?B 39-10259-1
Resource Conservation and Recovery Act
Subtitle C - Hazardous Waste Management
Section 3001 and 25C.14-Infectious Waste
Background Document
(U.S.) Environmental Protection Agency
Washington, DC
15 Dec 78
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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272-101
REPORT DOCUMENTATION
PAGE
1. REPORT NO.
EPA/530—SW—S3—055
4. Title and Subtitle
BACK. D0CU. RCRA SUBTITLE C (HAZARDOUS WASTE I1GMT) SECTIONS 3001 !< 250.14
INFECTIOUS WASTE
5. Report Date
12/15/73
6.
7. Author(s)
OFFICE OF SOLID WASTE
8. Performing Organization fiept. No
9. Performing Organization Name and Address
U.S. EPA
OFFICE OF SOLID WASTE
401 K STREET, SW
WASHINGTON. DC 20460
10. Project/Task/Work Unit No.
11. Contract(C) or Grant(G) No.
(0
(5)
12, Sponsoring Organization Name and Address
13. Type of Report J: Period Covered
DRAFT B.D. 12/73
15. Supplementary Notes
14.
Pa63-10259U
16. Abstract (Limit: 200 words)
This document provides background information and support tor regulations which have been designed to identify and list
hazardous waste pursuant to Section 3001 of the Resource Conservation and Recovery Act of 1976. This document pressnrs
the Agency's rationale in determining the definition of infectious hazardous waste.
17. Document Analysis a. Descriptors
b. idsntifiers/Gpsn-Endsd Terms
c. C0SATI Field/Group
IS. Availability Statement
19. Security Class (This Report!
2!. Ho. of Pages
UNCLASSIFIED
J1 lo
RELEASE UNLIMITED
20. Security Class (This Page)
22. Price
UNCLASSIFIED
f^1
See ANSI-239.13)
OPT
tONAL FORM 272 (4-7
(Formerly NTIS-35)
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DRAFT
BACKGROUND DOCUMENT
RESOURCE CONSERVATION AND RECOVERY ACT
SUBTITLE C - HAZARDOUS WASTE MANAGEMENT
SECTION 3001 - IDENTIFICATION AND LISTING OP
HAZARDOUS WASTE
SECTION 250.14 - HAZARDOUS WASTE LISTS
INFECTIOUS WASTE
DECEMBER 15,
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF SOLID WASTE
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Draft Background Document
Hazardous Waste Identification and Listing
Infectious Waste
Page
3.1 Introduction 1
3.2 Solid Waste/Disease Relationships 2
3.3 Indicator Organisms 3
3.4 The Source Approach 4
3.5 The Current State Approach 6
3.6 Related Federal Regulations 22
3.7 Epidemiological Evidence 26
3.8 Sources of Infectious Waste 28
3.9 Definitions 29
3.10 Rationale for Regulation of Health Care
Facilities Waste—Hospitals and Veterinary
Hospitals 33
3.11 Rationale for Regulation of Laboratory Waste ^9
3^^ Rational for Regulation of Unstabilized
Sewage Treatment Plant Sludge ^2
3^3 Methods for Biological Examination of Solid
/ Waste 83
3.14 References
Appendix
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Draft Eackgrour.d Document
Hazardous Waste Identification and Listing
Infectious Waste
3.1 Introduction
The purpose of this chapter of the background document
is to present the Agency's rationale in determining the
definition of infectious hazardous waste.
To date it has been the policy of the Agency under
Section 3001 of the Act, to define chemical and physical
hazardous waste characteristics such as toxicity, flammability,
and corrosivity, in quantitative terms? i.e. criteria have
been chosen that best quantify each hazardous characteristic,
with certain hazard levels specified for each tested parameter ..
(e.g., flashpoint for flammability, pH for corrosivity). For
enforcement purpose.s, this method of quantitatively defining
a hazardous waste is most desirable. It would follow then,
that a similar type of definition for "infectious characteristics"
would be the most useful one from a regulatory point of view.
Unfortunately, such quantification of infectious
characteristics is not possible, as will be discussed in
this document. Instead of specifying a certain number of
infectious agents allowed to be present in a waste, the
Agency has chosen to define infectious waste by specifying
the sources w bmxe disease microorganisms may occur. After
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clinical response in a host; yet for other disease agents it
is known that hundreds or even thousands of organisms are
necessary. Therefore setting a safe number of organisms for
solid waste would involve specifying a safe level for each
disease agent and providing a means to analyze for each one.
Unfortunately, dose levels for all disease agents are not known
at present and methods of environmental sampling and analysis
for many disease agents have not been developed.
3 .3 Indicator Organisms
Several EPA contacts have suggested the use of indicator
organisms such as Salmonella spp., fecal coliforms, or
S. aureus as an index of overall (i.e. viral, bacterial,
fungal, parasitic) biological hazard of a waste. The problems
associated with the use of indicator organisms have been
recognized by EPA. For water standards, the Office of Water
program Operations originally suggested the use of fecal
coliform as an indicator organism to determine the effectiveness
of the chlorination process (40 CFF. 133) . This standard was
later deleted (FR July 26, 1976) (15, with EPA recognizing that
fecal coliform is "not an ideal indicator of pathogenic (sic)
contamination" but is "a practical indicator of relative disease
causing potential."
While mi obial concentration standards may be applicable
in the evaluation of the efficacy of wastewater treatment systems,
their applicability as absolute quality standards remains to be
demonstrated. A problem is that in some situations, the die-
-3-
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degrees of severity from accidental inoculation or
injection or other means of.cutaneous penetration but
which are contained by ordinary laboratory techniques.
Class 3
Agents involving special hazard or agents
derived from outside the United States which require
a federal permit for importation unless they are
specified for higher classification. This class includes
pathogens which require special conditions for containment.
Class 4
Agents that require the most stringent conditions
for their containment because they are extremely hazardous
to laboratory personnel or may cause serious epidemic
disease. This class includes Class 3 agents from outside
the United States when they are employed in entomological
experiments or when other entomological experiments are
conducted in the same laboratory area.
Class 5
Foreign animal pathogens that are excluded from
the United States by law or whose entry is restricted
' by USDA administrative policy.
NOTE: It ha^"been pointed out that the current CDC list does not
include some agents of significance (e.g. Giardia, Ascaris,
Legionnaires bacterium) as well as it does include one
non-pathogen (Naegleria gruberi). The reader should keep
in mind that the list is periodically revised. The most
recently published list would be applicable.
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It is interesting to note that not cne of these definitions
attempts to quantify numbers of disease organisms that would
render a waste infectious and that it is these same States that
have promulgated criteria for physical/chemical characteristics
of hazardous waste on a quantitative basis similar to the
ones EPA is considering. The approach that the Agency is
taking to define infectious characteristics of waste, then,
and the deviance of this approach from that of defining
other characteristics of hazardous waste, is in line with
the thinking proposed by the most progressive State hazardous
waste management programs.
-7-
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TABLE ^
State Definitions Of Infectious Waste
State Agency
Legislative
Authority
(if any)
California Depatttoent
of Health
i
'O
I
fVssenobly Bill Ho.
1593: An Act to
Amend Section
25116. O*. 6.5.
Division 20, of
the Health and
Safety Code
Title of
Regulation/
Guideline/
Document
Definition(e)
"Infectious" means containing pathogenic
organians,or having baen exposed, or
reasonably being expected to have been
exposed, to contagious or infectious
disease. Articles which are "infectious"
include, but are not limited to, tte following:
(1) Wastes that contain pathologic speci-
mens, tissues, specimens or blood elements,
excreta or secretions fran humans or
animals at a hospital, medical clinic, re-
search center, veterinary institution, or
pathology laboratory.
(2) Surgical operating roan pathologic
specimens and articles attendant thereto
which may harbor or transmit pathogenic
organ!ems.
(3) Pathologic specimens and articles
attendant thereto fran outpatient areas
and emergency roans.
(4) Discarded equipment, instruments utensili.
and other articles which may harbor or tran
mit pathogenic organisms fran the roams of
patients with suspected or diagnosed com-
municable disease.
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TABLE _
State Definitions Of Infectious Waste
State Agency
Legislative
Auttority
(if any)
Title of
Regulation/
Guideline/
Document
Definition(s)
State of Marylanl
Department of
Health and Mental
Hygiene
Proposed Regula-
tions for Medical
Haste Disposal..
"Subcommittee Re-
port to the Task
Faroe on Medical
Waste Disposal -
Decanter 6, 1976"
Hie term medical wastes, eraexxtpassing
materials hitherto called "infectious"
"pathological", "ocntandnated", "special",
and "hazardous* shall be replaced with the
following new terms:
(1) llocpital Medical Wastes - shall mean all
solid waste generated within a hospital.
Blood ard blood products shall be included
in this solid waste category.
(2) Nursing Heme Medical Wastes - shall
be defined in categories, as follows;
(a) All disposable fomites from isola-
tion areas, all dressings, pledgets,
swabs, tongue depressors, plaster casti>,
body tissues, laboratory wastes, needles,
syringes, I.V. apparatus, and medicatiAjm;
(as permitted under Federal,
and local regulations).
State
(b)- Additional items which may be in-
cluded in the above category incluie
diapers and perineal pads.
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TAELK -
State Definitions Of Infectious Haste
State /^jc.Tcy
Legislative
AL'J.oricy
(it i.ny)
Title of
ftegolatiojv'
Guideline/
Docutimnt
Definition(s)
Minnesota Depart-
ment of Health,
Hftalth Facilities
Division.
Interpretive
Policies for the
Physical Plant:
Handling and Dis-
posal of Infect-
ious Haste
(Current DQH
Guidelines)
Infectious Waste;
(1) Hazardous Infectious Waste (same
as above). ¦
(2) General Infectious Waste (contaminated)
(a) Bandages, dressing, casts, cachete
tubing, and the like, which have in
oontact with wounds, burns, or
surgical incisions, but are not sus-
pected or have been not medically
identified as being of a hazardous
infectious nature.
(b) Discarded hypodermic needles and
syringes, scalpel blades, and
similar materials, when suspected
or identified to be of a hazardous
infectious nature.
(c) Incinerator ashes fran infectious
waste.
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State Definitions Of Infectious Waste
State Agency
Ijx'islativo
Auuuorily
(if any)
Minnesota BolHxIien
Control Agency,
(CENT.)
t n
Proposed, but to
no longer be part
of the hazardous
waste regulations,
W-l
Title of
Regulation/
Guideline/
DocLjtient
Def initiori (s)
(2) Surgical and obstetrical wastes,
pathological specimens, and disposal fcmites
from surgical operating roam, outpatient
areas, emergency rooms and similar areas
where such wastes are generated.
(3) Equipment, instruments, utensils,
and fanites of a disposable nature fran
the roans of patients with suspected or
diagnosed corounicable disease, or from
the rooms of patients who by nature or
their disease are required to be isolated
by the State Board of Health.
(4) Hypodermic needles and syringes,
scalpel blades, suture needles and simiiar
materials.
(5) Mixtures of any of the wastes in (1)
through (5) and other wastes that have
been oollected within the same container.
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£M5[£ a.
State Definitions Of Infectious teste
State Agency
Legislative
Authority
(it anv)
Pe^insylvania D^*rt-
ment of £ftvlrcn»ent
al Resources
~4
I
Texas Department of
Health Resources
State of Washington
Department of
Eoology
Pennsylvania Solid
¦ Management Act {35
(35 PS6-Q01), < •
PL 241
Title of
I^egulation/
Guideline/
Doctsnent
Definition (s)
Hazardous Haste
Management Profilfe
Ccmments to ANPR
Washington Adnin-
istrative Code
(KM!) Hazardous
teste Regulation,
Chapter 173-302
mr
VYlk'
General Claasiflcation of Hazardous Wastes
(1) Pathogenic Materials
(a) biological soli da
(b) laboratory wastes
(c)„ infectious wastes
(2) Otlier Hazardous Solid Waste
(a) diseased animals
Hazardous biological waste should include all
pathological vaste from chemical biological
and contagious wards as well as animals dead
of unknown disease and unstabilized domestic
sewage.
teste containing etiologic agents are toxic
serous wastes. Etiologic agent means
sle microorganism or its toxin, which
causes ot may cause human disease, and is
limited to those agents listed in 42 CFR
72.25(c) of the regulations of the
Department of HEW.
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Table 2A.
Areas/Sourcas Identified as Sources of Infectious Wastes, By state
Abattoir
Animal Caifxunds
Veterinary Hospitals
Health Services
Hospital, "pathological waste"
Emergency Rooms
Isolation Beans
Laboratory
Outpatient Areas
Pathology Laboratory
Surgical Operating Room
Medical Clinics
Nursing Hares
Research Center
Sewage Sludge
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
-19-
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B (Cent.)
ltm-s Id-'.ntrilled, By Siats;
Biological SolIda
Incinerator Ash Fran
Infectious Waste
Diseased Animals
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Haemophilus ducreyi, H. influenzae.
Hereilea vaqinicola.
Klebsiella—-all species and all serotypes.
Leptospira interrogans—all serotypes.
Listeria—allspecies.
Mima polymorpha.
Moraxella—»all species,
Mycobacterium—all species.
Mycoplasma—all species.
Neisseria gonorrhoeae, N. meningitidis.
PaateureTla—all species
Paeudomonaa pseudomallei.
Salmonella-'-'all speciea and all serotypes.
Shigella~-all species and all serotypes.
Sphacro"phorus necrophorus.
S taphylococcu'a aureus.
Streptobacilfua monTTiformis.
Streptococcus^pyoqenea.
Treponema careteura, T. pallidum, and T.
pertenue.
Vibrio £etua, V. comma, including biotype
El Tor, ana v[7 parahemolytlcua.
Yeracnia (Faateurella) peatia.
FUNGAL AGENTS
r
Actinomycetea (including Nocardla species,
Actinomyces speciea and Aracknla propi-
onlca).
Blaatomycea dermatitidis.
Coccidioldea"issmitls 7
Crvptococcus neoformaha.
Hiatoplaama capaulatumV
Paracoccidioides braaillenaia.
VIRAL, RICKETTSIAL, AND CHLAMYDIAL
AGENTS
Adenoviruses—human—all types.
Arboviruses.
Coxlella burnetii.
Coxaackie A and 3 viruses—all typea.
Cytomegaloviruses .
Dengue virua.
Echoviruaea—all typea.
Encephalomyocarditis virua.
Hemorrhagic teve'r" agents, including Crimean
Hachupo viruses
jiyj* *5
aerinea.
hemmorrfiaglc fever. (Congo) , Junin, and
and others as yet un*
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tested. EPA would prefer to rely on such a list as a way to
identify sources that may contain these et.iologic agents.
The CDC "Classification of Etiologic Agents on the Basis of
Hazard," a more complete list which includes animal etiologic
agents, will be used for source-identification purposes. (See
Appendix VI of the regulation.)
EPA has previously defined infectious waste in "Guidelines
for Thermal Processing and Land Disposal of Solid Waste,"
FR, August 14, 1974.(6) The definition, which is reprinted below,
is felt to be unenforceable, as are most State definitions of
infectious waste. Items specified in this definition would
be included in the "sources," under the proposed approach.
Also, this definition ignores the sewage sludge problem.
"Infectious waste" means:
(1) Equipment, instruments,
utensils, and fomites of a
disposable nature from the rooms
of patients who are suspected to
have or have been diagnosed as
having a communicable disease and
must, therefore, be isolated as
required by public health agencies;
(2) laboratory wastes such as
pathological specimens (e.g., all
tissues, specimens of blood elements,
excreta, and secretions obtained
from patients or laboratory animals)
and disposable fomites (any sub-
stance that may harbor or transmit
pathogenic organisms) attendant
thereto; (3) surgical operating
room pathologic specimens and dis-
posable fomites attendant thereto
and similar disposable materials
from outpatient areas and emergency
rooms.
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3.9 Definitions (8, 9, 10)
For clarification the later discussions, the following
definitions are provided:
ANIMAL WASTE - Waste generated from animal care or use;
including bedding, egestion, excretions, secretions, tissue,
remains, and any inedible by-products of animal processing for
food and fiber-production,
AUTOCLAVE - An apparatus for effecting sterilization by
steam under pressure. It is fitted with a gauge and a mechanical
system which automatically regulates the pressure and the
temperature to which the contents are subjected.
BACTERIA - Any of numerous unicellular microorganisms of
the class Schizomycetes, occuring in a wide variety of forms,
existing either as free-living organisms or as parasites, and
having a wide range of biochemical, sometimes pathogenic, properties.
ENTERIC - of or within the intestine.
ETIOLCGIC AGENT - A viable microorganism or its toxin which
causes, or may cause human disease. In the case of DOT Regulations,
etiologic agents axs (or are suspected to be) in relatively small
concentrated samples which are shipped to special laboratories for
.identificationJ
r
FOMITE - An inanimate object such as an article of clothing,
a dish, a toy, or a book, that is not itself corrupted but
is able to harbor pathogenic organisms which may by that means be
transmitted to others.
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PROTOZOAN - Any of the single-celled, usually microscopic
organisms of the phylum or subkingdor. Protozoa, which includes
the most primitive forms of animal life.
RICKETTSIA - Any of various microorganisms of the genus
Rickettsia, carried as parasites by many ticks, fleas, and lice.
Transmitted to man, they cause diseases such as typhus, scrub
typhus, and Rocky Mountain spotted fever.
SOLID WASTE - Any garbage, refuse, sludge from a waste
treatment plant, water supply treatment plant, or air pollution
control facility and other discarded material, including
solid, liquid, semisolid, or contained gaseous material result-
ing from industrial, commercial, mining, and agricultural
operations, and from community activities, not including solid
or dissolved material in domestic sewage, or solid or dissolved
material in domestic sewage, or solid or dissolved materials in
irrigation return flows or industrial discharges which are point
sources subject to permits under section 402 of the Federal Water
Pollution Control Act, as amended (86 Stat. 880) , or source,
special nuclear, or byproduct material as defined by the Atomic
Energy Act of 1954, as amended (68 Stat. 923).
SEWAGE Sludge - The residue resulting from wastewater
treatment.
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3.10 Rationale for Regulation of Health Care Facilities Waste
The nature of waste generated by health care facilities
is of concern to EPA due to a certain amount of potentially disease-
contaminated materials found in the waste that are not normally
found in other institutional solid wastes. Some studies have
stated that the type and numbers of bacteria and viruses found
in health-care solid waste are little different from that
found in wastes generated from dwelling units, offices,
factories and other institutions. Other researchers have
given a completely opposite view and stated that health care
facility wastes may be potentially dangerous to the environment
due to their infectious content. (11)
Both hospitals and veterinary hospitals (for more specific
breakdown by Standard Industrial Classification Code see §250.14
(b) of the regulations) are health care facilities that are
considered to be generators of infectious waste for purposes of
the regulation. EPA realizes that there are different problems
associated with the infectious wastes from the treatment of
people vs. animals and by no means does the Agency intend to
imply that these two types of health care facilities generate
the same types and amounts of waste or should treat or dispose
of their wastes by the same methods. A discussion of each
type-of health care facility and sources of waste associated
with them arelgiven below.
Hospitals
Theoretically, the difference between the biological
hazard of waste- generated in hospitsls, with their population
of "sick" people, and the waste generated by dwelling units
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incomplete at the time. It is these areas that infection
potential of ir.ost waste is unknown. So, at some point,
there is a reasonable possibility that infectious wastes can
be intermixed with other wastes.
Three surveys have been made which cover quite extensively
hospital practices with regard to waste collection and disposal
(Iglar and Bond, 1971? (13) Surchinal and Wallace, 1971;(14)
Esco/Greenleaf, 1972 (IS)}. The main interest, however, has been
in evaluating the overall waste collection and disposal
systems, with infectious wastes being considered as only one
aspect of the overall situation. This section is concerned
with discussing the infectious wastes which are identified in
the literature.
The composition of infectious wastes is well known.
They include items from surgery such as dressings, contaminated
disposable items, drapes, and human tissue (amputated limbs,
tissues, organs, placentas) ,* items from pathology and the
laboratory such as tissues, chemicals, bacteriological cultures,
urine, blood, and feces? animal remains and biological specimens;
and general infected material from the wards such as gauze
dressings and bandages, swabs, plaster casta, sputum cups,
paper tissues soaked with nose and throat secretions, and
wound drainage.
Some au ors distinguish between "pathological" wastes
and "hazardous" or "infectious" wastes (Litsky, et al., 1972). (16)
They call "pathological" materials those from surgery, labora-
tories, etc., and "hazardous" waste everything else—everything
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those traditionally considered to be sources cf infectious
waste, but also ward areas, doctors' offices, outpatient
clinics, and treatment rooms. Infectious waste averaged 43 percent
of the total waste in the hospitals studied, and the general patient
care areas generated almost three quarters of this infectious
waste.
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A survey in California (Anon, 1972b) (18) concluded that
it was possible to safely separate and collect infectious waste
within a hospital, but this does result in increased costs
of waste handling. With an average total waste per patient day
of 1C.25 lbs., the average infectious waste measured was
only 0.38 lbs.
Investigations by Bond and Michaelson (1964)(19) on the
effects of waste handling upon air and surface contamination
give some indication of what types of contamination to
expect. They found that soiled laundry handling had by far
the most significant influence on increased airborne bacteria.
Further investigations have been carried out on the solid
waste itself. Armstrong (1969) (20) looked at refuse chute3 with
respect to airborne bacteria. He found that placing the refuse
in bags reduces the number of airborne bacteria generated, and
that the possibility exists for the transmission of viable
organisms to other parts of the hospital by way of the refuse
chute.
Research at the University of West Virginia Medical Center
(Eurchinal and Wallace, 1971; (14) Wallace, et al., 1972; (21)
Smith, 1970j (22) Trigg, 1971 (23)) revealed that pathogenic
orga.nisaa can be present in hospital solid waste in significantly
Itions, and especially so if an organic substrate
is present. Coliform counts ranged from less than one per gram
of refuse at some stations to as high as 8.6 per gram. Fecal
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tamir.ated with viruses to established recovery tiroes and rates.
Vaccinia, Polio 1, Coxsaekie A-9, ar.d Influenza PR-8 were the
viral strains used for inoculation, Paper and cotton fabric
both held active viruses for long periods of time—'from 5 to
8 days in most cases. Virus titer decreased in most cases at a
steady rate with increasing time, implying that the agent
loses its viability upon incubation.
An air samplying program was carried out at the Los Angeles
County-USC Medical Center (Esco/Greenleaf, 19725.(15) Results are
given in Table 9 and substantiate the earlier findings of Eond
and Michaelson that laundry handling does generate considerably
greater aerosols than does trash handling.
Estimates of the total waste generated by hospitals vary
widely, ranging from about 10 Ibs/patient/day to as much as
40-50 lbs/patient/day (Litsky, et al., 1972? (16) Oviatt, 1969;(24)
Wallace, et al., 1972,* (21) Anon, 1972b (18) ? Small, 1971(25)).
Tables 10 and 11 give a breakdown of the types of wastes generated
and the disposal costs for seven California hospitals.. The great
variation is caused by the quantity of disposable items used.
The trend has been toward greater use of disposables because
of decreased danger of cross-infection and supposedly greater
economy. It has now become evident that "disposables" are
really merelyw"throw-aways"; and their actual disposal presents
a large problem. Even the cost advantage is open to question?
Table 12 indicates that disposables cost more to handle and
dispose of than reusables.
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Table 10
Breakdown of Daily Waste Production (Uxi/Day) By Types of Wastes (Esoo/Greenleaf, 1972)
Type of Waste
LMMJSC
Medical
Center
Long Beach
General
Hospital
Harbor
General
Hospital
Ranches Los
Amigos Hos-
pital
John
Wesley
Hospital
Olive
View
Hospital
Mir a
Lcma
Hospital
# of Beds
3000
428
715
1188
259
725
232
Sharps, Needles, Etc.
75
3
22
40
8
20
5
Path. & Surgical
1000
trace
156
4
115
6
trace
Soiled Linen
(Reusable)
45,000
3,740
13,600
16,320
2,900
5,630
1,120
Rubbish
16,200
540
6,569
2,760
717
1,722
362
Reusable Patient Items
trace
trace
traoe
trace
trace
traoe
trace
tton-ocnibustibles
1,500
75
465
725
80
250
80
Non-grindable (a) Garbage 1,800
150
660
875
160
475
110
Food Service Items
(Reusable)
9,000
1,400
2,400
4,200
800
2,500
600
Radiological
trace
—
trace
traoe
—
trace
—
Ash & Residue
trace
—
20
20
50
20
25
Animal Carcasses
25
—
220
20
10
23
—
Food Waste (Grindable)
2,600
330
950
1,100
210
1,860
150
Total Production
77,700
6,238
25,062
26,064
5,050
12,506
2,452
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Table U
Annual, Daily, and Unit Operatii. jsts (Esoo/Greenleaf, 1972)
UsC-USC Long Beach Harbor Rancho Los
John
Olive
Mira
Medical
»t-tiX-
General
Hospital
General
Hospital
Amigos
Hospital
Wesley
Hospital
View
Hospital
Lcma
Hospital
Quantity of Waste
Produced
Disposable*!
{Tonfl/DayJ^
11.60
0.55
4.53
2.77
0.68
2.19
0.37
Reusables
(Tons/day)
27.25
2.57
8.00
10.26
1.85
4.06
0.86
Total Waste
(Tons/Day)
38.85
3.
12.53
13.03
2.53
6.25
1.23
Cost of System Operation
Annual
$2,396,850
$223,600
$777,435
$656,340
$296,582
$750,585
$375,200
Daily
$
6,566
$ 612
$ 2,130
$ 1,798
$ 813
$ 2,056
$ 480
Average Daily Cost per Ton
Disposables
$
305
$ 325
$ 327
$ 364
$ 664
$ 516
$ 551
Reusables
110
168
82
77
195
229
322
Total Wastes
170
197
170
168
321
329
390
Average Daily Cost/Bed Patient [Calculated based cm total
nunber of patients not total nanber of beds!.
Disposables
$
1.76
$ 0.58
$ 2.73 $ 1.09
$ 2.65 $ 2.02
$ 1.42
Reusables
1.49
1.44
1.21
.85
2.13
1.65
1.91
Total Wastes
3.25
2.02
3.94
1.94
4.78
3.67
3.33
-------
Disposable items are found in all the areas of the
hospital, and have special application in burn therapy, aseptic
techniques, and isolation cases. Typical iteir.s are found in
Table 13. They are combinations of materials such as paper,
plastic, rayon, acrylic, cellulose, nylon, glass and metal.
The plastic content is much higher than the 2-3 percent found
in municipal solid waste; one study of infectious waste found
it to be 11.42 percent hard plastic and 7.09 percent soft
plastic (Anon, 1972b) . (18) Expenditures have risen from $3C
million in 1966 to $126 million in 1970, and may rise to an
estimated $900 million in 1978 (Fahlberg, 1973).(26) Further
estimates say that a hospital can double its waste output by
completely switching to disposable linen (Salkowski, 1970).(27)
Disposables add two problems to the waste treatment process;
first they increase the volume so that disposal systems are
taxed and second the plastic components are hard to degrade.
Also, it may be that some plasticizers are toxic. The John
Hopkins School of Hygiene and Public Health in Baltimore has
found that plasticizers in blood bags leach into the stored
blood and go on to lodge in lungs, spleen, liver, and
abdominal fat. Tests of embryonic heart cell cultures revealed
that the cells died when plastic tubing was substituted for
rubber (Anon,* 1971b) . (281
f
When a simple a change as supplying paper towels to
each patient's room was made at the Eaylor University Medical
-47-
-------
Center, it was found an additional wastebasket was then recuired
The maintenance cost from plugged toilets increased, and the
labor charge for emptying and washing wastebaskets increased by
3 0 percent, but the number of cloth towels used did not decrease
(Paul, 1964).(295 The pure bulk of the disposables presents the
problem that most authors comment on, but other hazards are also
present. Discarded needles and cutting edges remain a hazard to
collection personnel. Scavenging of the dumping areas for
useable items and play items for children show that spread
of infectious disease is a real hazard in the disposal of
disposables (Walter, 1964; (30) Mattson, 1974 (31)). Disease
organisms can also be introducted to a landfill in great
quantities via disposable linens and diapers (Ostertag and
Junghaus, 1965; (32) Peterson, 1974 (33)).
Some indication of the numbers of disposable hypodermic
needles used by individual hospitals can be obtained from
the literature. Michaelson and Vesley (1966)(34) found
from 14,000 to 833,000 used annually at various hospitals in
1966, and Eaker (1971)(35) found over 550,000 used annually
in 196S. There are proper ways to collect and destroy these
items, such as collecting them at the individual nursing
stations and returning them to central storage to be crushed
and. broken into fragments, then incinerated. They can also
be collected in special boxes and sent directly to the
incinerator, or collected at the nursing stations and sent
to central service to be autoclaved and melted into one
mass (Paul, 19641. (291 Seme hospitals have even tried
-49-
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Veterinary Hospitals
While veterinary hospitals have some of the waste disposal
problems which hospitals caring for people have, these problems
are mainly confined to disposing of dead animals, animal waste,
and waste generated during treatment of animals. Animal waste
includes waste generated from animal care or use, including
excretions, secretions, tissue, remains, and any inedible by-
products of animal processing for food and fiber production.
It has been pointed out to the Agency that the majority of
diseases that could be transmitted through improper disposal of
veterinary hospital waste are primarily ones that are transmitted
only from animal to animal. It is true that several hundred
diseases are transmitted from animal to animal, but more than
150 zoonotic diseases are transmitted between animals and man.
Decker and Steele (3 8a) report the human health problems
that are created by pathogenic zoonoses. Some of the most
significiant bacterial zoonoses are salmonellosis, staphlococcal
and streptococcal infectious, tetanus, tuberculosis, brucellosis,
leptospirosis, and colibacillosis. Animal wastes also play a
significant role in the distribution of fungal diseases by
providing nutrients for the survival and growth of fungi in
man's environment.
?
-51-
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soil associated with infected animals. Inhalation anthrax
results from inhalation of anthrax spores. Gastrointestinal
anthrax arises from ingestion of contaminated undercooked
meat. Anthrax spreads among herbivorous animals through
contaminated soil and feed and among omnivorous animals
through contaminated meat, bone meal or other feeds. Biting
flies and other insects are suspected of serving ag vectors.
Vultures have spread the organism from one area to another.
The spores of Bacillus anthracls, the infectious agent,
which resist environmental factors and disinfection, remain
viable in contaminated areas for many years after the source-
animal infection has terminated. (39)
Initial symptoms of inhalation anthrax are mild and
non-specific, resembling common upper respiratory infection;
acute symptoms of respiratory distress, fever and shock
follow in from 3 to 5 days, with death shortly thereafter.
Gastrointestinal anthrax is more difficult to recognize,
except that it tends to occur in explosive outbreaks? abdominal
distress is followed by fever, signs of septicemia, and death
in the typical case.
Untreated cutaneous anthrax has a fatality rate of from
5-20%, but with effective antibody therapy, few deaths
occur. (39) y.
SalmonellosiJ
Although this disease is discussed in the section on
sewage sludge, the important role that animals play in the
transmission of the disease shall be stressed here.
-53-
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Tuberculosis
Tuberculosis must still be considered as an important
disease related to animal wastes. While bovine tuberculosis
caused by Mycobacterium bovis has been effectively controlled
in this country, it is occasionaly found in some wild animals,
a3 well as in food animals and in pets,
Mycobacterium tuberculosis, the human type of tubercule
bacillus, is capable of infecting cattle swine, and household
pets.
Mycobacterium avium, the etiologic agent of tuberculosis
in gallinaceous birds, is capable of producing tuberculosis
in swine and of infecting cattle to such an extent that
reactions are produced in routine tuberculin testing of
cattle.
The bovine tubercle bacillus is transmitted to man
through respiratory secretions, feces, and milk. In those
few cases where infection of man with the bovine tubercle
bacillus is known, there usually is an occupational contact
with cattle. (38)
Brucellosis
Brucellosis is commonly an occupational disease of
those with close contact with cattle and swine and their
viscera and excreta. The disease in man and animals is
r
caused by any one of three 'species of Brucella.
-------
species host the Leptospira, including the domestic food-
producing species. Cattle and swine are the principal
domestic animals involved—leptospirosis occurs in epizootic
form in stables and feedlot herds. Dogs and rodents are
frequently infected.
Leptospirae are transmitted from the animal host to man
through a number of routes. Documented sources of human
infection are rice fields, swimming "holes", sewers, and a
number of occupations in which exposure to infected animals
is by direct contact. (38)
The disease in man shows a wide range of symptoms and
severity, depending on the species of leptospira involved,
exposure, and the health of the individual. It presents
symptoms similar to influenza, enteric viral infections,
infectious gastroenteritis, and a number of other diseases.
Fatality is low, but increases with advancing age and may
reach 20% or more in patients with jaundice and kidney
damage. (39)
Tularemia
The reservoir for Tularemia is normally wild animals,
but is occasionally found in sheep. Mode of transmission is
by inoculation of the skin, conjunctival sac or anal mucosa
with blood tissue while handling infected animals, as in
skinning, dressing, or performing necropsies; or by fluids
from infected flies, ticks,-or other animals, or through the
bite if arthropods including a species of deer fly. The
-57-
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3.11 Rationale for Regulation of Laboratory Waste
Data are generally not available that can be used to show
evidence of disease associated with laboratory waste. In a
recently published study at the University of Texas (Pike,
19751 (421, some waste/disease data can be extracted from the
50-year data base of published and unpublished cases of
laboratory-associated infections.
As shown in the reproduced table (Table 7), 4 6 cases of
laboratory-acquired infections related to the (waste) source
of discarded glassware are shown. Of these cases, 34 were
related to bacteria, 10 related to viruses, and 2 to rickettsiae.
Of the total number of reported laboratory-associated infections
studied, the 46 associated with discarded glassware represent
about 1% of the total.
The Center for Disease Control has determined that
certain microorganisms are of potential hazard to human
health and the environment, as published in the "Classification
of Etiologic Agents on the Basis of Hazard." Since it has
been determined by HEW that classes 2 through 5 are of
potential hazard, then any laboratory dealing with these
agents would be generating a potentially hazardous, infectious
waste. Given that roost hospitals and laboratories know
which organises are used in their work, the list is appended
f
-59-
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TABLE 7 - Distribution of Cases According to Proved or Pnobabla Source of Infection
Agenta
Sources
Bacteria
Viruses
Rickettsiae
Fungi
Chla-
mdiae
Parasites
Unspec-
ified
Totui
Accident —»t
378
174
45
33
14
38
21
703
Animal or ectoparasite
149
249
66
151
32
11
1
659
Clinical specimen
90
175
2
1
0
19
0
287
Discarded glassware
34
10
2
0
0
0
0
4 b
Human autopsy
56
9
4
0
0
1
5
75
Intentional Infection
14
1
0
0
0
4
0
19
Aerosol
101
92
217
88
22
2
0
522
Worked with the agent
381
213
100
62
43
28
0
827
Other
7
1
7
0
1
0
0
16
Unknown or not indicated
459
125
130
18
16
12
7
767
"total
1669
1049
573
353
128
115
34
3921
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In this bulletin general requirements for land application
of sludges are giver.. Reference is made to "Process Design
Manual for Sludge Treatment and Disposal" (EPA 625/1-74-006;
October 1974) which specifies in more detail the techniques for
sludge stabilization.
The bulk of the information presented in this section
of the background document is identical to that presented in
the background document for §257,4-5 (Land Criteria) to be
used for Section 4004 of RCRA. (45) Section 4004 regulations
will require sewage treatment plant sludge to be "stabilized"
to "reduce public health hazards."
Pathogenic organisms occuring in sewage sludge cover a
wide variety of bacteria, viruses and intestinal parasites.
Their individual presence, as well as their numbers, will
vary considerably from community to community depending upon
rates of disease in the contributing population. (46) Routes
of infection to humans and animals from sewage sludge may be
through direct contact with contaminated environments or
through the ingestion of contaminated food and water.
Bacteria
Among the bacteria that are commonly found in sewage
sludge, is the group referred to as 'the "enteric bacilli"
that natural!^ inhabit the gastronintestinal tract of humans.
In their virulence for humans, the enteric baccilli fall into
three general categories: pseudomonas species, salmonella
species, and shigella species.
-------
Shigella
The third category of enteric bacteria is the Shigella
genua. The shigella cause in humans a disabling disease
known as bacillary dysentery. This is an acute infection of
the large intestines, resulting i.n diarrhea, which, if
sufficiently severe, nay be accompanied, by bleeding from the
colon. All known species of the genus Shigella are pathogenic
for humans, with the following being the most common: S.
dyser.teriae, S. f lexneri, and S_;_ sonnei.
None of the enteric bacilli form spores. Spores are
resistant bodies produced by large number of bacterial
species that enable them to withstand unfavorable environmental
conditions such as heat, cold, desiccation and chemicals.
Since enteric bacilli are not spore formers, their survival
span outside of their normal environment (human intestinal
tract) is usually measured in days or months, compared to
years for spore forming bacteria. Most sludge stabilization
processes would create an unfavorable environment for enteric
bacilli to survive.
A pathogenic bacterium frequently found in sewage
sludge, although not an enteric organism, is the tubercle
bacillus Mycobacterium tuberculosis. This organism is
responsible f^r nearly all cases of pulmonary tuberculosis.
Tubercle bacilli are very hardy organisms, and can withstand
fairly extreme environmental conditions.
-6 5-
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Infectious hepatitis is an acute infectious disease
that causes fever, nausea, abdominal discomfort, followed by-
jaundice. It is caused by a resistant virus. The Hepatitis
virus is shed from the body through the feces, and fecal-
oral spread is probably the most common method of transmission.
Parasites
The third group of pathogenic organisms found in waste
water treatment sludges are the intestinal parasites. Those
parasites of concern to humans can be subdivided into two
categories: (.11 Protozoa, and (25 Helminths. Subgroups of
Protozoa group include amoebas, flagellates, and ciliates.
Subgroups of the Helminths include trematodes and nematodes.
Protozoa
At least five species of amoebae live in the intestinal
tract of humans, with Entamoeba histolytica being the only
proven pathogen. Infection with histolytica may produce
chronic diarrhea, amoebic hepatitis, abscess of the liver,
brain, lung, and ulceration of the skin. Amoebae have two
stages in their life cycles, a mobile form and a cyst form.
The cysts are infective upon passage from the body, and are
survive in a moist and cool environment. Giardia lamblia,
another protozoan, is also found in sewage sludge. Like the
amoeba, G. l^fcfclla is a parasite of the human intestinal
tract and is responsible for certain conditions such as
diarrhea or symptoms referable to the gall bladder.
Balantidium coli is the only ciliate human parasite
and is the largest of human protozoan parasites. It invades
_67-
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solium, I... saginatta, and Hvmenolepis nana. With the exception
of the species of Bymenolecls, infection with the common
human species results from eating raw or imperfectly cooked
beef, pork, or fish in which the larvae have developed.
Hymenolepis sp. on the other hand, need no intermediate
host. It is able to complete its entire life cycle in a
single host; thus, when eggs are ingested by man, the larvae
migrate into the lumen of the intestine.
Numerous studies report that pathogenic organisms
present in sludge are either killed or greatly reduced in
number when exposed to various stabilization methods used.
The specific number of ah organism necessary for the
establishment of the potential for disease is related to
various factors? etiologic agent, susceptibility of host
etc. However, there is evidence that with many pathogens
this dose may be rather high, in particular the enteric
pathogens. DuPont et. al (49) reported that approximately
105 Salmonella cells (including S typhi) are required to
cause a disease. This would tend to support the premise
that by reducing the number of pathogenic organisms in
sludge, the public health hazards associated with its use
would be greatly minimized.*
A revie^of the literature (7) has shown that there is a
paucity of epidemiological data linking disease transmission
of humans and animals directly to the landspreading of waste-
water treatment sludges. The data that do exist, indicate
-69-
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The stabilization process will reciuce the pathogen
population in sludge; the level of reduction will vary with
the process used and numerous ether variables, e.g., time,
temperature, pH etc. Since available epidemiological evidence
links disease transmission to the landspreading of unstabilized
sludge and not stabilized sludge, it ia evident that there
is a correlation between the concentration of pathogens in
the sludge and disease transmisssion.
Wastewater sludge stabilization is normally accomplished
by anaerobic and aerobic digestion, and lime treatment.
Lesser used methods include heat treatment, ponding and long
time storage, chlorination, and composting. The stabilization
of sludge by thermal irradiation is being addressed, but at
this time the process is still in the experimental state.
As previously mentioned, the extent to which pathogenic
organisms are reduced is related to the stabilization process
used as well as other variables. Not all stabilization
processes affect pathogenic organisms in the same manner,
therefore, some processes are more effective in reducing the
pathogen population than others. Also the levels of stabiliz-
ation within a particular process will vary as to their
effectiveness in reducing pathogenic organism numbers, e.g.,
anaerobic digestion of sludge for a two week period in the
-71-
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the eggs of A. lumfcricoides 0 to 4 5 percent.
Two groups (58,59) observed that there was 90 and 69
percent diminution of tubercle bacilli, while two others
(60,615 noted "survival" of M. tuberculosis after anaerobic
digestion.
McFinney et. al(621 found in their studies that approximately
93 percent of S. typhosa were removed after being exposed to
anaerobic digestion process for 20 days. Kenner (63) reported
that sludge treated by anaerobic digestion has been shown to
contain Salmonella and Pseudomonas organisms.
Cram (541 reported from his studies, that activated
sludge treatment does not affect the viability of E. histolytica
cysts or ascarid eggs. Aeration in the activated sludge
process for 5 months showed no effect on ascarid eggs except
a slow reduction in numbers (64), Kabler (53) reported that
studies indicate that activated sludge reduced S. typhosa
and strains of bacilli 91 to 99 percent.
if
-73-
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Enteric virus inactivation during the treatment of
wastewater by the activated sludge process has been reported
extensively in the literature. (G5-70) Carlson (71) et
al reported that after 6 months of aeration, polioviruses
were removed or inactivated to a point at which infectiousness
for mice was greatly reduced. Sproul (72) reported that
virus removal of 90 percent or mere has been obtained in a
number of studies with activated sludge process. Kelly et
al (731 reported that Coxsackie virus survived activated
sludge treatment.
Table 4
Removed of viruses by bench scale activated sltxige units
Coxsackie virus A9 Poliovirus 1
Mn. ia vim* Volatile Virus
solids Inactivated solids Inactivated
(mg/1) (Percent) (m?/l) (Percent)
1 fo5 $8.8 ifo 75
2 650 96.1 400 88
3 1,000 99.2 600 90
4. 1,100 99.1 600 91
5 1,500 97.4 1,200 92
6 1,500 99.4 1,200 91
7 4,000 94
Bacterial inhibition from caustic conditions has long been
known.(74) Studies have shown that Salmonella typhosa did
survive in concentrations in the range of pH 11.01-11.50
longer than two hours, while Shigella dysenterlae was destroyed
rapidly in al^J. pH range studies; pH 11.01-11.50 produced 100%
kill in 75 minutes. (75) However, the effectiveness of lime
treatment on parasitic ova and viruses has not been demonstrated.
-------
to sludge treatment are low pressure oxidation, heat drying
and pasteurization. During the low pressure oxidation (LPO)
process, the sludge temperature is elevated to between 3 50
and 400 F, pressure is raised to 180 to 210 psi, and the
retention time is between 20 and 30 minutes. The process
kills all pathogenic organisms due to the high temperature
achieved and the retention time. Over 26 U.S. cities are
currently using the LPO process.
Heat drying of sludge is presently being carried out in
a number of U.S. cities. However, the numbers are declining
because of cost of fuel necessary for the drying process,
and also because the market for heat dried sludge did not
develop as hoped. The temperature achieved during the heat
drying process kills moat bacteria.
Pasteurization is a process where the sludge is heated
to a specific temperature for a period of time that will
destroy pathogenic organisms. In most cases this is accomplished
by the use of steam. Currently, pasteurization is used only
in Europe.
While the technical literature presents some conflicting
data as to the degree that pathogenic organisms are reduced
by various sludge stabilization methods, it does generally
indicate that the stabilization process will reduce most
pathogenic organisms significantly. This reduction, in turn
minimizes the public health .risks associated with the
landspreading of stabilized sludges.
-77-
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in garden soil. Gudzhabidze (91} reported in the Soviet
Union that Ascaris ova survived 2-5 years in soil of irrigated
agriculture fields. The literature reviewed does not reveal
any studies in the United States where Ascaris ova survived
in sludge amended soils for more than one year.
Hess et al. (92) reported the survival of salmonellae on
grass contaminated with sludge for 40 to 58 weeks in a dry
atmosphere. McCarty and King (93) found that enteric pathogens
could survive and remain virulent for up to two months.
Rudolfs et. al. (94) concluded from field studies that the
survival of representatives of the Salmonella and Shigella
genera on tomato surfaces did not exceed seven days, even
when the organisms were applied with fecal organic material.
Ke attributed their short survival time to the lack of
resistant stages; thus making them more vulnerable to adverse
environmental conditions.
Martin (95), inoculating sterile virgin soils with E.
typhosa, found they died out rapidly, but in sterilized
contaminated soils growth occurred and the bacteria survived
for numerous months. Rudolfs (94) in his literature review,
found that the survival time of S. typhosa ranged from less
than 24 hours to more than two years in freezing moist
soil's, but generally less than 100 days.
Approximately 90 different enteric viruses have been
recovered from municipal sewage. However, there are few
-80-
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Fable 6
Survival times of Pathogenic MicroorganigBs in various media (8?)*
Organisms Medium Application* Survival time
Ascaris Ova
Soil
Soil
Plants and Fruits
Not stated
Sewage
AC
2-5 years
Up to 7 years
1 month
Endamoeba
Histolytica
cysts
Enteroviruses
Salmonella
Salmonella, other
than typhi
Shigella
f
Tubercle Raw 114
Soil
Tomatoes
Lettuce
Boots of bean
plants
Soil
Tomato & pea roots
Strawberries
Soil
Soil
Soil
Pea plant starts
Radish plant stems
Soil
Lettuce & endive
Soil
Soil
Lettuce
Radishes
Soil
Soil
Vegetables
Tomatoes
Soil
Potatoes
Carrots
Cabbage and
gooseberries
Streams
Harvested Fruits
Market tomatoes
Market apples
Tomatoes
Soil
Grass
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
Infected feces
Infected feces
Infected feces
AC
PC
PC
Sprinkled with
domestic sewage
Sprinkled with
danestic sewage
Sprinkled with
danestic sewage
Sprinkled with
danestic sewage
Not
PC
PC
PC
PC
PC
PC
stated
8 days
18-42 hours
18 hours
At least 4 days
12 days
4-6 days
6 hcurs
74 days
70 days
At least 4 days
14 days
4 days
Up to 20 days
1-3 days
2-110 days
Several months
18 days
53 days
74 days
15-70 days
2-7 weeks
Less than 7 days
40 days
40 days
10 days
5 days
30 minutes to 4
Minutes to 5 days
At least 2 days
At least 6 days
2-7 days
6 months
14-15 months
*Arti£ical Contamination
-79-
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published reports on the survival of viruses in soil, and
persistence on crops. Larkin et al, (96) described the
persistence of polioviruses for 14 to 30 days on lettuce and
radishes inoculated with sludge. According to Cliver (97)
the soil is generally not a very adverse environment for
viruses. Neither chemical nor biological inactivation
occurs very rapidly, but enteroviruses do lose infectiousness
as a function of time and temperature in the soil. Poliovirus
1, retained in sand from septic tank effluent, was inactivated
at a rate of 13 to 18 percent per cay at 20 to 25 C and at
1.1 percent per day at 6 C to 8 C. (97)
Rudolfs et al. (94) reported that unlike pathogenic
bacteria, the parasitic amoeba, Er.damoeba histolytica,
forms resistant cysts which enable the organism to survive
under adverse conditions. However, on the basis of laboratory
and field studies on the survival of Endamoeba histolytica
cysts, the cysts proved to be extremely sensitive to desiccation.
Rudolfs concluded from his studies that field-grown crops
contaminated with cysts of E^ histolytica are considered
safe in the temperate zone one week after contamination has
stopped and after two weeks in wetter tropical regions.
It has been shown in the general survey of the literature
(941 that certain parasite eggs, especially those of Ascarls,
are markedly?"resistant to external conditions. Yoshida (98)
found that mature eggs of luir.briocoidea were still viable
after five to six months under layers of soil in winter. He
-81-
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3.13 Methods for Biological Examination of Solid Waste
Bacteria
Mirdza L. Peterson of EPA has published "Methods for
Bacteriological Examination of Solid Waste and Waste- Effluents."
(104) After examining methods currently available for measuring
the bacteriological quality of solid waste, reliable methods
were established which are best suited to routinely measure,
under practical conditions, the bacteriological quality of
solid waste in and around waste processing areas. These methods
were not developed to be an all-inclusive battery of tests for
microorganisms in solid waste; rather, these methods test for
only a few of the possible microorganisms in the solid waste.
Three procedural lines of investigation were undertaken
in this effort: (15 to develop methods suitable for indicating
the sanitary quality of solid waste before and after processing'
or disposal; (2) to develop methods suitable for determining
the efficacy of operational procedures in removing or destroying
the microorganisms; and, (3) to develop methods suitable
for indicating the health hazard of solid waste in which
pathogenic species may be present in small numbers. Methods
presented in this publication are ones for determining:
total viable bacterial cell number, total coliforms, fecal
conforms, heat-resistant spores, and enteric pathogens,
especially Salmonella sp.
The determination of approximate total viable bacteria
multiplying at a temperature of 35 C may yield useful information
concerning the sanitary quality of a waste entering a processing
or a disposal site, and provide useful information in judging
-83-
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a long exposure time (1-1/2 to 2 hr!, even in an autoclave
(121 C) to be heated throughly so that the center reaches a
sporocidal temperature. Other reports (107) point out that
although internal air temperatures of municipal incinerators
usually range from 1200 to 1700 F (650 to 925 C) in continuous
operation, intermittent use, overcharging of the Incinerator,
and high moisture content of the waste may slow the process
and interfere with sterilization of the residue.
Fecal pollution of the environment by untreated and
improperly disposed waste may add enteric pathogenic bacteria
to a body of water or a water supply. The most common type
of pathogen which may be found in untreated waste is Salmonella.
The wide distribution of the many types of Salmonella in
many species of animals with which man has contact or may
use as food makes it difficult to prevent transmission to
man. (1085 Infections may occur through food, milk, or
water contaminated with infected feces or urine, or by the
actual ingestion of the infected animal tissues. (109) Salmonella
has been found in many water supplies (110), polluted waters
(111-113), raw municipal refuse and in incinerator residue (111-117)
General laboratory procedures, sample collection and
preparation procedures, and bacteriological examination
procedures fo* the organisms mentioned above can be found in
Appendix A-3,1.
Parasites
The FDA has recently prepared a methodology for Ascaris
determination in vegetable and sludge samples (118). The
-85-
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3.14
REFERENCES
1. U.S. Environmental Protection Agency. Water
Programs: Secondary Treatment Information.
Federal Register, 41(144); 30786-30789,
July 2?, lilt.
2. Cooper, R.C. and C.G. Golueke. Public Health
Aspects of On-Site Waste Treatment. Compost
Science, 18(31: 8-11.
3. U.S. Department of Health, Education, and Welfare,
Public Health Service, Center for Disease
Control of Eiosafety, Classification of
Etiologic Agents on the Basis of Hazard.
Atlanta, Georgia, July 1974. 4th edition 13p.
4. U.S. Department of Transportation, Materials
Transportation Bureau. Hazardous Materials
Regulations: Interim Publication. Federal
Register, 41(2295: 52086, November 26, 1976.
5. U.S. Department of Health, Education, and Welfare,
Public Health Service. Code of Federal Regulations,
42(72.25): 457-459, U.S. Government Printing
Office, 1976.
6. U.S. Environmental Protection Agency. Thermal
Processing and Land Disposal of Solid Waste:
Guidelines. Federal Register 39(158): 29328-29338,
August 14, 19 IT". .
7. Hanks, Thrift G. Solid Waste/Disease Relationships:
A Literature Survey. Public Health Service
Publication No. 999-UIH-6, Washington, U.S.
Government Printing Office. 1967. 179p.
8. Morris, William, ed. The American Heritage
Dictionary of the English Language. Boston,
Houghton Mifflin Company, 1976. 1550p.
9. Department of Health, Education, and Welfare,
National Institutes of Health. Recombinant DNA
Research Guidelines: Draft Environmental Impact
Statement, Federal Register, 41(176): 38426-
38483, September 9, 19^.
-87-
-------
21. Wallace, L,?.; Zaltzir.an, R. y Burchinal, J.C. 1972
Where solid waste comes from; where it should go.
Modern Hospitals, 118, (Feb.), 92-5.
22. Smith, R.J, 1970 Bacteriological Examination of
Institutional Solid Wastes. (M.S. Thesis) West
Virginia University.
23. Trigg, J.A. 1971 Microbial Examination of Hospital
Solid Wastes. (M.S. Thesis) West Virginia
University.
24. oviatt, V.R. 1969 How to dispose of disposables.
Med.-Surg. Rev. Second Quarter, 1969, p. 58.
25. Small, W.E. 1971 Solid waste: please burn, chop,
compact, or otherwise destroy this problem.
Modern Hospital, 117, (Sept.), 100-10.
26. Fahlberg, W.G. 1973 The hospital (disposable)
environment. In Phillips, G.B. and Miller, w.s.
ed. Industrial Sterilization. Duke University
Press, Durham, NC. p. 399-412.
27. Salkowski, M.D. 1970 Disposal of Single-Use Items
from Health Care Facilities? Report of the Second
National Conference, Sept. 23-24, 1970.
28. Anonymous 1971b Plastic leachate found harmful.
Journal of Environmental Health, 3£, (2), 196.
29. Paul, R.C. 1964 Crush, flatten, burn, or grind?
The not-so-simple matter of disposal. ¦ Hospitals,
J AHA,. 3J3, (1 Dec.), 99-101, 104-5
30. Walter, C.W. 1964 Disposables, now and tomorrow:
for the surgeon, many advantages, but still some
problems. Hospitals, JAHA, 38, (1 Dec.), 69, 70,
72.
31. Mattson, G. 1974 Handling potentially dangerous
throwawaya in Swedish hospitals. Solid Wastes
Management, 17, (2), 23, 46, 54.
32. - Ostertag. H. and Junghaus, W. 1965 Use and
elimination of disposable linen in hospitals
and convalescent homes. Stadtehygiene, 16_, (10) ,
213-8 (Ger,).
-89-
-------
43. U.S. Environmental Protection Agency, Municipal
Sludge Management: Environmental Factors; Technical
Bulletin. Pederal Register 22532-36, June 3, 1976.
42(2111: 57420-27, November 2, 1977.
44. U.S. Environmental Protection Agency, Office of
Technology Transfer, Process Design Manual for
Sludge Treatment and Disposal. EPA Publication
No. 625/1-74-006. Washington, U.S. EPA,
October 1974.
45. Office of Solid Waste, Background Document for
§4004, P.L. 94-580: §257.4-5, Land Criteria,
June 24, 1977 (Draft.)
46. Love, G.L., Tompkins E. and Galke, W.A. "Potential
Health Impact of Sludge Disposal on Land" Nat.
Conf. on Sludge Management and Disp. (1975)
47. Morbidity and Mortality Weekly Report, NCDC, PHS,
December 1976.
48. Malherbe, H.H.,-Cbolemly, M. (Quantitiative Studies
on Viral Survival in Sewage Purification Process
49. Dupont, H.L. and Hornick, R.B., Clinical Approach
to Infectious Diarrheas. Med., 52(1973), 265.
50. Sepp. E. The Use of Sewage for Irrigation. A
Literature Review. Bureau of Sanitary Engineering,
California State Department of Public Health, 1963.
51. Kreuz, A. Hygienic Evaluation of the Agricultural
Utilization of Sewage. Gesundheitsing. 76:206-
211, 1955.
52. Kroger, E. Detection of S. Barelly in Sewage Sludge
and Vegetables from an Irrigation Field after
an Epidemic. 8 Hyg. Infektkr. 139:202-207, 1954.
53. Kabler, P. Removal of pathogenic microorganisms
by sewage treatment processes. Sewage and
Industrial Wastes 31:1373, 1959.
54.' Cram, E.5., "The Effect of Various Treatment
Processes on the Survival of Helminth Ova
and Protozoan Cysts in Sewage." Sewage Works
Jour., 15, 6, 1119 (Nov. 1943).
-91-
-------
65. Clark, NA., et al., "Human Enteric Viruses in Water:
Source, Survival, and Removability." In Advances
in Water Pollution Research." Vol. 2, Pergamon
Press, London (1964).
66. England, B., et al., "Virological Assessment of-
Sewage Treatment at Santee, California." In
"Transmission of Viruses by the Water Route."
G. Berg (Ed.), Interscience Publishers, New York,
N.Y. (1967}.
67. Kelly, S.M., et al., "Removal of Enteroviruses from
Sewage by Activated Sludge." Jour. Water Poll.
Control Fed., 33, 1050 (1961).
68. Mack, W.N., et al., Entervorus Removed by Activated •
Sludge Treatment. Jour. Water Poll. Control Fed.
34, 1133 (19621
69. Lund, E., et al., "Occurrence of Enteric Viruses in
Wastewater after Activated Sludge Treatment."
Jour. Water Poll. Control Fed., 41, 169 (1969).
70. Clarke, N.A., et al., "Removal of Enteric Viruses
from Sewage by Activated Sludge Treatment."
Amer. Jour. Pub. Health, 51^1118 (1961).
71. Carlson, H.J., et al., "Effect of the Activated Sludge
Process of Sewage Treatment on Poliomyelitis Virus."
Amer. Jour. Pub. Health, 33, 1083 (1943).
72. Sproul, O.J. "Removal of. viruses ky Treatment
Processes" Inter. Conf. on Viruses in Water,
Mexico City, WHO-PAHO 1974.
73. Kelly, S.M., Clark, M.E., and Coleman, M.B.,
"Demonstration of Infectious Agents in Sewage."
Amer. Jour. Pub. Health, 45, 1438 (1955)
74. Morrison, S.M., Martin, K.L. and Humble, D.E.
"Lime Disinfection of Sewage Bacteria at Low
Temperature" EPA Contract No. 660/2-73-017.
75.-' Wattie, E., and C.W. Chambers. Relative Resistance
of CoMform Organisms and Certain Enteric
Pathogens to Excess-lime Treatment. J. Amer.
Water Works Asso. 35:709-720, 1943.
-93-
-------
88. Doran, J.W., Ellis, J.R., and McCalla, T.M. "Microbial
concerns when wastes are applied to land" Proc.
1970, Cornell Ag. Waste Management Conference.
89. Dunlop, S.C., July 1968. Survival of Pathogens and
related disease hazards. Presented at the
Symposium on the Use of Sewage Effluent for Irri-
gation, Louisiana Polytechnic Institute, P.uston,
Louisiana, July 1968.
90. Muller, G. "Investigations on the survival of
Ascaris eggs in garden soil," Zentralbl.
Bakteriol. 159:377 (1953).
91. Gudzfcabid2e, G.A. "Experimental observations on the
development and survival of Ascaris lumbricoides
eggs in soil of irrigated agricultural fields"
Med. Parazit., 28:578 (19 59); Abst. Soviet Med,
4:979 (19601.
92. Hess, E., Lott, G., and Breer, C., "Klarschlamn and
Freilandbiologie von Salmonellen," Zentralbl
Bakteriol. Hyg., 1 Abt. Crign. B. 158 (1974), 446.
93. McCarty, P.L., and King, P.H., "The Movement of
Pesticides in Soils," Proc. 21st Ind. Waste Conf.,
Purdue Univ., Lafayette, Indiana, (1966), 156.
94. Rudolfs, W., Falk, LL., and Ragotzkie, R.A.,
"Contamination of Vegetables Grown in Polluted
Soil I. Bacterial Contamination Sew. Ind. Wastes.
23(1951), 253.
95. Martin, S., Annual Reports of the Medical Officer
of the Local Government Board (1897-1900).
96. Larkin, E.P., Tierney, J.T., and Sullivan, R.,
Persistence of virus on sewage-irrigated
vegetables. Jour. Env. Eng. Div., Proc. Amer.
Soc. Civil Eng., 1976, 102: 29-35
97. Cliver, D.O. "Surface Application of Municipal
Sludges." Proceedings on Virsus Aspects of
Applying Municipal Wastes to Land. Symposium
June, 1976, University of Florida.
98. Yoshidaf S., "On the Resistance of Ascaris Eggs."
Jour. Parasit. 6, 132 (1920)
-95-
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&R.M
APPIHDXX a-3
Mathod* for Biological Examination of Solid Waataa
A** 3# 1 Bactariological Examination
3 # 2 1 ologleal Exas&ination
A-3.3 Oaf nai,nation of Aaearia spp. Igga
A-3.4 Datarmination of Fathoganic Fungi
1/1
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Culture media.
The um of dehydrated madia is recommended whenever possible, since these products offer the
advantages of good consistency from tot to lot, require less labor ia preparation, jmd are mors
economical. Each lot should be tested for performance before use.
Measurement of the final pH of a prepared culture medium should be accomplished colon-
metrically after autodaving and cooling. Acceptable pH range is 7.0 ± 0.1.
Media should be stored ia a cool, dry, and dark place to avoid dehydration, deterioration, and
adverse light effects. Storage la the refrigerator usually prolongs the shelf-life of most media. Media
should not be subjected to long periods of storage, because certain chemical reactions may occur in
a medium even at refrigerator temperatures.
Many of the media referred to below can be obtained from commercial sources in a dehydratad
form with complete information on their preparation. These media will therefore be listed but net
described in this section. Described in this section axe those media that are formulated from
ingredients or from dehydrated materials. Culture media (Difco or BBL products) am lirud as
follows:
Bacto-agar
Bismuth sulfite agar
JmOOu afa
Brain h*art broth
Brilliant green agar
Brilliant grsen lactose bQ*, 2 percent
fi-iagi it»«> manrrttnl agar
Dextrose
E C broth
Eosin methylene blue agar, Levine
Fluid thicgiycoUata medium
Gelatin
H-broth
Indole nitrite medium
KCN medium
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Bacteriological Examination
COLLECTION AND PREPARATION OF SAMPLES
Method for Collection of Solid Wat* or Semi-Solid Wait* SampUs
Equipment and mattriah.
Necessary items are as follow*:
1. Sample containers, specimen cups, sterile, 200-ml size (Falcon Plastics, Los Angeles)
2. Sampling tongs, stcrfle (stain 1cm steel, angled tips, 18 in. kx*g)
3. Shipping container, insulated, refri^snted, 6 by 12 in, ID.
4. Disposable glovee
Proctdur*.
1. Using sterile tongs, collect 20 to 40 random 100- to 200-« samples and place in sterile sampling
containers. When collecting samples from contaminated sources, veer dhpowbte doves and avoid
contaminating the outside of tte container.
2. Identify on tag **** Hm« tirf date of sampling. If incinerator residua saapiee are
taken, record operating temperatures of incinerator.
3. Deliver samples to laboratory. It is recommended that tike examination be started preferably
within 1 hr after coflactlaoj* *fi# Him »up»im between collection "«> exjnfatatioa at no
one exceed 8 tar.
Mtthod for ColUaUm of Liquid Samplm-Qumek md Industrial Wattrt or Lmefmt*
Equipment ami /notarial*.
Necessary items include a screw-capped, 250-mi, starSe sample bottle or a 16-ox, sterile plastic
Proetdurs.
Collect sample in bottle at plastic beg. leaving an air space in tiie container to facilitate mixing of
tiie sample before exanunatkaa. Hbm collecting samples from contaminated sources, wear disposable
gloves and avoid contaminating tte outride of the container.
Identify and deliver samples to labarataty. When shipping lemples to laboratory, protect coo-
tainen from crushing and maintain temperature below 10C during a maximum transport time
of 6 hr. Examine within 2 tte. If water sample contains residual chlorine, a dechlorination agent
such as sodium thioenUhSi it added to coBectioa bottles to neiifnlwe any rsafchiil chlorine and to
yiereut a conttaiatioa of tte hacfwfcrktal action of chlorine during the time the sample ts in
transit to tte laboratory. Enoogi aodtue thioentfata is added to tte dean sample bottle before
steriWiatfcM to proeida af ityuitreefe concentration of 100 mg per Uter ia tte srnpi*.
"If arapia is sMppad to a laboratory for aad rramfnsrtrt eanaec begin withfea 1 hr of cuOactlou, the
—rt'-'T —r *-- 'TfrtsMl iinl lawifili niletileil tiimr 10C doing tte zaaataatiacapart of 6 hr.Sodi samples
/ A I
-------
SAMPLING PROBE
VACUUM
PUMP
PHOSPHATE BUFFER
CARRYING CASE
Figure J. Portable sampler for micioorg*nkroa in incinerator slack emission.
-------
Solid or i«»l- solid
wail* *ainpl«
10 pi
200 qui
Hind with
1100 «l
buflarid wolor
1 Ml 0.1 »S
-1
-2
1 mI
lml 0.1 mI
lull
0.1*1
lml
0.1 ml
-6
-7
i—3
-4
Figtue 2. Preparation of decimal dilutions.
-------
Methods for Presence of Members of CoUform Group
The presence of fecal matter in waste and related materials is determined by the standard tests
for the coliform group described in Standard Methods for the Examination of Water and Waste
Water (3). The completed Most Probable Number
-------
Salanita brilliant
Graan/SuUa 270 ail
Saild wail* or rcttdw*
Sa m pi* 30 gm i
Incwbata at 39.S C end 41^5 C
1MB
Aaar
SS
Afar
BS
Agor
I
•4 plat** *ack<
BO
Afar
M«cC«nk*y'i
Afar
ln
-------
3. After incubation," airfTone loopf.il from each enrichment medium on each of four pUw of
ld™|!S^h^^0S'37eCCto m'm Wta «d pick «*icioua colonic, to triple w iron
*" V™1!;.,. .. 37 c for 24 hr and complete identification by appropriate method! aa
LSE^LS (m Isolation, preliminary identification, and biochemical Mh,
are described in rigors 3 and In Table 2.
Procedure to detect ptthcgens in quench or industrial water* and in leackate.
1. Place enough sterile diatomaceous earth on the screen of a stainless steel membrane filter
holder to form a 1-in. layer.
3 hSr With a «eril. spatula and pU=» into 90 ml of
mJS7tJoSsssl£*?ssrx- w <* *. ^ *. «.«»»i.f *¦*» uw
green/sulfa enrichment broth- Shake both flasks to mix.
4 Incubate both flasks In a water bath at 39.5 C for 16 to 18 hr.
S. aTdStcted in supf 3 throujh 5 of Procedure to Select Pathogena in Sobd Waau and
Incinerator Residue.
Method for Examination of Stack Effluents
As described in Methods for Collection of Incinerator Stack Effluents (using the Armstrong
sampler), the microorganisms are impinged into a 300-ml phosphate buffer
Filter 100 ml of the "Inoculated" phosphate buffer solution through a 0.45k HA membrane
-iter (3).
2. Transfer membrane Alter with sterile forceps to a culture plate containing tryptfease soy igar.
3. Incubate culture plate under constant saturated humidity for 20 hr(± 2 hr) at 3S C.
4. After incubation, remove cover from culture plate and determine colony count with the aid of
a low-power (10-15 magnifications) binocular, wide-field microscope. Characterize colonies using
specific isolation media.
5. Remove a 10-ral portion of the "inoculated** phosphate buffer solution and examine for viable
heat-resistant sports as directed in steps 1 through 6 of the procedure under Method to Determine
the Presence of Viable Heat-Resistant Spore Numbers.
Microbial counts are reported as organisms per cubic foot of air. If the sample is not taken
under Isokinetic conditions, the results are qualitative. If the stack velocity is known and remains
relatively constant, however, the flow me of the sampler can be adjusted to isokinetic conditions
to yield quantitative results.
Method for Examination of Dust
As described in Methods for Collection of Dust Samples, the Andersen sampler is used with two
types of media-tiypticase afy agar (TSA-BBL product) containing 5 percent sheep blood, and eosin
methylene blue agar (EM$btfco product}. The TSA/blood agar is used to isolate a wider range of
fastidious organisms such as Staphylococci, Streptococci, and DiplococsL The EMB agar is used to
isolate gram-negative bacteria. The plates are incubated aerobically at 37 C for 24 hr. (Preliminary
studies showed that few organisms in the dust would grow under anaerobic conditions.) Enumeration
of colonies is made with a Quebec colony counter. Microbial count is reported as organisms per
bic foot of air. At times, whea microbial counts are high, the sampling tine is 0.25 sin, thus yiekl*
i 0.25 cu ft air.
i 11
-------
REFERENCES
Hanks, T.G. Solid waste/disease relationships. U. S. Dept. of Health, Education, and Welfare,
Public Health Service PubL No. 999-UTH-6, CbiCTnn3.fi, National Center for Urban and Indus-
trial Health, 1967.
2. Armstrong, D.H. Portable sampler for microorganisms in incinerator stack emissions. Applied
Microbiology, 19 (1): 204-205, 1970.
3. American Public Health Association. Standard methods for the examination of water and
waste water. New York, American Public Health Association, 1971.
4. Andersen, AA. New sampler for the collection, sizing and enumeration of viable airborne
particle*. Journal of Bacteriology, 76:471-434, 1958.
5. Peterson, M.L. and FJ. Stutzenberger. Microbiological evaluation of incinerator operation.
MWnhirdrwcv.
o. American Public Health Association, Inc. Standard methods for the examination of dairy
products microbiological and chemical, New York, American Public Health Association, Inc,
1960.
7. Harris, AJi., and MJB. Coleman. Diagnostic procedures and reagents.. New York, American
Public Health Association, Inc. 1963.
8. Park, HJF., and P.W. Kabler. Revaluation of the significance of the colifonn bacteria. Journal
of American Wats Works Association, 56:931-936, 1964.
9. Smith, L., and &LA. Madison. A brief evaluation of two methods for total and fecal conforms
in municipal solid waste and related materials. Cincinnati, U. S. Environmental Protection
Agency, National Environmental Research Center. Unpublished data, 1972.
"n. Frobisher, M. Fundamentals of microbiology, 6th ed. Philadelphia, W. B. Saunders Cx, 1957.
p. 151-152.
. Barbeito, IL S. and G.G. Gremillion. Microbiological safety evaluation of an industrial refus*
incinerator. Applied Microbiology, 16:291-295, 1968.
12. Dauer, Cari C I960 Summary of disease outbreaks and a 10-year rmmm. Public Health Report,
76, no. 10, Oct. 1961. p 915.
13. Dubos, Rene. Bacterial and mycotic infections of man. Philadelphia, J. S. Lippiucott, 1958.
14. Weibel, S. R>, F.R. Dixon, R-B. Weidner, and LJ. McCabe. Witerborne-disease outbreaks
1946-1960. Journal of the American Water Works Association, 56:947-958, Aug^ 1964.
15. Spino, D.F. Bevated-tempezature techniques for the isolation of Salmonella from streams.
Applied Microbiology, 14:591, 1966.
16. Scarce, I,F, and MX. Peterson. Pathogens in streams tributary to the Great Lakes. In:
Proceedings; Ninth Conference on Great Lakes Research, Chicago, March 23-30, 1966.
Public No. 15. Ann Arbor, Univ. of Mich., 1966. p. 147.
17. Peterson, M.L. The occurrence of Salmonella in streams draining Lake Erie Basin. In: Proceed-
ings; Tenth Conference on Great Lakes Research, Toronto, Apr. 10-12, 1967, Ann Arbor, Univ.
of Mich., 1967. p. 79.
18. Peterson, M.L. and AJ. Klee. Studies on the detection of salmonella* in municipal solid
waste and incinerator residue. International Journal of Environmental Studies, a: 125-132, 1971.
19. Spino, D. Bacteriological study of the New Orleans East Incinerator. Cincinnati, U.S. Environ-
mental Protection Agency, National Environmental Research Center, 1971.
20. Edwards, P.R. andJVJL Ewing, Identification of Enterobactemceae. Minneapolis, Burgess
Publishing Cx, 197%
//J
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A-3.3 DETERMINATION OF ASCARIS spp. EGGS in SOLID WASTE
1. Materials
1.1 Balance: 10 g - 1 kg capacity.
1.2 Beakers: 150 ml & 600 ml
1.3 Bottle: 125 ml, Wheaton.
1.4 Bottle shaker.
1.5 Brush: B-8695 Scientific Product*.
1.6 Centrifuge: rotor radius 14.6 cm.
1.7 Centrifuge tube*: 15 ml and 50 ml.
1.8 Cheesecloth: FSN 3305-00-205-3496.
1.9 Counter: differential
1.10 Culture dish: with 2 mm grid.
1.11 Inverted microscope
1.12 Pip«ttes: Pasteur type and 5 ml serological.
1.13 Rubber bulb: ca. 2 ml
1.14 Tray: round, 10.5 inches diameter, 3 inches high
e.g., Beckman Instrument Co. 32-018.
2. Reagents
2.1 Saline: 0.85« NaCl in H20.
2.2 Nacconol: 0.4% of concentrate in H20
2.3 Hydrochloric acid: 2% solution in B2O.
-2.4 Solvent: alcohol:acetone:xylene in 1:1:2 ratios.
3.1 Vegetable Samples
3.1.1 The sample size for vegetables is 1 leg.
Leafy vegettables occuring in heads (cabbage, lettuce
-------
3.2.4 Rinse the bottle 3 times with 5 ml saline
and add each rinae to the beaker.
3.2.5 Transfer ths contents of the beaker to
seven 15 ml centrifuge tubes.
3.2.6 Rinse the beaker 3 times with 5 ml of
saline and add each rinse to the centrifuge tubes.
Centrifuqatlon Procedure
4.1 Centrifuge the tubes collected in 3.1 and/or 3.2
at 2,000 rps (radius 14.6 cm) for 4 minutes.
4.2 Remove and discard the supernatant.
4.3 Add 2 ml of saline to each tube.
4.4 Combine the sediments into one tube using a Pasteur
pipette to transfer the sediment and to rinse each tub*
3 times with 2 ml of saline. Each rinse is also added
to the collecting tube.
4.5 When the collecting tube is full, it is balanced
with a blank, centrifuged at 2,000 rpa for 4 minutes;
supernatant is discarded. Repeat if necessary.
4.6 Add saline to the 15 or 50 ml graduation mark on
the collecting" tube and resuspend the sedimant; centrifuge
at 2,000 rpa for 4 minutes.
4.7 Discard the supernatant; add 2 ml of saline and
~rest2ap*nd th« sediment.
4.3 Transfer the suspension to the culture dish; rinse
the tube 3 times with 2 ml of saline and add each rinse
to the culture dish. Add 3 ml of the 2% hydrochloric
acid to th* dish (to prevent mold growth) and cover the
dish.
-------
incubation (step 6 above) , fertilized eggs develop into
embryonated eggs which contain a second-stag# nematode
larva in a cuticular sheath. Types of Agcaris app.
eggs are illustrated in the following references.
8. References
8.1 Faust, E.C. Beaver, P.C., Jung, R.C. 1968. Animal
Agents and Vectors of Human Disease. Lea and Febiger,
Philadelphia.
8.2 Markell, E.X. and Voge, M. 1971. Medical Parasitology.
Saunders, W.B., Philadelphia.
1
-------
2.6 Incubate cultures for 4 weeks, making weekly
examination® (make smears of suspicious colonies;
identify fungi by cultural characteristics.)
3, Actldiona and Chloromycetin inoculation
3.1 Prepare two tubes of Sabouraud'a agar and two
tubes of Sabouraud1s agar containing 0.5 mg Actidione
per si and 0.05 g of Chloromycetin per liter.
3.2 Inoculate with a small portion of concentrated
sediment.
3.3 Incubate all tubes at 25 C and examine weekly.
3.4 At the end of 6 weeks make smears of suspicious
colonies and identify by cultural characteristics.
)
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