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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Sewage sludge -- Health aspects   ( lcsh )
Virus diseases   ( lcsh )
Soil microbiology   ( lcsh )
Environmental Engineering Sciences thesis Ph. D
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Thesis (Ph. D.)--University of Florida, 1982.
Bibliography:
Bibliography: leaves 265-284.
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Typescript.
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Vita.
Statement of Responsibility:
by Oscar Carlos Pancorbo.

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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.




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