOGICAL TREATMENT?
tNO
BIOTREATMENT OF HAZARDOUS WASTE
IS A CLEANUP ALTERNATIVE
FIGURE 3.1 Selection of Biotreatment as a Cleanup Option
31
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Selection of Biotreatment Options
Is the site environment compatible with biotreatment? (Figure 3 1, Step 1)
• Consider the following types of information
- climate (temperature, precipitation, wind direction)
- biota (plants, animals, microorganisms)
- soil (permeability, contamination)
- topography
- surface water (lakes, rivers, runoff)
- ground water (location, public use).
• Consider the following sources of information.
- public use records
- site environmental assessment
- environmental impact statements or management plans for other
activities
- governmental services (weather bureau, agricultural extension, soil
conservation district)
Can the waste be treated biologically? (Figure 3 1, Step 2)
• Consider the following types of information
- condition of waste containers (scaled, damaged)
- physicochemical characteristics (pH, solid, liquid)
- toxicity
- enhancement requirements to treat waste
- data about degradability of related compounds
- by-products of degradation
• Consider the following sources of information
- records (source of waste, condition)
- observable physical and chemical characteristics
- detailed chemical analysis
- available degradation data (journal articles, technical reports, expert
advice)
Is biotreatment prevented by regulation? (Figure 3 1, Step 3)
• Consider the following types of information:
- regulations applicable to the type of waste
- regulations applicable to the treatment process
- regulations applicable to transportation requirements
• Consider the following sources of information:
- local, state, and Federal laws, regulations, and officials
Will safety or environmental considerations preclude biological treatment?
(Figure 3 1. Step 4)
• Consider the following types of information
- site characteristics
- waste characteristics
32
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Selection of Bsotreatment Options
• Consider the following sources of information
- local, state, and Federal laws, regulations, and officials
- assessment of potential impacts of cleanup
Will public health and welfare considerations prevent timely biological
treatment? (Figure 3 1, Step 5)
• Consider the following types of information
- local population (density, proximity)
- avenues by which population may be affected
• Consider the following sources of information:
— risk assessment
- site environmental assessment
- environmental impact statements for other activities
3.2 CAN THE WASTE AT THE SITE BE TREATED BIOLOGICALLY?
Questions for determining whether or not a chemical can be biologically
degraded are outlined in Figure 3 2 Requirements for making decisions in
Steps 1 through 6 of Figure 3 2 are discussed below. The items listed below
each question are the types of information and sources of information needed
to answer the questions
Do published reports indicate the chemical has been successfully biotreated?
(Figure 3 2, Step 1)
• Consider the following types of information
- tables of chemical degradation rates
- published journals
- government publications
- technical reports
- acknowledged experts
Are reported rates of biodegradation sufficiently rapid foT a waste site or spill?
(Figure 3 2, Step 2)
• Consider the following types of information.
- time allowed to complete cleanup
- potential exposures over the cleanup period
• Consider the following sources of informatiorn
- published journals
- government publications
- technical reports
- acknowledged experts
- risk assessment of delayed treatment.
3.3
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Selection of Bsotreatment Options
NO
YES
__] 2
YES NO
I CAN SITE CONDITIONS BE
ENHANCED
J TO ACCELERATE
ARE PRELIMINARY FIELD
AND LABORATORY ASSESSMENTS
OF BIOTREATMENT
POSSIBLE?
YES
I ARE SUFFICIENT SITE DATA I
AVAILABLE TO ELIMINATE L °
THE NEED FOR FIELD AND [
LABORATORY ASSESSMENTI®
DOES SITE ASSESSMENT INDICATE
BIOTREATMENT IS POSSIBLE? J 5
YES
BIOTREATMENT
OF HAZARDOUS WASTE
IS A CLEANUP ALTERNATIVE
FIGURE 3.2 Flow Chart for Determining Whether or Not Chemicals Can be Treated
Biologically
Can site conditions be enhanced to accelerate biodegradation? (Figure 3 2,
Step 3)
• Consider the following types of information:
- site characteristics see Figure 3 1 (1)]
- biodegradation characteristics of waste [ see Figure 3 1(2) and Figure 3 2(2)1.
• Consider the following sources of information
- site environmental assessment
- environmental Impact statements for other activities
DO PUBLISHED REPORTS INDICATE
THE CHEMICAL HAS BEEN
SUCCESSFULLY BIOTREATED?
ARE REPORTED RATES OF
BIODEGRADATION SUFFICIENTLY
RAPID FOR CONSIDERED WASTE
SITE OR SPILL?
BIOLOGICAL
TREATMENT
NOT
ADVISED
OR
MORE
DATA
NECESSARY
34
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Selection of Biotreaiment Options
Are preliminary field and laboratory assessments of biotreatment possible?
(Figure 3 2, Step 4)
• Consider the following types of information
- treatment schedule (how soon must waste be cleaned up?)
- experimental data (for waste, for site)
- assessment techniques
• Consider the following sources of information.
- site environmental assessment
- environmental impact statements for other activities
Are sufficient site data available to eliminate the need for field and laboratory
assessment? (Figure 3 2, Step 5)
• Consider the following sources of information’
- data on analogous compounds
- previous studies at the site
- data from similar waste sites or spills.
Does site assessment indicate biotreatment is possible? (Figure 3 2 , Step 6)
• If assessment of the information discussed in Steps 1 through 5 results in a
positive response to Question 6, biotreatment should be included as a clean-
up alternative
3.3 WHAT QUESTIONS MUST BE ANSWERED TO EVALUATE
BIOTREATMENT OPTIONS?
Biotreatment options are outlined in Figure 2 1. Many specific questions need
to be considered when selecting a biotrea lment option Some of the questions
and the types of information necessary to answer these questions are listed
below ,
Natural Can the waste be secured or contained?
Biodegradation
(Figure Z1, (3A)(38)) • Consider the following types of information.
- mobility of waste in the environment
- volume of waste
- safety of contained waste
- duration of containment process
- regulations related to containment,
Can the waste or spill site be returned to previous use in a
reasonable time?
• Consider the following types of information’
- prior use of the site
- estimated biological and chemical half-life of the waste
- chemical concentrations that are safe (public health and
environ mental)
35
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Selection of Biotreatment Options
Natural Will natural biodegradation conditions remain constant over
Biodegradation time?
[ Figure 3.1, (3A)(3B)]
(contd) • Consider the following types of information.
- indigenous microorganisms
- chemical fate in secured site
- site characteristics
Land Treatment Can biotreatment be conducted at the selected site?
[ Figure 2.1, (5)]
• Consider the following types of information.
- population density near the site (land use, public access)
- toxicity of wastes to humans
- environmental conditions at the site (weather, wind direction)
- surface- and ground-water conditions on site
- wildlife access to site
- domestic animal access to site.
Is sufficient area available for offsite land treatment?
• Consider the following types of information
- site geology
- cost
- transportability of waste
- current land use
- adjacent land use
Are the soil, hydrology, topography, and climate suitable for
biotreatment?
• Consider the following types of information
- surface- and ground-water characteristics
- soil depth
- seasonal precipitation
- site climate
Can another site be found?
• Consider the following types of information
- transportation regulations
- distance to new site
- cost of transporting
- safety considerations
Are the waste volumes and transportation costs reasonable?
• Consider the following types of information
- waste characteristics
- available methods of transport
- transportation regulations
- total quantity of waste
36
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Selection of Biotreatment Options
Land Treatment Is composting a viable option at land farming site?
[ Figure 2.1, (5)J
(contd) • Consider the following types of information.
- ability of indigenous microorganisms to transform the waste
- energy demands
- characteristics of compost products
- disposal mechanism for composting products
Is land application a viable method?
• Consider the following types of information
- loading capacity of treatment site
- availability of treatment site
- natural site conditions (climate, geology, soil type)
Can composting and land application be combined to treat waste?
• Consider the following types of information
- site availability
- area of land required
- natural site conditions (soil type, climate, geology)
Will microbiological enhancement be required? [ Figure 2.1, (8)J
• Consider the following types of information
- natural conditions and indigenous microorganisms
- predicted degradation rates
- microorganism activity
Are nonbiological enhancement techniques necessary or
desirable? (Figure 2 1, (8)J
• Consider the following types of information.
- physical conditions
- chemical conditions
- available nutrients
Sioreactor Systems Can a bioreactor system be constructed on site?
[ Figure 2.1, (6)]
• Consider the following types of information
- site characteristics
- local populations
- regulatory restrictions
- cost
Can suspended solids be reduced to less than 1%?
• Consider the following types of information
- total dissolved solids
- availability of water for dilution
37
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Selection of Bsotreatment Options
Bioreactor Systems Can pre-treatment remove solids?
[ Figure 2.1,(6)]
(contd) • Consider the following types of informatioru
- available processes
- treatment requirements for solids.
Is sufficient land available to consider lagoon or pond systems?
• Consider the following types of information.
- local population density
- cost
— wildlife restrictions (waterfowl)
- domestic animal access to site
Will system disruption be unlikely to prevent use of an air acti-
vated sludge system?
• Consider the following types of information
- cost
- treatment requirements of sludge
- energy requirements
- shock loading potential of suspended solids
metal content of waste
biocrdal organic content of waste
Would the combination of trickling filter with activated sludge be
a viable process?
• Consider the following types of information
- potential system interruption (freezing, disruption of water
supply)
- microorganism sensitivity to shock loads
- organic load of waste
Are shock loadings unlikely to prevent use of a rotating disc system?
• Consider the following types of information
- potential system interruption (freezing, disruption of water
supply)
- system sensitivity to shock loading
- system modification requirements
- waste capacity.
Will microbiological enhancement be required?
• Consider the following types of information
- indigenous microorganisms
- degradation rates
38
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Selection of Biotreatment Options
Bioreactor Systems Are nonbiological enhancement techniques necessary or
[ Figure 2.1,(6)] desirable?
(contd)
• Consider the following types of informatiorr
- chemical and physical conditions
- toxicity of the wastes
- available nutrients
- adaptability of microorganisms to greater activity
Bloaccumulation Can the waste site or spill site be secured?
and Removal
[ Figure 2.1, ( )1 • Consider the following types of information
- area of site or spill
- public access
- wildlife access
- domestic animal access
- potential for offsite biotransport
Can bioaccumulating plants grow on the waste site?
• Consider the following types of information
- waste toxicity
- climate
- local flora
- compatibility of alien floia with site and waste characteristics.
Can bioaccumulated waste be taken elsewhere?
• Consider the following types of information
- local regulations
- available disposal sites
- characteristics of the accumulated waste
Enhancement Because of waste or site characteristics, it may be desirable to
[ Figure 2.1, (8)] enhance the conditions under which biodegradation occurs
Enhancement involves modifying specific physical, chemical, and
biological conditions to improve the effectiveness of biotreatment
Enhancement is applicable to all biotreatment options identified in
Figure 2 1
The following questions should be considered when selecting
enhancement options
Will natural conditions and indigenous microorganisms permit
timely removal of the hazardous waste through transformation,
degradation, and/or absorption?
• Consider the following types of information.
- waste degradation characteristics for site-specific conditions
- waste concentrations
- site recovery requirements
39
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Selection of B,otreatment Options
Enhancement Are physical and chemical conditions suitable for enhancement?
[ Figure 2.i,(8)] • Consider the following types of information
- soil
- climate
- nutrient availability
- site chemistry (aerobic, anaerobic, pH)
Will addition of nutrients and modification of physical and chemi-
cal characteristics enhance treatment?
• Consider the following types of information
- cost of modifications
- characteristics of the natural microbial community
Will addition of generic organisms enhance treatment?
• Consider the following types of information
- physical and chemical factors
- waste degradation rates with indigenous microorganisms
- waste concentrations
Will enriched microorganisms enhance treatment?
• Consider the following types of information
- availability of an enriched culture
- concentrations
Will adapted microorganisms enhance treatment?
• Consider the following types of information
- availability of an adapted culture
- waste concentrations
Will a combination of adapted and enriched microorganisms
enhance treatment?
• Consider the following types of information ’
- availability of culture
- waste concentrations
3 10
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4.0 SITE-SPECIFIC CONSIDERATIONS AT HAZARDOUS-WASTE
SITES OR SPILLS
Technical background information must be assessed before and after treat-
ment of a hazardous-waste site or spill Pre-treatment assessment provides data
necessary to begin cleanup activities and will often indicate whether a combi-
nation of physical, chemical, and biological methods is required for effective
waste removal If biotreatment is selected, decisions must be made about
whether to conduct cleanup of hazardous materials onsite or offsite, which
biotreatment to use, and whether or no enhancement techniques are
required
Post-treatment assessment helps ensure that no waste components still remain
a hazard to human health or the environment Data should be collected to
detect the presence of any residual hazardous materials or hazardous by-
products of the biotreatment process.
The purpose of conducting pre- and post-treatment activities is to ensure
human health and safety and environmental protection, and to provide data
for treatment decisions Because some of the data requirements and methods
used in pre- and post-treatment activities differ, this chapter is organized into
two subsections
• What information should be collected before treatment?
• What information should be collected after treatment?
4.1 WHAT INFORMATION SHOULD BE COLLECTED BEFORE
TREATMENT?
A hazardous-waste disposal site should be assessed before treatment methods
are applied to ensure that the most appropriate cleanup method is used Pre-
treatment assessment involves the following steps
1 Identify the chemicals and concentrations present
2 Determine the danger of those chemicals to human health and safety.
3 Qualify the environmental characteristics of the site.
4. Identify indigenous microorganisms.
5. Assess whether or not the waste can be ireated biologically
Identify Chemicals The first step in selecting remedial treatment is to determine the
and Concentrations types, concentrations, and distribution of waste materials present
Present It may be necessary to identify chemical mixtures and reaction
products Hazardous materials an be identified by the following
means
• records
• containers and packaging
41
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Site-Specific Considerations
Identify Chemicals • observable physical and chemical characteristics of the waste
and Concentrations • chemical analysis
Present (contd)
Records—Records can provide rapid and positive identification of
hazardous wastes, their location, amounts, and distribution at the
site. Records also indicate possible hazards that may arise during
waste recovery and handling (fire, corrosion, explosion) Records
usually are not available for uncontrolled or abandoned disposal
sites Older records may have been lost or destroyed Records may
be incomplete and may contain errors Waste manifests, which
describe each shipment of waste received) may be available at
controlled disposal sites
Containers and Packaging—Containers and packaging will usually
help identify hazardous-waste materials, however, labels and iden-
tification marks are not fool-proof means of identification because
the original contents may have been replaced with other materials
Identification marks are often lost with time Wastes at disposal
sites may be unconfined, leached into the soil, buried, or in
ground water Excavation may be required to expose buried con-
tainers or particulate wastes
Observable Characteristics—Observable physical and chemical
characteristics (odor, color, density, reaction) may be used to ten-
tatively identify unknown materials The approach is usually
limited to identifying broad classes of compounds and must not be
used to identify specific hazardous wastes Observable characteris-
tics are usually not suitable at abandoned or buried sites, or when
the wastes have entered soil or water
The Oil and Hazardous Materials-Technical Assistance Data Sys-
tem, OHM-TADS, is maintained by the EPA and can be used to
identify chemical substances based on their observable characteris-
tics Physical properties of the unknown material (physical state,
odor, color, turbidity, miscibility, reactions) are input to a compu-
ter system, which can perform a search for possible identities The
OHM-TADS system contains data on approximately 850 chemicals
and hazardous substances
Other sources of information can be used to identify chemicals on
the basis of their physical and chemical properties These include
The EPA Field Detection and Damage Assessment Manual for Oil
and Hazardous Materials Spills (EPA 1972) lists over 300 materials
and identifies them by odor, color, reaction, and other properties
Physical and Chemical Properties of Hazardous-Waste Constituents
(Dawson, English and Petty 1980) provides data on 250 chemicals
commonly found in hazardous-waste streams
U S Coast Guard Chemical Hazards Response Information System
(CHR iS) Manuals CG-446-1 and CG-446-2 (USCG 1974a, b) provide
observable characteristics of approximately 900 hazardous
chemicals.
42
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Site-Specific Considerations
Identify Chemicals Chemical Analysis—Chemical analysis of samples may be the only
and Concentrations way to identify hazardous-waste materials and determine the
Present (contd) extent of site contamination Collection of samples for chemical
analyses will depend largely on physical conditions at each loca-
tion In some cases, soil and water samples can be obtained
directly In other cases, overburden removal, excavation, well drill-
ing, and similar efforts will be necessary to obtain representative
samples Drainage or leachable liquids from sites where a variety of
wastes are buried can be collected and analyzed. Plant and animal
tissues can be analyzed to identify chemical wastes that have
bioaccumulated Backgrou rid levels of waste materials can be
determined by samples collected and analyzed from uncontami-
nated surrounding areas Analyses of samples taken before and
after site treatment will help establish the effectiveness of the
treatment effort
Analytical methods can be used to determine the types and con-
centrations of hazardous wastes present Advanced equipment and
trained manpower are usually required for analyzing hazardous
wastes Standard methods ol analysis for water and wastewater
(APHA 1951, EPA 1976) are usually inadequate for specific identifi-
cation of waste materials in the environment Hazardous wastes are
typically analyzed by six instrumental techniques discussed below
Gas chromatography (GC) and high-performance liquid chroma-
tography (HPIC) are used to analyze quantitatively the specific
organic materials in wastes For the most part, they are not suitable
for qualitative analysis of unknown materials in a sample unless
known standards are used for comparison Gas chromatography
and I-IPLC can be relatively expensive and time-consuming, but
they can detect the presence of contaminants in environmental
samples at levels in the ppb (parts per billion) range
Gas chromatography/mass spectrometry has been used primarily
for qualitative analysis Improvements in equipment now permit,
in some instances, use of GC/MS for quantitative analysis of
pollutants in environmental samples The GC/MS technique has
the ability to simultaneously identify and quantify organic com-
pounds present in environmental samples at the ppb level This
feature is particularly suitable when confirmation of contaminants
in a sample is required Also, quantitative GC/MS analysis can
eliminate error in quantification from the presence of an interfer-
ing contaminant The technique is expensive and time consuming,
therefore, its use should be based on the data needs required from
the individual sample or sample batches
Atomic absorption spectrometiy (AAS) and inductively coupled,
argon-plasma spectrophotmetry (ICAP) are used to quantify heavy
metals in environmental samples in the ppm or ppb concentration
range The AAS technique can quantify only a single compound at
any given time in a sample, whereas ICAP can obtain quantitative
43
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Site-Spec,fsc Considerations
Identify Chemicals information on several dozen metals from a single analysis of the
and Concentrations sample
Present (contd)
Chemical analysis with these analytical techniques can be used to
quantify or confirm the identity of compounds in hazardous-waste
materials Chemical analyses require time, funds, and a laboratory
Tables summarizing EPA-approved analytical procedures for 16
classes of organic chemicals and approved analytical procedures
for 80 hazardous materials are given in EPA (1982)
Determine Danger Workers at a hazardous-waste disposal site must be aware of the
of Chemicals to dangers from chemicals present and proceed accordingly Any
Worker Health and unidentified substance at a disposal site must be assumed to be
Safety hazardous unless firm evidence exists to the contrary
Two important characteristics of hazardous wastes are reactivity
and toxicity. The reactivity of chemicals at hazardous-waste sites
should be determined prior to treatment Numerous chemicals
and complex compounds may have been deposited at a site where
they subsequently may have mixed and reacted with each other
and with natural materials in the soil to form compounds with
properties different from the original components
Toxicity is a term used to define a substance’s capacity to produce
dysfunction, injury, malformation, or death in a living organism
Toxic responses in humans are generally a result of exposure by
inhalation, ingestion, or skin contact Exposures may be direct or
indirect Primary concern is with organic compounds or metals
that are moderately to severely toxic at normal use concentrations,
or that are carcinogenic (or mutagenic and teratogenic) at low
concentrations Identification of these types of compounds at
hazardous-waste sites is Important because many of them bio-
accumu late and resist biotransformation
The reactivity and toxicity of chemicals are described in several
data bases and handbooks. EPA’s data base provides rapid access to
information on the reactivity of over 850 chemicals; this includes
identification of binary reactions, corrosiveness, recommended
containers, flammability, explosiveness, and other dangerous
properties. The U S Coast Guard’s CHRIS Manual CG-446-2
(Arthur 0 Little, Inc 1972) contains information on approximately
900 chemicals, including reactivity with water and common mate-
rials (including containers), stability during transport, neutralizing
agents, polymerization reactions and inhibitors, flammability, and
flash points
Hatayama et al (1980) have prepared a manual on hazardous waste
compatibility that includes a reference chart and a classification of
common hazardous materials into 41 groups All binary reactions
are identified as heat generation, toxic gas generation, flammable
and nonflammable gas generation, fire and innocuous gas genera-
44
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Site-Specific Considerations
Determine Danger tron, explosion, violent polymerization, solubility of toxic sub-
of Chemicals to stances, and unknown but potentially hazardous reactions The
Worker Health and National Fire Protection Association has published the Manual of
Safety (contd} Hazardous Chemical Reactions (NFPA 1975), which identifies over
3500 hazardous reactions by reactant Sax (1979) identifies chemical
incompatibility of over 1000 different materials California
Hazardous-Waste Regulations (Calif State Depi Health 1977) list
about 800 chemicals that are considered to be hazardous or
extremely hazardous, and gives the dangerous properties of each
Information on other dangerous chemical properties of materials
that may be currently deposited at hazardous-waste sites is given
by the sources mentioned above (tJSCG 1974a,b, Hatayama et al
1980, NFPA 1975, Sax 1979) Additional publications with valuable
information include Prunkett (1966), Sittig (1979a, 1980), Verscheuren
(1977), and Arthur D Little, Inc (1972) Requests for quick informa-
tion, in event of an emergency, can be phoned to CHEMTREC
(Chemical Transportation Emergency Center-CMA, 800-424-9300).
This number is also listed in the chapter references
Lists of priority pollutants and hazardous materials published by
EPA and the U S Coast Guard can be consulted to determine if a
chemical or metal has been defined as hazardous These lists are
given in The Toxic Substances Control Act, P L. 96-510, Clean Air
Act, P 1. 91-604 as amended, and Federal Water Pollution Control
Act, P L 92-500 Lewis and Tatken (1980) provide a Registry of Toxic
Effects of Chemical Substances, which is updated quarterly on
microfiche and as a on-line data base A list of hazardous chemical
compounds prepared by the EPA is reproduced in Table 4 1 A list
of known or suspect carcinogenic materials issued by The National
Toxicology Program is reproduced in Table 4 2
A group of 129 chemical compounds and elements, referred to as
“priority pollutants,” were listed in section 307(a)(1) of the 1977
Clean Waler Act (33 U.S C 466 et seq., Committee Print H R 3199)
The priority pollutants were reviewed by Dryden et aI (1978), and
their environmental late in water was described by Callahan et al
(1979) The relative importance of each priority pollutant in envi-
ronrrienial compartments (water, sediment, biota) was established
by Chapman, Rom berg and Vigers (1982), and is summarized in
Table 4 3 The categories were derived by flow chart from three
main characteristics persistence, accumulation, and volatility
Chapman, Romberg and Vigers (1962) rated the pollutants in five
categories based on the flow chart shown in Figure 4.1 Category 1
pollutants (wastes) are persistent, accumulative, and nonvolatile,
and thus rate the highest environmental concern Category 2
pollutants are accumulative, persistent, and volatile Category 3
pollutants are persistent, non-accumulative, and non-volatile
Category 4 pollutants are persistent, non-accumulative, and vola-
tile Category 5 pollutants are non-persistent and rate the lowest
environmental concern
45
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Sate-Specific Considerations
TABLE 4.1 EPA List of Hazardous Chemical Compounds(a)
acenaphthene N-nitrosodiphenylamine
acrolein N-nitrosodi-n-propylamine
acrylonitrile pentachlorophenol
benzene bis(2-ethy lhexyl)phthalate
benzidine butyl benzyl phthalate
chlorobenzene di-n.butyl phthalate
1,2,4-trichlorobenzene di-n-octyl phthalate
hexachlorobenzene diethyl phthalate
1,2 -dich loroethane dimethyl phihalate
1,1, 1-trich loroethane benzo(a)anthracene
1,1 -dich loroethane (1,2-benzanthracene)
lj,2-trichloroethane benzo(a)pyrene (3,4-benzopyrene)
ch loroethane 3,4-benzofluoranthene
bis(chloromethyl) ether benzo(k)fluoranthane
bis(2-chloroethyl) ether (11,12-benzofluoranthene)
2-ch loroethyl vinyl ether (mixed) chrysene
2-ch lo ron a phi hale n e acenap ht hylene
2,4,6-trich iorophenol anthracene
p-chloro-m-cresol benzo(g,h,i)perylene
2 -chlorophenol (1,12-benzoperylene)
1,3-dichlorobenzene fluorene
3,3’-dichlorobenzidine phenanthrene
1,1 - di ch loroet hylene di benzo (a, h )an t h racene
1 ,2 -trans -dich loroethylene (1 ,2,5,6-dibenzanthracene)
2,4-dich lorophenol I ndeno(1,2,3-c,d)pyrene
1,2-dich loropropane )2,3 -o -phenylenepyrene)
1,2 -dich loropropylene pyrene
(1,3-dich loropropene) toluene
2,4 -dimethy lphenol vinyl chloride (chloroethylene)
2,4 -dinitrotoluene a ldrin
2 ,6 -dinitrotoluene ch lordane (technical mixture and
1,2-diphenyihydraz ine metabolites)
ethylbenzene a -endosulfan-Alpha
fluoranthene b-endosulfan-Beta
4-ch lorophenyl phenyl ether endosulfan sulfate
4-bromophenyl phenyl ether endrin aldehyde
bis(2-chloroisopropyl) ether heptach lor
bis(2-chloroethoxy) methane heptachlor epoxide
methyl chloride (ch loromethane) a-BHC-Alpha
bromoform (tribromomethane) b-BHC-Beta
dich lorobromome thane g -BHC-Delta
trichlorofluoromethane PCB-1 242 (Aroch lor 1242)
dich lorodif luoromethane PC8-1254 (Aroch lor 1254)
ch lorodibromomethane PCB-1221 (Aroch lor 1221)
hexach lorobutadiene PCB-1232 (Aroch lor 1232)
hexach lorocyclopentad iene PCB-1 248 (Aroch lor 1248)
isophorone PCB-1 260 (Arochlor 1260}
2-nitropheno l PCB-1016 (Aroch lor 1016)
4-nitrophenol 2,3,7,8-tetrachlorodibenzo-p-
4,6-dinitro-o-cresol dioxin (TCDD)
N-nitrosodimethylamine
(a) Source EPA, Federal Register 43, No 243, pp 58946-59028 (Dec 18, 1978)
Note: This list is considerably shorter than many lists of hazardous materials and should not be
considered to be inclusive
46
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Site-Specific Considerations
TABLE 4.2 Known or Suspect Carcinogenic Materials(a)
2-acetylaminofluorene indeno-(1,2,3 -cd )pyrene
acrylonitrile iron dextran
aflatoxins isopropyl.alcohol manufacturing
4-aminobiphenyl (strong acid process)
amitrole kepone
aramite lead acetate and lead phosphate
arsenic and related compounds lindane
asbestos me lphalan
auramine and its manufacture mirex
benz(a)anthracene mustard gas
benzene 2-naphthylamine
benzidine nickel, certain nickel compounds
benzo(b)Iluoranthene and and refining
benzo(j)fluoranthene N-nitrosodi-N-butyl -amine
benzo(a)pyrene N-nitrosodiet hanolamine
beryllium and certain beryllium compounds N-nitrosodiethylamine
N .N-bis(2 -ch loroethyl) 2naphtbyJamine N-nitrosodimethylamine
bis(chloromethyl) ether and its N-nitrosodi -N -propy lamine
technical grade N-nitroso-N-ethylurea
cadmium and certain cadmium compounds N-nitroso-N-methylurea
carbon tetrach loride N’-nitroso-methylvinylamine
ch lorambuc il N-nitrosomorpholine
chloroform N-nitrosonornicotine
chromium and certain related substances N-nitrosopiperidine
coke oven emissions N-nitrosopyrrolidine
p-cresidine N-nitrosoarcosine
cycasin oxymetholone
cyclophosphamide phenacetin
2,4-diaminotoluene phenazopyridine hydrochloride
dibenz(a,h)acridine phenytoin
dibenz(a,j)acridine PCBs
dibenz(a,h)-anthracene procarbazine and its hydrochloride
?H-dizenzo(c,g)carbazole b-propiolactone
dibenzo(a,h)pyrene reserpine
dibenzo(a.i)pyrene, saccharin
l 2-dibromo-3-chloropropane safrole
1,2-dibromoethane soots, tars and mineral oils
dichlorobenzidine streptozotocin
1,2-dich ioroethane TCDD
DES, 4-dimethyl aminoazobenzene thorium dioxide
dimethylcarbamoyl chloride o-toludine hydrochloride
dimethyl sulfate toxaphene
1 ,4-dioxane tris(I-aziridinyl)phosphine sulfide
formaldehyde tris(2,3-di bromopropyl) phosphate
hematite underground mining viny? chloride
hydrazobenzene
(a) Source National Toxicology Program, 2nd Annual Report on Carcinogens, April 30, 1982
47
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Site-Specific Considerations
TABLE 4,3 Compounds, Category Rank (see Figure 4 1), and Environmental
Compartments of EPA Priority Pollutants (Chapman, Romberg and Vigers
1982)
Category Environmental Compartment
Compound Rank Water Sediment Biota
Metals and inorganics
Antimony 3 X
Arsenic 1 X X
Asbestos 3 X
Beryllium 1 X X
Cadmium 1 X X
Chromium 1 X X
Copper 1 X X
Cyanides 5 X
Lead 1 x x
Mercury 1 X X
Nickel 1 X X
Selenium 1 X X
Silver 1 X X
Thallium 1 X X
Zinc I X X
Pesticides
Acrolein 2 X X
A ldrin 2 X X
Ch lordane 2 X X
DDD 1 X X
DDE 1 X X
DDT 1 X X
Dieldrin 1 X X
Endosulfan and endosulfan sulfate 3 X
Endrin and endrin aldehyde 1 X X X
Heptach lor 1 X X
Heptach lor epoxide 1 X X X
Hexachlorocyclohexane (a, /3. ô isomers) 3 X X
y-Hexachlorocyclohexane (Itndane) 3 X X
Isophorone 3 X
TCDD 1 X X
Toxaphene 1 X X
PCBs and related compounds
Polychlorinated biphenyls
(6 PCB aroch lors) 1 X X
2 -Chloronaphthalene 1 X X
Halogenated aliphatics
Ch loromethane (methyl chloride) 4 X
Dich loromethane (methylene chloride)4 X
Trich loromethane (chloroform) 4 X
Ch loroethane (ethyl chloride) 4 X
Tetrach loromethane (carbon tetrach loride) 4 X
1,1-Dich loroethane (ethylidine chloride 4 X
1,2-Dich loroethane (ethylene dich loride) 4 X
48
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Site-Specific Considerations
TABLE 4.3 (Conid)
Category Environmental Compartment
Compound Rank - Water Sediment Buota
Halogenated aliphatics (contd.)
1,1,1-Trichioroethane methyl chloroform) 4 X
1 ,1,2 -Tr uch loroethane 4 X
1,1,2 ,2 -Tetrach loroethane 4 X
Hexach loroethane 4 X
Chloroethene (vinyl chloride) 4 X
13-Duchloroethene (vunylidine chloride) 4 X
1,2 -Trans-dich loroethene 4 X
Trich loroethene 4 X
Tetrachloroethene (perch loroethylene) 4 X
1,2-Dichloropropane 4 X
1,3-Duchloropropene 4 X
Hexach lorobutadiene 1 X X
Hexach lorocyclopentadiene 1 X X
Bromomethane (methyl bromide) 4 X
Bromoduch loromethane 3 X X
Dubromoch loromethane 3 X
Tribromomethane (bromoform) 3 X X
aiDichlorodufluoromethane 4 X X
‘ 1 t Truchlorofluoromethane 4 X X
Ethers
aiBis(chloromethyl) ether 3 X
Bis(2-chloroethyl) ether 3 X
Bis(2-chloroisopropyl) ether 3 X
2-Chloroethy ) vinyl ether 4 X X
4-Chlorophenyl phenyl ether 1 X x
4-Bromophenyl phenyl ether 1 X X
Bix(2-chloroethoxy) methane 3 X
Monocyclic aromatics
Benzene 4 X
Chlorobenzene 2 X X
1,2-Dichlorobenzene (o-duchlorobenzene) 2 X X
1,3-Oichlorobenzene (m-dichlorobenzene) 2 X X
1,4-Dichlorobenzene (p-dichlorobenzene) 2 X
1,2,4-Trichlorobenzene 2 X X
Hexachlorobenzene 1 X X
Ethylbenzene 4 X
Nutrobenzene 3 X
Toluene 4 X
2,4-Dunitrotoluene 3 X
2,6-Dunitrotoluene 3 X
Phenols and cresols
Phenol 3 X
2-Ch lorophenol 3 X
2,4-Duch lorophenol 5 X
49
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Selection of Biotreatment Options
TABLE 4.3 (Contd)
Category Environmental Compartment
Compound Rank Water Sediment Biota
2,4 ,6-Trichloropheno l 3 X
Pentach lorophenol 1 x x
2-Nitrophenol 3 x
4-Nitrophenol 3 X
2.4-D initrophenol 3 X
2 ,4 -D imethy lphenol 1 X X
p.Chloro-rn-cresol 3 X
4,6-Dinitro-p-cresOI 3 X
Phthalate esters
Dumethyl phtha late 1 X X
Duethyl phihalate 1 X X
Di-n-butyl phthalate 1 X X
Di-n-octyl phthatate 1 X X
Bis(2-ethylhexyl) phihalate 1 X X
Butyt benzyl phihalate 1 X X
Polycyclic aromatics
Acenaphihene 1 X X
Acenaphihylene 1 X X
Anthracene 1 X X
Ben o(a)anthracene 1 X X
Benzo(b)ftuoranthene 1 X X
Benzo(k)fluoranihene 1 X X
Benzo(ghi)perylene 1 x x
Benzo(a)pyrene 1 X X
Chrysene 1 X X
Dibenzo(a,h)anthracene 1 X X
Fluoranthene 1 X X
Fluorene 1 X X
lndeno (1 ,2,3-cd)pyrene 1 X X
Naphtha lene 1 X X
Phenanthrene 1 X X
Pyrene 1 X X
Nitrosamines and miscellaneous
compounds
Dimethyt nitrosamine 3 X
Diphenyl nitrosamine 1 X X
Di-n-propyt nutrosamine 1 X X X
Benzidine 3 X
3,3’-Dichlorobenzidune 1 X X
1 ,2-Dipheny lhydrazine (hydrazobenzene) 1 X X
Acrylonitr ile 4 X X
(a) These compounds have been removed from the EPA priority pollutant list
4 10
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Selection of Biotrearment Options
NON-PERSISTENT VOLATILE
PRIORITY ] ACCUMULATIVE
POLLUTANT NON-VOLATILE
PERSISTENT VOLATILE
ACCUMULATIVE
NON-VOLATILE
FIGURE 4.1 A Flow Chart for Categorizing Priority Pollutants (modified from Chapman,
Romberg and Vigers 1982)
Identify the Physical and chemical features of a hazardous-waste disposal site
Environmental must be determined to evaluate the feasibilIty of biotreatment
Characteristics of methods Waste characteristics and worker health and safety have
the Site already been discussed Other factors to consider in evaluating the
general site environment include
• topography and size
• hydrology
• climate
• soil composition
• human activities
• biota
Topography and Size—The topography and size of a site, as well as
the types and quantities of toxic wastes present, will influence
decisions about the feasibility of various treatment methods Clean-
up activities can be conducted more efficiently on a relatively flat
site than on a sloping site Steep sites may lead to equipment oper-
ational problems, soil erosion, and excessive surface runoff If the
terrain is hilly or undulating, low areas may tend to collect surface
runoff and provide suitable locations for waste lagoons Sites
crossed by ravines and gullies are subject to substantial surface
runoff, if wastes are present in such locations, surface runoff
should be confined and controlled A certain amount of land
around a waste disposal site is desirable for a buffer strip, access
roads, soil stockpiles, construction of special biotreatment facilities
such as bioreactor systems, and related activities
Hydrology—Generally, any form of treatment (biological, chemical
or physical) must avoid contamination of surface and ground
water If a bioreactor system is used, it must be operated as an iso-
lated facility and designed and constructed to prevent seepage,
leachates or overflow (Fields and Lindsey 1975) Surface-water
flows must be controlled and confined, and other measures must
be taken to avoid contamination of navigable streams, lakes, reser-
voirs, and domestic or public water supplies
411
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Site-Specific Considerations
Identify the Depth to water table will influence contamination of ground water
Environmental beneath waste disposal sites Deep water tables beneath an
Characteristics of impermeable overburden are not apt to be contaminated If toxic
the Site (contd) wastes enter the water table, special isolation and removal work
may be needed The direction and rate of ground-water flow will
influence offsite spread of contaminated water Special under-
ground barriers and water-collection systems may be required
Climate—Seasonal temperature and rainfall (or total precipitation)
are the primary climatic factors to consider Air temperature and
moisture will influence the rate of microbial activity in bioreactor
systems exposed to the atmosphere Temperature and moisture
will also influence biotransformation rates in the soil, and the
uptake and bioaccumulation of hazardous materials by photosyn-
thetic organisms (bacteria, algae, and higher plants) Within limits
for most microorganisms in wastewater treatment facilities, the
higher the temperature and the greater the moisture, the greater
the rate of biotransformation activity Low temperature and dry
climates limit biological treatment Cold climates are unfavorable
for biotransformation of hazardous wastes, whereas warm or tern-
perate climates are favorable. Evaporation rates will affect the
movement of low-solubility contaminants to the atmosphere
(Mackay and Leinonen 1975) Wind forces and direction will inf lL’-
ence the spread of noise, dust, and odors during onsite cleanup
activities These factors will also affect possible offsite spread of
contaminated materials and exposure of onsite workers
Soil Composition—Physical and chemical characteristics of the soil
will influence biotransformation processes in several ways Soil
nutrients (e g, organic carbon, nitrogen, phosphorous, potassium,
iron and magnesium) are important to the activity of many photo-
synthetic organisms such as phototrophic bacteria, algae and larger
plants, and to the activity of microorganisms using organic com-
pounds as energy sources
Low pH usually inhibits the growth of most microorganisms in soil
and water, increases the solubility of metals, and affects the ion
exchange and absorption properties of the colloidal fraction of
soils (Fields and Lindsey 1975, Epstein and Chaney 1978) Clays are
more effective absorbers of heavy metals at higher pH levels The
cation exchange capacity of soils depends to a large extent on clay
content, but it increases in direct proportion to pH-dependent
charged particles such as hydrous metal oxides and organic matter
(Sittig 1979b)
Organic carbon may be a function of normal organic content
of the soil, or the chemicals and other materials present at a waste
disposal site Transformation activities of many microorganisms are
fueled directly or indirectly by organic carbon, other conditions
being favorable If concentrations of an organic compound are
low, the microorganisms may not obtain enough energy to be self-
412
-------
Site-Specific Considerations
Identily the sustaining, some microorganisms require less chemical energy
Environmental than others, and the phototrophs require sunlight (Chapter 6)
Characteristics of
the Site t contd’ Soil Permeability—Soil permeability depends on soil texture and
structure (Sittig 1979b). Fine-grained, poorly-structured soils have
the lowest permeabilities If a soil is already saturated with water,
its permeability will be low Impermeable soils will have more sur-
face runoff from precipitanon than other soils Hazardous waste is
most likely to leach from permeable soils Permeability will also
influence the depth of underground penetration of liquid wastes,
and the action of aerobic and anaerobic microbial processes
Human Activites—Population density near a waste disposal site will
influence onsite activities. Generally, disturbance of people must
be minimized Noise, odors, dust, and emission of toxic or irri-
tating gases must be kept minimal If population levels are high in
surrounding areas, biotreatrnent of hazardous wastes at another
location may be desirable Access to a disposal or cleanup site by
the general public should be discouraged or prevented Special
problems may be encountered when dealing with sites near hospi-
tals, schools, jails, retirement homes, parks, fishing areas, libraries,
colleges, training centers, and other facilities in heavy public use
Abandoned hazardous-waste sites may now be used for other pur-
poses Current use of the site may conflict with cleanup activities
Such a conflict normally can be resolved because protection of
people and the environment from toxic waste has priority over
most other land uses If cleanup is effective, the land can be used
for other purposes Biotreatment processes, because they are rela-
tively slow, may require a longer commitment of the site for clean-
up activity than is required for chemical and physical treatments
In some forms of biotreatment, particularly land treatment, there
are no residues that require further disposal In comparison, most
chemical and physical treatment processes leave some residue for
disposal, which requires commitment of land for this purpose
Biota—Biotransformation activities at hazardous-waste disposal
sites could lead to contamination of nearby vegetation and local
and migratory animals Risk of contamination is increased because
biotreatment usually takes longer than chemical and physical
treatments, and biotreatment is often conducted under open situa-
tions that require exposure of contaminated soil and liquids to air
and precipitation
Waste bioreactor systems that are exposed to the elements (acti-
vated sludge units or lagoons and ponds) should be surrounded by
a fence to block access of large animals Access of birds and small
animals also should be prevented when possible Burrowing
animals, including insects and small mammals, may become con-
taminated through contact with waste materials in soils at land
treatment sites Many of these small animals may travel offsite
4 13
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Sue-Specific Considerations
Identify the Where soil cultivation is practiced, grasses and other vegetation
Environmental may accumulate hazardous materials Thus, grazing by domestic
Characteristics of animals should be prohibited and use by game animals should be
the Site (contd) minimized Harvesting of plants and animals from a hazardous-
waste treatment site should be prohibited until post-treatment
monitoring provides assurance of safety
Sampling and chemical analyses of plants and animals at a
hazardous-waste site before and after treatment will demonstrate
the effectiveness of the method applied Some chemicals and
metals bioaccumulate and are persistent and may exist for years in
higher organisms
Identify Indigenous Indigenous microorganisms are those that occur naturally at a par-
Microorganisms ticular site The organisms may be capable of biotransforming the
waste materials by direct metabolic activity, indirect co-metabolic
activity, photosynthetic activity, or by some other method if they
are able to grow
Indigenous microorganisms should usually be evaluated as a com-
plex microbial community rather than as individual species This is
particularly true if the hazardous waste is represented by complex
mixtures rather than by physically separate toxic materials A single
species of organism, however, can sometimes be isolated directly
from one type of material, the organism can then be adapted to
use and transform the specific material
Indigenous microorganisms have several characteristics that enable
them to change toxic materials to innocuous materials They toler-
ate the toxic materials on which they occur, many have the ability
to attack, use, and degrade the toxic materials on which they live,
they are adapted to the physical and chemical conditions of the
site where they are found, they can be used to inoculate bioreac-
tor systems without extensive culture; and they can be cultured as
adapted and enriched populations for inoculation of composting
or bio reactor systems
Assess Whether Biotransformation data from the literature can be examined to
Waste Can Be answer the following questions about pre-treatment site
Treated Biologically assessment
• Is a chemical compound known to be biotransformable and, if
so, under what conditions?
• What are the relative rates and processes of biotransformation?
• What environmental features inhibit biotransformation?
• What concentrations of a waste inhibit biotransformation?
• What microorganisms or groups of organisms are most useful
for biotransforming a particular compound?
4 14
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Site-Specific Considerations
Assess Whether An extensive amount of literature is available that describes the
Waste Can Be biotreatment of chemical compounds Considerable information
Treated Biologically on such studies is avarlable in this guide and its references (for
(contd) example, see Table 6 2)
Progress has recently been made in defining biotransformation
pathways of a variety of synthetic compounds, pesticides, and
hazardous-waste materials in laboratory cultures of single micro-
organism species Studies of single species transformations in artifi-
cial media are relatively simple, because the test compound is
frequently present in proper concentrations, the microorganisms
consist of one species, and none of the complexing materials and
surface characteristic of soils, sediment, sewage, and surface water
are present In contrast, studies of environmental fate are more
difficult
Laboratory studies with pure cultures and test solutions provide a
wealth of background information on the biotransformation path-
ways of compounds that are of environmental concern, such as
pesticides and aliphatic and aromatic compounds. Laboratory
studies can be considered an exploratory phase of biotransforma-
tion processes They do not necessarily portray what happens in
outdoor ecological systems represented at hazardous-waste dis-
posal or accidental-release sites
Available biotreatment options usually employ mixed populations
of indigenous microorganisms rather than single-species popula-
tions Assessment of biotreatability should be based on analyses
that monitor or detect any disappearance of the chemical under
consideration Assessment should identify compounds formed by
the biotransformation process
4.2 WHAT INFORMATION SHOULD BE COLLECTED AFTER
TREATMENT?
Post-treatment monitoring, in many cases, will merge with treatment monitor-
ing Post-treatment monitoring, in a strict sense, refers to locations where bio-
treatment is applied to return a site to its former, natural condition However,
land farming practices and operation of bioreactor systems may involve long-
term operations during which more wastes are added as activities are con-
tinued Thus, monitoring is also necessary during biotreatment to ensure that
the effort is succeeding and that environmental components are not being
damaged
Data from post-treatment monitoring may also be compared with data from
pre-treatment monitoring to determine the effectiveness of treatment effort
Such comparisons aid in determining the cost-effectiveness of treatment
activities
4 15
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Site-Specific Considerations
Post-treatment site assessment involves the following activities
1. Gather technical background information.
2 Identify environmental components for monitoring
3 Develop a sampling program
4 Determine when monitoring should be terminated
Gather Technical Much of the technical background information obtained in pre-
Background treatment site assessment or during biotreatment activities will be
Information of value in post-treatment monitoring Post-treatment monitoring
will concern not only chemical compounds and waste mixtures
identified prior to biotreatment, but possible intermediate pro-
ducts and by-products resulting from biotreatment Since bio-
treatment involves the use of microorganisms, the following
questions should be examined
• Were the expected degradation reactions carried to completion
and the hazardous materials rendered harmless?
• Has sufficient time passed for the hazardous materials to be
biodegraded?
• Were undesirable intermediate products or by-products
produced?
• Do any environmental components (soil, water, sediment, air,
plants, animals) remain contaminated?
• What residual levels of the remaining hazardous materials are
acceptable to people and the environment?
• Did significant offsite movement of hazardous materials take
place?
Post-treatment monitoring consists largely of a planned sampling
and chemical characterization program Sampling designs are
largely site-specific For example, sampling the effluent from a bio-
reactor system is relatively simple to do, whereas sampling various
environmental components (air, soil, water, plants, wildlife) at an
abandoned waste disposal site is complex. (See Section 4 1 for a
discussion of chemical analysis)
Sampling schemes may consist only of periodic samples from a
single source for chemical analyses (e g , to monitor effluent from
a bioreactor system) Complex sampling designs to monitor several
environmental components over a period of time (e g , at waste
disposal sites) require the services of a statistician, preferably one
with a background in ecological monitoring Because analytical
procedures for low chemical concentrations have special require-
ments and may be costly, a chemist should also help establish a
practical sampling program
4 16
-------
Sire-Specific Considerations
Identify In most cases of post-treatment monitoring, it is not necessary to
Environmental sample nearby human populations to determine the effectiveness
Components (or of biological cleanup Human health problems will usually be
Monitoring examined before and during cleanup activities, if the hazardous
waste situation warrants Concern for human health will always be
paramount (EPA 1982)
The main environmental components to be considered for post-
treatment monitoring are
• air
• soil
• water
• sediment
• plants
• domestic animals
• wildlife
Each environmental component can be related to one or more
areas of concern Post-treatment monitoring to evaluate the effec-
tiveness of biotreatment and other cleanup activities must also
examine potential pathways that link the environmental compo-
nent to areas of concern A pathway becomes important only if the
cleanup effort was ineffective or partially effective, and an envi-
ronmental component remains contaminated with hazardous
materials
Monitoring is usually a complex and expensive task Only those
components of the waste site and surrounding environs that are
most important to the assessment should be monitored Figure 4 2
is a decision matrix by which the relative importance of environ-
mental components and areas of concern can be evaluated For
instance, if the contaminants appear in the soil only, the other
environmenta’ components need not be included in monitoring If
the land in question will revert to residential use after cleanup,
then the areas of concern would include 1) human densities and
distribution, 2) public resources and institutions, and 3) uses of
land Monitoring should be designed to assure that contaminant
levels be low enough to protect the areas of concern Evaluation
must be site-specific because the characteristics considered vary
widely from site to site, and sampling programs must be designed
accordingly
Eight major areas of concern given in Figure 4 2 must be
considered
(1) Human density and distribution
Are population densities large or small?
Are people scattered or grouped?
Do many people live or work near the treatment site?
Are population centers downwind?
4 17
-------
Size-Specific Considerations
A iR
SOIL
WATER
SED IMENT
PLANTS
DOMESTIC
ANIMALS
WILDLIFE
AREAS OF CONCERN
FIGURE 4.2 Decision Matrix for Planning Post-Treatment Monitoring Environmental
components should be sampled in relation to areas of concern
Identify
Environmental
Components for
Monitoring
(conS)
(2) Public resources and rnstitutions.
What public resources and institutions are located near the
treatment site? (parks, hospitals, libraries, retirement homes,
market centers)
(3) Uses of land
What are the dominant uses of the land surrounding a treat-
ment site? (recreational, commercial, agricultural, livestock)
(4) Uses of surface water
What uses are made of surface waters near the treatment site?
(fishing, swimming, boating, irrigation, livestock)
(5) Water supply sources
What are the sources of water for domestics municipal and
industrial users near a treatment site? (aquifers, wells,
reservoirs, rivers)
(6) Agriculture and forest production
What agricultural and forestry products are grown near a
treatment site? (crops, forage, animals, lumber, trees)
U,
I-
2
LU
2
0
a.
0
U
-J
4
I-
2
U i
2
0
>
2
L U
4 18
-------
Site-Specific Considerations
Identify (7) wildlife use and harvest
Environmental what wildlife species occur near the site?
Components for what forms are harvested as game animals?
Monitoring what forms are migratory?
(contd) (8) Other
what other critical areas might be adversely affected by
incomplete or ineffective biotreatment?
Air—Air quality may deteriorate during biodegradation of hazard-
ous materials if obnoxious gases are produced or chemical com-
pounds evaporate Once biotreatment is complete, these concerns
are normally resolved However, effective biotreatment often takes
more time than physical or chemical treatment, and gases may be
emitted over a period of years. where biotreatment is a continued
operation (land cultivation, composting, activated sludge system,
lagoons and ponds), some emission of gases may be unavoidable
Monitoring of air quality during and after biotreatment is war-
ranted by concerns that involve humans (see Figure 4 2, Areas of
Concern 1 and 2) Emissions must be controlled if large human
densities, public institutions, or public facilities are located down-
wind from the treatment site while the gaseous emissions may not
be hazardous in themselves, their presence may cause public
displeasure
Selected references on air monitoring are grouped in Section 4.3
Soil—Post-treatment monitoring of soil is necessary to document
low or harmless concentrations of former wastes before land can
be reclaimed and returned to normal uses Some hazardous mate-
rials such as metals are complexed in soil Future declines in soil
pH from acid precipitation may change harmless levels of hazard-
ous materials in soil, particularly certain metals, to levels capable of
environmental damage (EPA 1980, Haines 1981)
Soils may retain some toxic substances and toxic by-products after
biotreatment has been applied Retention is most likely to occur at
abandoned waste disposal sites and land treatment sites, where the
original hazardous materials were mixed with soil by composting,
layering, or tilling over large areas at various depths In such situa-
tions, isolated sections may contain wastes that escape effective
biotreatment
Soil samples are collected most efficiently at the surface where bio-
treatment is usually most effective If surface samples show that no
hazardous materials remain oi that residuals are at harmless levels,
surface runoff is not likely to be contaminated, crops may be
grown, and other human uses can be permitted Subsurface areas
may resist biotreatment more than surface areas, especially if mois-
ture and aeration is lacking Therefore, some subsurface or under-
ground locations may remain contaminated longer than others
Untreated or ineffectively treated locations below the surface may
4 19
-------
Sire-Specific Considerations
Identify eventually affect ground-water quality Future construction activity
Environmental may also expose subsurface layers that remain contaminated Deep
Components for locations in soil are difficult to sample effectively, but may be iden-
Monitoring tified through ground-water samples or by well-drilling and core-
(contd) sampling techniques
These considerations indicate that soil could contain some areas
with residual hazardous materials or by-products after biotreat-
ment is considered to be complete Such residuals could be harm-
ful to people in the future The sampling design for soils should be
designed to be adequate to detect the presence of such residuals.
If significant amounts of residuals are found, other methods of
treatment or long-term site isolation may be warranted
Selected references on soil monitoring are grouped in Section 4 3
Water—Water is relatively easy to sample and it provides a reliable
way to detect the presence of residual hazardous wastes or by-
products Water samples can be collected from surface runoff,
lea chate, ground water from land treatment sites or from liquid
effluents from bioreactor systems. Sampling wells can be drilled at
land treatment sites to determine if ground water is contaminated
with hazardous materials or their by-products
Ground water at abandoned waste disposal sites and accidental
spill sites is frequently contaminated Withdrawal of contaminated
ground water, followed by biotreatment and reinjection to the
ground, is one method useful for site cleanup Periodic chemical
analysis of ground-water samples will show the relative effective-
ness and progress of biotreatment efforts
Water represents a direct pathway for potential effects on human
health Areas of concern (Figure 4 2) include “uses of surface
water” and “water supply sources “Post-treatment monitoring
must include onsite and offsite sampling of these sources to pro-
vide assurance of continued safety to people and animals Sources
to monitor include aquifers, wells, springs, lakes, reservoirs, and
streams Domestic, municipal and industrial drinking water sources
must be examined for the presence of carcinogenic compounds
Information on public and private water supplies can be obtained
from such sources as public health departments, waler supply
companies, well drillers, and residents of the area Previous analy-
ses performed on water supplies may be identified and used as
baseline data for post-treatment assessment Knowledge of the
drainage basin will aid in selecting sampling locations in reservoirs,
lakes, and streams
Selected references on surface- and ground-water monitoring are
grouped in Section 4 3
Sediment—Sediment is a deposit of fine particulate matter by wind
or water Sediment can be deposited on land, in or near running
4 20
-------
Sit e-Specihc Considerations
Identify water, or beneath standing water Hazardous materials such as
Environmental metals can be complexed in sediment, as they are in soils. Deposits
Components for of sediment in waler may function as ecological “traps” for
Monitoring hazardous materials
(contd)
Selected references on soil and water monitoring are grouped in
Section 4 3 and include references for sediment monitoring
Plants—Plants will grow at field locations used for brotreatrnent
when waste concentrations in surface layers of the soil are not
phytotoxic (toxic to plants) Some organic compounds and metals
at low concentrations are bioaccumulated by terrestrial and
aquatic plants Former land-treatment and sludge-disposal sites
may be used to grow crops However, if trace amounts of toxic
materials remain in the soil, they may bioaccumulate and enter the
food chain when plants are consumed by people and animals
(Chaney et al in press, Chaney 1982)
When studying plants, the main areas of concern are with agricul-
ture and forest products Private gardens, orchards and small farm-
lots must be included in the sampling design if the area is
populated
Plant samples are easy to collect But concentrations of waste
materials or treatment by-products in plant tissues do not correlate
readily with concentrations in the environment (soil, water or air)
This is because several variables (including waste concentration
and chemical form, soil properties, and soil processes) influence
the kinetics of uptake reactions, particularly for metals (Cataldo
and Wildung 1978)
Therefore, the aim of post-treatment monitoring of plants should
be to detect concentrations of hazardous substances and treatment
by-products in leafy tissues that might be eaten by humans and
animals Analyses of plant tissues to quantify residual levels of
waste materials in soil and water are of limited value
Domestic Animals—Domestic animals using land treatment sites or
accidental release sites after biotreatment will be exposed to resid-
ual hazardous wastes or by-products that remain in the soil sur-
face, in plants, or in surface waters Exposure is usually by ingestion
or inhalation Post-treatment monitoring will be required to detect
possible bioaccumulation and possible harmful effects on animal
reproduction and health Many toxic materials that bio accumulate
and go undetected in domestic animals are readily transferred to
humans
Wildlife—Wildlife using land treatment sites and accidental release
sites after biotreatment will also be exposed to hazardous wastes or
by-products that remain The difference is in degree use of land
and water by domestic animals can usually be controlled, whereas
use by wild animals usually cannot Many forms of wildlife are
migratory and will use a site only temporarily Others reside at a
4 21
-------
Site-Specific Considerations
Identify site throughout the year Some migratory species such as birds will
Environmental use sites seasonally for breeding and rearing young
Components for
Monitoring Small mammals and insects will dig burrows in the soil, birds will
‘contd consume invertebrates in the soil, fish will live in water, and carni-
vores will consume prey Food-chain pathways for wildlife are
more varied and complex than those for domestic animals Game
animals and game fish harvested for human consumption might
not be subject to marketing restraints, whereas agricultural pro-
ducts usually are.
The aim of post-treatment monitoring of wildlife, however, is the
same as that for domestic animals, that is, to determine concentra-
tions of hazardous substances and treatment by-products in body
tissues that adversely affect their reproduction and health pro-
cesses, and concentrations that might be transferred to humans
State and local environmental groups will have information on
wildlife use The U S Fish and Wildlife Service and state depart-
ments of fish and game will have information on the biology, ecol-
ogy, and harvest of wildlife Collection of wildlife samples will
require permits or assistance from the Federal government (migra-
tory species), state departments of fish and game (resident species,
game or non-game animals), or both
Selected references on biological sampling (plant, domestic ani-
mals and wildlife) are grouped in Section 4 3
Develop a Sampling Post-treatment monitoring teams must assume that all results from
Program sampling of environmental components might be used in future
legal proceedings Therefore, sampling programs must be devel-
oped on a sound scientific basis and adhere to quality control
procedures. Numerous manuals exist which review methods and
designs to be used for obtaining statistically valid data from the
environment In general, the following items should be considered
when selecting a sampling design (EPA 1972)
• Is the method suitable for collecting statistically sound (time
and space) samples?
• Does the method provide for test and control samples?
• Is the method technically defensible, generally accepted, and
cited in standard texts?
• Can an adequate number samples be collected and analyzed
with the time and staff available?
• Will the method give quick preliminary results?
• Does the method sample the environmental components of
concern, with emphasis on foodchain pathways to humans?
4 22
-------
Site-Specific Con5 :derations
Develop a Sampling High standards of performance, care) analysis, and documentation
Program are as important as a standardized method of sampling to ensure
(contd) the quality of data Consistently uniform procedures are important
if the data will be statistically evaluated For this reason, a statisti-
cian and analytical chemist must both be involved in establishing
the sampling design before post-treatment monitoring begins
Selected references on sampling requirements, including statistical
considerations, are grouped in Section 4 3
Determine When Post-treatment monitoring begins when biotreatment activities are
Monitoring Should believed to have significantly reduced hazardous-waste materials,
Be Terminated or to have rendered them innocuous to humans and the environs
In many situations, monitoring will be conducted during biotreat-
ment to check on progress, to determine the effect of environ-
mental and microbiological enhancement efforts, and to adjust the
biotreatment process (operational monitoring)
Eventually, post-treatment monitoring must end Termination
occurs when the evidence collected and analyzed indicates that
the site no longer represents a hazard to humans and the sur-
rounding environs Where biotreatment is a continuing process, as
in most bioreactor systems, monitoring is an ongoing phase of the
operation that ends only when the operation terminates
A specific time to terminate post-treatment monitoring cannot be
established in advance A termination date will depend on the suc-
cess of the biotreatment process Success of biotreatment will
depend on numerous biotic and abiotic factors that vary over time
and from site to site If operational monitoring indicates limited
success, chemical and physical methods of waste detoxification,
waste removal, or waste isolation must be adopted
Post-treatment monitoring can be terminated when
• The wastes are removed or eliminated from the site
• The wastes are reduced to acceptable levels, and no toxic by-
products are produced
• The wastes are contained and possible future releases represent
minimal environmental risk
• Sufficient post-treatment monitoring data are obtained to estab-
lish that the site or waste materials are no longer hazardous
4 23
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Sue-Specific Conssderar,on5
4.3 CHAPTER REFERENCES
References, in addition to those cited in the text, are organized by area of
concern (see Figure 4 2) to give the user additional sources of information
on how to monitor selected environmental components
Site-Specific American Public Health Association (APHA), et al 1981 Standard
Considerations Methods for the Examination of Water and Wastewater, Vol 15
APHA, Washington, D C, p 1193
Arthur D Little, Inc 1972 Chemical Hazards Response Information
System (CHRIS), AD 757-472-3-4 Cambridge, Massachusetts
California State Department of Health 1977 Hazardous-Waste
Regulations Title 22, Register 77, No 24 Sacramento, California
(October 15, 1977).
Callahan, M A , et al 1979 Water Related Environmental Fate of
129 Priority Pollutants Vol I and II PB 80-204 373 and PB 80-204
381, NTIS, Springfield, Virginia.
Chapman, P M , C P Romberg and G A Vigers 1982 “Design of
Monitoring Studies for Priority Pollutants “/ Water Pollut Cont
Fed 54 292-297
Chemical Transportation Emergency Center-CMA (CHEMTREC)
Phone (800) 424-9300
Dawson, G W , C J English and S E. Petty 1980 Physical and
Chemical Properties of Hazardous-Waste Constituents U S Envi-
ronmental Protection Agency, Athens, Georgia
Dryden, F E , et al 1978 Priority Pollutant Treatability Review
Contract No 68-03-2579, U S Environmental Protection Agency,
Cincinnati, Ohio
EPA 1972 Field Detection and Damage Assessment Manual (or Oil
and Hazardous Materials Spills U S Environmental Protection
Agency, Washington, D C.
EPA 1973 Methods for the Collection and Analysis of Biological
Samples U S Environmental Protection Agency, Cincinnati, Ohio
EPA 1976 Manual of Methods for the Chemical Analysis of Water
and Wastes U S Environmental Protection Agency, Cincinnati,
Ohio
EPA 1978 “Hazardous Waste Proposed Guidelines and Regula-
tions and Proposal on Identifications and Testing “ Federal Register
43, No 243, pp 58946-59028, December 18, 1978
EPA 1979 Safety Manual for Hazardous-Waste Site Investigations
Office of Occupational Health and Safety, Washington, D C
(draft).
4 24
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Site-Specific Considerations
Site-Specific EPA 1982 EPA Field Guide for Scientific Support Activities Asso-
Considerations ciated With Super fund Emergency Response EPA-600/8-82-025,
(contd) Office Of Emergency and Remedial Response, U S Environmental
Protection Agency, Washington, D C
Epstein, E , and R I Chaney 1978 “Land Disposal of Toxic Sub-
stances and Water-Related Problems”] Water PoIIut Cont Fed
50 2037-2042
Fields, T, Jr, and A W Lindsey 1975 Landfill Disposal of Hazard-
ous Wastes A Review of Literature and Known Approaches
EPA/530/SW-165 Cincinnati, Ohio, PB-261 079, NTIS, Springfield,
Virginia
Franklin Research Center 1981 “Literature Study of the Biode-
gradability of Chemicals in Water “ Vol 1 Biodegradability, Predic-
tion, Advances In and Chemical Interferences With Wastewater
Treatment PB 82-1000843, NTIS, Springfield, Virginia.
Franklin Research Center 1981 “Literature Study of the Biode-
gradability of Chemicals in Water “ Vol 2, Permeated Index of
Chemicals, Microbial Populations, and Wastewater Treatment Sys-
tems with Bibliography PB 82-1000850, NTIS, Springfield, Virginia
Hatayama, H K , et al 1980 A Method for Determining the Com-
patibility of Hazardous Wastes EPA-600/2-80-076, U S. Environ-
mental Protection Agency, Cincinnati, Ohio
Lewis, R J , Jr , and R L Tatken, eds 1980 Registry of Toxic Effects
of Chemical Substances, Volumes 1 and 2 U S Department of
Health and Human Services, Cincinnati, Ohio
Mackay, D, and P J Leinonen 1975 “Rate of Evaporation of Low
Solubility Contaminants from Water Bodies to Atmosphere”
Environ Sci Technol 9 11781180
National Fire Protection Association (NFPA) 1975 Manual of
Hazardous Chemical Reactions NFPA-491M, Boston,
Massachusetts
Plunkett, E R 1966 Handbook of Industrial Toxicology Chemical
Publishing Company, mc, New York
Saegar, V W , and Q E Thompson 1980 “Biodegradability of Halo-
gen Substituted Diphenylmethanes “Environ Sci Technol
14 704-709
Sax, N I 1979 Dangerous Properties of Industrial Chemicals 4th
ed Van Nostrand, Reinhold, New York
Shuckrow, A J , A P Pajak and J W Osheka 1981 Concentration
Technologies for Hazardous Waste Treatment EPA-600/2-81-019,
U S Environmental Protection Agency, Cincinnati, Ohio, PB 81-
150583, NTIS, Springfield, Virginia
4 25
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Site-Specific Considerations
Site Specific Con- Sittig, M 1979a Hazardous and Toxic Effects of !ndustnal Chemi-
siderations (contd) cals Noyes Data Corporation, Park Ridge, New Jersey
Sittig, M 1979b Landfill Disposal of Hazardous Wastes and
Sludges Noyes Data Corporation, Park Ridge, New Jersey.
Sittig, M., ed 1980 Priority Toxic Pollutants Health Impacts and
Allowable Limits Noyes Data Corporation, Park Ridge, New Jersey
Tabak, H H , et al 1981 “Biodegradability Studies with Organic
Priority Pollutant Compounds “J Water Pollut Cont Fed
53 1503-1518
U S Coast Guard 1974a A Condensed Guide to Chemical
Hazards, CG-446--1 Washington, D C
U S Coast Guard 1974b Hazardous Chemical Data, CG-446-2
Washington D C
Verscheuren, K 1977 Handbook of Environmental Data on
Organic Chemicals Van Nostrand, Reinhold, New York
General Bowen, H J M 1979 Environmental Chemistry of the Elements
Post-Treatment Academic Press, New York
Cataldo, D A, and R E Wildung 1978 “Soil and Plant Factors
Influencing the Accumulation of Heavy Metals by Plants “ Environ
Health Persp 27 149-159
Chaney, R 1 1982 “Foodchain Pathways for Toxic Metals and
Toxic Organics in Wastes” In Environment and Solid Wastes
Characterization, Treatment, and Disposal Proceedings of 4th Life
Sciences Symposium, Oak Ridge National Laboratory, Oak Ridge,
Tennessee
Chaney, R L , et al “Effect of Sludge Qualities and Rate, Soil, pH,
and Time on Heavy Metal Residues in Leafy Vegetables” In Pro-
ceedings of 5th Annual Madison Waste Conference 1982 (in press)
EPA 1978 Microbiologicai Methods for Monitoring the Environ-
ment Water and Wastes EPA-600/8-78-017, Ii S. Environmental
Protection Agency, Cincinnati, Ohio.
EPA. 1980 Acid Rain EPA-600/9-79-036, Office of R&D, U S Envi-
ronmental Protection Agency, Washington, 0 C
EPA 1982 EPA Field Guide for Scientific Support Activities Asso-
ciated with Super fund Emergency Response EPA-600/8-25-025,
Office of Emergency and Remedial Response, U S Environmental
Protection Agency, Washington, D C.
Fritz, E 5, P J Rago and I P Murarka 1980 Strategy for Assessing
Impacts of Power Plants on Fish and Wildlife Populations
FWS/OBS-80/34, Biological Services Program, U S Fish and Wild-
life Service, Washington, D C
4 26
-------
Site-Specific Considerations
General Haines, T A 1981 “Acid Precipitation and Its Consequences for
Post-Treatment Aquatic Ecosystems A Review “ Trans Am Fish Soc 110 669-707
(contd) Maki, A W , K L Dickson and J Cairns, Jr., eds 1980 “Biotrans-
formation and Fate of Chemicals in the Aquatic Environment”
Am Soc Microbiol Washington, D C
Neeley, W B 1980 Chemicals in the Environment Marcel Dekker,
mc, New York
Air Monitoring Bowers, J F , J R Bjorklund and C S Cheney 1979. Industrial
Source Complex (ISC) Dispersion Model User’s Guide EPA-450/4-
79-031, U S Environmental Protection Agency, Research Triangle
Park, North Carolina
Briggs, G A 1973 Diffusion Estimation for Small Emissions ATDL
Contribution File No 79, Atmospheric Turbulence and Diffusion
Laboratory, Oak Ridge, Tennessee
Englemann, R J , and G A Sehmel 1976 Atmosphere-Surface
Exchange of Particulate and Gaseous Pollutants (1974) ERDA Sym-
posium Series 38, ERDA Technical Information Center, Oak Ridge,
Tennessee
EPA 1977 Users Manual for Single-Source (CRSTER) Model EPA-
450/2-77-013, U S Environmental Protection Agency, Research Tri-
angle Park, North Carolina
Neuman, J P 1980 Effects of Air Emissions on Wildlife Resources
FWS/OBS-80/40 1, U S Fish and Wildlife Service, Biological
Services Program, Supt of Documents, U S Govt Print Office
Washington, D C
Orgill, M M 1981 Atmospheric Studies in Complex Terrain, A
Planning Guide for Future Studies PNL-3656, Pacific Northwest
Laboratory, Richland, Washington
Pasquill, F 1974 Atmospheric Diffusion 2nd ed D. Van Nostrand
Company, Ltd, London, England
Pasquill, F 1971 “Atmospheric Dispersion of Pollution “ Quart I
Roya/ Meteor Soc 97 369-395
Shen, T T 1981 “Estimating Hazardous Air Emissions from Dis-
posal Sites “ Pollut Engr 13 31-34
Stern, A C ,ed 1977 Air Pollution, Volume II The Effects of Air
Pollution 3rd ed Academic Press, New York
Thibodeaux, I J 1981 “Estimating the Air Emissions of Chemicals
from Hazardous-Waste Landfills”) Haz Mat 4 235-244
Turner, D B 1967 Workbook of Atmospheric Dispersion Esti-
mates Public Health Service Publication 999-AP-26, Robert A Taft
Sanitary Engineering Center, Cincinnati, Ohio
4 27
-------
Site-Specific Considerations
Air Monitoring Turner, D. B , and J H Novak 1978 Users Guide for RAM EPA-
(contd) 600/8-78-016, U S Environmental Protection Agency, Research Tri-
angle Park, North Carolina.
Soil Monitoring Bension, R C , and R A Glaccum 1979 “Remote Assessment of
Pollutants in Soil and Groundwater.” In Proceedings of the 1979
Conference on Hazardous Material Risk Assessment, Disposal and
Management, Information Transfer, Inc., Miami Beach, Florida
Black, C A 1969 Methods of Soil Analysis Agronomy, No 9,
American Society of Agronomy, Madison, Wisconsin
Coperhaven, E D , and B K Wilkinson 1979 Movement of
Hazardous Substances in Soil A Bibliography, Volume 1 Selected
Metals EPA-600/9-79024a, U S Environmental Protection Agency,
Cincinnati, Ohio
Coperhaven, E D , and B K Wilkinson 1979 Movement of
Hazardous Substances in Soil A Bibliography, Volume 2 Pesti-
cides EPA-600/9-79-0246, U.S. Environmental Protection Agency,
Cincinnati, Ohio
Corps of Engineers 1972 Soil Sampling Department of the Army,
EM 1110-2-1907, Washington, D C
Fuller, W H 1978 Investigation of Landfill Leachate Pollutant
Attenuation by Soils EPA-600/2-78-158, Li S. Environmental Protec-
tion Agency, Cincinnati, Ohio
Maiver, B N , and C P Hale 1970 Laboratory Soils Testing
Department of the Army, EM 1110-2-1905, Washington, D C
Surface- and Barton, C M 1974 “Borehole Sampling of Saturated Uncemented
Ground-water Sands and Gravels” Groundwater 12 170-181
Monitoring Bird, R B , W E Stewart and E N Lightfoot 1960. Transport Phen-
omena John Wiley and Sons, New York
Clarke, J H et al 1980 A Model for Assessment of Environmental
Impact of Hazardous Materials Spills and Leaching Recra Envi-
ronmental and Health Services, Inc. Nashville, Tennessee
Bureau of Reclamation 1967 Water Measurement Manual 2nd
ed U S Government Printing Office, Washington, D.C.
Bureau of Reclamation 1977 Ground Water Manual U S
Government Printing Office, Washington, 0 C
EPA 1977 Procedures Manual for Ground Water Monitoring at
Solid Waste Disposal Facilities EPA/530/SW-611, U S Environ-
mental Protection Agency, Cincinnati, Ohio
EPA 1978 A Manual for Evaluating Contamination Potential of Sur-
face Impoundments EPA 570/9-78-003, U S Environmental Protec-
tion Agency, Washington, D C
4 28
-------
Sue-Specific Considerations
Surface- and EPA 1979 Handbook for Analytical Quality Control in Water and
Ground-water Wastewater Laboratories EPA-600/4-79-019, U S Environmental
Monitoring Protection Agency, Cincinnati, Ohio
(contd) EPA 1981 NEt C Manual for Groundwater/Subsurface Investiga-
tions at Hazardous Waste Sites EPA-330/9-81-002, National
Enforcement Investigations Center, Denver, Cororado
Everett, L G , et al 1976 Monitoring Groundwater Quality
Methods and Costs EPA-600/4-76-023, U S Environmental Protec-
tion Agency, Las Vegas, Nevada
Fenn, D, et al 1977 Procedures Manual for Groundwater Moni-
toring at Solid Waste Disposal Facilities EPA-530/SW-611, U S Envi-
ronmental Protection Agency, Cincinnati, Ohio
Freeze, R A , and J A Cherry 1979 Groundwater Prentice-Hall,
Englewood Cliffs, New Jersey.
General Electric Company 1980 Groundwater Monitoring
Business Growth Services, Schenectady, New York
Johnson Division 1975 Ground Water and Wells Johnson
Division 1 UOP, St Paul, Minneso la
Gibb, I P and R A Griffin 1979 Groundwater Sampling and
Sample Preservation Techniques (1st Annual Report) U S Envi-
ronmental Protection Agency, Cincinnati, Ohio
Mooij, H and F A Rovers 1976. Recommended Groundwater and
Soil Sampling Procedures Environmental Conservation
Directorate, Report EPS-4-EC-76-7, Ottawa, Ontario, Canada
Plumb, R H Jr 1981 Procedures for Handling and ChemicalAnaly-
sis of Sediment and Water Samples Tech Rept EPA/CE-81-1, US
Army Engineer Waterways Experiment Station, CE, Vicksburg,
Mississippi
USGS 1981 Catalogue of Information on Water Data—Index to
Water Data Acquisition Office of Water Data Coordination and
the National Water Data Exchange, U S Geological Survey,
Washington, D C
Biological EPA 1973 Methods for the Collection and Analysts of Biological
Monitoring Samples U S Environmental Protection Agency, Washington, D C
Fritz, E 5, P J Rago and I. Muiarka 1980 StrategyAssessing
Impacts of Power Plants on Fish and Shellfish Populations
FWS/OBS-80/34, Office of Biological Services, U S Fish and Wild-
life Service, Washingon, U C
Hocutt, C H , and R Stauffer. Jr 1980. Biological Monitoring of
Fish Lexington Books, Lexington, Massachusetts
Smith, J H , et al 1977 Environmental Pathways of Selected Chem-
icals in Freshwater Systems, Vol 1 Background and Experimental
4 29
-------
Site-Specific Considerations
Procedures EPA-600/7-77-13, U S Environmental Protection
Agency, Athens, Georgia
Sampling Box, G , W G Hunter and J S Hunter 1978 Statistics for Expert-
Requirements menters John Wiley and Sons, New York.
Cockran, W H 1977 Sampling Techniques 3rd ed. John Wiley
and Sons, New York p 428
De Vera, E R , et al 1980. Samplers and Sampling Procedures for
Hazardous Waste-Streams EPA-600/2-80-018, U S Environmental
Protection Agency, Cincinnati, Ohio
Dickson, K L , J Cairns, Jr and R. I Livingston 1978 Biological
Data in Water Pollution Assessment Quantitative and Statistical
Analyses STP 652, American Society for Testing and Materials, Phi-
ladelphia, Pennsylvania, p 184
EPA 1972 Field Detection and Damage Assessment Manual for Oil
and Hazardous Material Spills U S Environmental Protection
Agency, Washington, D C
Eberhardt, L I , and R 0 Gilbert 1975 “Biostatistical Aspects “ In
Environmental Impact Monitoring of Nuclear Power Plants, Source
Book of Monitoring Methods, Vol 2, pp 783-918, AIF/NE-SP-004,
Atomic Industrial Forum, Inc, New York
Holling, C S 1978 Adaptive Environmental Assessment John
Wiley and Sons, New York, p 377
Gilbert, R 0 1982. Some Statistical Aspects of Finding Hot Spots
and Buried Radioactivity PNL-SA-10274, Office of Health and Envi-
ronmental Research, U.S Department of Energy, Pacific Northwest
Laboratory, Richiand, Washington
McKenzie, D H, et al 1977 Design and Analysis of Aquatic Mont-
onng Program at Nuclear Power Plants PNL-2423, Pacific North-
west Laboratory, Richland, Washington
States, J B et al 1978 A System Approach to Ecological Baseline
Studies FWS/OBS-78/21 Biological Services Program, U S Fish and
Wildlife Service, Washington, 0 C
Ward, 0 V 1978 Biological Environmental Impact Studies Theory
and Methods Academic Press, New York.
Weber, C I. 1973 Biological Field and Laboratory Methods for
Measuring the Quality of Surface Water and Effluents EPA-670/4-
73-001, U S Environmental Protection Agency, Research Center,
Cincinnati, Ohio
Winer, B J 1971 Statistical Principles in Experimental Design 2nd
ed McGraw-Hill Book Co, New York
Zar, J H 1974 BiostatisticalAnalysis Prentice Hall, Englewood
Cliffs, New Jersey
4 30
-------
5.0 DESCRIPTION OF BIOTREATMENT OPTIONS
As discussed in Chapter 2, biotreatment options vary with environmental con-
ditions, local restrictions, state regulations, population density, and waste
characieristics This chapter describes some of the available treatment
methods, provides flow charts for selecting specific methods, and compares
the advantages and disadvantages of each option
Four treatment options are diagrammed in Figure 2 1 and discussed in the
following text
• natural biodegradation
• land treatment
• bioreactor systems
• bioaccumulation and removal
Also illustrated in Figure 2 1 and discussed in the following text are enhance-
ment techniques that can be applied to biotreatment options
5.1 NATURAL BIODEGRADATION
Most hazardous wastes in the environment, given sufficient time, will eventu-
ally be transformed or degraded by a combination of chemical, physical and
biological processes in some cases, it may take decades or centuries for the
wastes to degrade The hazardous nature of many wastes, their environmental
persistence and mobility, and their high volumes make rapid conversion to
innocuous compounds necessary One way to increase the rate of degradation
is to control the physical, chemical, and biological processes through bio-
treatment methods
Chemical degradation can result from oxidation-reduction, hydrolysis,
depolymerization, or isomerization Physic.al degradation can result from sun-
light and radiation Microbial degradation is influenced by the microbial popu-
lation and by soil conditions that affect their activity These conditions include
pH, moisture, temperature, exposure to air and sunlight, and presence of
organic matter The chemical nature of a waste and its structure can also
influence degradation
5.2. LAND TREATMENT
Solid or liquid wastes can be biologically treated by applying the wastes to the
land surface, by composting, or by combining both methods The concept of
land treatment described here does not include landfills, overland flow pre-
treatment, deep well injection, or soil reactor units Brown and Deuel (1980)
and Overcash and Pal (1979) describe the application of land treatment
methods to hazardous wastes These studies also discuss land treatment and
selection of potential land treatment Sites
Land treatment involves mixing or dispersing the wastes in soil in relatively
thin layers This has the effect of increasing the availability of the waste to
51
-------
Description of Riot reatmeni Options
microorganisms in the soil It assumes that some microorganisms will survive
the initial shock of being exposed to the wastes, will develop suitable enzyme
systems, and will biodegrade the compounds present In its most simple form,
land treatment uses microorganisms indigenous to the soil Under some condi-
tions, however, it may be necessary to add induced or adapted microbial
cultures
Land application rates can be determined for most industrial wastes The deci-
sion to proceed or not with land treatment is generally based on cost, not
technical feasibility Soil properties and waste composition determine the
application rate If land area is not sufficient to handle all of the waste at a
predetermined maximum rate of application, alternative disposal methods will
be necessary
To determine if a site is acceptable for land treatment, characteristics of the
surface water, ground water, climate, geology, topography, and soil must be
known Land treatment is an “open system” that potentially can lead to onsite
and offsite contamination of ground water, surface water, and air Therefore,
design, operation, and adequate monitoring are important factors for land
treatment Regulations for land treatment of hazardous wastes have been pub-
lished by the U S Environmental Protection Agency (EPA 1980)
Land Treatment The first step in designing a land treatment system is to determine
Systems and the capacity of the site to assimilate waste constituents Overcash
Limitations and Pal (1979) describe procedures to determine the ratio of waste
generation (kg/yr) to land capacity [ (kg/ha)/yr)] This ratio is the
area required for effective land treatment disposal
The form of waste may be liquid, slurry, sludge, or solid The differ-
ence in application of waste forms is primarily in the equipment
used to spread the material on the land Pre-treatment may be
needed to reduce the concentration of the land limiting constitu-
ent (LLC) The LLC is the waste component that first exceeds an
acceptable concentration in the soil (Overcash and Pal 1979) Pre-
treatment may allow a greater application rate or the use of less
land area A land application system should be designed to assimi-
late 100 percent of the LLC applied Wastes can usually be pumped
and handled as liquids if they contain less than 8 percent solids and
if particle sizes are smaller than 2 5 cm in diameter
Common land application methods include sprinkler irrigation,
overland flow, tank truck, and truck spreaders Most hazardous
wastes should be mixed with the soil by some tillage procedure to
ensure that the material remains where it is placed
An aerobic system is usually effective for biotreating most waste
compounds If loading rates are too high or if applications are too
frequent, anaerobic conditions will develop and slow the rate and
extent of biotransformation The same factors that influence the
decomposition of sewage sludge and crop residues in soils also
influence the decomposition rate of hazardous wastes Therefore,
52
-------
Description of Biotreatment Options
Land Treatment agricultural publications discussing disposal of sewage sludge on
Systems and land will explain the principles controlling the decomposition of
Limitations hazardous materials in soils
(contd)
There may be times during which application of hazardous or toxic
wastes to land is not possible For example, periods of high rainfall
will saturate the soil and periods of extended freezing or snow
cover will prevent mixing of waste and soil In these cases, interim
storage of the waste may be necessary in earth ponds, concrete
tanks, or in piles of relatively dry waste solids Waste integrity can
be maintained with covers, liners, and other barriers that prevent
moisture saturation, leaching, or surface runoff
The advantages and disadvantages of land treatment of hazardous
wastes are listed below For comparison with other methods dis-
cussed in this chapter, see Figure 5 1
Advantages:
• The treatment costs less than most physical or chemical pro-
cesses [ Costs associated with some land treatment activities are
discussed by EPA (1981)
• The method is relatively simple to apply
• The method is effective on wastes with relatively high metal
content
Disadvantages:
• Large areas of land may be required
• Degradation takes a long time and may never be complete for
many wastes
• Highly concentrated wastes may kill indigenous microorganisms
and sterilize the soil
• Toxic volatile compounds may be produced
• Toxic intermediate compounds, in rare instances, may form
• Heavy metals will remain in the soil
Composting Techniques for composting hazardous waste are still in early
development Wilson et al (1982a,b) describe composting as a pre-
treatment method of detoxifying wastes prior to land application
Compost ing is the microbial conversion of organic waste materials
in the presence of air and moisture into a product with the general
appearance and other characteristics of fertile soil The waste is
conditioned for composting by bulking materials (woodchips,
leaves, refuse) to render it permeable to air As the aerobic
microorganisms oxidize the sludge, they release heat and the
temperature of the waste rises
53
-------
Description of B,otreatmenf Options
MONEY
LAND
WATER
TIME (SETUP)
TIME
(DEGRADATION)
ENERGY
ENHANCEMENT
WASTE
CONCENTRATION
WASTE FORM
(LIQUID/SOLID)
LOAD RATE
OR GA NICS
METALS
VOLATILE S
ODORS
SLUDGE
SOLIDS
LIQUIDS
INTERMEDIATES
FREEZING
DRYING
SHOCK LOADING
CLOGGING
RECOVERY TIME
BIOTREATMENT OPTION
0
0
0
•
•
I
I
0
0
0
0
0
0
0
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S
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I
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5
0
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0
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00
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00
•
NA
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•
0
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0
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0
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0
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0
3
0
•
NA
NA
NA
NA
NA
3
0
0
0
NA
NA
• ADVANTAGE 0 ADVANTAGE/DISADVANTAGE 0 DISADVANTAGE
DEPENDENT ON SITE SPECIFIC
NA NOT APPLICABLE
FACTORS
FIGURE 5.1 Matrix Comparison of Advantages and Disadvantages of Biotreatment
Options for Hazardous Wastes
Composting
(contd)
/
I 1100
I-
C ,,
0
0
OIl
4
w
I-
U
z
0
‘I ,
4
0
C.,
I
U,
I-
U
0
0
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z
0
I-
0 .
uJ
I-
5.4
-------
Descripi ion of Biotreatment Options
Composting The most fundamental composting process involves placing the
(contd) waste or soil in windrow piles Aeration to enhance degradation by
aerobic microorganisms is achieved by periodically spreading or
turning the pile If pH levels are kept neutral to alkaline, metals
not absorbed on the soil or organic components of the waste pile
wiPI usually be present as precipitate This process is discussed by
Berkowitz, Funkhouser and Stevens (1978)
Several types of composting systems are available One innovative
system with promise for hazardous wastes is the Beltsville Aerated
Pile Composting Method (Wilson et al 1980), which has been used
primarily for composting sewage sludge. A mixture of waste and
bulking material is placed over a network of perforated pipes con-
nected to a blower that draws air down through the mass Exhaust
gases are scrubbed through a pile of finished compost to remove
odor before release to the atmosphere A blanket of compost over
the compostrng sludge prevents the escape of obnoxious odors
and insulates the pile to mci ease its temperature
The advantages and disadvantages of composting of hazardous
wastes are listed below For comparison with other methods dis-
cussed in this chapter, see Figure 5 1
Advantages
• The system has high tole:ance to microbially toxic chemicals
(i e , metals)
• Energy demands are low (Fuel costs for earth-moving equip-
ment are the most expensive requirement
• No sludge or brine disposal is required
• Most types of organic compounds are degraded
microbiologically
Disadvantages:
• Long retention or degradation periods are required
• Acclimation is needed for aerobic microorganisms
• Sufficient moisture must be present
Land treatment of hazardous waste, including composting, is most
effective for sites where
• Toxic wastes are present in relatively low concentrations
• The site does not have to he cleaned up or detoxified rapidly
• Soil moisture and temperature levels are favorable
Slow degradation is a problem only if the compounds are
extremely hazardous, if the site cannot be secured, or if there are
515
-------
Descnpt ion of Biotreatment Options
Composting
(contd)
land-use conflicts Good management practices can often acceler-
ate the degradation process to some extent Some waste materials
are highly toxic to microorganisms, or are present in sufficiently
high concentrations to sterilize the soil Control of application
rates and sound treatment practices would minimize this problem
If concentrated wastes from an abandoned site or a spill area can
be removed, they can be stored and applied to an approved land
treatment site at diluted or non-toxic concentrations Again, good
management practices and proper loading rates will resolve many
of these problems Application of water by sprinkler systems or
hoses will increase the water/waste ratio in soil or composts and
enhance degradation at many sites However, such dilution should
be avoided at sites where contamination of surface or ground
water is possible, or where seepage and runoff cannot be con-
trolled The course of action depends, to a large extent, on the site
and types of wastes present.
The decision process used to determine if land treatment methods
are practical for detoxification of hazardous wastes is shown in
Figure 5 2
FIGURE 5.2 Decision Process for Evaluating Land Application (Including Composting)
for Biotreatment of Hazardous Wastes
56
-------
Description of Bioireatrnent Options
5.3 BIOREACTOR SYSTEMS
Biological treatment of municipal and industrial wastes is relatively common
Microorganisms, however, have not been used extensively to transform or
degrade hazardous materials in bioreartor systems Microorganisms have
degraded many synthetic compounds tinder controlled laboratory conditions,
and their potential for similar activity in large-scale, engineered systems is
good
The advantages and disadvantages of bioreactor treatment of hazardous wastes
are listed below For comparison with other methods discussed in this chapter)
see Figure 5 1
Advantages:
• Degradation proceeds more rapidly than under field situations
• Degradation may proceed more rapidly than under land treatment activities
• Physical and chemical conditions are readily modified
Disadvantages:
• High construction, maintenance, and operational costs
• System disruptions must be prevented
• Sludges accumulate and must be removed
• Use is restricted to primarily liquid wastes
Application of biotreatment to hazardous waste liquids and leachates in bio-
reactors is considered by EPA (1982), Shuckrow, Pajak and Oshaka (1981), and
Berkowitz, Funkhouser and Stevens (1978) A study by SCS Engineers (1979a)
describes successful and unsuccessful biodegradation in wastewater treatment
processes, gives estimates of the time required to decompose different wastes,
and makes engineering and economic comparisons
Bioreactor systems to treat toxic or hazardous wastes must be designed to
• enhance oxygen transfer to microorganisms for aerobic systems
• enhance contact between wastes and microorganisms
• reduce toxic or inhibitory effects of the wastes
• prevent abrupt “shock” loadings
The most common bioreactor systems are air-activated sludge, trickling filter,
rotating disc, aerobic ponds and lagoons, and anaerobic ponds and lagoons A
general description, advantages, disadvantages, applications, and flow charts
for each system follow.
Air-Activated The air-activated sludge process involves pumping air into sludge
Sludge reactor tanks containing the waste The process relies on micro-
organisms being continuously circulated while in contact with
organic materials and oxygen [ he operator can modify aeration
57
-------
Description of Biotreacment Options
Air-Activated and mixing by changing the amount of air blown into the reactor
Sludge tanks Secondary clarifiers are used to separate activated sludge
(contd) solids from the mixed liquor, and to produce concentrated solids
for the return flow necessary to sustain the microbial population
Eventually, the sludge must be removed and deposited elsewhere
for the system to function.
Metals at low concentrations, non-toxic to microorganisms, are
concentrated and separated by the treatment Most insoluble
metals should be removed chemically by sedimentation before
treatment. Metals can have an adverse effect on metabolism of
microorganisms
Preliminary analysis of the waste helps determine if additional nutri-
ents are necessary. If the waste is deficient in either nitrogen or
phosphorus, the necessary nutrient is added Lime is almost always
added to maintain an alkaline pH and to precipitate metals
Problems with toxic compounds are low because the aeration unit
mixes and dilutes toxic components Following treatment, how-
ever, the resulting sludge may be hazardous because of sorption
and concentration of toxic compounds such as metals In addition,
the mixing action of the aeration unit may release volatile com-
pounds to the atmosphere Loss of volatile compounds can aid bio-
treatment but may also degrade air quality and affect personnel
safety (Berkowitz, Funkhouser and Stevens 1978)
Pre-treatment and post-treatment methods may be required to
meet discharge objectives (Shuckrow, Pajak and Touhill 1980)
The methods include precipitation of metals, carbon sorption,
ultrafiltration, chemical oxidation, wet air oxidation, ion exchange,
and electrochemical treatment
The advantages and disadvantages of air-activated sludge treat-
ment of hazardous wastes are listed below For comparison with
other methods discussed in this chapter, see Figure 5 1
Advantages:
• The method is acceptable for industrial wastewater treatment
• The method is adaptable to different types of waste streams
• The method is reliable
• The method can handle higher organic loads than many bio-
treatment processes
Disadvantages:
• The method is relatively expensive
• Toxic gases could be released during aeration
58
-------
Description of Biotreatment Options
Air-Activated • Sludge that can be high in metals and organic compounds is
Sludge produced
(contd)
• The method is sensitive to shock loads of suspended solids,
metals, and biocidal organic compounds
• Energy requirements are relatively high
The activated sludge process is sensitive to disruption from sus-
pended solids, oil, and grease The Environmental Protection
Agency (EPA 1982) recommends that suspended solids be less than
1 percent, with oil and grease less than 75 mg/I (preferably less
than 50 mg/I), for effective treatment The waste should also be
free of heavy metals at levels that inhibit microbial activity The
waste must also be free of any toxic organic compounds at levels
high enough to kill microorganisms or to prevent their growth If
this occurs, the wastes will not decompose or will do so slowly
Finally, the system must be cost-effective compared to alternative
methods The decision process used to determine if activated
sludge treatment may be applied to hazardous wastes is shown in
Figure 5 3
Trickling Filter Trickling filters have biological films grown on a fixed bed of
crushed rock or some other support medium Liquid waste is
applied to the top and passes over the slime layers, which consist
of a complex mixture of microorganisms Organic wastes are bio-
transformed by the microbial population, and biotransformation
products are returned to the liquid waste stream
Trickling filters can accept shock loads better than the activated
sludge process Various support media and depths of media are
used for different hydraulic loads The process requires that the
fitter bed be aerobic As a result, the wastewater to be treated
should contain less than 1 percent solids, that is, it should be
almost entirely liquid Higher solid concentrations cause the sup-
port media to plug and the system to become anaerobic
The trickling filter does not produce as high a quality of effluent as
the activated sludge process However, the system may be less
troublesome to operate and is less likely to strip volatile com-
pounds (Berkowitz, Funkhouser and Stevens 1978)
The advantages and disadvantages of trickling filter treatment of
hazardous wastes are listed below For comparison with other
methods discussed in this chapter, see Figure 5 1
Advantages:
• The method has short hydraulic residence times
• The method has low sensitivity to shock loads
• Suspended or colloidal matter can be removed
59
-------
Description of B,otreatment Options
Trickling Filter • The method can be used as a roughing filter to moderate
(contd) organic loads
Disadvantagest
• The method is applicable only to liquid waste streams
• Treatment capability is restricted in a one-pass-through
operation
• Odors may be a problem
• Flexibility and product control are relatively limited
• Microorganisms are slow to recover if disrupted
• After shutdown, the system recovers slower than the activated
sludge treatment
• Freezing temperatures may prevent operation
FIGURE 5.3 Decision Process for Evaluating Activated Sludge Biotreatmeni
5 10
-------
Description of Biotreament Options
Trickling Filter
(contd)
Rotating Disc
Because the trickling filter process involves continuous trickling of
water containing wastes over a support medium, the system should
not be disrupted Disruption may permit the medium to dry out,
thus destroying the microbial population Freezing temperatures
may also disrupt the system by solidifying the liquid waste and pre-
venting it from passing through the filter bed Pretreatment of
wastes may be necessary if odors are a problem Trickling filters are
usually used with other treatment methods because the short reten-
tion time does not generally produce an effluent low in toxic
components The decision process used to determine if trickling
filter treatment may be applied to biotransform hazardous liquid
wastes is shown in Figure 5 4
The rotating disc process is similar to the trickling filter process in
that both use a fixed-film growth of microorganisms to degrade
wastes A series of perforated disks are mounted on a horizontal
shaft and then placed in a tank having a contoured bottom The
discs are immersed approximately 40 percent The liquid trickles
from the void spaces and the biomass is aerated (Metry 1980) when
the disks are rotated in the tank.
PROCEED
WITH TRICKLING
FILTER
TREATMENT
FIGURE 5.4 Decision Process for Evaluating Trickling Filter Biotreatment
APPLY PRE-TREATMENT
TECHNIQUE OR
SELECT A DIFFERENT
TREATMENT METHOD
WILL THE METHOD
METHOD
HAS LIMITED
BE USED WITH NO
CAPABILITY
ADDITIONAL WASTE
IN ONE-PASS
TREATMENT?
——————J
5 11
-------
Descnpl;on of Biotteatmeni Options
Rotating Disc The advantages and disadvantages of rotating disc treatment of
(contd) hazardous wastes are listed below For comparison with other
methods discussed in this chapter, see Figure 5 1
Advantages:
• Operation is relatively flexible (Biomass, waste water and aera-
tion rate can be controlled by the rotation of the discs)
• Treatment capacity is relatively high (Retention time can be
controlled by varying tank size
• The system rarely clogs (Shearing forces continuously and uni-
formly strip excess growth
• The method can be modified to meet varied waste loadings.
Disadvantages:
• The method is applicable only to liquid waste streams
• More aeration may be needed for high organic loads
• Odors may be a problem
• Microorganisms are slow to recover if disrupted
• Organism activity recovers more slowly after a shock load than
in the activated sludge treatment
Rotating disks have been used for wastewater treatment in the
United States only since 1969, but their use is increasing
As is the case with trickling filters, the discs may dry and damage
the microbial population if the system is disrupted for any length
of time. Also, the method cannot operate when ice forms Addi-
tional aeration may be required for waste streams with high
organic loads, because aeration is provided only by disc rotation.
The decision process used to determine if rotating disc treatment
may be applied to hazardous wastes is shown in Figure 5 5
Aerobic and Lagoons and ponds used as bioreactor systems are large basins that
Anaerobic lagoons rely on long waste retention times and both natural and forced
and Ponds aeration for waste decomposition In the aerated basin, the wastes
are artificially aerated with diffused air or mechanical aerators
Basin treatments differ from activated sludge processes in that they
do not recycle biomass Waste lagoons and ponds are more sensi-
tive to high concentrations of inorganic materials and suspended
solids than are other biotreatment methods If there is no mixing,
suspended solids will settle, creating an excessive load and a
sludge blanket on the bottom
The advantages and disadvantages of lagoon treatment of hazard-
ous wastes are listed below For comparison with other methods
discussed in this chapter, see Figure 5 1
5 12
-------
Description ol Biotreatment Options
WILL
SYSTEM YES
BE FREQUENTLY
DISRUPTED?
NO
APPLY PRETREATMENT
WILL SYSTEM
TECHNIQUE OR
BE OPERATING YES —. SELECT
AT FREEZING
TEMPERATURES? ANOTHER
TREATMENT
METHOD
NO _________________
WILL ODORS
YES HIGH ORGANIC
BE LOADS MAY
OBJECTIONABLE? REQUIRE
SUPPLEMENTAL
NO AERATION
PROCEED —
WITH ROTATING — — — —
DISC TREATMENT
_____________ 5Y$TEM CAN HANDLE
HiGH ORGANIC SHOCK LOADS
AND LARGE FLOW VARIATIONS
FIGURE 5.5 Decision Process for Evalualing Rotating Disc Biotreatment
Aerobic and Advantages:
Anaerobic Lagoons
and Ponds • Operating costs are low
(contd) • The treatment is an effective final-phase method for waste
cleanup
• Energy requirements are low
Disadvantages:
• Only low-strength wastes can be treated
• Concentrations of suspended solids and metals must be low
• Requires large areas of land
• Freezing temperatures limit operations
• Flexibility is limited
• Volatile gases may be emitted
• Odors may be a problem
In general, lagoons and ponds can treat only low-strength wastes
Therefore, they should be used only after other treatments have
been applied
5 13
-------
Description of Bsoereatmern Options
Aerobic and Aerated basins may result in greater evaporation of waste
Anaerobic Lagoons compounds, which might impair air quality The large size of most
and Ponds basins, however, provides dilution and buffering of fluctuations in
(contd) waste loads Anaerobic lagoons and ponds are sensitive to waste
composition because anaerobic organisms are sensitive to envi-
ronmental changes The methane-forming bacteria are highly sensi-
tive to pH levels, operating best in a narrow range of pH of 6 8 to
7 5 (Berkowitz, Funkhouser and Stevens 1978) For both aerobic
and anaerobic basins, buildup of sludge may require periodic
shutdown for removal This residue, which may, in itself, be
hazardous, must be disposed of elsewhere at an environmentally
favorable site
The decision process to determine if lagoon and pond systems may
be used to biotransform hazardous wastes is shown in Figure 5 6
ARE
WASTES NO
LOW
STRENGTH?
YES
ARE WASTES
FREE OF NO
SUSPENDED
SOLIDS
AND METALS?
YES
IS A LARGE [ APPLY PRE-TREATMENT
LANDAREA NO I TECHNIQIJEOR
AVAILABLE SELECT A DIFFERENT
FOR WASTE I WASTE TREATMENT
TREATMENT? METHOD
YES
WILL SYSTEM BE
PROTECTED NO
FROM FREEZING
TEMPERATURES?
YES
WILL EMISSION
OF VOLATILE YE
GASES BE
OBJECTIONABLE?
NO
PROCEED
WITH LAGOON
OR POND
TREATMENT
FIGURE 5.6 Deciston Process for Evaluating Lagoon and/or Pond Treatment
5 14
-------
Descript son of Brat reatmenl Options
5.4 BIOACCUMULATION AND REMOVAl
Bioaccumulation occurs because algae and some higher plants can increase
the concentration of certain chemical substances in their cells to a level higher
than the levels in the adjacent environs Sorption of certain pesticides by
aquatic microorganisms and algae is also known to occur (Paris et al 1975)
Mass culture of the photosynthetic organisms (autotrophs) on hazardous
wastes, followed by harvest and disposal, is a possible biological cleanup
technique
The ability of algae to bioaccumu late waste materials is related to their high
adsorptive/absorptive nature, which is a function of their high surface-to-
volume ratio The potential use of algae and higher plants to bioaccumulate
should be considered when confronting certain waste disposal situations
Generally, only some metals and some organic materials can be bioaccumu-
lated . but bioaccumulation often plays a natural role in the environmental
transfer of chemical components (Jenkins 1975)
Little information is available on bioaccumulatson as a method of removing
hazardous wastes from the environment Most interest has been in the use of
algae to absorb excess nutrients (mainly nitrogen and phospherus) in stabilizing
ponds prior to release of treated water to streams or lakes (Fitzgerald and
Rohlich 1964, Sufferin, Fitzgerald and Szluba 1981) One limitation of bioaccu-
mulation is that only certain concentrations can be accumulated (especially of
the heavy metals) before autotrophs are inhibited or killed A number of inter-
acting factors influence the accumulation of heavy metals by plants (Cataldo
and Wildung 1978)
Tolerance of algae and higher plants to toxic metals and organic compounds
varies widely with species and environmental conditions Some chemicals will
inhibit bioaccumulation of other chemicals (Cushing and Rose 1970, Soeder
et al 1975, Hart 1977) Algae and plants can be killed directly by waste constitu-
ents, by waste degradation products and by-products, and by indirect effects
of the wastes on nutrient transfer (Chaney 1981b)
In theory, algae can be grown in bioreactor systems containing liquid wastes
Higher plants can be grown on land treatment sites, on the overburden of
abandoned hazardous-waste disposal sites, or on the site of accidental waste
release In all cases, the concentrations of hazardous wastes must be suffi-
csentl i low so that autotrophs will not be destroyed or their growth inhibited
Because of this requirement, bioaccumulatson is usually a final step in the
cleanup process
The advantages and disadvantages of bicaccumulation treatment of hazardous
wastes are listed below For comparison with other methods discussed in this
chapter, see Figure 5 1
Advantages:
• The method is relatively inexpensive if physical and chemical conditions are
suitable for autotrophic growth
5 15
-------
Description of Biorreatrnent Options
• The method can be used as final cleanup of an abandoned waste site, spill
site, or land treatment site
• The method can be used for pre- and post-treatment monitoring
• Algae and higher plants that bioaccumulate may be used for food, fertilizer,
and other uses if toxic levels do not preclude their use
Disadvantages:
• Waste concentrations cannot exceed levels that are lethal to autotrophs,
thus, the method is restricted to low-concentration wastes
• Use throughout the year is restricted to certain locations, because tempera-
ture and sunlight will affect growth rates
• Depending upon bioaccumulation levels, disposal of harvested biomass
may be a problem
In general, bioaccumulation offers few advantages over conventional waste
treatments However, the tendency of algae and some larger plants to accumu-
late hazardous materials may be of some value if combined with chemical and
physical treatment and with microorganism activities Some bioaccumulation
will be unavoidable at sites containing hazardous materials For example,
plants will grow on contaminated soil and composted materials, leachates will
drain into sumps and algae will grow in the liquid mix, and algae will grow in
waste-treatment lagoons and ponds Where natural bioaccumulation occurs,
human and wildlife access must be limited to control movement of waste
off site
Bioaccumulation by algae and some higher plants for hazardous-waste recov-
ery and removal cannot be used at all sites If used in field situations, the
method is limited to areas with suitable thermal and sunlight regimes present
throughout the year (i e, the sun belt) The method is restricted to those
hazardous wastes that are accumulated by algae and larger plants (some metals
and organic materials), and are not toxic or inhibitory to autotrophic growth.
Determining The decision process to determine if bioaccumulation and removal
Whether or not is a biotreatment option is shown in Figure 5.7. The following
Bioaccumulation information must be obtained to answer the questions in
and Removal is an Figure 5 7
Option
• wastes involved
• concentration of wastes in water or soil
• toxicity of wastes to algae and higher plants
at ambient concentrations
at accumulated concentrations
• environmental characteristics
physical - light, temperature, flow, etc
chemical - nutrient availability, pH, etc
biological - will mass culture be possible, will grazing animals
limit crop?
5 16
-------
Description of B,otreatment Options
FIGURE 5.7 Decision Process for Evaluating Bioaccumulation by Algae and Higher
Plants (Autotrophs)
Determining
Whether or not
Bioaccumulation
and Removal is an
Option
(contd)
• percent of waste removal necessary
• eventual disposal
ease of harvesting
disposition of biomass . Is the problem Just moved from one
place to another?
• accessibility of the site to humans and animals
5.5 ENHANCEMENT OF BIOTREATMENT PROCESSES
The objective of pre-treating hazardous wastes is to render them suitable for
biotransformation The wastes can be pre-treated by physical and/or chemical
methods to reduce their toxic or inhibitory effects on microorganisms, or to
alter their chemical composition to a form suitable for further microbial
transformation
Hazardous wastes may destroy or inhibit the activity of microorganisms The
main hazardous components of complex toxic waste mixtures, which may
occur at disposal sites, are
• heavy metal cations (Sb, As, Cd, Cr, Hg, Pb, Zn, Ni, Cu, V, P. Be, Se, Mn, Ti,
Sn, 13a, etc)
• heavy metal anions (chromates, chromites, arsenates, arsenites, etc)
• non-metallic toxic anions (cyanides, suif ides, thiocyanates, etc)
5 17
-------
Description of B,otreatment Options
S organics (hydrocarbons, organic acids, organic peroxides, esters, alcohols,
aldehydes, phenols, chlorocarbons, amines, analines, pyridines, organic sul-
fur compounds, organic phosphorous compounds, alkaloides, sterols, etc)
The most common form of pre-treatment is mixing with water In field situa-
tions where hazardous materials are dispersed in the soil, mixing occurs from
precipitation or ground-water infusion, or can be accomplished with overhead
sprinkler or underground injection systems Onsite application of water, how-
ever, must avoid contamination of surface-water and ground-watersupplies,
and prevent the spread of toxic materials beyond site boundaries Hence,
depending on ambient conditions, confinement systems may be required
Neutralization and precipitation are two other common practices used to
reduce the toxicity of hazardous wastes Since heavy metals remain in solution
over a certain acidic pH range, neutralization (pH adjustment) and precipita-
tion often occur together
Chemical and physical processes other than dilution, neutralization and pre-
cipitation may be necessary to reduce toxicity of waste materials prior to land
treatment or introduction to bioreactor systems Treatment processes common
to municipal and industrial waste streams are discussed by Landreth and
Rogers (1974), SCS Engineers (1979a), Arthur D Little, Inc (1977), Berkowitz,
Funkhouser and Stevens (1976), NATO (1981), and Shuckrow, Pajak and
Osheka (1981) In addition, chelating agents can be used to aid removal or
detoxification of heavy metals (Jenkins et al 1981, Muramoto 1981)
Potentially useful pre-treatment steps applicable to liquid wastes include
• chemical coagulation - Lime or alum can be added to form precipitates that
scavenge toxic substances such as heavy metals
• carbon sorption - Packed activated carbon can be added to the wastes to
reduce toxicant levels, particularly organics
• ultrafiltration or reverse osmosis - Large molecular compounds, which typi-
cally include the toxic or refractory components can be removed, while
leaving the biodegradable components
• steam stripping - Numerous, low molecular weight organic compounds can
be removed, but most of these are also biodegradable
• aeration, sedimentation, and filtration - Heavy metals can be precipitated to
facilitate removal by sedimentation or filtration.
• chemical oxidation - Certain materials can be detoxified, but most oxi-
dizable materials are readily biodegradable
Since the purpose of biotreatment is to transform materials from hazardous to
innocuous forms, post-treatment is generally not considered However, when
products arising from bioreactor units fail to reach acceptable environmental
standards, post-treatment will be required The need for post-treatment will
be determined through monitoring the treated waste materials (see
Chapter 4)
5 18
-------
Description of B:otreatmeni Options
Considerations for Enhancement should be considered when the biotransformation
Enhancing of rate is otherwise too slow to be practical Microbial degradation of
Biological Processes hazardous wastes can sometimes be enhanced in field situations
and bioreactor systems by adding exogenous microorganisms
(Atlas 1977, Daughton and 1-Isieb 1977, Barles, Daughton and Hsieh
1979, Kobayashi and Rittmann 1982)
Biotransformation processes can be slowed when degradation by
microorganisms is limited by their low numbers or when a waste
inhibits microorganism activity The rate at which microorganisms
degrade hazardous materials depends on their inherent ability and
on the physical and chemical properties of the environs that affect
their growth (viability) Rates of microbiological transformation
may be affected by physical and chemical factors such as the lack
of nutrients or extremes in pH, Eh, temperature, moisture content,
and salinity
Microbial activity in field situations or in a bioreactor system can
be increased by pre-treating the wastes, altering the physical and
chemical environs, and introducing cultured or engineered
microorganisms The waste environment is generally altered by
adding appropriate nutrients, chemically adjusting the pH, or
adjusting the Eh through flooding or cultivation Needs are identi-
fied by site-specific chemical analysis The natural microbial com-
munity is generally augmented by introducing enriched or
adapted microorganisms to degrade a specific waste This process
is sometimes called “biological seeding,” and it involves applying
select cultures of microorganisms to special wastes (SCS Engineers
1979a)
The initial biotransformation of a hazardous waste will usually
involve indigenous microorganisms that occur at the site Whether
or not these organisms can completely degrade the waste to
innocuous compounds will determine if other methods, including
microbial enhancement, should be used
Factors to consider when evaluating the usefulness of enhance-
ment in biotreatment options are grouped in Figure 5 8 The types
and sources of information necessary to answer the questions in
Figure 5 8 are listed throughout the following text
Are natural conditions and indigenous microorganisms suitable to
transform, degrade, and/or adsorb the hazardous wastes? [ see Fig-
ure s ô,(1)J
• Consider the following types and sources of information
- waste concentrations
- site characteristics (surface and ground water, soil, climate,
topography)
- site use characteristics
- local population characteristics
519
-------
YES WILL ADDITION OF
NUTRIENT AND MODIFICATION
OF PHYSICAL AND CHEMICAL
PARAMETERS BE SUFFICIENT?
NO
0
ARE NATURAL CONDITIONS AND
INDIGENOUS MICROORGANISMS
SUFFICIENT FOR TIMELY REMOVAL
OF THE HAZARDOUS WASTE THROUGH
TRANSFORMATION. DEGRADATION. AND/OR
ADSORPTION?
P1
I
ENHANCEMENT WILL FACILITATE
CLEANUP OF HAZARDOUS
WASTE SITE
U i
WILL ADDITION OF
GENERIC ORGANISM
BE SUFFICIENT?
NO
YES
YES
0
CD
0
0
0
CD
CD
0
0
®
WILL ENRICHED YES
MICROORGANISM
BE SUFFICIENT
NO
YES WILL A COMBINATION OF ADAPTED
AND ENRICHED MICROORGANISMS
BE SUFFICIENT?
r BIOLOGICAL ENHANCEMENT
IS NOT FEASIBLE
FIGURE 5.8 Decision Process for Determining if Enhancement is Required for Buotreatment
-------
Description of Bioireatmenr Options
Considerations for • Consider the following sources of information
Enhancing of - published reports (journal articles, technical reports, govern-
Biological Processes ment reports, expert advice)
(contd) - environmental impact assessments
Enhancement of Natural conditions at field locations and in bioreactor systems can
Site-Specific be altered to optimize growth and survival of microorganisms that
Conditions biotransform waste Optimization requires characterization of the
physical and chemical properties of the soil, water, geosubstrate,
ground water or bioreactor system, identification of the factors
that limit transformation rate, and selection of ways to alter factors
that limit microbial growth
Several physical and chemical conditions can influence the density
and composition of a microbial community
• moisture
• aeration
• temperature
• organic matter
• pH
• inorganic nutrients
Moisture—The moisture level influences biotransformation rates in
soils, sediments, and solid-waste treatment systems (composting)
It is not important in liquid media Most aerobic soil bacteria
usually occur when the moisture level is between 50 and 75 per-
cent of the soil’s retention capacity The moisture level may vary in
sediments and solid-waste treatment systems with rainfall and
dehydration Anaerobic systems are generally controlled by keep-
ing the medium saturated with water while limiting aeration
Optimal moisture levels can be maintained by sprinkler and drain-
age systems
Aeration—Aeration is necesary for aerobic biotransformation pro-
cesses Air should be forcibly injected into solid or semi-solid sub-
strates, as in aerobic composting Contaminated surface soils and
sediment can be aerated by regular cultivation Liquid waste
streams are easily aerated by mixing, spilling, air injection, and
other methods
Temperature—Temperature influences microbial activity at all
hazardous waste sites and in all biotreatment processes The
optimal temperature for most bacteria ranges from 25° to 35°C,
but bacteria can multiply at 15° to 45°C (Alexander 1977) Temper-
ature also affects the biotransformation rate For every 10°C
increase in temperature, the rate should increase about twofold
Temperature control is difficult under field conditions Microbial
activity in some situations (e g, composting) generates heat To
estimate the degradation rate, or the time to site recovery, the
5 21
-------
Description of Biotreatment Options
rnhancement of influence of temperature on microorganism activity must be con-
Site-Specific sidered Various procedures are used to maintain warm tempera-
Conditions tures in bioreactor systems
(contd)
Organic Matter—The number (biomass) of soil microorganisms
can, in many cases, be increased by adding organic matter such as
manure, crop residues, forest residues, and some industrial organic
wastes Commercial fertilizers can also be used (Jobson et al 1975)
An increase in biomass, however, may depend on other nutrients
Adding organic matter or nutrients is useful when more microor-
ganisms will increase the biotransformation rate In some cases,
adding organic matter may slow biotransformation activity and,
therefore, inhibit the removal of hazardous components The
value of organic matter or nutrients can often be determined from
simple laboratory studies, where the degradation is examined
under controlled conditions
pH—The growth of microorganisms is usually greatest within a pH
range of 6 to 8 Several methods are available to adjust the pH The
pH of soils, for example, is generally lowered by adding sulfur or
raised by adding lime (Tisdale and Nelson 1966) Most state agricul-
tural experiment stations and soil testing laboratories have quanti-
fied the lime requirements of major soil types in the areas they
serve For land areas contaminated by hazardous materials,
methods used to adjust pH for crops can be used to adjust pH for
indigenous microorganisms
Inorganic Nutrients—In many areas, microbiological transforma-
tion rates are slowed because one inorganic nutrient is absent or is
present in low quantities The nutrient is usually nitrogen or phos-
phorus Microbial activity can be increased by adding the appro-
priate inorganic nutrient
Physical and chemical factors should be considered when deter-
mining if enhancement is required for biotreatment The types of
information necessary to answer Questions (2) and (3) in Figure 5 8
are listed below
Are physical and chemical conditions suitable [ Figure 5 8 (2)]?
• Consider the following types of information
- moisture content
- oxygen content (aerobic conditions require sufficient
aeration, anaerobic conditions require little oxygen)
- temperature
- organic carbon
- pH
- inorganic nutrients
5 22
-------
Description of Bsoireatment Options
Enhancement of Will addition of nutrients and modification of physical and chem-
Site-Specific ical parameters improve microbial activity [ Figure 5 8 (3))?
Conditions
(contd) • Consider the following types of information
- cost
- degradation and transformation rates after modification
Addition of Generic Generic microorganisms are microbes that occur naturally and are
Microorganisms indigenous to the waste, the site, or the biotreatment system
Generic microorganisms are not intentionally adapted or enriched
for a specific waste material Sources of generic microorganisms
include surface soils, sediments, and sewage sludge In general, the
method involves amending the site or the biotransformation pro-
cess with indigenous microorganisms
Generic microorganisms can be used in land treatment or various
bioreactor systems In either application, generic microorganisms
are usually added when an increase in microbial biomass will
increase the degradation rate significantly The procedure is best
when indigenous microorganisms already present can degrade the
hazardous material Generic microorganisms are generally used to
begin biotreatment processes
Will addition of generic orgdnisms be sufficient to degrade or
transform the wastes (Figure 5 8 (4)]?
• Consider the following types of information
- physical and chemical characteristics
- concentrations of the hazardous wastes
- site recovery potential
- site characteristics (surface and ground water, soil, climate,
topography)
- toxic by-products potential
- human populations
- hazardous materials transportability
• Consider the following source of information
- published reports (journal articles, technical reports,
government reports, expert advice)
Culture and Enriched microorganisms are microbes cultured for transforming a
Introduction of specific hazardous waste
Enriched
Microorganisms Biotransformation rates can be inhibited because the microorga-
nisms that degrade a compound are only a small part of the total
microbial community, and because natural pathways leading to
degradation are limited
Increasing the ratio of effective microorganisms to total available
microorganisms can be accomplished by introducing cultures
enriched to transform the waste
5 23
-------
Description of Biotreatment Options
Culture and A microbial community (in soil, sediment, sewage sludge, or else-
Introduction of where) can be supplemented by adding microorganisms devel-
Enriched oped to use a specific waste as the sole source of carbon for
Microorganisms metabolic growth Both batch and continuous culture techniques
(contd) are suitable to grow enriched microorganisms
In batch enrichment, microorganisms are grown in a closed system
such as test tubes or bottles where the concentrations of nutrient
and metabolic products change during incubation Enrichment is
usually initiated by transferring a sample from a complex microbial
community to a culture medium that contains the waste as the sole
source of carbon Microorganisms unable to use the waste for
metabolic energy will disappear Repeated transfer of the culture
to fresh enrichment media after a period of growth will, in many
cases, lead to a stable microbial population capable of, and specifi-
cally enriched for, degrading the target waste
In continuous enrichment, microorganisms are grown in a chemo-
stat (Senior, Bull and Slater 1976), which continuously supplies the
nutrient that limits growth and removes metabolic products By
using the target waste as the nutrient that supplies carbon) a stable
microbial community may develop that will biotransform the
target waste
Several techniques can be used to increase biotransformation rates
of previously enriched cultures, to develop enriched cultures for
resistant organic compounds) or to apply in situations where nor-
mal enrichment procedures are unproductive Attempts to
increase the activity of enriched microorganisms in cultures have
been made by mutating an enriched culture and repeating the
enrichment process The strains that demonstrate an increased bio-
transformation rate are then identified (Wong, Leong and Dunn
1978)
When direct enrichment is difficult, pre-enrichment with related
organic substrates may aid the process An enrichment culture for
the specific organic substrate can then be prepared (Brilon et al.
1981)
A more elaborate procedure is plasmid-assisted molecular breed-
ing This is a recently developed technique (Kellogg, Chatterjee
and Chakrabarty 1981, Chatterjee, Kilbane and Chakrabarty 1982)
used to evolve a strain of bacteria that rapidly degrades 2, 4, 5-
trichlorophenoxyacetic acid (2,4,5-1) The technique is a modifica-
tion of the continuous culture (chemostat) enrichment procedure
The initial bacterial inoculum (soil) sediment) sewage sludge) is
amended with specific bacterial strains
The new strains contain plasmid-encoded pathways that may
evoke degradation of the target organic substrate Enrichment
5 24
-------
Description of Biotreatment Options
Culture and begins with a low concentration of substrate and a high concentra-
Introduction of non of a plasmid substrate Over the culture period, the concen-
Enriched tration of the plasmid substrate is lowered and the concentration
Microorganisms of the target substrate is raised. If successful, a strain of the
(contd) microorganism or a mixed microbial population capable of degrad-
ing the target waste will be produced Enriched cultures are not
readily available, and must be developed under laboratory
conditions
Initial efforts have been made by genetic engineering to produce
microorganisms that degrade specific organic mixtures such as oils
(Atlas 1977) These efforts to date have not produced microorga-
nisms that can degrade organic compounds when mixed microbial
populations (normally associated with natural systems or biotreat-
ment processes) are present However) genetic engineering, which
includes the biochemistry of gene splicing, microbial genetics, and
gene amplification, shows future promise
Enrichment and adaptation are not mutually exclusive In many
instances, both processes occur simultaneously For example,
when the substrate is changed) both adaptation and enrichment of
the microorganisms take place
Enriched cultures can be used as the initial inoculum for starting
various bioreactor systems In some cases, they can be used to
enhance biotransformation processes at field locations where
hazardous materials are present Enriched cultures are generally
used when the hazardous material is not degraded within suitable
time limits by generic (indigenous) microorganisms.
Will addition of enriched microorganisms be sufficient to degrade
the waste [ F igure 5 8 (5)]?
• Consider the following types of information
- physical and chemical characteristics
- hazardous-waste concentrations
- commercial availability of enriched culture
- potential for site recovery
- time necessary to prepare enriched culture
- site characteristics (surface and ground water, soil, climate,
topography)
- toxic by-products
- local human populations
- government regulations for use of enriched microbiological
cultures
- safety considerations (worker health and safety,
environmental)
- transportability of hazardous material
5 25
-------
Oescript ion of Biorreatmen Options
Culture and Adapted microorganisms are microbes cultured for site-specific
Introduction of physical and chemical conditions
Adapted
Microorganisms The activity of indigenous or enriched microorganisms at a
hazardous-waste site can be limited by nutrients, extremes in the
environment (e g , pH, Eh, temperature, salinity), or toxic compo-
nents (e g , trace metals, organic compounds, and degradation
products) Any one of these factors may depress microbial activity
If limiting conditions occur, microbiological growth can be
enhanced by either altering physical and chemical conditions (see
Enhancement of Site-Specific Conditions, p 5 21) or by introduc-
ing organisms specifically adapted to site-specific conditions
Adapted microorganisms are generally used when the wastes are
toxic to microorganisms or physical and chemical enhancement of
the site is not possible
A microbial population can be adapted by incrementally adjusting
the environs over time to the chemical and physical conditions
desired The initial microorgaisms are generally obtained from
soils, sediments, sewage sludge, and/or enrichment cultures Like
enrichment, adaptation is usually conducted in either batch or
continuous culture (chemostat) incubation systems
Batch adaptation may require gradual transfer of microorganisms
from an unadapted culture to a culture representing the desired
physical and chemical conditions When adaptation is difficult,
samples can be transferred through a series of batch cultures in
increments that approach final conditions In the latter situation,
each batch culture is allowed to reach a predetermined growth
level before a sample is transferred to the next incremental level
Complete adaptation occurs when the microorganisms actively
grow on a medium simulating physical and chemical conditions of
the waste site
Continuous culture (chemostat) systems have also been used for
adaptation By gradually altering the initial growth medium, condi-
(ions can be changed to the desired parameters A culture is
adapted if it actively grows when the target conditions are
attained Adapted cultures are not readily available and must be
developed under laboratory conditions
Adaptation and enrichment within a culture occur primarily by
two processes Both processes select microorganisms that can sur-
vive and increase as cultural conditions are changed These pro-
cesses also select for spontaneous mutations tha occur in a
cultured population In some cases, the mutants can grow under
the target conditions In this regard, adaptation may be enhanced
by inducing mutations among cultured microorganisms (Wong,
Leong and Dunn 1978) Mutations are induced conveniently by the
batch adaptation system
5 26
-------
Description of B,otreatrnenf Options
Culture and Adaptation and enrichment are not mutually exclusive In many
Introduction of instances, both processes occur simultaneously. For example,
Adapled when the substrate is changed, both adaptation and enrichment of
Microorganisms the microorganisms take place.
(cont Adapted microorganisms can be used as the initial inoculum for
starting various bioreactor systems In some cases, they can be
used to enhance indigenous biotransformation processes at aban-
doned hazardous-waste sites or accidental-release sites The use of
adapted cultures is generally considered when the hazardous
material is not degraded within suitable time limits by generic
(indigenous) microorganisms
Will addition of adapted microorganisms be sufficient to degrade
the wastes fFigure 5 8 (6)J?
• Consider the following types of information
- physical and chemical site characteristics
- hazardous-waste concentrations
- availability of adopted culture
- potential for site recovery
- time to prepare culture
- site characteristics (surface and ground water, soil, climate,
topography)
- toxic by-products
- local human populations
- government regulations
- safety of adapted culture (worker health and safety,
environmental)
Culture and Will a combination of adapted and enriched microorganisms be
Introduction of sufficient to degrade the waste [ Figure 5 8 (7)]?
Adapted and
The information to answer this question is similar to that given for
Enriched
adapted microorganisms [ Figure 5 8 (6) ] and enriched microorga-
Microorganisms
nisms [ Figure 5 8 (5)]
5.6 CHAPTER REFERENCES
References in addition to those cited in the text are provided
Biotreatment Alexander, M 1973 “Biotechnology Report Nonbiodegradable
Options and Other Recalcitrant Molecules “ Biotechno! Bioengr
15 611-647
Alexander, M 1977 Introduction to Soil Microbiology 2nd ed
John Wiley and Sons, New York, p 467
Alexander, M 1981 “Biodegradation of Chemicals of Environ-
mental Concern “Science 211 132-138
5 27
-------
Description of Biotreatment Options
Biotreatment American Public Health Association (APHA), et al 1980 Standard
Options Methods for the Examination of Water and Wastewater, Vol 15
(contd) APHA, Washington, D.C , p 1193
Arthur D Little, Inc 1977 Physical, Chemical and Biological
Treatment Techniques for Industrial Wastes, Vol I P8-275 054,
NTIS, Springfield, Virginia
Atlas, R M 1977 “Stimulated Petroleum Biodegradation “CRC
Cnt Rev Microbiol 5 371-386
Barles, R W , C G Daughton and D P H Hsieh 1979
“Accelerated Parathion Degradation in Soil Inoculated with
Acclimated Bacteria under Field Conditions “Arch Environ
Contam Toxico! 8647-660
Berkowitz, J B , J T Funkhouser and J I Stevens 1978 Unit
Operations For Treatment of Hazardous Industrial Wastes Noyes
Data Corporation, Park Ridge, New Jersey, p 920.
Brilon, C., et al 1981 “Enrichment and Isolation of Naphthalene
Sulfonic Acid-Utilizing Pseudomonads “Appi Environ Microbiol
42 39-43
Brown, K W, and L Deuel 1980 Hazardous-Waste Land
Treatment PB81-182 107, NTIS, Springfield, Virginia
Burnside, 0 C 1974 “Prevention and Detoxification of Pesticide
Residues in Soils” In Pesticides IQ Soil and Water, ed. W D.
Guenzis, pp 387-412 Soil Science Society of America Inc,
Madison, Wisconsin.
Callahan, M A, et al 1979 Water Related Environmental Fate of
129 Priority Pollutarns, Vol I and II EPA - 440/4-79-029a and EPA-
440/4-79-029b, U S Environmental Protection Agency, Washington,
DC, PB-80-204 373 and PB-80204-381, NTIS, Springfield, Virginia
Cataldo, 0 A , and R E. Wildung 1978 “Soil and Plant Factors
Influencing the Accumulation of Heavy Metals by Plants “ Environ
Health Persp 27 149159
Chaney, R L 1981a “Review and Preliminary Studies of Industrial
Land Treatment Practices” In Proceedings of the Seventh Annual
Researach Symposium on Land Disposal Hazardous Wastes, ed.
D W Schultz EPA, Cincinnati, Ohio
Chaney, R L 1981b “Incorporation of Hazardous Wastes Into
Soil—A Method for Treatment “Hazardous-Waste Management
Seminar, Edmonton, Alberta, November 3, 1981
Chaney, R L “Foodchain Pathways for Toxic Metals and Toxic
Organics in Wastes” Fourth Life Sciences Symposium, Oak Ridge
National Laboratory, Ann Arbor Science Pubis, Ann Arbor,
Michigan (in press)
5 28
-------
Description of Biotreatment Options
Biotreatment Chapman, P M , G P Romberg and C A Vigers. 1982 “Design of
Options Monitoring Studies for Priority Pollutants “J Water Pollut Cont
(contd) Fed 54 292 297
Chatter;ee, D K , J J Kilbane, and A M Chakrabarty 1982
“Biodegradation of 2,4,5-trichiorophenoxyacetic Acid in Soil by a
Pure Culture of Pseudomonas cepacsa “AppI Environ Microbiol
44514-516
Chesters, C, H B Pionke and T C Daniel 1974 “Extraction and
Analytical Techniques for Pesticides in Soil, Sediment, and Water
In Pesticides in Soil and Water, ed W D Guenzi, pp 451-550 Soil
Science Society of America Inc. Madison, Wisconsin
Cushing, C E, and F I Rose 1970 “Cycling of Zinc-65 by
Columbia River Periphyton in a Closed Lotic Microcosm “Limnol
Oceanogr 15 762-767
Daughton, C G , and D P H Hsieh 1977 “Accelerated Parathion
Degradation in Soil by Innoculation with Parathion-Utilizing
Bacteria “Bull Environ Contam Toxicol 18.48-56
DeWalle, F B, E S K Chian and J Brush 1979 “Heavy Metal
Removal with Completely Mixed Anaerobic Filter” Water Pollut
Cont Fed 51 22-36
EPA 1980 “Hazardous-Waste Management System Standards for
Owners and Operators of Hazardous-Waste Treatment, Storage,
and Disposal Facilities “Federal Register 45(98) 33154-33258
EPA 1982 Remedial Action at Waste Disposal Sites EPA-625/6-82-
006 U S Environmental Protection Agency
Epstein, E, and R L Chaney 1978 “Land Disposal of Toxic
Substances and Water - Related Problems I Water Pollut Corit
Fed 50 2037-2042
Fitzgerald, C P, and C A Rohlich 1964 “Biological Removal of
Nutrients from Treated Sewage Laboratory Experiments” Verh
Internat Verein Limnol 15.597-608
Cloyna, E F , and D I Ford 1970 Petrochemical Effluents
Treatment Practices Detailed Report PB-211 464, NTIS,
Springfield, Virginia
Gomaa, H M, and S D Faust 1974 “Removal of Organic
Pesticides from Water to Improve Quality” In Pesticides in Soil
and Water, ed W D Cuenzi, pp 413-450 Soil Science Society of
America Inc , Madison, Wisconsin
Hart, B A 1977 The Role of Phytoplankton in Cycling Cadmium in
the Environment U S Department of Commerce, PB-282 364,
NTIS, Springfield, Virginia
5 29
-------
Description of Biotreatment Options
Biotreatment Helling, C S ., P C Kearney and M Alexander 1971 “Behavior of
Options Pesticides in Soils “Advances in Agronomy Academic Press Inc,
(conid) New York,New York, p. 407
Hiltbold, A E 1974 “Persistence of Pesticides in Soil “ In Pesticides
in Soil and Water, ed W D Guenzi, pp 203-222 Soil Science
Society of America mc, Madison, Wisconsin
Jenkins, D W 1975 Biological Monitoring for Environmental
Pollutants U S Environmental Protection Agency, Cincinnati, Ohio
Jenkins, R L , et al 1981 “Metals Removal and Recovery from
Municipal Sludge “ I Water Pollw Cont Fed 53 25-32
Jobson, A , et al 1975 “Effect of Amendments on the Microbial
Utilization of Oil Applied to Soil “AppI Microbiol 27 166-171
Kaufman, D D 1974 “Degradation of Pesticides by Soil Micro-
organisms” In Pesticides in Soil and Water, ed W D Guenzi,
pp. 133-202 Soil Science Society of America Inc., Madison,
Wisconsin
Kellogg, 5 1, D K Chatterjee and A M Chakrabarty 1981
“Plasmid-Assisied Molecular Breeding New Technique for
Enhanced Biodegradation of Persistant Toxic Chemicals “Science
214 1133-1134
Kobayashi, H , and B E Rittmann 1982 “Microbial Removal of
Hazardous Organic Compounds” Environ Sci Technol
16 170A-182A
Landreth, R E , and C J Rogers 1974 Promising Technologies for
Hazardous Wastes EPA-670/2-74-088, U.S Environmental
Protection Agency, Cincinnati, Ohio, PB-238 145, NTIS, Springfield,
Virginia
Letey, J , and W J Farmer 1974 “Movement of Pesticides in Soil”
In Pesticides in Soil and Water, ed W D. Guenzi, pp 67-97 Soil
Science Society of America Inc , Madison, Wisconsin.
Muramoto, 5 1981 “Influence of Complexants (EDTA, DPTA) on
the Toxicity of Cadmium to Fish at Chronic Levels” Bull Environ
Contam Toxicol 2&641-
McCarty, P L , M Reinhardt and B E Rittmann 1981 “Trace
Organics in Groundwater “ Environ Sci Technol 1540-51
Metry, A A 1980 The Handbook of Hazardous Waste Manage-
ment Technomic Publishing Co , Westport, Connecticut, p 446
North Atlantic Treaty Organization 1981 NATO - CCMS Pilot
Study on Disposal of Hazardous Wastes Chemical, Physical and
Biological Treatment of Hazardous Wastes in NATO Countries
Report of Committee on Challenges of Modern Society PB82-114
539, NTIS, Springfield, Virginia, p 139
5 30
-------
Description of Biot real ment Options
Biotreatment Overcash, M R , ed 1981 Decomposition of Toxic and Nontoxic
Options Organic Compounds in Soils Ann Arbor Science, Ann Arbor,
(contd) Michigan, p 455
Overcash, M R , and D Pal 1979 Design of Land Treatment sys-
tems for Industrial Wastes—Theory and Practice Ann Arbor
Science, Ann Arbor, Michigan, p 684
Paris, D F , et al 1975 Microbial Degradation and Accumulation of
Pesticides in Aquatic Systems EPA-660/3-75-007, U S Environ-
mental Protection Agency, PB-241 293, NT 1S, Springfield, Virginia
Parr, J F , 1974 “Effects of Pesticides on Microorganisms in Soil
and Water” In Pesticides in Soil and Water, ed W D Guenzi, pp
315-340 Soil Science Society of America mc, Madison, Wisconsin
Saegar, V W , and Q E. Thompson 1980 “Biodegradability of
Halogen-Substituted Diphenylmethanes “ Environ Sc:. Technol
14 705-709
Schaefer, M 1980 Controlling Hazardous Wastes, Research Sum-
mary EPA-600/8-80-017, U S Environmental Protection Agency,
Cincinnati, Ohio, PB8O-194202, NTIS, Springfield, Virginia
SCS Engineers 1979a Selected Biodegradation Techniques for
Treatment and/or Ultimate Disposal of Organic Materials EPA-
600/2-79-006, U S Environmental Protection Agency, Cincinnati,
Ohio, P8-295 394, NTIS, Springfield, Virginia
SCS Engineers 1979b Disposal of Dilute Pesticide Solutions U S
Environmental Protection Agency, Cincinnati, Ohio, P6-297 985,
NTIS, Springfield, Virginia
Senior, E , A T Bull and J H Slater 1976 “Enzyme Evolution in a
Microbial Community Growing on the Herbicide Dalapon”
Nature 263 476-479
Shuckrow, A J , A P Pajak and C J Touhill 1980 Management of
Hazardous Waste Leachate U S Environmental Protection Agency,
SW 871, Cincinnati, Ohio
Shuckrow, A J , A P Pajak and J W Oshaka 1981 Concentration
Technologies for Hazardous Aqueous Waste Treatment EPA-
600/2-61-019, U S Environmental Protection Agency, Cincinnati,
Ohio, P881-150583, NTIS, Springfield, Virginia
Sikora, I J , et al 1980 “Metal Uptake by Crops Grown Over
Entrenched Sewage Sludge “I Ag Food Chem Nov-Dec
1980 1281-1285
Simon-Sylvestre, G , and J C Fournier 1979 “Effects of Pesticides
on Soil Microflora “Adv in Agronomy, Vol 31 Academic Press,
Inc, New York, New York, p 315
5 31
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Description of Biotreatmeni Options
Biotreatment Soeder, C J , et al 1978 Sorption and Concentration of Toxic
Options Minerals by Mass Cultures of Chlorococcales “ Mitt Internal
(contd) Verein Limnol 21 575-584
Sufferin, J 5, C M Fitzgerald and A I Szluha 1981 “Trace Metal
Concentrations in Oxidation Ponds “J Water Pollut Cont Fed
53 1599-1608
Tabak, H H., et al 1981 “Biodegradability Studies with Organic
Priority Pollutant Compounds “J Water PoIIut Cont Fed
53 1503-1518
Tisdale, S 1, and W I Nelson 1966 Soil Fertility and Fertilizers
The MacMillan Co, London, p 694
Wainwright, M. 1978 “A Review of the Effects of Pesticides on
Microbial Activity in Soils “J of Soil Sci 29 287-298
Weber, J B , and S B Weed 1974. “Effects of Soil on the Biological
Activity of Pesticides” In Pesticides in Soil and Water, ed W D
Guenzi, pp. 223-256 Soil Science Society of America Inc., Madison,
Wisconsin
Wentsel, R S , et al 1981 Restoring Hazardous Spill-Damaged
Areas Technique Identification/Assessment EPS-600/2-81 -208,
Municipal Environmental Research Laboratory, Cincinnati, Ohio,
PBB2-193 870, NTIS, Springfield, Virginia
Wilson, G B, et al 1980 Manual for Composting Sewage Sludge
by the Beltsville Aerated-Pile Method EPA-600/8-80-022, U S Envi-
ronmental Protection Agency, Cincinnati, Ohio
Wilson, C B , et al 1982a “Land Treatment of Industrial Wastes
Principles and Practices, Part 1” Bsocycle 1982 37-42
Wilson, G B., et al. 1982b “Land Treatment of Industrial Wastes
Principles nd Practices, Part II. ” Biocycle 1982.59-61
Wong, C 1, R W M Leong and N W. Dunn. 1978 “Mutation to
Increase Resistance to Phenol in Pseudomonas putida “Biotech
Bioengr 20 917-920
Wood, J M 1982 “Chlorinated Hydrocarbons Oxidation in the
Biosphere “ Environ Sci Technol 16291A-297A.
Wyss, A W , et al 1980 Closure of Hazardous Waste Surface
Impoundments SW-873, U S Environmental Protection Agency,
Cincinnati, Ohio, PP81-166 894, NTIS, Springfield, Virginia
5 32
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6.0 BIOTRANSFORMATION CONCEPTS
This chapter reviews information on mechanisms by which microorganisms
degrade or transform hazardous materials It also describes the functions and
limitations of microorganisms for biotreatment, Users of this guide with
limited backgrounds in microbiology may benefit from this overview of the
concepts
A combination of natural physical, chemical, and biological processes usually
interact to transform hazardous materials in the environment (Rogers and
Landreth 1975, Alexander 1981, Kobayashi and Ruttmann 1982) Of these, the
most important appear to be biological In some instances, physical and chem-
ical methods can be used to aid or enhance biological transformations (Fields
and Lindsey 1975, MacGregor and Metry 1977, Epstein and Chaney 1978, SCS
Engineers 1979b, Wyss, Willard and Adams 1980, Shuckrow, Pajak and Osheka
1981, North Atlantic Treaty Organization 1981)
Biological transformation is caused by the enzymatic activity of indigenous
microorganisms or, in some cases, by microorganisms specifically cultured or
genetically engineered Current evidence suggests that indigenous microbial
populations, particularly heterotrophic bacteria and fungi, are the chief agents
for changing chemical molecules in waters and soils
6.1 ROLE OF MICROORGANISMS IN BIOTRANSFORMATION
Microbial activity can reduce or eliminate hazardous materials by
• aerobically or anaerobically degrading them into innocuous forms
• aerobically degrading them to carbon dioxide and water (mineralization)
• anaerobically decomposing them to carbon dioxide and methane.
Some chemical compounds, however, ate not removed efficiently by existing
biological treatment techniques, either because they are metabolized very
slowly or because they are resistant to microbial degradation under prevailing
conditions in the environs (Kobayashi and Rittmann 1982)
Microorganisms are ubiquitous They induce biotransformation of natural and
synthetic compounds at rates that vary with environmental conditions and
compound structure Microorganisms that transform specific compounds can
be isolated, and many can be cultured, enriched, or adapted in the laboratory
Microorganisms may also be genetically engineered to transform certain com-
pounds The basis of biotransformation i the enzyme pathways of these
microorganisms, which can lead to appreciable changes in the molecular
structure of most organic and some inorganic compounds Nonenzymatic
reactions also occur, but they generally hold a lesser role in breaking down
synthetic compounds
Several groups of microorganisms, listed below in order of importance, bio-
transform hazardous materials
61
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Biotransformation Concepts
• heterotrophic bacteria
• fungi
• anaerobic bacteria
• actinomycetes
• phototrophic microorganisms
• oligotrophic bacteria
Each group has special characteristics that are useful for detoxifying
hazardous-waste materials These characteristics are summarized in Table 6 1
Heterotrophic Decomposition of synthetic organic compounds by heterotrophic
Bacteria bacteria is the most commonly studied biotransformation process
Characteristically, bacterial species are not identified individually
but are listed only by source of the inoculum (e g , sewage or soil)
In most cases a mixed population of species is involved The most
common heterotrophic bacteria isolated from areas contami-
nated by different organic compounds are represented by the
genera Pseudomonas, Achromobacter, Arthrobacter, Micrococcus,
V,br,o, A cinetobacter, Brevibacterium, Corynebactersum, and
Flavobacterium Corynebacteria spp are believed to be major
agents for breakdown of hydrocarbons and heterocyclic com-
pounds in aqueous environments Pseudomonas spp are ubiqui-
tous and able to biotransform many different synthetic
compounds For example, they are adaptable to biotransform dif-
ferent chlorobenzene isomers through plasmid transfer of genetic
material
Fungi Fungi have considerable ability to degrade or transform hydrocar-
bons of complex structure or long chain length Bacteria and
yeasts, on the other hand, show decreasing abilities to degrade
alkanes with increasing chain length Organisms in two orders of
fungi—Mucorales (such as Cunninghamella) and Moniliales
(Fusarsum, Aspergillus, and PenicsII:um)—s how high biotransfor-
mation potential For example, Fusarium oxysporum has the ability
to completely degrade DDT, no other microorganism with this
ability has been detected Because filamentous fungi and yeasts
have nonspecific enzyme systems for aromatic compounds, they
are believed to be more capable of degrading polychlorinated
biphenols (PCB5) than are bacteria However, fungus metabolism
often results in incomplete breakdown of a chemical structure In
this case, subsequent association with bacteria is necessary to
achieve mineralization
Anaerobic Bacteria Anaerobic transformation of organic matter to carbon dioxide and
methane involves groups of strictly anaerobic bacteria Little is
known about species composition and the changes that occur dur-
ing biotransformation However, evidence suggests that at least
four interacting groups of bacteria are involved
62
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8 ,otransIormauon Concepts
TABLE 6.1 A Summary of Microorganisms Involved in Biotransformation of Chemical
Compounds and Their SeIecti e Characteristics(a)
Microorganism
Selective Characteristicx(bf
Significance
Algae
Cyanobacteria (formerly
called blue-green algae)
Bacteria
l-teterotrophs faerobrcf
Anaerobic (fastidious)
Facultative anaerobes
Photosynthetic bacteria
Purple sulfur
Purple nonsulfur
Actinomycetes
Olugotrophs (may be from
almost any group above)
pH <5, ae-mae, high O iension,
pH C 5, moisture about 50%
ae-mae, light 600-700 nm, low
carbon flux
ae-mae. an, light 600-700 nm,
low carbon flux
ae. proper organic substrate,
gro sth factors as required, Eh
045 toO 2 V
an,Eh <-O2to-04V
mae-an, (h C-02V
an flight), mae (dark), Eh Oto
-02V, S 2to 8mM,04-1 mM,
light 800-890 nm at 1000-2000 lux,
high intensities near limit, low C
flux
an) Eh 0 to -02V, light 800-
890 nm, low carbon flux
ae, moisture 80-87%, temp
23-28°C, urea as nitrogen source
ae, carbon flux of Cl mg/Lid,
favorable attachment sites
Attacks and partially degrades complex
compounds not readily metabolized by other
organisms Wide range of nonspecific enzymes
Self-sustaining population, light is primary
energy source, partially degrades certain
complex compounds, photochemical reactions,
oxygenates effluent, supports growth of other
microbes, no aeration needed, effective in
bioaccu mulation of hydrophobic substances
See algae
For many compounds, degradation is more
complete and faster than under anaerobic
conditions High sludge production
Conditions for abtotic or biological reductive
dechlorination, certain detoxification reactions
not possible under aerobic conditions, no
aeration, little sludge produced
No aeration necessary, reductive dechlorination
possible
Self-sustaining population able to use light
energy, conditions right for reductive dechlori-
nation, no aeration
See purple sulfur bacteria, also nonspecific
enzymes
Universal scavengers with range of complex
organic substrates often not used by other
microbes
Removal of organic contaminants in trace
concentrations, many inducible enzymes for
multiple substrates
(a) From Kobayashi and Rittmann 11982)
(b) Possible characteristics for selection, not growth range
ae aerobic, mae = m croaerophilic (<02 atm oxygen), an = anaerobic
Fungi
Yeast
Mold
63
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Biotransformation Concepts
Anaerobic Bacteria • hydrolytic bacteria, which break down major biomass compo-
(conid) nents such as saccharides, proteins, and lipids
• H 2 -producing, acetogenic bacteria, which break down products
such as fatty acids and neutral end-products produced by
hydrolytic bacteria
• homoacetogenic bacteria, which break down multicarbon
compounds to acetic acid
• methanogenic bacteria, which use hydrogen and carbon
dioxide for growth
Anaerobic bacteria require oxygen-free conditions and oxidation-
reduction potentials of less than -o 2 V These organisms occur in
anaerobic sediments, in sewage sludge, and in stomachs (rumen)
of cattle The role of strictly anaerobic organisms in biotransforma-
tion is indicated by several detoxification reactions that occur in
the rumen of cattle These reactions include reductive dechlorina-
tion (or dehalogenation), a possible limiting factor in degradation
of certain compounds, nitrosamine degradation, a removal mech-
anism for a suspect carcinogen, reduction of epoxide groups in
various compounds to olefins, reduction of nitro groups, as found
in nitrophenol, and breakdown of aromatic structures
In addition to strict anaerobes, bacteria capable of living in anoxic
but not necessarily reducing environs, and bacteria that are both
aerobic and anaerobic, are important to biotransformation pro-
cesses These organisms are abundant in soil and sewage, and
occur at sites where reductive chlorination occurs
Actinomycetes Actinomycetes often occur in association with unusual chemical
compounds They can attack numerous complex organic materials
such as phenols, pyridines, glycerides, steroids, chlorinated and
nonchlorinated aromatic compounds; and even lignocellulose,
which very few microorganisms attack
The most common aquatic actinomycete is Nocardia spp , which
typically feed on lipids in activated sludge Actinomycetes can
grow under low nutrient levels (e g., in distilled water), tolerate a
wide temperature range, resist dessication, and exist under a wide
range of pH
Organic decomposition by actinomycetes generally results in
metabolites that can be mineralized in the presence of other
microorganisms Hence, mixed culture systems are necessary when
actinomycetes are used The number of nitrogenous compounds
that can be used by actinomycetes is limited, due to low cell syn-
thesis, most of the nitrogen in the substrate is liberated as ammo-
nia Low cell synthesis also tends to keep the populations of
actinomycetes low under natural conditions Actinomycetes may
be especially useful in treatment of contaminated soil where com-
posting would be practical
64
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Biot ran5(ormation Concepts
Phototrophic Algae, cyanobacteria (blue-green algae), and photosynthetic bac-
Organisms teria are phototrophic microorganisms Since they obtain energy
from sunlight and carbon by carbon dioxide fixation, they are able
to exist on substrates low in nutrients In addition, some cyanobac-
teria and photosynthetic bacteria are able to fix nitrogen and sur-
vive in soils where low nitrogen levels would not otherwise
support bacterial growth
In general, phototrophic organisms promote partial rather than
complete degradation, hence, interactions with other microorga-
nisms are important The prolific growth of phototrophs will pro-
mote the growth of heterotrophic organisms, which feed on the
metabolic products formed by the phototrophs Algae can readily
biotransform various synthetic compounds if proper densities of
bacteria are maintained (O’Kelley and Deason 1976, Matsumura
and Esaac 1979)
In addition to their capacity to break down organic materials, pho-
totrophic organisms are potentially useful because they can bioac-
cumulate hydrophobic compounds Bioaccumulation is a feature
shared by many different micro- and macroorgantsms Bioaccumu-
lation by phototrophs is increased because they can sustain rela-
tively large populations without needing organic matter in
concentrations high enough to serve as carbon and electron
donors In addition, some algae (especially Ch lorella and certain
diatoms) can be manipulated physiologically to develop large lipid
stores in which hydrophobic compounds are sequestered
Cyanobacteria in general, and the green algae Ch lorefla in particu-
lar, are tolerant of pollution and low concentrations of dissolved
oxygen Dunaliella can tolerate a wide range of salinity and can be
used where salt concentrations vary Some phototrophs, particu-
larly some species of cyanobacteria, appear to be easier to manipu-
late genetically than bacteria
Photosynthetic bacteria can metabolize numerous substances,
including simple sugars, alcohols, volatile fatty acids, tricarboxylic
acid (TCA) cycle intermediates, benzoates, and 1,3,5-
trihydroxybenzene These bacteria produce a range of inducible
enzymes, and they are widely used in wastewater processes that
treat a variety of organic compounds
Chromatiacae tolerate pollution and occur in waste-treatment
lagoons Rhodopseudomonas capsulata, a purple nonsulfur
bacteria, can degrade nitrosamines (tumor-inducing agents) to
innocuous compounds Some purple nonsulfur organisms are able
to grow anaerobically as phototrophs, but also can live aerobically
in the dark as heterotrophs This characteristic is typical of photo-
synthetic bacteria
Phototrophic microorganisms, because they are easy to grow selec-
tively, can grow under anaerobic conditions if light is available.
65
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Biotransformation Concepts
They normally occur, however, in only small numbers in waste-
water treatment systems
Oligotrophic Oligotrophic bacteria are microorganisms that can live under con-
Bacteria ditions of low carbon flux (less than one milligram of carbon per
liter per day) These organisms do not represent a taxonomic
group, but exist in almost any group of bacteria other than
chemotrophs Oligotrophs are obtained only from low nutrient
environments, oligotrophs can be readily adapted to high nutrient
levels, but readaptatron to low nutrient levels is difficult
Oligotrophs generally have a high surface-to-volume ratio and
high affinities for substrate They appear to prefer attachment to a
free-living existence and, thus, are common in biofilms An impor-
tant feature of oligotrophs is that they often appear to have multi-
ple inducible enzymes, can shift metabolic pathways, and can take
up and use mixed substrates One species of Clostridsum
can grow on at least 20 different substrates
Oligotrophs can potentially be used to remove trace concentra-
tions of organic contaminants from water, treat effluent from waste-
water treatment processes, or treat leachate from hazardous-waste
disposal sites Unlike bacteria, which cannot grow and live under
low-nutrient conditions, certain oligotrophs such as species of
Actinomycetes (Nocardia), Coryneforms, and Mycobacteria can
control their metabolism to survive as long as 30 days when no
nutrients are available
6.2 BIOTRANSFORMATION PROCESSES
Microorganisms are noted for two biochemical processes involving energy
transfer (metabolism)
• assimilation (anabolism) - where organic compounds are synthesized from
CO 2 . water, and other components; this process is endothemic and primar-
ily reductive
• dissimilation (catabolism) - where organic compounds are decomposed to
CO 2 and water, or partially decomposed to lower molecular weights (e g,
alcohols to aldehydes or acids), this process is exothermic, and involves
oxidation or reduction
The organisms that assimilate are autotrophic, while those that dissimilate are
heterotrophic Autotrophic organisms may assimilate photosynthetically with
sunlight for energy, or chemosynthetically with chemical reactions for energy,
whereby inorganic elements are oxidized Heterotrophic organisms obtain
energy from the decomposition of organic materials
Most degradation or transformation of organic compounds in the environ-
ment is done by heterotrophic microorganisms Heterotrophs may be either
aerobes, obligate anaerobes, or facultative anaerobes Aerobes require
molecular oxygen for respiration and synthesis, obligate anaerobes require the
66
-------
Bioiransformaicon Concepts
complete absence of oxygen, facultative anaerobes can grow either in the
presence or absence of oxygen Aerobic synthesis is faster than anaerobic syn-
thesis and has a higher growth yield of cells that produces more inert materials
(e g , sludge)
Dissimulation of organic compounds by heterotrophic microorganims can
occur by
• mineralization
• co-metabolism
Mineralization pathways characterize the microbial breakdown of several
classes of synthetic compounds (Alexander 1981) As organic material is con-
verted to inorganic material, the responsible microorganisms use some of the
substrate carbon and release enough energy to synthesize cell constituents for
growth Thus, mineralization is typically a growth process for microorganisms
Detoafication of a chemical is a common outcome of mineralization, but
sometimes the mineralized product (e g, nitrate or sulfide) is also of environ-
mental concern
Co-metabolism (co-oxidation) occurs when microorganisms are able to trans-
form a chemical compound without using it as a source of nutrients ( 1- lorvath
1972, Alexander 1981, Morrill, Mahilum and Mohuiddin 1982, Kobayashi and
Rittmann 1982) In other words, the microbial population is growing on one
substrate while performing a biotransformation on another
A number of important differences exist between mineralization (direct trans-
formation) and co-metabolism (indirect transformation) of a chemical
compound
Mineralization Co-metabolism
Microorganisms increase in No increase in numbers and
numbers and biomass biomass
Microorganisms transform waste Waste material is transformed
material directly indirectly
Waste transformation is rapid, rate Transformation is slow, rate does
increases with time not increase with time
Waste breaks down completely Waste breakdown is incomplete to
structurally similar compounds
Some of these relationships are shown in Figure 6 1
Mineralization and co-metabolism represent extremes The complete biotrans-
formation of most, if not all, hazardous organic wastes fall somewhere
between these extremes
Current knowledge of the chemistry of biotransformation and breakdown of
synthetic compounds by microorganisms is still limited Few biochemical
precedents exist for many of the compounds of environmental concern Inves-
tigation of metabolic pathways are often conducted in microbial cultures
because the studies are easier and the data are less equivocal (Alexander 1981)
67
-------
Biotransformation Concepts
200
80
z
0
0
60 .i
0
Af U .l
“U
C.)
uJ
>
a)” I—
-J
LU
0
LU
-J
0
>
p_ , 6
z
LU
LU IV
I -.
0
1
FIGURE 6.1 Hypothetical Model for Population Changes and Metabolism of a Chemical
Exposed to Mineralizing and Co-metabolizing Populations (from Alexander
1981)
Laboratory studies, however indicative, cannot always be used to predict
detoxification of hazardous materials in field situations
Information on chemical reactions resulting from the activities of micro-
organisms on an array of chemical compounds is given in Table 6 2
The biotransformation or biodegration of a compound may involve various
types of reactions The reactions represent sequences established in model
ecosystems or, in some instances, soil or water The chemical may be modified,
as indicated by the reaction type, or the reaction may apply to a late step in
the transformation or degradation process In some cases, the reaction may
not be microbial because some steps may be partially or wholly nonenzymatic
Furthermore, the types of product listed are found only outside of the cell and
at concentrations that are readily detectable
Not all chemical transformations that are effected by microorganisms in soil
and water are completely degradative A number of compounds are modified
to yield methyl, simple acyl, nitro, and nitroso derivatives and other interme-
diate products
TIME -
68
-------
Biotransformation Concepts
TASLE 6.2 Information on the Biotransformation of Chemical Compounds, Including
Reaction Type, Microorganisms Involved, Condition (Aerobic or
Anaerobic), and Transtormation Products
Microorganisms References
compound and Media Products ( see Sect 68 )
AEROBIC
Dehalogenatuon
R-ci-l 1 ci—RcH 2 0H
chiordane Soil Nocardropsis sp Pestui ide/ch lordane. chlorohydrin, etc (24 )
4 Chloroaniline east. Rhodosporisiium sp 4-amino phenol (45)
2 4-dichlorophenosyaceiic acid P,eudomonas sp 2-chiorophenosyaceiic acid (68 )
4-chioro-2 methyl P,eudomonas sp chio-ine lost after ring cleavage (75. 76)
phenosyaceiic acid
2 36 irichlorobenzoate Brevii,acreriijm sp 3 5-d chlorocasechol (103, 105)
2 4 S-irichlorophenosyac iic acid Brevibacrecium sp Dich(orocatechol (102)
Lindane (1 23 4 56 hesachloro Soil, tscherichia coli Di,sri tesra pentach(orobenzene (72, 130)
qclohexane) (cyclishexanes)
2 4-dichlorophenol Rhizoctonia praticola 2-chloro-1 4-benzoquunone (134)
I-laiogenated alkanes and faii acids Pseudomonas sp Not identified (144)
Kepone Pceudomonas aeruginosa Monohydrokepone, dihydrokepone (145)
mised culture
Fluorobenroic acids Pseudoniorsas sp The f’uorune is iosi after ring cleavage (159)
DOT Water Ounalieila sp RcC(, — RCOOI-1 (149)
,izmenells.jm sp
N serve Soil RCcI — RCOON (152)
DOT Aerobacreraerogenes RCCII — RCOOH (184)
Deamlmaiion
Benzylamine Pceudomonas put ida Benraldehyde (62)
Decarbosylation
4 5-dihydrosyisophshalaie Soil bacterium Prorocasechuare (64)
4 hydrossisophihalaie Pceudomonas puiida Proiocaiechuate (63)
o-phihalaie Pseuriomonas sp Proiocaiechuate (65)
Aicaiigenes sp
23 6-truchlorobenzoate Brevibacierium sp 2,3,5-trichorophenol (103, 105)
3 5-cyciohexaduene.1 .2 dioi-1 Alcaiigenes eutroplsus Casechol (153)
carbosylic acid
Dich lorofop-meihy) (herb cide) Soil Rcl-I(cH ( coot - i — RcH 2 cl-l, (167)
DOT Pseudomonas purida Ar 5 cii COOH — Ar 5 CH , (169)
3.(3i SLdichlorophenyi)5S Soil Ar NR COOt- I — Ar N (R)H (171)
dimethylosazolidine 2 .4 dione
(D000 (ungicide(
Methyl oxidation
Xylene Pseudomonas sp Toluic acid (57)
cresol Pseudornonas puiida Hydrosyben roates (100)
p-sy)ene Nocordia corollina p—ioluic acid (113)
Linaiool Pseudornas sp Oiiygenaiion at c- b methy (135)
Alkanes alkenes rung. carboxytuc acids, alcohols (129)
Pristane Co.’ynebacterium sp Pristanuc acid (141)
Alkanes Pulluiarsa pral!ulons carboxylic acids (133)
Aceione Mycobacierium vaccae Pyruvic acid (176)
i’iydroxyiation and Kelone Formation
Toluene sylenes Pseudomonas sp Benzo ,ite, ioluaies (190)
Alkanes Pseudomonas oleos’orans Aiiphatic alcohols. aldehydes. and acids (1. 2)
Benroic acid Tnchoaporon cuianeum 4-hydroxybenzoic acid (6. 53)
Rhodopseudornonas sp
ch lorophenols Arihrobacier sp Caiechoi (20)
69
-------
Biotransformation Concepts
TABLE 6.2 (Contd)
Microorganisms References
Compound and Media Products ( see Sect 6 8 )
Ilydroxylation and Ketone Formation (amid)
Naphthaline, 1 and 2-naphthalene Pseuclomonas sp 1, and 2-naphthol, naphthoquunone (25. 34, 35,
sullonic acid Cunninghamelia eiegans 36, 37, 58.
cyanobacterium microalgae 83, 114)
Biphenyl Pseudomonas putida 2,3 dihydroxybiphenyl (26)
Naphthalene Several fungal sp 1,2 naphthalene oxide (36)
Naphthalene Oscillatona sp (Cyano Cis-1.2-dihydroxy- (33)
bacterium( 1 ,2-dihydronaphthalene
Alkanes, cyclic alkanes, aromatics Methylococcus capsularus Alcohols (42)
3- and 4 hydroxphenyl acetic acid Escherichia coli 3,4-dihydroxyphenol acetic acid (43)
Niaphihalene Pseudomonas puiida 1 ,2-dihydroxy-1 ,2.dihydronaphthalene (47)
3-hydroxybenzoate Baci/lus sp 2,5 -dihydroxyben coate (48)
2-hydro syben roate Pseudomonas testosteroni 2,3-dihydroxybenzoate (55)
Yrezon )5-amino-4-chloro- Bacteria S amino-4-chloro-2-(2,3-cis-
2-phenyl-3(2Hlpytidarinonel dihydroxycyclo hexa-4.6-diene-l-yl(’ (59)
3 (2H(-pyridazinone
5-hydroxyisophthalic acid Soil bacterium 4,5-dihydroxyisophthalate (64)
4-chlorophenoxyaceiate Pseudomonas sp 4-chloro-2-hydroxyphenolacetate (67)
2 4-dichlorophenoxyaceiate Pxeudomonas sp 2,4 dichloro-6-hydroxypheflOx(i- (68)
acetic acid
o-phthalic acid Pseudomonas sp Protocatechuate (63)
Aicaligenes sp
Diphenylamine (DPA( Sludge 4-hydroxydiphenylamine (74)
4-chloro-2-methylphenoxyacetate Pseudomonas sp 5-Chloro-3-methylcatachol, 5-chloro- (75. 76(
acid o-cresol
p- sylene Pseydomonas putida 3.6-dimethyipyrocatechol (86)
Carboluron Soil 3-hydroxycarbofurans , 3-ketocarbolurans (90. 194)
p-cresol Pseudomonas putida p-hydroxybenzaldehyde (99)
2 36 irichlorobenroate Brevibacierium 2 .4,3-trichloro-4-hydroxybenzoate (10S(
Chlorobenzoate Mixed culture (sludge) Ch lorocatechol (106(
2-chlorobenroate Arrhrobacser sp 4-chlorocatechOl (107 1
Fluorobenzoates Mixed culture Fluorocatechol (106)
Benzoate anthranilate Pseudomonas aeruginosa. Catechols (110)
Micrococcus ureaei,
Pseudomonas Iluorescens
p-sylene Nocardia corallina 3,6-dimethylpyrocatechol (113)
3-ch lotobenroic acid Pseudomonas sp 3-hydroxybentoic. 25 dihydroxy- (115)
benzoic acids
Cliiorophenols Pseudomonas sp Chlorocatechols (123)
Benzoic acid ,4lca)igenes eutroplius 3,5-cyclohexadiene-1,2-diol- (155, 1%)
1-carboxylic acid
Hesazinone Soil 3,)4-hydroxycyclohexyl)-6-(dimethylamino) (154)
1-methyl-i ,3,5-triazine-2,4(1H.3H)-dione
1,3-diphenylbutane Bacillus sp. Pseudomona , tp 2-phenyl-4-hydroxyphenylbutane (164)
Cyclohexane carboxylic acid Alcafigenes sp p-hydroxybenzoate (175)
Naphihalene Soil bacterium (gram neg) 1 2-dihydro--’12-dihydroxynaphthalefle (183, 186)
7 12-dimethylbenz(o(anthracene Pseudomonas aeruginosa methyl-hydroxylated metabolites (191)
Benzoic acid Pseudomonas arvilla 1,2-diliydro.1,2-dihydroxybenzoic acid (193)
1- napht hal Water 4-hydroxy- 1-retralone (22 1
Denmert fungicide Soil R(R’)(R( CCH,— R(R’((R(CCHJOH (184)
B-oxidation
Cannabinoids Nocardia saimonicofor Reduction of a)iphatic side chains (2)
Phenylalkanoic acid Micrococcux ceriIicans Phenylacry)ic or pheny lproprionic acid (61(
Dicarbosylic acid Pseudomonas fluorescens Acetate and CC , (98 1
Ptistane Corynebacterium sp cr-methylglutaric and (141)
Cyclohexane butyric acid Arthrobacter sp Cyc)ohexaneacetic acid (146)
Eugenol Corynebactetium sp Protocatechuic acid (172)
w.(2,4-dichlorophenoxy)-alkanOic Soil Ar O(CHz)n Cf-I , CH,-COOH — (91 1
acid Ar O)CH 2 )n CCOH
6 10
-------
Biotransformation Concepts
TABLE 6.2 (Contd)
Microorganisms Relerences
compound and Media Products ( see Sect 68 )
Eposide formation
RCH • Cl -fR — R-CI-t-CH-R’
1 alkene Pseudomonas a(eovorans 1.2 epoxyalkanes (1.2. 132)
9 tO dihydoxv’9 lO-dihydro. cunnrnghamefla elegans Benzo(a(pyrene, 9,10-diol 7,8-epoxide (27, 28, 29, 30
benzo(a(pyrene
Aromatic hydrocarbons Fungi bacteria, yeast Arorratic osides (31, 32)
Linalool Pseudomonas 7,8-dihydro-7,8-eposy lina(ool (135)
1-ociene 1 7 octadiene P eudomonax oleosiorans 1,2 epoxioctane, 7,8-epoxy-i-octane (160, 174)
Qii,dat.on
Benrene P eudomonas sp e-benzene-dihydrodiol (8, 83, 84, 85)
Cyctohesane- 1 2 diol Acrnetobacter sp 2-hydrosycyclohesane-i.one (56)
AIL )tbenzenes Micrococcus cirihcanx Phen)lalkynoic acid (61)
1 1-diphen leihane Mised culiure Airapic acid (71)
Methylation
Meth)l arsenate Soil sewage (Cl-l 5 (Hg or (CH 3 ) 7 1-Ig (46)
cl- I, As(Q(Ol- I — (CH 5 ( 2 AsOH or (CH,), As
Pentach torophenol Tr,choclerma vwgalum ArOH — Ar OCH 1 , peniachloroanisole (49)
2 34 6-tetra and penta Aspergtllus sydow Scopuiari- AOH Ar OCHJ (50)
ch torophenol opts bres’icaufis Penecil(um
sp , Enierobacrer aerogenes &
soil bacteria
Mercuric ions (nterobac;er aerogenes soil H 2 4 — Cl-I, Hg — )CH ), I-ig (93)
Ether Formation
Diphenylmeihane Pseudomonas purida RCH R — (R(R) c-o-c R (170)
Acetylalion
2 6-dichtoro-4-nitroaniline Soil Pseudomonas cepacia 4-nit:o-2,6’-dichloroacetanilicie (4, 21, 3)
4’chtorobenzene Yeast Rhodosporidium 4-chtoro-2-hydrosyacetanitide (45)
2 4-dichtoroaniline Soil 3,4-dicl’loroacetanilide (181)
4-ch toroani line Fusarrum oxysporum 4-cIstoroacetanilide (119)
N-nilrosalion
Trimeihylamine (scherichia cob Dimethytnitrosamine (9, 10)
Streptococcus
Dimerizalion/polymerization
Dich loroana line Soil 3,3’,4,4’-tetrachloroazobenzene (14, iS)
2 ch lorophenol Rhizoctonsa praticola Oligomeric products (165)
2 ,4-dich torophenol Rhizoctonia pratico!a dimeric quinones (134)
2 and 4 ch lorophersol Rhizocsonia praticola Polymers (165. 166)
2,4’dichtorophenol Rhizoctonia pratico la Polymer’ (134)
3 4-dichtoroanihne Soil 3,3 ,4,4”ietrachloroazobenzene (181, 120)
246 irinitrotoluene Pseudomonax sp 2,2’,6,6’-ieiraniiro-4-azosytoluene (188)
2,6’dichtoro-4.nitroaniline Soil Unidenitlied dimer (4)
4-ch loroani line Fusarium oxysporum 2Ar NJI-l — Ar-F’kN-Ar (119)
Dinme st Soil R.S-R’t RSH — R-S-S .R (143)
Amino Osidation
4-ch loroani line Fusarium oxysporum R-NH 5 — R-NO, 4-chloronitrobenzene (119)
Diflubenziuon H 7 0 (1 12)
t-lesazinone Soil 3-(4-hydrosy cyclohexy)-6-(methylamino(- (154)
1 ‘methyl-1,3,5-triazene. 2,4(1H,3l-f)dione
Benaylisothiocyanate Enterobacter cioacae Ben sylamine (173)
Nitritottiacetic acid Pseudomonas sp Iminodiacetate ek (180)
Yrpazon Water 5-amino-4-chloro-3(2H)-pyridazinone (195)
Trimethylamine Morttere l la parrispora soil Dimethylamine (9, 10)
Thiron Rhizo biurn (CH 5 ) .N-C-Sl-l — (Cl-t 5 ) 2 NH (142)
Aldach lor Chaeromium globosum CH 5 R-)R’) N R’ — RNH 2 (179 )
6 11
-------
Biotransformation Concepts
TABLE 6.2 (Con ld)
Microorganisms References
compound and Media Products ( see Sect 6 8 )
Amino Osidation (contd)
Biphenyl Beijerinckia sf3 2.3 ”dihydroxybiphenyi (78)
Benzo(a lpyrene Beijeriitckia sp Dihydrodiols (79)
Benzo(alanthracene Beijerinckia sp Dihydrodiols (79)
Ethylbenzene Pseudomonas puuda 2,3-dihydroxy-1-ethyibenzene (80)
1-falogenated benzenes Pseudomonas pusida 3-haloginated catechols (81)
Toluene Psuedomonas puticla 2.3-dihydroxy-1-methylcyclohexa-4 ,6-diene (82. 124)
Resorcinol Azoiobacter vinelanthi Pyrogaliol (89)
Resorcinol Pxeudorrsonas putida Hydroxyquinol (39)
Cresols Pseudomonax pu ns -ia Methylcatechols (100)
Dibenzo-p-dioxin Pseudomonas sp, 1,2.dihydroxydsbenzo-p-dioxin (121. 122)
Beijersnckia sp
Benroate, methylbentote Pseudomonas ars’illa Catechol, methyl catechol (138)
Orcinol (3 5-dihydroxytoluene) Pseudomonas putsda 2.3.5 trihydroxytoluene (38)
Sulfur Osidatson
Aidicarb Soil Alcfecarb suifoxide, aldecarb suifone (7, 381
Temik Soil Temik suit oxide. Temik sulfone (44)
Parathion Mixed culture, water (137, 196)
Denmert Soil (143)
Reduction of double bond
A ldrin Water, algal culture -CC I = ccl - — -cHcl — CHCI— (149)
DDT Aerobacteraerogenes Ar 2 - CCHCI — Ar 1 -cH-cH 2 cl
Hydration of Double Bond
1-diphenylethylene Mixed culture 2,2 diphenylethanol (71)
Reduction of Nitro-groupx
4-ch loronitroben rene Rhodospotidium sp 4-ch loroaniline (45)
N-nitropendimethalin St-eptomycex sp Mixture of amino substituted products (128)
2 4-dinitrotoluene Mucroxpotium sp 2-amino-4-nitrotoluene, 4-amino.2-nitrotoluene 1139)
2 4 6.trinitrotcsluene Groundwater, Pxeudorrsonas Amino-dinitrotoluene (150, 188)
sp
2,6 -dichloro-4-nstroaniline Excherichsa coli, Pseudo- 2,5-dichloro-4-aminoaniline (3)
monax cepacia
Fenitrothion Water Amino fenitrothion
Parathion Mixed culture Amino parathion
Glycerol trinitrate Sludge Glycerol dinitrxte and mononstrate (185)
Chime Formation
Temik (insecticides) Soil RCHNOR’ — RCH n N — OH (44)
Nrtrile/amide metabolism
Acetonitrile Nocardia rhodochs’out Acetic acid, NH 3 (60)
Propionitrile Nocardia rhodochtoux Propionic acid, NH 3 (60)
Bromoxynil Fiexibacierium sp 2,4.dibromo-3-hydroxy-benzoate (168)
Benzonitrile Nocardia sp Bencoate, NH 3 (94)
Ester hydrolysis
Phthalate esters Nocardia sp , Arthobacter sp , Phthalic acid (66)
Pseudomonas sp
Phihalate esters Nocardia ereuthropofit Phthalic acid (126)
Malathion Water Malathion fl-mono acid (147, 148)
Butoxyxthyl esrer of 2,4-dichioro- Water 2,4 dichlorophenOxyacetic acid (147, 148)
phenosyacetic acid
Phthalate esters Water Phthalic acid (158)
Oichlorofap-methyl Soil R-ct-f 2 -cH 2 -cOOCH 3 — R-CH CH ,-CO 2 H (167)
612
-------
8 ,otransiormaf son Concepts
c-N-cleavage
Ethyilenediamineterraaceiic acid
Pyra xon
Anode hydrolysis
Propanil
Soil
Bacteria
References
( see Sect 6 8 )
18)
(59)
(41,14, 15 13)
cats
-------
Biot ransformat ion Concepts
TABLE 6.2 (Contd)
Microorganisms References
Compound and Media Products ( see Sect 6 8 )
ANAEROBIC
Dehalogenatlon
DDT Hydrogemonas sp DDD, DOMS, DSP (151)
Art hroba cter sp
Reduction of double bond
Phenol Soil n-caproic acid (11)
Senzoate Methanogenic bacteria Methane-3 (69)
Ferufic acid Methanogenuc bacteria Phenylpropionicacid-4 (97)
DDT i-lydrogemonaa sp
Arthobacter çp (151)
Aldrin Water, algal culture —CCI • CCI — — -Cl-ICI—CHCI— (149)
Reduction of nltro -group
2,6-d ,chloro-4-nitroaniline Flooded soil 2,6-dichloro .p -phenylinediam ine (4)
Parathion Flooded soil Amino parathion (88, 117, 401
Hesahydro-1,3,5-trinitro 1.3.5 Activated Sludge Hydrazine, methanol (140)
tria l me
Ring fission
Phenol Soil n-caproic acid (11)
Catechol. phenol Methanogenic culture Methane 196)
N-acyialion
Chloronated anilines Paracoccus sp Chlorinated acetoanilides (23)
2 4-dinitrotoluene Mucosporium sp 4-acetamide-2-nitrotoluene (139)
C-N Cleavage
Choline Vibro cholinscus Trimethylamine, EtOH + acetic acid (95)
Trilluralin Soil RN (Ali c Ia — R’NI-l AIls and/or RNI-I 2 (87)
Glyphosate Soil, water RNH, CN, R’ — RN1-4 2 ( 157)
Reduction of nitro-groups (continued)
Parathion Flooded soil Amino parathion (162)
2,4,6-trinitropheno l Pseudomonas acruginosa 2-amino-4,6-dinitrophenol (192)
Parathion Flooded soil, Amino parathion (40)
Flavobactersum sp
Dimeriration/poiymeiizatlon
Triliuralin Soil 2 ArNH 2 — ArN cN AR (87)
Dehalogenation (conlinued)
Trilluvalin Soil R”CFa — RCOOH (87)
Pentach lo rophenol Soil ArCI 3 — ArCI 1 OH (111)
DOT Soil RCCIJ — RCO0H (152)
6.3. RECALCITRANT COMPOUNDS
Many synthetic organic molecules are transformed very slowly or not at all
These molecules persist for years and become potential environmental
hazards Organic materials that persist in natural ecosystems because microor-
ganisms lack the ability to degrade or transform them are known as recalci-
trant (persistent) compounds (Alexander 1973, 1981, Horvath 1972)
6 14
-------
Biotransformation Concepts
Recalcitrant compounds in soil and water include plastics, other synthetic
polymers, chlorinated aromatic compounds of various types, pesticides, and
other industrial chemicals A recalcitrant compound is not necessarily hazard-
ous, but some are toxic and others are aesthetically undesirable Some recalci-
trant compounds are not toxic at concentrations found in soil and water, but
are subject to bioaccumulation because of their persistence
Many chemical substitutes, when attached to the parent organic compound,
may cause it to be recalcitrant Among these substituents are amine, methoxy,
sulfonate, and nitro groups, halogens, substitutions in the meta position of
benzene rings, ether linkages, and branched carbon chains (Campbell 1977)
In addition, larger molecules are generally less degradable than smaller ones
Tables 6 3, 6 4, and 6 5 list several synthetic materials that resist biodegradation
Some environmental concerns regarding recalcitrant compounds are that they
may
• persist for years
• accumulate over time
• prove aesthetically objectionable
• be transported for some distances
• prove to be hazardous
• be bioaccumulated or biomagnified
• be difficult to remove physically, chemically, or biologically
The persistence of, and lack of rapid microbial attack on, a chemical may be
due to chemical or physical causes Some molecules may completely resist
attack by microorganisms (e g , synthetic polymers such as polyethylene and
polyvinyl chloride) and persist for years or decades Some resistant compounds
may be acted upon enzymaticaliy, but the reaction is co-metabolic and, hence,
slow Some chemicals are easily transformed in one habitat and long-lived in
another, depending on environmental factors such as the presence or absence
of oxygen Some easily-transformed compounds may be rendered resistant
when complexed with resistant organic materials in soils
TABLE 6.3 Relative Persistence of Selected Pesticides in Soil Under Natural
Conditions(a)
Persistence
Pesticide Edwards (1973) Alexander (1973 )
Chlorinated hydrocarbon insecticides
Ch lordane 5 yr >15 yr
DDT 4yr > lSyr
BHC 3yr
Dieldrin 3 yr
Heptach lor 2 yr >14 yr
A ldrin 2 yr >15 yr
Endrin >14 yr
Lindane >15 yr
Toxaphene >14 yr
6 15
-------
Biotransformation Concepts
TABLE 6.3 (Contd)
Persistence
Pesticide Edwards (1973) Alexander (1973 )
Urea, triazines, and picloram herbicides
Propazine 18 mo 2-3 yr
Picloram 18 mo >5 yr
Simazine 12 mo —
Atrazine 10 no —
fenuron 8rno —
Diuron 8 mc >15 mo
Linuron 4 mo —
Prometryne 3 mo —
Monuron — 3yr
Phenoxy, toluidine, and nitrile herbicides
Trifluralin 6 mo >40 wk
2,4,5-T 5 mo >190 days
Dich lobenil 4 mc —
MCPA 3mo —
2,4-0 1 mc
Phosphate insecticides
Diazinon 12 wk
Disulfoton 4wk
Phorate 2 wk
Malathion 1 wk —
Parathion 1 wk >16 yr
Benzoic acid and amide herbicides
2,3,6-TBA 12 mo 2 yr
Bensulfide 10 no —
Diphenamide 8 mo
Amiben 3mo
CDAA 2mo —
Ducamba 2 no 4 yr
Carbamate and aliphatic acid herbicides
TCA l2wk
Dalapon Bwk
CtPC Bwk
CDEC 6wk
IPC 4wk
EPIC 4wk
Barban 2wk
Other pesticides
2- (2,4-DP) 30 days
Fenac >18 mo
fluometuron 195 days
Pentach loropheno l >5 yr
(a Adapted from Edwards (1975) and Alexander (1973), differences in listed persistence times for
identical compounds may reflect differences in test or environmental conditions, such as p 1- I
6 16
-------
Biotransformation Concepts
TABLE 6.4 Some Organic Compounds that Persist in Soils and Water for Relatively
Long Periods of Time(a)
Acetylethanolamine 2,2 -Dimethyl-1,3-propanediol
Acetylmorpholine 2,2-Dimeihylsuccinic acid
Aminobenzenesulfonic acids Diniirobenzenes
tert-Amyl alcohol Dio xane
Anthraquinonesulfcinic acid bis-2-l thoxyethyl eiher
tert-Butyl alcohol Ethylenediamineteiraacettc
Chloroanilines acid
Chloroniirobenzenes Meihcixyantlines
Chloropheno)s meia-substituted Morplioline
Diaminobenzenes Nitroanilines
2 6-Dibromohexanoic acid Nitroanisoles
Dichloroacetic acid Nitrobenzenesulfonic acids
2.3-Dichloropropionic acid Nitrotoluenes
Diethylaniline Pentaeryihritol
Diethylene glycol teri-Pentanol
Dieihyl eiher Phenoxyalkanecarboxylic acids, a-substituted
3.3-Diet hylglutaric a id 2-Phenylbutyric acid
3,3-Dimeihyl-1 -butano) Poly(ethylene glycol) 400
2.2-Dimethylglutaric acid Tetraethylene glycol
1 .1-Dimethy lhexanol Toluenesulfonic acid
Dumethylmalonic acid Triethanolamine
2.2-Dimethylocianoic acid Triethylene glycol
1.1-Dimethyl-1-pentanol Trimethylacetuc acid
2.2-Dimeihyl- 1-pent anol
(a) Adapted from Alecander (1973)
TABLE 6.5 Some Synthetic Polymers Resistant to Transformation by Microorganisms(a)
Aceiaie rayon (Estron) Poly (methyl methacrylate)
Acrylonitrile-vinyl chloride copolymer )Dynel) Polymonochlorotrifluoro-eihylene
Carboxymethylcellulose (high degree of Polystyrene
substitution) Poytetrafluorethylene (Teflon)
Cellulose acetate (fully acetylated) Polyurethane (polyether linked)
Cellulose acetaic-butyrate Poly (vinyl alcohol)
Cellulose acetate-propionate Poly (vinyl butyral)
Ethylcellu lose Poly (vinyl chloride)
Ethylene glycol terephihalate (Dacron) Poly (vinyl chloride)-acetate
Nylon Poly (vinylidene chloride)
Phenol-formaldehyde Resorcinol-formaldehyde
Polyacrylonitrile (OrIon) Siltrone resins
Polydich lorostyrene Vinylidene chloride-vinyl
Polyethylene )high-molecular-weightl chloride copolymer (Saran)
Polyisobutylene (high-molecular-weight) Zein formaldehyde (Vicara)
(a) Adapted from Alexander (1973)
617
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B,otransIormatiOfl Concepts
6.4 BIOACCUMULATION
Hazardous materials in the environment are often bioaccumulated by living
organisms Two terms, bioconcentration and biomagnification, are used to
describe this phenomena The words are often used interchangeably, but have
different meanings
• Bioconcentration—the uptake and retention of a chemical from the envir-
onment by an organism or tissue to the extent that the organism eventually
acquires levels of the chemical in its body that exceed levels in the envir-
onment (e g , transfer from environment to organism)
• Biomagnification—the uptake and retention of a chemical by an organism
or tissue from ingestion of other organisms containing the chemical so that
concentrations are magnified (e g, transfer from organism to organism)
Bioaccumulation is a characteristic shared by many different types of orga-
nisms (Table 6 6) Hazardous compounds that accumulate in the tissues of
larger animals and plants are generally those that are environmentally
persistent—in many cases, the recalcitrant molecules However, hazardous
materials other than those that are persistent may be bioaccumulated (e g,
benzene, toluene, naphthalene) Because of bioaccumulation, many of these
compounds can have deleterious effects on higher organisms, including
domestic animals and humans
Bioaccumulation by phototrophic organisms (algae, cyanobacteria, photosyn-
thetic bacteria, and higher plants) has limited application to hazardous-waste
treatment and removal processes Phototrophs, which use sunlight for energy,
can sustain relatively large populations without organic matter at levels high
enough to serve as carbon and electron donors (Kobayashi and Rittmann
1982) Under laboratory conditions, some algae can store hydrophobic com-
pounds in large lipid stores (Table 6 6) Once compounds accumulate in the
tissues, the algae can be harvested for destructive treatment or safe disposal
elsewhere Higher plants also may bioaccumulate hazardous materials from
soils
TABLE 6.6 Examples of Bioaccumulation of Syniheiic Compounds by
Microorganisms(a)
Microorganism Compound
Fungi
Aphanomyces euteiches, Fusarium solarn, DOT, dieldrin, PCNB (pentachloronitrObeflZene)
Pythium u!t,mum, Rhozocionia so lani
Aspergillus sp Toxaphene , methoxych lor, DOT
Actinomycetes
Strepiomyces albus, S aurefaciens. S griseus, Dieldrin
S vir,dochronio genes
6 18
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Biotransformation Concepts
TABLE 6.6 (Contd)
Microorganism Compound
Bacteria
Bacillus subtilus, Serratia marcesans, DDT, dieldrin, methoxychior
Agrobacrerium tumefaciens
Aerobacter aerogenes DDT
Flavo bacterium harrison: Toxaphene, methoxychlor
Cyanobacteria (blue-green algae)
Microcystis aeruginosa Benzene, toluene, chlorobenzene,
t2-dichlorobenzene, nitrobenzene,
naphthalene, 2,6-dinitrotoluene, phenanthrene,
di-N-butylphthalate, pyrene
Anacystis nidulans Malathion, carbaryl, parathion, DDT,
aidrin, dieldrin
Anabaena cylindrica Aldrin, dieldrin
Nostoc muscorum Dieldrin
Algae
Chlorella Toxaphene, meihoxychlor
Chioroccuam p ,( ) Dunaisella Mirex
tersolecca, (b) Chiamydamonas p ,(c)
Thalassiosira pseudornana,(d) Porphyridium
cruenturn(e)
N,tzsch,a(d) Mirex, rnethoxychlor, 2,4-DBE
Monoraph,diurn(d) Methoxychior
Euglena racilus , (C) Scenedesmus Parathion, DDT
ob!iqu,s(b)
Cylindrohea ciostenum(d) DDT
Selanastrum capncornaum(b) Benzene, toluene, chlorobenzene,
1,2-dichlorobenzene, nitrobenzene, naphthalene,
2,6-dinitrotoluene, phenathrene,
dr-n-butylphthalate, pyrene
(a) Adapted from Kobayashi and Rittmann (1982)
(b) Green alga
(C) Flagellate alga
(d) Diatom
(e) Rhodophyte (red algae)
6 19
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B,otransIormaflon Concepts
6.5 ACTIVATION
Enzymatic conversion of certain innocuous chemicals to products that are
hazardous to other organisms (animals, plants, and humans) is one possible
consequence of biotreatment Generation of toxicants from harmless chemical
precursors is called activation Activation is usually a mineralization sequence
but may also occur during co-metabolism, although the toxic intermediates
usually do not persist over long periods (Alexander 1981). Activation is a rela-
tively uncommon phenomenon, but it must be considered in biotreatment of
complex wastes or mixtures of complex wastes
Four possibilities may occur as a result of microbial activation
• a relatively non-toxic compound becomes toxic
• relatively short-lived compound becomes persistent
• a compound becomes both more toxic and more persistent
• a toxicant for one type of organism becomes toxic to another type of
organism
Microbial activation is evident in the methylation of inorganic mercury in
aquatic sediments to yield monodimethylmercury compounds which are more
hazardous and more readi!y bioaccumulated by aquatic animals Inorganic
arsenic may also be methylated, and the products of the microbial transforma-
tion are the far more toxic methyl arsines (Cox and Alexander 1973, Cheng
and Focht 1979)
In some cases, innocuous inorganic compounds may be converted to potential
carcinogens The reactions are apparently partly enzymatic and partly nonen-
zymatic, and are related to microorganism activities An example is the N-
nitrosation of secondary amines, which produces the relatively persistent and
toxic nitrosamines The organic precursor is nitrite, which is continually gener-
ated in soil and water from ammonium (or nitrate) during nitrification
Although the N-nitroso derivatives may endure in the environment for some
time, the dialkylamine precursors are readily transformed by microorganisms
(Ayanaba, Verstraete and Alexander 1973, Mills and Alexander 1976, Tate and
Alexander 1976)
A compound toxic to one type of organism may also be changed into products
that affect other organisms An example is the dehalogenation and oxidation
of pentachlorobenzyl alcohol, a compound used in Japan to control a fungus
that harms rice When plant residues containing the fungicide enter the soil,
tn- and tetrach lorinated benzoic acids are formed, which suppress the growth
of subsequent rice crops (Ishida 1972)
The phenomena of increased toxicity can occur with chemical detoxification
For example, the pesticide dimethoate can be entirely degraded by alkaline
hydrolysis, but the degradation product, mercaptoacetic acid, is almost as toxic
as the dimethoate (SCS Engineers 1979a)
6.20
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Biotransformation Concepts
6.6 METALS—SPECIAL CASES
Microorganisms have a minor role in converting metals from one form to
another in the environment Along with larger organisms, they sometimes par-
ticipate in uptake of metals Transfer of metals through soil and water depends
primarily on their interaction with other metals and their chemical form
(Epstein and Chaney 1978, Chaney 1981, 1982, Chaney et al 1982) The form of
metals can be altered in soils, and metals can be removed chemically from
municipal sludges (Jenkins et al 1981), or removed physically and chemically
from liquid waste streams (NATO 1981)
Copper , nickel, cadmium, lead, zinc, and, to a lesser extent, manganese and
cobalt behave similarly in soils In acid soils, these heavy metals can exist as
clivalent cations, for example Cu” and Zn In alkaline or neutral soils, they
may combine with a hydroxyl ion, for example Zn(OH) The hydrous oxide of
manganese and iron can control the availability of heavy metals by sorption
and desorption Organic materials bind metals more strongly at a soil p 1- I
below 7 5 Arsenic reacts in the soil with iron, aluminum, calcium, and magne-
sium ions, and its concentration in solution decreases with time Chromium
generally is oxidized or reduced to Cr and precipitates as an insoluble
hydroxide
The acidity of soil and water affects the solubility of heavy metals and trace
elements Solubility generally increases as pH decreases (e g , acidity
increases) For each unit rise in soil pH, there is about a 100-fold decrease in
the activity of zinc and copper Iron, manganese, and aluminum may be dis-
solved and become mobile in soil at low (acidic) pH As the solubility and
mobility of metals increases, the potential for their uptake and bioaccumula-
tion by plants also increases
Organic soils have a higher cation-retention capacity than do mineral soils
Thus, organic material tends to bind, or chelate, metal ions Chelation may be
more important than the simple cation exchange role of the organic matter,
and may involve greater metal selectivity Compared to mineral soils, metal
availablity is reduced in organic soils at lower pH
The cation exchange capacity (CEC) is important in binding all cations, includ-
ing the toxic metal cations A soil with a high CEC is safer for disposal of wastes
contaminated by metals than is soil with a low CEC
Toxic metals revert with time to chemical forms less available to plants
Reversion is usually rapid for zinc and, for metals in general, more rapid in
calcareous soils Soil p1 -i, phosphate, organic material, and the amount of toxic
metal added can affect the rate and extent of reversion Cobalt reversion
is related to the amount of manganese oxides in soil, but evidence on rever-
sion of other toxic metals is meager Reversion can be stopped with some
metals by prolonged soil submergence and especially by a decreased pH
Metals at hazardous-waste disposal sites are usually difficult to transform
through conventional biological treatments The reasons are fourfold
6 21
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Biotransformation Concepts
• Metals are generally not degraded (biologically or chemically) and tend to
persist for long periods of time in the environment
• Some metals are toxic to plants and animals, or cause adverse effects, at low
concentrations
• Some metals tend to be biologically concentrated by living organisms
• Some metals can be converted to a more toxic form in the environment
by bioprocesses
Both toxicity and biological concentration are highly species-dependent for
individual metals, and vary from relatively toxic to relatively innocuous for dif-
ferent metals Similarly, different metals behave differently in the environ-
ment Thus, identification of the metals present is important to planning
biotreatment
One feature common to all metals is their environmental persistence Unlike
organic pollutants, metals cannot undergo complete biological or chemical
degradation in nature (Buh ler 1973) Since metals are stable, they may be
transported for considerable distances by air and water, they may also pass
through, and accumulate in, food chains Consequently, metallic wastes tend
to remain indefinitely in the environment and can become long-term hazards
In terms of biotreatment at hazardous-waste sites, high levels of metals in the
soil may inhibit the breakdown of organic compounds by microorganisms
(Pajak et al 1977, Loveless and Painter 1968; Lamb et al 1964)
Inorganic material such as metals may be partially removed from soils and
liquids and concentrated in biomass by adsorption during biological treat-
ment Such inorganics, however, are not transformed or destroyed by biolog-
ical processes At higher concentrations, they may also inhibit microbial
activity and will remain a disposal problem
When land treatment is used to biotransform hazardous materials, the availa-
bility of heavy metals, their uptake, and their accumulation will depend upon
a number of factors (Epstein and Chaney 1978), including
• Soil pH - toxic metals are generally more available to plants below pH 6 5
• Organic matter - organic matter can chelate and complex heavy metals so
that they are less available to plants
• Soil phosphorus - phosphorus interacts with certain metal cations to
decrease their biological availability
• Cation exchange capacity - CEC is important for the binding of metal
cations Soils with high CEC are safer than soils with low CEC for land dis-
posal of sludge or other treated wastes containing metal residues
• Moisture, aeration, temperature - these factors affect activity and growth of
plants and, hence, uptake of metals
• Metal identity - Zn, Cu, Ni and other metals differ in their toxicity to plants
and their reactivity in soils
6 22
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Biotransformat ion Concepts
• Plant species - plant species and varieties differ widely in tolerance to
uptake of heavy metals Generally, vegetable crops are more sensitive
than grasses
• Plant parts - grains and fruits generally accumulate smaller amounts of
heavy metals than leafy tissues
• Plant age - the older leaves of plants will contain larger amounts of metals
than young leaves, and maximum metallic content occurs at the end of the
growing season
6.7 ENVIRONMENTAL INFLUENCES
Most hazardous-waste materials are degraded or transformed by hetero-
trophic microorganisms The organisms may coexist with other organisms and
partially depend upon them Heterotrophic organisms obtain some of their
oxygen from algae in waste stabilization ponds, as the algae photosynthesize
organic compounds and release oxygen Nitrifying bacteria, which are present
in activated sludge systems, are autotrophic and able to chemosynthesize
nitrate from ammonia
All microorganisms involved in degradation or transformation require carbon
as a food source Some organisms can use a wide range of organic substances
as their primary food source, while others are more specific All micro-
organisms require certain ratios of nutrients The essential nutrients are nitro-
gen, phosphorus, sulfur, potassium, calcium, and magnesium These are called
macronutrients because of the quantity in which they are required Micro-
nutrients are as essential as macronutrients, but they are needed only in trace
amounts Micronutrients essential for growth include iron, boron, copper,
manganese, zinc, chromium, and cobalt
Water is the medium of life for most microorganisms The organisms synthe-
size water-soluble organics Water is essential for plant life because it controls
temperature, dissolves nutrients, maintains correct pH values, and controls
osmotic pressure, hydrolysis reactions, oxidation reactions, and transport of
ions and compounds
In field situations, interactions among environmental factors such as dissolved
oxygen (aerobic or anaerobic metabolism), water, temperature, pH, presence
of other chemical compounds, and salinity often control biotransformation
processes (Alexander 1981, Kobayashi and Rittmann 1982) In addition, the
physical or chemical characteristics of a chemical compound (solubility, volatil-
ity, hydrophobicity, and octanol-water partition coefficient) affect the ability
of microorganisms to transform that compound Compounds that are insolu-
ble in water are less readily available to microorganisms than are soluble
compounds
Because of environmental conditions that differ, some chemicals are readily
biodegradable in one environment and persistent in another For example, the
absence of oxygen is associated with the resistance of carbohydrates to
microbial destruction This resistance is shown by peats, which are extremely
durable under water in anaerobic deposits In addition, certain classes of
6 23
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Biotransformation Concepis
chemicals are not readily transformed by microorganisms when attached to
the surface of colloidal materials Aeration, pH, soil moisture, and soil tempera-
ture affect the solubility and, hence, the availability and uptake of heavy
metals by plants (Epstein and Chaney 1978)
Most studies on biotransformation of chemical compounds have been con-
ducted in the laboratory (in vitro) under controlled conditions, with pure
microbe cultures and single substrates This approach is necessary to identify
biochemical pathways However, laboratory studies reflect only the potential
for degradation that would occur in the field situations The question remains,
“Will degradation occur in the field as it does in the laboratory?”
Biotransformation or degradation studies have also been conducted with soil
and sediment microcosms under representative environmental conditions (in
situ) for the following reasons
• Substances persisting in a pure culture will often be changed under mixed-
culture conditions (e g , waste treatment lagoons) due to co-metabolism
Also, mixed cultures are more apt to contain the required type of microor-
ganism or combinations of microorganisms
• Initial products from the activity of one microorganism may be broken
down further by a series of different microorganisms acting in sequence
(e g, products of co-metabolism undergo further enzymatic transformation
and are mineralized)
• A compound in the field may be present at such low levels that the availa-
ble nutrients are insufficient to initiate or sustain microbial activity (e g
there is too little energy flux)
• Physical conditions in the field may inhibit microorganisms from changing a
compound (e g, anaerobic conditions prevail where aerobic conditions are
necessary, pH is too high or too low, insufficient moisture)
• Antagonistic interactions between different species of microorganisms can
restrict the activity of the microorganism that will transform a chemical
compound (e g, bacteria are usually antagonistic to fungi)
• A compound that is readily changed or degraded experimentally may be
rendered resistant if complexed with resistant organic materials in the field
• Substances may be present in the field that inhibit microbial activity on a
specific compound or different compounds (e g, toxic levels of a metal)
If the factors controlling biotransformation of specific chemical compounds in
field situations are adequately known, it may be possible to modify them to
enhance microbial activity However, modification of environmental condi-
tions is done most efficiently in bioreactor systems
6 24
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BioiransIormauon Concepts
6.8 CHAPTER REFERENCES
References, in addition to those cited, in the text are provided to give the user
additional sources of information on biotransformation concepts
Biotranslormation Alexander, M 1973 “Nonbiodegradable and Recalcitrant Mole-
Concepts cules “Biotech B:oeng 15 611-647
Alexander, M 1977 Microbial Degradation of Pesticides Technical
Report, Defense Technical Information Center, Cameron Station,
Alexandria, Virginia, p 157
Alexander, M 1981 “Biodegradation of Chemicals of Environ-
mental Concern “Science 211 132-138
Ayanaba, A , W Verstraete and M Alexander 1973 “Formation of
Dimethylnitrosamine, a Carcinogen and Mutagen in Soils Treated
with Nitrogen Compounds “ Soil Sc: Soc Am Proc 37 564-568
Buhier, D R 1973 “Environmental Contamination of Heavy
Metals” In Heavy Metals in the Environment, pp. 1-36 Water
Resources Research Institute, Oregon State University, Corvallis,
Oregon
Campbell, R 1977 “Microbial Ecology” In Basic Microbiology, ed
J F Wilkinson Vol 5 John Wiley and Sons, New York
Chaney, R L 1981 “Potential Toxicity to Plants and Food-Chain
Resulting From Land Treatment of Hazardous Wastes “In Risk and
Discussion Analysis for Hazardous-Waste Disposal, pp 42-49
Hazardous Materials Control Institute, Baltimore, Maryland
Chaney, R L 1982 “Foodchain Pathways for Toxic Metals and
Toxic Organics in Wastes “ in Environment and Solid Wastes
Characterization, Treatment and Disposal, Ann Arbor Sciences
Pubis , Ann Arbor, Michigan
Cheng, C N , and D D Focht 1979 “Production of Arsine and
Methylarsines in Soil and Culture “AppI Environ Microbiol
38 494-498
Cox, D P and M. Alexander 1973 “Production of Trimethylarsine
Gas from Various Arsenic Compounds by Three Sewage Fungi
Bull Environ Contam Toxicol 9 84-88
CoIwell, R R and C S Sayler 1978 “Microbial Degradition of
Industrial Chemicals” In Water Pollution Microbiology Vol I, ed
R Mitchell, pp 111-124 John Wiley and Sons, New York.
Edwards, C A 1975 “Factors [ hat Affect the Persistence of Pesti-
cides in Plants and Soils “ Pure and AppI Chem 42 39-56
Epstein, E , and R I Chaney 1978 “Land Disposal of Toxic Sub-
stances and Water-Related Problems “J Water Pollut Cont Fed
50 2037-2042
6 25
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Biotransformation Concepts
Biotransformation Fields, T , Jr , and A W Lindsey 1975 Landfill Disposal of Hazard-
Concepts ous Wastes - A Review of Literative and Known Approaches U S
(contd) Environmental Protection Agency, Office of Solid Waste Manage-
ment Programs, Washington, 0 C , NTIS, Springfield, Virginia
Horvath, R S 1972 “Microbial Co-metabolism and the Degrada-
tion of Organic Chemicals in Nature” Bactenol Reviews
36 146-155
Ishida, M 1972 “Phytotoxic Metabolities of Pentachlorobenzyl
Alcohol “In Environmental Toxicity of Pesticides, eds
F Matsumura, C M Baush and T Misato, pp 281-306 Academic
Press, New York
Jenkins, R L ,et al 1981 “Metals Removal and Recovery from
Municipal Sludge “j Water Pollw Cont Fed 53 25-32
Kobayashi, H , and B E Rittmann 1982 “Microbial Removal of
Hazardous Organic Compounds “Environ Sci Technol
16 170A-183A
Lamb, J C, Ill, et al 1964 “A Technique for Evaluating the Biolog-
ical Treatability of Industrial Wastes “j Water Pollut Cont Fed
36 1263-1284
Landreth, R E , and C J Rogers 1974 Promising Technologies For
Treatment of Hazardous Wastes EPA-670 12-74-088, U S Environ-
mental Protection Agency, Cincinnati, Ohio, PB-238 145, NTIS,
Springfield, Virginia, p 37
Loveless, J E , and N A Painter 1968 “The Influence of Metal Ion
Concentration and pH Value on the Growth of a Nitrosomonas
Strain Isolated from Activated Sludge.”) Gen Microbiol 521-14
MacGregor, A , and A A Metry 1977 “Pros and Cons of Waste
Disposal Alternatives” In Natl Conf on Treatment and Disposal of
Industrial Wastewaters and Residues. pp 11-14 Houston, Texas
Maki, A W , K L Dickson and I Cairns, Jr , eds 1980. Biotransfor-
mation and Fate of Chemicals in the Aquatic Environment Ameri-
can Society for Microbiology, Washington, 0 C , p. 150
Matsumura, F and E G Esaac 1979 “Degradation of Pesticides by
Algae and Microorganisms “In Pesticides and Xenobiotic Metabo-
lism in Aquatic Organisms, eds N A.Q Khan, J J Leach and J
Menn, pp 371-387, American Chemical Society, Washington, D C
Mills, A L, and M. Alexander 1976 “Factors Affecting
Dimethynitrosamine Formation in Samples of Soil and Water “ 1
Environ Qual 5 437-440
Morrill, L C , B C Mahilum and S. H Mohuiddin 1982 Organic
Compounds in Soils Sorption, Degradation and Persistence Ann
Arbor Science Publishers, Ann Arbor, Michigan, p 326
6 26
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Biotrans formation Concepts
Biotransformation Qasim, S R and M L Stinehelfer 1982 “Effect of a Bacterial
Concepts Culture Product on Biological Kinetics “J Water Pollut Cont Fed
(contd) 54255- 260
North Atlantic Treaty Organization (NATO) 1981 NATO-CCMS
Pilot Study on Disposal of Hazardous Wastes Chemical, Physical,
and Biological Treatment of Hazardous Wastes in NATO Countries
Report of Committee on Challenges of Modern Society PBB2-
114539, NTIS, Springfield, Virginia, p 139
O’Kelley, J C , and I R Deason 1976 Degradation of Pesticides
by Algae U S Environmental Protection Agency, Office of
Research and Development, Athens, Georgia
Pajak, A P , et al 1977 Effect of Hazardous Material Spills on
Biological Treatment Processes EPA-600/2-77-239, U S Environ-
mental Protection Agency, Cincinnati, Ohio, PB-276 274, NTIS,
Springfield, Virginia
Rogers, C J , and R E Landreth 1975 Degradation Mechanisms
Controlling the Bioaccumulation of Hazardous Materials EPA-
670/2-75-005, U S Environmental Protection Agency, National
Environmental Research Center, Cincinnati, Ohio, PB-240 748,
NTIS, Springfield, Virginia
SCS Engineers 1979a Disposal of Dilute Pesticide Solutions U.S
Environmental Protection Agency, Office of Solid Wastes,
Washington, 0 C , PB-297 985, NTIS, Springfield, Virginia
SCS Engineers 197gb. Selected Biodegradation Techniques for
Treatment and/or Ultimate Disposal of Organic Materials EPA-
600/2-79-006, Municipal Environmental Research Laboratory,
Cincinnati, Ohio, PB-295 394, NTIS, Springfield, Virginia
Shuckrow, A J , A P Pajak and I W Osheka 1981 Concentration
Technologies for Hazardous Aqueous Waste Treatment EPA-
600/2-81-019, U S Environmental Protection Agency, Cincinnati,
Ohio, PB81-150 583, NTIS, Springfield Virginia
Tate III, R L , and M Alexander 1976 “Resistance of Nitrosamines
to Microbail Attack “J Environ Qual 5 131-133
Wyss, A W, H K Willard and R M Adams 1980. Closure of
Hazardous Waste Surface Impoundments Municipal Environ-
mental Research Laboratory, Cincinnati, Ohio, PB81-166 894, NTIS,
Springfield, Virginia
TABLE 6.2 1 Abbott, B J , and C T Hou 1973 “Oxidation of 1-Alkenes to
References 1,2-Epoxyalkanes by Pseudomonas oleovorans “AppI
(numbers refer Microbiol 26 86-91
to column 4 of 2 Abbott, B I, D S Fukuda and R A Archer 1977 “Microbio-
Ta e 6.2) logical Transformation of Cannabinoids “Experientia
33 718-720
6 27
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Biotransformation Concepts
TABLE 6.2 3 Alfen, N K V, and I Kosuge 1974 “Microbial Metabolism
References of the Fungicide 2,6-Dichloro-4-nitroaniline.” / Agric Food
(numbers refer Chem 22 221-224
to column 4 of
Table 6 2) Alfen, N K. V , and I Kosuge 1976 “Metabolism of the
(contd) Fungicide 2,6-Dichloro-4-nitroaniline in Soil “I Agric Food
Chem 24 584-588
5 Alvarez, C H , S W Page and V Ku 1982 “Biodegradation
of ‘ 4 C-tris (2,3-Dibromopropyl) Phosphate in a Laboratory
Activated Sludge System “ Bull Environ Contam Toxicol
18.85-90
6 Anderson, J J , and S Dagley 1980 “Catabolism of Aromatic
Acids in Trichosporon cutaneum “J Bacterso! 141 534-543
7 Andrawes, N R, W P Bagley and Richard A Hurett 1971
“Fate and Carryover Properties of Temik Aldicarb Pesticides
(2-Methyl-2-(methylthio) propionaldehyde O-(methyl-
carbamoyl) oxime) in Soil “1 Agrsc Food Chem
19 727-730.
8 Axell, B C, and P J Geary 1975 “Purification and Some
Properties of a Soluble Benzene-Oxidizing System from a
Strain of Pseudomonas “ B,ochem 1 146 173-183
9 Ayanaba, A. and M Alexander 1973 “Microbial Formation
of Nitrosamines in vitro “App! M,crobiol 25 862-868
10 Ayanaba, A, W Verstraete and M Alexander 1973 “Forma-
tion of Dimethylnitrosamine, a Carcinogen and Mutagen, in
Soils Treated with Nitrogen Compounds” Soil Sci Soc
Amer Proc 37 565-568
11 Bakker, G 1977 “Anaerobic Degradation of Aromatic Com-
pounds in the Presence of Nitrate” FEMS Letters 1.103-108
12 Bartha, R 1980 “Pesticide Residues in Humans “ASM News
46 356360
13. Bartha, R 1971 “Fate of Herbicide-Derived Chioroanilines in
Soil “J Agric Food Chem 19 385-387
14 Bartha, R , and D Pramer 1970 “Metabolism of Acyanilide
Herbicides “Adv App! Mscrobsol 13 317-341
15. Bartha, R, and D Pramer 1967 “Pesticide Transformation to
Aniline and Azo Compounds in Soil “Science 156 1617-1618
16 Ba jde, F 1, H L Pease and R F HoIt 1974 “Fate of Benovyl
on Field Soil and Terf “J Agric Food Chem 22413-418
17 Bayly, R. C , and S Dagley 1969 “Oxenoic Acids as Metabo-
lites in the Bacterial Degradation of Catechols” Biochem I
111 303-307
6 28
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Biotransformation Concepts
TABLE 6.2 18 Belly, R T , J J Lauff and C T. Goodhue 1975. “Degradation
References of Ethylenediaminetet.raacetic Acid by Microbial Populations
(numbers refer from an Aerated lagoon “App! Mscrobiol 29 787-794
to column 4o1 19 Bollag, J -M , et al 1968 2,4D Metabolism Enzymatic Degra-
dation of Chlorocatechols I Agric Food Chem 16 829-833
(contd)
20 Bollag, J -M , C S Helling and M Alexander 1968 “2,4-D
Metabolism Enzymatic Hydroxylation of Chlorinated Phen-
ols “I Agric Food Chem 16 826-828
21 Bollag, J -M, P Blattmann and I Loanio 1978 “Adsorption
and Transformation of Four Substituted Anilines in Soil “I
Agric Food Chem 26 1302-1306
22 Bollag, J -M, E J Czaplicki and R D Minard. 1975
“Bacterial Metabolism of 1-Naphthol “J Agric Food Chem
23 85-90
23 Bollag, J -M , a d S Russel 1976 “Aerobic Versus Anaerobic
Metabolism of Halogenated Anilines by a Parococcus sp”
Microbiof Eco! 3 65-73
24 Breman, R W, and F Matsumura 1981 “Metabolism of c ’s-
and trans-Chlordane by a Soil Microorganism “1 Agrsc
Food Chem 29 84-89
25 Brilon, C , W Beckmarin and H J Knackmuss 1981 “Catabo-
lism of Naphthalenesulfonic Acids by Pseudomonas sp A3
and Pseudomonas sp C22 “ App! Environ Microbiol
42 44-55
26 Catelani, et al 1973 “Metabolism of Biphenyl 2-Hydroxy-6-
oxo-phenylhexa-2,4-dienoate The Meta-Cleavage Product
from 2,3-Dihydroxybiphenyl by Pseudomonas putida “Bio-
chem J 134 1063-1066.
27 Cerniglia, C E , and D I Gibson 1980 “Fungal Oxidation of
(±)9,10-Dihydroxy 9,10-dihydrobenzo [ a] Purene Formation
of Diasteriomic benzo a)pyrene 9,10-diol 7,8-epoxides”
Proc Nat! Acad Sc, 77 4554-4558
28 Cerniglia, C E , and D I Gibson 1980 “Fungal Oxidation of
Benzo [ ajpyrene and (±)-Trans-7,8-dihydroxy-7,8-
dihydrobenzo(a)pyrene “J Biol Chem 255 5159-5163
29 Cerniglia, C E , and 0 1 Gibson 1979 “Oxidation of
Benzo(ajpyrene by the Filamentous Fungus Cunnsnghamel!a
elegans “J Bio! Chem 254 12174-12180
30 Ceriglia, C E , W Mahaffey and D I Gibson 1980 “Fungal
Oxidation of benzo [ a]pyrene formation of (±)-7,8-
Dihydroxy-7,8-Dihydrobenzo(a)pyrene by Cunninghamel!a
elegans “Biochem Biophys Res Comm 94 226-232
6 29
-------
B:ot ansIormat,ori Concepts
TABLE 6.2 31 Cernuglia, C E 1981 “Aromatic hydrocarbons Metabolism
References by Bacteria, Fungi and Algae” In Reviews of Biochemical
(numbers refer Toxicology, ed E Hodgsen, J R Bend and R M Phulpat,
to column 4 of pp 321-361 Vol 3 Elsevier/North Holland, New York
Table 6.2) 32 Cerniglra, C E , and S A Crow 1981 “Metabolism of
(contd)
Aromatic Hydrocarbons by Yeast Arch Microbiol 129 9-13
33 Cerniglia, C E , C V Baalen and 0. 1 Gibson 1980
“Metabolism of Naphthalene by the Cyanobacterium
Oscillatorta sp, Strain JCM “J Gen Microbsol 116 485-494
34 Cerniglia, C E , and D I Gibson 1977 “Metabolism of
Naphthalune by Cunninghamella elegans “App! Environ
Microbiol 34 363-370
35 Cerniglia, C E, D T Gibson and C V Baelin 1980 “Oxida-
tion of Naphthalene by Cyanobacterua and Mucroalgae “J
Gen Mscrobso! 116 495-500.
36 Cernuglia, et al “Fungal Transformation of Naphthalene
Arch Microbiol 177 135-143.
37 Cerniglia, C E, and D I Gibson 1978 “Metabolism of
Naphthalene by Cell Extracts of Cunnsghamella e!egans”
Arch Biochem Biophys 186 121-127
38 Chapman, P J ,and D. W Ribbons 1976 “Metabolism of
Resorcinylic Compounds by Bacteria Orcinol Pathway in
Pseudomonas putida “I Bacterio! 125 975-984
39 Chapman, P J , and D W Ribbons 1976 “Metabolism of
Resorcinyluc Compounds by Bacteria Alternative Pathways
for Resorcinol Catabolism in Pseudomonas putsda “J
Bactersol 125 985-998
40 Chin, W T, N Kucharcyzk and A F Smith 1973 “Parathion
Degradation in Submerged Rice Soils in the Phillippines”)
Agric Food Chem 21.504-507
41 Chisaka, H , and P C Kearney 1970 “Metabolism of Pro-
panil in Soils “J Agric Food Chem 18 854-858
42 Colby, J , D I Stirling and H Dalton 1977 “The Soluble
Methane Mono-oxygenase of Methylococcus capsulatus
(Both) Its Ability to Oxygenate N-Alkanes, N-Alkenes, Eth-
ers, and Alicylic Aromatic and Heterocyclic Compounds”
Biochem J 165 395-402
43 Cooper, R A , and M A Skinner 1980 “Catabolism of 3-and
4-Hydroxyphenylacetate by the 3,4-Dihydroxy Phenylacelate
Pathway in Escherichia coli “J Bacteriol 143 302-306
44 Coppedge, J R , et al 1967 “Fate of 2-Methyl-2- (methylthuo)
propronaldehyde O-(methyl-carbonyl) oxime (Temik) in
Cotton Plants and Soil “J Agric Food Chem 15 902
6 30
-------
Biotransformation Concepts
TABLE 6.2 45 Corbett, M D , and B R Corbett 1981 “Metabolism of 4-
References Ch loronutro benzene by the Yeast Rhodospondium sp”
(numbers refer App! Environ Microbial 41 942-949
to column 4 of 46 Cox, D P , and M Alexander 1973 “Production of
Table 6.2)
Trimethyarsine Gas from Various Arsenic Compounds by
con Three Sewage Fungi “Bull Environ Contam Toxicol 9 84-88
47 Cox, D P , and A L Williams 1980 “Biological Process for
Converting Naphthalene to cis-1,2-Duhydroxy-1 ,2-
dihydronaphthalene “App? Environ Microbiol 39 320-326
48 Crawford, R L 1975 “Degradation of 3-Hydroxybenzoate by
Bacteria of the Genus Bacillus “App! Microbiol 30 439-444
49 Cserjesu, A J , and E I Johnson 1972 “Methylation of Penta-
ch lorophenol by Tr,chciderma vargawm ‘ Can I Microbio!
18 45-49
50 Curtis, et al 1972 “2,3,4,6-Tetrachlouanisole Association with
Musty Taint in Chickens and Microbiological Formation
Nature 235 223-224
51 Dagley, 5, and D T Gibson 1964 “Degradation of Catechol
by Bacterial Enzymes “j Biol Chern 239.PC1284-PC1285
52 Dagley, 5, and D T Gibson 1965 “The Bacterial Degrada-
tion of Catechol “Biochem 1 95 466-474.
53 Dagley S 1971 “Catabolism of Aromatic Compounds by
Microorganisms “Adv Microbio! Phys 6 1-45
54 Dagley, S , W C Evans and D W Ribbons 1960 “New Path-
ways in the Oxidative Metabolism of Aromatic Compounds
by Micro-organisms “ Nature 188 560-566
55 Daumy, G 0 , A. S McColl and G C Andrews 1980 “Bio-
conversion of m-Hydroxybenzoate to 2,3-Dihydroxybenzoate
by Mutants of Pseudomonas tesrosceroni “1 Bacteriol
141 293-296
56 Davey, J F, and P W Trudgill 1977 “The Metabolism of
Transcyclohexan-1,2-diol by an Acineobacter Species “ Eur
I Biochem 74 115-127
57 Davey, J F , and D T Gibson 1974 “Bacterial Metabolism of
Para-and Metaxylene Oxidation of a Methyl Substituent.” I
Bacterial 119 923-929
58 Davis, J I , and W C Evans 1964 “Oxidative Metabolism of
Naphthalene by Soil Pseudomonads The Ring-fission Mech-
anism “ Biochem j 91 251-261
59 de Frenne, E , J Ebersp cher and F Lingens 1973 “The Bac-
terial Degradation of 5-Amino-4-chloro-2-phenyl-3(2H)-
pyridazinone” fur I Biochem 33 357-363
6 31
-------
Biotransformation Concepts
TABLE 6.2 60 DiGeronimo, and A D Antonine 1976 Metabolism of Ace-
References tonitrile and Propronitrile by Nocardia rhodochrous LL100-
(numbers refer 21 “App! Environ Microbiol 31 900-906
to column 4 of
Table 6 2) 61 Docuos, J 0 Jr, and J W Frankenfeld 1968 “Oxidation of
(contd) Alkylbenzenes by a Strain of Micrococcus cer,ficans Growing
on n-Paraffins “App! Microbiol 16 532-533
62 Durham, D R , and J J Perry 1978 “The Inducible Amine
Dehydrogenase in Pseudomonas putida NP and Its Role in
the Metabolism of Benzylamine “J Gen Mtcrob,oI 105 39-44
63 Elmorsi, E A , and D J Hopper 1977 The Purification and
Properties of 4-Hydroxyisophthalate Hydroxylase from
Pseudomonas putida NCIB 9866. Eur I Bsochem 76 197-208
64 Elsmorsi, E A, and D 1 Hopper 1979 “The Catabolism of 5-
Hydroxgisophthalate Acid by a Soil Bacterium “J Gen
Mtcrobsol 111 145-152
65 Engelhardt, G, P R Wallnbfer and H G Rast 1976
“Metabolism of o-Phthalic Acid by Different Gram-negative
and Gram-positive Soil Bacteria “Arch Microbiol
109 109-114
66 Engelhardt, G., and P R Wallnöfer 1978 “Metabolism of Di-
and Mono-N-butylphthalate by Soil Bacteria “App! Environ
Microbiol 35 243246
67 Evans, W C , et al 1971 “Bacterial Metabolism of 4-
Chiorophenoxyacetate” B,ochem 1 122 523-551
68 Evans, W. C, et al 1971 “Bacterial Metabolism of 2,4-
Dichlorophenoxyacetate “ Biochem 1 122 543-551
69 Ferry, J C ,and R. S Wolfe 1976. “Anaerobic Degradation of
Benzoate to Methane by a Microbial Consortium “Arch
Microbiol 107 33-40
70 Fisher, P R, J Appleton and J M Pembecton 1978 “Isola-
tion and Characterization of the Pesticide-degrading Plasmid
IP1 from A!cahgenes paradoxus “J Bacter:o! 135 798-804
71 Focht, D D, and H Joseph 1974 “Degradation of 1,1-
Diphenylethylene by Mixed Cultures” Can J Microbso!
20 631-635
72 Francis, A J., R J Sponggord and G. I Ouchi. 1975.
“Degradation of Lindane by Escherichia coil App! Micro-
biol 29 567-568
73 Gamor, Y, and J K Gaunt 1971 “Bacterial Metabolism of 4-
Chloro-2-methyl Phenoxyacetate Formation of Glyoxylate by
Side-Chain Cleavage “ Biochem 1122 527-531
6 32
-------
Biotransformation Concepts
TABLE 6.2 74 Gardner, A M , C H Aoarez and Y Ku 1982 “Microbial
References Degradation of 1 C-DiphenyIamine in a bboratory Model
(numbers refer Sewage Sludge System “Bull Environ Contam Toxico!
to column 4 of 28 91-96
Table 6.2) 75 Gaunt I K and W C Evans 1971 “Metabolism of 4-
(contc l)
Chloro-2-methyl-phenoxyacetate by a Soil Pseudomonad-
ring-fission, Lactonizing and Delactonizing Enzymes”
Biochem 1 122 533-542
76 Gaunt, J K , and W C Evans 1971 “Metabolism of 4-
Chloro-2-methylphencixyacetate by a Soil Pseudomonad—
Preliminary Evidence for the Metabolic Pathway “Biochem
1122 519-526
77 Gerjui, A J , and E I Johnson 1972 “Methylation of Penta-
chlorophenol by Trichoderma virgatuns “Can 1 Microbsol
18 45-49
78 Gibson, D T, et al 1973 “Oxidation of Biphenyl by a Beije-
rinckia Species.” Biochem Biophys Res Comm 50 211-219
79 Gibson, D T, et al 1975 “Oxidation of the Carcinogens
Benzo(a)pyrene and Benzo(a)anthracene to Dihydrodiols by
a Bacterium “Science 189 295-297.
80 Gibson, D T , et al 1973 “Initial Reactions in the Oxidation
of Ethylbenzene by Pseudomonas putida “Biochemistry
12 1520-1526
81 Gibson, D 1, et al 1968 “Oxidative Degradation of Aro-
matic Hydrocarbons by Microorganisms II Metabolism of
Halogenated Aromatic Hydrocarbons” Biochemistry
7 3795-3802
82 Gibson, D T ,et al 1970 “Formation of (+)-cis-2,3-
Dihydroxy-1-methylcydohexa-4,6-diene from Toluence by
Pseudomonas put ida “Biochemistry 9 1626-1630
83 Gibson, D 1 1971 “The Microbial Oxidation of Aromatic
Hydrocarbons “ CRC Ciitical Reviews in Microbiology
1 199-223
84 Gibson, D T , J R Koch and R E Kallio 1968 “Oxidation
Degradation of Aromatic Hydrocarbns by Microorganisms 1
Enzymatic Formation of Catechol from Benzene “Biochemis-
try 7 2653-2661
85 Gibson, D 1 1976 “Initial Reactions in the Bacterial Degrada-
tion of Aromatic Hydrocarbons “Zbl Bakt Hgg 162 157-168
86 Gibson, D T, V Mahadevan and J. F. Davey 1974 “Bacterial
Metabolism of Para- and meta-Xylene Oxidation of the
Aromatic Ring “J Bacteriol 119 930-936
6 33
-------
Biotransformation Concepts
TABLE 6.2 87 Golab, 1, W A Aithaus and H I Wootea 1979 “Fate of
References (‘ C)-trifluralin in Soil “J Agnc Food Chem 27.163-179
(numbers refer
to column 4 of 88 Graetz, D A , et al 1970 “Parathion Degradation in Lake
Table 6.2) Sediments”] Water Pollut Cont Fed 42 R76-R94
(contd) 89 Groseclose, E E. and D W Ribbons 1981 “Metabolism of
Resorcinylrc Compounds by Bacteria New Pathway for
Resorcinol Catabolism in Azctobacter vinelandi, “J
Bacteriol 146 460-466
90 Grunhalgh, R , and A Balanger 1981 “Persistance and
Uptake of Carbofuran in a Humic Mesisol and the Effects of
Drying and Storing Soil Samples on Residue Levels”) Agrsc
Food Chem 29 231-235
91 Gotenmann, et al 1964 “Beta Oxidation of Phenoxyalkanoic
Acids in Soil “Soil Sci Soc Am Proc 28 205-207
92 Haunes, J R., and M Alexander 1975. “Microbial Degrada-
tion of Polyethylene Glyols “App! M,crobtol 29 621-625
93 Hamdy, M K , and 0 R Noyes 1975 “Formation of Methyl
Mercury by Bacteria “App! Microbso! 30 424-432
94 Harper, D B 1977 “Microbial Metabolism of Aromatic
Nitriles Enzymology of C-N Cleavage by Nocardia sp
(Rhodochrousgroup)N Cl B 11216”Biochem 1165309-319
95 Hayward, H R , and I C Stadtman 1959 “Anerobic
Degradation of Choline 1 Fermentation of Choline by an
Anaerobic, Cytochromeproducing Bacterium, Vsbrio chohni-
cus N sp “I Bad er:ol 78 557-561
96 Healy, j B , Jr, and L Y Young 1978 “Catechol and Phenol
Degradation by a Methanogenic Population of Bacteria.”
App! Environ Microbsol 35 216-218
97 Healy, J B Jr, L Y Young and M. Reinhard 1980
“Methanogenic Decomposition of Ferolic Acid, A Model
Lignin Derivative “App! Environ Microbsol 39 436-444
98 Hoet, P P , and R Y Stanier 1970 “The Dissimilation of
Higher Dicarboxylic Acids by Pseudomonas fluorescens
Eur I B,ochem 1365-70
99 Hopper, D J 1976 “The Hydroxylation of P-Cresol and its
Conversion to P-Hydroxybenzaldehyde in Pseudomonas
put ide “B,ochem Bsophys Res Comm 69 462-468
100 Hopper, D I . and D C Taylor 1975 “Pathways for the
Degradation of M-Cresol and P-Cresol by Pseudomonas
putida “I Bactenol 122 16
6 34
-------
Biotransformation Concepts
TABLE 6.2 101 Horvath, R 5 1970 “Co-metabolism of Methyl- and Chloro-
References substituted Catechok by an Achromobacter sp Possessing a
(numbers refer New Meta-cleaving Oxygenase “ Biochem 1 119 871-676
to column 4 of 102 Horvath, R S 1971 “Microbial Cometabolium of 2,4,5-
Table 6.2)
Trichiorophenoxyacetic Acid Bull Environ Contam
(contd) Toxicoi 5 537-541
103 Horvath, R S 1972 “Cometabolism of the Herbicide, 2,3,6-
Trichlorobenzoate by Natural Microbial Populations “ Bull
Environ Contam Toxicol 7 273-276
104 Horvath, R S , and B W Koft 1972 “Degradation of Alkyl
Benzene Sulfonate by Pseudomonas Species “App!
Microbiol 23 407-414
105 Horvath, R S 1971 “Co-metabolism of the Herbicide 2,3,6-
Trichlorobenzoate “J Agrtc Food Chem 19 291-293
106. Horvath, R S 1973 “Enhancement of Co-metabolism of
Chlorobenzoates by the Co-substrate Enrichment Tech-
nique “App! Microbro! 25 961-963
107 Horvath, R S , and M Alexander 1970 “Co-metabolism of
M-Chlorobenzoate by an Art hrobacter “App! Microbiol
20 254-258
108 1-forvath, R S , and P Ilathman 1976 “Co-metabolism of
Fluerobenzoates by Natural Microbial Populations “App!
Environ Mtcrobio! 31 869-891
109 Hosoya, H , et al 1978 “Bacterial Degradation of Synthetic
Polymers and Oligomers with the Special Reference to the
Case of Polyethylene Glycol “Agric Biol Chem 42 1545-1552
110 Ichihara, A , et al 1962 “The Enzymatic Hydroxylation of
Aromatic Carboxylic Acids Substrate Specification of
Anthranilate and Benzoate Oxidases”) Bio! Chem
237 2296-2302
111 Ide, A ,et al 1972 “Decomposition of Pentachiorophenol in
Paddy Soil “Agric BtoI Chem 36 1937-1944
112 Ivie, C W, D I Bull and j A Veech 1980 “Fate of Diflu-
benzuron in Water “I Agric Food Chem 28 330-337
113 Jamison, V W, R L Raymond and J 0 Hudson 1969
“Microbial Hydrocarbon Co-oxidation Ill Isolation and
Characterization of an a, a, Dimethyl-cis, css-muconic Acid-
producing Strain of Nocardia corahna “App! Microbio!
17 853-856
114 Jeffrey, A M , et al. 1975 “Initial Reactions in the Oxidation
of Naphthalene by Pseudomonas putida “Biochemistry
14 575-584
6 35
-------
Biotransformation Concepts
TABLE 6.2 115 Johnston, H W, G G Briggs and M Alexander. 1972
References “Metabolism of 3-Chlorobenzoic Acid by a Pseudomoriad”
(numbers refer Soil Biol Biochem 4 187-190
to column 4 of
Table 6 2) 116 Jones, A S 1976 “Metabolism of Aldicarb by Five Soil
(contd) Fungi “1 Agric Food Chem 24115-117
117 Katan, J , I W Fuhremann and E P Lichtenstein 1976
“Binding of 1 14 C1 parathion in Soil A Reassessment of Pesti-
cide Persistance “Science 193 891-894
118 Kaufman, D D , and P C Kearney 1965 “Microbial Degrada-
tion of lsopropyl-N-3-chlorophenyl-carbamate and 2-
Chloroethyl-N-3-chlorophenyl Carbamate “App! Microbiol
13 443-446
119 Kaufman, D D, J R Plimmer and U I Klingebiel. 1973
“Microbial Oxidation of 4-Chioroaniline “I Agrsc Food
Chem 21127-132
120 Kaufman, D 0 , et al 1972 “3,3,’4,4’-Tetrachloroazoxyben-
zene from 3,4-Dichloroaniline in Microbial Culture”)
Agric Food Chem 20 916-917
121 Kearney, P E, E A Woolson and C P Ellington, Jr
“Persistance and Metabolism of Chlorodioxins in Soils”
Environ Sci Technol 6 1017-1018
122 Klecka, C M , and D T Gibson 1980 “Metabolism of
Dibenzo-p-dioxin and Chlorinated Dibenzo-p-dioxins by a
Beijerinckia Species “App! Environ Microbiol 39 288-296
123 Knackmuss, H J , and M. Hellwig 1978 “Utilization and Co-
oxidation of Chlorinated Phenols by Pseudomonas sp B13.”
Arch Microb:ol 1171-7
124 Kobal, V M , et al 1973 “X-ray Determination of the Abso-
lute Stereochemistry of the Initial Oxidation Product Formed
from Toluene by Pseudomonas putida 39/D.” / Amer
Chem Soc 95 4420-4421.
125 Ku, Y, and G H Alvarez 1982 “Biodegradation of [ ‘4C] tn-
p-cresyl Phosphate in a Laboratory Activated-sludge System
App! Environ M,crobsol 43 619-622
126 Kurane, R , I Suzuki and Y Takahara 1978. “Removal of
Phthalate Esters in Soil Column Inoculated with Microorga-
nisms “Agric Biol Chem 42 1469-1 478
127 Loos, M A, R N Roberts and M Alexander 1967 “Ferma-
non of 2,4Dichlorophenol and 2,4-Dichioroanisole from 2,4-
Dichlorophenoxyacetate by Arthrobacter sp “Can I
M:crobio! 13 691-699
636
-------
Biotransformation Concepts
TABLE 6.2 128 Lusby, W R , et al 1981 “Metabolism of N-Nitrosopen-
References dimethalin and N-Nitropendamethalin by a Streptomyces
(numbers refer Isolated from Soil “ I Agric Food Chem 29 245-250
to column 1’ ’ 129 Markovetz, A 1 , Jr , J Cazin and J E Allen 1968 “Assimila-
tion of Alkanes and Alkenes by Fungi “App! Microbio!
16 487-489
130 Mathur, S P . and J C Saha 1977 “Degradation of Lindane-
14C in a Mineral Soil and in an Organic Soil “Bull Environ
Con tam Toxico! 17 424-430
131 Matsumura, F , and C M Boush 1966 “Malathion Degrada-
tion by Trichoderma viride and a Psuedomonas sp “Science
153 1278-1280
132 May, S W, and B J Abbott 1973 “Enzymatic Epoxidation I I
Comparison Between the Expoxidation and Hydroxylation
Reactions Catalyzed by the w-Hydroglation System of
Pseudomonas oleovorans “J Biol Chem 248 1725-1730
133 Merdinger, E , and R I ’ Merdinger 1970 “Utilization of N-
Alkanes by Pullularia pu!!u!ans “App! Microbio! 20 651 -652
134 Minard, R D , S -y Liu and I -M BolIag 1981 “Oligomers
and Quinones from 2,4-Dich lorophenol “j Agric Food
Chem 29 250-253
135 Modyastha, K M , P K Bhattacharyya, and C S Vaidyana-
than 1977 “Metabolism of a Monoterpene Alcohol, Lina-
lool, by a Soil Pseudomonad “Can J Microbio!
23 230-239
136 Moody, R P, et al 1978 “The Fate of Fenitrothion in an
Aquatic Ecosystem “ Bu!! Environ Conta in Toxico! 19 8-14
137 Munnecke, 0 M, and D P H Hsieh 1976 “Pathways of
Microbial Metabolism of Parathion “App! Environ Micro-
biol 31 63-69
138 Murray, K ,et al 1972 “The Metabolism of Benzoate and
Methylbenzoates via the Metacleavage Pathway by Pseu-
domonas arvil!a mt-2” Eur I Biochem 28 301-310
139 McCormich, N C , J H Cornell and A M Kaplan 1978.
“Identification of Biotransformation Products from 2,4-
Dinitrotoluene “App! Environ Microbio! 35145-948
140 McCormich, N C , J H Cornell and A M Kaplan 1981
“Biodegradation of l-Iexahydro-1,3,5-Trinitio-1,3,5-trazine
App! Environ Microbiol 42 817-823
141 McKenna, E I , and R E Kallio 1971 “Microbial Metabolism
of the Jsoprenoid Alkane Pristane “Proc Nat! Acad S c
68 1552-1554
6 37
-------
Biotransformation Concepts
TABLE 6.2 142 Odeyemi, 0 , and M Alexander 1977 “Resistance of Rhizo-
References bium Stains to Phygon, Spergon and Thiram “App! Environ
(numbers reler Microbiol 33 764-790
to column 401 143 Ohkawa, H ,et al 1976. “Degradation of the Fungicide
(contd) Denmert (S-ri-butyl S’-p-tertbutylbenzyl N-3 Pyridyldithio-
carbon imidate, 5-1358) by Plants, Soils and Light “Agric Bio
Chem 40 943-951
144 Omori, 1, and M Alexander 1978 “Bacterial Dehalogena-
tion of Halogenated Alkanes and Fatty Acids “App! Environ
Microbsol 35 867-871
145 Orndorft, S A , and R R CoIwell 1980 “Microbial Trans-
formation of Kepone “App! Environ Microbio! 39 398-406
146 Ougham, H J , and P W Trudgill 1982 “Metabolism of
Cyclohexaneacetic Acid and Cyclohexanebutyric Acid by
Art hrobacder sp Strain CAl “J Bacterio! 150 1172-1182
147 Paris, D F , D L Lewis and N L Wolfe 1975 “Rates of
Degradation of Malathion by Bacteria Isolated from Aquatic
System “ Environ Sci Technol 9 135-138
148 Paris, D F , et al 1981 “Second-order Model to Predict
Microbial Degradation of Organic Compounds in Natural
Waters “App! Environ Microbiol 41 603-609
149 Patil, K C , F Matsumura and G M Boush 1972 “Metabolic
Transformation of DDT, Dieldrin, Aidrin, and Endrin by
Maine Microorganisms” Environ Sci Technol 6 629-632
150 Pereira, W E , et al 1979 “Isolation and Characterization of
TNT and its Metabolites in Ground Water by Gas
Chromatography-Mass Spectrometer-Computer Technique”
Bull Environ Contam Toxicol 21:554-562
151 Pfaender, F K, and M Alexander 1972 “Extensive Microbial
Degradation of DDT in vitro and DDT Metabolism by Natural
Communities “J Agric Food Chem 20 842-846
152 Redemann, C 1, R W Meike and J G Widofsky 1964 “The
Loss of 2-Chloro-6-(trichloromethyl)-pyridine from Soil “I
Agric Food Chem 12 207-209
153 Reiner, A M 1972 “Metabolism of Aromatic Compounds in
Bacteria. Purification and Properties of the Catechol-forming
Enzyme, 3,5-Cyclohexadiene-1,2-diol-1-carboxlic Acid
(NADP) Oxidoreductase (Decarboxylating) “I Biol Chem
247 4960-4965
154 Rhodes, R. C 1980 “Soil Studies with 1 C-Labeled
Hexazinone “I Agric Food Chem 28 311-315
6 38
-------
Biotrans(ormaiion Concepts
TABIF 6.2 155 Riener, A M , and G D Hegeman 1971 “Metabolism of
References Benzoic Acid by Bacteria Accumulation of (-)-3,5-
(numbers refer Cyclohexadiene-1,2-diol-1-carboxylic Acid by a Mutant
to column 4 of Strain of A!cahgenes eutrophus ‘ Biochemistry 10.2530-2536
Table 156 Riener, A M 1971 “Metabolism of Bcnzoic Acid by
con Bacteria 3,5-Cyclohe.adiene-1,2-diol-1-carboxylic Acid is
an Intermediate in the Formation of Catechol “ I Bacterio!
108 89-94
157 Rueppel, M L , et al 1977. “Metabolism and Degradation of
Glyphosate in Soil and Water “ I Agric Food Chern
25 517-528
158 Sanborn, I R , et a? 1975 “Plasticizers in the Environment
The Fate of di-N-octylphthalate (DOP) in Two Model Ecosys-
tems and Uptake and Metabolism of DOP by Aquatic
Organisms “Arch Env iron Contam Toxico! 3 244-255
159 Schreiber, A., et al 1980 “Critical Reactions in Fluoroben-
zoic Acid Degradation by Pseudomonas sp B13 “App!
Environ Microbiof 39 58-67
160 Schwartz . R D , and C J McCoy 1976 “Enzymatic Epoxida-
tion Synthesis of 7,8-Epoxy-1-octene, 1,2-7,8-diepoxyociane,
and 1,2-Epoxyoctane by Pseudomonas o!eovorans “App)
Environ Microbio! 31 78-82
161 Sethunathan, N 1973 “Degradation of Parathion in Flooded
Acid Soils “j Agric Food Chem 21 602-604
162 Sethumatan, N 1973 “Microbiol Degradation of Insecticides
in Flooded Soil and in Anaerbic Cultures.” Residue Rev
47 143-165
163 Siddaramappa, R , 1 < P Rajaram and N Sethunathan 1973
“Degradation of Parathion by Bacteria Isolated from Flooded
Soil “App! Microbiol 26 846-849
164 Sielicki, M , D D Focht and J P Martin 1978 “Microbial
Degradation of [ 14 C1 Polystyrene and 1,3-Diphenylbutane
Can I Mvcrobio! 24 798-803
165 Sjoblad, R. D , and I M Bollag 1977 “Oxidation Coupling of
Aromatic Pesticide Intermediates by a Fungal Phenol Oxi-
dase” App! Environ Mrcrobio) 33 906-910
166 Sjoblad, R D, R D Minard and I M Bollag 1976 “Poly-
merization of 1-Naphthol and Related Phenolic Compounds
by an Extracellular Fungal Enzyme” Pest Biochem Physio!
6 457-463
167 Smith, A E 1977 “Degradation of the Herbicide Dich lorfop -
Methyl in Prairie Soils”) Agnc Food Chem 25 893-898
6 39
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Biotransformation Concepts
TABLE 6.2 168 Smith, A E ,and D R Cullimore 1974 “The in vitro Degrada-
References non of the Herbicide Bromoxynil “Can I Mtcrobio!
(numbers refer 20 773-776
to column 4 of
Table 6 2) 169 Subba-Rao, R V and M Alexander 1977 “Cometabolism of
(contd) Products of 1,l,1,Truchloro-2,2-bis (p-chlorophenyl) Ethane
(DOT) by Pseudomonas put ida “j Agr Food Chem
25 855-858
170 Subba-Rao, R V , and M Alexander 1977. “Products from
Analogues of 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane
(DDT) Metabolites by Pseudomonas put ida “App! Environ
M,crobiol 33.101-108
171. Sumida, S, R Yoshihara and I Miyamoto 1973 “Degrada-
tion of 3’(3,’5’-Dichlorophenyl)-5,5-dimethyloxazolidlfle-
2,4-dione by Plants, Soil and Light “Agric B,o! Chem
37.2781-2790
172 Tadasa, K. 1977 “Degradation of Eugenol by a Micro-
organism “Agric Bso! Chem 41 925-929
173 Tang, C S, K Bhothipaksa and H A Frank 1972 “Bacterial
Degradation of Benzylisothiocyanate “App! Microbio!
23 1145-1148
174 Taniuchi, H ,and 0 Hayaishi 1963 “Studies on the Metabo-
lism of Kynurenic Acid III Enzymatic Formation of 7,8-
Dihydroxy-kynurenic Acid from Kynurenic Acid “I Biol
Chem 238 283-293
175 Taylor, D C ,and P W Trudgill 1978 “Metabolism of Cyclo-
hexane Carboxylic Acid by Alcaligenes Strain Wi “1
Bacteriol 134.401-411
176 Taylor, D C., et al. 1980 “The Microbial Metabolism of Ace-
tone”] Gen Microbiol 18 159-170
177 Tiedje, J M , and M Alexander 1969 “Enzymatic Cleavage
of the Ether Bond of 2,4-Dichlorophenoxyacetate”) Agric
Food Chem 17 1080-1085
178 Tiedje, J M , et al. 1969. “2,4-D MetabolismS Pathway of Deg-
radation of Chlorocatechols by Arthrobactes sp.”) Agric
Food Chem 171021-1026
179 Tiedje, J M , and M L Hagedorn 1975 “Degradation of
Alochor by a Soil Fungus, Chaetomium globosum “J Agric
Food Chemn 23 77-80
180 Tiedje, J M , et al 1973 “Metabolism of Nitrilotriacetate by
Cells of Pseudomonas Species “App! M,crobio! 25 811-818
6 40
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Biorrans format ion Concepts
TABLE 6.2 181 Vishwanathan, R I et al 1978 “Long-term Studies on the
References Fate of 3,4 Dichloroaniline 14C in a Plant-soil-system Under
(numbers refer Outdoor Conditions “J Environ S c Health B13 243-259
to column 4 of
Table 6 2) 182 Vlitos, A J , and L J King 1953 “Fate of Sodium 2,4-
(contd) Dichlorphenoxy-ethylsulfate in the soil “Nature 171 523-524
183 Walker, N , and G H Wiltshire 1953 “The Breakdown of
Naphthalene by a Soil Bacterium “J Gen Microbio!
8 273-276
184 Wedemeyer, G 1967 “Dichlorination of 1,1,1-Trichloro-2,2-
bis(p-chlorophenyl)ethane by Aerobacter aerogenes
1 Metabolic Products “App! Microbiol 15 569-574
185 Wendt, I M , J H Cornell and A M Kaplan 1978 “Microb-
ial Degradation of Glycerol Nitrates “App! Environ Micro-
b ,o! 36 693-699
186 Williams, P A , F A Catterall and K Murray 1975 “Metabo-
lism of Naphthalene, 2-Methylnaphthalene, Salicylate and
Benzoate by Pseudomonas Pc Regulation of Tangential
Pathways “J Bacterio! 124 679-685
187 Wolfe, N L, R G Zepp and D F Pariis 1978 “Carbaryl,
Propham, and Ch loropropham A Comparison of the Rates
of Hydrolysis and Photolysis with the Rate of Biolysis “ Water
Res 12 565-571
188 Won, W D , et al 1974 “Metabolic Disposition of 2,4 ,6-
Trinitrotoluene” App! Mscrobiol 27 513-516
189 Worsey, M J , F C H Franklin and P A Williams 1978
“Regulation of the Degiadative Pathway Enzymes Coded for
by the TOL Plasmid (pWWO) from Pseudomonas put i da mt-
2”J Bacteriol 134 757-764
190 Worsey, M I , and P A Williams 1975 “Metabolism of
Toluene and Xylenes by Pseudomonas putida (arvilla) mt-2
Evidence for a New Function of the 101 Plasmid “ I
Bacter,o! 124 7-13
191 Wyman, I F , et al 1979 “Conversion of 2,4,6-Trinitrophenol
to a Mutagen by Pseudomonas aeruginasa “App! Environ
Microbiol 37 222-226
192 Yamaguchi, M ,T Yamauchi and H Fujisawa 1975 “Studies
on Mechanism of Double Hydroxylation 1 Evidence for
Participating of NADHcytochrome c Reductase in the Reac-
tion of Benzoate 1,2-Dioxygenase (Benzoate Hydroxylase) “
Biochem Biophys Res Comm 67 264271
193 Vu, C -C , et al 1974 “Fate of Carbofuran in a Model Ecosys-
tem “ I Agric Food Chem 22.431-434
6 41
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Biotransformation Concepts
TABLE 6.2 194 Yu, c -c , et al 1975 “Fate of Pyrazon in a Model Ecosys-
References tern “I Agric Food Chem 23.309-311
(numbers refer
to column 4 of 196 Yu, C -C , and J R Sonborn 1975 “The Fate of Parathion in a
Table 6.2) Model Ecosystem “Bull Environ Contam Toxicol 13 543-550
(contd)
6 42
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