United States
            Environmental Protection
            Agency
            Municipal Environmental Research
            Laboratory
            Cincinnati OH 45268
EPA-600/8-80-034
August 1980
            Research and Development
&EPA
A Survey of
Pathogen Survival
During Municipal
Solid Waste and
Manure Treatment
Processes

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                RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and  application of en-
vironmental technology.  Elimination  of traditional grouping  was  consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

      1.   Environmental  Health Effects Research
      2.   Environmental  Protection Technology
      3.   Ecological Research
      4.   Environmental  Monitoring
      5   Socioeconomic Environmental Studies
      6.   Scientific and Technical  Assessment Reports (STAR)
      7.   Interagency  Energy-Environment Research and Development
      8.   "Special" Reports
      9.   Miscellaneous Reports

This report has been assigned to the "SPECIAL" REPORTS series. This series is
reserved for reports targeted to meet the technical information needs of specific
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.

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                                       EPA-600/8-80-034
                                       August 1980
A SURVEY OF PATHOGEN SURVIVAL DURING MUNICIPAL
  SOLID WASTE AND MANURE TREATMENT PROCESSES
                      by

                Sylvia A. Ware
             Ebon Research Systems
            Washington, D.C. 20001
           Contract No. 68-03-2460-5
               Project Officers

               Carlton C. Wiles
              Laura A. Ringenbach
  Solid and Hazardous Waste Research Division
  Municipal Environmental Research Laboratory
            Cincinnati, Ohio 45268
  MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
      OFFICE OF RESEARCH AND DEVELOPMENT
     U.S. ENVIRONMENTAL PROTECTION AGENCY
            CINCINNATI, OHIO 45268

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                             DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication.  Approval does not signify that the contents necessarily
reflect the views and policies of  the U.S.  Environmental Protection
Agency/ nor does mention of trade  names or  commercial products consti-
tute endorsement or recommendation for use.

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                                  FOREWORD
     The U.S. Environmental Protection Agency was created because of increasing
public and government concern about the dangers of pollution to the health and
welfare of the American people.  Noxious air, foul water, and spoiled land are
tragic testimonies to the deterioration of our natural environment.  The
complexity of the environment and the interplay of its components require a
concentrated and integrated attack on the problem.

     Research development is that necessary first step in problem solution; it
involves defining the problem, measuring its impact, and searching for solutions.
The Municipal Environmental Research Laboratory develops new and improved
technology and systems to prevent, treat, and manage wastewater and solid
and hazardous waste pollutant discharges from municipal and community sources,
to preserve and treat of public drinking water supplies, and to minimize the
adverse economic, social, health, and aesthetic effects of pollution.  This
publication is one of the products of that research and provides a most vital
communications link between the researcher and the user community.

     This report summarizes studies that evaluated pathogen survival during
solid waste treatment.  Methods discussed include incineration, composting,
landfill, and anaerobic digestion.
                                   Francis T. Mayo, Director
                                   Municipal Environmental Research Laboratory
                                      111

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                                  ABSTRACT
     Municipal solid waste  (MSW) and animal manures may contain microorganisms
that can cause disease in man and animals.  These pathogenic microorganisms
include enteric bacteria, fungi, viruses, and human and animal parasites.

     This report summarizes and discusses various research findings document-
ing the extent of pathogen  survival during MSW treatment.  The technologies
discussed are composting, incineration, landfill, and anaerobic digestion.
There is also a limited examination of the use of the oxidation ditch as a
means of animal manure stabilization.  High gradient magnetic separation
(HGMS), and gamma radiation sterilization are mentioned as future options,
especially for animal waste management.  Several standard methods for the
sampling, concentration, and isolation of microorganisms from raw and treated
solid waste are also summarized.

     This report was submitted in fulfillment of Contract No. 68-03-2460-5 by
Ebon Research Systems under the sponsorship of the U.S. Environmental
Protection Agency.  This report covers a period of March, 1978 to February,
1980 and work was completed as of March, 1980.
                                     IV

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                        CONTENTS
Foreword	   iii
Abstract	    iv
Figures	    vi
Tables   	    vi
Acknowledgements 	   vii

     1. Introduction 	     1
     2. Conclusions  	     5
     3. Recommendations  	     7
     4. Pathogen Survival during
          Specific Waste Treatment 	     8

         I.  Composting	     8
        II.  Incineration	    20
       III.  Sanitary Landfill 	    49
        TV.  Oxidation Ditch	    69
         V.  Anaerobic Digestion 	    74
        VI.  Other Processes	    78

     5. Analytical Methods	    80

References	    92

Appendix A:  Health Aspects of Solid Waste
              Disposal	   101

Appendix B:  Hospital Waste	,	   103

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                                FIGURES

Number

  1  Flow diagram of a high temperature combustion plant .  .   23
  2  A comparison of the efficiency of
       total bacteria removal from incinerators	30
  3  A comparison of the efficiency of
       heat resistant spore removal from incinerators. ...   31
  4  A comparison of the efficiency of total
       coliform removal from incinerators. .	32
  5  A comparison of the efficiency of fecal
       coliform removal from incinerators	33
  6  Air quality of various waste treatment facilities:
       average values of total bacteria. ... 	   44
  7  Air quality of various waste treatment facilities:
       average values of total coliforms 	   45
  8  Air quality of various waste treatment facilities:
       average values of fecal coliforms 	   46
  9  Air quality of various waste treatment facilities:
       average values of fecal streptococci	47
 10  Cross-section of an experimental sanitary landfill. .  .   52
 11  Change in total coliform bacteria
       with time of leaching	58
 12  Change in density of fecal coliforms
       with time of leaching .	   59
 13  Change in density of fecal streptococci
       with time of leaching ........... 	   60
                               VI

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                            TABLES
Number                                                      Page

  1  Some Pathogens and Indicator Organisms Found
       in Municipal Solid Waste. ........ 	   3
  2  Composition and Analysis of Composite Samples
       of Municipal Refuse 	 ....   4
  3  Amount of Soiled Diapers in Municipal Solid Waste ...   4
  4  Survival of Pathogens During Composting	  . .  10
  5  Species Investigated by Knoll 	  16
  6  Some Pathogenic Fungi Associated with Composting  ...  17
  7  Survival of Human Parasites Inserted in Refuse-
       Sewage Sludge Windrows	 .  19
  8  Survival of Dog Parasites Inserted in Refuse-
       Sewage Sludge Windrows	19
  9  Microorganisms Associated with Dust from
       Compost Operations.	 .  21
 10  Incinerator Characteristics	22
 11  Efficacy of Incinerator Operations in the
       Destruction of the Microflora Associated
       with Municipal Solid Wastes	 .  . .  24
 12  Efficacies of Various Incinerator Designs
       in the Destruction of Microflora Associated
       with Municipal Solid Wastes	  27
 13  Characterization of Gram-positive Cocci and
       Gram-negative Bacilli 	 ......  35
 14  Average Values of Selected Microorganisms
       Present in Microbiological Aerosols
       at an RDF Plant	 . .  . .  39
 15  Reported Aerosol Concentrations of Bacteria
       Found in Ambient Air and Various Working Locations. .-  39
 16  Ranking of Hi-Vol Samples Based on Average
       Bacterial Levels	  41
 17  Hi-Vol In-Plant Bacteria Count/Cubic Meter;
       High and Low Values (MPN) ..............  42
 18  Hi-Vol Ambient Bacteria Count/Cubic Meters
       High and Low Values (MPN) ..............  43
 19  Range of Chemical Composition of Sanitary
       Landfill Leachate 	 .............  53
 20  Occurence of Streptococci in Leachate .........  55
 21  Distribution of Fecal Coliforms and Fecal
       Streptococci in Leachates from the Upper Pipe ....  56
 22  Microflora of Municipal Solid Waste and
       Leachate from Simulated Landfills , . . .......  63
                              vii

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                      TABLES  (continued)
23  Operation of a Model Oxidation Ditch	71
24  Survival of Leptospira Pomona and
      and Salmonella TyphimuriunT"	72
25  Conditions Favorable to Destruction of Leptospires. . .  73
26  Disposition of Selected Biological Materials  	 104
                          viia

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                                ACKNOWLEDGMENTS


Ebon Research Systems would like to thank our Project Officers, Mr. Carlton C.
Wiles and Mrs. Laura A. Ringenbach, for their assistance throughout this
project.  We would also like to thank Dr. William L. West, Howard University
Medical School, and Dr. Lee Richman, Ebon Research Systems, who reviewed
the draft.

Table 3 is reproduced from Peterson, M.L. Soiled Disposable Diapers:  A Poten-
tial Source of Viruses.  American J. Public Health, 64:912, 1974.  With
permission of the copyright owner the American Public Health Association.

Tables 10 and 11 are reproduced from Peterson, M.L.,and F.J. Stutzenberger.
Microbiological Evaluation of Incinerator Operations.  Applied Microbiology,
18(1) 8-13, 1969.  With permission of the copyright owner the American Society
for Microbiology.

Table 14 is reproduced from two tables in Duckett, E.J.  Microbiological
Analyses of Dusts at the Equipment Test and Evaluation Facility.  Report No. TR
78-2.  National Center for Resource Recovery, Washington, D.C., March 1978.
With permission of the copyright owner the National Center for Resource
Recovery.

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                                    SECTION  1

                                   INTRODUCTION
PURPOSE OF THE REPORT

    The purpose of  this report  is to summarize and discuss research findings
documenting  the extent of pathogen  survial after waste treatment.  The technolo-
gies discussed are  composting,  incineration,  landfill, and anaerobic digestion
since  these  are common or currently discussed methods of treating solid waste.
There  is also a limited examination of the use of the oxidation ditch as a means
of animal manure  stabilization.  This examination is included since the oxida-
tion ditch has been recently promoted as a means of animal waste management, and
a certain amount  of literature  is available discussing its environmental
acceptability.  High-gradient magnetic separation (HOIS) and gamma radiation
sterilization are mentioned as  future options, especially for animal waste
management.  Appendix A discusses health aspects of solid waste disposal and
Appendix B briefly  examines disposal of hospital wastes.

    Given the heterogeneous nature  of MSW, it is difficult to sample raw waste
or residue in a representative  manner.  Methods of sampling, detecting, and
concentrating microorganisms isolated from waste or residue are not always
consistent or reliable, especially  for viruses.  Section 5 of this report
summarizes the analytical methods used in the studies discussed.

    Concentrations  of animal excreta present  in MSW have not been quantified.
From available studies, it would appear that  the greater proportion of fecal
matter in MSW is  of animal rather than human  origin (Peterson, 1971; Blannon and
Peterson, 1974; Cooper et al_.,  1974a; Duckett, 1978).

    The detection of various fecal  organisms  in MSW is evidence of fecal origin
and contamination.  While many  of these organisms are harmless, some are
etiologic agents  which cause disease in man and animals, i.e., they are patho-
gens.  Coliforms, streptococci, salmonella, and various parasites have been
isolated from MSW (see Table 1).  Enteroviruses have also been detected in MSW»
The presence of these microorganisms in waste, and their survival after various
methods of treatment is an indication that other pathogens (perhaps more
difficult to detect) may also be present in waste and survive treatment.  Hence,
a bacteriological and viral examination of raw or treated waste may include an
assay for:  total bacteria, total and fecal coliforms, fecal streptococci, heat
resistant spores, and enteroviruses (poliovirus, echovirus, coxsackievirus).

    The presence of pathogenic organisms in raw or treated waste is potentially
detrimental to public health.   [It's not the  fact that MSW is in the land that
it presents a potential problem.]    Hanks (1967) has documented possible health

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hazards associated with poor solid waste management  (see Appendix A).   However,
there is not one epidemiological study which can directly trace outbreaks of
disease to the presence of pathogens in MSW, though  there are  studies  relating
disease to water contamination by sewage sludge and  animal manures.  Though
various studies have shown that solid waste management  is a hazardous  occupation
in terms of injury and cardiovascular disease  (Sliepcevich, 1955; Anderson,
1964; Cimino, 1975), there is no adequately designed study which shows a
statistically significant difference in rates of respiratory,  gastrointestinal
or skin infections among sanitation workers, the group  most frequently exposed
to solid waste.  Hence, the health implications of the  studies documented in
this report are not conclusive.

MSW - CHARACTERIZATION

    The U.S. Environmental Protection Agency estimated  that in 1971, municipal
solid waste (MSW) generation in the United States was approximately
3.32 Ib/person/day (Lowe, 1974).  For a projected growth in solid waste genera-
tion of 3% to 4% annually, an estimated U.S. national average  of around
3.91 Ib/person/day in 1980, or an urban area average of 4.33 Ib/person/day
results.  Anderson (1972) has projected the generation  of over 220 million dry
tons of MSW in 1980.

    The composition of MSW is variable and depends on the geographical  location,
the season, and the nature of the community generating  the waste.  Table 2 shows
a representative analysis of MSW collected in a large city.  It should  be empha-
sized that this analysis is only an indication of the content  of MSW.   Neverthe-
less, the high percentage of paper products in the refuse is typical.   Human and
animal excreta are also found in MSW, the human excreta largely derived from
disposable diapers, and the animal excreta from household pet  litter.

    Peterson (1974) estimated that 0.65% to 2.5% wet weight of residual solid
waste was soiled disposable diapers (see Table 3).   She found  that approximately
33% of the diapers contained feces to an average value  of 60 g/lb (wet  weight)
of diapers, for an average of 0.2 g of feces/lb (wet weight) of solid waste.
Engelbrecht (1973)  also reported that an average of  33% of diapers from a
residential area of Cincinnati were stained with feces.

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                                   TABLE 1

               SOME PATHOGENS AND INDICATOR ORGANISMS POUND IN
                            MUNICIPAL SOLID WASTE
Organism
    Concentration*
                                   Reference
Fecal streptococci
Total colifonus
Fecal coliforms
Salmonella sp.

Heat resistant spores
Total coliforms
Fecal coliforms

Salmonella sp.
Heat resistant spores
Total coliforms
Fecal coliforms

Total coliform
Fecal coliform
Fecal streptococci
      10b (av.)
      108 (av.)
      107 (av.)
1.7 x 104  6.8 x 104
1.2 x 104  6.2 x 106
2.3 x 104    4 x 105
3.1
3.4 x
    x KP
      103
           1.9 x
           5.1 x
132 x 106t
       i6t
248 x 10*
                 10'
1.5 x 104  5.6 x 106
            86 x
            86 x
           396 x
                 10
                   6 = =
                   6 = =
                 10
                                Gaby, 1975
                                Peterson and
                                Stutzenberger,
                                1969

                                Peterson and
                                Klee, 1971

                                Peterson, 1971
                                Cooper et al.,
                                 1974a
* Counts/g
t Sample 1
={= Sample 2

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                                TABLE 2
  COMPOSITION AND ANALYSIS OF COMPOSITE SAMPLES OF MUNICIPAL REFUSE
                           (Kaiser,  1963)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28

Corrug. paper boxes
Newspaper
Magazine paper
Brown paper
Mail
Paper food cartons
Tissue paper
Wax cartons
Plastic coated paper
Vegetable food wastes
Citrus rinds and seeds
Meat scraps, cooked
Fried fats
Wood
Ripe tree leaves
Flower garden plants
Lawn grass green
Evergreen
Plastics
Rags
Leather goods
Rubber composition
Paint and oils
Vacuum cleaner catch
Dirt
Metals
Glass? ceramics, ash
Adjusted moisture

23.38%
9.40
6,80
5.57
2.75
2.06
1,98
0.76
0.76
2.29
1,53
2.29
2.29
2.29
2.29
1.53
1.53
1.53
0.76
0,76
0..38
0.38
0,76
Oo76
1.53
6,85
7.73
9.05
100.00
                               TABLE 3

      AMOUNT OF SOILED* DIAPERS IN MUNICIPAL SOLID WASTE, 1971
                          (Peterson, 1974)
Sampling

Area

A
A
B
B

Date

Feb
April
July
July
Total
Waste Separated
Ib (wet wt)
800
9,200
2,800
3,600
Mount of Diapers

Soiled

Feces-Contaminated
% total waste t
2.5
0.9
0.6f
0.8 +
1.0
0.3
0.2 "I"
0.3 t
* Contaminated with urine or feces
t Mean values from multiple samples

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                                    SECTION  2

                                   CONCLUSIONS


    From the literature reviewed,  composting appears to be an effective method
of destroying pathogenic organisms provided that sufficient time/temperatures
are achieved.  All organisms within the  compost should be exposed  to  thermophi-
lic temperatures of  50°C to 60°C for at  least  seven days.  There is some
evidence that parasitic cysts  and  ova (especially those of animal  rather  than
human origin) may survive long periods of composting.  It should be emphasized
that though parasites may remain morphologically intact after composting,  they
may not be viable.

    Various investigators have shown that the  extent to which microorganisms are
destroyed during incineration  is a function of both the design and the manner of
operation of the incinerator.  From available  studies it is concluded that all
pathogens should be  destroyed  provided that the entire solid waste charge
quickly reaches an appropriate temperature.  If an incinerator is  charged beyond
its capacity, if the waste is  too  tightly packed, or if operating  temperatures
are too low, then it is possible for pathogenic organisms to survive  incinera-
tion.  These organisms may remain  in unburned  residue, in quench water, or in
aerosolized form exitting the  stack,  especially if the stack is relatively
short.

    There is also a  possibility that pathogenic bacteria and viruses  may  survive
conditions within the sanitary landfill, and may be leached to ground or  surface
water.  However, there does appear to be a  significant decrease in viral  and and
bacterial content of leachate  with time  of  operation or leaching of the fill.
Also, the relatively high temperature (60°C) achieved during the first aerobic
stages of waste biodegradation is  inirnicable to many viruses and most pathogenic
bacteria.  It has also been shown  that the  chemical and physical characteristics
of the leachate contribute toward^j5oth~viral and~bacterial inactivatJonl~    ~

    Adsorption of viruses onto material  in  the fill is likely, and partly
explains the low rate of recovery  of viruses from landfill leachate.  It  is not
clear whether viruses are inactivated when  they are adsorbed onto  particulate
matter in the fill.

    No studies were  available  documenting the  possibility and extent  of pathogen
survival in anaerobically digested municipal solid waste.  Studies of digested
sewage sludge have shown survival  of pathogenic organisms at both  mesophilie
temperatures (viruses, bacteria, parasites), and thermophilic temperatures
(viruses).  For these studies, temperature, time of residence, and degree of
mixing were all factors determining the  extent of pathogen survival.
                                      5

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    From the limited material presented, it appears that prolonged survival of
some of the more common pathogens present in animal wastes is possible within
the oxidation ditch environment.  However, methods of operating field facilities
vary greatly, and the environmental characteristics of even one specific ditch
may change daily.

    Though various studies have shown that pathogenic organisms present in muni-
cipal solid waste may survive treatment, it should be emphasized that there is
not one sound epidemiological study correlating an outbreak of any infectious
disease in this country with the pathogen content of treated wastes.  Also,
there is no conclusive evidence that workers exposed to pathogen-containing raw
or treated municipal solid waste on a daily basis have a statistically signifi-
cant increase in gastrointestinal, respiratory, or skin infections as compared
to those of the populace at large.  Hence, the health significance of pathogen
survival in solid waste after treatment is not at all clear.

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                                   SECTION  3

                                RECOMMENDATIONS
•  It has been established that MSW  is contaminated with human and animal
   excreta, and contains fecal microorganisms some of which may be pathogens.
   However, the potential hazard presented by this contamination  is not clearly
   defined.

•  Methods for isolating viruses from raw and treated waste and leachate need  to
   be improved.  It  is often not clear whether samples are truly  negative for
   viruses, or that  their presence is undetected.

•  Given reports of  possible parasitic survival in composting sludge and
   refuse/sludge mixtures, there is  a need to clarify exactly how effective the
   process is in destroying human and animal parasitic ova and cysts.  One
   recent study reported that parasites remain morphologically intact for
   more than 49 days in refuse/sludge compost.  However, no determination was
   made of their viability.  Any future study conducted should make this
   determination to  remove ambiguities.

•  The extent to which pathogenic fungi survive composting should also be
   determined.

•  The most effective methods of operating an incinerator to ensure pathogen
   destruction should be clearly delineated.

•  The behavior of microbes in the sanitary landfill is not well  understood.
   This is especially true for viruses.  Any investigation to correlate specific
   chemical, biological, and physical characteristics of the fill environment
   with microbial inactivation would be very useful.

•  The extent to which a virus retains its infectivity when absorbed onto solids
   is also not well-established.  A  study of viral inactivation on solid waste
   components could  be conducted in  tandem with a study of factors effecting
   elution of virus  from the waste.

•  There is a need to determine the  effectiveness of anaerobic digestion as a
   means of pathogen destruction.  No relevant data for MSW were  found in the
   literature.  Reports on the extent of virus inactivation, and  bacterial and
   parasitic destruction in digesting sludge suggest that the conditions of
   anaerobic digestion may not be sufficient in effecting complete destruction
   of the microbes.

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                                    SECTION 4

             PATHOGEN SURVIVAL DURING SPECIFIC SOLID WASTE TREATMENTS
 I.  COMPOSTING

 The Process

     Biological deconposition of organic solids by aerobic microorganisms results
 in formation of a stable humus-like material and production of carbon dioxide
 and ammonia.  The process is termed composting and the humus-like material re-
 maining is compost.  Though composting may also be accomplished by anaerobic
 organisms this is a much slower process and intermediate products formed smell
 unpleasant.  Decay may be initiated by psychrophilic,  mesophilic, or
 thermophilic organisms,-  however, it usually proceeds through mesophilic to
 thermophilic digestion (45° to 60°C).

     The process reduces the mass and volume of the organic material while in-
 creasing its density (Finstein and Morris,  1975).   Thus,  composting waste
 material before land disposal will both extend the life of a landfill, and make
 it more physically stable (Finstein and Morris,  1975).  As an alternative to
 landfilling, a benefit is that the humus can be applied to the soil as both a
 conditioner and a fertilizer.

     A variety of solid wastes may be composted including municipal solid waste
 (MSW),  agricultural plant wastes,  manures and sewage solids.  Mixtures of dif-
 ferent wastes are often employed to provide a satisfactory nutrient balance for
 growth of microorganisms,  and to improve the physical  characteristics of the
 compost pile.

     Before composting,  the solid waste may be pretreated though this is not
 essential.   MSW,  for example, may be sorted,  magnetically separated, ground,  and
 undergo flotation,  ballistic separation,  or air classification.  The moisture
 content of MSW must be raised from about 30% to 60%e  This may be accomplished
 by  mixing with sewage sludge, which also adds nitrogen to lower the C/N ratio.
 Sewage  sludge  and animal  manures on the other hand must be dewatered prior to
 composting since  at high  water concentrations, air flow through the composting
 material is  impeded and aerobic conditions do not prevail.

    Various  composting techniques have been developed, principally in Europe  and
Asia, though to a limited  extent in the United States.  Material  to be conposted
may be  contained  in trenches in the ground,  loaded into rotating  drums,  silos,
vertical bins  or  open tanks,  stacked in open windrows  or  otherwise deployed.
Aeration may be accomplished by forcing air through the stack, or by mechanic-
ally turning or agitating  the compost  material.  Depending on the technique and

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nature of the feed material, the process may  be  completed  in  almost  any  period
from 4 to over 200 days.  A period of  curing  may follow digestion  to ensure  the
biological stability of the compost.   After curing,  the compost may  undergo
further screening and grinding treatment.

    Both batch and continuous processes have  been developed.   It is  possible to
process wastes continuously in a tiered silo  where  there is a temperature gradi-
ent from the top to the bottom deck.   This temperature difference  means  that
mesophilic organisms predominate at the top of the  silo, while thermophilic
organisms are found at the bottom.

    Continuous thermophilic composting is also possible where incoming waste is
heated by resident compost to eliminate the early mesophilic  growth  stage.
Inoculation with active thermophiles may be practiced in continuous  composting
(Finstein and Morris, 1975).  While most studies have indicated that microbial
inoculation of either mesophilic or thermophilic organisms is unnecessary, some
manufacturers inoculate their wastes prior to digestion (Wilde, 1958; Wiley,
1962; IPT, 1972; Finstein and Morris,  1975).


Survival of Pathogens

    Pathogen destruction has often been assumed  to  occur during composting
because of the relatively high temperatures achieved.  Some reports  of total
pathogen destruction during composting are based on knowledge of the time/temp-
erature required for thermal death, and the recorded temperature of  a particular
compost heap.  These reports do not always contain  evidence that sampling of the
compost was undertaken in order to confirm the destruction of specific micro-
organisms.  More recent studies have included pathogen survival tests during
composting of various wastes by different methods.   Some studies sample  for
pathogens naturally occuring in the wastes, while others inoculate known
pathogens into the compost to determine their viability during the composting
process.  Table 4 summarizes data from several of these pathogen survival tests.

    The survival of pathogenic organisms during  composting appears to depend  on
two factors;

    •  the temperature achieved and the time  of  maintenance of that  temperature
       (Morgan and Macdonald, 1969; Wiley and Westerberg, 1969; Gaby, 1975).

    •  antibiotic action or antagonistic effect  between organisms  (Knoll, 1959;
       Knoll, 1963)

It is generally agreed that most of the heat  generated during composting is
produced by respiration of aerobic microorganisms attacking the carbon
containing wastes (Finstein and Morris, 1975).   The  growth of these  microorgan-
isms depends on the moisture content and temperature of the compost,  as  well  as
the availability of air and nutrients.  Under favorable growth conditions,
temperatures in excess of 70°C are achieved within  less than  a week  (Finstein
and Morris,  1975).

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                                                TABLE 4
                                SURVIVAL OF PATHOGENS DURING COMPOSTING
Compost
Refuse/sew-
age sludge
Nightsoil &
"other
wastes"
Method
Open-pile,
2 turnings
per week
Not ident-
ified
Species
Not ident-
ified
Ascaris
eggs
Duration
(days)
6 to 8
weeks
50 to 60
Temp.
(°C)
65° to 66°
(max. )
60° to 66°
Comments
Pathogens destroyed;
species not identified;
no time/temperatures
2,000 Ascaris eggs/g
in fresh waste — only
100/g "largely"
degenerated or dead
after a week
Reference
Seabrook ,
1954
Stone,
1949
Refuse/
night-
soil
Refuse/
sludge
 Refuse/
 sludge
Layering of
equal wts. of
refuse/night-
soil in shallow
pits, top layer
of soil, no turn-
ing, anaerobic
Ascaris
eggs
10 to 15
 40°
(max.)
                             Salmonella sp.
                             Shigella sp.
Undisturbed open  Salmonella sp.
windrow, cultures
inoculated in gel-
atine capsules +
small bags of ground
refuse/sludge
                 50
             20° to 70°
              (range)
Decrease in viable
count irregular; after
3 to 5 months tended to
be negative though some
pits still gave viable
count.  After 6 months
all counts negative

No test bacteria re-
covered
Bhaskaran
e_t al.,
1957
                                    Knoll,
                                    1963
In ampules in-
serted into
windrows
S. paratyphi   7 to 9

S. paratyphi     257
                50°
               (max.)
                45°
               (max.)
           Destroyed

           Resistant up to 247
           days; complete de-
           struction 10 days
           later
                         Knoll,
                         1963

-------
Table 4 (continued)
Compost Method Species Duration
(days)
MSW/ Open windrow, Salmonella sp. 7 to 21
sewage mechanically Shigella sp.
sludge turned every few
days, moisture
content 50% to
60% Poliovirus 3 to 7
type 2
Human parasitic 7
cysts and ova
Dog parasitic 35
ova




Leptospira 2
Philadelphia
Histoplasma 26
capsulatum



Aspergillus 14
fumigatus


Temp.
(°C)
49° to 74°
(range).



49°
(max. )
60°
(max. )
72° (max.) on
day 20, 2 in.
depth

66° on day 27
mid-depth
49°
(max. )
49°
(max. )

60°
(max. )
36°
(day 14)
43°
(day 14)
Comments Reference
Disappeared whether Gaby,
naturally occurring or 1975
inserted under control-
led conditions

Inactivated 3 to 7
days after insertion
Disintegrated after
7 days exposure
Ova found intact after
35 days at both 2 in.
and mid-depth. Not
known if ova viable or
not

Very sensitive, did
not survive 2 days
One sample positive
taken from 2 in.
depth on day 26
No samples positive
after day 26
Positive at 2 in. depth

Positive at toe
No fungi isolated at day 35

-------
Table 4 (continued)
Compost
Swine
waste/
5% straw
Sludge/
refuse
Method
Thermophilic
windrow, mech-
anically turned
20 times per
week
American open
windrow system,
50% moisture
Species
Enteric
bacteria
Mycobacterium
tuberculosis

Duration
(days)
14
10
Temp.
60°
(max. )
65°
(av.)
Comments
Destroyed, early in-
crease in organisms
before thermophilic
temps, reached
No viable organisms
found in inserted
sample containers
Reference
Savage
et al . ,
1973
Morgan and
Macdonald,
1969
                                                                     averaging  65°  by
                                                                     third day
                                               21
 Refuse
 Night-
 soil
Windrow com-
posting, 2
turnings
Ascaris eggs

Endamoeba
histolytica
(cysts)

Endamoeba
coli (cysts)
                                   10
  3
weeks
                                                60°
                                              (day 21)
 65°
(av.)

 60°
(av.)
For one windrow
only, viable organ-
isms recovered through
day 17.  By day 21
no viable M. tuberculosis

No viable organisms
by day 10

Cysts more easily des-   Scott,
troyed than Ascaris.     1953
Ascaris eggs "usually"
destroyed by day 15

-------
Table 4 (continued)
Compost
Refuse
Method
Windrow
Species Duration
(days)
Mycobacterium phlei 9 months
(Sept.-
Salmonella abortus June)
Temp.
(°C)
87°
(av.)
Comments
All sjjecies perished
Reference
Jansen and
Kunst,
1953
                       equi

                       Micrococcus  aureus

                       Bacillus subtilis

                       M. phlei

                       B. subtilis

                       Salmonella
                       typhiitiurium

                       Pseudomonas
                       aeruginosa
3 months
(Dec.-
 March)
 68°
(max.)
All destroyed but
B. subtilis spores
in dry state still
viable
                        Erysipelotrix
                        rhusiopathiae

Raw     Composting bin  Salmonella
sewage   forced  out     newport
sludge   from bottom,
         controlled re-  Poliovirus type 1
         circulation
         around  bin      Ascaris lumbricoides

                         Candida albicans
            60° to 70°  All species destroyed
                        within 4 hours. Polio
                        virus type 1 inacti-
                        vated within 1 hour.
                                    Wiley and
                                    Westerberg,
                                          1969
                        C. albicans most resistant
                        still viable at 28 hours

-------
      Aerobic conditions may  be  promoted  by  turning  of  the  compost or by addition
  of a material  such as straw to produce  air pockets within the  decomposing waste.
  However,  too frequent turnings will  reduce the  temperature and slow the process.
  Savage  et al_.  (1973) have demonstrated  that with addition of 5% (wt/wt) straw
  and mechanical turning of swine waste 20 times  per week,  that  the temperature
  rises to  60°C  within three  days.   The more rapidly the  temperature rises to the
  thermophilic range, the  sooner the pathogenic organisms will be destroyed.

      Savage et  al.  also reported an increase of  Salmonella sp., fecal coliforms
  and fecal streptococci during  the  mesophilic stage of composting,  though all
  enteric bacteria were destroyed within  two weeks of attaining  thermophilic
  conditions.

      In  a  study of  refuse/sewage sludge  composting, Gaby (1975)  demonstrated that
  fecal streptococci maintained  populations  as high  as  10 /g even at temperatures
  of  55°  to 60°C.  He found a consistent  inverse  relationship between the number
  of  total  and fecal coliforms and the windrow temperature.   The coliforms
  appeared  to be more heat sensitive than the streptococci,  being reduced to less
  than detectable levels at 49°  to 55°C.  Though  salmonella species were often
  isolated  from  raw  sewage sludge, Gaby did  not detect  salmonella or shigella
  species in compost after seven days exposure.   All samples were negative for
 coagulase-positive staphylococci after  a day of composting except  for  one  sample
  isolated on the 49th day.

     The work of Gaby was part of a major study  to  establish the technical  feasi-
 bility of composting municipal refuse with or without sewage sludge  jointly
 undertaken at Johnson City by the Environmental Protection Agency  and  the
 Tennessee Valley Authority.   The project also included  insertion studies with
 Mycobacterium phlei (Stone and Wiles, 1975).  M. phlei  cultures were grown on
 Lowenstein Jensen  (L-J)  agar slants at  45°C for three days.  All  samples were
 prepared in duplicate (136 sets total)  and inserted into  12  windrows at  depths
 of 2 in.,  mid-depth (1 1/2 ft), and in  the toe on day 0 or day 14.  Duplicate
 slants were removed at selected times and subcultured by washing with  sterile
 phosphate  buffer (0.5 ml),  transferring to a new Lr-J slant,  and  incubating at
 37°C for at least 10 days.

     No samples  inserted  on day 0 at mid-depth were viable  after 14 days.
 Temperatures achieved at mid-depth averaged in excess of 60° for at least two to
 three weeks.  Generally  seven days at 53°C resulted in destruction of  the micro-
 organisms.  One viable sample was retrieved from mid-depth at 59°C on  the
 seventh  day of  composting, though later samples were not viable.

     The  higher  the  temperature recorded, the shorter the time exposure necessary
 to destroy the  pathogen.  Viable cells were found after 49 days at the depth of
 2 in. where the temperature  peaked at 33°C.  Viable cells  were also found up to
 21 days  where the maximum temperature recorded was 48°C.

    Morgan and  Macdonald  (1969) conducted survival  studies of the avirulent
Mycobacterium tuberculosis var. hominis  at  the  Johnson City plant.   Samples were
 inserted in the compost during  the  fall, winter, spring, and summer months.   M._
tuberculosis was normally destroyed by  the  14th  day of composting when  the


                                       14

-------
average temperature was 65°C.   For one windrow,  all microorganisms  were  des-
troyed by temperatures not exceeding  60°C.

    Several studies have compared the destruction of pathogens  in the  summer and
winter compost heap, where maximum temperatures  achieved might  be expected  to
differ.  Jansen and Kunst (1953) examined the  survival of a number  of  micro-
organisms placed in hermetically sealed  tubes, and buried in both a summer  and  a
winter dump.  They chose to study mainly non-pathogenic organisms with similar
resistance behavior to related  pathogens as a  "safety precaution."   Summer  dump
temperatures  (September to June) were shown to have risen above 87°C,  but did
not exceed 98°C (as shown by melting-point of  chemicals sealed  with the
cultures).  None of the microorganisms buried  (Mycobacterium phlei,  Salmonella
abortus equi, Micrococcus aureus, Bacillus subtilis) survived as dry or  wet
cultures.  Wet and dry controls kept  at  room temperature still  contained viable
cells in June.

    The maximum temperature of  the winter dump (December to March)  was about
68°C.  Mycobacterium phlei, Salmonella typhimurium, Pseudomonas aeruginosa  and
Erysipelotrix rhusiopathiae were all  destroyed during the process,  whether
buried as dry spores or in a liquid medium.  Bacillus subtilis  spores  were  still
viable, though B. subtilis in the liquid state was destroyed.   New  growth was
demonstrated  for all the controls kept in the  laboratory by the investigators.
Here, not only was there a demonstration of the  importance of temperature for
pathogen destruction, but an indication  that moisture content is also  important.

    Knoll (1959) also investigated survival of pathogens in both summer  and
winter windrows of mixed refuse/sludge (see Table 5).  Gelatin  capsules, inoc-
ulated with mixed and pure cultures of Salmonella sp., were placed  in  nylon bags
of MSW/sludge, and then buried  in the windrows in three different positions as
indicated below.  Temperatures  at the three locations were measured daily.
After 50 days (both summer and  winter windrows), none of the bacteria  were
recovered from the buried samples, though all  of the controls contained  viable
cells.  In a  later study, Knoll (1963) demonstrated that at average temperatures
of 45°C, S. typhi were viable up to 247  days after inoculation  of a refuse/
sludge windrow.

    While most studies have stressed  the importance of achieving thermophilic
temperatures as quickly as possible,  Strauch (1964) demonstrated that  this  is
not true for spore-formers.  Bacillus anthracis, for example, is more  easily
killed when at the vegetative stage of growth.   As germination  takes place  below
55°C, destruction of the organism is  certain if  the temperature is  kept  below
55°C for three days to permit germination, after which an increase  in
temperature (above 55°C) destroys the organism after three or more  weeks of ex-
posure (moisture content <40%).  When the temperature does not  exceed  55°C,
viable organisms have been isolated after 231  days (Golueke and McGauhey, 1970).

    While the interior of the compost heap may reach thermophilic temperatures,
the outer layers often do not reach 55°C.  Fungi colonies are known to persist
at the cooler outer edges (McGauhey et cd., 1953; Finstein and  Morris, 1975).
Many species of fungi have been detected in compost heaps, including human
pathogens (see Table 6).  When  the compost is  turned, the temperature  drops
temporarily.  There is the possibility that the  interior may be recolonized by

                                       15

-------
                                     TABLE 5

                          SPECIES INVESTIGATED BY KNOLL
                  Summer Compost
                                          Winter Compost
 Mixed
 Culture
Salmonella typhi
S. typhimurium
S. infantis
S. typhi
S. arechavaleta
S. litchfield
Pure
Culture
None indicated
S. para typhi B.
S. infantis

               Summer Compost
                                           Winter Compost
 the fungi or other microorganisms which may have settled on the compost from the
 air.  Chang and Hudson (1967)  noted reinvasion of the interior by thermophilic
 fungi at around 50°C.  As the compost cooled from a peak of 67°C, mesophilic
 fungi reappeared.   Gaby (1975) did not detect the pathogenic fungi Blastomyces
 dermatitidis and Histoplasma capsulatum in samples of MSW/sludge compost, though
 other genera were common,  especially during the cooling stage (Mucor, Rhizopus
 Penicillium, Aspergillus,  Cladosporium and Cephalotecium).  Seeded studies with
 various fungal species (Histoplasma capsulatum, Blastomyces dermatitidis,
 Geotrichum candidum and Aspergillus fumigatusT showed that none of the organisms
 survived 27 days in the windrow at temperatures up to 60°C (Gaby, 1975).  How-
 ever,  one viable sample of H._capsulatum was isolated after 26 days of exposure
 from the 2 in. depth of the windrow where temperatures did not exceed 49°C.
 Samples withdrawn from mid-depth at day 14 and day 24 were also positive for H.
 capsulatum.

     For poliovirus type 1  in raw sewage sludge, Wiley and Westerberg (1969)
 reported inactivation after only one hour at 60° to 70°C.  Gaby (1975)  reported
 that poliovirus type 2 was inactivated between three to seven days after
 insertion in MSW/sewage sludge compost.  The maximum temperature recorded for
 the  windrow  as 49°C.

     Knoll (1959, 1963)  demonstrated that elevated temperatures are but  one
 factor  accounting  for pathogen destruction during composting.   Antibiotic
 inhibitors or mutually antagonistic effects in the mixed culture of the compost
pile also result in  pathogen kill.   Knoll demonstrated this by inoculating pure
 cultures  of  Salmonella paratyphi B and S. cairo into a composting material held
at a constant temperature  of 50°C and relative humidity of 50%.   S. paratyjahd
were killed within two days, while S. cairo survived seven days.   Identical
cultures  of  the two  species were incubated at 50°C in the laboratory  (with 50%
                                       16

-------
                                    TABLE 6

             SOME * PATHOGENIC FUNGI ASSOCIATED WITH COMPOSTING
     (Golueke and McGauhey, 1970; Finstein and Morris,  1975; Gaby, 1975)
    Genera
  Species
   Disease
Torula
histolytica
Sporotrichum
schenckii
Candida
albicans
Geotrichum
 sp_.
Aspergillus
Mucor
Penicillium
 sp.
Cryptococcosis
(chronic, frequently
 fatal attacks on
 meninges and CNS,
 maybe lungs, viscera,
 skin and joints)

Sporotrichosis
(formation of nodules,
 ulcers and abscesses
 on skin and super-
 ficial lymph nodes)

Moniliasis
 (acute or subacute
  infection of skin or
  mucuous membranes
  may localize on skin
  nails, mouth, vagina,
  bronchi, lungs)

 Geotrichosis
 (lesions of mucous
  membranes resemb-
  ling thrush, in-
  fection of bronchi
  and lungs)
  Infections of
  external ear, granu-
  lomatous lesions in
  skin, nasal sinuses,
  bronchi, lungs.
* Not a complete listing.
                                      17

-------
 moisture).  S. paratyphi were destroyed within eight days, while S. cairo sur-
 vived in the incubator for 17 days.

     In a related experiment, Knoll again examined the survival of S. cairo and
 S. paratyphi B.  He prepared an aqueous extract from two day old refuse/sludge
 compost (at 50°C),  and sterilized the extract.  Pure extract, extract plus equal
 volumes of bouillon, and a bouillon control were inoculated with one of the
 species, and divided into four test series maintained at 37° and 50°C.  At 50°C,
 S. paratyphi disappeared from the extract plus bouillon after 10 days, from pure
 extract in 16 days and from the control after 30 days.  The more resistant S.
 cairo survived 14 days in the bouillon plus extract, 24 days in pure extract,
 and 30 days in the control.  Figures quoted above are the maximum values
 recorded;  for some extracts time-to-kill was much shorter.  Knoll has postulated
 that the bactericidal effects may be caused by several substances rather than
 one inhibitor.  However, Gaby (1975) was unable to demonstrate either
 bactericidal or bacteriostatic action for a wide variety of gram positive and
 gram negative bacteria found in composted solid waste/sludge mixtures.

     Many resistant forms of parasites are apparently destroyed during com-
 posting.  Stone (1949)  reported destruction of Ascaris eggs in night soil (60°
 to 66°C) after 50 to 60 days.  Bhaskaran et al. (1957) found that at 40°C,
 Ascaris eggs significantly decreased in viability in solid waste/night soil
 compost after 15 days.   However, some viable eggs were found in certain pits
 even after 6 months.  However, the compost was never turned and so anaerobic
 conditions prevailed.  Scott (1953) reported destruction of Ascaris eggs and
 cysts of Endamoeba  histolytica and Endamoeba coli within three weeks of windrow
 composting at an average temperature of 60°C.  The cysts were more easily
 destroyed  than the  Ascaris eggs which were usually destroyed by the 15th day.

     In parasite detection studies of refuse/sewage sludge compost, Gaby (1975)
 reported that 8 to  135  samples were positive for parasites (protozoa,  cestodes,
 and nematodes)  after 49 days of composting.  Three percent of all compost
 samples (day 0 to day 49)  contained one or more parasitic ova or cysts.  Though
 the parasitic forms remained morphologically intact, no determination was made
 of their viability.

     In related insertion studies,  Gaby demonstrated that while human parasites
 inserted into refuse/sludge windrows disintegrated within seven days (Table 7),
 dog parasites persisted in the compost for more than 35 days (see-Table 8).
 Ascaris, Trichuris,  Necatur,  Ancylostoma,  and Hymenolopsis were intact to the
 end of  the composting process.   Again no determination was made of their
 viability.

     Belding  (1958) has  shown that the ova of Trichuris trichuria, Necator ameri-
 canus, Ancylostoma duodenale,  Ascaris lumbricoides and Hymenolopsis diminuta are
 destroyed by  time/temperature conditions less severe than those prevalent during
 composting.   Keller  (1951)  found that temperatures of 54° to 55°C for  two hours
 inactivated Ascaris  ova in digested sludge.  Krige (1964) could not detect
Ascaris ova in municipal  compost where the temperature of the entire mass was
maintained at  60°C for  five days,  and stored for an additional 35 days  before
disturbing.   The work of  Scott reviewed previously also suggests  that the
remaining intact parasites found by Gaby could not have been  viable.

                                       18

-------
                                    TABLE  7

        SURVIVAL OF HUMAN PARASITES INSERTED IN REFUSE-SEWAGE SLUDGE
                         WINDROWS 9-5-68  TO  10-2-68
                                 (Gaby, 1975)
Day
0
7
14
21
28
Depth
Midpoint
Midpoint
Midpoint
Midpoint
Midpoint
Tenp
op

140
153
159
141
Cysts
^C E. nana E. histolytica Hookworm ova
-H-+ ++ +-H-
60
67
71
61 - -
(+++)  - Heavy infestation-many cysts or ova observed in wet mount.
(++)   - Several cysts or ova observed in wet mount.
(-)    - No cysts or ova observed in wet mount.

                                   TABLE 8

     SURVIVAL OF DOG PARASITES  INSERTED IN  REFUSE-SEWAGE SLUDGE WINDROWS
                              12-11-68 TO 1-20-69
                                 (Gaby, 1975)
                          Temp.                     Q/a	
 	Day	Depth	°F    °C    Hookworm	Tapeworm	Trichurius

       0   2", Mid     —     —

       6        2"    137.5   59

               Mid     —     —      -H-+          +++              x

      20        2"    159     71       X           +++             +++

               Mid     —     —      +-H-          +++             +++

      27        2"    120     49      +++

               Mid    150     66      +++

      35        2"    124     51      +++

	Mid             	x	X	x
 (+++) -  Heavy infestation;  large numbers of intact ova observed in wet
         mounts.
 (X)   -  Contaminated,  lost  or broken tube.
 (—)  -  Temperature  not  indicated.
                                      19

-------
  Conditions within  the  Composting  Plant

      Armstrong  and  Peterson  (1972) have  studied  the  microbial flora in and around
  a  MSW/sewage sludge  composting plant.   Methods  of sampling are discussed in
  Section  5.  Samples  were  taken during active  and inactive work periods.  Sta-
  phylococcus aureus,  gram  negative and gram positive bacilli and fungi were found
  at all locations sampled  (see Table  9 for locations and  colonies).   S. aureus
  and Alphahemolytic streptococcus  predominated during activity, with highest
  values of gram positive bacilli found at the  rejects hopper.   Fungi levels were
  high at  the leveling and metering gate, and the hand picking area.   No enteric
  coliforms were detected in the air.

      Total microbial  levels were as high at the  leveling  and metering gate and
  the rejects hopper areas as reported values in  factories (Winslow,  1926).
  Levels at other locations were not significantly different from factory levels
  and were much  lower  than reported values for  municipal incinerators and RDF
  plants (q.v. Incinerators).  Armstrong  and Peterson concluded that  the rela-
  tively low microbial counts resulted from "excellent housekeeping," the handling
  of only small  amounts of waste, and the newness of  the plant.

  Summary

      Composting appears an effective method of destroying pathogens  provided that
  sufficiently high  temperatures are maintained for long enough  periods of time.
 All  organisms within the compost must be exposed to  temperatures of 50°C to 60°C
  for  at least seven days.  This is facilitated by turning of the compost.  While
  it  seems that most pathogens are destroyed, some species may  survive.   Espec-
  ially of interest  in this regard are pathogenic fungi and parasitic ova.  It
 should be emphasized, that though pathogenic organisms may remain morphol-
 ologically intact after composting,  the viability of  the remaining  organisms  has
 not been established.

 II.  INCINERATION

 The Process

     Incineration is a method to reduce the volume and weight of municipal solid
 waste,  animal manures and  sewage sludge.  After burning  of  MSW, the  residue re-
 maining (10  to  15%  by volume of original change) is landfilled.  There are many
 designs of  incinerators from the older batch fed models  to more modern continu-
 ous rotary kilns.

     In  the conventional batch plant,  refuse is stored in  a giant refuse pit,
 typically 30 feet deep,  100  feet long and 20 feet wide (IRT, 1972).  The refuse
 is  delivered by overhead crane to a  feedhopper mechanism, which regulates feed-
 ing of  refuse onto  combustion grates  in the primary combustion chamber.  The
 grates  agitate  the  refuse  to present  maximum surface area for combustion,  in a
 conventional furnace, air  is kept  in  excess (150 to 200%) to keep furnace temp-
 eratures down to around  650°  to 1095°C.   Combustion gases from the primary
 combustion chamber  are  further incinerated in a secondary chamber.   Solids
 remaining and unburned material  are drenched with water in a residue bin.  Air
pollution control equipment  used may  be  electrostatic precipitators,  wet scrub-
bers  or bag filters.

                                      20

-------
                                                TABLE 9
                      MICROORGANISMS ASSOCIATED WITH DUST FROM COMPOST OPERATIONS*
                                     (Armstrong and Peterson,  1972)
(
w
3
u
b w
O D
rH OJ
Sampling area -c D
(0
ti
Receiving hopper (1) 2
Leveling and
metering gate (3) 5
Hand picking (6) 2
Rejects hopper (5) 3
Grinder throwback (8) 1
Premixer (9) 3
Postmixer (9) 3
Ground -waste
transfer (11) 2
Windrow (7 day old) 4
Curing and storage 2
	 jNun-operacing 	 1 i 	
H W W
4-> D OJ 0) 3
>i 0 > > 0
rH O -i-l •'-1 O
Q O 4-> 4-> Q
P y -i-i -H to -H O W
93 O W i— I tjn— I OD
£ 4-1 O -— 1 (UrH r- 1 i J-i
fOO) IU IO -H r— 1 .CD
£>-! Era E<0 Cn 03 difO
CU4J TOJ2 fOJD C 4J (C
r-l tf) VJ M 3 O 4-1
< O O £ H CO
002267

0 4 6 2 17 22
0 1 3 3 14 8
0 3 2 4 12 15
121385
0 4 4 2 13 1
0 2 1 3 9 10

1 3 2 3 11 12
0 5 5 2 16 5
002158
	 UJJtJLd Lilly 	 J
O
•H W
£3 Si SI
i— 1 O -H •«-!
O O 4-> 4->
SO -iH -H (0 -i-l
(1) O W i— 1 Dli— 1
x; 4-1 o 1-1 a; --H
1 DJ &••-* C -H
03 0) 1 O TO -H iH
.Ci-i6.<0 En3 en fa
Dj4-ifoJ3 n3J3 C 4-1
r^cn >-i M DO
rtj C3 O fcj c-i
0 8 5 9 29

5 11 14 11 63
1 8 15 14 46
3 18 11 8 55
0 6 2 2 15
1 7 5 3 17
3 12 5 4 34

1 5 10 6 34
2 18 10 3 38
2 11 2 2 25
*Microorganisms given in counts per 0.25 ft.

-------
     In the system diagrammed in Figure 1, MSW is periodically charged into the
 top of a vertical furnace which is supplementally heated by air from a super
 blast heater to 1425° to 1650°C in the gas igniter.  Slag from the base of the
 gasifier and the igniter flows into a water quench tank (not shown).  After
 cooling, the gases are cleaned of particulate matter in an emission control
 system.

     The design and operating characteristics of four incinerators investigated
 for pathogen survival are shown in Table 10.  These incinerators, located in
 Cincinnati and Chicago, were evaluated for efficacy in destroying bacteria by
 Peterson and Stutzenberger (1969).


                                   TABLE 10

                          INCINERATOR CHARACTERISTICS
                       (Peterson & Stutzenberger,  1969)
Incinerator
Characteristics
Design capacity*
No. of furnaces
Feed mechanism
Grate
I
500
2
Continuous
Traveling
II
500
4
Batch
Circular
III
1,200
4
Continuous
Rotary-kiln
IV
200
2
Batch
Reciprocating
 Operat.  Temp.   1,800°-2,000°F  1,800°-2,000°F  1,2000-1,700°F  1,800°-2,000°F
                                                   (primary)

                                                   650°-925°C

                  980°-1,090°C    980°-1,090°C  1,700°-2,200°F  980°-1,090°C
                                                (secondary)

                                                  925°-l,200°C

Duration of burn-  1.75-2.0       1.5-1.75          0.5-1.5          1.0
 ing  (hr)

Total burning rate    22            20               50              6.5
 (tons/hr)

Quench water recir-   No       No quench water     Yes        No quench water
 culated

Estimated volume    80-85%        80-85%           80-85%          80-85%
 reduction

 * Expressed as tons per 24 hr."

                                      22

-------
                             Incoming Municipal Refuse
              air
super
blast
heater
NJ
OJ
 gasifier
vertical
  	silo)
                                         slag
   gas
igniter
                                                      exhaust
   gas
cooler
                                              emission
                                                     control
               Figure 1.     Flew diagram for a high temperature combustion plant
                                              (IKT, 1972)

-------
Pathogen Survival

    Because of the high temperatures achieved,  it has often been assumed that
pathogen destruction  is complete on incineration.   Peterson and Stutzenberger
 (1969) showed that due to  inadequate incinerator design  and/or operations, that
significant numbers of coliforms and heat-resistant spore-formers survived in
the residue after incineration.

    It should be remembered that this residue  is destined  for landfill.   Of the
four  incinerators described in Table 10, fecal  coliform  levels were lowest for
incinerators # IV (< 1 count/g) (see Table 11).

    Incinerators # II and  # III also accomplished significant reductions of
fecal coliforms.  As  shown in Table 11, total cells and  total coliforms  detected
in incinerator # IV residue were also significantly lower  than in the other
three incinerators.   Note  that this incinerator was the  smallest in capacity,
and burned waste at a much slower rate  (6.5 tons/hour).  Table 11 gives  the bac-
terial populations found in both the solid waste charge  and the residues for
these incinerators.

                                    TABLE 11

          EFFICACY OF INCINERATOR OPERATIONS IN THE DESTRUCTION OF THE
               MICROFLORA  ASSOCIATED WITH MUNICIPAL SOLID  WASTES
                       (Peterson and Stutzenberger,  1969)
Material
Bacterial
Population*
Incinerator Design
I
Solid waste



Residue



Total cells
Heat resistant t
Total coliforms
Fecal Coliforms
Total cells
Heat resistant t
Total coliforms
Fecal coliforms
7.6
4.2
6.2
9.1
4.4
1.0
1.5
2.4
X
X
X
X
X
X
X
X
IO7
106

io4
107
10~"
IO4
10J
II
4.1
6.8
4.8
4.0
1.7
2.0
2,3
9
X
X
X
X
X
X
X

10^
10
IO6
10 b
IO6
IO4
IO2

5.6
2.7
5.4
1.2
1.2
3.9
4.1
5
III
X IO7
X IO4
X 10;?
X IO5
X IO6
X 10 3
X 101

IV
3.8 X
1.7 X
1.2 X
2.3 X
7.1 x
4.4 X
5
1

108
IO4
IO4
10 4
10 3
103


* Expressed as counts per gram.
t Expressed as spores per gram.

It was found that residues with a high bacterial population  (especially # I)
contained unburned vegetables, animal wastes and newspapers, while the residue
from incinerator # IV was more completely burned.

    In a related study, Peterson and Klee (1971) detected Salmonella  sp in solid
waste prior to incineration, and after incineration  in quenched residue and
quenchwater from incinerator # I.  They pointed out  that the recorded
temperatures achieved by the four incinerators were  theoretically  high enough  to
kill even the most heat resistant spores.  However,  because of the high survival

                                       24

-------
rate of both heat  susceptible  and heat resistant microorganisms,  it is clear
that masses of the charged waste do not reach these temperatures.

    The following  reasons for  incomplete combustion have been given:

         •  Organic waste is a poor conductor of heat.   Also,  as  the  charge
            burns, water is formed,  and the  temperature of the wet mass falls
            several hundred degrees.

         •  The  incinerators may be charged  beyond  their capacity.

         •  The  bulk density of the residue  may prevent complete  incineration
            and  thus sterilization does not  take place.

         •  The  forced  draft in the furnace  carries microbial aerosols up the
            stack  at high velocities.   Hence,  a sufficient time/temperature is
            not  achieved for sterilization.

         •  The  design  of the  furnace  may be inadequate.   For example,  if the
            stack  is short, there is an even greater chance that microbial
            aerosols will fail to achieve sterilization temperatures  before dis-
            charge.  The type  of internal conveyors used,  the design  of
            automatic vibrating grates,  and  even the lining of the furnace may
            all  be factors.

         •  The  contamination  of quenched residue by dust ever present in the
            incinerator environment is also  a possibility.

         •  Intermittent use of an incinerator results  in lower operating temp-
            eratures, as it takes time to raise the temperature of a  cold
            furnace.

    The significance of stack  height was investigated by Barbeito  and Gremillion
(1968) in a study  designed to  examine  the minimum temperatures required to pre-
vent release of  dry and wet spores of  Bacillus subtilis  var.  niger from the
stack exhaust of a municipal incinerator.  They mixed dry spores with animal
bedding and dumped them in the firebox.   The minimum temperatures  required to
ensure destruction of the dry  spores (concentration of  6  X 10 12 spores/ft3)  were
371°C for firebox  air and 196°C for firebrick refractory lining.   Retention time
was 26.5 seconds.  They found  that retention time of the  spores decreased with
increasing temperature, apparently due to increased velocity of expanding hot
air.

    A liquid suspension of B.  subtilis var.  niger spores was disseminated into
the firebox as an  aerosol.  At a concentration of 5.3 X 109 spores/ft3  air and
retention time of  41 seconds,  minimum  temperatures  to ensure destruction were
302°C for firebox  air and 196°C for refractory lining (A^C^SiC^). Barbeito and
Gremillion also  showed  that the concentration of spores  recovered  per cubic feet
of air sampled decreased by one log  as the spores recovered per cubic feet of
air sampled decreased by one log as  the  spores passed through the  temperature
gradients in the last 59 feet  of stack height.


                                       25

-------
     Peterson (1971),  in a progress report on studies of microbiological survival
 of the  incineration process,  collected data from eight incinerators including
 the four previously described in Table 10.  The incinerators were located in
 Cincinnati,  Chicago,  Memphis, Atlanta and New Orleans.

     A primary objective of this study was to develop and perfect sampling tech-
 niques.   The heterogeneous nature of MSW, and daily variations in its composi-
 tion, have made the task of sampling for indicator organisms an unreliable
 procedure.   It was postulated that the number of enteric bacteria in a given
 sample  of waste or residue followed a Poisson probability distribution.  Hence,
 it was  calculated  that  three  30 g subsamples (in duplicate)  were required for
 each raw refuse pile  and each incinerator residue examined to give a 95% pro-
 bability of  positive  sampling for enteric bacteria.

     Before incineration,  random samples of refuse were collected with sterile
 tongs and placed in sterile 200 ml specimen cups.  These samples were combined
 into one 2,000 to  4,000 g sample,  mixed,  and a final 200 g sub-sample was
 assayed for  total  viable bacterial cells,  total coliforms, fecal coliforms,  and
 heat-resistant spores.   Samples of quench water were similarly sampled and
 assayed (see Section  5).

     Air samples were  taken by an Anderson sampler.   Stack effluents were col-
 lected  after at least two hours of continuous normal operation using an impinge-
 ment method  adapted to  the incinerator design.   The  effluent passed through  a
 water-cooled sterile  stainless steel  tube drawn by a 1.0 ft3/minute vacuum pump
 (15 in.  mercury).   The  effluent was collected in a 30 ml aliquot of sterile
 0.067 M,  (pH 7.2)  phosphate buffer in a liter bottle.   A 10  ft3  sample was
 collected  in a 10  minute  run.   Total  viable bacterial count  in the eight raw
 refuse  samples prior  to incineration  ranged from 4.0 x 10  to 6.8 x l(r
 counts/g;  total coliform densities were from 3.4 x 10^ to 5.1 x  107  counts/g,
 with fecal coliform count of  1.5 x 104 to 8.1 x 105  counts/g.  The high fecal
 coliform density was  interpreted as indicating fecal contamination of the waste.

     None of  the incinerator residues  were sterile, though there  were significant
 differences  in the microbial  quality  of each residue.   As found  in previous
 studies  (Peterson  and Stutzenberger,  1969;  Peterson  and Klee, 1971), overall
 performance  of incinerator #  1  (continuous feed,  traveling grate) was poorest,
 with total viable  cells  (9.0  x  107  counts/g),  heat-resistant spores (1.9 x 105
 counts/g), total coliform count (1.2  x 105 counts/g),  and fecal  coliforms
 (4.7 x 103 counts/g).  Fecal  coliform densities were less than 10 counts/g of
 residue for  both incinerator  #  IV and # V.   Low total viable cells,  spores and
 coliforms were  also obtained  from incinerators # VI, # VII,  and  # VIII (see
 Table 12).

    Quench water from incinerator  # VI contained less than 1 x 102 counts/ml of
 total coliforms and 1 x 102 counts/ml of  fecal coliforms.  The total coliform
 concentration  in quench water  from incinerator VIII  was the  same as for incin-
erator VI.   Fecal  coliform concentration  for quench  water from incinerator VII
was less than 1 x  10  counts/ml.

    Quench water from incinerator  # VIII  contained total and fecal  coliform
counts of 2.9 x 104 and 1.7 x  104  counts/100 ml respectively.  Stack emissions


                                      26

-------
             Incinerator  Number
             Design         of
                          Samples
                          Tested
                                                                         TABLE 12
                                             EFFICACIES OF VARIOUS INCINERATOR DESIGNS IN THE DESTRUCTION OF
                                                    MICROFLORA ASSOCIATED WITH MUNICIPAL SOLID WASTES
                                                         (Data expressed in arithmetic averages)
                                            	        (Peterson, 1971)
                                                          Total Bacterial Count
                              Solid Waste
                                                                  Residue
Quench Waters
                      Total     Heat      Total   Fecal   Total    Heat     Total   Fecal     Total     Heat-   Total   Fecal
                      Viable  Resistant   Coli-   Coli-   Viable Resistant  Coli-   Coli-     Viable  Resist-  Coli-   Coli-,
                      Cells"''   Spores     forms^f  forms'^  Cellst   Spores   forms^  forms^   Cells  §    ant     fortnsT forms!
                     	Spores	
-J
Continuous
feed, travel-
ing grate      4

   II
Batch feed,
circulating
grate          3

   III
Continuous
feed, rotary
kiln           6

   IV
Batch feed,
reciprocating
grate          3

   V
Continuous
feed, conical
burner         4
                                   l.lxlO8 2.7xl05    3.0xl06  2.6xl05 9.0xl07  1.9xl05  1.2xl05 4.7xl03    —
                                   4.5x108 l.lxlO5    6.7xl06  S.lxlO5 l.lxlO7  2.9xl04  2.8xl02
                                    7.8xl07 3.8xl04   1.6xl06  1.2xl06 2.3xl06  9.7xl03   1.2xl02 2.0X101   —
                                    4.8xl08  3.1xl04   l.lxlO6 6.3xl05 1.3xl04  5.6xl03     < 4     <
                                    6.8xl08  1.9xl06   5-lxlO7 S.lxlO6 1.3xl06  l.lxlO5   2.GxlO1 3.8x10

-------
              TABLE 12 (continued)
Incinerator Number
Design of
Samples Total
Tested Viable
Cellst
Total Bacterial Count
Solid Waste
Heat-
Resistant
Spores
Total
Coli-
formsf
Fecal
Coli-
formsf
Total
Viable
Cellst
Residue
Heat-
Basistant
Spores

Total
Coli-
formsf

Fecal
Coli-
formsf




Quench Waters
Total
Viable
Cells?
Heat
Resist-
ant
Spores
Total
Coli-
formsf
Fecal
Coli-
forms T
00
   VI
Continuous
feed, rotary
kiln           2

   VII
Continuous
feed, rotary
kiln           2

   VIII
Continuous
feed, recipro-
cating grate   3
                                     5.4xl07  3.5xl04   1.3xl07 5.6xl06 S.SxlO1  6-OxlO1   a.OxlO2  <1.0xl02 l.OxlO3 2.0xl02  <1.0xlo2  l.Oxlt?
                                     4.0x106  2.5x104   3.4xl03 l.SxlO4  2.7x103  l.OxlQl   
-------
were sampled for incinerator  # V only.  While most microorganisms  were removed
by scrubbing, a few gram-positive bacilli  (2 counts/ft3  air)  escaped.

    This study again demonstrates that  the design and  operation of a given
incinerator is an important factor  in destruction of pathogenic organisms  which
may be present in the solid waste charge.  Of the incinerators  evaluated,  the
performance of incinerator #  IV  (batch  feed reciprocating grate) was superior in
reduction of total and  fecal  coliforms.  Incinerator # IV also  showed  a four log
order decrease in total viable cells.   Incinerator # VI (continuous feed,  rotary
kiln) showed a six log  order  reduction  in  total viable cells  as did incinerator
# VIII (continuous feed, reciprocating  grate).  Figures 2, 3, 4, and 5 illus-
trate this difference in performance for the incinerators evaluated by Peterson
and Stutzenberger (1969), Peterson  (1971), and Spino (1971).

    Spino (1971) compared the efficiency of pathogen destruction of the New
Orleans East incinerator with the other seven described above.   The New Orleans
East incinerator averaged a six  log reduction of  total bacterial count in  the
residue (3.8 x 108 to 2.1 x 102  counts/g).  The most efficient  furnace in  the
Cincinnati - Chicago studies  averaged a 37 thousand-fold reduction (4.8 x  108  to
1.3 x 104 counts/g) while the other three  furnaces averaged a 1 to 40  fold
reduction (6.6 x 108 to 2.4 x 104 counts/g).  The Memphis incinerator  gave a 27
thousand fold reduction (6.6  x 108  to 2.4  x 104 counts/g).  No  coliforms were
detected in the New Orleans East residue,  but they were recovered  in residues
from the other seven facilities.  There was an average of 100 thousand-fold
reduction of aerobic spores for  the New Orleans East incinerator compared  to 10
thousand and one thousand fold from the Cincinnati - Chicago  and Memphis studies
respectively.  Salmonella give and  Salmonella St. Paul detected in New Orleans
East refuse were not found in the incinerator residue.

Air Quality - the Incinerator and the Refuse Processing Plant

    A major concern for all operations  involved in the handling and processing
of solid waste is the quantity of dust  generated.  A number of  studies have
shown that various microorganisms,  some pathogenic, are associated with this
dust (Peterson, 1971; Armstrong  and Peterson, 1972; Glysson et.  al., 1974;  Fiscus
et aU, 1977; Duckett,  1978).

    Airborne bacteria are carried by dust  and by  water droplet  nuclei.   Dust
particles carrying bacteria may  settle  fairly rapidly,  but any  activity may  re-
suspend the particles in the  working environment.  As  Glysson et al^.,  have
pointed out, the bacteria transported by dust are probably too  large for any
lung penetration (10/u), and, hence, the dustborne bacteria may present less of
a respiratory hazard than the smaller water droplet carried bacteria.   Bacteria
transported by water droplets may remain air-suspended for some time and are
small enough (0.5 to 5/u) to  penetrate  lung tissue.  Larger bacteria would be
trapped in nasal mucosa, and  would  eventually find their way  into  the  gastro-
intestinal tract.
    Glysson et al. studied the air  quality of two incinerators  and a transfer
station.  Air~quality was evaluated using  an Andersen  air sampler  to separate
air particles automatically into six aerodynamic  sizes. Samples were  collected
five feet from the floor and  at  important  activity centers within  the  plant.
Colonies were developed for 24 hours at 37°C on trypticase soy  agar (TSA).  As

                                      29

-------
             9 .
U)
O
      g
      rd en
      -P CQ
       -H
      8
        5-1
        O
     rH
      p -p
      B tC
      M-l -P
      O d
6 .
             5  _
             1  .
                                   II   II
                                   III   III
IV
IV
                                                                             V
VI
      Figure 2.  A comparison of the efficiency of total bacteria removal of incinerators
                     (Peterson and Stutzenberger, 1969; Peterson, 1971, Spino, 1971)
        I   Continuous  feed,  traveling grate

      II   Batch  feed,  circular grate

     III   Continuous,  rotary kiln
                                                IV Batch,  reciprocating

                                                V Continuous,  conical

                                                VI Continuous,  reciprocating
                                                                                  Total  column
                                                                                  is bacterial
                                                                                  concentration
                                                                                  in MSW

                                                                                  Bacterial cone.
                                                                                  in residue
                                                                                  incinerators

-------
 s
     en
     
-------
U)
NJ
        .8
        u w
        O CJ
        o 3
        68
        o q
        IH -H
                                          Total column is
                                          total coliforms
                                          in MSW charge

                                          Total coliforms
                                          in residue
                                        II
III  III
IV  IV
V
VI
             Figure 4.   A comparison of the efficiency of total coliform removal of  incinerators
                             (Peterson and Stutzenberger, 1969;  Peterson,  1971; Spino,  1971)
               I  Continuous feed,  traveling grate

              II  Batch feed, circular grate

             III  Continuous, rotary kiln
               IV  Batch, reciprocating

                V  Continuous,  conical

               VI  Continuous,  reciprocating

-------
CO
            !
             88
            M-l
              •H

              *
            M-l
            O

            £<
            •H
            -P
                Total column is
                fecal coliforms
                in MSW charge

                Fecal coliforms
                in residue
                                                              IV   IV

                  Figure 5.   A comparison of the efficiency of fecal coliform removal of  incinerators
                                  (Peterson and Stutzenberger, 1969; Peterson,  1971; Spino,  1971)
                    I  Continuous feed, traveling rate

                   II  Batch feed, circular grate

                  III  Continuous, rotary kiln
IV  Batch, reciprocating

 V  No data given for incinerator

VI  Continuous, reciprocating

-------
expected, for all facilities the bacterial  content  of the air was very dependent
on activity within the plant.

    For the smallest incinerator (125 tons/day,  TPD),  highest bacterial levels
were recorded after washing the charging  floor of the incinerator
(334 counts/ft3).  Refuse was discharged  directly onto the concrete floor from
packer trucks, and pushed into the charging hopper  of the furnace (end-charged,
batch-fed) by a rubber-tired front-end loader.   Emptying of the trucks was
associated with high bacterial levels (183, 141.2,  172.3, 207.2 counts/ft3),  as
was charging of the hopper (240.8 and 117.5 counts/ft3).   Lowest values were
recorded for periods of little or no activity (14.5,  23.8 counts/ft3 ).   Changes
in manner of operation resulted in significant drops  of bacterial levels.   For
instance, when the trucks were emptied onto a previously wetted floor,  levels
were only 32.6 counts/ft3.  Dumping and moving bagged refuse gave an average  air
quality of 52.1 counts/ft3.

    A variety of colonies were counted without any  attempt being made to dif-
ferentiate between organisms.  Most colonies developed on the first stages of
the sampler, though the presence of smaller bacteria  (i.e.,  those likely to
present respiratory tract hazard) was also  confirmed  by viable counts on stage
five and six plates (25% particles in hazard size range).

    The presence of staphylococcus colonies was  suspected for stages 2, 3, and
4, and confirmed by culturing 33 suspected  colonies and testing for mannitol
fermentation using standard M-staphylococcus broth.   Sixteen (16)  colonies were
mannitol positive, and on microscope examination were seen to be gram positive
staphylococc i.

    In a later study of air quality at the Southeast  Oakland incinerator (600
TPD) and at a large refuse transfer station, Glysson  e_t al.  identified alpha  and
beta hemolytes and E. coli in dust samples.  Sampling methods were similar to
the previous study.  Total colonies were  developed  on TSA with 5% defibrinated
sheep blood (see Section 5) for 24 hours  at 37°C.   E.  coli was developed on
eosin methylene blue (EMB) agar for 24 hours at  37°C.

    Again, significant densities of total bacteria  were associated with tipping
and removal of unbagged refuse.  A total  bacterial  count of  1,950 counts/ft^  was
recorded on the receiving/dumping floor as the truck  was emptying refuse.
Levels of 800 and 670 counts/ft3 were recorded near the ashhopper-residue
discharge area and the charging floor, respectively.   Values for the transfer
station were similarly high when trucks were emptied  of trash.   A value of 2210
counts/ft3 was recorded at the far end of the storage pit during trash dumping.

    Despite the high total bacterial levels recorded  by Glysson e_t al., at both
the incinerator and the transfer station  E. coli values were surprisingly low
(see also Peterson,  1971)  with a maximum  value recorded of 3 counts/ft3.  This
may be explained by the inability of E. coli to  survive for  long when in the  dry
state.

    In Peterson's study (1971), dust samples were taken from the waste  dumping
areas,  the charging floors and the residue areas of six of the  incinerators pre-
viously described.  Data are summarized in Table 13.   Incinerator #  VI  which
                                      34

-------
                      TABLE 13

   CHARACTERIZATION OF GRAM - POSITIVE COCCI AND
GRAM - NEGATIVE BACILLI ISOLATED FROM 0.25 CU FT3 AIR
                (Peterson, 1971)
Sampling
area

Dumping
floor
Incinera-
tor:
I
II
III
IV
V
VI
Charging
floor
Incinera-
tor:
I
II
III
IV
V
VI
Residue
area
Incinera-
tor:
I
II
III
IV
V
VI

Total





27
34
45
43
6
2




55
43
7
34
4
1




48
22
7
6
0
0
Gram
jitaphylococcus
aureus





2
1
2
0
0
1




1
1
0
1
0
0




0
0
4
0
0
0
- positive cocci
Staphylococcus
epidermis





19
18
25
23
0
0




44
13
4
17
0
0




34
7
0
3
0
0
Diplocooccus
pneumoniae





0
1
1
0
0
0




1
0
0
0
0
0




0
0
0
0
0
0
a-hemolytic
streptococci





6
14
17
20
6
1




9
29
3
16
4
1




14
15
3
3
0
0
                       35

-------
TABLE 13 (continued)
Sampling
area
Dumping
floor
Incinera-
tor:
I
II
III
IV
V
VI
Charging
floor
Incinera-
tor:
I
II
III
IV
V
VI
Residue
area
Incinera-
tor:
I
II
III
IV
V
VI

Gram
Aerobacter
Total




82
66
28
40
100
0




31
28
22
24
17
4




25
3
11
1
7
0
species




29
26
8
26
0
0




12
10
1
13
0
2




15
0
0
1
4
0
- negative bacilli
Escherichia Klebsiella
coli




2
6
2
4
0
0




2
1
1
0
0
2




1
0
1
0
0
0
pneumoniae




1
2
0
1
0
0




0
0
0
2
0
0




0
0
0
0
0
0
Others*




50
32
18
9
100
0




17
17
20
9
17
0




9
3
10
0
3
0
*Proteus, Pseudomonas,  Alcaligenes
                                    36

-------
showed the lowest level of microbial cells was newer  and more  hygienically man-
aged than the other incinerators  sampled.  Staphylococcus aureus,  Diplococcus
pneumoniae (both pathogenic organisms associated with skin  and upper  respiratory
tract ailments) were found in small quantities in  the incinerator  dust.   Large
quantities of Staphylococcus epidermis were detected  in dust from  the dumping
and charging room floors of four  of the  incinerators,  and in dust  from the
residue area of three of the incinerators.  Alpha-hemolytic streptococci  were
detected in dust samples from all of the incinerators especially in samples col-
lected from the dumping and charging room floors.   Small amounts of E.  coli were
found in the dust of five incinerators,  indicating fecal contamination of the
wastes.  Such contamination is obviously one way in which pathogenic  organisms
may be transmitted to man and the environment.

    The microbiological content of dusts at a pilot resource recovery plant in
Washington, D.C., was examined by Duckett (1978).   The plant can process  10 to
15 tons/hour of MSW, and includes front-end recovery  of glass,  aluminum,  and
ferrous metal.  Three samples were collected during operating  and  nonoperating
periods in-plant at each of three locations:  (i)  near the  primary shredder;
(ii) near the aluminum eddy current separator; and, (iii) in the densified
refuse-derived fuel (RDF) room.   These sites were  considered very  likely  to have
high levels of microbial aerosols, and were each chosen to  represent  a "worst
possible" situation.  A total of  18 samples were collected  using an Andersen
2000 Impactor Preseparator connected with a Sierra Instruments 216 Ambient
Cascade Impactor.

    Some samples were assayed immediately after collection, while  others  were
stored over night.  Samples were  prepared by washing  off each  filter  with 0.1%
of peptone dilution water containing 0.1% Triton X-100.  Subsequent serial dilu-
tions were prepared from the original solution using  the same  general dilution
medium.  Samples were assayed for total  aerobes, total and  fecal coliforms,  and
fecal streptococci using the standard methods described by  the American Public
Health Association (APHA, 1971)  (see also Section  5).

    Large numbers of viable cells were found on all plates.  Levels of non-
respirable aerobic organisms were often  so high that  only "greater than"
estimates could be made.  These estimates ranged from <1,400 x 103 counts per  m3
air to >8,500 x 103 counts/m3 air.  Highest levels were found  in the  RDF  room
during activity periods.

    The combined respirable fraction was also highest in the RDF room during
operation, when the level peaked  at >7,000 x 103 counts/m3  air (other values -
490 x 103 and 630 x 103 counts/m3).  A maximum level  of 2,300  x 103 counts/m3
air for combined respirable size  fractions was found  near the  primary shredder
during operation.  The highest level of  respirable bacteria at site  (b) was much
lower (200 x 103 counts/m8 air).

    Coliforms were either absent  or present in much smaller concentrations.
Fecal coliforms were found in six samples, with the highest level  recorded
during activity in the RDF room  (>39 x 103 counts/m3  air).  Highest total coli-
form count was in the same sample (>40 x 103 counts/m3 air).   More than 90% of
the fecal coliforms collected were in the nonrespirable fraction of the dusts.


                                     37

-------
     Levels of fecal streptococci tended to be higher than levels of fecal
 coliforms.  Fecal streptococci were isolated from 15 of the 18 samples.  Again
 highest levels were found in the RDF room during operation (> 45 x 103 counts/m3,
 >110 x 103 counts/m3).   Overall average values for all organisms were found in
 Table 14.  Table 15 gives overall average values for other worksites to permit
 comparison.

     The fungus,  Aspergillus fumigatus,  was detected in three samples (four
 plates),  and Staphylococcus aureus in seven samples (11 plates) (Duckett, 1978).
 Both organisms were found in respirable and non-respirable size fractions.  No
 Salmonella or Shigella  were isolated.  Klebsiella pneumoniae was found in one
 respirable size sample  collected from the RDF room.  Citrobacter, Enterobacter,
 and Arizona were found  in occasional samples.   The types of organism found and
 the ratio of fecal coliforms to fecal streptococci are indicative of fecal con-
 tamination of the wastes by animals other than humans.

     Duckett stressed that various sources of error make these results approxi-
 mate rather than absolute.   Some of these sources of error are applicable to
 other studies discussed.   Possible sources of error mentioned by Duckett were:
          •  Preferential suppression of pathogens by non-pathogenic organisms
             because of  the  higher survival and growth rates of the latter;

          •  Deterioration of samples stored overnight resulting in lower rates
             of recovery;

          •  Use  of dilution and  growth  media of a non-specific nature  (i.e.,
             media for bacterial  growth  not necessarily the  preferred media for
             each organism detected);

          •  Incomplete  dispersion of clumped cells;

          •  Incomplete  elution of cells  from dust particles;

          •  The  presence  of microbial growth inhibitors.

     As  can be  seen from Tables 14 and 15,  concentrations of total  aerobes  for
 both operating and non-operating modes  are much higher than ambient  values.  The
 level of  total aerobes during operation  is very much higher than the given
 values  for factories, incinerators,  a sewage treatment plant, a resource
 recovery plant, and spray irrigation.  These very high average values are, how-
 ever, of  the same  log order as the highest  inplant value reported  by Fiscus et
 a!U  (1977) in  their study of the microbiological  quality of air in the St. Louis
 RDF plant.  The average value for all inplant locations at  the RDF plant was a
 log order lower.

    Fiscus et al.  conducted an assessment of the  relative bacteria and virus
 emissions at the St. Louis Refuse Processing Plant and other waste handling
 facilities.  The St. Louis plant was operational  from  1972  to  1976 treating
 272 Mg/day of solid waste to produce RDF.  The  other facilities tested were a
municipal incinerator, a wastewater  treatment plant, a refuse transfer station,
 and a sanitary landfill.  Testing was also undertaken  in downtown  St. Louis, and
for a refuse collection packer truck.


                                      38

-------
                                  TABLE 14

            AVERAGE VALUES OF SELECTED MICROORGANISMS PRESENT IN
                  MICROBIOLOGICAL AEROSOLS AT AN RDF PLANT
                               (Duckett, 1978)
Mode of Opera-
tion
Nonoperating
Operating
Size of Average overall concentration (counts/np)
Bacteria
All
Respirable §
Nonrespirable #
All
Respirable §
Nonrespirable #
TA*
10,240
5,370
4,875
4.5 x 10 6
1.9 x 106
2.6 x 106
FST
141
36
106
3.1 x 104
9,925
21,370
FC +
36
0
36
8,620
1,340
7,280
* TA - Total aerobes, measured by Most Probable Number  (MPN) method
t FS - Fecal streptococci, measured by MPN method
f FC - Fecal coliforms, measured by MPN method
§ Respirable    - <10 /urn
# Nonrespirable -  10 to 80 /urn

                                  TABLE 15

        REPORTED AEROSOL CONCENTRATIONS OF BACTERIA FOUND IN AMBIENT
                     AIR AND VARIOUS WORKING LOCATIONS*
Environment
    Concentrat ion (counts/m 3)
 Total         Fecal         Fecal
aerobes     streptococci   coliforms
                                                           Reference
Urban air 495
Urban air
Country air
Schools/offices
Factories
Incineration 1,410
- 6,005
2,540
1,980
3,360
3,990
-27,830
Hers & Winkler,
Winslow, 1926
Winslow, 1926
Winslow, 1926
Peterson &
Stutzenberger,
1973




1969
Resource re-
 covery plant  4,730 -12,720
Sewage treat-
 ment plant      320 -42,390
Spray irri-
 gation          106 -10,600
            13,990-19,075  140-2,440 Diaz et al., 1976

                                     Rickey & Reist, 1975

                                     Bausum et al., 1976
* See also the work of Fiscus et al., 1977.
                                      39

-------
    The purposes of the study were several:

         «  To provide comparative data on airborne  bacterial  and viral levels
            for the facilities selected;

         ©  To identify any correlation between bacterial  concentration and
            particulate size;

         «  To evaluate the efficiency of particulate and  microorganism removal
            by a pilot scale mobile filter unit provided by  EPA;  and,

         •  To determine the efficiency of removal of microorganisms,  trace
            metals and asbestos from the RDF environment by  the air classifier-
            system.

    Testing was conducted over a three or four day period  for  each facility,
partly because of cost considerations.  Samples were collected using Hi-Vol
ambient air filters and Andersen agar plate impactors stationed at in-plant and
property line locations.  Additionally, at the RDF plant,  emissions from the  air
classifier system were evaluated for microorganisms, trace metals and  asbestos,

    Samples were assayed according to the standard methods described in Section
5 (q.v.).  As all virus assays were negative for reasons not determined,  no com-
parisons were possible for viral counts.  Though assays were made for  Salmonella
sp. , Staphylococcus aureus, and Klebsiella sp. , results were generally negative,
indicating that either these species were present in very  low  concentrations,  or
if present, were not viable.

    Hi-Vol results for bacteria present are given below by rank order  (see  Table
16) .  The range of bacterial levels for both in-plant and  ambient locations is
shown in Tables 17 and 18.  These tables give maximum and  minimum values  for
each facility, but do not distinguish between different sampling  points in  the
same facility.  Average values for individual sampling points  are shown in
Figure 6 for total bacteria, Figure 7 for total calif orms, Figure 8 for fecal
coliforms, and Figure 9 for fecal streptococci.

    For in-plant samples, the total bacterial count was highest at the RDF  plant
(1.68 x 10 6 counts/m 3 in the control room), and lowest at  the  landfill (95.6
counts/m3 air for working face East) (see Table 17).  Total  and fecal  coliforms
were highest in the packer truck (352 counts/m3 for both), while  fecal strepto-
cocci levels were greatest at the waste transfer station (6,340 counts/m3 air  at
the truck ramp).
        range of in-plant and ambient values for each facility was  large, making
it difficult to interpret results.  The range of airborne bacterial levels was
greatest downwind of the RDF plant,  Since this plant was located upwind and
immediately adjacent to the incinerator, and the property-line sampling points
for both facilities were identical, it does not seem realistic to separate
ambient values for one plant from another.

    Total bacterial colonies as measured by the Andersen agar plate samples
followed a similar pattern to the Hi-Vol assays , though the results are not

                                     40

-------
                                  TABLE 16

        RANKING* OF HI-VOL SAMPLES BASED ON AVERAGE BACTERIAL LEVELS
                            (Fiscus et al., 1977)
Total bacteria    Total coliform
                     Fecal coliform     Fecal streptococci
(a) In-plant

RDF plant
Packer truck
Incinerator
Waste transfer
WWTP
Landfill
Packer truck
RDF plant
Waste transfer
Incinerator
Landfill
WWTP1"
Packer truck
RDF plant
Waste transfer
Incinerator
Landfill
WWTP
Waste transfer
Packer truck
RDF plant
Incinerator
Landfill
WWTP
(b) Ambient

  Upwind and downtown
RDF plant
Incinerator
Downtown
Waste transfer
WWTP
Landfill
RDF plant
Downtown
Incinerator
WWTP
Waste transfer
Landfill
RDF plant
Downtown
Waste transfer
Incinerators
WWTP
Landfill
RDF plant
Incinerator
Waste transfer
Downtown
WWTP
Landfill
  Downwind and downtown
RDF plant
Incinerator
Downtown
WWTP
Waste transfer
Landfill
RDF plant
Waste transfer
Incinerator
Landfill
Downtown
WWTP
RDF plant
Waste transfer
Incinerator
WWTP
Downtown
Landfill
RDF plant
Incinerator
Waste transfer
Downtown
WWTP
Landfill
* In descending order
t WWTP - Wastewater treatment plant
                                      41

-------
                                            TABLE 17

             HI-VOL IN-PLANT BACTERIA COUNT/CUBIC METER:   HIGH  AND  LOW VALUES  (MPN)*
                                      (Fiscus et al.,  1977)
Total Bacteria
Location
Packer truck
RDF plant
Waste transfer
station
Incinerator
Wastewater
treatment
Sanitary
Low
13,500
3,820
2,870
15,300
<473
<95.6
High
114,000
1,630,000
30,550
239,000
174,000
2,480
Total Col i form
Low
3.76
0.755
2.07
<0.017
<0.020
<0.020
High
>352
>213
153
18.6
0.755
16.3
Fecal Col i form
Low
1.24
0.755
0.143
<0.017
<0.020
<0.020
High
>352
30.4
10.3
4.86
<0.061
16.3
Fecal Streptococci
Low
235
10.5
14.3
2.13
<0.946
<0.956
High
411
478
6.340
411
<1.79
6.70
Most probable number method, see Section 5.

-------
                                                            TABLE 18

                             HI-VOL AMBIENT BACTERIA COUNT/CUBIC METER:   HIGH AMD LCW VALUES (MPN)*
                                                     (Fiscus et al., 1977)
Total Bacteria
Location
Incinerator
Upwind
Downwind
RDF Plant
Upwind
Downwind
Waste transfer
station
Upwind
Downwind
Wastewater
treatment
Upwind
Downwind
Sanitary
landfill
Upwind
Downwind
Down town
1
2
Low
470
1,900

2,830
949


< 477
< 469


< 477
< 477


239
< 95.2

<497
712
High
6,045
12,400

14,400
78,800


2,910
3,820


2,700
5,720


944
1,430

1,820
4,780
Total Col i form
Low
0.061
0.038

0.312
0.104


< 0.020
< 0.020


< 0.020
< 0.020


< 0.020
< 0.020

0.199
0.029
High
0.225
5.16

2.33
51.2


0.224
22.9


0.447
1.05


0.211
3.16

0.655
0.59
Fecal Col i form
Low
< 0.019
< 0.020

< 0.020
< 0.020


<0.020
< 0.018


< 0.020
< 0.020


< 0.020
< 0.020

< 0.020
< 0.019
High

-------
id
•H
I
•d
5
6j


5




4 -


3 .




2


1


0
         tit
         as
   Truck
                         04
            Bf
!
                  I       A

                Incinerator
                                   en
                                     en
                I      A

               PDF plant
 I      A

Landfill
  I       A         I       A

Waste transfer   Wastewater plant
        I - In-plant, A - Ambient
       Figure 6.   Air quality of various waste treatment  facilities - total bacteria, average valuss
                                             (Fiscus et al., 1977)

-------
Ln
      8
     1
     1
             0
           10
10
            -2
           10
                               1
                                                                          -p -p
                                                                          ll } I
                                                                          o p
                                                                          trH rH
                                                                          *w m



                                                                        ill!
                                                                        JH -H -H  a,
                                                                        EH H EH  !J
                 Truck
                              I      A        I       A

                             RDF plant        Landfill

                I - In-plant,  A - Antoient
I       A
Incinerator
 I       A       I        A

Waste transfer Wastewater plant
             Figure 7.  Air quality of various waste treatirent facilities - total coliforms, average values
                                                  (Fiscus et al., 1977)

-------
           102
CTl
       8   10
             0
       •H
       H
       8
       u
       fi
           10'
             ,-1
           10
-2
       ::•
      m
      m<
                 Bft'-
                         I      A       I       A       I       A
                Truck     Incinerator    RDF plant      Landfill
                         I - In-plant, A - Anbient
                                                           I       A         I         A
                                                           Waste  transfer   Wastewater plant
             Figure 8.   Air quality of various waste treatment facilities- fecal colifonns,  average  values
                                                  (Fiscus et al./ 1977)

-------
--J
           10"2
A
I       A
 RDF plant
                 Truck    Incinerator

                       I - In-plant, A - Anbient
I      A
Landfill
I       A
Waste transfer
 I       A
Wastewater plant
        Figure 9.  Air quality of various waste treatment facilities - fecal streptococci, average values
                                              (Fiscus et al.f 1977)

-------
directly comparable because of differences in the nature  and methodology of the
sampling.  One obvious difference between the two. was the very high values of
total bacteria found in the pressroom of the sewage  treatment  plant during dump-
ing of the filter cake (1.55 x 105 counts/m3).  The  Hi-Vol  samples were taken
over a longer period of time (six hours), and hence  the effect of this activity
is not seen for the Hi-Vol samples.

    Because the results were difficult to interpret, a statistical analysis of
the data was undertaken.  Analysis of variance procedures showed  that RDF plant
concentrations were either significantly higher (95% confidence level)  than the
other locations, or there was no statistically significant  difference.   The RDF
plant values were never significantly lower than the other  facilities.   Ambient
fecal streptococci values were significantly higher  at the  RDF plant than the
other locations, but in-plant samples were not significantly different.   Since
only a few replicate samples were collected, and the range  of  values was so high
for each site, a greater number of samples might present  a  very different set of
statistical conclusions.

    Some of the Hi-Vol and the Andersen samples were examined  microscopically.
Gram-positive and gram-negative rods predominated with some gram-negative cocci
and actinomycetes.

    Particulate tests on the air classifier system at the RDF  plant showed un-
controlled particulate emissions of 14.2 to 17.8 kg/hr.   Total bacteria in the
emissions averaged 5.3 x 107 counts/g (approximately the  same  concentration as
found in raw, shredded refuse).  The pilot scale mobile filter, taking  a side-
stream drawoff of the air classifier emissions, removed 99.95% of the particu-
late mass, 99.6% of total bacteria, and at least 99.9% of total coliforms.   It
seems clear that a filter system on the air classifier discharge  should be very
efficient at removing particulate matter and the associated bacteria.

    Andersen samples do not give an indication of the number of bacteria carried
by a particle (which may vary), but do indicate the number  of  particles carrying
bacteria.  The results of the Andersen samples showed that  the smaller  sized
bacteria, as transported by suspended particulate matter, represented a signifi-
cant percentage of the total bacterial count for all facilities tested.

    As previously mentioned, the smaller sized bacteria present a greater
respiratory hazard than the larger bacteria, which are more likely ^to enter the
gastrointestinal tract via the upper respiratory system.  The  health implica-
tions of high dust levels in solid waste processing plants  are, however,
unclear.  One epidemiological study reported an increase  in chronic bronchitis
among sanitation workers, but did not control for smoking habits  (Ducel et al.,
1976).  An earlier study showed that cardiovascular disease, arthritis,  and skin
complaints were some of the occupational hazards of New York City refuse col-
lectors (Sliepcevich, 1955).  Cimino (1975) was unable to show that respiratory
diseases, skin infections, or asthma were significantly more prevalent  among New
York sanitation workers than other working groups, but did  confirm the  high rate
of cardiovascular disease.

Summary

    It is clear from the studies completed to date that various bacteria may

                                     48

-------
survive incineration.  Possible reasons for this survival  in spite of high  tem-
peratures achieved within the furnace have been reviewed.  The degree to which
both heat-tolerant and heat-susceptible organisms do survive incineration varies
greatly, depending on the design and operation of each furnace.  The detection
of Salmonella sp. and their survival in residue after incineration is indicative
of the potential health hazard associated with incomplete  sterilization of  solid
wastes.  The presence of fecal coliforms in waste, residue, and quench water  is
indicative of fecal contamination of MSW; and spread of pathogenic organisms
from feces is obviously a possibility where sterilization  is incomplete.

    As shown in these studies, dust and associated bacteria levels are very high
within the incinerator and RDF plant environment if proper dust control measures
are not taken.  Total bacteria levels at an RDF plant during activity are
especially high, and may exceed a million counts/m3 air if uncontrolled.  It  is
clear that certain precautions should be taken when working in such a dust-laden
environment.  Various dust control systems have been evaluated for the Environ-
mental Protection Agency-  The St. Louis study has already been mentioned.  To
recapitulate, the attachment of a baghouse to a side stream drawoff of the  air
classifier system removed 99.95% of the particulate mass,  99.6% of total
bacteria, and at least 99.9% of total coliforms.  This same device was tested at
an RDF plant in Houston, Texas.  Dust and associated bacteria removal
efficiences were up to 99.6% (Freeman, 1978).

    In addition to installation of a properly designed dust control system, all
activities which tend to aerate or agitate the refuse should be kept to a
minimum and where possible circumvented.  For example, use of a conveyer to
carry refuse through the plant would reduce dust levels.   Bagging of refuse is
also a useful procedure.  Glysson et al. (1974) recommended the use of dust
masks by personnel during high activity periods, and also  suggested that some
form of wet-vacuuming would help control dust generation.  The introduction of
handling procedures that reduce human exposure to the dust should obviously be
encouraged as a precautionary measure for all facilities processing solid waste.

III.  SANITARY LANDFILL

The Process

    Land disposal is the final method of disposing of solid wastes whether  or
not they have been previously treated.  This is because other methods of waste
treatment (incineration, composting, anaerobic digestion,  etc.) reduce weight
and volume of the waste, but still leave a residue.  Sanitary landfill is an
environmentally acceptable way of land-disposing of solid  wastes.  The selection
of a site for the fill is critical, and should include the following
considerations:

         •  hydrogeological survey of the area to determine the potential for
            vertical and horizontal leaching from the fill,

         •  access roads to the area,

         •  availability of cover materials; and,

         •  grades for proper drainage.

                                     49

-------
    There are several appropriate methods of operating  a  fill  depending on the
topography of the area and the climate.  Where the water  table is low,  trenches
are dug and layers of rubbish and excavated earth are alternated in the fill.
For unprocessed municipal waste, the rubbish is covered with soil daily (6 in.
layer).  For shredded waste or residues from previous treatments (incineration,
digestion, etc.), it is not necessary to cover with soil  daily.

    Where the water table is high, refuse is layered above  the ground to heights
of 50 to 60 feet, when a final two-foot layer of soil covers the mound.  When
the terrain is uneven, a ramp or slope method is used.  Rubbish  is layered on
the slope and covered with soil lying above the fill.

    The rate and degree of bacterial activity within the  fill  depends on the
following factors (Engelbrecht and Amirhor, 1975):

         •  the moisture content of the fill,

         •  the temperature,

         •  the composition and properties of the waste material,

         •  microorganisms present in the waste or soil cover, and

         •  overall environment of the fill including oxygen availability.

    The moisture content of MSW is usually in the range of  20  to 35%.  The
moisture content of the fill may be increased by rainfall,  runoff from the sur-
rounding area, or seepage into the fill.  At 40 to 60%  moisture,  microorganisms
within the fill are very active.

    Though initially aerobic conditions and, hence, aerobic bacteria  prevail in
the fill, after a few days the limited oxygen supply -is exhausted,  and
facultative and anaerobic microorganisms predominate.   The  initial exothermic,
aerobic bioreaction may result in a temperature high of around 60°C (Farguhar
and Rovers, 1973) though the temperature high is also dependent  upon  the initial
temperature of the fill material.  Subsequent chemical  and  biological reactions
are primarily endothermic.

    Temperatures within the fill slowly decrease from the initial peak value,
and within the fill essentially anaerobic conditions prevail.  Biological action
by mesophilic organisms decomposes the organic matter to  produce methane gas,
water, and the biodegraded residue.  The activity is analogous to the anaerobic
digestion process suggested for methane recovery from wastes.

    While sanitary landfill may be an environmentally acceptable means of solid
waste disposal, methods of operating fills are not always sound.   Hazards
associated with improper landfill practices may be aesthetic (unsightly appear-
ance, blowing paper, etc.), obvious health dangers (accumulation,  rats, insects,
birds), or involve spontaneous combustion of escaping gas.
                                      50

-------
Survival of Pathogens

    The degree of hazard presented  by pathogenic organisms which may be present
in a landfill depends on three basic  factors:

         •  the  initial concentration and nature of contaminant in the waste;

         •  the  extent of  survival  of pathogens within the fill;  and,

         •  possible leaching of pathogens from the fill  into the environment.

    As already established, MSW and other solid wastes may be heavily  contami-
nated by a variety of pathogenic microorganisms.   Composted material destined
for landfill may contain pathogenic fungi and/or parasites (Gaby, 1975).   Simi-
larly, the sludge from anaerobic digestion,  especially the mesophilic
modification, may not be sterile.   Given the initial contamination of  the  solid
waste to be landfilled, the survival  of  enteric organisms  expected to  be present
in waste (fecal  coliforms, fecal streptococci,  Salmonella  sp.  enteroviruses)
must be examined in the environment of the fill,  as well as possible leaching of
pathogens from the site.

    Excess moisture in the fill may result in  formation of a  leachate  containing
water soluble chemicals, biological species, and suspended particulate matter.
Leachate may seep from the fill at  the surface,  or percolate  through the fill
and the surrounding soil.  Where drainage pipes are installed,  the leachate may
be collected and treated if required.  The amount of leachate produced by  a fill
depends principally on the composition of the  solid waste,  the hydrological con-
ditions and the  nature and gradient of the soil cover (Engelbrecht and Amirhor
1975).  Note that not all  landfills generate leachate.  The physical and
chemical characteristics of leachate  vary widely (see Table 19).   While the
chemical properties of the leachate influence  its biological  composition,  it is
not possible with present knowledge to make a  direct correlation  between the two
sets of properties (Engelbrecht and Arnirhoir, 1975).

    Various studies have indicated  that  there  is a significant bacterial popu-
lation associated with leachate, but  that this population  decreases in density
with time of operation or  time of leaching (Quasim and Burchinal,  1970;  Blannon
and Peterson, 1974; Cooper et al.,  1974a;  Engelbrecht et cd.,  1974;  Engelbrecht
and Amirhor, 1975; Glotzbecker and  Novello,  1975).

    Blannon and  Peterson (1974) studied  the occurrence and survival of fecal
coliforms and fecal streptococci in leachates  collected from  an experimental
field-scale sanitary landfill over  an 11 month period.  The trench-fill sampled
was 140 feet long by 30 feet wide (see Figure  10).   The fill  contained 435 tons
(wet weight) of  compacted  solid waste (1,035 Ib/yd3).   The bottom of the fill
was lined with a clay soil liner (18  in.).   The first leachate was collected
approximately six weeks after placement.   Additional samples  were collected
weekly from two  pipes lying one within and one below the clay liner.  Tempera-
tures at the center of the fill peaked at 60°C after a week,  and  gradually
decreased to 10° to 16°C after 11 months.   The edges of the fill  were  some 5° to
9°C lower than the center.
                                     51

-------
                             140 ft
                                                                     8% ft
                                                                Polyethene, 6 mil
                                                                Hypalon liner, 30 mil
Figure 10.  Cross-section of an experimental sanitary landfill
                 (Blannon and Peterson, 1974)

-------
                          TABLE 19




RANGE OF CHEMICAL COMPOSITION OF SANITARY LANDFILL LEACHATE"
Constituent
COD
BOD 5
TOC
pH
TS
TDS
TSS
Specific Conductance
Alkalinity (CaCOa)
Hardness (CaCOs)
Total-P
Ortho-P
NH.^N
NO 3 + N02-N
Ca
Cl
Na
K
S04
Range of Valuest
40-89,520
81-33,360
256-28,000
3.7-8.5
0-59,200
584-44,900
10-700
2,810-16,800
0-20,850
0-22,800
0-130
6.5-85
0-1,106
0.2-10.29
50-7,200
4.7-2,467
0-7,700
28-3,770
1-1,558
                                            (continued)
                              53

-------
TABLE 19  (continued)
             Constituent                         Range of Valuest


             Na                                  0.09-125

             Mg                                    17-15,600

             Fe                                     0-2,820

             Zn                                     0-370

             Cu                                     0-9.9

             Cd                                < 0.03-17

             Pb                                < 0.10-2.0
* All constituents ariTlsxpressedTh mg/1 except pH and specific conductance
  (/u noh/cm).
1" Summary Report:   Municipal Solid Waste Generated Gas and Leachates.
  Internal Report, U.S.  Environmental Protection Agency, National Environ-
  mental Research  Center,  Cincinnati, Ohio,  1974.  Cited in Engelbrecht and
  Amirhor, 1975.
                                     54

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    Organisms were determined by  the most  probable number technique (MPN)
considered to be more accurate  than the membrane  filter method (MF).   Smith
(1972) and Glotzbecker  (1974) have compared  the two methods  and shown that
density of total and fecal coliforms in leachate  may be 2 or 3 logs higher using
the MPN method.  However, Engelbrecht et al_.  (1974) have called for comparison
of the two techniques with a control system  included in the  experimental design,
and with different suspending media.

    A total of 60 streptococcal strains were identified (see Table  20).  Fecal
streptococci and fecal  coliforms  were detected in leachates  from both pipes.
Organisms found in the  upper pipe were believed to have leached from  the cooler
edges of the fill.  The large number of organisms found in the lower  pipe
indicated that the clay soil liner was a poor filter.   During the first two
month period, average fecal streptococci densities in  samples from  the upper  and
lower pipes were 48 million and 460,000 organisms/100  ml respectively.  In the
upper pipe, fecal streptococci densities exceeded 10,000 organisms/100 ml  during
the final sampling period.  Fecal streptococci densities averaged 2,900
organisms/100 ml from the lower tube after 11 months.

                                    TABLE  20

                    OCCURENCE OF  STREPTOCOCCI IN  LEACHATE
                         (Blannon & Peterson, 1974)
     Species
Strains
Sanitary Significance
S.Faecalis var. liquefaciens   58.33   35


atypical S. Faecalis var.      8.33    5


S. faecalis biotype II        15       9

S. faecalis biotype III        5       3



S. durans                      5       3

S. equinus                     8.33    5
            none— wide spread in
             environment

            none— associated with
              vegetation
            Indicative of fecal contami-
              nation of the waste by
              warm-blooded animals
    Initial fecal coliform densities averaged 1.5 million/100 ml  (upper
tube) and 280,000 organisms/100 ml  (lower tube).  Fecal coliform  densities
dropped fairly rapidly in leachates from both tubes.   In the upper  tube
values varied from 30 to 180 organisms/100 ml for the  last six months of  the
investigation.  Fecal coliform densities in leachates  from the lower tube
averaged 20 organisms/ml for the last 2 months of the  survey (see Table 21).
                                     55

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    Blannon and Peterson found that the ratio of  fecal  coliforms  to fecal
streptococci in the leachate ranged from  0.011  to 0.272 for  four  samples
collected during the initial five weeks of leaching.  One  sample  was found
with a ratio of 6.202.  Human waste would be expected to have  a fecal
coliform to fecal streptococci ratio of 4 or more, while the ratio for
warm-blooded animals would be less than 0.6 (Geldreich  and Kenner,  1969).
Since the ratio of fecal colifonus to fecal streptococci reported by Blannon
and Peterson is low, the leachate was most likely contaminated by animal
excreta rather than human feces.

                                  TABLE 21

  DISTRIBUTION OF FECAL COLIFORMS AND FECAL STREPTOCOCCI IN  LEACHATES FROM
                               THE UPPER  PIPE
                         (Blannon & Peterson, 1974)
Collection Date
8-31-71
9-1-71
9-13-71
9-20-71
9-27-71
Densities/lOOml
Fecal col i forms Fecal Streptococci
2.6 x 106
4.9 x 106
2 x 103
9 x 103
3.3 x 104
2.4 x 108
7.9 x 105
7.9 x 104
3.3 x 104
1.70x 105
Ratio
FC/FS
0.011
6.202
0.025
0.272
0.194
    In a related study, Glotzbecker and Novello  (1975) compared the survival
rates of poliovirus and bacterial indicators in the same experimental fill,
and in an operating municipal landfill.  Over a 4 1/2 month period, fecal
coliform densities in the municipal landfill leachate dropped from 4.6 x 102
to 23 counts/100 ml (MPN).  Fecal streptococcal levels dropped from 1.5 x 103
to 1.1 x HT counts/100 ml.

    Survival studies were conducted using Escherichia coli ATCC 11229, Strep-
tococcus faecalis SEC and poliovirus 1 Mahoney (LP) EKP-42.  Within three
hours at 10°C, there was a 99.9% reduction of E. coli in the experimental
landfill leachate.  S. faecalis survived just over five hours.  Survival
times were much higher for the municipal landfill.  At 10°C, 99.9% reduction
of E. coli took 56 days, and for S. faecalis more than 100 days.  At 20°C,
survival time of E. coli dropped to 21 days, and S. faecalis to 35 days.  The
leachates collected were transported in sample bottles containing EDTA to
protect against inactivation of bacteria on transport from the fills to the
laboratory.  Addition of EDTA to the cell culture improved recovery of
viruses (plaque method), a finding in agreement with other studies (Cooper et
al., 1974a; Sobsey et al., 1975).

    Poliovirus survival was less variable for the two fills.  On day 90 at a
temperature of 10°C, there was 8.9% viral recovery for the municipal landfill

                                     56

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and 36% for the experimental  trench.   On  day 90  at  20°C,  recovery from the
municipal fill was 0.01% and  0.43% respectively.  There was  a much more rapid
decrease in viral concentrations  at  20°C  than at 10°C indicating  the  suscepti-
bility of the viruses to inactivation  at  the higher temperature.

    Engelbrecht (1973) also found a  decrease in  bacterial population  with
temperature.  He showed that  the  inactivation of Salmonella  typhimurium,
fecal streptococci, and polioviruses was  more rapid at 55°C  than  at 22°C for
a simulated landfill (lysimeter).

    It was also found that inactivation of  both  the bacteria and  the  viruses
was pH dependent.  At pH 7.0  and  55°C, fecal streptococci were stable in
leachate for up to eight days,  but were reduced  in  density by four logs
within 24 hours at pH 5.3.  S.  typhimurium  was more stable at both pH values
than fecal coliforms, and more  stable  than  the fecal streptococci at  pH 5.3.

    Since landfill leachates  commonly  have  pH values between 5.0  and  5.5
because of formation of organic acids  (Fungaroli  and Steiner,  1971),  these
results seem to suggest that  the  known pathogen,  S.  typhimurium,  is more
stable in the landfill leachate than the  indicator  organism.However,  as the
rate of die-away of the Salmonella was still high (four logs within eight
days at pH 5.3, 55°C), and its  density should be  lower than  that  of the indi-
cator organism, survival of S.  typhimurium  seems  unlikely in the  environment
of the fill.

    The rate of virus inactivation was also greater at the lower  pH and
higher temperature.  No viruses could  be  detected at either  pH 7  or pH  5.3
after a few minutes at 55°C.  At  22°C, the  rate of  inactivation was much
slower (30% to 50% in 100 hours at pH  7,  and 95%  in 100 hours at  pH 5.3).
Though the poliovirus seems sensitive  to  low pH values, some other entero-
viruses are acid stable.

    In a later study, Engelbrecht et_ al_.  (1974) examined  the decrease of bac-
terial population with time of  leaching from a simulated  landfill.  They
reported that the total plate count  of bacteria  in  leachate  samples decreased
logarithmically over a 50 day period following first appearance of the  leach-
ate.  Total and fecal coliforms persisted for 40  to 60 days,  and  then rapidly
disappeared.

    It was observed that initial  densities  of total coliform,  fecal coliform,
and fecal streptococci reported by Engelbrecht et_ al.  were several logs lower
than values reported by other investigators (Quasim and Burchinal, 1970;
Blannon and Peterson, 1974; Cooper et aJU,  1974b).   Figures  11, 12, and 13
illustrate these differences  which may have resulted from different methods
of analyzing the samples.  Engelbrecht et al.  used  the membrane filter  tech-
nique, while the other studies  shown used the MPN method.  Also a factor is
that the MSW had probably started to biodegrade prior to  filling  of the lysi-
meter.  The waste was collected in a residential  area of  Cincinnati,  randomly
selected and segregated, milled to a 1 in.  size  for placement in  the  lysi-
meter, and placed in cold storage in barrels for  several  days prior to  ship-
ping to Urbana for this study.  The  lysimeter was filled  with the waste five
days after collection.  A temperature  high  of 65°C  in the waste pile  strongly


                                     57

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   I
   -p
   E'
    o
    u
   •r)
    cn
(0
"S
u
             10
          10
             10
              10
              10
                            O—0 From Cooper et ot . ,  1974b
                            0—Q From Engctorecht et ol . ,  1974
                                  From Qosim And Burchinol ,  19 70
                                50           100

                            Time of  Leaching  (days)
Figure  11.   Change in  total coliform bacteria with time of  leaching
                     (Engelbrecht and Amirhor, 1975)
                                   58

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I
If
•5!
§
Q
 -
a
     10
8    10
10
                               From Cooper e' Jl., 19 74a
                               From Enqelbrecht et ol.j 1974

                            D  UpPCr Pif>e From  Blonnon And Peterson.  1974
                        	•  Lover Pipe
                    50
                                                   <_O[
                                                  JL.
                                                       •
                                                     <0.l
                                                    ~5P-+
                          100       150        200       250


                                  Time of Leaching  (days)
	oW
      300
   Figure 12.   Change in density of  fecal coliforms with time of leaching

                         (Engelbrecht and Amirhor, 1975)
                                        59

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      10'
 CO
 -p

•H
cn
      10'
      10-
      10
      10'
      102
               O   O From Cooper etfli ,j 1974 t>
               &	a From Engelbrecht ejal-, 1974
               D-—0 Upper Pipe
               •	• Lower Pipe
                                                            3|Qnfx)n An
-------
suggests that aerobic  activity  had  already begun prior to placement in the
lysimeter.  Hence, partial  thermal  inactivation of bacteria is also a possi-
bility.

    In an attempt to describe bactericidal and  virucidal  factors within the
fill further, Engelbrecht and Amirhor  (1975)  reexamined conditions affect-
ing survival of S. typhimurium,  fecal  streptococci,  and poliovirus in the
fill.  Leachate samples were collected from a lysimeter containing milled  MSW
(3,358 Ib), and from two landfills  (one closed,  the other operational).

    Again it was found that inactivation was more rapid at pH  5.4 than at  pH
7, and increased with  increase  in temperature.   At 22°C,  the effect of the
lower pH was more marked than at 55°C.   For S.  typhimurium at  22°C and pH  5.4,
there was a four log decrease in 48 hours  (4.15 x KP  cfu/ml to 60 cfu/ml).   '
At pH 7.0 for the same time and temperature,  S.  typhimurium decreased approx-
imately 3 logs (4.5 x  105 to 5  x 102 cfu/ml).   After only four hours at 55°C,
S. typhimurium concentration was less  than 1 cfu/ml at both pH values.

    For fecal streptococci, values  dropped by six logs after 48 hours at pH
5.4 (2.99 x 106 to 4 cfu/ml), and by four  logs  at pH 7.0.   Fecal strepto-
cocci were very sensitive to the 55°C  temperature at both pH values
(<1 cfu/ml after four hours).   Poliovirus  inactivation was also very rapid at
pH 5.4 and 55°C (<10 PFU/ml after 15 minutes compared  to  1.30  PFU/ml at pH 7).

    Engelbrecht and Amirhor also showed that both the  dilution of leachate
with water, and the age of  material in the fill influenced the activation
capacity.  Reduction in activation  capacity was found  to  vary  directly with
degree of dilution.  For these  studies,  inactivation of bacteria in leachate
from the older fill was generally less than for leachate  from  the younger
fill.

    An attempt was made to  correlate the chemical and  biological composition
of the leachate.  Using ultrafiltration techniques to  fractionate the leach-
ate, it was found that the  degree of inactivation was  greatest for a fraction
identified as 500 MW permeate (i.e., leachate containing material with molec-
ular weights greater than 500).  This  fraction  contained  relatively high con-
centrations of iron and zinc cations,  and  short chained fatty  acids.

    Reverse osmosis (RD) further fractionated the 500  MW  permeate.   The  RO
permeate had a more pronounced  effect  on the stability of both the "S.  typhi-
murium and the poliovirus,  but  no apparent effect on fecal streptococci.
Conversely, the RO retentate had a  significant  effect  on  the fecal strepto-
cocci but no obvious effect on  S. typhimurium.   Inactivation of poliovirus by
RO retentate proceeded slowly for .the  first ten days (50%  inactivation),
after which the rate of inactivation increased  considerably.

    RO permeate contained greater concentrations of very  low molecular weight
fatty acids, and metal ions than the RO retentate.   It was suggested that  the
high concentration of fatty acids,  and iron and  zinc cations was partly re-
sponsible for inactivation of the S. typhimurium and the  poliovirus.   It was
not clear why the rate of inactivation of  the fecal streptococci was greater
in the RO retentate.

                                     61

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    Results of inactivation tests with both bacteria and the poliovirus in
synthetic salt solution confirmed that high concentrations of  iron  and  zinc
were associated with rapid inactivation, especially of the bacteria.

    More recently, Riley et al_. (1977) have investigated the possibility that
the aliphatic acids present in raw leachate exert a bacteriostatic  action on
coliforms.  Chromatographic studies (paper and gas) established the presence
of acetic, proprionic, butyric, n-valeric, isovaleric, and n-caproic acids in
leachate collected from an aluminum pipe at the base of an operating waste
tip.  Assay of this leachate on nutrient agar plates seeded with E. coli pro-
duced small, indistinct zones of growth inhibition.  If the acids were  re-
moved by ether extraction, then the "stripped" leachate did not inhibit coli-
form growth, while the acid concentrates gave sharp zones of inhibition meas-
uring 33 mm in diameter.

    An on-going study of the chemical and microbiological characteristics of
landfill leachates is now being conducted by Scarpino (1978).  The  primary
objective of this study is to determine the health and environmental signif-
icance of the persistence of fecal streptococci in landfill leachates.   The
project is divided into two phases:  (i) to verify analytical methods and to
determine the presence of indicator organisms in leachate, and (ii) to  study
the relationship between the extent of waste decomposition and the microbial
population dynamics.  First published results of his investigation will  be
available by the end of the year.

    Cooper et al. (1974a) studied the effect of adding disposable diapers on
the composition of leachate from lysimeters simulating sanitary landfills and
open dumps.  Sixteen "fills" and "open dumps" were loaded with unsorted
refuse that had been hammermilled 24 hours previously (densities of refuse
856 to 1000 lb/yd3 in the fills, 676 741 lb/yd3 in the open dumps).  Various
amounts of diapers and feces were added to 12 of the lysimeters.  The con-
centration of viruses added to each of the lysimeters was also varied.   The
controls were two simulated sanitary landfills and two simulated open dumps
which received no feces, diapers, or viruses.  Two fills were brought to
field capacity by weekly additions of water over a 16 week period, while the
rest were saturated with water over a two week period.  Maximum temperatures
achieved were 29° to 38°C for the top 14 in. depth of fill, and 46° to  57°C
in the open dumps.

    The total aerobic count decreased fairly steadily in leachate from  lysi-
meters with and without added human infant feces and diapers.  The  addition
of feces and diapers resulted in no noticeable difference in the rate of
biodegradation of the MSW.  The refuse itself had an extremely high level of
fecal streptococci (108/100 ml) and coliforms (105 to 106/100 ml).  Total and
fecal coliform densities were similar, indicating widespread fecal  contami-
nation of the wastes.  The ratio of fecal coliforms to fecal streptococci in
the fresh waste indicated the presence of animal excreta in the MSW.  Table
22 gives the initial and final bacterial concentrations in the leachate
samples.
                                     62

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                                  TABLE 22

  MICROFLORA OF MUNICIPAL SOLID WASTE AND LEACHATE FROM SIMULATED LANDFILLS
                           (Cooper et a^., 1974a)

  Organism	Concentration	

Total aerobic count               Initial counts of 106 to 10 7 counts/100 ml
                                  declined 2 to 3 logs over 20 weeks

Total coliform                    Initial counts of 105 to 106 counts/100 ml
                                  declined rapidly to 10  within 8 to 10
                                  weeks

Fecal coliform                    Composed 50 to 90 +% of total coliform
                                  count, behaved similarly

Fecal steptococci                 Initial counts very high, about 108/100 ml
                                  declining to 104 to 105 /100 ml at 20 weeks


    Cooper e_t al_.  (1974a, 1974b) also found that the appearance of poliovirus
type 1 in the leachates was sporadic over a 20 week period.  Virus recovery
efficiency using EDTA and diluting the preclarified leachate with water 8:1
averaged 60%.  No viruses were recovered from the two lysimeters brought to
field capacity over a 16 week period.  For lysimeters brought to field
capacity within two weeks, there was very low recovery of viruses from the
first samples (average 2.5 PFU/5 ml).  These results were considered suspect
by the investigators, so several lysimeters were emptied, refilled, and
reseeded with viruses.  For the second test run, there were three consecutive
weekly recoveries of 150, 2,310, and 380 PFU/gallon."  All further samples
were negative for  the presence of viruses.  No viruses were detected in
leachate from the open dumps, where, as previously mentioned, temperatures
averaged almost 20°C higher than the lysimeters.

    Other studies on virus survival in the landfill environment seem to agree
with Cooper  et a^. that few or no viruses appear in the leachate  (Peterson,
1971a; Engelbrecht, 1973; Engelbrecht et a^., 1974; and Sobsey et. al_. , 1975).
Peterson detected poliovirus type 3 (150 PFU/100 ml) in one of 13 samples of
leachate from a lysimeter containing raw MSW.  Here again, the lysimeter was
brought to field capacity more rapidly than would be considered normal, pro-
ducing leachate after only three days.  Peterson also examined the survival
of poliovirus type-1 in a full scale sanitary landfill.  Viruses were seeded
onto ground  MSW which was placed in nylon bags, and inserted  into a working
fill during  normal operation.  The bags were withdrawn periodically; on
examination  no viruses were detected.  Peterson also determined that there
was no virus migration in the fill, and concluded that at 40° to  60°C sur-
vival time of poliovirus type 1 in the fill would be about two to four days.

    Engelbrecht et al. (1974) studied the inactivation of poliovirus type 1
and reovirus in a  simulated landfill.  They added viruses to  the  supernatant

-------
of centrifuged leachate samples, and  found  that poliovirus  type  1  was not
significantly inactivated by initial  leachates collected  (days 36  to 47 of
lysimeter operation).  Poliovirus inactivation by older samples  of leachate
was more rapid.  However, as the leachates  became cloudy, and the  samples
were not treated with EDTA before virus assay, it was not certain  if the
viruses were inactivated, or adsorbed onto  particulate matter in the leachate
sample.  In contrast, reovirus was significantly inactivated after 36 days of
operation, and disappeared from the day 47  leachate after only 10  minutes
exposure.

    Sobsey et a^. (1975) studied the  survival of enteroviruses when inocul-
ated into simulated refuse contained  in two lysimeters.  Both laboratory and
field strains of poliovirus type 1 and echovirus type 7 were investigated.
None of these viruses were detected in leachate samples collected  over a four
month period.  The viruses were assayed by  the plaque technique  (small
volumes) and by the tissue culture technique  (large volumes).  The pH of the
lysimeters ranged from 5.4 to 6.1.

    When viruses were added to the leachates, they were recovered  with an
efficiency of 50% or more when concentrated 10- to 20-fold.  This  indicates
that the non-detection of viruses in  leachate samples was not due  to
deficiences in the detection method used.

    The possibility of virus adsorption to MSW waste components  was examined
by investigating the short-term interactions of viruses with paper,  glass,
lawn waste, and foodscraps in (a) a salt solution (pH 5.5), and  (b)  in dis-
tilled water.  The salt solution contained NaCl (2.5 g/l)t  CaCfe  (5.5 g/l),
MgCl2 (5 g/l)/ NH4C1 (0.75 g/1)/ and  H3P04  (0.7 g/l)r and hence  was similar
in composition to the major inorganic components of typical leachate.

    While there was negligible adsorption in distilled water alone,  for salt
solution (composition and concentration as  indicated above) high percentages
of both viruses were adsorbed onto the solid waste components.   Once the
viruses were solids-adsorbed, only a  small proportion was removed  by eluting
with either salt solution or glycine  (pH 11.5).

    The non-appearance of viruses in  the leachate could also be  explained if
the lysimeters were acting as plug-flow reactors.  In this  case, the lysi-
meters would not have been operational for a long enough period  of time to
allow viruses to appear in the leachate.

    Inactivation studies showed that  survival of field-strain poliovirus in
composite leachates from both lysimeters was highly temperature  dependent.
At 20°C, 95% inactivation of viruses  took two weeks; at 37°C, 97%  inacti-
vation was achieved within six days.  The rates of inactivation  were differ-
ent for the two lysimeters for reasons not determined (q.v. Glotzbecker and
Novello, 1975).

    After termination of the experiment, the lysimeter contents  were assayed
and found to contain no viruses.  It  was concluded that viruses  may be re-
tained in a landfill for a long enough period to ensure inactivation.   How-
ever, it was recommended that the mechanism of virus inactivation  be studied
further, both in the laboratory and in the field.
                                      64

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    It should be pointed out that adsorption of viruses onto  solids  does  not
rrean that viruses are inactivated.   Several studies have demonstrated  that
viruses (including coliphage T2, poliovirus type  1, and enteroviruses)  retain
their infectious nature upon adsoption  (Carlson et al., 1968; Moore  et  al.,
1975; Schaub and Sagik, 1975).

Movement of Microorganisms through Soil

    Given the possible survival and  leachage of bacteria and viruses from a
fill, the extent to which the organisms were retained, survive, or move through
various types of soil is obviously of paramount concern.  Factors which
influence the behavior of microorganisms within the soil include  (Gilbert et
al., 1976):

         •  soil texture/composition,

         •  soil moisture,

         •  salt concentrations,

         •  PH,

         •  climate (rainfall and temperature),

         •  nutrient availability; and,

         •  antagonisms.

    Glotzbecker and Novello (1975) compared the survival of E. coli  and
poliovirus in a sandy and a clay soil.  They percolated leachate seeded with
E. coli (108 organisms/100 ml) down  a clay soil column, and leachate contain-
ing JII_co]LL (108 organisms/100 ml) and poliovirus (105/100 ml) down  a sandy
soil column.  Leachates and percolates were examined for fecal coliform and
viruses.

    The clay soil was most effective in retaining E. coli (3 fecal coliforms
/100 ml).  Percolated leachate (640  ml) was collected  for 119 days.  This
volume was 1.8 times the calculated  pore volume of the soil.  E. coli
detected in percolate from the sandy soil column decreased from 1% (day 4) to
9% (day 14), to just over 0.1% on day 26.  An increase from day 14 to 20 was
thought to be due to channeling in the soil (cf. Wellings et a^., 1975, also
reported breakthrough of viruses present in the soil as a result of  fractures
and channels present in the strata).

    Overall, more than 99% of E. coli disappeared from the leachate.  Polio-
virus concentration in the percolate increased from 1% (day 4) to about 8%
(day 20), decreasing to 0.7% after 35 days.

    Duboise et al. (1976) investigated poliovirus survival and movement in
sandy forest~soTT and confirmed the  findings of previous investigators
(Wellings et aU , 1975, Schaub et al., 1975) that virus movement may be quite
considerable after rainfall.  Movement of poliovirus I (Chat) was monitored


                                      65

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through nonsterile core samples of a sandy  forest  soil.   The  core sairples
were loaded with either distilled water or  dechlorinated  final  effluent from
an activated sludge treatment plant, to simulate alternate  rainfall  and land
irrigation respectively.  The level of poliovirus  applied was around 2 x 107
PFU of virus suspended in 20 ml of dechlorinated final effluent (Duboise et
al., 1976).

    Application of dechlorinated effluent ("irrigation")  enhanced retention
compared to the distilled water ("rainfall") for both continuous  and inter-
mittent loading.  The importance of pH was  examined by adjusting  the final
effluent to various pH values, and using as an eluate.  At  pH 9,  virus
release was enhanced for both final effluent and water loadings.   At pH 5.5,
virus retention was enhanced.  Intermittent loading also  improved virus
retention.

    As the amount of "rainfall" was increased in volume,  there  was an accom-
panying increase in the percentage of viruses detected in the eluates from
the soil cores.  When the cores were eluted with 500 ml of  distilled water,
viruses, detected in the eluates ranged from 15 to 20% of the initial
concentrat ion.

    It was shown that most of the viruses were retained in  the  top layer of
soil by adsorption onto ions contained in the effluent, up  to the limits of
the soil's ion exchange capacity.  Addition of distilled  water  resulted in  a
dilution of ions and increased movement of  viruses through  the  soil.   Accord-
ing to Duboise, this suggests that an optimal range for poliovirus release
was exceeded.  It also suggests that secondary bands of virus adsorption less
concentrated than the top soil layer might  move down through  the  soil after
intermittent rainfall.  This movement would be limited by the depth  of the
soil, pH, ion concentration, soil moisture, etc.

    To determine the effect of temperature, poliovirus survival and  migration
in the soil cores was investigated for 84 days at 4°C and 20°C.   There was  no
apparent change in migratory ability over the 84 day period.  After  42 days
at 20°C, no viruses were detected at a 10 cm depth (concentration 2  x 10 3
PFU/g soil at day 0).  After 84 days at 20°C, viral concentration was 1.5
PFU/g soil at 1 cm depth (concentration at  day 0 - 4.6 x  105  PFU/g soil).
Virus survival was greater at 4°C.  After 84 days, concentration  at  1 cm was
2.2 x 105 PFU/g soil, and concentration at  10 cm was 4.0  x  102 PFU/g soil.
Initial concentrations were 4.5 x 105 and 2.0 x 103 PFU/g soil  at 1  cm and  10
cm depth respectively.

    Damgaard-Larsen et a^. (1977)  studied the movement of coxsackie  virus and
tritiated water through four lysimeters containing clay (two), neutral sand
(one) and acidic sandy soil (one).  They seeded municipal sludge  with the
virus (initial concentration 106 TCID SQ/g), and dug the  sludge into each
lysimeter soil.  The pH of the clay soils ranged from 7.1 to  7.4, pH of the
neutral sand was from 6.0 to 6.4, and pH of the acid sand from  4.8 to 5.4.
The lysimeters (125 cm depth) were exposed  to natural rainfall  over  a five
month period (December to May, total rainfall of 300 mm).   The minimum air
temperature was -12°C, and the maximum temperature was 25.7°C.
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    No viruses were  found  in  leachate  from any of the lysimeters at any time.
After 23 weeks, no viruses were detected  in any soil  sample.   Tritium tracing
showed that there was some water  penetration of the neutral  sand by the begin-
ning of April; a month later  tritium activity in the  leachate was about 10
times the background activity.  This represents a water breakthrough of about
1% of the total rainfall.

    This study and a similar  investigation by Lance et al.  (1976)  tend to
support the findings of Duboise et  aJU  discussed previously.   Lance et al.
also examined movement of viruses in soil columns (250 cm depth)  flooded with
secondary sewage effluent.  Effluent containing 3 x 104 ppu/ml of poliovirus
type 1 (LSc) was passed through piping packed with loamy sand from an area of
the dry Salt River bed used over  a  four year period for groundwater recharge
of secondary effluent.  The same  soil  had been flooded with  sewage effluent
in the laboratory for a further three  years.

    Viruses were not detected in  samples  below 160 cm.   Flooding of the col-
umn with the effluent did not result in virus desorption, nor in saturation
of the soil surface with viruses.   Like Duboise et al_.,  Lance et a^.  found
that flooding with deionized  water  increased the movement of  viruses down the
column.

    This movement was retarded by adding  chloride to  the deionized water.
Drying the column reduced or  completely prevented desorption  of  virus
depending on the time allowed for drying.   It was concluded  that viruses
should be inactivated by passage  through  sandy soil provided  that flooding
does not take place within 24 hours of land application.

    The effect of alternate flooding and  drying cycles on virus  and enteric
bacteria mobility in land-applied secondary sewage effluent  was  examined by
Gilbert et a^.  (1976).  The effluent was  percolated through  six  soil basins
(fine loamy sand/coarse sand/gravel/clay  to a depth of 75 m),  and water
samples were obtained from a  series of observation wells (6.1 to 76.2 m deep)
after flooding every two months for a  year.  The average infiltration rate
was 90 m/year.

    Poliovirus types 2 and 3, echovirus types 7 and 15,  coxsackievirus B4,
and reovirus types 1 and 2 were all isolated from the sewage effluent.  The
viral concentrations ranged from  158 to 7,475 PFU/100 liters.  No viruses
were detected in the renovated water from any of the  wells.

    Salmonella sp. were also  commonly  found in the sewage effluent (17 to 26
counts/100 ml), but were never detected in the renovated water.   Fecal coli-
forms, fecal streptococci, and total bacteria decreased by 4  logs (99.9%)
after passage of the wastewater through 9 m of soil.   While  it was clear that
the microorganisms were retained  by the soil, the survival rate  of the adsor-
bed viruses and bacteria was  not  determined.

    This project (Flushing Meadows) has been continually operated for eight
years during which time viruses and enteric bacteria  were either non-detectable,
or reduced to very low levels after filtering of the  sewage  effluent through
the soil recharge basins.


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    Bitton et al_. (1976) studied adsorption of poliovirus  type  1  and bacter-
iophage T2 onto soil columns covered with a constant head  of either tap water
or secondary sewage effluent (3 cm).  The sandy soil columns were removed
from a cypress dome, normally kept  covered with secondary  effluent.   The tap
water was adjusted to pH 6.5, while the pH of the effluent ranged from pH 6.2
to 7.6.

    Viruses were loaded on the columns either by suspending in  tap water or
effluent and percolating through the soil at a constant  flow rate,  or by
placing a dose of the viruses on top of the soil, allowing them to soak com-
pletely in, and then eluting with secondary effluent.

    For poliovirus type 1, adsorption was higher in the  presence  of tap water
(99.7%) than in the presence of secondary effluent (66.6%).  For  bacterio-
phage T2 suspended in tap water (initial concentration 3 x 104  PFU/ml),
adsorption on the sandy columns was about double (98.4%) that for T2 suspended
in secondary effluent.

    It was also found that the secondary effluent desorbed greater concentra-
tions of both viruses than the tap  water.  It was postulated that organic
matter present in the secondary effluent (total organic  carbon  27 to 37 mg/1)
in some way interfered with adsorption of the viruses on the soil columns.
Interfering substances have been shown to hinder virus adsorption onto various
materials (Carlson et a^., 1968; Oliver, 1968).

    At the cypress dome site itself there is a clay loam layer  (28%  clay)
below the sand, while at the center of the dome is a layer of black  organic
mud (61 cm).  These layers may contribute to virus removal on site.   However,
Wellings ^t aJL (1975) have isolated animal viruses from 305 cm deep wells  in
the cypress dome indicating possible damage or structural  inadequacies in the
clay layer.

    All these studies indicate that movement of microorganisms  through soil
is highly dependent upon a number of factors, and may be minimal  or  consid-
erable depending on the environmental factors discussed  above.  Survival and
movement of viruses in particular is of concern given that adsorbed  viruses
may not be inactivated, but may be desorbed after a heavy  rainfall  to move  as
a band through the soil.

Summary

    There is obviously a possibility that pathogenic bacteria and viruses may
survive conditions within the sanitary landfill, and enter ground or surface
water through leachate penetration of the soil.  However,  there does appear
to be a significant decrease in viral and bacterial content of  leachate with
time of operation or leaching of the fill.  Also, the relatively  high tempera-
ture (60°C) achieved during the first aerobic stages of  waste biodegradation
is inimicable to many viruses and most pathogenic bacteria.  It has  also been
shown that the chemical and physical characteristics of  the leachate contribute
toward both viral and bacterial inactivation.
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    Adsorption of viruses onto material  in  the  fill  is  likely,  and  partly
explains the low rate of recovery of viruses  from landfill  leachate.   If
leachate does contain viruses, there is  a possibility that  the  viruses would
be retained by the soil for a long enough period  of  time  to become  inactivated.
This would be especially true in very dry climates,  or  where rain is  intermit-
tent and light.

IV.  OXIDATION DITCH

The Process

    Aerobic treatment of animal wastes may  take place in  a  pond,  lagoon or
ditch.  When manures are held in a shallow  container, dissolved oxygen is
probably sufficient for aerobic stabilization of  the wastes.  Otherwise,  oxy-
gen must be added by means of floating aerators.

    In the oxidation ditch, the waste slurry  is circulated  by use of  a hori-
zontal shaft rotor.  In addition to preventing  settling out of  solids,  the
rotor aerates the waste.  The ditch may  be  built  directly below cattle penned
in a feedlot.  Here, the ditch is covered with  slatted  flooring,  and  the animal
waste drops directly into a liquid medium present in the  ditch.

    Stabilization of wastes may be partially  or completely  accomplished
depending on the method of operation.  If only  partial  stabilization  is
achieved, the waste is generally held in an external lagoon until final dis-
posal.  Several investigators mention the need  for a holding basin  to permit
flocculation and settlement of solids prior to  containment  within the ditch
(Diesch et_ al^., 1973).  Environmental conditions  vary considerably  from one
operation to another, and within one ditch  depending on the volume  and the
nature of its contents.  Variables, such as loading  rate, pH, suspended
solids, biochemical oxygen demand (BOD), chemical oxygen  demand (COD),  dis-
solved oxygen, and temperature fluctuate considerably.  One operational field
ditch is described in the section below.  In  1967, there  were over  400 oxida-
tion ditches operational in the U.S.

    Several advantages to this mode of treatment  include:

         •  relatively odor free operation;

         •  storage prior to land disposal;

         •  reduction of BOD and COD; and,

         •  economy of labor possible where slatted  floors  used.

Survival of Pathogens

    As previously indicated, the increased  confinement  of livestock in feedlots
has resulted in increased difficulty in  the hygienic disposal of  wastes. Though
it is well-known that more than 150 diseases  can  be  transmitted to  man by ani-
mals, there is little quantitative data  to  describe  the extent  of this trans-
mission.  However, there has been several studies to develop methods  of


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detection and rates of  survival  of the more widespread and hazardous patho-
genic organisms  found in animal  wastes.

    Diesch e_t aJU  (1973) studied detection and survival of Leptospira pomona
and SalmonelTaTyphimurium in a  laboratory model (1:10 scale) of an opera-
tional field oxidation  ditch.  Variables  investigated were pHf dissolved
oxygen, temperature, and level of  total solids.

    The operations of the field  unit  oxidation ditch were previously studied
to determine essential  environmental  and  working parameters for operation of
the simulated model.  Briefly, the field  unit consisted of a continuous chan-
nel of dimensions 172 feet x 7 feet x 4 1/2 feet deep.  The unit was lined
with concrete to prevent percolation  through the soil, and roofed with a
slatted floor.   Water was added  to the ditch,  and oxygen was supplied to the
level of 0.5 to  1 ppm of dissolved oxygen in the manure and sludge.   The
entire unit was  contained in a rigid  steel  building,  and housed from 36 to 45
head of cattle.  Waste  generated by the cattle dropped through the slats into
the ditch.

    Severe foaming occurred within the ditch over the winter (November to
January).  The possibility of aerosol transmission of pathogens could not be
ruled out.  An initial  plan to collect and  study effluents discharged from
the field unit was abandoned.  Over a three-year period,  no effluents were
discharged.

    Four cattle  (average weight  1100  Ib)  died  of idiopathic toxicosis.   These
deaths occurred  hours after the  rotor malfunctioned  and according to Diesch
et al. may have  been due to emission  of toxic  gases  from the inadequately
aerated manure during this period.

    Two laboratory models were designed to  simulate  the conditions prevailing
in the field unit.  Both models  were  cooled by circulating ethylene  glycol
through a stainless steel trough within the ditch.   The cooler-condenser unit
was installed below each ditch which  was  insulated with two in.  thick rigid
styrofoam.  The  ditches also had a plexiglass  cover  to protect the operators
against aerosols.  Table 23 gives  the operational  parameters for Model  A.

    Model B was  identical to Model A  except that Model B  had a storage  pit to
facilitate separation and removal  of  solid  materials.   Two designs of rotor
were tested in Model B.   The brush-type rotor  was  abandoned because  of  too
low water velocities.  Cooling of  the ditch was  required  during winter
operation (heated building) and  slight heating during summer.

    Survival times of Leptospira pomona and Salmonella typhimurium in the
manure slurry and in effluent and  sludge  from  the  settling are given in Table
24.  As can be seen from these data,  aeration  of manure in the ditch
generally resulted in greater survival of the  microorganisms than in the non-
aerated effluents and sludge.  However, for S. typhimurium at 2°C survival
was longer in the sludge (87 days) and effluent  (66 days)  than in the slurry
(47 days).
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                                   TABLE 23

              OPERATION OF A MODEL OXIDATION DITCH  (MODEL A DATA)
                             (Diesch et al.,  1973)


              Temp:   surtmer*                        13° to 25°C
                     winter                         1.7° to 6.4°C

              PH                                    6.8 to 8.4

              Total  solids (TS)                    5,802-135,333 mg/1

              TS attempted range                  5,000-10,000 mg/1

              Dissolved oxygen                       1 to 5 ppm

              Initial volume of manure               113 liters

              Daily  loading of manure                2.2 Ib/dayt

* Dow-Corning Antifoam A Spray added to prevent foaming in summer.

t Added regularly up to 10,000 mg/1 TS; then added  intermittently to maintain
  at this level.  Addition of unchlorinated well water was necessary to keep
  TS within desired range.

    Burrows and Rankin  (1970), in a study of pathogen survival in cattle
slurry, also found that certain bacteria (including Salmonella sp.) survived
for a longer period of time in an aerated cattle slurry than in non-aerated
samples.  The bacteria studied were Salmonella typhimurium, Salmonella dublin,
Brucella abortus, Staphylococcus aureus, and Escherichia coli.  Slurry was
collected from five farms where it was stored in polyethene bins  (9 gallons
capacity).  Each bin was seeded with one of the cultures (106 to  107 viable
units/ml) and then covered.  Sanples were collected daily after agitation of
the slurry.

    All microorganisms decreased in density with time.  In most cases, bac-
teria could not be counted 10 weeks after seeding  (direct counting and enrich-
ment techniques), though viable organisms were still detected after 12 weeks.
Organisms in the slurry from one particular farm survived for a longer time
than organisms in the other slurry samples.  The slurry tank on this farm was
emptied three times a week, so the slurry in the bins was coirparatively fresh
at all times and probably contained a greater amount of dissolved oxygen.
Also, the slurry had a higher solids content (4.5%  as opposed to  0.2% to 2.6%),
and a slightly lower pH (7.2 as opposed to 7.6).

    Other investigators have also shown that survival of leptospires is
longer in aerated sludges.  Chang et al. (1948) indicated that survival of
leptospires in domestic sewage was 12 to 14 hours.  When the sewage was
aerated, survival time  rose to two to three days.   Other conditions influen-
cing survival of leptospires are summarized in Table 25.


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                               TABLE 24




       SURVIVAL OF LEPTOSPIRA PCMONA and SALMONELLA TYPHIMURIUM
(Diesch et al.f 1973)
Substrate Organism Temp. Duration
Cattle Leptospira 20°C 138 days
pomona
5 days
14 days
2°C 18 days
9 days
11 days
Salmonella
typhimurium 20°C 17 days
14 days
2°C 47 days
87 days
66 days

Comments
*Minnesota summer temp.
survival time in slurry
some organisms still
viable
Survival time in effluent
Survival time in sludge in
model settling chamber
Slurry winter tempera-
tures, some organisms
still viable
Survival in effluent
In sludge in model set-
tling chamber
Slurry survival time
Effluent and sludge
Slurry survival time
Sludge survival time
Effluent surival time
*Maximum time measured.
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                                  TABLE 25

             CONDITIONS FAVORABLE TO DESTRUCTION OF LEPTOSPIRES
               Condition                         Reference
    Presence of other microorganisms             Noguchi,  1918
                                                 Chang et  al_., 1948
                                                 Okazaki & Ringen, 1957

    Non-aeration                                 Chang et  al., 1948
                                                 Diesch et al., 1973

    pH    < 5 to 6                               Okazaki & Ringen, 1957
          > 7                                    Smith and Turner, 1961
          < 8.4                                  Stockard  et  al., 1968

    Temperatures   <  7°C                         Okazaki & Ringen, 1957
                   > 36°C                         Ryu and Liu, 1966

    Lack of Water                                Okazaki & Ringen, 1957


    Derbyshire (1976) studied survival of enteroviruses in a  series of field
and laboratory experiments, and  found that  the viruses were more rapidly
inactivated in aerated liquid pig manure than in untreated pig manure.  Samp-
ling of raw and aerated manure concentrates over a  ten week period consistently
showed the concentration of the  former to be three  logs greater than  the
latter.

    In related laboratory studies,  raw liquid pig manure was  seeded with
swine enterovirus, and continuously aerated and stirred for 71 days at 22°C.
The controls were  seeded untreated  manure.  The virus was  present up  to 14
days in the aerated samples, and 71 days in the control.   The mechanism of
inactivation was not identified.  Viruses were recovered by concentrating the
waste by PE-60 adsorption, and isolating the viruses from  the concentrates on
pig kidney cell cultures.

Summary

    From the limited material presented, it appears that prolonged survival
of some of the more common pathogens present in animal wastes is possible
within the oxidation ditch environment.  However, as pointed  out by Diesch
et al. (1973), methods of operating field facilities vary  greatly, and the
environmental characteristics of even one specific  ditch may  change on a
daily basis.  Given that units may  be badly designed or poorly managed,
pathogens may not  only survive but  thrive under certain conditions.
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V.  ANAEROBIC DIGESTION

The Process

    Anaerobic digestion involves biodegradation of the cellulosic  fraction of
solid wastes by at least three sets of organisms:  cellulolytic, acetogenic,
and methanogenic bacteria.  Initially, the cellulose is converted  to  short
chain volatile acids.  The methane-forming bacteria then convert the  products
of acidogenesis to a mixture of methane and carbon dioxide.   The process  can
be used for stabilization of MSW, manures, agricultural wastes, and sewage.

    Anaerobic digestion has received a great deal of attention recently as a
means of producing methane gas.  However, the process is still in  the pilot
plant and demonstration stages for solid wastes, though it is a common method
for stabilizing sewage sludge.

    Digestion can occur at mesophilic (30° to 40°C) or thermophilic (50°  to
60°C) temperatures.  The detention time in the digester depends on the temp-
erature of digestion and the design of the fermenter.  At lower temperatures,
detention time is longer.  Generally, thermophilic processes  would take from
5 to 15 days with complete mixing of the feed.  A completely  mixed mesophilic
digester might take from 15 to 30 days to stabilize the waste.  This  is the
process as currently applied to municipal sewage sludges.

    The batch load digester employs two completely mixed reactors, one of
which ferments while the other is fed, resulting in an increase in
efficiency.  Like the completely mixed process, it is necessary to premix and
dilute the manure, resulting in an improvement in biodegradation and  gas  pro-
duction.  The plug-flow longitudinal reactor does not mix contents, and
operates at 20° to 30°C for 30 to 50 days.  It is common practice  to  add
nutrients containing nitrogen and phosphorus to the digesting wastes, and to
balance the pH to the optimum value for growth of methanogenic bacteria by
addition of alkali.

Survival of Pathogens

    Literature reports discussing the advantages of digestion of solid waste
point out that the sludge remaining is odor-free, does not attract flies,  ands
is biologically stable.  It is interesting to note that while many researchers
cite possible destruction of pathogens, studies to confirm or deny this
assumption are not yet available.

    As Gaby (1975) pointed out, most of the pathogenic bacteria found in  raw
refuse are facultative anaerobes, and could survive landfill  conditions.   It
might be suspected that pathogens could survive digestion at  mesophilic tem-
peratures, or even at thermophilic temperatures if the retention time is  very
short, and the contents of the digester are not well mixed.

    Since no studies were available documenting the extent of possible
bacterial and viral survival in digested solid waste, it was  decided  that an
examination of the survival of microorganisms in digested sludge would give
some perspective to this potential problem.

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    Inactivation of viruses  in digested  sludge may  be  caused  by one  or more
of tiie following factors  (Palfi, 1973; Warl  and Ashley,  1976):

         •  heat denaturation of the protein coat;

         •  enzymatic action on the protein  coat;

         •  natural die-off; and,

         •  cleavage of viral proteins followed by  nicking of
            encapsulated  RNA.

    A completely mixed digester may give poor results  for virus destruction
because some influent may exit early with  the effluent.  Also, the tempera-
ture may not be constant  throughout the  digester.   Even  for a high even temp-
erature, adsorption of viruses onto particulate matter may protect from
inactivation by heat.  However, there seems  to be general agreement  that
virus inactivation in digested sewage sludge is primarily a function of
temperature and residence time.

    Palfi (1973) has examined virus survival in digested sewage sludge before
and after modifications in digester operation.  Hence, he was able to  compare
different operating conditions though he was not able  to control  the vari-
ables.  Initially, the digester operated with a retention period  of  21 days
at 30°C, continuous inflow and outflow of  sludge, and  constant mixing.

    After year-round sampling, he  found  that 28 out of 74 samples (38%)
contained one or more virus  strains.  Reovirus (35%) and echovirus type 11
(15%) were present in the highest  number.  Forty (40)  strains were identified
in all.  Viral densities  ranged from 2.3 to  175.5 MPNCU/100 ml (most probable
number of cytopathogenic  units).   The average density  was 39  MPNCU/100 ml for
the November to December  period.   The viruses were  determined by  the MPN-
method using 5-5 primary  monkey kidney tube  cultures.

    Operating conditions  were modified so  that the  sludge was digested in
three stages.  Retention  time was  20 and 10  days at stages one and two
respectively with complete mixing  of sludge.  Retention  at stage  three was an
additional 10 days without mixing.  The  temperature was  raised to 33°C.

    For winter sampling,  13  of 82  samples  (15%) were positive for viruses.
Only 14 strains were identified.   More than  half the positive samples
contained poliovirus type 3.  The  average  viral concentration was 5.4
MPNCU/100 ml; the range was  2.3 to 19 MPNU/100 ml.

    Lund (1971) has also  isolated  viruses  from sewage  sludge  digested  for 50
to 60 days at 50°C.  She  noted an  increase in heat  stability  of poliovirus
compared to other types of enterovirus.  Neither Palfi nor Lund could  deter-
mine virus recovery efficiency, as their data were  drawn from operational
experience.

    Ward and Ashley (1976) have studied  the  inactivation of radioactive carbon
labeled poliovirus in digested sludge under  controlled laboratory conditions.


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They found that while the virus was  fully recovered  from the sludge,  its in-
fectivity was inversely proportional  to  the  time  and temperature of
incubation.  The decrease in infectivity ranged from greater than 1 log/day
at 28°C to about 1 log/5 days at 4°C.  Infectivity of both free and solids-
associated virus was found to be identical.  The  process was investigated at
low temperatures to minimize the effects of  heat  inactivation.   Raw sludge
was shown to have no virucidal activity, and, hence,  it  appears that
virucidal agents were produced by the bacteria of digestion.

    In related studies, Ward et cd.  (1976) confirmed the heat protective
effect described by Lund for poliovirus.  At temperatures of 43°C, 47°C, and
51°C, poliovirus was bound to solids  in  both raw  and digested sludge.   The
inactivation was more rapid, however, in digested sludge.

    Suspending raw sludge solids in the  supernatant  of digested sludge
produced irreversible inactivation of viruses.  At 61°C,  five minutes  contact
with the supernatant improved the inactivation of raw sludge from 98%  to
99.9% for poliovirus type 1 (Chat).  At  51°C, five minutes contact improved
the inactivation of raw sludge from 96%  to 99.99% for poliovirus type  1
(Mahoney).  For poliovirus type 2 (712) at 51°C,  there was an improvement
from 48% inactivation in raw sludge to 99% inactivation  with addition  of the
supernatant.

    Ward et: al. pointed out that as they experienced  difficulties in recover-
ing viruses from the digested waste, that the actual  degree of  inactivation
might be considerably less than indicated.

    Given that solid waste may be contaminated with various parasites  (Gaby,
1975), there exists the possibility of the survival of nematodes,  protozoa,
and cestodes in digested solid waste.  Again, there  are  no available studies
for MSW, though there have been a series of  investigations of parasite sur-
vival in anaerobically digested sewage sludge.

    Ova of Ascaris 1. suum in sewage sludge  survived  conditions simulating
the anaerobic digestion process, and later became embryonated (Fitzgerald and
Ashley, 1977).  Sludge appeared to inhibit development,  while protecting the
ova.  Ova failed to embryonate when held at  38°C  for  21  or 25 days in  sludge,
but after removal and air exposure in 1% formalin for 58 days at 22°C, up to
90% of the ova embryonated.  Ova held in physiological saline for 25 days
failed to embryonate.  Under the same conditions,  oocysts  of Eimeria stiedai
were destroyed within five days.

    The sludge used in this study was collected from four  different sources.
One of the four systems was lethal to the ova, illustrating the highly vari-
able nature of sludge.

    Fox and Fitzgerald (1977) have found the eggs or  cysts of 10 genera of
parasites in anaerobically digested sewage sludge.   The  range of egg or cyst
counts per 100 g of digested sludge from four treatment  plants  was:

         Ascaris lumbricoides            11 to 120 counts/100 g    (0 to 113)


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         Toxocara sp.                     101  to  340           (0  to  279)

         Tpxascaris leonina                 0  to   16           (0  to   37)

         Trichuris sp.                      0  to   23           (0  to   23)

         Cestodes (Taenia sp.
           and Hymenolepis sp.)             0  to   33

         Coccidia (Eimeria sp.
           and Isospora sp.)                0  to  372           (0  to   50)

         Entamoeba coli                     0  to   34

The values in parenthesis refer  to  the  concentration of  the parasites  in raw
sewage for five treatment plants.

    The viability of the nematode ova was determined by  Arther and  Fitzgerald
(1977).  Ova were isolated from  fresh sludge  by  means of sugar flotation.
Extracted ova were placed in 1.5% formalin, and  aerated  for 21 to 28 days at
22°C.  Ova were then removed from the formalin,  when it  was found on exami-
nation that larval development had  occurred for  A. lumbricoides  (58%), T.
leonina (57%), Tbxocara sp.  (48%),  and  Trichuris sp. (33%).   Studies are
continuing to determine the ability of  the  ova to infect suitable animal hosts.

    Digested sewage sludge may contain  pathogenic bacteria as well  as  viruses
and parasites.  Hess and Breer (1974) found that 81.9% of 136 sanples  of an-
aerobically digested sludge contained Salmonella sp.  Maximum concentration
was 106 organisms/liter with a mode of  103/liter.(The  average  concentra-
tion of Salmonella sp. in raw sludge was  10^  organisms/liter.)   The average
value of E. coli in digested sludge (104  samples)  was 107/liter, and in  raw
sludge 10y organisms /liter.

    Hess and Breer also demonstrated survival of Salmonellae  in  digested
sludge for up to 72 weeks after  spreading on  pastureland.  They  found  a
direct correlation between the spreading  of unsanitized  sludge on land,  and
an outbreak of salmonellosis in  cattle  after  grazing upon the pasture.

    Wizigmann and Wiirshing   (1974)  have also  demonstrated survival  of  Salmon-
ellae, enterococces, and total enteric  bacteria  after anaerobic  digestion of
sludge.  As MSW and animal manures  may  also contain these organisms, there
remains the possibility that they may survive in digested solid  waste.   Given
the disease control measures practiced  on the modern farm, it seems possible
that MSW, of variable and uncertain composition,  is more likely  to  contain
problem organisms than manures.

Summary

    No studies are yet available documenting  the possibility  and extent  of
pathogen survival in anaerobically  digested solid waste.  Studies of digested
sewage sludge have indicated survival of  pathogenic microorganisms  at  both
mesophilic temperatures (viruses, bacteria, and  parasites), and  thermophilic


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temperatures (viruses).  An increase in heat stability of poliovirus has been
noted by several researchers.

    It would appear that survival of pathogens present in solid waste (MSW
and manures) is a possibility, depending upon the design and operating para-
meters of the digester.  Temperature, time of residence, and mixing  of the
digesting waste are all determining factors.
VI.  OTHER PROCESSES

Magnetic Separation

    High^gradient magnetic separation (HQ^S) is a new technique to extract
weakly paramagnetic submicron particles from wastewater and slurries  (DeLatour
and Kolim, 1976).  The treatment relies on addition of a strongly magnetic
seeding agent like magnetite to the water.  A chemical coagulant (any aluminum
or ferric salt at 3 to 20 ppm) is also added, and the suspension agitated for
up to five minutes.  The coagulum formed is removed in the HQYIS.

    DeLatour and Kolim demonstrated complete removal of total coliform and
fecal coliform bacteria from river bottom sludge after addition of 5 ppm of
A13+ (form not indicated), and 1000 ppm Fe^ and application of a magnetic
field of 1000 gauss.  Concentrations of the controls were 20,000
organisms/100 ml for total coliforms, and 1,200 organisms/100 ml for fecal
coliforms.  Flow rate of the sludge was 30 gpm/ft2.  Total coliforms averaged
35/100 ml in surface water treated with 4 ppm Al3+(form not indicated), and
1000 ppm Fe304 with a field of 10 k gauss.  Flow rate was 300 gpm/ft2 and the
control contained 22,000 organisms/100 ml.

    Bitton and Mitchell (1974) demonstrated H(M3 as a means of removing
Escherichia coli bacteriophage T-y from water.  The virus was adsorbed onto
magnetite in the presence of calcium chloride by passing the virus-containing
water through a filter placed in a magnetic field.  The process accomplished
99% removal when the magnetite concentration was 400 ppm.  Metcalf (1976) has
demonstrated the feasibility of removing poliovirus from water using HOIS.

    It was not determined if there have been any attempts to remove pathogens
from solid waste systems by HGMS, but presumably animal slurries might be
amenable to this approach considering their high water content (e.g., cow
manure may be 85% water).

Gamma Radiation Sterilization

    Radiation sterilization of agricultural or municipal solid wastes has
been suggested as the first stage of resource recovery (Padova et a^., 1974).
Since animal wastes may contain high densities of bacteria including
Salmonellae, refeeding of these wastes may be hazardous.  Poultry waste, for
example, contains from 7 x 105 to 3.6 x 106 total bacteria/g with a coliform
count from 6 x 102 to 1 x I03/g (Carmi and Ashbel, 1974).  Blair and Knight
(1973)  have found Salmonellae in 33% of poultry samples analyzed.
                                     78

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    Irradiation of poultry wastes prior  to  refeeding was  suggested  by Jackson
(1970-1971).  He has described  a plant to treat  6.0  tons of poultry  slurry
daily.  At a moisture content of 82%  to  83%,  a dose of 1.2 x  105  rad "should"
kill "most" of the bacteria present,  and enhance solids separation.

    Simon and Tamasi (1974) have also described  a plant to irradiate animal
wastes to produce a nutritive and microbiologically safe  feed.  They reported
destruction of Salmonellae in liquid  swine  manure at 0.4  Mrad.

    It has also been suggested  that irradiation  of  MSW could  yield  an animal
feed.  Padova et aJU (1974) described a  proposed pilot plant  to grind and
separate 75 to 150 tons of garbage daily, yielding  50 to  100  tons of putres-
cible matter.  Samples from this plant would  undergo irradiation, and be
tested as a potential feed.

    An additional use of  irradiation-cured  MSW has  been proposed  by Feates
and George (1974).  They  have treated compacted  polymer-impregnated MSW with
gamma radiation to produce formed structural  materials suggested  as a replace-
ment for concrete, wood,  or aluminum  in  building.

    More data is available for  irradiation  of sewage sludges  than for solid
waste.  Van den Berg et. a^. (1974) have  described the complete kill of  fecal
coliforms in digested~~sludge at a dose of 120 Krad.  At a water content re-
duced 50%, irradiation alone reduced  fecal  coliforms by only  one  log at doses
of 240 Krad.

    Wizigmann and Wursching (1974) demonstrated  that after irradiation  of
sludge (60Co 260 Krad in  210 minutes), total  bacterial and enterococcal
counts were reduced by two logs and five logs, respectively.  Salmonellae
were found in two of 40 samples of irradiated sludge (and 16  of 25  samples of
digested sludge).  The destruction of Ascaris cysts and ova by irradiation
was unconfirmed by the investigators.

Summary

    It is not clear how effective, and how  technically and economically feas-
ible, the above processes might be.   However, they  represent  future options
in solid waste management, and  illustrate the efforts that are being made to
improve waste management  practices.
                                      79

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                                  SECTION 5

                             ANALYTICAL METHODS

    Analytical methods for use in the studies discussed in Section 4 are
tabulated below.  As previously mentioned, many of these methods are
standard.  Each procedure described is detailed; the references at the foot
of each page allude to the studies which used this technique or a close
modification.  The publication Methods for Bacteriological Examination of
Solid Waste and Waste Effluents by Dr. Peterson of the National Environmental
Research Center, Cincinnati, was an invaluable source of information for this
section.  Interested readers are referred to this publication for further
details of general laboratory procedures and bacteriological methods.
                PROCEDURE - COLLECTION OF SOLID WASTE SAMPLES
Organisms                               n.a.
Methodology

    Using sterile tongs collect 20 to 40 random samples (100-200 g).  Place
    in sterile containers.  If source contaminated, wear disposable gloves.
    Avoid external contamination of the container.  Identify samples in-
    cluding all relevant data — time, date,  location, temperature, etc.
    Send to laboratory.
Limitations or Precautions

    Examine within 1 hour after collection; in any case, no later than  30
    hours after collection.  Maintain  temperature of collection as closely
    as possible.
Additional Comments
References:
                                        Peterson and Stutzenberger,  1969
                                        Peterson, 1971
                                        Peterson, 1972.
                                      80

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     PROCEDURE - COLLECTION OF LIQUID SAMPLES, QUENCH WATER, OR LEACHATE

Organisms                               n.a.
Methodology
    If source contaminated, wear rubber gloves.  Collect  in sterile bottle or
    plastic bag leaving air space  for mixing the sample prior to assay.
    Identify samples with all relevant data.

Limitations or Precautions
    Protect containers from damage while shipping.  Examine within 4 hours.
    Maintain collection temperature as nearly as possible.  Sample contains
    residual chlorine, add 100 mg/1 sodium thiosulfate prior to collection of
    sample to neutralize chlorine.

Additional Comments
    Peterson (1971) concentrated quench water samples on  a diatomaceous earth
    layer.

References:Peterson and Stutzenberger, 1969.
                                        Peterson, 1971
	Peterson, 1972.	
            PROCEDURE - COLLECTION OF  INCINERATOR STACK EFFLUENTS

Organisms                               n.a.
Methodology
    Use Armstrong portable sampler mounted on steel plate with attached
    sampling assembly and vacuum pump  as described by Peterson (1972).  Dry
    heat sterilize prior to use.  Draw effluents through sterile stainless
    steel water-cooled probe at a rate of 1 ft3 of sample/minute (vacuum 14.3
    on of water).  Collect sample for  10 minutes (10 ft3).  Identify  and ex-
    examine with 48 hours.

Limitations or Precautions
    Reduce frothing by inserting end of probe entering the sample above the
    buffered solution (1.27 cm).  Insert probe into various stack locations
    to give representative samples.  Keep the probe dry since organisms may
    be adsorbed in wall moisture.

Additional Comments
    Peterson (1971) sampled stack effluents at the primary outlet, the
    electrostatic precipitator outlet  and the scrubber stack.

References:
                                        Peterson and Stutzenberger, 1969
                                        Peterson, 1971
     ^	Peterson, 1972.	

                                     81

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                   PROCEDURE -  COLLECTION OF DUST SAMPLES
             AND DETECTION OF GRAM POSITIVE AND NEGATIVE BACILLI

Organisms                            Gram Positive and  Gram Negative Bacilli
Methodology
    Draw air through sterile portable Anderson  sanpler  at rate of 1 ft? /min-
    ute (vacuum of 15 in. Hg).  Plates  in sanpler are trypticase soy agar
    (TSA) containing 5% sheep blood for respiratory tract and  skin bacteria
    (gm positive bacilli).  Use 6 plates  per sample. Use eosin methylene blue
    agar for detection of intestinal tract bacteria (gm negative bacilli).
    After collection withdraw plates, cover and incubate aerobically at 35°
    0.5°C.

Limitations or Precautions
    Maintain aseptic conditions throughout collection.

Additional Comments
    Sample run for 15 seconds (Armstrong  and Peterson,  1972) to give well
    separated 1-30 colonies.

References:
                                        Peterson,  1971
                                        Armstrong and Peterson,  1972.
                                        Peterson,  1972.
    PROCEDURE - PREPARATION OF SOLID AND SEMI-SOLID SAMPLES FOR ANALYSES

Organisms                               n.a.
Methodology
    Composite all random samples in a beaker  (5000 ml), mixing  well.   Trans-
    fer weighed 200 g subsample into sterile blender.  Homogenize  for  15  sec-
    onds at 17,000 rpm with sterile phosphate buffered solution (1800  ml).
    Prepare series of serial decimal solutions  (10~1, 10~2, 10~3,  10~4) by
    repeated dilution of 1 ml solution in 9 ml  phosphate buffered  water.

Limitations or Precautions
    Make sure each solution is homogenized prior to sampling  for subsequent
    dilution by shaking vigorously 25 times.

Additional Comments

References:""
                                        Peterson and Stutzenberger,  1969
                                        Peterson, 1971
	Peterson, 1972	

                                     82

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               PROCEDURE - BACTERIAL COUNT BY AGAR POUR PLATE
Organisms	Bacteria	
Methodology
    Pipe the prepared solution of sample  (0.1 ml, 1 ml etc.) onto
    duplicate trypticase soy agar plates with 7% defibrinated sheep blood
    (TSA + blood).  Add 10-12 ml of melted tryptose glucose extract agar to
    petri dish.  Mix well by rotating or tilting.  Solidify as rapidly as
    possible.  Invert plates and incubate for   24 ±  2 hours at 35°±  0.5°C.
    Count plates with 30-300 colonies using Quebeck colony counter.  Express
    results in counts per gram of waste (wet weight) per 100 ml water.

Limitations or Precautions
    Accurate to only 2 significant figures.

Additional Comments

References:
                                        Peterson and Stutzenberger, 1969.
                                        Peterson, 1971
	    	      	Peterson, 1972.	
              PROCEDURE - BACTERIAL COUNT BY AGAR STREAK PLATE
Organisms	Bacteria
Methodology
    Pipe the 0.1 ml sample of serially diluted samples on surface of labeled
    duplicate blood agar plates.  Plates contain TSA + blood spread evenly
    over surface with sterile glass spreader.  Invert and incubate for 24±  2
    hours at 35° ± 0.5°C.  Count plates with 30-300 colonies.

Limitations or Precautions

Additional Comments

References:
                                        Peterson, 1971
            	   Peterson, 1972.	

                                      83

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                     PROCEDURE - DETECTION OF COLIPORMS
                    MOST PROBABLE NUMBER (MPN) TECHNIQUE
Organisms                               Total coliforms


Methodology

    Presumed Positive Test;  Inoculate serial dilutions of sample into  5
    large tryptose broth fermentation tubes.  Incubate at 35°±  0.5°C   for  24±
    2 hours.  If no gas present, incubate up to 48±  3 hours.

    Confirmed Test;  Use a sterile platinum loop (3 nun diameter) to transfer
    medium from presumed positive tubes to a fermentation tube containing a
    brilliant green lactose bile broth (2%).  Incubate at 35°±  0.5°C for
    48±  3 hours.  The presence of gas in any tube indicates a confirmed pos-
    itive test.

    Completed Test;  Streak samples from all confirmed positive tests on
    eosin methylene blue agar plates as soon as gas is detected.  Incubate
    for 24 ±  2 hours at 35°±  0.5°C.  Transfer one or more colonies from
    plate to lactose tryptose broth in fermentation tubes and nutrient  agar
    slants.  Incubate as before.  Prepare gram stained smears from agar
    slants if gas detected in lactose broth.  Examine smears under oil  immer-
    sion.  Compute MPN as indicated in APHA 1976.


Limitations or Precautions
Additional Comments

    MPN best technique for quantifying coliforms in solid waste.  Peterson
    (1971) also used membrane filter technique for 3 incinerators in the 8
    incinerator study.  Spino (1971) also used membrane filter technique in
    the New Orleans Incinerator Study.


References:
                                        Peterson and Stutzenberger, 1969
                                        Peterson, 1971
                                        Peterson, 1972
                                        Blannon and Peterson, 1974
                                        Glotzbecker and Novello, 1975
                                      84

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                      PROCEDURE -  DETECTION OF COLIFORMS
Organisms	Fecal  Coliforms
Methodology               ~      ——	
    Presumptive test as  indicated on  previous  page.   Transfer  3 mm  loop of
    broth from positive  presumptive test to  an E.G.  broth  fermentation tube.
    Incubate in water bath  for  24 hours at 44.5°± 0.5°C.  Gas production is
    a positive indication of  fecal coliforms.

Limitations or Precautions
    Tubes must be placed in water bath  within  30  minutes of preparation.

Additional Comments

References:~"Peterson  and  Stutzenberger,  1969
                                         Peterson, 1971
                                         Peterson, 1972
                                         Blannon and Peterson,  1974
                                         Gaby,  1975
	       Glotzbecker and Novello, 1975
        PROCEDURE - DETECTION OF VIABLE HEAT-RESISTANT SPORE FORMERS

Organisms
    Heat-resistant spore-forming microorganisms capable of surviving 80°C
    for at least 30 minutes.

Methodology
    Transfer volumes of original and diluted samples  (10 ml) into screw-
    capped test tubes.  Heat for 30 minutes at 80°C.  Cool in cold water for
    5 minutes.  Determine viable heat resistant spores by agar pour plate
    method described previously.

Limitations or Precautions
    Water line must be 1 1/2 in. above level of samples in tubes.

Additional Comments

References:  ~~                        Peterson, 1972.
                                        Peterson and Stutzenberger, 1969
         	Peterson, 1971	^^

                                     85

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            PROCEDURE - DETECTION OF ENTERIC PATHOGENIC ORGANISMS
             IN QUENCH WATER, INDUSTRIAL WATERS AND IN LEACHATE
Organisms                               Shigella and Salmonella sp.

Methodology
    Filter sanple (800 ml) through a 1 in. layer of sterile diatomaceous
    earth on a stainless steel membrane filter holder.  Add half the clay to
    90 ml of Selenite F enrichment broth and half to 90 ml of Selenite bril-
    liant green sulfa enrichment broth.  Shake both bath flasks well and
    incubate for 16-18 hours in a water bath at 39.5°C.  Streak one loopful
    from each enrichment on four sets of plates of Salmonella-Shigella (SS)
    agar, bismuth sulfite (BS) agar, eosin methylene blue (EMB) agar to
    brilliant green agar and McConkey's agar.  Incubate plates for 24-48
    hours at 37°C.  Transfer characteristic colonies to triple sugar iron
    (TSI) agar slants.  Incubate overnight at 37°C.  Further identify accord-
    ing to methods of Edwards and Ewing (1962).

Limitations or Precautions

Additional Comments

Reference:Spino, 1966
                                        Peterson and Klee, 1971
                                        Peterson, 1972.       	
             PROCEDURE - DETECTION OF FECAL BACTERIA IN COMPOST

OrganismFecal Streptococci

Methodology
    Incubate on KF streptococcal broth for 48 hours at 35°C.

Limitations or Precautions

Additional Comments
    KF streptococcal broth is more conducive than KF streptococcal agar when
    broth tested simultaneously.  Samples were prepared as described by
    Peterson, 1972 (q.v.).

Reference:Gaby, 1975\
                                      86

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         PROCEDURE - DETECTION OF GRAM POSITIVE BACTERIA  IN COMPOST

Organisms	Coagulase Positive Staphylococci
Methodology            "             ~	
    Prepare serial decimal dilutions of compost suspensions.  Plate on Staph
    110 medium and incubate at 37°C  for 24 to  48 hours.   Small round glist-
    ening low colonies subcultured and later tested for coagulase activity.

Limitations or Precautions

Additional Comments
    In Gaby's study, Chapman-Stone, TPEY and 5% blood agar were also compared
    as media for isolating Staphylococci.  Colonies grown on Staph 110 were
    easiest to recognize.

Reference:~~Gaby,  1975.
            PROCEDURE - DETECTION OF PATHOGENIC FUNGI IN COMPOST

Organisms	Various Species	
Methodology
    Add compost (5 g) to sterile physiological saline (100 ml).  Shake to
    suspend and centrifuge for 15 minutes at 2500 rpm.  Decant supernatant
    and mix sediment with penicillin (10,000 units) and streptomycin  (10 mg).
    After 20 minutes standing at room temperature, inoculate 3 white Swiss
    mice intraperitoneally each with 0.5 ml of sediment.   After 3 weeks,
    sacrifice mice.  Remove and mince spleen and portion of liver. __ Use small
    portions to inoculate 2 tubes of Sabouraud's agar (I) and 2 tubes of
    Sabouraud's agar plus cycloheximide (0.5 mg/ml) and chloromycetin (0.05
    mg/1) (II).  Incubate for 4 weeks at 25°C, examining weekly.  Prepare
    smears of likely colonies and identify by standard cultural methods.
    Also inoculate I and II with portion of the concentrated sediment.  Incu-
    bate for 6 weeks at 25°C and identify by cultural characteristics.

Limitations or Precautions

Additional Comments

Reference:         ~~                   Gaby, 1975
                                      87

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                PROCEDURE - DETECTION OF PARASITES IN COMPOST
Organisms                               Hookworms, Tapeworms, Wireworms
                                        Ova and Cysts
Methodology
    Preparation of Sample;  Place compost (2 g) in flask (250 ml) containing
    saline (20-30 ml) and glass beads to cover flask bottom.  Shake to emul-
    sify, tilt flask and allow sedimentation to occur.

     (a) Direct mount
         Prepare iodine mount using 0.05 ml of sediment for direct micro-
         scopic examination.

     (b) Brine flotation method
         Mix suspension and strain through wire mesh funnel into centrifuge
         tube (50 ml).  Wash mesh with saline (15-25 ml).  Centrifuge for 1
         to 2 minutes at 2000 rpm.  Decant supernatant and resuspend sedi-
         ment in saline.  (Save 10 ml for suspension for formalin-ether
         method). Centrifuge remaining 10 ml as before, decant supernatant,
         resuspend in brine.  Transfer into shell vial, fill with brine to
         lip and cover with slide.  Avoid overflow and airpockets.  After 15
         minutes remove slide, fit with cover glass, seal edges with Vaspar
         and examine microscopically.

     (c) Formalin-ether sedimentation
         Centrifuge 10 ml suspension from brine flotation method.  Decant
         supernatant.  Add 10 ml of 10% formalin, mix and allow to stand for
         5 minutes.  Add ether (3 ml), shake for 30 seconds and centrifuge at
         1500 rpm for 1 to 2 minutes.  Four layers are formed.  Decant top 3
         layers and prepare an iodine mount from the bottom sediment.
Limitations or Precautions
Additional Comments
Reference:                              Gaby, 1975.
                                     88

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         PROCEDURE - ASSAY OF VIRUSES  IN LEACHATE THROUGH SANDY SOIL

Organisms-Poliovirus type 1 (Chat)

Methodology    ~~             "       ~
    Cell Culture;  Grow HeLa cells on  culture dishes  (60 mm) containing Eagle
    minimal essential medium, penicillin G  (200 units/ml), streptomycin (16.6
    units/ml), gentamicin (25 ug/ml) and Fungizone (0.5 ug/ml).

    Virus Assay;  Add 0.2 ml leachate  to HeLa cell monolayers.  Incubate at
    37°C for 48 hours.  Stain with 2%  neutral red in Hanks balanced salt
    solution for 2 to 3 hours.  Count  plaques, express findings in plaque
    forming units/ml.

Limitations or Precautions
    Assay samples within 24 hours after collection.

Additional Comments
    In Duboise study, poliovirus was eluted from soil samples  (5 g) with
    tryptose phosphate broth by vortexing energetically for 30 seconds.  The
    eluates were centrifuged at 12,000Xg for 10 minutes and the supernatant
    was assayed as described above.

Reference:Duboise et al, 1976^
         PROCEDURE - PREPARATION OF CELL CULTURES FOR VIRAL ANALYSIS

Organisms
                                      Poliovirus type 1 (field & lab strain)
                                      Echovirus type 2 (field & lab strain)

Methodology
    Grow BSC 1 cells in Eagle minimum essential medium (I) with 10% fetal
    calf serum, 0.08% NaHCO , streptomycin  (100 ug/ml) and pencillin (100
    units/ml).  Maintain cells in I containing 2% fetal calf serum, 0.12%
    NaHC03, streptomycin (100 ug/ml) and penicillin (100 units/ml).  Grow
    baboon kidney cells as described by Melnick and Werner (1969).

Limitations or Precautions

Additional Comments
    BSC 1 cells are a continuous line derived from African green monkey
    kidney cells.  Glotzbecker and Novello  (1975) added EDTA to the cell
    culture inoculum prior to plaque assay.

Reference:        ~~~Sobsey et al., 1975
                                        Glotzbecker and Novello, 1975
                                     89

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          PROCEDURE - PREPARATION OF CELL CULTURES FOR VIRAL ASSAY
                            (ALTERNATIVE CULTURE)

OrganismsPoliovirus I

Methodology
    Cell Culture:  Grow the HeLa cells in monolayers or minimum essential
    medium containing fetal calf serum (5-10%), penicillin  (100 units/ml) and
    streptomycin (100 ug/ml).  Disperse cells in 0.02% EDTA (10 ml volumes)
    in Ca2+ and Mg2+ free phosphate buffered saline.  Centrifuge for 5 minutes
    at 1000 g.  Resuspend cells in growth medium  (cone. 5 x 10^ cells/ml).
    Inoculation to bottles for plaque assay (50 ml aliquots in 32 oz pres-
    cription bottles, 8 ml in 3 oz, 4 ml in 1 oz).  Monolayers form after 3
    to 4 days incubation at 37°C.

Limitations or Precautions

Additional Comments

Reference:Cooper et al., 1974.
               PROCEDURE - ASSAY OF VIRUSES - PLAQUE TECHNIQUE
Organisms                               Poliovirus I

Methodology
    Pour aliquots of leachage onto drained cell sheets in 32 oz bottles.
    Leave to adsorb at 31°C for 45-60 minutes.  Pour off sample and spread
    overlay medium (50 ml) onto cell sheets.  Overlay medium is 1.5% agar
    (Difco), 3% gamma globulin free PCS, MgCl2 (25 mM), DEAF-dextran (0.02%),
    NaHC03 (0.15%) in MEM without phenol red.  After agar hardened, invert
    medium and incubate 2 days at 37°C.  Stain for counting by adding 3.5 ml
    for 0.08% neutral red.  Count after 4 hours expressing result as plaque
    forming units/100 ml.

Limitations or Precautions

Additional Comments
    Freezing samples containing viruses prior to concentration adversely
    affected viral recovery in Cooper's study.

Reference:Cooper et al., 1974a.
                                     90

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           PROCEDURE - ASSAY OF VIRUSES - TISSUE CULTURE TECHNIQUE


Organisms                           Poliovirus type 1 (field and lab strains)
                                    Echovirus type 7 (field and lab strains)


Methodology
    Make leachate isotonic and purify by membrane filtration (0.22 urn poro-
    sity nitrocellulose filter pretreated with Tween 80 to prevent virus
    adsorption).  Dilute 1:4 in maintenance medium described previously.
    Inoculate into 1 or 16 oz bottles of BSC-1 cells.  Incubate at 37°C
    examining for cytopathic effects over 10 days.  Filter samples of cell
    lysate showing cytopathic effects through 0.22/urn porosity cellulose
    ester filter (treated with Tween 80).  Identify viruses serologically.
    Estimate viral count from total sample volume inoculated and fraction of
    bottles with cytopathic effects due to each virus.
Limitations or Precautions


Additional Comments
    Sobsey et_ al.  (1975) also used plaque techniques for small sample volumes
    (0.2 ml sample in 1 oz bottle of BSC-1 monolayers).


Reference:                              Sobsey et al., 1975
                                      91

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                                 APPENDIX A

                   HEALTH ASPECTS OF  SOLID  WASTE DISPOSAL


    In a study of digested  sludge, Palfi (1973) found that approximately 8%
of all samples analyzed contained poliovirus type 3 within weeks of a local
immunization campaign.  Poliovirus type 3 and echovirus type 2 were found  in
11% of all fecally stained  diapers examined by Peterson (1974).  There exists
the possibility that live attenuated  viruses used in immunization programs
(e.g., the Sabin polio vaccine) may revert  back to virulent wild virus strains
in the environment (Melnick, 1960).

    Live strains of virus for vaccination are obtained by laboratory manipula-
tion of wild strains to increase the  ratio  of attenuated to virulent particles
(Melnick, 1960).  The attenuated particles, which are selected from the mixture,
still cause the antigen/antibody formation  necessary to protect from virulent
strains, but do not produce the disease.  The effectiveness of any live
attenuated poliovirus vaccination program depends on the genetic stability of
the non-virulent vaccine.   In vitro tissue  culture markers are used to
distinguish between attenuated and virulent strains.  For example, viruses
processing the d+ marker grow more rapidly  than d- viruses at low bicarbonate
concentrations in agar.  MS+ viruses  grow more rapidly than MS- strains on
monkey stable kidney cells.  T+ strains grow more readily at 40°C than T-
strains.  Generally, the virulent wild polio viruses possess d+, MS+ and T+
markers as opposed to the d-, MS~P T- highly attenuated strains.

    Melnick (1960) showed that a significant proportion of vaccinated children
(Sabin's attenuated poliovirus types  1, 2,  and 3) excreted viruses which had
reverted to wild types.  He found that d-T- markers reverted to d+T+ in 19%
of the children vaccinated, and that  d-T- reverted to d+T- in 54% of the
children.  Peterson (1974)  also detected wild strains of poliovirus in her
examination of fecally contaminated disposable diapers found in MSW.  This
indicates that either wild  polio strains were circulating in the area, or  at-
tenuated vaccine strains reverted in  the wild.  Upon repeated human passage,
these strains may increase  in neurovirulence and attack unprotected members
of the community (Melnick,  1960; Peterson,  1974).

    Various pathogenic bacteria have  also been isolated from municipal solid
waste.  For example, Salmonella sp. was isolated from refuse by both Spino
(1971), and Gaby (1975).  Salmonella  species are the most common cause of
acute food poisoning in man, and outbreaks  of typhoid and paratyphoid fevers
have been traced to salmonella organisms.   In the United States in 1970,
there were approximately 24,000 reported human cases of salmonellosis.  Many
more cases are not reported or not diagnosed.  It has been estimated that
there are at least two million cases  per annum in this country alone.

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    There are over 1,000 distinct serotypes, all of which  are  pathogenic to
man and/or animals.  S. typhimurium is the most widespread, and  infects both
man and animals.  Infected cattle may excrete 10 million microorganisms per
gram of feces (Diesch, 1973).  Salmonella contaminated manure  may erode from
feedlots into streams and rivers, where it may infect fish, mollusks,  and
other wild animals.  The organisms may also be air-transmitted in dust.

    Shigellosis (bacillary dysentary) is an acute bowel infection characterized
by gastrointestinal cramps, fever, and diarrhea.  The organisms  are excreted
by infected individuals and animals, and may be disseminated by  fecal  pollution
of the water supply; aerosol transmission; transfer of bacilli to flies;  and
the handling of food by infected workers.

    Leptospirosis (Weil's disease, spirochetal jaundice) is a  widespread zoon-
osis caused by a variety of serotypes of the genus Leptospira.   Leptospires
are shed in the urine of infected animals (average concentration 1 x lO^/ml)
for periods of several months.  The organisms may live in  the  environment for
several weeks.  L. icterohemorrhagiae, L. pomona, and L. canicola are  the
commonest strains found in this country"It is associated with  poor manage-
ment of animal wastes rather than MSW.

    The disease may be transmitted to men and animals through  ingestion of
contaminated food and water or by inoculation through broken skin.  There
have been "several" reported outbreaks in this country as  a result of
swimming in contaminated water (Diesch and McCullough, 1966).  Certain
occupational groups are more exposed to the disease and, hence,  have a higher
than average incidence of leptospirosis.  These groups include farmers,  sewer
men, abattoir workers, rat-catchers, and veterinarians.

    Brucellosis is also associated with the above occupations  and the  poor
management of infected wastes.  Brucella abortus commonly  infects cattle and
hogs, and is found in the abortus of infected animals; Br. suis  attacks  hogs.
The disease is transmitted to man by direct contact, or by ingestion of  in-
fected milk.  It has been reported that the organisms have survived in feces
at 80°C for a year, though they are destroyed with a few hours of exposure to
direct sunlight (Stableforth, 1959).

    Human and animal parasitic ova and cysts may also be found in slurries
and in MSW as a result of fecal contamination (Gaby, 1975).  Nematodes such
as Ascaris lumbricoides, Tricuris trichium, and Trichostrongylus sp. may
cause toxocariasis in humans.  Ingestion of nematode ova by a  child may
result in ocular toxocariasis with retinal glaucoma (Powell, 1974).  Protozoa
such as Entamoeba histolytica, Entamoeba coli,  and Giardia Iambiia cause
amebiasis, a colitis characterized by passage of bloody mucoid stools.

    As stated previously, there have been studies linking  disease outbreaks
to contamination of water supplies by sewage sludge and animal slurries
(Powell, 1974).   It should again be emphasized that there  is not one sound
epidemiological study correlating an outbreak of any infectious  disease  in
this country with the pathogen content of municipal waste.
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                                  APPENDIX B

                                HOSPITAL WASTE


    Some  investigators  believe  that the pathogenic  organisms  found  in hos-
pital waste differ  from those present in MSW only in  their  concentrations.
Others think  that hospital  wastes are much more  dangerous due to  their
"infectious"  nature (Ross Hoffmann Associates, 1974a, 1974b).   Research has
indicated  that  up to 15% of raw hospital waste is "potentially infectious"
with from 2%  to 8%  containing pathogens.   Staphylococcus aureus is  the most
common pathogen, but the waste  also contains signficant counts of streptococci,
coliforms, Candida  albicans, and Pseudomonas sp.  (Ross Hoffmann Associates,
1974a, 1974b).   Airborne transmission of microorganisms is  especially signifi-
cant in the hospital environment.

    Considering the well-documented pathogenic nature of much hospital waste,
it is interesting to examine the methods of solid waste handling  and disposal
used in hospitals.   Most hospitals take special  care  in handling  infectious
waste by double bagging in  waxed or plastic waste bags.  They may also color-
code the bags according to  degree of hazard.  These bags may  be hand-carried
or chute-delivered  to a central disposal area.   Pneumatic systems for
transporting  bagged waste have  been associated with high concentrations of
pathogens due to broken bags, etc.  Grieble et aU  (1974) described one chute
hydropulping waste  disposal system as a "reservoir  of enteric bacilli and
Pseudomonas in  a modern hospital."

    After collection, disposal  remains a problem.   Iglar and  Bond (1973) sur-
veyed over 100  hospitals for methods of disposing of  all solid waste, includ-
ing biological  material.  Biological material included afterbirth,  amputated
tissue, autopsy tissue,  animal  carcasses,  and bacteriological cultures.  In-
cineration was  the  preferred form of disposal, but  some biological  material
was hauled away with mixed  solid waste usually to a landfill  (see Table  Bl)
Some hospitals  autoclave biological wastes or treat them with formalin prior
to disposal.  Some  biological material (afterbirth) is used by drug firms for
experimental work.   Some hospitals even disposed of untreated wastes in dumps,
or fed the garbage  to hogs,  both of which are illegal and environmentally
unacceptable practices.

    Grinders have also  been used in hospitals for treatment of biological
waste.  Jopke and Hass  (1968) reported that regardless of design, grinders
generate bacterial  aerosols which,  however,  could be  minimized with an
exhaust system.  After  grinding,  the waste is discharged into the sewage
                                     103

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system.  The local municipal sewage facilities may have problems  in handling
this heavy load of suspended solids.

    Iglar and Bond confirmed the findings of previous researchers that
hospital incinerators are often poorly operated.  The staff may not be
competent, incinerators are often overcharged, and they may be used only
periodically so that necessary kill temperatures are not achieved (see also
Section 4, this report).  They also found that the necessity of compliance
with air pollution-particulate emission standards has apparently  forced sev-
eral hospitals to abandon incineration.

    It does appear from this very brief examination of the solid  waste dis-
posal practices of the American hospital, that there are no special methods
used by hospitals to dispose of highly infectious wastes that are applicable
to the management of MSW.  Similar problems seem to exist for both types of
waste.  The most common method of treating hospital wastes is incineration,
and for a variety of reasons, reviewed previously, incineration is not always
an efficient method of destroying those pathogens present in municipal waste.

                                  TABLE Bl

                DISPOSITION OF SELECTED BIOLOGICAL MATERIALS
                           (Iglar and Bond, 1973)
                               % Hospitals (Full-Scale Stage)
Type of
Biological
Material
Inciner-
ation
On-Site Burial
Hauling Away Use by
with Other Drug
Waste Firm
Not
Appli-
Other cable*
Afterbirth             26

Amputated tissue       50

Autopsy tissue         31

Animal carcasses       15

Bacteriological
  cultures             69
 9

34
 7

11

 4

 1


21
                        51
3

6
13

24

27

83
* No material of indicated type discarded.
  Refers to tissue replaced in body.
                                     104

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
  REPORT NO
 EPA-600/8-80-034
                                                           3. RECIPIENT'S ACCESSION-NO.
  TITLE AND SUBTITLE
  A Survey of  Pathogen Survival During Municipal  Solid
  Waste and Manure  Treatment Processes
             5. REPORT DATE
               August 1980 (Issuing Date)
                                                           6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
                                                           8. PERFORMING ORGANIZATION REPORT NO.
  Sylvia A. Ware
9. PERFORMING ORGANIZATION NAME AND ADDRESS
  Ebon Research  Systems
  1542 Ninth  Street, N.W.
  Washington,  D.C.   20001
             10. PROGRAM ELEMENT NO.
               1DC618
             11. CONTRACT/GRANT NO.


               68-03-2460-5
 12,^P£)JvlSQRLN_Q AQ.ENC_XN AME AND ADDRESS
  Municipal Environmental Research Laboratory--Cin.,OH
  Office of Research  and Development
  U.S. Environmental  Protection Agency
  Cincinnati, Ohio   45268
             13. TYPE OF REPORT AND PERIOD COVERED
               Final Research
             14. SPONSORING AGENCY CODE
               EPA/600/14
15. SUPPLEMENTARY NOTES
  Project Officers:   Carlton C. Wiles and  Laura A.  Ringenbach  (513) 684-7871
 16. ABSTRACT

    Municipal solid  waste (MSW) and animal manures may contain microorganisms  that can
    cause disease  in man and animals.  These  pathogenic microorganisms  include enteric
    bacteria, fungi, viruses, and human and animal  parasites.

    This report  summarizes and discusses various  research findings documenting the
    extent of pathogen  survival during MSW treatment.   The technologies discussed  are
    composting,  incineration, landfill, and anaerobic  digestion.  There is also  a
    limited examination of the use of the oxidation ditch as a means of animal manure
    stabilization.   High gradient magnetic separation  (HGMS), and gamma radiation
    sterilization  are mentioned as future options,  especially for animal waste
    management.  Several  standard methods for the sampling, concentration, and
    isolation of microorganisms from raw and  treated solid waste are also summarized.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                              b.IDENTIFIERS/OPEN ENDED TERMS
                           c.  COSATI Field/Group
    Microorganisms
    Bacteria
    Refuse Disposal
    Viruses
    Wastes
    Manure
 Compost
 Incineration
 Landfill
 Samp!ing
 13B
18. DISTRIBUTION STATEMENT


        Release to Public
19. SECURITY CLASS (This Report)

  Unclassified
                                                                         21. NO. OF PAGES
115
20. SECURITY CLASS (This page)
  Unclassified
                           22. PRICE
EPA Form 2220-1 (9-73)
                                            105
                      1 U S GOVERNMENT PRINTING OFFICE 1<*SO -6 57 - 1 6 5/006 1

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