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Fate of viruses following sewage sludge application to soils

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Fate of viruses following sewage sludge application to soils
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Pancorbo, Oscar Carlos, 1953-
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viii, 285 leaves : ill. ; 28 cm.

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Activated sludge ( jstor )
Chemicals ( jstor )
Poliovirus ( jstor )
Porosity ( jstor )
Sewage sludge ( jstor )
Sludge ( jstor )
Sludge digestion ( jstor )
Sludge treatment ( jstor )
Soils ( jstor )
Viruses ( jstor )
Dissertations, Academic -- Environmental Engineering Sciences -- UF
Environmental Engineering Sciences thesis Ph. D
Sewage sludge -- Health aspects ( lcsh )
Soil microbiology ( lcsh )
Virus diseases ( lcsh )
City of Gainesville ( local )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1982.
Bibliography:
Bibliography: leaves 265-284.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Oscar Carlos Pancorbo.

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University of Florida
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FATE OF VIRUSES FOLLOWING SEWAGE SLUDGE
APPLICATION TO SOILS









By

OSCAR CARLOS PANCORBO


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY




UNIVERSITY OF FLORIDA


1982


















This dissertation is dedicated to my parents,
and to Ambrosina, Adrianne and Amanda.















ACKNOWLEDGEMENTS


The author would like to acknowledge and sincerely thank the

chairman of his doctoral committee, Dr. Gabriel Bitton, for his wisdom,

patience and encouragement during the course of this study, and for his

assistance in developing this dissertation. The author is also grateful

to the other members of the committee, Dr. Thomas L. Crisman, Dr. Dale

A. Lundgren, Dr. George E. Gifford, Dr. Samuel R. Farrah, and Dr. Allen

R. Overman, for the advice and guidance they extended to him.

The author also expresses his thanks to other faculty members,

including Dr. James M. Davidson and Dr. John Cornell, for their advice

on various phases of this study.

The author is indebted to Mr. Orlando Lanni for his excellent

technical assistance.

The assistance of Mr. Albert White, Kanapaha wastewater treatment

plant, City of Gainesville, Florida, is acknowledged. The author also

wishes to thank Dr.Gerald H. Elkan, Department of Microbiology, North

Carolina State University, Raleigh, for the loan of the hydrostatic

pressure chamber.

Special thanks are extended to fellow students, in particular, Mr.

Phillip R. Scheuerman, for their insights during the course of this study.

This work was supported by grant No. R804570 from the United States

Environmental Protection Agency.

Finally, the author acknowledges with gratitude his wife, Ambrosina,

for her understanding and patience during his graduate study.

iii














TABLE OF CONTENTS



Page
ACKNOWLEDGEMENTS ---------------------------------------------- iii

ABSTRACT ---------------------------------------------------- vii

CHAPTER

I INTRODUCTION---------------------------------------------

II LITERATURE REVIEW ---------------------------------------- 4

Viral Pathogens Found in Raw Wastewater -------------- 4
Removal of Viruses by Wastewater Treatment Processes 5
Primary Sedimentation ----------------------------5
Activated Sludge --------------------------------- 13
Removal of Viruses by Sludge Treatment Processes --- 16
Viruses in Raw Sludges --------------------------- 16
Sludge Treatment Processes ----------------------- 18
Viral and Other Health Hazards Associated
with Treated Sludges --------------------------- 42
Final Sludge Disposal ----------------------------43
Fate of Sludge-Associated Viruses in Soils --- 49
III EFFECT OF SLUDGE TYPE ON POLIOVIRUS ASSOCIATION
WITH AND RECOVERY FROM SLUDGE SOLIDS --------------------- 50

Introduction -----------------------------------------50
Materials and Methods ----------------------------- 52
Virus and Viral Assays ---------------------------52
Sludges ----------------------------------------- 56
Association of Seeded Poliovirus
with Sludge Solids ----------------------------- 59
Recovery of Seeded Poliovirus from
Sludge Components ------------------------------ 65
Statistical Treatment of Data -------------------- 67
Results and Discussion ------ --------------------- 67
Association of Seeded Poliovirus
with Sludge Solids -----------------------------67
Recovery of Solids-Associated Viruses ------------ 75












CHAPTER Page

IV POLIOVIRUS TRANSPORT STUDIES INVOLVING SOIL
CORES TREATED WITH VIRUS-SEEDED SLUDGE UNDER
LABORATORY CONDITIONS ----------------------------------- 82

Introduction --------------------------------------- 82
Materials and Methods ------------------------------ 83
Virus and Viral Assays ------------------------- 83
Primary Wastewater Effluent --------------------- 84
Sludges ---------------------------------------- 84
Association of Seeded Poliovirus
with Sludge Solids --------------------------- 91
Rain Water ------------------------------------- 92
Soils --------------------------------------- 94
Poliovirus Transport Studies -------------------- 94
Effect of Soil Bulk Density
on Poliovirus Transport ----------------------- 108
Results and Discussion ------------------------------ 116
Poliovirus Suspended in 0.01 N CaCi2 ------------ 117
Poliovirus Suspended in Diluted
Anaerobically Digested Sludge ----------------- 120
Poliovirus Suspended in Undiluted
Anaerobically Digested Sludge ----------------- 150
Conditioned-Dewatered Sludge -------------------- 158
Chemical Sludges ------------------------------ 161
Lime-Stabilized, Chemical Sludges --------------- 170

V RETENTION AND INACTIVATION OF ENTEROVIRUSES
IN SOIL CORES TREATED WITH VIRUS-SEEDED SLUDGE
AND EXPOSED TO THE NORTH-CENTRAL FLORIDA ENVIRONMENT ---- 178

Introduction -------------------------------------- 178
Materials and Methods ------------------------------- 179
Viruses and Viral Assays ------------------------ 179
Sludges --------------------------------------- 181
Association of Seeded Viruses
with Sludge Solids ---------------------------- 181
Soil ----------------------------------------- 182
Fate of Viruses in Soil Cores -------------------- 183
Virus Recovery Procedures ------------------------ 190
Measurement of Environmental Parameters ---------- 193












CHAPTER Page


Results and Discussion ----------------------------- 196
Association between Seeded Enteroviruses
and Sludge Solids ---------------------------- 197
First Survival Experiment (7 October
1977-12 October 1977) ------------------------ 197
Second Survival Experiment (2 June
1978-24 August 1978) ------------------------- 200
Third Survival Experiment (11 October
1978-20 January 1979) ------------------------ 211

VI MONITORING OF INDIGENOUS ENTEROVIRUSES AT TWO
SLUDGE DISPOSAL SITES IN FLORIDA ------------------------ 222

Introduction --------------------------------------- 222
Materials and Methods ------------------------------- 224
Sludge Disposal Sites --------------------------- 224
Virus Recovery Procedures ----------------------- 236
Viral Assays ----------------------------------- 242
Weather Data ----------------------------------- 242
Results and Discussion ------------------------------ 244
Kanapaha Sludge Disposal Site ------------------- 244
Jay Sludge Disposal Site ------------------------ 244

VII EFFECT OF HYDROSTATIC PRESSURE ON THE
SURVIVAL OF POLIOVIRUS SEEDED IN GROUNDWATER
AND SEAWATER ------------------------------------------ 250

Introduction --------------------------------------- 250
Materials and Methods --------------------- -------- 251
Virus and Viral Assays -------------------------- 251
Water Samples ----------------------------------- 251
Poliovirus Exposure to Hydrostatic Pressures -- 252
Results and Discussion ------------------------------ 254

VIII CONCLUSIONS ------------------------------------------- 258

APPENDIX: COMPOSITION OF MEDIA AND SOLUTIONS USED
IN ENTEROVIRUS ASSAYS ----------------------------------- 260

BIBLIOGRAPHY -------------------------------------------------- 265

BIOGRAPHICAL SKETCH -------------------------------------------- 285















Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

FATE OF VIRUSES FOLLOWING SEWAGE SLUDGE
APPLICATION TO SOILS

By

Oscar Carlos Pancorbo

August, 1982


Chairman: Gabriel Bitton
Major Department: Environmental Engineering Sciences


In recent years, land disposal of sewage sludge has been viewed

as a viable alternative to other disposal practices. However, there

is growing concern over the contamination of groundwater and surface

waters with microbial pathogens, particularly viruses, present in

digested sludge. The major objective of this study was to assess the

potential health risk, from viral pathogens, of sludge application to

soils.

Poliovirus type 1 (LSc) was found to be largely associated with

digested, conditioned-dewatered, chemical and lime-stabilized, chemical

sludge solids. Sludge type was found to affect, however, the degree

of association between seeded poliovirus and sludge solids. For

example, the degree of association between poliovirus and sludge solids











was significantly greater for aerobically digested sludges (95%)

than for activated sludge mixed liquors or anaerobically digested

sludges (72% and 60%, respectively). The effectiveness of the glycine

method in the recovery of solids-associated viruses was also found to

be affected by sludge type. Significantly lower mean poliovirus recovery

was found for aerobically digested sludges (15%) than for mixed liquors

or anaerobically digested sludges (72% and 60%, respectively).

Poliovirus transport studies involving soil cores treated with

virus-seeded sludge were conducted under controlled laboratory and

saturated flow conditions. A Red Bay sandy loam displayed a substantially

greater retention capacity for poliovirus in anaerobically digested sludge

than a sandy soil (i.e., Eustis find sand). The Red Bay sandy loam was

shown to completely retain poliovirus following the application of

conditioned-dewatered, chemical and lime-stabilized, chemical sludge.

Undisturbed soil cores of Eustis fine sand were treated with several

inches of virus-seeded (poliovirus and echovirus type 1-Farouk) sludge

during a two-year period. The soil cores were exposed to natural conditions

and soil temperature, soil moisture and rainfall were monitored. Both

viruses were found to be rapidly inactivated in the sludge during the

drying process on top of the soil cores. Monitoring of the/top inch of

soil revealed that both viruses were inactivated with time and were

undetectable after 35 days. Soil leachates collected after natural

rainfall (unsaturated flow conditions) were negative for both viruses.

Indigenous enterovirus were not detected in topsoil and groundwater

samples from two sludge disposal sites in Florida.


viii














CHAPTER I
INTRODUCTION

In the United States, the Water Pollution Control Act of 1972

(PL 92-500), as recently amended, requires acceptable methods for the

utilization and disposal of wastewater effluents and sludges

(Willems 1976). It now appears that land disposal of wastewater

effluents and sludges is a viable and attractive alternative to other

disposal practices. Land spreading of wastewater effluents and sludges

has many advantages, including the addition of plant nutrients, water

conservation, improvement of soil physical properties, and increased

soil organic matter. However, concern has been raised over the con-

tamination of groundwater and surface waters with nitrates, heavy

metals, and microbial pathogens, particularly viruses (Bitton 1975;

Bitton 1980b; Burge and Marsh 1978; Gerba et al. 1975).

Viruses are generally associated with wastewater solids (Cliver

1976; Lund 1971) and a significant fraction of these viruses is

transferred to sludge as a result of wastewater treatment processes.

Sludge treatment processes, such as anaerobic digestion, do not

completely inactivate or remove viruses (Bertucci et al. 1977).

Therefore, the application of anaerobically digested sludge

onto land can lead to groundwater contamination as a result of virus

transport through the soil matrix. The movement of sludge-associated

viruses is probably limited due to the immobilization of sludge solids









in the top portion of the soil profile (Cliver 1976). However, there

are "free" viruses which have not become associated with the sludge

solids or which dissociated from these solids as a result of changes

in the physico-chemical properties within the soil matrix, and which

may move through the soil to contaminate groundwaters. The movement

of these individual particles through the soil has been reviewed by

Bitton (1975) and Gerba et al. (1975), and is dependent on the type of

soil, flow rate, degree of saturation of pores, pH, conductivity, and

the presence of soluble organic materials.

A multidisciplinary project designed to study the effect of

sludge application on crops, land, animals, and groundwater was under-

taken by researchers at the University of Florida, and was funded by

the U.S. Environmental Protection Agency (Edds et al. 1980). A virus

study was included since virtually little is known on the survival and

movement of sludge-bound viruses in soils. The major objective of this

study was to assess the potential health risk, from viral pathogens, of

sludge application to soils. This objective was achieved by studying

the following:

1. Effect of sludge type on poliovirus association

with and recovery from sludge solids (Chapter III)

2. Poliovirus transport studies involving soil cores

treated with virus-seeded sludge under laboratory

conditions (Chapter IV)

3. Retention and inactivation of enteroviruses in soil

cores treated with virus-seeded sludge and exposed

to the North-Central Florida environment (Chapter V)










4. Monitoring of indigenous enteroviruses at two

sludge disposal sites in Florida (Chapter VI)

5. Effect of hydrostatic pressure on the survival of

poliovirus seeded in groundwater and seawater (Chap-

ter VII--this research was conducted in order to

determine virus survival in the groundwater

environment).
This research has allowed the determination of the persistence and

possible movement of pathogenic viruses in soils treated with wastewater

sludge. The information gained from this study is of value in the

ultimate assessment of the potential risk of viral infection to humans

associated with land disposal of sludges.














CHAPTER II
LITERATURE REVIEW

Viral Pathogens Found in Raw Wastewater

The pathogens found in raw wastewater fall into one of the

following four groups: bacteria, protozoa, helminthic parasites, and

viruses. Several reviews have appeared in the literature that address

all the pathogens found in sewage (Burge and Marsh 1978; Foster and

Engelbrecht 1973; Elliott and Ellis 1977). Herein, the emphasis will be

on the viral pathogens present in raw wastewater.

Over 100 types of viruses are found in raw wastewater (Bitton

1980b; Burge and Marsh 1978; Foster and Engelbrecht 1973; Elliott and

Ellis 1977). The most important virus groups are the enteroviruses

(i.e., polioviruses, coxsackieviruses and echoviruses), reoviruses,

adenoviruses, infectious hepatitis agent (viral hepatitis type A--Hall

1977), and viral gastroenteritis agents [variously designated as duo-

viruses, rotaviruses, reovirus-like agents or Norwalk agent (parvo-

virus)--Chanock 1976]. These organisms cause such diseases as polio-

myelitis, aseptic meningitis, myocarditis, enteritis, jaundice,

infectious hepatitis, and gastroenteritis (Burge and Marsh 1978; Foster

and Engelbrecht 1973).

Indigenous virus concentrations ranging from 500 to 80,000

plaque-forming units (PFU)/z were measured by Buras (1974) in raw sewage

from Haifa, Israel. Dugan et al. (1975) found between 27 and 19,000









PFU/A of virus in raw sewage from the Mililani (Oahu, Hawaii) sewage

treatment plant. Mack et al. (1962) found a maximum of 62,800 PFU/j

of virus in raw sewage. In Austin, Texas, raw sewage, Moore et al.

(1977) reported virus concentrations between 140 and 1,490 PFU/X.

Wellings et al. (1974, 1975) found virus concentrations ranging from

54 to > 161 PFU/k in raw sewage from two locations in Florida. Clearly,

a wide range of virus concentrations and types is found in raw waste-

water. The virus concentration detected in raw wastewater depends on

the geographical location, season of the year (Lund et al. 1969) and

virus recovery method used (Buras 1974; Foster and Engelbrecht 1973).


Removal of Viruses by Wastewater Treatment Processes

Wastewater treatment processes (Fair et al. 1968; Zoltek and
Melear 1978) vary in their ability to remove pathogenic viruses.

Several authors have reviewed the literature on virus removal by waste-

water treatment processes (Berg 1973a; Elliott and Ellis 1977; Foster

and Engelbrecht 1973; Grabow 1968; Kollins 1966; Malina 1976; Sproul

1976). In this section, the emphasis will be placed on the two treat-

ment processes that generate the most sludge. These processes are

primary sedimentation, with or without chemical addition, and acti-

vated sludge (i.e., secondary treatment).

Primary Sedimentation

Primary sedimentation is the most common and sometimes the only

treatment prior to final disposal of wastewater (Kollins 1966;

Grabow 1968). The detention time of wastewater in this treatment









process is usually only some hours (Fair et al. 1968). The capacity

of primary sedimentation to remove viruses is at best minimal (Foster

and Engelbrecht 1973; Kollins 1966; Grabow 1968; Sproul 1976). Clarke

et al. (1961) found only 3% removal of seeded poliovirus type 1

(Mahoney) from raw sewage during a three-hour settling period. Chemical

flocculation using alum, ferric chloride or lime followed by sedimenta-
tion (i.e., intermediate or chemical treatment), however, has been shown

to be very effective in the removal of viruses from raw sewage (Berg

1973a; Lund 1976; Malina 1976; Grabow 1968; Sproul 1976). The removal
of viruses by chemical flocculation has been tested in the laboratory

using suspending media of varying composition (see review by Berg 1973a).
Several laboratory studies, using suspending media other than
raw sewage, have shown that seeded viruses are effectively removed
during flocculation. Change et al. (1958) reported 86.3 to 98.7% removal

of seeded coxsackievirus A2 after flocculation at pH 6.2 in distilled

water-SiO2-NaHCO3 buffer with 40 to 100 ppm of alum [as A12(S04)3],
respectively. The removal of this virus by alum flocculation conformed

to the Freundlich isotherm and, therefore, these investigators concluded

that the removal mechanism was adsorption. Approximately 60% of the
virus associated with the aluminum flocs was. recovered following
elution with 0.1 M NaHCO3, at a final pH 8.5 (see Chang et al. 1958).
In a similar study involving phosphate precipitation from water, Brunner

and Sproul (1970) found 89 to >98% removal of seeded poliovirus type 1

(Sabin) from distilled water-phosphate medium following alum [68 mg/k
as A12(S04)3] flocculation. The removal measured was attributed to the










adsorption of the virus to the aluminum phosphate flocs and, therefore,

the removal efficiency increased with a corresponding increase in the

quantity of phosphate precipitated. These investigators also found

that the removal efficiency of poliovirus was influenced by pH with

maximum removal (i.e.., >96%) observed at pH 6.4, and a reduction in

removal at pH 5.1 and 7.3. Moreover, only 40% of the polioviruses

associated with the aluminum phosphate flocs were recovered when the

flocs were dissolved in 0.1 N NaOH, final pH 8.3 to 9.3 (see Brunner and

Sproul 1970). These investigators attributed their lack of recovery

of the adsorbed viruses to viral inactivation during the precipitation

process. However, it is likely that the method used to dissolve the

flocs was inefficient in the recovery of the adsorbed viruses. Brunner

and Sproul (1970) also studied the removal of poliovirus (93 to >97%)

from filtered wastewater effluent during phosphate precipitation with

alum. In this medium, virus removal increased with decreasing pH from

6.9 to 5.0. This was attributed to an increase in suspended solids

removal with decreasing pH. Bacteriophages have also been effectively

removed (93 to >99%) from water following flocculation with alum

(Brunner and Sproul 1970; Chang et al. 1958; Chaudhuri and Engelbrecht

1972; York and Drewry 1974).

In addition to alum, ferric chloride has also been tested as a
coagulant in the removal of viruses from water. Chang et al. (1958)

reported 96.6 and 98.1% removal of seeded coxsackievirus A2 after

flocculation at pH 6.2 in distilled water-SiO2-NaHCO3 buffer with 20 and

40 ppm of ferric chloride (as FeCl3), respectively. Similarly, Sobsey










et al. (1977) studied the flocculation of poliovirus type 1 (LSc) by

ferric chloride. These investigators added the virus to a membrane

concentrate (i.e., 0.5 M glycine buffer) of turbid estuary water and

proceeded to concentrate the virus by flocculation. At a concentration

of 0.001 M FeCl3, >99% of the seeded viruses were removed from the

supernatant at pH values from 3.5 to 7.5. However, maximum precipita-

tion was observed at pH 3.5. Bacteriophages have also been removed

efficiently from water (>99% removals) by flocculation with ferric

chloride (Change et al. 1958; York and Drewry 1974). In fact, bacterio-

phage removals consistently exceeded those of enteroviruses (Chang et al.

1958). Chang et al. (1958) found ferric chloride to be more efficient,

on a molar basis, than alum in the removal of coxsackievirus A2 from

water by flocculation. Furthermore, these researchers noted that the

flocs formed with ferric chloride were more compact and settled more

rapidly than those formed with alum. Chang et al. (1958) were unsuc-

cessful in recovering the coxsackieviruses associated with the iron

flocs by eluting with 0.1 M NaHCO3, final pH 8.5. It is likely that

this solution was inadequate as an eluent for the recovery of the

adsorbed viruses, although viral inactivation during the flocculation

process cannot be ruled out. In contrast, 74% of the polioviruses asso-

ciated with the iron flocs were recovered by Sobsey et al. (1977) follow-

ing elution with fetal calf serum (FCS) at pH 8.0.

Calcium hydroxide (i.e., lime) is another chemical frequently
used in flocculation tests for the removal of viruses from water.

Sproul (1972) has reviewed the literature on virus removal by water-

softening precipitation processes involving lime as the coagulant.









Removals in excess of 99% have been reported for poliovirus type 1

during excess lime-soda ash softening at pH 10.8 to 11.2 (Wentworth

et al. 1968). Enteroviruses are actually inactivated under the high

pH conditions achieved rather than merely removed by the flocculation
process (Sproul 1972). The inactivation of enteroviruses under alka-

line pH is believed to be caused by the denaturation of the protein

coat and the subsequent disruption of the structural integrity of the

virus (Sproul 1972). Lime flocculation followed by sedimentation or/and

sand filtration is frequently used as a tertiary (advanced) treatment

of secondary wastewater effluents (Malina 1976). This treatment
effectively removes suspended solids and phosphates from secondary

effluents (Berg et al. 1968). Several studies (field and laboratory)

have been undertaken to determine the removal efficiency of viruses from

secondary effluents by lime flocculation. Brunner and Sproul (1970)

performed laboratory precipitation tests involving the addition of

lime [as Ca(OH)2] to filtered wastewater effluent until the pH was

raised to between 9.5 and 10.9. Poliovirus type 1 (Sabin) removals
ranging from 88 to 94% were achieved. Generally, virus removal

increased with a corresponding increase in pH and in the quantity of

phosphate precipitated. In a similar laboratory study, Berg et al.

(1968) showed that 70 to >99% of poliovirus type 1 (LSc) seeded in

secondary wastewater effluent was removed by lime flocculation [with
300 mg/k (pH 10.2) to 500 mg/t (pH 11.0) of Ca(OH)2, respectively]

followed by sedimentation. These investigators also observed that
virus removal increased as the pH achieved by the addition of lime
increased. Additional experimentation confirmed that poliovirus was









inactivated by the high pH produced and that the viral inactivation

rate increased as the pH was raised from 10.1 to 11.1 (see Berg et al.

1968). Therefore, these researchers concluded that the removal of

poliovirus by this treatment process resulted from a combination of

viral inactivation and physical separation of the virus by the floccu-

lation process. The effectiveness of lime flocculation in the removal

of viruses from secondary effluents has also been shown in the field.

At a wastewater reclamation plant, Grabow et al. (1978) showed that >99.9%

of indigenous enteric viruses in activated sludge effluent were removed

by lime flocculation at pH 9.6 to 11.2 followed by sedimentation.

Coliphages, enterococci and coliform bacteria were also effectively

removed by the lime flocculation process (see Grabow et al. 1978).

Chemical flocculation using alum, ferric chloride or lime is

frequently combined with sedimentation in the primary treatment of raw

sewage (U.S. Environmental Protection Agency 1973). Among the advantages

of this treatment process are low capital costs, minimal space require-

ments and high reliability (Weber et al. 1970). In laboratory-scale

experiments, Shuckrow et al. (1971) showed that the flocculation of

raw sewage with alum was highly efficient in removing suspended solids,

total organic carbon (TOC) and chemical oxygen demand (COD). Similar

results were obtained by Weber et al. (1970) when lime or ferric chloride

were used as coagulants. Substantial removals of phosphates, nitrates,

and organic color are also achieved by this treatment process (Sproul

1976; Weber et al. 1970). As reviewed in the previous paragraphs,

numerous studies have shown that viruses are effectively removed from










water and wastewater effluents by flocculation with alum, ferric

chloride or lime. Unfortunately, only a few studies have been con-

ducted to determine the effectiveness of chemical flocculation in

removing viruses from raw sewage (see reviews by Berg 1973a; Lund 1976;

Malina 1976; Sproul 1976). In laboratory-scale pilot plants, Lund and

R0nne (1973) studied the fate of indigenous enteric viruses in raw

sewage following flocculation with alum [75 to 175 mg/X as A12(S04)3],

ferric chloride (25 to 35 mg/k as FeCl3) or lime (added lime until pH
10.5 was maintained). These investigators noted appreciable removals

of viruses in all flocculation experiments. The viruses were found

concentrated in the chemical sludges produced. Furthermore, no viral

inactivation was observed in the chemical sludges. In another labora-

tory study, Sattar et al. (1976) showed that 99.995% of poliovirus
type 1 (Sabin) seeded in raw sewage was inactivated during flocculation

with lime at pH 11.5. No viruses were recovered in the supernatants

following flocculation and an average of only 0.005% of the total
viral input was recovered in the lime sludges by eluting with 10% FCS

in saline, pH 7.2. During storage at 28C, further viral inactivation
was noted in the lime sludges and no viruses could be detected in the

sludge samples after 12 hours. The inactivation rate of seeded polio-

virus type 1 (Sabin) during lime flocculation of raw sewage was
reduced as the pH of the solution was decreased. Sattar and Ramia

(1978) found that 92.4 and 97.2% of the total inputs of poliovirus

were inactivated (i.e., recovered only 7.6 and 2.8% in the supernatants
and sludges combined) during lime flocculation at pH 9.5 and 10.5,










respectively. In these studies (Sattar et al. 1976; Sattar and Ramia

1978), it was pointed out that the method used (i.e., elution with

10% FCS in saline, pH 7.2) to recover viruses from the lime sludges

may not have been completely effective. In spite of this possible

limitation, the authors concluded that substantial viral inactivation

can be achieved during the flocculation of raw sewage with lime at high

pH.

The literature presented herein indicates that chemical floccu-

lation, under optimal laboratory conditions, can remove large quanti-

ties of viruses from raw sewage and from other water samples. Virus

removal efficiency depends on the coagulant, coagulant dose, water

type, pH, virus type and flocculation procedure employed. Since it

is difficult to ensure optimum floc formation routinely in practice,

Grabow (1968) hypothesized that, under field conditions, significant

virus removals by flocculation are not likely. As proposed by Berg

(1973b), more research is needed before a conclusion can be reached as

to the virus removal capacity of chemical flocculation in actual prac-

tice.

The effluents from the primary sedimentation units generally

still contain demonstrable quantities of enteric viruses (Sattar and

Ramia 1978). These primary effluents usually undergo further treatment

by, for example, the activated sludge process. Large quantities of

sludge (i.e., 2,400 to 5,000 gallons of sludge per million gallons

of wastewater treated) are produced during the primary sedimentation

process (U.S. Environmental Protection Agency 1974). When chemical










flocculation is combined with primary sedimentation, even larger quan-
tities of sludge (i.e., 5000 to 38,000 gallons of sludge per million

gallons of wastewater treated) are produced (U.S. Environmental Protec-

tion Agency 1974). These raw primary sludges (in particular, the

chemical sludges) contain significant amounts of enteric viruses (Berg

and Berman 1980; Lund 1976; Lund and Ronne 1973; Nielsen and Lydholm

1980; Sattar and Ramia 1978; Sproul 1976). In fact, the indigenous

enteric viruses are concentrated in the chemical sludges (Lund and R0nne

1973). Sattar and Ramia (1978) readily detected indigenous viruses in

all the primary lime sludge samples they tested. The sludge samples

were obtained from a wastewater treatment plant in Canada which employed

lime flocculation of raw sewage at a pH of approximately 10. Clearly,

even under such virucidal conditions of alkaline pH, viruses were

recovered from the sludges produced. Thus, all primary sludges should

be considered to represent potential health hazards, and should be

handled with care during further treatment and final disposal (Brunner

and Sproul 1970; Lund 1976; Sattar and Ramia 1978).


Activated Sludge

The activated sludge process (i.e., secondary or biological treat-

ment) is perhaps the most effective wastewater treatment process in the

removal of viruses. Several authors have reviewed the literature on virus

removal by this treatment process (Berg 1973a; Foster and Engelbrecht 1973;

Grabow 1968; Kollins 1966; Malina 1976; Sproul 1976). In laboratory-

scale pilot plants, 90% or more of the seeded enteroviruses were removed

by the activated sludge process (Clarke et al. 1961; Malina et al. 1975).









In these studies, the seeded viruses were transferred to the settled

sludges (Clarke et al. 1961; Malina et al. 1975). Clarke et al. (1961)

recovered only a small fraction of the viruses (i.e., poliovirus type

1--Mahoney or coxsackievirus A9) theoretically associated with the

settled sludge. These researchers concluded that the viruses were

inactivated when adsorbed to sludge particles. However, their failure

to adequately recover the adsorbed viruses can partly be attributed

to the poor recovery method used (i.e., used buffer solutions and

versene as eluents). In a laboratory-scale activated sludge unit,

Malina et al. (1975) effectively recovered poliovirus type 1 (Mahoney)

from the settled sludge by using a better eluent, distilled deionized

water. Moreover, poliovirus associated with the sludge particles was

observed to be inactivated over time. These investigators measured the

viral inactivation rate in the settled sludge and found that the rate

conformed to the following equation:


Ct = Cie- k2t (2-1)


where Ci. = virus associated with sludge initially (PFU/mg of

dry sludge solids)

Ct = virus associated with sludge at time t (PFU/mg of

dry sludge solids)

t = time (min)

k2 = rate constant (min-1)

The rate constant (i.e., k2) varied with mixed liquor suspended solids

(MLSS) concentration. For example, rate constants of 3.17 x 10-3 and









2.5 x 10-3 min-1 were determined for MLSS concentrations of 1,590 and

3,140 mg/k, respectively (see Malina et al. 1975).

As far as indigenous viruses are concerned, much less work has
been done on their removal by the activated sludge process. Moore et

al. (1977, 1978) and Farrah et al. (1981b) readily recovered indigenous

enteroviruses from the mixed liquor suspended solids of activated sludge

plants in Texas, Illinois, Montana and Oregon, and Florida, respectively.

Since in these three studies most of the viruses detected were directly

associated with the solids, ift can be hypothesized that a large frac-

tion of these viruses would be removed during subsequent secondary

sedimentation of the sludge solids. The effectiveness of the activated

sludge process in removing indigenous viruses from wastewater was

confirmed by Lund et al. (1969) and Moore et al. (1977) in field studies

in Denmark and the United States (Austin, Texas), respectively.

In-spi.te of the substantial virus removal capacity of the
activated sludge process, effluents from this treatment method routinely

contain demonstrable indigenous viruses (Buras 1974; Dugan et al.

1975; England et al. 1965; Gilbert et al. 1976a, 1976b; Merrell and

Ward 1968; Moore et al. 1977; Vaughn et al. 1978; Wellings et al.

1974, 1976a, 1978). Further treatment by chlorination (Dugan et al.

1975; England et al. 1965; Merrell and Ward 1968; Vaughn et al. 1978;

Wellings et al. 1974, 1978), or by tertiary processes such as oxidation

pond, denitrification followed by sand filtration, or alum floccula-

tion (England et al. 1965; Merrell and Ward 1968; Vaughn et al. 1978;

Wellings et al. 1978) often does not eliminate all indigenous viruses

from activated sludge effluents. In addition to viruses, secondary










effluents also contain bacterial pathogens (Foster and Engelbrecht

1973), parasites (Hays 1977) and a variety of hazardous chemicals

(e.g., nitrates, phosphates, chromium, cadmium, mercury, zinc, copper,

polychlorinated biphenyls and phthalates; see Lee 1976). Therefore,

secondary effluents should be regarded as potential health hazards in

their final disposal.

During the activated sludge process, large quantities of sludge

(i.e., 14,000 to 19,000 gallons of sludge per million gallons of

wastewater treated) are also produced (U.S. Environmental Protection

Agency 1974). The volume of waste activated sludge (i.e., secondary

sludge) produced is usually much greater than the volume of sludge gener-

ated during primary sedimentation, due to the greater moisture content

of the former (U.S. Environmental Protection Agency 1974). In fact,

when a wastewater treatment plant is upgraded to activated sludge

treatment, the capacity for excess sludge handling (i.e., sludge

treatment and disposal) must be significantly increased (U.S. Environ-

mental Protection Agency 1974). Indigenous enteric viruses are

routinely found in raw secondary sludges (Berg and Berman 1980; Lund

1976; Lund and R0nne 1973; Nielsen and Lydholm 1980). Therefore, all

secondary sludges should be considered to represent potential health

hazards, and should be handled with care during further treatment and

final disposal.

Removal of Viruses by Sludge Treatment Processes

Viruses in Raw Sludges

As reviewed above, raw sludges produced during the primary and

secondary treatment of wastewater contain substantial quantities of









enteric viruses. The indigenous virus titer of raw sludge has been
measured by several investigators and expressed in different units.

In raw primary sludge, indigenous enteric virus concentrations of 2.4
to 15 PFU/ml, -<6.9 x 10 PFUJ/ml, 6.9 to 215 PFU/g dry wt. of total

suspended solids (TSS), 7.9 x 102 to 4.3 x 103 PFU/g dry wt. TSS and

10 to 1,000 50% tissue culture infective dose (TCID50)/ml were measured
by Cliver (1975), Nath and Johnson (1980), Turk et al. (1980), Moore et

a]. (1978) and Lund (1976), respectively. In mixtures of primary

(1/3) and secondary (2/3) raw sludge from the City of Los Angeles
Hyperion treatment plant, Berg and Berman (1980) found indigenous

enteric virus concentrations ranging from 3.8 to 116 PFU/ml. Nielsen

and Lydholm (1980) detected 0.1 to 9.0 TCID50/mg TSS of indigenous
enteric viruses in raw sludge (primary and secondary) from three

Danish wastewater treatment plants. Secondary sludges have been shown
to contain 10-to 100-fold less virus than primary sludges (Lund 1976;

Lund and Ronne 1973). Clearly, a wide range of virus concentrations is

found in raw sludges. The indigenous virus concentration detected in
raw sludge depends on the sludge type, geographical location, season

of the year (Berg and Berman 1980) and virus recovery method used (Nath

and Johnson 1980). Moreover, a large fraction of the indigenous

viruses in raw sludges have been shown to be associated with the sludge
solids (Cliver 1975; Lund 1976; Lund and R0nne 1973; Nath and Johnson
1980). Indigenous enteric viruses are strongly associated with fecal

solids in raw wastewater (Bitton 1980a; Cliver 1975; Cliver 1976;
Wellings et al. 1976a) and tend to remain associated with solids during










wastewater treatment processes (Cliver 1976). Furthermore,

indigenous viruses are believed to be mostly embedded within the

sludge solids rather than merely surface adsorbed (Wellings et al.

1976a). This association between indigenous viruses and sludge solids

has significant implications on viral survival during subsequent

sludge treatment.


Sludge Treatment Processes

A variety of sludge treatment processes is routinely used in

treatment plants and they are shown in Figure 2-1. From Figure 2-l,it

can also be seen that several often overlapping functions are achieved

by each sludge treatment process. The order of the sludge treatment

processes shown in Figure 2-1 is as most often used in treatment

plants (Fair et al. 1968; Malina 1976; U.S. Environmental Protection

Agency 1974). However, it should be pointed out that not all sludge

treatment processes shown in Figure 2-1 are employed at every treatment

plant. Moreover, the order of the sludge treatment processes may be

varied from that shown in Figure 2-1 depending on the existing condi-

tions. Characteristics of the sludge treatment processes have been

reviewed by several authors (Dick 1978; Fair et al. 1968; Malina 1976;

U.S. Environmental Protection Agency 1974, 1978a, 1978b; Yates 1977).

Some work has been done on the removal of viruses (i.e., by physical

separation and/or inactivation of viral particles) from raw sludges

(i.e., primary, chemical, and/or secondary sludges) by sludge treatment

processes andis reviewed below.











FIGURE 2-1. Outline of sludge treatment processes and their functions
Adapted from Fair et al. (1968), Malina (1976) and
United States Environimental Protection Agency (1974,
1978a, 1978b).







TREATMENT PROCESSES

Raw Sludge----
THICKENING
1. Gravity settling
2. Flotation
3. Centrifugation


STABILIZATION
1. Anaerobic digestion
2. Aerobic digestion
3. Composting
4. Lime or chlorine treatment
5. Heat treatment

CONDITIONING
1. Chemical (ferric chloride,
ferrous sulfate, lime, alum
or organic polymers) treat-
2. Heat treatment

DEWATERING
1. Rotary vacuum filter
2. Centrifugation
3. Drying beds
4. Drying lagoons
i --
HEAT DRYING
1. Flash dryer
2. Tray dryer
3. Spray dryer
4. Multiple hearth

REDUCTION
1. Incineration
2. Wet air oxidation
3. Pyrolysis


FINAL DISPOSAL
1.- Ocean disposal
2. Sanitary landfill
3. Land application


FUNCTIONS


1. Water removal (i.e., in-
crease in solids content)
2. Volume reduction
3. Blending (e.g., primary
with secondary sludge)


1. Pathogen destruction
2. Odor control
3. Putrescibility control
4. Volatile sludge solids
reduction
5. Volume reduction


1. Improves subsequent
dewatering
2. Stabilization




1. Water removal
2. Volume reduction
3. Reduces fuel requirements
Lfor incinerating/drying



1. Water removal
2. Volume reduction
3. Sterilization


1. Volatile sludge solids
destruction
2. Water removal
3. Volume reduction
4. Sterilization


1. Disposal (ocean and land-
fill)
2. Utilization (i.e., land
reclamation or use on
cropland)










Stabilization-digestion. Raw sludges are frequently stabilized

using anaerobic (Fair et al. 1968; U.S. Environmental Protection Agency

1974, 1978a) or aerobic (U.S. Environmental Protection Agency 1974,

1978a; Yates 1977) digestion. These digestion processes reduce the

odor, putrescibility potential and pathogen content of raw sludges

(U.S. Environmental Protection Agency 1974, 1978b) and thereby achieve

sludge stabilization (see Figure 2-1).

There are several different design-types of anaerobic sludge

digestion and they employ either no heating, heating at mesophilic

(30-35C) temperatures, or heating at thermophilic (approximately 50C)

temperatures (U.S. Environmental Protection Agency 1974). In general,

the rate of sludge stabilization increases as the anaerobic digestion

temperature is increased. Consequently, the sludge digestion time

is usually reduced as the anaerobic digestion temperature is increased

(U.S. Environmental Protection Agency 1974).

Several authors have reviewed the literature on viral inactiva-

tion during anaerobic digestion of raw sludges (Berg 1973a; Bitton 1978;

Cliver 1976; Foster and Engelbrecht 1973; Moore et al. 1977, 1978). In

laboratory and field studies, enteroviruses have been shown to be in-

activated during anaerobic digestion (Bertucci et al. 1977; Berg and

Berman 1980; Cliver 1975; Eisenhardt et al. 1977; Moore et al. 1977,

1978; Nielsen and Lydholm 1980; Palfi 1972; Sattar and Westwood 1979).

During anaerobic digestion (35C) of sludge in laboratory-scale units,

Eisenhardt et al. (1977) measured the inactivation rate of seeded









coxsackievirus B3 at 2 loglo units per 24 hours. In similar laboratory

anaerobic digesters, Bertucci et al. (1977) observed the inactivation

rates of enteroviruses [i.e., poliovirus l (Sabin), coxsackievirus A9

(Griggs), coxsackievirus B4 (JVB) and echovirus 11 (Gregory)] seeded in

sludge to follow a first-order reaction pattern and to significantly

differ for the four viruses tested.

Ward and his collaborators have done a great deal of work on
the inactivation of enteric viruses seeded in raw and anaerobically

digested sludges. Poliovirus type 1 (CHAT) and poliovirus type

1 (Mahoney) seeded in anaerobically digested sludge (6% sludge solids,

pH 8.0) became largely associated (67 and 65% of the total virus

added, respectively) with the sludge solids and both were inacti-

vated at rates of approximately 1 loglo units/5 days at 4C, >2 loglo

units/3 days at 20C, and >1 log1o units/day at 28C (Ward and

Ashley 1976). Anaerobically digested sludge displayed no detectable

virucidal activity against enteroviruses when adjusted to pH values

between 4.5 and 7.5 (Ward and Ashley 1977a). Similarly, there was no

appreciable inactivation of seeded poliovirus type 1 in 5 days at 20C
in raw sludge maintained at its naturally low pH of 6.0 (Ward and

Ashley 1976). However, the virucidal activity against poliovirus in

raw sludge significantly increased as the pH of the sludge was raised

above 7.5 (Ward and Ashley 1977a). Thus, Ward and Ashley (1977a)

demonstrated that the uncharged form of ammonia, which exists mostly

at pH values above 8, was the causative agent in the irreversible

inactivation of enteroviruses in anaerobically digested sludge and in









raw sludge adjusted to pH values above 7.5. Moreover, this agent was

shown to be present mainly in the sludge supernatant (i.e., produced

by centrifuging anaerobically digested sludge at 18,000 x g for 20

minutes) rather than in the sludge solids (Fenters et al. 1979; Ward

and Ashley 1976). Microbial activity in anaerobically digested sludge

supernatant was found not to affect the inactivation rates of seeded

poliovirus 1 (Sabin), echovirus 6 and coxsackievirus B4 (Fenters et al.

1979). The mechanism of inactivation of poliovirus type 1 (CHAT) in
anaerobically digested sludge was found to be cleavage of the two

largest viral coat proteins (i.e., breakdown of VP-1 and VP-2) followed

by nicking of the encapsulated RNA (Ward and Ashley 1976). At the

thermophilic temperature of 43C, the inactivation rate of seeded

poliovirus type 1 (CHAT) was significantly lower in raw and anaerobi-

cally digested sludge than in phosphate-buffered saline (PBS) (Ward
et al. 1976). Ward et al. (1976) proposed that poliovirus was protected

from heat inactivation by a component found in the sludge solids. In

the case of anaerobically digested sludge, however, this protective

effect was always less than that observed for raw sludge and was

largely reversed at higher temperatures (i.e., 47 and 51C) due to the

presence of the virucidal agent, uncharged ammonia (Ward et al. 1976).

Thus, at the higher temperatures, the inactivation rates of poliovirus

in anaerobically digested sludge were similar to those in PBS (Ward

et al. 1976). Ward et al. (1976) also demonstrated that enteroviruses

in anaerobically digested sludge were irreversibly inactivated (RNA

molecule hydrolyzed) during heating at temperatures of approximately










50C. At the low natural pH of the raw sludge, the virucidal agent

was not present (Ward and Ashley 1976, 1977a). Therefore, only the

protective effect attributed to the sludge solids was observed for

this sludge type at all temperatures tested (i.e., 43 to 51C) (Ward

et al. 1976). Ionic detergents were later identified as the components

in raw and anaerobically digested sludge solids which protected polio-

virus type 1 (CHAT) and other enteroviruses from heat inactivation

(Ward and Ashley 1977c, 1978a, 1979). In contrast, these ionic deter-

gents cationicc more active than anionic) were shown to be responsible

for reducing the heat required to inactivate reovirus type 3 (Dearing)

in raw and anaerobically digested sludge (Ward and Ashley 1977c, 1978a).

Ward and Ashley (1979) demonstrated that two ionic organic detergents,

sodium dodecyl sulfate and dodecyltrimethylammonium chloride in buffer

solutions, were potent virucidal agents for reovirus, but that their

virucidal effects were strongly pH dependent. The virucidal activity

against reovirus displayed by these ionic detergents was greater in

alkaline than in acid conditions (Ward and Ashley 1977c). Further

research (Ward and Ashley 1979) revealed that the inactivation pattern

of reovirus as a function of pH at 45C in anaerobically digested

sludge was qualitatively similar to that found in buffer solutions

containing ionic detergents.

The literature reviewed in the paragraph above indicates that

the inactivation of enteric viruses in anaerobically digested sludge is

a complex phenomenon. It is clear, however, that the anaerobic diges-

tion process raises the pH of raw sludge and thereby produces in the











digested sludge the uncharged form of ammonia which is virucidal for

enteroviruses. The presence of this virucide coupled with thermophilic

digestion temperatures (i.e., approximately 50C) results in the

rapid inactivation of enteroviruses in anaerobically digested sludge.

Furthermore, the protective effect towards enteroviruses attributed to

ionic detergents in the sludge solids and observed at low pH in raw

sludge is largely overcome by the virucidal ammonia in anaerobically

digested sludge (Ward and Ashley 1977c) particularly at thermophilic

temperatures. Reoviruses are also inactivated in anaerobically digested

sludge at thermophilic temperatures and at the naturally high pH values

attained by the digestion process (Ward and Ashley 1979).

As presented above, the inactivation of viruses during anaerobic

digestion has been studied in the laboratory using sludge artificially

contaminated with virus. The validity of such research has been ques-

tioned because it is believed that, unlike indigenous viruses, the

seeded viruses become mostly adsorbed to the surface of sludge solids

(Moore et al. 1977; Nielsen and Lydholm 1980). Due to their strong

association with fecal solids in raw wastewater (Bitton 1980a; Cliver

1975, 1976; Wellings et al. 1976a), indigenous viruses are believed to

end up, during wastewater treatment, mostly embedded within sludge solids

rather than merely surface adsorbed (Wellings et al. 1976a). Conse-

quently, Moore et al. (1977) proposed that indigenous viruses in sludge

are less susceptible than seeded viruses to the environmental stresses

(e.g., chemical and heat inactivation) encountered during sludge treat-

ment by virtue of the former's more insulated environment. Evidence to










support this hypothesis has been obtained from studies involving the

anaerobic digestion of sludge. At the East Pearl treatment plant in

Boulder, Colorado, Moore et al. (1978) found total reductions of

indigenous enteroviruses in primary raw sludge of only 2 logo units

(i.e., 99%) during 100 days of anaerobic digestion (40 days at 37C

in digester no. 1 followed by 60 days in unheated digester no. 2).

Significantly higher inactivation rates were measured for seeded entero-

viruses by Bertucci et al. (1977) and Eisenhardt et al. (1977) during

anaerobic digestion of sludge. For example, Eisenhardt et al. (1977)

found the inactivation rate of seeded coxsackievirus B3 to be 2 loglo

units per 24 hours. The inactivation rates of seeded enteroviruses were

drastically reduced when the viruses were incorporated into the sludge

during sludge production rather than simply mixed with the final

sludge sample. Moore et al. (1977) reported an inactivation rate of

approximately 2 logo units per 15 days for poliovirus naturally

incorporated (and assumed embedded) into wasted sludge during activated

sludge treatment in a continuous flow, bench-scale unit and then sub-

jected to anaerobic digestion at 30C. In fact, these investigators

detected poliovirus in the sludge undergoing anaerobic digestion even

after 30 days. Clearly, enteroviruses embedded within sludge solids

are afforded some protection from virucidal chemical agents (e.g.,

uncharged ammonia in the liquid fraction of anaerobically digested

sludge--see Ward and Ashley 1977a) and/or physical stresses (e.g.,

heat) encountered during the anaerobic digestion of sludge, as well as

during other sludge treatment processes.









Although the anaerobic digestion process appears capable of

removing considerable quantities of viruses from sludge, a fraction of

the viruses initially present will, nevertheless, survive this diges-

tion process [see reviews by Berg (1973a), Bitton (1978), Cliver

(1976), Foster and Engelbrecht (1973), and Moore et al. (1978)].
Indigenous viruses have been routinely detected in anaerobically

digested sludge (Berg and Berman 1980; Cliver 1975; Farrah et al. 1981a;

Moore et al. 1978; Nielsen and Lydholm 1980; Palfi 1972; Sattar and
Westwood 1979; Sagik et al. 1980; Turk et al. 1980; Wellings et al.

1976a). Moreover, the indigenous virus titer of this sludge type has
been measured by several investigators and expressed in different

units. Anaerobically digested sludge sampled at various locations

throughout the United States displayed indigenous enteric virus con-

centrations of 0 to 8 PFU/ml, <0.014 to 4.1 PFU/ml, 7 to 40 PFU/g
dry wt. TSS, 1.1 to 17 PFU/g dry wt. TSS, 0.2 to 17.0 PFU/g dry wt. TSS

and 2 to 7 TCID50/g dry wt. TSS as measured by Cliver (1975), Berg and

Berman (1980), Moore et al. (1978), Sagik et al. (1980), Turk et al.

(1980) and Farrah et al. (1981a), respectively. Nielsen and Lydholm

(1980) found 0 to 600 TCID50/g dry wt. TSS of indigenous enteric

viruses in anaerobically digested sludge from three Danish wastewater

treatment plants. Evidently, the indigenous virus concentration
detected in anaerobically digested sludge depends on the virus con-
centration in the raw sludge, and thereby, on the geographical location
(Sagik et al. 1980) and on the season of the year (Berg and Berman
1980; Moore et al. 1978). The indigenous virus titer found in










anaerobically digested sludge also depends on the virus removal

efficiency of the anaerobic digestion procedure employed. For example,

Berg and Berman (1980) reported that, at the City of Los Angeles

Hyperion treatment plant, thermophilic anaerobic digestion (20 days at

approximately 49C) was superior to mesophilic anaerobic digestion

(20 days at approximately 35C) in the removal of indigenous viruses

from raw sludge. In the laboratory, Ward et al. (1976) confirmed that

the inactivation of seeded poliovirus type 1 (CHAT) in anaerobically

digested sludge was accelerated under thermophilic temperatures.

Although little is known about the viral-inactivating capacity

of the aerobic digestion process, several investigators have reported

that, as in the case of anaerobic digestion, not all indigenous entero-

viruses are eliminated from sludge during aerobic digestion (Farrah

et al. 1981a, 1981b; Hurst et al. 1978). Farrah et al. (1981a, 1981b)

measured indigenous enterovirus titers ranging from 1.7 to 260 TCID50/g

dry wt. TSS in aerobically digested sludge from three wastewater treat-

ment plants in Florida. In sludges from two wastewater treatment

plants in Pensacola, Florida, Farrah et al. (1981a) showed that aerobi-

cally digested sludge contained larger indigenous viral titers than

anaerobically digested sludge.

Clearly, both anaerobically and aerobically digested sludges can
contain substantial quantities of enteric viruses. Therefore, all

digested sludges should be considered to represent potential health

hazards, and should be handled with care during further treatment and

final disposal (Palfi 1972).










Stabilization-composting. Composting is a biological, aerobic,
thermophilic (approximately 60C) process frequently used to stabilize

(see Figure 2-1) raw sludges (U.S. Environmental Protection Agency 1974,

1978b). As such, this process reduces the odor, putrescibility poten-

tial and pathogen content of raw sludges (U.S. Environmental Protection

Agency 1974, 1978b). Bitton (1980b) described in detail the sludge

composting process and reviewed the literature on pathogen destruction

during this sludge treatment procedure. Pathogenic parasites and

bacteria as well as bacteriophage f2 have been shown to be inactivated

during sludge composting (Bitton 1980b). Ward and Ashley (1978b)

demonstrated that seeded poliovirus type 1 (CHAT) was heat-inactivated

(43C) at a significantly greater rate in composted sludge than in

dewatered raw sludge held at the same sludge solids content of 40%. In

the case of seeded reovirus, the reverse was observed (i.e., greater

inactivation rate in dewatered raw sludge; see Ward and Ashley 1978b).

These viral inactivation patterns were attributed to the effects of

sludge solids-associated, ionic detergents (Ward and Ashley 1978b).

These detergents were previously shown to influence differently the

heat inactivation rate of enteroviruses and reoviruses in sludge.

Whereas enteroviruses in sludge were protected from heat inactivation

by ionic detergents, reoviruses were inactivated at an accelerated

rate (Ward and Ashley 1977c, 1978a, 1979). During composting, however,

the ionic detergents in raw sludge were shown to be substantially

degraded (Ward and Ashley 1978b). Thus, viral inactivation rates were

markedly different in composted sludge as compared to raw sludge and










for enteroviruses versus reoviruses (Ward and Ashley 1978b). Although

most enteroviruses are rapidly inactivated during sludge composting,

reoviruses apparently are capable of surviving this sludge treatment

process (Ward and Ashley 1978b). Clearly, sludge compostingdoes not

yield a virus-free product, and therefore, all composted sludges should

be handled with care during further treatment and final disposal.

Stabilization-lime treatment. Lime treatment at pH 11.0 to 11.5

is another practice frequently employed to stabilize (see Figure 2-1)

raw sludges (Farrell et al. 1974; U.S. Environmental Protection Agency

1974, 1978a). During periods when digesters are out of service or when

sludge quantities exceed digester design capacity, lime treatment is an

effective alternate method of sludge stabilization (Farrell et al. 1974;

U.S. Environmental Protection Agency 1978a). Due to the large quanti-

ties of chemical sludges (e.g., alum and iron) usually produced, lime

treatment is particularly suited for the stabilization of these sludge

types (Farrell et al. 1974). At relatively low costs, lime stabilization

reduces the odor, putrescibility potential and pathogen content of raw

sludges (Farrell et al. 1974; U.S. Environmental Protection Agency 1974,

1978a, 1978b). However, the effectiveness of this sludge stabilization

procedure is apparently dependent upon the pH achieved and maintained.

Farrell et al. (1974) demonstrated adequate stabilization of chemical

sludges during lime treatment at pH 11.5 for 30 minutes (pH was main-

tained above 11 for 24 hours). Further research has indicated, however,

that the pH must be maintained above 12 for 30 minutes (pH remaining

above 11 for at least 14 days) during liming in order to ensure effec-

tive sludge stabilization (U.S. Environmental Protection Agency 1974,










1978a). The lime dosage required to exceed, for example, pH 12 for

30 minutes has been found to be affected by the sludge type, chemical

composition of the sludge and percent sludge solids (Farrell et al.

1974; U.S. Environmental Protection Agency 1978a). In addition to

achieving stabilization, lime treatment also conditions the sludge (see

Figure 2-1) such that subsequent sludge dewatering is improved (Farrell

et al. 1974; U.S. Environmental Protection Agency 1974).

Most bacterial pathogens in raw sludge have been shown to be

destroyed during lime stabilization (Farrell et al. 1974; U.S. Environ-

mental Protection Agency 1974, 1978a). Fecal streptococci, however,

remain viable during liming (U.S. Environmental Protection Agency

1978a). Moreover, regrowth of bacterial organisms can occur if the

pH of the lime-stabilized sludge is allowed to drop rapidly below 11

(Farrell et al. 1974; U.S. Environmental Protection Agency 1974).

Under ideal conditions, lime treatment is superior to anaerobic diges-

tion in the inactivation of bacterial pathogens in raw sludge (U.S.

Environmental Protection Agency 1978a). As a result, the bacterial

pathogen concentrations in lime-stabilized sludges are 10 to 1,000

times lower than in anaerobically digested sludges (U.S. Environmental

Protection Agency 1978a).

As far as enteric viruses are concerned, no research has been

conducted on their fate during lime stabilization of raw sludge

(Farrell et al. 1974). However, since seeded poliovirus type 1 has

been shown to be inactivated during the flocculation of raw sewage with

lime at pH 11.5 and to be undetectable (i.e., apparently fully










inactivated) after 12 hours in the lime sludge produced (Sattar et al.

1976), it can be hypothesized that substantial quantities of entero-

viruses are probably inactivated during the lime stabilization of

raw sludges (i.e., primary, chemical and secondary sludges). This

hypothesis has yet to be confirmed experimentally. Until new informa-

tion becomes available, lime-stabilized sludges should be regarded as

potentially containing pathogenic enteric viruses, and,therefore, should

be handled with care during further treatment and final disposal.

Stabilization-heat treatment. Heat treatment is yet another

process which has been used to stabilize (see Figure 2-1) raw sludges

(U.S. Environmental Protection Agency 1974, 1978b). Two types of heat

treatment have been used for sludge stabilization, and they are pasteuriza-

tion at approximately 70C and low-pressure (180 to 210 psi) oxidation

at approximately 200C (U.S. Environmental Protection Agency 1974).

Pasteurization at 70C for 30 to 60 minutes destroys most pathogens

in raw sludge including enteric viruses (U.S. Environmental Protection

Agency 1974). Due to the extremely high temperatures (i.e., 200C)

employed during low-pressure oxidation, all pathogens, including

enteric viruses, in raw sludge are undoubtedly destroyed (U.S. Environ-

mental Protection Agency 1974, 1978b). Under the most ideal conditions,

however, low-pressure oxidation has displayed a poor capacity to reduce

the odor and the putrescibility potential of raw sludges (U.S. Environ-

mental Protection Agency 1978b). As shown in Figure 2-1 and described

below, heat treatment by the low-pressure oxidation process also

conditions the sludge such that subsequent sludge dewatering is










improved (U.S. Environmental Protection Agency 1974, 1978a). It should

be pointed out that the heat treatment of sludge is an energy-intensive

process. Consequently, in actual practice, the applicability of this

treatment procedure is limited due to high energy costs.

Conditioning-chemical treatment. As shown in Figure 2-1,

stabilized sludge is frequently conditioned with organic polymers or

inorganic chemicals (i.e., ferric chloride, ferrous sulfate, lime or

alum) in order to facilitate water removal by subsequent sludge de-

watering processes (U.S. Environmental Protection Agency 1974, 1978a).

These flocculants provide charge neutralization and thereby aggregate

the sludge particles such that a porous, free-draining cake structure

is produced (U.S. Environmental Protection Agency 1974, 1978a). Con-

sequently, chemical conditioning improves sludge dewaterability and

sludge solids capture during dewatering procedures (U.S. Environmental

Protection Agency 1974).

Although never tested, it can be hypothesized that indigenous

enteric viruses in sludge are probably associated with the sludge-

particle aggregates produced during the chemical conditioning of sludge.

Thus, enteric viruses can be expected to be concentrated in the con-

ditioned-dewatered sludge. Except in the case of lime treatment, no

significant viral inactivation is likely to result from chemical con-

ditioning.

Conditioning of sludge with lime is routinely undertaken in

conjunction with ferric chloride (U.S. Environmental Protection Agency

1974). As shown in Figure 2-1 and described above, lime treatment










also provides stabilization of the sludge (U.S. Environmental Protection

Agency 1974, 1978a, 1978b). As such, lime conditioning of sludge

reduces odors and the pathogen, including viral, content of sludge

(U.S. Environmental Protection Agency 1974, 1978b; also see pages 30 to

32 in this chapter). Until more information becomes available, however,

lime-conditioned sludges should not be regarded as virus free.

Conditioning-heat treatment. Heat treatment at temperatures of

300 to 500F (i.e., 150 to 260C) and pressures of 150 to 400 psi for

periods of 15 to 40 minutes is another conditioning process which

facilitates sludge dewatering (see Figure 2-1) (U.S. Environmental Pro-

tection Agency 1974, 1978a). Such heat treatment solubilizes and

hydrolyzes the smaller and more highly hydrated sludge particles which

are then removed from the bulk sludge sample and end up in the cooking

liquor (U.S. Environmental Protection Agency 1974). Consequently, heat-

conditioned sludge displays a reduced affinity for water and an

improved dewatering capacity (U.S. Environmental Protection Agency 1974,

1978a). As shown in Figure 2-1 and described above, heat treatment

also leads to the stabilization of sludge (U.S. Environmental Protec-

tion Agency 1974, 1978b). Due to the high temperatures employed, all

pathogens, including enteric viruses, in sludge are destroyed during

heat conditioning (U.S. Environmental Protection Agency 1974, 1978b).

Unfortunately, high energy costs often make heat conditioning of sludge

impractical.

Dewatering-drying beds. In the United States and Europe,

sandbed drying (see Figure 2-1) is the most widely used method for

sludge dewatering (U.S. Environmental Protection Agency 1974). Drying










beds consist of 6 to 9 inches (ca. 15 to 23 cm) of sand underlaid with

approximately 12 inches (ca. 31 cm) of graded gravel or stone (U.S.

Environmental Protection Agency 1974). Criteria for the design of

drying beds can be found in the literature (U.S. Environmental Protec-

tion Agency 1974, 1978a). In drying beds, water removal from sludge

is accomplished first by drainage filtratee is collected by underdrain

system and is returned to the plant for further treatment) and then

followed by evaporation (U.S. Environmental Protection Agency 1974).

The effectiveness of sludge dewatering in drying beds is influenced by

weather conditions (e.g., precipitation, solar radiation, air tempera-

ture, and relative humidity), sludge characteristics (e.g., primary

sludge dries faster than secondary sludge, digested sludge dries faster

than raw sludge, and digested sludge dries faster than lime-stabilized

sludge) and prior use of sludge conditioning (e.g., proper chemical

conditioning can reduce sludge dewatering time by 50% or more) (U.S.

Environmental Protection Agency 1974, 1978a). Sludge solids contents

ranging from 45% for well-digested sludge to 90% for chemically

conditioned sludge can be achieved on drying beds (U.S. Environmental

Protection Agency 1974). As with other dewatering processes, sandbed

drying reduces the volume of sludge to be further treated and disposed

of (U.S. Environmental Protection Agency 1974). Such dewatering,

therefore, reduces the fuel requirements of, for example, sludge

incineration (U.S. Environmental Protection Agency 1974).

During the air drying of sludge, it has also been demonstrated

that enteric viruses are inactivated. Working with raw sludge (pH 6 or










less; lacking virucidal ammonia), Ward and Ashley (1977b) found a

gradual reduction in the titer of seeded poliovirus type 1 (CHAT)

during the air drying of sludge at 21C from 5% to 65% sludge solids

content. However, when the sludge was allowed to dry to a solids

content of 83% or greater, these investigators observed a dramatic

decrease in poliovirus titer of greater than three orders of magni-

tude in 4 days (similar results were also obtained using seeded cox-

sackievirus Bl and reovirus 3). Ward and Ashley (1977b) went on to

demonstrate that viral RNA is released during the air drying of sludge

and this results in irreversible viral inactivation. Moreover, the

evaporation process itself, and not some virucidal agent (note that

the raw sludge used lacked the virucidal form of ammonia), was found

responsible for poliovirus inactivation during the air drying of

sludge at 21C (Ward and Ashley 1977b). In fact, the inactivation

rate of poliovirus incorporated into dewatered raw sludge was signifi-

cantly lower than the inactivation rate of poliovirus seeded in raw

sludge and allowed to air dry (Ward and Ashley 1977b). During heat

treatment at 47C or 51C, the inactivation rate of poliovirus type 1

(CHAT) incorporated into dewatered raw sludge significantly declined as

the sludge solids content was increased from 5% to 80% (Ward and Ashley

1978b). Sludge solids content (or sludge moisture content) itself was

shown to have an insignificant effect on the rate of poliovirus inac-

tivation by heat (Ward and Ashley 1978b). Sludge solids-associated

ionic detergents, however, were found to protect poliovirus in sludge

from heat inactivation and to be concentrated during sludge dewatering










(Ward and Ashley 1978b). Hence, the greater protection from heat

inactivation afforded to poliovirus as the sludge solids content was

increased (Ward and Ashley 1978b). The ionic detergents in raw sludge

were shown to be substantially degraded during the composting process

and as a result, seeded poliovirus was heat-inactivated (39C or 43C)

at a greater rate in composted sludge than in raw sludge held at the

same sludge solids content (Ward and Ashley 1978b). Other enteroviruses

(e.g., poliovirus 2, coxsackievirus A13 and coxsackievirus Bl) have

also been shown to be protected from heat inactivation in dewatered raw

sludge (Ward and Ashley 1978b).

From the research presented above, it can be concluded that

substantial viral inactivation would occur, during the air drying of raw

sludge at 21C, only when sludge solids contents above 80% are achieved

(Ward and Ashley 1977b). In actual practice, however, such sludge

solids contents are rarely attained for raw sludge during sandbed drying

(U.S. Environmental Protection Agency 1974). Furthermore, raw sludge is

not routinely subjected to air drying because of the odors, insect

pests, unsatisfactory drying rate and other problems associated with

this practice (U.S. Environmental Protection Agency 1974). Thus, sand-

bed drying is normally restricted to well-digested sludge (U.S. Environ-

mental Protection Agency 1974). It can be hypothesized that entero-

viruses are probably more rapidly inactivated in anaerobically digested

sludge than in raw sludge when subjected to air drying. This is

because anaerobically digested sludge usually contains virucidal

ammonia (Ward and Ashley 1976, 1977a). This agent has been shown to










reverse the protective effect towards enteroviruses attributed to ionic

detergents associated with sludge solids (Ward and Ashley 1977c, 1978a,

1979; Ward et al. 1976). Moreover, since the evaporation process
itself was found primarily responsible for viral inactivation during

the air drying of sludge (Ward and Ashley 1977b), and digested sludge

has been shown to air dry at a more rapid rate and to a greater extent

than raw sludge (U.S. Environmental Protection Agency 1974), it follows

that viral inactivation would probably be greater in anaerobically

digested sludge than in raw sludge. Ward and Ashley (1978b) also ob-

served the inactivation rate of poliovirus type 1 (CHAT) seeded in

dewatered raw sludge to increase with a corresponding increase in tem-

perature. Consequently, viral inactivation is likely to be substan-

tially accelerated during the air drying of sludge at higher temperatures

than the 21C employed by Ward and Ashley (1977b). Under themost ideal

conditions, however, sludge dewatering by air drying in sandbeds is not
likely to yield a virus-free product. In Florida, for example, Wellings et

al. (1976a) detected 24 PFU of echovirus type 7 in 250 grams of air-

dried (for 13 days) sludge obtained from drying beds. Due to the pos-

sible viral hazard, all air-dried sludges should be handled with care
during further treatment and final disposal (Wellings et al. 1976a).

Dewatering-drying lagoons. Lagoons have also been commonly
used in the United States for sludge dewatering (see Figure 2-1) (Fair

et al. 1968; U.S. Environmental Protection Agency 1974). Bitton (1980b)
reported that 264,000 dry tons of treated sludge (or 4.5% of the total

sludge available) are discharged yearly into lagoons in the United








States. Due to the great potential for odor problems associated with

sludge lagoons, only well-stabilized sludge has been recommended for

dewatering in lagoons (U.S. Environmental Protection Agency 1974).

Criteria for the design of drying lagoons can be found in the litera-

ture (Sanks et al. 1976; U.S. Environmental Protection Agency 1974).

Particularly important are the design criteria intended for the protec-

tion of groundwater supplies. For example, the bottom of sludge lagoons

must be at least 18 inches above the maximum groundwater table in order

to prevent groundwater contamination (U.S. Environmental Protection

Agency 1974). In drying lagoons,water removal from sludge is primarily

achieved by evaporation (U.S. Environmental Protection Agency 1974).

Therefore, the effectiveness of sludge dewatering in drying lagoons is

mostly influenced by weather conditions (e.g., maximum drying rate in

hot, arid climate) and by sludge depth (e.g., drying rate increases as

the sludge depth decreases) (U.S. Environmental Protection Agency 1974).

The little information available indicates that sludge dewatering in

drying lagoons is an extremely slow process. Sludge held in a lagoon

at depths of 2 to 4 feet, for example, required three years to dewater

from 5% solids content to 45% solids content (U.S. Environmental Pro-

tection Agency 1974).

Long-term lagooning has been found to destroy a significant

fraction of the pathogens in digested sludge (U.S. Environmental Pro-

tection Agency 1978b). In lagoons that had stopped receiving addi-

tional quantities of digested sludge prior to their investigations,

Sattar and Westwood (1979) and Farrah et al. (1981a) confirmed that

indigenous enteric viruses associated with lagooned sludge are inac-

tivated at a measurable rate. Under the warm temperatures of late










spring in Florida, Farrah et al. (1981a) found, for example, that

the enterovirus titer of lagooned sludge dropped from 80 TCID50/g

of dry sludge to low or undetectable levels in approximately 6 weeks

(note that similar decline was also observed for fecal coliforms in

the lagooned sludge). In contrast, Sattar and Westwood (1979) detected

enteric viruses in 39% of the sludge samples obtained from a lagoon

in Ottawa, Canada, over a 14-month period (i.e., from April 1975 to

May 1976). These investigators were able to recover viruses from sludge

samples taken from the lagoon after 8 months. Clearly, indigenous

viruses were inactivated at a much slower rate in the Canadian sludge

lagoon (Sattar and Westwood 1979) than in the Floridian sludge lagoon

(Farrah et al. 1981a). Apparently, lower Ottawa temperatures (Sattar

and Westwood 1979) contributed to greater viral persistence in the

Canadian sludge lagoon. Whereas temperature appears to be an important

factor affecting the inactivation rate of viruses in sludge lagoons,

sludge drying is unlikely to have a significant effect. This is

because, in lagoons, sludge dries at such a slow rate that substantial

viral inactivation cannot be expected to result from the drying process

itself. In addition to sludge treatment (i.e., drying and further di-

gestion), lagoons also provide a temporary method of sludge storage

(Fair et al. 1968; U.S. Environmental Protection Agency 1974). Ulti-

mately, however, lagooned sludge must be disposed of and the method of

choice is usually land application (U.S. Environmental Protection Agency

1974). Although the research presented above indicates that long-term









lagooning substantially reduces the viral content of digested sludge,

indigenous enteric viruses have, nevertheless, been routinely detected

in lagooned sludge undergoing land application (Farrah et al. 1981a;

Sattar and Westwood 1979; Turk et-al. 1980). Consequently, all lagooned

sludges should be handled with care during final disposal in order to

avoid possible viral hazards (Sattar and Westwood 1979).

Heat drying and reduction. Numerous heat drying and reduction

processes (see Figure 2-1) are currently used for the removal of water

from and for the reduction in the volume of sludge (U.S. Environmental

Protection Agency 1974, 1978b). Reduction processes also destroy a

major portion of sludge solids (U.S. Envrionmental Protection Agency

1974). Due to the extremely high temperatures employed in these

treatment processes, all pathogens, including enteric viruses, in

sludge are destroyed (U.S. Environmental Protection Agency 1974, 1978b).

Rising energy costs, however, are making these sludge treatment

processes impractical (U.S. Environmental Protection Agency 1974, 1978b).

It should be pointed out that the heat-dried sludge or ash produced

by the drying or reduction processes, respectively, require final dis-

posal.

Sludge irradiation. The treatment of sludge with ionizing
radiation has recently been shown to be highly effective in destroying

the pathogens, including enteric viruses, present in sludge (see

review by Bitton 1980b). In particular, thermoradiation (i.e., ionizing

radiation combined with moderate heat) has been demonstrated by Ward

(1977) to rapidly inactivate poliovirus type 1 (CHAT) seeded in raw










sludge. The combined heat and radiation treatments appeared to have a

synergistic effect on the survival of poliovirus in raw sludge (Ward

1977). Due to its usual lack of pathogens, irradiated sludge has been

recommended for use as an animal feed supplement or for other agricul-

tural purposes (Bitton 1980b). It is worth noting, however, that sludge

irradiation is an energy-intensive process that has yet to become widely

used in the treatment of sludge (note that it is not included in

Figure 2-1 as a standard sludge treatment process).

Final sludge disposal. Methods for final sludge disposal will

be dealt with in a subsequent section.

Viral and Other Health Hazards
Associated with Treated Sludges

As described above, most sludge treatment processes do not

yield a virus-free product. With the possible exception of sludges

treated at high temperatures, treated sludges represent potential health

hazards due to the presence of viral pathogens (see review presented

above). In addition to viruses, treated sludges (e.g., digested

sludges) also contain bacterial pathogens (Foster and Engelbrecht 1973;

Kowal and Pahren 1978), parasites (Hays 1977; Kowal and Pahren 1978;

Little 1980; Pahren et al. 1979), toxic metals such as cadmium, copper,

nickel, lead, zinc, and chromium (Chaney 1980; Jones and Lee 1978; Kowal

and Pahren 1978; Pahren et al. 1979), and toxic organic residues such

as aldrin, dieldrin, chlordane, heptachlor, lindane, toxaphene, poly-

chlorinated biphenyls, and benzo(a)pyrene (Dacre 1980; Jones and Lee 1978;

Kowal and Pahren 1978; Pahren et al. 1979). Clearly, the biological










and chemical properties of sludge are quite complex (Peterson et al.

1973; U.S. Environmental Protection Agency 1978b). Due to the numerous

health hazards associated with treated sludges, final sludge disposal

should be handled with the utmost of care.


Final Sludge Disposal

In 1979, municipal treatment plants in the United States were

producing approximately 4.5 billion dry kg of sludge per year and

this.is expected to rise to 8 billion by the early 1980s (Pahren et al.

1979). Such large quantities of sludge coupled with the health hazards

associated with sludge make final sludge disposal the most difficult

of the sludge treatment processes (see Figure 2-1). Final sludge dis-

posal is usually accomplished by either ocean dumping, sanitary landfill

or land application (see Figure 2-1) (U.S. Environmental Protection

Agency 1974, 1978b). Sludge incineration is not technically a final

disposal method since ash is produced which requires disposal (in

Figure 2-1, incineration is classified as a sludge reduction process).

In actual practice, however, sludge incineration is considered a

disposal method (Pahren 1980; Pahren et al. 1979; U.S. Environmental

Protection Agency 1978b).

Incineration. Of the total amount of sludge disposed of in

1979 nationally, approximately 35% was incinerated (Pahren et al. 1979).

In the future, however, sludge disposal by incineration is likely to

be significantly curtailed due to air pollution, high energy costs and

other problems associated with this disposal practice (Pahren 1980;

Pahren et al. 1979; U.S. Environmental Protection Agency 1974, 1978b).










Moreover, sludge incineration destroys a potentially valuable resource

which could be utilized, for example, on agricultural land (Pahren

1980).

Ocean dumping. For years, seacoast communities have been dis-

charging digested sludge offshore in deep water (Fair et al. 1968;

U.S. Environmental Protection Agency 1974). Approximately 15% of the

sludge disposed of in 1979 nationally was dumped in the ocean (Pahren

et al. 1979). By the end of 1981, however, ocean disposal of sludge

will be prohibited by the U.S. federal government (Cowlishaw and Roland

1973; Pahren 1980; Pahren et al. 1979; U.S. Environmental Protection

Agency 1974). The primary reason for prohibiting ocean dumping is that

such a practice has been found to have long-term adverse effects on

the ocean environment and on marine life (Cowlishaw and Roland 1973).

Sanitary landfill. The burial of sludge (i.e., sludge covered

by a soil depth greater than the plow layer) in a sanitary landfill

is another popular and acceptable method for sludge disposal (U.S.

Environmental Protection Agency 1974, 1978b). Of the total amount of

sludge disposed of in 1979 nationally, approximately 25% was buried in

sanitary landfills (Pahren et al. 1979). In order to prevent odor,

pathogen, operational and other problems, disposal in sanitary land-

fills is usually restricted to well-stabilized, dewatered (>15% solids

content for sludge-only landfills) sludge (U.S. Environmental Protec-

tion Agency 1974, 1978b). The disposal of sludge in improperly

managed landfills can result in groundwater pollution (Pahren 1980).

Land application. Land application of sludge is receiving

increased attention and will probably be the predominant sludge










disposal method of the future (U.S. Environmental Protection Agency

1974, 1978b). Of the total amount of sludge disposed of in 1979

nationally, approximately 25% was applied to land (Pahren et al. 1979).

In addition to achieving the goal of disposal, the application of

sludge tostrip-mined land and to cropland provides resource utiliza-

tion in land reclamation and crop production, respectively (U.S. Environ-

mental Protection Agency 1974, 1978a, 1978b).

Sludgeapplication benefits cropland in several ways. Nutrients

which are abundantly present in municipal sludge are utilized effec-

tively by growing plants (Cowlishaw and Roland 1973; Pahren 1980; Pahren

et al. 1979; U.S. Environmental Protection Agency 1974, 1978a, 1978b).

Sludge application also improves several soil properties which are

important for crop growth. For example, sludge addition increases the

water content, water retention capacity, cation exchange capacity

(CEC), organic carbon content and stable aggregate content of soils

(Epstein 1975; Epstein et al. 1976). The application of lime-

stabilized sludge increases soil pH and can, therefore, increase sig-

nificantly the productivity of acidic soils such as those found in many

humid regions (Brady 1974). Land application of sludge involving

resource utilization (e.g., cropland application) has distinct advan-

tages over disposal-only methods and is favored by the U.S. Environmental

Protection Agency (Pahren 1980; U.S. Environmental Protection Agency

1978a). It has been estimated that only 1.3% of cultivated lands would

be required for the application of all the sludge and animal waste

produced in the United States (U.S. Environmental Protection Agency

1978b).










The practice of applying sludge to land is not without its

problems. Water movement, for example, has been found to be restricted

(i.e., saturated hydraulic conductivity declined) in sludge-treated

soils (Epstein 1975). Such a reduction in the soil conductivity has

been attributed to the clogging of soil pores by microbial decomposition

products (Epstein 1975). If improperly applied, sludge has been found

to inhibit the growth of a previously planted crop. For example, lime-

stabilized sludge was reported to form a filamentous mat on the soil

surface which resulted in the partial inhibition of previously planted

wheat (U.S. Environmental Protection Agency 1978a). No matting or

crop inhibition was observed when lime-stabilized sludge was incorporated

into the soil prior to planting (U.S. Environmental Protection Agency

1978a).

Undoubtedly, the major drawback of sludge application to land

is the possible dissemination of pathogens and toxic chemicals leading

to adverse effects on human and animal life (Burge and Marsh 1978;

Elliott and Ellis 1977; Foster and Engelbrecht 1973; Kowal and Pahren

1978; Pahren 1980; Pahren et al. 1979). In particular, enteric

viruses present in sludge could potentially move through the soil

matrix and contaminate groundwater supplies (see reviews by Berg 1973b;

Bitton 1975; Bitton 1980a; Bitton et al. 1979b; Burge and Marsh 1978;

Burge and Parr 1980; Cliver 1976; Duboise et al. 1979; Elliott and

Ellis 1977; Foster and Engelbrecht 1973; Gerba et al. 1975; Moore et al.

1978; Sagik 1975). Due to the poor mixing and slow flow (generally

<1 ft /day) conditions found in aquifers, many pollutants entering the










groundwater environment are not appreciably diluted and can persist

for long periods of time (Lee 1976).

Although sludge can be applied to land in several forms (i.e.,

liquid, dewatered or cake-dried), the application of sludge in the

liquid form is usually preferred because of its simplicity (U.S.

Environmental Protection Agency 1974, 1978b). For example, dewatering

processes are not required and inexpensive transfer systems (e.g.,

tank trucks) can be employed for handling liquid sludges (U.S. Environ-

mental Protection Agency 1978b). Liquid sludge is usually applied to

land using one of the following methods: spray irrigation, surface

spreading-ridge and furrow irrigation, surface spreading--followed by

sludge incorporation into the topsoil within 2 to 14 days, and subsurface

injection (U.S. Environmental Protection Agency 1974, 1978b). There

are potential problems associated with each of these application methods.

While spray irrigation is a flexible method that requires minimum soil

preparation,dangerous aerosols containing pathogens are generated during

the spraying of sludge (U.S. Environmental Protection Agency 1974).

The spreading of sludge on the soil surface can lead to the contamina-

tion of surface waters via runoff and/or soil erosion (U.S. Environ-

mental Protection Agency 1974). However, if the applied sludge is

promptly incorporated into the topsoil, surface water pollution, odor

and aesthetic problems are largely eliminated (U.S. Environmental Pro-

tection Agency 1974). The injection of sludge below the soil surface

avoids many of the problems associated with other application methods

(e.g., aerosols, runoff and odors) (U.S. Environmental Protection Agency










1974). Due to the more favorable subsurface environment, however,

pathogens (e.g., viruses) may persist longer in subsurface-injected

sludge than in surface-applied sludge (Moore et al. 1978).

Whatever the application method employed, the disposal of

sludge on land should be undertaken in accordance with local, state,

and federal regulations and recommendations (Manson and Merritt 1975;

U.S. Environmental Protection Agency 1974, 1978b; Wright 1975).

Generally, only stabilized sludge is recommended for land application

(U.S. Environmental Protection Agency 1974). Application rates of

sludge to cropland vary depending upon sludge composition, soil charac-

teristics, climate, vegetation and cropping practices (U.S. Environ-

mental Protection Agency 1974), but should not exceed 20 dry tons of

sludge solids/acre/year (44.8 dry metric tons/ha/year) or 46.8 m3 /ha/

day (liquid rate) (Manson and Merritt 1975; U.S. Environmental Protec-

tion Agency 1974). In order to prevent groundwater and surface water

contamination, as well as other potential problems, Manson and Merritt

(1975) recommended that sludge disposal sites conform to the following

standards:

1. High water tables should be no closer to the soil

surface than 4 ft (1.22 m)

2. Isolation from surface waters by a minimum distance

of 200 ft (61 m)

3. Maximum slope of 5% in order to prevent excessive

surface runoff

4. A crop that can be harvested is the preferred

ground cover










5. A minimum distance of 250 ft (ca. 76 m) to the

nearest residence

6. Access to the sludge disposal site should be

restricted

Sludge disposal on properly managed sites in the United States has been

successful and has not led to significant problems (Manson and Merritt

1975; U.S. Environmental Protection Agency 1974).


Fate of Sludge-Associated Viruses in Soils

Literature pertaining to the survival and possible movement of

enteric viruses in sludge-treated soils is presented in the Introductions

to Chapters IV through VI.














CHAPTER III
EFFECT OF SLUDGE TYPE ON POLIOVIRUS
ASSOCIATION WITH AND RECOVERY FROM SLUDGE SOLIDS


Introduction
The degree of association between viruses and sludge solids

is a critically important factor in the assessment of the fate of

these pathogens following sludge disposal on land. Yet, the nature of

the association between viruses and sludge solids has not been adequately

explored, partly because of the lack of virological methods. Recently,

however, methods have been developed for the recovery of viruses from

wastewater sludges. Practical methods for the recovery of viruses

from sludge samples involve two steps. Because viruses in sludges

have been found to be solids associated (Abid et al. 1978; Glass et al.

1978; Hurst et al. 1978; Lund 1971; Ward and Ashley 1976; Wellings et

al. 1976a), the first step of an effective method, therefore, consists

of releasing both surface-adsorbed and solids-embedded viruses. The

second step consists of concentrating the eluted viruses prior to viral

assays.

Various chemicals have been used to elute viruses from sludge

solids, namely 0.1% sodium lauryl sulfate in 0.05 M glycine, pH 7.5

(Abid et al. 1978), tryptose phosphate broth (Moore et al. 1978),

3% beef extract, pH 9.0 (Wellings et al. 1976a), 3% beef extract,

ambient pH (Glass et al. 1978; Sattar and Westwood 1976), 0.05 M glycine

buffer, pH 11.0 (Hurst et al. 1978), 2% fetal calf serum in Earle's










balanced salt solution, pH 9.5 (Subrahmanyan 1977), and 10% fetal
calf serum (Sattar and Westwood 1976). Elution of solids-associated

viruses may be aided by sonication (Abid et al. 1978; Glass et al.
1978; Wellings et al. 1976a), shaking on a wrist-action shaker

(Sattar and Westwood 1976), homogenization in a blender (Glass et al.

1978; Moore et al. 1978; Subrahmanyan 1977), or magnetic stirring

(Abid et al. 1978; Hurst et al. 1978). Eluted viruses have been con-
centrated by organic flocculation at low pH (Abid et al. 1978; Glass

et al. 1978; Hurst et al. 1978), hydroextraction (Wellings et al.
1976a) or adsorption to bentonite clay (Turk et al. 1980).

Several of the methods proposed for the recovery of viruses
from sludges do not contain a concentration step but simply involve
the elution of viruses from sludge solids (Moore et al. 1978; Sattar

and Westwood 1976; Subrahmanyan 1977). The use of these methods is

limited to raw sludges and other sludges containing large amounts of
viruses. The concentration method proposed by Wellings et al. (1976a)

involving hydroextraction is cumbersome and requires considerably more
time to perform than organic flocculation. The procedure developed

by Abid et al. (1978) is not practical, since it does not adequately
concentrate the viruses eluted from sludge solids. Therefore, the

best methods proposed to date for virus recovery from sludges appear to
be those of Glass et al. (1978) and Hurst et al. (1978). Working with
anaerobically digested sludge, Glass et al. (1978) obtained an overall
recovery of poliovirus type 1 (CHAT) of 31%. Hurst et al. (1978) found
that poliovirus type 1 (LSc) could be recovered from activated sludge










samples with an overall efficiency of 80%. It is difficult to compare

these two methods, since they were evaluated with different sludge

types.

The research reported in subsequent chapters of this disserta-

tion deals primarily with the fate of enteroviruses following sludge

disposal on land. In the course of conducting this research, the viral

(i.e., indigenous and seeded) content of different sludge types had to

be determined. Consequently, it was imperative to determine if viruses

could be effectively recovered from the solids of different sludge types.

The glycine method developed by Hurst et al. (1978) was evaluated for

its effectiveness in recovering poliovirus type 1 (LSc) from different

sludge types. The sludge types used were activated sludge mixed liquor,

and anaerobically and aerobically digested sludges. It is the aim of

this chapter to show that sludge type is a factor that can strongly

influence the degree of association between viruses and sludge solids,

as well as the recovery of sludge solids-associated viruses by the gly-

cine method (Hurst et al. 1978).


Materials and Methods
Virus and Viral Assays

Poliovirus type 1 (strain LSc) was used in the research

reported in this chapter. This virus strain is a non-neurotropic

variant of the Mahoney strain, and is avirulent for mice and monkeys by

all routes. This strain is identical to the virus designated as at-

tenuated Sabin strain (Cooper 1967; Hahn 1972; World Health Organiza-

tion 1968). Some general properties of polioviruses are shown in









Table 3-1. Stocks of the virus were prepared by infecting monolayer

cultures of AV3 (a continuous line of human amnion), BGM (a continuous

line of Buffalo green monkey kidney--Barron et al. 1970; Dahling et al.

1974) or MA-104 (a continuous line of fetal rhesus monkey kidney) cells

in a 32 oz (128 cm2) glass bottle (rubber-lined, screw-capped--Brockway

Glass Co., Inc.). After allowing an adsorption period of 60 minutes

with tilting at 15-minute intervals, the cells were overlaid with 40 ml

of Eagle's minimal essential medium (MEM) supplemented with 10% fetal

calf serum (FCS), 250 U/ml penicillin, and 125 ig/ml streptomycin (see

Appendix for more details on the composition of this and other media

used). After approximately 48 hours of incubation at 37C, the overlay

medium was decanted and then centrifuged at. 270 x g for 15 minutes at

4C to remove cell debris. The resulting supernatant containing the

virus was distributed in 1, 2 or 5 ml aliquots and immediately frozen

at -70C. The virus was kept at -70C until used.

Poliovirus was assayed by the plaque technique (Cooper 1967) on

AV3, BGM or MA-104 cell monolayers prepared as follows. Confluent

cell monolayers were grown in 32 oz glass bottles using Eagle's MEM

supplemented with 10% FCS, 250 U/ml penicillin and 125 ug/ml strepto-

mycin (i.e., growth medium; see Appendix). Each cell monolayer was

then washed three times with 10 ml of the pre-trypsin solution (see
Appendix). This treatment removes all traces of serum (contains

trypsin inhibitors), as well as Ca2 and Mg12 ions (enhance adsorption

of cells to glass). To remove the cells from the glass bottle, 10 ml

of the standard trypsin-versene solution (see Appendix) was then added









General properties of polioviruses


Property


Nucleic acid

Molecular weight of nucleic acid (daltons)

Particle diameter (nm)

Particle morphology

Particle isoelectric point

Stability at 25C

Stability at pH 3.0

Stability in ether


RNAab (single-stranded)

2 x 106ab

27 to 30a'b'c

Icosahedrala',b

4.5 and 7.0d

Relatively stable

Stablea,b,e

Stable


aFrom Davis et al. (1973).

bFrom Hahn (1972).

CFrom Schwerdt and Schaffer (1955).

dFrom Mandel (1971). The data were obtained using poliovirus type
1 (strain Brunhilde).
eFrom Bachrach and Schwerdt (1952).


Value


TABLE 3-1.










and allowed to spread over the entire monolayer for 30 to 60 seconds.

This solution was subsequently decanted and the cell culture was

allowed to rest at room temperature until the cells came off the glass

(approximately 5 minutes). Growth medium (10 ml) was then added, and

pipetted twice up and down to dislodge the cells from the glass and

break up clumps. An additional 190 ml of the growth medium was added

to the content of the 32 oz glass bottle. This cell suspension was

then distributed in 5 ml aliquots to 40 glass (2 oz--20 cm2) or plastic

(25 cm2) bottles. After approximately 48 hours of incubation at 37C,

these small bottles contained confluent cell monolayers and were ready

for use in viral assays.

Experimental samples were diluted, if necessary, prior to assay

in either Eagle's MEM containing 5% calf serum (rarely used) or

phosphate-buffered saline (PBS) containing 2% FCS (both solutions

contained 250 U/ml penicillin, 125 pg/ml streptomycin, and phenol red;

see Appendix). All samples from each experiment were assayed on only

one cell line (i.e., AV3, BGM or MA-104) using the procedure described

below. A 0.6 ml aliquot of each diluted or undiluted sample was

inoculated in fractions of 0.2 ml into three drained cell monolayers.

Following inoculation, a 60-minute adsorption period with tilting at

15-minute intervals was allowed. The infected monolayers were then

overlaid with 4 ml of 1% methyl cellulose in Eagle's MEM supplemented

with 5% FCS, 250 U/ml penicillin, 125 pg/ml streptomycin, and 117 ug/ml

kanamycin (see Appendix). After incubation at 37C for approximately

48 hours, the cell monolayers were stained with either crystal violet










or neutral red (see Appendix). Plaques were subsequently counted with

the unaided eye as suggested by Cooper (1967). In several experiments,

the plaques were counted using an Omega photographic enlarger B22

(Simmon Brothers, Woodside, New York). Each tabulated viral count

represents the average of triplicate counts. The numbers of viruses were

expressed as plaque-forming units (PFU).


Sludges

A variety of wastewater sludges was used in this study as listed

in Table 3-2. The sludges were obtained from four wastewater treatment

plants located in Gainesville and Pensacola, Florida. In addition, a

lagooned sludge sampled at the West Florida Agricultural Experiment

Station (Jay, Florida) was also used. The treatments the sludges

received before being sampled are also shown in Table 3-2. One of the

sludge types used was activated sludge mixed liquor. The term "mixed

liquor" refers to the suspension undergoing treatment in an activated

sludge unit. The abbreviated sludge designations (see Table 3-2) will

be used to identify sludges in the rest of this dissertation. The

sludges were collected in sterile Nalgene bottles, transported to the

University of Florida (Gainesville) laboratory and then immediately

refrigerated. All sludge samples were used within 30 days of sampling

and most samples were used within 3 days. At the time of use, a

sludge sample was first allowed to come to room temperature. The pH

and solids content of the sludge was then determined. The pH was

measured using a digital pH meter model 125 from Corning (Corning, New

York). The solids content was determined by drying in an oven at 105C








Sources of the wastewater sludges used in this study


Wastewater Abbreviated Sludgeb Sludge treatment
treatment sludge type
plant designation used Digestion Digestion Additional
generating procedure time treatment
the sludge (days)

Campus plant UML Mixed liquor _d --.
(Univ. of Florida) UDA Digested Aerobic 39 --

Main street plant GML Mixed liquor ..--
(Gainesville, GDA90 Digested Aerobic 90 --
Florida) GDA180 Digested Aerobic 180 --
GDAN Digested Anaerobic 60 --

Montclair plant PDA Digested Aerobic 30 Conditioned with
(Pensacola, (and dewatered) Magnafloce 1563c
Florida) and then dewatered

Main street plant PDAN Digested Anaerobic 60 Conditioned with
(Pensacola, (and dewatered) Magnafloce 2535c
Florida) and then dewatered

Montclair and Main LAG Lagoonf --
street plants
(Pensacola, Florida)

aAll plants treated municipal wastewater with the exception of the Main street plant of Pensacola
which treated a mixture of municipal (2/3) and industrial (1/3) wastewater.
bThe sludges used were generated during the secondary treatment (activated sludge or tricking filter)
of wastewater.


TABLE 3-2.




CThe term "mixed liquor" refers to the suspension undergoing treatment in an activated sludge unit.
Although they contain activated sludge solids, mixed liquors are not to be confused with settled sludges
which must be treated and ultimately disposed of.
dA dash means that the sludge treatment was either not applicable or not performed.

eThese are cationic polymers supplied to the treatment plants by the American Cyanamid Company,
Wayne, N.J.


fThis sludge is a mixture of sludge from
Florida. The mixture was kept in a lagoon at
before ultimately being disposed of on land.
study.


the Montclair (1/3) and Main street (2/3) plants of Pensacola,
the West Florida Agricultural Experiment Station (Jay, Florida)
It was the lagooned sludge which was sampled and used in this










for 24 hours a measured volume of sludge and was expressed as a per-

centage on a weight (grams) to volume (milliliters) basis. Most of

the sludges used were not autoclaved or decontaminated in any other

way. Due to uncontrollable contamination of cell monolayers during

viral assays, two sludge samples (GDAN and PDAN--see Table 3-2) had to

be sterilized by autoclaving at 121C with applied pressure of 15 psi

for 15 minutes prior to use. During sludge treatment, digested sludges

are frequently subjected to heat (i.e., 150 to 260C under pressures of

150 to 400 psi) as a conditioning step which improves subsequent

sludge dewatering (U.S. Environmental Protection Agency 1978a; also

see Figure 2-1). Thus, autoclaved sludge can be considered to be heat-

conditioned sludge. As seen in Figure 3-1, the solids of autoclaved,

anaerobically digested sludge (GDAN--see Table 3-2) settled in 2

hours while those of nonautoclaved sludge remained dispersed. This

increase in the rate of settling of sludge solids probably accounts for

the improved-dewatering property of heat-conditioned sludge. Apart

from the effect on the rate of settling of sludge solids, autoclaving

probably did not significantly alter other sludge properties which

affect sludge-virus interactions. For example, Ward and Ashley (1977a)

found that autoclaving does not destroy the enterovirus-inactivating

capacity of anaerobically digested sludge. Furthermore, autoclaving

did not affect the degree of association between poliovirus and

anaerobically digested sludge solids as determined below.

Association of Seeded Poliovirus with Sludge Solids

One milliliter of poliovirus stock in PBS containing 2% FCS was
added directly to 100, 500, or 1,000 ml of sludge while stirring the











FIGURE 3-1. Effect of autoclaving on the rate of settling of anaerobi-
cally digested sludge solids

An aliquot of anaerobically digested sludge (GDAN--
see Table 3-2; solids content, conductivity and pH
equal to 2.0%, 3,250 umho/cm at 25C and 8.3,
respectively) was autoclaved at 121C with applied
pressure of 15 psi for 15 minutes and compared to an
aliquot of the sludge which had not been autoclaved.
Approximately 21 ml each of autoclaved sludge
and nonautoclaved sludge were added to graduated
cylinders A and B, respectively. After 2 hours,
the solids of the autoclaved sludge had settled
while those of the nonautoclaved sludge remained
dispersed.












suspension using either a magnetic stirrer or pipette (magnetic stirring

could not be used for some sludges which had high solids contents).

Poliovirus was seeded in the sludge samples at concentrations approxi-

mately 1,000 times greater than the indigenous virus levels measured

in the sludges used. Therefore, indigenous viruses present in the

sludge samples did not affect the results presented herein. Magnetic

stirring (or frequent mixing with a pipette) was continued for 10

minutes to 60 minutes. Following the contact period, an aliquot of the

unfractionated sludge (i.e., sludge sample without solids separated)

was diluted in PBS containing 2% FCS and assayed directly for seeded

viruses by the plaque technique. This assay was performed in order

to determine if the direct viral assay after the contact period would

agree with the calculated virus input based on the added volume of

poliovirus stock of known titer. From Table 3-3, it can be seen that

poliovirus was recovered with a mean efficiency of 109%',, 104%, and

98% from mixed liquors, aerobically digested sludges and anaerobically

digested sludges, respectively, following dilution and subsequent

direct assay on cell cultures. Thus, poliovirus type 1 (LSc) added

to sludge was recovered effectively after a contact period of 10 to

60 minutes without any significant inactivation. Direct inoculation

of unfractionated sludge into cell cultures has been found'to be toxic

to cells (Nielsen and Lydholm 1980; Subrahmanyan 1977). In my research,

the diluted sludge samples were not toxic to the cell cultures. Simi-

larly, Hurst et al. (1978) diluted virus-seeded activated sludge in

Tris buffer and then successfully inoculated it directly into cell

cultures without causing cell toxicity. Some of the sludges used in my






TABLE 3-3.


Recovery of poliovirus type 1 from unfractionated sludge by dilution and subsequent
direct assay on cell cultures


Sludge Sludges No. of Calculated Mean recovery
type used experimental virus of calculated
trials input virus input
(mean, total PFU) (% SE )

Mixed liquor UML, and 2 4.8 x 106 108.9 6.6
GML
Aerobically UDA, 11 8.2 x 107 103.6 15.7
digested PDA,
GDA90, and
GDA180
Anaerobically GDAN, 10 2.1 x 108 98.1 9.0
digested PDAN, and
LAGu

apoliovirus was added to 1000 ml of sludge while stirring the mixture using a magnetic stirrer.
The calculated virus input was based on the volume of poliovirus stock of known titer added to the
sludge. Magnetic stirring was continued for 10 min to 60 min.


bFollowing the contact period, an aliquot of
were not separated by centrifugation) was diluted
ml, 125 pg of streptomycin per ml and phenol red,


the unfractionated sludge (i.e., the sludge solids
in PBS containing 2% FCS, 250 U of penicillin per
and assayed by direct inoculation into cell cultures.


cAbbreviation for standard error.
dThe lagoon sludge is a mixture of aerobically digested sludge (1/3) and anaerobically digested
sludge (2/3), and consequently, its properties are close to those of anaerobically digested sludges
(see Table 3-2). Therefore, lagoon sludges were placed in the population of anaerobically digested
sludges.









study did, however, produce cell culture contamination when diluted and

then directly inoculated into cell cultures. For these sludges (i.e.,

when not decontaminated by autoclaving), the initial virus present was

determined based on the amount of virus stock of known titer added to

the sludge. From the results shown in Table 3-3, it is believed that

the determination of the initial virus added to the sludge was accurately

achieved by either direct viral assay of the sludge or based on the

amount of virus stock of known titer added to the sludge. The total

sludge volume was subsequently centrifuged at 1,400 x g for 10 minutes

at 4C (only an aliquot of sludges GDA180 and GDAN was centrifuged).

An aliquot of the sludge supernatant produced was assayed for viruses.

Thus, allowing the calculation of the "viable unadsorbed virus" frac-

tion as shown below:

viable unadsorbed virus in sludge supernatant (total PFU) 1
virus (%) virus in unfractionated sludge (total PFU)

(3-1)

Furthermore, the "sludge solids-associated virus" fraction was also

estimated as shown below:

solids-associated virus (%) = 100 viable unadsorbed virus (%) (3-2)

For sludges GDA180 and GDAN, viral assays were performed, as described

above (i.e., of unfractionated sludge and of sludge supernatant), several

times throughout a 12-hour to 8-day period. During this period, the

virus-seeded sludge was left at room temperature undisturbed, and was

stirred only prior to obtaining a sample for viral assay.









Recovery of Seeded Poliovirus from Sludge Components

The sludge supernatant and sludge solids generated were

separately subjected to the virus recovery methodology described below.

In several experiments, the sludge supernatants were not processed for

virus concentration. These supernatants were simply adjusted to neutral

pH and assayed for viruses as described previously. The number of

viruses thus found were included in the "overall virus recovery" values

reported. In those experiments in which the sludge supernatants were

subjected to the virus concentration procedure, the supernatants were

processed like the sludge solids eluates described below.

The sludge solids-associated viruses were eluted and further

concentrated using a modification of the glycine method developed by

Hurst et al. (1978). The solids were mixed with five volumes of 0.05 M

glycine buffer, pH 11.5. The pH of the mixture was adjusted to between

10.5 and 11.0 by the addition of 1 M glycine buffer, pH 11.5. The

samples were vigorously mixed for 30 seconds using a magnetic stirrer

and centrifuged at 1,400 x g for 5 minutes at 4C (all centrifuga-

tion was performed using a Sorvall RC5-B centrifuge, Ivan Sorvall

Inc., Norwalk, Connecticut). The supernatants (i.e., the sludge solids

eluates) were recovered, adjusted to neutral pH by the addition of 1 M

glycine buffer, pH 2.0, and assayed for eluted viruses. The entire

procedure described above was performed in less than 10. minutes. Thus,

poliovirus was subjected to the high pH of 10.5 to 11.0 for no more

than .10. minutes. Both Hurst et al. (1978) and Sobsey et al. (1980b)

observed no appreciable inactivation in D.10. minutes of poliovirus type 1










(LSc) seeded in 0.05 M glycine buffer, pH 10.5 to 11.0. Therefore, it

is believed that there was no significant inactivation of poliovirus

during the elution procedure. It should be noted, however, that this

elution method is not practical for the recovery of reoviruses and

rotaviruses from sludge. These virus types are rapidly inactivated

when subjected to such high pH values (Sobsey et al. 1980b). The

viruses in the sludge solids eluates were concentrated by organic

flocculation (Katzenelson et al. 1976b) as follows. The eluates were

adjusted to pH 3.5 by the addition of 1 M glycine buffer, pH 2.0,%and

the flocs produced were pelleted by centrifugation at 1,400 x g for

20 minutes at 4C. The supernatants and pellets produced were treated

separately. The supernatants were assayed for viruses and then passed

through a series of 3.0, 0.45, and 0.25 im Filterite filters (Filterite

Corp., Timonium, Maryland) in a 47-mm holder. Adsorbed viruses were

eluted from the filters with 7 ml of PBS containing 10% FCS, pH 9.0.

The filter eluates were adjusted to neutral pH by the addition of 1 M

glycine buffer, pH 2.0, and assayed for viruses. The filtrates (i.e.,

the fluids having passed the filters) were adjusted to neutral pH by

the addition of 1 M glycine buffer, pH 11.5, and assayed for viruses.

The pellets previously obtained by centrifuging the samples at pH 3.5

were mixed with five volumes of PBS containing 10% FCS, pH 9.0. The

mixtures were adjusted to pH 9.0 by the addition of 1 M glycine buffer,

pH 11.5, vortexed for 30 seconds and then centrifuged at 14,000 x g

for 10 minutes at 4C. The supernatants were adjusted to neutral pH

by the addition of 1 M glycine buffer, pH 2.0,,and assayed for viruses.










Viral assays were performed at various steps in the procedure in order

to determine the efficiency of the individual steps. Each "overall

virus recovery" value reported was determined from the viruses recovered

in the filter concentrate, pellet concentrate and sludge supernatant

(concentrated or not).

Statistical Treatment of Data

Statistical treatment of the data was performed with the use of

a Hewlett-Packard calculator model 9810A and Statistics Package V-6

(Hewlett-Packard Company, Loveland, Colorado).

Results and Discussion

Association of Seeded Poliovirus
with Sludge Solids

Poliovirus seeded in the various sludge samples rapidly became

associated with the sludge solids. However, no statistically signifi-

cant linear correlation was found between the percent solids .contents .

of the sludges studied and the degree of virus association by the sludge

solids (Table 3-4). This lack of correlation was found within each

sludge type and for all sludge types combined. Thus, the sludge solids

content was shown not to affect the association of virus with sludge

solids, at least in the range of solids contents studied (i.e., 0.5%

through 2.9%). This allowed sludges of different solids contents, but
belonging to the same sludge type, to be grouped together in the same

category.

The mean percent of solids-associated viruses for activated

sludge mixed liquors, anaerobically digested sludges, and aerobically









Effect of sludge type on the association between poliovirus type 1 and sludge solids


Sludge Sludge Sludge parameters Virusb in Virus in Viable Solids- Meang
type used -unfractionatedc sludge unadsorbede associated associated
pH Solidsa sludge supernatant virus virus virus for
content (total PFU) (total PFU) (%) (%) each sludge
(%) type
(% SEh)

Mixed UML 6.4 1.6 8.4 x 106 5.0 x 106 59.5 40.5 57 17A
liquor GML 6.9 0.5 1.3 x 106 3.4 x l05 26.2 73.8

Aerobically UDA 4.8 1.5 6.5 x 106 l.l x 106 16.9 83.1 95 2.0B
digested UDA 4.8 1.5 6.5 x 106 6.4 x 105 9.8 90.2

UDA 6.1 1.3 2.0 x 107 1.3 x 106 6.5 93.5
UDA 6.1 1.3 2.0 x 107 9.6 x 105 4.8 95.2
UDA 6.5 1.3 9.6 x 106 4.0 x 104 0.4 99.6
GDA90 5.8 1.0 1.3 x 107 4.3 x 105 3.3 96.7
GDA180 5.0 1.3 8.2 x 108 4.8 x 106 0.6 99.4
PDA 5.8 2.8 4.1 x 106 5.7 x 103 0.1 99.9

Anaerobically GDAN 8.3 2.0 1.O x lO7 4.4 x 106 44.0 56.0 70 8.4A
digested PDAN 7.2 1.4 6.0 x 106 3.2 x 106 53.3 46.7

PDAN 6.4 1.9 5.5 x 106 1.4 x 106 25.5 74.5
LAGi 7.3 2.9 1.8 x 106 2.9 x 105 16.1 83.9
LAG1 6.9 2.9 1.9 x 109 1.7 x 108 8.9 91.1


TABLE 3-4,




aA two-tailed, t-test concerning the population correlation coefficient (r) revealed that at the 0.01
level there was no significant linear correlation (i.e., r = 0) between the solids content (%) of the sludge
and the "solids-associated virus (%)" values. This lack of correlation was found within each sludge type
and for all sludge types combined.
bThe virus was added to the sludge while stirring the suspension using a magnetic stirrer. Magnetic
stirring was continued for 10 to 60 min and then an aliquot of the virus-seeded sludge was obtained for
viral assay.
CThe sludge solids were not separated prior to assaying.
dThe sludge was clarified by centrifugation at 1400 x g for 10 min at 4C and the supernatant was
subsequently assayed.
eThe "viable unadsorbed virus (%)" values were calculated as shown in the Materials and Methods section.

fThe "solids-associated virus (%)" values were estimated as shown in the Materials and Methods section.

gMean values displaying different superscript capital letters are significantly different at the 0.01
level when subjected to analysis of variance using Duncan's test.
hAbbreviation for standard error, A
to
iThe lagoon sludge is a mixture of aerobically digested sludge (1/3) and anaerobically digested sludge
(2/3), and consequently, its properties are close to those of anaerobically digested sludges (see Table 3,2).
Therefore, lagoon sludges were placed in the population of anaerobically digested sludges.









digested sludges was 57%, 70%, and 95%,. respectively (Table 3-4).

Ward and Ashley (1976) have obtained similar results with anaerobically
digested sludge. These investigators have shown that 65% and 67% of

seeded poliovirus type 1 (Mahoney) and poliovirus type 1 (CHAT),
respectively, were associated with anaerobic sludge solids after a con-

tact period of 15 minutes. The association between seeded poliovirus

and sludge solids was significantly greater for aerobically digested
sludges than for mixed liquors or anaerobically digested sludges. No

statistically significant difference was found between the mean

percent of solids-associated viruses for mixed liquors and anaerobically
digested sludges. The lagoon sludges mentioned in Table 3-4 are a

mixture of aerobically digested sludge (1/3) and anaerobically digested

sludge (2/3), and consequently, were placed in the category of

anaerobically digested sludges. However, the association of poliovirus

with lagoon sludge solids was greater than the association of this virus

with other anaerobic sludge solids tested (see Table 3-4). Apparently,

the presence of aerobic sludge solids (1/3) in lagoon sludge accounts

for the greater ability of lagoon sludges to bind viruses.

The reason for the greater association of seeded viruses with
aerobic sludge solids is still unknown. However, aerobically digested

sludges generally displayed lower pH values (ranging from 4.8 to 6.5)
than mixed liquors (6.4 and 6.9) or anaerobically digested sludges
(ranging from 6.4 to 8.3) (see Table 3-4). Virus adsorption to surfaces
is promoted at low pH. Other parameters of aerobically digested sludges,
as yet unidentified, could also account for the greater ability of these









sludges to bind viruses. However, we have shown that the sludge solids
content does not account for the differences observed in the virus-

binding capacity of different sludge types. In the range of sludge

solids contents studied, there appears to be sufficient sites for virus
binding and, therefore, this sludge parameter is not a limiting factor

in determining the association of viruses with sludge solids.

The degree of association between seeded poliovirus, and aerobic
(see Table 3-5) or anaerobic (see Table 3-6) sludge solids remained

fairly constant over a contact period of 8 days or. 12 hours,
respectively. Clearly, varying the contact time would not have sig-

nificantly affected the results presented above on the virus-binding

capacity of different sludge types. The inactivation rate of poliovirus

seeded in sludge was also determined from the data shown in Tables 3-5

and 3-6. In anaerobically digested sludge, the total amount of
infectious poliovirus present in the sludge (i.e., virus in unfrac-

tionated sludge) steadily declined over a 12-hour period (Table 3-6).
In this sludge type, there was approximately a 50% reduction in the
poliovirus titer in 12 hours (i.e., approximately 1 loglo reduction/36

hours) at room temperature (see Table-3-6). Ward and Ashley (1976)
measured similar inactivation rates for poliovirus type 1 (CHAT and

Mahoney) seeded in anaerobically digested sludge. In contrast to
anaerobically digested sludge, there was no significant inactivation
of poliovirus seeded in aerobically digested sludge during a 7-day

contact period at room temperature (see Table 3-5). The low viral-

inactivating capacity of aerobically digested sludge has also been








TABLE 3-5.


Effect of contact time on the association between
poliovirus type 1 and aerobically digested sludge solids


Contact Virus in Virus Viable d Solids-
time unfractionatedb in sludge unadsorbedd associated
sludge supernatantc virus virus
(total PFU) (total PFU) (%) (%)

30 min 8.9 x O108 4.2 x 106 0.5 99.5
60 min 8.2 x O108 4.8 x 106 0.6 99.4
2 days 1.2 x 109 1.3 x 107 1.1 98.9
4 days 1.2 x 109 4.3 x 106 0.4 99.6
5 days 8.0 x 108 8.8 x 106 1.1 98.9
6 days 5.8 x O108 1.9 x 106 0.3 99.7
7 days 8.2 x 108 2.5 x O106 0.3 99.7
8 days 6.9 x O108 2.5 x 106 0.4 99.6

aThe virus was added to 1000 ml of aerobically digested sludge
(GDAl80--see Table 3-2; solids content and pH equal to 1.3% and 5.0,
respectively) while stirring the suspension using a magnetic stirrer.
Magnetic stirring was continued for 60 min. For the remainder of the
experimental trial, the virus-seeded sludge was left at room temperature
undisturbed, and was stirred only prior to obtaining a sample for viral
assay.
bThe sludge solids were not separated prior to assaying.

CThe sludge was clarified by centrifugation at 1400 x g for 10 min
at 4C and the supernatant was subsequently assayed.
dThe "viable unadsorbed virus (%)" values were calculated as shown
in the Materials and Methods section.
eThe "solids-associated virus (%)" values were estimated as shown
in the Materials and Methods section.








TABLE 3-6, Effect of contact time on the association between poliovirus
type 1 and anaerobically digested sludge solids


Contact Virusa in Virus Viable Solids-
time unfractionatedb in sludge unadsorbedd associated
(hours) sludge supernatant virus virus
(total PFU) (total PFU) (%) (%)

0 1.1 x 107 4.6 x 106 41.8 58.2

0.5 1.1 x 107 3.5 x 106 31.8 68.2

1.0 1.0 x 107 4.4 x 106 44.0 56.0

6.0 9.4 x 106 2.9 x 106 30.9 69.1

8.5 6.5 x 106 2.3 x 10O6 35.4 64.6

12.0 5.3 x 106 2.5 x 106 47.2 52.8

aThe virus was added to 1000 ml of anaerobically digested sludge
(GDAN--see Table 3-2- solids content and pH equal to 2.0% and 8.3,
respectively) while stirring the suspension using a magnetic stirrer.
Magnetic stirring was continued for 60 min. For the remainder of the
experimental trial, the virus-seeded sludge was left at room temperature
undisturbed, and was stirred only prior to obtaining a sample for viral
assay.
bThe sludge solids were not separated prior to assaying.

CThe sludge was clarified by centrifugation at 1400 x g for 10 min
at 4C and the supernatant was subsequently assayed.
dThe "viable unadsorbed virus (%)" values were calculated as shown
in the Materials and Methods section.
eThe "solids-associated virus (%)" values were estimated as shown
in the Materials and Methods section.









demonstrated in the field. Farrah et al. (1981a) found that aerobi-

cally digested sludge contained larger indigenous viral titers than

anaerobically digested sludge. The uncharged form of ammonia has

been shown to exist in sludge (i.e., tested raw and anaerobically

digested sludge) mostly at pH values above 8 and to display virucidal

activity against enteroviruses (Ward and Ashley 1976, 1977a). Due to

the typically low pH of aerobically digested sludge (see Tables 3-4

and 3-5), it can be hypothesized that the virucidal ammonia is probably

largely absent from this sludge type. Therefore, the low viral-

inactivating activity observed in the aerobically digested sludge

(GDA180 sludge, pH 5.0) employed in this study (see Table 3-5) was to

be expected. Similarly, Ward and Ashley (1976) found no appreciable

inactivation of seeded poliovirus type 1 in .5 days:. at: 20C in raw

sludge maintained at its naturally low pH of 6.0. Naturally high pH

values, on the other hand, have been found in anaerobically digested

sludge and consequently, this sludge type has demonstrated substantial

viral-inactivating activity due to the presence of the virucidal

ammonia (Ward and Ashley 1976, 1977a). The results presented in Table

3-6 confirm that enteroviruses (i.e., used poliovirus type 1) seeded in

anaerobically digested sludge (i.e., used GDAN sludge, pH 8.3) are

inactivated at a significant rate.

It is emphasized that the results and conclusions presented
above pertain only to the virus used, poliovirus type 1 (LSc). Other

enterovirus may, in fact, display different patterns of adsorption to

sludge solids. Research has shown that only 20.7% of seeded echovirus









type 1 (Farouk) became associated with lagoon sludge solids after a

contact period of 60 minutes (see Table 3-4). From Table 3-4 it can

be seen that a larger fraction of poliovirus type 1 (83.9% and 91.1%)

became associated with lagoon sludge solids. Goyal and Gerba (1979)

have also shown that seeded echovirus type 1 (Farouk) does not adsorb

well to a sandy loam soil. It is clearly established that virus type

is a factor that affects virus adsorption to surfaces, including sludge

solids. It was the aim of this chapter, however, to show that sludge
type is also a critical factor influencing the degree of association of

viruses with sludge solids.

Recovery of Solids-Associated Viruses

Seeded viruses that became associated with sludge solids were

eluted and further concentrated according to the glycine method. In

Table 3-7, it can be seen that significantly lower mean poliovirus

recovery was found for aerobically digested sludges (15%) than for

mixed liquors or anaerobically digested sludges (72%. and 60%-:.,

respectively). The mean poliovirus recoveries from mixed liquors and

anaerobically digested sludges were not significantly different statis-

tically. The recovery of solids-associated viruses was not dependent

upon the volume of liquid sludge (100, 500, or 1,000 ml) processed. The

mean poliovirus recovery from mixed liquors (72%).-. was similar to the

recovery (80%) of the same virus reported by Hurst et al. (1978). These

researchers worked with activated sludge, which is the same sludge type

as our mixed liquors.

It is clearly established that the effectiveness of the glycine
method in recovering solids-associated viruses is reduced for aerobically








TABLE 3-7. Effect of sludge type on the recovery of poliovirus type 1 from sludge using
of the glycine method


a modification


Sludge Sludge Sludge parameters Volume Virusb Overall Meand virus
type used of sludge added to virus recovery
Solids processed sludge recovery for each
pH content (ml) (total PFU) (%) sludge type
(%) (% SEe)

Mixed liquor UML 6.4 1.6 100 8.4 x 106 84.3*f 72 12A
GML 6.9 0.5 1000 1.3 x 106 60.3

Aerobically UDA 4.8 1.5 500 6.5 x 106 18.9* 15. + 2.7B
digested UDA 4.8 1.5 500 6.5 x 106 11.9*

UDA 6.1 1.3 500 2.0 x 107 8.0*
UDA 6.1 1.3 500 2.0 x 107 5.9*
UDA 6.5 1.3 1000 9.6 x 106 15.5
GDA90 5.8 1.0 1000 1.3 x 107 14.1
PDA 5.8 2.8 1000 4.1 x 106 26.9

Anaerobically PDAN 7.2 1.4 100 6.0 x 106 59.9 60 2.1A
digested PDAN 6.4 1.9 1000 5.5 x 10O6 63.9*

LAGg 7.3 2.9 1000 1.8 x 106 56.7


aThe procedure used was a modification
in the Materials and Methods section.


of the method developed by Hurst et al. (1978) and is described


bThe virus was added to sludge while stirring the suspension using a magnetic stirrer. Magnetic
stirring was continued for 10 min to 60 min and then the total sludge volume was centrifuged at 1400 x g for
10 min at 4C. The sludge supernatant and sludge solids generated were separately subjected to the virus
recovery methodology.




CThe "overall virus recovery (%)" values were determined from the viruses recovered in the final con-
centrates and were based on the amount of viruses (total PFU) added to the sludge as 100%.
dMean values displaying different superscript capital letters are significantly different at the 0.01
level when subjected to analysis of variance using Duncan's test.
eAbbreviation for standard error.

fThe separated supernatants of the sludges with an asterisk were not processed for virus concentration.
These supernatants were simply adjusted to neutral pH and assayed for viruses. The viruses recovered were
included in the "overall virus recovery (%)" values displayed.
gThe lagoon sludge is a mixture of aerobically digested sludge (1/3) and anaerobically digested
sludge (2/3), and consequently, its properties are close to those of anaerobically digested sludges
(see Table 3.2). Therefore, lagoon sludges were placed in the population of anaerobically digested
sludges.









digested sludges. The reason for the reduced recovery of viruses is

that the elution step (i.e., mixing the sludge solids with five volumes

of 0.05 M glycine buffer, pH 10.5 to 11.0, followed by rapid mixing for

30 seconds) is not effective for this sludge type. For example, the

mean elution of solids-associated viruses for this sludge type was only

24%. In contrast, the mean elution of solids-associated viruses from

mixed liquors and anaerobically digested sludges was 76% and 80%,

respectively. The elution of viruses that was measured for mixed liquor

solids (i.e., 76%). closely approaches the value of 84% reported by

Hurst et al. (1978) for the elution of poliovirus type 1 from activated

sludge solids. The reason for the poor elution of solids-associated

viruses from aerobic sludges has not been determined. However, this

sludge type was able to bind a larger fraction of poliovirus than mixed

liquors or anaerobically digested sludges (see Table 3-4). In order to

understand the mechanisms) involved, the chemical and physical nature

of the solids of different sludge types should be studied. All other

virus adsorption-elution steps of the glycine method (i.e., virus

concentration steps) were equally effective in poliovirus recovery for

all sludge types tested.

Several methods have been proposed for the recovery of sludge
solids-associated viruses and these methods are summarized in Table 3-8.

Of the methods evaluated with enteroviruses seeded in sludge, all were

tested for virus recovery efficiency using only one sludge type (see

Table 3-8). The research presented above shows that sludge type in-

fluences the recovery of poliovirus from sludge solids. Although the







TABLE 3-8, Summary of the methods developed for the recovery of viruses from sludges


Concentration Evaluation of the method
Eluenta technique for for virus recovery Reference
u s e d v i r u s i n e l u a t e V s y-u dd e y s s
Virus type(s) used Sludgeb type(s) used


Sodium lauryl sulfate
(0.1%) in 0.05 M
glycine, pH 7.5

Beef extract (3%),
ambient pH


Glycine buffer (0.05 M),
pH 11.0


Tryptose phosphate
broth


Organic
flocculation


Organic
flocculation


Organic
flocculation


None


Poliovirus 1
(Sabin)
Echovirus 7

Poliovirus 1
(CHAT)


Poliovirus 1 (LSc)
Coxsackievirus B3
(Nancy)
Echovirus 7
(Wallace)
Indigenous


Indigenous


Anaerobically
digested (14 days)


Anaerobically
digested
Dewatered, composted

Activated
Returned
Aerobically digested
(thickened, dewatered
Dried sludge (aerobi-
cally digested) from
a sludge disposal
site

Raw (primary)
Anaerobically
digested (40 days)
Anaerobically
digested (100 days)


Abid et al. (1978)


Glass et al. (1978)


Hurst et al.


(1978)


Moore et al. (1978)


Glycine buffer, pH 11.0,
beef extract or dis-
tilled water
Beef extract (10%), pH 7.0,
or Tris buffer, pH 9.0


Adsorption to
bentonite
clay


None


Indigenous


Indigenous


Raw (primary
Anaerobically
digested
Raw (primary and
secondary)


Turk.etal. (1980)


Nielsen and
Lydholm (1980)




Anaerobically
digested (30 days)
Anaerobically
digested (22-25 days)
Dried sludge (anaero-
bically digested)
sampled at the soil
surface 1 day to 4
months after land
application


Beef extract (3%) or
fetal calf serum (10%)
both in saline, pH 7.2
Fetal calf serum (10%)
in saline, pH 7.2


Fetal calf serum (2%)
in Earle's balanced
salt solution, pH 9.5

Beef extract (3%),
pH 9.0


None


None


None


Hydroextraction


Indigenous


Indigenous


Poliovirus 1
(Sabin)


Indigenous


Raw


Raw
Anaerobically
digested (20 days)
Lagoon-dried (anaero-
bically digested)


Digested


Digested (40 days)
Digested (> 60 days)
Dried sludge cake from
a sludge spray site


Sattar and Westwood
(1976)

Sattar and Westwood
(1979)


Subrahmanyan (1977)


Wellings et al.
(1976a)--


aThe chemicals listed were used to elute viruses from sludge solids.

bFor some digested sludges, the type of digestion (i.e., aerobic or anaerobic) and/or the digestion
time were not given in the literature.
cThe effectiveness of this method in recovering seeded viruses was evaluated using activated sludge
only. The method was subsequently used for recovering indigenous viruses from all the sludge types listed.










results pertain only to poliovirus type 1 (LSc) and to the glycine

method used to recover the solids-associated viruses, the indication

is that sludge type is an important factor that should be considered

when assessing the effectiveness of virus recovery methods. Research

conducted using sludge artificially contaminated with virus has been

questioned because it is believed that, unlike indigenous viruses, the

seeded viruses become mostly adsorbed to the surface of sludge

solids (Moore et al. 1977; Nielsen and Lydholm 1980). Indigenous

viruses are believed to be mostly embedded within the sludge solids

rather than merely surface adsorbed (Wellings et al. 1976). In the

case of wastewater-suspended solids, however, Stagg et al. (1978)

demonstrated that most (85%) of the indigenous, solids-associated

coliphages were adsorbed to the surface of sewage solids rather than

embedded. The exact nature of the association between seeded or

indigenous viruses and sludge solids has not yet been conclusively

determined. Whereas research performed with virus-seeded sludge may

not completely simulate natural conditions, valuable information can,

nevertheless, be obtained in less time and at lower cost than when

working with indigenous viruses. Using seeded viruses, the research

reported herein has elucidated the role of sludge type in the recovery

of viruses from sludge solids. The results of this study lead one

to suggest that future methods developed for the recovery of viruses

from sludges be evaluated for the various sludge types likely to be

tested. There is clearly a need for a method that can be shown to be

effective in the recovery of viruses from a variety of sludge types.














CHAPTER IV
POLIOVIRUS TRANSPORT STUDIES INVOLVING
SOIL CORES TREATED WITH VIRUS-SEEDED SLUDGE
UNDER LABORATORY CONDITIONS

Introduction
The application of wastewater sludge to land is receiving
increased attention and will probably be the predominant sludge dis-

posal method of the future (U.S. Environmental Protection Agency 1974,

1978b). There is some concern, however, that this practice may result

in the contamination of groundwater supplies with pathogenic viruses

(see reviews by Berg 1973b; Bitton 1975, 1980a; Bitton et al. 1979b;

Burge and Marsh 1978; Burge and Parr 1980; Cliver 1976; Duboise et

al. 1979; Elliott and Ellis 1977; Foster and Engelbrecht 1973;

Gerba et al. 1975; Moore et al. 1978; Sagik 1975). Unfortunately,

the transport pattern (i.e., movement or retention) of sludge-

associated viruses in soils has not been adequately evaluated. The few

studies that have been conducted indicate that enteroviruses seeded in
anaerobically digested sludge are effectively retained by the soil

matrix (Damgaard-Larsen et al. 1977; Moore et al. 1978; Sagik 1975).

Since both seeded and indigenous viruses have been found to be associated

with the sludge solids (Abid et al. 1978; Glass et al. 1978; Hurst et al.

1978; Lund 1971; Ward and Ashley 1976; Wellings et al. 1976a; also see

Table 3,4), it follows that viruses are probably immobilized along with

the sludge solids in the top portion of the soil profile (i.e., during

surface spreading of sludge) or at the injection site within the soil









matrix (i.e., during subsurface injection of sludge) (Cliver 1976).

Moreover, it appears that viruses are not readily dissociated from sludge

solids in the soil environment (Burge and Parr 1980; Sagik 1975).

In this chapter, results of poliovirus (type 1, strain LSc)
transport studies involving soil cores treated with virus-seeded sludge

are presented. These studies were conducted under controlled laboratory

conditions. The effect of sludge type (used anaerobically digested

sludge, conditioned-dewatered sludge, chemical sludge and lime-stabilized,

chemical sludge), soil type (used a Red Bay sandy loam and a Eustis fine

sand), soil core type (used laboratory-packed soil columns and undis-

turbed soil cores) and application regime (virus-seeded sludge was

applied continuously or in a spiked fashion) on the transport of sludge-

associated poliovirus in soil cores was evaluated. The capacity of rain

water to elute poliovirus from the sludge-soil matrix was also investi-

gated. All soil cores were leached under saturated flow conditions.

The information gained from this study should shed further light on the

role of mineral soils in retaining sludge-associated viruses.


Materials and Methods
Virus and Viral Assays

Poliovirus type 1 (strain LSc) was used in the research reported
in this chapter. Some general properties of polioviruses are shown in

Table 3-1. Stocks of the virus were prepared as described in Chapter

III (see page 53). The virus was kept at -70C until used. Poliovirus

was assayed by the plaque technique as described in Chapter III (see

pages 53-56). Each viral count shown represents the average of









triplicate counts. The numbers of viruses were expressed as plaque-

forming units (PFU).

Primary Wastewater Effluent

Primary wastewater effluent was obtained from the University

of Florida campus wastewater treatment plant, Gainesville, Florida. The

detention time of raw wastewater in the primary settlers was approxi-

mately 2 hours. No chlorine residual was found in the primary effluent

sample used (i.e., by the orthotolidine test). The sample of primary

effluent used was collected, and its pH and conductivity were measured

as described below for digested sludges.

Sludges

Several sludge types were used in the research reported in this

chapter and they are described below.

Digested sludges. Anaerobically digested sludges sampled at

the Main Street wastewater treatment plants of Gainesville and Pensacola,

Florida (GDAN and PDAN, respectively--see Table 3-2), were used. The

sludges were collected and sludge parameters (i.e., pH and solids con-

tent) were measured as described in Chapter III (see page 56). The

sludge conductivity was measured using a Beckman conductivity bridge

model RC 16B2 (Beckman Instruments, Fullerton, California). Due to

uncontrollable contamination of cell cultures during viral assays, these

sludge samples had to be sterilized by autoclaving at 121C with applied

pressure of 15 psi for 15 minutes prior to use. As explained earlier

(see page 59), autoclaving did not significantly affect the sludge-virus










interactions and the autoclaved sludge can simply be considered to be

heat-conditioned. The sludges were used undiluted or diluted (1:50,

vol./vol.) with either distilled water or 0.01 N calcium chloride.

The pH and conductivity of each diluted sludge sample was also

measured as described above for undiluted sludge.

Sludge liquor. Anaerobically digested sludge liquor was pro-

duced by centrifuging (all centrifugation was performed using a Sorvall

RC5-B centrifuge, Ivan Sorvall Inc., Norwalk, Connecticut) GDAN sludge

(see above) at 14,000 x g for 10 minutes at 4C. This procedure was

performed again on the decanted supernatant and this yielded the clear

sludge liquor. Chemical parameters for this sludge liquor were deter-

mined by the Analytical Research Laboratory, Soil Science Department,

University of Florida, Gainesville, and are presented in Table 4-1.

The sludge liquor (containing 0.01 N calcium chloride) was also used

to dilute (1:50, vol./vol.) the GDAN sludge. Lagoon sludge (LAG--see

Table 3-2; a mixture of 1/3 aerobically digested sludge and 2/3

anaerobically digested sludge) liquor was also used and it was produced

by the centrifugation of lagoon sludge as outlined above for GDAN

sludge. The lagoon sludge liquor was passed through a series of

0.45- and 0.25-pm Filterite filters (Filterite Corp., Timonium, Maryland)

in a 47-mm holder and then adjusted to pH 8.0 using 0.01 N NaOH prior

to use. The pH and conductivity of each sludge liquor sample was

measured as described above for undiluted digested sludges.

Conditioned-dewatered sludge. Poliovirus-seeded, GDAN sludge

(see above) was conditioned with 1200 mg/t of the cationic polymer,








TABLE 4-1, Chemical parameters
liquor


Parametera Sludgeb liquor
value
(ppm)

Soluble salts 703
Na 63
K 29
Ca 20
Mg 15
Al 0
Fe 1.05


for the anaerobically digested sludge


Parametera Sludgeb liquor
value
(ppm)

As 0
Cd 0
Cr 0
Cu 0.05
Ni 0
Pb 0
Zn 0.04


aChemical parameters were determined by the Analytical
Laboratory, Soil Science Department, University of Florida,


Research
Gainesville.


bAnaerobically digested sludge (GDAN--see Table 3-2 ; solids con-
tent, conductivity and pH equal to 2.0%, 3250 umho/cm at 25C and 8.3,
respectively) was centrifuged at 14,000 x g for 10 min at 4C. This
procedure was performed again on the decanted supernatant and this
yielded a clear sludge liquor.









Hercufloc #871 (Hercules Co., Atlanta, Georgia). The polymer was added

to 1000 ml of the virus-seeded sludge while mixing rapidly on a mag-

netic stirrer. Mixing was continued slowly for an additional 5 minutes.
The entire sludge sample was then centrifuged at 320 x g (i.e., 1400

rpm) for 10 minutes at 25C. The supernatant was decanted, assayed for

viruses, and discarded. The dewatered sludge volume and sludge solids

content (i.e., as percent, by method described above for digested

sludges) was measured. The conditioned-dewatered sludge produced
(i.e., in duplicate) was then assayed for viruses as described below.

The procedure employed above in the conditioning and dewatering of the

sludge was identical to that used at the Main Street wastewater treat-
ment plant, Gainesville, Florida (Dr. DuBose, Main Street plant, per-

sonal communication; see Figure 6-1).
Chemical sludges. The chemical sludges were precipitated from

1000 ml of poliovirus-seeded, raw sewage using the general procedure
of Sattar et al. (1976) and the coagulants, alum (i.e., aluminum sul-

fate),.ferric chloride or lime (i.e., calcium hydroxide). The raw sewage

used was obtained from the University of Florida campus wastewater treat-

ment plant or from the Main Street wastewater treatment plant, both being
located in Gainesville, Florida. The raw sewage samples were collected
and sewage parameters (i.e., pH and conductivity) were measured as
described above for sludge samples. The raw sewage samples were sterilized

by autoclaving at 121C with applied pressure of 15 psi for 15 minutes

prior to use. Viral assays were made before and after the addition

of coagulant. The final concentration in sewage of alum was 300 mg/A









[as Al2(S04)3* 18 H20] and of ferric chloride was 50 mg/k (as FeC13).
Similar concentrations of these coagulants were used by Wolf et al.

(1974) in the precipitation of an activated sludge effluent as a
tertiary treatment process. Following the addition of alum or ferric

chloride, the pH of the solution was adjusted using 0.2 N HCl to 6.0 or

5.0, respectively, in order to achieve maximum flocculation (Fair et al.

1968). The coagulant, lime, was added until a pH of 11.1 to 11.3 [i.e.,
final concentration in sewage of 150 to 250 mg/Z of Ca(OH)2] was

achieved (according to Sattar et al. 1976). Following the addition
of the coagulants, the sewage samples were mixed on a magnetic stirrer
rapidly for 10 minutes and slowly for 5 minutes. The flocculated

sewage samples were then transferred to Imhoff cones and 60 minutes
was allowed for the formation and settling of the chemical sludges
(see Figure 4-1). The supernatants in the Imhoff cones were assayed

for viruses and discarded. The sludge volume and sludge solids content
(i.e., as percent, by method described above for digested sludges) was

measured for each chemical sludge. The chemical sludges produced (i.e.,
in duplicate for the lime sludge, and in triplicate for alum and ferric

chloride sludges) were then assayed for viruses as described below.

Lime-stabilized, chemical sludges. A sample of alum sludge
and a sample of ferric chloride sludge, produced and assayed for viruses
as described above, were lime-stabilized according to the procedure of
Farrell et al. (1974). The sludges were treated with an aqueous slurry

of lime [5% (wt./vol.) stock of Ca(OH)2] until a pH of 11.5 was achieved
and maintained for 5 minutes. The final concentration of lime [as











FIGURE 4-1.


Chemical sludges (i.e., lime and alum) were precipitated
from poliovirus-seeded, raw sewage and are shown after
settling for 60 minutes in Imhoff cones




90
!s









Ca(OH)2] added to the alum and ferric chloride sludge was 1389 and 625

mg/k, respectively. A contact time of 30 minutes was allowed while

mixing the suspension on a magnetic stirrer. After 30 minutes of mixing,

the pHs of the sludges were measured and had dropped to 11.1 or 11.3.

The lime-stabilized, chemical sludges were then assayed for viruses as

described below.

Association of Seeded Poliovirus with Sludge Solids

Poliovirus stock in phosphate-buffered saline (PBS) containing

2% fetal calf serum (FCS) (see Appendix for more details on the compo-

sition of this solution) was added directly to 0.01 N calcium chloride,

primary wastewater effluent, anaerobically digested sludge (diluted or

undiluted) and sludge liquor at the rate of 1 ml of virus stock per

1000 ml of solution and while stirring the suspension using a magnetic

stirrer (see Chapter III, page 59). After a 1-minute mixing period,

an aliquot of the samples containing low solids contents (i.e., 0.01 N

calcium chloride, primary wastewater effluent, diluted anaerobically

digested sludge, and sludge liquor) was diluted in PBS containing 2%

FCS and assayed directly for seeded viruses by the plaque technique.

This viral assay was performed in order to determine the amount of

virus present in these samples initially and was repeated at the end

of experimental trials in order to assess any viral inactivation which

might have occurred. No attempt was made to determine the degree of

association between seeded poliovirus and the small quantity of solids

present in these samples.

The association of poliovirus with sludge solids was deter-
mined for undiluted anaerobically digested sludge (seeded with










poliovirus and suspension magnetically mixed for 10 to 60 minutes),

conditioned-dewatered sludge (poliovirus transferred to this sludge

during dewatering process), chemical sludges (poliovirus transferred to

chemical sludges during precipitation of virus-seeded, raw sewage) and

lime-stabilized, chemical sludges. The procedures used are outlined

in detail in Chapter III (see page 62). Briefly, an aliquot of the

unfractionated sludge (i.e., sludge sample without solids separated)

was diluted in PBS containing 2% FCS and assayed directly for viruses by

the plaque technique. This method (i.e., sludge dilution and subsequent

direct assay on cell cultures) has been previously shown to be highly

efficient in the recovery of poliovirus from unfractionated sludge

(see Chapter III, page 62 and Table 3-3). The unfractionated sludge

assay was performed in order to determine the total amount of virus

present in the sludge sample. An aliquot of the sludge was subsequently

centrifuged at 1400 x g for 10 minutes at 4C. The sludge supernatant

produced was assayed for viruses. The "viable unadsorbed virus" and

"sludge solids-associated virus" fractions were calculated as shown

in Chapter III (see page 64).


Rain Water

Rain water was collected next to the Environmental Engineering

Sciences building at the University of Florida, Gainesville. Chemical

parameters for the rain water used were determined by Hendry (1977)

and are presented in Table 4-2. The rain water was sterilized by auto-

claving at 121C with applied pressure of 15 psi for 15 minutes prior

to use.




Full Text
TABLE 5-9. Analysis for the presence of poliovirus type 1 (Sabin) and echovirus type 1 (Farouk) in soil
leachates collected after natural rainfall from soil cores which had been treated with 2.5 cm
of seeded liquid sludge (11 October 1979-20 January 1979)
Dates
Cumulative
Soil
Cumulative
Cumulative
Cumulative
Cumulative
Range of
Range of
of
rainfall3
coreb
leachate
number of
virus
percent of
conductivi ty
pH values
leachates
collected
(cm)
number0
volume
(ml)
pore .
volumes0
breakthrough
(total PFU)
total virus
applied
values of
leachates
collected
(pmho/cm)
of
leachates
collected
01 Dec. 78-
20 Jan. 79
24.95
SCI
400
(20.4)e
1.7
0
0
140-625
5.8-7.0
01 Dec. 78-
20 Jan. 79
24.95
SC2
385
(19.6)
1.6
0
0
135-710
6.3-7.0
ro
01 Dec. 78-
20 Jan. 79
24.95
Cl
750
(4.0)
0.3
0
0
375-800
6.3-6.8
LO
01 Dec. 78-
20 Jan. 79
24.95
C2
980
(5.2)
0.5
0
0
190-975
6.1-6.3
01 Dec. 78-
20 Jan. 79
24.95
C3
920
(4.9)
0.4
0
0
280-1200
5.9-6.9
28 Dec. 78-
20 Jan. 79
24.95
C4
410
(2.2)
0.2
0
0
560-875
6.0-6.9


231
FIGURE 6-3.
Scheme for sludge disposal at the West Florida Agricul
tural Experiment Station, Jay, Florida


TABLE 4-8.
Retention of poliovirus type 1 by a packed column of Red Bay sandy loam subsoil when sus
pended in anaerobically digested sludge diluted (1:50) with 0.01 N CaCl2 and after subsequent
application of rain water
No. of pore
volumes9
eluted
Poliovirus
eluted
(PFU/ml)
% of Influent^
poliovirus
concentration
% of Total PFU
having been
applied at each
pore volume
(cumulative)
Conductivity
of pore volume
collected
(pmho/cm at 25C)
pH of pore
volume
collected
SIudgec
diluted (1:50) with
0.01 N CaCl2 and seeded with poliovirus
1/3
0
0
0
1260
4.8
2/3
0
0
0
1280
5.2
1
0
0
0
1260
4.9
1 1/3
0
0
0
1270
4.9
1 2/3
0
0
0
1300
4.9
2
0
0
0
1320
5.0
2 1/3
0
0
0
1330
5.0
2 2/3
0
0
0
1340
4.9
3
0
0
0
1350
4.9
3 1/3
0
0
0
1350
4.9
3 2/3
0
0
0
1360
4.7
4
0
0
0
1360
4.9
4 1/3
0
0
0
1360
4.9
4 2/3
0
0
0
1360
4.9
5
0
0
0
1360
4.9
5 1/3
0
0
0
1360
4.9
5 2/3
0
0
0
1360
4.9
6
0
0
0
1360
4.9
6 1/3
0
0
0
1370
5.0
6 2/3
0
0
0
1370
5.0
7
7.5
0.1
0.005
1370
5.0
7 1/3
0
0
0.005
1370
5.0
7 2/3
0
0
0.005
1370
5.0
8
0
0
0.004
1370
5.0
8 1/3
15
0.2
0.01
1370
5.0


255
TABLE 7-2.
Effect of hydrostatic
of groundwater
pressure on
the temperature and pH
Pressure3
Mean
Mean pH
(psi)
temperature
(C)
14.7
24
8.1
(atmospherf
c)
3,000
25
8.3
aPressurization time was 24 hours.


TABLE 5-6. Analysis for the presence of poliovirus type 1 (Sabin) in soil leachates collected after natural
rainfall from large soil cores of Eustis fine sand which had been treated with 2.5 cm of seeded
liquid sludge (2 June 1978-24 August 1978)
Dates
of
leachates
collected
(1978)
Cumulative
rainfal1
(cm)
Soil
core3
number
Cumulative
leachate
volume
(ml)
Cumulative
number of
pore .
volumes0
Cumulative
virus
breakthrough
(total PFU)
Cumulative
percent of
total virus
applied
Range of
conductivity
values of
leachates
collected
(pmho/cm at 25C)
Range of
pH values
of
leachates
collected
05 June-
24 Aug.
51.05
Cl
1544
(8.2)c
0.7
0
0
114-1200
6.3-7.8
05 June-
24 Aug.
51.05
C2
1135
(6.0)
0.5
0
0
106-1360
5.7-7.2
a0ne inch (or 2.5 cm) of lagooned sludge seeded with a total of 6.1 x 10^ PFU of poliovirus was
applied on top of soil cores. The soil cores were exposed to natural conditions.
^One pore volume fore the large soil cores equals 2178 ml.
Q
Values in parentheses represent the number of centimeters of cumulative leachate volume.


284
Wright, R. L. 1975. An overview-wastewater treatment disposal sys
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Wulff, H., and T. D. Y. Chin. 1972. Echoviruses. In Strains of
Human Viruses, pp. 41-65. Edited by M. Majer and S. A. Plotkin.
S. Karger, New York, N.Y.
Yates, T. 1977. Conditioning and land application of aerobically
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Yeager, J. G., and R. T. O'Brien. 1979a. Enterovirus inactivation in
soil. Appl. Environ. Microbiol. 38: 694-701.
Yeager, J. G., and R. T. O'Brien. 1979b. Structural changes associated
with poliovirus inactivation in soil. Appl. Environ. Microbiol.
38: 702-709.
York, D. W., and W. A. Drewry. 1974. Virus removal by chemical coagu
lation. J. Am. Water Works Assoc. 66: 711-716.
Young, R. H. F., and N. C. Burbank, Jr. 1973. Virus removal in Hawaiian
soils. J. Am. Water Works Assoc. 65: 598-604.
Zenz, D. R., J. R. Peterson, D. L. Brooman, and C. Lue-Hing. 1976.
Environmental impacts of land application of sludge. J. Water
Pollut. Control Fed. 48: 2332-2342.
ZoBell, C. E., and A. B. Cobet. 1962. Growth, reproduction, and death
rates of Escherichia coli at increased hydrostatic pressures.
J. Bacteriol. 84: 1228-1236.
ZoBell, C. E., and F. H. Johnson. 1949. The influence of hydrostatic
pressure on the growth and viability of terrestrial and marine
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Zoltek, J., Jr., and E. L. Melear. 1978. Wastewater treatment-
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268
Chang, S. L., R. E. Stevenson, A. R. Bryant, R. L. Woodward, and
P. W. Kabler. 1958. Removal of coxsackie and bacterial viruses
in water by flocculation. 1. Removal of coxsackie and bac
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testing conditions. Am. J. Public Health 48: 51-61.
Chanock, R. M. 1976. Nonbacterial gastroenteritis. Bull. Pan Am.
Health Organ. 10: 202-204.
Chaudhuri, M., and R. S. Engelbrecht. 1972. Removal of viruses from
water by chemical coagulation and flocculation. J. Am. Water
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Clark, C. S., H. S. Bjornson, J. W. Holland, T. L. Huge, V. A. Majeti,
and P. S. Gartside. 1980. Occupational hazards associated
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Edds, and J. M. Davidson. Ann Arbor Science Publishers, Inc.,
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Clarke, N. A., R. E. Stevenson, S. L. Chang, and P. W. Kabler. 1961.
Removal of enteric viruses from sewage by activated sludge
treatment. Am. J. Public Health 51: 1118-1129.
Cliver, D. 0. 1975. Virus association with wastewater solids.
Environmental Letters 10: 215-223.
Cliver, D. 0. 1976. Surface application of municipal sludges. In
Proceedings of the Symposium on Virus Aspects of Applying
Municipal Waste to Land, pp. 77-81. Edited by L. B. Baldwin,
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Florida.
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Recycling Treated Municipal Wastewater and Sludge through
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279
Sadovski, A. Y., B. Fattal, D. Goldberg, E. Katzenelson, and H. I.
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N.Y. Acad. Sci. 61: 740-753.




216
0.13 cm of rain fell on 11 October 1978 to 1 November 1978 (see Table
5-8). This was the period during which virus survival was monitored.
Both poliovirus and echovirus were not detectable in soil after eight
days of exposure to natural conditions in the dry fall season (see
Table 5-8). The two enteroviruses were completely inactivated some
time between the eighth and the 21st day (see Table 5-8). Soil leach
ates were also monitored and a summary of the data is displayed in
Table 5-9. Neither poliovirus nor echovirus was detected in the
leachates from all the soil cores (see Table 5-9).
It appears from these studies that, under conditions prevailing
in North-Central Florida, enteroviruses are rapidly inactivated during
sludge application to soils. Their inactivation in the soil appears
to be affected more by desiccation than by soil temperature. Under
ideal conditions (warm and dry), a rapid decline of virus was observed
in the sludge drying on top of the soil and in the top 2.5 cm of soil.
Other investigators have shown that virus survival in sludge-treated
soils is prolonged by low temperatures (Damgaard-Larsen et al_. 1977;
Nielsen and Lydholm 1980). Soil leachates collected after natural
rainfall were negative for both poliovirus and echovirus. Virus
studies in sludge-amended soils have dealt mainly with the transport
and survival patterns of enteroviruses and more work is needed on the
behavior of other enteric viruses, namely rotaviruses, in soils
receiving wastewater sludges.


14
In these studies, the seeded viruses were transferred to the settled
sludges (Clarke et al_. 1961; Malina etal_. 1975). Clarke etal_. (1961)
recovered only a small fraction of the viruses (i.e., poliovirus type
l--Mahoney or coxsackievirus A9) theoretically associated with the
settled sludge. These researchers concluded that the viruses were
inactivated when adsorbed to sludge particles. However, their failure
to adequately recover the adsorbed viruses can partly be attributed
to the poor recovery method used (i.e., used buffer solutions and
versene as eluents). In a laboratory-scale activated sludge unit,
Malina et al_. (1975) effectively recovered poliovirus type 1 (Mahoney)
from the settled sludge by using a better eluent, distilled deionized
water. Moreover, poliovirus associated with the sludge particles was
observed to be inactivated over time. These investigators measured the
viral inactivation rate in the settled sludge and found that the rate
conformed to the following equation:
where C. = virus associated with sludge initially (PFU/mg of
dry sludge solids)
C^ = virus associated with sludge at time t (PFU/mg of
dry sludge solids)
t = time (min)
k^ = rate constant (min~^)
The rate constant (i.e., k^) varied with mixed liquor suspended solids
_ o
(MLSS) concentration. For example, rate constants of 3.17 x 10 and


TABLE 4-17. Association of poliovirus type 1 with anaerobically digested sludge conditioned with a
cationic polymer and subsequently dewatered by centrifugation
Experiment Virus3 in Virus in sludge supernatant Virus in unfractionatedc, dewatered sludge
sludgeb
(total PFU)
Total PFU
Recovery^
(%)
Total PFU
Recovery^
(%)
Sludge
volume
(ml)
Sludge solidse
content
(%)
1
2.8 x 107
2.4 x 106
8.6
2.5 x 107
89.3
320
5.1
2
3.6 x 107
2.4 x 106
6.7
2.6 x 107
72.2
310
4.7
aVirus in the sludge before the addition of polymer. The cationic polymer used was Hercofloc #871
(Hercules Co., Atlanta, Georgia) and it was used at a concentration of 1200 mg/1 in the sludge.
^The anaerobically digested sludge (GDAN--see Table 3-2) used had a solids content of 2.0%, a con
ductivity of 3950 ymho/cm at 25C and a pH of 6.0. The sludge was autoclaved prior to use. The sludge was
conditioned-dewatered as follows. The polymer was added to 1000 ml of virus-seeded sludge while mixing
rapidly on a magnetic stirrer. Mixing was continued slowly for an additional 5 min. The entire sample was
then centrifuged at 320 x g for 10 min at 25C. The supernatant was decanted, assayed for viruses and dis
carded. The dewatered sludge produced was then assayed for viruses. The dewatered sludges from experiments
no. 1 and 2 were applied to Red Bay sandy loam columns 1 and 2, respectively, as shown in Table
cThe sludge solids were not separated prior to assaying.
^Percent recoveries were calculated based on the amount of viruses (total PFU) present in the sludge
before the addition of polymer at 100%.
p
Sludge solids content was expressed as a percentage on a weight to volume basis.


FIGURE 6-2. Diagram of the Kanapaha sludge disposal site, Gainesville, Florida
Conditioned-dewatered sludge from the Main Street wastewater treatment plant (Gaines
ville) was applied to the 10 acres (4.05 ha) of land as shown in Figure 6-1. From
August 1977 to February 1978 (i.e., last month of sampling in this study), a total of
3.7 inches (9.4 cm) of sludge were applied to the site. The soil found at this site
belongs to the Lochloosa series. A 200-g composite topsoil sample [40 grams per
sampling point (sampling points designated by triangles)] was obtained from each 5
acre plot and analyzed for the presence of indigenous enteroviruses. Groundwater from
the center well [60 ft (ca. 18 m)] in the disposal site was also monitored for
indigenous enteroviruses.


ro
-P*


8
et al_. (1977) studied the flocculation of poliovirus type 1 (LSc) by
ferric chloride. These investigators added the virus to a membrane
concentrate (i.e., 0.5 M glycine buffer) of turbid estuary water and
proceeded to concentrate the virus by flocculation. At a concentration
of 0.001 M FeCl^, >99% of the seeded viruses were removed from the
supernatant at pH values from 3.5 to 7.5. However, maximum precipita
tion was observed at pH 3.5. Bacteriophages have also been removed
efficiently from water (>99% removals) by flocculation with ferric
chloride (Change et al_. 1958; York and Drewry 1974). In fact, bacterio
phage removals consistently exceeded those of enteroviruses (Chang et al.
1958). Chang et aK (1958) found ferric chloride to be more efficient,
on a molar basis, than alum in the removal of coxsackievirus A2 from
water by flocculation. Furthermore, these researchers noted that the
floes formed with ferric chloride were more compact and settled more
rapidly than those formed with alum. Chang et aJL (1958) were unsuc
cessful in recovering the coxsackieviruses associated with the iron
floes by eluting with 0.1 M NaHCO^, final pH 8.5. It is likely that
this solution was inadequate as an eluent for the recovery of the
adsorbed viruses, although viral inactivation during the flocculation
process cannot be ruled out. In contrast, 74% of the polioviruses asso
ciated with the iron floes were recovered by Sobsey et al_. (1977) follow
ing elution with fetal calf serum (FCS) at pH 8.0.
Calcium hydroxide (i.e., lime) is another chemical frequently
used in flocculation tests for the removal of viruses from water.
Sproul (1972) has reviewed the literature on virus removal by water
softening precipitation processes involving lime as the coagulant.


81
results pertain only to poliovirus type 1 (LSc) and to the glycine
method used to recover the solids-associated viruses, the indication
is that sludge type is an important factor that should be considered
when assessing the effectiveness of virus recovery methods. Research
conducted using sludge artificially contaminated with virus has been
questioned because it is believed that, unlike indigenous viruses, the
seeded viruses become mostly adsorbed to the surface of sludge
solids (Moore etal_. 1977; Nielsen and Lydholm 1980). Indigenous
viruses are believed to be mostly embedded within the sludge solids
rather than merely surface adsorbed (Wellings et al_. 1976). In the
case of wastewater-suspended solids, however, Stagg et al_. (1978)
demonstrated that most (85%) of the indigenous, solids-associated
coliphages were adsorbed to the surface of sewage solids rather than
embedded. The exact nature of the association between seeded or
indigenous viruses and sludge solids has not yet been conclusively
determined. Whereas research performed with virus-seeded sludge may
not completely simulate natural conditions, valuable information can,
nevertheless, be obtained in less time and at lower cost than when
working with indigenous viruses. Using seeded viruses, the research
reported herein has elucidated the role of sludge type in the recovery
of viruses from sludge solids. The results of this study lead one
to suggest that future methods developed for the recovery of viruses
from sludges be evaluated for the various sludge types likely to be
tested. There is clearly a need for a method that can be shown to be
effective in the recovery of viruses from a variety of sludge types.


150
The sludge liquor employed in these experiments represents
an artificial system probably never encountered in the environment.
However, under field conditions, rain water may leach the sludge
liquor from the applied sludge, which then may affect virus transport
through the soil. The implications of this possible phenomenon in
field situations deserve further investigation.
Poliovirus Suspended in Undiluted
Anaerobically Digested Sludge
The movement of sludge-associated viruses through soil was
next studied under more realistic conditions. Poliovirus was added
to undiluted anaerobically digested sludge while stirring the mixture
on a magnetic stirrer. One inch (2.5 cm) of seeded sludge was then
applied on top of undisturbed cores of Eustis fine sand and Red Bay
sandy loam. The sludge was allowed to soak in (in one experiment the
sludge was allowed to air dry for 24 hours) and then worked under.
The soil cores were then eluted with either 0.01 N CaCl2 or rain
water.
Red Bay sandy loam. Undisturbed soil cores of Red Bay sandy
loam were treated with poliovirus-seeded sludge as described above.
The applied sludge was allowed to air dry for 24 hours, under field
conditions, before being mixed with the top 2.5 cm of soil to simulate
field practices. The soil columns were then eluted with three to four
pore volumes of rain water. Table 4-15 shows that there was a virus
breakthrough in the second or the first pore volume collected. This
virus breakthrough represented 0.1 and 0.2% of the total PFU applied to


148
with the adsorption of poliovirus to sludge and soil particles. Fur
thermore, Figure 4-7 shows that the passage of ten pore volumes
(2250 ml) of sludge diluted with sludge liquor brought to a final CaCl2
concentration of 0.01 N resulted in the gradual increase in the soil
solution pH from 5.8 to the pH of the leaching solution, 8.3. Thus,
the soil was unable to buffer the pH and this increase in pH into the
basic range further prevented viral adsorption to the soil.
Eustis fine sand. Experiments involving poliovirus suspended
in sludge liquor were also performed using Eustis fine sand columns.
The sludge liquor employed in these studies was produced by centrifug
ing lagoon sludge (LAGsee Table 3-2; 2/3 anaerobically digested and
1/3 aerobically digested sludge), and subsequently passing the result
ing supernatant through a series of 0.45 and 0.25 ym Filterite filters
and then adjusting the sludge liquor to pH 8.0. The filtration pro
cedure did not remove all bacterial cells from the sludge liquor.
This was confirmed by microscopic examination of the sludge liquor at
a magnification of lOOOx. Figure 4-8 shows that poliovirus break
through occurred at the first pore volume, and by the tenth pore
volume, 100% of the applied virus had appeared in the leachate from
a 10-cm column of this soil. Further research by Overman et al_. (un
published data) has confirmed that this sludge liquor strongly inter
feres with the adsorption of poliovirus type 1 and echovirus type 4
to the Eustis fine sand. Moreover, these investigators demonstrated
that this sludge liquor was not able to elute substantial numbers of
previously adsorbed viruses (i.e., poliovirus and echovirus).


235
TABLE 6-2. Some characteristics of the Troup and Orangeburg soil
series
found at
the Jay
site
Soil3
Soil
horizon
Depth
(cm) -
l
Mechanical composition (%)
PH
- (in 1:1
water)
Sand
(2-
3.05 nm)
Silt
(0.05-
0.002 mm)
Clay
(<0.002 mm)
Troup
Ap
0-15
79.0
13.7
7.3
6.1
loamy
sand
A21
15-41
79.0
13.2
7.8
5.8
A22
41-71
80.0
12.2
7.8
5.4
A23
71-91
80.5
12.5
7.0
5.4
A24
91-117
80.3
11.9
7.8
5.4
Bit
117-132
76.0
9.6
14.4
5.4
B21t
132-147
62.0
10.0
28.0
5.2
Orangeburg
Ap
0-20
77.5
13.7
8.8
4.9
sandy
loam
Bit
20-36
69.6
13.6
16.8
6.1
B211
36-64
63.5
12.6
23.9
6.3
B22t
64-119
56.9
8.6
34.5
6.3
B23t
119-185
61.8
8.1
30.1
5.6
aData were adapted from Calhoun et al_. (1974).


254
Results and Discussion
Initially, it was important to determine the effect of elevated
pressure on the temperature and pH of groundwater. As shown in Table 7-2,
the pH and temperature of groundwater was not markedly changed following
pressurization at 3000 psi for 24 hours.
The inactivation of poliovirus in seawater subjected to 1000 psi
of hydrostatic pressure was found to increase as the pressurization time
was increased from 2 to 24 hours (see Table 7-3), After 24 hours of
exposure to 1000 psi of hydrostatic pressure, only 15.6% of seeded polio
virus was recovered. Clearly, poliovirus in seawater was inactivated at
an accelerated rate under 1000 psi of pressure relative to the control at
atmospheric pressure. No such inactivation was observed for poliovirus
in groundwater even after exposure to as high as 4000 psi of hydrostatic
pressure (see Table 7-4). Thus, hydrostatic pressures in groundwater are
not likely to increase viral inactivation.
High hydrostatic pressures have been previously shown to inacti
vate bacterial enzyme systems (Morita 1967; Morita and ZoBell 1956), to
retard the growth of mesophilic terrestrial bacteria (Horvath and Elkan
1978; ZoBell and Cobet 1962; ZoBell and Johnson 1949), and to accelerate
the death rate of mesophilic bacteria (Baross et al_. 1975; Morita 1967;
ZoBell and Cobet 1962). Although tobacco mosaic virus (Lauffer and Dow
1941), and and T4 bacteriophages (Hedn 1964) have been demonstrated
to be inactivated at pressures in excess of 1900 atm, little work had
previously been done on the effect of hydrostatic pressure on animal
viruses. The research presented above is only preliminary and more work
is needed on the effect of hydrostatic pressure on viruses.


161
for example, digested sludge is conditioned with a cationic polymer
(Hercofloc #871, Hercules Co., Atlanta, Ga.) and subsequently dewatered
by centrifugation. The fate of poliovirus seeded in anaerobically
digested sludge was determined following conditioning and dewatering
of sludge as described above. As shown in Table 4-17, in two experi
ments, 72.2% and 89.3% of poliovirus initially added to the sludge was
found in the conditioned-dewatered sludge. Moreover, in both experi
ments, 99.9% of the virus recovered from the conditioned-dewatered
sludge was found associated with the sludge solids (see Table 4-18).
The application of the conditioned-dewatered sludge containing polio
virus to columns of Red Bay sandy loam and the subsequent elution
with rainwater did not result in any virus breakthrough (see Table
4-19). These results show that viruses present in conditioned-
dewatered sludge are effectively retained by soils.
Chemical Sludges
In addition to biological sludges, chemical sludges may also
be produced during wastewater treatment. These sludges may be produced
during primary treatment when coagulation (using alum, ferric chloride,
or lime) is combined with sedimentation to upgrade the removal
efficiency of the treatment process (i.e., intermediate treatment) or
during advanced wastewater treatment (tertiary treatment) (Malina et al.
1976). Previous research has demonstrated that viruses are concen
trated in alum and ferric chloride sludges (Lund and R0nne 1973; Wolff
et al_. 1974). However, in lime sludges, viruses have been found to be
effectively inactivated (Lund and Rtfnne 1973; Sattar et al_. 1976).


Soil bulk density (dry grams/cm )
111
FIGURE 4-4. Soil moisture content-bulk density curve for the Red
Bay sandy loam subsoil
Air-dried samples of Red Bay sandy loam subsoil
(i.e., consisting of the A2 and Bit horizonssee
Table 4-3) were adjusted to moisture contents
ranging from 7.3% to 14.4% (wt./wt.). The soil
samples were then compacted using the procedure
of Wilson (1950).


149
The mechanisms(s) of sludge liquor interference with virus
adsorption to soils is (are) not well understood. It is known that
water-soluble "humic substances" interfere with the sorptive capacity
of soil and sediments toward viruses. The decrease in virus retention
is due to humic fractions with a molecular weight of less than 50,000
(Bitton et al_. 1977; Scheuerman et al. 1979). Anaerobically digested
sludge contains fulvic acid fractions (Baham et al_. 1978; Holtzclaw
et al_. 1976, 1978; Sposito and Holtzclaw 1977; Sposito et al_. 1976,
+2
1978) that are known to complex Ca ions, an important and ubiquitous
metal cation in soil solution (Sposito et aj_. 1978). This complexa-
tion phenomenon may aid in the inhibition of virus adsorption to soils.
Other mechanisms are probably involved in this inhibition process.
When poliovirus was suspended in sludge liquor that had been filtered
through a 0.22 ym Millipore filter, the percent breakthrough of this
virus in Eustis fine sand columns was significantly reduced (i.e.,
4%) as compared to the breakthrough (34%) following the application of
Filterite-treated sludge liquor (Overman et al_., unpublished data).
Microscopic examination showed that Filterite-treated sludge liquor
contained substantial numbers of bacterial cells whereas no bacteria
were found in Mi Hi pore-treated sludge liquor. It is then possible
that these bacterial cells may compete with viruses for adsorption
sites on the surface of the soil particles. This is a mere speculation
that needs to be demonstrated under more controlled conditions. Com
petitive adsorption between viruses and bacteria has not been reported
in the literature.


No. of pore volumes eluted
Conductivity Poliovirus eluted (expressed as cumulative % of total PFU having
(x 102 pmho/cm at 25C) been applied at each pore volume)
pH Poliovirus eluted (x 1(P PFU/ml)


196
on a wet-weight basis by drying in an oven at 105C for 24 hours
a measured weight of wet soil from the top inch of the soil cores and
was expressed as a percentage as follows:
Soil moisture (!) = et. soil weight (a) -dry soil weight (g)
wet soil weight (gj
x 100
(5-1)
Unfortunately, soil moisture could not be measured more frequently
because it could not be automated. The rainfall was measured next to
the soil cores with a farm rain gauge (model no. 510, Science Asso
ciates, Inc., Princeton, N.J.) attached to the wooden box as seen in
Figure 5-1. The rainfall was measured after each rain event in centi
meters. Chemical parameters for the rainfall at the experimental site
are presented in Table 4-2. In the first survival experiment that began
on 7 October 1977, measurement of some environmental parameters (i.e.,
temperature and rainfall) could not be carried out at the experimental
site due to the lack of the necessary equipment. In this case, the
data from the weather station of the Department of Agronomy, University
of Florida, were used. This station is approximately one mile from the
experimental site.
Results and Discussion
In the previous chapter (i.e., Chapter IV), virus transport
through soils was evaluated under controlled laboratory conditions.
It appeared necessary, however, to study virus transport and survival
under more natural conditions. Undisturbed soil cores were used to
assess viral transport and survival under field conditions. In these


43
and chemical properties of sludge are quite complex (Peterson et ^1_.
1973; U.S. Environmental Protection Agency 1978b). Due to the numerous
health hazards associated with treated sludges, final sludge disposal
should be handled with the utmost of care.
Final Sludge Disposal
In 1979, municipal treatment plants in the United States were
producing approximately 4.5 billion dry kg of sludge per year and
this .is expected to rise to 3 billion by the early 1980s (Pahren et al.
1979). Such large quantities of sludge coupled with the health hazards
associated with sludge make final sludge disposal the most difficult
of the sludge treatment processes (see Figure 2-1). Final sludge dis
posal is usually accomplished by either ocean dumping, sanitary landfill
or land application (see Figure 2-1) (U.S. Environmental Protection
Agency 1974, 1978b). Sludge incineration is not technically a final
disposal method since ash is produced which requires disposal (in
Figure 2-1, incineration is classified as a sludge reduction process).
In actual practice, however, sludge incineration is considered a
disposal method (Pahren 1980; Pahren et aK 1979; U.S. Environmental
Protection Agency 1978b).
Incineration. Of the total amount of sludge disposed of in
1979 nationally, approximately 35% was incinerated (Pahren et a]_. 1979).
In the future, however, sludge disposal by incineration is likely to
be significantly curtailed due to air pollution, high energy costs and
other problems associated with this disposal practice (Pahren 1980;
Pahren e* aj_. 1979; U.S. Environmental Protection Agency 1974, 1978b).


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
ABSTRACT vii
CHAPTER
I INTRODUCTION 1
II LITERATURE REVIEW 4
Viral Pathogens Found in Raw Wastewater 4
Removal of Viruses by Wastewater Treatment Processes 5
Primary Sedimentation 5
Activated Sludge 13
Removal of Viruses by Sludge Treatment Processes 16
Viruses in Raw Sludges 16
Sludge Treatment Processes 18
Viral and Other Health Hazards Associated
with Treated Sludges 42
Final Sludge Disposal 43
Fate of Sludge-Associated Viruses in Soils 49
ttt EFFECT OF SLUDGE TYPE ON POLIOVIRUS ASSOCIATION
1 WITH AND RECOVERY FROM SLUDGE SOLIDS 50
Introduction 50
Materials and Methods 52
Virus and Viral Assays 52
Sludges 56
Association of Seeded Poliovirus
with Sludge Solids 59
Recovery of Seeded Poliovirus from
Sludge Components 65
Statistical Treatment of Data 67
Results and Discussion 67
Association of Seeded Poliovirus
with Sludge Solids 67
Recovery of Solids-Associated Viruses 75
TV


245
TABLE 6-3. Indigenous enteroviruses associated with sludges from
the Main Street wastewater treatment plant, Gainesville,
Florida
Sampling date
Sludge3 type
Viruses detected
(TCID5Qb/g dry wt.)
December 1977 and
February 1978
Wasted
11 to 25
December 1977 and
Aerobically digested0
(90 days)
0.3 to 1.2
February 1978
December 1977,
January 1978, and
February 1978
Conditioned-dewateredd
0
aSludge samples of 1 to 4 liters were centrifuged at 1400 x g
for 10 min at 4C. The sludge supernatants produced were discarded.
The sludge solids were tested for the presence of viruses.
bRefers to the 50% tissue culture infective dose.
identified as sludge GDA90 in Table 3-2.
^Sludge aerobically digested for 180 days (GDA180--see Table 3-2)
is conditioned with a cationic polymer and then dewatered by centrifu
gation at the Main Street plant. The conditioned-dewatered sludge
was applied to the Kanapaha site (see Table 6-4 for data on the viral
monitoring of the Kanapaha site).


282
Wang, D.-S., J. C. Lance, and C. P. Gerba. 1980b. Evaluation of
various soil water samplers for virological sampling. Appl.
Environ. Microbiol. 39: 662-664.
Ward, R. L. 1977. Inactivation of poliovirus in wastewater sludge
with radiation and thermoradiation. Appl. Environ. Microbiol.
33: 1218-1219.
Ward, R. L., and C. S. Ashley. 1976. Inactivation of poliovirus in
digested sludge. Appl. Environ. Microbiol. 31: 921-930.
Ward, R. L., and C. S. Ashley. 1977a. Identification of the virucidal
agent in wastewater sludge. Appl. Environ. Microbiol. 33: 860-
864.
Ward, R. L., and C. S. Ashley. 1977b. Inactivation of enteric viruses
in wastewater sludge through dewatering by evaporation. Appl.
Environ. Microbiol. 34: 564-570.
Ward, R. L., and C. S. Ashley. 1977c. Discovery of an agent in waste-
water sludge that reduces the heat required to inactivate
reovirus. Appl. Environ. Microbiol. 34: 681-688.
Ward, R. L., and C. S. Ashley. 1978a. Identification of detergents
as components of wastewater sludge that modify the thermal
stability of reovirus and enteroviruses. Appl. Environ. Micro
biol. 36: 889-897.
Ward, R. L., and C. S. Ashley. 1978b. Heat inactivation of enteric
viruses in dewatered wastewater sludge. Appl. Environ.
Microbiol. 36: 898-905.
Ward, R. L., and C. S. Ashley. 1979. pH modification of the effects
of detergents on the stability of enteric viruses. Appl.
Environ. Microbiol. 38: 314-322.
Ward, R. L., C. S. Ashley, and R. H. Moseley. 1976. Heat inactivation
of poliovirus in wastewater sludge. Appl. Environ. Microbiol.
32: 339-346.
Weber, W. J., Jr., C. B. Hopkins, and R. Bloom, Jr. 1970. Physico
chemical treatment of wastewater. J. Water Pollut. Control
Fed. 42: 83-99.
Wellings, F. M., A. L. Lewis, and C. W. Mountain. 1974. Virus survival
following wastewater spray irrigation of sandy soils. In
Virus Survival in Water and Wastewater Systems, pp. 253-260.
Edited by J. F. Malina, Jr., and B. P. Sagik. Center for
Research in Water Resources, University of Texas, Austin, Texas.


91
Ca(OH)2^ added to the alum and ferric chloride sludge was 1389 and 625
mg/2,, respectively. A contact time of 30 minutes was allowed while
mixing the suspension on a magnetic stirrer. After 30 minutes of mixing,
the pHs of the sludges were measured and had dropped to 11.1 or 11.3.
The lime-stabilized, chemical sludges were then assayed for viruses as
described below.
Association of Seeded Poliovirus with Sludge Solids
Poliovirus stock in phosphate-buffered saline (PBS) containing
2% fetal calf serum (FCS) (see Appendix for more details on the compo
sition of this solution) was added directly to 0.01 N calcium chloride,
primary wastewater effluent, anaerobically digested sludge (diluted or
undiluted) and sludge liquor at the rate of 1 ml of virus stock per
1000 ml of solution and while stirring the suspension using a magnetic
stirrer (see Chapter III, page 59). After a 1-minute mixing period,
an aliquot of the samples containing low solids contents (i.e., 0.01 N
calcium chloride, primary wastewater effluent, diluted anaerobically
digested sludge, and sludge liquor) was diluted in PBS containing 2%
FCS and assayed directly for seeded viruses by the plaque technique.
This viral assay was performed in order to determine the amount of
virus present in these samples initially and was repeated at the end
of experimental trials in order to assess any viral inactivation which
might have occurred. No attempt was made to determine the degree of
association between seeded poliovirus and the small quantity of solids
present in these samples.
The association of poliovirus with sludge solids was deter
mined for undiluted anaerobically digested sludge (seeded with


TABLE 5-3. Association between seeded enteroviruses and sludge solids
Virus9
used
Sludge
used
Sludge parameters
Virus in .
unfractionated0
sludge
(total PFU)
Virus in
sludge
supernatant0
(total PFU)
Viable
unadsorbed
virus
(%)
Solids-
associated
virus
(%)
Unfractionated
sludge applied
to soil cores0
as described in
PH
Conductivity
(ymho/cm at
25C)
Sol ids
content
(%)
Poliovirus
type 1
Aerobic6
5.0
--
1.3
3.9 x 108
6.1 x 108
2.3 x 106
7
0.6
99.4
Table 5-4
Lagoon1
6.9
3500
2.9
5.4 x 107
8.9
91.1
Table 5-5
Lagoon
7.0
1525
7.0
8.6 x 108
4.1 x 107
4.8
95.2
Table 5-8
Echovirus
type 1
Lagoon
7.0
1525
7.0
2.9 x 106
2.3 x 106
79.3
20.7
Table 5-8
aVirus was added to sludge while stirring the suspension using a magnetic stirrer. Magnetic stirring
was continued for 10 to 60 min and then an aliquot of the virus-seeded sludge was obtained for viral assay.
bThe sludge solids were not separated prior to assaying. The values shown represent the total virus
PFU contained in 479 ml of sludge.
cThe sludge was clarified by centrifugation at 1 ,400 x g for 10 min at 4C and the supernatant was
subsequently assayed.
^Undisturbed large soil cores received 2.5 cm or 479 ml of virus-seeded sludge.
eAerobically digested sludge (GDA180--see Table 3-2) was sampled at the Main Street wastewater treat
ment plant of Gainesville, Florida.
f
Lagoon sludge (LAG--see Table 3-2) is a mixture of aerobically digested sludge (1/3) and anaerob
ically digested sludge (2/3). The mixture was kept in a lagoon at the West Florida Agricultural Experiment
Station (Jay, Florida) before ultimately being disposed of on land.


247
TABLE 6-5. Analysis of topsoil samples from the Jay site for the
presence of indigenous enteroviruses
Soil
plot no.
Amount of lagooned
sludgeb applied yearly
[acre-in (ha-cm)]
Total number of
grams of topsoilc
sampled
Viruses
detected
1
15 (15.4)
800
0
32
15 (15.4)
800
0
42
0
800
0
61
15 (15.4)
800
0
aThe locations of these soil plots at the Jay site are shown in
Figure 6-4.
bThe viral content of lagooned sludge is shown in Table 6-6.
CA composite topsoil sample of 100 wet grams was taken from each
soil plot monthly from June 1978 through January 1979 and was tested for
the presence of viruses.


FIGURE 4-8. Movement of poliovirus type 1 through a 10 cm packed column of Eustis fine sand
subsoil when suspended in filtered-pH adjusted lagoon sludge liquor
One pore volume for the column used equals 71 ml. The laboratory-packed
column was 10 cm in length and 4.8 cm internal diameter. The sample of
Eustis fine sand subsoil used consisted mainly of the A21 and A22 horizons
(see Table 4^3), The column was conditioned with 2 pore volumes of
filtered-pH adjusted sludge liquor. The poliovirus was then suspended in
the filtered-pH adjusted sludge liquor at a concentration of 4.5 x 10^
PFU/ml and applied to the column. All solutions were applied continuously
to the column at approximately 3 ml/min using a peristaltic pump (Buchler,
Fort Lee, N.J.). The sludge liquor was produced by centrifuging lagoon
sludge (LAG--see Table 3-2; solids content, conductivity and pH equal to
2.9%, 3500 ymho/cm at 25C and 6.9, respectively) at 14,000 x g for 10 min
at 4C. The sludge liquor (i.e., the supernatant from the previous cen
trifugation) was passed through a series of 0.45 and 0.25 ym Filterite
filters (Filterite Corp., Timonium, Md.) in a 47 mm holder and then adjusted
to pH 8.0 using 0.01 N NaOH. The conductivity of the filtered-pH adjusted
sludge liquor was 1800 ymho/cm at 25C.


28
anaerobically digested sludge also depends on the virus removal
efficiency of the anaerobic digestion procedure employed. For example,
Berg and Berman (1980) reported that, at the City of Los Angeles
Hyperion treatment plant, thermophilic anaerobic digestion (20 days at
approximately 49C) was superior to mesophilic anaerobic digestion
(20 days at approximately 35C) in the removal of indigenous viruses
from raw sludge. In the laboratory, Ward et aj_. (1976) confirmed that
the inactivation of seeded poliovirus type 1 (CHAT) in anaerobically
digested sludge was accelerated under thermophilic temperatures.
Although little is known about the viral-inactivating capacity
of the aerobic digestion process, several investigators have reported
that, as in the case of anaerobic digestion, not all indigenous entero
viruses are eliminated from sludge during aerobic digestion (Farrah
et aj_. 1981a, 1981b; Hurst £t al_. 1978). Farrah et al_. (1981a, 1981b)
measured indigenous enterovirus titers ranging from 1.7 to 260 TCID^/g
dry wt. TSS in aerobically digested sludge from three wastewater treat
ment plants in Florida. In sludges from two wastewater treatment
plants in Pensacola, Florida, Farrah et al_. (1981a) showed that aerobi
cally digested sludge contained larger indigenous viral titers than
anaerobically digested sludge.
Clearly, both anaerobically and aerobically digested sludges can
contain substantial quantities of enteric viruses. Therefore, all
digested sludges should be considered to represent potential health
hazards, and should be handled with care during further treatment and
final disposal (Palfi 1972).


70
digested sludges was 57%, 70%, and 95%, respectively (Table 3-4).
Ward and Ashley (1976) have obtained similar results with anaerobically
digested sludge. These investigators have shown that 65% and 67% of
seeded poliovirus type 1 (Mahoney) and poliovirus type 1 (CHAT),
respectively, were associated with anaerobic sludge solids after a con
tact period of 15 minutes. The association between seeded poliovirus
and sludge solids was significantly greater for aerobically digested
sludges than for mixed liquors or anaerobically digested sludges. No
statistically significant difference was found between the mean
percent of solids-associated viruses for mixed liquors and anaerobically
digested sludges. The lagoon sludges mentioned in Table 3-4 are a
mixture of aerobically digested sludge (1/3) and anaerobically digested
sludge (2/3), and consequently, were placed in the category of
anaerobically digested sludges. However, the association of poliovirus
with lagoon sludge solids was greater than the association of this virus
with other anaerobic sludge solids tested (see Table 3-4). Apparently,
the presence of aerobic sludge solids (1/3) in lagoon sludge accounts
for the greater ability of lagoon sludges to bind viruses.
The reason for the greater association of seeded viruses with
aerobic sludge solids is still unknown. However, aerobically digested
sludges generally displayed lower pH values (ranging from 4.8 to 6.5)
than mixed liquors (6.4 and 6.9) or anaerobically digested sludges
(ranging from 6.4 to 8.3) (see Table 3-4). Virus adsorption to surfaces
is promoted at low pH. Other parameters of aerobically digested sludges,
as yet unidentified, could also account for the greater ability of these


FIGURE 4-10. Movement of poliovirus type 1 through an undisturbed
core of Eustis fine sand (conditioned with 0.01 N CaCl2)
following the application of 2.5 cm of seeded anaerobi-
cally digested sludge and the subsequent elution with
rain water
One pore volume for the core used equals 234 ml.
The undisturbed soil core was 33 cm in length and
5.0 cm internal diameter; consists of the Ap and
A21 horizons of the Eustis fine sand (see Table
4-3). The core was initially conditioned with
5 pore volumes of 0.01 N CaCl2 (conductivity and
pH equal to 1210 umho/cm at 25C and 6.4, respec
tively). One inch or 2.5 cm (51.6 ml) of anaerobi
cally digested sludge (GDAN--see Table 3-2;
solids content, conductivity and pH equal to
2.0%, 3250 ymho/cm at 25C and 8.3, respectively)
seeded with a total of 9.8 x 105 PFU of poliovirus
was applied to the core, allowed to soak in and
then, was worked under 2.5 cm. Elution with rain
water was subsequently undertaken. This solution
was applied from an inverted, self-regulated,
1 liter Erlenmeyer flask set to maintain a 2.5 cm
hydraulic head on the core. The flow rate through
the core was measured at 3.9 ml/min. The rain
water was collected next to the Environmental
Engineering Sciences building at the University
of Florida, Gainesville. See Table 4-2 for
chemical characteristics of the rain water.


105
The bulk density is defined as the mass of dry soil in a unit volume
consisting of soil solids and pores (Blake 1965; Brady 1974). The
particle density is defined as the mass of a unit volume of soil solids
3
(Brady 1974). The bulk density of the Red Bay sandy loam was 1.45 g/cm
(used this value for both laboratory-packed columns and undisturbed
cores), and that of the Eustis fine sand was 1.56 or 1.61 g/cm (for
laboratory-packed columns or undisturbed cores, respectively). An
3 3
average value of 2.60 g/cm (range from 2.52 to 2.69 g/cm ) was used for
particle density. These values of particle density are typical for
mineral soils (Brady 1974). The pore volume of each core was then cal
culated as shown below:
pore volume (ml) =
pore space (%)
100
total volume (ml)
of core
(4-2)
An aliquot of each leachate sample was diluted (i.e., if neces
sary) in PBS containing 2% FCS and assayed directly for viruses by the
plaque technique. In order to detect small numbers of viruses, the
leachates from laboratory-packed soil columns which had received
chemical sludges and lime-stabilized, ferric chloride sludge were
concentrated by membrane filtration (Farrah et al_. 1976; Hill et al.
1971; Shuval and Katzenelson 1972; Sobsey et al_. 1973; Sobsey et al.
1980b). These soil leachates were collected in 1/2 pore volume frac
tions which were assayed individually for viral infectivity as
described above. Pore volumes 0.5 through 5.0 (and 5.5 through the


251
Morita and ZoBell 1956; Zobell and Cobet 1962; ZoBell and Johnson 1949)and
tobacco mosaic virus (Johnsonet al. 1948; Lauffer and Dow 1941). In
this chapter, the effect of elevated hydrostatic pressure on the survival
of poliovirus seeded in groundwater was investigated, For comparative
purposes, virus survival in seawater under elevated pressure was also
studied.
Materials and Methods
Virus and Viral Assays
Poliovirus type 1 (strain LSc) was used in the research reported
in this chapter. Some general properties of polioviruses are shown in
Table 3-1. Stocks of poliovirus were prepared as described in Chapter
m (see page 53 ). Viral stocks were kept at -70C until used. Polio
virus was assayed by the plaque technique as described in Chapter III
(seepages 53-56). Each viral count shown represents the average of tripli
cate counts. The numbers of viruses were expressed as plaque-forming
units (PFU).
Water Samples
The seawater used in this study was sampled at the Mantanzas Inlet
on Florida's east coast. The groundwater sample was obtained from a
1200-foot deep well at the Kanapaha wastewater treatment plant,
Gainesville, Florida. The water samples were collected in sterile
Nalgene carboys, transported to the University of Florida (Gainesville)
laboratory and then immediately refrigerated. No chlorine residual was
found in these water samples (i.e., by the orthotolidine test). The pH
of each water sample was measured using a digital pH meter model 125
from Corning (Corning, New York). The conductivity of each water


TABLE 5-7. Analysis for the presence of poliovirus type 1 (Sabin) in soil leachates collected after
natural rainfall and after experimental leaching with rain water from small soil cores of
Eustis fine sand which had been treated with 2.5 cm of seeded liquid sludge (2 June 1978-
24 August 1978)
Small soil
core3
number
Date
of
leachate
collected
(1978)
Cumulative
rainfal1
(cm)
Leachate
volume
(cm)
Cumulative
leachate
volume
(ml)
Virus
breakthrough
(total PFU)
Cumulative
percent of
total virus
applied
Conductivity
of soil
leachate
collected
(ymho/cm
at 25C)
pH of
soil
leachate
collected
SCI
06 Jun.
6.03
16
16 (0.1)b
0
0

5.5
06 Jun.
(L.R.)C
24.88
317
333 (1.4)
3.9 x 102
0.0006
720
6.0
08 Jun.
26.13
19
352 (1.5)
6.7
0.0006

7.2
13 Jun.
28.88
46
398 (1.7)
0
0.0006
100
7.1
23 Jun.
(L.R.)
67.52
360
758 (3.2)
0
0.0006
430
5.8
10 Jul.
74.25
80
838 (3.6)
0
0.0006
151
6.9
10 Jul.
(L.R.)
89.54
320
1158 (4.9)
0
0.0006
310
6.4
13 Jul.
91.06
20
1178 (5.0)
0
0.0006
425
6.8
03 Aug.
112.44
145
1323 (5.7)
0
0.0006
84
6.5
24 Aug.
123.41
145
1468 (6.3)
(74.8)d
0
0.0006
69
6.7


158
undisturbed core was initially conditioned with rain water. When an
identical, undisturbed core was conditioned with 0.01 N CaCl^, only
10.6% of the total poliovirus applied was detected in the effluent
by the 8.5 pore volume (see Figure 4-10). Conditioning with calcium
chloride enhanced the adsorption of viruses to the soil. Thus, it was
found that the nature of the conditioning solution can affect the virus
breakthrough pattern later obtained.
It appears then that Eustis fine sand, under saturated flow,
does not retain viruses effectively (in Chapter V,data are presented
on virus transport through Eustis fine sand cores under unsaturated
flow and under field conditions). There is a dramatic difference
between the virus "retention potential" of the Red Bay sandy loam
(99.8%) and the Eustis- fine sand (70.7%). It is postulated that the
low adsorptive capacity of the Eustis fine sand is a direct result of
its low clay content (only 3.2%, average for the Ap and A21 horizons,
see Table 4-3). In the case of the Eustis fine sand, sludge applica
tion could lead to ground water contamination with pathogenic viruses.
However, the results presented for the Red Bay sandy loam indicate
that, under appropriate conditions, sludge application could be
undertaken without threatening the quality of ground water with
viruses.
Conditioned-Dewatered Sludge
At many wastewater treatment plants around the country,
digested sludges are conditioned with polymers prior to dewatering.
At the Main Street wastewater treatment plant in Gainesville, Florida,


10
inactivated by the high pH produced and that the viral inactivation
rate increased as the pH was raised from 10.1 to 11.1 (see Berg et al.
1968). Therefore, these researchers concluded that the removal of
poliovirus by this treatment process resulted from a combination of
viral inactivation and physical separation of the virus by the floccu
lation process. The effectiveness of lime flocculation in the removal
of viruses from secondary effluents has also been shown in the field.
At a wastewater reclamation plant, Grabow et al_. (1978) showed that >99.9%
of indigenous enteric viruses in activated sludge effluent were removed
by lime flocculation at pH 9.6 to 11.2 followed by sedimentation.
Coliphages, enterococci and coliform bacteria were also effectively
removed by the lime flocculation process (see Grabow et al_. 1978).
Chemical flocculation using alum, ferric chloride or lime is
frequently combined with sedimentation in the primary treatment of raw
sewage (U.S. Environmental Protection Agency 1973). Among the advantages
of this treatment process are low capital costs, minimal space require
ments and high reliability (Weber et aj_. 1970). In laboratory-scale
experiments, Shuckrow et al_. (1971) showed that the flocculation of
raw sewage with alum was highly efficient in removing suspended solids,
total organic carbon (TOC) and chemical oxygen demand (COD). Similar
results were obtained by Weber et aj_. (1970) when lime or ferric chloride
were used as coagulants. Substantial removals of phosphates, nitrates,
and organic color are also achieved by this treatment process (Sproul
1976; Weber et al_. 1970). As reviewed in the previous paragraphs,
numerous studies have shown that viruses are effectively removed from


248
indigenous enteroviruses from the digested sludges added to the lagoon
and also from the lagooned sludge (see Table 6-6). However, these
investigators observed a rapid decline in the number of enteroviruses
associated with lagooned sludge which had been applied to land. In
fact, indigenous enteroviruses were almost undetectable in lagooned
sludge allowed to dry for only two days on the soil surface at the Jay
site (60% sludge solids contentsee Table 6-6). It follows that
enteroviruses are not likely to be detected in topsoil samples. Thus,
allowing the sludge to dry on top of the soil before being mixed with
the soil results in the inactivation of all or most of the viruses
present (this was also demonstrated using seeded viruses in Chapter V)
This may be an advantage over sludge injection into soils (Moore et al
1978), where viruses can survive for longer periods of time. Despite
the numerous advantages of sludge injection (aesthetic acceptability,
and minimal odor and runoff), surface spreading of sludge may result
in the inactivation of viruses at accelerated rates. It should be
pointed out that Farrah et al_. (1981a) were unable to detect any
viruses in groundwater samples obtained from the Jay site (see Table
6-6). It appears that at this sludge disposal site, as was the case
at the Kanapaha site described above, enteroviruses pose a minimal
hazard with respect to soil and groundwater contamination.


230
TABLE 6-1. Some characteristics of the Lochloosa soil series found
at the Kanapaha site
Soil3
horizon
Depth
(cm)
Mechanical composition (%)
PH
(in 1:1
water)
Sand
(2-
0.05 mm)
Silt
(0.05-
0.002 mm)
Clay
(<0.002 mm)
Ap
0-18
88.9
7.8
3.3
4.9
A21
18-43
91.2
4.0
4.8
5.1
A22
43-71
89.2
4.5
6.3
5.2
Bit
71-81
79.3
4.7
16.0
5.1
B21tg
81-89
67.4
4.7
27.9
4.8
B22tg
89-145
66.8
3.6
29.6
4.6
B3g
145-175
59.2
2.6
38.2
4.8
aTypifying pedon is Lochloosa fine sand. Data were adapted from
Calhoun et al_. (1974).


136
TABLE 4-14. Distribution of poliovirus type 1 in the soil profile of
a 27-cm packed column of Eustis fine sand which had
received virus-seeded, anaerobically digested sludge
diluted (1:50) with distilled water
Depth in
column3 (cm)
Poliovirus recovered from soi1b
PFU/g of
wet soil
PFU/soi1
section
% of Total
PFU applied0
Top Sludged
4,434
4.9
X
104
0.4
0-3
2,720
2.9
X
K>5
2.6
3-5
2,940
2.5
X
105
2.3
5-7
2,266
2.2
X
105
2.0
7-9
966
7.3
X
104
0.7
9-11
1,694
1.3
X
105
1.2
11-13
1,106
9.8
X
104
0.9
13-15
734
6.4
X
104
0.6
15-17
874
6.8
X
104
0.6
17-19
260
2.2
X
104
0.2
19-21
614
4.0
X
104
0.4
21-23
534
5.2
X
104
0.5
23-25
46
3.4
X
103
0.03
25-27
60
5.8
X
103
0.05
aThe laboratory-packed column was 29 cm in length and 4.8-cm
internal diameter; the column was filled only 27 cm with soil (2 cm
left on top for the packed sludge solids). The column was treated
with virus-seeded diluted sludge as described in Table 4-12. The
soil column was then sectioned and virus was eluted from each soil
section.


a
The cumulative rainfall (cm) values represent the total rainfall from the beginning of the experiment
on 11 October 1978.
^One inch (2.5 cm) of lagooned sludges seeded with a total of 8.6 x 10^ (9.3 x 10^ for the small core)
PFU of poliovirus or 2.9 x 10 (3.1 x 1CP for the small core) PFU of echovirus was applied on top of soil
cores. The soil cores were exposed to natural conditions.
c
Echovirus was seeded in the sludge applied to small core 1, core 1, and core 2. Poliovirus was
seeded in the sludge applied to small core 2, core 3, and core 4.
d0ne pore volume for the large soil cores and the small soil cores equals 2178 ml and 234 ml, respec
tively.
0
Values in parentheses represent the number of centimeters of cumulative leachate volume.


51
balanced salt solution, pH 9.5 (Subrahmanyan 1977), and 10% fetal
calf serum (Sattar and Westwood 1976). Elution of solids-associated
viruses may be aided by sonication (Abid et a]_. 1978; Glass et al.
1978; Wellings et al_. 1976a), shaking on a wrist-action shaker
(Sattar and Westwood 1976), homogenization in a blender (Glass et al.
1978; Moore et aj_. 1978; Subrahmanyan 1977), or magnetic stirring
(Abid et a]_. 1978; Hurst et al_. 1978). Eluted viruses have been con
centrated by organic flocculation at low pH (Abid et al_. 1978; Glass
et aj_. 1978; Hurst et al_. 1978), hydroextraction (Wellings et al.
1976a) or adsorption to bentonite clay (Turk et al_. 1980).
Several of the methods proposed for the recovery of viruses
from sludges do not contain a concentration step but simply involve
the elution of viruses from sludge solids (Moore et al_. 1978; Sattar
and Westwood 1976; Subrahmanyan 1977). The use of these methods is
limited to raw sludges and other sludges containing large amounts of
viruses. The concentration method proposed by Wellings et al_. (1976a)
involving hydroextraction is cumbersome and requires considerably more
time to perform than organic flocculation. The procedure developed
by Abid et £1_. (1978) is not practical, since it does not adequately
concentrate the viruses eluted from sludge solids. Therefore, the
best methods proposed to date for virus recovery from sludges appear to
be those of Glass et aj_. (1978) and Hurst et al_. (1978). Working with
anaerobically digested sludge, Glass et al_. (1978) obtained an overall
recovery of poliovirus type 1 (CHAT) of 31%. Hurst et al_. (1978) found
that poliovirus type 1 (LSc) could be recovered from activated sludge


TABLE 4-20. Association of poliovirus type 1 with chemical sludges precipitated from virus-seeded,
raw sewage
Sludge3 Experi- Virus in sewageb Virus Virus in Sludge
type ment -- in unfractionated0 was
no. Before addition After addition supernatant sludge
of coagulant of coagulant0
Total
PFU
Total
PFU
Recovery6
(%)
Total
PFU
Recovery6
(%)
Total
PFU
Recovery6
(2)
Sludge
volume
(ml)
Sludge
sol ids'
content
(*)
Limed
Applied
to soil
column
no.
Alum
1
5.3
X
107
9
--
9.4
X
,o6
17.7
1.2
X
io7
22.6
20

No
1.
Table
4-22
2
4.5
X
107
4.5
x 107
100.U
1.6
X
io6
3.6
4.3
X
io7
95.6
50
0.3
No
2,
Table
4-22
3
3.9
X
107

--
4.7
X
10b
1.2
1.1
X
io7
28.2
21
1.3
Yes,
Table
4-23
Ferric
1
4.3
X
107
7.7
x 106
17.9
4.0
X
10b
0.9
4.7
X
,06
10.9
31
0.5
No
1,
Table
4-22
chloride
2
4.5
X
107
7.3
x 106
16.2
9.3
X
10b
2.1
4.4
X
io6
9.8
33
0.4
No
2,
Table
4-22
3
3.9
X
107
9.8
x 106
25.1
9.3
X
10b
2.4
5.2
X
106
13.3
31
0.7
Yes,
Table
4-23
--
Lime
1
5.8
X
107


1.0
X
io5
0.2
7.5
X
IO4
0.1
30
--
No
1,
Table
4-22
2
4.5
X
107
0
0
0
0
0
0
37
1 .0
No
2,
Table
4-22
aThe chemical sludges were precipitated from 1000 ml of virus-seeded, raw sewage using the concentrations of coagulants shown below. Virus
assays were made before and after the addition of coagulants. Following the addition of alum and ferric chloride, the pHs of the solutions were
adjusted to 6.0 and 5.0, respectively, in order to achieve maximum flocculation. The coagulant, lime, was added until pHs of 11.1 and 11.3 were
achieved in experiments no. 1 and no. 2, respectively. Following the addition of the coagulants, the sewage samples were mixed on a magnetic
stirrer rapidly for 10 min and slowly for 5 min. The flocculated sewage samples were then transferred to Imhoff cones and 60 min was allowed
for the formation and settling of the sludges. The supernatants in the Imhoff cones were assayed for viruses and discarded. The sludges
produced were then assayed for viruses.
bThe sewage used was sampled at the University of Florida campus sewage treatment plant (only used for experiments no. 1-alum and no.1-lime;
conductivity and pH equal to 520 imiho/cm at 25C and 9.0, respectively), or at the Main street sewage treatment plant of Gainesville, Florida
(used for all other experiments; conductivity and pH equal to 770 umho/cm at 25C and 8.9, respectively). The sewage samples were sterilized by
autoclaving prior to use.
cThe final concentrations in sewage of the coagulants used were 300 mg/1 of Al(SO^)j-18 I^O, 50 mg/1 of FeCl^, and 150 or 250 mg/1 of
Ca(0H)2 (for experiments no. 1 and no. 2, respectively).
^The sludge solids were not separated prior to assaying.
ePercent recoveries were calculated based on the amount of viruses (total PFU) present in the sewage before the addition of coagulants as 100X.
^Sludge solids content was expressed as a percentage on a weight to volume basis.
9A dash means not done.
os
os


252
sample was measured using a YSI model 33 S-C-T meter (Yellow Springs
Instrument Co., Inc., Yellow Springs, Ohio). The conductivity and pH
of each water sample are shown in Table 7-1. The water samples were
neither filtered nor autoclaved prior to use.
Poliovirus Exposure to Hydrostatic Pressures
Poliovirus was added to 75 ml of either seawater or groundwater
(water sample was temperature acclimated for experimental trial) and
the solution was mixed for 1 minute. The water sample was then assayed
in order to determine the initial virus concentration. Following the
initial viral assay, 65 ml of the virus-seeded water sample was pressur
ized (i.e., to between 500 and 4000 psi) directly in a pressure chamber
(virus-free and temperature acclimated for experimental trial) described
by Horvath and Elkan (1978). The remaining 10 ml of virus-seeded water was
left at atmospheric pressure (i.e., 14.7 psi). An atmospheric pressure
control sample was always run with each elevated pressure trial. The
elevated and atmospheric pressure water samples were then placed at the
same temperature (i.e., 2C or 24C) for a known period of time (2, 8 and
24 hours). At the end of the experimental trial, the pressure was
released, and the water sample was transferred to sterile glassware
and assayed for poliovirus. The atmospheric pressure water sample was
also assayed for poliovirus. Two experimental trials were run at each
elevated pressure condition tested. Poliovirus recovery after exposure
to elevated pressure was expressed as a percentage of the viral titer
in the atmospheric pressure sample.


29
Stabi1 ization-composting. Composting is a biological, aerobic,
thermophilic (approximately 60C) process frequently used to stabilize
(see Figure 2-1) raw sludges (U.S. Environmental Protection Agency 1974,
1978b). As such, this process reduces the odor, putrescibility poten
tial and pathogen content of raw sludges (U.S. Environmental Protection
Agency 1974, 1978b). Bitton (1980b) described in detail the sludge
composting process and reviewed the literature on pathogen destruction
during this sludge treatment procedure. Pathogenic parasites and
bacteria as well as bacteriophage f2 have been shown to be inactivated
during sludge composting (Bitton 1980b). Ward and Ashley (1978b)
demonstrated that seeded poliovirus type 1 (CHAT) was heat-inactivated
(43C) at a significantly greater rate in composted sludge than in
dewatered raw sludge held at the same sludge solids content of 40%. In
the case of seeded reovirus, the reverse was observed (i.e., greater
inactivation rate in dewatered raw sludge; see Ward and Ashley 1978b).
These viral inactivation patterns were attributed to the effects of
sludge solids-associated, ionic detergents (Ward and Ashley 1978b).
These detergents were previously shown to influence differently the
heat inactivation rate of enteroviruses and reoviruses in sludge.
Whereas enteroviruses in sludge were protected from heat inactivation
by ionic detergents, reoviruses were inactivated at an accelerated
rate (Ward and Ashley 1977c, 1978a, 1979). During composting, however,
the ionic detergents in raw sludge were shown to be substantially
degraded (Ward and Ashley 1978b). Thus, viral inactivation rates were
markedly different in composted sludge as compared to raw sludge and


18
wastewater treatment processes (Oliver 1976). Furthermore,
indigenous viruses are believed to be mostly embedded within the
sludge solids rather than merely surface adsorbed (Weilings et al.
1976a). This association between indigenous viruses and sludge solids
has significant implications on viral survival during subsequent
sludge treatment.
Sludge Treatment Processes
A variety of sludge treatment processes is routinely used in
treatment plants and they are shown in Figure 2-1. From Figure 2-1,it
can also be seen that several often overlapping functions are achieved
by each sludge treatment process. The order of the sludge treatment
processes shown in Figure 2-1 is as most often used in treatment
plants (Fair et al_. 1968; Malina 1976; U.S. Environmental Protection
Agency 1974). However, it should be pointed out that not all sludge
treatment processes shown in Figure 2-1 are employed at every treatment
plant. Moreover, the order of the sludge treatment processes may be
varied from that shown in Figure 2-1 depending on the existing condi
tions. Characteristics of the sludge treatment processes have been
reviewed by several authors (Dick 1978; Fair et aK 1968; Malina 1976;
U.S. Environmental Protection Agency 1974, 1978a, 1978b; Yates 1977).
Some work has been done on the removal of viruses (i.e., by physical
separation and/or inactivation of viral particles) from raw sludges
(i.e., primary, chemical, and/or secondary sludges) by sludge treatment
processes and is reviewed below.


117
transport under optimal conditions (i.e., in the presence of 0.01 N
CaCl2) and under more realistic conditions involving sludge applica
tion to soils.
Poliovirus Suspended in 0.01 N CaCl2
Red Bay sandy loam. The "retention potential" of this soil
towards poliovirus was first determined, under optimal conditions, in
the presence of 0.01 N CaCl2. As shown in Table 4-6, more than 99.99%
of the viral load was removed, presumably due to adsorption, after
10 pore volumes of solution had passed through the soil. The pH of the
soil solution varied from 5.1 to 6.3 and the conductivity was around
1300 ymhos/cm (see Table 4-6). A shift from calcium chloride to rain
water did not result in any appreciable release of soil-bound viruses
although the conductivity of the soil solution decreased from 1320 to
54 ymhos/cm. It is well known that divalent cations, at appropriate
concentrations, enhance the adsorption of viruses to soils (Bitton
1975; Drewry and Eliassen 1968; Gerba et al_. 1975; Lefler and Kott
1974). Rain water may be important in the redistribution and transport
of viruses through the soil matrix. This has been suggested in the
field (Weilings et aj_. 1975) and demonstrated in the laboratory
(Duboise et al_. 1976; Lance et £]_. 1976). However, it was found that
rain water did not significantly affect the desorption of viruses from
a soil containing 28% clay (Scheuerman jit a_l_. 1979).
Eustis fine sand. The "retention potential" of this soil
towards poliovirus was also evaluated, as described above for the Red
Bay sandy loam, in the presence of 0.01 N CaCl2.
It was observed


FIGURE 4-6. Movement of poliovirus type 1 through a 10 cm packed
column of Red Bay sandy loam subsoil when suspended
in anaerobically digested sludge diluted (1:50) with
sludge liquor containing 0.01 N CaCl2
One pore volume for the column used equals 80 ml.
The laboratory-packed column was 10 cm in length
and 4.8 cm internal diameter. The sample of Red
Bay sandy loam subsoil used consisted mainly of
the A2 and Bit horizons (see Table 4-3). The
column was conditioned with 2 pore volumes of
sludge liquor containing 0.01 N CaCl2- Poliovirus
was then suspended in the diluted sludge at a con
centration of 4.0 x 10^ PFU/ml and applied to the
column. All solutions were applied continuously
to the column at approximately 5 ml/min using a
peristaltic pump (Buchler, Fort Lee, N.J.). The
anaerobically digested sludge (GDAN--see Table
3-2) used had a solids content of 2.0%, a con
ductivity of 3250 ymho/cm at 25C and a pH of 8.3.
The conductivity of sludge diluted (1:50) with
sludge liquor containing 0.01 N CaCl2 was 2500
ymho/cm at 25C and the pH was 7.5. The sludge
liquor was produced by centrifuging GDAN sludge
at 14,000 x g for 10 min at 4C. This procedure
was performed again on the decanted supernatant
and this yielded the clear sludge liquor (see
Table 4-2 for chemical parameters).


FIGURE 5-6. Daily soil temperature (2.5 cm below the soil surface of
a large core of Eustis fine sand) for the duration of
the survival experiment that began 11 October 1978
Soil temperature was measured at the experimental
site (i.e., next to the Environmental Engineering
Sciences building, University of Florida, Gaines
ville) using a thermocouple placed at the 2.5-cm
depth in a large soil core of Eustis fine sand
(LC4--see Figures 5-1 and 5-2). The soil tempera
ture was monitored every hour at the 2.5-cm depth,
as well as at the surface, 10-cm depth, and 20-cm
depth. From Table 4-3, it can be seen that all
temperature readings were made in the Ap horizon
of the soil. No significant difference was found
between temperature readings at the surface, 2.5-
cm depth, 10-cm depth, and 20-cm depth. Therefore,
only the soil temperature at the 2.5-cm depth is
reported.


17
enteric viruses. The indigenous virus titer of raw sludge has been
measured by several investigators and expressed in different units.
In raw primary sludge, indigenous enteric virus concentrations of 2.4
to 15 PFU/ml, <6.9 x 102 PFU/ml, 6.9 to 215 PFU/g dry wt. of total
suspended solids (TSS), 7.9 x 102 to 4.3 x 103 PFU/g dry wt. TSS and
10 to 1,000 50% tissue culture infective dose (TCID5Q)/ml were measured
by Oliver (1975), Nath and Johnson (1980), Turk et al_. (1980), Moore et
al. (1978) and Lund (1976), respectively. In mixtures of primary
(1/3) and secondary (2/3) raw sludge from the City of Los Angeles
Hyperion treatment plant, Berg and Berman (1980) found indigenous
enteric virus concentrations ranging from 3.8 to 116 PFU/ml. Nielsen
and Lydholm (1980) detected 0.1 to 9.0 TCID^/mg TSS of indigenous
enteric viruses in raw sludge (primary and secondary) from three
Danish wastewater treatment plants. Secondary sludges have been shown
to contain 10- to 100-fold less virus than primary sludges (Lund 1976;
Lund and R0nne 1973). Clearly, a wide range of virus concentrations is
found in raw sludges. The indigenous virus concentration detected in
raw sludge depends on the sludge type, geographical location, season
of the year (Berg and Berman 1980) and virus recovery method used (Nath
and Johnson 1980). Moreover, a large fraction of the indigenous
viruses in raw sludges have been shown to be associated with the sludge
solids (Cliver 1975; Lund 1976; Lund and R0nne 1973; Nath and Johnson
1980). Indigenous enteric viruses are strongly associated with fecal
solids in raw wastewater (Bitton 1980a; Cliver 1975; Cliver 1976;
Wellings et aj_. 1976a) and tend to remain associated with solids during


CHAPTER II
LITERATURE REVIEW
Viral Pathogens Found in Raw Wastewater
The pathogens found in raw wastewater fall into one of the
following four groups: bacteria, protozoa, helminthic parasites, and
viruses. Several reviews have appeared in the literature that address
all the pathogens found in sewage (Burge and Marsh 1978; Foster and
Engelbrecht 1973; Elliott and Ellis 1977). Herein, the emphasis will be
on the viral pathogens present in raw wastewater.
Over 100 types of viruses are found in raw wastewater (Bitton
1980b; Burge and Marsh 1978; Foster and Engelbrecht 1973; Elliott and
Ellis 1977). The most important virus groups are the enteroviruses
(i.e., polioviruses, coxsackieviruses and echoviruses), reoviruses,
adenoviruses, infectious hepatitis agent (viral hepatitis type AHa11
1977), and viral gastroenteritis agents [variously designated as duo-
viruses, rotaviruses, reovirus-1ike agents or Norwalk agent (parvo
virus)Chanock 1976]. These organisms cause such diseases as polio
myelitis, aseptic meningitis, myocarditis, enteritis, jaundice,
infectious hepatitis, and gastroenteritis (Burge and Marsh 1978; Foster
and Engelbrecht 1973).
Indigenous virus concentrations ranging from 500 to 80,000
plaque-forming units (PFU)/£ were measured by Buras (1974) in raw sewage
from Haifa, Israel. Dugan et^ al_. (1975) found between 27 and 19,000
4


25
digested sludge the uncharged form of ammonia which is virucidal for
enteroviruses. The presence of this virucide coupled with thermophilic
digestion temperatures (i.e., approximately 50C) results in the
rapid inactivation of enteroviruses in anaerobically digested sludge.
Furthermore, the protective effect towards enteroviruses attributed to
ionic detergents in the sludge solids and observed at low pH in raw
sludge is largely overcome by the virucidal ammonia in anaerobically
digested sludge (Ward and Ashley 1977c) particularly at thermophilic
temperatures. Reoviruses are also inactivated in anaerobically digested
sludge at thermophilic temperatures and at the naturally high pH values
attained by the digestion process (Ward and Ashley 1979).
As presented above, the inactivation of viruses during anaerobic
digestion has been studied in the laboratory using sludge artificially
contaminated with virus. The validity of such research has been ques
tioned because it is believed that, unlike indigenous viruses, the
seeded viruses become mostly adsorbed to the surface of sludge solids
(Moore et al_. 1977; Nielsen and Lydholm 1980). Due to their strong
association with fecal solids in raw wastewater (Bitton 1980a; Oliver
1975, 1976; Wellings et^ a]_. 1976a), indigenous viruses are believed to
end up, during wastewater treatment, mostly embedded within sludge solids
rather than merely surface adsorbed (Wellings et al_. 1976a). Conse
quently, Moore et a]_. (1977) proposed that indigenous viruses in sludge
are less susceptible than seeded viruses to the environmental stresses
(e.g., chemical and heat inactivation) encountered during sludge treat
ment by virtue of the former's more insulated environment. Evidence to


53
Table 3-1. Stocks of the virus were prepared by infecting monolayer
cultures of AV3 (a continuous line of human amnion), BGM (a continuous
line of Buffalo green monkey kidney--Barron et al_. 1970; Dahling et al.
1974) or MA-104 (a continuous line of fetal rhesus monkey kidney) cells
in a 32 oz (128 cm ) glass bottle (rubber-lined, screw-capped--Brockway
Glass Co., Inc.). After allowing an adsorption period of 60 minutes
with tilting at 15-minute intervals, the cells were overlaid with 40 ml
of Eagle's minimal essential medium (MEM) supplemented with 10% fetal
calf serum (FCS), 250 U/ml penicillin, and 125 ug/ml streptomycin (see
Appendix for more details on the composition of this and other media
used). After approximately 48 hours of incubation at 37C, the overlay
medium was decanted and then centrifuged at 270 x g for 15 minutes at
4C to remove cell debris. The resulting supernatant containing the
virus was distributed in 1, 2 or 5 ml aliquots and immediately frozen
at -70C. The virus was kept at -70C until used.
Poliovirus was assayed by the plaque technique (Cooper 1967) on
AV3, BGM or MA-104 cell monolayers prepared as follows. Confluent
cell monolayers were grown in 32 oz glass bottles using Eagle's MEM
supplemented with 10% FCS, 250 U/ml penicillin and 125 yg/ml strepto
mycin (i.e., growth medium; see Appendix). Each cell monolayer was
then washed three times with 10 ml of the pre-trypsin solution (see
Appendix). This treatment removes all traces of serum (contains
+2 +2
trypsin inhibitors), as well as Ca and Mg ions (enhance adsorption
of cells to glass). To remove the cells from the glass bottle, 10 ml
of the standard trypsin-versene solution (see Appendix) was then added


15
-3 -1
2.5 x 10 min were determined for MLSS concentrations of 1,590 and
3,140 mg/£, respectively (see Malina et aj_. 1975).
As far as indigenous viruses are concerned, much less work has
been done on their removal by the activated sludge process. Moore et
al. (1977, 1978) and Farrah et^ al_. (1981b) readily recovered indigenous
enteroviruses from the mixed liquor suspended solids of activated sludge
plants in Texas, Illinois, Montana and Oregon, and Florida, respectively.
Since in these three studies most of the viruses detected were directly
associated with the solids, it can be hypothesized that a large frac
tion of these viruses would be removed during subsequent secondary
sedimentation of the sludge solids. The effectiveness of the activated
sludge process in removing indigenous viruses from wastewater was
confirmed by Lund et al_. (1969) and Moore et^ al_. (1977) in field studies
in Denmark and the United States (Austin, Texas), respectively.
In.spite of the substantial virus removal capacity of the
activated sludge process, effluents from this treatment method routinely
contain demonstrable indigenous viruses (Buras 1974; Dugan et al.
1975; England et al_. 1965; Gilbert et al_. 1976a, 1976b; Merrell and
Ward 1968; Moore et al_. 1977; Vaughn et al_. 1978; Wellings et al.
1974, 1976a, 1978). Further treatment by chlorination (Dugan et al.
1975; England et^ a^. 1965; Merrell and Ward 1968; Vaughn et al_. 1978;
Wellings et al_. 1974, 1978), or by tertiary processes such as oxidation
pond, denitrification followed by sand filtration, or alum floccula
tion (England et al_. 1965; Merrell and Ward 1968; Vaughn et_ aj_. 1978;
Wellings et al_. 1978) often does not eliminate all indigenous viruses
from activated sludge effluents. In addition to viruses, secondary


97
in each column while allowing the free movement of viruses (i.e.,
did not adsorb viruses in soil leachates). The columns were packed
uniformly at a constant rate while gently tapping to prevent soil
subsidence (Drewry and Eliassen 1968). The field bulk densities of
3
1.45 g/cm for the Red Bay sandy loam (i.e., average for the A2 and
Bit horizons--see Table 4-3) and 1.56 g/cm for the Eustis fine sand
(i.e., average for the A21 and A22 horizons--see Table 4-3) were repro
duced in the soil columns by packing the appropriate grams of dry soil
3
in the measured volume of each column (i.e., 180.86 cm for the 10-cm
3
column and 488.33 cm for the 27-cm column). The soil columns were
then placed in soil column holders (supplied by Soil Moisture Equipment
Corp., Santa Barbara, California) as shown in Figures 4-2 and 4-3 for
10-cm and 27-cm columns, respectively. The air in the columns was then
displaced by flushing with carbon dioxide for approximately 60 minutes
in order to ensure subsequent uniform wetting of the soil. The soil
columns were then conditioned by passing 2 to 5 pore volumes of non-
seeded 0.01 N calcium chloride, distilled water, rain water, or sludge
liquor using a peristaltic pump (Buchler, Fort Lee, New Jersey). The
conditioning solution used was identical or similar to the test
solution. For example, soil columns ultimately receiving virus-seeded,
anaerobically digested sludge diluted (1:50, vol./vol.) with 0.01 N
calcium chloride were previously conditioned with 0.01 N calcium
chloride. For soil columns receiving undiluted sludge (e.g., chemical
sludge), rain water was used as the conditioning solution. Following
conditioning, poliovirus was suspended in 0.01 N calcium chloride, sludge


37
(Ward and Ashley 1978b). Hence, the greater protection from heat
inactivation afforded to poliovirus as the sludge solids content was
increased (Ward and Ashley 1978b). The ionic detergents in raw sludge
were shown to be substantially degraded during the composting process
and as a result, seeded poliovirus was heat-inactivated (39C or 43C)
at a greater rate in composted sludge than in raw sludge held at the
same sludge solids content (Ward and Ashley 1978b). Other enteroviruses
(e.g., poliovirus 2, coxsackievirus A13 and coxsackievirus Bl) have
also been shown to be protected from heat inactivation in dewatered raw
sludge (Ward and Ashley 1978b).
From the research presented above, it can be concluded that
substantial viral inactivation would occur, during the air drying of raw
sludge at 21C, only when sludge solids contents above 80% are achieved
(Ward and Ashley 1977b). In actual practice, however, such sludge
solids contents are rarely attained for raw sludge during sandbed drying
(U.S. Environmental Protection Agency 1974). Furthermore, raw sludge is
not routinely subjected to air drying because of the odors, insect
pests, unsatisfactory drying rate and other problems associated with
this practice (U.S. Environmental Protection Agency 1974). Thus, sand-
bed drying is normally restricted to well-digested sludge (U.S. Environ
mental Protection Agency 1974). It can be hypothesized that entero
viruses are probably more rapidly inactivated in anaerobically digested
sludge than in raw sludge when subjected to air drying. This is
because anaerobically digested sludge usually contains virucidal
ammonia (Ward and Ashley 1976, 1977a). This agent has been shown to


267
of Measurement and Sampling, pp. 374-390. Edited by C. A. Black,
D. D. Evans, J. L. White, L. E. Ensminger, F. E. Clark, and R. C.
Dinauer. American Society of Agronomy, Inc., Madison, Wiscon
sin.
Brady, N. C. 1974. The Nature and Properties of Soils. 8th edition.
Macmillan Publishing Co., Inc., New York, N.Y.
Breindl, M. 1971. The structure of heated poliovirus particles.
Gen. Virol. 11: 147-156.
Brown, K. W., S. G. Jones, and K. C. Donnelly. 1980. The influence of
simulated rainfall on residual bacteria and virus on grass
treated with sewage sludge. J. Environ. Qual. £: 261-265.
Brunner, D. R., and 0. J. Sproul. 1970. Virus inactivation during
phosphate precipitation. J. Sanitary Eng. Div., ASCE 96: 365-
379.
Buras, N. 1974. Recovery of viruses from waste-water and effluent by
the direct inoculation method. Water Res. 8: 19-22.
Burge, W. D., and N. K. Enkiri. 1978. Virus adsorption by five soils.
J. Environ. Qual. 1_: 73-76.
Burge, W. D., and P. B. Marsh. 1978. Infectious disease hazards of
landspreading sewage wastes. J. Environ. Qual. _7: 1-9.
Burge, W. D., and J. F. Parr. 1980. Movement of pathogenic organisms
from waste applied to agricultural lands. In Environmental
Impact of Nonpoint Source Pollution, pp. 107-124. Edited by
M. R. Overcash and J. M. Davidson. Ann Arbor Science Publishers,
Inc., Ann Arbor, Michigan.
Calhoun, F. G., V. W. Carlisle, R. E. Caldwell, L. W. Zelazny, L. C.
Hammond, and H. L. Breland. 1974. Characterization Data for
Selected Florida Soils. Soil Science Research Report No. 74-1.
Institute of Food and Agricultural Sciences, University of
Florida, Gainesville, Florida.
Carlson, G. F., Jr., F. E. Woodard, D. F. Wentworth, and 0. J. Sproul.
1968. Virus inactivation on clay particles in natural waters.
J. Water Pollut. Control Fed. 40: R89-106.
Chaney, R. L. 1980. Health risks associated with toxic metals in
municipal sludge. In SludgeHealth Ris-ksof Land Application,
pp. 59-83. Edited by G. Bitton, B. L. Damron, G. T. Edds, and
J. M. Davidson. Ann Arbor Science Publishers, Inc., Ann Arbor,
Michigan.


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103
into the soil to the desired depth according to the procedure of Blake
(1965). Care was taken not to compress the soil in the pipes during
sampling, and, thereby, to preserve the natural structure and packing
of the soil as nearly as possible in the pipes. This was accomplished
by ensuring that the elevation of the soil inside the pipes was the same
as the elevation of the surface soil outside the pipes during sampling
(Blake 1965). The soil around the pipes was then removed with a shovel
and the soil cores were gently removed. In the laboratory, soil ex
tending beyond the bottom end of each core was trimmed with a spatula.
A polypropylene screen (105-ym pore sizesee above) and a spout were
secured at the bottom of each undisturbed soil core. Red Bay sandy
loam undisturbed cores were sampled at the West Florida Agricultural
Experiment Station at Jay. These soil cores were 54 cm in length
(pipes were 60 cm in length) and had an internal diameter of 5.0 cm,
and,thereby, consisted of the Al, A2, Bit and B21t horizons of the sandy
loam (see Table 4-3). The Red Bay sandy loam cores were not conditioned
prior to use. Eustis fine sand undisturbed cores were sampled at
the agronomy farm, University of Florida, Gainesville. These soil
cores were 33 cm in length (pipes were 40 cm in length) and had an in
ternal diameter of 5.0 cm, and thereby,consisted of the Ap and A21 hori
zons of the fine sand (see Table 4-3). The Eustis fine sand cores were
conditioned with either 5 pore volumes of rain water or 0.01 N calcium
chloride. All undisturbed soil cores were then treated with undiluted
anaerobically digested sludge (GDANsee above) which had been seeded
with poliovirus. The poliovirus concentrations in the anaerobically


27
Although the anaerobic digestion process appears capable of
removing considerable quantities of viruses from sludge, a fraction of
the viruses initially present will, nevertheless, survive this diges
tion process [see reviews by Berg (1973a), Bitton (1978), Oliver
(1976), Foster and Engelbrecht (1973), and Moore et al_. (1978)].
Indigenous viruses have been routinely detected in anaerobically
digested sludge (Berg and Berman 1980; Oliver 1975; Farrah et al_. 1981a
Moore et al_. 1978; Nielsen and Lydholm 1980; Palfi 1972; Sattar and
Westwood 1979; Sagik et al_. 1980; Turk et al_. 1980; Wellings et al.
1976a). Moreover, the indigenous virus titer of this sludge type has
been measured by several investigators and expressed in different
units. Anaerobically digested sludge sampled at various locations
throughout the United States displayed indigenous enteric virus con
centrations of 0 to 8 PFU/ml, <0.014 to 4.1 PFU/ml, 7 to 40 PFU/g
dry wt. TSS, 1.1 to 17 PFU/g dry wt. TSS, 0.2 to 17.0 PFU/g dry wt. TSS
and 2 to 7 TCID^g/g dry wt. TSS as measured by Oliver (1975), Berg and
Berman (1980), Moore et ajk (1978), Sagik et a/h (1980), Turk et al.
(1980) and Farrah et_ al_. (1981a), respectively. Nielsen and Lydholm
(1980) found 0 to 600 TCID^g/g dry wt. TSS of indigenous enteric
viruses in anaerobically digested sludge from three Danish wastewater
treatment plants. Evidently, the indigenous virus concentration
detected in anaerobically digested sludge depends on the virus con
centration in the raw sludge, and thereby, on the geographical location
(Sagik et al_. 1980) and on the season of the year (Berg and Berman
1980; Moore et al_. 1978). The indigenous virus titer found in


239
ultracentrifugation at 120,000 x g for 2 hours at 5C in a Tl-60 rotor
using a Beckman model L3-50 ultracentrifuge (Beckman Instruments,
Fullerton, California). The pellets produced were suspended in 1 ml of
FCS. The concentrated samples were sterilized by passage through
0.25-ym Filterite filters in 13-mm holders and were then assayed for
viruses as described below.
Groundwater. Groundwater from a 60-ft (ca. 18-m) well on the
Kanapaha site (see Figure 6-2) was monitored for indigenous entero
viruses. Each groundwater sample of 100 gallons (ca. 384 liters) was
hand pumped into a 100-gallon tank and was concentrated by membrane fil
tration (Farr ah et aU 1976; Hill et al_. 1971; Shuval and Katzenelson
1972; Sobsey et^ al_. 1973; Sobsey eit aj^. 1980b) in the field (see
Figure 6-5) as follows. The water was adjusted to pH 3.5 by the
addition of 0.2 N HC1 and adjusted to 0.0005 M aluminum chloride. The
treated water was then passed through a 10-in (ca. 25-cm), 0.25-ym
pore size Filterite filter. The filter was then treated with 800 ml
of 0.05 M glycine buffer, pH 11.5. The glycine solution was permitted
to remain in contact with the filter for 1 minute, was removed and
then was adjusted to neutral pH by the addition of 1 M glycine buffer,
pH 2.0. The neutralized sample was transported to the laboratory, and
within 1 hour, it was adjusted to pH 3.5 by the addition of 1 M glycine
buffer, pH 2.0, and passed (without prior centrifugation) through a
series of 3.0- and 0.45-ym Filterite filters in a 47-mm holder. Ad
sorbed viruses were eluted from the filters with 7 ml of PBS contain
ing 10% FCS, pH 9.0. The filter eluate was adjusted to neutral pH by


104
digested sludge samples and the degree of poliovirus association with
the sludge solids were determined as described earlier in this chapter.
Following viral assays, one inch or 2.5 cm (51.6 ml) of poliovirus-
seeded sludge was applied to each soil core, allowed to soak in and
then was worked under 2.5 cm. In one experiment with two Red Bay sandy
loam cores, the applied sludge was allowed to air dry for 24 hours
(the cores were placed on the roof of the Environmental Engineering
Sciences building, University of Florida, Gainesville) before being
worked under 2.5 cm. Following incorporation of the sludge solids into
the top inch of soil, the soil cores were eluted with either 0.01 N
calcium chloride or rain water. These solutions were applied from
inverted, self-regulated, 1-liter Erlenmeyer flasks set to maintain
a 2.5-cm hydraulic head on the cores (Sanks et al_. 1976). After perco
lation through the soil, leachates from the undisturbed soil cores were
collected in sterile screw-capped bottles and assayed for viral
infectivity as described below. The percolation rate of fluid through
each undisturbed soil core was determined by measuring the time required
to collect a known volume of leachate.
Leachates from transport studies. The leachates from
laboratory-packed soil columns and undisturbed soil cores were collected
in pore volumes or fractions of pore volumes. The pore volume of each
soil core (or column) is the volume within the core which is not
occupied by the soil particles (Brady 1974) and is determined by first
calculating the percent pore space of the soil as shown below:
pore space (%) = 100 -
bulk density lnn
particle density
(4-1)


Soil temperature (C)
207


I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
George E. Gifford
Professor of Immunology and Medical Microbiology
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy,
^5 O^nnoujJl.
Samuel R. Farrah
Associate Professor of Microbiology and
Cell Science
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy,
't'V /v O U
Allen R. Overman""
Professor of Agricultural Engineering


115
TABLE 4-5. Characteristics of the 10-cm columns of Red Bay sandy
loanusubsoil packed at bulk densities of 1.45 and 1.60
g/cnr
Bulk density9
Particle density13
Pore space0
n ; d
Pore volume
(g/cm3)
(g/cm3)
(*)
(ml)
1.45
2.60
44.2
79.9
1.60
2.60
38.5
69.6
aThe sample of Red Bay sandy loam subsoil used consisted mainly of
the A2 and Bit horizons (see Table 4-3). The bulk densities shown were
produced in the soil columns (i.e., 10 cm in length and 4.8-cm internal
diameter) by packing the appropriate grams of air-dried soil in the
column volume.
bThis particle density value is typical for mineral soils (Brady
1974).
p
Calculated using Equation (4-1).
Calculated using Equation (4-2).


Orangeburg *- * Lucie * -* Troup
234
480 ft
72
71
70
69
68
67
66
65
64
63
62
61
A
3
6
15
0
9
12
0
3
6
9
m
12
15
49
50
51
52
53
54
55
56
57
58
59
60
15
9
12
3
0
6
12
15
6
9
3
0
48
47
46
45
44
43
42
/41
40
39
38
37
3
6
0
15
9
12 c
r J
3
6
9
12
15
25
26
27
28
29
<
4
47
#30
31
32

33
34
35
36
15
9
12
3
6
12
15
6
9
3
0
24
23
22
21
20
19
18
17
16
15
14
13
3
6
0
15
9
12
0
3
6
9
12
15
1
2
3
4
5
6
7
8
9
10
11
12

15
9
12
3
0
6
12
15
6
9
3
0
-
%
4k Soils monitored in this study
Wells (40-60 ft) monitored by Farrah eta]_. (1981)
720 ft


67
Viral assays were performed at various steps in the procedure in order
to determine the efficiency of the individual steps. Each "overall
virus recovery" value reported was determined from the viruses recovered
in the filter concentrate, pellet concentrate and sludge supernatant
(concentrated or not).
Statistical Treatment of Data
Statistical treatment of the data was performed with the use of
a Hewlett-Packard calculator model 9810A and Statistics Package V-6
(Hewlett-Packard Company, Loveland, Colorado).
Results and Discussion
Association of Seeded Poliovirus
with Sludge Solids
Poliovirus seeded in the various sludge samples rapidly became
associated with the sludge solids. However, no statistically signifi
cant linear correlation was found between the percent solids contents ..
of the sludges studied and the degree of virus association by the sludge
solids (Table 3-4). This lack of correlation was found within each
sludge type and for all sludge types combined. Thus, the sludge solids
content was shown not to affect the association of virus with sludge
solids, at least in the range of solids contents studied (i.e., 0.5%
through 2.9%). This allowed sludges of different solids contents, but
belonging to the same sludge type, to be grouped together in the same
category.
The mean percent of solids-associated viruses for activated
sludge mixed liquors, anaerobically digested sludges, and aerobically


238
any possible cytotoxicity and were then assayed for viruses as described
below.
Composite topsoil samples (100 wet grams) were obtained from
the plots shown in Figure 6-4 at the Jay site. Indigenous enteroviruses
were recovered from these soil samples using the procedure described
by Bitton et (1979a). This method consisted of mixing each 100-g
sample of wet soil with 200 ml of 0.5% (wt./vol.) isoelectric casein
(Difco Laboratories, Detroit, Michigan), pH 9.0. If necessary, the pH
of the mixture was adjusted to between 9.0 and 9.2 by the addition of
5 M Trizma base (Sigma Chemical Co., St. Louis, Missouri). The samples
were vigorously shaken by hand for 30 seconds and then shaken on a
rotating shaker for 15 minutes. The samples were subsequently centri
fuged at 1400 x g for 4 minutes at 4C. The supernatants (i.e., the
soil eluates) were recovered and immediately adjusted to neutral pH
by the addition of 1 M glycine buffer, pH 2.0. Viruses in the soil
eluates were concentrated by organic flocculation (Katzenelson et al.
1976b) as follows. The eluates were adjusted to pH 4.4 by the addition
of 1 M glycine buffer, pH 2.0. The floes produced were pelleted by
centrifugation at 160 x g for 1 minute at 4C. The supernatants were
discarded. The pellets were mixed with 2 ml of 0.15 M Na^HPO^, pH 9.0.
The mixtures were adjusted to neutral pH by the addition of 1 M glycine
buffer, pH 11.5, and then magnetically stirred until the pellets were
completely resolubilized. The samples were subsequently centrifuged
at 14,000 x g for 10 minutes at 4C. The supernatants were adjusted to
neutral pH (i.e., if necessary) and FCS was added to a final concentra
tion of 2%. Viruses in these samples were further concentrated by


109
(see Table 4-3). In the laboratory-packed columns, the field bulk
densities were accurately reproduced as detailed earlier. The undis
turbed soil cores were obtained in a manner that preserved the natural
structure and bulk density of the soil (see page 102 above). In fact,
undisturbed soil cores are frequently taken in order to measure the
field bulk density of a soil (Blake 1965). However, the soil in
undisturbed cores can be compressed during sampling and thereby result
in an increase in bulk density as compared to the field bulk density
(Blake 1965; Funderburg et al_. 1979). For example, compression of the
soil is likely to occur when the soil is wet during sampling (Blake
1965). Although the undisturbed cores used in this study were carefully
sampled so as to not compress the soil, it was deemed important to
determine what effect, if any, compression of the soil (i.e., increase
in bulk density) might have on the transport of poliovirus. Laboratory-
packed soil columns of Red Bay sandy loam subsoil (consisted mainly of
the A2 and Bit horizons--see Table 4-3) were used in these experiments
and they were prepared at different bulk densities as described below.
Soil moisture content-bulk density curve. If the compactive force
is held constant, the density to which a given soil can be compacted
increases with a corresponding increase in the soil moisture content
up to the optimum moisture level (Felt 1965). Increases in soil
moisture content beyond this level result in reductions in the soil
bulk densities achieved (Felt 1965). A soil moisture content-bulk
density curve was produced for the Red Bay sandy subsoil using the pro
cedure of Wilson (1950). Briefly, the procedure consisted of adjusting


90


21
Stabilization-digestion. Raw sludges are frequently stabilized
using anaerobic (Fair et al_. 1968; U.S. Environmental Protection Agency
1974, 1978a) or aerobic (U.S. Environmental Protection Agency 1974,
1978a; Yates 1977) digestion. These digestion processes reduce the
odor, putrescibility potential and pathogen content of raw sludges
(U.S. Environmental Protection Agency 1974, 1978b) and thereby achieve
sludge stabilization (see Figure 2-1).
There are several different design-types of anaerobic sludge
digestion and they employ either no heating, heating at mesophilic
(30-35C) temperatures, or heating at thermophilic (approximately 50C)
temperatures (U.S. Environmental Protection Agency 1974). In general,
the rate of sludge stabilization increases as the anaerobic digestion
temperature is increased. Consequently, the sludge digestion time
is usually reduced as the anaerobic digestion temperature is increased
(U.S. Environmental Protection Agency 1974).
Several authors have reviewed the literature on viral inactiva
tion during anaerobic digestion of raw sludges (Berg 1973a; Bitton 1978;
Oliver 1976; Foster and Engelbrecht 1973; Moore et aj_. 1977, 1978). In
laboratory and field studies, enteroviruses have been shown to be in
activated during anaerobic digestion (Bertucci et al_. 1977; Berg and
Berman 1980; Oliver 1975; Eisenhardt et al_. 1977; Moore et aj_. 1977,
1978; Nielsen and Lydholm 1980; Palfi 1972; Sattar and Westwood 1979).
During anaerobic digestion (35C) of sludge in laboratory-scale units,
Eisenhardt et al_. (1977) measured the inactivation rate of seeded


272
Gerba, C. P., and J. C. Lance. 1978. Poliovirus removal from primary
and secondary sewage effluent by soil filtration. Appl.
Environ. Microbiol. 36: 247-251.
Gerba, C. P., C. Wallis, and J. L. Mel nick. 1975. Fate of wastewater
bacteria and viruses in soil. J. Irrigation and Drainage Div.,
ASCE 101: 157-174.
Gilbert, R. G., C. P. Gerba, R. C. Rice, H. Bouwer, C. Wallis, and
J. L. Mel nick. 1976a. Virus and bacteria removal from waste-
water by land treatment. Appl. Environ. Microbiol. 32: 333-338.
Gilbert, R. G., R. C. Rice, H. Bouwer, C. P. Gerba, C. Wallis, and
J. L. Melnick. 1976b. Wastewater renovation and reuse: Virus
removal by soil filtration. Science 192: 1004-1005.
Gilcreas, F. W., and S. M. Kelly. 1955. Relation of coliform-organism
test to enteric-virus pollution. J. Am. Water Works Assoc. 47:
683-694.
Glass, J. S., R. J. Van Sluis, and W. A. Yanko. 1978. Practical
method for detecting poliovirus in anaerobic digester sludge.
Appl. Environ. Microbiol. 35: 983-985.
Goyal, S. M., and C. P. Gerba. 1979. Comparative adsorption of human
enteroviruses, simian rotavirus, and selected bacteriophages
to soils. Appl. Environ. Microbiol. 38: 241-247.
Grabow, W. 0. K. 1968. The virology of waste water treatment. Water
Res. 2: 675-701.
Grabow, W. 0. K., I. G. Middendorff, and N. C. Basson. 1978. Role of
lime treatment in the removal of bacteria, enteric viruses
and coliphages in a wastewater reclamation plant. Appl. Environ.
Microbiol. 35: 663-669.
Green, K. M. 1976. Sand Filtration for Virus Purification of Septic
Tank Effluent. Doctor of Philosophy Thesis, University of
Wisconsin, Madison, Wisconsin.
Hahn, E. E. A. 1972. Polioviruses. In Strains of Human Viruses,
pp. 155-176. Edited by M. Majer and S. A. Plotkin. S. Karger,
New York, N.Y.
Hall, W. T. 1977. Identification, Isolation and Characterization of
the Infectious Hepatitis (Hepatitis A) Agent. EPA-600/1-77-
049. Health Effects Research Laboratory, U.S. Environmental
Protection Agency, Cincinnati, Ohio.
Hamann, S. D. 1963. The ionization of water at high pressures, J.
Phys. Chem. 67: 2233-2235.


Mean monthly air temperature (F)
FIGURE 6-6. Weather data for the 'West Florida Agricultural Experiment Station, Jay, Florida
Data were collected at the station and qan be converted to metric units
with the following: C = 5/9 (F 32) and 1 in = 2.54 cm.
20
15
10
5
0
Total monthly precipitation (inches)


41
lagooning substantially reduces the viral content of digested sludge,
indigenous enteric viruses have, nevertheless, been routinely detected
in lagooned sludge undergoing land application (Farrah et al_. 1981a;
Sattar and Westwood 1979; Turk et_ al_. 1980). Consequently, all lagooned
sludges should be handled with care during final disposal in order to
avoid possible viral hazards (Sattar and Westwood 1979).
Heat drying and reduction. Numerous heat drying and reduction
processes (see Figure 2-1) are currently used for the removal of water
from and for the reduction in the volume of sludge (U.S. Environmental
Protection Agency 1974, 1978b). Reduction processes also destroy a
major portion of sludge solids (U.S. Envrionmental Protection Agency
1974). Due to the extremely high temperatures employed in these
treatment processes, all pathogens, including enteric viruses, in
sludge are destroyed (U.S. Environmental Protection Agency 1974, 1978b).
Rising energy costs, however, are making these sludge treatment
processes impractical (U.S. Environmental Protection Agency 1974, 1978b).
It should be pointed out that the heat-dried sludge or ash produced
by the drying or reduction processes, respectively, require final dis
posal .
Sludge irradiation. The treatment of sludge with ionizing
radiation has recently been shown to be highly effective in destroying
the pathogens, including enteric viruses, present in sludge (see
review by Bitton 1980b). In particular, thermoradiation (i.e., ionizing
radiation combined with moderate heat) has been demonstrated by Ward
(1977) to rapidly inactivate poliovirus type 1 (CHAT) seeded in raw


ACKNOWLEDGEMENTS
The author would like to acknowledge and sincerely thank the
chairman of his doctoral committee, Dr. Gabriel Bitton, for his wisdom,
patience and encouragement during the course of this study, and for his
assistance in developing this dissertation. The author is also grateful
to the other members of the committee, Dr. Thomas L. Crisman, Dr. Dale
A. Lundgren, Dr. George E. Gifford, Dr. Samuel R. Farrah, and Dr. Allen
R. Overman, for the advice and guidance they extended to him.
The author also expresses his thanks to other faculty members,
including Dr. James M. Davidson and Dr. John Cornell, for their advice
on various phases of this study.
The author is indebted to Mr. Orlando Lanni for his excellent
technical assistance.
The assistance of Mr. Albert White, Kanapaha wastewater treatment
plant, City of Gainesville, Florida, is acknowledged. The author also
wishes to thank Dr.Gerald H. El kan, Department of Microbiology, North
Carolina State University, Raleigh, for the loan of the hydrostatic
pressure chamber.
Special thanks are extended to fellow students, in particular, Mr.
Phillip R. Scheuerman, for their insights during the course of this study.
This work was supported by grant No. R804570 from the United States
Environmental Protection Agency.
Finally, the author acknowledges with gratitude his wife, Ambrosina,
for her understanding and patience during his graduate study.


FIGURE 4-11. Movement of poliovirus type 1 suspended in primary
wastewater effluent through 10-cm columns of Red Bay
sandy loam subsoil packed at bulk densities of 1.45
and 1.60 g/cm3
One pore volume for the columns packed at a bulk
density of 1.45 or 1.60 g/cm^ equals 80 or 70 ml,
respectively (see Table 4-5). The laboratory-
packed columns were 10 cm in length and 4.8-cm
internal diameter. The sample of Red Bay sandy
loam subsoil used consisted mainly of the A2 and
Bit horizons (see Table 4-3). The columns were
conditioned with 2 pore volumes of nonseeded
primary wastewater effluent. Poliovirus was then
suspended in the primary wastewater effluent at
the concentrations shown in the figure in paren
theses and applied to the columns. All solutions
were applied continuously to the columns at
approximately 3.5 ml/min using a peristaltic pump
(Buchler, Fort Lee, N.J.). The primary wastewater
effluent sample used displayed a pH of 7.5 and a
conductivity of 340 ymho/cm at 25C. The mean pH
and conductivity (ranges indicated by vertical
lines) of each pore volume were calculated from
the individual values obtained for the 6 columns.


151
TABLE 4-15. Retention of poliovirus type 1 by undisturbed cores of
Red Bay sandy loam following the application of 2.5 cm
of seeded anaerobically digested sludge (air dried 24
hrs) and the subsequent elution with rain water
No. of Poliovirus l of Total Conductivity pH of pore
pore eluted PFU applied*3 of pore volume volume
volumes3 (total PFU) (cumulative) collected collected
eluted (ymho/cm
at 25C)
Column 1
1/3
0
0
245
4.4
2/3
0
0
195
4.7
1
0
0
125
4.8
1 1/3
0
0
106
5.1
1 2/3
1.1 x 103
0.2
80
5.2
2
0
0.2
82
5.1
2 1/3
0
0.2
77
5.1
2 2/3
0
0.2
72
5.0
3
0
0.2
66
5.0
3 1/3
0
0.2
66
5.0
Column 2
1/3
5.2 x 10
0.1
232
4.8
2/3
0
0.1
177
5.2
1
0
0.1
157
5.5
1 1/3
0
0.1
115
6.0
1 2/3
0
0.1
98
5.6
2
0
0.1
87
5.4
2 1/3
0
0.1
75
5.6
2 2/3
0
0.1
69
5.5
3
0
0.1
62
5.4
3 1/3
0
0.1
58
5.5


23
raw sludge adjusted to pH values above 7.5. Moreover, this agent was
shown to be present mainly in the sludge supernatant (i.e., produced
by centrifuging anaerobically digested sludge at 18,000 x g for 20
minutes) rather than in the sludge solids (Fenters et al_. 1979; Ward
and Ashley 1976). Microbial activity in anaerobically digested sludge
supernatant was found not to affect the inactivation rates of seeded
poliovirus 1 (Sabin), echovirus 6 and coxsackievirus B4 (Fenters et al.
1979). The mechanism of inactivation of poliovirus type 1 (CHAT) in
anaerobically digested sludge was found to be cleavage of the two
largest viral coat proteins (i.e., breakdown of VP-1 and VP-2) followed
by nicking of the encapsulated RNA (Ward and Ashley 1976). At the
thermophilic temperature of 43C, the inactivation rate of seeded
poliovirus type 1 (CHAT) was significantly lower in raw and anaerobi
cally digested sludge than in phosphate-buffered saline (PBS) (Ward
et al_. 1976). Ward et al_. (1976) proposed that poliovirus was protected
from heat inactivation by a component found in the sludge solids. In
the case of anaerobically digested sludge, however, this protective
effect was always less than that observed for raw sludge and was
largely reversed at higher temperatures (i.e., 47 and 51C) due to the
presence of the virucidal agent, uncharged ammonia (Ward et aj_. 1976).
Thus, at the higher temperatures, the inactivation rates of poliovirus
in anaerobically digested sludge were similar to those in PBS (Ward
et al_. 1976). Ward et al_. (1976) also demonstrated that enteroviruses
in anaerobically digested sludge were irreversibly inactivated (RNA
molecule hydrolyzed) during heating at temperatures of approximately


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
FATE OF VIRUSES FOLLOWING SEWAGE SLUDGE
APPLICATION TO SOILS
By
Oscar Carlos Pancorbo
August, 1982
Chairman: Gabriel Bitton
Major Department: Environmental Engineering Sciences
In recent years, land disposal of sewage sludge has been viewed
as a viable alternative to other disposal practices. However, there
is growing concern over the contamination of groundwater and surface
waters with microbial pathogens, particularly viruses, present in
digested sludge. The major objective of this study was to assess the
potential health risk, from viral pathogens, of sludge application to
soils.
Poliovirus type 1 (LSc) was found to be largely associated with
digested, conditioned-dewatered, chemical and lime-stabilized, chemical
sludge solids. Sludge type was found to affect, however, the degree
of association between seeded poliovirus and sludge solids. For
example, the degree of association between poliovirus and sludge solids
vi 1


T79
transport in sludge-amended soils and noted that viruses were completely
retained by the soils under study.
In this chapter, the survival and transport of viruses in
sludge-treated soils were evaluated under field conditions. Undisturbed
soil cores were used and environmental parameters (i.e., soil tempera
ture, soil moisture, and rainfall) were monitored. The protocol of
sludge disposal to soil was similar to that practiced at sludge dis
posal sites. Virus survival and transport were monitored during three
different runs, using the same soil cores.
Materials and Methods
Viruses and Viral Assays
Poliovirus type 1 (strain LSc) and echovirus type 1 (strain
Farouk-prototype strain according to Wulff and Chin 1972) were used in
the research reported in this chapter. Some general properties of
echoviruses are shown in Table 5-1 (see Table 3-1 for general proper
ties of polioviruses). Stocks of echovirus were prepared as described
for poliovirus in Chapter III (see page 53 ). Viral stocks were con
centrated by either ultracentrifugation or by the method developed by
Farrah et al_. (1978) that involved blending with trichlorotrifluoro-
ethane (Freon 113, DuPont DeNemours Co., Wilmington, Delaware) followed
by concentration on Filterite filters (Filterite Corp., Timonium, Mary
land). The concentrated viruses were kept at -70C until used. Echo-
virus was assayed by the plaque technique as described for poliovirus
in Chapter III (seepages 53-56 ). Each viral count shown represents


Sludge treatment at the Main Street wastewater
Kanapaha sludge disposal site treatment plant, Gainesville, Florida
;2;26
Wasted sludge
From the activated sludge unit
y
Aerobic digestion
Two digesters in series with total de
tention time of approximately 180 days
(90 days each)
y
Sludge conditioning
Using 1200 mg/2, of the cationic polymer,
Hercofloc #871 (Hercules Co., Atlanta,
Georgia)
\
t
Sludge dewatering
By centrifugation at 1400 rpm for 10
minutes
N
f
Sludge di
Application to 10 acr
land. Site charact
1. Soil belongs to
2. 1.27 cm/min pe
3. 50 ft (ca. 15 i
4. 128 cm mean ann
sposal
es (4.05 ha) of
eristics:
Lochloosa series
rcolation rate
n) to water table
jal rainfall
y
Sludge application procedure'""
The conditioned-dewatered sludge was
spread on the soil and immediately
disced into the soil. In the presence
of a cover crop, the sludge was applied
as a top dressing on the crop.


FIGURE 4-3. Laboratory-packed soil columns (27 cm in length) used in poliovirus transport studies
The soil columns (4.8 cm internal diameter) were packed as described in the
Materials and Methods section, and were then placed in soil column holders.
The soil column holders were supplied by the Soil Moisture Equipment Corp.
(Santa Barbara, California). Leaching solutions were applied to the soil
columns using a peristaltic pump (Buchler, Fort Lee, N.J.) as shown.


56
or neutral red (see Appendix). Plaques were subsequently counted with
the unaided eye as suggested by Cooper (1967). In several experiments,
the plaques were counted using an Omega photographic enlarger B22
(Simmon Brothers, Woodside, New York). Each tabulated viral count
represents the average of triplicate counts. The numbers of viruses were
expressed as plaque-forming units (PFU).
Sludges
A variety of wastewater sludges was used in this study as listed
in Table 3-2. The sludges were obtained from four wastewater treatment
plants located in Gainesville and Pensacola, Florida. In addition, a
lagooned sludge sampled at the West Florida Agricultural Experiment
Station (Jay, Florida) was also used. The treatments the sludges
received before being sampled are also shown in Table 3-2. One of the
sludge types used was activated sludge mixed liquor. The term "mixed
liquor" refers to the suspension undergoing treatment in an activated
sludge unit. The abbreviated sludge designations (see Table 3-2) will
be used to identify sludges in the rest of this dissertation. The
sludges were collected in sterile Nalgene bottles, transported to the
University of Florida (Gainesville) laboratory and then immediately
refrigerated. All sludge samples were used within 30 days of sampling
and most samples were used within 3 days. At the time of use, a
sludge sample was first allowed to come to room temperature. The pH
and solids content of the sludge was then determined. The pH was
measured using a digital pH meter model 125 from Corning (Corning, New
York). The solids content was determined by drying in an oven at 105C


242
the addition of 1 M glycine buffer, pH 2.0, and assayed for viruses
as described below.
Viral Assays
Samples were serially diluted, if necessary, in PBS containing
2% FCS (see Appendix for more details on the composition of this
solution) and then assayed for indigenous enteroviruses on BGM cell
cultures prepared as described in Chapter III (see pages 53 and 55).
Inoculated cell cultures were examined for cytopathic effects for
up to three weeks. Cell cultures showing cytopathic effects were
passed and viral isolates were titered according to the procedures
of Farrah et al_. (1981a). The 50% tissue culture infective dose
(TCID50) was determined for samples containing indigenous entero
viruses.
Weather Data
Kanapaha site. Weather data were not available for this sludge
disposal site.
Jay site. Weather data were collected at the West Florida
Agricultural Experiment Station, Jay, Florida, and kindly provided by
the station's staff. Mean monthly air temperature (maximum and mini
mum) and total monthly precipitation from September 1977 through March
1979 are reported in Figure 6-6.


FIGURE 5-4. Daily soil temperature (2.5 cm below the soil surface of
a large core of Eustis fine sand) for the duration of
the survival experiment that began 2 June 1978
Data were measured at the experimental site (i.e.,
next to the Environmental Engineering Sciences
building, University of Florida, Gainesville)
using a thermocouple placed at the 2.5-cm depth in
a large (33 cm in length and 15.5 cm internal
diameter) undisturbed soil core of Eustis fine sand
(see Table 4-3 ; consists of the Ap and A21 hori
zons of this soil). The soil temperature was
monitored every hour at the 2.5-cm depth, as well
as at the surface, 10-cm depth, and 20-cm depth.
From Table 4-3 it can be seen that all tempera
ture readings were made in the Ap horizon of the
soil. No significant difference was found between
temperature readings at the surface, 2.5-cm depth,
10-cm depth, and 20-cm depth. Therefore, only
the soil temperature at the 2.5-cm depth is
reported.


264
0.2 grams KH2PO4
1000 ml deionized water
Autoclave.
10. PBS, containing 2% fetal calf serum at pH 7.4 (this solution was
also used to make virus dilutions.).:
To 490 ml of PBS, add the following aseptically:
10 ml fetal calf serum (International
Sci.)
0.5 ml phenol red stock (0.5%)
0.5 ml streptomycin-penicillin stock
(lOOOx)
11. NaCl. stock (used to bring an undiluted sample to isotonicityi.e.
0.85 grams NaCl per 100 ml):
17.0 grams NaCl
100 ml deionized water
Autoclave. Dilute 1/20 in final sample.


66
(LSc) seeded in 0.05 M glycine buffer, pH 10.5 to 11.0. Therefore, it
is believed that there was no significant inactivation of poliovirus
during the elution procedure. It should be noted, however, that this
elution method is not practical for the recovery of reoviruses and
rotaviruses from sludge. These virus types are rapidly inactivated
when subjected to such high pH values (Sobsey et al_. 1980b). The
viruses in the sludge solids eluates were concentrated by organic
flocculation (Katzenelson et a]_. 1976b) as follows. The eluates were
adjusted to pH 3.5 by the addition of 1 M glycine buffer, pH 2.0, and
the floes produced were pelleted by centrifugation at 1,400 x g for
20 minutes at 4C. The supernatants and pellets produced were treated
separately. The supernatants were assayed for viruses and then passed
through a series of 3.0, 0.45, and 0.25 pm Filterite filters (Filterite
Corp., Timonium, Maryland) in a 47-mm holder. Adsorbed viruses were
eluted from the filters with 7 ml of PBS containing 10% FCS, pH 9.0.
The filter eluates were adjusted to neutral pH by the addition of 1 M
glycine buffer, pH 2.0, and assayed for viruses. The filtrates (i.e.,
the fluids having passed the filters) were adjusted to neutral pH by
the addition of 1 M glycine buffer, pH 11.5, and assayed for viruses.
The pellets previously obtained by centrifuging the samples at pH 3.5
were mixed with five volumes of PBS containing 10% FCS, pH 9.0. The
mixtures were adjusted to pH 9.0 by the addition of 1 M glycine buffer,
pH 11.5, vortexed for 30 seconds and then centrifuged at 14,000 x g
for 10 minutes at 4C. The supernatants were adjusted to neutral pH
by the addition of 1 M glycine buffer, pH 2.0,, and assayed for viruses.


FIGURE 5-
. Hourly soil temperature (2.5 cm below the soil surface
of a large core of Eustis fine sand) profile for
17 June 1978
Soil temperature was measured at the experimental
site (i.e., next to the Environmental Engineering
Sciences building, University of Florida, Gaines
ville) using a thermocouple placed at the 2.5-cm
depth in a large soil core of Eustis fine sand
(LC4see Figures 5-1 and 5-2). The soil tempera
ture was monitored every hour at the 2.5-cm depth,
as well as at the surface, 10-cm depth, and 20-cm
depth. From Table 4-3, it can be seen that all
temperature readings were made in the Ap horizon
of the soil. No significant difference was found
between temperature readings at the surface, 2.5-
cm depth, 10-cm depth, and 20-cm depth. Therefore,
only the soil temperature at the 2.5-cm depth is
reported. The average temperature for the day was
24.2C. The maximum temperature was 30.8C and
it occurred at 2 P.M. The minimum temperature was
18.9C and it occurred at 6 A.M.


155
from sludge solids as a result of changes in the physico-chemical
properties within the soil matrix--see Table 3-6) will move in the
soil solution or be retained by the soil particles as govered by pH,
flow rate, conductivity, and the presence of soluble organic materials
(Bitton 1975; Gerba et al_. 1975). Of particular importance in the
retention of these "free" viruses by the soil is the nature of the
soil itself and, more specifically, the clay content of the soil. Due
to their large surface area, the clay minerals in soils comprise the
fraction most active in retaining viruses (Carlson et al_. 1968). The
Red Bay sandy loam studied displayed a clay content ranging from 13.6%
in the A1 horizon to 36.2% in the B21t horizon (see Table 4-3). The
dominant clay in the Al, A2, and Bit horizons was vermiculite, while in
the B21t horizon, it was gibbsite. The retention of "free" poliovirus
during sludge application to the Red Bay sandy loam is attributed to
the adsorptive capacity of the clay fraction found throughout the soil
profile and accumulated in the deeper horizons (e.g., Bit and B21t).
Moreover, this soil contained iron oxides which are also effective in
retaining viruses (Bitton 1980a). The interaction between iron oxides
and viruses in soils deserves further study.
Eustis fine sand. Similar application of sludge to an undis
turbed core of Eustis fine sand and subsequent elution with rain water
resulted in the breakthrough of 29.3% of the total (5.7 x 105 PFU)
poliovirus applied (see Figure 4-9). A peak in conductivity (400
umho/cm at 25C) was found at the 1.5 pore volume and this was probably
due to sludge leachates passing through the soil matrix. This


275
Kowal, N. E., and H. R. Pahren. 1978. Health effects associated with
wastewater treatment and disposal. J. Water Pollut. Control
Fed. 50: 1193-1200.
Laak, R., and D. M. McLean. 1967. Virus transfer through a sewage
disposal unit. Can. J. Public Health 58: 172-176.
Lance, J. C., and C. P. Gerba. 1980. Poliovirus movement during high
rate land filtration of sewage water. J. Environ. Qual. 9: 31-
34.
Lance, J. C., C. P. Gerba, and J. L. Melnick. 1976. Virus movement
in soil columns flooded with secondary sewage effluent. Appl.
Environ. Microbiol. 32: 520-526.
Landry, E. F., J. M. Vaughn, and W. F. Penello. 1980. Poliovirus
retention in 75-cm soil cores after sewage and rainwater
application. Appl. Environ. Microbiol. 40: 1032-1038.
Landry, E. F., J. M. Vaughn, M. Z. Thomas, and C. A. Beckwith. 1979.
Adsorption of enteroviruses to soil cores and their subsequent
elution by artificial rainwater. Appl. Environ. Microbiol. 38:
680-687.
Lauffer, M. A., and R. B. Dow. 1941. The denaturation of tobacco mosaic
virus at high pressures. J. Biol. Chem. 140: 509-518.
Lee, G. F. 1976. Potential problems of land application of domestic
wastewaters. In Land Treatment and Disposal of Municipal and
Industrial Wastewater, pp. 179-192. Edited by R. L. Sanks and
T. Asano. Ann Arbor Science Publishers, Inc., Ann Arbor,
Michigan.
Lefler, E., and Y. Kott. 1974. Virus retention and survival in sand.
In Virus Survival in Water and Wastewater Systems, pp. 84-91.
Edited by J. F. Malina, Jr., and B. P. Sagik. Center for
Research in Water Resources, University of Texas, Austin, Texas.
Little, M. D. 1980. Agents of health significance: Parasites. In
Sludqe--Health Risks of Land Application, pp. 47-58. Edited by
G. Bitton, B. L. Damron, G. T. Edds, and J. M. Davidson. Ann
Arbor Science Publishers, Inc., Ann Arbor, Michigan.
Lund, E. 1971. Observations on the virus binding capacity of sludge.
In Proceedings of the 5th International Water Pollution Research
Conference, July-August 1970, pp. 1-24/1-1-24/5. Edited by
S. H. Jenkins. Pergamon Press Ltd., London.
Lund, E. 1976. Disposal of sludges. In Viruses in Water, pp. 196-
205. Edited by G. Berg, H. L. Bodily, E. H. Lennette, J. L.
Melnick, and T. G. Metcalf. American Public Health Associa
tion, Inc., Washington, D.C.


16
effluents also contain bacterial pathogens (Foster and Engelbrecht
1973), parasites (Hays 1977) and a variety of hazardous chemicals
(e.g., nitrates, phosphates, chromium, cadmium, mercury, zinc, copper,
polychlorinated biphenyls and phthalates; see Lee 1976). Therefore,
secondary effluents should be regarded as potential health hazards in
their final disposal.
During the activated sludge process, large quantities of sludge
(i.e., 14,000 to 19,000 gallons of sludge per million gallons of
wastewater treated) are also produced (U.S. Environmental Protection
Agency 1974). The volume of waste activated sludge (i.e., secondary
sludge) produced is usually much greater than the volume of sludge gener
ated during primary sedimentation, due to the greater moisture content
of the former (U.S. Environmental Protection Agency 1974). In fact,
when a wastewater treatment plant is upgraded to activated sludge
treatment, the capacity for excess sludge handling (i.e., sludge
treatment and disposal) must be significantly increased (U.S. Environ
mental Protection Agency 1974). Indigenous enteric viruses are
routinely found in raw secondary sludges (Berg and Berman 1980; Lund
1976; Lund and R0nne 1973; Nielsen and Lydholm 1980). Therefore, all
secondary sludges should be considered to represent potential health
hazards, and should be handled with care during further treatment and
final disposal.
Removal of Viruses by Sludge Treatment Processes
Viruses in Raw Sludges
As reviewed above, raw sludges produced during the primary and
secondary treatment of wastewater contain substantial quantities of


32
inactivated) after 12 hours in the lime sludge produced (Sattar et al.
1976), it can be hypothesized that substantial quantities of entero
viruses are probably inactivated during the lime stabilization of
raw sludges (i.e., primary, chemical and secondary sludges). This
hypothesis has yet to be confirmed experimentally. Until new informa
tion becomes available, lime-stabilized sludges should be regarded as
potentially containing pathogenic enteric viruses, and, therefore, should
be handled with care during further treatment and final disposal.
Stabilization-heat treatment. Heat treatment is yet another
process which has been used to stabilize (see Figure 2-1) raw sludges
(U.S. Environmental Protection Agency 1974, 1978b). Two types of heat
treatment have been used for sludge stabilization, and they are pasteuriza
tion at approximately 70C and low-pressure (180 to 210 psi) oxidation
at approximately 200C (U.S. Environmental Protection Agency 1974).
Pasteurization at 70C for 30 to 60 minutes destroys most pathogens
in raw sludge including enteric viruses (U.S. Environmental Protection
Agency 1974). Due to the extremely high temperatures (i.e., 200C)
employed during low-pressure oxidation, all pathogens, including
enteric viruses, in raw sludge are undoubtedly destroyed (U.S. Environ
mental Protection Agency 1974, 1978b). Under the most ideal conditions,
however, low-pressure oxidation has displayed a poor capacity to reduce
the odor and the putrescibility potential of raw sludges (U.S. Environ
mental Protection Agency 1978b). As shown in Figure 2-1 and described
below, heat treatment by the low-pressure oxidation process also
conditions the sludge such that subsequent sludge dewatering is


TABLE 7-4. Effect of hydrostatic pressure on the survival of poliovirus type 1 seeded in groundwater at
24C
Pressure9
(psi)b
Initial
virus
concentration
(PFU/ml)
Final
virus concentration
(PFU/ml)
Poliovirus
recovery at
elevated
pressure
{% of
control)
Mean Poliovirus
recovery
for each
pressure
(% + SE)
Initial
Final
At atmospheric
pressure
(control)
At elevated
pressure
500
350
1.7
X
104
1.6
X
104
1.5
X
104
93.8
90.7 3.2
500
420
1.6
X
104
1.6
X
104
1.4
X
104
87.5
1,000
820
1.9
X
104
1.0
X
104
1.0
X
104
100.0
95.5 + 4.6
1,000
900
2.0
X
104
1.1
X
104
1.0
X
104
90.9
2,000
1,900
1.3
X
104
1.1
X
104
8.8
X
103
80.0
85.5 + 5.5
2,000
1,800
1.6
X
104
1.1
X
104
1.0
X
104
90.9
3,000
2,300
1.6
X
104
1.5
X
io4
1.1
X
104
73.3
82.5 + 9.2
3,000
2,300
1.8
X
104
1.2
X
104
1.1
X
104
91.7
4,000
2,700
2.0
X
104
1.2
X
104
1.3
X
104
108.0
100.0 + 7.6
4,000
2,800
2.0
X
104
1.4
X
104
1.3
X
104
92.9
aPressurization time was 24 hours.
b0ne atm per 14.7 psi.


262
Solution II [1% versene (i.e., EDTA) stock in Gey's A]:
2.0 grams ethylenediamine-
tetraacetic acid (EDTA)
10 ml 2 M NaOH (8g/100 ml)
20 ml Gey's A (lOx)
170 ml glass distilled water
Dispense and autoclave.
Solution III (2.5% trypsin stock):
1.0 gram trypsin (Difco Laboratories,
Detroit, Michigan; 1:250)
100 ml glass distilled water
Sterilize by cold filtration. Dispense in 5 ml
aliquots to screw-capped test tubes and freeze for
storage.
Solution IV (standard trypsin-versene solution):
100 ml Gey's A (lx)
4 ml Gey's C (20x)
4 ml stock trypsin (2.5%)
4 ml stock versene (1% in Gey's A)
This solution is good for only one day. This
solution is used to remove the cells from the
32-ounce bottles in which they have been growing
prior to their distribution to plaque bottles.
6. Methyl cellulose overlay for cell cultures (1% methyl cellulose
plus 5% FCS):
Solution I: 300 ml glass distilled water
6 grams methyl cellulose
(1500 centipoise)
Autoclave and then allow to cool to room temperature,
shaking vigorously every hour to avoid layering.
Refrigerate.
Solution II (2x Eagle's MEM):
350 ml glass distilled water
120 ml Eagle's MEM (lOx) with Hanks'
salts (International Sci.)


74
demonstrated in the field. Farrah et al_. (1981a) found that aerobi
cally digested sludge contained larger indigenous viral titers than
anaerobically digested sludge. The uncharged form of ammonia has
been shown to exist in sludge (i.e., tested raw and anaerobically
digested sludge) mostly at pH values above 8 and to display virucidal
activity against enteroviruses (Ward and Ashley 1976, 1977a). Due to
the typically low pH of aerobically digested sludge (see Tables 3-4
and 3-5), it can be hypothesized that the virucidal ammonia is probably
largely absent from this sludge type. Therefore, the low viral-
inactivating activity observed in the aerobically digested sludge
(GDA180 sludge, pH 5.0) employed in this study (see Table 3-5) was to
be expected. Similarly, Ward and Ashley (1976) found no appreciable
inactivation of seeded poliovirus type 1 in 5 days at; 20C in raw
sludge maintained at its naturally low pH of 6.0. Naturally high pH
values, on the other hand, have been found in anaerobically digested
sludge and consequently, this sludge type has demonstrated substantial
viral-inactivating activity due to the presence of the virucidal
ammonia (Ward and Ashley 1976, 1977a). The results presented in Table
3-6 confirm that enteroviruses (i.e., used poliovirus type 1) seeded in
anaerobically digested sludge (i.e., used GDAN sludge, pH 8.3) are
inactivated at a significant rate.
It is emphasized that the results and conclusions presented
above pertain only to the virus used, poliovirus type 1 (LSc). Other
enterovirus may, in fact, display different patterns of adsorption to
sludge solids. Research has shown that only 20.7% of seeded echovirus


121
TABLE 4-7. Retention of poliovirus type 1 by a packed column of
Eustis fine sand subsoil when suspended in 0.01 N
CaCl 2
No. of pore
volumes3
eluted
Poliovirus
eluted
(PFU/ml)
% of Influent
poliovirus
concentration
1
0
0
2
0
0
3
4.2
0.06
4
0
0
5
4.2
0.06
6
0
0
7
0
0
8
25
0.3
9
21
0.3
10
0
0
11
0
0
a0ne pore volume for the column used equals 71 ml. The labora
tory-packed column was 10 cm in length and 4.8 cm internal diameter.
The sample of Eustis fine sand subsoil used consisted mainly of the
A21 and A22 horizons (see Table 4-3), The column was conditioned
with 5 pore volumes of 0.01 N CaC^- The solution was applied con
tinuously to the column at approximately 5 ml/min using a peristaltic
pump (Buchler, Fort Lee, N.J.).
bPoliovirus was seeded in the influent (i.e., 0.01 N CaCl2) at
at concentration of 7.3 x 10^ PFU/ml. The conductivity of 0.0T
N CaCl2 was 1210 pmho/cm at 25C and the pH was 6.4.


15
0
0
0.005
62
5.8
16
0
0
0.005
74
6.5
17
0
0
0.005
61
6.5
18
0
0
0.005
60
6.3
19
0
0
0.005
59
6.6
20
0
0
0.005
54
6.7
One pore volume for the column used equals 80 ml. The laboratory-packed column was 10 cm in length
and 4.8 cm internal diameter. The sample of Red Bay sandy loam subsoil used consisted mainly of the A2
and Bit horizons (see Table 4-3 ). The column was conditioned with 5 pore volumes of 0.01 N CaCl2. All
solutions were applied continuously to the column at approximately 5 ml/min using a peristaltic pump
(Buchler, Fort Lee, N.J.).
bPoliovirus was seeded in the influent (i.e., 0.01 N CaCl2) at a concentration of 2.4 x 104 PFU/ml.
cThe conductivity of 0.01 N CaC12 was 1210 ymho/cm at 25C and the pH was 6.4.
^Rain water was collected next to the Environmental Engineering Sciences building at the University of
Florida, Gainesville. See Table 4-2 for chemical characteristics of the rain water.


138
distilled water, on the other hand, show that poliovirus was distributed
throughout the length of the column with slightly higher concentrations
in the top 3 cm of soil (2.6% of the total PFU applied--see Table
4-14). Thus, the results presented indicate that, because of the
appropriate ionic environment, poliovirus was adsorbed in the top of
the column receiving sludge diluted with 0.01 N calcium chloride. How
ever, sludge diluted with distilled water affected the absorption
process and allowed virus to move down the column, ultimately appearing
in the effluent (see Table 4-12).
Poliovirus Suspended in Sludge Liquor
The centrifugation of anaerobically digested sludge resulted in
a supernatant that will be referred to as sludge liquor. This liquor
had a conductivity of 1580 ymhos/cm at 25C and its pH was 8.1. Other
characteristics of this liquor are displayed in Table 4-1. In order to
stress the importance of sludge solids in virus movement through soils,
soil column experiments were undertaken which involved viruses sus
pended in sludge liquor.
Red Bay sandy loam. Poliovirus was suspended in sludge liquor
and subsequently was applied continuously to a 10-cm column of Red Bay
sandy loam subsoil. In Figure 4-5, the breakthrough curve obtained is
shown and it is seen that 33.7% of the total virus applied had appeared
in the leachate by the seventh pore volume. The conductivity of the
leachates did not vary significantly from the conductivity of the
sludge liquor (i.e., 1580 ymho/cm at 25C). The pH of the leachates
varied from approximately 5.0 to 6.3. Moreover, it is seen in Table 4-1


No. of pore volumes eluted
Poliovirus eluted (expressed as cumulative % of total PFU having
been applied at each pore volume)
Poliovirus eluted (x 10^ PFU/ml)
ZH


36
less; lacking virucidal ammonia), Ward and Ashley (1977b) found a
gradual reduction in the titer of seeded poliovirus type 1 (CHAT)
during the air drying of sludge at 21C from 5% to 65% sludge solids
content. However, when the sludge was allowed to dry to a solids
content of 83% or greater, these investigators observed a dramatic
decrease in poliovirus titer of greater than three orders of magni
tude in 4 days (similar results were also obtained using seeded cox
sackievirus B1 and reovirus 3). Ward and Ashley (1977b) went on to
demonstrate that viral RNA is released during the air drying of sludge
and this results in irreversible viral inactivation. Moreover, the
evaporation process itself, and not some virucidal agent (note that
the raw sludge used lacked the virucidal form of ammonia), was found
responsible for poliovirus inactivation during the air drying of
sludge at 21C (Ward and Ashley 1977b). In fact, the inactivation
rate of poliovirus incorporated into dewatered raw sludge was signifi
cantly lower than the inactivation rate of poliovirus seeded in raw
sludge and allowed to air dry (Ward and Ashley 1977b). During heat
treatment at 47C or 51C, the inactivation rate of poliovirus type 1
(CHAT) incorporated into dewatered raw sludge significantly declined as
the sludge solids content was increased from 5% to 80% (Ward and Ashley
1978b). Sludge solids content (or sludge moisture content) itself was
shown to have an insignificant effect on the rate of poliovirus inac
tivation by heat (Ward and Ashley 1978b). Sludge solids-associated
ionic detergents, however, were found to protect poliovirus in sludge
from heat inactivation and to be concentrated during sludge dewatering


165
Experiments were undertaken to assess the capacity of the Red Bay
sandy loam subsoil to retain viruses following the application of
chemical sludges (alum, ferric chloride, and lime). The aim of this
research was to determine if there is a risk of groundwater contamina
tion with viruses when chemical sludges are disposed on land.
The chemical sludges (alum, ferric chloride, and lime) were
precipitated from poliovirus-seeded, raw sewage. As shown in Table
4-20, variable fractions of the seeded viruses were recovered from
the chemical sludges produced (22.6% to 95.6% for alum sludges,
9.8% to 13.3% for ferric chloride sludges, and 0% to 0.1% for lime
sludges). Since the viruses became embedded in the sludges produced
during the flocculation process, it is likely that not all viruses
present in the sludges were recovered. It is generally believed that
embedded viruses are difficult to elute. It does appear, however,
that due to the high pH of the lime sludges, most of the viruses
originally seeded in the raw sewage were inactivated (see Tables
4-20 and 4-21). The association between poliovirus and chemical
sludge solids was then evaluated. It was found that from 97% to 100%
of input virus was associated with alum, ferric chloride, and lime
sludge solids (Table 4-21). In one sample (experiment no. 2) of
lime sludge, all viruses were inactivated due to the high pH (pH 11.3)
generated during the process. The application of these virus-seeded
sludges to soil columns of Red Bay sandy loam did not result in any
virus breakthrough following leaching with two to ten pore volumes
of rainwater (Table 4-22). It is worth stressing that no virus could


127
TABLE 4-10. Movement or retention of poliovirus type 1 when suspended in
anaerobically digested sludge diluted (1:50) with distilled
water or 0.01 N CaClp respectively, and applied to 10 cm
packed columns of Eustis fine sand subsoil
No. of pore Poliovirus
volumes3 eluted
eluted (PFU/ml)
% of Influent*3 Conductivity
poliovirus of pore
concentration volume collected
(ymho/cm at 25C)
pH of pore
volume
collected
Column
l--applied
sludgec
diluted (1 :50) with
distilled water
1
5.0
x 101
0.1
46
7.0
2
3.3
x 101
0.1
102
6.8
3
1.4
x 103
2.4
250
6.6
4
7.4
x 103
12.5
280
5.9
5
1.7
x 104
28.2
300
5.8
6
2.1
x 104
36.2
310
5.8
7
2.4
x 104
39.9
310
5.5
Column
2--applied
siudgec
diluted (1:50) with
0.01 N CaCl2
1
0
0
1230
5.1
2
0
0
1290
5.2
3
0
0
1310
5.2
4
0
0
1320
5.2
5
0
0
1360
5.3
6
0
0
1300
5.3
1250 5.4
7
0
0




132
TABLE 4-12. Movement or retention of poliovirus type 1 when suspended in
anaerobically digested sludge diluted (1:50) with distilled
water or 0.01 N CaCl2> respectively, and applied to 27 cm
packed columns of Eustis fine sand subsoil
No. of pore Poliovirus
volumes9 eluted
eluted (PFU/ml)
% of Influentb
poliovirus
concentration
Conductivity
of pore
volume collected
(ymho/cm at 25C)
pH of pore
volume
collected
Column
1applied sludge0
diluted (1:50) with distilled water
0.5
0
0
29
6.6
1.0
0
0
29
6.3
1.5
0
0
51
6.2
2.0
0
0
61
6.2
2.5
0
0
104
6.5
3.0
0
0
77
6.5
3.5
2.5 x 102
1.8
78
5.6
4.0
2.7 x 102
1.9


Column
2--applied sludge0
diluted (1:50)
with 0.01 N CaCl2
0.5
0
0
1180
4.8
1.0
0
0
1270
4.8
1.5
0
0
1270
4.7
2.0
0
0
1300
4.8
2.5
0
0
1260
4.9
3.0
0
0
1300
4.9


TABLE 5-8. Survival of poliovirus type 1 and echovirus type 1 following suspension in liquid sludge and
subsequent application to large soil cores of Eustis fine sand exposed to natural conditions
(11 October 1978-1 November 1978)
Sampling
date
(1978)
Days
after the
beginning
of
experiment
Cumulative
rainfall9
(cm)
Sludge^
solids
content
{%, wt/wt)
Soil
moisture
[%, wt/wt)
No. of viruses
(PFU/g dry weight of sludge or
soil)
Echovirus
Poliovirus
LC1
LC2
LC3
LC4
LIQUID SLUDGE SAMPLE
11 Oct.
0
0
7.0C
__d
8.6 x 104
8.6 x 104
2.6 x 107
2.6 x 107
DRYING SLUDGE SAMPLE
14 Oct.
3
0
38.0
--
1.6 x 104
1.3 x 104
1.3 x 106
2.9 x 106
Sludge mixed with the top 2.5 cm
of soil on day 3
SOIL SAMPLES (top 2.5 cm)
14 Oct. 3 0 7.5 2.8 x 102 1.9 xlO2 4.3 x 104 1.9 xlO4
6.9 x 101 1.6 x 101 3.5 x 103 2.1 x 102
16 Oct.
5
0
3.0


246
TABLE 6-4. Analysis of topsoil and groundwater samples from the
Kanapaha site for the presence of indigenous entero
viruses
Sampling date
Cumulative amount of
sludge3 applied by
the sampling date
[in (cm)]
Sample
Viruses
detected
December 1977
2.6 (6.5)
Topsoilb
0
Groundwater0
0
January 1978
2.6 (6.5)
Topsoilb
0
Groundwater0
0
February 1978
3.7 (9.3)
Topsoilb
0
Groundwater
0
Application of conditioned-dewatered sludge (see Table 6-3) to
the Kanapaha site began in August 1977.
^Composite topsoil samples (200 wet grams) were obtained monthly
from the Kanapaha site as shown in Figure 6-2 and were tested for the
presence of viruses.
cGroundwater (100 gallons or 384 liters) from a 60-ft (ca. 18-m)
well on the Kanapaha site (see Figure 6-2) was monitored monthly for the
presence of viruses.


172
TABLE 4-24. Association between poliovirus type 1 and lime-stabilized,
chemical sludge solids
Sludge3
type,
1 ime-
stabilized
Virus in ,
unfractionatedb
sludge
(total PFU)
Virus in
sludge
supernatant0
(total PFU)
Viable
unadsorbed6
virus
(%)
Solids-
associated6
virus
(%)
Alum
0
0
0
0
Ferric _
chloride
6.2 x 10
0
0
100.0
aThe chemical sludges were precipitated from virus-seeded, raw
sewage. The methods used to produce these sludges and to determine the
amount of viruses present in the sludges are described in Table
The chemical sludges were then stabilized with Ca(0H)o as described in
Table 4-23.
bThe sludge solids were not separated prior to assaying.
cThe sludge was clarified by centrifugation at 1400 x g for 10 min
at 4C and the supernatant was subsequently assayed.
*The "viable unadsorbed virus (%)" values were calculated as shown
in the Materials and Methods section.
eThe "sludge solids-associated virus (%)" values were estimated as
shown in the Materials and Methods section.


FIGURE 6-5. Concentration of groundwater by membrane filtration for the detection of indigenous
enteroviruses at the Kanapaha site
Groundwater from a 60-ft (ca. 18-m) well is shown being hand pumped into a small
bucket (A). Once 100 gallons (ca. 384 liters) of water are collected in the large
tank shown (B), the water is treated as described in the Materials and Methods
section and then passed through a 10-in (ca. 25-cm), 0.25-ym pore size Filterite fil
ter (placed in the filter housing C as shown). The filter is subsequently treated
for virus recovery as described in the Materials and Methods section.




94
Soils
The soils studied were a Red Bay sandy loam sampled at the West
Florida Agricultural Experiment Station, Jay, and a Eustis fine sand
sampled at the agronomy farm, University of Florida, Gainesville. The
Red Bay sandy loam has been classified as a Rhodic Paleudult, fine-
loamy, siliceous, thermic while the Eustis fine sand was classified as a
Psammentic Paleudult, sandy, siliceous, hyperthermic (Calhoun et al.
1974). Some characteristics of these soils are shown in Table 4-3.
The percent organic matter in these two soils was measured at less
than 1% except for the Al horizon of the Red Bay sandy loam which was
found to contain 4.3% organic matter (Calhoun et a]_. 1974).
Poliovirus Transport Studies
Poliovirus transport (i.e., movement or retention) in soil cores
treated with virus-seeded sludge was studied under laboratory conditions.
Two types of soil cores were used as described below.
Laboratory-packed soil columns. Laboratory-packed soil columns
were prepared using subsoil samples of Red Bay sandy loam (consisted
mainly of the A2 and Bit horizons--see Table 4-3) and Eustis fine sand
(consisted mainly of the A21 and A22 horizons--see Table 4-3). Each
subsoil sample was screened by hand to remove rocks and large organic
matter, and was then allowed to air dry. The soils were not autoclaved
or sterilized in any other way. The dry soils were then carefully
packed into acrylic plastic columns 10 cm or 29 cm in length (packed
10 cm or 27 cm with soil, respectively) and 4.8-cm internal diameter.
A polypropylene screen (105-ym pore size) was used to support the soil


55
and allowed to spread over the entire monolayer for 30 to 60 seconds.
This solution was subsequently decanted and the cell culture was
allowed to rest at room temperature until the cells came off the glass
(approximately 5 minutes). Growth medium (10 ml) was then added, and
pipetted twice up and down to dislodge the cells from the glass and
break up clumps. An additional 190 ml of the growth medium was added
to the content of the 32 oz glass bottle. This cell suspension was
2
then distributed in 5 ml aliquots to 40 glass (2 oz-r-20 cm ) or plastic
2
(25 cm ) bottles. After approximately 48 hours of incubation at 37C,
these small bottles contained confluent cell monolayers and were ready
for use in viral assays.
Experimental samples were diluted, if necessary, prior to assay
in either Eagle's MEM containing 5% calf serum (rarely used) or
phosphate-buffered saline (PBS) containing 2% FCS (both solutions
contained 250 U/ml penicillin, 125 yg/ml streptomycin, and phenol red;
see Appendix). All samples from each experiment were assayed on only
one cell line (i.e., AV3, BGM or MA-104) using the procedure described
below. A 0.6 ml aliquot of each diluted or undiluted sample was
inoculated in fractions of 0.2 ml into three drained cell monolayers.
Following inoculation, a 60-minute adsorption period with tilting at
15-minute intervals was allowed. The infected monolayers were then
overlaid with 4 ml of 1% methyl cellulose in Eagle's MEM supplemented
with 5% FCS, 250 U/ml penicillin, 125 pg/ml streptomycin, and 117 pg/ml
kanamycin (see Appendix). After incubation at 37C for approximately
48 hours, the cell monolayers were stained with either crystal violet


CHAPTER VII
EFFECT OF HYDROSTATIC PRESSURE ON THE
SURVIVAL OF POLIOVIRUS SEEDED IN GROUNDWATER AND SEAWATER
Introduction
At several disposal sites in the United States receiving wastewater
effluent, indigenous enteroviruses have been recovered from groundwater
(Dugan et al_. 1975; Schaub and Sorber 1977; Vaughn et al. 1978; Wellings
et aj_. 1974 and 1975). Although indigenous enteroviruses were not
detected in the groundwater from the two sludge disposal sites described
in Chapter VI (see pages 244 to 249; also see Farrah et aK 1981a),
poliovirus type 2 was isolated from a 28-foot (8.5-m) deep well and a 58-
foot (17.7-m) deep companion well at a sludge disposal site in St. Peters
burg, Florida (Wellings et at. 1978). In addition to theapplication of
wastes to land, poor engineering practices (e.g., wells not properly
sealed and cesspools near wells) have also been demonstrated to result in
the contamination of groundwater with viral and other pathogens (Allen
and Geldreich 1975 ; Mack et aK 1972; Robeck 1979).
It appears, therefore, that viruses sometimes find their way into
our groundwater supplies. Unfortunately, we know little about the sur
vival of viruses in the groundwater environment. In this environment,
elevated hydrostatic pressures are likely to be encountered (McNabb and
Dunlap 1975). Such elevated pressures have been found to affect water
chemistry (Disteche 1959; Hamann 1963; Hamann and Strauss 1955; Horne
and Johnson 1966), and the survival of bacteria (Baross et al_. 1975;
Heden 1964; Horvath and Elkan 1978; Jannasch et al. 1976;
250


FIGURE 6-1. Scheme for sludge disposal at the Kanapaha site,
Gainesville, Florida


Soil temperature (C)


236
Virus Recovery Procedures
Sludge. Samples (1 to 4 liters) of wasted sludge, aerobically
digested sludge (90 days) and dewatered-conditioned sludge from the
Main Street wastewater treatment plant (Gainesville) were collected
in sterile Nal gene bottles and transported to the laboratory. The
pH and solids content of each sludge sample was determined as described
in Chapter III (seepages 56-59).The sludge samples (i.e., total volume)
were then centrifuged at 1400 x g for 10 minutes at 4C. The sludge
supernatants were discarded. The sludge solids-associated viruses were
eluted and further concentrated using a modification of the glycine
method developed by Hurst et al_. (1978). This method is detailed in
Chapter III (see pages 65 to 67).The filter and pellet concentrates
produced were then assayed for viruses as described below.
Soil. Composite topsoil samples (200 wet grams) from the
Kanapaha site were obtained as shown in Figure 6-2. Indigenous entero
viruses were recovered from these soil samples using the procedure
described by Hurst and Gerba (1979). This method consisted of mixing
each 200-g sample of wet soil with 600 ml of 0.25 M glycine buffer,
0.05 M ethylenediaminetetraacetic acid (EDTA), pH 11.5. If necessary,
the pH of the mixture was adjusted to between 11.0 and 11.5 by the
addition of 1 M glycine buffer, pH 11.5. The samples were vigorously
shaken by hand for 30 seconds and then shaken on a rotating shaker for
4.5 minutes. The samples were subsequently centrifuged at 1400 x g
for 4 minutes at 4C (all centrifugation was performed using a Sorvall
RC5-B centrifuge, Ivan Sorvall Inc., Norwalk, Connecticut). The


CHAPTER VIII
CONCLUSIONS
Based on the findings of this study, the following conclusions can
be drawn:
1. Poliovirus type 1 (LSc) was largely associated with digested,
conditioned-dewatered, chemical (alum, ferric chloride and lime)
and lime-stabilized, chemical sludge (alum and ferric chloride)
sol ids.
2. Sludge type was found to affect the degree of association between
seeded poliovirus and sludge solids. The mean percent of solids-
associated viruses for activated sludge mixed liquors, anaerobic
ally digested sludges and aerobically digested sludges was 57,
70 and 95, respectively. The degree of association between polio
virus and sludge solids was significantly greater for aerobically
digested sludges than for the other two sludge types.
3. A smaller fraction of echovirus type 1 (Farouk) was associated
with lagooned sludge solids than was the case for poliovirus.
4. The effectiveness of the glycine method in the recovery of solids
associated viruses was found to be affected by sludge type. Sig
nificantly lower mean poliovirus recovery was found for aerobic
ally digested sludges (15%) than for mixed liquors or anaerobic
ally digested sludges (72% and 60%, respectively).
5. Poliovirus transport studies involving soil cores treated with
virus-seeded sludge were conducted under controlled laboratory
and saturated flow conditions. A Red Bay sandy loam displayed
a substantially greater retention capacity for poliovirus in
anaerobically digested sludge than a sandy soil (i.e., Eustis
fine sand). The Red Bay sandy loam soil was shown to completely
retain poliovirus following the application of conditioned-
dewatered, chemical (alum, ferric chloride and lime) and lime-
stabilized, chemical (alum and ferric chloride) sludge.


The term "mixed liquor" refers to the suspension undergoing treatment in an activated sludge unit.
Although they contain activated sludge solids, mixed liquors are not to be confused with settled sludges
which must be treated and ultimately disposed of.
dash means that the sludge treatment was either not applicable or not performed.
eThese are cationic polymers supplied to
Wayne, N.J.
the treatment plants by the American Cyanamid Company,
fThis sludge is a mixture of sludge from
Florida. The mixture was kept in a lagoon at
before ultimately being disposed of on land,
study.
the Montclair (1/3) and Main street (2/3) plants
the West Florida Agricultural Experiment Station
It was the lagooned sludge which was sampled and
of Pensacola,
(Jay, Florida
used in this


278
surveillance studies. Abstracts of the Annual Meeting, Ameri
can Society for Microbiology. Abstract Q144, p. 218.
Overman, A. R. 1975. Effluent irrigation as a physicochemical hydro-
dynamic problem. Florida Sci. 38: 215-222.
Pahren, H. R. 1980. Overview of the problem. In Sludqe--Health Risks
of Land Application, pp. 1-5. Edited by 6. Bitton, B. L.
Damron, G. T. Edds, and J. M. Davidson. Ann Arbor Science
Publishers, Inc., Ann Arbor, Michigan.
Pahren, H. R., J. B. Lucas, J. A. Ryan, and G. K. Dotson. 1979. Health
risks associated with land application of muncipal sludge.
J. Water Pollut. Control Fed. 51: 2588-2601.
Palfi, A. 1972. Survival of enteroviruses during anaerobic sludge
digestion. In Proceedings of the 6th International Water
Pollution Research Conference, June 1972, pp. A/13/26/1 -
A/13/26/6. Edited by S. H. Jenkins. Pergamon Press Ltd.,
London.
Peterson, J. R., C. Lue-Hing, and D. R. Zenz. 1973. Chemical and
biological quality of municipal sludge. In Recycling Treated
Municipal Wastewater and Sludge through Forest and Cropland,
pp. 26-37. Edited by W. E. Sopper and L. T. Kardos. Pennsyl-
vania State University Press, University Park, Pennsylvania.
Plotkin, S. A., and M. Katz. 1965. Minimal infective doses of viruses
for man by the oral route. In Transmission of Viruses by the
Water Route, pp. 151-166. Edited by G. Berg. Interscience
Publishers, New York, N.Y.
Quisenberry, V. L., and R. E. Phillips. 1976. Percolation of surface-
applied water in the field. Soil Sci. Soc. Am. J. 40: 484-
489. ~
Quisenberry, V. L., and R. E. Phillips. 1978. Displacement of soil
water by simulated rainfall. Soil Sci. Soc. Am. J. 42: 675-
679. ~
Robeck, G. G. 1969. Microbial problems in ground water. Ground Water
7: 33-35.
Robeck, G. G., N. A. Clarke, and K. A. Dostal. 1962. Effectiveness
of water treatment processes in virus removal. J. Am. Water
Works Assoc. 54: 1275-1292.
Romero, J. C. 1970. The movement of bacteria and viruses through
porous media. Ground Water 8: 37-48.



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81,9(56,7< 2) )/25,'$


20
TREATMENT PROCESSES FUNCTIONS


197
experiments, environmental parameters (i.e., temperature, soil
moisture, and rainfall) were monitored. The protocol of sludge dis
posal to soil was similar to that practiced at sludge disposal sites.
Virus survival and transport were monitored during three different runs,
using the same cores. The survival monitoring was terminated when
viruses were not detectable in soil samples.
Association between Seeded
Enteroviruses and Sludge Solids
Prior to studying virus transport through soil cores, it was
necessary to assess the extent of virus association with sludge solids.
Poliovirus was added to aerobically digested sludge and to lagooned
sludge (2/3 anaerobic and 1/3 aerobic sludge), while echovirus was
added to lagooned sludge only. Following magnetic stirring for 10 to
60 minutes, the fraction of sludge solids-associated virus was deter
mined. As shown in Table 5-3, more than 90% of poliovirus was found
associated with sludge solids (aerobic or lagooned sludge). On the
other hand, only 20.7% of seeded echovirus was observed to be associated
with lagooned sludge solids (see Table 5-3). The virus-seeded sludge
was then applied to the undisturbed soils cores. The association
between viruses and sludge solids may be instrumental in virus retention
during sludge application to land.
First Survival Experiment (7
October 1977-12 October 1977 )
During this period, the soil temperature was not monitored.
However, air temperature data were obtained from the weather station
of the Department of Agronomy, University of Florida (Figure 5-3). It


CHAPTER Pacje
IV POLIOVIRUS TRANSPORT STUDIES INVOLVING SOIL
CORES TREATED WITH VIRUS-SEEDED SLUDGE UNDER
LABORATORY CONDITIONS 82
Introduction 82
Materials and Methods 83
Virus and Viral Assays 83
Primary Wastewater Effluent 84
Sludges 84
Association of Seeded Poliovirus
with Sludge Solids 91
Rain Water 92
Soi 1 s 94
Poliovirus Transport Studies 94
Effect of Soil Bulk Density
on Poliovirus Transport 108
Results and Discussion 116
Poliovirus Suspended in 0.01 N CaC^ 117
Poliovirus Suspended in Diluted
Anaerobically Digested Sludge 120
Poliovirus Suspended in Undiluted
Anaerobically Digested Sludge 150
Conditioned-Dewatered Sludge 158
Chemical Sludges 161
Lime-Stabilized, Chemical Sludges 170
V RETENTION AND INACTIVATION OF ENTEROVIRUSES
IN SOIL CORES TREATED WITH VIRUS-SEEDED SLUDGE
AND EXPOSED TO THE NORTH-CENTRAL FLORIDA ENVIRONMENT 178
Introduction 178
Materials and Methods 179
Viruses and Viral Assays 179
Sludges 181
Association of Seeded Viruses
with Sludge Solids 181
Soil 182
Fate of Viruses in Soil Cores 183
Virus Recovery Procedures 190
Measurement of Environmental Parameters 193
v


107
in the research reported herein); and b can be set at any value between
1/a and the final pore volume number collected. For cores receiving
poliovirus in a spiked fashion (i.e., all at once at the beginning of
the experimental trial; used this procedure in both laboratory-packed
soil columns and undisturbed soil cores), the quantity of poliovirus
leached was expressed as the cumulative percent of the total viral PFU
applied and it was calculated by the following equation:
poliovirus eluted at pore
volume b (as cumulative %
of total PFU applied)
pore volume (ml)
a
poliovirus applied (total PFU)
x 100 (4-4)
where pv, a, and b are defined as in Equation (4-3) above.
The pH and conductivity of each soil core leachate were measured
as described above for undiluted digested sludges.
Distribution of virus in the soil. The distribution of polio
virus in the soil profile was studied in two 27-cm laboratory-packed
columns of Eustis fine sand. These soil columns were prepared and
treated with virus-seeded, diluted (1:50 with 0.01 N calcium chlor
ide or distilled water) anaerobically digested sludge as described
above. The leachates from these columns were collected and assayed for
viruses as described earlier. Following leaching, the soil profile in
y poliovirus eluted in
pv=l/a each Porevo^ume (PFU/ml)


221
The results presented above show that the movement of viruses in
sludge-amended soil cores exposed to natural conditions is limited due
to the following factors:
1. Viruses were retained with the sludge solids on top
of the soil profile.
2. Viruses were inactivated in the natural environment
(laboratory leaching studies involving viruses are
usually completed in one day and neglect to take
viral inactivation into account see Figure 4-9).
3. Rain water moved through the soil cores under natural,
unsaturated flow conditions.
Several investigators have demonstrated that,under natural
conditions, rain water flows rapidly down macropores causing only
partial displacement of the initial soil water and contributing greatly
to groundwater recharge (Elrick and French 1966; McMahon and Thomas
1974; Nielsen and Biggar 1961; Quisenberry and Phillips 1976, 1978;
Thomas and Phillips 1979; also see review by Burge and Parr 1980).
Under these conditions, it can be hypothesized that viral particles
associated with the immobile, matrix, soil water (i.e., in micropores)
in the sludge-amended cores were probably bypassed by the fast moving
rain water. In laboratory studies involving saturated flow conditions,
the reverse would probably occur; that is, the viral particles would
move rapidly with the soil water in the macropores and this was
demonstrated in Figure 4-9.


183
Fate of Viruses in Soil Cores
The survival and transport (i.e., movement or retention) of
poliovirus and echovirus in soil cores treated with virus-seeded
sludge was studied under natural conditions.
Undisturbed soil cores. Undisturbed soil cores (Blake 1965;
Sanks et £1_. 1976) of Eustis fine sand were used and they were
obtained by driving polyvinyl chloride pipes into the soil at the
agronomy farm, University of Florida, Gainesville, as described in
Chapter IV (see pages 102-103). The undisturbed soil cores were
obtained in a manner that preserved the natural structure and bulk den
sity of the soil as found in the field (see Table 4-3). The soil cores
were 33 cm in length (pipes were 40 cm in length) and had an internal
diameter of 5.0 cm (small soil cores) or 15.5 cm (large soil cores),
and thereby consisted of the Ap and A21 horizons of the fine sand
(see Table 4-3). Two small soil cores and four large soil cores were
employed. A polypropylene screen (105-ym pore size) which supported
the soil while allowing the free movement of water and viruses (i.e.,
did not adsorb viruses in soil leachates), and a spout were secured
at the bottom of each small soil core. Porous ceramic cups attached to
spouts, on the other hand, were installed at the bottom of the large
soil cores. The porous ceramic cups used were 6.9 cm long, and had a
wall thickness of 0.23 cm and a pore diameter of 1.4 to 2.1 pm (no.
2131, Soil Moisture Equipment Corp., Santa Barbara, California). These
cups restricted the movement of water somewhat and consequently produced
an artificial groundwater table in the bottom part of the soil during


FIGURE 4-9. Movement of poliovirus type 1 through an undisturbed
core of Eustis fine sand (conditioned with rain water)
following the application of 2.5 cm of seeded anaerobi
cally digested sludge and the subsequent elution with
rain water
One pore volume for the core used equals 234 ml.
The undisturbed soil core was 33 cm in length and
5.0 cm internal diameter; consists of the Ap and
A21 horizons of the Eustis fine sand (see Table
4-3). The core was initially conditioned with
5 pore volumes of rain water. One inch or 2.5 cm
(51.6 ml) of anaerobically digested sludge (GDAN
see Table 3-2; solids content, conductivity and
pH equal to 2.0%, 3250 yrnho/cm at 25C and 8.3,
respectively) seeded with a total of 5.7 x 105
PFU of poliovirus was applied to the core, allowed
to soak in and then,was worked under 2.5 cm.
Elution with rain water was subsequently under
taken. This solution was applied from an inverted,
self-regulated, 1 liter Erlenmeyer flask set to
maintain a 2.5 cm hydraulic head on the core. The
flow rate through the core was measured at 3.9
ml/min. The rain water was collected next to the
Environmental Engineering Sciences building at the
University of Florida, Gainesville. See Table 4-2
for chemical characteristics of the rain water.


269
Dahling, D. R., G. Berg, and D. Berman. 1974. BGM, a continuous cell
line more sensitive than primary rhesus and African green
kidney cells for the recovery of viruses from water. Health
Laboratory Science 11: 275-282.
Damgaard-Larsen, S., K. 0. Jensen, E. Lund, and B. Nissen. 1977. Sur
vival and movement of enterovirus in connection with land
disposal of sludges. Water Res. 11: 503-508.
Davis, B. D., R. Dulbecco, H. N. Eisen, H. S. Ginsberg, and W. B. Wood.
1973. Microbiology. Harper and Row, Hagerstown, Maryland.
Dazzo, F. B., and D. F. Rothwell. 1974. Evaluation of porcelain cup
soil water samplers for bacteriological sampling. Appl.
Microbiol. 27: 1172-1174.
Derbyshire, J. B., and E. G. Brown. 1978. Isolation of animal viruses
from farm livestock waste, soil and water. J. Hyg. 81: 295-302.
Dick, R. I. 1978. Sludge treatment, utilization and disposal.
J. Water Pollut. Control Fed. 50: 1096-1131.
Distfeche, A. 1959^ pH measurements with a glass electrode withstanding
1500 kg/crn hydrostatic pressure. Rev. Sci. Instr. 30: 474-478.
Drewry, W. A., and R. Eliassen. 1968. Virus movement in groundwater.
J. Water Pollut. Control Fed. 40: R257-271.
Duboise, S. M., B. E. Moore, and B. P. Sagik. 1976. Poliovirus sur
vival and movement in a sandy forest soil. Appl. Environ.
Microbiol. 31: 536-543.
Duboise, S. M., B. E. Moore, C. A. Sorber, and B. P. Sagik. 1979.
Viruses in soil systems. Crit. Rev. Microbiol. 7_: 245-285.
Dugan, G. L., R. H. F. Young, L. S. Lau, P. C. Ekern, and P. C. S. Loh.
1975. Land disposal of wastewater in Hawaii. J. Water Pollut.
Control Fed. 47: 2067-2087.
Edds, G. T., 0. Osuna, and C. F. Simpson. 1980. Health effects of
sewage sludge for plant production or direct feeding to cattle,
swine, poultry or animal tissue to mice. In SludgeHealth
Risks of Land Application, pp. 311-325. Edited by G. Bitton,
B. L. Damron, G. T. Edds, and J. M. Davidson. Ann Arbor Science
Publishers, Inc., Ann Arbor, Michigan.
Edmonds, R. L. 1976. Survival of coliform bacteria in sewage sludge
applied to a forest clearcut and potential movement into
groundwater. Appl. Environ. Microbiol. 32: 537-546.
Eisenhardt, A., E. Lund, and B. Nissen. 1977. The effect of sludge
digestion on virus infectivity. Water Res. 11: 579-581.


72
TABLE 3-5. Effect of contact time on the association between
poliovirus type 1 and aerobically digested sludge solids
Contact
Virus3 in
Virus
Viable
Sol ids-
time
unfractionatedb
in sludge
unadsorbedd
associated1
sludge
supernatant0
virus
virus
(total PFU)
(total
PFU)
(%)
(%)
30 min
8.9 x 108
4.2 x
106
0.5
99.5
60 min
8.2 x 108
4.8 x
106
0.6
99.4
2 days
1.2 x 109
1.3 x
107
1.1
98.9
4 days
1.2 x 109
4.3 x
106
0.4
99.6
5 days
8.0 x 108
8.8 x
106
1.1
98.9
6 days
5.8 x 108
1.9 x
106
0.3
99.7
7 days
8.2 x 108
2.5 x
106
0.3
99.7
8 days
6.9 x 108
2.5 x
106
0.4
99.6
aThe virus was added to 1000 ml of aerobically digested sludge
(GDA180--see Table 3-2; solids content and pH equal to 1.3% and 5.0,
respectively) while stirring the suspension using a magnetic stirrer.
Magnetic stirring was continued for 60 min. For the remainder of the
experimental trial, the virus-seeded sludge was left at room temperature
undisturbed, and was stirred only prior to obtaining a sample for viral
assay.
bThe sludge solids were not separated prior to assaying.
cThe sludge was clarified by centrifugation at 1400 x g for 10 min
at 4C and the supernatant was subsequently assayed.
^The "viable unadsorbed
in the Materials and Methods
virus (%)" values were calculated as shown
section.
eThe "solids-associated
in the Materials and Methods
virus (%)"
section.
values were estimated as shown


131
TABLE 4-11. Continued.
No. of pore
volumes3
eluted
Poliovirus
eluted
(PFU/ml)
% of Influent^
poliovirus
concentration
Conductivity
of pore
volume collected
(ymho/cm at 25C)
pH of pore
volume
collected
Shift to nonseeded 0.01
N CaCl2e
15
0
0
60
6.5
16
0
0
1010
5.2
17
22
0.1
1240
5.2
18
0
0
1320
5.2
19
0
0
1320
5.1
Shift to nonseeded rain
waterd
20
0
0
1200
5.1
21
0
0
350
5.9
22
0
0
88
6.5
23
0
0
64
6.8
24
0
0
58
6.8
a0ne pore volume for the column used equals 71 ml. The laboratory-
packed column was 10 cm in length and 4.8 cm internal diameter. The
sample of Eustis fine sand subsoil used consisted mainly of the A21 and A22
horizons (see Table 4-3). The column was conditioned with 5 pore volumes
of 0.01 N CaClo. All solutions were applied continuously to the column at
approximately 5 ml/min using a peristaltic pump (Buchler, Fort Lee, N.J.).
^Poliovirus was seeded in the influent fi.e., sludge diluted with
0.01 N CaCl2) at a concentration of 2.6 x 10*+ PFU/ml.
CThe anaerobically digested sludge (PDANsee Table 3-2) used had a
solids content of 1.4% and a pH of 7.2. Chemical parameters were not
measured for the sludge diluted (1:50) with 0.01 N CaCl2.
dRain water was collected next to the Environmental Engineering
Sciences building at the University of Florida, Gainesville. See Table
4-2 for chemical characteristics of the rain water.
eThe conductivity and pH of 0.01 N CaCl2 was 1210 ymho/cm at 25C
and 6.4, respectively.


CHAPTER I
INTRODUCTION
In the United States, the Water Pollution Control Act of 1972
(PL 92-500), as recently amended, requires acceptable methods for the
utilization and disposal of wastewater effluents and sludges
(Willems 1976). It now appears that land disposal of wastewater
effluents and sludges is a viable and attractive alternative to other
disposal practices. Land spreading of wastewater effluents and sludges
has many advantages, including the addition of plant nutrients, water
conservation, improvement of soil physical properties, and increased
soil organic matter. However, concern has been raised over the con
tamination of groundwater and surface waters with nitrates, heavy
metals, and microbial pathogens, particularly viruses (Bitton 1975;
Bitton 1980b; Burge and Marsh 1978; Gerba et afL 1975).
Viruses are generally associated with wastewater solids (Cl i ver
1976; Lund 1971) and a significant fraction of these viruses is
transferred to sludge as a result of wastewater treatment processes.
Sludge treatment processes, such as anaerobic digestion, do not
completely inactivate or remove viruses (Bertucci et al_. 1977).
Therefore, the application of anaerobically digested sludge
onto land can lead to groundwater contamination as a result of virus
transport through the soil matrix. The movement of sludge-associated
viruses is probably limited due to the immobilization of sludge solids
1


FIGURE 5-2. Photograph of a large soil core of Eustis fine sand
(LC4) shown with thermocouples placed at the soil sur
face and at depths of 2.5, 10, and 20 cm
The thermocouples were used to monitor the soil
temperature. Details on the procedures used to
prepare this soil core appear on pages 183 to
184 and on pages 193 to 196.


266
Berg, 6., R. B. Dean, and D. R. Dahling. 1968. Removal of poliovirus
1 from secondary effluents by lime flocculation and rapid sand
filtration. J. Am. Water Works Assoc. 60: 193-198.
Bertucci, J. J., C. Lue-Hing, D. Zenz, and S. J. Sedita. 1977. Inac
tivation of viruses during anaerobic sludge digestion. J. Water
Pollut. Control Fed. 49: 1642-1651.
Bitton, G. 1975. Adsorption of viruses onto surfaces in soil and water.
Water Res. 9_: 473-484.
Bitton, G. 1978. Survival of enteric viruses. In Water Pollution
Microbiology. Volume 2, pp. 273-299. Edited by R. Mitchell.
John Wiley and Sons, Inc., New York, N.Y.
Bitton, G. 1980a. Adsorption of viruses to surfaces: Technological and
ecological implications. In Adsorption of Microorganisms to
Surfaces, pp. 331-374. Edited by G. Bitton and K. C. Marshall.
John Wiley and Sons, Inc., New York, N.Y.
Bitton, G. 1980b. Introduction to Environmental Virology. John
Wiley and Sons, Inc., New York, N.Y.
Bitton, G., M. J. Charles, and S. R. Farrah. 1979a. Virus detection
in soils: A comparison of four recovery methods. Can. J.
Microbiol. 25: 874-880.
Bitton, G., J. M. Davidson, and S. R. Farrah. 1979b. On the value of
soil columns for assessing the transport pattern of viruses
through soils: A critical outlook. Water, Air, and Soil Pollu
tion 12: 449-457.
Bitton, G., and S. R. Farrah. 1980. Viral aspects of sludge applica
tion to land. American Society for Microbiology News 46: 622-
625.
Bitton, G., N. Lahav, and Y. Henis. 1974. Movement and retention of
Klebsiella aerogenes in soil columns. Plant and Soil 40: 373-
m~.
Bitton, G., N. Masterson, and G. E. Gifford. 1976. Effect of a
secondary treated effluent on the movement of viruses through a
cypress dome soil. J. Environ. Qua!. J5: 370-375.
Bixby, R. L., and D. J. O'Brien. 1979. Influence of fulvic acid on
bacteriophage adsorption and complexation in soil. Appl.
Environ. Microbiol. 38: 840-845.
Blake, G. R. 1965. Bulk density. In Methods of Soil Analysis. Part
1. Physical and Mineralgica! Properties, Including Statistics


2
in the top portion of the soil profile (Oliver 1976). However, there
are "free" viruses which have not become associated with the sludge
solids or which dissociated from these solids as a result of changes
in the physico-chemical properties within the soil matrix, and which
may move through the soil to contaminate groundwaters. The movement
of these individual particles through the soil has been reviewed by
Bitton (1975) and Gerba et al_. (1975), and is dependent on the type of
soil, flow rate, degree of saturation of pores, pH, conductivity, and
the presence of soluble organic materials.
A multidisciplinary project designed to study the effect of
sludge application on crops, land, animals, and groundwater was under
taken by researchers at the University of Florida, and was funded by
the U.S. Environmental Protection Agency (Edds et al_. 1980). A virus
study was included since virtually little is known on the survival and
movement of sludge-bound viruses in soils. The major objective of this
study was to assess the potential health risk, from viral pathogens, of
sludge application to soils. This objective was achieved by studying
the following:
1. Effect of sludge type on poliovirus association
with and recovery from sludge solids (Chapter III)
2. Poliovirus transport studies involving soil cores
treated with virus-seeded sludge under laboratory
conditions (Chapter IV)
3. Retention and inactivation of enteroviruses in soil
cores treated with virus-seeded sludge and exposed
to the North-Central Florida environment (Chapter V)


152
TABLE 4-15. Continued.
No. of
pore
volumes9
eluted
Poliovirus
el uted
(total PFU)
% of Total,
PFU applied0
(cumulative)
Conductivity
of pore volume
collected
(pmho/cm
at 25C)
pH of pore
volume
collected
3 2/3
0
0.1
54
5.6
4
0
0.1
54
5.7
4 1/3
0
0.1
49
6.4
a0ne pore volume for these cores equals 471 ml. The undisturbed
soil cores were 54 cm in length and 5.0 cm internal diameter; consists
of the Al, A2, Bit and B21t horizons of the Red Bay sandy loam (see
Table 4-3). The cores were not conditioned.
b0ne inch or 2.5 cm (51.6 ml) of anaerobically digested sludge
(GDANsee Table 3-2 ; solids content, conductivity and pH equal to
2.0%, 3250 umho/cm at 25C and 8.3, respectively) seeded with a total
of 5.1 x 10$ PFU of poliovirus was applied to each of the cores. The
cores were then placed on the roof of the Environmental Engineering
Sciences building at the University of Florida, Gainesville. The ap
plied sludge was allowed to air dry for 24 hrs and then was worked
under 2.5 cm. Elution with rain water was subsequently undertaken.
This solution was applied from an inverted, self-regulated, 1 liter
Erlenmeyer flask set to maintain a 2.5 cm hydraulic head on the cores.
The flow rate through the cores was measured at 2.4 ml/min. The rain
water was collected next to the Environmental Engineering Sciences
building. See Table 4-2 for chemical characteristics of the rain
water.


45
disposal method of the future (U.S. Environmental Protection Agency
1974, 1978b). Of the total amount of sludge disposed of in 1979
nationally, approximately 25% was applied to land (Pahren et al_. 1979).
In addition to achieving the goal of disposal, the application of
sludge to strip-mined land and to cropland provides resource utiliza
tion in land reclamation and crop production, respectively (U.S. Environ
mental Protection Agency 1974, 1978a, 1978b).
Sludg.eapplication benefits cropland in several ways. Nutrients
which are abundantly present in municipal sludge are utilized effec
tively by growing plants (Cowlishaw and Roland 1973; Pahren 1980; Pahren
et al_. 1979; U.S. Environmental Protection Agency 1974, 1978a, 1978b).
Sludge application also improves several soil properties which are
important for crop growth. For example, sludge addition increases the
water content, water retention capacity, cation exchange capacity
(CEC), organic carbon content and stable aggregate content of soils
(Epstein 1975; Epstein et aj_. 1976). The application of lime-
stabilized sludge increases soil pH and can, therefore, increase sig
nificantly the productivity of acidic soils such as those found in many
humid regions (Brady 1974). Land application of sludge involving
resource utilization (e.g., cropland application) has distinct advan
tages over disposal-only methods and is favored by the U.S. Environmental
Protection Agency (Pahren 1980; U.S. Environmental Protection Agency
1978a). It has been estimated that only 1.3% of cultivated lands would
be required for the application of all the sludge and animal waste
produced in the United States (U.S. Environmental Protection Agency
1978b).


205
on 2 June 1978. Figure 5-5 shows the hourly soil temperature profile
on 17 June 1978. A minimum in soil temperature was observed at 6 a.m.
and a maximum at 2 p.m.
With regards to rainfall, the study period was very wet with
13.63 cm of cumulative rainfall measured from 2 June through 7 July
1978 (see Table 5-5). Poliovirus survival was monitored in two soil
cores which had been treated with virus-seeded sludge (see Table 5-5).
In contrast to the first survival experiment (see Table 5-4), there was
no drastic decline in virus numbers in the drying sludge prior to the
sludge being mixed with the top 2.5 cm of soil. Soil monitoring
revealed that poliovirus could be detected for up to 35 days in both
soil cores. It is difficult to correlate virus survival with soil
moisture since this parameter was not continuously monitored. Heavy
rainfall, however, did not allow the soil (or sludge on the soil sur
face) to dry for an extended period of time and this probably con
tributed to longer virus survival (see Table 5-5).
Monitoring of soil leachates from 5 June to 24 August 1978 did
not reveal any virus, despite their concentration by membrane filtra
tion (see Table 5-6). Although 51 cm of rain fell during the study
period, this represented only 0.5 to 0.7 pore volume. This is the
reason why we conducted parallel studies with smaller cores (5 cm i.d.
instead of 15.5 cm i.d.) which were also exposed to natural conditions,
and treated with virus-seeded sludge and then leached with rainwater
(the experimental leaching was continued until approximately one pore
volume of leachate was collected) in addition to natural rainfall. In
these core studies, some virus breakthrough was observed, but this


49
5. A minimum distance of 250 ft (ca. 76 m) to the
nearest residence
6. Access to the sludge disposal site should be
restricted
Sludge disposal on properly managed sites in the United States has been
successful and has not led to significant problems (Manson and Merritt
1975; U.S. Environmental Protection Agency 1974).
Fate of Sludge-Associated Viruses in Soils
Literature pertaining to the survival and possible movement of
enteric viruses in sludge-treated soils is presented in the Introductions
to Chapters IV through VI.


170
be detected in any of the soil leachates despite the concentration of
the leachates by membrane filtration (160-fold concentration).
Lime-Stabilized, Chemical Sludges
Large quantities of chemical sludge are usually produced during
primary treatment and may be stabilized with the use of lime (Farrell
et aj_. 1974). The effect of lime stabilization on the infectivity of
poliovirus present in chemical sludges (alum and ferric chloride ) was
investigated next. The fate of poliovirus following the application of
lime-stabilized, chemical sludges to soil columns was also studied.
The stabilization of chemical sludges (alum and ferric chloride
sludges) with lime resulted in almost comDlete inactivation of polio
virus (Tables 4-23 and 4-24), It was, thus, not surprising to observe
that the virus did not break through when the stabilized sludges were
applied to soil columns of Red Bay sandy loam which were subsequently
leached with ten pore volumes of rainwater (Table 4-25).
These results show that lime stabilization of chemical sludges
can effectively inactivate viruses. Moreover, as in the case of
conditioned-dewatered sludge and chemical sludges, viruses remaining
in lime-stabilized, chemical sludges can be effectively retained by soils
following sludge disposal on land.
Effect of Soil Bulk Density on Poliovirus Transport
The effect of soil bulk density on poliovirus transport was studied
using laboratory-packed soil columns of Red Bay sandy loam subsoil. The
air-dried soil was packed into l.Q-cm acrylic plastic columns at bulk
densities of 1.45 and 1.60 g/cm^, Poliovirus was suspended in primary


SC2
06 Jun.
6.03
40
40
(0.2)
06 Jun.
(L.R.)
22.34
282
322
(1.4)
08 Jun.
23.59
21
343
(1.5)
13 Jun.
26.34
54
397
(1.7)
23 Jun.
(L.R.)
58.61
365
762
(3.3)
10 Jul.
65.34
68
830
(3.5)
10 Jul.
(L.R.)
81.90
340
1170
(5.0)
13 Jul.
83.42
19
1189
(5.1)
03 Aug.
104.80
140
1329
(5.7)
24 Aug.
115.77
160
1489
(6.4)
(75.9)
0
0
770
6.8
3.9 x 102
0.0006
370
6.5
1.0 x 101
0.0006
7.3
0
0.0006
118
7.3
0
0.0006
480
6.3
0
0.0006
148
7.0
0
0.0006
160
6.7
0
0.0006
462
7.2
0
0.0006
82
6.6
0
0.0006
80
6.9
ro
CJ
a0ne inch (or 2.5 cm) of lagooned sludge seeded with a total of 6.6 x 10^ PFU of poliovirus was
applied on top of soil cores. The soil cores were exposed to natural conditions.
bValues in parentheses represent the cumulative number of pore volumes eluted. One pore volume for
the small soil cores equals 234 ml.
Q
The small soil cores were priodically leached with rain water applied from inverted, self-regulated,
1-liter Erlenmeyer flasks set to maintain a 2.5-cm hydraulic head on the columns. The experimental
leaching was continued until approximately one pore volume of leachate was collected and these leachates
are designated L.R. (or leaching with rain).
^Values in the second parentheses represent the number of centimeters of cumulative leachate
volume.


This dissertation was submitted to the Graduate Faculty of the College
of Engineering and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
August, 1982
/ G .
Dean, College of Engineering
Dean for Graduate Studies and Research


249
TABLE 6-6*- Analysis of sludge and groundwater samples from the Jay
site for the presence of indigenous enteroviruses
Sample3
Viruses detected
(TCID5Qb/g dry wt.)
Viruses identified
Diqested sludge0 added
to the sludge lagoon
Aerobic
Anaerobic
2 to 260
14 to 260
2 to 7
Poliovirus 1, 2, and 3,
echovirus 1 and 7, and
coxsackievirus B4
Lagooned sludqed (3%)e
<0.1 to 100
Poliovirus 1 and 2,
echovirus 7 and 15,
and coxsackievirus B4
Lagooned sludge applied
to land
<0.01 to 4.6
Poliovirus 1, echovirus
1,4, and 7, and
coxsackievirus B4
Day 0 (9%)e
Day 2 (60%)e
Day 9 (81%)e
1.4 to 4.6
0.10 to 0.72
<0.01 to 0.02
f
Groundwater
0
None
. aData were adapted from Farrah et al,(1981a).
bRefers to the 50% tissue culture infective dose.
cAerobically digested and anaerobically digested sludge from the
Montclair and Main Street wastewater treatment plants of Pensacola,
Florida, and Pensacola, r Florida, respectively (PDA and PDAN, respec
tively; see Table 3-2). Digested sludge samples were obtained from 17
February 1978 to 12 February 1979.
dLagooned sludge samples were obtained on a monthly basis from 17
February 1978 to 24 January 1979.
eSludge solids content was expressed as a percentage on a weight
to volume basis.
^Groundwater from several wells at the Jay site (see Figure 6-4)
was monitored for the presence of viruses. Over a l.ryear period, a
total of 5,950 liters (1,100 to 2,650 liters per well) of groundwater
was tested.


128
TABLE 4rl0. Continued.
No. of pore
volumesa
eluted
Poliovirus
eluted
(PFU/ml)
% of Influent^
poliovirus
concentration
Conductivity
of pore
volume collected
(pmho/cm at 25C)
pH of pore
vol ume
col lected
8
0
0
1300
5.5
9
0
0
1400
5.4
a0ne pore volume for the columns used equals 71 ml. The laboratory-
packed columns were 10 cm in length and 4.8 cm internal diameter. The
sample of Eustis fine sand subsoil used consisted mainly of the A21 and
A22 horizons (see Table 4-3). Columns 1 and 2 were conditioned with
5 pore volumes of distilled water and 0.01 N CaCl2, respectively. All
solutions were applied continuously to the columns at approximately
5 ml/min using a peristaltic pump (Buchler, Fort Lee, N.J.).
^Poliovirus was seeded in the influents of column 1 (i.e., sludge
diluted with distilled water) and column 2 (i.e., sludge diluted^with
0.01 N CaCl2) at concentrations of 5.9 x 10^ PFU/ml and 2.6 x 10H
PFU/ml, respectively.
cThe anaerobically digested sludge (PDAN--see Table 3-2) used had
a solids content of 1.4% and a pH of 7.2. Chemical parameters were not
measured for the sludge diluted (1:50) with distilled water or 0.01 N CaC^.


93
TABLE 4-2. Chemical parameters for the rain water used in this study
Parameter
Value3
Parameter
Value3
pH
4.46
K
0.15
Conductance13
23.9
Ca
0.50
TOC
5.20
Mg
0.07
TKN
0.69
Cl"
0.71
nhJ-n
0.10
s4'2
1.84
NO" -N
0.17
Cd
5.7
Ortho-P
0.016
Pb
15.2
Total-P
0.032
Cu
39.4
Na
0.33
Zn
28.2
aData were adapted from Hendry (1977). The values shown repre
sent average weighted concentration of individual rain events col
lected next to the Environmental Engineering Sciences building,
University of Florida, Gainesville, from June 1976 to May 1977.
^Value in ymho/cm. The values for Cd, Pb, Cu, and Zn are in
yg/1. All other values except pH are in mg/1.


TABLE 3-3. Recovery of poliovirus type 1 from unfractionated sludge by dilution and subsequent
direct assay on cell cultures
Sludge
type
Sludges
used
No. of
experimental
trials
Calculated
virus3
input
(mean, total PFU)
Mean recovery
of calculated
virus input
(% SEC)
Mixed liquor
UML, and
GML
2
4.8 x 106
108.9 6.6
Aerobically
digested
UDA,
PDA,
GDA90, and
GDA180
11
8.2 x 107
103.6 15.7
Anaerobically
digested
GDAN,
PDAN, and
LAGd
10
2.1 x 108
98.1 9.0
aPoliovirus was added to 1000 ml of sludge while stirring the mixture using a magnetic stirrer.
The calculated virus input was based on the volume of poliovirus stock of known titer added to the
sludge. Magnetic stirring was continued for 10 min to 60 min.
^Following the contact period, an aliquot of the unfractionated sludge (i.e., the sludge solids
were not separated by centrifugation) was diluted in PBS containing 2% FCS, 250 U of penicillin per
ml, 125 yg of streptomycin per ml and phenol red, and assayed by direct inoculation into cell cultures.
Abbreviation for standard error.
^The lagoon sludge is a mixture of aerobically digested sludge (1/3) and anaerobically digested
sludge (2/3), and consequently, its properties are close to those of anaerobically digested sludges
(see Table 3-2). Therefore, lagoon sludges were placed in the population of anaerobically digested
sludges.


7
adsorption of the virus to the aluminum phosphate floes and, therefore,
the removal efficiency increased with a corresponding increase in the
quantity of phosphate precipitated. These investigators also found
that the removal efficiency of poliovirus was influenced by pH with
maximum removal (i.e.., >96%) observed at pH 6.4, and a reduction in
removal at pH 5.1 and 7.3. Moreover, only 40% of the polioviruses
associated with the aluminum phosphate floes were recovered when the
floes were dissolved in 0.1 N NaOH, final pH 8.3 to 9.3 (see Brunner and
Sproul 1970). These investigators attributed their lack of recovery
of the adsorbed viruses to viral inactivation during the precipitation
process. However, it is likely that the method used to dissolve the
floes was inefficient in the recovery of the adsorbed viruses. Brunner
and Sproul (1970) also studied the removal of poliovirus (93 to >97%)
from filtered wastewater effluent during phosphate precipitation with
alum. In this medium, virus removal increased with decreasing pH from
6.9 to 5.0. This was attributed to an increase in suspended solids
removal with decreasing pH. Bacteriophages have also been effectively
removed (93 to >99%) from water following flocculation with alum
(Brunner and Sproul 1970; Chang et al_. 1958; Chaudhuri and Engelbrecht
1972; York and Drewry 1974).
In addition to alum, ferric chloride has also been tested as a
coagulant in the removal of viruses from water. Chang et al_. (1958)
reported 96.6 and 98.1% removal of seeded coxsackievirus A2 after
flocculation at pH 6.2 in distilled water-SiO^-NaHCO^ buffer with 20 and
40 ppm of ferric chloride (as FeClg), respectively. Similarly, Sobsey


274
Hurst, C. J., C. P. Gerba, and I. Cech. 1980a. Effects of environ
mental variables and soil characteristics on virus survival in
soil. Appl, Environ. Microbiol. 40: 1067-1079.
Hurst, C. J., C. P. Gerba, J. C. Lance, and R. C. Rice. 1980b. Sur
vival of enteroviruses in rapid-infiltration basins during
the land application of wastewater. Appl. Environ. Microbiol.
40: 192-200.
Jannasch, H. J., C. 0. Wirsen, and C. D. Taylor. 1976. Undecompressed
microbial populations from the deep sea. Appl. Environ.
Microbiol. 32: 360-367.
Johnson, F. H., M. B. Baylor, and D. Fraser. 1948. The thermal
denaturation of tobacco mosaic virus in relation to hydrostatic
pressure. Arch. Biochem. 19: 237-245.
Johnson, F. H., and D. H. Campbell. 1946. Pressure and protein de
naturation. J. Biol. Chem. 163: 689-698.
Jones, R. A., and G. F. Lee. 1978. Chemical agents of potential
health significance for land disposal of municipal wastewater
effluents and sludges. In Proceedings of the Conference on
Risk Assessment and Health Effects of Land Application of
Municipal Wastewater and Sludges, pp. 27-60. Edited by B. P.
Sagik and C. A. Sorber. The University of Texas at San
Antonio, San Antonio, Texas.
Katzenelson, E., I. Buium, and H. I. Shuval. 1976a. Risk of com
municable disease infection associated with wastewater irriga
tion in agricultural settlements. Science 194: 944-946.
Katzenelson, E., B. Fattal, and T. Hostovesky. 1976b. Organic
flocculation: An efficient second-step concentration method
for the detection of viruses in tap water. Appl. Environ.
Microbiol. 32: 638-639.
Klute, A. 1965. Laboratory measurement of hydraulic conductivity
of saturated soil. In Methods of Soil Analysis. Part .
Physical and Mineralgica! Properties, Including Statistics of
Measurement and Sampling, pp. 210-221. Edited by C. A. Black,
D. D. Evans, J. L. White, L. E. Ensminger, F. E. Clark, and
R. C. Dinauer. American Society of Agronomy, Inc., Madison,
Wisconsin.
Koch, G. 1960. Influence of assay conditions on infectivity of heated
poliovirus. Virology 12: 601-603.
Kollins, S. A. 1966. The presence of human enteric viruses in sewage
and their removal by conventional sewage treatment methods.
Advances in Appl. Microbiol. 8_: 145-193.


5
PFU/£ of virus in raw sewage from the Mililani (Oahu, Hawaii) sewage
treatment plant. Mack et aj_. (1962) found a maximum of 62,800 PFU/2.
of virus in raw sewage. In Austin, Texas, raw sewage, Moore et al.
(1977) reported virus concentrations between 140 and 1,490 PFU/,.
Wellings et aj_. (1974, 1975) found virus concentrations ranging from
54 to > 161 PFU/S, in raw sewage from two locations in Florida. Clearly,
a wide range of virus concentrations and types is found in raw waste-
water. The virus concentration detected in raw wastewater depends on
the geographical location, season of the year (Lund et al_. 1969) and
virus recovery method used (Buras 1974; Foster and Engelbrecht 1973).
Removal of Viruses by Wastewater Treatment Processes
Wastewater treatment processes (Fair et al_. 1968; Zoltek and
Mel ear 1978) vary in their ability to remove pathogenic viruses.
Several authors have reviewed the literature on virus removal by waste-
water treatment processes (Berg 1973a; Elliott and Ellis 1977; Foster
and Engelbrecht 1973; Grabow 1968; Kollins 1966; Malina 1976; Sproul
1976). In this section, the emphasis will be placed on the two treat
ment processes that generate the most sludge. These processes are
primary sedimentation, with or without chemical addition, and acti
vated sludge (i.e., secondary treatment).
Primary Sedimentation
Primary sedimentation is the most common and sometimes the only
treatment prior to final disposal of wastewater (Kollins 1966;
Grabow 1968). The detention time of wastewater in this treatment


189
indicated by the data in Table 5-2, substantial retention of polio
virus by the ceramic cups at the bottom of the large soil cores is
quite likely. In light of the research by Wang et al_. (1980b), a
significant fraction of echovirus type 1 would probably also be lost
during passage through the ceramic cups on the large soil cores.
Due to the possible viral loss, the entire leachate volume from the
large soil cores must be evaluated for the presence of viruses.
Application of virus-seeded sludge to soil cores. Sludge
seeded with poliovirus was applied to two large soil cores (i.e.,
LC3 and LC4see Figure 5-1) in October 1977, June 1978, and October
1978. Poliovirus-seeded sludge was also applied to two small soil
cores (i.e., SCI and SC2see Figure 5-1) in June 1978 and to one
small core (i.e., SC2) in October 1978. Sludge seeded with echovirus
was applied to two other large soil cores (i.e., LC1 and LC2see
Figure 5-1) and to one small core (i.e., SCI which had received polio
virus-seeded sludge in June 1978) in October 1978. Each soil core
was treated with 2.5 cm of virus-seeded sludge which is equivalent
3
to a liquid application rate of 254 m /ha. The applied sludge was
allowed to soak in and dry on top of the soil for one to four days.
During this period, the drying sludge solids on the soil surface of
the large soil cores were monitored for the presence of viruses, as
described below. Following the drying period, the sludge resting on
the soil surface was mixed with the top 2.5 cm of soil. The top 2.5
cm of soil in the large soil cores was then monitored for the presence
of seeded viruses as described below. Soil monitoring was continued


285
BIOGRAPHICAL SKETCH
Oscar Carlos Pancorbo was born November 27, 1953, in Mantanzas,
Cuba. There, he attended parochial school until his immigration to
the United States in October 1961. In May 1971, he graduated from
Immaculata-La Salle High School in Miami, Florida. In August 1974,
he graduated from the University of Florida with a Bachelor of Science
in zoology with Honors. Following graduation, he enrolled in the
Graduate School of the University of Florida and in June 1976, was
awarded the degree of Master of Science in environmental engineering
sciences. Since August 1981, he has been employed as an Assistant
Professor in the Department of Environmental Health at East Tennessee
State University, Johnson City.
Oscar Carlos Pancorbo is a member of the American Society for
Microbiology, American Association for the Advancement of Science,
Florida Academy of Sciences, and of the Honor Societies of Phi Kappa
Phi (General Scholarship), Tau Beta Pi (Engineering) and Epsilon Nu
Eta (Environmental Health). He is married to the former Ambrosina
Pita and they have two daughters, Adrianne and Amanda.


130
TABLE 4-11. Retention of poliovirus type 1 by a 10 cm packed column of
Eustis fine sand subsoil when suspended in anaerobically
digested sludge diluted (1:50) with 0.01 N CaCl2, and after
subsequent application of rain water and 0.01 N CaCl2
No. of pore
volumes9
eluted
Poliovirus
eluted
(PFU/ml)
% of Influentb
poliovirus
concentration
Conductivity
of pore
volume collected
(pmho/cm at 25C)
pH of pore
volume
collected
Sludge0 diluted (1:50) with 0.01 N CaCl2 and seeded with poliovirus
1
0
0
1280
4.7
2
0
0
1335
4.9
3
0
0
1380
4.9
4
0
0
1400
5.0
5
0
0
1430
5.1
6
0
0
1430
5.2
7
0
0
1430
5.3
8
0
0
1420
5.2
9
0
0
1420
5.2
Shift to nonseeded rain
water0*
10
0
0
1360
5.1
11
0
0
315
5.0
12
0
0
150
5.0
13
0
0
88
6.6
14
0
0
63
6.6


APPENDIX
COMPOSITION OF MEDIA AND SOLUTIONS
USED IN ENTEROVIRUS ASSAYS
1. Gey's Balanced Salt Solution (BSS) is the common diluent for cell
cultures:
Gey's A (10x): 70 grams NaCl
3.7 grams KC1
3.01 grams Na2HP04 12H20
0.237 grams KH2PO4
100 ml 0.1% phenol red
10 grams glucose
900 ml glass distilled water
5 ml chloroform, as a preservative
This stock solution of Gey's A is stored at room
temperature unautoclaved, and is diluted 1:10 and
autoclaved when needed.
Gey's B (20x): 0.42 grams MgCl2 6H2O
0.14 grams MgS04 7H20
0.34 grams CaCl2
100 ml glass distilled water
Gey's C (20x): 2.25 grams NaHC03
100 ml glass distilled water
Bubble CO2 into Gey's C until pH is less than 7.6.
Dispense and tightly cap.
Gey's B and C are autoclaved without further dilution.
To make the complete Gey's Balanced Salt Solution (BSS) add:
90 parts Gey's A (lx)
5 parts Gey's B (20x)
5 parts Gey's C (20x)
2. Hepes buffer (1 M) stock solution:
47.7 grams Hepes
190 ml Gey's A (lx)
260


244
Results and Discussion
Kanapaha Sludge Disposal Site
Although indigenous enteroviruses were readily recovered from
the wasted sludge solids of the Main Street wastewater treatment plant
(Gainesville, Florida), aerobic digestion for 90 days reduced the
solids-associated viruses in sludge to almost undetectable levels (see
Table 6-3). At this treatment plant, the sludge was further digested
aerobically for an additional 90 days (i.e., total digestion time of
180 days), conditioned with a cationic polymer, dewatered by centrifu
gation, and then disposed of at the Kanapaha site. As shown in Table
6-3, indigenous enteroviruses could not be recovered from the
conditioned-dewatered sludge solids. In light of this fact, it is
understandable that no indigenous enteroviruses were ever detected in
topsoil and groundwater samples from the Kanapaha site (see Table 6-4).
Thus, apparently by increasing the sludge digestion time at the waste-
water treatment plant, the viral hazard of sludge disposal on land was
eliminated.
Jay Sludge Disposal Site
Lagooned sludge (2/3 anaerobic and 1/3 aerobic) has been
applied to land for many years at the Jay site. The sludge is allowed
to dry on top of the soil for 2 to 14 days, and then is turned under
the soil. As shown in Table 6-5, indigenous enteroviruses were not
detected in 100-g topsoil samples obtained, over an eight-month
period, from plots at the Jay site which received 15 acre-inches of
lagooned sludge yearly. Farrah et al_. (1981a) readily recovered


126
pertaining to the movement of poliovirus through the Red Bay sandy
loam treated with sludge which had been diluted in distilled water.
No virus breakthrough was detected in the soil leachates following
percolation of 8.5 pore volumes. The application of sludge diluted
with distilled water resulted in a gradual soil clogging and the per
colation experiment was ended when only 8.5 pore volumes had been
collected. In this particular experiment, the specific conductance was
around 90 ymhos/cm and was probably sufficient to promote virus adsorp
tion to the soil.
Eustis fine sand. The transport pattern of poliovirus suspended
in anaerobically digested sludge diluted in distilled water or 0.01 N
calcium chloride was next evaluated using columns of Eustis fine sand.
As shown in Table 4-10, poliovirus suspended in sludge diluted in
distilled water was found to rapidly move thorugh the soil and appear
in the column effluent. Breakthrough occurred by the first pore
volume (0.1%) and reached a maximum at the seventh pore volume of 39.9%
of the influent poliovirus concentration. Fractions beyond the seventh
pore volume could not be collected because of clogging of the column.
The results for sludge diluted with 0.01 N calcium chloride, on the
other hand, show no virus breakthrough by the ninth pore volume beyond
which the column became clogged (see Table 4-10). Thus, it was observed
that the change in the ionic composition of sludge when diluted in
distilled water allowed for rapid virus transport through the soil
column. It is postulated that a reduction in the specific conductance
of the sludge diluted in distilled water resulted in poor virus


1S7
As shown in Table 5-2, 38.7% (mean for the two ceramic cups) of polio
virus suspended in rain leachate was lost (presumably retained by
ceramic cups) during passage through the porous ceramic cups. Other
investigators have found substantially greater retention of poliovirus
type 1 by similar porous ceramic cups (i.e., 75% to 99.7%) when the
virus was seeded in dechlorinated tapwater or in unchlorinated activated
sludge effluent (Sobsey 1976; Wang et al_. 1980b). These solutions
displayed greater conductivity values [e.g., 580 umho/cm for tapwater
and 787 ymho/cm for activated sludge effluent (Wang jit al_. 1980b)] than
that found for the rain leachate (i.e., 18 ymho/cm--see Table 5-2)
employed in this study. Consequently, the abundantly present salts
in tapwater and activated sludge effluent promoted greater viral
adsorption to the ceramic material. Wang et al_. (1980b) also demon
strated that echovirus type 1 (strain V239--isolated from groundwater)
seeded in tapwater or activated sludge effluent was retained (i.e.,
30% to 86%) by ceramic cups but to a lesser degree than observed for
poliovirus type 1. Since the strain of echovirus type 1 used was
previously shown to adsorb poorly to soil, it is likely that this
virus did not adsorb efficiently to the ceramic material either (Wang
et al_. 1980b). In addition to viruses, fecal coliforms in water have
also been reported to be retained by ceramic cups (Dazzo and Rothwell
1974). It is worth noting that the rain leachate used herein to
evaluate viral retention by ceramic cups closely approximates the
chemical composition of soil water actually passing the ceramic cups
installed at the bottom of the large soil cores (see above). As
p


aOne pore volume for the column used equals 225 ml. The laboratory-packed column was 29 cm in length
and 4.8 cm internal diameter; the column was filled only 27 cm with soil (2 cm left on top for packed
sludge). The sample of Red Bay sandy loam subsoil used consisted mainly of the A2 and Bit horizons (see
Table 4-3 ). The column was conditioned with 5 pore volumes of distilled water. All solutions were ap
plied continuously to the column at approximately 5 ml/min using a peristaltic pump (Buchler, Fort Lee,
N.J.).
^Poliovirus was seeded in the influent (i.e., sludge diluted with distilled water) at a concentration
of 1.1 x 10^ PFU/ml. The anaerobically digested sludge (GDAN--see Table 3-2) used had a solids content
of 2.0%, a conductivity of 3250 ymho/cm at 25C and a pH of 8.3. The conductivity of sludge diluted (1:50)
with distilled water was 210 ymho/cm at 25C and the pH was 7.4.


No. of pore volumes eluted
Conductivity
(x l(r ymho/cm at 25C)
Poliovirus eluted (expressed as cumulative % of total PFU having
been applied at each pore volume)
1 1 ho ro i i * i > ro r\jr\j
ocnocn or'0-P*cr>cooro-t=0'>Ooorv>-fa.


31
1978a). The lime dosage required to exceed, for example, pH 12 for
30 minutes has been found to be affected by the sludge type, chemical
composition of the sludge and percent sludge solids (Farrell et al.
1974; U.S. Environmental Protection Agency 1978a). In addition to
achieving stabilization, lime treatment also conditions the sludge (see
Figure 2-1) such that subsequent sludge dewatering is improved (Farrell
et aJL 1974; U.S. Environmental Protection Agency 1974).
Most bacterial pathogens in raw sludge have been shown to be
destroyed during lime stabilization (Farrell et aj_. 1974; U.S. Environ
mental Protection Agency 1974, 1978a). Fecal streptococci, however,
remain viable during liming (U.S. Environmental Protection Agency
1978a). Moreover, regrowth of bacterial organisms can occur if the
pH of the lime-stabilized sludge is allowed to drop rapidly below 11
(Farrell et a_l_. 1974; U.S. Environmental Protection Agency 1974).
Under ideal conditions, lime treatment is superior to anaerobic diges
tion in the inactivation of bacterial pathogens in raw sludge (U.S.
Environmental Protection Agency 1978a). As a result, the bacterial
pathogen concentrations in lime-stabilized sludges are 10 to 1,000
times lower than in anaerobically digested sludges (U.S. Environmental
Protection Agency 1978a).
As far as enteric viruses are concerned, no research has been
conducted on their fate during lime stabilization of raw sludge
(Farrell et aQ_. 1974). However, since seeded poliovirus type 1 has
been shown to be inactivated during the flocculation of raw sewage with
lime at pH 11.5 and to be undetectable (i.e., apparently fully


46
The practice of applying sludge to land is not without its
problems. Water movement, for example, has been found to be restricted
(i.e., saturated hydraulic conductivity declined) in sludge-treated
soils (Epstein 1975). Such a reduction in the soil conductivity has
been attributed to the clogging of soil pores by microbial decomposition
products (Epstein 1975). If improperly applied, sludge has been found
to inhibit the growth of a previously planted crop. For example, lime-
stabilized sludge was reported to form a filamentous mat on the soil
surface which resulted in the partial inhibition of previously planted
wheat (U.S. Environmental Protection Agency 1978a). No matting or
crop inhibition was observed when lime-stabilized sludge was incorporated
into the soil prior to planting (U.S. Environmental Protection Agency
1978a).
Undoubtedly, the major drawback of sludge application to land
is the possible dissemination of pathogens and toxic chemicals leading
to adverse effects on human and animal life (Burge and Marsh 1978;
Elliott and Ellis 1977; Foster and Engelbrecht 1973; Kowal and Pahren
1978; Pahren 1980; Pahren et^ al_. 1979). In particular, enteric
viruses present in sludge could potentially move through the soil
matrix and contaminate groundwater supplies (see reviews by Berg 1973b;
Bitton 1975; Bitton 1980a; Bitton et^ al_. 1979b; Burge and Marsh 1978;
Burge and Parr 1980; Oliver 1976; Duboise et al_. 1979; Elliott and
Ellis 1977; Foster and Engelbrecht 1973; Gerba et al_. 1975; Moore et al.
1978; Sagik 1975). Due to the poor mixing and slow flow (generally
<1 ft /day) conditions found in aquifers, many pollutants entering the


This dissertation is dedicated to my parents,
and to Ambrosina, Adrianne and Amanda.


190.
until viruses could no longer be detected. The small soil cores were
not used to study viral survival in sludge-treated soil but rather
were used to evaluate viral transport as described before.
Leachates from soil cores. Leachates from all sludge-treated
soil cores were collected during natural rainfall as shown in Figure
5-1. In June and July 1978, the small soil cores (i.e., SCI and SC2
see Figure 5-1) treated with poliovirus-seeded sludge were periodically
leached with rain water (see Table 4-2 for chemical characteristics)
applied from inverted, self-regulated, 1-liter Erlenmeyer flasks set
to maintain a 2.5-cm hydraulic head on the cores (Sanks et al_. 1976).
The artificial leaching consisted of applying the rain water at a flow
rate of approximately 3.9 ml/min until approximately one pore volume
(234 ml for small soil cores) of leachate was collected. All leachate
samples (i.e., natural or artificial) were promptly taken into the
laboratory where their volumes were accurately measured [reported volume
in ml, cm, and pore volumes (calculated according to equations 4-1
and 4-2)]. The pH and conductivity of each leachate sample were then
measured using the procedures described above for sludges. Finally,
each leachate sample was concentrated by membrane filtration and
assayed for seeded viruses as described below.
Virus Recovery Procedures
Sludge. Samples of drying sludge were obtained from the
surface of the large soil cores (one sample per soil core per sampling
date). THe solids content of each sludge sample was then determined as
described below. Seeded viruses (i.e., poliovirus or echovirus) were


95
TABLE 4-3. Some characteristics of the soils under study
Soil9
Soil
horizon
Depth
(cm)
Description
Mechanical composition (%)
Sand
(2-
0.05 mm)
Silt
(0.05-
0.002 mm)
Clay
(< 0.002 mm)
Red
Bay
A1
0-15
Dark brown
fine sandy loam
66.0
20.4
13.6
sandy
loamc
A2
15-30
Yellowish-red
sandy loam
61.0
20.0
19.0
Bit
30-48
Red sandy
clay loam
56.0
15.4
28.6
B21t
48-97
Red light
sandy clay
52.4
11.4
36.2
Eustis
fine ,
Ap
0-25
Dark gray
fine sand
94.8
2.4
2.8
sand0
A21
25-58
Light yellowish-
brown fine sand
94.4
2.0
3.6
A22
58-102
Light yellowish-
brown fine sand
94.3
2.3
3.4
A23
102-135
Light yellowish-
brown fine sand
94.7
1.6
3.7
A24
135-163
Yellowish-brown
fine sand
93.9
1.3
4.8
aAdapted from Calhoun et al_. (1974).
^Identification by x-ray diffraction.
cSample was taken in Santa Rosa County, Florida; West Florida
Agricultural Experiment Station, Jay.
dSample was taken in Alachua County, Florida; agronomy farm,
University of Florida, Gainesville.


280
Shuckrow, A. J., G. W. Dawson, and D. E. Olesen. 1971. Treatment of
raw and combined sewage. Water and Sewage Works 118: 104-111.
Shuval, H. I., and E. Katzenelson. 1972. The detection of enteric
viruses in the water environment. In Water Pollution Micro
biology. Volume 1, pp. 347-361. Edited by R. Mitchell.
John Wiley and Sons, Inc., New York, N.Y.
Sobsey, M. D. 1976. Field monitoring techniques and data analysis.
In Proceedings of the Symposium on Virus Aspects of Applying
Municipal Waste to Land, pp. 87-96. Edited by L. B. Baldwin,
J. M. Davidson, and J. F. Gerber. Institute of Food and Agri
cultural Sciences, University of Florida, Gainesville, Florida.
Sobsey, M. D., C. H. Dean, M. E. Knuckles, and R. A. Wagner. 1980a.
Interactions and survival of enteric viruses in soil materials.
Appl. Environ. Microbiol. 40: 92-101.
Sobsey, M. D., C. P. Gerba, C. Wallis, and J. L. Melnick. 1977. Con
centration of enteroviruses from large volumes of turbid
estuary water. Can. J. Microbiol. 23: 770-778.
Sobsey, M. D., J. S. Glass, R. J. Carrick, R. R. Jacobs, and W. A.
Rutala. 1980b. Evaluation of the tentative standard method
for enteric virus concentration from large volumes of tap
water. J. Am. Water Works Assoc. 72: 292-299.
Sobsey, M. D., C. Wallis, M. Henderson, and J. L. Melnick. 1973.
Concentration of enteroviruses from large volumes of water.
Appl. Microbiol. 26: 529-534.
Sposito, G., and K. M. Holtzclaw. 1977. Titration studies on the
polynuclear, polyacidic nature of fulvic acid extracted from
sewage sludge-soil mixtures. Soil Sci. Soc. Am. J. 41: 330-
336.
Sposito, G., K. M. Holtzclaw, and J. Baham. 1976. Analytical proper
ties of the soluble, metal-comp!exing fractions in sludge-soil
mixtures: II. Comparative structural chemistry of fulvic acid
Soil Sci. Soc. Am. J. 40: 691-697.
Sproul, 0. J. 1972. Virus inactivation by water treatment. J. Am.
Water Works Assoc. 64: 31-35.
Sproul, 0. J. 1976. Removal of viruses by treatment processes. In
Viruses in Water, pp. 167-179. Edited by G. Berg, H. L. Bodily
E. H. Lennette, J. L. Melnick, and T. G. Metcalf. American
Public Health Association, Inc., Washington, D.C.
Stagg, C. H., C. Wallis, C. H. Ward, and C. P. Gerba. 1978. Chlori
nation of solids-associated coliphages. Prog. Water Techno!.
10: 381-387.


No. of pore volumes eluted
Conductivity
(x ICr ymho/cm at 25C)
Poliovirus eluted (expressed as cumulative % of total PFU applied)
O
r>o
GJ
co
cn
ro
cn
Oo
ro
-£=
ro
co co
o co
PH
cn


aOne pore volume for these columns equals 225 ml. The laboratory packed columns were 29 cm in length
and 4.8 cm internal diameter; the columns were filled only 27 cm with soil (2 cm left on top for packed
sludge), and were conditioned with 2 pore volumes of rain water. Thesampleof Red Bay sandy loam subsoil
used consisted mainly of the A2 and Bit horizons (see Table 4-3).
^The chemical sludges were precipitated from virus-seeded, raw sewage. The methods used to produce
these sludges and to determine the amount of viruses present in the sludges are described in Table
The sludges were applied to the soil columns, allowed to soak in, and then, worked under 2.5 cm. Elution
with rain water was subsequently undertaken. The rain water was applied continuously to the columns at
approximately 5 ml/min using a peristaltic pump (Buchler, Fort Lee, N.J.). The rain water was collected
next to the Environmental Engineering Sciences building at the University of Florida, Gainesville. See
Table .4-2 for chemical characteristics of the rain water.
cSoil leachates were collected in 1/2 pore volume fractions and assayed individually for viral in-
fectivity. In order to detect small numbers of viruses, pore volumes 0.5 through 5.0 (and 5.5 through
the final pore volume) were subsequently combined and concentrated 160-fold by membrane filtration. The
concentrates were assayed for poliovirus. This concentration procedure was only performed on the leachates
from the columns receiving alum (Column 2) and ferric chloride sludge.


22
coxsackievirus B3 at 2 log.|0 units per 24 hours. In similar laboratory
anaerobic digesters, Bertucci et al_. (1977) observed the inactivation
rates of enteroviruses [i.e., poliovirus 1 (Sabin), coxsackievirus A9
(Griggs), coxsackievirus B4 (JVB) and echovirus 11 (Gregory)] seeded in
sludge to follow a first-order reaction pattern and to significantly
differ for the four viruses tested.
Ward and his collaborators have done a great deal of work on
the inactivation of enteric viruses seeded in raw and anaerobically
digested sludges. Poliovirus type 1 (CHAT) and poliovirus type
1 (Mahoney) seeded in anaerobically digested sludge (6% sludge solids,
pH 8.0) became largely associated (67 and 65% of the total virus
added, respectively) with the sludge solids and both were inacti
vated at rates of approximately 1 1 og-jq units/5 days at 4C, >2 log^
units/3 days at 20C, and >1 log-jg units/day at 28C (Ward and
Ashley 1976). Anaerobically digested sludge displayed no detectable
virucidal activity against enteroviruses when adjusted to pH values
between 4.5 and 7.5 (Ward and Ashley 1977a). Similarly, there was no
appreciable inactivation of seeded poliovirus type 1 in 5 days at 20C
in raw sludge maintained at its naturally low pH of 6.0 (Ward and
Ashley 1976). However, the virucidal activity against poliovirus in
raw sludge significantly increased as the pH of the sludge was raised
above 7.5 (Ward and Ashley 1977a). Thus, Ward and Ashley (1977a)
demonstrated that the uncharged form of ammonia, which exists mostly
at pH values above 8, was the causative agent in the irreversible
inactivation of enteroviruses in anaerobically digested sludge and in


65
Recovery of Seeded Poliovirus from Sludge Components
The sludge supernatant and sludge solids generated were
separately subjected to the virus recovery methodology described below.
In several experiments, the sludge supernatants were not processed for
virus concentration. These supernatants were simply adjusted to neutral
pH and assayed for viruses as described previously. The number of
viruses thus found were included in the "overall virus recovery" values
reported. In those experiments in which the sludge supernatants were
subjected to the virus concentration procedure, the supernatants were
processed like the sludge solids eluates described below.
The sludge solids-associated viruses were eluted and further
concentrated using a modification of the glycine method developed by
Hurst et al_. (1978). The solids were mixed with five volumes of 0.05 M
glycine buffer, pH 11.5. The pH of the mixture was adjusted to between
10.5 and 11.0 by the addition of 1 M glycine buffer, pH 11.5. The
samples were vigorously mixed for 30 seconds using a magnetic stirrer
and centrifuged at 1,400 x g for 5 minutes at 4C (all centrifuga
tion was performed using a Sorvall RC5-B centrifuge, Ivan Sorvall
Inc., Norwalk, Connecticut). The supernatants (i.e., the sludge solids
eluates) were recovered, adjusted to neutral pH by the addition of 1 M
glycine buffer, pH 2.0, and assayed for eluted viruses. The entire
procedure described above was performed in less than 10. minutes. Thus,
poliovirus was subjected to the high pH of 10.5 to 11.0 for no more
than .10. minutes. Both Hurst et al_. (1978) and Sobsey et al_. (1980b)
observed no appreciable inactivation in .10. minutes of poliovirus type 1


102
liquor or diluted anaerobically digested sludge and was subsequently
applied continuously to soil columns at approximately 5 ml/min using
the peristaltic pump. As detailed above, influent poliovirus concen
tration was determined from viral assays made at the beginning and end
of each column experiment. In several experiments, a shift was made to
the application of nonseeded 0.01 N calcium chloride or rain water con
tinuously (at approximately 5 ml/min) by the peristaltic pump in order
to determine if these solutions could elute adsorbed viruses. The
undiluted sludge samples (i.e., conditioned-dewatered sludge, chemical
sludges and lime-stabilized, chemical sludges) could not be applied to
the soil columns via the peristaltic pump because of the high solids
contents. These sludges were applied directly on top of the soil
columns, were allowed to soak in, and then, were worked under 2.5 cm.
The poliovirus concentrations in these sludges was determined as
described earlier. Following the application of the sludges, the
covers on the soil columns (i.e., the top plates of the soil column
holders) were replaced and the soil columns were then leached with non
seeded rain water. The rain water was applied continuously to the
soil columns at approximately 5 ml/min using the peristaltic pump.
After percolation through the soil, the leachates from all laboratory-
packed columns were collected in sterile screw-capped bottles and
assayed for viral infectivity as described below.
Undisturbed soil cores. Undisturbed soil cores (Blake 1965;
Sanks et a]_. 1976) were also used in poliovirus transport studies.
Undisturbed cores were obtained in driving polyvinyl chloride pipes


TABLE 4-22. Retention of poliovirus type 1 by packed columns of Red Bay sandy loam subsoil following
the application of chemical sludges and the subsequent leaching with rain water
SIudge
Total no.
Sludge^
Sludge
Total no. of poliovirus, PFU
Range of
Range of
type
of pore
vol ume
sol ids
conductivity
pH values
volumesa
applied
applied
Contained in
Passing
values for
for pore
eluted
(ml)
(g)
the sludge
through
pore volumes
volumes
applied
the soilc
collected
collected
(umho/cm
at 25C)
Alum Column 1
5.0
15
-
9.0
X
106
0
43 -
142
5.4 -
5.5
Column
2
10.0
43
0.1
3.7
X
107
0
30 -
240
5.0 -
- 5.6
Ferric
Column
1
chloride
10.0
22
0.1
3.3
X
106
0
32 -
- 175
5.5 -
- 5.7
Column
2
9.0
25
0.1
3.3
X
106
0
31 -
187
4.8 -
5.1
Lime
Column
1
2.0
24
-
6.0
X
104
0
95
- 130
5.4
- 5.6
Column
2
\
10.0
32
0.3
0
0
27
- 145
5.6 -
- 6.9


61
A
B


134
TABLE 4-13. Distribution of poliovirus type 1 in the soil profile
of a 27-cm packed column of Eustis fine sand which had
received virus-seeded, anaerobically digested sludge
diluted (1:50) with 0.01 N CaC^
Deptjj in
column5 (cm)
Poliovirus recovered from soi1b
PFU/g of
wet soil
PFU/soi1
section
% of Total
PFU applied0
Top Sludged
38,200
2.1 x 105
1.6
0-3
1,000
9.9 x 104
0.8
3-5
80
8.1 x 104
0.6
5-7
0
0
0
7-9
0
0
0
9-11
0
0
0
11-13
0
0
0
13-15
0
0
0
15-17
0
0
0
17-19
0
0
0
19-21
0
0
0
21-23
0
0
0
23-25
0
0
0
25-27
0
0
0
aThe laboratory-packed column.was 29 cm in length and 4.8-cm
internal diameter; the column was filled only 27 cm with soil (2 cm
left on top for the packed sludge solids). The column was treated
with virus-seeded diluted sludge as described in Table 4-12. The
soil column was then sectioned and virus was eluted from each soil
section.


114
TABLE 4-4. Effect of soil bulk density on the saturated hydraulic
conductivity of the Red Bay sandy loam subsoil
Bulk
density ^
(dry g/cm )
Saturated hydraulic conductivity*3
Permeability
classd
ml/min
cm/hrc
1.45
33
73
Very rapid
1-60
a. 2
18
Rapid
1.70
0.35
0.77
Moderately slow
1.85
oe
0
Very slow
2.00
oe
0
Very slow
aThe sample of Red Bay sandy loam subsoil used consisted mainly of
the A2 and Bit horizons (see Table 4-3). The bulk densities shown were
produced in soil columns (i.e., 10 cm in length and 4.8-cm internal
diameter; packed 5.0 cm with soil) as described in the Materials and
Methods section.
bThe saturated hydraulic conductivity of each soil column was
measured using the "constant-head" method of Klute (1965).
Calculated using Equation (4-5).
According to Klute (1965).
eNo leachate passed through these columns in a 2-hour period.


30
for enteroviruses versus reoviruses (Ward and Ashley 1978b). Although
most enteroviruses are rapidly inactivated during sludge composting,
reoviruses apparently are capable of surviving this sludge treatment
process (Ward and Ashley 1978b). Clearly, sludge composting does not
yield a virus-free product, and therefore, all composted sludges should
be handled with care during further treatment and final disposal.
Stabilization-1ime treatment. Lime treatment at pH 11.0 to 11.5
is another practice frequently employed to stabilize (see Figure 2-1)
raw sludges (Farrell et al_. 1974; U.S. Environmental Protection Agency
1974, 1978a). During periods when digesters are out of service or when
sludge quantities exceed digester design capacity, lime treatment is an
effective alternate method of sludge stabilization (Farrell et al_. 1974;
U.S. Environmental Protection Agency 1978a). Due to the large quanti
ties of chemical sludges (e.g., alum and iron) usually produced, lime
treatment is particularly suited for the stabilization of these sludge
types (Farrell et al_. 1974). At relatively low costs, lime stabilization
reduces the odor, putrescibility potential and pathogen content of raw
sludges (Farrell et al_. 1974; U.S. Environmental Protection Agency 1974,
1978a, 1978b). However, the effectiveness of this sludge stabilization
procedure is apparently dependent upon the pH achieved and maintained.
Farrell et al_. (1974) demonstrated adequate stabilization of chemical
sludges during lime treatment at pH 11.5 for 30 minutes (pH was main
tained above 11 for 24 hours). Further research has indicated, however,
that the pH must be maintained above 12 for 30 minutes (pH remaining
above 11 for at least 14 days) during liming in order to ensure effec
tive sludge stabilization (U.S. Environmental Protection Agency 1974,


167
TABLE 4-21. Association between poliovirus type 1 and chemical sludge
sol ids
Sludge3
type
Experiment
no.
Virus in
unfractionated0
siudge
(total PFU)
Virus in
sludge
supernatant0
(total PFU)
Viable
unadsorbed0
virus
(%)
Solids-
associated6
virus
(%)
Alum
1
1.2 x 107
3.6 x 105
3.0
97.0
2
4.3 x 107
1.5 x 105
0.3
99.7
3
h-
o
X
5.0 x 104
0.5
99.5
Ferric
1
4.7 x 106
6.5 x 104
1.4
98.6
chloride
2
4.4 x 106
1.2 x 105
2.7
97.3
3
5.2 x 106
7.0 x 104
1.3
98.7
Lime
1
7.5 x 104
0
0
100.0
2
0
0
0
0
aThe chemical sludges were precipitated from virus-seeded, raw sewage.
The methods used to produce these sludges and to determine the amount of
viruses present in the sludges are described in Table 4-20.
^The sludge solids were not separated prior to assaying.
cThe sludge was clarified by centrifugation at 1400 x g for 10 min at
4C and the supernatant was subsequently assayed.
dThe "viable unadsorbed virus (%)" values were calculated as shown in
the Materials and Methods section.
eThe "solids-associated virus (%)" values were estimated as shown in
the Materials and Methods section.


FIGURE 4-5. Movement of poliovirus type 1 through a 10 cm packed
column of Red Bay sandy loam subsoil when suspended
in anaerobically digested sludge liquor
One pore volume for the column used equals 80 ml.
The laboratory-packed column was 10 cm in length
and 4.8 cm internal diameter. The sample of Red
Bay sandy loam subsoil used consisted mainly of
the A2 and Bit horizons (see Table 4-3). The
column was conditioned with 2 pore volumes of sludge
liquor. Poliovirus was then suspended in the sludge
liquor at a concentration of 1.9 x 10^ PFU/ml and
applied to the column. All solutions were applied
continuously to the column at approximately 5 ml/min
using a peristaltic pump (Buchler, Fort Lee, N.J.).
The sludge liquor was produced by centrifuging
anaerobically digested sludge (GDAN--see Table 3-2;
solids content, conductivity and pH equal to
2.0%, 3250 umho/cm at 25C and 8.3, respectively)
at 14,000 x g for 10 min at 4C. This procedure
was performed again on the decanted supernatant
and this yielded the clear sludge liquor. The
conductivity of the sludge liquor was 1580 ymho/
cm at 25C and the pH was 8.1 (see Table 4-1
for other chemical parameters).


261
10 ml Gey's B (20x)
16 ml 2 M NaOH (8g/100 ml)
Dispense and autoclave.
3.Streptomycin-penicillin (lOOOx) stock solution:
Solution I: 1.0 gram streptomycin
8 ml Gey's A (lx)
Solution II: 106 units of penicillin
4 ml of Solution I
Solution II contains 125 mg of streptomycin and
2.5 x 10$ units of penicillin per ml which is
lOOOx of what is required. Therefore, it must
be diluted 1:1000 in the final solution.
4.Eagle's Minimal Essential Medium (MEM) using Gey's BSS plus 10%
fetal calf serum (FCS) (i.e., growth medium):
300 ml Gey's A (lx)
20 ml Gey's B (20x)
20 ml Gey's C (20x)
8 ml MEM essential amino acids (50x)
(International Scientific, Gary,
Illinois)
4 ml vitamins (lOOx) (International
Sci.)
4 ml glutamine (lOOx) (International
Sci.)
0.4 ml streptomycin-penicillin stock
(lOOOx)
40 ml FCS (International Sci.)
5.Solutions required for the removal of cells from glass (trypsini-
zation):
Solution I (pre-trypsin wash):
300 ml Gey's A (lx)
5 ml Gey's C (20x)
Dispense and autoclave. This solution removes
all traces of serum (which contains trypsin inhibi
tors) as well as Ca+2 and Mg+2 ions.


211
represented only 0.0006% of the total viral input (see Table 5-7).
These data show that some virus breakthrough can be achieved, under
saturated flow, in the small cores. This had also been demonstrated
in laboratory studies (see Figure 4-9). Under unsaturated flow in
the large cores, however, no such breakthrough occurred (see Table 5-6).
Third Survival Experiment
(11 October 1978-20 January 1979)
From the results of the first two survival experiments, it
became apparent that with regard to transport pattern, poliovirus type
1 (LSc) would not be the ideal model virus since it has a high affinity
for sludge solids (see Table 5-3) and is subsequently immobilized,
along with the sludge solids, at the top of the soil profile. A virus
with less affinity for sludge solids would perhaps be more suitable
for transport studies. Goyal and Gerba (1979) previously found that
echovirus type 1 (Farouk) poorly adsorbed to soil when compared to
poliovirus type 1. The association between lagooned sludge solids,
and poliovirus type 1 (LSc) and echovirus type 1 (Farouk) was, therefore,
investigated (Table 5-3). As presented above (see Table 5-3), echo-
virus was less adsorbed (20.7%)to sludge solids than poliovirus
(95.2%). Lagooned sludge was, thus, seeded with either of these two
enteroviruses and then applied to soil cores on 11 October 1979. Viral
presence in the soil and leachates was monitored for 21 and 101 days,
respectively.
During the study period, the average soil temperature, as
monitored with thermocouples placed in a soil core (LC4--see Figure 5-2),
ranged from 18C to 27C (Figure 5-6). With regard to rainfall, only


120
(see Table 4-7) that the maximum breakthrough per pore volume was 0.3%.
This table also shows that approximately 99.3% of the viral load was
retained by this soil following leaching with 10 pore volumes of
seeded 0.01 N CaC^- As in the case of the Red Bay sandy loam (see
Table 4-6), this sandy soil has displayed a substantial capability of
removing poliovirus suspended in a calcium chloride solution.
Poliovirus Suspended in Diluted An
aerobically Digested Sludge
Red Bay sandy loam. Anaerobically digested sludge (2% solids
content, w/v), diluted 1 to 50 (v/v) with 0.01 N calcium chloride or
distilled water, was seeded with poliovirus and thoroughly mixed to
bring about the adsorption of the virus to the sludge particles. The
virus-sludge mixtures were then pumped onto the top of Red Bay sandy
loam columns. The dilution of the sludge (1:50) with calcium chloride
or distilled water was necessary in order to facilitate its delivery
by the pump. Under these conditions, 10 pore volumnes of the diluted
sludge corresponded to the application of 2.5 cm (1 inch) of anaerob
ically digested sludge containing 2% solids (w/v). Table 4-8 describes
the movement of poliovirus suspended in sludge diluted in 0.01 N calcium
chloride. No virus breakthrough was detected prior to the seventh pore
volume. At the tenth pore volume, only 0.01% of the total virus applied
appeared in the soil effluent. The subsequent addition of two pore
volumes of sterile rain water (which is the equivalent of 25 cm of
rain) did not elute adsorbed viruses (see Table 4-8). In fact, at the
12th pore volume, the percent of total viruses applied which appeared
in the leachate remained at 0.01. Table 4-9 displays the results


24
50C. At the low natural pH of the raw sludge, the virucidal agent
was not present (Ward and Ashley 1976, 1977a). Therefore, only the
protective effect attributed to the sludge solids was observed for
this sludge type at all temperatures tested (i.e., 43 to 51C) (Ward
et al_. 1976). Ionic detergents were later identified as the components
in raw and anaerobically digested sludge solids which protected polio
virus type 1 (CHAT) and other enteroviruses from heat inactivation
(Ward and Ashley 1977c, 1978a, 1979). In contrast, these ionic deter
gents (cationic more active than anionic) were shown to be responsible
for reducing the heat required to inactivate reovirus type 3 (Dearing)
in raw and anaerobically digested sludge (Ward and Ashley 1977c, 1978a).
Ward and Ashley (1979) demonstrated that two ionic organic detergents,
sodium dodecyl sulfate and dodecyltrimethyl ammonium chloride in buffer
solutions, were potent virucidal agents for reovirus, but that their
virucidal effects were strongly pH dependent. The virucidal activity
against reovirus displayed by these ionic detergents was greater in
alkaline than in acid conditions (Ward and Ashley 1977c). Further
research (Ward and Ashley 1979) revealed that the inactivation pattern
of reovirus as a function of pH at 45C in anaerobically digested
sludge was qualitatively similar to that found in buffer solutions
containing ionic detergents.
The literature reviewed in the paragraph above indicates that
the inactivation of enteric viruses in anaerobically digested sludge is
a complex phenomenon. It is clear, however, that the anaerobic diges
tion process raises the pH of raw sludge and thereby produces in the


192
described by Bitton et al_. (1979a). This method consisted of mixing
each 10-g soil sample with 20 ml of 0.5% (wt./vol.) isoelectric
casein (Difco Laboratories, Detroit, Michigan),. pH 9.0. If necessary,
the pH of the mixture was adjusted to between 9.0 and 9.2 by the
addition of 5 M Trizma base (Sigma Chemical Co., St. Louis, Missouri).
The samples were vigorously vortexed for 30 seconds and then shaken on

a rotating shaker for 15 minutes. The samples were subsequently cen
trifuged at 1,400 x g for 4 minutes at 4C. The supernatants (i.e.,
the soil eluates) were recovered and immediately adjusted to neutral
pH by the addition of 1 M glycine buffer, pH 2.0. Viruses in the soil
eluates were concentrated by organic flocculation (Katzenelson et al.
1976b) as follows. The eluates were adjusted to pH 4.4 by the addition
of 1 M glycine buffer, pH 2.0. The floe produced were pelleted by
centrifugation at 160 x g for 1 minute at 4C. The supernatants were
discarded. The pellets were mixed with 2 ml of 0.15 M Na2HP04, pH 9.0.
The mixtures were adjusted to neutral pH by the addition of 1 M glycine
buffer, pH 11.5, and then magnetically stirred until the pellets were
completely resolubilized. The samples were subsequently centrifuged
at 14,000 x g for 10 minutes at 4C. The supernatants were adjusted
to neutral pH (i.e., if necessary), adjusted to a final concentration
of 2% FCS and assaysed for eluted viruses as described above. The
numbers of viruses recovered were expressed as PFU per g dry weight of
soil.
Leachates. Leachate samples were concentrated by membrane
filtration (Farrah et al_. 1976; Hill et al. 1971; Shuval and Katzenelson


FIGURE 5-1. Photograph of the soil cores of Eustis fine sand used
in this study
Details on the procedures used to prepare these
soil cores appear on pages 183 to 184. Four large
soil cores (i.e., LC1 through LC4) and two small soil
cores (i.e., SCI and SC2) were employed as seen
in the photograph.


TABLE 7-3. Effect of pressurization time on the survival of poliovirus type 1 seeded in seawater at 2C
Pressuri
zation
time
(hr)
Pressure (psi)a
Initial Final
Initial
virus
concentration
(PFU/ml)
Final virus concentration
(PFU/ml)
Poliovirus
recovery at
elevated
pressure
{% of
control)
Mean
poliovirus
recovery
for each
pressure
(% + SE)
At atmospheric
pressure
(control)
At elevated
pressure
2
1,000
1,000
3.5 x 103
3.1 x 103
2.9 x
103
93.5
92.0 + 1.5
1,000
1,000
4.2 x 103
4.2 x 103
3.8 x
103
90.5
8
1,000
1,000
4.5 x 103
2.7 x 103
2.3 x
103
85.2
76.0 + 9.3
1,000
1,000
3.4 x 103
3.0 x 103
2.0 x
103
66.7
24
1,000
800
3.5 x 103
3.5 x 103
5.7 x
102
16.3
15.6 + 0.7
1,000
920
4.0 x 103
2.9 x 103
4.3 x
102
14.8
a0ne atm per 14.7 psi.


129
adsorption to the sludge and soil particles. Poliovirus retained by a
Eustis fine sand column treated with sludge diluted with 0.01 N calcium
chloride was not eluted with rain water (see Table 4-11). It appears,
therefore, that adsorbed viruses in sludge-treated soils are not readily
displaced and transported further down the soil profile by a solution
low in ionic strength such as rain water.
The distribution of poliovirus within Eustis fine sand columns
was investigated next. For this purpose, 29-cm columns were packed
with 27 cm of this soil, and poliovirus was subsequently applied to the
soil surface while suspended in sludge diluted (1:50, v/v) in distilled
water or 0.01 N CaC^. Table 4-12 shows the transport patterns of
poliovirus in 27-cm soil columns that had been treated with the diluted
sludges. Similar trends were previously observed with the 10-cm
columns (see Table 4-10). No virus breakthrough was observed in the
column treated with sludge diluted with 0.01 N CaCl2, whereas the
column receiving sludge diluted with distilled water was found to
display virus in the leachate after the third pore volume (see Table
4-12). The experiment was stopped by the fourth pore volume because
the column receiving the sludge diluted with distilled water became
clogged. The columns were subsequently sectioned (i.e., after allowing
ponded water to soak in overnight) to study virus distribution within
the soil (see Tables 4-13 and 4-14). It can be seen that in the
column treated with sludge diluted with 0.01 N calcium chloride, polio
virus was found in the packed sludge and in the top 5 cm of soil (see
(Table 4-13). The results for the column receiving sludge diluted with


86
TABLE 4-1, Chemical parameters for the anaerobically digested sludge
1iquor
Parameter3
Sludge*3 1 iquor
value
(ppm)
Parameter3
Sludge*3 liquor
val ue
(ppm)
Soluble salts
703
As
0
Na
63
Cd
0
K
29
Cr
0
Ca
20
Cu
0.05
Mg
15
Ni
0
A1
0
Pb
0
Fe
1.05
Zn
0.04
aChemical parameters were determined by the Analytical Research
Laboratory, Soil Science Department, University of Florida, Gainesville.
bAnaerobically digested sludge (GDANsee Table 3-2 ; solids con
tent, conductivity and pH equal to 2.0%, 3250 ymho/cm at 25C and 8.3,
respectively) was centrifuged at 14,000 x g for 10 min at 4C. This
procedure was performed again on the decanted supernatant and this
yielded a clear sludge liquor.


FIGURE 6-4. Diagram of the sludge disposal site at the West Florida
Agricultural Experiment Station, Jay, Florida
Lagooned sludge (LAG--see Table 3-2) has been ap
plied, for some years, to 8 acres (345,600 ft^ or
3.24 ha) of land at the West Florida Agricultural
Experiment Station. As depicted in the figure,
the disposal site is divided into 72 plots (plot
number is shown on the top of each plot) of 40 by
120 ft (ca. 12 by 36 m) which received from 0 to 15
acre-inches (0 to 15.4 ha-cm) of sludge per year
(acre-inches applied per year is shown in the
bottom of each plot). The soil series found at
the disposal site are Troup, Lucie, and Orangeburg.
The topsoils in plots numbered 1, 32, 42, and 61
(designated by triangles) were monitored for the
presence of indigenous enteroviruses. Groundwater from
3 wells in the disposal site (designated by
hexagons) and one well near the sludge lagoon (not
shown) also was monitored for enteroviruses by
Farrah et al_. (1981a). The water table at these
well sites was 40 to 60 ft (ca. 12 to 18 m) below
the surface.


no
air-dried soil to moisture contents ranging from 7.3% to 14.4% (wt./wt.)
using rain water and then compacting each sample in the Harvard compac
tion apparatus (Soiltest Inc., Evanston, Illinois). Each soil sample
was added to the compaction mold (known volume) in 3 layers with a
compactive force of 10 tamps applied per layer (at different positions)
using a 20-lb (9.1-kg) tamper (Soiltest Inc., Evanston, Illinois; see
Wilson 1950). The soil sample was then ejected from the mold, wet
weighed, and dried to constant weight. The exact soil moisture content
and dry compacted bulk density were calculated for each sample. The
soil moisture content-bulk density curve obtained for the Red Bay sandy
loam subsoil is shown in Figure 4-4. For the compactive force used, a
3
maximum bulk density of 1.96 g/cm was achieved when the soil moisture
content was 12.4% (i.e., optimum moisture content). It should be noted
that cohesionless soils (i.e., sands) are compacted to maximum density
by simply vibrating the air-dried soil (Felt 1965). However, the
increase in bulk density achieved by maximum compaction is much smaller
for sands than for finer textured soils (Brady 1974; Freeze and Cherry
1979). This phenomenon was observed with the Eustis fine sand subsoil
(consisted mainly of the A21 and A22 horizons--see Table 4-3) for which
3
the maximum bulk density attained was only 1.70 g/cm Thus, the Red
Bay sandy loam subsoil was used here because of the greater range in bulk
densities which could be produced with this soil.
Saturated hydraulic conductivity. Prior to initiating polio
virus transport studies, it was important to determine the effect com
paction (i.e., increase in bulk density) of the Red Bay sandy loam


153
the soil columns. Afterwards, no virus was detected in the leachates,
even after the passage of 77.5 to 93 cm of rain (equivalent to 1,570
to 1,884 ml of rain water). The observed breakthrough of poliovirus
when the first pore volume had percolated through column 2 is not
surprising due to the fact that the columns were not initially satur
ated. A soil column treated with 2.5 cm of sludge and subsequently
eluted with 0.01 N calcium chloride was used as a control. From
Table 4-16, it is clear that no virus could be detected in the column
leachates. As discussed earlier, the presence of calcium chloride in
the soil solution readily enhances virus adsorption to the soil matrix.
However, rain water was able to transport 0.1 to 0.2% of the total
applied viruses through the soil profile. This breakthrough would
probably be lower if the soil was. allowed to dry for a longer period
of time under field conditions (see Chapter V).
The results presented above show that the Red Bay sandy loam
studied is effective in retaining viruses during sludge application.
Virus associated with sludge solids will be retained at the surface
of the soil matrix and will be inactivated with time due to environ
mental factors (e.g., temperature, drying and solar radiation). In
Table 3-6, the adsorption of poliovirus to anaerobic sludge solids
during a 12-hour contact period is found to range from 52.8% to 69.1%.
Thus, the effectiveness of virus retention by soils during sludge
application is partly attributed to the capacity of sludge solids to
bind viruses in the top of the soil profile. However, viable "free"
virus (i.e., viruses not associated with sludge solids or dissociated


TABLE 3^8, Summary of the methods developed for the recovery of viruses from sludges
Eluent3
used
Concentration Evaluation of the method
technique for for virus recovery Reference
virus in eluate r
Virus type(s) used Sludge0 type(s) used
Sodium lauryl sulfate
(0.1%) in 0.05 M
glycine, pH 7.5
Organic
flocculation
Poliovirus 1
(Sabin)
Echovirus 7
Anaerobically Abid et_ al_. (1978)
digested (14 days)
Beef extract (3%),
ambient pH
Organic
flocculation
Poliovirus 1
(CHAT)
Anaerobically Glass e]t aK (1978)
digested
Dewatered, composted
Glycine buffer (0.05 M), Organic
pH 11.0 flocculation
Poliovirus 1 (LSc)
Coxsackievirus B3
(Nancy)
Echovirus 7
(Wallace)
Indigenous
Activated0 Hurst et_ £l_. (1978)
Returned
Aerobically digested
(thickened,dewatered)
Dried sludge (aerobi
cally digested) from
a sludge disposal
site
Tryptose phosphate None Indigenous
broth
Glycine buffer, pH 11.0,
beef extract or dis
tilled water
Adsorption to
bentonite
clay
Beef extract (10%), pH 7.0, None
or Tris buffer, pH 9.0
Indigenous
Indigenous
Raw (primary)
Anaerobically
digested (40 days)
Anaerobically
digested (100 days)
Raw (primary
Anaerobically
digested
Raw (primary and
secondary)
Moore frt aj_. (1978)
Turk etal. (1980)
Nielsen and
Lydholm (1980)


96
TABLE 4-3. Extended.
Dominant
clayb
PH
(in 1 :1
water)
Bulk density
(g/cm3)
Saturated
hydraulic
conductivity
(cm/hr)
Vermicul ite
5.3
1.26
12.1
Vermiculite
5.4
1.43
15.9
Vermiculite
5.6
1.48
20.2
Gibbsite
5.1
1.60
21.6

6.7
1.62
28.0

6.5
1.59
27.2

6.5
1.54
34.0

6.3
1.55
52.6
6.0
1 .53
54.2


277
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Moore, B. E., B. P. Sagik, and C. A. Sorber. 1977. An assessment of
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in primary wastewater sludges. Abstracts of the Annual Meeting,
American Society for Microbiology. Abstract Q141, p. 217.
Nielsen, A. L., and B. Lydholm. 1980. Methods for the isolation of
virus from raw and digested wastewater sludge. Water Res. 14:
175-178.
Nielsen, D. R., and J. W. Biggar. 1961. Miscible displacement in soils:
I. Experimental information. Soil Sci. Soc. Am. Proc. 25: 1-5.
Ottolenghi, A. C., V. V. Hamparian, J. D. Pollack, and B. U. Bowman.
1980. Sewage sludge application to farmland--human health


177
TABLE 4-26. Total amount of poliovirus type 1 detected in ten pore
volumes of leachate from 10-cm columns of Red Bay sandy
loam subsoil packed at bulk densities of 1.45 and 1.60
g/cm3
Bulk density3
(g/cm3)
Column no.
At pore volume
no. 10
Poliovirus
applied
(total PFU)
Poliovirus
breakthrough
(%)b
Meanc poliovirus
breakthrough for
each bulk density
(%b + SEd)
1.45
1
2.2 x 106
12
7.3 + 2.9
2
1.7 x 106
7.9
3
1.7 x 106
2.0
1.60
4
2.0 x 106
5.6
2.6 1.6
5
2.0 x 106
1.6
6
2.0 x 106
0.5
aComplete data and experimental procedures are shown in
Figure 4-11.
^Expressed as cumulative percent of total PFU applied at pore
volume no. 10.
cThe two mean values shown are not significantly different at the
0.05 level when subjected to a two-tailed ,t-test.
Abbreviation for standard error.


87
Hercufloc #871 (Hercules Co., Atlanta, Georgia). The polymer was added
to 1000 ml of the virus-seeded sludge while mixing rapidly on a mag
netic stirrer. Mixing was continued slowly for an additional 5 minutes.
The entire sludge sample was then centrifuged at 320 x g (i.e., 1400
rpm) for 10 minutes at 25C. The supernatant was decanted, assayed for
viruses, and discarded. The dewatered sludge volume and sludge solids
content (i.e., as percent, by method described above for digested
sludges) was measured. The conditioned-dewatered sludge produced
(i.e., in duplicate) was then assayed for viruses as described below.
The procedure employed above in the conditioning and dewatering of the
sludge was identical to that used at the Main Street wastewater treat
ment plant, Gainesville, Florida (Dr. DuBose, Main Street plant, per
sonal communication; see Figure 6-1).
Chemical sludges. The chemical sludges were precipitated from
1000 ml of poliovirus-seeded, raw sewage using the general procedure
of Sattar et al_. (1976) and the coagulants, alum (i.e., aluminum sul
fate),.ferric chloride or lime (i.e., calcium hydroxide). The raw sewage
used was obtained from the University of Florida campus wastewater treat
ment plant or from the Main Street wastewater treatment plant, both being
located in Gainesville, Florida. The raw sewage samples were collected
and sewage parameters (i.e., pH and conductivity) were measured as
described above for sludge samples. The raw sewage samples were sterilized
by autoclaving at 121C with applied pressure of 15 psi for 15 minutes
prior to use. Viral assays were made before and after the addition
of coagulant. The final concentration in sewage of alum was 300 mg/£


191
eluted from 1-g (wet weight) samples of drying sludge using the glycine
procedure developed by Hurst et^ al_. (1978). This method consisted of
mixing each 1-g sludge sample with 5 ml of 0.05 M glycine buffer,
pH 11.5. If necessary, the pH of the mixture was adjusted to between
10.5 and 11.0 by the addition of 1M glycine buffer, pH 11.5. The
samples were vigorously vortexed for one minute and centrifuged at
14,000 x g for 5 minutes at 4C (all centrifugation was performed
using a Sorvall RC5-B centrifuge, Ivan Sorvall Inc., Norwalk, Con
necticut). The supernatants (i.e., the sludge solids eluates) were
recovered, adjusted to neutral pH by the addition of 1 M glycine buffer,
pH 2.0, and assayed for eluted viruses as described above. Further
viral concentration was not required. The entire procedure described
above was performed in less than 10 minutes. Thus, poliovirus and
echovirus were subjected to the high pH of 10.5 to 11.0 for no more
than 10 minutes. Both Hurst et al_. (1978) and Sobsey et al_. (1980)
observed no appreciable inactivation in 10 minutes of poliovirus type
1 (LSc) seeded in 0.05 M glycine buffer, pH 10.5 to 11.0. Therefore,
it is believed that there was no significant inactivation of poliovirus
or echovirus during the elution of sludge solids. The numbers of
viruses recovered were expressed as PFU per g dry weight of sludge.
Soil. Soil samples were obtained from the top 2.5 cm of soil
in the large soil cores (one sample per soil core per sampling date).
The moisture content of each soil sample was then determined as
described below. Seeded viruses (i.e., poliovirus or echovirus) were
eluted from 10-g (wet weight) samples of soil using the procedure


181
the average of triplicate counts. The numbers of viruses were expressed
as plaque-forming units (PFU).
Sludges
Two sludge types were used in these experiments: aerobically
digested sludge (GDA 180see Table 3-2) sampled at the Main Street
wastewater treatment plant of Gainesville, Florida, and lagooned sludge
(LAGsee Table 3-2) sampled at the West Florida Agricultural Experiment
Station, Jay, Florida. The lagooned sludge is a mixture of aerobically
digested sludge (1/3) and anaerobically digested sludge (2/3) from the
Montclair and Main Street wastewater treatment plants of Pensacola,
Florida, respectively (see Table 3-2). The mixture was kept in a lagoon
at the experiment station before ultimately being disposed of on land.
The sludges were collected and sludge parameters (i.e., pH and solids
content) were measured as described in Chapter III (see page 56). The
sludge conductivity was measured using a Beckman conductivity bridge
model RC 16B2 (Beckman Instruments, Fullerton, California). The sludges
used were not autoclaved or decontaminated in any other way.

Association of Seeded Viruses with Sludge Solids
Poliovirus or echovirus stock in phosphate-buffered saline (PBS)
containing 2% fetal calf serum (FCS) (see Appendix for more details
on the composition of this solution) was added directly to sludge at
the rate of 1 ml of virus stock per 1 ,000 ml of sludge and while stirring
the suspension using a magnetic stirrer. Magnetic stirring was con
tinued for 10 to 60 minutes and then the association of viruses with
sludge solids was determined using the procedure outlined in detail in


I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Bitton, Chairman
issor of Environmental
Engineering Sciences
I certify that I have read this study and that in my opinion
if conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
as L. Crisman'
Associate Professor of Environmental Engineering
Sciences
I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Professor of Environmental Engineering Sciences


TABLE 4-25. Retention of poliovirus type 1 by packed columns of Red Bay sandy loam subsoil following the
application of lime-stabilized, chemical sludges and the subsequent elution with rain water
Sludge
type,
1 ime-
stabilized
Total no.
of pore
volumesa
eluted
Sludgeb
volume
applied
(ml)
Sludge
sol ids
applied
(g)
Total no. of poliovirus, PFU
Range of
conductivity
values for
pore volumes
collected
(ymho/cm
at 25C)
Range of
pH values
for pore
volumes
collected
Contained in
the sludge
applied
Eluted
from the
soil0
Alum
10.0
12
0.2
0
0
25 118
5.7 6.9
Ferric
chloride
10.0
22
0.2
4.3 x 103
0
30 255
5.4 5.7
a0ne
pore volume for
these columns equals
225 ml. The
laboratory-packed columns were 29
cm in length
and 4.8 cm internal diameter; the columns were filled only 27 cm with soil (2 cm left on top for packed
sludge), and were conditioned with 2 pore volumes of rain water. The sample of Red Bay sandy loam subsoil
used consisted mainly of the A2 and Bit horizons (see Table 4^-3).
^The chemical sludges were precipitated from virus-seeded, raw sewage. The methods used to produce
these sludges and to determine the amount of viruses present in the sludges are described in Table
The chemical sludges were then stabilized with Ca(0H)2 as described in Table 4-23. The lime-stabilized,
chemical sludges were applied to the soil columns, allowed to soak in, and then, worked under 2.5 cm.
Elution with rain water was subsequently undertaken. The rain water was applied continuously to the
columns at approximately 5 ml/min using a peristaltic pump (Buchler, Fort Lee, N.J.). The rain water was
collected next to the Environmental Engineering Sciences building at the University of Florida, Gainesville.
See Table 4-2 for chemical characteristics of the rain water.
c
Soil leachates were collected in 1/2 pore volume fractions and assayed individually for viral in-
fectivity. In order to detect small numbers of viruses, pore volumes 0.5 through 5.0 (and 5.5 through 10.0)
were subsequently combined and concentrated 160-fold by membrane filtration. The concentrates were assayed
for poliovirus. This concentration procedure was only performed on the leachates from the column receiving
lime-stabilized, ferric chloride sludge.


13
flocculation is combined with primary sedimentation, even larger quan
tities of sludge (i.e., 5000 to 38,000 gallons of sludge per million
gallons of wastewater treated) are produced (U.S. Environmental Protec
tion Agency 1974). These raw primary sludges (in particular, the
chemical sludges) contain significant amounts of enteric viruses (Berg
and Berman 1980; Lund 1976; Lund and R0nne 1973; Nielsen and Lydholm
1980; Sattar and Ramia 1978; Sproul 1976). In fact, the indigenous
enteric viruses are concentrated in the chemical sludges (Lund and R0nne
1973). Sattar and Ramia (1978) readily detected indigenous viruses in
all the primary lime sludge samples they tested. The sludge samples
were obtained from a wastewater treatment plant in Canada which employed
lime flocculation of raw sewage at a pH of approximately 10. Clearly,
even under such virucidal conditions of alkaline pH, viruses were
recovered from the sludges produced. Thus, all primary sludges should
be considered to represent potential health hazards, and should be
handled with care during further treatment and final disposal (Brunner
and Sproul 1970; Lund 1976; Sattar and Ramia 1978).
Activated Sludge
The activated sludge process (i.e., secondary or biological treat
ment) is perhaps the most effective wastewater treatment process in the
removal of viruses. Several authors have reviewed the literature on virus
removal by this treatment process (Berg 1973a; Foster and Engelbrecht 1973;
Grabow 1968; Kollins 1966; Malina 1976; Sproul 1976). In laboratory-
scale pilot plants, 90% or more of the seeded enteroviruses were removed
by the activated sludge process (Clarke et al_. 1961; Malina et al. 1975).


FIGURE 2-1.
Outline of sludge treatment processes and their functions
Adapted from Fair et al_. (1968), Malina (1976) and
United States Environmental Protection Agency (1974,
1978a, 1978b).


75
type 1 (Farouk) became associated with lagoon sludge solids after a
contact period of 60 minutes (see Table 3-4). From Table 3-4 it can
be seen that a larger fraction of poliovirus type 1 (83.9% and 91.1%)
became associated with lagoon sludge solids. Goyal and Gerba (1979)
have also shown that seeded echovirus type 1 (Farouk) does not adsorb
well to a sandy loam soil. It is clearly established that virus type
is a factor that affects virus adsorption to surfaces, including sludge
solids. It was the aim of this chapter, however, to show that sludge
type is also a critical factor influencing the degree of association of
viruses with sludge solids.
Recovery of Solids-Associated Viruses
Seeded viruses that became associated with sludge solids were
eluted and further concentrated according to the glycine method. In
Table 3-7, it can be seen that significantly lower mean poliovirus
recovery was found for aerobically digested sludges (15%) than for
mixed liquors or anaerobically digested sludges (72% and 60%:,:,,
respectively). The mean poliovirus recoveries from mixed liquors and
anaerobically digested sludges were not significantly different statis
tically. The recovery of solids-associated viruses was not dependent
upon the volume of liquid sludge (100, 500, or 1,000 ml) processed. The
mean poliovirus recovery from mixed liquors (72%:);. was similar to the
recovery (80%) of the same virus reported by Hurst et al_* (1978). These
researchers worked with activated sludge, which is the same sludge type
as our mixed liquors.
It is clearly established that the effectiveness of the glycine
method in recovering solids-associated viruses is reduced for aerobically


TABLE 4-19. Retention of poliovirus type 1 by packed columns of Red Bay sandy loam subsoil following the
application of conditioned-dewatered sludge and the subsequent leaching with rain water
Total no. of
pore volumes3
eluted
Sludgeb
volume
applied
(ml)
Sludge
solids
applied
(g)
Total no. of poliovirus, PFU
Range of
conductivity
values for
pore volumes
collected
(pmho/cm at
25C)
Range of pH
values for
pore volumes
collected
Contained in
the sludge
applied
Passing
through
the soi1c
Column 1
10.0
17
0.9
1.3 x 106
0
41 148
5.5 6.7
Column 2
10.0
17
0.8
1.4 x 106
0
37 175
5.6 6.5
a0ne pore volume for these columns equals 225 ml. The laboratory packed columns were 29 cm in length
and 4.8 cm internal diameter; the columns were filled only 27 cm with soil (2 cm left on top for packed
sludge), and were conditioned with 2 pore volumes of rain water. The sample of Red Bay sandy loam subsoil
used consisted mainly of the A2 and Bit horizons (see Table 4-3).
^The methods used to produce the dewatered sludges and to determine the amount of viruses present in
the dewatered sludges are described in Table 4-17. The dewatered sludges were applied to the soil columns,
allowed to soak in, and then, worked under 2.5 cm. Elution with rain water was subsequently undertaken.
The rain water was applied continuously to the columns at approximately 5 ml/mn using a peristaltic pump
(Buchler, Fort Lee, N.J.). The rain water was collected next to the Environmental Engineering Sciences
building at the University of Florida, Gainesville. See Table 4-2 for chemical characteristics of the
rain water.
c
Soil leachates were collected in 1/2 pore volume fractions and assayed individually for viral infec-
tivity. Concentration of soil leachates was not performed.


TABLE 4-6.
Retention of poliovirus type 1 by a packed column of Red Bay sandy loam subsoil when suspended
in 0.01 N CaC^ and after subsequent application of rain water
No. of pore
volumes3
eluted
Poliovirus
eluted
(PFU/ml)
l of Influent^
poliovirus
concentration
% of Total PFU
having been
applied at each
pore volume
(cumulative)
Conductivity
of pore volume
collected
(ymho/cm at 25C)
pH of pore
volume
col 1ected
0.01 N CaCl ^
seeded with poliovirus
1
0
0
0
1260
6.3
2
0
0
0
1300
6.1
3
11
0.046
0.02
1340
5.4
4
0
0
0.01
1380
6.3
5
0
0
0.009
1320
5.2
6
0
0
0.008
1320
5.4
7
0
0
0.007
1360
5.1
8
0
0
0.006
1340
5.1
9
0
0
0.005
1320
5.1
10
0
0
0.004
1350
5.2
Shift to nonseeded rain water^
11
2
0.008
0.005
1320
6.4
12
0
0
0.005
740
5.3
13
0
0
0.005
168
4.8
14
0
0
0.005
73
6.0


108
each column was separated into 2- to 3-cm sections. The sludge solids
resting on top of the soil were also separated and considered as one
section. Each soil (or sludge solids) section was well mixed in a
sterile beaker with a spatula, wet weighed, and then a representative
sample (10 grams of wet soil; total known amount of top wet sludge
solids) was taken and subjected to the following virus recovery method
ology. Each sample was mixed with 3% (wt./vol.) beef extract (Difco
Laboratories, Detroit, Michigan), buffered with Tris(hydroxymethyl)
aminomethane (Sigma Chemical Co., St. Louis, Missouri) at pH 9.0 in the
proportion of 1 gram of wet soil (or wet sludge solids) per 2 ml of
eluent. This solution has been found to be effective in the elution of
poliovirus type 1 from soil (Bitton et a]_. 1979a) and from sludge solids
(Farrah et al_. -1981b). The mixtures were then vortexed for 30 seconds
and sonicated for 3 minutes at maximum deflection (60 watts) using a
Branson son.ifijar (Branson Instruments Inc., Danbury, Connecticut). The
samples were then centrifuged at 1900 x g for 10 minutes at 4C. The
supernatants were adjusted to neutral pH. An aliquot of each super
natant produced was then diluted (i.e., if necessary) in PBS containing
2% FCS and assayed directly for viruses by the plaque technique. The
amount of poliovirus recovered was expressed as PFU per soil (or sludge
solids) section.
Effect of Soil Bulk Density on Poliovirus Transport
The poliovirus transport studies described above were conducted
with laboratory-packed soil columns and undisturbed soil cores displaying
similar bulk densities as found in the field for the two soils studied


9
Removals in excess of 99% have been reported for poliovirus type 1
during excess lime-soda ash softening at pH 10.8 to 11.2 (Wentworth
et al_. 1968). Enteroviruses are actually inactivated under the high
pH conditions achieved rather than merely removed by the flocculation
process (Sproul 1972). The inactivation of enteroviruses under alka
line pH is believed to be caused by the denaturation of the protein
coat and the subsequent disruption of the structural integrity of the
virus (Sproul 1972). Lime flocculation followed by sedimentation or/and
sand filtration is frequently used as a tertiary (advanced) treatment
of secondary wastewater effluents (Malina 1976). This treatment
effectively removes suspended solids and phosphates from secondary
effluents (Berg et al_. 1968). Several studies (field and laboratory)
have been undertaken to determine the removal efficiency of viruses from
secondary effluents by lime flocculation. Brunner and Sproul (1970)
performed laboratory precipitation tests involving the addition of
lime [as CaiOH)^] to filtered wastewater effluent until the pH was
raised to between 9.5 and 10.9. Poliovirus type 1 (Sabin) removals
ranging from 88 to 94% were achieved. Generally, virus removal
increased with a corresponding increase in pH and in the quantity of
phosphate precipitated. In a similar laboratory study, Berg et al.
(1968) showed that 70 to >99% of poliovirus type 1 (LSc) seeded in
secondary wastewater effluent was removed by lime flocculation [with
300 mg/i, (pH 10.2) to 500 mg/i, (pH 11.0) of CaiOH^ respectively]
followed by sedimentation. These investigators also observed that
virus removal increased as the pH achieved by the addition of lime
increased. Additional experimentation confirmed that poliovirus was


48
1974). Due to the more favorable subsurface environment, however,
pathogens (e.g., viruses) may persist longer in subsurface-injected
sludge than in surface-applied sludge (Moore et aj_. 1978).
Whatever the application method employed, the disposal of
sludge on land should be undertaken in accordance with local, state,
and federal regulations and recommendations (Manson and Merritt 1975;
U.S. Environmental Protection Agency 1974, 1978b; Wright 1975).
Generally, only stabilized sludge is recommended for land application
(U.S. Environmental Protection Agency 1974). Application rates of
sludge to cropland vary depending upon sludge composition, soil charac
teristics, climate, vegetation and cropping practices (U.S. Environ
mental Protection Agency 1974), but should not exceed 20 dry tons of
sludge solids/acre/year (44.8 dry metric tons/ha/year) or 46.8 m /ha/
day (liquid rate) (Manson and Merritt 1975; U.S. Environmental Protec
tion Agency 1974). In order to prevent groundwater and surface water
contamination, as well as other potential problems, Manson and Merritt
(1975) recommended that sludge disposal sites conform to the following
standards:
1. High water tables should be no closer to the soil
surface than 4 ft (1.22 m)
2. Isolation from surface waters by a minimum distance
of 200 ft (61 m)
3. Maximum slope of 5% in order to prevent excessive
surface runoff
A crop that can be harvested is the preferred
ground cover
4.


188
TABLE 5-2. Retention
of poliovirus type 1
by porous ceramic cups
Influent3 virus
concentration
(PFU/ml)
Effluent^ virus
concentration
(PFU/ml)
Virus
retained by
ceramic cupc
(%)
2.7 x 104
1.8 x 104
33.3
2.5 x 104
1.4 x 104
44.0
Mean: 38.7
aPoliovirus was suspended in a rain leachate (conductivity and
pH equal to 18 ymho/cm at 25C and 6.0, respectively) from an undis
turbed core of Eustis fine sand, and passed through sterile, porous,
ceramic cups with the use of a vacuum pump at a flow rate of approx.
2 ml hr"1 cm"2.
^Concentration of virus after passage through a porous ceramic
cup.
The porous ceramic cups used were 6.9 cm long and had a wall
thickness of 0.23 cm (no. 2131, Soil Moisture Equipment Corp., Santa
Barbara, California). The cups used had a pore diameter of 1.4 to
2.1 ym.


TABLE 5-5. Survival of poliovirus type 1 following suspension in liquid sludge and subsequent application
to large soil cores of Eustis fine sand exposed to natural conditions (2 June 1978-7 July 1978)
Sampling
date
(1978)
Days
after the
beginning
of
experiment
Cumulative
rainfall3
(cm)
Sludge^
sol ids
content
(%, wtywt*)
Soil
moisture
(%, wt/wt)
No. of viruses
(PFU/g dry weight of
sludge or soil)
LC3
LC4
LIQUID SLUDGE SAMPLE
02 June
0
0
2.9C
_d
4.4 x 107
4.4 x 107
DRYING SLUDGE SAMPLES
03 June
1
1.80
23.1
--
2.5 x 106
4.9 x 106
06 June
4
6.03


1.0 x 106
5.0 x 105
Sludge mixed with
the top 2.5 cm
of soil on day 4
SOIL SAMPLES (top 2,5 cm)
06 June 4 6.03
07 June 5 7.03
14.7 3.3 x 104 4.1 x 104
0.9 8.7 x 104 7.4 x 104
9.9 2.5 x 103 2.4 x 103
09 June
7
7.28



ro
rv>
co


163
TABLE 4-18. Association between poliovirus type 1 and conditioned-
dewatered sludge solids
Experiment
no.
Virus in
unfractionateda
sludge^
(total PFU)
Virus in
sludge
supernatant0
(total PFU)
Viable
unadsorbedd
virus
(%)
Solids-
associated6
virus
(%)
1
2.5 x 107
1.1 x 104
0.04
99.9
2
2.6 x 107
3.1 x 104
0.1
99.9
aThe sludge solids were not separated prior to assaying.
^The methods used to produce the dewatered sludges and to determine
the amount of viruses present in the dewatered sludges are described in
Table 4-17.
cThe sludge was clarified by centrifugation at 1400 x g for 10 min
at 4C and the supernatant was subsequently assayed.
^The "viable unadsorbed virus (%)" values were calculated as shown
in the Materials and Methods section.
eThe "solids-associated virus (%)" values were estimated as shown
in the Materials and Methods section.


92
poliovirus and suspension magnetically mixed for 10 to 60 minutes),
conditioned-dewatered sludge (poliovirus transferred to this sludge
during dewatering process), chemical sludges (poliovirus transferred to
chemical sludges during precipitation of virus-seeded, raw sewage) and
lime-stabilized, chemical sludges. The procedures used are outlined
in detail in Chapter III (see page 62). Briefly, an aliquot of the
unfractionated sludge (i.e., sludge sample without solids separated)
was diluted in PBS containing 2% FCS and assayed directly for viruses by
the plaque technique. This method (i.e., sludge dilution and subsequent
direct assay on cell cultures) has been previously shown to be highly
efficient in the recovery of poliovirus from unfractionated sludge
(see Chapter III, page 62 and Table 3-3). The unfractionated sludge
assay was performed in order to determine the total amount of virus
present in the sludge sample. An aliquot of the sludge was subsequently
centrifuged at 1400 x g for 10 minutes at 4C. The sludge supernatant
produced was assayed for viruses. The "viable unadsorbed virus" and
"sludge solids-associated virus" fractions were calculated as shown
in Chapter III (see page 64). .
Rain Water
Rain water was collected next to the Environmental Engineering
Sciences building at the University of Florida, Gainesville. Chemical
parameters for the rain water used were determined by Hendry (1977)
and are presented in Table 4-2. The rain water was sterilized by auto
claving at 121C with applied pressure of 15 psi for 15 minutes prior
to use.


54
TABLE 3-1. General properties of polioviruses
Property
Value
Nucleic acid
Molecular weight of nucleic acid (daltons)
RNAa,b (single-stranded)
6a,b '
2 x 10b
Particle diameter (nm)
27 to 30a,bc
Particle morphology
Icosahedral9,b
Particle isoelectric point
4.5 and 7.0b
Stability at 25C
Relatively stable9
Stability at pH 3.0
Stablea,b,e
Stability in ether
Stableb
aFrom Davis et aj_. (1973).
bFrom Hahn (1972).
cFrom Schwerdt and Schaffer (1955).
^From Mandel (1971). The data were obtained using poliovirus type
1 (strain Brunhilde).
p
From Bachrach and Schwerdt (1952).


FIGURE 4-2.
Laboratory-packed soil columns (10 cm in length) used in poliovirus transport studies
The soil columns (4.8 cm internal diameter) were packed as described in the
Materials and Methods section, and were then placed in soil column holders.
The soil column holders were supplied by the Soil Moisture Equipment Corp. (Santa
Barbara, California). Leaching solutions were applied to the soil columns using
a peristaltic pump (Buchler, Fort Lee, N.J.) as shown.


CHAPTER III
EFFECT OF SLUDGE TYPE ON POLIOVIRUS
ASSOCIATION WITH AND RECOVERY FROM SLUDGE SOLIDS
Introduction
The degree of association between viruses and sludge solids
is a critically important factor in the assessment of the fate of
these pathogens following sludge disposal on land. Yet, the nature of
the association between viruses and sludge solids has not been adequately
explored, partly because of the lack of virological methods. Recently,
however, methods have been developed for the recovery of viruses from
wastewater sludges. Practical methods for the recovery of viruses
from sludge samples involve two steps. Because viruses in sludges
have been found to be solids associated (Abid et al_. 1978; Glass et al.
1978; Hurst et al_. 1978; Lund 1971; Ward and Ashley 1976; Wellings et
al. 1976a), the first step of an effective method, therefore, consists
of releasing both surface-adsorbed and solids-embedded viruses. The
second step consists of concentrating the eluted viruses prior to viral
assays.
Various chemicals have been used to elute viruses from sludge
solids, namely 0.1% sodium lauryl sulfate in 0.05 M glycine, pH 7.5
(Abid et al_. 1978), tryptose phosphate broth (Moore et al_. 1978),
3% beef extract, pH 9.0 (Wellings et al_. 1976a), 3% beef extract,
ambient pH (Glass et aj_. 1978; Sattar and Westwood 1976), 0.05 M glycine
buffer, pH 11.0 (Hurst et al_. 1978), 2% fetal calf serum in Earle's
50


40
spring in Florida, Farrah et aj_. (1981a) found, for example, that
the enterovirus titer of lagooned sludge dropped from 80 TCID^g/g
of dry sludge to low or undetectable levels in approximately 6 weeks
(note that similar decline was also observed for fecal coliforms in
the lagooned sludge). In contrast, Sattar and Westwood (1979) detected
enteric viruses in 39% of the sludge samples obtained from a lagoon
in Ottawa, Canada, over a 14-month period (i.e., from April 1975 to
May 1976). These investigators were able to recover viruses from sludge
samples taken from the lagoon after 8 months. Clearly, indigenous
viruses were inactivated at a much slower rate in the Canadian sludge
lagoon (Sattar and Westwood 1979) than in the Floridian sludge lagoon
(Farrah et al_. 1981a). Apparently, lower Ottawa temperatures (Sattar
and Westwood 1979) contributed to greater viral persistence in the
Canadian sludge lagoon. Whereas temperature appears to be an important
factor affecting the inactivation rate of viruses in sludge lagoons,
sludge drying is unlikely to have a significant effect. This is
because, in lagoons, sludge dries at such a slow rate that substantial
viral inactivation cannot be expected to result from the drying process
itself. In addition to sludge treatment (i.e., drying and further di
gestion), lagoons also provide a temporary method of sludge storage
(Fair etaj_. 1968; U.S. Environmental Protection Agency 1974). Ulti
mately, however, lagooned sludge must be disposed of and the method of
choice is usually land application (U.S. Environmental Protection Agency
1974). Although the research presented above indicates that long-term


33
improved (U.S. Environmental Protection Agency 1974, 1978a). It should
be pointed out that the heat treatment of sludge is an energy-intensive
process. Consequently, in actual practice, the applicability of this
treatment procedure is limited due to high energy costs.
Conditioning-chemical treatment. As shown in Figure 2-1,
stabilized sludge is frequently conditioned with organic polymers or
inorganic chemicals (i.e., ferric chloride, ferrous sulfate, lime or
alum) in order to facilitate water removal by subsequent sludge de
watering processes (U.S. Environmental Protection Agency 1974, 1978a).
These flocculants provide charge neutralization and thereby aggregate
the sludge particles such that a porous, free-draining cake structure
is produced (U.S. Environmental Protection Agency 1974, 1978a). Con
sequently, chemical conditioning improves sludge dewaterability and
sludge solids capture during dewatering procedures (U.S. Environmental
Protection Agency 1974).
Although never tested, it can be hypothesized that indigenous
enteric viruses in sludge are probably associated with the sludge-
particle aggregates produced during the chemical conditioning of sludge.
Thus, enteric viruses can be expected to be concentrated in the con-
ditioned-dewatered sludge. Except in the case of lime treatment, no
significant viral inactivation is likely to result from chemical con
ditioning.
Conditioning of sludge with lime is routinely undertaken in
conjunction with ferric chloride (U.S. Environmental Protection Agency
1974). As shown in Figure 2-1 and described above, lime treatment


73
TABLE 3-6, Effect of contact time on the association between poliovirus
type 1 and anaerobically digested sludge solids
Contact
time
(hours)
Virus3 in
unfractionatedb
sludge
(total PFU)
Virus
in sludge
supernatant0
(total PFU)
Viable
unadsorbed6
virus
(%)
Solids-
associated6
virus
(%)
0
o
X
r
4.6 x 106
41.8
58.2
0.5
1.1 x 107
3.5 x 106
31.8
68.2
1.0
1.0 x 107
4.4 x 106
44.0
56.0
6.0
9.4 x 106
2.9 x 106
30.9
69.1
8.5
6.5 x 106
2.3 x 106
35.4
64.6
12.0
5.3 x 106
2.5 x 106
47.2
52.8
aThe virus was added to 1000 ml of anaerobically digested sludge
(GDANsee Table 3-2> solids content and pH equal to 2.0% and 8.3,
respectively) while stirring the suspension using a magnetic stirrer.
Magnetic stirring was continued for 60 min. For the remainder of the
experimental trial, the virus-seeded sludge was left at room temperature
undisturbed, and was stirred only prior to obtaining a sample for viral
assay.
dThe sludge solids were not separated prior to assaying.
cThe sludge was clarified by centrifugation at 1400 x g for 10 min
at 4C and the supernatant was subsequently assayed.
dThe "viable unadsorbed virus (%)" values were calculated as shown
in the Materials and Methods section.
eThe "solids-associated virus (%)" values were estimated as shown
in the Materials and Methods section.


184
periods of rainfall. Similarly, Robeck et al_. (1962) simulated a
groundwater table in a sand column that was sealed at the bottom.
The capacity of the porous ceramic cups to retain viruses was evaluated
as described below. All six soil cores were exposed to natural condi
tions outside the Environmental Engineering Sciences building at the
University of Florida, Gainesville. The soil cores rested on a wooden
box such that soil leachates produced during natural rainfall could be
collected (see Figure 5-1). Unlike the small soil cores, the large soil
cores were insulated by surrounding them with duct insulation as shown
in Figure 5-1. All soil cores were treated with virus-seeded sludge
as described below.
Porous ceramic cups. The retention capacity of porous ceramic
cups towards viruses was evaluated using poliovirus suspended in a rain
leachate from a small undisturbed core (see above) of Eustis fine sand.
The rain leachate was produced in the laboratory by passing rain water
continuously through the small soil core. The rain water was applied
from an inverted, self-regulated, 1-liter Erlenmeyer flask set to main
tain a 2.5-cm hydraulic head on the soil core (Sanks et al_. 1976).
Leachate from the soil core (400 ml) was seeded with poliovirus. The
virus-seeded leachate was then divided into two fractions of 200 ml and
each fraction was passed through a sterile ceramic cup (no. 2131see
characteristics above) with the use of a vacuum pump at a flow rate of
-1 -2
approximately 2 ml hr cm The concentration of poliovirus in rain
leachate was determined before and after passage through the porous
ceramic cups in order to claculate the percent retention of the virus.


FIGURE 4-1. Chemical sludges (i.e., lime and alum) were precipitated
from poliovirus-seeded, raw sewage and are shown after
settling for 60 minutes in Imhoff cones


253
TABLE 7-1. Conductivity
Water sample
and pH of water samples used in this study
Conductivity
(umho/cm at 25C)
pH
Seawater
40,000
7.7
Groundwater
475
7.9


42
sludge. The combined heat and radiation treatments appeared to have a
synergistic effect on the survival of poliovirus in raw sludge (Ward
1977). Due to its usual lack of pathogens, irradiated sludge has been
recommended for use as an animal feed supplement or for other agricul
tural purposes (Bitton 1980b). It is worth noting, however, that sludge
irradiation is an energy-intensive process that has yet to become widely
used in the treatment of sludge (note that it is not included in
Figure 2-1 as a standard sludge treatment process).
Final sludge disposal. Methods for final sludge disposal will
be dealt with in a subsequent section.
Viral and Other Health Hazards
Associated with Treated Sludges
As described above, most sludge treatment processes do not
yield a virus-free product. With the possible exception of sludges
treated at high temperatures, treated sludges represent potential health
hazards due to the presence of viral pathogens (see review presented
above). In addition to viruses, treated sludges (e.g., digested
sludges) also contain bacterial pathogens (Foster and Engelbrecht 1973;
Kowal and Pahren 1978), parasites (Hays 1977; Kowal and Pahren 1978;
Little 1980; Pahren et al_. 1979), toxic metals such as cadmium, copper,
nickel, lead, zinc, and chromium (Chaney 1980; Jones and Lee 1978; Kowal
and Pahren 1978; Pahren et a]_. 1979), and toxic organic residues such
as aldrin, dieldrin, chlordane, heptachlor, lindane, toxaphene, poly
chlorinated biphenyls, and benzo(a)pyrene (Dacre 1980; Jones and Lee 1978;
Kowal and Pahren 1978; Pahren et al_. 1979). Clearly, the biological


52
samples with an overall efficiency of 80%. It is difficult to compare
these two methods, since they were evaluated with different sludge
types.
The research reported in subsequent chapters of this disserta
tion deals primarily with the fate of enteroviruses following sludge
disposal on land. In the course of conducting this research, the viral
(i.e., indigenous and seeded) content of different sludge types had to
be determined. Consequently, it was imperative to determine if viruses
could be effectively recovered from the solids of different sludge types.
The glycine method developed by Hurst et aj_. (1978) was evaluated for
its effectiveness in recovering poliovirus type 1 (LSc) from different
sludge types. The sludge types used were activated sludge mixed liquor,
and anaerobically and aerobically digested sludges. It is the aim of
this chapter to show that sludge type is a factor that can strongly
influence the degree of association between viruses and sludge solids,
as well as the recovery of sludge solids-associated viruses by the gly
cine method (Hurst et^ al_. 1978).
Materials and Methods
Virus and Viral Assays
Poliovirus type 1 (strain LSc) was used in the research
i
reported in this chapter. This virus strain is a non-neurotropic
variant of the Mahoney strain, and is avirulent for mice and monkeys by
all routes. This strain is identical to the virus designated as at
tenuated Sabin strain (Cooper 1967; Hahn 1972; World Health Organiza
tion 1968). Some general properties of polioviruses are shown in


141
that the sludge liquor contained high levels of Na, K, Ca, Mg, and
soluble salts which would be conducive to virus adsorption to soil.
In spite of this favorable ionic environment, a dramatic virus break
through (33.7%) occurred. These data support the contention that the
sludge liquor contained substances which strongly interfered with virus
adsorption to this soil. Furthermore, in this experiment, sludge solids
which can bind viruses in the top of the soil matrix were not added.
In a similar experiment, poliovirus was suspended in sludge diluted
with sludge liquor brought to a final calcium chloride concentration
of 0.01 N and subsequently applied to the soil column. In Figure 4-6,
the breakthrough of poliovirus is seen to have been reduced to 22.6%
(from 33.7% with only sludge liquor) as a direct result of the presence
of sludge solids. When the length of the soil column was increased
to 27 cm from 10 cm, there was a further decrease in poliovirus break
through from 22.6% to 8.1% (see Figure 4-7). Dilution of sludge in its
own liquor prevents extreme changes in the sludge properties. For
example, the pH was unchanged (8.3) and the conductivity was only
slightly reduced from 3250 to 2600 yrnho/cm at 25C. A direct compari
son can be made between the experiment in which 27-cm columns
received sludge diluted with sludge liquor brought to a final CaC^
concentration of 0.01 N (Figure 4-7) and the experiment in which simi
lar size columns were treated with sludge diluted in 0.01 N calcium
chloride (Table 4-8). For these experiments, breakthroughs of polio
virus detected in the soil leachates at the tenth pore volume cor
responded to 8.1% and 0.01% of the total virus applied, respectively.
Quite clearly, the sludge liquor contained substances which interfered


BIBLIOGRAPHY
Abid, S. H., C. Lue-Hing, and S. Sedita. 1978. Development of a Method
for Concentrating Enteroviruses in Anaerobically Digested
Sludge. Report 78-13. The Metropolitan Sanitary District of
Greater Chicago, Chicago, Illinois.
Allen, M. J., and E. E. Geldreich. 1975. Bacteriological criteria for
ground-water quality. Ground Water 13: 45-51.
Bachrach, H. L., and C. E. Schwerdt. 1952. Purification studies on
Lansing poliomyelitis virus: pH stability, CNS extraction and
butanol purification experiments. J. Immunol. 69: 551-561.
Bagdasaryan, G. A. 1964. Survival of viruses of the enterovirus group
(poliomyelitis, ECHO, coxsackie) in soil and on vegetables.
J. Hyq. Epidemiol. Microbiol. Immunol. 8; 497-505.
Baham, J., N. B. Ball, and G. Sposito. 1978. Gel filtration studies
of trace metal-fulvie acid solutions extracted from sewage
sludges. J. Environ. Qua!. 7_: 181-188.
Baross, J. A., F. J. Hanus, and R. Y. Morita. 1975. Survival of human
enteric and other sewage microorganisms under simulated deep-
sea conditions. Appl. Microbiol. 30: 309-318.
Barron, A. L., C. Olshevsky, and M. M. Cohen. 1970. Characteristics
of the BGM line of cells from African green monkey kidney.
Archiv fiir die gesamte Virusforschung 32: 389-392.
Benarde, M. A. 1973. Land disposal and sewage effluent: Appraisal of
health effects of pathogenic organisms. J. Am. Water Works
Assoc. 65: 432-440.
Berg, G. 1973a. Removal of viruses from sewage, effluents and waters.
1. A review. Bull. W.H.O. 49: 451-460.
Berg, G. 1973b. Removal of viruses from sewage, effluents and waters.
2. Present and future trends. Bul 1. W.H.O. 49: 461-469.
Berg, G., and D. Berman. 1980. Destruction by anaerobic mesophilic
and thermophilic digestion of viruses and indicator bacteria
indigenous to domestic sludges. Appl. Environ. Microbiol. 39:
361-368.
265


59
for 24 hours a measured volume of sludge and was expressed as a per
centage on a weight (grams) to volume (milliliters) basis. Most of
the sludges used were not autoclaved or decontaminated in any other
way. Due to uncontrollable contamination of cell monolayers during
viral assays, two sludge samples (GDAN and PDAN--see Table 3-2) had to
be sterilized by autoclaving at 121C with applied pressure of 15 psi
for 15 minutes prior to use. During sludge treatment, digested sludges
are frequently subjected to heat (i.e., 150 to 260C under pressures of
150 to 400 psi) as a conditioning step which improves subsequent
sludge dewatering (U.S. Environmental Protection Agency 1978a; also
see Figure 2-1). Thus, autoclaved sludge can be considered to be heat-
conditioned sludge. As seen in Figure 3-1, the solids of autoclaved,
anaerobically digested sludge (GDAN--see Table 3-2) settled in 2
hours while those of nonautoclaved sludge remained dispersed. This
increase in the rate of settling of sludge solids probably accounts for
the improved-dewatering property of heat-conditioned sludge. Apart
from the effect on the rate of settling of sludge solids, autoclaving
probably did not significantly alter other sludge properties which
affect sludge-virus interactions. For example, Ward and Ashley (1977a)
found that autoclaving does not destroy the enterovirus-inactivating
capacity of anaerobically digested sludge. Furthermore, autoclaving
did not affect the degree of association between poliovirus and
anaerobically digested sludge solids as determined below.
Association of Seeded Poliovirus with Sludge Solids
One milliliter of poliovirus stock in PBS containing 2% FCS was
added directly to 100, 500, or 1,000 ml of sludge while stirring the


26
support this hypothesis has been obtained from studies involving the
anaerobic digestion of sludge. At the East Pearl treatment plant in
Boulder, Colorado, Moore et aj_. (1978) found total reductions of
indigenous enteroviruses in primary raw sludge of only 2 log-jg units
(i.e., 99%) during 100 days of anaerobic digestion (40 days at 37C
in digester no. 1 followed by 60 days in unheated digester no. 2).
Significantly higher inactivation rates were measured for seeded entero
viruses by Bertucci ert al_. (1977) and Eisenhardt et a]_. (1977) during
anaerobic digestion of sludge. For example, Eisenhardt et al_. (1977)
found the inactivation rate of seeded coxsackievirus B3 to be 2 log-^
units per 24 hours. The inactivation rates of seeded enteroviruses were
drastically reduced when the viruses were incorporated into the sludge
during sludge production rather than simply mixed with the final
sludge sample. Moore et al_. (1977) reported an inactivation rate of
approximately 2 1 og-jq units per 15 days for poliovirus naturally
incorporated (and assumed embedded) into wasted sludge during activated
sludge treatment in a continuous flow, bench-scale unit and then sub
jected to anaerobic digestion at 30C. In fact, these investigators
detected poliovirus in the sludge undergoing anaerobic digestion even
after 30 days. Clearly, enteroviruses embedded within sludge solids
are afforded some protection from virucidal chemical agents (e.g.,
uncharged ammonia in the liquid fraction of anaerobically digested
sludge--see Ward and Ashley 1977a) and/or physical stresses (e.g.,
heat) encountered during the anaerobic digestion of sludge, as well as
during other sludge treatment processes.


Conductivity
Conductivity Poliovirus eluted (expressed as cumulative% of total PFU having
(x 10 umho/cm at 25C) been applied at each pore volume)


aRainfal1 data were collected at the weather station of the Department of Agronomy, University of
Florida, Gainesville. This station is approximately 1 mile from the experimental site (i.e., next to the
Environmental Engineering Sciences building, University of Florida, Gainesville).
l o
D0ne inch or 2.5 cm (254 ni /ha) of aerobically digested sludge (see Table 5-3 for sludge character
istics) seeded with a total of 3.9 x 10PFU of poliovirus (or 6.3 x 107 PFU/g dry weight of sludge--see
Table 5-3) was applied to large soil cores of Eustis fine sand (LC3 and LC4--see Figure 5-1). The large
soil cores were 33 cm in length and 15.5 cm internal diameter; they consisted of the Ap and A21 horizons
of the Eustis fine sand (see Table 4-3). The seeded sludge was allowed to soak in and dry on top of the
soil for 1 day or 3 days before being mixed with the top 2.5 cm of soil. The drying sludge solids and
the soil were monitored for the presence of seeded viruses as detailed on pages 192 to 194.
cIn the case of the liquid sludge, sludge solids content was expressed as a percentage on a weight-to-
volume basis.
dA dash means not done or not applicable.


35
beds consist of 6 to 9 inches (ca. 15 to 23 cm) of sand underlaid with
approximately 12 inches (ca. 31 cm) of graded gravel or stone (U.S.
Environmental Protection Agency 1974). Criteria for the design of
drying beds can be found in the literature (U.S. Environmental Protec
tion Agency 1974, 1978a). In drying beds, water removal from sludge
is accomplished first by drainage (filtrate is collected by underdrain
system and is returned to the plant for further treatment) and then
followed by evaporation (U.S. Environmental Protection Agency 1974).
The effectiveness of sludge dewatering in drying beds is influenced by
weather conditions (e.g., precipitation, solar radiation, air tempera
ture, and relative humidity), sludge characteristics (e.g., primary
sludge dries faster than secondary sludge, digested sludge dries faster
than raw sludge, and digested sludge dries faster than lime-stabilized
sludge) and prior use of sludge conditioning (e.g., proper chemical
conditioning can reduce sludge dewatering time by 50% or more) (U.S.
Environmental Protection Agency 1974, 1978a). Sludge solids contents
ranging from 45% for well-digested sludge to 90% for chemically
conditioned sludge can be achieved on drying beds (U.S. Environmental
Protection Agency 1974). As with other dewatering processes, sandbed
drying reduces the volume of sludge to be further treated and disposed
of (U.S. Environmental Protection Agency 1974). Such dewatering,
therefore, reduces the fuel requirements of, for example, sludge
incineration (U.S. Environmental Protection Agency 1974).
During the air drying of sludge, it has also been demonstrated
that enteric viruses are inactivated. Working with raw sludge (pH 6 or


263
50 ml Gey's C (20x)
60 ml FCS (International Sci.)
25 ml Hepes buffer (1 M) stock
solution
12 ml glutamine (lOOx) (International
Sci.)
1.2 ml streptomycin-penicillin stock
(lOOOx)
Combine equal amounts of Solutions I and II to make the methyl
cellulose overlay. To 500 ml of the methyl cellulose overlay,
0.175 ml of kanamycin stock (i.e., stock supplied as a liquid at a
concentration of 1 gram per 3 ml).
7.Crystal violet:
Solution I: 20 grams crystal violet
200 ml absolute ethanol
Allow this solution to sit overnight.
Solution II: 8 grams ammonium oxalate
800 ml distilled water
Mix Solutions I and II, and dilute 1:10 with tap water. This
stain is used to make the plaques on the cell monolayer visible
to the naked eye. In some experiments, the cells were stained,
instead, with 0.5 ml of 0.5% neutral red.
8.Eagle's MEM using Gey's BSS plus 5% calf serum and 0.03 M Hepes
buffer at pH 7:
This solution was used to make some virus dilu
tions. The solution is made by substituting, in
Solution 4 above, 20 ml of calf serum (International
Sci.), 12 ml of 1 M Hepes buffer stock solution
and 8 ml of Gey's A (lx) for 40 ml of fetal calf
serum.
9.Phosphate-buffered saline (PBS) at pH 7.4-7.6:
8.0 grams NaCl
0.2 grams KC1
1.15 grams ^HPO^


TABLE 3-7. Effect of sludge type on the recovery of poliovirus type 1 from sludge using a modification
of the glycine method
Sludge
type
Sludge
used
Sludge parameters
Volume
of sludge
processed3
(ml)
Virusb
added to
siudge
(total PFU)
Overa11
virus
recovery0
(%)
Mean^ virus
recovery
for each
sludge type
(% SEe)
PH
Solids
content
(%)
Mixed liquor
UML
6.4
1.6
100
8.4
X
106
84.3*f
72 12a
GML
6.9
0.5
1000
1.3
X
106
60.3
Aerobically
UDA
4.8
1.5
500
6.5
X
!06
18.9*
15. < 2.7b
digested
UDA
4.8
1.5
500
6.5
X
106
11.9*
UDA
6.1
1.3
500
2.0
X
107
8.0*
UDA
6.1
1.3
500
2.0
X
107
5.9*
UDA
6.5
1.3
1000
9.6
X
106
15.5
GDA90
5.8
1.0
1000
1.3
X
107
14.1
PDA
5.8
2.8
1000
4.1
X
106
26.9
Anaerobically
PDAN
7.2
1.4
100
6.0
X
106
59.9*
60 2.1A
digested
PDAN
6.4
1.9
1000
5.5
X
106
*
63.9
LAGS
7.3
2.9
1000
1.8
X
106
56.7
aThe procedure used was a modification of the method developed by Hurst et^ aK (1978) and is described
in the Materials and Methods section.
bThe virus was added to sludge while stirring the suspension using a magnetic stirrer. Magnetic
stirring was continued for 10 min to 60 min and then the total sludge volume was centrifuged at 1400 x g for
10 min at 4C. The sludge supernatant and sludge solids generated were separately subjected to the virus
recovery methodology.


47
groundwater environment are not appreciably diluted and can persist
for long periods of time (Lee 1976).
Although sludge can be applied to land in several forms (i.e.,
liquid, dewatered or cake-dried), the application of sludge in the
liquid form is usually preferred because of its simplicity (U.S.
Environmental Protection Agency 1974, 1978b). For example, dewatering
processes are not required and inexpensive transfer systems (e.g.,
tank trucks) can be employed for handling liquid sludges (U.S. Environ
mental Protection Agency 1978b). Liquid sludge is usually applied to
land using one of the following methods: spray irrigation, surface
spreading--ridge and furrow irrigation, surface spreading--followed by
sludge incorporation into the topsoil within 2 to 14 days, and subsurface
injection (U.S. Environmental Protection Agency 1974, 1978b). There
are potential problems associated with each of these application methods.
While spray irrigation is a flexible method that requires minimum soil
preparation, dangerous aerosols containing pathogens are generated during
the spraying of sludge (U.S. Environmental Protection Agency 1974).
The spreading of sludge on the soil surface can lead to the contamina
tion of surface waters via runoff and/or soil erosion (U.S. Environ
mental Protection Agency 1974). However, if the applied sludge is
promptly incorporated into the topsoil, surface water pollution, odor
and aesthetic problems are largely eliminated (U.S. Environmental Pro
tection Agency 1974). The injection of sludge below the soil surface
avoids many of the problems associated with other application methods
(e.g., aerosols, runoff and odors) (U.S. Environmental Protection Agency


44
Moreover, sludge incineration destroys a potentially valuable resource
which could be utilized, for example, on agricultural land (Pahren
1980).
Ocean dumping. For years, seacoast communities have been dis
charging digested sludge offshore in deep water (Fair et al_. 1968;
U.S. Environmental Protection Agency 1974). Approximately 15% of the
sludge disposed of in 1979 nationally was dumped in the ocean (Pahren
et alL 1979). By the end of 1981, however, ocean disposal of sludge
will be prohibited by the U.S. federal government (Cowlishaw and Roland
1973; Pahren 1980; Pahren et aj_. 1979; U.S. Environmental Protection
Agency 1974). The primary reason for prohibiting ocean dumping is that
such a practice has been found to have long-term adverse effects on
the ocean environment and on marine life (Cowlishaw and Roland 1973).
Sanitary landfill. The burial of sludge (i.e., sludge covered
by a soil depth greater than the plow layer) in a sanitary landfill
is another popular and acceptable method for sludge disposal (U.S.
Environmental Protection Agency 1974, 1978b). Of the total amount of
sludge disposed of in 1979 nationally, approximately 25% was buried in
sanitary landfills (Pahren et al_. 1979). In order to prevent odor,
pathogen, operational and other problems, disposal in sanitary land
fills is usually restricted to well-stabilized, dewatered (>]5% solids
content for sludge-only landfills) sludge (U.S. Environmental Protec
tion Agency 1974, 1978b). The disposal of sludge in improperly
managed landfills can result in groundwater pollution (Pahren 1980).
Land application. Land application of sludge is receiving
increased attention and will probably be the predominant sludge


ro
co
o
o
a
o
D
<
o
£Z
3
fD
t/>
n>
c+
fD
Q-
cn
Mean pH
Mean
conductivity
(pmho/cm at
25C)
Poliovirus eluted (expressed as cumulative % of total PFU
having been applied at each pore volume)
CO
co
cr>
r
T
00
T
o
T
ro
oo
c
o_
a>
3
(/)
o
o
o
o
o
o '
o ^
o
O
O'"
o
ro
1 ro
*ro
1 o^rvo
ro
1 ro


c:
c
C O
c
c
C Ul "
co
3 co
3 co
3 *
3 *
3 -o
3
3
3
3 CO
3
3
3 CO
X
X
X
^X
X
X
\
cr>
cn
o
CO
ro
' O
i
j
1
3 1
J
i
3 _
o
o
o
coo
o
o
CO
CO
*T0
CO
O
CO
O
cn
o
i


TABLE 5-4. Survival of poliovirus type 1 following suspension in liquid sludge and subsequent application
to large soil cores of Eustis fine sand exposed to natural conditions (7 October 1977-12 October
1977)
Sampling Days
date after the
(1977) beginning
of
experiment
Cumulative
rainfall3
(cm)
Sludge
solids
content
(%, wt/wt.)
Soil
moisture
(%, wt-/wt>)
No. of viruses
(PFU/g dry weight of
sludge or soi1)
LC3
LC4
LIQUID SLUDGE SAMPLE
07 Oct. 0
0
1.3C
__d
6.3 x 107
6.3 x 107
DRYING SLUDGE SAMPLE
10 Oct. 3
0.08
38.0


1.0 x 103
Sludge mixed
with the top 2.5
cm of soil on
day 1 (LC3) or
on day 3 (LC4)
SOIL SAMPLES (top 2.5 cm)
08 Oct. 1
0


4.4 x 104

10 Oct. 3
0.08
--
11.3
2.2 x 103
4.9 x 101
12 Oct. 5
0.23


0
0


11
water and wastewater effluents by flocculation with alum, ferric
chloride or lime. Unfortunately, only a few studies have been con
ducted to determine the effectiveness of chemical flocculation in
removing viruses from raw sewage (see reviews by Berg 1973a; Lund 1976;
Malina 1976; Sproul 1976). In laboratory-scale pilot plants, Lund and
R0nne (1973) studied the fate of indigenous enteric viruses in raw
sewage following flocculation with alum [75 to 175 mg/Jl as A12(S0^)3],
ferric chloride (25 to 35 mg/i, as FeCl3) or lime (added lime until pH
10.5 was maintained). These investigators noted appreciable removals
of viruses in all flocculation experiments. The viruses were found
concentrated in the chemical sludges produced. Furthermore, no viral
inactivation was observed in the chemical sludges. In another labora
tory study, Sattar et al_. (1976) showed that 99.995% of poliovirus
type 1 (Sabin) seeded in raw sewage was inactivated during flocculation
with lime at pH 11.5. No viruses were recovered in the supernatants
following flocculation and an average of only 0.005% of the total
viral input was recovered in the lime sludges by eluting with 10% FCS
in saline, pH 7.2. During storage at 28C, further viral inactivation
was noted in the lime sludges and no viruses could be detected in the
sludge samples after 12 hours. The inactivation rate of seeded polio
virus type 1 (Sabin) during lime flocculation of raw sewage was
reduced as the pH of the solution was decreased. Sattar and Ramia
(1978) found that 92.4 and 97.2% of the total inputs of poliovirus
were inactivated (i.e., recovered only 7.6 and 2.8% in the supernatants
and sludges combined) during lime flocculation at pH 9.5 and 10.5,


TABLE 4-9.
Retention of poliovirus type 1 by a packed column of Red Bay sandy loam subsoil when sus
pended in anaerobically digested sludge diluted (1:50) with distilled water
No. of pore
volumes3
eluted
Poliovirus
eluted
(PFU/ml)
% of Influent
poliovirus
concentration
% of Total PFU
having been
applied at each
pore volume
(cumulative)
Conductivity
of pore volume
collected
(umho/cm at 25C)
pH of pore
volume
collected
0.5
0
0
0
66
6.0
1.0
0
0
0
72
5.6
1.5
0
0
0
87
5.9
2.0
0
0
0
94
5.8
2.5
0
0
0
95
6.1
3.0
0
0
0
97
5.7
3.5
0
0
0
96
5.6
4.0
0
0
0
93
6.1
4.5
0
0
0
92
5.8
5.0
0
0
0
92
5.8
5.5
0
0
0
90
5.8
6.0
0
0
0
89
5.8
6.5
0
0
0
87
5.8
7.0
0
0
0
88
5.9
7.5
0
0
0
86
5.9
8.0
0
0
0
88
5.8
8.5
0
0
0
93
5.9


Anaerobically
digested (30 days)
Anaerobically
digested (22-25 days)
(
Dried sludge (anaero
bically digested)
sampled at the soil
surface 1 day to 4
months after land
application
Beef extract (3%) or
fetal calf serum (10%)
both in saline, pH 7.2
None
Indigenous
Raw
Sattar and Westwood
(1976)
Fetal calf serum (10%)
in saline, pH 7.2
None
Indigenous
Raw
Anaerobically
digested (20 days)
Lagoon-dried (anaero
bically digested)
Sattar and Westwood
(1979)
Fetal calf serum (2%)
in Earle's balanced
salt solution, pH 9.5
None
Poliovirus 1
(Sabin)
Digested
Subrahmanyan (1977)
Beef extract (3%),
pH 9.0
Hydroextraction
Indigenous
Digested (40 days)
Digested (> 60 days)
Dried sludge cake from
a sludge spray site
Wei lings et al.
(1976a)
aThe chemicals listed
were used to elute
viruses from sludge
solids.
^For some digested sludges, the type of digestion (i.e., aerobic or anaerobic) and/or the digestion
time were not given in the literature.
cThe effectiveness of this method in recovering seeded viruses was evaluated using activated sludge
only. The method was subsequently used for recovering indigenous viruses from all the sludge types listed.


270
Elliott, L. F., and J. R. Ellis. 1977. Bacterial and viral pathogens
associated with land application of organic wastes. J. Environ.
Qua!. 6: 245-251.
Elrick, D. E., and L. K. French. 1966. Miscible displacement patterns
on disturbed and undisturbed soil cores. Soil Sci. Soc. Am.
Proc. 30: 153-156.
England, B., R. E. Leach, B. Adame, and R. Shiosaki. 1965. Virologic
assessment of sewage treatment at Santee, California. In
Transmission of Viruses by the Water Route, pp. 401-417. Edited
by 6. Berg. Interscience Publishers, New York, N.Y.
Epstein, E. 1975. Effect of sewage sludge on some soil physical
properties. J. Environ. Qua!. 4j 139-142.
Epstein, E., J. M. Taylor, and R. L. Chaney. 1976. Effects of sewage
sludge and sludge compost applied to soil on some soil physical
and chemical properties. J. Environ. Qua!. 422-426.
Evans, J. 0. 1973. Research needs--land disposal of municipal sewage
wastes. In Recycling Treated Municipal Wastewater and Sludge
through Forest and Cropland, pp. 455-462. Edited by W. E.
Sopper and L. T. Kardos. Pennsylvania State University Press,
University Park, Pennsylvania.
Fair, 6. M., J. C. Geyer, and D. A. Okun. 1968. Water and Wastewater
Engineering. Volume 2. Water Purification and Wastewater
Treatment and Disposal. John Wiley and Sons, Inc., New York,
N.Y.
Farrah, S. R., G. Bitton, E. M. Hoffmann, 0. Lanni, 0. C. Pancorbo,
M. C. Lutrick, and J. E. Bertrand. 1981a. Survival of entero
viruses and coliform bacteria in a sludge lagoon. Appl.
Environ. Microbiol. 41: 459-465.
Farrah, S. R., C. P. Gerba, C. Wallis, and J. L. Melnick. 1976.
Concentration of viruses from large volumes of tap water using
pleated membrane filters. Appl. Environ. Microbiol. 31: 221-
226.
Farrah, S. R., S. M. Goyal, C. P. Gerba, R. H. Conklin, C. Wallis,
J. L. Melnick, and H. L. DuPont. 1978. A simple method for
concentration of enteroviruses and rotaviruses from cell culture
harvests using membrane filters. Intervirology 9: 56-59.
Farrah, S. R., P. R. Scheuerman, and G. Bitton. 1981b. Urea-lysine
method for recovery of enteroviruses from sludge. Appl.
Environ. Microbiol. 41: 455-458.


276
Lund, E., C. E. Hedstrom, and N. Jantzen. 1969. Occurrence of enteric
viruses in wastewater after activated sludge treatment.
Water Pollut. Control Fed. 41: 169-174.
Lund, E., and V. R0nne. 1973. On the isolation of virus from sewage
treatment plant sludges. Water Res. 7_: 863-871.
Lutrick, M. C., and J. E. Bertrand. 1976. Agronomic and cattle studies
with municipal liquid digested sludge. Proc. Institute Environ.
Sciences 22: 528-532.
Mack, W. N., J. R. Frey, B. J. Riegle, and W. L. Mallmann. 1962.
Enterovirus removal by activated sludge treatment. J. Water
Pollut. Control Fed. 34: 1133-1139.
Mack, W. N., Y.-S. Lu, and D. B. Coohon. 1972. Isolation of polio
myelitis virus from a contaminated well. Health Services
Reports 87: 271-274.
Malina, J. F., Jr. 1976. Viral pathogen inactivation during treatment
of municipal wastewater. In Proceedings of the Symposium on
Virus Aspects of Applying Municipal Waste to Land, pp. 9-23.
Edited by L. B. Baldwin, J. M. Davidson, and J. F. Gerber.
Institute of Food and Agricultural Sciences, University of
Florida, Gainesville, Florida.
Malina, J. F., Jr., K. R. Ranganathan, B. P. Sagik, and B. E. Moore.
1975. Poliovirus inactivation by activated sludge. J. Water
Pollut. Control Fed. 47: 2178-2183.
Mandel, B. 1971. Characterization of type 1 poliovirus by electro
phoretic analysis. Virology 44: 554-568.
Manson, R. J., and C. A. Merritt. 1975. Land application of liquid
municipal wastewater sludges. J. Water Pollut. Control Fed. 47:
20-29.
Marzouk, Y., S. M. Goyal, and C. P. Gerba. 1979. Prevalence of entero
viruses in ground water in Israel. Ground Water 17: 487-491.
McFeters, G. A., G. K. Bissonnette, J. J. Jezeski, C. A. Thomson, and
D. G. Stuart. 1974. Comparative survival of indicator bac
teria and enteric pathogens in well water. Appl. Microbiol.
27: 823-829.
McMahon, M. A., and G. W. Thomas. 1974. Chloride and tritiated water
flow in disturbed and undisturbed soil cores. Soil Sci. Soc.
Am. Proc. 38: 727-732.
McNabb, J. F., and W. J. Dunlap. 1975. Subsurface biological activity
in relation to ground-water pollution. Ground Water 13: 33-44.


200
is seen that air temperature was as high as 31C and as low as 15C.
The survival of poliovirus, under natural conditions, following sus-
g
pension in aerobically digested sludge (3.9 x 10 total PFU or 6.3 x
10^ PFU/g dry weight of sludge) and subsequent application to soil
cores is shown in Table 5-4. No virus could be recovered in the soil
samples after three days. The sludge was left on top of the soil for
three days (i.e., large soil core no. 4--LC4), and mixed thereafter
with the top 2.5 cm of soil. During that time period, there was more
than a four 1 g-|q reduction in virus numbers in the drying sludge
(see LC4Table 5-4). It is worth noting that during this first
experiment, the rainfall was low (0.23 cm after five days--see Table
5-4) and the sludge solids increased from 1.3% to 38%. Dessication
was probably the major factor which caused the rapid decline of polio
virus in the drying sludge and in the soil.
Second Survival Experiment
(2 June 1978-24 August 1978)
The second survival experiment was initiated in the summer when
the weather is generally warm and wet in the Gainesville area. Tempera
ture data were collected with thermocouples placed at the surface of
the soil, and at the 2.5-, 10-, and 20-cm depths. Data analysis showed
that there was no significant difference between soil temperature
readings at these different depths. Therefore, only the soil
i
temperature at the 2.5-cm depth is shown in Figure 5-4. The average
temperature ranged from 23.5C to 29C during a 35-day period beginning


Soil temperature (C)
t
2 June 78
Days after sludge application to the soil


12
respectively. In these studies (Sattar et al_. 1976; Sattar and Ramia
1978), it was pointed out that the method used (i.e., elution with
10% FCS in saline, pH 7.2) to recover viruses from the lime sludges
may not have been completely effective. In spite of this possible
limitation, the authors concluded that substantial viral inactivation
can be achieved during the flocculation of raw sewage with lime at high
pH.
The literature presented herein indicates that chemical floccu
lation, under optimal laboratory conditions, can remove large quanti
ties of viruses from raw sewage and from other water samples. Virus
removal efficiency depends on the coagulant, coagulant dose, water
type, pH, virus type and flocculation procedure employed. Since it
is difficult to ensure optimum floe formation routinely in practice,
Grabow (1968) hypothesized that, under field conditions, significant
virus removals by flocculation are not likely. As proposed by Berg
(1973b), more research is needed before a conclusion can be reached as
to the virus removal capacity of chemical flocculation in actual prac
tice.
The effluents from the primary sedimentation units generally
still contain demonstrable quantities of enteric viruses (Sattar and
Ramia 1978). These primary effluents usually undergo further treatment
by, for example, the activated sludge process. Large quantities of
sludge (i.e., 2,400 to 5,000 gallons of sludge per million gallons
of wastewater treated) are produced during the primary sedimentation
process (U.S. Environmental Protection Agency 1974). When chemical


38
reverse the protective effect towards enteroviruses attributed to ionic
detergents associated with sludge solids (Ward and Ashley 1977c, 1978a,
1979; Ward et al_. 1976). Moreover, since the evaporation process
itself was found primarily responsible for viral inactivation during
the air drying of sludge (Ward and Ashley 1977b), and digested sludge
has been shown to air dry at a more rapid rate and to a greater extent
than raw sludge (U.S. Environmental Protection Agency 1974), it follows
that viral inactivation would probably be greater in anaerobically
digested sludge than in raw sludge. Ward and Ashley (1978b) also ob
served the inactivation rate of poliovirus type 1 (CHAT) seeded in
dewatered raw sludge to increase with a corresponding increase in tem
perature. Consequently, viral inactivation is likely to be substan
tially accelerated during the air drying of sludge at higher temperatures
than the 21C employed by Ward and Ashley (1977b). Under the most ideal
conditions, however, sludge dewatering by air drying in sandbeds is not
likely to yield a virus-free product. In Florida, for example, Wellings et
al. (1976a) detected 24 PFU of echovirus type 7 in 250 grams of air-
dried (for 13 days) sludge obtained from drying beds. Due to the pos
sible viral hazard, all air-dried sludges should be handled with care
during further treatment and final disposal (Wellings et al_. 1976a).
Dewatering-drying lagoons. Lagoons have also been commonly
used in the United States for sludge dewatering (see Figure 2-1) (Fair
et al_. 1968; U.S. Environmental Protection Agency 1974). Bitton (1980b)
reported that 264,000 dry tons of treated sludge (or 4.5% of the total
sludge available) are discharged yearly into lagoons in the United


isa
TABLE 5-1. General properties of echoviruses
Property
Value3
Nucleic acid
RNA (single-stranded)
Molecular weight
of nucleic acid (daltons)
2 x 106
Particle diameter (nm)
17 to 30
Morphology
Icosahedral
Stability at 4C
Stable for 1 to 2 years
Stability at pH 3.0
Stable for 3 hours at 25C
Stability in ether
Stable
aAll data were obtained from Wulff and Chin (1972).


71
sludges to bind viruses. However, we have shown that the sludge solids
content does not account for the differences observed in the virus
binding capacity of different sludge types. In the range of sludge
solids contents studied, there appears to be sufficient sites for virus
binding and, therefore, this sludge parameter is not a limiting factor
in determining the association of viruses with sludge solids.
The degree of association between seeded poliovirus, and aerobic
(see Table 3-5) or anaerobic (see Table 3-6) sludge solids remained
fairly constant over a contact period of 3 days or 12 hours,
respectively. Clearly, varying the contact time would not have sig
nificantly affected the results presented above on the virus-binding
capacity of different sludge types. The inactivation rate of poliovirus
seeded in sludge was also determined from the data shown in Tables 3-5
and 3-6. In anaerobically digested sludge, the total amount of
infectious poliovirus present in the sludge (i.e., virus in unfrac
tionated sludge) steadily declined over a 12-hour period (Table 3-6).
In this sludge type, there was approximately a 50% reduction in the
poliovirus titer in 12 hours (i.e., approximately 1 1 og-jQ reduction/36
hours) at room temperature (see Table-3-6). Ward and Ashley (1976)
measured similar inactivation rates for poliovirus type 1 (CHAT and
Mahoney) seeded in anaerobically digested sludge. In contrast to
anaerobically digested sludge, there was no significant inactivation
of poliovirus seeded in aerobically digested sludge during a 7-day
contact period at room temperature (see Table 3-5). The low viral-
inactivating capacity of aerobically digested sludge has also been


TABLE 3-2.
Sources of the wastewater sludges used in this study
Wastewater
treatment
plant3
generating
the sludge
Abbreviated
sludge
designation
SIudgeb
type
used
Sludge treatment
Digestion
procedure
Digestion
time
(days)
Additional
treatment
Campus plant
UML
Mixed liquorc
__d
_ _
_ _
(Univ. of Florida)
UDA
Digested
Aerobic
39

Main street plant
GML
Mixed liquor0
_ _
(Gainesville,
GDA90
Digested
Aerobic
90

Florida)
GDA180
Digested
Aerobic
180
--
GDAN
Digested
Anaerobic
60

Montclair plant
(Pensacola,
Florida)
PDA
Digested
(and dewatered)
Aerobic
30
Conditioned with
Magnafloce 1563c
and then dewatered
Main street plant
(Pensacola,
Florida)
PDAN
Digested
(and dewatered)
Anaerobic
60
Conditioned with
Magnafloce 2535c
and then dewatered
Montclair and Main
street plants
(Pensacola, Florida)
LAG
Lagoonf
aAll plants treated municipal wastewater with the exception of the Main street plant of Pensacola
which treated a mixture of municipal (2/3) and industrial (1/3) wastewater.
^The sludges used were generated during the secondary treatment (activated sludge or tricking filter)
of wastewater.


271
Farrell, J. B., J. E. Smith, Jr., S. W. Hathaway, and R. B. Dean.
1974. Lime stabilization of primary sludges. J. Water Pollut.
Control Fed. 46: 113-122.
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1. Physical and Mineralgica! Properties, Including Statistics
of Measurement and Sampling, pp. 400-412. Edited by C. A.
Black, D. D. Evans, J. L. White, L. E. Ensminger, F. E. Clark,
and R. C. Dinauer. American Society of Agronomy, Inc., Madison,
Wisconsin.
Fenters, J., J. Reed, C. Lue-Hing, and J. Bertucci. 1979. Inactiva
tion of viruses by digested sludge components. J. Water Pollut.
Control Fed. 51: 689-694.
Filmer, R. W., and A. T. Corey. 1966. Transport and Retention of
Virus-Sized Particles in Porous Media. Sanitary Engineering
Paper No. 1. Colorado State University, Fort Collins, Colorado.
Floyd, R., and D. G. Sharp. 1977. Aggregation of poliovirus and
reovirus by dilution in water. Appl. Environ. Microbiol. 33:
159-167.
Floyd, R., and D. G. Sharp. 1978a. Viral aggregation: Quantitation
and kinetics of the aggregation of poliovirus and reovirus.
Appl. Environ. Microbiol. 35: 1079-1083.
Floyd, R., and D. G. Sharp. 1978b. Viral aggregation: Effects of
salts on the aggregation of poliovirus and reovirus at low pH.
Appl. Environ. Microbiol. 35: 1084-1094.
Floyd, R., and D. G. Sharp. 1979. Viral aggregation: Buffer effects
in the aggregation of poliovirus and reovirus at low and high
pH. Appl. Environ. Microbiol. 38: 395-401.
Foster, D. H., and R. S. Engelbrecht. 1973. Microbial hazards in
disposing of wastewater on soil. In Recycling Treated Munici
pal Wastewater and Sludge through Forest and Cropland, pp. 247-
270. Edited by W. E. Sapper and L. T. Kardos. Pennsylvania
State University Press, University Park, Pennsylvania.
Freeze, R. A., and J. A. Cherry. 1979. Groundwater. Prentice-Hall,
Inc., Englewood Cliffs, New Jersey.
Funderburg, S. W., B. E. Moore, C. A. Sorber, and B. P. Sagik. 1979.
Method of soil column preparation for the evaluation of viral
transport. Appl. Environ. Microbiol. 38: 102-107.
Gainesvi11 e-Alachua County Regional Utilities. 1976. Application for
Permit Demonstration Sludge Farming Project Utilizing Digested
Sewage Sludge as a Resource Material. Regional Utilities Board,
Gainesville, Florida.


283
Wellings, F. M., A. L. Lewis, and C. W. Mountain. 1976a. Demonstra
tion of solids-associated virus in wastewater and sludge.
Appl. Environ. Microbiol. 31: 354-358.
Wellings, F. M., A. L. Lewis, and C. W. Mountain. 1976b. Viral con
centration techniques for field sample analysis. In Proceedings
of the Symposium on Virus Aspects of Applying Municipal Waste
to Land, pp. 45-51. Edited by L. B. Baldwin, J. M. Davidson,
and J. F. Gerber. Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, Florida.
Wellings, F. M., A. L. Lewis, and C. W. Mountain. 1978. Assessment
of health risks associated with land disposal of municipal
effluents and sludge. In Proceedings of the Conference on Risk
Assessment and Health Effects of Land Application of Municipal
Wastewater and Sludges, pp. 168-179. Edited by B. P. Sagik
and C. A. Sorber. The University of Texas, San Antonio,
Texas.
Wellings, F. M., A. L. Lewis, C. W. Mountain, and L. V. Pierce. 1975.
Demonstration of virus in groundwater after effluent discharge
onto soil. Appl. Microbiol. 29: 751-757.
Wentworth, D. F., R. T. Thorup, and 0. J. Sproul. 1968. Poliovirus
inactivation in water-softening precipitation processes.
J. Am. Water Works Assoc. 60: 939-946.
Wesner, G. M., and D. C. Baier. 1970. Injection of reclaimed waste-
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203-210.
Willems, D. G. 1976. Land treatment of wastewaters institutional and
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Municipal and Industrial Wastewater, pp. 1-15. Edited by R. L.
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Wilson, S. D. 1950. Small soil compaction apparatus duplicates field
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World Health Organization. 1968. The Work of WHO Virus Reference
Centres and the Services They Provide. World Health Organiza
tion, Geneva, Switzerland.


aA two-tailed, t-test concerning the population correlation coefficient (r) revealed that at the 0.01
level there was no significant linear correlation (i.e., r = 0) between the solids content (%) of the sludge
and the "solids-associated virus (%)" values. This lack of correlation was found within each sludge type
and for all sludge types combined.
bThe virus was added to the sludge while stirring the suspension using a magnetic stirrer. Magnetic
stirring was continued for 10 to 60 min and then an aliquot of the virus-seeded sludge was obtained for
viral assay.
cThe sludge solids were not separated prior to assaying.
dThe sludge was clarified by centrifugation at 1400 x g for 10 min at 4C and the supernatant was
subsequently assayed.
eThe "viable unadsorbed virus (%)" values were calculated as shown in the Materials and Methods section
'The "sol ids-associated virus (%)" values were estimated as shown in the Materials and Methods section.
gMean values displaying different superscript capital letters are significantly different at the 0.01
level when subjected to analysis of variance using Duncan's test.
^Abbreviation for standard error.
]The lagoon sludge is a mixture of aerobically digested sludge (1/3) and anaerobically digested sludge
(2/3), and consequently, its properties are close to those of anaerobically digested sludges (see Table 3-2)
Therefore, lagoon sludges were placed in the population of anaerobically digested sludges.


171
TABLE 4-23. Inactivation of poliovirus type 1 following lime stabilization
of chemical sludges
Sludge3
Concentration
pH, 30 min
Virus in un
Virus in
type
Pf
after the
fractionated0
unfractionated
lime0 used
addition
sludge,
sludge, 30 min
(mg/1)
of lime
before
1iming
after liming
Total PFU Total PFU Recoveryd
Alume
1389
11.3
i
X
o
0
0
Ferric
chloridee
625
11.1
5.2 x 106
6.2 x 103
0.1
aThe chemical sludges were precipitated from virus-seeded, raw sewage.
The methods used to produce these chemical sludges and to determine the
amount of viruses present in the sludges are described in Table 4-20.
bAn aqueous slurry of lime (5% Ca(0H)2) was added to the chemical
sludges shown until a pH of 11.5 was achieved and maintained for 5 min. The
final concentrations of Ca(0H)2 used arppear in the table above. A contact
time of 30 min was allowed while mixing the suspension on a magnetic stirrer.
cThe sludge solids were not separated prior to assaying.
^Percent recoveries shown were calculated based on the corresponding
unfractionated sludge assay before liming as 100%.
eThe lime-stabilized, chemical sludges were applied to columns of Red
Bay sandy loam subsoil (see Table 4-25).


3
4. Monitoring of indigenous enteroviruses at two
sludge disposal sites in Florida (Chapter VI)
5. Effect of hydrostatic pressure on the survival of
poliovirus seeded in groundwater and seawater (Chap
ter VII--this research was conducted in order to
determine virus survival in the groundwater
environment).
This research has allowed the determination of the persistence and
possible movement of pathogenic viruses in soils treated with wastewater
sludge. The information gained from this study is of value in the
ultimate assessment of the potential risk of viral infection to humans
associated with land disposal of sludges.


281
Subrahmanyan, T. P. 1977. Persistence of enteroviruses in sewage
sludge. Bull. W.H.O. 55: 431-434.
Thabaraj, G. J. 1975. Land-spreading of secondary effluents. Florida
Sci. 38: 222-227.
Thomas, G. W., and R. E. Phillips. 1979. Consequences of water movement
in macropores. J. Environ. Qual. 8: 149-152.
Tierney, J. T., R. Sullivan, and E. P. Larkin. 1977. Persistence of
poliovirus 1 in soil and on vegetables grown in soil previously
flooded with inoculated sewage sludge or effluent. Appl.
Environ. Microbiol. 33: 109-113.
Turk, C. A., B. E. Moore, B. P. Sagik, and C. A. Sorber. 1980. Recovery
of indigenous viruses from wastewater sludges, using a bentonite
concentration procedure. Appl. Environ. Microbiol. 40: 423-425.
United States Environmental Protection Agency. 1973. Physical-Chemical
Wastewater Treatment Plant Design. EPA-625/4-73-002a. Environ-
mental Research Information Center, Technology Transfer, Cin
cinnati, Ohio.
United States Environmental Protection Agency. 1974. Process Design
Manual for Sludge Treatment and Disposal. EPA-625/1-74-006.
Environmental Research Information Center, Technology Transfer,
Cincinnati, Ohio.
United States Environmental Protection Agency. 1977. Process Design
Manual for Land Treatment of Municipal Wastewater. EPA-625/
1-77-008. Environmental Research Information Center, Technology
Transfer, Cincinnati, Ohio.
United States Environmental Protection Agency. 1978a. Sludge Treatment
and Disposal. Volume 1, Sludge Treatment. EPA-625/4-78-012.
Environmental Research Information Center, Technology Transfer,
Cincinnati, Ohio.
United States Environmental Protection Agency. 1978b. Sludge Treatment
and Disposal. Volume 2. Sludge Disposal. EPA-625/4-78-012.
Environmental Research Information Center, Technology Transfer,
Cincinnati, Ohio.
Vaughn, J. M., E. F. Landry, L. J. Baranosky, C. A. Beckwith, M. C.
Dahl, and N. C. Delihas. 1978. Survey of human virus occurrence
in wastewater-recharged groundwater on Long Island. Appl.
Environ. Microbiol. 36: 47-51.
Wang, D.-S., C. P. Gerba, and J. C. Lance. 1980a. Movement of entero
viruses through soil columns. Abstracts of the Annual Meeting,
American Society for Microbiology. Abstract Q103, p. 211.


137
TABLE 4-14. Continued
bEach soil section was mixed well, and a 10-gram wet sample was
taken and mixed with 20 ml of 3% beef extract, Tris buffered at pH 9.0.
This mixture was then vortexed for 30 sec and sonicated for 3 min.
The sample was then centrifuged at 1900 x g for 10 min at 4C. The
supernatant was subsequently assayed for viruses.
c 7
The amount of virus applied to the soil column was 1.1 x 10
total PFU (see Table 4-12). Overall recovery of the virus applied was
13.0% [i.e., 12.5% found in the soil and 0.5% found in the soil leachates
(see Table 4-12)].
dRefers tothesludge solids resting on top of the soil. The total
amount of these solids was separated, subjected to the same virus
elution method as the soil (added eluent at the proportion of 2 ml per
gram of wet sludge solids) and was considered as one section.


62
suspension using either a magnetic stirrer or pipette (magnetic stirring
could not be used for some sludges which had high solids contents).
Poliovirus was seeded in the sludge samples at concentrations approxi
mately 1,000 times greater than the indigenous virus levels measured
in the sludges used. Therefore, indigenous viruses present in the
sludge samples did not affect the results presented herein. Magnetic
stirring (or frequent mixing with a pipette) was continued for 10
minutes to 60 minutes. Following the contact period, an aliquot of the
unfractionated sludge (i.e., sludge sample without solids separated)
was diluted in PBS containing 2% FCS and assayed directly for seeded
viruses by the plaque technique. This assay was performed in order
to determine if the direct viral assay after the contact period would
agree with the calculated virus input based on the added volume of
poliovirus stock of known titer. From Table 3-3, it can be seen that
poliovirus was recovered with a mean efficiency of 109%, 104%, and '
98% from mixed liquors, aerobically digested sludges and anaerobically
digested sludges, respectively, following dilution and subsequent
direct assay on cell cultures. Thus, poliovirus type 1 (LSc) added
to sludge was recovered effectively after a contact period of 10 to
60 minutes without any significant inactivation. Direct inoculation
of unfractionated sludge into cell cultures has been found1 to be toxic
to cells (Nielsen and Lydholm 1980; Subrahmanyan 1977). In my research,
the diluted sludge samples were not toxic to the cell cultures. Simi
larly, Hurst et al_. (1978) diluted virus-seeded activated sludge in
Tris buffer and then successfully inoculated it directly into cell
cultures without causing cell toxicity. Some of the sludges used in my


112
subsoil would have on the rate of water movement through the soil.
The soil was packed into acrylic plastic columns (10 cm in length and
4.8-cm internal diameter; packed 5.0 cm with soil) at bulk densities
3
of 1.45, 1.60, 1.70, 1.85, and 2.00 dry g/cm A polypropylene screen
(105-ym pore size) was used to support the soil in each column. The
3
columns at bulk densities of 1.45 and 1.60 g/cm were packed with
air-dried soil while tapping on the outside of the columns. The amount
of tapping was increased to achieve the higher bulk density (i.e.,
3
1.80 g/cm ). The columns at bulk densities of 1.70, 1.85, and 2.00
3
g/cm were packed with moist soil [12.4% (wt./wt.) moisure content was
adjusted with rain water]. As shown above, 12.4% is the optimum
moisture content for the compaction of this soil. The moist soil
was compacted in the soil columns in 3 layers with 13, 20, or 33 tamps
applied per layer [using a 20-lb (9.1-kg) tamper as described by
Wilson (1950)--see above] in order to obtain a bulk density of 1.70,
1.85, or 2.00 g/cm respectively. The saturated hydraulic conduc
tivity of each soil column was then measured using the "constant-head"
method of Klute (1965). Briefly, the procedure consisted of applying
a constant, hydraulic head of 2.5 cm to each saturated soil column using
rain water and then measuring the volume of leachate collected in a
measured time. When possible, the leachate sample was collected
within 30 minutes of the beginning of leaching as recommended by Klute
(1965). The saturated hydraulic conductivity was calculated by the
following equation (Klute 1965):
K = (Q/At)(L/AH)
(4-5)


237
supernatants (i.e., the soil eluates) were recovered and immediately
adjusted to neutral pH by the addition of 1 M glycine buffer, pH 2.0.
The entire procedure described above was performed in approximately
10 minutes. Viruses in the soil eluates were concentrated by organic
flocculation (Katzenelson et cfL 1976b) as follows. The eluates were
adjusted to pH 3.5 by the addition of 1 M glycine buffer, pH 2.0, and
to 0.06 M aluminum chloride. The floes produced were pelleted by
centrifugation at 8000 x g for 10 minutes at 4C. The supernatants
and pellets produced were treated separately. The supernatants were
passed through a series of 3.0-, 0.45- and 0.45-um Filterite filters
(Filterite Corp., Timonium, Maryland) in a 47-mm holder. Adsorbed
viruses were eluted from the filters with 7 ml of phosphate-buffered
saline (PBS) containing 10% fetal calf serum (FCS), pH 9.0. The filter
eluates were adjusted to neutral pH by the addition of 1 M glycine
buffer, pH 2.0, and assayed for viruses as described below. The
pellets previously obtained by centrifuging the samples at pH 3.5 (and
at 0.06 M aluminum chloride) were mixed with five volumes of PBS
containing 10% FCS, pH 9.0. The mixtures were adjusted to pH 9.0 by
the addition of 1 M glycine buffer, pH 11.5, vortexed for 30 seconds and
then centrifuged at 8000 x g for 10 minutes at 4C. The supernatants
were adjusted to neutral pH by the addition of 1 M glycine buffer, pH
2.0. Viruses in these samples were further concentrated by hydro
extraction at 4C using polyethylene glycol (i.e., Carbowax PEG 20,000
from Fisher Scientific Co., Pittsburgh, Pennsylvania). The hydroex-
tracted samples were dialyzed against PBS at 4C in order to remove


8 2/3
0
0
0.01
1370
5.0
9
0
0
0.01
1370
5.0
9 1/3
0
0
0.01
1370
5.0
9 2/3
0
0
0.01
1370
5.0
10
0
0
0.01
1370
5.0
Shift to
nonseeded rain water^
10 1/3
0
0
0.01
1360
5.1
10 2/3
0
0
0.01
1360
5.1
11
0
0
0.01
1360
5.0
11 1/3
0
0
0.01
1200
5.1
11 2/3
0
0
0.01
800
5.0
12
7.5
0.1
0.01
400
5.1
aOne pore volume for the column used equals 225 ml. The laboratory packed column was 29 cm in length
and 4.8 cm internal diameter; the column was filled only 27 cm with soil (2 cm left on top for packed
sludge). The sample of Red Bay sandy loam subsoil used consisted mainly of the A2 and Bit horizons (see
Table 4^3). The column was conditioned with 5 pore volumes of 0.01 N CaCl2. All solutions were applied
continuously to the column at approximately 5 ml/min using a peristaltic pump (Buchler, Fort Lee, N.J.).
^Poliovirus was seeded in the influent (i.e., sludge diluted with 0.01 N CaClo) at a concentration of
7.3 x 103 PFU/ml.
cThe anaerobically digested sludge (GDAN--see Table 3-2) used had a solids content of 2.0%, a
conductivity of 3250 ymho/cm at 25C and a pH of 8.3. The conductivity of sludge diluted (1:50) with
0.01 N CaCl2 was 1350 ymho/cm at 25C and the pH was 6.5.
^Rain water was collected next to the Environmental Engineering Sciences building at the University of
Florida, Gainesville. See Table 4-2 for chemical characteristics of the rain water.


39
States. Due to the great potential for odor problems associated with
sludge lagoons, only well-stabilized sludge has been recommended for
dewatering in lagoons (U.S. Environmental Protection Agency 1974).
Criteria for the design of drying lagoons can be found in the litera
ture (Sanks et a]_. 1976; U.S. Environmental Protection Agency 1974).
Particularly important are the design criteria intended for the protec
tion of groundwater supplies. For example, the bottom of sludge lagoons
must be at least 18 inches above the maximum groundwater table in order
to prevent groundwater contamination (U.S. Environmental Protection
Agency 1974). In drying lagoons,water removal from sludge is primarily
achieved by evaporation (U.S. Environmental Protection Agency 1974).
Therefore, the effectiveness of sludge dewatering in drying lagoons is
mostly influenced by weather conditions (e.g., maximum drying rate in
hot, arid climate) and by sludge depth (e.g., drying rate increases as
the sludge depth decreases) (U.S. Environmental Protection Agency 1974).
The little information available indicates that sludge dewatering in
drying lagoons is an extremely slow process. Sludge held in a lagoon
at depths of 2 to 4 feet, for example, required three years to dewater
from 5% solids content to 45% solids content (U.S. Environmental Pro
tection Agency 1974).
Long-term lagooning has been found to destroy a significant
fraction of the pathogens in digested sludge (U.S. Environmental Pro
tection Agency 1978b). In lagoons that had stopped receiving addi
tional quantities of digested sludge prior to their investigations,
Sattar and Westwood (1979) and Farrah et al_. (1981a) confirmed that
indigenous enteric viruses associated with lagooned sludge are inac
tivated at a measurable rate. Under the warm temperatures of late


273
Hamann, S. D., and W. Strauss. 1955. The chemical effects of pressure.
Part 3. Ionization constants at pressures up to 12,000 atm.
Trans. Faraday Soc. 51: 1684-1690.
Hays, B. D. 1977. Potential for parasitic disease transmission with
land application of sewage plant effluents and sludges. Water
Res. 11: 583-595.
Hedn, C.-G. 1964. Effects of hydrostatic pressure on microbial sys
tems. Bacterio!, Rev. 28: 14-29.
Hendry, C. D. 1977. The Chemistry of Precipitation in North-Central
Florida. Master of Science Thesis, University of Florida,
Gainesville, Florida.
Hiatt, C. W. 1964. Kinetics of the inactivation of viruses. Bac
terio!. Rev. 28: 150-163.
Hill, W. F., Jr., E. W. Akin, and W. H. Benton. 1971. Detection of
viruses in water: A review of methods and application. Water
Res. 5: 967-995.
Holtzclaw, K. M., D. A. Keech, A. L. Page, G. Sposito, T. J. Ganje,
and N. B. Ball. 1978. Trace metal distributions among the
humic acid, the fulvic acid, and precipitable fractions
extracted with NaOH from sewage sludges. J. Environ. Qual. 7:
124-127.
Holtzclaw, K. M., G. Sposito, and G. R. Bradford. 1976. Analytical
properties of the soluble, metal-complexing fractions in
sludge-soil mixtures: I. Extraction and purification of fulvic
acid. Soil Sci. Soc. Am, J. 40: 254-258.
Horne, R. A., and D. S. Johnson. 1966. The viscosity of water under
pressure. J. Phys. Chem. 70: 2182-2190.
Horton, J. H., and R. H. Hawkins. 1965. Flow path of rain from the
soil surface to the water table. Soil Science 100: 377-383.
Horvath, E., and G. H. El kan. 1978. Method for correcting laboratory
model deep-well disposal system data for hydrostatic pressure
effects. Appl. Environ. Microbiol. 35: 1221-1222.
Hurst, C. J., S. R. Farrah, C. P. Gerba, and J. L. Melnick. 1978.
Development of quantitative methods for the detection of
enteroviruses in sewage sludges during activation and following
land disposal. Appl. Environ. Microbiol. 36: 81-89.
Hurst, C. J., and C. P. Gerba. 1979. Development of a quantitative
method for the detection of enteroviruses in soil. Appl.
Environ. Microbiol. 37: 626-632.


FIGURE 4-7. Movement of poliovirus type 1 through a 27 cm packed
column of Red Bay sandy loam subsoil when suspended
in anaerobically digested sludge diluted (1:50) with
sludge liquor containing 0.01 N CaCl2
One pore volume for the column used equals 225 ml.
The laboratory-packed column was 29 cm in length
and 4.8 cm internal diameter; the column was filled
only 27 cm with soil (2 cm left on top for packed
sludge). The sample of Red Bay sandy loam subsoil
used consisted mainly of A2 and Bit horizons (see
Table 4-3). The column was conditioned with 2
pore volumes of sludge liquor containing 0.01
N CaClo Poliovirus was then suspended in the
diluted sludge at a concentration of 7.0 x 103
PFU/ml and applied to the column. All solutions
were applied continuously to the column at approxi
mately 5 ml/min using a peristaltic pump (Buchler,
Fort Lee, N.J.). The anaerobically digested sludge
(GDAN--see Table 3-2) used had a solids content
of 2.0%, a conductivity of 3250 ymho/cm at 25C
and a pH of 8.3. The conductivity of sludge
diluted (1:50) with sludge liquor containing 0.01
N CaClp was 2600 ymho/cm at 25C and the pH was
8.3. The sludge liquor was produced by centri
fuging GDAN sludge at 14,000 x g for 10 min at 4C.
This procedure was performed again on the decanted
supernatant and this yielded the clear sludge
liquor (see Table 4-2 for chemical parameters).


223
could not be detected in the sludge after 3 months of drying on the
soil surface (see Hurst et al_. 1978). When sludge was injected below
the soil surface, viral persistence was found to be prolonged. At a
sludge injection site in Butte, Montana, for example, Moore et al_. (1978)
recovered indigenous enteric viruses (1.1 PFU per gram dry wt. of soil
obtained from the sludge injection depth--approximately 15 cm) from soil
sampled 6 months (mostly fall and winter seasons; thus, low temperatures
were encountered) after sludge injection had been discontinued. Viral
inactivation was significantly accelerated in injected sludge which
had seeped to the soil surface at the Butte site and had been subjected
to air drying (see Moore et aK 1978). Indigenous enteric viruses at
sludge disposal sites have been shown not to be transported to surface
waters (Zenz et al_. 1976) or groundwater (Farrah et al_. 1981a).
In this chapter, results of viral monitoring at two sludge
disposal sites in Florida are presented. The City of Gainesville
(Florida) sludge disposal site adjacent to Lake Kanapaha was monitored
(monitored the sludge applied to the site, topsoil and groundwater) for
indigenous enteroviruses on a monthly basis from December 1977 through
February 1978. Topsoil from the sludge disposal site at the West
Florida Agricultural Experiment Station, Jay, Florida, was also moni
tored for indigenous enteroviruses on a monthly basis from June 1978
through January 1979. The applied sludge and groundwater at the Jay
site were monitored for indigenous enteroviruses by Farrah et al.
(1981a) and their results are sunmarized herein. The information gained
from viral monitoring at these two sludge disposal sites should be of


23 June
21
10.45

0.2
4.7
17.1
07 July
35
13.63

0.2
1.3
0.8
aRainfal1 data were measured at the experimental site as described on page 196.
b 3
u0ne inch or 2.5 cm (254 rn /ha) of lagooned sludge (see Table 5-3 for sludge characteristics) seeded
with a total of 6.1 x 108 PFU of poliovirus (or 4.4 x 107 PFU/g dry weight of sludge--see Table 5-3) was
applied to large soil cores of Eustis fine sand (LC3 and LC4--see Figure 5-1). The large soil cores were
33 cm in length and 15.5 cm internal diameter; they consisted of the Ap and A21 horizons of the Eustis fine
sand (see Table 4-3). The seeded sludge was allowed to soak in and dry on top of the soil for 4 days before
being mixed with the top 2.5 cm of soil. The drying sludge solids and the soil were monitored for the
presence of seeded viruses as detailed on pages 192 to 194. In addition to the large soil cores, the virus-
seeded sludge was also similarly applied to two small soil cores (SCI and SC2--see Figure 5-1) for the
purpose of evaluating viral transport only. Leachates from all soil cores were monitored for viruses as
shown in Tables 5-6 and 5-7.
cIn the case of the liquid sludge, sludge solids content was expressed as a percentage on a weight-to-
volume basis.
dA dash means not done or not applicable.


113
where
K = saturated hydraulic conductivity (in cm/hr)
3
Q = volume of leachate (in cm )
o
A = cross-sectional area of soil sample (18.09 cm for
the soil columns in this study)
t = time to collect volume of leachate (in hr)
L = length of soil sample (5.0 cm)
AH = hydraulic head difference (7.5 cm)
From Table 4-4, it is clear that the saturated hydraulic conductivity
of the Red Bay sandy loam subsoil decreased as the bulk density of the
3 3
soil increased from 1.45 to 2.00 g/cm At bulk densities of 1.70 g/cm
or greater, the saturated hydraulic conductivities were drastically
reduced (see Table 4-4). Therefore, soil columns packed a these high
bulk densities (i.e.,>1.70 g/cm ) could not be used in subsequent
poliovirus transport experiments because of the large amount of time
required to collect an adequate volume of leachate.
Poliovirus transport. The effect of soil bulk density on
poliovirus transport was studied using laboratory-packed soil columns
of Red Bay sandy loam subsoil. The air-dried soil (not autoclaved)
was packed into acrylic plastic columns (10 cm in length and 4.8-cm
internal diameter; packed 10 cm with soil) at bulk densities of 1.45
3
and 1.60 g/cm while tapping onr the outside of the columns. The charac
teristics of the soil columns packed at these bulk densities are shown
in Table 4-5. As the bulk density of the soil was increased from 1.45
3
to 1.60 g/cm there was a corresponding decrease in the percent pore


was significantly greater for aerobically digested sludges (95%)
than for activated sludge mixed liquors or anaerobically digested
sludges (72% and 60%, respectively). The effectiveness of the glycine
method in the recovery of solids-associated viruses was also found to
be affected by sludge type. Significantly lower mean poliovirus recovery
was found for aerobically digested sludges (15%) than for mixed liquors
or anaerobically digested sludges (72% and 60%, respectively).
Poliovirus transport studies involving soil cores treated with
virus-seeded sludge were conducted under controlled laboratory and
saturated flow conditions. A Red Bay sandy loam displayed a substantially
greater retention capacity for poliovirus in anaerobically digested sludge
than a sandy soil (i.e., Eustis find sand). The Red Bay sandy loam was
shown to completely retain poliovirus following the application of
conditioned-dewatered, chemical and lime-stabilized, chemical sludge.
Undisturbed soil cores of Eustis fine sand were treated with several
inches of virus-seeded (poliovirus and echovirus type 1-Farouk) sludge
during a two-year period. The soil cores were exposed to natural conditions
and soil temperature, soil moisture and rainfall were monitored. Both
viruses were found to be rapidly inactivated in the sludge during the
drying process on top of the soil cores. Monitoring of the/top inch of
soil revealed that both viruses were inactivated with time and were
undetectable after 35 days. Soil leachates collected after natural
rainfall (unsaturated flow conditions) were negative for both viruses.
Indigenous enterovirus were not detected in topsoil and groundwater
samples from two sludge disposal sites in Florida.
viii


182
Chapter III(See page 62). Briefly, an aliquot of the unfractionated
sludge (i.e., sludge sample without solids separated) was diluted
in PBS containing 2% FCS and assayed directly for viruses by the
plaque technique. This method (i.e., sludge dilution and subsequent
direct assay on cell cultures) has been previously shown to be highly
efficient in the recovery of poliovirus from unfractionated sludge
(see Chapter III, page 62 and Table 3-3). Similar efficient
recovery of echovirus from unfractionated sludge was also observed.
The unfractionated sludge assay was performed in order to determine the
total amount of virus present in the sludge sample. An aliquot of the
sludge was subsequently centrifuged at 1,400 x g for 10 minutes at 4C.
The sludge supernatant produced was assayed for viruses. The "viable
unadsorbed virus" and "sludge solids-associated virus" fractions were
calculated as shown in Chapter III (see page 64 ). Following the
initial viral assays, virus-seeded sludge was applied to soil cores
as described below.
Soil
The soil used in the research reported in this chapter was a
Eustis fine sand sampled at the agronomy farm, University of Florida,
Gainesville. This soil was classified as a Psammentic Paleudult, sandy,
siliceous, hyperthermic (Calhoun et aj_. 1974). Some characteristics of
this soil are shown in Table 4-3. The percent organic matter in this
soil was measured at less than 1% (Calhoun et aK 1974).


CHAPTER V
RETENTION AND INACTIVATION OF ENTEROVIRUSES
IN SOIL CORES TREATED WITH VIRUS-SEEDED SLUDGE
AND EXPOSED TO THE NORTH-CENTRAL FLORIDA ENVIRONMENT
Introduction
Although numerous studies have been conducted to determine the
survival (Bagdasaryan 1964; Derbyshire and Brown 1978; Duboise et al.
1976; Green 1976; Lefler and Kott 1974; Hurst et £l_. 1980a, 1980b; Moore
et al_. 1977; Sobsey £t al_. 1980a; Yeager and O'Brien 1979a, 1979b) and
transport pattern (Bitton ert al_. 1976; Drewry and Eliassen 1968;
Duboise et al_. 1976; Funderburg et al_. 1979; Gerba and Lance 1978;
Greene 1976; Laak and McClean 1967; Lance and Gerba 1980; Lance et al.
1976; Landry et aj_. 1980; Lefler and Kott 1974; Robeck et al_. 1962;
Schaub and Sorber 1977; Scheuerman et al_. 1979; Sobsey et aj_. 1980a;
Young and Burbank 1973) of viruses following water or wastewater appli
cation to soils, few have been undertaken to assess viral persistence
and transport in sludge-treated soils (see reviews by Bitton 1975,
1978, 1979b, 1980a, 1980b; Duboise et aj_. 1979; Foster and Engelbrecht
1973). Viruses have been found to be inactivated in sludge allowed to
dry on the soil surface (Brown et aj_. 1980; Hurst et al_. 1978; Nielsen
and Lydholm 1980). Other investigators have demonstrated that, under
cold winter temperatures, viruses can persist in sludge-amended soils
for as long as six months (Tierney et aj_. 1977; Damgaard-Larsen et al.
1977). Damgaard-Larsen et^ £]_. (1977) used lysimeters to study viral
178


224
value in the ultimate assessment of the actual viral risk of sludge
application to soils.
Materials and Methods
Sludge Disposal Sites
Two sludge disposal sites were monitored for indigenous entero
viruses and they are described below.
Kanapaha site. The City of Gainesville (Florida) sludge dis
posal site (10 acres or 4.05 ha) adjacent to Lake Kanapaha has been in
operation since August 1977. At the time of this study, the sludge
applied to this site originated at the Main Street wastewater treatment
plant, Gainesville. At this treatment plant, wasted sludge undergoes
180 days of aerobic digestion. The digested sludge (GDA180--see
Table 3-2) is conditioned with a cationic polymer and then dewatered
by centrifugation (U.S. Environmental Protection Agency 1974, 1978a; also
see Figure 2-1). The conditioned-dewatered sludge was transported by
tank truck to the Kanapaha site for ultimate disposal. The schematic
of sludge treatment and final disposal at the Kanapaha site is shown
in Figure 6-1. The conditioned-dewatered sludge was spread out onto
the soil and immediately disced into the soil except when a cover crop
was present. In the presence of a cover crop, the sludge was applied
as a top dressing on the crop. Coastal bermudagrass was utilized
during the summer months while ryegrass was used in the winter months
(Gainesville-Alachua County Regional Utilities 1976).
The Kanapaha site is depicted in Figure 6-2. As shown, a 60-ft
(ca. 18-m) deep well in the center (west) of the site was monitored for


174
wastewater effluent (pH of 7.5 and conductivity of 340 pmho/cm at 25C)
and applied continuously to the soil columns at approximately 3.5 ml/min
using a peristaltic pump. As shown in Figure 4-11, poliovirus moved
faster and appeared in the leachates in greater numbers in columns
packed at a bulk density of 1.45 g/cnr(i.e., columns 1 and 2) than in
3
columns packed at a bulk density of 1.60 g/cm Only column 3 at a
3
bulk density of 1.45 g/cm displayed slow movement through the soil
typical of the soil columns at a bulk density of 1.60 g/cm (see
Figure 4-11). It appears that in the columns displaying large viral
breakthroughs (i.e., columns at bulk density of 1.45 g/cm ), the
virus-seeded primary effluent followed a less circuitous path through
the soil resulting in less opportunity for poliovirus adsorption to
the soil particles. In spite of this, however, no significant
statistical difference was found between the fractions of polio
virus eluted at the tenth pore volume in soil columns packed at the
2 different bulk densities (see Table 4-26).


154
TABLE 4-16. Retention of poliovirus type 1 by an undisturbed core of
Red Bay sandy loam following the application of 2.5 cm of
seeded anaerobically digested sludge and the subsequent
elution with 0.01 N CaC^
No. of
pore
volumes3
eluted
Poliovirus
eluted
(total PFU)
% of Total,
PFU applied^
(cumulative)
Conductivity
of pore volume
collected
(ymho/cm at
25C)
pH of pore
volume
collected
1/3
0
0
650
4.5
2/3
0
0
1360
4.5
1
0
0
1570
4.8
1 1/3
0
0
1660
5.2
1 2/3
0
0
1720
4.8
2
0
0
1750
5.0
2 1/3
0
0
1760
4.9
2 2/3
0
0
1770
4.9
3
0
0
1770
4.8
3 1/3
0
0
1770
4.8
3 2/3
0
0
1770
4.8
4
0
0
1770
4.8
4 1/3
0
0
1770
4.7
a0ne pore volume for this core equals 471 ml. The undisturbed
soil core was 54 cm in length and 5.0 cm internal diameter; consists of
the Al, A2, Bit and B21t horizons of the Red Bay sandy loam (see
Table 4-3). The core was not conditioned.
^One inch or 2.5 cm (51.6 ml) of anaerobically digested sludge
(GDANsee Table 3-2; solids content, conductivity and pH equal to
2.0%, 3250 umho/cm at 25C and 8.3, respectively) seeded with a total
of 2.2 x 105 PFU of poliovirus was applied to the core, allowed to
soak in and then, worked under 2.5 cm. Elution with 0.01 N CaCl2
(conductivity and pH equal to 1210 ymho/cm at 25C and 6.4, respectively)
was subsequently undertaken. This solution was applied from an in
verted, self-regulated, 1 liter Erlenmeyer flask set to maintain a
2.5 cm hydraulic head on the core. The flow rate through the core was
measured at 3.5 ml/min.


CHAPTER VI
MONITORING OF INDIGENOUS ENTEROVIRUSES
AT TWO SLUDGE DISPOSAL SITES IN FLORIDA
Introduction
Wastewater effluents and sludges are being disposed of on land
with increasing frequency (U.S. Environmental Protection Agency 1974,
1977, 1978b). It has been suggested, however, that this practice may
result in the dissemination of viral pathogens throughout the disposal
site (e.g., soil and groundwater) and adjacent areas (e.g., surface
waters) (Burge and Marsh 1978; Elliott and Ellis 1977; Foster and
Engelbrecht 1973; Kowal and Pahren 1978; Pahren 1980; Pahren et al.
1979). Consequently, numerous studies have been conducted to determine
the fate of indigenous enteric viruses following wastewater effluent
application to land at several locations in the United States (Dugan
et al_. 1975; England et aK 1965; Gilbert et al_. 1976a, 1976b; Merrell
and Ward 1968; Schaub and Sorber 1977; Vaughn et aj_. 1978; Wellings
et al_. 1974, 1975, 1978). In contrast, viral persistence and transport
at sludge disposal sites has not been adequately investigated (see
review by Bitton et aj_. 1979b). Hurst jst al_. (1978) found that indige
nous enteroviruses (mostly echovirus type 7) were inactivated at the
rate of 2 1 og-jQ per week in aerobically digested-dewatered sludge
undergoing further drying in piles on land (temperature range: 20 to
31C). These investigators demonstrated that viral inactivation in the
sludge piles was directly related to the loss of moisture. Viruses
222


106
final pore volume) were subsequently combined and concentrated 160-fold
as follows. The leachate sample was adjusted to pH 3.5 by the addition
of 1 M glycine buffer, pH 2.0, and adjusted to a final concentration
of 0.0005 M aluminum chloride. The treated sample was then passed
through a series of 3.0- and 0.45-pm Filterite filters in a 47-mm
holder. Adsorbed viruses were eluted from the filters with 7 ml of PBS
containing 10% FCS, pH 9.0. The filter eluate was adjusted to neutral
pH by the addition of 1 M glycine buffer, pH 2.0, and assayed for
viruses by the plaque technique.
The quantity of poliovirus detected in each soil leachate was
expressed as a percentage of the amount of virus applied to the soil.
For cores receiving poliovirus continuously (i.e., laboratory-packed
soil columns), the quantity of poliovirus leached was expressed as
the cumulative percent of the total viral PFU having been applied at
each pore volume and it was calculated by the following equation:
poliovirus eluted at pore
volume b (as cumulative %
of total PFU having been
applied at pore volume b)
pore volume (ml)
a
x
pv=b
I
pv=l/a
poliovirus eluted in
each 1/a pore volume (PFU/ml)
b x pore volume (ml)
influent poliovirus
concentration (PFU/ml)
x 100 (4-3)
where pv represents the pore volume number; the leachate samples were
collected and assayed in 1/a pore volume fractions (a set at 1, 2, or 3


135
TABLE 4-13. Continued.
bEach soil section was mixed well, and a 10-gram wet sample was
taken and mixed with 20 ml of 3% beef extract, Tris buffered at pH 9.0.
This mixture was then vortexed for 30 sec and sonicated for 3 min.
The sample was then centrifuged at 1900 x g for 10 min at 4C. The
supernatant was subsequently assayed for viruses.
c 7
The amount of virus applied to the soil column was 1.3 x 10
total PFU (see Table 4-12). Overall recovery of the virus applied was
3.0% [i.e., 3.0% found in the soil and 0% found in the soil leachates
(see Table 4-12)].
^Refers to the sludge solids resting on top of the soil. The
total amount of these solids was separated, subjected to the same virus
elution method as the soil (added eluent at the proportion of 2 ml per
gram of wet sludge solids) and was considered as one section.


259
6. The stabilization of chemical sludges (alum and ferric chloride
sludges) with lime resulted in almost complete inactivation of
seeded poliovirus.
7. Undisturbed soil cores of Eustis fine sand were treated with
several inches of virus-seeded (poliovirus and echovirus) sludge
during a two-year period. The soil cores were exposed to natural
conditions and soil temperature, soil moisture and rainfall were
monitored. Both viruses were found to be rapidly inactivated in
the sludge during the drying process on top of the soil cores.
Monitoring of the top inch of soil revealed that both viruses
were inactivated with time and were undetectable after 35 days.
The inactivation of both viruses in the soil appears to be affect
ed more by soil moisture. Soil leachates collected after natural
rainfall (unsaturated flow conditions) were negative for both
viruses except on one occasion (only 0.0006% of total poliovirus
applied was found in leachate) when heavy rainfall occurred
immediately after liquid sludge application to the soil.
8. Indigenous enteroviruses were not detected in topsoil and ground-
water samples from two sludge disposal sites in Florida. It
appears that,at these two sludge disposal sites, enteroviruses
pose a minimal hazard with respect to soil and groundwater
contamination.
9. Poliovirus in seawater was found to be inactivated when subjected
to 1000 psi of hydrostatic pressure for 24 hours. No such in
activation was observed for this virus in groundwater even after
exposure to as high as 4000 psi of hydrostatic pressure.
This research has allowed the determination of the persistence and possible
movement of pathogenic viruses in soils treated with wastewater sludge.
The information gained from this study is of value in the ultimate assess
ment of the potential risk of viral infection to humans associated with
land disposal of sludges.


Q
The "overall virus recovery (%)" values were determined from the viruses recovered in the final con
centrates and were based on the amount of viruses (total PFU) added to the sludge as 100%.
dMean values displaying different superscript capital letters are significantly different at the 0.01
level when subjected to analysis of variance using Duncan's test.
Abbreviation for standard error.
fThe separated supernatants of the sludges with an asterisk were not processed for virus concentration.
These supernatants were simply adjusted to neutral pH and assayed for viruses. The viruses recovered were
included in the "overall virus recovery (%)" values displayed.
gThe lagoon sludge is a mixture of aerobically digested sludge (1/3) and anaerobically digested
sludge (2/3), and consequently, its properties are close to those of anaerobically digested sludges
(see Table 3-2) Therefore, lagoon sludges were placed in the population of anaerobically digested
siudges.


CHAPTER IV
POLIOVIRUS TRANSPORT STUDIES INVOLVING
SOIL CORES TREATED WITH VIRUS-SEEDED SLUDGE
UNDER LABORATORY CONDITIONS
Introduction
The application of wastewater sludge to land is receiving
increased attention and will probably be the predominant sludge dis
posal method of the future (U.S. Environmental Protection Agency 1974,
1978b). There is some concern, however, that this practice may result
in the contamination of groundwater supplies with pathogenic viruses
(see reviews by Berg 1973b; Bitton 1975, 1980a; Bitton et al_. 1979b;
Burge and Marsh 1978; Burge and Parr 1980; Cliver 1976; Duboise et
al. 1979; Elliott and Ellis 1977; Foster and Engelbrecht 1973;
Gerba et al_. 1975; Moore et^ al_. 1978; Sagik 1975). Unfortunately,
the transport pattern (i.e., movement or retention) of sludge-
associated viruses in soils has not been adequately evaluated. The few
studies that have been conducted indicate that enteroviruses seeded in
anaerobically digested sludge are effectively retained by the soil
matrix (Damgaard-Larsen et al. 1977; Moore et aj_. 1978; Sagik 1975).
Since both seeded and indigenous viruses have been found to be associated
with the sludge solids (Abid et_ al_. 1978; Glass et^ aj_. 1978; Hurst et al.
1978; Lund 1971; Ward and Ashley 1976; Wei lings et a]_. 1976a; also see
Table 3^4), it follows that viruses are probably immobilized along with
the sludge solids in the top portion of the soil profile (i.e., during
surface spreading of sludge) or at the injection site within the soil
82


FATE OF VIRUSES FOLLOWING SEWAGE SLUDGE
APPLICATION TO SOILS
By
OSCAR CARLOS PANCORBO
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1982


6
process is usually only some hours (Fair et aK 1968). The capacity
of primary sedimentation to remove viruses is at best minimal (Foster
and Engelbrecht 1973; Kollins 1966; Grabow 1968; Sproul 1976). Clarke
et al_. (1961) found only 3% removal of seeded poliovirus type 1
(Mahoney) from raw sewage during a three-hour settling period. Chemical
flocculation using alum, ferric chloride or lime followed by sedimenta
tion (i.e., intermediate or chemical treatment), however, has been shown
to be very effective in the removal of viruses from raw sewage (Berg
1973a; Lund 1976; Malina 1976; Grabow 1968; Sproul 1976). The removal
of viruses by chemical flocculation has been tested in the laboratory
using suspending media of varying composition (see review by Berg 1973a).
Several laboratory studies, using suspending media other than
raw sewage, have shown that seeded viruses are effectively removed
during flocculation. Change et al_. (1958) reported 86.3 to 98.7% removal
of seeded coxsackievirus A2 after flocculation at pH 6.2 in distilled
water-Si02"NaHC03 buffer with 40 to 100 ppm of alum [as A^iSO^Jg],
respectively. The removal of this virus by alum flocculation conformed
to the Freund!ich isotherm and, therefore, these investigators concluded
that the removal mechanism was adsorption. Approximately 60% of the
virus associated with the aluminum floes was. recovered following
elution with 0.1 M NaHCOg, at a final pH 8.5 (see Chang et al_. 1958).
In a similar study involving phosphate precipitation from water, Brunner
and Sproul (1970) found 89 to >98% removal of seeded poliovirus type 1
(Sabin) from distilled water-phosphate medium following alum [68 mg/£
as A12(S0^)flocculation. The removal measured was attributed to the


TABLE 3-4. Effect of sludge type on the association between poliovirus type 1 and sludge solids
SIudge
type
Sludge
used
Sludge parameters
pH Solids3
content
(%)
Virusb in
unfractionatedc
sludge
(total PFU)
Virus in
sludge .
supernatant3
(total PFU)
Viable
unadsorbede
virus
(%)
Solids-
associated
virus
(%)
Mean9
' associated
virus for
each sludge
type
(% SEh)
Mi xed
UML
6.4
1.6
8.4
X
106
5.0
X
106
59.5
40.5
57 17A
1 iquor
GML
6.9
0.5
1.3
X
106
3.4
X
105
26.2
73.8
Aerobical ly
UDA
4.8
1.5
6.5
X
106
1.1
X
106
16.9
83.1
95 2.0B
digested
UDA
4.8
1.5
6.5
X
106
6.4
X
105
9.8
90.2
UDA
6.1
1.3
2.0
X
107
1.3
X
106
6.5
93.5
UDA
6.1
1.3
2.0
X
107
9.6
X
105
4.8
95.2
UDA
6.5
1.3
9.6
X
106
4.0
X
104
0.4
99.6
GDA90
5.8
1 .0
1.3
X
107
4.3
X
105
3.3
96.7
GDA180
5.0
1.3
8.2
X
108
4.8
X
106
0.6
99.4
PDA
5.8
2.8
4.1
X
106
5.7
X
103
0.1
99.9
Anaerobically
GDAN
8.3
2.0
1.0
X
107
4.4
X
106
44.0
56.0
70 8.4A
digested
PDAN
7.2
1.4
6.0
X
106
3.2
X
106
53.3
46.7
PDAN
6.4
1.9
5.5
X
106
1.4
X
106
25.5
74.5
LAG1
7.3
2.9
1.8
X
106
2.9
X
105
16.1
83.9
LAG1
6.9
2.9
1.9
X
109
1.7
X
108
8.9
91.1


193
1972; Sobsey ert al_. 1973; Sobsey et al_. 1980b) as follows. Each
leachate sample was adjusted to pH 3.5 by the addition of 1 M glycine
buffer, pH 2.0, and adjusted to a final concentration of 0.0005 M
aluminum chloride. The treated water was then passed through a series
of 3.0-, 0.45-, and 0.25-um Filterite filters in a 47-mm holder.
Adsorbed viruses were eluted from the filters with 7 ml of PBS con
taining 10% FCS, pH 9.0. The filter eluate was adjusted to neutral
pH by the addition of 1 M glycine buffer, pH 2.0, and assayed for
seeded viruses as described above. The quantity of poliovirus or
echovirus detected in each leachate sample was expressed as total PFU
and as a percentage of the amount of virus applied to the soil (i.e.,
cumulative percent of the total viral PFU applied was calculated
according to Equation 4-4).
Measurement of Environmental Parameters
The soil temperature, soil moisture, and rainfall were
monitored. The soil temperature was monitored every hour using ther
mocouples placed at the soil surface and at depths of 2.5, 10, and 20
cm on one of the large soil cores as shown on Figure 5-2. The
thermocouples were connected to an Esterline Angus Key Programmable
Data Acquisition System (Model PD-2064, Esterline Angus Instrument
Corporation, Indianapolis, Indiana) which printed voltage (millivolts)
at each thermocouple every hour. The voltages measured were later
converted to temperature readings with the use of a computer. The
soil moisture was monitored only when a sample of soil was obtained
for viral assay. Soil moisture content was determined gravimetrically


CHAPTER Page
Results and Discussion 196
Association between Seeded Enteroviruses
and Sludge Solids 197
First Survival Experiment (7 October
1977-12 October 1977) 197
Second Survival Experiment (2 June
1978-24 August 1978) 200
Third Survival Experiment (11 October
1978-20 January 1979) 211
VIMONITORING OF INDIGENOUS ENTEROVIRUSES AT TWO
SLUDGE DISPOSAL SITES IN FLORIDA 222
Introduction 222
Materials and Methods 224
Sludge Disposal Sites 224
Virus Recovery Procedures 236
Viral Assays 242
Weather Data 242
Results and Discussion 244
Kanapaha Sludge Disposal Site 244
Jay Sludge Disposal Site 244
VIIEFFECT OF HYDROSTATIC PRESSURE ON THE
SURVIVAL OF POLIOVIRUS SEEDED IN GROUNDWATER
AND SEAWATER 250
Introduction 250
Materials and Methods 251
Virus and Viral Assays 251
Water Samples 251
Poliovirus Exposure to Hydrostatic Pressures 252
Results and Discussion 254
VIIICONCLUSIONS 258
APPENDIX: COMPOSITION OF MEDIA AND SOLUTIONS USED
IN ENTEROVIRUS ASSAYS 260
BIBLIOGRAPHY 265
BIOGRAPHICAL SKETCH 285
vi


34
also provides stabilization of the sludge (U.S. Environmental Protection
Agency 1974, 1978a, 1978b). As such, lime conditioning of sludge
reduces odors and the pathogen, including viral, content of sludge
(U.S. Environmental Protection Agency 1974, 1978b; also see pages 30 to
32 in this chapter). Until more information becomes available, however,
lime-conditioned sludges should not be regarded as virus free.
Conditioning-heat treatment. Heat treatment at temperatures of
300 to 500F (i.e., 150 to 260C) and pressures of 150 to 400 psi for
periods of 15 to 40 minutes is another conditioning process which
facilitates sludge dewatering (see Figure 2-1) (U.S. Environmental Pro
tection Agency 1974, 1978a). Such heat treatment solubilizes and
hydrolyzes the smaller and more highly hydrated sludge particles which
are then removed from the bulk sludge sample and end up in the cooking
liquor (U.S. Environmental Protection Agency 1974). Consequently, heat-
conditioned sludge displays a reduced affinity for water and an
improved dewatering capacity (U.S. Environmental Protection Agency 1974,
1978a). As shown in Figure 2-1 and described above, heat treatment
also leads to the stabilization of sludge (U.S. Environmental Protec
tion Agency 1974, 1978b). Due to the high temperatures employed, all
pathogens, including enteric viruses, in sludge are destroyed during
heat conditioning (U.S. Environmental Protection Agency 1974, 1978b).
Unfortunately, high energy costs often make heat conditioning of sludge
impractical.
Dewatering-drying beds. In the United States and Europe,
sandbed drying (see Figure 2-1) is the most widely used method for
sludge dewatering (U.S. Environmental Protection Agency 1974). Drying


133
TABLE 4-12. Continued.
No. of pore
volumes3
eluted
Poliovirus
eluted
(PFU/ml)
% of Influent
poliovirus
concentration
Conductivity
of pore
volume collected
(umho/cm at 25C)
pH of pore
volume
collected
3.5
0
0
1310
5.0
4.0
0
0
1310
5.0
a0ne pore volume for the columns used equals 192 ml. The laboratory-
packed columns were 29 cm in length and 4.8 cm internal diameter; the
columns were filled only 27 cm with soil (2 cm left on top for packed
sludge). The sample of Eustis fine sand subsoil used consisted mainly of
the A21 and A22 horizons (see Table 4-3). Columns 1 and 2 were condi
tioned with 5 pore volumes of distilled water and 0.01 N CaClp, respective
ly. All solutions were applied continuously to the columns at approxi
mately 5 ml/min using a peristaltic pump (Buchler, Fort Lee, N.J.).
Following elution, the columns were sectioned as seen in Tables 4-13 and
4-14.
bPoliovirus was seeded in the influents of column 1 (i.e., sludge
diluted with distilled water) and column 2 (i.e., sludge diluted with
0.01 N CaC^) at concentrations of 1.4 x 10^ PFU/ml and 1.7 x 10^ PFU/ml,
respectively.
cThe anaerobically digested sludge (PDANsee Table 3-2) used had a
solids content of 1.4% and a pH of 7.2. Chemical parameters were not
measured for the sludge diluted (1:50) with distilled water or 0.01 N CaC^.


232
Lagooned sludge (LAG--see Table 3-2) was subsequently spread on 8
acres (3.24 ha) of land at the Jay site. A diagram of the field used
for sludge disposal at Jay is shown in Figure 6-4. The field site was
divided into 72 plots of 40 by 120 ft (ca. 12 by 36 m) which received
from 0 to 15 acre-inches (0 to 15.4 ha-cm) of sludge per year (see
Figure 6-4). The applied sludge was allowed to dry on the soil surface
for 2 to 14 days and was then turned under the soil (see Figure 6-3).
The soil series found at the site are Troup, Lucie, and Orange
burg (see Figure 6-4). The Troup series has been classified as a
Grossarenic Paleudult, loamy, siliceous, thermic, while the Orangeburg
series was classified as a Typic Paleudult, fine-loamy, siliceous,
thermic (Calhoun et al_. 1974). Some characteristics of the typifying
pedons, Troup loamy sand and Orangeburg sandy loam, are shown in
Table 6-2. No information was available on the Lucie soil series.
The topsoil from plots numbered 1, 32, and 61 which received 15 acre-
inches (15.4 ha-cm) of sludge per year was monitored for indigenous
enteroviruses on a monthly basis from June 1978 through January 1979.
In addition, the topsoil from the plot numbered 42 which received no
sludge was monitored for viruses as a control. Indigenous enteroviruses
in the Pensacola sludges (PDA and PDANsee Table 3-2) added to the
sludge lagoon and in the lagooned sludge (LAG--see Table 3-2) were
monitored by Farrah et^ aj_. (1981a). Also, groundwater from wells on
the Jay site (see Figure 6-4) was analyzed for the presence of indigenous
enteroviruses by Farrah et^ al_. (1981a).


116
space and pore volume of the 10-cm soil column (see Table 4-5).
Three columns were prepared at each bulk density. A polypropylene
screen (105-ym pore size) was used to support the soil in each column.
The soil columns were placed in soil column holders and treated with
carbon dioxide as described earlier (see page 97), The soil columns
were then conditioned by passing 2 pore volumes of nonseeded primary
wastewater effluent using the peristaltic pump. Following conditioning,
poliovirus was suspended in primary wastewater effluent (as described
on page 91) and subsequently applied continuously to the soil columns
at approximately 3.5 ml/min using the peristaltic pump. As detailed
earlier (see page 91 ), influent poliovirus concentration was determined
from viral assays made at the beginning and end of each column experiment.
After percolation through the soil, the column leachates were collected
in pore volumes using sterile screw-capped bottles. The poliovirus
content, pH and conductivity of the leachates were determined as
described earlier (see pages 104-107).
Statistical treatment of data. Statistical treatment of the
data was performed with the use of a Hewlett-Packard calculator model
9810A and Statistics Package V-6 (Hewlett-Packard Company, Loveland,
Colorado).
Results and Discussion
Prior to conducting field experiments, it appeared necessary
to study the transport pattern (i.e., movement or retention) of sludge-
associated viruses in the soils under consideration, Eustis fine sand
and Red Bay sandy loam. Experiments were undertaken to study viral


78
digested sludges. The reason for the reduced recovery of viruses is
that the elution step (i.e., mixing the sludge solids with five volumes
of 0.05 M glycine buffer, pH 10.5 to 11.0, followed by rapid mixing for
30 seconds) is not effective for this sludge type. For example, the
mean elution of solids-associated viruses for this sludge type was only
24%. In contrast, the mean elution of solids-associated viruses from
mixed liquors and anaerobically digested sludges was 76% and 80%,
respectively. The elution of viruses that was measured for mixed liquor
solids (i.e., 76%) ) closely approaches the value of 84% reported by
Hurst et al_. (1978) for the elution of poliovirus type 1 from activated
sludge solids. The reason for the poor elution of solids-associated
viruses from aerobic sludges has not been determined. However, this
sludge type was able to bind a larger fraction of poliovirus than mixed
liquors or anaerobically digested sludges (see Table 3-4). In order to
understand the mechanism(s) involved, the chemical and physical nature
of the solids of different sludge types should be studied. All other
virus adsorption-elution steps of the glycine method (i.e., virus
concentration steps) were equally effective in poliovirus recovery for
all sludge types tested.
Several methods have been proposed for the recovery of sludge
solids-associated viruses and these methods are summarized in Table 3-8.
Of the methods evaluated with enteroviruses seeded in sludge, all were
tested for virus recovery efficiency using only one sludge type (see
Table 3-8). The research presented above shows that sludge type in
fluences the recovery of poliovirus from sludge solids. Although the


85
interactions and the autoclaved sludge can simply be considered to be
heat-conditioned. The sludges were used undiluted or diluted (1:50,
vol./vol.) with either distilled water or 0.01 N calcium chloride.
The pH and conductivity of each diluted sludge sample was also
measured as described above for undiluted sludge.
Sludge liquor. Anaerobically digested sludge liquor was pro
duced by centrifuging (all centrifugation was performed using a Sorvall
RC5-B centrifuge, Ivan Sorvall Inc., Norwalk, Connecticut) GDAN sludge
(see above) at 14,000 x g for 10 minutes at 4C. This procedure was
performed again on the decanted supernatant and this yielded the clear
sludge liquor. Chemical parameters for this sludge liquor were deter
mined by the Analytical Research Laboratory, Soil Science Department,
University of Florida, Gainesville, and are presented in Table 4-1.
The sludge liquor (containing 0.01 N calcium chloride) was also used
to dilute (1:50, vol./vol.) the GDAN sludge. Lagoon sludge (LAGsee
Table 3-2; a mixture of 1/3 aerobically digested sludge and 2/3
anaerobically digested sludge) liquor was also used and it was produced
by the centrifugation of lagoon sludge as outlined above for GDAN
sludge. The lagoon sludge liquor was passed through a series of
0.45- and 0.25-pm Filterite filters (Filterite Corp., Timonium, Maryland)
in a 47-mm holder and then adjusted to pH 8.0 using 0.01 N NaOH prior
to use. The pH and conductivity of each sludge liquor sample was
measured as described above for undiluted digested sludges.
Conditioned-dewatered sludge. Poliovirus-seeded, GDAN sludge
(see above) was conditioned with 1200 mg/£ of the cationic polymer,


84
triplicate counts. The numbers of viruses were expressed as plaque
forming units (PFU).
Primary Wastewater Effluent
Primary wastewater effluent was obtained from the University
of Florida campus wastewater treatment plant, Gainesville, Florida. The
detention time of raw wastewater in the primary settlers was approxin
mately 2 hours. No chlorine residual was found in the primary effluent
sample used (i.e., by the orthotolidine test). The sample of primary
effluent used was collected, and its pH and conductivity were measured
as described below for digested sludges.
Sludges
Several sludge types were used in the research reported in this
chapter and they are described below.
Digested sludges. Anaerobically digested sludges sampled at
the Main Street wastewater treatment plants of Gainesville and Pensacola,
Florida (GDAN and PDAN, respectively--see Table 3-2), were used. The
sludges were collected and sludge parameters (i.e., pH and solids con
tent) were measured as described in Chapter III (see page 56). The
sludge conductivity was measured using a Beckman conductivity bridge
model RC 16B2 (Beckman Instruments, Fullerton, California). Due to
uncontrollable contamination of cell cultures during viral assays, these
sludge samples had to be sterilized by autoclaving at 121C with applied
pressure of 15 psi for 15 minutes prior to use. As explained earlier
(see page 59), autoclaving did not significantly affect the sludge-virus


19 Oct.
8
O
1.0
01 Nov. 21 0.13
1.0
5.6 x 101 6.3 x 101
3.6 x 103 2.1 x 102
aRainfall data were measured at the experimental site as described on page 196.
b 3
One inch or 2.5 cm (254 m/ha) of laqooned sludge (see Table 5-3 for sludge characteristics) seeded
with a total of 2.9 x 10 PFU (or 8.6 x 10^ PFU/g dry weight of sludge) or a total of 8.6 x 108 PFU (or
2.6 x 10? PFU/g dry weight of sludge) of echovirus or poliovirus, respectively (see Table 5-3), was
applied to large soil cores of Eustis fine sand (LC1 through LC4--see Figure 5-1). The large soil cores
were 33 cm in length and 15.5 cm internal diameter; they consisted of the Ap and A21 horizons of the Eustis
fine sand (see Table 4-3). The seeded sludge was allowed to soak in and dry on top of the soil for 3
days before being mixed with the top 2.5 cm of soil. The drying sludge solids and the soil were monitored
for the presence of seeded viruses as detailed on pages 192 to 194. In addition to the large soil cores,
virus-seeded sludge was also similarly applied to small soil cores (33 cm in length and 5.0 cm internal
diameter) of Eustis fine sand for the purpose of evaluating viral transport only. Leachates from all soil
cores were monitored for viruses as shown in Table 5-9/ Cf
00
Q
In the case of the liquid sludge, sludge solids content was expressed as percentage on a weight-to-
volume basis.
^A dash means not done or not applicable.


No. of pore volumes eluted
Conductivity
(x 1q2 ymho/cm at 25C)
Poliovirus eluted (expressed as cumulative % of total PFU applied)
oojct> ioro o 1 rv> gj 4^oicn ca
PH
CT>
O


229
indigenous enteroviruses. The water table at the site has been found
to be 50 ft (ca. 15 m) below the soil surface (Gainesvi11 e-Alachua
County Regional Utilities 1976). Other characteristics of the Kanapaha
site are shown in Figure 6-1. The groundwater flows in a northwesterly
direction as shown in Figure 6-2. The topsoil at the Kanapaha site was
also monitored for indigenous enteroviruses (see Figure 6-2). The soil
found at the site belongs to the Lochloosa series (Gainesville-Alachua
County Regional Utilities 1976). This soil series is classified as an
Aquic Arenic Paleudult, loamy, siliceous, hyperthermic (Calhoun et al.
1974). Some characteristics of the typifying pedon, Lochloosa fine sand,
are shown in Table 6-1.
The conditioned-dewatered sludge, topsoil, and groundwater from
this site were monitored for indigenous enteroviruses on a monthly
basis from December 1977 through February 1978. Wasted sludge and
aerobically digested sludge (digested 90 days; GDA90 in Table 3-2) from
the Main Street wastewater treatment plant (Gainesville, Florida) were
also tested for the presence of indigenous enteroviruses in December
1977 and February 1978.
Jay site. Aerobically digested (1/3) and anaerobically digested
(2/3) sludge from the Montclair and Main Street wastewater treatment
plants of Pensacola, Florida, respectively, were transported by tank
truck and discharged into a sludge lagoon located at the West Florida
Agricultural Experiment Station, Jay, Florida (PDA and PDAN, respec
tively; see Table 3-2). Characteristics of the sludge lagoon and the
scheme for sludge disposal at the Jay site are shown in Figure 6-3.


Air temperature (C)
199
Days after sludge application to the soil
7 October 77
FIGURE 5-3. Daily air temperature (5 ft above ground) for the dura
tion of the survival experiment that began 7 October
1977
Data were collected at the weather station of the
Department of Agronomy, University of Florida,
Gainesville. This station is approximately 1 mile
from the experimental site (i.e., next to the
Environmental Engineering Sciences building,
University of Florida, Gainesville). The mean,
maximum temperature for the period was 30.5C and
the mean, minimum temperature for the period was
16.9C.


FIGURE 3-1. Effect of autoclaving on the rate of settling of anaerobi
cally digested sludge solids
An aliquot of anaerobically digested sludge (GDAN--
see Table 3-2; solids content, conductivity and pH
equal to 2.0%, 3,250 ymho/cm at 25C and 8.3,
respectively) was autoclaved at 121C with applied
pressure of 15 psi for 15 minutes and compared to an
aliquot of the sludge which had not been autoclaved.
Approximately 21 ml each of autoclaved sludge
and nonautoclaved sludge were added to graduated
cylinders A and B, respectively. After 2 hours,
the solids of the autoclaved sludge had settled
while those of the nonautoclaved sludge remained
dispersed.


83
matrix (i.e., during subsurface injection of sludge) (Oliver 1976).
Moreover, it appears that viruses are not readily dissociated from sludge
solids in the soil environment (Burge and Parr 1980; Sagik 1975).
In this chapter, results of poliovirus (type 1, strain LSc)
transport studies involving soil cores treated with virus-seeded sludge
are presented. These studies were conducted under controlled laboratory
conditions. The effect of sludge type (used anaerobically digested
sludge, conditioned-dewatered sludge, chemical sludge and lime-stabilized,
chemical sludge), soil type (used a Red Bay sandy loam and a Eustis fine
sand), soil core type (used laboratory-packed soil columns and undis
turbed soil cores) and application regime (virus-seeded sludge was
applied continuously or in a spiked fashion) on the transport of sludge-
associated poliovirus in soil cores was evaluated. The capacity of rain
water to elute poliovirus from the sludge-soil matrix was also investi
gated. All soil cores were leached under saturated flow conditions.
The information gained from this study should shed further light on the
role of mineral soils in retaining sludge-associated viruses.
Materials and Methods
Virus and Viral Assays
Poliovirus type 1 (strain LSc) was used in the research reported
in this chapter. Some general properties of polioviruses are shown in
Table 3-1. Stocks of the virus were prepared as described in Chapter
III (see page 53). The virus was kept at -70C until used. Poliovirus
was assayed by the plaque technique as described in Chapter III (see
pages 53-56). Each viral count shown represents the average of


LOL


88
[as Al2(S0^)3*18 H^O] and of ferric chloride was 50 mg/£ (as FeCl3).
Similar concentrations of these coagulants were used by Wolf et al.
(1974) in the precipitation of an activated sludge effluent as a
tertiary treatment process. Following the addition of alum or ferric
chloride, the pH of the solution was adjusted using 0.2 N HC1 to 6.0 or
5.0, respectively, in order to achieve maximum flocculation (Fair et al.
1968). The coagulant, lime, was added until a pH of 11.1 to 11.3 [i.e.,
final concentration in sewage of 150 to 250 mg/2, of Ca(0H)2] was
achieved (according to Sattar et al_. 1976). Following the addition
of the coagulants, the sewage samples were mixed on a magnetic stirrer
rapidly for 10 minutes and slowly for 5 minutes. The flocculated
sewage samples were then transferred to Imhoff cones and 60 minutes
was allowed for the formation and settling of the chemical sludges
(see Figure 4-1). The supernatants in the Imhoff cones were assayed
for viruses and discarded. The sludge volume and sludge solids content
(i.e., as percent, by method described above for digested sludges) was
measured for each chemical sludge. The chemical sludges produced (i.e.,
in duplicate for the lime sludge, and in triplicate for alum and ferric
chloride sludges) were then assayed for viruses as described below.
Lime-stabilized, chemical sludges. A sample of alum sludge
and a sample of ferric chloride sludge, produced and assayed for viruses
as described above, were lime-stabilized according to the procedure of
Farrell et al_. (1974). The sludges were treated with an aqueous slurry
of lime [5% (wt./vol.) stock of Ca(0H)2J until a pH of 11.5 was achieved
and maintained for 5 minutes. The final concentration of lime [as


64
study did, however, produce cell culture contamination when diluted and
then directly inoculated into cell cultures. For these sludges (i.e.,
when not decontaminated by autoclaving), the initial virus present was
determined based on the amount of virus stock of known titer added to
the sludge. From the results shown in Table 3-3, it is believed that
the determination of the initial virus added to the sludge was accurately
achieved by either direct viral assay of the sludge or based on the
amount of virus stock of known titer added to the sludge. The total
sludge volume was subsequently centrifuged at 1,400 x g for 10 minutes
at 4C (only an aliquot of sludges GDA180 and GDAN was centrifuged).
An aliquot of the sludge supernatant produced was assayed for viruses.
Thus, allowing the calculation of the "viable unadsorbed virus" frac
tion as shown below:
viable unadsorbed = virus in sludge supernatant (total PFU) 1nn
virus (%) virus in unfractionated sludge (total PFU) x uu
(3-1)
Furthermore, the "sludge solids-associated virus" fraction was also
estimated as shown below:
solids-associated virus (%) = 100 viable unadsorbed virus (%) (3-2)
For sludges GDA180 and GDAN, viral assays were performed, as described
above (i.e., of unfractionated sludge and of sludge supernatant), several
times throughout a 12-hour to 8-day period. During this period, the
virus-seeded sludge was left at room temperature undisturbed, and was
stirred only prior to obtaining a sample for viral assay.


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