-------
and filtration dewatered.  The final sludge treatment process
employed was aerated static pile composting and resulted in an
annual compost production of 70,000 cubic yards.  The sludge was
composted for 21 days with wood chips added as a bulking agent.
The compost was cured for 30 days prior to distribution.  Wood
chips were recycled.  The final sludge product was distributed
predominantly in bulk although a small portion was distributed  in
bags by a scata agency.  The product was used by homeowners (28%),
nurseries (!%)/ landscapars (33%), and others (37%), including
contractors, institutions, and top soil dealers.

III-B-4—
     Treated wastewater was estimated to come from domestic (90%)
and industrial (10%) sources.  Secondary sludges were produced
which were anaerobically digested and filtration dewatered.  The
final sludge treatment process employed was aerated static pile
composting and resulted in an annual compost production of 9,200
cubic yards.  The sludge was composted for 21 days with wood chips
added as a bulking agent.  The compost was cured for 30 days prior
to distribution.  Wood chips were recycled.  The final sludge
product was distributed entirely in bulk.  The product was used by
homeowners (49%), nurseries (2%), iandscaoers (35%), and others
(14%).

III-J-1—
     This facility treated 10 mgd of wastewater which was estimated
to come from 95% domestic and 5% industrial sources.  Primary,
secondary, and chemical sludges were produced which were lime
treated and anaerobically digested prior to filtration dewatering.
The final sludge treatment process employed was an aerated windrow
composting process and resulted in an annual compost production of
4,200 cubic yards.  The sludge was composted for 21 days with
recycled compost added as a bulking agent.  The compost was cured
for 30 days prior to distribution.  The final sludge product was
distributed 95% in bulk.  The product was used by farmers (20%),
homeowners (5%), landscapers (65%), and others (10%).

IV-B-1—
     Treatment plant IV-B-1 treated 4.5 mgd of wastewater which was
estimated to come from 35% domestic and 65% industrial sources.
ieeendary sludges were produced which were anaerobically digested
and then dewatered by centeifugation.  The final sludge treatment
process employed was aerated static pile composting and resulted in
an annual compost production of 6,500 cubic yards.  The sludge was
composted for 23 days with wood chips added as a bulking agent.
The compost was cured for 23 days prior to distribution.  Wood
chips were recycled.  The final sludge product was distributed
almost entirely in bulk.  The product was used by farmers (10%),
homeowners (40%), nurseries (20%), and landscapers (30%).

IV-D-1—
     This municipality treated 201 mgd of wastewater at a number of


                                 16

-------
treatment plants which was estimated to come from 95% domestic and
<5% industrial sources.  Primary and secondary sludges were
produced which were anaerobically digested and dewatered by
centrifugation.  The final sludge treatment process employed was
drying beds and resulted in an annual sludge production of 20,000
dry tons.  The final sludge product was distributed entirely in
bulk by an outside company.  The product was used by farmers.


     Municipality IV-F-1 treated 2.6 mcd of wastewacer which was
estimated to come entirely from domestic sources.  Secondary
sludges were produced which were aerobically digested.  The final
sludge treatment process employed was drying beds.  The final
sludge product was distributed in bulk by the operating agency.
The product was used by homeowners (60%) and nurseries (40%).

IV-I-1—
     This facility treated 13 mgd of wastewater which was estimated
to come from 95% domestic and 5% industrial sources.  Secondary
sludges were produced which were dewatered by centrifugation.  The
final sludge treatment process employed was a heat drying process
and resulted in an annual sludge production of 3,000 dry tons.  The
final sludge product was distributed entirely in bulk by the
operating agency „  The product was used by farmers.
     Treatment plant V-B-1 treated 1.5 mgd of wastewater which was
estimated to come from 90% domestic and 10% industrial sources.
Primary and secondary sludges were produced which were thickened
and filtration dewatered.  The final sludge treatment process
employed included both windrow and aerated static pile composting
and resulted in an annual compost production of 900 dry tons.  The
sludge was composted with wood chips, sawdust, recycled compost,
corn shueks and leaves added as bulking agents.  The material was
composted, cured? and stored for t total of 120 days prior to
distribution.  Wood chips were recycled.  The final sludge product
was distributed entirely in bulk by the operating agency.  The
product was used by farmers (5%), homeowners (90%), nurseries
(2.5%), and landscapes  (2.5%).

V-K-1—
     This municipality treated 6,4 mgd of wastewater which was
estimated to come from 32% industrial, 25% domestic, and 43% other
sources.  Primary and secondary sludges were produced which were
thermally conditioned with a commercial system and filtration
dewatsred.  The sludge treatment process resulted in an annual
sludge production of 3300 cubic yards.  The final sludge product
was not distributed? the product was disposed in a landfill.  This
facility was included to evaluate the effects of thermal
pretreatmsnt.

-------
7I-D-1—
     Municipality IV-D-1 treated  112 mgd of wastewater  which was
estimated  to come from 10% industrial and 90% domestic  sources.
Primary and secondary sludges wera produced which  were
anaerobically digested.  The final sludge treatment  process
employed was drying beds and resulted in an annual sludge
production of 30,000 dry tons.  The dried sludge was stored  prior
to distribution.  The final sludga product was  distributed in bulk
by the operating agency.  Tha product was used  by  the Park and
Highways Departments.

7II-D-I —
     This municipality treated 38 mgd of wastewater  which  was
estimated to come from 55% industrial and 45% domestic  sources.
Primary and secondary sludges wera produced which  were
anaerobically digested and dewatered by a combination of filtration
and lagooning.  The final sludge  treatment process employed  was
windrow drying and resulted in an annual sludge production of 5,000
dry tons.  The first three samples received from this site were
composites of sludge that had been windrow dried and stored  for  two
or more years prior to distribution.  Sludge processing was  changed
at the facility to include the addition of 20%  cement kiln dust  to
the wet sludge.  The remaining three samples contained  the kiln
dust and represented recently processed sludge.  The final sludge
product was distributed in bulk to other city agencies  after a two
year holding period.

7II-A-2—
    Municipality 7II-A-2 treated  16.5 mgd of wastewater which was
estimated to come from 20% industrial and 30% domestic  sources.
Primary and secondary sludges were produced which  were
anaerobically digested and filtration dewatered.   The final  sludge
treatment process employed was windrow composting  and resulted in
an annual compost production of 3100 dry tons.  The  sludge was
composted for 42 days without any added bulking agents.  The
compost was cured for 1.5 years prior to distribution.  The  final
sludge product was distributed entirely in bulk by the  operating
agency.  The product was used by homeowners (10%), landscapers
(45%) and governmental agencies (45%).

71II-D-1—
     This facility treated 2.0 mgd of wastewater which  was
estimated to come from 90% domestic and 10% other  sources.
.Secondary sludges were anaerobically digested.  The  final  sludge
treatment process employed was drying beds.  The final  sludge
product was distributed in bulk by the operating agency.   The
product was used by homeowners (35%) and landscapers (15%).

VII-P-l—
    Treatment plant VIII-F-1 treated a variable flow of 4  to 10  mgd
of wastewater which was estimated to come from  99% domestic  and  1%
industrial sources.  Secondary sludges were produced which were


                                  13

-------
aerobically digested.  Tha final sludge treatment process employed
was drying beds and resulted in an annual sludge production of 260
dry tons.  The dried sludge was stored prior to distribution.  The
final sludge product was distributed in bulk by the operating
agency.  The product was used entirely by homeowners.

VIII-H-1—This municipality treated 7 mgd of wastewater which was
estimated to come from 2% industrial and 90% domestic sources.
Secondary sludges wara produced which were anaerobically digescsd
and filtration dewatsred.  Tha sludge was composted by a private
company and used to produce a line of commercially marketed
products.  The sludge was composted for 180 days with wood chips,
sawdust, and proprietary ingredients added as bulking agents.  The
final sludge product was distributed 50% in.bulk and 50% in bags.
The product was used by homeowners (70%), nurseries (10%),
landscapers (15%), and others (5%).


VIII-J-1—
    Municipality VIII-J-1 treated 150 ragd of wastewater which was
estimated to come from 90% domestic and 10% industrial sources.
Primary and secondary sludges were produced which were
anaerobically digested and dewatered by centrifugation.  The final
sludge treatment process employed was aerated windrow composting
and resulted in an annual compost production of 30, 000 dry tons.
The sludge was composted for 25 days with wood chips and other
materials added as bulking agents.  The final sludge product was
distributed in bulk by the operating agency .  The product was used
by farmers, homeowners, nurseries, landscapers, and others.

IX-A-10—
    This municipality treated 3.5 mgd of wastewater which was
estimated to come from 8% industrial, 69% domestic, and 23%
commercial and institutional sources.  Primary and secondary
sludges were produced which were anaerobically digested and
dewatered by a combination of gravity thickening, drying beds, and
eentrifugation*  Ths final sludge treatment process employed was
windrow composting and resulted ia an annual compost preduetian of
20,000 cubic yards.  The sludge was composted for 60 days with rice
hulls .added as a bulking agent.  The compost was cured for 30 days
prior to distribution.  The final sludge product was distributed  in
bulk by an outside contractor.  The product was used by homeowners
(10%), nurs@sies (20%), and landscapers (70%).

IX-B-1—
    This regional facility treated 80 mgd of wastewater which was
estimated to come from 13% industrial, 65% domestic, and 22%
commercial seurcss.  Primary and secondary sludges were produced
which vert anaesobically digested and dewatered by centrifugation.
The finai sludge treatment process employed was aerated static pile
composting and resulted in an annual compost production of 125,000
cubic yards.  The sludge was composted for 25 days with wood chips
added as a bulking agent.  The compost was cured for 30 days prior


                                 19

-------
to distribution.  Wood chips were recycled.  The final sludge
product was distributed 95% in bulk and 5% in bags by an outside
contractor.  The product was usad by homeowners (70%), nurseries
(5%), and landscapers (25%).

IX-0-1—
    Municipality IX-D-1 treated 155 mgd of wastewatar which was
estimated to coma from 7% industrial and 93% domestic scurcas.
Primary and secondary sludges ware produced which wera
anaerobically digested.  The final sludge treatment process
employed was drying beds and resulted in an annual sludge
production of 50,000 cubic yards.  The final,sludge product was
stockpiled.  Marketing was planned for the future.

IX-D-2—
    Treatment plant IX-D-2 treated 150 mgd of wastewater which was
estimated to come from 8% industrial and 92% domestic sources.
Primary and secondary sludges were produced which were
anaerobically digested and dried in lagoons.  The final sludge
treatment process employed was drying beds and resulted in an
annual sludge production of 30,000 dry tons.  The final sludge
product was distributed entirely in bulk by an outside contractor.
The product was used farmers.


IX-D-3—
     This facility treated 155 mgd of wastewater which was
estimated to come from 5% industrial and 95% domestic sources.
Primary and secondary sludges were produced which were
anaerobically digested and dried in lagoons.  The final sludge
treatment process employed was drying beds and resulted in an
annual sludge production of 40,000 dry tons.  The final sludge
product was distributed in bulk by an outside contractor.  The
product was used by farmers (90%) and landacapers (10%).
                                 20

-------
Table 4.
Bimonthly Sampling Sites*
  Sita Code
      iiocation
Process
Classitication
X •
2.
3 .
4.
5 .
6 .
7.
a.

9.
10.
11.
12.
13.
14.
15.
16.

17.
18.
19.
20.
21.
22.
23.
24.
1-8-1
II-C-1
in-a-3
III-3-4
III-J-1
-1—7 _ r» _ l
* V 23 -.
IV-D-1
IV-P-1

IV-I-1
V-B-1
V-K-1
VI-D-1
VII-D-1
VII-A-2
VIII-D-1
VIII-F-1

VIII-H-1
VIII-J-1
IX-A-10
IX-B-1
IX-D-1
IX-D-2
IX-D-3
X-C-1
Maine
New Jersey
Maryland
Virginia
Virginia
North Carolina
Florida
Florida
' >
Georgia
Indiana
Indiana
Texas
Kansas
Kansas
Colorado
Dtah

Montana
Colorado
California
California
California
Arizona
Arizona
Oregon
Static Pile
In-Vessel
Static Pila
Static Pile
Aerated Windrow
Static Pile
Drying Bed
Aerobic Digestion-
Air Dried
Heat Drying
Static Pile
Thermal/Filter Press
Air Dried
Air Dried
Windrow
Air Dried
Aerobic Digestion-
Air Dried
Proprietary
Aerated Windrow
Windrow
Static Pile
Air Dried
Air Dried
Air Dried
In-Vessel
PFRP
PFRP
PFR?
??RP
PFRP
PFRF
PSR?

PSRP
PPRP
PFRP
PPRP
PSRP
PSRP
PFRP
PSRP

PSRP
PFRP
PFSP
PFRP
PFRP
PSRP
PSRP
PSRP
PFRP
     * Sampled once every two months from April 1986 - April 1987.
                   t
Note;  Air dried sludges were anaerobically digested unless other-"
       wise stated.

3f—/" — 1 =»-.
A v=> i^™
     Municipality X-C-1 treated 71 mgd of wastewater which was
estimated to come from 40% industrial and 60% domestic sources.
Primary and secondary sludges were produced which were
anaerobieally digested and filteration dewatered. The final sludge
treatment process employed was an in-vess«l composting system and
resulted in a proposed manual compost production of 60,000 cubic
yards.  The sludge was composted for 15 days with sawdust added as
a bulking agent,  The compost was cured for 15 days prior to
distribution.  The final sludge product was distributed in bulk by
an outside contractor.  The product was used by homeowners (!%)/
nurseries (4%), landscapers (70%), and other users  (2S%).
                                 21

-------
SELECTION OP MICROORGANISMS FOR TESTING.


The microorganisms salectsd for quantitative analysis during this
project can generally be divided into two broad groups, the
indicator organisms, and the pathogens.  In some cases, however,
that separation is not distinct.  For example, the caliform
bacteria are commonly used as indicator bacteria but a subgroup of
the California, the enterotoxigenic S. coli, are pathogens.
Nevertheless, the basic separation is useful and reflects the
primary reason for determining the presence of the organisms.
Table 5 lists the organisms included for analysis.

Table 5.  Microorganisms Selected for Analysis	'   	
 Indicator Groups                         Pathogenic Microorganisms

Total coliform                         Enterotoxigenic E. coli
Fecal coliform         '                Total enteric bacteria
Fecal streptococci                     Salmonella
Aerobic plate count                    Campylobacter
Anaerobic Plate count                  Yersinia
Total Fungi                            Ascaris'ova
Thermophilic fungi                     Total parasites
Bacteriophage                          Total enteric viruses
     The indicator groups, collectively, served two purposes in
this study.  The first was to determine if there was a correlation
between standard or non-standard indicators and the occurrence of
any pathogens detected.  The use of indicator organisms, such as
coliforms, fecal streptococci and plate count organisms has been a
long established practice in sanitary microbiology/ however, the
significance of indicators in treated sludges was uncertain.  The
second purpose of the indicator groups was.to ascertain if post
treatment bacterial or fungal regrowth occurred to any significant
extent.

     The pathogens listed in Table 5 were selected on the basis of
three-criteria:  (a) enteric pathogens previously demonstrated in
sludges, (b) enteric pathogens of current epidemiological concern
whose presence or fate in sludge was unknown and (c) pathogens of
potential concern due to the nature of exposure associated with
home use of sludge products.

     Many of the tests for the microorganisms listed in Table 5 are
standard tests.  However, some of the procedures and associated
terminology are non-standard and should be defined to avoid
confusion.  The following definitions apply.  Laboratory methods
are described in Section 5.

     Total and Thermophilic Fungi - The term total fungi, as used
here, is somewhat of a misnomer.  Mesophilic and thermophilic fungi


                                 22

-------
would be more accurate descriptive terms.  No one medium or
incubation temperature is adequate to recover all of the fungi
potentially present.  An evaluation of fungal media and incubation
temperatures was conducted during the initial stage of the study to
select the most productive media and temperatures.  Results of this
evaluation are detailed in Appendix A.  In many cases, there is a
great deal of overlap between the fungi in the two groups.  At
other times, the two populations may be mutually exclusive.  It is
posible to have a substantial number of thermcphilic fungi and no
total fungi/ or vice-versa.

     Bacteriophage - The bacterial virus measured in this study was
the coliphage that infects E!. coli C.

     Snterotoxigenic E. coli - The initial proposal indicated that
the potential health risk associated with enteropathogenic E. eoli
would be addressed by screening a representative portion of E. coli
isolates with commercially prepared antisera for the most common
pathogenic serotypes.  A 1970 publication by the American Public
Health Association listed 11 serogroups that were most frequently
associated with E. coli diarrhea.  More recent publications (Sack,
1975) however, indicated that there was little relationship between
serotype and pathsgenieity of a strain.  Conversations with the Los
Angeles County-University of Southern California Medical Center
clinical laboratory and the Los Angeles County Public Health
Department laborateey revealed that these laboratories no longer
assigned any clinical or public health significance to the presence
of the classic "pathogenic" serogroups.

     The cholera-like symptoms produced by pathgogenic E. coli
strains are usually the result of enterotoxins produced~by the
organisms.  Two general types of enterotoxins are produced:
heat-stable (ST) and heat-labile (LT).  Almost all human strains
studied have been shown to produce both types (Sack, 1975).  The
ability to produce enterotoxins is carried by plasmids.  E. eoli
serotypes might eentain, or conversely lose, the enterotoxin=>
controlling plassaid.  It has been speculated that many serotypes
originally described as enteropathogenic were enteretoxigenie.

     Because of the discrepancy between enteropathogenic serotypes
and enterotoxigenic properties, there is confusion in defining
these terms „  Sack (1975) recognized two basic types of
diarrheagenie E» eoLii  (a) enterotexigenic strains, and (b)
invasive strain's.  Mere recently, a World Health Organization (WHO)
working group (1980) defined three forms:  (a) enters texigenic,, (b)
enteropathogenic, and (c) enteroinvasive.  The enterotexigenic and
enteroinvasive groups are the same as described by Sack.  The
enteropathogenic group defined by WHO includes E. coli strains  •
shown to produeg enteritis but where no virulence factors have been
identified.  The enttreinvasive E. eoli strains are biochemically
and antigeniea-lly similar to Shigella.  It is likely that many
outbreaks due to these organisms"have been reported as shigellosis
(WHO 1980).  From a purely clinical point of view, the difference
is probably moot.


                                 23

-------
     In order to assess potential health risk associated with
diarrheagenic E. coli, this study tested S. coli populations for
enterotoxigenic strains.  The term enterotoxigenic will be used
exclusively and will refer to those S. coLL strains producing ST
and LT snterotoxins.

     Total Enteric Bacteria - This refers to a plate count
performed on a common enteric isolation medium, MacConkey agar.
The purpose of this procedure was to dstact: enteric bacteria that
were not analyzed by a more specific test.  Ths plate count data
reported for this test includes ail organisms capable of growing on
MacConkey agar.  These include many bacteria that are not members
of the Snterobacteriaceae,  and therefore are not classic enteric
bacilli.

     Total Parasites - This category includes the sum. of all
parasitic helminth ova and protozoan cysts detected in a sample.
                                 24

-------
                             SECTION 5

                       MATERIALS AND METHODS


 SAMPLING PROCEDURES

     Compost or sludge samples were collected packed and shipped by
personnel at each participating municipality.  Sampling kits were
provided for each sample collected.  The sampling kit included two
sterile 5 inch x 14 inch Whirl-Pale bags for microbiological samples,
a 1 quart glass jar with a teflon lined lid for chemistry samples,
a sterile scoop, polyfoam refrigerant packs, sampling instructions, a
sample collection form, and a Freeze Safe insulated mailing container.
The sampling instructions and documentation form are illustrated in
Figures 1, 2, and 2,

     When the sample arrived in the laboratory, sample
documentation and labelling were checked, then the Whirl-Pak bag
microbiology samples were stored at 4 C and the 1 qt. glass jar
chemistry sample was stored in a -20 C freezer.  Microbiological
analyses were usually started within 5 days of sample collection.

MICROBIOLOGICAL METHODS

     The microbiological methods which were used for this study
are presented in outline form.  All references to Standard Methods
refer to the sixteenth edition (APHA, 1985).  All media were
prepared, and reactions interpreted, according to manufacturers'
instructions, unless otherwise stated.

    Sample Preparation

        Dewatered sludge samples or compost samples were suspended
        in an appropriate diluent in order to inoculate broths
        and/or plates for microbiological analyses.

        A.  Preparing suspension (two blender jars were prepared,
            one for bacterial tests and one for parasite tests)

            1.  50 g of sample was weighed into a sterile 1 qt.
                stainless steel Waring blender jar.

            2.  500 mL sterile phosphate buffered dilution water
                (Standard Methods APHA, 1985) containing 0.1%
                Tween 30 (acts as dispersant) was added.


                                 25

-------
Figure 1.    Forsi Latter  Detailing  Sludge  Sampling Procedures
                        Procedures  for  PQ7W  Sludge  Sampling
      The  sampling  of  sludge  at  your  waatawater  treatment  facility  should  be
      performed at  the location  specified  previously.
                                                                                    !
      Irrespective  of  where  the  sludge  samples are  actually  collected,  it  is
      important that three basic objectives  be kept in mind:   (1) Samples
      should be representative of the bulk material from which they are
      collected,   (2)  Sludge  character  or  quality should not be altered as a         |
      result of the sampling, and (3) Proper QA  procedures appropriate  for          •
      collecting  samples  for  microbiological analyses  should be adhered to.
      It is important  that all procedures  employed  relative  to sample
      collection  are properly documented in  a study plan or  field log.   (See
      sample collection form  and instructions.)

      Factors such  as  accessibility and physical characteristics of the sludge
      should be considered when  selecting  a  sampling device  and/or  procedure.
      The  sampling  device should b« elsas  and constructed  of an inert or
      unreactive  substance such  ac stainless steel  or  teflon,   the  sampling
      method will vary depending on the type of  sample requested.   Dried
      sludge in either a  "cake"  fona  or within a drying b«d  sheuid  be easily
      accessible  and can  be  sampled using  either a  trowel, scoop, shovel or
      auger.  Availability and'e&se of  ua« will  probably be  the determining
      factor.  A  shovel or auger as®  better  suited  for sampling from a  deeper
      bed  of material, or a stock pile.  Again,  it  should  be emphasized th&t
      whatever sample? is used,  proper  cleaning  procedures should be followed.

      For  purposes  of  this sampling program?  it  will be necessary to fill  a 5
      inch X 14 inch sterile  Whirl-Pak  bag and % 1  quart glass jar  having  a
      teflon lined  lid.   The  bag should m filled stoeue 2/3  full, and the  jar
      should be filled as completely  as possible0   % sterile scoop  is provided
      to fill the sample  containers.  Preservatives must not be added to any
      of the samples.  Samgl&s should be rsfriferatsd  and  shiop«d as soon  as
      possible.                                              **

      Lastly, it  is important that all  samplas are  properly  labeled and
      packaged prior to shipment.  Osing a felt  marking pen,  label  the  bag and
      jar  with the  site eod«, date, and time sample was collected.   The
      samples should be packaged with & Fre®se«Psk  and svery attempt should be
      made to ensure that ths sample  bottle  will net: be broken during ermnsit.
      The  insulated box containing the  samples sheuld  also be  taped, labeled,
      and  shipped via  Federal Express.

      Any  questions regarding sampling  should be directed  to Bill tanks at the
      Los  Angeles County  Sanitation Districts (213)  S85-9S72.
                                       26

-------
Figura  2.    Instructions .Sent  to  Participating  Facilities
            INSTRUCTIONS FOR COMPLETING SAMPLE COLLECTION FORM
                   (Oaa 4 aacarata tosxi for each sampia)

      Samola Information

      1.  3ampla I. D. !  Ths identifying information written on the lac«si
         on ciia sample container. "This information includes sits code,
         sample number if more than one sample collected on game day,
         data, and tiaie sample colleetad.  Your site code is: 	
      2.  Sampling Site?  Thw name of treatment plant and/or city and
         state.

      3.  Field Sampling Manager:  The name of person collecting sample.

      4 and 5.  Already completed.

      5.  Source Sampled;  The point the sample was collected from,  such
         as, " stockpile at treatment plant* or 'bag of Grow Fast garden
         food" or "drying bad", .etc.

      7.  Quantity Sampled;  i.e., "2/3 of whirl pak bag* and/or
         "1 o.t.      ~
     3.  Sample Description?  i.e., "sir dried digested sludge" or
         "windrow compos'te? sludge" or "in-vessel composted sludge".
         Also indicate if composite* sample or single grab sample.

     9.  Other Information t  Air temperature t  Self explanatory,

                   Brief description of prevailing weather conditions
         when samples collected, i.e., "cold, snow" or "intermittent
         rain" or "hot humid" etc.   Pile Temperature:  if sludge is in a
         stockpile and is self heating, or if sludge is above ambient
         temperature for any reason, measure sample temperature at a
         representative point or depth.  Other is any other information
         you think may be pertinent regarding samples,  such as, "flocks
         of seagulls feeding on tops of stockpiles", or "extremely heavy
         rains three days ago".  Any information about conditions that
         could affect the sample would be helpful.

 II.  Handling and Shipping

     1.   Describe Sample Treatment  Prior to Shipping:  Briefly
         describe how you collected the sample.   Foe axantpla,  "TJsing a
         clean shovel, removed approximately one foot of material from
         surface of stockpile*  With clean scoop,  collected sample and
         plaeed in sample
     2 and 3,   Self explanct@ry

     4.   Comments;   Any comments  or observations concerning  sampling
         that  might influence laboratory results.

     5.   UPS,  Federal  Express,  Etc.

     6.   Already Completed.

 III.   Arrival

      This section  is  filled  out  by  Laboratory.
                                    27

-------
Figure 3.    Sample  Collection  Form Sent to Participants
                             SAMPLE COLLECTION FORM

     I.   Sample Information

     1. Sample 1.0. (Code)            Collection Data

     2. Sampling Sita
     3. ?iald Sampling Manager (on sita)
     4. Contractor  L.A. County Sanitation Dist.   Contract No. ca-912589-010
     5. EPA Project Officar  H. Jakubowaki  Program Name  Oceurranca of	
       Pathogens In Distribution and Marketing Municipal Sludges	
    6. Source Sampled
     7. Quantity Sampled/Units

     3. Sample Description
    9. Other Information aa Applicable    Air tsiap ____ Pile Temp

       Weather	 Other	
    II.  Handling and ShiOBing

     1. Dssesibtt Sample Treatment Prier fee
     2. Piald Storage and Shipping  Coraditiens"
                 Container                          geagerstu.
                 Mti3.ri Pa* Bag                       	Ambient
                _Gla«s  With  Taflere  kid  Liner  	Packed  with  Pre«g8  Psk
     3.  Date and  Tims,  Shipped

     4»  Co«i8«ats	
     5.  node and Carries  £os Shipping 	
     6,  Sa«pl«  Shippsd  tot    San  Joes Creek  Water Quality  Laboratory
                             1965 So. Workman Mill Rd. , wh'ittier^ CA 50501
                             Attentions W. A.
    III.  Arrival  (Lab uc« only) Date _       Tima            sy
    Late Job  No.           Charge No.                     Pro-j. N©~
    R®qu»«€sd  bys ^_^_ia=mm^_^_aM_^___,__„_ Report
         and Tia* - Geate Saapias	/	       _/_
    Sample                      ~'''~

-------
         3.  The mixture was blended at medium to high speed
             for 1 minute.

     3.  Total Solids (TS) were determined for the original
         sample according to procedures described in Standard
         Methods (APEA, 1985).  TS results were used to
         calculate final results from microbiological analyses
         i.e-., number of microorganisms/g dry weight sample.

Tests for Standard Indicator Organisms

     A.  Total coliform, fecal coliform, fecal streptococci.

         1.  Tubes of appropriate media for most probable
             number (MPN) tests were inoculated from the
             sample suspension.

         2.  Dilutions to be inoculated were selected
             based on experience of the anticipated range
             of bacteria in the samples.

         3.  Tests were performed and MPNs computed as
             described in Standard Methods (APEA, 1985).

         4.  Osing TS data, final results were calculated
             and expressed as MPN/g dry weight.

     B.  Aerobic and anaerobic plate count.

         1.  0.1 mL of dilutions from the sample suspension
             were inoculated onto duplicate plates of pre-
             dried plate count agar for each dilution.


         2.  Dilutions to be inoculated were selected
             based on experience of the anticipated
             range of bacteria in the samples.

         3.  Spread plate method tests were performed
             as described in Standard Methods (APEA, 1985).

         4c  Anaerobic plates were incubated in "Gas
             Fmk" mnaefofeie jars, per manufacturer's
             instructions*

         5.  Plates were incubated at 35 C for 48 hrs.

         6.  Colonies were counted per Standard Methods
             (APEA, 1985) guidelines.

         7.  TS results were used to calculate and report
             final results as colony forming units (CPU)/g.
                              29

-------
Tests for Non-Standard Indicator Organisms

      A.  Total Fungi

          0.5 mL and G.Q5 mL of sample suspension were
          inoculated onto predried plates of modified Rose
          Bengal agar and V-8 juica agar in triplicate for
          each dilution.  Additional dilutions were necessary
          for some samples.

             a.  Modified Rose Bengal agar
                 Cooke Rose Bengal Agar (Difco)    36.0 g/L
                 Tergitol NP-10 (Baker)             0.1 mL/L
                 To autoclaved, cooled medium, add  0.1 g/L
                   chlcramphenicol (dissolve 0.1 g
                   chloramphenicol in 4 mL ethanol).

             b.  V-3 juice agar
                 V-8 Juice cocktail            200 mL
                 Calcium carbonate               3*0 g
                 Yeast extract                   2.0 g
                 Agar                           20,0 g
                 Deioniged (DI) water          720 mL
                 Tergitol NP10 (Baker)           0*1 mL

          To auteclaved cooled medium, add 20 mL/L Pen-=
          Strep stock (1 million units Penicillin G and
          1.0 g Stratomyein sulfate per 100 mL); and
          150 ppm Benlate  (Dupont) (0.16 g Benlate 60% wettable
          powder in 10 raL sterile D.I. water)? rinse into
          medium with 40 mL sterile D.I. water.

          2.  Fungal plates were incubated at 35 C for 48 hrs.
              while exposed to light.

          3o  Differential colony counts were done for each
              medium based on colony morphelogy*

          4.  Fungal colonies exhibiting adequate morphological
              characteristics were identified.

          5.  Fungal colonies not showing adequate reproductive
              characteristics were transferred to Potato
              Dextrose agar (Difeo) and cultured for
              identification.

          6.  Fungal colonies were identified using standard
              keys (Barnett at al 1972, Raper at al 1965).

          7.  Differential counts were added together  for total
              fungus counts.

          8.  Final results were calculated and expressed as
              CFU/g dry weight.


                               30

-------
     B.  Thermophiiic Fungi

         1.  Platas of Rosa Bengal agar and V-8 juice agar wera
             inoculated as described under Total Fungi.

         2.  Plates were incubated at 44 C for 48 hrs.

         2,  Colonies wera counted and identified a.s described
             undar Total Fungi.

         4.  Final results were calculated and expressed as
             CFU/g dry weight.

     C.  Bactariophage
         1.  40 g of sample was weighed into 400 mL of sterile
             3% beef extract, pH 9.5 containing 0.4 mL/L
             antifoam 3 (Baker).

         2.  The mixture was blended in a sterile stainless
             steel Waring blender jar for 3 min. at high speed.

         3.  A portion of blended sample was poured into a 250
             mL centrifuge bottle and centrifuged at 10,000
             RPM (700 X g )for 15 minutes in a refrigerated
             centrifuge.

         4.  A portion of supernatant was collected and
             filtered through a 0.2 urn porosity Mi11ex
             (Millipore Corp.) filter pretreated with sterile
             3% beef extract.

         5.  Samples were assayed by the soft agar overlay
             plaque assay technique (Adams 1959).   ' •

             a.  Prepared plates of Trypticase soy agar (TSA)
                 (3SL) were used as the base medium.

             b.  0.5 mL of sample (or dilution), 0.5 mL of a
                 12 hr. culture of S. coli C (ATCC 13706, grown
                 at 37 C on a shake?) and 1 mL of soft agar
                 (Trypticase soy broth with 1% agar added) were
                 mixed and poured over base medium.  Duplicate
                 plates were prepared of each dilution.

             c.  Plates were incubated 18-24 hrs. at 37 C.

             d.  Plaque forming units (PFU) were counted
                 and calculated as PFU/g dry weight.
Tests for Pathogenic Bacteria

     A.  Enterotoxigenic E. coli


                              31

-------
    1.  Positive EC tubes from the 10 mL inocula of the
        fecal coliform test were streaked onto M-Endo
        LES agar plates and incubated at 35 C for
        18-24 hours.

    2.  Representative sheen producing colonies were
        picked and inoculated onto TSA slants (B3L).
        Cultures were incuba-ad 18-24 hrs. at 35 C.
        Cultures were saved £or entsrotcxin assay.

    3.  One day before enterotoxin assay, cultures were
        transferred to trypticase soy broth and incu,bated
        18-24 hrs. at 35 C.

    4.  Presumptive E. eoli cultures were tested for
        enterotoxin production with the Y-l adrenal cell
        culture test described by Sack (1975).

    5.  With each enterotoxin assay, "blind" positive and
        negative controls were included.

        a*  Positive controls (provided by R. Sack)
            EC eoli 408.3
            E* eoU TC268C2
            E. eoTI K108C3

        b.  Negative centrols
            E« eoli K{12)HFR
            E. COll' B
            E. COll C

    6.  Sensitivity of Y-l cells was tested by deter-
        mining the greatest dilution of cholera toxin
       • necessary to produce morphological changes in
      ..the Y-l cell line*

B.  Total Enteric Bacteria

    1.  0.1 mL of sample dilutions from the sample
        suspension were inoculated onto duplicate
        plates of peedried MacConkey CS agar  (Difco)f
        with agar increased to 1.5%.  Quebec grid
        dish@s (Lab~Tek 4018) were used.

    2.  Spread plate method tests were performed as
        described in Standard Methods (APEA, 1985).

    3.  Plates were incubated at 35 C for 48 hrs.

    4.  Colonies were counted per Standard Methods  (APH&,
        1985) guidelines and calculated and expressed as
        CFO/g dry weight.
                         32

-------
    5.  Colonies were picked from the grid pattern on
        the dish for identification, according to the
        following guidelines:

        a.  total count <50	pick all grids
        b.  total count >50, <100 .  .   .pick 1/2 grids
        c.  total count >100, <300.  .   -pick 1/4 grids

    6.  Picks were straakad  to TSA containing 1.1
        dextrose and 0.03 g/'L brom thymol blue/ co
        assure pure cultures.

    7.  Cultures were identified, using Minitek
        Snterobacteriaceae or Nonfermenter test kits,
        according to manufacturer's instructions.

    3.  Which kit to inoculate was based on oxidase
        reaction and acid formation from dextrose on
        the modified TSA plate.

        a.  oxidase    acid  from    test kit
            	    dextrose     	

              +            +•        Ncnfennenter
              +            -        Nonfermenter
                           *        Enterobacteriaceae
                                    Nonfermenter

        b.  One of the recommended controls was run with
            each set of samples.  Control cultures were
            varied.

        c.  Isolates were reported as percent of the
            total picks.

C.  Salmonella

    1.  Tubes of SBG sulfa enrichment (Oifco) were
        inoculated for M?N tests from the sample
        suspension (Walker and Yanko 1987).  SBG
        sulfa enrichment broth was prepared fresh by
        heating in a water bath te SO-»7Q C for 30 tain.
        instead of boiling for 10 min. as indicated in
        manufacturer's instructions.

    2.  SBG inoculated tubes were incubated 20-24 hrs.
        at 37 C.

    3.  Growth in SBG was streaked to plates of xylose-
        lysine desoxycholate (XLO) agar  (Oifco) and
        modified lysine iron agar (MLIA) (Difco base,
        modified as described by Rappold et al, 1979).
                         33

-------
4.  S3G enrichment broths were reincubateci an
    additional 24 hrs.

5.  Isolated colonies exhibiting typical salmonellae
    morphology were picked to slants of triple sugar
    iron agar (TSI) and lysine iron agar (LIA) (both
    Difco) and urease test broth (3BL).

6.  Cultures showing the correct biochemical reactions
    wara confirased by agglutination with Salmonella
    polyvalent 0 antiserum (Difco).

7.  When examining the primary isolation plates (XLR,
    MLIA) it was determined whether the pattern of
    presumptive salmonellae isolations followed a
    logical dilution distribution.

    a.  If misses occurred, 1 mL from the
        corresponding 48 hr.  SBG enrichment tube was
        inoculated into a fresh tube of SBG enrichment
        and incubated 24 hrs. at 37 Cc

    b.  These secondary enrichment tubes ware
        streaked to isolate salmonellae as described
        shove.

    c.  MPNs were computed from enrichment tubes
        confirmed to contain salmonellae and reported
        as MPN/g dry weight.

Campylobacter

1. -The sample suspension was swabbed and streaked
    on plates of Campy Agar (Gibeo) (Blaser et al
    1979, Ottolenghi at al 1987).

2.  Plates were incubated at 42 C for 48 hrs* in
    a jar ported for gas flow.  A mieroaerophilic
    atmosphere was maintained with a flow of gas (5%
    oxygen, 10% carbon dioxide 83% nitrogen).
    (Sergey's 1984). Gas was warmed and humidified
    by bubbling through a flask of DI water in the
    incubator.

3.  Campy plates were examined for the presenes of.
    possible Campylobacter colonies.

4.  Presumptive colonies were examined or tested
    for the following!

    a»  microseooic morphology (Standard Methods,
        APHA 1985)
    b.  eytoehrome osidase (Sergey's, 1984)
    c.  satalase  (Sergey's, 1985)

-------
    5.  Isolates exhibiting correct reactions were
        confirmed by the following tests (Bergev's
        1984):

        a.  growth at 25 C (Brucella agar slant, Gibco)
        b.  hippurate hydrolysis (Gibco)
        c.  nalidixic acid sensitivity (30 micrograra
            disk, Difco)
        d.  indole production (SIM, Difco)
        e.  hydrogen sulfide reaction (TSI)

S.  Yarsinia                  ,.

    1.  Tubes of Peptone sorbitol bile salts (PS3) broth
        (Weagant et al 1983a) inoculated for MPN tests
        from the sample suspension

        a.  PSB Enrichment Broth
            Na2HP04 anhydrous       3.23 g
            NaH2PO4 H20             1.2  g
            NaCl                     5.0  g
            D-Sorbitol              10.0  g
            Bile Salts No. 3         1.5  g
            Peptone                  5.0  g
            Deionized Water          1000 mL

        Mix ingredients and dissolve.  Dispense 10 mL
        portions into tubes and autoclave.

        b.  Prepare double strength media for 10 mL
            inocula.

    2.  Enrichment tubes were incubated in a refrigerator
        for four weeks at 4-5 C.

    3.  After three weeks, turbid tubes were streaked to
        Yersinia selective agar (Difco) and incubated
        for 48 hr. at 24 C.

        a.  Potential Yersinia colonies were picked to TSI
            Dife©}, LAIA (Difco Lysine Iron agar plus 10%
            L- Argiaine) (Weagant 1936) and TSA (BBL)

            TSI-   acid/acid no gas, no hydrogen sulfide
                   (some alkaline/acid)
            LAIA - alkaline/acid, no gas/ no hydrogen
                   sulfide
            TSA -  colonies <1.0 mm (24 hr.  37 C).

        b.  Colonies exhibiting correct presumptive
            reactions were confirmed by the following
            tests (Sergey's 1984):
                         35

-------
 r
f,  "•
                      Motility 25 C*                        +•
                      Motility 37 C*
                      Ornithine decarboxylase (Difco)        +
                      Urease (Urea agar base, Difco)         •*•
                      Simmons citrate (Difco)

                  *  (Edwards & Swing motility test medium)

              4.   Enrichment tubes ware rastreakad to  Yersinia
                  selective agar again aftar four weeks.

              5,   Enrichment tubes confirmed to contain Yersinia
                  during earlier weeks were not retested.

              6.   MPNs were computed from confirmed Yersinia positive
                  enrichment tubes and reported as MPN/g dry weight.
Tests For Parasites

    A.  Protozoan Cysts (adapted from Reimers et_ a^ 1981,
        see Appendix A)

        1.  A second blender jar of sample suspension was
?.,-';    _.   prepared as described in Sample Preparation.
  J-
   >,   2«  100 mL of homogenized sample was measured into  a
            100 mL centrifuge tube and centrifuged at 1250
            RPM (400 x g) for 3 min.

        3.  Supernatant was poured off and the pellet
            resuspended in sine sulfate (Sp. Gr. 1.20).

        4c  Tubes were centrifuged at 1250 (400 x g) HPM
            for 3 min.

        5*  Osing a pasteur pipet, the surface of the sine
            sulfate was carefully aspirated and transferred
            to a 15 isL conical centrifuge tube.
                      tube was filled with deionized water and
                  eantrifuged at 1400 RPM (480 x g) for 3 min.
                              was poured off and pellet re-
                  suspended in tube 1/2 full of acid-alcohol
                  solution (0.1 N sulfric acid in 35% ethanol
                  solution).

              8*   Approximately 3 mL of ether was added.

              9.   The tube was capped with a rubber stopper and
                  inverted several times, venting each time.

             .0.   The tube was centrifuged at 1800 RPM (660 x g)
                  for 3 min.

                                   36

-------
   11.  The acid-alcohol, ether and plug was poured off
        and the tube inverted over a paper towel to
        prevent reagent from running back into tube.

   12.  After well drained, two drops of DI water were
        added to the pellet and mixed.  Two smears were
        made on microscope slides,

        a.  Slides wera allowed to dry.
        b.  They were fixed in Schaudinn's solution
            for a minimum of 1 hr.
        c.  Slides were stained with trichrome stain.

   13.  Slides were examined microscopically with a
        high dry objective (45X) and 15X eyepieca.

B.  Helminth Ova (adapted from Reimers et_ al 1981)

    1.  The remaining volume of homogenized sample
        (after removing lOOmL for protozon test) was
      .  measured and poured through a 43 mesh seive
        placed in a large funnel over a two liter
        beaker.

    2.  Sample was washed through the sieve with several
        rinses of warm tap water catching the washings
        in the beaker.

    3.  The washed sample in the beaker was allowed
        to settle overnight.

    4.  The supernatant was siphoned off to just above
        the settled layer of solids in beaker.

    5.  The settled material in the beaker was mixed by
        swirling and poured into two 100 mL centrifuge
        tubes.

    6.  The beaker was rinsed two or three times and
        rinsings poured into two 100 mL centrifuge tubes.

    7.  The tubes were balanced and centrifuged at 12SQ
        RSM (400 x g) for 3 min.

    3.  The supernatant was poured off and pellet
        resuspended thoroughly in zinc sulfate solution
        (Sp. Gr. 1.20).

    9.  Zinc sulfate was centrifuged at 1250 RPM for
        for 3 min.

   10.  The zinc sulfate supernatant was poured into a
        500 mL Erlenmeyer flask, diluted with deionized
        water, covered and allowed to settle 3 hrs. or
        overnight.
                         37

-------
        11.  The supernatant was aspirated off to just
             above the settled material.

        12.  The sediment was resuspended by swirling and
             pipetted into two to four 15 mL conical centrifuge
             tubes.

        13.  The flask was rinsed with deionizsd water  two  to
             three times and rinse water pipetted into  tubes.

        14.  Tubes were centrifuged at 1400  (480 x g) RPM
             for 3 rain.
', t

        15.  Pallets were combined into one  tube and centrifuged
             at 1400 RPM (480 x g) for 3 min.

        15.  Pellets were resuspended in acid alcohol
             solution and processed as described in
             Protozoan Cysts.

        17.  After completion of step A.11,  the pellet
             was resuspended in 0.1% sulfuric acid and
             poured into Nalgene tubes with  loose caps.

        18.  Tubes were incubated in a slant rack at 26  C
             far three to four weeks.

             a.  Control ova dissected from  an adult Ascaris
                 lusabrieoides var. sums were also incubated.

             b.  When the majority of control ova had
                 effibryemated, samples were examined.

        19.  Concentrates were examined microscopically using
             a Sedgewiek Rafter cell to enumerate detected  ova.

             a.  Viability was noted based on presence  of
                 essbryonated ova and whether or not larval
                 forms could be induced to move.

             b0  Ova were identified and reported as ova/g
                 dry weight.

Enteric Viruses

     A.  Processing Samples  (Glass et al_ 1978)

         1,  40 g of sample  was weighed into 400 mL of  sterile
             3% beef extract, neutral pH, containing 0.4 mL/L
         2.  The mixture was  blended  in  a  sterile  stainless
             steel  Waring  blender  jar  for  3  min. at  high speed.
                               38

-------
    3.  The suspension was then disrupted by sonic  treat-
        ment  (Lab-Line modal 9100 ultrasonic generator  with
        9106  horn at 170 W for 2 min.)

    4.  The suspension was centrifuged  in 250 mL
        centrifuge bottles at 10,000 RPM  (7,000 X g)
        for IS min. in a refrigerated centrifuge.

    5.  The supernatant was recovered and pH adjusted
        to 3.5 with 6N HC1 while mixing.

    6.  The pH 3.5 supernatant was mixed for 30 min.

    7.  Th'e supernatant was centrifuged again as in
        step  4.

    8.  The supernatant was then discarded.

    9.  Pellets were dissolved and combined in 0.15
        M N32HPO4 (6 to lOmL) producing a
        concentrated eluate.

B.  Detoxification of Sample (Glass et, al 1973)

    1.  Oithizone in chloroform was prepared as described
        in Standard Methods (APHA, 1985).

    2.  An equal volume of dithizone solution was added
        to the concentrated eluant above.

    3.  The dithizone-eluant mixture was mixed for  2 min.
        on a  vortex mixer.

    4.  The mixture was centrifuged 15 min. at 15,000 RPM
        (40,000 .X g).

    5.  The upper aqueous phase was recovered and two
        drops of 1% calcium chloride was added.

    S.  Filtered air was gently bubbled through the
        sample for approximately 3 to 4 hrs. to remove
        residual chloroform.

C.  Virus Assay

    1.  The final concentrate was subsampled for
        distribution to contract laboratories in addition
        to testing in the Districts' laboratory.

        a.  1.5 mL was sent to Pierre Payment, University
            of Quebec, for immunoperoxidase assay.

        b.  1.0 mL was saved for selective pooling  for
            Charles Gerba, University of Arizona,
                         39

-------
        Rotavirus assay.

    c.   The remainder of  sample (4,5 to 7.5 mL)  was
        used in the Districts'  assay procedures.

2.   1.0 mL of sample was  initially assayed for
    plaqueabla virus en Buffalo graen monkey
    kidney (BGM)  mcnolayers.

    a.   0.2 mL of sanpla  was  added to each of
        five drained 2 02.  bottles containing
        BGM monolayers.

    b.   A 2-3 hr. adsorption  period was allowed at
        37 C with frequent  rocking.

    c.   Sample was poured off and cells overlayed with
        Eagle Mminimura Essential Medium containing 1.2%
        purified agar (Difco) 5% fetal bovine serum,
        25 mM magnesuim chloride, 100 units/mL
        pencillin, 0.7S ug/mL streptomycin, 0.017%
        neutral red, 1% milk  and 0.2% sodium
        bicarbonate.

    d.   Bottles were observed for up to 7 days for
        plaque formation.

3.   Iff  the plaque assay was negative, the remainder
    of  sample was assayed by  liquid overlay .tech-
    nique on BGM cells and rhabdomyosarcoma
    (RD) cells.

    a.   Remaining sample  was  divided between two
        drained 16 oz. bottles containing BGM
    b,   A 2=3 hr. adsorption period was allowed at
        37 C with firequent rocking.

    Ce   The sample was aspirated with a pipet and to
        ona bottle, 40 mL Hank's Maintenance Medium
        with 5% lactalbumin hydrolysate, 0.5%
        tryptose, 0.455% HE?£S buffer, 0.0525% sodium
        bicarbonate 100 ug/mL. streptomycin, 100 units
        peneillin and 0.75 ug/mL Fungizone was added.

    d.   To the second bottle, 10 ug/mL trypsin was
        added to the se-rum free medium to enhance
        reovirus and rotavirus (M.D. Sobsey and
        P. Payment personal communication).

    e.   The aspirated sample was reinoculated into two
        16 02. bottles of drained RD cells.
                     40

-------
        f.  RD calls were treated as described in steps
            b. through d.

        g.  Bottles were observed at 24 hr. and every
            48 hrs. for up to 14 days.

    4.  For blind passaga, negative bottles from cho
        preceding step ware fraeae/thaw iysad and the
        lysace retested as previously described.

    5.  Any bottles showing cytopathic effect (CPE)
        in step 3 or 4 were freeze/thaw lysed and passed
        to confirm virus CPE.

    6.  If the plaque assay in step 2 showed "polio-like"
        plaques within 43 hrs., the sample was reacted
        with polyvalent polio antisera before proceeding
        with steps 3 through 5.

0.  Propagation of Cell Cultures

    1.  3GM and RD calls were grown in Eagle MEM (Alpha
        Modified) with 10% fatal bovine serum, 0.2%
        sodium bicardonate, 100 ug/mL streptomycin, 100
        units/mL penicillin and 60 mcg/raL Tylosin.

    2.  3GM and RD cells were maintained in Hank's
        Maintenance Medium with 5% lactalbumin
        hydolysate 0.5% Tryptose, 0.455% HEPES buffer,
        0.0525% sodium bicarbonate 10Q ug/mL Streptomycin,
        100 units/mL penicillin and 0.75 ug/mL Fungizone.
    3.  Cell cultures were trypsinized and subcultured
        weekly.

    4.  All cell cultures were grown and maintained at
        37 C.

E.  Contract Testing

    Standard entetie virus analyses were performed in the
    project laboratory.  In addition to the conventional
    tissue culture assays, two specialized virus assays
    were conducted by other research laboratories.

    Composited sample concentrates were sent to Dr.
    Charles Gerba at the University of Arizona for
    rotavirus testing and aliquots of individual virus
    concentrates were sent to Dr. Pierrre Payment at the
    University of Quebec for virus testing by the
    imnmnoperoxidase-HISG method.  Procedures for the
    rotavirus assay (Smith and Gerba 1982) and the
    immunoperoxidase assay (Payment and Trudel 1985) have
    been described elsewhere.

                         41

-------
          A number of Yersinia cultures derived from project
          samples were tasted for pathogenicity by the New York
          State Department of Health.   Yersinia characterization
          procedures have also bean described elsewhere
          (Shayegani 1986).
Data Analyses
      Statistical analyses were perfomed wich 5MDP Statistical
      Software on an IBM mainframe computer system.   All
      statistical computations were performed with log (base 10}
      transformed data co more closely approximate a normal
      distribution.  Data files were maintained in an in-house
      laboratory data management system that permitted direct
      transfer of files to the BMDP Program.  Test results for
      indicator organisms were reported and tabulated in the log
      format to facilitate data handling.  Log mantissas were
      carried to three places in order to maintain the correct
      two significant figures for the cardinal values of the
      microbiological data.  T-distributions were analyzed using
      Bonferroni t statistics which apply to both balanced and
      unbalanced cases (Miller 1981).

      Data for the fungal populations and total enteric bacterial
      populations were summarized in tables and bar  graphs in
      Section S showing the relative diversity of populations.
      Fungal populations are often difficult to quantify and
      correspondingly troublesome to analyse.  The fungal
      populations consisted of a few fungi that were detected
      regularly and numerous other fungi that occurred randomly.
      Two assumptions were-made when analysing these data?  (1)
      If a given genus-species was detected once in  a sample, it
      was considered part of a population.  Those times it was
      not detected, it was assumed present, but below the
      detection limit.  (2) If a given genus-species detected
      from any sample was not detected in samples from a
      particular sampling site, it was assumed that  fungus was
      not pare of the population at that site.  The  average
      concentration for each genus-species present was
      calculated*  "Less than18 values were assigned  a value of
      one half the detection limit (Gleit 1985).  Populations  •
      were then normalised to the least common fungus, ie each
      individual concentration was divided by .the lowest value.
      In this way, the least common fungus is assigned a value of
      1 and all other fungi obtain a positive ratio  value greater
      than 1.  This provides a relatively simple, albeit rough
      approximation, of the distribution of the populations.

      In the case of the total enteric bacteria, the data were
      distributed based on the number of times a bacterial colony
      was identified as a particular genus-species,  i.e., the
      results are presented as a frequency distribution.
                               42

-------
The values plotted in the bar graphs represent the ratio of
the number of isolations of any given bacteria to the least
commonly isolated bacteria in the sample.  Again, the
intent was to provide a picture of how the populations
distributed within a sample and varied between samples.
                         43

-------
                              SECTION 6

                        RESULTS  AND  DISCUSSION

  MICROBIOLOGICAL  RESULTS  -  WEEKLY SITES

  Indicator  organisms

      The  data  collected during  this portion of .the  study  indicated
 that  microbial concentrations in compost were highly  variable,
 sometimes tanging as  much as  ten orders of magnitude.   Despite
 the variability and  range of  the data, normal probability plots
 showed  that the log  (base 10) transformed data followed a normal
 distribution.   Basic  descriptive statics for the  indicator
 organisms are  summarized  in Tables  6 through 13 and the geometric
 means are grouped by  faciltiy in Tables 14 and 15.

      Comparative  analysis of  the two static pile  facility sampling
 points, III-B-1 (giveaway bin)  and  III-B-2 (screened  compost),
 indicated that all means  were significantly different  (significant
^at 95%  CL)  except the thermophilic  fungi.  The giveaway bin
'contained lower bacterial and coliphage concentrations  and higher
 fungal  levels  than the screened compost.  The lower bacterial
 levels  could have been a  result of  the greater amount of  wood in
 the giveaway bin  compost.   Since the test results were  expressed on
 a per gram  basis,  data for  the  giveaway bin actually  represented a
 smaller amount of sludge  which  may  account for the  difference.   To
 test  this theory,  the amount  of wood chips in giveaway  bin compost
 samples vs  screened compost samples was estimated.  Representative
 samples from each site were thoroughly washed through a #14 USA
 standard  sieve with openings  of 1.4mm.  After washing,  the retained
 wood  chips  were collected from  the  screen and the dry weight
 determined.  In this  manner it  was  estimated that the screened
 compost contained 28% dry weight wood chips (>1.4mm)  and  the
 giveaway  bin contained 78%  dry  weight wood chips  (>1.4mm).  Using
 these results  the III-B-1 and III-B-2 data sets were  recalculated
 to the  adjusted bases and ANOVA and pairwise "t"  tests  were rerun.
 Results of  this comparison  are  summarized in Table  16.

      Normalizing  the  data for the difference in wood  chips resulted
 in bringing the means of  the  bacterial groups closer  together and
 increased the  difference  between the fungal populations.   Fecal
 coliform, fecal streptococci  and coliphage concentrations were
 still significantly lower and fungal populations were significantly


                                 44

-------
                o <
                u r
                  0.
 z

 a
 >u


 •j
 VI

 O
IU
J
               Si
               i- ik
 < z
 >» IU

 25
               at O
               O u
               c
               tu vu

               z <
Z



S
a
t-
          z
          a
               -J O

               & U
               4 IU

               J tt

               IAS *•

               Ik VI
               U -I

               IU O
IU
a

z
                            n
                            o
                            a
                       <-•   a
                    a
                    31
                    •o
                            a   o<   a>

                            a   —   —

                            in   —   o
             «   in   o
             n   m   t»
             e   —   —
                            in    -
                            '•a    3
                    a
                    o
                    a
N   in   -   o    -
T   *   m   —    a>
a   a   —   «    in

r-   —   a   «    in
             _    ie    <-    «    —
             —    p*    ^    p»    a
             «    CB    ~    e    n

             p-    o    a    a        m
             <>>    r>    N
               —    in
               m    in
               0    04
                            IS
                            a
                         u

                         c
                         3  Z

                         O  •<
                                               a

                                               a
                                                    0
                                     w

                                     V)
                                i   i












S
IU
IU
z
VI
1

pe
Q
0.
i
u
IU
sJ
Z
U
5
M
0
ft
1
t
»

It!
tn
j
IA
to
P-?
2
S
£
O
S
{=>
^
IJ

2



pa

M
ffi
K









a

D
a.
a.
'J
















a
3
£
u
IS
0






















Mg


i
§















]
1— t u
-• O
0 <
U£
S -

IU Z
r p

J «•
« J
i- Z
1- Ik


u

< z
H> IU
O ^
H= Z




uz
il
S
IU IU
z •*
a.



IU f
1- Z
5§
«MJ U
& U



-id.
< IU
U Z
Ik M
X
z
o
-i Ik

iu a
Ik U

3
&
Jifc

>• a)
OS
h-U

K
IU
h"
IU
X

z



T r* f^ f^
ia in eg —

n - a in
a ts 3 is

*O O •" (8

•T — o a
* at <3 f*
in n in in
o « M a
•r - o in



at a a aa
10 a - a
n r» — n
a a a a





f> n ^ a
in ia o a

e e — «
 »
at « pt n
10 - e »


u

z iu S
H» Q • 3
vu as
S Z • • -
o < a iu x
IU IU 1- <
OS vi wt S


S)
-------
















K
I/I
3
a
2


*
^
'.*>
o
a
a
 U


Z
IU

u
X

a


en a a a
a o - a
-

CM - a a



a a •» «
— — - n
. — a 9






n •- CM —
C^ CM 9 f*
— 9 n i»
9 - a a

9 cn * at
~ ft f* r*»
a i- - a
9 a a a»




9 ^ tn ^
a 9 a »
<» CM n u 2 a

O 4 Q LU X
IU IU h- -4
u a 1/1 i/i a


3)

9
a

a

•a
a
at
at
-------

























t-
u
3
Q
Q
oe
0,

a
IU
(3
<^
ffl
*7
i
X

JA5
~
^
s

M
Z


Of
e
s
0

u

_
-•"

Q


«
ffl
•4








a
3
u.
a

^
O
-J













9
3
tic.

O





























3
V,
Z.
i

o















. UJ
~ tj
-J <
o r
u a



S 1J
^f =
f™ u.

J <-

a a
H- U.




u
< c
1- IU
a ^
^ z




^B
U <£
B* 3
a a
3 U
05
U4 U|
g ,3
4 «J
&>

IU f»
1- ^
< 3
a, w




j a.
4 IU
u nc
LU P»
!L (A


%
A

« »
UJ Q
u, t»

I
Q
•a! %

C O
»= w

3£
I4S
Ui
S

K
<*
a,


M r^ N to in
to « •* r* r- (M
a o — — (H in

- - a >r a

a u» e i M
01 31 O3 — C fN
o ^ •- — a \f>
r-* *- a -o a

c« P» n a -
n a P* a v P<
m IB M o to in
X
N - a « o -
z

_ j
j
3
0) P* <£ U» 01
N n —  S
a
e
O
in & f* 'n 31 4
c! 31 is m gt ft (j
N * *^ ^ ffl "* S
n - a a a z
1 0.

00
1C C Q P9 58 "*

i». O !•« « « Wi IU
(ej PI e r*. Q ia
1 *
a!
u • =t
B»3 ^i t/J
5 0 • 1 1 a,
tu S 3 a Q

o < a iu x z

S s ^ wi a s z
n
3
u.
o.

J
o












gi
"*»
J
u
(J
o
J





























a
S
Q.
s




















! UJ
- O
«* <
o r
u a

Ct ' J
•ju Z
•w* -^
* — '
^- u.

< t3
Si
>— (4.

U
M
-J flt
^ tu
5£






fo
z
U 3

ffl W
a
X IU
z 5
 Z
< 2
0, U




j a,
.^ w
u e
IU 1=
UB trt


g
- 01
T 10 O OT Cn —
m « - a a in

a u> n  co n
3t «  a a is



(*8 — ^ (0 P«4

— « - *• (O -
at a a Q 
-------




















X

a

H1
V)
i
3
<
VJ
^
u
2
§
z
0>

o

u
o

a
in
'
*
^
—
IU
VI
VI
2
Vf
.*
2
•s
z
o
£
0

4
U'
M}
i
0")




«

IU
a
*•










3

3
Ik
a
3
J















3
•s.
3
U.
u
8































OS
Xt
z
i

i
-s





















-IS
J 4
3 r
u a



13
uj 2
Z 3
H> Ik

«j **
•». i3
t~ Z
a 3
^" Ik


u
t^
j z
4 IU
^v ^a
O Z
(- IU





z
U 3
-O
ou
0
Z IU
IU 1—
Z 4
4 -J
&


WH>
K 2
« 3
j ^
& *j




j a
•t IU
-J Z
Ik VI


s

o
-i Ik
4 "•»
U -1
IU Q
It W


y

Q
^ Ik
< J
00



z
IU
1-
IU
a

z
^
a




in P* — f* 31 r-4
a in f* •• to in
« — O «S Q



t-i » aj a 01 —

t . . .
c* — a in a

a to o a a
31 ^ a to a
" 7 ~ * * S
o ^ a >n a
X
H«
a
« to m - -
ut a a ^ a iu
^ to a n a « «i
	 m a
« a s a « ~
3
a
».
u
3
a
o
3,
te a t o« in
a M « r» a a
r* a — m » M u
	 m 13
p> - a a M o

a
a
• i
n en a a a i
O * « 10 f- X
a * a f» N n. -
a o 3 a r> iu
M
tfl
VI
a
VI
a 9 V fNl Ul a*
>1 » - . < .-1 (M Z
a m P* ci t~ m 4
us — a « - z
3
z
o
H
to  uv
z u a a
K Q • 3 3 Ik
iu a a a o
3 Z • •• ->
a 4 a iu x z
IU IU H- 4 « O
o a vi vi a a z

>^,
3
j.
a

Z 4
4 U
a.


IU H-
1- Z
4 3
j O
a u




— a
4 IU
U Z
Ik VI


a
z
0
-1 Ik

U -i
IU O-
Ik U


a
z
3
.J ik
< •"
O 0
t- u


z
IU
IU
a

z
^
&

a a n T a

— in r< a to in
to — a 10 a


_'5 C*J > d CJ5
M ^ 35 33 01 —
:n ^ — m u in
t*s ™ a 'ji a



f. - - - a
a a a a a r»
a n <•« to  a o a a>




ji T us m us
to « — a* m —
 V)
• z iu a a
K Q • 3 3 Ik
iu a a a o
a z • . -. -
o 4 a 
-------
                   _! U
                   o <
                   u r
                      a
 i/i
 z
 s

 u

. a
  S
  a
 Hi
 J


 &


 U


 H>
 1/1

 3

 Z

 5

 a •


 §
                   I- Z
                   O 3
                   I- Ik
                   -I X
                   < IU
                   c z
u ^
— o
at u
o
z at
IU K>
Z <
« u
   a,
                   IU K


                   « 3
                  ,JO
                   O.U
_1 0,
< us
u e
                   IU O
                   Ik U
                   Jtfc
                   <•=
                   C3S
                   (" U
                                  a    a



                                  in    »
                                       in
                                       a
                     01
                     •a
                                                 vt
                                                 Z
                                                 IU
                                                 u

                                                 tr
                                   u
                                   4
                                                 K
                                                 a
              (=5
              a
us


us
      in
      a>
                                                 tf
                                                 O

                                                 i
                                                                   o <
                                                                   u r
                                                                   .j  —
                                                                   ^  13
                                                                   I-  2
                                                                   O  3
                                                    j a
                                                    < IU
                                                    t- t-
                                                    o z
                                                    )- IU
   z
U 3



O
QC I&3
U *»
z <
4 J
   a.
                                                    IU (-
                                                    *• Z
                                                    « 3
U Z
IU »•=
t V)
                                                                                     U =!
                                                                                     Q Q
                                                                                       aj
                                                                                       31
                                                                                       ^r    a
                                                                                       —    o
                                                                              n
                                                                              in
                                                                                                                     31
                                                                                                                     31
                                                                                          
-------
Table 16   Significance of Differences Between Means of Original
           and Adjusted Basis Data frcn Static Pile Sites III-3-:
           and III-3-2.
TEST ORIGINAL 3ASIS
MEAN DIFF SIG
Total Coliform Log MPN/g
Fecal Colifora Log MPN/g
Fecal Strep Log MPN/g
Plate Count Log CFU/g
An. Plate Count Log CFU/g '
Total Enteric Log CFU/g
Total Fungi Log CFU/g
Therm. Fungi Log CFU/g
Coliphage Log PFU/g
-0.91 ***
-1.37 ***
-1.64 ***
-0.52 ***
-0.76 ***
-0.43 **
0.98 ***
0.31
-1.22 ***
ADJUSTED
BASIS
MEAN DIFF SIG
-0.40
-0.85
-1.12
OoOO
-0.24
0.09
1.49
Q.83
-0.71

**
***



***
***
**
Nomenclature
  1% Significance  ***

  5% Significance  **

 10% Significance  *

  No Significance
                                 50
-------
higher in the giveaway bin samples.  The more generalized measures,
total coliform and plate count populations, were no longer
significantly different.  It does appear that there is a difference
in the microbial populations at these two sampling points, although
in the case of the bacterial groups and coliphage, the differences
are not as great as the original data indicates.  The difference
may reflect the presence of older material in the giveaway bin.
The bin was filled with compost on a demand basis.  During winter
months, samples from the bin may have reflected older and dryer
compost than the screened compost samples.  The higher fungal
populations in the giveaway bin material would also be consistent
with the presence of older material.  The data distribution can be
visualized in the confidence intervals shown in Figure 4 where M
is the mean and U and L are the upper and lower limits of the 95%
confidence interval.

     The data from the windrow facility sites IX-A-1 through
IX-A-6 were analyzed by ANOVA; a significant difference was
indicated (P<.99).  Pairwise t tests were run (Bonferroni) and
it was observed that the dafca had a tendency to group ito two
strata.  The first strtum included the completed compost, with
either sawdust (IX-A-1) or recycled compost (IX-A-2) used as the
bulking agent.  The mean densities for all microbial groups were
higher in sawdust compost than in recycle compost except the fecal
streptococci which were higher in the recycle.  None of these
differences were statistically significant.  The average
concentration of organisms in the unamended bagged product, (IX-A-3;
was higher than in the final compost material but lower than the
amended bagged products.  The unamended bagged product contained
significantly higher levels of fecal eoliform, fecal streptococci
and plate count bacteria than in the recycle compost (IX-A-2) but
only the fecal streptococci were significantly higher in the
bagged product compared to the sawdust compost (IX-A-1).  When
compared to the bagged amended products (IX-A-4, IX-A-5 and
IX-6), the unamended bagged material was significantly, lower
in 21 out of 27 comparisons*  Although the -concentrations of
microorganisms in IX-A-3 generally tended to group more closely
with the final field compost than with the other bagged products,
the unamended bagged material appeared to represent an intermediate
level between the two basic density groupings.  The mean
differences and significance levels are summarised in Table 17.

     The second general grouping of data consisted ©f these bagged
products in which the compost was blended with other materials,
IX-A-4 through IX-A-6.  The average concentration of organisms in
the second stratum were significantly higher than those in the
first.  Within the second stratum, the mean concentrations of the
individual bacterial groups increased in the following orders •
IX-A-4 < IX-A-5 < IX-A-6.  Some of the individual differences were
significant while others were note  Mean differences and
significance levels for these sites are also shown in Table 17.
The confidence intervals of the data are shown in Figure S where


                                 51
-------
FIGGBS 4
          7 DISTRIBUTION 0? SITE III-3-1 AND  III-3-2  INDICATOR  ORGANISMS
          3STORE AND AFTER ADJUSTING DATA FOR WOOD CHIP  CONTENT

                           95% CONFIDENCE INTERVALS
                  SAMPLE
                                -TOTAL COLIFORM-
                                3S?CRS ADJUSTMENT

1
2





i_
2



3172
III-3-1
rii-s-2




SITS
III-B-1
ni-a-2



MEAN 3122
5.02 43 £,
6.93 45
r fv* uw f — *
UJG Mrs)/ 3— *•————
5.44
SAMPLE
MEAN SIZE
6.53 43 L
7.07 45
r /^* unit? /I-TM. A^««^Mu
UGG rtirri/  «!>•• <^^<>iS»33 <^ ^ ^ ^ ^
5.39 5.69 5.99 7.29 7.59
                  SAMPLE
                                -FECAL COLIFORM-
                                3EFORE ADJUSTMENT
1
2
1
2
SITE
III-3-1
III-3-2
SITE
III-3-1
III-3-2
MEAN
5.03
6.45
MEAN
5.74
6.59
r.rvi a
SIZE
48 L
45
4.45
SAMPLE
SIZE
48 L
45
row /»_...*.____..
M a
L M 1

4. 95 5.45 5.95 6.45
AFTER ADJUSTMENT
M 0
L M a

                       5.12
                                 5.32
5.92
5.32
          5.72
                                                                          a
                                                                          —+
                                                                          5.95
7.12
   SITE
1 III-3-1
2 III-3-2
   SITS
1 III-B-1
2 tII-8-2
                  SAMPLE
            MEAN   SIZE
            5.26    48    L_
            6.39    45

            LOG MPS/g—+•—-
                      4.70

                  SAMPLE
            MEAN   SX1S
            5.92    48    L,
            7.04    4S

            LOG MPN/g—+	
                      5.36
                                  -FECAL STSE?-
                                BEFOHS ADJUSTMENT
                                 S.20      5.70

                                       ADJUSTMENT
6.20
                    5.70
                    7.20
L M

5 5.76 6.15 5.56 6.96
(

r
                                      52
-------
FIGURE 4 CONT'D.
                              -AEROBIC PLATS COUNT-


1
2





1
2




SITE
III-3-1
III-3-2




SITS
III-3-1
in-a-2



SAMPLE
MEAN SIZE
9.03 43 L
9.55 45


3.30
SAMPLE
MEAN SIZE
9.59 43 L
9.S9 45
•

9.44
BEFORE ADJUSTMENT

M 0
L M U


3.33 9. la 9.34 9.52 9.
AFTER ADJUSTMENT

M 0
L M U


9.54 9.S4 9.74 9.34 9.
                  SAMPLE
  -ANAEROBIC PLATS COUNT-
     BEFORE ADJUSTMENT
1
2

1
2

SITS
iii-a-i
m-a-2

SZT2
m-a-i
m-a-2

MEAN SIZE
7.31 48 L
8.57 45

LOG CSTJ/ g— *~— - ••»•=•
7.50
SAMPLE
MEAN ' SIZE
80 47 48 L
8.71 45
LOG C?0/g~+ 	 • 	
8. IS
M 0


7.75 3.00
As JT&n ftOwUaXMESli
M
L
S.31 8.46

L M 0

8.2S 3.50 8.75
0
	 M " a
3. SI 8.76 3.91
                  SAMF&S
-TOTAL SNTESIC PLATS COONT-
     3SFORE ABJOSTMENT
1
2
1
2
SITS
IIX-8-1
III-B-2
SIT2
m-a-i
III-3-2
MEM SIZS
7.94 48 L
3.37 45
LOG CStl/g— *—-—-*—
7.S2
SAMPLE
MSAM SI2S
3. 50 48 L
8o51 45 L
M 0
L M 0
7.82 8.02 8o22 8=42 8«62
AFTER ADJUSTMENT
M D
M a
             LOG
                       3.20
      8.3S
3.30
8,-,6S
8.9S
                                     53
-------
FIGUHS 4 CQOT'D.
                                  -TOTAL FUNGI-


4t
2




1
2



SITS
III-3-1
III-3-2



SITE
in-a-i
iii-a-2


SAMPLE BEFORE ADJUSTMENT
MEAN SI 22
5.03 45 L M
4.05 43 w M ~


3.44 3.34 4.24 4.54 3.04
SAMPLE AFTEH ADJUSTMENT
MEAN SIZS
5.69 46 L M
4.20 43 L M a


3.60 4.10 4.60 5.10 5.60


a



5.44


	 a



• 6.10
                               -THEHMOPHILIC FUNGI-
                                BEFORE ADJUSTMENT
1
2
1
2
1
2
1
2
SITS
iii-a-i
m-a-2
SITE
rii-a-i
rii-a-2
SIT2
III-S-l
m-a-2
SITS
m-a-i
iii-a-2
MEAN
5.01
4.70
LOG C
MEAN
5.67
4.34
LOG C
MEAN
2.03
3.25
LOG i
MEAN
2.69
3.40
LOG 1
SIZS
45
43 L
4.36
SAMPLE
SIZS
45
43 L
MTTT /1-tMM^MM.,
.ru/g— -*---
4.53
SAMPL2
SIZE
48 L
45
1.60
SAMPLE
SIZS
48 L
45
»TO/g— 1~~ •
2.28


4.56
AJT2H
M

L M
M CJ

4.76 4.36 5.16
ADJUSTMENT
L M
a

4.33 5.13 5.43 5.73
-COLIPHAGB-
3E7CRS ADJUSTMENT
M a

2.00
AFTS8
M

2.58
L M
.
2.40 2.30 3.20
ADJUSTMENT
a
L M

2.38 3ol8 3.48
U

5.36
a
6.03
0

3.60
U
3.78
                                    54
-------
TAILS U PAIIM1SS SiCMIVICAHCC Or HKAM DlfTgBKMCgS Or IHOICATOK
OftOAMISMa IK HIKDBOtl SAWLCSi II- A- 1 TfULOUCB U-A-4
^
p
•"•«.
3
§
Z
5

t f KCAIU PECAL PLATS AMASKOB 1C TOTAL TOTAL TIIBftUOi 'U t L i C COL 1
POKM COLIFORM STRKF COUMT flATC COUNT ENTERiC FUNGI KUitCl PUAC
II
us
J en
en
a
5
0
a*
s
z
CJ
CM
a
CM
z
en
hi
CM
3
X
a
a
8
i
Q
C*
3
u
1
Q
1
tn
s
CM
a
3






,7777, 7?,, 7

, , i . . i , i i , , i i ,


I i I I t I I t I I I t t





M«n.«m«a» .««
1 t 1 1 1 1 9 1 t 1 t > 1 t

t . '< i i i 1 r r i , . i i
l i *f i i i "f i • i i i i i i



< *
* * <
« « « «
* e e e e
a « « * 
-------
FIGURE 5  T DISTRIBUTION OF  SITS  IX-A-1  THHOCGH IX-A-6 INDICATOR ORGANISMS

                           35* CONFIDENCE INTERVALS
   SITS
1 IX-A-L
2 IX-A-2
3 IX-A-3
4 IX-A-4
5 IX-A-5
5 IX-A-5
   SITS
1 IX-A-1
2 IX-A-2
3 IX-A-3
4 IX-A-4
3 IX-A-5
5 IX-A-5
   S2T2
1 IX-A-1
2 IX-A-2
3 IX-A-3
4 IX-A-4
5 IX-A-5
S IX-A-5
SAMPLE
MEAN SIZS
2.35 37
2.03 13 L
3.30 32
3.11 51
5.03 32
6.77 51
0.30
SAMPLE
MEAN SIZS
2.21 37
1.20 13 Z,
3.23 32
4.55 51
5.54 32
5.50 51
UX !lrrt/g— —+——."
0.0
SAMPLZ
MEAN SIZZ
2.30 37 L
3.41 18
4. 95 52
5,11 51
5.34 52
5.27 51
IM& iWM/g— —*———•
1.30
SAMPLE
MEAN SIZS
3.3S 37
3.02 13 L
3.73 52
9.11 51
9.00 52
9.33 51
f fV fffVl ^rfTr™H.±i.r.«tr..»««
7.52
-TOTAL COL I FORM-
L 	 M 	 a
u M a
L M a
' L~~~ M 0
~ "!T_M 	 a
1.30 3,30 4.30 S,30 7.30
-FECAL COLIFORM-
L M a
L M a
~ L M a
L M a
" 5"~~M 	 0
1.50 3.00 4.50 5.00 7.50
-FECAI, STSE?-
M a
E~ M a
L M a
L M a
L~ M a
L M 	 a
2.30 3.30 4.30 3.30 5.30
-AEHOBIC PLATS COONT-
L M a
M 0
L M a
L M a
L M a
L^^w^^a
7.92 8.32 3.72 9.12 9.52
-------
FIGURE 5 CONT'D.
   SITE
  IX-A-1
  •IX-A-2
  IX-A-3
  IX-A-4
  IX-A-5
  IX-A-S
   SITS
1 IX-A-1
2 IX-A-2
3 IX-A-3
4 IX-&-4
5 IX-A-S
6 IX-A-6
   SITS
  IX-A-1
  IX-A-2
  IX-A-3
  IX-A-4
  IX-A-S
   SITS
  IX-A-1
  IX-A-2
  IX-A-3
  IX-A-4
  IX-A-S
  IX-A-5
SAMPLE -ANAEHOBIC PLATS COCHT-
KEAN SIZE
5.59 37 L M 0
5.13 13 L M U
S.73 52 L M 0
7.79 51
7.71 52
7.35 51
5.2S S.38 6.4S 7.03
SAMPLE -TOTAL ENTERIC PLATE COpNT-
HEAN SIZE
7.07 37 L M 0
7.03 13 L M a
7.23 52 L M 0
8.23 51 L
8.46 52 ~
3.75 51


S.4S S.9S 7.43 7.95
SAMPL2 -TOTAL FUNGI -
MEAN SIZE
2.08 36 L M a
Io38 18 l> M a
2. S3 52 ' L M a
3.50 47 ~ T
3.36 SO L 1
4.01 47
LOG ci-a/g— f— -«• -- — »+— - — — *. — 	 	 «— —
0.5S 1.3S 2.15 2.96
SAMPLE -THSHMOPHILIC FUNGI-
MEAN SZZS
2.21 37 L M 0
1.1S 18 L MO " " " '
2.09 52 L M 3
3o63 48 "~~ '
2.93 51 L M
3 = 53 51 ~~~~ "
LOG CfC/g— +— — — -+— -.——*—-.— ~*-—
0.63 1.33 2.03 2.73
SAMPLS -COLI?HAG2=
MSAH SIZE
1.40 37 L M tJ
0.98 18 LM0 ~"
1.88 52 L M 0
2*S4 SI L M
2.96 52 rT~~~
3.18 51 L_
LOG PFO/g—* 	 -——+—. — — +. 	 -«-— *— ^
0.78 1.38 1.98 2.58





L M 'J
L 	 H 	 u
" ~ "* " """" •• >>~™^ f-t
^ 	 ^
7.58 8.28

', J



M a
L M a
L_M_0

—
8.45 3.95





M 0
1 0
!•__ 	 M 	 _tJ
«.•__=»+_„_— «,__«,*
3.76 4.56





L M a
o «_____=
"TTL^M a
.«_K,»»
-------
the mean (M) and upper (U) and lower  (L) limits of  the  95%  con-
fidence intervals are graphed.  The tendency of the data  to divide
inco the groups is apparent in this Figure.  The significance  of
the mean differences can be readily visualized.  Overlapping
significance intervals generally indicate the means are not
different at tha 95% confidence level.  Intervals that  do not
overlap indicate the respective means are significantly different.
(When viewing this figure/ only consider the lines, not tha
letters "C" and "L1' when considering overlap.  Overlapping  con-
fidence intervals may give a general indication of  significance
but are not based on the same statistical procedure used  to test
for significance.  In this case the overlapping confidence
intervals correspond well with the significance tests summarized
in Table 17.)


     Figure 5 clearly indicates that the modified bagged  compost
products contain significantly (95% CL) higher concentrations  of
microorganisms than the final windrow compost.  These data  suggest
that adding amendments to compost, such as rice hulls and forest
products, may stimulate bacterial and fungal growth.  Coliphage
populations also increased significantly further indicating the
presence of an actively metabolizing bacterial population.

     The most commonly occurring fungus, by far, was Asgergillus
fumigatus.   Phialoghora sp. and Mucor sp. were also commonly
isolated.  Absidia sp. occurred often at the static pile  facility
but not at the windrow site.  None of the isolated  fungi  were-  of
profound significance healthwise.

     The occurrence of Aspergillus fumigatus has been cited as a
possible concern (Milner et al 1977).  This ubiquitous  fungus  is a
common allergen and may be an opportunistic pulmonary pathogen.
Densities of A. fumigatus are tabulated in Table 18.  A.  fumigatus
concentrations" varied, from approximately 5 CFU/g to 5500  CFU/g.
In general, the aspergilli concentrations measured  in the various
compost products were similar to those reported by  Milner et al
(1977) at Beltsville but more diverse fungal populations  were
observed during this study.

     The windrow composts contained significantly lower
concentrations of aspergilli than the static pile composts.  This
is probably due to the greater amount of wood chips used  and the
recycling of wood chips in the static pile system.  The effect of
wood materials can also be seen at the windrow facility.  Site
IX-A-2, windrow compost without any sawdust, contained  86%  less A.
fumigatus than IX-A-1 samples which were composted  with sawdust."
Aspergilli levels also increased in the commercially bagged
products that contained increasing amounts of cellulosic  materials.

     Although A. fumigatus is a relatively common fungus, these
data support the cautionary measures suggested by Milner  et al
(1977) concerning dust control at composting sites.
                                 53
-------
     The relative abundance of the mean fungal populations for the
one year sampling period is shown in Figures 6 through 13.  This
species distribution does not substantially distinguish between
fungi that were detracted infrequently and those that were detected
regularly but at very low levels.  It does allow one to see the
relative distribution and diversity of the fungal populations and
now the populations varied among the sampling sites.  These
relationships are relative, not quantitative, as described in tha
methods section.

     The data are also shown numerically in Tables 19 and 20 along
with the percentage of samples from which each species was
isolated.  The relative abundance number is a simple ratio of the
occurrence of each species relative to the least isolated species
at each site.  A species with a relative abundance number of 40 was
40 times more abundant than a species with an abundance number of
1.  Similarly, a species with an abundance number of 40 was twice
as abundant as one with a value of 20.
Table 18     Aspergillus fuiaigatus Portion of Thermophilic and
             Total Fungi - Geometric Means
Site
in-a-i
ui-a-2
IX-A-l
IX-A-2
IX-A-3
IX-A-4
IX-A-5
IX-A-6

Aagergillus
fumigatus

3.737
3.270
1*538
0,670
1.735
2.813
2.720
3.028
LOG CFU/g
Thermophilic
Fungi
5.010
4.698
2.206
1.146
2.090
3. 625
2.932
3.52S

Total
Fungi
5.034
4.054
2.077
1.378
2.532
3.499
3.299
4.007
    The weekly sampling for one year at the two large cemposting
facilities was primarily intended to provide an adequate data  base
to assess microbial variabilty.  It was also hoped that seasonal
effects, if present, would be detected.  No readily apparent
seasonal trends were observed with the exception of Yersinia,  which
will be discussed in the section about pathogens.


                                 59
-------
  S3
  -i



  Ul
  H

  35
  z
  3
  uu
 r
 a.

 I
 ac
 iu

 H
uu
H
55
 i


1

u.

u



I
2
, o
s
• s
^1
• -w
_.
- ™

01
|
a
?
jf
u
TP
H
ll
i
X
J


J.


. ,»1 ,M 	 	 .1
• « >
: 3 3
a U
' ; c
. 3
. CM
. 9










I
1
V
>








1
)
<



•



. ... .11 J 	 1
- a
• 3
• 3
• 3
• S
- S
L «
• n
- s
- 3
- S
- 2
MB
° a

Ul
U
z
a
§
<
>
P
3

                                                                                                    CQ
                                                                                                    cn


                                                                                                    'ra


                                                                                                    I

                                                                                                    'o


                                                                                                    u

            o


            (3

           15
           cc

           <0
           OS

           uu
                                                   60
-------
03


U4

to
 I

O
2
M^
01

U
 a.
 O

 s
 Ul
 X
              I
                    35 Ss 855353585
         <£_•-/! O.t/5O.CCC/iCO
                             13*1
5 35 35 S5g!
:a»,S**
   !Sc

	 • -I 	 J 	 -,.,,••
                       ui
                       o
                   ' »  I
                   - 3  a
                       3
                       <

                   t*  ^
                    -  5
                    <«>  ^
                    =  z
                                                     C*
                                       ;ss<
                                   HSFB1!
                                   il=la
ills
!!»!
                                                                CD

                                                                 I
                                                                 "o
ul
 !

5
                                                     • a
                                                     • a
                                                     - s
                                                     • s
                                                         i
                                                        Ul
                                                     a  >
                                                     8  5
                                                     - 2
                                                                 "a
                                                                &
                                                                u.
                                 61
-------
 X
 UJ
 35
  i
 3
 2:
 u.
 o
 3
 £
 a.
 O
 cc
 Ul
 X
I
UJ
5s
 i

1
u.
J

©
- s
- 3

- S  O


r »  I
I. 2  a
     <
     >
                                                    UJ
                                               r
••   •>•  lT,a^jf
                          csinosi srwas
                                                        •  3
                                                   a
 "  tl|
 3  >
                                                                      c
                                                                      il
                                                            o
                                                            u


                                                            oc
                                                            2
                                                            en
                                                            il
                                    61
                                    ±.
-------
 X
 111
 H
 35
 t
 3
 2
 u.
£
111
!•=
          SagSSSSSSftftfc
     ••a
- s
     a
  •*   3
  •y   to
!- s   >
L ~>   i-
L ~   ^
                                                                       X
                                                                       ffl
                                                                       55
                                                                      I
                                                                      "©
X
US
H
31
 I
3
3
tfe,
                                                          s
                                                          «
                                                          s
• S
 S
 3
 s-
              (S
              •a
              i
                                                             U
             as
              ss
                                  63
-------
 X
 01
 35
  I
 0
 z
 LL.
 o
 j
 5
 &.
 o
 cc
 UJ
x
UJ
H
(n
 t
O
O
                                 (SlVlOSf SHN3S
a
OQ
•a
IU
cc



                                                                                           x
                                                                                            o
                                                                                           515
                                                                                           u.
                                                                                           "o
                                                                                            (0
                                                                                           •o
                                                                                           I
                                                                                           <
                                                                                           IT
           2
           OJ
                 V]   uj
                                 031V1OSI
                                                64
-------
X

111


WJ
o
z
3
u.
O
o

cc
til

                                                                                                          (
                                                                                                           ss
                                                                                                           c
                                                                                                          I
                                                                                                          "o

X

118

SI


5




<


I
                                                                                             UJ
                                                                                             U
                                                                                            o
                                                                                            us

 §
•3
 OS
«MC
 9
(£
                                                                                                          S
                                                      65
-------
 in
 tu
                                                             r S
f- 3
r3
  !  s
                                                                  UJ
                                                                 a
                                                                 -
 ~
 u.
li
 0.
 o

 CC'
 tu
in
Ul

WJ
 I

3

u.
_J


I
                                                              a

                                                              3

                                                              3
                                                             a
                                                             y
                                                             y
                                                                          in
                                                                           i

                                                                          X

                                                                           o

                                                                          55

                                                                          '5

                                                                          I

                                                                          "o

                                                                           u

                                                                          I
              <0


              13
              "3
              ec
              1
              Q)
                                       66
-------
 u
 Hi

 35
 i

 5
 o


 CL
 O


 tu

 p
   > S; — g— U5*ui
'•'•Sd^iSi^^s
SS'-'sSa-^ino
"S *«>S5siH3^
— 3^ ^ * "*, 7? <^ do m. /J ^ ^ oe ea> Si _»  ^ PA -«*>
ui
U


Q
                                                                  UI
                                                                           X
                                                                           ffl

                                                                           55
         I
         **3D
          O
          V
          u

          CO
         T3
                                                                            9

          I
          at
                                        67
-------


























H
z
i
u

111
<
J
a.
I
3
ib

J

W
O
*"
a
a
z
a
IU
^*
^
u
o
VI
™
3
§
ik
j

^,
a














TABLE 1






7
i
X












«•
t
1
X













CM
1
ca
i
M
£












I
a
i
M
**

















"41
I '-U U
> 2

< z
'•U CU
X <
LU
> z

< z
J 3
iu a
z <

in
»IU
^ «J
— a.
via
a. vi

*

ui
IUU
> z
x <
< Z
uia
OS <


IU

•• IU
«• a.
wi 3
O ^
a. vi
*

IU
IUU
> z

£
vi a
a <
a vi










VI
111
111
a.
VI
I/I
IU


n
a
-






~





OO OP*

— — — to






OT aa







a
a
«« J
{•J






O c« —
M ^ . P*






U-H.O
z - ao-
I a. 3 iu -i
u iu r oo


o
Q
-












^ a
O HI
_ —






a »
ov






a -








— a







(M CI
^ ••

-I0«





a in n










a.
VI

3 vi vi ai
333 "•
5Si i
« IU VI • —
a - 3 z a> vi tu
vi z
Q Z Z Z >-
-! a o o o
1 U U  1
z aa aa i


a
01
-






—





a
— !O
J —
^





a —







us
-







fS,







e
(N

- <0





(S B*
^4









t/l
 O
Z VI 
-------




























K>
1
O
U
fm,
=J
a.

S
i
it,
j
<^
)a>
a
N>
a
a
Uc
a
>• -
^
j
o
„
X
3
«.

J

N0
9





(
?
®
a








»
l
1
X
M










K5
|
1
X












i
^
1
X
M









1
<
J
X












UJ
IU ZJ
> z

i- Q
< Z
-1 3
IU 01
c <

u
> ul
— IU
!~ -1
- a.
tn 3
0 <
IU
IU U
> Z

H Q
< Z
IU 81
OS <

IU
•* IU
» a.
vi a
a <
a, vi
*
IU
IU U
> z

< z
J 3
IU OS
S <

IU
21"

^> ^
» a,
ui a



tu
UJ U
> z

U OS
S 4


IU
> VI
=4 IU
f= J
c«9 a,
93






VJ
GENUS/ SPEC 8 S.


— 
to
PI






T at f
— • f^



n to n
an »
. > .
<™ •* "• «
_




PI a 





N« IN





a f» « »
a a PI  3
J iZ D U
3 USIU «
aa»«
he •• 14 iu >
ZZZHO,
VI
tfs «««« irt
333-39
-J J J J 3
S « g BE r-
IM tti IU l-J Itf
a. a a. a. <
V> V> Wl VI 2


f* f»
£•5 O

f< —








•CJt ^
•«



 w w






w ® ^
c~ «•





a fa v in
a v *- o
"* *• <=° ^






p| fjf 1Q fl|





a.

3 l/l M 1
HAPLOSPOHANGl
MUCOR HIEMAL!
MUCOR PUSILLU
MUCOH SP.
MVROTHECIUM S


Ul
a

PI eo —
w «•







*••* J*T !N
rt (*?



<0 O
0 0

p*. W <-





\& ffl ^
« v




f*s i*5 






.V S N






38 V
CM« 0
«» f* ••






as Q w





w?

a. • N ** • S
RHIZOPUS SP.
SCOPULARIOPSl
SEPEOON8UM SP
SVCEPHALASTRUI
TORULA SP.





Cj
d







a







T
n




 «• *M






P«!§1 Ml





US
X
tu
a, u
VI US •
s • OE a. w
0. 3 < £ a
o u o « j
vt o u ee u
f — a o »
&. 5 u j
u ui i a a


o *™ to
— ir) r-

— h. 









C4 O Q
an am






ffl Q}
3S -V
nt0




ts®
CO •»





fta BD
-» 9
w-






«n





.
&
V)
§MM a!
--> 3 wi
O J J
55^ 1
BE IU W! -a
O» 3%U
"I Z
e« K « M.
J OO GO
a, 
Z3 3 S 3


f" ** P*B
in o a
»
IV -4 w






oo •» «r






a a
— o
« •
•=> ••









* O*







9 © O
-> e s^
 O
3 I W» =
v% @ a v) •
=» 3 << 0.
3JJ5 « ^
«* 2 ^
a, J o r <
O 3 C3 a. _j
N a, iu iu 3
- ® a u tr
r u iu > a
e v> u> «i K>


M
«

^ ts
i^





f in
^





in a
a in

•* n









» «
CB






(3 «•

(«) CS!




V V






E@ "^
US ®
« W






SS








Q
Ik
UNIDEN1
YEAST
71
-------
    The indicator data were divided into subsets based on the
calendar seasons and tested for significant differences.
Unfortunately other events occurred that confounded the data and
make it difficult to determine the cause of any observed effects.
For example, the method of composting was changed at the windrow
facility during the late summer and fail periods.  During that
period little sawdust was used; recycled compost was the
predominant bulking agent.  Mean indicator concentrations were  lower
during the summer and fail but it is not clear if season (climatic
conditions) or composting procedure was the significant variable.

    The static pile facility data were also examined for
seasonality.  The climatic variation from season to season was  more
pronounced at this site.- Again differences were detected but the
observed variations were not logical.  Other operational factors
appeared to confound any seasonal effects.  Although seasonality
may be a factor during composting, it was not possible to isolate
seasonal effects as independent variables affecting indicator
populations during the course of this study.

Pathogenic bacteria

 Salmonellae—

     Salmonella so.  were detected regularly in compost products
from both of theTacilities.  Salmonellae data are summarized in
Table 21.  The populations of Salmonellae followed patterns similar
to those observed for the indicator bacteria.  At the static pile
facility, the number of samples with detectable levels of
Salmonellae was notably less from the giveaway bin compared to  the
screened compost.  The average concentration of salmonellae was
also significantly lower in the giveaway bin samples.  This
difference remained significant when the data were adjusted for the
variation in wood chip content.

     The occurrence of salmonellae in the samples from the windrow
facility was different from that at the static pile site.  Pew
salmonellae were detected in the finished compost from the windrow
facility (IX-A-1 and IX-A-2).  The same was true of the bagged
product that contained only screened compost (IX-A-3).  On the  few
occasions that salmonellae were detected in IX-A-3, the levels  were
quite low.  In contrast, the bagged products IX-A-4, IX-A-5 and
IX-A-6 which contained mixtures of compost and amendments,
frequently contained salmonellae and often at relatively high
concentrations.  The mean concentrations of salmonellae shown in
Table 21 only marginally reflect the increased number of positive
samples from the amended compost.  Although the mean concentrations
for the amended products (IX-A-4, IX-A-5 and IX-A-6) were higher
than the unamended material, the mean concentrations were still
relatively low.  The same is true for the mean concentrations
of salmonellae at the two static pile sites, III-B-1 and III-B-2.
Table 22, which shows how these Salmonella populations were
distributed, gives a different perspective to the data.  In the
ease of site III-B-2, sixteen percent of the samples contained


                                 72
-------
salmonellae at a level greater than 10,000 MPN/g.  Applying this
figure to the annual production at that site would estimate that
34,000 dry tons of compost were distributed which contained greater
chan 10,000 salmonellae per gram.  When applied to the bagged
products IX-A-5 and IX-A-5, the data in Table 17 would predict that
approximately 193,000 bags of product contained greater than 1,000
salmonellae per gram and 26,000 bags of product contained greater
than 10,000 saimonellae per gram based on proprietary marketing data,

     The occurrence of salnonellae in these samples was much
greater than expected.  Studies conducted az Beltsville detected
salmonellae in only four of 31 compost samples from 30 facilities
(Hussong et, al 1985).  With one exception the concentrations were
very low.  The apparent difference between their results 'and these
data may be due to either one or both of two factors.  First, this
study concentrated much more intensive sampling at two facilities.
If only one sample had been collected from each of the sampling
points in this study, there is a reasonable probability that little
or no salmonellae would have been detected.  The extent of the

Table 21     Occurrence of Salmonella at Weekly Sites
                                     Mean            Range
Site                Positive         MPN/g           MPN/g


III-B-1                48               1           <0.1 - 8,700

III-3-2                80              44           <0ol - 85,000

IX-A-1                  8              <0.2         <0.1'- 34

IX-A-2                  0              <0.2             N.A.  .

IX-A-3                 17      '        
-------
Table 22      Percentage of Samples Containing Salmonellae
              Concentrations Graacer Than Given Log. Increments


MPN/g 	    Percent	

    III-S-1  III-3-2   IX-A-1 IX-A-2 IX-A-3 IX-A-4 IX-A-5 IX-A—5
>DLa
>1
>10
>100
>1,000
>10,QQO
>100,000
43
40
27
20
4
0
0
30
78
64
42
27
IS
0
3
6
' 3
0
0
0
0
0
0
0
0
0
0
0
17
15
6
4
2
0
0
47
41
24
6
0
0
0
69
62
58
27
10
2
0
65
59
53
37
20
2
0
a > detection limit of test, 0.2 MPN/g

salmonellae presence was only revealed by the large number of
samples spread over a long period of time.

     Second, the laboratory methods used in the two studies may
also have been a factor.  Eussong et §!_ (1985) and this laboratory
both found conventional methods inadequate for quantifying
salmonellae in sludge and compost.  Both laboratories developed
modified methods which have been described elsewhere (Hussong et al
1985, Walker and Yanko 1987).  The salmonellae procedures usedTn
these two studies were quite different and may have been a factor
in the results; however, the respective methods had not been
compared for this study.

     The pattern of salmonellae isolations is somewhat difficult to
explain and to some extent, contradictory.  Site III-B-2 at the
static pile facility whiett represented the final compost product,
contained the highest levels of salmonellae.  It is unknown if
these salmonellae initially survived the high temperature aeration
phase or if the population was reduced during the aeration stage
and then regrew during the curing/storage period or if the compost
was contaminated by -external sources after production.  Salmonella
regrowth would not be consistent with the Beltsville data (Hussong
et_ al_ 1985) that demonstrated salmonellae seeded into compost died
off at a fairly rapid rate.  If the earlier assertion that the
material in the giveaway bin, on average, represented older
compost, the lower concentrations of salmonellae in the bin would
be consistent with the Beltsville research.  Subsequent
contamination of the compost by animals (rodents, birds) after
production was not evaluated in this study.

                                 74
-------
     On che other hand, results from the windrow facility clearly
suggest: regrowth of salmonellae-  The finished compost from the
windrow contained very little salmonellae, as was also the case
with the bagged product containing only screened compose.  When
rice hulls or forest by-products were added to the compost and
aged, the salmonellae populations increased in the final products.
This increase occurred concurrent with increases in the indicator
bacteria, heterotrcphic plate count populations and fungal
populations, contrary co the conclusions of Husscng et_ al_ (1985)
that the active indigenous flora of compost establishes a barrier
to colonization by salmonellae.

     This project concentrated on testing only end products;
however other research at the windrow facility (Hay 1986) examined
microbial populations in the amendment materials (rice hulls and
forest byproducts) and found high coliform populations but no
salmonellae.  Although contamination at the bagging facility was
found to be a probable factor, nutrient related regrowth was the
only plausible explanation for the high levels of salmonellae
detected in many of the modified compost products (Hay 1986).

     The laboratory experiments by Russ and Yanko (1981)
demonstrated regrowth of indigenous salmonellae in compost in the
presence of the competing microbial populations; the effect was
transient and die off subsequently occurred.  The Beltaville
research (Hussong et al 198S) showed regrowth in irradiation
sterilized compost~b~ut die off occurred in untreated compost when
seeded with laboratory cultures of £._  typhimurium and S^  newport.
Perhaps the apparent difference between these studies is related t©
the specific salmonellae involved.  This project and the laboratory
experiments by Russ and Yanke (1981) identified Salmonella only to
the genus level.  The.salmonellae species measured may have been
those that had already survived anaerobic digestion and composting
and may have been better adapted than laboratory cultures to
compete with the indigenous' flora in compost.  Another possible
explanation for the differnees in the results may relate to the
experimental design itself.  Although composting does not sterilize
compost, it is probable that all mesophilic nonsporeforming
bacterial populations are greatly reduced during the high
temperature phase.  The simultaneous seeding of sterile compost
with coliforms and salmonellae, as in the Beltsville experiments
(Hussong et al 1985), may actually approximate the population
distribution of these organisms immediately following thermophilie
composting.  At that point it is unlikely that salmonellae
populations would have been selectively reduced while coliform
populations remain high.  Given appropriate conditions, the
salmonellae and coliforms, as well as other mesophilic populations,
may regrow at the same time.  If the ather populations increased
without an increase in salmonellae, the indigenous populations
would then probably inhibit salmonellae as suggested by Hussong et
aJL (1985).  The key question is why salmonella® repopulation
appears to occur in some cases and not others.  Salmonellae
                                 75
-------
repopulaMon is clearly a phenomenon that needs to be better
understood in order to be properly managed.

     In viewing the individual salmonellae results tabulated in
Appendix C, two trends were noted.  Salmonellae isolations occurred
regularly at the giveaway bin (III-B-1) with no discernible pattern
whereas isolations from the screened compost (ZII-B-2) increased
dramatically from August through December.  In contrast,
saimoneilae isolations occurred much more frequently in the bagged
products (IX-A-4, IX-A-5 and IX-A-6) from January through July but
decreased notably from August through December.  These observed
changes did not correlate with any apparent seasonal factors.

     Operational changes occurred at both of the facilities that
may have been related to the patterns of salmonellae isolations.
The static pile composting facility was physically relocated in the
spring of the project year.  In addition, the facility experienced
a municipal employee strike during the summer at which time the
composting operation was not monitored as closely as usual.

     At the windrow facility, more stringent guidelines, including
laboratory testing, were instituted for determining when a
composted windrow was to be released to the commercial producer.
The commercial producer also used sludges from other sources and it
is not known which sludges were contained in bags of final product,
further confounding data analysis at this site.

     The actual effect of these various factors is unsubstantiated
but may have been significant.  In any case, the results suggest
that some form of monitoring may be necessary to detect changes and
and assure product quality.  This subject will be discussed later
in the report.

     Although some salmonellae were higher than would be considered
desirable,  no consistent overt health hazard was apparent
considering estimated infective doses for Salmonella infections.
Use of these compost products in home vegetable gardens however,
may increase exposure risk.  It is also not known what infective
doses may be applicable to young children, the elderly or
immunologically suppressed individuals.

    Rates oc Salmonella infections have been increasing in the
United Statas in recent years.  Salmonellosis clearly remains a
disease of concern.  Nevertheless, the salmonellae are fairly
ubiquitous and it is common knowledge that salmonellae may be found
in food products such as chicken and turkey.  The WHO (1981) has
suggested that it may be impossible to eliminate salmonellae from
the environment and that the best control measures may be increased
education about food handling practices.

    It is difficult to assess the potential health significance of
the salmonellae detected in the compost products.  There are no
known cases of salmonellosis traceable to the use of sludge based


                                 76
-------
soil amendments.  The studies by Ottolenghi et_al (1987) found no
apparent risk to farm families using anaerobTcaily digested sludges
in agricultural applications.  These sludges were shown to contain
salmoneilae.  Available data suggest that saimcneliae may persist
in sludge amended soil for up to five months but that a 90%
reduction occurs within three weeks (Sorber and Moore 1986).  There
is some evidence that specific Salmonella serotypes may selectively
grow in compost and that these strains are less commonly associated
wich clinical infections (unpublished data).

    Ail of these factors appear to mitigate the health significance
of the salmonellae data.  At the same time the question raised by
these results should not be ignored.  Additional research to better
understand the reasons for the high salmonellae levels is
warranted.  Relatively simple management practices may
significantly alleviate any potential hazards associated with
salmonellae in compost.
                                 77
-------
Yersinia —

       The genus name Yersinia is used here to refer to Yersinia
  entarocplitica and closely related species.   Yersinia data for the
  weexiy sites are summarized in Table 23.  There was a significant
  difference between the two facilities sampled weekly for yersiniaa
  isolations.  Yersinia were isolated infrequently and only at lew
  levels from finished compost and bagged compost samples at the
  windrow facility.  High concentrations of Yersinia were observed
  in many of the static pile samples.  The mean concentrations of
  Yersinia at III-B-1 and III-B-2 were not very high when averaged
  over the year; however, the pattern of isolations was consistent
  with an hypothesized seasonal occurrence.  The seasonality of
  the data is depicted in Figure 14 where it can be seen that the
  Yersinia populations were high during the winter and spring
  months" and were not detectable in summer and early fall months.
  It is unknown if the Yersinia occurred seasonally in the sewage
  or if chey proliferated at some point in the treatment process.
  Langland (1983) suggested that Yersinia may grow in sewage
  sludge since it was more frequently isolated in stored sludge.
  It would seem plausible that the ability of the Yersinia to grow
  at low temperatures was related to the occurrence of the organism
  during periods of cold weather.  Temperature may also have been
  a factor in the absence of Yersinia at the windrow facility which
  was located in a more temperate climatic region.


  Table 23             Occurrence of Yersinia at Weekly 'Sites
SITS
III-B-1
III-B-2
IX- A- 1
IX-A-2
IX- A- 3
IX-A-4
IX-A-S
IX-A-6
1 Samples
Positive
27
42
3
0
2
6
4
2
Mean
MPN/g
0.6
6
<0.2
<0.2
<0.2
<0.2
<0.2
<0.2
Range
MPN/g
<0.1 - 54,000
<0.1 - 2,500,
<0.1 - 0.7
N.A.
<0.1 - 1
<0.1 - 3.3
<0.1 - 2.4
<0.1 - 0.5

,000
000






-------
                S
                Ul


                S


                CO
                     u
                     Ul
                     O
                     z
o
o

&
u

M

ea



yji


03


©
cc
a,

Q
UJ
                               ©
                               C£
                               Ul
                               Q

                               <
                               E
                               ©
                               as
                               <
                               UJ
79
-------
     The  fata of Yersinia enterocolitica in compost and dried
 sludge was  unknown and  only  limited  information was available  about
 the occurrence of Yerainia in sludges.  Seattle researchers  (Metro
 1983) reported high levels (10°-1Q9  per g wet weight) of
 Yersinia  enterocolitica in anaerobically digested sludges  and
 suggested the prevalent biotypes were avirulent.  Langeland
 (1933) isolated Y. enterocolitica, Y_. intermedia/ Y. kristsnsenij.
 and ocher Yersinia from 51%  of 35 samples cf stcrad sewage sludge.
 The isolates belonged to 17  different serogroups, but 42%  of the
 cultures  were non-typable.   The serogroups most commonly associated
 with disease, 0:3, 0:8  and 0:9 were  not detected.  Langeland's  data
 underscores one of the  difficulties  in evaluating the Yersinia
 results.  Most environmental strains of Y_. enterocolitica
 are avirulent but disease outbreaks  have been associated
 with environmental sources (Shayegani 1986).  With large numbers
 of Yersinia present in  sludge or compost, these materials
 could constitute a reservoir of pathogenic Yersinia strains.

     The  laboratory tests used to isolate and enumerate the
 yersiniae do not discriminate between virulent and non-virulent
 strains.  Virulence is  governed by chromosoraally determined
 characteristics and related  to the presence of a plasmid.  In vitro
 and in vivo tests for characteristics associated with pathogenicity
 are available but were  outside the scope of this study.
 Nevertheless, the large numbers of Yersinia detected in some
 samples clearly presented a  concern  that needed to be addressed.
 The New York State Department of Health (NYSDOH), which had
 previously conducted studies examining environmental reservoirs of
 pathogenic Yersinia (Shayegani 1986), agreed to characterize a
 number of Yersinia isolates  from this project.  Results of these
 tests are shown in Table 24.

     Twenty-eight randomly selected cultures from the static pile
 composting sites were thoroughly characterized using 36 'biochemical
 tests and were serogrouped with antisera 0:1 through Os34  prepared
 in the NYSDOH laboratory.  Seventeen were identified as Y.
 enterocolitica, seven as Y.  kristensenii, two as Y. frederiksenii
 and two as Y. intermedia."" Most of the Y. enterocolitica cultures
 were serologically non-groupable.  Three representative cultures of
 the non-groupable Y. enterocolitica, the two serogrouped Y.
 enterocolitica and one  serogrouped Y. frederiksenii were Eested for
 pathogenicity markers.   None of the~isolates were positive in the
 pathogenicity tests summarized in Table 25-.  The non-serogroupable
 strains of Y. entrocolitiea  are not usually considered pathogenic.

     For  a project of this magnitude it was necessary to use a
shortened confirmation  scheme in order to adapt Yersinia isolation
 techniques to a quantitative multiple tube procedure.  Although
abbreviated, it was originally thought that the quantitative method
used for  this study was  reasonably specific for Y. enterocolitica,
as indicated in the literature cited for the methods (Weagant 1983
a&b).   The comprehensive identification of isolates performed by


                                 80
-------
Table 24    Identification of Random Yjsrsinia_  Isolates  from
            Stacic Pile Compost Samples
Sample
Date
02/01/86
03/14/86
03/21/86
03/28/86
03/28/86
04/25/86
11/24/36
12/01/86
12/23/86
12/30/86
01/06/87
Site
III-8-2
III-B-2
III-B-2
III-B-1
III-B-2
III-B-1 •
III-B-1
III-B-1
III-B-2
III-B-2
III-B-2 •
Isolate Identificacion
NO.
1
2
1
2
1
1
2
I
I
2
1
2
3
4
1
2
1
2
3
4
1
2
3
4
5
€
7
1
Y.
Y.
Y.
Y.
Y.
Y.
Y.
Y.
Y.
Y.
Y.
Y.
Y.
Y.
Y.
YT
Y.
Y.
Ye
Y.
Y.
Y.
Y.
Y.
YT
Y.
Y.
Y.

enterocolitica
intermedia
enterocolitica
kristensenii
f rederiksenii
enter ocolitica
kristensenii
f rederlk'senii
kristensenii
kristensenii
enter ocolitica
enterocolitica
enterecolitica
intermedia
kristensenii
kristensenii
enterocolitica
enteeocolitica
entereeelitica
kristensenii
enterocolitica
enterocolitica
enterocolitica
enterocolitica
enterocolitica
enterocolitica
enterocolitica
enterocolitica

Serogroup
NG
SG
NG
0:19
0:16,29
NG
0:11,23
0:16,29
0:11,23
0:11,23
NG
NG
NG
0:4,16,
0:11,24
0:11,24
NG
NG
NG
Os29
NG
NG
0:4,33
0:14
NG
NG
NG
NG

,24
,24
,24
20
NG « not groupable
                                  SI
-------
Table 25    Pathcgenisity Tasting of Salec-ad Yersinia Isolates

                                          Pathogenicity Tests
Isolate	Identification	AA CaDep HeLa Mice Plasmid
03/14/86 III-B-2#l  Y.enterocolitica NG             ND  -     14Md
03/21/86 III-S-2*!  Y.frederiksenii 0:16,29 -
03/23/36 III-B-ltt  Y.enterocolitica NG     -
12/23/36 ni-B-2#l  Y.enterocolitica NG     -
12/30/36 III-B-2#3  Y.enterocolitica Os4,33 -
12/30/36 IH-B-2#4  Y.enterocolitica 0:14   -

   AA      -  Autoagglutination at 37 C
   CaDep   -  Calcium dependence at 37 C
   HeLa    -  HeLa cell adherence
   Mice    -  M .use lethality with 10^ bacteria injected
              iat raperLtoneally
   Pldsmid -  42 megadalton plasiuid considered responsible for
              pathogenicity
   NG      -  ncngroupabie
   ND      -  not done
                                 32
-------
NYSDOH revealed that the quantitative test measured Y. enteroco-
litica, Y. kristensenii, Y. fr ed e r ikseni ij. and Y. intermedia.The
apparent reason for the discrepancy is that the taxonomy of the
Yersinia group is fairly recent.  The other species identified by
NYSDOH were, until recently, considered part of the Y. enteroco-
litica group, usually categorized as atypical or unusual Y.
encerocolitica (Sergey's Manual, 1984).  This discrimination,
however, is reasonably significant because the pathogenic strains
are in the true Y. encerpcplitica.  The other species, J_.
kristensenii/ Y. frederikseni i and Y.  intarmedia, are considered
non-pathogenic environmental organisms.

     The citrate (Simmons) test at 25 C included in the screening
procedures should have eliminated Y. intermedia.  The fact that
NYSDOH did identify some isolates as Y. intermedia probably
reflected the more extensive characteFization and an atypical
citrate reaction.  The addition of a Voges-Proskauer test at 25 C
and an acid from rhamnose reaction to the confirmation tests used
during this study would have more reliably limited the measured
population to Y. enterocolitica.  However, extensive biochemical
testing would be necessary to completely assure the accuracy of
each identification.

     The characterization of Yersinia isolates perfermed by NYSDOH
suggests that the high levels of this organism detected at the
static pile site probably do not constitute a public health risk;
but the number of isolates actually tested was very small relative
to the quantity of Yersinia measured in some compost samples.
These data do not definitively establish that virulent Y.
enterocolitiea were not present or that sludges do not serve as a
reservoir of pathogenic Yersinia strains.  Additional research will
be necessary to fully address these questions and understand the
ecology of yersiniae in affected sludge treatment systems.
                                 83
-------
 "oxiaeruc E. Coli —
     A relatively small number of toxigenic strains of S. coll
were detected.  A total of 14 toxigenic E. coll were isolated from
all six of the windrow facilicy sampling sites and 3 toxigenic
cultures were detracted in samples from the -static pile facility,


Table 26 Enterotoxigenic E. coli Positive Samples from Weekly Sices
Site
III-B-I


IX-A-2
IX-A-3



IX-A-4

IX-A-5
IX-A-S

.Date
06-17-86
10-27-86
11-24-86
11-06-86
06-12-86
07-24-86
11-13-86
12-04-86
02-20-86
09-18-86
06-26-86
05-22-86
01-22-87
f Toxigenic
Isolates
1
1
1
1
2
2
2
1
1
1
2
1
1
Fecal Coliform
Log MPN/g
7.184
2.419
2.431
2.851
1.294
1.475
4.176
2.602
5.990
6 . 146
7.157
7.637
5.204
     Quantitatively, it appeared the toxigenic strains represented
an insignificant portion of the fecal coliform population.  It was
originally thought that toxigenic strains, if present, would
probably represent a relatively small fraction of the E. coli
population.  For that reason only fecal coliform tubes~~resulting
from the largest inoculum (IQmL) of the coliform MPN teat were
tested for the presence of toxigenic E. coli.  The positive fecal
coliform tubes were streaked to Sndo agar and five typical E. coli
colonies were picked and tested for toxin production.  If all five
colonies were toxin negative, the original MPN tube was considered
negative for the MPN titer.  When all three of the IQmL inoculum


                                 84
-------
tubes were negative  for toxin producing strains, as was usually the
case, it was  indicated that toxigenic S. coli populations were
less than the detection limit of the test.

     Reexanunation of the data suggests that this approach may
have grossly underestimated the levels of toxigenic E. coli.  If
the results are expressed as percentage of E. coli colonies that
were toxigenic, a substantially different population estimate
results.  A total of 3,915 E. ccli colonies  from windrow facility
sampla were tasted and 14 were toxin positive (0.25%); 1395
colonies from the static pile facility samples were tested and 3
were positive (0.22%).  Data from the bi-monthly sampled
facilities, which will be discussed later in the report, revealed
that 0.375 of these  colonies were toxin positive.  These
percentages were all relatively close; an average of 0.32% of
slightly more than 7000 E. coli colonies picked were toxigenic
strains.  If one assumes the fecal coliform  test measured
predominantly E. coli populations and multiples the mean fecal
coliform results by  the average percentage of toxigenic colonies,
the estimated numbers of toxigenic E. coli were significantly
higher than originally reported (Appendix C), as shown in Table 27.


               Table 27 Mean Toxigenic E. coli Levels
          Estimated  by Percentage Toxin Positive Colonies
                     Times Fecal Coliform Counts


            Possible Toxigenic E. coli Densities - MPN/g


III-B-1  TIT-B-2  IX-A-1  IX-A-2  IX-A-3  IX-A-4  IX-A-5  IX-A-6
 290     10,000   0.5      0.05     5.4    140     1400    10,000


     It would appear that the Table 27 estimates may be a  more
accurate assessment of the levels of toxigenic E. coli and that
the original procedure was inadequate to 'detect~"thes@ levels.
Given-that tne toxigenic E. eoli represent approximately 0.3% of
the total E. eoli and that the fecal coliforms were predominately
§« eeli; the initial inoculum in a coliforms MPN test would contain
a ratio ofi toaeigenie E. coli to fecal coliforms of approximately
1:300.  Again, assuming the populations all grow at the same rate
in the lauryl tryptose medium and in EC medium, thus maintaining
the Is 300 ratio when streaked to the Endo agar, picking 5  colonies
would result in 60:1 odds against selecting a toxigenic colony.

     It became apparent that the original procedure for quantifying
•che cojcigenic E. co 1,_i could not have enumerated a level of 0,3%
toxigenic strains,,  However, 0.3% of the tested colonies were toxin
positive and therefore, it is reasonable to assume that 0.3% of  the
E. ££_U populations were toxigenic.
-------
     The estiiaar.es in Table 27 were based on the assumption  chac
the fecaL coiifrorm test measured predominantly E. coli.
Unfortunately fecal colifonn populations were not characterized
during the course of the study.  Even if only half the fecal
coliforms were E. coli /  trhe density of toxigenic strains would
still be high in some samples.  Considering the relatively high
densities of facal coliforras occurring in seme samples,  it  appears
additional research would be warranted to address potential  helath
risks associated with toziganic E. coli.
                                 86
-------
Campylobactor—

       There have been no published reports on the detection of
  indigenous Campylobactor in seweage or sludge.   However, this
  organism is a significant cause of human enteritis and there are
  numerous animal reservoirs for the infection as well (Blaser and
  Keller 1981).  Therefore, since it is likely that this enteric
  bacterial pathogen is entering sewage, attempts were made to detect
  It :n tr.e D & M products.

       No Campy j._3acter were isolated during the course of this
  study, however the available rnetnodology for detecting
  campylobacters in compost proved to be relatively inadequate.  As
  .described in Appendix A, experiments with Campylobacter seeded into
  compost suspensions estimated a detection limit of approximately
  1000 CPU/ml.  This translates to a detection limit of approximately
  20,000 CPU per dry gram of compost.  All attempts to improve
  recovery (described in Appendix A) were unsuccessful.  Ottolenghi
  and Hamparian (1987) reported recovering seeded Campylobacter from
  anaerobically digested sludge and estimated a detection limit of
  approximately 150 CPU/ml based on two tests.  It is not known why
  lower levels of seeded Campylobacter could be recovered from the
  liquid sludges,  but it may be related to characteristics of the
  sludges compared to compost suspensions used in the respective
  seeding experiments or to differences in the C. jejurai cultures
  used for seeding.  Qttoleghi and Hamparian (1987) used a clinical
  isolate and this study used an &TCC culture.

       Essentially all Campylobacter seeded omtp sterile compost
  suspensions could be recovered indicating the compose was not
  innerently toxic to the C. jejuni.  The enrichment media and Campy
  Blood Agar medium did not appear to be selective enough to allow
  low levels of Campylobacter to be detected in the presence of the
  background compost populations.

       Airhough Octolenghi and Hamparian (1987) were able to detect
  somewr.cc lower levels of seeded Campylobacter e  they isolated no
  Campy i-obacter spp. from 99 samples of sludge.  These authors
  ccuc: ..:sd t-nat the high sensitivity of this organism to oxygen make
  its presence highly ^nlikely in aerobically digested sludges.  The
  same would certainly be crue of aerobic composting systems which
  peasant in even more hostile environment.  Considering the
  relatively fastidious nature of Campylobacter and its
  susceptioility co drying (Doyle and Roman 1982)? it is extremely
  doubtful that Camgylobacter spp. would persist through any
  composting or sludge drying process.
                                   87
-------
S_n_terbacter iace.se—

     The quantitative values for tha undifferentiated total enteric
plate counts were included with the data summaries of the indicator
groups  (Table 6 - 13).  The primary purpose of this test however,
was  to  screen for enteric pathogenic bacteria that might have been
present in  large numbers but were not texted with dedicated
procedure.  A number of colonies were picked from the countable
dilution of the enteric plate count and identified with the Minite.k
Systems, a  commerically available clinical test kit.  The
limitations of this approach are discussed in Appendix A.

     Sixty-eight different species or groups of bacteria were
identified  which were almost equally divided between the
Enterobacteriaceae and the non-fermenter groups.  These data are
not quantitative per se, but the relative occurrence of isolates
can be  distributed by isolation frequency.  Figures 15 through 22
show the relative occurrence of bacterial isolated from the sample
sites at the windrow and static pile composting facilities.  The
data are summarized numberically in Tables 28 and 29.  In general,
the majority of bacteria detected .were pseudomonads.  Beyond that,
the other dominant genera and relative occurrence ratios varied
providing further evidence that these samples contained active
microflora  that adapted and changed with conditions.  In addition
to the pseudomonads, the dominant genera isolated were
Acinetobacter, Alcaligenes, Achromobacter and Moraxella.  The
validity of the Moraxella identifications is somewnat weak.  The
identification of these bacteria i's based more on the absence of
reactions rather than its ability to utilize specific substrates.
There are alos a number of groups of unnamed bacteria that closely
resemole Moraxella (Lennete L974).  There is a reasonable
probaoility that tne isolates could have been Pseudomonas diminuta
or other Pseudomonas species.  For purposes of this study the
differentiation is moot; any of the potential variants would be
considered of low pacnogenicity and opportunistic pathogens at
oest.

     The same is basically true of most of the bacteria regularly
identified during the enteric screening.  Many may be opportunistic
pathogens but none are associated with endemic or epidemic disease.
Early in the study a few isolates were identified as shigellae.  At
first it was considered surprising, but not impossible that a
Shigella would be detected.  Subsequently, more cultures were
identiried as shigellae and the validity of the identifications was
questioned.  Additional testing confirmed that these isolates were
not shigellae.  The first few isolates had been discarded before
the additional tests were instituted and tnerefore, were not
confirmed as Shigella.  Considering that all subsequent isolates
which were  identified as shigellae by Minitek were subsequently
shown to be other organisms, it is highly unlikely that tha
earlier, discarded isolates were Shigella.  The difficulties of
adapting clinical test kits to testing environmental isolates and  '
tne Shigella problem is discussed in greater detail in Appendix A.
-------
Ul


55
 i

w
OS
UJ
at

z
ai
u.

O
CO


UJ

55
 i
IU
u
<

S
Ul

O

S
O
Eg
Ul
UJ
                    i* 3
                    
-------
ca
UJ
>_

35

 i

en
cc
UJ
H


Ul


e
Ul
u.


o
03
ui

35
 i
ui

!U
ui

o

01
o
QE
IU













ll





L_|








































|






















M j
I
1 i
i i
1 1
JWU












H






• §
• § tu
o y
— UJ
Str
cc
"" 2
- 3 H
i- 3 o
L a ui
r r- >
- 2 P



— a
r! 3
- ? cc
- S
- a
_ o
            -_ =•«> «!-•»<*.— _=usu5a:
                          03J.V1OS! SHN39
      S*S85a
      5±isr2sns
                                                         3i3

                          yj     ***

                                s







                          031V1OS!
                                                       ^£15

                                                         III
                                                         S^s
                                                         25 ^ Crt
r -

• i

• §
  o


• I

  3

  3
  g

  3

  S

3
u


>

3
                                                                            .2
                                                                            35

                                                                            S
                                                                            03
                                                                           tu
                                                                            0)
                                                                            u


                                                                            I

                                                                            (J
                                                                            o
                                                                           cc

                                                                           to
                                                                           T-

                                                                            CD



                                                                           I
                                        90
-------
UJ


«5
UJ

cc
UJ
u.

o
                                               UJ
                                               o

                                               111
                                               cc
                                               e

                                               u
                                               u
                                               o
                                               iy
                                                    u
                                                    cc
X
                                                     !9


                                                     I
                                                     "5
                                                     m
                                                     ffl

                                                     o

                                                     5


                                                     u
                                                          o

                                                          ID
                                                          i
                                                          i
31
 i

as

UJ
                                               o
                                                     13
                                                     (£
UJ
                                                          3
                                                          OS
ea
O
OE
UJ

z
IjJ
    . I I ¥f I I I I I T I I I I I  I I I T • .  . . I I I I  I *FT

sss
                                s^;5^
                                  liisgliisss
                    a
            Si-Ss8
                       issgSaa^aww-

^|Sg
 |25|

 wliS
  ISs
  §li
                          sriNis
                             91
-------
<

X

UJ
y~
35



S
UJ


UJ

£
\u
u.

o
2
<«•

<

X

UJ

55
 i

us

Ul
o


£
UJ

o
<
-------

UJ
h-

UJ


s.
UJ
u.


O
X

!U

51


ui

tu
u


E
UJ
H=
O

as
O
E
tu
t-

1AJ

     siil|l£?plSll=i|2S3ig*p32S
-------
X

UJ


55
 i


£2
01
H-

01


QC
01
U.


O
x

UJ

35
 i
ui

01
u

£
UJ
CQ
O
ce
iu

                                                                                                      X

                                                                                                      ra"
                                                                                                      CD
                                                                                                      UJ
                                                                                                      I
                                                                                                      u

                                                                                                     i
                                                                                                      >
                                                                                                     'a
                                                                                                     o
                                                                                                     CM

                                                                                                      O

                                                                                                      3
                                  03JLV1OSI
                                                   94
-------
 in

 <

 X

 ii!


 £5
 y?
 oc
 UJ


 U!
 S
 £
 IU
 Ik

 O
irt
Ui


03


IU


UJ
O

ca
O
£
LU


Ul
                                           ia^>-:oj«s
                                       j^ —~;   IM ^~ MB *-^ uy —^ ^5 ^s ^s ^^


                                         g
                                                    •<• *•* ^ ae ^P ^i ^c ^^ ^" — —   r™1 *>4 f— ac
                                     »
                                                                                                           to
x
m
 O

 ffl

at

13

I

"o

 u
O

 03


IS
"3
M
 as

 3
 as
IE
                                                SHN39
                                                    95
-------
<0



X

Ul


35
J/3
IS
LU

z
Ul


c
01
u.

o
CO

<

X

IU


35

 i

IU

tu
CJ


£
Ul

u

CO
o
as
UJ
L
A
c
>
!• ..
a
si
c
.L.L

.
I
- a
- g
- S
. o
u
u
c
•i)
_1
Ul
e
                                    "

                              SHN3O
X


.2"

«



03

o
                                                                  ,o
       o

       i
       3
       U
       U
       O

       §
       cv
       OS
                                                 <,,<«,1,
                                   i|pilliiis
-e«S3
                                    
-------























(-
z
i
u

IU

^
^
a.
u
z
IU

z
Ul
.J
<
^»
9
to,

i
a
u.

o
IU
1—
J
«*

Ul '

IU
U
2
s
IU
u
^
s
o
£

z









a
j
ffi
t=







i
*

X
"*











w
i
^
i
X















M
1
e
i
M
tat











*3
)
gj
1
ort
M
BO














uj
UJ (j
— UJ
v- z
-1 3
u u
z u
0
UJ
"> */'
Mr Of
— 1
in a
O <
3. *
*
IU
Ul U
•> x
HZ
< Z
-13
Ul U
zu
Q
IU
> wi
MIU
1— J
— 0.
ui a
0 <

*

IU
U1U
> X
1- Z
< s •
J 3
IU U
* a

IU
» Ul
1— J
M &
ui a
2<
Ul
>« .
IU
UIU
> z
— IU

< z
i «j
Ul U
zu

Ul
> w
« IU
JgO J
M9 ft,
VB 3
0 <
QL Ul







V!
U

1
$



09







—
*•






«*  r*


5/|
U! 3
3 U

< M H> VIM
ZU. < 3 —
>- u. z tna
•• o o oe z
Z 2UU3
* a — z
eg ce < a u.
\tt IU
W W W IU Ul
«<;>=, to H>
a auuu
00 « « <
iu a) a o o
z z ce ce z
< < wou



















n «>•









w w








?





OA
BaS y$
at ** vt
M ua K
< Z m
< 2^JJ
w» >O3
U >
oseE >»
IU U| < < US
S «5S 2
< « u G <
o o s s <
ww r z 2
tt* IH us tjy s






































« w «e
t=






N W (0
a»















N ®






IW
^
•I kd
U lU Q X
o < a <
x w za!o
oo a «« s
J «J W aj «J
tti IU IU IU U
Irt VJ V3 W <
C9 ffi (8 63 t3
j ^2j|



















C3









S*J








* f» »







W ^ V







• ^ a.




' •



n "9 w



JP
IU
MM
(J
< ix w
U. OB •>
x IU 1—
ut -i a z
Mlrt < 1- <
J«OI- 3
a < < at tn
Z -1 < < <<
3 > U u (j
zz z
id us lu tu IU
3 3 aae
Uj VU •• «= -s
H- 1- > S >
O O Q O O
Z S Z X Z
a, a. a, a, a,






































a» f 10 •«•







^ ^ v tf







^ N








V e*a





Ul
»w>
!= Z —
Ul » IU Ul
< a- z «
z »u ui e
3 OC < U UJ
N IU U. I/I U.
» to W UJ a
ce x 3 u «
=» < a

j j •« < *
m iu BO M i^
§O « < •<
3CCZZ
4 4 tU U UJ
Ul I/I VI (/S VP



to —







eg (O







U3 «









w n








w v ^ w







•* r* w w







« w w
w







^ e^ f»s
w



a <

e=3
(^ ^ (3d
•« J -1
« U -J -1
z «• < ui o
iu Z vuo u
u. |«> < •= o
- 3 a r z
O * ffi >"
Q«J 39 £
s a, s ui ui
z z a o us
sis!!!


































































a.
13

«

c=:
gm
O
U
NTERO
UJ
ISSMI&
l£
97
-------

























1-
z
3
3
U
IU
t-

a
u
X
IU
t-
z
IU

j
^
i-
2

1
X
u.

a
IU
1—
o
VI
M

IU
IU
u

M
X
IU
u

a
a
X
01
H
Z
Ol









a
1
CJ
a
Ol
-i
a
H








as

x
••












in
i
x
i
X
"














•»
1
1
X














C*9
1
1
X




















Oj
xi y
> Z
— IU
>- X
< X
-1 3
IU U
X U
0
^ wl
—  z
— ut
t- X
X X
-1 3
iu u
X U
a

UI
^ UJ
1- J
— a
vi a
22
x

IU

> z
— 01
— X
< X
J 3
IU U
o

IU
M IU
h- J
— a
via
o •<
0. VI
*

IU
01 CJ
> Z
"x
< X
U 3
Ol O
X U
o
IU

>•• (U
1- -J

vi a
0 <
0. VI

X








ECIES
a.
VI
^
VI
3
UI
(3




— n a •» a
— V CM — ft
*m





a n CM a oo








lflf» r» CM CM (0
•• • tfl ftM
X 0, < 3 —
H-u. z v><3
-O OX Z
zai j iu 3
< -1 < > IU
xx < a *
Ol UI
UU IU IU IU
< < H H t-
n ca u u u
H- K CO CB ca
01 ui o o a
z z x x x
U U MM ••
< < u u u
1
J


— M
M CM






CM CM
— —







CM CM U)
CM








CM PI F^
«







•™ n ^*
(M







CM » »
••







CM









CM







OT
Z
Wl < IU
IU X <
x o< at •»
iu a x ^
< O 3
XX M
vuui < < tu
1- H- _ M >
uu i r J
a o « z <
x x 01 01 M
ui  < Ol • X
x N z a o
o o at vi a
j _j _j j _i
_i j j _i _i
m m m m ^
VI UI VI VI <
o co ca ca a
01 m iu ui x
J J -1 J O
* * x x a




•~







,-,








ffl — — CM Ifl
M








•* M (S V 38








*•








^1








fl « (O 49
tS V








ex -
cr j < < <
a > u uu
z z z
3 sa a a
Ol Ol -• M M
H H. > > >
a o o o o i
X X X X X 1
a. a a. a. a. i




o
•^






T



























CM V








CM ^




























VI
— vt
M Z —
Ol M Ol I/I
< a M z <
Z - cj iu x
a z < u oi
M 01 o. yi o.
_> t- -« IU M
X Z 3 U X
< Ol OX O
M < a
< < -i a o
j -i •< < <
ZZHK^
o o < < <
a a x x x
J .J X X X
< < Ol O) IU
I/I VI VI VI VI
1



CM







0<



























ca n CM








7 CM (M


























.
a. -<
VIU

CM < 1-
< J M
x u -a -t
x >•» < 01 a
U! I UI U U
o. H- < — a
- 30 IX
x a M vi ui
a > a i—
§-j 3 a z
a. x 01 iu
« < s <
t- 1- >- o. Z
< •< < Z —
x x x a vi
x x x u x
01 01 01 Z 01
vi vi vi 3 >










































































X
(5

^
u

H-
O

93
-------

























HB
Z
3
o
u

Ul
2
a.
u

s
t—
z
IU

-!
£;
s


o
K
u.

ul
fc-
3
o
VI


I/I
E
IU
1-
2
3
IS

tfc
z
s
z












OS
IU
a
t-







!M
1
*
X











tm
1
1
X















C*€
P
OS
1
tsa
"












Of
1
18
1
MS
0)


















UJ
UJ U
> z
-* IU
i- Z
< cr
-1 3
IU U
oc u
o
IU
-> vt
t~ «•
vi a
o <
*
IU
IU U
> z
M Ul

< 05
-1 3
IU U
•3

tu
^ VI
•4 \U

— a,
vi X
£ Si
>i

IU
IU(J
» z
i= ae
« K
J 3
IU U
aeu
Q

IU
> V9
«=»«
k» *J
«= a.
vi X
0 «
a. vi
i*
IU
ul u
> Z
» IU

< a
-I 3
IU U
as u
a
m
^ tfj
» IU

-» a,

o <
a^ vs
IS







ut
IU

w

!3



<«•*•»







CO — -O
—






— « w « -^

BB







)*• « (O « ^
•• ^>






98 0S OS 4)
j p= ta =
n - to







a> w •* <•  ••
Cg £ % *J VI
ui iu iu a
U U U IUU
< « < u z
ffl ffi 33 > 1U
gg g g (S3 (^
2 *^ S J10 W
<< < < <



(S —







- o
1— —






OJ W — (A
^








«« n c0







a (M M «
dD







« C«M h-
«







w N
•=>








* us
1=







1
I
Q
»it-N a
1 1 1 l 3
Uc. w < IU m
IU
a. Ob 9.4,1-
3 3 3 3 U
eaoo «
s oe K x n
^






u? a n

0*







^ — n
— (0






to ds N w
ENi W
a>






W « W «
N tn







» a ^









0» Ifl «








u •<
-^ u
> 10 —
3 IU !-
£ Z X
ft, as m o
10 j 03
> at iu
§Z IU <
ui - < < H (-
< a: s vi _!
< U OU.
< ta tn v> vi
j « < « <
J Z Z Z Z
IU O O O O
ae 3 3 3 3
3Q OO ©
iu a a ffi e
H 33 3 3
VI Ul IU 01 UJ
< I/I VI VI VI
a. a. a, a a,



n •*







£5 {£J







N oi a n a
•• w








n ai 7 not
M> ^






 -1
-• -J <
a. a >- >-o
a •=• ui us a
I- a a.



T N n
^






r-4 — -a
p* —






P. f= (O
CB
«>







Ui to 03
Kl






— » as w

«•






^ 99 1* N
^







— e< — to

«•







IN r. rx tS
(O






« VI
Ut Z VI
J IU —
KJ *" £
O Ik IU 3
a ui M u
3 ae H. —
tut- • 3 vi
M 3 a, >-
IO 
o t-
J <
< a
4U
sz
5s
to «<
— s
> 13
99
-------
























h-


U
IU
H
J
u

f^
IU

IU

J
<

a


a
a
x
u.
a
IU
H
J
a
vi
"*
VI
Z
tu
t-
z
tu
a
X
IU
u.
1
z









a
)M

rt
u
31
IU
-J
01







 z
>- X
< X
U 3
IU U
X U
o
'.U
> VI
M IU
H1 vj
— 0.
It 3
X
IU
IU U
> z
H X
< Z

IU U
X U
o
Ul
> m
MIU
si
a <
a. vi
it
IU
IUU
> z
» IU
>- z
< z
-13
IUU
xu
o

III
> V)
M IU
1- J
— IX
in x
235

*
'U
IUU
> z
1- Z
< x
-1 3
IUU
XU
9

IU

M tU
t-= J
v» a
o •<
a, vi








VI
IU

u
IU
GENUS/SP
i

i
a> — — n
n ^ ^ 9







o "O ss a
« n




n  —
vlirt x X •
a a
X Z Z U VI
IU IU IU O
HI K H H VI
UUU IUIU
ACHHOMOBA
ACHROUOBA
ACHROMOBA
ACTINOUVC
ALCALIGCN



r» a








a T





a in
••







9 CO







a COM w









o cor* M







a *  > X

a. a. a. a. t-
3 3 3 3 U
ao o o <
5ce x x a
tjo a o
u u uu <
a ffl a o -i
u u u u u. i



<•» (N . ni
a




o o
04 ^
04






2E







•• C*
— . a








* u>
- «






r» «








3 <
3 IU H>
K Z >
• > IU -J
3, a. (39
vt j 9 a
> Z IU
X Z IU <
3 IU • < Z
— 2 a.1
x a vi < <
IU J J
H < < J -
U -1 U IU IU
< -1 _( C I
a iu iu 3 3
9 X X IU IU
><<>->-
< X X I/I VI
-i 9 9 « <
it X a a. a.



V - 3 •< -9
-1 Z fl. X 3
3 tu IU — J
X < u 9 u.

< I/I VI VI VI
J X < < <
PASTEUREL
pseuoouoH
PSEUOOUON
PSEUOOUOH
PSEUOOUON



n to r» a
T SB







-^ T -T —

— J •<
— a «• — j
J 9 1- |- <
0, X H f-9
O «• IU Ul Q
H U X X 3
J 3 U U IU
a a. a. a. a.

I/I VI VI I/I I/I

z z z z z
99999
9 9 Q 9 9
33333
(U tu tu IU IU
VI VI VI I/I VI
a, a. a. a a



n <•> 01
CO l~
••






 31 »
a M







» ^ 9 « V







P* 01 <*t ®
a «








»r* — 4
(•) ^






^ to r*.
a —











VI VI I/I VI VI

O O 9 9 9
99999
33333
IU IU IU IU IU
VI VI VI VI VI
a, a. i a. a



_
••







•o





in








us







us









^







— u>










CM 
M MO
J 1-
VIBH1O ALi
GRAM-NEGA
100
-------
     Examining Figures 15-22 does illustrate the dynamic nature of
the bacterial populations.  Sice IX-A-2 samples, which consist of
compose only, show the lowest diversity and fewer bacterial dominate
the population.  IX-A-1 samples contained sawdust during the
composting process; bacterial diversity and dominant species both
increased.  The commercially bagged products, IX-A-3 through IX-A-6
continue tc increase in diversity and number as more species are
represented in the dominant portions of the populations.

     An obvious drawback to the total enteric procedure was that it
only detected those bacteria occurring in greatest numbers.  The
total enteric plate counts were reasonably consistent throughout
the study averaging approximately 10s CFU/g (Tables 6 - 13).
After a sample was diluted to the countable range for the plate
count, an enteric pathogen present at levels of 101 to 105
CFU/g would not likely be detected.  This was illustrated by the
Salmonella and Yersinia data.  These organisms were detected
frequently in substantial numbers with dedicated test procedures;
however, they were seldon detected in the total enteric assay.

     In general the total enteric test provided some information
about the diversity of the predominant non-=fermenter and
Enterobacteriaceae populations in compost but was of little value
for assessing potential risks from pathogenic enteric bacteria.
                                101
-------
 Parasites

      Helminth ova  were  detected  regularly  in  samples  from  both  the
 windrow  composting facility and  the  static pile  facility.   The  most
 common ova  detected were  Trjichurig and Ascaris,  in  that  order.
 Overall, 74%  of  the windrow sica  samples wera positive  for
 Trichuris and 45%  contained Ascaris  ova,   The stacic  pile  samples
 were 49% positive  for TrichurT-s'and;  38% contained Ascaris.
 Toxocara and  Hymenolepis  ova were observed infrequently  in  samples
 from both facilities.   No other ova  or protozoan cysts were
 detected.   Ova densities  in positive samples  ranged from 0.1 to 2
 ova/g dry weight.

      Measurements  of the  size of  the Trichuris ova indicated that
 many of these ova  were  of  non-human  origin.There are many
 different species  of Trichuris which are generally considered host
 specific.  The ova produced by adult Trichuris sp. are
 structurally  similar but  often vary  in size (Levine 1980).  For
 example Trichuris  trichiura  ova, the human parasite, measure 50-54
 X 22-23 micrometers whereas  T. campanula ova, found in cats,
 average 71-81 x 31-36 micrometers.   Many of the Trichuris ova
 detected were larger than  those of T. trichiura suggesting sources
 other than human infections.       ~~

      None of the Ascaris ova examined during this study were found
 to be viable by embryonation testing.  Viability was reported only
 for the Ascaris ova listed  in the data tables in Appendix C because
 the positive controls used to judge adequate conditions for
 embryonation were Ascaris ova.   Nevertheless,  Triefauris ova would
 be expected  to embryonate under the same conditions  and time
 (Kaneshiro 1985,  Brown 1975).  None of  the  examined  Tricfauris ova
 embryonated  or showed earlier developmental stages.   It can be
 reasonably assumed that  all examined Trichuris ova were also
 non-viable.

      It  is not known if  the almost exclusive  detection of Trichuris
 and Ascaris  ova reflects the ubiquity of  these infections,  the
 resistance -of  these ova  to destruction,  or  a  combination of both.
 The heat  generated  during  composting  processes would be expected to
 render ova non-viable, but the  inactivated  ova may still  be
 relatively resistant to  degradation within  the time  frame of the
 composting process.  There is some evidence that  Trichuris  and
 Ascaris ova  can persist  intact  longer than  other  helminth ova and
 of  the two,  that  Tricfauris is the  hardier.  Taylor (1955)  reported
 finding larger numbers of  Trichuris and Ascaris ova  in fecal
material  excavated  from  a  large pit that was part of Roman  ruins
 located in Winchester, England.  The  material  in  the pit  was
approaching  the consistency of peat.  Based on other artifacts,  the
 ruins were estimated to  be llth or early 12th  century.  As many  as
 5,700 eggs per gram of Trichuris trichiura  and 600 of  Ascaris
 lumbricoides were detected.It"was suggested  that the high  egg
count could  be explained by the concentration of nonfermentable
material during the passage of time and that the  ratio of Trichuris
to Ascaris reflected the relative ability of the ova to persist.


                                102
-------
Phe absence of other helminth ova, such as any strongyloid egg, was
considered due to their failure to resist disintegration.

     When examining the reported parasite data tabulated in
Appendix C, it is apparent that more ova were detected from January
until approximately June compared to after June.  This was true
at both the windrow site and the static pile site suggesting the
possiblity of a systematic analytical problem.  No procedural
changes wara incorporated which would explain a decrease in
recovery.  Both composting facilities instituted operational
changes during the course of the study which may somehow be related
to the decrease in detected ova, but the apparent sudden change in
recovery of ova is unexplained.  An examination of ova data from
the bimonthly sites during the same periods showed that fewer ova
were detected during the second half of the sampling program at
these sites too, however the difference was relatively
insignificant.

     In summary, no potential hazard from parasites was found
associated with compost from either the windrow or the static pile
facility.  O'Donnel et al (1984) studied the survival of helminth
ova seeded into sludges and tested both viability and infactivity.
They reported that even if most of the eggs appeared to be
nonviable, a few were still capable of causing infections in test
animals.  These authors ware dealing with large numbers of ova that
had been seeded into samples.  Only a portion of the sample
concentrates were examined prior to infecting the test animals.
The suggestion that viability testing might not completely address
potential infectivity is clearly applicable to the data developed
during this study.  However, in view of the large sample size, the
low concentrations of indigenous ova and the complete absence of
indications of viability/ any theoretical risk must indeed be
small.

     The fact that no protozoan cysts were detected probably
reflects the inability tb recover these forms from compost as much
their absence in the samples.  Results of seeding experiments with
Giardia cysts are discussed in Appendix A.  Although recovery of
cysts was poor, it still remains highly improbable that protozoan
cysts .could survive conditions capable of destroying helminth ova
such as Ascaris.
                                103
-------
Enteric Viruses

     Virus casting of sludge and compost samples is very method
dependent.  There are two basic components to the virus test:   (1)
elation and .concentration of the viruses, and (2) detection of  the
viruses.  The elution/concentration procedures used during this
project had been thoroughly evaluated and found particularly
effective for high solids samples such as compost (Goyal e_c aj^
1S34).  The conventional assays performed in the project laboratory
were run on two complementary cell lines using both plaque and
liquid overlay procedures.  The BGM cell line had been previously
evaluated in this laboratory (unpublished results) and found highly
receptive to indigenous viruses in sludge cake.  In addition to  the
conventional assays, sample concentrates were also tested for
rotaviruses at the University of Arizona using an immunofluorescence
test procedure (Smith & Gerba 1982) and for a broad spectrum of
viruses using a newly developed immunoperoxidase procedure (Payment
and Trudel 1985) at the University of Quebec.

     In spite of intensive efforts devoted to detecting viruses,
only two indigenous virus isolates were confirmed during the study.
The 5/2/36 giveaway bin sample and the 5/8/36 bagged product
(IX-A-6) sample contained untypable picornavirus.  Viruses were
detected in six ocher samples but these isolates appeared to be
contaminants resulting from "blind seeds" submitted to the
laboratory as part of the quality assurance program.  The virus
isolations and quality assurance samples are compared in Table  30.

     Examining the chronology of the introduction of seeded samples
and the subsequent, isolation of the seed strain from other samples
strongly indicates laboratory contamination during the sample
processing or assay procedures.  It is highly unlikely that the
only viruses isolated would be the same as seed virus and then only
occur after the seed was used in the laboratory.

     These results emphasize the importance of segregating high
tite-red laboratory viruses from environmental samples.  Even with
experienced personnel, low level contamination is difficult to
control.  Blind spikes do have value from a quality control
perspective, but in this case the problems outweighed the
advantages.   Other samples known to contain low levels of naturally
occurring viruses could be used as positive controls for virus
methods.  These samples could be processed separately from other
samples and still provide adequate assurance that the virus methods
are working.

     Discounting the apparent contaminants, only the two isolates
of untypable picornavirus remain.  This virus was present at a
level below the quantitative limit for the plaque assay, less
than 2.3 PPU/g.   It was detected by the liquid overlay portion of
the assay.  It appeared to be an ECHO type virus since'it cross
reacted with a number of the ECHO antisera in the Lim
Benyesh-Melnick pool (Lim e_t al_ I960}.  It did not react with the


                               . 1(14
-------























1/1
3 '
C
>(«
tutu
> t-

(0 (J

w «J
SO,
:a
IMS

36 3C
&• «
a a.

za
oz
— <

«=! US
IU J,
95 &>
X

s
o
i
fcD










'















Q
ra
IU
u
3}
ho










a
'.U
VI
a
j
O
X
a. >
«OO
B <
«!§
-i 0
15
<
^
« a
i>
«s

3
as
5







u

^
^
VJ






IU
k.
<
O

a
n
a
n
n
n
a
c->

as
CM


o
<*

*

M

a
n

a

 * 3
- k
e ;
• i
e '

a. 4
s :
e $
= i
ia i
t
< '
•3
« 5« >




e 
i
a
» 9




3 1
9 9
» II
« •
1 1
3 t
-


























.











<3»
*> 1




^ ^



^ ^
a s
x >
a c
u <.



«

3
«
J * -1
e



e
§ •
a
i •
« »
1 9
< 
« X >




S US q
B 0! g
9 f» f

B f» 9
3 e e






































•










r
9
e
3
j




«
9

k >
C



L
i


9
9
4
«
0 9




9 S
9 9
• ^
< «
i <
3 ;

















4
















-



»


















P ^
a a

€ J
3 C
i C,







9 •<

C .

( >




9 5
I 9
r *
3 -
»» ^
B «
s e












4




'
















a
•>






















r ^
s a

e >
3 C
J (.







f U

c •

c >




9 0
9 a
=> i
D f
* ^
B C
3 C





a






•












































r ^
s a

C >
3 C
J C.






«
s
(
(
H
< •




9 a
9 9
=* "^
t 9
« C
* ^
e «
3































iok

























r
>

e
i






B

9

•
•




9
1
S'
1
3
•
'









































I •
t— r*
3
o
t- n.
i- <
ooc
Z IU
U)
••
X Z >

IU Ul
U VI •
> - < a
99 U. IU
- Z l-
WIU » U
3 HI IU
K Ok a K
> (-> O
<« u
r o in a
 e
» 0> IU
u. z a
ZX iu tu
O — Z Ul
cj ^ 3 vt

•< «r T
*^ «^ stf
10S
-------
 specific ECHO 11 antisera that will identify the laboratory strain.

      The two untypable virus isolates and the virus contaminants
 were detected with the conventional tissue culture assay.  No
 viruses were detected in project samples using the immunoperoxidase
 assays or the rotavirus tests performed in other laboratories.  The
 immunoperoxidase assay did detect viruses in four of eight blind
 positive controls submitted along with tha other samples.  All
 positive controls contained concentrations of viruses greater than
 the theoretical detection limit.  In one case a polio virus seed
 was detected with BGM cells (150 MPN/mL) but was not detected with
 MA-104 cells.  The immunoperoxidase assay had been reported to be
 more sensitive than standard tissue culture tests for detecting
 enteroviruses from water samples (Payment and Trudel 1985).  It is
 not known why four of the positive controls were not detected.  In
 one case,  shipping delays may have been a factor.

     No rotavirus spiked samples were included with the samples for
 rotavirus assay.  The contract laboratory,  however,  did run
 positive controls with the assays.   One concentrate  from
 anaerobically digested sludge that was shown to contain indigenous
 enteroviruses was included with the samples  tested for
 rotaviruses;  no  rotaviruses  were detected in this sample either.

     Since most of the project samples  were  negative  for viruses,  it
 is  not possible  to  assess  the value of either  the immunoperoxidase
or  rotavirus  assays for  testing compost and  sludge samples.
Nevertheless,  considering  the overall  virus  assay protocol, only
two  low  level virus isolations  from the approximately  500  samples
collected during this study  is  essentially  insignificant.   No
detectable virus hazard  was  found  associated with composted sludge,
or  for that matter/ any  treated sludge tested during this  study.
                                106
-------
  Indicator/Pathogen  Correlations

      One goal of  this  study  was  to assess  the value of standard and
  non standard indicator  groups  for predicting the occurrence of
  pathogens or conversely, assuring the absence of pathogens.  Sinca
  no viable parasites  and only two virus  isolates were detected,
  examining the occurrence of  these agents relative to indicator
  populations was precluded.   Coiiphaga densities variad with
  coliforn populations suggesting  that phage, as measured in this
  study, would not  be  a good surrogate for antaric viruses in treated
  sludges.  The interrelationships of the  indicator groups themselves
 and their relationship  to the occurrence of the potentially
 pathogenic bacteria  can be analyzed.

      Tables 31 and 32 show correlation matrices for the grouped
 data from all of  the windrow composting site samples (IX-A-1
 through IX-A-6) and  the static pile samples (III-B-1, 2)
 respectively.  As might be expected, the strongest correlation
. occurred between  total  coliform and fecal coliform (0.974 and 0.910
 for the windrow and static pile sites,  respectively.  The fecal
 streotoccocci populations also correlated with the coliform groups
  (r > 0.75) .

      Due to the large number of samples, the critical r values were
 fairly low  fo.r  the correlations,  0.126  for the windrow  samples and
 0.205  for the static pile sites.   Therefore, at the 95% confidence
 level  a correlation was detected any time the test r value was
 greater than the critical value.   The correlation matrices show
 that many of the microbiological populations correlated with each
 other  but that  the strength of  the correlations were often weak.
 Overall,  population correlations  were stronger  at the windrow site
 than the static pile site.   This  may reflect the apparent regrowth
 phenomenon  observed with the windrow products where most
 populations  appeared to increase  simultaneously.

     The conforms and  fecal streptococci  were  the best indicators
 for  the occurrence of salmonella®.   Fecal  coliforms,  followed  by
 total  coliforms  and fecal  streptococci  produced the strongest
 correlations with  the windrow facility  samples.   At the static p.ile
 site,  s-almonellae  correlated best with  fecal streptococci,  fecal
 coliform and total colifons in  that  order.

    All of the  data from both facilities were grouped to  further
 examine the  relationship between  doliforms  and  fecal  streptococci
 versus  salmonellae.   Scatter  plots and  regression  analyses  of  these
data are  shown  in  Figures  23  through  25. Data points  below  the
salmonellae  detection limit  are shown on the scatter  plots  (cutoff
at -1 on  y axis) but  were  not included  in  calculation of  the
 regression line.   It  is  reasonable to assume that  these points
would have fallowed a similar, distribution  pattern  if the  test
orocedures could have measured  lower concentrations.
                                 107
-------
     Although  there  is  a  graat  deal  of  scatter  in  the  data,  there  is
 clearly  a  relationship between'indicator  concentration and
 salmonellae density.   Osing  the  derived regression equations,  it
 can be predicted  that  salmonellae populations  would be below the
 analytical detection limit  (<0.2 MPN/g) when indicator concen-
 trations were  240 M?N/g,  43  MPN/g and  73  MPN/g respectively
 for total  colifcria, fecal coliform, and fecal  streptococci.

     Currently  there are  no standards or guidelines that would
 designate an acceptable  level  of salmonellae.   Compost or sludge
 products containing no detectable salmonellae  when tested with
 acceptable analytical  techniques performed by  competent personnel
 would probably represent a conservative margin  of safety given
 current knowledge about  infective doses ana the ubiquity of
 salmonellae in the environment.

      Therefore, in addition  to the standard linear correlations of
 the quantitative data, the ability of  the coliforms and fecal
 streptococci to predict  the  occurrence of salmonellae in compost
 were examined from another approach.  The indicator bacterial data
 were divided into log increment  groups.  Indicator values with a
 log mantissa greater than .699 were rounded up to the next higher
 log increment; values with a log mantissa less than .699 were
 rounded down.   All of the data from both of the facilities were
 grouped in this manner.  Then the probability of salmonellae being
 detected in a  sample was determined for each group.   The
 probability of salmonellae being detected at each increment  of
 indicator bacteria was plotted and is  shown in Figures 26-28.   As
 can be seen,  the concentration of indicator bacteria  was very
 stongly related to the probability of  salmonellae being  detected.
 Linear regression lines were computed  for  these data  points  but the
 relationship  is actually  S shaped since the probability  limits  of 1
 and 0  cannot be exceeded.  A more sophisticated non linear analysis
 of  these  data  could  possibly be performed  and would likely indicate
 that  there  would be  some  finite probability of  detecting
 salmonellae when indicator desities  were  very low.  Nevertheless,
 it  was felt that  the linear  regression  extrapolated to the zero
 probability level  provided a  reasonable estimate of the  indicator
 concentrations  that  would be  assoicated with  a  very low probability
 of  salmonellae  being detected.   These values  were:  total  coliform
 240 MPN/g,  fecal  colifo«8 47  MPN/g and  fecal  streptococci  150 MPN/g,
 Examining the  data used to construct this  relationship revealed
 that no salmonellae  isolations  occurred when  the indicator
 densities were  below the  extrapolated levels.   Although  the  derived
 indicator concentrations  do  not guarantee  the complete absence  of
 salmonellae in  compost, they  would appear  to  provide a reasonably
 conservative level of assurance that salmonellae would not be
 detected using  the analytical procedures employed during this
 study.

    Interestingly, the  two approaches to analyzing the data
produced very similar results for the total and fecal  coliform
densities that would indicate no  detectable salmonellae.  The
predicted fecal streptococci  levels varied by a factor of  2 but
 that difference is not  great  considering the orders of magnitude
spread of data.

                                108
-------



M
'O
1
1
X


i
3
a
E
«•

<
I
X
VI
LLJ
H-
M
(/I

O
z


VI
O
a.
3
a
u
3
O

a

s
i

VI
2
2
U.I
4
s

a.

j
<
Ml
(J
a
o

a
a
Q.
(J

3
u.
a

X
i
3

i

^
j
IU
s


u
_
Cl

IU
a
h»
Z
O
3 n

«c
VI


z

V) V
IZ -
u

3
I
3 N
S —
LU
r
a
3
Ik —
1 -
K
0



tu
a
< 0



i_
j*
IU

t-
o

OTU
IU ft.
3 1
3 IE t0
« IU
> <
t <



u
a, a
1
OS
IU


a.
IU
et v

1
;l

Ik

J M
a
ik1


J N
O
"l
t-












0
0
a

—



a —
O r-
O es

- O

o r* nusnsi
O O ^ A ^ F*^ 43 09
o (O ^ 0
O *° O (O O ^ W (* 1*
O«'»es

U (S3
Ik Ife &, 9. 1- Z tU
x ~ IU 0 IZ 3 IZ Z
3 O t-= IIU l<9 l« «» 3
uiui iiuzoror-ti
^°lfcilfe ^^^CL^^^tfl


















•".
^"
or
i
M
Ml
•M


VI
IU
f_
Ml
I/I

J
~

(J

^
<
VI
I

o
as
Uj
_ S

o oe
0 <

a
n -;
a. <<
» »
e o
o
IQ Q
<~ fca
a
a o
— u
« -=
O 3
O Ik
n S
3.
X
f=

Z
IU

*-
O

W9 y
u a.
3 1
J 06 ®
4 IU
Z
t. <



(J
t Ifl
1
Qe
^ •

9>
IU
a v

1
ik

ik

acj "5
O
(J
U.

U_
!U M
O
U(
!=>












O
o
a

—



O 1-
O 3!
O O

- 3

O - O
a N -t
000

- 0 0

o N  n
Q O — T •»
— o o o o



o a « 09 r* —
O (O IO — - (N
o in c< N ui n
— QOOO O



o f a 100 n a
<3 (s a <»i33U)COQ^^>
Q «= ^ CS ^ eg ^ qg Q Q
0-g<0t8&<£ — <- rt(ffl
-ooas aooos


a*-^OYtor^oc«(0tfl
ad9r%^tAP^(O04C4ntn
-OQOQSQGOae




(?«e^'!*'ifi«F*.a — w-vra

(j j 3
u. u. a, a >- z u.
« - uy) CJ IZ 2 !Z Z
o o H- itu lo^iosS a
! i tiuzorazuj^
^»=lt.4.<
i.
j
^
u
j;
ce
u
109
-------
                                Figure  23

          Total  Colifcr^i vs Salmonella  - Sca~~ar
             . H- ....-*•.
                                .+.,..+.
                M > 1S3
                R *%458
                P < .001
SAIJ4OM
LOG  2
MPN/g
      1   *
              *....*. ...*.... + ... .•*•....•!•. ...>....*....*....>....>.......
                  .900      2.70      4. SO     5.30      3.10      9.30
             0.00      1.30      3.60      5.40      7.20     9.30

                              TOTAL COL I FORM
                                LOG MPN/g
                                   LINE —   — SMS--
                          1.3843 *.496S7*X    1.5733
                                    110
-------
                              Figure  24

          'ecal CoHform. vs Salmonella  - Scatter
                M > 163
                R =• .463
                P < .001
      3   •*•
SALMON
LOG  2
HPN/g
.*. OOO*. ...*....*. 0. .*....*..,.*.. ..*... „*....*...«+
     olOO      2.70      4oSO      §=,30      8.10
0.00      1.80     3«SO      3,40      7.20      9.
                                                               9.90
                                   COLI?ORM
                               LOS HPM/9

                       «-RS68SSS10M LINE--   — SMS—
                                            1.S633
                                   111
-------
                             Figure  25
       Fecal  Streptococci vs Salmonella -  Scatter Plot
                                            .*....•*•.
               H * 1S3
               R =• .497
               P < .001
SALMON
LOG  2
MPN/g
            •»'^*t»««^»«« • * * • • o^*»*»»**»«**^» •• • ^ • • • • ^" • « • » T* a » • • T* » • • «^« • » • ^* t •
                 .30       2.4       4.0       5.5        7.2       8.3
            0.0      l.S       3.2       4.3       S.4       8.0

                           fECAL Sra£2TOCOCOJS
                               LOG  MPN/g

                                 LINE—   ~sns—
                               +.50871*X    1.4989
                                  112
-------
lAIHSOd dQ JU.I1
       113
-------
LU
                                      114
-------
115
-------
      Yersinia  populations  did  not  corralats  as  well  as  salmonellae
 did with  the standard  indicators.  Although  a statistically
 significant correlation  did  exist  between Yersinia occurrences and
 all other groups,  the  strengths of the correlations  were  relatively
 weak.  Only scattered  isolations of Yersinia occurred at  the windrow
 sites so  the correlation coefficients are of little  significance,
 Substantial nuabsrs of Yjarsin-La wsra dstactad at  the static oils
 sites but no strong correlations developed; Yersinia populations
 correlated best with the total enteric plate count.  The  relatively
 poor correlations  are probably the result of the  seasonality of the
 Yersinia data which was  not  generally reflected with the  other
 organisms.

      In summary, there was a strong relationship  between  the
 densities of standard fecal  indicator bacteria and the occurrence
 of salmonellae implying  that the indicator bacteria may have
 value for process control monitoring.   The actual value of
 bacterial monitoring may be questionable if salmonellae are capable
 of repopulating under certain conditions following the composting
 process.   In this case point of sampling would be critical to
 assessing the quality of treated sludges utilized in 0 & M
 programs.   Monitoring would need to be as close to the user as
 possible  and include amended products  where applicable.

      As mentioned in the earlier dicussion about salmonellae,  a
 voluntary  coliform monitoring program  was instituted at  the windrow
 facility.   In  addition to meeting minimum recommended time and
 temperature  requirements (40 CFR 257.4),  compost is  not  released
 to the commercial producer  until total  coliforms have been reduced
 to a  median  of  10 MPN/gdw,  based on previously  published
 recommendations (Haug  1930}.   Continued  monitoring of the
 commercial products (unpublished data)  indicates that the  levels  of
 salmonellae  have been  substantially reduced  in  the bagged  products
 since instituting the  field monitoring program.

    It is  not entirely  clear  why  salmonellae  levels  declined  in the
 bagged products after  instituting  the coliform monitoring.  Few
 salmonellae  were detected in  the  final compost  (IX-A-1 & IX-A-2)
 prior to initiating the coliform monitoring program.  Achieving the
 low coliform level  sometimes  requires longer  composting  time  than
 dictated by  time/temperature  requirements  alone.   It  may be that
 the low coliform level  reflects a degree of stabilization  that
 limits the potential for  salmonellae regrowth.   If this  is the
 case, it is possible that other measures of stabilization  may also
 suffice.   It should also  be noted that a small number of windrows
 never met  the 10 MPN/g median coliform level  even  with extended •
 composting time  (additional 3 to 4  weeks).  These  windrows  were
 tested for salmonellae and  released if negative.   Although  this
study indentified a correlation between bacteria indicator
concentrations and  salmonellae levels, the critical factors
determining whether or not  salmonellae repopulation can occur have
                                116
-------
not been identified.  Bacterial monitoring may indicate the
potential for salmonellae being present and may also serve as an
indicator of regrowth potential, but better knowledge of the
mechanism involved with the survival and regrowth of enteric
bacteria in'compost is the key to effectively controlling this
phenomenon.
                               117
-------
 MICROBIOLOGICAL RESULTS - BIMONTHLY SITES

      The final sludge product produced at twenty-four treatment
 facilities was sampled every other month for one year.  The
 sampling sites were described in detail in Section 4.  Faciiitias
 sampled were well distributed around the continental U.S.  and
 included composting facilities, drying bed operations and heat
 creacment processes.  The treacment plants and basic processes were
 summarized in Tables 2 and 4.

      The basic process groupings represent generalized
 descriptions.  Other treatment variables combined with climatic
 variations resulted in not having any of the sampling sites
 represent truly identical treatment processes.


 Indicator Organisms

      Individual data for each of the twenty-four  facilities is
 tabulated in Appendix C.   Table 33  lists the mean values  for  the
 indicator groups  at each site.   It  was  not feasible to compare
 sites individually because with twenty-four  sites there were  276
 pairwise comparisons possible.   No  single  site  was  highest  or
 lowest  for  all organisms.   The  lowest mean coliform densities were
 measured at III-J-1,  an  aerated windrow site.   Site IX-D-1, a
 drying  bed  facility had  the  lowest  fecal steptococci  counts.   Site
 IV-I-1,  the heat  drying  site, had the lowest aerobic  and  anaerobic
 plate counts,  total enteric  plate count and  fungal  counts.  The
 lowest  coliphage  densities occurred jointly  at  IV-I-1 and IX-D-3.
 The highest microorganism  levels occurred  at two  sites.   The
 greatest mean  densities  of fecal streptococci and aerobic and
 anaerobic plate count  bacteria  were measured at 7-K-l,  the
 thermally conditioned  sludge.   Site IV-B-1,  a static  pile
 composting  facility, produced the highest  concentrations  of
 California,  total  enterics, fungi and coliphage.

      After  tabulating  the  geometric means  for each  sampling site,
 the-data were  grouped  by treatment  process.  The  means  for products
 produced  by  each are shown in Table 34.  No  one process was lowest
 or highest  for  all  of  the  microorganism groups tested.  In order to
provide  some overall rating, the nine processes were  ranked for
microorganism  density  for  each  test, with  1  representing  the  lowest
density of microorganisms  and 9  being the highest.  These rankings
are listed  in  Table 35.  The position numbers were  then averaged
 for each process.  The numbers represent only the average number of
times a process yielded the lowest,  highest or intermediate
concentration of the indicator organisms listed in  Table  34.   In
this manner, the processes were  arranged and are  listed in Table 36
from lowest to highest combined  concentration of organisms.
                                118
-------
I
a
w
<

&
i
Irt

5
Z
IU
U

o
u
IU
a
a i
•» 1 1 01
3 - a
'A. -1 it
a. or
i ^
Z -
a a
I "5
w
»— u.

< 3
fr- Z
O«
^
i- ik
u
-J ae
< IU
Ov
£
O »-. u
*<•>
U
! «i
§*° t)

as us
x •*
e,
tu k=

< 3
a 3
-1 O,

u ae
U WS
S5
a o
-. j a,
z « -=
a wo
as w
3
~* a
§

Ml

o r* 33 v r*
 <•> ca to
— 7 a 10 01
- - o - a
t ™ ct a  id <3
ci a f »
T " ": ";

b r* i a
« r»* c^ f*
O* <%i CM C3
10 « — a
•T (*5 ^ ^f
* CH "" 9
N « 
i/i a z
l/f O M Q
tu •» a
> a t-
1 Z 4
Z Z IU IU
- 4 4 Z

(N
d
'"!
o
31
10
a
w
a
a
-'




in
a
C4
a






^
*•
a

a




a



n
en
o

r»






X
^
^
IU

z
a
0
z
a.












a
IU
z
z

i
*
a
IU
a,
a
X
I/I
a

M
z
4
i
§
4

a
s
it.
a
z
IU
u
z
O
U
z
4
IU
U
X
H-
IU

a
IU
0
n

HI

a















i
t

































a
z






































                                                                                 o  r
                                                                                 u  a.
                                                                                               i3
                                                                                               u z
                                                                                 -j —
                                                                                 < a
                                                                                 t- z
                                                                                 o ^
                                                                                 t- a.
                                                                                 J X
                                                                                 4 IU
                                                                                              O Z
                                                                                              I- IU
a
Ziu

4 4
Z -J
4 a.
                                                                                JO
                                                                                a,u
                                                                                JO,

                                                                                4 IU
                                                                                U «
                                                                                IUK
                                                                                H. VI
   3
   z
   O
-J H.

U -I
iua
u. u
                                                                                             oo
                                                                                             h-U
                                                                                IU
                                                                                u
                                                                                a
                                                                                s
                                                                                a
                                                                                                                                —    en    •
                                                                                                                         Z
                                                                                                                         a
                                                                                                                    >
                                                                                                                    z    z
                                                                                                                    a    -

                                                                                                                    Of
                                                                                                              u    —     I
                                                                                                              IU    4
                                                                                                                                                  a
                                                                                                                                                  z    >
                                                                                                                                                  Q    z
                                                                                                                                                  U    4
                                                                                                                                       a   z    -i    w
                                                                                                                                       —   Q<    —
U    irt    (3         _     .
•»    iu    •»    a          a    z
t-    >    Q         t-    z    a.
41          X    4    IU    O
>-    Z    Z    iu    IU    I    Z
                                                                              120
-------
 Table  36.  Processes Ranked From Lowest to Highest Indicator
            Microorganism Density
          Process                                    Index
    Heat Drying                                       1.6

    Aerated Windrow Composting                        3.1

    Anaerobic Digestion - Air drying                  4.0

    Windrow Composting                                4.2

    In-vessel Composting                              4.8

    Proprietary Composting                            Sol

    Thermal Conditioning (Dewatered)                  7.0

    Aerobic Digestion - Air drying                    7,3

    Static Pile Composting                            7.9

     The products were ranked by process for illustrative purposes
only.   The significance of these rankings is unclear.   The natural
assumption would be that lower concentrations of indicator and
background raieroogansisms would be desirable.  As previously
mentioned, Hussong et al (1985) suggested the opposite  was true  and
that high levels of ""Indigenous microflora,  including  califorms,
would  suppress salmonellae;  however,  results from this  study appear
to  differ  from those findings.
     Perhaps  the  most  surprising  aspect  of  these  data was  how  well
the anaerobically digested,  air dried  sludges  ranked compared  to
the composted sludges.   Only the  heat  dried sludge- and  aerated
windrow' facility  samples eentained  lower  overall  concentrations of
indicator organisms.

    On  the other  hand*  the  relatively  poor  ranking  of the  static
pile products should not be  viewed  as  an  indictment of  static  pile
systems, per  St.   The  composts included  in  the static pile ranking
ranged  from products that centained very  low densities  of
miroorganissns (IXI-B~3)  to those  whese products contained  among the
highest  concentrations  of microorganisms  (IV-=B-1  and IX-B-1),  The
full range of data from th@s@ sites can be  examined by  looking at
all "B"  designated sites in  Table 33 and  Appendix C.  Investigation
of the  reasons for these differences may  provide  a  better
understanding of  operation variables.
                                121
-------
      The  fungi  isolated  at  the  bimonthly sitas were similar to
 those detected  at  the weekly  sites.  The only fungus detected of
 any potential health significance was Aspergjllus fumigatus.  The
 average concentrations of A.  fumigatus at eacn site are shown in
 Table 37  and grouped by  process  in Table 38.  The static pile
 composts  contained ths grea'esst  concentrations of A. fuatigatu3_.
 Table 37
Occurrence of Aspergillus fumigatus at 3i-Monthly
Sampling Sites (Geometric Means)
Site
%
Positive
I-B-1
II-C-1
iii-a-3
III-B-4
III-J-1
IV-B-1
IV-0-1
IV-P-1
83
17
50
57
33
100 1,
33
33
Mean
CFU/g
470
5
21
140
6
300,000
330
S
Site
%
Positive
I7-I-1
7-B-l
7-8-1
7T-D-1
VII-D-1
VI I -A- 2
7III-D-1
7TII-P-1
33
100 10
17
100 2
50
•33
33
33
Mean
CFU/g
3
,000
2
,000
250
230
410
10
Site %
Positive
VIII-H-1 83
7III-J-1 67
IX-A-10 50
1X-B-1 100
IX-D-1 17
IX-0-2 50
IX-0-3 83
X-C-1 50
Mean
CFU/g
1500
120
15
380,000
4
10
15,000
40
Table  38      Occurrence of Aspergillus fumigatus Grouped by
              Treatment Process   .          "
Process
                                          CFU/g
           Geometric Mean
Range
Heat Treated

Aerobic  Dig/DB

In-Vessel Composting

Aerated  Windrow

Windrow

Anaerobic Dig/DB

Prop. Compost

Static Pile
                       3

                       9

                     14

                     27

                     65

                    270

                   1500

                   4400
  - 40

  - 2,500

  - 22,000

  - 4,700

  - 29,000

  - 32,000

  - 21,000

  - 18,000,000
                                122
-------
 Pathogens

      Salmonellae and  Yersinia were  randomly isolated from a
 relatively small number of  samples  from the bimonthly sites.  A
 total of 144 samples  (6 from each site) were collected for this
 part of the project.  Salmoneliae were detected in 27 samples and
 I'ersinia were isolated in 15,  Samples containing these bacteria
 are iisced in Tables  39 and 40.
 Table 39.
Salmonella Positive Samples
Bimonthly Sites
Samples Containing
Salmonellae
II
III
IV
IV
IV
V
VIII
VIII
IX
IX
IX
IX
X
- c -
- B -
- B -
- p «
*y T «•>
- K -
- D -
- H -
- a -
- D -
«=> T^ •=&
- .D -
f°9
*=' W>
1
4
1
1
1
1
1
1
1
1
2
3
1
No. of Positive Concent rat ion(s)
Samples (N=6) MPN/g
1
1
3
2
1
5
4
1
1
1
2
4
1
810
0.8
0.8; 170; 370,000
71; 2,200
0.4
3.1; 3.8; 18; 24; 110
1; 2; 64; 390
1
0.6
140
0.1; 500
0.1; 2; 17; 48
0.2
     Thirteen  of  the twenty-four  facilities  that were sampled
bimonthly had  one ot more  samples  with detectable salmonellae,
Salmonellae were  isolated  from 50% or more of  the samples at four
of the sites»   Two of these  sites  were drying  bed plants, one was a
thermal conditioning process and  one was a static pile composting
operation.  Salmonella®  densities  in these samples ranged from Q.I
to 370,000 MPN/g,   The data  indicate that the  occurrence of
salmonellae observed at  the  weekly sites did not necessarily
represent isolated situations.  Detectable levels of salmonellae
occurred in roughly 20%  of the  bimonthly samples but in most cases
the densities  were low.
                                123
-------
 Table 40.
Yersinia Positive Samples
Bimonthly Sites
 Samples Containing
     Yersinia
             Mo. of Positive
               Samples(N=6)
   Concentrations)
        MFN/g
-r
III
IV
IV
V
V
VII
VIII
VIII
IX
X
- a -
- a -
- a -
- i -
- a -
- K -
- A -
- D -
- F -
- B -
- C -
1
4
1
1
1
1
2
1
1
1
1
2
1
2
1
1
1
1
2
I
2
1
0.2; 0.5
0.4
1; 4200
0?1
100
1
2.7
0.5; 3,9
1
0.4; 0.6
52
          Yersinia positive samples mostly contained  very  low
concentrations of yersiniae.   in all  but  one  case/ the  Yersinia
isolations  occurred during the period from December  through May,
providing further evidence for the seasonal occurrence  of Yersinia
as hypothesized earlier.

     As previously described ,  many of the Yersinia isolates were
sent to the New York State Department of  Health  for  detailed
identification.   Results  of these tests are shown in Table 41.
Table 41   Identification of Yersinia  Isolates from the Bi-Monthly
           Sites
SITE Sample
Date
1-3-1 ' 8-18-86
12-22-36
iri-B-4 2-23-87
IV-B-1 2-09-87
IV-I-1 2-25-87
V-B-1 2-9-87
V-K-1 12-8-86
VIII-D-1 5-5-86
IX-B-1 1-26-87
X-C-1 3-9-87
Isolate
NO.
1
1
2
1 .
1
1
1-4
1
1
2
1
1-5
6-14
(a) NG - Non Groupable
(b) NT - Not Tested
Identification Serb-type Pathogenicity
Test
Y. enterocolitica
Y. enterocolitica
Y. frederiksenii
Y» frederiksenii
Y . enterocolitica
Y. intermedia
Y. enterocolitica
Y. intermedia
Y .enterocolitica
Y. frederiksenii
Y. frederiksenii
Y. kristensenii
Y. kristensenii
0 : 8 Neg
0:3, 14 Neg
0:29 NT(b)
Os 16, 29 Neg
NG{a) Neg
0 : 12 Neg
NG Neg { c )
0:4, 16 Neg
NG Neg
NG NT
0:16, 29 Neg
0:16, 29 Neg(d)
NG NT
(c) 1 of 4 isolates tested
(d) 1 of 5 isolates tested
                                124
-------
      A  number  of  the  Yersinia  isolates  were also  tested  for
 pathogen!city.  The 0:8  serotype  detected  at  site I-B-1  is the most
 common  pathogenic sertoype  occurring  in the U.S.; however/ none of
 the  isolates tested,  including the  0:8  serotype were positive in
 the  pathogenicity tests.  Environmental yersiniae isolates
 generally have not been  found  to  test positive in pathogenicity
 tests regardless  of their serogroups- (M. Shayegani, personal
 communication).   The  factors mediating  pathogenicity in  the
 Yersinia ara not  thoroughly understood.  It is not known if
 potentially pathogenic Yersinia,  such as che  0:8  serotype can
 revert  to the pathogenic form.

      Toxigenic E.  coli were detected in four  samples, one each
 from III-B-4, IV-D-1, IV-P-1 and  IX-B-1.   The previous discussion
 about toxigenic E. coli colonies  suggests  the densities of
 toxigenic strains  may be higher than indicated by the data shown in
 the tables in Appendix C.

      As at the weekly sites, no Campylobacter were isolated.
' Available evidence strongly suggests that  campylobacters would not
 survive in either  composting systems or drying beds.

      Intact helminth ova were  detected  in  at least one or more
 samples from every facility except IX-D-2.   The predominant ova
 detected were Tricfauris, Asearls and Toacocara. .  One potentially
 viable  Trichuris ovum was observed in the May 12,  1986 sample from
 site VIII-P-1.   The ovum contained.a fully developed  embryo.   The
 embryo  was  not  observed to move within the ovum and movement  could
 not be  induced  with intense light from the microscope illuminator.

      The sludge treatment process employed at site VIII-P-1 was
 aerobic digestion followed by air drying and storage.   It is
 possible that conditions during aerobic digestion  were suitable for
 embryonation to occur  but that the embryo died during the ensuing
 two year period of drying and storage.   Embryo movement would have
 confirmed viability but since no movement could be induced?  the
 co-ndition of the  developed ovum was  uncertain.

      No  other detected ova showed any signs of development  or
 indications  of  viability after incubation.   Most of the Trichuris
 ©va detected from the  bimonthly samples  were also  outside the size
 range for Trichuria trieniura again  suggesting non-human orgin.
Also, as was the  case  for the weekly sites, no protozoan cysts  were
observed in  any of the bimonthly samples.

      The previous  discussion about viruses  is  applicable to the
bimonthly sites.   A polio 1  virus  was  isolated from one sample
 (III-J-1, 5-29-86) but it was  almost assuredly a laboratory
contaminanto  Enterie  viruses  were not  detected  in any  other
oimonthly sample..   Givtn  available analytical  technology,  there
would appear  to be little if any concern associated with  enteric
viruses  in properly treated  sludges,
                                 125
-------
     The  small  number  of samples  from each  site  and  large  variation
 in  the  data  preclude meaningful statistical analysis of  data  from
 the bimonthly sites.  The results did reveal that most of  the
 sludge  products examined contained few detectable enteric  pathogens
 but that  bacterial  pathogens  may  sometimes  be  a  concern.

     The  data also  suggest  that anaerobic digestion  and  air drying
 may be  an acceptable low cost sludge  treatment process for
 eliminating  microbiological health hazards.  Digestion and drying
 parameters and  storage  time need  to be  clearly defined to qualify
 air drying as an acceptable process.  Although microbial levels in
 dried sludges compared  favorably  with composted  sludges  examined
 during  this  study,  the  dried  sludges  have not  received the
 additional stabilization that occurs  during  composting.  The
potential for bacterial regrowth  in remoistened  air  dried sludges
 is unknown.
                                126
-------
                               REFERENCES


  1.   Adams, M. H. 1953. Bacteriophages.   Intsrscianca, Sew York, M.I,

  2.   Akin, S. W., W. Jakubowski, J. B. Lucas, and H. R. Pahran 1977.
      Health hazards associated with wastewater effluents and sludge.
      In:  Proceedings of the Conference  on Risk Assessment and
      Health Effects of Land Application  of Municipal Wastewater
      Sludges (3. P. Sagik and C. A. Sorber, eds.) pp. 9-25,  The
      University of Texas at San Antonio, San Antonio, TX.

.  3.   American Public Health Association, American Water Works Assoc.
      Water Pollution Control Federation.  198S. Standard Methods
      for Examination of Water and Wastewater, 16th ed. American
      Public Health Association, Washington, D.C.

  4c   American Public Health Association. 1970.   Control of
      Comnunicable Diseases in Man,  llth  ed.  (A°  S.  Benenson,
      Ed.)/ A.P.H.A., New York*

  5.   Anjello,  L.  1958.   Soil as a natural reservoir  for human
      pathogenic fungi.   Science 123s876-°879.

  6.   Barnett,  H.  L.  and B.  B.  Hunter.  1972.  Illustrated Genera
      of  Imperfect  Fungi,  third edition.   Burgess  Publishing  Company,
      Minneapolis,  Minnesota, pp.  1-239.
           i
  7.   Sercovier,  H.,  J.  Braulfe,  S. Cohen,  R. Melis, T.  Lambert,  and
      H.  H.  Mollaret.   1984c   A new  isolation  medium  far the
      recovery  of Yersinia  enterocolitica from environmental  sources.
      Current Microbiology,  Vol.  10,  pp.  121-124.

  8.   Bergey's  Manual  of Systematic  Bacteriology,  Vol.  1. 1984.  N. R.
      Krieg,  ed., Williams  and  Wilkins, Baltimore.

  9=   Beuehat,  L. R.  1985=   Efficacy  of media  and  methods for
      detecting  and enumerating  Campylobaeter  jejuni  in refrigerated
      chicken meat. Appl. Environ. Microbiol 50;934~939.

10.   Blaser, M. J,f  I.  D. Berkowitz, F.  M.  LaForce,  J.  Cravens,
      L. B. Heller, W. Wang.  1979. Campylobaeter
      enteritiss  clinical and  epidemiologic features.   Ann.  Intern.
     Med.  91:179-85.
                                 127
-------
 11.  Blaser, M.  J., H.  L.  Hardesty,  B.  Powers, W.  L. Wang  1980a.
      Survival of Campylobacter  jejuni  in  biological iailieus.
      Journal of  Clinical Microbiology  !!_: 309-313.

 12.  Blaser, M.  J., P.  M.  LaForce, N.  A.  Wilson, W. L. Wang. 1980b.
      Reservoirs  for human  caaspyiobactariosis.  Journal of  Infectious
      Disease.  141.:665-669.

 13.  Blaser/ M.  J. and  L.  B. Heller. 1981.  Campylobactar  enteritis.
      New England Journal of Medicine,  305:1444-1451.

 14.  Boik/ R. J. 1979.  Interactions, partial interactions and
      interaction contrasts in the analysis of variance.
      Psychological Bulletin, 86;1084-1089.

 15.  Brandon, J. R. 1978.  Parasites in sludge/soil systems.
      Sandia Laboratories, Albuquerque, N.M., Available from NTIS,
      Springfield, 7A.

 16.  Brandon, J. R.,  W.  D.  Surge, and N. E. Enkiri. 1977.
      Inactivation by ionizing radiation of Salmonella enteritidis
      serotype montevideo growth in composted sewage sludge.Appl.
      Environ. Microbiol.  33.: 1011-1012.

 17.  Brown,  Harold W.  1975. Basic clinical parasitology,  fourth
      edition.  (0. L.  Belding,  ed.)  Appleton-Century-Crofts.
      New  York.   pp. 112-116.

 18.   Burrows,  W.  1968. Textbook of Microbiology - The  Pathogenic
      Microorganisms,   p. 708.  W.B.  Saunders Co., Philadelphia.

 19.   Craun,  G.  P. 1979.   Waterborne  disease - A status  report
      emphasizing outbreaks  in groundwater  systems.   Groundwater
      r7:183.

 20.   D'Aoust, J.  Y. and  H.  Pivnick.  1976.   Small  infectious
      doses of Salmonella.   Lancet,  1, 366.

 21.   Doyle,  Michael, P., and  0.  J. Roman.  1982.   Recovery of
      Caaroylobacter  coll  from  inoculated foods  by  selective
      enrichment.   Appl.  Environ.  Microbiol.   4_3; 1343-1353.

22.   Doyle, Michael, P., and  D.  J. Roman.   1982.  Sensitivity of
      Campylobacter  jejuni to  drying.  Journal  of  Pood Protection.
      45_: 507-510.

23.   Dudley, D. J., M. N. Guentzel, M.  J.  Ibarra, B. S. Moore and
     B.  P. Sagik.  1980.  Enumeration  of  potentially pathogenic
     bacteria from  sewage sludges.  Appl.  Environ.  Microbiol.
      39:118-126.
                                 128
-------
 24.  SPA. 1978. Microbiological Methods for Monitoring the
      Environment - Water and Wastes. S. Bordner and J. Winter,
      eds.  EPA-600/8-78-017.

 25.  Farrell, J. B. 1986.  Risk of infectious disease from use of
      sludge on land and methods to reduce these risks. (PB86-156378)
      Available from NT IS., Springfield, VA 22161

 25.  Feachem, Richard G., D, J. Bradlay, H. Garelick, and D. D. Mara
      1983.  Sanitation and Disease:  Health Aspects of Excreta and
      Wastewater Management.  World Bank Studies in Water Supply
      and Sanitation 3. John Wiley and Sons, New York, N.Y.

 27.  Geldreich, E. 1972.  Waterborne pathogens.  In:  Water
      Pollution Microbiology (R. Mitchell ed.) John Wiley & Sons,
      Inc.  New York.  pp. 207-241.

 28.  Gerba,  C. P.  1983.  Pathogens,  In:   Utilization of Municipal
      Wastewater and Sludge on Land (A.  L.  Page, T. L. Gleason,
      J.  E. Smith,  I.  K.  Iskander,  and L. E. Sommers,  eds.)'
      pp.  147-187,  University of California, Riverside, CA.

 29 o   Glass,  J. S.,  R.  J. Van Sluis  and W.  A.  Yanko. 1978.   Practical
      method  for detesting poliovirus in anaerobic  digester sludge.
      Appl. and Environ.  Microbiol.  35:983-985.

 30.   Gleit,  A. 1985,   Estimation for small normal  data sets  with
      detection limits.   Environ. Sci.  Technol.  19:1201-1206.

 31.   Gcldsehraidt ,  M. Co  and DuPont,  H.  L.  1976.  Snteropathogenic
      Sscheriehia eolit   Lack of correlation of  serotype  with
      pathogenicitye  The Journal of  Infectious  Diseases.
      133(2) ; 153-156
                                                           •
 32.   Goldstein, N,  1983.   Marketing  composted sludge.  Bioeycle,
      Nov-Dee,,  p.  29-31.

 33 .   Goyal,  S.  M. s  S»  A.  Schaub, F.  M.  Well ings, D. Berman,  J.  S.
      Glass,  C<=  J. Hurst,  D.  A.  Brashear, C. A.  Sorber, B.  E.
      Moore,  G«  Bitton? P.  H.  Gibbs,  and S.  R. Farrah.  1984.   Round
      robin investigation of  methods  for  recovering human enteric
      viruses  from sludge.  Aopl. and Environ.  Microbiol*
34.  Haug, R.R. 1980. Compost Engineering, Principles and Practice.
     Ann Arbor Science Publisher Co., Inc., Ann Arbor, MI.

35.  Hay, J. C. 1986 .  Compost Salmonellae Study, Phase I, II, and
     III.  Agency Report:  County Sanitation Districts of Los Angeles
     County, Carson,
                                 129
-------
 36.  Hays,  3.  D.  1977.   Potential  for  parasitic  disease  transmission
      with  land application  cf  sawage plant  effluents  and sludges.
      Watar  Research.   11_: 583-595.

 37.  Head,  Carol  3., Deborah A. Whitty, and Samuai Ratnam.  1982.
      Comparative  study of selective media for  recovery of Yersinia
      anterocQliclea.  Journal  of Clinical Microbiology.  Vol. 16,
      NO . 4. o.
 38.  Heisick, Judy. 1985.  Comparison of enrichment broths for
      isolation of Campylobacrer jejuni.  Acpliad and Environmental
      Microbiology.  5£:1313-1314.

 39.  Hunt, J. M., D. W. Francis, J. T. Peeler, and J. Lovett. 1985.
      Comparison of methods for isolating Campylobacter jejuni from
      raw milk.  Applied and Environmental Microbiology.  SO;535-536»

 40.  Hussong, David, W. 0. Surge, and N. K. Enkiri.  1985.
      Occurrence, growth, and suppression of salmonellae in composted
      sewage sludge.  Aoplied and Environmental Microbiology.
      50(4):887-893.
 41.   Kaneshiro, Edna S., and Gerald Stern. 1985.  Survival of
      parasite eggs in stored sludge. (PBS6-137143).   Available
      from OTIS, Springfield, VA 22161.

 42.   Kott,  Y. 1966.   Estimation of low numbers of Sscherichia cpli
      bacteriophage by use of the most probable number method.
      Appl.  Microbiol. 14(2);141-144.

 43.   Kowal,  N.  E.  1983.   An overview of public health effects.   In:
      Utilization of  Municipal Wastswater and Sludge  on Land (A.  L.
      Page,  T. L. Gleason, J. E. Smith,  I.  K.  Iskander,  and
      L.  E.  Sommers,  ed.), pp. 329-394,  University of California,
      Riverside, CA.

 44.   Langeland, Gunnar.  1983.  Yersinia enterocolitica and Yersinia
      encerocolitica-like bacteria in drinking water  and sewage
      sludge.   Acta path,  microbiol.  immunol.  scand.  91.-179-185.

 45.   Lennette,  Edwin  a.,  S.  a.  Spaulding,  J.  P.  Truant.  1974.  Manual
      of  Clinical Microbiology,  Second Edition.   American Society
      for  Microbiology, Washington,  O.C.  pp.  291-294.

 46.   Levine,  N.D.  1980.   Nematode  Parasites  of  Domestic Animals
      and  of Man.   Burgess Publishing Company.  Minneapolis, MN.

47.   Lim, K.  A.  and Benyesh-Melnick,  M.  1969.  Typing  of viruses by
      combinations  of  antiserum  pools.   Application to  typing  of
      enteroviruses (coxsackie and ECHO).   J.  Immunology.  84.309-317.

48.   Lipson,  A.  1976.   Infecting dose  of  Salmonella.   Lancet, 1_:969.
                                 110
-------
 49.   Mentznig, L. 0.  1981.  Waterborne outbreak of Campylobacter
      enteritis in central Sweden.  Lancet.   ii;352-254.

 50.   Metro.  1983.  Metro sludge quality monitoring report and
      literature review.  Metro Water Quality Division.  Seattle, WA.

 51.   Miller,  R. G. 1S81.  Simultaneous statistical inference.
      Springer-Veriag, New York.

 52.   Milner,  P. D.,  P. 3. Marsh, R.  3.  Sr.owden and J. P. Parr.
      1977.   Occurrence of Aspergillus fuaiqatus during composting
      of  sewage sludge.  Appl.  Environ.  Microbiol. 34;765-772.

 53.   O'Donnell? Cathlene J.,  K. B.  Meyer, J.  V. Jones', T.  Benton,
      E.  S.  Kaneshiro, J. S. Nichols, and P.  W.  Schaefer  III.   1984.
      Survival of parasite eggs upon  storage  in  sludge.  Applied  and
      Environmental Microbiology.  48;618-625.

-54.   Oosterora, J., M. J. G. M. Vereijken, G.  B. Engels.  1981.
      Campylobacter isolation.  The Veterinary  Quarterly.  3_: 104

 55.   Ottolenghi,  Abramo C., and Vincent V. Hamparian.   1987.
      Multiyear study of sludge application te farmlands   prevalence
      of  bacterial  enteric pathogens  and antibody status  of  farm
      families.  Applied and Environmental Microbiology.
      53(5);1118-1124.

 56.   Page,  A.  L.,  T.  L.   Gleason, J.  E.  Smith,  I.  K.  Iskandar  and
      L.  E.  Sonnners.   1983.   Proceedings of the  1983  workshop on
      utilization of  municipal  wastewater and  sludge  on land,
      University of California,  Riverside, CA.

57.   Payment,  Pierre, and Michel Trudel.  1985.   Immunoperoxidase
      method with human immune  serum  globulin  for broad-spectrum
      detection of  cultivable human enteric viruses;  application to
      enumeration of  cultivable viruses in environmental  samples«
      Applied  and Environmental  Microbiology.  5£U,)sl3Q8.

58-.   Raper, K.  B.  and D.  I. Fennel.  1965.  The  Genus Aapergillus,
      Robert E.  Krieger Publishing Co.,  Malabar,  FL., pp.  1-686.

59.   Reimers,  R. S.,  M.  D.  Little,  A.  J. Snglande,  0. B.
      Leftwieh,  D.  D,  Bowman„ and R.  ?.  Wilkinson.  1981o  Parasites
      in  southern sludges  and disinfection by standard  sludge
      treatment.  Tulane  Onivarsity,  New  Orleans,  LA.   Available  from
      NTIS,  Springfield,  7A.

60e  Rogol,  M., B. Shpak, D. Rofehman,  and I. Sechtsr. '1985.  Enrich-
     ment medium for  iselation  of Camgylobacter  jejuni -  Campylobacter
     coli.  Applied.   Environ. Microbiol. 5_p_;125~126.
                                 131
-------
 SI.  Rosef, Olav and George  Kapperad.  1933.  House  flies  (Musca
      domestica) as possible  vectors  of Campylobactar  fetus  subsp.
      jejuni."Applied and  Environmental Microbiology."  4_5_: 331-383.

 62.  Rothenberg, P. J., N. J. Stern, and D. C. Westhoff.  1984.
      Selected environmental  broths for recovery of  Campy 1 o_bacter
      jejuni from foods.   Appl. and Snviron. Microbioi. 48;78-30.

 63.  Russ, C. P. and W. A. Yanko.  1981.  Factors affecting
      salmonellae repopulation in composted sludges.  Appl. Snviron.
      Microbioi.  41:597-602.

 64.  Sack, R. B. 1975a.   Human diarrheal disease caused by
      enterotoxigenic Sscherichia cpli.  Ann. Rev. Microbioi
      2£:333.

 65.  Schiemann, D.  A. 1982.  Development of a two-step enrichment
      for  recovery of Yersinia enterocolitica from food.  Vol 43,
      No.  1., p. 14-27.

 66.  Seppf E.  1980.   Pathogen survival in sludge stabilization
      processes.  California Department of Health Services, Sanitary
      Engineering Section,  Berkeley,  Ca.

 67.  Shayegani,  Mehdi,  W.  3.  Stone,  I.   DeForge,  T.  Root,  L. M.
      Parsons,  and P.  Maupin.   1986.   Yersinia entarocolitica and
      related species  isolated from wildlife in New York State.
      Applied and Environmental Microbiology.   52(3);42Q-424.

 63.   Smith,  E.  M. and C. P. Gerba.   1982.   Development of  a  method
      for  detection of human rotavirus in  water  and sewage.  Appl.
      Environ.  Microbioi.   43;1440-1450.

 69.   Sorber, C.A. and 3.S. Moore  1986.  Survival  and  transport of
      pathogens  in sludge-amended  soil:  a  critical  literature review.
      Available  from NTIS,  Springfield,  7A.   Cooperative Agreement
      No.  CR811918-01-0.

 70.   Swaminathan, B./ M. C. Harmon and  I. J. Mehlman.   1982.  A
      review- Yerainia enterocolitica.   Journal of  Applied
      Bacteriology, 52,: 151-183.

71.   Vogt, R. L., H.  E. Sours, T.  Barret, R. A. Peldman,  R. J.
      Dickinson,  and L. Witherell.  1982.  Campylobacter enteritis
      associated  with contaminated water.  Annals of  Internal
      Medicine.   96:292-296.

72.  Walker, A.  S., and W.  A. Yanko.  1987.  SBG Sulfa  enrichment
      for  the quantitative  isolation of salmonellae from sewage and
      compost.  In:  Analytical Techniques and Residuals Management  in
     Water Pollution Control.  Water Pollution Control  Federation
     Specialty Conference  Series, June 1-2, 1987, pp. 331-337.
                                 132
-------
 73.   Ward,  R. L., G. A. McPeters and J.  G.   Yeager.   1984.
      Pathogens in sludga:  occurrence,  inactivation,  and potential
      for regrowth.  Sandia National Laboratories,  Albuquerque,  New
      Mexico.   Available from NTIS,  Springfield,  VA.

 74.   Weagant, S.  D., and C.  A.  Kaysner,  1983a.   Modified enrichment
      broth  for isolation of  Yersinia ent erocolitica  from nonfood
      sources.  Applied and Environmental Microbiology.  45(2);463-471.

 75.   Weagant, S.  D.  19S3b.  Medium  for presumptive identification of
      Yersinia enterocolitica.   Appl and  Environ. Microbiol.
      45(2);472-473.

 76.   WHO Euro Reports and Studies.  1981.  The Risk to Health of
      Microbes in  Sewage Sludge  Applied to Land.  j>_4:l-27 World  Health
      Organization.

 77.   WHO Scientific  Working  Group.  1980.  Escharichia coli diarrhoea.
      Bull.  World Health Org.   5£;23-36.

 78.   Yanko, W.  A., C.  D.  MeGee,  and J= S. Glass. 1983.   The fate
      of  viruses during composting'of sewage  solids.   A  review of the
      problem  and  results of  field studies,.   las  windrow and static
      pile composting of municipal sewage  sludges,  M.  D.  laeaboni,
      J.  R. Livingston,  T.  J. LaBrun.  Los Angeles  County
      Sanitation Districts  for EPA,  Wast©water Research  Division,
      Municipal  Environmental Research Laboratory,  Cincinnati, Ohio.

79.   Yeager,  J. G. and  R.  L. Ward.  1981.  Effects  of  moisture
      content  on long-term  survival  and regrewth of bacteria in
     wastewater sludge.  Appl. Environ.  41:1117-1122.
                                 133
-------
            APPENDIX A
MICROBIOLOGICAL METHODS EVALUATION
          AND DEVELOPMENT
                   134
-------
                              FUNGI


      Methods for enumerating fungi were thoroughly evaluated.  It
 should be kept in mind that the term  "total fungal count" is
 somewhat of a misnomer and that no one medium or incubation
 temperature will be appropriate to recover all of the fungi
 potentially present.  Dr. Martin Stoner (California Polytechnic
 University at Pomona, mycology consultant for the project)
 suggested several media to evaluate which included the following:

      1)  Alpha-cellulose agar - isolation of cellulose degrading
          fungi, regularly supports growth of Gliocladium and
          Trichoderma,
      2)  Diet-food medium with antibiotics and surfactant-general
          isolation medium, especially for the recovery of
          Penicilliuai,  Gliocladiua,  and allied genera.

      3)  Peptone-pentachloronitrobensene mediua (PCHB) - very
          selective for Fusarium sp.

      4}  Potato dextrose agar  - with antibiotics and surfactant,
          general isolation medium for fungi.

      5)  7-8  vegetable juice agar  -  selective isolation of
          zygomycetss and water  molds.

      6)  Rose bengal 'agar - recommended in Standard  Methods
          (APEA,  1985)  for fungal  plate counts.

      Early  in the  evaluation, nine different  compost  samples  were
plated  on the above  media to get a general assessment  of  populations
present and to  practice  fungal  identification procedures.  The
preliminary experiments  revealed that  few  fungi  were  detected from
compost samples with alpha-cellulose  agar  or  PCNB medium  (both
media are selective  for  cellulose degrading fungi).   At this  point
a decision was made  to eliminate the  alpha-cellulose  and  PCNB from
further evaluation.

     The  remaining media  were compared quantitatively  in  a series
of five experiments.   Samples used were composites of  compost
products  from sites  IX-A-3, IX-A-4 and IX-A-5.   Each medium was
incubated at  three temperatures:  room termperature  (13 -  23  C), 35
C and 42  C.  The results  are summarised in Table A-l.  Overall the
rose bengal medium yielded  the greatest number of fungal colonies
and the greatest diversity of species  and  35 C incubation was the


                                135
-------
 most productive temperature.  Although the rose bengal medium was
 clearly the most productive medium in this comparison, other
 factors also need to be considered in the final analysis.  Among
 these are the groups of fungi selected by the medium/temperature
 combination and the colonial morphology and growth characteristics
 of the fungi on the medium.

      Most of the fungi isolated from the compost samples were
 members of three groups, the genera AspergijJLus and Paecilomycgs,
 and the Class Zygomycatas; these produced vary characteristic
 morphological structures.  The V-8 juice agar was quantitatively
 the least productive medium, but was very specific for the
 Zygomycetes.  In a similar vein, the 35C incubation temperature was
 most productive/ however, 42-44C will select for the therraotolerant
 fungi and this group may have some indicator or correlative value
 or may have associated health implications.

      The final media selection attempted to  optimize and balance
 these various considerations.  The Cooke rose bengal medium (Difco)
 with O.OSg/L ehloramphenicol and 0.1 percent Tergitol incubated at
 35 C was used for .the "total fungal count."   V-8 juice agar was
 used in conjunction with the rose bengal at  35 C to select for
 Zygomycetes.

      1C was originally proposed that eaegall  medium incubated at 44
 C  would be used for thermotolerant fungi.  A subsequent comparison
 (unpublished data)  found the rose bengal medium superior for this
 purpose due to  bet'ter  suppression of the bacterial population.
 Cooke rose bengal with ehloramphenieol and 0.01 percent Tergitol,
 incubated  at  44 C was  used for  the thermotolerant  fungi,  including
 Aspergillus  fumigatus.

      Fungal  identifications were based on a  macroscopic to
 microscopic progression.   The first  step was  the macroscopic
 observations  of colonial  morphology,  color,  texture and other
 qualitative  features.   This was  followed by  a  microscopic
 examination of  the  reproductive  structures.   In general,  the actual
 form  of  reproductive parts  or other  specified  components  usually
 take  precedence  over color,  colony texture,  or  other  more
 qualitative features that  are subject to variation  with
 environmental conditions.

      The observed fungal  characteristics were  then  compared  to
 those listed  in  one or  more ta^enomic keys (Raper  et  al 1965,
 Barnett et al 1972).  The  keys used to identify fungi were not
 entirely consistent and occasionally more than  one  approach  in  a
keying system had to be used.  The final determinations of fungal
 identification were based on  the closest or most accurate  species
description.  When an  isolate could net be identified,  it  was
subcultured and  sent to the consultant for evaluation.
                                136
-------
Table A-l    Summary of Total Fungal Counts on Selected Media
             at Three Incubation Temperatures a
 Incubation              CFU/mL Compost Suspension  	 Grand
    Temp	Diet Food  Pot Dex  Hose Bengal  V-8 Juice  Mean Temp c
 Room Temp    190       170        230          17         ISO
 (18-23C)
 35  C         700       490        700         190         520
 42  C         230       280        810          31         350
 Grand Mean   390       310        580          79        	
  Media d

 a.   Samples were composites of sites IX-A-3,  IS-A-4 and IX-A-5.
 b.   Each value  is the arithmetic mean of  15 determinations
     (5 trials,  triplicated plates in each trial at countable
     dilution).
 c.   Arithmetic  mean for  all media at the  given  temperature.
 d.   Arithmetic.mean for  all temperatures  for  a  given medium.
                                 137
-------
                             COLIFHAGS


      Methods for detecting coliphaga in sludges had to be developed
 for this s.tudy. f'.The investigation was divided into three tasks.
 The first5- task involved controlling bacterial contamination of the
 sample without causing a phage titer loss.  The second task was to
 maximize the recovery of phage from the solids using various
 mechanical manipulations and eluents.  The third task was to
 compare four strains of E. coli to determine which would recover
 the greatest number of coliphage from sludge samples.

      Initial decontamination studies using primary and secondary
 wastewater examined diluting the sample, centrifugation (ISC head
 870;  2000 x g,  10 min; 15,000 x g, 10,  30 and 45 min)  chloroform
 treatment (APHA 1985), non treated 0.22 micrometer porosity filters,
 and filtration through filters pretreated with 10 ml of 3% beef ex-
 tract.   The arithmetic mean indigenous  phage titers fro® § trials
 assayed by the plaque technique (Adams  1959) are demons t rat eel in
 Figure  A-l.   The samples using the dilution and centrifugation
 techniques were not adequately decontaminated.  Indigenous phage
 titers  of 370 PFO/mL were observed using an untreated  filter,  1,800
 PPU/mL  using chloroform decontamination, and 3,500 ?FD/mL using a
 treaed  filter "for decontamination.

      Because decontamination of the sample by filtration removed
 particulate  matter which would be significant in sludge samples,  the
 next  set  of  experiments examined eluting particulate associated
 phage and centrifuging to remove the solids in primary wastewater.
 Beef  extract (3%  w/v)  was added to the  wastewater«   The mixture was
 blended in a Waring blender,  sonicated  as described by Glass et al
 (1978)  and divided into centrifuged (ISC 1872 3500  x g,  15 rain*)
 and non egntrifuged portions.   These were split and decontaminated
 by chloroform or  filtration (0.22 micrometer porosity  Millex).,
 Wastewater without beef extract added was also blended and
 sonicated? it was divided into 3  portions and 1 portion
 decontaminated  with chloroform,  1 portion filtered  through a Millex
 and 1 portion filtered through a  beef extract treated  Millex.

    Figure A-2  shows that  without beef  extract present,  relative
 recovery  from the primary  wastewater  was  the same as observed  from
 the first series  of experiments?  phage  adsorbed to  untreated
 filters and  were  partially inactivated  by chloroform.   When  beef
 extract was  added to the  wastewater,  phage recovery increased  in
all cases indicating phage were eluted  from the solids.  None  of
 the results  for the beef  extract  treated  samples were


                                138
-------
 significantly different (n=5,  P=<0.05)  indicating  that  cantri-
 fuging  did  not affect  the aluted  phage.   These  results  also
 suggest that  beef  extract may  mitigate  the  detrimental  effects
 of chloroform treatment.

      Next the effect of mechanical manipulation was  examined
 independently by keeping  the addition of  beef extract constant and
 varying combinations of blanding/ sonication and cantrifugation.
 The results,  shown  in  Figure A-3  show only  that a  somewhat higher
 phage titer was obtained  using the condition with  fewest
 manipulations.

      In  the next set of experiments anaerobically  digested sludge
 samples were  blended,  sonicated and centrifuged and eluents were
 varied.  The  eluents compared  were: 3% beef extract at pH 7.2; 3%
 beef extract  at pH  9.5; Trypticase soy broth; 0.25 M, pH 11.5
 glycine with  0.05 M EDTA; and  5%  casein at pH 9.5.  The mean
 recoveries of  3 experiments are presented in Figure A-4.  The only
 significant difference  between eluents was that high pfl glycine was
 the least efficient.  All other eluents worked  equally as well with
 a  trend toward the  pH 9.5 beef extract giving somewhat better
 recovery.

      At this point a different approach to controlling unwanted
 bacterial growth was evaluated.  Eluents were assayed using an MPN
 technique described by Kott (1966).   Lauryl Tryptose broth (LT3), a
 somewhat selective medium for colifonns, was used in hopes of
 controlling  unwanted baterial growth.   A nonselective medium,
 Trypticase soy broth (TSB), was also used for comparison.   The
 experimental design is shown in Figure A-5.

      The interaction between the  five  eluents and the two  media
 used  in the  MPN assay as well as  the decontamination with
 chloroform and filtration (treated filters with  assay by plaque
 technique) was examined by comparing these data  in  an ASS
 matrix  (3oik 1979).   Average values  were obtained for each
 condition.   Data for the eluents  independent of  decontamination
 were  averaged  by collapsing across the  rows  and  average  values for
 the decontamination techniques  independent of  eluent  were  averaged
 by collapsing  down  the  columns. The row and column means  are  known
 as marginal  means and are  represented  in Figures A-6  and A-7.
 Slution  from solids  was best obtained using  the  9.5 pa beef extract
 and decontamination using  a treated  filter and plaque assay gave
 the most efficient  recovery of  phage.  The MPN assay  procedure with
 either medium  did not give results comparable  to a  direct
 decontamination and  plaque assay technique.

     The final task  of  the investigation was a comparison  of the
 host strains E.  coli B,  E.  coli K12Hfr,  E. coli  C-3000 and 3.  coli
 C.   Indigenous phage in T6 anaerobically~digested sludge eluates
were simultaneously  assayed  with all four  cell cultures.  The
 total number of plaques  obtained from each strain is  represented
 in Figure A-8.   Here it  can  be  seen that E. coli B  was the
 least effective host cell  with  increasing  recoveries  from


                                139
-------
K12Hfr, C-3000 and E.  coll C.  S.  coli C gave a statistically
significant  (P=>0.05)  higher 'recovery value.

     To summarize, the method  selected that optimized coliphage
detection from composted sewage solids was to blend the sample in
pH 9.5, 3% beef extract, centrifuge out the particulates,
decontaminate the sample using a pretreatad 0.22 micrometer
porosicy filtar and assay the  sanipla on S. cadi C using the
plaque technique,
                                140
-------





3,300 -

3.00O-
? 2300-
z

21 2JQQO-
D
i 1.500-
z
uS 1,000-
2
500-








Q
Jr
i
< I i c
Z 3 Z *"
O d O Ul
o o o o











Q
£
tu
£ °=
i w
Z u.
If- -;
a
Ul
H- CC
< UJ
1— i

2
§
§
i
o







t- - .
I'.:-- •
5-.^."™'-
p? .'-""'•
r^v;...'
L










;•





f
i

^













FIGURE A-1 PHAGE T7TEH - DECONTAMINATION INTERACTION
                                             Ul
5,500-
5,000 -
4,500-
S 4.000-
Z 3300-
2 3,000-
| 2,500-
5 2.000-
Ul
2 1,500-
500-









8-
<
Z u.
Fr»-' j


|
§
tr
o
j
fc
fSt
a
<: tu





ss

^
:
i-%
^ff





1 1 J
NO SESF EXTRACT






il
i~ .'
?


!'•" "-"
B : C






"3
»
?."• '• '
r
V-
i, ""~
*. ," ""
r. ~-;:
2
^ o





ui S
00
-


^ ;
'-





ST!
*•
c
rt-


f
1






i i i i i
BESF EXTRACT (3% WT7VOU
       FIGURE A-2 PART1CULATE ASSOCIATION -
                 DECONTAMINATION INTERACTION
                         141
-------
                BEEF EXTRACT
r ,uww -
8,000-
5,000 -
4,000-
3,000-
24300-

1,000-
0-






i,~ '•-*•%—'"-
*• *""*••'' ' "i~ r
l^^gj;
M-S^^v?^"
iMjji






Ki
|S':JS?
S*v "f "%rf
EJ. i»3 " " ' ' ' "".",.
isi





;
. .^. ,...^
.,

-





BLENDED BLENDED BLENDED
SONICATED SONICATED 
-------
 CO
 05
 05
^^•^v

Q.
2

O)
   o
   o

   oil

   Q

   O
   O


Z <0
Q. ^>
Su.
ui O



S!
o
0
o

p

cc

o
Z

a

o


-------
CO SO *t O J"
CO €0 r- T- C5
C c
0 -
"•••• £^ i
—rth^J *^W ^
00 •"•
W M
(^3 **• U3
o o 2
(Ipi^P ^^B ^^M
£ 0
c
O "5 £
•43 2 &
C s S
1 "
"c
o m
m £ -I
jSf ®*
1 a
"S §
- H
yj
(O
i
M T
\ '










*rf
dBO
P
k /







'


**
0
fft
9
\ /








•

«
T
i /










^je
•
^
01
J^ O

O
*



0
2

2 2 .£ f e
2 X "5 CM * iq J»QU^ J «5
3 S «»• ^ ^^OjZwO
05 H-^ ®3! ©X««5*S««-O3^
•M * m^ M, •*• ^a ^5 2! i« ZL a™
ll ^Sa^aggz^a
MM »B «^( ^V , _,,»
144
-------
  5,000 •
i
4,000 -
3,000-
2,000 -
1,000-


_







r — i


i
&~" ~*
». . -*•
4c
|~ '









?- "
$ *".
t". ^
•*>"•
t ""-'









p
i-j^*"1 ""
•K- >
t'T'- «-
F"'








1"
l~ .;
„'
"".^

~ *






n


•MM^MH



r-









*~ \ *
".'

* .
r **








1




-







        3LYCME BE CASEIN TSB  9E  MPN  MPN CHCU FILTERED
         OH 9^ pH 7^ pH 3.5 pH 7.3 pH 9.5 (LTB) (T38)
         EDTA
                  ELUENTS           DECONTAMINATION
  FIGURE A-7  ELUENT - DECONTAMINATION INTERACTION
90,000-]
30,000-
0 70.000-
n
Z 80,000-
o
g 50,000-
LU
O 40,000-
O
C 30,000-
3
£ 20,000-

10,000-

0-








•






















r ."T.-.;,^»
^•v$:ts§
fc4:~"^^>
ra:, • 'v^-,. — ,.
j
E.COU9




















s
?~ ..'"•' —".-'•
f.t«a"V^,
^^''S--
p;f""^-"-r'T-'
«= **' "."_••-
^ »^ • •
P*- * -r"'-*" '
^ - * „.









'
5
J"-. '. ,'
s • • "
f"' v»^ '1,' ~
r-'Cf^"?.'
^S^"
gfe^^
Er:.-1--; \,r
|^4_ -.-.-.^ :












I-SS.
^- " 7'-';-;
r "~

, ;

,


t- - ' •' *1
j. * -." - .
K ,->-^.; : ^
F' * •"
^ ' -
i.

















1 1 I 1
£. COU E.COU S. COLI C
K^Hfr C-3000
FIGURE A-8 COL1PHAGE RECOVERY ON FOUR HOST CELLS
                          145
-------
                    SSTEHOTQXIGENIC  SSCEERICEIA COLI
    Research  into  the  causes of  acute diarrhea has implicated E. coli
 as one of  the causative microorganisias.  A World Health Organisation
 (WHO) working group in 1980 defined three groups of 2. coli as
 important  diarrheal pathogens?   (a) the enterotoxgenTc E. coli  (ETSC;
 which produce enterocoxins that  cause diarrhea in infants/ young
 children and adults in developing countries as well as travelers to
 those countries;  (b)  enteropathogenic E. coli which have been re-
 sponsible  for frequent outbreaks of inrantile diarrhea in many parts
. of the world and  are  known to belong to specific serotypes; and (c)
 eraeroinvasive E. coli which are tissue invasive and have a.
 pathological behavior similar to Shigella.

      The cholera-like symptoms produced by the ETEC are the result
 of enterotoxins which have been generally divided into either
 heat-stable (ST) or heat labile  (LT) categories.  The LT is
 immunologically related to the cholera toxin.  The ST is a
 relatively low molecular weight non-antigenic enterotoxin.  The
 ability of any E. coli to produce these esterotoxins is carried by
 plasmids.  Plasmid mediation of toxin production means that
 probably any E.  coli strain might contain or lose the controlling
 plasxnid thus nil ing out significance of a particular serotype as
 being a causal agent for acute diarrhea.  This was confirmed by
 Goldschmidt and DuPont (1976), who showed a lack of correlation
 between classical enteropathogenic serotypes of E. coli and
 virulence properties in animal models.   At this time,  there is no
 suitable laboratory method for differentiating the enteropathogenic
 or enteroinvasive strains.   Thert are adequate methods available to
 test  for the enterotoxin produced by the enterotoxigenic E«  eoli.
      In order to assess the toxin producing E. eoli populations in
 this  study, a plan was developed to test the~E.  coli population in
 each  of the samples.   The in vitro cell culture assay for
 enterotoxin described  by Saek  (1975)  was selected as•the diagnostic
 tool  for this purpose.  In this test whole bacterial cultures are
 briefly exposed  to Y-l adrenal cells in tissue culture.   A rounding
 response of the  Y-l cells within 18 to  24 hours is considered
 evidence of toxin production  by the bacterial culture.   Sack's  test
 was selected because  it is  relatively  non subjective,  accurate,  did
 not require removal of the  baeterial cells by filteration or
 inactivatioR with chloroform,  produced  results in 18 to  24 hours,
 and growing the  cells  in  ssierotiter plates permitted screening  a
 large number  of  samples efficiently.


                                 146
-------
       Sample  testing was  initially delayed  due  to  difficulties
 encountered  with the Y-i cell culture.   The  first vial  of  frozen
 cells  received from the  American  Type Culture  Collection  (ATCC)
 contained  only a•few viable  ceils even  though  the vial  had been
 handled according to directions.   Before ordering a  second vial, a
 technical  representative from ATCC and  the person growing  these
 calls  in Dr.   Sack's laboratory at John Hopkins were consulted.  As
 a result scms  changas were mads in the  handling procedures.
 Typically, cells are frozen  with  a protective  freezing
 additive,either  glyce rol or  dimethyl sulfoxide (DMSO).  Standard
 thawing procedure  is to  change the culture medium 24 hours  after
 thawing a vial  to  remove the  freezing additive.   It  was pointed out
 by the ATCC  representative that a  characteristic  of  the Y-l line is
 a delayed attachment to  the  flask  following thawing.  He
 recommended  that  the first medium  change be postponed until 43
 hours after  thawing.  Dr.  Sack's  cell  culture technician  indicated
 that she thought  the cells were extremely  sensitive  to osmotoc
 pressure and recommended that  upon  thawing the vail, the first 10
 mL of fresh medium should be added  drop by drop after which the
 remaining medium could be added.  This procedure was in lieu of
 adding the contents  of the frozen vial directly to a flask
 containing the entire amount of medium.   Both modifications were
 tried with the next vial, and  it did grow.   It was the virologist's
 opinion,  however, that the initial vial simply did not contain many
 viable cells.

      After getting the cells to grow from frozen stock,
 subculturing  problems were encountered.   Occasionally,  the cells
 would not  grow following trypsinization.  The-trypsin with EDTA was
 suspected  and switched to 0.1% plain trypsin.  This change helped.

      During the period of cell culture problems with the Y-l cells,
 Vero, BGMK, V-79, HeLa and MAI04  cell lines were evaluated as
 substitutes.   Dr. Sack's laboratory provided three toxigenic E.  coli
 strains that  had been stable for  toxin production  for many
 years,  and some cholera  toxin, all of which were used as positive
 controls for  the cell line evaluations.   No other  cell  line gave as
 clear cut  a response to  the  toxin  as did the  Y-l cells.
 Eventually, the Y-l line began to  grow well;  finding a  substitute
 cell  line  was abandoned.

      With  the assay system established,  the procedure for  screening
 samples was examined. An initial  experiment  where the samples were
 obtained from EC tubes of the fecal coliform  test  failed because
 the sterile EC  medium caused  cell  morphology  changes  and cell
mortality.  An  attempt to obtain cultures for  testing from  the
 lauryl  tryptdse broth failed  because the diverse bacterial
population overwhelmed the antibiotics in the culture medium.

      Apparently these problems  were unique  to the  compost samples
since Dr. Sack  did  not experience'similar problems using the same
 technique.  The  following approach  was eventually  selected:  streak
positive EC tubes  from the 10  mL inoculum of  the fecal coliform
test  onto M-Sndo  LSS  plates;  incubate at 3SC  for 18-24 hours; pick

                                147
-------
 five representative sheen producing colonies from each plate and
 inoculate onto trypticase soy agar slants (B3L);  incubate the
 slants 18-24 hours at 35C, store the slants at 5C prior to toxin
 assay (Cultures were expected to ba stored 2 to 3 weeks prior to
 conducting the toxin assay.   Due to cell culture  problems with the
 Y-l  cells, a testing backlog developed and some cultures were
 stored as long as 16 weeks.)? one day before the  assay/ pool the 5
 slants from aach sample by making transfers into  a single tube of
 trypcicasa soy broth and incubata 18-24 hours at  35C;  inoculate 50
 aiicroliters c£ the 18-24 hour broth Guitars into  triplicata
 microtiter plate wells containing a 72 hour monolayer  of ¥-1 calls.
 After a 15 minute exposure time aspirate the inoculum,  wash with
 PBS,  and add fresh medium; observe the cells at 24 and  43 hours for
 characteristic rounding.  Every assay contained three  positive
 control strains of toxigenic E.  coli,  and three confirmed negative
 cultures from our own culture""collection.


      Based on estimates by Geldreich (1972),  it was assumed that
 the  toxigenic E.  coli populations would be low when compared to
 the  fecal  colilbrm population and therefore would probably only be
 detected in low dilutions  in a dilution test.   Only EC  tubes
 resulting  from 10 ml inocula were examined.   For  enumeration
purposes,  an  MFH value was computed based  on single dilution
calculations.

      A  compost  sample spiked with 3 mL of  an IS hr  TS3  broth culture
of a  toxin  producing E.  coli strain was tested with the assay
procedure.  The  spike'was  detected  so  the  method  was applied to
each  of  the 500  samples  in the study.
                                148
-------
                    TOTAL SOTEHIC PLATS COUNT

          IDENTIFICATION OF  ISOLATES FROM MACCONKEY AGAR


      The total enteric plate count data were obtained by the spread
 plate technique.  Blended sample, serially diluted was plated on
 MacConkey agar and incubated 43 hours.  Data were reported as log
 CFU/mL.

      Colonies were picked off the primary spread plate medium and
 transferred onto a trypticase soy agar (TSA~) medium supplemented
 with ten percent dextrose and containing a bromthymol blue
 indicator.   This step was added to insure isolate purity and to aid
 in distinguishing fermcnters from nonfermenters.  Isolated colonies
 on the dextrose medium were tested for oxidase reaction and
 simultaneously inoculated into fermenter/nonfermenter broth for use
 in the BBL  Mini tele System for the Identification of
 Enterobacteriaceae and Nonfermenters.

      Oxidase positive isolates were inoculated onto the
 nonfermenter panel.   Oxidase negative, dextrose negative colonies
 were also placed on the nonfermenter panel.   Oxidase negative,  .
 dextrose positive colonies  were inoculated onto the
 Enterobacteriaceae II panel.  Dextrose utilization could not be
 determined  as oxidative or  fermentative on the dextrose plate
 medium and  due to the necessity for expediency,  standard
 Oxidation/Fermentation (OF)  tests were not run.   Therefore,  the
 dextrose well in the  Enterobacteriaceae II panel was  overlaid with
 sterile  mineral oil as a double check  on  the dextrose reaction.   A
 few  oxidase negative  colonies utilizing dextrose on the plates  were
 actually nonfermenters and  were inoculated onto  the wrong  panel.
 Oil  overlay of the dextrose well indicated when  the dextrose
 utilization was nonfermantative?  these organisms were reinoculated
 to the correct panel.

      Errors  in some early identifications  occurred due  to  this  lack
of distinction between oxidative  and fermentative use of dextrose.
Some  isolates  gave  reactions on  Minitek Enterobacteriaceae  II
typical  of  Shigella sp.  These  isolates were  submitted  to  the Los
Angeles  County Public  Health Department, and  most were  identified
as nonfermenters,  thus failing  to confirm  their  identity as
shigellae.  A  few were identified as Citrobacter  so.

     After  implementing  the  oil overlay of dextrose,  the only other
unusual  isolates obtained were of the  genus Bordetella.  Additional

                                 149
-------
 tests to confirm  the  identification were recommended by 3BL.  In
 most cases  isolates identified as Bordetella brqnchiseptica were
 non-motile  and therefore did not confirm as Bordetella^Those
 isolates identified as Bordetella parapertussis were non-motile and
 additional  tests  were needed to~confirm that they were not
 Bordetella  parapertussis.  Other tests utilized were gelatin
 hydrolysis, heiaolysis, and tetrazolium reduction.  All of the
 Bordetella  pa_ra_per_t_u,ssi_3 isolates were non-hemclytic, thus failing
 to confirm  as Sordatella.

      Many samples contained organisms which formed pin point
 colonies on MacConkey agar at 48 hours and subsequently did not
 grow when picked  and transferred to the dextrose plating medium.
 These small colonies were not present on MacConkey at 24 hours and
 were most likely  not true "MacConkey positive" organisms and should
 not have been included in the identification.  These organisms
 still appear as a percentage of the total sample and are designated
 "pick did not grow".

      The first method investigated for the identification of
 isolates from MacConkey agar was one similar to that used for the
 identification of fungal isolates.   Fungal colonies that were
 morphologically alike were counted and one of each type identified.
 The inability to discriminate between colonies based on morphology
 made this method impractical for the identification of bacterial
 isolates.   Isolates to be identified were then obtained by plating
 the sample on MacConkey agar in petri dishes imprinted with a
 Quebec  colony counting grid.   Those  colonies lying within the
 twelve  squares were transferred to  dextrose medium and later
 identified.

      In  the  early  stages  of  testing,  it  was discovered that picking
 colonies from the  grid areas  on the  petri  dish could result in over
 40  isolates  per  sample which  would have  been too time consuming and
 costly  to  identify using  the  identification system we had chosen
 (BBL  Minitek System for  the  Identification  of  Enterobaeteriaeeae
 and  Nonfermenters).  The  number of grids picked was  decreased and
 depended upon the  colony  count  obtained.  Ranges were defined as
 followsi  All  twelve grids were picked if  the  enteris plate count
 was  between  30 and 50  CFD, half of the grids were  picked  if the
 enteric  plate  count was between SI and 100  CPU and one  fourth of
 the grids were picked  if  the  enteric  plate  count was  between  101
 and  300  CPU,   These  ranges were selected to economize on  both time
     Since  isolates  were  picked  from  the  "countable plate18,  enteric
organisms detected in  other  tests  (Yersinia  and  Salmonella)  were
often not identified using Minitek.   The  most probable  reason  for
missing these  isolates was that  the countable range for  the  total
enteric plate  eount  occurred at  a  dilution much  higher  than  the
level at which specific enterics were detected in other  tests.  A
sample with 1000 Salmonella  per  100 wL of blended sample may not
have Salmonella as a 'kinitek isolate  if the  total enteris plats
count was orders of  magnitude higher.

                                 150
-------
       The method for obtaining the total enteric plate count and
  identification of isolates from MacConkey agar underwent  changes as
  problems were encountered.  Many isolates gave profile numbers  not
  in  the  BBL directory.   These profile numbers  were called  into 3BL
  technical services for  idantification.   Each  identification is
  accompanied by a confidence value and biotypa validity.

       The confidence value is the likelihood that an  unknown
  organism is a given species and is  expressed  as a percentage.
  Therefore,  if an unknown organism is identified as a member of  a
  species  in a group,  the confidence  value is the percent probability
  that  it  is that  species.

       The biotype validity is an expression of  the "typicality"  of
  the isolate compared to the species  in  which  it is placed.   It  is
  the ratio  of likelihood of the  isolate  to that  which would  have
 been  obtained if  the results had  agreed with  its  respective
. probability.   A  biotype validity  of  one indicates  that the  results
 of the isolate matched  exactly  the  results that would have  been
 expected  for  the  species  to which it  belongs.

      According to BBL technical services, a good  identification
 should have  a  confidence  value  no lower  than 35 percent.  The
 biotype validity could  vary from  1 to over 100,000 depending upon
 the number of  biochemical  tests that match the  identification and
 the number of  strains in  the Minitek database.

      Several of the identifications obtained had low confidence
 values and/or a high biotype validity.  As an example, Pseudomonas
 stutzeri, profile number  644215, had a confidence value of 66.08%
 and a biotype validity of 1481.  This could be due to the fact that
 Minitek  test kits were designed for use in a clinical setting.
 Environmental isolates therefore, may not be identified as
 accurately since few strains are included in the Minitek database.
 On the other hand, since the Minitek database  does contain
 clinically significant strains, there is a positive bias toward
 identification of an isolate as a pathogen.   Therefore it  is
 probable that if an enteric pathogen were isolated, it would be
 identified as such.  In  practice, several isolates were identified
 as pathogens.  Supplemental tests were conducted to support  or
 refute those identifications.

     Although the certainty of individual identifications  is
 variable, with the large number of isolates  identified a good
 profile  of species present can be obtained.
                                 151
-------
                           CAMPYLOBACTER


       A  preliminary literature review found no references  concerning
 campylobacters in sludge;  however,  much work  had been directed
 toward  detecting these organisms in food.   Numerous  media
 modifications  had been reported to  improve Campylobacter  recoveries
 from  various  types of samples.   These references were evaluated  and
 the most  promising were selected for laboratory  evaluation.

       Many difficulties were encountered with  the procedures  for
 Campylobacter  isolation and enumeration.   The first  series of
 experiments was  designed to select  an enrichment broth.   Pour
 enrichment media which had been described  in  the literature  (cited
 in Table  A-2)  were compared.  These media  isted  with results of  the
 comparison in  Table A-2.   The experiment was  set up  in two parts.
 First,  the growth of  Campylobaeter  in the  enrichment was  compared.
 Each  SO raL volume of  broth was  inoculated  with equal amounts'of
 suspension of  C.  jejuni and incubated in anaerobic jars with Campy
 Paks  (BBL  for  24  hours at  42  C.   The Camgylobacter concentration in
 each  broth was then determined  by each  broth  was then determined by
^plate count on commercially prepared Campy Agar  (Gibco).

      The second  part  of the experiment  compared  the  ability of the
 enrichment .media  to inhibit growth  of background organisms present
 in sludge.  Again,  each 50  mL bottle of broth was inoculated with §
 IBL of a 20% 'compost suspension  and  incubated  for  24  hours a-t 42  C
 in a microaerophilie  atmosphere  (Campy-Fak).

      Beginning and  final concentrations for both  experiments are
 summarized in Table A-2.  After  24  hours incubation,   Campylobacter
 was not detected  in two of  the  enrichment media.   The Doyle and
 Roman (1982)  broth  (DHB) yielded  the highest number  of
 Campylobacter.  Inhibition  of background, organism growth was not
 signxricantly different in  any of the media.  Although loss of the
 Campylobacter in  two  of the enrichment media seemed  unusual,
 recently puelished  comparisons of Campylobacter enrichments
 (Heisick 1985, Rothenberg et  al 1984, Beuchat 1985)   also reported
 DRB to be superior  or  equal to others compared.  Therefore, the DRB
 enrichment medium was  selected  for  further evaluation.

      The next series of experiments was designed to.  determine
 quantitative  recovery of Campylobacter from spiked compost.  A
 compost  suspension was  seeded with Campylobaeter.  DRB was
 inoculated in a multiple tube dilution"test and incubated for 24
 hours  at 42 G in jars with a microasrophilic atmosphere {BBL
 Carnpy-Pak) „  The enrichment tubes were streaked to commercially

                                 152
-------
 prepared Campy  Agar  (Gifaco)  and incubated  under  the  same
 conditions.   No Camgylobacter  was  decected,  however,  che  seed  was
 fairly  low  (10  CFU/raL).   The experiment was  repeated  with a  higher
 seed concentration  (50 CFO/raL)  and numerous  positive  controls.
 Again.,  no Camglyobacter  was  detected.  It  was  noted  that  positive
 control pla-as,  i.e., plates straaksd with a Campvlj3bac_ter cultura,
 that wars in  jars along  with plates sfcraaksd from compost
 enrichment tubes, also did not  grow.  Same control platss that
 incidentally  were in a jar by  themselves did grow Campylobacter.
 It was  then hypothesized that  the  background population in the
 compost, when streaked to the Campy plates, used up the oxygen in
 the raicroaerophilic. atmosphere  causing the jars to go anaerobic.
 The anaerobic environment in .turn  prevented the Campylobacter from
 growing.  Additional experiments confirmed this hypothesis.

      To compensate, a gas cylinder was ordered containing the
 recommended atmosphere,  5% oxygen, 10% carbon dioxide, and 35%
 nitrogen.   The anaerobic jars were ported and set up with self
 closing quick connect fittings.  A gas line was run into  the 42 C
 incubator  and the gas was warmed and humidified by bubbling through
 a  flask of water held in the incubator.   One reference reported
 that a  constant flow of gas was a superior method for recovering
 Campylobacter from milk  (Hunt et al 1985).   It  was hoped that
 maintaining  the proper incubation atmsophere would alleviate the
 initial recovery problems.

      Recovery experiments with Campylobacter  seeded into compost
 suspensions  were then resumed.   The new  system  was shown to maintain
 microaerophilic  conditions,  nevertheless  levels of 240 CPU/mL
 of  Campylobacter seeded into  sludge could  not be detected.  When
 the seed level was increased  to 10-3,  the Campylobacter could
 be  detected  from the  higher dilution tubes  of the MFN test but  not
 from the lower dilution tubes.   This pattern  suggested that the
 large background population  in  the  sludge  still overwhelmed
 Campylobacter in spite  of the enrichment medium.

     At  this  point  it was decided to  evaluate a modification  of  the
 enrichment medium.  The current edition of  Standard Methods  (APEA)
 discusses Campylobacter isolation from water  and  recommends the
 addition of 1.5% ox bile  to the enrichment  medium.  Interestingly,
 the  reference  cited foe this  aodifiemtioR was not  a technical
 paper;  it was  a  letter  to the editor  in a veterinary  journal
 (Oosteron et  al  1981.  More recent  publications which  discuss
enrichment and isolation  of Campylobacter  (Heiaick 1985) made no
mention  of using ox bile  enrichments so it  had  not been incorporated
 into the enrichments evaluated  for  this study.
                                153
-------
      Since  part  of  the  recovery  problem  appeared to be  related to
 competition from other  organisms in  the  sludge, it was  decided to
 evaluate modifications  to  the  DRB enrichment  (ORE) that might help
 inhibit background  organisms.  Sergey's  Manual  (1984) indicated
 that most Campylobacter  species  are  resistant to 1.0% bile.  Also,
 ox bile or  bile  salts is commonly employed  in enteric media.
 Considering the  Standard Methods (APHA 1985) recommendation, the
 DRB broth was modified  with  the  addition of 1.5 g/L bile salts
 (a more purified and concentrated facia of ox bile).

      Initial experiments confirmed the observation of Oostarom at. a_l
 (1981), that the addition  of bile to Campylobactar media greatly
 stimulated  the growth of the Campylobactsr; growth in DRB broth with
 bile salts  added increased by  more than  an order of magnitude.

      A second modification of  the isolation procedure was designed
 to take advantage of the small size and  active motility of the
 organism.    Campylobacter isolation procedures for water recommend
 prefiltering the  sample  through  a  0.6 micrometer porosity filter.
 Campylobacter will pass  through  the filter while most other
 bacteria will be  retained.  Unfortunately, sludge and compost
 suspensions cannot be filtered.

      It was noted in our lab, however, that in broth cultures
 incubated  in the mxcroaerephilie atmosphere, the Caatgylobacter
 tend to grow in the upper portion of the culture tube or bottle*
 Also,  when compost suspension is  inoculated into a broth medium?
 the compost solids tend  to settle out in the tube during 18-24 hr.
 stationary incubation.   The "clarified,  supernate"  medium can be
 withdrawn  from the tube and passed through a 0.6 micrometer filter
 at that point.

      By use of  this procedure,  it was hoped that any viable
 Campylobacter in the sludge suspension would migrate to  the upper
 portion or  the  ORB broth while  the sludge solids settled out.  At
 the same time,  the stimulatory  effect of  the bile  salts  might help
 the organism grow in the presence of  the  competing  population.
 Then,  even  if the ratio  sf  background population to Caispylobaeter
 was still  too high for  the  Campylobacter  t©  be detected  on
 isolation media*  the filtration and reinoculation off  filtrate into
 fresh enrichment  medium  should  permit the Camgvlobacter  to  grow  to
 levels  where they could  b«  readily detecte37~~"

     Experiments  with pure  cultures confirmed  that  each  step  of
 this procedure was conceptually valid.  Again, when low  numbers of
 Campylobacter wera seeded into  sludge or  compost, they were not
 recovered.   If the seed  consentration was high enough to reeovtr
 the Campylobactar by direct plating techniques,  they were generally
 also recovered from enrichment  broths.  When the sludge  was seeded
 at  a level below  that detectable  by direct plating  (<103 CFU/mL),
 the available enrichment media  appear  inadequate to select  for
 the Campylobacter in the presence of  the  large diverse sludge
populations.


                                154
-------
        An exact threshold  for recovery was not determined.  Low level
   seeds in these experiments generally meant approximately 100 or
   less organisms per mL.   Seeded campylobacters were usually
   recovered by direct plating of compost suspensions containing
   around 1,000 or mora calls per mL.  Compost suspensions used in the
   seeding experiments contained in excess of a million background
   organisms par mL.

        The purpose of enrichment is to provide a cultural environment
   which will favor the growth of a particular bacterium making it
   easier to isolate from a mixed population.   Available enrichment
   media for Campylobacter were apparently unable to provide a
   suitable growth environment with compost suspensions.  Beuchat
   (1935)  reported similar results from an evaluation of media and
   methods for detecting and enumerating Campylobacter jejuni  in
   refrigerated chicken meat.  He indicated that,without exception,
   direct  plating of samples on isolation agars was superior to the
   MPN method for enumerating C.  jejuni in refrigerated chicken.
Table A-2.  Comparison of Enrichment Media for  Isolation  of  Campylobacter
Sfedium
                      Caarovlobacter  CFU/raL
                      Initial
                                    24  hrs
    £
    hi
    Background
 Organisms CPU/mL
 Initial,   24hrs.
Rosef & Kapperud
(1983)

Doyle and Roman
(1982)
                         74
                         74
Thioglycollate Broth    74
Plus Antibiotics(GIBCO)
3 EM
(Rogol et al 1985)
                        74
44,000


 7,000


  <100
              1.4 x 10^1.0 x 10'
1.4 x 1069.2 x 107
                                                1.4 x 10°3.9 x 10
1.4 x 10Q2.0 x 10
                                                                 a
                                  155
-------
                                EMTSaOCOLITICA
      A literature search was conducted to evaluate procedures for
 recovering Yersinia from sewage sludge.  No procedures were found,
 however many references contained procedures for recovering
 Yersinia from food and water.  Selenite-F and more recently
 Peptone-Sorbitol-Bile Salts (PSB) (Weagant et_ al, 1983a) were
 recommended enrichment broths.  Other enrichment procedures were
 reported but most were more complex and many of the enrichment
 broths were found too selective for the various serogroups of
 Yersinia (Schiemann, 1982).  Since Yersinia can grow faster at cold
 temperatures than other bacteria, incubation was recommended at 4 C
 for up to 4 weeks.  Initial tests with primary sludge demonstrated
 that PSB enrichment broth was somewhat more selective than
 Selenite-F.  PSB and Selenite-F enrichment were incubated at 4
 degrees C for up to 4 weeks.  Fresh debate red primary sludge was
 used as the sample.   Enrichment broths were streaked ont© Yersinia
 Selective agar (YSA, also known as CIN agar)  at the end of weeks 1,
 1,  3,  and 4.   Plates were incubated 48 hours at 26 degrees C.   At
 weeks  1 and 2 there was very little growth from PSB enrichment tubes
 and light to moderate growth from the Selenite-F enrichment tubes.
 At  weeks  3  and 4, the Selenite-F tubes showed heavy growth whereas
 the PSB tubes yielded moderate growth.  By week 4 the plates from
 the Selenite-? enrichment tubes had a lot of  large mueeid type
 colonies  covering the plate.  The PSB broth plates did not have
 these  large mucoid colonies.  At weeks 3  and  4, Yersinia- type
 colonies  were observed on plates from .both enrichment broths?.
 however the colonies from the  Selenite-F  broth  were not well iso-
 lated  due to  the  large raucoid  colonies.   Colonies from the PSB
 tubes  were  isolated  fairly  well.   As a result of this observation,
 it  was  decided  to use PS3 as the enrichment medium.

     YSA was reported by far to be the best primary isolation
medium  (Head,  et  alf  1982). One report indicated that a  new
medium, SABY-4, was  very good  for isolating Yersinia  from
environmental  type samples  (Bereovier,  et al , 1984)  .   An
experiment  was  set up to compare  YSA and~~BABY-4.   Dewatered primary
sludge  was  used and  PSB was the enrichment medium.  The procedure
for  BABY-4  was  to incubate  one day anaerobically followed by two
days aerobieally  at  26  degrees  C.  The procedure recommended for
YSA  is  2  days aerobieally at 26 degrees C.  Osing  the  enrichment
procedure established  previously,  a  3  tube MPN  was  set  up.   At  the
end  of  each week,  the  first 2  rows of  the enrichment  tubes  were
streaked  to YSA and  BABY -=4.  After the recommended  incubation
period  for  both plating  media,  it  was  evident that  BABY-4 was  not
as good a differential  medium  as  YSA.  On BABY-4  there  were many


                                 156
-------
 Yerainia-type colonies that did not confirm,  whereas on YSA,
 Yersinia were more distinctive.  Whan 3A3Y-4  was incubated without
 the anaerobic step, the plates were overgrown with other bacteria
 and no Yersinia were evident.

      Incubation of tha enrichment tubas was originally established
 at  1 to 4 weeks at 4 C.   At ths end of each week 'the enrichment
 tubes were piatad on tha primary isolation iaadiun.   Early in  the
 study it was  determined that Yersinia were never isolated during
 the first two weeks of incubation;pTating was subsequently only
 performed at  the end of  weeks 3 and 4.

      The manufacturer of Yersinia Selective Agar states  to incubate
 their  medium  for 48 hours at 25 C.   With compost samples better
 isolation was achieved after 24 hours.  The Yersinia colonies were
 small  but were easier to differentiate from other  background
 bacteria.-

      Pinally,  recovery experiments  were conducted  using  the PSB
 enrichment  at 4  C and YSA~as the selective isolation medium.
Results  are listed in Table A-3.  Average  recovery was 110% and
 ranged  from 40%  to 175%.
                                157
-------
                                           o
                                           a
      a
      3
      2
      a
                    o    a
                    a    -
                          o
                          o
       a.

      z
       X
       L.
                    o    a   o    a   m
                    to    n   n    &   T
                    ••    P*   0)    P5
                    o    O   n
                    rt    rt   Qi
                    o    n    o    o
                    10    A    
-------
                  GIARDIA  CYSTS AND ASCAHIS OVA


      The parasite ova procedures used for this study were
 essentially the same as those used and documented by other
 researchers for sludge samples i.e., a zinc sulfate density
 gradient separation followed by an acid-alcohol/ether
 sedimentation.  Recovery  experiments were conducted using seeded
 Ascaris ova and are reported in Table A-4.  Average recovery of
 seeded ova was 33% and ranged from 33%-to 92%.

      Since the same procedure was going to be used for cysts,
 recovery experiments were also conducted with preserved Giardia
 muris cysts.

      •Initial experiments examined methods for enumerating the
 cysts.   The Sedgewick Rafter cell used for helminth ova was not
 satisfactory due to the small size of the cysts and the inability
 to use  high power objectives with a Sedgewick Rafter cell.   A
 hemocytometer was found adequate for counting cysts, as a fairly
 large number of cysts was present.

      An experiment  was then set up to determine the recovery of
 Giardia cysts from  compost.  A ten percent suspension of compost
 (350  tnL)  was seeded with 2.0 x 10 5  G. muris  cysts.   The sample
 was processed by the procedure used~for  Aacaris ova.  The final
 concentrate volume  was 1 mL.  One tenth  milliliter  was counted
 yielding  a  recovery of less than  one percent.   Counting cysts
 proved  to be quite  difficult.

      A  second experiment  was designed to  test  the presence  or
 absence of  cysts in seeded compost  containing  decimal  dilutions of
 cysts»  Four 200 mL aliquots of  ten  percent  compost  suspension were
 seeded  respectively with  12*500 cysts/gdw.  After processing  and
 concentrating  the seeded  samples  by  the procedure used for  Ascaris
 ova,  portions  of the final concentrate were  fixed to microscope
 slides, stained with trichrome stain  and  examined microscopically.
 The trichrome  stain  made  it much  easier to see the cysts  in the
presence  of  other micro-particulates  that  end  up  in  the
 concentrate.   Nevertheless,  detectability was  still  quite poor.
Cysts were detected  from  the 1,250 cysts/gdw sample  but not  from
 the lower concentration samples.

      It was  obvious  at this  point that the separation/concentration
procedure used  for helminth  ova was not effective for Giardia
cysts.  Another  experiment was conducted  to determine where the


                                159
-------
 cysts were lost during the concentration procedure.  The first step
 of the ova procedure was to blend the compost sample with phosphate
 buffered dilution water containing a wetting agent/dispersant
 (Tween 80) and wash the suspension through a 48 mesh sieve.  The
 resulting washed suspension was allowed to settle overnight during
 which time the ova settled to the bottom.  This step was examined
 using Giardia cysts.  A compost sample was seeded with 2 X 10°
 cysts and processed as usual.  After overnight settling
 (approximately IS hours}  the supernatant was siphon decanted and
 saved.  Both the sediment and che.supernace were cantrifuged and
 resuspandad in zinc sulfata.  The resulting concentrates were then
 titered.   The supernate contained 1.3 X 10° cysts (65%) and the
 sediment  contained 1.8 X 10^ cysts (9%).  Twenty-six percent of
 the cysts were lost or unaccounted for due to anaytical error.
 This experiment demonstrated that a majority of the cysts did not
 settle out overnight and were lost when the supernatant was
 discarded.

      The  method for detecting helminth ova was shown to be an
 effective procedure for ova.  It appeared to be relatively
 ineffective for cysts.   Alternate techniques for detecting cysts
 that circumvent the gravity settling step were evaluated.  One
 approach  that was discussed and showed some promise was to take a
 100  mL aliquot from the blended sample and centrifuge it directly
 before zinc sulfate flotation.   A presence/absence  test was set up
 using  this approach.   Four  compost  suspensions were seeded with
 cysts  at  concentrations o£  8,300 cysts gdw,  830  cysts/gdw,  83
 cysts/gdw,  and 8.3  eyats/gdw respectively.   One  hundred ailliliter
 grab samples  were taken directly from each blender  jar  and
 centrifuged*   The resulting pellet  was resuspended  in zinc sulfate
 and  the surface  layer was collected by skimming  with.a  pasteur
 pipet.  Resulting concentrates  were fixed to slides,  triehrosie
 stained and examined microscopically.   The  large particulates
 usually removed  by  the  sieve  interfered with the microscopic
 observation.   In  spite  of this  difficulty,  cysts were detected-at
 the  83 cysts/gdw  eoncentration  but  not at  the  8.3 cysts/gdw level.

     Although  these data represented  a significant  improvement in
decectability  of  cysts, the  detection  limit  still appeared  to be
 relatively  high.  A truly efficient method of  recovering  Giardia
cysts  from  compost was  not  available  for  this  project.
                                160
-------
         Table A-4.   Ascaris Ova Recovery of Sasdsd
                     Ova From Compost Suspension
                  Ova/mL	        %Recovery
Sample      Seed'Recovered
A            40                  34                    85

B            47                  43                    91

C            53                  49                    34

0            66                  57                    36

E            25                  23                    92
                              161
-------
                  COMPARISON OP BGMK AND MAI04 CELLS


     The sensitivity of Buffalo Green Monkey Kidney (3GMK) and
 Embryonic Rhesus Monkey Kidney (MA104) calls were compared for
 enumerating enteric viruses in sewage and sludge samples.  The BGMK
 cell line was obtained from Dr. T.G. Metcalf in 1979.  Several
 clones of BGMK had baen evaluated in Metcalf'5 laboratory and this
 clone had been determined to be the most sensitive for detecting
 enteric viruses (T.G. Metcalf,.personal communication).  BGMK
 cells were used until reaching passage 175 and then replaced with
 lower passage cells from frozen stock.  MA104 cells were obtained
 at passage 64 in May 1985 from Montgomery Laboratories, Pasadena,
 California.

     Each cell line was simultaneously inoculated with concentrates
 from wastewater and anaerobieally digested sludge.  Cell cultures
 were maintained and samples were concentrated and assayed as
 previously described (Glass et al 1978).   Nineteen wastewater
 samples and twelve sludge samples were tested.   In addition four
 stock enteric virus suspensions (Polio I,  ECHO 1,  ECHO 7 and
 Coxsaekie B6)  were titered ©n both cell lines.   The plaques counted
 on each cell line  were totaled for the wastewater and sludge
 samples;  stock virus titers were calculated and expressed as
 PFO/mL.  -Results are shown in Table AS.

 Table AS     BGMK - MAI04  Comparison

             "   ~™          ™"    ~~   Plague  Forming Quits'
Samole
Wastewater
Sludge
Poli© 1
ECHO 1
ECHO 7
Coxsackie B6
n
19
12
1
1
1
1
BGMK
306
515
7.0 x
2.3 x
1.9 x
1.6 x

Iflf
10
10*
107

9.5
1.1
1.4
7.0
MAI 04
142
310
x 10?
x 10
x Iflf
x 105
(a)  Total number  of  PFU  for  wastewater  and  sludge  samples  and
     PFO/mL  for  stock viruses„

     The BGMK cell line produced  approximately  twice  as many
plaques as the MAI04  cells  from the wastewater  and  sludge samples.
The BGMK cells also produced  higher titers for  the  SCSO viruses
and the Coxsmekie  virus.  The Coxsackie  virus titer was more  than
an order of  magnitude higher  on the BGMK cells.  The  MA104  calls
produced a slightly higher  titer  for  the polio  1 stock.  Overall,
the BGMK cell line was mare sensitive than the  MA104  cells.   BGMK
was selected for assaying sludge  and  compost samples  during this
itudy.
                                162
-------
           APPENDIX 3
MICROBIOLOGICAL QUALITY ASSURANCE
              163
-------
                   MICROBIOLOGICAL QUALITY ASSURANCE


 GENERAL PHOC2D05LSS

      Microbiological quality assurance practices for this project
 consisted of three main components.  First, standard microbiology
 laboratory quality assurance procedures, as described in Standard
 Methods (APEA, 1985) and the EPA Manual, Microbiological Methods "
 fgr_ Monitoring the Environment (EPA, 1978), such as recording
 incubator and water bath temperatures daily and maintaining media
 preparation and sterilization records, were followed.   Second,  ten
 percent of the project samples were run in duplicate.   Precision
 criteria were calculated from fifteen duplicate samples as
 described in Standard Methods (APHA, 1985).  The differences
 between sample duplicates were compared to the precision criteria
 to  indicate possible analytical problems.   The final aspect of  the
 quality assurance program involved "blind  spiking"  of  a portion of
 the  duplicate samples.

 Routine Quality Assurance

     Routine QA practices indicated there  were not  any undetected
 equipment,  media or  'reagent  problems during the course of the
 project that would have affected the reliability of the
 microbiological data.

 Duplicate  Analyses

     Ten percent of  the samples  were run in duplicate  and compared
 to pre-established precision criteria.   The initial precision
 criteria were  calculated from compost  samples  collected  from  a
 single  source  and therefore  were  not entirely  representative  of  all
 samples'tested during  the study.   The  precision  criteria  were later
 r@ealeula.ted with data from  fifteen  randomly selected  project
duplicates  representing all  sample types and compared  to  the
 initial values.   These results are shown in Table B-l.
                                164
-------
Table B-l
Comparison of Initial and Recalculated Precision
Criteria

TEST
Total Coliforia
Fecal Coliform
Pecal Streptococci
Plate Count-Aerobic
Plate Count-
Anaerobic
Total Fungi
Thermophilic Fungi
Total Enteric
Bacteria
Salmonella
Yersinia
Total Parasites
As carls
Bacteriophage
INITI
Criterion
Value
0.9414
1.1121
1.0722
0.2492
0.1799
b
b
0,6219
1.S210
1.1462
0,6674
0.9192
0.6949
AL
Number a
Pass/Fail
38/2
34/1
42/3
31/1
26/6
«-
— .
34/0
13/1
3/0
28/0
15/0
13/4
RECALCULATED
Criterion
Value
1.1988
1.1288
1.6722
0.3812
0.6452
0.7161
0.5957
0.4376
1.1315
c
0.5702
0.729€
1.2669
Number
Pass/Fai.
39/1
34/1
44/1
32/0
30/2
16/3
18/1
34/0
13/1
27/1
14/1
17/0
aThe numerator  plus  the  denominator  represent 10% of all  tests
 performed  in that category where positive  results were obtained

bNct determined initially.

cNot enough positive samples  to  recalculate.
                           165
-------
       It.  can  be  seen  that  the precision  criteria generally  reflected
 a greater  spread  of  data  using  the  actual project samples  compared
 to  initial criteria  values  derived  from a single source prior to
 the beginning of  the sampling program.  Exceptions were the  total
 enteric  bacteria,  Salmonella/ total  parasites and the Ascaris
 casts.   Initial values were not  calculated for the fungal  tests.
 Any time a duplicate failed tc  fall  within the as-ablishsd
 criterion, analytical techniques and general QA data were  reviewed.
 Mo systematic errors were detracted  to explain the cases cf excess
 variation.

      Overall/ precision was  acceptable.  The cases of excess
 variation  ranged from 3.8 percent to 6.5 percent of the duplicates
 depending  on which set of criterion  values was used.  Inherent
 characteristics of two of tha tests  explained a major portion of
 the variation.  The  anaerobic plate  counts occasionally contained
 large spreading colonies.  One or two of these colonies often
 obscured many smaller colonies.   The presence of these colonies
 frequently resulted  in excess variation between duplicates.  A
 similar situation occurred with the fungi.  Certain species of
 fungi obscured an entire plate.   If the anaerobic plate count and
 fungal variation is discounted,  unexplained excess variation
 occurred in slightly less than two percent of the duplicates.

      The total number of duplicate analyses shown in Table 3-1 were
 different for the various tests.  Samples  that were negative were
 not  included  in  the comparison resulting in notably fewer
 duplicates  for the pathogens.  Also, duplicates  that were  seeded
 with blind  spikes were affected  by the  spike  and were not  included
 in  the precision evaluation.

 Blind  Spikes

     A screw  cap culture  tube containing approximately  5 mL of
 sterile compost  suspension was supplied  to the laboratory  and added
 to  the duplicate sample  suspension in the  second  blender jar.
 Occasionally/  the  tube contained a  microorganism  spike.  Staff did
 not  know  which tubes  were  seeded.   Spikes  were added  at estimated
 concentrations ranging from  approximately  1000  to  100/000  units/mL.
 Concentrations were estimated from  pretitered stock  suspensions  for
 parasites and viruses and  by dilution procedures  for  the bacteria
 (Standard Methods,  &PH& 198S, p  336).  Spikes were not  titered
 separately.  Recovery was  reported  as plus or minus.

     Spike  recoveries are  listed  in  Table  3-2.  Salmcnellae,
Yersinia/ Ascaris  ova, polio virus  and Coxsackie virus were
detected  each time  they were spiked.  CampyJLobacter was detected  in
three of  five spikes/  toxigenic  E.  coli  was detected  in two of
four samples, Giardia cysts  were~"detected  in  three of eight spikes
and cA.hu  virus was  detected  in one of two spiked samples.

     Based  on preliminary .recovery studies or previous
experimentation in  the laboratory/ the blind  spike recoveries
followed expected patterns.   The Campylobacter and Giardia cyst


                                166
-------
 procedures were known to be low sensitivity tests.   Quantitative
 recovery experiments were not conducted for the  toxigenic E.  coli;
 however,  it would be expected that recovery would be related  to  the
 ratio  of toxigenic strains to non-toxigenic E. coli.   The greater
 that ratio, the less likely it would be that a toxigenic  strain
 would  be picked for subsequent testing.

     Ths  missed ECHO virus seed was unexpected.  Tha  first ECHO
 seed was  detected but the second was not.   It is possible that the
 frozen virus  stock used for seeding had lost infactivity.   Tha ECHO
 II stock  was  retitered a month before and  five months  after the
missed seed.   Infective viruses were detected in the  earlier
 retitecing  of the stock but not in the latter.


Table B-2 Detection of Blind Spikes
Organism                     __	Number of Spikes
                                DetectedNot Detected
Salmonella so
Salmonella typhi
Yersinia enterocolitica
Campylobacter jejuni
Shigella soneii
Toxigenic E. coli
Ascaris ova
Giardia Cysts
ECHO II virus
Polio I virus
Coxsaekie B4 virus
2
I
6
3
0
2
6
3
1
2
2
0
0
Q
2
1
2
0
5
la
0
0
  aSeed  stock  may have been dead
                                167
-------
   Quality Assurance Summary
      A general summary of the analytical parameters for testa used
 during tills study ara summarized in Table 3-3.  Certain aspects of
 the microbiological quality assurance presented definite problems.
 Duplicate precision was inherently difficult with some of the
 plating tests, particularly the anaerobic plate count and the
 fungal tests.   The problems associated with large spreading
 bacterial colonies and the growth characteristics of a few fungi
 clearly interfered with the use of standard quantitative precision
 comparisons.
Table  3-3.
Analytical Parameters Associated with Microbiological
Tests Used for Occurrence of Pathogens Project
Test
          Calculated
     Detection Limit3
       Recovery of
Seeded Organisms (%)
Total Coliform
Fecal Coliform
Fecal Streptococci
Aerobic Plate Count
Anaerobic Plate
  Count
Total Fungi
Thermophilic Fungi
Bacteriophage
Snteropathogenic
  E. Coli
ToFal Enteric
  Plate Count
Salmonella
Camgylobacter
         0.5 MPN/g
         0.5 MPN/g
         0.5 MPN/g
         170 CFD/g
         170 CFU/g

          33 CFU/g
          33 CFU/g
          17 PFU/g
          0.7 MPN/g

         170 CFU/g

          0.2 MPN/g
        1000 CFU/g*
          ND°
          NO
          NO
          NAC
          NA

          MA
          NA
          NO
          NO

          NA

          105
Parasites (Ova)
Parasites (Cysts)
Enteric Viruses
0.2 OVA/g
NA
0.05 lU/g
88a

30f
 * Based on average 60% TS; individual samples may vary.
 ° Not determined.
 *: Not applicable.
   Ascaris ova.
 c Giardia' cysts.
 c Glass et al 1978.
 5 EstimatedTrom recovery experiments.
                                168
-------
      These difficulties,  however,  were probably compensated for  by
 the  size of the data base collected for this study.

      The large range of the observed data also made  the blind
 seeding experiments difficult to conduct and evaluate.   This was
 readily seen with the toxigenic E.  coli.  If a seed  concentration
 ot 10,000 - 100,000 toxigenic Z. coli was introduced into a sample
 containing a low density  of facal  coliforms, the seed essentially
 swamped the population and all that was measured was essentially
 the  seed organism.   On the orher hand,  if the fecal  coiiforms were
 at a  concentration  of eight to nine orders of magnitude/  the seed
 might  represent less than one percent of the population.

      The use of virus seeds also resulted in apparent contamination
 problems.   The laboratory usually  segregated seeding experiments
 from  actual sample  testing.   That  practice was not followed for  the
 blind  seeds and a low level of contamination occurred.  The pH
 electrode used during sample processing was suspected as  the source
 of the sample to sample contamination but that was not  definitively
 established.   This  writer would not recommend the use of  blind
 virus  seeds for environmental monitoring studies.  Similar  sample
 types  containing low indigenous virus populations, sueh as  raw
 sludges  for this project,  could be  used as  positive  controls  for
 the virus methods and alleviate the problems  associated with
working  concurrently with seed stocks and negative samples.
                                169
-------
    APPENDIX C
MICROBIOLOGICAL DATA
        170
-------
3

IU
o
4,
U

at
         ».«   3

         r (3   uu
         i Z   u
         O ^   t3
         an.   o
         CE     J
         4J
         •< U  U.

         I- Z  U

         C i  'J

         I- u.  O
         u z
         «• 3

         §s
         I
         IU Hi
         < h-
         Z <

           a.
         U 3
         -O
         I  CS T ft
aa i» *• —
-/ * -r •» m
A
3
T at — -a ft
a  <••»
T IO Ul f T


a ta M in n
a in in in in
*• *•*«"•

a ain>> r»
m a c< aa
r- a r- a  aa a

a
n — » in »
— aaa a
a ft ft — —


a
a •r a a a
T in at in —


a
aa — a T
a — a — as

a
v n-a»«
-ma — a
§ssss
M"NM£
VI VI I/I VI I/I
aaaaa
aaaaa
- _ Q - -
m in a a a
aaaaa

a v a us r»
M - am -
n T a -r m
A

a a a >N —
a o a f y>
& v 
aaaaa


in a mn «r
«<-»««

a a
to a mn T
(>• » n — a
ci •* i* i» m

a a
wn — — a
uitaa<- a a
r> rx n r> a
n «
*o ;o 13 sn w

a
•T — M a —
a m ft — T
-r a n ma
a •• »• a a
a
amoi aas
— -r no M
aaaaa

a
a a n r» «•
a * — (*• a
o«a «» »


a a
naa aw
— •/ -r — a
a — — m t
aaa •* n

a a
n a » M M
— » -r a a
a — in r» v
a a a v m
a
- °r mace
a eaa r»
m ex o a n
a r. m a aa
a — n m a
m a a a a
VI VI VI VI V»
aaaaa
aaaaa
« a r» N a
- - ft OO
aaaaa
aaaaa

— -J9 i/i ta a
0> — J! T S
a m . a — na
a a m T p»
a a r» a a

a
o — i* aa
a •• m — a
a ^* a m p»
ma en o 3»
T - -i r-i •-<
p* :£i 
a
a a
na — a —
a — m r» *
f* m ao a
a T f» v ft
a
a a
f -to - -
— a r» » n
— «r — a «f
a ex r» n o*
a
a a
- a a
r» a n ft a
•r a - a n
!•• a a a a
M CM CM fM fS
I/I VI VI I/I I/I
to co a a a
aaaaa
a r» n a «r
a a - - -
                                                                171
-------
>
<

<
u
>
«
t-
,
JO    Z
< it    a,

m J    t2

   u    J
          JO   Z
          < u.   a,
          o J
          < —-
          i- J
          oo
             O



             (3
             O
   IU



   a
                        » O - M l»
                        O 31 ir V r*
                        IN 
                        to 
                       via «»r> a
                       eoesese
                                  •»
                       9»09«O O
                       •> 1 1 T?
                       ws w« «ffi ws 

                                       •a in v
                             a « —
                             a » ra
                                       » •» a
                                       ooo
                             «r « cs
                             o e -
  pa a P»
  — w «
  »«,•«.•»,

i  a s o
                                                                             172
-------
z

a






HI
VI
a
u

ui
t-
Vt
01

H


VI
a
u
        •* 2:
        *- <
        ou
        ^c —


        3 i
         5  5
         ui  &

         Ul
       !    3
         !U  (9
  j


  VI
        i-u u.
          _^8
         iua-i
u

z j
iu a
       x •
       a fin n n
aaaaa

pt •& n PI ^




a
ooo o o
aaa -«
-OT


o * 04 c* a
ta P»
«
V
» * V * O
o
a-««o
 a i* a P»

a
aa - Aa
a i» a FO  aai»
aaaai*

a — — - —
a a na a
a n * n n
a a P» aa
o an tar*
v v \nia\a
I/I VI I/I VI VI
aaaaa
aaaaa
na <>« A »
a-a ?* N a
a a a o «






a

a

a
a a M «• —
— a
V
a
f» o« maw
aar» — a
a
a a a a ui

a
ia a a « o
IA Ma an
31 r« ui a a
ai — n in a
to a a a a
n N CM M M
Ul VI I/I VI VI
aaaaa
M A r» <* a
- -M aa
aaa A A
a a a a «
I
i
i



~

*;



O -OO -
— fin
V
« in  — f » r»
VI VI l/| VI VI
aaaaa
aaaaa
aM A a v
— ft f* a —
A A A a a
9


a


O

9

fl
a
v v v y
a a
A V NP» A
r» a » a I*
a a
a a P* ii> ID

a a a
Aaaaa
f. a i~ a o
aaaaa
v « y
r» a n« a
A — T » a
r> a a a A
I/I I/I VI Ul VI
a a a a a
aaaaa
-------
1 *
t
»
»
*
*
*
•
*
»
*
*
»
*
*

*
»
ul
>-
3
1/9
%

K
W5
H
&
J
&
s
<
t/»
9
e
e
0
»
»
®


©
«
»
9
#
»
9
®
£
*
e
•
s
i/>
J « 'J
< iX -x
a u >
*- in a
<
in
U4
-J 1- 13
< ~ -v
•3 < rf
i-  ut
a. «
<
u
<
-j

z •»
i §
.1 a
vt
f<3
{jt**.
J — OE 3
< « UiU.
t- 1U(- U
K Z < O
U« J
u
BV BBS
Z J (9
 M « 01 ai
o • • o a
V V
CD 
-••CO
V V

Pi





a
oof <•»<•> —
— P4 ffl O OS
» O»ffl O> 1O
«« in mm

e e a
r» O O O —
r* O O O O
»eo an


»a e net
r» coos O —
n na> v •»
o> ssaso o
re c* w n <*s
V9 VI Vi VlUt
te ® 98 S Pa
« «B ffl 3 >,
ft (V i* CO ~
3» 0 O)
o a o
V V V
3> «> a>
o 
-------
<
IU
a.

u

t-
<
H
v>
i
IK
H
o
u
a
•< i
u wi
J — ui O
K 111 3 •
252=!
< _.

U 4
o >
§ °

z o
U
1
t/i

£ <
u 2
-* o
QE
(A
IU M 1J
-1 « >.
a < <
«j >
53 °
<3
a

H>
<

- rt <•»<•» » to
ooooo










— — — M d

 a — a
P«  r» «
(0 as rtiai*
N M o* ev P«
« V» tfl V> WI
c£ to<0  i* « e «
ai — « ta  V» VI
c0 «  ai
1-5 a a a a
<
v ifl (O
to r* f»« r* r*
C* (S (X t* C«
VI Vt Ut I/I VI
rg (0  a» <»«
0 0 - - -
                                                  175
-------







Z

S

2

i

(A
O
a.
a
a
u .

a)
i'
a,

i
V9


1
1
•n
US
tf$


a

^B
Z
o
u
Hi
J
35 1
<« 1
>» 1
*
*
*
*
*
*
*
*
*
VI


VI

£

tfV

-



J


X

^




"*

•
«
*
.
«



»
«
,

e

*

»
»
«


• !

UUI
KUJ 3 •
a >- &3-
)-Z- •
a: o
i i
X O
0

ft
at >.
0 >
Z O
IU
>



in

C (5
3 i
o 5
- 0
K

U)
U! -. (3
J s ^
83. >
> <
Q
Z
(9
Q






s
<
"


ooo o o



etaaia a





oaoo o









axaa « a
oa a so





C3 S3 91 9i O
a o o Q Q



49 Q g <*5 {<
nnSf ?
9 SB Ol G Q
N «•« WPS
n •? •» *)
intn ui vt M


?Sllt
•" a n o «
© a «« n a
N S>4 W W «
o>
000





o oo









3» 3» B)
a o a





3» <*<»
o a a



^ ^ ^
a» «5 f»
««
a, « =
©s©
     < a,

     z a   iu
     o      u
     uu.   z
       Q   IU
          a
 •°   "» < W! OS
     f Qg !W Ul
«-»   W» tU H> >•
^   O > tt£ £
«   a, •€ ae«
tie
*->   i  i  i   i
9
z   •« a a i
                        176
-------







1ST - SCRECNCC
s
2
a
u
(U
.j
a,
M*
t—
<
*•
v»
w
tt
1
•*
•M
N*
.0
>-
I/I
M
u
'XI
J
<
J-
*
•
•
*
*
*
•
*
*
»
*
*
*
*
•
(SI
M
J
3
(A
31
ts
H-
wl
tu
»•
HI
U
a
a
«f
wi
•
*
•
•
•
*
*>
»
e
»
»
»
•
*
•
*
»
•
*
•
2 «
=!- 5
51 S
a1*. 9
 Wt
i
g
-J

H
<

aaaa» — p»
t- i» a Oir»
id «r ainuo
r»atna so — '& n
f y» «* ^ &
a T t «««>
— o« — «
»<•«•<» —
»«
P»<^I^ <•«
«r» VI VI
« o a a  » N «
a
•r •- QJ P* t*>
^ 41 CM -a  a
r» a 
a * a — o
r» otaoiai
a a
r-c«BaitO
M r«a M a
a — to » o
nr«iai0ia
a aa
r» «»r»«
UO  r»  U> 
a » to o» <»
is 'O as a a»
T M 10 n *t
O
71 — -a f» —
ai « u> 31 to
ao 3» o o
a M r> r> a
9 ia M M ia
• v* id —
a « v a a
a
fv M t«> — a
a 10 f» » 10
aar> n —
m T »»w
a » P« » r»
r»ooa •»

a a a a a
a
— » a v n
(O — tB M M
a — <•» r«. «
» ^r ^ in »
a
a r» a a o
a r a a f»
a n in ao
a a a a a
» a f» n 01
M nca *- M
t* n vv> at
at 0101 ota
a
a K en
in m KI aM
acocor»
id a ** *~ ui
 a •» '8
- « in in •«•
V \
a m 
a — » o a
in » — f» r«
!•• a a f» f»
aacaaia
r» ao o ia
a a a r> M
asa»a»38«
a
« f» 
s o — M M
ts oicn en a
a a a a —
— — « o» a
f> 3» as a —
ui fl n a in

o a f- to 
a nan a
f» «r» «t»
« — « — —
noi — as in
a «> a ia a
f» uia« r»
ar» a aa
n a a 01 o
•a nt in in  « M n a
a — » r» »••
\a 
ar» ar> a
a
a
a«M tan
naaaa
M aaaot
ar» aaca
n MM * ia id
«-««-
a ft f» 31 g>
T a n nu>
M M M M M
I/I I/I VI VI VI
aaaaco
4 09 41 tf Q
a * -an
— M a o M
— — «>« M M
a o — S3 —
*» a «* 9 o
9 u> a tn n
» » -a ui »
- 3 ia n n
— a 31 to CN
« — aa> a
9 a - a«
r« O »  f»
a id « ia us
a
v + at a a
id in r» • i» a — *
» 
-------
     

     IU

     c
         u
         <
         §
a.


u
a
IU
z
IU
u
e
u
ut-
u>
a
IU
-j
a

u
    IU

    t-
        a
tu



1/1 O
I/I
IU
 z
ae x
IU

<
a
.j
i =
a A.





wn««=«
• aiaaia
r-ai r» p.<»

a
f« P» n  us ffl P»
 (O
WOWf*' W
9 po noc<
- — N n N
a o a o a
P, _nM_

a - p> oaj
a«an an

on «n~
f» f* « n •
ia f» as <- n
in usia CO IO »


a* osus ea
as •*• ffl O M
in =• «in at
re «IM « «•
* a
10 e to a
-= <9 n
N » fl> n v
10 to (* to V
n ma a at


a
v « — a  e> o «»
— to v o in
» •> PSPJCT
«<>««««<<<(
W9 WI W»  0» 3)
« •» W <«J 1
i
i
i
i
i
i
i
T n <>< at N i
O O OO - I
ui ut M en 1/1 i
i
a 
-------
a
w

u
41
«
u
                                                 iu
                                                               < a:

                                                                i  t
                                                           o
                                                           z   a a

                                                           3
                                                           O
                                                           u.
           uui
          j — m 43
         HIM 3
         OK« 3


           !U > —
ul
M
&   <9
1U   >.
J   <
O   >
Z   9
           £
           u   >
           -»   o
           «
         33
           a
»IO* » P»
aaaaa

— <*••(»•«>

-<>» — « —

— <>4«1O —


V V V » V
f»« KldSlO
» — aean
coaa
aa---
<^< c< ty c< N
-mm
VI VI VI Vt VI
«««««
aaaaeea
« r« « a i-
ex N a *• —
— — <•» « n
ao a a a
ta• — •••-<«•


« v y v v
aiaaaia
/» a r» c« n
atataan
»•»»»«
r< MM (« M
I/I VI I/I Ul VI
o9 OXBP- in
ia r« 41 •* n
ia in uxato
(H (N M  -> 1
I/IM tflWI VI
o<
^ a aa a
a a a a a
tea lainr*
oa aao








y » y v »
«•* o«a
a on  — v
(OtO to l~ P"
M f< M  1 -)
I/I Wl I/I I/I (rt
CO CO 40 (O tO
a aai to«
« » o r» n
e —  O •"
a n r» a> a>
•r o ft <•» m
Q A Q Ol 01
«  •» T I 1
Ifl Wl I/I I/I VI
« * C«« M M
f V V V V
* :n M a> N
a — 31 uj r-
* «r * a n
a o a a —
n n n n n
-i -> -)-)->
i/l I/I VI VI l/t
ca («» r» ,•* f*
31 9 fO O 98
a 
-------

















31DA3=
OE
i
«
G
4
O
(J

i
§j
Z
X
PS
1
<
I
X
••
H»
Ul
V
U
IU
-I
<
»B
•

^
*
*
*
*
*
*
*
*

.
:

VI
8
h»


IU
u
a.
a
•
*
0
*
9


»
e
*
*
e
»

u
IJJ *"J "^
Q. Z U
o a a
a *. o
Z -i
X
J "> 3
SI C
i- u. O
K
U Z U
no 3
I U
z 5 -j
X
z o
ui 5
9 «J Ik
O *.»«
o
09 — s» r* «
r*« a CD r- (0
a) a> at r» 

n — 
vor^ a "
SS««M
«««««>
C6 «=> ^ ^ ^>
eaot a o o

S ri I*i J^ S
o a a> a a

aoooo
o a o oa
Y V V V V
a a ca
o  a r»
rae «« w
a»« - we

aa
« 098505 —
(SOB a!
V V
aa
N MS =•
» ^ n

tfj fM ^J PV
«««>f«»e
as SB ws OT (n

T •«• T «3 ffl
v *• at — v
N M N N (X
tea 18 « as
G9 39 09 S3 OB
as as « e te
o — 
X
Ul
tu
- o a -i i- o
c*3 Q a *•• vn y
V V 2,
a
<
o as o -2
v r» in z a
IU
tr
Ul
a i-
U IU
OB » r» a <
(•i w ^ a «j
030809 > Ul
a —
u
«
j
"ft « JO

ra FS «» a a
a < a
U M >.
tew us j «•« 3
** t£> H> IU H U
e>« « H g «s o
y
u
M M
o =, = z j a
10 V OB a O Z
«« ' x.l
O IU
^3 (3
< >»
** . i 5
O ^ « « tS,
A S
U «i
r» u) « 2
^ w ^
V Q « O

Ul WIM
s as eg
n -
— c« a <
— •> (X

o< o<
-

31 O r»  oc o ^
T f* ffl (B «J
^ Ifi P* « —
fX M 
to is 
— n a — PC
— p» as asaa
a a a a a
aa



ID
PI (X — — -3
•- CX
a
fl — tN  o P*
* us 




f* 01 f« — —

V
fM CV « ES) W





« W M W W


(O C4 «y C4 09
i^ P«» C*5 ^ (O







tU r% e* — «e
*• ^ T 
-------












Ul
u

u
UJ
oe
i
v»
O
g •
u

2
X
a
z

3

W
i
i


.«
>it
Ml
s
J_
z
a
u

u
J
9

*
*
*
*
*
«
*
* *
VI
-J
3
t/t

vu
OB

tff

H-
(M
j

(X

vt



*


*

*
0
*

A
*
ft

^ .
*

*
*

•


.
9
»
1
1


U VI
J Ml IU O
t- IU 3 •
a i-« a
N> Z - •

OS O
< X
u <
a >
x a
0
I/I
M
IU >.
z a
41
>

Ml
3 -x
U >
- a
X





i/i .
m M t3
j oe x
a < <
< u >
Ml I/I O





a'
z
a
a






i




KHad r>«
aoooo














» — K»MC«


















« MM M «
I/I V» VI l/ltfl




sssss
10 — !•• <*> —
_ ro ai tn«
*a a ea a a



oooao




a
• a • -o

a
• a • -a





«Ct-N 9







a

• o • - ^
f* iu Z
— < OE 3
ID
H III
333 § aaa
PI cO V O
^ O8 O O
--M
181
-------
(-
U1
3
wt

I

L.
«1
O
1
Z
n
u
1U
_i
-< a
j -»
r a ».
a. z u


Z
a
J - 3
t J U.
w £ u


•ROB 1C
• COUNT
iCFU/G
«c t- o
Z < J
< -1
3.
H-
z o
yt ZQ
i*» co « a «q


a
m a sn « *»
c« inn n K>
a
» — to w3»o»
e
aaoo o
m N « ON
P.O. *> <3 «S

ainniaiD
o w w PS w

a
•r mo coo
nn f. raen
« f« (Sf« U)
N o ® e «i
o « o n o
V V T » »
(«• in f>
r* O o> O <•»


n co — «j •»
"? 'f O ^ • •*


is.  » o
in — — OQ
toaaaa
o« <>• aj to
n o> «»
too « o «
r. — O  a cs ®
SǤ2g
e« PS PS m f?
a
n — a o a
f* O f* tB tr
— 10O - N


a
— O 31 C 31
- O S> C3 V
M m «» — M


a
a — « — a>
a — a T a
r- — » uj n
n r» o w P»
X
a a
«a o o •
ffe O O ^
V
an — as
m* n a

a
«fN«S
af» M rois8
a « «« to
f» p€ o ft rj
is « M r» «
«««««
r* « o f* v
« a — — (M
?5 ^ •* T V
cfl c< — 
n r< a) rs o
ffl   a o —
to » as a co
— M — •»
M — f» «1 Ifl
a v n N in
Of» on »
n O O O O


a
n a a a a
— a o o o
a a a o o


o a
a r» a o —
N in r* <£ r*
O <•< O Q a
a<0 f» »i»
a a
in r- m — n
v a n n  04 


o o o - —
a ra o c o
a a a !•> «
'

a
« s> in * —
«r ^» ta r* aa
n o N — 

to in ts N «—
is a « ft n
»• V •»
u to « o a
{•Jifl o f 
e?
a
«
10
*
PS
tfl
«
n
OT
fx.
a
M
M
O
                                                     182
-------
3


VI


 I
VI

a
o
CE
IU
J

a .
           oo
           i- vl
             Ul
             '.JJ


           J -    °
           hw (/I    c.;

           O <    ot
           J— i^    O
             z   ?
             -   z
             vi   a.
          <     H«
          a     •<
          a     -i
          j     a
          >     VI
             u   a

             z   z

             i   i
               «3
          J-053

          < « IUO.
            ua j
          z-i   a
            Sa   >.
            u   z


          a u
                 a-
a
— \n IH — a
10 —
V




a
MF»MNO>
A
» a oa r-
idioataia



ioaa o. , x x> x,
a i^ <0 pi a
N r« a — M
M M ft ft ft
aaaaa
en i- n ft t
ft
V
T M
a
AN M MM



a

0
«OI tO T r*
V


aMOMg


oaio <•» « a
r« 04 a M M

VI VI VI I/I I/I
«««« a M


ar» a n »
n 10 P» to • X,
a am M 0)
n a ^ 04 M
» in w iota
aaaaa
a
M — M IO ••
¥ V V
a
M — « x x >,
IBM a con
a — -Ma
to to to a »»
aaaaa
in — — — —
V V V
T Ul 00 — 1-
f4 M — M M



O
C«l  M M

a aa
01 r> eg a M



-aa--
aoa a a
a a a n«


M aM » ft
a eon ^ a
a — n n uj
M P4 M C4 M
(O «« 
-------
      3


      VI
              j » iu «9
VI
•s
v>
O
a,
3
      !•=>

      B9

      118

      (=
m

J

8,

S

««

tn
               «
               i    i
               IU


               X
               •«
               91
            t« n


            IS <
Q
Hi

a
               a
 s»
»tO WQ —
«>« W W  P- co —
a o o a •





O3 W O$ N Wl

« V M * (O
P*« — W
W f*.f>4 W M

-e^ttdB
a<-4 PU

fl9 IO «(O «
f* M a r« w
— «  «"
C* « « N P«
T> Tf -5
wtuiuttn vi
caa 
A f» a» f» w
O l» T CO 1O
«« «  » oa
o — !•) nio
<»• «M ey r» r»
-) -» -m
Wl tft IA Wt Ut
0 CO to fiO 49
o a w o oo
O f» » 0 t
— — P4 ft O
r» r« h. m 0»
ooooo
f»  a
a o a o a







r* — -• w —
v v v

^ tf>  rj TO
n -) n -) •>
 — « irt
— — no —
c* w w «- -*
— — — a a
0
9
o







n
V
M

«
^i
to
«
-i
t/i
f^
SQ

                                                                                                                                                                       a v
                                                                                                                                                                       < &
                                                                                                                                                                       >- 3
                                                                                                                                                                       z a    a
                                                                                                                                                                       o       *-
                                                                                                                                                                       u u.    z
                                                                                                                                                                          o    a
                                                                                                                                                                       iu    03
                                                                                                                                                                       J IU IU
                                                                                                                                                                       CB 13 t-= II

                                                                                                                                                                       VI S UJ f—
                                                                                                                                                          1/1    «    irt aj ^ •->
                                                                                                                                                                IA    O > us z
                                                                                                                                                                -«    a. < i 3
                                                                                                                                                                IU
                                                                                                                                                                k=    I   I   I  I
                                                                                                                                                          r-    O
                                                                                                                                                                2    « ffl S X

                                                                                                                                                                O
                                                                                                                                                                a
                                                                                             134
-------
a
a
tE
a
1U
a
CJ

ca
a
IU
J
CO
              '/I
            -I -
            •< cs
            +- <
            ou
            >- i/l
              •11
           J l-   (S
           o <    «>
           >- cs    a
              -I    «
              IU    >•

              11
           < x uj u.
           t- iu >- u
                •
du    z

x  •    x
OIU
             o
             •J
             a
   111
   I-
   <



r* r» ^ •*• —





w « p. — —
 ix> a to id

« con * «
— cao v a
•v rw >
•• CM <•» a ••
— — — ft ft
o aoo o


"
a
mow--
"


a

a
a «-•<•• —
a ....
0
*i o«in

0
W —CM «• —
P» « CM V ft
(XJ f» f*» ^ V
«—><<••=» —
<<« i» a n a
gj — Q on
(N f to (»> as
(<< M N C« N
 ia n » f»
— * 
<«• C« f» — *
r»«f» 
nm r« a at
M M «• « «
I/I W» Vt Irt Wl
cO«««
atutvn/t n• ft
a ca n r» a>
t» — en in P»
• » — r-
— — <<< no
P» i^ r* r^ <&
aoooo
i
i


•~ -• 'J) Jl t33







aa
» r« u) «(»
«f» tBca in
p* ^ r» ?* 4
co in i— <•» p»
cam m m in

§CN a o a
a a a a
ocaaa a
— — — — —
— a co T ca
as ca 
-------






























*





t-
u
§
e
0.

a
1

a

i
X
s
U)


Q
«
5
u
Sfl
U
IU
~i
a

i *
•

*
*
*
*
*

*
*

*
*
*
•

•

*
l/>


J
3

1/1


>•=

i/l
"

IU
J
^
a
•*
1/8

e

9

«
•
•

I
*
"

•
•

*
•

*
*

•
,

•


t/l
_j ** c5
< 2 ^
t- < <
ou >
tf»
'a
J i- <3
w V) u
O < •*
t- Z 9
a.
- o
5 z
Ul CL
cr a
Ul
K
IU
U IU
< )™
a <
a -i
-i a

<
tU •x
Z Z
0 a
a a
Wl
u — -^

j ee IK u.
as 
N


V V V V




o oo es o
ra « « sg «



rt P* A 46 tO
^ ® O (^ N
88- WO
r> omcsei
N f>«« N W
-) -5 T T 1







«•=««-
« a wo o
e « »i f*. i»




r» f» « «a «



V V V V


e o e a

o a o r» a
« « n r» n



^ iSl !ffi fll (B
fte Q Q ^| (3
ra o to n f
asesai oo

1 "> •» T T


« as « * i*
a • e « e
» i. 5 « «
Q 00 OB (^ Q
•X -^ •«>•».>,








— •"



V V
M«M











P0 (V
. (




n M
in v
P* a




?» i«>



v y


a a

90
n n



p» *
-------
CJ

a
o
at
a

a
iu
o
a

C8
 «
            <   X
            CJ   <
            O   >
           VI

           a   d
           >u   -»

           ^   5
           Z   9
           VI
IS   <3
3   ^

u   2
«   o
0!
         S3
O
           J
           a
           IU
           H
           <
           a
»«•»»(•»
aoaoa








- IN no —
— — — MM
ffloeoo
» T » » «
oa oo a







— a <•> — —
y v


 
oosoo
in ui — in m
a a -a a

a


fl


a
* - — — -
v v y
e


01 e0 «r ^ f*.
f* M Q P* C«
— gi « a —
tN « (N M  "1 T -1 1
irt u> ut v»ut
«f
V V V


i» in v «• N N <<•
1 TT T •>
UtWIWIUt Ul
O <
O n itf « ca
M ««« N M ft
•t ~> ^ •> ")
W< WIU1VIUI
ti a ffl
KI N a ga n
o — -NO
 f» «
a — n in r»
N r« «< N C4
-)-)->-> -)
I/I 01 tf) WI VI
as en o as a
O f» * — f»
" — ft no
r+ r» f» f» a
aaa a a
in ui in 10 in
a o a a a







- — a ds a
V V V V V


- -n  ui T -5 T
VI I/I Wt U1 Ul
co oa < O —
o en co en ca
a a a o a
ui in r» P-  to
- « o a —
a o> a o o
a o — — —
                                                                    139
-------
a
IU
o
a
            uut
            -. iu a
         K III 3  •
         Oi-z 3
           u
           a
           a.   a
           IU   X
           _j   <
           o   •>
           z   o
           IU

           >
           ui
         IU n
         ffl •*    <
         < u    >
         «• M « « N
1 Tl -S "?
W»«8
iE tO « « *'
IB as ffl as a
CJ O (O P5 to
N « a — «
N. -s» •*»%.***
00 — — —
f» f» f, ffl CO
O Q <3 O O




V V V V V
— — N fS —
V
^ <» ^ft 1=1 e«i
V V V V V
<(= \otffl as IB
r» «> aato
nntan v
asasoso a
N P« N n n
•mm
WtWttflM WJ
to*® «f»
« w® o«
» ~«- a
a — — n o
** "N. *s, -V X
N C4 P4 M •»
•• «5 OT> ^ Q
as te
00




V V
V V
V V
r« V
» if*
V O
oo
nei
T T
VI Vt
f»»»
tea
IBM
-• w
•Si X
oe
IM
*

a,




a

£
                                                              Q
                                                            IU Ui
                                                     «<»   o:.   . _
                                                     —   a. <«3
                                                     O


                                                     a
                                                     o
< aa Q a
                                                                       190
-------
*
*
*
*
#
*

*
*
*
-J
3
ul
(if
X
Ml
HI
Ut
a
a
ui

*
*
*
*
*
*
*
*
*
*
*
*

*
U
J >.
£z u
uj "*
j — -3
t~ r u

•ROOIC 1
: COUNT f
icpu/a LC
< >- o
Z < -J
< J
CX
t-
Z !3
U 3 x,
-a 3
au u.
o u
III)- O
^ < j
a.
13
jo. z
< ui a.
ucs a
Itl/l O
a ca
CE x.
u — a
it a a
a «
CS -x
< U. 9.
>-— a
t-o o
U -J
VI
-1 0
1- J X
so
o
§
-J
>-

-i
•r O M « o
% as n fx x  us  — M 
a
a rx x n U1 o CM in
tOfM CO <0CM
a
fM COM CM 01
rtia — MM
r- fx o infx
a

(• ca coca ca
a
»»<»»•»
eg --a ft » —
o ix m caca
a CM a M M
— a co o» —
CAM M vi vt
cacoco cecg
r» n o r» »
CM O — — M
ooooo
fx f» -a -a a
T 31 O t CM
co ia — n ^
V
3 co 3 in n
us co a cr» to
^
V
9 ** U) ^ O
id in » ca M
a caca ca co
0 10 •"• •• O»
p* f* r» « to
^ — co ~ «
« 10 — M »
a
C4 Q 9 <9
rx  O C3
n in M t —
V
a a o a o
T a a a a
—
V V V V
O
— ca a fx T
C0 M IX c^ (fl
ix CO CO <0 CO
a
to in co  1 T T
I/I VI VI VI UI
co coca coco
aa cacaca
M 01 cano
— — M o —
CO <0c0fx ix
o a ooo
a
V O T  o
ca to ca m ca
a nrx 01 ca
M M M M M
T T T T ~)
VI UI VI Ut VI
a to a to co
a aa aa
fx » — fx T
— M n o —
fx ix rx a a
00000
a a in o a
a o to o o
a o ix o r»
V V
a o a co «•
o o a - ~
'
V V V
a
to a ca ca ca
» in fx fx n
— rx M o a
to in  to
a
a CM o - -
a r» T a »
no o»no
a ca fx 01 ca
a aa
to to co O a rx
in M T CM n
a
y3 Q ?x T ••
'
A
a
a to — CM co
in in a a T
in' rx ca ta ca
a
acaann
o ca can ca
o a ca ca ai
a
aco n mcacao
a
O « CBCO*
in ca •* ^ a
CO T r- tn«
a
§ CM m n CM
a m in ca
ix » r- m to
a 01 mn o
ca n r» ix n
uj a a a co
in a rx 31 T
o ta T T T
- f T rx a>
M CM M M CM 1
1 I -t ~> -1 1
I/I I/I UI VI VI 1
1
(O CO tOCO CO 1
aa aco co i
in CM ca ton i
M O C3 — CM 1
ca o o a a i
o — — — — i
191
-------
x

3
u

as
a
IU

i
•*
X
i
u
Us

a
 its
 a. z
 a 3
 3 u.
 ae
 iu
           -J —   r
           < O   4.
          O 3
          t- u.
 u z   d

 a o   3
 a u   u.
 s      u
 Ul IU   O
 < H-   O
 Z <   -I
 U 3   ^
 -a   s
 au   a.
 o      u
 x in   a
 
i- u
o o
                 a
  iu


  a
                       to o t <* a
                       in a T o o
                       ^ a o>  r-
                      me  -.

               in 10  a
-------
Ul
u
I-
u
I
a
a
IU
I
•V
 I
     J
     3
     VI
     iu
     oe
     Ul
     IU
     Ul
     J
     a.
     t
IU
H*
(J
<
a
a

i
Ul
a
<
£
a
u
o

-vi a
4.1
IU
j I- J
• < «4
• FU/G
a
j
a
a
J

t-

— as ex a a

to as ex ;x —
\ f
ex ex ex ex ex



txamesco
ex a
in ex <•»*•«
ea a so » r»


a T i» f» —
ae»<3 ear*
IX T » V »
in n * «p»
* coeaa —

VI Ul ut VI VI
ce co eoaa
ce rao ex a
ex m ta a ai
exexexexex
ui vi ui in vl
•O tO 'O tO to
CO  a a
a
efl a — — ^
— a tB a ex
to a ai m to
aaaaa
r- a a  T
a co f f 9
— v T r» 31
ex ex ex ex ix
VI Vt VI VI VI
10 c0 a a a
a ce a a a
in ex en a n
ex a a — ex
en a a a a
a - 	 	
                                                                  193
-------
*
»
*

•
»
*
*
»
*

*

*

*

'*
»
*
*
»
*
I/I

H

•J
a

VI

e

k=
VI
iid
*-



iti
_j

a,
a
<

v>


a

e
»
»
»
e

•
»
e
»
#
»
9

»
*
»
e
•
*
VI
-i i- a
< a: >.
o u >
(- w) O
VI
u
_J 1- IS

>• '-^ u
O *C 94
t- x a
•*
a

- o
» z
wi a.
ae a
IU
X
IU
1-
u tu
a <
o u
-1 O

a. —
U
U 13
UJ ^
Z 2
i 1
OT
y — >.

< nil a.
t- 1U K O
2 z < O
we «i



u
aa
z •» a
"o z
wa  ar» e






eeta in a ~ ao
e a a a a
m a«o a

V V V V

« a 8» f» 10
— «* •* a «

N M N P*  —
. . . o •




V V / V

	 
aaiaaai






 s a a
f5 US ^ T T
OS SSQO O

M MMM VI
5B Cg IB® OS
— « — 
-------
u
••
ce
X
a.
a
01
i—

VI
a
CJ
C9
           UUI
         l-iu 3  •
         OHCB3
         HZ-  •
           a   ca
           <   >«
           u   <
           o   >

           I   °
           ut
           H«
           ix   o
           IU   >.
           -I   <

           i   Q-
           IU
•»   o
CC
           t/l
         IU«   13
         JOB   ^
         a <   <

         2%   §
           IU
           i
wiaio m
ooooa


•» ^ w — -^

^
^ — M — —

m M/ w « <•»
.re _<* — .
V V V V V
•a n » aj i*
4 tM tM r« w
-)->->->->
vt in vi vi «t
10 co — O«<"»
CM v  -5 1 -) 1
at wsi/» «i wt
cO tO to (0  -1 1 ->
utvu/nntn
C0 CO < O •• — N
« * ^ •» ^
OOC300
CO f» CO P» <8
OOO 00






l/> <•• — — CO
V
y v v y v
s»«* ^> O
(• o n T o
id r- am -
M N N f<  IO tO
f  T> -I
I/I I/I 
C>« CN M CN (N
T 1 T T T
 r* r» 
oaa o a






— — — — ID
y y
v y v y y
at r* ta o co
ce « a co in
— o in  d> a»
ooa a a
 T
a  co ft
CM O O — 
-------












X
3

1
Hi
u
s

u
3
a
a
x
a,
a
(3
a
s

i
<
i
at
Ml
1/9


Q
.
N°
S
a
u


tfl
h-


U
1
z
<




+

*
*
*




9

*
e
9
o

»

^

»


#
•

i

uvi
^" Ui 3 •
O i-ae 3
I- Z- •
IU > —

.
u <
a >
0 °
L_
ut
a a
IU -x
i ^
*
=

V)
5 o
3 >s,
Z <
U >
- S
l^



Wl
_! « >»
5 U >
— OT a


s

o
a












tote « ma
oao o o




A OB •** — 9
O O * • O




ta as — — «
oo • • o





9 ® -* — Q
O Q • • 0








® ® ^ «™ fl
as • • o





near* o
— ^ ^ © &
ffi® A 08 <»
PS « N W M
-J =5 1 1 -5
(rttft «rt «9 V!



ce to is a as
«9 ffl ® 55
o« PS to «r
ra a — <»e o
O s^ ^ "^ W


OT «Of»f» ^.
OOOOO




— — <-««
. . .Q "!




— — •= gj _
• • -O •





— — » 38 —
. . . S3 .








~ °°. "". « ~





«f» owe
ss MS a as
nio T T T
31 OS Q Q O
<"« M m n «
1 T -5 1 -»
Ifi V9 t^f M W



e to a F>= p»
e a a tea
— a — « M
--no -
(•« M ft a, _


a
a




_





„
•





OB















US
to
to
o
n
•^
 IU
« at

 i  i
          u •«»
          ZZ
          ut a.
          s a
          U!
          it n
          Z Z
          w 3
           i  i

          -> 3
                  196
-------
p>
CJ

a
o
ce
a


1
to
 i
t-
M

VI
ca
CJ
- CJ
J -•
1 3 u.
3. Z U
tu Q
r
j
-i — 3
-C 'J Ik
1- Z U
h- Ik O

h-
 10 a <*>
— — a CM 10
•»ncM CM *•  ten ta<8
10 ta » » »
co CM loaca
 IO T a
•r to p> 10 a
ap» ea car*

ta
aiocn»<3
a to ca CM CM

ta
taujea — ea
aeoioan
ca
to •• v p» a
co to en T a
!•• r* p» r* ea

n eo ea en

ca
•a —  o
CO eO d3 O 'f
» IO T IO IO


o ca « CM co
a to a i" —
a a v a n
 r» r* r»
ta
loaacM M
to a n «r a
10 — CM eo co


— ea n CM 10
1010 1010 t0
ea f» « to ea
a r» a n >r
n to r> a a
CM CM CM CM CM
VI VI VI VI VI
» as to cn 03
o ea 10 CM a
n a — CM CM
» 10 10 10 10
aaaaa
r> co — T —
a r» .3 — a
a *••*!•• co
— n 10 «c «


— 
ea
to — f»ajee
ta a
»ar» O to
» I0CM V •
•• — a —
 r«
— — CM n a
aaaaa
 « caaea
a -a to a
a n ca n
V 10 ea
y

* 31 — as —
iota ea to  O O en CN
CM — •» CM CM
y v
a
— » a *• CM
a a — en a
» CM in ui eo
ca ea P. m P-

a
CM i0 *- n *r
n a n to CM
ea a eaa ea

n a
a a — — — i
                                                               197
-------

























































X
a
i=
l/l
3

3

2
O
U
p*
u
Uj
u
ffl
< t
!-» 1
*

*

»
»
*

•

*
*
*

»
•»
*

*

*

•

•

t
»



W»

h-

J

3
vt

UJ

X




>-
VI

IU

»•





IU
J

&

a
<

vs




»

*

*

*

»
*

»

*

«
»

*

»

»
*

*

«


*
*

•
•
u

J

51
a 2
z
tu
z
y*


J —
'< 'J
t: 3
Pi j.




*-
u z

9 O
O-
<
a

i .
cj i a
x, i
3 i aotao n
u. s a o in a to
u s a o N on
a i •» *• w N c«
_j tp
I V V
II
t
J
•j i a
3 i a a — a -
•1.1 a a c o »-
u i 
j i
t
i
1
i
<3 i a
•x i
z i «*• — <-«
a. i aaioata
31 an» X, X, «x X
1 O O — — —
I
1

>r — — CM ui
a « e w -
« «?>« T
^r o« « n  ••
O d fx n o
C4BQ Oin
» f- O « fx





_


— « •«•«««
s f» ^ •» a






a

no «• nn
V O W CD a»o o
i •? -s -s •=»
WS W8 B9 Wl Ml



« «« lOf*
% (8 9 O OS

oo
— n

V




a »
o —
a -
- t^j
^ m
KI <>«
U) US








•= 91
KI O
to 10









0)10

7 {0
a a
P7CJ
•^ ^
-------
*
*
»
*
*
*
*


;
3
Ul
X

Ul
IU

IU
J
3
VI

•
•
•
•
•
•
*
*
*
9

*
I/I
H. < <
OU >
t- VI 3
Ul
'U
*- Ul 'J
O -t a
i- cs a
a.

- (3
- Z
ui a
« a
IU
IX
IU
U IU
a <
3 £
u

J <3
IU •»
z z
I/I
 o a <•» —
ta ca ta a o

» OCM IOM
o — o as en
CM » 10 r» a)
CM CM CM CM CM
VI «l VI I/I VI
a a 03 oo os
o N an o
CM CM O •" CM
oaooo
o ~ n *~ CM


"•


CM — ~

a
fM CM CM CM ft

a


a
oaa oo
» n r* o r»
eg •• ca •*
a

a
a a o a o
a co 0» r» a)
*««»«




a
a  a T r~ —
ncM o (M —

ca r» Br>coa,

p» f« i»a(O
-


CM a f» CM 10
T as r» ia f«

- in CM a a
0 i-or» CM
-CM «(oa
CHCMMNCM
Ul VI VI VI VI
a a a as
a — — CM a
3J co cO 
oo o a a
a

,.:.-
•*•
v
3J
CHC4C4N (N




a
M C4 Q Q d
• • m v •
v v
a
«• CM a r» <•»
in a a a a
oiaia — at
ar» aa «

a


a
o a *»in —
o o o
0 0 - - -
199
-------








X
••
X
1CT/ SAWDUST
a
o
oe
a,
a
at
1
s
i
«£
I
*
Ml
IK
H>
•=
O

H- «-=<««!«
y
n n in * *
-5 -i -» -5 -S
I«U9 «» «9 MS
V — « — «
O — — rt O
« PJ «>« C« = 1
^^ i^


V V
N M



— f>
y
MQ
* «


a
OS
'87 SJ30488 -« 9
'87 SJ3065.6 i
£N
SI O
200
-------
 3.
 OL
01
3
 i
x
IU
-1
aa
           K IU 3  •
           Oi-
              x   o
              a
              VI
              M
              a.   o
              iu   >>
              -i   <

              i   a
              IU
-   o
X
              VI
           III"   o
           -1 IS   -^
           a <   <
           < u   >
           m VI   a
              a
              a

ooooo

a


0


Q
a
""* (M CJ -^

« N
aaooa

oa a oo

a


a


Q
0


* «0* WO*
04 V Of* Ok
O4 04 O4 04 04
") -? -^ -) n
V) VI V) V) VI
3 f* 03 rt a
04 O4 Q — O4
a o o Q a

o a ooa








V


Q 04 Q C4 O4
-1 -I T 1 T
VI VI VI I/I V>
a a; a) to  
VI M VI VI VI
^ CO CO CO to
ar» T -f«
- — M n o
a a a a a

a a o a a











«
-) T -1 -) 1
VI VI I/I VI C/l
a 
a a a a a
<
oo a o o











f*» (0 r* ai o
ifl o to rf ifl
1 -5 -5 1 I
VI VI VI VI VI
Q CO CO CO 03
ca ui N en 10
- M a a —
a> en a a o
o o — - —
                                                                                 201
-------


















X
3
1-
- as
»- z- .
UJ > •»
fi (3
u <
o >
X &
o
V)
& (3
w ^
J <
0 >
z e

E
2 O
i <
u >
— e





«»'
, u
J S ^
a < <
< U 3>



§

^
O
J





1-








a
too a a> f»
a o a a o



e

. o >o •



ffi

. * .



39
US f**> ~* G» ifl
• e -o -



V V



ffi

. .


V V V V V

\a T — a a

g> " V v a
p» etaaaa
N f« «<« M N
M»«SW»W»W»


<0««0«
a ffl » at a
« ffl «D fi <8
N n a — M
as -— - i







r~ 
a =• — no
M f« N N <-
— — — — s





1
(O Q
aa
















. * " '



V V








V V

« IU Z
.IU

 I   <
       OS O 3
            202
-------
 Ul


 3,
U

a
a
x
a,

a
Ul
 I
x
a
u
ui
a
a
          r a   u.
          a 2   u
          as   a
          a u.   a
          u z   a

          ao   3
          a u   u.
          QC     U
          Ul Ul   O
          < H   a
          z <   -:
          < u
            jj   (3
          U 3   >.
          -a   3
            So   ^
                 U
          at ai   13
          m i-   a
                 u

          -JO,   Z
          <

«««• N W
VIVIUtVIV*
•<»<<««
a a oo a
a
ia — 
'jj t>j o \a o
a a r» — *
aj «r  a
.a — 
A
0)
O t •» T »
a in a» 10 nr«M m
IN -r iacM so
r» UJ  f* ai M r»
n f« » T a
p» 4 A p» l f*
VI I/I I/I VI VI
fl ^ (X
3> « rt — ffl
* n * • (S» rf3 C*J O
* n  31
 •• rt » »
aaea »«
« ia

a can — a
<•» ai — 10 
r* O n n o
a 4 (>4
I/I Ul VI VI VI
 in » a
f» « «  —
m tool * r>
«a»o»t»ai
a
at n
ca — a r~ »
- M  p»  ^
no ci P>  u>
«r» «««•«
f« o n 

a aa f» »  —
•a UT o -a :o
3> «> :•» as  P» r» a»  »
no — u»oi ai at

Mifloiao*
•atoanaio
atiaco » •»
^ <0 10 f» P*

M «x ia  f* P*

Kt to » — a
o w » — n
to 
N M W <>< M
VI Ul Wl VI VI
to to to to a
as aj aj com
« «<
V V

a» — to a f
f. rs ~ a f»
a r» •» a to
^ n n — to
V A
a
n 3) (•• » »
" 01 — «• O
» n at at
a
at o O M -
at » 10 — «
i^ at m « at
r» f» in in »
a
a to to M —
- •
— to r» a fi
« inotQ a
o i» » m *

— <>• a « -9
a  to
a
« « 3» <•* to
r» o 01 o a
a n P" ia to
» m ci«  O «
•- — *• 04 a
«o -  3) O
* .-• ia n a
•T CM tfl <*1 C4


O « /M IN 33
c: to s ijj —
c» ^ vr « r*
» M  n

to  ^ ^
^ (O 31 r* 
-------
 u
 _J

 ii
 03
 tl
 O 3
 I- Ik
 uz   a
 "3   x
 aa   a
 QU   U.
 X      U
 HI tU   O
 <>-   o
 Z <   -J
 < J
   z    o

23    5

S°    5
ce ui    d
UJ I-    O
< <    -i
ja.
«ui
uce
1UH-
   cc
-JO

 I
o.
P- I
°d
  at
JQ
OO
to m
  u


  Q
             ia a» in f f»
             CN 1O UJ T P-
             el 3> o> a CJ
             *N 01 O W <•!
             i9 to r» co aa
               a

               a
             a\an T i

             ap«na<
               a

               OB

             - a ino s
             a i» man
             « n N us N
             » as as as «
             v or*  •• <
            o as® in i
             — T  e ra
             « (>• N M <<«
             "> i "V ") ~s
            « o -> M o
            a - - —
to >T r» is P)
to o « a) n
III W IO A 0
— 
                            •T T W •»
                            a —1»« a
                            10 o — in P»
                            a>nui
-------
J
u


I
I
a.

a
IU
a
 i
 i
x
IU





VI
a
u


IU


a
            VI
          -i —   J

          < as   -»
          - x   <
          QCJ   >
          I— vi   O
            -u
         .J I-
         :3 <

         t- OS
           VI

           K
           III
             .
u o  z
-     a.
x  •  a
Oiu
  o
  u
  V-


  a



CO «3 (I* CO tfl
^ ™
C« M<0<*4 C4



oo oo o
no ai»
a
n»i«- — id
M*(OO«
inio-* — to
<*«<»««
r» r> a»r m f» oi
aao — <•«
(M (* N MM
Vtl/l Ul VK/I
aaa a a
no a no
ft n O <- M
— — M « f« t
OOOOO I
9


a
in ss a a in
M —
a
MM e>4 M M



a
— — nMQ
» M
»
a
p- » — 10 f»
n 10 -
aoiaidia
— P» ^ f. f»

n « as  Vt VI V)
CO IS  m — m 03
""
r* c* is o* IN



o«a»o
n f» o
» —
01 ar»OOO>
N a n n a
a to ai — n
<>• (M  W>
qj a ta to 10
a a a a a
n a f» 4 «

w 01 — f <«•
r- n « ai
w 01 a n o
— — M O —
(O to to r» r»
oaooo
i
i a


a
— KI f» U3 —

a
— 0< f4 ft C4

a

e
— a  — o n n
«(*» — »
*»  M — "
V
» <0 10 <0 »
a n ^ oi 01
- n 10 r- at
(* f4 f4 f4 ft
ui ui in ut ui
a a a a a
N T — r«» T
— M n a "
r* r* ^ a a
a o a a a



n ^ — — 3>

N 
-------
































X
U*
s
IU
^
K
i
^
^
U
I
as
&
a
IU
a
u

s


i
i
X
„
IU
M
wi

a

|E3
S
a
u

&
U
iu

* 1
*
*
^

*
*

+
*
*
*
*
*


•


j - a
< ac ^
ou >
1- -
a J
a a
J. 

a.
u
J
-i (S
ill ->
Z Z
So.
a
^
at
< (3
U w ^
^ ** (£ 3
< e iu ib
1— 111 >> U
I- Z < O



U
M M
2-1 O
111 O ^
au z
X • 2
am
^


ui
t3 (3
Z 3
& - u.
-= 0.
o o




o


a
a







i—


'a
at ifl — — —


V V V V
a

a> at «r » —



a
(MMMMN





a






a
r«n M e a

10 «

y y

a

— 







a a
a e a e —
a e « ft
T 1 -S 1 -1




<9  — 0)


V V

N W N N M












a N N M a
t ...


V



c*) ifl n <^ .^
o — na»
at 9 «<««<>««=





-------
X

3
u
-j
3.
 a
 a
 OE
 a.

 a
 IU
I
U
IU

a
           H IU 3  •
           OI-OC3
             *    c5
             ^    ^
             cj    <
             O    >
             X    O
             o
3.
Ill
             >
§   1
             1A
           IU —
           JZ
           a •<
           < u
           — ul
             (3
             O
IU
H

3
10 » tO » »
oa oaa









a to o ai ta
UjaO —  ««
-I 1 1 T T
I/I VI I/I MM
a to  a a a a
CM O —  o r»  co   1 1 1
en M M M vi
tO co to CO  o>
o o o o o
00000


. .0 • •

. -a • •

V V


r» a o> -  co n
CN 0 0 - P«
« O O O O
a — — — —
                                                                              207
-------
§











X


li)
J
a,
J^
j
3
a
-N
H-
U


as
e.
e
1

ffl


<8
1
i
X
UJ
M
VI



a
©

u
(U

ffl
*=
*
*
*
*
*
*
*
*
*
*
fr-
J
3
VI

IU
ae

>=>

K>

ul

J


X

^

WB


&

9
»
*
e

*
s

*
t>

*
^

^

•
»
•
•
4


*
l
l



 C3
Hi ^
o >
Z 0
*
i
~


(^
c^
z a
E «
u >
•» o

1-



-  ® W P»
•9 ^ ^ O (^
® oeeoa as

WS^W^^
«««««
® © © « 9
3 Je ?s 3 i
« © -> « O
a *=«,«» N
(» OD 0 0 «B




r* i^ «!*«. (0
o o o a o



















3J «= g) ™ OS




V V V










a as ««• ~a
a a o — as
nw v  .
      u a.    z Z
         a    '-u a,
      u    a OE a
      J 4U Ul IU
      33 a K ik »
      _ < ut ae
      "I Z U IU K
•o    u> Ul K I— •>»
VI    O >IUZ Z
—    ft, ««- 3
tu
t-    1   !  i  I   I
O
z    •< a e "j a
-------




















1
0
I
>-
M
VI
a
u
tu
J
<
H
*
*

*
»
»
-»
*
*
»
«
»
*
»

*

Z
l»
Irt
(-
IU
_)
3
<
W1

*
*
•
»
•
*


*
*
*
»
»
*
•
»
»
*
*
*
- 1
1
a i a
*-. i
— i- 3 • o io — <« >a VB
«c a u. i a ii) «r -a s» !"•
k- 2: u i <•« N <5 •• «i an
!— O. C t — T •*• M -. 1
aa 3 i a w« a » —
a o i » N <•• w *r in
< >- a i r» i*  a i 
3
VI
«
H>
Vt
K
ttl
J
a
<
VI

*
*
*
*
»
*


*
*
*
»
*
*
*
*
»
*
*
*
t/t
-J *- 'J
t- < <
H- »» a
<
VI
•-a
~ i- (3
< — -X
:- i '_i
v. cs O
«e
a.
5 5
M £
e a
IU
>
X
IU
i—
U IU
a <
a -t
> i/i
a ""
<
u
<
j
. j o
I I
VI
< a
(J -. -s
-at 3
< 01 h-- ui a j
u
Z J (3
du z
x • a
a iu
ui <3
2 5
£ *.
3 1
« 3
u
a
«
a
j

K
<

9
« N ^ M — f*

v v y
3
M « C4C* <•><•»

a
»»—<>• ?J P4
T 1* <•» * * »

* a
a
vt
a. 
UI
CC 4
1 i
- 9
Z
i/»
Ud n (2
ffl < <

> •«
a
u
a

v-
<







a a o • • •

a
(M _ 0« N — CN

a
ft — f4ft " ft


a
ft •* ft ft — ft


a
« — «<»« — «



VI
a.
o J
^
u. z
M — f^NB O i
— a f- « oif* aa
n 01 Z
— < S 3
IU
H III
<0 
-------



















i

-J

trt
a
t-
M
N=
ty
j
s
<<
w
»
O
e
»
*
»
«
3>
*
«
*
»
»
9
$
»
9
»
«
1
* t
2 0
j ».
Z a u.
a z u
u. a
a -J
K
J
«c a a.
t- a. 3
-j
1—
u z - o
z << -i
&
Z C9
23 3
a U Ifc

tU 1= O
< < J
a,
a
V,
-is. r
< a a
US X
iu *•> 5
(fc at O
j
X es
..3 z
JSS %
its O
(J J
m .
je z
5 j o
h-o a
U J
 W?
a
a
o

H»
X

a
10 a> c ui «o a
- m o « » a
— v — t t —
u
in o a o a a
T to a tno a
M — — SO — —
V V V
a
a*
— van » s>
O ««COO»3»
r« m r- co r» T
a
a
O SJ — 0> » Q
f. - UJO  1C P! <•>
f» f» « -US •
M » « IS
V W— V
X
a a
•——wow
oao •« •
to  CO P^. Q W
•re w w N <°> w
M W W W W (/(
ffl 
Wl
H
ttl
.J
X
<
w?
»
•
*
»
*
0
e
*
*
»
e
*
»
*
e
*
*
*
»
*
- * o
<
0.
z ?
2 z
CC X
IU
oc
tu
H
U Ul
< 1-
o 2
3 Q
> irt
a, —
CAt
kLMQNELLA
MPN/G
W1
< a
u — -»
-- lUffl J
u
S~ (9
a u z
iui s
iu «« —
e
«Of.•,  * 4 W •» CM -»
* « 35 Q C* «
S O O — — Q
»
*
*
»
*
*
#
.»
»
»
*
*

»
VI
H
J

V)
X
H
wv
H1
tu
u
2
<
VI
«
*
•
*
*
•
•
*
*
*
•
«
»
«
•
»
*
»
•
*








U 0
_J p- tU tf

ess?
rOXOCARA f
V
OVA/G i
i/i
M
& O
IU »»
J <
i §
IU
>
£
in
c o
z «
u >
-• o
B
VI
HI «•» 'J
a < <
M VI O
> <
i
a
O
j

*-
<









e~ tor* . M — — *
•o • • -o

a
«. ^ fSQ ao  f«. N — — K)


Ul
a.
3
a a
•x
U. S
nr«Bi(!9K> o i
— «p»io — » ax
« 10 — r» »r 04 iu U!
n» VI Ul I/I -" UJ )= -=
VJ > (tt Z
« < cs 3
Oi
t- iii
 o 
-------




























«
i
M*
»*
M*
It!
VI
U
U
32
<
H t
•
•


«
*
4
»
*
«.
-»
*
*
•

»
vl
h>
3
vi
X

*»
VI
i-

UJ
J
a
<
vl


*
*
*
»
*
•
*
*
*
*
*
•
»
*
•
*
*
*
*
2 a
-- ^

1 Z •->
a a. 3
e -
P
u
*fl


2s: o
j
H
u 2 (3
— 3 •••
MO 3
ce u
< t- o
Z < -1
a.
i-
r a
U 3 •>
a u u.
o u
a: u o
'u v- a
< < J
OL
(3
•x

u i a
u. vi a
j
x a
X ^

Ul U <3
u. a o
•>


HO O
u u
Ul •
j a
t- J M
so
1- VI
i
(5
a
j

H>
«
Q



* o ® to c o
0* fS W !»» — —


Q *•* ^r T 10
— f-s fN jn «f —
a a
CM tO (O 04 W «
n n ia
id a f» f» f» r»
a o
c* at *«<><
aso v w — «t
ajasto aoa
to  VI
a. -
<
u
<
j

§z
a a
-i
Vt
«3
{J — N»

< Ul (" -i-»
<






en *•


f« — 04 *- M N






Cfl d •*» Pfl M




31 CO to n Q a
r% 


o o — a - —
cna o a a a
M o (*» a « «

rt p» r*  * « «n
<*S * ifl f» <3t —
CM M M C« N PJ
VI U) M Wl VI VI
a co .

u
J
a
<
VI


*
*
*
*
*
*
*
«
*
»
*
»
*
*
*
•
*
•
*












J »« UJ (J
<&'•*->+
KUI 3 .
OK* a
l~Z~ .
<

u <
3 °
VI

J <
o >
z a
w.
>
VI
**

5 5
^ i
«
VI
IU "• (J
-J ae -s
s < <
< u >
53 a
i
§

^
<












t
i
o o a o a a








•






a
V
en p* /^ c* — «r a.
naaiuidip* aa
— m as ^ n n lu
nT«r»a>— H-II
ft ft ct ft et n - in
VIV)V)V> ^» 1- —
VI IU Z
-• S3
01
H > 1
(O to 9 to 03 f» O
a 33 aim 
-------






























*
f
«
f
•=3
w
«
"a
iu
K
I/I
PS
u
m
=J
<
H>
! »
*


*
»
-*
»

»
•
*
*
*
•

»
(/»
H>


VI
Cg

H-
V*
>-

01
J
X
<
vt


*
*
»
»
»
•
»
»
»
»
*
*
s>
e
»
«
»
*
*
(J
«t O
-J >*

a z o
3 u. 0
X -1
&
"..

r- Z -
U Z (3
«• 3 "•
§0 a
0* U
< f- O
z < -i
a.
t-
z a
us ^.
§u s

U* t- O
< < J
&
(5
•x»

UOE a
Ik V) O
J
a o
§>.

»

5 » s
K O O
u _>
1/1

§0 *
!•= Irt
O
O
a
h>
g

i3

— O f  f"
N m — u> e"> »
V
Q e
OB o •» 18 a r»
W ««'- f* 01
coo  -


V»
s

>»
V*
>-

w
J
a
<
i/»


*
*
*
*
*
e>
«
»
•
•
»
»
«
*
*
*
e
*
*
W)

H < <
K ft Q
<
- v> u
i- £z a
<
a.
2 a
5 1
K a
IU
X.
H
(J Uj
a <

> Srt
a> M
<
u
 — — w v
r» f» «r» ««
te

N

e
a a a a en «
« « n n tn w
0 0 gB 0 ^ f>0
« s w « tn«
«« SS^1 O W
U3 e tB es v ®
« T tef» G —
« « M W P> «
T *) "1 1 "J ->
(rt tfl V> VI VI t/B
^ « ts® -=©
*
»


*
*
ft
»
*
•
*
*
*
*
»

*
t/»
H

^
U*
«

t«=
M
h-

IU
u
a
<
V9


*
«
*
*
*
*
»
«
•
•
*
•
*
•
»
*
*
*
*











J M (U <3

O^-«3
>- Z •« •
£ a

U <
X O
o
Ul
•*

-J <
z o
UA
VI
i 5
i 5
- o
a
tf»

a < <
•o W» O
> <
1
(3
0
w
•—
2












aa csoe a


83

•

a



®



e



 o
anes-ran a
m s ifi as •tf ts iu uj
n^iopoO— i3>-
(MNMMnn - «l/i
•2T1-51T a aj
«1 OT WS !« I/I  « IU 1-
W« > IU
>- 
-------

























1
1
tu
VI
n
u
UJ
J 1
< t
i- 1
*


*
*
*
»
*
*
*
*

.
(/I


VI
OS
1-

H-

UJ

a
VI
*
*
•

*
*
*
*
*
*
*
*
*



3 x a i - cs f« — —
a: -/ i
IU IV V V
Z I
'r* i
•J i
*s i
-^ ** 3 i rt o c*i o (*• f*
>- z u i a a 33 a T P?
<» u. O' •• c*9 P* «•• ~ CN
_J I
1 V V
H" 1
u z a i
•« 3 >« 1
BO 3 1 «CO N COM 10
Z  a . 1

u •« a i — *>
it a a i ft —
U J 1
i
i
ad! a a
OC >. 1
j a z i p- m » ao -
H -• a i — .— a »
o j 3 	
v- a a i f» M
U J 1
i
i
i
t
VI 1

So * I SSS2SS
f- VJ |
1
1
1
1
t
1
a i
i w n co CD - « a j
u
(3U Z
— 0.
x • a
a .
< 3

3 S
a ^
u
a
a



«


V V V

a
•

CO



a



« ca •• ^ to a
«<>••»«««

r.  r- •»

a a



3<3I C3 Ul O 
z
vt
I 5
li
z


a < <
< u >

s
13
a

<









\
i
o a o o o o


as


a

*

0

33



VI
tX
Q O
U. Z
aaintoaco a a.
ton- ii
CN«ln .. .«M
VI > IU Z
>* < Z 3
IU
>- III
•J to 
-------
























i
>
•*
«">
us
*•«
VI
^
u
IU
-3
< i
fna
*
*

*
«
«
*
*
«
•
4»
*
«
»
»
*
u»
K


VI
2

*-
Ul
H*

tu
u
a
<
ut

»
*
*
»

*
»
»
e
*
#•
»
»
»
»
*
*
•
t
i
* i
(J
- (5
_i x
- -. 3
a. 2 u
a u. Q
X _i
UJ
X
^
a
•••*
-1 - 3
*— ~ '-)
w u. o
.J
»•
o z a
— 3 ~»
a o a
a u u.
I U
< l- O
2 < J
< -1
1
^
z a
U 3 >.
a u ik

UK O
< < J
a,
cj
-S,

u ee 3
u. in a
J
a a
K ^-


uu o O
U -1
3 a
S -s
JO Z

>•» a o
U -i
V)

i- -, »«
>= 01
<3
i

>«>
•<

a a ft o f» f
r- N r
— 71 T T in in
a &  ai

««a>ajffl«i
a
a N ui on —
w* w« * * •* ™
O -3> O> O» 0> 35


as f»  9


3> m c. &e* —

f C9 « « W tfS
W O 08 US® f*
— ta a ^ © —
N N W W M «
W? V9 VI W» tf> VI
ca « «e ® © p*
CB 

1U
J
X
<
Ul

*
«
•
*

9
»
$
»
*
»
»
»
»
€)
«>
*
*
»
I/I
j - a
H- < <
i- 1/1 O
<
in
u
j i- a
w Irt - 
K
IU
H
y u
03 <

> Irt
a. -
<
u
<
j

§r
X
j
U9
«

< tu H u
o z «es
»- IU OS J
u
Z J «
au x
a tu
u a
a ^.
r £
* i
3 o
o 3
u
o'
a
o
«x

h>
<

— — M » — —

y v v v
P5 — M T — —
-
V •/ /
O w w r« w —
a ....
w
V V V V •





« a
l«»
«
o

a <-<3> (3S •-
w M r* w w w
tf9M«A V* V8 tfl
« « ce««p»
e« eeeaaa
—«-«««
w -» — e o Q
^ 

^
(A
H

tu
-J
3
<
Ul

#
*
»
*

e
»
»
*
»
*
*
*
*
9
«>
*
*
»






J i-* UJ O

at- a: =
t= z — •
5 a

a >
§ °
Ul
OBJ

-J <
o
IU
>
£
Ul
•«*
a: a
1 <
•• a
%
Ul
U we C3
O -< <
« irt O
> <
3

H
<







o o o a a o



M





-
n — <» — — —



*^


'
v a a n  o a> «7 a> f» o
— in 8> T n n lu
n » in r» a> — K
(NNMnMn •• ft
VII/>I/IV>I/1VI -=> K
ai iU
« «
IU
1- 1
o to  o> o
04 — — o o a a
v <0 a a N N
a a a — - o
214
-------





















1
a
i
IU
VI
•a
Ui
2
*
*
*
*
•
9
*
*
*
*

*
*
H-

^
VI
0*

t-
VI
IU
J
VI

.
*
*
*
*
*
*
-
•
•
» l

Q£ (J ( PI 10 ^ M P4 ^
IU IU t3 1 . * i • • •
< t- oir»iaui<8r» O
<
ISI
IU
-i 1- J
•t. «* *-
t- vl U
K a o
<
3.
<
i
- Z
VI 3»
c a
IU
«
IU
I—
•o tu
3 *
3 a
> VI
a. —
U
^
J

z z
a a
VI
< a
-3 3
j a: iu ik
< IU H U
o z < o
u
z j a
o u z
O IU
iu (3
a •>.
5 2
" §
0 J
u
a
o

5

s
• a o -a •
V V V
a
. ' / , . a •
a


a



a



a a
a * 
S
VI

— o
«
VI
•u- o
a < <
» VB O
a
j









3 a o a a a


g
^ 2 2 ** 2 2


a
— en — P« OH
P4P4C4O4PIP! ~  IU
>^ < is
IU
1— 1 1
-------


















!
IL,
1
>
Ul
Ml
vi
3
U
w
J

^«
§!fl
>»
au ik
O u
HI H 9
« < J
a.
(3
-JO. Z
u a a
Ik, V» O
j
X (9
Z x,
-SO Z
u » a
U.O O
U J
Z (9
K •*
-JO Z

HO O
U W
VI
j a
t= J !«
K W*
i
o
a

H
<

a
to n N n 03 n
ia B o o f o
to » « a — 01
T» — <*(•»
V V
a
cs -• •* to *~ n
— 3) n m r> a
^ •'3> T Is* \C5 S3
IO M 89 * tf>
A V
a a
u>f» ajooo
T — n NOO
air- a van
a e
R O O O> 05 f
f. o u) r* « »
csa as® «!«
ffi
«*
0 OT «* «Sf5 G
ew — «s««
«io •» «•*«
a
» « w ia w '=
woo » TO
f.  n
o oo —e e
*
•
*
•
*
•
&
•t
#
«
•
•
•

•
vi
h-
3
VI

K
VI
US
h»
iU
J
0.
3
<
Wl

«•
$
»
9
»
*
»
ft
*
»
e
*
€>
*
*
e
»
»
«
t/»
-J "• O
< ee v
H < <
H» VI O
<
wj
:^
^ I- O
< - ^
!•• V M
i— CK Q
<
Q.
2 a
5 z
 VI
X -
<
u
<
J
u O
I f
i 1
j
<
VI
«3
B
<

ca
C3 N O M !M V3
n • « — — «s
<0 ' '

a
— oar. — M
noa •» an
(<« — «••<<•<«<
0>P» M tf] ->*>
p* e  d> — •= «
o oo *• G ©
»
4
*
*
*
*
k
»
*
*
*
*
»

*
V)
t~
3
vt
(E
k-
w»
HI
(-
IU
_J
&•
X
<
vt

*
*
#
*
*
»
*
*
*
•
*
*
«
*
»
*
»
*
*






J *-. OJ (J
< a wi ^»
2SS3.
<
Z 19
u «t
x a
o
t»
Ml
X a
j <
X Q
I
Z
VI
m
ac o
u i
s °
i/t
•U — (3
n < <
-vi o
> <
O
!

^*
<







• a • • • o

a
r*, a. « ^ ^ IQ

V V V V
e
r. «= n P« w to

V V V V V
a
f«* — « W W C@


o
r*. — rt « « (9


VI
&
3
Q
Ik
as »» (<< m — t=» o
p» a w n  W) WS VI "=> Hi K
vi > iu
« 05 CE
IU
i- C 1
to 
-------
























1
1
M}
Hi
1/1
u
IM
<8

»

*
*
*
*
»
*
*


,
-


VI
«

1—
VI
h-
Ol
J
VI

*
IP
*


»
*
*
*
*
*
*
e
*
.
1 U
1 - (J
_i >-.
& Z U
a u. o
a -
-!•
P
J - 3
< O a.
— Z '-'
h- u. C
U Z (3

S U
< V- O
Z < J
a.
Z 13
U 3 ^
OU II.

til K O
tX
>»

 <*3 V3 IN tO —
Jl T T O T O
v y y v
a a

<•) — <•< o> o <•»
in v> m in  as aoiops


M — 


U»
s

h-
VI
H-
IU
U
a
<
VI

*
•
*
*
*


*
»
«
*
»
»
*
•
#
*
*
»
J ~ -J
(- •< •<
(- Irt O
HI
01
-J >- 'J
>- OS 3
a.
5 *^
- z
z a
IU
z
IU
H>
U IU
a <
2 a
> VI
a. —
<
u
<
j

§z
a
j
VI
«3
-«3
_l z iu u.
< IU h- U
Q z < a
t- 01 as j
u
Z J (3
iu a -v
(3 cj Z
x • a
o m
PHAGE 1
•PFU/G
j a
u
z
1

h~
<
t



«~ — • '& 'J3 <3 fN
• • a a o •
v y v











a o
c^ O rt *• 41 ^
	 «c-«



G OOQ G O
<3 a a a o o
o Q o a o a

N — • « U» « ^
HI ffl (O T V CO
v»
OOOO - W
ifl r* a »- — N i
o o a — a o i
9
9

*
9
»
»
t»
*
»
*
*
*
»


»
VI
h-


f/l
X

H>
VI
K
U
J
a
<
vt

*
•
*
*
»


*
•
*
*
»
*
•
•
*
•
»
*









_J r- tU tf
i 1U 3
252=!
<
S Q
U <

VI
M
a. <3
IU --
J <
z a
IU
VI
OS cj
I i
- a
X
I/I
UJ «• (3
j S --
«u >
5 <
a
(3
a

i-
<










o o o o a o



M • (g ^ j gg


— — (O -u
CNC* iu Z
— < Z 3
IU
>- III
co to to to f» ^ a
a aao -<>« o
lA ^ flj •• — W
o o a — o o
217
-------






















as
t
>
01
\n
9
U
01
-1
<
*-
*
*

»
*
»
*
*
«
*
4

»
(/»
V-


v»
e
He
V9
W
tu
J
a
<
wi

»
«
*
«
*
&
«
«.
®-
»
*
*
«
»
»
»
»
*
2 a
_i -.
— -. 3
a. z u
a u. o
a -i
UJ
(-
(3
•*.
i- z u
03 0
t- n. a
a z 19
— 3 •>
a a 3
K U
< i- a
Z < -1
/Y
K>
z a
U 3 -s
a u v.

|U H> O
< < -J
;3
j a, z
 Q
U » X
u. O O
U J
2 ^
..g Z
k- - a
K O O
U -J
I/I
J O
I- J «l
I- «I
i
13
a

K
<

(•• — r» a> n i-
in 3 -r o « — «o
e 
* v p- « a 10
use v n
PSCTWCTOt —
a — a> » t»o
r- M a 01 8» «
«»«»»«
o
— e ION m *
««f» N O »

v otoinma
— «P» inasr*
n* <««« «
MS we ouitfltn


3
ut
CE
>•

H-
Ul
J
a
<
- 2 a
<
5.
= «
M Z
x a
Ul
ct
IU
K
U UJ
a <

> Ul
(L M
<
U
<
J
-i -3
2 Z
a a
VI
< O
U — v.
—  .
5 £
nl r\
3 o
O J
u
a
(j
a
_!

k-
<

- 3j _ 	 5,

V V V
T SI — — — CO
V V V
n N — <<« c«4O






es os «= « c« «


— n * am to
* «-
^ e lam « OB
«- w r«> m as r*»
PS «8 •• T MS "V
w T « ^. os «
P4 W N W W «
tfS W W « Ml W*
sS « « 
® e 
tu
u
2
trt

e
•
*
«
*
*
•
*
*
^
€>
*
e
•
*







j « (U <3

O t- 0! 3
h- Z -« •
1 0
i i
S e
w
Ul
4 a
a S
i 1
UJ
>
V)
i s
i 5
« °
in
UJ «. (S
ffl < <
55 s
o
(5
O

>-







•
a o o o o a


= P» ^ > — e<


~ M — =- ~- ft


n « — — — P*


_  «• «i a» p= a
n  IU
•=» 4 S
i!
1= 1 1
« is « «
o o o — — o
218
-------



















r
*
i
>
IU
H»
h*
U1
*
u
(U
J
<
H
*
*
*

*
»
*
*
*
*
*
*
*
*
%
VI
>-
a
i/i
«
»•
vi
i—
tu
u
a
<
VI
*
*
*
•
•
•
*
*
*
»
*
»
*
*
*
»
*
*
•
u
- (3
i- 3
Z O u.
3. S —
,»
~v
J — 3
«c tf a.
t- 3 '-'
•3 .•= £
•HOBIC 1
: COUNT f
iCfll/G LI
Z < J
< U
a.
i—
Z CJ
»i i
ffl U 0.
a .
-1 O Z
< iu a.
HO o
u -*
i/i -
-ia
so "
H= VB
i
<3
O
J
H
<

\
ci o a - —
T a 3 a o a
M o a n ^ rj


.•o a =3 - - -a
9 a a a s —
<*> o a « en m
V V V V
,. 0
» «-ab T o r»
— 01 « — a in
«<» a — n 01
3jr. 33 (jj CD a
a
a — f» « « v
3»« O 3» « 
r- M a O a r»
tn• i»
aa
«n — «a —
nann na
f» n » a *
aa

i«.»»* in 10
O31 CMP« -  id ai r»
— \o — » n n
n T < N M i
.u
^f J
< «. ^
1— i" U
O •< -rf
<
i
1 1
ac a
01
>
i
IU
h.
U 111
a <
O J
> VI
fc
<
CJ
d o
z z
a a
-i
(A
«d
u — ^
-«3
< 01 h- U
3 Z < 0
t- 01 a -
u
z~ a
tu O ^
a »
! s
— a
i *
o ^
u
o
d
-j
K
<

— ia n — 
V V V V V


a — «F* f»
o is n * « •»
— •" — — — -
« oif» » n -5
Vt I/I VI V* VI W1
a co coco « — — ao —
v ie a a - - z -. .
2 «
U <
5 °
VI
X O
-J <
Z O
LU
r
VI
i ^
s <
— o
a:
VI
IU N. O
a < <
.« a
a
a
a
t-
<







a a aa • •

PI -• •- — « rt


N — — — <•« C*


f» mno« N

M — — — <«4 M


ia 01 f» t n  pw Q
— m — » n n u
« »  — i-
NtMNNNn •• in
-i -> -i T •> -i u
VII/II/II/II/II/I '^ >•
VI IU
— I
IU
H 1
•a 
-------





















t
a
i
•*
>
iu
K
M
W$
©
w
u
544
«J
<
(->
*
•
»
*
*
*
*
»
*
#
•
*
»

*
V)
h»


1/1
a:
H
in
K
IU
-1
a
4
VI

•
•
*
*
*
*


*
ft
*
•
»
4
•
»
«
»
»
•
U
- C3
-f ^
r ^ £
13 U
x. a
X -j
*
o
"•x
-J ~ 3
< -3 ^
!- .£ U
*~ J. Q
t»
u z o
no 3
cr * u
< >- o
z < a
X
1-
§t3
>»
_ Q 3
09 U *.

IU 1- O
< < J
•J
0*
o
*s
JO. Z
UK 3
-
«

— tsi ~* <•> <* a
ft r* at ta f M
T a t to o  » o« a
• o ^ ^ » o
a n o na
— <<• — «•
« |J v p- « »
ina«M » t» —
— — in taetft
a
M .= «r — ««
a?=. f«. a oe a


Q« r*o (fi T
••«««•»«
r* os o <- * P*
C3 <

U)
k-
IU
J
X
<
VI

»
e
*
*
*
*


»
«>
«
*
«
»
e
G
*
•
'yl
w « O
< x -^
h- < <
r- V» O
crt
UJ
-J f» C3
< M ^.
•- VI U
25 o
«c
a.
- O
5 z
i a
U
K
IU
^.
U UJ
ID <

> K1
a. -»
<
u
3 o
§£
i
»
0 — — — ffltH
e e««« »
is <9  « e ~ «
a s o — oo
»
•
*
,
•
»
»
*
tt
*
*
*

*
V)
H


V9
X
>>•
tft
K>
_
tu
j
x
<
(A

«
«
*
K>
•
»


*
*
*
•
«
*
*
*
o
*
*
»







j - iu ta
. _ , ^
ffl »- K 3
*- Z- •
<
e a
u <
o >
§ a
VI
i <9
IU -x
Q 5
' 1 I
IU
r
VI
S ta
i 5
- 1
s
(/%
Iti m O
e <  <
Q
O
O
K>








a o o a o Q


— -«(»«—

— — a> r» « —
• -OOQ •

— — 01 ^ 
a
§ d
^
3 a f- a in 
TTTI-J-J KIMK
40 Irt 
irt > tU Z
— «5 « S
IU
»= III
ta to < r-. o o> 03 n Q
— O O <•< O M o
u> f» a» a •= «••
o o o — o o
220
-------
















t
a
\u
wi
ct
u
UJ
H"
*
*
*
*
*
#
•
*
*
VI
3
VI
ae

VI
Ul
3
^
VI
*
*
•
*
*
*
*
*
*
*
*
6,
»
*
JTAL TH£HMQPMILIC
JNG1 FUNGI
.CFU/G UkttCfU/G
i- J. O
~t
u z a
x u
< H a
r < j
a.
H
§a
^

XUl (9
u H a
a.
'J
**
-ia. z
u x a

3 tS
-10 z
"o o
U -1
I SJ
-JO Z
a" 3
HO O
u u
Ul
-JO
So1 "
("VI
a
a
a



JT c*l P* O W O
d> w •• 71 ^ a
v v v
31 ai o — T n
a f. T a iaa
/^ av
a a
3333?-
r» «f» 
*
»
•
•
.
HAl. TOTAL
iSiTtS ASCAHIS
I.C/G OVA/G
H- X O
a.
IM O
5 J
x a
IU
X
Ul
1-
u tti
§ 2

<
J
-J (9
fz
a
VI
< O
J X Ul U.
az < o
I-UIBJ
CJ
Z -i O
StJ Z
Out
u cj
f|
3 1
0 =.
u
1
13
o



• • • • a o
V V V
55 "J? '-*3 %*9 33 33
V








aa
10 V »f» OO
<>• a n » a o
r* ^ to *• — —
v v v

«OOf» O O
« a o » a a

at — ton •» «
wo a  »»«<•» i
OOO — OO 1
1 »
1
•
*
*
•
•
»
*
*
*
+
•
»
«
•

•
Ul
H
a
VI
X

VI
H
Ul
u
a.
a
•
*
*
»
»
•
»
*
*
•
•
•
*
*
*
»
*
*
*
*





j — u ta
h- Ul 3 •
HZ- •
X 0
U <
0 °
ui
a a
IU ->
z a
Ul
a
r
Ul
« -J
Z <
- 0
*
VI
Ul fc* c3
ffl < <
< u >
— vi o
> <
i
a

H-
<






a a a • a o

r« o« 


"~o -a o
V V V V

— — a — a a


i/i
a.
a o
*v
U« Z
<9Mme*)Y(O a a>
PJ o  IU Z
— < as 3
Ul
H III
DO 0303 sj as z aaa
-------


























f«
1
<
1
M
>
liS
K>
WS
Pfl
ee
u
a*
.j
<
>» t
*
*


*
»
*
*
*
*
•
*
*
*

•»
1/9
H=


tfl
ce
K>
tf?
i-
iti
_i
a
«
(A


*
»
*
»
*
©
»
*
*
ft
*
9
»
9
d
*
*
*
«
U
M (3
-J >*
-3 2
a. 2 u
2 Ik. O
X -
UJ
£
^3
*••
H- Z U
— U. O
-J
u r a
•* 3 'v
o a a
x u
< >- a
r < -i
%
h*
Z (3
U 3 >.
g] U U.

Ul H O
< < -i
1
O
^
-10. Z
u oc a
u. 10 9
-1
X O
^§ 5
u- a
11.9 9
U -i
a a
« >.


1- Q O
U -J
tn

)- J X
O9
K  10 r« n
>aa T
W— —N _
X X
ar» loouxa
tan o v
« —


04 ^ M ® « -V
^ W •- — —

**• '^ *** ^
«J ^ V O V «
ia to teio 10 ^

C9 lO«
z
H»
M
K
01
J
3
<
i/i


*
*
*
*

*
*
»
*
*
*
»
•
•
*
•
*
*
t/i

>- < <
H-  fl


«<»• OB 9 » «*>
r* Q ee <*s  W
i ** -» m -)
w wt w» wi wi «t
« to 
— - o o -> o
tors. « - — «
a o o -a o
*
»


*
-»
*
*
»
*
»
«
*
•
•

•
V)
h-


vt
&
t-
tfl
1—
IU
_J
a
<
ta


»
e
e
»

e>
ft
•
»
»
0
€>
ft
ft
9
»
ft
ft
»









_i« uiO

e >- cs a
t- z -. •
« e
u <<
X O
g
in
a. o
j <
Z 0
UJ
>
£
V)
»•

•«
u 2
MA Q
%
m
i »
C8 < <
•( U >
» ui o
> <«
1
 a a
e — 
-i^^-ITT lUh-
«/» VS «5 vt W Ul •*» h* «
V! U Z
— as
IU
H> ! 1
!O to <8 » 9
-------

















J
a
(U

uj

*
*

*
*
»
*
*
*

VI
a
trt
cc
VB
IU
III

VI
*
»
»

*
*
*
*
*
*
*
*
fj 1
•* (j t a
j ^ i
a i u i ai » 1
SO 3 I a O — 3»«cO
 co r- r. r» co
Z < -11
< -1 1
1
z a i a
So u. i ex « m — a r»
u i M «e< » in —
iu)- Oi c»cBt» en t» a
a, i
i
a
a i a
•x i
-10. Z 1 <* a tt
U J 1
t
i
a
a (3 i o a"
§>. i
Z i r» a <*• 01 0 «t
 *r r» <•» P»
HO O I r- « co co 10 »
U -J I
I
i
i
VI 1
•^ Q 1 CW td 
O i in « t*i as v . %
H t IflO «O <»«
< 1 O « W N W C«
I U3 - •< < i
H vl o I
< 1
IV V V V
1
1
t
vi i en
u i
-JH i3i — tntNtn— T

>- as o i
< i
a. iv v v
i
i
i
i a a
< i
•• ^f C9d**d?lul
- Z 1 «
x a i
> 1 V V V V
1
1
IX
IU 1
i- i a
U IU 1
S 3!
> VI 1
& - 1
< 1
» 1 "" OKO
z z i a
< 1 V V
VI 1
1
1
 oi
i
i
i
u i a
Z -1 13 1 eO P» « CO — «
HI O •« I • • . •
(3U Z 1
Q IU 1
1
1
IU O 1
o >. i a
< 3 1
X u_ i «  IUO
at- a 3
HZ— •
:P1S TOXOCAHA C
V
'G OVA/G 1
-J <
Z O
'U
>
VI
1 -
s 5
- 0
CE
VI
ee < <
-> vi 3
C3
0










a a aa • •

a
. . a • ~ •
V V V V
a
^

a
— — a r« — »
. . a • • •

a
•" — CD M — »
. . a • • •

VI
a.
i
'A.
- ft r* o » m o
a en in us o f> a
in CB  IU
— < x
IU
t- 1 1
to 
-------



















\
Ho
)
tx#
«
>
«
K1
ws
WS
qp
w
u
&
-J
ffl

ttl
-»
/%
^
x
<
ys

6
«>
*>
c
©
&
«
*
•
e>
»
*
©
»
0
»
»
§>
9
*
«
2 o
.j •»
r a u.
a z u
O 3 t3
a t 9
Z U
'.u
1™
O
^
< <3 U.
1- Z U
O^ t-i
~i W
1- U. O
u
u Z ta
— O v.
a a 3
O U tt
z u
Ul IU O
< >- o
z < u
<3l
Z 53
-a a
** w ^3
a u u.
o u
Z IU  vi O
^
X <3
OS. ^
JO 2
< it. a
£!@ O
U -J
a o
jS 5
2- a
2^ 3
U -J
V!
j a
53 *
dU J«*
W W
t= v»
©
13
ly
<

a to
o M e N — o
•r uj a o  o  rr to a>
CO p» — o ^ t*J
O M Ul M — 0}
» n N \a in n
a
a a a
— o a» g> CN a
» — a> M T  ft *•
*\a r~ fi*><3
matt* w N
a
e s s e «B
n» » ve v
(O Q • •« •
ffl » 10
r« =.
V V V
@ as
* p» as «» » p*
as e » « « ««<•««««
"5 "^ *S •? "3 t
1/1 at vt vtvt -
^
3
vt
ttfi
«
*>
Ul
til
J
{^

»
*
9
9
•^
•
e
0
9
0
«
9
•
•
«
4
*
tf»
-i » a
< « ^
i~ < <
C3 tj >
H- VI O
<
VI
'U
J t- «
< «• >.
"f _ a.
- 1
a 3
u
O
o
^
IB

a a
in — a r. — uj
• -oo -o
V V V
a a
a — a> f* — o
• -o o • o
V V V
a a
— M — — <•» —


a e


e e
CS <« ™ » (X -•


s a a a
» » n«o «M
a «aa» « 10
« v a»n — as
r» K f» f» a e
«a ss
® * IS S!!ffl(8
•

e a
«  « OS -
««««««
•» as as® - as
P=> <» US — T r«
« V *« O —
w w w M rj rt
T ^ "? m ^
w? w» w M« «rt M
 p»
« «« «0ea
N G W fl> P* «
• *> o w a o
tfl f* « e •= w
G a a — o o
»
*
»
»
*
»
*
«
*
*
»
•
•»
*

•
vt
•j
u»
*
R.
w»
K
Ul
J
&
 «
iy ^ =a
ae ^
< x>
u <
S 0
^
vt
M9
a. o
vu >>
-i <
§>
O
u
u)
3£ O
1 i
2 1
01
tsi)
l/»
W M (j
«^ 9E *•*
a  <
I
0
^
k>







^ (6 Ul (fl P*  a a
T«iS8IO'-»r^ IUUJ
« «• IO O O — 'J >- II
NMtHCinn - < in
-5T11TT • OSIUI—
vitrtviutvtvi ^^ iu>«m
V! > IU Z
« « ffi 3
IU
»- III
0 -j3 <0 e ?• f» o
co o « aa « a z aaa
N O W 03 f* n O
» — O N O O O
10 !«. si o •- n
<3 o o — eo
224
-------
























^
.*•
1
IK
>
UJ
U1
HI
W
U
tit
-J
<
H»
1 •
+

*
*
*
*
*
*
*
*
0
»
*

»
1/1
)-
3
t/l
OS
)•
t/f
h-
tu
U
a
<
t/9


*
•
*
*
•
»
*
«
*
*
«
*
»
»
»
•
*
•
•
2 „: •
j •». i
-, ^ 3 i n « 10 — a «
& z u i o rr- 3 i ui rx  
^ t
1 V
i
i
1— I
u z j i a
- 3 ^ 1
a a 3 i « o » » — <••
a: u i «- O i « f» «<0 f» r-
z < _i i
0. 1
i
i- i
§. i
§. l
ja. 21 iaao — «a
uac a i r» o <•< ?• f» a
it » a i . i

>-•* a i r*f»
i na ai o a m
St ajio r« » w —
i c< v in r» o> •>
u i N (M n is ct n
i vim u> ui uiui
i
i «
< i o — o MO a
t v 
HI
U
9
<
Ul


*
»
•
*
»
*
»
*
*
*
*
*
•
•
•
*
•
•
*
VI
- - (3
K •< <
I- «1 O
<
VI
u
-11- (3
>- irt U
»- « o
a
; j-
2 2
ae a
01
QC
1U
t-
CJ 111
§ 2
> vt
a. —
<
-U
o z < a
H- iu a j
u
•M *4

au z
3« *
UJ (3
cj >.
5 £
- s
0 J
u
§

K
2

1
1
(M M — M — M


« ^ *r 01 — M

v v y v





m^r^ « A^


«t f» 100*0 •-
ia 
« IOP» » n —
(>• T 10 f» at —
M N c< r« N n
V> VI I/I VI VI Vt
.
5
t«
«
H.
VI
K
HI
U
<
w»


*
•
»
*
*
»
*
«
»
»
»
•
*
*
*
•
•
•
*
t
I








j _ m<3
< « vt »»
O»-a: 3
1- Z - •
« <3
U <
a *
vt
a. o
^ <
i 3
tu
>
W9
M

X
s °
VI
1U » u]
ffl < <
< u >
»ut a
> <
a'
i

i—
<










•oaoo •

p« M n<>« — • — r« — <«4


V)
O
n  i* -
VI > UJ
— < as
IU
H i i
-------
*
*
*
#
*
*
*
*

*
I
3
V,
OS
W?

IU

a
V*

e
*
»
e
*
*
e
i
« i
2 a
s 3 a.
a. z u
so. a
S -i
IU
a
«t '.3 Ik
w, x 0
w. it O
-i
u z a
— 3 *N
as a 3
* u
< K O
X « -1
a,
»_
z a
U 3 ».
ffl U Ik
o u
iu K 5
a
a
-%
u « a
IM i— a
Ik VI O
a a
j§ 1

Ik O O
a a
.,§ 5

H-O S
U J
VI
u a*

je vj
i
3
5

39 3J O
V V
a if) rr IN — o
!•) «! iJJ O I- <0
o f» s>  n
T •» N
a a
inm  e as
f» «p us »•> as o
N N M n M CI
"5 -5 •> T "5 1
WIMWIVKAW!
CO CQ <0 (Q tS p» ,
CO Q C0 (8 Q d3
^ CO « w v p*
O O W W W W
© £3 
VI
H
Ul
-J
(A

*
»
«
e
«
«>
s>
e
&
*
«
*
*
e
»
«
9
e
©
»
< at ^
H < <
H fl O
<
tu
-j *- a
1- VI U
>~ as o
<
** O
M z
 VI
a ••
^
u
J «
z z
§ '
.
< IU 1— U
o z < o
t- u a j
u
z " a

x ° m
O U£
 — *• W
y v
M — ' => M n rs





)N «=«"P4 Cd«



M Ul tO r»  ffj M
« — io — OGB
-9 *1 T ^ •» n
w» M» vj tfi = cfl ® fX T f*
O @ N (M W N
©o©o — S
•
*
»
»
*
*
»
»
*
»
•»
»
»
»

*
VI
^,
3
v?

K
«
^>
tu

S

»
*
»
*
*
•
•
*
*
*
*
»
*
*
*
*
*
»
*






J — 01 13
o i- ce 3
t- Z - •
<
ae cs
U «£
a >
X O
a
VI
k«
& cj
J <
Z 9
IU
>
Vft
ws
K CS
r <
- o
oe
iu » (9
a < <
MO V) Q
a
o
<







o oa a o •



^ ^ A ^ ^ W
•




— — 81 — — M

V V V V tf V
z
— ut as o (•< v a
- a ^ — « PJ aa
* — in — a a iu
N»«p"aso i— it
N W N W N PJ *• Vfi
V! tui
— eg s
K 1 1
« to co ia  — —
a a a o — o
226
-------






















0
I
<
1
X
!U
M
VI
i>»
N
u
LJ
J
02
<
w
•
*

•
*
*
*
»
»
»
*
%
•
*

•
VI
H


VI
OE
t-
VI
H-
ui
-i
a
<
v>

*
•
*
*
•
*

»
*
*
»
*
*
•
•
*
*
*
•
*
U 1
- (J 1 ' a
-J -x f
-.*• 3 ! •* « — — 3 —
o. x ^ i o« f* ^ t"i o f*
a u. a i !•»« — — - —
OE -J 1
U4 | V V V
2 1
M 1
r
•j i -a
-* i
J— 3 l » » - — a -
< 13 u. i vcoooa
K z u i n « ?i « o ci
.— U. 3 ! fM « — ~ — —
-j i
t V V V
1
1
u z  a» 
j i
i a
i
a
a a i a a
X -. 1
jo z i »— v
U J 1
i
i
a
i
x  —
J 1 — tfl U
»- « o
<
1
; ^
5 z
S a
1U
«
IU
u u
a 2

> VI
a. -
<
u
<
J O
z z
§ x
VI
< C9
U *• ^
« « 3
< III *• U
a z «•»<•><•« M «


a



a
(•«<•> rt e« o« <>i


a .a
•• a <* «•*<>• m
«»•««»
i»^«r- a a
a


a
a
USFfefk — — f»
» v » nn ^

» a a« — a
«oec» ««
ac* x -x x ^
T « « OC40I
o a oa - o
' *
•*

»
*
*
»
»
*
«
•
»
*
»

•
VI
M


VI
X
H
U1
t—
IU
J
a
<
VI

«
»
»
*
*
*

*
*
*
*
•
»
»
»
»
*
*
*
»







J M U| (3

255 =
<
5 5
a 5
3 °
Ul
% a
J <
i a
'•U
>
z
Ul
f"" <3
<
s s
VI
a" a
a< <
«j >
> <
a
!

h-
<
a







ao aa a a


a
«<<«(»<«• — —


a
r» e<« ft ft — —


a
M M N M — ^


a
(N (N (V (N — —


VI
a
i ^
a. z
— at-n
<>«c>«N«(««n •• xu>
ut in wi ui vi vi "» lu i- —
Ul > 111 Z
~ < S. 3
111
k- III
» — N a
- aawa a a
•^ ^ •x •* •** N. a.
-------



















1
®
1
X
BOO
UJ
H
vs
ao
(S
u
IU
«!
<
^>
»
*
»
*
*
»
*
*
*
*
*
•
»
»

•»
(A
H


t/»
oe
H
i#9
*»
Ul
.J
a.
§
'<
w

•
*
»
e
#
•
*
«
»
*
»
»
»
*
*
»
*
*
*
i u
« 13
J x.
53 £
a z  r- a o>
f a r» *• sr  — o v « N
f« 10 a aoi o«
a o
N <>• a o  * 1 P' o*
<»»<»<»«(»
a a
n — ->o«^
« w 
io««r siinin
ffi Q
» <»na> — f»
n 
(>< v m r» ai o
N « <«« N (S n
-) -5 •» T t T
MUtOTUt ««M


»
»
&
e
»
•
•
«
e
»
e
»
»
»
0
»
e
»
*
V)
-i - a
«e « ^
•- 
a
IU
H»
U IU
 V)
X
<
u
<
_i <3
Z Z

w>
«3
u-» •>.
-« 3
j x iu ib
< 111 t- U
o z < o
H> iu a j
u
Z™ (3
a u z
§« s
-
|

03
h-  — — «
<~  — -.(V
O ® O — « N
-> o o — rt N
'•O — « — O  VI *fl
tO « * « (0 P*
«« co «a«
f* N « M V GO
o ao* p-e w w
* (0 *> 0> •- ••
O O O Q ^ O
*
*
*
»
*
»
*
»
*
*
*
*
*
*

*
(A
h*>


tA
*
H
M
H-
VU
-J
3,
9
<
wn

•
•
•
*
•
•
»
»
*
»
*
*
*
*
*
*
•
*
*






_j — ty c3

252=!
<
I 13
u <
§ °
)LEPJS 1
/A/a
z o
IU
2
1
tf)
Z O
r <
S °
V)
iu » (3
« < <
>• vi s
> «
O
a
j
>-
g




', J

a «
o o o o o o


a
— -oio <3>-
NWCM<<«P«PS ~ <«?
1-5-?1T-» SlU
VKAI/IOTtflM A  IU
— < K
IU
H> 1 1
 a
aoeaattceo z ffl a
P* OS ® 
-------





















1
a
i
X
1U
VI
A
N
u
IU
-I
<
H-
*
»
*
»
»
»
»
*
•
*
*
»
»

*
vi
K


VI
«
K
VI
K
Ul
J
3
<
VI

•
*
*
•
*
•
*
*
»
•
*
•
»
*
*
»
»
*
»
f U
- (5
J N,
Z c* u.
a z u
a u. o
a; -i
i—
o
**
J — 3
< 13 It.
1- Z U
>- u. O
J
si ^
a a 3
at u
« t- o
Z « -1
&
z a
.
<3U Ik

IU t- O
< < -I
a.
(9
JO. Z
uac a
ui H a
u. ut o
j
3 (3
.
JO Z

>-o o
U J
i/i
JO
S^ *
>- ut
a
3

H-
<
fl

I
I ' a
> o a a v v
'.o o a o *> <>4 — IB
d>oiiiia ato
nn
a
a — oiaioa
rt r» f» to v r*
ftf»ftia VtWI Wt
[fl  *
*
*
*
*
»
*
*
»
-»
»
•
»

*
VI
f-


VI
*
t—
VI
)-
IU
j
X
<
VI

0
*
*
*
*
*
»
•
•
»
•
*
*
*
*
*
*
*
• 1
VI
-J~ 13
< a *%
>- <« <
1- v> O
<
1/5
j£ tf
< » -,
>- ^ U
K « O
<
2 «,
5 z
CE a
IU
X
•u
t-
U IU
a <

> V*
a
<
u
<
J
J .
-063
-J IX IUU.
•< lUt-U
o z < o
I-IU « _l
u
r " o
du z
< . a
O IU
Ul O
2 5
r <*.
j O
3 J
o
§

1—
<

a
— M «- (^ T aj

a
?9 fy yj *• «r -•
• • • • a
,n ~» — PI •

a
N — p» — — —


a



a
« — — a — —

a
!•«««« —
a or» <•« <» a
r. p» T v  o
ft ft f» N ft n
vnnmututut
- z - •
at o
u <
a >
X O
0
w>
& O
IU •x
^ 5
z o
IU
1
Wt
* <3
1 i
- o
as
VI
!U» ^
O < <
< U >
>s °
o
Q
O
J

h-
<









a o oo o a


a
n « — « iv —

a
— — — wr« —
. . .00 •

0
P4 «• to  c« as
 a ut-ii
CSC«O) •• w>«
Ul > IU Z
*~ < ac 3
IU
1- III
cO a to « » 
-------

























(X
1
a
X
m
1—
a
3
h- 1
! »
*
*
-
*
*

»
U*


tfl
oe


u,


u
3
VI


*
*
*
*
*
*
*
*
*
*
•
*
-. <3
_; ^
S3 Z
O 3 O
a u. a
s u
< cj iu

u z d
— 3 -»
no 3
ce u
< t- a
2 < J
^-
z d
•• O 3
ea u u.
Si
tu i— O
< < u
a
o
j a z


it° •
JO Z
< u. a.

U J
s d
-JO Z


-0 §
U J
l/i"
JO
< •»
i- J i«
O O
v- in
a
9


CD
a* T 
t- in o
V)
>- « 0

- z
ce a
IU
ce
IU
u iu

a j
a «
u
«j
-i <3


i
l/»

a z < a
H- IU 03 -J
M k«


x • x
a iu
iu d
d ^>
Z *•
J O
0 J
u
d
8


9
« t a a a> o>
• a a o a a

a
• o a o a s

ca


0




*3»


fi SO
<*s (s e« 
-» wi a
1
O
J









OS
a a a o o Q


*:....



a
• aaao o


0



a
N « a a CD ai
•e aaa a


a
a d
IS. Z
« — — ««•-. o a
nacotncgco @x
(VWWO4NW ••  -m •» ^ siui-
vt > iu z
— < ce 3
IU
H* ) 1 1
* « — N !>« Q
a a a — — a
230
-------




















«
I
Q
X
M
U
M
t/>
c»
u
ui
-j
<
H>
»
»
*
*
•
*
»
*
*
*
»
*
*
*


*
t/t
>-


vi
c
>-
UI
K
III
J
a
<
vi

*
*
»
»
«
*
*
*
»
•
*
*
*
*
*
*
*
*
* t
(J '
M & i ' n as
J N. 1
•* K 3 t r» p* I.N « «- ^
Z '-3 U. 1 O <* W f M «
as u i ua ^ « r» «B ai
an. o i a» a> in * •*
X -1 1
S | v
h- 1
J
•j : a a
•%, !
_j— 31 —— — — «•?
< -J il. 1 .3 T O O l*» 43
H r ui r» a « i»> a o»
(- u. a i ia 10 to * ia
-j i
1 V
1
1
k- 1
u z 'J i a a a
-3 xi

x u i in « « a - i
z a i aa a
(J 3 >.l
SO u. i r* f< a>«« T
<* * * "^ ***
*i h- a i to UI 1
1
t
1
1
1
1
a i
z i ctaootoo
i — co a caca
y- i taaetp- ft n
< 1 — O OW — —
i ia r* «a — «
i ao a -oo
*
*
*
«
*
*
»
*
*
*>
»
*
*
*


*
VI
H


VI
X
>»
(A
H>
UI
U
a
<
i/i

*
•
•
*
•
*
*
•
*
*
*
*
•
•
•
•
*
»
*
t/i
j - ^
< 06 ^
H < <
t- u» O
<
'J>
>il
-it- 'J
<< -. X
1— w» U
i- a a
a.
<

5 z
 VI
4
CA»
U-MONELI-A
UPH/C
VI
 a a
ia a N r-» CM rs
— OO M — —
m ^ «o •* «
o a a - a o i
»
*
*
»
*
*
»
•9
»
*
*
*
4
»


*
W1
V-


vt
ce
K
VI
1-
Ul
J
a
<
VI

*
*
•
*
*
*
*
»
*
*
*
*
*
*
•
•
*
*
*
1







j M 
»UI 3
> •<
ci
o
o
j

K
<








a
o oa ooo


a a
a — u is a a
a -oooo
a a
a — ia  a a co a a a.
- 03 o f* o a> a a
p-aiui — VT tutu
n» IU Z
•~> < CE 3
HI
H> III
-------




















i
u
X
01
u;

*
*
*
*
»
*
4
*
*
*

3

X
t-
Ul

111
J
a
Ul

.
•
•

•
*
*
*
*
»
•
e
»
•
U
~ 3 u.
a. z u
a u- o
£ -I
-
J « 3

(-0. O
-1
h-
u r a
— 3 >>
§O 3
Z U
55 3
< u
a.
t-
r a
U 3 •*•
-0 3
au u.
a u
m ^* a
CL.
«J &, 2
u OE a
ik> wt o
^
a a

U -J
3 ^
J Q Z
I— — ' 2
U -J



M Wt
a
§


— o n \a  ~ -

V V V
a a
a>a> — net at

mar* a CD r.
a a
onoffl o -
r»a»a»iB3i a»
a
a a
es  o>
« — 
-


in
J
S
<
V5

*
€>
»
0
*
*
*
*
*
*
e
*
•
*
4
V)
J - O
< tS -x
fr- < •<
H- (/> Q
01
UJ
J >- C3
J— VI U
< w
a.

-DBACTER
VER!
M-AfE Ml
> la
a
^
U
cJ
-i O

a
-3 3
< ui t- U
o z  « M —
. . a • • •
V V V V V
CO
C3 — » « N —
-
a

V V V V V
a

e
FS « ft ft a ft


a
 98 B*> « tfl &
<0 «D O P* O'® ^-,
OQ


Q
fi

 58 O •• «
o Q e — a cs
*
. •
*
»
4
*

Ul
3
V)
as
»
(A

UJ
--r
a
Ul

4
»
4
4
*
*
4
*
4
4
4
4
4
4
4
4





U Vt
< SS*s
O H- CE 3
t- r- •
£ a
u <
x a
o
a. a

z a
>
 ttl Z
h= 
-------