Pathogens and Vector Attraction in Sewage Sludge


&EPA

United States
Environmental Protection
Agency

EPA/600/R-22/194 | January 2023 | www.epa.gov/research

Pathogens and Vector
Attraction in Sewage Sludge

Office of Research and Development
Washington, D.C.

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EPA/600/R-22/194
January 2023

PATHOGENS AND VECTOR
ATTRACTION IN SEWAGE SLUDGE

Edited and Prepared by

Laura Boczek, Ronald Herrmann

U.S. Environmental Protection Agency
Center for Environmental Solutions and Emergency Response
Office of Research and Development
26 W. Martin Luther King Drive
Cincinnati, OH 45268

Elizabeth Resek, Tess Richman

U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
1200 Pennsylvania Avenue, N.W.
Washington, DC 20460

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NOTICE

This document has been reviewed in accordance with U.S. Environmental Protection Agency policy

and approved for publication. Any mention of trade names, manufacturers or commercial
products does not imply an endorsement by the United States Government or the U.S.
Environmental Protection Agency. EPA and its employees do not endorse any commercial
products, services, or enterprises.

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FOREWORD

The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the Nation's land,
air, and water resources. Under a mandate of national environmental laws, the Agency strives to
formulate and implement actions leading to a compatible balance between human activities and the
ability of natural systems to support and nurture life. To meet this mandate, EPA's research program is
providing data and technical support for solving environmental problems today and building a science
knowledge base necessary to manage our ecological resources wisely, understand how pollutants affect
our health, and prevent or reduce environmental risks in the future.

The Center for Environmental Solutions and Emergency Response (CESER) within the Office of Research
and Development (ORD) conducts applied, stakeholder-driven research and provides responsive technical
support to help solve the Nation's environmental challenges. The Center's research focuses on innovative
approaches to address environmental challenges associated with the built environment. We develop
technologies and decision-support tools to help safeguard public water systems and groundwater, guide
sustainable materials management, remediate sites from traditional contamination sources and emerging
environmental stressors, and address potential threats from terrorism and natural disasters. CESER
collaborates with both public and private sector partners to foster technologies that improve the
effectiveness and reduce the cost of compliance, while anticipating emerging problems. We provide
technical support to EPA regions and programs, states, tribal nations, and federal partners, and serve as
the interagency liaison for EPA in homeland security research and technology. The Center is a leader in
providing scientific solutions to protect human health and the environment.

Gregory Sayles, Ph.D., Director

Center for Environmental Solutions and Emergency Response

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TABLE OF CONTENTS

1: Introduction	1

2: Sewage Sludge Pathogens	7

3: Domestic Septage	19

4: Class A Pathogen Requirements	22

5: Processes to Further Reduce Pathogens (PFRPs)	35

6:	Class B Pathogen Requirements for Material Applied to Agricultural Land, a Forest,

or a Reclamation Site	47

7: Processes to Significantly Reduce Pathogens (PSRPs)	56

8: Equivalency and EPA's Pathogen Equivalency Committee	65

9: Requirements for Reducing Vector Attraction	75

10: Sampling Procedures and Analytical Methods	87

11: References/Additional Resources	112

12: Appendix A	A-l

13: Appendix B	B-l

14: Appendix C	C-l

15: Appendix D	D-l

16: Appendix E	E-l

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17:	Appendix F	F-l

18:	Appendix G	G-l

19:	Appendix H	H-l

20:	Appendix I	1-1

21:	AppendixJ	J-l

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ACRONYMS AND ABBREVIATIONS

EPA	Environmental Protection Agency

POTW	Publicly Owned Treatment Works

CWA	Clean Water Act

CFR	Code of Federal Register

PSRP	Processes that Significantly Reduce Pathogens

PFRP	Processes to Further Reduce Pathogens

EQ	Exceptional Quality

VAR	Vector Attraction Reduction

MPN	Most Probable Number

CFU	Colony Forming Units

PFU	Plaque Forming Units

QAPP	Quality Assurance Project Plan

SRT	Solids Retention Time

MCRT	Mean Cell Residence Time

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ACKNOWLEDGMENTS

This document was originally produced by the U.S. Environmental Protection Agency (EPA) Pathogen
Equivalency Committee (PEC), specifically members James E. Smith Jr., Robert Brobst, Robert K. Bastian,
and Mark C. Meckes provided significant contributions to the original document published in 1999, and
the revised document in 2003. Along with the preparers of this document Laura Boczek, Ronald
Herrmann, Elizabeth Resek, and Tess Richman this version has been further updated with the assistance
of the following reviewers Chris Impellitteri, EPA (ORD) and Cassandra Kirk EPA (OW) and editorial
contributions from Jeongwon Ryu.

This document has been organized differently from the previous versions to help remove redundancy
and duplication that existed in those versions, as well as improve the flow and allow the reader an easier
time navigating through the document. In addition to formatting changes this document has been
updated to clarify Alternatives 3 and 4 specifically for Class A pathogen reduction. The methods section
has been updated to include the U.S. EPA methods for testing biosolids for the presence of fecal
coliforms and Salmonella.

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1: INTRODUCTION

1.1 Sewage Sludge and Regulations

Sewage sludge (often termed "biosolids") results from the treatment of domestic sewage in a wastewater
treatment facility. When applied to land at the appropriate agronomic rate biosolids provide several
benefits including nutrient and water addition, and soil structure enhancement1. Land application of
biosolids also can have economic and waste management benefits including conservation of landfill
space, reduction of demand on non-renewable resources like phosphorus and reduction of farm costs for
fertilizers. There are over 14,600 publicly owned treatment works (POTWs) servicing over 238 million
people across the U.S.2 Additionally, there are more than 60 million people in the U.S. that have private
sewage systems (septic systems)3. While data reported to EPA are limited, Figure 1 is based on 20194
electronic reporting to EPA5 by over 2,200 facilities.

Biosolids Use & Disposal from POTWs in 2019

Figure 1. Distribution of biosolids use and disposal based on 2019 EPA electronic reporting data. It should
be noted that smaller facilities treating less than 1 million gallons per day and private wastewater
treatment facilities are not represented in Figure 1. However, according to a 2007 North East Biosolids
and Residuals Association (NEBRA) report these facilities generate about eight percent of the total flow

1 Various studies sited at: U.S. Department of Agriculture Agricultural Research Service.

2 EPA Clean Watersheds Needs Survey 2012: Report to Congress.

3 EPA Office of Wastewater Management.

4 For facilities with a (1) Class 1 management facilities (any publicly owned treatment works (POTW) with approved
pretreatment program); (2) major POTWs (POTWs with a design flow rate greater than or equal to one million
gallons per day); and (3) POTWs that serve 10,000 people or more or otherwise required to report by EPA or
permitting authority.

5 EPA Enforcement and Compliance History Online.

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generated in the United States6,7. These smaller treatment facilities tend to store solids in lagoons,
transport untreated solids to larger wastewater treatment plants, and generally use the lowest-cost and
easiest methods of disposal such as landfilling given the economies of scale involving beneficial use of
biosolids.

Biosolids can be applied to agricultural land (e.g., pastures and cropland), disturbed areas (e.g., mined
lands and construction sites), plant nurseries, forests, recreational areas (e.g., parks and golf courses),
cemeteries, highway and airport runway medians, and home lawns and gardens.

Section 405(d) of the Clean Water Act (CWA) requires EPA to:

•	Establish numeric limits and management practices that protect public health and the
environment from the reasonably anticipated adverse effects of chemical and microbial
pollutants during the use or disposal of sewage sludge.

•	Review biosolids (sewage sludge) regulations every two years to identify additional toxic
pollutants that occur in biosolids (i.e., biennial reviews) and set regulations for those pollutants
if sufficient scientific evidence shows they may harm human health or the environment.

As required by Section 405(d) of the CWA, EPA developed a regulation to protect public health and the
environment from any reasonably anticipated adverse effects of pollutants that might be present in
sewage sludge. This regulation, The Standards for the Use or Disposal of Sewage Sludge, was published
on February 19, 1993 (58 FR 9248).

The Standards for the Use oir Di.spo.sal of Sewage Sludge, found in 40 CFIR Part 503 or "Part 503",
establishes requirements for the final use or disposal of sewage sludge when it is: 1) applied to land as a
fertilizer or soil amendment; 2) placed in a surface disposal site, e.g. sewage sludge-only landfills; or 3)
incinerated in a sewage sludge incinerator. Sewage sludges that are used as alternative daily,
intermediate, or final cover at municipal solid waste landfills are regulated under 40 CFR Part 258. 40 CFR
Part 503, Subpart B land application requirements include:

•	Applicability (§ 503.10)

•	Special definitions (§ 503.11)

•	General requirements (§ 503.12)

•	Pollutant limits (§ 503.13)

•	Management practices (§ 503.14)

6	NEBRA (July 20, 2007) 'A national biosolids regulation, quality, end use & disposal survey', North East Biosolids and
Residuals Association, Tamworth, NH. Available

at: https://staticl.squarespace.eom/static/54806478e4b0dc44el698e88/t/5488541fe4b03c0a9b8ee09b/14182205
75693/NtlBiosolidsReport-20J uly07.pdf

7	h ttps:://'www „ b i osol i d sd a ta „ o rg

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• Operational standards - pathogens and vector attraction reduction (§ 503.15)

• Frequency of monitoring (§ 503.16)

• Recordkeeping (§503.17)

• Reporting (§ 503.18)

All Part 503 requirements apply to publicly- and privately-owned treatment works that generate or treat
domestic sewage sludge and to anyone who uses or disposes of sewage sludge. The requirements of Part
503 are self-implementing and must be followed even without the issuance of a permit. In addition,
persons using or disposing biosolids are subject to state and potentially county or local biosolids
management regulations.

Complete information on requirements for pathogen and vector attraction reduction can be found in 40
CFR Part 503, Subpart D. These requirements are designed to reduce the presence of, and potential for
contact with, the disease bearing microorganisms (pathogens) in sewage sludge applied to the land or
placed on a surface disposal site. Requirements are divided into two areas:

• (§503.32) Requirements designed to control and reduce pathogens in biosolids; and

• (§503.33) Requirements designed to reduce the ability of the biosolids to attract vectors (insects
and other living organisms that can transport biosolids pathogens away from the land application or
surface disposal site).

This document is intended as a resource to pathogen and vector attraction reduction for anyone involved
with the treatment of sewage sludge for land application. For information on other land application
requirements (applicability, special definitions, general requirements, pollutant limits, management
practices, frequency of monitoring, recordkeeping, and reporting) please consult Part 503.

1.2 Definitions

Terms used throughout this guidance are consistent with Part 503, however terminology not defined by
Part 503 also may be used. Examples include, but are not limited to, the term "biosolids", operational
parameters and biosolids management. The following glossary provides common terms used in this
document:

Applier - The applier is the individual or party that land applies treated sewage sludge (biosolids). This
may include farmers, municipalities, private enterprises and contractors of these parties.

Biosolids - Biosolids are treated sewage sludge that meet Part 503 requirements for land application.
Typically intended to be land applied as a soil amendment or fertilizer.

Exceptional Quality (EQ) Biosolids - Class A "Exceptional Quality" or "EQ" biosolids is treated sewage
sludge that meets the pollutant concentrations in § 503.13(b)(3), the Class A pathogen requirements in §
503.32(a) and one of the vector attraction reduction requirements in §§ 503.33(b)(1) through (b)(8). As
such, Class A EQ biosolids meets the most stringent pollutant, pathogen, and vector attraction reduction
requirements under EPA's regulations. Biosolids in this category are not subject to the Part 503 general

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requirements (§ 503.12) and management practices (§ 503.14) for land application. It should be noted
that the term "Exceptional Quality (EQ)" is not used in Part 503 (Table 1.0), however it is commonly
understood within the regulatory and regulated sectors.

Table 1.0 Minimum Requirements for Land Application



Pollutant Limit Requirement
§503.13(a)(l)-(a)(4) and
§503.13(b)(l)-(b)(4)

Pathogen Requirements

§503.13(a)
§503.32(a)(3)-(a)(8) and
§503.32(b)(2)-(b)(4)

Vector Attraction
Reduction Requirement
§503.15(c)
§503.33(b)(l)-(b)(10)

Class A
Exceptional
Quality (EQ)

Ceiling
Concentrations

(a)(1)

Pollutant
Concentration (b)(3)

Any Class A Alternative
(a) (3 )-(a)(S)

Any Alternative l-(b)(l)-(b)(8)

Class A

Ceiling
Concentrations

(a)(1)

Pollutant
Concentration (a)(2)-
(a)(4)or(b)(3)

Any Class A Alternative
(a)(3)-(a)(8)

Alternative 9 or 10
(b)(9)or (b)(10)

Cumulative Pollutant
Loading Rates (a)(4) or
(b)(4)

Any Alternative
l-10(b)(l)-(b)(10)

Annual Pollutant
Loading Rates
(a)(4) or (b)(4)

Any Alternative
1-8 (b)(l)-(b)(8)

Class B

Ceiling
Concentrations

(a)(1)

Pollutant
Concentration
(a)(2)-(a)(4) or (b)(3)

Any Class B Alternative
(b)(2)-(b)(4)

Any Alternative
1-10 (b)(l)-(b)(10)

Cumulative Pollutant
Loading Rates
(a)(2) or (b)(2)

Any Alternative
1-10 (b)( 1 )-(b)( 10)

Class A Biosolids - Sewage sludge intended for land application that has been either treated or tested for
the presence of microbial pathogens through the use of process indicator organisms e.g. fecal coliforms
or salmonella, enteric viruses, and viable helminth ova. The term Class A biosolids typically refers only to
the pathogen monitoring and / or treatment of the sewage sludge.

Class B Biosolids - Sewage sludge intended for land application that has been either treated or tested for
the presence of microbial pathogens. Class B materials when land applied must adhere to management
practices, including public access, crop harvest, and animal grazing restrictions once they are land applied
because they could contain pathogens. The term Class B biosolids typically refers to the level of pathogen
destruction or treatment.

Detectable Limits - Minimum measured concentration at which an analyte can be detected. The
detectable limit for any given analyte varies depending on the lab methodology used and the volume of
material analyzed.

Domestic Septage - Domestic septage is either liquid or solid material removed from a septic tank,
cesspool, portable toilet, Type III marine sanitation device, or similar treatment works that receives only
domestic sewage. Domestic septage does not include liquid or solid material removed from a septic tank,

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holding tank, cesspool, or similar treatment works that receives either commercial wastewater or
industrial wastewater and does not include grease removed from a grease trap at a restaurant.

Domestic Sewage - Domestic sewage is waste and wastewater from humans or household operations
that is discharged to or otherwise enters a treatment works.

Indicator Organism - A bacterium, group of bacteria, virus, or protozoa that are used to estimate the level
of pathogens present. These organisms are used to help determine if the sewage sludge treatment was
able to sufficiently reduce the microbial populations including potential pathogens. Fecal coliform is an
indicator used in sewage sludge regulations.

Non-Public Contact Sites - Sites that are not frequently visited or used by the public such as agricultural
land, forests, and reclamation sites.

Pathogen - A bacterium, virus, or other microorganism that can cause disease.

Preparer - The entity who prepares sewage sludge is either the person who generates biosolids during the
treatment of domestic sewage in a treatment works or the person who changes the quality of sewage
sludge received from a treatment works prior to land application or derives a material from sewage
sludge (§ 503.9(r)). Any time the quality of sewage sludge is changed, a material is derived from sewage
sludge and the preparer becomes a generator. Examples of materials derived from biosolids include, but
are not limited to, the mixing of multiple sources of sewage sludge (as from wastewater treatment
facilities), biosolids treated by composting (where sewage sludge is mixed with bulking agents or other
admixtures), pelletizing, or drying, and mixtures of non EQ biosolids with other materials (e.g., biosolids
blended with soil).

Process to Further Reduce Pathogens (PFRP) - PFRP terminology was used in the original sewage sludge
regulations prior to the Part 503 regulations. A PFRP is a process that can consistently reduce the density
of the microbial population including all bacterial, viral, and protozoan pathogens to below detectable
levels. PFRP processes follow Class A criteria for pathogens, vector attraction reduction, and land
application.

Process to Significantly Reduce Pathogens (PSRP) - PSRP terminology was used in the original sewage
sludge regulations prior the Part 503 regulations. A PSRP is a process that can consistently reduce the
density of the microorganisms in sewage sludge by 2 log or greater, or by a factor of 100. PSRP processes
establish treatment parameters for Class B biosolids.

Product - The term "product" is sometimes used in this document in discussions regarding material
distribution. A product is the treated sewage sludge (biosolids) for final use or disposal that has met Part
503 requirements. Product can refer to any biosolids that meet land application requirements. Treatment
includes, but is not limited to, thickening, stabilization, and dewatering of sewage sludge.

Sewage Sludge - Sewage sludge is defined in Part 503 as "solid, semi-solid, or liquid residue generated
during the treatment of domestic sewage in a treatment works. Sewage sludge includes, but is not limited
to, domestic septage; scum or solids removed in primary, secondary, or advanced wastewater treatment
processes; and any material derived from sewage sludge. Sewage sludge does not include ash generated

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during the firing of sewage sludge in a sewage sludge incinerator or grit and screenings generated during
preliminary treatment of domestic sewage in a treatment works". The distinction between untreated
sewage sludge and treated sewage sludge (biosolids) is made throughout this document.

Vectors - Organisms such as insects and rodents, that can spread disease by carrying and transferring
pathogens.

1.3 References

Alberici, T.M., W.E. Sopper, G.L Storm, and R.H. Yahner. 1989. Trace metals in soil, vegetation, and voles from
mine land treated with sewage sludge. Journal of Environmental Quality 18:115-120.

Anderson, T.J., and G.W. Barrett. 1982. Effects of dried sewage sludge on meadow vole (Microtus pennsylvanicus)
populations in two grassland communities. Journal of Applied Ecology 19:759-772.

Bastian, R.K. 1997. The biosolids (sludge) treatment, beneficial use, and disposal situation in the USA. European
Water Pollution Control Journal, Vol 7, No. 2, 62-79. B.L., H.R. Wilson, M.F. Hall, W.L Johnson, O. Osuna, R.L
Suber, and G.T. Edds. 1982. Effects of feeding dried municipal sludge to broiler type chicks and laying hens. Polut.
Sci. 61:1073-1081.

Hegstrom, LJ. and S.D. West. Heavy metal accumulation in small mammals following sewage sludge application to
forests. Journal of Environmental Quality 18:345.

Keinholz, E.W. 1980. Effects of toxic chemicals present in sewage sludge on animal health. P 153-171 in Sludge -
Health risks of land application. Ann Arbor Science Publishers.

Keinholz, E.W., G.M. Ward, D.E. Johnson, J. Baxter, G. Braude, and G. Stern. 1979. Metropolitan Denver sludge fed
to feedlot steers. J. Anim. Sci. 48:735-741.

National Research Council. 1996. Use of reclaimed water and sludge in food crop production. Washington, D.C.

USEPA/USDA/FDA. 1981. Land Application of Municipal Sewage Sludge for the Production of Fruits and
Vegetables; A Statement of Federal Policy and Guidance. SW-905. U.S. EPA, Office of Solid Waste, Washington,
D.C. 21pp.

USEPA. 1984. EPA policy on municipal sludge management. Federal Register, 49(114):24358-24359. June 12,

1984.

USEPA. 1991. Interagency Policy on beneficial use of municipal sewage sludge on federal land. Federal Register,
56(138):33186-33188. July 19, 1991.

USEPA. 1992. Technical support document for Part 503 pathogen and vector attraction reduction requirements in
sewage sludge. NTIS No.: PB89-136618. Springfield, VA: National Technical Information Service.

USEPA. 1995. Part 503 implementation guidance. EPA 833-R-95-001. Washington, D.C.

WEF/U.S. EPA. 1997. Biosolids: A short explanation and discussion. In Biosolids Fact Sheet Project.

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2: SEWAGE SLUDGE PATHOGENS

2.1	Introduction

This chapter provides general information on pathogens associated with sewage sludge, biosolids and
wastewater treatment processes. Biosolids preparers should have a basic knowledge of microbiology so
that they can understand what is expected to meet Part 503 requirements. Basic knowledge will also
help address questions regarding pathogens and the protection of public health and the environment.
The Part 503 regulations are designed to significantly reduce or eliminate the risk from pathogens present
in treated sewage sludges.

2.2	Pathogens

A pathogen is an organism capable of causing disease to a host. For that capability to be realized, three
main criteria must be met: a susceptible host, a route of exposure, and an infectious agent. Without
meeting these criteria, exposure to a pathogen will have no effect on human health. Treatment processes
and management practices described in this document protect public health by addressing one or more
of the criteria required for disease to occur. Throughout this document, "pathogen" refers only to living
organisms, except where specified.

Pathogens that propagate in the enteric or urinary systems of humans and are discharged in feces or
urine pose the greatest risk to public health regarding the management of sewage sludge and/or
biosolids. Pathogens are also found in the urinary and enteric systems of other animals and may
propagate in non-enteric settings. However, because this document is concerned with the regulation of
sewage sludge and biosolids, this chapter focuses on the pathogens most found in the human enteric
system.

2.3	Pathogens in Sewage Sludge

The four major types of human pathogenic organisms (bacteria, viruses, protozoa, and helminths) all may
be present in domestic sewage. The actual species and quantity of pathogens present in the domestic
sewage from a particular municipality (and the sewage sludge produced when treating the domestic
sewage) depend on the health status of the local community and may vary substantially at different
times. The level of pathogens present in treated sewage sludge (biosolids) also depends on the reductions
achieved by the wastewater and sewage sludge treatment processes.

Wastewater treatment processes typically have 2 treatment trains which consist of the liquids also known
as effluents, and the solids called sewage sludges. Pathogens can occur in both the liquids as well as
settle out with the solids during wastewater treatment. Biological wastewater treatment processes such
as lagoons, trickling filters, and activated sludge treatment may substantially reduce the number of
pathogens in the wastewater (USEPA, 1989). These processes may also reduce the number of pathogens
in sewage sludge by creating adverse conditions for pathogen survival.

Nevertheless, the resulting biological sewage sludges may still contain sufficient levels of pathogens to
pose a public health and environmental concern. Table 2.1 lists some principal pathogens of concern that
may be present in wastewater and sewage sludge. These organisms and other pathogens can cause

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infection or disease if humans and animals are exposed to sufficient levels of the organisms or pathogens.
The levels, called infectious doses, vary for each pathogen and each host.

Table 2.1 Principal Pathogens of Concern in Domestic Sewage and Sewage Sludge

Organism

Disease/Symptoms

Bacteria

Salmonella sp.

Salmonellosis (food poisoning), typhoid fever

Shigella sp.

Bacillary dysentery

Yersinia sp.

Acute gastroenteritis (including diarrhea, abdominal pain)

Vibrio cholerae

Cholera

Campylobacter jejuni

Gastroenteritis

Escherichia coli (pathogenic
strains)

Gastroenteritis

Enteric Viruses

Hepatitis A virus

Infectious hepatitis

Norwalk and Norwalk-like viruses

Epidemic gastroenteritis with severe diarrhea

Rotaviruses

Acute gastroenteritis with severe diarrhea

Enteroviruses

Poliovi ruses

Poliomyelitis

Coxsackieviruses

Meningitis, pneumonia, hepatitis, fever, cold-like symptoms, etc.

Echoviruses

Meningitis, paralysis, encephalitis, fever, cold-like symptoms, diarrhea, etc.

Reovirus

Respiratory infections, gastroenteritis

Astroviruses

Epidemic gastroenteritis

Caliciviruses

Epidemic gastroenteritis

Protozoa

Cryptosporidium

Gastroenteritis

Entamoeba histolytica

Acute enteritis

Giardia lamblia

Giardiasis (including diarrhea, abdominal cramps, weight loss)

Balantidium coli

Diarrhea and dysentery

Toxoplasma gondii

Toxoplasmosis

Helminth Worms

Ascaris lumbricoides

Digestive and nutritional disturbances, abdominal pain, vomiting
May produce symptoms such as coughing, chest pain, and fever due to
worms migrating within the body

Ascaris suum

Trichuris trichiura

Abdominal pain, diarrhea, anemia, weight loss

Toxocara canis

Fever, abdominal discomfort, muscle aches, neurological symptoms

Taenia saginata

Nervousness, insomnia, anorexia, abdominal pain, digestive disturbances

Taenia solium

Nervousness, insomnia, anorexia, abdominal pain, digestive disturbances

Necator americanus

Hookworm disease

Hymenolepis nana

Taeniasis

Source: Kowal (1985) and USEPA (1989).

2.4 Survivability of Pathogens

Wastewater generally contains significantly high concentrations of pathogens which may enter the
wastewater system from industries, hospitals, and infected individuals. Wastewater treatment removes
many of these pathogens from the influent through processes like solids settling and thereby

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concentrating the pathogens in the sewage sludge. Pathogens that do not settle out with the solids
remain in the liquid phase of wastewater and are further subjected to other processes used in secondary
and tertiary treatments of wastewater. Like any other living organisms, pathogens thrive only under
certain conditions. Outside of these set conditions survivability decreases. Each pathogen species has a
different tolerance to different conditions; pathogen reduction requirements are therefore based on the
need to reduce all pathogenic populations. Some of the factors that influence the survival of pathogens
include pH, temperature, competition from other microorganisms, sunlight, contact with host organisms,
proper nutrients, and moisture level. Table 2.2 shows a comparison of the survival of bacteria, viruses,
and parasites in different sewage sludge treatments.

Table 2.2 Summary of the Effects of Sewage Sludge Treatment on Pathogens (Log Reductions Shown*)

„ . ... Parasites

PSRP Treatment Bacteria Viruses

(protozoa and helminths)

Anaerobic Digestion

0.5-4.0

0.5-2.0

0.5

Aerobic Digestion

0.5-4.0

0.5-2.0

0.5

Composting (PSRP)

2.0-4.0

Air Drying

0.5-4.0

Lime Stabilization

0.5-4.0

4.0

0.5

*A 1-log reduction (10-fold) is equal to a 90 percent reduction.
Class B processes are based on a 2-log reduction.

2.5 Units for Measuring Microorganisms

Density of microorganisms in Part 503 is defined as number of microorganisms per unit mass of total
solids (dry weight). Ordinarily, microorganism densities are determined as number per 100 milliliters of
wastewater. While the use of units of volume is sensible for wastewater, it is less sensible for sewage
sludge. Many microorganisms in sewage sludge are associated with the solid phase. When sewage sludge
is diluted, thickened, or filtered, the number of microorganisms 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 requires that sewage sludge solids content
always be determined when measuring microorganism densities.

Under Part 503, the density limits for the microorganisms are expressed as numbers of plaque forming
units (PFUs), colony forming units (CFUs), or most probable number (MPNs) per 4 grams dry weight
sewage sludge. 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 (e.g., 0.25/g or 0.75/g). However, density limits for
fecal coliforms, the indicator organisms, are given on a "per gram" basis because these organisms are
much more numerous than pathogens.

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2.6 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 (numbers) under a microscope. Viruses are
usually counted in plaque-forming units (PFU). Each PFU represents an infection zone where a single
infectious 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 statistical estimate of numbers in a
sample. The sample is diluted at least once into tubes containing nutrient medium. The tubes are
maintained under conditions favorable for bacterial growth. The original bacterial density in the sample
is estimated based on the number of tubes that show growth and the level of dilution in those tubes.

2.7 Pathogen Reduction

Pathogen reduction can be achieved by treating sewage sludge prior to use or disposal and through
natural attenuation. Many sewage sludge treatment processes are available that use a variety of
approaches to reduce pathogens and alter the sewage sludge so that it becomes a less effective medium
for microbial growth and vector attraction (Table 2.3). Processes vary significantly in their effectiveness.
For example, some processes (e.g., lime stabilization) may effectively reduce 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 operated. 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.

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Table 2 3. General Approaches to Controlling Pathogens and Vector Attraction in Sewage Sludge

Treatment Approach

Effectiveness

Process Examples3

Application of high
temperatures (tem-
peratures may be generated
by chemical, biological, or
physical processes).

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.

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 (biological and physical
heat)b

Application of radiation

Depends on dose. Sufficient
doses can reduce bacteria,
viruses, protozoan cysts, and
helminth ova to below detectable
levels. Viruses are most resistant
to radiation.

Gamma and high-energy electron beam
radiation.

Application of chemical
disinfectants

Substantially reduces bacteria
and viruses and vector attraction.
Probably reduces protozoan
cysts. Does not effectively reduce
helminth ova unless combined
with heat.

Lime stabilization

Reduction of the sewage
sludge's volatile organic
content (the microbial food
source).

Reduces bacteria. Reduces vector
attraction.

Aerobic digestion
Anaerobic digestion
Composting15

Removal of moisture from
the sludge.

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.

Air or heat drying

aSee Chapters 5 and 7 for a description of these processes. Many processes use more than one approach to reduce
pathogens.

Effectiveness depends on design and operating conditions.

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2.8 Monitoring Indicator Species

Sewage sludge may contain numerous species of pathogenic organisms and conducting an analysis for
each species is impractical and unnecessary. Therefore, the Part 503 microbiological requirements are
largely based on the use of indicator organisms for the possible presence of pathogens. These
requirements test for both the representative and hardiest of known viral, helminth, and bacterial species
to represent the larger set of pathogens. The indicator and representative organisms are ones having
been found to respond to treatment processes and environmental conditions in a similar manner to other
organisms. Monitoring the levels of these organisms, therefore, provides information about the survival
of the larger group.

The microbiological indicators that are required for monitoring under Part 503 include helminth ova,
enteric virus, fecal coliforms and Salmonella spp. Helminth ova, enteric viruses, and Salmonella spp. are
considered pathogens. These organisms are not always found in untreated or treated sewage sludge.

Even though these organisms are not always present in sewage sludge, they remain as process indicators
because they represent microbes that are diverse and can be environmentally resistant to treatments.

Fecal coliforms are enteric bacteria that are used as indicators for the presence of bacterial pathogens.
Although fecal coliforms themselves are usually not harmful to humans, their presence indicates that
pathogens may be present. They are abundant in human feces and therefore are always present in
untreated sewage sludge. They are easily and inexpensively measured, and their densities decline in
about the same proportion as enteric bacterial pathogens during sludge processing (USEPA, 1992).

Salmonella sp. may be found in untreated sewage sludges. Salmonella typically has a low infectious dose
and can cause disease with ingestion of a lower amount of these organisms than many other bacterial
pathogens. The problem with using Salmonella sp. as an indicator of treatment is that in many instances
Salmonella occurs variably, and in such low numbers that it is below the detection limits for the methods
used to isolate it from sewage sludge or biosolids. Using this indicator can give a false sense that the
sewage sludge was adequately treated when there is no way of knowing if Salmonella was in the
untreated material.

Tests required by Part 503 for helminth ova are employed to determine their presence and viability. The
only helminth ova viability that can be determined is that of Ascaris sp., the hardiest of known helminth
species. It follows that if conditions are such that Asacaris cannot survive, the same conditions would
prevent the survival of other helminth species such as Toxacara, Trichuris, and Hymenolepis.

Enteric viruses represent a class of many diverse different viruses that can be found in the intestinal
system of humans. Collectively they are known as enteric viruses. Part 503 testing for viruses
simultaneously monitors for several enterovirus species that are presumed to be good representatives for
other types of enteric viruses. Enteric viruses are not the only viruses that are found in sewage sludge.

2.9 Bacteria Regrowth

One of the primary concerns for biosolids preparers is regrowth of pathogenic bacteria. Some bacteria
are unique in that they can multiply outside of a host. The processes outlined in Part 503 and in this
document have been demonstrated to reduce pathogens, but even very small populations of certain

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bacteria can rapidly proliferate under the right conditions. For example, regrowth can occur in sewage
sludge in which the competitive bacterial populations have been essentially eliminated through
treatment. Some examples of these treatments include pasteurization, radiation, and some drying
technologies. During these treatments many, if not all, the bacteria can be effectively killed off, and
during those processes the carbon biomass is often broken down into smaller fragments which can
become a food source for other and or surviving microorganisms. If these food sources come into
contact with pathogens, they can cause exponential growth of the bacterial population (Chen et al. 2011,
Gibbs, 2007, Qi, 2008, Sidu et al. 2001, Zaleski et al. 2005). Regrowth does not occur for viruses,
helminths, and protozoa because they cannot regrow outside their specific host organism(s). Once
reduced by treatment, their populations do not increase. Part 503 contains specific requirements
designed to ensure that regrowth of bacteria has not occurred prior to use or disposal.

2.10 Potential Exposure to Pathogens

Humans and animals could be exposed to pathogens directly by contact with untreated or improperly
treated sewage sludge, mishandled biosolids, or indirectly by consuming drinking water or food con-
taminated by sewage sludge pathogens. Insects, birds, rodents, and even farm workers could contribute
to these exposure routes by transporting sewage sludge and biosolids including pathogens away from the
site. Potential routes of exposure include:

2.11 Direct Contact

Examples of direct contact include:

• Touching the sewage sludge or biosolids which may lead to infection of the skin, or pathogens
may find their way into the mouth through contaminated hands.

• Walking through an area such as a field, forest, or reclamation area shortly after biosolid
application.

• Handling soil from fields where biosolids have been applied.

Direct contact could occur through an inhalation exposure of microbes that become airborne (via
aerosols and dust) during sewage sludge spreading or by strong winds, plowing, or cultivating the soil
after application (Pillai, 2007). However, additional studies show the exposure of inhalation due to land
application of biosolids is minimal. (Herrmann et al. 2017, Tanner et al. 2008, Brooks et al. 2005)

2.12 Indirect Contact

Examples of indirect contact include:

• Consumption of pathogen-contaminated crops grown on biosolid-amended soil.

• Consumption of pathogen-contaminated milk or meat from animals contaminated by grazing in
pastures or fed crops grown on biosolid-amended fields.

• Ingestion of drinking water or recreational waters contaminated by runoff from nearby land
application sites or by organisms from biosolid migrating into ground-water aquifers.

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• Contact with biosolids or pathogens transported away from the land application or surface
disposal site by rodents, insects, or other vectors, including grazing animals or pets.

Part 503 requirements place barriers in the pathway of exposure either by reducing the number of
pathogens in biosolids or by preventing direct or indirect contact with any pathogens possibly present in
the biosolids.

2.13 Site Restrictions

Treatment of Class A biosolids results in biosolid material that is pathogen free (see Chapter 4), therefore
it can be distributed, and land applied without any restrictions to the public. Class B biosolids are not
treated to the same extent as Class A, therefore a significantly reduced, but measurable number of
pathogens may be present in these materials (see Chapter 6),Therefore management practices and site
restrictions are required for Class B land application such that they can still be safely land applied. Class B
materials used in conjunction with proper management practices provides the same safety level as land
application with Class A biosolids.

While the site restrictions required in Part 503 are sufficient to protect the public from health impacts,
workers exposed to Class B biosolids might benefit from several additional precautions. For example,
dust masks should be worn for the spreading of dry materials and workers should wash their hands
carefully after working with Class B biosolids. Other recommended practices for workers handling
biosolids include (CDC, 2002):

• Wash hands before eating, drinking, smoking, or using the restroom.

• Use gloves when touching biosolids or sewage sludge or surfaces exposed to biosolids or sewage
sludge.

• Remove excess sewage sludge or biosolids from shoes prior to entering an enclosed vehicle.

• Keep wounds covered with clean, dry bandages.

• If contact with biosolids or sewage sludge occurs, wash contact area thoroughly with soap and
water.

• Trucks that are used in the transportation of Class B biosolids and then immediately used for
transport of harvested crops should be thoroughly cleaned prior to loading the crops.

2.14 Vector Attraction Reduction

Insects, birds, rodents, and domestic animals may transport sewage sludge and pathogens from sewage
sludge to humans, unless properly mitigated as per the regulations. Vectors may be attracted to sewage
sludge as a food source, and the reduction of the attraction of vectors to sewage sludge are designed to
prevent the spread of pathogens. This requirement is equally important as pathogen reduction and is a
focus of Part 503. Vector attraction reduction (VAR) can be accomplished in two ways: 1) treating the
sewage sludge to the point at which vectors will no longer be attracted to the sewage sludge; and 2)
placing a barrier between the sewage sludge and vectors. VAR must occur simultaneously to the

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pathogen reduction processes or after pathogen reduction. VAR is not permitted to be done prior to
pathogen reduction to meet the requirements of Part 503. The technological and management options
for vector attraction reduction are discussed in Chapter 9.

2.15 References and Additional Resources

Ahmed, Anise U., and Darwin L. Sorensen. 1995. Kinetics of pathogen destruction during storage of dewatered
biosolids. Water Environment Research, Vol. 67, No.2:143-150.

Ault, Steven K., Michael Schott, 1993. Aspergillus, aspergillosis, and composting Operations in California, Technical
Bulletin No. 1. California Integrated Waste Management Board.

Beuchat, Larry, and J.H. Ryu. 1997. Produce handling and processing practices. Emerging Infectious Diseases,
Centers for Disease Control and Prevention. Vol 3, No. 4.

J.P., B.D. Tanner, K.L Josepheson, C.P. Gerba, C.N. Haas, I.L Pepper. 2005 A National Sudy on the Residential
Impact of Biological Aerosols from the Land Application of Biosolids. J. Appl. Microbiol., 99. 310-322.

Casson, L.W., et al. 1992. HIV survivability in water. Water Environmental Research, Vol 64: 213-215.

CDC 2002. Guidance for Controlling Potential Risks to Workers Exposed to Class B Biosolids.

Chen, Y. C., Higgins, M. J., Beightol, S. M., Murthy, S. N., & Toffey, W. E. (2011). Anaerobically digested biosolids
odor generation and pathogen indicator regrowth after dewatering. Water research, 45(8), 2616-2626.

Farzadegan, Homayoon. 1991. Proceedings of a Symposium: Survival of HIV in environmental waters. Baltimore,
MD. National Science Foundation and the Johns Hopkins University.

Farrell, J.B., G. Sternard, A.D. Venosa. 1985. Microbial destructions achieved by full-scale anaerobic digestion.
Paper presented at Municipal Wastewater Sludge Disinfection Workshop, Kansas City, Mo. Water Pollution
Control Federation October 1985.

Gibbs, R. A., C.J.Hu, G.E. Ho, I.Unkovich. 1997. Regrowth of faecal coliforms and salmonellae in stored biosolids
and soil amended with biosolids. Water Science and Technology, 35, 269-275.

Gupta, Phalguni. 1991. HIV survivability in wastewater. Proceedings of a Symposium: Survival of HIV in
Environmental Waters. Baltimore, MD. National Science Foundation and the Johns Hopkins University.

Haines, John, 1995. Aspergillus in compost: Straw man or fatal flaw? BioCycle, April 1995 (32-35).

Haug, Roger T. 1993. The practical handbook of compost engineering. Lewis Publishers.

Hay, Johnathan C., 1996. Pathogen destruction and biosolids composting. BioCycle, Vol. 37 No.6:67-76.

Herrmann, R.F., R.J. Grosser, D. Farrar, R.B. Brobst. 2017. Field studies measuring the aerosolization of endotoxin during
the land application of Class B biosolids. Aerobiologia 33,417-434.

Jenkins, M.B., D.D. Bowman, and W.C. Ghiorse. 1998. Inactivation of Cryptosporidium parvum oocysts by
ammonia. Appl. Envir. Microbiol. 64, No.2, 784-788.

15

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Kindzierski, W.B., R.E. Roberts, and N.J. Low. 1993. Health effects associated with wastewater treatment,
disposal, and reuse. Water Environment Research, Vol. 65: 599-606.

Kowal, N.E. 1985. Health effects of land application of municipal sludge. Pub. No.: EPA/600/1-85/015. Research
Triangle Park, NC: U.S. EPA Health Effects Research Laboratory.

Kowal, N.E. 1994. Pathogen risk assessment: Status and potential application in the development of Round II
regulations. Proceedings of the June 19-20,1994 Specialty Conference. The Management of Water and
Wastewater Solids for the 21st Century: A Global Perspective. Water Environment Federation. Alexandria, VA.

Lang, N. L., M.D. Bellett-Travers, S.R Smith. 2007. Field investigations on the survival of Escherichia coli and
presence of other enteric micro-organisms in biosolids-amended agricultural soil. Journal of Applied
Microbiology, 103(5), 1868-1882.

Martin, J.H., Jr., H.F. Bastian, and G. Stern. 1990 Reduction of enteric microorganisms during aerobic sludge
digestion. Wat. Res. 24(11):1377-1385.

Meckes, M.C., E.W. Rice, C.H. Johnson, and S. Rock, 1995 "Assessment of the Bacteriological Quality of Compost
from a Yard Waste Processing Facility," Comp. Sci. & Util. 3:3

Millner, P.D., S.A. Olenchock, E. Epstein, R. Rylander, J. Haines, J. Walker, B.L Ooi, E. Home, and M. Maritato.
1994. Bioaerosols associated with composting facilities. Compost Science and Utilization. 2(4):6-57.

Moore, B.E. 1993. Survival of human immunodeficiency virus (HIV), HIV-infected lymphocytes, and poliovirus in
water. Applied and Environmental Microbiology. Vol. 59:1437-1443.

Morbidity and Mortality Weekly Report. 1996. Outbreak of E.Coli 0157:H7 infections associated with drinking
unpasteurized commercial apple juice. Centers for Disease Control and Prevention. Vol. 45, No. 44.

Obeng, L. 1985. Health aspects of water supply and sanitation. In Information and Training for Low-Cost Water
Supply and Sanitation. Ed D. Trattles. World Bank. Washington, D.C.

Pell, Alice. 1997. Manure and microbes: Public and animal health problem? Journal of Diary Science 80:2673-
2681.

Pillai, Suresh D. 2007. Bioaerosols from Land Applied Biosolids: Issues and needs. Water Env. Research Vol 79
270-278.

Ponugoti, Prabhaker R., Mohamed F. Dahab, Rao Surampalli. 1997. Effects of different biosolids treatment
systems on pathogen and pathogen indicator reduction. Water Environment Research, Vol. 69:1195-1206

Qi, Y., S.K Dentel, D.S. Herson. 2008. Effect of total solids on fecal coliform regrowth in anaerobically digested
biosolids. Water research, 42(14), 3817-3825.

Scheuerman, P.R., S.R. Farrah, and G. Bitton. 1991. Laboratory studies of virus survival during aerobic and
anaerobic digestion of sewage sludge. Water Resources 25:241-245.

Sidhu, J., R.A. Gibbs, G.E. Ho, I. Unkovich. 2001. The role of indigenous microorganisms in suppression of
Salmonella regrowth in composted biosolids. Water research, 35(4), 913-920.

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Smith, James E. and J. B. Farrell. 1996. Current and future disinfection - Federal Perspectives. Presented at Water
Environment Federation 69th Annual Conference & Exposition.

Soares, Hugo M., Beatriz Cardenas, David Weir, and Michael S. Switzenbaum. 1995. Evaluating pathogen regrowth
in biosolids compost. BioCycle, Vol. 36. No.6:70-76.

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, sludge, and soils. Boca Raton, FL: CRC Press

Sorber, C.A. ,B.E. Moore, D.E. Johnson, H.J. Harding, R.E. Thomas. 1984. Microbiological aerosols from the
application of liquid sludge to land. Journal WPCF Vol. 56, No.7:830-836.

Stadterman, A.M. Sninsky, J.LSykora and W. Jakubowski. 1995. Removal and inactivation of Cryptosporidium
oocysts by activated sludge treatment and anaerobic digestion. Wat. Sci. Tech. 31, No. 5-6, 97-104

Tan, L., M.A. Williams, M.K. Khan, H.C. Champion, N.H. Nielsen. 1995. Risk of transmission of Bovine Spongiform
Encephalopathy to humans in the United States. JAMA. 281, 24, 2330.

Tanner, B.D.,J.P. Brooks, C.P. Gerba, C.N. Haas, K.L Josephson, I.L Pepper. 2008. Estimated occupational risk
from bioaerosols generated during land application of Class B biosolids. J of Envi. Quality. Vol. 37 2311-2321.

USEPA. 1983. Enteric virus removal in wastewater treatment lagoon systems (Project Summary, EPA/600/S1-83-
012)." Research Triangle Park, NC: U.S. EPA/Health Effects Research Laboratory.

USEPA. 1985. Health effects of land application of municipal sludge (EPA/600/1-85/015). Research Triangle Park,
NC: U.S. EPA/Health Effects Research Laboratory.

USEPA. 1986. Inactivation of enteric pathogens during aerobic digestion of wastewater sludge (Project Summary,
EPA/600/SO-86/047). Cincinnati, OH: U.S. EPA/Water Engineering Research Laboratory.

USEPA. 1989. Technical support document for pathogen reduction in sewage sludge. NTIS No.: PB89-136618.
Springfield, VA: National Technical Information Service.

USEPA. 1991. Preliminary risk assessment for viruses in municipal sewage sludge applied to land (Project
Summary, EPA/600/SR-92/064). Washington, DC: U.S. EPA/Office of Health & Environmental Assessment.

USEPA. 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.

Ward, R.L, G.A. McFeters, and J.G. Yeager. 1984. Pathogens in sludge: Occurrence, inactivation, and potential for
regrowth. Sandia National Laboratories, Albuquerque, NM. SAND83-0557, TTC-0428, UC-41. U.S. DOE Contract
C E AC04-76D P00789.

WEF/USEPA. 1997. Can Aids be transmitted by biosolids?" in WEF/U.S. EPA Biosolids Fact Sheet Project.

West, P. A., "Human pathogenic viruses and parasites: Emerging pathogens in the water cycle," Journal of Applied
Bacteriology JABAA4, Vol.70, No. Supp, p 107S-114S, 1991.

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Yanko, William A. 1988. Occurrence of pathogens in distribution and marketing municipal sludges. (EPA/600/1-
87/014) County Sanitation District of Los Angeles and EPA/Health Effects Research Laboratory.

Yeager, J.G. and R.L Ward. 1981. Effects of moisture content on long-term survival and regrowth of bacteria in
wastewater sludge. Appl. Environ. Microbiol. 41(5):1117-1122.

Zaleski, K. J., K.LJosephson, C.P. Gerba, I.L. Pepper. 2005. Survival, growth, and regrowth of enteric indicator and
pathogenic bacteria in biosolids, compost, soil, and land applied biosolids. J. Residuals Sci. Technol, 2(1), 49-63.

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3: DOMESTIC SEPTAGE

3.1	Introduction

This chapter discusses Part 503 pathogen reduction and vector attraction requirements for domestic
septage. States and local entities may have additional and/or more stringent requirements.

3.2	Domestic Septage

Domestic septage is either liquid or solid material removed from a septic tank, holding tank, cesspool,
portable toilet, Type III marine sanitation device, or similar treatment works that receives only domestic
sewage. Domestic septage does not include liquid or solid material removed from a septic tank, cesspool,
or similar treatment works that receives either commercial wastewater or industrial wastewater and does
not include grease removed from a grease trap at a restaurant. If domestic septage is mixed with
commercial septage it is no longer regulated under Part 503. Domestic septage contains a variety of the
same pathogens present in wastewater treatment facilities, is typically more liquid in nature, and usually
contains lower levels of heavy metals and other pollutants (USEPA 1996). Septage can contain valuable
resources such as nitrogen and phosphrous that make land application of these residuals acceptable.

3.3	Pathogen Reduction

The Part 503 regulations cover land application of domestic septage to ensure further protection of
public health and the environment. 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 treated sewage sludge (biosolids) applied to these types of land (Class A
requirements). Separate, pathogen reduction requirements exist for domestic septage applied to non-
public contact sites (e.g. agricultural land, forests, or reclamation sites). These requirements include site
restrictions to reduce the potential for human exposure to domestic septage and to allow for pH
adjustment or environmental attenuation with site restrictions only on harvesting crops. No pathogen
requirements apply if domestic septage is placed on a surface disposal site.

Under Part 503.32(c), pathogen reduction in domestic septage applied to these non-public contact sites:
agricultural land, forest, or reclamation sites8 may be reduced in one of two ways:

•	If no treatment of the septage is done prior to application the material must be directly injected
into the soil or incorporated within six hours of application. This allows for the natural microbial
population to further reduce the pathogens present in these materials.

•	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, The Part 503 regulation uses the
term alkali in the broad sense to mean any substance that causes an increase in pH.

8 Class B sewage sludge requirements apply to domestic septage applied to all other types of land. No pathogen-
related requirements apply to domestic septage placed on a surface disposal site.

19

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If the pH is below 12, either initially or after 30 minutes, additional alkali material needs to 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 at least or greater than 12.

If domestic septage is not applied soon after pH adjustment and 30-minute latent period, it is
recommended that the pH be retested and additional alkali be added to the domestic septage to raise
the pH to 12, if necessary.

3.4 Vector Attraction Reduction

Domestic septage also must meet vector attraction reduction requirements (VAR). 40 CFR Part 503 lists
12 different options for meeting the VAR component of biosolids land application (Chapter 9). The choice
of vector attraction options may affect the duration of site restrictions in some cases. Specifically, if
Option 9 or 10 (injection or incorporation) is used to reduce vector attraction, the restriction on
harvesting for food crops grown below the soil surface (e.g., potatoes and carrots) is increased from 20
months to 38 months for domestic septage.

Option 12 for vector attraction reduction applies to materials that are treated with alkali substances for
pathogen reduction. This vector attraction reduction requirement is slightly less stringent than the alkali
addition requirement 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 in 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. 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.

3.5 Site Restrictions

Domestic septage that is land applied outside of Class A treatments must also maintain site restrictions
where the land is restricted for public access and other activities as outlined in Part 503. See chapter 6 for
further details on site restrictions.

3.6 References

Bonner, A.B. and D.O Cliver. 1987. Disinfection of viruses in septic tank and holding tank wast by calcium
hydroxide (Lime). Unpublished report, Small Scale Waste Management Project. U. of Wisconsin. Madison,
Wl.

USEPA. 1980. Samplers and sampling procedures for hazardous waste streams. Report No.: EPA/600/2-
80/018. Cincinnati, OH: Office of Research and Development.

USEPA. 1993. POTW sludge sampling and analysis guidance document. EPA 833-B-89-100. Office of
Water.

USEPA. 1993. Domestic Septage Regulatory Guidance: A Guide to the EPA 503 Rule. EPA 832-B-92-005.
Office of Water

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USEPA. 1995. Process Design Manual: Land Application of Sewage Sludge and Domestic Septage. EPA
625/R-95/001. Office of Research and Development.

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4: CLASS A PATHOGEN REQUIREMENTS

4.1 Introduction

This chapter discusses the Class A pathogen requirements in Subpart D of 40 CFR Part 503. The implicit
goal of the Class A pathogen requirements is to reduce all the pathogens present in sewage sludge
(including enteric viruses, pathogenic bacteria, and viable helminth ova) to below detectable levels. Once
Class A requirements are met along with pollutant limits and a suitable vector attraction reduction option
materials are suitable for unrestricted use, including biosolids that are sold or given away in a bag or
other container, bulk biosolids applied to a lawn or home garden and bulk biosolids applied to other types
of land.

There are six alternative requirements for demonstrating Class A pathogen reduction. Two of these
alternatives provide continuity with 40 CFR Part 257 (the regulation that governed sewage sludge prior to
Part 503) by allowing use of Processes to Further Reduce Pathogens (PFRPs) and equivalent technologies
(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 by using the organisms as listed in Table 4.1:

Table 4.1 Microbial indicators for Class A biosolids treatment

Microbial Indicator

Class A regulatory Limit

Fecal coliforms or
Salmonella sp.

less than 1000 MPN per gram total solids (dry weight basis)
less than 3 MPN per 4 grams total solids (dry weight basis)

Enteric viruses9

less than 1 PFU per 4 grams total solids (dry weight basis)

Viable helminth ova

less than 1 viable helminth ova per 4 grams total solids (dry weight basis)

For the following sections, the title of each section provides the number of the Subpart D requirement discussed in
the section. The regulatory language can be found in Appendix B. Chapter 10 provides guidance on the sampling and
analysis needed to meet the Class A microbiological monitoring requirements.

9 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 viruses. Since the objective of the Part 503 regulation is
to reduce all enteric viruses to less than 1 PFU per 4 grams total solids sewage sludge, this document refers to
"enteric viruses" when discussing this requirement, although the detection method enumerates only enteroviruses.

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Class A Product or Process

A product is considered Class A once it meets the pollutant limits, pathogen
monitoring standards and one of the vector attraction reduction requirements.

Pathogen reduction methods must be met before vector attraction reduction or
simultaneously. This order can't be modified.

4.2 Vector Attraction Reduction to Occur Simultaneously or After Class A Pathogen
Reduction [503.32(a)(2)]

Although vector attraction reduction (VAR) and pathogen reduction are separate requirements, they are
often related steps of a process. Chapter 9 discusses the VAR options in greater detail.

The order of Class A pathogen reduction requirements in relation to VAR is a critical component of the
regulations. Part 503.32(a)(2) requires that Class A pathogen reduction be accomplished prior or
simultaneously to VAR. This requirement on the order of VAR and pathogen reduction is necessary to
prevent the growth of bacterial pathogens after sewage sludge is treated. Contamination of biosolids
with a bacterial pathogen after one of the Class A pathogen reduction alternatives has been conducted
may result in extensive bacterial regrowth unless: a) an inhibitory chemical is present, b) the biosolids are
too dry to allow bacterial growth, c) little food remains for the microorganisms to consume, or d) an
abundant population of non-pathogenic bacteria is present. Vegetative cells of non-pathogenic bacteria
suppress the growth of pathogenic bacteria by "competitive inhibition" due to competition for nutrients.
It should be noted that vector attraction reduction by alkali addition [503.3(b)(6)] or drying [503.3(b)(7)
and (8)] is based on the characteristic of the biosolids (pH or total solids) remaining elevated. Should the
pH drop or the biosolids absorb moisture, the biosolids may be more hospitable to microorganisms, and
pathogenic bacteria, if introduced, may grow. Therefore, it is required that biosolids treated with these
methods be stored appropriately to maintain dryness and/or elevated pH.

Biological treatment processes like anaerobic digestion, aerobic digestion, and composting produce
changes in the sewage sludge so that it satisfies one of the vector attraction reduction requirements
[503.3(b)(1) through (5)]. These processes repress bacterial growth by minimizing the food supply and
providing competition for the remaining food possibly used by non-pathogenic organisms. The pathogen
reduction alternative must precede the vector attraction reduction process; otherwise, the large number
of beneficial non-pathogenic bacteria would be killed, and growth of pathogenic bacteria could occur.
Certain pathogen reduction processes such as composting accomplish VAR by a biological process
simultaneously with thermal reduction of pathogens. A non-pathogenic bacterial community survives
which adequately suppresses growth of pathogenic bacteria.

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4.3 Monitoring of Fecal Coliform or Salmonella sp. to Detect Growth of Bacterial
Pathogens [503.32(a) (3)-(8)]

The goal of Class A processes is to reduce the level of pathogens to below detectable levels. The Class A
processes listed in 40 CFR Part 503 have been shown to sufficiently reduce pathogen levels in biosolids.
Favorable conditions for the regrowth of pathogenic bacteria following Class A treatments include
adequate moisture, absence of an inhibitory chemical, and inadequate reduction of nutrients in the
treated sewage sludge.

Because Class A biosolids may be used without site restrictions, all Class A material must be tested to
show that the microbiological requirements are met at the time when it is ready to be used or disposed.
In addition to meeting process requirements, Class A biosolids must meet one of the following
requirements:

Eitherthe density of fecal coliforms in the sewage sludge be less than 1,000 MPN10 per gram total solids
(dry weight basis), cvthe density of Salmonella sp. bacteria in the sewage sludge be less than 3 MPN per 4
grams of total solids (dry weight basis).

Although Part 503 does not specify the number of samples that should be taken to show compliance with
Class A density requirements, sampling programs should provide adequate representation of the
biosolids generated. Chapter 9 provides guidance for calculating the number of samples that should be
taken per sampling event. Unlike Class B biosolids, compliance with Class A requirements is not based on
a geometric mean value. Each sample analyzed must comply with the numerical requirements.

The microbiological requirement must be met either:

• At the time of use or disposal11, or

• At the time the biosolids are prepared for sale or give away in a bag or other container for land
application, or

• At the time Class A EQ biosolids or material that meets Class A EQ requirements are derived from
sewage sludge12

When a facility stores material before it is distributed for use or disposal, microbiological testing
should take place after storage, just prior to application or distribution. This will ensure that the
material meets the microbiological requirements for Class A materials upon application.

In each case, the timing represents the last practical monitoring point before the biosolids are applied to
the land or placed on a surface disposal site. Biosolids that are sold or given away cannot be monitored

10The membrane filter method is not allowed for Class A because, at the low fecal coliform densities expected, the
filter would have too high a loading of sewage sludge solids to permit a reliable count of the number of fecal
coliform colonies.

11 Minus the time needed to test the biosolids and obtain the test results prior to use or disposal (see Chapter 10).

12 See the applicability requirements in 40 CFR § 503.10 and the EPA memorandum Land Application Requirements
for Class A Exceptional Quality Treated Sewage Sludge (November 5, 2020) for more information. See appendix J for
the EPA memo.

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just prior to actual use or disposal instead, monitoring is required immediately prior to bagging or
distribution.

As discussed in Chapter 10, the timing of pathogen sampling is also a function of laboratory turnaround
time. Obtaining results for fecal coliform and Salmonella sp. analysis may take several days if tests are
performed in-house, but commercial labs may require more time to process and report results. It is not
unusual for laboratories to have a turnaround time of 2 weeks, even for simple tests such as fecal
coliform analysis. If this is the case, this time should be factored into the sampling program so that
results can be obtained before biosolids are distributed for use or disposal.

4.4 Monitoring Fecal Conforms or Salmonella sp.

Fecal coliforms are used in Part 503 as an indicator organism because reduction in fecal coliforms
correlates to reduction in Salmonella sp. and other organisms. The requirements were based on
experimental work by Yanko (1987) and correlations developed from Yanko's data by Farrell (1993) which
show that low levels of fecal coliforms correlate with a very low level of Salmonella sp. detection in
composted sewage sludge (USEPA, 1992).

Anecdotal reports suggest that some composting facilities may have difficulty meeting this requirement
even when Salmonella sp. are not detected. This might be expected under several circumstances. For
example, severe thermal treatments of sewage sludge during composting can eliminate Salmonella sp.
yet leave residual fecal coliforms. If the sewage sludge has been poorly composted and thus is a good
food source, fecal coliforms may grow after the compost cools down from thermophilic temperatures,
however because the Salmonella sp. are absent, they cannot grow.

Chapter 2 mentions that Salmonella in most situations is not an ideal treatment indicator organism
because it is found in low densities in most raw sludges, and therefore its absence in the treated sewage
sludge does not correlate to proper treatment. Also, the methods for Salmonella sp. are not as robust
and are variable compared to the methods for measuring fecal coliforms. While it may be possible to
meet the regulatory requirements by testing and meeting the regulatory limits for Salmonella sp., it is
recommended that the pathogen reduction process be reviewed to determine at what point fecal
coliforms potentially are not being reduced or are being reintroduced into treated biosolids and ensure
that process requirements are being fulfilled. Also, treated sewage sludge should not be tested for both
Salmonella and fecal coliform and subsequently only report Salmonella values if fecal coliform levels are
too high. Both values should be reported. High fecal coliform values would suggest the process is not
robust enough to kill off all the bacterial pathogens.

4.5 Monitoring for Enteric Virus and Viable Helminth Ova

Alternatives 3, 4, and 6 require additional microbial indicator testing for enteric viruses and viable
helminth ova. The specific methods that must be used for these tests are outlined in Chapter 10, as well
as referenced in Appendices F and G. The method required for enteric viruses only enumerates
enteroviruses which are presumed to be a good indicator for all enteric viruses. Since the objective of the
Part 503 regulation is to reduce all enteric viruses to less than 1PFU per 4 grams total solids within the
sewage sludge, this document refers to "enteric viruses" when discussing the method for this
requirement. Tests for enteric viruses and viable helminth ova take a substantial amount of time. It can

lry

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take four weeks to determine whether helminth ova are viable, and two weeks or longer for enteric virus
analysis results. In situations where analyses of these organisms are required by Part 503, the biosolids
must be held and not land applied until the test results show compliance. This will require the ability to
store material until the test results are available. Finding laboratories that perform these tests can also
be a challenge, laboratories must be familiar with the tests allowed in the regulation. Deviation from
these methods can invalidate the results and render the material to be out of compliance with the
regulations.

4.6 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 pathogens. Four different time-temperature regimes are included in Alternative 1.

Each regime is based on the percent solids of the sewage sludge and on operating parameters of the
treatment process. Experimental evidence (USEPA, 1992) demonstrates that these four time-temperature
regimes reduce the pathogenic organisms to below detectable levels.

The four time-temperature regimes are summarized in Table 4.2. They involve two different
time-temperature equations. The equation used in Regimes A through C results in requirements that are
more stringent than the requirement obtained using the equation in Regime D. For any given time, the
temperature calculated for the Regime D equation will be 3 Celsius degrees (5.4 Fahrenheit degrees)
lower than the temperature calculated for the Regimes A through C equation.

The time-temperature relationships described for Alternative 1 are based on research conducted to
correlate the reduction of various pathogens in sewage sludge to varying degrees of thermal treatment.
The resulting time-temperature relationship which is the basis for Alternative 1 is shown in Figure 4-1.
These requirements are similar to the FDA requirements for treatment of eggnog, a food product with
flow characteristics similar to those of liquid sewage sludge. The Regimes A through D differ depending
on the characteristics of sewage sludge treated and the type of process used because of the varying
efficiency of heat transfer under different conditions.

Therefore, testing of temperatures throughout the sewage sludge mass and agitating the material to
ensure uniformity is appropriate. For processes such as thermophilic digestion, it is important that the
digester design not allow for short circuiting of untreated sewage sludge as this would render the digestor
process out of compliance with Alternative 1.

These time-temperature regimes are not intended to be used for composting (the time-temperature
regime for composting is covered in Alternative 5: Processes to Further Reduce Pathogens).

It is mandatory for all sewage
sludge particles to meet the
time-temperature regime.

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Table 4.2 The Four Time-Temperature Regimes for Alternative 1 (Thermally Treated Sewage Sludge)

503.32(a)

3)]









A

5 03.32(a)(3)(ii) (A)

Sewage sludge with at least 7% solids
(except those covered by Regime B)

D= 131,700,000/10 ai400t
t > 50°C (122°F)2
D > 0.0139 (i.e., 20 minutes)3

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

D = 131,700,000/10°'1400t
t :> 50°C (122°F)2
D >: 1.74 X 10~4 (i.e., 15 seconds)5

C

503.32(a)(3)(ii)(C)

Sewage sludge with less than 7% solids
treated in processes with less than 30
minutes contact time

D= 131,700,000/10al4°ot
D £1.74 X 10 ~4 (i.e., 15 seconds)
and D < 0.021 (i.e. 30 minutes)6

D

503.32(a)(3)(ii)(D)

Sewage sludge with less than 7% solids
treated in processes with at least 30
minutes contact time

D = 50,070,000/10al4°ot
t i> 50°C (122° F)2
D ^ 0.021 (i.e. 30 minutes)7

'D = time in days; t = temperature (°C).

2The restriction to temperatures of at least 50°C (122°F) is imposed because information on the time-temperature
relationship at lower temperatures is uncertain.

3 A 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 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.

5Time-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.

27

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100

90

80

o



i—

3

2



Cl
£



I—

60

50

01	2	34567

Logarithm of Time (Log Sec)

Figure 4.1 EPA's time-temperature relationship for thermal disinfection compared
with time-temperature relationships.

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 structure that inhibits the mixing
that contributes to a non-uniform distribution 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 information 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. Time at the
desired temperature is readily determined for batch or plug flow operations, or even laminar flow in
pipes.

Vector Attraction Reduction

Thermally treated sewage sludge must be treated by an additional vector attraction reduction process
since thermal treatment does not necessarily break down the volatile solids in sewage sludge. Vector
attraction reduction can be met by further processing the sewage sludge with pH adjustment or heat
drying (Options 6 and 7), or by meeting one of the other options (Options 8-11). Options 1 through 5
are not applicable to thermally treated sludge unless the sludge was subject to biological digestion after
or during thermal treatment (for example: Option 1 could be utilized for a thermophilic anaerobic or
aerobic digestion if volatile solids reduction is satisfied across the digestion process).

28

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4.1 Alternative 2: Sewage Sludge Treated in a High pH-High Temperature Process
(Alkaline Treatment) [503.32(a)(4)]

This alternative describes conditions of a high temperature-high pH process that has proven effective in
reducing pathogens to below detectable levels. The process conditions required by Part 503 are:

• Elevating pH to greater than 12 and maintaining the pH for more than 72 hours.

• Maintaining the temperature above 52°C (126°F) throughout the sewage sludge 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, high temperature, and reduced moisture for prolonged time periods
allow a variance to a less stringent time-temperature regime than for the thermal requirements under
Alternative 1. The pH of the sewage sludge is measured at 25°C (77°F) or an appropriate correction is
applied (see Section 10.7).

Because the elevated pH and temperature regimes must be met by the entire sewage sludge mass,
operational protocols include monitoring pH and temperature at various points in a batch and agitating
the sewage sludge during operations to ensure consistent temperature and pH are appropriate.

Vector Attraction Reduction

The pH requirement of VAR Option 6 is met when the pathogen requirement by Alternative 2 is achieved.
Compliance with the pathogen requirement by Alternative 2 exceeds the pH requirements of VAR Option
6.

4.8 Alternative 3: Sewage Sludge Treated in Other Processes [503.32(a)(5)]

This alternative applies to sewage sludge treated by processes that do not meet the process conditions
required by Alternatives 1 and 2 and can be used while testing under Alternative 6. This requirement
relies on comprehensive monitoring of bacteria, enteric viruses and viable helminth ova to demonstrate
adequate reduction of pathogens:

• Eitherthe density of fecal coliforms in the sewage sludge must be less than 1000 MPN per gram
of total solids (dry weight basis), or the Salmonella sp. bacteria in sewage sludge must be less
than three MPN per four grams of total solids (dry weight basis) at the time the sewage is used or
disposed, at the time the sewage sludge is prepared for sale or given away in a bag or other
container for land application, or at the time Class A EQ biosolids or material that meets Class A
EQ requirements are derived from sewage sludge.

• The density of enteric viruses in the sewage sludge after pathogen treatment must be less than
one PFU per four grams of total solids (dry weight basis).

• The density of viable helminth ova in the sewage sludge after pathogen treatment must be less
than one per four grams of total solids (dry weight basis).

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Testing for enteric viruses and viable helminth ova can be complicated by the fact that they are often 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 sewage 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 one PFU per four grams total solids (dry weight
basis) and/or the density of viable helminth ova is greater than or equal to one per four grams total solids
(dry weight basis), see section 4.15 for testing frequency. At this point, the treated sewage sludge is
analyzed to see if these organisms survived treatment. If enteric virus densities are below detection
limits, the sewage sludge meets Class A requirements for enteric viruses and will continue to do so if the
treatment process is operated under the same conditions 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 helminth ova and will continue to do so if 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 helminth ova reduction
have been successfully demonstrated (see Section 7.4). The minimum frequency of monitoring under this
alternative is the same as the times listed in Table 1.1, however, it may be necessary to sample more
frequently to adequately capture the operational conditions of the treatment process.

Vector Attraction Reduction

Meeting vector attraction reduction depends on the process by which pathogen reduction is met. For
example, sewage sludge subjected to long-term storage may meet vector attraction reduction through
volatile solids reduction (Options 1-3). Sewage sludges may also undergo additional processing or be
applied followingthe requirement in Options 8-11.

4.9 Alternative 4: Sewage Sludge Treated in Unknown Processes [503.32(a)(6)]

Alternative 4 is intended to be utilized for treatment processes that are "unknown". In these situations,
there is no specific treatment regime that is applied that meets one of the other Class A Alternatives. This
may include lagoons, or other processes with an undefined or inconsistent treatment process. Under this
alternative the sewage sludge must meet the following limits at the time the biosolids (or material
derived from sludge) are used or disposed, at the time the sewage sludge is prepared for sale or given
away in a bag or other container for land application, or at the time Class A EQ biosolids or material that
meets Class A EQ requirements are derived from sewage sludge.

• The density of enteric viruses in the sewage sludge must be less than one PFU per four grams of
total solids (dry weight basis).

• The density of viable helminth ova in the sewage sludge must be less than one per four grams of
total solids (dry weight basis).

• In addition, as for all Class A biosolids, the sewage sludge must meet fecal coliform or Salmonella
sp. limits.

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4.10 Vector Attraction Reduction

Like Alternative 3, meeting vector attraction reduction for Alternative 4 depends on the process by which
pathogen reduction is met. For example, sewage sludge subject to long-term storage may meet vector
attraction reduction through volatile solids reduction (Options 1-3). Sewage sludge may also undergo
additional processing or be applied following the requirement in Options 8-11.

4.11 Alternative 5: Use of PFRP [503.32(a)(7)]

Alternative 5 states that sewage sludge is considered to be Class A if:

• It has been treated in one of the Processes to Further Reduce Pathogens (PFRPs) listed in Table
4.3. The material must also be tested for either fecal coliforms or Salmonella sp. The density of
fecal coliforms in the sewage sludge must be less than 1,000 MPN per gram total solids (dry
weight basis), or the density of Salmonella sp. bacteria in the sewage sludge less than 3 MPN per
4 grams total solids (dry weight basis). These tests need to be conducted at the time the sewage
sludge is used or disposed or at the time the sewage sludge is prepared for sale or given away in a
bag or other container for land application, or at the time Class A EQ biosolids or material that
meets Class A EQ requirements are derived from sewage sludge.

Table 4.3 describes the sewage sludge treatment processes allowed under Alternative 5.

For all PFRP processes, the goal of temperature monitoring should be to represent all areas of a batch or
pile and to ensure that temperature profiles from multiple points in the process all meet mandated
temperatures. In some instances, it may be possible to monitor representative areas of a batch, pile, or
reasonable worst-case area to ensure compliance. Chapter 5 contains more guidelines about the
operation of PFRP processes.

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Table 4.3. Processes to Further Reduce Pathogens (PFRPs) Listed in Appendix B of 40 CFR Part 5031

PFRP PFRP Description

Composting

Using either the within-vessel composting method or the static aerated pile composting
method, the temperature of sewage sludge is maintained at 55°C (131°F) or higher for 3
consecutive days.

Using the windrow composting method, the temperature of the sewage sludge is
maintained at 55°C (131°F) or higher for 15 consecutive 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.

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 bulb temperature of the gas in contact with the
sewage sludge as the sewage sludge leaves the dryer exceeds 80°C (176°F).

Heat Treatment

Liquid sewage sludge is heated to a temperature of 180°C (356°F) or higher for 30 minutes.

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).

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]).

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]).

Pasteurization

The temperature of the sewage sludge is maintained at 70°C (158°F) or higher for 30
minutes or longer.

1 Chapter 5 provides a detailed description of these technologies.

4.12 Alternative 6: Use of a Process Equivalent to PFRP [503.32(a)(8)]

Under Alternative 6, sewage sludge is considered Class A sewage sludge if:

• It is treated by any process equivalent to a PFRP, and either the density of fecal coliforms in the
sewage sludge is less than 1,000 MPN per gram total solids (dry weight basis), or the density of
Salmonella sp. bacteria in the sewage sludge is less than three MPN per four 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 Class A EQ biosolids or material that meets Class A EQ requirements are derived from
sewage sludge13.

Facilities that meet Alternative 6 for pathogen reduction must still meet vector attraction reduction

requirements.

13 See the applicability requirements in 40 CFR § 503.10 and the EPA memorandum Land Application Requirements
for Class A Exceptional Quality Treated Sewage Sludge (November 5, 2020) for more information. See appendix J for
the EPA memo.

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4.13 Processes Recommended as Equivalent

Processes recommended to be equivalent to PFRP are shown in Table 8.1. Products of all equivalent
processes must still meet the Class A fecal coliform or Salmonella sp. requirements.

4.14 Equivalency Determination

Part 503 gives the permitting authority responsibility for determining equivalency under Alternative 6.
The EPA's Pathogen Equivalency Committee (PEC) is available as a resource to provide guidance and
recommendations on equivalency determinations to both the permitting authority and the regulated
community (see Chapter 8).

4.15 Frequency of Testing

Part 503 sets minimum sampling and monitoring requirements. Table 1.1 in Chapter 1 describes the
minimum frequency at which the sewage sludge must be sampled and analyzed for pathogens or vector
attraction reduction to meet regulatory requirements. In addition to meeting these requirements, EPA
recommends that sewage sludge generators consider the potential public health impacts and possible
liability issues when designing a sampling program. In some cases, it may be appropriate to sample more
frequently than what is required under Part 503.

4.16 Important Considerations for Class A treated Materials

Care must be taken that once pathogen reduction treatments are performed no other additives or
additional treatments to the material that may potentially contain pathogenic microorganisms encounter
the treated material. This includes common practices where wastewater influent or effluent is added
back to the treated solids in the case of dewatering or prior to digestion. Influent or effluent is sometimes
used as a diluent to decrease the solids content prior to or afterdigestion, or as a matrix that is used to
reconstitute polymers that aid in dewatering. Typical wastewater influents or effluents are either not
treated or treated using a process that would not comply with Class A sewage sludge pathogen
requirements, as such they may contain pathogens or indicators that are above the allowable limits for
Class A materials. In fact, wastewater effluent regulatory requirements use different process indicator
organisms and different testing requirements from sewage sludges. Therefore, effluent that is mixed
with Class A treated materials can compromise the integrity of the treated biosolids, by contaminating
the product with pathogenic organisms. It is also not acceptable to use "treated wastewater effluent" for
these purposes, unless the effluent is treated in a Class A manner as outlined in 40 CFR Part 503.

If the generator assumes they have a Class A product for pathogens, but recent testing results show that
the product meets Class B standards for pathogens instead, then distribution of that product as Class A
would constitute a violation of Part 503. Therefore, it is recommended that the product remains on site
until lab results are available. Also, it is advisable to store biosolids in discrete batches and take multiple
samples per sampling event. This will allow better identification of piles that may be out of compliance
and will allow for the distribution of material that is correctly identified as Class A.

If pathogen testing shows that a product distributed as Class A material is actually a Class B product,
entities that received the product should be notified. The facility may even consider recalling the
biosolids from the users of that product.

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If the biosolids product has already been distributed to public access areas, including homes, gardens,
parks, or other public areas, the biosolids preparer may consider testing the soil. If the testing indicates
unacceptable levels of pathogens, corrective actions may be necessary.

4.17 References

Barker, T.A. 1970. Pasteurization and sterilization of sludges. Proc. Biochem, August, p 44-45.

Farrell, J.B., J.E. Smith, Jr., S.W. Hathaway, and R.B. Dean. 1974. Lime stabilization of primary sludges. J.
WPCF 46(1):113-122.

Farrell, J.B. 1993. Fecal pathogen control during composting, p 282-300 in "Science & Engineering of
Composting: Design, Environmental, Microbiological, and Utilization Aspects." edit. H.A. J. Hoitink and
H.M. Keenerg.

Foess, Gerald W., and Ronald B. Sieger. 1993. Pathogen/vector attraction reduction requirements of the
sludge rules. Water/Engineering & Management, June, p 25

IRGRD (International Research Group on Refuse Disposal) 1968. Information Bulletin No. 21-31. August
1964 - December 1967. p 3230 - 3340. Reprinted by US Dept. HEW, Bureau of Solid Waste Management
(1969).

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. Reduction of enteric microorganisms during aerobic
sludge digestion. Wat. Res. 24(11):1377-1385.

Schafer, P.L., J.B. Farrell, W.R. Uhte, and B. Rabinowitz. 1994. Pre-pasteurization, European and North
American assessment and experience, p 10-39 to 10-50 in "The Management of Water and Wastewater
Solids for the 21st Century: A Global Perspective." Conference Proceedings, June 19-22, 1992. Water
Environment Federation.

USEPA. 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.

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: National Technical Information Service.

34

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5: PROCESSES TO FURTHER REDUCE PATHOGENS
(PFRP)

5.1	Introduction

Processes to Further Reduce Pathogens (PFRPs) is terminology that was used in the previous sewage
sludge regulations prior 40 CFR Part 503. This distinction was given to processes that were able to reduce
pathogens below detectable levels. There are seven PFRPs specifically listed in Part 503: composting, heat
drying, heat treatment, thermophilic aerobic digestion, beta ray irradiation, gamma ray irradiation, and
pasteurization. When these processes are operated under the conditions specified in the regulations,
pathogenic bacteria, enteric viruses, and viable helminth ova are reduced to below detectable levels as
defined in 40 CFR Part 503. In addition to these seven PFRP's listed in Part 503, the Pathogen Equivalency
Committee (PEC) recommends alternative processes for PFRP to the proper permitting authority.

This chapter provides detailed descriptions of the seven PFRPs listed in Part 503, Alternative 5. Because
the purpose of these processes is to produce Class A biosolids, the pathogen reduction process must be
conducted concurrent to or prior to the vector attraction reduction process (see Section 4.2). Table 5.1
includes the PFRP approvals granted under Alternative 6 at the end of this chapter.

Under Part 503.32(a)(7), sewage sludge treated by PFRPs are Class A with respect to helminth ova, enteric
viruses, and pathogenic bacteria. In addition, Class A biosolids must be monitored for fecal coliform or
Salmonella sp. bacteria at the time of use or disposal, at the time the biosolids are prepared for sale or
give away in a bag or other container for land application, or at the time the biosolids are prepared to
meet the requirements for "exceptional quality" sludge in 503.10(b),(c),(e) or (f) to ensure that growth of
bacteria has not occurred (see Section 4.3, Appendix J). As mentioned earlier in Chapter 4 microbial
sampling for all Class A products including PFRPs must occur as close to the time of use and disposal as
possible. Part 503 doesn't list exact times for testing because sewage sludge processing and land
application events vary, and this allows for flexibility that is necessary to ensure public health is
maintained during land application events. It is also worth noting that the addition of all additives to the
sewage sludge process such as anti-dust sprays, polymers, and dewatering methods should occur prior to
sampling events, because the addition of these products or processes may impact the microbial quality of
the material.

5.2	Composting

Composting is the controlled, aerobic decomposition of organic matter which produces a humic-like
material. Sewage sludge that is composted is generally mixed with a bulking agent such as wood chips to
allow air to pass more easily through the composting material, thereby creating aerobic conditions.

There are three commonly used methods of composting: windrow, static aerated pile and within-vessel.

35

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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 three
consecutive days.

•	Using the windrow composting method, the temperature of the sewage sludge is maintained at
55°C (131°F) or higher for 15 consecutive 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.

For aerated static pile and in-vessel composting processes, temperatures should be taken at multiple
points at a range of depths throughout the composting medium. Points which are likely to be slightly
cooler than the center of the pile, such as the toes of piles, also should be monitored. Because the entire
mass of sewage sludge must attain the required temperatures for the required duration, the temperature
profiles from every monitoring point, not just the average of the points, should reflect PFRP conditions.

It has been found that points within 0.3 m (1 foot) of the surface of aerated static piles may be unable to
reach PFRP temperatures, and for this reason it is recommended that a 0.3 m (1 foot) or greater layer of
insulating material be placed over all surfaces of the pile. Finished compost is often used for insulation. It
must be noted that because the insulation will most likely be mixed into the composted material during
post-processing or curing, compost used as an insulation material must be a Class A material so as not to
reintroduce pathogens into the composting sewage sludge.

For windrow composting, the operational requirements are based on the same time-temperature
relationship as aerated static pile and in-vessel composting. The material in the core of the windrow
attains at least 55°C and must remain at that temperature for three consecutive days. Windrow turning
moves new material from the surface of the windrow into the core so that this material may also undergo
pathogen reduction. After five turnings, all material in the windrow must have spent 3 days at the core of
the pile. The time-temperature regime takes place over a period of at least 15 consecutive days during
which time the temperature in the core of the windrow is at least 55°C. See Appendix H for additional
guidance.

Pathogen reduction is a function of three parameters:

•	Ensuring that all sewage sludge is mixed into the core of the pile at some point during active
composting

•	Ensuring that all sewage sludge particles spend 3 consecutive days in the core during which time
the temperatures are at 55°C

•	Preventing growth of pathogenic bacteria in composted material

Ensuring that all material is mixed into the core of the pile, depends on the configuration of the windrows
and the turning methodology. Pile size and shape as well as material characteristics determine how much
of the pile is in the "hot zone" at any given time. Additional turning and maintenance of temperatures

36

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after the mandated 15 days are recommended, depending on the windrow configuration. For example,
the Los Angeles County Sanitation District found that as many as 12-15 turnings were necessary to reduce
pathogens in windrow composted sewage sludge (Personal Communication, Ross Caballero, Los Angeles
County Sanitation District, 1998).

It is important that once that material is in the pile core it be subject to the full time-temperature regime
necessary to reduce pathogens. Therefore, the turning schedule and the recovery of the core zone to
55°C are important factors. If pile turning is not evenly distributed throughout the 15-day period, some
material may not spend adequate time in the core of the pile. Additionally, pile temperatures generally
drop off immediately after turning; if temperatures in the pile core do not quickly recover to 55°C (within
24 hours), the necessary pathogen reduction period of 3 days will not be achieved.

Because of the operational variability, pathogen reduction in windrow composting has been found to be
less predictable than pathogen reduction in aerated static pile or in-vessel composting. In order to
improve pathogen reduction, the following operational guidelines are recommended.

• Windrow turning should take place after the pile core has met pathogen reduction temperatures
for three consecutive days. Windrow turnings should be evenly spaced within the 15 days so that
all material remains in the core zone for three consecutive days; allowing additional time as
needed for the core temperature to come up to >55°C.

• Pathogen reduction temperatures (55°C) must be met for 15 consecutive days at the pile core.

• Temperatures should be taken at approximately the same time each day in order to demonstrate
that 55°C has been reached in the pile core within 24 hours after pile turning.

• Testing frequency should be increased; a large sewage sludge windrow composting operation
recommends testing each windrow for Salmonella sp. before piles are distributed (Personal
Communication, Ross Caballero, Los Angeles County Sanitation District, 1998). Samples are taken
after turning is completed, and piles which do not comply with Class A requirements are retained
on site for further composting.

Vector Attraction Reduction

VAR Option 5 is the most appropriate for composting operations. This option requires aerobic treatment
(e.g., composting) of the sewage sludge for at least 14 consecutive days at over 40°C (104°F) with an
average temperature of over 45°C (113°F). This is usually easily attained by sewage sludge composting.

The PFRP and VAR requirements can be met concurrently in composting. For within-vessel or aerated
static pile composting, the temperature profile should show PFRP temperatures at each of the
temperature monitoring points for three consecutive days, followed by a minimum of 11 more days
duringwhich time the average temperature of the pile complies with VAR requirements. For windrow
piles, the compliance with PFRP temperatures will also fulfill VAR requirements.

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PFRP temperatures should be met before or at the same time that VAR requirements are fulfilled in order
to reduce the potential for pathogen regrowth. However, continued curing of the composting material
will most likely further prevent the growth of pathogenic bacteria from taking place.

Like all microbiological processes, composting can only take place with sufficient moisture (45-60%).
Excessive aeration of composting piles or arid ambient condition may dry composting piles to the point at
which microbial activity slows or stops. The cessation of microbial activity results in lowered pile
temperatures which can easily be mistaken for the end-point of composting. Although composting may
appear to have ended, and compost may even meet vector attraction reduction via Option 7, overly dried
compost can cause both odor problems and vector attraction if moisture is reintroduced into the material
and microbial activity resumes. It is therefore recommended that the composting process be maintained
at moisture levels between 45-60% (40-55% total solids) (Epstein, 1997).

Although not mandated by Part 503, compost is usually maintained on site for longer than the required
PFRP and VAR duration. In order to produce a high-quality, marketable product, it has been found that a
curing period, or the period during which the volatile solids in the sewage sludge continue to decompose,
odor potential decreases, and temperatures decrease into the mesophilic (40-45°C) range, is necessary.
Depending on the feedstock and the PFRP selected, the curing period may last an additional 30 - 50 days
after regulatory requirements are met.

In general, compost is not considered marketable until the piles are no longer self-heating. It is important
to note that compost piles that are cooled by excessive aeration or that do not self-heat because the
material is too dry to support microbial activity may not actually be fully decomposed.

It has been found that further reduction of organic material takes place during the curing phase of
composting (Epstein, 1997). Therefore, microbiological testing should take place at the end of the curing
process when compost is prepared for sale or distribution. Compost which is stored on site for extended
periods of time until it can be sold or distributed must be tested for compliance with microbiological
limits when it is to be used or disposed.

5.3 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 viruses, bacteria, and helminth ova to below
detectable levels. Four processes are commonly used for heat drying sewage sludge: flash dryers, spray
dryers, rotary dryers and steam dryers.

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Vector Attraction Reduction

No further processing is required because the PFRP requirements for heat drying also meet the
requirements of VAR Option 8 for vector attraction reduction (the percent solids must be at least 90%
before mixing the sewage sludge with other materials). This fulfills the requirement of Option 7 if the
sewage sludge being dried contains no unstabilized solids.

Drying of sewage sludge to 90% solids deters the attraction of vectors, however unstabilized dried
biosolids that are rewetted may become odorous and attract vectors. Therefore, it is recommended that
materials be used or disposed while the level of solids remains high and that dried material be stored and
maintained under dry conditions.

Some operators have found that maintaining stored material at solids levels above 95% helps to deter
reheating because microbiological activity is halted. However, storage of materials approaching 90% total
solids can lead to spontaneous combustion with subsequent fires and risk of explosion. While there is
little likelihood of an explosion occurring with storage of materials like pellets, precautionary measures
such as maintaining proper oxygen levels and minimizing dust levels in storage silos and monitoring
temperatures in material can reduce the risk of fires.

5.4 Heat Treatment

Heat treatment processes are used to disinfect sewage sludge and reduce pathogens to below detectable
levels. The processes involve heating sewage sludge under pressure for a short period of time. The
sewage sludge becomes sterilized and bacterial slime layers are solubilized, making it easier to dewater
the remaining sewage sludge solids.

The Part 503 PFRP description for heat treatment is:

• Liquid sewage sludge is heated to a temperature of 180°C (356°F) or higher for 30 minutes.

Vector Attraction Reduction

Heat treatment in most cases must be followed by vector attraction reduction. VAR Options 6 to 11 (pH
adjustment, heat drying, or injection, incorporation, or daily cover) may be used (see Chapter 8). Options
1 through 5 would not typically be applicable to heat treated sludge unless the sludge was digested or
otherwise stabilized during or after heat treatment (e.g., through the use of wet air oxidation during heat
treatment).

5.5 Thermophilic A erobic Digestion

Thermophilic aerobic digestion is a refinement of the conventional aerobic digestion processes discussed
in Section 7.2. 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

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biodegradable volatile solids content of the sewage sludge can be reduced by up to 70% in a relatively
short time. The digested sewage sludge is effectively pasteurized due to the high temperatures.
Pathogenic viruses, bacteria, viable helminth ova and other parasites are reduced to below detectable
limits if the process is carried out at temperatures exceeding 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 description of thermophilic aerobic digestion is:

• 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 consecutive days at 55°C to 60°C (131°F to 140°F).

The thermophilic process requires significantly lower residence times (i.e., solids retention time) than
conventional aerobic 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), respectively. This is due to the dramatic increase in temperatures compared to
mesophilic aerobic digestion. Residence time is normally determined by dividing the volume of sewage
sludge in the vessel by the volumetric flow rate. Facility operation should minimize the potential for
bypassing by withdrawing treated sewage sludge before feeding, and feeding no more than once a day.

Complete-mix reactors with continuous feeding may not be adequate to meet Class A pathogen
reduction because of the potential for bypassing or short-circuiting of untreated sewage sludge. These
types of systems are difficult to demonstrate complete mix, as well as account for the short circuiting that
will occur. Since every particle must be treated in a Class A process any short circuiting would invalidate
this process.

Vector Attraction Reduction

Vector attraction reduction must be demonstrated. Although all options, except VAR Options 2, 4, and 12
are possible, VAR Options 1 and 3 which involve the demonstration of volatile solids loss are the most
suitable. VAR Option 2 is appropriate only for anaerobically digested sludge, and VAR Option 4 is not
possible because it is not yet known how to translate SOUR measurements obtained at high
temperatures to 20°C [68°F].

5.6 Beta Ray and Gamma Ray Radiation

Radiation can be used to disinfect sewage sludge. Radiation 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 million volts. Both types of radiation destroy pathogens that they penetrate if the doses are
adequate.

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The Part 503 PFRP descriptions for irradiation systems are:

Beta Ray Irradiation

• Sewage sludge is irradiated with beta rays from an accelerator at dosages of at least 1.0 megarad
at room temperature (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 (20°C [68°F]).

The effectiveness of beta radiation in reducing pathogens depends on the radiation dose, which is
measured in 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.
Since organic matter is not destroyed with the use of this process, sewage sludge must be properly stored
after processing to prevent contamination.

Vector Attraction Reduction

Radiation treatment must be followed by vector attraction reduction. The appropriate options for
demonstrating vector attraction reduction are the same as for heat treatment (see Section 9.4), namely
VAR Options 6 to 11. Options 1-5 are not applicable unless the sewage sludge is subsequently digested.

5.1 Pasteurization

Pasteurization involves heating sewage sludge to above a predetermined temperature for a minimum
time period. For pasteurization, the Part 503 PFRP description is:

• The temperature of the sewage sludge is maintained at 70°C (158°F) or higher for 30 minutes or
longer.

Pasteurization reduces bacteria, enteric viruses, and viable helminth ova to below detectable values.
Sewage sludge can be heated by heat exchangers or by steam injection. Sewage sludge is pasteurized in
batches to prevent recontamination that might occur in a continuous process. Sewage sludge must be
properly stored after processing because the organic matter has not been stabilized and therefore odors
and growth of pathogenic bacteria can occur if sewage sludge is re-inoculated.

In theory, quicklime can be used to meet the requirements for pasteurization of sewage sludge. The
water in the sludge slakes the lime, forming calcium hydroxide, and generates heat. However, it is
difficult to ensure that the entire mass of sewage sludge comes into contact with the lime and achieves
the required 70oC for 30 minutes. This is particularly true for dewatered sewage sludges. Processes
must be designed to 1) maximize contact between the lime and the sewage sludge, 2) ensure that
adequate moisture is present, 3) ensure that heat loss is minimal, and 4) if necessary, provide an auxiliary
heat source. Pasteurization cannot be accomplished in open piles.

In addition, in order for pasteurization to be conducted properly, facility operators must be trained with
regard to 1) the proper steps to be taken to ensure complete hydration of the alkaline reagent used, 2)
the evaluation of the slaking rate of the lime based alkaline material required for their particular process,

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specifying the reactivity rate required, 3) the proper measurement of pH, 4) an awareness of the effect of
ammonia gassing off and how this affects the lime dose, and 5) the necessity for maintaining sufficient
moisture in the sewage sludge/alkaline mixture during the mixing process to ensure the complete
hydration of the quicklime and migration of hydroxyl ions throughout the sewage sludge mass. This is
done to ensure that the entire sewage sludge mass is disinfected.

EPA -sponsored studies showed that pasteurization of liquid sewage sludge at 70°C (158°F) for 30
minutes inactivates parasite ova and cysts and reduces the population of measurable viruses and
pathogenic bacteria to below detectable levels (USEPA, 1979). This process is based on the
pasteurization of milk which must be heated to at least 63°C (145°F) for at least 30 minutes.

Vector Attraction Reduction

Pasteurization must be followed by a vector attraction reduction process unless the vector attraction
reduction conditions of VAR Option 6 (pH adjustment) have been met. The VAR options appropriate for
demonstrating vector attraction reduction are the same as those for heat treatment (see Section 9.4),
namely VAR Options 6 to 11. VAR Options 1 to 5 are not applicable unless the sludge is subsequently
digested.

5.8 Equivalent Processes

Under Class A Alternative 6, sewage sludge treated in processes that are determined to be equivalent to
PFRP are considered to be Class A Processes because they consistently demonstrated microbial removal
and as well as pathogen levels below detection. Chapter 8 discusses how the Pathogen Equivalency
Committee makes a recommendation of equivalency. Table (5.1) lists all the PFRP processes found to be
equivalent and approved for use in the US, either on a national or site-specific basis.

Table 5.1 EPA Approved PFRP Equivalencies
| Applicant /	Equivalency Type /

Process Name

Date Received

Process Description

Magna Management
(Tucson, AZ)
MagnaGrow Process

!	Site-Specific PFRP Equivalency

:	September 2013

!	Green Valley Wastewater Treatment

=	facility located at 2201 North Old

i	Nogales Highway Green Valley, AZ

i	85614

:	Gallons per dry ton; and Sodium or Potassium Hydroxide is added

;	until the pH of the mixture in the reactor is above 12. Percent total

:	solids of sludge can range between 5% and 25%. The batch reactor

;	is a specially designed rotomixer. The sludge is kept in the tightly

:	sealed rotomixer for 24 hours from the starting time, after which

Metam sodium is added to sludge based on sludge total solids in

the generated product is neutralized for beneficial reuse. Since the
sludge is kept above pH of 12 for 2 hours and the pH does not fall
below 11.5 for at least 22 hours, then the treatment process

satisfies the requirements of Part 503.33(b) (6) (i.e., add base to pH
greater than 12, not to fall below this pH for two hours, and not to
fall below pH 11.5 for 22 more hours, with only a single application
of base).

BCR Environmental
(Jacksonville, FL)
Neutralizer Process

Conditional National Equivalency
November 2010

Waste activated sludge total solids concentration of less than or

equal to 4% is treated at temperatures greater than or equal to 15°
C (59° F) with an oxidizing agent (chlorine dioxide) at an oxidation
reduction potential (ORP) of greater than or equal to +100 mV with
a contact time at +100 mV ORP > 1 hour. Such treatment in

followed by addition of sodium nitrite at 1500 mg/L and acid to

42

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Applicant/ Equivalency Type /

Process Description

Process Name Date Received





achieve a pH of < to 2.3 s.u. for six hours. The ORP during this
second treatment stage must be > +100 mV and nitrite contact
time at specified pH and ORP is at least six hours.

Columbus Water Works
(Columbus, GA)

Columbus Biosolids Flow-
Through Thermophilic
Treatment (CBFT3) Process

Conditional Site-Specific PFRP
Equivalency [Conditionality: 1) once
built the fluid dynamics of the full-scale
CFMD must be verified to be consistent
with that of the laboratory-scale CFMD;
2) helminth and enteric viruses must
continue to be monitored on the full-
scale process in addition to and at the
same frequency as, the regulatory
requirement for fecal coliform
or Salmonella spp. monitoring until data
statistically supports the required 2 and
3-log reductions, respectively.]

October 2005

The process consists of four stages: 1) sludge preheat tank; 2)
continuously fed, mixed digester (CFMD) operated at a minimum
temperature of 53°C and a residence time of > 6.0 days; 3) a plug-
flow reactor or series of batch tanks that provide a contact time of
at least 30 minutes at a temperature of > 60°C; 4)a mesophilic
digester. It is further necessary that the limited conditions under
which the process was tested are maintained at full-scale
operation, namely that: 1) Columbus's co-thickened mixed primary
and waste activated sludge contains 6.0 & ± 1.0 to 2.0% total solids
of which > 50% is volatile; 2) the average ammonium-nitrogen
content of the digesting sludge in the CFMD is > 920 mg/L; 3) the
pH in the digester is > 7.3 and < 8.3; 4) total volatile acid
concentrations are between 1,000 and 2,250 mg/L; 5) thorough
heating of the sludge is verified throughout process operations by
continuous temperature monitoring.

Burch Biowave, Inc.
(Fredericktown, OH)1
Burch Biowave™ Process

Acknowledgement as a Class A, Alt. 1

process

March 2005

A thin layer of dewatered sludge (> 7% total solids) is conveyed
through a system of microwave generators (75-100 kW) which heat
sludge to > 80°C for 6-14 minutes. These conditions exceed the
time and temperature requirements for Class A, Alternative 1 [D =
131,700,000/1001400t where D = time required in days; t =
temperature in °C (Regime B)]. Heated air and an exhaust blower
assist in drying the sludge to 75 - 90% solids.

Schwing Bioset, Inc.
(Houston, TX)

Bioset Process

National PFRP Equivalency
August 2011

Dewatered municipal sludge solids between six to thirty-five
percent total solids by weight are mechanically mixed with calcium
oxide (quicklime) to achieve a pH of greater than or equal to twelve
standard units. Sulphamic acid is added to, and mixed with the
sludge/quicklime to promote an exothermic reaction which
increases the temperature of the mixture to equal to or greater
than 55°C (131°F). The sludge/quicklime/sulphamic acid mixture is
then directed to a pressurized plug flow reactor for a minimum
solids retention time of forty minutes at a minimum temperature of
55°C (131°F).

ONDEO Degremont
(Richmond, VA) (formerly
held by Lyonnaise des
Eaux (Le Pecz-Sur-
Seine,France))

Two-Phase Thermo-Meso
Feed Sequencing
Anaerobic Digestion
(2 PAD™)

Conditional National PFRP Equivalency
[Conditionality: Helminth and enteric
viruses must continue to be monitored
on the full-scale process in addition to
and at the same frequency as, the
regulatory requirement for fecal
coliform or Salmonella spp. monitoring
until data statistically supports the
required 2 and 3-log reductions,
respectively.]

September 2002

Sewage sludge is treated in the absence of air in an acidogenic
thermophilic reactor and a mesophilic methanogenic reactor
connected in series. The mean cell residence time shall be at least
2.1 days (± 0.05 d) in the acidogenic thermophilic reactor followed
by 10.5 days (± 0.3 d) in the mesophilic methanogenic reactor.
Feeding of each digester shall be intermittent and occurring 4 times
per day every 6 hours. The mesophilic methanogenic reactor shall
be fed in priority from the acidogenic thermophilic reactor.

Between two consecutive feedings temperature inside the
acidogenic thermophilic reactor should be between 49°C and 55°C
with 55°C maintained during at least 3 hours. Temperature inside
the mesophilic methanogenic reactor shall be constant at least
37°C.

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Applicant /

Equivalency Type /

Process Description

Ultraclear, Marlboro, NJ

Microbiological Conditioning and Drying
Process (MVCD)

In this process, sludge cake passes through several aerobic-
biological type stages (Composting is an example) where different
temperatures are maintained for varying times. Stage 1 occurs at
35°C for 7-9 hours; stage 2 occurs at 35-45°C for 8-10 hours; stage
3 occurs at 45-65°C for 7-10 hours; and the last stage is
pasteurization at 70-80°C for 7-10 hours. In addition one of two
conditions described below must be met:

Condition 1: Dewatered sludge cake is dried by direct or indirect
contact with hot gases, and moisture content is reduced to 10% or
lower. Sludge particles reach temperatures we// in excess of 80°C
or the wet bulb temperature of the gas stream in contact with the
sludge at the point where it leaves the dryer is in excess of 80°C. OR
Condition 2: A) Using the within-vessel, static aerated pile, or
windrow composting methods, the sludge is maintained at
minimum operating conditions of 40°C for 5 days. For 4 hours
during the period the temperature exceeds 55°C; {Note: another
PSRP-type process should be substituted for that of composting};
and B) Sludge is maintained for at least 30 minutes at a minimum
temperature of 70°C.

Synox Corp.

National PFRP Equivalency

Operation occurs in a batch mode under the following conditions:

Pori International, Inc.
(Baltimore, MD)

Pori Process

National PFRP Equivalency
May 1992

Sludge is preheated to 82°C (180°F) using recovered steam. Sulfuric
acid is added to reduce the pH to 3. The mixture is then pressurized
to 100 psig achieving temperatures of > 165°C (330°F) for a
treatment time of 1 hour. Lime slurry is used to neutralize pH

CBI Walker, Inc.

Conditional National PFRP Equivalency

Sludge is introduced intermittently into a vessel, amounting to 5 to
20% of its volume, where it is heated by both external heat
exchange and by the bio-oxidation which results from vigorously
mixing air with the sludge (pasteurized) and has a nominal
residence time of 18 to 24 hours. Time between feedings of
unprocessed sludge can range from 1.2 (@ ~ 65°C) to 4.5 (@ ~
60°C) hours. Exiting sludge is heat exchanged with incoming
unprocessed sludge. Thus, the sludge is cooled before it enters a
mesophilic digester.

Fuchs Gas Und
Wassertechnik, Gmbh
(Mayen, Germany)2
Autothermal Thermophilic
Aerobic Digestion (ATAD)

Conditional National PFRP Equivalency
[Conditionality: l)Time and temperature
in the first vessel must be > 30 minutes
and > 50°C, and controlled by the
equation D = 50,070,000/1001400t(where
D = time required in days; t =
temperature in °C) for sludges of < 7%

ATAD is a two-stage autothermal aerobic digestion process. The
stages are of equal volume. Treated sludge amounting to 1/3 the
volume of a stage is removed every 24 hours from the second stage
as a product. An equal amount then is taken from the first stage
and fed to the second stage. Similarly, an equal amount of
untreated sludge is then fed to the first stage. In the 24-hour period
between feedings, the sludge in both stages is vigorously agitated

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Equivalency Type /

Process Description

Date Received



solids; 2) Operations of the reaction
vessel during the time-temperature
periods must be either plug flow or
batch mode.]

November 1992

and contacted with air. Bio-oxidation takes place and the heat
produced increase the temperature. Sludge temperature in the
reactors averages between 56 and 57°C for > a 16-hour period,
while the overall hydraulic residence time is 6 days

K-F Environmental
Technologies, Inc.
(Pompton Plains, NJ)1
Type of Sludge Drying
Process

Acknowledgement of PFRP by meeting
current regulations under 40 CFR 257,
App. II

November 1992

Sludge is heated to a minimum temperature of 100°C and indirectly
dried to below 10% moisture using oil as a heat transfer medium.
The final discharge product has exceeded a temperature of 80°C
and is a granular, dry pellet that can be land applied, incinerated, or
landfilled. In addition the following conditions must be met:
Dewatered sludge cake is dried by direct or indirect contact with
hot gases, and moisture content is reduced to 10% or lower. Sludge
particles reach temperatures well in excess of 80°C or the wet bulb
temperature of the gas stream in contact with the sludge at the
point where it leaves the dryer is in excess of 80°C.

International Process
Systems, Inc.
(Glastonbury, CT)1
Type of Composting
Process

Conditional National PFRP Equivalency
[Conditionality: Process operation is to
be controlled so that the composting
mass passes through a zone in the
reactor in which the temperature of the
compost is at least 55°C throughout the
entire zone, and the time of contact in
this zone is at least three days.]

April 1991

IPS developed a unique within-vessel composting reactor using
forced-aeration and bed-agitation to create an optimal aerobic
environment. Long rectangular vessels are loaded at one end. An
agitator/mixer assembly rides across the top of the vessel, mixing &
conveying material down the vessel at a rate of approximately 12
ft/day. Finished compost reaches the opposite end of the vessel in
18 days having passed through five zones of treatment with
average temperatures > 60°C.

ATW, Inc.

(Santa Barbara, CA)
Alkaline Stabilization /
Pasteurization

PFRP Equivalency
Prior to 1989

Manchak process uses quicklime to simultaneously stabilize and
pasteurize biosolids. Quicklime, or a combination of quicklime and
fly ash, is mixed with dewatered sludge at a predetermined rate in a
confined space. An instant exothermic reaction is created in the
product wherein the pH is raised in excess of 12 after two hours of
contact, in addition, the temperature is raised in excess of 70°C for
> 30 minutes

N-Viro Energy Systems,
Ltd.

(Toledo, OH)

Advanced Alkaline
stabilization with
subsequent accelerated
drying

National PFRP Equivalency
January 1988

Method 1: Fine alkaline materials (cement kiln dust, lime kiln dust,
quicklime fines, pulverized lime, or hydrated lime) are uniformly
mixed by mechanical aeration mixing into liquid or dewatered
sludge to raise the pH to > 12 for 7 days. If the resulting sludge is
liquid, it is dewatered. The stabilized sludge cake is then air dried
(while pH remains > 12 for > 7 days) for > 30 days and until the cake
is > 65% solids. A solids concentration of > 60% is achieved before
the pH drops below 12. The mean temperature of the air
surrounding the pile is > 5°C (41°F) for the first 7 days.

Method2: Now in 40 CFR 503 as Class A, Alternative 2

Scarborough Sanitation
District

(Scarborough, ME)1
Fly ash composting

Site-Specific PFRP Equivalency
March 1987

Traditional static aerated pile composting using fly ash as the
bulking agent. Thus, heat (at least in part) is generated through
chemical reaction with the fly ash and not through biological
reactions as would a typical composting process. Time and
temperature requirements for Class A static aerated piles were
exceeded with operating conditions of 60 to 70°C reached within
24 hours and maintained for 14 days. Equivalency was
recommended on this basis.

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5.9 References

Caballero, Ross. 1984. Experience at a windrow composting facility: LA County site technology transfer.
USEPA, Municipal Environmental Research. Cincinnati, Ohio.

Composting Council. 1994. Compost facility operating guide: A reference guide for composting facility and
process management. Alexandria, Virginia.

Epstein, Eliot. 1997. The science of composting. Technomic Publishing Company.

Farrell, J.B. 1992. Fecal pathogen control during composting, presented at International Composting
Research Symposium, Columbus, Ohio.

Haug, Roger T. 1993. The practical handbook of compost engineering. Lewis Publishers.

lacaboni, M.D., J.R. Livingston, and T.J. LeBrun. 1984. Windrow and static pile composting of municipal
sewage sludges. Report No.: EPA/600/2-84-122 (NTIS PB84-215748).

USEPA. 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.

USDA/USEPA. 1980. Manual for composting sewage sludge by the Beltsville aerated-pile method. Report
No.: EPA/600/8-80-022.

WEF/ASCE. 1998. WEF Manual of Practice No. 8, Design of Municipal Wastewater Treatment Plants. Pub.
WEF (Alexandria, VA) and ASCE (New York, NY).

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: National Technical Information Service.

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6: CLASS B PATHOGEN REQUIREMENTS FOR
MATERIAL APPLIED TO AGRICULTURAL LAND,
A FOREST, OR A RECLAMATION SITE

6.1 Introduction

This chapter discusses the Class B requirements in Subpart D of 40 CFR Part 503. The implicit goal of the
Class B pathogen requirements is to reduce the pathogens load in the sewage sludge. As mentioned in
Chapter 2, Part 503 uses microbial indicators as one option to determine if sewage sludges have been
properly treated with respect to pathogen destruction. In the case of Class B materials, the assumption is
2 million MPN or CFU of fecal coliforms in the final product equates to a 2 log reduction of the overall
fecal coliform population in the raw sludge. It is further assumed that a 2 log reduction in the bacterial
population would show a 1 log reduction in the virus population (Kowal 1985). There is no assumption of
any protozoan removal using the fecal coliform indicator level.

Land application of these materials whether meeting PSRP requirements or fecal coliform monitoring,
includes proper barriers and site restrictions to allow for the natural attenuation of pathogens present in
these biosolids. The site restrictions are discussed in further detail in section 6.5 of this chapter. The
degree of the site restriction access is dependent upon the particular use of that land that received the
Class B material. For instance, land that will be used for planting crops will have different requirements
than land that will be used for animal grazing. The purpose of the site restrictions is to allow further
reduction of the pathogen population in the applied biosolids through environmental conditions such as
sunlight, desiccation, and natural attenuation. It is also important that treatment workers, land appliers
and haulers, and farm workers understand the inherent risk with Class B land application and use proper
personal protective equipment (PPE) and precautions (CDC 2002).

There are three alternative requirements for demonstrating Class B pathogen reduction. As with Class A
biosolids, Class B biosolids must address vector attraction. The choice of vector attraction options may
affect the duration of the site restrictions in some cases.

6.2 Sewage Sludge Alternative 1: Monitoring of Fecal Coliform [503.32(b)(2)]

Alternative 1 requires that seven samples of treated sewage sludge be collected and that the geometric
mean fecal coliform density of these samples be less than 2 million CFU or MPN per gram of biosolids (dry
weight basis). This approach uses fecal coliform density as an indicator of the average density of bacterial
and viral pathogens.

A geometric mean of at least seven samples is required with this alternative. The use of seven samples is
expected to reduce the standard error to a reasonable value. The standard deviation can be a useful
predictive tool. A standard deviation of greater than 1 log for the fecal coliform density indicates a wide
range in the densities of the individual samples. This may be due to sampling variability or variability in

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the laboratory analysis, or it may indicate that the treatment process is not consistent in its reduction of
pathogens. A high standard deviation can therefore alert the preparer that the sampling, analysis, and
treatment processes should be reviewed.

Each of the multiple samples taken for fecal coliform analysis should be taken at the same point in the
process so that treatment of each sample is equal. Generally, a log standard deviation between duplicate
samples under 0.3 is acceptable for lab analyses (see Table 6.1).

Table 6.1 Calculating the Geometric Mean for Class B Alternative 1

j Directions for performing geometric mean analysis on Class B Biosolids

• Take seven samples over a 2-week period.

• Analyze samples for fecal coliform using the membrane filter or MPN dilution
method.

• Take the log (Base 10) of each result.

• Take the average (arithmetic) of the logs.

• Take the anti-log of the arithmetic average. This is the geometric mean of the
results.

| Example: The results of analysis of seven samples of sewage sludge are shown below. The
i second column of the table shows the log of each result.

Fecal Coliform
(MPN/dry gram
sewage sludge)

Sample Number (MPN/dry gram Lt>g

Sample 1 6.4 xlO6 6.81

Sample 2 4.8 x 104 4.68

Sample 3 6.0 xlO5 5.78

Sample 4 5.7 xlO5 5.76

Sample 5 5.8 xlO5 5.76

Sample 6 4.4 xlO6 6.64

Sample 7 6.2 xlO7 7.80

Average (Arithmetic) 6.18

Antilog (geometric mean) 1.5 xlO6*

Log standard deviation 1.00*

*Note that this sewage sludge would meet Class B fecal coliform requirements even though several of the
analysis results exceed the 2.0 x 106/dry gram limit. Duplicate analyses on the same sample would give a

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much lower standard deviation. Variability is inflated by differences in feed and product over a 2-week
sampling period

Process parameters including retention time and temperature should be examined to verify that the
process is running as specified. Monitoring equipment should be calibrated regularly.

The seven samples should be taken over a 2-week period in order to represent the performance of the
facility under a range of conditions. For small facilities that are required to sample infrequently, sampling
should be performed under worst case conditions, for example, during the winter when the climatic
conditions are the most adverse.

When lagoons are infrequently dredged, the lagoon can be cordoned off into seven equal sections and a
sample taken from each section and the geometric mean calculated. Samples could be composited within
each section to increase representativeness.

Vector Attraction Reduction

Meeting the requirements for VAR depends on the process by which the pathogen reduction level is met.
Chapter 9 discusses VAR in more detail (Table 9.2).

6.3 Sewage Sludge Alternative 2: Use of a Process to Significantly Reduce Pathogens
(PSRP) [503.32(b)(3)]

Under Alternative 2, biosolids are considered to be Class B for pathogen reduction if they are treated
using one of the "Processes to Significantly Reduce Pathogens" (PSRPs) Table7.1. The biological PSRP
processes are sewage sludge treatment processes that have been demonstrated to result in a 2-log
reduction in fecal coliform density (see Chapter 7).

Microbial Monitoring

Unlike the comparable Class A requirement (see Section 4.8), this Class B alternative does not require
microbiological monitoring. However, monitoring of process requirements such as time, temperature,
and pH required.

Vector Attraction Reduction

Meeting the requirements for VAR depends on the process by which the pathogen reduction level is met.
Chapter 9 discusses VAR in more detail.

6.4 Sewage Sludge Alternative 3: Use of Processes Equivalent to PSRP
[503.32(b)(4)]

Under Class B Alternative 3, sewage sludge treated by any process determined to be equivalent to a PSRP
is considered to be Class B biosolids. A list of processes that have been recommended as equivalent to
PSRP are shown in Table 8.1.

Part 503 gives the regulatory authority responsibility for determining equivalency. The Pathogen
Equivalency Committee is available as a resource to provide guidance and recommendations on
equivalency determinations to the regulatory authorities (see Chapter 8).

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Microbial Monitoring

Vector Attraction Reduction

Meeting the requirements for VAR depends on the process by which the pathogen reduction level is met.
Chapter 9 discusses VAR in more detail.

6.5 Site Restrictions for Land Application of Biosolids [503.32(b)(5)]

Potential exposure to pathogens in Class B biosolids via food crops is a function of three factors: 1.)
presence of pathogens in land applied biosolids. 2.)Transfer of these pathogens to the harvested crop,
and lastly 3.)ingestion of the crop prior to removal of pathogens due to crop processing protocols.
Elimination of one of these steps eliminates the pathway by which public health may be affected. As
stated previously, biosolids that meet the Class B requirements may contain reduced but still significant
densities of pathogenic bacteria, viruses, protozoans, and viable helminth ova. Thus, site restrictions are
used to allow time for further reduction in the pathogen population. Harvest restrictions are imposed to
eliminate the possibility that food will be harvested and ingested before pathogens which may be present
on the food have died off. Harvest restrictions vary, depending on the type of crop, because the amount
of contact a crop will have with biosolids or pathogens in biosolids varies.

The site restrictions are primarily based on the survival rate of viable helminth ova, one of the hardiest
pathogens that may be present in sewage sludge. The survival of pathogens, including the helminth ova,
depends on exposure to the environment. Some of the factors that affect pathogen survival include pH,
temperature, moisture, cations, sunlight, presence of soil microflora, and organic material content. On
the soil surface, helminth ova have been found to die off within four months, but survival is longer if
pathogens are within the soil. Helminth ova have been found to survive in soil for several years (Smith,
1997; Kowal 1985).

Site restrictions also take the potential pathways of exposure into account. For example, crops that do
not contact the soil, such as oat or wheat, may be exposed to biosolids, but pathogens on crop surfaces
have been found to be reduced very quickly (30 days) due to exposure to sunlight, desiccation, and other
environmental factors. Crops that touch the soil, such as melons or cucumbers, may also come into
contact with biosolids particles, but pathogens in this scenario are also subject to the harsh effects of
sunlight and rain and will die off quickly. Crops grown in soil such as potatoes are surrounded by biosolids
amended soil, and pathogen die-off is much slower below the soil surface.

These pathways should be considered when determining which site restriction is appropriate for a given
situation. The actual farming and harvesting practices as well as the intended use of the food crop should
also be considered. For example, oranges are generally considered a food crop that does not touch the
ground. However, some oranges grow very low to the ground and may come into contact with soil. If the
oranges that have fallen to the ground or grew touching the ground are harvested for direct consumption
without processing, the 14-month harvest restriction for crops that touch the soil should be followed.
Orange crops which do not touch the ground at all would not fall under the 14-month harvest restriction;

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harvest would be restricted for 30 days under 503.32(b)(5)(iv) which covers food crops that do not have
harvested parts in contact with the soil. For similar situations, the potential for public health impacts
must be considered. Harvest practices such as the use of fallen fruit or washing or processing crops
should be written into permits so that restrictions and limits are completely clear. Figure 6.1 illustrates
the steps of exposure that should be considered when making a decision about harvest and site
restrictions. In addition, several examples of permit conditions are included. The site restrictions for land
applied Class B biosolids are summarized below. Note that the restrictions apply only to the harvesting of
food crops, but not to the planting or cultivation of crops and the time periods are from the time of
application to the time of harvest.

6.6 Food Crops with Harvested Parts That Touch the biosolid/Soil Mixture

503.32(b)(5)(l): Food crops with harvested parts that touch the biosolid/soil mixture and are totally above
the land surface shall not be harvested for 14 months after application of biosolids.

This time frame is sufficient to enable environmental conditions such as sunlight, temperature, and
desiccation to further reduce pathogens on the land surface. Note that the restriction applies only to
harvesting. Food crops can be planted at any time before or after biosolids application, as long as they
are not harvested within 14 months after sludge application. Examples of food crops grown on or above
the soil surface with harvested parts that typically touch the sewage sludge/soil mixture include lettuce,
cabbage, melons, strawberries, and herbs. Land application should be scheduled so that crop harvests
are not lost due to harvest restrictions.

6.1 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 incorporation into the soil.

Pathogens on the soil surface will be exposed to environmental stresses which greatly reduce their
populations. Helminth ova have been found to die off after 4 months on the soil surface (Kowal, 1994).
Therefore, a distinction is made between biosolids left on the soil surface for four months and biosolids
which are injected, disced, or plowed into soil more quickly.

6.8 Examples of Site Restrictions for Questionable Food Crop Situations

Tree Nut Crops - Nuts which are washed, hulled, and dehydrated before being distributed for
public consumption must follow the 30-day restriction. Nuts which are harvested from the
ground and sold in their shell without processing are subject to the 14-month restriction.

Sugar Beets - Sugar beets aren't expected to be eaten raw. If the beets are transported off site
and considerable biosolids amended soil is carried off with them, the restrictions apply. If
biosolids are left on the soil surface for 4 months or longer before being incorporated, the 20-
month restriction applies. If biosolids are incorporated within 4 months of application, the 38-
month restriction applies.

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Tomatoes (and peppers) - Fruit often comes in contact with the ground. Tomatoes are sold
both to processors and to farm stands. Tomatoes may be eaten raw by the public without
further processing. The 14-month restriction applies.

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 four months prior to incorporation into the soil.

Exposure of the surface of root crops such as potatoes and carrots to viable helminth ova is a principal
concern under these circumstances. Four months is considered the minimum time for environmental
conditions to reduce viable helminth ova in biosolids on the land surface. Class B biosolids incorporated
into the soil surface less than four months after application 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. Thirty-eight months
after biosolids application is usually sufficient to reduce helminth ova to below detectable levels.

6.9 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)(l-iii) This would include crops with
harvested parts that do not typically touch the biosolids/soil mixture and which are not collected from the
ground after they have fallen from trees or plants. The restriction also applies to all feed and fiber crops.
These crops may be exposed to pathogens when biosolids are applied to the land. Harvesting of these
crops could result in the transport of biosolids pathogens from the growing site to the outside
environment. After 30 days, however, any pathogens in biosolids that may have adhered to the crop
during application will likely have been reduced to non-detectable levels. Hay, corn, soybeans, or cotton
are examples of a crop covered by this restriction.

6.10 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.

Biosolids can adhere to animals that walk on biosolids amended land and thereby be brought into
potential contact with humans who come in contact with the animals (for example, horses and milking
cows allowed to graze on a biosolids amended pasture). Thirty days is sufficient to substantially reduce
the pathogens in surface applied biosolids, thereby significantly reducing the risk of human and animal
contamination.

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6.11	Turf Harvesting

503.32(b)(5)(vi): Turf grown on land where sewage sludge is applied shall not be 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.

The one-year waiting period is designed to significantly reduce pathogens in the soil so that subsequent
contact of the turf layer will not pose a risk to public health and animals. A permitting authority may
reduce this time -period in cases in which the turf is not used on areas with high potential for public
access.

6.12	Public Access

503.32(b)(5)(vii): Public access to land with a high potential for public exposure shall be restricted for one
year after application of the sewage sludge.

As with the turf requirement above, a one-year waiting period is necessary to protect public health and
the environment in a potential high-exposure situation. A baseball diamond, playground, public park,
soccer field, and overflow parking lot are examples of land with a high potential for public exposure. The
land gets heavy use and contact with the soil is substantial (children or ball players fall on it and dust is
raised which is inhaled and ingested).

503.32(b)(5)(viii): Public access to land with a low potential for public exposure shall be restricted for 30
days after application of the sewage sludge.

A farm field used to grow corn or soybeans is an example of land with low potential for public exposure.
Even farm workers and family members walk about very little on such fields. Public access restrictions do
not apply to farm workers, but workers should be aware of the public health implications of land
application and the land application schedule and should follow good hygiene practice during the 30-day
period.

53

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Does sewage sludge comply with Class B
requirements?

No

Must be diverted from land application.

Yes





Does sewage sludge comply with Class A
requirements?

Yes

Sludge can be land-applied without site
restrictions.

No





Is the sewage sludge applied to a food
crop?

No

Site restrictions for sod farms grazing
animals, or public access should be
followed.

Yes





Does the food crop touch the ground or will
fruit that falls on the ground be harvested?

No

Harvest may not take place until 30 days
after application.

Yes





Is It possible that harvested food will be
eaten raw or handled by the public?

No

Permitting authority may use discretion to
reduce waiting period from 14 months to
30 days, depending on the application.

Yes





Is the edible part of the crop grown below
the surface of the land?

No

Harvest may not take place until 14 months
after application.

Yes





Does the sewage sludge remain on the
surface of the land for more than 4 months
after application?

Yes

Harvest may not take place until 20 months
after application.

No





Harvest may not taken place until 38
months after application.





Figure 6.1. Decision tree for harvesting and site restrictions.

54

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6.13 References

Farrell, J.B., G. Stern, and A.D. Venosa. 1985. Microbial destructions achieved by full-scale anaerobic
digestion. Workshop on Control of Sludge Pathogens, Series IV. Alexandria, VA: Water Pollution Control
Federation.

Farrell, J.B., B.V. Salotto, and A.D. Venosa. 1990. Reduction in bacterial densities of wastewater solids by
three secondary treatment processes. Res. Jour. WPCF 62(2): 177-184.

Gerba, C.P., C. Wallis, and J.L. Melmick. 1975. Fate of wastewater bacteria and viruses in soil. J. Irrig.

Drain Div. Am. Soc. Civ. Engineers. 101:157-174.

Kowal, N.E. 1985. Health effects of land application of municipal sludge. Pub. No.: EPA/600/1-85/015.
Research Triangle Park, NC: U.S. EPA Health Effects Research Laboratory.

Kowal, N.E. 1994. Pathogen risk assessment: Status and potential application in the development of
Round II regulations. Proceedings of the June 19-20, 1994 Speciality Conference. The Management of
Water and Wastewater Solids for the 21st Century: A Global Perspective. Water Environment Federation.
Alexandria, VA.

Moore, B.E., D.E. Camann, G.A. Turk, and C.A. Sorber. 1988. Microbial characterization of municipal
wastewater at a spray irrigation site: The Lubbock infection surveillance study. J. Water Pol I ut. Control
Fed. 60(7): 1222-1230.

Smith, J.E., Jr. 1988. Fate of pathogens during the sewage sludge treatment process and after land
application. In Proceedings of the January 21-22, 1998 California Plant and Soil Conference: Agricultural
challenges in an urbanizing state, Sacramento, CA.

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 in sludge-amended soil, a critical
literature review. Report No.: EPA/600/2-87/028. Cincinnati, OH: Office of Research and Development.

Storey, G.W. and R.A. Phillips. 1985. The survival of parasite eggs throughout the soil profile. Parasitology.
91:585-590.

USEPA. 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.

55

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7: PROCESSES TO SIGNIFICANTLY REDUCE
PATHOGENS (PSRP)

7.1 Introduction

Processes to Significantly Reduce Pathogens (PSRPs) was a distinction given to processes that can reduce
the microbial populations in treated sewage sludge by a factor of 10 or greater. This terminology was
first introduced into the sewage sludge regulations prior to 40 CFR Part 503.Once Part 503 was adopted
these were added under Alternative 2 and Alternative 3 for Class B materials. There are five PSRPs listed
under Alternative 2 (see Table 7.1). When operated under the conditions specified outlined in the
regulations, PSRPs reduce fecal coliform densities to less than 2 million CFU or MPN per gram of total
solids (dry weight basis) and reduce Salmonella sp. and enteric virus densities in sewage sludge by
approximately a factor of 10 (Farrell, et al., 1985).

Additionally, the Pathogen Equivalence Committee (PEC) can recommend a process as PSRP equivalent
under Alternative 3, more detail on the PEC and PSRP testing is provided in Chapter 8. Table 7.2 lists PSRP
equivalencies approved by EPA by under Alternative 3.

Although theoretically two or more PSRP processes, each of which fails to meet its specified
requirements, could be combined for effectively reducing pathogens (e.g., partial treatment in digestion
followed by partial treatment by air drying) it cannot be assumed that the pathogen reduction
contribution of each of the operations will result in the 2-log reduction in fecal coliform necessary to
define the combination as a PSRP. Therefore, to comply with Class B pathogen requirements, one of the
PSRP processes must be conducted as outlined in this chapter, or fecal coliform testing must be
conducted in compliance with Class B Alternative 1. The biosolids preparer also has the option of
applying for PSRP equivalency for the combination of processes. Achieving PSRP equivalency enables the
preparer to eliminate the requirement of monitoring for fecal coliform density.

Table 7.1. Processes to Significantly Reduce Pathogens (PSRPs) Listed in Appendix B of 40 CFR Part 503

PSRP

Aerobic

Digestion

Air Drying

Anaerobic
Digestion

Composting

Lime

Stabilization

Regulatory Conditions to maintain PSRP

Sewage 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).
Sewage 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 temperature is above 0°C
(32°F).

Sewage sludge is treated in the absence of air 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 15 days at 35°C to 55°C (131°F) and 60 days at 20°C (68°F).

Using either the within-vessel, static aerated pile, or windrow composting methods, the temperature
of the sewage sludge is raised to 40°C (104oF) 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).
Sufficient lime is added to the sewage sludge to raise the pFH of the sewage sludge to 12 for > 2 hours
of contact.

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7.2 A erobic Digestion

In aerobic digestion, sewage sludge is biochemically oxidized by bacteria in an open or enclosed vessel. To
supply aerobic microorganisms with enough oxygen, either the sewage sludge must be agitated by a
mixer, or air must be forcibly injected. Under proper operating conditions, the volatile solids in sewage
sludge are converted to carbon dioxide, water, and 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 are then separated from the clarified supernatant. 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:

Sewage sludge is agitated with air or oxygen to maintain aerobic 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).

For temperatures between 15°C (59°F) and 20°C (68°F) use the relationship between time and
temperature provided below to determine the required mean cell residence time.

= 1.08 - (20 -T)

40 days

t = Time (units) at temperature (°C)

T = Temperature in °C

The regulation does not differentiate between batch, intermittently fed and continuous operation, so any
method is acceptable. The mean cell residence time is considered the residence time of the sewage
sludge solids. The appropriate method for calculating residence time depends on the type of digester
operation used (see Appendix D).

Continuous-Mode, No Supernatant Removal: For continuous-mode digesters where no supernatant is
removed, nominal residence times may be calculated by dividing liquid volume in the digester by the
average daily flow rate in or out of the digester.

Continuous-Mode, Supernatant Removal: In systems where the supernatant is 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 systems, the flow rate of the sewage sludge out of the digester is used to calculate
residence times.

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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 requirement, 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 completely-mixed reactors in series are
more effective in reducing pathogens than is a single well-mixed reactor at the same overall residence
time. The residence time required for this type of system to meet pathogen reduction goals may be 30%
lower than the residence time required in the PSRP definition for aerobic digestion (see Appendix D).
Flowever, since lower residence times would not comply with PSRP conditions required for aerobic
digestion in the regulation, approval of the process as a PSRP by the permitting authority would be
required.

Other: Digesters are frequently operated in unique ways that do not fall into the categories above.
Appendix D provides information that should be helpful in developing a calculation procedure for these
cases. Aerobic digestion carried out according to Part 503 requirements typically reduces bacterial
organisms by 2-log and viral pathogens by 1-log. Flelminth 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 sewage sludges is demonstrated either when the
percent volatile solids reduction during sewage sludge treatment equals or 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 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 9).

7.3 A naerobic Digestion

Anaerobic digestion is a biological process that uses bacteria 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 that may or may not be heated. Because the biological activity consumes most of the
volatile solids needed for further bacterial growth, microbial activity in the treated sewage sludge is
limited. Anaerobic digestion is one of the most widely used treatments for sewage sludge treatment,
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 intermittently. The

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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 temperature, though heat is sometimes
added to accelerate biological activity. High-rate systems use a combination of active mixing and carefully
controlled, elevated temperature to increase the rate of volatile solids destruction. These systems
sometimes use pre-thickened sewage sludge introduced at a uniform rate to maintain constant
conditions in the reactor. Operating conditions in high-rate systems foster more efficient sewage sludge
digestion.

The PSRP description in Part 503 for anaerobic digestion is:

• Sewage sludge is treated in the absence of oxygen 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).

• Straight-line interpolation to calculate mean cell residence time is allowable when the
temperature falls between 35 °C and 20 °C.

Anaerobic digestion that meets the required residence times and temperatures typically reduces bacterial
and viral pathogens by 90% or more. Viable 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).

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%, which
satisfies vector attraction reduction requirements. Alternatively, vector attraction reduction can be
demonstrated by Option 2 of the vector attraction reduction requirements, which requires that additional
volatile solids loss during bench-scale anaerobic batch digestion of the sewage sludge for 40 additional
days at 30°C to 37°C (86°F to 99°F) be less than 17% (see Section 8.3). The SOUR test is an aerobic test
and cannot be used for anaerobically digested sewage sludge.

7.4 Air Drying

Air drying allows partially digested sewage sludge to dry naturally in the open air. Wet sewage sludge is
usually applied to a depth of approximately 23 cm (nine inches) onto sand drying beds, or even deeper on
paved or unpaved basins. The sewage sludge is left to drain and dry by evaporation. 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 air-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. During the drying/storage period in the bed, the sewage sludge is undergoing physical,
chemical, and biological changes. These include biological decomposition of organic material, ammonia
production, and desiccation.

The PSRP description in Part 503 for air drying is:

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Sewage sludge is dried on sand beds or on paved or unpaved basins. The sewage sludge dries for a
minimum of three months. During two of the three months, the ambient average daily temperature is
above 0°C (32°F).

Although not required by Part 503, it is advisable to ensure that the sewage sludge drying beds are
exposed to the atmosphere 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 by 1-log and bacteria by approximately 2-log. Viable
helminth ova also are reduced, except for some hardy species that remain substantially unaffected.

Vector Attraction Reduction

Frequently sand-bed drying follows an aerobic or anaerobic digestion process that does not meet the
specified process requirements and does not produce 38% volatile solids destruction. However, it may be
that the volatile solids reduction produced by the sequential steps of digestion and drying will meet the
vector attraction reduction requirement of 38% volatile solids reduction. If this is the case, vector
attraction reduction requirements are satisfied.

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 (VAR Option 1, see Section 9.2), 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 VAR Option 7 in
Section 9.8, and VAR Option 8 in Section 9.9).

7.5 Composting

Composting involves the aerobic decomposition of organic material using controlled temperature,
moisture and oxygen levels. Composting can yield either Class A or Class B biosolids, depending on the
time 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 carbon. 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 reduce
pathogens for production of Class B biosolids 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, and the
composted biosolids are "cured" for an additional period.

The three most common composting methods in the United States are windrow, aerated static pile, and
within-vessel composting. Windrow composting involves stacking the sewage sludge/bulking agent
mixture into long piles, or windrows, generally 1.5 to 2.7 meters high (5 to 9 feet) and 2.7 to 6.1 meters
wide (9 to 20 feet). These rows are regularly turned or mixed with a turning machine or front-end loader
to fluff up the material and increase porosity which allows better convective oxygen flow into the

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material. Turning also breaks up compacted material and reduces the moisture content of the
composting media. 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 reduction.

Aerated static pile composting uses forced-air rather than mechanical mixing 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 odor control. 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 but they share two basic techniques: the process
takes place in a reactor vessel where the operating conditions can be carefully controlled, and active
aeration meets the system's high oxygen demand. Agitated bed systems (one type of within-vessel
composting) depend on continuous or periodic mixing within the vessel followed by a curing period.

Pathogen reduction during composting depends on time and temperature variables. Part 503 provides
the following definition of PSRP requirement for pathogen reduction during composting:

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, aerated static pile, or windrow methods, reduce
bacterial pathogens by 2-log and viral pathogens by 1-log.

A process time of only 5 days is not long enough to fully break down the volatile solids in sewage sludge,
so the composted sewage sludge produced under these conditions will not be able to meet any of the
requirements for reduced vector attraction. In addition, sewage sludge that has been composted for only

5	days may still be odorous. Breakdown of volatile solids 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 during which time pile turning or active aeration may continue.

Vector Attraction Reduction

Vector attraction reduction must be conducted in accordance with Option 5, or compost must be
incorporated into soil (VAR Options 9,10) when land applied. This option requires 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).

7.6 Lime Stabilization

The lime stabilization process is relatively straightforward. Lime that is either hydrated lime (Ca(OFI)2),
quicklime (CaO), or lime containing kiln dust or fly ash is added to sewage sludge in sufficient quantities to

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raise the pH above 12 for 2 hours or more after contact. Lime stabilization can reduce bacterial and viral
pathogens by 99% or more. However, such alkaline conditions have little effect on helminth ova.

The Part 503 PSRP description for lime stabilization states:

Sufficient lime is added to the sewage sludge to raise the pH of the sewage sludge to 12 after 2 hours of
contact.

For the Class B lime stabilization process, the alkaline material must be a form of lime. Use of other
alkaline materials must first be demonstrated to be equivalent to a PSRP. Elevation of pH to 12 for two
hours is expected to reduce bacterial and viral density effectively.

Lime may be introduced to liquid sewage sludge in a mixing tank or combined with dewatered sewage
sludge, providing the mixing is complete and the sewage sludge cake is moist enough to allow aqueous
contact between the sewage sludge and lime.

Mixing must ensure that 1) the entire mass of sewage sludge comes into contact with the lime and
undergoes the increase in pH, and 2) samples are representative of the overall mixture (see ChapterlO).
The pH should be measured at several locations to ensure that itis raised throughout the sewage sludge.

A variety of lime stabilization processes are currently in use. The effectiveness of any lime stabilization
process for controlling pathogens depends on maintaining the pH at levels that reduce microorganisms in
the sewage sludge. Field experience has shown that the application of lime stablized material after the
pH has dropped below 10.5 can create odor problems. Therefore, it is recommended that biosolids
application take place while the pH remains elevated. If this is not possible and odor problems develop,
alternative management practices in the field including injection, incorporation, or top dressing the
applied biosolids with additional lime, may be necessary. Alternate management practices (e.g., adding
additional lime, drying, and composting) may also be necessary if the biosolids have not left the
wastewater treatment plant

Vector Attraction Reduction

For lime-treated sewage sludge, vector attraction reduction is best demonstrated by Option 6 of the
vector attraction reduction requirements. This option requires that the sewage 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 9.7).

7.1 Alternative 3 Equivalent Processes

Table 7.2. below lists the processes that the EPA's Pathogen Equivalency Committee has recommended
as being equivalent to PSRP. Information on the PEC and how to apply for equivalency are discussed in
Chapter 8.

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Table 7.2. EPA Approved PSRP Equivalencies

. Equivalency
Applicant/ ,

Type/ ^ . .
Process ^ Process Description
Date

Name

Received

BCR Environmental
CleanB™
(Jacksonville, FL)

National PSRP
Equivalency
October, 2015

The CleanB™ process is a plug-flow, chemical oxidation/aeration process
that utilizes chlorine dioxide to achieve Class B PSRP equivalent waste
activated sludge disinfection. Chemical addition consists of the addition
of site-generated chlorine dioxide into a closed contact chamber and
providing 10 minutes of contact time.

East Bay Municipal
Utilities District
Low MCRT
Treatment Process

Site Specific PSRP
Equivalency
November 2010

Single stage completely mixed thermophilic anaerobic digestion process
operated at a temperature of 50 ± 3° C and a mean cell residence time
of > 240 hours (10-day simple moving average) for treatment of primary
sludge, waste-activated sludge, and high-strength non-hazardous organic
materials. Digester feeding is semi-continuous and draw-off is
intermittent, both occurring at any time.

Synox Corp.
(Jacksonville, FL)
OxyOzonation

National PSRP
Equivalency
August 1989

Batch process where sludge is acidified to pH 3.0 by sulfuric acid;
exposed to 1 lb. Ozone/1000 gallons of treated sludge under 60 psig
pressure for 60 minutes; 100 mg/L of sodium nitrite and held for > 2
hours; and stored at < pH 3.5. Limitations imposed were for total solids
to be < 4%; temperature must be > 20°C; and total solids must be < 6.2%
before nitrite addition.

N-Viro Energy
Systems, Ltd.
(Toledo, OH)
Alkaline Addition to
achieve Lime
Stabilization

National PSRP
Equivalency
April 1987

Use of cement kiln dust and lime kiln dust (instead of lime) to treat
sludge by raising the pH. Sufficient lime or kiln dust is added to sludge to
produce a pH of 12 for at least 12 hours of contact.

Comprehensive
Materials
Management, Inc.
(Houston, TX)
Cement Kiln Dust to
achieve Lime
Stabilization

National PSRP
Equivalency
March 1987

Use of kiln dust (instead of lime) to treat sludge pH to at least 12 after 2
hours of contact.Dewatered sludge is mixed with cement kiln dust in an
enclosed system then hauled off for land application.

Ned K. Burleson and
Associates, Inc.

(Fort Worth, TX)
Mid-Range

Temperature Aerobic
Digestion

National PSRP
Equivalency
Prior to 1989

Typical aerobic digestion for 20 days at 30°C (86°F) or 15 days at 35°C
(95°F). This is above regulation temperatures for PFRP (15 - 20°C), but
below regulation temperatures for PFRP (55 - 60°C).

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7.8 References

Berg G. and D. Berman. 1980. Destruction by anaerobic mesophilic and thermophilic digestion of viruses
and indicator bacteria indigenous to domestic sludges. Appl. Envir. Microbiol. 39 (2):361-368.

Farrell, J.B., G. Stern, and A.D. Venosa. 1990. Microbial destructions achieved by full-scale anaerobic
digestion. Paper presented at Municipal Wastewater Sludge Disinfection Workshop. Kansas City, MO.
Water Pollution Control Federation, October 1995.

USEPA. 1992. Technical support document for reduction of pathogens and vector attraction in sewage
sludge. EPA 822/R-93-004.

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8: EQUIVALENCY AND EPA'S PATHOGEN
EQUIVALENCY COMMITTEE

8.1 Introduction

One way to meet the pathogen reduction requirements of Part 503 is to treat sewage sludge in a process
"equivalent to" the processes to further reduce pathogens (PFRP) or processes to significantly reduce
pathogens (PSRP) (see Tables 4.3 and 7.1):

• Under Class A Alternative 6, sewage sludge that is treated in a process equivalent to PFRP
and meets the Class A microbiological requirement (see Section 4.3) is considered to be a
Class A biosolids 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 biosolids with respect to pathogens (see Section 5.4).

Under Part 503, equivalency pertains only to pathogen reduction. However, like all Class A and B
biosolids, sewage sludges treated by equivalent processes must also meet a separate vector attraction
reduction requirement (see Chapter 9).

8.2 Equivalency Determination

To be equivalent, a treatment process must be able to consistently reduce pathogens to levels
comparable to the reduction achieved by the listed PFRPs or PSRPs. These levels are the same levels
required of all Class A and B biosolids. The process continues to be equivalent as long as it is operated
under the same conditions (e.g., time, temperature, pH) that produced the Part 503 required reductions.
The permitting authority is responsible for determining equivalency under Part 503. The permitting
authority and facilities are encouraged to seek guidance from EPA's Pathogen Equivalency Committee
(PEC) in making equivalency determinations. The PEC makes both site-specific and national equivalency
recommendations. The recommendation is then given to the proper permitting authority, and they will
ultimately decide if the process can be deemed equivalent. The PEC's recommendations is not an
endorsement of any process by EPA.

8.3 Benefits of Equivalency

A determination of equivalency can be beneficial to a facility because it reduces the microbiological
monitoring burden in exchange for greater monitoring of process parameters. For example, a facility
meeting Class A requirements by sampling for enteric viruses and viable helminth ova in compliance with
Alternative 4 may be able to eliminate this monitoring burden if they are able to demonstrate that their
treatment process adequately reduces these pathogens on a consistent basis14. Similarly, a facility
meeting Class B Alternative 1 requirements by analyzing sewage sludge for fecal coliform may be able to
eliminate the need for testing if the process is shown to reduce pathogens to the same extent as all PSRP

14 A determination of PFRP equivalency will not reduce the monitoring required for Salmonella sp. or fecal coliform
because all Class A biosolids, even biosolids produced by equivalent processes, must be monitored for Salmonella
sp. or fecal coliform (see Section 4.3)

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processes. Equivalency is also beneficial to facilities which may have low cost, low technology systems
capable of reducing pathogen populations. Options such as long-term storage, air drying, or low
technology composting have been considered by the PEC.

Because equivalency status allows a facility to eliminate or reduce microbiological sampling, it is
imperative that the treatment processes deemed equivalent undergo rigorous review to ensure that the
Part 503 requirements are met. Obtaining a recommendation of equivalency necessitates a thorough
examination of the process and an extensive sampling and monitoring program. The time needed to
review an application is contingent on the completeness of the initial application. Sewage sludge
preparers wishing to apply for equivalency should review this chapter carefully and discuss the issue with
the regulatory authority to determine if equivalency is appropriate for their situation.

8.4 Recommendation of Site-Specific Equivalency

Equivalency may be site-specific meaning the equivalency applies only to a particular operation run at a
specified location under the conditions that are written in the equivalency determination. It cannot be
assumed that the same equivalency process will work for all types of sewage sludges or would be
effective in other facilities with different geographical characteristics. A facility that would attempt to
utilize a site-specific equivalency at a new location would need to go through the equivalency process as a
new equivalency determination. Once an equivalency is granted, any modification of the process would
require new testing to prove the modification has no effect on the pathogen destruction performance.

8.5 Recommendation of National Equivalency

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. A recommendation of national
equivalency can be useful for treatment processes that will be marketed, sold, or used at different
locations in the United States. Such a recommendation may be useful in getting PFRP or PSRP equivalency
determinations from different permitting authorities across the country. National equivalencies are
granted only after the process is demonstrated successfully in multiple locations, or with different
conditions such that it can be determined that the process is not influenced by geographic location.

8.6 Role of the Pathogen Equivalency Committee

The EPA created the PEC in 1985 to make recommendations to EPA management on applications for
PFRP and PSRP equivalency under Part 257 (Whittington and Johnson, 1985). The PEC consists of
scientists with diverse expertise who review the equivalency determinations. See Appendix I for a copy of
the memo.

8.1 Guidance and Technical Assistance on Equivalency Determinations

The PEC continues to review and make recommendations 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 503. It is not necessary to consult the PEC on sampling and monitoring programs if

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a protocol is already approved under one of the Class A alternatives. The PEC does not write
recommendations or approvals for processes that meet an existing alternative. The PEC will evaluate if it
is necessary for a process to go through the equivalency or if it already meets one of the existing
alternatives thus eliminating the need for an equivalency determination.

Equivalency determinations made by the PEC are
RECOMMENDATIONS, the permitting authority has the ultimate
decision on granting approval of a process as equivalent. The
Equivalency process is lengthy and expensive. A
recommendation for Equivalency is not considered an
endorsement by EPA.

8.8 Overview of the PEC's Equivalency Recommendation Process

The first point of contact for any equivalency determination, recommendation, or other guidance is
usually the permitting authority. This is the regional EPA office or the State in cases in which
responsibility for the Part 503 program has been delegated to the state. 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 must submit a Quality
Assurance Project Plan (QAPP) which includes information on sewage sludge characteristics, process
characteristics, climate, and other factors that may affect pathogen reduction or process efficiency as
described in Section 8.5. The committee evaluates this information considering current knowledge
concerning sewage sludge treatment and pathogen reduction and endorses the QAPP as a proper way to
evaluate the process for an equivalency determination.

If the PEC recommends that a process is equivalent to a PFRP or PSRP, the operating parameters and any
other conditions critical to adequate pathogen reduction are specified in the recommendation. The
equivalency recommendation applies only when the process is operated under the specified conditions.

If the PEC finds that it cannot recommend equivalency, 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 the applicant, if necessary, to ensure that the appropriate data are gathered in
an acceptable manner. The committee then reviews the revised application when the additional data are
submitted.

8.9 Basis for PEC Equivalency Recommendations

As mentioned in Section 8.1, to be determined equivalent, a treatment process must consistently and
reliably reduce pathogens in sewage sludge to the same levels achievable by the listed PFRPs or PSRPs.
The applicant must identify the process operating parameters (e.g., time, temperature, pH) that result in
these reductions.

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8.10 PFRP Equivalency

To be equivalent to a PFRP a treatment process must be able to consistently reduce sewage sludge
pathogens to below detectable limits. For purposes of equivalency, the PEC is concerned only with the
ability of a process to demonstrate that enteric viruses and viable helminth ova have been reduced to
below detectable limits. The PEC requires that an equivalent process determination reduce enteric
viruses by 3 log(baselO), and viable helminth ova by 2 log(base 10). In many situations these organisms
are not in a high enough density in raw sewage sludges, therefore it is necessary to spike the untreated
material with these indicator organisms to achieve the proper log reductions. The equivalency
determination does not require any demonstration of log reduction with the bacterial indicators fecal
coliforms or Salmonella because Part 503 requires ongoing monitoring of all Class A biosolids for fecal
coliform or Salmonella sp. (see Section 4.3). Thus, to demonstrate PFRP equivalency, the treatment
process must be able to consistently show that enteric viruses and viable helminth ova are below the
detectable limits shown below:

PFRP Microbial Indicator

Detection Limit

Enteric viruses

less than 1 plaque-forming unit per 4 grams
total solids sewage sludge (dry weight basis)

Viable helminth ova

less than 1 per 4 grams total solids sewage
sludge (dry weight basis)

8.11 PSRP Equivalency

A PSRP equivalency determination requires the demonstration of treatment reducing fecal coliforms by 2
log (base 10) and the density of these organisms cannot exceed 2 million CFU or MPN per dry gram of
material. This 2 log reduction is based upon data from conventional biological and chemical treatment
processes such as digestion and lime stabilization that a 2 log reduction in the indicator class fecal
coliforms is equivalent to at least a 1 log reduction in other pathogenic bacteria, viruses and protozoans
that may be present in the sludge. Since these pathogenic organisms are present in lower concentrations
than fecal coliforms it is expected that a 1 log reduction is adequate treatment when combined with the
other site restrictions imposed on these materials during land application. (Farrell et al., 1985, Farrah et
al., 1986,USEPA, 1989c).

The data submitted must be scientifically sound 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 reductions under the variety of climatic and other conditions that may be
encountered at different locations in the United States. As mentioned in the PFRP section this is achieved
by demonstration and different locations.

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8.12 Guidance on Demonstrating Equivalency for PEC Recommendations

Many of the applicants seeking equivalency do not receive a recommendation from the PEC. The most
common reason for this is incomplete applications or insufficient microbiological data. The review
process can be both lengthy and expensive, but it can be expedited and simplified if the applicant is
aware of the type of data that will be required for the review and submits a complete plan for
demonstrating equivalency in a timely fashion.

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 Part 503 (see Tables 4.3 and 7.1), 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 condition required in Part 503 for lime stabilization) but uses a
substance other than lime to raise pH could possibly qualify as a PSRP equivalent. 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 microbiological data (listed below) are needed to
demonstrate process equivalency:

• A description of the various parameters (e.g., sewage sludge characteristics, process
operating parameters, climatic factors) that influence the microbiological characteristics of
the treated sewage sludge (see Section 9.5 for more detail on relevant parameters).

• Sampling and analytical data to demonstrate that the process has reduced microbes to the
required levels (see Section 8.3 for a description of levels).

• A discussion of the ability of the treatment process to consistently operate within the
parameters necessary to achieve the appropriate reductions.

Sampling and Analytical Methods

Sewage sludge that is submitted as the final compliance sample should be sampled using accepted,
regulatory approved techniques for sampling and analyzed using the methods required by Part 503 (see
Chapter 10). The PEC will consider allowances for alternative methods for enteric virus and viable
helminth ova. These methods must be clearly described in the QAPP and endorsed by the PEC prior to
testing. The applicant must provide documentation that the modified or alternative method is
comparable with the methods prescribed in Part 503. The PEC reserves the right to determine such an
allowance. The sampling program should demonstrate the quality of the sewage sludge that will be
produced under a range of conditions. Therefore, sampling events should include a sufficient number of
samples to adequately represent product quality, and sampling events should be designed to reflect how

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the operation might be affected by changes in conditions including climatic and sewage sludge quality
variability. Sampling can also include surrogates in addition to the regulatory approved organisms such as
coliphage and aerobic endospores, as further demonstration that processes are able to remove
pathogens.

Data Quality

The quality of the data provided is an important factor in EPA's equivalency recommendation. The most
important step that ensures the proper data quality and collection is by first obtaining QAPP that is
reviewed and accepted by the PEC. The QAPP will outline the specific parameters of the process and
provide details on testing, and data collection portion of the equivalency determination. This first step
will allow the PEC to give feedback on the proposed testing and let an applicant know if the data collected
will provide the necessary detail to make an equivalency determination. The analysis should be
conducted using an independent and experienced laboratory that is familiar with biosolids sampling and
analytical methods.

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.

Equivalency at pilot scale

The PEC will only consider proof at full-scale, however, a pilot-scale operation can be used and in some
instances is encouraged for organisms that require spikes (enteric virus, and viable helminth ova). In
these situations, the PEC requires that a pilot-scale test occur simultaneously to a full-scale test
operation. In these cases, the pilot scale operation will be evaluated for all the organisms and other
physical or chemical parameters that would be evaluated in the full scale. The full-scale test may only
include a portion of the indicators in addition to all of the physical and chemical parameters. If it is
determined that the pilot-scale operates identically to the full-scale system, then the data for the spiked
organisms will be accepted from the pilot test and is not needed for full scale demonstration. This testing
approach needs to clearly be described in the QAPP, and the permitting authority and the PEC will need
to approve this type of testing prior to initiation of equivalency. The QAPP must specify where data is
obtained from a pilot-scale operation, and to discuss why and to what extent this simulates full-scale
operation.

The conditions of the pilot-scale operation should be at least as rigorous as those of a 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
parameters from the full-scale operation that might reduce the comparability of the pilot-scale test
results will invalidate any PEC equivalency recommendations and permitting authority equivalency deter-
minations and will require a retest under more similar conditions.

8.13 Guidance on Application for Equivalency Recommendations

The following outline and instructions are provided as guidance for preparing applications for equivalency
recommendations by EPA's PEC.

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Applications should include the original PEC endorsed QAPP followed by a summary fact sheet.
The fact sheet will summarize key information about the process, and other important facts that
will be considered as part of what is required for measuring or monitoring after an equivalency is
granted. The report should also include the full name of the treatment facility and the processes
used should be provided. The application should indicate whether it is for recommendation of a
PFRP or PSRP equivalency. And a site-specific or national equivalency. It is then necessary to give a
proper process description which includes the type of sewage sludge used in the process 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 wastewater and sewage 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.15 The earliest point at which sewage sludge treatment can be defined is
the point at which the sewage sludge is collected from the wastewater treatment process. Sufficient
information should be provided for a mass balance calculation (e.g., actual or relative volumetric flows
and solids concentration in and out of all streams, additive 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:

Sewage Sludge Characteristics

• Total and volatile solids content of sewage sludge before and after treatment

• Proportion and type of additives (diluents) in sewage sludge

• Chemical characteristics (as they affect pathogen survival/destruction, e.g., pH)

• Type(s) of sewage sludge (unstabilized vs. stabilized, primary vs. secondary, solids content,
domestic, industrial, etc.)

• Wastewater treatment process performance data (as they affect sewage sludge type, sewage
sludge age, etc.)

• Quantity of treated sewage sludge

• Sewage sludge age

• Sewage sludge detention time

15 When 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.

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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 sewage sludge

•	Mixing procedures

•	Duration and type of storage (e.g., aerated vs. nonaerated)

Climate

•	Ambient seasonal temperature range

•	Precipitation

•	Humidity

The application should include a description of how the process parameters are monitored including
information on monitoring equipment. Process uniformity and reliability should also be addressed. Actual
monitoring data should be provided whenever appropriate.

The report will include the treated sludge characteristics including the type of treated sewage sludge as
well as the sewage sludge monitoring program for pathogens. This can be completed by answering the
following questions in the report:

•	How and when are samples taken?

•	Parameters needed for samples analyzed?

•	What protocols are used for analysis?

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• What are the results?

• How long has this program been in operation?

The report should give a detailed description of the sample techniques that were used during the testing.
The PEC will evaluate the representativeness of the samples and the adequacy of the sampling
techniques. For a recommendation of national PFRP equivalency, samples of untreated and treated
sewage sludge are usually needed (see Sections 8.3, 4.6, and 11.4). The sampling points should
correspond to the beginning and end of the treatment process as defined previously under the section
Process Description. Chapters 10 and 11 provide guidance on sampling. Samples should be representative
of the sewage sludge in terms of location of collection within the sewage sludge pile or batch. The
samples taken should include samples from treatment under the least favorable operating 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.

• Number of composite samples compiled.

• Total solids of each sample.

• Air temperature at time of sampling.

• Temperature of sample at time of sampling.

• Sample handling, preservation, packaging, and transportation procedures.

• The amount of time that elapsed between sampling and analysis.

The report will also include analytical methods and results section, this is necessary to determine if the
proper analytical techniques were used to evaluate the equivalency. This is done by identifying the
analytical techniques used and the laboratory(s) performing the analysis. 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.

The report should include a quality assurance section that describes how the quality of the analytical data
has been ensured. Subjects appropriate to address are how the samples are representative; the quality
assurance program; the qualifications of the in-house or contract laboratory used; and the rationale for
selecting the sampling technique.

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Finally, the application should describe why, in the applicant's opinion, the process qualifies for PSRP or
PFRP equivalency. 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 consistently.
Complete references should be provided for any data cited. Applications for a recommendation of
national equivalency should discuss why the process effectiveness is expected to be independent of the
location of operation. Lastly a copy of the complete laboratory report(s) for any sampling and analytical
data should be attached as an appendix. Any important supporting literature references should also be
included as appendices.

8.14 Pathogen Equivalency Committee Recommendations

The EPA biosolids webpage (https://www.epa.gov/biosolids/examples-equivalent-processes-pfrp-and-psrp)
list processes that the PEC has recommended for use nationally as equivalent to PSRP or PFRP
respectively. Tables 5.1 and 7.2 list the approved PSRP, and PFRP equivalencies. As such individuals
having an interest in any of the processes are encouraged to contact either the PEC or the applicant for
greater detail on how the process must be operated to be PFRP or PSRP respectively.

8.15 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. NTIS Publication No. PB86-183084/A5. National Technical Information Service. Springfield. Virginia.

Farrell, J.B., G. Stern, and A.D. Venosa. 1985. Microbial destructions achieved by full-scale anaerobic
digestion. Workshop on control f Sludge pathogens. Series IV. Water Pollution Control Federation.
Alexandria, Virginia.

Smith, James E. Jr. and J.B. Farrell. 1996. Current and future disinfection - Federal perspectives. Presented
at Water Environment Federal 69th Annual Conference & Exposition.

Whittington, W.A., and E. Johnson. 1985. Application of 40 CFR Part 257 regulations to pathogen
reduction preceding land application of sewage sludge or septic tank pumpings. Memorandum to EPA
Water Division Directors. USEPA Office of Municipal Pollution Control, November 6.

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9: REQUIREMENTS FOR REDUCING VECTOR
ATTRACTION

9.1 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 organisms 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.

Suitable methods for measuring vector attraction directly are not available currently. Vector attraction
reduction is accomplished by employing one of the following:

• Biological processes that breakdown volatile solids, reducing the available food nutrients for
microbial activities and odor producing potential

• Chemical or physical conditions that stop microbial activity

• Physical barriers between vectors and volatile solids in the sewage sludge

40 CFR Part 503 does not provide an option for vector attraction equivalency, therefore there is no
equivalency committee comparable to the PEC. As a result, the specific options listed in Part 503 are the
only available means for demonstrating vector attraction reduction. In addition to pathogen reduction
alternatives, one of these options for VAR must be met in order to fully comply with 40 CFR Part 503 for
land application.

The term stability is often used to describe sewage sludge. Although it is associated with vector
attraction reduction, stability is not regulated by Part 503. Regarding sewage sludge, stability is generally
defined as the point at which food for rapid microbial activity is no longer available. Sewage sludge which
is stable will generally meet vector attraction reduction (VAR) requirements. The converse is not
necessarily true; meeting VAR requirements does not ensure sewage sludge stability. Because stability is
related to odor generation and the continued degradation of sewage sludge, it is often considered an
important parameter when producing biosolids for sale or distribution. Table 9.1 lists some of the
common methods for measuring stability.

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Table 9.1 Stability Assessment

Process

Monitoring Methods

Composting

C02 respiration, 02 uptake

Heat Drying

Moisture content

Alkaline Stabilization

pH; pH change with storage; moisture; ammonia evolution;
temperature

Aerobic Digestion

SOUR; volatile solids reduction, additional volatile solids reduction

Anaerobic Digestion

Gas production; volatile solids reduction, additional volatile solids
reduction

Part 503 contains 12 options for demonstrating a reduction in vector attraction of sewage sludge. These
requirements are designed to either reduce the attractiveness of sewage sludge to vectors (Options 1
through 8 and Option 12) or prevent the vectors from coming into contact with the sewage sludge
(Options 9 through 11). VAR options are summarized in Table 9.2. Guidance on when and where to
sample sewage sludge to meet these requirements is provided in Chapter 10.

Table 9.2 Vector Attraction Reduction Options

Requirement

What is Required

Most Appropriate For:

Option 1
503.33(b)(l)

At least 38% reduction in volatile
solids during sewage sludge
treatment

Sewage sludge processed by:
Anaerobic biological treatment
Aerobic biological treatment

Option 2
503.33(b)(2)

Less than 17% additional volatile
solids loss during bench-scale
anaerobic batch digestion of the
sewage sludge for 40 additional
days at 30°C to 37°C (86°F to
99°F)

Only for anaerobically digested
sewage sludge that cannot meet
the requirements of Option 1

Option 3
503.33(b)(3)

Less than 15% additional volatile
solids reduction during
bench-scale aerobic batch
digestion for 30 additional days
at 20°C (68°F)

Only for aerobically digested
liquid sewage sludge with 2% or
less solids that cannot meet the
requirements of Option 1 - e.g.,
sewage sludges treated in
extended aeration plants.
Sludges with > 2% solids must be
diluted.

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Requirement

What is Required

Most Appropriate For:

Option 4
503.33(b)(4)

SOUR at 20°C (68°F) is 1.5 mg
oxygen/hr/g total sewage sludge
solids

Liquid sewage sludges from
aerobic processes run at
temperatures between 10 to 30°
C. (should not be used for
composted sewage sludges).

Option 5
503.33(b)(5)

Aerobic treatment 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)

Composted sewage sludge
(Options 3 and 4 are likely to be
easier to meet for sewage
sludges from other aerobic
processes)

Option 6
503.33(b)(6)

Addition of sufficient alkali to
raise the pH to at least 12 at 25°C
(77°F) and maintain a pH >12 for
2 hours and a pH >11.5 for 22
more hours

Alkali-treated sewage sludge
(alkaline materials include lime,
fly ash, kiln dust, and wood ash)

Option 7
503.33(b)(7)

Percent solids >75% prior to
mixing with other materials

Sewage sludges treated by an
aerobic or anaerobic process
(i.e., sewage sludges that do not
contain unstabilized solids
generated in primary wastewater
treatment)

Option 8
503.33(b)(8)

Percent solids >90% prior to
mixing with other materials

Sewage sludges that contain
unstabilized solids generated in
primary wastewater treatment
(e.g., heat-dried sewage sludges)

Option 9
503.33(b)(9)

Sewage sludge is injected into
soil so that no significant 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.

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

Option 10
503.33(b)(10)

Sewage sludge is incorporated
into the soil within 6 hours 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 the pathogen reduction
process.

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

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Option 11
503.33(b)(ll)

Sewage sludge placed on a Sewage sludge or domestic

surface disposal site must be septage placed on a surface
covered with soil or other disposal site

material at the end of each
operating day.

Option 12
503.33(b)(12)

pH of domestic septage must be Domestic septage applied to

raised to >12 at 25°C (77°F) by agricultural land, a forest, or a

alkali addition and maintained at reclamation site or placed on a

>12 for 30 minutes without surface disposal site
adding more alkali.

9.2 Monitoring for Vector Attraction Reduction

Not all the vector attraction reduction options listed in this chapter require lab testing. Specifically,
Options 5,9,10, and 11 are technology descriptions. These technologies must be maintained throughout
the year in the manner described in the regulation.

The remaining vector attraction reduction options are based on laboratory testing for volatile solids
reduction, moisture content or oxygen uptake reduction. Some of the options can only be used with
certain sludge processes. For example, the oxygen uptake rate test is only appropriate for a sludge from
any aerobic digestion or wastewater treatment process. Other options such as the 38 percent reduction
in volatile solids, can be applied to a variety of biological sludge treatment processes. In any case, the
technology aspect of the option or the process by which vector attraction reduction is being attained
must be documented. Monitoring for vector attraction reduction should be performed at a minimum
according to the required monitoring schedule.

Some tests for vector attraction reduction 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 in the material tested and the performance of the
vector attraction reduction process as affected by the changes in feedstock. It is suggested in Section 9.14
that facilities maintain a sampling program that involves sampling at evenly spaced time intervals
throughout an established monitoring period. The on-going samples can be used to calculate running
averages of volatile solids reduction which are more representative than single samples or an attempt to
correlate feed sludge and sludge product. As is the case for the microbiological tests, these vector
attraction reduction tests should be conducted over approximately 2 weeks to minimize the expected
effect of these variations. The 2-week period can be the same 2-week period during which the
microbiological parameters for pathogen reduction are being determined.

The longer VAR tests present a similar problem as monitoring for microbiological quality. Tests such as
the additional 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 variability in the performance of the treatment process.
Storing samples taken during this period until the equipment becomes available is not an option, because

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samples cannot be stored for more than a limited time period (see Section 9.6). In such circumstances,
the preparer may wish to run the vector attraction reduction tests more frequently than required to
demonstrate ongoing compliance with the requirements. More frequent testing will indicate if the facility
is performing consistently and will reduce the need for multiple samples during the sampling period.

The preparer may wish to conduct composite sampling which combines samples taken within a 24-hour
period to better represent sludge quality (See Section 10.6). Since some of the bench scale tests may be
affected by long-term storage of samples, compositing should be limited to a 24-hour period. If
composting is done, the composite should be held at < 5°C during compositing and the assay must begin
immediately upon completion of the composite.

Preparers should discuss specific facility parameters with the permitting authority to design a sampling
program that is appropriate. It should be notes that Options 1-5 can be met prior to storage and need
not be retested after storage.

9.3 Option 1: Reduction in Volatile Solids Content [503.33(b)(1)]

This option is intended for use with biological treatment systems only. Historically, volatile solids
reduction has been used as a measure of proper digestion, which is why it should only be used with
biological process and not chemical addition or other physical processes. Under Option 1, reduction of
vector attraction is achieved if the mass of volatile solids in the sewage sludge is reduced by at least 38%.
This is the percentage of volatile solids reduction that can easily be attained by 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 percent volatile solids reduction can include any additional volatile solids
reduction that occurs before the biosolids leave the treatment works, such as might occur when the
sewage sludge is processed on drying beds or in lagoons.

The starting point for measuring volatile solids in sewage sludge is at the point at which sewage sludge
enters a sewage sludge treatment process. This can be problematic for facilities in which wastewater is
treated in systems like oxidation ditches or by extended aeration. Sewage sludges generated in these
processes are already substantially reduced in volatile solids content by their long exposure to oxidizing
conditions in the process. If sewage sludge removed from these processes is further treated by anaerobic
or aerobic digestion to meet VAR requirements, it is unlikely that the 38% reduction required to meet
Option 1 can be met. In these cases, use of Options 2,3, or 4 is more appropriate.

The end point where volatile solids are measured to calculate volatile solids losses can be at any point in
the process. Volatile solids continue to degrade throughout sewage sludge treatment; however it is
recommended that samples be taken at the end of treatment. Information on methods of calculation is
provided in Appendix C.

9.4 Option 2: Additional Digestion of Anaerobically Digested Sewage Sludge
[503.33(b)(2)]

Under this option, 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 in the laboratory in a bench-scale unit at 30°C to 37°C (86°F to 99°F) for an additional 40

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days. Procedures for this test are presented in Appendix D. As noted in Appendix D, the material balance
method for calculating additional volatile solids reduction will likely show greater reductions than the Van
Kleeck method.

Frequently, return activated 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 anaerobic digestion for a period of time, they are adequately reduced in vector attraction, but
because they entered the digester with volatile solids already partially reduced, 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. It is not
necessary to demonstrate that Option 1 cannot be met before using Option 2 or 3 to comply with VAR
requirements.

This additional anaerobic digestion test may have utility beyond use for sewage sludge from the classical
anaerobic digestion process. The regulation states that the test can be used for a previously
anaerobically digested sewage sludge. One possible application is for sewage sludge that is to be
removed from a wastewater lagoon. Such sewage sludge may have been stored in such a lagoon for
many years, during which time it has undergone anaerobic digestion and lost most of its volatile solids. It
is only recognized by the regulations as a sewage sludge when it is removed from the lagoon. If it were to
be further processed by anaerobic digestion, the likelihood of achieving 38% volatile solids reduction is
very low. The additional anaerobic digestion test which requires a long period of batch digestion at
temperatures between 30 and 37°C is an appropriate test to determine whether such a sewage sludge
has the potential to attract vectors.

9.5 Option 3: Additional Digestion of Aerobically Digested Sewage Sludge
[503.33(b)(3)]

Under this option, aerobically digested sewage sludge with 2% or less solids is considered to have
achieved satisfactory vector attraction reduction if it loses less than 15% additional volatile solids when it
is aerobically batch-digested in the laboratory in a bench-scale unit at 20°C (68°F) for an additional 30
days. Procedures for this test and the method for calculating additional volatile solids destruction 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). Liquid sludges with
greater than 2% solids can be diluted to 2% solids with unchlorinated effluent, and the test can then be
run on the diluted sludge. This option should not be used for non-liquid sewage sludge such as
dewatered cake or compost.

This option is appropriate for aerobically digested sewage sludges that cannot meet the 38% volatile
solids reduction required by Option 1. These include sewage sludges from extended aeration and
oxidation ditch processes, where the nominal residence time of sewage sludge leaving the wastewater
treatment 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.

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As was suggested for the additional anaerobic digestion test, the additional aerobic digestion test may
have application to sewage sludges that have been aerobically treated by other means than classical
aerobic digestion.

9.6 Option 4: Specific Oxygen Uptake Rate (SOUR) for Aerobically Digested Sewage
Sludge [503.33(b)(4)

For an aerobically digested sewage sludge with a total solids content equal to or less than 2% which has
been processed at a temperature between 10 - 30° C, reduction in vector attraction can also be
demonstrated using the SOUR test. The SOUR of the sewage sludge to be used or disposed must be less
than or equal to 1.5 mg of oxygen per hour per gram of total sewage sludge solids (dry weight basis) at
20°C (68°F). 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 volatile matter in the sewage sludge that
is being oxidized. The SOUR definition in Part 503 is based on the total solids primarily to reduce the
number of different determinations needed and for consistency with application rates, which are
measured in total solids per unit area. Generally, the range in the ratio of volatile solids to total solids in
aerobically digested sewage sludges is not large. The 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.

This test is based on the fact that if the aerobically treated 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 it.
Frequently, aerobically digested sewage sludges are circulated through the aerobic biological wastewater
treatment process 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. The SOUR test should not be used on sewage sludge
products such as heat or air-dried sludge or compost. Because of the reduction of microbial populations
that occur in these processes, the SOUR results are not accurate and should not be used. SOUR testing
on sewage sludges with a total solids content below 0.5% may give inaccurately high results. According to
the publication by Farrell, et al. (1996) several investigators indicate such an effect. Farrell, et al. (1996)
also note that storage for up to two hours did not cause a significant change in the SOUR measurement.
It is therefore suggested that a dilute sewage sludge could be thickened to a solids content less than 2%
solids and then tested, provided that the thickening period does not exceed two hours.

The SOUR test requires a poorly defined temperature correction at temperatures differing substantially
from 20°C (68°F). SOUR cannot be applied to sewage sludges digested outside the 10-30° C range (50-
86°F). The actual temperature of the sewage sludge tested cannot be adjusted because temperature
changes can cause short-term instability in the oxygen uptake rate, and this would invalidate the results
of the test (Benedict, et al. 1973; Farrell, et al. 1996). Guidance on performing the SOUR test and on
sewage sludge-dependent factors are provided in Appendix D.

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It should be noted that the limit on the use of the SOUR test for sewage sludges at different solids and
temperature levels is due to the lack of research and data on different sewage sludges

9.1 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, but not exclusively, to composted sewage sludge. These processed sewage sludges generally
contain substantial amounts of partially decomposed organic bulking agents, in addition to sewage
sludge. This option must be used for composted sewage sludge; other options are either not appropriate
or have not adequately been investigated for use with compost.

Part 503 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 as long as temperature
requirements can be met and the sewage sludge is maintained in an aerobic state for the treatment
period, but other methods such as Options 3 and 4 are likely to be easier to meet for this type of sewage
sludge.

If composting is used to comply with Class A pathogen requirements, the VAR time-temperature regime
must be met along with or after compliance with the pathogen reduction time-temperature regime.

9.8 Option 6: Addition of Alkali [503.33(b)(6)]

Sewage sludge is considered to have undergone adequate vector attraction reduction 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 two hours

• Maintain a pH of at least 11.5 without addition of more alkali for an additional 22 hours

The pH should be measured in a slurry ideally at 25°C. For more information on making a slurry, see
Section 10.7. It is acceptable to measure the pH by adjusting for the temperature by using the following
calculation:

0.03 pH units X (Tmeas — 25°C)

Correction Factor =

1.0°C

Where Tmeas = the measured temperature in degrees centigrade
Actual pH = Measured pH +/-the Correction factor

As noted in Section 6.6, the term "alkali" means a substance that causes an increase in pH. Raising
sewage sludge pH through alkali addition reduces vector attraction by reducing or stopping biological
activity. Flowever, this reduction in biological activity is not permanent. If the pH drops, surviving bacteria
become biologically active and the sewage sludge will again putrefy and potentially attract vectors. The
more soluble the alkali, the less likely it is that there will be an excess present when a pH of 12 is reached.
Consequently, the subsequent drop in pH with time will be more rapid than if a less soluble alkali is used.

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The conditions required under this option are designed to ensure 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. The requirement of raising the pH to 12
increases the probability that the material will be used before pH drops to a level at which putrefaction
can occur.

Raising the pH to 12 and maintaining this pH for two hours and a pH of 11.5 for an additional 22 hours
ensures that the pH will stay at adequately high levels to prevent putrefaction before disposal in all but
unusual cases. In any event, it is prudent to apply the treated sludge in a timely manner in a thin layer or
incorporate it into the soil for the prevention of odors and vector attraction.

More information on alkali addition and measurement of pH are included in Chapter 10.

9.9 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 reduction
must be achieved by removing water, not by adding inert materials.

It is important that the sewage sludge not contain unstabilized solids because the partially degraded food
scraps likely to be present in such a sewage sludge could attract birds, some mammals, and possibly
insects, even if the solids content of the sewage sludge exceeds 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 sewage sludge
could equilibrate to a lower solids content and attract vectors. Proper management should be conducted
to prevent this from happening.

9.10 Option 8: Moisture Reduction of Sewage Sludge Containing Unstabilized Solids
[503.33(b)(8)]

Vector attraction of any sewage sludge is 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
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.

Because sewage sludge meeting vector attraction reduction with this option may contain unstabilized
solids, material that absorbs moisture or is rewet may putrefy and attract vectors. Proper storage and
use of this material should be considered to prevent potential pathogen growth and vector attraction.

9.11 Option 9: Injection [503.33(b)(9)]

Vector attraction reduction can be achieved by injecting 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

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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 sewage sludge, which reduces the mobility and odor of
the sewage sludge. Odor is usually present at the site during the injection process, but it quickly dissipates
when injection is complete.

The special restriction requiring injection within 8 hours for Class A sewage sludge is needed because
these sewage sludges are likely devoid of actively growing bacteria and are thus an ideal medium for
growth of pathogenic bacteria (see Section 4.3). If pathogenic bacteria are present (survivors or
introduced by contamination), their numbers increase slowly for the first 8 hours after treatment, but
after this period their numbers can rapidly increase. This kind of explosive growth is not likely to happen
with Class B sludge because high densities of non-pathogenic bacteria are present which suppresses the
growth of pathogenic bacteria. In addition, the use of Class B biosolids requires site restrictions which
reduce the risk of public exposure to pathogens. Consequently, this special requirement is not needed
for Class B biosolids.

9.12 Option 10: Incorporation of Sewage Sludge into the Soil [503.33(b)(10)]

Under this option, sewage sludge applied to the land surface or placed on a surface disposal site must be
incorporated into the soil within six hours after application to or placement on the land. If the sewage
sludge is Class A with respect to pathogens, 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/100th or less of
the mass of soil in the plow layer (approximately the top six inches of soil). 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 that have not already met the vector attraction reduction requirements of the regulation by one
of the other options.

The six 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
soaks into the first inch or two of topsoil.

9.13 Option 11: Covering Sewage Sludge [503.33(b)(11)]

Under this option, sewage sludge placed on a surface disposal 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.

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9.14 Option 12: Raising the pH of Domestic Septage [503.33(b)(12)]

This option applies only to domestic septage applied to agricultural land, forest, a reclamation site, or
surface disposal site. Vector attraction 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 pathogen reduction for domestic septage (see Section 6.6.). When this option is used, every
container (truckload) must be monitored to demonstrate that it meets the requirement. As noted in
Section 6.6, "alkali" refers to a substance that causes an increase in pH.

This vector attraction reduction requirement is slightly less stringent than the alkali addition requirement
for sewage sludge. The method is geared toward the practicalities of the use or disposal of domestic
septage which is typically treated by lime addition in the domestic septage hauling truck and land applied
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.

If domestic septage is not applied soon after pH adjustment, it is recommended that pH be retested and
additional alkali be added to the domestic septage to raise the pH to 12 if necessary. Alternatively, if pH
has dropped and the domestic septage begins to putrefy, it is advisable to cover or incorporate the
domestic septage to prevent vector attraction

9.15 Number of Samples and Timing

Unlike pathogenic bacteria, volatile solids cannot regenerate once they are destroyed, so samples can be
taken at any point along the process. However, since volatile solids are destroyed throughout treatment,
it is recommended that samples be taken at the end of processing.

Facilities that use Option 2 or 3 to demonstrate vector attraction reduction must schedule sampling to
leave ample time to complete the laboratory tests before sewage sludge is used or disposed. A suggested
procedure would be to take several samples at evenly spaced time intervals during the period between
the required monitoring dates and calculate running averages comprised of at least four volatile solids
results. This has the advantage of not basing the judgement that the process is performing adequately
(or inadequately) on one or two measurements that could be erroneous because of experimental error or
a poorly chosen sample inadvertently taken during a brief process upset. It also provides an important
quality control measure for process operations. Since Part 503 does not specify a sampling program, it is
recommended that sewage sludge preparers consult with the regulatory authority regarding sampling
schedules.

9.16 References

Benedict, A.H., and D.A. Carlson. 1973. Temperature acclimation in aerobic bio-oxidation systems. Jour.
WPCF: 45(1), 10-24. January.

Farrell, Joseph B., Vinayak Bhide, James E. Smith, Jr. 1996. Development of EPAs new methods to
quantify vector attraction of wastewater sludges. Water Environment Research, Volume 68, Number 3.

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Fisher, W.J. 1984. Calculation of volatile solids destruction during sludge digestion. Pp 514-528 in Bruce,
A., ed Sewage sludge stabilization and disinfection. Published for Water Research Center. Chichester,
England: E. Harwood, Ltd.

Smith, J. E.,Jr.,and J. B. Farrell, Vector attraction reduction issues associated with the Part 503 Regulations
and Supplemental Guidance, Proceedings of the Water Environment Federations Conference,
International Management of Water and Wastewater Solids for the 21st Century: A Global perspective,
June 19-22, 1994, Washington, DC, pp 1311-1330.

Switzenbaurm, Michael S., L.H. Moss, E. Epstein, A. B. Pincince, J.F. Donovan. 1997. Defining biosolids
stability: a basis for public and regulatory acceptance. Water Environment Research Foundation.

WEF/ASCE. 1998. WEF Manual of Practice No. 8, Design of Municipal Wastewater Treatment Plants. Pub.
WEF (Alexandria, VA) and ASCE (New York, NY).

WERF. 1997. Defining biosolids stability: A basis for public and regulatory acceptance. Pub. WERF.
Alexandria, VA.

USEPA 1992. Technical support document for reduction of pathogens and vector attraction in sewage
sludge. EPA 822/R-93-004. EPA, Washington, D.C.

86

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10: SAMPLING PROCEDURES AND ANALYTICAL
METHODS

10.1 Introduction

Part 503 Subpart D pathogen and vector attraction reduction requirements call for monitoring and
analysis of the sewage sludge to ensure that microbiological quality and vector attraction reduction meet
specified requirements. The purpose of this chapter is to describe procedures for obtaining
representative samples and ensuring their quality and integrity. It also summarizes the analytical methods
required under Part 503 and directs the reader to other sections of this document that describe some of
those methods.

Sampling personnel will benefit also from reading expanded presentations on the subject. The WERF
project entitled "An Investigation into Biosolids Sampling and Handling Methods for EPA-Approved
Microbial Detection Techniques" provides the most comprehensive data with respect to the Part 503
microbial methods and sewage sludge sampling (WERF 2008). Other useful documents for additional
information regarding sampling are "Standard Methods" (APHA, 2020), "Principles of Environmental
Sampling" (Keith, 1988), "Sludge Sampling and Analysis Guidance Document" (USEPA, 1993), and ASTM
Standard E 300-86, "Standard Practice for Sampling Industrial Chemicals" (ASTM, 1992a) and are highly
recommended. The latter publication provides an in-depth description of available sampling 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 inappropriate 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 contamination during sampling.

Although extensive sampling is time consuming and facility operators are often under pressure to reduce
costs, it is strongly recommended that multiple samples be included in a sampling plan so that the
variable quality of sludge can fully be understood. Daily, weekly and seasonal fluctuations that occur in
wastewater treatment works and sludge quality make it difficult to adequately represent sludge quality
with minimal sampling. Therefore, multiple samples should be taken for any sampling event and samples
should be taken over a minimum 2-week period in order to best represent the performance of a sludge
treatment process.

Sampling events are intended to reflect the typical performance of the treatment works. Conditions
should be as stable 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.
For some process changes, long periods of time (as much as 3 months if anaerobic digestion is part of the
process train) may be required before steady state operation is reestablished.

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10.2 Monitoring for Microbiological Quality

To meet the Part 503 pathogen reduction requirements, sewage sludges may have to be monitored to
determine densities of fecal coliforms, Salmonella sp., enteric viruses and/or viable helminth ova.
Monitoring for these microorganisms presents special problems, primarily caused by the length of time it
takes to obtain microbiological test results. This is a function of the time it takes to deliver the samples to
a laboratory, have the tests conducted and obtain the results. Microbiological analyses require a
substantially longer period than conventional physical and chemical analyses. The approximate time to
complete specific microbiological analyses is summarized as follows.

• Fecal coliform (MPN), two-four days

• Salmonella sp. (MPN) five to seven days

• Enteric viruses, 14 days

• Viable helminth ova, 28 days

Variations in the microbiological quality of the treated sludge and intrinsic variation in the analytical
methods are generally large enough that a single measurement of a microbiological parameter is inade-
quate to determine whether a process meets or fails to meet a requirement. The Pathogen Equivalency
Committee recommends that the monitoring event include at least seven samples taken over a period of
approximately 2 weeks (see Section 10.7). Based on the reliability of the treatment process and historic
test results, there may be times when a reduction in this monitoring recommendation is justified.

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 generated is unknown. As discussed in Section 4.10, classification of
sludge as Class A or B is based on the most recent test results available. Therefore, material can continue
to be distributed under its classification as Class A or B until more recent analytical results are available.
However, it is recommended that material generated during the monitoring event be retained on site until
results from the monitoring event area va liable. This will prevent misclassified sludge from being
erroneously distributed.

For example, consider a facility producing a Class A sludge that is sampled for Salmonella sp. analysis
every quarter. All historic data have shown the facility to be in compliance with Class A standards
including the most recent set of lab analyses from the January monitoring event. Under these results,
materials are distributed as Class A products even throughout April when a subsequent monitoring event
takes place. This is acceptable because material is still classified under the most recent available lab
result. However, suppose the April results show non-compliance with Class A standards. Despite the fact
that the preparer complied with regulations, it is possible that some sub-standard material was
inadvertently distributed for Class A use.

In order to avoid this situation, it is recommended that the sludge processed during the monitoring event
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). This may take up to 3 weeks in the case

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of fecal coliform or Salmonella sp. analysis and much longer if sludge is being analyzed for helminth ova
or viruses. Contingencies for this type of situation should be discussed with the regulatory authority and
included in permit conditions and operational plans. For more discussion on the timing of sampling and
distribution, see Section 4.10.

10.3 Comparison of Feed Sludge and Sludge Product Samples

In some instances, it is necessary to compare the quality of the feed sludge to the final treated product to
determine vector attraction reduction methods, as well as microbial reduction that occurs during the
treatment process. There are many factors that go into ensuring that comparison of feed sludge to
treated sludges are accurate (e.g., feed rate, flow rate, solids retention time (SRT), mean cell residence
time (MCRT), etc.) All of these factors need to be considered when planning how to properly sample
across the treatment process.

Samples taken after the process has reached steady state operation are considered as corresponding.
Obtaining samples that correspond can be difficult for sewage sludge treatment processes that treat
sludge in fully mixed reactors with long residence times (e.g., anaerobic digestion). For example, as
mentioned in Section 11.3, it can take up to 3 months for an anaerobic digester to achieve steady state
operation after some substantive change in feed sludge or process condition is made.

Many of the treatment processes that might be considered for demonstrating equivalency to PFRP are
either batch or plug flow processes. In theory it is relatively simple to obtain corresponding samples - it is
only necessary 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 seldom
easy. Consider for example, the difficulty of obtaining good correspondence of feed and treated sludge
for a composting operation in which the feed sewage sludge is to be compared to composted sludge that
has been stored for 3 months.

Taking multiple samples and appropriately compositing the samples of feed and treated sludge averages
out the composition of these sewage sludges and reduces the correspondence problem. It is the
regulatory authority's task to determine how many samples should be taken and how much data is
necessary to demonstrate reduction of microorganisms in corresponding samples. As indicated in Section
11.6, limitations on the periods of time over which microbiological samples can be collected limit the
utility of compositing.

10.4 The Effect of Sludge Processing Additives on Monitoring

Many sewage sludge dewatering and stabilization processes introduce other substances into the sludge.
With the exception of large bulky additives such as wood chips, there is no need to modify sampling and
analytical procedures. As discussed below, additives such as wood chips can complicate sample
preparation and analysis and are best removed prior to analysis.

Lime, ferric chloride, paper pulp, and recycled sludge ash are frequently used to aid in dewatering.
Disinfection by alkaline treatment requires the addition of lime or other alkaline materials to increase the
temperature of the sewage sludge cake to disinfecting temperature. These materials also reduce the
microbial densities by dilution and increase solids content. However, the change in microbial density

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caused by dilution may not be substantial. 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.

Polymers are probably the most common additive used during the dewatering process. Polymers that are
in a powdered form must be reconstituted with a liquid prior to use. Polymers should not be
reconstituted with "treated effluent". Effluents are not treated to the same standards that are required
by Part 503 and these materials may contain significant microbial loads. When polymers reconstituted
with effluents are combined with treated sewage sludge they can contaminate these materials, as well as
concentrate microbes through the dewatering process. This can give an elevated microbial level which
can result in the material being out of compliance with the regulatory limits.

Human health can be directly impacted through contact with treated sewage sludge. Therefore, it is
imperative that the final product integrity with respect to pathogen levels be considered when handling
the material after sludge treatment. This includes the proper timing for pathogen sampling, as required
by Part 503, to occur as close to the time of disposal, or application. This requires that the treated sludge,
regardless of the mass of other materials added, meet the microbial standards for Class A or Class B
biosolids.

For some sludges, particularly those treated by composting (these usually will be Class A biosolids), the
amount of additive can be considerable. Nevertheless, the regulation requires that the biosolids meet the
standard which means that no correction need be made for dilution. The issues of sampling and analytical
procedures for employment are different when considering wood chips or other materials which are
often added to sludge as a bulking agent for composting.

Large additives are removed 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 consists of a mix of sludge solids
and fibrous wood-chip residues from blending. Another reason for removing the wood chips prior to
microbial analysis is that any potential exposure concerns when using the compost is relative to the
sewage sludge content and not the wood chips.

When analyzing composted materials that contain a considerable amount of wood chips it is necessary to
remove these large pieces prior to analysis. This step can be performed using a sterilized sieve and the
size of the sieve needed depends on the dimensions of the wood chips, but the same sieve size should be
used for each sampling event.

10.5 Collecting Representative Samples

Sludge quality varies depending on the inputs to the wastewater system. In addition, the process is
subject to ambient conditions which vary daily as well as seasonally. The goal of a sampling program is to
adequately represent the sludge quality overall taking into consideration processing variables. This is
completed with frequency of sampling events and the number of samples taken during each sampling
event being considered carefully. This section discusses the issue of variability and how sampling
frequency and composite sampling can improve the quality of data collected. A sampling plan is
recommended for all sampling events to assure representative samples.

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Random Variability

Virtually all sewage sludge treatment processes will experience 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. Cyclic variation
can be minimized by sampling on randomly selected days and times-of-day within a given week. For Class
B fecal coliform analysis ONLY, variability is minimized by taking the geometric mean of analytical results.
In the case of Class A biosolids, all samples must meet the fecal coliform or Salmonella sp. numerical limit.

Seasonal Variability

For some sewage sludge treatment processes, performance is poorer during certain parts of the year due
to seasonal variations in temperature, sun radiation, and precipitation. For example, aerobic digestion
and some composting operations can be adversely affected by low ambient temperature. 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 at intervals of 2 or 3 months, 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. It may also be beneficial to
initially conduct sampling more frequently than the required minimum (e.g., on a quarterly basis) in order
to determine the range of sludge quality. Process criteria of PSRPs and PFRPs should be discussed by the
facility with the regulatory authority and specific requirements should be included in permit conditions.

Composite Sampling

Composite sampling, or the combination of several grab samples to better represent a large quantity of
sludge, is frequently practiced in wastewater treatment. Composites may consist of grab samples taken
over time (typically for continuous flow processes) or from random locations in a vessel or pile (typically
for batch processes). Since the purpose of composite sampling is to provide representation of a large
quantity of sludge, certain factors should be considered: the number and distribution of grab samples,
the locations from where the samples are taken, and the process of combining the grab samples.

The following is an example of a sampling procedure for compositing a continuous flow process. A small
stream of wastewater or sludge is drawn off at a rate proportional to the flow of the mainstream being
sampled and collected as a single sample. Typically, times of collection are for one shift (8 hours) or one
day (24 hours). In this case, the accumulated sample represents a volume-averaged sample over the
period of time the sample is drawn. The sample is chilled during the period it is being collected to prevent
chemical/microbiological change until it can be brought back to the laboratory for analysis.

Composite sampling from stockpiled solid material involves taking multiple grab samples from a range of
locations in the stockpile. Samples should be taken from different interior sections of the pile which may
represent material produced in different time periods. Grab samples should all be of the same size so
that the composite is an equal representation of all grab samples. The grab samples should be mixed
thoroughly, and a subsample pulled from the mixture. In the case of monitoring a lagoon when dredging
the sludge for land application after a number of years, the lagoon can be cordoned into seven areas and
a composite sample can be taken from each section or as described in Section 10.9.

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Composite sampling is useful for any type of sampling, but the protocol must be modified when microbial
analyses are intended. Samples must be taken over a shorter period of time so that microbial populations
do not undergo significant changes during the sampling event. For example, a composite time-average
sample can be obtained by combining a series of small samples taken once every 5 minutes for a period
of an hour. A composite sample for bacterial and viral testing could be taken over an hour or less under
most circumstances without compromising the results. Composite sampling over 24 hours or longer if
special precautions 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 oxidants). In addition to limiting the sampling period, sterile
equipment must be used when taking grab samples or compositing the samples for microbiological
analysis to prevent introducing pathogenic bacteria.

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., aerobic or anaerobic digestion). This subject is discussed in Appendix
D which presents procedures for three methods to demonstrate reduced vector attraction.

Requirements for Sampling Equipment and Containers

Sampling containers may be made of glass or plastic that does not contain a plasticizer (Teflon,
polypropylene, and polyethylene are acceptable). Pre-sterilized bags are especially useful for thick
sewage sludges and free-flowing solids. Stainless steel containers are acceptable, but steel or zinc coated
steel vessels are not appropriate. In addition to providing guidance on appropriate containers for specific
analyses, analytical laboratories will typically provide sample containers. Sampling containers used for
microbiological analyses must be sterile. Sampling tools that come in contact with the sample should be
constructed of stainless steel, which is easily cleaned and sterilized. Tools made of wood are difficult to
sterilize because of porosity and should not be used.

Equipment

The sampling equipment used is primarily dependent on the type of material being sampled. For
relatively high solids content biosolids, a hand trowel or scoop may be adequate, whereas, collecting
stratified samples from a lagoon requires more sophisticated and specialized equipment. Automated
sampling equipment that is commonly used for wastewater should not be used since it can cause solids
separation during sampling and it is difficult to clean, resulting in cross contamination. Sampling
equipment should be constructed of non-corrosive materials such as stainless steel, Teflon, or glass that
can be thoroughly cleaned and sterilized. Sampling equipment should be dedicated for this task and
should not be used for other applications. Equipment should be cleaned well with detergent and a nylon
scrub brush after each use and stored in a dedicated location. The types of sampling equipment and their
applications are presented in Table 10.1. The use of this equipment is discussed in greater detail in
Sections 10.6 and 10.7.

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Table 10.1: Equipment used for Collecting Sewage Sludge Samples

Equipment

Application

Composite Liquid Waste
Sampler (Coliwasa)

The Coliwasa is a device employed to sample free-flowing sewage sludges
contained in drums, shallow tanks, pits, and similar containers. It is especially
useful for sampling wastes that consist of several immiscible liquid phases. The
Coliwasa consists of a glass, plastic, or metal tube equipped with an end closure
that can be opened and closed while the tube is submerged in the material to be
sampled.

Weighted Bottle

This sampling device consists of a glass or plastic bottle, sinker, stopper, and a line
that is used to lower, raise, and open the bottle. The weighted bottle is used for
sampling free flowing sewage sludges and is particularly useful for obtaining
samples at different depths in a lagoon. A weighted bottle with line is built to the
specifications in ASTM Method D270 and E300.

Dipper

The dipper consists of a glass or plastic beaker clamped to the end of a two- or
three-piece telescoping aluminum or fiberglass pole that serves as the handle. A
dipper is used for obtaining samples of free-flowing sewage sludge that are difficult
to access. Dippers are not available commercially and must be fabricated.

Sampling Thief

A thief consists of two slotted concentric tubes, usually made of stainless steel or
brass. The outer tube has a conical pointed tip that permits the sampler to
penetrate the material being sampled. The inner tube is rotated to open and close
the sampler. A thief is used to sample high solids content materials such as
composted and heat dried biosolids for which particle diameter is less than one-
third the width of the slots. Thief samplers are available from laboratory supply
companies.

Trier

A trier consists of a tube cut in half lengthwise with a sharpened tip that allows the
sampler to cut into sticky materials such as dewatered cake and lime stabilized
biosolids. A trier is used to sample moist or sticky solids with a particle diameter
less than one-half the diameter of the trier. Triers 61 to 100 cm long and 1.27 to
2.54 cm in diameter are available from laboratory supply companies

Auger

An auger consists of sharpened spiral blades attached to a hard metal central shaft.
An auger can be used to obtain samples through a cross section of a biosolids
stockpile. Augers are available at hardware and laboratory supply stores.

Scoops and Shovels

Scoops are used to collect samples from sewage sludge or biosolids stockpiles,
shallow containers, and conveyor belts. Stainless steel or disposable plastic scoops
are available at laboratory supply houses. Due to the difficulty of sterilizing shovels
and other large sampling equipment, this type of equipment should only be used
for accessing the center of stockpiles and should not be used for collecting the
actual sample.

Sterilization

The containers and tools used for sampling must be sterilized if the biosolids product is to be analyzed for
Class A and Class B microbiological parameters. Alternatively, pre-sterilized, disposable scoops, and other
sampling devices can be purchased, alleviating the need to sterilize sampling equipment. After the
samples are collected, the sampling equipment should be cleaned well with soap and water and put away

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until the next sampling event. Equipment should be dedicated to sampling and not used for other
activities. Only equipment that touches the actual sample must be sterilized. Equipment such as shovels
or heavy equipment used to access a sludge pile interior does not need to be sterilized, but should be
clean, as long as another sterile sample collection device (such as a hand trowel) is used to access and
collect the actual sample. Sterilization is not required when collecting samples of sewage sludge to be
used in vector attraction reduction tests, but all equipment must be clean.

Either steam or a sterilizing solution such as sodium hypochlorite (household bleach) should be used for
sterilizing equipment. If bleach is used, equipment must be rinsed thoroughly to prevent residual bleach
from influencing the microbial population in the sample. Equipment should be thoroughly washed with
water, soap, and a brush prior to sterilization. If an autoclave or large pressure cooker is available,
enclose the sampling tool in a kraft paper bag and place the bag in the autoclave. A minimum of 30
minutes at a temperature of 121°C is required for sterilization. The kraft paper bag keeps the sampling
device from becoming contaminated in the field. A steam cleaner can also be used to sterilize sampling
equipment. Place the equipment in a heat resistant plastic bucket and direct steam onto the equipment
for a minimum of 10 minutes. When done, place the sterilized equipment in a kraft paper bag. Many
facilities do not have an autoclave or steam cleaning equipment and will need to purchase presterilized
collection bottles or bags. In these situations, it is acceptable to use a sterilizing solution to sterilize some
equipment (e.g., collection spoons or shovels). This protocol should not be used for sample bottles due
to the large potential for disinfectant residual to affect the microbial population. A 10% household bleach
solution (1-part bleach, 9-parts water) is readily available and works well. However, bleach is corrosive
and may also affect the microbial population of a sample and does need to be adequately removed from
the equipment prior to sample collection. Make up the 10% solution in a clean plastic bucket. Immerse
each piece of clean equipment in the solution for a minimum contact time of a minute. Rinse the
equipment in another bucket containing sterile or boiled water. Let the equipment air dry for a few
minutes or dry with sterile paper or cloth towels. After drying, place the equipment in a paper bag. Sterile
plastic bags obtained from a scientific equipment supplier can also be used for short-term sterile
equipment storage.

10.6 Laboratory Selection

A very important, but often overlooked component of pathogen and vector attraction monitoring is
selecting an appropriate analytical laboratory. The analysis of sewage sludge or biosolids for indicator
and pathogenic organisms is more complex than water analysis. Solid samples such as biosolids are
prepared differently than water samples and typically contain a much higher background microbial
population than water contains. Biosolids products such as compost can be very heterogeneous,
requiring special sample preparation procedures. It is therefore important to use a laboratory that has
developed an expertise through the routine analysis of biosolids products.

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To ensure that a laboratory has adequate experience with biosolids analyses, the following information
should be obtained and reviewed:

• For how long has the laboratory been analyzing biosolids for the specified parameters?

• Approximately how many biosolids samples does the laboratory analyze per week or month?

• For how many wastewater treatment facilities is the laboratory conducting the specified
analyses?

• A list of references.

• Does the laboratory have a separate and distinct microbiology lab?

• Does the laboratory have microbiologists on staff? Request and review their resumes.

• Who will perform the analyses?

• Is the laboratory familiar with the analytical procedures including sample preparation, holding
times, and QA/QC protocols?

A laboratory tour and reference check are also recommended. A good laboratory should be responsive,
providing requested technical information in a timely manner. It is the biosolids generator's responsibility
to provide accurate analytical results. Consequently, the selection of an appropriate laboratory is an
important component of developing a biosolids monitoring plan.

10.7 Safety Precautions

Sewage sludge that is being sampled should be presumed 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, 2017) in Sections 1060 and 1090.

Individuals performing sampling (usually employees of wastewater treatment works) should receive
training in the microbiological hazards of sewage sludge and in safety precautions to take when sampling.
Laboratory personnel should be aware that the outside of every sample container is probably
contaminated with microorganisms, some of which may be pathogens. Personal hygiene and laboratory
cleanliness are also extremely important. Several safety practices that should be standard procedures
during sample collection and analysis are:

• Gloves should be worn when handling or sampling untreated sewage sludge or treated biosolids.

• Personnel taking the samples should clean sample containers, gloves, and hands before
delivering the samples to others.

• Hands should be washed frequently and always before activities that involve hand-to-mouth
contact.

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• Photocell-activated or foot-activated hand washing stations are desirable to reduced spreading of
contamination to others.

• Employees should train themselves to avoid touching their lips or eyes.

• Mouth pipetting should be forbidden.

Employees involved in sample collection (or any other activity where they are exposed to wastewater or
sewage sludge) should review their immunization history. At a minimum, employees should be
immunized against tetanus. However, employees should consider immunization for other diseases,
particularly hepatitis A and B. Employees should also consider having a blood sample analyzed to
determine if they still have active antibodies for the various immunizations they received as children.

Personnel analyzing sewage sludge or biosolids samples should receive training in awareness and safety
concerning biohazards. Because microbiological laboratories have safety programs, this subject is not
covered in depth in this document. A facility's sampling plan should include a section on microbiological
hazards and appropriate safety practices or, alternatively, refer the reader to another document where
this information is presented.

10.8 Sampling Frequency and Number of Samples Collected

The primary objective of biosolids monitoring is to assure that all biosolids produced meet the regulatory
requirements related to land application. It is not feasible to sample and analyze every load of biosolids
leaving a facility, nor is it necessary. However, a sampling plan does need to adequately account for the
variability of the biosolids. The plan should entail collecting a sufficient number of samples at an
adequate frequency. The minimum sampling frequency and number of samples to be analyzed as listed
in 40 CFR Part 503 are listed in table 10.2.

Table 10.2 Testing frequency required by Part 503.
j Frequency of Monitoring for Land Application and Surface Disposal

i Amount of Biosolids1 (metric tons dry solids per 365-day period) = Minimum 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)

1Either the amount of bulk biosolids applied to the land, or the amount of sewage sludge received by a
person who prepares biosolids that is sold or given away in a bag or other container for application to the
land (dry weight basis), or the amount of biosolids (excluding domestic septage) placed on a surface
disposal site.

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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)

The number of samples which must be analyzed for compliance with Class A microbiological parameters
is not specified, however, it is strongly recommended that multiple samples per sampling event be
analyzed. The number of samples taken must be sufficient to adequately represent biosolids quality. It
must be noted that for Class A biosolids, analytical results are not averaged; every sample analyzed must
meet the Class A requirements.

To meet Class B Alternative 1 requirements, seven samples must be taken, and the geometric mean of
results must meet the 2.0 x 106 MPN/CFU fecal coliform per dry gram limit (see Chapter 6). It is
recommended that the samples be taken over a two-week period to adequately represent variability in
the sewage sludge.

The actual sampling and analysis protocols are typically developed by the facility and reported to the
regulatory authority which can require a more stringent sampling and analysis protocol than what is
stipulated in Part 503. In some cases, the regulatory authority may initially require a more stringent
monitoring schedule that may be relaxed once product consistency is established. There may also be
requirements that analysis be conducted by certified laboratories. The biosolids preparer should carefully
consider the treatment process, analytical variability, end-use and other factors when determining the
frequency and number of samples to be analyzed. Collecting samples more frequently or analyzing more
samples will help to ensure the product meets the regulatory criteria and that pathogen and vector
attraction reduction goals have been met.

It is also recommended that additional sampling be conducted for heterogeneous biosolids products. A
single grab sample may adequately represent the sewage sludge in a digester that is being mixed but
might not adequately represent tons of compost product stored in several stockpiles. Likewise, a facility
that conducts a single annual analysis should consider more frequent monitoring, particularly if the
analytical results from the annual analysis are near the regulatory limit. It is a facility's responsibility, and
in the facility's best interest, to develop a monitoring plan that assures product quality.

10.9 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 flow readily into a sample bottle. They
are heterogeneous, and concentration gradients develop upon standing. Generally settling is slow and is
overcome by good mixing.

Liquid sewage sludge may be flowing in pipes, undergoing processing, 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 10.7.

Filling Containers

Liquid sewage sludge samples are usually transferred into sterile wide mouth bottles or flexible plastic
containers. Sewage sludge can generate gases, and pressure may build up in the container. Consequently,

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the bottle or container is generally not filled completely allowing at least 1 inch of headspace. If the
sewage 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 microorganisms in the sewage sludge. Conversely, if
the sewage sludge is to be used for the additional anaerobic digestion test for vector attraction reduction,
it is important that it not be exposed to oxygen more than momentarily. Consequently, sample bottles for
the anaerobic digestion test must be filled to the top. Bottles should have closures that can pop off or
made of flexible plastic that 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. Preferably the transfer should be made by use of a ladle rather than by
pouring since some settling can occur in the pail, particularly with primary or mixed sewage sludges of
solids contents below about 3%.

Collecting Liquid Sewage Sludge Samples

Liquid sewage should be sampled downstream of a pump that serves to mix the sewage sludge
thoroughly. The sample is taken through a probe facing upstream in the center of the discharge pipe and
is withdrawn at the velocity of the liquid at the center line of the pipe. This approach generally is not
possible with sewage sludge that is not liquid because fibrous deposits can 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 downstream 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.

The collection of a representative sample often requires the use of time compositing procedures. 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 composited into a single sample. If the sample is being analyzed for bacteria,
viruses, or vector attraction reduction, the composite should be prepared within an hour of collecting the
first individual grab sample. Holding times are listed in 40 CFR Part 136 for the specific method that is
used for each sample. These holding times must be observed for testing to be compliant with the
regulations. A longer holding time might allow microbiological changes to occur in the first sample taken.

Sampling Sewage Sludge in Tanks

The purpose of the sampling is to determine the properties of the entire mass of the sewage sludge. This
requires that the tank be well-mixed, otherwise many subsamples must be taken throughout the tank and
composited. Large tanks, even if they are well-mixed, have the potential for developing gradients in
composition. An enclosed tank such as an anaerobic digester must be sampled through pipelines entering
the digester. A minimum of three taps on a side wall of the enclosed tank is recommended. The sample

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tap pipe should project several feet into the tank. Precautions must be taken to minimize contamination
from sample collection lines. When a sample is taken, enough material must be withdrawn to thoroughly
flush the line before the sample is collected. This helps flush any contaminants in the sample line and
assure that a representative sample is collected from the tank. The sample line should be back-flushed
with water after the sample is withdrawn to clean out residual sewage sludge and prevent microbial
growth. Sampling should be conducted when the tank is being agitated. An open tank such as an aerobic
digester can be sampled by drawing a vacuum on a vacuum-filtering flask connected to a rigid tube
lowered to the desired level in the tank. A weighted sampling bottle may also be used to sample the
liquid at the desired depth in the tank (see ASTM E30086, Par. 21, in ASTM [1992a]).

10.10 Sampling Thick Sewage Sludges

If sewage sludges are above 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 in
lagoons to 15% solids. At these concentrations they will not flow easily and require a substantial
hydrostatic head before they will flow into a sample bottle.

Sampling sewage sludge stored in lagoons may be very difficult depending on the objectives of sampling
and the nature of the sewage sludge in the lagoon. The thickened sewage sludge solids are generally
nonuniformly distributed in all three dimensions. It is desirable 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 the nonuniformity of the solids deposit into account. 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 sewage 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 sewage sludge layer may be useful. Weighted bottle samplers (ASTM, 1992a) that can be opened
at a given depth can be used to collect samples at a desired depth. Samples at three depths should be
taken and composited. Most likely the sewage sludge will be as much as twice as high in solids content at
the bottom of the sewage sludge layer as at the top. Compositing of equal volumes of samples from top,
middle and bottom 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 for microbiological and VAR
parameters because there is no practical way of separately removing layers of sewage sludge from a
lagoon. Determining whether there are gradients with length and width makes more sense because
sewage sludge could be removed selectively from part of a lagoon, leaving behind the unacceptable
material.

Sewage sludges from dewatering equipment such as belt filter presses and centrifuges can have a solids
content up to 35% and even higher following some conditioning methods. High solids content sewage
sludges are easy to sample if they are on moving conveyors, as described in Section 9.5. However, if the
sewage sludge is stored in piles, obtaining a representative sample requires more planning and a greater
overall effort. As a result of different environmental conditions between the pile surface and interior, a
gradient will develop over time in the sewage sludge storage pile. The sampling methodology used needs
to address this potential gradient between the pile surface and interior. Sampling devices such as augers
(a deeply threaded screw) are used on high solids cakes (ASTM, 1992a) to collect a cross sectional sample.

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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. Alternatively, a shovel can be used to collect subsamples for
compositing from the pile interior. The pile should be sampled in proportion to its mass; more samples
are taken where the pile is deeper.

10.11 Sampling Dry Sewage Sludge

For purposes of this discussion, "dry" sewage sludge contains as much as 60% water. This includes heat
dried and composted sewage sludge, and sewage sludge from dewatering processes such as pressure
filtration, that produce 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 sewage sludge is best sampled when it is being transferred, 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.

Collecting a representative sample from a stockpile containing a dried sewage sludge poses a greater
challenge than collecting the sample from a conveyor. The settling and classification of the material and
the different environments between the pile edge and interior must be considered. When a material
comprised of discrete particles is formed into a pile, classification occurs. If the particles are homogene-
ous in size and composition a representative sample can be easily obtained (assuming the sample is
collected within 24 hours of pile construction). However, if the particles are of a different size or
composition an unequal distribution of the particles may result during settling. For example, a
composted sewage sludge may be heterogeneous with respect to particle composition even when
oversized bulking agents have been removed. It is important that the edges and interior of such piles are
properly weighted as part of the sample collection procedure. A sampling grid that prevents bias, such as
that presented in ASTM E300-86, Item 31.4 (ASTM, 1992a), should be used.

The heterogeneous nature and presence of large particles in some composted sewage sludge can cause
problems in sampling. For example, most augers and sampling thiefs will be ineffective in getting a repre-
sentative sample from the interior of a pile containing large wood chips and fine composted sewage
sludge. There may be no substitute for digging with a shovel to get to the desired location.

Stockpile sampling is made more difficult by the constant evolution of the characteristics of stored
material. Immediately after a sewage sludge stockpile is constructed, physical, chemical, and biological
changes begin to occur within and on the surface of the stockpile. Within a period as short as 24 hours,
the characteristics of the surface and outer part of the pile can differ substantially from that of the pile
interior. The outer part of a pile tends to remain at or near ambient temperature, loses moisture through
evaporation, and volatilizes some compounds such as ammonia. In contrast, pile interiors retain heat
(achieving temperatures that can be 40°C greater than the pile surface) and lose little moisture or
chemical compounds through evaporation and volatilization. As a result, the level of microbial growth
and activity within the pile and on the pile surface will differ. The potential for growth of fecal coliform
bacteria in mesophilic regions of the pile is of particular concern. If a sewage sludge stockpile is more

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than one day old the sample should be collected from a pile cross section. This is especially important
when there is a large temperature gradient between the pile surface and interior.

10.12 Temperature, pH, and Oxygenation Control for Microbial Tests

Table 10.2 summarizes allowed microbial methods along with the maximum holding times and
temperatures for sewage sludge samples for microbial analyses. All samples should be cooled to
appropriate temperatures immediately after they are collected to minimize changes in indicator organism
and pathogen populations. 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
practical size of the sample collection container. A gallon sample bottle takes much longer to cool than a
quart bottle. Use of bottles no larger than a quart is recommended, particularly if the sewage sludge
being sampled is from a process operated at above ambient temperature. Granular solids and thick
sewage sludges take a long time to cool, so use of containers smaller than one quart is advised in these
situations. For rapid cooling, place the sample container in a slurry of water and ice. Placing the sample
container in a cooler containing 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 refrigerator. The presence or
absence of oxygen is not a serious concern for the microbiological tests if the samples are promptly
cooled.

Table 10.2. Analytical Methods Required Under Part 503

Analysis Methodology Maximum Holding

Time3/Temperature

Enteric Viruses

American Society for Testing and
Materials Method D 4994-89 (ASTM,
1992b)1(Appendix F of this document)

-18°C(0°F); up to 2
weeks

Fecal Coliform

EPA Method 1680, EPA Method 1681
(Appendix E of this document) Preferred
methods by US EPA

Standard Methods Part 9221 C E (APHA,
2006) or Part 9222 D (APHA, 1997)2

4°C (39.2°F) (do not
freeze); 8 hours with
the exception of Class A
composted, and Class B
Aerobically or
Anaerobically digested
materials may have a 24
hr maximum hold time
as listed in 40 CFR part
136.

Salmonella sp.
Bacteria

EPA 1682 (Appendix E of this document)

4°C (39.2°F) (do not
freeze); 8 hours

Viable Helminth Ova

Yanko (1987) (see Appendix G of this
document)

4°C (39.2°F) (do not
freeze); 1 month

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Analysis Methodology Maximum Holding

Time3/Temperature

Specific Oxygen
Uptake Rate (SOUR)

Standard Methods Part 2710B
(APHA,1992)

20°C (sewage sludge
must be digested in the
10-30°C range); 2 hours

Total, Fixed, and
Volatile Solids

Standard Methods Part 2540G
(APHA,1992)

Percent Volatile
Solids Reduction

Appendix C of this document

Appendix H of this document presents a detailed discussion of this method.

2Method SM-9221D, the membrane filtration procedure is also allowable for Class B biosolids. See
Appendix F of this document for recommended sample preparation procedures and a discussion of the
reporting of results.

3Time between sampling and actual analysis, including shipping time

EPA conducted a hold time study for analysis of fecal coliform and Salmonella using EPA Methods 1680,
1681, and 1682 (USEPA, 2014 and 2006). The results of that study determined that a holding time longer
than 8 hours could result in significantly lower numbers of fecal coliforms and Salmonella in the final
biosolid material, except for composted Class A, and aerobically or anaerobically digested Class B
products. The results of this study have been used to determine the hold times that are listed in 40 CFR
Part 136, and as such they supersede any other contradicting hold times that are present in any other
approved method. The sample should never be frozen.

Proper planning and coordination with the courier service and analytical laboratory are essential if
bacterial analyses are to be conducted within the proper hold time of sample collection. The laboratory
needs to be notified several days in advance so they can be prepared to initiate the analysis within several
hours of receiving the sample. If they are not notified, the laboratory may not be adequately prepared,
and another day may lapse before the samples are analyzed. Careful coordination with the laboratory
needs to ensure that the tests for fecal coliform and Salmonella start no longer than 8 hours after time of
collection, unless allowed a longer hold time to 24 as permitted in 40 CFR Part 136. The holding times for
enteric virus and viable helminth ova are much longer and as such are typically not a problem for
coordinating with the lab.

Follow-up with the laboratory is important to determine the actual sample holding time and temperature
of the sample when it was received. This information can be used to improve the overall sample
collection and transfer procedure. Feedback received from the lab regarding sample condition and
holding times may also provide an explanation for erroneous or unexpected test results.

The requirement for prompt chilling of samples is appropriate for viruses as well as bacteria. There are far
fewer laboratories capable of carrying out virus tests than can conduct bacterial analyses, so time

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between sample collection and analysis can routinely exceed the hold times. Fortunately, viruses are not
harmed by freezing. Typically, virology laboratories store samples at -70°C (-94°F) before analysis.

Samples can be frozen in a -18°C (0°F) freezer and stored for up to 2 weeks without harm. Samples should
be frozen, packed in dry ice, and shipped overnight to the analytical laboratory.

Viable helminth ova are only slightly affected by temperatures below 35°C (95°F), provided chemicals
such as lime, chlorine, or ammonia are not 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 not
well understood.

Vector Attraction Reduction Tests

For the vector attraction reduction tests that measure oxygen 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 provides 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.

Depending on whether the sewage sludge is from an aerobic process or anaerobic digestion, the
presence or lack of oxygen will determine which vector attraction reduction test is appropriate and
therefore how the sample should be handled. For aerobic sewage sludges, a lack of oxygen will interfere
with the metabolic rate of the aerobic microorganisms in the sample. Similarly, presence of oxygen will
seriously affect or even kill the anaerobic organisms that convert organic matter to gases in anaerobic
digestion. With samples taken for SOUR analysis, it has been the experience of some investigators that if
the test is not run almost immediately after collection (within about 15 minutes), erroneous results are
obtained. The additional aerobic digestion test is more "forgiving" (because it is a long-term test and
shocked bacteria can revive); up to 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.

No pH adjustment is to be made for any of the vector attraction reduction tests. 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 9.7).

pH Adjustment and Sewage Sludges

For addition of alkali to sewage sludges, the pH requirement is part of both the PSRP process description
(see Section 6.3) and the requirement of a vector attraction option (see Section 9.7). Monitoring is
required from 1 to 12 times a year (see Table 3.4 in Chapter 3), and the process must meet the
prescribed operating conditions throughout the year.

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

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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 measurement 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 second measurement is made 24
hours after addition of alkali. If the sludge is still in the liquid state, the pH measurement is made in the
same fashion. However, 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
measurement. Acceptable procedures for preparing the sample and measuring pH are given by USEPA,
1986. The procedure requires adding 20 ml_ of distilled water (containing 0.01 M CaCI2) to 10 g of sludge
cake, mixing occasionally 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 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 tankfor pH measurement is
sufficient.

If alkali is added to sludge cake, more sampling is suggested. Typically, alkali (usually lime) is added to
sludge cake in a continuous process. The sludge from the dewatering process discharges continuously to
a 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 composite samples collected for pH measurement must be reduced in size for the pH
measurement. The alkaline-treated sludge may be discharged from the mixing devices in the form of
irregular balls that can be up to 5 to 7.6 cm (2 or 3 inches) in diameter. If the discharged biosolids are ball
shaped and the alkali has not penetrated the entire ball, one or both of these goals is not met for the
material inside the ball. The entire ball 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 approach is to sample only the interior of the balls.

Vector Attraction Reduction Tests

Testing samples for vector attraction reduction is different from testing for microbes in that it is not
necessary to use sterilized equipment. There is no concern for microbial contamination with these
samples. There are, however, some important points to consider for VAR sampling. When sampling to

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measure for volatile solids reduction it is important to keep the aerobic samples aerobic and to prevent
the anaerobic samples from coming into contact with air, which is done by filling the bottle completely
with no air space once capped. Subsamples for the anaerobic tests can be collected into individual
bottles at the sampling location. As noted previously, 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 in size for a representative
composite sample, must be avoided. One acceptable sample size reduction procedure is to flush a large
sterile 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 thoroughly to accomplish the blending.
Analytical laboratories generally can perform this size reduction procedure.

10.13 Packaging and Shipment

Proper packaging and shipment are important to ensure that the samples arrive in good condition
(proper temperature, no spillage) within the specified time frame.

Sealing and Labeling Sample Containers

Sample containers should be securely taped to avoid contamination and sealed (e.g., with gummed
paper) so it is impossible to open the container without breaking the seal. Sealing ensures that sample
integrity is preserved until the sample is opened in the laboratory. A permanent label should be affixed to
each sample container. Sterile collection bottles or bags that are used and shipped out on ice also should
be placed in individual sealable bags to avoid contamination with the ice that is used during shipping.
Never place a bottle directly into ice, this will cause contamination of the sample inside the bottle. At a
minimum the following information should be provided on each sample container:

• Type of analysis to be performed (e.g., Salmonella sp., fecal coliform bacteria, enteric virus, or
viable helminth ova)

• Sample identification code (if used) or a brief description of the sample (that distinguishes it from
other samples) if no sample code system is used

• Sample number (if more than one sample was collected at the same point on the same day)

Other information may include:

• Facility name, address and telephone number

• Date and time the sample was taken

• Facility contact person

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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

•	Requested preprocessing (subsampling, compositing, particle size reduction)

•	Requested analyses

•	Sample code number for each sample (if used)

•	Signatures of the persons involved in the chain of possession

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 forms can be obtained from
the laboratory and should be used even if the laboratory is on-site and part of the treatment facility.

Shipment Container

A soundly constructed and insulated shipment box is essential to provide the proper environment for
preserving the sample at the required temperature and to ensure the sample arrives intact. Small plastic
cased coolers are ideal for sample shipping. It is recommended that the outside of the shipment
container be labeled with the following information:

•	The complete address of the receiving laboratory (including the name of the person responsible
for receiving the samples and the telephone number)

•	Appropriate shipping label that conforms to the courier's standards

•	Number of samples included (e.g., "This cooler contains 10 samples")

•	The words "Fragile" and "This End Up"

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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 "tempered" to warm it up to the melting point of ice (0°C [32°F]) before it
is placed around the sample. Additional packing material (bubble wrap, Styrofoam peanuts, balled-up
newspaper) should be placed in the shipping container to fill in empty space to prevent sample containers
from moving and potentially breaking or spilling during shipping. It is recommended also that the courier
be contacted to determine if there are any special shipping requirements for these types of samples.

Adherence to Holding and Shipment Times

Adherence to sample preservation and holding time limits described in Section 10.11 is critical. Samples
that are not processed within the specified time and under the proper conditions can yield erroneous
results and are out of regulatory compliance. Make sure the analytical laboratory reports the date and
time when the samples arrived, and total holding time (period from when the sample was collected to the
initiation of analysis). This information will be valuable for improving future sample events and
maintaining quality control.

10.14 Documentation
Sampling Plan

It is recommended that all procedures used in sample collection, preparation and shipment be described
in a sampling plan. At a minimum, a sampling plan should provide the following information:

•	Sample collection locations

•	Volume of sample to be collected

•	Sample compositing procedures

•	Days and times of collection

•	Required equipment

•	Instructions for labeling samples and ensuring chain of custody

•	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 (USEPA, 1980).

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Sampling Log

All information pertinent to a sampling event should be recorded in a bound log book, preferably with
consecutively numbered pages. At a minimum, the following information should be recorded in the log
book.

Purpose of sampling event
Date and time of sample collection
Location where samples were collected

Grab or composite sample (for composite samples, the location, number, and volume of
subsamples should be included)

Name of the person collecting the sample(s)

Type of sewage sludge

Number and volume of the sample taken

Description of sampling point

Date and time samples were shipped

10.15 Analytical Methods

40 CFR Part 503.8(b) and 40 CFR Part 136 specify methods that must be used when analyzing for enteric
viruses; fecal coliform; Salmonella sp.; viable helminth ova; specific oxygen uptake rate; and total, fixed
and volatile solids. Table 10.2 lists the required methods.

Table 10.2 Analytical methods required by 40 CFR Part 503, and 40 CFR Part 136
| Method	Appendix in this document

i Calculating volatile solids reduction	Appendix B

i Conducting additional digestion and specific oxygen	; Appendix C

i uptake rate (SOUR) tests

Determination of residence time in digesters

Sample preparation and analytical methods -- fecal
coliform and Salmonella sp.

Analytical method -- enteroviruses in sewage sludge

Analytical method -- viable helminth ova

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10.16 Quality A ssurance

Quality assurance involves establishing a sampling plan and implementing quality control measures and
procedures for ensuring that the results of analytical and test measurements are correct. A complete
presentation of this subject is beyond the scope of this manual. A concise description of quality assurance
is found in Standard Methods and is strongly recommended. 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: certification 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 Part 503 these elements cannot be met
completely, but they should be kept in mind as a goal.

Microbial Tests

For the microbiological tests, quality assurance is needed to verify precision and accuracy. Quality
assurance for microbiological methods is discussed in Part 9020 of Standard Methods. The quality control
approach suggested is recommended for the microbiological tests required by Part 503. Part 9020B-4,
Analytical Quality Control Procedures state that precision be initially established by running multiple
duplicates. Additional duplicates (5% of total samples) should be run during testing to determine
whether precision is being maintained.

Spiking and recovery tests are an important part of quality assurance. EPA methods 1680, 1681, and
1682 list the spiking tests that should be used for the bacterial indicator tests. Yanko (1987) found that
spiking is useful for the viable helminth ova test with. With any of the EPA methods listed above, the
density of the measured pathogens should be at levels that are relevant to Part 503. For example, for
viable helminth ova, samples should be spiked to density levels of approximately 100 per gram.

For both commercial and in-house laboratories, quality assurance procedures should be incorporated
into the sampling and analytical methods and assessed routinely. Communication with the analytical
personnel is an important part of developing a good sampling and analysis protocol. The sewage sludge
preparer should review quality assurance data along with analysis results to ensure that laboratory
performance is acceptable.

Vector Attraction Reduction Tests

It is not possible to test for accuracy for any of the vector attraction reduction tests because standard
sewage sludges with consistent qualities do not exist. Standard Methods give guidance on precision and
bias. However, for some of the vector attraction reduction options, this information was not available or
was approximate. Section 10.7 provides guidance on the number of samples to take. The procedures for
three of the vector attraction options developed for Part 503 (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.

109

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10.17 References

APHA. 1997. Standard methods for the examination of water and wastewater. 16th ed. Washington DC:
American Public Health Association.

APHA. 2006. Standard methods for the examination of water and wastewater 21st ed. Washington DC:
American Public Health Association.

APHA. 2017. Standard methods for the examination of water and wastewater 23rd ed. Washington DC:
American Public Health Association.

ASTM. 1992a. Annual book of ASTM standards. Philadelphia, PA: American Society for Testing and
Materials.

ASTM. 1992b. Standard practice for sampling industrial chemicals. Philadelphia, PA: American Society for
Testing and Materials.

Kent, R.T., and K.E. Payne. 1988. Sampling groundwater monitoring wells: Special quality assurance and
quality control considerations, p 231-246 in Keith, L.H. Principles of Environmental Sampling. American
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Keith, L.H., ed. 1988. Principles of environmental sampling. American Chemical Society.

USEPA. 2014. Method 1680: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation
using Lauryl Tryptose Broth (LTB) and EC Medium. EPA-821-R-14-009. Office of Water.

USEPA. 2006.Method 1681: Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple Tube Fermentation
using A-l medium. EPA-821-R-06-013. Office of Water.

USEPA. 2006. Method 1682: Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-
Vassiliadis (MSRV) Medium. EPA-821-R-06-14. Office of Water.

USEPA 2006. Assessment of the Effects of Holding Time on Fecal Coliform and Salmonella Concentrations
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USEPA. 1980. Samplers and sampling procedures for hazardous waste streams. Report No.: EPA/600/2-
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USEPA. 1993. POTW sludge sampling and analysis guidance document. EPA 833-B-89-100. Office of
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USEPA. 1993. Sewage sludge sampling techniques (video).

USEPA. 1999. Biosolids Management Handbook. U.S. EPA Region VIII, P-W-P, 999 18th Street Denver CO
80202-2466.

WERF. 2008. An Investigation into Biosolids Sampling and Handling Methods for USEPA-Approved
Microbial Detection Techniques. Pub. WERF. Alexandria, VA

110

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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: National Technical Information

111

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11: REFERENCES/ADDITIONAL RESOURCES

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Bastian, R.K. 1997. The biosolids (sludge) treatment, beneficial use, and disposal situation in the USA.
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Benedict, A.H., and D.A. Calrson. 1973. Temperature acclimation in aerobic bio-oxidation systems. J.
WPCF 45(1):10 - 24.

Berg G. and D. Berman.1980. Destruction by anaerobic mesophilic and thermophilic digestion of viruses
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Counts, C.A. and A.J. Shuckrow. 1975. Lime stabilized sludge: its stability and effect on agricultural land.
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Ltd. Essex, England:

Engineering News Record, August 13, 1987. No AIDS Threat in Sewage. Issue 47

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Farrell, J.B., G. Stern, and A.D. Venosa. 1985. Microbial destructions achieved by full-scale anaerobic
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Federation.

Farrell, J.B., B.V. Salotto, and A.D. Venosa. 1990. Reduction in bacterial densities of wastewater solids by
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Farrell, J. B., V. Bhide, and J. E. Smith Jr. 1996. Development of EPA's new methods to quantify vector
attraction of wastewater sludges. Water Environ. Res. 68 (3): 286-294.

Farzadegan, H. 1991. Proceedings of a Symposium: Survival of HIV in Environmental Waters. Baltimore,
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Feldman, K., 1995. Sampling for Airborne Contaminants. BioCycle 36(8): 84-86

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Ltd. Chichester, England.

Fox, C.J., P.R. Fitzgerald, and C. Lue-Hing. 1981. Sewage organisms: a color atlas. Metropolitan Water
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Gerba, C.P., C. Wallis, and J.L. Melmick. 1975. Fate of wastewater bacterial and viruses in soil. J. Irrig.
Drain Div. Am. Soc. Civ. Engineers. 101:157-174.

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 viruses from sludge. Applied & Environ. Microbiol. 48:531-538.

Gover, N. 1993. HIV in wastewater not a recognized threat, other pathogens can be. National Small
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Gupta, P. 1991. HIV Survivability in Wastewater. Proceedings of a Symposium: Survival of HIV in
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Haines, J., 1995. Aspergillus in compost: Straw man or fatal flaw? BioCycle, 1995 36 (4): 32-35.

Harding, H.J., R.E. Thomas, D.E. Johnson, and C.A. Sorber. 1981. Aerosols generated by liquid sludge
application to land. Rept. No. EPA-600/1-81-028. USEPA, Office of Research and Development.
Washington, DC.

Haug, R. T. 1993. The practical handbook of compost engineering. Lewis Publishers.

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Hay, J. C., 1996. Pathogen destruction and biosolids composting. BioCycle, 37 (6):67-76

Helsel, D.R. 1990. Less than obvious: statistical treatment of data below the detection limit. Environ. Sci.
Technol. 24(12): 1767-1774.

lacaboni, M.D., J.R. Livingston, and T.J. LeBrun. 1984. Windrow and static pile composting of municipal
sewage sludges. Report No.: EPA/600/2-84-122 (NTIS PB84-215748).

Johns Hopkins School of Hygiene and Public Health. 1991. HIV transmission in the environment: What are
the risks to the public's health? Public Health News.

Johnson, R.W., Blatchley, E.R. Ill, and D.R. Mason. 1994. HIV and the blood borne pathogen regulation:
Implications for the wastewater industry. Water Environ. Res. 66: 684-691.

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 K.E. Payne. 1988. Sampling groundwater monitoring wells: Special quality assurance and
quality control considerations, pp. 231-246 In Keith, L.H., (ed.) Principles of Environmental Sampling.
American Chemical Society.

Kindzierski, W.B., R.E. Roberts, and N.J. Low. 1993. Health effects associated with wastewater treatment,
disposal, and reuse. Water Environ. Res. 65: 599-606.

Kowal, N.F. 1985. Health effects of land application of municipal sludge. Pub. No.: EPA/600/1-85/015.
Research Triangle 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. Stem. 1990. Reductions of enteric microorganisms during aerobic
sludge digestion. Water. Res. 24(11):1377-1385.

Millner, P.D., S.A. Olenchock, E. Epstein, R. Rylander, J. Haines, J. Walker, B.L. Ooi, E. Home, and M.
Maritato 1994. Bioaerosols associated with composting facilities. Compost Sci. and Util. 2(4):6-57.

Moore, B.E., D.E. Camann, G.A. Turk, and C.A. Sorber. 1988. Microbial characterization of municipal
wastewater at a spray irrigation site: The Lubbock infection surveillance study. J. WPCF. 60(7): 1222-1230.

Moore, B.E. 1993. Survival of human immunodeficiency virus (HIV), HIV-infected Lymphocytes, and
Poliovirus in Water. Applied and Environ. Microbiol. 59:1437-1443.

Newman, M.C. and P.M. Dixon. 1990. UNCENSOR: A program to estimate means and standard deviations
for data sets with below detection limit observations. Am. Envir. Laboratory 2(2):26-30.

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Obeng, L. Health aspects of water supply and sanitation. In Information and Training for Low-Cost Water
Supply and Sanitation. D. Trattles.(ed.) World Bank. Washington, D.C.

Olivieri, V. P., L. Cox, M. Sarao, J. L. Sykora, and P. Gavagahn. 1989. Reduction of selected indicator and
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Residuals Management Conference, San Diego, CA.

Ponugoti, PrabhakerR., Mohamed F. Dahab, Rao Surampalli. 1997. Effects of different biosolids
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Reimers, R.S., M.D. Little, T.G. Akers, W.D. Henriques, R.C. Badeaux, D.B. McDonnell, and K.K. Mbela.
1989. Persistence of pathogens in lagoon-stored sludge. Rept. No. EPA/600/2-89/015 (NTIS No.
PB89-190359/AS). Cincinnati, OH: U.S. EPA Risk Reduction Engineering Laboratory.

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Environment Federation.

Scheuerman, P.R., S.R. Farrah, and G. Bitton. 1991. Laboratory studies of virus survival during aerobic and
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Smith Jr, J. E., and J. B. Farrell. 1994. Vector attraction reduction issues associated with the Part 503
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Smith Jr., J E. and J. B. Farrell. 1996. Current and future disinfection - Federal perspectives. Presented at
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biosolids compost. BioCycle, 36(6):70-76.

Sobsey, M.D., and P.A. Shields. 1987. Survival and transport of viruses in soils. Model studies pp. 155-
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Sorber, C.A., and B.E. Moore. 1986. Survival and transport of pathogens in sludge-amended soil, a critical
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USEPA. 1980. Samplers and sampling procedures for hazardous waste streams. Report No.:
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USEPA. 1986. Test methods for evaluating solid waste: method 9045A, soil and waste pH, Revision 1, Nov.
1990. Washington, D.C.: Office of Solid Waste and Emergency Response, U.S. EPA. U.S. Supt. of
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USEPA. 1986. Inactivation of enteric pathogens during aerobic digestion of wastewater sludge (Project
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USEPA. 1988. National sewage sludge survey database. National Computer Center. Research. Triangle
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Whittington, W.A., and E. Johnson. 1985. Application of 40 CFR Part 257 regulations to pathogen
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117

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12: APPENDIX A

The text in Appendix A has been taken from the previously published document "Control of Pathogens
and Vector Attraction in Sewage Sludge" (July 2003, EPA 625-R-92-013). Page numbers will be
inconsistent with the previous text.

A.l

-------
Subpart D of the Part 503 Regulation
[Code of Federal Regulations]

[Title 40, Volume 21, Parts 425 to 699]
[Revised as of July 1,1998]

From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR503.30]

TITLE 40 - PROTECTION OF
ENVIRONMENT

CHAPTER I - ENVIRONMENTAL
PROTECTION AGENCY (Continued)

PART 503 - STANDARDS FOR THE USE OR
DISPOSAL OF SEWAGE SLUDGE-Table of
Contents

Subpart D-Pathogens and Vector

Attraction Reduction
Sec. 503.30 Scope.

(a)	This subpart contains the requirements for a sewage
sludge to be classified either Class A or Class B with re-
spect to pathogens.

(b)	This subpart contains the site restrictions for land on
which a Class B sewage sludge is applied.

(c)	This subpart 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.

Sec. 503.31 Special definitions.

(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 decomposi-
tion 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 microor-
ganisms 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 lo-
cated in a populated area (e.q., a construction site located
in a city).

(e)	Land with a low potential for public exposure is land
that the public uses infrequently. Tnis 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 organ-
isms. These include, but are not limited to, certain bacte-
ria, protozoa, viruses, and viable helminth ova.

(g)	pH means the logarithm of the reciprocal of the hy-
drogen ion concentration.

(h)	Specific oxygen uptake rate (SOUR) is the mass of
oxygen consumed per unit time per unit mass of total sol-
ids (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.

(I) Volatile solids is the amount of the total solids in sew-
age sludge lost when the sewage sludge is combusted at
550 degrees Celsius in the presence of excess air.

A.2

-------
Sec. 503.32 Pathogens.

(a) Sewage sludge-Class A. (1) The requirement in Sec.
503.32(a)(2) and the requirements in either Sec.
503.32(a)(3), (a)(4), (a)(5), (a)(6) (a)(7) or (a)(8) shall be
met for a sewage sludge to be classified Class A with re-
spect to pathogens.

(2)	The Class A pathogen requirements in Sec. 503.32

(a)(3)	through (a)(8) shall be met either prior to meeting or
at the same time the vector attraction reduction require-
ments in Sec. 503.33, except the vector attraction reduc-
tion requirements in Sec. 503.33 (b)(6) through (b)(8), are
met.

(3)	Class A-Alternative 1. (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-
sis), or the density of Salmonella sp. bacteria in the sew-
age sludge shall be less than three Most Probable Num-
ber 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 Sec. 503.10

(b),	(c), (e), or (f).

(ii) 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 be 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 an immiscible liquid.

D =

131,700,000

Eq. (2)

10 0.1400t
Where,

D=time in days.

t=temperature 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 immis-
cible liquid, the temperature of the sewage sludge shall be
50 degrees Celsius or higher; the time period shall be 15
seconds or longer; and the temperature and time period
shall be determined using equation (2).

(C)	When the percent solids of the sewage 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 pe-
riod is 30 minutes or longer, the temperature and time pe-
riod shall be determined using equation (3).

D

50,070,000

10'

,0.1400t

Eq.3

Where,

D=time in days.

t=temperature 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-
sis), or the density of Salmonella sp. bacteria in the sew-
age sludge shall be less than three Most Probable Num-
ber 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 Sec. 503.10

(b),	(c), (e), or (f).

(ii)(A) The pH of the sewage sludge that is used or dis-
posed 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 pe-
riod 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 (di^ weight ba-
sis), or the density of Salmonella sp. bacteria in 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 sew-
age 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 Sec. 503.10 (b),

(c),	(e), or (f).

(ii)(A) The sewage sludge shall be analyzed prior to
pathogen treatment to determine whether the sewage
sludge contains enteric viruses.

(B)	When the density of enteric viruses in the sewage
sludge prior to pathogen treatment is less than one Plaque-
forming Unit per four grams of total solids (dry weight ba-
sis), the sewage sludge is Class A with respect to enteric
viruses until the next monitorinq episode for the sewaqe
sludge.

(C)	When the density of enteric viruses in the sewage
sludge prior to pathogen treatment is equal to or greater

A.3

-------
than one Plaque-forming Unit per four grams of total sol-
ids (dry weight basis), the sewage sludge is Class A with
respect to enteric viruses when the density of enteric vi-
ruses 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 treat-
ment 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 patho-
gen 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 docu-
mented 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 sew-
age sludge prior to pathogen treatment is less than one
per four grams of total solids (dry weight basis), the sew-
age 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 sew-
age 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 helm-
inth ova when the density of viable helminth ova in the
sewage sludge after pathogen treatment is less than one
per four grams of total solids (dry weight basis) and when
the values or ranges of values for the operating param-
eters for the pathogen treatment process that produces
the sewage sludge that meets the viable helminth ova den-
sity requirement are documented.

(D)	After the viable helminth ova reduction in paragraph

(a)(5)(iii)(C)	of this section is demonstrated for the patho-
gen treatment process, the sewage sludge continues to
be Class A with respect to viable helminth ova when the
values for the pathogen treatment process operating pa-
rameters are consistent with the values or ranges of val-
ues documented in paragraph (a)(5)(iii)(C) of this section.

(6) Class A - Alternative 4. (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-
sis), or the density of Salmonella sp. bacteria in the sew-
age sludge shall be less than three Most Probable Num-
ber 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 Sec. 503.10

(b),	(c), (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 con-
tainer 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 Sec. 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 applica-
tion to the land; or at the time the sewage sludge or mate-
rial derived from sewage sludge is prepared to meet the
requirements in Sec. 503.10 (b), (c), (e), or (f), unless oth-
erwise specified by the permitting authority.

(7)	Class A - Alternative 5. (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-
sis), or the density of Salmonella sp. bacteria in the sew-
age sludge shall be less than three Most Probable Num-
ber 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 Sec.
503.10 (b), (c), (e), or (f).

(ii) Sewage sludge that is used or disposed shall be
treated in one of the Processes to Further Reduce Patho-
gens 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 ba-
sis), or the density of Salmonella, sp. bacteria in the sew-
age sludge shall be less than three Most Probable Num-
ber 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 Sec.
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 Fur-
ther Reduce Pathogens, as determined by the permitting

authority.

(b) Sewage sludge-Class B. (1)(i) The requirements in
either Sec. 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 Sec. 503.32(b)(5) shall be met
when sewage sludge that meets the Class B pathogen
requirements in Sec. 503.32(b)(2), (b)(3), or (b)(4) is ap-
plied to the land.

A.4

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(2)	Class B - Alternative 1. (i) Seven samples of the sew-
age 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 be 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 deter-
mined 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 be 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 appli-
cation of sewage sludge when the sewage sludge remains
on the land surface for four months or longer prior to incor-
poration into the soil.

(iii)	Food crops with harvested parts below the surface
of the land shall not be harvested for 38 months after ap-
plication of sewage sludge when the sewage sludge re-
mains 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 be 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 Sec.
503.32 (b)(5) shall be met when domestic septage is ap-
plied 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 Sec. 503.32 (b)(5)(i) through (b)(5)(iv) shall
be met.

Sec. 503.33 Vector attraction reduction.

(a)(1)	One of the vector attraction reduction requirements
in Sec. 503.33 (b)(1) 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 Sec. 503.33 (b)(1) 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 Sec. 503.33 (b)(1) through (b)(8) shall be met when sew-
age sludge is sold or given away in a bag or other con-
tainer for application to the land.

(4)	One of the vector attraction reduction requirements
in Sec. 503.33 (b)(1) through (b)(11) shall be met when
sewage sludge (other than domestic septage) is placed
on an active sewage sludge unit.

(5)	One of the vector attraction reduction requirements
in Sec. 503.33 (b)(9), (b)(10), or(b)(12) shall be met when
domestic septage is applied to agricultural land, forest, or
a reclamation site and one of the vector attraction reduc-
tion requirements in Sec. 503.33 (b)(9) through (b)(12) shall
be met when domestic septage is placed on an active sew-
age sludge unit.

(b)(1)	The mass of volatile solids in the sewage sludge
shall be reduced by a minimum of 38 percent (see calcu-
lation procedures in "Environmental Regulations and Tech-
nology - Control of Pathogens and Vector Attraction in
Sewage Sludge," EPA/625/R-92/013,1992, U.S. Environ-
mental Protection Agency, Cincinnati, Ohio 45268).

(2)	When the 38 percent volatile solids reduction require-
ment in Sec. 503.33 (b)(1) cannot be met for an anaerobi-
cally digested sewage sludge, vector attraction reduction
can be demonstrated by digesting a portion of the previ-
ously digested sewage sludge anaerobically in the labora-
tory in a bench-scale unit for 40 additional days at a tem-
perature 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 less
than 17 percent, vector attraction reduction is achieved.

(3)	When the 38 percent volatile solids reduction require-
ment in Sec. 503.33 (b)(1) cannot be met for an aerobi-
cally digested sewage sludge, vector attraction reduction
can be demonstrated by digesting a portion of the previ-
ously digested sewage sludge that has a percent solids of
two percent or less 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

A.5

-------
by less than 15 percent, vector attraction reduction is

achieved.

(4)	The specific oxygen uptake rate (SOUR) for sewage
sludge treated in an aerobic process shall 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 de-
grees Celsius.

(5)	Sewage sludge shall be treated in an aerobic pro-
cess for 14 days or longer. During that time, the tempera-
ture of the sewage sludge shall be higher than 40 degrees
Celsius and the average temperature of the sewage sludge
shall be 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 waste-
water 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 treat-
ment process shall be equal to or greater than 90 percent
based on the moisture content and total solids prior to mix-
ing 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 sew-
age 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 sur-
face within eight hours after being discharged from the
pathogen treatment process.

(10)	(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.

(11)	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 pro-
cess.

(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.

A.6

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13: APPENDIX B

The text in Appendix B has been taken from the previously published document "Control of Pathogens
and Vector Attraction in Sewage Sludge" (July 2003, EPA 625-R-92-013). Page numbers will be
inconsistent with the previous text.

B.l

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Determination of Volatile Solids Reduction by Digestion

Introduction

Under 40 CFR 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 vola-
tile solids in the sewage sludge by 38% or more 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 com- parison is
between the sewage sludge entering the digester and the
sewage sludge leaving the digester. The remain- der of
the discussion is limited to this circumstance, ex- cept for
the final section of this 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) ob-
served 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 discus-
sion of the ways that volatile solids reduction may be cal-
culated and their limitations. 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
approach but requires more information than is currently

collected at a wastewater treatment plant. The approxi-
mate mass balance equation assumes steady state con-
ditions. The "constant ash" equation requires the assump-
tion 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 ex-
amples of considerable complexity and illustrates that dif-
ferent methods frequently yield different results.

Fischer's paper is extremely thorough and is highly rec-
ommended for someone trying to develop a deep under-
standing of potential complexities in calculating volatile
solids reduction. However, it was not written as a guid-
ance document for field staff faced with the need to calcu-
late volatile solids reduction. The nomenclature is precise
but so detailed that it makes comprehension difficult. In
addition, two important troublesome situations that com-
plicate the calculation of volatile solids reduction - grit depo-
sition in digesters and decantate removal - are not explic-
itly discussed. Consequently, this presentation has been
prepared to present guidance that describes the major pit-
falls likely to be encountered in calculating percent volatile
solids reduction.

It is important to note that the calculation of volatile sol-
ids 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 repre-
sentative, 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.

B.2

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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
constant ash equation. This equation gives identical re-
sults to the 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 meth-
ods 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 "s" in subscripts. Simi-
larly, "bottoms" is used in place of "sludge" to avoid use of
"s" in subscripts.

Method Full Mass Balance

The full mass balance method must be used when steady
conditions do not prevail over the time period chosen for
the calculation. The chosen time period must be substan-
tial, at least twice the nominal residence time in the di-
gester (nominal residence time equals average volume of
sludge in the digester divided by the average volumetric
flow rate. Note: when there is decantate withdrawal, vol-
ume of sewage sludge withdrawn should be used to cal-
culate 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 composi-
tions. If input compositions have been relatively constant
for a long period of time, then the time period can be short-
ened.

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),
discharging 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
solids + accumulation of volatile solids in the digester. (1)

Loss of volatile solids is calculated from Equation 1.
FVSR is calculated by Equation 2:

FVSR = loss in volatile solids

sum of volatile solids inputs	(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
volumes, initial and final volumes in the digester, and vola-
tile 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 volatile solids in the digester over the time period of the
test, an approximate mass balance (AMB) may be used.
The basic relationship is stated simply:

volatile solids input rate = volatile solids output rate + rate
of loss of volatile solids.	(3)

The FVSR is given by Equation 2.

No Decantate, No Grit Accumulation (Problem 1)

Calculation of FVSR is illustrated for Problem 1 in Table
B-1, which represents a simple situation with no decantate
removal and no grit accumulation. An approximate mass
balance is applied to the digester operated under constant
flow conditions. 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	(4)

Loss = 100(50-30) = 2000	(5)

FVSR = Loss

FYf	(6)

FVSR = 2000 = 0.40

(100) (50)	(7)

Nomenclature is given in Table B-1. Note that the calcu-
lation did not require use of the fixed solids concentra-
tions.

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) devia-
tion divided by arithmetic average) than the fractional vola-
tile solids (VS is the fraction of the sewage sludge solids

B.3

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Table B-1. Quantitative Information for Example Problems 1,2,3

Problem Statement Number

Parameter

Symbol

Units

Nominal Residence Time

Time period for averages

—

Feed Sludge

Volumetric flow rate

m3/d

100

Volatile solids concentration

kg/m3

Fixed solids concentration

VSf

kg/m3

Fractional volatile solids

kg/kg

0.746

Mass flow rate of solids

kg/d

6700

Digested Sludge (Bottoms)

100

49.57

Volumetric flow rate

m3/d

Volatile solids concentration

kg/m3

41.42

Fixed solids concentration

kg/m3

23.50

Fractional volatile solids

VSb

kg/kg

0.638

0.667

0.638

Mass flow rate of solids

kg/d

4700

4500

Decantate

Volumetric flow rate

m3/d

50.43

Volatile solids concentration

kg/m3

12.76

Fixed solids concentration

kg/m3

—

7.24

Fractional volatile solids

vsd

kg/kg

—

0.638

Mass flow rate of solids

kg/d

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.

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 YfYbdata will show larger deviations than if it were cal-
culated by the Van Kleeck equation using VSf -VSb data.

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 sus-
pending the grit, and it tends to settle. If agitation is inad-
equate to keep the grit particles in suspension, they will
accumulate in the digester. The approximate mass bal-
ance can be used to estimate accumulation of fixed sol-
ids.

For Problem 1, the balance yields the following:
FXf = BXb + fixed solids loss (8)

(100)(17) = (100)(17) + Fixed Solids Loss (9)

Fixed Solids Loss = 0 (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 con-
sume 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.

Grit Deposition (Problem 2)

The calculation of fixed solids is repeated for Problem 2.
Conditions in Problem 2 have been selected to show grit
accumulation. Parameters are the same as in Problem 1
except for the fixed solids concentration (Xb) and param-
eters related to it. Fixed solids concentration in the sew-
age 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 balance on fixed solids (input
rate = output rate + rate of loss of fixed solids) is presented
in Equations 11-13.

FXf = BXb + Fixed Solids Loss (H)

Fixed Solids Loss = FXf- BXb (12)

Fixed Solids Loss = (100)(7) - (100)(15) = 200 kg/d (13)

The material balance, which only looks at inputs and
outputs, informs us that 200 kg/d of fixed solids have not
appeared in the outputs as expected. Because fixed sol-
ids are not destroyed, it can be concluded that they are
accumulating in the bottom of the digester. The calcula-
tion of FVSR for Problem 2 is exactly the same as for Prob-
lem 1 (see Equations 4 through 7) and yields the same
result. The approximate mass balance method gives the

B.4

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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 know-
ing 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., accumulation in the digester)
is zero.

Selecting a basis for F of 100 m3/d
Volume balance: 100 = B + D	(14)

Fixed solids balance: 100 Xf + BXb + DXd	(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
Problem 3 and solving for D and B,

(100)(17) = (100 - D)(23.50) + (D)(7.24)	(16)

D = 40.0 m3/d, B = 60.0m3/d	(17)

The FVSR can now be calculated by drawing a volatile
solids balance:

FYf = BYb + DYd + loss
FVSR= !°ss_ = FYf-BYb-DYd

FY <

FYf

FVSR = (100) (50)- (60) (41.42) -(40)(12.76) = 0 40
(100) (50)

Unless information is available on actual volumes of
decantate and sewage sludge (bottoms), it is not possible
to determine 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 biologi-
cal reactions that convert them to carbon dioxide and meth-
ane. 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 accumulation of volatile sol-
ids. FVSR calculated by Equations 18 through 20 will be
overestimated if the volatile solids accumulation 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 Equa-

tions 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 calculated assuming no grit accumu-
lation (Problem 3-see previous discussion), and measured
quantities are compared below:

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) - (50.43)(12.76)

(100)(50)

0.461

(21)

If it had been assumed that there was no grit accumula-
tion, FVSR would equal 0.40 (see Problem 3). It is pos-
sible to determine 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)

(18)

(19)

(20)

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 = (100)(17) - (49.57)(23.50) - (50.43)(7.24)

FXf

(100)(17)

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 accu-
mulation 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 deriva-
tion 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 volatiie sol-
ids mass balances around the digestion system. Consider
a digester operated under steady state conditions with
decantate and bottom sewage sludge removal. A total sol-
ids mass balance and a volatile solids mass balance are:

Mf = Mb + Md + (loss of total solids)

(25)

B.5

-------
Mf • VSf - Mb • VSb + Md • VSd + (loss of volatile solids)

(26)

where

Mf, Mb, andMd are the mass of solids in the feed, bot-
toms, 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
concentration 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 Equations 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),

FVSR = Loss of vol. solids = <|_

Mf x VSf

(Mb + Md) VSb
Mf x VSf

(27)

From Equation 25, recalling that we have assumed that
loss of total solids equals loss of volatile solids,

Mb + Md + Mf - loss of vol. solids

Substituting for Mb + Md into Equation 27,
FVSR = 1 - (Mf"loss °fv0'- solids) • VSb

Mf • VSf

Simplifying further,

1- (1/VSf -FVSR)
Solving for FVSR,
FVSR = vsf" vsb

VSb

VSf-(VSf-VSb)

(28)

(29)

(30)

(31)

This is the form of the Van Kleek equation found in WPCF
Manual of Practice No. 16 (WPCF, 1968). Van Kleeck
(1945) presented the equation in the following equivalent

form:

FVSR = 1 - VSb x (1 -VSf)

VSf x (1-VSb)

(32)

Approximate Mass

Balance (AMB)
Van Kleeck (VK)

1

0.40
0.40

2

0.40
0.318

3

0.40
0.40

0.461
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 short-
comings 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 equa-
tions will all be averages. For the material balance meth-
ods, the averages should be weighted averages accord-
ing 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 addi-
tions

Addition

1

2

3

4

Volume

12	m3
8 m3

13	m3
10 m3

Total Solids
Concentration

72 kg/m3
50 kg/m3
60 kg/m3
55 kg/m3

VS

0.75
0.82
0.80
0.77

Weighted by Mass

(33)

VS av =

12x72x0.75 + 8x50x0.82
+ 13 x 60 x 0.80 + 10 x 55 x 0.77
12 x 72 + 8 x 50+ 13 x 60 + 10 x 55

0.795

(34)

Weighted by Volume

vs av _ 12 x 0.75 + 8 x 0.82 +13 x 0.80 + 10 x 0.77
12+8+13+10

= 0.783

Arithmetic Average

Vg av _ 0.75 + 0.82 + 0.80 + 0.77 = 0.785
4

(35)

(36)

The Van Kleeck equation is applied below to Problems 1
through 4 in Table C-1 and compared to the approximate
mass balance equation results:

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 vola-
tile solids reduction for all approaches to digestion, even

B.6

-------
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 concentration and the volumes of all streams
added to or withdrawn from 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 "equiva-
lent" method that shows that the sewage sludge has un-
dergone the proper volatile solids reduction 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
treatment equivalent to a 38% volatile solids reduction if
volatile solids reduction after batch digestion of the sew-
age 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 composi-
tion, 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 accumu-
lation of grit is substantial and FVSR is calculated assum-
ing 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 pre-
sented. The equation is never valid when there is grit ac-
cumulation because it assumes the fixed solids input equals
fixed solids output. Fortunately, it produces a conservative
result in this case. Unlike the AMB method it does not pro-
vide a convenient way to check for accumulation of grit. It
can be used when decantate is withdrawn, provided VSb
equals VSd. Just how significant the difference between
these VS values can be before an appreciable error in
FVSR occurs is unknown, although it could be determined
by making up a series of problems with increasing differ-
ences 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 sub-
stantial, 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 de-

viation divided by arithmetic average) than volatile solids
and fixed solids concentrations. 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 approach may turn out to be advanta-
geous. The AMB method could be used first to determine
if grit accumulation is occurring. If grit is not accumulating,
the Van Kleeck equation could be used. If decantate is
withdrawn, the Van Kleeck equation is appropriate, par-
ticularly if the decantate is low in total solids. If not, and if
VSd differs substantially from VSb, it could yield an incor-
rect 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
calculate the volatile solids loss from the point at which
the sewage 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 balance approach, because flow
rates are not uniform. The full mass balance could be used
in principle, but practical difficulties 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 impossibility.
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 cor-
respondence between the incoming sewage sludge and
the treated sewage sludge to which it is being compared
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 Require-
ments in Sewage Sludge. Office of Water, U.S. EPA,
Washington, DC. NTIS No PB93-110609. Natl. Techni-
cal Information Service, Springfield, VA.

Fischer, W.J. 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, En-
gland.

B.7

-------
Van Kleeck, L.W. 1945. Sewage Works J., Operation of Water Pollution Control Federation. 1968. Manual of Prac-
Sludge Drying and Gas Utilization Units. 17(6): 1240- tice No. 16, Anaerobic Sludge Digestion. Washington,
1255.	DC.

B.8

-------
14: APPENDIX C

The text in Appendix C has been taken from the previously published document "Control of Pathogens
and Vector Attraction in Sewage Sludge" (July 2003, EPA 625-R-92-013). Page numbers will be
inconsistent with the previous text.

C.l

-------
Guidance on Three Vector Attraction Reduction Tests

This appendix provides guidance for the vector attrac-
tion reduction Options 2,3, and 4 to demonstrate reduced
vector attraction (see Chapter 9 for a description of these
requirements).

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 param-
eters including volatile solids content while carrying out
additional digestion of anaerobically digested sludge from
several treatment 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 procedures 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 representa-
tive sample of the sewage sludge to be evaluated to
determine additional volatile solids destruction. Keep
the sample protected from oxygen and maintain it at
the temperature of the digester. 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 determination. 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 watersealed bubbler by means of a ver-
tical glass tube. The tube should be at least 30-cm
long with enough water in the bubbler so that an in-
crease 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 tempera-
ture 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 con-
trolled within + 0.15°C (0.27°F).

•	Each flask should be swirled every day to assure ad-
equate 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 evi-
dence that gas is being produced and passing through
the bubbler.

•	After 20 days, withdraw five flasks at random. Deter-
mine 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 with-
drawn. Use a consistent procedure. If holdup on walls
appears excessive, a minimal amount of distilled wa-
ter 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. De-
termine total and volatile solids content using the en-
tire sample from each flask for the determination. Use
the same precautions as in the preceding step to re-
move virtually all of the sludge, leaving only material
with the same approximate composition as the mate-
rial removed.

C.2

-------
Total and volatile solids contents are determined using
the procedures of Method 2540 G of Standard Methods
(APHA, 1992).

Mean values and standard deviations of the total solids
content, the volatile solids content, and the percent vola-
tile 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 Ap-
pendix 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 sub-
stantiate 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, pro-
vided accuracy and reproducibility can be demonstrated.
The approach described above was developed because
Farrell and Bhide (1993) in their preliminary work experi-
enced much difficulty in withdrawing representative
samples from large digesters even when care was taken
to stir the digesters thoroughly before sampling. If an al-
ternative 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 reduction.

Variability in flow rates and nature of the sludge will re-
sult in variability in performance of the plant-scale digest-
ers. It is advisable to run the additional digestion test rou-
tinely so that sufficient data are available to indicate aver-
age performance. The arithmetic mean of successive tests
(a minimum of three is suggested) should show an addi-
tional volatile solids reduction of < 17%.

Calculation Details

Appendix C, Determination of Volatile Solids Reduction
by Digestion, describes calculation methods to use for di-
gesters 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 ending 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 equa-
tion calculations can be made in the manner described in
Appendix B.

The calculation of the volatile solids reduction (and the
fixed fractional solids reduction [FFSR]) by the mass bal-
ance method shown below has been refined by subtract-
ing 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 usually is ignored, because the amount is

small in relation to the total digesting mass, and mass be-
fore and after digestion are assumed to be the same. Con-
sidering the inherent difficulty in matching mass and com-
position entering to mass and composition 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 sol-
ids and the water in the sludge)- and are indicated by, the
symbols lowercase y and x. This is different from the us-
age in Appendix C where concentrations are given in mass
per unit volume, and are indicated by the symbols upper-
case y and x. This change has been made because masses
can be determined more accurately 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 con-
verted to gases that escape or to volatile compounds that
distill off when the sludge is dried. Any production or con-
sumption of water by the biochemical reactions in diges-
tion is assumed to be negligible. The data collected (vola-
tile solids and fixed solids concentrations of feed and di-
gested sludge) allow mass balances to be drawn on vola-
tile 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
fractional fixed solids reduction (m.b. method) are given
below:

yf(1-xb)-yb(1-xf)

FVSR(m.b.)= 	

YfC-Xb-Yb)	(1a)

ux xf(i-yb)-xb(i-yf)

FFSR m.b. =	(1b)

xf(1-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:

FVSR(m.b.)

(yf-YbVYf C-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-di-
gested by Farrell and Bhide (1993), the fixed solids reduc-

C.3

-------
tions were about one-third of the volatile solids reductions.
When the FVSR(m.b.) calculated by Equation 1a averaged
15%, the FVSR(m.b.) calculated by Equation 2 averaged
14.93%, which is a trivial difference.

The disappearance of fixed solids unfortunately has a
relatively large effect on the calculation of FVSR by the
Van Kleeck equation. The result is lower than it should be.
For five sludges that were batch-digested by Farrell and
Bhide (1993), the FVSR calculated by the Van Kleeck
method averaged 15%, whereas the FVSR (m.b.) calcu-
lated by Equation 1a or 2 averaged about 20%. When the
desired endpoint is an FVSR below 17%, this is a sub-
stantial 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 re-
quired, with the requirement adjusted upward by an ap-
propriate 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
digesting sludge. The procedure required by the Part 503
regulation for this test is presented in 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 reli-
able method for indicating sludge stability provided tem-
perature effects 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
02/hr/g VSS (volatile suspended solids). The authors in-
vestigated several alternative methods for indicating sta-
bility 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 digest-
ers operated at temperatures above 10°C (50°F), SOUR
fell to about 2.0 mg 02/hr/g VSS after a total sludge age of
60 days and to 1.0 mg 02/hr/g VSS after about 120 days
sludge age. These authors state that a SOUR of less than
1.0 mg 02/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 elimi-
nate odor generation from aerobically digested sludges.

Since the industry is not being deluged with complaints
about odor from aerobic 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 protective. Farrell and Bhide (1993) reviewed the
data these authors obtained with four sewage sludges from
aerobic treatment processes and concluded that a stan-
dard of 1.5 mg 02/hr/g TS at 20°C (68°F) would discrimi-
nate between adequately stabilized and poorly stabilized
sludges. The "adequately digested" sludges were not to-
tally trouble-free, i.e., it was possible under adverse con-
ditions to develop odorous conditions. In all cases where
the sludge was deemed to be adequate, minor adjustment
in plant operating conditions created an acceptable sludge.

The SOUR requirement is based on total solids rather
than volatile suspended solids. This usage is preferred for
consistency 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 ra-
tional since the entire sludge solids and not just the vola-
tile 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 recom-
mended value and 2.1 times higher than Ahlberg 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 di-
gesting sludge will be close to ambient temperature, which
can range from 5°C to 30°C (41 °F to 86°F). In this tem-
perature range, SOUR increases with increasing tempera-
ture. Consequently, if a requirement for SOUR is selected,
there must be some way to convert SOUR test results to a
standard temperature. Conceivably, the problem could be
avoided if the sludge were simply heated or cooled to the
standard temperature before running the SOUR test. Un-
fortunately, this is not possible, because temperature
changes in digested sludge cause short-term instabilities
in oxygen uptake rate (Benedict and Carlson [1973], Farrell
and Bhide [1993]).

Eikum and Paulsrud (1977) recommend that the follow-
ing equation be used to adjust the SOUR determined at
one temperature to the SOUR for another temperature:

(S0UR)t1/(S0UR)tc = e(T1"T2)	(3)

where:

(SOUR)n = specific oxygen uptake rate at T1
(SOUR)T2 = specific oxygen uptake rate atT2

9 = the Streeter-Phelps temperature sensitivity
coefficient

C.4

-------
These authors calculated the temperature sensitivity
coefficient using their data on the effect of temperature on
the rate of reduction in volatile suspended solids with time
during aerobic 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 vola-
tile solids disappearance. Another problem is that the
coeffficient 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 temperatures above
15°C (59°F) for aerobic digestion of waste activated slud-
ges, whereas Eikum and Paulsrud (1977) determined 0 to
equal 1.112 for primary sludges. Grady and Lim (1980)
reviewed the data of several investigators and recom-
mended that 0 = 1.05 be used for digestion of waste-acti-
vated sludges when more specific information is not avail-
able. Based on a review of the available information and
their own work, Farrell and Bhide (1993) recommend that
Eikum and Paulsruds' temperature 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 02/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 tem-
perature at which the aerobic digestion is occurring in the
treatment works and corrected to 20°C (68°F) by the fol-
lowing equation:

SOUR20 = SOURT x 0 (20"T>	(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 re-
quired to limit the possible error in the SOUR caused by
selecting an improper temperature coefficient. Farrell and
Bhide's (1993) results indicate that the suggested values
for 0 will give a conservative value for SOUR when trans-
lated 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 directions be followed if a respirom-
eter is used. No further reference to respirometric meth-
ods 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
followed with one exception. The total solids concentra-

tion instead of the volatile suspended solids concentration
is used in the calculation of the SOUR. Total solids con-
centration is determined by Standard Method 2540 G.
Method 2710 B cautions that if the suspended solids con-
tent 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 02/g/h. It is pos-
sible to verify that mixing is adequate by running repeat
measurements at several stirrer bar speeds. If stirring is
adequate, 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 probably 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 waste-
water 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 mineral 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 deter-
mine 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 im-
portant. The sample should be a composite of grab samples
taken within a period of a few minutes duration. The sample
should be transported to the laboratory expeditiously and
kept under aeration if the SOUR test cannot be run imme-
diately. The sludge should be kept at the temperature of
the digester from which it was drawn and aerated thor-
oughly 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 inap-
propriate 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 re-
sult in variability in performance of the plant-scale digest-
ers. It is advisable to run the SOUR test routinely so that
sufficient data are available to indicate average perfor-
mance. The arithmetic mean of successive tests-a mini-
mum of seven over 2 or 3 weeks is suggested-should give
a SOUR of < 1.5 mg 0?/hr/g total solids.

3. Additional Digestion Test for

Aerobically Digested Sewage Sludge

Background

Part 503 lists several options that can be used to dem-
onstrate reduction of vector attraction in sewage sludge.
These options include reduction of volatile solids by 38%
and demonstration of the SOUR value discussed above
(see also Chapter 9). These options are feasible for many,
but not all, digested sludges. For example, sludges from
extended aeration treatment works that are aerobically di-

C.5

-------
gested usually cannot meet this requirement because they
already are partially reduced in volatile solids content by
their exposure to long aeration times in the wastewater
treatment process.

The specific oxygen uptake test can be utilized to evalu-
ate aerobic sludges that do not meet the 38% volatile sol-
ids reduction requirement. Unfortunately, this test has a
number of limitations. 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 un-
der all possible conditions of use, such as for sludges of
more than 2% solids.

A straightforward approach for aerobically treated slud-
ges that cannot meet either of the above criteria is to de-
termine 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 gen-
eration that attracts vectors. Such a test necessarily takes
many days to complete, because time must be provided
to get measurable biodegradation. Under most circum-
stances, 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
possible to show compliance over a period of time.

The additional digestion test for aerobically digested slud-
ges 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, BOD, and SOUR-declined
smoothly and approached asymptotic values with time as
sludge was aerobically digested. Any one of these param-
eters potentially could be used as an index of vector at-
traction reduction for aerobic sludges. SOUR has been
adopted (see above) for this purpose. Farrell and Bhide
(1993) have shown that the additional volatile solids re-
duction that occurs when sludge is batch digested aerobi-
cally 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 slud-
ges investigated by Jeris et al. (1985), the relationship
between SOUR and additional volatile solids reduction
showed that the SOUR was approximately equal to 1.5
mg 02/hr/g (the Part 503 requirement for SOUR) when
additional volatile solids reduction was 15%. The two re-
quirements 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 accessible and are easily scraped clean of ad-
hering 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 par-
ticle-free air is supplied to the bottom of the digester through
porous stones at a rate sufficient to thoroughly mix the
sewage sludge. This will supply adequate oxygen 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 sec-
ondary effluent at the start of the test. The requirement
stems from the results of Reynolds (1973) and Malina
(1966) which demonstrate that rate of volatile solids re-
duction decreases as the feed solitis concentration in-
creases. Thus, for example, a sludge with a 2% solids con-
tent 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 vola-
tile solids reduction. However, the long duration of the test
should provide adequate time for recovery and demon-
stration of the appropriate reduction in volatile solids con-
tent.

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 reduction. Even if the bacteria are
shocked and do not recover completely, 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 at-
tract 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 temperature
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. Con-
sequently, it is unlikely to attract vectors.

The digester is charged with about 12 liters of the sew-
age sludge to be additionally digested, and aeration is com-
menced. The constant flow of air 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 temperature of the digesting sludge should be
approximately 20°C (68°F). If the temperature of the labora-

C.6

-------
tory 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 succes-
sive weeks. Before sampling, makeup water is added (this
will generally 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 contents of both samples are
determined preferably by Standard Method 2540 G (APHA,
1992). Percent volatile solids is calculated from total and
volatile solids content. Standard Methods (APHA, 1992)
states that duplicates should agree within 5% of their av-
erage. 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 and by a mass balance method. The mass
balance (m.b.) equations become very simple, 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-YbWYf	(5a)

FFSR(m.b.) = (Xf - Xb) / Xf	(5b)

where:

y and x = mass fraction of volatile and fixed solids, re-
spectively (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
densities are the same. Very little error is introduced by
this assumption.

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 procedure 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 volatile solids reduction than the calcu-
lation made by using the Van Kleeck equation.

4. References

Ahlberg, N.R. and B.I. Boyko. 1972. Evaluation and de-
sign of aerobic digesters. Jour. WPCF 44(4):634-643.

Benedict, A.H. and D.A. Carlson. 1973. Temperature ac-
climation in aerobic big-oxidation systems. Jour. WPCF
45(1): 10-24.

Eikum, A., and B. Paulsrud. 1977. Methods for measuring
the degree of stability of aerobically stabilized slud-
ges. Wat. Res. 11:763-770

EPA. 1992. Technical support document for Part 503 patho-
gen and vector attraction reduction requirements in
sewage sludge. NTIS No.: PB93-110609. Springfield,
VA: National Technical Information Service.

Farrell, J.B., V. Bhide, and Smith, J. E. Jr. 1996.

Development of methods EPA's new methods to
quantifying vector attraction of wastewater sludges,
Water Envir. Res.,68,3,286-294
APHA (American Public Health Association). 1992. Stan-
dard methods for the examination of water and waste-
water. 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. Lim. 1980. Biological waste-
water treatment: theory and applications. Marcel

Dekker, New York.

Jeris, J.S., D. Ciarcia, E. Chen, and M. Mena. 1985. De-
termining the stability of treated municipal sludge. EPA
Rept. No. 600/2-85/001 (NTIS No. PB 85-1471891
AS). U.S. Environmental Protection Agency, Cincin-
nati, 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 Re-
search, Vol. 2, pp 143-163. Water Pollution Control
Federation, Washington, DC.

Reynolds, T.D. 1973. Aerobic digestion of thickened waste-
activated sludge. Part 1, pp. 12-37, in Proc. 28th
Industr, Waste Conf., Purdue University.

C.7

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15: APPENDIX D

The text in Appendix D has been taken from the previously published document "Control of Pathogens
and Vector Attraction in Sewage Sludge" (July 2003, EPA 625-R-92-013). Page numbers will be
inconsistent with the previous text.

D.l

-------
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 tempera-
tures 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 residence 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 digest-
ers.

Approach

The discussion has to be divided into two parts: resi-
dence time for batch operation and for plug flow, and resi-
dence 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 follow-
ing equation:

0 = V/q	(1)

where

0 = 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 0 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 par-
ticles escape very soon after entry whereas others circu-
late in the reactor for long periods before escaping. The
average time in the reactor is given by the relationship:

0n

Z(8s x 0)
X(5s)

where

8s = an increment of sludge solids that leaves the reactor
0 = 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:

e„ = vc«

qca

(3)

where

V = reactor volume
q = flow rate leaving
Cv = 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 reac-
tor agitation, allow a supernatant layer to form, decant the
supernatant, and resume operation. Under these condi-
tions, 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 qC 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 residence time equation:

Qn _ mass of solids in the digester
mass flow rate of solids leaving

(4)

(2)

Using this form, residence time for the important operat-
ing 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 in-
crease residence time and part removed as product. The

calculation follows Equation 4 and is identical with the SRT
(solids retention time) calculation used in activated sludge
process calculations. 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 Equa-
tion 5:

D.2

-------
9n = VCy

pCp	(5)

where

p = flow rate of processed sludge leaving the system
Cp = solids concentration in the processed sludge

The subscript p indicates the final product leaving the

system, not the underflow from the thickener. This approach
ignores any additional residence time in the thickener since
this time is relatively short and not at proper digestion con-
ditions.

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 op-
erating modes:

Case 1

•	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:

9n= VOy ~ V
qcq q

The concentration terms in Equation 4 cancel out be-
cause Cv equals Cq.

Case 2a

•	Complete-mix reactor

•	Sludge is introduced in daily batches of volume (Vj)
and solids concentration (Cj)

•	Vessel contains a "heel" of liquid sludge (Vf) at the
beginning of the digestion step

•	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:

0n = SVjCj x time that batch i remains in the reactor

SVjCj

The following problem illustrates the calculation:

Let Vh = 30 m3 (volume of "heel")

Vd= 130 m3 (total digester volume)

Vj = each day 10 m3 is fed to the reactor at the begin-
ning of the day
Cj =12 kg/m3

Vf is reached in 10 days. Sludge is discharged at the
end of Day 10.

Then 0n =(10-12-10+10-12-9 +...+10-12-1)

(10-12 + 10-12 + ... 10-12)

0n = 10*12*55 = 5.5 days

10-12-10

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 = 30m3, and Vd = 130m3

Day Vi (m3)	Solids Content (kg/m3) Decantate (m3)

1

10

10

0

2

10

15

0

3

10

20

0

4

10

15

0

5

10

15

0

6

10

10

0

7

10

20

0

8

10

25

0

9

10

15

10

10

10

10

0

11

10

15

10

12

10

20

0

0n = (10-10-12+10-15-11+10-20-10+...
..+10-10-3+10-15-2+10-20-1)

(10-10+10-15+10-20+...

+10-10+10-15+10-20

0n = 11,950/1,900 = 6.29d

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 sev-
eral days.

A conservative 0n can be calculated by simply adding
the number of extra days of operation to the 0n calculated

D.3

-------
for Case 2. The same applies to any other cases followed
by batch mode operation.

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 re-
turned 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

qf = 10 m3/d (feed stream)

Cf = 40 kg solids/m3

q = 5 m3/d (existing sludge stream)

Cv = 60 kg solids/m3

0n = 100x60 = 20d
5x60

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

thickened, some sludge is recycled, and some is drawn
off as product

This mode of operation is sometimes used in aerobic
digesters. 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 m3/d

Solids content of sludge from the digester = 13.3 kg/m3

Flow rate of sludge from the thickener = 4 m3/d

Solids content of sludge from the thickener = 40 kg/m3

Flow rate of sludge returned to the digester = 2 m3/d

Flow rate of product sludge = 2 m3/d

9n= 100x13.3= 16.6 d
2x40

The denominator is the product of the flow rate leaving
the system (2 m3/d) and the concentration of sludge leav-
ing the thickener (40 kg/m3). Notice that flow rate of sludge
leaving the digester did not enter into the calculation.

Comments on Batch and Staged Operation

Sludge can be aerobically digested using a variety of
process configurations (including continuously fed single-
or multiple-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). Single-stage completely mixed re-
actors with continuous feed and withdrawal are the least
effective of these options for bacterial and viral destruc-
tion, because organisms that have been exposed 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
operation, 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 com-
pared to single-stage operation. If the kinetics of the reduc-
tion in pathogen densities are known, it is possible to esti-
mate how much improvement can be made by staged op-
eration.

Farrah et al. (1986) have shown that the declines in den-
sities of enteric bacteria and viruses follow first-order ki-
netics. 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 improved operating
mode. Since not all factors involved in the decay of micro-
organisms 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 required be reduced to 70% of the time
required for single-stage aerobic digestion in a continu-
ously mixed reactor. This allows a 30% reduction in time
instead of the 50% estimated from theoretical consider-
ations. 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
reduction.

If the plant operators desire, they may dispense with the
PSRP time-temperature requirements of aerobic digestion
but instead demonstrate experimentally that microbial lev-
els in the product from their sludge digester are satisfacto-
rily reduced. Under the current regulations, fecal coliform
densities must be less than or equal to 2,000,000 CFU or
MPN per gram total solids. Once this performance is dem-
onstrated, the process would have to be operated between
monitoring episodes at time-temperature conditions at least
as severe as those used during their tests.

D.4

-------
References

Farrah. S.R., G. Bitton, and S.G. Zan. 1986. Inactivation
of enteric pathogens during aerobic digestion of waste-
water sludge. EPA Pub. No. EPA/600/2-86/047. Wa-
ter Engineering Research Laboratory, Cincinnati, OH.

NTIS Publication No. PB86-183084/AS. National Tech-
nical 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.

D.5

-------
16: APPENDIX E

Method 1630: Fecal coliforms in .Sewage .Sludge (Biosolids) by Multiple-Tube Fermentation using Lauiryll
Tryptose Birol	and IEC Medium

Method 1681: Fecal Coliforms in .Sewage Sludge (Biosolids) by Multiple Tube Fermentation using A-l
Medium || July 2006

Method 1632: Salmonella in .Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis
(MSRV) Medium

E.l

-------
17: APPENDIX F

The text in Appendix F has been taken from the previously published document "Control of Pathogens
and Vector Attraction in Sewage Sludge" (July 2003, EPA 625-R-92-013). Page numbers will be
inconsistent with the previous text.

F.l

-------
Method for the Recovery and Assay of Total Culturable Viruses from Sludge

1.	Introduction

1.1.	Scope

This chapter describes the method that must be
followed to produce Class A sludge when virus monitoring
under 40 CFR Part 503 is required. The method is designed
to demonstrate that sludges meet the requirement that
human enteric viruses (i.e., viruses that are transmitted via
the fecal-oral route) are less than one plaque-forming unit
(PFU) per 4 g of total dry solids.

1.2.	Significance

More than 100 different species of pathogenic human
enteric viruses maybe present in raw sludge. The presence
of these viruses can cause hepatitis, gastroenteritis and
numerous other diseases. Hepatitis A virus and noroviruses
are the primary human viral pathogens of concern, but
standard methods for their isolation and detection have not
been developed. The method1 detailed in this chapter
detects total culturable viruses, which primarily include the
human enteroviruses (e.g., polioviruses, coxsackieviruses,
echoviruses) and reoviruses.

1.3.	Safety

The sludges to be monitored may contain pathogenic
human enteric viruses. Laboratories performing virus analy-
ses are responsible for establishing an adequate safety plan
and must decontaminate and dispose of wastes according
to their safety plan and all applicable regulations. Aseptic
techniques and sterile materials and apparatus must be
used throughout the method.

2.	Sample Collection

For each batch of sludge that must be tested for
viruses, prepare a composite sample by collecting ten
representative samples of 100 mL each (1,000 mL total)
from different locations of a sludge pile or at different times
from batch or continuous flow processes. Combine and mix
thoroughly all representative samples for a composite.
Batch samples that cannot be assayed within 24 hours of
collection must be frozen at -70°C; otherwise, they should
be held at 4°C until processed. If representative samples
must be frozen before they can be combined, then thaw,
combine and mix them thoroughly just prior to assay. Then
remove a 50 mL portion from each composite sample for

'Method D4994-89, ASTM(1992)

solids determination as described in section 3. The remain-
ing 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.

Freeze/thawing biosolids may result in some virus

loss.

3.	Determination of Total Dry Solids2

3.1.	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.

3.2.	Place the pan and its contents into an oven main-
tained at 103-105°C for at least one hour.

3.3.	Cool the sample to room temperature in a desiccator
and weigh again.

3.4.	Repeat the drying (1 h each), cooling and weighing
steps until the loss in weight is no more than 4% of the
previous weight.

3.5.	Calculate the fraction of total dry solids (T) using the
formula:

= (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).

4.	Total Culturable Virus Recovery from
Sludge

4.1. Introduction

Total culturable viruses 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

2Modified from EPA/600/4-84/013(R7), September 1989 Revision (section 3). This
and other cited EPA publications may be requested from the Biohazard Assessment
Research Branch, National Exposure Research Laboratory, U.S. Environmental Pro-
tection Agency, Cincinnati, Ohio, USA 45268.

F.2

-------
bind free virus to solids. Virus is then eiuted from the solids
and concentrated prior to assay.

4.2. Conditioning of Suspended Solids

Conditioning of sludge binds unadsorbed total cultura-
ble viruses present in the liquid matrix to the sludge solids.

Each analyzed composite sample (from the portion re-
maining after solids determination) must have an initial total
dry solids content of at least 16 g. This amount is needed
for positive controls and for storage of a portion of the
sample at-70°C as a backup in case of procedural mistakes
or sample cytotoxicity.

4.2.1 Preparation

(a)	Apparatus and Materials

(a.1) Refrigerated centrifuge capable of attaining 10,000
xg and screw-capped centrifuge bottles with 100 to 1000 mL
capacity.

Each bottle must be rated for the relevant centrifugal

force.

(a.2) A pH meter with an accuracy of at least 0.1 pH unit,
equipped with a combination-type electrode.

(a.3) Magnetic stirrer and stir bars.

(b)	Media and Reagents

Analytical Reagent or ACS grade chemicals (unless
specified otherwise) and deionized, distilled water (dH20)
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.

(b.1) Hydrochloric acid (HCI) — 1 and 5 M.

Mix 10 or 50 mL of concentrated HCI with 90 or 50 mL
ofdH20, respectively.

(b.2) Aluminum chloride (AICI3 • 6HzO) — 0.05 M.

Dissolve 12.07 g of aluminum chloride in a final
volume of 1000 mL of dH20. Autoclave at 121 °C for 15
minutes.

(b.3) Sodium hydroxide (NaOH) — 1 and 5 M.

Dissolve 4 or 20 g of sodium hydroxide in a final
volume of 100 mL of dH20, respectively.

(b.4) Beef extract (Difco Product No. 0115-17-3 or
equivalent).

Prepare buffered 10% beef extract by dissolving 10 g
beef extract, 1.34 g Na2HP04 ¦ 7H20 and 0.12 g citric acid
in 100 mL of dH20. 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) for virus elution. This beef extract tends
to elute cytotoxic materials from sludges.

(b.5) HOCI —0.1%

Add 19 mL of household bleach (Clorox, The Clorox
Co., or equivalent) to 981 mL of dH20 and adjust the pH of
the solution to 6-7 with 1 M HCI.

(b.6) Thiosulfate — 2% and 0.02%

Prepare a stock solution of 2% thiosulfate by dissolv-
ing 20 g of thiosulfate in a total of 1 liter of dH20. Sterilize
the solution by autoclaving at 121°C for 15 minutes.
Prepare a working solution of 0.02% thiosulfate just prior to
use by mixing 1 mL of 2% thiosulfate with 99 mL of sterile
dH20.

4.2.2 Conditioning Procedure

Figure 1 gives a flow diagram for the procedure to
condition suspended solids.

(a) Calculate the amount of sample to condition.

Use a graduated cylinder to measure the volume. If
the volumes needed are not multiples of 100 mL (100, 200,
300 mL, etc.), add sterile water to bring the volume to the
next multiple of 100 mL. Each sample should then be
aliquoted into 100mL 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.

(a.1) Calculate the amount needed to measure the
endogenous total culturable virus in a composite sludge
sample using the formula:

where Xts equals the milliliters of sample required to obtain
12 g of total solids and T equals the fraction of total dry sol-
ids (from section 3).3

(a.2) Calculate the amount needed for a recovery control
for each sludge composite from the formula:

4

Xpc = -

where Xpc equals the milliliters of sample required to obtain
4 g of total solids.

Add 400 plaque forming units (PFU) of a Sabin
poliovirus stock to the recovery control sample. Use a virus
stock that has been filtered through a 0.2 fjm filter (see
Section 4.3.1) prior to assay to remove clumped virus
particles.

(a.3) Place 30 mLof 10% buffered beef extract and 70 mL
of dHzO into a 250 mL beaker with stir bar to serve as a
negative process control.

(a.4) Freeze any remaining composite sample at-70°C for
backup purposes.

3This formula is based upon the assumption that the density of the liquid in sludge is
1 g/mL. If the fraction of total dry solids is too low (e.g., less than 0.02), then the
volume of sludge collected must be increased.

F.3

-------
SUSPENDED SOLIDS (PER 100 mL)

I Mix suspension on magnetic stirrer.

Am Add 1 mL of 0.05 M AICi3.

SALTED SOLIDS SUSPENSION

Continue mixing suspension.

I Adjust pH of salted suspension to 3.5 ±0.1
I with 5 M HCI.

~ Mix vigorously for 30 minutes.

pH-ADJUSTED SOLIDS SUSPENSION

Centrifuge salted, pH-adjusted suspension
I at 2,500 *g for 15 minutes at 4°C.

. I • Discard supernatant.

Retain solids.

SOLIDS

Figure 1. Flow diagram of method for conditioning suspended
solids

(b) Perform the following steps on each 100 mL aliquot
from steps 4.2.2a.1 to 4.2.2a.3.

(b.1) Place the beaker on a magnetic stirrer, cover loosely
with aluminum foil, and stir at a speed sufficient to develop
vortex. Add 1 mL of 0.05 M AICI3 to the mixing aliquot.

The final concentration of AICI3 in each aliquot is
approximately 0.0005 M.

(b.2) Place a combination-type pH electrode into the
mixing aliquot. Adjust the pH of the aliquot to 3.5 ± 0.1 with
5 M HCI. Continue mixing for 30 minutes.

The pH meter must be standardized at pH 7 and 4.
When solids adhere to an electrode, clean it by moving up
and down gently in the mixing aliquot.

After adjusting the pH of each sample, rinse the
electrode with dH20 and sterilize it with 0.1% HOCI for five
minutes. Neutralize the HOCI by submerging the electrode
in sterile 0.02% thiosulfate for one to five minutes.

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
HCI. If the pH drifts down, readjust it with 5 M NaOH. Use
1 M acid or base for small adjustments. Do not allow the pH
to drop below 3.4.

(b.3) Pour the conditioned aliquot into a centrifuge bottle
and centrifuge at 2,500 *g for 15 minutes at 4°C.

To prevent the transfer of the stir bar into the centri-
fuge 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. If a
large enough centrifuge bottle is available, the test sample
aliquots may be combined into a single bottle at this step.
If there is more than one recovery control aliquot, they may
also be combined into another centrifuge bottle.

(b.4) Decant the supernatant into a beaker and discard.
Replace the cap onto the centrifuge bottle. Elute the solids
by following the procedure described in section 4.3.

4.3. Elution of Viruses from Solids

4.3.1	Apparatus and Materials

In this and following sections only apparatus and
materials which have not been described in previous
sections are listed.

(a)	Membrane filter apparatus for sterilization — 47 mm
diameter Swinnex filter holder and 60 mL slip-tip syringe
(Millipore Corp. Product No. SX00 047 00 and Becton
Dickinson Product No. 1627 or equivalent).

(b)	Disc filters, 47 mm diameter— 3.0, 0.45, and 0.2 |_im
pore size filters (Mentec America, Filterite Div., Duo- Fine
series, Product No. 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 0.2 /jm 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 °C
for 15 min.

Filters stacked in tandem as described tend to clog
more slowly when turbid material is filtered through them.
Prepare several filter stacks.

4.3.2	Elution Procedure

A flow diagram of the virus elution procedure is given
in Figure 2.

(a)	Place a stir bar and 100 mL of buffered 10% beef
extract into the centrifuge bottle containing the solids (from
section 4.2.2b.4).

If the test and control samples are divided into more
than one centrifuge bottles, the solids should be combined
at this step.

Place the centrifuge bottle on a magnetic stirrer, and
stir at a speed sufficient to develop a vortex for 30 min at
room temperature.

To minimize foaming (which may inactivate viruses),
do not mix faster than necessary to develop vortex.

(b)	Remove the stir bar from each bottle with a long
sterile forceps or a magnet retriever and centrifuge the
solids-eluate mixture at 10,000 *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.

F.4

-------
SOLIDS

¦ Add 100 mL of buffered 10% beef extract,
I adjust to pH 7.0 ±0.1 if necessary.

w Mix resuspended solids on magnetic stirrer for
30 minutes to elute viruses.

RESUSPENDED SOLIDS

Centrifuge resuspended solids for 30 minutes
I at 4°C using a centrifugal force of 10,000 *g

*ly Discard solids.

Retain eluate (supernatant).

ELUATE

Filter eluate through 47 mm Filterite filter
I stack of 3.0, 0.45 and 0.2 |_im pore sizes
I with the 0.2 |_im pore size on support screen
\l/ of filter and remaining filters on top in
order of increasing pore size.

FILTERED ELUATE

Figure 2. Flow diagram of method for elution of virus from solids.

Centrifugation at 10,000 *g is normally required to
clarify the sludge samples sufficiently to force the resulting
supernatant through the filter stacks.

(c) Place a filter holder that contains filter stacks (from
section 4.3.1b) onto a 250 mL Erlenmeyer receiving flask.
Load 50 mL syringes with the supernatants from step 4.3.2c.
Place the tip of the syringe into the filter holder and force the
supernatant through the filter stacks into 250 mL receiving
flasks.

Prior to use, pass 15 mL of 3% beef extract through
each filter holder to minimize non-specific adsorption of
viruses. Prepare 3% beef extract by mixing 4.5 mL of 10%
beef extract and 10.5 mL of dH20. 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 filter stack to force
residual eluate from the filters. Continue the filtration proce-
dure with another filter holder and filter stack. Discard con-
taminated filter holders and filter stacks. This procedure
may be repeated as often as necessary to filter the entire
volume of supernatant. Disassemble each filter holder and
examine the bottom 0.2 ym filters to be certain they have
not ruptured. If a bottom filter has ruptured, repeat the step
with new filter holders and filter stacks.

Proceed immediately to section 4.4.

4.4. 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 total cul-

turable viruses in the eluate. The step significantly reduces
costs associated with labor and materials.

Floe formation capacity of the beef extract reagent
must be pretested. Because some beef extract lots may not
produce sufficient floe, each new lot must be pretested to
determine virus recovery. This maybe performed by spiking
100 mL of dH20 with a known amount of poliovirus in the
presence of a 47 mm nitrocellulose filter. This sample
should be conditioned using section 4.2 above to bind virus
to the filter. Virus should then be eluted from the filter using
the procedure in section 4.3, and concentrated and assayed
using the following procedures. Any lot of beef extract not
giving a overall recovery of at least 50% should not be used.

4.4.1	Media and Reagents

In this and following sections only media and reagents
which have not been described in previous sections are
listed.

(a) Sodium phosphate, dibasic (Na2HP04 • 7H20) —
0.15 M.

Dissolve 40.2 g of sodium phosphate in a final volume
of 1000 mL. Autoclave at 121°C for 15 minutes.

4.4.2	Virus Concentration Procedure

A flow diagram for the virus concentration procedure
is given in Figure 3.

(a)	Pour the filtered eluates from the test sample,
recovery control and negative process control from section
4.3.2d into graduated cylinders, and record their volumes.
Transfer the samples into separate 600 mL beakers and
cover them loosely with aluminum foil.

(b)	For every 3 mL of beef extract eluate, add 7 mL of
dH20 to the 600 mL beakers. Add stir bars to each 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.

(c)	Recordthetotalvolumeofthedilutedeluates. Place
the beakers onto a magnetic stirrer, cover loosely with
aluminum foil, and stir at a speed sufficient to develop
vortex.

To minimize foaming (which may inactivate viruses),
do not mix faster than necessary to develop vortex.

(d)	For each diluted, filtered beef extract, insert a sterile
combination-type pH electrode and then add 1 M HCI slowly
until the pH of the extract reaches 3.5 ± 0.1. Continue to stir
for 30 minutes at room temperature.

The pH meter must be standardized at pH 4 and 7.
Sterilize the electrode by treating it with 0.1% HOCI for five
minutes. Neutralize the HOCI by treating the electrode with
0.02% sterile thiosulfate for one to five minutes.

F.5

-------
FILTERED ELUATE

¦	Add sufficient volume ofdH20 to filtered eluate
I to reduce concentration of beef xtract from

*1# 10% to 3%. Record total volume of the

~	diluted beef extract.

DILUTED, FILTERED ELUATE

Mix diluted eluate on a magnetic stirrer.

I Adjust the pH of the eluate to 3.5 ±0.1 with
I M HCI. A precipitate (floe) will form.

~	Continue mixing for 30 minutes.

FLOCCULATED ELUATE

Centrifuge flocculated eluate at 2,500 *g for

¦	15 minutes at 4°C.

I Discard supernatant.

Retain floe.

FLOC FROM ELUATE

Add 0.15 M Na2HP04 to floe, using 1/20th of
the recorded volume of the diluted 3% beef
I extract.

I Mix suspended floe on magnetic stirrer until
floe dissolves.

Adjust to a pH of 7.0 to 7.5.

DISSOLVED FLOC

¦	See section 5 for virus assay procedure.

y

ASSAY DISSOLVED FLOC FOR VIRUSES

Figure 3. Flow diagram of method for concentration of viruses
from beef extract eluate.

A precipitate will form. If the pH is accidentally re-
duced 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.

(e)	Pour the contents of each beaker into 1,000 mL
centrifuge bottles. Centrifuge the precipitated beef extract
suspensions at 2,500 *g for 15 minutes at 4°C. Pour off
and discard the supernatants.

To prevent the transfer of the stir bar into a centrifuge
bottle, hold another stir bar or magnet against bottom of the
beaker when decanting contents.

(f)	Place stir bars into the centrifuge bottles that con-
tains the precipitates. To each, add a volume of 0.15 M
Na2HP04 • 7H20 equal to exactly 1/20 of the volume record-
ed in step 4.4.2c. If the precipitate from a sample is in more
than one bottle, divide the 1/20th volume equally among the
centrifuge bottles containing that sample. Place the bottles

onto a magnetic stirrer, and stir slowly until the precipitates
have dissolved completely.

Support the bottles as necessary to prevent toppling.
Avoid foaming which may inactivate or aerosolize viruses.
The precipitates may be partially dissipated with sterile spa-
tulas before or during the stirring procedure.

(g)	Measure the pH of the dissolved precipitates.

If the pH is above or below 7.0-7.5, adjust to that
range with either 1 M HCI or 1 M NaOH.

(h)	Freeze exactly one half of the dissolved precipitate
test sample (but not the positive and negative controls) at -
70°C. This sample will be held as a backup to use should
the sample prove to be cytotoxic. Record the remaining test
sample volume (this volume represents 6 g of total dry
solids). Refrigerate the remaining samples immediately at
4°C until assayed in accordance with the instructions given
in section 5 below.

If the virus assay cannot be undertaken within 24
hours, store the remaining samples at -70°C.

5. Assay for Plaque-forming Viruses4

5.1.	Introduction

This section outlines procedures for the detection of
viruses in sludge by use of the plaque assay system. The
system uses an agar medium to localize virus growth follow-
ing attachment of infectious virus particles to a cell mono-
layer. 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, whose number is proportional to the
amount of infectious virus particles inoculated.

The detection methodology presented in this section
is geared towards laboratories with a small-scale virus as-
say requirement. Where the quantities of cell cultures,
media and reagents set forth in 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.

5.2.	Plaque Assay Procedure

5.2.1	Apparatus and materials.

(a) Waterbath set at 50 ± 1°C.

Used for maintaining the agar temperature (see
section 5.2.2j).

5.2.2	Media and Reagents.

(a) ELAH — 0.65% lactalbumin hydrolysate in Earle's
base.

Dissolve 6.5 g of tissue culture, highly soluble grade
lactalbumin hydrolysate (Gibco BRL Product No. 11800 or

'Modified from EPA/600/4-84/013(Rl 1), March 1988 Revision

F.6

-------
equivalent) in 1 L of Earle's base (Gibco BRL Product No.
14015 or equivalent) prewarmedto 50-60°C. Sterilize ELAH
through a 0.22 /im filter stack and store for up to two months
at 4°C.

(b)	Wash medium—Add 1 mL of penicillin-streptomycin
stock (see section 6.4.2e.1 for preparation of antibiotic
stocks), 0.5 mL of tetracycline stock and 0.2 mL of fungi-
zone stock per liter to ELAH immediately before washing of
cells.

(c)	HEPES — 1 M (Sigma Chemical Product No.
H-3375 or equivalent).

Prepare 50 mLof a 1 M solution by dissolving 11.92g
of HEPES in a final volume of 50 mL dH20. Sterilize by
autoclaving at 121°C for 15 min.

(d)	Sodium bicarbonate (NaHC03) — 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 dH20.
Sterilize by filtration through a 0.22 ym filter.

(e)	Magnesium chloride (MgCI2 • 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 dH20.
Sterilize by autoclaving at 121 °C for 15 min.

(f)	Neutral red solution — 0.333%, 100 mL volume
(GIBCO BRL Product No. 630-5330 or equivalent).

Procure one 100 mL bottle.

Some neutral red solutions are cytotoxic. All new solu-
tions should be tested prior to their use for assaying sludge
samples. Testing may be performed by assaying a stock of
poliovirus with known titer using this plaque assay proce-
dure.

(g)	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.

(h)	Preparation of Medium 199.

The procedure described is for preparation of 500 mL
of Medium 199 (GIBCO BRL Product No. 400-1100 or equi-
valent) at a 2X concentration. This procedure will prepare
sufficient medium for at least fifty 6 oz glass bottles or eighty
25 cm2 plastic flasks.

(h.1) Place a three inch stir bar into a one liter flask. Add
the contents of a 1 liter packet of Medium 199 into the flask.
Add 355 mL of dHzO. Rinse medium packet with three
washes of 20 mL each of dHzO and add the washes to the
flask.

Note that the amount ofdH20 is 5% less than desired
final volume of medium.

(h.2) Mix on a magnetic stirrer until the medium is com-
pletely dissolved. Filterthe reagent under pressure through
a filter stack (see section 6.2.6).

Test each lot of medium to confirm sterility before the
lot is used (see section 6.5). Each batch may be stored for
two months at 4°C.

(i) Preparation of overlay medium for plaque assay.

The 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 section 5.2.2j.

(i. 1) Add 79 mLof Medium 199 (2X concentration) and 4 mL
of serum to a 250 mL flask.

(1.2)	Add the following to the flask in the order listed, with
swirling after each addition: 6 mL of 7.5% NaHC03, 2 mL of
1 % MgCI2, 3 mL of 0.333% neutral red solution, 4 mL of 1 M
HEPES, 0.2 mLof penicillin-streptomycin stock (see section
6.4.2e for a description of antibiotic stocks), 0.1 mL of
tetracycline stock, and 0.04 mL of fungizone stock.

(1.3)	Place flask with overlay medium in waterbath set at 36
± 1°C.

(j) Preparation of overlay agar for plaque assay.

(j. 1) Add 3 g of agar (Sigma Chemical Product No. A-9915
or equivalent) and 100 mL of dHzO to a 250 mL flask. Melt
by sterilizing the agar solution in an autoclave at 121 °C for
15 min.

(j.2) Cool the agar to 50°C in waterbath set at 50 ± 1°C.

(k) Preparation of agar overlay medium.

(k.1) Add 2 mL of 10% skim milk to overlay medium pre-
pared in section 5.2.2i.

(k.2) 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 min.

5.2.3 Procedure for Inoculating Test Samples.

Section 6.6 provides the procedures for the prepara-
tion of cell cultures used for the virus assay in this section.

BGM cell cultures used for virus assay are generally
found to be at their most sensitive level between the third
and sixth days after initiation. Those older than seven days
or which are not 100% confluent should not be used.

(a)	Decant and discard the growth medium from pre-
viously prepared cell culture test vessels.

To prevent splatter, a gauze-covered beaker may be
used to collect spent medium.

The medium is changed from one to four hours before
cultures are to be inoculated and carefully decanted so as
not to disturb the cell monolayer.

(b)	Replace discarded medium with an equal volume of
wash medium (from section 5.2.2b) on the day the cultures
are to be inoculated.

F.7

-------
Table 1. Guide for Virus Inoculation, Suspended Cell Concentration and
Overlay Volume of Agar Medium

Volume of Volume of Agar
Virus Inoculum Overlay Medium Total Number of
Vessel Type	(ml_)	(ml_)	Cells

1 oz glass
bottle1

0.1

5

1 x 107

25 cm2 plastic
flask

0.1-0.5

10

2 x 107

6 oz glass bottle

0.5-1.0

20

4 x 107

75 cm2 plastic
flask

1.0-2.0

30

6 x 107

1Size is given in oz only when it is commercially designated in that unit.

To reduce shock to cells, prewarm the wash 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 wash medium to the
side of cell culture test vessel opposite the cell monolayer.

(c)	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 monolayers are to be inoculated.

(d)	Decant and discard the wash medium from cell cul-
ture test vessels.

Do not disturb the cell monolayer.

(e)	Inoculate BGM cultures with the test sample and
positive and negative process control samples from section
4.4.2h. Divide each sample onto a sufficient number of BGM
cultures to ensure that the inoculum volume is no greater
than 1 mL for each 40 cm2 of surface area. Use Table 1 as
a guide for inoculation size.

Avoid touching either the cannula or the pipetting de-
vice to the inside rim of the cell culture test vessels to avert
the possibility of transporting contaminants to the remaining
culture vessels.

If the samples are frozen, thaw them rapidly by placing
them in warm water. Samples should be shaken during the
thawing process and removed from the warm water as soon
as the last ice crystals have dissolved.

(e. 1) Inoculate BGM cultures with the entire negative pro-
cess control sample using an inoculum volume per vessel
that is appropriate for the vessel size used.

(e.2) Inoculate two BGM cultures with an appropriate vol-
ume of 0.15 M Na2HP04 • 7HzO preadjusted to pH 7.0-7.5
and seeded with 20-40 PFU of poliovirus. These cultures
will serve as a culture sensitivity control.

(e.3) Remove a volume of the test sample concentrate
exactly equal to 1 /6th (i.e., 1 g of total dry solids) of the vol-
ume recorded in section 4.4.2h. Seed 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 5.2.5b).

(e.4) Inoculate BGM cultures with the entire recovery con-
trol sample using an inoculum volume per vessel that is
appropriate for the vessel size used.

(e.5) 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 por-
tion (even if diluted to reduce cytotoxicity) onto BGM
cultures using an inoculum volume per vessel that is appro-
priate for the vessel size used. Inoculation of the entire
volume is necessary to demonstrate a virus density level of
less than 1 PFU per 4 g total dry solids.

(f)	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.

(g)	Incubate the inoculated cell cultures at roomtemper-
ature for 80 min to permit viruses to adsorb onto and infect
cells and then proceed immediately to section 5.2.4.

It may be necessary to rock the vessels every 15-20
min during the 80 min incubation to prevent cell death in the
middle of the vessels from dehydration.

5.2.4 Procedure for Overlaying Inoculated Cul-
tures with Agar.

If there is a likelihood that a test sample will be toxic to
cell cultures, the cell monolayer should be treated in accor-
dance with the method described in section 5.2.5b.

(a)	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 1).

The preparation of the overlay agar and the agar over-
lay medium must be made far enough in advance so that
they will be at the right temperature for mixing at the end of
the 80 min 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.

(b)	Place cell culture testvessels, monolayer side down,
on a level stationary surface at room temperature (22-25°C)
so that the agarwill remain evenly distributed as it solidifies.
Cover the vessels with a sheet of aluminum foil, a tightly
woven cloth, or some other suitable coverto reduce the light
intensity during solidification and incubation. Neutral red
can damage or kill tissue culture cells by light-induced
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 com-
plete and an erroneous virus assay will result.

Agar will fully solidify within 30 min.

F.8

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(c) After 30 min, invert the cell culture test vessels and
incubate them covered in the dark at 36.5 ± 1°C.

5.2.5 Plaque Counting Technique.

(a)	Count, mark and record plaques in cell culture test
vessels on days one, two, three, four 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.

(b)	Determine if samples are cytotoxic by macroscopic
examination ofthe appearance of the cell culture monolayer
(compare negative, positive and recovery controls from
section 5.2.3e with seeded and unseeded test samples)
after one to four days of incubation at 36.5 ± 1°C. Samples
show cytotoxicity if cell death is observed on test and
recovery control samples prior to its development on
positive controls. Cytotoxicity should be suspected when
the agar color is more subdued, generally yellow to yel-
low-brown. This change in color results in a mottled or
blotchy appearance instead ofthe evenly diffused "reddish"
color observed in "healthy" cell monolayers. Cytotoxicity
may also cause viral plaques to be reduced in 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 (section 5.2.3e). If
samples are cytotoxic, do not proceed to the next steps.
Re-assay a small amount of the remaining sample using
1:2, 1:4 and 1:8 dilutions. Then re-assay the remaining
sample as specified in section 5.2.3 using the dilution which
removes cytotoxicity and the specified number of flasks
times the reciprocal ofthe dilution.

A small amount of sample maybe tested for cytotoxici-
ty prior to a full assay.

(c)	Examine cell culture test vessels as in step 5.2.5a on
days six, eight, twelve and sixteen.

If no new plaques appear at 16 days, proceed with
step 5.2.6; otherwise continue to count, mark and record
plaques every two days until no new plaques appear be-
tween counts and then proceed with step 5.2.6.

Inoculated cultures should always be compared to un-
inoculated control cultures so that the deterioration of the
cell monolayers is not recorded as plaques. If experience
shows that cultures start to deteriorate prior to 16 days, a
second layer of agar can be added after 7 days as de-
scribed in section 5.2.4.

If negative process controls develop plaques or if pos-
itive controls fail to develop plaques, stop all assays until the
source of the problem is corrected.

Samples giving plaque counts that are greater than 2
plaques per cm2 should be diluted and replated.

5.2.6 Virus Plaque Confirmation Procedure

The presence of virus in plaques must be confirmed
for all plaques obtained from sludge samples. Where more
than ten plaques are observed, it is allowable to confirm at
least ten well-separated plaques per sample or 10% ofthe
plaques in a sample, whichever is greater. Flasks may be
discarded after samples are taken for plaque confirmation.

(a)	Apparatus, Materials and Reagents

(a. 1) Pasteur pipettes, disposable, cotton plugged — 229
mm (9 inches) tube length and rubber bulb — 1 mL capa-
city.

Flame each pipette gently about 2 cm from end ofthe
tip until the tip bends to an approximate angle of 45°. Place
the pipettes into a 4 liter beaker covered with aluminum foil
and sterilize by autoclaving or by dry heat.

(a.2) 16 x 150 mm cell culture tubes containing BGM
cells.

See section 6.6 for the preparation of cell culture

tubes.

(a.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).

(a.4) Freezer vial, screw-capped (with rubber insert) or
cryogenic vial — 0.5-1 dram capacity.

(b)	Procedure for obtaining viruses from plaque.

In addition to plaques from sludge samples, perform
the procedure on at least three negative regions of negative
process control flasks and at least three plaques from pos-
itive control flasks.

(b.1) 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.2) 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 ofthe pipette. Gently force the tip of
the pipette through the agartothe surface ofthe vessel, and
scrape some ofthe cells from the edge ofthe plaque.

Repeatedly scratch the surface and use gentle suction
to insure that virus-cell-agar plug enters the pipette.

(b.3) Remove the pipette from the plaque bottle and tightly
replace the cap or stopper.

(c)	Procedure for inoculating cell cultures with agar
plugs from negative control samples and from plaques.

(c.1) Prepare plaque conformation maintenance medium
by adding 5 mL of serum and 5 mL of dHzO per 90 mL of
wash medium (section 5.2.2b) on day samples are to be
tested.

(c.2) Pour the spent medium from cell culture tubes and
discard the medium. Replace the discarded medium with 2

F.9

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mL of the plaque conformation 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 medi-
um to 36.5 ± 1 °C before placing on cell monolayer.

To prevent disturbing cells with the force of the liquid
against the cell monolayer, add the maintenance medium to
the side of 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.

(c.3) Remove the cap from a cell culture tube and place
the tip of a Pasteur pipette containing the agar plug from
section 5.2.6b.3 into the maintenance medium in the cell
culture tube. Force the 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 pro-
cedure and to avoid scratching the cell sheet with the
pipette.

Squeeze bulb repeatedly to wash contents of pipette
into the maintenance medium.

(c.4) Place the cell culture tubes in the drum used with the
tissue culture roller apparatus. 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 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 enteric virus infections. How-
ever, uninfected cells round up during mitosis and a sample
should not be considered positive unless there are signifi-
cant clusters of rounded-up cells over and beyond what is
observed in the uninfected controls. If there is any doubt
about the presence of CPE or if CPE appears late (i.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.

If tubes receiving agar plugs from negative controls
develop CPE or tubes receiving agar plugs from positive
controls fail to develop CPE, stop all assays until the source
of the failure is identified and corrected.

Tubes developing CPE may be stored in a -70°C
freezer for additional optional tests (e.g., the Lim Benyesh-
Melnick identification procedure 5

(c.5) Determine the fraction of confirmed plaques (C) for
each sludge sample tested. Calculate "C" by dividing the
number of tubes inoculated with agar plugs from plaques

5For more information see EPA/600/4-84/013(R12), May 1988 Revision

that developed CPE by the total number of tubes inoculated
(i.e., if CPE was obtained from 17 of 20 plaques, C = 0.85).

5.2.7	Calculation of virus titer.

If more than one composite sample was assayed,
average the titer of all composite samples and report the
average titer and the standard deviation for each lot of
sludge tested.

(a)	If the entire remaining portion of a test sample was
inoculated onto BGM cultures as described in section
5.2.3e.5, calculate the virus titer (V) in PFU per 4 g of total
dry solids according to the formula:

V = 0.8 x = P x = C

where P is the total number of plaques in all test vessels for
that sample and C equals the fraction of confirmed plaques.

(b)lf	the sample was diluted due to high virus levels
(e.g., when the virus density of the input to a process is be-
ing determined; see section 5.2.5c), calculate the virus titer
(V) in PFU per 4 g total dry solids with the formula:

P

F=0.8x= j x = DxSxC

where P is the total number of plaques in all test vessels for
dilution series, I is the volume (in mL) of the dilution inocu-
lated, D is reciprocal of the dilution made on the inoculum
before plating, S is the volume of the remaining portion of
the test sample (as recorded in section 5.2.3e.5) and C is
the fraction of confirmed plaques.

5.2.8	Calculate the percent of virus recovery (R) using the
formula:	p

where P is the total number of plaques on all test vessels
inoculated with the recovery control.

6. Cell Culture Preparation and
Maintenance6

6.1. Introduction

This section outlines procedures and media for cultur-
ing the Buffalo Green monkey (BGM) kidney 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 characteris-
tics of this line were described by Barron etal. (1970). Use
of BGM cells for recovering viruses from environmental
samples was described by Dahling et at. (1974). The media
and methods recommended 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 Biohazard Assessment Research Branch, National
Exposure Research Laboratory, U. S. Environmental Protec-
tion Agency, Cincinnati, Ohio, USA 45268. Although BGM

6Modified from EPA/600/4-84/013(R9), January 1987 Revision

F.10

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cells will not detect all enteric viruses that maybe present in
sludges, the use of this cell line alone is sufficient to meet
the requirements of 40 CFR Part 503.

6.2. Apparatus and Materials

6.2.1	Glassware, Pyrex (Corning Product No. 1395 or
equivalent).

Storage vessels must be equipped with airtight clo-
sures.

6.2.2	Autoclavable inner-braided tubing with metal
quick-connect connectors or with screw clamps for
connecting tubing to equipment to be used under
pressure.

Quick-connect connectors can be used only after
equipment has been properly adapted.

6.2.3	Positive pressure air, nitrogen or 5% C02
source equipped with pressure gauge.

Pressure sources from laboratory airlines and pumps
must be equipped with an oil filter. The source must not
deliver more pressure to the pressure vessel than is recom-
mended by manufacturer.

6.2.4	Dispensing pressure vessel — 5 or 20 liter
capacity (Millipore Corp. Product No. XX67 OOP 05
and XX67 OOP 20 or equivalent).

6.2.5	Disc filter holders — 142 mm or 293 mm diame-
ter (Millipore Corp. Product No. YY30 142 36 and
YY30 293 16 or equivalent).

Use only pressure type filter holders.

6.2.6	Sterilizing filter stacks — 0.22 pm pore size
(Millipore Corp. Product No. GSWP 142 50 and
GSWP 293 25 or equivalent). Fiberglass prefilters
(Millipore Corp. Product No. AP15 142 50 or AP15
293 25 and AP20 142 50 or AP20 293 25 or equiva-
lent).

Stack AP20 and AP15 prefilters and 0.22 /jm mem-
brane filter into a disc filter holder with AP20 prefilter on top
and 0.22 /im membrane filter on bottom.

Always disassemble the filter stack after use to check
the integrity of the 0.22 /im filter. Refilter any media filtered
with a damaged stack.

6.2.7	Positively-charged cartridge filter — 10 inch
(Zeta plus TSM, Cuno Product No. 45134-01-600P or
equivalent). Holder for cartridge filter with adaptorfor
10 inch cartridge (Millipore Corp. Product No. YY16
012 00 or equivalent).

6.2.8	Culture capsule filter (Gelman Sciences Prod-
uct No. 12140 or equivalent).

6.2.9	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.

6.2.10	Screw caps, black with rubber liners (Brock-
way Product No. 24-414 for 6 oz bottles7 or equiva-
lent).

Caps for larger culture bottles usually supplied with
bottles.

6.2.11	Roller apparatus (Bellco Product No. 7730 or
equivalent).

6.2.12	Incubator capable of maintaining the temper-
ature of cell cultures at 36.5 ± 1 °C.

6.2.13	Waterbath, equipped with circulating device
to assure even heating at 36.5 ± 1°C.

6.2.14	Light microscope, with conventional light
source, equipped with lenses to provide 40X, 100X,
and 400X total magnification.

6.2.15	Inverted light microscope equipped with lens-
es to provide 40X, 100X, and 400Xtotal magnification.

6.2.16	Cornwall syringe pipettors, 2, 5 and 10 mL
sizes (Curtin Matheson Scientific Product No. 221-
861, 221-879, and 221-887 or equivalent).

6.2.17	Brewer-type pipetting machine (Curtin Mathe-
son Scientific Product No. 138-107 or equivalent).

6.2.18	Phase contrast counting chamber (hemocy-
tometer) (Curtin Matheson Scientific Product No.
158-501 or equivalent).

6.2.19	Conical centrifuge tubes, sizes 50 mL and
250 mL.

6.2.20	Rack fortissue culture tubes (Bellco Product
No. 2028 or equivalent).

6.2.21	Bottles, aspirator-type with tubing outlet, size
2,000 mL.

Bottles for use with pipetting machine.

6.2.22	Storage vials, size 2 mL.

Vials must withstand temperatures to -70°C.

6.3. Media and Reagents

6.3.1 Sterile fetal calf, gammagobulin-free newborn
calf or iron-supplemented calf serum, certified free of

7Size is given in oz only when it is commercially designated in that unit.

F.11

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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 should be
heat-inactivated at 56°C for 30 min and stored at 4°C for
short term use.

6.3.2	Trypsin, 1:250 powder (Difco Laboratories
Product No. 0152-15-9 or equivalent) ortrypsin, 1:300
powder (BBL Microbiology Systems Product No.
12098 or equivalent).

6.3.3	Sodium (tetra) ethylenediamine tetraacetate
powder (EDTA), technical grade, (Fisher Scientific
Product No. S657-500 or equivalent).

6.3.4	Thioglycollate medium (Difco Laboratories
Product No. 0257-01-9 or equivalent).

6.3.5	Fungizone (amphotericin B, Sigma Chemical
Product No. A-9528 orequivalent), Penicillin G (Sigma
Chemical Product No. P-3032 orequivalent), dihydro-
streptomycin sulfate (ICN Biomedicals Product No.
100556 orequivalent), and tetracycline (ICN Biomed-
icals Product No. 103011 orequivalent).

Use antibiotics of at least tissue culture grade.

6.3.6	Eagle's minimum essential medium (MEM) with
Hanks' salts and L-glutamine, without sodium bicar-
bonate (GIBCO BRL Product No. 410-1200 or equiva-
lent).

6.3.7	Leibovitz's L-15 medium with L-glutamine
(GIBCO BRL Product No. 430-1300 orequivalent).

6.3.8	Trypan blue (Sigma Chemical Product No. T-
6146 or equivalent).

Note: This chemical is on the EPA list of proven or
suspected carcinogens.

6.3.9	Dimethyl sulfoxide (DMSO; Sigma Chemical
Product No. D-2650 or equivalent).

6.3.10	Mycoplasma testing kit (Irvine Scientific Prod-
uct No. T500-000 or equivalent).

6.4. Preparation of Cell Culture Media

6.4.1 General Principles

(a)	Equipment care — Carefully wash and sterilize
equipment used for preparing media before each use.

(b)	Disinfection ofworkarea — Thoroughly disinfect sur-
faces on which the medium preparation equipment is to be
placed. Many commercial disinfectants do not adequately
kill total culturable viruses. To ensure thorough disinfection,
disinfect all surfaces and spills with either a solution of 0.5%
(5 g per liter) iodine in 70% ethanol or 0.1% HOCI.

(c)	Aseptic technique — Use aseptic technique when
preparing and handling media or medium components.

(d)	Dispensing filter-sterilized media — To avoid post-
filtration contamination, dispense filter-sterilized media into
storage containers through clear glass filling bells in a micro-
biological laminar flow hood. If a hood is unavailable, use
an area restricted solely to cell culture manipulations.

(e)	Coding media —Assign a lot numberto 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.

(f)	Sterility test — Test each lot of medium and medium
components to confirm sterility as described in section 6.5
before the lot is used for cell culture.

(g)	Storage of media and medium components — Store
media and medium components in clear airtight containers
at 4°C or -20°C as appropriate.

(h)	Sterilization of NaHC03-containing solutions — Ster-
ilize media and other solutions that contain NaHC03 by
positive pressure filtration.

Negative pressure filtration of such solutions increases
the pH and reduces the buffering capacity.

6.4.2 Media Preparation Recipes

(a)	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 dissolu-
tion in dHzO 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.

(b)	Procedure for the preparation of EDTA-trypsin.

The procedure described is for the preparation of 10
liters of EDTA-trypsin reagent. It is used to dislodge cells
attached to the surface of culture bottles and flasks. This
reagent, when stored at4°C, retains its working strength for
at least four months. The amount of reagent prepared
should be based on projected usage over a four-month
period.

(b.1) Add 30 g of trypsin (1:250) or 25 g of trypsin (1:300)
and two liters of dHzO to a six liter flask containing a three
inch stir bar. Place the flask onto a magnetic stirrer and mix
the trypsin solution rapidly for a minimum of one hour.

Trypsin remains cloudy.

(b.2) Add four liters of dHzO and a three-inch stir bar into
20 liter clear plastic carboy. Place the carboy onto a mag-
netic stirrer and stir at a speed sufficient to develop a vortex
while adding the following chemicals: 80 g NaCI, 12.5 g

F.12

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EDTA, 50 g dextrose, 11.5 g Na2HP04 • 7HzO, 2.0 g KCI,
and 2.0 g KH2P04.

Each chemical does not have to be completely dis-
solved before adding the next one.

(b.3) Add four more liters of dHzO to carboy.

Continue mixing until all chemicals are completely dis-
solved.

(b.4) Add the two liters of trypsin from step 6.4.2b. 1 to the
prepared solution in step 6.4.2b.3 and mix for a minimum of
one hour. Adjust the pH of the EDTA-trypsin reagent to 7.5
-7.7.

(b.5) 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 preHlter (section 6.2.7) can be used in
line with the culture capsule sterilizing filter (section 6.2.8)
as an alternative to a filter stack (section 6.2.6).

(c)	Procedure forthe preparation ofMEM/L-15 medium.

The procedure described is for preparation of 10 liters
of MEM/L-15 medium.

(c. 1) Place a three inch stir bar and four liters of dHzO into
20 liter carboy.

(c.2) Place the carboy onto a magnetic stirrer. Stir at a
speed sufficient to develop a vortex and then add the con-
tents of a five liter packet of L-15 medium to the carboy.
Rinse the medium packet with three washes of 200 mL each
of dHzO and add the rinses to the carboy.

(c.3) Mix until the medium is evenly dispersed.

L-15 medium may appear cloudy as it need not be
totally dissolved before proceeding to step 6.4.2c. 4.

(c.4) Add three liters of dHzO to the carboy and the con-
tents of a five liter packet of MEM medium to the carboy.
Rinse the MEM medium packet with three washes of 200
mL each of dHzO and add the rinses to the carboy. Add 800
mL of dHzO and 7.5 g of NaHC03 and continue mixing for
an additional 60 min.

(c.5) Transfer the MEM/L-15 medium to a pressure can
and filter under positive pressure through a 0.22 |_im steriliz-
ing filter. Collect the medium in volumes appropriate forthe
culturing of BGM cells (e.g., 900 mL in a 1 liter bottle) and
store in tightly stoppered or capped containers at 4°C.

Medium may be stored for periods of up to two
months.

(d)	Procedure for preparation of trypan blue solution.

The procedure described is for the preparation of 100
mL of trypan blue solution. Ids used in the direct determina-
tion of the viable cell counts of the BGM stock cultures. /\s
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.

(d.1) Add 0.5 g of trypan blue to 100 mL of dHzO in a 250
mL flask. Swirl the flask until the trypan blue is completely
dissolved.

(d.2) Sterilize the solution by autoclaving at 121 °C for 15
minutes and store in a screw-capped container at room tem-
perature.

(e) Procedure for preparation of stock antibiotic solu-
tions.

If not purchased in sterile form, stock antibiotic solu-
tions must be filter-sterilized by the use of 0.22 ym mem-
brane 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.

(e.1) Preparation of penicillin-streptomycin stock solution.

The procedure described is for preparation often 10
mL aliquots of penicillin-streptomycin stock solution at
concentrations of 1,000,000 units of penicillin and 1,000,000
fjg of streptomycin per 10 mL unit. The antibiotic concentra-
tions listed in step 6.4.2e.1.1 may not correspond to the
concentrations obtained from other lots or from a different
source.

(e. 1.1) Add appropriate amounts of penicillin G and dihydro-
streptomycin sulfate to a 250 mL flask containing 100 mL of
dHzO. Mix the contents of the flasks on magnetic stirrer un-
til the antibiotics are dissolved.

For penicillin supplied at 1435 units per mg, add 7 g of
the antibiotic.

For streptomycin supplied at 740 mg per g, add 14 g
of the antibiotic.

(e.1.2) Sterilize the antibiotics by filtration through 0.22 |_im
membrane filters and dispense in 10 mL volumes into
screw-capped containers.

(e.2) Preparation of tetracycline stock solution. Add 1.25
g of tetracycline hydrochloride powder and 3.75 g of ascor-
bic acid to a 125 mL flask containing 50 mL of dHzO. Mix
the contents of the flask on a magnetic stirrer until the antibi-
otic is dissolved. Sterilize the antibiotic by filtration through
a 0.22 |_im membrane filter and dispense in 5 mL volumes
into screw-capped containers.

(e.3) Preparation of amphotericin B (fungizone) stock
solution. Add 0.125 g of amphotericin B to a 50 mL flask
containing 25 mL of ddHzO. Mix the contents of the flask on
a magnetic stirrer until the antibiotic is dissolved. Sterilize
the antibiotic by filtration through 0.22 |_im membrane filter
and dispense 2.5 mL volumes into screw-capped contain-
ers.

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6.5.	Procedure for Verifying Sterility of
Liquids

There 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 compo-
nents has been demonstrated. BGM cell lines should be
monitored every six months for mycoplasma contamination
according to test kit instructions. Cells that are contami-
nated should be discarded.

6.5.1	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 seven days to determine whether growth of contam-
inating organisms has occurred.

Vessels that contain thioglycollate medium must be
tightly sealed before and after medium is inoculated.

6.5.2	Visual Evaluation of Media for Microbial Contami-
nants. Incubate media at 36.5 ± 1°C for at least one 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.

6.6.	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.

6.6.1 Vessels and Media for Cell Growth

(a) The BGM cell line grows readily on the inside sur-
faces of glass or specially treated, tissue culture grade plas-
tic vessels. 16 to 32 oz (or equivalent growth area) flat-sid-
ed, 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-sid-
ed 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.

(b) 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 anti-
biotics (100 mL of serum, 1 mL of penicillin-streptomycin
stock, 0.5 mL of tetracycline stock and 0.2 mL of fungizone
stock per 900 mL of MEM/L-15). Prepare maintenance
medium by supplementing MEM/L-15 with antibiotics and
2% or 5% serum (20 or 50 mL of serum, antibiotics as
above for growth medium and 70 or 50 mL of dHzO, respec-
tively).

6.6.2 General Procedure for Cell Passage

Pass stock BGM cell cultures at approximately seven
day intervals using growth medium.

(a)	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.

(b)	Add to the cell cultures a volume of warm EDTA-
trypsin reagent equal to 40% of the volume of medium
replaced.

See Table 2 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.

(c)	Allowthe EDTA-trypsin reagentto 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 five min).

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 neces-
sary as prolonged contact can alter or damage the cells.

(d)	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.

(e)	Centrifuge cell suspension at 1,000 *g for 10 min to
pellet cells. Pour off and discard the supernatant.

Do not exceed this speed as cells maybe damaged or
destroyed.

F.14

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TABLE 2. Guide for Preparation of BGM Stock Cultures

.. .T	EDTA-Trypsin Media Volume Total No. Cells to

esse ype Volume (mL)1	(mL)2	Plate per Vessel

16 oz glass flat
bottles3

10

25

2.5 x

106

32 oz glass flat
bottles

20

50

5.0 x

106

75 cm2 plastic
flat flask

12

30

X
O
CO

106

150 cm2 plastic
flat flask

24

60

X

o

CD

106

690 cm2 glass
roller bottle

40

100

X
O

107

850 cm2 plastic
roller bottle

50

120

8.0 x

107

1The volume required to remove cells from vessels.

2Serum requirements: growth medium contains 10% serum; maintenance medium
contains 2-5% serum. Antibiotic requirements: penicillin-streptomycin stock solution,
1.0 mL/ liter; tetracycline stock solution, 0.5 mL/liter; fungizone stock solution, 0.2
mL/liter.

3Size is given in oz only when it is commercially designated in that unit.	

(f)	Suspend the pelleted cells in growth medium (see
section 6.6.1b) and perform a viable count on the cell
suspension according to procedures in section 6.7.

Resuspend pelleted cells in sufficient volumes of medi-
um 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 proce-
dure. The quantity of medium used for resuspending
pelleted cells varies from 50 to several hundred mL, de-
pending upon the volume of the individual laboratory's need
for cell cultures.

(g)	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 Brew-
er-type pipetting machine dispenser.

Calculate the dilution factor requirement using the cell
count established in section 6.7 and the cell and volume
parameters given in Table 2 for stock cultures and in Table
3 for virus assay cultures.

/\s 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 two hundred 25 cm2
cell culture flasks weekly from a low-level passage of the
line would require the preparation of six roller bottles (sur-
face area 690 cm2 each): two to prepare the six roller bottles
and four to prepare the 25 cm2 flasks.

(h)	Except during handling operations, maintain BGM
cells at 36.5 ± 1°C in airtight cell culture vessels.

6.6.3 Procedure for Changing Medium on Cultured Cells
— Cell monolayers normally become 95 to 100% confluent
three to four 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. Mainte-
nance medium with 5% serum should be used when

monolayers are not yet 95% to 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.

6.7. Procedure for Performing Viable Cell
Counts

With experience a fairly accurate cell concentration
can be made based on the volume of packed cells. How-
ever, viable cell counts should be performed periodically as
a quality control measure.

6.7.1	Add 0.5 mL of cell suspension (or diluted cell sus-
pension) to 0.5 mL 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 be-
tween 20 and 50. This range is equivalent to between 6.0
x 70s and 1.5 * 1Cf cells per mL o f 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.

6.7.2	Disperse cells by repeated pipetting.

Avoid introducing air bubbles into the suspension, be-
cause air bubbles may interfere with subsequent filling of the
hemocytometer chambers.

6.7.3	With a capillary pipette, carefully fill a hemocyto-
meter chamber on one side of a slip-covered hemo-
cytometer slide. Rest the slide on a flat surface for about
one min to allow the trypan blue to penetrate the cell
membranes of nonviable cells.

Do not under or over fill the chambers.

6.7.4	Under 100X total magnification, countthe 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 clear in color. Do not count cells
that are blue.

Table 3. Guide for Preparation of Virus Assay Cell Cultures

,, . t	Volume of Medium Final Cell Count per

Uocco Tuno	.	1

1 oz glass bottle2

4

9.0 x 105

25 cm2 plastic flask

10

3.5 x 106

6 oz glass bottle

15

5.6 x 106

75 cm2 plastic flask

30

1.0 x 107

16 mm x 150 mm
tubes

2

4.0 x 104

1Serum requirements: growth medium contains 10% serum; maintenance medium
contains 2-5% serum. Antibiotic requirements: penicillin-streptomycin stock solution,
1.0 mL/liter; tetracycline stock solution, 0.5 mL/liter; fungizone stock solution, 0.2
mL/liter.

2Size is given in oz only when it is commercially designated in that unit.

F.15

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6.7.5 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 4000,
and where necessary, multiplying the resulting product by
the reciprocal of the dilution.

6.8. Procedure for Preservation ofBGM Cell
Line

An adequate supply ofBGM 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.

6.8.1	Preparation of Cells for Storage

The procedure described is for the preparation of 100
cell culture vials. Cell concentration per mL must be at least
1 x 1CP.

Base the actual number of vials to be prepared on
usage of the line and the anticipated time interval require-
ment between cell culture start-up and full culture produc-
tion.

(a)	Prepare cell storage medium by adding 10 mL of
DMSO to 90 mL of growth medium (see section 6.6.1b).
Sterilize cell storage medium by passage through an 0.22
l_im sterilizing filter.

Collect sterilized medium in 250 mL flask containing a
stir bar.

(b)	Harvest BGM cells from cell culture vessels as
directed in section 6.6.2. Count the cells according to the
procedure in section 6.7 and resuspend them in the cell
storage medium at a concentration of 1 * 106 cells per mL.

(c)	Place the flask containing suspended cells on a
magnetic stirrer and slowly mix for 30 min. Dispense 1 mL
volumes of cell suspension into 2 mL vials.

6.8.2	Procedure for Freezing Cells

The freezing procedure requires slow cooling of the
cells with the optimum rate of-1°C per min. 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 recom-
mended by the manufacturers.

(a)	Place the vials in a rack and place the rack in refrig-
erator at 4°C for 30 min, in a -20°C freezer for 30 min, 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.

(b)	Rapidly transfer vials into boxes or other containers
for long-term storage.

To prevent substantial loss of cells during storage,
temperature of cells should be kept constant after-70°C has
been achieved.

6.8.3 Procedure for Thawing Cells

Cells must be thawed rapidly to decrease loss in cell
viability.

(a)	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% iodine in
70% ethanol.

(b)	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 3). Use two vials of
cells for 6 oz bottles and one vial for 25 cm2 flasks.

(c)	Incubate BGM cells at 36.5 ± 1°C. After 18 to 24 h
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 section
6.6.

7. Bibliography and Suggested Reading

ASTM. 1998. Standard Methods for the Examination of
Water and Wastewater (L.S. Clesceri, A.E. Greenberg
and A.D. Eaton, ed), 20th Edition. United Book Press,
Baltimore, MD.

Barron, A.L., C. Olshevsky, and M.M. Cohen. 1970. Charac-
teristics of the BGM line of cells from African green
monkey kidney. Archiv. Gesam. Virusforsch. 32: 389-392.

Berg, G., D. Berman, and R.S. Safferman. 1982. A Method
for concentrating viruses recovered from sewage sludges.
Can. J. Microbiol. 28:553-556.

Berg, G., R.S. Safferman, D.R. Dahling, D. Berman, and
C.J. Hurst. 1984. USEPAManualofMethodsforVirology.
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. 3:
283-291.

Brashear, D.A., and R.L. Ward. 1982. Comparison of
methods for recovering indigenous viruses from raw
wastewater sludge. Appl. Environ. Microbiol. 43:1413-
1418.

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. 51:790-812.

Dahling, D.R., G. Berg, and D. Berman. 1974. BGM, a
continuous cell line more sensitive than primary rhesus
and African green kidney cells forthe recovery of viruses
from water. Health Lab. Sci. 11:275-282.

Dahling, D. R., G. Sullivan, R. W. Freyberg and R. S.
Safferman. 1989. Factors affecting virus plaque confirma-
tion procedures. J. Virol. Meth. 24:111-122.

F.16

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Dahling, D. R., R. S. Safferman, and B. A. Wright. 1984.
Results of a survey of BGM cell culture practices. Envi-
ron. Internat. 10:309-313.

Dulbecco, R. 1952. Production of plaques in monolayer
tissue cultures by single particles of an animal virus. Proc.
Natl. Acad. Sci. U.S.A. 38:747-752.

Eagle, H. 1959. Amino acid metabolism in mammalian cell
cultures. Science. 130:432-437.

Hay, R. J. 1985. ATCC Quality Control Methods for Cell
Lines. American Type Culture Collection, Rockville, MD.

Hurst, C. J. 1987. Recovering viruses from sewage sludges
and from solids in water, pp. 25-51. In G. Berg (ed),
Methods for Recovering Viruses from the Environment.
CRC Press, Boca Raton, FL.

Katzenelson, E., B. Fattal, and T. Hostovesky. 1976.
Organic flocculation: an efficient second-step concentra-
tion method forthe detection of viruses in tap water. Appl.
Environ. Microbiol. 32:638-639.

Lennette, E. H. and N. J. Schmidt (ed.). 1979. Diagnostic
Procedures for Viral, Rickettsial and Chlamydial Infec-
tions, 5th ed. American Public Health Association, Inc.,
Washington, D.C.

Safferman, R. S., M. E. Rohrand T. Goyke. 1988. Assess-
ment of recovery efficiency of beef extract reagents for
concentrating viruses from municipal wastewater sludge
solids by the organic flocculation procedure. Appl. Envi-
ron. Microbiol. 54:309-316.

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.

Ward, R. L., and C. S. Ashley. 1976. Inactivation of polio-
virus in digested sludge. Appl. Environ. Microbiol.
31:921-930.

F.17

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18: APPENDIX G

The text in Appendix G has been taken from the previously published document "Control of Pathogens
and Vector Attraction in Sewage Sludge" (July 2003, EPA 625-R-92-013). Page numbers will be
inconsistent with the previous text.

G.l

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Test Method for Detecting, Enumerating, and
Determining the Viability of Ascaris Ova in Sludge

1.0 Scope

1.1	This test method describes the detection, enu-
meration, and determination of viability of Ascaris ova
in water, wastewater, sludge, and compost. These patho-
genic intestinal helminths occui in domestic animals and
humans. The environment may become contaminated
through direct deposit of human or animal feces or
through sewage and wastewater discharges to receiv-
ing waters. Ingestion of water containing infective As-
caris ova may cause disease.

1.2	This test method is for wastewater, sludge, and
compost. It is the user's responsibility to ensure the va-
lidity of this test method for untested matrices.

1.3	This standard does not purport to address all
ofthe safety problems, ifanv. associated with its use. It
is the responsibility of the user of this standard to es-
tablish appropriate safety and health practices and de-
termine the applicability of regulatory limitations prior
to use. For specific hazard statements, see section 9.

2.0 Referenced Documents

2.1 AMMStandards;

° D 1129 Terminology Relating to Water1
° D 1193 Specification for Reagent Water2
° D 2777 Practice for Determination of Precision
and Bias of Applicable Methods of committee
D-19 on Water3

3.0 Terminology

(Definitions and Descriptions of Terms must be ap-
proved by the Definitions Advisor.)

3.1 Definitions - For definitions of terms used in
this test method, refer to Terminology D 1129.

3.2 Descriptions of Terms Specific to This Stan-
dard:

3.2.1	The normal nematode life cycle consists of
the egg, 4 larval stages and an adult. The larvae are
similar in appearance to the adults; that is, they are typi-
cally worm-1 ike in appearance.

3.2.2	Molting (ecdysis) of the outer layer (cuticle)
takes place after each larval stage. Molting consists of
2 distinct processes, the deposition of the new cuticle
and the shedding ofthe old one or exsheathment. The
cuticle appears to be produced continuously, even
throughout adult life.

3.2.3	A molted cuticle that still encapsulates a larva
is called a sheath.

3.2.4	Ascaiid egg shells are commonly comprised
of layers. The outei tanned, bumpy layer is referred to
as the mammillated layer and is useful in identifying
Ascaris eggs. The mammillated layer is sometimes
absent. Eggs that do not possess the mammillated layer
are referred to as decorticated eggs.

3.2.5	A potentially infective Ascaris egg contains a
third stage larva4 encased in the sheaths of the first
and second larval stages.

4.0 Summary of Test Method

4.1 This method is used to concentrate pathogenic
Ascaris ova from wastewater, sludge, and compost.
Samples are processed by blending with buffered wa-
ter containing a surfactant. The blend is screened to
remove large particulates. The solids in the screened
portion are allowed to settle out and the supernatant is
decanted. The sediment is subjected to density gradi-
ent centrifugation using magnesium sulfate (specific
gravity 1.20). This flotation procedure yields a layer likely

1 Annual Book of ASTM Standards, Vol 11.01.
2Annual Book of ASTM Standards, Vol 11.01.
3Annual Book of ASTM Standards, Vol 11.01.

4P L. Geenen, J. Bresciani, J. Boes, A. Pedersen, L. Eriksen,
H P. Fagerholm, and P. Nansen (1999)The morphogenesis
of Ascaris suum to the infective third-stage larvae within
the egg, J. Parasitology 85(4) 616-622.

G.2

-------
to contain Ascaris and some other parasitic ova, if
present, in the sample. Small particulates are removed
by a second screening on a small mesh size screen.5
The resulting concentrate is incubated at 26EC until
control Ascaris eggs are fully embryonated. The con-
centrate is then microscopically examined for the cat-
egories of Ascaris ova on a Sedgwick-Rafter counting
chamber.

5.0 Significance and Use

5.1	This test method is useful for providing a quan-
titative indication ofthe level of Ascaris ova contamina-
tion of wastewater, sludge, and compost.

5.2	This test method will not identify the species of
Ascaris detected nor the host of origin.

5.3	This method may be useful in evaluating the
effectiveness of treatment.

6.0 Interferences

6.1 Freezing of samples will interfere with the buoy-
ant density of Ascaris ova and decrease the recovery
of ova.

7.0 Apparatus

7.1	A good light microscope equipped with
brightfield, and preferably with phase contrast and/or
differential contrast optics including objectives ranging
in power from 10X to 45X.

7.2	Sedgwick-Rafter cell.

7.3	Pyrex beakers, 2 L. Coat with organosilane.

7.4	Erlenmeyer flask, 500 mL, Coat with

organosilane.

7.4	A centrifuge that can sustain forces of at least
660 X G with the rotors listed below.

7.4.1	A swinging bucket rotorto hold 100 or250 ml
centrifuge glass or plastic conical bottles.

7.4.2	A swinging bucket rotorto hold 15 ml conical
glass or plastic centrifuge tubes.

7.5	Tylersieves.

7.5.1 20 or 50 mesh.

5This method is based on a protocol published by Bowman,
D.D., M D. Little, and R.S. Reimers (2003) Precision and
accuracy of an assay for detecting Ascaris eggs in various
biosolid matrices. Water Research 37(9):2063-2072.

7.5.2	400 mesh, stainless steel, 5 inch in diameter.

7.5.3	A large plastic funnel to support the sieve.
Coat with organosilane.

7.6	Teflon spatula.

7.7	Incubator set at 26EC.

7.8	Large test tube rack to accommodate 100 or
250 ml centrifuge bottles.

7.9	Small test tube rack to accommodate 15 ml
conical centrifuge tubes.

7.10	Centrifuge bottles, 100 or 250 ml. Coat with
organosilane.

7.11	Conical centrifuge tubes, 15 mL. Coat with

organosilane.

7.12	Pasteur pipettes. Coat with organosilane.

7.13	Vacuum aspiration apparatus.

7.13.1	Vacuum source.

7.13.2	Vacuum flask, 2 L or larger.

7.13.3	Stopper to fit vacuum flask, fitted with a glass

or metal tubing as a connector for 1/4 inch tygon tub-
ing,

7.14	Spray bottles (16 fl oz.) (2).

7.14.1	Label one "Water".

7.14.2	Label one "1%7X".

8.0 Reagents and Materials

8.1 Purity of Reagents — Reagent grade chemi-
cals shall be used in all tests. Unless otherwise indi-
cated, it is intended that all reagents shall conform to
the specifications of the Committee on Analytical Re-
agents of the American Chemical Society6. Other
grades may be used, provided it is first ascertained that
the reagent is of sufficiently high purity to permit its use
without lessening the accuracy of the determination.

'Reagent Chemicals. American Chemical Specifications.
American Chemical Society, Washington, D.C. For sugges-
tions on testing of Reagents not listed by the American
Chemical Society, see Analar Standards for Laboratory
Chemicals. BHD Ltd., Poole, Dorset, U.K. and the United
States Pharmacopeia and National Formulary. U.S. Phar-
maceutical Convention, Inc. (USPC).

G.3

-------
8.2	Purity of Water—Unless otherwise indicated,
references to water shall be understood to mean re-
agent water conforming to Specification D 1193, Type I

8.3	Preparation of Reagents — Prepare reagents
in accordance with Practice E200.

8.3.1	Phosphate-buffered water (1 L = 34 0 g
KH2P04, pH adjusted to 7.2 ± 0.5 with 1 N NaOH).

8.3.2	1 % (v/v) 7X ("ICN" laboratory detergent) (1 L
= 999 mL phosphate-buffered water, 1 ml 7X "ICN",
Adjust pH to 7,2 ± 0.1 with 1N NaOH),

8.3.3	Magnesium sulfate, sp, gr. 1,20. (1 L = 215.2
g MgSO, , check specific gravity with a hydrometer; ad-
just as necessary to reach 1.20).

8.3.4	Organosilane. For coating glassware. Coat
all glassware according to manufacturer's instructions.

8.3.5	Fresh Ascaris ova for positive control, puri-
fied from Ascaris infected pig fecal material.

9.0 Precautions

9.1 When handling Ascaris ova and biosolids, per-
sonal protective measures must be employed to pre-
vent infection. Prevention of infection in humans is a
matter of good personal hygiene. Wear a laboratory

coat at ail times in the laboratory. In addition, latex or
nit rile gloves and splash protection safety glasses should

always be worn in the laboratory. Mouth pipetting is
strictly forbidden. Contaminated pipettes are never laid
down on the bench top but are immediately placed in a
pipette discard container which has disinfectant in it.
Contaminated equipment is separated as it is used into
containers for disposable materials and containers for
re-cycling. After these containers which are always
autoclave pans, are full, they are autoclaved for 30 min-
utes at 121 EC and 15 pounds/in2. Contaminated glass-
ware is never washed until after it has been autoclaved.
Eating, drinking, and smoking in the laboratory is not
permitted. Likewise, refrigerators are not to be used
for storing lunches or other items for human consump-
tion. If infective Ascaris ova are ingested they may cause
disease.

10.0 Sampling

10.1	Collect 1 liter of compost, wastewater, or
sludge in accordance with Practice D 1066, Specifica-
tion D 1192, and Practices D 3370, as applicable.

10.2	Place the sample containers) on wet ice or

around chemical ice and ship back to the laboratory for
analysis within 24 hours of collection.

10.3	Store the samples in the laboratory refriger-
ated at 2 to 5EC. Do not freeze the samples during
transport or storage.

11.0 Preparation of Apparatus

11.1	Test the centrifuge with a tachometer to make
sure the revolution's per minute correlate with the speed
gauge.

11.2	Calibrate the incubator temperature with a
NIST traceable thermometer.

11.3	Microscope.

11.3.1	Clean the microscope optics.

11.3.2	Adjust the condenser on the microscope, so

Kohler illumination is established.

12.0 Procedure

12.1	The percentage moisture of the sample is de-
termined by analyzing a separate portion of the sample,
so the final calculation of ova per gram dry weight can
be determined. The concentration of ova in liquid sludge
samples may be expressed as ova per unit volume.

12.2	Initial preparation:

12.2.1	Dry or thick samples: Weigh about 300 g
(estimated dry weight) and place in about 500 ml water
in a beaker and let soak overnight at 4 - 10EC. Transfer
to blender and blend at high for one minute. Divide
sample into four beakers.

12.2.2	Liquid samples: Measure 1,000 ml or more
(estimated to contain at least 50 g dry solids) of liquid
sample. Place one half of sample in blender. Add about
200 mL water. Blend at high speed for one minute trans-
fer to a beaker. Repeat for other half of sample.

12.3	Pour the homogenized sample into a 1000

mL tall form beaker and using a wash bottle, thoroughly
rinse blender container into beaker. Add 1 % 7X to reach
900 ml final volume.

12.4	Allow sample to settle four hours or overnight
at 4 -10EC. Stir occasionally with a wooden applicator,
as needed to ensure that material floating on the sur-
face settles. Additional 1 % 7X may be added, and the
mixture stirred if necessary.

12.5	After settling, vacuum aspirate supernatant

to just above the layer of solids. Transfer sediment to
blender and add water to 500 ml, blend again for one
minute at high speed.

12.6	Transfer to beaker, rinsing blender and add
1% 7X to reach 900 ml. Allow to settle for two hours at
4 - 10EC, vacuum aspirate supernatant to just above
the layer of solids.

G.4

-------
12.7	Add 300 ml 1% 7X and stir for five minutes
on a magnetic stirrer.

12.8	Strain homogenized sample through a 20 or
50 mesh sieve placed in a funnel over a tall beaker.
Wash sample through sieve with a spray of 1 % 7X from
a spray bottle.

12.9	Add 1% 7X to 900 mL final volume and allow
to settle for two hours at 4 -10EC.

12.10	Vacuum aspirate supernatant to just above
layer of solids. Mix sediment and distribute equally to
50 mL graduated conical centrifuge tubes. Thoroughly
wash any sediment from beaker into tubes using water
from a wash bottle. Bring volume in tubes up to 50 ml
with water.

12.11	Centrifuge for 10 minutes at 1000 X G.
Vacuum aspirate supernatant from each tube down to
just above the level of sediment. (The packed sediment
in each tube should not exceed 5 mL. If it exceeds this
volume, add water and distribute the sediment evenly
among additional tubes, repeat centrifugation, and
vacuum aspirate supernatant.)

12.12	Add 10 to 15 mL of MgSO, solution (spe-
cific gravity 1.20) to each tube and mix for 15 to 20 sec-
onds on a vortex mixer. (Use capped tubes to avoid
splashing of mixture from the tube.)

12.13	Add additional MgSO, solution (specific
gravity 1.20) to each tube to bring volume to 50 mL.
Centrifuge for five to ten minutes at 800 to 1000 X g.
DO NOT USE BRAKE.

12.14	Allow the centrifuge to coast to a stop with-
out the brake. Pourthe top 25 to 35 mL of supernatant
from each tube through a 400 mesh sieve supported in
a funnel over a tall beaker.

12.15	Using a water spray bottle, wash excessive
flotation fluid and fine particles through sieve.

12.16	Rinse sediment collected on the sieve into
a 100 mL beaker by directing the stream of water from
the wash bottle onto the upper surface of the sieve.

12.17	After thoroughly washing the sediment from
the sieve, transfer the suspension to the required num-
ber of 15 mL centrifuge tubes, taking care to rinse the
beaker into the tubes. Usually one beaker makes one
tube.

12.18	Centrifuge the tubes forthree minutes at 800
X G, then discard the supernatant.

12.19	If more than one tube has been used for the
sample, transfer the sediment to a single tube, fill with
water, and repeat centrifugation.

12.20	Aspirate the supernatant above the solids.

12.21	Resuspend the solids in 4 mL 0.1 N H S04
and pour into a 20-mL polyethylene scintillation vial or
equivalent with loose caps.

12.22	Before incubating the vials, mark the liquid
level in each vial with a felt tip pen. Incubate the vials,
along with control vials containing Ascaiis ova mixed
with 4 mL0.1 N H .SO,, at26EC forthree to fourweeks.
Everyday orso, check the liquid level in each vial. Add
reagent grade water up to the initial liquid level line as
needed to compensate for evaporation. After 18 days,
suspend, by inversion and sample small aliquots of the
control cultures once every 2 - 3 days. When the ma-
jority of the control Ascaiis ova are fully embryonated,
samples are ready to be examined.

12.23	Examine the concentrates microscopically
using a Sedgwick-Rafter cell to enumerate the detected
ova. Classify the ova as either unembryonated, em-
bryonated to the first, second, or third larval stage. In
some embryonated Ascaris ova the larva may be ob-
served to move. See Figure 1 for examples of various
Ascaris egg categories.

13.0 Calculation

13.1	Calculate % total solids using the % mois-
ture result:

% Total solids = 100% - % moisture

13.2	Calculate catagories of ova/g dry weight in
the following manner:

Ova/g dry wt = (NO) x (CV) x (FV)

(SP) x (TS)

Where:

NO = no. ova

CV = chamber volume(= 1 mL)

FV = final volume in mL
SP = sample processed in mL org
TS = % total solids

14.0 Report

14.1 Report the results as the total number oMs-
caris ova, number of unembryonated Ascaris ova, num-
ber of 1st, 2nd, or 3rd stage larva; reported as number
of Ascaris ova and number of various larval Ascaris ova
perg dry weight.

15.0 Keywords

Ascaris, ova, embryonation, viability assay, helminth.

G.5

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Notice

The PEC was consulted in a recent (1998-1999) pi-
lot study by Lyonnaise des Eaux concerning the use of

a microscope in making helminth ova counts for differ-
ent types of sludge. Solids and debris present in the

sludge being viewed with the microscope were found to
impair ones ability to count. Dilution of raw sludge and
digested sludge, however, with phosphate-buffered
water priorto analyzing them significantly improved the
numberofova that could be counted. Rawsludges were
diluted by a factor of 20 and digested sludges by a fac-
tor of 5. QA/QC procedures were followed to validate
this procedure. The PEC should be consulted for more
details.

[revised May 15, 2003]

G.6

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Figure A1.1. Ascaris ovum: potentially non-fertile,
note bumpy mammilated outer layer.

Figure A1.3. Ascaris ovum: decorticated, unembryonated.
Note the outer mammilated layer is gone

Figure A1.4. Ascaris ovum: decorticated and embryonated.

Figure A1.2. Ascaris ovum: fertile, note the bumpy
outer mammilated layer.

G.7

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Figure A1.5. Ascaris ovum: decorticated, embryonated.

Figure A1.6. Ascaris ovum with second stage iarva;
note the first stage larval sheath at the anterior end of
the worm

G.8

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19: APPENDIX H

The text in Appendix H has been taken from the previously published document "Control of Pathogens
and Vector Attraction in Sewage Sludge" (July 2003, EPA 625-R-92-013). Page numbers will be
inconsistent with the previous text.

H.l

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The Biosolids Composting Process

Introduction

Composting is the biological decomposition of organic
matter under controlled aerobic conditions. The objectives
of composting are to reduce pathogens to below detect-
able levels, degrade volatile solids, and produce a usable
product. Pathogen reduction is a function of time and tem-
perature. Composted biosolids is one way to meet 40 CFR
Part 503 pathogen (and vector attraction) reduction require-
ments. Composted biosolids can meet either a "Process
to Significantly Reduce Pathogens" (PSRP/Class B) or a
"Process to Further Reduce Pathogens" (PFRP/Class A)
standard, depending upon the operating conditions main-
tained at the facility. Process and operational consider-
ations must be taken into account when a facility desires
to meet the pathogen and vector attraction requirements
of 40 CFR 503. The 40 CFR Part 503 regulations require
composted biosolids applied to the land to meet specific
pollutant limits, site restrictions, management practices,
and pathogen and vector attraction reduction processes,
depending upon whether they: 1) are applied to agricul-
tural land, forest, a public contact site, or a reclamation
site; 2) are sold or given away in a bag or other container;
or 3) are applied to a lawn or home garden. Discussions
provided here are presented in summary form; it is recom-
mended that the facility seek additional details in develop-
ing a compost operation.

Composting Process Description

The addition of a bulking agent to sewage sludge pro-
vides optimum conditions for the composting process,
which usually lasts 3 to 4 weeks. A bulking agent acts as a
source of carbon for the biological process, increases po-
rosity, and reduces the moisture level. The composting
process has several phases, including the active phase,
the curing phase, and the drying phase.

Active phase. During the active or stabilization phase,
the sewage sludge/bulking agent mix is aerated and the
sewage sludge is decomposed due to accelerated biologi-
cal activity. The biological process involved in composting
can raise the temperature up to 60°C or more. At these
high temperatures, all of the disease-causing pathogens
are destroyed. Windrow systems must meet this condition
by achieving 55°C for a minimum of 15 consecutive days
during which time the windrow is turned five times. The

critical requirement is that the material in the core of the
compost pile be maintained at the required temperatures
(55°C) for the required time (3 days). Therefore, the first
phase typically lasts 21 days. Aeration is accomplished in
one of two ways: 1) by mechanically turning the mixture
so that the sewage sludge is exposed to oxygen in the air;
or 2) by using blowers to either force or pull air through the
mixture.

Curing phase. After the active phase, the resulting ma-
terial is cured for an additional 30 days to 180 days. At this
time, additional decomposition, stabilization, pathogen
destruction, and degassing takes place. Composting is
considered complete when the temperature of the com-
post returns to ambient levels. Depending upon the extent
of biodegradation during the active phase and the ultimate
application of the finished product, the curing phase may
not be carried out as a separate process.

Drying phase. After curing, some operations add another
step called the drying phase which can vary from days to
months. This stage is necessary if the material is to be
screened to either recover the unused bulking agent for
recycling or for an additional finished product. If the prod-
uct is to be marketable, the final compost should be 50%
to 60% solids.

There are two main process configurations for the
composting process:

Unconfined composting. This process is conducted in
long piles (windrows) or in static piles. Operations using
unconfined composting methods may provide oxygen to
the compost by turning the piles by hand or machine or by
using air blowers which may be operated in either a posi-
tive (blowing) or negative (suction) mode. For windrows
without blower aeration, it is typical to turn the windrow
two or three times a week, using a front-end loader. Prop-
erly operating aerated static piles do not require turning.

Confined (in-vessel) composting. This process is car-
ried out within an enclosed container, which minimizes
odors and process time by providing better control over
the process variables. Although in-vessel composting has
been effective for small operations, typically these opera-
tions are proprietary and therefore will not be described
any further in this fact sheet.

H.2

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Operational Considerations

The key process variables for successful composting are
the moisture content and carbon to nitrogen (C:N) ratio of
the biosolids/bulking agent mixture, and temperature and
aeration of the compost pile. Other process parameters
such as volatile solids content, pH, mixing and the materi-
als used in the compost also affect the process.

Bioso/ids/Bu/king Agent Mixture Moisture Content. Mois-
ture control is an important factor for effective composting.
Water content must be controlled for effective stabiliza-
tion, pathogen inactivation, odor control and finished com-
post quality (Benedict, 1988). The optimum moisture con-
tent of the mix is between 40% and 60%. At less than 40%
water, the material is too fluid, has reduced porosity and
has the potential for producing septic conditions and odors;
above 60% solids, the lack of moisture may slow down the
rate of decomposition. Since typical dewatered sewage
sludge or biosolids are often in the range of 15% to 20%
solids for vacuum filtered sewage sludge or biosolids and
20% to 35% solids for belt press or filter pressed sewage
sludge or biosolids, the addition of drier materials (bulking
agents) is usually essential.

Biosolids/Bulking Agent Mixture Carbon to Nitrogen Ra-
tio. Microorganisms need carbon for growth and nitrogen
for protein synthesis. For efficient composting, the carbon
to nitrogen (C:N) ratio of the biosolids/bulking agent mix-
ture should be in the range of 25:1 to 35:1

Oxygen levels. For optimum aerobic biological activity,
air within the pile should have oxygen levels of between
5% and 15%. Lower levels of oxygen will create odors and
reduce the efficiency of the composting. Excessive aera-
tion will cool the pile, slow the composting process, and
will not provide the desired pathogen and vector attraction
reduction.

Conventional windrows obtain necessary oxygen through
the natural draft and ventilation induced from the hot, moist
air produced during active composting and from the peri-
odic windrow turning. Where blowers are used for aera-
tion, it is typical to provide at least one blower per pile.

Biosolids/Bulking Agent Mixture Volatile Solids Con tent.
The volatile solids content of the biosolids/bulking agent
mix should be greater than 50% for successful composting
(EPA, 1985). This parameter is an indicator of the energy
available for biological activity and therefore compostability.

Bioso/ids/Bu/king Agent Mixture pH. The pH of the
biosolids/bulking agent mix should be in the range of 6 to
9 for efficient composting (EPA, 1985). Higher pH mixtures
may result if lime stabilized biosolids are used. They can
be composted; however, it may take longer for the
composting process to achieve the temperatures needed
to reduce pathogens.

Biosolids and Bulking Agent Mixing. Uniform mixing is
necessary in order to assure that moisture concentration
is constant through the pile and that air can flow throughout.

Type of Biosolids. The type of biosolids used
may have an effect on the composting process.
Composting can be accomplished with unstabilized
biosolids, as well as anaerobically and aerobically digested
biosolids. Raw sludge has a greater potential to cause
odors because they have more energy available and will,
therefore, degrade more readily. This may cause the com-
post pile to achieve higher temperatures faster unless suf-
ficient oxygen is provided and may also cause odors (EPA,
1985).

Material for Bulking Agents. Materials such as wood
chips, sawdust and recycled compost are usually added
as "bulking agents" or "amendments" to the compost mix-
ture to provide an additional source of carbon and to con-
trol the moisture content of the mixture. Other common
bulking agents used by facilities around the country include
wood waste, leaves, brush, manure, grass, straw, and
paper (Goldstein, 1994). Because of their cost, wood chips
are often screened out from the matured compost, for re-
use. Although sawdust is frequently used for in-vessel
composting, coarser materials such as wood chips, wood
shavings, and ground-up wood are often preferred because
they permit better air penetration and are easier to remove.
Recycled compost is often used as a bulking agent in wind-
rows, especially if bulking agents must be purchased. How-
ever, its use is limited because the porosity decreases as
the recycle ages (EPA, 1989). The amount of biosolids
and bulking agent which must be combined to make a suc-
cessful compost is based on a mass balance process con-
sidering the moisture contents, C:N ratio, and volatile sol-
ids content.

Compost Pile Size. In general, assuming adequate aera-
tion, the larger the pile the better. A larger pile has less
surface area per cubic yard of contents and therefore re-
tains more of the heat that is generated and is less influ-
enced by ambient conditions. In addition, less cover and
base material (recycled compost, wood chips, etc.) is
needed as well as the overall land requirements for the
compost operation. Larger piles tend to retain moisture
longer. The surface area to volume ratio has an effect on
the temperature of the pile. Assuming other factors are
constant (e.g., moisture, composition, aeration), larger piles
(with their lower surface area to volume ratio), retain more
heat than smaller piles. Ambient temperatures have a sig-
nificant impact on composting operations (Benedict, 1988).

A typical aerated static pile for a large operation would
be triangularly shaped in cross section about 3 meters(m)
high by 4.5 to 7.5 m wide (15 to 25 feet) at the base by 12
to 15 m long (39 to 50 feet) (Haug, 1980). One survey
study indicates that extended aerated static pile (where
piles are formed on the side of older piles) heights were
typically 12 to 13 feet high. Minimum depths of base and
cover materials (recycled compost, wood chips, etc.) were
12 and 18 inches, respectively (Benedict, 1988).

In windrow composting, the compost mix is stacked in
long parallel rows. In cross section, windrows may range
from rectangular to trapezoidal to triangular, depending

H.3

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upon the material and the turning equipment. Atypical trap-
ezoidal windrow might be 1.2 m (4 feet) high by 4.0 m (13
feet) at its base and 1.0 m (3 feet) across the top (Haug,
1980).

Monitoring and Sampling of the Compost
Pile

Unless the entire composting mass is subject to the
pathogen reduction temperatures, organisms may survive
and repopulate the mass once the piles or windrows are
cooled. Therefore it is crucial that temperatures be attained
throughout the entire pile. For aerated static piles or in-
vessel systems using static procedures such as tunnels or
silos, temperature monitoring should represent points
throughout the pile, including areas which typically are the
coolest. In aerated static piles this is usually the toes of
the pile (Figure 1). Temperatures should be taken at many
locations and at various depths to be assured that the core
of the pile maintains the required temperature. Records of
the temperature, date, and time should be maintained and
reviewed on an ongoing basis. Microbial analysis should
at a minimum be taken in a matter to represent the entire
compost pile. Operational parameters such as moisture,
oxygen as well as the others should be monitored at a
frequency necessary to assure that the compost opera-
tion is operating within acceptable ranges.

For composting, vector attraction reduction (VAR) is
achieved through the degradation of volatile solids. The
extent to which the volatile solids are degraded is often
referred to as compost stability. Stabilization requires suf-
ficient time for the putrescible organic compounds and for
other potential food sources for vectors to decompose.
Under this vector attraction reduction option, the Part 503
requires that biosolids be maintained under aerobic condi-

tions for at least 14 days, during which time temperatures
are over 40°C(104°F), and the average temperature is
over 45°C (113°F) (503.33(b)(5). These criteria are based
on studies which have shown that most of the highly pu-
trescible compounds are decomposed during the first 14
days of composting and that significant stability is achieved
at mesophilic (<45°C ) temperatures.

Recommendations for Specific
Technologies

Aerated static pile - Aerated static piles should be cov-
ered with an insulation layer of sufficient thickness to en-
sure that temperatures throughout the pile, including the
pile surface, reach 55°C. It is recommended that the insu-
lation layer be at least 1 foot thick. Screened compost is a
more effective insulation than unscreened compost or wood
chips. Screened compost also provides more odor control
than the other two materials.

Air flow rate and the configuration of an aeration system
are other factors which affect temperature. Air flow must
be sufficient to supply oxygen to the pile, but excessive
aeration removes heat and moisture from the composting
material. The configuration of an aeration system is also
important. Aeration piping too close to pile edges may re-
sult in uneven temperatures in the pile and excessive cool-
ing at the pile toes. If holes in the perforated piping are too
large or not distributed properly, portions of the pile may
receive too much air and be too cool as a result.

Windrows - Compliance with the pathogen reduction
requirements for windrows depends on proper windrow size
and configuration. If windrows are too small, the high sur-
face area to volume ratio will result in excessive heat loss
from the pile sides. Turning must ensure that all material

A 1 foot thick insulation layer is recommended to ensure that the
entire pile reaches pathogen reduction temperatures.

/ \

Pile toes are usually the coolest part of an aerated static pile.

Figure 1a. Aerated static pile.

H.4

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Pile Core

Material turned into the pile core reaches pathogen reduction temperatures.
Operations must ensure that all material is turned into the core at some point
during composting and that core temperatures rise to 55 degrees after turning.

Figure 1b. Windrow.

in a windrow be introduced into the pile core and raised to
pathogen reduction temperatures. This is most easily
achieved with a windrow turning machine.

In-Vessel systems- It is difficult to provide guidance for
these systems as there are numerous types with varying
configurations. Two key factors that apply to all in-vessel
systems are aeration and available carbon. As with aer-
ated static piles, the air flow configuration and rate can
affect the distribution of aeration to different parts of a
composting mass and the temperature profile of a pile.
Many in-vessel systems use sawdust as an amendment.
This may not provide sufficient energy if the volatile solids
in the biosolids are low.

Requirements for Class A/Class B Compost

For class A biosolids, aerated static pile, conventional
windrow and in-vessel composting methods must meet the
PFRP requirements, including the following temperature/
time requirements:

•	Aerated static piles and in-vessel systems must be
maintained at a minimum operating temperature of
55°C (131 °F) for at least 3 days; and

•	Windrow piles must be maintained at a minimum op-
erating temperature of 55°C (131°F) for 15 days or
longer. The piles must be turned five times during this
period.

For class B biosolids, aerated static pile, conventional
windrow and in-vessel composting methods must meet the
PSRP requirements, including the following temperature/
time requirements:

•	The compost pile must be maintained at a minimum of
40°C for at least five days; and

•	During the five-day period, the temperature must rise
above 55°C for at least four hours to ensure pathogen
destruction. This is usually done near the end of the
active composting phase in order to prevent inactivat-
ing the organic destroying bacteria.

To meet 40 CFR Part 503 vector attraction reduction
requirements using the "aerobic process" alternative,
composting operations must ensure that the process lasts
for 14 days or longer at a temperature greater than 40°C.
In addition, the average temperature must be higher than
45°C.

Additional References

Benedict, Arthur et al., Composting Municipal Sludge: A
Technology Evaluation, Pollution Technology Review
No. 152, Noyes Data Corporation, Park Ridge, New
Jersey, 1988.

BioCycle, Managing Sludge by Composting, JG Press Inc.,
Emmaus, PA, 1984.

Goldstein, N. et al., "1994 Biocycle Biosolids Survey."
Biocycle: Journal of Composting and Recycling, De-
cember 1994.

Haug, Roger T., Compost Engineering, Principles and Prac-
tice, Ann Arbor Science Publishers, Ann Arbor, Michi-
gan, 1980.

Information Transfer Inc., 1977 National Conference on
Composting of Municipal Residues and Sludges, Au-
gust 23-25,1977, Information Transfer, Inc., Rockville,
Maryland, 1978.

Jensen, Ric, Research Encourages Biosolids Re-use, En-
vironmental Protection, December 1993.

The BioCycle Guide to In-Vessel Composting, JG Press
Inc., Emmaus, PA. 1986.

U.S. EPA, A Plain English Guide to the EPA Part 503
Biosolids Rule. Office of Wastewater Management.
EPA/832/R-93/003. September 1994.

U.S. EPA Guidance for NPDES Compliance Inspectors,
Evaluation of Sludge Treatment Processes. Office of
Wastewater Enforcement and Compliance, Office of
Water. November 1991.

H.5

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U.S. EPA, Summary Report, In-Vessel Composting of Mu-
nicipal Wastewater Sludge. EPA/625/8-89/016. Sep-
tember 1989.

U.S. EPA, Seminar Publication: Composting of Municipal Waste-
water Sludges. Center for Environmental Research Infor-

mation, Office of Research and Development. EPA/625/4-
85/014. August 1985.

U.S. EPA, Environmental Regulations and Technology, Use
and Disposal of Municipal Wastewater Sludge. EPA/
625/10-84/003.1984.

H.6

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20: APPENDIX I

The text in Appendix I has been taken from the previously published document "Control of Pathogens and
Vector Attraction in Sewage Sludge" (July 2003, EPA 625-R-92-013). Page numbers will be inconsistent
with the previous text.

1.1

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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON. D.C. 20460

JUN | 5 1993

OFFICE OF
WATER

MEMORANDUM

SUBJECT:

The Role of the Pathogen Equivalency Committee Under
the Part 503 Standards for the Use or Disposal of
Sewage Sludge

FROM:

Michael B. Cook, Direct
Office of Wastewater Enforc

TO:

PURPOSE

James A. Han Ion, Acting Director
Office of Science & Technology

Water Division Directors
Regions I - X

ce



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

Figure 11-2. Role of the PEC under Part 503.

I-2

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21: APPENDIX J

J.l

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UNITED ST A lbs I \\ IRONMl N I \l PKOI ECTION AGENCY

\\ \sni\cnov nx ^-ton

OFFICE OF WATER

A Exceptional Quality Treated Sewage Sludge

Digitally signed by DAVID

ROSS

Dale; 2020.11.05
18:56:07 -05'00*

TO:	Regional Administrators

Regions 1-10

The Environmental Protection Agency's (EPA) Biosolids Program has received several requests foi
clarification on Class A Exceptional Quality (HQ) treated sewage sludge land application requirements
The purpose of this memorandum is to provide EPA's interpretation of 40 C hR ), regarding whether certain land application requirements apply to entities, ^
including a treatment works or a soil blender, that derive material from Class A EQ sewage sludge,

HP A regulations set out treatment standards for different classes of sewage sludge and different ^
management and land application requirements, depending on the class of sewage sludge or material
derived from that sludge. Class A "Exceptional Quality" or "HQ" sludge is treated sewage sludge that
meets the pollutant concentrations in § 503.13(b)(3), the Class A pathogen requirements in * Mb. 3*1 a)
and one of the vector attraction reduction requirements in §§ 503.33(b)! 1) through (b)(8). As such, t lass
A EQ sewage sludge meets the most stringent pollutant, pathogen, and vector attraction reduction
requirements under EPA's regulations. Class A. and Class B sewage sludge meet less stringent
requirements than Class A EQ.

The Standards for the Use or Disposal of Sewage Sludge are set out in 40 C 1 R Part M)3. Subpart B (40
CFR § 503.10) provides requirements for land application of sewage sludge, including when these
requirements apply; and management practices, monitoring, recordkeeping and reporting requirements.
This memorandum clarifies the land application applicability provisions found at 40 CER §§ M)3.10(e),
(f). and (g). Section 503.10(e) provides that seven of the nine land application requirements apply when
Class A EQ sewage sludge is produced and then distributed or sold in a bag or other container. Section
503.10( f) provides that seven of the nine land application requirements apply when a Class A EQ
material is produced and then distributed or sold in a bag or other container. Section 503.10(g) provides

MEMORANDUM

SI'B.I EC T: Land Application Requirements for Class

FROM; David P. Ross	DAVID

Assistant Administrator	ROSS

' There arc similar provisions for bulk sewage sludge (us opposed to in a bag or other container! at 40 CFR §§ 503.10(1*), (c),
and Id); however. Class A F.Q sewage .sludge is rarely distributed as bulk material That said, th.s interpretation would
equally apply to those provisions if such a scenario arose.

-------
that none of the nine land application requirements apply when a material is derivedfrom Class A EQ
sewage sludge and then that material is distributed or sold in a bag or other container.

Under EPA's biosolids regulations (40 CFR §§ 503.10(e), (f), and (g)), any preparer of sewage sludge
(including a treatment works or a soil blender) that (1) produces Class A EQ sewage sludge, (2) derives
a material from that Class A EQ sewage sludge, and (3) sells or gives that material away in a bag or
other container is exempt from all land application requirements {i.e., it benefits from the exclusions
under 40 CFR § 503.10(g)), even if that preparer began the process with non-Class A EQ sewage sludge.
Note that such a preparer would remain subject to 40 CFR § 503.10(e) or § 503.10(f) for the initial Class
A EQ sewage sludge or material derived from non-Class A EQ sewage sludge {i.e., the preparer would
have to demonstrate that the initial sludge or material meets Class A EQ standards).

This interpretation is reasonable, fair, and protective of human health and the environment. It ensures
that any preparer, e.g., a treatment works or a soil blender, that derives a material from Class A EQ
sewage sludge is subject to the same regulatory requirements. This interpretation is reasonable because
it focuses on the quality of the sewage sludge and/or material derived from sewage sludge, rather than
on the actor who is managing or treating the material to ensure that Class A EQ quality is achieved. This
interpretation avoids creating inequities between treatment works and private contractors {e.g., soil
blenders) that are taking the same action - deriving a material from Class A EQ sewage sludge and then
selling or giving away that material in a bag or other container. This interpretation is also protective of
human health and the environment as such protections depend not on who is taking the actions but on
what actions are being taken - treating sewage sludge to Class A EQ standards before deriving a
material from it, and then selling or giving away that material in a bag or other container.

If you have any questions, please contact Elizabeth Resek at (202) 566-1228 or

Resek.Elizabeth@epa.eov.

cc: Water Management Division Directors, Regions 1-10

2

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Penalty for Private Use
$300

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