&EPA
United Slates
Environmental Protection
Agency
EPA/600/R-08/035F | February 2011 | www.epa.gov/ncea
Problem Formulation for
Human Health Risk
Assessments of Pathogens
in Land-applied Biosolids
National Center for Environmental Assessment
Office of Research and Development, Cincinnati, OH 45268

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E PA/600/R-08/035 F
Feburary 2011
Problem Formulation for
Human Health Risk
Assessments of Pathogens in
Land-applied Biosolids
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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NOTICE
The U.S. Environmental Protection Agency (EPA) through its Office of Research
and Development funded and managed this research with support from the Oak Ridge
National Laboratory (IAG DW89922097-01-0) and ICF, Inc. (Contract No. EP-C-09-
009). The document underwent internal peer review in October 2007 and was later
released for external review as a predissemination draft for a 45-day public comment
period from September 19, 2008 to November 3, 2008 (73 FR 54400). An external,
independent panel of experts reviewed the document and comments received during
the public comment period and a workshop open to the public conducted on
November 19, 2008 in Cincinnati, Ohio. These proceedings where arranged and
managed by Eastern Research Group, Inc. under Contract No. EP-C-07-025. The
document was updated and revised by EPA based on the comments received from the
expert panel review and the public. It has been subjected to the Agency's peer and
administrative review and been approved for publication as an EPA document. Mention
of trade names or commercial products does not constitute endorsement or
recommendation for use.
ABSTRACT
This document provides concepts and planning considerations for conducting
human health risk assessments on potential pathogens in land-applied biosolids. As
one component of U.S. EPA's Action Plan for setting new priorities for it's biosolids
program, this document summarizes the existing literature; defines critical pathogen
stressors; develops conceptual models linking the most likely stressors, pathways and
health responses of concern; evaluates the overall quality and utility of available risk
assessment data; highlights existing tools and methodologies; and provides an outline
of an Analysis Plan that identifies gaps in knowledge and research and methods
needed to provide more scientifically defensible assessments relevant to U.S. EPA's
decision needs.
Preferred Citation:
U.S. Environmental Protection Agency (U.S. EPA). 2011. Problem Formulation for Human Health Risk
Assessments of Pathogens in Land-applied Biosolids. National Center for Environmental Assessment,
Cincinnati, OH. EPA/600/R-08/035F.
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TABLE OF CONTENTS
Page
LIST OF TABLES	vi
LIST OF FIGURES	vii
LIST OF ABBREVIATIONS	viii
PREFACE	ix
AUTHORS, CONTRIBUTORS AND REVIEWERS	x
EXECUTIVE SUMMARY	xii
1.	INTRODUCTION	1
1.1. Scope	4
2.	HAZARD IDENTIFICATION	5
2.1.	SOURCE	6
2.1.1.	Spatial Extent of Source	7
2.1.2.	Reproduction	7
2.1.3.	Matrix	7
2.1.4.	Treatment	8
2.1.5.	Site Restrictions	9
2.1.6.	Vector Attraction Reduction	10
2.2.	PATHOGENS	10
2.2.1.	Bacteria	16
2.2.2.	Viruses	19
2.2.3.	Protozoa	22
2.2.4.	Helminths	23
2.2.5.	Endotoxins	24
2.2.6.	Emerging Pathogens	25
2.2.7.	Multiple Hazards	26
3. DEVELOPMENT OF CONCEPTUAL MODELS, ENDPOINTS AND SCENARIOS. 27
3.1.	PREAPPLICATION PROCESSES	30
3.2.	APPLICATION	30
3.2.1.	Methods of Land Application of Biosolids	30
3.2.2.	Rates of Land Application of Biosolids	32
3.2.3.	The Timing of Land Applications of Biosolids	34
3.2.4.	Regional Application Issues	34
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TABLE OF CONTENTS cont.
Page
3.3.	FATE AND TRANSPORT OF PATHOGENS	35
3.3.1.	Pathogen Survival, Growth and Death	35
3.3.2.	Pathogen Transport	37
3.3.3.	Vector Transport	40
3.4.	HUMAN ROUTES OF EXPOSURE	41
3.4.1.	Inhalation	41
3.4.2.	Ingestion	42
3.4.3.	Dermal Exposure	45
3.5.	REGULATORY RESTRICTIONS	46
3.6.	FACTORS THAT AFFECT INFECTION AND DISEASE	46
3.6.1.	Human Factors	48
3.6.2.	Additional Susceptibility Factors	50
3.6.3.	Pathogen Factors	51
3.7.	INFECTION AND DISEASE	51
3.8.	SCENARIOS	52
3.8.1.	Scenario 1. Neighboring Residences and Schools	53
3.8.2.	Scenario 2. Residents (Farm Families)	53
3.8.3.	Scenario 3. Pica Child	53
3.8.4.	Scenario 4. Drinking Water Consumers of Groundwater	57
3.8.5.	Scenario 5. Drinking Water Consumers of Surface Water	57
3.8.6.	Event Related and Regional Aspects of Scenarios	57
4.	SCREENING OUT ELEMENTS OF THE CONCEPTUAL MODEL	61
5.	CONSIDERATIONS FOR DEVELOPING AN ANALYSIS PLAN	64
5.1.	INTRODUCTION	64
5.2.	MANAGEMENT NEEDS	65
5.2.1.	Assessment Endpoints	65
5.2.2.	Data and Data Quality	66
5.3.	PLAN FOR CHARACTERIZATION OF EXPOSURE	67
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TABLE OF CONTENTS cont.
Page
5.3.1.	Measures of Exposure	67
5.3.2.	Detection of Pathogens	68
5.3.3.	Use of Indicator Species	70
5.3.4.	Background Levels of Pathogens	72
5.3.5.	Environmental Fate of Pathogens	74
5.3.6.	Transport of Pathogens	76
5.3.7.	Contact with Crops	80
5.3.8.	Uptake and Dosage	81
5.3.9.	Exposure Factors	81
5.4.	PLAN FOR CHARACTERIZATION OF EFFECTS	82
5.4.1.	Measures of Effect	82
5.4.2.	Establishing Cause and Effect	83
5.4.3.	Dose-Response Models for Infection	84
5.4.4.	Predicting Disease	90
5.5.	PLAN FOR RISK CHARACTERIZATION	93
5.5.1.	Screening Risk Assessment	93
5.5.2.	Weight of Evidence	94
5.5.3.	Uncertainty Analysis	94
REFERENCES	96
APPENDIX: LITERATURE REVIEW	125
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LIST OF TABLES
No.	Title	Page
1	Site Restrictions for Class B Biosolids	11
2	Example Pathogens of Potential Concern in Sewage Sludge and Biosolids	14
3	Estimated Biosolids Application Rates for Different Land Uses	33
4	Environmental Factors Positively or Negatively Affecting the Survival of
Pathogenic Microbes	36
5	Pathways of Exposure and Applicable Use Restrictions for Class B Biosolids	44
6	Minimum Time Interval between Application and Harvest, Grazing or Public
Access to Lands Applied with Class B Biosolids	47
7	Suitability of Select Agents as Indicators of Post-Treatment Risk for Viruses in
Biosolids (Modified from Smith et al., 2005b)	73
8	Survival Times of Pathogens in Soil and on Plants Modified from Gerba and
Smith (2005)	75
9	Examples of Dose-Response Models for Microbial Agents	86
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LIST OF FIGURES
No.	Title	Page
1	General Framework for Assessing the Risks of Human Diseases Following
Exposure to Pathogens	
2	General Conceptual Model of the Potential Risks from Pathogens in
Land-Applied Biosolids	
3	Pathogen Fate Conceptual Model	
4	Disease Factors Conceptual Model	
5	Scenario 1: Neighboring Residences and Schools (or Adjacent Property)
Conceptual Model	
6	Scenario 2: Resident (Farm Family) Conceptual Model	
7	Scenario 3: Pica Child Conceptual Model	
8	Scenario 4: Groundwater Conceptual Model	
9	Scenario 5: Surface Water Conceptual Model	
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CFR
DNA
HIV
HPC
ICC-PCR
ILSI
MAD
NRC
PCR
PM-io
PSRP
Ro
RNA
RT-PCR
SDWA
U.S. EPA
UV
LIST OF ABBREVIATIONS
Code of Federal Regulations
Deoxyribonucleic acid
Human immunodeficiency virus
Heterotrophic plate counts
Integrated cell-culture PCR
International Life Sciences Institute
Mesophilic anaerobic digestion
National Research Council
Polymerase chain reaction
Particulate matter with a diameter between 2.5 and 10 micrometers
Process to significantly reduce pathogens
Reproductive number (R naught)
Ribonucleic acid
Reverse transcriptase PCR
Safe Drinking Water Act
United States Environmental Protection Agency
Ultraviolet
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PREFACE
The U.S. Environmental Protection Agency (EPA), Office of Research and
Development (ORD), National Center for Environmental Assessment (NCEA) prepared
this document for Federal, State, and Local risk managers and assessors, contractors,
and other interested parties who may be interested in conducting microbial risk
assessments on land-applied biosolids.
In 2002, the National Research Council (NRC) released a report entitled:
"Biosolids Applied to Land: Advancing Standards and Practices" which was a review of
EPA's regulation "The Standards for the Use or Disposal of Sewage Sludge" (otherwise
known as the "Part 503 Rule" found in Title 40 of the Code of Federal Regulations
published in 1993). In 2003, EPA released a final action plan for setting new priorities
for the biosolids program which included the Agency's response to the NRC report
called "Standards for the Use or Disposal of Sewage Sludge; Final Agency Response to
the National Research Council Report on Biosolids Applied to Land and the Results of
EPA's Review of Existing Sewage Sludge Regulations" (68 FR 75531).
This present report follows-up on one project listed in EPA's action plan;
specifically, to provide a problem formulation and analysis plan relating to uncertainties
associated with conducting quantitative microbial risk assessments on land-applied
biosolids (68 FR 75540). In particular, this work focuses on critical human health
assessment endpoints and potential pathogens that should be considered; develops
conceptual models linking the most likely stressors, pathways and health responses of
concern; evaluates the overall quality and utility of available risk assessment data, tools
and methodologies; and develops an analysis plan suggesting additional research and
methods for improving risk assessments in this topical area and supporting EPA's
decision needs.
This report is based on the results of a literature review and summary first
conducted in 2008 and then updated again in 2010 based on feedback received from an
expert panel of independent, external peer reviewers and from public comments. The
intent of this document is to improve the planning of future risk assessments of land-
applied biosolids and to assist in the design of specific research to fill current data gaps
on this important environmental topic.
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AUTHORS, CONTRIBUTORS AND REVIEWERS
AUTHORS
Rebecca Efroymson
Environmental Sciences Division
Oak Ridge National Laboratory
Oak Ridge, TN 37831
Audrey Ichida
ICF International
Fairfax, VA
Anthony Armstrong
Environmental Sciences Division
Oak Ridge National Laboratory
Oak Ridge, TN 37831
Joy Lee
ICF International
Fairfax, VA
CONTRIBUTORS
Michael Troyer
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
Glenn Suter II
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
INTERNAL REVIEWERS
Michael Broder
Office of the Science Advisor
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC 20460
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AUTHORS, CONTRIBUTORS AND REVIEWERS cont.
James Smith
National Risk Management Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
Richard Stevens
Health and Ecological Criteria Division
Office of Science and Technology
Office of Water
U.S. Environmental Protection Agency
Washington, DC 20460
EXTERNAL PEER REVIEWERS
Joseph Eisenberg
Department of Epidemiology
University of Michigan
Charles Menzie
Director, Eco Sciences Practice
Exponent
Ian Pepper
The Environmental Research Lab
University of Arizona
Jeffrey Soller
Soller Environmental
Marylynn Yates
Professor of Environmental Microbiology
University of California-Riverside
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EXECUTIVE SUMMARY
Approximately 3.4 million tons of biosolids, dry weight, are land-applied annually
to farms, forests, rangelands, mine lands, and other land use types (Pepper et al., 2006;
NRC, 2002). Biosolids are defined by the U.S. Environmental Protection Agency (EPA)
(U.S. EPA, 1995) as "the primarily organic solid product yielded by municipal
wastewater treatment processes that can be beneficially recycled" as soil amendments.
Concerns for potential human health effects from land-applied biosolids can be
addressed through the conduct of a risk assessment. This report focuses on the
systematic planning step (a "problem formulation" defining the major factors to be
considered) for risk assessments of pathogens in land-applied biosolids. This report
follows the common problem formulation steps of hazard identification, conceptual
model development, and the development of an analysis plan. A review of pathogens in
biosolids literature forms the basis of this report. The intended use of this document is
to assist in the development of future risk assessments and to identify specific research
needed to fill current data gaps.
Policy Background
In 1993, EPA's Standards for the Use or Disposal of Sewage Sludge (Title 40 of
the Code of Federal Regulations Part 503) standardized the land application practices
of biosolids. Risk-based limits were established only for chemicals. Regulations for
pathogens in biosolids were developed based on existing methods for detection. Since
the promulgation of the Part 503 rule, a body of scientific data and methodologies has
become available for re-examination of EPA's management of microbial pathogens in
land-applied biosolids. In 2002, the National Research Council (NRC) convened a
review of the Part 503 rule by the Committee on Toxicants and Pathogens in Biosolids
applied to land and published a report entitled, Biosolids Applied to Land: Advancing
Standards and Practices. The report identified a critical need to update the scientific
basis of the Part 503 rule and made recommendations for EPA to improve the biosolids
program. This problem formulation document represents one of many responses to the
NRC report since that time (U.S. EPA, 2003b).
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Scope
This document focuses on potential microbial risks to human health by
pathogens in land-applied biosolids. The particular product of concern is Class B
biosolids originating from human waste, not Class A biosolids which are treated to
reduce the numbers of pathogenic organisms to below limits of detection.
Considerations of animal waste are excluded from the scope of this problem
formulation. Pathogens that may be present in Class B biosolids include bacteria,
viruses, protozoa, and helminthes (see Section 2, Hazard Identification). Exposure
routes of inhalation, ingestion, and dermal exposure leading to human infection and
diseases are discussed in Section 3, but issues relating to occupational risks do not fit
under the regulatory authority of EPA and, as such, are not included in this report. The
conceptual models illustrated and narrated here are meant to be generic and broadly
useful, and can be adapted for use in site-specific risk assessments. Overall,
five scenarios of common public concern are described:
1)	Neighboring residences and schools
2)	Farm Residents
3)	Pica child
4)	Drinking water consumers of groundwater
5)	Drinking water consumers of surface water
The following assumptions are made in the conceptual models developed for this
generic problem formulation document:
•	Class B biosolids have higher potential levels of human pathogens than Class A
biosolids
•	All sewage treatment practices meet standards
•	All sewage treatment technologies operate as intended
•	Storage facilities are bounded by effective physical barriers
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• Modifications to the conceptual models presented here; for example, to relax or
add to the assumptions above, or to account for other concerns (e.g., secondary
transmission, etc.), can be considered on a case-by-case basis by users of this
document
This document provides useful suggestions, but does not serve as guidance for
how one would conduct any specific risk assessment. The analysis plan chapter (see
Section 5) describes exposure measures, detection methods, data quality needs,
dose-response models, and uncertainty analysis. Research needs for future defensible
risk assessments are identified without prioritization in the Appendix.
Utility
Major products of this problem formulation document are the generic conceptual
models illustrating key relationships between potential pathogens in land-applied
biosolids and human health. Risk assessors may use these to develop conceptual
models more applicable to their particular scenarios. Risk managers may also use this
document to help develop the meaningful questions that risk assessors need to address
in order to reach a health protective outcome. The analysis plan will benefit researchers
as a summary of the measures, methods, and data needs for improving risk
assessment. Researchers will also likely find the Appendix useful as an organized
summary of current, peer-reviewed scientific studies relevant to land-applied biosolids.
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1. INTRODUCTION
In January 2004, the U.S. Environmental Protection Agency (U.S. EPA) released
a final action plan for setting new priorities for the biosolids program, which included the
Agency's response to the National Research Council (NRC) report entitled Biosolids
Applied to Land: Advancing Standards and Practices (NRC, 2002). This report serves
as an important step in the Agency's response to the NRC report. Presented here is a
development of a problem formulation and analysis plan for conducting quantitative
microbial risk assessments on land-applied biosolids. For risk assessment the NRC
recommends seeking stakeholder input. For this document stakeholder input was
gathered through public comments and peer review.
This report summarizes the existing literature on the microbial risks to humans
posed by pathogens in land-applied biosolids (see the Appendix); defines critical
microbial hazards; develops conceptual models linking the most likely hazards,
pathways and health responses of concern; evaluates the overall quality and utility of
available risk assessment data, tools and methodologies; and develops an analysis plan
which identifies the research and methods required for providing a scientifically
defensible risk assessment relevant for EPA's decision needs.
"Problem formulation is a systematic planning step that identifies the major
factors to be considered in a particular assessment" (U.S. EPA, 2003a). It was
developed for ecological risk assessment and subsequently adopted for cumulative
human health risk assessments (U.S. EPA, 1998,2003a). The principal products of
problem formulation are a conceptual model and an analysis plan (U.S. EPA, 2003a).
As a guide to problem formulation development, this report organizes current
literature and presents conceptual models for a generic framework. For use in specific
cases, further refining of information is recommended. Two particular audiences may
find this document most helpful. First, assessors who seek to assess risks to human
health from land-applied biosolids can use this generic problem formulation as a basis
for developing their own problem formulations. It can point users to information
sources, serve an introduction to the relevant literature, and facilitate the design process
for different types of risk assessments. Second, researchers and research planners can
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use the research needs identified in this report to select and prioritize research projects
related to pathogens in biosolids. It can also help researchers to understand how to
design their studies so as to generate results relevant to risk assessment.
There are many examples of microbial risk assessments conducted by Federal
agencies. The risk assessment performed for the Long Term 2 Enhanced Surface
Water Treatment Rule, for example, has all the major features of a quantitative
microbial risk assessment using the human health chemical risk assessment framework
also suggested here (U.S. EPA, 2006b).
Figure 1 illustrates a generalized framework for human health risk assessments
(adapted from ILSI, 2000). Note that the document you're reading now focuses on the
Problem Formulation (Model Development) stage shown in Figure 1. Some of the
major sources of information on risk assessment frameworks evaluated during the
development of this problem formulation document include the following:
•	National Academy of Sciences, National Research Council, Risk Assessment in
the Federal Government: Managing the Process (NRC, 1983);
•	National Academy of Sciences, National Research Council, Science and
Decisions: Advancing Risk Assessment (NRC, 2009);
•	EPA Office of Water/International Life Sciences Institute (ILSI) Risk Science
Institute Revised Framework for Microbial Risk Assessment (ILSI, 2000);
•	EPA Guidelines for Ecological Risk Assessment (U.S. EPA, 1998); and
•	EPA Lessons Learned on Planning and Scoping for Environmental Risk
Assessments (U.S. EPA, 2002a).
Problem formulation was developed as part of the ecological risk assessment
framework. However, it has been adapted to human health risk assessment in various
contexts including cumulative risk assessment (U.S. EPA, 2003a) and microbial risk
assessment (ILSI, 2000). The National Academy of Sciences has recommended that
human health risk assessors emulate ecological risk assessors in problem formulation
(NRC, 2009).
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MICROBIAL RISK ASSESSMENT
Risk characterization
Characterization of
exposure
Characterization of
human health effects
PROBLEM FORMULATION
(MODEL DEVELOPMENT)
FIGURE 1
General Framework for Assessing the Risks of Human Diseases Following
Exposure to Pathogens
Source: Adapted from ILSI (2000).
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1.1. SCOPE
This report focuses on land applied Class B biosolids, which are defined by a
combination of treatment requirements and subsequent site restrictions. Class A
biosolids are not specifically considered in this document because they are treated to
reduce the numbers of pathogenic organisms to below limits of detection. Furthermore,
Class A biosolids generally have a reduced level of biodegradable components that
could attract vectors and are thus generally unregulated for use in commerce. It is
assumed here that, based on applicable treatment regimes, Class B biosolids have a
greater potential for higher levels of microbial pathogen hazards than Class A biosolids.
Occupational exposure is not addressed in this document because the Occupational
Safety and Health Administration is the primary agency responsible for regulating
human health risks in occupational settings. Studies of untreated animal manures are
also beyond the scope of this report. This problem formulation document also focuses
on microbial hazard risk assessment planning and therefore does not include
discussions of chemical or physical hazards potentially associated with land applied
biosolids.
Although EPA's Guidelines for Ecological Risk Assessment (U.S. EPA, 1998)
were considered during the development of this document, the scope of this document
is limited to human health and does not include any ecological effects of land applied
biosolids. Furthermore, scenarios that include noncompliance with existing regulations
are not included.
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2. HAZARD IDENTIFICATION
Hazards1 are agents that may adversely affect human health or other
assessment endpoints. The identification of hazards is a necessary precursor to
developing conceptual models, especially for risk assessments of a complex substance
like biosolids. Similar to what EPA (1998) describes to be important questions to
answer in ecological risk assessments, a human health risk assessment for microbial
pathogens in biosolids should consider the following:
1.	What is the source of the pathogens?
2.	What is the spatial extent of the source?
3.	What types of hazards are present: bacterial, viral, or others?
4.	What are the modes of action of the hazards?
Essentially, hazards and their sources should be identified and characterized well
enough to inform the development of conceptual models, as well as, the more detailed
exposure pathways covering all reasonable exposure scenarios relevant to a particular
case. For example, pathogens in bioaerosols come from a different source than those
that remain in biosolids-amended soil particles. The resultant differences in pathogen
fate and transport and other factors can all be discussed in problem formulation
documentation.
This report focuses on pathogens and endotoxins originating in biosolids. For
use in the development of a specific risk assessment, assessors should include, in
addition to descriptions of microorganisms in biosolids, aspects of the biosolids matrix
that affect pathogenicity, and dimensions of the source that affect how exposure is
modeled or monitored.
This chapter describes the source of biosolids, including the components of the
mixture, the extent of the source, the matrix, the Class B treatment process, site
1 In human health risk assessment the term "hazard" has been adopted and in ecological risk assessment
the term "stressor" is used.
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restrictions, and vector attraction reduction options. Following the description of the
source, this chapter provides pertinent information about bacterial, viral, protozoan, and
helminth pathogens, as well as endotoxins that may be present in biosolids and may
cause adverse effects to human health.
2.1. SOURCE
Approximately 3.4 million tons of biosolids, dry weight, are land-applied annually
to farms, forests, rangelands, mine lands and other land use types. Only about 0.1 % of
available agricultural land in the United States is treated with biosolids (Pepper et al.,
2006; NRC, 2002). These soil amendments are thought to have beneficial nutrients for
plant growth as well as components to improve the physical properties of soils. The
EPA did not use the term biosolids in the Part 503 rule, but EPA (1995) defines
biosolids as "the primarily organic solid product yielded by municipal wastewater
treatment processes that can be beneficially recycled" as soil amendments. The NRC's
definition of biosolids is "sewage sludge treated to meet the land-application standards
in the Part 503 rule or any other equivalent land application standards" (NRC, 2002).
Pathogen standards are technologically based requirements "aimed at reducing the
presence of pathogens and potential exposures to them by treatment or a combination
of treatment and use restrictions" (NRC, 2002).
Biosolids are complex mixtures that contain organic and inorganic compounds
and organisms from wastewaters of households, commercial and industrial facilities, as
well as compounds added or formed during wastewater treatment processes (NRC,
2002). Inorganic and organic contaminants in biosolids are also described in NRC
(2002) and may include metals, trace elements, polychlorinated biphenyls, dioxins,
pharmaceuticals, surfactants and other contaminants. Class B biosolids are host to a
microbial community that potentially includes human pathogens, whereas Class A
biosolids have been treated to reduce the numbers of human pathogens to below
detectable levels, Thus, risks to human health from potential pathogens in Class B
biosolids are the primary focus of this problem formulation.
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2.1.1.	Spatial Extent of Source
Risk assessors need to characterize the areal extent of biosolids application or
storage that is specific to a particular risk assessment. Biosolids may be localized (e.g.,
a pile of stored biosolids is a continuous point source), more diffuse (e.g., a land applied
field is a continuous source that may be classified as a point source or a nonpoint
source), or mobile (e.g., a biosolids applicator is a short term source that moves from
location to location). Pathogen transport models may be specific to the spatial extent of
the source and include considerations for proximity of pathogens to humans. Large
piles of biosolids that serve as temporary storage before placement can represent
continuous, localized sources of pathogen-containing bioaerosols (described below)
(Dowd et al., 2000). Similarly, bioaerosols can be created during the transport of
biosolids from one location to another at a site, during the 'front-end loading' or
"shoveling" of biosolids from one pile to another, or from the lifting of biosolids-amended
soil particles by strong winds (Pillai, 2007). Areas of application may be large fields or
more localized windrows. If the risk assessment is intended to estimate cumulative risk,
then biosolids application in adjacent fields (to the site where maximum exposure
occurs) over time may be pertinent. A risk assessment may even address larger spatial
scales, for example, the entire area treated with biosolids either nationally, or by state.
2.1.2.	Reproduction
In addition to providing physical reservoirs of pathogens, biosolids and
biosolids-amended soils can serve as sources of additional pathogens if some of the
bacterial organisms reproduce (Zaleski et al., 2005a). Evidence about regrowth or lack
of regrowth of particular species in Class B biosolids is important information to include
in a conceptual model.
2.1.3.	Matrix
Four principal biosolids-containing matrices are possible sources of pathogens:
liquid biosolids, solid biosolids, biosolids-amended soil and bioaerosols created from
biosolids. Bioaerosols emerged as an important potential source of pathogens during
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the literature review for this report and are therefore important to consider during the
problem formulation stage of risk assessments pertaining to land-applied biosolids.
1.	Liquid biosolids. Liquid biosolids are the texture of muddy water and usually
contain 2-8% solids (Paez-Rubio et al., 2007). They are expensive to transport.
2.	Soiid biosoiids. Biosolids cake (15-30% solids content) (Paez-Rubio et al., 2007;
Meckes, 2011) is dewatered biosolids with the texture of a wet sponge (Virginia
Department of Health, 1999).
3.	Biosolids-amended soil. Over repeated applications, biosolids-amended soil has
different physical properties from soil alone. The altered physical properties of
soil include increased water holding capacity, water infiltration and stability of soil
aggregates (Brown and Henry, 2002).
4.	Bioaerosols, Bioaerosols are aerosolized biological particles that vary from
0.02-100 pm in diameter. They are formed when dewatered biosolids are
loaded into application equipment or when liquid and dewatered biosolids are
spread onto land (Paez-Rubio et al., 2007). The following information comes
from references in Pillai and Ricke (2002) and Pillai (2007). The size,
composition and concentration of microbial populations comprising aerosols vary
with biosolids source, method of application and meteorology and other
environmental conditions at the biosolids application site. Bioaerosols generated
from water sources (e.g., liquid biosolids) usually have a thin layer of moisture
surrounding clusters of microorganisms. Bioaerosol particles have a net charge
that depends on the source characteristics and can affect deposition rates.
Factors that control bioaerosol transport include the size, density and shape of
particles or droplets, as well as wind speed, relative humidity and temperature.
When some aerosolized bacteria are exposed to high relative humidity, they sorb
water, which protects the cells from inactivation by ultraviolet (UV) light (Peccia
et al., 2001).
2.1.4. Treatment
A description of the sewage sludge treatment process provides risk assessors
with information about the potential pathogen content of biosolids. Treatment methods
are intended to reduce the volume and organic content of biosolids and to reduce the
number of pathogens while retaining beneficial properties for fertilization and other soil
amendment and land reclamation purposes (NRC, 2002). The Part 503 rule defines
two categories of biosolids: Class A biosolids, which have no detectable concentrations
of pathogens, and Class B biosolids, which have detectable concentrations of
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pathogens (U.S. EPA, 1993). The example conceptual models presented in this report
focus on Class B biosolids, which are defined by a combination of treatment
requirements and site restrictions. The treatment of these biosolids must meet one of
three criteria: fecal coliform count of less than 2 x 106/gram of dry solids at the time of
disposal, treatment by a process to significantly reduce pathogens (PSRP), or treatment
by a process equivalent to a PSRP. In the absence of standardized methodologies for
developing risk-based processes, five operational-based processes in the Part 503 Rule
(listed below) were determined to be PSRPs, based on their resulting fecal coliform
concentrations less than 2 x 106/gram of dry solids and their ability to reduce
Salmonella and enteric virus levels by a factor of 10 (U.S. EPA, 1999):
1.	Aerobic digestion at specific combinations of time and temperature
2.	Air drying for three months, with average ambient daily temperatures above
freezing for at least two months
3.	Anaerobic digestion for specific combinations of time and temperature
4.	Composting for specific combinations of time and temperature
5.	Lime stabilization to give a pH greater than 12 after 2 hours of contact
Fecal conforms are enteric bacteria that are used as indicators of the potential for
the presence of bacterial pathogens. Salmonella species are human pathogens.
Changes in indicator and pathogen loads before and after the promulgation of the
Part 503 rule have been documented (Pepper et al., 2008b). In this problem
formulation, it is assumed that treatment requirements and site restrictions meet
standards. If sewage sludge is dewatered, thickening agents such as ferric chloride,
lime, or polymers are added (NRC, 2002).
2.1.5. Site Restrictions
Site restrictions also provide information about the content of biosolids to which
humans are exposed, because, although pathogens attenuate overtime, soil amended
with Class B biosolids may not be considered free from pathogens for at least one year
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following treatment (Gibbs et al., 1997). Site restrictions are required to reduce contact
with Class B biosolids until environmental exposures such as heat and desiccation have
decreased concentrations of bacterial, viral and helminth pathogens to below detectable
concentrations equivalent to those in Class A biosolids (NRC, 2002). Natural
attenuation also incorporates biological factors such as competition, predation,
hyperparasitism (growth of a secondary microorganism in or on the primary pathogen or
parasite) and antibiosis (Smith et al., 2005a). Site restrictions to public access, grazing
and harvesting are included (see Table 1).
2.1.6. Vector Attraction Reduction
The Part 503 rule requires that one of twelve management options be used to
control disease vectors. These are described in detail in the rule and in NRC (2002):
volatile solids reduction, specific oxygen uptake rate, anaerobic bench-scale test,
aerobic bench-scale test, aerobic process for compost, pH adjustment, drying without
primary solids, drying with primary solids, injection, and incorporation. The first
eight options are process-based options, the first five of which are intended to contribute
to long-term stabilization through the degradation of putrescible organics. Injection of
biosolids and incorporation within six hours of application are considered physical
barriers to vector attraction.
2.2. PATHOGENS
A variety of bacterial, viral, protozoan, and helminth pathogens may be present in
Class B biosolids. The relative quantity of pathogen shedding in the contributing human
population will influence the prevalence of pathogens in biosolids. Therefore, fecal
borne pathogens that are rare in the United States might be less of a concern than
endemic pathogens and a pathogen that is responsible for a community outbreak
situation. In a study conducted over 20 years (1986-2006), Zerzghi et al. (2009)
reported no bacterial or viral pathogens were detected in soil samples collected from
biosolid amended plots in December (10 months after the last land application)
demonstrating that pathogens introduced via Class B biosolids survived in soil only
transiently.
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TABLE 1
Site Restrictions for Class B Biosolids (40 CFR 503)*
40 CFR 503.14 Management Practices
(a)	Bulk sewage sludge shall not be applied to the land if it is likely to adversely affect a threatened or
endangered species listed under Section 4 of the Endangered Species Act or its designated critical
habitat.
(b)	Bulk sewage sludge shall not be applied to agricultural land, forest, a public contact site, or a
reclamation site that is flooded, frozen, or snow-covered so that the bulk sewage sludge enters a wetland
or other waters of the United States, as defined in 40 CFR 122.2, except as provided in a permit issued
pursuant to section 402 or 404 of the CWA.
(c)	Bulk sewage sludge shall not be applied to agricultural land, forest, or a reclamation site that is
10 meters or less from waters of the United States, as defined in 40 CFR 122.2, unless otherwise
specified by the permitting authority.
(d)	Bulk sewage sludge shall be applied to agricultural land, forest, a public contact site, or a reclamation
site at a whole sludge application rate that is equal to or less than the agronomic rate for the bulk sewage
sludge, unless, in the case of a reclamation site, otherwise specified by the permitting authority.
40 CFR 503.32[b] [5] Pathogens
(i)	Food crops with harvested parts that touch the biosolids/soil mixture and are totally above the land
surface shall not be harvested for 14 months after application of biosolids.
(ii)	Food crops with harvested parts below the surface of the land shall not be harvested for 20 months
after application of biosolids when the biosolids remain on the land surface for four months or longer prior
to incorporation into the soil.
(iii)	Food crops with harvested parts below the surface of the land shall not be harvested for 38 months
after application of biosolids when the biosolids remain on the land surface for less than four months prior
to incorporation into the soil.
(iv)	Food crops, feed crops and fiber crops shall not be harvested for 30 days after application of
biosolids.
(v)	Animals shall not be grazed on the land for 30 days after application of biosolids.
(vi)	Turf grown on land where biosolids is applied shall not be harvested for one year after application of
the biosolids when the harvested turf is placed on either land with a high potential for public exposure or a
lawn, unless otherwise specified by the permitting authority.
(vii)	Public access to land with a high potential for public exposure shall be restricted for one year after
application of biosolids.
(viii)	Public access to land with a low potential for public exposure shall be restricted for 30 days after
application of biosolids.
*http://yosemite. epa.gov/r10/water. nsf/NPDES%20Permits/Sewage%20S825/$FILE/503-032007.pdf.
CFR = Code of Federal Regulations; CWA = Clean Water Act.
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Risk assessors should consider and list the range of possible pathogens in the
problem formulation, though it may be necessary to focus on only a limited number
based on the specifics of each individual risk assessment. For example, the source,
transport and fate of pathogens in applied biosolids may be considered, including:
(1) the origin of the human waste, (2) the likelihood of each pathogen to survive sewage
treatment, and (3) their fate in soil. For additional considerations about these and other
pathogen factors, see the EPA Office of Water's Draft Protocol for Microbial Risk
Assessment to Support Human Health Protection for Water-Based Media (U.S. EPA,
2009a). Many of the potential pathogens in Class B biosolids and the diseases they
may cause are summarized in Table 2. Due to the location and objective of each study
cited, researchers who list principal pathogens of concern in sewage sludge and/or
biosolids do not always list the same organisms (Dudley et al., 1980; NRC, 2002; Gerba
and Smith, 2005; Pepper et al., 2006, 2010; Epstein, 2006; Yanko, 2005). Unlike
chemical hazards, pathogens can multiply and some bacteria can reproduce outside of
the host organism under favorable environmental conditions. The types and levels of
pathogens in biosolids are determined by the incidence of infection within the biosolids
generating community and the type of treatment process used (Straub et al., 1993).
The biosolids matrix (i.e., biosolids, biosolids-amended soil, bioaerosols, or biosolids
particles in water) may affect the fate of pathogens, and therefore determine exposure.
Additional factors to consider for conducting risk assessments may include the
accessibility, attenuation, and the bioavailability of the pathogens, as well as site factors
such as underlying geology, soil type, and depth to groundwater. Considerations for
screening pathogens from inclusion are presented for each class of pathogens
throughout the remainder of this section. It should be noted that, in general, pathogens
without dose-response data are not candidates for quantitative microbial risk
assessment. However, qualitative microbial risk assessment would still be possible.
References that provide helpful information for prioritizing pathogens of concern include
Sidhu and Toze (2009) and Smith et al. (2005a).
For risk assessors who are familiar with chemical risk assessment it is important
to note that pathogens differ from chemicals in several ways that are important for risk
assessment. Some of these differences include:
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•	Microbial Growth and Death—Some pathogens can multiply in the environment
and all multiply in hosts. Different species, and even different strains within a
species, grow and die in unique patterns. In contrast, although chemicals can
bioaccumulate and bioconcentrate, they are not known to multiply in the
environment or hosts. Both chemicals and pathogens can decrease due to
environmental factors; chemicals can be transformed or degrade and pathogens
can die.
•	Detection Methodologies—Generally, methods for detecting chemical pollutants
are sufficiently sensitive to detect and quantify concentrations well below the
levels that are known to have human health effects. This is not necessarily the
case for pathogens. Theoretically, a single pathogenic organism can cause
infection (and lead to illness). Analytical methods for detecting low levels of
pathogens (e.g., one organism) are not sufficiently developed to be reliable.
•	Genetic Diversity of Pathogens—Microorganisms are genetically diverse and
allelic ratios in a population can change significantly within a few generations. In
addition, microbial genomes can evolve quickly (within days or weeks) through
mutation or horizontal gene transfer. Pathogens in biosolids would presumably
be reflective of the pathogen status of the generating community.
•	Host Immunity and Susceptibility—Infection and illness due to pathogens is, in
some cases, highly dependent on the immune status of the individual, which can
fluctuate based on time since last exposure, presence of concurrent infections
(e.g., human immunodeficiency virus [HIV]), and a number of other factors (e.g.,
life stages, gender, genetics). Although body weight, age, and metabolic
capacity differences are considered in the development of chemical criteria,
genetic and acquired differences in susceptibility are not usually considered.
Secondary Transmission—Microbial infections can be transmitted from an
individual to other susceptible individuals, and even to some animals. With the
exception of the mother-fetus relationship, chemicals in tissues of exposed individuals
are not known to transmit to other individuals. Chemicals that are on exposed
individuals' clothing or skin can be transferred to household and other contacts. But this
type of chemical exposure would not be amplified compared to the primary exposed
individual, whereas pathogen secondary transmission can amplify the disease
incidence.
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TABLE 2
Example Pathogens of Potential Concern in Sewage Sludge and Biosolids
Class
Organism
Disease or Symptoms
Bacteria
Listeria
monocytogenes
Meningitis, encephalitis, septicemia, intrauterine or cervical
infections with abortion

Helicobacter pyiori
Stomach ulcers, gastritis, increased risk of stomach cancer

Campylobacter
jejuni
Gastroenteritis

Pathogenic
Escherichia coli
Gastroenteritis, hemolytic uremic syndrome

Shigella spp.
Bacillary dysentery

Salmonella spp.
Salmonellosis (food poisoning), typhoid/paratyphoid fever

Yersinia spp
Yersiniosis (gastroenteritis)

Legionella spp.
Severe respiratory illness, mild flulike illness
Viruses
Astroviruses
Gastroenteritis

Rotaviruses
Gastroenteritis

Caliciviruses
Gastroenteritis

Adenoviruses
Respiratory diseases, gastroenteritis

Hepatitis virus A-E
Infectious hepatitis, liver inflammation, hepatic cancer
Helminth
Parasites
Taenia spp.
Nervousness, enteric distress, abdominal pain, anorexia,
insomnia

Ascaris
lumbricoides
Digestive disturbances, abdominal pain, transitory liver and
lung disease

Trichuris spp.
Gastrointestinal distress, anemia

Toxocara canis
Fever, abdominal discomfort, neurological symptoms
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Table 2 (cont.)
Class
Organism
Disease or Symptoms
Protozoan
Parasites
Cryptosporidium
parvum
Diarrhea
Giardia lamblia
Fever, diarrhea
Cyclospora
Diarrhea, nausea, vomiting and abdominal cramps
Microsporidia
Diarrhea
Entamoeba
histolytica
Dysentary, colitis
Baiantidium coii
Diarrhea, constipation, abdominal pain
Sources: Dudley et al. (1980), Gerba and Smith (2005), Epstein (2006), NRC (2002), Pepper et al. (2006)
and Bowman and Fayer (2005).
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2.2.1. Bacteria
2.2.1.1.	Salmonella
All serotypes of this genus are pathogenic to humans and cause symptoms
ranging from mild gastroenteritis to severe disease and death. In the United States,
salmonellosis is mainly due to foodborne transmission because the bacteria found in
beef and poultry are able to grow in foods (Pepper et al., 2006). Salmonella can
apparently survive during sewage treatment and grow in biosolids under some
conditions (Sahlstrom et al., 2006). Class A biosolids allows for growth of Salmonella
under anaerobic concentrations (Zaleski et al., 2005a). Because of this potential for
growth, Pepper et al. (2006) argue that Salmonella are the bacteria of greatest concern
in Class B biosolids, although Skanavis and Yanko (1994) concluded a low probability
of infection in most scenarios. In 40 Code of Federal Regulations (CFR) 503,
Salmonella are the bacterial pathogen indicators for biosolids quality.
2.2.1.2.	Escherichia coli 0157.H7
Escherichia coli is found in the intestinal tract of humans and most warm-blooded
animals, and most strains are not pathogenic. However, several strains can cause
gastroenteritis. The greatest concern in the United States is enterohemorrhagic E. coli
of the serotype 0157:H7 (Pepper et al., 2006). The organism has been spread in
contaminated drinking water, through recreational water exposure, and contaminated
food (Yanko, 2005; Pepper et al., 2006; Barker et al., 1999). Cattle manure is the most
significant source of exposure, but the organism has been detected in biosolids too
(Lytle et al., 1999; Pepper et al., 2006).
2.2.1.3.	Campylobacter jejuni
This pathogen is the principal cause of bacterial diarrheal illness in the United
States (Mead et al., 1999). Food is the major source of infection. Little research has
been conducted to investigate the occurrence of Campylobacter in sewage sludges,
biosolids, or the environment (Yanko, 2005), though a few studies of raw and treated
sludge are reviewed in Pepper et al. (2006).
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2.2.1.4.	Shigellas pp.
Bacteria of this genus are closely related to E. coli. The bacteria are frequently
found in water contaminated with human sewage and are transmitted by the fecal-oral
route. Salads, raw vegetables, milk and dairy products and poultry sometimes are
contaminated with Shigella (Pepper et al., 2006). Shigella is more infective than most
enteric bacteria and secondary attack rates are high among children (Haas et al., 1999).
In addition to transmission in day care facilities, Shigella has also been transmitted
through unchlorinated wading pools, water fountains, food items such as parsley and
bean dip, men who have sex with men (CDC, 2008a), and surface waters due to bather
shedding (WHO, 2004). However, Shigella spp, do not survive well in the environment
or after treatment of biosolids. Therefore, they are not likely to be a significant problem
(Pepper et al., 2006).
2.2.1.5.	Yersinia Spp.
These bacteria cause gastroenteritis with diarrhea or vomiting, fever and
abdominal pain. Yersinia enterocolitica has been detected in environmental sources
such as ponds and lakes, though the major source of infection in the United States is
pork products (Pepper et al., 2006). Waterborne outbreaks have also occurred. In
Japan, infections of Y. pseudotuberculosis from contaminated water and foods have
been reported. The bacterium has been detected in raw, digested and dewatered
biosolids (Straub et al., 1993), but little information is available about background levels
or survival in soils or waters (Pepper et al., 2006).
2.2.1.6.	Listeria monocytogenes
This bacterium causes foodborne diseases, primarily in immunocompromised
people such as pregnant women. It can cause encephalitis, meningitis and intrauterine
or cervical infections (Epstein, 2006). L. monocytogenes has been detected in activated
and anaerobically digested biosolids (Watkins and Sleath, 1981; DeLuca et al., 1998).
The bacterium is widespread in the environment (Yanko, 2005).
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2.2.1.7.	Helicobacter pylori
This bacterium is the principal cause of stomach ulcers and is associated with
increased risk of stomach cancer.2 H. pylori may be the most common cause of
bacterial infection in humans (up to 90% of some populations are infected, Epstein
2005), though rates of infection are decreasing (Yanko, 2005). The source of many
infections is vegetables irrigated with untreated wastewater (Brown, 2000). The
digestive tract of humans is apparently the main reservoir of H. pylori (Yanko, 2005).
Whether H. pylori is present in Class B biosolids is unknown (Pepper et al., 2006).
2.2.1.8.	Legionella
Infections with Legionella can result in a life-threatening respiratory illness,
Legionnaires' Disease, especially in immunocompromised people or the elderly, or a
mild illness called Pontiac Fever. Outbreaks of Legionella usually occur through
airborne transmission of bacteria from hot water in building cooling towers or other
aerosolizing devices (Yanko, 2005). High concentrations have been measured in
biosolids at a food industry sewage treatment plant where workers contracted Pontiac
Fever (Gregersen et al., 1999; Yanko, 2005). Moreover, Yanko (2005) speculates that
the bacteria should grow well in "warm, self-composting organic masses." However,
there is no known case of either Legionnaires' Disease or Pontiac Fever associated with
the production or land application of biosolids.
2.2.1.9.	Screening Bacterial Pathogens
Some bacteria may be excluded from consideration in risk assessments of
pathogens in biosolids. For example, some experts believe that Staphylococcus aureus
"are not a likely source of...human exposure or infection" (Pepper et al., 2006). In a
study of 23 biosolids samples (16 Class B samples) from 15 U.S. sites, none contained
S. aureus (Rusin et al., 2003a). Similarly, analyses of 37 air samples were also
negative for the bacterium (Rusin et al., 2003a). However these studies have been
criticized because of the technical difficulties associated with organic matter-associated
2 National Cancer Institute, available online at:
http://www.cancer.gov/cancertopics/factsheet/risk/h-pylori-cancer (accessed 6/30/10).
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pathogen extraction (Lewis and Gattie, 2003). Although there is little information on the
fate of Vibrio cholera in biosolids treatment or land application, Yanko (2005)
recommends that the low incidence of this disease in the United States (0-5 cases per
year) is a good justification for focusing research on other pathogens.
2.2.1.10. Ranking Bacterial Pathogens
Risk assessors may also prioritize bacterial pathogens of concern in risk
assessments of land-applied biosolids. A workgroup of biosolids experts developed
methods for evaluating 20 potential pathogens in biosolids (see Chapter 4 in Smith et al.
[2005a]). They considered several factors such as a pathogen's public health
significance (number of infections or severity of disease), prevalence in biosolids and
sewage sludge, survival during wastewater treatment and the availability of appropriate
analytical methods. Similar criteria might be used by risk assessors in the problem
formulation.
2.2.2. Viruses
Over 140 types of enteric viruses are excreted by humans and are likely to be
present in municipal wastewater. Viruses are almost always detected in Class B
biosolids and by definition are not detected in Class A biosolids (Pepper et al., 2008a;
Gerba et al., 2002). Examples covered here include enteroviruses, rotaviruses,
caliciviruses, adenoviruses, astroviruses, and picornaviruses that may cause Hepatitis A
and E.
2.2.2.1. Enteroviruses
Enteroviruses include polioviruses, coxsackieviruses, and echoviruses. Both
fecal-oral and respiratory routes of infection are common. Enteroviruses are commonly
isolated from untreated biosolids. Generally, they are reduced by 90% or more during
the aerobic and anaerobic digestion of sludge which produces Class B biosolids
(Pepper et al., 2006).
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2.2.2.2.	Rotaviruses
These are the only double-stranded ribonucleic acid (RNA) viruses transmitted
through water to humans (NRC, 2002). Along with caliciviruses, rotaviruses are the
leading cause of gastroenteritis in the United States (Monroe et al., 2000; CDC, 2008b)
and a major cause of hospitalization of children in the United States (Gerba et al.,
1996a). These viruses cause waterborne and foodborne outbreaks in the United
States. They have been detected in wastewater, but little information is available
regarding their occurrence in biosolids (NRC, 2002).
2.2.2.3.	Caliciviruses
Caliciviruses may be the leading cause of water and foodborne illness in the
world and are a leading cause of viral gastroenteritis (Monroe et al., 2000). The
two genera are the Norovirus (previously known as Norwalk viruses) and Sapovirus (an
example species being Sapporo virus) (NRC, 2002).3 Recently researchers have
demonstrated that a highly differentiated 3-D cell culture model can support the natural
growth of human noroviruses, which is a major breakthrough in tools that will enable the
future study of noroviruses (Straub, 2007).
2.2.2.4.	Adenoviruses
These common and persistent viruses in wastewater (NRC, 2002) are the
second most common cause of childhood viral diarrhea (Gerba et al., 1996a). NRC
(2002) provides references indicating that recreational and drinking waters are
pathways of exposure for adenoviruses. Adenoviruses are present in untreated sewage
sludge (Gerba et al., 2002). Enteric adenoviruses have been detected in Class B
biosolids (NRC, 2002; Pepper et al., 2008a), and adenovirus type 40 has been detected
in anaerobically digested biosolids (NRC, 2002). Along with hepatitis A virus,
adenovirus is the most thermally resistant virus (Gerba et al., 2002). Using polymerase
chain reaction (PCR) human adenovirus genomes were found in 88% of the Class B
3 Virus taxonomy: available online at: http://talk.ictvonline.org/files/ictv_documents/rn/msl/1231.aspx
(accessed 6/30/10).
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biosolids sampled (Viau and Peccia, 2009). Overall, little is known about the removal of
adenoviruses by Class B treatment processes (Gerba et al., 2002).
2.2.2.5.	Astroviruses
These viruses are a cause of gastroenteritis, primarily in children. Foodborne
and waterborne outbreaks have occurred in the past. They have been found in
biosolids (Chapron et al., 2000), though little is known about their removal by Class B
treatment processes (Gerba et al., 2002).
2.2.2.6.	Hepatitis A
This picornavirus is responsible for infectious hepatitis. This pathogen is
transmitted by food and water, and primarily infects the liver. The highest infection rate
is among children 5-14 years old (CDC, 1999). Along with adenoviruses, Hepatitis A is
the most thermally resistant virus (Gerba et al., 2002). No information is available on
the prevalence of Hepatitis A in biosolids.
2.2.2.7.	Hepatitis E
This picornavirus, transmitted by the fecal-oral route, has been responsible for
major waterborne disease outbreaks in developing countries but has also been reported
frequently in travelers to those regions. It is the major cause of acute viral hepatitis in
developing countries (Gerba, 2005). Symptoms include jaundice, fatigue, abdominal
pain and nausea. Hepatitis E is a more serious infection than Hepatitis A, with case
fatalities of 2-3% in the general population and 20-30% in pregnant women (Haas
et al., 1999). No information is available on the prevalence of Hepatitis E in biosolids.
2.2.2.8.	Screening Viral Pathogens from Consideration
Some viruses may be excluded from consideration in biosolids by pathogen risk
assessors. For example, a workgroup on viruses in biosolids concluded that
blood-borne viruses such as HIV would be likely to be inactivated during wastewater or
biosolids treatment (Smith et al., 2005b). This workgroup also concluded that
lipid-containing viruses have low viability in water and may not survive wastewater or
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biosolids treatment. However, they recommended that lipid-containing viruses such as
rhinoviruses, influenza viruses and herpes viruses not be excluded from consideration
until it is known whether any survive treatment (Smith et al., 2005b).
2.2.3. Protozoa
Cryptosporidium and Giardia are the predominant diarrhea-causing protozoan
parasites transmitted through food and water in the United States. These parasites of
the small intestine have infective, environmentally resistant stages called cysts (for
Giardia) or oocysts (for Cryptosporidium). Pepper et al. (2006) review studies in which
Cryptosporidium and Giardia have been detected in sewage sludge and biosolids.
Oocysts do not survive under low moisture or high heat conditions, and therefore would
be expected to be inactivated during treatment and land application (Whitmore and
Robertson, 1995). This expectation has been confirmed by Bowman et al. (2000), who
found that these protozoa died within days of Class B biosolids treatment. However
Pepper et al. (2006) suggest that new cell culture methods are needed to assess
protozoan oocyst viability and confirm that these organisms do not present a hazard in
biosolids.
Additional protozoa could be present in sewage sludge and/or biosolids
(Bowman and Fayer, 2005). Cyciospora causes diarrhea, nausea, vomiting and
abdominal cramps. Toxoplasma gondii causes neurologic flu-like symptoms, retinitis
and if mothers are infected for the first time while pregnant the fetus can suffer death,
brain damage, hydrocephaly, and numerous less severe symptoms (APHA, 2004).
Some genera of Microsporidia cause diarrhea. Entamoeba histolytica causes severe
dysentery and extra-intestinal abscesses. Balantidium coli causes diarrhea and
constipation, but Bowman and Fayer (2005) suggest that their presence is less likely in
biosolids than that of other protozoa. Life histories of all of these species, as well as
potential effects of biosolids treatment, are summarized in Bowman and Fayer (2005).
Bowman and Fayer (2005) consider the potential hazards of various protozoa by
summarizing information on settling rates in wastewater and considering potential
resistance to disinfection. "Soft-shelled" protozoa (Balantidium, Entamoeba and
Giardia) will probably persist in effluents but not in biosolids. The Apicomplexan
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protozoa (Cryptosporidium, Cyclospora, Toxoplasma) probably react similarly (but
sometimes uncertainly) to the effects of different disinfection methods but settle at
different rates. Microsporidia have not been studied much in the context of biosolids
treatment (Bowman and Fayer, 2005).
2.2.4. Helminths
Several helminth species potentially occur in biosolids. Eggs of many helminth
species probably settle in wastewater, are resistant to sewage treatment methods, and
end up in biosolids (Bowman and Fayer, 2005).
2.2.4.1.	Trichuris trichiura
Trichuris (whipworm) is a genus of nematode that is parasitic in the cecum and
large intestine of mammals. It causes diarrhea. Human infections result from ingestion
of infected eggs. Eggs in wastewater would be expected to settle rapidly and be found
in sewage sludge wherever infected people are present in the community (Bowman and
Fayer, 2005). Eggs are not likely to be damaged by the quantities of ultraviolet, ozone,
or chlorine used for disinfection in wastewater treatment processes.
2.2.4.2.	Ascaris lumbricoides
Ascaris is a genus of nematode that is parasitic in the small intestine. Adult
worms may develop within the small intestine and cause digestive disturbances.
Transitory liver and lung disease is caused by larval migration (Bowman and Fayer,
2005). Human infections with Ascaris lumbricoides result from ingestion of infectious
Ascaris eggs usually from soil or produce grown in soil containing Ascaris eggs (APHA,
2004). Although Ascaris eggs appear to be present at very low densities in biosolids
and perhaps even in raw sewage sludge (NRC, 2002), the eggs of Ascaris are an
indicator in biosolids because of their resistance to most treatment processes and
representativeness of helminth egg viability.
2.2.4.3.	Taeniid Tapeworm Eggs
The life histories of taeniid tapeworms require a carnivore final host in which the
small intestine is infected (Bowman and Fayer, 2005). For Taenia solium the final host
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is humans or pigs. For Taenia saginata, eggs passed in the stool of humans are only
infectious to cattle. Human infection is from the ingestion of raw or undercooked beef
containing the larval stage (APHA, 2004). The adult worms cause few or no symptoms
in humans, but eggs can develop to a larval stage (cysticercus) that can cause central
nervous system and enteric symptoms. Although Taenia species are usually acquired
from ingestion of infected beef or pork, the eggs of this pathogen have been detected in
some biosolids (Barbier et al., 1990).
2.2.5. Endotoxins
Endotoxins are nonspecific lipopolysaccharide-protein complexes created from
the cell walls of gram-negative bacteria (DeLuzio and Friedman, 1973). They consist of
polysaccharide chains connected by a core oligosaccharide to a lipid portion, consisting
of a series of long-chain fatty acids, connected by amide and ester linkages to a
phosphorylated diglucosamine structure (Epstein, 2006). They may become airborne
when dried, pulverized to micrometer and submicrometer size particles, and agitated
(Smith et al., 2005a). In the bloodstream these toxins may cause a broad range of
physiological effects, including fever, coughing, breathlessness, flu-like symptoms,
inflammation, and shock (Yanko, 2005; Pepper et al., 2006; Epstein and Moss, 2006).
Endotoxins are relatively heat stable (Epstein, 2006).
Endotoxins have been measured in studies of air at composting plants, though
no evidence of residential impact was found because levels decreased to background
concentrations beyond site boundaries (Clark et al., 1983; Pepper et al., 2006).
Ambient levels of dust-associated endotoxin are high (Smith et al., 2005a; Pepper et al.,
2006). Endotoxin levels in Class B biosolids are similar to concentrations in animal
manures and composts (Brooks et al., 2006). Farming activities, such as driving a
tractor across a field, can result in comparable levels of aerosolized endotoxins as those
from land application of biosolids (Brooks et al., 2004a). In fact, most bacteria
aerosolized during land application are soil borne in origin (Brooks et al., 2007b). In
contrast, low concentrations of endotoxins were present in groundwater at two sites
where wastewater was applied to land (Yanko, 2005).
24

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2.2.6. Emerging Pathogens
The lists of pathogens covered in this document should not be considered
exhaustive. New pathogens are continually being identified or found in new areas for
several reasons such as: changes in the way foods are produced, the global
transportation of food and people, advances in molecular biology that permit the
identification of new pathogens and their sources, the evolution of pathogens, aging
demographics, and the use of microbial risk assessment to quantify risks from
environmentally transmitted pathogens (Gerba and Smith, 2005). Emerging pathogens
are novel pathogens that have not previously been characterized or established and
have only recently been considered hazards of concern in particular media. Gerba
et al. (2002) designated E. coli 0157:H7, H. pylori and L. monocytogenes as newly
emerging bacterial pathogens of potential concern in biosolids. Yanko (2005) points out
that many of these emerging bacterial pathogens do not fit the classic fecal-oral
transmission pattern. The NRC listed Mycobacterium, E. coli 0157:H7, Legionella,
Listeria and Microsporidia as emerging pathogens likely to be present in biosolids and
Adenovirus, Norovirus, Astrovirus, Hepatitis A, Rotavirus and Hepatitis E as emerging
viral pathogens likely to be present as well (NRC, 2002). Gerba (2005) listed several
emerging viruses without speculating which are likely to be in biosolids, including:
picobirnaviruses, picotrinaviruses, coronaviruses, and toroviruses. Yates and Yates
(2007) added selected bacteria, viruses and parasites to water and/or microcosms to
simulate Class A and Class B treatment. They observed that organisms surviving in the
highest numbers or numbers representing the highest risk included E. coli, Clostridium
perfringens spores, Listeria innocua and bacteriophage phi X174.
NRC (2002) identified criteria for selecting emerging pathogens for which
additional information on occurrence, persistence, and risk is justified, and for which
additional regulations may be needed. These criteria are useful for selecting pathogens
on which to focus the hazard characterization in a risk assessment:
•	Reliable viability assay
•	Wastewater-related disease-causing agents
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•	Extent of existing data on probability of surviving biosolids treatments (organisms
surviving at high pH above 11-12 and heat resistance are of greatest concern)
•	Extent of survival in the environment
Based on these criteria, NRC (2002) recommended E. coli 0157:H7,
adenovirus 40, astrovirus, hepatitis A virus and rotavirus in biosolids as priorities for
analysis. Caliciviruses would have been selected as a priority if methods for assessing
viability were available (NRC, 2002). With the recent publication on a dose-response
relationship for noroviruses (Teunis et al., 2008a), caliciviruses are now ready to
become priority pathogen for analysis. Legionella also merits investigation, but current
detection methods are inefficient, difficult to use and expensive (NRC, 2002).
2.2.7. Multiple Hazards
Microbial risk assessors typically assume that microbial pathogens act
independently of each other and that the probability of an adverse effect from one type
of pathogen is independent of the probability of an adverse effect from another.
However, microbial risk assessors may want to consider exposures to pathogens in
biosolids at offsite locations or other sources that are not the direct subject of a biosolids
risk assessment. This may allow for estimation of the risks of infectious disease from
biosolids combined with other sources of the same infectious disease.
There is no evidence to suggest that pathogens and chemicals such as metals in
biosolids have interactive effects in humans. However, Lewis et al. (2002) suggested
that chemical contaminants in biosolids might irritate the skin and mucous membranes,
thereby weakening the first line of defense in the human host, leading to an increase in
pathogen host susceptibility. In addition, other constituents in biosolids (e.g., chemicals,
metals) may have effects on human immune status (Germolec et al., 1991). Modeling
tools have not been developed that include nuances of human immune status due to
factors either associated with biosolids or not associated with biosolids. However, the
potential for such effects could be discussed when characterization of host susceptibility
is presented.
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3. DEVELOPMENT OF CONCEPTUAL MODELS, ENDPOINTS AND SCENARIOS
A conceptual model for a risk assessment is a representation of the assumed
relationships between sources and effects (Suter, 1999) or between hazards and
assessment endpoints (U.S. EPA, 1998). Multiple models may be developed for
multiple scenarios. The written descriptions of the risk hypotheses, accompanied by
diagrams (termed conceptual models) that illustrate the key relationships, are among
the primary products of the problem formulation (U.S. EPA, 1998). Conceptual models
"provide a framework for prediction and are the template for generating more risk
hypotheses." They form the basis for developing quantitative exposure and effects
models for the risk assessment. The models tend to emphasize exposure pathways,
including indirect exposures, over mechanisms of effects. Conceptual models have
been developed for human health risk assessments of pathogens in biosolids that
include detailed source descriptions, transport pathways and routes of exposure
(Colford et al., 2003; Eisenberg et al., 2004, 2005, 2006).
For this report, EPA developed conceptual models illustrating the potentially
important human exposure pathways for pathogens in biosolids that have been applied
to land. These models are developed in response to NRC's assertion that "EPA should
develop a conceptual site model to identify the major and minor exposure pathways
(including secondary transmission) by which humans might come into contact with
pathogens in biosolids" (NRC, 2002). The models are applicable to biosolids
amendments to cropland, pasture land, forests, mineland (for reclamation), or other
uses. The conceptual models presented here are limited to primary transmission, i.e.,
exposure of humans to pathogens from biosolids without an intermediate human host.
Secondary transmission is infection by pathogens that were shed by infected people.
This problem formulation does not provide detailed advice concerning incorporating
estimates of secondary infection because the process is not unique to pathogens in
biosolids. This does not mean that secondary transmission of pathogens in this context
is assumed to be unimportant. For example, pathogens with a high basic reproductive
number (R0), have a high potential for secondary transmission4 (Heffernan et al., 2005)
4 A helpful explanation of R0 can be found at http://wiki.medpedia.com/lnfluenza_Disease_Transmission.
27

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and might merit consideration of susceptible, infected, and resistant (SIR)
populations-type modeling approaches (Smieszek, 2009) for problem formulation. A
tiered strategy for a risk assessment could include initial screening for data on R0, and
use of primary disease modeling for pathogens of low transmissibility and consideration
of also including secondary transmission modeling for pathogens of high
transmissibility. A variety of mathematical models have been formulated,
mathematically analyzed, and applied to infectious disease transmission (Hethcote,
2000).
The conceptual models presented in this report are not meant to imply that the
risk assessor must assume that adverse health effects are caused by exposure to
pathogens in land-applied biosolids. Rather, the models present potential pathways for
consideration and supporting evidence on a case-by-case basis. In addition, the
conceptual models do not consider contributions from background pathogens not
associated with biosolids.
This chapter first presents an overall, general conceptual model for risks from
pathogens in land-applied biosolids (see Figure 2), as well as a narrative description of
the model. The model is a cascade of processes and states (Suter, 1999) that indicates
the mechanisms by which the pathogen hazards potentially contact human hosts to
produce infection and disease. A description of the source (methods and rates of land
application), environmental fate and transport processes, routes of exposure, host
susceptibility factors, infection, and disease are also included. Next, five additional
exposure scenarios are presented to demonstrate how Figure 2 (the general conceptual
model) can be tailored to more specific scenarios of interest. All the conceptual models
presented here may be modified as more knowledge is available on a case-by-case
basis.
Each conceptual model contains routes of exposure considered to be potentially
significant in many instances. Additional routes may be considered when there are
different concerns, or more specific concerns requiring more detail. For example,
indirect routes (not included here) involving human consumption of livestock, dairy
products, wildlife, fish or shellfish exposed to pathogens could be added. Conversely,
28

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Pathogens in
sewage sludge
Treatment
Pathogens in Land Applied Biosolids
Pathogens in
bioaerosols
Pathogens in surface
applied biosolids
Pathogens in biosolids
below surface
Pathogens in
biosolids-soil mixture
Pathogens on crops
Pathogens on
food
Pathogens in
surface water
Pathogens in
groundwater
Application—
spreading or
spraying
Loading,
unloading
Application
injection
Storage
Transport
Aerosolization
Incorporation
in soil
Deposition
Surface runoff
and erosion
Wind
Vector
transport
Leaching
erosion
Irrigation
Water flow
Human infection
FIGURE 2
General Conceptual Model of the Potential Risks from Pathogens in
Land-Applied Biosolids
Legend: Green rectangles: source of pathogens, white hexagons: human actions, white
parallelograms: natural process, blue circles: exposure pathways, yellow rectangles with
rounded corners: potential responses.
29

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some routes may be deleted if enough information is available to rule them out
scientifically.
3.1.	PREAPPLICATION PROCESSES
Various treatment processes are not separate boxes in the conceptual model
because all treatment technologies are assumed to be operating as intended,
generating Class B biosolids (see Figure 2). Human actions in the conceptual model
include storage; transport within a site; loading and unloading; land application by
spreading, spraying, incorporation or injection in soil; and irrigation (see Figure 2).
Biosolids storage, transport within a site, and loading and unloading processes
have been observed to generate bioaerosols (Pillai, 2007; Paez-Rubio et al., 2007;
Figure 2). Biosolids are stored when the ground is frozen, during inclement weather,
periods of equipment breakdown, or crop growth periods (Evanylo, 1999). Dewatered
biosolids are stockpiled, and liquid biosolids may be stored in digesters, tanks, lagoons
or drying beds (Evanylo, 1999). Regulations may specify the type of storage facility for
long-term storage and require a barrier to prevent the erosion of biosolids or the surface
runoff or leaching of pathogens. However, if risk assessors determine that leaks of
biosolids or pathogens from storage facilities are feasible, then additional pathways can
be included in the conceptual model (e.g., arrows between "Storage" and "Surface
runoff and erosion" and/or "Leaching" in Figure 2).
3.2.	APPLICATION
3.2.1. Methods of Land Application of Biosolids
The three major methods of biosolids application are injection, surface
application without incorporation into soil, and surface application with incorporation into
soil. Methods depend on the water content of biosolids, land use, site topography,
quantity of debris, presence of obstructions such as trees, presence of waterways,
climate, the availability of application equipment (NRC, 2002; Brown and Henry, 2002),
and state or local regulations (e.g., Solano County, California requires incorporation of
biosolids into soil). The application method is an important determinant of bioaerosol
generation, chemical odor, and ultraviolet inactivation of pathogens (NRC, 2002).
30

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Subsurface injection of liquid biosolids involves small-diameter injection tubes to
minimize soil disturbance or disking if soil turnover is desired in farm management
practices (NRC, 2002). Injection is typically at a depth of 6-9 inches (15-23 cm) and
usually occurs before planting or after harvest (NRC, 2002). Injection reduces odor and
risk of runoff to surface water (NRC, 2002) as well as prevents aerosolization of
biosolids (see Figure 2). As would be expected, Gerba et al. (2002) found that injected
biosolids presented a much lower risk of infection from ingestion than surface-applied
biosolids without incorporation. Hence, injection is treated separately from surface
application in the conceptual model (see Figure 2). Injection can be used on slopes up
to a 15% grade (Evanylo, 1999), dependent on state or local laws. This application
method serves as a physical barrier that satisfies vector-control requirements
(U.S. EPA, 1993). Injection or soil incorporation is rarely used for pasture or hay crops.
Application under any circumstance is prohibited for any land use when the ground is
frozen (U.S. EPA, 1993).
Surface application involves the application of liquid biosolids or cake solids to
the soil surface. Liquid biosolids are typically pumped and sprayed through a cannon or
spray nozzle. Solid biosolids are flung from a manure-type spreader or dumped from a
truck. Where application is to a forest, a portion of the sprayed biosolids may coat tree
surfaces prior to washing down to soil surfaces. In some climates and at high depths of
biosolids (thick land applications), drying of the material may require a complete
summer period. Drying can be promoted by seeding with a grass such as annual rye or
wheat that can germinate and survive in fairly anaerobic conditions (Brown and Henry,
2002). In contrast to injection, surface application is commonly used for hay crops and
winter applications. Stabilization of biosolids to meet vector-control requirements must
occur through treatment prior to surface application. Surface application may permit
ultraviolet inactivation of viruses (NRC, 2002). Spreading of dewatered biosolids may
sometimes produce higher bioaerosol emission rates than spraying of liquid biosolids
(Paez-Rubio et al., 2007).
Incorporation of cake biosolids into soil through plowing or disking at a depth of
6-9 inches (15-23 cm) may follow surface application (NRC, 2002) and partial drying
(Evanylo, 1999). The method is usually used before planting or after harvest (NRC,
31

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2002). Surface application with incorporation is generally limited to soils with less than
a 7% slope (Evanylo, 1999), additional state and local laws notwithstanding.
Incorporation serves as a physical barrier that satisfies vector-control requirements
(U.S. EPA, 1993).
Application methods vary with region and type of biosolids. In the arid and
semiarid southwest, liquid anaerobic-digested biosolids are typically injected into the
soil subsurface (NRC, 2002). On pasture land, the material tends to be applied to the
soil surface, as incorporation is more difficult than on crop land (NRC, 2002). Similarly,
incorporation is not common in forests. In many agricultural lands, biosolids cakes are
disked into soil (NRC, 2002).
3.2.2. Rates of Land Application of Biosolids
Biosolids are applied at a rate equal to or less than the agronomic rate (the key
determinant being the nitrogen requirements of crops, trees, or other vegetation). Rates
of application are generally calculated on a dry weight basis. Information on application
rates from the 1980s is summarized in Table 3. Notably, the rate of application at
reclamation sites is usually much higher than that at farm sites (NRC, 2002). However,
agricultural sites are more likely to involve multiple applications (NRC, 2002). EPA has
predicted that cumulative pollutant loading limits for the application rates in Table 3 will
be reached after 100 years for agriculture, 55 years for forest, 32 years for public
contact, and 13 years for reclamation, assuming annual applications (NRC, 2002;
U.S. EPA, 1992). Applications are assumed to cease when cumulative loading limits
are reached. Time to reach cumulative loading limits assumes a maximum allowed
concentration at a maximum application rate. In reality, applications may be more
sporadic. Problem formulation may consider actual application rates if available, rather
than predicted maximum rates. For example, land application data by county is
available from the following seven States: Colorado, Florida, Maryland, New Jersey,
New York, Virginia, and Wisconsin (U.S. EPA, 2002c). This group of states includes a
large importer of biosolids (Virginia); a large exporter of biosolids (New York); and a
State to which EPA has delegated the biosolids program (Wisconsin).
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TABLE 3
Estimated Biosolids Application Rates for Different Land Uses
Land Use
No.
Observations
Mean Application Rate
(metric tons/ha/yr of
dry wt)
Standard
Deviation
75th Percentile
(metric tons/ha/yr of
dry wt)
Agriculture
87
6.8
105
16
Forest
2
26
26
34
Public
Contact
11
19
122
125
Reclamation
7
74
148
101
Sources: NRC (2002) and EPA (1992).
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3.2.3.	The Timing of Land Applications of Biosolids
The timing of land applications of biosolids is another factor that determines
exposure. In agricultural operations, application is scheduled around tillage, planting,
and harvesting, and is also influenced by the physical properties of specific crops,
climate regimes, and soil factors (Evanylo, 1999). The State of Virginia recommends
that biosolids applied to land between fall and spring have a vegetation cover to
minimize the runoff of pathogens and nutrients, and the erosion of sediment-bound
biosolids (Evanylo, 1999). However, spray irrigation is not recommended for applying
biosolids to forage, row crops, or young tree stands during the growing season,
because adherence to leaves can reduce photosynthesis (Evanylo, 1999; McFarland,
2000). Workers who apply biosolids may tend to avoid periods of rain, because their
vehicles and equipment may compact or create ruts in soils that reduce crop yields
(Evanylo, 1999).
Although rain may be avoided when applying biosolids, literature reviewed for
this document did not reveal whether heavy winds might be similarly avoided.
Meteorology should certainly be considered when modeling the transport of
land-applied biosolids.
3.2.4.	Regional Application Issues
Exposure factors that vary by region include: methods of biosolids application,
climate, soils, and land available for application in juxtaposition to human populations.
A few regional differences in application methods and timing have already been
described too in previous sections. Climatic differences contribute to differences in fate
and transport of pathogens in biosolids and biosolids-amended soil. For example,
pathogen survival tends to be highest in cool, moist soils, such as those in the
northeastern United States (Pepper et al., 1993). Hot, dry soils as in the southwestern
United States contribute to pathogen mortality (see section below on fate and transport
of pathogens). Deficits in rainfall may be counteracted by irrigation in drier climates.
Groundwater contamination by pathogens from biosolids is most likely in
coarse-textured, sandy soil or land underlain by high permeability karst (NRC, 2002).
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The number of people potentially affected by pathogens in biosolids also varies
regionally. Potential exposure at the human population level increases as the density of
people increases because of greater sewage sludge output, greater needs to find land
application sites, higher rates of biosolids applications, and more residents and children
potentially exposed near their homes and schools. In the arid southwestern United
States, farms are often located far from cities, so fewer residents would be expected to
be exposed to pathogens in biosolids (NRC, 2002). However, members of small rural
communities closer to urban areas where biosolids are generated and then applied
rurally, may have higher individual exposures.
3.3. FATE AND TRANSPORT OF PATHOGENS
3.3.1. Pathogen Survival, Growth and Death
Unlike chemical hazards, biological hazards have the potential to reproduce or to
die. Thus, conceptual models need to consider factors affecting the survival and growth
of pathogens in biosolids, biosolids-amended soils, and bioaerosols (see Figure 3).
Environmental factors affecting the survival of viruses, bacteria and protozoa are
presented in Table 4 (Bujoczek et al., 2001; Gerba et al., 2002; Pepper et al., 2006;
NRC, 2002). Most enteric pathogenic bacteria are nonspore-formers and relatively
sensitive to environmental factors such temperature, desiccation and ultraviolet
exposure. Some fecal bacteria have been found to be persistent in biosolids (Vilanova
and Blanch, 2005). Salmonella, E. coll and fecal coliforms are capable of regrowth in
moist conditions following treatment (Lang and Smith, 2007; Lang et al., 2007).
Regrowth of pathogens can occur in Class A biosolids where biological competition is
low compared to Class B biosolids (Zaleski et al., 2005a). However, Zaleski et al.
(2005b) demonstrated that pathogens (Salmonella and indicators) decreased in
numbers when soil was amended with biosolids. Yates and Yates (2007) observed that
E. coll was able to grow in soil columns with spiked biosolids at 22°C, and
bacteriophage phi X174 exhibited the greatest potential for soil transport. More
transport was observed in sand rather than loam soils.
Pathogen survival and reproduction are depicted in Figure 3. Note that
temperature and moisture are the primary variables affecting the survival of enteric
35

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Pathogen abundance in
applied/in co rpo rated
biosolids
Pathogen
abundance in
bioaerosols
Aerosoiization
Survival and
reproduction
Survival and
reproduction
/	PH>	\
/	moisture,
ultraviolet light,
temperature (e.g., freezing, heating)
particle size (e.g., clay content),
\ indigenous microflora,
\	desiccation	/
Pathogen
abundance in
site media
FIGURE 3
Pathogen Fate Conceptual Model
Legend: Green rectangles: sources of pathogen, white parallelograms: natural
processes, white triangles: pathogen response (may be positive or negative), white circle:
physical environment factors that affect the fate of pathogens.
TABLE 4


Environmental Factors Positively or Negatively Affecting the Survival of Pathogenic
Microbes
Parameter
Survival Time
Virus
Bacteria
Protozoa
Temperature Increasing
-
-
-
Soil Moisture Decreasing
-
-
-
Rate of Desiccation Increasing
-
-
-
Clay Content Increasing
+
+
Not known
pH range of 6-8
+
+
+
Sources: NRC (2002), Pepper et al. (2006).
36

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viruses in soil (Gerba et al., 2002). In addition to the mechanisms in Table 4, ultraviolet
light has the potential to attenuate pathogens, especially those that have been
aerosolized (Paez-Rubio and Peccia, 2005; Pepper et al., 2006). Viruses vary
considerably in their ability to survive outside a host organism. Ascaris eggs may
survive several years in soils that are not very wet or very dry (NRC, 2002). Little is
known about the viability of protozoa following land application of biosolids (NRC,
2002). Even less is known about the survival and reproduction of pathogens in
bioaerosols than about their survival in biosolids or biosolids-amended soil. For more
information about the fate of emerging pathogens, a study by Yates and Yates (2007)
may be consulted.
3.3.2. Pathogen Transport
Pathogens may be transported from biosolids through various environmental
media such as air, soil, and water. In addition to the application process, storage,
site-to-site transportation, and loading and unloading are human processes that could
mobilize pathogens for transport (see Figure 2). Several mechanisms of transport are
possible: aerosolization followed by aerial transport and deposition, erosion, surface
runoff and leaching to surface and ground water resources (see Figure 2).
3.3.2.1. Aerial Transport
Yates and Yates (2007) observed no microorganisms in air samples collected in
a field setting where biosolids were spiked with pathogens (E, coli, Clostridium
perfringens spores, Listeria innocua and bacteriophage phi X174) and applied to
monitored soil columns. However, the land application of biosolids may generate
bioaerosols either through agitation of the soil during application or following a series of
weathering events of deposited biosolids in association with specific climatic conditions
(see hazard characterization). Biosolids left on the soil surface or lightly incorporated
may be subjected to conditions that lead to drying of the material, rendering it friable.
Particulates generated from the friable material are capable of becoming airborne along
with the associated pathogens. Bioaerosol droplets or particles may also be generated
at the site of biosolids application, storage, site-to-site transport, or loading and
37

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unloading processes, including shoveling biosolids from one pile to another (Straub et
al., 1993; Pillai, 2007; Brooks et al., 2007a; Figure 2). Such bioaerosols may potentially
move to downwind locations. Wind can erode and resuspend biosolids previously
applied to the soil surface (see Figure 2). In contrast, injection of biosolids is typically a
barrier to the aerosolization of biosolids (Smith et al., 2005a, Figure 2).
The disking process, one example of "incorporation in soil" in Figure 2, can be a
"substantial source of biosolids-derived aerosols" (Paez-Rubio et al., 2006). The
emission rate of pathogens during disking of biosolids may be greater than rates during
the spreading of dewatered biosolids by side slinger or spraying liquid biosolids
(Paez-Rubio et al., 2006). Aerosol emission rates from dewatered biosolids may be
higher than those for liquid biosolids (Paez-Rubio et al., 2007). In one study, loading
and unloading operations were responsible for the highest predicted annual risks of
infection by Coxsackievirus A21 at a distance of 30.5 m (Brooks et al., 2005b).
The launch patterns of bioaerosols from localized sources of biosolids have a
conical dispersion form, whereas bioaerosols originating from more spatially extensive
fields have a particulate-wave type of dispersion (NRC, 2002; Tanner et al., 2008).
Both the application and incorporation processes, as well as site-to-site transport
provide moving sources of aerosols. In addition to the source, the physical properties of
aerosols and environmental settings affect the dispersal and settling of bioaerosols.
Physical properties include the size, density, and shape of droplets or particles.
Precipitation, relative humidity, temperature, and air currents can affect dispersal and
deposition of aerosolized biosolids (Pillai, 2007).
Evidence from Tanner et al. (2005) suggests that under some conditions,
aerosolized viruses may be transported farther than aerosolized gram-negative
bacteria, which would affect relative exposure patterns.
3.3.2.2. Erosion and Runoff to Surface Water
Water-borne exposure to pathogens from biosolids is driven by precipitation
sufficient to move the organisms from the site of application to surface water as runoff
(NRC, 2002). The movement of pathogens associated with applied biosolids to surface
water depends on the numerous environmental properties of the area where the
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biosolids are applied as well as those of adjacent lands. The NRC noted that EPA did
not adequately consider the potential for contamination of neighboring properties or
surface water by runoff in the Part 503 rule (NRC, 2002). Smith et al. (2005b) identified
the monitoring of pathogens in runoff from land application of biosolids to be a research
priority, because little is known about this transport pathway.
Where biosolids are applied to the soil surface, runoff may transport particles to
surface waters down-gradient (Straub et al., 1993), at least "in principle" (NRC, 2002).
Disking operations also break up and mix the biosolids with soil, which increases the
potential for erosion and runoff but buries the amendment and dilutes the initial numbers
of pathogens. Selvaratnam and Kunberger (2004) reported evidence of a correlation
between farmland (amended with treated sewage sludge) runoff and higher incidence of
antibiotic resistance and fecal coliforms in downstream surface water. Runoff of
pathogens to surface water is expected to be higher where the biosolids are left on the
surface (e.g., in forests) compared with incorporation into agricultural soils. Edmonds
(1976) found that sewage sludge applied to forest land was unlikely to contaminate
groundwater or nearby springs and rivers through vertical movement of the bacteria
through the soil. However, the study also indicated that stormwater runoff from
forest-applied sludge could potentially contaminate surface waters.
3.3.2.3. Leaching to Groundwater
Following precipitation, microorganisms may infiltrate soil and contaminate
groundwater (Straub et al., 1993). The NRC noted that EPA did not adequately
consider the potential for contamination of groundwater by runoff in the Part 503 rule
(NRC, 2002). The transport of microorganisms through soils is affected by both abiotic
and biotic factors, including the presence of biosolids, soil characteristics, water flow
rates, adhesion processes, filtration effects, the physiological state and mobility of
pathogen cells and predation (NRC, 2002). Viruses have a greater potential to be
transported to groundwater than other pathogens, although sorption to colloids and
biosolid particles limits this potential (NRC, 2002; Chetochine et al., 2006). Transport of
larger organisms (bacteria, protozoa, helminths) is less likely, but possible if flows occur
through cracks or macropores of soils (NRC, 2002). Transport of pathogens to
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groundwater is most likely where soils are sandy and coarse-textured or where karst
topography is present (NRC, 2002). Eisenberg et al. (2008) predicted that human
health risks due to groundwater exposure are lower than risks from exposure to
aerosolized biosolids. Studies that examine pathogen occurrence in groundwater
(Borchardt et al., 2003) and survival and inactivation of pathogens in groundwater are
available (John and Rose, 2005).
3.3.2.4. Sorption to Crops
Pathogens from biosolids could become sorbed to root crops with particles from
the biosolids-soil mixture (see Figure 2). Although crops are generally washed before
eating, a fraction of biosolids-amended soil will remain sorbed to the crop (estimated at
10% by Gale [2005b]). Additional pathogens might become sorbed to root crops
following runoff from biosolids-amended fields to neighboring fields. Leaf crops might
become contaminated with pathogens deposited from bioaerosols or from rain splash
(see Figure 2). Leaf or root crops could become contaminated with pathogens via
irrigation with contaminated surface water or groundwater (see Figure 2). Exposure to
contaminated crops is discussed in Section 3.4.2.
3.3.3. Vector Transport
The transport of pathogens from biosolids by insects, birds, pets, and other
vectors is possible. For example, flies might become contaminated, leaving trace
pathogens on food that is ingested by humans (Graczyk et al., 2001). This potential
pathway is included in Figure 2. No information is available on the extent to which land
application of biosolids attracts flies or other potential vectors, such as mosquitoes or
birds (NRC, 2002). Pets are a potential vector, resulting in dermal, oral (hand to mouth)
or respiratory exposures. It is unclear whether procedures in the Part 503 rule that are
intended to discourage vectors are effective or not (NRC, 2002). Similarly, it is unclear
whether vectors are involved in the transmission of pathogens to humans from
land-applied biosolids (NRC, 2002).
40

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3.4. HUMAN ROUTES OF EXPOSURE
Potential routes of exposure to pathogens originating in biosolids include
inhalation, ingestion, and dermal exposure (see Figure 2). Problem formulation might
consider these exposure routes in the context of bioaerosols or water that come in
contact with biosolids, or direct contact with land applied biosolids after the time period
required by Class B regulations. Potential exposures associated with noncompliant
behaviors are not covered in this document, but could be considered by appropriately
modifying or adapting the conceptual models presented here for other scenarios of
interest.
3.4.1. Inhalation
Routes of exposure for aerosolized pathogens are not well characterized but
likely involve a combination of both inhalation and ingestion pathways (Pillai, 2007,
Figure 2). Pathogens can be physically transported away from the site of land
application on aerosolized particles of biosolid material. Large aerosolized particles
(between 5 and 20 pm) can deposit in the upper respiratory tract. Clearance of these
particles results in oral exposures. Smaller particles penetrate deep into the lungs and
may be lodged and retained by the alveoli (Pillai, 2007). Several reports of respiratory
symptoms associated with biosolids application (e.g., Herr et al., 2003; Gavett and
Koren, 2001; George et al., 2001) indicate inhalation is the most probable route of
exposure to smaller particles. In one study that investigated bioaerosols emitted during
the spreading of dewatered Class B biosolids onto farm land, the diameters of most
emitted particles were of inhalable and possibly respirable size (Paez-Rubio et al.,
2007). Because of the high volume of air that is inhaled daily, Pillai and Ricke (2002)
assert that inhalation is the predominant route of exposure for aerosolized pathogens
that may result in adverse health effects. It should be noted that not all pathogens can
infect tissues exposed during inhalation.
The NRC (2002) determined that the inhalation pathway was among the routes
of exposure that was not adequately assessed by EPA in the development of the
Part 503 rule. They noted that inhalation of dust was presumed by EPA to occur only
on-site and that controlling site access was thought to prevent that route of exposure
41

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(NRC, 2002). Brooks et al. (2005a,b) investigated the potential for inhalation of
pathogens by off-site residents. However, the literature search did not locate many
studies of inhalation of biosolids-derived aerosols or pathogens by off-site residents.
Thus, inhalation of pathogens by off-site residents needs more consideration. For more
information about the potential health effects of inhalation hazards, see Shusterman
(1992) and Lewis etal. (2001).
3.4.2. Ingestion
Ingestion of biosolids-related pathogens may occur via several exposure
scenarios including; direct and incidental ingestion of surface or groundwater containing
pathogens originating from biosolids; ingestion of pathogens which are sorbed to crops
and food items after application of biosolids in agricultural fields; incidental ingestion of
pathogens associated with surface-applied biosolids and biosolids mixed with soil; and
ingestion of bioaerosols containing pathogens (see Figure 2).
Ingestion of biosolids in soil occurs through the transfer of pathogens to the
mouth from contaminated hands or crops or though inhalation followed by swallowing
(Gerba et al., 2002; Figure 2). Larger particles in contact with the respiratory tract can
be cleared from the tract and swallowed. Researchers vary in their estimation of the
percentage of inhaled pathogens ingested (Pillai, 2007).
Ingestion of groundwater or surface water is a potential route of exposure to
biosolids-derived pathogens. Untreated surface water contaminated with pathogens
from biosolids might be ingested while swimming, potentially allowing for greater
consumption of pathogens than a domestic tap supplying water treated in accordance
with the Safe Drinking Water Act (SDWA).
Food consumption is a potential direct route of exposure to pathogens, especially
involving ingestion of foods not subjected to cooking or washing. Biosolids are applied
to agricultural soil to improve its fertility and to enhance crop yields. The application of
biosolids to soil along with consumption of food grown on amended fields provides an
avenue of exposure to pathogens through the food chain. Reasonable exposure
scenarios involve the adherence of the pathogens to the plant (i.e., roots, stems,
42

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leaves), particularly the edible portion of the plant, and subsequent consumption by
individuals.
At least three exposure scenarios may result in the ingestion of pathogens
associated with biosolids when applied in crop settings. Each exposure scenario differs
with respect to the portion of the plant that is eventually consumed. The first scenario
involves the deposition of aerosolized material on the surface of the aboveground
portions of the plant (see Figure 2). This exposure may arise during biosolids
application. In this scenario, biosolids may be applied by spreading or spraying the
material onto the soil with the resulting generation of airborne pathogens from the
biosolids (see Figure 2). Pathogens and biosolids material subsequently land on and
adhere to the aboveground portion of the plant that is intended for consumption.
Compliance with current regulations makes pathogen ingestion from crops a more
unlikely exposure pathway for farm residents (see the section on regulatory restrictions
below). Part 503 regulations provide for time restrictions between application to the
field and harvesting of plants (see Table 5). However, harvesting of plants in nearby
fields where pathogen deposition from air or surface runoff may occur is not restricted.
Additionally, the placement of microorganisms on the aboveground portion of the plant
subjects the pathogens to environmental stressors such as UV radiation and
desiccation, both of which diminish the viability and spread of the pathogens. Moreover,
the types of foods that may be affected by the deposition of aerosolized material are
grains and some vegetables which normally undergo preparation and cleaning
processes to reduce pathogen viability prior to consumption. Although this scenario
might constitute a minor pathway, it should be considered in a problem formulation if
appropriate.
The second exposure scenario associated with crops addresses plant
consumption in which the palatable portion is aboveground, but is expected to come in
contact with the soil. This scenario includes some fruits and vegetables such as
melons, cucumbers, and tomatoes. This scenario allows for extended contact with soil
while the plant develops with the possibility of infection of the plant through a lesion or
by adherence to the plant surface. Many of the crops that fall into this category include
vegetables that are consumed without prior food preparation other than normal
43

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TABLE 5
Pathways of Exposure and Applicable Use Restrictions for Class B Biosolids
Pathways
Part 503 Required Use Restriction
Handling soil from fields where biosolids have
been applied
No public access3 to application until at least
1 year after Class B biosolids application
Handling soil or food from home gardens where
biosolids have been applied
Class B biosolids may not be applied on
home gardens
Inhaling dustb
No public access to application sites until at
least 1 year after Class B biosolids application
Walking through fields where biosolids have
been applied13
No public access to fields until at least 1 year
after Class B biosolids application
Consuming crops from fields on which biosolids
have been applied
Site restrictions that prevent the harvesting of
crops until environmental attenuation has
taken place
Consuming milk or animal products from
animals grazing on fields where biosolids have
been applied
No animal grazing for 30 days after Class B
biosolids have been applied
Ingesting surface water contaminated by runoff
from fields where biosolids have been applied
Class B biosolids may not be applied within
10 meters of any waters to prevent runoff
from biosolids-amended land
Ingesting inadequately cooked fish from water
contaminated by runoff from fields where
biosolids have been applied, affecting the
surface water
Class B biosolids may not be applied within
10 meters of any waters to prevent runoff
from biosolids-amended land
Contact with vectors that have been in contact
with biosolids
All land-applied biosolids must meet one of
the vector-attraction-reduction options
aPublic-access restrictions do not apply to farm workers. If there is low probability of public exposure to
an application site, the public-access restrictions apply for only 30 days. However, application sites that
are likely to be accessed by the public, such as ballfields, are subject to 1-year public-access
restrictions.
"Agricultural land is private property and not considered to have a high potential for public access.
Nonetheless, public-access restrictions are applied.
Source: Taken from NRC (2002), which adapted the table from EPA (1999).
44

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washing, which may not apply to all households. However, as the area of contact is
with the soil surface, it is anticipated that the pathogens would be exposed to higher
levels of environmental stressors which would reduce the viability of pathogens. The
third scenario applies to crops that have the palatable portion below the soil surface.
Examples include root vegetables, such as potatoes, carrots, and yams. This scenario
poses a concern for several reasons. First, this exposure scenario involves direct
contact to pathogens with the greatest potential for long-term survival, i.e., those that
are found below the soil surface. Furthermore, because the food portion of the plant
develops in close contact with the soil, it has the greatest potential for retaining
pathogens on the plant surface (Chale-Matsau and Snyman, 2006). Finally, some
tubers may be ingested with little or no preparation that would remove or inactivate
pathogens on the edible plant surface. For example, carrots are usually eaten raw.
They may be washed or skinned prior to eating, but the amount of preparation varies
considerably.
Part 503 regulations address these exposure scenarios for Class B biosolids
through appropriate grazing, harvesting, and public access restrictions. Existing
regulations establish temporal restrictions on the harvesting and consumption of food
grown on land receiving Class B biosolids. Planting is indirectly restricted because the
harvesting restrictions are 30 days to 38 months, depending on the part of the plant that
is harvested, and some crops require less than 38 months from planting to harvesting.
Nonetheless the potential remains for consuming food harvested from amended
plots. As presented in the section on regulatory restrictions (below), Part 503
regulations require a waiting time of either 20 or 38 months for crops whose harvested
portion is below ground; and shorter periods for crops where the above-ground portion
is harvested. Pathogens capable of surviving over this period of time can adhere to the
surface of the harvested portion of the plant, and with inadequate food preparation
steps, could be consumed.
3.4.3. Dermal Exposure
Dermal contact constitutes a direct method of transfer of pathogens in biosolids
to receptors (see Figure 2). Dermal transmission of pathogens would occur primarily
45

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through skin abrasions or cuts, either through contact with contaminated soil or surface
water.
Dermal contact may also occur during occupational exposure or during
unintended contact with biosolids that have moved from the original site of application
(e.g., through aerial dispersion or runoff). Workers most likely come in contact with
biosolids when processing, loading, and applying them.
Recreation during the summer months may be another possible exposure
scenario to consider. For example, swimming might permit dermal contact (as well as
the ingestion or inhalation) of pathogens in surface waters contaminated by biosolids
applied to the surrounding landscape. To assess dermal exposures, the risk assessor
would need information on the amount of material adhering to the skin and
dose-response values for the pathogens of interest, as well as data, on the distribution
and numbers of pathogens in biosolids and their potential for regrowth or reproduction.
3.5.	REGULATORY RESTRICTIONS
Many site restrictions related to land application of biosolids are intended to
reduce exposure to pathogens and chemicals in the material (see Table 5). As such,
these restrictions will affect the plausibility of various exposure pathways in a particular
conceptual model. Time intervals required prior to site access are summarized in
Table 6. Particular states may also have varying regulatory criteria for: required
distances between land-applications and surface waters or wetlands, slope restrictions,
depths to groundwater and bedrock, soil permeability rates, distances to residences,
schools, health care facilities or recreation areas, and distances to private or public
water-supply wells (NRC, 2002).
3.6.	FACTORS THAT AFFECT INFECTION AND DISEASE
Several host and pathogen characteristics affect the probability or intensity of
disease (see Figure 4). For planning of a specific risk assessment, attributes of
pathogens need to be considered together with attributes of the host and environment.
For example:
46

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TABLE 6


Minimum Time Interval between Application and Harvest, Grazing or Public Access to
Lands Applied with Class B Biosolids
Criteria
Injection
Surface
Application
Surface With
Incorporation
Harvest
Food crops whose
harvested parts may contact
biosolids-amended soil
14 months
14 months
14 months

Food crops whose
harvested parts grow in soil
38 months
20 or 38
months*
38 months

Food, feed and fiber crops
30 days
30 days
30 days
Grazing
Animal grazing
30 days
30 days
30 days
Public Access
High potential for exposure
1 year
1 year
1 year

Low potential for exposure
30 days
30 days
30 days
The 20-month interval prior to harvesting applies if the biosolids stay on the surface for 4 months or
longer prior to incorporation. The 38-month interval applies if the biosolids stay on the surface for less
than 4 months prior to incorporation.
Source: Modified from: NRC (2002) and 40 CFR Part 503.
47

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Pathogens, noninfectious organisms,
endotoxins, irritants, odors
Host acquired
immunity
Pathogen
infectivity
Pathogen
virulence
Human infection
~-	~}
r

A

Disease

V

J
FIGURE 4
Disease Factors Conceptual Model
Legend: Blue box: hazards; purple diamonds: host factors; green diamonds: pathogen
factors; yellow rounded squares: host outcomes; solid lines: "leads to"; dashed lines:
"influences"; T-line: "inhibits."
48

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1)	Host immunity may be important and differs for different pathogens
2)	Person to person transmission can be an aspect of a particular setting within
which biosolids are being applied and in those cases can be included in the risk
assessment
3)	Human activities (see Figure 2) can affect biosolids and the differences in
locations between generation and application can be important. Thus,
"community factors" are a potentially important aspect of assessing risks
associated with applications of biosolids.
Other key human and pathogen factors are described below.
3.6.1. Human Factors
Three host factors discussed in NRC (2002) are concomitant exposures, genetic
factors and acquired immunity. Based on ILSI (2000), the Draft Protocol for Microbial
Risk Assessment to Support Human Health Protection for Water-Based Media
(U.S. EPA, 2009a) includes the following host characteristics. Each has the potential to
influence exposure and health effects (U.S. EPA, 2009a):
•	Immune status (also see Section 3.6.1.3)
•	Age (also see Section 3.6.2)
•	Concurrent illness/medical treatment
•	Genetic background (also see Section 3.6.1.2)
•	Pregnancy
•	Nutritional status
•	Previous exposure
•	Social/behavioral traits
Each of these factors may impact a risk assessment concerning host
susceptibility to disease and severity of illness, and would be useful for consideration on
a case-by-case basis. Differential susceptibility within the population may be explicitly,
implicitly, or not considered. Transparency is enhanced when risk assessors can
document how and why each host factor is considered or not.
49

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3.6.1.1.	Concomitant Exposures
Various hazards such as pathogens, noninfectious organisms, cellular
components, irritants, and odors may influence individual immunity, other aspects of
susceptibility, or the nature or intensity of disease (see Figure 4; Schiffman et al., 2000).
Synergistic effects might result from combined exposures to these hazards (NRC, 2002,
Figure 4). For example, endotoxins may combine with particles and allergenic
components to promote the development of respiratory diseases and systemic effects
(NRC, 2002).
3.6.1.2.	Genetic Factors
Genetic factors influence individual immunity as well as other aspects of disease
susceptibility (see Figure 4). Genetic factors such as a predisposition to asthma attacks
can be a factor in determining whether infection proceeds to disease (Bracken et al.,
2002). No information is available on the role of genetic factors in contributing to health
effects due to bioaerosols from land-applied biosolids (NRC, 2002).
3.6.1.3.	Acquired Immunity
Acquired immunity is the result of previous exposure to pathogens and is part of
the immunity box in Figure 4. Acquired immunity can reduce the fraction of illness in a
population exposed to pathogens (NRC, 2002). Genetic factors also contribute to the
immune status of an individual. The dynamics of immunity are not well understood for
most pathogens. Loss of immunity to pathogens is also a possible result of exposure to
other pathogens, or biological or chemical hazards (see Figure 4).
3.6.2. Additional Susceptibility Factors
For public health risk assessment purposes, exposed populations are evaluated
based on age (children, adults, geriatrics). In addition, sensitive subpopulations may be
evaluated based on gender, ethnicity, baseline health status (immunocompromised,
hereditary diseases, etc.) or any other site-specific health characteristic of the
potentially exposed population that warrants special consideration.
50

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3.6.3. Pathogen Factors
Infectivity and virulence are pathogen factors that can also influence infection
and disease (see Figure 4). Infectivity is the relationship between the quantity of
pathogens ingested or inhaled or in contact with skin and the probability of infection.
For many pathogens, risk assessors have typically assumed a no-threshold effect (i.e.,
assumed that one organism is sufficient to produce infection in some portion of an
exposed population or subgroup) (Haas et al., 1999, also see Analysis Plan chapter for
discussion of thresholds). Virulence is a measure of the severity of the disease that the
pathogen is capable of causing.5
3.7. INFECTION AND DISEASE
Two primary, broad endpoints of risk assessments for pathogens in land-applied
biosolids are human infection and disease (see Figures 2 and 4-9). Infection is the
process by which a microorganism multiplies or grows in or on the host. Clinical
diseases are evidenced by signs or symptoms. Soller and Eisenberg (2008) provide
parameter values for the proportion of infected individuals with symptomatic responses
for enteroviruses, rotavirus, Cryptosporidium, Giardia lamblia, Salmonella, E. coli
0157:H7, Shigella, and a composite value (minimum of 10, median of 40, maximum of
75%).
A variety of diseases may arise from exposure to enteric viruses (i.e.,
enterovirus, rotavirus, adenovirus) such as gasteroenteritis, respiratory illness,
cardiovascular disease and central nervous system disorders. Likewise, the enteric
bacteria associated with biosolids such as Salmonella, Shigella, Campylobacter, E. coli
and Listeria have been identified as causative agents of illness in exposed humans.
Infections of enteric bacteria have resulted in gastrointestinal illness, dysentery, arthritis,
Reiter and Guillain-Barre syndrome, and neuromuscular paralysis (see Table 2). The
protozoans of concern Giardia, Cryptosporidium and Entamoeba, produce cysts and
oocysts which have been shown to be environmentally stable and somewhat resistant
to disinfectants. Thus, they are recognized as significant human pathogens with the
5 Some authors include infectivity in the definition of virulence, but in this context virulence is separate
from infectivity. For further discussion of risk assessment terminology see EPA (2007).
51

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potential to cause gastrointestinal illness exhibited by diarrhea, dehydration and weight
loss and, in the case of Cryptosporidium and immunocompromised individuals, mortality
too. Potential effects of particular pathogens found in biosolids are described in the
hazard characterization chapter (see Section 2.2).
Public health endpoints may include the prevalence (total number of cases in a
population) or incidence (number of new cases in a population during a specific time
interval) of disease (or morbidity). For example, severity (e.g., number of days lost to
illness) may be another property of disease that is of interest to the risk assessor.
Mortality is an additional, potential endpoint. For further discussion on issues relating to
the severity of illness, see EPA (2009a).
3.8. SCENARIOS
Risk assessors may describe scenarios that do not include all the pathways
shown in Figure 2. For example, in this section, five exposure scenarios representing
common public concerns are presented to demonstrate how Figure 2 may be modified
to address more specific scenarios of interest. These include the following:
1.	Neighboring residences and schools adjacent to a site applied with biosolids
2.	Residents of a site where biosolids are applied (e.g., farm families)
3.	A pica child exposed to biosolids
4.	Drinking water consumers of groundwater aquifer supplies underlying sites
applied with biosolids (i.e., particularly those with highly permeable soils or
shallow water tables)
5.	Drinking water consumers of surface waters downstream from sites where
biosolids are applied
EPA's Exposure Factor's Handbook supplies exposure estimates for many types of
behaviors that may be included in these and other scenarios assessors may investigate
(U.S. EPA, 1997).
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3.8.1.	Scenario 1. Neighboring Residences and Schools
Individuals potentially exposed to biosolids-derived pathogens may reside on
lands adjacent to farms, forests, reclaimed minelands, or other lands where biosolids
are applied. Similarly, schoolchildren may be exposed to eroded soils or bioaerosols
from land-applied biosolids. The conceptual model for this scenario (see Figure 5)
adapts most of the pathways from the general conceptual model (see Figure 2). The
primary source processes that do not appear in this scenario are storage, transport and
loading and unloading activities (see Figure 5). For this example it is assumed that the
biosolids were stored, loaded, and unloaded in an enclosed facility, so exposure from
these activities need not be addressed. Other scenarios could be developed to assess
the loading and unloading of biosolids in open facilities, or cases where leaks or runoff,
for example, may occur.
3.8.2.	Scenario 2. Residents (Farm Families)
Individuals potentially exposed to biosolids-derived pathogens may reside on
farms where biosolids are applied. The conceptual model for this scenario (see
Figure 6) adapts all of the potential pathways from the general conceptual model
including storage, transport and loading and unloading activities (see Figure 2).
However, a specific model for farm families might include pathways by which
biosolids-amended soil is tracked into the residence (e.g., contaminated boots, work
clothes, or equipment that is returned to the barn). Recreational hikers in forests where
biosolids have been applied might also bring pathogens home on their clothing.
3.8.3.	Scenario 3. Pica Child
Soil ingestion is the consumption of soil as the result of various behaviors such
as consuming soil directly or contacting dirty hands or contaminated crops. Moreover,
soil-pica, the scenario considered here, is the recurrent ingestion of unusually high
amounts of soil (i.e., on the order of 1-5 grams per day). Groups at risk of soil-pica
behavior are generally any child aged 6 years and younger including children within that
same age range in farm families or at schools discussed in Scenarios 1 and 2. Noting
that soil ingestion is a normal behavior among children, evaluation of all types of soil
ingestion is included in the soil-pica scenario (see Figure 7).
53

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Pathogens in Land Applied Biosolids
Aeroso ization

Application-
spreading or
spraying
Pathogens in
bioaerosols
Pathogens in surface
applied biosolids
Incorporation
in soil
Application
injection


Pathogens in biosolids
below surface
Pathogens in
biosolids-soil mixture
Deposition
Surface runoff
and erosion
Wind
erosion
Vector
transport
Leaching
Pathogens on
food
Pathogens in
surface water
Irrigation /<
Water f ow
Dermal
exposure
nha ation
Ingestion
Human infection
Pathogens in
groundwater
FIGURE 5
Scenario 1: Neighboring Residences and Schools (or Adjacent Property)
Conceptual Model
Legend: Green rectangles: sources of pathogen, white hexagons: human actions, white
parallelograms: natural processes, blue circles: exposure pathways, yellow rectangles
with rounded corners: potential responses.
54

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Pathogens in Land Applied Biosolids
Inhalation
Inhalation
Ingestion
Application-
spreading or
spraying
Loading,
unloading
Application-
injection
Storage
Transport
Aerosolization A
Incorporation
in soil
Deposition
Surface runoff
and erosion
Wind
Vector
transport
Leaching
erosion
Irrigation >4
Water flow
Pathogens in
bioaerosols
Pathogens in surface
applied biosolids
Pathogens in biosolids
below surface
Pathogens in
biosolids-soil mixture
Pathogens on
food
Pathogens in
surface water
Pathogens in
groundwater
Disease
Human infection
FIGURE 6
Scenario 2: Resident (Farm Family) Conceptual Model
Legend: Green rectangles: sources of pathogen, white hexagons: human actions, white
parallelograms: natural processes, blue circles: exposure pathways, yellow rectangles
with rounded corners: potential responses.
55

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Incorporation
in soil
Pathogens in surface
applied biosoiids
Pathogens in
biosoiids-soii mixture
Pathogens in Land Applied Biosoiids
s	/
^ _ *
Human infection
Ingestion
Disease
FIGURE 7
Scenario 3: Pica Child Conceptual Model
Legend: Green rectangles: sources of pathogen, White hexagons: human actions, White
parallelograms: natural processes, Blue circles: exposure pathways, Yellow rectangles
with rounded corners: potential responses.
56

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3.8.4.	Scenario 4. Drinking Water Consumers of Groundwater
Leaching of pathogens to groundwater is of potential concern following the
injection of biosolids in the subsurface, or after surface applications to porous soils or
Karst topography overlying an aquifer or well. Studies conducted on porous soils have
demonstrated that pathogens in water can move with the liquid through different vertical
horizons in the soil (Chetochine et al., 2006). Groundwater aquifers serve as the sole
source of water in many communities and therefore may be used for both farming and
domestic purposes. For example, groundwater may be consumed and used in food
preparation (e.g., when washing fruits or vegetables or cooking). Cooking, in particular,
can account for a significant reduction or elimination of most pathogens. Groundwater
is also used in such communities for bathing and other household activities. This
scenario emphasizes groundwater consumption (see Figure 8). Risk assessors may
choose to consider groundwater sources covered by the SDWA separately from private
wells, which are not regulated by the SDWA.
3.8.5.	Scenario 5. Drinking Water Consumers of Surface Water
The use of down-gradient surface waters as a source of potable water may result
in exposure to biosolids-related pathogens (see Figure 9). The major pathways of
potential exposure to pathogens would be erosion of biosolids particles and surface
runoff from treated sites (see Figure 9). Additionally, pathogens might be carried to
surface water through interactions with contaminated groundwater, or possibly
deposited to surface water following aerial transport. Treatment of water before
consumption can greatly reduce the potential for exposure pathogens. Water supplies
regulated by SDWA require between 3-log and 5-log removal/inactivation efficacies
prior to public consumption.6
3.8.6.	Event Related and Regional Aspects of Scenarios
These scenarios are merely representative and not meant to be exhaustive.
Different scenarios will certainly occur in different regions or may be more important to
consider depending on the circumstances. For example, surface water drinking
6 Maximum contaminant levels under SDWA http://www.epa.gov/safewater/contaminants/index.html.
57

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Pathogens in Land Applied Biosolids
Application-
spreading or
spraying
Application-
injection
Incorporation
in soil
Pathogens in	Pathogens in biosolids
biosolids-soil mixture	below surface
Leaching
Pathogens in
groundwater
i
Jf
Human infection
Ingestion
Disease
FIGURE 8
Scenario 4: Groundwater Conceptual Model
Legend: Green rectangles: sources of pathogen, White hexagons: human actions, White
parallelograms: natural processes, Blue circles: exposure pathways, Yellow rectangles
with rounded corners: potential responses.
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Pathogens in Land Applied Biosolids
Ingestion
Pathogens in
groundwater
Deposition
Pathogens in
bioaerosols
Pathogens in surface
applied biosolids
Pathogens in biosolids
below surface
Pathogens in
biosolids-soil mixture
Pathogens in
surface water
Disease
Water flow
Application—
spreading or
spraying
Aeroso ization
Surface runoff
and erosion
Wind
erosion
Incorporation
in soil
Irrigation
Human Infection
Leaching
FIGURE 9
Scenario 5: Surface Water Conceptual Model
Legend: Green rectangles: sources of pathogen, White hexagons: human actions, White
parallelograms: natural processes, Blue circles: exposure pathways, Yellow rectangles
with rounded corners: potential responses.
59

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scenarios would be less applicable to arid regions if application is during the dry
season. Scenarios involving aerosolization of pathogens in biosolids would be more
applicable to windy regions. Development of region-specific risk assessments and
research would benefit from modifying the scenarios based on the relevant
circumstances of each individual case.
In addition, risk assessors may want to consider scenarios that include relatively
common events, such as storms, or possibly rarer events such as floods, hurricanes,
and tornados. For arid regions, seasonal factors may influence runoff and erosion
(Moffet et al., 2005). Consideration of the timing of biosolids application may be
important if the region has notable wet weather and/or dry weather patterns. As
mentioned previously, application of biosolids to frozen ground is not permitted.
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4. SCREENING OUT ELEMENTS OF THE CONCEPTUAL MODEL
On a case by case basis, the general conceptual model (see Figure 2) may be
examined to determine if sufficient information is available to screen out unlikely
hazards, scenarios, routes of exposure, or endpoints from consideration in risk
assessments of pathogens in biosolids. This effort should help simplify the model to
focus on the most pertinent factors and should not be confused with the screening-level
risk assessment process that is site-specific and part of the analysis phase rather than
the problem formulation.
Very little information is available to directly compare the relative importance of
different exposure pathways. Academic studies tend to emphasize a single exposure
pathway rather than a comparison of multiple pathways. However, the literature review
(see the Appendix) suggests that certain pathogens and exposure pathways tend to be
less likely, for example:
•	Endotoxin. Brooks et al. (2007a) found that biosolids-amended soil did not have
higher levels of endotoxin than unamended soil. Levels of endotoxin in
aerosolized soil were sometimes above those associated with aerosolized,
biosolids-amended soil, calling into question whether biosolids were the primary
source of the endotoxin (Brooks et al., 2006).
•	Staphylococcus aureus. A broad study of 15 sites across the United States
found that S. aureus was detected in raw sewage samples but not in biosolids
(Rusin et al., 2003a).
•	Certain protozoa. Gerba et al. (2002) determined that microsporidia and
Cyclospora would not be likely to survive under high temperatures of anaerobic
digestion or under conditions of low moisture in Class B biosolids treatment.
•	Certain bacterial or viral pathogens in bioaerosols. Pathogens and indicator
bacteria were only rarely found in aerosolized samples in a study of land
application of biosolids in Tucson, AZ. These included coliforms and coliphages,
which were present at high densities in biosolids. The authors suggested that
only microorganisms in the aqueous phase of biosolids were able to aerosolize;
others remained sorbed to the solid phase (Brooks et al., 2004a). Furthermore,
Tanner et al. (2005) determined bioaerosol emission rates and plume
characteristics during spray application of liquid Class B biosolids. They did not
detect coliphages or coliform bacteria just downwind of the biosolids application,
though pathogens sprayed in inoculated groundwater were detected. The
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researchers concluded that the presence of biosolids reduces aerosolization of
microorganisms relative to application of inoculated groundwater. The duration
of exposure to any pathogens (below detection limits) downwind of biosolids
application is brief (Tanner et al., 2005).
However, insufficient evidence exists to support any broad generalizations about
negligible elements at this time.
Brooks et al. (2005b) undertook a study to estimate risks of microbial infection of
residents near biosolids application sites. At 10 sites (five in Arizona, five elsewhere in
the United States) amended with either liquid or solid Class B biosolids, they measured
heterotrophic plate counts of bacteria, total coliform bacteria, E. coli, Clostridium
perfringens, coliphage, enteroviruses, hepatitis A virus and norovirus in aerosol samples
downwind from application sites. The study distinguished between loading, unloading,
land application and background operations. Of the microorganisms evaluated, the
greatest risk of infection was from coxsackievirus A21 from loading operations, having a
4 x 10~4 chance of infection (Brooks et al., 2005b). Based on this work, Pepper et al.
(2006) concluded that the overall community risk of infection from bioaerosols during
land application was relatively negligible.
Some evidence (below) might support a decision to screen out certain exposure
pathways in Figure 2 from general or regional consideration in the future. However,
more evidence is needed to support such a judgment.
•	Groundwater pathway. Because of the large size of bacteria, soil (especially
fine-textured soil) can act as a filter to limit bacterial transport (NRC, 2002). Soil
would also be expected to limit the transport of larger protozoa and helminths
(NRC, 2002). A review of the literature has concluded that few pathogens (even
viruses) from biosolids leach to groundwater, except perhaps if biosolids are
applied over karst topography (Pepper et al., 2006; Edmonds, 1976). Although
Gerba (2005) acknowledges that of the pathogens in biosolids, viruses have the
greatest potential for contamination of groundwater, Pepper et al. (2006)
concluded that "groundwater contamination from land-applied biosolids does not
appear to be likely." Sandy soils with low cation exchange capacity deserve
more study.
•	Root crop ingestion pathway. A United Kingdom study of infection from
consumption of root crops grown on biosolids-amended soils found that risks to
humans was low. Seven pathogens were included in the study: salmonellas,
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Listeria monocytogenes, Campylobacters, Escherichia coii 0157,
Cryptosporidium parvum, Giardia and enteroviruses (Gale, 2005b). However,
United Kingdom biosolids may not be comparable to Class B biosolids in the
United States.
Regulations might also allow a risk assessor to screen out potential pathways of
exposure in the general case. For example, if biosolids must be stored in enclosed
facilities, the generation of bioaerosols from that source (and exposure to neighboring
residents) would not be likely.
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5. CONSIDERATIONS FOR DEVELOPING AN ANALYSIS PLAN
5.1. INTRODUCTION
The analysis plan is the final stage of problem formulation. It summarizes the
measures, methods, and data needs for conducting the exposure, health effects and
risk assessment steps. Providing a rational for the selection of measures, methods and
tools is essential for transparency. All of these should be described in the context of the
sources, pathways, environmental media, and human health endpoints described in the
conceptual model. The emphasis is on uncertainties and variables to which the risk
assessment is sensitive, if known. A rigorous analysis plan is especially necessary if
there is no established protocol for conducting a particular type of risk assessment
(U.S. EPA, 1998), as with human health risk assessment of biosolids-derived
pathogens.
The analysis plan evaluates risk hypotheses to determine how they will be
assessed (U.S. EPA, 1998,2003a). A rationale for selecting or eliminating risk
hypotheses has been presented in Guidelines for Ecological Risk Assessment
(U.S. EPA, 1998). An analysis plan for a risk assessment of pathogens in biosolids can
be designed to eliminate negligible pathways in the conceptual model (e.g., refer to
Chapter 4 of this document). Available data are described, as well as new data that, if
collected, might enhance the risk assessment. The feasibility of collecting new data can
also be discussed. There are no definitive guidelines on what constitutes adequate
data. A transparent description of available data and data needs helps reviewers and
users of a risk assessment to understand judgments made. Lack of transparency can
be more damaging to a risk assessment's utility than judgments and assumptions that
lead to a healthy scientific debate. The analysis plan addresses both measurements
and models. The plan also describes where parameters of interest may be extrapolated
from existing data.
This chapter is structured as an analysis plan might be structured for a specific
risk assessment on land-applied biosolids. Following the introduction, management
needs, including parameters requiring estimation, and data quality objectives are
discussed. Then the plan for the characterization of exposure, including the selection of
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measures of exposure, the detection of microbes, the issue of background levels of
pathogens and the estimation of fate, transport, uptake, and dosage are discussed.
The plan for the characterization of effects follows, including the selection of measures
of effect, establishing cause and effect and dose-response models for infection.
Methods for predicting disease, including the existence of infection thresholds and the
role of immunity and epidemiological methods are also discussed. Finally, the plan for
risk characterization is set forth, including the issue of standards, the possibility of tiered
analysis, the weight-of-evidence approach, probabilistic assessment and uncertainty
analysis.
The emphasis in this chapter is on aspects of analysis plans that are unique to
risk assessments for biosolids-derived pathogens rather than risk assessments for
pathogens in general. Therefore, some dose-response and epidemiological information
is deemphasized. However, many opportunities for research, observational studies,
and methods development are identified to improve and provide more defensible risk
assessments. Finally, because this is a generic framework for an analysis plan, it does
not contain the level of detail that would be expected in an analysis plan for a specific
site or a particular regulatory action. This report does not provide site-specific advice on
how to prioritize data needs, models, or assessment endpoints.
5.2. MANAGEMENT NEEDS
Risk managers have two fundamental requirements of risk assessors. The
assessment process must estimate risks to endpoints that are important to the decision,
and the results must have sufficient quality to be reliable. That is, risk managers should
be able to understand the reliability of the results and determine if that reliability
supports the decision to be made.
5.2.1. Assessment Endpoints
In any risk assessment, the assessment endpoint is an explicit expression of the
value that should be protected. In health assessments, the endpoint is a property of
human health. National level risk assessments for pathogens in biosolids would likely
be conducted by EPA's Office of Water, and therefore, risk managers from this office
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would determine the appropriate assessment endpoints. These may include
population-level endpoints or individual-level endpoints. For example, it may be
desirable to estimate the probability of infection (individual endpoint), number of
infections during a period of time (population endpoint), number of infections during an
outbreak (population endpoint), disease incidence (population endpoint), or related
endpoints. The endpoint may be cumulative (estimating risk from pathogens of all
sources) or may focus on only those infections or illnesses that are estimated to result
from pathogens in biosolids. The risk manager may also specify levels of infection or
disease that are acceptable or that require regulatory action. If applicable, these levels,
as well as other properties of the assessment endpoint, should be described in the
analysis plan. A purpose of the analysis plan is to set forth methods for estimating the
assessment endpoint. The assessment endpoints will allow EPA to determine the level
of public health and environmental protection from pathogens in biosolids afforded by
40 CFR 503,determine protective buffer distances, or evaluate current operational
standards and management practices.
5.2.2. Data and Data Quality
EPA (1998) recommends that risk assessors consider several general questions
related to the selection of data for the assessment:
•	How relevant will the results be to the assessment endpoint(s) and conceptual
model(s)?
•	Are there sufficient data of high quality to conduct the analyses with confidence?
•	How will the analyses help establish cause-and-effect relationships?
•	How will results be presented to address managers' questions?
•	Where are uncertainties likely to become a problem?
The analysis plan also specifies data quality objectives for the risk assessment.
The Superfund program provides a good model for specifying the type of information
that is needed to ensure data quality, specifying necessary and optimal levels of data
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quality, and identifying the means
of obtaining this information from
risk managers (U.S. EPA, 1994).
These steps are described in
Text Box 1.
In addition, in compliance
with the Information Quality Act
(Public Law 106-554), EPA
published Guidelines for
Ensuring and Maximizing the
Quality, Objectivity, Utility, and
Integrity, of information
disseminated by the
Environmental Protection Agency
(U.S. EPA, 2002b). EPA's
Information Quality Guidelines
build on ongoing efforts to
improve the quality of the data
and analyses that support EPA's
various policy and regulatory
decisions and programs.
Text Box 1.
Recommended Steps for Specifying Data Quality Objectives
(modified from U.S. EPA, 1994).
1.	State the Problem. Clearly specify the question that
relates to pathogens in biosoiids. Is the concern a generic
national problem? Or is it a site-specific one? Has an
infection or disease been observed where the cause is
unknown? Or is the risk manager concerned with future
prediction?
2.	Identify the Decision. Identify the decision that must be
made to solve the problem. For example, are new
regulations required to prevent unacceptable risk to human
health?
3.	Identify Inputs to the Decision. Identify the information
needed to make the decision and measurements,
simulations, and other analyses that must be undertaken
to provide that information. These are the major
components of the analysis plan.
4.	Define the Assessment Boundaries. Specify the
conditions to be assessed, including the spatial area, the
time period and the exposure scenarios to which the
decision will apply and for which inputs must be generated.
5.	Develop Decision Rules. Define conditions under which
an action, such as the promulgation of new regulations, wiii
be taken.
6.	Specify Acceptable Limits of Decision Error. Define error
rates that are acceptable to the risk manager.
7.	Optimize the Design. Design a study in which new data
are collected and design the use of existing data in
exposure or effects models, such that the expected
variance in parameters results in an acceptable limit in
decision error.
5.3. PLAN FOR CHARACTERIZATION OF EXPOSURE
5.3.1. Measures of Exposure
The first step to planning the characterization of exposure is selecting the
measures of exposure. In a human health risk assessment, these are measurable
characteristics of pathogens that are used to quantify exposure to humans or contact
with particular organ systems. Measures of exposure include several variables such as:
(1) concentrations of particular pathogens in environmental media or components of
these media (biosoiids, biosolids-amended soil, air, water, clay, aerosols), (2) duration
of each exposure (daily versus per event), and (3) number and frequency of discrete
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exposures over a specified time period.7 Measures of exposure to microbial pathogens
may also include inputs to models of fate, transport, or exposure (e.g., doses to
humans), as described below.
5.3.2. Detection of Pathogens
Following the selection of measures of exposure, the detection of pathogens is
the first type of analysis considered in the analysis plan. As stated in the literature
review (see the Appendix), one of the major data gaps related to pathogens in biosolids is
a recent national survey regarding levels of particular pathogens in sewage sludge and
biosolids. Appropriate sampling and analytical methods are also needed for detecting
and quantifying particular pathogens in sewage sludge (biosolids). In developing
standard measurement methods for pathogens in biosolids, careful evaluation should
be placed on method precision, accuracy, representativeness, sensitivity, and
reproducibility of measurement methods. This information is needed to support
national-scale human health risk assessments of biosolids. In site-specific risk
assessments, it is possible to analyze the biosolids, amended soil, water, air or
bioaerosol of concern to estimate pathogen levels, though these methods have high
levels of uncertainty. The only feasible current option for national scale risk
assessments is to conduct an analysis of pathogens in biosolids at several application
sites that are thought to be representative of such sites across the country. EPA
randomly samples at many biosolids sites (74 between 2006-2007) in many states (35
between 2006-2007). 145 analytes are tested and reported in the Targeted National
Sewage Sludge Survey (U.S. EPA, 2009b). However, no microbial agents are included
in the Targeted National Sewage Sludge Survey.
5.3.2.1. Bacteria
Smith et al. (2005b, Chapter 4) describe detection and enumeration capabilities
for bacterial pathogens that involve general or selective enrichment combined with
7 Chemical risk assessments often define exposure units as low level daily exposure over a 70-year life
span. Microbial risk assessments often define exposure units as per event (e.g., per meal, per serving,
per swimming event). The unit of exposure definition should be clear about whether daily exposure is
compounded over a lifetime.
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selective culturing or PCR and molecular identification techniques. However, these
experts acknowledge that the use of these methods to detect all potential pathogens in
a sample might be too costly or require too much effort to be practical. Thus, the use of
indicator organisms is recommended if adequate indicators and appropriate analytical
methodology are available (Smith et al., 2005b, Chapter 4) (also see the section on the
Use of Indicator Species below). Recent research on species-specific biosensors may
also produce useful products for detecting pathogens in biosolids (e.g., Guntupalli et al.,
2007).
Organic matter and high bacterial counts reduce recovery fraction for pathogens
in biosolids or amended soils (Rusin et al., 2003b). The analysis plan should indicate
the recovery rates for the detection technologies that will be used. For example,
recovery percentages of bacterial pathogens in aerosols that are reported in the
literature are currently about 10% (Lubick, 2007). Rusin et al. (2003a) had a recovery
efficiency of 8.7% for Staphylococcus aureus in Class B biosolids. EPA has new
standardized analytical methods for fecal coliforms and Salmonella (Federal Register
57 14219).
5.3.2.2. Viruses
Sampling and detection of viruses that are present at high levels in biosolids is
much easier than demonstrating conclusively that viral agents are not present (NRC,
2002). The primary determinant of the ease of detection of viruses is whether they can
be cell-cultured. All of the viral pathogens listed in the hazard characterization chapter
can be cell-cultured (NRC, 2002; Straub et al., 2007). Methods used to recover viruses
from sewage sludge have been optimized for the enteroviruses rather than for other
enteric viruses (Goyal et al., 1984; Gerba and Smith, 2005). Therefore, risk assessors
need to be aware that there is high uncertainty regarding concentrations of
nonenteroviruses in raw sewage sludge and treated biosolids (Smith et al., 2005b,
Chapter 8). In addition to the interlaboratory variabilities on methodology, other
disadvantages of cell culture methods include the high cost, long time required for
positive results (up to one month) and the presence of potentially toxic organic
compounds and inorganic elements in sewage sludge.
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PCR is an alternative family of methods for identifying viruses. These analyses
are quick, relatively inexpensive and sensitive. Direct reverse transcriptase PCR
(RT-PCR) detects nucleic acid sequences from active and inactive viral particles, and
thus may overestimate exposure (Straub et al., 1994). Integrated cell-culture PCR
(ICC-PCR) amplifies viruses in cell culture and amplifies viral RNA through enzymatic
PCR. The NRC (2002) thought that ICC-PCR would be the method of choice because
of the potential for cell culture alone to underestimate human exposure and for RT-PCR
to overestimate exposure (Reynolds et al., 1996; NRC, 2002).
5.3.2.3.	Helminths
Various assays for helminth eggs in biosolids are available, but no standard
assay exists, mainly because quality-assurance and quality-control studies have not
been published for many study protocols (NRC, 2002). Candidate methods are
referenced in NRC (2002), each with different recovery percentages for Ascaris eggs.
Many do not adequately consider sample preservation and pretreatment. Some of
these are not very accurate. The Tulane assay is discussed with recovery percentages,
but this assay may not be valid for detecting helminths such as Trichuris trichiura that
have eggs of different densities from Ascaris (NRC, 2002).
5.3.2.4.	Protozoa
Methods for detecting helminths may be applicable to protozoa if final sieve size
of the collection filters is adjusted to the smaller size of Giardia and Cryptosporidium.
Viability and infectivity assays for protozoa that are available for the analysis plan
include vital dye staining, animal infectivity, cell culture or PCR. Recovery rates from
biosolids are low; 10% recovery rate using the sedimentation technique and less than
3% using the flotation technique. On average 3.2-16.3% Cryptosporidium oocysts and
2.4-41.7% Giardia cysts are recovered (NRC, 2002).
5.3.3. Use of Indicator Species
Because of the wide range of pathogens found in human feces, domestic
wastewater and biosolids, direct monitoring and quantification of all of the pathogens in
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biosolids may not be practical for a site-specific risk assessment (Nappier et al., 2006).
Indicator species are abundant and are typically nonpathogenic microorganisms that
may be used to indicate the presence of a suite of pathogens. For example, fecal
coliform density and Salmonella are used as indicators of wastewater treatment
efficiency (40 CFR 136). Tests for indicator microorganisms should be relatively simple
and routine (NRC, 2002). However, most indicators have been chosen to indicate
treatment effectiveness rather than measures of pathogens that are quantitative and are
more closely related to public health (Smith et al., 2005b, Chapter 4). Suggestions for
criteria for selecting indicator organisms in water matrices are presented in Text Box 2.
Tanner et al. (2005) cite research in their laboratory and other literature to show
that (a) there is approximately one human pathogenic bacterium per 1000 coliform
bacteria in biosolids and (b) one human enteric virus in Class B biosolids per
1000 coliphage.
Bacteria and helminths.
Indicators of a range of
pathogens in biosolids are
needed. It may not be
feasible for individual risk
assessors to develop these
indicators in the analysis plans
for individual risk
assessments. Given the
resistance of spore-forming
bacteria to desiccation,
indicators of these bacterial
pathogens would need to
behave similarly. The NRC
(2002) discusses Clostridium
perfringens as a potential
indicator of the efficiency of
disinfection. In particular, they
Text Box 2.
Criteria for Selecting an Ideal Indicator Organism in Water
Matrices (adapted from Gerba [2009] and NRC [2004]).
Biological Attributes
•	The organism should be useful for aii types of water.
•	The organism should be present whenever enteric
pathogens are present.
•	The organism should be present in greater numbers than
pathogens (relates to ease of detection).
•	The organisms should have a similar or longer survival
time than the hardiest enteric pathogen.
•	The organisms should have similar or greater transports
to pathogens.
•	The organism should not grow in water.
•	The density of the indicator organism should have some
direct relationship to the degree of fecal pollution and
correlate with health risk.
•	The organism should be a member of the intestinal
microflora of warm-blooded animals.
Methods Attributes
•	The testing method should be easy to perform with timely
results.
•	Method should be specific to desired target organism
•	Method should have broad applicability in different water
types.
•	Method should have adequate precision.
•	Method should have adequate sensitivity.
•	Method should provide quantifiable results.
•	Method may measure viability or infectivity.
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provide references suggesting that its spores might be a surrogate for eggs of Ascaris
because of its resistance to similar chemical and physical disinfection agents.
Furthermore, Dowd et al. (1997) recommend thermotolerant Clostridia as indicators of
fecal contamination in bioaerosols. Pillai et al. (1996) found that Clostridia and H2S
producers were detected on glass impingers at locations near biosolids-amended sites
where traditional bacterial indicators (fecal coliforms and fecal streptococci) were not.
Thus Clostridium perfringens may be a useful surrogate for a range of pathogens in the
analysis plan. Risk assessors may consider indicators of anaerobic pathogens, but
genera such as Bifidobacterium and Bacterioides cannot be reliably detected and
therefore cannot be routinely monitored (NRC, 2002).
Viruses, Smith et al. (2005b, Chapter 5) summarize the suitability of selected
agents as indicators of treatment performance and post-treatment risk for viruses. Only
the latter is relevant here and is presented in Table 7. Bacteriophages are the only
potential indicator viruses mentioned in NRC (2002) because of their presence in
sewage. Somatic coliphage infects strains of E. coii and can be detected using simple,
inexpensive methods (NRC, 2002). Lime is also included as a potential indicator of
post-treatment risk for survived viruses in Smith et al. (2005b), presumably because
enteric viruses should be eliminated with extended alkaline treatment. At this time,
these indicators are qualitative. Risk assessors would need to do substantial testing to
quantify relationships between these indicators and pathogens of potential concern.
Risk assessors may consider the use of a model organism for the development of a risk
assessment. For an example of how model organisms can be used in risk
assessments, see Soller (2006).
5.3.4. Background Levels of Pathogens
The analysis plan should assess background levels of pathogens through
measurement or extrapolation from regional values if available. Background levels of
pathogens are levels in environmental media (soil, water, or air) not amended with or
contaminated by biosolids. Background levels are due to colonization of media at the
regional scale. For example, endospore-forming bacteria such as Clostridium
perfringens are very common in soil. The risk assessment is only concerned with the
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TABLE 7
Suitability of Select Agents as Indicators of Post-Treatment Risk for
Viruses in Biosolids (Modified from Smith et al., 2005b)
Agent
Suitability
Adenoviruses
?
Ascaris
Yes
Coliphages
Yes
Clostridium perfringens spores
Yes
Enterococci
No
Enteroviruses
Yes
E. coli
No
Fecal coliforms
No
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incremental risk from pathogens in biosolids or the cumulative risk from pathogens in
biosolids-amended soil, rather than the risk from pathogens in soil alone.
Background levels of pathogens (and/or pathogenic factors) may be confounding
contributors to risk. For example, in a study of aerosolized endotoxin concentrations
downwind from a biosolids-amended site, Brooks et al. (2006, 2007b) found that levels
of endotoxin and diversity of bacteria in aerosolized soil were sometimes above those
associated with biosolids amended-soil, calling into question whether biosolids were the
primary source of the endotoxin.
5.3.5. Environmental Fate of Pathogens
The survival or regrowth of pathogens should be estimated if the risk assessment
is prospective (i.e., concerned with forecasting), and environmental media cannot be
sampled at the time of interest. Regulations that limit contact with biosolids do not
prevent environmental processes in the conceptual model such as aerosolization or
erosion (see Figure 2) and the death or multiplication of pathogens (see Figure 3).
Therefore, the analysis plan may include a plan for estimating pathogen fate. Most
studies of the fate of pathogens in sewage sludge are concerned with predicting the
reduction or inactivation of pathogens by treatment processes (e.g., Epstein, 2006;
Gantzer, 2001). Straub et al. (1993) reviewed available studies of survival of pathogens
in soil and sewage sludge and Pepper et al. (2008a) examined detection of viable
pathogens in biosolids; both are pertinent to this analysis plan discussion. Gerba and
Smith (2005) provide survival times of pathogens on soil and plants (see Table 8).
Microorganism regrowth is also a possibility (Zaleski et al., 2005a,b).
Risk assessors should not use survival data from enteric organisms such as
E. co//and Salmonella to estimate the much longer survival rates of bacterial pathogens
that form spores or are encapsulated (such as Mycobacterium spp.). Instead, a spore
forming organism like Clostridium perfringens may be a more appropriate indicator
(Karpowicz et al., 2009) because it is typically found throughout a wide range of
environmental conditions, including extreme temperatures, relative humidity, and UV
levels (Brooks et al., 2004a).
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TABLE 8
Survival Times of Pathogens in Soil and on Plants
Modified from Gerba and Smith (2005)
Pathogen
Soil
Plants
Highest
Maximum
Typical
Maximum
Highest
Maximum
Typical
Maximum
Bacteria
1 year
2 months
6 months
1 month
Viruses
6 month
3 months
2 months
1 month
Protozoa
10 days
2 days
5 days
2 days
Helminths
7 years
2 years
5 months
1 month
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5.3.6. Transport of Pathogens
The conceptual model in Figure 2 describes several transport processes,
including wind erosion, surface runoff and water erosion, aerial dispersal of bioaerosols,
deposition on crops, leaching to groundwater and vector transport. Applying biosolids
can affect the rate at which soil erodes (Moffet et al., 2005). The analysis plan needs to
provide a plan for answering the questions of how far and in what concentrations
pathogens will travel. Models are available for most transport processes, though they
may have some limitations as described below.
5.3.6.1.	Water Erosion
Water erosion is typically modeled using the universal soil loss equation or its
modifications. Average annual soil erosion is the product of a rainfall erosivity index,
soil erodibility factor, topographic factor, cropping factor and conservation practice factor
(Wischmeier and Smith, 1978). The soil erodibility factor estimates the cohesive nature
of a soil type and resistance to transport from raindrop impact and surface flow. While
this factor is available for various soil types, to our knowledge, it has not been measured
for biosolids or biosolids-amended soils. The crop management factor is specific to
agricultural systems and can include tillage but could be adapted to forest, greenway,
mineland, or other biosolids application sites. Significant soil disturbance resulting from
tracked vehicles could be incorporated in the soil erodibility or crop management
factors. A limitation is that this equation is not applicable to a specific storm or year. If
erosion is expected to be a significant transport process, these analyses would need to
be part of the analysis plan.
5.3.6.2.	Surface Runoff and Aqueous Transport
Methods for estimating surface runoff should be described separately from
erosion models in the analysis plan. For example, Montemagno et al. (2004) describe a
modeling strategy for estimating surface water contamination by pathogens from
agricultural sources, using the specific example of oocysts of Cryptosporidium. Both
surface runoff and water erosion are simulated.
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For site-specific assessments, it may be desirable to use a spatially explicit
model to simulate transport from land to streams and through a watershed to
recreational areas or water intakes. Better Assessment Science Integrating Point and
Nonpoint Sources (http://www.epa.gov/waterscience/basins/) provides an integrated
system for such assessments. Alternatively, simple models of dilution and transport in a
generic stream can be used.
5.3.6.3.	Wind Erosion
Wind erosion should be considered in areas where wind speeds are often above
the 19.3 km/h required to initiate soil movement (Brady, 1974). Wind erosion of soils
and landscapes is governed by 11 primary variables: soil erodibility, knoll erodibility,
surface crust stability, soil ridge roughness, wind velocity, surface soil moisture,
distance across field, sheltered distance, quantity of vegetative cover, kind of vegetative
cover and orientation of vegetative cover (Woodruff and Siddoway, 1965). The Wind
Erosion Equation, developed by Woodruff and Siddoway (1965) groups many of these
variables into an erodibility factor (which increases with percentage of soil particles
greater than 0.84 mm diameter), a ridge roughness factor, a climatic factor, a field
length factor and a vegetative cover factor. Clearly, the erodibility factor could be
specific to biosolids, but the climatic factor, which incorporates soil moisture, would also
be affected by biosolids added to the surface of soil or incorporated in soil. Again, this
equation is not applicable to a specific year or wind event. Also, the Wind Erosion
Equation provides a measure of dislodged soil; the equation provides no estimates of
the travel distance of the soil (Batie, 1983).
5.3.6.4.	Aerial Transport of Bioaerosols
To estimate bioaerosol transport, a risk assessor should understand the
mechanism of transport, the release rates of the different microbes, the dispersion of
the bioaerosols, and the deposition of the microorganisms (Baertsch et al., 2007; Pillai,
2007). These quantities depend on whether pathogens are aerosolized during
particular types of biosolids application or following application. Pathogens in
bioaerosols and their transport may be measured or modeled. The analysis plan may
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include measurement of pathogens in air as a source term for a dispersion model or
near the human receptors of interest.
The sampling of bioaerosols involves the removal and concentration of biological
particles from the air (Pillai and Ricke, 2002). Sampling bioaerosols poses a particular
challenge, compared to sampling of biosolids. Impaction, impingement, gravity settling,
filtration and electrostatic precipitation are options for concentrating microorganisms
from bioaerosols, but efficiencies of collection can be low or uncertain (NRC, 2002; Pillai
and Ricke, 2002). Where molecular assays are feasible, collection methods do not
have to preserve the viability of microbes for identification. It should be noted that
culture methods require viability. If molecular methods are used, a description of the
targets that are detected should be discussed in the context of how they compare to
culture methods (Pillai and Ricke, 2002). Although there is a standard method for
assessing occupational exposures to bioaerosols in indoor environments, no
comparable standard exists for outdoor environments (NRC, 2002). Due to insufficient
testing of available methods, no recommendation has been established for a particular
sampling method for bacteria in bioaerosols. Risk assessments are more robust if the
authors describe methods and acknowledge caveats for testing sampling efficiencies of
their equipment in the analysis plan. Risk assessors should also be aware that during
transport, deposition, and sampling, bacteria as well as other pathogens can be
desiccated or inactivated, resulting in failure to culture and an underestimation of the
number of viable cells. The analysis plan should specify how sampled pathogens will
be handled.
Furthermore, determining an appropriate spatial distribution of samples is a
challenge for sampling bioaerosols. If tens of acres are amended with biosolids,
substantial micrometeorological differences may result from differing topography,
vegetation, and mechanical agitation (NRC, 2002). Wind direction and speed may vary
during the sampling time. The orifices of bioaerosol samplers downwind may collect too
small of a volume of air to obtain detectable levels of bacteria, even if they are present
in bioaerosols. In addition, efficiency of samplers decreases with increased wind speed.
Thus, appropriate statistical analysis (Spicerand Gangloff, 2000) and appropriate
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numbers of replicates are uncertain. These issues should be addressed in the analysis
plan.
Models are available to estimate transport of pathogens in bioaerosols (Dowd
et al., 2000; Brooks et al., 2004b, 2005a; Eisenberg et al., 2006). "Point-source"
transport models are appropriate for localized sources of biosolids, such as a storage
pile, and "area-source" models are more appropriate for predicting concentrations of
pathogens downwind from a large biosolids-amended field in which including the length
and width of the field more accurately estimates aerosol loading rates (Dowd et al.,
2000). Dowd et al. (2000) modified a standard point-source transport model to
incorporate the expected reduction in microbial concentration with increased distance
from the source. Variables included the inactivation rate of the microorganism, mean
wind speed, diffusion constants, downwind distance from source and height of sample.
Typically, the risk assessor needs to back-calculate the rates of release of
microorganisms from the source using sampling data, because measurement is
extremely difficult (Dowd et al., 2000). A mathematical error in Dowd et al. (2000) was
corrected in Brooks et al. (2004b).
An empirical model is another option for estimating aerosolized pathogen
concentrations with distance from the source. Brooks et al. (2005a) derived a linear
regression model that estimated coliphage concentrations at various distances from the
spray application location, normalized for initial microbial concentration and wind speed.
The researchers conducted field tests with coliphage MS-2 added to water and sprayed
with a biosolids spray application truck. Temperature was also observed to influence
aerosol concentration (Brooks et al., 2005a). The relationship these researchers
derived may not be applicable to other biosolids, application methods or regions, but the
development of similar empirical models may be an objective of the analysis plan.
Correlations have been developed between microbial levels in biosolids and their
concentrations emitted during disking (Paez-Rubio et al., 2006) and spreading with a
slinger side-spreader (Paez-Rubio et al., 2007). These types of reconstructions permit
risk assessors to avoid difficulties of detecting pathogens in aerosols.
Indicator species may be used to estimate transport of related pathogens. For
example, the ratio between the concentration of indicator virus in aerosols and the
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concentration in biosolids was used to estimate a value for airborne enteric virus
(Coxsackievirus) in Dowd et al. (2000).
Even allowing for sampling limitations and recovery efficiency issues,
measurement is probably superior to models (which are validated using measurements
in any case). Many of the physicochemical interactions between pathogens and
biosolids and between pathogens and other components of bioaerosols are difficult to
model. For example, viruses have been observed to sorb strongly to biosolids particles
but to aerosolize more easily if present in the liquid fraction of biosolids (Brooks et al.,
2004a). The transport of large dust particles is not usually modeled. Moreover, during
application, the aerosol plume at each location is detectable for only a short period of
time (e.g., less than one minute per pass of a spray applicator in Tanner et al. [2005]).
Potentially complicating factors include variation in terrain, topography, vegetation,
micrometeorological conditions, biosolid composition, and biosolids land application
processes. Also, the bioaerosol transport reconstruction in Paez-Rubio et al. (2006)
tended to result in a lower concentration than what was measured. Thus, risk
assessors should justify selection of particular models in the analysis plan.
5.3.7. Contact with Crops
Biosolids and associated pathogens can deposit to crop leaves following erosion,
aerial transport, or rain splash. Pathogen residues on root and leaf crops can be
measured or the deposition processes can be modeled. Because of the uncertainty of
modeling, direct measurement of pathogens on select crops would be preferred. If
measurement is not possible, risk assessors can estimate the biosolids residues on root
and leaf crops based on standard crop exposure assumptions (U.S. EPA, 1997), though
these assumptions do not account for aerosolized pathogens depositing directly on
leaves. Gale (2005b) offers assumptions that 10% of root crops were consumed
unwashed or that 90% of soil was removed by washing prior to consumption.
Gale (2005a,b) describes ramifications of using the arithmetic mean root crop
concentration as an input to dose-response models. This statistic often overestimates
the number of people who are exposed to pathogens, because where pathogens are
spatially clustered, many individuals are not exposed. Thus, the analysis plan should
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indicate that the arithmetic mean exposure concentration (if used) may give a
conservative estimate of the number of people exposed.
5.3.8.	Uptake and Dosage
The analysis plan should include methods for estimating inhalation, ingestion,
and dermal exposure when consideration of those routes of exposure is appropriate
(see conceptual model discussion). For example, the dose of aerosolized pathogens to
a person during a period of time may be estimated by measuring or modeling
concentrations of microbes at a specific distance from the source and the inhalation rate
over a period of time.
5.3.9.	Exposure Factors
EPA does not have standard exposure factors for use in risk assessments of
pathogens in biosolids. However, many of the exposure factors and assumptions
described in EPA's Exposure Factors Handbook (U.S. EPA, 1997) and Child-specific
Exposure Factors Handbook (U.S. EPA, 2008), which was designed for use in human
exposure assessments for chemical contaminants, are pertinent. These include general
exposure factors (e.g., drinking water intake rates, soil ingestion rates including for the
pica child scenario, inhalation rates, body weight, body surface area), food ingestion
factors (e.g., fruit and vegetable intake rates and water contents) and activity factors
(e.g., time spent outdoors). This and other risk assessment guidance is available from
the Risk Assessment Information System (U.S. DOE, 2006).
Some of the exposure factors in EPA (1997) may not be pertinent to risk
assessments for pathogens in biosolids. For example, activity factors that estimate time
spent outdoors may not be as relevant for a risk assessment of bioaerosols generated
during biosolids application as the duration of the application process. The percentage
of inhaled particles that would be ingested should be specific to biosolids-generated
aerosols. Pepper et al. (2006) describe studies that use a factor of 10%, and Brooks
etal. (2005b) uses 50%. Haasetal. (1999) recommend exposure factors that are
relevant to risk assessments for pathogens. While many of these factors are analogous
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to those in EPA (1997), others are more pertinent to risk assessments for pathogens
(e.g., proportion of pathogens that are transferred to and from hands).
5.4. PLAN FOR CHARACTERIZATION OF EFFECTS
5.4.1. Measures of Effect
A measure of effect is a measurable quantity that is used to estimate the effects
of exposure (to biosolids-derived pathogens) on the assessment endpoint. In this
problem formulation, assessment endpoints include aspects of human health estimated
at the individual level or population level. The analysis plan describes the measures of
effect for the risk assessment. Suter et al. (2000) summarized considerations in
selecting measures of effect for ecological risk assessments of chemical contaminants.
These considerations were adapted here for pathogens in biosolids and at least the first
two considerations are necessary to meet the definition of a measure of effect:
•	Corresponds to an assessment endpoint (usually infection or illness)
•	Endpoint is quantifiable or binary (e.g., infected or not infected)
•	Makes use of existing data
•	Is readily measured/detected (e.g., illness endpoint can be diagnosed, likelihood
of disease given infection has been characterized, infection can be directly
measured)
•	Is of appropriate temporal and spatial scale (symptoms that occur within a
timeframe where causality is relatively easier to establish versus chronic
sequelae that occur far removed in time from infection)
•	Is appropriate to the exposure route (pathogen can infect the tissues that are
relevant for the exposure route)
•	Is diagnostic of particular pathogens (pathogens can be grouped based on
symptom sets)
•	Shows low variability, increasing the likelihood of detecting an effect (Variability
in response of the population is not a problem if it is well characterized, e.g., rate
of movement between susceptible, infected, and resistant status is known.
Variability in pathogen infectivity and virulence can introduce significant
uncertainty into estimates. Particularly if strain/serovar/isolate variations are
known to be highly variable.)
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•	Is broadly applicable to different locations (For national level microbial risk
assessment information on prevalence of strains with different properties is
important. Local immunity may also increase variability and therefore
uncertainty.)
•	Is a standard test or measurement method
Measures of effect are derived from laboratory studies (e.g., rat or mouse
ingestion or bioaerosol inhalation studies) or epidemiological studies designed around
biosolids application or disease outbreaks (controlled human clinical studies involving
ingestion or inhalation are likely rare or nonexistent). Studies of disease outbreaks are
often used to validate measures derived from animal models. The most applicable data
would come from studies with biosolids, but other studies of pathogens can provide
relevant data, especially in the absence of studies of biosolids. Additional health
endpoints such as psychological effects may be considered in the problem formulation
stage of a specific risk assessment.
Measures of effect in this problem formulation for biosolids-derived pathogens
may include probability of infection (individual measure), number of infections during a
period of time (population measure), number of infections during an outbreak
(population measure), disease incidence (population measure) or related measures.
5.4.2. Establishing Cause and Effect
As noted later in the literature review (see the Appendix), a causal association
between exposures to pathogens in biosolids and adverse effects on human health has
not been documented based on principles of epidemiology. However, some experts
point out this may be "too high a bar" to achieve before demonstrating more genuine
concerns about potential effects. Risk assessors should examine relevant data both
supporting and refuting a cause-and-effect relationship. This is most important in
locations where biosolids are being implicated for observed disease symptoms.
Principles for establishing causality are described in Hill (1965). These include
strength of association, consistency of association (e.g., observation of the symptoms
near multiple biosolids application sites), specificity of association, relationships
between timing of application and onset of symptoms, biological gradient
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(dose-response relationship), plausibility of the causative relationship, coherence of
evidence, observation in experiments and analogy to known associations (e.g.,
occupational exposures to pathogens in biosolids). Hill's principles may be used to
determine whether land application of biosolids may cause particular diseases. The
analysis plan for site-specific risk assessments where disease has been observed might
include methods that are not pertinent to national-scale assessments. For example,
deoxyribonucleic acid (DNA) fingerprinting methods can be used to determine whether
pathogens isolated from sick individuals have originated from land-applied biosolids
(Dowd and Pillai, 1999; NRC, 2002). Santo Domingo etal. (2007) provide methods to
track sources of fecal pollution. Although there are no EPA approved methods, there
are standardized DNA fingerprinting methods used in the PulseNet labs that monitor
foodborne pathogens.8 Epidemiological studies are discussed below. Risk assessors
for site-specific human health assessments might also benefit from reviewing EPA's
guidance for identifying ecological stressors to specific aquatic ecosystems in the
Stressor Identification Guidance Document (U.S. EPA, 2000) and the supporting Causal
Analysis/Diagnosis Decision Information System Web site (http://www.epa.gov/caddis/)
to see how approaches used there might apply to pathogens in land-applied biosolids.
5.4.3. Dose-Response Models for Infection
Empirical effects models quantify the relationship between the dose of a
microbial agent and frequency of a particular adverse outcome, such as infection,
disease, or mortality. These models may assume a minimum infective dose greater
than one organism (which for microbial pathogens is supported by little evidence, see
below) or a no-threshold continuous dose-response function. These empirical models
allow risk assessors to estimate risk at low doses of pathogens. The equations are
derived from exposure of humans or animal models to various concentrations of
pathogens. For a more detailed treatment of dose-response models, see the Draft
Protocol for Microbial Risk Assessment to Support Human Health Protection for
Water-Based Media (U.S. EPA, 2009a).
8 The standard lab protocols are published on the Centers for Disease Control and Protection website:
http://www.cdc.gov/pulsenet/protocols.htm (accessed 6/30/10).
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Microbial dose-response models mathematically represent the measure of the
dose that yields the probability of a given adverse effect. For microbes, the models are
required to be biologically plausible and should consider effects due to microbial strain
variations (Coleman et al., 2004) and that a population of humans exposed to infectious
microbes will receive a distribution of actual doses (Haas et al., 1999).
Several dose-response models have been used to assess human health risk
from microbial agents. These models include exponential dose-response, beta-Poisson
dose-response and simple and variable threshold models. These models have been
used to assess risk from waterborne and foodborne exposures to microbial agents and
recently in risk assessments of pathogens in dewatered, land-applied biosolids (Dowd
et al., 2000; Brooks et al., 2004b, 2005b; Eisenberg et al., 2004). Table 9 provides
examples of dose-response models for microbial agents that may be associated with
biosolids. All of these examples, except for endotoxins, pertain to the endpoint of
infection rather than disease. Only the endotoxins use a threshold model which is
consistent with their chemical nature; doses of all of the other microbial agents were as
whole organisms. Further reading and examples of critically analyzed dose-response
curves for microbial agents that may be associated with biosolids are presented in
Chapter 9 of Quantitative Microbial Risk Assessment (Haas et al., 1999) and in studies
by Coleman and Marks (2000), Teunis et al. (2005), and EPA (2009a).
Infective doses reported for various bacteria, viruses, and protozoan and
helminth parasites are tabulated in Epstein (2006) and Gutierrez (2005). However,
Haas et al. (1999) argue that most evidence supports the independent action (or
single-organism) hypothesis that even a single organism can initiate an infection in a
susceptible host. Risk assessors might view reported infective doses as doses where
infection becomes likely rather than actual thresholds.
Dose-response models represent major information gaps for risk assessments
related to pathogens in biosolids (Coleman and Marks, 1998). Most dose-response
models have been developed from human or animal feeding studies or from
investigations of outbreaks caused by contaminated food without apparent biosolids
involvement (Haas et al., 1999; Teunis et al., 2008b). Dose-response relationships are
not available for all of the pathogens potentially found in biosolids (see hazard
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TABLE 9
Examples of Dose-Response Models for Microbial Agents
(adapted from U.S. EPA, 2009a)
Organism
Measure of
Exposure
Model
Endpoint
Reference
Adenovirus 4
Dose
Exponential
Human
infection
Crabtree et al.
(1997), Haas etal.
(1999)
Campylobacter
jejuni
Dose
Beta-Poisson
Human
infection
Haas etal. (1999),
Medema and Smeets
(2004), Teunis et al.
(1996)
Campylobacter
jejuni
Dose
Hypergeometric
beta-Poisson
Infection
Illness:
conditional on
infection
Teunis et al. (2005)
Coxsackievirus
B3
Dose
Exponential
Human
infection
Dowd et al. (2000),
Brooks etal. (2004b),
Haas etal. (1999)
Cryptosporidium
parvum
Dose
Exponential
Human
infection
Dupont et al. (1995),
Haas etal. (1996,
1999), Okhuysen et
al. (1999), EPA
(2006b)
Cryptosporidium
parvum
Dose
Beta-Poisson
Human
infection
Englehardt and
Swartout (2004)
Cryptosporidium
parvum
Dose
Beta-Poisson
Gastroenteric
illness
Englehardt and
Swartout (2006)
Echovirus 12
Dose
Exponential
Human
infection
Haas etal. (1999)
Echovirus 12
Dose
Beta-Poisson
Human
infection
Teunis et al. (1996),
Regli etal. (1991),
Rose and Sobsey,
(1993), Rose and
Gerba (1991)
Endamoeba coli
Dose
Beta-Poisson
Human
infection
Haas etal. (1999)
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TABLE 9 cont.
Organism
Measure of
Exposure
Model
Endpoint
Reference
E. coli (0111)
Dose
Beta-Poisson
Human
infection
Ferguson and June
(1953), Haas etal.
(1999)
E. coli (055)
Dose
Beta-Poisson
Human
infection
June et al. (1953),
Haas etal. (1999)
E. coli 0157:H7
Dose
Beta-Poisson
Human
infection
(Teunis et al., 2008b)
E. coli 0157:H7
Dose
Hypergeometric
beta-Poisson
Human
infection
(Teunis et al., 2004)
Endotoxin
Concen-
tration in air
Threshold
Decreased
lung efficiency,
Organic Toxic
Dust
Syndrome
Baker et al. (1986)
Enteric virus
Dose
Beta-Poisson
Human
infection
Gerba etal. (2002)
Giardia lamblia
Dose
Exponential
Human
infection
Haas etal. (1999),
Regli etal. (1991),
Rose and Gerba
(1991), Rose etal.
(1991), Teunisetal.
(1996)
Hepatitis A virus
Dose
Exponential
Human
infection
Haas etal. (1999)
Legionella
Dose
Exponential
Human
infection
Armstrong and Haas
(2008)
Norovirus
Dose
Hypergeometric
function; one-hit
Human
infection
Teunis et al. (2008a)
Poliovirus I
Dose
Beta-Poisson
Human
infection
Regli etal. (1991),
Rose and Sobsey
(1993),
Rose and Gerba
(1991)
Poliovirus I
Dose
Exponential
Human
infection
Haas etal. (1999),
Regli etal. (1991)
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TABLE 9 cont.
Organism
Measure of
Exposure
Model
Endpoint
Reference
Poliovirus III
Dose
Beta-Poisson
Human
infection
Rose and Sobsey
(1993), Regli etal.
(1991), Rose and
Gerba (1991)
Rotavirus
Dose
Exponential
Beta-Poisson
Log-probit
Human
infection
Ward etal. (1986),
Haas etal. (1999),
Gerba etal. (1996b)
Regli etal. (1991),
Rose and Gerba
(1991),
Rose and Sobsey
(1993)
Rotavirus
Dose
Hypergeometric
bet-Poisson
Human
infection
Teunis and Havelaar
(2000)
Salmonella spp.
Dose
Beta-Poisson
Human
infection
Rose and Gerba
(1991)
Salmonella spp.
Dose
Gompertz log
Human
infection
Coleman and Marks
(2000), Coleman et
al. (2004), Solleret
al. (2007)
Salmonella spp.
Dose
Generalized
linear mixed
models and
fractional
polynomials of
dose
Human
infection
Bollaerts etal. (2008)
Salmonella
serovar Anatum
Dose
Beta-Poisson
Human
infection
McCullough and
Eisele (1951), Haas
etal. (1999)
Salmonella
serovar Typhi
Dose
Beta-Poisson
Human
infection
Dowd et al. (2000),
Brooks etal. (2004b),
Haas etal. (1999),
Rose and Gerba
(1991)
Salmonella
serovar Typhi
Dose
Fractional
polynomials
Human
infection
Namata etal. (2008)
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TABLE 9 cont.
Organism
Measure of
Exposure
Model
Endpoint
Reference
Shigella
Dose
Beta-Poisson
Human
infection
Haas etal. (1999)
Vibrio cholera
Dose
Beta-Poisson
Human
infection
Haas etal. (1999)
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characterization chapter). Dose-response relationships are also not available for
inhaled microorganisms (NRC, 2002). As stated in the literature review (see the
Appendix), the percentage of inhaled pathogens that are ingested is unknown.
Dose-response models are also not available for dermal exposure. Furthermore, few
dose-response models are available for disease.
5.4.4. Predicting Disease
Existing risk assessment studies for pathogens in biosolids estimate risk of
human infection rather than risk of disease (see literature review in the Appendix). If
limited by existing data, risk assessments for diseases caused by pathogens in
biosolids would be highly uncertain.
Disease is a function of a "triad," the interaction of pathogen, host, and
environment. All three factors figure into assessing the incidence of disease in
individuals, and the problem formulation should include a plan for the analysis of all
three aspects. The pathogen is the causative agent of the disease. Whereas
chemicals are generally assumed to elicit human-comparable responses in appropriate
animal models, pathogens are more host-specific. Pathogens can elicit adverse
responses either through their own biological activity within the host or through the
production of toxic byproducts.
The second aspect of disease is the host condition. The disease manifestation
can vary considerably among infected individuals based on nutritional and health status,
and immune profile. Individuals in good health with a history of prior exposure to similar
strains of pathogens are less likely to exhibit pronounced symptoms than individuals in
poor health or without prior exposure. Host susceptibility can be considered on an
individual level or a population level (Balbus et al., 2000). Immunity is one of the most
important parameters influencing the risk from pathogens in biosolids, based on the
Eisenberg et al. (2004) model. The analysis plan should specify whether groups of
individuals of particular immune status are assessment endpoint entities in the risk
assessment. Protocols to incorporate immune status or other pathogen susceptibility
factors (pregnancy, age) into risk assessments have not been robustly developed
(NRC, 2002).
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The environment aspect of the triad refers to conditions which promote or retard
the ability of the organism to survive in various media and which contribute or limit the
spread of the organisms to a receptor. For the most part, the environment is addressed
in the exposure components of the conceptual model and is pertinent to infection rather
than disease. An assessment of disease incidence cannot proceed without an
understanding of these factors and how they influence individual components of the
model.
5.4.4.1.	Risk Assessment Model
Colford et al. (2003) and Eisenberg et al. (2004, 2005, 2006) developed
methodologies to assess risks to human health from pathogens in biosolids and
biosolids-amended soil. While many of the processes in the model are those described
in this chapter (fate, transport, uptake, exposure pathways to groundwater and
aerosols), not all aspects of those models may be needed depending on the particular
risk assessment. Eisenberg et al. (2008) argue that raw sludge data on pathogen levels
combined with fate and transport modeling for treatment effects is a helpful tool.
Eisenberg et al. (2004, 2005, 2006) also modeled secondary transmission, which is
important for estimating the total burden of disease. However, secondary transmission
of pathogens is not unique to pathogens found in biosolids and, as such, is not
discussed further here. This document is concerned first and foremost about any
potential risks of primary infection because secondary transmission is not a concern
unless a primary infection has occurred. If secondary transmission is suspected, other
sources of information may be investigated on how to address that. For a few
resources regarding secondary transmission refer to Section 3.
5.4.4.2.	Role of Epidemiology
Epidemiological assessments of land-applied biosolids would provide much
needed information concerning the potential for adverse impact to human health
following land application of biosolids. Presently, few data exist to provide insight as to
whether a causative association exists between applied biosolids and adverse health
effects. Temporal and spatial relationships between the time of application and onset of
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symptoms or other indicators would identify key routes of exposure to assess the
validity of the conceptual models presented here and to prioritize different exposure
scenarios. Epidemiological assessments would focus on studies or disease reports
(clustering of illness cases) that can draw a link between those individuals living in close
proximity to sites of application and members of farm families and workers who apply
biosolids to determine if those individuals have a higher incidence of disease over time.
Risk assessments which use epidemiological studies of sites on or near places of
biosolids application would be based on the collection of several key data sets. First,
the data should indicate whether individuals living on or near lands receiving biosolids
have a higher incidence of infection compared with cohorts at more distant locations.
Second, data should identify temporal relationships between time and duration of
application and onset of symptoms. Such relationships could indicate potential route of
exposure—rapid onset may suggest aerosol exposure, whereas delayed disease may
indicate an alternate exposure route. Third, data should establish a concordance of
symptoms which could also help to determine the route of exposure and whether a
single or multiple pathogens are responsible for the effects. Collectively, this
information would help to determine if there is a significant microbial risk associated with
the use of Class B biosolids and, if so, to help to refine conceptual models and to
identify the primary data and methods needed for the risk assessment.
Additionally, epidemiological information for biosolids amendments should focus
on plausible exposure scenarios and the characterization of potentially exposed
cohorts. First, identifying the exposure settings provides a link between biosolids
application and environmental transport of pathogens and exposure points for human
contact. Second, data on potentially exposed populations should be identified using
information on proximity to the site of biosolids application, climatic conditions, and
temporal relationships between posited exposures and the onset of infection or clinical
symptoms. The selection of appropriate cohorts is important along with the availability
of supporting medical information, such as isolates of pathogens and/or serology
demonstrating infection within a time frame that corresponds with a plausible exposure
scenario (e.g., time of application, environmental transport, exposure point, exposure
route, infection, etc.).
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Risk assessors should be aware of the difficulties in conducting an
epidemiological study of biosolids exposure. For example, various symptoms may be
associated with one pathogen, and various pathogens can cause similar symptoms
(Simmonds, 2005). In addition, low level health effects are very difficult to detect with
epidemiological studies because very large population sizes would need to be studied
to observe effects. However, preliminary work has been done to scope epidemiological
designs to assess acute health effects and community-level exposure to treated sewage
sludge (Class B biosolids) in North Carolina and Virginia (Heaney et al., 2006).
5.5. PLAN FOR RISK CHARACTERIZATION
The analysis plan should include a plan for conducting the risk characterization,
which is the phase of risk assessment that integrates the characterization of exposure
and the exposure-response relationships to estimate the likelihood of health effects
endpoints. Care should be taken that the units between the exposure parameters and
the dose-response parameters agree or the uncertainty introduced by differences in
units can be qualitatively or quantitatively characterized. For example, exposure age
groups may encompass different age groups than dose-response data. If "healthy
adults" is defined as age 18-55 with specific "health" indicators determined by doctor's
examination (e.g., in a clinical trial) for the dose-response parameter and the exposure
parameter population group is defined as "adults age 16-60" then the nuances of those
differing groupings and the possible effects those differences have on the risk
characterization should be discussed.
5.5.1. Screening Risk Assessment
The analysis plan describes whether the risk assessment will include a
screening-level risk characterization to eliminate pathways, pathogens, or scenarios that
are clearly not of concern. A screening analysis typically makes use of effects
standards or benchmarks, but no acceptable level of pathogens in biosolids has been
established. Screening analysis can also eliminate pathways using qualitative
information (e.g., obvious lack of contact between pathogens and residents in an area
devoid of residences). A risk assessor with sufficient resources could develop critical
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distances for potential risk associated with the bioaerosol transport pathway, and thus
eliminate scenarios where there are no people within the critical distance. Screening
analysis is usually conducted for information-rich risk assessment topics, which is not
the expected situation for risk assessments for pathogens in biosolids.
5.5.2.	Weight of Evidence
If multiple lines of evidence are expected, the analysis plan should explain how
these results would be weighed. Each line of evidence links an exposure estimate with
an effects estimate, and qualitative or quantitative weights may be given to the
combined risk estimate. Evidence from measures of pathogen levels in aerosols might
be weighted more than evidence from modeled estimates based on measures of
biosolids-amended soils. Evidence from well-designed epidemiological studies might
be weighted more than evidence from rodent studies that have not been corroborated
with epidemiological evidence. Suter et al. (2000) provide the following criteria for
weighing evidence: relevance to the assessment endpoint, demonstrated relationship
between exposure and response, temporal scope of evidence compared to temporal
variance, spatial scope of evidence compared to spatial area of interest, data quality,
number of observations, and uncertainty of evidence. Given the paucity of exposure
and effects data for risk assessments of land-applied biosolids, weight-of-evidence
procedures may not be possible or infrequent.
5.5.3.	Uncertainty Analysis
Uncertainty analysis is the component of the risk characterization that reveals the
uncertainties of the exposure or risk estimate in quantitative or qualitative terms. The
management goal of uncertainty analysis may be simply to describe uncertainties, to
rank uncertainties, or to calculate a probabilistic endpoint. In the case of pathogens in
biosolids, probabilistic endpoints might be generated from variability and uncertainty in
measurements of pathogens in biosolids, outputs of transport models, or outputs of
dose-response models. Haas et al. (1999) divided uncertainty into parameter
uncertainty, which is related to measurement, and model uncertainty, which is related to
the structure of the equations (e.g., whether an important factor was missing from the
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model or not). The uncertainties associated with the sampling and modeling methods
are described above in the relevant sections. When new data are needed and cannot
be obtained, risk pathways that cannot be assessed are a source of uncertainty and
should be described in the analysis plan. Risk assessors need to distinguish between
pathways that are unquantifiable and pathways that are deemed negligible based on
evidence. For further information in uncertainty analysis see EPA (1997, 2009a).
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REFERENCES
APHA (American Public Health Association). 2004. Control of Communicable
Diseases Manual, 18th ed., D. Heymann, Ed. APHA, Washington, DC. (APHA, 2004,
635109)
This is a book that summarizes information for most pathogens.
Armstrong, T.W. and C.N. Haas. 2008. Legionnaires' disease: Evaluation of a
quantitative microbial risk assessment model. J. Water Health. 6(2):149—166.
(Armstrong and Haas, 2008, 449404)
Dose-response information for Legionella.
Baertsch, C., T. Paez-Rubio, E. Viau and J. Peccia. 2007. Source tracking aerosols
released from land-applied class B biosolids during high wind events. Appl. Environ.
Microbiol. 73(14):4522-4531. (Baertsch et al., 2007, 597302)
Used DNA-based source tracking to confirm that wind is a possible mechanism for the
aerosolization and off-site transport of land-applied biosolids.
Baker, J., S. Curtis, O. Hogsettand F. Humenik. 1986. Safety in Swine Production
Systems. Pork Industry Handbook. PIH-104 Cooperative Extension Service, Water
Quality and Waste Management, North Carolina Cooperative Extension Service. North
Carolina State University. Available at
http://www.bae.ncsu.edu/programs/extension/publicat/wqwm/pih104.html. (Baker et al.,
1986, 635112)
Dose-response information for Endotoxin.
Balbus, J., R. Parkin and M. Embrey. 2000. Susceptibility in microbial risk assessment:
Definitions and research needs. Environ. Health Perspect. 108(9):901 —905.
(Balbus et al., 2000, 635113)
Risk assessments should be clear about individual and population-level definitions of
susceptibility.
Barbier, D., C. Perrine, C. Duhamel, R. Doublet and P. Georges. 1990. Parasitic
hazard with sewage sludge applied to land. Appl. Environ. Microbiol. 56(5):1420-1422.
(Barbier et al., 1990. 598355)
This reference documents a modified FAUST technique that allows highly regular recovery of
Taenia saginata eggs from sewage sludge and quantification.
Barker,J., T.J. Humphrey and M.W.R. Brown. 1999. Survival of Escherichia coli 0157
in a soil protozoan: Implications for a disease. FEMS Microbiol. Lett. 173(2):291 -295.
(Barker et al., 1999, 104890)
E. coli 0157 survives and replicates in a common environmental protozoan, Acanthamoeba
polyphaga. As protozoa are widely distributed in soils and effluents, they may constitute an
important environmental reservoir for transmission of E. coli 0157 and other pathogens.
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Batie, S.S. 1983. Soil Erosion: Crisis in America's Croplands? The Conservation
Foundation, Washington, DC. (Batie, 1983. 598377)
This reference documents the concern of soil erosion, the nature and extent of soil erosion, the
effects of erosion, techniques for reducing soil erosion, and soil conservation.
Bollaerts, K., M. Aerts, C. Faes, K. Grijspeerdt, J. Dewulf and K. Mintiens. 2008.
Human salmonellosis: Estimation of dose-illness from outbreak data. Risk. Anal.
28(2):427-440. (Bollaerts et al., 2008, 635117)
Dose-response information for Salmonella.
Borchardt, M.A., P.D. Bertz, S.K. Spencer and D.A. Battigelli. 2003. Incidence of
enteric viruses in groundwater from household wells in Wisconsin. Appl. Environ.
Microbiol. 69(2):1172-1180. (Borchardt et al., 2003, 635118)
This reference estimates the incidence of viruses in Wisconsin household wells located near
septage land application sites or rural subdivisions served by septic systems to be approximately
8% and transient in nature.
Bowman, D.D. and R. Fayer. 2005. Concerns related to protozoan and helminth
parasites in biosolids and animal wastes. In: Contemporary Perspectives on Infectious
Disease Agents in Sewage Sludge and Manure, J.E. Smith, Jr., P. Millner, W.
Jakubowski and N. Goldstein Eds. The J.G. Press, Inc., Emmaus, PA. p. 127-162.
(Bowman and Fayer, 2005, 625218)
This reference documents protozoa and helminths of human health concern found in municipal
biosolids or animal wastes.
Bowman, D.D., R.S. Riemers, M.D. Little, M.B. Jenkins, W.S. Bankston and M.M.
Atique. 2000. Assessment and comparison of Ascaris egg and Cryptosporidium oocyst
inactivation with respect to biosolids processing. In: Proceedings of the Water
Environment Federation, Residuals and Biosolids Management 2000, Alexandra, VA:
Water Environment Federation, p. 111 -122. (Bowman et al., 2000, 625193)
This reference documents a higher level of resistance to inactivation by Ascaris eggs compared
to Cryptosporidium oocysts.
Bracken, M.B., K. Belanger, W.O. Cookson, E. Triche, D.C., Christiani and B.P.
Leaderer. 2002. Genetic and perinatal risk factors for asthma onset and severity: A
review and theoretical analysis. Epidemiol. Rev. 24(2):176—189. (Bracken et al., 2002,
This reference documents the genetic risk factors for asthma in the U.S. population.
Brady, N.C. 1974. The Nature and Properties of Soils. Macmillan Publishing, Co.,
New York, NY. (Brady, 1974, 070386)
This reference is a book that covers topics ranging from the physical properties of soil, to soil
organisms, to the erosion and regulations concerning soil.
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Brooks, J.P., B.D. Tanner, K.L. Josephson, C.P. Gerba and I.L. Pepper. 2004a.
Bioaerosols from the land application of biosolids in the desert southwest USA. Water
Sci. Technol. 50:7-12. (Brooks et al., 2004, 598384)
This reference documents the emission of bioaerosols during land application of Class B
biosolids in and around Tuscon, AZ. Bacteria and viruses may sorb to biosolids (contains high
percentage of organic matter) and may not be readily aerosolized.
Brooks, J.P., C.P. Gerba and I.L. Pepper. 2004b. Biological aerosol emission, fate and
transport from municipal and animal wastes. J. Residuals Sci. Technol. 1:16-28.
(Brooks et al., 2004, 635124)
This reference documents the mathematical error in the Dowd et al. (2000) paper.
Brooks, J.P., B.D. Tanner, C.P. Gerba, C.N. Haas and I.L. Pepper. 2005a. Estimation
of bioaerosol risk of infection to residents adjacent to a land applied biosolids site using
an empirically derived transport model. J. Appl. Microbiol. 98(2):397-405.
(Brooks et al., 2005, 598450)
This reference documents the development of an empirically derived transport model for use in
predicting downwind concentrations of viruses and bacteria during land application of liquid
biosolids and for use in subsequent microbial risk assessments.
Brooks, J.P., B.D. Tanner, K.L. Josephson, C.P. Gerba, C.N. Haas and I.L. Pepper.
2005b. A national study on the residential impact of biological aerosols from the land
application of biosolids. J. Appl. Microbiol. 99(2):310-322. (Brooks et al., 2005,
This reference documents the overall incidence of aerosolized micro-organisms from the land
application of biosolids and subsequently determined that microbial risks of infection were low for
residents close to biosolids application sites.
Brooks, J.P., B.D. Tanner, C.P. Gerba and I.L. Pepper. 2006. The measurement of
aerosolized endotoxin from land application of Class B biosolids in Southeast Arizona.
Can. J. Microbiol. 52(2):150-156. (Brooks et al., 2006, 598721)
This reference documents the presence of aerosolized endotoxin downwind of a biosolids land
application site. Levels were within ranges of concern suggested by other studies.
Brooks, J.P., S.L. Maxwell, C. Rensing, C.P. Gerba and I.L. Pepper. 2007a.
Occurrence of antibiotic-resistant bacteria and endotoxin associated with the land
application of biosolids. Can. J. Microbiol. 53(5):616-622. (Brooks et al., 2007,
598939)
This reference discusses the prevalence of antibiotic-resistant bacteria and endotoxin in soil and
found that land application of biosolids did not significantly increase the concentration of
endotoxins in soil.
Brooks, J.P., C.P. Gerba and I.L. Pepper. 2007b. Diversity of aerosolized bacteria
during land application of biosolids. J. Appl. Microbiol. 103(5): 1779-1790. (Brookset
al., 2007, 635125)
Molecular methods (including community DNA extraction, 16S rRNA PCR amplification, cloning
and sequencing) in this reference demonstrate that most bacteria aerosolized during land
application are actually of soilborne origin.
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Brown, L.M. 2000. Helicobacter pylori: epidemiology and routes of transmission.
Epidemiol. Rev. 22(2):283-297. (Brown, 2000. 599159)
This reference is a review of epidemiology and routes of transmission of Helicobacter pylori.
Consumption of raw or uncooked vegetables is related to the risk of Helicobacter pylori infection.
Brown, S. and C. Henry. 2002. Using Biosolids for Remediation and Reclamation of
Disturbed Soils. Center for Urban Horticulture, University of Washington, Seattle, WA.
Available at http://www.brownfieldstsc.org/pdfs/BiosolidsWhitePaper-UWash.pdf
(accessed 6/30/10). (Brown and Henry, 2002, 635132)
This is an overview of many practical aspects of biosolids application.
Bujoczek, G., R.S. Reiners and J.A. Olaszkiewicz. 2001. Abiotic factors affecting
inactivation of pathogens in sludge. Water Sci. Technol. 44:79-84. (Bujoczek et al.,
2001,603087)
This reference documents methods for biosolids treatment that results in effective inactivation of
pathogens.
CDC (Centers for Disease Control and Prevention). 1999. Prevention of Hepatitis A
Through Active or Passive Immunization: Recommendations of the Advisory Committee
on Immunization Practices (ACIP). MMWR48(RR12):1-37. Available at
http://www.cdc.gov/mmwr/preview/mmwrhtml/rr4812a1 .htm. (CDC, 1999, 625219)
This reference is a recommendation for the vaccination of children as the most effective way to
reduce hepatitis A incidence nationwide overtime.
CDC (Centers for Disease Control and Prevention). 2008a. Shigella Surveillance:
Annual Summary, 2006. U.S. Department of Health and Human Services, Atlanta,
Georgia. November. Available at
http://www.cdc.gOv/ncidod/dbmd/phlisdata/shigtab/2006/ShigellaAnnualSummary2006.p
df. (CDC, 2008, 646950)
This reference is an annual summary of Shigella surveillance that contains surveillance data on
reported laboratory-confirmed Shigella infections in the United States.
CDC (Centers for Disease Control and Prevention). 2008b. Delayed Onset and
Diminished Magnitude of Rotavirus Activity—United States, November 2007-May 2008.
MMWR 57:1-4. Available at
http://www.cdc.gov/mmwr/preview/mmwrhtml/mm57e625a1 .htm. (CDC, 2008, 635134)
This reference documents the ongoing rotavirus activity in the United States during the
2007-2008 season. Rotavirus activity during this season appears both substantially delayed in
onset and diminished in magnitude, compared with previous years.
Chale-Matsau J.R. and H.G. Snyman. 2006. The survival of pathogens in soil treated
with wastewater sludge and in potatoes grown in such soil. Water Sci. Technol,
54(5):269-277. (Chale-Matsau and Snyman, 2006, 459031)
Other than E. agglomerans, all the other species identified were found to be mainly either plant or
soil pathogens. The E. agglomerans are not primary pathogens but secondary opportunistic
pathogens, particularly in immunocompromised individuals. These results suggest that growing
high risk crops using wastewater sludge contaminated soil may lead to limited infestation of
produce with primary pathogens.
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Chapron, C.D., N.A. Ballester and A.B. Margolin. 2000. The detection of astrovirus in
sludge biosolids using an integrated cell culture nested PCR technique. J. Appl.
Microbiol. 89:11-15. (Chapron et al., 2000, 606788)
This reference demonstrates the prevalence and frequency of astrovirus in sludge and validated
the ICC-RT-PCR/nested PRC technique as a useful tool to detect viruses in sludge.
Chetochine, A., M.L. Brusseau, C.P. Gerba and I.L. Pepper. 2006. Leaching of phage
from Class B biosolids and potential transport through soil. Appl. Environ. Microbiol.
72(1 ):665-671. (Chetochine et al., 2006, 624915)
This reference documents the sorption of viruses within biosolids resulting in less mobility and
decreased transport of viruses through soil. Overall, only 8% of total viruses were available for
transport.
Clark, C.S., R. Rylanderand L. Larsson. 1983. Levels of gram-negative bacteria,
Aspergillus fumigatus, dust and endotoxin at compost plants. Appl. Environ. Microbiol.
45(5):1501-1505. (Clark et al., 1983. 025350)
This reference documents the level of airborne gram-negative bacteria, Aspergillus fumigatus,
dust and endotoxin measured at compost plants. A. fumigatus exceeded 10(6)/m3, whereas the
number of gram-negative bacteria was usually lower. Dust levels were moderate, and endotoxin
levels were well below 0.5 micrograms/m3.
Coleman, M. and H. Marks. 1998. Topics in dose-response modeling. J. Food Protect.
61 (11): 1550—1559. (Coleman and Marks, 1998, 635141)
Criteria for development of surrogate dose-response models for pathogens for which human data
are lacking; Risk assessment methods.
Coleman, M. and H. Marks. 2000. Mechanistic modeling of salmonellosis. Quant.
Microbiol. 2(3):227-247. (Coleman and Marks, 2000, 635140)
Risk assessors should routinely apply a variety of model forms in addition to the commonly used
beta-Poisson model to depict more fully the uncertainty of the "true" dose response model.
Coleman, M., H. Marks, N.J. Golden and H.K. Latimer. 2004. Discerning strain effects
in microbial dose-response data. J. Toxicol. Env. Heal. A. 67(8-10):667-685.
(Coleman et al., 2004, 635142)
Risk assessment tool.
Colford, J.M., Jr., D.M. Eisenberg and J.N.S. Eisenberg. 2003. A dynamic model to
assess microbial health risks associated with beneficial uses of biosolids—Phase 1.
Water Environment Research Foundation Report 98-REM-1 IWA Publishing, London,
U.K. (Colford et al., 2003, 635143)
Risk assessment tool.
Crabtree, K.D., C.P. Gerba, J.B. Rose, and C.N. Haas. 1997. Waterborne adenovirus:
A risk assessment. Water Sci. and Technol. 35(11—12):1 —6. (Crabtree et al., 1997,
-635144)
Dose-response information for Adenovirus.
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Dahab, M.F. and R.Y. Surampalli. 2002. Effects of aerobic and anaerobic digestion
systems on pathogen and pathogen indicator reduction in municipal sludge. Water Sci.
Technol. 46(10):181 -187. (Dahab and Surampalli, 2002, 624910)
This reference documents the evaluation of the effectiveness of anaerobic treatment systems in
reducing pathogen density levels under the Part 503 rule. The results indicate that Class B
sludge requirements under the rule are reasonable and can be achievable by the existing
treatment systems while Class A sludge requirements under the same rule may not be easily
achieved by the existing treatment systems.
DeLuca, G., F. Zanetti, P. Fateh-Moghadm and S. Stampi. 1998. [Occurrence of
Listeria monocytogenes in sewage sludge.] Zentralbl Hyg. Umweeltmed.
201 (3):269-277. [German] (De Luca etal., 1998, 624916)
This reference reports an investigation of Listeria in the sludge produced by the Bologna (Italy)
treatment plant during the various phases of treatment. The highest frequency and
concentrations of Listeria species (100% and 2743 MPN/g dry matter) was found in activated
sludge and the lowest (63% and 6 MPN/g dry matter) was found in digested sludge.
DeLuzio, N.R. and T.J. Friedman. 1973. Letter: Bacterial endotoxins in the
environment. Nature. 224(5410):49-51. (DeLuzio and Friedman, 1973, 624918)
This reference documents the detection of gram-negative endotoxins in deionized water, New
Orleans tap water, and comparison to other cities.
Dorn, C.R., C.S. Reddy, D.N. Lamphere, J.V. Gaeuman and R. Lanese. 1985.
Municipal sewage sludge application on Ohio farms: Health effects. Environ. Res.
38(2):332-359. (Dorn et al., 1985, 624924)
A 3-year prospective epidemiologic study that was conducted on 47 farms receiving annual
applications of treated sludge and 46 control farms in three geographic areas of Ohio. The
estimated risks of respiratory illness, digestive illness, or general symptoms were not significantly
different between sludge farm and control farm residents.
Dowd, S.E. and S.D. Pillai. 1999. Identifying the sources of biosolid derived pathogen
indicator organisms in aerosols by ribosomal DNA fingerprinting. J. Environ. Sci. Hlth.
A. 34(5):1061-1074. (Dowd and Pillai, 1999, 624927)
This reference documents the identification of aerosolized Clostridia isolate sources at a
commercial biosolid land application site using a ribosomal DNA fingerprinting method.
Dowd, S.E., K.W. Widmer and S.D. Pillai. 1997. Thermotolerant Clostridia as an
airborne pathogen indicator during land application of biosolids. J. Environ. Qual.
26(1 ):20-48. (Dowd etal., 1997. 624926)
This reference demonstrates the use of thermotolerant Clostridia as a reliable indicator to
determine the presence of microbial pathogens and biosolid derived microbial populations in
aerosols being generated from biosolid application programs.
Dowd, S.E., C.P. Gerba, I.L. Pepper and S.D. Pillai. 2000. Bioaerosol transport
modeling and risk assessment in relation to biosolid placement. J. Environ. Qual.
29:343-348. (Dowd et al., 2000, 624930)
This reference documents a study designed to generate biosolids-derived microbe release rate
data for use in modeling bioaerosol transport. An error in calculation was subsequently corrected
in Brooks et. al. (2004b).
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Dudley, D.J., M.N. Guentzel, M.J. Ibarra, B.E. Moore and B.P. Sagik. 1980.
Enumeration of potentially pathogenic bacteria from sewage sludges. Appl. Environ.
Microbiol. 39:118-126. (Dudley et al., 1980, 104895)
Enumerated Klebsiella, Staphylococcus, gram-negative enteric bacteria, and commonly used
indicator organisms, Salmonella, Mycobacterium sp., fluorescent Pseudomonas sp., Clostridium
perfringens, and Shigella in three domestic wastewater sludges. Aspects of methods are
discussed.
Dupont, H., C. Chappell, C. Sterling, P. Okhuysen, J.B. Rose and W. Jakubowski.
1995. Infectivity of Cryptosporidium parvum in healthy volunteers. N. Engl. J. Med.
332:855-859. (DuPont et al., 1995, 624933)
Dose-response information for Cryptosporidium pan/um.
Edmonds, R.L. 1976. Survival of coliform bacteria in sewage sludge applied to a forest
clearcut and potential movement into groundwater. Appl. Environ. Microbiol.
32(4):537-546. (Edmonds, 1976. 104898)
Groundwater contamination from vertical movement of potential pathogens appears unlikely, but
hazards from surface runoff and direct handling in the first year may arise.
Eisenberg, J.N.S., J.A. Soller, J. Scott, D.M. Eisenberg and J.M. Colford, Jr. 2004. A
dynamic model to assess microbial health risks associated with beneficial uses of
biosolids. Risk Anal. 24(1 ):221-236. (Eisenberg et al., 2004, 624944)
This reference documents a methodology for assessing risks to human health from pathogen
exposure using a population-based model that explicitly accounts for properties unique to an
infectious disease process, specifically secondary transmission and immunity. The applicability
of this risk-based method was demonstrated using a case study example in which the route of
exposure was direct consumption of biosolids-amended soil and the pathogen present in the soil
was enterovirus.
Eisenberg, J.N.S., J.A. Soller, J. Scott, D.M. Eisenberg and J.M. Colford, Jr. 2005. A
dynamic model to assess microbial health risks associated with beneficial uses of
biosolids. In: Contemporary Perspectives on Infectious Disease Agents in Sewage
Sludge and Manure, Compost Science and Utilization, J.E. Smith, Jr., P. Millner, W.
Jakubowski, and N. Goldstein, Ed. The J.G. Press, Inc., Emmaus, PA. p. 177-194.
(Eisenberg et al., 2005, 635158)
Risk assessment tool.
Eisenberg, J.N.S., K. Moore, J.M. Colford, Jr. 2006. Application of a dynamic model to
assess microbial health risks associated with beneficial uses of biosolids. Water
Environment Research Foundation, Alexandria, VA. (Eisenberg et al., 2006, 625220)
This reference is a book that demonstrates the application of dynamic models to assess microbial
health risks associated with beneficial uses of biosolids.
Eisenberg,J.N., K. Moore, J.A. Soller, D. Eisenberg, and J.M. Colford, Jr. 2008.
Microbial risk assessment framework for exposure to amended sludge projects.
Environ. Health Perspect. 116(6):727—733. (Eisenberg et al., 2008, 635155)
Method for assessing risks to human health from exposure to pathogens in land-applied
biosolids.
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Englehardt, J.D. and J. Swartout. 2004. Predictive population dose-response
assessment for Cryptosporidium parvurrr. Infection endpoint. J. Toxicol. Env. Heal A
67(8-10):651 -666. (Englehardt and Swartout, 2004, 625221)
Dose-response information for Cryptosporidium pan/um.
Englehardt, J.D. and J. Swartout. 2006. Predictive Bayesian microbial dose-response
assessment based on suggested self-organization in primary illness response:
Cryptosporidium parvum. Risk Anal. 26(2):543-554. (Englehardt and Swartout, 2006,
Dose-response information for Cryptosporidium parvum.
Epstein, E. 2006. Pathogens and pathogenic substances in biosolids and manures. J.
Residuals Sci. Technol. 3:71 -77. (Epstein, 2006, 624949)
This reference is a review of pathogens and pathogenic substances in biosolids and manures that
may pose a public health concern.
Epstein, E. and L.H. Moss. 2006. A comparison of characteristics of manures,
biosolids, and mineral fertilizers. J. Residuals Sci. Technol. 3:35-42.
(Epstein and Moss, 2006, 624954)
This reference is a comparison of manures, biosolids, and mineral fertilizer characteristics.
Evanylo, G.K. 1999. Agricultural land application of biosolids in Virginia: Managing
biosolids for agricultural use. Virginia Cooperative Extension, Virginia Tech,
Blacksburg, VA. (Evanylo, 1999, 625223)
This reference summarizes pertinent information on agricultural use of biosolids application.
Information includes agronomic rates, application methods, and storage.
Ferguson, W.W. and R.C. June. 1952. Experiments on feeding adult volunteers with
Escherichia coii 111 B4: A coliform organism associated with infant diarrhea. Am. J.
Hyg. 55(2):155—169. (Ferguson and June, 1952, 625224)
Dose-response information for E. coli.
Gale, P. 2003. Using event trees to quantify pathogen levels on root crops from land
application of treated sewage sludge. J. Appl. Microbiol. 94:35-47. (Gale, 2003,
624974)
This reference documents a modeling approach to predict the incremental exposure of root crops,
at point of harvest to enteric pathogens from sewage sludge applied to agricultural land according
to current regulations and guidance (Safe Sludge Matrix). Land application of sewage sludge
treated by conventional processes (achieving 2-log removal) is predicted to increase the
exposures of root crops to Salmonella and Cryptosporidium by counts of 0 070 and 0 033 kg-1,
respectively.
Gale, P. 2005a. Pathogens in biosolids—microbiological risk assessment. In:
Contemporary Perspectives on Infectious Disease Agents in Sewage Sludge and
Manure. Compost Science and Utilization, J.E. Smith, Jr., P. Millner, W. Jakubowski,
and N. Goldstein, Ed. The J.G. Press, Inc., Emmaus, PA. p. 177-194. (Gale, 2005,
625225)
This reference is a book chapter that examines microbiological risk assessment of biosolids
derived pathogens.
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Gale, P. 2005b. Land application of treated sewage sludge: Quantifying pathogen risks
from consumption of crops. J. Appl. Microbiol. 98(2):380-396. (Gale, 2005, 624976)
This reference assesses risks to human health from the consumption of vegetable crops grown
on agricultural land to which treated sewage sludge has been properly applied.
Gale, P. and G. Stanfield. 2001. Towards a quantitative risk assessment for BSE in
sewage sludge. J. Appl. Microbiol. 91 (3):365-369. (Gale and Stanfield, 2001, 624973)
This reference documents a quantitative risk assessment to determine the risk of BSE
transmission to humans and cattle through the application of sewage sludge to agricultural land.
The study finds that risks to humans through consumption of vegetable crops are acceptably low.
Risks to cattle are higher because of higher exposure to soil and greater susceptibility. The
model demonstrates that sewage sludge alone cannot sustain the BSE epidemic in the UK cattle
herd.
Gantzer, C.P., P. Gaspard, L. Galvez, A. Huyard, N. Dumouthier and J. Schwartzbrod.
2001. Monitoring of bacterial and parasitological contamination during various
treatment of sludge. Water Res. 35(16):3763-3770. (Gantzer et al., 2001, 104901)
Impact of various sludge treatments on pathogens (Salmonella and nematode eggs) and
indicators (E. coli, Enterococci, sulfite-reducing anaerobes spores). Various treatment processes
were analyzed: 4 biological, 3 chemical, 1 heat, 2 storage and 4 sampling campaigns were
carried out.
Gattie, D.K. and T.J. McLaughlin. 2004. A high-level disinfection standard for
land-applied sewage sludges (biosolids). Environ. Health Perspect. 112(2):126—131.
(Gattie and McLaughlin, 2004, 635170)
Complaints associated with land-applied sewage sludges primarily involve irritation of the skin,
mucous membranes, and the respiratory tract accompanied by opportunistic infections.
Gavett, S.H., and H.S. Koren. 2001. The role of particulate matter in exacerbation of
atopic asthma. Int. Arch. Allergy Immunol. 124(1-3):109-112. (Gavett and Koren,
2001.016168)
Results of these studies suggest that transition metals in ambient particulate matter promote the
formation of reactive oxygen species and subsequent lung injury, inflammation, and airway
hyperresponsiveness leading to airflow limitation and symptoms of asthma.
George, C.L., H. Jin, C.L. Wohlford-Lenane, et al. 2001. Endotoxin responsiveness
and subchronic grain dust-induced airway disease. Am. J. of Physiol. Lung C.
280(2):L203-L213. (George et al., 2001, 104906)
Findings demonstrate that subchronic inhalation of grain dust extract results in the development
of chronic airway disease only in mice sensitive to endotoxin but not in mice that are genetically
hyporesponsive to endotoxin, suggesting that endotoxin is important in the development of
chronic airway disease.
Gerba, C.P. 2005. Enteric viruses in biosolids. In: Contemporary Perspectives on
Infectious Disease Agents in Sewage Sludge and Manure. Compost Science and
Utilization, J.E. Smith, Jr., P. Millner, W. Jakubowski, and N. Goldstein, Ed. The J.G.
Press, Inc., Emmaus, PA. (Gerba, 2005, 625226)
This reference characterizes enteric viruses found in biosolids.
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Gerba, C.P. 2009. Environmental indicators. In: Environmental Microbiology, 2nd ed.,
R.M. Maier, I.L. Pepper, and C.P. Gerba, Ed. Academic Press, NY. p .485-498.
(Gerba, 2009, 635174)
A reference book including a discussion about environmental indicators.
Gerba, C.P. and V. Smith, Jr. 2005. Sources of pathogenic microorganisms and their
fate during land application of wastes. J. Environ. Qual. 34:42-48. (Gerba and Smith,
2005, 624982)
This reference is a review that identifies: (1) the types of wastes applied to land in which
pathogens may be present, (2) current concerns with the risks of these practices, and (3) future
research needs.
Gerba, C.P., J.B. Rose and C.N. Haas. 1996a. Sensitive populations: Who is at
greatest risk? Intl. J. Food Microbiol. 30(1-2):113-123. (Gerba etal., 1996, 080225)
This reference is a review that identifies groups of individuals who would be at the greatest risk of
serious illness and mortality from water and foodborne enteric microorganisms. Groups include
the very young, the elderly, pregnant women, and the immunocompromised.
Gerba, C.P., J.B. Rose, C.N. Haas and K.D Crabtree. 1996b. Waterborne rotavirus: A
risk assessment. Water Res. 30(12):2929-2940. (Gerba et al., 1996, 635177)
Dose-response information for Rotavirus.
Gerba, C.P., I.L. Pepper and L.F. Whitehead, III. 2002. A risk assessment of emerging
pathogens of concern in the land application of biosolids. Water Sci. Technol.
46(10):225-230. (Gerba et al., 2002, 624979)
This reference is a risk analysis that assesses the most likely emerging pathogens to survive
treatments for Class B biosolids before land application. The study concludes that Adenoviruses
and Hepatitis A virus are the most thermally resistant viruses that can survive prolonged periods
of time in the environment. And injection of liquid biosolids into the soil results in significant
reduction of the concentration of the viruses. This reference also provides dose-response
information for enteric viruses.
Germolec, D.R., R.S H. Yang, M.F. Ackerman et al. 1991. Toxicology studies of
chemical mixtures of 25 groundwater contaminants: Immune suppression in B6C3F
mice. Fund. Appl. Toxicol. 13(3):377-387. (Germolec et al., 1991, 635179)
Mixture of 25 common groundwater contaminants frequently found near toxic waste dumps, as
determined by EPA surveys, tested on mice. These results suggest that longterm exposure to
contaminated groundwater may represent a risk to the immune system in humans.
Gibbs R.A., C.J. Hu, G.E. Ho and L. Unkovich. 1997. Re-growth of faecal coliforms
and salmonellae in stored biosolids and soil amended with biosolids. Water Sci.
Technol. 35(11-12):269-275. (Gibbs et al., 1997, 635180)
Soil amended with biosolids could not be considered free from pathogens for at least 1 year
following amendment.
Goyal, S.M., S.A. Schaub, F.M. Wellings et al. 1984. Round robin investigation of
methods for recovering human enteric viruses from sludge. Appl. Environ. Microbiol.
48(3):531 -538. (Goyal etal., 1984, 624983)
This reference documents the selection of a tentative standard method for detection of viruses in
sludge.
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Graczyk, T.K., R. Knight, R.H. Gilman and M.R. Cranfield. 2001. The role of non-biting
flies in the epidemiology of human infectious diseases. Microbes Infect. 3(3):231 -235.
(Graczyk et al., 2001, 635182)
This reference is a review on the role of nonbiting flies in the epidemiology of human infections
and diseases. Topics include the feeding and reproductive habits, subceptible populations, and
transmission in hospital environments.
Gregersen, P., K. Grunnet, S.A. Uldum, B.H. Andersen and H. Madsen. 1999. Pontiac
fever at a sewage treatment plant in the food industry. Scand. J. Work Environ. Health
26(3):291 -295. (Gregersen et al., 1999, 624984)
This reference documents an investigation of workers contracting Pontiac fever after repairing a
decanter for sludge concentration at a sewage treatment plant. It was concluded that the fever
was caused by L. pneumophila emitted to the environment by the uncovered decanter.
Guntupalli, R., R.S. Lakshmanan, J. Hu et al. 2007. Rapid and sensitive
magnetoelastic biosensors for the detection of Salmonella typhimurium in a mixed
microbial population. J. Microbiol. Meth. 70(1 ):112—118. (Guntupalli et al., 2007,
This reference reports the employable performance of a wireless, magnetoelastic biosensor
designed to selectively detect Salmonella typhimurium in a mixed microbial population.
Gutierrez, S. 2005. Why a workshop on emerging infectious disease agents and
issues associated with animal manures, biosolids, and other similar by-products? In:
Compost Science and Utilization, J.E. Smith, Jr., P. Millner, W. Jakubowski, and N.
Goldstein, Ed. The J.G. Press, Inc., Emmaus, PA. p. 25-31. (Gutierrez, 2005,
This reference provides tabulated information on pathogen infectious doses.
Haas, C.N., C.S. Crockett, J.B. Rose, C.P. Gerba and A.M. Fazil. 1996. Assessing the
risks posed by oocysts in drinking water. J. Am. Waterworks Assoc. 88(9):131—136.
(Haas et al., 1996. 635186)
Dose-response information for Cryptosporidium pan/um.
Haas, C.N., J.B. Rose and C.P. Gerba. 1999. Quantitative Microbial Risk Assessment.
John Wiley and Sons, Inc., New York, NY. (Haas et al., 1999, 625228)
This book is a commonly cited source of dose-response relationships and other tools and
methods for microbial risk assessment.
Heaney, C., W. Steven and A. Lowman. 2006. Study design features of a longitudinal
investigation of acute health effects and community-level Class B biosolids exposure:
Subject recruitment and exposure assessment. Epidemiology. 17(6) (Suppl. S):
S443-S443. Available at http://journals.lww.eom/epidem/Fulltext/2006/11001/Study_
Design_Features_of_a_Longitudinal.1189.aspx. (Heaney et al., 2006, 625233)
This reference presents strengths and weaknesses of two study design features of a proposed
investigation of acute health effects and community-level exposure to treated sewage sludge
(Class B biosolids) in North Carolina (NC) and Virginia (VA).
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Heffernan, J.M., R.J. Smith and L.M. Wahl. 2005. Perspectives on the basic
reproductive ratio. J. R. Soc. Interface 2(4):281-293. (Heffernan et al., 2005, 635189
This reference is a review of the basic reproductive ratio, R0. The review includes a survey of
recent use of R0 in assessing emerging diseases, such as severe acute respiratory syndrome
and avian influenza, a number of recent livestock diseases, and vector-borne diseases malaria,
dengue and West Nile virus.
Herr, C.E.W., A. zur Nieden, M. Jankofsky, N.I. Stilianakis, R.H. Boedeker and T.F.
Eikmann. 2003. Effects of bioaerosol polluted outdoor air on airways of residents: A
cross section study. Occup. Environ. Med. 60:336-342. (Herr et al., 2003, 104909)
Residents exposed to bioaerosol pollution were shown to report irritative respiratory complaints
similar to mucous membrane irritation independently of perceived odors.
Hethcote, H.W. 2000. The mathematics of infectious diseases. SIAM Rev. Ind. Appl.
Math 42(4):599-653. (Hethcote, 2000, 635191)
Risk assessment methods.
Hill, A.B. 1965. The environment and disease: Association or causation? Proc. R.
Soc. Med. 58:295-300. (Hill, 1965. 071664)
A classic article summarizing several aspects to consider in interpreting causation.
Hinckley, G.T., C.J. Johnson, K.H. Jacobson etal. 2008. Persistence of pathogenic
prion protein during simulated wastewater treatment. Environ. Sci. Technol.
42(14):5254-5259. (Hinckley et al., 2008, 104896)
Results suggest that if prions were to enter municipal wastewater treatment systems, most would
partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in
treated biosolids.
Horswell, J., J. Hewitt, J. Prosser et al. 2010. Mobility and survival of Salmonella
typhimurium and human adenovirus from spiked sewage sludge applied to soil columns.
J. Appl. Microbiol. 108(1):104-114. (Horswell etal., 2010, 635194)
This reference documents that the presence of sewage sludge can significantly influence the
transport and survival of bacterial pathogens in soils, probably because of the presence of
organic matter. Environmental contamination by virus is unlikely because of strong soil
adsorption.
ILSI (International Life Sciences Institute). 2000. Revised Framework for Microbial Risk
Assessment. ILSI Risk Science Institute, Washington, DC. (ILSI, 2000, 625240)
This reference provides a comprehensive framework for use in microbial risk assessments.
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Jin, Y., J.T. Sims and K. Kniel. 2008. Effect of Land Application of Wastes on the Fate
and Transport of Pathogens in Soil. U.S. Department of Agriculture, National Research
Initiatives (NRI). Available at
http://www.reeis.usda.gov/web/crisprojectpages/207060.html. (Jin et al., 2008, 635196)
Objectives: (1) determine the survival potential of selected viruses in representative manures and
biosolids, and on plants grown in soils amended with these materials; (2) measure sorption and
desorption of viruses by manures, biosolids, and soils; (3) examine the soil factors and
manure/biosolids application methods controlling the leaching potential of viruses using large
undisturbed soil columns containing benchmark soil series of the Mid-Atlantic U.S.; and
(4) elucidate the mechanisms of virus retention and transport during leaching under saturated and
unsaturated flow conditions in controlled laboratory column studies.
John, D.E. and J.B. Rose. 2005. Review of factors affecting microbial survival in
groundwater. Environ. Sci. Technol. 39(19):7345-7356. (John and Rose, 2005,
This reference is a review that quantitatively examines a number of published studies that
evaluated survival and inactivation of public-health-related microorganisms in groundwater.
June, R.C., W.W. Ferguson and M.T. Worfel. 1953. Experiments in feeding adult
volunteers with Escherichia coli 55 B5: A coliform organism associated with infant
diarrhea. Am. J. Hyg. 57:222-236. (June et al., 1953, 624987)
Dose-response information for E. coli.
Karpowicz, E., A. Novinscak, F. Barlocher et al. 2009. qPCR quantification and genetic
characterization of Clostridium perfringens populations in biosolids composted for
2 years. J. Appl. Microbiol. 108(2):571 —581. (Karpowicz et al., 2009, 635199)
This reference reports a study to improve the understanding of C. perfringens persistence in
composted biosolids by monitoring its presence and studying its genetic diversity. The study
found that composting did not significantly decrease the number of C. perfringens cells. High
genetic diversity of C. perfringens isolates present in composted biosolids is reported for the first
time.
Khuder S., S.A. Milz, M. Bisesi, R. Vincent, W. McNulty and K. Czaikowski. 2007.
Health survey of residents living near farm fields permitted to receive biosolids. Arch.
Environ. Occup. Health. 62(1 ):5—11 .(Khuder et al., 2007, 104914)
Survey findings suggest an increased risk for certain respiratory, gastrointestinal, and other
diseases among residents living near farm fields on which the use of biosolids was permitted.
Lang, N.L. and S.R. Smith. 2007. Influence of soil type, moisture content and biosolids
application on the fate of Escherichia coli in agricultural soil under controlled conditions.
J. Appl. Microbiol. 103(6):2122-2131. (Lang and Smith, 2007, 635203)
Evaluates survival of E.coli of biosolid origin once land applied.
Lang, N.L., S.R. Smith, D.M. Bellett-Travers, E.B. Pike and C.L. Rowlands. 2003.
Decay of Escherichia coli in soil following the application of biosolids to agricultural land.
Water Environ. J. 17(1):23-28. (Lang et al., 2003, 598358)
This reference presents a field experiment on decay of E. coli in a sandy loam soil, amended with
enhanced and conventionally treated biosolids following spring and autumn application of sewage
sludge.
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Lang, N.L., M.D. Bellett-Travers and S.R. Smith. 2007. Field investigations on the
survival of Escherichia coli and presence of other enteric microorganisms in biosolids-
amended agricultural soil. J. Appl. Microbiol. 103(5):1868-1882. (Lang et al., 2007,
Evaluates survival of E. coli of biosolid origin once land applied.
Lewis, D.L. and D.K. Gattie. 2002. Pathogen risks from applying sewage sludge to
land. Environ. Sci. Technol. 36(13):287A-293A. (Lewis and Gattie, 2002, 598361)
This reference provides an overview for issues related to pathogen risks from applying sewage
sludge to land.
Lewis, D.L. and D.K. Gattie. 2003. Comment on "Evidence for the absence of
Staphylococcus aureus in land applied biosolids". Environ. Sci. Technol. 37(24):5836.
(Lewis and Gattie, 2003, 635205)
Comments on Rusin et al. (2003a).
Lewis, D.L., S. Shepherd, D.K. Gattie, S. Sanchez and M. Novak. 2001. Enhanced
susceptibility to infection from exposure to gases emitted by sewage sludge: A case
study. In: Proceedings of the Water Environment Federation, Innovative Uses of
Biosolids and Biosolids Management. Water Environment Federation, Alexandria, VA.
p. 392-399. Available at
http://www.ingentaconnect.eom/content/wef/wefproc/2001/00002001/00000004/art0004
2. (Lewis et al., 2001, 104919)
This reference documents symptoms exhibited by residents living in a Greenland, NH
neighborhood where Class B biosolids were applied in 1995. The residents experienced severe
irritation of the eyes, skin, and mucous membrane followed by respiratory and gastrointestinal
illnesses.
Lewis, D.L., D.K. Gattie, M.E. Novak, S. Sanchez and C. Pumphrey. 2002. Interactions
of pathogens and irritant chemicals in land-applied sewage sludges (biosolids). BMC
Pub. Health. 2:11. Available at http://www.biomedcentral.eom/1471-2458/2/11. (Lewis
et al., 2002, 598363)
This reference documents the reported symptoms of residents of biosolids land application sites
and suggests that an increased risk of infection may occur when allergic and nonallergic
reactions to endotoxins and other chemical components irritate skin and mucus membranes and
thereby compromise normal barriers to infection.
Lubick, N. 2007. Estimating aerosolized contaminants from Class B biosolids.
Environmental Science and Technology Online News, (accessed 4/4/07). (Lubick,
2007, 635209)
Lytle, D.A., E.W. Rice, C.H. Johnson and K.R. Fox. 1999. Electrophoretic mobilities of
Escherichia coli 0157:H7 and wild-type Escherichia coli strains. Appl. Environ.
Microbiol. 65(7):3222-3225. (Lytle et al., 1999, 598366)
This reference documents the electrophoretic mobilities (EMs) of E. coli 0157:H7 and wild-type
strains. The EPMs of E. coli 0157:H7 strains differed from those of wild-type strains. As the
suspension pH decreased, the EPMs of both types of strains increased.
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McCullough, N.B. and C.W. Eisele. 1951. Experimental human salmonellosis: I.
Pathogenicity of strains of Salmonella meleagridls and Salmonella anatum obtained
from spray dried whole egg. J. Infec. Dis. 88:278-289.(McCullough and Eisele, 1951,
Dose-response information for Salmonella.
McFarland, M.J. 2000. Biosolids Engineering. McGraw Hill, New York, NY.
(McFarland, 2000, 598369)
This is a general reference book covering broad but detailed aspects of biosolids too.
Mead, P.S., L. Slutsker, V. Dietz et al. 1999. Food related illness and death in the
United States. Emerg. Infect. Dis. 5(5):607-625. (Mead et al., 1999, 635213)
This CDC reference is widely cited for foodborne disease rates.
Meckes, M. 2011. E-mail from M. Meckes, Office of Research and Development,
National Risk Management Laboratory, Cincinnati, OH to M. Troyer, Office of Research
and Development, National Center for Environmental Assessment, Cincinnati, OH,
January 20, 2011.
Research experience in this laboratory is that solids content in biosolids cake is frequently
15-25%.
Medema, G. and P. Smeets. 2004. The interaction between quantitative microbial risk
assessment and risk management in the water safety plan. Kiwa Water Research/Delft
University. (Medema and Smeets, 2004, 635214)
Dose-response information for Campylobacter jejuni.
Moffet, C.A., R.E. Zartman, D.B. Wester and R.E. Sosebee. 2005. Surface biosolids
application: effects on infiltration, erosion, and soil organic carbon in Chihuahuan Desert
grasslands and shrublands. J. Environ. Qual. 34(1 ):299—311. (Moffet et al., 2005,
This reference documents the measurement of infiltration and erosion of surface application of
biosolids. Infiltration increased with increasing biosolids application rate. Soil erosion was
reduced by the application of biosolids; however, the extent of reduction in erosion depended on
the initial erodibility of the site. Surface application of biosolids has important hydrological
consequences on runoff and soil erosion in desert grasslands that depend on the rate of biosolids
applied, and the site and biosolids characteristics.
Monroe, S.S., T. Ando and R.I. Glass. 2000. Introduction: human enteric
caliciviruses—an emerging pathogen whose time has come. J. Infect. Dis.
181 (Suppl. 2):S249-S251. (Monroe et al., 2000, 635216)
This reference provides the background information on how caliciviruses became an important
human pathogen of concern and lays out the current state of the field.
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Montemagno, C.D., L.L. Yeghiazarian and P. Kalita. 2004. Field calibration and
berification of a pathogen transport model. Water Environment Research Foundation,
Alexandria, VA.. Available at
http://www.werf.org/AM/Template. cfm?Section=Preparing_a_Report&Template=/CM/Co
ntentDisplay.cfm&ContentFilelD=22. (Montemagno et al., 2004, 635217)
This reference documents an integrated modeling strategy to quantify the risk of surface drinking
water contaminated by waterborne pathogens, in particular the oocysts of C. pan/um, from
agricultural nonpoint pollution sources.
Namata, H., M. Aerts, C. Faes and P. Teunis. 2008. Model averaging in microbial risk
assessment using fractional polynomials. Risk Analysis 28(4):891 -905. (Namata et al.,
2008, 635218)
Dose-response information for Salmonella (Tyhpi).
Nappier, S.P., M.D. Aitken and M.D. Sobsey. 2006. Male-specific coliphages as
indicators of thermal inactivation of pathogens in biosolids. Appl. Environ. Microbiol.
72:2471 -2475. (Nappier et al., 2006, 598373)
This reference documents the use of coliphase as indicators of thermal inactivation of pathogens
in biosolids. The approach began by isolating coliphases from municipal wastewater sludge from
biosolid samples after thermophilic anaerobic digestion to evaluate the susceptibility of specific
groups to thermal inactivation. IF+ RAN phages were found to be the majority organism present
in digested biosolids, likely reduced via a mechanism different from heat inactivation. Therefore,
III F+ RNA coliphages should function as a potential indicator.
NRC (National Research Council) 1983. Risk Assessment in the Federal Government:
Managing the Process. National Academy Press, Washington, DC. (NRC, 1983,
This report is often referred to as the "Red Book" and is commonly cited as a risk assessment
framework for chemicals.
NRC (National Research Council). 2002. Biosolids Applied to Land: Advancing
Standards and Practices. National Academy Press, Washington, DC. (NRC, 2002,
598314)
This reference evaluates the standards and practices of biosolids land application.
Recommendations were made to use improved risk assessment methods to establish standards
for chemical and pathogen hazards; conduct a new national survey of chemicals and pathogens
in sewage sludge; establish a framework for an approach to implement human health
investigations; and to increase the resources devoted to EPA's biosolids program.
NRC (National Research Council). 2004. Indicators for Waterborne Pathogens.
National Academies Press, Washington, DC. (NRC, 2004, 635221)
This report discusses water quality indicators, ecology of waterborne pathogens and indicators,
attributes of indictors, and monitoring approaches.
NRC (National Research Council). 2009. Science and Decisions: Advancing Risk
Assessment. National Academy Press, Washington, DC. (NRC, 2009, 180073)
This report expands on the 1983 NRC "Red Book" framework by developing planning and
scoping and problem formulation as well as decision making frameworks for chemical risk
assessment.
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Nwachuku, N. and C.P. Gerba. 2004. Emerging waterborne pathogens: Can we kill
them all? Curr. Opin. Biotechnol. 15(3):175—180. (Nwachuku and Gerba, 2004,
This reference is a review that focuses on the need to better understand why children are at
greater risk from environmentally transmitted pathogens and the need for special consideration
for this age group when developing environmental standards.
Okhuysen, P.C., C.L. Chappell, J.H. Crabb, C.R. Sterling and H.L. DuPont. 1999.
Virulence of three distinct Cryptosporidium parvum isolates for healthy adults. J. Infect.
Dis. 180(4):1275-1281. (Okhuysen et al., 1999, 635223)
Dose-response information for Cryptosporidium pan/um.
Paez-Rubio, T. and J. Peccia. 2005. Estimating solar and nonsolar inactivation rates of
airborne bacteria. J. Environ. Eng. 131 (4):612—617. (Paez-Rubio and Peccia, 2005,
This reference documents the pilot-scale bioaerosol reactor experiments that independently
measure the solar and nonsolar (absence of solar radiation) inactivation rates of airborne
Mycobacterium parafortuitum and E. coli. E. coli was more susceptible to airborne decay than
M. parafortuitum at all relative humidity (RH) levels tested. RH strongly influenced solar and
nonsolar airborne inactivation rates in both bacteria. These inactivation rates for both bacteria
were greatest at moderate RH levels.
Paez-Rubio, T., X. Hua, J. Anderson and J. Peccia. 2006. Particulate matter
composition and emission rates from the disk incorporation of Class B biosolids into
soil. Atmos. Environ. 40(36):7034-7045. (Paez-Rubio et al., 2006, 119151)
Field studies were conducted at Central Arizona biosolids land application sites to characterize
the physical, chemical, and biological content of aerosols produced during biosolids disking and
the content of bulk biosolids and soil from which the aerosols emanate. Source aerosol
concentrations and calculated emission rates reveal that disking is a substantial source of
biosolids-derived aerosols.
Paez-Rubio, T., A. Ramarui, J. Sommer, H. Xin, J. Anderson and J. Peccia. 2007.
Emission rates and characterization of aerosols produced during the spreading of
dewatered Class B biosolids. Environ. Sci. Technol. 41 (10):3537-3544.
(Paez-Rubio et al., 2007, 598702)
This reference documents the measurement of aerosol emission rates produced during the
spreading of dewatered Class B biosolids onto agricultural land. Rates were determined in
multiple independent experimental runs by characterizing both the source aerosol plume
geometry and aerosol concentrations of PM10, total bacteria, heterotrophic plate count bacteria
(HPC), two types of biosolids indicator bacteria, endotoxin, and airborne biosolids regulated
metals. Based on the land application rates of spreaders used in this study, an estimated
7.6 ± 6.3 mg of biosolids were aerosolized for every 1 kg (dry weight) applied to land.
Peccia, J., H.M. Werth, S. Miller and M. Hernandez. 2001. Effects of relative humidity
on the ultraviolet induced inactivation of airborne bacteria. Aerosol Sci. Technol.
35:728-740. (Peccia et al., 2001, 598841)
This reference assesses the response of aerosolized Serratia marcescens, Bacillus subtilis, and
Mycobacterium parafortuitum to ultraviolet irradiation at different relative humidity (RH) levels in a
0.8 m3 completely-mixed chamber. When RH exceeded approximately 50%, sorption increased
markedly and a sharp concurrent drop in UV-induced inactivation rate was observed.
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Pepper, I.L., K.L. Josephson, R.L. Bailey, M.D. Burr and C.P. Gerba. 1993. Survival of
indicator organisms in Sonoran Desert soil amended with sewage sludge. J. Environ.
Sci. Heal. A 28(6): 1287-1302. (Pepper et al., 1993, 635230)
This study documented the survival period of fecal coliforms after field land application and also
regrowth after rainfall events.
Pepper, I.L., J.P. Brooks and C.P. Gerba. 2006. Pathogens in biosolids. Adv. Agron.
90:1-41. (Pepper et al., 2006, 599024)
This reference is a book chapter review of the human pathogens as potential hazards associated
with biosolids and its land application.
Pepper, I.L., H. Zerzghi, J.P. Brooks and C.P. Gerba. 2008a. Sustainability of land
application of Class B biosolids. J. Environ. Qual. 37:58-67. (Pepper et al., 2008,
635231)
This reference documents the sustainability of long term land application of biosolids. Viable
pathogens were not detected in soil 6 months after the application of biosolids, even after
20 annual applications.
Pepper, I.L., J.P. Brooks and C.P. Gerba. 2008b. Pathogens and indicator organisms
in Class B biosolids before and after the Part 503 Sludge Rule. In: Proceeds of the
Water Environment Federation's Technical Exhibition and Conference WEFTEC 2008:
Session 51 through Session 60. Water Environment Federation, Alexandria, VA.
p. 3955-3961. (Pepper et al., 2008, 635228)
Indicator loads are similar to loads prior to the promulgation of the Part 503 Rule, whereas
pathogen loads have decreased.
Pepper, I.L., J.P. Brooks, R.G. Sinclair, R.L. Gurian, C.P. Gerba. 2010. Pathogens and
indicators in United States Class B biosolids: National and historic distributions. J.
Environ. Qual. 39(November-December). E-pub doi:10.2134/jeq2010.0037. Available
online http://www.virginiabiosolids.com/pdf/Pathogens%20in%20biosolids-2010.pdf.
(Pepper et al., 2010, 697223)
Incidence of indicator organisms and pathogens in Class B biosolids from 18 wastewater
treatment plants across the United States.
Pillai, S.D. 2007. Bioaerosols from land applied biosolids: Issues and needs. Water
Environ. Res. 79(3):270-278. (Pillai, 2007. 624872)
This reference provides an overview for the issues and needs related to bioaerosols from land
applied biosolids. The focus is on current information and technology gaps related to estimating
public health risks.
Pillai, S.D. and S.C. Ricke. 2002. Bioaerosols from municipal and animal wastes:
Background and contemporary issues. Can. J. Microbiol. 48:681-696.
(Pillai and Ricke, 2002, 621261)
This reference is a review that synthesizes the information related to bioaerosols and addresses
the contemporary issues associated with bioaerosols from municipal and animal wastes, with a
focus on pathogens.
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Pillai, S.D., K.W. Widmer, S.E. Dowd and S.C. Ricke. 1996. Occurrence of airborne
bacteria and pathogen indicators during land application of sewage sludge. Appl.
Environ. Microbiol. 62(1):296-299. (Pillai et al., 1996, 615793)
This reference documents the occurrence of airborne bacteria and pathogen indicators during
land application of sewage sludge. Indicators such as H2S producers and pathogenic Clostridia
were present in locations having significant physical agitation of the sludge material.
Regli, S., J.B. Rose, C.N. Haas and C.P. Gerba. 1991. Modeling the risk from Giardia
and viruses in drinking-water. J. Am. Water Works Assoc. 83:76-84. (Regli et al.,
1991, 635235)
Dose-response information for Echovirus and Giardia lamblia.
Reynolds, K.A., C.P. Gerba and I.L. Pepper. 1996. Detection of infectious
enteroviruses using cell culture/PCR procedure. Appl. Environ. Microbiol.
62(4):1424-1427. (Reynolds et al., 1996. 635236)
This is a key reference for ICC-PCR. First research group to demonstrate the method's
advantages.
Rose, J.B. and C.P. Gerba. 1991. Use of risk assessment for development of microbial
standards. Water Sci. Technol. 24:29-34. (Rose and Gerba, 1991, 635237)
Dose-response information for Echovirus, Giardia lamblia, Poliovirus, and Salmonella.
Rose, J.B. and M.D. Sobsey. 1993. Quantitative risk assessment for viral
contamination of shellfish and coastal waters. J. Food Protect. 56(12): 1043—1050.
(Rose and Sobsey, 1993, 635239)
Dose-response information for Echovirus and Poliovirus.
Rose. J.B., C.N. Haas and S. Regli. 1991. Risk assessment and control of waterborne
giardiasis. Am. J. Pub. Health. 81 (16):709-713. (Rose et al., 1991. 635238)
Dose-response information for Giardia lamblia.
Rusin, P.A., S.L. Maxwell, J.P. Brooks, C.P. Gerba and I.L. Pepper. 2003a. Evidence
for the absence of Staphylococcus aureus in land applied biosolids. Environ. Sci.
Technol. 37(37):4027-4030. (Rusin etal.,2003, 624873)
This reference documents an investigation to determine if Staphylococcus aureus is present in
biosolids using samples from 15 biosolids land application sites across the U.S. The results
suggest that biosolids are not a likely source of S. aureus human exposure or infection.
Rusin, P.A., S.L. Maxwell, J.P. Brooks, C.P. Gerba and I.L. Pepper. 2003b. Response
to comment on "Evidence for the absence of Staphylococcus aureus in land applied
biosolids". Environ. Sci. Technol. 37(24):5836. (Rusin et al., 2003, 624875)
This is a rebuttal to D.L. Lewis and D.K. Gattie (2003).
Sahlstrom L., B. de Johg and A. Aspan. 2006. Salmonella isolated in sewage sludge
traced back to human cases of salmonellosis. Lett. Appl. Microbiol. 43(1 ):46—52.
(Sahlstrom et al., 2006, 104921)
This study demonstrates that Salmonella spp. isolated in sewage treatment plants (STP) originate
from infected humans and survive treatment at STP. It also highlights the risk of spreading
resistant Salmonella strains from sewage sludge to the environment.
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Santo Domingo, J.W., D.G. Bambie, T.A. Edge and S. Wuertz. 2007. Quo vadis
source tracking? Towards a strategic framework for environmental monitoring of fecal
pollution. Water Res. 41 (16):3539-3552. (Santo Domingo et al., 2007, 624876)
This reference examines different viewpoints associated with the practical use of microbial source
tracking (MST) to identify critical research gaps, proposes a priority-based timeline to address
them, and outlines emerging technologies that will likely impact the future of source tracking.
Schiffman, S.S., J.M. Walker, P. Dalton et al. 2000. Potential health effects of odor
from animal operations, wastewater treatment facilities and recycling byproducts. J.
Agromed. 7(1 ):7-81. (Schiffman et al., 2000, 635244)
This report summarizes the conclusions from the Workshop regarding the potential mechanisms
responsible for health symptoms from ambient odors.
Selvaratnam, S. and J.D. Kunberger. 2004. Increased frequency of drug-resistant
bacteria and fecal coliforms in an Indiana creek adjacent to farmland amended with
treated sludge. Can. J. Microbiol. 50(8):653-656. (Selvaratnam and Kunberger, 2004,
These results suggest that surface runoff from the farmland treated with sludge is strongly
correlated with higher incidence of Amp(R) and fecal coliforms at one test site.
Shusterman, D. 1992. Critical review; The health significance of environmental odor
pollution. Arch. Environ. Health. 47: 76-87. (Shusterman, 1992, 076187)
Noxious environmental odors may trigger symptoms by a variety of physiologic mechanisms,
including exacerbation of underlying medical conditions, innate odor aversions, aversive
conditioning phenomena, stress-induced illness, and possible pheromonal reactions.
Sidhu, J.P. and S.G. Toze. 2009. Human pathogens and their indicators in biosolids: A
literature review. Environ. Int. 35(1 ):187—201. (Sidhu and Toze, 2009, 635247)
This reference is a review that summarizes reported literature on the numbers and fate of enteric
pathogens and indicators in biosolids. The advantages and limitations of the use of conventional
and alternative index and model microorganisms for the prediction of pathogen presence in
biosolids are also discussed.
Simmonds, C. 2005. Pathogens in biosolids: Risks and regulations. In: Contemporary
Perspectives on Infectious Disease Agents in Sewage Sludge and Manure, Compost
Science and Utilization, J.E. Smith, Jr., P. Millner, W. Jakubowski, N. Goldstein and R.
Rynk, Ed. The J.G. Press, Inc., Emmaus, PA. p. 231-238. (Simmonds, 2005, 624879)
This reference is a book chapter that focuses on the risks posed by pathogens in biosolids and
the guidelines that have arisen in Australia and the U.S. to manage these risks.
Skanavis, C. and W.A. Yanko. 1994. Evaluation of composted sewage sludge based
soil amendments for potential risks of salmonellosis. J. Environ. Health. 56(7):19.
(Skanavis and Yanko, 1994, 104931)
The distribution of salmonellae serotypes did not suggest a strong correlation between the
occurrence of Salmonella in the compost products and Salmonella infections in the community.
Analysis of exposure demonstrated that the probability of infection was low in most scenarios.
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Smieszek, T. 2009. A mechanistic model of infection: Why duration and intensity of
contacts should be included in models of disease spread. Theor. Biol. Med. Model.
6:25. Available at http://www.tbiomed.eom/content/6/1/25. (Smieszek, 2009, 635250)
This reference documents the difference in modeling outcomes between a mechanistic model
and a constant per-contact transmission probability. In particular, cases with many different
contacts (super-spreaders) have much lower expected numbers of secondary cases when using
the mechanistic model.
Smith, J.E., P D. Millnerand N. Goldstein. 2005a. Highlights, insights, and
perspectives on infectious disease agents in sewage sludge and animal manure in the
United States. In: Contemporary Perspectives on Infectious Disease Agents in Sewage
Sludge and Manure, Compost Science and Utilization, J.E. Smith, Jr., P. Millner, W.
Jakubowski, N. Goldstein and R. Rynk, Ed. The J.G. Press, Inc., Emmaus, PA.
p. 3-23. (Smith et al., 2005, 624882)
This reference is a book chapter that highlights the core principles and findings from the
workshop in 2001 and to provide a historical, policy, and regulatory framework on issues related
to infectious disease agents in sewage sludge and animal manure in the United States.
Smith, J.E., Jr., P. Millner, W. Jakubowski, N. Goldstein and R. Rynk. 2005b.
Contemporary Perspectives on Infectious Disease Agents in Sewage Sludge and
Manure, Compost Science and Utilization. The J.G. Press, Inc., Emmaus, PA. (Smith
et al., 2005, 624883)
This reference provides a comprehensive evaluation of facts and critical gaps in knowledge about
the growth, survival and dissemination of infectious agents from wastewater sludge and animal
manures and the prospects for disinfection with a variety of existing treatment technologies.
Soller, J.A. 2006. Use of microbial risk assessment to inform the national estimate of
acute gastrointestinal illness attributable to microbes in drinking water. J. Water Health.
4(Suppl 2): 165—186. (Soller, 2006, 635253)
Use of model organism in microbial risk assessments. Risk assessment tool.
Soller, J.A. and J.N.S. Eisenberg. 2008. An evaluation of parsimony for microbial risk
assessment models. Environmetrics 19(1)61-78. (Soller and Eisenberg, 2008,
635254)
Mathematical equations for dynamic risk assessment models.
Soller, J.A., J.N.S. Eisenberg, D.M. Eisenberg et al. 2006a. Research digest: use of
risk assessment to evaluate human health risks associated with pathogens in biosolids.
Water Environment Research Foundation, Alexandria. (Soller et al., 2006, 624885)
This research digest summarizes the findings of the results of the two phases of WERF project
98-REM-1. The risk assessment framework provides a mechanism to discuss biosolids
management microbial risk using a common metric for comparison of treatment methods,
management alternatives, and to set risk-based standards for microbial contaminants in biosolids.
Soller, J.A., A.W. Olivieri, J.N.S. Eisenberg, J. DeGeorge, R.C. Cooper and G.
Tchobanoglous. 2006b. A public health evaluation of recreational water impairment. J
Water Health. 4(1 ):1 -19. (Soller et al.. 2006. 104932)
Disease transmission model method.
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Soller, J.A., E.Y. Seto and A.W. Olivieri. 2007. Application of microbial risk assessment
techniques to estimate risk due to exposure to reclaimed waters. WateReuse
Foundation, Final Project Report, WRF-04-011. (Soller et al., 2007, 635256)
Dose-response information for Salmonella.
Spicer, R.C. and J.J. Gangloff. 2000. Limitations in application of Spearman's rank
correlation to bioaerosols sampling data. Am. Ind. Hyg. Assoc. J. 61 (3):362-366.
(Spicer and Gangloff, 2000, 624887)
This reference documents the model simulations comparing two zones of microbial data from the
same environment. The simulations indicated that nonparametric statistical treatment of
bioaerosol data as currently recommended for building assessment purposes has limitations. An
inordinately high Type II error (failure to reject a null hypothesis which is actually not true) is
especially apparent when there are small numbers of samples.
Stine, S.W., I. Song, C.Y. Choi and C.P. Gerba. 2005. Effect of relative humidity on
preharvest survival of bacterial and viral pathogens on the surface of cantaloupe,
lettuce, and bell peppers. J. Food Protect. 68(7):1352-1358. (Stine et al., 2005,
This reference documents the comparative effects of humidity on the preharvest survival of
microbial pathogens on cantaloupe, lettuce, and bell peppers. C. perfringens may be an
acceptable indicator of bacterial contamination and survival in various environments and on
different types of crops.
Straub, T.M., I.L. Pepper and C.P. Gerba. 1993. Hazards from pathogenic
microorganisms in land-disposed sewage sludge. Rev. Environ. Contam. Toxicol.
132:55-91. (Straub et al., 1993. 624899)
This reference provides an overview of the characteristics of microbial pathogen hazards in
sewage sludge.
Straub, T.M., I.L. Pepper, M. Abbaszadegan and C.P. Gerba. 1994. A method to
detect enteroviruses in sewage sludge-amended soil using the PCR. Appl. Environ.
Microbiol. 60(3):1014-1017. (Straub et al., 1994, 635260)
PCR can detect enteroviruses in land applied biosolids long after they are not detected by cell
culture. The implication of this is that PCR will detect infectious or non-infectious virus, thereby
overestimating exposure to infectious virus.
Straub, T.M., K. Honerzu Bentrup, P. Orosz-Coghlan et al. 2007. In vitro cell culture
infectivity assay for human noroviruses. Emerg. Infect. Dis. 13(3):396-403.
(Straub et al., 2007, 635259)
This paper documents that noroviruses, a member of the caliciviruses has been grown in cell
culture for the first time.
Suter, G.W., II. 1999. Developing conceptual models for complex ecological risk
assessments. Hum. Ecol. Risk Assess. 5(2):375-396. (Suter, 1999, 624900)
This reference presents a strategy for creating conceptual models for complex ecological risk
assessments that are complete, comprehensible, and efficient.
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Suter, G.W., II, R.A. Efroymson, B.E. Sample and D.S. Jones. 2000. Ecological Fisk
Assessment for Contaminated Sites. Lewis Publishers/CRC Press, Boca Raton, FL.
(Suter etal., 2000, 624901)
This is a general reference book on ecological risk assessment.
Tanner, B.D. 2004. Aerosolization of microorganisms and risk of infection from reuse
wastewater residuals. Ph.D. Dissertation, Graduate School of Medicine, The University
of Arizona, Tucson, AZ. (Tanner, 2004, 624902)
This reference documents 3 experiments conducted to characterize the concentration of
microorganisms in biosolids, the plume of aerosols created during land application of biosolids,
and the occupational risk of infection due to pathogens aerosolized during land application of
biosolids in the United States.
Tanner, B.D., J.P. Brooks, C.N. Haas, C.P. Gerba and I.L. Pepper. 2005. Bioaerosol
emission rate and plume characteristics during land application of liquid Class B
biosolids. Environ. Sci. Technol. 39(6):1584-1590. (Tanner et al., 2005, 624903)
This reference documents bioaerosol emission rates and plume characteristics of bioaerosols
generated during land application of liquid Class B biosolids. A comparison of aerosolization
rates of coliphages and total coliform bacteria between liquid biosolids land application and
inoculated groundwater during land application. The results indicate that some property of
biosolids reduces aerosolization of microorganisms relative to groundwater and aerosolization of
coliphages and coliform bacteria after liquid biosolids have been applied to land does not occur at
detectable levels.
Tanner, B.D., J.P. Brooks, C.P. Gerba, C.N. Haas, K.L. Josephson and I.L. Pepper.
2008. Estimated occupational risk from bioaerosols generated during land application
of Class B biosolids. J. Environ. Qual. 37:2311 -2321. (Tanner et al., 2008, 635265)
This paper gives additional information on bioaerosols generated during land application.
Teunis, P.F.M. and A.H. Havelaar. 2000 The beta-poison model is not a single hit
model. Risk Anal. 20(4):513-520. (Teunis and Havelaar, 2000, 635269)
Dose-response information for Rotavirus.
Teunis, P.F., O.G. van der Heijden, J.W.B. van der Giessen and A.H. Havelaar. 1996
The dose-response relation in human volunteers for gastro-intestinal pathogens. The
Netherlands: RIVM (National Institute of Public Health and the Environment) Report No.
284550002. (Teunis and Havelaar, 1996. 635270)
Dose-response information for Giardia lamblia.
Teunis, P., K. Takumi and K. Shinagawa. 2004. Dose response for infection by
Escherichia coli 0157:H7 from outbreak data. Risk Anal. 24(2):401-407. (Teunis et
al., 2004, 635267)
Dose-response information for E. coli 0157:H7.
Teunis, P.F., W. Van den Brandhof, M. Nauta, J. Wagenaar, H. Van den Kerkhof and
W. Van Pelt. 2005. A reconsideration of the Campylobacter dose-response relation.
Epidemiol. Inf. 133(4):583-592.(Teunis et al., 2005, 635268)
Risk assessment tool.
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Teunis, P.F., C.L. Moe, P. Liu et al. 2008a. Norwalk virus: How infectious is it? J. Med.
Virol. 80(8):1468-1476. (Teunis et al., 2008, 635271)
Risk assessment tool: pathogen specific but broader application possible.
Teunis, P.F., I.D. Ogden and N.J. Strachan. 2008b. Hierarchical dose response of E.
coli 0157:H7 from human outbreaks incorporating heterogeneity in exposure.
Epidemiol. Inf. 136(6):761 -770. (Teunis et al., 2008, 635272)
Dose-response method.
U.S. DOE (Department of Energy). 2006. Risk Assessment Information System
(RAIS). Office of Environmental Management, Washington, DC. Available at
http://rais.ornl.gov/. (U.S. DOE. 2006. 624905
This is an online resource of risk assessment tools.
U.S. EPA (U.S. Environmental Protection Agency). 1992. Technical Support Document
for Land Application of Sewage Sludge, Vol. II. Appendices. Eastern Research Group,
Lexington, MA. Office of Water, Washington, DC. EPA 822/R-93-001 b. (U.S. EPA,
1992. 635289)
This document provides the scientific discussion that supports the Part 503 Rule.
U.S. EPA (U.S. Environmental Protection Agency). 1993. The Standards for the Use or
Disposal of Sewage Sludge. Final Rules. EPA 822/Z-93-001. 40 CFR Parts 257, 403,
and 503. Federal Register, 58(32):9248-9415. Available at
http://nepis.epa.gov/EPA/html/Pubs/pubtitleOW.htm. (U.S. EPA, 1993. 624909)
This is Title 40: Protection of Environment PART 503—STANDARDS FOR THE USE OR
DISPOSAL OF SEWAGE SLUDGE.
U.S. EPA (U.S. Environmental Protection Agency). 1994. Guidance for the Data Quality
Objectives Process. Quality Assurance Management Staff, Washington, DC.
EPA/600/R-96/055. Available at
http://www.epa.gov/wastes/hazard/correctiveaction/resources/guidance/qa/epaqag4.pdf.
(U.S. EPA, 1994, 624925)
This is EPA's guidance on data quality objectives.
U.S. EPA (U.S. Environmental Protection Agency). 1995. A Guide to the Biosolids Risk
Assessments for the EPA Part 503 Rule. Office of Wastewater Management,
Washington, DC. EPA 832/B-93-005. Available at
http://www.epa.gov/owm/mtb/biosolids/503rule/index.htm. (U.S. EPA, 1995. 624928)
This is a risk assessment guide for biosolids.
U.S. EPA (U.S. Environmental Protection Agency). 1997. Exposure Factors
Handbook, Office of Research and Development, National Center for Environmental
Assessment, Washington, DC. Available at http://www.epa.gov/ncea/pdfs/efh/front.pdf.
(U.S. EPA, 1997. 635276)
This is EPA's resource for quantitative information on human exposure patterns.
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U.S. EPA (U.S. Environmental Protection Agency). 1998. Guidelines for Ecological
Risk Assessment. Office of Research and Development, Washington, DC. EPA
630/R-95-002F. Available at
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=12460. (U.S. EPA, 1998. 042805)
This is an EPA guideline for conducting ecological risk assessments.
U.S. EPA (U.S. Environmental Protection Agency). 1999. Environmental Regulations
and Technology: Control of Pathogens and Vector Attraction in Sewage Sludge
(including domestic septage). Office of Research and Development, Washington, DC.
EPA 625/R-92/013. Available at
http://www.epa.gov/nrmrl/pubs/625r92013/625R92013.pdf. (U.S. EPA, 1999. 624938)
U.S. EPA (U.S. Environmental Protection Agency). 2000. Stressor Identification
Guidance Document. Office of Water and Office of Research and Development,
Washington, DC. EPA 822/B-00/025. Available at
http://www.epa.gov/waterscience/biocriteria/stressors/stressorid.pdf. (U.S. EPA, 2000,
This is a guidance document on stressor identification for ecological risk assessment.
U.S. EPA (U.S. Environmental Protection Agency). 2002a. Lessons Learned on
Planning and Scoping for Environmental Risk Assessments. EPA Science Policy
Council, Washington, DC. Available at http://www.epa.gov/spc/pdfs/handbook.pdf.
(U.S. EPA, 2002. 635283)
This document outlines planning and scoping and includes case studies with descriptions of how
stakeholders were engaged.
U.S. EPA (U.S. Environmental Protection Agency). 2002b. Guidelines for Ensuring and
Maximizing the Quality, Objectivity, Utility, and Integrity, of Information Disseminated by
the Environmental Protection Agency. U.S. Environmental Protection Agency,
Washington, DC. EPA/260/R-02/008. Available at
http://www.epa.gov/quality/informationguidelines/documents/EPA_lnfoQualityGuidelines
.pdf. (U.S. EPA, 2002, 635281)
This is EPA guidance document in response to OMB's guidance on data quality.
U.S. EPA (U.S. Environmental Protection Agency). 2002c. Land Application of
Biosolids. Office of Inspector General status report 2002-S-000004. Available at
http://www.epa.gov/oig/reports/2002/BIOSOLIDS_FINAL_REPORT.pdf. (U.S. EPA,
2002, 635282)
Status report on land application of biosolids. Issues examined include: (1) EPA and state
biosolids program staff, (2) delegation of the biosolids program to the states, (3) extent to which
biosolids are land applied in seven states, (4) responding to and tracking health complaints,
(5) risk assessment and pathogen testing concerns, (6) EPA's relationship with a professional
association, and (7) public acceptance concerns.
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U.S. EPA (U.S. Environmental Protection Agency). 2003a. Framework for Cumulative
Risk Assessment. Office of Research and Development, National Center for
Environment Assessment, Washington, DC. EPA/630/P-02/001F. Available at
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=54944. (U.S. EPA, 2003. 192145)
This reference is an information document that focuses on describing various aspects of
cumulative risk whether or not the methods or data currently exist for adequate analysis or
evaluation.
U.S. EPA (U.S. Environmental Protection Agency). 2003b. Standards for the use or
Disposal of Sewage Sludge; Agency Response to the National Research Council
Report on Biosolids Applied to Land and the Results of EPA's Review of Existing
Sewage Sludge Regulations. Office of Wastewater Management, Washington, DC.
Fed. Regist. 68(68):17379-17395. Available at
http://edocket.access.gpo.gov/2003/pdf/03-8654.pdf. (U.S. EPA, 2003. 624988)
This is EPA's response to the 2002 NRC report on biosolids.
U.S. EPA (U.S. Environmental Protection Agency). 2006a. National Primary Drinking
Water Regulations:Long Term 2 Enhanced Surface Water Treatment Rule; Final Rule,
40CFR Parts 9, 141 and 142. Fed. Regist. 71 (3):654-786. Available at
http://edocket.access.gpo.gov/2006/pdf/06-4.pdf. (U.S. EPA, 2006, 635284)
Example of secondary transmission modeling.
U.S. EPA (U.S. Environmental Protection Agency). 2006b. National Primary Drinking
Water Regulations: Ground Water Rule; Final Rule. 40CFR Parts 9, 141 and 142. Fed.
Regist. 71(216):65574-65660. Available at
http://edocket.access.gpo.gov/2006/pdf/06-8763.pdf. (U.S. EPA, 2006. 646951)
Includes risk assessment methods for microbes, specifically Cryptosporidium.
U.S. EPA (U.S. Environmental Protection Agency). 2007. Thesaurus of Terms Used in
Microbiological Risk Assessment. EPA Office of Water, Washington, DC. Available at
http://www.epa.gov/waterscience/criteria/humanhealth/microbial/thesaurus/microbial-
thesaurus.6.pdf. (U.S. EPA, 2007. 635818)
This is a collection of risk assessment definitions from U.S. and international sources.
U.S. EPA (U.S. Environmental Protection Agency). 2008. Child-specific Exposure
Factors Handbook. Office of Research and Development. EPA/600/R-06/096F.
Available at http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=199243. (U.S. EPA,
2008, 196062)
This document has quantitative information on exposures experienced by children.
U.S. EPA (U.S. Environmental Protection Agency). 2009a. Draft Protocol for Microbial
Risk Assessment to Support Human Health Protection for Water-Based Media. Office
of Water, Washington, DC. Available at
http://yosemite.epa.gov/sab/sabproduct.nsf/368203f97a15308a852574ba005bbd01/dad
b7c7d689ea5c58525753600614bba!OpenDocument. (U.S. EPA, 2009, 635274)
This is a collection of microbial risk assessment tools including details on a framework for
conducting MRA.
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U.S. EPA (U.S. Environmental Protection Agency). 2009b. Targeted National Sewage
Sludge Survey. Overview Report. Office of Water, Washington, DC.
EPA/822/R-08-014. Available at _http://www.epa.gov/waterscience/biosolids/tnsss-
overview.pdfhttp://www.epa.gov/waterscience/biosolids/tnsss-overview.html. (U.S.,
2009, 646954)
This is a document that reports the results of biosolids monitoring. Pathogens are not included.
Viau, E. and J. Peccia. 2009. Survey of wastewater indicators and human pathogen
genomes in biosolids produced by Class A and Class B stabilization treatments. Appl.
Environ. Microbiol. 75(1 ):164-74. (Viau and Peccia, 2009, 635290)
This reference reports on a survey using quantitative PCR (qPCR) and culture assays to detect
environmentally resistant bacterial and viral pathogens and biosolid indicator organisms for
36 biosolid grab samples. Human adenovirus genomes were found in 88% of the Class B
samples and 70 to 100% of the Class A samples.
Vilanova, X. and A.R. Blanch. 2005. Distribution and persistence of fecal bacterial
populations in liquid and dewatered sludge from a biological treatment plant. J. Gen.
Appl. Microbiol. 51 (6):361 -368. (Vilanova and Blanch, 2005, 104933)
Comparison of bacteria in municipal sewage and their derived sludge; antibiotic-resistant strains
persisted in sludge.
Virginia Department of Health. 1999. The biosolids lifecycle. Division of Wastewater
Engineering. Available at http://www.biosolids.state.va.us/.
(Virginia Department of Health, 1999, 624960)
This is a reference tool website.
Ward, R.L., D.L. Bernstein, E.C. Young, J.R. Sherwood, D.R. Knowlton and G.M. Schiff.
1986. Human rotavirus studies in volunteers: Determination of infectious dose and
seriological response to infection. J. Infec. Dis. 154(5):871-880. (Ward et al., 1986,
-624961)
Dose-response information for Rotavirus.
Watkins, J. and K.P. Sleath. 1981. Isolation and enumeration of Listeria
monocytogenes from sewage, sewage sludge, and river water. J. Appl. Bacteriol.
50(1 ):1-9. (Watkins and Sleath, 1981. 624963)
This reference documents the presence of L. monocytogenes in sewage and sewage sludge in
considerable numbers and that this organism survives longer than Salmonella spp. on land
sprayed with sewage sludge.
Whitmore, T.N. and L.J. Robertson. 1995. The effect of sewage sludge treatment
processes on oocysts of Cryptosporidium parvum. J. Appl. Bacteriol. 78(1 ):34-38.
(Whitmore and Robertson, 1995. 635662)
The viability of Cryptosporidium oocysts decreased within the range 20-40% in sludge-treated
soil mesocosms over 30 days. The survival results obtained, however, indicated that oocysts
would survive well beyond this period.
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WHO (World Health Organization). 2004. Waterborne Zoonoses: Identification,
Causes, and Control. J.A. Cotruvo, A. Dufour, G. Rees, J. Bartram, R. Carr, D.O. Cliver,
G.F. Craun, R. Fayer and V.P.J. Gannon, Ed. Geneva: WHO. Available at
http://www.who.int/water_sanitation_health/diseases/zoonoses/en/index.html. (WHO,
2004, 646957)
A detailed review of waterborne zoonotic pathogens.
Willert,C. and P. Eng. 2005. Biosolids pellet review study: human health and ecological
risk assessment. Prepared for Toronto Public Health, Toronto, Ontario, Canada by
Jacques Whitford Limited. Available at
http://www.toronto.ca/health/hphe/pdf/abtp_presentation2.pdf. (Willert and Eng, 2005,
This reference documents the technical study of biosolids pellets in assessing human health and
ecological risks.
Wischmeier, W.H. and D. Smith. 1978. Predicting Rainfall Erosion Losses: A Guide to
Conservation Planning. Science and Education Administration, USDA, Hyattsville, MD.
Agriculture Handbook No. 537. (Wischmeier and Smith, 1978, 624965)
This reference documents a procedure for predicting soil loss using an empirical equation that is
believed to be applicable wherever numerical values of it factors are available.
Woodruff, N.P. and F.H. Siddoway. 1965. A wind erosion equation. Soil Sci. Soc. Am.
Proc. 29:602-608. (Woodruff and Siddoway, 1965, 624968)
This reference reports an equation for use in determining wind erosion of soil. The tool is useful
for estimating the potential erosion level and the impact of specific field conditions.
Yanko,W.A. 2005. Bacterial pathogens in biosolids—emerging issues. In:
Contemporary Perspectives on Infectious Disease Agents in Sewage Sludge and
Manure, Compost Science and Utilization, J.E. Smith, Jr., P. Millner, W. Jakubowski,
and N. Goldstein, Ed. The J.G. Press, Inc., Emmaus, PA. p. 35-49. (Yanko, 2005,
This is a book chapter summarizing emerging pathogens of concern.
Yates, M.V., and S. Yates. 2007. Assessing the Fate of Emerging Pathogens in
Biosolids. WERF Report: Protecting human health.01-HHE-3. IWA Publishing, London,
U.K. (Yates and Yates, 2007, 104935)
Research on the fate of emerging pathogens during biosolids treatment and subsequent
application on land. Specific objectives were to: (1) assess the fate of several emerging
pathogens (e.g., Adenoviruses, Hepatitis A virus, E. coli 0157:H7, Listeria, and Cryptosporidium)
during Class A and Class B biosolids treatment processes; (2) based on the results of
Objective 1, assess the fate and transport of the most significant organisms in biosolids applied to
soil columns; and (3) assess the potential for selected pathogens to survive and be transported at
a field site.
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Zaleski, K.J., K.L. Josephson, C.P. Gerba and I.L. Pepper. 2005a. Survival, growth,
and regrowth of enteric indicator and pathogenic bacteria in biosolids, compost, soil,
and land applied biosolids. J. Residuals Sci. Technol. 2:49-63. (Zaleski et al., 2005,
This reference evaluates the potential for conversion of Class B to Class A biosolids with respect
to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. The results
suggest that the use of concrete-lined beds created a situation in which moisture added as rainfall
accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from
external sources.
Zaleski, K.J., K.L. Josephson, C.P. Gerba and I.L. Pepper. 2005b. Potential regrowth
and recolonization of Salmonellae and indicators in biosolids and biosolid-amended soil.
Appl. Environ. Microbiol. 71(7):3701-3708. (Zaleski et al., 2005, 624980)
This reference is a review on issues related to the survival and potential regrowth of pathogenic
and indicator bacteria in biosolids, compost, soil, and land-applied biosolids.
Zerzghi, H., C.P. Gerba, J.P. Brooks, I.L. Pepper. 2009. Long-term effects of land
application of Class B biosolids on the soil microbial populations, pathogens, and
activity. J. Residuals Sci. Technol. 7:51 -61. (Zerzghi et al., 2010, 635670)
This reference documents a 20-year study that showed no long-term adverse effects at 20 annual
biosolids land application sites and concludes that land application of biosolids at this particular
site was sustainable throughout the 20-yr period, with respect to soil microbial properties.
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APPENDIX
LITERATURE REVIEW
This appendix presents a literature review that summarizes the available
information on microbial risks to humans posed by land-applied biosolids. The review is
organized in terms of summary points, research and data gaps, relevant aspects of the
National Research Council (NRC, 2002) recommendations on biosolids, and data and
information available for phases of risk assessments (e.g., fate, transport, uptake,
infectivity, risk assessment, causal analysis). Although some studies of pathogens in
manures may be relevant to biosolids (e.g., models of pathogen transport),
investigations of these untreated materials are beyond the scope of this report. This
literature review was completed prior to developing the other chapters in this report.
SUMMARY POINTS
•	The range of pathogens that may be present in biosolids is well understood, but
the current national distribution of these pathogens, the variation with type of
sewage sludge treatment, and standard analytical methods for detecting and
quantifying pathogens in biosolids are not well understood or developed.
•	Many analytical methods for detecting and quantifying pathogens focus on
detecting deoxyribonucleic acid sequences rather than viable cultures.
•	The use of indicator organisms to represent pathogens of concern has the
potential to introduce large uncertainties into estimates of exposure.
•	The use of pathogen-specific dose-response relationships coupled with the
uncertainties of indicator organism modeling (the above point) could compound
the difficulty in conducting a meaningful and scientifically defensible risk
assessment.
•	Risk assessments of pathogens in biosolids have been performed, but the
emphasis has been on the use of particular transport models to quantify risks
from a few pathogens to individuals at a distance from particular biosolids
application sites. Eisenberg et al. (2004, 2005, 2006, 2008) have developed
some risk assessment tools that may be useful for national-scale or other broad
risk assessment.
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•	Some conceptual models for human health risk assessments of pathogens in
biosolids that include detailed source descriptions, transport pathways and routes
of exposure have been developed (Colford et al., 2003).
•	Epidemiological studies of biosolids application sites are generally lacking and
are problematic to conduct.
•	An epidemiologic causal association between exposures to biosolids and
adverse effects on human health has not been documented.
•	Although the Unites States Environmental Protection Agency (EPA) has standard
exposure factors and effects levels relevant to chemicals, some standard
exposure factors and effects levels needed for risk assessments of pathogens in
biosolids are not available.
•	EPA is currently reviewing a standard quantitative microbial risk assessment
framework for use in risk assessments of pathogens in water media, which was
also designed with biosolids in mind (U.S. EPA, 2009a, ILSI, 2000).
•	Dose-response relationships used in risk assessments of pathogens in biosolids
have been derived from nonbiosolids studies (e.g., food or water matrix
ingestion), and it is unclear to what extent these relationships may be expected
to apply to biosolids, particularly for the inhalation pathway.
•	The science of biosolids exposure analysis is still under development and studies
of the effects of pathogens in biosolids are limited.
•	Little information is available to support the elimination of exposure scenarios or
pathways from consideration at all sites where biosolids have been applied.
Information may support the screening of exposure pathways from consideration
at particular sites.
•	Bioaerosol emissions from biosolids have been studied most rigorously in
Arizona; few data exist for other regions.
•	Exposure assumptions vary in existing risk assessments for bioaerosols
generated from biosolids.
•	Existing risk assessment studies of pathogens in biosolids at specific sites
estimate risk of infection rather than risk of disease.
Many of the research and monitoring gaps related to human health risk assessments
of biosolids are described in key papers and are summarized in Table A-1. These
include aspects of problem formulation, exposure assessment, and effects assessment.
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TABLE A-1

Research, Monitoring, Assessment and Modeling Needs Related to Risk Assessment for
Land Application of Biosolids
Need
Reference
Hazard Characterization
New national survey of pathogens in sewage sludge
NRC (2002)
Research on incidence of prions in biosolids
Pepper et al. (2006)
Research to assess utility of additional indicator microoganisms
such as Clostridium perfringens
NRC (2002)
Research to assess metabolic status of aerosolized pathogens
and environmental and biological factors that influence this
metabolic state
Pillai and Ricke
(2002)
Research to assess potential for pathogen reproduction within
bioaerosols
Pillai and Ricke
(2002)
New indicators for viruses in biosolids (judged by cited workgroup
to be a medium priority)
Virus workgroup in
Smith et al. (2005b)
Measures of Exposure (quantifying pathogens)
Improvement (e.g., analytical specificity, sensitivity, accuracy),
standardization, validation of detection methods for bacteria,
viruses, protozoan parasites, helminth parasites in biosolids
Smith et al. (2005a),
NRC (2002), EPA
(2003b)
Standardized methods for measuring and characterizing
pathogens in bioaerosols
NRC (2002), Pillai
(2002)
Molecular, immunological, immuno-magnetic separation and
culture techniques for detection of low numbers of pathogens
Smith et al. (2005a)
Standardization and validation of assays for detecting and
enumerating waterborne protozoan parasites (Cryptosporidium,
Cyciospora, Toxoplasma, Microsporidia, Balantidium, Giardia and
Entamoeba), fecal conforms, Salmonella spp., enteric viruses and
helminth eggs in biosolids matrices
Smith et al. (2005a)
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TABLE A-1 cont.
Need
Reference
Measurement of occurrence, survival, fate and transport of cysts
of protozoans and worms/nematodes, as well as viruses or
surrogates with respect to different treatment and land
application scenarios
Smith et al. (2005a)
Evaluation of the usefulness of surrogates and models to
determine presence or survival of infectious agents before and
after treatment and land application
Smith et al. (2005a)
Measurement of antibiotic resistance determinants in bacteria in
biosolids
Smith et al. (2005a)
Measurements of post-treatment pathogen concentrations,
confirmation that Class B treatment combined with use
restrictions result in below-detection pathogen concentrations
NRC (2002), Gerba
(2005)
Creation of matrix of virus concentrations in different types of
biosolids, by source of sewage sludge and type of treatment
(judged by cited workgroup to be a medium priority)
Virus workgroup in
Smith et al. (2005b)
Measures of Exposure (fate and transport)
Research on the fate and transport of bioaerosols from land
application or spray irrigation
Smith et al. (2005a),
NRC (2002)
Better bioaerosol dispersion and viability models
Pillai and Ricke
(2002)
Improved bioaerosol samplers that are designed not only for
bacterial collection, but also for virus and endotoxin collection
Pillai (2007)
Research to assess transport and fate of viruses in land applied
biosolids (judged by cited workgroup to be a medium priority)
Virus workgroup in
Smith et al. (2005b)
Monitoring of pathogens at various points in the environmental
transport process from the biosolids source to the site of
exposure
Eisenberg et al.
(2004)
Relationships between pathogen survivorship and environmental
factors
Eisenberg et al.
(2004)
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TABLE A-1 cont.
Need
Reference
Development of site-specific atmospheric dispersion models
(and research supporting parameter development) to identify
appropriate bioaerosol sampling locations depending on
micrometeorological conditions
Pillai (2007)
Research on effect of harvest and grazing restrictions on
pathogen fate and transport
NRC (2002)
Monitoring to assess potential exposures from runoff from land
application of biosolids (judged by cited workgroup to be a
medium priority)
Parasite workgroup
in Smith et al.
(2005b)
Research to assess fate of viruses most resistant to temperature
and high pH treatment processes, i.e., hepatitis A and
adenoviruses
Pepper et al. (2006)
Monitoring to assess potential for regrowth of E. coli 0157:H7
after treatment processes
Pepper et al. (2006)
Measurement of fate of Cryptosporidium oocysts during
treatment and after soil amendment in a variety of environments
Pepper et al. (2006)
Relevance of correlations between indicator and endpoint
microorganisms in biosolids to relationships in aerosols
Brooks et al.
(2005b)
Measures of Exposure (biotic uptake)
Research to assess adequacy of 30-day waiting period for
grazing following land application of Class B biosolids (judged by
cited workgroup to be a medium priority)
Virus workgroup in
Smith et al. (2005b)
Measures of Exposure (human parameters)
Research on exposure of workers and off-site residents to
biosolids and biosolids components (bioaerosols, dust)
Smith et al. (2005a)
Virus workgroup in
Smith et al. (2005b)
Information on actual ingestion and inhalation rates, as well as
duration of exposure (e.g., percent of inhaled bacteria that are
swallowed)
Gerba and Smith
(2005), Brooks et al.
(2005b)
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TABLE A-1 cont.
Need
Reference
Determination of route of exposure of humans to aerosolized
pathogens
Pillai (2007)
Information on household-level transmission of pathogens
Eisenberg et al.
(2004)
Information on human transmission of pathogens (such as
nontyphi Salmonella) by inhalation of bioaerosols and associated
dose-response relationships
Pepper et al. (2006)
Dose-Response Relationships
Development of relationships between ingested doses and
severity and duration of effects, including species and
subspecies differences in infectivity
NRC (2002)
Validation of animal-derived dose-response relationships for
humans
NRC (2002)
Tests of models used to extrapolate dose-response relationships
derived at high doses to low doses
NRC (2002)
Development of relationships between treatment process
conditions (time, temperature, pH, chemical doses, holding
times), pathogen indicator concentrations and maximum
acceptable pathogen concentrations
NRC (2002)
Research on the role of chemical irritants in affecting
pathogen-related risks
Lewis et al. (2002)
Research on infectivity of aerosolized microbial pathogens,
especially enteric pathogens
Pillai and Ricke
(2002), Pillai (2007)
Determination of infective doses for parasites
Parasite workgroup
in Smith et al.
(2005b)
Research on minimum infective doses (minimum number of
infectious units required to cause an infection), especially for
immunocompromised individuals
Lewis and Gattie
(2002)
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TABLE A-1 cont.
Need
Reference
Research on how different pathogen strains interact in the
development of immunity
Eisenberg et al.
(2004)
Risk Assessment
Quantitative microbial risk assessment methods
NRC (2002)
Sensitivity analyses to determine what critical information is
needed to reduce uncertainty in microbial risk assessments
NRC (2002)
Risk assessment of Ascaris ova, which requires data on levels of
viable ova in biosolids and survival under different environmental
conditions (many limits for use of agricultural land after land
application of Class B biosolids are determined by survival of
Ascaris ova)
Pepper et al. (2006)
Risk assessment on Class B biosolids and vectors (e.g., flies) for
virus transmission (judged by cited workgroup to be a high
priority)
Virus workgroup in
Smith et al. (2005b)
Risk assessment for exposure of public to Class B biosolids,
including scenarios where food crops are grown or harvested
(judged by cited workgroup to be a high priority)
Virus workgroup in
Smith et al. (2005b)
Population-based risk model related to biosolids properties and
properties of pathogens from biosolids
Eisenberg et al.
(2004)
Research on management alternatives such as riparian buffers
Smith et al. (2005a)
Validation of health risk models using epidemiological studies
Pillai and Ricke
(2002), Pillai (2007)
Causal Analysis
Demonstration of causal association between biosolids
exposures and adverse health outcomes
NRC (2002)
Framework for establishing causation in human health
investigations, including (1) studies in response to unusual
exposures and unusual occurrences of disease, (2) preplanned
studies to characterize exposures of workers and communities,
and (3) epidemiological studies of biosolids use
NRC (2002)
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TABLE A-1 cont.
Need
Reference
Epidemiological studies on exposed populations such as those
who apply biosolids including farmers and communities near
land application sites
NRC (2002), Dowd
etal. (2000)
Rapid response investigations of reported health effects
potentially resulting from land application of biosolids
EPA (2003b) from
Water
Environmental
Research
Foundation (WERF)
Biosolids Research
Summit
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NRC RECOMMENDATIONS
The NRC was asked by EPA to evaluate "technical methods and approaches
used to establish the chemical and pathogen standards for biosolids, focusing
specifically on human health protection and not ecological or agricultural issues" (NRC,
2002). NRC recognized the need to reduce uncertainty about potential for adverse
human health effects from exposure to biosolids (NRC, 2002).
Many of the committee's recommendations are pertinent to a problem
formulation for risk assessment of land application of biosolids. The Committee on
Toxicants and Pathogens in Biosolids Applied to Land was asked to perform the
following pathogen-related tasks:
•	"Review the current standards for pathogen elimination in biosolids and their
adequacy for protecting public health. Consider (a) whether all appropriate
pathogens were considered in establishing the standards; (b) whether enough
information on infectious dose and environmental persistence exists to support
current control approaches for pathogens; (c) risks from exposure to pathogens
found in biosolids; and (d) new approaches for assessing risks to human health
from pathogens in biosolids."
•	"Explore whether approaches for conducting pathogen risk assessment can be
integrated with those for chemical risk assessment. If appropriate, recommend
approaches for integrating pathogen and chemical risk assessments."
Biosolids management practices and recent risk assessment methods were
reviewed. The committee reviewed evidence of human health responses to biosolids
including anecdotal allegations of disease, reviewed risk assessments and technical
data used to develop pathogen standards, and examined management practices of the
Part 503 rule. Peer-reviewed literature and government reports on human health
effects of biosolids and treated wastewater were reviewed and described in a table in
the NRC report, with no attempt to verify other allegations. The committee noted that a
cause and effect relationship between biosolids and adverse health effects has not
been documented (NRC, 2002) (see Table A-1). Overarching recommendations
included: (1) supplementing technological approaches with risk assessments to
establish regulatory criteria for pathogens in biosolids; (2) conducting a new national
survey of pathogens in sewage sludge; and (3) developing a framework for establishing
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causation in human health investigations, including (a) studies in response to unusual
exposures and unusual occurrences of disease, (b) preplanned studies to characterize
exposures of workers and communities and (c) epidemiological studies of biosolids use
NRC (2002, Table A-1). Furthermore, the committee recommended that EPA assess
the reliability of biosolids treatment processes, monitor compliance with pathogen
standards, conduct environmental hazard surveillance, and study human exposure and
health.
More specific recommendations of the NRC committee included the use of new
indicator organisms, such as Clostridium perfringens in regulation of land application of
biosolids (see Table A-1). Moreover, the committee recommended that site restrictions,
buffer zones and holding periods for applications of Class B biosolids be specific to
geographic and site-specific conditions that affect fate and transport of pathogens. The
committee recommends verification of site restrictions to determine if they meet their
intended pathogen levels (see Table A-1).
Regarding risk assessment, the committee recommended that a conceptual site
model should be used to identify all potential routes of exposure (NRC, 2002). The
committee found that it is not yet possible to integrate pathogen risk assessment with
chemical risk assessment, given the data gaps and paucity of risk assessment methods
for complex mixtures. Furthermore, they noted that several exposure pathways were
not adequately addressed in the 1993 Part 503 pathogen requirements, including the
inhalation pathway, the potential for surface-water contamination by runoff, groundwater
contamination and secondary transmission of disease (NRC, 2002). In particular,
pathogen transport and survival in bioaerosols is highly uncertain (see Table A-1).
Many of these research, monitoring and assessment gaps are included in Table A-1.
PATHOGENS
Extensive information is available describing pathogens that may be present in
Class B biosolids as well as their potential effects. Pathogens include bacteria, enteric
viruses, protozoan pathogens, helminths, and others. Articles that provide detailed
information on these classes of pathogens include Epstein (2006), Epstein and Moss
(2006), Pepper et al. (2006), NRC (2002), Straub et al. (1993) and chapters in Smith
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et al. (2005b). The list of potential pathogens is long, but little information is available to
eliminate particular agents. However, researchers contributing to the Smith et al.
(2005b) volume selected and provided criteria for selecting the most significant
bacterial, viral and parasitic pathogens.
Many of the articles above provide information on indicators of pathogens in
biosolids. Dowd et al. (1997) recommend thermotolerant Clostridia as indicators of fecal
contamination in bioaerosols. Pillai et al. (1996) found that Clostridia and H2S (hydrogen
sulfide) producers were better indicators of airborne biosolids-derived material than
traditional bacterial indicators (fecal coliformsand fecal streptococci).
The primary information gap related to hazard characterization is recent
national-scale data on the distributions of concentrations of pathogens in biosolids, with
respect to method of treatment, acceptable analytical methods for detecting and
quantifying pathogens and other variables (see Table A-1). Epstein and Moss (2006)
cite references regarding probable numbers of fecal conforms and Salmonella spp. in
Class B biosolids. Dahab and Surampalli (2002) found that existing treatment systems
do achieve Class B requirements under the U.S. Part 503 rule, while Class A may not
be easily achieved.
Biosolids experts distinguish between traditional and emerging pathogens, and
Gerba et al. (2002) reviewed the latter. A committee of experts convened at the
Workshop on Emerging Infectious Disease Agents and Issues associated with Sewage
Sludge, Animal Manures and Other Organic By-Products in Cincinnati, OH, June 2001,
concluded that emerging pathogens do not exhibit survival or other properties that are
very different from those exhibited by traditional pathogens (Smith et al., 2005a).
Pepper et al. (2006) reviewed studies of various traditional and emerging pathogens
and summarized which have been detected in biosolids and which have not been
detected in biosolids or not studied.
One recent study found that biosolids were not a likely source of Staphylococcus
aureus exposure or infection (Rusin et al., 2003a). Helminths are probably the most
persistent of enteric pathogens (Pepper et al., 2006; Straub et al., 1993). Little research
on the survival of protozoan parasites (e.g., Cryptosporidium species, Giardia) in
biosolids-amended soil has been conducted.
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It is impossible to test biosolids for all possible pathogens (Smith et al., 2005a).
Enteric viruses and helminth ova have been selected as indicators of treatment efficacy
because they are resistant to treatment and can be quantified (Smith et al., 2005a).
Chapter 4 in Smith et al. (2005b) provides detection/analytical capabilities and
recommendations for bacterial pathogens in biosolids.
MEASURES OF EXPOSURE
Numerous factors determine human exposure to pathogens in biosolids. These
include health status of contributors, method of treatment, percent solids, friability,
exposure to heat and ultraviolet. The literature search did not include an exhaustive
search for articles on factors that influence the fate of pathogens. The review below
presents a sampling of articles on the topic.
Detection of Pathogens
The detection of pathogens in environmental samples such as
biosolids-amended soil is inefficient. For example, Rusin et al. (2003a) had a recovery
efficiency of 8.7% for Staphylococcus aureus in Class B biosolids. Organic matter and
high bacterial counts reduce recovery fraction for pathogens (Rusin et al., 2003b).
Decay of Pathogens
Lang et al. (2003) studied the decay of E. coli in biosolids-amended sandy loam
soil and quantified indigenous E. coli in control soils in the United Kingdom. Stine et al.
(2005) studied survival of bacterial and viral pathogens on the surface of fruit and
vegetable crops, but not in a biosolids matrix. Straub et al. (1993) reviews studies of
survival of pathogens in soil and sewage sludge.
Lewis and Gattie (2002) assert that models typically use data from experiments
from enteric organisms such as E. coli and Salmonella to estimate bacterial survival
rates. They point out that these microorganisms are short-lived compared to those that
form spores or are encapsulated (such as Mycobacterium spp.).
Gerba et al. (2002) investigated which emerging pathogens are likeliest to
survive Class B biosolids treatments. Literature was reviewed (1) relating pathogen
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survival to temperature and environmental variables, (2) documenting pathogen
occurrence in biosolids and (3) describing dose-response models for pathogens. The
study concluded that adenoviruses and hepatitis A were heat resistant viruses and
therefore likely to survive long periods in the environment. Escherichia coli 0157:H7
and Listeria monocytogenes are emerging bacterial pathogens that can survive
anaerobic digestion and can sometimes regrow following land application of biosolids.
In contrast, the parasites microsporidia and Cyciospora would not survive under high
temperatures of anaerobic digestion or under conditions of low moisture.
Reactivation and Regrowth of Pathogens
Zaleski et al. (2005a) asked "Does regrowth occur following reintroduction or
recolonization of pathogens after land application or during storage under favorable
conditions?" The authors note that regrowth of indicator bacteria and Salmonella in
biosolids has been observed under certain moisture, temperature and substrate
conditions, and when indigenous bacteria are low. Moreover, pathogens in biosolids
may be reduced if they are stored at certain moisture and temperature ranges. In
biosolids-amended soils, increased moisture may lead to survival and regrowth of
bacterial pathogens. In one study the use of concrete-lined beds for storage during
desiccation allowed moisture from rainfall to accumulate in the beds, leading to growth
of fecal conforms and salmonellae added from external sources (Zaleski et al., 2005b).
Furthermore, survival rates of bacteria are higher in soil of finer textures (Zaleski et al.,
2005a).
Aerial Transport of Pathogens
Pathogens have rarely been measured in biosolid aerosols (see Table A-1).
Pillai and Ricke (2002) reviewed factors controlling bioaerosol transport, as well as
bioaerosol sampling methods and culture-based approaches to the detection and
characterization of specific components of bioaerosols.
Brooks et al. (2004a) measured bioaerosol emissions during land application of
Class B biosolids in the region of Tucson, AZ. The objective was to develop empirical
models of the fate and transport of bioaerosols. Pathogens and indicator bacteria were
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only rarely found in aerosolized samples. These included coliforms and coliphages,
which were present at high densities in biosolids, and animal viruses, which were not
detected in biosolids. Clostridium perfringens was detected only in a small fraction of
aerosol samples, but these were present under various weather conditions. The
authors suggest that only microorganisms in the aqueous phase of biosolids were able
to aerosolize; others remained sorbed to the solid phase (Brooks et al., 2004a).
In another study, Brooks et al. (2006) measured aerosolized endotoxin
concentrations downwind of a single biosolids-amended site. Levels were generally
within limits previously proposed in occupational exposure studies, though peak
concentrations occasionally exceeded these limits. Levels of endotoxin in aerosolized
soil were sometimes above those associated with biosolids amended-soil, calling into
question whether biosolids were the primary source of the endotoxin. Additional studies
of bioaerosol transport that included a risk assessment component are described in the
section on risk assessment.
Tanner et al. (2005) determined bioaerosol emission rates and plume
characteristics during spray application of liquid Class B biosolids. They did not detect
coliphages or coliform bacteria just downwind of the biosolids application (approximately
a 2-m distance away), though bacteria that had been added to groundwater and
sprayed were detected. The researchers concluded that the presence of biosolids
reduces aerosolization of microorganisms relative to application of inoculated
groundwater. Even if bacteria had been present below detection limits, the duration of
exposure to any pathogens just downwind of biosolids application would be expected to
be brief because of the moving applicator (Tanner et al., 2005).
Paez-Rubio et al. (2006) investigated the content of bioaerosols produced during
the disking of biosolids on an application site in Central Arizona. Biosolids source
emission factors (number of microorganisms or mass of biotoxins per area) and
emission rates (number of microorganisms or mass of biotoxins per time) were
measured for total bacteria, culturable heterotrophic bacteria (heterotrophic plate counts
[HPC]), total coliforms, sulfite-reducing Clostridia, and endotoxin, as well as particulate
matter 10 micrometers (PM10). The authors presented a correlation between microbial
concentrations emitted during disking and their content in biosolids. Disking was
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determined to be a "substantial source of biosolids-derived aerosols" and might be of
greater potential concern than other application methods. The emission rate during
disking of biosolids was greater than rates that had been measured during spreading of
dewatered biosolids by side slinger or spraying of liquid biosolids. For example, total
coliform emissions during disking were about two times greater than emissions
associated with spreading dewatered biosolids and at least two orders of magnitude
greater than maximum emission rates reported by Tanner et al. (2005) during spraying
of liquid biosolids (Paez-Rubio et al., 2006). The authors provide a framework for
reconstructing aerosol concentrations and emission rates.
In a related study, Paez-Rubio et al. (2007) measured bioaerosol emission rates
from the spreading of Class B biosolids with a side-slinging applicator in Arizona.
Concentrations of pathogens in bioaerosols were reconstructed from concentrations in
bulk biosolids and PMi0. Aerosol emission rates of several bacterial indicators were
correlated with their concentrations in bulk biosolids. Aerosol emission rates of
dewatered biosolids were one to two orders of magnitude higher than those reported for
liquid biosolids. Diameters of emitted particles suggest that most were inhalable and
possibly respirable. The authors assert that their work "move[s] aerosol studies beyond
indicator measurements by estimating specific toxic compound or pathogen aerosol
concentrations based on more easily obtained PM10 measurements and bulk biosolids
analysis—where detection limits are much lower due to the large sample size possible."
J. Peccia, one of the authors, notes that rates of recovery of pathogens in aerosols that
are reported in the literature are currently only about 10% (Lubick, 2007). The authors
acknowledge that the relationship between source emission rates and bulk biosolids
concentration that they present is limited to the type of spreader they used (i.e., a
"ProTwin Slinger" side discharge spreader, the most common spreader for biosolids of
the 20-30% solids content range).
Leaching to Groundwater
A review of the literature has concluded that few pathogens from biosolids leach
to groundwater (Pepper et al., 2006). For example, Chetochine et al. (2006) measured
the numbers and leaching potential of coliphage MS-2, specific to E. coli, from Class B
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biosolids. Much of the phage was sorbed to or associated with solid particles.
Following serial extraction, less than 8% of the phage initially present in the biosolids
leached from biosolids-amended soil. The phage was not appreciably retained in a
column containing a sandy porous medium. Horswell et al. (2010) tested soil cores and
concluded that environmental contamination by virus is unlikely because of strong soil
adsorption.
Y. Jin, J. Sims and K. Kniel of the University of Delaware were awarded a
U.S. Department of Agriculture (USDA) grant from 2006-2009 to study the fate and
transport of viruses in biosolids and their potential to contaminate groundwater and
foodcrops as a result of land application of biosolids (Jin et al., 2006).
Erosion and Surface Runoff
The literature search did not find information on these mechanisms of transport
of pathogens in biosolids.
Pathogens on Crops
Studies of pathogens on crops are described in the section on risk assessment.
Also, the DA grant described above that was awarded to Y. Jin, J. Sims and K. Kniel of
the University of Delaware includes an investigation of the contamination of crops (Jin
et al., 2006).
RISK ASSESSMENT
Risk Assessment Process
Risk assessments of pathogens in biosolids have been performed by various
investigators, but the emphasis has been on the use of particular transport models to
quantify exposure and risk, rather than the process of planning and conducting a broad
risk assessment. One recent risk assessment of biosolids application found that the
science of assessing risk from environmental exposure to biological agents, as well as
acceptable levels is "under development at the present time" (Willert and Eng, 2005).
Therefore, the focus of that study was altered from the quantification of risk to the
effectiveness of a peptization process to destroy biological agents of potential concern.
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Soller et al. (2006a) described general methods for conducting health risk
assessments of pathogens in biosolids that were developed as part of a Water
Environment Research Foundation project. The methods included characteristics of an
infectious disease process, including the consideration of multiple transmission
pathways, disease transmission models, and the presence of immunity (Soller et al.,
2006a,b). Soller et. al.'s framework for evaluating human risks associated with
microbes in biosolids included an exposure characterization component (quantifying
pathogen levels in the environment) and a health effects component. A schematic
diagram displayed several Class A and Class B sludge treatment processes as well as
environmental variables affecting exposure (time, temperature and moisture). They
described the tradeoff between site-specific monitoring data and more general data on
treatment effectiveness and fate and transport of pathogens from points earlier in the
waste stream. A conceptual health effects model was also included in the report. This
model, first published in Eisenberg et al. (2004), contained six epidemiological states:
(1) susceptible state, (2) exposed state (asymptomatic and infectious), (3) carrier state 1
(asymptomatic but infectious), (4) diseased state, (5) carrier state 2 (previously
symptomatic, now asymptomatic and infectious) and (6) protected state (postinfectious
and noninfectious and some level of immunity). Soller et al. (2006a) also included a
table of data required to parameterize a basic health effects model.
Although Soller et al. (2006a) included information and diagrams useful for
developing a problem formulation for pathogens in biosolids, they did not organize it as
a problem formulation. These elements are found in the Guidelines for Ecological Risk
Assessment (U.S. EPA, 1998).
The International Life Sciences Institute developed a framework for microbial risk
assessment related to human exposures and waterborne pathogens (ILSI, 2000). The
framework describes the stages of risk assessment, including problem formulation, but
does not provide or cite scientific advice regarding particular pathogens or exposure
pathways.
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Pathways for Bioaerosol Exposure
One of the primary research needs identified by the NRC was human exposure
to pathogens in bioaerosols (NRC, 2002). Researchers at the University of Arizona
conducted several major studies to help understand community and worker risk of
infection from bioaerosols, as well as to develop methods for modeling transport of
pathogens and human exposure (Brooks et al., 2004a, 2005a,b, 2006, Tanner et al.,
2008). Prior to that study, the same group of researchers studied bioaerosols in West
Texas (Dowd et al., 2000). Conclusions were that community risks were relatively
negligible, with worker risks somewhat higher.
Dowd et al. (2000) sampled bioaerosols emitted from anaerobically digested,
dewatered biosolids applied in west Texas. The study generated bacterial and virus
release rates from large biosolids piles where they were stored prior to application and
fields where biosolids were sprayed. Levels of Salmonella and an indicator virus
(coliphage) were measured. The ratio between the concentration of indicator virus in
aerosols and the concentration in biosolids was used to estimate a value for airborne
enteric virus (Coxsackievirus). Microbial transport models (a point source model and an
aerial source model) were used to generate downwind concentrations. Dose-response
models were used to estimate risk to workers on site and nearby residents at least
10 km away. The pathway was assumed to consist of inhalation and swallowing of the
pathogen. The single hit exponential model [p = 1 — exp (—rN)] was used to describe
the probability of infection by Coxsackievirus B3, and the Beta-distribution model
(p = 1 - [1 + (N/(3)(21/a-1)]~a) was used to describe the risk of infection by Salmonella
serovar Typhi, where p = probability of infection, N = number of organisms inhaled, (3 is
the ID5o, and a and rare parameters that describe the dose-response curve. The
authors indicated that several sources of conservatism must be considered when
evaluating these risk estimates (e.g., the wind does not always come from the same
direction, Dowd et al., 2000). Brooks et al. (2004b) corrected a mathematical error in
Dowd et al. (2000) which resulted in the newly calculated risk estimates being much
lower than reported in Dowd et al. (2000). Citing comments by Brooks et al. (2004a) on
the improved efficiency of modern wastewater treatment plants, Pepper et al. (2006)
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argue that a more realistic estimate of infectivity is five orders of magnitude lower than
Dowd's worst case estimates.
Brooks et al. (2005b) undertook a study to estimate risks of microbial infection of
residents near biosolids application sites. At 10 sites throughout the United States that
were amended with either liquid or solid Class B biosolids (five sites in Arizona, two in
Washington State, one in Virginia, one in Texas and one in Illinois), they measured HPC
bacteria, total coliform bacteria, E. coli, Clostridium perfringens, coliphage,
enteroviruses, hepatitis A virus and norovirus in aerosol samples downwind from
application sites. The study distinguished between loading, unloading, land application
and background operations. In general, risks of infection were determined to be low,
with the greatest risk of infection, 4 x 10~4, from coxsackievirus A21 released during
loading operations.
Brooks et al. (2005b) cited a dissertation of Tanner (2004) in reporting that the
risk of infection to a biosolids handler can reach as high as 34% annually from exposure
to coxsackievirus A21 and 2% annually from exposure to Salmonella species. This
study assumed exposure on a daily basis (250 days per year).
Brooks et al. (2005a) developed an empirical transport model for viruses
aerosolized during land application of liquid biosolids. Data were generated from
collections of bioaerosols in field tests with coliphage MS-2 added to water and sprayed
with a biosolids spray application truck. Risks of infection for residents adjacent to land
application sites were also calculated at 10~7 (realistic) to 10~5. Conservative annual
risks were calculated at no more than seven times that value. A second goal of the
study was to develop a transport model for bacteria, but E. coli used in the study did not
typically survive the aerosolization process.
Based on Brooks' studies, Pepper et al. (2006) concludes that overall community
risk of infection from bioaerosols during land application was relatively negligible.
Occupational risks during land application were higher than community risks but were
still low (Brooks et al., 2004). Pillai (2007) cautions against extrapolating these results
to different source materials, regions, or even parts of a region. Pathogens in biosolids
might be more desiccated or inactivated from exposure to ultraviolet light than in other
parts of the country.
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In a study of bioaerosol emission rates from the spreading of Class B biosolids in
Arizona, measured source endotoxin concentrations that were greater than reported
conservative thresholds for mucous membrane irritation, and most exceeded the
threshold for acute bronchial constriction (Paez-Rubio et al., 2007).
Pathways for Groundwater Contamination
Based on a review of the literature such as Chetochine et al. (2006, above),
Pepper et al. (2006) conclude that groundwater contamination from land-applied
biosolids is not likely, and therefore human health risks are likely negligible. By
extension, pathways by which pathogens in groundwater may contaminate land or
surface water via springs or other interactions are also unlikely to be significant for
pathogens from biosolids.
Ingestion of Soil
Gerba et al. (2002) used a beta-Poisson model (P = 1 - [1 + N/p-aJ) from Haas et
al. (1999) to assess the risk of infection and illness from enteric viruses following land
application of Class B biosolids, assuming that exposure was from ingestion of
biosolids-amended soil. They focused on rotavirus and echovirus 12. Gerba et al.
(2002) determined that direct ingestion of biosolids, if they were spread across the
surface of the soil, would result in an annual risk from a one time exposure exceeding
1 x 10~4. They assumed no natural attenuation of virus. Injection of biosolids into the
soil results in a risk below this level.
Consumption of Vegetation
Most of the information on risks from the crop ingestion pathway is from the
United Kingdom. Consumption of root crops is assumed to represent the worst case
scenario because they contain higher proportions of soil than leafy crops and they are
often consumed uncooked (Gale, 2005a). Gale (2003) estimated the exposure of root
crops to Cryptosporidium and Salmonella species from biosolids applied to agricultural
land in accordance with the United Kingdom's Safe Sludge Matrix. An approach using
event trees combined with empirical data was used to estimate pathogen levels in raw
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sewage sludge, in treated sludge and biosolids mixed with topsoil and root crops.
Expert opinion suggested that up to 2% of root crops by weight may be soil at the point
of harvest. Monte Carlo simulations were performed to model variation in Salmonella
levels on root crops, assuming a Poisson-log-normal distribution of bacterial counts.
Gale (2005b) conducted risk assessments to estimate the number of humans in
the United Kingdom at risk from consumption of root crops obtained from areas where
biosolids were applied according to the Safe Sludge Matrix regulations. (Gale [2005a]
presents a subset of that study.) Seven classes of pathogens were the focus of the
study: salmonellas, Listeria monocytogenes, Campylobacters, Escherichia coli 0157,
Cryptosporidium parvum, Giardia and enteroviruses. The study showed that if linear
decay were assumed to occur and if the treatment process (mesophilic anaerobic
digestion [MAD]) were assumed to be 100% efficient, potential risks from the
seven classes of pathogens were essentially eliminated. If pathogen decay in treated
soil was assumed not to occur, then 50 Giardia infections were expected in the United
Kingdom and less than one infection per year resulting from the other six pathogens.
Also if the MAD process was 99% or lower, substantially more infections from Giardia
and possibly E. coli 0157 were predicted.
Gale and Stanfield (2001) calculated risks to humans from consumption of
vegetable crops contaminated with the bovine spongiform encephalopathy agent in
sewage sludge in the United Kingdom. Hinkley et al. (2008) suggest that prions survive
wastewater treatments and Pepper et al. (2006) identified the incidence of prions in
biosolids as a research priority in the United States (see Table A-1).
Proliferation of Antibiotic Resistance
In addition to risks to human health from specific pathogens, another relevant
indirect health issue is the possible proliferation of antibiotic resistant bacteria. The
potential risk is that human pathogenic strains become resistant to overused antibiotics,
which can no longer treat the pathogen. Pepper et al. (2006) ask the question "Can
antibiotic resistant genes be transferred from nonpathogenic bacteria to human
pathogenic strains?" Brooks et al. (2004a, 2007a) concluded that Class B biosolids had
an equal or lower incidence of antibiotic resistant bacteria compared to unamended soil.
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The NRC (2002) did not "believe that land-applied biosolids have any substantial
potential to alter the prevalence of antibiotic resistance among pathogenic organisms."
Infectivity
Gerba and Smith (2005) describe broad risk assessment principles for land
application of wastes based on a quick review of the literature, as well as their own
experience and expertise. They note that information on infectivity of enteric pathogens
is available from many human feeding or inhalation studies.
Dose-response data suggest that a threshold infectious dose does not exist for
enteric pathogens (Gerba and Smith, 2005). Infectivity of enteric viruses is greater than
infectivity of enteric bacteria. Of known human enteric viruses, rotavirus is the most
infectious, causing 10-15% ofthose ingesting the virus to become infected. Half of the
people infected with an enteric pathogen become ill. Mortality is typically less than 1 %,
but greater for infants, young children, the elderly and immunocompromised people
(Gerba and Smith, 2005). A recently developed norovirus dose-response model by
Teunis et al. (2008a) includes the above enteric virus characteristics and incorporates
additional parameters that may be used for enteric virus risk assessments.
Nwachuku and Gerba (2004) address the susceptibility of children to pathogens,
including increased sensitivity and increased exposure. Other reasons why children are
at greater potential risk from pathogens in biosolids include the following:
•	immature immune system;
•	intestinal mucosa more permeable to water;
•	proportionally less extracellular fluid than adults;
•	physiological deficiency in Immunoglobulin A; and
•	reduced stomach acid and pepsin secretion.
For example, children appear to be the most sensitive population to
enteroviruses. Studies have not been conducted to estimate relative infectivity of
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enteric pathogens for children and adults. However, reduced stomach acid and pepsin
secretion could make children more likely to be infected than adults for a given dose.
Disease Risk
Existing empirical studies of biosolids do not estimate disease risk. However,
risks of disease might be assumed to be 10% that of infectious risk, though this quantity
varies with microorganism (Haasetal., 1999). Sollerand Eisenberg (2008) provide
parameter values for the proportion of the infected individuals with symptomatic
responses for enteroviruses, rotavirus, Cryptosporidium, Giardia lamblia, Salmonella, E.
coli 0157:H7, Shigella, and a composite value (minimum of 10, median of 40, maximum
of 75%).
Dynamic Risk Model
In assessing microbial risks, one may choose either a static or a dynamic risk
model (Soller et al., 2008; U.S. EPA, 2006a). Eisenberg et al. (2004) developed a
deterministic, dynamic model for estimating risks from pathogens in biosolids. In
addition to infectivity, their model considered person-to-person transmission, immunity,
asymptomatic infection and incubation period. The model contains six disease states:
(1) susceptible state, (2) exposed state (asymptomatic and infectious), (3) carrier state 1
(asymptomatic but infectious), (4) diseased state, (5) carrier state 2 (previously
symptomatic, now asymptomatic and infectious) and (6) protected state (postinfectious
and noninfectious and some level of immunity). Processes that were not accounted for
include climate, behavior and various environmental factors that are not well
understood. Three types of risks were estimated: individual-level single event risk,
individual-level annual risk and population level attributable risk (Eisenberg et al., 2006).
The model was demonstrated in a case study involving the direct ingestion of
enterovirus. Sensitivity analysis of simulations in the case study showed that the
four most important factors in determining the risk attributable to biosolids were: (1) the
relative contribution of biosolids toward exposure, relative to other pathways; (2) the
rate of pathogen shedding by infectious people; (3) the rate of person-to-person
transmission and (4) immunity. Risk attributable to biosolids was "low" if the rate of
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pathogen shedding was relatively high or low or if person-to-person transmission was
relatively "high." These were not necessarily intuitive results. The simulations resulted
in a decision tree for classifying risk associated with biosolids as high or low.
EXPOSURE ASSUMPTIONS
EPA does not have standard exposure factors for use in risk assessments of
pathogens in biosolids. Risk assessment results described above are highly dependent
on human exposure factors, and these vary from study to study. For example, because
human transmission of aerosols containing Salmonella has not been demonstrated,
researchers make different assumptions about the percentage of inhaled particles that
would be ingested. Pepper et al. (2006) describe studies that use 10%, and Brooks
etal. (2005b) uses 50%.
Very little information is available that would allow us to compare the relative
importance of different exposure pathways. Academic studies tend to emphasize a
single exposure pathway rather than a comparison of multiple pathways. Many studies
have found low risk. For example, a British study by Gale (2005b) concluded that risk to
human health from consumption of vegetation crops contaminated with pathogens in
biosolids is low. Moreover, a study of bioaerosols in Arizona found that risk of infection
of residents from bioaerosols generated during land application of biosolids was rather
negligible at 10 km, though if residents were assumed to reside closer, estimated risks
would have been higher (Brooks et al., 2005b; Pepper et al. 2006). Based on a review
of the literature, Pepper et al. (2006) conclude that "groundwater contamination from
land-applied biosolids does not appear to be likely." Moreover, it is argued that
regrowth of pathogens in biosolids-amended soil may be ignored because of the
biological competition in Class B biosolids (Pepper et al., 2006; Zaleski et al., 2005a,b).
However, insufficient information is available to ignore particular exposure pathways at
all sites.
CAUSAL ANALYSIS
"Causal association between biosolids exposures and adverse health outcomes
has not been documented" (NRC, 2002). Gattie and McLaughlin (2004) investigated
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public complaints and concluded that irritants associated with volatile chemicals and
dust blowing from biosolids treated land (e.g., bacterial toxins, lime, organic amines)
may cause nearby residents to be more susceptible to infections. Lewis et al. (2002)
recorded symptoms reported by 48 residents near 10 biosolids application sites in the
United States and Canada. The wide range of symptoms included various
combinations of coughing, burning eyes, sore throat, burning lungs, headache,
congestion, difficulty breathing, flu-like symptoms, fever, nausea/vomiting, diarrhea,
sinusitis, staphylococcal infection, pneumonia, skin rash, nosebleed and fatigue. The
researchers did not establish cause and effect between biosolids and reported adverse
effects. They suggested that chemical contaminants in biosolids might irritate the skin
and mucous membranes and thus increase pathogen host susceptibility (Lewis et al.,
2002). Another survey conducted by Khuder et al. (2007) suggests a higher risk of
certain respiratory, gastrointestinal, and other diseases among residents living near
farm fields on which biosolids were applied.
In contrast, Dorn et al. (1985) conducted a health effects study of 47 biosolids
application sites (annual applications) and 46 control sites on farms in Ohio. Estimated
risks of respiratory illness, digestive problems or other general symptoms did not differ
between biosolids and nonbiosolids farms. The authors cautioned readers when
considering the results in the context of larger acreages, higher application rates or
biosolids containing larger concentrations of pathogens.
NRC (2002) summarized studies of sewer workers and others exposed to raw
sewage to identify potential hazards from biosolids. The committee also summarized a
survey study in which workers who loaded, unloaded and applied Class B biosolids had
a history of gastrointestinal illness. However, it was later determined that the biosolids
did not meet Class B requirements.
Simmonds et al. (2005) describe the difficulties of conducting an epidemiological
study of biosolids exposure. Few people who are exposed are expected to become
infected, and even fewer to manifest symptoms of disease. Also, various symptoms
may be associated with one pathogen, and various pathogens can cause similar
symptoms.
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Preliminary work has been done to scope epidemiological designs to assess
acute health effects and community-level exposure to treated sewage sludge (Class B
biosolids) in North Carolina and Virginia (Heaney et al., 2006).
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