v>EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
EPA/625/R-92/013
December 1992
Environmental
Regulations and
Technology
Control of Pathogens and
Vector Attraction in
Sewage Sludge
-------
-------
EPA/625/R-92/013
December 1992
Environmental Regulations and Technology
Control of Pathogens and Vector Attraction
in Sewage Sludge
(Including Domestic Septage)
Under 40 CFR Part 503
This guidance was prepared by
U.S. Environmental Protection Agency
Office of Research and Development
Office of Science Planning and Regulatory Evaluation
Center for Environmental Research Information
Cincinnati, OH 45268
Office of Water
Office of Science and Technology
Sludge Risk Assessment Branch
Washington, DC 20460
Printed on Recycled Paper
-------
This document was produced by the U.S. Environmental
Protection Agency (EPA's) Pathogen Equivalency Committee
(PEC), consisting of Robert Bastian, Joseph Farrell, G. Shay
Fout, Walter Jakubowski, Norman Kowal, Mark Meckes, and
J.E. Smith, Jr. Joseph Farrell contributed significantly to the
document's preparation. Among his contributions were data and
other information and the writing of Chapters 7 and 8 and
Appendices C, D, and E. Mark Meckes contributed the expla-
nation of the fecal coliform test. G. Shay Fout and Daniel R.
Dahling wrote Appendix H, with contributions from colleagues
in the Virology Branch, Microbiology Research Division, of
EPA's Environmental Monitoring and Support Laboratory. The
contributions of Robert M. Southworth of EPA's Office of
Water, who critically reviewed the document, are especially
appreciated. Jan Connery of Eastern Research Group, Inc., in
Lexington Massachusetts, prepared and edited the document
under the committee's direction and from information and data
supplied by the committee. Other EPA regional, office, and
laboratory personnel also contributed information and sugges-
tions for improving this document. Their assistance is sincerely
appreciated.
This report has been reviewed by the U.S. Environmental
Protection Agency and approved for publication. The process
alternatives, trade names, or commercial products are only ex-
amples and are not endorsed or recommended by the U.S.
Environmental Protection Agency. Other alternatives may exist
or may be developed.
This guidance was published by
U.S. Environmental Protection Agency
Office of Research and Development
Office of Science, Planning and Regulatory Evaluation
Center for Environmental Research Information
Cincinnati, OH 45268
COVER PHOTOGRAPH: Application of sewage sludge
compost to the White House lawn.
11
-------
Contents
Abbreviations and Acronyms viii
Chapter 1 Introduction 1
1.1 What Is Sewage Sludge? 1
1.2 U.S. Regulation of Sewage Sludge 1
1.3 What's in This Document? 2
Chapter 2 Protection of Public Health and the Environment from Sewage Sludge Pathogens 5
2.1 What Are the Pathogens of Concern? 5
2.2 How Does Exposure Occur? 5
2.3 How Well Do Pathogens Survive in the Environment? 6
2.4 How Can the Public and Animals Be Protected? 6
2.5 How Can Pathogen Reduction Be Measured? 7
2.6 What Units Are Used to Measure Microorganism Density Under Part 503? 8
Chapter 3 Overview of Part 503 Subpart D Requirements, Their Applicability, and
Related Requirements 11
3.1 Introduction 11
3.2 Pathogen Reduction Requirements 11
3.3 Vector Attraction Reduction Requirements [503.33] 11
3.4 Applicability of the Requirements [503.15] 12
3.5 Frequency of Monitoring 12
3.6 Recordkeeping Requirements [503.17 and 503.27] 12
3.7 Reporting Requirements for Sewage Sludge [503.18 and 503.28] 13
3.8 Permits and Direct Enforceability [503.3] 14
3.9 Compliance Period [503.2] 14
Chapter 4 Class A Pathogen Requirements • 17
4.1 Introduction 17
4.2 Vector Attraction Reduction to Occur With or After Class A Pathogen Reduction
[503.32(a)(2)] 17
4.3 Monitoring of Fecal Coliform or Salmonellae to Detect Regrowth [503.32(a)(3)-(8)] 17
4.4 Alternative 1: Thermally Treated Sewage Sludge [503.32(a)(3>] 18
4.5 Alternative 2: Sewage Sludge Treated in a High pH—High Temperature Process
(Alkaline Treatment) [503.32(a)(4>] 19
4.6 Alternative 3: Sewage Sludge Treated in Other Processes [503.32(a)(5>] 19
4.7 Alternative 4: Sewage Sludge Treated in Unknown Processes [503.32(a)(6>] 20
4.8 Alternative 5: Use of PFRP [503.32(a)(7)] 20
4.9 Alternative 6: Use of a Process Equivalent to PFRP [503.32(a)(8>] 21
Chapter 5 Class B Pathogen Requirements and Requirements for Domestic Septage Applied to
Agricultural Land, a Forest, or a Reclamation Site 23
5.1 Introduction 23
5.2 Sewage Sludge Alternative 1: Monitoring of Fecal Coliform [503.32(b)(2>] 23
5.3 Sewage Sludge Alternative 2: Use of PSRP [503.32(b)(3>] 24
5.4 Sewage Sludge Alternative 3: Use of Processes Equivalent to PSRP [503.32(b)(4>] 24
5.5 Site Restrictions [503.32(b)(5)] 25
m
-------
5.6 Domestic Septage [503.32(c)] 26
Chapter 6 Requirements for Reducing Vector Attraction 27
6.1 Introduction 27
6.2 Option 1: Reduction in Volatile Solids Content [503.33(b)(l)] 27
6.3 Option 2: Additional Digestion of Anaerobically Digested Sewage Sludge [503.33(b)(2)J.. 27
6.4 Option 3: Additional Digestion of Aerobically Digested Sewage Sludge [503.33(b)(3)] ... 27
6.5 Option 4: Specific Oxygen Uptake Rate (SOUR) for Aerobically Digested Sewage Sludge
[503.33(b)(4)] 28
6.6 Option 5: Aerobic Processes at Greater Than 40°C [503.33(b)(5>] 29
6.7 Option 6: Addition of Alkali [503.33(b)(6>] 29
6.8 Option 7: Moisture Reduction of Sewage Sludge Containing No Unstabilized Solids
[503.33(b)(7>] 29
6.9 Option 8: Moisture Reduction of Sewage Sludge Containing Unstabilized Solids
[503.33(b)(8)] 29
6.10 Option 9: Injection [503.33(b)(9)] 29
6.11 Option 10: Incorporation of Sewage Sludge into the Soil [503.33(b)(10)] 30
6.12 Option 11: Covering Sewage Sludge [503.33(b)(ll>] 30
6.13 Option 12: Raising the pH of Domestic Septage [503.33(b)(12)] 30
Chapter 7 Meeting the Quantitative Requirements of the Regulation 31
7.1 Introduction 31
7.2 Process Conditions 31
7.3 Monitoring Events: Needs and Duration. 32
7.4 Correspondence of Samples 33
7.5 Adjusting for Diluents 33
7.6 Representative Samples 33
7.7 Regulatory Objectives and Number of Samples That Should Be Tested 34
Chapter 8 Sampling Procedures and Analytical Methods 41
8.1 Introduction 41
8.2 Safety Precautions 41
8.3 Sampling Free-Flowing Sewage Sludges 41
8.4 Sampling Thick Sewage Sludges 42
8.5 Sampling Dry Sewage Sludges 43
8.6 Control of Temperature, pH, and Oxygenation After Sample Collection 43
8.7 Sample Compositing and Size Reduction 44
8.8 Requirements for Sample Containers and Sampling Tools 44
8.9 Packaging and Shipment 45
8.10 Documentation 45
8.11 Analytical Methods 46
8.12 Quality Assurance 46
Chapter 9 Processes to Significantly Reduce Pathogens (PSRPs) 49
9.1 Introduction 49
9.2 Aerobic Digestion 49
9.3 Anaerobic Digestion 51
9.4 Air Drying 52
9.5 Composting 52
9.6 Lime Stabilization 54
9.7 Equivalent Processes 54
Chapter 10 Processes to Further Reduce Pathogens (PFRPs) 55
10.1 Introduction 55
10.2 Composting 55
10.3 Heat Drying 55
10.4 Heat Treatment 56
10.5 Thermophilic Aerobic Digestion 56
10.6 Beta Ray and Gamma Ray Radiation 57
IV
-------
10.7 Pasteurization 58
10.8 Equivalent Processes 58
Chapter 11 Role of EPA's Pathogen Equivalency Committee in Providing Guidance Under Part 503 59
11.1 Introduction 59
11.2 Overview of the PEC's Equivalency Recommendation Process 61
11.3 Basis for PEC Equivalency Recommendations 61
11.4 Guidance on Demonstrating Equivalency for PEC Recommendations 66
11.5 Guidance on Application for Equivalency Recommendations 67
11.6 Examples of Recommendations 68
Chapter 12 References 71
Appendix A EPA Regional and State Sludge Coordinators and Map of EPA Regions 73
Appendix B Subpart D of the Part 503 Regulation 81
Appendix C Determination of Volatile Solids Reduction by Digestion 85
Appendix D Guidance on Three Vector Attraction Reduction Tests 93
Appendix E Determination of Residence Time for Anaerobic and Aerobic Digestion 99
Appendix F Sample Preparation for Fecal Coliform Tests and Salmonella sp. Analysis 103
Appendix G Kenner and Clark (1974) Analytical Method for Salmonella sp. Bacteria 107
Appendix H Method for the Recovery and Assay of Enteroviruses from Sewage Sludge 117
Appendix I Analytical Method for Viable Helminth Ova 147
-------
Figures
1-1. Generation, treatment, use, and disposal of sewage sludge 1
1-2. EPA policy on sludge management 2
9-1. Aerobic digestion. ...,, 50
9-2. Two-stage anaerobic digestion (high rate) 51
9-3. Static aerated pile composting 53
10-1. Schematic representation of cobalt-60 (gamma ray) irradiation facility at Geiselbullach, Germany.. 57
10-2. Beta ray scanner and sludge spreader. 57
11-1. When is application for PFRP or PSRP equivalency appropriate? 60
11-2. Role of the PEC under Part 503 62
A-l. EPARegions 74
VI
-------
Tables
2-1. Principal Pathogens of Concern in Municipal Wastewater and Sewage Sludge 6
2-2. Survival Times of Pathogens in Soil and on Plant Surfaces 7
2-3. General Approaches to Controlling Pathogens and Vector Attraction in Sewage Sludge 7
3-1. Subpart D Requirements for Bulk Sewage Sludge 12
3-2. Subpart D Requirements for Sewage Sludge Sold or Given Away in a Bag or Other Container ... 13
3-3. Subpart D Requirements for Domestic Septage Applied to Agribultural Land, a Forest, or a
Reclamation Site or Placed on a Surface Disposal Site 13
3-4. Frequency of Monitoring for Land Application and Surface Disposal 13
3-5. Summary of Pathogen-Related and Vector Attraction Reduction-Related Recordkeeping
Requirements for Land Application of Sewage Sludge 14
3-6. Summary of Pathogen-Related and Vector Attraction Reduction-Related Recordkeeping
Requirements for Surface Disposal of Sewage Sludge 15
4-1. The Four Time-Temperature Regimes for Alternative 1 (Thermally Treated Sewage
Sludge) [503.32(a)(3)] 19
4-2. Processes to Further Reduce Pathogens (PFRPs) Listed In Appendix B of 40 CFR Part 503 21
4-3. A Partial List of Processes Recommended as Equivalent to PFRP Under Part 257 21
5-1. Processes to Significantly Reduce Pathogens (PSRPs) Listed in Appendix B
of 40 CFR Part 503 24
5-2. A Partial List of Processes Recommended as Equivalent to PSRP Under Part 257 24
6-1. Summary of Requirements for Vector Attraction Reduction Under Part 503 28
7-1. True Geometric Mean Needed If Standard Fecal Coliform Density of 2 Million CFU Per
Gram Is to Be Rarely Exceeded 35
8-1. Analytical Methods Required Under Part 503 46
vu
-------
Abbreviations and Acronyms
BOD
°C
CFR
CPU
cm
EPA
oF
FFSR
FFVSR
FR
FVSR
g
gpm
hr
kg
L
log
m3
m.b.
mg
MOD
min
mL
MPN
no.
02
OWEC
PEC
PFRP
PFU
psig
PSRP
RSC
SM
SOUR
sp.
SRAB
SSC
TS
TSS
VS
VSS
biological oxygen demand
degrees centigrade
Code of Federal Regulations
colony-forming unit
centimeter(s)
U.S. Environmental Protection Agency
degrees Fahrenheit
fractional fixed solids reduction
fractional fixed volatile solids reduction
Federal Register
fractional volatile solids reduction
gram(s)
gallons per minute
hour(s)
kilogram(s)
liter(s)
logarithm
cubic meter(s)
mass balance
milligram(s)
million gallons/day
minute(s)
milliliter(s)
most probable number
number
oxygen
EPA Office of Wastewater Enforcement and Compliance
EPA Pathogen Equivalency Committee
process to further reduce pathogens
plaque-forming unit
pounds per square inch gauge
process to significantly reduce pathogens
EPA Regional Sludge Coordinator
"Standard Methods for the Examination of Water and Wastewater," 18th edition
specific oxygen uptake rate
species
EPA Sludge Risk Assessment Branch
State Sludge Coordinator
total solids
total suspended solids
volatile solids
volatile suspended solids
vm
-------
Chapter 1
Introduction
1.1 What Is Sewage Sludge?
Sewage sludge—the residue generated during treatment of
domestic sewage (Figure 1-1)—is used as a soil conditioner
and partial fertilizer in the United States and many other coun-
tries. It is applied to agricultural land (pastures and cropland),
disturbed areas (mined lands, construction sites, etc.), plant
nurseries, forests, recreational areas (parks, golf courses, etc.),
cemeteries, highway and airport runway medians, and home
gardens (see photographs on pages 3 and 4). Certain publicly
owned treatment works (POTWs) own or have access to land
dedicated solely to disposal of sewage sludge—a practice re-
ferred to as surface disposal. The U.S. Environmental Protec-
tion Agency (EPA), the primary federal agency responsible for
sewage sludge management, encourages the beneficial use of
sewage sludge (Figure 1-2). A 1988 survey found that as much
as 33% of the sewage sludge generated in the United States
was being applied to land (EPA, 1988).1
Sewage sludge has beneficial plant nutrients and soil con-
ditioning properties; however, it may also contain bacteria, vi-
ruses, protozoa, parasites, and other microorganisms that can
cause disease. Land application and surface disposal of sewage
sludge create a potential for human exposure to these organisms
through direct and indirect contact. To protect public health
from these organisms and from the pollutants that some
sludges2 contain, many countries now regulate the use and
disposal of sewage sludge.
1.2 U.S. Regulation of Sewage Sludge
In the United States, the use and disposal of sewage sludge
(including domestic septage) are regulated under 40 CFR Part
503.3 This regulation, promulgated on February 19, 1993, was
issued under the authority of the Clean Water Act as amended
in 1977 and the 1976 Resource Conservation and Recovery Act
(RCRA). For most sewage sludge,4 the new regulation replaces
'Domestic septage—the material removed from septic tanks and other on-site treatment
systems that receive only domestic sewage—is a form of sewage sludge and therefore may
also be applied to the land or disposed in a surface disposal site.
2In this document, the term "sludge" always refers to sewage sludge.
Because domestic septage is a form of sewage sludge, any use of the term "sewage
sludge" or "sludge" in this document includes domestic septage.
Sewage sludge generated at an industrial facility during the treatment of domestic sewage
commingled with industrial wastewater in an industrial wastewater treatment facility is
still covered under 40 CFR Part 257 if the sewage sludge is applied to the land.
INDUSTRIAL
WASTEWATER
GENERATION
SEWAGE SLUDGE
TREATMENT
• Digestion
• Drying
« Composting
• Lime stabilization
• Heat treatment
• Etc.
USE
DISPOSAL
Land Application
• Agricultural land
• Strip-mined land
• Forests
• Plant nurseries
• Cemeteries
• Parks, gardens
• Landfill cover
» Lawns and home
• gardens
Figure 1-1. Generation, treatment, use, and disposal of sewage sludge.
-------
The U.S. Environmental Protection Agency (EPA)
will actively promote those municipal sludge man-
agement practices that provide for the beneficial use
of sludge while maintaining or improving environ-
mental quality and protecting human health. To im-
plement this policy, EPA will continue to issue
regulations that protect public health and other en-
vironmental values. The Agency will require states
to establish and maintain programs to ensure that
local governments utilize sludge management tech-
niques that are consistent with federal and state
regulations and guidelines. Local communities will
remain responsible for choosing among alternative
programs; for planning, constructing, and operating
facilities to meet their needs; and for ensuring the
continuing availability of adequate and acceptable
disposal or use capacity.
Figure 1-2. EPA policy on sludge management. Source: EPA, 1984.
40 CFR Part 257—the original regulation governing the use
and disposal of sewage sludge—in effect since 1979.
Protection of Public Health and the Environment
In the judgment of the Administrator of EPA, Subpart D
of the Part 503 regulation protects public health and the envi-
ronment through requirements designed to reduce the potential
for contact with the disease-bearing microorganisms (patho-
gens) in sewage sludge applied to the land or placed on a
surface disposal site. These requirements are divided into:
• Requirements designed to control and reduce pathogens hi
sewage sludge.
• Requirements designed to reduce the ability of the sewage
sludge to attract vectors (insects and other living organisms
that can transport sludge pathogens away from the land ap-
plication or surface disposal site).
Subpart D includes both performance- and technology-
based requirements. It is designed to provide a more flexible
approach than Part 257, which required sewage sludge to be
treated by specific listed or approved treatment technologies.
Under Part 503, treatment works may continue to use the same
processes they used under Part 257, but they now also have the
freedom to modify conditions and combine processes with each
other, as long as the treated sewage sludge meets the applicable
requirements.
Environmental Effects of Pathogens in Sewage Sludge
A major environmental concern (other than effects on pub-
lic health) associated with land application of sewage sludge is
the effect of pathogens on animals. Certain human pathogens
can cross species lines and infect animals, particularly warm-
blooded animals. Little information is available on whether
these pathogens pose a risk to wildlife. Available information
on the impact of sludge pathogens on grazing animals suggests
that the Part 503 Subpart D requirements for pathogen control
(which include restrictions on grazing) protect grazing animals
(EPA, 1992).
1.3 What's in This Document?
This document describes the federal requirements concern-
ing pathogens in sewage sludge applied to land or placed on a
surface disposal site, and it provides guidance concerning those
requirements. The document is intended for:
• Owners and operators of treatment works treating domestic
sewage.
• Developers or marketers of sewage sludge treatment processes.
• Groups that distribute and market sewage sludge products.
• Individuals involved in applying sewage sludge to land.
• Regional, state, and local government officials responsible
for implementing and enforcing the Part 503 Subpart D
regulation.
• Consultants to these groups.
• Anyone interested in understanding the federal requirements
concerning pathogens in sewage sludge.
Chapter 2 of this document discusses why pathogen control
is necessary to protect public health and the environment, and
Chapters 3 through 6 describe the current federal requirements
under Subpart D of Part 503. Chapters 7 and 8 discuss sampling
and analysis to meet the quantitative requirements of Part 503.
Chapters 9 and 10 describe the sewage sludge treatment proc-
esses listed under Part 503. Chapter 11 discusses the kind of
support EPA's Pathogen Equivalency Committee can provide
to permitting authorities.
The appendices provide additional information on:
• Determination of volatile solids and residence time for di-
gestion.
• Sample preparation and analytical methods for meeting the
Part 503 pathogen reduction requirements.
• Tests for demonstrating reduced vector attraction.
Also, Appendix A lists EPA and state sludge coordinators, and
Appendix B contains Subpart D of the Part 503 regulation.
Many sewage sludges also contain heavy metals that .may
pose public health and environmental concerns. The federal
regulation under 40 CFR Part 503 includes requirements de-
signed to limit the amount of heavy metals in sewage sludge
applied to land or placed on a surface disposal site. This docu-
ment focuses on pathogen-related requirements and does not
discuss the heavy metal requirements.
-------
Highway median strip in Illinois after land application of dried
sludge. (Photo courtesy of Metropolitan Water Reclamation District of
Greater Chicago)
Flower beds amended with sludge compost in Tulsa, Oklahoma.
(Photo courtesy of City of Tulsa, Oklahoma)
Injection of liquid sludge into sod.
Oat field showing sludge-treated (right) and untreated (left) areas.
(Photo courtesy of City of Tulsa, Oklahoma)
-------
Mine apdl land before sludge treatment Note sparse, weedy
growth Incapable of supporting grazing cattle. (Photo courtesy of
City of Tutea, Oklahoma)
Corn grown on sludge-treated soil (right) and untreated soil (left).
Mlno spoil land after sludge treatment. Note lush vegetative cover
on reclaimed soil which will support grazing. (Photo courtesy of City
of Tulsa, Oklahoma)
Cross-section of Douglas fir tree showing how sludge application
increases tree growth. Note increased size of outer rings indicat-
ing more rapid growth after sludge application. (Photo courtesy of
Metro Silvigrow)
-------
Chapter 2
Protection of Public Health and the Environment from
Sewage Sludge Pathogens
2.1 What Are the Pathogens of Concern?
Municipal wastewater generally contains four major types
of human pathogenic (disease-causing) organisms: bacteria, vi-
ruses, protozoa, and helminths (parasitic worms) (EPA, 1985).
The actual species and density of pathogens present in waste-
water from a particular municipality (and the sewage sludge
produced when treating the wastewater) depend on the health
status of the local communityT,-and may vary substantially at
different times. The level of pathogens present in sewage sludge
also depends on the reductions achieved by the wastewater and
sewage sludge treatment processes.
The pathogens hi wastewater are primarily associated with
insoluble solids. Primary wastewater treatment processes con-
centrate these solids into sewage sludge, so untreated or raw
primary sewage sludges have higher densities of pathogens than
the incoming wastewater. Biological wastewater treatment
processes such as lagoons, trickling filters, and activated sludge
treatment may substantially reduce the number of pathogens in
the wastewater (EPA, 1989).
Nevertheless, the resulting biological sludges may still
contain sufficient levels of pathogens to pose a public health
and environmental concern.1 Table 2-1 lists some principal
pathogens of concern that may be present hi wastewater and/or
sewage sludge. These organisms and other pathogens can cause
infection or disease if humans and animals are exposed to suf-
ficient levels of the organisms or pathogens. The levels—called
infectious doses—vary for each pathogen and each host.
Some of the common pathogens of concern that appear in
municipal wastewater and sludge are shown in the photographs
on pages 9 and 10. These include ascarids (Ascaris lumbri-
coides and Toxocard), whipworms (Trichuris sp.), tapeworms
(Hymenolepis sp. and Taenia sp.), amoeba (Entamoeba coli),
and giardia (Giardia lamblia). As shown hi these photographs,
several color staining procedures are needed to identify the
organisms and the different structures within the organisms.
The photograph of Giardia lamblia depicts specimens stained
with Lugol's iodine solution, showing two nuclei, a median
body, and axonemes in each. In addition, scientists use a blue
filter when photographing the pathogenic organisms through a
'AS mentioned in Chapter 1, a major environmental concern (other than effects on public
health) is the potential effect of some human pathogens on animals.
microscope. This filter is necessary to show the natural color
of the organisms.
2.2 How Does Exposure Occur?
When sewage sludge is applied to land or placed on a
surface disposal site, humans and animals can be exposed to
pathogens directly by coming into contact with the sewage
sludge, or indirectly by consuming drinking water or food con-
taminated by sewage sludge pathogens. Insects, birds, rodents,
and even farm workers can contribute to these exposure routes
by transporting sewage sludge and sewage sludge pathogens
away from the site. Potential routes of exposure include:
Direct Contact
• Inadvertent contact with sewage sludge.
• Walking through an area—such as a field, forest, or recla-
mation area—shortly after sewage sludge application.
• Handling soil and raw produce from fields or home gardens
where sewage sludge has been applied.
• Inhaling microbes that become airborne (via aerosols, dust,
etc.) during sewage sludge spreading or by strong winds,
plowing, or cultivating the soil after application.
Indirect Contact
• Consumption of pathogen-contaminated crops grown on
sewage sludge-amended soil or of other food products that
have been contaminated by contact with these crops.
« Consumption of pathogen-contaminated milk or other food
products from animals grazing in pastures or fed crops
grown on sewage sludge-amended fields.
• Ingestion of drinking water or recreational waters contami-
nated by runoff from nearby land application sites or by
organisms from sewage sludge migrating into ground-water
aquifers.
• Consumption of inadequately cooked or uncooked patho-
gen-contaminated fish from water contaminated by runoff
from a nearby sewage sludge application site.
-------
Contact with sewage sludge or pathogens transported away
from the land application or surface disposal site by rodents,
insects, or other vectors, including grazing animals.
Table 2-1. Principal Pathogens of Concern in Municipal Wastewater
and Sewage Sludge
Organism
Disease/Symptoms
Bacteria
Salmonella sp.
ShigeKa sp.
Yorsinla sp.
Vibrio choleraa
Campytobacter Jejunl
Eschorichla coll
(pathogenic strains)
Enteric Viruses
Hepatitis A virus
Nomalk and
Norwalk-like viruses
Rotaviruses
Enteroviruses
Poliovirusos
Coxsackiovirusos
Echovifusos
Roovirus
Astroviruses
Callch/irusos
Protozoa
Ciyptospotidlum
Enlamoeba hlstolytlca
Gfardla lamblla
Balanlfdium coll
Toxoplasma gondff
Helminth Worms
Ascaris lumbricoidcs
Ascaris suum
TrtcfHJris trfchlura
Toxocara canls
Taonta saglnala
Taenla solium
Nocator amerteanus
Hymonolcpis nana
Salmonellosis (food poisoning).
typhoid fever
Bacillary dysentery
Acute gastroenteritis (including
diarrhea, abdominal pain)
Cholera
Gastroenteritis
Gastroenteritis
Infectious hepatitis
Epidemic gastroenteritis with severe
diarrhea
Acute gastroenteritis with severe
diarrhea
Poliomyelitis
Meningitis, pneumonia, hepatitis, fever,
cold-like symptoms, etc.
Meningitis, paralysis, encephalitis,
fever, cold-like symptoms, diarrhea, etc.
Respiratory infections, gastroenteritis
Epidemic gastroenteritis
Epidemic gastroenteritis
Gastroenteritis
Acute enteritis
Giardlasis (including diarrhea,
abdominal cramps, weight loss)
Diarrhea and dysentery
Toxoplasmosis
Digestive and nutritional disturbances,
abdominal pain, vomiting, restlessness
May produce symptoms such as
coughing, chest pain, and fever
Abdominal pain, diarrhea, anemia,
weight loss
Fever, abdominal discomfort, muscle
aches, neurological symptoms
Nervousness, insomnia, anorexia,
abdominal pain, digestive disturbances
Nervousness, insomnia, anorexia,
abdominal pain, digestive disturbances
Hookworm disease
Taeniasis
Source: Kowal (19B5) and EPA (1989).
2.3 How Well Do Pathogens Survive in the
Environment?
The potential for exposure diminishes over tune as envi-
ronmental conditions such as heat, sunlight, desiccation, and
other microorganisms destroy pathogens that may be present in
sewage sludge. Table 2-2 summarizes the survival rates of four
types of pathogenic organisms on soil and on plants. Because
protozoan cysts on soil and plants are rapidly killed by envi-
ronmental factors, the threat to public health and animals from
protozoa in sewage sludge is minunal. Bacteria, viruses, and
helminths (particularly helminth eggs, which are the hardiest
part of the helminth life cycle) are of much greater concern.
For this reason, Part 503 contains requirements for the reduc-
tion of bacteria, viruses, and helminths in sewage sludge, but
does not contain requirements for the reduction of protozoa.
Regrowth of Bacteria
Some bacteria are unique among sewage sludge pathogens
in their ability to regrow. Even very small populations of certain
bacteria can rapidly proliferate under the right conditions, e.g.,
hi sewage sludges where the bacterial populations have been
essentially eliminated through treatment (see Section 2.4). 'Vi-
ruses, helminths, and protozoa cannot regrow outside their spe-
cific host organism(s). Once reduced by treatment, their
populations stay reduced. Part 503 contains specific require-
ments designed to ensure that regrowth of bacteria has not
occurred prior to use or disposal.
2.4 How Can the Public and Animals Be Protected?
Public health and animals can be protected from sewage
sludge pathogens in several ways:
• Reduce the number of pathogens in sewage sludge through
treatment and/or environmental attenuation.
• Reduce transport of pathogens by reducing the attractiveness
of the sewage sludge to disease vectors (insects, birds, ro-
dents, and other living organisms that can transport patho-
gens).
• Limit human and animal contact with the sewage sludge
through site restrictions to allow natural die-off to reduce
pathogen levels to low levels.
Part 503 uses a combination of all these approaches (see Chap-
ters 3 through 6 for a description of the requirements).
Pathogen Reduction
Reduction in the number of pathogens can be achieved
technologically—by adequately treating sewage sludge prior to
use or disposal—and through environmental attenuation (see
Section 2.3 above). Many sewage sludge treatment processes
are available that use a variety of approaches to reduce patho-
gens and alter the sewage sludge so that it becomes a less
effective medium for microbial growth and vector attraction
(Table 2-3). They vary significantly in their effectiveness. For
example, some processes (e.g., high pH conditions) may com-
-------
Table 2-2. Survival Times of Pathogens in Soil and on Plant Surfaces'1
Soil
Plants
Pathogen
Bacteria
Viruses
Protozoan cystsd
Helminth ova
Absolute Maximum3
1 year
1 year0
10 days
7 years
Common Maximum
2 months
3 months
2 days
2 years
Absolute Maximumb
6 months
2 months
5 days
5 months
Common Maximum
1 month
1 month
2 days
1 month
"For survival rates, see Sorber and Moore (1986).
bGreater survival time is possible under unusual conditions such as consistently low temperatures or highly sheltered conditions (e.g., helminth ova
below the soil in fallow fields) (Jakubowski, 1988).
cSobsey and Shields, 1987.
dl_ittle, if any, data are available on the survival times of Giardia cysts and Cryptosporidum oocysts.
Source: Kowal, 1985.
Table 2-3. General Approaches to Controlling Pathogens and Vector Attraction in Sewage Sludge
Approach Effectiveness
Process Examples3
Kill pathogens with high temperatures (tem-
peratures may be generated by chemical, bio-
logical, or physical processes).
Kill pathogens with radiation.
Kill pathogens using chemical disinfectants.
Inhibit pathogen growth by reducing the sew-
age sludge's volatile organic content (the mi-
crobial food source).
Inhibit pathogen survival by removing moisture
from the sludge.
Depends on time and temperature. Sufficient
temperatures maintained for sufficiently long
time periods can reduce bacteria, viruses,
protozoan cysts, and helminth ova to below
detectable levels. Helminth ova are the most
resistant to high temperatures.
Depends on dose. Sufficient doses can
reduce bacteria, viruses, protozoan cysts,
and helminth ova to below detectable levels.
Viruses are most resistant to radiation.
Substantially reduces bacteria and viruses
and vector attraction. Probably reduces
protozoan cysts. Does not effectively reduce
helminth ova unless combined with heat.
Reduces bacteria. Reduces vector attraction.
Reduces viruses and bacteria. Reduces
vector attraction as long as the sewage
sludge remains dry. Probably effective in
destroying protozoan cysts. Does not
effectively reduce helminth ova unless
combined with other processes such as
high temperature.
•Composting (uses biological processes to
generate heat).
• Heat drying and heat treatment (use physical
processes to generate heat, e.g., hot gases,
heat exchangers).
• Pasteurization (physical heat, e.g., hot
gases, heat exchangers).
•Aerobic digestion (biological heat).b
•Anaerobic digestion (physical heat).b
•Gamma and high-energy electron beam
radiation.
• Superchlorination.
• Lime stabilization.
•Aerobic digestion.
•Anaerobic digestion.
•Composting."
•Air or heat drying.
aSee Chapters 9 and 10 for a description of these processes. Many processes use more than one approach to reduce pathogens.
bEffectiveness depends on design and operating conditions.
pletely destroy bacteria and viruses but have little or no effect
on helminth eggs. The effectiveness of a particular process can
also vary depending on the conditions under which it is oper-
ated. For example, the length of time and the temperature to
which sewage sludge is heated is critical to the effectiveness of
heat-based treatment processes.
Part 503 lists sewage sludge treatment technologies that
are judged to produce sewage sludges with pathogens suffi-
ciently reduced to protect public health and animals. The regu-
lation also allows the use of any other technologies that produce
a sewage sludge with adequately reduced pathogens as demon-
strated through microbiological monitoring. The Part 503 re-
quirements also include site restrictions to allow environmental
factors to further reduce pathogens in treated sewage sludge
that is used or disposed.
2.5 How Can Pathogen Reduction Be Measured?
Under Part 503, microbiological analysis of sewage sludge
(see photographs on pages 9 and 10) is an important means of
determining the effectiveness of a sewage sludge treatment
-------
process in reducing pathogens. Methods have not yet been
developed to detect all pathogens that may occur in sewage
sludge, and it would be impractical to run all the tests that do
exist. For this reason, Part 503 requires monitoring for repre-
sentative pathogens and nonpathogenic indicator organisms, as
described below.
Nonpathogenic Indicators
As detailed in Chapters 4 and 5, some of the Part 503
requirements call for monitoring of fecal coliform bacteria.
These bacteria are commonly used as indicators of the potential
presence of pathogens in sewage sludges. They are abundant
in human feces and therefore are always present in untreated
sewage sludges. They are easily and inexpensively measured.
Although fecal coliforms themselves are usually not harmful to
humans, their presence indicates the presence of fecal waste
which may contain pathogens.
Direct Monitoring for Pathogens
Part 503 also requires direct monitoring for the three more
common types of pathogens—bacteria, viruses, and viable
helminth ova. For viable helminth ova, a single test is available
that monitors forAscaris ova and thereby serves as an indicator
for several other helminth species (Toxocara, Trichuris, and
Hymenolepis—see photographs on pages 9 and 10). The Ana-
lytical Method for Viable Helminth Ova, provided in Appendix
I, involves extraction, concentration, and incubation of recov-
ered ova versus control Ascaris ova to determine viability,
rather than the traditional staining techniques or membrane and
dilution tube culture techniques used for many other pathogens.
For viruses, a test is available that simultaneously monitors for
several enterovirus species (a subset of enteric viruses—see
Table 2-1), which are presumed to be good indicators for other
types of enteric viruses. No such test is available for bacteria.
When direct monitoring of pathogenic bacteria is important,
Part 503 requires monitoring of Salmonella sp. Salmonellae are
bacteria of great concern in sewage sludge. They are also good
indicators of reduction of other bacterial pathogens because
they are typically present in higher densities than are other
bacterial pathogens and are at least as hardy.
2.6 What Units Are Used to Measure Microorganism
Density Under Part 503?
Use of Units of Mass Versus Units of Volume
Density of microorganisms in Part 503 is defined as num-
ber of microorganisms per unit mass of total solids (dry
weight). Ordinarily, microorganism densities are determined as
number per 100 milliliters of wastewater or sewage sludge.
While the use of units of volume is sensible for wastewater, it
is less sensible for sewage sludge. The microorganisms hi
sewage sludge are associated with the solid phase. When sew-
age sludge is diluted, thickened or filtered, the number of mi-
croorganisms per unit volume changes markedly, whereas the
number per unit mass of solids remains almost constant. This
argues for reporting their densities as the number present per
unit mass of solids, which would require that sewage sludge
solids content always be determined when measuring microor-
ganism densities.
A second reason for reporting densities per unit mass of
solids is that sewage sludge application to the land is typically
measured and controlled in units of mass of dry solids per unit
area of land. If pathogen densities are measured as numbers per
unit mass of solids, the rate of pathogen application to the land
is thus directly proportional to the mass of dry sewage sludge
solids applied.
Different Methods for Counting Microorganisms
The methods and units used to count microorganisms vary
depending on the type of microorganism. Viable helminth ova
are observed and counted as individuals under a microscope.
"viruses are usually counted in plaque-forming units (PFU).
Each PFU represents an infection zone where a single infec-
tious virus has invaded and infected a layer of animal cells. For
bacteria, the count is in colony-forming units (CFU) or most
probable number (MPN). CFU is a count of colonies on an agar
plate or filter disk. Because a colony might have originated
from a clump of bacteria instead of an individual, the count is
not necessarily a count of separate individuals. MPN is a sta-
tistical estimate of numbers in an original sample. The sample
is diluted at least once into tubes containing nutrient medium;
there are several duplicates at each dilution. The original bac-
terial density hi the sample is estimated based on the number
of tubes that show growth.
Part 503 Density Limits
Under Part 503, the density limits for the pathogens are
expressed as numbers of PFUs, CFUs, or MPNs per 4 grams
total solids sewage sludge (see Section 4). This terminology
came about because most of the tests started with 100 mL of
sewage sludge which typically contained 4 grams of sewage
sludge solids. Also, expressing the limits on a "per gram" basis
would have required the use of fractions (i.e., 0.25/g or 0.75/g).
Density limits for fecal coliform, however, are given on a "per
gram" basis because these organisms are much more numerous
than pathogens.
-------
Ascaris lumbricoides (or var. suum) eggs, 65 |im, from anaerobi-
cally digested sludge. Two-cell stage. (Photos on this page courtesy
ofFoxetal., 1981)
Toxocara sp. egg, 90 m|i, from raw sewage.
Ascaris lumbricoides (or var. suum) eggs, 65 urn, from anaerobi-
cally digested sludge.
Trichuris sp egg, 80 nm, from anaerobically digested sludge.
-------
Taonla sp. ovum. (Photo courtesy of Fox et al., 1981)
Giardia lamblla cysts. (Photo courtesy of Frank Schaefer, U.S. EPA,
Risk Reduction Engineering Laboratory, Cincinnati, Ohio)
Hymenolepls (tapeworm) ova. (Photo courtesy of Fox et al., 1981)
Preparing compost for pathogen analysis. (Photo courtesy of U.S.
Department of Agriculture, Beltsville, Maryland)
Entamocba coll cyst, 15 urn, from anaeroblcally digested sludge.
(Photo courtesy of Fox et al., 1981)
10
-------
Chapter 3
Overview of Part 503 Subpart D Requirements, Their Applicability,
and Related Requirements
3.1 Introduction
The Subpart D (pathogen and vector attraction reduction)
requirements of the 40 CFR Part 503 regulation apply to sew-
age sludge (both bulk sewage sludge and sewage sludge that is
sold or given away in a bag or other container for application
to the land) and domestic septage applied to the land or placed
on a surface disposal site. The regulated community includes
persons who generate or prepare sewage sludge for application
to the land, as well as those who apply it to the land. Included
is anyone who:
• Generates sewage sludge that is land applied or placed on a
surface disposal site.
• Derives a material from sewage sludge.
• Applies sewage sludge to the land.
• Owns or operates a surface disposal site.
A sewage sludge cannot be applied to land or placed on a
surface disposal site unless it has met the two basic types of
requirements in Subpart D:
• Requirements to ensure reduction of pathogens.
• Requirements to reduce the potential of the sewage sludge
to attract vectors (rodents, birds, insects, and other organ-
isms that can transport pathogens).
These two types of requirements are separated in Part 503
(they were combined in Part 257), which allows flexibility in
how they are achieved. Compliance with the two types of re-
quirements must be demonstrated separately. Therefore, dem-
onstration that a requirement for reduced vector attraction has
been met does not imply that a pathogen reduction requirement
also has been met, and vice versa.
This chapter provides an overview of these requirements,
their applicability, and the requirements related to frequency of
monitoring and recordkeeping. Where relevant, the titles of the
sections in this chapter include the number of the Subpart D
requirement discussed in the section. Chapters 4 through 6
provide detailed information on the pathogen and vector attrac-
tion reduction requirements.
3.2 Pathogen Reduction Requirements
Sewage Sludge [503.32(a) and (b)]
The pathogen reduction requirements for sewage sludge
are divided into two categories: Class A and Class B. These
requirements use a combination of technological and microbio-
logical requirements to ensure reduction of pathogens.
The implicit goal of the Class A requirements is to reduce
the pathogens in sewage sludge (including enteric viruses,
pathogenic bacteria, and viable helminth ova) to below detect-
able levels. The implicit goal of the Class B requirements is to
ensure that pathogens have been reduced to levels that are
unlikely to pose a threat to public health and the environment
under die specific use conditions. For Class B sewage sludge
that is applied to land, site restrictions are imposed to minimize
the potential for human and animal contact with the Class B
sewage sludge for a period of time following land application
until environmental factors have further reduced pathogens.
Class B sludges cannot be sold or given away in bags or other
containers for application to the land. There are no site restric-
tions for Class A sewage sludge.
Domestic Septage [503.32(c)J
As mentioned in Chapter 1, domestic septage is a form of
sewage sludge. The requirements for domestic septage vary
depending on how it is used or disposed. Domestic septage
applied to a public contact site, lawn, or home garden must
meet the same requirements as other forms of sewage sludge.
Separate, less complicated requkements for pathogen reduction
apply to domestic septage applied to agricultural land, forests,
or reclamation sites. These requirements include site restric-
tions to reduce the potential for human contact and to allow for
environmental attenuation, or pH adjustment with site restric-
tions only on harvesting crops. No pathogen requkements apply
if domestic septage is placed on a surface disposal site.
3.3 Vector Attraction Reduction Requirements
[503.33]
Subpart D specifies 12 options to demonstrate reduced
vector attraction. These are referred to in this document as
Options 1 through 12. Table 6-1 summarizes these options, and
Chapter 6 provides more detailed information.
11
-------
Reduction Through Treatment
Options 1 through 8 apply to sewage sludge that has been
treated in some way to reduce vector attraction (e.g., aerobic
or anaerobic digestion, composting, alkali addition, drying).
These options consist of operating conditions or tests to dem-
onstrate that vector attraction has been reduced in the treated
sludge.
Option 12 is a requirement to demonstrate reduced vector
attraction in domestic septage through elevated pH. This option
applies only to domestic septage.
Reduction Through Barriers
Options 9 through 11 are "barrier" methods. These options
require the use of soil as a physical barrier (i.e., by injection,
incorporation, or as cover) to prevent Vectors from coming hi
contact with the sewage sludge. They include injection of sew-
age sludge below the land surface, incorporation of sewage
sludge into the soil, and placement of a cover over the sewage
sludge. Options 9 through 11 apply to both sewage sludge and
domestic septage. Option 11 may only be used at surface dis-
posal sites.
3.4 Applicability of the Requirements [503.15]
The applicability of the pathogen and vector attraction re-
duction requirements is covered in Part 503.15. Tables 3-1 to
3-3 summarize the applicability of the Subpart D requirements
to sewage sludge and domestic septage.
3.5 Frequency of Monitoring
Sewage Sludge [503.16(a)and 503.26(a)]
The Class A and Class B pathogen requirements and the
vector attraction reduction Options 1 through 8 (the treatment-
related methods) all involve some form of monitoring. The
minimum frequency of monitoring for these requirements is
given in Part 503.16(a) for land application and Part 503.26(a)
for surface disposal. The frequency depends on the amount of
sewage sludge used or disposed annually (see Table 3-4). The
larger the amount used or disposed, the more frequently moni-
toring is required.
Domestic Septage [503.16(b) and 503.26(b)J
One of the options that can be used for demonstrating both
pathogen reduction and vector attraction reduction in domestic
septage is to elevate pH to >12 for 30 minutes (see Sections
5.6 and 6.13). When this option is used, each container of
domestic septage (e.g., each tank truck load) applied to the land
or placed on a surface disposal site must be monitored for pH.
3.6 Recordkeeping Requirements [503.17 and
503.27]
Recordkeeping requirements are covered hi Part 503.17 for
land application and Part 503.27 for surface disposal. Records
Table 3-1. Subpart D Requirements for Bulk Sewage Sludge1
Land Application
Applied to
Agricultural
Land, a Forest,
a Public
Contact Site,2
or a
Reclamation
Site3
Applied to
a Lawn
or Home
Garden
Surface Disposal
Pathogen Class A or
Requirements Class B with
site restrictions
Class A4
Vector
Attraction
Reduction
Requirements
Options 1-107
Options
1-87'8
Class A or Class B
excluding the site
restrictions6 unless
the unit is covered at
the end of each
operating day, in
which case no
pathogen
requirements apply8
Options 1-117
1Bulk sewage sludge is sewage sludge that is not sold or given away
in a bag or other container for application to the land.
^Public contact site is land with a high potential for contact by the public,
e.g., public parks, ball fields, cemeteries, plant nurseries, turf farms, and
golf courses.
^Reclamation site is drastically disturbed land (e.g., strip mine, construc-
tion site) that is reclaimed using sewage sludge.
4The regulation does not permit use of a sludge meeting Class B re-
quirements on lawns or home gardens, because it would not be feasible
under these circumstances to impose the site restrictions that are an
integral part of the Class B requirements.
5Class B site restrictions are excluded here because the management
practices in Part 503 Subpart C already impose similar site restrictions
to reduce exposure to pollutants in sewage sludge.
6No pathogen requirement applies when daily cover isolates the sewage
sludge and allows the environment to reduce the pathogens in the
sewage sludge.
7See Chapter 6 for a description of these options.
8The two vector attraction reduction requirements that cannot be met
when bulk sewage sludge is applied to a lawn or a home garden are
injection of the bulk sewage sludge below the land surface and incor-
poration of bulk sewage sludge into the soil. Implementation of these
requirements for bulk sewage sludge applied to a lawn or a home
garden would be difficult, if not impossible.
are required for both sewage sludge and domestic septage. All
records must be retained for 5 years except when the cumula-
tive pollutant loading rates hi Part 503 Subpart B (Land Appli-
cation) of Part 503 are used.1 In that case, certain records must
be kept indefinitely. Some records must be reported to the
permitting authority (see Section 3.7).
Land Application
Records must be kept to ensure that the sewage sludge
meets the applicable pollutant limits, management practices,2
Cumulative pollutant loading rates are not related to pathogen control and therefore are
not covered in this document
Pollutant limits and management practices are not related to the pathogen requirements
and therefore are not covered in this document.
12
-------
Table 3-2. Subpart D Requirements for Sewage Sludge Sold or Given
Away in a Bag or Other Container
Land Application
Surface Disposal
Pathogen
Requirements
Vector Attraction
Reduction
Requirements
Class A1
Options 1-82
N/A
N/A
1Class B requirements do not apply to sewage sludge that is sold or
given away because it is not feasible to impose tie Class B site restric-
tions when sewage sludge is widely distributed in bags or other con-
tainers.
^nly the treatment-related options for vector attraction reduction apply
to sewage sludge that is sold or given away in bags or other containers
for application to the land, because enforcement of the barrier options,
which are implemented at the site of application, would be impossible.
See Chapter 6 for a description of these options.
Table 3-3. Subpart D Requirements for Domestic Septage Applied to
Agricultural Land, a Forest, or a Reclamation Site1 or Placed
on a Surface Disposal Site
Application to Agricultural
Land, a Forest, or a Surface
Reclamation Site2 Disposal
Pathogen
Requirements
Vector Attraction
Reduction
Requirements
Class B site restrictions
only or a pH adjustment
(pH >12 for 30 minutes)
plus restrictions
concerning crop
harvesting
Options 9,10,124
No pathogen
requirement3
Options 9-124
1For application to all other types of land, domestic septage must meet
the same requirements as other forms of sewage sludge (see Tables
3-1 and 3-2).
^Reclamation site is drastically disturbed land (e.g., strip mine, construc-
tion site) that is reclaimed using sewage sludge.
3There is no pathogen requirement for domestic septage placed on a
surface disposal site because site restrictions for grazing of animals,
public access, and crop growing are already imposed by the Part 503
Subpart C management practices to reduce exposure to pollutants in
domestic septage placed on a surface disposal site.
4See Chapter 6 for a description of these options.
one of the pathogen requirements, one of the vector attraction
reduction requirements and, where applicable, the site restric-
tions associated with land application of Class B sludge. When
sewage sludge is applied to land, the person preparing the sew-
age sludge for land application and the person applying bulk
sewage sludge must keep records.3'4 The person applying sew-
age sludge that was sold or given away does not have to keep
records. Table 3-5 summarizes the recordkeeping requirements
for land application.
Person as defined under Part 503.9 may be an individual, association, partnership, corpo-
ration, municipality, state or federal agency, or an agent or employee of a state or federal
agency.
"^hen sewage sludge is prepared by one person, and another person who places it in a
bag or other container for sale or give-away for application to the land changes the quality
of that sewage sludge, both persons must keep the records required of preparers (see Table
3-5).
Table 3-4. Frequency of Monitoring for Land Application and Surface
Disposal
Amount of Sewage Sludge1 (metric
tons dry solids per 365-day period) Frequency
Greater than zero but less than 2902
Equal to or greater than 290 but
less than 1,5002
Equal to or greater than 1,500 but
less than 15.0002
Equal to or greater than 15.0002
Once per year
Once per quarter (four
times per year)
Once per 60 days (six
times per year)
Once per month (12
times per year)
1£/f/jerthe amount of bulk sewage sludge applied to the land, or the
amount of sewage sludge received by a person who prepares sewage
sludge that is sold or given away in a bag or other container for appli-
cation to the land (dry weight basis), or the amount of sewage sludge
(excluding domestic septage) placed on a surface disposal site.
2290 metric tons = 320 tons (approximately 0.9 tons/day for a year)
1,500 metric tons = 1,653 tons (approximately 4.5 tons/day for a year)
15,000 metric tons = 16,534 tons (approximately 45 tons/day for a year)
Surface Disposal
When sewage sludge is placed on a surface disposal site,
the person preparing the sludge and the owner/operator of the
surface disposal site must keep records. In the case of domestic
septage applied to agricultural land, forest, or a reclamation site
or placed on a surface disposal site, the person applying the
domestic septage and the owner/operator of the surface disposal
site may be subject to pathogen-related recordkeeping require-
ments, depending on which vector attraction reduction option
was used. Table 3-6 summarizes the pathogen-related record-
keeping requirements for surface disposal.
Certification Statement
In every case, recordkeeping involves signing a certifica-
tion statement that the requirement has been met. Parts 503.17
and 503.27 of the regulation contain the requked certification
language.
3.7 Reporting Requirements for Sewage Sludge
[503.18 and 503.28]
Reporting requirements for sewage sludge are found in
Part 503.18 for land application and Part 503.28 for surface
disposal. These requirements apply to Class I sludge manage-
ment facilities5 and to publicly owned treatment works either
with a design flow rate equal to or greater than 1 million gallons
per day and/or that serve 10,000 or more people. These facilities
must submit to the permitting authority the records they are
requked to keep as "preparers" of sewage sludge (see Tables
3-5 and 3-6) and/or as the owner/operators of surface disposal
sites (see Table 3-6) on February 19 of each year. There are no
reporting requkements associated with the use or disposal of
domestic septage.
5 A Class I sludge management facility is any publicly owned treatment works (POTW) re-
quired to have an approved pretreatment program under 40 CFR 403.8(a) [including any
POTW located in a state that has assumed local program responsibilities under 40 CFR
403.10(e)] and any treatment woiks treating domestic sewage classified as a Class I sludge
management facility by EPA or the state sludge management program because of the po-
tential for its sewage sludge use or disposal practices to adversely affect public health and
the environment.
13
-------
Table 3-5. Summary of Pathogen-Related and Vector Attraction Reduction-Related Recordkeeping Requirements for Land Application
of Sewage Sludge^
Required Records
Who Must Keep
Records?
Description
of How
Class A
Pathogen
Requirement
Was Met
Description
of How
Class B
Pathogen
Requirement
Was Met
Description of
How the Class
BSite
Restrictions
Were Met at
Each Site
Where Sewage
Sludge Was
Applied
Description of How
Pathogen
Requirement for
Domestic Septage
Applied to
Agricultural Land,
a Forest, or a
Reclamation Site
Was Met
Description of
How Vector
Attraction
Reduction
Requirement
Was Met
Certification
Statement
That the
Requirement
Was Met
Sowago Sludge—Pathogen Requirements
Person preparing Class •
A bulk sowaga sludge
Person preparing Class •
A sowago sludge for
sa!o or give away In a
bag or other container
Person preparing Class •
B sewage sludge
Person applying Class B
sowago sludge
Sewage Sludge—Vector-Attraction Reduction Requirements
Person preparing
sowaga sludge that
moots one of the
treatment-related
vector attraction
reduction
requirements
(Options 1-8)
Person applying sewage
sludgo If a
barrior-retated option.
(Options 9-11) Is used
to moot the vector
attraction reduction
requirement
Domestic Septage
Person applying
domestic septage to
agricultural land,
a forest, or a
reclamation site
'Other recordkeeping requirements, not covered in this document, apply to pollutant limits and management practices.
3.8 Permits and Direct Enforceability [503.3]
Permits
Under Part 503.3(a), the requirements in Part 503 may be
implemented through (1) permits issued to treatment works
treating domestic sewage by EPA or by states with an EPA-ap-
provcd sludge management program, and (2) by permits issued
under Subtitle C of the Solid Waste Disposal Act; Part C of the
Safe Drinking Water Act; the Marine Protection, Research, and
*Sec 40 CFR Paslt 122,123, and SOI; 54 FR 187l6VMay 2,1989; and 58 FR 9404/Febru-
uy 19,1993, for regulations establishing permit requirements and procedures, as well as
requirement* for states wishing to implement approved sewage sludge management pro-
grams as cither pan of their NFDES programs or under separate authority.
Sanctuaries Act of 1972; or the Clean Air Act. Treatment works
treating domestic sewage should submit a permit application6
to the approved state program, or, if there is no such program,
to the EPA Regional Sludge Coordinator (see Appendix A).
Direct Enforceability '
Under Part 503.3(b), the requirements of Part 503 automat-
ically apply and are directly enforceable even when no permit
has been issued.
3.9 Compliance Period [503.2]
Compliance with the Part 503 requirements must be
achieved as expeditiously as possible. Full compliance must be
14
-------
Table 3-6. Summary of Pathogen-Related and Vector Attraction Re-
duction-Related Recordkeeping Requirements for Surface
Disposal of Sewage Sludge1
Required Records
Who Must Keep
Records?
Description
of How
Class A or B
Pathogen
Requirement
Was Met
Description
of How
Vector
Attraction
Reduction
Requirement
Was Met
Certification
Statement
That the
Requirement
Was Met
achieved no later than February 19, 1994, unless compliance
requires construction of new pollution control facilities, in
which case full compliance must be achieved no later than
February 19, 1995. Monitoring, recordkeeping, and reporting
requirements related to pathogens are effective on July 20,
1993.
Sewage Sludge—Pathogen Requirements
Person preparing • •
the sewage
sludge
Sewage Sludge—Vector Attraction Reduction Requirements
Person preparing • •
sewage sludge
that meets one
of the
treatment-related
vector attraction
reduction
requirements
(Options 1-8)
Owner/operator of • •
the surface
disposal site if a
barrier-related
option (Options
9-11) is used to
meet the vector
attraction
reduction
requirement
Domestic Septage
Person who places • •
domestic
septage on the
surface disposal
site if the
domestic
septage meets
Option 12 for
vector attraction
reduction
Owner/operator of • •
the surface
disposal site if a
barrier-related
option (Options
9-11) is used to
meet the vector
attraction
reduction
requirement
1Other recordkeeping requirements, not covered in this document, apply
to pollutant limits and management practices.
15
-------
-------
Chapter 4
Class A Pathogen Requirements
4.1 Introduction
This chapter discusses the Class A pathogen requirements
in Subpart D of the 40 CFR Part 503 regulation. Sewage sludge
that is sold or given away in a bag or other container for
application to land must meet these requirements (see Section
3.4). Bulk sewage sludge applied to a lawn or home garden
also must meet these requirements. Bulk sewage sludge applied
to other types of land may meet these requirements.
There are six alternative requirements for demonstrating
Class A pathogen reduction. Two of these alternatives provide
continuity with 40 CFR Part 257 by allowing use of Processes
to Further Reduce Pathogens (PFRPs) and equivalent technolo-
gies (see Sections 4.8 and 4.9). Any one of these six alternatives
may be met for the sewage sludge to be Class A with respect
to pathogens. The implicit objective of all these requirements
is to reduce pathogen densities to below detectable limits which
are:
Salmonella sp.
enteric viruses
viable helminth ova
less than 3 per 4 grams total solids
sewage sludge
less than 1 per 4 grams total solids
sewage sludge
less than 1 per 4 grams total solids
sewage sludge
One of the vector attraction reduction requirements (see
Chapter 6) also must be met when sewage sludge is applied to
the land or placed on a surface disposal site.
This chapter discusses the Class A pathogen requirements.
These include:
A requirement concerning the relationship of pathogen re-
duction to reduction of vector attraction (Section 4.2).
The six alternatives for Class A pathogen reduction (Sections
4.4 to 4.9), each of which includes a requirement to monitor
for regrowth (Section 4.3).
Enteric viruses are monitored using a method that detects several enterovirus species—a
subset of enteric viruses. This method is presumed to be a good indicator of enteric vi-
ruses. Since the objective of the regulation is to reduce all enteric viruses to less than 1 per
4 grams total solids sewage sludge, this document refers to "enteric viruses" when dis-
cussing this requirement, although, in reality, the detection method enumerates only en-
tero viruses.
The title of each section provides the number of the Sub-
part D requirement discussed in the section. The exact regula-
tory language can be found in Appendix B, which reproduces
Subpart D. Chapters 7 and 8 provide guidance on the sampling
and analysis needed to meet the Class A microbiological moni-
toring requirements.
4.2 Vector Attraction Reduction to Occur With or
After Class A Pathogen Reduction [503.32(a)(2)]
The order of Class A pathogen reduction in relation to the
reduction of vector attraction is important when certain vector
attraction reduction options are used. Part 503.32(a)(2) requires
that Class A pathogen reduction be accomplished before or at
the same time as vector attraction reduction, except for vector
attraction reduction by alkali addition [503.33(b)(6)] or drying
[503.33(b)(7) and (8)] (see Chapter 6).
This requirement is necessary because Class A sludges
have very low bacterial densities (below detectable levels).
Bacterial regrowth (see Section 2.3) is possible unless a deter-
rent remains in the sewage sludge after the pathogen reduction
process. Drying and alkali addition provide such a deterrent.
So do the nonpathogenic bacterial populations left in the sew-
age sludge when the pathogen reduction process and the vector
attraction reduction process occur at the same time, and when
pathogen reduction occurs before vector attraction reduction.
4.3 Monitoring of Fecal Coliform or Salmonellae to
Detect Regrowth [503.32(a)(3)-(8)]
The potential for regrowth of pathogenic bacteria in Class
A sludges makes it important to ensure that substantial regrowth
has not occurred. For this reason, all the Class A pathogen
requirement alternatives require that:
• Either the density of fecal coliform in the sewage sludge be
less than 1,000 MPN2 per gram total solids (dry weight
basis),
• Or the density of Salmonella sp. bacteria in the sewage be
less than 3 MPN per 4 grams of total solids (dry weight
basis).
^The membrane filter method is not allowed here because, at the low fecal coliform densi-
ties expected, the filter would have too high a loading of sludge solids to permit a reliable
count of the number of fecal coliform colonies.
17
-------
This requirement must be met either:
* At the time of use or disposal,3 or
• At the time the sewage sludge is prepared for sale or give
away in a bag or other container for land application, or
• At the time the sewage sludge or material derived from the
sewage sludge is prepared to meet the requirements in
503.10(b), 503.10(c), 503.10(e), or 503.10(f).4
In each case, the timing represents the last practical moni-
toring point before the sewage sludge is applied to the land or
placed on a surface disposal site. Sewage sludge that is sold or
given away cannot be monitored just prior to actual use or
disposal; instead monitoring is required as it is prepared for
sale or give away. Sewage sludge that meets the 503.10(b, c,
d, or e) requirements is of very high quality with regard to both
pollutants and pathogens and is therefore not subject to further
control. For this reason, the regrowth requirement must be met
at the time the sewage sludge is prepared to meet the 503.10
requirements, which in most cases is the last time the sewage
sludge is controlled with respect to the Part 503 requirements.
The fecal coliform requirement is based on experimental
work by Yanko (1987) which shows that this level of fecal
coliform correlates with a very low level of salmonellae detec-
tion in composted sludge (EPA, 1992). Anecdotal reports sug-
gest that some composting facilities may have difficulty
meeting this requirement even when salmonellae are never de-
tected. This might be expected under several circumstances. For
example, very severe thermal treatment of sewage sludge dur-
ing composting can totally eliminate salmonellae yet leave re-
sidual fecal coliforms. If the product has been poorly
composted and thus is a good food source, fecal coliforms may
have regrown after the compost cooled down from thermophilic
temperatures. Because the salmonellae are absent, they cannot
regrow. An even more probable circumstance could occur if the
sewage sludge had been treated with lime before composting.
Lime effectively destroys salmonellae in sewage sludge and
leaves surviving fecal coliforms (Farrell et al., 1974). Under
conditions favorable for regrowth, the fecal coliforms can re-
grow to levels higher than 1,000 MPN per gram. For this rea-
son, all the Part 503 Class A alternatives allow use of a test (in
lieu of the fecal coliform test) to determine that Salmonella sp.
are below detectable limits.
4.4 Alternative 1: Thermally Treated Sewage Sludge
[503.32(a)(3)]
This alternative may be used when the pathogen reduction
process uses specific time-temperature regimes to reduce patho-
gens. Under these circumstances, time-consuming and expen-
sive tests for the presence of specific pathogens can be avoided.
It is only necessary to demonstrate that:
'Minus the time needed to test the sewage sludge and obtain the test results prior to use or
disposal (sec Section 7.3).
uw 503,10(bXcXc) and (f) requirements arc not discussed in this document
• Either fecal coliform densities are below 1,000 MPN per
gram of total solids (dry weight basis), or Salmonella sp.
bacteria are below detection limits (3 MPN per 4 grams total
solids [dry weight basis]) at the time the sewage sludge is
used or disposed, at the time the sewage sludge is prepared
for sale or give away in a bag or other container for land
application, or at the time the sewage sludge or material
derived from the sewage sludge is prepared to meet the
requirements in 503.10(b), 503.10(c), 503.10(e), or
503.10(f).
• And the required time-temperature regimes are met.
Microbiological Requirement
The microbiological portion of the requirement is designed
to ensure that the microbiological reductions expected as a
result of the time-temperature regimes have actually been at-
tained. This requirement uses the low level of fecal coliform or
nondetection of salmonellae as an indicator of the destruction
of pathogenic bacteria, enteric viruses, and helminths (based on
research by Lee et al. [1989], Yanko [1987], and Martin et al.
[1990], and discussed by Farrell [1992]). The microbiological
requirement also ensures (as described in Section 4.3) that re-
growth of bacterial pathogens has not occurred.
Time-Temperature Requirement
Four different time-temperature regimes are given in Al-
ternative 1. Each regime is based on the percent solids of the
sewage sludge and on operating parameters of the treatment
process. Experimental evidence (EPA, 1992) demonstrates that
these four tune-temperature regimes reduce the pathogenic or-
ganisms to below detectable levels.
The four time-temperature regimes are summarized in Ta-
ble 4-1. They involve two different time-temperature equations.
The equation used in Regimes A through C results in require-
ments that are more stringent than the requirement obtained
using the equation in Regime D. For any given time, the tem-
perature calculated for the Regime D equation will be 3°C
(37°F) lower than the temperature calculated for the Regimes
A through C equation.
A more conservative equation is required for sewage
sludges with 7% or more solids (i.e., those covered by Regimes
A and B) because these sewage sludges form an internal struc-
ture that inhibits the mixing that contributes to uniform distri-
bution of temperature. The more stringent equation is also used
in Regime C (even though this regime applies to sewage
sludges with less than 7% solids) because insufficient informa-
tion is available to apply the less stringent equation for times
less than 30 minutes.
The time-temperature requirements apply to every particle
of sewage sludge processed. Tune at the desired temperature is
readily determined for batch operations, turbulent flow in pipes,
or even laminar flow in pipes (time of contact is one-half the
contact time calculated from the bulk throughput rate). Time of
contact also can be calculated for a number of completely
mixed reactors in series, but for the very large reductions
18
-------
Table 4-1. The Four Time-Temperature Regimes for Alternative 1 (Thermally Treated Sewage Sludge) [503.32(a)(3)]
Regime Part 503 Number Applies to:
Required Time-Temperature
Relationship1
503.32(a)(3)(ii)(A) Sewage sludge with at least 7% solids (except those covered by
Regime B)
503.32(a)(3)(ii)(B) Sewage sludge with at least 7% solids that are small particles
heated by contact with either warmed gases or an immiscible liquid4
503.32(a)(3)(ii)(C) Sewage sludge with less than 7% solids treated in processes with
less than 30 minutes contact time
503.32(a)(3)(ii)(D) Sewage sludge with less than 7% solids treated in processes with
at least 30 minutes contact time
D = 131,700,000/10a1400t
t>50°C(122°F)2
D 2. 0.0139 (i.e., 20 minutes)3
D = 131,700,000/10°-14001
t > 50°C (122°F)2
D > 1.74 x 10"4 (i.e., 15 seconds)5
D=131,700,000/10a1'loot
1.74 x 10-4 (i.e., 15 seconds)
< D < 0.021 (i.e., 30 minutes)6
D = 50,070,000/1 Oa1400t
t>50°C(122°F)2
D > 0.021 (i.e., 30 minutes)7
1D = time in days; t = temperature.
^he restriction to temperatures of at least 50°C (122°F) is imposed because information on the time-temperature relationship at lower temperatures
is uncertain.
3A minimum time at 20 minutes is required to ensure that the sewage sludge has been uniformly heated.
4Two examples of sewage sludge to which this requirement applies are:
— Sewage sludge cake that is mixed with previously dried solids to make the entire mass a mixture of separate particles, and is then dried by
contact with a hot gas stream in a rotary drier.
— Sewage sludge dried in a multiple-effect evaporator system in which the system sludge particles are suspended in a hot oil that is heated by
indirect heat transfer with condensing steam.
sTime-at-temperature of as little as 15 seconds is allowed because, for this type of sewage sludge, heat transfer between particles and the heating
fluid is excellent. Note that the temperature is the temperature achieved by the sewage sludge particles, not the temperature of the carrier medium.
6Time-at-temperature of as little as 15 seconds is allowed because heat transfer and uniformity of temperature is excellent in this type of sewage
sludge. The maximum time of 30 minutes is specified because a less stringent regime (D) applies when time-at-temperature is 30 minutes or more.
7Time-at-temperature of at least 30 minutes is required because information on the effectiveness of this time-temperature regime for reducing
pathogens at temperatures of less than 30 minutes is uncertain.
requked to reduce densities to below detection limits, this type
of processing would require so many reactors in series as to be
totally impractical.
4.5 Alternative 2: Sewage Sludge Treated in a High
pH-High Temperature Process (Alkaline
Treatment) [503.32(a)(4)]
This alternative describes conditions of a particular high
temperature-high pH process that has proven effective in re-
ducing pathogens to below detectable levels. The process con-
ditions required by the regulation are:
• Elevating pH to greater than 12 for more than 72 hours.
• Maintaining the temperature above 52°C (126°F) for at least
12 hours during the period that the pH is greater than 12.
• Air drying to over 50% solids after the 72-hour period of
elevated pH.
The hostile conditions of high pH and high temperature for
prolonged time periods allow a variance to a less stringent
time-temperature regime than for the thermal requirements un-
der Alternative 1. The pH of the sludge is measured at 25°C
(77°F) or an appropriate correction is applied (see Section 7.7).
As with all the Class A alternatives, microbiological moni-
toring for fecal coliforms or salmonellae is requked (see Sec-
tion 4.3) to ensure that pathogens have been reduced and
regrowth has not occurred.
4.6 Alternative 3: Sewage Sludge Treated in Other
Processes [503.32(a)(5)]
This alternative applies to sewage sludge produced by
processes that do not meet the process conditions requked by
Alternatives 1 and 2. This requkement relies on comprehensive
monitoring of bacteria, enteric viruses, and viable helminth ova
to demonstrate adequate reduction of pathogens:
• Either the density of fecal coliform in the sewage sludge
must be less than 1,000 MPN per gram of total solids (dry
weight basis), or the density of Salmonella sp. bacteria in
sewage sludge must be less than 3 MPN per 4 grams of total
solids (dry weight basis) at the time the sewage sludge is
used or disposed, at the time the sewage sludge is prepared
for sale or give away in a bag or other container for land
application, or at the time the sewage sludge or material
derived from the sewage sludge is prepared to meet the
requkements in 503.10(b), 503.10(c), 503.10(e), or
503.10(f).
• The density of enteric viruses in the sewage sludge after
pathogen treatment must be less than 1 PFU per 4 grams of
total solids (dry weight basis).
19
-------
• The density of viable helminth ova in the sewage sludge
after pathogen treatment must be less than 1 per 4 grams of
total solids (dry weight basis).
Testing for enteric viruses and viable helminth ova can be
complicated by the fact that they are sometimes not present in
the untreated sewage sludge. In this case, an absence of the
organisms in the treated sewage sludge does not demonstrate
that the process can reduce them to below detectable limits. For
this reason, Alternative 3 requires that the feed sewage sludge
be analyzed for enteric viruses and viable helminth ova. If these
organisms are not detected in the feed sewage sludge, the sew-
age sludge is presumed to be acceptable as a Class A material
until the next monitoring episode. Monitoring is continued until
enteric viruses and/or viable helminth ova are detected in the
feed sewage sludge (i.e., the density of enteric viruses is greater
than or equal to 1 PFU per 4 grams total solids [dry weight
basis] and/or the density of viable helminth ova is greater than
or equal to 1 per 4 grams of total solids [dry weight basis]). At
this point, the treated sewage sludge is then analyzed to see if
these organisms survived treatment. If enteric viruses densities
are below detection limits, the sewage sludge meets Class A
requirements for enteric viruses and will continue to do so as
long as the treatment process is operated under the same con-
ditions that successfully reduced the enteric virus densities. If
the viable helminth ova densities are below detection limits, the
process meets the Class A requirements for enteric viruses and
will continue to do so as long as the treatment process is
operated under the same conditions that successfully reduced
the viable helminth ova densities. Thus, it is essential to monitor
and document operating conditions until adequate enteric virus
and viable helminth ova reduction have been successfully dem-
onstrated. Samples of untreated and treated sewage sludge must
correspond (see Section 7.4).
Tests for enteric viruses and viable helminth ova take sub-
stantial time: 4 weeks to determine whether helminth ova are
viable, and 2 weeks or longer for enteric viruses. The treatment
works operator does not know whether the feed sewage sludge
has enteric viruses or helminth ova until at least 2 to 4 weeks
after the first samples for testing feed densities are taken. This
problem can be overcome by sampling both the feed and the
treated sewage sludge during each monitoring episode and pre-
serving the treated sewage sludge samples until the results of
the feed analysis indicate whether analysis of the treated sew-
age sludge is necessary. For enteric viruses, the sewage sludge
should be stored frozen, unless the sample can be processed
within 24 hours, in which case the samples may be stored at
4°C (39°F). For viable helminth ova, the sewage sludge should
be stored at about 4°C (39°F) (see Section 8.6).
4.7 Alternative 4: Sewage Sludge Treated in Unknown
Processes [503.32(a)(6)]
This requirement is similar to Alternative 3, except there
is no option to substitute monitoring of effective operating
parameters for microbiological monitoring. The sewage sludge
must meet the following limits at the time the sewage sludge
(or material derived from sludge) is used or disposed, at the
time the sewage sludge is prepared for sale or give away in a
bag or other container for land application, or at the time the
sewage sludge or material derived from the sewage sludge is
prepared to meet the requirements in 503.10(b), 503.10(c),
503.10(e), or 503.10(f):
• Either the density of fecal coliform in the sewage sludge
must be less than 1,000 MPN per gram of total solids (dry
weight basis), or the density of Salmonella sp. bacteria in
sewage sludge must be less than 3 MPN per 4 grams of total
solids (dry weight basis).
• The density of enteric viruses in the sewage sludge must be
less than 1 PFU per 4 grams of total solids (dry weight
basis).
• The density of viable helminth ova in the sewage sludge
must be less than 1 per 4 grams of total solids (dry weight
basis).
This requirement applies in the following situations:
• A sewage sludge treatment process is unknown.
• The sewage sludge was produced with the process operating
at conditions less stringent than the operating conditions at
which the sewage sludge could qualify as Class A under
other alternatives.
• The past history of the sewage sludge is not completely
known.
The requirements for enteric viruses and viable helminth
ova may be modified by the permitting authority. An example
of this situation would be a pile of sewage sludge that had been
stored for many years. If fecal coliform densities are suffi-
ciently low, enteric virus survival is unlikely. In such a case,
the permitting authority may reduce the requirement to test for
enteric viruses, but would probably insist on measuring viable
helminth ova densities.
4.8 Alternative 5: Use of PFRP [503.32(a)(7)]
Alternative 5 provides continuity with the 40 CFR Part 257
regulation. This alternative states that sewage sludge is consid-
ered to be Class A if:
• It has been treated in one of the Processes to Further Reduce
Pathogens (PFRPs) listed in Appendix B of the regulation,
and
• Either the density of fecal coliform in .the sewage sludge is
less than 1,000 MPN per gram total solids (dry weight ba-
sis), or the density of Salmonella sp. bacteria in the sewage
sludge is less than 3 MPN per 4 grams total solids (dry
weight basis) at the time the sewage sludge is used or dis-
posed, at the time the sewage sludge is prepared for sale or
give away in a bag or other container for land application,
or at the time the sewage sludge or material derived from
the sewage sludge is prepared to meet the requirements in
503.10(b), 503.10(c), 503.10(e), or 503.10(f).
20
-------
To meet this requirement, the sewage sludge treatment
processes must be operated according to the conditions listed
in Appendix B of the regulation.
The Appendix B list of PFRPs is reproduced in Table 4-2.
This list is very similar to the PFRP technologies listed in 40
CER Part 257, with two major differences:
• All requirements related to vector attraction reduction have
been removed.
• All the "add-on" processes listed in Part 257 are now full-
fledged PFRPs.
Under this alternative, treatment processes classified as
PFRP under 40 CFR Part 257 can continue to be operated;
however, microbiological monitoring must now be performed
to ensure that the pathogen density levels are below detection
Table 4-2. Processes to Further Reduce Pathogens (PFRPs) Listed in
Appendix B of 40 CFR Part 5031
1. Composting
Using either the within-vessel composting method or the static
aerated pile composting method, the temperature of the sewage
sludge is maintained at 55°C (131°F) or higher for 3 days.
Using the windrow composting method, the temperature of the
sewage sludge is maintained at 55°C (131°F) or higher for 15 days
or longer. During the period when the compost is maintained at 55°C
(131°F) or higher, there shall be a minimum of five turnings of the
windrow.
2. Heat Drying
Sewage sludge is dried by direct or indirect contact with hot gases to
reduce the moisture content of the sewage sludge to 10% or lower.
Either the temperature of the sewage sludge particles exceeds 80°C
(176°F) or the wet bulk temperature of the gas in contact with the
sewage sludge as the sewage sludge leaves the dryer exceeds 80°C
(176°F).
3. Heat Treatment
Liquid sewage sludge is heated to a temperature of 180°C (356°F)
or higher for 30 minutes.
4. Thermophilic Aerobic Digestion
Liquid sewage sludge is agitated with air or oxygen to maintain
aerobic conditions and the mean cell residence time (i.e., the solids
retention time) of the sewage sludge is 10 days at 55°C (131°F) to
60°C (140°F).
5. Beta Ray Irradiation
Sewage sludge is irradiated with beta rays from an electron
accelerator at dosages of at least 1.0 megarad at room temperature
(ca. 20°C [68"F])-
6. Gamma Ray Irradiation
Sewage sludge is irradiated with gamma rays from certain isotopes,
such as Cobalt 60 and Cesium 137, at dosages of at least 1.0
megarad at room temperature (ca. 20°C [68°F]).
7. Pasteurization
The temperature of the sewage sludge is maintained at 70°C (158°F)
or higher for 30 minutes or longer.
1Chapter 10 provides a detailed description of these technologies.
limits and to ensure that regrowth of Salmonella sp. bacteria
does not occur between treatment and use or disposal.
4.9 Alternative 6: Use of a Process Equivalent to
PFRP[503.32(a)(8)]
The 40 CFR Part 257 regulation allowed any treatment proc-
ess to be determined equivalent to a PFRP. Under Alternative 6,
sewage sludge is considered to be a Class A sewage sludge if:
• It is treated by any process equivalent to a PFRP, and
• Either the density of fecal coliform in the sewage sludge is
less than 1,000 MPN per gram total solids (dry weight ba-
sis), or the density of Salmonella sp. bacteria in the sewage
sludge is less than 3 MPN per 4 grams total solids (dry
weight basis) at the time the sewage sludge is used or dis-
posed, at the time the sewage sludge is prepared for sale or
give away in a bag or other container for land application,
or at the time the sewage sludge or material derived from
the sewage sludge is prepared to meet the requirements in
503.10(b), 503.10(c), 503.10(e), or 503.10(f).
Processes Already Recommended as Equivalent
Table 4-3 lists some of the processes recommended to be
equivalent to PFRP under Part 257. Since these processes have
Table 4-3. A Partial List of Processes Recommended as Equivalent to
PFRP Under Part 2571
Operator
Process Description
Scarborough Static pile aerated "composting" operation that
Sanitary uses fly ash from a paper company as a
District bulking agent. The process creates pile
Scarborough, temperatures of 60°C to 70°C (140°F to
Maine 158°F) within 24 hours and maintains these
temperatures for up to 14 days. The material
is stockpiled after 7 to 14 days of "composting"
and then marketed.
Mount Holly Zimpro 50-gpm low-pressure wet air oxidation
Sewage process. The process involves heating raw
Authority primary sewage sludge to 177°C to 204°C
Mount Holly, (350°F to 400°F) in a reaction vessel under
New Jersey pressures of 250 to 400 psig for 15 to 30
minutes. Small volumes of air are introduced
into the process to oxidize the organic solids.
Miami-Dade Anaerobic digestion followed by solar drying.
Water and Sewage sludge is processed by anaerobic
Sewer digestion in two well-mixed digesters operating
Authority in series in a temperature range of 35°C to
Miami, Florida 37°C (95°F to 99°F). Total residence time is 30
days.'The sewage sludge is then centrifuged
to produce a cake of between 15% to 25%
solids. The sewage sludge cake is dried for 30
days on a paved bed at a depth of no more
than 46 cm (18 inches). Within 8 days of the
start of drying, the sewage sludge is turned
over at least once every other day until the
sewage sludge reaches a solids content of
greater than 70%. The PFRP recommendation
was conditional on the microbiological quality
of the sewage sludge.
'These processes were all recommended for site-specific equivalency
(see Section 11.1).
21
-------
already been recommended as equivalent, the sewage sludges
produced by these processes should meet the Class A pathogen
requirements as long as they meet the microbiological require-
ments.
An Approach to Determining Equivalency
One procedure likely to be used for determining equiva-
lency under Part 503 is Alternative 3. This alternative enables
a treatment works to demonstrate, through microbiological
monitoring, that a sewage sludge treatment process effectively
reduces enteric viruses and viable helminth ova to below de-
tectable levels. Once these reductions have been demonstrated,
no further enteric virus or viable helminth ova monitoring is
required as long as the process continues to be operated under
the conditions that produced the reduction (see Section 4.6).
The only further monitoring that must be conducted is the fecal
coliform or salmonellae monitoring required of all Class A
alternatives. Thus, for all practical purposes, a process that
successfully demonstrates all the requirements in Alternative 3
can be considered equivalent to a PFRP.
Who Determines Equivalency?
Part 503 gives the permitting authority responsibility for
determining equivalency under Alternative 6. The EPA's Patho-
gen Equivalency Committee (PEC) is available as a resource
to provide guidance and recommendations on equivalency de-
terminations to both the permitting authority and the regulated
community (see Chapter 11).
22
-------
Chapter 5
Class B Pathogen Requirements and Requirements for Domestic Septage Applied to
Agricultural Land, a Forest, or a Reclamation Site
5.1 Introduction
Class B pathogen requirements can be met in three differ-
ent ways. The implicit objective of all three alternatives is to
ensure that pathogenic bacteria and enteric viruses are ade-
quately reduced in density, as demonstrated by a fecal coliform
density in the treated sludge of 2 million MPN or CPU per
gram total solids sewage sludge (dry weight basis).1 Viable
helminth ova are not necessarily reduced in a Class B sludge.
Unlike a Class A sludge, which is essentially pathogen-
free, a Class B sludge contains some pathogens. For this reason,
site restrictions that restrict crop harvesting, animal grazing,
and public access for a certain period of time until environ-
mental factors have further reduced pathogens have to be met
when a Class B sewage sludge is applied to land. Where ap-
propriate, these restrictions are designed to ensure sufficient
reduction in viable helminth ova—the hardiest of pathogens—
since these pathogens may not have been reduced during sludge
treatment.
The Class B requirements apply to bulk sewage sludge that
is applied to agricultural land, a forest, a public contact site, or
a reclamation site. Sewage sludge that is placed on a surface
disposal site also must meet the Class B pathogen requirement,
unless the active sewage sludge unit on which the sewage
sludge is placed is covered at the end of each operating day
(see Table 3-1).
The requirements for pathogens in domestic septage ap-
plied to agricultural land, forest, or a reclamation site are dif-
ferent from the Class B requirements for sewage sludge applied
to those types of land. Domestic septage applied to other types
of land (e.g., a public contact site) must meet the pathogen
requirements and site restrictions for sewage sludge. No patho-
gen-related requirements apply to domestic septage placed on
a surface disposal site.
'Farrell et al. (1985) have shown that if a processed sewage sludge has a fecal coliform
density of 2 million MPN or CPU per gram, pathogenic viruses and bacteria are reduced.
A comparison of suspended solids densities in entering wastewater to suspended solids
densities in treated sewage sludge shows that this density of fecal coliform in treated
sludge represents a 100-fold (2-log) reduction in fecal coliform density, and is expected to
correlate with an approximately 1.5-log (apprbximately^32-fold) reduction in salmonellae
density and an approximately 13-log (20-fold) reduction in the density of enteric viruses.
Class B sewage sludge and domestic septage also must
meet a vector attraction reduction requirement (see Chapter 6).
Sections 5.2 to 5.4 discuss the three alternative Class B
pathogen requirements for sewage sludge. Section 5.5 discusses
the site restrictions and Section 5.6 presents the requirements
for domestic septage applied to agricultural land, forests, or
reclamation sites. The title of each section provides the number
of the Subpart D requirement discussed in the section. The
exact regulatory language can be found hi Appendix B, which
reproduces the Subpart D regulation. Chapters 7 and 8 provide
guidance on the sampling and analysis necessary to meet the
Class B microbiological requirements.
5.2 Sewage Sludge Alternative 1: Monitoring of Fecal
Coliform [503.32(b)(2>]
Alternative 1 requires that seven samples of treated sewage
sludge be collected at the time of use or disposal, and that the
geometric mean fecal coliform density of these samples be less
than 2 million CPU or MPN per gram of sewage sludge solids
(dry weight basis). .
This approach uses fecal coliform density as an indicator
of the average density of bacterial and viral pathogens. Over
the long term, fecal coliform density is expected to correlate
with bacterial and viral pathogen density in sewage sludge
treated by biological treatment processes (EPA, 1992).
This alternative requires analysis of multiple samples dur-
ing each monitoring episode because the methods used to de-
termine fecal coliform density (membrane filter methods and
the MPN dilution method) have poor precision and sewage
sludge quality varies. Use of at least seven samples is expected
to reduce the standard error to a reasonable value.
The standard deviation can be a useful predictive tool. A
relatively high standard deviation for the fecal coliform density
indicates a wide range in the densities of the individual samples.
The wider this range (i.e, the higher the standard deviation),
the less the treatment process can be relied on to consistently
produce sewage sludge that will meet the requirement. A high
standard deviation (e.g., a log standard deviation > 0.3) can
therefore alert treatment workers of the potential need for proc-
ess modifications to improve consistency and reliability.
23
-------
5.3 Sewage Sludge Alternative 2: Use of PSRP
[503.32(b)(3)]
Class B Alternative 2 provides continuity with the 40 CER
Part 257 regulation. Under this alternative, sewage sludge is
considered to be Class B if it is treated in one of the "Processes
to Significantly Reduce Pathogens" (PSRPs) listed in Appendix
B of Part 503. The listed PSRPs are reproduced in Table 5-1
and described in detail in Chapter 9. They are similar to the
PSRPs listed in the Part 257 regulation, except that all condi-
tions related to reduction of vector attraction have been re-
moved. Under this alternative, sewage sludge treated by
processes that are PSRPs under 40 CFR Part 257 are Class B
with respect to pathogens. Unlike the comparable Class A re-
quirement (see Section 4.8), this Class B alternative does not
require microbiological monitoring.
Table 5-1. Processes to Significantly Reduce Pathogens (PSRPs)
Listed in Appendix B of 40 CFR Part 5031
1. Aoroblc Digestion
Sowago sludge Is agitated with air or oxygen to maintain aerobic
conditions for a specific mean cell residence time (i.e., solids
retention time) at a specific temperature. Values for the mean cell
residence time and temperature shall be between 40 days at 20°C
(68'F) and 60 days at 15°C (59°F).
2. Air Drying
Sowago sludge Is dried on sand beds or on paved or unpaved
basins. Ths sewage sludge dries for a minimum of 3 months. During
2 of Iho 3 months, the ambient average daily temperature is above
O'C (32«F).
3. Anaerobic Digestion
Sewage sludge Is treated in the absence of air for a specific
moan cell residence time (I.e., solids retention time) at a specific
temperature. Values for the mean cell residence time and
tomporature shall be between 15 days at 35°C to 55°C (131°F)
and 60 days at 20"C (68°F).
4. Composting
Using either the within-vossel, static aerated pile, or windrow
composting methods, the temperature of the sewage sludge is raised
to 409C (104»F) or higher and remains at 40°C (104°F) or higher for
5 days. For 4 hours during the 5-day period, the temperature in the
compost pile exceeds 55"C (131°F).
5. LImo Stabilization
Sufficient lime Is added to the sewage sludge to raise the pH of the
sowage sludge to 12 after 2 hours of contact.
'Chapter 9 provides a detailed description of these technologies.
5.4 Sewage Sludge Alternative 3: Use of Processes
Equivalent to PSRP [503.32(b)(4>]
The former Part 257 regulation allowed the sewage sludge
to be treated by a process determined to be equivalent to a
PSRP. Under Alternative 3, sewage sludge treated by any proc-
ess determined to be equivalent to a PSRP is considered to be
a Class B sewage sludge.
Table 5-2 provides a partial list of processes that were
recommended as equivalent to PSRP under 40 CER Part 257.
Because these processes are already recommended as equiva-
Table 5-2. A Partial List of Processes Recommended as Equivalent to
PSRP Under Part 2571
Operator
Process Description
Town of Telluride,
Colorado
Comprehensive
Materials
Management,
Inc.
Houston, Texas
N-Viro Energy
Systems, Ltd.
Toledo, Ohio
Public Works
Department
Everett,
Washington
Haikey Creek
Wastewater
Treatment Plant
Tulsa, Oklahoma
Ned K. Burleson &
Associates, Inc.
Fort Worth,
Texas
Combination oxidation ditch, aerated storage,
and drying process. Sewage sludge is treated
in an oxidation ditch for at least 26 days and
then stored in an aerated holding tank for up
to a week. Following dewatering to 18%
solids, the sewage sludge is dried on a paved
surface to a depth of 2 feet (0.6 m). The
sewage sludge is turned over during drying.
After drying to 30% solids, the sludge is
stockpi|ed prior to land application. Together,
the drying and stockpiling steps take
approximately 1 year. To ensure that PSRP
requirements are met, the stockpiling period
must include one full summer season.
Use of cement kiln dust (instead of lime) to
treat sewage sludge by raising sewage sludge
pH to at least 12 after 2 hours of contact.
Dewatered sewage sludge is mixed with
cement kiln dust in an enclosed system.
Use of cement kiln dust and lime kiln dust
(instead of lime) to treat sewage sludge by
raising the pH. Sufficient lime or kiln dust is
added to sewage sludge to produce a pH of
12 for at least 12 hours of contact.
Anaerobic digestion of lagooned sewage
sludge.-Suspended solids had accumulated in
a 30-acre (12-hectare) aerated lagoon that
had been used to aerate wastewater. The
lengthy detention time in the lagoon (up to 15
years) resulted in a level of treatment
exceeding that provided by conventional
anaerobic digestion. The percentage of fresh
or relatively unstabilized sewage sludge was
very small compared to the rest of the
accumulation (probably much less than 1% of
the whole).
Oxidation ditch treatment plus storage.
Sewage sludge is processed in aeration
basins followed by storage in aerated sludge
holding tanks. The total sewage sludge
aeration time is greater than the aerobic
digestion operating conditions specified in the
Part 503 regulation of 40 days at 20°C (68°F)
to 60 days at 15°C (59°F). The oxidation ditch
sludge is then stored in batches for at least
45 days in an unaerated condition or 30 days
under aerated conditions.
Aerobic digestion for 20 days at 30°C (86°F)
or 15 days at 35°C (95°F).
1AII processes were recommended for site-specific equivalency, except
the N-Viro System, which was recommended for national equivalency
(see Section 11.1 for definition of site-specific and national equivalency).
lent, the sewage sludge treated by these processes is Class B
with respect to pathogens.
Part 503 gives the permitting authority responsibility for
determining equivalency. The Pathogen Equivalency Commit-
tee is available as a resource to provide guidance and recom-
mendations on equivalency determinations to the permitting
authorities (see Chapter 11).
24
-------
5.5 Site Restrictions [503.32(b)(5>]
Sewage sludge that meets the Class B requkements may
contain reduced but still significant densities of pathogenic bac-
teria, viruses, and viable helminth ova. Thus, site restrictions
are needed to further reduce pathogenic organisms if the sewage
sludge is applied to land. These requirements are based on
scientific data regarding how rapidly pathogens die off on the
soil surface or within the soil (EPA, 1992). The site restrictions
for Class B sewage sludges are summarized below. The regu-
latory language is given in italics.
Food Crops with Harvested Parts That Touch the
Sewage Sludge/Soil Mixture
503.32(b)(5)(i): Food crops with harvested parts that touch the
sewage sludge/soil mixture and are totally above the land sur-
face shall not be harvested for 14 months after application of
sewage sludge.
This time frame is sufficient to enable environmental con-
ditions such as sunlight, temperature, and desiccation, to reduce
pathogens on the land surface to below detection limits. Note
that the restriction applies to harvesting. Growing of these food
crops (such as melons, cucumbers, or strawberries) can begin
prior to 14 months after application, as long as the crops are
not ready for harvesting earlier than 14 months after sewage
sludge application.
Food Crops with Harvested Parts Below the Land
Surface
503.32(b)(5)(ii): Food crops with harvested parts below the
surface of the land shall not be harvested for 20 months after
application of sewage sludge when the sewage sludge remains
on the land surface for 4 months or longer prior to incorpora-
tion into the soil.
For example, for a September 1994 harvest, sewage sludge
could be applied to the soil surface up to the end of December
1992, plowed or disked into the soil in April 1993, and the crop
could be harvested in September 1994. Examples of crops with
harvested parts below the land surface are potatoes, radishes,
and carrots.
503.32(b)(5)(iii): Food crops with harvested parts below the
surface of the land shall not be harvested for 38 months after
application of sewage sludge when the sewage sludge remains
on the land surface for less than 4 months prior to incorpora-
tion into the soil.
Contamination of the surface of root crops with viable
helminth ova is a principal concern under these circumstances.
Four months is considered the minimum time for environ-
mental conditions to reduce viable helminth ova in sewage
sludge on the land surface to negligible levels. Sewage sludge
incorporated into the soil surface less than 4 months after ap-
plication may contain significant numbers of viable helminth
ova. Once incorporated into the soil, die-off of these organisms
proceeds much more slowly; therefore, a substantially longer
waiting period is required to protect public health.
Food Crops, Feed Crops, and Fiber Crops
503.32(b)(5)(iv): Food crops, feed crops, and fiber crops shall
not be harvested for 30 days after application of sewage sludge.
This restriction covers food crops that are not covered by
503.32(b)(i-iii) (i.e., it covers food crops that do not have har-
vested parts that touch the sewage sludge/soil mixture or that
are below the land surface). The restriction also applies to all
feed and fiber crops. These crops may become contaminated
when sewage sludge is applied to the land. Harvesting of these
crops could result in the transport of sewage sludge pathogens
from the growing site to the outside environment. After 30 days,
however, any pathogens in sewage sludge that may have ad-
hered to the crop during application will likely have been re-
duced to negligible levels. Hay is an example of a crop covered
by this restriction.
Animal Grazing
503.32(b)(5)(v): Animals shall not be allowed to graze on the
land for 30 days after application of sewage sludge.
Sewage sludge can adhere to animals that walk on sew-
age-sludge-amended land and thereby be brought into potential
contact with humans who come in contact with the animals (for
example, riding horses and milking cows allowed to graze on
a sewage-sludge-amended pasture). Thirty days is sufficient to
substantially reduce the pathogens in surface-applied sewage
sludge, thereby significantly reducing the risk of human and
animal contamination.
Turf Growing
503.32(b)(5)(vi): Turf grown on land where sewage sludge is
applied shall not be harvested for 1 year after application of
the sewage sludge 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.
The 1-year waiting period is designed to significantly re-
duce pathogens in the soil so that subsequent contact of the turf
layer will not pose a risk to public health and animals. A per-
mitting authority may reduce this time period in low-risk ap-
plications, e.g., turf applied by the commercial grower's staff
to lawns that will not experience public traffic immediately
after application.
Public Access
503.32(b)(5)(vii): Public access to land with a high potential
for public exposure shall be restricted for 1 year after applica-
tion of the sewage sludge.
As with the turf requirement above, a 1-year waiting period
is necessary to protect public health and animals in a potentially
high-exposure situation. A baseball diamond or a soccer field
is an example of land with a high potential for public exposure.
The land gets heavy use and contact with the soil is substantial
(players fall on it and dust is raised which is inhaled and
ingested).
25
-------
503,32(b)(5)(viii): Public access to land with a low potential
for public exposure shall be restricted for 30 days after appli-
cation of the sewage sludge.
A farm field used to grow corn or soybeans is an example
of a low potential for public exposure. Even farm family mem-
bers walk about very little on such fields.
5.6 Domestic Septage [50332(c>]
Under Part 503.32(c), pathogen reduction in domestic sep-
tage applied to agricultural land, a forest, or a reclamation site2
may be reduced in one of two ways:
• Either all the Class B site restrictions under 503.32(b)(5)—
see Section 5.5, above—must be met,
• Or the pH of the domestic septage must be raised to 12 or
higher by alkali addition and maintained at pH 12 or higher
for 30 minutes without adding more alkali, and the site
restrictions on crop harvesting in 503.32(b)(5)(i-iv) must be
met (see Section 5.5). The regulation uses the term alkali in
the broad sense to mean any substance that causes an in-
crease in pH.
The pH requirement applies to every container of domestic
septage applied to the land, which means that the pH of each
container must be monitored.
The first alternative reduces exposure to pathogens in land-
applied domestic septage while environmental factors attenuate
pathogens. The second alternative relies on alkali treatment to
reduce pathogens and contains the added safeguard of restrict-
ing crop harvesting, which prevents exposure to crops grown
on domestic septage-amended soils.
'diii A or B sewage sludge requirements apply to domestic septage applied to all other
types of Und. No pathogen-related requirements apply to domestic septage placed on a
surface disposal site.
26
-------
Chapter 6
Requirements for Reducing Vector Attraction
6.1 Introduction
The pathogens in sewage sludge pose a disease risk only
if there are routes by which the pathogens are brought into
contact with humans or animals. A principal route for transport
of pathogens is vector transmission. Vectors are any living or-
ganisms capable of transmitting a pathogen from one organism
to another either mechanically (by simply transporting the
pathogen) or biologically by playing a specific role in the life
cycle of the pathogen. Vectors for sewage sludge pathogens
would most likely include insects, rodents, and birds.
The Part 503 regulation contains 12 options, described
below and summarized in Table 6-1, for demonstrating reduced
vector attraction of sewage sludge. These requirements are de-
signed to either reduce the attractiveness of sewage sludge to
vectors (Options 1 through 8 and 12) or prevent the vectors
from coming hi contact with the sewage sludge (Options 9
through 11). Guidance on when and where to sample sewage
sludge to meet these requirements is provided in Chapter 7.
As mentioned in Chapter 3, compliance with the vector
attraction reduction requirements must be demonstrated sepa-
rately from compliance with the pathogen reduction require-
ments. Therefore, demonstration of adequate vector attraction
reduction (e.g., through reduction of volatile solids by 38% as
described below) does not demonstrate achievement of ade-
quate pathogen reduction.
Volatile solids reduction is typically achieved by anaerobic
or aerobic digestion. These processes degrade most of the bio-
degradable material to lower activity forms. Any biodegradable
material that remains characteristically degrades slowly—so
slowly that the vectors that would be attracted to unprocessed
sewage sludge are not drawn to it.
6.3 Option 2: Additional Digestion of Anaerobically
Digested Sewage Sludge [503.33(b)(2>]
Under this option, an anaerobically digested sewage sludge
is .considered to have achieved satisfactory vector attraction
reduction if it loses less than 17% additional volatile solids
when it is anaerobically batch-digested hi the laboratory in a
bench-scale unit at 30°C to 37°C (86°F to 99°F) for an addi-
tional 40 days. Procedures for this test are presented in Appen-
dix D.
Frequently, sewage sludges have been recycled through the
biological wastewater treatment section of a treatment works
or have resided for long periods of time in the wastewater
collection system. During this time they undergo substantial
biological degradation. If they are subsequently treated by an-
aerobic digestion for a period to time, they are adequately re-
duced in vector attraction, but because they entered the digester
already partially stabilized, the volatile solids reduction after
treatment is frequently less than 38%. The additional digestion
test is used to demonstrate that these sewage sludges are indeed
satisfactorily reduced in vector attraction.
6.2 Option 1: Reduction in Volatile Solids Content
[503.33(b)(l>]
Under this option, reduction of vector attraction is achieved
if the mass of volatile solids in the sewage sludge is reduced
by at least 38% during sludge treatment. (This is the amount
of volatile solids reduction that can be attained at the "good
practice" recommended conditions for anaerobic digestion of
15 days residence time at 35°C [95°F] in a completely mixed
high-rate digester.) The volatile solids reduction can include
any additional volatile solids reduction that occurs before the
sewage sludge leaves the treatment works, such as might occur
when the sewage sludge is processed on drying beds or in
lagoons, or when sewage sludge is composted. Volatile solids
reduction is calculated by a volatile solids balance around the
digester or by the Van Kleek formula (Fisher, 1984). Guidance
on methods of calculation is provided in Appendix C.
6.4 Option 3: Additional Digestion of Aerobically
Digested Sewage Sludge [503.33(b)(3>]
Under this option, an aerobically digested sewage sludge
with 2% or less solids is considered to have achieved satisfac-
tory vector attraction reduction if it loses less than 15% addi-
tional volatile solids when it is aerobicalTy batch-digested in
the laboratory in a bench-scale unit at 20°C (68°F) higher for
an additional 30 days. Procedures for this test are presented in
Appendix D. The test can be run on sewage sludges up to 2%
solids and does not require a temperature correction for sewage
sludges not initially digested at 20°C (68°F). Sewage sludges
with greater than 2% solids can be diluted to 2% solids with
effluent, and the test can then be run on the diluted sludge.
This option is appropriate for aerobically digested sewage
sludges that cannot meet the 38% volatile solids reduction re-
27
-------
Table 6-1. Summary of Requirements for Vector Attraction Reduction Under Part 503
Requirement What Is Required?
Most Appropriate for:
Option 1 At least 38% reduction in volatile solids during sewage
503.33(b}(1) sludge treatment
Option 2 Less than 17% additional volatile solids loss during bench-
503.33(b){2) scale anaerobic batch digestion of the sewage sludge for
40 additional days at 30°C to 37°C (86°F to 99°F)
Option 3 Less than 15% additional volatile solids reduction during
G03.33(b)(3) bench-scale aerobic batch digestion for 30 additional days
at 20°C (68°F)
Option 4 SOUR at 20°C (68°F) is £1.5 mg oxygen/hr/g total
SQ3.33(b){4) sewage sludge solids
Option 5 Aerobic treatment of the sewage sludge for at least 14
503.33{b)(5) days at over 40°C (104°F) with an average temperature
of over 45"C (113°F)
Option 6 Addition of sufficient alkali to raise the pH to at least 12 at
503.33{b}{6) 25°C (77°F) and maintain a pH £12 for 2 hours and a pH
£11.5 for 22 more hours
Option 7 Percent solids £75% prior to mixing with other materials
503.33{bX7)
Option 8 Percent solids £90% prior to mixing with other materials
503.33(b)(8)
Option 9 Sewage sludge is Injected into soil so that no significant
503.33(b)(9) amount of sewage sludge is present on the land surface
1 hour after Injection, except Class A sewage sludge
which must be injected within 8 hours after the pathogen
reduction process.
Option 10 Sewage sludge is incorporated into the soil within 6 hours
503.33(b)(10) after application to land or placement on a surface
disposal site, except Class A sewage sludge which must
be applied to or placed on the land surface within 8 hours
after fie pathogen reduction process.
Option 11 Sewage sludge placed on a surface disposal site must be
503.33(fa){11) covered with soil or other material at the end of each
operating day.
Option 12 pH of domestic septage must be raised to £12 at 25°C
503.33(b)(12) (77°F) by alkali addition and maintained at &12 for 30
minutes without adding more alkali.
Sewage sludge processed by:
• Anaerobic biological treatment
• Aerobic biological treatment
• Chemical oxidation
Only for anaerobically digested sewage sludge that cannot
meet the requirements of Option 1
Only for aerobically digested sewage sludge with 2% or less
solids that cannot meet the requirements of Option 1—e.g.,
sewage sludges treated in extended aeration plants
Sewage sludges from aerobic processes (should not be
used for composted sludges)
Composted sewage sludge (Options 3 and 4 are likely to be
easier to meet for sludges from other aerobic processes)
Alkali-treated sewage sludge (alkalies include lime, fly ash,
kiln dust, and wood ash)
Sewage sludges treated by an aerobic or anaerobic process
(i.e., sewage sludges that do not contain unstabilized solids
generated in primary wastewater treatment)
Sewage sludges that contain unstabilized solids generated
in primary wastewater treatment (e.g., any heat-dried
sewage sludges)
Sewage sludge applied to the land or placed on a surface
disposal site. Domestic septage applied to agricultural land,
a forest, or a reclamation site, or placed on a surface
disposal site
Sewage sludge applied to the land or placed on a surface
disposal site. Domestic septage applied to agricultural land,
forest, or a reclamation site, or placed on a surface disposal
site
Sewage sludge or domestic septage placed on a surface
disposal site
Domestic septage applied to agricultural land, a forest, or a
reclamation site or placed on a surface disposal site
quired by Option 1. These include sewage sludges from ex-
tended aeration and oxidation ditch plants, where the nominal
residence time of sewage sludge leaving the wastewater treat-
ment processes section generally exceeds 20 days. In these
cases, the sewage sludge may already have been substantially
reduced in biological degradability prior to aerobic digestion.
6.5 Option 4: Specific Oxygen Uptake Rate (SOUR)
for Aerobically Digested Sewage Sludge
[503.33(b)(4)]
For an aerobically digested sewage sludge, reduction in
vector attraction can also be demonstrated if the SOUR of the
sewage sludge to be used or disposed is determined to be equal
to or less than 1.5 mg of oxygen per hour per gram of total
sewage sludge solids (dry weight basis) at 20°C (eS0!7).1 This
test is based on the fact that if the sewage sludge consumes
very little oxygen, its value as a food source for vectors is very
low and therefore vectors are unlikely to be attracted to the
sewage sludge.
Frequently aerobically digested sewage sludges are circu-
lated through the aerobic biological wastewater treatment proc-
'SOUR is defined in Part 503 as the mass of oxygen consumed per unit time per unit mass
of total solids (dry weight basis) in the sewage sludge. SOUR is usually based on total
suspended volatile solids rather than total solids, because it is assumed that it is the vola-
tile matter in the sewage sludge that is being oxidized. The SOUR definition in Part 503 is
based on total solids primarily to reduce the number of different determinations needed.
Generally the range in the ratio of volatile solids to total solids in aerobically digested
sewage sludges is not large. The standard required for SOUR based on total solids will
merely be slightly lower than if it had been based on volatile suspended solids to indicate
the same endpoint.
28
-------
ess for as long as 30 days. In these cases, the sewage sludge
entering the aerobic digester is already partially digested, which
makes it difficult to demonstrate the 38% reduction required by
Option 1.
The oxygen uptake rate depends on the conditions of the
test and, to some degree, on the nature of the original sewage
sludge before aerobic treatment. It should be noted that the
SOUR method may be unreliable at solids content above 2%
and that it requires a poorly defined temperature correction at
temperatures differing substantially from 20°C (68°F). Guid-
ance on performing the SOUR test and on sewage sludge-de-
pendent factors are provided in Appendix D.
6.6 Option 5: Aerobic Processes at Greater Than 40°C
[503.33(b)(5)]
The sewage sludge must be aerobically treated for 14 days
or longer during which time the temperature must be over 40°C
(104°F) and the average temperature higher than 45°C (113°F).
This option applies primarily to composted sewage sludge.
These processed sewage sludges generally contain substantial
amounts of partially decomposed organic bulking agents, in
addition to sewage sludge. Application of the other options for
aerobic sewage sludges described above to composted products
is either impossible (because the percent volatile solids reduc-
tion of the sewage sludge fraction cannot be assessed) or has
not been adequately investigated in terms of reliability.
The regulation does not specifically mention or limit this
option to composting. This option can be applied to sewage
sludge from other aerobic processes such as aerobic digestion,
but other methods such as Options 3 and 4 are likely to be
easier to meet for these sludges.
6.7 Option 6: Addition of Alkali [503.33(b)(6>]
Sewage sludge is considered to be adequately reduced in
vector attraction if sufficient alkali is added to:
• Raise the pH to at least 12.
• Maintain a pH of at least 12 without addition of more alkali
for 2 hours.
• Maintain a pH of at least 11.5 without addition of more alkali
for an additional 22 hours.
As noted in Section 5.6, the term "alkali" is intended to
mean a substance that causes an increase in pH. Raising sewage
sludge pH through alkali addition reduces vector attraction by
reducing or stopping biological activity. However, this reduc-
tion hi biological activity is not permanent. If the pH drops, the
surviving bacterial spores become biologically active and the
sewage sludge will again putrefy and potentially attract vectors.
(The more soluble the alkali, the faster this is likely to happen.)
The conditions required under this option are designed to en-
sure that the sewage sludge can be stored for at least several
days at the treatment works, transported, and applied to soil
without the pH falling to the point where biological activity
results in vector attraction.
6.8 Option 7: Moisture Reduction of Sewage Sludge
Containing No Unstabilized Solids [503.33(b)(7)]
Under this option, vector attraction is considered to be
reduced if the sewage sludge does not contain unstabilized
solids generated during primary wastewater treatment and if the
solids content of the sewage sludge is at least 75% before the
sewage sludge is mixed with other materials. Thus, the reduc-
tion must be achieved by removing water, not by adding inert
materials.2
It is important that the sewage sludge not contain unsta-'
bilized solids because the partially degraded food scraps likely
to be present in such a sewage sludge would attract birds, some
mammals, and possibly insects, even if the solids content of
the sewage sludge exceeded 75%.
The way dried sewage sludge is handled or stored before
use or disposal can create or prevent vector attraction. If dried
sewage sludge is exposed to high humidity, the outer surface
of the sludge could equilibrate to a lower solids content and
attract vectors. Steps should be taken to prevent this from hap-
pening.
6.9 Option 8: Moisture Reduction of Sewage Sludge
Containing Unstabilized Solids [503.33(b)(8>]
Vector attraction of any sewage sludge is considered to be
adequately reduced if the solids content of the sewage sludge
is increased to 90% or greater. This extreme desiccation deters
vectors in all but the most unusual situations. As noted in the
footnote for Option 7, the solids increase should be achieved
by removal of water and not by dilution with inert solids.
Drying to this extent severely limits biological activity and
strips off or decomposes the volatile compounds that attract
vectors.
6.10 Option 9: Injection [503.33(b)(9>]
Vector attraction reduction can be demonstrated by inject-
ing the sewage sludge below the ground. Under this option, no
significant amount of the sewage sludge can be present on the
land surface within 1 hour after injection, and, if the sewage
sludge is Class A with respect to pathogens, it must be injected
within 8 hours after discharge from the pathogen-reduction
process.
Injection of sewage sludge beneath the soil places a barrier
of earth between the sewage sludge and vectors. The soil
quickly removes water from the sludge, which reduces the mo-
^The moisture reduction may be achieved by adding active materials that remove water by
reaction [e.g., CaO reacting with water to form Ca(OH)i], by adsorption, or as water of
crystallization (e.g., formation of CaSO.»»2H2O). The best way to determine whether the
material added is active or inert is to subject the sewage sludge/solid mixture to a drying
determination at mild conditions since the objective is to determine available water.
Method 2540B in "Standard Methods" (APHA, 1992) is appropriate. The sewage sludge
or mixture is dried at 103°C to 105°C (217°F to 221°F). Drying time should be more than
1 but less than 2 hours.
29
-------
bility and odor of the sewage sludge. Odor is usually present
al the site during the injection process, but it quickly dissipates
when injection is complete.
:•
Special restrictions apply to Class A sludges because these
sewage sludges are a medium for regrowth (see Section 4.3).
During the first 8 hours of regrowth, levels of pathogenic bac-
teria should still be quite low; however, after this point, the
regrowth of pathogenic bacteria may rapidly increase.
6.11 Option 10: Incorporation of Sewage Sludge
into the Soil [503.33(b)(10)]
Under this option, sewage sludge applied to the land sur-
face or placed on a surface disposal site must be incorporated
into the soil within 6 hours after application to or placement on
the land. If the sewage sludge is Class A with respect to patho-
gens, the time between processing and incorporation after ap-
plication or placement must not exceed 8 hours—the same as
for injection under Option 9.
When applied at agronomic rates, the loading of sewage
sludge solids typically is about l/200th of the mass of soil in
the plow layer. If mixing is reasonably good, the dilution of
sewage sludge in the soil surface is equivalent to that achieved
with soil injection. Odor will be present and vectors will be
attracted temporarily, as the sewage sludge dewaters on the soil
surface. This attraction diminishes and is virtually eliminated
when the sewage sludge is mixed with the soil. The mixing
method applies to liquid sewage sludges, dewatered sewage
sludge cake, and even to dry sewage sludges.
The 6 hours allowed to complete the mixing of sewage
sludge into the soil should be adequate to allow for proper
incorporation. As a practical matter, it may be wise to complete
the incorporation in a much shorter time. Clay soils tend to
become unmanageably slippery and muddy if the liquid sewage
sludge is allowed to soak into the first inch or two of topsoil.
6.12 Option 11: Covering Sewage Sludge
[503.33(b)(ll)]
Under this option, sewage sludge placed on a surface dis-
posal site must be covered with soil or other material at the end
of each operating day. Daily covering reduces vector attraction
by creating a physical barrier between the sewage sludge and
vectors, while environmental factors work to reduce pathogens.
6.13 Option 12: Raising the pH of Domestic Septage
[503.33(b)(12>]
This option applies only to domestic septage. Vector attrac-
tion is reduced if the pH is raised to at least 12 through alkali
addition and maintained at 12 or higher for 30 minutes without
adding more alkali. (These conditions also accomplish patho-
gen reduction for domestic septage—see Section 5.6.) When
this option is used, every container must be monitored to dem-
onstrate that it meets the requirement. As noted in Section 5.6,
"alkali" refers to a substance that causes an effect similar to an
alkali; that is, it causes an increase in pH.
This vector attraction reduction requirement is slightly less
stringent than the alkali addition method for sewage sludge.
The method is geared to the practicalities of the use or disposal
of domestic septage, which is typically treated by lime addition
to the domestic septage hauling truck. The treated septage is
typically applied to the land shortly after lime addition. During
the very short time interval, the pH is unlikely to fall to a level
at which vector attraction could occur.
30
-------
Chapter 7
Meeting the Quantitative Requirements of the Regulation
7.1 Introduction
The Part 503 regulation contains operational standards for
pathogen and vector attraction reduction. This chapter suggests
ways to satisfy these requirements.
The regulation provides only minimal guidance on the
amount of information that must be obtained during a monitor-
ing event to prove that a standard has been met or to demon-
strate that process conditions have been maintained. The final
decision is up to the permitting authority. Sufficient information
should be provided to the permitting authority to enable a quali-
fied reviewer to determine if the requirements have been met.
This chapter suggests how much information will satisfy this
need. For purposes of this discussion:
• A sampling event is defined as the period during which the
samples needed for monitoring are collected.
• A monitoring event includes the sampling period and the
period to analyze the samples and provide the results needed
to determine compliance.
7.2 Process Conditions
Sufficient information must be collected about sludge
processing conditions so that a regulator can be reasonably
certain that the process is being operated as claimed. How this
information is collected and how much information is needed
depend on the process. The following example illustrates the
kind of information and the level of detail that are appropriate.
Consider the case of a treatment works that meets the pathogen
reduction requirement for a Class B sludge by using anaerobic
digestion conducted at the PSRP conditions of 35°C (95°F)
with a 15-day residence time. To meet the pathogen reduction
requkement, the monitoring results must demonstrate that the
35°C (95°F) temperature and 15-day residence time are main-
tained whenever the process is being used to produce sewage
sludge that will meet the Class B requkement. The following
are suggested as reasonable monitoring requkements for this
situation:
The treatment works operator should measure the temperature of
the sludge in the digester at least every shift Preferably, tempera-
ture should be continuously recorded. Information should be
available on typical uniformity of temperature within the digester.
The temperature measuring device must be calibrated. To calcu-
late the residence time, the volume of the digester, depth of liquid
in the digester, and flow rate into or out of the digester must be
known. Mixing equipment should be capable of mixing the entire
contents of the digester. Information should be available on the
true working volume of the digester. Since poorly maintained
digesters are sometimes as much as one quarter filled with stag-
nant grit and scum, there should be evidence that the digester is
cleaned on a reasonable schedule. The flow rate can be deter-
mined by means of a flow meter, or by observing rise or fall of
liquid level during charging of feed or withdrawal of the treated
sewage sludge and the number of feedings or withdrawals per
day. If a flow meter is used, there should be records showing that
the meter is calibrated periodically and its readings are correct
Residence time in the digester is determined from the volume of
sludge in the digester and the daily average flow rate. The tem-
perature-time requkements of the PSRP process of anaerobic
digestion must be equaled or exceeded, because the regulation
requires that all sewage sludge that is used or disposed meet the
requirements. The 35°C (95°F) temperature requkement is some-
times difficult to meet Lower temperatures and longer residence
times may be used, as determined by a linear interpolation be-
tween 35°C (95°F) and 15 days and 20°C (68°F) and 60 days. It
is suggested that a running average temperature and a running
average residence time be computed, and that the average tem-
perature and time must meet or exceed the requirements of the
linear time-temperature relationship. The period of the running
average must be approximately equal to the average residence
time. Whenever the time-temperature relationship is not met, the
sludge does not meet Class B requkements and must be diverted
from agricultural use.
Other processes will have different requkements. For ex-
ample, some treatment works collect liquid sewage sludge in
batches, treat it with lime and either dewater it or apply it to
the land in the liquid state. They often import sewage sludges
or domestic septage and treat them in the same fashion. If
feedstock changes substantially in character from batch to
batch, records of performance will have to be kept on every
batch. If sludge is consistent in quality and records demonstrate
this to be true, it may be necessary to check only every thkd
batch to determine if pH at 2 hours and at 24 hours meets the
requkements of the regulation for pathogen and vector attrac-
tion reduction (see Sections 5.3 and 6.7 for requkements). If
records show that the treated sewage sludge is virtually never
off specification, the measurement frequency could be changed
to one randomly selected batch a day. (This determination
would have to be made through a permit, or at least with the
agreement of the regulator.) For other processes, such as static
pile composting, one of several piles constructed in a day could
be monitored, probably with several thermocouples at different
31
-------
elevations in the pile, to demonstrate conformance for the
whole day's production.
Frequently, processes do not conform to process condi-
tions. In such cases, the operator should keep records showing
that the treated sludge produced was either recycled to be proc-
essed again or diverted in some manner for use or disposal
consistent with the sludge quality (e.g., disposal in a landfill
with daily cover or, if the sludge meets the Class B require-
ments, application as a Class B [rather than a Class A] sludge).
73 Monitoring Events: Needs and Duration
Monitoring events are intended to reflect the average per-
formance of the treatment works. Conditions should be as sta-
ble as possible before the monitoring event. Day-to-day
variations in feed rate and quality are inevitable in sewage
sludge treatment, and the processes are designed to perform
satisfactorily despite these variations. However, major process
changes should be avoided before monitoring events, because
long periods of time—as much as 3 months if anaerobic diges-
tion is part of the process train—are required before steady state
operation is established.
Monitoring for Microbiological Quality
To meet the Part 503 pathogen reduction requirements,
sewage sludges may have to be monitored to determine densi-
ties of fecal coliforms, Salmonella sp., enteric viruses, and
viable helminth ova Monitoring for these microorganisms pre-
sents special problems, primarily caused by the length of time
it takes to be sure that the treated sludge meets the Class A and
Class B microbiological density requirements. Variations in the
microbiological quality of the treated sludge and intrinsic vari-
ation in the analytical methods are generally large enough that
a single measurement of a microbiological parameter is inade-
quate for deciding whether a process meets or fails to meet a
requirement
The Pathogen Equivalency Committee recommends that
the monitoring event include several samples taken over a pe-
riod of approximately 2 weeks (see Section 7.7). In addition,
the microbiological tests themselves take time to complete. The
MPN test for fecal coliform takes about 4 days to complete.
The MPN test for salmonellae takes about 5 days, but the
difficulty of conducting the test may require the use of an
off-site laboratory, so it could take 7 days to get results. Enteric
virus determinations take over 2 weeks and, in almost all cases,
must be done by an outside laboratory. The situation is similar
for viable helminth ova analysis, except the test takes about 4
weeks to conduct Thus, the time required for a monitoring
event could range from 3 to 7 weeks. During this time, the
quality of the treated sewage sludge is unknown. This means
that the sludge processed during the monitoring event should
either be stored until it is demonstrated that the processed
sludge meets the quality requirements for use as a Class A or
B sludge, or—if the sludge is being monitored for Class A
requirements—used or disposed as a Class B sludge (provided
it meets the Class B requirements). Another option is to com-
plete the sampling, which might take 2 weeks, and store all the
sewage sludge produced during sampling (or, if possible, divert
it to a Class B use), and then shut down until all the analytical
results have been reported back to the treatment works operator.
This latter course is only practicable for processes that produce
on-specification sludge shortly after start-up.
Monitoring for Vector Attraction Reduction
Not all the vector attraction reduction options listed in the
regulation (see Chapter 6) require special monitoring. Four of
the methods (treatment of sewage sludge in an aerobic process
for 14 days or longer, injection below the surface of the land,
incorporation of sludge into the land, and placement of sludge
on a surface disposal site and covering it at the end of each
day) are technology descriptions. These technologies have to
be maintained throughout the year in the manner described in
the regulation. Examples of the kind of information needed to
demonstrate adequate performance are provided in Section 7.2.
The remaining vector attraction reduction options all have
performance requirements that are monitoring goals. All have
some technology element. For example, the oxygen uptake rate
test is only appropriate for a sludge from an aerobic digestion
or wastewater treatment process. Even the 38% volatile solids
reduction requirement (see Section 6.2) has a technology ele-
ment (though none is mentioned in the regulation), since what-
ever was done to reduce the volatile solids content by at least
38% must be continued. The technology aspects of these op-
tions must be documented kt the manner described in Section
7.2. The measures of performance are a monitoring requirement
and must be evaluated according to the required monitoring
schedule (see Table 3-4 hi Chapter 3).
Monitoring for vector attraction reduction presents similar
problems to monitoring for microbiological quality. Some of
the tests can be conducted within a few hours while others can
take more than a month. For the tests that can be conducted
within a few hours, the sampling event must be more than a
few hours to account for the variability hi the feedstock (the
incoming sludge) and the performance of the vector attraction
reduction process as affected by the changes in feedstock. Just
as for the microbiological tests, these vector attraction reduction
tests should be conducted over approximately 2 weeks to mini-
mize the expected effect of these variations. The 2-week period
can be the same 2-week period during which the microbiologi-
cal parameters are being determined.
Some vector attraction reduction tests—such as the addi-
tional digestion tests—take more than a month to complete.
Unless the treatment works has several sets of duplicate testing
equipment, it will be impossible to run these tests on enough
samples during a 2-week sampling period to assess the vari-
ability hi the performance of the treatment process. Storing
samples taken during this period until the equipment becomes
available is not an option, because samples cannot be stored for
more than a limited time period (see Section 8.6). In such
circumstances, if treatment works operation is fairly steady, the
monitoring event should be started as much as a month early
so that enough information can be provided to adequately
assess treatment works performance (see also discussion under
"Additional Digestion Tests" in Section 7.7).
32
-------
7.4 Correspondence of Samples
The enteric virus and viable helminth ova requirements
under Class A Alternatives 3 and 4 and some of the vector
attraction reduction methods (e.g., percent volatile solids reduc-
tion) involve taking input and output samples that correspond
(i.e., they are "before processing" and "after processing" sam-
ples of the same batch of sludge). Obtaining samples that cor-
respond can be difficult for sewage sludge treatment processes,
such as anaerobic digestion, that characteristically treat sludge
in fully mixed reactors with long residence times. As mentioned
in Section 7.3, it can take up to 3 months to demonstrate that
an anaerobic digester has reached steady state after some sub-
stantive change in feed sludge or process conditions. Samples
will correspond only when a process has reached steady state.
Almost all the treatment processes that might be used to
reduce pathogens under Class A Alternatives 3 and 4 are either
batch processes or plug flow continuous processes. In theory it
is relatively simple to obtain correspondence—it is only neces-
sary to calculate the time for the input material to pass through
the system and sample the downstream sludge at that time.
Achieving accurate correspondence in practice, however, is sel-
dom easy. Consider, for example, the difficulty of obtaining
good correspondence of feed and treated sludge for a compost-
ing operation in which the feed sewage sludge is to be com-
pared to composted sludge that has been stored 3 months.
Appropriate compositing of the samples of feed and treated
sludge averages out the composition of these sewage sludges
and reduces the correspondence problem. As indicated in Sec-
tion 7.6, limitations on the periods of time over which micro-
biological samples can be collected limit the utility of
compositing.
7.5 Adjusting for Diluents
Sewage sludge processing often introduces other sub-
stances into the sludge. For example, polymers, lime and ferric
chloride, paper pulp, and recycled sludge ash are frequently
added to aid in dewatering. Lime is sometimes added to in-
crease the temperature of the sewage sludge cake to disinfecting
temperature, and wood chips are added to absorb moisture and
provide air channels in the sewage sludge cake being com-
posted. These materials reduce the microbial densities by dilu-
tion and increase solids content, although the change is not as
much as might be thought. For example, an increase in mass
of 20% would result in a reduction in the log density of a
microbiological parameter of only 0.079.
Because risk is directly related to mass of treated sludge,
it is appropriate not to be concerned whether the sludge has
been diluted or not by treatment, but to be concerned about the
numbers of pathogenic organisms in the treated sewage sludge
per unit mass of that treated sludge. This is the approach taken
by the Part 503 regulation, which requires that the treated
sludge meet the standards for Class A or Class B sludge. The
treated sludge includes any additives, so no correction is needed
for dilution effects.
For some sludges, particularly those treated by composting
(these usually will be Class A sludges), the amount of additive
can be considerable. Nevertheless, the regulation requires that
the treated sludge meet the standard, which-means that no cor-
rection need be made for dilution.
In many composting installations, wood chips are used as
the bulking agent. Sometimes the compost is sold or given
away without screening out the wood chips. Although the regu-
lation requires that the treated sludge must meet the standard,
it is appropriate to remove the wood chips when the microbial
analyses are carried out. The primary reason is to improve the
accuracy of the microbial measurements. The wood chips are
so big (typically 4 cm x 4 cm x 1 cm) that a very large sample
would have to be taken and blended to get a representative
subsample. Sample reliability is reduced when the sample con-
sists of a mix of sludge solids and fibrous wood-chip residue
from blending. Another reason for removing the wood chips
prior to microbial analysis is that the exposure of users to the
compost is related to the fine particle content and not to large
physically distinct wood chips. For example, a user who han-
dles the compost gets his or her hands covered with compost
particles that do not include wood chips. Similarly, the user
might breathe in a dust of compost particles that would not
include the wood chips. In these cases, what matters is the
number of pathogenic organisms present in the material that the
user is exposed to.
7.6 Representative Samples
Except when the investigator is searching for the maximum
range of a parameter, a sample, even a grab sample, should
be chosen to be representative of the sewage sludge being
sampled. Specific procedures for sampling are discussed in
Chapter 8.
Random Variability
Virtually all sewage sludge treatment processes will expe-
rience a certain amount of short-term random or cyclic variation
in the feed sludge and in process performance. Evaluation of
average performance over a 2-week time period is suggested
as a reasonable approach to account for these variations. It
permits selection of sampling days to avoid a day-to-day effect
and at least allows a comparison between adjacent weeks.
Seasonal Variability
For some sewage sludge treatment processes, performance
is poorer during certain parts of the year due to seasonal vari-
ations in such factors as temperature, sunshine, and precipita-
tion. For example, aerobic digestion and some composting
operations can be adversely affected by low ambient tempera-
ture. In such cases, it is critical that process performance be
evaluated during the time of year when poorest performance is
expected. If a treatment works is evaluated four or more times
a year, there is no problem, because all seasons of the year will
be covered. For small treatment works that are evaluated only
once or twice a year, it is important to monitor in the time of
year where performance is expected to be poorest, to avoid
approving a process that is not performing adequately for much
of the year. Process criteria of PSRPs and PFRPs must be
33
-------
evaluated and recorded continuously, with the kind of records
outlined in Section 7.2.
Composite Sampling
Composite sampling is frequently practiced in wastewater
treatment. A small stream of wastewater or sludge is drawn off
at rate proportional to the flow of the main stream being sam-
pled and collected as a single sample. Typically, times of col-
lection are for one shift (8 hours) or one day (24 hours). In this
case, the accumulated sample represents a volume-average
sample over the period of time the sample is drawn. The sample
is chilled during the period it is being collected to prevent
chemical change until it can be brought back to the laboratory
for analysis. This is an excellent sampling procedure, but it
must be modified when microbial analyses are intended. A
composite time-average sample can be obtained by combining
a series of small samples taken, for example, once every 5
minutes for a period of an hour. A composite sample for bac-
terial and viral testing could be taken over an hour or less under
most circumstances without compromising the results. Com-
posite sampling over 24 hours, or even longer if special pre-
cautions are taken, is possible for viable helminth ova provided
the ova in the sample are not exposed to thermal or chemical
stress (e.g., temperatures above 40°C [104°F] or the addition
of certain chemicals such as ammonia, hydroxides, and oxi-
dants).
Composite sampling may be possible for samples to be
used in some of the procedures to determine whether vector
attraction reduction is adequate. It may not be appropriate for
those procedures that depend on bacterial respiration (i.e., aero-
bic or anaerobic digestion). This subject is discussed in Appen-
dix D which presents procedures for three methods to
demonstrate reduced vector attraction.
7.7 Regulatory Objectives and Number of Samples
That Should Be Tested
The Part 503 regulation requires that stated goals for patho-
gen reduction and vector attraction reduction be met Sufficient
information should be collected in a monitoring event to dem-
onstrate that the requirements have been met, but excessive
efforts to collect information should be avoided. Unfortunately,
the daily, weekly, and seasonal fluctuations that occur hi influ-
ent to treatment works make it difficult to minimize the infor-
mation-gathering task. Based on judgment, 2 weeks is
suggested as a reasonable period for assessing the performance
of a sewage sludge treatment process.
If the impact of the treated sludge (i.e., the disease risk it
poses and the potential it creates for heavy metal uptake by
crops) could be based on an average performance or composi-
tion and a composite collected over 2 weeks would remain
stable, it might be possible to simply measure a parameter once
to adequately assess performance. This is possible for certain
chemical analyses. Unfortunately, with a few exceptions (see
below), composites for periods exceeding an hour cannot be
made with microbiological and vector attraction reduction pa-
rameters.
For the most part, several measurements must be made and
averaged to determine the average performance over a 2-week
period. Part 503 requires the geometric mean of seven samples
to demonstrate Class B pathogen reduction, but does not spec-
ify the number of samples required to demonstrate achievement
of the other pathogen reduction requirements or of the vector
attraction reduction requirements. Based on judgment, seven
samples are also suggested as adequate to demonstrate average
performance for all other cases, except when compositing over
several days is possible. However, if the performance of the
sludge treatment process is highly variable, or if correspon-
dence between feed and product is difficult to establish, more
than seven tests must be made to determine a reliable average.
The various parameters that must be measured to meet the
various pathogen reduction and vector attraction reduction re-
quirements are considered below to assess the range of accu-
racy that can be expected when seven measurements are made,
whether compositing can reduce the number of measurements
needed, or whether variance is so large than more than seven
measurements are needed. This discussion begins with the
Class B pathogen reduction requirement (see Section 5.2), be-
cause this is the only case where the regulation actually defines
the number of samples to be tested.
Class B: Monitoring for Fecal Coliform Densities
Part 503 requires that seven samples be taken to demon-
strate compliance with the fecal coliform levels required of
Class B sludges. Seven samples were judged adequate to ac-
count for the short-term fluctuations in treated sludge quality
and allow determination of average performance. Variance of
fecal coliform determinations is known to be high, but analysis
(presented below) showed that if seven samples are averaged,
the error band about the mean value is sufficiently compressed
that treatment works with adequately treated sludge would not
have difficulty meeting the standard. If the mean value does
not meet the standard, the material is not a Class B sludge and
must be disposed of otherwise until the standard is met.
The regulation requires that the geometric mean fecal coli-
form density of the seven samples be less than 2 million CPU
or MPN per gram of total solids sewage sludge (dry weight
basis). If a treatment works were producing a treated sewage
sludge with a true mean density of exactly 2 million fecal
colifbrm per gram, measured values of the fecal coliform den-
sity would cluster around 2 million per gram, but half would
be below and half would be above it. Half the time, the treat-
ment works would appear not to be meeting the requirement.
The true mean density must be below 2 million per gram to be
confident that the experimentally determined average will be
below 2 million per gram. Just how much below depends on
the standard error of the average.
Use of at least seven samples is expected to reduce the
standard error to a reasonable value. In tests on extended aera-
tion sludges, Farrell et al. (1990) obtained a standard deviation
of the logarithm of the fecal coliform density (s) of 0.3 using
the membrane filter method. This included the variability in the
analysis as well as variability over time (approximately a year).
Standard error for the average of seven measurements (S.E.-
34
-------
s/n) is 0.11. Using the normal probability distribution, the true
mean must be below 1.30 million if the geometric mean of
seven measurements is to be below 2 million 95% of the time
(see Table 7-1 for details of this calculation). If the standard
deviation were higher, the true mean would have to be even
lower to be reasonably confident that the geometric mean
would be below 2 million per gram. Thus, efforts should be
made to reduce variability. Steps that can be taken are:
• Reduce the standard error by increasing the number of meas-
urements used to determine the geometric mean.
• Reduce process variability.
• Improve sampling and analytical techniques.
What action to take to reduce the geometric mean depends
on the process. For anaerobic or aerobic digestion, some sug-
gested steps are to increase temperature, increase residence
time, use a draw-and-fill feeding procedure rather than fill-and-
draw or continuous feeding, and increase the time between
withdrawal and feeding. After an attempt at improvement, the
Table 7-1. True Geometric Mean Needed If Standard Fecal Coliform
Density of 2 Million CPU Per Gram Is to Be Rarely Exceeded
Assumptions
• The fecal coliform densities of the sewage sludge are log nor-
mally distributed. (The arithmetic mean of the logarithms of the
fecal coliform densities is the mean of the distribution. The geo-
metric mean is the antilog of the log mean.)
• The goal is to ensure that the measured mean value does not
exceed the density requirement more than once in 20 monitoring
events.
• The standard deviation of the log density is 0.30.
Calculation
To predict the expected frequency of a measurement using the
normal probability distribution, the variable x is converted to the
standard measure (u — see below) and its probability of occurrence
is obtained from tabulated values of the probability distribution. In
this case, the reverse is carried out. A certain probability of
occurrence is desired and the value of the standard measure is read
from the tables. From the normal distribution table (single-sided), u is
1.645 when P = 0.05 (one in 20),
Where: P = the proportion of the area under the curve to the
right of u relative to the whole area under the
curve.
and: u = the standard measure
u = (X-M.)/SJC
Where: ji = true log mean
x = log mean of the several measurements
Sj< = s/VrT
n = number of measurements that are averaged
s = standard deviation of a single measurement of
log mean density
The logarithm of the fecal coliform density requirement (2 million
CFU/g) is x (x= 6.301). This is the number that should not be
exceeded more than once in 20 monitoring episodes. Substituting
into Equation 1 and calculating n,
1.645= (6.301-fi)/(0.3/V7)
H = 6.114
Antilog 6.114 = 1.3 million CFU/g.
evaluation should be repeated. If the process continues to fail,
more substantial changes to the process may be appropriate.
Class A: Monitoring for Fecal Coliform Densities
Part 503 requires that, to qualify as a Class A sludge,
sewage sludge must be monitored for fecal coliform (or Sal-
monella sp.—see below) and have a density of less than 1,000
MPN fecal coliform per gram of total solids sewage sludge (dry
weight basis). The regulation does not specify the number of
samples that have to be taken during a monitoring event or, if
several samples are taken, how to determine the average fecal
coliform density.
Available information indicates that the variability of the
MPN test for fecal coliform that must be used for the Class A
sludges is high. Yanko's (1987) quality assurance data on du-
plicate samples show high variability, indicating that several
measurements are needed to get reasonable precision. For ex-
ample, the 95% confidence interval of the geometric mean for
a sample with a measured fecal coliform density of 1,000
MPN/g is 224 to 4,470 MPN/g (calculated from the standard
deviation of the log fecal coliform density, s = 0.30 with 13
degrees of freedom, determined from Yanko's 1987 report). If
seven measurements were taken, the confidence interval would
be much smaller—580 to 1,740 MPN/g. The spread between
the lower and upper limits has been reduced from a factor of
20 to a factor of 3.0 by increasing the number of measurements
from one to seven. The advantage of taking several samples is
obvious. Note that these confidence intervals are based on the
variability that resulted when duplicates drawn from a larger
sample were analyzed and do not include additional variability
caused by fluctuations in feedstock or process performance.
The measured fecal coliform density provides an estimate
of the likelihood of salmonellae detection and, if detected, the
expected density. Yanko (1987) obtained a good correlation
between fecal coliform density and salmonellae detections in
his extensive investigation of composts derived from sewage
sludge. Fraction detected is less than 10% when fecal coliform
density is less than 1,000 MPN/g. Yanko also obtained a good
correlation between fecal coliform density and salmonellae
density for those samples for which salmonellae were detected.
That correlation predicts that, for fecal coliform densities less
than 1,000 MPN/g, salmonellae densities will be less than 1.0
MPN/g. Thus, at fecal coliform densities <1,000 MPN/g, sal-
monellae detections will be infrequent and, if detected, densi-
ties are expected to be below 1 MPN/g.
The standard deviation for the fecal coliform measurement
for Class A sludges will probably be the same or somewhat
higher than for Class B sludges. Thus, the number of samples
taken should be the same or, in rare cases, more than for Class
B sludges. It is suggested, then, that the sampling event extend
for 2 weeks or more and that at least seven samples be collected
and analyzed.
What action to take to reduce average density in case the
fecal coliform requirement is not met depends on the process.
For aerated deep-pile composting, thicker insulating layers on
the pile, a longer period of temperature above 55°C (131°F),
35
-------
improved efforts at eliminating cross-contamination between
feed and treated sludge, and longer maturing times are sug-
gested.
Class A: Monitoring for Salmonellae Densities
Part 503 allows Salmonella sp. to be monitored instead of
fecal coliforms (see Section 4.3). The density of the Salmonella
sp. must be below detection limits of 3 MPN/4 g of total sewage
sludge solids (dry weight basis). The Salmonella sp. determi-
nation is superficially similar to the fecal coliform test, but it
is much more expensive and requires a high experience level.
In all likelihood, the salmonellae tests would have to be carried
out by a contract laboratory.
Yanko (1987) found that the standard deviations of the
MPN procedures, as determined from results on duplicates,
were nearly identical for both the fecal coliform and salmonel-
lae methods. In light of this information, one may conclude that
if the seven measurements are appropriate to determine a mean
fecal coliform density, the same number should be used for
determining a mean density for salmonellae.
The determination of a mean density for salmonellae is
complicated by the fact that most of the measurements will
show densities below detection limits (3 MPN/4 g), and some
way must be used to include these measurements into the av-
erage. The calculation of averages for "censored" data (a sub-
stantial portion of the data is below detection limits) is of
considerable interest in analytical chemistry, and numerous ap-
proaches to constructing a proper average have been suggested
(Newman and Dixon, 1990; Helsel, 1990). These methods are
useful when a third or less of the measurements are nondetect-
able, but with treated sludge, most of the measurements will be
nondetectable. Rather than over-interpret scarce data, it is sug-
gested that an arithmetic mean be determined for the data
points, considering the nondetectable measurements as 0
MPN/4 g. The arithmetic mean is chosen rather than the geo-
metric mean because it relates better to the true number of
salmonellae in a given mass of treated sludge than does the
geometric mean. If the arithmetic mean density is greater than
3 MPN/4 g, the process does not reduce pathogens to the speci-
fied densities.
Class A: Monitoring for and Demonstration of Enteric
Virus and Viable Helminth Ova Reduction
The accuracy of monitoring results demonstrating the ab-
sence of enteric viruses and helminth ova is influenced by the
variability in the influent to the treatment works and the inher-
ent error in the experimental method. Information on method
error for both enteric viruses and helminth ova is available only
on standard deviations calculated from duplicate samples.
Goya! et al. (1984) report that, in their comparison of methods
for determining enteroviruses, the log standard deviation for the
virus determination in sewage sludge was 0.26 (47 degrees of
freedom). A review of the work of Reimers et al. (1989) indi-
cates that, in the range of 5 to 100 viable Ascaris ova per 10
grams sewage sludge solids, standard deviation was about half
the number of viable ova. This is equivalent to a log density of
0.3, which is about the same as for fecal coliform. Thus, there
is no unusually high variability in the basic test methods that
would require an increased number of samples to minimize this
effect.
Deciding how many samples to take for enteric viruses and
viable helminth ova is more difficult than for fecal coliform
and salmonella, because enteric viruses and viable helminth ova
often are not present in untreated sludge. For this reason, the
interpretation of the density determinations for these organisms
hi treated sludge depends on the quality of the feed sludge. If
no enteric viruses or viable helminth ova are detected in the
feed sludge, then the absence of these organisms in correspond-
ing samples of treated sludge does not indicate in any way
whether the process is or is not capable of reducing these or-
ganisms to below detectable limits. The ability of a process to
reduce these organisms to below detectable limits is indicated
when analysis shows that these organisms were present in the
feed sludge and were not present in corresponding samples of
treated sludge. One important questions is: What fraction of the
total pairs of corresponding samples must show positive in the
feed sludge and negative in the treated sludge to provide con-
vincing evidence that the process consistently reduces enteric
viruses and viable helminth ova to below detectable levels?
This is a difficult question to answer.
Because viable helminth ova are relatively stable microor-
ganisms, compositing is suggested as a way to obtain meaning-
ful representative samples and analytical results. If precautions
are taken, such as cooling the sample promptly to close to 0°C
(32°F) and destroying or neutralizing any added chemicals such
as strong bases that were added as part of the pathogen-reduc-
ing process, composites can be collected over a 2-week period.
Corresponding composites of feed and treated sludge can be
compared, with a much lower likelihood of not finding viable
helminth ova in the feed sewage sludge. Because the analytical
method itself has a high variance (see above), approximately
four duplicates of the composite should be tested.
For enteric viruses, the same approach may be used as
suggested above for viable helminth ova. Precautions are taken
to cool the sample and destroy or neutralize any chemicals
added in the pathogen-reducing process. Samples are collected
on separate days and are promptly frozen at 0°F (-18°C), or
-94°F (-70°C) if storage will be for more than 2 weeks. When
the samples are to be analyzed, the individual samples are
thawed and composited, and viral densities determined.
The density of both viable helminth ova and enteric viruses
in processed sludge must be computed from the results of sev-
eral measurements. Most of these measurements are expected
to show below detectable densities. The averaging procedure
suggested above for salmonellae should be followed. The arith-
metic mean is determined considering the nondetectable meas-
urements as zero per 4 grams. If the arithmetic mean is above
1 CPU (for viruses) or 1 viable helminth ovum (for helminths)
per 4 grams, the process does not meet the Part 503 operational
standard.
36
-------
Vector Attraction Reduction Tests
Reduction in Volatile Solids
One way to demonstrate reduction in volatile solids re-
quires measurement of volatile solids of the sewage sludge
before and after sludge treatment. The sampling point for the
"after treatment" measurement can be immediately leaving the
processing unit or at the point of use or disposal, provided there
has been no significant dilution downstream with inert solids.
Farrell and Bhide (1993) have determined the standard
deviation of the percent volatile solids (%VS) determination for
separate samples withdrawn from pilot-scale digesters to be
0.65% (total solids content ranged from 2% to 5%). Conven-
tional statistical procedures (see Davies and Goldsmith, 1972)
were used to calculate the standard error of the percent volatile
solids reduction (%VSR), which is calculated from the %VS
of the untreated and treated sludge. The standard error of the
%VSR when calculated by the Van Kleeck equation (see Ap-
pendix D) is 2.0% in the range of interest (38% VSR). The 95%
confidence limits of the %VSR are ±4%, which is excessive.
If the %VSR is the average of seven determinations, the con-
fidence interval is reduced to ±1.5%, which is a more accept-
able value.
The most difficult problem with the %VSR determination,
as discussed above in Section 7.4, is getting correspondence of
the influent sludge with the effluent sludge. If there has been a
significant change in an inlet concentration or flow rate, achiev-
ing correspondence can require several months of monitoring
inlet and outlet volatile solids concentrations. If conditions have
been steady and feed compositions have been fluctuating about
an average value for a long period, data taken over a 2-week
period would be adequate to establish steady state perform-
ance.1 This implies that data have been collected beforehand
that demonstrate that sewage sludge composition has been sta-
ble for a long period before the 2-week sampling period. It
appears that regular collection of data for some months before
the sampling period is unavoidable to demonstrate stable per-
formance before the testing period. Fortunately, the total and
volatile solids determinations are not costly, and they provide
valuable operating information as well.
Total and volatile solids content of a sample do not change
significantly over the course of a day, particularly if the sludge
is cooled. Time composites collected over a course of a day can
be used for these determinations. Seven or more determinations
are recommended to reduce the error band around the mean to
minimize the chance that a process that actually has a greater
volatile solids reduction than 38% might show an average that
is below this value.
Additional Digestion Tests
The essential measurement in the additional digestion tests
for aerobic and anaerobic sludges (see Sections 6.3 and 6.4) is
the percent volatile solids content (%VS) from which the per-
'Note that, unlike the plug flow case, there should be no displacement in time between
comparisons of input and output for fully mixed reactors. Only when there has been a sig-
nificant change is it necessary to wait a long time before the comparisons can be made.
cent volatile solids reduction is calculated (%VSR). Using the
standard deviation of 0.65% determined by Farrell and Bhide
(see above), the standard error of the %VSR when calculated
by the Van Kleeck equation (see Appendix D) is 2.5% in the
range of interest (15% VSR). The 95% confidence limits of the
%VSR are ±5%. The tests (see Appendix D) require substantial
internal replication which shrinks these confidence limits. Sam-
ples should also be taken to account for the variability in the
process. The 2-week sampling period suggested for the micro-
biological tests may be excessively restrictive if several sam-
ples are to be evaluated. The equipment needed for the test is
not expensive but the units take up substantial bench space. It
is unlikely that a treatment works will want to have more than
two sets of test equipment. Since the tests take 30 to 40 days,
it is not possible to run more than one set of tests (two in a set)
within a monitoring event. It is suggested that these tests be
routinely carried out during the year and the results be consid-
ered applicable to the monitoring period. It is estimated on a
best judgment basis that five tests are needed to account for
variability in the feed sludge and hi the treatment process itself.
Specific Oxygen Uptake Rate Test
The specific oxygen uptake rate test (SOUR, see Appendix
D) can be completed in the laboratory in a few minutes, so there
is no difficulty in completing the test during a monitoring event.
The test requkes the SOUR determination to be made on two
subsamples of a given sample. Farrell and Bhide (1993) found
that, in the target SOUR value of 1.5 mg O2/hr/g, sludge solids
replicates agreed within about ±0.1 mg O2/hr/g. Since the test
is easy to run, it is suggested that seven tests within the 2-week
sampling event will adequately define the SOUR. Arithmetic
average of the tests should be computed and compared against
the Part 503 SOUR value.
Raise pH to 12
There are two options in the regulation that reduce vector
attraction by pH adjustment. In the first, sludge is raised in pH
by alkali addition so that pH is >12 for 2 hours after alkali
addition and, without further alkali addition, remains at pH
>11.5 for an additional 22 hours (see Section 6.7). The second
method is for domestic septage. The pH is raised to pH >12 by
alkali addition and, without further addition of alkali, remains
at >12 for 30 minutes (see Section 6.13). As noted in Section
5.6, the term alkali is used in the broad sense to mean any
substance that increases pH.
The pH requirement in the regulation was established using
data obtained at room temperature (Counts and Shuckrow,
1975; Ronner and Cliver, 1987), which is presumed to have
been 25°C (77°F). Consequently, pH should be measured at
25°C (77°F) or measured at the existing temperature and con-
verted to 25°C (77°F) by use of a temperature-versus-pH con-
version table determined experimentally for a treated sludge
that meets the pH requirements. The correction is not trivial for
alkaline solutions—it is about -0.03 pH unit/°C (-0.017 pH
unit/°F) for aqueous calcium hydroxide with a pH of about
12—and should not be ignored. Note that temperature-compen-
sated pH meters only adjust instrument parameters and do not
compensate for the effect of temperature on the pH of the
solution.
37
-------
Septage. Each container of domestic septage being treated
with alkali addition must be monitored. The pH is monitored
just after alkali addition and a half hour or more after alkali
addition. Bonner and Cliver (1987) suggest that alkali (they
used slaked lime) be added to the septic tank or to the septic
tank truck while domestic septage is being pumped from a
septic tank into the tank truck. If slaked lime is used, a dose of
0.35 Ib per 10 gallons (4.2 g per liter) is sufficient to raise the
pH to 12 for a typical domestic septage of about 1% solids
content The agitation from the high velocity incoming stream
of septage distributes the lime and mixes it with the domestic
septage. The pH is measured when the truck loading is com-
plete. The truck then moves to the use or disposal site. Agitation
generated by the motion of the truck helps in mixing and dis-
tributing the lime. The pH is again measured at the use or
disposal site. The time should be at least a half hour after the
addition of lime. The sample may be obtained through the top
entry of the tank truck, using, for example, a stainless steel cup
welded to a long handle to collect the sample. The pH is most
conveniently measured with alkaline pH paper in the pH range
of 11 to 13. The pH paper can age and become contaminated.
It is best to use strips from two separate containers. If they do
not agree, compare with a third batch and reject the one that
disagrees with the others, Accuracy of these measurements is
within ±0.1 pH unit. If the pH is below 12, either initially or
after 30 minutes, more lime should be added and mixed in.
After an additional waiting period of at least 30 minutes, the
pH must again be measured to ensure that it is greater than 12.
Savage Sludges. For addition of alkali to sewage sludges,
the pH requirement is part of both the PSRP process description
(see Section 5.3) and the requirement of a vector attraction
option (see Section 6.7). Monitoring is required from 1 to 12
times a year (see Table 3-4 in Chapter 3), and the process must
meet the processing operating conditions throughout the year.
For vector attraction reduction, the pH requkement has to be
met at least during the required monitoring episodes. For patho-
gens, the assumption is that the pH requirement is met all the
time.
Alkali is sometimes added to liquid sludge and sometimes
to dewatered sludge. The pH requirements as stated in the
regulation apply in the same way for both liquid and dewatered
sludge. For the first measurement of pH in liquid sludge 2 hours
after addition of alkali, it is assumed that the alkali and the
sludge have been mixed together for a sufficient time to reach
equilibrium (not considering the gradual changes that occur
over substantial periods of time). Consequently, the pH meas-
urement can be made directly in the liquid sludge. The pH
measurement is made preferably with a pH meter equipped with
a temperature compensation adjustment and a low-sodium glass
electrode for use at pH values over 10. The pH electrode is
inserted directly in the sludge for the reading. The use of an
alkaline range pH paper is acceptable if the paper is calibrated
by use of pH standards. The second measurement is made 24
hours after addition of alkali. If the sludge is still hi the liquid
state, the pH measurement is made in the same fashion. How-
ever, if the process includes a dewatering step immediately
following the alkali addition and the sludge is now a dewatered
cake, the cake must be made into a slurry for the pH measure-
ment. Acceptable procedures for preparing the sample and
measuring pH are given by Block (1965) and by EPA (1986).
The procedure requires adding 20 mL of distilled water (con-
taining 0.01 M CaCl2) to 10 g of sludge cake, mixing occasion-
ally for half an hour, waiting for the sample to clarify if
necessary, and then measuring pH. The important step is the
mixing step that allows the alkali-treated dewatered sludge to
come into equilibrium with the added water.
Number of Samples. The accuracy of pH meters and of pH
paper is within ±0.1 pH unit. More than one sample is necessary
if the domestic septage or sludge is not well mixed. If the lime
has been added gradually over the period in which septage is
being pumped into a tank truck, mixing is considered adequate
and a single measurement taken at the top of the tank truck is
sufficient. If alkali has been added to liquid sludge in a tank at
a treatment plant, tests are easily run to establish how much
mixing is required to produce a uniform pH in the sludge. If
this adequate mixing time is used, a single sample withdrawn
from the tank for pH measurement is sufficient.
If alkali is added to sludge cake, more sampling is sug-
gested. Typically, alkali (usually lime) is added to sludge cake
in a continuous process. The sludge from the dewatering proc-
ess discharges continuously to a continuous mixer, from which
it discharges to a pile or to a storage bin. Lime is metered into
the mixer in proportion to the sludge flow rate. The flow rate
and compositions of the sewage sludge can vary with time. To
demonstrate compliance on a given day, several time-composite
samples each covering about 5 minutes should be collected, and
the pH measured. This procedure should be repeated several
times during the course of a 2-week sampling event.
For sludge cake, the composites collected for pH measure-
ment must be reduced in size for the pH measurement. Typi-
cally, the alkaline-treated sludge is discharged from the mixing
devices in the form of irregular balls that can be up to 2 to 3
inches (5 to 7.6 cm) in diameter. It is important that the sludge
to which the environment will be exposed has been treated to
reduce pathogens and vector attraction to the desired level. If
the alkali has not penetrated the entire pellet, one or both of
these goals might not be met for the material on the interior of
the pellet How much risk is involved when the interior is not
as well treated as the exterior cannot be directly measured or
accurately estimated. To be conservative, the entire pellet
should be at the proper pH. It is suggested that the composite
be thoroughly mixed and that a subsample be taken for analysis
from the mixed composite. An even more conservative ap-
proach is to sample only the interior of pellets, but then the test
tends to become subjective (i.e., how close should one come to
the skin of the pellet when taking out the inside of it?) and
somewhat unrealistic. It seems more likely that the environment
will be exposed to the average composition of the pellet rather
than the ulterior alone, so the average composition should be
analyzed.
Percent Solids Greater Than or Equal to 75% and 90%
The monitoring requirement for these vector attraction op-
tions (see Sections 6.8 and 6.9) is simply measurement of total
solids. This measurement is described in Standard Methods
(APHA [1992], Standard Method 2540 G). Standard Methods
38
-------
states that duplicates should agree within ±0.5% of their aver-
age. For 75% solids, this would be ±3.8%, which appears un-
realistically high. For a continuous process, a time-composite
sample can be taken over the course of a day, and duplicate
analyses carried out on this composite. This is possible because
biological activity essentially ceases at high solids content, and
further changes will not occur if the sludge is kept from further
drying. Approximately seven such composites over the course
of a 2-week sampling period would provide adequate sampling.
Some drying processes—such as drying sludge on sand
drying beds—are batch processes. In such cases, it may be
desirable to ascertain that the sludge meets the vector attraction
reduction requirements before removing the sludge from the
drying area. This can be done by taking two separate space-
composites from the dried sludge, analyzing each of them in
duplicate, and removing the sludge only if it meets the required
solids content.
39
-------
-------
Chapter 8
Sampling Procedures and Analytical Methods
8.1 Introduction
Many of the Part 503 Subpart D pathogen and vector at-
traction reduction requirements call for monitoring and analysis
of the sewage sludge to ensure that microbiological quality and
vector attraction reduction meet specified requirements (see
Chapters 4 to 6 for a description of the requirements). This
chapter describes procedures that should be followed in obtain-
ing samples and insuring their quality and integrity. It also
summarizes the analytical procedures required under Part 503,
and directs the reader to other sections of this document that
describe some of those procedures.
Sampling personnel will also benefit from reading ex-
panded presentations on the subject. Especially recommended
are "Standard Methods" (APHA, 1992), "Principles of Envi-
ronmental Sampling" (Keith, 1988), "Samplers and Sampling
Procedures for Hazardous Waste Streams" (EPA, 1980),
"Sludge Sampling and Analysis Guidance Document" (EPA,
1993), and ASTM Standard E 300-86, "Standard Practice for
Sampling Industrial Chemicals" (ASTM, 1992a). The latter
publication provides an in-depth description of available sam-
pling devices and procedures.
When referring to other publications, it is important to note
that most guidance on specific sampling techniques is directed
toward chemical analysis. Procedures described may be inap-
propriate for microbiological sampling because they expose the
samples to possible contamination, or may be appropriate only
after some modification to reduce the risk of microbial con-
tamination during sampling.
8.2 Safety Precautions
Sewage sludges that are being sampled should be pre-
sumed to contain pathogenic organisms, and should be handled
appropriately. Both the sampler and the person carrying out the
microbiological analysis must take appropriate precautions.
Safety precautions that should be taken when sampling and
when analyzing the samples are discussed in Standard Methods
(APHA, 1992) in Sections 1060A and 1090C.
Individuals performing sampling (usually employees of
wastewater treatment works) should receive training in the mi-
crobiological hazards of wastewater and sludge and in safety
precautions to take when sampling. For example, gloves should
be worn when handling or sampling untreated or treated sewage
sludges. The person taking the samples should clean the sample
containers, gloves, and his or her hands before delivering the
samples to others. Hands should be washed frequently. Photo-
cell-activated or foot-activated hand washing stations are desir-
able to reduced spreading of contamination to others.
Employees should train themselves to avoid touching their lips
or eyes. At a minimum, employees should be immunized
against tetanus.
Personnel analyzing sludge samples should also receive
training in awareness and safety concerning biohazards. Since
microbiological laboratories have safety programs, this subject
is not covered in depth here. Laboratory personnel should be
aware that every sample container is probably contaminated on
the outside with microorganisms, some of which may be patho-
gens. Personal hygiene and laboratory cleanliness are extremely
important. Mouth-pipetting should be forbidden.
8.3 Sampling Free-Flowing Sewage Sludges
Sewage sludges below about 7% solids behave, at worst,
like moderately viscous liquids such as an SAE 20 lubricating
oil. They flow freely under small pressure gradients, and will
flow readily into a sample bottle. They are heterogeneous, and
concentration gradients develop upon standing. Generally set-
tling is slow and is overcome by good mixing.
Liquid sludges may be flowing in pipes, undergoing proc-
essing, or stored in concrete or metal tanks, in tank cars or tank
trucks, or in lagoons. This section describes procedures for
sampling from these various situations, except for lagoons,
which are discussed in Section 8.4.
Filling Containers
Liquid sludge samples are usually transferred into wide-
mouth bottles or flexible plastic containers. Sludges can gener-
ate gases and pressure may build up in the container.
Consequently, the bottle or container is generally not filled. If
the sludge is to be used for the oxygen uptake test, the sample
bottle should not be more than half full, to provide some oxygen
for respiration of the sludge.
On the other hand, if the sludge is to be used for the
additional anaerobic digestion test for vector attraction reduc-
tion, it is important that it not be exposed to oxygen more than
41
-------
momentarily. Consequently, the bottles must be filled. They
should be provided with a closure that can pop off, or else be
collected in a flexible plastic container than can both stretch
and assume a spherical shape to relieve any internal pressure
that develops.
The containers used to collect the samples can be wide-
mouth bottles that can be capped, or pails. If a pail is used and
only part of its contents will be taken as a sample, the sample
should be transferred to a bottle at the sampling location. Pref-
erably, the transfer should be made by use of a ladle rather than
by pouring, since some settling can occur in the pail, particu-
larly with primary or mixed sludges of solids contents below
about 3%.
Sampling Flowing Streams
If liquid sewage sludges are to be sampled, it is most
desirable to sample them as they are being transferred from one
vessel to another. Preferably this is done downstream of a pump
that serves to mix the sludge thoroughly. Ideally, the sample is
taken though a probe facing upstream in the center of the dis-
charge pipe and is withdrawn at the velocity of the liquid at the
center-line of the pipe. This approach generally is not possible
with sludge, because fibrous deposits probably would build up
on the probe and plug up the pipeline. Sampling through a side
tap off the main discharge pipe is adequate only if the flow is
turbulent and the sample point is over ten pipe diameters down-
stream from the pipe inlet (e.g., for a 3-inch [7.6-cm] pipe, 30
inches [76 cm] downstream) or the tap is downstream from a
pump. For any kind of a slurry, the fluid at the wall contains
fewer particles than the bulk of the fluid in the pipe. The sample
should be withdrawn fast enough so that it minimizes the
amount of thinned-out fluid from the outside pipe wall that
enters the sample.
If the sludge discharges into the open as it is transferred
from one vessel to another, it can be sampled by passing a
sample container through the discharge stream. The container
should be large enough to catch the whole stream during the
sampling interval, rather than, for example, just sampling the
center or the edge of the discharge. The sample container could
be a pail or a beaker at the end of an extension arm.
Sample volume should be about three times what is needed
for the analyses planned. Samples should be time-composited.
For example, if a digester is being sampled during a withdrawal
that takes about 15 minutes, a sample can be taken during the
first, second, and third 5-minute period. The three separate
samples should be brought back to the laboratory and compo-
sited into a single sample. The maximum allowable time for a
composite for either bacteria, viral, or vector attraction reduc-
tion test samples is about 1 hour; a greater time might allow
microbiological changes to occur in the first sample taken.
Composite sampling over 24 hours is possible for viable
helminth ova provided the ova in the sample are not exposed
to chemical or thermal stress—for example, temperatures above
40°C (104°F) or certain chemicals such as ammonia, hydrox-
ides, and oxidants.
Sampling Sewage Sludge in Tanks
The purpose of the sampling is to determine a mass-aver-
age property of the sludge, rather than, for example, to find out
if there is a gradient hi the property at various points in the
tank. This requires that the tank be well-mixed, otherwise many
subsamples must be taken throughout the tank and composited.
If tanks are large, even well-mixed tanks containing sludge
show gradients in composition. An enclosed tank such as an
anaerobic digester must be sampled through pipelines entering
the vessel. A minimum of three taps on a side wall of the tank
is recommended. The sample tap pipe should project several
feet into the tank. Preferably the sample line should be back-
flushed with water after the sample is withdrawn. When a sam-
ple is withdrawn, enough material must be withdrawn to
thoroughly flush the line before a sample is collected. The
sampling should be done when the tank is being agitated. An
open tank such as an aerobic digester can be sampled by draw-
ing a vacuum on a vacuum-filtering flask connected by a tube
to the desired level in the tank. A weighted sampling bottle may
also used to sample the liquid at three depths (see ASTM E300-
86, Par. 21, in ASTM [1992a]).
8.4 Sampling Thick Sewage Sludges
If sewage sludges are above about 7% solids, they take on
"plastic" flow properties; that is, they require a finite shear
stress to cause flow. This effect increases as the solids content
increases. Solids may thicken hi lagoons to 15% solids. At these
concentrations, they will not flow easily and require a substan-
tial hydrostatic head before they will flow into a sample bottle.
Sampling of lagoons ranges from difficult to very difficult,
depending on the objectives of the sampling and the nature of
the sludge in the lagoon. The thickened sludge solids are gen-
erally nonuniformly distributed in all three dimensions. It is
deskable first to map the distribution of depth with length and
width to determine where the sampling should be concentrated.
A length-width grid should be established that takes into ac-
count the nonuniformity of the solids deposit. ASTM E300-86,
Figure 19 (ASTM, 1992a), shows a grid for sampling a uniform
deposit in a railroad car.
The layer of water over the sludge complicates the use of
many types of tube samplers because the overlying water
should not be included in the sample. A thief sampler (ASTM,
1992a) that samples only the sludge layer may be useful.
Weighted bottle samplers (ASTM, 1992a) that can be opened
up at a desired depth can be used to collect samples at a desired
depth. Samples at three depths could be taken and composited.
Most likely the sludge will be as much as twice as high in solids
content at the bottom of the sludge layer as at the top. Compo-
siting of equal volumes of samples from top, middle, and bot-
tom produces an excellent mass-average sample and adjusts for
this difference in solids content. Generally there is no point in
determining the gradient with depth in a parameter, because
there is no practical way of separately removing layers of
sludge from a lagoon. Determining whether there are gradients
with length and width makes more sense because, if desired,
sludge could be removed selectively from part of a lagoon,
leaving behind the unacceptable material.
42
-------
Sludges from dewatering equipment such as belt filter
presses and centrifuges can reach 35% solids and even higher
solids following some conditioning methods. They are easy to
sample if they are on moving conveyors, but if they are stored
in piles, the sampling problem becomes very difficult. Sam-
pling devices such as augers (a deeply threaded screw) are used
on high solids cakes (ASTM, 1992a). The auger is "turned into
the pile and then pulled straight out. The sample is removed
from the auger with a spatula or other suitable device." The
pile should be sampled in proportion to its mass—that is, more
samples are taken where the pile is deeper.
8.5 Sampling Dry Sewage Sludges
For purposes of this discussion, "dry" sewage sludges in-
clude sludges that may contain as much as 60% water. They
include heat-dried sludges, composted sludges, and sludges
from dewatering processes such as pressure filtration that pro-
duce a cake which is usually handled by breaking it up into
pieces. Some centrifuge cakes are dry enough that they are
comprised of small pieces that remain discrete when piled.
Dry sludges are best sampled when they are being trans-
ferred, usually on conveyors. Preferably material across the
entire width of the conveyor is collected for a short period of
time. Several of these across-width samples are collected and
combined into a time-composite sample. If the entire width of
the conveyor cannot be sampled, the sample is collected from
various points across the breadth of the conveyor, and a space-
and time-composited sample is collected.
When a material comprised of discrete particles is formed
into a pile, classification occurs. If the particles are homogene-
ous, this creates no problem, but sometimes the particles are of
different composition. For example, a composted sludge may
be heterogeneous even when oversize bulking agents have been
removed. It is important that the edges of such piles and the
center be properly weighted. ASTM E300-86, Item 31.4
(ASTM, 1992a) suggests a sampling grid for a pile that pre-
vents bias.
The heterogeneous nature of some composted sludges
causes another problem in sampling. For example, most augers
and sampling thiefs will be ineffective in getting a repre-
sentative sample of the interior of a pile containing large wood
chips and fine composted sludge. There may be no substitute
for digging with a shovel to get to the desired location.
Even processed sewage sludge is not inert; in the presence
of air it oxidizes slowly. Temperatures can rise to substantial
levels. For example, a storage pile of compost or dried sludge
may be at room temperature on the outside but could be at 60°C
(140°F) at a depth of 2 feet (0.6 m) within the pile. The micro-
biological content of samples from the surface and from the
ulterior of the pile may be considerably different. When there
is a large temperature gradient in a storage pile, it is important
to include an analysis of the sludge from the cooler section of
the pile where the chance of regrowth of bacterial populations
is greatest. In any case, samples from a large pile should be
taken at various depths and along its length.
8.6 Control of Temperature, pH, and Oxygenation
After Sample Collection
Samples for Microbial Tests
All samples for microbial analyses should be cooled to
water-ice temperatures when collected or very soon thereafter.
For example, enteric viral and bacterial densities are noticeably
reduced by even 1 hour of exposure to temperatures of 35°C
(95°F) or greater. The requirement for cooling limits the prac-
tical size of the sample collection container. A gallon sample
bottle will take much longer to cool than a quart bottle. Use of
bottles no bigger than a quart is recommended for most sam-
ples, particularly if the sludge being sampled is from a process
operated at above ambient temperature. Granular solids and
thick sludges will take a long time to cool, so a small container
is advised. For rapid cooling, placing the sample container in
a slurry of water and ice produces excellent results. Bagged ice
or "blue ice" is effective in maintaining low temperatures but
several hours can elapse before this kind of cooling reduces
sample temperature to below 10°C (50°F) (Kent and Payne,
1988). The same is true if warm samples are placed in a refrig-
erator.
Standard Methods (APHA, 1992) states that for bacterial
species, which include fecal colifbrm and salmonellae, if sam-
ples are not to be tested within 1 hour after collection, they
should be cooled to below 10°C (50°F) during a maximum
transport time of 6 hours. The authors then admit that this tune
can be unrealistic, particularly if material must be mailed or
air-expressed to a testing laboratory. They then say that the time
between collection and analysis should not exceed 24 hours. If
samples are brought to 4°C (39.2°F) by prompt chilling, 24
hours between sampling and analysis should not adversely af-
fect the results. Samples for bacteriological analyses should not
be frozen.
The requirement for prompt chilling of samples is appro-
priate for viruses as well as bacteria. There are far fewer labo-
ratories capable of carrying out virus tests than bacteria, so time
between analysis and sampling could routinely exceed 24
hours. Fortunately, viruses are not harmed by freezing. Typi-
cally, virology laboratories store samples at -70°C (-94°F) be-
fore analysis. Deep freezers are not ordinarily available in a
wastewater treatment works, but samples can be frozen in a
-18°C (0°F) freezer and stored for up to 2 weeks without harm.
They then can be packed in dry ice and shipped to the analyzing
laboratory.
Viable helminth ova are only slightly affected by tempera-
tures below 35°C (95°F), provided chemicals such as lime,
chlorine, or ammonia have not been utilized in the treatment
process. Nevertheless, chilling to 4°C (39.2°F) is advised. If
the samples are held at this temperature, a period of a month
can elapse between sampling and analysis. Freezing should be
avoided because the effect of freezing on helminth ova is in-
completely understood.
If the sludge is treated by a chemical such as lime, the lime
may have to be removed (in this case by neutralization) imme-
diately after sampling if the microbial tests are to be valid.
43
-------
Failure to remove the lime extends the treatment time, and the
high pH may interfere with the microbial test.
The presence or absence of oxygen is not a serious concern
for the microbiological tests if the samples are promptly cooled.
Vector Attraction Reduction Tests
For the vector attraction reduction tests that measure oxy-
gen uptake, or additional anaerobic or aerobic digestion (see
Appendix D), the samples must be kept at the temperature at
which they were collected. This sometimes can be done just by
collecting a large sample in a large container. Covering the
sample with an insulating blanket or placing it in an insulated
box will be adequate protection against temperature change in
most cases. Desired temperature can be maintained in the box
by adding a "hot water bottle" or a bag of blue ice.
For any of the vector attraction reduction tests, no adjust-
ment of pH is to be made. For those vector attraction processes
that utilize lime, the only requirement is to measure pH after
the time periods indicated in the vector attraction reduction
option (see Section 6.7).
Lack of oxygen for aerobic sludges will interfere with the
metabolic rate of the aerobic microorganisms hi the sample.
Similarly, presence of oxygen will seriously affect or even kill
the anaerobic organisms that convert organic matter to gases in
anaerobic digestion. For the oxygen uptake rate test, care must
be exercised not to deprive the sample of air for more than an
hour. The additional aerobic digestion test is more "forgiving"
(because it is a long-term test and shocked bacteria can revive),
so perhaps 4 hours of shortage of oxygen can be tolerated. For
the additional anaerobic digestion test, the sample containers
should be filled to exclude air. In any subsequent operations
where there is a freeboard in the sample or testing vessel, that
space should be filled with an inert gas such as nitrogen.
8.7 Sample Compositing and Size Reduction
The amount of sample collected will exceed the amount
needed foe analysis by a large margin. The sample generally
must be reduced to a manageable size for the analyst to handle.
Sample size reduction is more difficult for samples for micro-
bial tests than for vector attraction reduction tests, because care
must be exercised to rninimize opportunity for microbial con-
tamination.
drawn into the pipette slowly and the tip moved through the
sample to minimize selective collection of liquid over solid
particles.
Sample size reduction for thick sludges is difficult, because
they cannot be mixed by shaking. Stirring with a mechanical
mixer or a paddle is often inadequate (recall how long it takes
to mix a can of paint). A satisfactory approach is to hand mix
a composite of any subsamples, and then take a large number
of small grabs from the large sample to form the smaller sample
for the analyst.
Dry solids samples can generally be mixed adequately by
shaking, but the individual particles are frequently large and
must be reduced hi size to get a representative sample. If the
particles are large and a number of subsamples must be com-
bined into a large composite, it may be necessary to reduce the
particle size before they are composited. This can be done in a
sterile covered chopper, blender, or grinder. The individual sub-
samples are then combined and mixed by shaking, rotating, and
tumbling. A smaller composite is then prepared by combining
a number of grabs from all parts of the combined sample. Some
other methods used to reduce size, such as "coning and quar-
tering" (ASTM, 1992a) cannot be used for microbiological
samples because it is difficult to avoid contaminating the sam-
ple when using these procedures.
Vector Attraction Reduction Tests
The lack of a need to prevent microbial contamination
makes compositing and size reduction easier for vector attrac-
tion reduction tests than for microbial tests. There is a need to
keep the aerobic samples aerobic and to prevent the anaerobic
samples from coming into contact with air. Subsamples for the
anaerobic tests can be collected into individual bottles at the
sampling location. As noted above, these sample bottles should
be filled completely and capped. A brief exposure to air will
not cause a problem, but any prolonged exposure, such as might
occur when several subsamples are being blended together and
reduced hi size for a representative composite sample, must be
avoided. One approach is to flush a sterile large bottle with
nitrogen, then pour in the subsamples and blend them together
with nitrogen still bleeding into the vessel. Alternatively, the
nitrogen-filled vessel could be flushed with more nitrogen after
the admission of the subsamples, capped, and then shaken thor-
oughly to accomplish the blending.
Microbial Tests
For freely flowing liquids, samples can be adequately
mixed in the sample bottles by shaking the bottles. There must
be room in the bottle for adequate mixing. Compositing of
smaller samples is accomplished by pouring them into a larger
bottle with adequate freeboard and mixing it by shaking or
stirring it thoroughly with a sterile paddle. Pouring off a small
part of the contents of a large container into a smaller bottle is
a poor procedure, because the top layer of any slurry always
contains fewer solids than lower layers. Sampling with a pipette
with a wide bore is an acceptable alternative, provided the bore
of the pipette is as wide as possible. The sample should be
8.8 Requirements for Sample Containers and
Sampling Tools
Materials of Construction
Sampling containers may be of glass or plastic that does
not contain a plasticizer (teflon, polypropylene, and polyethyl-
ene are acceptable). Plastic bags are especially useful for thick
sludges and free-flowing solids. Pre-sterilized bags are avail-
able. Stainless steel containers are acceptable, but steel or zinc-
coated steel vessels are not appropriate.
44
-------
Sterilization
The containers and tools used for sampling should be ster-
ilized if the material is to meet the Class A microbiological
requirements. Conservative microbiological practice also re-
quires sterilization of containers and sampling tools to be used
for collecting samples to be tested for meeting the Class B
requirements. All equipment should be scrupulously clean.
Sterilization is not required when collecting samples of sewage
sludge to be used in vector attraction reduction tests, but all
equipment must be clean.
Sterilization of the larger tools for sample collection can
frequently be accomplished in large laboratory sterilizers. A
shovel can be sterilized by enclosing the blade in a kraft paper
bag, sealing the bag around the handle, and placing the entire
shovel in the sterilizer. The handle gets contaminated when it
is touched, but the bag around the blade is not removed until
the shovel is used to take the sample, leaving it sterile. In other
cases, a trowel could be sterilized and a clean but unsterile
shovel used to get close to the desired sampling spot. The sterile
trowel is used for the sample collection.
For larger devices, it is possible to clean the device very
carefully, direct a jet of atmospheric pressure steam over the
surface that would contact the sample for a few minutes, and
then enclose the device in a sterile bag. In some cases, the
device need not be covered but can simply be closed up until
it is used to collect the sample. A tube or thief sampler could
be sterilized by running atmospheric pressure steam through it
for 10 minutes. This procedure does not give assurance of total
sterilization, but may be the only possible option.
8.9 Packaging and Shipment
Proper packaging and shipment are important to ensure
preservation.
Taping and Sealing
Sample containers should be securely taped to avoid con-
tamination, and sealed (e.g., with gummed paper) so it is im-
possible to open the container without breaking the seal.
Sealing ensures that sample integrity is preserved until the sam-
ple is opened in the laboratory. It is recommended that perma-
nent labels be affixed to the samples. Suggested information
for a sample label includes:
• Type of sewage sludge (e.g., "air-dried digested sludge" or
"windrow composted sludge").
• Amount sampled.
• Type of sample (grab or composite).
• Type of analysis to be performed (e.g., Salmonella sp., fecal
coliform bacteria, enteric virus, or viable helminth ova).
• Date and time the sample was taken.
• Sample identification code (if used) or a brief description of
the sampling point and treatment process if no sample code
system is used.
• Sample number (if more than one sample was collected at
the same point on the same day).
• Facility name and address.
• Facility contact person.
• Name of the person collecting the sample.
Other information, such as sewage sludge pile temperature
at representative depth (e.g., if above ambient temperature),
may also be helpful (Yanko, 1987).
Shipment Container
A soundly constructed and insulated shipment box is es-
sential to provide the proper environment for the preserving
sample at the required temperature. It is recommended that the
outside label of the shipment container include:
• The complete address of the receiving lab (including the
name of a responsible person).
• A number clearly indicating how many samples are in-
cluded.
• The words "Fragile" and "This End Up."
To maintain a low temperature in the shipment box, a
blue-ice type of coolant in a sealed bag should be included in
the box. If the blue ice has been stored in a 0°F (-18°C) freezer
(e.g., a typical household freezer), the coolant should be "tem-
pered" to warm it up to the melting point of ice (0°C [32°F])
before it is placed around the sample.
Adherence to Holding and Shipment Times
Adherence to sample preservation and holding time limits
described in Section 8.6 is critical. Samples that are not proc-
essed within the specified time and under the proper conditions
can yield erroneous results, especially with the less stable mi-
croorganisms (i.e., bacteria).
8.10 Documentation
Sampling Plan
It is recommended that all sampling procedures be docu-
mented in a sampling plan that identifies the sampling points,
volumes to be drawn, days and times of collection, required
equipment, instructions for labelling samples and ensuring
chain of custody, and a list of contact persons and telephone
numbers in case unexpected difficulties arise during sampling.
If a formal sampling plan is not available, a field log that
includes instructions and a sample collection form may be used
(EPA, 1980).
45
-------
Sampling Log
It is suggested that all information pertinent to a sampling
be recorded in a bound log book, preferably with consecutively
numbered pages. Suggested entries in the log book include, at
a minimum:
• Purpose of sampling.
• Location of sampling.
• Grab or composite sample.
» Name and address of the field contact.
• Type of sewage sludge.
• Number and volume of the sample taken.
• Description of sampling point
• Date and time of collection.
A good rule of thumb is to record sufficient information
so that the sampling situation can be reconstructed without
reliance on the collector's memory.
Chain of Custody
To establish the documentation necessary to trace sample
possession from the time of collection, it is recommended that
a chain-of-custody record be filled out and accompany every
sample. This record is particularly important if the sample is to
be introduced as evidence in litigation. Suggested information
for the chain-of-custody record includes, at a minimum:
• Collector's name.
• Signature of collector.
• Date and time of collection.
• Place and address of collection.
• Signatures of the persons involved in the chain of posses-
sion.
8.11 Analytical Methods
Part 503.8(b) of the Part 503 regulation specifies methods
that must be used when analyzing for enteric viruses, fecal
coliform, salmonellae, viable helminth ova, specific oxygen
uptake rate, and total, fixed, and volatile solids. Table 8-1 lists
the required methods. Complete references for these methods
can be found in Chapter 12. Appendix F presents sample prepa-
ration methods for fecal coliform tests and Salmonella sp.
analysis and discusses reporting of results. Appendix G pro-
vides the required analytical method for Salmonella sp. Appen-
dix H presents a detailed discussion of the required method for
analysis of enteroviruses from sewage sludge, and Appendix I
Table 8-1. Analytical Methods Required Under Part 503
Enteric Viruses
Fecal Coliform
Salmonella sp. Bacteria
Viable Helminth Ova
Specific Oxygen
Uptake Rate (SOUR)
Total, Fixed, and
Volatile Solids
Percent Volatile Solids
Reduction
American Society for Testing and
Materials Method D 4994-89 (ASTM,
1992b)1
Standard Methods Part 9221 E or Part
9222 D (APHA, 1992)2
Standard Methods Part 9260D (APHA,
1992)2 or
Kenner and Clark (1974) (see
Appendix G of this document)2
Yanko (1987) (see Appendix I of this
document)
Standard Methods Part 271 OB (APHA,
1992)
Standard Methods Part 2540G (APHA,
1992)
Appendix C of this document
1Appendix H of this document presents a detailed discussion of this
method.
2See Appendix F of this document for recommended sample preparation
procedures and a discussion of the reporting of results.
provides the required analytical method for viable helminth
ova.
Part 503 also refers to this document for the method to be
used when calculating percent volatile solids reduction (see
Appendix C of this document). Appendix D provides guidance
on how to conduct the additional digestion tests to demonstrate
reduced vector attraction in anaerobically and aerobically di-
gested sewage sludge. Appendix D also provides guidance on
adjusting the specific oxygen uptake rate (SOUR) determined
at the temperature at which aerobic digestion is occurring in
the treatment works to a SOUR for 20°C (68°F).
8.12 Quality Assurance
Quality assurance comprises establishing a sampling plan,
quality control measures, and procedures for ensuring that the
results of analytical and test measurements are correct. A com-
plete presentation of this subject is beyond the scope of this
manual. A concise treatment of quality assurance is found in
Standard Methods (APHA, 1992) and is especially recom-
mended. Parts 1000 to 1090 of Standard Methods are relevant
to the entire sampling and analysis effort. Part 1020 discusses
quality assurance, quality control, and quality assessment.
Standard Methods (Part 1020B) states that "a good quality
control program consists of at least seven elements: certifica-
tion of operator competence, recovery of known additions,
analysis of externally supplied standards, analysis of reagent
blanks, calibration with standards, analysis of duplicates, and
maintenance of control charts." For most of the tests to be
carried out to meet the pathogen and vector attraction reduction
requirements of the Part 503 regulation, these elements cannot
be met completely, but they should be kept in mind as a goal.
46
-------
Microbial Tests
For the microbiological tests, quality assurance is needed
to verify precision and accuracy. Quality assurance for micro-
biological methods is discussed in Part 9020 of Standard Meth-
ods. The quality control approach they suggest is recommended
for the microbiological tests required because of Part 503. In
Part 9020B-4, Analytical Quality Control Procedures, it is sug-
gested that precision be initially established by running a num-
ber of duplicates, and that thereafter duplicates (5% of total
samples) be run to determine whether precision is being main-
tained.
To estimate accuracy, spiking and recovery tests are
needed. The spiking should be to density levels significant to
the Part 503 regulation. For example, for viable helminth ova,
samples should be spiked to density levels of under 100 per
gram. Yanko (1987) did not find spiking useful for bacterial
tests or viral tests, although it was effective for the helminth
ova test. For viruses, instead of spiking, he recommends dem-
onstrating the effectiveness of recovery on primary sewage
sludges that typically contain viruses at low but consistent lev-
els (for example, primary sludges from large cities).
Vector Attraction Reduction Tests
It is not possible to test for accuracy for any of the vector
attraction reduction tests, because standard sludges with con-
sistent qualities do not exist. Standard Methods gives guidance
on precision and bias. However, for some of the vector attrac-
tion reduction options, this information was not available or
was approximate. Section 7.7 provides guidance on the number
of samples to take. The procedures for the three vector attrac-
tion options developed for the Part 503 regulation (additional
anaerobic and aerobic digestion and the specific oxygen uptake
rate test), which are presented in Appendix D, have internal
quality control procedures that include replication. Since the
tests are newly proposed, the data are insufficient to judge
whether agreement between replicates is adequate. This kind of
information will be communicated as experience with these
options accumulates.
47
-------
-------
Chapter 9
Processes to Significantly Reduce Pathogens (PSRPs)
9.1 Introduction
Processes to Significantly Reduce Pathogens (PSRPs) are
listed in Appendix B of Part 503. There are five PSRPs: aerobic
and anaerobic digestion, air drying, composting, and lime sta-
bilization. Under Part 503.32(b)(3), sewage sludge treated in
these processes is considered to be Class B with respect to
pathogens (see Section 5.3).
When operated under the conditions specified in Appendix
B, PSRPs reduce fecal coliform densities to less than 2 million
CPU or MPN per gram of total solids (dry weight basis) and
reduce salmonellae and enteric virus densities in sewage sludge
by approximately a factor of 10 (Farrell et al., 1985).
This level of pathogen reduction is required, as a mini-
mum, by the Part 503 regulation if the sewage sludge is applied
to agricultural land, a public contact site, a forest, or a recla-
mation site or placed on a surface disposal site.1 Because Class
B sludges do contain some pathogens, land application of a
Class B sludge is allowed only if crop harvesting, animal graz-
ing, and public access are limited for a period of tune following
application of a Class B sewage sludge so that pathogens can
be further reduced by environmental factors (see Section 5.5).
The PSRPs listed in Part 503 are essentially identical to
the PSRPs that were listed under the 40 CFR Part 257 regula-
tion, except that all requirements related solely to reduction of
vector attraction have been removed. Vector attraction reduc-
tion is now covered under separate requirements (see Chapter
6) that include some of the requirements that were part of the
PSRP requirements under Part 257, as well as some new op-
tions for demonstrating vector attraction reduction. These new
options provide greater flexibility to the regulated community
in meeting the vector attraction reduction requirements.
This chapter provides detailed descriptions of the PSRPs
listed in Appendix B. Since the conditions for the PSRPs, par-
ticularly aerobic and anaerobic digestion, are designed to meet
pathogen reduction requirements, they are not necessarily the
same conditions as those traditionally recommended by design
texts and manuals.
92 Aerobic Digestion
In aerobic digestion, sewage sludge is biochemically oxi-
dized by bacteria in an open or enclosed vessel (see photo). To
supply these aerobic microorganisms with enough oxygen to
carry out their task, either the sewage sludge must be agitated
by a mixer or air must be forcibly injected (Figure 9-1). Under
proper operating conditions, the volatile solids in sewage
sludge are converted to carbon dioxide, water, and nitrate
nitrogen.
Aerobic systems operate in either batch or continuous
mode. In batch mode, the tank is filled with untreated sewage
sludge and aerated for 2 to 3 weeks or longer, depending on
the type of sewage sludge, ambient temperature, and average
oxygen levels. Following aeration, the stabilized solids are al-
lowed to settle and then separated from the clarified super-
natant. The process is begun again by inoculating a new batch
of untreated sewage sludge with some of the solids from the
previous batch to supply the necessary biological decomposers.
In continuous mode, untreated sewage sludge is fed into the
digester once a day or more frequently; thickened, clarified
solids are removed at the same rate.
The PSRP description in Part 503 for aerobic digestion is:
'Unless the active sewage sludge unit is covered at the end of each operating day, in
which case no pathogen requirement applies.
Digester in Vancouver, Washington.
49
-------
AERODIGESTER
SETTLING
TANK
RAW
SLUDGE
f- ^ > X OXIDIZED
K \sA OVERFLOW
4 ^-——-r-^,. I TO TREATMENT WORKS
I |^
x>iis>w£srt.**.'i>;wy
RETURN SLUDGE
TO AERODIGESTER
STABILIZED
SLUDGE
TO DISPOSAL
Figure 9-1. Aerobic digestion.
Sewage sludge is agitated with air or oxygen to maintain aero-
bic conditions for a specific mean cell residence time at a
specific temperature. Values for the mean cell residence time
and temperature shall be between 40 days at 20°C (68°F) and
60 days at 15°C (59°F).
The regulation does not differentiate between semi-batch
and continuous operation, so either method is acceptable. The
mean cell residence time is the residence time of the sewage
sludge solids. The appropriate method for calculating residence
time depends on the type of digester operation used:
• Continuous-Mode, No Supernatant Removal. For con-
tinuous-mode digesters where no supernatant is removed,
nominal residence times may be calculated by dividing liq-
uid volume in the digester by the average daily flow rate in
or out of the digester.
• Continuous-Mode, Supernatant Removal. In systems
where the is supernatant removed from the digester and
recycled, the output volume of sewage sludge can be much
less than the input volume of sewage sludge. For these sys-
tems, the flow rate of the sewage sludge out o/the digester
is used to calculate residence times.
• Continuous-Mode Feeding, Batch Removal of Sewage
Sludge. For some aerobic systems, the digester is initially
filled above the diffusers with treated effluent, and sewage
sludge is wasted daily into the digester. Periodically, aeration
is stopped to allow solids to settle and supernatant to be
removed. As the supernatant is drawn off, the solids content
in the digester gradually increases. The process is complete
when either settling or supernatant removal is inadequate to
provide space for the daily sewage sludge wasting require-
ment, or sufficient time for digestion has been provided. The
batch of digested sewage sludge is then removed and the
process begun again. If the daily mass of sewage sludge
solids introduced has been constant, nominal residence time
is one-half the total time from initial charge to final with-
drawal of the digested sewage sludge.
• Batch or Staged Reactor Mode. A batch reactor or two or
more well-mixed reactors in series are more effective in
reducing pathogens than is a single well-mixed reactor at the
same overall residence time. Reduction in residence time of
30% from the times given in the PSRP definition for aerobic
digestion is recommended. The basis for this recommenda-
tion is given in Appendix E. These times are less than the
PSRP conditions requked for aerobic digestion in the regu-
lation; consequently, approval by the regulatory authority is
requked.
• Other. Digesters are frequently operated in unique ways that
do not fall into the categories above. Appendix E provides
information that should be helpful in developing a calcula-
tion procedure for these cases.
Aerobic digestion carried out according to the Part 503
conditions typically reduces bacterial and vkal pathogens by
90% (i.e., a factor of 10). Helminth ova are reduced to varying
degrees, depending on the hardiness of the individual species.
Aerobic digestion typically reduces the volatile solids content
(the microbes' food source) of the sewage sludge by 40% to
50%, depending on the conditions maintained in the system.
Vector Attraction Reduction
Vector attraction reduction for aerobically digested sludges
is demonstrated either when the percent volatile solids reduc-
tion during sludge treatment exceeds 38%, or when the specific
oxygen uptake rate (SOUR) at 20°C (68°F) is less than or equal
to 1.5 mg of oxygen per hour per gram of total solids, or when
50
-------
additional volatile solids reduction during bench-scale aerobic
batch digestion for 30 additional days at 20°C (68°F) is less
than 15% (see Chapter 6).
Thermophilic aerobic systems (operating at higher tem-
peratures) capable of producing Class A sludge are gaining in
popularity as operators and researchers learn how to control
and stabilize this comparatively delicate process. These systems
are described more fully in Section 10.5.
9.3 Anaerobic Digestion
Anaerobic digestion is a biological process that uses bac-
teria that function in an oxygen-free environment to convert
volatile solids into carbon dioxide, methane, and ammonia.
These reactions take place in an enclosed tank (see Figure 9-2)
that may or may not be heated. Because the biological activity
consumes most of the volatile solids needed for further bacterial
growth, the sewage sludge is stabilized. Currently, anaerobic
digestion is one of the most widely used treatments for sewage
sludge stabilization, especially in treatment works with average
wastewater flow rates greater than 19,000 cubic meters/day (5
million gallons per day).
Most anaerobic digestion systems are classified as either
standard-rate or high-rate systems. Standard-rate systems take
place in a simple storage tank with sewage sludge added inter-
mittently. The only agitation that occurs comes from the natural
mixing caused by sewage sludge gases rising to the surface.
Standard-rate operation can be carried out at ambient tempera-
ture, though heat is sometimes added to speed the biological
activity.
High-rate systems use a combination of active mixing and
carefully controlled, elevated temperature to increase the rate
of sewage sludge stabilization. These systems sometimes use
pre-thickened sewage sludge introduced at a uniform rate to
maintain constant conditions in the reactor. Operating condi-
tions in high-rate systems foster more efficient sewage sludge
digestion.
The PSRP description hi Part 503 for anaerobic digestion
Sewage sludge is treated in the absence of air for a specific
mean cell residence time at a specified temperature. Values for
the mean cell residence time and temperature shall be between
15 days at 35°C to 55°C (95°F to 131°F) and 60 days at 20°C
(68°F).
Section 9.2 provides information on calculating residence
times. Anaerobic digestion that meets the required residences
times and temperatures typically reduces bacterial and viral
pathogens by 90% or more. Helminth ova are not substantially
reduced under mesophilic conditions (32°C to 38°C [90°F to
100°F]) and may not be completely reduced at temperatures
between 38°C (100°F) and 50°C (122°F).
Vector Attraction Reduction
Anaerobic systems reduce volatile solids by 35% to 60%,
depending on the nature of the sewage sludge and the system's
operating conditions. Sewage sludges produced by systems that
meet the operating conditions specified under Part 503 will
typically have volatile solids reduced by at least 38%. Alterna-
tively, vector attraction reduction can be demonstrated by Op-
tion 2 of the vector attraction reduction requirements, which
requires that additional volatile solids loss during bench-scale
anaerobic batch digestion of the sludge for 40, additional days
at 30°C to 37°C (86°F to 99°F) be less than 17% (see Section
6.3).
FIRST STAGE
(completely mixed)
SECOND STAGE
(stratified)
Figure 9-2. Two-stage anaerobic digestion (high rate).
51
-------
Sludge drying operation. (Photo credit East Bay Municipal Utility Dis-
trict)
9.4 Air Drying
Air drying allows partially digested sewage sludge to dry
naturally in the open air (see photo above). Wet sewage sludge
is usually applied to a depth of approximately 23 cm (9 inches)
onto sand drying beds, or even deeper on paved or unpaved
basins. The sewage sludge is left to drain and dry by evapora-
tion. Sand beds have an underlying drainage system; some type
of mechanical mixing or turning is frequently added to paved
or unpaved basins. The effectiveness of the drying process
depends very much on the local climate: drying occurs faster
and more completely in warm, dry weather, and slower and less
completely in cold, wet weather.
The PSRP description in Part 503 for air drying is:
S&vage sludge is dried on sand beds or on paved or unpaved
basins. The sewage sludge dries for a minimum of 3 months.
During 2 of the 3 months, the ambient average daily tempera-
ture is above 0"C (32°F).
In addition, it is advisable to ensure that the sewage sludge
drying beds are exposed to the atmosphere (i.e., not covered
with snow) during the 2 months that the daily temperature is
above 0°C (32°F). Also, the sewage sludge should be at least
partially digested before air drying. Under these conditions, air
drying will reduce the density of pathogenic viruses and bac-
teria by approximately 90%. Helminth ova are reduced, except
for some hardy species that remain substantially unaffected.
Vector Attraction Reduction
Air-dried sewage sludge typically is treated by aerobic or
anaerobic digestion before it is placed on drying beds. Usually,
the easiest vector attraction reduction requirement to meet is a
demonstration of 38% reduction in volatile solids (Option 1—
see Section 6.2), including the reduction that occurs during its
residence on the drying beds.
In dry climates, vector attraction reduction can be achieved
by moisture reduction (see Option 7 in Section 6.8, and Option
8 in Section 6.9).
9.5 Composting
Composting involves the aerobic decomposition of organic
material using controlled temperature, moisture, and oxygen
levels. Composting results hi a highly stabilized, humus-like
material. Several different composting methods are currently in
use in the United States. The three most common are windrow,
aerated static pile, and within-vessel composting, all described
below. Composting can yield either a Class A or Class B sludge,
depending on the tune and temperature variables involved in
the operation.
All composting methods rely on the same basic processes.
Bulking agents such as wood chips, bark, sawdust, straw, rice
hulls, or even finished compost are added to the sewage sludge
to absorb moisture, increase porosity, and add a source of carb-
on. This mixture is stored (in windrows, static piles, or enclosed
tanks) for a period of intensive decomposition, during which
temperatures can rise well above 55°C (131°F). Depending on
ambient temperatures and the process chosen, the time required
to produce a high-quality sewage sludge can range from 2 to 4
weeks. Aeration and/or frequent mixing or turning are needed
to supply oxygen and remove excess heat. Following this active
stage, bulking agents may or may not be screened from the
completed compost for recycling (see photo below), and the
composted sewage sludge is "cured" for an additional period.
Composted sludge Is screened to remove the bulking agent prior
to land application.
Windrow composting involves stacking the sewage
sludge/bulking agent mixture into long piles, or windrows, gen-
erally 0.9 to 1.8 m high (3 to 6 feet) and 1.8 to 4.9 m wide (6
to 16 feet). These rows are regularly turned or mixed (e.g.,
using a front-end loader) to ensure a steady oxygen supply for
the microorganisms and to reduce moisture content (see photo,
next page). Active windrows are typically placed in the open
air, except in areas with heavy rainfall. In colder climates,
winter weather can significantly increase the amount of time
needed to attain temperatures needed for pathogen control.
52
-------
Compost mixing equipment turns over a windrow of compost for
solar drying prior to screening. (Photo credit: East Bay Municipal
Utility District)
Aerated static pile composting uses forced-air rather than
mechanical mixing (see Figure 9-3) to both supply sufficient
oxygen for decomposition and carry off moisture. The sewage
sludge/bulking agent mixture is placed on top of either (1) a
fixed underlying forced aeration system, or (2) a system of
perforated piping laid on the composting pad surface and
topped with a bed of bulking agent. The entire pile is covered
with a layer of cured compost for insulation and noxious odor
containment. Pumps are used to blow air into the compost pile
or suck air through it. The latter provides greater odor control
because the compost-air can be easily collected and then filtered
or scrubbed.
Within-vessel composting systems vary greatly in design.
They share, however, two basic techniques: the process takes
place in a reactor vessel where the operating conditions can be
carefully controlled (see photo below), and active aeration
meets the system's high oxygen demand. Agitated bed systems
(i.e., within-vessel composting) depend on continuous or peri-
odic mixing within the vessel, followed by a curing period
outside of it.
Taulman Weiss in-vessel composting facility in Portland, Oregon.
Pathogen destruction during composting depends on time
and temperature variables (see photo, next page). Part 503 pro-
vides the following definition of PSRP requirement for patho-
gen destruction during composting:
Air
Air
Composted
Sludge
Bulking Agent/
Sludge Mixture
Porous Base:
Wood Chips or
Compost
Filter Pile of
Composted Sludge
Figure 9-3. Static aerated pile composting.
53
-------
Compost operator measures compost pile temperature as part of
process monitoring. (Photo credit East Bay Municipal Utility District,
Oakland, California)
Using either the within-vessel, static aerated pile, or -windrow
composting methods, the temperature of the sewage sludge is
raised to 40°C (104°F) or higher and remains at 40°C (104°F)
or higher for 5 days. For 4 hours during the 5-day period, the
temperature in the compost pile exceeds 55°C (131°F).
These conditions, achieved using either within-vessel, aer-
ated static pile, or windrow methods, reduce bacterial and viral
pathogens, but not helminth ova or other parasites, by more
than 90% (10-fold).
A process time of only 5 days is not long enough to fully
stabilize the sewage sludge solids, so the composted sewage
sludge produced under these conditions will not be able to meet
any of the requirements for reduced vector attraction. Complete
stabilization may require 14 to 21 days for within-vessel; 21 or
more days for aerated static pile; and 30 or more days for
windrow composting. Many treatment works allow the finished
sewage sludge to further mature or cure for at least several
weeks following active composting.
Vector Attraction Reduction
Vector attraction reduction must be demonstrated for com-
posted sewage sludge. In a few cases, this can be demonstrated
by Option 1 of the vector attraction reduction requirements
(38% reduction in volatile solids—see Section 6.2). However,
in most cases, Option 5 is more appropriate. This option re-
quires aerobic treatment (i.e., composting) of the sewage sludge
for at least 14 days at over 40°C (104°F) with an average
temperature of over 45°C (113°F).
9.6 Lime Stabilization
For 2,000 years, lime has been used to deodorize and sta-
bilize night soil and manure. Today, lime treatment is gaining
popularity as an effective option for controlling pathogens in
sewage sludge. The process is relatively straightforward:
lime—either hydrated lime, Ca(OH)2; quicklime, CaO; or lime-
containing kiln dust or fly ash—is added to sewage sludge in
sufficient quantities to raise the pH above 12 after 2 hours of
contact, as specified in the Part 503 PSRP description for lime
stabilization:
Sufficient lime is added to the sewage sludge to raise the pH
of the sewage sludge to 12 after 2 hours of contact.
Lime may be introduced to liquid sewage sludge in a mix-
ing tank or combined with dewatered sewage sludge, providing
the mixing is intimate and the sewage sludge cake is moist
enough to allow aqueous contact between the sewage sludge
and lime.
A variety of lime stabilization processes—some patented—
are currently in use. The growing popularity of this treatment
means new techniques will undoubtedly be developed in the
-future. The effectiveness of any lime stabilization process for
controlling pathogens depends on maintaining the pH at levels
that reduce microorganisms in the sewage sludge and also later
inhibit bacterial growth should contamination occur after treat-
ment. Lime stabilization can reduce bacterial and viral patho-
gens 99 percent or more. Such alkaline conditions have little
effect on hardy species of helminth ova, however.
Lime stabilization does not reduce volatile solids. If the
pH of lime-stabilized sewage sludge drops below 11, remaining
pathogenic bacteria or those introduced by animal vectors may
grow rapidly to substantial densities, given the rich food source.
Thus long-term storage of alkali-treated sewage sludge requires
either additional treatment with lime to maintain elevated pH,
drying, or further treatment to reduce volatile solids (e.g., com-
posting).
Vector Attraction Reduction
For lime-treated sludge, vector attraction reduction is dem-
onstrated by Option 6 of the vector attraction reduction require-
ments. This option requires that the sludge pH remain at 12 or
higher for at least 2 hours, and then at 11.5 or more for an
additional 22 hours (see Section 6.7).
9.7 Equivalent Processes
Under Class B Alternative 3, sewage sludges treated in
processes that are determined to be equivalent to PSRP are
considered to be Class B. Table 5-2 in Chapter 5 lists some of
the processes that the EPA's Pathogen Equivalency Committee
has recommended as being equivalent to PSRP under Part 257.
Chapter 11 discusses how the PEC makes a recommendation
of equivalency.
54
-------
Chapter 10
Processes to Further Reduce Pathogens (PFRPs)
10.1 Introduction
Processes to Further Reduce Pathogens (PFRPs) are listed
in Appendix B of Part 503. There are seven PFRPs: compost-
ing, heat drying, heat treatment, thermophilic aerobic digestion,
beta ray irradiation, gamma ray irradiation, and pasteurization.
When these processes are operated under the conditions speci-
fied in Appendix B, they produce sewage sludges with patho-
genic bacteria, enteric viruses, and viable helminth ova reduced
to below detectable levels. Reduction of vector attraction must
occur during or after PFRP treatment (see Section 4.2).
Under Part 503.32(a)(7), sewage sludge treated in these
processes is considered to be Class A with respect to pathogens.
Class A sludges can be used in any land application situation
(including lawns and gardens) without restriction; however,
Class A sludges must be monitored for fecal coliform or Sal-
monella sp. bacteria at the time of use or disposal, at the time
the sewage sludge is prepared for sale or give away in a bag
or other container for land application, or at the time the sewage
sludge or material derived from the sewage sludge is prepared
to meet the requirements in 503.10(b), 503.10(c), 503.10(e), or
503.10(f) to ensure that regrowth of bacteria has not occurred
(see Section 4.3).
The PFRPs listed in Part 503 are essentially identical to
the PFRPs listed under the 40 CFR Part 257 regulation, except
that all requirements related solely to reduction of vector at-
traction have been removed. This chapter provides detailed
descriptions of the seven PFRPs listed in Part 503.
10.2 Composting
As described in Chapter 9, composting reduces sewage
sludge, which has generally been mixed with a bulking agent,
to a humus-like material through biological degradation. There
are three commonly used methods of composting: windrow,
static aerated pile, and within-vessel.
To be considered a PFRP under Part 503, the composting
operation must meet certain operating conditions:
Using either the within-vessel composting method or the static
aerated pile composting method, the temperature of the sewage
sludge is maintained at 55°C (131°F) or higher for 3 days.
Using the windrow composting method, the temperature of the
sewage sludge is maintained at 55°C (131°F) or higher for 15
days or longer. During the period when the compost is main-
tained at 55°C (131°F) or higher, there shall be a minimum of
five turnings of the windrow.
In general, within-vessel composting attains the required
conditions in approximately 10 days. The static-pile and wind-
row processes generally require about 3 weeks. If the condi-
tions specified by the regulation are met, all pathogenic viruses,
bacteria, and parasites will be reduced to below detectable lev-
els. However, composting under these conditions may not ade-
quately reduce vector attraction. Longer composting periods
may be necessary to fully stabilize the sewage sludge (see
Section 9.5).
Under some conditions, it may be difficult to meet the
Class A monitoring requirement for fecal coliforms even when
Salmonella sp. bacteria are not present. Sewage sludge treat-
ment involving high heat, for example, can reduce salmonellae
to below detectable levels while leaving some, fecal coliforms
intact. If volatile solids remain in the sludge, coliforms can later
regrow to significant numbers. The same thing can happen
when sewage sludge is pre-treated with lime before compost-
ing. It may be necessary, therefore, to test composted sewage
sludge directly for salmonellae, rather than using fecal coli-
forms as an indicator of pathogen control.
Vector Attraction Reduction
The options for demonstrating vector attraction reduction
for both PFRP and PSRP composting are the same, and are
discussed in Section 9.5.
10.3 Heat Drying
Heat drying is used to reduce both pathogens and the water
content of sewage sludge. The Part 503 PFRP description of
heat drying is:
Sewage sludge is dried by direct or indirect contact with hot
gases to reduce the moisture content to 10% or lower. Either
the temperature of the sewage sludge particles exceeds 80°C
(176°F) or the wet bulb temperature of the gas in contact with
the sewage sludge as it leaves the dryer exceeds 80°C (176°F).
Properly conducted heat drying will reduce pathogenic vi-
ruses, bacteria, and helminth ova to below detectable levels.
55
-------
Four processes are commonly used for heat drying sewage
sludge: flash dryers, spray dryers, rotary dryers, and the Carver-
Greenfield process (EPA, 1979). Hash dryers used to he the
most common heat drying process installed at treatment works,
but current practice favors rotary dryers.
Flash Dryers
Flash dryers pulverize sewage sludge in the presence of
hot gases. The process is based on exposing fine sewage sludge
particles to turbulent hot gases long enough to attain at least
90% solids content.
Spray Dryers
A spray dryer typically uses centrifugal force to atomize
liquid sewage sludge into a spray that is directed into a drying
chamber. The drying chamber contains hot gases that rapidly
dry the sewage sludge mist Some spray drying systems use a
nozzle to atomize sewage sludge.
Rotary Dryers
Rotary dryers function as horizontal cylindrical kilns. The
drum rotates and may have plows or louvers that mechanically
mix the sewage sludge as the drum turns. There are many
different rotary kiln designs, utilizing either direct heating or
indirect heating systems. Direct heating designs maintain con-
tact between the sewage sludge and the hot gases. Indirect
heating separates the two with steel shells.
Carver-Greenfield Process
The Carver-Greenfield process is a patented multiple-effect
evaporative oil-immersion process in which dewatered sewage
sludge is mixed with a light oil. This mixture is pumped through
a series of evaporators that selectively remove the water in
sewage sludge, which has a lower boiling point than the oil
carrier. The oil maintains the mixture in a liquid state, even
when virtually all the water has been removed. The product of
this process, an oil and dry sewage sludge mixture, is put
through a centrifuge to separate the dry sewage sludge solids
from the oil. The recovered oil can be reused in the process.
Vector Attraction Reduction
The PFRP requirements for heat drying also meets the
requirements of Option 8 for vector attraction reduction (i.e.,
the percent solids must be at least 90% before mixing the sludge
with other materials). It exceeds the requirement of Option 7 if
the sludge being dried contains no unstabilized solids.
10.4 Heat Treatment
Heat treatment processes are used both to stabilize and
condition sewage sludge. The processes involve heating sewage
sludge under pressure for a short period of time. The sewage
sludge becomes sterilized and bacterial slime layers are solu-
bilized, making it easier to dewater the remaining sewage
sludge solids. The Part 503 PRFP description for heat treatment
is:
Liquid sewage sludge is heated to a temperature of 180°C
(356°F) or higher for 30 minutes.
If operated according to these requirements, the process
effectively reduces pathogenic viruses, bacteria, and helminth
ova to below detectable levels. Sewage sludge must be properly
stored after processing because organic matter has not been
reduced and, therefore, regrowth of pathogenic bacteria can
occur if treated sewage sludge is reinoculated.
Two processes have been used for heat treatment: the
Porteous and the Zimpro process. In the Porteous process the
sewage sludge is preheated and then injected into a reactor
vessel. Steam is also injected into the vessel under pressure.
The sewage sludge is retained in the vessel for approximately
30 minutes after which it is discharged to a decant tank. The
resulting sewage sludge can generally be concentrated and de-
watered to high solids concentrations. Further dewatering may
be desirable to facilitate sewage sludge handling.
The Zimpro process is similar to the Porteous process.
However, air is injected into the sewage sludge before it enters
the reactor and the vessel is then heated by steam to reach the
required temperature. Temperatures and pressures are approxi-
mately the same for the two processes.
Vector Attraction Reduction
Heat treatment must be followed by a vector attraction
reduction process. Vector attraction reduction Options 6 to 11
may be used (see Chapter 6). Options 1 to 5 are not applicable
unless the sludge is subsequently digested.
10.5 Thermophilic Aerobic Digestion
Thermophilic aerobic digestion is a refinement of the con-
ventional aerobic digestion processes discussed in Section 9.2.
In this process, feed sewage sludge is generally pre-thickened
and an efficient aerator is used. In some modifications, oxygen
is used instead of air. Because there is less sewage sludge
volume and less air to carry away heat, the heat released from
biological oxidation warms the sewage sludge in the digester
to as high as 60°C (140°F).
Because of the increased temperatures, this process
achieves higher rates of organic solids reduction than are
achieved by conventional aerobic digestion, which operates at
ambient air temperature. The biodegradable volatile solids con-
tent of the sewage sludge can be reduced up to 70% in a
relatively short time. The digested sewage sludge is effectively
pasteurized due to the high temperatures. Pathogenic vkuses,
bacteria, and parasites are reduced to below detectable limits if
temperatures exceed 55°C (131°F).
This process can either be accomplished using auxiliary
heating of the digestion tanks or through special designs that
allow the energy naturally released by the microbial digestion
process to heat the sewage sludge. The Part 503 PFRP descrip-
tion of thermophilic aerobic digestion is:
56
-------
Liquid sewage sludge is agitated with air or oxygen to maintain
aerobic conditions and the mean cell residence time of the
sewage sludge is 10 days at 55°C to 60°C (131°F to I40°F).
The thermophilic process requires significantly lower resi-
dence times (i.e., solids retention time) than conventional aero-
bic processes designed to qualify as a PSRP, which must
operate 40 to 60 days at 20°C to 15°C (68°F to 59°F), respec-
tively. Residence time is normally determined by dividing the
volume of sewage sludge in the vessel by the volumetric flow
rate. Operation should minimize the potential for bypassing by
withdrawing treated sludge before feeding, and feeding no
more than once a day.
Vector Attraction Reduction
Vector attraction reduction must be demonstrated. Al-
though all options, except Options 3 and 12 are possible, Op-
tions 1 and 2 are the most suitable. (Option 3 is not possible
because it is not yet known how to translate SOUR measure-
ments obtained at high temperatures to 20°C [68°F].)
10.6 Beta Ray and Gamma Ray Radiation1
Radiation can be used to disinfect sewage sludge. Radia-
tion destroys certain organisms by altering the colloidal nature
of the cell contents (protoplasm). Gamma rays and beta rays
are the two potential energy sources for use in sewage sludge
disinfection. Gamma rays are high-energy photons produced by
certain radioactive elements. Beta rays are electrons accelerated
in velocity by electrical potentials in the vicinity of 1 millions
volts. Both types of radiation destroy pathogens that they pene-
trate if the doses are adequate.
The Part 503 PFRP descriptions for irradiation systems are:
Although the two types of radiation function similarly to
inactivate pathogens, there are important differences. Gamma
rays can penetrate substantial thicknesses of sewage sludge and
can therefore be introduced to sewage sludge by either piping
liquid sewage sludge into a vessel that surrounds the radiation
source (Figure 10-1) or by carrying composted or dried sewage
sludge by hopper conveyor to the radiation source. Beta rays
have limited penetration ability and therefore are introduced by
passing a thin layer of sewage sludge under the radiation source
(Figure 10-2).
Figure 10-1. Schematic representation of cobalt-60 (gamma ray) irra-
diation facility at Geiselbullach, Germany.
Source: EPA, 1979.
Beta ray irradiation—Sewage sludge is irradiated with beta
rays from an accelerator at dosages of at least 1.0 megarad at
room temperature (ca. 20°C [68°F]).
Gamma ray irradiation—Sewage sludge is irradiated with
gamma rays from certain isotopes, such as Cobalt 60 and
Cesium 137 [at dosages of at least 1.0 megarad] at room
temperature (ca. 20°C [68°F]).2
The effectiveness of beta radiation in reducing pathogens
depends on the radiation dose, which is measured hi rads. A
dose of 1 megarad or more will reduce pathogenic viruses,
bacteria, and helminths to below detectable levels. Lower doses
may successfully reduce bacteria and helminth ova but not
viruses. Sewage sludge must be properly stored after processing
because organic matter has not been reduced and therefore
regrowth of pathogenic bacteria can occur if sewage sludge is
reinoculated.
INPUT
(UNTREATED OR
DIGESTED SLUDGE)
INCLINED
FEED RAMP
ELECTRON BEAM
SCANNER
HIGH ENERGY
DISINFECTION
ZONE
SLUDGE
• RECEIVING
TANK
OUTPUT
(DISINFECTED
SLUDGE)
Current usage by physicists reserved the term "beta rays" for high-energy electrons emit-
ted by radioactive elements. More properly, the regulation should have used the term
"high-energy electrons" instead of "beta rays."
2The 1.0 megarad dose was inadvertently omitted from the final Part 503 regulation.
Figure 10-2. Beta ray scanner and sludge spreader.
Source: EPA, 1979.
57
-------
Vector Attraction Reduction
Radiation treatment must be followed by a vector attraction
reduction process. The appropriate options for demonstrating
vector attraction reduction are the same as for heat treatment
(see Section 10.4), namely Options 6 to 11. Options 1 to 5 are
not applicable unless the sludge is subsequently digested.
10.7 Pasteurization
Pasteurization involves heating sewage sludge to above a
predetermined temperature for a minimum time period. For
pasteurization, the Part 503 PERP description is:
Ttie temperature of the se\vage sludge is maintained at 70°C
(158°F) or higher for 30 minutes or longer.
Pasteurization reduces bacteria, enteric viruses, and helminth
ova to below detectable values.
Sewage sludge can be heated by heat exchangers or by
steam injection. Although sewage sludge pasteurization is un-
common in the United States, it is widely used in Europe. The
steam injection method is preferred because it is more effective
at maintaining even temperatures throughout the sludge batch
being processed. Sewage sludge is pasteurized in batches to
prevent recontamination that might occur in a continuous proc-
ess. Sewage sludge must be properly stored after processing
because the organic matter has not been stabilized and therefore
odors and regrowth of pathogenic bacteria can occur if sewage
sludge is reinoculated.
In Europe, serious problems with regrowth of Salmonella
sp. have occurred, so pasteurization is rarely used now as a
terminal treatment process. Pre-pasteurization followed by
mesophilic digestion has successfully replaced the use of pas-
teurization after digestion in many European communities.
Vector Attraction Reduction
Pasteurization must be followed by a vector attraction re-
duction process. The options appropriate for demonstrating
vector attraction reduction are the same as those for heat treat-
ment (see Section 10.4), namely Options 6 to 11. Options 1 to
5 are not applicable unless the sludge is subsequently digested.
10.8 Equivalent Processes
Under Class A Alternative 6, sewage sludge treated in proc-
esses that are determined to be equivalent to PERP are consid-
ered to be Class A with respect to pathogens (assuming the
treated sewage sludges also meet the Class A regrowth require-
ment). Table 4-2 in Chapter 4 lists some of the processes that
were found, based on the recommendation of EPA's Pathogen
Equivalency Committee, to be equivalent to PERP under Part
257. Chapter 11 discusses how the PEC makes a recommenda-
tion of equivalency.
58
-------
Chapter 11
Role of EPA's Pathogen Equivalency Committee in
Providing Guidance Under Part 503
11.1 Introduction
One way to meet the pathogen control requkements of Part
503 is to treat sludge in a process "equivalent to" the PFRP or
PSRP processes listed in Appendix B of the regulation (see
Tables 4-2 and 5-1 for a list of these processes):
• Under Class A Alternative 6, sewage sludge that is treated
in a process equivalent to PFRP and meets the Class A
regrowth requirement (see Section 4.3) is considered to be
a Class A sludge with respect to pathogens (see Section 4.9).
• Under Class B Alternative 3, sewage sludge treated by a
process equivalent to PSRP is considered to be a Class B
sludge with respect to pathogens (see Section 5.4).
These alternatives provide continuity with the Part 257
regulation, which requked that sewage be treated by a PSRP,
PFRP, or equivalent process prior to use or disposal. There is
one major difference between Part 257 and Part 503 with re-
spect to equivalency. Under Part 257, a process had to be found
equivalent in terms of both pathogen reduction and vector at-
traction reduction. Under Part 503, equivalency pertains only
to pathogen reduction. (However, like all Class A and B
sludges, sewage sludges treated by equivalent processes must
also meet a separate vector attraction reduction requkement
[see Chapter 6]).
What Constitutes Equivalency?
To be equivalent, a treatment process must be able to con-
sistently reduce pathogens to levels comparable to the reduction
achieved by the listed PSRPs or PFRPs. (These levels, de-
scribed in Section 11.3, are the same levels requked of all Class
A and B sludges.) The process continues to be equivalent as
long as it is operated under the same conditions (e.g., time,
temperature, pH) that produced the required reductions.
Equivalency is site-specific—that is, equivalency applies only
to that particular operation run at that location under the speci-
fied conditions, and cannot be assumed for the same process
performed at a different location, or for any modification of the
process. Processes that are able to consistently produce the
requked pathogen reductions under the variety of conditions
that may be encountered at different locations across the coun-
try may qualify for a recommendation of national equivalency,
i.e, a recommendation that the process will likely be equivalent
wherever it is operated in the United States.
Who Determines Equivalency?
The permitting authority is responsible for determining
equivalency under Part 503. The permitting authority often
seeks guidance from EPA's Pathogen Equivalency Committee
(PEC) in making equivalency determinations. The PEC is re-
sponsible for making national equivalency recommendations.
What Are the Benefits of Equivalency?
A determination of equivalency is beneficial when it re-
duces the microbiological monitoring burden, i.e., when less
monitoring is requked to demonstrate equivalency than is re-
quired under the other Class A or B alternatives for meeting the
pathogen reduction requkements of Part 503. Figure 11-1 indi-
cates when application for equivalency may be appropriate.
PFRP Equivalency
Equivalency is not beneficial for processes akeady covered
under Class A Alternatives 1, 2, or 5 (see Chapter 4 for a
description of these alternatives). For processes not covered by
Alternatives 1, 2, or 5, a determination of PFRP equivalency
can reduce the enteric virus and viable helminth ova monitoring
burden1 in certain cases (see Section 11.3 for details of when
PFRP equivalency may be beneficial).
PSRP Equivalency
A determination of equivalency to PSRP eliminates the
fecal coliform monitoring requked under Class B Alternative 1
for nonequivalent processes.
Recommendation of National Equivalency
A recommendation of national equivalency can be useful
for treatment processes that will be marketed, sold, and/or used
at different locations in the United States. Such a recommen-
dation may be useful in getting PFRP or PSRP equivalency
determinations from different permitting authorities across the
country.
A determination of PFRP equivalency will not reduce the monitoring required for sal-
monellae or fecal coliform since all Class A sludges—even sludges produced by equiva-
lent processes—must be monitored for salmonellae or fecal coliform (see Section 4.3).
59
-------
NO
Is your process capable of
consistently reducing enteric
viruses and viable helminth ova to
below detectable levels?
Is your process capable of
consistently reducing the density of
fecal coliforms to below 2 million
CPU or MPN per gram total sewage
sludge solids?
YES
Your process Is '
unlikely to be
equivalent to
PSRP
Site-specific
PSRP
equivalency
may be useful
I
Are you a developer of a sewage
sludge treatment process that has
been or will be marketed and sold in
different areas of the United States?
I
NO
recommendation
of national
equivalency is
unnecessary
NO
YES
YES
i
Is your process covered under Class
A Alternative 1,2, or 5?
Site-specific
PFRP
equivalency
may be useful
(see Section
11.3)
YES
Equivalency is
unnecessary
Are you a developer of a sewage
sludge treatment process that has
been or will be marketed and sold in
different areas of the United States?
Is the effectiveness of your process
Independent of the variety of
climatic and other conditions that
may be encountered in different
locations In the United States?
INO
4E ^
I"1 ^
Your process is
unlikely to be
recommended
as equivalent on
a national level
INO
Is the effectiveness of your process
independent of the variety of climatic
and other conditions that may be
encountered in different locations in
the United States?
^^^^^^^^^^^^^^iiS^i^M^^^-J^^^^^^^-^S.
I
YES
YES
A recommendation of national PSRP
equivalency may be useful
A recommendation of national PFRP
equivalency may be useful
Figure 11-1. When Is application for PFRP or PSRP equivalency appropriate?
Role of the Pathogen Equivalency Committee
The U.S. Environmental Protection Agency created the
Pathogen Equivalency Committee (PEC) in 1985 to make rec-
ommendations to EPA management on applications for PSRP
and PFRP equivalency under Part 257 (Whittington and
Johnson, 1985). The PEC consists of approximately six mem-
bers with expertise in microbiology, wastewater engineering,
statistics, and sludge regulations. It includes representatives
from EPA's Office of Research and Development and Office of
Water.
Guidance and Technical Assistance on Equivalency
Determinations
The PEC will continue to review and make recommenda-
tions to EPA management on applications for equivalency under
Part 503. Its members also provide guidance to applicants on
the data necessary to determine equivalency, and to permitting
authorities and members of the regulated community on issues
(e.g., sampling and analysis) related to meeting the Subpart D
(pathogen and vector attraction reduction) requirements of Part
60
-------
503. Figure 11-2 elaborates on the role of the PEC under Part
503.
National Equivalency Recommendations
The PEC can also recommend that a process be considered
equivalent on a national level if the PEC finds that the process
consistently produces the required pathogen reductions under
the variety of conditions that may be encountered at different
locations across the country.
What's in This Chapter?
This chapter explains how the PEC makes equivalency
recommendations and describes how to apply for PEC guid-
ance. The guidance in this chapter may also prove useful for
permitting authorities in establishing the information they will
need to make equivalency determinations.
11.2 Overview of the PEC's Equivalency
Recommendation Process
The first point of contact for any equivalency determina-
tion, recommendation, or other guidance is usually the permit-
ting authority. Appendix A provides a list of the Regional
Sludge Coordinators (RSCs) and the State Sludge Coordinators
(SSCs). If PEC involvement is appropriate, the permitting
authority will coordinate contact with the PEC.
The PEC considers each equivalency application on a case-
by-case basis. Applicants submit information on process oper-
ating parameters and/or the sewage sludge, as described in
Section 11.5. The committee evaluates this information in light
of current knowledge concerning sewage sludge treatment and
pathogen reduction, and recommends one of five decisions
about the process or process sequence:
• It is equivalent to PFRP.
• It is not equivalent to PFRP.
• It is equivalent to PSRP.
• It is not equivalent to PSRP.
• Additional data or other information are needed.
Most processes are recommended for site-specific equiva-
lency. To receive a recommendation for national equivalency,
the applicant must demonstrate that the process will produce
the desired reductions in pathogens under the variety of condi-
tions that may be encountered at different locations across the
country. Processes affected by local climatic conditions or that
use materials that may vary significantly from one part of the
country to another are unlikely to be recommended as equiva-
lent on a national basis unless specific material specifications
and process procedure requirements can be identified.
If the PEC recommends, based on the information submit-
ted, that a process is equivalent to PSRP or PFRP, the operating
parameters and any other conditions critical to adequate patho-
gen reduction are specified. The equivalency recommendation
applies only when the process is operated under the specified
conditions.
If the committee finds that it cannot recommend equiva-
lency, the committee provides an explanation for this finding.
If additional data are needed, the committee describes what
those data are and works with the permitting authority and/or
the applicant, if necessary, to ensure that the appropriate data
are gathered in an acceptable manner. The committee then re-
views the revised application when the additional data are sub-
mitted.
11.3 Basis for PEC Equivalency Recommendations
As mentioned in Section 11.1, to be determined equivalent,
a treatment process must consistently and reliably reduce patho-
gens in sludge to the same levels achievable by the listed PSRPs
or PFRPs. The applicant must identify the process operating
parameters (e.g., time, temperature, pH) that are necessary and
sufficient for producing these reductions.
PFRP Equivalency
To be equivalent to PFRP, a treatment process must be able
to consistently reduce sewage sludge pathogens to below de-
tectable limits. For purposes of equivalency, the PEC is con-
cerned only with the ability of a process to reduce enteric
viruses and viable helminth ova to below detectable limits,
because Part 503 requires ongoing monitoring of sludge pro-
duced by PFRP-equivalent processes for fecal coliform or Sal-
monella sp. (see Section 4.3) to ensure that Salmonella sp. are
reduced to below detectable limits (i.e., to less than 3 MPN per
4 grams total solids sewage sludge [dry weight basis]). Thus,
to demonstrate PFRP equivalency, the treatment process must
be able to consistently reduce enteric viruses and viable
helminth ova to below detectable limits, which are:
enteric viruses
viable helminth ova
less than 1 plaque-forming unit per
4 grams total solids sewage sludge
(dry weight basis)
less than 1 per 4 grams total solids
sewage sludge (dry weight basis)
There are two ways these reductions can be demonstrated:
• Direct monitoring of treated and untreated sewage sludge
for enteric viruses and viable helminth ova.
• Comparison of the operating conditions of the process with
the operating conditions of one of the listed PFRPs.
In practice, the monitoring approach to demonstrating site-
specific PFRP equivalency offers no advantages as a means to
fulfill Class A requkements because owners and operators can
achieve the same outcome by performing the monitoring re-
quired under Class A Alternative 3 (see Section 4.6). Use of
Alternative 3 may be simpler than seeking equivalency, since
Alternative 3 does not require the involvement of the PEC. The
monitoring approach may be of value, however, when seeking
61
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON. D.C. 20460
JUN | 5 1993
OFFICE OF
WATER
MEMORANDUM
SUBJECT:
FROM:
The Role of the Pathogen Equivalency Committal* Under
the Part 503 Standards for the USB or Disposal of
Sewage Sludge
ce
TO:
PURPOSE
Michael B. Cook, Directd
Office of Wastewater Enforce
James A. Hanlon, Acting Directo
Office of Science & Technology
Water Division Directors
Regions I - X
This memorandum explains the role of the Pathogen
Equivalency Committee (PEC) in providing technical assistance and
recommendations regarding pathogen reduction equivalency in
implementing the Part 503 Standards for the Use or Disposal of
Sewage. The PEC is an Agency resource available to assist your
permit writers and regulated authorities. This information
should be sent to your Regional Sludge Coordinators, Municipal
Construction Managers, Permits and Enforcement Coordinators, and
Solid Waste Offices, State Sludge Management Agencies and others
concerned with sewage sludge management.
BACKGROUND
The PEC Under Part 257
The Criteria for Classification of Solid Waste Facilities
and Practices (44 FR 53438, September 13, 1979), in 40 CFR Part
257 required that sewage sludge disposed on the land be treated
by either a Process to Significantly Reduce Pathogens (PSRP) or a
Process to Further Reduce Pathogens (PFRP). A list of PSRPs and
PFRPs were included in Appendix II to Part 257.
In 1985, the PEC was formed to provide technical assistance
and recommendations on whether sewage sludge treatment processes
not included in Appendix II to Part 257 were equivalent to PSRP
or PFRP. Under Part 257, the PEC provided technical assistance
to both the permitting authority and to members of the regulated
Rgure 11-2. Role of the PEC under Part 503.
62
-------
community. The PEC membership bas included representatives from
the Office of Research & Development (ORD), office of Wastewater
Enforcement & Compliance (OWEC), and the Office of Science &
Technology (OST) with extensive experience in microbiology,
sludge process engineering, statistics and regulatory issues.
The PEC recommendations regarding the equivalency of processes
were forwarded to the office of Science and Technology, which
notified applicants about the PEG'S recommendations. Final
decisions on equivalency were made by the permitting authority.
The Part 503 Sewage Sludge Standards
The 40 CFR Part 503 Standards for the Use or Disposal of
Sewage Sludge were published in the Federal Register on
February 19, 1993 (58 FR 9248) under the authority of section 405
of the Clean Water Act, as amended. Part 503 establishes
requirements for sewage sludge applied to the land, placed on a
surface disposal site, or fired in a sewage sludge incinerator.
Along with the 40 CFR Part 258 Municipal Solid Waste (HSW)
Landfill Regulation (56 FR 50978, October 9, 1991), which
established requirements for materials placed in MSW landfills,
the Part 503 requirements for land application of sewage sludge
and placement of sewage sludge on a surface disposal site,
replaces the requirements for those practices, including the
requirement to treat the sewage sludge in either a PSRP or a
PFRP, in Part 257.
The Part 503 regulation addresses disease-causing organisms
(i.e., pathogens) in sewage sludge by establishing requirements
for sewage sludge to be classified either as Class A or Class B
with respect to pathogens as an operational standard. Class A
requirements are met by treating the sewage sludge to reduce
pathogens to below detectable limits, while the Class B
requirements rely on a combination of treatment and site
restrictions to reduce pathogens. The site restrictions prevent
exposure to the pathogens and rely on Natural Environmental
processes to reduce the pathogens in the sewage sludge to below
detectable levels. In addition to pathogen reduction, a vector
attraction reduction requirement has to be met when sewage sludge
is applied to the land or placed on a surface disposal site.
Vector attraction reduction requirements are imposed under
Part 503 to reduce the potential for spreading of infectious
disease agents by vectors (i.e., flies, rodents, and birds). A
series of alternative methods for meeting the vector attraction
reduction requirement are provided in the rule.
All sewage sludges that are to be sold or given away in a
bag or other container for land application, or applied to lawns
or home gardens must meet Class A pathogen control and vector
attraction reduction requirements. All sewage sludge intended
for land application must meet at least the Class B pathogen
control and vector attraction reduction requirements. Surface
disposal of sewage sludge requires that Class A or Class B
requirements, along with one of the vector attraction reduction
practices, be met unless the sewage sludge is covered with soil
or other material daily.
Figure 11-2. Role of the PEC under Part 503 (continued).
63
-------
A series of options are provided in the Part 503 regulation
for meeting the specific requirements for the two classes of
pathogen reduction. One of the class A alternatives is to treat
the sewage sludge by a process equivalent to a PFRP and one of
the Class B alternatives is to treat the sewage sludge by a
process equivalent to a PSRP. The permitting authority must
decide whether a process is equivalent to a PFRP or a PSRP, which
is the same approach used under Part 257.
THE PEC UNDER 503
Part 503 provides specific criteria and procedures for
evaluating bacterial indicators (Fecal coliforms and Salmonella
sp.), enteric virus and viable helminth ova as well as vector
attraction reduction. The PEC will continue to support the
permitting authority and members of the regulated community under
the new Part 503 regulation in evaluating equivalency situations
and providing technical assistance in matters such as sampling
and analysis. Specifically the PEC:
• will continue to provide technical assistance to the
permitting authority and regulated community, including
recommendations to the permitting authority about
process equivalency. The PEC also will make both site-
specific and national (i.e., a process that is
equivalent anywhere in the United States where it is
installed and operated) recommendations on process
equivalency .
will submit recommendations on process equivalency to
the Director, Health and Ecological Criteria Division,
Office of Science and Technology, who will review those
recommendations and then notify the applicant and
appropriate permitting authorities of our
recommendation.
For site-specific recommendations, requests for PEC review
or assistance should be made through the appropriate Federal
permitting authority (e.g., the state sludge regulatory authority
for delegated programs or the EPA Regional sludge Coordinator for
non-delegated programs). For national recommendations, requests
for PEC review or assistance can also be made through the
Director, Health and Ecological Criteria Division (WH-586),
Office of Science & Technology, U.S. EPA, 401 M St., SW,
Washington, D.C. 20460 or directly to the PEC chairman. The
current PEC Chairman is: Dr. James E. Smith, Jr., U.S. EPA,
CERI, (Center for Environmental Research Information) 26 W Martin
Luther King Dr., Cincinnati, OH 45268 (Tele: 513/569-7355).
Additional information and guidance to supplement the
pathogen reduction requirements of Part 503 and the procedures to
use to reach the PEC and the assistance provided by the PEC is
provided in "Control of Pathogens and Vector Attraction in Sewage
Figure 11-2. Role of the PEC under Part 503 (continued).
64
-------
Sludge" (EPA 625/R-92/013), which will be updated from time to
time by the PEC. This document is an update of the 1989 document
"Control of Pathogens in Municipal Wastewater Sludge"
(EPA/625/10-89/006), and is available from CERI.
If there are any questions about this memorandum/ please
contact Bob Bastian from OWEC at 202/260-7378 or Dr. Smith at
CERX.
Figure 11-2. Role of the PEC under Part 503 (continued).
65
-------
a national PFRP equivalency recommendation—something that
cannot be automatically achieved through the use of Alternative
3. In this case, applicants may wish to submit microbiological
monitoring data similar to that required under Alternative 3 as
part of the package of information (see Section 11.5) required
to demonstrate national PFRP equivalency.
The process comparison approach to demonstrating
equivalency is discussed in Section 11.4.
PSRP Equivalency
For PSRP equivalency, the treatment process must consis-
tently reduce the geometric mean of the fecal colifonn density
in seven samples of sewage sludge per sampling episode to less
than 2 million CFU or MPN per gram of total solids (dry weight
basis). Sufficient demonstrations of the required reductions are
needed to ensure that the process can reliably produce the re-
quired reductions under all the different types of conditions that
the process may operate. For example, for processes that may
be affected by daily and seasonal variations in the weather, four
or more sets of samples taken at different times of the year and
during different precipitation conditions (including worst-case
conditions) will be needed to make this demonstration. For
national equivalency recommendations, the demonstration must
show that the process can reliably produce the desired reduc-
tions under the variety of climatic and other conditions that may
be encountered at different locations in the United States.
11.4 Guidance on Demonstrating Equivalency for
PEC Recommendations
As described below, equivalency can be demonstrated in
one of two ways:
* By comparing operating conditions to existing PFRPs or
PSRPs.
* By providing performance and microbiological data.
Comparison to Operating Conditions for Existing
PSRPs or PFRPs
If a process is similar to a PSRP or PFRP described in the
Part 503 regulation (see Tables 4-2 and 5-1), it may be possible
to demonstrate equivalency by providing performance data
showing that the process consistently meets or exceeds the
conditions specified in the regulation. For example, a process
that consistently produces a pH of 12 after 2 hours of contact
(the PSRP conditions required in Part 503 for lime stabilization)
but uses a substance other than lime to raise pH could qualify
as a PSRP. In such cases, microbiological data may not be
necessary to demonstrate equivalency.
Process-Specific Performance Data and
Microbiologic Data
In all other cases, both performance data and microbiologi-
cal data (listed below) are needed to demonstrate process
equivalency:
• A description of the various parameters (e.g., sludge char-
acteristics, process operating parameters, climatic factors)
that influence the microbiological characteristics of the
treated sludge (see Section 11.5 for more detail on relevant
parameters).
• Sampling and analytical data to demonstrate that the process
has reduced microbes to the required levels (see Section 11.3
for a description of levels).
• A discussion of the ability of the treatment process to con-
sistently operate within the parameters necessary to achieve
the appropriate reductions.
Sampling and Analytical Methods
Sewage sludge should be sampled using accepted, state-
of-the-art techniques for sampling and analyzed using the meth-
ods required by Part 503 (see Chapters 7 and 8).
Data Quality
The quality of the data provided is an important factor in
EPA's equivalency recommendation. The following steps can
help ensure data quality:
• Use of accepted, state-of-the-art sampling techniques.
• Obtaining samples that are representative of the expected
variation in sludge quality.
• Developing and following quality assurance procedures for
sampling.
• Using an independent, experienced laboratory to perform the
analysis.
Since processes differ widely in their nature, effects, and
processing sequences, the experimental plan to demonstrate
that the process meets the requirements for PSRP or PFRP
equivalency should be tailored to the process. Field verification
and documentation by independent or third-party investigators
is desirable. EPA will evaluate the study design, the accuracy
of the data, and the adequacy of the results for supporting the
conclusions drawn.
Can Pilot-Scale Data Be Submitted?
Operation on a full scale is desirable. However, if a pilot-
scale operation truly simulates full-scale operation, testing on
this reduced scale is possible. In such cases, it is important to
indicate that the data were obtained from a pilot-scale opera^-
tion, and to discuss why and to what extent this simulates
full-scale operation. Any data available from existing full-scale
operations would be useful.
The conditions of the pilot-scale operation should be at
least as severe as those of full-scale operation. The arrangement
of process steps, degree of mixing, nature of the flow, vessel
sizing, proportion of chemicals used, etc. are all part of the
requirement. Any substantial degree of departure in the process
66
-------
parameters of the full-scale operation that might reduce the
severity of the procedure will invalidate any PEC equivalency
recommendations and permitting authority equivalency deter-
minations and will require a retest at the new condition.
11.5 Guidance on Application for Equivalency
Recommendations
The following outline and instructions are provided as
guidance for preparing applications for equivalency recommen-
dations by EPA's Pathogen Equivalency Committee.
Summary Fact Sheet
The application should include a brief fact sheet that sum-
marizes key information about the process. Any important ad-
ditional facts should also be included.
Introduction
The full name of the treatment works and the treatment
process should be provided. The application should indicate
whether it is for recommendation of:
• PSRP or PFRP equivalency.
• Site-specific or national equivalency.
Process Description
The type of sludge used in the process should be described,
as well as other materials used in the process. Specifications
for these materials should be provided as appropriate. Any
terms used should be defined.
The process should be broken down into key steps and
graphically displayed in a quantified flow diagram of the waste-
water and sludge treatment processes. Details of the wastewater
treatment process should be provided and the application
should precisely define which steps constitute the beginning
and end of sewage sludge treatment.2 The earliest point at
which sewage sludge treatment can be defined as beginning is
the point at which the sludge is collected from the wastewater
treatment process. Sufficient information should be provided
for a mass balance calculation (i.e., actual or relative volumetric
flows and solids concentration in and out of all streams, addi-
tive rates for bulking agents or other additives). A description
of process parameters should be provided for each step of
the process, giving typical ranges and mean values where
appropriate. The specific process parameters that should be
discussed will depend on the type of process and should include
any of the following that affect pathogen reduction or process
reliability:
2When defining which steps constitute the "treatment process," bear in mind that all steps
included as part of a process equivalent to PSRP or PFRP must be continually operating
according to the specifications and conditions that are critical to pathogen reduction. Thus,
the operational and monitoring burden may be greater for a multi-step process.
Sewage Sludge Characteristics
• Total and volatile solids content of sludge before and after
treatment
• Proportion and type of additives (diluents) in sludge
• Chemical characteristics (as they affect pathogen sur-
vival/destruction—e.g., pH)
• Type(s) of sludge (unstabilized vs. stabilized, primary vs.
secondary, etc.)
• Wastewater treatment process performance data (as they af-
fect sludge type, sludge age, etc.)
• Quantity of treated sludge
• Sludge age
• Sludge detention time
Process Characteristics
• Scale of the system (e.g., reactor size, flow rate)
• Sewage sludge feed process (e.g., batch vs. continuous)
• Organic loading rate (e.g., kg volatile solids/cubic
meter/day)
• Operating temperature(s) (including maximum, minimum,
and mean temperatures)
• Operating pressure(s) if greater than ambient
• Type of chemical additives and their loading rate
« Mixing
• Aerobic vs. anaerobic
• Duration/frequency of aeration
• Dissolved oxygen level maintained
• Residence/detention time
• Depth of sludge
• Mixing procedures
• Duration and type of storage (e.g., aerated vs. nonaerated)
Climate
• Ambient seasonal temperature range
• Precipitation
• Humidity
67
-------
The application should include a description of how the
process parameters are monitored, as well as process uniformity
and reliability. Actual monitoring data should be provided
whenever appropriate.
Description of treated Sewage Sludge
The type of treated sludge should be described, as well as
the sludge monitoring program for pathogens (if there is one).
How and when are samples taken? What is analyzed for? What
are the results? How long has this program been in operation?
Sampling Technique(s)
The PEC will evaluate the representativeness of the sam-
ples and the adequacy of the sampling techniques. For a rec-
ommendation of national PFRP equivalency, samples of
untreated and treated sludge are usually needed (see Sections
11.3, 4.6, and 7.4). The sampling points should correspond to
the beginning and end of the treatment process as defined pre-
viously under Process Description, above. Chapters 7 and 8
provide guidance on sampling. Samples should be repre-
sentative of the sewage sludge hi terms of location of collection
within the sludge pile or batch. The samples taken should in-
clude samples from treatment under the least favorable operat-
ing conditions that are likely to occur (e.g., wintertime).
Information should be provided on:
• Where the samples were collected from within the sewage
sludge mass. (If samples were taken from a pile, include a
schematic of the pile and indicate where the subsamples
were taken.)
• Date and time the samples were collected. Discuss how this
timing relates to important process parameters (e.g., turning
over, beginning of drying).
• Sampling method used.
• How any composite samples were compiled.
• Total solids of each sample.
* Ambient temperature at time of sampling.
• Temperature of sample at time of sampling.
• Sample handling, preservation, packaging, and transporta-
tion procedures.
• The amount of time that elapsed between sampling and
analysis.
Analytical Methods
Identify the analytical techniques used and the labora-
tory(s) performing the analysis.
Analytical Results
The analytical results should be summarized, preferably in
tabular form. A discussion of the results and a summary of
major conclusions should be provided. Where appropriate, the
results should be graphically displayed. Copies of original data
should be provided in an appendix.
Quality Assurance
The application should describe how the quality of the
analytical data have been ensured. Subjects appropriate to ad-
dress are: why the samples are representative; the quality as-
surance program; the qualifications of the in-house or contract
laboratory used; and the rationale for selecting the sampling
technique.
Rationale for Why Process Should Be Determined
Equivalent
Finally, the application should describe why, in the appli-
cant's opinion, the process qualifies for PSRP or PFRP equiva-
lency. For example, it may be appropriate to describe or review
particular aspects of the process that contribute to pathogen
reduction, and why the process is expected to operate consis-
tently. Complete references should be provided for any data
cited. Applications for a recommendation of national equiva-
lency should discuss why the process effectiveness is expected
to be independent of the location of operation.
Appendices
A copy of the complete laboratory report(s) for any sam-
pling and analytical data should be attached as an appendix.
Any important supporting literature references should also be
included as appendices.
11.6 Examples of Recommendations
Tables 4-3 and 5-2 list processes that the PEC has recom-
mended as equivalent to PSRP or PFRP. Two of these proc-
esses are discussed below.
Raising Sewage Sludge pH Using an Alternative
Chemical
The PEC evaluated a treatment process used by a Texas-
based company for recommendation as equivalent to PSRP. The
process was similar to lime stabilization except that cement kiln
dust was used instead of lime to raise sewage sludge pH. The
data provided by the applicant showed that the process reliably
raised sludge pH to greater than 12 for at least 2 hours, so the
PEC recommended that the process be determined equivalent
to PSRP.
Use of a Chemical to Generate Heat During
"Composting"
The Scarborough Sanitary District in Maine requested rec-
ommendation of its sewage sludge treatment process as a PFRP.
The process was described as composting using fly ash as a
68
-------
bulking agent. The applicant provided time and temperature
data demonstrating that the piles reached temperatures of 60°C
to 70°C (140°F to 158°F) within 24 hours and maintained this
temperature range for up to 14 days. The process exceeded the
PFRP requirements for static aerated pile composting. How-
ever, the PEC found that the process might not in fact be a
composting process since it worked by adding an inorganic
agent (fly ash) that produced high temperatures. The regulatory
requirements for composting were based on the generation of
heat by the biological processes that occur when an organic
bulking agent is used. Thus, a determination of equivalency was
necessary.
The applicant provided information on the location of the
samples from the compost pile, so that the PEC could determine
that sufficient temperatures were maintained throughout the pile
to provide adequate pathogen destruction. The PEC recommended
that the process be determined equivalent to PFRP because it met
the time/temperature conditions that result in the reduction of
pathogens in sewage sludge to below detectable limits.
69
-------
-------
Chapter 12
References
APHA. 1992. Standard methods for the examination of water
and wastewater. 18th ed. Washington, DC: American Pub-
lic Health Association.
ASTM. 1992a. Annual book of ASTM standards. Philadelphia,
PA: American Society for Testing and Materials.
ASTM. 1992b. Standard practice for recovery of viruses from
wastewater sludges. Section 11—Water and Environmental
Technology in ASTM (1992a).
Block, C.A. 1965. Methods of soil analysis. Part 2: chemical
and microbiological properties. Madison, WI: Amer. Soc.
Agronomy.
Bonner, A.B. and D.O. Cliver. 1987. Disinfection of viruses in
septic tank and holding tank waste by calcium hydroxide
(Lime). Unpublished report, Small Scale Waste Manage-
ment Project Madison, WI: University of Wisconsin.
Counts, C.A. and A J. Shuckrow. 1975. Lime stabilized sludge:
its stability and effect on agricultural land. Kept. EPA-
670/2-75-012, pub. U.S. EPA.
Davies, O.L. and P.L. Goldsmith, ed. 1972. Statistical Methods
in Research and Production. Essex, England: Longman
Group Ltd.
EPA. 199_. POTW sludge sampling and analysis guidance
document. 2nd edition. Washington, DC: Office of Waste-
water Enforcement and Compliance. To be published.
EPA. 1992. Technical support document for Part 503 pathogen
and vector attraction reduction requirements hi sewage
sludge. NTIS No.: PB93-11069. Springfield, VA: National
Technical Information Service.
EPA. 1989. Technical support document for pathogen reduction
hi sewage sludge. NTIS No.: PB89-136618. Springfield,
VA: National Technical Information Service.
EPA. 1988. National sewage sludge survey database. Research
Triangle Park, NC: National Computer Center.
EPA. 1986. Test methods for evaluating solid waste: method
9045A, soil and waste pH, Revision 1, Nov. 1990. Wash-
ington, DC: Office of Solid Waste and Emergency Re-
sponse, U.S. EPA. (avail. U.S. Supt. of Documents).
EPA. 1985. Health effects of land application of municipal
sludge. EPA Pub. No. 600/1-85/015. Research Triangle
Park, NC: EPA Health Effects Research Laboratory.
EPA. 1984. EPA policy on municipal sludge management. Fed-
eral Register 49:24358, June 12, 1984.
EPA. 1980. Samplers and sampling procedures for hazardous
waste streams. Report No.: EPA/600/2-80/018. Cincinnati,
OH: Municipal Environmental Research Laboratory.
EPA. 1979. Process design manual for sludge treatment and
disposal. Report No.: EPA/625/1-79/001. Cincinnati, OH:
Water Engineering Research Laboratory and Center for
Environmental Research Information.
Farrell, J.B. 1992. "Fecal pathogen control during composting,"
presented at International Composting Research Sympo-
sium, Columbus, OH, May 27-29 (1992). Proceedings to
be published by Ohio State University.
Farrell, J.B. and V. Bhide. 1993. New methods to quantify
vector attraction reduction of wastewater sludges. To be
published.
Farrell, J.B., B.V. Salotto and A.D. Venosa. 1990. Reduction in
bacterial densities of wastewater solids by three secondary
treatment processes. Res. J. WPCF 62(2):177-184.
Farrell, J.B., G. Stern, and A.D. Venosa. 1985. Microbial de-
structions achieved by full-scale anaerobic digestion.
Workshop on Control of Sludge Pathogens, Series IV. Al-
exandria, VA: Water Pollution Control Federation.
Farrell, J.B., I.E. Smith, Jr., S.W. Hathaway, and R.B. Dean.
1974. Lime stabilization of primary sludges. J. WPCF
46(1):113-122.
Fisher, W.J. 1984. Calculation of volatile solids destruction
during sludge digestion, pp. 514-528 in Bruce, A., ed. Sew-
age sludge stabilization and disinfection. Published for
water Research Centre. Chichester, England: E. Harwood,
Ltd.
71
-------
Fox, C.J., P.R. Fitzgerald, and C. Lue-Hing. 1981. Sewage
organisms: a color atlas. Metropolitan Water Reclamation
District of Greater Chicago, Chicago, Illinois. (Photos in
Chapter 2 reproduced with permission of the Metropolitan
Water Reclamation District of Greater Chicago.)
Goyal, S.M., S.A. Schaub, F.M. Wellings, D. Berman, J.S.
Glass, C.J. Hurst, D.A. Brashear, C.A. Sorber, B.E. Moore,
G. Bitton, P.H. Gibbs, and S.R. Farrah. 1984. Round robin
investigation of methods for recovering human enteric vi-
ruses from sludge. Applied & Environ. Microbiology
48:531-538.
Helsel, D.R. 1990. Less than obvious: statistical treatment of
data below the detection limit. Envir. Sci. Technol.
24(12):1767-1774.
Jakubowski, W. 1988. Ascaris ova survival in land application
conditions. U.S. EPA Administrator's Item Deliverable
2799 (May, 1988). Unpublished.
Keith, L.H., ed. 1988. Principles of Environmental Sampling.
American Chemical Society.
Kenner, B.A. and H.P. Clark. 1974. Detection and enumeration
of Salmonella and Pseudomonas aeruginosa. J. WPCF
46(9):2163-71.
Kent, R.T. and KJE. Payne. 1988. Sampling groundwater moni-
toring wells: Special quality assurance and quality control
considerations, pp. 231-246 in Keith, L.H., Principles of
environmental sampling. American Chemical Society.
Kowal, NJF. 1985. Health effects of land application of munici-
pal sludge. Pub. No.: EPA/600/1-85/015. Research Trian-
gle Park, NC: U.S. EPA Health Effects Research
Laboratory.
Lee, K.M., C.A. Brunner, J.B. Farrell, and A.E. Eralp. 1989.
Destruction of enteric bacteria and viruses during two-
phase digestion. J. WPCF 61(8):1422-1429.
Martin, J.H., Jr., H.E. Bostian, and G. Stern. 1990. Reductions
of enteric microorganisms during aerobic sludge digestion.
Wat. Res. 24(11):1377-1385.
Newman, M.C. and P.M. Dixon. 1990. UNCENSOR: A pro-
gram to estimate means and standard deviations for data
sets with below detection limit observations. Am. Envir.
Laboratory 2(2):26-30.
Reimers, R.S., M.D. Little, T.G. Akers, W.D. Henriques, R.C.
Badeaux, D.B. McDonnell, and K.K. Mbela. 1989. Persist-
ence of pathogens in lagoon-stored sludge. Rept. No.
EPA/600/2-89/015 (NTIS No. PB89-190359/AS). Cincin-
nati, OH: U.S. EPA Risk Reduction Engineering Labora-
tory.
Ronner, A.B. and D.O. Cliver. 1987. Disinfection of viruses in
septic tank and holding tank waste by calcium hydroxide
(Lime). Unpublished report, Small Scale Waste Manage-
ment Project. Madison, WI: University of Wisconsin.
Sobsey, M.D., and P.A. Shields. 1987. Survival and transport
of viruses in soils: Model studies, pp. 155-177 in V.C. Rao
and J.L. Melnick, eds. Human viruses in sediments,
sludges, and soils. Boca Raton, FL: CRC Press.
Sorber, C.A., and B.E. Moore. 1986. Survival and transport of
pathogens hi sludge-amended soil, a critical literature re-
view. Report No.: EPA/600/2-87/028. Cincinnati, OH: Of-
fice of Research and Development.
Whittington, W.A., and E. Johnson. 1985. Application of 40
CER Part 257 regulations to pathogen reduction preceding
land application of sewage sludge or septic tank pumpings.
Memorandum to EPA Water Division Directors. U.S. EPA
Office of Municipal Pollution Control, November 6.
Yanko, W.A. 1987. Occurrence of pathogens in distribution and
marketing municipal sludges. Report No.: EPA/600/1-
87/014. (NTIS PB88-154273/AS.) Springfield, VA: Na-
tional Technical Information Service.
72
-------
Appendix A
EPA Regional and State Sludge Coordinators1 and Map of EPA Regions
REGIONAL SLUDGE COORDINATORS
Thelma Hamilton
U.S. EPA - Region I
Municipal Facilities Branch (WMT-2111)
Water Management Division
John F. Kennedy Federal Building, Room 2103
Boston, MA 02203
617-565-3564; fax 617-565-4940
Alia Roufaeal
U.S. EPA - Region H
Water Management Division
26 Federal Plaza, Room 837
New York, NY 10278
212-264-8663; fax 212-264-9597
Stephanie Kordzi
U.S. EPA - Region VI
Water Management Division
Allied Bank Tower at Fountain Place
1445 Ross Avenue
Dallas, TX 75202-2733
214-655-7175; fax 214-655-6490
John Dunn
U.S. EPA - Region VJJ.
Construction Grants Branch
Water Management Division
726 Minnesota Avenue
Kansas City, KS 66101
913-551-7594; fax 913-551-7765
Ann Carkhuff
U.S. EPA - Region HI
Water Management Division (3WM53)
841 Chestnut Street
Philadelphia, PA 19107
215-597-9406; fax 215-597-8541
Vince Miller
U.S. EPA-Region IV
Technology Transfer Unit
Water Management Division
345 Courtland Street
Atlanta, GA 30365
404-347-2391; fax 404-347-3269
John Colletti
U.S. EPA - Region V
Water Division
Technology Section (5WQP-16J)
77 West Jackson Boulevard
Chicago, EL 60604
312-886-6106; fax 312-886-7804
'As of December 1992.
Bob Brobst
U.S. EPA - Region VIE
Water Management Division/Municipal
Facilities Branch
999 18th Street, Suite 50-0
Denver, CO 80202-2413
303-293-1627; fax 303-294-1386
Lauren Fondahl
U.S. EPA-Region DC
Pretreatment Permits and Compliance
Water Management Division
75 Hawthorne Street (W-5-2)
San Francisco, CA 94105
415-744-1909; fax 415-744-1235
Laura Felten (WD-134)
U.S. EPA - Region X
Municipal Facilities Branch
1200 Sixth Avenue
Seattle, WA 98101
206-553-1647; fax 206-553-0165
73'
-------
[Boston
New York
Philadelphia
VIRGIN ISLANDS
PUERTO RICO
Figure A-1. EPA Regions.
STATE SLUDGE COORDINATORS2
Region 1
Connecticut
Warren Herzig
Department of Environmental Protection
Water Compliance Unit
122 Washington Street
Hartford, CT 06106
203-566-2154
Maine
Brian Kavannah/Steven Page
Department of Environmental Protection
Bureau of Solid Waste Management
State House, Station 17
Augusta, ME 04333
207-582-8740
JA« of December 1992.
Massachusetts
Dennis Dunn
Department of Environmental Protection
Division of Water Pollution Control
1 Winter Street
Boston, MA 02108
617-556-1130
New Hampshire
Selena Makofsky, Supervisor
Water Supply and Pollution Control Division
Department of Environmental Services
Sludge and Septage Management Section
P.O. Box 95
6 Hazen Drive
Concord, NH 03301
603-271-3398
Rhode Island
Chris Campbell, Senior Environmental Planner
Department of Environmental Management
Division of Water Resources
291 Promenade Street
Providence, RI 02908-5657
401-277-3961
74
-------
Vermont
George Desch, Chief
Residuals Management Section
Department of Environmental Conservation
Building 9 South
103 South Main Street
Waterbury, VT 05676
802-244-8744
Region 2
New Jersey
Mary Jo M. Aiello, Chief
Bureau of Pretreatment and Residuals
Wastewater Facilities Regulation Program (CN-029)
Department of Environmental Protection
Trenton, NJ 08625-0029
609-633-3823
New York
Ed Dasatti, Supervisor
Residuals Management Section
Division of Solid Waste
Department of Environmental Conservation
50 Wolf Road
Albany, NY 12233-4013
518-457-2051
Puerto Rico
Tomas Rivera, Director
Water Quality Area
Environmental Quality Board
P.O. Box 11488
Santurce, Puerto Rico 00916
809-767-8073
Region 3
Delaware
Ronald E. Graeber
Department of Natural Resources and
Environmental Control
Division of Water Resources
Waste Utilization Program
P.O. Box 1401
89 Kings Highway
Dover, DE 19903
302-736-5731
District of Columbia
James R. Collier
DCRA Environmental Control Division
Water Hygiene Branch
5010 Overlook Avenue, SW.
Washington, D.C. 20037
202-404-1120
Maryland
Simin Tirgari, Chief
Solid Waste Program
Sewage Sludge Compliance
Department of the Environment
2500 Broening Highway
Baltimore, MD 21224
301-631-3318
Pennsylvania
Stephen Socash
Municipal & Residual Waste Permits Section
Bureau of Waste Management
P.O. Box 2063
Harrisburg, PA 17120
717-787-1749
Virginia
Cal M. Sawyer, Director
Division of Wastewater Engineering
Department of Health
109 Governor Street, Room 927
Richmond, VA 23219
804-786-1755
Virgin Islands
Leonard G. Reed, Jr., Assistant Director
Environmental Protection Division
Department of Planning and Natural Resources
45 ANisky, Suite 231
St. Thomas, VT 00802
809-774-3320
West Virginia
Clifton Browning
Department of Commerce, Labor &
Environmental Protection
Division of Environmental Protection
1201 Greenbrier Street
Charleston, WV 25311
304-348-2108
75
-------
Martin Ferguson
Office of Water Resources Management
State Water Control Board
2111 North Hamilton Street
Richmond, VA 23230
804-367-6136
Mississippi
Glen Odoms
Department of Environmental Quality
Office of Pollution Control
P.O. Box 10385
Jackson, MS 39289-0385
601-961-5159
Region 4
Alabama
Cliff Evans, Environmental Engineer
Water Division
Municipal Waste Branch
Department of Environmental Management
1751 Congressman W.L. Dickinson Drive
Montgomery, AL 36130
205-271-7816
Florida
Julie Gissendanner
Bureau of Water Facilities Planning & Regulation
Domestic Waste Section
Department of Environmental Regulation
Twin Towers Office Building
2600 Blairstone Road
Tallahassee, FL 32399-2400
904-488-4524
Georgia
Mike Stevens
Department of Natural Resources
Municipal Permitting Program
4244 International Parkway, Suite 110
Atlanta, GA 30354
404-362-2680
KentucJty
Arthur S. Curtis, Jr.
Division of Water
Ft. Boone Plaza
18 Reilly Road
Frankfort, KY 40601
502-564-3410
MarkCrim
Division of Waste Management
Ft. Boone Plaza
18 Reilly Road
Frankfort, KY 40601
502-564-6716
North Carolina
Dennis Ramsey
Water Quality Section
Division of Environmental Management
P.O. Box 29535
512 No. Salisbury Street
Raleigh, NC 27626-0535
919-733-5083
South Carolina
Jeffrey DeBessonet
Domestic Wastewater Division
Bureau of Water Pollution Control
Department of Health and Environmental Control
2600 Bull Street
Columbia, SC 29201
803-734-5300
Mike Montebello
Domestic Wastewater Division
Bureau of Water Pollution Control
Department of Health and Environmental Control
2600 Bull Street
Columbia, SC 29201
803-734-5226
Tennessee
Roger Lemasters, Bio-Solids Coordinator
Division of Water Pollution Control
Department of Health & Environment
L & C Annex
401 Church Street
Nashville, TN 37243-1534
615-532-0649
Region 5
Illinois
Al Keller
Environmental Protection Agency
2200 Churchill Road
Springfield, JJL 62706
217-782-0610
76
-------
Indiana
Dennis Lassiter, Supervisor
Land Application Group
Office of Water Management
Department of Environmental Management
105 South Meridian
Indianapolis, IN 46206
317-232-8732
Michigan
Bob Deatrick
Waste Characterization Unit
Waste Management Division
Department of Natural Resources
John A. Hanna Building
P.O. Box 30241
Lansing, MI 48909
517-373-8411
Minnesota
Jorja A. DuFresne
Land Treatment Team
Municipal Section
Water Quality Division
Pollution Control Agency
520 Lafayette Road
St. Paul, MN 55155
612-296-9292
Region 6
Arkansas
Jamal Solaimanian
Department of Pollution Control & Ecology
P.O. Box 9583
Little Rock, AR 72219
501-562-7444
Bob Makin
Division of Engineering
Bureau of Environmental Health Services
Department of Health
Division of Engineering (MS 37)
4815 Markham
Little Rock, AR 72201
501-661-2623
Louisiana
Ken Fledderman
Construction Grants Unit
Department of Environmental Quality
11720 Airline Highway
Baton Rouge, LA 70814
504-295-8900
Hoa Van Nguyen
Solid Waste Division
Department of Environmental Quality
P.O. Box 44307
Baton Rouge, LA 70804
504-765-0249
Ohio
Paul Novak, Chief
Permits Section
Division of Water Pollution Control
P.O. Box 1049
1800 Water Mark Drive
Columbus, OH 43266-0149.
614-644-2001
Wisconsin
John Melby
Department of Natural Resources
P.O. Box 7921
Madison, WI 53707
608-267-7666
Robert Steindorf
Department of Natural Resources
P.O. Box 7921
Madison, WI 53707
608-266-0449
New Mexico
Arun Dhawan
Environment Department
1190 St. Francis Drive
Santa Fe, MM 87503
505-827-2811
Oklahoma
David Hardgrave/Danny Hodges
State Department of Health
P.O. Box 53551
1000 N.E. 10th Street
Oklahoma City, OK 73152
405-271-5205
Texas
Louis Herrin
Construction Grants Division
Water Commission
P.O. Box 13087 - Capital Station
Austin, TX 78711-3087
512-463-1087
77
-------
Region 7
Iowa
Darrell McAllister, Chief
Surface and Groundwater Protection Bureau
Department of Natural Resources
Wallace Building
900 East Grand Avenue
Des Moines, IW 50309
515-281-8869
Kansas
Rodney Geisler
Forbes Held
Department of Health & Environment
Topeka, KS 66620
913-296-5527
Missouri
Ken Arnold
Water Pollution Control Program
Department of Natural Resources
P.O. Box 176
Jefferson City, MO 65102
314-751-6624
Nebraska
Rudy Fielder
Water Quality Division
Department of Environmental Control
P.O. Box 98922
Statehouse Station
Lincoln, NB 68509-8922
402-471-4239
Region 8
Colorado
Phil Hegeman
Water Quality Control Division
Department of Health
4210 East llth Avenue
Denver, CO 80220
303-692-3598
North Dakota
Sheila McClenathan
Division of Water Supply and Pollution Control
Department of Health
1200 Missouri Avenue
Bismark, ND 58505
701-221-5210
South Dakota
Bill Geyer
Division of Water Quality
Department of Water & Natural Resources
Joe Foss Building
523 East Capitol
Pierre, SD 57501-3181
605-773-3151
Utah
Paul Krautl
Bureau of Water Pollution Control
P.O. Box 16690
Salt Lake City, UT 84116-0690
801-538-6146
Wyoming
Larry Robinson
Water Quality Division
Department of Environmental Quality
Herschler Building, 4th floor West
122 West 25th Street
Cheyenne, WY 82002
307-777-7075
Region 9
Arizona
Krista Gooch
Solid Waste Unit, Room 402
Office of Waste Programs
Department of Environmental Quality
2501 North Fourth Street, Suite 14
Flagstaff, AZ 86004-3770
602-773-9285
Montana
Scott Anderson
Water Quality Bureau
Department of Health & Environmental Sciences
Cogswell Building (A-206)
Helena, MT 59620
406-444-2406
California
Steve Austrheim-Smith
Special Waste Section
Integrated Waste Management Board
8800 Cal Centre Drive
Sacramento, CA 95826
916-255-2343
78
-------
Ron Duff, Chief
Regulatory Unit
Division of Water Quality
State Water Resources Control Board
P.O. Box 944213
Sacramento, CA 94244-2130
916-675-0775
Idaho
Martin Bauer, Chief
Construction Permits Bureau
Division of Environmental Quality
Department of Health and Welfare
1410 North Hilton
Boise, ID 83706
208-334-5898
Hawaii
Dennis Tulang, Chief
Wastewater Treatment Works
Construction Grants Branch
Department of Health
P.O. Box 3378
633 Halekauwila Street, 2nd Floor
Honolulu, ffl 96813
808-548-6769
Nevada
Jim Williams, Chief
Bureau of Water Pollution Control
Department of Conservation and Natural
Resources
Division of Environmental Protection
Capitol Complex
333 West Nye Lane
Carson City, NV 89710
702-687-5870
Jerry Yoder
Construction Permits Bureau
Division of Environmental Quality
Department of Health and Welfare
1410 North Hilton
Boise, ID 83706
208-334-5866
Oregon
Mark Ronayne
Sludge Program Coordinator
Municipal Wastewater Section
Department of Environmental Quality
811 S.W. Sixth Avenue
Portland, OR 97204
503-229-6442
Mike Downs, Administrator
Water Quality Division
Department of Environmental Quality
811 S.W. Sixth Avenue
Portland, OR 97204 '
503-229-6099
Region 10
Alaska
Deena Henkins, Chief
Wastewater and Water Treatment Section
Department of Environmental Conservation
410 Willoughby Avenue
Juneau, AK 99801
907-465-5312
Washington
Kyle Dorsey, Biosolids Coordinator
Ecology Solid Waste Program
Rowe 6 - Building 4
4224 Sixth Avenue SE
Olympia, WA 98404-8711
206-459-6307
Tom Eaton, Manager
Ecology Solid & Hazardous Waste Program
Rowe 6 - Building 4
4224 Sixth Avenue
Olympia, WA 98404-8711
206-459-6316
79
-------
-------
Appendix B
Sufopart D of the Part 503 Regulation
9398 Federal Register / Vol. 58, No. 32 / Friday, February 19, 1993 / Rules and Regulations
Subpart D—Pathogens and Vector
Attraction Reduction
§503.30 Scope.
(a) This subpart contains the
requirements for a sewage sludge to be
classified either Class A or Class B with
respect to pathogens.
(b) This subpart contains the site
restrictions for land on which a Class B
sewage sludge is applied.
(c) This siibpart contains the pathogen
requirements for domestic septage
applied to agricultural land, forest, or a
reclamation site.
(d) This subpart contains alternative
vector attraction reduction requirements
for sewage sludge that is applied to the
land or placed on a surface disposal site.
S 503.31 Special definition*.
(a) Aerobic digestion is the
biochemical decomposition of organic
matter in sewage sludge into carbon
dioxide and water by microorganisms in
the presence of air.
(b) Anaerobic digestion is the
biochemical decomposition of organic
matter in sewage sludge into methane
gas and carbon dioxide by
microorganisms in the absence of air.
(c) Density of microorganisms is the
number of microorganisms per unit
mass-of total solids (dry weight) in the
sewage sludge.
(d) Land with a high potential for
public exposure is land that the public
uses frequently. This includes, but is
not limited to, a public contact site and
a reclamation site located in a populated
area (e.g, a construction site located in
a city).
(e) Land with a low potential for
public exposure is land that the public
uses infrequently. This includes, but is
not limited to, agricultural land, forest,
and a reclamation site located in an
unpopulated area (e.g., a strip mine
located in a rural area).
(f) Pathogenic organisms are disease-
causing organisms. These include, but
are not limited to, certain bacteria,
protozoa, viruses, and viable helminth
ova.
(g) pH means the logarithm of the
reciprocal of the hydrogen ion
concentration.
(h) Specific oxygen uptake rate
(SOUR) is the mass of oxygen consumed
per unit time per unit mass of total
solids (dry weight basis) in the sewage
sludge.
(i) Total solids are the materials in
sewage sludge that remain as residue
when the sewage sludge is dried at 103
to 105 degrees Celsius.
(j) Unstabilized solids are organic
materials in sewage sludge that have not
been treated in either an aerobic or
anaerobic treatment process.
(k) Vector attraction is the
characteristic of sewage sludge that
attracts rodents, flies, mosquitos, or
other organisms capable of transporting
infectious agents.
(1) Volatile solids is the amount of the
total solids in sewage sludge lost when
the sewage sludge is combusted at 550
degrees Celsius in the presence of
excess air.
81
-------
Federal Register / Vol. 58, No. 32 / Friday, February 19, 1993 / Rules and Regulations 9399
5503.32 P«thosj9n«.
(a) Sewage sludge—Class A. (1) The
requirement in § 503.32(a)(2] and the
requirements in either § 503.32(a)(3).
(aU4j. (a){5). (a)(6). (a){7). or (a)(8) shall
bo met for a sewage sludge to be
classified Class A with respect to
pathogens.
(2) The Class A pathogen
requirements in §503.32 (a)(3) through
(a){8) shall be met either prior to
meeting or at the same time the vector
attraction reduction requirements in
§ 503.33, except the vector attraction
reduction requirements in § 503.33
(10(0) through (b)(8), are met.
(3) Class A—Alternative 1. (i) Either
the density of fecal coliform in the
sewage sludge shall be loss than 1000
Most Probable Number per gram of total
solids (dry weight basis), or the density
of Salmonella sp. bacteria in the sewage
sludge shall bo less than three Most
Probable Number per four grams of total
solids (dry weight oasis) at the time the
sewage sludge is used or disposed; at
(he time the sewage sludge Is prepared
for sale or give away in a oag or other
container for application to the land; or
at the time the sewage sludge or
material derived from sewage sludge is
prepared to meet the requirements in
§ 503.10 (b).(c), (e). or (f).
(i!) The temperature of the sewage
sludge that is used or disposed shall be
maintained at a specific value for a
period of time.
(A) When the percent solids of the
sewage sludge is seven percent or
higher, the temperature of the sewage
sludge shall bo 50 degrees Celsius or
higher; the time period shall be 20
minutes or longer; and the temperature
and time period shall be determined
using equation (2), except when small
particles of sewage sludge are heated by
either warmed gases or on immiscible
liquid.
131,700.000
Eq.(2)
Whore,
Dstimo In days.
Utompcraturo In degrees Celsius.
(B) When the percent solids of the
sewage sludge is seven percent or higher
and small particles of sewage sludge are
heated by either warmed gases or an
immiscible liquid, the temperature of
the sewage sludge shall be 50 degrees
Celsius or higher; the time period shall
bo 15 seconds or longer; and the
temperature and time period shall be
determined using equation (2).
(C) When the percent solids of the
suwogo sludge is less than seven percent
and the time period is at least 15
seconds, but less than 30 minutes, the
temperature and time period shall be
determined using equation (2).
(D) When the percent solids of the
sewage sludge is less than seven
percent; the temperature of the sewage
sludge is 50 degrees Celsius or higher;
and the time period is 30 minutes or
longer, the temperature and time period
shall be determined using equation (3).
50,070,000
] 00.1400,
Eq. (3)
Whore.
D=tlmo In days.
t=tomperature in degrees Celsius.
(4) Class A—Alternative 2. (i) Either
the density of fecal coliform in the
sewage sludge shall be less than 1000
Most Probable Number per gram of total
solids (dry weight ba^is), or the density
of Salmonella sp. bacteria in the sewage
sludge shall be less than three Most
Probable Number per four grains of total
solids (dry weight basis) at the time the
sewage sludge is used or disposed; at
the time the sewage sludge is prepared
for sale or give away in a bag or other
container for application to the land; or
at the time the sewage sludge or
material derived from sewage sludge is
prepared to meet the requirements in
§ 503.10 (b), (c), (e), or (f).
(ii) (A) The pH of the sewage sludge
that is used or disposed shall be raised
to above 12 and shall remain above 12
for 72 hours.
(B) The temperature of the sewage
sludge shall be above 52 degrees Celsius
for 12 hours or longer during the period
that the pH of the sewage sludge is
above 12.
(C) At the end of the 72 hour period
during which the pH of the sewage
sludge is above 12, the sewage sludge
shall be air dried to achieve a percent
solids in the sewage sludge greater than
50 percent.
(5) Class A—Alternative 3. (i) Either
the density of fecal coliform in the
sewage sludge shall be less than 1000
Most Probable Number per gram of total
solids (dry weight basis), or the density
of Salmonella sp. bacteria in sewage
sludge shall be less than three Most
Probable Number per four grains of total
solids (dry weight basis) at the time the
sewage sludge is used or disposed; at
the time the sewage sludge is prepared
for sale or give away in a bag or other
container for application to the land; or
at the time the sewage sludge or
material derived from sewage sludge is
prepared to meet the requirements in
§ 503.10 (b), (c), (e), or(f).
(ii) (A) The sewage sludge shall be
analyzed prior to pathogen treatment to
determine whether Use sewage sludge
contains enteric viruses.
(B) When the density of enteric
vinises in the sewage sludge prior to
pathogen treatment is less than one
Plaque-forming Unit per four grams of
total solids (dry weight basis), the
sewage sludge is Class A with respect to
enteric viruses until the next monitoring
episode for the sewage sludge.
(C) When the density of enteric
viruses in the sewage sludge prior to
pathogen treatment is equal to or greater
than one Plaque-forming Unit per four
grains of total solids (dry weight basis),
the sewage sludge is Class A with
respect to enteric viruses when the
density of enteric viruses in the sewage
sludge after pathogen treatment is less
than one Plaque-forming Unit per four
grams of total solids (dry weight basis)
and when the values or ranges of values
for the operating parameters for the
pathogen treatment process that
produces the sewage sludge that meets
the enteric virus density requirement
are documented,
(D) After the enteric virus reduction
in paragraph (a)(5)(ii)(C) of this section
is demonstrated for the pathogen
treatment process, the sewage sludge
continues to be Class A with respect to
enteric viruses when the values for the
pathogen treatment process operating
parameters are consistent with the
values or ranges of values documented
in paragraph (a)(5)(ii)(C) of this section.
(iii)(A) The sewage sludge shall be
analyzed prior to pathogen treatment to
determine whether the sewage sludge
contains viable helminth ova.
(B) When the density of viable
helminth ova in the sewage sludge prior
to pathogen treatment is lass than one
per four grams of total solids (dry
weight basis), the sewage sludge is Class
A with respect to viable helminth ova
until the next monitoring episode for
the sewage sludge.
(C) When the density of viable
helminth ova in the sewage sludge prior
to pathogen treatment is equal to or
greater than one per four grams of total
solids (dry weight basis), the sewage
sludge is Class A with respect to viable
helminth ova when the density of viable
helminth ova in the sewage sludge after
pathogen treatment is less than one par
four grams of total solids (dry weight
basis) and when the values or ranges of
values for the operating parameters for
the pathogen treatment process that
produces the sewage sludge that meats
the viable helminth ova density
requirement era documented.
(D) After the viable helminth ova
reduction in paragraph (a)(5)(iii)(C) of
this section is demonstrated for the
pathogen treatment procuss, the sewage
82
-------
9400 Federal Register / Vol. 58, No. 32 / Friday, February 19, 1993 / Rules and Regulations
sludge continues to be Class A with
respect to viable helminth ova when the
values for the pathogen treatment
process operating parameters are
consistent with the values or ranges of
values documented in paragraph
(a)(5)(iii)(C) of this section.
(6) Class A—Alternative 4. (i) Either
the density of fecnl coliform in the
sewage sludge shall be less than 1000
Most Probable Number per gram of total
solids (dry weight basis), or the density
of Salmonella so. bacteria in the sewage
sludge shall be less than three Most
Probable Number per four grams of total
solids (dry weight basis) at the time the
sewage sludge is used or disposed; at
tlio time the sewage sludge is prepared
for sale or give away in a bag or other
container for application to the land; or
nt the time the sewage sludge or
material derived from sewage sludge is
prepared to meet the requirements in
§ 503.10 (b), (cj, (e), or (f).
(ii) The density of enteric viruses in
the sewage sludge shall be less than one
Plaque-forming Unit per four grams of
total solids (dry weight basis) at the time
the sewage sludge is used or disposed;
at the time the sewage sludge is
prepared for sale or give away in a bag
or other container for application to the
land; or at the time the sewage sludge
or material derived from sewage sludge
is prepared to moot the requirements in
§ 503.10 (b), (c), (e), or (f), unless
otherwise specified by the permitting
authority.
(iii) The density of viable helminth
ova in the sewage sludge shall be less
than one per four grams of total solids
(dry weight basis) at the time the sewage
sludge is used or disposed; at the time
the sewage sludge is prepared for sale or
give away in a bag or other container for
application to the land; or at the time
the sewage sludge or material derived
from sewage sludge is prepared to meet
the requirements in §503.10 (b), (c), (e),
or (0, unless otherwise specified by the
permitting authority.
(7) Class A—Alternative 5. (i) Either
She density of fecal coliform in the
sewage sludge shall ho less than 1000
Most Probable Number per gram of total
solids (dry weight basis), or the density
of Salmonella, sp. bacteria in the sewage
sludge shall be less than three Most
Probable Number per four grams of total
solids (dry weight basis) at the time the
sewage sludge is used or disposed; at
the time the sewage sludge is prepared
for sale or given away in a bag or other
container for application to the land; or
at the time the sewage sludge or
material derived from sewage sludge is
prepared to meet the requirements in
§503.10(b), (c), (o).or(f).
(ii) Sewage sludge that is used or
disposed shall be treated in one of the
Processes to Further Reduce Pathogens
described in appendix B of this part.
(8) Class A—Alternative 6. (i) Either
the density of fecal coliform in the
sewage sludge shall be less than 1000
Most Probable Number per gram of total
solids (dry weight basis), or the density
of,Salmonella, sp. bacteria in the sewage
sludge shall be less than three Most
Probable Number per four grams of total
solids (dry weight basis) at the time the
sewage sludge is used or disposed; at
the time the sewage sludge is prepared
for sale or given away in a bag or other
container for application to the land; or
at the time the sewage sludge or
material derived from sewage sludge is
prepared to meet the requirements in
§503,10(b), (c), (e),or(f).
(ii) Sewage sludge that is used or
disposed shall be treated in a process
that is equivalent to a Process to Further
Reduce Pathogens, as determined by the
permitting authority.
(b) Sewage sludge—Class B. (l)(i) The
requirements in either § 503.32(b)(2),
(b)(3), or (b)(4) shall be met for a sewage
sludge to be classified Class B with
respect to pathogens.
(ii) The site restrictions in
§ 503.32(b)(5) shall be met when sewage
sludge that meets the Class B pathogen
requirements in § 503.3Zfb)(2), (b)(3), or
(b)(4) is applied to the land.
(2) Class B—Alternative 1.
(i) Seven samples of the sewage
sludge shall be collected at the time the
sewage sludge is used or disposed.
(ii) The geometric mean of the density
of fecal coliform in the samples
collected in paragraph (b)(2)(i) of this
section shall bo less than either
2,000,000 Most Probable Number per
gram of total solids (dry weight basis) or
2,000,000 Colony Forming Units per
gram of total solids (dry weight basis).
(3) Class B—Alternative 2. Sewage
sludge that is used or disposed shall be
treated in one of the Processes to
Significantly Reduce Pathogens
described in appendix B of this part.
(4) Class B—Alternative 3. Sewage
sludge that is used or disposed shall be
treated in a process that is equivalent to
a Process to Significantly Reduce
Pathogens, as determined by the
permitting authority.
(5) Site Restrictions, (i) Food crops
with harvested parts that touch the
sewage sludge/soil mixture and are
totally above the land surface shall not
bo harvested for 14 months after
application of sewage sludge.
(ii) Food crops with harvested parts
below the surface of the land shall not
be harvested for 20 months after
application of sewage sludge when the
sewage sludge remains on the land
surface for four months or longer prior
to incorporation into the soil.
(iii) Food crops with harvested parts
below the surface of the land shall not
be harvested for 38 months after
application of sewage sludge when the
sewage sludge remains on the land
surface for less than four months prior
to incorporation into the soil.
(iv) Food crops, feed crops, and fiber
crops shall not be harvested for 30 days
after application of sewage sludge.
(v) Animals shall not be allowed to
graze on the land for 30 days after
application of sewage sludge.
(vi) Turf grown on land where sewage
sludge is applied shall not he harvested
for one year after application of the
sewage sludge when the harvested turf
is placed on either land with a high
potential for public exposure or a lawn;
unless otherwise specified by the
permitting authority.
(vii) Public access to land with a high
potential for public exposure shall be
restricted for one year after application
of sewage sludge.
(viii) Public access to land with a low
potential for public exposure shall be
restricted for 30 days after application of
sewage sludge.
(c) Domestic septage. (1) The site
restrictions in § 503.32(b)(5) shall be
met when domestic septage is applied to
agricultural land, forest, or a
reclamation site; or
(2) The pH of domestic septage
applied to agricultural land, forest, or a
reclamation site shall be raised to 12 or
higher by alkali addition and, without
the addition of more alkali, shall remain
at 12 or higher for 30 minutes and the
site restrictions in § 503.32 (b)(5)(i)
through (b)(5)(iv) shall be met.
§ 503.33 Vector attraction reduction.
(a)(l) One of the vector attraction
reduction requirements in § 503.33
(b)(l) through (b)(10) shall be met when
bulk sewage sludge is applied to
agricultural land, forest, a public contact
site, or a reclamation site.
(2) One of the vector attraction
reduction requirements in § 503.33
(b)(l) through (b)(8) shall be met when
bulk sewage sludge is applied to a lawn
or a home garden.
(3) One of the vector attraction
reduction requirements in § 503.33
(b)(l) through (b)(8) shall be met when
sewage sludge is sold or given away in
a bag or other container for application
to the land.
(4) One of the vector attraction
reduction requirements in § 503.33
(b)(l) through (b)(ll) shall be met when
sewage sludge (cither than domestic
83
-------
Federal Register / Vol. 58, No. 32 / Friday, February 19, 1993 / Rules and Regulations 9401
soptogo) is placed on on active sewage
sludge unit.
(5) Ono of the vector attraction
reduction requirements in § 503.33
(b)(9), (b)(10), or (b)(12) shall be mot
whon domestic septage is applied to
ngrlculturnl land, forest, or a
reclamation site and one of the vector
attraction reduction requirements in
§ 503.33 (b){9) through (b)(12) shall be
mot whon domestic septage is placed on
an active sewage sludge unit.
(b)(l) The mass of volatile solids in
the sewage sludge shall be reduced by
a minimum of 38 percent (sea
calculation procedures in
"Environmental Regulations and
Technology—Control of Pathogens and
Vector Attraction in Sewage Sludge",
EPA-625/R-92/013.1992, U.S.
Environmental Protection Agency,
Cincinnati. Ohio 45268).
(2) When the 38 percent volatile
solids reduction requirement in
§503.33(b)(l) cannot be met for an
onaoroblcally digested sewage sludge,
vector attraction reduction can be
demonstrated by digesting a portion of
the previously digested sewage sludge
onneroblcally in the laboratory in a
bench-scale unit for 40 additional days
til a temperature between 30 and 37
degrees Celsius. When at the end of the
40 days, the volatile solids in the
sewage sludge at the beginning of that
period is reduced by loss than 17
percent, vector attraction reduction is
achieved.
(3) When the 38 percent volatile
solids reduction requirement in
§ 503.33(b)(l) cannot be met for an
aoroblcnlly digested sewage sludge,
vector attraction reduction can be
demonstrated by digesting a portion of
(ho previously digested sewage sludge
that has a percent solids of two percent
or loss aerobically in the laboratory in
a bench-scale unit for 30 additional days
at 20 degrees Celsius. When at the end
of the 30 days, the volatile solids in the
sewage sludge at the beginning of that
period is reduced by loss than 15
percent, vector attraction reduction is
achieved.
(4) The specific oxygen uptake rate
(SOUR) for sewage sludge treated in an
aerobic process shell be equal to or less
than 1.5 milligrams of oxygen per hour
per gram of total solids (dry weight
basis) at a temperature of 20 degrees
Celsius.
(5) Sewage sludge shnll he treated in
an aerobic procnss for 14 days or longer.
During that time, the temperature of the
sewage sludge shall be higher thnn 40
degrees Celsius and the average
temperature of the sewage sludge shall
bo higher than 45 degrees Celsius.
(6) The pH of sewage sludge shall be
raised to 12 or higher by alkali addition
and, without the addition of more alkali,
shall remain at 12 or higher for two
hours and then at 11.5 or higher for an
additional 22 hours.
(7) The percent solids of sewage
sludge that does not contain
unstabilized solids generated in a
primary wastewater treatment process
shall be equal to or greater than 75
percent based on the moisture content
and total solids prior to mixing with
other materials.
(8) The percent solids of sewage
sludge that contains unstabilized solids
generated in a primary wastewater
treatment process shall be equal to or
greater than 90 percent based on the
moisture content and total solids prior
to mixing with other materials.
(9)(i) Sewage sludge shall be injected
below the surface of the land.
(II) No significant amount of the
sewage sludge shall be present on the
land surface within one hour after the
sewage sludge is injected.
(iii) When the sewage sludge that is
injected below the surface of the land is
Class A with respect to pathogens, the
sewage sludge shall be injected below
the land surface within eight hours after
being discharged from the pathogen
treatment process.
(I0)(i) Sewage sludge applied to the
land surface or placed on a surface
disposal site shall be incorporated into
the soil within six hours after
application to or placement on the land.
(ii) When sewage sludge that is
incorporated into the soil is Class A
with respect to pathogens, the sewage
sludge shall be applied to or placed on
the land within eight hours after being
discharged from the pathogen treatment
process.
(11) Sewage sludge placed on an
active sewage sludge unit shall be
covered with soil or other material at
the end of each operating day.
(12) The pH of domestic septage shall
be raised to 12 or higher by alkali
addition and, without the addition of
more alkali, shall remain at 12 or higher
for 30 minutes.
84
-------
Appendix C
Determination of Volatile Solids Reduction by Digestion
Introduction
Under 40 CER Part 503, the ability of sewage sludge to
attract vectors must be reduced when sewage sludge is applied
to the land or placed on a surface disposal site. One way to
reduce vector attraction is to reduce the volatile solids in the
sewage sludge by 38% or more (see Section 6.2 of this docu-
ment). Typically, volatile solids reduction is accomplished by
anaerobic or aerobic digestion. Volatile solids reduction also
occurs under other circumstances, such as when sewage sludge
is stored in an anaerobic lagoon or is dried on sand beds. To
give credit for this extra loss in volatile solids, the regulation
allows the untreated sewage sludge to be compared with the
treated sewage sludge that leaves the treatment works, which
should account for all of the volatile solids reduction that could
possibly occur. For most processing sequences, the processing
steps downstream from the digester, such as short-term storage
or dewatering, have no influence on volatile solids content.
Consequently, the appropriate comparison is between the sew-
age sludge entering the digester and the sewage sludge leaving
the digester. The remainder of the discussion is limited to this
circumstance, except for the final section of mis appendix,
which compares incoming sewage sludge with the sewage
sludge leaving the treatment works.
The Part 503 regulation does not specify a method for
calculating volatile solids reduction. Fischer (1984) observed
that the United Kingdom has a similar requirement for volatile
solids reduction for digestion (40%), but also failed to prescribe
a method for calculating volatile solids reduction. Fischer has
provided a comprehensive discussion of the ways that volatile
solids reduction may be calculated and their h'mitations. He
presents the following equations for determining volatile solids
reduction:
• Full mass balance equation
• Approximate mass balance equation
• "Constant ash" equation
• Van Kleeck equation
The full mass balance equation is the least restricted ap-
proach but requires more information than is currently collected
at a wastewater treatment plant. The approximate mass balance
equation assumes steady state conditions. The "constant ash"
equation requires the assumption of steady state conditions as
well as the assumption that the ash input rate equals the ash
output rate. The Van Kleeck equation, which is the equation
generally suggested in publications originating in the United
States (WPCF, 1968), is equivalent to the constant ash equation.
Fischer calculates volatile solids reduction using a number of
examples of considerable complexity and illustrates that differ-
ent methods frequently yield different results.
Fischer's paper is extremely thorough and is highly rec-
ommended for someone trying to develop a deep understanding
of potential complexities in calculating volatile solids reduc-
tion. However, it was not written as a guidance document for
field staff faced with the need to calculate volatile solids reduc-
tion. The nomenclature is precise but so detailed that it makes
comprehension difficult. In addition, two important trouble-
some situations that complicate the calculation of volatile solids
reduction—grit deposition in digesters and decantate re-
moval—are not explicitly discussed. Consequently, this pres-
entation has been prepared to present guidance that describes
the major pitfalls likely to be encountered in calculating percent
volatile solids reduction.
It is important to note that the calculation of volatile solids
reduction is only as accurate as the measurement of volatile
solids content in the sewage sludge. The principal cause of error
is poor sampling. Samples should be representative, covering
the entire charging and withdrawal periods. Averages should
cover extended periods of time during which changes in process
conditions are minimal. For some treatment, it is expected that
periodic checks of volatile solids reduction will produce results
so erratic that no confidence can be placed in them. In this case,
adequacy of stabilization can be verified by the method de-
scribed under Options 2 and 3 in Chapter 6—periodically batch
digest anaerobically digested sewage sludge for 40 additional
days at 30°C (86°F) to 37°C (99°F), or aerobically digested
sewage sludge for 30 additional days at 20°C (68°F). If the
additional VS reduction is less than 17% for the anaerobically
digested sewage sludge or less than 15% for the aerobically
digested sewage sludge, the sewage sludge is sufficiently stable
(see Sections 6.3 and 6.4).
Equations for FVSR
The equations for fractional volatile solids reduction
(FVSR) that will be discussed below are the same as those
developed by Fischer (1984), except for omission of his con-
stant ash equation. This equation gives identical results to the
85
-------
Van Kleeck equation so it is not shown. Fischer's nomenclature
has been avoided or replaced with simpler terms. The material
balance approaches are called methods rather than equations.
The material balances are drawn to fit the circumstances. There
is no need to formalize the method with a rigid set of equations.
In the derivations and calculations that follow, both VS
(total volatile solids content of the sewage sludge or decantate
on a dry solids basis) and FVSR are expressed throughout as
fractions to avoid the frequent confusion that occurs when these
terms are expressed as percentages. "Decantate" is used in place
of the more commonly used "supernatant" to avoid the use of
4ls" in subscripts. Similarly, "bottoms" is used in place of
"sludge" to avoid use of "s" in subscripts.
Full Mass Balance Method
The full mass balance method must be used when steady
conditions do not prevail over the tune period chosen for the
calculation. The chosen time period must be substantial, at least
twice the nominal residence time in the digester (nominal resi-
dence time equals average volume of sludge in the digester
divided by the average volumetric flow rate. Note: when there
is decantate withdrawal, volume of sewage sludge withdrawn
should be used to calculate the average volumetric flow rate).
The reason for the long time period is to reduce the influence
of short-term fluctuations in sewage sludge flow rates or com-
positions. If input compositions have been relatively constant
for a long period of time, then the time period can be shortened.
An example where the full mass balance method would be
needed is where an aerobic digester is operated as follows:
• Started with the digester 1/4 full (time zero)
• Raw sewage sludge is fed to the digester daily until the
digester is full
* Supernatant is periodically decanted and raw sewage sludge
is charged into the digester until settling will not occur to
accommodate daily feeding (hopefully after enough days
have passed for adequate digestion)
• Draw down the digester to about 1/4 full (final time), dis-
charging the sewage sludge to sand beds
The full mass balance is written as follows:
Sum of total volatile solids inputs in feed streams during the
entire digestion period = sum of volatile solids outputs in
withdrawals of decantate and bottoms + loss of volatile sol-
ids H- accumulation of volatile solids in the digester. (1)
Loss of volatile solids is calculated from Equation 1. FVSR
is calculated by Equation 2:
The accumulation of volatile solids in the digester is the
final volume in the digester after the drawdown times final
volatile solids concentration less the initial volume at time zero
times the initial volatile solids concentration.
To properly determine FVSR by the full mass balance
method requires determination of all feed and withdrawal vol-
umes, initial and final volumes in the digester, and volatile
solids concentrations in all streams. In some cases, which will
be presented later, simplifications are possible.
Approximate Mass Balance Method
If volumetric inputs and outputs are relatively constant on
a daily basis, and there is no substantial accumulation of vola-
tile solids in the digester over the time period of the test, an
approximate mass balance (AMB) may be used. The basic re-
lationship is stated simply:
volatile solids input rate = volatile solids output
rate + rate of loss of volatile solids.
The FVSR is given by Equation 2.
(3)
No Decantate, No Grit Accumulation (Problem 1)
Calculation of FVSR is illustrated for Problem 1 in Table
C-l, which represents a simple situation with no decantate re-
moval and no grit accumulation. An approximate mass balance
is applied to the digester operated under constant flow condi-
tions. Because no decantate is removed, the volumetric flow
rate of sewage sludge leaving the digester equals the flow rate
of sewage sludge entering the digester.
Applying Equations 3 and 2,
FYf=BYb + loss
Loss = 100(50-30) = 2000
Loss
FVSR =
FVSR = -
FY,
2000
(100) (50)
- = 0.40
(4)
(5)
(6)
(7)
FVSR =
loss in volatile solids
sum of volatile solids inputs
(2)
Nomenclature is given in Table C-l. Note that the calcu-
lation did not require use of the fixed solids concentrations.
The calculation is so simple that one wonders why it is so
seldom used. One possible reason is that the input and output
volatile solids concentrations (Yf and Yb) typically will show
greater coefficients of variation (standard deviation divided by
arithmetic average) than the fractional volatile solids (VS is the
fraction of the sewage sludge solids that is volatile—note the
difference between VS and Y). If this is the case, the volatile
solids reduction calculated by the approximate mass balance
method from several sets of Y^Yb data will show larger devia-
tions than if it were calculated by the Van Kleeck equation using
VSrVSb data.
86
-------
Table C-1. Quantitative Information for Example Problems1'2'3
Problem Statement Number
Parameter
Nominal residence time
Time period for averages
Feed Sludge
Volumetric flow rate
Volatile solids
concentration
Fixed solids concentration
Fractional volatile solids
Mass flow rate of solids
Digested Sludge (Bottoms)
Volumetric flow rate
Volatile solids
concentration
Fixed solids concentration
Fractional volatile solids
Mass flow rate of solids
Decantate
Volumetric flow rate
Volatile solids
concentration
Fixed solids concentration
Fractional volatile solids
Mass flow rate of solids
Symbol
6
—
F
Y,
x,
vs,
M,
B
Yb
xb
vsb
Mb
D
Yd
Xd
vsd
Md
Units
d
d
m3/d
kg/m3
kg/m3
kg/kg
kg/d
m3/d
kg/m3
kg/m3
kg/kg
kg/d
m3/d
kg/m3
kg/m3
kg/kg
kg/d
1
20
60
100
50
17
0.746
6700
100
30
17
0.638
4700
0
—
—
—
—
2
20
60
100
50
17
0.746
6700
100
30
15
0.667
4500
0
—
—
—
—
3
20
60
100
50
17
0.746
6700
41.42
23.50
0.638
12.76
7.24
0.638
4
20
60
100
50
17
0.746
6700
49.57
41.42
23.50
0.638
50.43
12.76
7.24
0.638
Conditions are steady state; all daily flows are constant. Volatile solids are not accumulating in the digester, although grit may be settling out in
the digester.
Numerical values are given at 3 or 4 significant figures. This is unrealistic considering the expected accuracy in measuring solids concentrations
and sludge volumes. The purpose of extra significant figures is to allow more understandable comparisons to be made of the different calculation
methods.
3AII volatile solids concentrations are based on total solids, not merely on suspended solids.
Grit deposition can be a serious problem in both aerobic
and anaerobic digestion. The biological processes that occur in
digestion dissolve or destroy the substances suspending the grit,
and it tends to settle. If agitation is inadequate to keep the grit
particles in suspension, they will accumulate in the digester.
The approximate mass balance can be used to estimate accu-
mulation of fixed solids.
For Problem 1, the balance yields the following:
FXf = BXb + fixed solids loss (8)
Grit Deposition (Problem 2)
The calculation of fixed solids is repeated for Problem 2.
Conditions in Problem 2 have been selected to show grit accu-
mulation. Parameters are the same as in Problem 1 except for
the fixed solids concentration (Xb) and parameters related to it.
Fixed solids concentration hi the sewage sludge is lower than
in Problem 1. Consequently, VS is higher and the mass flow
rate of solids leaving is lower than in Problem 1. A mass bal-
ance on fixed solids (input rate = output rate + rate of loss of
fixed solids) is presented in Equations 11-13.
(100)(17) = (100)(17) + Fixed Solids Loss
Fixed Solids Loss = 0
(9)
(10)
The material balance compares fixed solids in output with
input. If some fixed solids are missing, this loss term will be a
positive number. Because digestion does not consume fixed
solids, it is assumed that the fixed solids are accumulating in
the digester. As Equation 10 shows, the fixed solids loss equals
zero. Note that for this case, where input and output sewage
sludge flow rates are equal, the fixed solids concentrations are
equal when there is no grit accumulation.
FXf = BXb + Fixed Solids Loss
Fixed Solids Loss = FXf - BXb
Fixed Solids Loss
= (100)(7) - (100)(15) = 200 kg/d
(11)
(12)
(13)
The material balance, which only looks at inputs and out-
puts, informs us that 200 kg/d of fixed solids have not appeared
in the outputs as expected. Because fixed solids are not de-
stroyed, it can be concluded that they are accumulating in the
bottom of the digester. The calculation of FVSR for Problem 2
is exactly the same as for Problem 1 (see Equations 4 through
87
-------
7) and yields the same result. The approximate mass balance
method gives the correct answer for the FVSR despite the
accumulation of solids in the digester. As will be seen later, this
is not the case when the Van Kleeck equation is used.
Decantate Withdrawal, No Grit Accumulation (Problem 3)
In Problem 3, decantate is withdrawn daily. Volatile and
fixed solids concentrations are known for all streams but the
volumetric flow rates are not known for decantate and bottoms.
It is impossible to calculate FVSR without knowing the relative
volumes of these streams. However, they are determined easily
by taking a total volume balance and a fixed solids balance,
provided it can be assumed that loss of fixed solids (i.e., accu-
mulation in the digester) is zero.
Selecting a basis for F of 100 m3/d,
Volume balance: 100 = B + D
Fixed solids balance: 100 Xf+BXb
(14)
(15)
Because the three Xs are known, B and D can be found.
Substituting 100-D for B and the values for the Xs from Prob-
lem 3 and solving for D and B,
(100)(17) = (100 -D)(23.50) + (D)(7.24)
D M0.0m3/d, B = 60.0 m3/d
(16)
(17)
The FVSR can now be calculated by drawing a volatile
solids balance:
FYf+BYb+DYd-Hoss
loss FYf-Byb-DYd
FVSR:
FYr
FYf
(18)
(19)
-(40) (12.76)
Unless information is available on actual volumes of de-
cantate and sewage sludge (bottoms), it is not possible to de-
termine whether grit is accumulating in the digester. If it is
accumulating, the calculated FVSR will be in error.
When the calculations shown in Equations 18 through 20
are made, it is assumed that the volatile solids that are missing
from the output streams are consumed by biological reactions
that convert them to carbon dioxide and methane. Accumulation
is assumed to be negligible. Volatile solids are less likely to
accumulate than fixed solids, but it can happen. In poorly mixed
digesters, the scum layer that collects at the surface is an accu-
mulation of volatile solids. FVSR calculated by Equations 18
through 20 will be overestimated if the volatile solids accumu-
lation rate is substantial.
Decantate Withdrawal and Grit Accumulation (Problem 4)
In Problem 4, there is suspected grit accumulation. The
quantity of B and D can no longer be calculated by Equations
14 and 15 because Equation 15 is no longer correct. The values
of B and D must be measured. All parameters in Problem 4 are
the same as in Problem 3 except that measured values for B
and D are introduced into Problem 4. Values of B and D cal-
culated assuming no grit accumulation (Problem 3—see pre-
vious discussion), and measured quantities are compared
below:
B
D
Calculated
60
40
Measured
49.57
50.43
The differences in the values of B and D are not large but
they make a substantial change in the numerical value of FVSR.
The FVSR for Problem 4 is calculated below:
FVSR =
(100)(50) - (49.57)(41.42) - (50.43X12.76)
(100)(50)
= 0.461
(21)
If it had been assumed that there was no grit accumulation,
FVSR would equal 0.40 (see Problem 3). It is possible to de-
termine the amount of grit accumulation that has caused this
change. A material balance on fixed solids is drawn:
FXf = BXb + DXd + Fixed Solids Loss
(22)
The fractional fixed solids loss due to grit accumulation is
found by rearranging this equation:
Fixed Solids Loss FXf - BXb - DXd
FXf
FXf
(23)
Substituting in the parameter values for Problem 4,
Fixed Solids Loss _ (100X17) - (49.57)(23.50) - (50.43 )(7.24)
FXf (100X17)
= 0.100 (24)
If this fixed solids loss of 10 percent had not been ac-
counted for, the calculated FVSR would have been 13% lower
than the correct value of 0.461. Note that if grit accumulation
occurs and it is ignored, calculated FVSR will be lower than
the actual value.
The Van Kleeck Equation
Van Kleeck first presented his equation without derivation
in a footnote for a review paper on sewage sludge treatment
processing in 1945 (Van Kleeck, 1945). The equation is easily
derived from total solids and volatile solids mass balances
around the digestion system. Consider a digester operated under
steady state conditions with decantate and bottom sewage
sludge removal. A total solids mass balance and a volatile solids
mass balance are:
88
-------
f=Mb + Md + (loss of total solids)
(25)
Mf • VSf=M • VSb + Md • VSd + (loss of volatile solids) (26)
where
Mf, Mb, andMj are the mass of solids in the feed,
bottoms, and decantate streams.
The masses must be mass of solids rather than total mass
of liquid and solid because VS is an unusual type of concen-
tration unit—it is "mass of volatile solids per unit mass of total
solids."
It is now assumed that fixed solids are not destroyed and
there is no grit deposition in the digester. The losses in Equa-
tions 25 and 26 then comprise only volatile solids so the losses
are equal. It is also assumed that the VS of the decantate and
of the bottoms are the same. This means that the bottoms may
have a much higher solids content than the decantate but the
proportion of volatile solids to fixed solids is the same for both
streams. Assuming then that VSb equals VSd, and making this
substitution in the defining equation for FVSR (Equation 2),
Loss of vol. solids , (Mb + Md)VSb
— 1 —"
MfxVSf
MfxVSf
(27)
From Equation 25, recalling that we have assumed that loss
of total solids equals loss of volatile solids,
Mb + Md 4- Mf - loss of vol. solids
Substituting for Mb + Md into Equation 27,
FVSR=1
— loss of vol. solids) • VSb
Mf • VSf
(28)
(29)
Simplifying further,
l-(l/VSf-FVSR)-VSb
Solving for FVSR,
FVSR =
vsf-vsb
VSf-(VSfxVSb)
(30)
(31)
This is the form of the Van Kleeck equation found in
WPCF Manual of Practice No. 16 (WPCF, 1968). Van Kleeck
(1945) presented the equation in the following equivalent form:
FVSR=1-
VSbx(l-VSf)
VSfX(l-VSb)
(32)
The Van Kleeck equation is applied below to Problems 1
through 4 in Table C-l and compared to the approximate mass
balance equation results:
Approximate Mass 0.40
Balance (AMB)
Van Kleeck (VK) 0.40
2
0.40
0.318
3
0.40
4
0.461
0.40 0.40
Problem 1: No decantate and no grit accumulation. Both
methods give correct answer.
Problem 2: No decantate but grit accumulation. VK is
invalid and incorrect.
Problem 3: Decantate but no grit accumulation. AMB
method is valid. VK method is valid only if VSb equals VSd.
Problem 4: Decantate and grit accumulation. AMB method
valid only if B and D are measured. VK method is invalid.
The Van Kleeck equation is seen to have serious shortcom-
ings when applied to certain practical problems. The AMB
method can be completely reliable, whereas the Van Kleeck
method is useless under some circumstances.
Average Values
The concentrations and VS values used in the equations
will all be averages. For the material balance methods, the
averages should be weighted averages according to the mass of
solids in the stream in question. The example below shows how
to average the volatile solids concentration for four consecutive
sewage sludge additions.
Addition
1
2
3
4
Volume
12m3
8m3
13m3
10m3
Total Solids
Concentration
72 kg/m3
50 kg/m3
60 kg/m3
55 kg/m3
VS
0.75
0.82
0.80
0.77
(33)
For the Van Kleeck equation, the averages of VS are re-
quired. Properly they should be weighted averages based on the
weight of the solids in each component of the average, although
an average weighted by the volume of the component, or an
arithmetic average may be sufficiently accurate if variation in
VS is small. The following example demonstrates the calcula-
tion of all three averages.
Weighted by Mass
12x72x0.75 + 8x50x0.82
+ 13 x 60 x 0.80 + 10 x 55 x 0.77
VSav =
12x72 + 8x50 + 13x60 + 10x55
= 0.795
Weighted by Volume
12 x 0.75 + 8 x 0.82 + 13 x 0.80 + 10 x 0.77
(34)
VSav = -
12 + 8+13 + 10
(35)
= 0.783
89
-------
Arithmetic Average
... 0.75 + 0.82 + 0.80 + 0.77
VSav= -.
= 0.785
(36)
In this example the arithmetic average was nearly as close
as the volume-weighted average to the mass-weighted average,
which is the correct value.
Which Equation to Use?
Full Mass Balance Method
The full mass balance method allows calculation of volatile
solids reduction for all approaches to digestion, even processes
in which the final volume in the digester does not equal the
initial volume and where daily flows are not steady. A serious
drawback to this method is the need for volatile solids concen-
tration and the volumes of all streams added to or withdrawn
ftom the digester, as well as initial and final volumes and
concentrations in the digester. This can be a daunting task,
particularly for the small treatment works that is most likely to
run digesters in other than steady flow modes. For treatment
works of this kind, an "equivalent" method that shows that the
sewage sludge has undergone the proper volatile solids reduc-
tion is likely to be a better approach than trying to demonstrate
38% volatile solids reduction. An aerobic sewage sludge has
received treatment equivalent to a 38% volatile solids reduction
if the specific oxygen uptake rate is below a specified maxi-
mum. Anaerobically digested sewage sludge has received treat-
ment equivalent to a 38% volatile solids reduction if volatile
solids reduction after batch digestion of the sewage sludge for
40 days is less than a specified maximum (EPA, 1992).
Approximate Mass Balance Method
The approximate mass balance method assumes that daily
flows are steady and reasonably uniform in composition, and
that digester volume and composition do not vary substantially
from day to day. Results of calculations and an appreciation of
underlying assumptions show that the method is accurate for
all cases, including withdrawal of decantate and deposition of
grit, provided that in addition to composition of all streams the
quantities of decantate and bottoms (the digested sewage
sludge) are known. If the quantities of decantate and bottoms
are not known, the accumulation of grit cannot be determined.
If accumulation of grit is substantial and FVSR is calculated
assuming it to be negligible, FVSR will be lower than the true
value. The result is conservative and could be used to show that
minimum volatile solids reductions are being achieved.
Van Kleeck Method
The Van Kleeck equation has underlying assumptions that
should be made clear wherever the equation is presented. The
equation is never valid when there is grit accumulation because
it assumes the fixed solids input equals fixed solids output.
Fortunately, it produces a conservative result in this case. Un-
like the AMB method it does not provide a convenient way to
check for accumulation of grit. It can be used when decantate
is withdrawn, provided VSb equals VS^. Just how significant
the difference between these VS values can be before an ap-
preciable error in FVSR occurs is unknown, although it could
be determined by making up a series of problems with increas-
ing differences between the VS values, calculating FVSR using
the AMB method and a Van Kleeck equation, and comparing
the results.
The shortcomings of the Van Kleeck equation are substan-
tial, but the equation has one strong point: The VS of the
various sewage sludge and decantate streams are likely to show
much lower coefficients of variation (standard deviation di-
vided by arithmetic average) than volatile solids and fixed sol-
ids concentration. Reviews of data are needed to determine how
seriously the variation in concentrations affect the confidence
interval of FVSR calculated by both methods. A hybrid ap-
proach may turn out to be advantageous. The AMB method
could be used first to determine if grit accumulation is occur-
ring. If grit is not accumulating, the Van Kleeck equation could
be used. If decantate is withdrawn, the Van Kleeck equation is
appropriate, particularly if the decantate is low in total solids.
If not, and if VSd differs substantially from VSb, it could yield
an incorrect answer.
Volatile Solids Loss Across All Sewage Sludge
Treatment Processes
For cases when appreciable volatile solids reduction can
occur downstream from the digester (for example, as would
occur in air drying or lagoon storage), it is appropriate to cal-
culate the volatile solids loss from the point at which the sew-
age sludge enters the digester to the point at which the sewage
sludge leaves the treatment works. Under these circumstances,
it is virtually never possible to use the approximate mass bal-
ance approach, because flow rates are not uniform. The full
mass balance could be used in principle, but practical difficul-
ties such as measuring the mass of the output sewage sludge
(total mass, not just mass of solids) that relates to a given mass
of entering sewage sludge make this also a practical impossi-
bility. Generally then, the only option is to use the Van Kleeck
equation, because only the percent volatile solids content of the
entering and exiting sewage sludge is needed to make this
calculation. As noted earlier, this equation will be inappropriate
if there has been a selective loss of high volatility solids (e.g.,
bacteria) or low volatility solids (e.g., grit) in any of the sludge
processing steps.
To make a good comparison, there should be good corre-
spondence between the incoming sewage sludge and the treated
sewage sludge to which it is being compared (see Section 7.4).
For example, when sewage sludge is digested for 20 days, then
dried on a sand bed for 3 months, and then removed, the treated
sludge should be compared with the sludge fed to the digester
in the preceding 3 or 4 months. If no selective loss of volatile
or nonvolatile solids has occurred, the Van Kleeck equation (see
Equation 31) can be used to calculate volatile solids reduction.
References
EPA. 1992. Technical Support Document for Part 503 Pathogen
and Vector Attraction Reduction Requirements in Sewage
Sludge. Office of Water, U.S. EPA, Washington, DC. NTIS
90
-------
No.: PB93-11069.
Springfield, VA.
Natl. Technical Information Service,
Fischer, WJ. 1984. Calculation of volatile solids during sludge
digestion. In: Bruce, A., ed. Sewage Sludge Stabilization
and Disinfection, pp. 499-529. Water Research Centre, E.
Horwood Ltd., Chichester, England.
Van Kleeck, L.W. 1945. Sewage Works J., Operation of Sludge
Drying and Gas Utilization Units. 17(6): 1240-1255.
Water Pollution Control Federation. 1968. Manual of Practice
No. 16, Anaerobic Sludge Digestion. Washington, DC.
91
-------
-------
Appendix D
Guidance on Three Vector Attraction Reduction Tests
This appendix provides guidance for the vector attraction
reduction Options 2, 3, and 4 to demonstrate reduced vector
attraction (see Chapter 6 for a description of these require-
ments).
1. Additional Digestion Test for Anaerobically
Digested Sewage Sludge
Background
The additional digestion test for anaerobically digested
sewage sludge is based on research by Jeris et al. (1985). Farrell
and Bhide (1993) explain in more detail the origin of the time
and volatile solids reduction requirements of the test.
Jeris et al. (1985) measured changes in many parameters
including volatile solids content while carrying out additional
digestion of anaerobically digested sludge from several treat-
ment works for long periods. Samples were removed from the
digesters weekly for analysis. Because substantial amount of
sample was needed for all of these tests, they used continuously
mixed digesters of 18 liters capacity. The equipment and pro-
cedures of Jeris et al., although not complex, appear to be more
elaborate than needed for a control test. EPA staff (Farrell and
Bhide, 1993) have experimented with simplified tests and the
procedure recommended is based on their work.
Recommended Procedure
The essentials of the test are as follows:
« Remove, from the plant-scale digester, a representative sam-
ple of the sewage sludge to be evaluated to determine addi-
tional volatile solids destruction. Keep the sample protected
from oxygen and maintain it at the temperature of the di-
gester. Commence the test within 6 hours after taking the
sample.
• Flush fifteen 100-mL volumetric flasks with nitrogen, and
add approximately 50 mL of the sludge to be tested into each
flask. Frequently mix the test sludge during this operation
to assure that its composition remains uniform. Select five
flasks at random, and determine total solids content and
volatile solids content, using the entire 50 mL for the deter-
mination. Seal each of the remaining flasks with a stopper
with a single glass tube through it to allow generated gases
to escape.
• Connect the glass tubing from each flask through a flexible
connection to a manifold. To allow generated gases to escape
and prevent entry of air, connect the manifold to a water-
sealed bubbler by means of a vertical glass tube. The tube
should be at least 30-cm long with enough water in the
bubbler so that an increase in atmospheric pressure will not
cause backflow of air or water into the manifold. Maintain
the flasks containing the sludge at constant temperature
either by inserting them in a water bath (the sludge level in
the flasks must be below the water level in the bath) or by
placing the entire apparatus in a constant temperature room
or box. The temperature of the additional digestion test
should be the average temperature of the plant digester,
which should be in the range of 30°C to 40°C (86°F to
104°F). Temperature should be controlled within + 0.15°C
(0.27°F).
• Each flask should be swirled every day to assure adequate
mixing, using care not to displace sludge up into the neck
of the flask. Observe the water seal for the first few days of
operation. There should be evidence that gas is being pro-
duced and passing through the bubbler.
• After 20 days, withdraw five flasks at random. Determine
total and volatile solids content using the entire sample for
the determination. Swirl the flask vigorously before pouring
out its contents to minimize the hold up of thickened sludge
on the walls and to assure that any material left adhering to
the flask walls will have the same average composition as
the material withdrawn. Use a consistent procedure. If hold-
up on walls appears excessive, a minimal amount of distilled
water may be used to wash solids off the walls. Total re-
moval is not necessary, but any solids left on the walls
should be approximately of the same composition as the
material removed.
• After 40 days, remove the remaining five flasks. Determine
total and volatile solids content using the entire sample from
each flask for the determination. Use the same precautions
as in the preceding step to remove virtually all of the sludge,
leaving only material with the same approximate composi-
tion as the material removed.
Total and volatile solids content are determined using the
procedures of Method 2540 G of Standard Methods (APHA,
1992).
93
-------
Mean values and standard deviations of the total solids
content, the volatile solids content, and the percent volatile
solids are calculated. Volatile solids reductions that result from
the additional digestion periods of 20 and 40 days are calculated
from the mean values by the Van Kleeck equation and by a
material balance (refer to Appendix C for a general description
of these calculations). The results obtained at 20 days give an
early indication that the test is proceeding satisfactorily and will
help substantiate the 40-day result.
Alternative approaches are possible. The treatment works
may already have versatile bench-scale digesters available. This
equipment could be used for the test, provided accuracy and
reprodueibility can be demonstrated. The approach described
above was developed because Farrell and Bhide (1993) in their
preliminary work experienced much difficulty in withdrawing
representative samples from large digesters even when care was
taken to stir the digesters thoroughly before sampling. If an
alternative experimental setup is used, it is still advisable to
carry out multiple tests for the volatile solids content in order
to reduce the standard error of this measurement, because error
in the volatile solids content measurement is inflated by the
nature of the equation used to calculate the volatile solids re-
duction.
Variability in flow rates and nature of the sludge will result
in variability in performance of the plant-scale digesters. It is
advisable to run the additional digestion test routinely so that
sufficient data are available to indicate average performance.
The arithmetic mean of successive tests (a minimum of three
is suggested) should show an additional volatile solids reduc-
tion of £17%.
Calculation Details
Appendix C, Determination of Volatile Solids Reduction
by Digestion, describes calculation methods to use for digesters
that are continuously fed or are fed at least once a day. Although
the additional anaerobic digestion test is a batch digestion, the
material balance calculations approach is the same. Masses of
starting streams (input streams) are set equal to masses of end-
ing streams (output streams).
The test requires that the fixed volatile solids reduction
(FVSR) be calculated both by the Van Kleeck equation and the
material balance method. The Van Kleeck equation calculations
can be made in the manner described in Appendix C.
The calculation of the volatile solids reduction (and the
fixed fractional solids reduction [FFSR]) by the mass balance
method shown below has been refined by subtracting out the
mass of gas lost from the mass of sludge at the end of the
digestion step. For continuous digestion, this loss of mass usu-
ally is ignored, because the amount is small in relation to the
total digesting mass, and mass before and after digestion are
assumed to be the same. Considering the inherent difficulty in
matching mass and composition entering to mass and compo-
sition leaving for a continuous process, this is a reasonable
procedure. For batch digestion, the excellent correspondence
between starting material and final digested sludge provides
much greater accuracy in the mass balance calculation, so in-
clusion of this lost mass is worthwhile.
In the equations presented below, concentrations of fixed
and volatile solids are mass fractions—mass of solids per unit
mass of sludge (mass of sludge includes both the solids and the
water in the sludge)—and are indicated by the symbols lower-
case y and x. This is different from the usage in Appendix C
where concentrations are given in mass per unit volume, and
are indicated by the symbols uppercase y and x. This change
has been made because masses can be determined more accu-
rately than volumes in small-scale tests.
In the material balance calculation, it is assumed that as
the sludge digests, volatile solids and fixed solids are converted
to gases that escape or to volatile compounds that distill off
when the sludge is dried. Any production or consumption of
water by the biochemical reactions in digestion is assumed to
be negligible. The data collected (volatile solids and fixed sol-
ids concentrations of feed and digested sludge) allow mass
balances to be drawn on volatile solids, fixed solids, and water.
As noted, it is assumed that there is no change in water mass—
all water in the feed is present in the digested sludge. Fractional
reductions in volatile solids and fixed solids can be calculated
from these mass balances for the period of digestion. Details
of the calculation of these relationships are given by Farrell and
Bhide (1993). The final form of the equations for fractional
volatile solids reduction (mass balance [m.b.] method) and frac-
tional fixed solids reduction (m.b. method) are given below:
(la)
(Ib)
FFSR(m.b.) =
yf(l-xb-yb)
xf(i-yb)-xb(l-yf)
xf(l-xb-yb)
where:
y = mass fraction of volatile solids in the liquid sludge
x = mass fraction of fixed solids in the liquid sludge
f = indicates feed sludge at start of the test
b = indicates "bottoms" sludge at end of the test
If the fixed solids loss is zero, these two equations are
reduced to Equation 2 below:
.) = (yf-yb)/vfa-yb)
(2)
If the fixed, solids loss is not zero but is substantially
smaller than the volatile solids reduction, Equation 2 gives
surprisingly accurate results. For five sludges batch-digested by
Farrell and Bhide (1993), the fixed solids reductions were about
one-third of the volatile solids reductions. When the
FVSR(m.b.) calculated by Equation la averaged 15%, the
FVSR(rab.) calculated by Equation 2 averaged 14.93%, which
is a trivial difference.
The disappearance of fixed solids unfortunately has a rela-
tively large effect on the calculation of FVSR by the Van Kleeck
equation. The result is lower than it should be. For five sludges
94
-------
that were batch-digested by Farrell and Bhide (1993), the FVSR
calculated by the Van Kleeck method averaged 15%, whereas
the FVSR (m.b.) calculated by Equation la or 2 averaged about
20%. When the desked endpoint is an FVSR below 17%, this
is a substantial discrepancy.
The additional digestion test was developed for use with
the Van Kleeck equation, and the 17% requirement is based on
results calculated with this equation. In the future, use of the
more accurate mass balance equation may be required, with the
requirement adjusted upward by an appropriate amount. This
cannot be done until more data with different sludge become
available.
2. Specific Oxygen Uptake Rate
Background
The specific oxygen uptake rate of a sewage sludge is an
accepted method for indicating the biological activity of an
activated sewage sludge mixed liquor or an aerobically digest-
ing sludge. The procedure required by the Part 503 regulation
for this test is presented hi Standard Methods (APHA, 1992)
as Method 2710 B, Oxygen-Consumption Rate.
The use of the specific oxygen uptake rate (SOUR) has
been recommended by Eikum and Paulsrud (1977) as a reliable
method for indicating sludge stability provided temperature ef-
fects are taken into consideration.. For primary sewage sludges
aerobically digested at 18°C (64°F), sludge was adequately
stabilized (i.e., it did not putrefy and cause offensive odors)
when the SOUR was less than 1.2 mg O2/hr/g VSS (volatile
suspended solids). The authors investigated several alternative
methods for indicating stability of aerobically digested sludges
and recommended the SOUR test as the one with the most
advantages and the least disadvantages.
Ahlberg and Boyko (1972) also recommend the SOUR as
an index of stability. They found that, for aerobic digesters
operated at temperatures above 10°C (50°F), SOUR fell to
about 2.0 mg O2/hr/g VSS after a total sludge age of 60 days
and to 1.0 mg O2/hr/g VSS after about 120 days sludge age.
These authors state that a SOUR of less than 1.0 mg O2/hr/g
VSS at temperatures above 10°C (50°F) indicates a stable
sludge.
The results obtained by these authors indicate that long
digestion times—more than double the residence time for most
aerobic digesters in use today—are needed to eliminate odor
generation from aerobically digested sludges. Since the indus-
try is not being deluged with complaints about odor from aero-
bic digesters, it appears that a higher SOUR standard can be
chosen than they suggest without causing problems from odor
(and vector attraction).
The results of long-term batch aerobic digestion tests by
Jeris et al. (1985) provide information that is helpful in setting
a SOUR requirement that is reasonably attainable and still pro-
tective. Farrell and Bhide (1993) reviewed the data these
authors obtained with four sewage sludges from aerobic treat-
ment processes and concluded that a standard of 1.5 mg O2/hr/g
TS at 20°C (68°F) would discriminate between adequately sta-
bilized and poorly stabilized sludges. The "adequately di-
gested" sludges were not totally trouble-free, i.e., it was
possible under adverse conditions to develop odorous condi-
tions. In all cases where the sludge was deemed to be adequate,
minor adjustment in plant operating conditions created an ac-
ceptable sludge.
The SOUR requirement is based on total solids rather than
volatile suspended solids. This usage is preferred for consis-
tency with the rest of the Part 503 regulation where all loadings
are expressed on a total solids basis. The use of total solids
concentration in the SOUR calculation is rational since the
entire sludge solids and not just the volatile solids degrade and
may exert some oxygen demand. Making an adjustment for the
difference caused by basing the requirement on TS instead of
VSS, the standard is about 1.8 times higher than Eikum and
Paulsrud's recommended value and 2.1 tunes higher than Ahl-
berg and Boykos' recommendation.
Unlike anaerobic digestion, which is typically conducted
at 35°C (95°F), aerobic digestion is carried out without any
deliberate temperature control. The temperature of the digesting
sludge will be close to ambient temperature, which can range
from 5°C to 30°C (41°F to 86°F). In this temperature range,
SOUR increases with increasing temperature. Consequently, if
a requirement for SOUR is selected, there must be some way
to convert SOUR test results to a standard temperature. Con-
ceivably, the problem could be avoided if the sludge were sim-
ply heated or cooled to the standard temperature before running
the SOUR test. Unfortunately, this is not possible, because
temperature changes in digested sludge cause short-term insta-
bilities in oxygen uptake rate (Benedict and Carlson [1973],
Farrell and Bhide [1993]).
Eikum and Paulsrud (1977) recommend that the following
equation be used to adjust the SOUR determined at one tem-
perature to the SOUR for another temperature:
(SOUR)T1/(SOUR)T2 = e0"-1^ (3)
where:
(SOUR)xi = specific oxygen uptake rate at Tj
(SOUR)x2 = specific oxygen uptake rate at T2
0 = the Streeter-Phelps temperature sensitivity
coefficient
These authors calculated the temperature sensitivity coef-
ficient using then: data on the effect of temperature on the rate
of reduction in volatile suspended solids with time during aero-
bic digestion. This is an approximate approach, because there
is no certainty that there is a one-to-one relationship between
oxygen uptake rate and rate of volatile solids disappearance.
Another problem is that the coefficient depends on the makeup
of each individual sludge. For example, Koers and Mavinic
(1977) found the value of 0 to be less than 1.072 at tempera-
tures above 15°C (59°F) for aerobic digestion of waste acti-
vated sludges, whereas Eikum and Paulsrud (1977) determined
© to equal 1.112 for primary sludges. Grady and Lim (1980)
95
-------
reviewed the data of several investigators and recommended
that © = 1.05 be used for digestion of waste-activated sludges
when more specific information is not available. Based on a
review of the available information and their own work, Farrell
and Bhide (1993) recommend that Eikum and Paulsruds' tem-
perature correction procedure be utilized, using a temperature
sensitivity coefficient in the range of 1.05 to 1.07.
Recommended Procedure for Temperature Correction
A SOUR of 1.5 mg O2/hr/g total solids at 20°C (68°F) was
selected to indicate that an aerobically digested sludge has been
adequately reduced in vector attraction.
The SOUR of the sludge is to be measured at the tempera-
ture at which the aerobic digestion is occurring in the treatment
works and corrected to 20°C (68°F) by the following equation:
SOURM = SOURT x ©P0-^ (4)
where
0 = 1.05 above 20°C (68°F)
1.07 below 20°C (68°F)
This correction may be applied only if the temperature of
the sludge is between 10°C and 30°C (50°F and 86°F). The
restriction to the indicated temperature range is required to limit
the possible error in the SOUR caused by selecting an improper
temperature coefficient Farrell and Bhide's (1993) results in-
dicate that the suggested values for © will give a conservative
value for SOUR when translated from the actual temperature
to 20°C (68°F).
The experimental equipment and procedures for the SOUR
test are those described in Part 2710 B, Oxygen Consumption
Rate, of Standard Methods (APHA, 1992). The method allows
the use of a probe with an oxygen-sensitive electrode or a
respirometer. The method advises that manufacturer's direc-
tions be followed if a respirometer is used. No further reference
to respirometric methods will be made here. A timing device is
needed as well as a 300-mL biological oxygen demand (BOD)
bottle. A magnetic mixer with stirring bar is also required.
The procedure of Standard Method 2710 B should be fol-
lowed with one exception. The total solids concentration in-
stead of the volatile suspended solids concentration is used in
the calculation of the SOUR. Total solids concentration is de-
termined by Standard Method 2540 G. Method 2710 B cautions
that if the suspended solids content of the sludge is greater than
0.5%, additional stirring besides that provided by the stirring
bar be considered. Experiments by Farrell and Bhide (1993)
were carried out with sludges up to 2% in solids content without
difficulty if the SOUR was lower than about 3.0 mg O2/g/h. It
is possible to verify that mixing is adequate by running repeat
measurements at several stirrer bar speeds. If stirring is ade-
quate, oxygen uptake will be independent of stirrer speed.
The inert mineral solids in the wastewater in which the
sludge particles are suspended do not exert an oxygen demand
and properly should not be part of the total solids in the SOUR
determination. Ordinarily, they are such a small part of the total
solids that they can be ignored. If the ratio of inert dissolved
mineral solids in the treated wastewater to the total solids in
the sludge being tested is greater than 0.15, a correction should
be made to the total solids concentration. Inert dissolved min-
eral solids in the treated wastewater effluent is determined by
the method of Part 2540 B of Standard Methods (APHA, 1992).
This quantity is subtracted from the total solids of the sludge
to determine the total solids to be used in the SOUR calculation.
The collection of the sample and the time between sample
collection and measurement of the SOUR are important. The
sample should be a composite of grab samples taken within a
period of a few minutes duration. The sample should be trans-
ported to the laboratory expeditiously and kept under aeration
if the SOUR test cannot be run immediately. The sludge should
be kept at the temperature of the digester from which it was
drawn and aerated thoroughly before it is poured into the BOD
bottle for the test. If the temperature differs from 20°C (68°F)
by more than ±10°C (±18°F), the temperature correction may
be inappropriate and the result should not be used to prove that
the sewage sludge meets the SOUR requirement.
Variability in flow rates and nature of the sludge will result
in variability in performance of the plant-scale digesters. It is
advisable to run the SOUR test routinely so that sufficient data
are available to indicate average performance. The arithmetic
mean of successive tests—a minimum of seven over 2 or 3
weeks is suggested—should give a SOUR of < 1.5 mg O2/hr/g
total solids.
3. Additional Digestion Test for AerobicaUy Digested
Sewage Sludge
Background
Part 503 lists several options that can be used to demon-
strate reduction of vector attraction in sewage sludge. These
options include reduction of volatile solids by 38% and dem-
onstration of the SOUR value discussed above (see also Chap-
ter 6). These options are feasible for many, but not all, digested
sludges. For example, sludges from extended aeration treatment
works that are aerobically digested usually cannot meet this
requirement because they already are partially reduced in vola-
tile solids content by their exposure to long aeration times in
the wastewater treatment process.
The specific oxygen uptake test can be utilized to evaluate
aerobic sludges that do not meet the 38% volatile solids reduc-
tion requirement. Unfortunately, this test has a number of limi-
tations. It cannot be applied if the sludges have been digested
at temperatures lower than 10°C (50°F) or higher than 30°C
(86°F). It has not been evaluated under all possible conditions
of use, such as for sludges of more than 2% solids.
A straightforward approach for aerobically treated sludges
that cannot meet either of the above criteria is to determine to
what extent they can be digested further. If they show very little
capacity for further digestion, they will have a low potential for
additional biodegradation and odor generation that attracts vec-
96
-------
tors. Such a test necessarily takes many days to complete, be-
cause time must be provided to get measurable biodegradation.
Under most circumstances, this is not a serious drawback. If a
digester must be evaluated every 4 months to see if the sewage
sludge meets vector attraction reduction requirements, it will
be necessary to start a regular assessment program. A record
can be produced showing compliance. The sludge currently
being produced cannot be evaluated quickly but it will be pos-
sible to show compliance over a period of tune.
The additional digestion test for aerobically digested
sludges in Part 503 is based on research by Jeris et al. (1985),
and has been discussed by Farrell et al. (EPA, 1992). Farrell
and Bhide (1993) explain in more detail the origin of the time
and volatile solids reduction requirements of the test.
Jeris et al. (1985) demonstrated that several parameters—
volatile solids reduction, COD, BOD5, and SOUR—declined
smoothly and approached asymptotic values with time as
sludge was aerobically digested. Any one of these parameters
potentially could be used as an index of vector attraction re-
duction for aerobic sludges. SOUR has been adopted (see
above) for this purpose. Farrell and Bhide (1993) have shown
that the additional volatile solids reduction that occurs when
sludge is batch digested aerobically for 30 days correlates
equally as well as SOUR with the degree of vector attraction
reduction of the sludge. They recommend that a sewage sludge
be accepted as suitably reduced in vector attraction when it
shows less than 15% additional volatile solids reduction after
30 days additional batch digestion at 20°C (68°F). For three
out of four sludges investigated by Jeris et al. (1985), the rela-
tionship between SOUR and additional volatile solids reduction
showed that the SOUR was approximately equal to 1.5 mg
O2/hr/g (the Part 503 requirement for SOUR) when additional
volatile solids reduction was 15%. The two requirements thus
agree well with one another.
Recommended Procedure
There is considerable flexibility in selecting the size of the
digesters used for the additional aerobic digestion test. Farrell
and Bhide (1993) used a 20-liter fish tank. A tank of rectangular
cross-section is suggested because sidewalls are easily accessi-
ble and are easily scraped clean of adhering solids. The tank
should have a loose-fitting cover that allows air to escape. It is
preferable to vent exhaust gas to a hood to avoid exposure to
aerosols. Oil and particle-free air is supplied to the bottom of
the digester through porous stones at a rate sufficient to thor-
oughly mix the sewage sludge. This will supply adequate oxy-
gen to the sludge, but the oxygen level in the digesting sludge
should be checked with a dissolved oxygen meter to be sure
that the supply of oxygen is adequate. Oxygen level should be
at least 2 mg/L. Mechanical mixers also were used to keep
down foam and improve mixing.
If the total solids content of the sewage sludge is greater
than 2%, the sludge must be diluted to 2% solids with secon-
dary effluent at the start of the test. The requirement stems from
the results of Reynolds (1973) and Malina (1966) which dem-
onstrate that rate of volatile solids reduction decreases as the
feed solids concentration increases. Thus, for example, a sludge
with a 2% solids content that showed more than 15% volatile
solids reduction when digested for 30 days might show a lower
volatile solids reduction and would pass the test if it were at
4%. This dilution may cause a temporary change in rate of
volatile solids reduction. However, the long duration of the test
should provide adequate time for recovery and demonstration
of the appropriate reduction in volatile solids content.
When sampling the sludge, care should be taken to keep
the sludge aerobic and avoid unnecessary temperature shocks.
The sludge is digested at 20°C (68°F) even if the digester was
at some other temperature. It is expected that the bacterial
population will suffer a temporary shock if there is a substantial
temperature change, but the test is of sufficient duration to
overcome this effect and show a normal volatile solids reduc-
tion. Even if the bacteria are shocked and do not recover com-
pletely, the test simulates what would happen to the sludge in
the environment. If it passes the test, it is highly unlikely that
the sludge will attract vectors when used or disposed to the
environment. For example, if a sludge digested at 35°C (95°F)
has not been adequately reduced in volatile solids and is
shocked into biological inactivity for 30 days when its tempera-
ture is lowered to 20°C (68°F), it will be shocked in the same
way if it is applied to the soil at ambient temperature. Conse-
quently, it is unlikely to attract vectors.
The digester is charged with about 12 liters of the sewage
sludge to be additionally digested, and aeration is commenced.
The constant flow of ah" to the aerobic digestion test unit will
cause a substantial loss of water from the digester. Water loss
should be made up every day with distilled water. Solids that
adhere to the walls above and below the water line should be
scraped off and dispersed back into the sludge daily. The tem-
perature of the digesting sludge should be approximately 20°C
(68°F). If the temperature of the laboratory is maintained at
about 22°C (72°F), evaporation of water from the digester will
cool the sludge to about 20°C (68°F).
Sewage sludge is sampled every week for five successive
weeks. Before sampling, makeup water is added (this will gen-
erally require that air is temporarily shut off to allow the water
level to be established), and sludge is scraped off the walls and
redistributed into the digester. The sludge in the digester is
thoroughly mixed with a paddle before sampling, making sure
to mix the bottom sludge with the top. The sample is comprised
of several grab samples collected with a ladle while the digester
is being mixed. The entire sampling procedure is duplicated to
collect a second sample.
Total and volatile solids content of both samples are deter-
mined preferably by Standard Method 2540 G (APHA, 1992).
Percent volatile solids is calculated from total and volatile sol-
ids content. Standard Methods (APHA, 1992) states that dupli-
cates should agree within 5% of their average. If agreement is
substantially poorer than this, the sampling and analysis should
be repeated.
Calculation Details
Fraction volatile solids reduction is calculated by the Van
Kleeck formula (see Appendix C) and by a mass, balance
97
-------
method. The mass balance (m.b.) equations become very sim-
ple, because final mass of sludge is made very nearly equal to
initial mass of sludge by adjusting the volume by adding water.
These equations for fractional volatile solids reduction (FVSR)
and fractional fixed solids reduction (FFSR) are:
FVSR(m.b.) = (yf-yb)/yf
FFSR(m.b.) = (xf - xb) / xf
<5a)
(5b)
where:
y and x = mass fraction of volatile and fixed solids,
respectively (see previous section on
"Calculation details" for explanation of "mass
fraction")
f and b = subscripts indicating initial and final sludges
This calculation assumes that initial and final sludge den-
sities are the same. Very little error is introduced by this as-
sumption.
The calculation of the fractional fixed solids reduction is
not a requirement of the test, but it will provide useful infor-
mation.
The test was developed from information based on the
reduction in volatile solids content calculated by the Van Kleeck
equation. As noted in the section on the additional anaerobic
digestion test, for batch processes the material balance proce-
dure for calculating volatile solids reduction is superior to the
Van Kleeck approach. It is expected that the volatile solids
reduction by the mass balance method will show a higher vola-
tile solids reduction than the calculation made by using the Van
Kleeck equation.
4. References
Ahlberg, N.R. and B.I. Boyko. 1972. Evaluation and design of
aerobic digesters. Jour. WPCF 44(4):634-643.
Benedict, A.H., and D.A. Carlson. 1973. Temperature acclima-
tion in aerobic bio-oxidation systems. Jour. WPCF 45(1):
10-24.
Eikum, A., and B. Paulsrud. 1977. Methods for measuring the
degree of stability of aerobically stabilized sludges. Wat.
Res. 11: 763-770
EPA. 1992. Technical support document for Part 503 pathogen
and vector attraction reduction requirements in sewage
sludge. NTIS No.: PB93-11069. Springfield, VA: National
Technical Information Service.
Farrell, J.B. and V. Bhide. 1993. Development of methods for
quantifying vector attraction reduction. (Manuscript in
preparation.)
APHA (American Public Health Association). 1992. Standard
methods for the examination of water and wastewater.
Greenberg, A.E., L.S. Clesceri, and A.D. Eaton (eds.).
APHA, AWWA, and WEF, Washington, DC.
Grady, C.P.L., Jr., and H.C. Lira. 1980. Biological wastewater
treatment: theory and applications. Marcel Dekker, New
York.
Jeris, J.S., D. Ciarcia, E. Chen, and M. Mena. 1985. Determin-
ing the stability of treated municipal sludge. EPARept. No.
600/2-85-001 (NTIS No. PB 851-147189/AS). U.S. Envi-
ronmental Protection Agency, Cincinnati, Ohio.
Koers, D.A., and D.V. Mavinic. 1977. Aerobic digestion of
waste-activated sludge at low temperatures. Jour. WPCF
49(3): 460-468.
Malina, Jr., J.F. 1966. Discussion, pp. 157-160, in paper by D.
Kehr, "Aerobic sludge stabilization in sewage treatment
plants." Advances in Water Pollution Research, Vol. 2, pp
143-163. Water Pollution Control Federation, Washington,
DC.
Reynolds, T.D. 1973. Aerobic digestion of thickened waste-ac-
tivated sludge. Part 1, pp. 12-37, in Proc. 28th Industr.
Waste Conf., Purdue University.
98
-------
Appendix £
Determination of Residence Time for Anaerobic and Aerobic Digestion
Introduction
The PSRP and PFRP specifications in 40 CFR 257 for
anaerobic and aerobic digestion not only specify temperatures
but also require minimum mean cell residence times of the
sludge in the digesters. The mean cell residence time is the time
that the sludge particles are retained in the digestion vessel
under the conditions of the digestion. The calculation of resi-
dence time is ordinarily simple but it can become complicated
under certain circumstances. This appendix describes how to
make this calculation for most of the commonly encountered
modes for operating digesters.
Approach
The discussion has to be divided into two parts: residence
time for batch operation and for plug flow, and residence time
for fully mixed digesters. For batch operation, residence time
is obvious—it is the duration of the reaction. For plug flow, the
liquid-solid mixture that is sludge passes through the reactor
with no backward or forward mixing. The time it takes the
sludge to pass through the reactor is the residence time. It is
normally calculated by the following equation:
9 = V/q (1)
where
6 = plug flow solids residence time
V = volume of the liquid in the reactor
q = volume of the liquid leaving the reactor
Normally the volume of liquid leaving the reactor will
equal the volume entering. Conceivably, volume leaving could
be smaller (e.g., because of evaporation losses) and residence
time would be longer than expected if 6 were based on inlet
flow. Ordinarily, either inlet or outlet flow rate can be used.
For a fully mixed reactor, the individual particles of the
sludge are retained for different time periods—some particles
escape very soon after entry whereas others circulate in the
reactor for long periods before escaping. The average time in
the reactor is given by the relationship:
where
8s = an increment of sludge solids mat leaves the reactor
9 = time period this increment has been in the reactor
0n = nominal average solids residence time
When the flow rates of sludge into and out of the com-
pletely mixed vessel are constant, it can be demonstrated that
this equation reduces to:
vcv
(3)
where
V = reactor volume
q = flow rate leaving
Cy = concentration of solids in the reactor
Cq = concentration of solids in exiting sewage sludge
It is important to appreciate that q is the flow rate leaving
the reactor. Some operators periodically shut down reactor agi-
tation, allow a supernatant layer to form, decant the supernatant,
and resume operation. Under these conditions, the flow rate
entering the reactor is higher than the flow rate of sludge
leaving.
Note that in Equation 3, VCV is the mass of solids in the
system and qCq is the mass of solids leaving. Ordinarily Cv
equals Cq and these terms could be canceled. They are left in
the equation because they show the essential form of the resi-
dence time equation:
mass of solids in the digester
mass flow rate of solids leaving
(4)
(5s x 6)
S(Ss)
(2)
Using this form, residence time for the important operating
mode in which sludge leaving the digester is thickened and
returned to the digester can be calculated.
In many aerobic digestion installations, digested sludge is
thickened with part of the total volume returned to increase
residence time and part removed as product. The calculation
follows Equation 4 and is identical with the SRT (solids reten-
tion time) calculation used in activated sludge process calcula-
tions. The focus here is on the solids in the digester and the
solids that ultimately leave the system. Applying Equation 4
for residence time then leads to Equation 5:
99
-------
vcv
(5)
where
p a flow rate of processed sludge leaving the system
Cp = solids concentration in the processed sludge
The subscript p indicates the final product leaving the sys-
tem, not the underflow from the thickener. This approach ig-
nores any additional residence tune in the thickener since this
time is relatively short and not at proper digestion conditions.
Sample Calculations
In the following paragraphs, the equations and principles
presented above are used to demonstrate the calculation of
residence time for several commonly used digester operating
modes:
ZvjCj x time that batchi remains in the reactor
0n=
The following problem illustrates the calculation:
Let vh = 30 m3 (volume of "heel")
Vd = 130 m3 (total digester volume)
V; = each day 10 m3 is fed to the reactor at the
beginning of the day
Ci=12kg/m3
Vf is reached in 10 days. Sludge is discharged at the end
of Day 10.
(10-12-10+10- 12-9 +•• - + 10-12- 1)
Then 6n = - - -
(10 • 12 + 10 • 12 + • • • + 10 • 12)
en=
10 • 12 • 55
10 • 12 • 10
= 5.5 days
Casel
* Complete-mix reactor
• Constant feed and withdrawal at least once a day
• No substantial increase or decrease in volume in the reactor
(V)
• One or more feed streams and a single product stream (q)
The residence time desired is the nominal residence time.
Use Equation 3 as shown below:
« VCV V
The concentration terms in Equation 4 cancel out because
Cy equals Cq.
Case 2a
• Complete-mix reactor
• Vessel contains a "heel" of liquid sludge (Vh) at the begin-
ning of the digestion step
• Sludge is introduced in daily batches of volume (Vj) and
solids concentration (Q)
• When final volume (Vf) is reached, sludge is discharged
until Vh remains and the process starts again
Some aerobic digesters are run in this fashion. This prob-
lem is a special case involving a batch reaction. Exactly how
long each day's feeding remains in the reactor is known, but
an average residence time must be calculated as shown in
Equation 2:
Notice that the volume of the digester or of the "heel" did
not enter the calculation.
Case 2b
Same as Case 2a except:
• The solids content of the feed varies substantially from day
to day
• Decantate is periodically removed so more sludge can be
added to the digester
The following problem illustrates the calculation:
Let Vh = 30 m3, and Vd = 130 m3
Day v, (m3) Solids Content (kg/m3) Decantate (m3)
1
2
3
4
5
6
7
8
Q
10
11
12
10
10
10
10
10
10
10
10
10
10
10
10
10
15
20
15
15
10
20
25
15
10
15
20
0
0
0
0
0
0
0
0
10
0
10
0
= (10-10-12+10- 15- 11 + 10-20- 10 + -• •
••• + 10-10-3 + 10- 15-2+10-20-1)
(10- 10+10- 15 + 10-20 + '
15 +10 • 20)
9n= 11,950/1,900 +6.29 d
+ 10 • 10 + 10 •
100
-------
The volume of "heel" and sludge feedings equaled 150 m3,
exceeding the volume of the digester. This was made possible
by decanting 20 m3.
Case 3
Same as Case 2 except that after the digester is filled it is
run in batch mode with no feed or withdrawals for several days.
A conservative 6n can be calculated by simply adding the
number of extra days of operation to the 9n calculated for Case
2. The same applies to any other cases followed by batch mode
operation.
Flow rate of sludge from the thickener = 4 m /d
Solids content of sludge from the thickener = 40 kg/m
Flow rate of sludge returned to the digester = 2 m3/d
Flow rate of product sludge = 2 m /d
100 x 13.3
2x40
= 16.6 d
The denominator is the product of the flow rate leaving the
system (2 m3/d) and the concentration of sludge leaving the
thickener (40 kg/m3). Notice that flow rate of sludge leaving
the digester did not enter into the calculation.
Case 4
• Complete-mix reactor
• Constant feed and withdrawal at least once a day
• No substantial increase or decrease of volume in the reactor
• One or more feed streams, one decantate stream returned to
the treatment works, one product stream; the decantate is
removed from the digester so the sludge in the digester is
higher in solids than the feed
This mode of operation is frequently used in both anaero-
bic and aerobic digestion in small treatment works.
Equation 3 is used to calculate the residence time:
Let V = 100 m3
% = 10 m3/d (feed stream)
Q = 40 kg solids/m3
q = 5 m3/d (existing sludge stream)
Cv = 60 kg solids/m3
Case 5
• Complete-mix reactor
• Constant feed and withdrawal at least once a day
• Volume in digester reasonably constant
• One or more feed streams, one product stream that is thick-
ened, some sludge is recycled, and some is drawn off as
product
This mode of operation is sometimes used in aerobic di-
gesters. Equation 5 is used to calculate residence time.
Let V = 100 m3
Feed flow rate = 10 m3/d
Feed solids content = 10 kg/m3
Flow rate from the digester = 12 m /d
Solids content of sludge from the digester = 13.3 kg/m
Comments on Batch and Staged Operation
Sludge can be aerobically digested using a variety of proc-
ess configurations (including continuously fed single- or mul-
tiple-stage completely mixed reactors), or it can be digested in
a batch mode (batch operation may produce less volatile solids
reduction for a primary sludge than the other options because
there are lower numbers of aerobic microorganisms in it). Sin-
gle-stage completely mixed reactors with continuous feed and
withdrawal are the least effective of these options for bacterial
and viral destruction, because organisms that have been ex-
posed to the adverse condition of the digester for only a short
time can leak through to the product sludge.
Probably .the most practical alternative to use of a single
completely mixed reactor for aerobic digestion is staged opera-
tion, such as use of two or more completely mixed digesters in
series. The amount of slightly processed sludge passing from
inlet to outlet would be greatly reduced compared to single-
stage operation. If the kinetics of the reduction in pathogen
densities are known, it is possible to estimate how much im-
provement can be made by staged operation.
Farrah et al. (1986) have shown that the declines in densi-
ties of enteric bacteria and viruses follow first-order kinetics.
If first-order kinetics are assumed to be correct, it can be shown
that a one-log reduction of organisms is achieved in half as
much time in a two-stage reactor (equal volume in each stage)
as in a one-stage reactor. Direct experimental verification of
this prediction has not been carried out, but Lee et al. (1989)
have qualitatively verified the effect.
It is reasonable to give credit for an unproved operating
mode. Since not all factors involved in the decay of microor-
ganisms densities are known, some factor of safety should be
introduced. It is recommended then that for staged operation
using two stages of approximately equal volume, the time re-
quired be reduced to 70% of the time required for single-stage
aerobic digestion in a continuously mixed reactor. This allows
a 30% reduction in time instead of the 50% estimated from
theoretical considerations. The same reduction is recommended
for batch operation or for more than two stages in series. Thus,
the time required would be reduced from 40 days at 20°C
(68°F) to 28 days at 20°C (68°F), and from 60 days at 15°C
(59°F) to 42 days at 15°C (59°F). These reduced times are also
more than sufficient to achieve adequate vector attraction re-
duction.
101
-------
If the plant operators desire, they may dispense with the
PSRP time-temperature requirements of aerobic digestion but
instead demonstrate experimentally that microbial levels in the
product from their sludge digester are satisfactorily reduced.
Under the current regulations, fecal coliform densities must be
less than or equal to 2,000,000 CPU or MPN per gram total
solids. Once this performance is demonstrated, the process
would have to be operated between monitoring episodes at
time-temperature conditions at least as severe as those used
during their tests.
References
Farrah, S.R., G. Bitton, and S.G. Zan. 1986. Inactivation
of enteric pathogens during aerobic digestion of wastewater
sludge. EPA Pub. No. EPA/600/2-86/047. Water Engineering
Research Laboratory, Cincinnati, OH. NT1S Publication No.
PB86-183084/A5. National Technical Information Service,
Springfield, Virginia.
Lee, K.M., C.A. Brunner, J.B. Farrell, and A.E. Eralp.
1989. Destruction of enteric bacteria and viruses during two-
phase digestion. Journal WPCF 61(6):1421-1429.
102
-------
Appendix F
Sample Preparation for Fecal Conform Tests and Salmonella sp. Analysis
1. Sample Preparation for Fecal Coliform Tests
LI Class B Alternative 1
To demonstrate that a given domestic sewage sludge sam-
ple meets Class B pathogen requirements under Alternative 1,
the density of fecal coliform from seven samples of treated
sewage sludge must be determined and the geometric mean of
the fecal coliform density must not exceed 2 million Colony
Forming Units (CPU) or Most Probable Number (MPN) per
gram of sewage sludge solids (dry weight basis). The solids
content of treated domestic sludge can be highly variable.
Therefore, an aliquot of each sample must be dried and the
solids content determined in accordance with procedure 2540
G. of the 18th edition of Standard Methods for the Examination
of Water and Wastewater (APHA, 1992), hereafter referred to
as SM.
Sludge samples to be analyzed in accordance with SM
9221 E (Fecal Coliform MPN Procedure) and 9222 D (Fecal
Coliform Membrane Filter Procedure) may require dilution
prior to analysis. An ideal sample volume will yield results that
accurately estimate the fecal coliform density of the sludge.
Detection of fecal coliform in undiluted samples could easily
exceed the detection limits of these procedures. Therefore, it is
recommended that the following dilution scheme be used.
For Liquid Samples:
1. Use a sterile pipette to transfer 1.0 mL of well-mixed
sample to 99 ml. of sterile buffered dilution water (see
SM Section 9050C) in a sterile screw cap bottle, and
mix by vigorously shaking the bottle a minimum of 25
times. This is dilution "A." A volume of 1.0 mL of this
mixture is 0.010 mL of the original sample.
2. Use a sterile pipette to transfer 1.0 mL of dilution "A"
to a second screw cap bottle containing 99 ml. of sterile
buffered dilution water, and mix as before. This is di-
lution "B." A volume of 1.0 mL of this mixture is
0.00010 mL of the original sample. Use a sterile pipette
to transfer 1.0 mL of dilution "B" to a sterile screw cap
bottle containing 99 mL of sterile buffered dilution
water, and mix as before. This is dilution "C." Go to
Step 3 for MPN analysis or Step 5 for membrane filter
(MF) analysis.
3. For MPN analysis, follow procedure 9221 E in SM.
Four series of five tubes will be used for the analysis.
Inoculate the first series of five tubes each with 10.0
mL of dilution "B." This is a 0.0010 dilution of the
original sample. The second series of tubes should be
inoculated with 1.0 mL of dilution "B" (0.00010). The
third series of tubes should receive 10.0 mL of "C"
(0.000010). Inoculate a fourth series of five tubes each
with 1.0 mL of dilution "C" (0.0000010). Continue the
procedure as described in SM.
4. Refer to Table 9221.IV in SM to estimate the MPN
index/100 mL. Only three of the four series of five
tubes will be used for estimating the MPN. Choose the
highest dilution that gives positive results in all five
tubes, and the next two higher dilutions for your esti-
mate. Compute the MPN/g according to the following
equation:
MPN fecal coliform/g =
IPX MPN index/100 mL
largest volume X % dry solids tested
Examples:
In the examples given below, the dilutions used to deter-
mine the MPN are underlined. The number in the numerator
represents positive tubes; that in the denominator, the total
number of tubes planted; the combination of positives simply
represents the total number of positive tubes per dilution.
0.0010 0.00010 0.000010 0.0000010 Combination
Example mL mL m mL of Positives
a
b
c
5/5
5/5
0/5
5/5
3/5
1/5
3/5
1/5
0/5
0/5
0/5
0/5
5-3-0
5-3-1
0-1-0
For each example we will assume that the total solids
content is 4.0%.
For Example a:
The MPN index/100 mL from Table 9221.4 is 80. There-
fore:
For Example b:
103
-------
The MPN index/100 mL from Table 9221.4 is 110. There-
fore:
MPN/g =
10x110
0.0010x4.0
= 2.8 x 10s
For Example c:
The MPN index/100 mL from Table 9221.4 is 2. Therefore:
MPN/g:
10x2
0.0010x4.0
= 5.0xl03
5. Alternately the membrane filter procedure may be used
to determine fecal coliform density. Three individual
filtrations should be conducted in accordance with SM
9222 D using 10.0 mL of dilution "C," and 1.0 mL and
10.0 mL of dilution "B." These represent 0.000010,
0.00010, and 0.0010 mL of the original sample. Incu-
bate samples, and count colonies as directed. Experi-
enced analysts are encouraged to modify this dilution
scheme (e.g., half log dilutions) in order to obtain fil-
ters which yield between 20 and 60 CPU.
6. Compute the density of CPU from membrane filters
that yield counts within the desired range of 20 to 60
fecal coliform colonies:
coliform colonies/g =
coliform colonies counted x 100
mL samplex % dry solids
For Solid Samples:
1. In a sterile dish weigh out 50.0 grams of well-mixed
sample. Whenever possible, the sample tested should
contain all materials which will be included in the
sludge. For example, if wood chips are part of a sludge
compost, some mixing or grinding means may be
needed to achieve homogeneity before testing. One ex-
ception would be large pieces of wood which are not
easily ground and may be discarded before blending.
Transfer the sample to a sterile blender. Use 450 mL
of sterile buffered dilution water to rinse any remaining
sample into the blender. Cover and blend on high speed
for 2 minutes. One milliliter of this sample contains
O.10 g of the original sample.
2. Use a sterile pipette to transfer 11.0 mL of the blender
contents to a screw cap bottle containing 99 mL of
sterile buffered dilution water and shake vigorously a
minimum of 25 times. One milliliter of this sample
contains 0.010 g of the original sample. This is dilution
"A."
3. Follow the procedures for fluid samples starting at Step
2.
Examples:
Seven samples of a treated sludge were obtained prior to
land spreading. The solids concentration of each sample was
determined according to SM. These were found to be:
Sample No.
1
2
3
4
5
6
7
Solids Concentration (%)
3.8
4.3
4.0
4.2
4.1
3.7
3.9
The samples were liquid with some solids. Therefore the
procedure for liquid sample preparation was used. Furthermore,
the membrane filter technique was used to determine if the fecal
coliform concentration of the sludge would meet the criteria for
Class B Alternative 1. Samples were prepared in accordance
with the procedure outlined above. This yielded 21 individual
membrane filters (MFs) plus controls. The results from these
tests are shown in Table 1.
Table 1. Number of Fecal Coliform Colonies on MF Plates
Sample No.
1
2
3
4
5
6
7
0.000010
mL Filtration
0
2
0
0
0
0
0
0.00010
mL Filtration
1
18
8
5
1
1
1
0.0010 mL
Filtration
23
TNTC
65
58
17
39
20
Whenever possible, the coliform density is calculated us-
ing only those MF plates that have between 20 and 60 blue
colonies. However, there may be occasions when the total num-
ber of colonies on a plate will be above or below the ideal
range. If the colonies are not discrete and appear to be growing
together, results should be reported as "too numerous to
count"(TNTC). If no filter has a coliform count falling in the
ideal range (20 to 60), total the coliform counts on all countable
filters and report as coliform colonies/g. For sample number 2
the fecal coliform density is:
coliform colonies/g =
(2 + 18) X 100
(0.000010 + 0.00010) X 4.3
= 4.2xl06
Sample number 3 has two filters that have colony counts
outside the ideal range also. In this case, both countable plates
should be used to calculate the coliform density/g. For sample
number 3, the fecal coliform density is:
coliform colonies/g =
(8 + 65) x 100
(0.00010 +0.0010) X 4.0
= 1.6 x 106
Except for sample number 5, all the remaining samples
have at least one membrane filter within the ideal range. For
these samples, use the number of colonies formed on that filter
to calculate the coliform density. For sample number 1, the fecal
coliform density is:
104
-------
coliform colonies/g =
23 x 100
O.OOlOx 3.8
= 6.0xl05
Coliform densities of all the samples were calculated and
converted to logio values to compute a geometric mean. These
calculated values are presented in Table 2.
Table 2. Coliform Density of Sludge Samples
Sample No. Coliform Density
1
2
3
4
5
6
7
6.0 X 105
4.2 X 106
1.6 X106
9.0 X 105
4.0 X 10s
1.0 X 10s
5.1 X 10s
5.78
6.62
6.20
6.14
5.60,
6.02
5.71
The geometric mean for the seven samples is determined
by averaging the Iog10 values of the coliform density and taking
the antilog of that value:
(5.78 + 6.62 + 6.20 + 6.14 + 5.60 + 6.02 + 5.71) / 7 = 6.01
The antilog of 6.01 = 1.0 x 106
Therefore, the geometric mean fecal coliform density is
below 2 million and the sludge meets Class B pathogen require-
ments under Alternative 1.
1.2 Class A Alternative 1
Part 503 requires that, to qualify as a Class A sludge,
treated sewage sludge must be monitored for fecal coliform (or
Salmonella sp.) and have a density of less than 1,000 MPN
fecal coliform per gram of total solids (dry weight basis). The
regulation does not specify total number of samples. However,
it is suggested that a sampling event extend over 2 weeks and
that at least seven samples be collected and analyzed. The
membrane filter procedure may not be used for this determina-
tion. This is because the high concentration of solids in such
sludges may plug the filter or, render the filter uncountable. The
total solids content for each sample must be determined in
accordance with procedure 2540 G of SM.
For Liquid Samples:
1. Follow procedure 9221 E in SM. Four series of five
tubes will be used for the analysis. Use a sterile pipette
to inoculate the first series of five tubes with 10.0 mL
of well-mixed sample per tube (it may be convenient
to use a sterile pipette with a large diameter opening
capable of transferring sludge solids). The second se-
ries of tubes should receive 1.0 mL of well-mixed sam-
ple. Use a sterile pipette to transfer 1.0 mL of sample
to 99 mL of sterile buffered dilution water (see SM
Section 9050 C) in a sterile screw cap bottle, and mix
by vigorously shaking the bottle a minimum of 25
times. This is dilution "A." Use a sterile pipette to
inoculate the third series of tubes with 10.0 mL of
dilution "A." The fourth series of tubes should be in-
oculated with 1.0 mL of dilution "A." Complete the
procedure as described hi SM.
2. Calculate the MPN as directed hi Step 4 above.
For Solid Samples:
1. Using aseptic techniques, weigh out five portions of
10.0 grams each of well-mixed sample. For example,
if wood chips are part of a sludge compost, some mix-
ing or grinding means may be needed to achieve ho-
mogeneity before testing. One exception would be
large pieces of wood that are not easily ground and may
be discarded before blending. Transfer each portion to
MPN tubes containing 10.0 mL of double-strength
Lauryl Tryptose Broth (71.2 g/L). The second series of
MPN tubes should contain 10.0 mL of single-strength
Lauryl Tryptose Broth (35.6 g/L). Using aseptic tech-
nique, weigh out five portions of 1.0 g each of well-
mixed sample and transfer each to individual tubes
prepared for the second series of MPN tubes. Please
note that some solids will not separate easily and/or
may float. Since gas fermentation tubes are used for
this procedure shaking is not practical. Therefore, it is
recommended that a sterile loop be used to gently sub-
merge solids in the broth. Prepare dilution "A" as de-
scribed above under "Class B Alternative 1, For Solid
Samples." Inoculate the third series of MPN tubes with
10.0 mL of dilution "A." The fourth series of five tubes
must receive 1.0 mL of dilution "A" in each tube.
Continue with the procedure hi Section 9221 E of SM.
2. Calculate the MPN as directed in Step 4 above.
2. Sample Preparation for Salmonella Sp. Analysis
As an alternative to fecal coliform analysis, Salmonella sp.
quantification may be used to demonstrate that a sludge meets
Class A criteria. Sludges with Salmonella sp. densities below 3
MPN/4 g (dry weight basis) meet Class A criteria. The analyti-
cal method presented hi Appendix G of this document, de-
scribes the procedure used to identify Salmonella sp. in a water
sample. To use this MPN procedure for sewage sludges, the
sample preparation step described here should be used, and the
total solids content of each sample must be determined accord-
ing to Method 2540 G in SM.
_ For Liquid Samples:
1. Follow the same procedure used for liquid sample
preparation for fecal coliform analysis described under
Section 1.2 above (Class A Alternative 1). However,
the enrichment medium used for this analysis should
be dulcitol selenite broth (DSE) as described in Appen-
dix G, and only three series of five tubes should be
used for this MPN procedure. Use a sterile open-tip
pipette to transfer 10.0 mL of sample to each tube in
the first series. These tubes should contain 10.0 mL of
double-strength DSE broth. Each tube in the second
series should contain 10.0 ml. of single- strength DSE
broth. These tubes should each receive 1.0 mL of sam-
ple. The final series of tubes should contain 10.0 mL
105
-------
of double-strength DSE broth. These tubes should each
receive 10.0 niL of dilution "A" as described above.
Complete the MPN procedure as described hi Appen-
dix G.
2. Refer to Table 9221.IV in SM to estimate the MPN
index/100 mL. Calculate the MPN/4 g according to the
following equation:
Salmonella sp. MPN/ g =
Example:
MPN index/lOOmLx 4
% dry solids
If one tube in the first series was identified as being posi-
tive for Salmonella sp. and no other tubes were found to be
positive, from Table 9221.IV one finds that a 1-0-0 combination
of positives has an MPN index/100 mL of 2. If the mass of dry
solids for the sample was 4.0%, then:
2x4
Salmonella sp. MPN/ g=-rr- = 2
For Solid Samples:
1. Follow the procedure for solid sample preparation for
fecal coliform analysis described under Section 1.2
(Class A Alternative 1) above. However, the enrich-
ment medium used for this analysis should be dulcitol
selenite broth (DSE) as described hi Appendix G, and
only three series of five tubes should be used for this
MPN procedure. Use aseptic technique to weigh out
and transfer 10.0 g of well-mixed sample to each screw
cap tube in the first series, shake vigorously to mix.
These tubes should contain 10.0 mL of double-strength
DSE broth. Each tube in the second series should con-
tain 10.0 mL of single-strength DSE broth. These tubes
should receive 1.0 g of sample, mix as noted above.
The final series of tubes should contain 10.0 mL of
double-strength DSE broth. These tubes should receive
10.0 mL of dilution "A" as described above. Loosen
caps before incubating the tubes. Complete the MPN
procedure as described in Appendix G.
2. Refer to Table 9221.IV in SM to estimate the MPN
index/100 mL. Calculate the MPN/4 g according to the
following equation:
Salmonella sp. MPN/ g =
MPN index/lOOmL x 4
% dry solids
106
-------
Appendix G
Kenner and Clark (1974) Analytical Method for Salmonella sp. Bacteria*
Detection and enumeration of Salmonella
and Pseudomonas aeruginosa
BERNARD A. KENNER AND HAROLD P. CLARK
THE FEDERAL WATER POLLUTION CON-
TROL AMENDMENTS of 1972*-4 may
well require the quantification and enu-
meration of pathogens such as Salmonella
species in all classes of waters. The re-
quirements are described by Shedroff.5
One of the continuing programs of the
Environmental Protection Agency (EPA)
is a research project concerned with the
development of practical laboratory meth-
ods for the isolation, quantification, and
enumeration of pathogens from polluted
waters. This paper reports a monitoring
method developed for the simultaneous
isolation and enumeration of Salmonella
species and Pseudomonas aeruginosa from
potable waters, reuse waters, treatment
plant effluents, receiving waters, and
sludges.
The method described herein, and de-
veloped by Kenner,6 is practical because
readily available bacteriological media,
chemicals, and equipment are all that are
required to obtain the desired results.
These results are the establishment of the
absence or presence of Salmonella species
(pathogenic hazardous bacteria) and/or
Pseudomonas aeruginosa (potential patio-
gens) that affect persons who are in a
debilitated condition and are very com-
mon as infectious agents in hospitals be-
cause of their resistance to antibiotic
therapy.7"9 Potable waters have also been
shown to contain Ps. aeruginosa.6'10 The
sources of these potential pathogens are
human and animal feces and waste-
waters.11- 12
When the monitoring method was used,
it was found that 100 percent of municipal
wastewaters and treatment plant sludges
contained both of these potential patho-
gens. Ps. aeruginosa has been found in
potable water supplies of large and small
municipalities where insufficient residual
chlorine is evident. Also important is the
fact that these organisms may be found in
the absence of fecal coliforms, whereas
negative indicator tests may give a false
sense of security. It is believed by the
authors that these organisms may be better
indicators than fecal coliforms of pollu-
tion in potable, direct reuse, bathing, and
recreational waters.
MATERIALS AND METHODS
The monitoring method uses a multiple
tube (MPN) procedure in which dulcitol
selenite broth (DSE) 13 is used for primary
enrichment medium, and is modified by
the use of sodium acid selenite (BBL).
The formula is proteose peptone (Bacto),
0.4 percent; yeast extract (Bacto), 0.15
percent; dulcitol, 0.4 percent; BBL, 0.5
percent; Na2HPO4, 0.125 percent; and
KH2PO4, 0.125 percent in distilled water.
The constituents are dissolved in a sterile
flask, covered with foil, and heated to
88 °C in a water bath to obtain a clear
sterile medium that does not require ad-
justment of pH. Productivity for Salmo-
nella, species is enhanced by the addition
of an 18-hr, 37 °C culture of Salmonella
paratyphi A (10 percent by volume) in
single-strength DSE broth, killed by heating
to 88°C.
Concentration of bacteria from large
volumes of water is necessary when pota-
ble, direct reuse, receiving waters, and
treatment effluents are being monitored.
Vol. 46, No. 9, September 1974 2163
*Copyright © 1974 Water Environment Federation. Reprinted with permission.
107
-------
KENNER AND CLARK
TABLE I.—Retentive Characteristics of Several Glass Fiber Filter Papers*
Compared with Membrane Filters
Filter
Milliporc (MF) HAWG 047 HA 0.45 n, white,
grid, 47 mm, Millipore Filter Corp.
984H Ultra Glass Fiber Filter, 47 mm,
Reeve Angel Corp.
GF/F Glass Paper Whatman.J 47 mm,
Reeve Angel Corp.
GF/D Glass Paper Whatman, t 47 mm,
Reeve Angel Corp.
934AH Glass Fiber Filter, 47 mm,
Reeve Angel Corp.
GF/A Glass Paper Whatman, 47 mm,
Reeve Angel Corp.
Total Bacteriaf
Filtered
1,376
1,229
2,698
2,622
1,049
1,066
Number Passing
Filter
0
25
6
2,166
198
680
Percentage
Retention
100
98
99.8
17.4
81
36
* The 984H Ultra Glass Fiber Filter is flexible when wet, readily allows filtration of large volumes of water,
can readily be bent double with forceps, and, when placed into primary enrichment broth, disintegrates when
tube is shaken and releases entrapped bacteria.
t Enteric bacteria, E. coli, 0.5 X 1-3 ft.
t A new paper filter GF/F has better retentive properties than the 984H, and has same properties (tested
Oct. 1973).
Concentration is attained by filtration
through glass fiber filters ° in a membrane
filter apparatus. After the desired volume
of water is filtered through the ultra filter,
the flexible filter is folded double with
sterile forceps and inserted into a suitable
volume of single-strength DSE medium
contained in a test tube located in the
first row of the multiple tube setup. The
tube should then be shaken to cause the
filter to disintegrate (Table I and Figure
1). To obtain MPN results per one 1 or
per 10 1, 100 ml or 1,000 ml of sample,
respectively, are filtered for each tube of
DSE medium in the first row of the five-
tube MPX setup. Additional dilutions are
made by transferring material from tubes
in the first row to tubes farther back in
the setup.
Obtaining results on a per 1-gal (3.8-1)
basis requires filtration of 380 ml, and on
a per 10-gal (38-1) basis requires filtration
of 3,800 ml for each tube in the first row.
Where concentration of bacteria is not
usually required, as in municipal waste-
waters, sludges, or primary effluents, the
regular transfer of 10 ml of sample to each
• Reeve Angel 984H ultra glass fiber filter, 47
mm, Reeve Angel &. Co., Inc., Clifton, N. J.
Mention of trade names does not constitute en-
dorsement or recommendation by EPA.
2164 Journal WPCF
tube in the first row of the setup into 10
ml of double-strength DSE is made, 1 ml
of sample in 9 ml of single-strength. DSE
in the second row, and so on. The MPN
table in "Standard Methods" " is used to
read directly the results per volume of
sample.
Incubation temperature of 40° ± 0.2°C
for 1 and 2 days is critical to obtain opti-
mum recovery of Salmonella sp. and
Pseudomonas aeruginosa when DSE broth
is used for primary enrichment. After
primary incubation at 40° C, surface 1'oop-
fuls (scum) (7 mm platinum or nichrome
wire loop) are removed from each multi-
ple-tube culture and streaked on each of
two sections of a divided plate of Xylose
lysine desoxycholate agar (XLD) 1!i in order
to isolate colonial growth. The numbered
plates are inverted and incubated at 37°C
for a period not to exceed 24 hr.
Commercial dehydrated XLD agars (BBL
and Difco) are satisfactory if they are re-
constituted in distilled water in sterile
foil-covered flasks and heated to 88° or
92 °C, respectively. The agar is then
cooled to 55° to 60°C and distributed in
sterile petri dishes. This laboratory pre-
fers 10-ml portions in each section of a
divided sterile disposable plastic dish
(Figure 1).
108
-------
PATHOGEN DETECTION
i
Sterile
polypropylene
container
Filter funnel
for 47-mm 984 H
filter pad
vacuum
flask
After filtration filter-pad is
folded double with forceps
5 1000ml pad inserted into 20ml
1xDSE broth for each of 5-tubes
in 1st row
) 2ml to
8ml 1xDSE in
2nd row
I
1 ml from
2nd row to
9ml 1xDSE /
3rd row etc.
^Completed MPN incubated at 40C for
1- and 2- days-Secondary medium streaked for
isolated colonies from surface MPN tubes with
7 mm Nichrome 22 gauge loop
6
extract
I blue
1000A
XLD Agar plate invert plates incubate 37C 20-24 hrs
Loosen caps
H
Loosen cap & incubate
6-20 hrs
_-, 40C
Chloroform
King A
Tech Agar
Pick Black centered
colonies to KIA
slants
1000 A
Pink colonies —-
rarely
Salmonella sp.
'Streak and Stab butt
f Red
t no-change
s
slant
["H2S
I Black
T Yellow
Acid
LButt
Blue Green
Ps. aeruginosa
Slide Serology
Salmonella "O" poly A-
or Salmonella "H" poly a
Incubate Slants at 35-
Pick flat erose edge 37C 18-20 hr.
grayish alkaline colonies
to Tech Agar streak & stab,
, typical slant
^'for isolated pure strains
Test
,-l f Urease I
-z \ Negative
FIGURE 1.—Procedure for isolation of pathogens,
Positive incubated XLD plate cultures
contain typical clear, pink-edged, black-
centered SaZmoneZZa colonies, and flat,
mucoid, grayish alkaline, pink erose-edged
Ps. aeragmosa. The SaZmoneZZa colonies
are picked to Kligler iron agar (KIA) or
Triple sugar iron agar slants for typical
appearance, purification, and identity tests.
Ps. fleragmosa colonies are picked to King
A agar slants (Tech agar BBL) for obtain-
ing the bluegreen pyocyanin confirmation
•at40°C (Figure 1).
Typically, Salmonella sp. slant cultures
(streaked and stabbed), incubated over-
Vol. 46, No. 9, September 1974 2160
109
-------
KENNER AND CLARK
TABLE II.—Advantage of Ultra-filter 984H Use in Monitoring Suspected
Waters for Salmonella species
Type of Sample
Stormwater runoff
Stormwatcr runoff
Activated sludge effluent
Municipal wastewater
Municipal wastewater
Activated sludge effluent
Mississippi River water,
mile 403.1
Municipal wastewater
Salmonella
(no./100 ml)
4.5
<3.0 •
<3.0
6.2
<3.0
<3.0
43
3.0
Serotypes 'Found
(no./ 100 ml)
5. bareilly1
none
none
Arizona3
none
none
5. ohiow
S. cholerasuis
var. kunzendorf2
Salmonella
(no./gal)
210
7.3
3.6
1,500
110
28
>11,000
21
Serotypes Found
(no./gal)
5. kottbus™
S. bareilly11
S. Java*
S. muenchent
S. group G4
Arizona4
5. anatum?
S. newporfi
S. san diego1
S. worthington2
S, anatum*
S. derby1
S. newport3
S. Uocklef
S, newport3
S. ohio1*
S. derby*
S. meleagridis*
S. cholerasuis
var. kunzendorf6
5. newport*
night at 37°C, give an unchanged or alka-
line red-appearing slant; the butt is black-
ened by HaS, is acid-yellow, and has gas
bubbles, except for rare species. Typical-
appearing slant cultures are purified by
transferring them to XLD agar plates for
the development of isolated colonies. The
flat or umbonated-appearing colonies with
large black centers and clear pink edges
then are picked to KIA slants (streaked and
stabbed), incubated at 37°C, and urease
tested before the identification procedure
(Figure 1). Urease-negative tubes are re-
tained for presumptive serological tests
and serotype identification.
Typical Tech agar slant cultures for Ps.
aeruginoca that are incubated at 40°C
overnight turn a bluegreen color from
pyocyanin, a pigment produced only by
this species. A reddish-blue color is caused
by the additional presence of pyorubin.
The blue pigment is extractable in chloro-
form and is light blue in color after a few
hours at room temperature. No further
tests are necessary. The count is read di-
rectly from the MPN table.
216G Journal WPCF
JUSTIFICATION FOR PROCEDURES
Choice of primary enrichment medium
and secondary isolation agar. Most of the
enrichment media described in contem-
porary literature were designed for the
isolation of pathogens from clinical speci-
mens from ill persons or from samples of
suspected foods, and they work quite well
for those types of samples. When they
are used, however, for the isolation of
pathogens from polluted waters and other
types of environmental samples, such as
soils, they do not prove adequate. En-
richment media that were tested and found
wanting in regard to detection and selec-
tivity were tetrathionate broth (TT), with
and without brilliant green at 41.5°C;
selenite cystine broth at 37°C; selenite
F broth at 37 °C; selenite brilliant green,
with and without sulfa, at 37° and 41.5°C;
and Gram-negative broth (GN) at 40° and
41.5°C.
None of the media named worked well
at 37°C for the isolation of Salmonella sp.,
and isolation from wastewaters only oc-
110
-------
PATHOGEN DETECTION
TABLE III.—Percentage of Colony Picks from DSE-XLD Combination Positive
for Salmonella species
Liquid Samples
Municipal wastewater
Stockyard wastewater
Rivers
Mississippi
Ohio
Stormwater runoffs
Activated sludge biological
effluent
Trickling filter effluent
Package plant effluent
Package plant sludge
Chlorinated primary outfall
Creek 1 mile (1.6 km) below
package plant outfall
Home cisterns
Dupont R-O
Feed
Reject
Product-negative
Raw primary sludge
Primary activated sludge
Anaerobic digester sludge
Anaerobic digester sludge
(28 days)
Activated secondary sludge
Total
No.
15
1
8
2
20
7
6
2
2
2
2
1
1
4
1
3
1
6
84
Total Picks
from XLD
315
36
110
18
386
103
83
41
17
37
17
20
16
80
15
78
9
189
1,570
No.
Positive
250
36
84
14
306
78
55
37
13
16
10
14
8
66
13
65
3
155
1,223
No.
Negative
65
0
26
4
80
25
28
4
4
21
7
6
8
14
2
13
6
• 34
347
Percentage
Positive
Range of
Salmonella
counts/ 100 ml
79 3.0-1,500
100
76
78
79
76
66
90
76
43
59
.70
50
83
87
83
33
82
average 78
2,100
1.5->300
0.2-1.5
0.1-1,100
0.35-140
1.8-620
43-240
3-43
4.5-12
0.26-1,1
4.3
0.91 '
13-700
23
79-170
2
11->11,000
curred by chance and was purely qualita-
tive. Of the above-named media used in
preliminary tests, selenite brilliant green
sulfa broth (SBGS) at 41.5°C gave the
best isolation of Salmonella sp. from waste-
waters (with and without the addition of
S. typhimurium in known numbers). Of
thirteen wastewater samples tested in SBGS
at 41.5°C, six contained Salmonella or 46
percent were positive. With DSE broth at
40°C, 28 of 28, or 100 percent of waste-
water samples, gave positive results.
Studies were not continued on SBGS me-
dium when it was noted that some lots of
commercially available SBGS seemed to be
selective for Salmonella sp. while others
were not. The medium was then pre-
pared according to the original formula16
with six different lots of brilliant green
(certified), only one of which was selec-
tive. The use of brilliant green agar as
a selective medium is subject to the same
variability, according to Read and Reyes.17
The main reasons for rejection of XT,
with and without brilliant green, and for
selenite broth's using brilliant green agar
. and XLD agar as secondary media are not
only fewer isolations of Salmonella sp., but
also the poor selectivity of these combina-
tions when they are used for monitoring
polluted waters. These combinations' poor
selectivity at 41.5°C is apparent in the
results of Dutka and Bell,18 where the TT
broth-xLD combination yielded 26 percent
confirmation of colonial picks, and selenite
broth-EGA and selenite broth-XLD gave 55
and 56 percent confirmations, respectively.
The authors had similar results. The GN-
XLD combination was poorest for water
samples at 40° and 41.5°C, yielding less
than 10 percent isolations from waste-
waters.
Effect of incubation temperature on iso-
lation of Salmonella sp. In a study of
26 wastewater samples that was conducted
with the DSE multiple tube setups at three
Vol. 46, No. 9, September 1974 - 2167
111
-------
KENNER AND CLARK
TABLE IV. — Serotypes of Salmonella
Found in
Polluted Waters
Serotype
1 lyphimitriuni*
2 derby
3 cabana
4 chtftcr
5 H«?P0rt
6 kallbui
7 bSoeklfy
8 itifanlii
v eHterititiiS
10 Quotum
11 keiilflbtm
12 ui<»iJiii<(ii«
13 paratyptti B
14 iHiiioij
15 thniafson
16 litiitffstmie
17 nioiifiFii/fo
18 mueitfhen
19 oraaienberg
20 TQM tiifffO
21 6
M)
id
•iy
35
39
27
40
.
* Rank in human occurrence Table I, Martin
Rank in
Human
Occurrence*
1 and 6f
12
20
19
4
,; ';, — t
9
e
13
6
14
17
4
16
18
15
18
23
~j
25
~%
24
— t
— t "
' T
__f
23
— t
__
22
— t
f
— |
7
8
21
2S*
J
i
— t
10
__*
20
_
— t
24
and Kwing.13
t Jro|inr,ulon of .V. lyphimuriiim and var. Copenhagen not
done alter initial identification*.
(ScroiypM occurring in humane. 1965-1971. Center for
Disease Control, Salmonella Survi-illance. Annual Summary
1971. Table IX. U. S. DUliW, PUS DIIEW Publ. N'o. (HSM)
73-8184 (Oct. 1972).
21 GS Journal V\
7PCF
different temperatures, it was found that
100 percent of the samples contained Sal-
monella sp. and Ps. aeruginosa at 40°C.
At 41.5° C, however, only 50 percent or 13
of the samples yielded Salmonella sp., and
at 37 °C only 8 percent or 2 of the samples
yielded Salmonella sp.
Effect of enhancement of DSE broth with
a killed culture of S. paratyphi A. In a
study of 84 samples of activated sludge
effluents, trickling filter effluents, package
plant effluents, and stream waters, DSE
broth enhanced with a. killed culture of
S. paratyphi A in DSE broth (10 percent
by volume) yielded isolations in 64 sam-
ples or 74 percent isolated Salmonella sp.,
compared with 48 samples or 57 percent
isolations when the DSE broth was used
without enhancement. An improved iso-
lation of 17 percent was achieved with
enhanced DSE broth.
Ultra-filter. The advantages of ultra-
filter use in testing water samples are illus-
trated in Table II.
RESULTS AND DISCUSSION
Of importance to those who must use
bacteriological tests to obtain Salmonella
sp. and Ps. aeruginosa counts from waters
is the amount of work that must be done
to secure accurate results. Table III pre-
sents the percentage of colony picks made
with the described method that proved to.
be Salmonella sp. If there are black-
centered colonies on the XLD plates, more
than 75 percent of the picks will prove to
be Salmonella sp.; thus, the method leads
to less unproductive work. When other
methods were used, the authors have at
times had to pick 50 black-centered col-
onies to obtain only 5 Salmonella sp.
strains. This type of unproductive work
has given the search for pathogens in the
environment an undeserved bad reputation,
and it has caused some to give up.
In Table II it may readily be seen that
in many cases the fault with many tests
has been the testing of an insufficient vol-
ume of sample. Many people think that
it involves too much work, and that only
expensive fluorescent antibody techniques
will work. The problem is, however, to
112
-------
PATHOGEN DETECTION
TABLE V.—Percentage of Various Types of Water Samples Positive for Salmonella species
Type of Sample
Municipal wastewaters
Municipal primary effluents
(chlorinated)
Activated sludge effluents (clarified)
Activated sludge effluents
Before chlorination
After chlorination, 1.4-2.0 mg/1
residual, 5 min contact
Trickling filter effluents
Package plant effluents
Creek 1 mile (1.6 km) below package
plant
Ohio River above Cincinnati public
landing
\Vabash River
Mississippi River
Streams collective
Stormwater runoff after heavy rain
Farm wells
Home cisterns suburban
Septic tank sludges
Totals
Number of
Samples
28
9
40
5
8
26
15
3
20
4
4
31
6
. 4
5
6
183
Number
Positive
28
5
29
4
0
15
7
3
9
3
3
18
3t
0
2
3
114
Number
Negative
0
4
11
1
8
11
8
0
11
1
1*
13
3t
4
3
3
69
Percentage
Positives
100.0
56.0
72.5
80.0
0.0
57.7
46.7
100.0
45.0
75.0
75.0
58.0
50.0
0.0
40.0
50.0
* Municipal intake.
f Positive by per-gallon technique.
t Negative by per-100 ml technique.
concentrate the bacteria in a 10-gal (38-1)
sample or a 100-gal (380-1) sample of
potable or reuse water to obtain results,
and still not require even more expensive
filtration or centrifugation equipment. It
also seems unrealistic to test only extremely
small samples of the water being examined,
because they may not be representative.
Table IV contains a list of Salmonella
serotypes isolated from polluted waters and
ranked according to the frequency of sero-
type isolations. It will be noted that all
of the serotypes except S. typhi were iso-
lated from environmental samples by the
monitoring method, and that only 6 of the
65 serotypes reported were not reported
as occurring in humans in the U. S. over
the period from 1965 to 1971.
Table V summarizes the percentage of
various types of water samples positive
for Salmonella sp. Of interest is the fact
that 100 percent of the municipal waste-
waters tested contain Salmonella sp., that
56 percent of chlorinated primary effluents
tested contain the pathogens, and that 100
percent of chlorinated secondary effluents
are negative for pathogens. There are
more studies scheduled for testing of sec-
ondary and tertiary effluents to obtain
minimal chlorine residuals. Calabro et
al.20 reported that more than 50 attempts
at isolating Salmonella sp. from septic tank
samples using SBGS-BGSA combinations were
unsuccessful.
Table VI summarizes the isolation of
Ps. aeruginosa from potable water supply,
that is, wells, cisterns, and small municipal
water supply. It should be noted that
fecal coliforms were not detected in most
of these samples. Fecal streptococci counts
were higher than fecal coliform counts
where both tests were used. Ps. aeruginosa
were present in all but three of the tests,
and Salmonella sp. were isolated from
two different cistern samples.
It is of importance to the user of patho-
gen tests that the test be quantitative. In
initial studies on the DSE-XLD combination,
it was important to know if the enrichment
broth would support the growth of a wide
Vol. 46, No. 9, September 1974 2169
113
-------
KENNEB AND CLABK
TABLE VI.—Isolation of Pseudomonas aeruginosa from Potable Water Supply
Type of Sample
Well 8/16/71
Well 8/25/71
Well 3/27/72
Well 3/27/72
(chlorinated)
Well 8/23/72
Well 10/ 4/72
Suburban cisterns
8/ 4/72
10/ 9/72*
ll/ 6/72*
ll/ 6/72
11/26/72
Municipal supplies
Population served 54,700
3/17/71
6/21/71
7/19/71
6/19/72
10/ 9/72
5/ 8/72
Population served 14,000
5/ 8/72
10/24/72
Population served < 10,000
11/27/72
Ps. aeruginosa
Isolation •
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
0
+
0
Indicators/100 ml
Total
Conforms
4
22
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
~
Fecal
Coliforms
—
—
<1
<1
0.25
<2
180
15
<2
<2
3
<1
<1
<1
0.26
<1
<1
<1
<1
<1
Fecal
Streptococci
—
—
—
— •
62
46
—
156
22
2
28
—
—
—
—
<1
18
<1
* Salmonella sp. also present in samples.
range of Salmonella serotypes. Laboratory
cultures of S. paratijphi A, S. typhimurium,
S. bredeney, S. oranienberg, S. pullorum, S.
anatum, S. give, and S. worthington were
tested in three enrichment broths. The
time required to isolate each of the above
cultures from an estimated 10 to 20 orga-
nisms/100 ml in buffer water was 48 to 72
hr for S. paratijphi A in TT broth, 24 hr for
DSE broth, and 36 to 48 hr for SBGS broth.
The rest of the cultures were isolated in es-
timated numbers in 14 to 24 hr in TT and
DSE broths. In SBGS broth, S. typhimurium,
S. bredeney, S. anatum, S. give, and S.
worthington required 36 to 48 hr incuba-
tion, and S. pullorum and S. oranienberg
required 48 to 72 hr incubation.
It is impossible to know if 100 percent
of Salmonella sp. in a polluted water
sample are isolated. In tests where lab-
2170 Journal WPCF
oratory cultures have been added in low
numbers to wastewater and treatment
effluent samples, all of the numbers added
were detected, as well as the Salmonella
sp. that were naturally occurring. The
higher the quality of the water (for ex-
ample, secondary or tertiary treatment
effluent, or even potable waters), the better
the possibility of isolation of all the Salmo-
nella serotypes present, as well as Ps.
aeruginosa, a potential pathogen.
SUMMARY
A practical laboratory method is pre-
sented for the simultaneous isolation and
enumeration of Salmonella sp. and Pseudo-
monas aeruginosa from all classes of waters,
including potable water supplies, with a
minimum of interfering false positive iso-
lations. The method allows for the testing
114
-------
PATHOGEN DETECTION
of large volumes of high quality waters,
wherein the absence of indicator bacteria
(that is, total coliforms, fecal coliforms,
and fecal streptococci), may give a false
sense of security because of the low
volumes of water usually tested. Justifica-
tion for each step of the procedural method
is presented.
ACKNOWLEDGMENTS
Credits. The technical assistance of
Pauline C. Haley in performing the neces-
sary serology for identifying many of the
Salmonella serotypes reported is gratefully
acknowledged.
Authors. Bernard A. Kenner is super-
visory research microbiologist, and Harold
P. Clark is biological technician, Waste
Identification and Analysis Activity of the
Advanced Waste Treatment Research Lab-
oratory, Natonal Environmental Research
Center, U. S. Environmental Protection
Agency, Cincinnati, Ohio.
REFERENCES
1. Federal Water Pollution Control Amendments,
PL 92-500, 86 Stat. 816, 33 U. S. Code
Sec. 1151 at seq. (1972).
2. FWPCA, Section 504 as amended (1972).
3. FWPCA, Section 307 (a) (1972).
4. FWPCA, Section 311 (1972).
5. Quality Assurance Division, Office of Research
and Monitoring, U. S. EPA, "Proc. 1st
Microbiology Seminar on Standardization
of Methods." EPA-R4-73-022 (Mar. 1973).
6. Kenner, B. A., et al., "Simultaneous Quantita-
tion of Salmonella species and Pseudomonas
aeruginosa. I. Polluted Waters. II. Persist-
ence of Pathogens in Sludge Treated Soils.
III. Analysis of Waste Treatment Sludges
for Salmonella species as a Surveillance
Tool." U. S. EPA, National Environ-
mental Research Center, Cincinnati, Ohio
(Sept. 1971).
7. Moody, M. R., et al., "Pseudomonas aeruginosa
in a Center for Cancer Research. I. Dis-
tribution of Intraspecies Types from
Human and Environmental Sources." Jour.
Inf. Diseases, 125, 95 (1972).
8. Edmonds, P., et al., "Epidemiology of Pseudo-
monas aeruginosa in a Burns Hospital. Sur-
veillance by a Combined Typing System."
Appl. Microbiol, 24, 219 (1972).
9. "New Hospital Controls Urged to Stem
Pseudomonas Rise." Clin. Lab. Forum
(Eli Lilly), 2, 1 (May-June 1970).
10. Reitler, R., and Seligmann, R., "Pseudo-
monas aeruginosa in Drinking Water."
Jour. Appl. Bacterial., 20, 145 (1957).
11. Ringen, L. M., and Drake, C. H., "A Study
of the Incidence of Pseudomonas aeruginosa
from Various Natural Sources." Jour.
Bacterial., 64, 841 (1952).
12. Drake, C. H., "Evaluation of Culture Media
for the Isolation and Enumeration of
Pseudomonas aeruginosa." Health Lab.
Set., 3, 10 (1966).
13. Raj, H., "Enrichment Medium for Selection
of Salmonella from Fish Homogenate."
^ Appl. Microbiol, 14, 12 (1966).
14. "Standard Methods for the Examination of
Water and Wastewater." 13th Ed., Amer.
Pub. Health Assn., New York, N. Y. (1971).
15. Taylor, W. I., "Isolation of Shigellae. I.
Xylose Lysine Agars; New Media for Iso-
lation of Enteric Pathogens." Tech. Butt.
Reg. Med. Technol, 35, 161 (1965).
16. Osborne, W. W., and Stokes, J. L., "A Modi-
fied Selenite Brilliant-Green Medium for
the Isolation of Salmonella from Egg Prod-
ucts." Appl. Microbiol., 3, 295 (1955).
17. Read, R. B., Jr., and Reyes, A. L., "Variation
in Plating Efficiency of Salmonellae on
Eight Lots of Brilliant Green Agar." Appl.
Microbiol, 16, 746 (1968).
18. Dutka, B. J., and Bell, J. B., "Isolation of
Salmonellae from Moderately Polluted
Waters." Jour. Water Poll. Control Fed.,
45, 316 (1973).
19. Martin, W. J., and Ewing, W. H., "Prevalence
of Serotypes of Salmonella." Appl. Micro-
biol, 17, 111 (1969).
20. Calabro, J. F., et al." Recovery of Gram Nega-
tive Bacteria with Hektoen Agar." Jour.
Water Poll. Control Fed., 44, 491 (1972).
-Vol. 46, No. 9, September 1974 2171
115
-------
-------
Appendix H
Method for the Recovery and Assay of Enteroviruses from Sewage Sludge
I. INTRODUCTION
The Class A requirements of 40 CFR Part 503 can be met on the basis of several alternatives which specify
the final densities of pathogenic organisms after a pathogen reduction process or in sewage sludge at the time of
distribution. Where required under Part 503 (see Chapter 4), human enteric viruses (i.e., viruses that are
transmitted via the fecal-oral route) must be less than 1 plaque-forming unit (PFU) per 4 g of total dry solids.
The method required to demonstrate this virus density is described below.
Chapters 7 and 8 of this document describe the quantitative and sampling criteria. In some cases the
collection of four or more composite samples is recommended. A composite sample should be prepared by
collecting 10 representative samples of 60 mL each (600 mL total) from different locations of a batch sludge
pile or on different days over a period of several weeks when testing a process sludge sample. Batch samples
that cannot be assayed within 8 hours of collection must be frozen; otherwise, they should be held at 4°C until
processed. Just prior to assay, frozen samples for each composite are thawed, combined, and mixed
thoroughly. A 50 mL portion is removed from each sample for solids determination as described in Section n
below. The remaining portion is held at 4°C while the solids determination is being performed, or frozen for
later processing if the assay cannot be initiated within 8 hours.
The "enteric viruses" specified for testing in the Part 503 regulation consist of more than 100 virus types,
with hepatitis A virus and Norwalk virus being the primary human viral pathogens of concern. At the present
time, however, standard methods for isolating and detecting these and many other enteric viruses have not been
developed. The method detailed in Section TTT below has been tested and approved for isolating the enterovirus
group (e.g., polioviruses, coxsackieviruses, echoviruses) of enteric viruses.1 This method and the standard
procedures for virus quantitation which follow it should be rigorously followed. Enteroviruses are assayed
using a BGM cell line (Section IV) and the plaque assay technique (Section V). Occasionally, components
isolated from sludges along with enterovirus may show cytotoxic effects on BGM cells, leading to false negative
results. Samples showing cytotoxicity should be reassayed using the method given in Section VI. Cytotoxic
and other components of sludge can also produce false positive results, requiring that all potential viral isolates
be confirmed as infectious virus. A method to confirm virus isolates is given hi Section VII.
Aseptic techniques and sterile materials and apparatus are to be used throughout all sections of the virus
procedure described below. Virus-contaminated materials must be sterilized by autoclaving at 121 °C for 15
minutes before discarding.
n. DETERMINATION OF TOTAL DRY SOLIDS2
1. Weigh a dry weighing pan that has been held in a desiccator and is at a constant weight. Place the 50 mL
sludge portion for solids determination into the pan and weigh again.
2. Place the pan and its contents into an oven maintained at 103-105°C for at least 1 hour.
3. Cool the sample to room temperature in a desiccator and weigh again.
'Method D4994-89, ASTM (1992).
2Modified from EPA/600/4-84/013(R7), September 1989 Revision (Section 3). This and other cited EPA
publications may be requested from the Virology Branch, Environmental Monitoring Systems Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio 45268.
117
-------
4. Repeat the drying (1 hour each), cooling, and weighing steps until the loss in weight is no more than 4% of
the previous weight.
5. Calculate the fraction of total dry solids (T) using the formula: T = (A-C)/(B-C), where A is the weight of
the sample and dish after drying, B is the weight of the sample and dish before drying, and C is the weight of
the dish. Record the fraction of dry solids (T) as a decimal (e.g., 0.04).
HI. ENTEROVBRUS RECOVERY FROM SLUDGES3
1. INTRODUCTION
Enteroviruses in sludge will primarily be associated with solids. Although the fraction of virus associated
with the liquid portion will usually be small, this fraction may vary considerably with different sludge types.
To correct for this variation, samples will first be treated to bind free virus to solids. Virus is then eluted from
the solids and concentrated prior to assay.
The procedures in this section require dispensing the entire sample volume into a centrifuge bottle. If
bottles of sufficient capacity are unavailable, the sample should be divided and then recombined after
centrifugation.
2. CONDITIONING OF SUSPENDED SOLIDS
Conditioning of sludges binds unadsorbed enterovlruses present in the liquid matrix to the sludge solids.
2.1 Preparation
2.1.1 Apparatus and materials
(a) Refrigerated centrifuge capable of attaining 10,000 x g and screw-capped centrifuge bottles with 100 to
1,000 mL capacity.
Each bottle must be rated for the relevant centrifugal force.
(b) A pH meter with an accuracy of at least 0.1 pH unit, equipped with a combination-type electrode.
(c) Magnetic stirrer and stir bars.
2.1.2 Media and reagents
Analytical reagent or ACS grade chemicals (unless specified otherwise) and deionized, distilled water
(ddHyO) should be used to prepare all reagents. All water used must have a resistance of greater than 0.5
megohms-cm, but water with a resistance of 18 megohms-cm is preferred.
(a) Hydrochloric acid (HC1) — 1 and 5 M.
Mix 10 or 50 mL of concentrated HCl with 90 or 50 mL ofddH2O, respectively.
(b) Aluminum chloride (A1C13 • 6tLff) — 0.05 M.
Dissolve 12.07 g of aluminum chloride in a final volume of 1,000 mL ofddHfl. Autoclave at 121°C
for 15 minutes.
(c) Sodium hydroxide (NaOH) — 1 and 5 M.
Dissolve 4 or 20 g of sodium hydroxide in a final volume of 100 mL ofddHf), respectively.
3Modified from EPA/600/4-84/013(R7), September 1989 Revision.
118
-------
2.2 Conditioning procedure — Each analyzed composite sample (from the portion remaining after solids
determination) must have an initial total dry solids content of at least 12 g. This amount is necessary to allow
storage of one half of the sample at -70°C as a backup in case of procedural mistakes or sample cytotoxicity
(see Section VI) and to use a portion of each sample for a positive control.
Figure 1 gives a flow diagram for the procedure to condition suspended solids.
Calculate the amount of sample needed from the formula: X = 12/T, where X = the milliliters of sample
required to obtain 12 g, and T =• the fraction of total dry solids (from Section II).4 Use a graduated cylinder
to measure the volume. IfXis not a multiple of 100 mL (100, 200, 300 mL, etc.), sterile water should be
added to bring the volume to the next multiple of 100 mL. Each sample should then be aliquoted into 100 mL
portions before proceeding. Samples must be mixed vigorously just before aliquoting because solids begin to
settle out as soon as the mixing stops. Each aliquot should be placed into a 250 mL beaker containing a stir
bar.
CAUTION: Always avoid the formation of aerosols by slowly pouring samples down the sides of vessels.
2.2.1 The following steps must be performed on each 100 mL aliquot.
2.2.2 Place the beaker on a magnetic stirrer, cover loosely with aluminum foil, and stir at a speed sufficient to
develop a vortex. Add 1 mL of 0.05 M A1C13 to the mixing aliquot.
The final concentration ofAlCl3 in each aliquot is approximately 0.0005 M.
2.2.3 Place a combination-type pH electrode into the mixing aliquot. Adjust the pH of the aliquot to 3.5 +
0.1 with 5 M HC1. Continue mixing for 30 minutes.
The pH meter must be standardized at pH 7 and 4. When solids adhere to electrodes, clean electrodes by
moving them up and down gently in the mixing aliquot.
The pH of the aliquot should be checked at frequent intervals. If the pH drifts up, readjust it to 3.5 ± 0.1
with 5 M HCl. If the pH drifts down, readjust it with 5 M NaOH. Use 1 M add or base for small adjustments.
Do not allow the pH to drop below 3.4.
2.2.4 Pour the conditioned aliquot into a centrifuge bottle and centrifuge at 2,500 x g for 15 minutes at 4°C.
To prevent the transfer of the stir bar into the centrifuge bottle when decanting the aliquot, hold another stir
bar or magnet against the bottom of the beaker. Solids that adhere to the stir bar in the beaker may be removed
by manipulation with a pipette. It may be necessary to pour the aliquot back and forth several times from the
centrifuge bottle to the beaker to obtain all the solids in the bottle.
2.2.5 Decant the supernatant into a beaker and discard. Replace the cap onto the centrifuge bottle. Elute
viruses from the solids by following the procedure described below under "Elution of Viruses from Solids."
3. ELUTION OF VIRUSES FROM SOLIDS
3.1 Apparatus and materials
In this and following sections only apparatus and materials which have not been described in previous
sections are listed.
3.1.1 Membrane filter apparatus for sterilization — 47 mm diameter Swinnex filter holder and 50 mL slip-tip
syringe (Millipore Corp., product no. SXOO 047 00, and Becton Dickinson, product no. 1627, or equivalent for
filter holder only).
4This formula is based upon a reliable assumption that the density of the liquid in sludge is 1 g/mL. Only 550
mL of the original sample is available for analysis at this point. If the fraction of total dry solids is below 0.022,
550 mL will not be sufficient and the amount of initial sample collected will have to be increased to equal the
volume of X + 50 mL.
119
-------
SUSPENDED SOODS (per 100 mL)
Mix suspension on magnetic stirrer.
Add 1 mL of 0.05 M A1C13.
V
SALTED SOODS SUSPENSION
Continue mixing suspension.
Adjust pH of salted suspension
to 3.5 ±0.1with5MHCl.
Mix vigorously for 30 minutes.
pH-ADJUSTED SOLIDS SUSPENSION
Centrifuge salted, pH-adjusted
suspension at 2,500 x g for
15 minutes at 4°C.
Discard supernatant.
Retain solids.
SOLIDS
Figure 1. Flow Diagram of Method for Conditioning Suspended Solids
3.1.2 Disc filters, 47 mm diameter — 3.0, 0.45, and 0.2 /tm pore size filters (Mentec America, Filterite Div.,
Duo-Fine series (product nos. 8025-030, 8025-034, and 8025-037, or equivalent). Filters may be cut to the
proper diameter from sheet filters.
Disassemble a Swinnex filter holder. Place the filter with a 0.25 \un pore size on the support screen of the
filter holder and stack the remaining filters on top in order of increasing pore size. Reassemble and tighten
filter holder. Wrap filter stack in foil and sterilize by autoclaving at 121 °Cfor 15 min.
Filters stacked in tandem as described tend to clog more slowly when turbid material is filtered through
them. Prepare several filter stacks.
3.2 Media and Reagents
In this and following sections only media and reagents -which have not been described in previous sections
are listed.
3.2.1 Beef extract powder (BBL Microbiology Systems, product no. 12303, or equivalent).
Prepare buffered 10% beef extract by dissolving 10 g beef extract powder, 1.34 g NaJHPO^ • 7H2O and
0.12 g citric acid in 100 mL ofddH2O. The pH should be about 7.0. Dissolve by stirring on a magnetic
stirrer. Autoclave for 15 minutes at 121° C.
Do not use paste beef extract (Difco Laboratories, product no. 0126) or beef extract V powder (BBL
Microbiology Systems, product no. 97531) for virus elution. These beef extracts may elute cytotoxic materials
from sludges.
3.3 Elution Procedure
A flow diagram of the virus elution procedure is given in Figure 2.
3.3.1 Place a stir bar and 100 mL of buffered 10% beef extract into the centrifuge bottle containing the solids
(from Subsection 2.2.5). If more than one aliquot per sludge sample was processed, the solids should be
combined at this step.
120
-------
3.3.2 Place the centrifuge bottle on a magnetic stirrer, and stir at a speed sufficient to develop a vortex for 30
minutes at room temperature.
To minimize foaming (which may inactivate viruses), do not mix faster than necessary to develop a vortex.
3.3.3 Remove the stir bar from the bottle with a long forceps or a magnet retriever and centrifuge the
solids-eluate mixture at 10,000 x g for 30 minutes at 4°C. Decant supernatant fluid (eluate) into a beaker and
discard the solids.
Determine if the centrifuge bottle is appropriate for the centrifugal force that will be applied.
Centrifugation at 10,000 x g is normally required to clarify the sludge samples sufficiently to force the
resulting supernatant through the filter stacks. It may be possible to use 2,500 x gfor some samples.
3.3.4 Place a filter holder that contains a filter stack (from Subsection 3.1.2) onto a 250 mL Erlenmeyer
receiving flask. Load a 50 mL syringe with supernatant from Step 3.3.3. Place the tip of the syringe into the
filter holder and force the supernatant through the filter stack into a 250 mL receiving flask.
Take care not to break off the tip of the syringe and to minimize pressure on the receiving flask because
such pressure may crack or topple the flask. If the filter stack begins to clog badly, empty the loaded syringe
into the beaker containing unfiltered eluate, fill the syringe with air, and inject air into the filter stack to force
residual eluate from the filters. Continue the filtration procedure with another filter holder and filter stack.
Discard contaminated filter holders and filter stacks. Step 3.3.4 may be repeated as often as necessary to filter
the entire volume of supernatant. Disassemble each filter holder and examine the bottom 0.25 pm filters to be
certain they have not ruptured. If a bottom filter has ruptured, repeat Steps 3.3.4 with new filter holders and
filter stacks.
Proceed immediately to Subsection 4.
4. CONCENTRATION OF VIRUSES FROM ELUATES BY ORGANIC FLOCCULATION
This organic flocculation concentration procedure (Katzenelson et al., 1976) is used to reduce the number of
cell cultures needed for assays by concentrating enteroviruses in the eluate. Although the overall virus recovery
may be diminished by this step, organic flocculation will result in considerable cost reductions in labor and
materials.
Floe formation capacity of the powdered beef extract reagent must be pretested. Some powdered beef
extracts may not produce sufficient floe, resulting in significantly reduced virus recoveries. Beef extract
reagents may be pretested by spiking 100 mL of ddH2O with a known amount of virus in the presence of a 47
mm nitrocellulose filter. This sample should be conditioned using Subsection 2 above to bind virus to the filter.
Virus should then be elutedfrom the filter using the procedure in Subsection 3, and concentrated using the
following procedure. Any lot of beef extract not giving a overall recovery of at least 50% should be discarded
or supplemented with floe from paste beef extract or beef extract V. The procedure for the preparation of the
additional floe is described in Subsection 4.1.3. The overall recovery of virus should also be at least 50% when
supplementation is required.
4.1 Media and Reagents
4.1.1 Sodium phosphate, dibasic (Na-jHPC^ • 7H2O) — 0.15 M.
Dissolve 40.2 g of sodium phosphate in a final volume of 1,000 mL. Autoclave at 121 °Cfor 15 minutes.
4.1.2 Paste beef extract (Difco Laboratories, product no. 0126, or equivalent) or beef extract V (BBL
Microbiology Systems, product no. 97531, or equivalent) — 3%.
Prepare a 3% paste beef extract or beef extract V stock solution by dissolving 30 g of. beef extract in 1,000
mL ddH2O. Autoclave the stock solution at 121° C for 15 minutes and use at room temperature. From this
stock solution, one 330 mL aliquot is removed for each sample requiring supplementation with beef extract floe.
Although the stock solution may be stored at 4°Cfor an extended time period, it is advisable to prepare the
solutions on a weekly basis, thereby lessening the possibility ofmicrobial contamination.
121
-------
SOLIDS
Add 100 mL of buffered 10% beef extract, adjust
to pH 7.0 + 0.1 if necessary.
Mix resuspended solids on magnetic stirrer for
30 minutes to elute viruses.
RESUSPENDED SOLIDS
Centrifuge resuspended solids for 30 minutes
at 4°C using a centrifugal force of 10,000 x g
Discard solids.
Retain eluate (supernatant).
ELUATE
Filter eluate through 47 mm Filterite filter
stack of 3.0, 0.45, and 0.25 /on pore sizes
with the 0.25 jtm pore size on support screen
of filter and remaining filters on top hi
order of increasing pore size.
FILTERED ELUATE
Figure 2. Flow Diagram of Method for Elution of Virus from Solids
4.1.3 Preparation of Floe from 3 % Beef Extract Reagent
A flow diagram for the procedure to prepare floe is given in Figure 3.
(a) Place a stir bar and 330 mL of the 3 % beef extract stock solution into a 600 mT, beaker and cover
loosely with aluminum foil. Place the beaker onto a magnetic stirrer, and stir at a speed sufficient to
develop a vortex.
(b) Insert a combination-type pH electrode into the beef extract stock solution. Add 1 M HC1 to flask
slowly until the pH of beef extract reaches 3.5 + 0.1.
The pH meter must be standardized at pH 4 and 7.
A precipitate will form.
(c) Remove the electrode and pour the contents of the beaker into a 1,000 mL centrifuge bottle. Centri-
fuge the precipitated beef extract suspensions at 2,500 x g for 15 minutes at 4°C. Pour off and
discard the supernatant.
To prevent the transfer of the stir bar into the centrifuge bottle, hold another stir bar or magnet
against the bottom of the beaker while decanting the contents.
(d) Retain the floe in the centrifuge bottle at 4°C for subsequent mixing with the non-flocculating buffered
beef extract (Subsection 4.2.4).
4.2 Virus Concentration Procedure
A flow diagram for the virus concentration procedure is given in Figure 4.
122
-------
3 % BEEF EXTRACT REAGENT
Autoclave at 121 °C for 15 minutes (if
stored, cool and hold at 4°C).
Use at room temperature.
V
STERILE:
BEEF EXTRACT STOCK
Add a 330 ml, portion of the beef extract
stock to a beaker containing a stir bar.
Place the beaker onto a magnetic stirrer and mix.
Adjust the pH of the beef extract stock to 3.5 +0.1
with 1 M HC1.
Continue mixing for 30 minutes.
FLOCCULATED BEEF EXTRACT
Centrifuge flocculated beef extract at
2,500 x g for 15 minutes at 4°C.
Discard supernatant.
Retain floe.
BEEF EXTRACT FLOG
Figure 3. Flow Diagram of Method for Preparation of Floe from Beef Extract Reagent
4.2.1 Pour the filtered eluate (from Subsection 3.3.4) into a graduated cylinder, and record the volume.
Transfer into a 60O mL beaker and cover loosely with aluminum foil.
4.2.2 For every 3 mL of beef extract eluate, add 7 mL of ddH2O to the 600 mL beaker.
The concentration of beef extract is now 3%. This dilution is necessary because 10% beef extract often
does not process well by the organic flocculation concentration procedure.
4.2.3 Record the total volume of the diluted eluate.
Proceed directly to Subsection 4.2.5 only if the powdered beef extract reagent used for the virus elution
process (in Subsection 3) is known to form sufficient floe to efficiently concentrate virus without the additional
floe. Where additional floe is required, add the diluted eluate to the floe as described in Subsection 4.2.4.
4.2.4 Pour the eluate from the beaker into the centrifuge bottle containing floe from Subsection 4.1.3(d).
Disperse the floe manually using a pipette until it is dissolved in the eluate and then transfer the eluate/fioc
mixture into a 600 mT. beaker.
4.2.5 Place a stir bar into the beaker that contains the diluted, filtered beef extract. Place the beaker on a
magnetic stirrer, cover loosely with aluminum foil, and stir at a speed sufficient to develop a vortex.
To minimize foaming (which may inactivate viruses), do not mix faster than necessary to develop a vortex.
123
-------
FILTERED ELUATE
Add sufficient volume of .ddRjO to
filtered eluate to reduce concentration of beef
extract from 10% to 3%. Record total volume
of the diluted beef extract.
Add the diluted eluate to the previously prepared
floe (see Figure 3), if the powdered beef
extract reagent has been determined to produce
insufficient floe when processed by the organic
flocculation procedure. Disperse manually using
a pipette until the floe is dissolved.
\J
DILUTED, FILTERED ELUATE
Mix diluted eluate on a magnetic stirrer.
Adjust the pH of the eluate to 3.5 ±0.1
with 1M HC1. A precipitate (floe) will form.
Continue mixing for 30 minutes.
FLOCCULATED ELUATE
Centrifuge flocculated eluate at 2,500 x g for
15 minutes at 4°C.
Discard supernatant.
Retain floe.
FLOG FROM ELUATE
Add 0.15 M Na2HPO4 to floe, using l/20th of the
recorded volume of the diluted 3 % beef extract.
Mix suspended floe on magnetic stirrer until floe
dissolves.
Adjust to a pH of 7.0-7.5.
DISSOLVED FLOC
See Section V for virus assay procedure.
ASSAY DISSOLVED FLOC FOR VIRUSES
Figure 4. Flow Diagram of Method for Concentration of Viruses from Beef Extract Eluate
4.2.6 Insert a combination-type pH electrode into the diluted, filtered beef extract. Add 1 M HC1 to the flask
slowly until the pH of the beef extract reaches 3.5 ±0.1. Continue to stir for 30 minutes at room temperature.
Tlie pH meter must be standardized at pH 4 and 7.
A precipitate will form. If the pH is accidentally reduced below 3.4, add 1 M NaOH until it reaches 3.5 ±
0.1. Avoid reducing the pH below 3.4 because some inactivation of virus may occur.
124
-------
4.2.7 Remove the electrode from the beaker, and pour the contents of the beaker into a 1,000 ml. centrifuge
bottle. Centrifuge the precipitated beef extract suspensions at 2,500 x g for. 15 minutes at 4°C. Pour off and
discard the supernatant.
To prevent the transfer of the stir bar into a centrifuge bottle, hold another stir bar or magnet against the
bottom of the beaker when decanting contents.
4.2.8 Place a stir bar into the centrifuge bottle that contains the precipitate. Add a volume of 0.15 M
Na2HPO4 • 7H2O equal to exactly 1/20 of the volume recorded in Subsection 4.2.3. Place the bottle onto a
magnetic stirrer, and stir slowly until the precipitate has dissolved completely.
Support the bottle as necessary to prevent toppling. Avoid foaming, which may inactivate or aerosolize
viruses. The precipitate may be partially dissipated with a spatula before or during the stirring procedure.
4.2.9 Measure the pH of the dissolved precipitate.
If the pH is above or below 7.0-7.5, adjust to that range with either 1 M HCl or 1 M NaOH.
4.2.10 Freeze exactly one half of the dissolved precipitate sample at -70°C. This sample will be held as a
backup to use should the sample prove to be cytotoxic (see Section VI). Record the remaining sample volume
(this volume represents 6 g of total dry solids). Refrigerate the sample immediately at 4°C and maintain at that
temperature until it is assayed in accordance with the instructions given in Section V below.
If the virus assay cannot be undertaken within 8 hours, store the remaining sample at -70° C.
IV. CELL CULTURE PREPARATION AND MAINTENANCE5
1. INTRODUCTION
This section outlines procedures and media for culturing the Buffalo green monkey (BGM) cell line and is
intended for the individual who is experienced in cell culture preparation. BGM cells are a continuous cell line
derived from African green monkey kidney cells. The characteristics of this line were described by Barron et
al. (1970). Use of BGM cells for recovering viruses from environmental samples was described by Dahling et
al. (1974). The media and methods recommended in Sections M and IV are the results of the BGM cell line
optimization studies by Dahling and Wright (1986). The BGM cell line can be obtained by qualified
laboratories from the Virology Branch, Environmental Monitoring Systems Laboratory, U.S. Environmental
Protection Agency, Cincinnati, Ohio 45268.
BGM cells are highly susceptible to many enteric viruses (Dahling et al,, 1984; Dahling and Wright, 1986);
however, these cells are not sensitive for detecting all enteric viruses that may be present in environmental
samples. The use of several cell lines would be required to maximize the number of viruses recovered from
environmental samples. Since it is difficult to specify the type of cell lines which would be best for a particular
sludge sample, the use of the BGM cell line only will be sufficient to meet the requirements of the 40 CFR Part
503 regulation.
2. MEDIUM PREPARATION
2.1 Apparatus and Materials
2.1.1 Glassware, Pyrex (Corning, product no. 1395, or equivalent).
Storage vessels must be equipped with airtight closures.
2.1.2 Autoclavable inner-braided tubing with metal quick-disconnect connectors or with screw clamps for
connecting tubing to equipment to be used under pressure.
Quick-disconnect connectors can be used only after equipment has been properly adapted.
5Modified from EPA/600/4-84/013(R9), January 1987 Revision.
125
-------
2.1.3 Positive pressure air, nitrogen or 5% -CO2 source equipped with pressure gauge.
Pressure sources from laboratory air lines and pumps must be equipped with an oil filter. The source must
not deliver more pressure to the pressure vessel than is recommended by the manufacturer.
2.1.4 Dispensing pressure vessel — 5 or 20 L capacity (Millipore Corp., product nos. XX67 OOP 05 and
XX67 OOP 20, or equivalent).
2.1.5 Disc filter holders — 142 mm or 293 mm diameter (Millipore Corp., product nos. YY30 142 36 and
YY30 293 16, or equivalent).
Use only pressure-type filter holders.
2.1.6 Sterilizing filter stacks — 0.22 ^m pore size (Millipore Corp., product nos. GSWP 142 50 and GSWP
293 25, or equivalent). Fiberglass prefilters (Millipore Corp., product nos. AP15 142 50 or APIS 293 25 and
AP20 142 50 or AP20 293 25, or equivalent).
Stack AP20 and APIS prefilters and 0.22 pm membrane filter into a disc filter holder with AP20 prefilter on
top and 0.22 fan membrane filter on bottom.
Ahvays disassemble the filter stack after use to check the integrity of the 0.22 (am filter. Refilter any media
filtered with a damaged stack.
2.1.7 Positively-charged cartridge filter — 10 inch (Zeta plus TSM, Cuno Div., product no. 45134-01-600P,
or equivalent). Holder for cartridge filter with adaptor for 10 inch cartridge (Millipore Corp., product no.
YY16 012 00, or equivalent).
2.1.8 Culture capsule filter (Gelman Sciences, product no. 12140, or equivalent).
2.1.9 Cell culture vessels — Pyrex, soda, or flint glass or plastic bottles and flasks or roller bottles (e.g.,
Brockway, product no. 1076-09A, 1925-02, Corning, product no. 25100-25, 25110-75, 25120-150, 25150-1750,
or equivalent).
Vessels must be made from clear glass or plastic to allow observation of the cultures and be equipped with
airtight closures. Plastic vessels must be treated by the manufacturer to allow cells to adhere properly.
2.1.10 Screw caps, black with rubber liners (Brockway, product no. 24-414 for 6 oz bottles,6 or equivalent).
Caps for larger culture bottles usually supplied with bottles.
2.1.11 Roller apparatus (Belco, product no. 7730, or equivalent).
2.1.12 Incubator capable of maintaining the temperature of cell cultures at 36.5 ± 1°C.
2.1.13 Waterbath, equipped with circulating device to ensure even heating at 36.5 + 1°C.
2.1.14 Light microscope, with conventional light source, equipped with lenses to provide 40X, 100X, and
400X total magnification.
2.1.15 Inverted light microscope equipped with lenses to provide 40X, 100X, and 400X total magnification.
2.1.16 Cornwall syringe pipettors, 2, 5, and 10 mL sizes (Curtin Matheson Scientific, product nos. 221-861,
221-879, and 221-887, or equivalent).
2.1.17 Brewer-type pipetting machine (Curtin Matheson Scientific, product no. 138-107, or equivalent).
is given in oz only when it is commercially designated in that unit.
126
-------
2.1.18 Phase contrast counting chamber (hemocytometer) (Curtin Mathespn Scientific, product no. 158-501,
or equivalent).
2.1.19 Conical centrifuge tubes, sizes 50 mL and 250 mL.
2.1.20 Rack for tissue culture tubes (Bellco Glass, product no. 2028, or equivalent).
2.1.21 Bottles, aspirator-type with tubing outlet, size 2,000 mL.
Bottles for use with pipetting machine.
2.1.22 Storage vials, size 2 mL.
Vials must withstand temperatures to -70°C.
2.2 Media and Reagents
2.2.1 Sterile fetal calf, gamma globulin-free newborn calf, or iron-supplemented calf serum, certified free of
viruses, bacteriophage and mycoplasma (GIBCO BRL, or equivalent).
Test each lot of serum for cell growth and toxicity before purchasing. Serum should be stored at -20° C for
long-term storage. Upon thawing, each bottle must be heat-inactivated at 56°Cfor 30 minutes and stored at
4°Cfor short-term use.
2.2.2 Trypsin, 1:250 powder (Difco Laboratories, product no. 0152-15-9, or equivalent) or trypsin, 1:300
powder (BBL, Microbiology Systems, product no. 12098, or equivalent).
2.2.3 Sodium (tetra) ethylenediamine tetraacetate powder (EDTA), technical grade (Fisher Scientific, product
no. S657-500, or equivalent).
2.2.4 Thioglycollate medium (Difco Laboratories, product no. 0257-01-9, or equivalent).
2.2.5 Fungizone (amphotericin B, Sigma Chemical, product no. A-9528, or equivalent), penicillin G (Sigma
Chemical, product no. P-3032, or equivalent), and dihydrostreptomycin sulfate (ICN Biomedicals, product no.
100556, or equivalent), tetracycline (ICN Biomedicals, product no. 103011, or equivalent).
Use antibiotics of at least tissue culture grade.
2.2.6 Eagle's minimum essential medium (MEM) with Hanks' salts and L-glutamine, without sodium
bicarbonate (GIBCO BRL, product no. 410-1200, or equivalent).
2.2.7 Leibovitz's L-15 medium with L-glutamine (GIBCO BRL, product no. 430-1300, or equivalent).
2.2.8 Trypan blue (Sigma Chemical Co., product no. T-6146, or equivalent).
Note: This chemical is on the EPA list of proven or suspected carcinogens.
2.2.9 Dimethyl sulfoxide (DMSO; Sigma Chemical Co., product no. D-2650, or equivalent).
2.2.10 Mycoplasma testing kit (Irvine Scientific, product no. T500-000, or equivalent).
3. PREPARATION OF CELL CULTURE MEDIA
3.1 General Principles
3.1.1 Equipment care — Carefully wash and sterilize equipment used for preparing media before each use.
127
-------
3.1.2 Disinfection of work area — Thoroughly disinfect surfaces on which the medium preparation equipment
is to be placed. Many commercial disinfectants do not adequately kill enteroviruses. To ensure thorough
disinfection, disinfect all surfaces and spills with either a solution of 0.5% (5 g per liter) Ij in 70% ethanol or
0.1% HOC1. HOC1 can be prepared by adding 19 mL'of household bleach (Clorox, The Clorox Co., or
equivalent) to 981 mL of ddH2O and adjusting the pH of the solution to 6-7 with 1 M HC1.
3.1.3 Aseptic technique — Use aseptic technique when preparing and handling media or medium components.
3.1.4 Dispensing filter-sterilized media — To avoid post-filtration contamination, dispense filter-sterilized
media into storage containers through clear glass filling bells in a microbiological laminar flow hood. If a hood
is unavailable, use an area restricted solely to cell culture manipulations.
3.1.5 Coding media — Assign a lot number to and keep a record of each batch of medium or medium
components prepared. Place the lot number, the date of preparation, the expiration date, and the initials of the
person preparing the medium on each bottle.
3.1.6 Sterility test — Test each lot of medium and medium components to confirm sterility as described in
Subsection 4 before the lot is used for cell culture.
3.1.7 Storage of media and medium components — Store media and medium components hi clear, airtight
containers at 4°C or -20°C as appropriate.
3.1.8 Sterilization of NaHCO3-containing solutions — Sterilize media and other solutions that contain NaHCO3,
by positive pressure filtration.
Negative pressure filtration of such solutions increases the pH and reduces the buffering capacity.
3.2 Media Preparation Recipes
3.2.1 Sources of cell culture media.
Commercially prepared liquid cell culture media and medium components are available from several
sources. Cell culture media can also be purchased in powder form that requires only dissolution in ddH^O and
sterilization. Media from commercial sources are quality controlled. The conditions specified by the supplier
for storage and expiration dates should be strictly observed. However, media can also be prepared in the
laboratory directly from chemicals. Such preparations are labor intensive, but allow quality control of the
process at the level of the preparing laboratory.
3.2.2 Procedure for the preparation of EDTA-trypsin.
The procedure described is for the preparation of 10 L of EDTA-trypsin reagent. It is used to dislodge
cells attached to the surface of culture bottles and flasks. This reagent, when stored at 4°C, retains its working
strength for at least 4 months. The amount of reagent prepared should be based on projected usage over a 4
month period.
(a) Add 30 g of trypsin (1:250) or 25 g of trypsin (1:300) and 2 L of ddHjO to a 6 L flask containing a 3
inch stir bar. Place the flask onto a magnetic stirrer and mix the trypsin solution rapidly for a
minimum of 1 hour.
Trypsin remains cloudy.
(b) Add 4 L of ddH2O and a 3 inch stir bar into 20 L clear plastic carboy. Place the carboy onto a
magnetic stirrer and stir at a speed sufficient to develop a vortex while adding the following chemicals:
80 g NaCI, 12.5 g EDTA, 50 g dextrose, 11.5 g Na^PO,, • 7H2O, 2.0 g KC1, and 2.0 g KH2PO4.
Each chemical does not have to be completely dissolved before adding the next one.
128
-------
(c) Add 4 more liters of ddH2O to carboy.
Continue mixing until all chemicals are completely dissolved.
(d) Add the 2 L of trypsin from step (a) to the prepared solution hi step (c) and mix for a minimum of 1
hour. Adjust the pH of the EDTA-trypsin reagent to 7.5-7.7.
(e) Filter reagent under pressure through a disc filter stack and store the filtered reagent in tightly
stoppered or capped containers at 4°C.
The cartridge prefilter (Subsection 2.1.7) can be used in line with the culture capsule sterilizing filter
(Subsection 2.1.8) as. an alternative to a filter stack (Subsection 2.1.6).
3.2.3 Procedure for the preparation of MEM/L-15 medium.
The procedure described is for preparation of 10 L ofMEM/L-15 medium.
(a) Place a 3 inch stir bar and 4 L of ddH2O into 20 L carboy.
(b) Place the carboy onto a magnetic stirrer. Stir at a speed sufficient to develop a vortex and then add
the contents of a 5 L packet of L-15 medium to the carboy. Rinse the medium packet with 3 washes
of 200 mT. each of ddH2O and add the rinses to the carboy.
(c) Mix until the medium is evenly dispersed.
L-15 medium may appear cloudy as it need not be totally dissolved before proceeding to step (d).
(d) Add 3 L of ddH2O to the carboy and the contents of a 5 L packet of MEM medium to the carboy.
Rinse the MEM medium packet with three washes of 200 mL each of ddl^O and add the rinses to the
carboy. Add 800 mL of ddH2O and 7.5 g of NaHCO3 and continue mixing for an additional 60
minutes.
(e) Transfer the MEM/L-15 medium to a pressure can and filter under positive pressure through a 0.22
jtun sterilizing filter. Collect the medium in volumes appropriate for the culturing of BGM cells (e.g.,
900 mT. hi a 1 L bottle) and store in tightly stoppered or capped containers at 4°C.
Medium may be stored for periods of up to 2 months.
3.2.4 Procedure for preparation of trypan blue solution.
The procedure described is for the preparation of 100 mL of trypan blue solution. It is used in the direct
determination of the viable cell counts of the BGM stock cultures. Since trypan blue is on the EPA suspect
carcinogen list, particular care should be taken in its preparation and use so as to avoid skin contact or
inhalation. The wearing of rubber gloves during preparation and use is recommended.
(a) Add 0.5 g of trypan blue to 100 mL of ddH2O in a 250 mL flask. Swirl the flask until the trypan blue
is completely dissolved.
(b) Sterilize the solution by autoclaving at 121 °C for 15 minutes and store hi a screw-capped container at
room temperature.
3.2.5 Procedure for preparation of stock antibiotic solutions.
If not purchased in sterile form, stock antibiotic solutions must be filter-sterilized by the use of 0.22 pm
membrane filters. It is important that the recommended antibiotic levels not be exceeded when planting cells as
the cultures are particularly sensitive to excessive concentrations at this stage.
Antibiotic stock solutions should be placed in screw-capped containers and stored at -20°C until needed.
Once thawed, they may be refrozen; however, repeated freezing and thawing of these stock solutions should be
avoided by distributing them in quantities that are sufficient to support a week's cell culture work.
129
-------
(a) Preparation of penicillin-streptomycin stock solution.
The procedure described is for preparation of ten 10 mL aliquots of penicillin-streptomycin stock
solution at concentrations of 1,000,000 units of penicillin and 1,000,000 ^g of streptomycin per 10 mL
unit. The antibiotic concentrations listed in step (a. 1) may not correspond to the concentrations obtained
from other lots or from a different source.
(a.l) Add appropriate amounts of penicillin G and dihydrostreptomycin sulfate to a 250 mL flask
containing 100 mL of ddH2O. Mix the contents of the flasks on magnetic stirrer until the
antibiotics are dissolved.
For penicillin supplied at 1,435 units per mg, add 7 g of the antibiotic.
For streptomycin supplied at 740 mg per g, add 14 g of the antibiotic.
(a.2) Sterilize the antibiotics by filtration through 0.22 /tm membrane filters and dispense in 10 mL
volumes into screw-capped containers.
(b) Preparation of tetracycline stock solution. Add 1.25 g of tetracycline hydrochloride powder and 3.75
g of ascorbic acid to a 125 mL flask containing 50 mL of ddHp. Mix the contents of the flask on a
magnetic stirrer until the antibiotic is dissolved. Sterilize the antibiotic by filtration through a 0.22 /tm
membrane filter and dispense in 5 mL volumes into screw-capped containers.
(c) Preparation of amphotericin B (fungizone) stock solution. Add 0.125 g of amphotericin B to a 50 mL
flask containing 25 mL of ddHjO. Mix the contents of the flask on a magnetic stirrer until the
antibiotic is dissolved. Sterilize the antibiotic by filtration through 0.22 /tm membrane filter and
dispense 2.5 mL volumes into screw-capped containers.
4. PROCEDURE FOR VERIFYING STERILITY OF LIQUIDS
Tliere are many techniques available for verifying the sterility of liquids such as cell culture media and
medium, components. The two techniques described below are standard in many laboratories. The capabilities
of these techniques are limited to the detection of microorganisms that grow unaided on the test medium utilized.
Viruses, mycoplasma, and microorganisms that possess fastidious growth requirements or that require living
host systems will not be detected. Nonetheless, with the exception of a few special contamination problems, the
test procedures and microbiological media listed below should prove adequate. Do not add antibiotics to media
or medium components until after sterility of the antibiotics, media, and medium components has been
demonstrated. The BGM cell line used should be checked every 6 months for mycoplasma contamination
according to test kit instructions. Cells that are contaminated should be discarded.
4.1 Procedure for Verifying Sterility of Small Volumes of Liquids. Inoculate 5 mL of the material to be tested
for sterility into 5 mL of thioglycollate broth. Shake the mixture and incubate at 36.5 + 1°C. Examine the
inoculated broth daily for 7 days to determine whether growth of contaminating organisms has occurred.
Vessels that contain thioglycollate medium must be tightly sealed before and after medium is inoculated.
4.2 Visual Evaluation of Media for Microbial Contaminants. Incubate media at 36.5 ± 1°C for at least 1 week
prior to use. Visually examine and discard any media that lose clarity.
A clouded condition that develops in the media indicates the occurrence of contaminating organisms.
S. PROCEDURES FOR PREPARATION AND PASSAGE OF BGM CELL CULTURES
A laminar flow biological safety cabinet should be used to process cell cultures. If a biological safety
cabinet is not available, cell cultures should be prepared in controlled facilities used for no other purposes.
Viruses or other microorganisms must not be transported, handled, or stored in cell culture transfer facilities.
130
-------
5.1 Vessels and Media for Cell Growth
5.1.1 The BGM cell line grows readily on the inside surfaces of glass or specially treated, tissue culture grade
plastic vessels. 16 to 32 oz (or equivalent growth area) flat-sided, glass bottles, 75 or 150 cm2 plastic cell
culture flasks, and 690 cm2 glass or 850 cm2 plastic roller bottles are usually used for the maintenance of stock
cultures. Flat-sided bottles and flasks that contain cells in a stationary position are incubated with the flat side
(cell monolayer side) down. If available, roller bottles and roller apparatus units are preferable to flat-sided
bottles and flasks because roller cultures require less medium than flat-sided bottles per unit of cell monolayer
surface. Roller apparatus rotation speed should be adjusted to one-half revolution per minute to ensure that cells
are constantly bathed in growth medium.
5.1.2 Growth and maintenance media should be prepared on the day they will be needed. Prepare growth
medium by supplementing MEM/L-15 medium with 10% serum and antibiotics (100 mL of serum, 1 mL of
penicillin-streptomycin stock, 0.5 mL of tetracycline stock, and 0.2 mT. of fungizone stock per 900 mT, of
MEM/L-15). Prepare maintenance medium by supplementing MEM/L-15 with antibiotics and 2% or 5% serum
(20 or 50 mT. of serum, antibiotics as above for growth medium, and 70 or 50 mL of ddH2O, respectively).
5.2 General Procedure for Cell Passage
Pass stock BGM cell cultures at approximately 7 day intervals using growth medium.
5.2.1 Pour spent medium from cell culture vessels, and discard the medium.
To prevent splatter, a gauze-covered beaker may be used to collect spent medium.
Before discarding, autoclave all media that have been in contact with cells or that contain serum.
5.2.2 Add to the cell cultures a volume of warm EDTA-trypsin reagent equal to 40% of the volume of medium
replaced.
See Table 1 for the amount of reagents required for commonly used vessel types.
To reduce shock to cells, warm the EDTA-trypsin reagent to 36.5 ± 1°C before placing it on cell
monolayers. Dispense the EDTA-trypsin reagent directly onto the cell monolayer.
5.2.3 Allow the EDTA-trypsin reagent to remain in contact with the cells at either room temperature or at 36.5
+ 1°C until cell monolayer can be shaken loose from inner surface of cell culture vessel (about 5 minutes).
If necessary, a sterile rubber policeman (or scraper) may be used to physically remove the cell sheet from
the bottle. However, this procedure should be used only as a last resort because of the risk of cell culture
contamination inherent in such manipulations. The EDTA-trypsin reagent should remain in contact with the
cells no longer than necessary as prolonged contact can alter or damage the cells.
5.2.4 Pour the suspended cells into centrifuge tubes or bottles.
To facilitate collection and resuspension of cell pellets, use tubes or bottles with conical bottoms.
Centrifuge tubes and bottles used for this purpose must be able to withstand the g-force applied.
5.2.5 Centrifuge cell suspension at 1,000 x g for 10 minutes to pellet cells. Pour off and discard the
supernatant.
Do not exceed this speed as cells may be damaged or destroyed.
5.2.6 Suspend the pelleted cells in growth medium (see Subsection 5.1.2) and perform a viable count on the
cell suspension according to procedures hi Subsection 6.
Resuspend pelleted cells in sufficient volumes of medium to allow thorough mixing of the cells (to reduce
sampling error) and to minimize the significance of the loss of the 0.5 mL of cell suspension required for the cell
counting procedure. The quantity of medium used for resuspending pelleted cells varies from 50 to several
hundred mL, depending upon the volume of the individual laboratory's need for cell cultures.
131
-------
Table 1. Guide for Preparation of BGM Stock Cultures
Volume of EDTA-
Vessel Trypsin Used to
Type Remove Cells (mL)
Total No. Cells
Volume of to Plate
Medium (mL)* per Vessel
16 oz** glass
flat bottles
32 oz glass
flat bottles
75 cm2 plastic
flat flask
150 cm* plastic
flat flask
690 cm* glass
roller bottle
850 cm2 plastic
roller bottle
10
20
12
24
40
50
25
50
30
60
100
120
2.5 x 10s
5.0 x 10s
3.0 x 106
6.0 x 106
7.0 x 107
8.0 x 107
*Serum requirements: growth medium contains 10% serum;
maintenance medium contains 2-5% serum.
Antibiotic requirements: penicillin-streptomycin stock solution, 1.0 mL/L;
tctracycline stock solution, 0.5 mL/L; fungizone stock solution,
0.2 mL/L.
**Size is given in oz only when it is commercially designated in that unit.
5.2.7 Dilute the cell suspension to the appropriate cell concentration with growth medium and dispense into
cell culture vessels with either a Cornwall-type syringe or Brewer-type pipetting machine dispenser.
Calculate the dilution factor requirement using the cell count established in Subsection 6 and the cell and
volume parameters given in Table Ifor stock cultures and in Table 2 for virus assay cultures.
As a general rule, the BGM cell line can be split at a 1:3 ratio. However, a more suitable inoculum is
obtained if low passages of the line (passages 100-150) are split at a 1:2 ratio and higher passages (generally
above passage 250) are split at a 1:4 ratio. To plant 200 25 cm2 cell culture flasks weekly front a low-level
passage of the line would require the preparation of 6 roller bottles (surface area 690 cm2 each): 2 to prepare
the 6 roller bottles and 4 to prepare the 25 cm2 flasks.
5.2.8 Except during handling operations, maintain BGM cells at 36.5 + 1°C in airtight cell culture vessels.
5.3 Procedure for Changing Medium on Cultured Cells
Cell monolayers normally become 95-100% confluent 3-4 days after seeding with an appropriate number of
cells, and growth medium becomes acidic. Growth medium on confluent stock cultures should then be replaced
with maintenance medium containing 2% serum. Maintenance medium with 5% serum should be used when
monolayers are not yet 95-100% confluent but the medium in which they are immersed has become acidic. The
volume of maintenance medium should equal the volume of discarded growth medium.
132
-------
Table 2. Guide for Preparation of Virus Assay Cell Cultures
Vessel Type
1 oz** glass bottle
25 cm2 plastic flask
6 oz glass bottle
75 cm2 plastic flask
16 mm x 150 mm tubes
Volume of
Medium* (mL)
4
10
15
30
2
Final Cell Count
per Bottle
9.0 x 10s
3.5 x 106
5.6 x 106
1.0 x 107
4.0 x 10"
*Serum requirements: growth medium contains 10% serum.
Antibiotic requirements: penicillin-streptomycin stock solution, 1.0 mL/L;
tetracycline stock solution, 0.5 mL/L; fungizone stock solution,
0.2 mL/L.
**Size is given in oz only when it is commercially designated in that unit.
6. Procedure for Performing Viable Cell Counts
With experience, a fairly accurate cell concentration can be made based on the volume of packed cells.
However, viable cell counts should be performed periodically as a quality control measure.
6.1 Add 0.5 mL of cell suspension (or diluted cell suspension) to 0.5 mT. of 0.5% trypan blue solution in a test
tube.
To obtain an accurate cell count, the optimal total number of cells per hemocytometer section should be
between 20 and 50. This range is equivalent to between 6.0 x itf and 1.5 x l(f cells per mL of cell suspension.
Thus, a dilution of 1:10 (0.5 mL of cells in 4.5 mL of growth medium) is usually required for an accurate count
of a cell suspension.
6.2 Disperse cells by repeated pipetting.
Avoid introducing air bubbles into the suspension, because air bubbles may interfere with subsequent filling
of the hemocytometer chambers.
6.3 With a capillary pipette, carefully fill a hemocytometer chamber on one side of a slip-covered
hemocytometer slide. Rest the slide on a flat surface for about 1 minute to allow the trypan blue to penetrate
the cell membranes of nonviable cells.
Do not under or overfill the chambers.
6.4 Under 100X total magnification, count the cells in the four large corner sections and the center section of
the hemocytometer chamber.
Include in the count cells lying on the lines marking the top and left margins of the sections, and ignore
cells on the lines marking the bottom and right margins. Trypan blue is excluded by living cells. Therefore, to
quantify viable cells, count only cells that are dear in color. Do not count cells that are blue.
133
-------
6.5 Calculate the average number of viable cells in each mL of cell suspension by totaling the number of viable
cells counted in the five sections, multiplying this sum by 4,000, and where necessary, multiplying the resulting
product by the reciprocal of the dilution.
7. PROCEDURE FOR PRESERVATION OF BGM CELL LINE
An adequate supply of BGM cells must be available to replace working cultures that are used only
periodically or become contaminated or lose virus sensitivity. Cells have been held at -70°C for more than 15
years with a minimum loss in cell viability.
7.1 Preparation of Cells for Storage
Tlie procedure described is for the preparation of 100 cell culture vials. Cell concentration per mL must be
at least 1 x ICf.
Base the actual number of vials to be prepared on usage of the line and the anticipated time interval
requirement beMeen cell culture start-up and full culture production.
7.1.1 Prepare cell storage medium by adding 10 mL of serum and 10 mL of DMSO to 80 mL of growth
medium (see Subsection 5.1.2). Sterilize cell storage medium by passage through an 0.22 jitrn sterilizing filter. „
Collect sterilized medium in a 250 mL flask containing a stir bar.
7.1.2 Harvest BGM cells from cell culture vessels as directed in Subsections 5.2.1 through 5.2.5. Count the
cells according to the procedure in Subsection 6 and resuspend them in the cell storage medium at a
concentration of 1 x 10s cells per mL.
7.1.3 Place the flask containing suspended cells on a magnetic stirrer and slowly mix for 30 minutes.
Dispense 1 mL volumes of cell suspension into 2 mL vials.
7.2 Procedure for Freezing Cells
Tlie freezing procedure requires slow cooling of the cells with the optimum rate of -1 °C per minute. A slow
cooling rate can be achieved using the following method or by using the recently available freezing containers
(e.g., Nalge Company, product no. 5100-0001, or equivalent) as recommended by the manufacturers.
7.2.1 Place the vials in a rack and place the rack hi refrigerator at 4°C for 30 minutes, hi a -20°C freezer for
30 minutes, and then in a -70 °C freezer overnight. The transfers should be made as rapidly as possible.
To allow for more uniform cooling, wells adjoining each vial should remain empty.
7.2.2 Rapidly transfer vials into boxes or other containers for long-term storage.
To prevent substantial loss of cells during storage, the temperature of cells should be kept constant after
-70°C has been achieved.
7.3 Procedure for Thawing Cells
Cells must be thawed rapidly to decrease loss in cell viability.
7.3.1 Place vials containing frozen cells into a 36°C water bath and agitate vigorously by hand until all ice has
melted. Sterilize the outside surface of the vials with 0.5% I2 in 70% ethanol.
7.3.2 Add BGM cells to either 6 oz tissue culture bottles or 25 cm2 tissue culture flasks containing an
appropriate volume of growth medium (see Table 2). Use two vials of cells for 6 oz bottles and one vial for 25
cm2 flasks.
7.3.3 Incubate BGM cells at 36.5 ± 1°C. After 18 to 24 hours replace the growth medium with fresh growth
medium and then continue the incubation for an additional five days. Pass and maintain the new cultures as
directed in Subsection 5.
134
-------
V. CELL CULTURE PROCEDURES FOR ASSAYING PLAQUE-FORMING VIRUSES6
1. INTRODUCTION
This section outlines procedures for the detection of viruses in sludge by use of the plaque assay system, as
described by Dulbecco (1952), Dulbecco and Vogt (1954), Hsiung and Melnick (1955), and Dahling and Wright
(1986). The system uses an agar medium to localize virus growth following attachment of infectious virus
particles to a cell monolayer. Localized lesions of dead cells (plaques) developing some days after viral
infection are visualized with the vital stain (neutral red), which stains only live cells. The number of circular
unstained plaques are counted and reported as plaque-forming units (PFU), whose number is proportional to the
amount of infectious virus particles inoculated. The procedures outlined below describe the plaque assay
technique for enterovirus enumeration of sludge sample concentrates using the BGM cell line.
The detection methodology presented in this section is geared toward laboratories with a small-scale virus
assay requirement. Where the quantities of cell cultures, media, and reagents set forth hi the section are not
sufficient for processing the test sample concentrates, the prescribed measures may be increased proportionally
to meet the demands of more expansive test regimes.
2. PLAQUE ASSAY PROCEDURE
2.1 Apparatus and Materials
2.1.1 Waterbath set at 50 + 1°C.
Used for maintaining the agar temperature (see Subsection 2.2.10).
2.2 Media and Reagents
2.2.1 ELAH — 0.65% lactalbumin hydrolysate in Earle's base (Gibco BRL, product no. 320-1250, or
equivalent).
ELAH can be purchased in liquid form or prepared by dissolving 6.5 g per liter of tissue culture, highly
soluble grade lactalbumin hydrolysate in Earle's base (Gibco BRL, product no. 31O-4O1O, or equivalent)
prewarmed to 50-60°C. Sterilize ELAH prepared in-house through a 0.22 pm filter stack. ELAH prepared in-
house may be stored for 2 months at 4°C.
2.2.2 Maintenance medium — Add 1 mL of penicillin-streptomycin stock (see Section IV, Subsection 3.2.5 for
preparation of antibiotic stocks), 0.5 mL of tetracycline stock, and 0.2 mL of fungizone stock per liter to ELAH
immediately before washing of cells.
2.2.3 HEPES — 1 M (Sigma Chemical Co., product no. H-3375, or equivalent).
Prepare 50 mL of a 1 M solution by dissolving 11.92 g of HEPES in a final volume of 50 mL ddH£).
Sterilize by autoclaving at 121 °Cfor 15 minutes.
2.2.4 Sodium bicarbonate (NaHCO3) — 7.5% solution.
Prepare 50 mL of a 7.5% solution by dissolving 3.75 g of sodium bicarbonate in a final volume of 50 mL
ddH2O. Sterilize by filtration through a 0.22 nm filter.
2.2.5 Magnesium chloride (MgCl2 • 6H20) — 1.0% solution.
Prepare 50 mL of a 1.0% solution by dissolving 0.5 g of magnesium chloride in a final volume of 50 mL
ddH2O. Sterilize by autoclaving at 121 °Cfor 15 minutes.
2.2.6 Neutral red solution — 0.333%, 100 mL volume (GIBCO BRL, product no. 630-5330, or equivalent).
Procure one 100 mL bottle.
'Modified for EPA/600/4-84/013(Rll), March 1988 Revision.
135
-------
2.2.7 Bacto skim milk (Difco Laboratories, product no. 0032-01, or equivalent).
Prepare 100 mL of 10% skim milk in accordance with directions given by manufacturer.
2.2.8 Preparation of Medium 199.
Tlie procedure described is for preparation of 500 mL of Medium 199 (GIBCO BKL, product no. 400-1100,
or equivalent) at a 2X concentration. This procedure will prepare sufficient medium for at least fifty 6 oz glass
bottles or eighty 25 en? plastic flasks.
(a) Place a 3 inch stir bar into a 1 L flask. Add the contents of a 1 L packet of Medium 199 into the
flask. Add 355 mL of ddH2O. Rinse medium packet with 3 washes of 20 mL each of ddH2O and add
the washes to the flask.
Note that the amount ofddH2O is 5% less than desired for the final volume of the medium.
(b) Mix on a magnetic stirrer until the medium is completely dissolved. Filter the reagent under pressure
through a filter stack (see Section IV, Subsection 2.1.6).
Test each lot of medium to confirm sterility before the lot is used (see Section IV, Subsection 4). Each
batch may be stored for 2 months at 4°C.
2.2.9 Preparation of overlay medium for plaque assay.
Tlie procedure described is for preparation of 100 mL of overlay medium and will prepare sufficient media
for at least ten 6 oz glass bottles or twenty 25 oz plastic flasks when mixed with the agar prepared in Subsection
2.2.10.
(a) Add 79 mL of Medium 199 (2X concentration) and 4 mL of serum to a 250 mL flask.
(b) Add the following to the flask in the order listed, and swirl after each addition: 6 mL of 7.5%
NaHCOj, 2 mL of 1% MgC^, 3 mL of 0.333% neutral red solution, 4 mL of 1 M HEPES, 0.2 mL
of penicillin-streptomycin stock (see Section IV, Subsection 3.2.5 for a description of antibiotic
stocks), 0.1 mL of tetracycline stock, and 0.04 mT. of fungizone stock.
(c) Place flask with overlay medium hi waterbath set at 36 + 1°C.
2.2.10 Preparation of overlay agar for plaque assay.
(a) Add 3 g of agar (Sigma Chemical Co., product no. A-9915, or equivalent) and 100 mL of ddH2O to a
250 mL flask. Melt by sterilizing the agar solution hi an autoclave at 121 °C for 15 minutes.
(b) Cool the agar to 50°C in waterbath set at 50 ± 1°C.
2.2.11 Preparation of agar overlay medium.
(a) Add 2 mL of 10% skim milk to overlay medium prepared in Subsection 2.2.9.
(b) Mix equal portions of overlay medium and agar by adding the medium to the agar flask.
To prevent solidification of the liquified agar, limit the portion of agar overlay medium mixed to that
volume which can be dispensed in 10 minutes.
2.3 Procedure for Inoculating Test Samples.
Section TV, Subsection 5 provides the procedures for the preparation of cell cultures used for the virus assay
in this section.
Cell cultures used for virus assays are generally found to be at their most sensitive level between the third
and sixth days after initiation. Those older than seven days should not be used.
136
-------
2.3.1 Decant and discard the growth medium from previously prepared cell culture test vessels.
To prevent splatter, a gauze-covered beaker may be used to collect spent medium.
The medium is changed from 1-4 hours before cultures are to be inoculated and carefully decanted so as not
to disturb the cell monolayer.
2.3.2 Replace discarded medium with an equal volume of maintenance medium on the day the cultures are to
be inoculated.
To reduce shock to cells, prewarm the maintenance medium to 36.5 ± 1 °C before placing it onto the cell
monolayer.
To prevent disturbing cells with the force of the liquid against the cell monolayer, add the maintenance
medium to the side of the cell culture test vessel opposite the cell monolayer.
2.3.3 Identify cell culture test vessels by coding them with an indelible marker. Return the cell culture test
vessels to a 36.5 ± 1°C incubator and hold at that temperature until the cell monolayer is to be inoculated.
2.3.4 Decant and discard the maintenance medium from cell culture test vessels.
Do not disturb the cell monolayer.
2.3.5 Thaw (if frozen) the remaining sample from Section HI, Subsection 4.2.10 and inoculate each BGM cell
monolayer with a volume of test sample concentrate appropriate for the cell surface area of the cell culture test
vessels used.
Inoculum volume should be no greater than 1 mLfor each 40 cm2 of surface area. Use Table 3 as a guide
for inoculation size.
Avoid touching either the cannula or the pipetting device to the inside rim of the cell culture test vessels to
avert the possibility of transporting contaminants to the remaining culture vessels.
(a) Inoculate 2 BGM cultures with an appropriate volume of 0.15 M Na2HPO4 • 7H2O (see Section ffl,
Subsection 4.1.1) preadjusted to pH 7.0-7.5. These cultures will serve as negative controls.
(b) Inoculate 2 BGM cultures with an appropriate volume of 0.15 M NajHPO4 • 7H2O preadjusted to pH
7.0-7.5 and spiked with 20-40 PFU of poliovirus. These cultures will serve as a positive control for
the plaque assay.
(c) Remove a volume of the test sample concentrate exactly equal to l/6th (i.e., 1 g of total dry solids) of
the volume recorded in Section IH, Subsection 4.2.10. Spike this subsample with 20-40 PFU of
poliovirus. Inoculate the subsample onto one or more BGM cultures using a inoculum volume per
vessel that is appropriate for the vessel size used. These cultures will serve as controls for
cytotoxicity (see Section VI).
(d) Record the volume of the remaining 5/6th portion of the test sample. This remaining portion
represents a total dry solids content of 5 g. Inoculate the entire remaining portion (even if diluted to
reduce cytotoxiciry) onto BGM cultures. Inoculation of the entire volume is necessary to demonstrate
a virus density level of less than 1 PFU per 4 g total dry solids.
2.3.6 Rock the inoculated cell culture test vessels gently to achieve uniform distribution of inoculum over the
surface of the cell monolayers. Place the cell culture test vessels on a level, stationary surface at room
temperature (22-25 °C) so that the inoculum will remain distributed evenly over the cell monolayer.
137
-------
Table 3. Guide for Virus Inoculation, Suspended Cell Concentration
and Overlay Volume of Agar Medium
Volume of Virus Volume of Agar Total Numbers
Vessel Type Inoculum (mL) Overlay Medium (mL) of Cells
1 oz* glass 0.1 5
bottle
25 cm2 plastic 0.1-0.5 10
flask
6 oz glass 0.5-1.0 20
bottle
75 cm2 plastic 1.0-2.0 30
flask
Ix 107
2x 107
4x 107
6x 107
*Size is given in oz only when it is commercially designated in that unit.
2.3.7 Incubate the inoculated cell cultures at room temperature for 80 minutes to permit viruses to adsorb onto
and infect cells and then proceed immediately to Subsection 2.4.
It may be necessary to rock the vessels every 15-20 minutes during the 80 minute incubation to prevent cell
death in the middle of the vessels from dehydration.
2.4 Procedure for Overlaying Inoculated Cultures with Agar
If there is a likelihood that a test sample will be toxic to cell cultures, the cell monolayer should be treated
in accordance with the method described in Section VI.
2.4.1 To each cell culture test vessel, add the volume of warm (42-46°C) agar overlay medium appropriate for
the cell surface area of the vessels used (see Table 3).
Tfte preparation of the overlay agar and the agar overlay medium must be made far enough in advance that
they will be at the right temperature for mixing at the end of the 80 minute inoculation period.
To prevent disturbing cells with the force of the liquid against the cell monolayer, add the agar overlay
medium to the side of the cell culture test vessel opposite the cell monolayer.
2.4.2 Place cell culture test vessels, monolayer side down, on a level, stationary surface at room temperature
(22-25°C) so that the agar will remain evenly distributed as it solidifies. Cover the vessels with a sheet of
aluminum foil, a tightly woven cloth, or some other suitable cover to reduce the light intensity during
solidification and incubation. Neutral red can damage or kill tissue culture cells by light-induced the
crosslinking of nucleic acids.
Care must be taken to ensure that all caps on bottles and flasks are tight; otherwise, the gas seal will not be
complete and an erroneous virus assay will result.
Agar willfully solidify within 30 minutes.
2.4.3 After 30 minutes, invert the cell culture test vessels and incubate them covered in the dark at 36.5 +
leC.
138
-------
2.5 Virus Quantitation
2.5.1 Plaque counting technique.
(a) Count, mark, and record plaques in cell culture test vessels on days two, three, four, six, eight, and
twelve after adding the agar overlay medium. Plaques should be counted quickly using a lightbox
(Baxter, product no. B5080-1, or equivalent) in a darkened room. Most plaques should appear within
1 week.
Depending on the virus density and virus types present in the inoculated sample, rescheduling of virus
counts at plus or minus one day may be necessary. Virus titers are calculated from the total plaque
count. Note that not all plaques will be caused by viruses. The presence of virus must always be
confirmed using the procedure in Section VII.
(b) Examine cell culture test vessels on day sixteen.
If no new plaques appear at 16 days, discard the vessels; otherwise, continue to count, mark, and
record plaques every 2 days until no new plaques appear between counts.
Inoculated cultures should always be compared to uninoculated control cultures so that the
deterioration of the cell monolayers is not recorded as plaques.
Samples giving plaque counts that are greater than 2 plaques per cm2 should be diluted and replated.
2.5.2 Calculation of virus titer.
(a) If the entire remaining portion of the test sample was inoculated onto BGM cultures as described in
Subsection 2.3.5(d), sum the total number of plaques in all test vessels for each sample. Multiply the
sum by 0.8 to obtain the titer in PFU per 4 g of total dry solids. Average the titer of all composite
samples and report the average titer and the standard deviation for each lot of sludge tested.
(b) If the sample was diluted due to high virus levels (e.g., when the virus density of the input to a
process is being determined), calculate the virus titer (V) in PFU per 4 g total dry solids with the
formula: V = 0.8 x (P/I) x D x S, where P is the total number of plaques in all test vessels for each
sample, I is the volume (in mL) of the dilution inoculated, D is reciprocal of the dilution made on the
inoculum before plating, and S is the volume of the remaining portion of the test sample (as recorded
in Subsection 2.3.5(d)). Average the composite samples and report as in step (a).
VI. METHOD FOR THE REDUCTION OF CYTOTOXICITY IN SAMPLE CONCENTRATES7
The procedure described in this section reduces the cytotoxicity of cytotoxic sludge samples. However, the
procedure may result in a titer reduction of up to 30% and should be applied only to inocula known to be or
expected to be toxic.
1. VIRUS RECOVERY AND ASSAY OF SAMPLES
Process and store sludge samples as described in Section III. Inoculate samples onto cell monolayers as
described in Section V, Subsection 2.3.
2. REDUCTION OF TOXICFTY OF SAMPLE CONCENTRATE
2.1 Apparatus and Materials
2.1.1 Cell culture bottles.
See Section IV for the preparation of cell culture bottles.
7Modified from EPA/600/8-84/013(R8), April 1986 Revision.
139
-------
2.2 Media and Reagents
2.2.1 Washing solution.
(a) To a flask containing a stir bar and an appropriate volume of ddH2O, add NaCl to a final
concentration of 0.85% (weight/volume; e.g., 0.85 g in 100 mL). Mix the contents of the flask on a
magnetic stirrer at a speed sufficient to dissolve the salt. Remove the stir bar and autoclave the
solution at 121°C for 15 minutes. Cool to room temperature.
The volume of the NaCl washing solution required will depend on the number of bottles to be
processed and the cell surface area of the bottles used for the plaque assay.
(b) Add 2% (volume/volume; e.g., 2 mL per 100 mL) serum to the sterile salt solution. Mix thoroughly
and store at 4°C.
Although the washing solution may be stored at 4°Cfor an extended time period, it is advisable to
prepare solutions on a weekly basis, thereby lessening the possibility ofmicrobial contamination.
2.3 Procedure for Cytotoxicity Reduction
2.3.1 Decant and discard the inoculum from inoculated cell culture bottles after the 80 minute inoculation
period. Add 0.25 mL of the washing solution (Subsection 2.2. l(b)) for each cm2 of cell surface area into each
bottle.
To reduce thermal shock to cells, warm the washing solution to 36.5 + 1 °C before placing it on the cell
monolayer.
To prevent disturbing cells with the force of liquid against the cell monolayer, add washing solution to the
side of the cell culture bottle opposite the cell monolayer. Also, avoid touching either the cannula or syringe
needle of the pipette or the pipetting device to the inside rim of the cell culture bottles to avert the possibility of
transporting contaminants to the remaining culture bottles.
2.3.2 Rock the washing solution gently across the cell monolayer a minimum of 2 times. Decant and
discard the spent washing solution in a manner that will not disturb the cell monolayer.
It may be necessary to gently rock washing solution across the monolayer more than twice if the sample is
oily and difficult to remove from the cell monolayer surface.
2.3.3 Continue by performing the agar overlay procedure for plaque assay hi Section V, Subsections 2.4
and 2.5.
If this procedure fails to reduce cytotoxicity with a particular type of sludge sample, eluates prepared from
another lot of the sludge sample may be diluted 1:2 to 1:4 before repeating the procedure. This dilution
requires that 2-4 times more culture vessels be used.
3. DETERMINATION OF CYTOTOXICITY
Uninoculated cell cultures should always be processed to serve as controls in later comparisons to
determine the reduction in sensitivity or survival of the BGM cells attributable to the toxicity of the samples.
3.1 Determine cytotoxicity by macroscopic examination of the appearance of the cell culture monolayer
(compare control from Section V, Subsection 2.3.5(a) with test samples) after 3-5 days of incubation at 36.5 ±
1°C. Cytotoxicity should be suspected when the agar color is more subdued, generally yellow to yellow-brown.
This change in color results hi a mottled or blotchy appearance instead of the evenly diffused "reddish" color
observed in "healthy" cell monolayers.
3.2 Cytotoxicity may also cause viral plaques to be reduced hi number or to be difficult to distinguish from the
surrounding monolayer. To determine if this type of cytotoxicity is occurring, compare the two types of
positive controls (see Section V, Subsection 2.3.5(c) and (d)).
140
-------
VH. VIRUS PLAQUE CONFIRMATION PROCEDURE
1. INTRODUCTION
This section describes a procedure for confirming virus plaques in cell cultures adhering to glass or plastic
surfaces. Where large numbers of plaques are observed and confirmation of each plaque is not practical, select
at least 10 well-separated plaques per sample or 10% of the plaques in a sample, whichever is greater.
2. RECOVERY OF VIRUS FROM PLAQUE
2.1 Apparatus, Materials, and Reagents
2.1.1 Pasteur pipettes, disposable, cotton plugged — 229 mm (9 inches) tube length and rubber bulb — 1 ml.
capacity.
Flame each pipette gently about 2 cm from the end of the tip until the tip bends to an approximate angle of
45°. Place the pipettes into a 4L beaker covered with aluminum foil and dry heat sterilize for not less than 1
hour at 170°C.
2.1.2 16 x 150 mm cell culture tubes containing BGM cells.
See Section TV, Subsection 5 for the preparation of cell culture tubes.
2.1.3 Tissue culture roller apparatus — 1/5 rpm speed (New Brunswick Scientific, product no. TC-1, or
equivalent) with culture tube drum for use with roller apparatus (New Brunswick Scientific, product no.
ATC-TT16, or equivalent).
2.1.4 Freezer vial, screw-capped (with rubber insert) or cryogenic vial — 0.5-1 dram capacity.
2.2 Procedure for Virus Confirmation
2.2.1 Procedure for obtaining viruses from plaque.
A decision to test the plaque material for viruses immediately or to store the material at -70° C for later
testing must be made before proceeding further. Whenever possible, the plaque material should be tested
immediately, because storage at -70°C may result in some reduction in confirmation counts.
(a) Place a rubber bulb onto the upper end of a cotton-plugged pasteur pipette and then remove the
screw-cap or stopper from a plaque bottle.
(b) Squeeze the rubber bulb on the pasteur pipette to expel the air and penetrate the agar directly over the
edge of a plaque with the tip of the pipette. Gently force the tip of the pipette through the agar to the
surface of the vessel, and scrape some of the cells from the edge of the plaque.
Repeatedly scratch the surface and use gentle suction to ensure that the virus-cell-agar plug enters the
pipette.
(c) Remove the pipette from the plaque bottle and tightly replace the cap or stopper.
If the sample is to be tested in cell culture immediately, proceed to Subsection 2.2.2(b). If sample
must be stored, proceed to Subsection 2.2.2(c).
2.2.2 Procedure for inoculating viruses obtained from plaques onto cell cultures.
(a) Cell culture processing.
If at all feasible, use a laminar flow hood while processing cell cultures. Otherwise, use an area
restricted solely to cell culture manipulations. Viruses or other microorganisms must not be transported,
handled, or stored in cell culture transfer facilities.
141
-------
(a. 1) Prepare plaque confirmation maintenance medium by adding 5 mL of serum and 5 mL of
ddH2O per 90 mL of antibiotic-supplemented ELAH (see Section V, Subsection 2.2.2) on day
samples are to be tested.
(a.2) Pour the spent medium from cell culture tubes and discard the medium. Replace the discarded
medium with 2 mL of the plaque confirmation maintenance medium. Label the tubes with
sample and plaque isolation identification information.
To prevent splatter, a gauze-covered beaker may be used to collect spent medium.
To reduce shock to cells, warm the maintenance medium to 36.5 ± 1 °C before placing it on
the cell monolayer.
To prevent disturbing cells with the force of the liquid against the cell monolayer, add the
maintenance medium to the side of the cell culture test tube opposite the cell monolayer. Note
that cells will be only on the bottom inner surface of the culture tube relative to their position
during incubation.
(b) Procedure for samples tested immediately.
(b. 1) Remove the cap from a cell culture tube and place the tip of a pasteur pipette containing the
virus-cell-agar plug from Subsection 2.2. l(c) into the maintenance medium in the cell culture
tube. Force the virus-cell-agar plug from the pasteur pipette by gently squeezing the rubber
bulb. Withdraw and discard the pipette, and replace and tighten down the screw-cap on the
culture tube.
Tilt cell culture tube as necessary to facilitate the procedure and to avoid scratching the cell
sheet with the pipette.
Squeeze bulb repeatedly to wash contents of pipette into the maintenance medium.
(b.2) Place the cell culture tube in the drum used with the tissue culture roller apparatus along with
three additional culture tubes which have not been inoculated with agar sample to serve as
negative controls. Add 0.1 mL of ELAH containing 20-40 PFU of polivirus to each of three
more culture tubes to serve as positive controls.
(b.3) Incubate the cell cultures at 36.5 + 1°C while rotating at a speed of 1/5 rpm. Examine the
cells daily microscopically for 1 week, starting with day 3, for evidence of cytopathic effects
(CPE).
CPE may be identified as cell disintegration or as changes in cell morphology. Rounding-up of
infected cells is a typical effect seen with enterovirus infections. However, uninfected cells round
up during mitosis and a sample should not be considered positive unless there are significant
clusters ofrounded-up cells over and beyond what Li observed in the uninfected controls. If there
is any doubt about the presence of CPE or if CPE appears late ft. e., on day 6 or 7), the
conformation process should be repeated by transferring 0.2 mL of the medium in the culture tube
to a freshly prepared tube.
Incubation ofBGM cells in roller apparatus for periods greater than 1 week is not
recommended as cells under these conditions tend to die-off if held longer.
Tubes developing CPE may be stored in a -70° C freezer for additional optional tests (e.g., the
Lim Benyesh-Melnick identification procedure).*
(c) Procedure for samples to be stored at -70 °C before testing.
Place 0.1 mL of antibiotic supplemented ELAH from Section V, Subsection 2.2.2 in a freezer vial for
each plaque to be confirmed.
8For more information see EPA/600/4-84/013(R12), May 1988 Revision.
142
-------
(c. 1) Remove cap from vial containing ELAH. Place the tip of the pasteur pipette containing the
virus-cell-agar plug from Subsection 2.2.1 Step (c) into the vial. Force the virus-cell-agar plug
from the pasteur pipette into the ELAH by gently squeezing the rubber bulb.
Squeeze the bulb repeatedly to wash the contents of the pipette into the ELAH.
(c.2) Withdraw the pipette from the vial, replace and tighten down the screw-cap, and discard the
pipette. Store the vial at -70°C. Place 3 additional vials containing 0.1 mL of ELAH that
have not been inoculated with an agar sample to serve as negative controls and 3 vials
containing 0.1 mL of ELAH with 20-40 PFU of poliovirus to serve as positive controls at
-70°C.
(c.3) When confirmation is to be completed, prepare cell culture tubes in accordance with Step (a)
above and thaw each frozen sample quickly in warm water (30-37 °C).
(c.4) Remove the caps from each tube and transfer the entire contents of the vial containing the
thawed sample into a cell culture tube using a pipette.
Tilt cell culture tube as necessary to facilitate the procedure and to avoid scratching the cell
sheet with the pipette.
Place the tip of the pipette into the maintenance medium in the cell culture tube. Squeeze the
bulb repeatedly to wash any of the remaining test sample into the maintenance medium.
(c.5) Withdraw the pipette from the cell culture tube. Replace and tighten down the screw-cap on
the tube, discard the pipette and sample vial, and continue with Step (b3) above.
VHI. BIBLIOGRAPHY AND SUGGESTED READING
ASTM. 1992. Standard Methods for the Examination of Water and Wastewater (A. E. Greenberg, L. S.
Clesceri, and A. D. Eaton, eds.), 18th edition. American Public Health Association, Washington, DC.
Barren, A. L., C. Olshevsky, and M. M. Cohen. 1970. Characteristics of the BGM line of cells from
African green monkey kidney. Archiv. Gesam. Virusforsch. 52:389-392.
Berg, G., D. Berman, and R. S. Safferman. 1982. A Method for concentrating viruses recovered from
sewage sludges. Can. J. Microbiol. 25:553-556.
Berg, G., R. S. Safferman, D. R. Dahling, D. Berman, and C. J. Hurst. 1984. U.S. EPA Manual of Methods
for Virology. U.S. Environmental Protection Agency Publication No. EPA/600/4-84/013, Cincinnati, OH.
Berman, D., G. Berg, and R. S. Safferman. 1981. A method for recovering viruses from sludges. J. Virol.
Methods. 5:283-291.
Brashear, D. A., and R. L. Ward. 1982. Comparison of methods for recovering indigenous viruses from
raw wastewater sludge. Appl. Environ. Microbiol. 45:1413-1418.
Cooper, P. D. 1967. The plaque assay of animal viruses, pp. 243-311. In K. Maramorosch and H.
Koprowski (eds.), Methods in Virology, Vol. 3. Academic Press, New York, NY.
Dahling, D. R., and B. A. Wright. 1986. Optimization of the BGM cell line culture and viral assay
procedures for monitoring viruses in the environment. Appl. Environ. Microbiol. 57:790-812.
Dahling, D. R., G. Berg, and D. Berman. 1974. BGM, a continuous cell line more sensitive than primary
rhesus and African green kidney cells for the recovery of viruses from water. Health Lab. Sci.
17:275-282.
143
-------
Dahling, D. R., R. S. Safferman, and B. A. Wright. 1984. Results of a survey of BGM cell culture
practices. Environ. Internal. 70:309-313.
Dahling, D. R., G. Sullivan, R. W. Freyberg, and R. S. Safferman. 1989. Factors affecting virus
plaque confirmation procedures. J. Virol. Meth. 24:111-122.
Dulbecco, R. 1952. Production of plaques hi monolayer tissue cultures by single particles of an animal
virus. Proc. Natl. Acad. Sci. U.S.A. 38:747-752.
Dulbecco, R., and M. Vogt. 1954. Plaque formation and isolation of pure lines with poliomyelitis viruses.
J. Exp. Med. 99:167-182.
Eagle, H. 1959. Amino acid metabolism in mammalian cell cultures. Science. 130:432-437.
Farrah, S. R. 1982. Isolation of viruses associated with sludge particles, pp. 161-170. In C.P. Gerba and S.
M. Goyal (eds.), Methods in Environmental Virology. Marcel Dekker, Inc., New York, NY.
Farrah, S. R., P. R. Scheuerman, and G. Bitton. 1981. Urea-lysine method for recovery of enteroviruses
from sludge. Appl. Environ. Microbiol. 41:455-458.
Freshney, R. I. 1983. Culture of Animal Cells: A Manual of Basic Technique. Alan R. Liss. Inc., New
York, NY.
Goddard, M. R., J. Bates, and M. Butler. 1981. Recovery of indigenous enteroviruses from raw and
digested sewage sludges. Appl. Environ. Microbiol. 42:1023-1028.
Hay, R. J. 1985. ATCC Quality Control Methods for Cell Lines. American Type Culture Collection,
Rockville, MD.
Hsiung, G. D., and J. L. Melnick. 1955. Plaque formation with poliomyelitis, coxsackie and orphan (echo)
viruses in bottle cultures of monkey epithelial cells. Virology 7:533-535.
Hurst, C. J. 1987. Recovering viruses from sewage sludges and from solids hi water, pp. 25-51. In G. Berg
(ed.), Methods for Recovering Viruses from the Environment. CRC Press, Boca Raton, FL.
Hurst, C. J., and T. Goyke. 1983. Reduction of interfering cytotoxicity associated with wastewater sludge
concentrates assayed for indigenous enteric viruses. Appl. Environ. Microbiol. 46:133-139.
Katzenelson, E., B. Fattal, and T. Hostovesky. 1976. Organic flocculation: an efficient second-step
concentration method for the detection of viruses hi tap water. Appl. Environ. Microbiol. 52:638-639.
Kedmi, S., and B. Fattal. (1981) Evaluation of the false-positive enteroviral plaque phenomenon occurring hi
sewage samples. Water Res. 75:73-74.
Laboratory Manual in Virology. 1974. 2nd edition. Ontario Ministry of Health, Toronto, Ontario, Canada.
Leibovitz, A. 1963. The growth and maintenance of tissue-cell cultures hi free gas exchange with the
atmosphere. Amer. J. Hyg. 75:173-180.
Lcnnette, E. H., and N. J. Schmidt (ed.). 1979. Diagnostic Procedures for Viral, Rickettsial and Chlamydial
Infections, 5th ed. American Public Health Association, Inc., Washington, DC.
144
-------
Lund, E., and C.-E. Hedstrom. 1966. The use of an aqueous polymer phase system for enterovirus
isolations from sewage. Am. J. Epidemiol. 54:287-291.
Nielsen, A., and B. Lydholm. 1980. Methods for the isolation of virus from raw and digested wastewater
sludge. Water Res. 74:175-178.
Pancorbo, O. C., P. R. Scheuerman, S. R. Farrah, and G. Bitton. 1981. Effect of sludge type on poliovirus
association with and recovery from sludge solids. Can. J. Microbiol. 27:279-287.
Paul, J. 1975. Cell and Tissue Culture. 5th edition. Churchill Livingstone, London, Great Britain.
Rao, V. C., and J. L. Melnick. 1987. Human Viruses in Sediments, Sludges, and Soils. CRC Press, Boca
Raton, FL.
Rovozzo, G. C., and C. N. Burke. 1973. A Manual of Basic Virological Techniques. Prentice-Hall, Inc.,
Englewood Cliffs, NJ.
Safferman, R. S., M. E. Rohr, and T. Goyke. 1988. Assessment of recovery efficiency of beef extract
reagents for concentrating viruses from municipal wastewater sludge solids by the organic flocculation
procedure. Appl. Environ. Microbiol. 54:309-316.
Sattar, S. A., and J. C. N. Westwood. 1976. Comparison of four eluents in the recovery of indigenous
viruses from raw sludge. Can. J. Microbiol. 22:1586-1589.
Sattar, S. A., and J. C. N. Westwood. 1979. Recovery of viruses from field samples of raw, digested, and
lagoon-dried sludges. Bull. World Health Org. 57:105-108.
Stetler, R. E., M. E. Morris and R. S. Safferman. 1992. Processing procedures for recovering enteric viruses
from wastewater sludges. J. Virol. Meth. 40:67-76.
Subrahmanyan, T. P. 1977. Persistence of enteroviruses in sewage sludge. Bull. World Health Org.
55:431-434.
Turk, C. A., B. E. Moore, B. P. Sagik, and C. A. Sorber. 1980. Recovery of indigenous viruses from
wastewater sludges, using a bentonite concentration procedure. Appl. Environ. Microbiol. 40:423-425.
Ward, R. L., and C. S. Ashley. 1976. Inactivation of poliovirus in digested sludge. Appl. Environ.
Microbiol. 37:921-930.
Waymouth, C., R. G. Ham, and P. J. Chappie. 1981 The Growth Requirements of Vertebrate Cells In
Vitro. Cambridge University Press, Cambridge, Great Britain.
Wellings, F. M., A. L. Lewis, and C. W. Mountain. 1976. Demonstration of solids-associated virus in
wastewater and sludge. Appl. Environ. Microbiol. 37:354-358.
145
-------
-------
Appendix I
Analytical Method for Viable Helminth Ova*
388, 389, 390, 391, 392, 393. HELMINTH OVA
INTRODUCTION
Parasitic infections present a potential health risk associated
with use of sludge due to the existence of highly resistant
stages of the organisms and low infective doses. Ascaris ova are
the most commonly isolated nematode ova in sludge. Others may
include Trichuris. Toxocara. Hymenolepis and Taenia. In 1973,
ascariasis was estimated to affect four million people in the
United States. Ova from the parasitic helminths enter sewage
from the feces of infected individuals.
Ascaris ova are probably the most resistant of the ova or cysts
found in sludge. This fact, along with the common occurrence of
Ascaris ova, make their, a good indicator for the fate of parasites
as a group. The described test procedure was developed for solid
and semi-solid samples. It is not suitable for water or sewage.
A total solids analysis is also required to express the final
results as ova/g dry weight.
Procedure 388,389.390.391.392,393: Helminth Ova fll-25-91)
1. Scope and Application
1.1 This procedure determines CSDLAC parameter numbers 388,
Total Parasites; 389, Total Ascaris; 390, Viable
Ascaris; 391, Trichuris; 392, Hvmenolepis; 393,
Toxocara.
1.2 This procedure is applicable to composted sewage sludge
and other solid and semi-solid materials.
2. Summary of Procedure
2.1
This procedure identifies, quantifies and determines
the viability of several types of ova from intestinal
parasites. Solid samples are processed by blending
with buffered water containing a surfactant. The blend
is screened to remove large particles. The solids in
the screened portion are allowed to settle out and the
supernatant decanted off. The sediment is subjected to
density gradient centrifugation using zinc sulfate
(specific gravity 1.20). This flotation procedure
yields a layer most likely to contain Ascaris and some
other parasitic ova. Proteinaceous material is removed
using an acid-alcohol/ether extraction step and the
resulting concentrate is incubated at 26°C until
control ova of Ascaris lumbricoides var. suum are fully
embryonated. The concentrate is then microscopically
examined for parasite ova using a Sedgwick-Rafter
counting chamber.
388,389,391,392,393-1
2/92
This is an expanded version of the Yanko (1987) method referenced in the Part 503 regulation. This expanded version provides additional detail and presents the method in a step-by-step
fashion as provided in pages 393-1 to 393-6 of "Laboratory Section Procedures for the Characterization of Water and Wastes," 4th edition, published by the Sanitation Districts of Los An-
geles County, Los Angeles, California, 1989.
147
-------
5.
Sample Handling and Preservation
3.1 Solid samples are collected in sterile bags such as
Whirl-Pak bags. Liquid sludge samples are collected in
clean screw cap containers such as Nalgene bottles or
jars.
3.2 Samples not analyzed promptly are stored at 0°C to 4°C.
Adavantages and Limitations
4.1 Concentration of the sample increases the probability
that ova will be detected if they are in the sample.
4.2 Ascaris ova as an indicator is advantageous since they
are relatively large and easy to identify.
4.3. Seeded studies have indicated the recovery for this
test to be approximately ninety per cent.
4.4 The test uses standard microbiological equipment.
4.5 The test requires intensive training to enable the
analyst to identify ova in a complex mixture of debris
and to determine viability.
4.6 The test may take up to 5 weeks to complete including
sample processing and incubation.
4.7 Numerous transfers of the sample to new vessels may
decrease the recovery of indigenous ova.
Apparatus
5.1 Standard light microscope.
5.2 Sedgwick-Rafter cell.
5.3 2 L Pyrex beakers.
5.4 Table top centrifuge.
5.5 Rotor to hold four 100 mL centrifuge tubes, preferably
glass or teflon.
5.6 Rotor to hold eight 15 mL conical centrifuge tubes,
preferably glass or teflon.
5.7 48 mesh Tyler sieve.
5.8 Large plastic funnel to support sieve.
388,389,391,392,393-2
2/92
148
-------
5.9 Teflon spatula.
5.10 Large test tube rack to accommodate 100 mL centrifuge
tubes.
5.11 Small test tube rack to accommodate 15 mL conical
centrifuge tubes.
5.12 Number "0" rubber stoppers.
5.13 Wooden applicator sticks.
5.14 Vacuum source.
5.15 Vacuum flask, 2 L or larger.
5.16 Stopper to fit vacuum flask fitted with glass or metal
tubing as a connector for 1/4 inch tygon tubing.
5.17 Pasteur pipets.
5.18 Incubator at 26°C.
Reagents
6.1 Phosphate-buffered water. Prepare stock phosphate
buffer solution by dissolving 34.0 g potassium
dihydrogen phosphate (KH2PO4) in 500 mL distilled
water, adjusting to pH 7.2 ± 0.5 with 1 N NaOH, and
diluting to 1 L with distilled water.
Add 1.25 mL stock phosphate buffer solution and 5.O mL
magnesium chloride solution (81.1 g MgCl,.6H,O/L
distilled water) to 1 L distilled water.
Prepare phosphate buffer working solution containing
,0.1% (v/v) Tween 80. Adjust the pH to 7.2 ± 0.1 with
1 N NaOH.
6.2 Tween 80.
6.3 Zinc sulfate solution, sp. gr. 1.20. Weigh 454 g
ZnSO^ into 1 L DI H2O. Dissolve and check specific
gravity with a hydrometer. Adjust specific gravity to
1.2 as necessary.
6.4 0.1 N H2S04 in 35% ethyl alcohol.
6.5 Ethyl ether, reagent grade.
Procedure
388,389,391,392,393-3
2/92
149
-------
7.1 Weigh 50 g (wet weight) of compost and blend at high
speed for 1 min. with 450 mL phosphate buffered water
(PBW) containing 0.1 percent Tween 80 to achieve a ten
percent suspension. If the sample is a liquid sludge,
pour it directly into a blender jar (400 to 500 mL) and
add Tween 80 to 0.1% v/v prior to blending as above.
Record volume tested.
7.2 The % moisture of the sample is determined by the
Analytical group on a separate portion of the sample
for use in the final calculation of ova/g dry weight.
The concentration of ova in liquid sludge samples may
be expressed as ova per unit volume.
7.3 Pour the homogenized sample through a 48 mesh Tyler
sieve held on a large funnel over a 2 L beaker.
7.4 Wash the sample through the sieve with several rinses
of warm tap water. Washings are caught in the beaker.
7.5 Allow the screened and washed sample to settle
overnight.
7.6 Siphon off the supernatant to just above the settled
layer of solids.
7.7 Mix the settled material by swirling and then pour it
into two 100 mL centrifuge tubes.
7.8 Rinse the beaker two or three times and pour the
rinsings into two 100 mL centrifuge tubes.
7.9 Balance the tubes and centrifuge at 1250 RPM (400 x G)
for 3 min.
7.10 Pour off the supernatant and resuspend the pellet
thoroughly in zinc sulfate solution, specific gravity
1.20.
7.11 Centrifuge the zinc sulfate suspension at 1250 RPM for
3 min.
7.12 Pour the zinc sulfate supernatant into a 500 mL
Erlenmeyer flask, dilute to at least half the
concentration with deionized water, cover and allow to
settle 3 h or overnight.
7.13 Aspirate the supernatant to just above the settled
material.
7.14 Resuspend the sediment by swirling and pipette into two
to four 15 mL conical centrifuge tubes.
388,389,391,392,393-4
2/92
150
-------
7.15 Rinse the flask two to three times with deionized water
and pipette the rinse water into the tubes.
7.16 Centrifuge the tubes at 1400 RPM (480 x G) for 3 min.
7.17 Combine the pellets into one tube and centrifuge at
1400 RPM for 3 min.
7.18 Resuspend the pellets in 7 mL acid alcohol solution
(0.1 N H2SO4 in 35% EtOH) and add 3 mL of ether.
7.19 Cap the tube with a rubber stopper and invert several
times, venting each time.
7.20 Centrifuge the tube at 1800 RPM (660 x G) for 3 min.
7.21 Resuspend the pellet in 4 mL 0.1 N H2SO4 and pour into
Nalgene tubes with loose caps.
7.22 Incubate the tubes at 26°C for three to four weeks.
7.22.1
7.22.2
Simultaneously incubate control ova dissected
from an adult Ascaris lumbricoides var. suum.
When the majority of control ova are
embryonated, samples are ready to be
examined.
7.23 Examine concentrates microscopically using a
Sedgwick-Rafter cell to enumerate detected ova.
7.23.1 Note viability based on the presence of
embryonated ova whose larval forms can be
induced to move when the light intensity is
increased.
7.23.2 Identify the ova and report as ova/g dry
weight.
8. Calculation
8.1 Calculate % total solids using the % moisture result:
% Total Solids = 100% - % moisture
8.2 Calculate ova/g dry weight in the following manner:
Ova/g dry wt = (No. ova) x (Cell factor/# transects) x
(mL final volume)
(% cone)x( mL sample screened)x
388,389,391,392,393-5
2/92
151
-------
(% total solids)
Where: Transect = One microscope field diameter width
across the length of a Sedgwick-Rafter cell
Cell Factor = # of transects to examine entire
Sedgwick-Rafter. This is dependent on microscope
model and magnification.
Cell factor is also equal to the number of
transects per mL since the Sedgwick-Rafter cell
contains 1 mL.
9. Quality Assurance Guidelines
9.1 Run duplicate tests every tenth sample.
10. Precision and Accuracy
10.1 Precision criterion was established as per Standard
Methods. 17th Ed., 1989, 9020B.4b, pp. 9-17 to 9-18.
10.2 The current established precision criterion is 0.5702T
10.3 There is currently no means of assessing the accuracy
of the method.
11. References
11.1 Theis, J. H., V. Bo1ton and D. R. Storm (1978).
"Helminth Ova in Soil and Sludge from Twelve U.S. Urban
Areas". J. Water Pollution Control Fed. 50:2485-2493.
11.2 Reimers, R. S., et al, 1981. "Parasites in Southern
Sludges and Disinfection by Standard Sludge Treatment".
Tulane Univ., New Orleans, LA. NTIS Publication #
PB82-102344, Springfield, VA.
11.3 Yanko, W. A. "Occurrence of Pathogens in Distribution
and Marketing Municipal Sludges". County Sanitation
Districts of Los Angeles County, Whittier, CA. NTIS
Publication # PB88-154273-AS, pp. 159-161.
11.4 Standard Methods for the Examination of Water and
Wastewater. 17th Ed., 1989, 9020 B and 9050 C.
388,389,391,392,393-6
2/92
152
-------
-------
------- |