EPA-600/1-79-015
March 1979
VIRAL AND BACTERIAL LEVELS RESULTING
FROM THE LAND APPLICATION OF DIGESTED SLUDGE
by
Metropolitan Sanitary District
of Greater Chicago
Chicago, Illinois 60611
and
IIT Research Institute
Chicago, Illinois 60616
Contract No. 68-02-2223
Project Officer
Walter Jakubowski
Field Studies Division
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects
Research Laboratory, Office of Research and Development, U.S.
Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
11
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FOREWORD
The U.S. Environmental Protection Agency was created because
of increasing public and government concern about the dangers of
pollution to the health and welfare of the American people.
Noxious air, foul water, and spoiled land are tragic testimony
to the deterioration of our natural environment. The complexity
of that environment and the interplay between its components
require a concentrated and integrated attack on the problem.
Research and development is that necessary first step in
problem solution and it involves defining the problem, measuring
its impact, and searching for solutions. The primary mission of
the Health Effects Research Laboratory in Cincinnati (HERL) is
to provide a sound health effects data base in support of the
regulatory activities of the EPA. To this end, HERL conducts a
research program to identify, characterize, and quantitate harm-
ful effects of pollutants that may result from exposure to
chemical, physical, or biological agents found in the environment.
In addition to valuable health information generated by these
activities, new research techniques and methods are being
developed that contribute to a better understanding of human
biochemical and physiological functions, and how these functions
are altered by low-level insults.
This report provides an assessment of microbiological levels
in surface and ground waters and in aerosols at a land reclama-
tion site receiving large quantities of anaerobically digested
sludge, The accumulation of data on microbiological contaminant
levels as a result of land application will enable a determina-
tion of potential health effects associata&ovith such practices.
R,
Director
Health Effects Research Laboratory
111
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ABSTRACT
Surface waters, ground waters, sludge, soils and aerosols
were intensively sampled at a 15,000 acres (6,070 hectares)
Central Illinois land reclamation site during 1975 and 1976.
The site has received large quantities of anaerobically digested
sludge (M% solids) for several years (1971-1978). These samples
were analyzed for viral and bacterial components to determine
the impact of large scale sludge application on the environment.
Conventional techniques (MPN and membrane filtration) were used
to measure the bacterial parameters. Viruses in water were first
concentrated using a modified aluminum hydroxide, continuous-flow
centrifugation procedure. The concentrates were assayed for
virus using Buffalo Green Monkey (BGM) cells to demonstrate
plaque-forming units (pfu) or virus concentration. Viruses were
identified by serum neutralization tests. Bacterial viruses
(coliphage) were assayed using a combination of MPN or direct
plaque assay with Escherichia coli C3000 as host. Soil and sludge
samples were assayed for virus by first concentrating the viruses
using a polyethylene glycol procedure and subsequent virus assay
of the concentrate using BGM cells. Aerosols from a sludge spray
application site were captured using either Litton high volume
samplers (LVAS) or Andersen six-stage impactors. Fluid from LVAS
units was assayed directly for total bacteria, coliphage or animal
virus as required.
Sixty-eight (68) water samples from streams, reservoirs,
wells and runoff were processed for bacteria and viruses during
the fifteen months of this study. Big Creek water samples up-
stream (S-l) and downstream (S-2) of the site show that the down-
stream site is lower in total coliform S (TC) than the upstream
site, while there are no differences in fecal coliform (FC) or
fecal streptococcus (FS) levels. Water samples from Reservoir
3 (R-3) which drains approximately 5,000 acres (2,023 hectare)
of land to which sludge has been applied indicate TC levels
higher than those in R-10, a control reservoir which drains
untreated land, with no differences between FC and FS. Of the
68 water sample concentrates, six contained virus which were
confirmed by subpassage. Three of these were found to be con-
taminated and contained poliovirus 1, sabin strain. Two of the
other positive samples were from stream site S-l and contained
echovirus 1 and an unidentified isolate. The other positive
sample was from stream site S-2 and contained an unidentified
virus isolate. No viruses could be confirmed in any well water
IV
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samples. Bacteriological data from the well water samples was
of an indeterminate nature.
No animal virus could be detected in any sludge or soil
samples nor could virus be detected in the sludge spray source
used for surface application. No virus were confirmed in runoff
water from fields to which sludge was applied.
Aerosol studies performed during three separate sampling
periods were difficult to assess. Statistical analysis of the
data indicated that LVAS samplers gave total bacterial counts
from 104-106 colony forming units (cfu)/m3 and for coliphage
from 0-2.2x10-* pfu/m3. The Andersen six-stage sampler gave total
viable counts in the range of 5.8x10-'- - 6.6xl03 cfu/m3, downwind
of the sludge spray apparatus, upwind values for the LVAS samplers
ranged from 1.5x10^ - 5.5xl02 cfu/m3 and 0-1.2xl02 pfu/m3 for
total bacteria and coliphage respectively. Upwind values taken
with Andersen six-stage samplers ranged from 4.6xlQl - 3.6xl02
cfu/m3.
Four (4) ambient air samples (1 upwind, 3 downwind) had
detectable confirmed virus levels. Confirmations were based on
second or third blind passage in homologous cells. Aerosol data
were subjected to rigorous statistical analysis.
Laboratory studies were conducted during the course of these
investigations to determine the efficiency of the two virus con-
centration techniques and to simulate virus travel in the Fulton
County soils. The Al(OH)3 continuous-flow centrifugation tech-
nique had a recovery efficiency for seeded water samples of 67%
using Hep 2 cell cultures to 333% in BGM cell cultures, with
poliovirus type 1 as test organism. The polyethylene glycol the
hydroextraction procedure recovery efficiency ranged from 13.7 -
44.4% using BGM cell cultures and poliovirus type 1. Poliovirus
type 1 and echovirus 7 were shown to adsorb very readily to
Fulton County soil; with penetration through a saturated soil
limited to the top 1 or 2 cm. Assuming identical recovery
efficiencies of seeded viruses in soil columns at the beginning
and end of these experiments one can estimate virus inactivation
of 85 and 82% for poliovirus 1 and echovirus 7 respectively.
This report was submitted in fulfillment of contract
No. 68-02-2223 by the U.S. Environmental Protection Agency.
v
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CONTENTS
Foreword iii
Abstract iv
Figures viii
Tables ix
Abbreviations and Symbols xi
Acknowledgement xii
1. Introduction 1
2 . Summary 6
3. Materials and Methods 7
4 . Experimental Procedures 15
5. Results and Discussion 21
References 53
Appendices
A. Statistics of comparisons of sludge
application to bacterial populations 56
B. Statistical evaluation of aerosol
data - Table 14 62
VII
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FIGURES
Number Page
1 Typical field design with runoff water
capture system 3
2 Map Fulton County land reclamation site 4
3 Aerodynamic size of total bacteria - containing
particles 50
Vlll
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TABLES
Number Page
1 Some Characteristics of the Fulton County
Soil Used in Soil Columns 18
2 Efficiency of Al(OH)3 - Continuous Flow Centrifuga-
tion Technique as Determined from Fulton County
Water Samples Seeded with Poliovirus Type 1 22
3 Bacteriological Data from the Fulton County
Surface Water Analysis 24
4 Virological Data from Fulton County Surface
Water Analysis 26
5 Bacteriological Data from the Fulton County
Well Water Analysis 29
6 Virological Data from Fulton County Well Water
Analysis 30
7 Bacteriological Data from the Fulton County
Runoff Water Analysis 32
8 Virological Data from Fulton County Runoff
Water Analysis 33
9 Frequency of Virus Isolation from Water Samples 36
10 Efficiency of Polyethylene Glycol Hydro Extraction
Technique for Concentrating Viruses from
Different Sludge Samples 38
11 Virological Examination of Fulton County Sludge
Lagoon Samples 39
12 Virological Examination of Fulton County Sludge
Incorporated Field and Runoff Basin 13-1 Samples.... 40
13 Soil Column Studies: Virus Penetration and
Adsorption by Fulton County Soil - 1976 43
IX
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TABLES (CONT'D)
Number
14 Concentrations of Animal Virus, Coliphage, and
Total Bacteria Detected in Aerosols at MSDGC
Sludge Irrigation Site - May, July, August,
September, 1976 45
Al Levels of Bacteria in Reservoirs RIO, R3, and
B-13-7 during August 1975 to September 1975 57
A2 Levels of Bacteria at Stations SI and S2 during
the period August 1975 to September 1976 « 58
Bl Occurrence of Animal Virus Upwind versus Downwind 63
x
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LIST OF ABBREVIATIONS
ABBREVIATIONS
A6S
BGM
cf s
cf u
cpe
EDTA
E-MEM
FCS
g
HBSS
HIFCS
iu
LVAS-M
MPN
mpnpfu
MSDGC
PAB
PABA
PBS
pfu
TCID50
TSA
v/v
w/v
Anderson six-stage air sampler
Buffalo Green Monkey
-- cubic feet per second
colony forming units
cytopathic effect
disodium ethylenediamine tetraacetic acid
minimum essential medium with Earle's salts
-- fetal calf serum
-- gravity
Hank's balanced salt solution
-- heat-inactivated fetal calf serum
international UNF
large volume air sampler, Litton-model M
most probable number
-- most probable number of plaque forming units
Metropolitan Sanitary District of Greater Chicago
-- phage assay broth
-- phage assay broth agar
phosphate buffered saline
plaque forming unit
50% end-point tissue culture infectious dose
trypticase soy agar
-- volume per volume
-- weight per volume
XI
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ACKNOWLEDGEMENT
The cooperation of the Illinois Institute of
Technology Research Institute (IITRI) Life Sciences
Division is gratefully acknowledged.
The efforts of the Maintenance and Operations
Department of the Metropolitan Sanitary District
of Greater Chicago and personnel of the Soil Sci-
ences Section, Research Division, Research and De-
velopment Department of the Metropolitan Sanitary
District of Greater Chicago, in the construction
of the pilot sludge lagoon and sampling at the
Fulton County site, are greatly appreciated.
The Technical Services Division, Research
and Development Department of the Metropolitan
Sanitary District of Greater Chicago, especially
Dr. K. C. Rao, is hereby acknowledged for their
efforts in the statistical evaluation of the viral,
bacterial and aerosol data in this report.
Mr. Walter Jakubowski, Project Officer, pro-
vided valuable guidance.
XII
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SECTION 1
INTRODUCTION
The use of anaerobically digested municipal sludge as a fer-
tilizer and soil amendment is becoming a widely accepted method
of sludge disposal both in Europe and the United States (1-3).
By this practice, plant nutrients are recycled, and an economical
non-polluting sludge disposal method can result.
The Metropolitan Sanitary District of Greater Chicago (MSDGC)
has implemented a program whereby processed sludge is used for the
reclamation of strip mined fields and fertilization of row crops
in Fulton County, Illinois. Waste activated sludge and some pri-
mary sludge are thickened, anaerobically digested, shipped by barge
200 miles down the Illinois Waterways and finally lagooned before
land application. The product of this treatment is an organically
stabilized liquid fertilizer (4) .
Despite the extensive processing, the possibility of increased
virus and bacteria in the environment resulting from sludge appli-
cation is of concern to the MSDGC and other agencies. For this
reason, the various processes employed in the production of liquid
fertilizer are being examined in an effort to model the fate of
viruses and bacteria initially present in sewage. In addition,
an extensive monitoring program is being established to determine
the possible presence of sludge-associated bacteria and viruses
in the environment as a result of the land application of this
liquid fertilizer.
Enteric bacteria and viruses are present in sewage. The
activated sludge sewage treatment process results in the adsorp-
tion of bacteria and viruses to the activated sludge floe (5). A
portion of this floe, the waste activated sludge, is later anaero-
bically digested. Enteric bacteria and viruses associated with
the waste activated sludge would, therefore, be carried into the
anaerobic digestion unit process. Following an average digestion
period of 14 days and shipment to Fulton County, the processed
material is lagooned for at least 60 days before being applied to
strip mined fields and placed on land by spray irrigation of sub-
surface injection into the soil with disc equipment. For spray
irrigation, water-winch brand sprayers were used. Orifices of 1-
1/2" to 2" and up to 90 psi sludge pressure were used. Each field
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which receives sludge is completely bermed and drained solely
into a runoff capture basin which was designed to hold at least
the 100 year storm. Each basin had a control structure so that
only regulated release of runoff water into receiving waters was
possible (Figure 1).
The objective of this study was the assessment of the effects
of the land application of digested lagooned sludge upon the viral
and bacterial content of contingent surface waters, ground water,
soil and air at the Fulton County land reclamation site. The
Fulton County site is representative of land reclamation programs
(1-3) in areas of similar soil and environmental characteristics.
The overall study consisted of two components. The first
component was concerned with the development of methodologies
for the concentration and isolation of viruses from water, sludges
and soils. Upon establishment of the appropriate methodologies,
the various elements of the second component were initiated.
The second component of the study included the monitoring of
various environments at the land reclamation site for viruses and
indicator bacteria. The environments monitored included 3 sur-
face water sites, 3 ground water sites, a site associated with a
field runoff basin, lagooned sludge, a field where lagooned sludge
was injected into the soil and 4 fields where aerosols were
generated during the spray application of lagooned sludge.
In addition, a laboratory study was conducted to assess the
movement of two types of viruses seeded onto soil columns in the
laboratory. The soils used in this study were taken from the land
reclamation site.
The specific work items established for the entire program
were:
1. Surface Water Monitoring - Two stream sites (S-l and
S-2) and one reservoir site (R-3) (see Figure 2) were
monitored for viruses and indicator organisms monthly
during the study. The surface water monitoring was to
estimate the transfer of viruses possibly contained in
sludge to surface drainage. To this end, also, an MSDGC
land application area (Field 13) was selected and close-
ly monitored for viruses. Sludge applied to fields,
runoff water from rain events, and release water and
sediments from the field runoff capture basin (B-13)
were examined for viruses. In addition, the stream into
which the runoff capture basin discharged was examined
both upstream and downstream of the basin discharge
(see Figure 2).
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Figure 2. Map Fulton County Land Reclamation Site
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2. Methodology Development and Verification - Procedures for
concentrating and isolating viruses from water, soil and
sludge were developed and verified.
3. Determination of Virus Survival in Sludge Lagoons -
Virus levels, in fresh digested sludge, were monitored
over a 2 month period to determine the die-away rate.
These studies were carried out utilizing a small pilot
lagoon constructed and filled with fresh digested sludge
for this purpose.
4. Virus Movement in Soil - Laboratory study - Soil
columns were set up with Fulton County soil. Sludge
seeded with known amounts of 2 human viruses were
applied to these columns, and rainfall was simulated by
addition of water to the columns daily for a period of
5 days. Virus in the leachate and at various depths
within the column were determined.
5. Virus Movement in Soil - Groundwater study - Wells in
the vicinity of the sludge holding lagoons at the MSDGC
Fulton County site (see Figure 2) were monitored to
assess virus transport from the sludge to the groundwater.
6. Aerosol Studies - During application of lagooned sludge
to Fields 2, 3, 17, and 19 (see Figure 2), aerosols were
captured and the animal virus, coliphage and bacterial
levels were determined. Large volume air samplers as
well as Anderson six-stage samplers were employed for
these determinations. Sludge source samples were also
assayed for virus and bacteria.
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SECTION 2
SUMMARY
1. After digestion and storage in open lagoons, no viruses
were detected in sludge used at the Fulton County site.
2. Bacterial and viral analysis of surface water and runoff
water indicates that the land application of sludge did
not affect the quality of these waters.
3. Well water analysis indicate no effect of sludge applica-
tion upon the microbiological quality or groundwater at
the Fulton County site.
4. Statistical analyses of aerosol data indicate that prior
conditions influence downwind recovery of animal virus.
5. Only four (4) of the twenty-two (22) aerosol samples
showed detectable animal viruses, no viruses were detected
in the sludge source, and all detected viruses were of
the same serotype-poliovirus 1. The possibility of con-
tamination of these samples coulu uot be ruled out.
6. Coliphage levels decreased exponentially as the square root
of the distance of the sampler from the spray source.
7. Bacterial counts in aerosols appeared to be directly in-
fluenced by the wind velocity and inversely affected by
distance and temperature, when estimates are made using
Andersen six-stage samplers.
8. Bacterial counts in aerosols appeared to be directly
influenced by temperature and distance, when estimates are
made using Litton high-volume samplers.
9. Movement of seeded poliovirus 1 and echovirus 7 was limited
to the top 1.3 cm of soil columns in laboratory experiments,
10. Relative humidity did not seem to affect estimates of
bacterial aerosols using either sampler.
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SECTION 3
MATERIALS AND METHODS
CELL CULTURES AND MEDIA
The continuous African green monkey kidney cell line desig-
nated Buffalo Green Monkey (BGM) was obtained in its 136th passage
from Dr. Flannagan of the State University of New York at Buffalo
or International Biological Laboratories (Rockville, Md.). Embry-
onal rhabdomyosarcoma; Human, cell cultures (RD) were obtained
from the American Type Culture Collection (Bethesda, Md.). Epi-
dermoid carcinoma, larynx; Human, cells (HEP 2) were obtained from
the Illinois Department of Public Health.
RD cell cultures were passaged on growth medium consisting
of minimum essential medium with Earle's salts (E-MEM) contain-
ing 10% (v/v) fetal calf serum (PCS) and 50 ug/ml of gentaraicin
and 2.5 ug/ml amphotericin B. BGM cell cultures were passaged on
the same medium except on alternate passages 100 ug/ml chloro-
tetracycline was added to suppress possible mycoplasma contamina-
tion. Monolayer cultures were maintained with E-MEM supplemented
with 2% (v/v) heat inactivated fetal calf serum (HIFCS) and 200
iu/ml penicillin G, 200 ug/ml streptomycin sulfate and 2.5 ug/ml
of amphotericin B. Plaque assay medium consisted of E-MEM supple-
mented with 2% (v/v) HIFCS, nonessential amino acids, 0.0017%
(w/v) neutral red, 50 ug/ml gentamicin and 5 ug/ml amphotericin B
and 1.5% (w/v) Noble agar or purified agar (Difco Laboratories,
Detroit, Mich.).
Stock cultures were maintained in Bellco roller bottles with
a surface area of 1100 cm2 at a speed of about 2 R.P.M. at 35° -
37°C and were passaged weekly with a split ratio of 1:4. To
passage the cell cultures, confluent monolayers of cells growing
in roller bottles were washed 3 times with Hank's balanced salt
solution without calcium and magnesium, (HBSS w/o Ca+2 + Mg+2).
Following this, HBSS w/o Ca+2 + Mg+2, containing 0.25% (w/v)
trypsin and 0.02% (w/v) disodium ethylenediamine tetraacetic acid
(EDTA), was added to the cultures and the cultures were rotated
at about 2 R.P.M. until the cells sloughed off the flask. The
cell contents of one roller bottle were then suspended in 1 liter
of the growth medium and 250 ml of the suspension were added to
each of 4 roller bottles to continue the seed cells. The roller
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bottles were prepared by gassing them in a 5% (v/v) C02 incubator
immediately following washing and sterilizing.
For the production of petri dish cultures and microtiter cul
tures for virus assay, the cell contents of a roller bottle were
suspended in 1 liter of the growth medium except that the anti-
biotic constituents were 250 iu/ml penicillin G and 250 ug/ml
streptomycin sulfate. This cell suspension was dispensed in
12 ml amounts into 100 mm diameter plastic petri dishes or in 0.2
ml amounts into the wells of disposable flat bottom microtiter
tissue culture plates (Cook Engineering) . The petri dish and
microtiter cultures were incubated in a humidified incubator
under 5% (v/v) CC>2> The cultures generally became confluent with
in 3 to 5 days at which time they were used for virus assays.
LABORATORY VIRUS STOCKS
Poliovirus 1, Sabin strain, (Abbott Laboratories, North
Chicago, Illinois) and coxsackievirus B4 (University of Illinois
Medical School, Chicago, 111.) were propagated in BGM cell cul-
tures. Harvested materials were stored in 1.5 ml aliquots at
-70°C. The infectivity titers of the viruses were 108-5 and
50% endpoint tissue culture infectious doses (TCID5g)/ml respec-
tively. Virus preparations were passed through 0.22 urn Millipore
or Nuclepore filters prior to use. Millipore filters were pre-
treated with 1 ml of HIFCS to minimize virus adsorption. Nucle-
pore filters required no pretreatment.
INFECTIVITY TITRATIONS
Stock viruses and virus isolates were serially diluted 10-
fold in maintenance medium. A 0.1 ml amount of each dilution was
inoculated in quadruplicate into microtiter plates containing
monolayered BGM cells. The cultures were incubated at 37 °C in a
5% (v/v) C02 atmosphere and were observed daily for one week for
cytopathic effect (CPE) . Infectivity titers were determined by
the method of Reed and Muench (6) .
PLAQUE ASSAY TECHNIQUE
Monolayered BGM cells in 100 mm tissue culture dishes
(Corning) were inoculated with 0.1 to 1 ml of the appropriate
sample or dilution of a sample and were allowed to stand for 2
hours at room temperature for virus adsorption. For the assay of
viruses contained in environmental samples, plates were washed
twice with Hank's balanced salt solution (HBSS) containing 2.5
ug/ml amphotericin B, 200 iu/ml penicillin G and 200 ug/ml strep-
tomycin sulfate. Cultures were then overlayed with 15 ml of
plaque assay medium. The plates were incubated at 37° under 5%
8
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(v/v) CC>2 for at least 8 days and were observed daily for plaque
formation. As a control, an appropriate dilution of poliovirus 1
(50-100 plaque forming units (pfu)/ml) was plaqued in each
experiment. The test was assumed valid when 50-100 plaques were
observed on the control plates. Sample toxicity controls consist-
ing of 2 ml aliquots of each concentrate mixed with 1 ml of con-
trol poliovirus were also included in each assay. Positive
plaques were subpassaged at least once in BGM cells before re-
cording a sample as positive or negative for virus.
IDENTIFICATION OF VIRUS ISOLATES
Serum neutralization tests were performed in Falcon multiwell
dishes (Falcon Plastics) containing 24 wells. The dishes con-
tained 24-hour cultures of BGM or RD cells. Twenty-five antibody
units in 0.025 ml of Lim Benyesh-Melnick horse antiserum pools A
through H (National Institute of Allergy and Infectious Disease)
were mixed with 100-320 TCID5Q per 0.025 ml of viral isolate and
incubated at 37°C for 2 hours. A 0.05 ml amount of each virus-
serum mixture was added in duplicate to wells containing 1 ml of
E-MEM with 0.5% (v/v) FCS. Plates were incubated at 37°C in 5%
(v/v) C02 and observed daily for CPE. Specific neutralization
tests were conducted by the same methods using horse antisera to
echovirus 9, Hill; exhovirus 17, CHHE; echovirus 21, Farine; and
poliovirus 3, Leon; and Rhesus monkey antisera to poliovirus 1,
Brunhilde; and poliovirus 2, Lansing.
Physical Characterization
Ether sensitivity was determined by exposing a 1:10 dilution
of a virus isolate to 20% ethyl ether for 18 hours at 40°C.(7)
Acid resistance was determined by making a 1:10 dilution of
the virus isolate in HBSS, then lowering the pH to 3.0 for 3 hours
at room temperature.(8)
Temperature markers were differentiated by inoculating con-
firmed poliovirus isolates into BGM cell cultures and incubating
at 37°C and 40°C to differentiate virulent and attenuated strains.
(9)
MYCOPLASMA DETECTION
Concentrates of cell cultures and selected virus isolates
were tested for presence of mycoplasma.(10) One milliliter of
each sample was inoculated into 10 ml of Difco PPLO (pleuropneu-
monia-like organism) broth without crystal violet, supplemented
with Difco Mycoplasma Supplements. A 0.1 ml aliquot of the test
sample was also inoculated onto duplicate petri dishes containing
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mycoplasma broth with 0.9% (w/v) Oxide lonagar No. 2. One plate
was incubated aerobically and the other anaerobically at 37°C.
After 4 days of incubation, broth cultures were transferred to
fresh petri dishes and broth cultures and incubated. These broth
cultures were transferred to agar plates after an additional
4 days of incubation. All plates were examined microscopically
(300x) at 2 to 3 day intervals for 14 days before being discarded
as negative. Presumptive Mycoplasma would be confirmed by Dienes
stain retention, demonstration of subsurface colony growth, and
ability to grow when subcultured. Mycoplasma were not detected
during the study.
CONCENTRATION OF VIRUSES FROM WATER SAMPLES
For the water samples collected August 18, 1975 and September
23, 1975, 2 virus concentration techniques were used. Viruses
from 4 liter creek samples were concentrated approximately 1000
fold by means of the aluminum hydroxide (Al (OH) 3) procedure (11).
Solids removed from the prefiltration filter by means of a spatula
were suspended in 15 to 25 ml of a medium containing 0.05M glycine
at pH9 and 3% (w/v) disodium EDTA. The mixture was sonified in a
sonic water bath (Cole-Parmer, Model 8845-6) for 20 minutes. For
sonication the material was placed in 16mm by 150mm screw cap
tubes and suspended in the sonic water bath by means of a wire.
The above preparation was then centrifuged at approximately 3000
x g for 30 minutes prior to filter sterilization of the supernatant
employing a serum treated 25 mm Millipore type HA (0.45 um) filter.
For treatment of the above filter, 20 ml of 10% HIFCS contained in
phosphate buffered saline (PBS) were passed through the filter with
a syringe. Sterilized filtrates were maintained at -70°C until
virus assay. Viruses from 20 liter reservoir samples were concen-
trated by the PE 60 method(12). Solids obtained by prefiltration
were treated as described above for the Al(OH)3 procedure.
After September 23, 1975, viruses from all water samples were
concentrated using an A1(OH)3- continuous flow centrifugation
technique. A Sorval Model RC 2B centrifuge and Sorval KSB contin-
uous flow system were used. All metal apparatus that came in con-
tact with the sample was autoclaved or disinfected with Roccal.
Rubber tubing was autoclaved or replaced. Samples were adjusted
to pH 6.0 and 10 ml of freshly prepared A1(OH)3 were added per
liter of water sample. After a 2-hour mixing period in an ice
bath during which pH was held constant, the sample was centrifuged
at 27,000 x g at a flow rate of 200 ml/min. The sediment was re-
suspended in pH 10.5 glycine buffer (0.05M) containing 2% (w/v)
disodium-EDTA and 10% (v/v) HIFCS and adjusted to pH 9.0 with
0.. 5M NaOH while stirring constantly for 10 minutes. The concen-
trate was sonicated (Branson sonifier Model S 125) for 10 to 20
sec. at 4.5 amps, mixed for 20 min to elute the virus and centri-
10
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fuged at 27,000 x g (Sorval RC 2B with SS 34 rotor) for 15 min.
to remove particulates. The supernatant was collected and
filtered through a HIFCS treated, 25 mm, 0.45 urn Millipore or
0.2 um Nucleopore membrane filter. The filtrate was mixed with
an equal volume of HBSS containing 3% (w/v) beef extract, 1%
(w/v) gelatin and 5% (w/v) MgSO4 to stabilize the virus.(13)
Aliquots were frozen at -70°C until assayed.
CONCENTRATION OF VIRUSES FROM SLUDGE AND SLUDGE-SOIL SAMPLES
Viruses in sludge and sludge-soil samples were concentrated
by a modification of a Polyethylene glycol hydroextraction method.
(14) A 200 ml volume of sludge or a 200 gram quantity of sludge-
soil sample was diluted to twice its volume by adding glycine
buffer to a final concentration of 0.05 M. Tween 80 was added
to give a concentration of 0.5% (v/v) and the mixture was adjusted
to pH 10.5 with 1M NaOH. The mixture was stirred for 15-20
minutes on a magnetic stirrer at 4°C and then sonified for 3
minutes with a Branson W 350 sonifier. The power setting (output
control) was 7 and percent duty cycle setting was 60%. The
standard probe was used. The sonified mixture was centrifuged
at 9400 x g at 4°C for 30 minutes in a Sorval RC 5 refrigerated
centrifuge using an HS-4 rotor. The supernatant was adjusted
to pH 7.5 with 2N HCL and twice treated with Freon 113 (Dupont).
For Freon treatment, an equal volume of Freon 113 was added to
the supernatant and the mixture was stirred for 30 minutes at
4°C. The aqueous layer was removed with a pipette, without
disturbing the interphase, and centrifuged at 9.400 x g for 60
minutes at 4°C. Gentamicin and amphotericin B were added to the
supernatant to give final concentrations of 62.5 ug/ml and 6.25
ug/ml respectively and the supernatant was dialyzed for 18-24
hours at 4°C against polyethylene glycol (Carbowax 6000, McKesson
Chemical). The concentrate was collected in a sterile tube. The
inner surface of the dialysis bag was twice washed with 15-25 ml
of E-MEM containing 2% (v/v) HIFCS. The washings were pooled with
the concentrate. The concentrated mixture was sonified for 30
seconds at a power setting (output control) of 7 and the per-
cent duty cycle set at 60%. A microtip probe was used. During
sonication the tube containing the sample was held in an ice-
water mixture. The sonified mixture was centrifuged in a Sorval
RC 5 with a SS-34 rotor at 40,000 x g for 60 minutes at 4°C. The
supernatant was maintained at -20°C until assayed for viruses.
CONCENTRATION OF MICROORGANISMS FROM AIR
Air samples for animal virus and coliphage assay were col-
lected by methods that were essentially those described by Fannin,
et al.(15) Bacterial samples were collected by these methods when
(a) Andersen samplers were not available to the project and
11
-------
(b) when the sludge spray apparatus was shut down prior to
Andersen sample collection but after the commencement of large-
volume air sample. The Large Volume Air Samplers, Model-M (LVAS-
M), (Litton Systems, Inc.) were operated at an air sampling rate
of 1 m3/min. with 14.0-15.0 kilovolts through the electrostatic
precipitators and a sampling fluid flow rate of 7 to 9 ml/min.
depending upon environmental conditions. These samplers contained
qutomatic devices to recirculate the sampling fluid through the
LVAS-M while replacing water lost through evaporation. A sampling
fluid, consisting of 30 ml of PBS, containing 2% (v/v) of a 1%
(w/v) phenol red solution, 0.03% (v/v) GE Antifoam 10, and 2%
(v/v) HIPCS was used. The reservoir containing this fluid was
kept in an ice bath during the sampling period.
After sampling, the fluid was collected in sterile Vacutainer
tubes and kept on ice until frozen at the end of each sampling
day. After each field sampling period, the samples were stored
at -70°C until assayed.
Between samples, the following disinfection protocol was
used: LVAS tubing was washed with at least 400 ml of a greater
than 25% 7X detergent solution, 1000 ml sterile distilled water,
and 500 ml sterile triple distilled water.
Nontubing attachments to each sampler were autoclaved (15
Ibs., 15 min.) prior to each use. When collection of total bac-
teria with the LVAS was intended, 0.1 ml of final wash water was
plated on trypticase (TSA) by the spread plate procedure. No
growth was observed on these plates.
Processing of Samples
Each LVAS-M air sample, contained in sampling fluid, was
thawed and the volume measured. After thorough mixing of the
entire sample, the initial pH was measured and recorded. The pH
was then adjusted to 9.0 with 0.1 N NaOH and the sample was soni-
cated for 15 sec. with a Branson S 125 Sonifier at 4.5 amps using
the standard probe.
Following centrifugation at 940 x g for 30 minutes, the sam-
ple was filtered through a 0.45 urn Millipore filter pretreated
with HIFCS. The pH was then adjusted to 7.0 with 0.1 N HC1 and
the sample was placed in 2.5 ml aliquots in sterile Vacutainer
tubes which were frozen at -70°C until assayed.
Portions of liquid sludge source samples for coliphage assay
were processed by initial Vortex mixing followed by sonication as
described above. The sample was then centrifuged at 940 x g for
30 minutes and the supernatant filtered through a 0.45 urn Milli-
pore filter pretreated with HIFCS.
12
-------
Sludge source samples were processed for animal virus isola-
tion employing the procedure described in the previous section.
Sample Assay
Samples were plaque-assayed for animal viruses in BGM cells
as described. Prior to air sample inoculation, monolayers of BGM
cells were washed twice with HBSS. Aliquots taken in experiments
to evaluate virus concentration and isolation techniques were
assayed in BGM or Hep 2 cell culture.
Coliphage and total bacterial assays were run using the
following protocol. Cultures of Escherichia coli C3000 grown for
4 hours in phage assay broth (PAB) were used for coliphage assays.
PAB was prepared by adding distilled water to 8.0 gm nutrient
broth; 5.0 gm NaCl; 0.20 gm MgS04.7-H20; 0.05 gm MnSC^-I^O to a
final volume of 1 liter. After dissolving these ingredients, 0.15
gm CaCl2 was added. The phage assay broth agar (PABA) was pre-
pared by adding 7 or 15 gm Difco agar to 1000 ml of PAB. Soft
agar (0.7%) was used for preparation of a seeded host lawn or for
coliphage plaquing over a hard agar (1.5%) base.
The procedure described by Chang, et al (16) was used for
most probable number plaque forming unit (mpnpfu) calculations.
Coliphage assays on air sample concentrates were made by the most
probable number (MPN) procedure as described by Fannin, et al (14)
or, when endpoints were not reached with this method, by plaquing
10-fold dilutions by the soft agar overlay method.(17)
For the MPN procedure, fourfold dilutions of each sample were
inoculated into five replicate tubes of 10 ml of PAB. To each of
the inoculated tubes, 0.1 ml of a 4-hour culture of E. coli C3000
was added. Following Vortex mixing, the tubes were Tncubated
overnight. Each tube was assayed for phage growth by spotting
with a sterile wooden applicator stick a drop from each tube onto
a freshly seeded lawn of a 4-hour E. coli C3000 - soft PABA (0.7%
agar) suspension on top of an agar base. The soft agar overlay
was performed by inoculating 2.5 ml of melted soft PABA at 45°C
with 0.1 ml of a 4-hour culture of E. coli C3000. One ml of the
assay dilution was inoculated into this host-PABA suspension,
mixed on a Vortex, and poured onto a prepared hard PABA base
plate. The mixture was gently tilted to cover the entire agar
plate surface. All plates were incubated for 5 to 8 hrs. and
observed for lysis or plaque formation. Positive and negative
phage controls were included with all tests.
Total bacteria from LVAS-M sampling fluid were assayed on
13
-------
TSA in duplicate by the spread plate method and incubated for
about 24 hours at 37°C. Plates taken with Anderson six-stage
air sampler (A6S) were incubated under similar conditions.
Results were reported as colony forming units (cfu).
ENUMERATION OF BACTERIA
The indicator bacteria: Fecal coliforms, total coliforms
and fecal streptococci in surface and well water samples were
enumerated according to Standard Methods.(18)
STATISTICAL ANALYSES
Statistical methods and analyses of bacteriological data
are presented in Appendix A, and of virological data in Appendix
B.
14
-------
SECTION 4
EXPERIMENTAL PROCEDURES
WATER SAMPLES
Surface and ground water samples were collected and pro-
cessed for virus isolation and identification, and enumeration
of indicator bacteria. The stations S-l and S-2 were respec-
tively the points where Big Creek enters and leaves the MSDGC
land reclamation property. Station R-3 is at the outlet of
Evelyn Reservoir which drains a large portion of the sludge
treated area (Figure 2).
Before July, 1976, samples from Big Creek (S-l and S-2)
were obtained by means of automatic samplers (Pro Tech, Model
CG 125) and were 24-hour composites. Samples from Evelyn
Reservoir (R-3) were grab samples. Because no viruses were
detected, sample volumes for S-l and S-2 were increased from
4 liters to about 20 liters after the June, 1976 sample. Because
the automatic composite samplers could not collect 20 liters of
sample, the grab method was used. Approximate four liter grab
samples were collected from the outfall of basin B-13-1 (B-13-lc),
above the discharge (B-13-la) and 10 feet downstream of the
basin discharge (B-13-lb). Runoff basin B-13-1 discharges into
Evelyn Creek which drains R-3. Twenty to 40 liter water samples
were collected from Well 6, Well 12 and Well 14 (Figure 2).
The wells were sampled in order to assess the movement of viruses
from the sludge lagoon to the ground water. Wells 12 and 14 are
in very close proximity to over 260 acres of sludge holding basins
some of which are over 50 feet deep. In the original work plan,
Well 12 was to be monitored on a bimonthly basis. The pump in
the well was malfunctioning at the beginning of the study, however,
and Well 14 was monitored instead. Well 6 was a control well far
removed from the sludge application and sludge holding basin areas.
Direct field runoff was collected from Field 13 (RT-13),
by burying a two-inch deep trough in the upper soil in the field.
Two rainfall events occurred during the course of the study for
which water samples were collected and processed.
Water samples were collected, packed in wet ice, and shipped
to Chicago. They were received within 24 hours. The samples
15
-------
were processed immediately upon receipt or after 24 hours of
storage at 4°C.
Virus assay of these concentrated water samples was performed
using the plaque technique. The total number of virus plaques
was recorded.
Virus plaques, selected on the basis of size and morphology,
were picked, resuspended in maintenance medium and frozen at
-70°C for subpassage. A minimum of 10 plaques were picked from
plates inoculated with a concentrate. All plaques were picked
if less than 10 were observed.
When CPE was observed, cells and supernatant fluids were
harvested and frozen for identification. After infectivity ti-
trations, attempts were made to identify the agents by serum
neutralization tests or by physical characterization.
Samples were processed by methods developed in the MSDGC
laboratory in conjunction with Illinois Institute of Technology
Research Institute (IITRI). To evaluate the efficiency of the
Al(OH)3-continuous flow centrifugation technique, four typical
ground and surface waters were inoculated with known concentra-
tions of stock poliovirus 1 and coxsackievirus B4. Concentrates
were assayed for virus using the plaque assay or infectivity
titrations.
SLUDGE AND SLUDGE-SOIL SAMPLES
In this phase of the work, the existence of viruses in
anaerobically digested sludge and sludge-soil environments was
assessed.
Pilot Sludge Holding Basin
Virus survival, with time, was examined in a field pilot
sludge holding basin (41m x 27m x 6m). The pilot holding
basin was filled with sludge piped 10.4 miles from barges in
Liverpool, Illinois on the Illinois River. Samples were obtained
from the lagoon as it was filled and at weekly intervals there-
after. After the sludge in the basin separated into a superna-
tant and sediment fraction (approximately 1 week after filling
the basin) both fractions were sampled simultaneously. Twelve
samples were examined for virus content.
Field 17
Sludge-soil samples were collected at approximately weekly
16
-------
intervals from Field 17 (Figure 2) after sludge was incorporated
into its topsoil. Three samples were examined for virus content.
Runoff Basin
Because of infrequent rainfall, the runoff basin (B-13) was
discharged only once during the study. Therefore, to supplement
the runoff basin information, the sediment on the bottom of run-
off basin 13-1 (B-13-1) was sampled periodically and processed
for viruses. Four samples were examined for virus content
during June, September, and October.
VIRUS MOVEMENT IN SOIL - LABORATORY STUDY
The purpose of this study was to estimate the extent of
virus penetration into water-saturated columns of Fulton County
soil. Table 1 lists some characteristics of the soil used.
Poliovirus 1, and echovirus 7, suspended in sludge or distilled
water were each applied to the surfaces of columns containing soil,
The columns (12.1 cm in diameter by 30.5 cm in length) were made
of plexiglass and contained 3.5 kg of soil each. The soil was
sieved to exclude particles greater than 2 mm in diameter. Prior
to the application of viruses in water or sludge, the columns
were saturated with distilled water. The columns were prepared
by adding 100 ml of distilled water per day to the tops of the
columns and recording the eluate volumes. After one to two weeks,
the daily eluate volumes stabilized at approximately 75 to 95 ml
per day. The balance was assumed lost by evaporation. For
poliovirus 1, 100 ml of digested sludge seeded with 4.3 x
pfu were applied to three columns, 100 ml of unseeded digested
sludge were applied to a fourth column and 100 ml of distilled
water seeded with 4.3 x 10? pfu were applied to a fifth column.
For echovirus 7, 100 ml of digested sludge seeded with 2.6 x 108
pfu were applied to four columns, 100 ml of unseeded digested
sludge were applied to a fifth column and 100 ml of distilled
water seeded with 2.6 x 108 pfu were applied to a sixth column.
Immediately after adding the sludge and distilled water for
each virus, the contents of one of the columns containing sludge
and seeded virus was completely mixed and an unmeasured aliquot
of approximately 2.0 gm was taken, suspended in 2 ml of distilled
water and volumetrically equally split for solids determination
and virus assay. For virus estimation, the split aliquot was
suspended in 4 ml of HBSS containing 3 % (w/v) beef extract, 1%
(w/v) gelatin, 2.5 ug/ml amphotericin B and 200 ug/ml gentamicin.
Distilled water was added to each remaining column at a rate of
100 ml/day for 5 days. This is equivalent to rainfall of 1.70
inches.
17
-------
TABLE 1. SOME CHARACTERISTICS OF
THE FULTON COUNTY SOIL USED IN
SOIL COLUMNS(18)
Parameter Soil
pH 7.4
Organic carbon, % 0.61
Electrical Conductivity 1.29
Cation Exchange Capacity 14.3
NH4-N, ug/g 6.59
N02+N03-N, ug/g 1.77
0.1N HC1 extractable
Zn, ug/g 31.7
Cd, ug/g 0.20
Cu, ug/g 4.79
Ni, ug/g 6.7
Texture loam, silty clay
loam, clay loam
Bulk density, g/cc 1.61
18
-------
In order to estimate possible virus penetration through the
entire length of the columns, daily for 5 days, 1 ml of eluate
from all of the remaining columns was collected for virus assay.
The eluate samples were diluted in 4 ml of BBSS containing 3%
(w/v) beef extract: 1% (w/v) gelatin, 2.5 ug/ml amphotericin B
and 200 ug/ml gentamicin.
After 5 days, for each virus, a column containing seeded
sludge was thoroughly mixed and sampled as described above for the
first columns. A comparison of the virus content of these mixed
columns with the virus content of the above described columns,
sampled five days previously gave an estimate of virus inactiva-
tion in the columns during the study.
For each virus, a seeded sludge column was frozen solid and
the plexiglass casing was broken off. Aliquots of soil were
removed with a 3/8" cork borer at the circumference of the
columns at approximate depths of 1.3 cm, 2.6 cm, 5.1 cm, 10.2 cm,
20.4 cm and the bottom of the columns. The aliquots removed were
treated as described for the soil samples taken from the mixed
columns at the beginning and the end of the experiments.
AEROSOL SAMPLES
Samples for total bacteria assay were taken with the LVAS-M
during the May 24 sampling period. After ASS samplers became
available for the project, they were used in all subsequent tests
(excluding 3 samples during the August 30 sampling period where
the sludge sprayers shut down prior to A6S aerosol collection).
Each sampler was loaded with 6 plates, each containing 27 ml of
TSA. The A6S were selected for use in order to obtain an aero-
dynamic size distribution of total bacteria-containing particles
at the location of LVAS-M sampling. The total bacterial counts
indicate the relative concentrations of microbial contamination
arising from direct spraying of sludge as well as other nonspeci-
fic sludge application activities. During each sample collection
period, wind direction was observed and velocity was measured with
a hand-held anemometer. Relative humidity was measured with a
battery-operated psychrometer. Air temperature, sky condition,
precipitation, and daylight or darkness were also noted. Atmo-
spheric stability classes were estimated by the method referenced
by Lighthart and Frisch.(20) Efforts were made to take upwind
samples following downwind sampling. Due to periodic shifts in
wind direction, these samples were occasionally subjected to
downwind influences from the spray source. Sludge source samples
for assay of animal virus, coliphage, and total bacteria were
taken from an outlet at the sludge pump station near the end of
each sampling day.
19
-------
The source sample was assayed for total bacteria within 2
hours after collection. All samples for coliphage and animal
virus assay were kept on ice until they were frozen at the end of
each sampling day and kept frozen at -70°C until initial process-
ing and final assay.
Aerosol source sludge samples were collected and processed
in conjunction with the aerosol study to be described.
All sludge and sludge-soil samples were kept frozen at -20°C
until they were processed. Seven aerosol source sludge samples
were examined for virus content.
20
-------
SECTION 5
RESULTS AND DISCUSSION
EFFICIENCY OF THE Al(OH)3~CONTINUOUS
FLOW CENTRIFUGATION TECHNIQUE
During preliminary methodology development, a number of
efficiency trials were performed using seeded stock poliovirus
type 1 or coxsackievirus B4. For these initial experiments,
virus titers were determined by the TCID5Q endpoint method.(6)
As compared to a plaque assay the endpoint method is very impre-
cise. (21) Further evaluation of the methodology was carried out
employing the plaque method for virus assay. Though the first
experiments aided in gaining familiarity with the physical aspects
of the system, the data was discounted and is not contained herein.
Table 2 shows the results of efficiency studies performed
employing the aluminum hydroxide-continuous flow centrifuge
technique and the plaque assay method. Three different water
sources were employed in the trials. Four of the trials were
run with a relatively high seed of poliovirus type 1 (7.8 x 10^
pfu - 4.9 x 10? pfu/sample) and one was run with a much lower
virus seed (3.6 x 103 pfu/sample).
The apparent recoveries of greater than 100% may have
resulted from disaggregation of virus clumps during the experi-
mental manipulations and/or from the experimental error associated
with the virus assay.
ISOLATION OF VIRUSES AND BACTERIA FROM WATER SAMPLES
A total of 68 water samples were processed for virus isola-
tion employing the aluminum hydroxide techniques. Tables 3
through 8 list the samples, the bacteriological data and the
virological data obtained for each sampling date throughout the
study. Concentration factors for water samples ranged from
approximately 100 to 1000 fold depending upon the amount of par-
ticulate matter suspended in the original sample. Many of the
R-3, S-l and S-2 samples contained soil, algae and other parti-
culate matter. Well water samples usually contained large
amounts of iron oxide. Runoff trough and runoff basin waters
contained soil and sludge particles.
21
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Reservoir R-3 drains much of the area that is sludge treated.
Big Creek also drains much of the treated area. Sampling stations
S-l and S-2 are respectively the points where Big Creek enters
and exits the MSDGC property: Reservoir R-10 does not receive
any runoff from sludge treated fields. Basin B-13-1 in a runoff
basin that receives runoff directly from a sludge treated field
(Field 13).
Statistical comparisons of bacteriological data from surface
waters (Table 3), runoff basin water (Table 7), and reservoir
R-10 (Table Al, data from MSB Log books) are presented in
Appendix A. These comparisons establish relationships among the
various types of surface waters as follows:
The bacterial levels (TC, FC, and FS) in a reservoir
(R-3 receiving runoff from sludge treated fields, are higher
than the bacterial levels in a reservoir (R-10) receiving
runoff from fields receiving no sludge.
Basin runoff water (B-13-1) (see Table 7) is higher in
total coliforms than is the water from R-3; while there is
no difference between the two in the levels of fecal coli-
forms and fecal streptococcus. This is not considered a
degradation of water quality since in Illinois the only
criterion is the fecal coliform count.
A ranking of B-13-1, R-3, and R-10 with respect to some
bacterial indicator organisms is possible, with the levels
of total coliform and fecal streptococcus increasing in the
order R-10, R-3, and B-13-1. There is no statistical
difference among the surface water types with respect to
fecal coliform.
Comparisons between R-3 and R-10 are difficult to make
because of the difference in size between the two and the size of
the area drained by each reservoir. R-3 is a large reservoir
formed by damming Evelyn Creek while R-10 is a strip-mine lake.
It is more productive to compare R-10 with a similar strip-mine
lake, R-12, which drains a sludge application area. Recent infor-
mation (22) indicates that R-12 and R-10 are virtually indistin-
guishable in terms of indicator organisms.
Although one can rank B-13-1, R-3, and R-10 in terms of some
indicator organisms (see above) it is difficult to interpret this
ranking with respect to water quality. The water in B-13-1 is
runoff from an adjacent field to which sludge has been applied.
At any given time various indicator counts may be higher than
those in R-3 and R-10. This runoff water is not released, however,
until the FC counts are less than 494/lOOml. This value is set
by the Illinois Environmental Protection Agency and serves to
protect the reservoirs receiving the runoff from a deterioration
in water quality.
23
-------
TABLE 3. BACTERIOLOGICAL DATA FROM THE
FULTON COUNTY SURFACE WATER ANALYSIS
Bacteria (cfu/1)
Sample
Source
S-l
S-2
R-3
S-l
S-2
R-3
S-l
S-2
R-3
S-l
S-2
R-3
S-l
S-2
S-l
S-2
R-3
S-l
S-2
R-3
S-l
S-2
R-3
S-l
S-2
R-3
Date
8-8-75
8-8-75
8-8-75
9-22-75
9-22-75
9-22-75
10-23-75
10-23-75
10-23-75
11-5-75
11-5-75
11-5-75
12-10-75
12-10-75
1-14-76
1-14-76
1-14-76
2-5-76
2-5-76
2-5-76
3-4-76
3-4-76
3-4-76
4-8-76
4-8-76
4-8-76
Total
Coliform
2.0x10^
1.5x10:?
2.0X10-3
6.0x10;!
4.3x10^
6. 0x10 J
7.5xl04
e.oxio;:
2.3xlOb
NA
NA
NA
3.1x10^
1.0xlOJ
1.5x10^
2.3x10^
<1.0xl03
1.9xl05
NA .
2.4xl04
4.2xl06
1.8x10°
1.2xlOZ
3.8x10;?
1.2xl05
9.0xl03
Fecal
Coliform
1.2x10^
1.7x10;
4.0xl02
9.0xl04
2.8xl04
2.0x10^
4.0x10^
1.0x10^
<1.0xl02
NA
NA
NA
3.9x105
l.OxlO3
>6.0xlo3
2.3x10^
<1.0xl02
S.lxlO3
2.2xl03
l.OxlO2
1.3x10^
2.3x10^
1.0x10^
1.9xl04
3.1xl04
<2.0xl01
Fecal
Streptococcus
2.3xl04
3.2x10^
l.OxlO2
1.4xl03
2.3xl03
7.0x10^
2.5xl03
4.5x10^
<1.0xl02
NA
NA
NA
5.0X103
l.OxlO2
1.7x104
1.8x10^
2.0xl02
2.1xl03
S.OxlO2,
l.OxlO2
8.6xl04
7.0X104
1.0x10^
3.8xl03
4.0xl02
<5.0xl01
(continued)
24
-------
TABLE 3 (continued)
Bacteria (cfu/1)
Sample
Source
S-l
S-2
R-3
S-l
S-2
R-3
S-l
S-2
R-3
S-l
S-2
S-l
S-2
R-3
S-l
S-2
R-3
Date
5-13-76
5-13-76
5-13-76
6-16-76
6-16-76
6-16-76
7-14-76
7-14-76
7-14-76
7-21-76
7-21-76
8-5-76
8-5-76
8-5-76
9-14-76
9-14-76
9-14-76
Total
Coliform
l.OxlOJj
l.OxlOr1
<2.0xlCr
1.9xlO-j
9.0xl04
2.0xl03
l.SxlO4,
6.0x107,
5.0xl02
1.2x10^
1.4xl04
1.7xl05
4.9X104,
5.8xl02
5.1x105
6.5xl04
8.4xl02
Total
Coliform
2.2x10^
1.9xl04
<5.0xlO±
5.6x10^
3.6x10^
l.OxlO2
3.9xlQ3
<5.0xlOi
l.OxlO2
1.5x10^
6.0xl03
7.5xl03
6.0x103
1.6x10^
1.5x10^
5.2xlOJ
1.6xl02
Fecal
Streptococcus
1.0x10:?
e.oxiof
<5.0xl01
l.SxlO3
l.OxlO3
3.0xl02
2.4xl03
2.4xl03
3.5xl03
l.lxlO3
l.SxlO3
9.1xl03
7.5x103
6.6xl02
2.1xl03
4.0xl03
S.OxlO1
NA - Lab error - Data not available.
25
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28
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TABLE 5. BACTERIOLOGICAL DATA FROM THE
FULTON COUNTY WELL WATER ANALYSIS
Bacteria (cfu/1)
Sample
Source
W-14
W-14
W-14
W-6
W-14
W-12
W-12
W-12
W-14
W-12
W-12
W-6
W-12
W-12
W-12
W-12
W-14
Date
10-30-75
11-20-75
12-18-75
12-18-75
1-6-76
1-22-76
2-9-76
3-16-76
3-18-76
3-30-76
4-13-76
4-27-76
5-20-76
7-7-76
7-22-76
9-8-76
9-22-76
Total
Coliform
NA
NA
<1.0xl03
NA
l.OxlO2
S.OxlO2
<1.0xl03
<1.0xl02
<1.0xl02
<1.0xl02
NA
NA
<5.0xl01
<5.0xl01
l.OxlO2
<2.0xl01
1.6xl02
Fecal
Coliform
NA
NA
<1.0xl02
NA
<1.0xl02
<1.0xl02
<1.0xl02
<1.0xl02
<1.0xl02
<1.0xl02
<1.0xl02
<1.0xl02
<5.0xl01
<5.0xl01
<1.0xl01
<2.0xl01
<2.0xl01
Fecal
Streptococcus
NA
NA
<1.0xl02
NA
<1.0xl02
<1.0xl02
<1.0xl02
<1.0xl02
<1.0xl02
<1.0xl02
NA
NA
<5.0xl01
l.OxlO2
<1.0xl01
<2.0xl01
5.2xl02
NA - Lab error - Data not available
29
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Sample
Source
TABLE 7. BACTERIOLOGICAL DATA FROM THE
FULTON COUNTY RUNOFF WATER ANALYSIS
Date
Total
Coliform
Bacteria(cfu/1)
Fecal
Coliform
Fecal
Streptococcus
B-13-1
B-13-1
B-13-la
B-13-lb
B-13-lc
RT-13
RT-13
B-13-1
11-12-75
11-18-75
12-3-75
12-3-75
12-3-75
2-23-76
8-4-76
9-22-76
4.0xl04
3.7xl05
l.OxlO3
7.0xl04
5.2xl04
3.0xl02
1.9xl04
7.0xl03
S.OxlO2
l.OxlO2
<1.0xl02
S.OxlO2
1.3xl03
<1.0xl02
2.0xl02
<1.0xl02
3.5xl03
1.4xl03
1.2xl04
2.7xl04
l.SxlO5
4.0xl02
1.3xl04
5.7xl03
32
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33
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The stream sampling stations S-l and S-2 (see Figure 2, and
Table 3) were chosen to reflect the quality of the water in a
stream at the point of entry and exit, respectively, from the
MSDGC property. With respect to any given parameter then,
stations S-l and S-2 would be expected to reflect the effects of
sludge application activities on the stream.
Statistical comparisons between station S-l and S-2 (see
Appendix A) indicate that the levels of total coliforms at S-2
are significantly lower than at S-l. There do not appear to be
any differences in fecal coliform or fecal streptococcus levels
between the two stations. These comparisons indicate that the
sludge application to the MSDGC property is not resulting in a
decrease in water quality in the stream (Big Creek) which drains
the majority of the sludge application area. Some consideration
must be given to the flow patterns at the two stream sampling
sites (S-l and S-2) and at R-3 which discharges into Evelyn Creek.
The mean flows at S-l, S-2, and R-3 during the 1976 water year
were 22.7, 32.2, and 3.64 cubic feet per second (cfs) respectively.
The flow at R-3 represents only 38% of the difference in flow
between S-l and S-2, thus 72% of this difference comes from
stormwater runoff, seepage and other non-gaged streams or sources
which are not monitored for indicator organisms. The actual
conditions, therefore, seem to rule out erosion of stream quality
due to the sludge application activities of the MSDGC and are
consistent with the statistical comparisons.
No viruses could be detected in any well samples (Table 6).
No statistical analysis of bacteriological data for well water
(Table 5) was attempted due to the indeterminate nature of the
data.
Samples collected on October 23, 1975 from S-l, S-2, and R-3
yielded viruses (Table 4) that were identified as poliovirus
type 1 by serum neutralization tests. They were identified as
vaccine strain by temperature sensitivity tests. A 3 to 4 log
reduction in titer was noted when virus was propagated at 40°C
compared to 37°C. Subsequent studies indicated that these
isolates were laboratory contaminants.
An evaluation of the data in Table 4 shows that the number of
poliovirus 1 isolates in the S-l, S-2, and R-3 samples of 10/23/
75 is striking. Since we had performed an efficiency study on
10/22/75 by inoculating 7.8 x 105 pfu poliovirus 1 into 3 liters
of R-3 water, we felt there was a source of viral contamination
in the concentration system itself. Thus an efficiency study
was repeated in which 10 liters of W-12 water were seeded with
4.9 x 107 pfu poliovirus 1. The following day, 39 liters of
unseeded W-12 water were processed and 7.2 x 10^ pfu poliovirus
1 was recovered, indicating a definite carryover of virus in
the centrifugation system. In an attempt to pinpoint the source
34
-------
of contamination, 1 ml of approximately 6 x 10^ pfu poliovirus 1
was added to 3 liters of tap water, then processed. The pro-
cessing equipment was dismantled and disinfected as usual. With
the exception of the flowmeter, all parts were soaked overnight
in lj 1000 Roccal, then soaked and washed in hot water containing
7X cleaning solution (Linbro), rinsed twice in tap water and
once in double distilled water and dried. The equipment was
reassembled and sterile saline added. No virus was found in the
centrifuge head. However, 5.5 x 10^ pfu poliovirus was found
in the tubing. Untreated fluids in the flowmeter at the inflow
position contained more than 102 pfu virus after 24 hours at
room temperature. As a result, after December, 1975 and for the
duration of the program, all parts were autoclaved or, as in the
case of the rubber tubing, replaced to rectify this problem. In
addition, the flowmeter was changed to an outflow position.
These data point out the need for extreme caution when decontam-
inating equipment to be employed in these types of environmental
studies.
Assay of water concentrates from site S-l (1-14-76) yielded
an echovirus type 1 as identified by serum neutralization tests.
Two other confirmed isolates from S-l (11/5/75) and S-2 (1/14/76)
could not be completely identified. Both 3.2 x 102 and 3.2 x 104
TCID5Q of these isolates were totally neutralized by 25 antibody
units of all eight of the Lim Benyesh-Melnick serum pools. On
the other hand, they were not neutralized by monospecific
poliovirus 1, 2, and 3 or echovirus 9, 12, and 21 antisera. Both
isolates were 0.2 um in size as determined by their passage
through a Nuclepore 0.2 um filter. They were ether and acid
stable and negative for mycoplasma. The isolates could be
enteroviruses; adenoviruses or reoviruses.
Plaque-like lesions were also observed during assay of 14
other samples. These lesions varied in size and shape, some best
described as "bulls eye" type or central stained cells surrounded
by a circle of unstained cells surrounded by stained cells. All
areas were monitored microscopically to assure that confluent
monolayers were present beneath the agar overlay. These lesions
were picked and blind-passaged twice in fluid BGM and/or RD
cell cultures. Inasmuch as CPE was not observed in either
passage during 14 days of incubation, these samples were con-
sidered negative for viruses.
Table 9 summarizes the data from the virus isolation studies.
Six of the 68 sample concentrates were found to contain virus
which could be subpassaged in cell culture, titered and iden-
tified. Three of these six positive samples (S-l, S-2 and R-3
all from the 10/23/75 sampling date) were determined to be con-
taminants and contained poliovirus 1, Sabin strain. The other
three positive samples (S-l, 11/5/75; S-l and S-2, 1/14/76)
contained virus other than the poliovirus 1, Sabin strain.
35
-------
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EVALUATION OF POLYETHYLENE GLYCOL HYDROEXTRACTION
TECHNIQUE FOR CONCENTRATING VIRUSES FROM SLUDGE
Different types of sludge samples were seeded with polio-
virus 1, Sabin strain, 9.46 x 106 to 2.24 x 107 pfu and pro-
cessed for virus concentration using the polyethylene glycol
(PEG) hydroextraction technique. As shown in Table 10, virus
recoveries ranged from 13.7% to 44.4% in a sludge-soil mixture
and anaerobic digester draw-off I samples, respectively. In
other attempts to evaluate the methodology, the virus recoveries
were 19.9% and 21.6% in sludge samples from holding basin and
anaerobic digester draw-off II, respectively.
DETECTION OF INDIGENOUS VIRUSES IN SLUDGE SAMPLES
Sludge samples from the field pilot sludge lagoon, sludge
which was incorporated into field 17 sediment from runoff
basin 13-1 and sludge applied via spray application were pro-
cessed and assayed for viruses.
Virus Survival in Sludge Lagoon
A pilot lagoon (41m x 27m x 6m) was constructed and filled
from the same sludge source as the full scale lagoons existing
at the Fulton County site. The purpose of this study was to
determine what quantities of virus if any, are present in such
sludge and also to monitor the survival of viruses in lagooned
sludge. As shown in Table 11, no virus was detected in either
supernatant or sediment samples collected over a two month
period. Since no virus was detected in either supernatants or
sediments for 6 sampling periods (4 initial consecutive sampling
and 2 later consecutive sampling periods); efforts to isolate
viruses from this source were discontinued.
Determination of Viruses in Sludge Incorporated Fields and
Basin 13-1 Samples
Two hundred gram sludge-soil samples from a sludge incor-
porated field were processed to viruses to 13-15 ml of final
volume and were assayed in BGM cell cultures. Plaque-like lesions
(28-40) were observed in each sample (Table 12). One half of
the plaques from each sample were picked and passaged in BGM
tube cultures. None of the plaques induced cytopathogenic effect
on second passage in BGM cells. These results indicated that
the plaques were produced by "non-viral" material. Similarly,
when 200-400 ml of sludge samples from runoff basin 13-1 were
concentrated and assayed for plaques, none of the "plaques" which
developed could be confirmed as viruses upon subpassage (Table 12),
37
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TABLE 11. VIROLOGICAL EXAMINATION OF FULTON
COUNTY SLUDGE LAGOON SAMPLES
Sample
Number
01-S1
02-SL
03-SL
04-SL
08-SL
09-SL
Concentrated Volume* (ml) Number of Plaques
Date
6-28
7-15
7-23
7-29
8-25
9-2
Supernatant
12
18
35
14
24
12
Sediment Supernatant Sediment
20
10
18
19
15
25
* Initial volume of all supernatant and sediment samples was
200 ml.
39
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TABLE 12. VIROLOGICAL EXAMINATION OF FULTON COUNTY
SLUDGE INCORPORATED FIELD AND RUNOFF BASIN 13-1 SAMPLES
Sample
Number
*
01-IP
02-IP
03-IP
**
01-B13
02-B13
03-B13
04-B13
Date
7-19-76
8-3-76
8-18-76
6-21-76
9-9-76
10-14-76
10-22-76
Sample
Initial
200 g
200 g
200 g
200 ml
200 ml
400 ml
400 ml
Size Number of
Final Plaques
15
15
13
50
26
52
51
ml 34
ml 28
ml 40
ml 29
ml 4
ml 58
ml 17
Plaques
Confirmed
0
0
0
0
0
0
0
* IP - Sludge-soil mixture samples from sludge incorporated
field.
** B13 - Bottom sediment samples from Runoff holding basin 13-1.
40
-------
Employing the sludge virus isolation system consisting of hydro-
extraction concentration and BGM assay, no viruses were isolated
from the sludge examined.
Virus Detection in Sludge Applied Via Spraying
Seven different sludge samples (200 ml each) from the spray
application system were concentrated and assayed for viruses
as described. None of the samples were confirmed for viruses
(Table 12). Two samples developed 12 plaque-like lesions. Nine
of the 12 plaques were blind passaged 3 times. In no case was
CPE observed.
As above, employing the virus isolation methodology de-
scribed, no viruses were confirmed as being present in any of the
sludge samples examined.
Virus Movement In Soil - Laboratory Study
Poliovirus type 1 (4.3 x 107 pfu), or echovirus 7 (2.6 x
108 pfu), suspended in sludge or distilled water, were each
applied to the surfaces of five columns containing soil. The
columns were constructed of plexiglass and each contained 3.5 kg
of Fulton County soil, sieved to exclude particles greater than
2 mm in diameter. Prior to the application of viruses in water
or sludge, the columns were saturated with distilled water, and
then 100 ml of distilled water was added to each column daily
for one to two weeks until the eluate volume stabilized at from
75-95 ml per day. The balance was lost by evaporation.
For poliovirus type 1, 100 ml of digested sludge seeded
with 4.3 x 107 pfu were applied to three (3) columns. Two
other columns were treated with 100 ml of unseeded digested
sludge, and 100 ml of distilled water seeded with 4.3 x 107
pfu, respectively. Echovirus 7, 2.6 x 108 pfu in distilled
water or sludge, was applied to similar soil columns. Immediately
after addition of the sludge, or distilled water with or without
virus, the contents of one column containing sludge and seeded
virus was completely mixed and assayed for virus concentration
and dry weight. The recoveries of poliovirus 1 and echovirus
7 immediately after mixing were 70 & 8% respectively (see Table
13). This loss may reflect the method used to recover the
viruses from the soil.
Distilled water was added to each remaining column at a rate
of 100 ml per day for five (5) days. This is equivalent to a
rainfall of 1.70 in over five (5) days. One (1) ml of eluate
was assayed daily for virus assay. After five (5) days, in
each experiment, a column containing seeded sludge was thoroughly
41
-------
mixed, as described, and assayed for virus and dry weight. In
addition, after five (5) days, a seeded sludge column was frozen
solid, (in a freezer), and the plexiglass casing removed.
Aliquots of soil were taken with a 3/8" cork borer from approxi-
mate depths of 1.3 cm, 2.6 cm, 5.1 cm, 10.2 cm, 20.4 cm and the
bottom of the soil columns. No soil samples were assayed for
viruses from the columns receiving unseeded sludge.
The results of these soil-column studies are shown in
Table 13.
No viruses were detected in any of the column eluates from
either the poliovirus 1 or the echovirus 7 experimental runs,
indicating that the viruses did not penetrate the entire soil
column. The initial concentrations of viruses in the thoroughly
mixed column were: poliovirus 1, 8.6 x 103 pfu/gram; and
echovirus 7, 6.1 x 103 pfu/gram. After five days the virus
concentrations in the mixed columns were 1.3 s 103 pfu/gram and
1.1 x 103 pfu/gram for poliovirus 1 and echovirus 7 respectively
(see Table 13).
If one assumes that the efficiency of recovery of viruses
from the soil was the same at the end of the study as at the
beginning of the study for the mixed columns, these findings
suggest an inactivation over the period of 84.9% and 82.0% for
poliovirus 1 and echovirus 7 respectively.
At a depth of 1.3 cm virus concentrations were 1.7 x 103
pfu/gram and 9.1 x 102 pfu/gram for poliovirus 1 and echovirus
7 respectively. In neither case were viruses isolated from
depths greater than 1.3 cm (Table 13).
As shown in Table 1 the texture of the soil used in the
study was clay loam and had a cation exchange capacity of
14.3 meg/gm. These soil characteristics may have accounted for
the minimal virus movement in the columns.
In a recent study in which coxsackievirus type B-3 was
added to municipal sludges which were placed on lysimeters
containing sandy soil or clay soil, the virus remained bound
to sludge placed in the top soil. (23) These findings are in
agreement with those presented herein.
AEROSOL STUDIES
Aerosol studies were performed in an effort to assess the
microbial contribution to the air downwind of the sprayers.
Generally, only one field was receiving sludge by spraying at
any particular time. The selection of the field sites for spray-
ing was made by the Maintenance and Operations Department of
MSDGC and was not influenced by the presence of the personnel
42
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43
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engaged in the aerosol studies.
These studies were performed during three separate sampling
periods: the weeks of May 24, July 19, and August 30, 1976.
Conditions during each of these sampling periods were varied
in wind direction and velocity, temperature and relative humidity.
Wind velocity ranged from 0.5 to 4.3 meters per second, tempera-
ture from 20.1 to 30.6°C, and relative humidity from 31% to 87%.
The July 19 sampling period was restricted by rain, which pre-
vented sludge application during much of that week. The samples
collected during that period were preceded by heavy rainfall.
Air sampling was performed in the vicinity of the particular
field to which sludge was applied by spraying.
Field sampling sites were selected on the basis of accessi-
bility and wind direction relative to the location of the sludge
spray gun. During sludge application, the spray gun moved con-
tinually, and this resulted in variable distances and locations
of the sampling sites relative to the aerosol source. Average
estimated distances of sampling locations from the sludge sprayer
ranged from 50 to 450 m. The duration of each air sampling
period was influenced by sprayer shut down, (resulting from
mechanical breakdown or completion of sludge application at a
particular site), movement of the sprayer to a location that
would not permit representative downwind (or upwind) sampling,
or by dramatic shifts in wind direction. Attempts were made to
obtain control samples upwind from the spray source. However,
it was not always possible to determine the nature of activities
that were occurring upwind from a "control" sample. In some
cases sludge incorporation activities occurred on fields near
the site of sludge spray irrigation but were visually isolated
from the site of sampling.
Rough terrain in and surrounding each sludge application
field prevented the kind of mobility that would have been
necessary to readily readjust sampling positions following
significant wind direction alterations. Since periodic shifts
in wind direction could not be quantitatively accounted for, re-
ported sampling times should be interpreted as representing maxi-
mum periods of downwind sampling.
Table 14 summarizes the results of the aerosol studies.
The Litton high-volume sampler (LVAS) indicated bacterial
concentrations in the air sampled of 104 - 106 cfu/m3 and for
coliphage of 0 - 2.2x10^ pfu/m3. These estimates were made at
varying distances downwind of the sludge spray apparatus from
50 - 450 meters (see Table 14). Upwind values ranged from
l.SxlO2 - 5.5x102 cfu/m3 for total bacteria and 0 - 1.2xl02 pfu/
m-5 for coliphage, both of these parameters being estimated from
LVAS sampler fluid.
44
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Total colony forming units (cfu) estimated with the Andersen
six-stage sampler ranged from 4.6 x 101 - 3.6 x 102 cfu/m3 for
upwind control samples and from 5.8 x IQl - 6.6 x 1()3 for samples
taken downwind at varying distances (see Table 14).
Sludge spray source samples were also examined for the
viral and bacterial parameters. No animal virus were found in
any of the spray source samples, while coliphage and total
bacteria ranged from 0 - 7.0 x 104 pfu/1 and 2.2 x 107 - 1.2 x
1C)10 cfu/1 respectively.
The data from 24 Andersen six-stage samplers was pooled to
provide an estimate of the size distribution of the particles
captured by these instruments. The greatest percentage of viable
particles captured was in the 9.2 urn size range. A cumulative
average of approximately 40% of the particles collected were
in the 5.5 um or less size range (see Figure 3). Within this
size range the highest percentage of viable particles was between
1.0 - 2.0 um (see Figure 3).
It can be seen from the data in Table 14 that the combination
of environmental variables which can be expressed as stability
class (Lighthart & Frisch, (20)) had little or no effect on the
recovery of coliphage or bacteria whether sampled with the high-
volume sample or the Andersen six-stage impactor.
Although animal viruses were not detected in the sludge spray
source, 4 ambient air samples had detectable virus levels. Of
these 4 samples one was an upwind (control) sample.
A total of six plaques were confirmed (on second or third
blind passage) as animal virus. Each of these viruses was
identified as poliovirus type 1. Akin and Jakubowski (24) have
discussed the problems of contamination which threaten this type
of work, and since all of the viruses identified in the ambient
air samples were of the same type (poliovirus 1) as that used
for experiments conducted simultaneously in the same laboratory
this possibility cannot be dismissed.
All of the available data for animal virus isolations, total
bacteria, coliphage and the environmental information shown in
Table 14, was subjected to rigorous statistical analysis.
This statistical analysis is presented in Appendix B. From
this analysis the following inferences may be drawn:
1. The presence of animal virus downwind of the sludge
spray source during active application periods is not
independent of the background conditions. There is
evidence which indicates that the spray application is
not the only source of virus.
49
-------
70
STANDARD DEVIATION
60
MEAN (Data From 24
Andersen Samplers)
50
40
30
UJ
o
ac
UJ
o. 20
10
1
>9.2 5.5- 3.3- 2.0- 1.0- SI.O
9.2 5.5 3.3 2.0
AERODYNAMIC SIZE (urn)
Figure 3. Aerodynamic size of total bacteria -
containing particles
50
-------
2. The levels of coliphage decrease exponentially with
distance of the sampler from the spray source.
3. The Andersen six-stage sampler and the Litton high-
volume sampler provide different estimates for total
bacterial counts.
4. Wind velocity, temperature and relative humidity seem
to have little influence on downwind concentrations of
the coliphage and bacterial parameters.
5. If one assumes that the sludge spray source remains
constant, then temperature becomes a determining factor
in the levels of total bacteria captured in aerosols
downwind.
Some discussion of these results is in order in light of
previous work with microbial aerosols.
A considerable amount of research effort has been expended
in measuring microbial aerosols emanating from wastewater treat-
ment plants of various types.(25) Although these studies do not
exactly correspond to what we have attempted here they are instruc-
tive for what has been accomplished.
Only two later studies have actually tried to determine
the presence of animal viruses in the aerosol from wastewater
reclamation plants, Fannin in the U.S. (26) and Telsch and
Katznelson (27) in Israel. Fannin attempted, unsuccessfully, to
capture animal viruses from both trickling filter and waste
activated sludge plants in Michigan. Telsch and Katznelson re-
port capture of confirmed echovirus 7 in four out of twelve
samples collected over a two week period. The samples were
collected 40m downwind of a spray irrigation sprinkler which
used secondary effluent. The Israeli workers effectively sampled
more air than did Fannin by about a factor of 3.5. Fannin con-
ceded in his discussion that the quantity of air which he sampled
may not have been sufficient. No effort was made by the Israeli
workers to quantitate the virus content of the air captured.
Crude calculations, however, can be made using their data. These
calculations give a minimum estimate of 3.4 x 10~2 virus mpn/m^.
This figure is some 2,000 times higher than the minimum estimate
of animal virus in waste treatment plant aerosols made by Fannin
(1976). These startling differences might be explainable on
three counts: 1) Fannin (26) sampled aerosols from WRP having
a much lower aerosolization efficiency than the spray irrigation
apparatus utilized by Telsch and Katznelson (27). 2) Sewage in
Israel is much more concentrated (10-20x) than sewage in the U.S.
thus concentrations in the effluent could also be expected to be
higher. 3) The effluent source is a large university hospital.
Comparisons are difficult to make, however, since the Israeli
51
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workers made no estimates of viral concentrations in their irri-
gation spray source.
In the present study the data are equivocal with respect
to virus concentrations downwind of the sludge spray source (see
Table 14). The animal virus concentrations appear to decrease
with distance downwind of the sludge spray source. However, one
upwind control sample was also positive for animal virus, at a
concentration equivalent to that found at the intermediate down-
wind distance. To further confound the issue no animal virus
was demonstrable in any of the sludge source samples (see Table
14). Furthermore, no animal virus was demonstrable in any sludge
or soil specimens taken throughout the entire 15 months of the
present study (see Tables 11, 13, and 14). Downwind air samples
taken at the same time and at similar distances downwind were
negative for animal virus (see Table 14). These similar samples
processed more air than did the positive samples in two out of
three instances (see Table 14).
The bacteriological data in Table 14 are also inconsistent,
in that, at times air concentrations at the greatest downwind
distances are greater than those at intermediate distances. Such
inconsistency may be more apparent than real as all of the samples
at the several distances on a given day may not have been taken
simultaneously nor for the same length of time.
Sorber et al (28) compared total bacterial count from LEAP
high volume samplers with the total bacterial count from Andersen
six-stage impactors. The estimates of aerosol strength were
essentially similar for both types of sampler. The data in the
present study (Table 14) indicates that the LVAS sampler and the
Andersen six-stage impactor may, in fact, be sampling different
populations of particles.
52
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REFERENCES
1. Lue-Hing, C., Lynam, B.T., Peterson, J.R. and Gschwind, J.G.,
Chicago Prairie Plan - A Report on Eight Years of Municipal
Sewage Sludge Utilization. In Land as a Waste Management
Alternative. Proceedings of the 1976 Cornell Agricultural
Waste Management Conference, Ann Arbor Science, Ann Arbor,
Michigan. Raymond G. Loehr, Editor (1977) .
2. Ardern, D.A., The Agricultural Use of Municipal Sludge.
In Land a_s a Waste Management Alternative. Proceedings of
the 1976 Cornell Agricultural Waste Management Conference,
Ann Arbor Science, Ann Arbor, Michigan. Raymond G. Loehr,
Editor (1977).
3. Cliver, D.O., Surface Application of Municipal Sludges in
Virus Aspects of Applying Municipal Waste to Land, Symposium
Proceedings, Edited by L.B. Baldwin, J.M. Davidson and
J.F. Gerber, University of Florida (1976) .
4. Sedita, S.J., P. O'Brien, J.J. Bertucci, C. Lue-Hing, and
D.R. Zenz, Public Health Aspects of Digested Sludge Utili-
zation. In Land a£ a Waste Management Alternative; Pro-
ceedings of the 1976 Cornell Agricultural Waste Management
Conference. Raymond G. Loehr, Editor. Ann Arbor Science
Publishers Inc. (1977) .
5. Malina, J.F., Jr., Ranganathan, B.P. Sagik and Moore,
Poliovirus Inactivation by Activated Sludge J.W.P.C.F., 47
(1975) .
6. Reed, L.J. and Muench. A Simple Method of Estimating Fifty
Percent Endpoints. Amer. J. Hyg. 27:493-497 (1938) .
7. Diagnostic Procedures for Viral and Rickettsial Infections.
Fourth Edition, E.H. Lennette and N.J\Schmidt, Ed. Amer.
Publ, Hlth. Assocl, N.Y. p. 537 (1969) .
8. Viral and Rickettsial Infections iri Man. Fourth Edition,
F.L. Horsfall and I. Tamm, Ed. J.B. Lippincott Co.,
Philadelphia. p. 428 (1965) .
9. Hsuing, G.D. and Henderson, J.R., Diagnostic Virology, Yale
University Press New Haven, p. 37 (1964) .
53
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10. Barile, M., Contamination in Tissue Culture, J. Fogh Ed.,
Academic Press, N.Y. p. 135. (1973) .
11. Wallis, C. and Melnick, J.L. Concentration of Viruses on
Aluminum and Calcium Salts, Am. J. Epidemiol. 85, p. 459.
(1967) .
12. Wallis, C. , Grinstein., Melnick, J.L., and Fields, J.E.
Concentration of Viruses from Sewage and Excreta on Insol-
uble Polyelectrolytes, Appl. Micro 18, p. 1007. (1969) .
13. Wallis, C., Melnick, J.L., Rapp, F., Different effects of
MgCl2 and MgSC>4 on tne thermostability of viruses. Virology
26:694. (1965) .
14. Wellings, F.M., Lewis, A.L., and Mountain, C.W., "Demon-
stration of Solids - Associated Virus in Wastewater and
Sludge." Applied and Environmental Microbiol. 31: 354-
358. (1976) .
15. Fannin, K.F., Spendlove, K.W., Cochran, K.W., Gannon, J.J.,
Airborne Coliphages From Wastewater Treatment Facilities.
Appl. Environ. Microbiol. 3_1:705-710. (1976) .
16. Chang, S.L., Berg, G., Busch, K.A., Stevenson, R.E.,
Clarle, M.A. and Kabler, P.W., Application of the "Most
Probable Number" Method for Estimating Concentrations of
Animal Viruses by the Tissue Culture Techniques. Virology
^:27-42. (1958) .
17. Adams, M.H., Bacteriophages Interscience, New York. (1959) .
18. Standard Methods for the Examination of Water and Wastewater
13th Edition, APHA, AWWA, WPCF. (1971) .
19. Peterson, J.R., Pietz, R.I., Lue-Hing, C., Water, Soil and
Crop Quality of Illinois Coal Mine Spoils Amended with
Sewage Sludge. In Symposium on Municipal Waste Water and
Sludge Recycling on Forest Land and Disturbed Land. Penn.
State Univ. Press. (In Press) .
54
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20. Lighthart, B., Frisch, A.S., Estimation of Viable Airborne
Microbes Downwind From a Point Source. Appl. and Environ.
Microbiol. 31:700-704. (1976).
21. Davis, B.D., Dulbecco, R., Eisen, H.N., Ginsberg, H.S.,
Wood, W.B., McCarty, M., Microbiology 2nd Ed. Harper and
Row (1973).
22. Lue-Hing, C., S.J. Sedita and B.C. Rao. Report: Viral and
Bacterial Levels Resulting from the Land Application of
Digested Sludge. R&D Department, MSDGC, Report No. 77-21.
Presented at Sumposium on Municipal Wastewater and Sludge
Recycling on Forest Land and Disturbed Land, Philadelphia,
Pa. March 21-23, 1977.
23. Damgaard-Larsen, S., Jensen, K.O., Lunde, E., Nissen, B.,
Survival and Movement of Enteroviruses in Connection with
Land Disposal of Sludges. Water Research 11:503-508 (1977).
24. Akin, E.W., and W. Jakubowski. Viruses in Finished Water.
Presented at the American Water Works Assoc. Water Quality
Tech. Conf., San Diego, California (December, 1976).
25. Hickey, John L.S. and Parker C. Reist. Health significance
of airborne microorganisms from wastewater treatment pro-
cesses. Part I: Summary of investigations. J.W.P.C.F.
47(12) -.2741-2756 (1975).
26. Fannin, Kerby Frank. An Assessment of the Airborne Emission
of Selected Viruses by Wastewater Treatment Facilities.
Ph.D. Dissertation, University of Michigan, 1976.
27. Teltsch, B. and E. Katznelson. Airborne Enteric Bacteria
and Viruses from Spray Irrigation with Wastewater. Appl.
and Environ. Microbiol. 35(2);290-296 (1978).
28. Sorber, Charles A., Howard J. Bausum, Stephen A. Schaub and
Mitchell J. Small. A study of bacterial aerosols at a
wastewater irrigation site. J.W.P.C.F. 48(10) ;2367-2379
(1976).
55
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APPENDIX A
This section consists of statistical analyses pertaining
to certain comparisons that reflect the effect of sludge appli-
cation to land on the bacterial populations. The results are
presented in separate subsections each of which is arranged to
address itself to a particular aspect of the problem. The data
corresponding to the levels of viral populations are not amenable
to similar statistical analysis as the observed values are not
different from zero. In what follows, we use the terms control
fields for those fields that did not receive any sludge and the
treated fields for those fields that received sludge.
The data presented in Table Al pertaining to R-10 were
obtained from the log books of MSDGC while the data pertaining
to R3 and B-13-1 were obtained from Tables 3 and 7 of the text.
Effect of Runoff from Treated Fields on the Reservoirs
Reservoir R-3 receives runoff from sludge treated fields.
Reservoir R-10 is a control field. If the bacterial populations
present in the fields are transported by runoff, then the levels
of these populations in the reservoirs that receive runoff from
sludge treated fields will increase significantly when compared
to those reservoirs that receive runoff from the control fields.
To measure such an effect we employ the Wilcoxon rank sum
statistic to test the hypothesis:
H: There is no difference in the levels of bacteria
between R-3 and R-10.
against the alternative hypothesis
H]_: The levels of bacteria in R-3 are higher than those
in R-10.
If n and m are the number of observations on R-3 and R-10
respectively, then using the large sample approximation we obtain
the statistic
W - Err (W)
Z =
SDH (W)
56
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58
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where:
W = sum of the ranks associated with R-3
EH (W) = m (nH-n+D/2
SDR (W) = mn (m+n+l)/12
which is a standard normal variable. Also, for small values of
n and m, the small sample exact probabilities of the statistic W
are available. Using such tables we can evaluate P[W>WO] where
W is any fixed value. Table Al contains under the captions
A and B, the statistics resulting from comparisons of R-3 and
R-10;and R-3 and B-13-1, respectively. It can be seen from
Column A that there exist significant increases in the levels
of bacteria between R-3 and R-10 with respect to each of the
variables - total coliform, fecal coliform and fecal strepto-
coccus. Thus the runoff from treated field increases the
bacterial levels in the reservoirs more than that from control
fields. The small sample evaluations also confirm the same
hypothesis in rending small probabilities (see P (WT>W) in
column A).
It is conceivable that the proximity of a reservoir re-
ceiving runoff from the treated field might affect the bacterial
populations in that as the distance increases the density of
the bacterial population in the runoff decreases. Basin B-13-1
receives runoff directly from the treated fields and is located
nearest to a set of treated fields while R-3 is located farther
away. Informally we imply that the quality of runoff reaching
R-3 is a diluted version of what is received by B-13-1 as far
as the bacterial populations are concerned. A comparison of
the levels of bacterial populations of R-3 and B-13-1 would
provide evidence as to the tenability of this hypothesis.
Column B of Table Al gives the values of the statistics that
test the hypothesis
H: There is no difference between the levels of bacterial
populations of R-3 and B-13-1.
against the alternative hypothesis
Hj_: The bacterial density in B-13-1 is higher than that
of R-3.
The Wilcoxon rank sum statistic is computed £s in the previous
case. The results shows that there is no difference between
B-13-1 and R-3 as far as fecal coliform and fecal streptococcus
are concerned; however the total coliform levels in B-13-1 are
higher than those in R-3.
59
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Finally, it is of interest to find if there is any ordering
of the bacterial levels among R-10, R-3, and B-13-1. In parti-
cular, we wish to test the hypothesis
H: There is no difference among the levels of bacteria
of R-10, R-3, and B-13-1.
against the alternative hypothesis
H]_: The levels of bacteria are in increasing order of
magnitude relative to R-10, R-3, and B-13-1.
The above hypothesis can be tested by the Jonckheere statistic:
W = A Uij
i<: J
where U^-; is the number of times the values in the itn sample are
preceded by the values in the j tn sample. For the present prob-
lem we employ the large sample approximation and obtain the
standard normal variable
Z -
W ~ E
H
SDH (W)
where EH (W) and SDH (W) are computed in the following way. If
nj_, r\2r ar*d n-^ demote the number of observations from the reser-
voirs R-10, R-3, and B-13-1, and N(=ni+n2+n3) denotes the total
number of observation then
3
EH (W) = (N2 - I n-:2)/4
J
SDH (W) = ([N2(2N+3) - Z nj2 (2nj+3)]/72)
Column C in Table Al gives the values of Z. With respect to
the variables total coliform and fecal streptococcus the al-
ternative hypothesis H^ holds, while with respect to fecal
coliform it seems that there is no evidence to reject the null
hypothesis.
Effect of Sludge Application on the Stream
Sampling stations S-l and S-2 were chosen so as to monitor
the quality of water in a stream at the point of entry and exit
of the property of the district. The differences, if they exist,
60
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between the stations S-l and S-2 with respect to any given
variable (parameter) should reflect the effects of sludge
application to the fields of MSDGC, with subsequent runoff to
R-3 and to the stream.
Table A2 summarizes the data collected at stations S-l and
S-2. The Wilcoxon rank sum test is applied to test the hypothesis
H: There is no difference between S-l and S-2 with respect
to the levels of bacteria
against the alternative
H^: The levels of bacteria are higher at S-2 when compared
to S-l
The results show that the levels of total coliform are signifi-
cantly lower at S-2 than at S-l while there are no differences
in the levels of fecal coliform and fecal streptococcus between
the stations. This indicates that sludge application to the
fields of MSDGC is not deteriorating the stream as far as these
variables are concerned.
61
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APPENDIX B
This section consists of the details pertaining to the
statistical analysis of the data presented in Table 14 of the
text. The data on coliphage and total bacterial counts were
analyzed to obtain only functional relationships that might
exist between the observed count and the distance of the sampler
from the spray, and environmental factors such as wind velocity,
temperature and relative humidity. The data on animal viruses
were analyzed to test the effect of upwind conditions on down-
wind conditions. Each of these analyses is presented below:
A. Analysis of Animal Virus Data
Animal virus data were obtained by analyzing the samples
collected employing the Litton sampler. These were placed at
various distances upwind and downwind of the sludge sprayer.
Upwind measurements are expected to reflect the conditions
prior to spraying while the downwind measurements are expected
to shed light on the rate at which the levels of animal virus
decrease with increasing distance of the sampler from the source
of spray.
For the purposes of this analysis we will call the-event
of detection of the presence of animal virus in a sample a
success and the non-detection or absence of animal virus in a
sample a failure. First, we will examine the data to test
whether or not upwind conditions (conditions prior to spraying)
affect the conditions downwind. If the upwind condition does
not influence the downwind condition, then the probability of
detecting the presence of animal virus downwind of the sprayer
whether or not the virus is detected upwind remains the same.
To express this in probabilistic terms, let A and B denote the
event of obtaining a success downwind and upwind respectively;
while A and B denote the events of obtaining a failure downwind
and upwind respectively. Let P (A/B) denote the conditional
probability of obtaining a success downwind given that a success
has also been realized upwind; and a corresponding interpreta-
tion holds for P (A/B). If the downwind condition is independent
of the upwind condition then:
(1) P (A/B) = P (A/B)
62
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To test whether the data can support the hypothesis in
equation (I)/ we must make the following assumptions:
(a) For each downwind observation there exists a
corresponding upwind observation. For this
study, on any given day, several observations
were made downwind while only one observation
was made upwind. This assumption implies that
repetitive upwind measurements would yield
the same result and thus one can pair corre-
sponding upwind, downwind observations.
(b) The observations made at the various distances
downwind of the spray are indistinguishable
in that we ignore the effect of distance on
the observation. This implies that we can
consider the downwind observations as a
random sample.
Under these two assumptions we can derive the table below
from the data presented in Table 14 of the text:
TABLE Bl
OCCURRENCE OF ANIMAL VIRUS
UPWIND VERSUS DOWNWIND
Upwind Downwind
Present (A) Absent (A) Total
Present (B)
Absent (B)
[X] 1
[Y] 1
[X1] 0
[Y1] 10
[m] 1
[n] 11
Total [T] 2 [T1] 10 [N] 12
The letters within the brackets in Table Bl denote the
general values for the entries. We reject the hypothesis pre-
sented in equation 1 if the conditional probability of X=x given
X + Y = T is small. That is, if P[X=x/X+Y=t] is small. From
Table Bl above, we found that P[X=1/X+Y=2] = 0.1666. Given that
the marginal totals are fixed, the only other possible outcome
of this experiment would have been one in which X = 0, X1 = 1,
Y = 2, and Y1 = 9. For this outcome we find that P[X=0/X+Y=2] =
0.8333. Of these two likely events, the present experiment
yield an outcome that is less probable which throws doubt on the
63
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validity of the hypothesis of Equation (1). We are thus inclined
to believe that the upwind conditions affect the downwind con-
ditions, which implies that the spray is not the only source
that might contribute animal viruses to the air currents.
It would have been of considerable interest for this experi-
ment to study the effect of distance from the spray source on
recovery of animal virus. Although such an analysis can be
pursued using the available data, the validity of the results
would be suspect in the light of the upwind versus downwind
results which indicate that the sprayer may not be the only
source which contributes animal virus.
The data for coliphage and total bacterial count were
analyzed by regression techniques. The "goodness of fit" of
a model, as far as these analyses are concerned, is judged by
the square of the multiple correlation coefficient R. The
various functions of the independent variables were selected to
enter the model if R2 were "significantly" improved; otherwise,
the independent variable was dropped from the model in order to
maintain a certain level of parsimony of the model. The models
thus presented are the best in this sense and they are empirical
models because of the regression technique.
B. Analysis of Coliphage and Total Bacterial Count Data
The aerosol samples collected on September 1, 1976 (pre-
sented in Table 14) were analyzed for coliphages by a method
different from the one employed for previously collected samples.
These five samples were collected at various distances from the
spray source. Since the same sludge was sprayed, we can study
the effect of distance of sampler from the spray source on the
levels of coliphages captured in the sampler.
Let Y denote the level of coliphages captured in aerosol
samples collected at a distance X from the spray source. Then
based on above mentioned five observations, we obtain that
loge Y is linearly related to X through the equation.
(1) loge Y = 10.2178 - 0.0225 X
This regression equation explains 95.6 percent (percent R2) of
the variation in loge Y. Thus, one can infer that if the sludge
source remains the same, the levels of coliphages captured at
a distance X from the sprayer exponentially decrease with X.
The data collected on the other days are not amenable to such
analysis because the distances at which the sampler was positioned
were not selected in any schematic way.
However, it is desirable to know about the dispersion of
the coliphages and total bacteria irrespective of the levels of
64
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these parameters in sludge sources; that is, if the levels in
sludge source are fixed, it is desirable to study the way in
which the other environmental factors, such as wind velocity,
distance of the sampler from the spray source, temperature and
relative humidity, influence the levels observed in aerosols.
Let Xi denote the level of either coliphages or the total
bacteria in the sludge source. Let Y denote the level of corres-
ponding parameter in the aerosol samples collected at a distance
X2 from the sprayer source. From the analyses of the data pre-
sented in Table 14, the regression model of the type
(2)
= B
0
B2
seems to explain the variation of Y adequately for either set
of data (coliphages or total bacterial count). Here e's are
normal random variables with mean zero and variance a^.
The model in (2) is suggested to an extent by the model
obtained through the analyses of coliphage data and presented
in equation (1) above, which says that a transformation of the
form YP should be used and that the value of p should be small.
The validity of the model presented in equation (2) is examined
by using the data presented in Table 14. In analyzing the data
the values of p, q, and r are selected by trial and error such
that the value of R2 is improved. From the data the regression
coefficients BQ, B]_, and B2 have been estimated. The data per-
taining to coliphages, total bacterial count obtained by Litton
sampler and that obtained by Andersen six-stage sampler are
analyzed separately and the results are given below. The number
of observations employed for the regression analyses is denoted
by n.
Coliphage data:
p = 0.075
B0 = 0.2326
Total bacterial count data:
Litton Sampler:
p = 0.075
n
q
R2 =
n
q
22
1.25
1.4024 x 10~6
0.7554
r = 0.5
B2 = 0.0147
12
0.75
r = 0.5
65
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B0 = 1.8724
B]_ = 4.37 x 1CT8
R2 = 0.6911
B2 = 0.0187
Andersen six-stage sampler:
p = 0.075
B0 = 1.8892
n = 10
q = 0.75
B]_ = 3.03 x 10~8
R2 = 0.4216
r = 0.5
B2 = 0.0197
From these results the model presented in equation (2)
seems to explain adequately the levels of organisms in aerosols.
Also, it appears that the results obtained by the Litton sampler
and those obtained by the Andersen sampler differ significantly.
Ideally, the parameters p, q, and r need to be estimated from
the data, however, the number of samples collected are not
sufficient enough to estimate as many as six parameters.
Further, attempts have been made to find if the other
environmental factors such as wind velocity, temperature and
relative humidity influence the levels found in aerosols. It
appears, from the data, these factors have little effect on
Y in that the value of R2 is not improved to any significant
extent. From this finding it can be concluded that during the
sampling period'these three factors remained relatively (with
respect to the variation in the aerosol levels) stationary; and
the observed variations within each of these factors can be
considered insignificant enough to induce a noticeable effect
in the aerosol levels.
The levels of total bacterial count in the sludge source
varies from 2.2 x 107 cfu/1 to 1.2 x 1010 cfu/1. If this varia-
tion can be considered not significant when compared to the
variability of the method of estimation, then we can assume
that the levels of total bacterial count remains stationary during
the period of sampling. Under this assumption, it was found
that temperature influences the levels of total bacterial count
observed in the levels of aerosols captured downwind. Such a
result indicates that it is important to have the sludge source
in the model, as an independent variable.
66
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REFERENCES
1. Lehmann, E.L., Statistical Hypotheses, John Wiley and Sons,
Inc. New York. (1959).
2. Hollander, M. and D.A. Wolfe. Nonparametrie Statistical
Methods. John Wiley and Sons, Inc. New York. (1973).
67
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORT NO.
EPA-600/1-79-015
3. RECIPIENT'S ACCESSION-NO.
4. TITLE ANDSUBTITLE
Viral and Bacterial Levels Resulting from the Land
Application of Digested Sludge
5. REPORT DATf
March 197_9_ _
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Metropolitan Sanitary District of Greater Chicago
Chicago, Illinois 60611 &
III Research Institute
Chicago, Illinois 60616
10. PROGRAM ELEMENT NO.
]RA607A(bJ
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Lab. - Cinn, OH
Office of Research & Development
U. S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final Report - 8/75 to 12/76
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
Project Officer - Walter Jakubowski, (513) 684-7385
16. ABSTRACT Surface and ground waters, sludge, soils and aerosols were sampled at a land
reclamation site. The site has received large quantities of anaerobically digested
sludge for several years. Samples were analyzed for viral and bacterial components to
determine the impact of large scale sludge application on the environment. Sixty-eight
water samples from streams, reservoirs, wells and runoff were processed for bacteria
and viruses. Water samples upstream (S-l) and downstream (S-2) of the site show that
the downstream site is lower in total coliform (TC) than the upstream site, while there
are no differences in fecal coliform (FC) or fecal streptococcus (FS) levels. Water
samples from Reservoir 3 which drains approximately 5,000 acres of land to which sludge
has been applied indicate TC levels higher than those in a control reservoir which
drains untreated land, with no differences between FC and FS. Six samples contained
virus which were confirmed by subpassage. Three of these were found to be contaminatec
and contained poliovirus 1. Two of the other positive samples were from stream site S-
and contained echovirus 1 and an unidentified isolate. The other positive sample was
from stream site S-2 and contained an unidentified virus isolate. No animal viruses
could be confirmed in any well water, sludge or soil samples nor in runoff water from
fields to which sludge was applied. LVAS samplers gave total bacterial counts up to
cfu/nr and coliphage up to 2.2x10^ pfu/m . Andersen sampling gave total viable counts
up to 6.6x10^ cfu/mj downwind of the sludge spray apparatus. Upwind values for the
LVAS samplers ranged to 5.5x10 cfu/m and 1.2x10 pfu/m^ for total bacteria and coli-
phage respectively. Upwind Andersen samples ranged to 3.6x10
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Aerosols
Sludge disposal
Sewage sludge
Land application
Virus transport
Virus survival
Indicator bacteria
68G
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
80
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
68
*USGPO: 1979-657-060/1633
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