E P A/600/R-08/035A
February 2008
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
This report is an external draft for review purposes only and does not constitute
Agency policy. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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TABLE OF CONTENTS
Page
LIST OF TABLES	vi
LIST OF FIGURES	vii
LIST OF ABBREVIATIONS	viii
AUTHORS, CONTRIBUTORS AND REVIEWERS	ix
1.	INTRODUCTION	1
2.	STRESSOR CHARACTERIZATION	3
2.1.	SOURCE	4
2.1.1.	Spatial Extent of Source	5
2.1.2.	Reproduction	5
2.1.3.	Matrix	6
2.1.4.	Class B Treatment	6
2.1.5.	Site Restrictions	8
2.1.6.	Vector Attraction Reduction	8
2.2.	PATHOGENS	10
2.2.1.	Bacteria	10
2.2.2.	Viruses	16
2.2.3.	Protozoa	19
2.2.4.	Endotoxins	22
2.2.5.	Emerging Pathogens	23
2.2.6.	Multiple Stressors	24
3.	DEVELOPMENT OF CONCEPTUAL MODELS, ENDPOINTS AND
SCENARIOS	26
3.1.	PREAPPLICATION PROCESSES	29
3.2.	APPLICATION	30
3.2.1.	Methods of Land Application of Biosolids	30
3.2.2.	Rates of Land Application of Biosolids	33
3.2.3.	Timing of Land Application of Biosolids	34
3.2.4.	Regional Application Issues	34
3.3.	FATE AND TRANSPORT OF PATHOGENS	35
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TABLE OF CONTENTS cont.
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3.3.1.	Pathogen Survival, Growth and Death	35
3.3.2.	Pathogen Transport	38
3.3.3.	Vector Transport	42
3.4.	HUMAN ROUTES OF EXPOSURE	42
3.4.1.	Inhalation	43
3.4.2.	Ingestion	43
3.4.3.	Dermal Exposure	47
3.5.	REGULATORY RESTRICTIONS	48
3.6.	FACTORS THAT AFFECT INFECTION AND DISEASE	48
3.6.1.	Human Factors	48
3.6.2.	Additional Susceptibility Factors	52
3.6.4.	Pathogen Factors	53
3.7.	INFECTION AND DISEASE	53
3.8.	SCENARIOS	54
3.8.1.	Scenario 1. Neighboring Residences and Schools	55
3.8.2.	Scenario 2. Residents	55
3.8.3.	Scenario 3. Pica Child	58
3.8.4.	Scenario 4. Drinking Water Consumers of Groundwater	59
3.8.5.	Scenario 5. Drinking Water Consumers of Surface Water	59
3.8.6.	Regional Aspects of Scenarios	62
4.	SCREENING OUT ELEMENTS OF THE CONCEPTUAL MODEL	63
5.	ANALYSIS PLAN	66
5.1.	INTRODUCTION	66
5.2.	MANAGEMENT NEEDS	67
5.2.1.	Assessment Endpoints	68
5.2.2.	Data and Data Quality	69
5.3.	PLAN FOR CHARACTERIZATION OF EXPOSURE	69
5.3.1.	Measures of Exposure	69
5.3.2.	Detection of Pathogens	70
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TABLE OF CONTENTS cont.
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5.3.3.	Use of Indicator Species	73
5.3.4.	Background Levels of Pathogens	75
5.3.5.	Environmental Fate of Pathogens	77
5.3.6.	Transport of Pathogens	78
5.3.7.	Contact with Crops	84
5.3.8.	Uptake and Dosage	85
5.3.9.	Exposure Factors	85
5.4.	PLAN FOR CHARACTERIZATION OF EFFECTS	86
5.4.1.	Measures of Effect	86
5.4.2.	Establishing Cause and Effect	87
5.4.3.	Dose-Response Models for Infection	88
5.4.4.	Predicting Disease	91
5.5.	PLAN FOR RISK CHARACTERIZATION	95
5.5.1.	Screening Risk Assessment	95
5.5.2.	Weight of Evidence	96
5.5.3.	Uncertainty Analysis	97
6. REFERENCES	98
APPENDIX A LITERATURE REVIEW	107
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LIST OF TABLES
No.	Title	Page
1	Site Restrictions for Class B Biosolids	9
2	Example Pathogens of Potential Concern in Sewage Sludge and Biosolids	11
3	Estimated Biosolids Application Rates for Different Land Uses	33
4	Environmental Factors Positively or Negatively Affecting the Survival of
Pathogenic Microbes	37
5	Pathways of Exposure and Applicable Use Restrictions for Class B
Biosolids	49
6	Minimum Time Interval between Application and Harvest, Grazing or
Public Access to Lands Applied with Class B Biosolids	50
7	Suitability of Select Agents as Indicators of Post-Treatment Risk for Viruses
in Biosolids, Modified from Smith et al. (2005b)	76
8	Survival Times of Pathogens in Soil and on Plants Modified from Gerba
and Smith (2005)	78
9	Examples of Dose-Response Models for Microbial Agents	90
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LIST OF FIGURES
No.	Title Page
1	General Conceptual Model	28
2	Pathogen Fate Conceptual Model	36
3	Disease Factors Conceptual Model	51
4	Adjacent Property Conceptual Model	56
5	Resident Conceptual Model	57
6	Pica Child Conceptual Model	58
7	Groundwater Conceptual Model	60
8	Surface Water Conceptual Model	61
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LIST OF ABBREVIATIONS
CFR	Code of Federal Regulations
HPC	heterotrophic plate counts
ICC-PCR	integrated cell-culture PCR
NRC	National Research Council
PCR	polymerase chain reaction
PSRP	process to significantly reduce pathogens
RT-PCR	direct reverse transcriptase PCR
U.S. EPA	United States Environmental Protection Agency
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AUTHORS, CONTRIBUTORS AND REVIEWERS
AUTHORS
Rebecca Efroymson
Environmental Sciences Division
Oak Ridge National Laboratory
Oak Ridge, TN 37831
Anthony Armstrong
Environmental Sciences Division
Oak Ridge National Laboratory
Oak Ridge, TN 37831
CONTRIBUTORS
Glenn Suter II
National Center for Environmental Assessment
Office of Research and Development
U.S. Environment Protection Agency
Cincinnati, OH 45268
Michael Troyer
National Center for Environmental Assessment
Office of Research and Development
U.S. Environment Protection Agency
Cincinnati, OH 45268
INTERNAL REVIEWERS
Michael Broder
Office of the Science Advisor
Office of Research and Development
U.S. Environment Protection Agency
Washington, DC 20460
James Smith
National Risk Management Research Laboratory
Office of Research and Development
U.S. Environment Protection Agency
Cincinnati, OH 45268
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AUTHORS, CONTRIBUTORS AND REVIEWERS cont.
Richard Stevens
Health and Ecological Criteria Division
Office of Science and Technology
Office of Water
U.S. Environment Protection Agency
Washington, DC 20460
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1. INTRODUCTION
In January 2004, the United States 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 Practice (NRC, 2002). This report
is an important step in the Agency's response because it addresses the development of
a problem formulation and analysis plan relating to uncertainties associated with
conducting quantitative microbial risk assessments on land-applied biosolids. This
report summarizes the existing literature (Appendix A); 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, tools and methodologies; and develops an analysis plan which
identifies the research and methods required for providing a scientifically defensible risk
assessment relevant for U.S. 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 was 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).
This generic problem formulation should serve two audiences. First, assessors
who must 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
serve as a template, an information source and an introduction to the relevant literature.
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1	Second, the research needs identified in this report can be used by researchers and
2	research planners to select and prioritize research projects related to pathogens in
3	biosolids. It can also help researchers to understand how to design their studies so as
4	to generate results that will be relevant to risk assessment.
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2. STRESSOR CHARACTERIZATION
Stressors are chemical, physical or biological agents that may adversely affect
human health or other assessment endpoints. The description of stressors is a
necessary precursor to developing conceptual models, especially for risk assessments
of a complex substance like biosolids. U.S. EPA (1998) describes several questions
that a stressor characterization for an ecological risk assessment should answer.
These points are modified for human health risk assessments for pathogens.
1.	What is the source of the pathogens?
2.	What is the spatial extent of the source?
3.	What types of stressors are present: physical, chemical or biological?
4.	What are the modes of action of the stressors?
Essentially, sources and stressors must be characterized well enough to inform
decisions about the conceptual models and exposure pathways within them that are
needed to characterize all reasonable exposure scenarios. For example, pathogens in
bioaerosols have different fates from those that remain in biosolids-amended soil
particles, and the problem formulation should describe these differences.
This report focuses on pathogens and endotoxins originating in biosolids. In
addition to descriptions of microorganisms in biosolids, the assessor should include
aspects of the biosolids matrix that affect pathogenicity and dimensions of the source
that affect how exposure is modeled or monitored. Studies of untreated manures are
beyond the scope of this report.
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This chapter describes the biosolids source, including the components of the
mixture, the extent of the source, the matrix, the Class B treatment process, site
restrictions and vector attraction reduction options. Following the description of the
source is 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 (Pepper et al., 2006;
NRC, 2002). These soil amendments have nutrients for plant growth as well as
components that improve physical properties of soils. The U.S. EPA did not use the
term biosolids in the Part 503 rule, but U.S. 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 a complex mixture that contains 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
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(2002) and may include metals, trace elements, PCBs, dioxins, pharmaceuticals,
surfactants and other contaminants.
2.1.1.	Spatial Extent of Source
Risk assessors need to characterize the areal extent of biosolids application or
storage that is the subject of the risk assessment. Biosolids may be localized or more
diffuse sources of infectious microbes. Pathogen transport models may be specific to
the spatial extent of the source. 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 over
time may be pertinent. At the extreme, a risk assessment may address the entire area
treated with biosolids 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 as some of the organisms
reproduce (Zaleski et al., 2005a). Evidence about reproduction or lack of reproduction
of particular species is important information for the conceptual models.
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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 are of particular interest in this problem formulation.
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.	Solid biosolids. Biosolids cake (usually 20-30% solids content) (Paez-Rubio et
al., 2007) 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 (University of Washington, 2002).
4.	Bioaerosols. Bioaerosols are aerosolized biological particles that vary from 0.02
to 100 |jm 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 light (Peccia et al.,
2001).
2.1.4. Class B Treatment
A description of the sewage sludge treatment process provides risk assessors
with information about the potential pathogen content of biosolids. Treatment methods
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are intended to reduce the volume and organic content of biosolids and to reduce the
number of pathogens, but to retain 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
pathogens (U.S. EPA, 1993). This report focuses 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 l06/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 PSRPs. Five
processes in the Part 503 Rule were determined to be PSRPs, based on their resulting
fecal coliform concentrations less than 2 * 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 and
5.	Lime stabilization to give a pH greater than 12 after 2 hours of contact.
Fecal coliforms are enteric bacteria that are used as indicators of the likelihood of
the presence of bacterial pathogens. Salmonella species are human pathogens. In this
problem formulation, it is assumed that treatment requirements and site restrictions
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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 pathogens attenuate over time. 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 (Table 1).
2.1.6.	Vector Attraction Reduction
The Part 503 rule requires that one of ten 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
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TABLE 1
Site Restrictions for Class B Biosolids (Copied from NRC (2002), Adapted from 40
CFR 503.32[b][5])
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.
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.
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.
Food crops, feed crops and fiber crops shall not be harvested for 30 days after
application of biosolids.
Animals shall not be grazed on the land for 30 days after application of biosolids.
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.
Public access to land with a high potential for public exposure shall be restricted for
one year after application of biosolids.
Public access to land with a low potential for public exposure shall be restricted for 3
days after application of biosolids.
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biosolids and incorporation within 6 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. 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. Many of these organisms and the diseases they cause are summarized
in Table 2. Researchers who list principal pathogens of concern in sewage sludge
and/or biosolids do not always list the same organisms (NRC, 2002; Gerba and Smith,
2005; Pepper et al., 2006; Epstein, 2006; Yanko, 2005). As biological stressors,
pathogens can multiply, and many 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 a community and the type of treatment
process (Straub et al., 1993). The biosolids matrix (i.e., whether humans are exposed
to biosolids, biosolids-amended soil, bioaerosols, or biosolids particles in water) may
affect the fate of pathogens, and therefore determine exposure.
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 U.S.,
salmonellosis is mainly due to foodborne transmission because the bacteria found in
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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 pylori
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
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1
Table 2 (cont.)
Class
Organism
Disease or Symptoms
Helminth
Parasites
(cont.)
Trichuris spp.
Gastrointestinal distress, anemia
Toxicocara canis
Fever, abdominal discomfort, neurological
symptoms
Protozoan
Parasites
Cryptosporidium
parvum
Diarrhea

Giardia lamblia
Fever, diarrhea

Cyclospora
Diarrhea, nausea, vomiting and abdominal
cramps

Microsporidia
Diarrhea

Entamoeba
histolytica
Dysentary, colitis

Balantidium coli
Diarrhea, constipation, abdominal pain
2
3	Sources: Gerba and Smith (2005), Epstein (2006), NRC (2002), Pepper et al. (2006)
4	and Bowman and Fayer (2005).
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beef and poultry are able to grow in foods (Pepper et al., 2006). As of 1998, there was
no known association of biosolids with foodborne outbreaks of Salmonella (Yanko,
2005). However, Salmonella can apparently grow in biosolids under some conditions
(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. They are the
40 CFR 503 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 U.S. 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 food (Yanko,
2005; Pepper et al., 2006). Cattle are the most significant source of exposure, but the
organism has been detected in biosolids (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 U.S.
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.	Shigella Spp.
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
polluted with Shigella (Pepper et al., 2006). The pathogen has a low infectious dose.
Shigella does not survive well in the environment or after treatment of biosolids.
Therefore, they are unlikely 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 U.S. 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 montocytogenes
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. montocytogenes 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. 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 Legionnaires' Disease 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. Experts believe that Staphylococcus aureus "are not a likely
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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). 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 U.S. (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 prioritize bacterial pathogens for inclusion in their risk
assessments of land application of biosolids. A workgroup of biosolids experts
developed methods for evaluating 20 potential pathogens in biosolids (Chapter 4 in
[Smith et al., 2005]). They considered their 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 may be present in
municipal wastewater and possibly biosolids (Gerba et al., 2002).
2.2.2.1. Enteroviruses
The enteric viruses most often detected in polluted waters are the enteroviruses,
though this may be an artifact of the ease of detection in animal cell culture (Pepper et
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al., 2006). These include poliovirus, Coxsackie virus, echovirus and enteroviruses
69-91. 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 Class B processes such as aerobic and anaerobic sludge digestion
(Pepper et al., 2006).
2.2.2.2.	Rotaviruses
These are the only double-stranded RNA viruses transmitted through water to
humans (NRC, 2002). Along with caliciviruses, rotaviruses are the leading cause of
gastroenteritis in the U.S. (Monroe et al., 2000) and a major cause of hospitalization of
children in the U.S. (Gerba et al., 1996). These viruses cause waterborne and
foodborne outbreaks in the U.S. 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 Norwalk viruses and the Sapporo viruses (NRC, 2002). Little is known
about their environmental occurrence and fate because caliciviruses have not yet been
grown in cell culture (Gerba et al., 2002; NRC, 2002).
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., 1996). The
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mortality of immunocompromised people (e.g., organ transplant, cancer chemotherapy
patients) ranges from 53%-69% (Gerba et al., 1996). 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 (Sabalos, 1998;
NRC, 2002), 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). Little more is known about removal 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 still 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, is transmitted by food and
water and primarily infects the liver. The highest infection rate is among children 5 to 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.
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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
in frequent 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 to 3% in the general population and 20 to 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 by risk assessors of
pathogens in biosolids. 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 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 protozoan parasites
transmitted through food and water in the U.S. that cause diarrhea. These parasites of
the small intestine have environmentally resistant stages called cysts or oocysts.
Pepper et al. (2006) review studies in which Cryptosporidium and Giardia have been
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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. 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). Cyclospora causes diarrhea, nausea, vomiting and
abdominal cramps. Toxoplasma gondii causes neurologic flu-like symptoms, retinitis
and severe disfunction in fetuses if mothers are infected for the first time while pregnant.
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
protozoa (Cryptosporidium, Cyclospora, Toxoplasma) probably react similarly (but
sometimes uncertainly) to the effects of different disinfection methods but settle at
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different rates. Microsporidia have not been studied much in the context of biosolids
treatment (Bowman and Fayer, 2005).
2.2.3.1.	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).
2.2.3.2.	Trichuris trichuria
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 usual quantities of ultraviolet,
ozone, or chlorination disinfection methods.
2.2.3.3.	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 infected
eggs. The eggs of Ascaris were chosen as an indicator organism in biosolids because
of their resistance to most treatment processes and representativeness of helminth egg
viability.
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2.2.3.4. 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 and Taenia
saginata, the final host is a human or pig, and the intermediate host is a pig or cow,
respectively. The adults cause little effect in humans, but eggs can cause enteric
distress. Although Taenia species are usually acquired from ingestion of beef, the eggs
of this pathogen have been detected in some biosolids (Barbieret al., 1990).
2.2.4. 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
phosphorolated diglucosamine structure (Epstein, 2006). They may become airborne
when dried, pulverized to micron and submicron 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 air at composting plants, though there was
no evidence of residential impact 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
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manures and composts (Brooks et al., 2006). Farming activities, such as driving a
tractor across a field, result in comparable levels of aerosolized endotoxins as those
from land application of biosolids (Brooks et al., 2004). Low concentrations of
endotoxins were present in groundwater at two sites where wastewater was applied to
land (Yanko, 2005).
2.2.5. 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: 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 pathogens that have only
recently been considered stressors of concern in particular media. Gerba et al. (2002)
designated E. coli 0157:H7, H. pylori and L. montogenes 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 bacterial pathogens likely to be present in biosolids and Adenovirus, Norwalk
virus, Astrovirus, Hepatitis A, Rotavirus and Hepatitis E as emerging viral pathogens
likely to be present (NRC, 2002). Gerba (2005) listed several emerging viruses without
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speculating which are likely to be in biosolids: picobirnaviruses, picotrinaviruses,
coronaviruses and toroviruses.
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, suggested by C. Gerba of the
University of Arizona, are useful for selecting pathogens on which to focus the stressor
characterization in a risk assessment.
•	Reliable viability assay
•	Wastewater-related disease-causing agents
•	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. The
committee would have selected caliciviruses as a priority, but methods of assessing
viability are not available (NRC, 2002). Similarly, Legionella merits investigation, but
current detection methods are inefficient, difficult to use and expensive (NRC, 2002).
2.2.6. Multiple Stressors
It may be reasonable to assume that microbial pathogens act independently of
each other and that the probability of an adverse effect from one pathogen is
independent of the probability of an adverse effect from another. However, assessors
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1	of cumulative risks should consider exposures to offsite pathogens in biosolids or other
2	sources that are not the direct subject of a biosolids risk assessment.
3	There is no evidence to suggest that pathogens and chemicals such as metals in
4	biosolids have interactive effects in humans. However, Lewis et al. (2002) speculated
5	that chemical contaminants in biosolids might irritate the skin and mucous membranes
6	and thus increase pathogen host susceptibility.
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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 stressors 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 are much
more common in ecological risk assessment than in human health risk assessment, and
conceptual models for human health risk assessments of pathogens in biosolids that
include detailed source descriptions, transport pathways and routes of exposure have
not been developed previously.
In this report we develop 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
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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 advice concerning 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.
Some of the primary differences between conceptual models for pathogen risk
assessments and conceptual models for chemical risk assessments are that: (a) some
microorganisms can reproduce in the environment, (b) host factors such as individual
immunity and genetic factors influence disease and (c) infection may occur via person-
to-person transmission (Soller et al., 2006), though that transmission pathway is not
treated here.
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 biosolids. A causal association between exposures to biosolids and
adverse effects on human health has not been documented.
In this chapter we first present a general conceptual model for risks from
pathogens in land-applied biosolids (Figure 1), 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 stressors potentially contact human hosts to
produce infection and disease. We describe the source (methods and rates of land
application), environmental fate and transport processes, routes of exposure, host
susceptibility factors, infection and disease. Then we describe five exposure scenarios,
along with related generic conceptual models, that are of interest
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Pathogens in
sewage sludge
-*(j reatment^-
Pathogens in Class B Biosolids
Storage
Transport
3Z
Loading,
unloading
Application -
Spreading or
Spraying
\
Application ¦
Injection
-W Aerosolization
/
2
v
Pathogens in
bioaerosols
Pathogens in surface
applied biosolids
Incorporation
in soil


Pathogens in biosolids
below surface
Pathogens in
biosolids-soil mixture
Deposition
Surface runoff
and erosion
vector
transport
Leaching
Pathogens on
food
Pathogens in
surface water
Irrigation >4
A
Water flow
Dermal
Exposure
nha ation
Ingestion
Pathogens in
groundwater
(
Human Infection
v
t—
f \
Disease

FIGURE 1
General Conceptual Model
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for assessing risks from the land application of biosolids. The generic conceptual
models presented here may be modified as more knowledge is available on a case-by-
case basis.
The model contains routes of exposure that are considered to be potentially
significant in many instances. However, some additional routes may be considered
when there is a particular concern. For example, indirect routes involving human
consumption of livestock, dairy products, wildlife, fish or shell fish that are exposed to
pathogens from biosolids were not included as too indirect and hypothetical. However,
such routes should be considered if they are an important issue for stakeholders at a
site.
Site-specific conceptual models that make use of these generic models would be
needed for site-specific risk assessments. Site-specific conceptual models can be
generated from these generic models by eliminating routes that are impossible or highly
improbable at the site, adding routes that are peculiar to the site and adding details. In
the next chapter, we screen out pathways that usually contribute negligible human
exposures to biosolids-derived pathogens.
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 (Figure 1). Additional human processes in the conceptual
model include storage, transport within a site, loading and unloading and land
application (Figure 1).
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Biosolids storage, transport within a site and loading and unloading processes
are included in the general conceptual model because these processes have been
observed to generate bioaerosols ([Pillai, 2007; Paez-Rubio et al., 2007], Figure 1).
Biosolids are stored during winter, inclement weather, periods of equipment breakdown,
or crop growth periods (Evanylo, 1999). Regulations may specify the type of storage
facility for long-term storage, and this problem formulation assumes that a barrier is
present to prevent erosion of biosolids or surface runoff or leaching of pathogens.
Thus, there is no arrow between storage and surface runoff and erosion or leaching in
Figure 1. However, if risk assessors determine that leaks of biosolids or pathogens
from storage facilities are feasible, then additional pathways from the storage facility
must be included in the conceptual model. Dewatered biosolids are stockpiled, and
liquid biosolids may be stored in digesters, tanks, lagoons or drying beds (Evanylo,
1999).
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 and the availability of application equipment (NRC, 2002; University of
Washington, 2002), and state or local regulations (e.g., Solano County, California
requires incorporation of biosolids into soil). The application method is an important
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determinant of bioaerosol generation, chemical odor and ultraviolet inactivation of
pathogens (NRC, 2002).
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 to 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 preventing aerosolization of
biosolids (Figure 1). 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 (Figure 1). Injection can be used on slopes up to 15
percent (Evanylo, 1999), dependent on state or local laws. This application method
serves as a physical barrier that satisfies vector-control requirements (NRC, 2002).
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 (NRC, 2002;
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, 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
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survive in fairly anaerobic conditions (University of Washington, 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 permits 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
to 9 inches (15 to 23 cm) may follow surface application (NRC, 2002) and partial drying
(Evanylo, 1999). The method is usually used before planting or after harvest (NRC,
2002). Surface application with incorporation is generally limited to soils with less than
a 7 percent slope (Evanylo, 1999), additional state and local laws notwithstanding.
Incorporation serves as a physical barrier that satisfies vector-control requirements
(NRC, 2002).
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).
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3.2.2. Rates of Land Application of Biosolids
Biosolids are applied at a rate equal to or less than the agronomic rate (nitrogen
needed by 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). U.S. 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.
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 U.S. EPA (1992).
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3.2.3.	Timing of Land Application of Biosolids
The timing of land application of biosolids is another factor that determines
exposure. In agricultural operations, application is scheduled around tillage, planting
and harvesting and is also influenced by properties of crops, climate and soil factors
(Evanylo, 1999). Most applications are performed when plants are ready to use the
nitrogen in biosolids so as to minimize leaching to groundwater (Evanylo, 1999). The
State of Virginia recommends that biosolids applied to land between fall and spring
have a vegetation cover to minimize runoff of pathogens and nutrients and 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 avoid periods of rain, because
vehicles may compact or create ruts in soils that reduce crop yields (Evanylo, 1999).
Although rain is avoided during application of biosolids, we have found no
evidence that heavy winds are similarly avoided. Meteorology should be considered in
the modeling of transport of 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 relation to human populations. A few
regional differences in application methods and timing are described above. Climatic
differences contribute to differences in fate and transport of pathogens in biosolids and
biosolids-amended soil. Pathogen survival tends to be highest in cool, moist soils, such
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as those in the northeastern U.S. Hot, dry soils as in the southwestern U.S. contribute
to pathogen mortality (see section below on fate and transport of pathogens).
Differences in rainfall are 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).
The number of people potentially affected by pathogens in biosolids also varies
regionally. Potential exposure increases as the density of people increases because (1)
greater sewage sludge output leads to a greater need to find land application sites and
to apply biosolids at higher rates and (2) the greater density of people means more
residents and children potentially exposed near their homes and schools. In the arid
southwestern U.S., farms are often located far from cities, so fewer residents would be
expected to be exposed to pathogens in biosolids (NRC, 2002).
3.3. FATE AND TRANSPORT OF PATHOGENS
3.3.1. Pathogen Survival, Growth and Death
As stated in the stressor characterization chapter, unlike chemical stressors,
biological stressors have the potential to reproduce or to die. Thus, conceptual models
need to consider factors affecting survival and growth in biosolids, biosolids-amended
soils and bioaerosols (Figure 2). The environmental factors affecting 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 non-
spore-formers and relatively sensitive to environmental factors such temperature,
desiccation and ultraviolet exposure. Although Salmonella, E. coli and fecal coliforms
are capable of regrowth in moist conditions following treatment, regrowth is typically
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Aerosolization
i l
Survival and
reproduction
Survival and
reproduction
/	pH'	\
/	Moisture,	>
Ultraviolet light,
Temperature (e.g., freezing, heating)
Particle size (e.g., clay content),
\ Indigenous microflora, j
\	Desiccation	r
k i
Pathogen
abundance in
bioaerosols
Pathogen
abundance in
site media
Pathogen abundance in
applied/incorporated
biosolids
1
2
3
4
5
6
FIGURE 2
Pathogen Fate Conceptual Model
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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 dessication increasing
-
-
-
Clay content increasing
+
+
Not known
pH range of 6-8
+
+
+
2
3	Sources: NRC (2002) Pepper et al. (2006).
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limited to Class A biosolids where biological competition is low compared to Class B
biosolids (Pepper et al., 2006).
Pathogen survival and reproduction are depicted in Figure 2. Temperature and
moisture are the primary variables affecting survival of enteric 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.
3.3.2. Pathogen Transport
Pathogens may be transported from biosolids to various media. In addition to the
application process, storage, site-to-site transportation and loading and unloading are
human processes that could mobilize pathogens for transport (Figure 1). Several
mechanisms of transport are possible: aerosolization followed by aerial transport and
deposition, erosion, surface runoff and leaching to groundwater (Figure 1).
3.3.2.1. Aerial Transport
Land application of biosolids can generate bioaerosols either through agitation
during application or following a series of weathering events of deposited biosolids in
association with specific climatic conditions (see stressor characterization). Biosolids
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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 are generated at the site of biosolids application,
storage, site-to-site transport and loading and unloading processes, including shoveling
biosolids from pile to pile (Straub et al., 1993; Pillai, 2007, Figure 1). Bioaerosols are
potentially transported to downwind locations. Wind can resuspend biosolids that have
been previously applied to the soil surface through the wind erosion process in Figure 1.
Injection is a barrier to aerosolization of biosolids (Smith et al., 2005a, Figure 1).
The disking process, marked as "incorporation in soil" on Figure 1, 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
spreading of dewatered biosolids by side slinger or spraying of 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). 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
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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.
3.3.2.2.	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
biosolids were applied as well as those of adjacent lands. 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 cropped soils. The NRC noted that U.S.
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.
3.3.2.3.	Erosion to Surface Water
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
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potential for erosion and runoff but buries the amendment and dilutes the initial numbers
of pathogens. However, we have found no studies of microbial contamination of
surface water where biosolids have been applied.
3.3.2.4.	Leaching to Groundwater
Following precipitation, microorganisms may infiltrate soil to contaminate
groundwater (Straub et al., 1993). The NRC noted that U.S. 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 adhesion processes, filtration effects, physiological state of
the cells, soil characteristics, water flow rates, predation, intrinsic cell mobility and
presence of biosolids (NRC, 2002). Viruses have a greater potential to be transported
to groundwater than other pathogens, though sorption to colloids and biosolids particles
limits this potential (NRC, 2002). Transport of larger organisms (bacteria, protozoa,
helminths) is less likely but possible if preferential flow occurs through cracks and
macropores (NRC, 2002). Transport of pathogens to groundwater is most likely where
soils are sandy and coarse-textured or where karst topography is present (NRC, 2002).
However, we have found no studies of microbial contamination of groundwater where
biosolids have been applied.
3.3.2.5.	Sorption to Crops
Pathogens from biosolids could become sorbed to root crops with particles from
the biosolids-soil mixture (Figure 1). Although crops are generally washed before
eating, a fraction of biosolids-amended soil will remain sorbed to the crop (estimated at
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10% by Gale [2005b]). This pathway is likely the dominant route to crops. 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 rainsplash (Figure 1). Leaf or root crops could become
contaminated with pathogens via irrigation with contaminated surface water or
groundwater (Figure 1).
3.3.3. Vector Transport
Vector transport of pathogens from biosolids is possible. For example, flies
might become contaminated, leaving trace pathogens on food that is ingested by
humans. This potential pathway is included in the general conceptual model (Figure 1).
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 (NRC, 2002). Similarly, it is unclear whether vectors are involved in the
transmission of pathogens to humans from biosolids (NRC, 2002).
3.4. HUMAN ROUTES OF EXPOSURE
Potential routes of exposure to pathogens originating in biosolids include
ingestion, inhalation and dermal exposure (Figure 1). Whereas all of these routes are
feasible, none has been implicated in disease. Risk assessors should consider all of
these potential routes, unless fewer routes are specified in a scenario of interest.
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3.4.1.	Inhalation
The route of exposure of humans to aerosolized pathogens is uncertain,
involving a combination of inhalation and ingestion (Pillai, 2007, Figure 1). Large
aerosolized particles (between 5 and 20 |jm) can deposit in the upper respiratory tract.
Clearance of these particles results in oral exposures. Smaller particles penetrate deep
into the lungs, with many retained by the alveoli (Pillai, 2007). Thus, 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 to
aerosolized pathogens that may result in adverse health effects.
The NRC (2002) determined that the inhalation pathway was among the routes
of exposure that was not adequately assessed by U.S. EPA in the development of the
Part 503 rule. They noted that inhalation of dust was presumed by U.S. EPA to occur
only on-site and that controlling site access was thought to prevent that route of
exposure (NRC, 2002). We did not locate studies of inhalation of biosolids-derived
aerosols or pathogens by off-site residents. Thus, inhalation of pathogens by off-site
residents needs more consideration.
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
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pathogens that originated in biosolids; ingestion of pathogens which are sorbed to crops
and food items after application of biosolids in agricultural fields; incidental ingestion
pathogens associated with surface-applied biosolids and biosolids mixed with soil, and
ingestion of bioaerosols containing pathogens (Figure 1).
Ingestion of biosolids in soil occurs through the transfer of pathogens to the
mouth from contaminated hands or crops and or though inhalation followed by
swallowing (Gerba et al., 2002, Figure 1). 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 organisms that are ingested (Pillai, 2007).
Ingestion of groundwater or surface water is a potential route of exposure to
biosolids-derived pathogens (see scenario descriptions below). Untreated surface
water contaminated with pathogens from biosolids might be ingested while swimming,
potentially allowing for greater consumption of pathogens than domestic consumption
from a tap.
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, leaves),
particularly the edible portion of the plant, and consumption by individuals.
Three exposure scenarios may result in ingestion of pathogens associated with
biosolids when applied in crop settings. The exposure scenarios differ with respect to
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the portion of the plant that is intended for consumption. The first scenario involves the
deposition of aerosolized material on the surface of the aboveground portions of the
plant (Figure 1). 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 (Figure 1). 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 on crops an
unlikely exposure pathway for farm residents (see section on regulatory restrictions,
below). Part 503 regulations provide for time restrictions between application to the
field and harvesting of plants. However, harvesting of plants in nearby fields where
pathogen deposition from the air or runoff may have occurred 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 of the pathogens. Moreover, the types
of foods that may be affected by deposition of aerosolized material are grains and some
vegetables which normally undergo preparation to reduce pathogen viability prior to
consumption. Although this scenario might constitute a minor pathway, it should be
considered in the problem formulation.
A second exposure scenario 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
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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 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.
A third scenario applies to crops that have the palatable portion below the soil
surface. An example is tubers; crops for which the roots serve as the consumable
portion of the plant, 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 the pathogens on
the plant surface. 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 planting, harvesting and consumption
of food grown on land receiving Class B biosolids. 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
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38 months for crops whose harvested portion is below ground; 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, can be consumed.
3.4.3. Dermal Exposure
Dermal contact constitutes a direct method of transfer of pathogens in biosolids
to receptors (Figure 1). Dermal exposure to pathogens would occur primarily through
skin abrasions, either through contact with contaminated soil or surface water.
Dermal contact may occur during occupational exposure or during unintended
contact with biosolids that have moved from the site of application (e.g., through aerial
dispersion or runoff). Workers will most likely come in contact with biosolids during
processing, loading and application which can lead to penetration of the pathogens
through skin or existing cuts or abrasions. However, this problem formulation is
concerned with residents and other community receptors rather than workers (Figure 1).
A possible exposure scenario may occur as the result of recreation during the
summer months. For example, swimming in surface waters would permit dermal
contact with pathogens, as well as ingestion or inhalation.
To assess dermal exposures, the risk assessor would need information on the
amount of material adhering to 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.
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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 (Table 5). These
restrictions affect the credibility of exposure pathways in the conceptual model. Time
intervals required prior to site access are summarized in Table 6. Particular states may
have regulatory criteria for distances to 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 (Figure 3).
3.6.1. Human Factors
The three host factors that are discussed in NRC (2002) are concomitant
exposures, genetic factors and acquired immunity. Age is an additional determinant of
susceptibility.
3.6.1.1. Concomitant Exposures
Various stressors 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 (Figure 3). Synergistic effects might
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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 applied15
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 with 10
meters of any waters 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
2
3	aPublic-access restrictions do not apply to farm workers. If there is low probability of public
4	exposure to an application site, the public-access restrictions apply for only 30 days.
5	However, application sites that are likely to be accessed by the public, such as ballfields, are
6	subject to 1-year public-access restrictions.
7	Agricultural land is private property and not considered to have a high potential for public
8	access. Nonetheless, public-access restrictions are applied.
9	Taken from NRC (2002), which adapted the table from U.S. EPA (1999).
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1
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
2
3	*The 20-month interval prior to harvesting applies if the biosolids stay on the surface for
4	4 months or longer prior to incorporation. The 30-month interval applies if the
5	biosolids stay on the surface for less than 4 months prior to incorporation.
6	Modified from: NRC (2002) and 40 CFR Part 503.
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Infectivity
Genetic
or Acquired
Immunity
Genetic
factors
Human nfection
Susceptibility
to pathogens
Virulence
Pathogens,
non infectious organisms
endotoxins,
irritants,
odors
r

\

Disease



V
2
3
4	FIGURE 3
5
6	Disease Factors Conceptual Model
7
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result from combined exposures to these stressors (NRC, 2002, Figure 3). 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 (Figure 3). Genetic factors such as a predisposition to asthma attacks can
be a factor in determining whether infection proceeds to disease. 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 3. 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 stressors (Figure 3).
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,
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hereditary diseases, etc.) or any other site-specific health characteristic of the
potentially exposed population that warrants special consideration.
3.6.4. Pathogen Factors
Infectivity and virulence are two pathogen factors that can also influence infection
and disease (Figure 3). Infectivity is the relationship between the quantity of pathogens
ingested or inhaled or in contact with skin and the probability of infection. There is
probably no minimal infectious dose for enteric pathogens (Haas et al., 1999, also see
Analysis Plan chapter). Virulence is a measure of the severity of the disease that the
pathogen is capable of causing.
3.7. INFECTION AND DISEASE
Two primary, broad endpoints of risk assessments for pathogens in land-applied
biosolids are human infection and disease (Figure 1). Infection is the process by which
a microorganism multiplies or grows in or on the host. Clinical diseases are evidenced
by signs or symptoms.
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. The protozoans of
concern Giardia, Cryptosporidium and Entamoeba, produce cysts and oocysts which
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have been shown to be environmentally stable and somewhat resistant to disinfectants.
Thus, they are recognized as significant human pathogens with the potential to cause
gastrointestinal illness exhibited by diarrhea, dehydration and weight loss. Potential
effects of particular pathogens in biosolids are described in the stressor characterization
chapter.
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 (morbidity). Mortality is an additional, potential endpoint. Severity
(e.g., number of days lost to illness) may be another property of disease that is of
interest to the risk assessor.
3.8. SCENARIOS
Risk assessors may describe scenarios that do not include all of the pathways in
Figure 1. We consider five example exposure scenarios that represent common public
concerns, and we present conceptual models for each. These do not include
occupational scenarios, which are under the purview of the Occupational Safety and
Health Administration. The scenarios considered here include:
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.	Pica child playing on a site recently applied with 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); and
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5. Drinking water consumers of surface waters downstream from sites where
biosolids are applied.
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 generic conceptual model for this scenario (Figure 4)
adapts most of the pathways from the general conceptual model (Figure 1). The
primary source processes that do not appear in this scenario are storage, transport and
loading and unloading activities (Figure 4). 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.
3.8.2.	Scenario 2. Residents
Individuals potentially exposed to biosolids-derived pathogens may reside on
farms where biosolids are applied. The generic conceptual model for this scenario
(Figure 5) adapts all of the potential pathways from the general conceptual model
(Figure 1). 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.
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Pathogens in Class B Biosolids

Application ¦
Spreading or
Spraying
Aerosolization
Pathogens in
bioaerosols
Incorporation
in soil
Application ¦
Injection
Pathogens in surface
applied biosolids


Pathogens in biosolids
below surface
Pathogens in
biosolids-soil mixture
Deposition
Surface runoff
and erosion
Vector
transport
Leaching
Pathogens on
food
Pathogens in
surface water
Irrigation
7-
Pathogens in
groundwater
Water flow
Dermal
Exposure
Inhalation
Ingestion
/ ¦>

r


Human Infection


Disease






V

y
1
2
3
4
5
FIGURE 4
Adjacent Property Conceptual Model
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Pathogens in Class B Biosolids
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
Dermal
Exposure
Inhalation
Application -
Spreading or
Spraying
Loading,
unloading
Application
Injection
Storage
Transport
W Aerosolization
Incorporation
in soil
Deposition
Surface runoff
and erosion
Wind
Erosion
Vector
transport
Leaching
Water flow
Human Infection
Disease
1
2	FIGURE 5
3
4	Resident Conceptual Model
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3.8.3. Scenario 3. Pica Child
Soil ingestion is the consumption of soil as the result of various behaviors such
as visiting treated fields and forests and consuming soil directly and indirect exposure
from 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 to 5 grams per day). Groups at risk of soil-pica behavior are generally
children aged 6 years and younger. Noting that soil ingestion is a normal behavior
among children, evaluation of all types of soil ingestion is included in the soil-pica
scenario (Figure 6).
Incorporation
in soil
Pathogens in surface
applied biosolids
Pathogens in
biosolids-soil mixture
Pathogens in Class B Biosolids
N	/
^ /
Human Infection
Ingestion
Disease
FIGURE 6
Pica Child Conceptual Model
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3.8.4.	Scenario 4. Drinking Water Consumers of Groundwater
Leaching to groundwater is of potential concern following injection of biosolids in
the subsurface or following surface application to porous soils overlying an aquifer or
well. Most drinking water aquifers contain geologic water but may be recharged
following significant precipitation. Soils that are uniformly porous throughout the profile
permit movement of water to aquifers or wells. Studies conducted on porous soils have
demonstrated that pathogens in water can move with the liquid through soil horizons.
Aquifers serve as the sole source of water in many communities and therefore may be
used for both farming and domestic purposes. As such, the water may be consumed,
used in food preparation (either during washing or cooking, the latter would account for
significant reduction or elimination of most pathogens), bathing and other household
activities. This scenario emphasizes groundwater consumption (Figure 7).
3.8.5.	Scenario 5. Drinking Water Consumers of Surface Water
The use of downgradient surface waters as a source of potable water may result
in exposure to biosolids-related pathogens (Figure 8). The major pathways of potential
exposure to pathogens would be erosion of biosolids particles and surface runoff from
treatment sites (Figure 8). Additionally, pathogens might be carried to surface water in
groundwater, and small quantities of pathogens might deposit to surface waters
following aerial transport. Treatment of water before consumption greatly reduces the
potential for exposure.
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Pathogens in Class B Biosolids

Application -
Spreading or
Spraying

Application
Injection
Incorporation
in soil
Pathogens in
biosolids-soil mixture


Pathogens in biosolids
below surface
Leaching
~1
Pathogens in
groundwater
Ingestion




N
Human Infection


Disease

v J

v
y
FIGURE 7
Groundwater Conceptual Model
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Pathogens in Class B Biosolids
Application -
Spreading or
Spraying
Application -
Injection
Aerosolization
Pathogens in
bioaerosols
Pathogens in surface
applied biosolids
Incorporation
in soil
Pathogens in biosolids
below surface
Pathogens in
biosolids-soil mixture
Deposition
Surface runoff
and erosion
Wind
Erosion
Leaching
Irrigation
Pathogens in
surface water
Pathogens in
groundwater
Water flow
v
Ingestion
Human Infection
Disease
1
2	FIGURE 8
3
4	Surface Water Conceptual Model
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1	3.8.6. Regional Aspects of Scenarios
2	These scenarios and others may occur in various regions. Surface water
3	drinking scenarios would be less applicable to arid regions. Scenarios involving
4	aerosolization of pathogens in biosolids would be more applicable to windy regions.
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4. SCREENING OUT ELEMENTS OF THE CONCEPTUAL MODEL
In this chapter we examine the general conceptual model (Figure 1) to determine
if sufficient information is available to screen out unlikely stressors, scenarios, routes of
exposure, or endpoints from consideration in risk assessments of pathogens in
biosolids. This effort 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 that would allow us to compare directly the
relative importance of different exposure pathways. Academic studies tend to
emphasize a single exposure pathway rather than a comparison of multiple pathways.
However, our reading of the literature (see literature review, Appendix A) suggests that
certain pathogens and exposure pathways may tend to be unimportant. However,
insufficient evidence exists to support broad generalizations about negligible elements
at this time.
Will this caveat in mind, risk assessors may find it easier to screen out some of
the following stressors in site-specific risk assessments:
•	Endotoxin. Brooks et al. (2007) 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 U.S. found that S.
aureus was detected in raw sewage samples but not in biosolids (Rusin et al.,
2003a).
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1	• Certain protozoa. Gerba et al. (2002) determined that microsporidia and
2	Cyclospora would not be likely to survive under high temperatures of anaerobic
3	digestion or under conditions of low moisture in Class B biosolids treatment.
4	• Certain bacterial or viral pathogens in bioaerosols. Pathogens and indicator
5	bacteria were only rarely found in aerosolized samples in a study of land
6	application of biosolids in Tucson, AZ. These included coliforms and coliphages,
7	which were present at high densities in biosolids. The authors suggested that
8	only microorganisms in the aqueous phase of biosolids were able to aerosolize;
9	others remained sorbed to the solid phase (Brooks et al., 2004). Furthermore,
10	Tanner et al. (2005) determined bioaerosol emission rates and plume
11	characteristics during spray application of liquid Class B biosolids. They did not
12	detect coliphages or coliform bacteria just downwind of the biosolids application,
13	though pathogens sprayed in inoculated groundwater were detected. The
14	researchers concluded that the presence of biosolids reduces aerosolization of
15	microorganisms relative to application of inoculated groundwater. The duration
16	of exposure to any pathogens (below detection limits) downwind of biosolids
17	application is brief (Tanner et al., 2005).
18
19	Brooks et al. (2005b) undertook a study to estimate risks of microbial infection of
20	residents near biosolids application sites. At 10 sites (five in Arizona, five elsewhere in
21	the U.S.) amended with either liquid or solid Class B biosolids, they measured
22	heterotrophic plate counts (HPC) bacteria, total coliform bacteria, E. coli, Clostridium
23	perfringens, coliphage, enteroviruses, hepatitis A virus and norovirus in aerosol samples
24	downwind from application sites. The study distinguished between loading, unloading,
25	land application and background operations. In general, risks of infection were
26	determined to be low, with the greatest risks, that of infection by coxsackievirus A21
27	from loading operations having a 4 * 10"4 chance of infection. Based on this work,
28	Pepper et al. (2006) concluded that the overall community risk of infection from
29	bioaerosols during land application was relatively negligible.
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1
Some evidence (below) might support a decision to screen out certain exposure
2	pathways (Figure 1) from general or regional consideration in the future. However,
3	more evidence is needed to support such a judgment.
4
5	• Groundwater pathway. Because of the large size of bacteria, soil (especially
6	fine-textured soil) can act as a filter to limit bacterial transport (NRC, 2002). Soil
7	would also be expected to limit the transport of larger protozoa and helminths
8	(NRC, 2002). A review of the literature has concluded that few pathogens (even
9	viruses) from biosolids leach to groundwater (Pepper et al., 2006). Although
10	Gerba (2005) acknowledges that of the pathogens in biosolids, viruses have the
11	greatest potential for contamination of groundwater, Pepper et al. (2006)
12	concluded that "groundwater contamination from land-applied biosolids does not
13	appear to be likely." Sandy soils with low cation exchange capacity deserve
14	more study.
15	• Root crop ingestion pathway. A United Kingdom study of infection from
16	consumption of root crops grown on biosolids-amended soils found that risks to
17	humans was low. Seven pathogens were included in the study: salmonellas,
18	Listeria monocytogenes, Campylobacters, Escherichi coli 0157, Cryptosporidium
19	parvum, Giardia and enteroviruses (Gale, 2005b). United Kingdom biosolids
20	may not be comparable to Class B biosolids in the U.S.
21
22	Regulations might also allow a risk assessor to screen out potential pathways of
23	exposure in the general case. For example, if biosolids must be stored in enclosed
24	facilities, the generation of bioaerosols from that source (and exposure to neighboring
25	residents) would not be likely.
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5. 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 analysis phase of the risk
assessment, i.e., the characterization of exposure and the characterization of effects.
Methods are described to characterize the source, pathways, environmental media and
human endpoints. The emphasis is on 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). The rationale for selecting or eliminating risk
hypotheses is set forth (U.S. EPA, 1998). An analysis plan for a risk assessment of
pathogens in biosolids must be designed to eliminate negligible pathways in the
conceptual model. Available data are described, as well as new data that should be
collected to conduct the risk assessment and the feasibility of their collection. The
analysis plan describes both measurements and models. The plan also describes
where parameters of interest may be extrapolated from existing data. Extrapolation
allows the use of data collected from other locations or for other microbial pathogens
where similar problems exist.
This chapter is structured as an analysis plan might be structured for a risk
assessment on land-applied biosolids. Following the introduction, we discuss
management needs, including parameters requiring estimation and data quality
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objectives. Then we discuss the plan for the characterization of exposure, including the
selection of measures of exposure, the detection of microbes, the issue of background
levels of pathogens and the estimation of fate, transport, uptake and dosage. 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 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 of the dose-response and epidemiological
information is deemphasized. Furthermore, because of the numerous research gaps,
we identify research, observational studies and methods development that should be
performed to complete a defensible risk assessment to support regulatory actions.
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 mangers 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.
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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. Many risk assessments for pathogens in biosolids will be conducted by
U.S. EPA's Office of Water, and therefore, risk managers from this office will determine
the appropriate assessment endpoints. These may include population-level endpoints
or individual-level endpoints. 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 U.S. 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 validate the current operational standards and
management practices.
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5.2.2. Data and Data Quality
U.S. EPA (1998) recommends that risk assessors ask 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 uncertainties are 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
quality, and identifying the means of obtaining this information from risk managers (U.S.
EPA, 1994). These steps are described in Text Box 1.
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. Measures of exposure are measures of stressor existence and
movement in the environment and their contact or co-occurrence with the assessment
endpoint entity. More specifically, in a human health risk assessment these are
measurable characteristics of pathogens that are used to quantify exposure of humans
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or contact with particular organ
systems. Measures of
exposure include
concentrations of particular
pathogens in environmental
media or components of these
media (biosolids, biosolids-
amended soil, air, water, clay,
aerosols). 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.
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 biosolids. 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, will 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.
5.3.2. Detection of Pathogens
Following the selection of measures of exposure, the detection of pathogens is
the first type of analysis required in the analysis plan. As stated in the literature review
(Appendix A), 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 analytical methods are also needed for detecting and quantifying particular
pathogens in sewage sludge and biosolids. This information is needed to support
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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 current option for national scale risk assessments is to
conduct analysis of pathogens in biosolids at several application sites that are thought
to be representative of such sites across the country.
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
selective culturing or polymerase chain reaction (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) (see section on 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
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efficiency of 8.7% for Staphylococcus aureus in Class B biosolids. U.S. EPA has new
standardized analytical methods for fecal coliforms and Salmonella (FR 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. Of the viral pathogens listed in the stressor characterization chapter,
astroviruses, rotaviruses, hepatitis A and E and adenoviruses can be cell-cultured,
whereas human caliciviruses cannot (NRC, 2002). 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 non-
enteroviruses in raw sewage sludge and treated biosolids (Smith et al., 2005b, Chapter
8). And risk assessors should indicate in the analysis plan that risks from caliciviruses
cannot be determined at this time. 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.
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. Integrated cell-culture PCR (ICC-PCR) amplifies viruses in
cell culture and amplifies viral RNA through enzymatic PCR. ICC-PCR is the
recommended method for viral risk assessment because of the potential for cell culture
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alone to underestimate human exposure and for RT-PCR to overestimate exposure
(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
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. Recoveries from biosolids are low, e.g., 10% for
the sedimentation technique, less than 3% for the flotation technique, 3.2-16.3% for
Cryptosporidium oocysts and 2.4-41.7% for Giardia cysts (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 typically non-pathogenic 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). 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. However, this
estimate is not helpful for pathogen-specific risk assessments, because the identity of
the pathogen is an important determinant of risk.
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 provide references
suggesting that its spores might be a surrogate for eggs of Ascaris suum 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
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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 Bactericides 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. Because somatic coliphage infects strains of E. coli, it can be detected using
simple, inexpensive methods (NRC, 2002). Lime is also included as a potential
indicator of post-treatment risk for 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.
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
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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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perfringens are very common in soil. The risk assessment is only concerned with the
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 may be significant contributors to risk. For
example, in a study of aerosolized endotoxin concentrations downwind from a biosolids-
amended site, Brooks et al. (2006) found that 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.
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 (Figure 1) and the death or multiplication of pathogens (Figure 2). Therefore,
the analysis plan may include a plan for estimating pathogen fate. Most models 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). Straub et al.
(1993) reviewed available studies of survival of pathogens in soil and sewage sludge
that are pertinent to this analysis plan discussion. Gerba and Smith (2005) provide
survival times of pathogens on soil and plants (Table 8).
Risk assessors should not use survivorship data from enteric organisms such as
E. coli and Salmonella to estimate the much longer survival rates of bacterial pathogens
that form spores or are encapsulated (such as Mycobacterium spp.).
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TABLE 8
Survival Times of Pathogens in Soil and on Plants
Modified from Gerba and Smith (2005)
Pathogen
Soil
Plants
Absolute
Maximum
Typical
Maximum
Absolute
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
5.3.6. Transport of Pathogens
The conceptual model in Figure 1 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. 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 have some limitations.
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
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(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.
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. BASINS (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.
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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 is
controlled 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 and is a
function of the 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 would 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 must understand the release
rates of the different microbes, the dispersion of the bioaerosols and the deposition of
the microorganisms (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
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analysis plan may 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, as they did when culture methods were
required for identification (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, and some of the indoor samplers
that rely on external vacuum and power sources cannot be carried to remote sites
(NRC, 2002). Insufficient testing of available methods has occurred to recommend a
particular sampling method for bacteria in bioaerosols, but we recommend that
assessors describe methods for testing sampling efficiencies of their equipment in the
analysis plan. Risk assessors should also be aware that during transport, deposition
and sampling, bacteria 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,
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vegetation and mechanical agitation (NRC, 2002). Wind direction and speed may vary
during the sampling time. The orifices of bioaerosol samplers downwind may be too
small to obtain detectable levels of bacteria, even if they are present in bioaerosols.
Thus, appropriate statistical analysis (Spicer and Gangloff, 2000) and appropriate
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., 2005a). "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).
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
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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
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.,
2004). 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]).
Complicating factors include variation in terrain, topography, vegetation,
micrometeorological conditions, biosolid composition and biosolids land application
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processes (Pillai, 2007). 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 the use of particular models in the analysis plan.
5.3.7. Contact with Crops
Pathogen residues on root and leaf crops can be measured. Biosolids and
associated pathogens can deposit to crop leaves following erosion, aerial transport or
rainsplash, and these processes can be modeled. Because of the ease of
measurement and uncertainty of modeling, we recommend that pathogens on select
crops be measured. 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
indicate that the arithmetic mean exposure concentration (if used) may give a
conservative estimate of the number of people exposed.
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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
U.S. 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 the Exposure Factors Handbook (U.S. EPA, 1997), 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 U.S. 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
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aerosols. Pepper et al. (2006) describe studies that use a factor of 10%, and Brooks et
al. (2005b) uses 50%. Haas et al. (1999) recommend exposure factors that are relevant
to risk assessments for pathogens. While many of these factors are analogous to those
in U.S. 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 are adapted here for pathogens in biosolids.
•	Corresponds to an assessment endpoint
•	Relates to the human health endpoint in a quantifiable manner
•	Makes use of existing data
•	Is readily measured
•	Is of appropriate temporal and spatial scale
•	Is appropriate to the exposure pathway
•	Is appropriate to the mode of action
•	Is diagnostic of particular pathogens
•	Shows low variability, increasing the likelihood of detecting an effect
•	Is broadly applicable to different locations
•	Is a standard test or measurement method
The first two considerations are necessary to meet the definition of a measure of effect.
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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.
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 in the literature review (Appendix A), a causal association between
exposures to pathogens in biosolids and adverse effects on human health has not been
documented. Risk assessors should examine relevant data (and perhaps conduct
epidemiological studies) supporting or refuting a cause-and-effect relationship. This is
most important in locations where biosolids are being implicated for 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 (dose-
response relationship), plausibility of the causative relationship, coherence of evidence,
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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 causes 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, 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 et al. (2007) provide methods to track sources of fecal pollution.
Epidemiological studies are discussed below. Risk assessors for site-specific human
health assessments might also benefit from guidance for identifying stressors to specific
aquatic ecosystems in the Stressor Identification Guidance Document (U.S. EPA, 2000)
and CADDIS (http://www.epa.gov/caddis/).
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.
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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 that a population of humans
exposed to infectious microbes will receive a distribution of actual doses (Haas et al.,
1999). Also, infectious microbes have the ability to propagate within a susceptible host
at an appropriate location within the body (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 food-borne exposures to microbial agents and
recently in risk assessments of pathogens in dewatered, land-applied biosolids (Dowd
et al., 2000; Brooks et al., 2005b; Eisenberg et al., 2004). Table 9 provides examples of
dose-response models for microbial agents that may be associated with biosolids.
Almost all of these examples pertain to the endpoint of infection rather than disease.
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).
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. Risk
assessors might view reported infective doses as doses where infection becomes likely
rather than actual thresholds.
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TABLE 9
Examples of Dose-Response Models for Microbial Agents
Organism
Measure of
Exposure
Model
Endpoint
Reference
Rotavirus
Dose
Exponential
Beta-Poisson
Log-probit
Human
Infection
Ward etal. (1986),
Haas et al. (1999)
Cryptosporidium
parvum
Dose
Exponential
Human
Infection
Dupont et al.
(1995)
Cryptosporidium
parvum
Dose
Beta-Poisson
Human
Infection
Englehardt and
Swartout (2004)
Cryptosporidium
parvum
Dose
Beta-Poisson
Gastroenteric
illness
Englehardt and
Swartout (2006)
Enteric virus
Dose
Beta-Poisson
Human
Infection
Gerba et al. (2002)
Salmonella
serovar Anatum
Dose
Beta-Poisson
Human
Infection
McCullough and
Eisele (1951),
Haas et al. (1999)
Coxsackievirus
B3
Dose
Exponential
Human
Infection
Dowd et al. (2000)
Salmonella
serovar Typhi
Dose
Beta-Poisson
Human
Infection
Dowd et al. (2000)
E.coli (0111)
Dose
Beta-Poisson
Human
Infection
Ferguson and
June (1952), Haas
etal. (1999)
E. coli (055)
Dose
Beta-Poisson
Human
Infection
June et al. (1953),
Haas et al. (1999)
Endotoxin
Concentration
in air
Threshold
Decreased lung
efficiency,
Organic Toxic
Dust Syndrome
Baker et al. (1986)
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Dose-response models represent major information gaps for risk assessments
related to pathogens in biosolids. 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). Dose-
response relationships are not available for all of the pathogens potentially found in
biosolids (see stressor characterization chapter). Dose-response relationships are not
available for inhaled microorganisms (NRC, 2002). As stated in the literature review
(Appendix A), 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 Appendix A). 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 analysis of all three
aspects. The pathogen is the causative agent of the disease. Whereas chemicals are
generally assumed to elicit comparable responses in appropriate animal models as do
humans, pathogens are more host-specific. Pathogens can elicit adverse responses
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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. Immunity is one of the most important
parameters influencing the risk from pathogens in biosolids, based on Eisenberg et al.'s
(2004) model. The analysis plan should specify whether groups of individuals of
particular immune status are assessment endpoint entities in the risk assessment.
However, validated protocols are not available to incorporate immune status or other
pathogen susceptibility factors (pregnancy, age) into risk assessments (NRC, 2002).
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
Eisenberg et al. (2004, 2005, 2006) developed a methodology to assess risks to
human health from pathogens in biosolids-amended soil. While many of the processes
in the model are those described in this chapter (fate, transport, uptake), others may not
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be needed. For example, Eisenberg et al. modeled the attenuation of organisms in
sewage sludge, but it is just as easy to measure concentrations in biosolids as in
sewage sludge. Thus, that component of their model is unnecessary. Eisenberg et al.
also modeled secondary transmission, which is important for estimating the total burden
of disease. However, secondary transmission of pathogens is not unique to the
biosolids context, and it is not discussed in this problem formulation, which is concerned
with risks of primary infection.
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 time of application and onset of
symptoms or other indicators would identify key routes of exposure to assess the
validity of the conceptual models presented here and to prioritize 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. 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.
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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 will 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.).
Risk assessors should be aware of the difficulties in conducting an
epidemiological study of biosolids exposure. In theory, it is unlikely that land application
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of properly treated Class B biosolids would result in adverse health impacts. 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 (Simmonds, 2005). However, a
recent conference abstract indicates that an epidemiological study of biosolids exposure
is underway (Heaney et al., 2007).
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.
5.5.1. Screening Risk Assessment
The analysis plan must describe 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 pathogen levels in biosolids that would result in a very
low and acceptable dosage of pathogens are not available. 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 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
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information-rich risk assessment topics, which risk assessments for pathogens in
biosolids are not expected to be.
5.5.2. Weight of Evidence
If multiple lines of evidence are expected, the analysis plan should explain how
these results will be weighed. For example, an unvalidated animal model might predict
a certain infection rate, but epidemiological evidence might show that the only disease
outbreak was probably associated with a local crop to which biosolids was not applied.
In this case, the latter evidence might be given a higher weight. 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
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 be infrequent.
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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
model). 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.
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1	APPENDIX A
2	LITERATURE REVIEW
3
4
5	This appendix presents a literature review that summarizes the available
6	information on microbial risks to humans posed by land-applied biosolids. The review is
7	organized in terms of summary points, research and data gaps, relevant aspects of the
8	NRC (2002) recommendations on biosolids, and data and information available for
9	phases of risk assessments (e.g., fate, transport, uptake, infectivity, risk assessment,
10	causal analysis). Although some studies of pathogens in manures may be relevant to
11	biosolids (e.g., models of pathogen transport), investigations of these untreated
12	materials are beyond the scope of this report. This literature review was completed
13	prior to the other chapters in this report.
14
15	SUMMARY POINTS
16	• The range of pathogens that may be present in biosolids is well understood, but
17	the current national distribution of these pathogens, the variation with type of
18	sewage sludge treatment, and analytical methods for detecting and quantifying
19	pathogens are not well understood or developed.
20	• Many analytical methods for detecting and quantifying pathogens focus on
21	detecting DNA sequences rather than viable cultures.
22	• The use of indicator organisms to represent pathogens of concern has the
23	potential to introduce large uncertainties into estimates of exposure.
24	• Risk assessments of pathogens in biosolids have been performed, but the
25	emphasis has been on the use of particular transport models to quantify risks
26	from a few pathogens to individuals at a distance from particular biosolids
27	application sites rather than the process of planning and conducting a national-
28	scale or other broad risk assessment. A formal problem formulation for
29	pathogens in biosolids has not been undertaken.
30	• Conceptual models for human health risk assessments of pathogens in biosolids
31	that include detailed source descriptions, transport pathways and routes of
32	exposure have not been developed previously.
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1	• A causal association between exposures to biosolids and adverse effects on
2	human health has not been documented.
3	• Epidemiological studies of biosolids application sites are generally lacking and
4	are problematic to conduct.
5	• Although the U.S. EPA has standard exposure factors and effects levels relevant
6	to chemicals, some standard exposure factors and effects levels needed for risk
7	assessments of pathogens in biosolids are not available.
8	• U.S. EPA does not have a standard quantitative microbial risk assessment
9	framework for use in risk assessments of pathogens in biosolids.
10	• Dose-response relationships used in risk assessments of pathogens in biosolids
11	have been derived from non-biosolids studies, and it is unclear how applicable
12	these relationships are to biosolids, particularly for the inhalation pathway.
13	• Although the science of biosolids exposure analysis is still under development,
14	studies of effects of pathogens in biosolids are limited.
15	• Little information is available to support the elimination of exposure scenarios or
16	pathways from consideration at all sites where biosolids have been applied.
17	Information may support the screening of exposure pathways from consideration
18	at particular sites.
19	• Bioaerosol emissions from biosolids have been studied most rigorously in
20	Arizona; few data exist for other regions.
21	• Exposure assumptions vary in existing risk assessments for bioaerosols
22	generated from biosolids.
23	• Existing risk assessment studies of pathogens in biosolids at specific sites
24	estimate risk of infection rather than risk of disease.
25
26	Many of the research and monitoring gaps related to human health risk assessments
27	of biosolids are described in key papers and are summarized in Table A-1. These
28	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
Stressor 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 Gudged 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, helminthic parasites in biosolids
Smith et al. (2005a),
NRC (2002), U.S.
EPA (2003b)
Standardized methods for measuring and characterizing
pathogens in bioaerosols
NRC (2002), Pillai
(2002)
Molecular, immunological, immuno-magnetic separation and
culture (IMSC) 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,
Cyclospora, Toxoplasma, Microsporidia, Balantidium, Giardia
and Entamoeba), fecal coliforms, 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 Gudged 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 non-
typhi 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 Gudged 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
et al. (2000)
Rapid response investigations of reported health effects
potentially resulting from land application of biosolids
U.S. EPA (2003b)
from WERF
Biosolids Research
Summit
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
NRC RECOMMENDATIONS
The NRC was asked by U.S. 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
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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) (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 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 U.S. 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 (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 (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
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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 (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 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 coliforms and fecal streptococci).
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The primary information gap related to stressor 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 (Table A-1). Epstein and Moss (2006) cite
references regarding probable numbers of fecal coliforms and Salmonella spp. in Class
B biosolids. Dahab and Surampalli (2002) found that existing treatment systems do
achieve Class B requirements under the US 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 UV. We have not conducted 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
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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
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 montocytogenes are emerging bacterial pathogens that can survive
anaerobic digestion and can sometimes regrow following land application of biosolids.
In contrast, the protozoan parasites microsporidia and Cyclospora 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
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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 coliforms 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 (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. (2004) 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
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. Clostridum 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., 2004).
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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 (HPC), total coliforms,
sulfite-reducing Clostridia, and endotoxin, as well as PMi0. The authors presented a
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correlation between microbial concentrations emitted during disking and their content in
biosolids. Disking was 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
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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
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.
Y. Jin, J. Sims and K. Kniel of the University of Delaware were awarded a USDA
grant from 2006 to 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.
Erosion and Surface Runoff
We did not find information on these mechanisms of transport of pathogens in
biosolids.
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Pathogens on Crops
Studies of pathogens on crops are described in the section on risk assessment.
Also, the USDA 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.
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" (Jacques Whitford Limited,
2004). 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.
Soller et al. (2006) 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 and the presence of immunity. 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
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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. (2006) also included a table of data required to parameterize a
basic health effects model.
Although Soller et al. (2006) 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 (ILSI) developed a framework for
microbial risk assessment related to human exposures to waterborne pathogens (ILSI,
2000). The framework describes the stages of risk assessment, including problem
formulation, but without providing or citing scientific advice regarding particular
pathogens or exposure pathways.
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Bioaerosol Pathways
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 a major study 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., 2004, 2005a,b, 2006). 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 ID50, and a
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and r are parameters that describe the dose-response curve. Under one of the wind
speeds in the study (2 m/s), the risk of bacterial and viral infection of workers exposed
for one hour at a distance of 100 m is 2E-2 and 3E-2, respectively. Under these
conditions, residents at 10 km from the biosolids source were found to be at no risk from
aerosolized viruses and low risk of infection from bacteria (2E-4). Under some more
moderate and high wind conditions, especially where exposures were for 8 hours or
more at distances of 500 m or less from the source, risks of infection of workers (or
others) from bioaerosols were close to 1.0. 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). Citing
comments by Brooks et al. (2004) on the improved efficiency of modern wastewater
treatment plants, Pepper et al. (2006) 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 U.S. 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 noravirus 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 * 10"4, from coxsackievirus A21 released during
loading operations.
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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 risk 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.
In a study of bioaerosol emission rates from the spreading of Class B biosolids in
Arizona, measured source endotoxin concentrations were greater than reported
conservative thresholds for mucous membrane irritation, and most exceeded the
threshold for acute bronchial constriction (Paez-Rubio et al., 2007).
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Groundwater Pathways
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 from Haas et al. (1999, P = 1 -
[1 + N/(3-a]) 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
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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
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 or 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
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sewage sludge in the United Kingdom. Pepper et al. (2006) identified the incidence of
prions in biosolids as a research priority in the U.S. (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. (2004) and Brooks et al. (2007) concluded that Class
B biosolids had an equal or lower incidence of antibiotic resistant bacteria compared to
unamended soil. 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
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infectious, causing 10-15% of those 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).
Nwachuku and Gerba (2004) address the susceptibility of children to pathogens,
including increased sensitivity and increased exposure. Reasons that children are at
greater potential risk from pathogens in biosolids are
•	immature immune system;
•	intestinal mucosa more permeable to water;
•	proportionally less extracellular fluid than adults;
•	physiological deficiency in IgA;
•	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
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
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 (Haas et al., 1999).
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Dynamic Risk Model
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 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.
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EXPOSURE ASSUMPTIONS
U.S. 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 et al. (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.
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CAUSAL ANALYSIS
"Causal association between biosolids exposures and adverse health outcomes
has not been documented" (NRC, 2002). Lewis et al. (2002) recorded symptoms
reported by 48 residents near 10 biosolids application sites in the U.S. 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 speculated that chemical
contaminants in biosolids might irritate the skin and mucous membranes and thus
increase pathogen host susceptibility (Lewis et al., 2002).
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 non-biosolids 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.
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1	Simmonds et al. (2005) describe the difficulties of conducting an epidemiological
2	study of biosolids exposure. Few people who are exposed are expected to become
3	infected, and even fewer to manifest symptoms of disease. Also, various symptoms
4	may be associated with one pathogen, and various pathogens can cause similar
5	symptoms.
6	A recent abstract indicates that a health effects study of biosolids exposure is
7	underway (Heaney et al., 2007).
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