ENTEROVIRUSES IN WATER AND WASTEWATER

by

WILLIAM RAYMOND MORRIS

Regional Laboratory, Severn Trent Water, Coventry

and

Department of Microbiology, University of Surrey

September 1986

Submitted as part fulfilment for the degree of Doctor of Philosophy.

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ABSTRACT

Procedures for the detection of cytopathic enteroviruses in the water

cycle were assessed. A method was developed and applied to a wide

range of field samples. It depended upon concentration of viruses by

adsorption to epoxy-bound fibre-glass filter tubes with subsequent

elution of viruses with beef extract solution. Viruses were further

concentrated by organic flocculation and infectivity assayed by a

suspended cell plaque method using BGM cells.

I

Cytopathic enteroviruses, limited to serotypes of polioviruses,

coxsackie group B viruses and echoviruses, were isolated from treated

and untreated potable waters, surface waters, raw sewages and

wastewater effluents. The relationships of physicochemical and

bacteriological pollutants to viral contamination were examined and

indicated that as levels of bacteria rose the likelihood of viruses

also being present increased. However, the correlation was poor

because viruses were found in the absence of indicator bacteria and

conversely.

The virological quality of the Severn and Trent river catchments

revealed that two tributaries, the river Stour and the river Tame,

were the major sources of viral pollution in each catchment

respectively. Storage of river waters, destined for further treatment

as drinking water, for at least fifty days resulted in significant

reductions of the numbers of viruses. Similarly, long-term lagooning

of wastewater effluents resulted in a substantial decline in virus

titres. The value of other forms of wastewater treatment,

sedimentation, activated sludge, percolating filtration and sand

filtration, effected unpredictable’ reductions of virus numbers.

The case for surveillance of virus levels in the water cycle is

stressed.

1

ACKNOWLEDGEMENTS

It is with much gratitude that I recognise the unstinting support and

enthusiasm of Michael Butler who first persuaded me to prepare a

thesis on water virology. Throughout the gestation period of the

dissertation he has been constructively critical and instrumental in

making sense out of chaos. Similarly, John Leahy, acting as my

industrial supervisor encouraged the research and was instrumental in

persuading the Severn-Trent Water Authority to provide both finance

and facilities to enable the work to be carried out. I am grateful to

him and the Authority for permission to utilise field data gained

while in the Authority's employ.

Many colleagues involved in water virology worldwide have taken an

interest in my work and have contributed to much discussion.

Additionally, several have kindly made available their unpublished

findings. The PHLS Communicable Disease Surveillance Centre has

kindly allowed me to quote from their weekly provisional summaries

published as the Communicable Disease Report. Bacteriological and

chemical analyses were conducted by Paul Finch and Keith Bamford (and

staff) respectively, while Dave Sharp and Tommy Timmons played a major

role in the processing of field samples. The original draft of much

of the thesis was prepared by Pauline Smith, while Christine Croot

typed the final document.

Finally, I thank my wife who repeatedly kept reminding me that I had

spent long enough on the thesis and that it was about time I finished

it'.

CONTENTS

Page NumberTitle page AbstractAcknowledgements Contents List of tables List of figures

SECTION Is INTRODUCTION

The importance of water 1-1Water resources 1-3Monitoring of microbiological quality 1-7Viral epidemiology 1-12Minimal infective dose 1-15Faecally excreted viruses 1-18Waterborne viral diseases 1-23Factors affecting virus survival in water 1-27Viruses in water and wastewater 1-31Virus removal by water and wastewater treatment 1-36The project 1-3 9

SECTION 2; MATERIALS AND METHODS

Viruses 2-1Monodispersion of virus 2-1Cell lines 2-2Propagation of cell lines 2-2Adjuncts 2-4Assay of viruses 2-4Sample collection 2-5Filters 2-7Concentration of water samples 2-7Assay of field samples - wastewaters 2-9Assay of field samples - other waters 2-9Identification of field isolates 2-9Bacteriological analyses 2-10Coliphage determinations 2-10Chemical analyses 2-11

SECTION 3A: LABORATORY INVESTIGATIONS - RESULTS

Efficiency of the concentration procedure 3A-1Evaluation of the field concentrator 3A-3Evaluation of flocculation 3A-3Evaluation of the microtitration assay 3A-4Susceptibility of selected cell lines 3A-5Evaluation of focal and quantal assays - cell

presentation 3A-8Sensitivity of cell culture types to different

enterovirus serotypes 3A-13The sensitivity of mixed cell cultures 3A-13Optimisation of the suspended cell assay system 3A-16

SECTION 3B: LABORATORY INVESTIGATIONS - DISCUSSIONPage Number

Filter evaluations 3B-1Secondary concentration 3B-5Cell cultures 3B-7Cell presentation 3B-11Optimisation of the BGM suspended cell plaque assay 3B-13

SECTION 4: FIELD INVESTIGATIONS - RESULTS AND DISCUSSION

Viruses in drinking water Viruses in groundwaters Viruses in river watersViruses in potable waters from storage reservoirs Viruses in wastewatersValue of indicator systems for predicting the

presence of enteroviruses Enterovirus serotypes identified

SECTION 5: GENERAL DISCUSSION

The need for surveillance of viruses in thewater cycle 5-1

The practice of virus surveillance 5-3Is there a need for viral standards? 5-7Communications 5-10Concluding Remarks 5-12

Papers published References citedAppendix A: Outbreaks of waterborne disease Appendix B: Isolations of viruses from the water cycle Appendix C: Materials Appendix D: Virological data

4-14-34-54-144-17

4-284-34

LIST OF TABLES

Page Number

1 Quality data for representative source waters 1-62 Microbiological standards for drinking waters 1-93 Microbiological standards for bathing waters 1-104 Transmission of virus infections 1-145 Human enteric viruses in the water cycle 1-186 Factors contributing to waterborne disease 1-247 Survival times of enteroviruses in various environments 1-278 Factors affecting virus survival in water 1-289 Materials capable of adsorbing viruses 1-29

10 Physicochemical characteristics of test waters 3A-111 The influence of different concentration procedures on the

recovery of poliovirus type 2 from tap and river waters 3A-212 The value of organic flocculation 3A-413 Reproducibility of the microtitration assay 3A-514 Infectious titres of cytopathogenic viruses in different

cell lines 3A-615 .Titration of viruses in BGM cells after non-cytopathogenic

replication in certain other cells 3A-716 Titres of infectious virus in river water concentrates 3A-1017 Sensitivity of different cell lines in the suspended cell

plaque assay to infectious viruses in effluents 3A-1118 Serotypes in effluent detected by ten cell lines 3A-1419 Mixed cell assay of effluent 3A-1520 Effect of time of staining on detection of viruses in

effluents 3A-1921 Effect of gelling agents on plaquing efficiency 3A-2022 Effect of gelling agents on virus detection in wastewaters 3A-2223 Effect of magnesium chloride on plaque numbers and

serotypesin wastewater effluents 3A-2624 Effect of 5-iodo-2'-deoxyuridine on detection of viruses ^

in effluents 3A-2925 Efficiency of large volume concentrator for recovery of

poliovirus 2 from tapwater 4-126 Range and levels of viruses in rivers in the Severn-Trent

Water Authority 1979 - 1983 4-627 Effectof the Tame lake on physicochemical and

microbiological determinants 4-1028 Detection of viruses in stored waters 4-1529 Virus levels in river and stored waters 4-1630 Effect of sedimentation on levels of virus in wastewater 4-2031 Effect of percolating filtration on levels of viruses in

wastewater 4-2032 Effect of activated sludge on levels of viruses in

wastewater 4-2233 Effect of tertiary treatments on levels of viruses in

wastewater 4-2234 Overall reduction of naturally occurring enteroviruses

by two wastewater treatment works 4-2435 Incidence of virus in effluents at five works 1980 4-2736 Relationship between levels of cytopathic enteroviruses

and physicochemical determinants 4-2937 Enterovirus serotypes 1979 - 1982 4-35

LIST OF FIGURES

Page Number

1 The river Severn 1-52 The river Trent 1-53 Underground sources in STWA 1-74 Water routes for virus transmission 1-155 Seasonality of virus diseases 1-226 Viral diseases from consumption of shellfish in the UK 1-357 Large volume field concentrator 2-68 Time of appearance of maximum plaque counts in different

cell lines 3A-129 Poliovirus 1 plaquing in suspended cell cultures at

different cell concentrations 3A-1710 Effect of cell concentration on poliovirus 1 plaque size 3A-1711 Effect of adsorption time on poliovirus 1 plaquing 3A-1812 Effect of gelling agents on plaque size 3A-2113 Effect of serum on poliovirus 1 plaquing 3A-2214 Effect of DEAE-dextran on plaque numbers 3A-2415 Effect of DEAE-dextran on coxsackievirus A9 plaque size 3A-2416 Effect of semicarbazide on plaque numbers 3A-2517 Effect of semicarbazide on coxsackievirus A9 plaque size 3A-2518 Effect of magnesium chloride on plaquing by three viruses 3A-2719 Effect of magnesium chlorine on virus detection in

effluents 3A-2720 Effect of protamine sulphate on plaquing 3A-2821 Effect of IDU on virus titres 3A-3022 Effect of IDU on virus detection in effluents 3A-3023 Virus levels in the river Severn 1979 - 1981 4-724 Virus levels in river Worfe (Cosford) 1979 - 1981 4-825 Virus levels in river Avon (Tewkesbury) 1979 - 1981 4-826 Virus levels in the river Stour 1979 - 1981 4-827 Microbiological quality of the river Tame 1982 4-1128 Flow diagram for Finham WRW 4-1829 Flow diagram for Monkmoor WRW 4-1830 Virus levels in Finham sewage 1982 4-1931 Enterovirus levels in effluents 1980 - 1981 4-2632 Virus levels in effluents from five works 1980 4-2633 Relationships between levels of enteroviruses and

indicators 4-3034 Serotypes 1979 - 1981 4-3235 Coxsackie B viruses in waters and wastewaters 1979 - 1981 4-3636 Polioviruses in waters and wastewaters 1979 - 1981 4-36

SECTION 1 : INTRODUCTION

The Importance of Water

In 1974 the international conference on 'Viruses in Water' (Berg et

al, 1976) was introduced by E E Alvarez of the Mexican Department of

Health and Welfare with the words

"Water is important in any nation's development and survival.

The importance of this element of the environment, essential for

the existence of life on earth, is comparable only to that of

oxygen in the air. While the human race does not as readily take

note of the lack of good air, water has clearly been a

determining factor in the survival of many people."

The relevance of this statement is emphasised by the effect of drought

conditions in Africa in the 1980's with many millions of people

suffering starvation due to the lack of water to grow food crops.

Even when water is readily available it is not always possible to

guarantee that its use will not have a detrimental effect on the

consumer. Alvarez points out:-

"The situation is even more drastic when one considers that the

quality of water consumed is often not very good. Moreover, the

ready contamination of water with physical, chemical and

especially biological pollutants frequently makes water a source

of illness and hazard instead of a source of health. The cholera

and typhoid epidemics that occur in various parts of the world

point to the bacteriological problems water presents. One may

also cite various non-bacterial gastrointestinal diseases

originating from the same source that cause countless deaths. In

any case, although it is possible to control bacterial

transmission by water, viruses in water are an unknown that

humanity deeply fears.'1

The importance of water for the transmission of gastroenteritis was

emphasised in September 1980 when the United Nations launched their

campaign for good quality drinking water for all the world's

population by 1990, the 'Water Decade'. In the introduction to this

scheme (Bourne, 1980) it was pointed out that:-

".... eighty per cent of all diseases in the world are.

water-related and at any time 400 million human beings have

gastroenteritis."

Obviously, the figures relate to all types of gastrointestinal

complaints and refer, in general, to those regions with poor

sanitation and lacking a clean, accessible drinking water supply. In

the more developed countries the incidence of waterborne diseases is

substantially lower, but, as will be described later, it is disturbing

that outbreaks still occur despite the availability of advanced

treatment procedures and intensive quality control monitoring.

Water Resources

The health and well-being of man are thus dependent on both the

quality and quantity of the water available to meet demands. The

ultimate amount of fresh water available to a country is finite but,

with increasing population, more and more water must be abstracted

from the environment in order to make up that additional increment

required. Most of the water used returns to the environment in the

form of contaminated wastes containing a range of both chemical and

biological pollutants. However, the natural hydrological cycle has

become increasingly short-circuited in urban, industrial and arid

zones with the consequent paradoxical situation arising of more and

more water being needed but less and less being available at the

required quality. One particular development which has profound

consequences is that the effluent from one community commonly becomes

the raw water source for potable supply to another downstream, a

situation commented upon by the World Health Organisation (WHO)

committee on the re-use of wastewater (WHO, 1973):-

"We are already faced with the situation where some of our rivers

are now so loaded with such vast amounts of wastes of all kinds

that by the time they reach the sea almost all or all of the flow

has been pumped out for municipal or industrial use at least once

and returned to the river. This means that many of our sources

of so-called fresh water are, in effect, partially diluted

wastewaters which have undergone varying degrees of treatment."

t

Such multiple use of river water can be illustrated by the river

Severn (Figure 1). A total of 354 megalitres (Ml) of water is removed

daily at six abstraction points with wastewater being returned at many

more discharge points at a rate of 586 Ml per day. By many standards

the river is of good quality throughout its entire length (354km) with

only about 7% of the flow having been made up by effluent. In

contrast, the river Trent (264km) has up to 20% of its flow

attributable to wastewater discharges and is, at this time, not used

for potable supply although some of its cleaner tributaries are

(Figure 2; STWA, 1983).

A number of factors require consideration when a source of water is

selected. First, it is obvious that it must be capable of meeting the

demands of the population, taking into account average seasonal and

daily variations in requirements as well as being adequate for

projected population growth. Second, the quality of the raw water

must be such that the chosen treatment can process the water to the

required hygienic standards. Third, the source must be protected from

pollutants in order to minimise the demands on the treatment. Last,

cost will dictate which of a number of suitable sources may be the

most appropriate to use and adequate surveys of potential new sources

are essential in order to define appropriate water treatment regimes

along with the necessary pollution control measures to protect the raw

water source (Cox, 1969).

River waters provide much of the raw potable supply in the United

Kingdom. For example, the Severn Trent Water Authority derives about

60% from this source. However, the role of underground sources

ABSTRACTIONS (MLD) DISCHARGES (MLD)

80

347021

654820

5070128

R STOUR5050

15274 0

8489

R AVON7750

2 5 1TTTl MEAN DAILY — -I FLOW (MLD) 959800

SEA

MLD = MEGALITRES PER DAY

FIGURE 2. THE RIVER TRENT.

DISCHARGES (MLD) DISCHARGES (MLD)

154

1 8 21166

R TAME

7 9 3R DOVE

4 855

R DERW ENT4389

H 65R SOAR.

R EREWASH2107 5

893825 212R IDLE '

82MEAN DAILY FLOW (MLD)

9500

SEA

(’groundwater1) in providing suitable water is significant. Such

reserves are usually of high quality when compared to river water

(Table 1) . However, adequate aquifer protection policies must be

enforced because groundwaters are prone to ingress of contamination by

agricultural practices, past as well as present, industrial activity

and municipal land filling.

TABLE 1 Quality Data for Representative Source Waters

(STWA, 1983)

Colour (Hazen scale)Turbidity (Formazin scale) Electroconductivity (us at 20°C)p H -iAlkalinity (mgl x CaCC^)Total Hardness (mgl--*- CaCO3 )Free/Saline ammonia (mgl--*- N)Nitrate (mgl--*- N)Chloride (mgl- -

(mgl--*- Pb)~1 Fe)(mql-1

Sodium (mgl Na)Magnesium (mgl- - Mg) Sulphate (mgl--*- SO4 )

(100cm-3)

Lead Iron (mgl Manganese

Cl)

Mn)

Coliform bacteria Escherichia coli (100cm-3)

36,3,

Notes: 1 Abstraction of the river Severn2 Abstraction from Bunter sandstone3 Abstraction from Oolitic limestone

Mythe^ Bromsberrow^ 3Caswell

20 1 119 0.7 1.2

478 400 5677.8 6.8 7.3

118 101 265119 187 314

0.2 0.01 0.015.1 12.7 2.5

49 28 180.01 0.01 0.010.03 0.01 0.020.09 0.01 0.01

32 13 912 5 560 30 41

100 0 7500 0 4

The importance of such underground waters is illustrated by the number

of aquifers in the Severn Trent Water Authority which provide about

700 Ml daily out of a total requirement of 1,800 Ml (Figure 3).

Underground sources, because of their general good quality, tend to

receive only minimal treatment, usually low level disinfection,

although in some parts of the world, for example Denmark, no treatment

of any kind is carried out (Lund, 1984 personal communication). In

such instances prevention of pollution is of paramount importance.

UNDERGROUND SOURCES IN STWA.

FIGURE 3

MAIN AQUIFERS

MAIN POPULATION CENTRES

Monitoring of Microbiological Quality

In assessing the quality of drinking water, the consumer relies

completely upon the senses. Various constituents may affect the

appearance, smell or the taste of the water and the consumer will

evaluate the quality and the acceptability of the water essentially on

these criteria. Water that contains suspended matter, is highly

coloured or has an objectionable taste will be regarded as being

dangerous to drink and will be rejected. However, it is no longer

acceptable to use such criteria as the sole means of determining the

health hazards posed and the absence of sensory effects does not

guarantee the safety of water for drinking (WHO, 1984).

The relative priorities assigned to the many substances which are

included in standards for drinking waters often depend on local

circumstances. Some, such as pH and colour, may not be related to

health, but may have been used over a long period to ensure the

'wholesomeness1 of the water. The microbiological quality of drinking

water, however, is of the greatest importance and must never be

compromised in order to provide aesthetically pleasing and acceptable

water.

Effective programmes to control drinking water quality depend,

ideally, upon the existence of adequate legislation supported by

regulatory standards and codes that specify the quality of the water

to be supplied and practices to be followed when selecting water

sources, treatment and distribution. The precise nature of the

legislation will be dictated by national, constitutional and other

considerations.

Until fairly recently, monitoring of drinking water for the presence

of microbiological contamination, using the so-called ’indicator'

bacteria, was carried out only by the more industrially developed

nations as their demand for water of high quality increased. However,

the need to use sources of water containing many contaminants, and in

some cases the direct reuse of wastewaters, has necessitated the

greater control of quality by improved treatment. Along with this,

new standards of hygiene have been introduced to take into account

pathogens other than bacteria, notably viruses and protozoa. The WHO

have recently published guidelines for the levels of microbiological

contamination which would be acceptable for drinking waters' (WHO,

1984) while the European Economic Community have issued a directive

laying out similar standards for its member countries (EEC, 1980).

This latter document became statutory on 18 July 1985.

TABLE 2 Microbiological Standards for Drinking Waters

(EEC, 1980)

0 0 0

human consumption should not contain

Table 2 shows the mandatory levels for microbial pollutants in

drinking water but it is noticeable that the pathogenic bacteria,

viruses and zoonoses are only referred to in general terms:-

"Water intended for human consumption should not contain

pathogenic organisms. If it is necessary to supplement the

microbiological analysis intended for human consumption, the

samples should be examined not only for the bacteria referred to

... but also for pathogens including salmonella, staphylococci,

enteroviruses, parasites etc."

Total coliforms lOOcrn-^ Faecal coliforms lOOcm- Faecal streptococci lOOcm""^ PLUS: water intended forpathogenic organisms.

It is now difficult to see, when such a recommendation is presented,

how water undertakings can avoid examination for pathogens and the

past reliance almost entirely on the indicator concept must be

actively questioned. WHO (1984) categorically says that:-

"Where virological facilities can be provided, it is desirable to

examine the raw water sources and the finished drinking water for

the presence of viruses. This will provide base-line data to

evaluate the health risk faced by the population."

The question of the need to examine waters for the presence of viruses

will be discussed later.

Microbial contamination of waters used for bathing or other

recreational purposes have also been subject to standardisation. The

establishment of 'Euro-beaches', those reaching EEC standards, has

been used by many resorts in their publicity campaigns. In these

cases, surprisingly, both bacterial and viral standards have been

imposed (Table 3).

TABLE 3 Microbiological Standards for Bathing Waters

(EEC, 1975)

Guideline Mandatory

Total coliforms lOOcnf^ Faecal coliforms lOOcnf^ Faecal streptococci lOOcm- Salmonellae 1”-*- Enteroviruses 101-"*-

500100100

10,0002,000

00

In contrast, wastewater standards do not usually take into account the

levels of micro-organisms. In the UK the primary aim of treating

wastewater has been to produce an effluent which does not adversely

affect the river into which it is discharged. In many cases this

means that effluents should not affect the biological population of

the river and should not affect the value of the waterway as a

recreational amenity. Consequently, standards have been restricted to

those determinants which would affect natural river populations,

namely biological oxidation demand, ammonia and suspended solids.

Elevated levels of these could lead to fish kill and destruction of

other river fauna and flora with resultant degradation and

putrefaction of organic matter. It is only in recent years that

statutory limits have been imposed with the introduction of the

Control of Pollution Act (HMSO, 1974) with Part II now the statutory

basis for water pollution control.

Existing wastewater standards do not normally include defined limits

for associated microbiological populations. A reason for this

omission partly reflects the questionable belief that risk of

infection has been perceived as deriving solely from the consumption

of contaminated drinking water with such an event being unlikely to

occur during bathing. Furthermore, in temperate zones water

temperatures are usually low enough for most of the year to deter

people from swimming, a situation, incidentally, which is not true in

the pan-tropical areas where exposure to sewage and

sewage-contaminated water may be a normal daily event.

Another and more pragmatic reason for the absence of microbiological

standards in wastewaters is that effective disinfection is difficult

to achieve without causing problems associated with the chemical

residual (Rappaport et al , 1979) . A further pertinent aspect of

microbial contamination is that there is little epidemiological

evidence for sewage-associated diseases. In addition, the problems

associated with the identification of major routes of disease

transmission is compounded in many countries by generally low levels

~of personal hygiene. Nonetheless, it is reasonable to deduce that

efficient pathogen destruction in sewage treatment would be a major

contribution in preventing disease transmission from this source.

Therefore, the role of microbiological monitoring, in particular of

pathogens, is of obvious relevance.

Viral Epidemiology

The study of viral epidemics includes the examination of circumstances

in which both infection and disease arise in a population and also

investigates those factors which may influence disease frequency,

spread and distribution. The distinction between infection (the

multiplication of an agent in a suitable host, determined largely by

factors governing exposure to the agent and the susceptibility of the

host) and disease (the response of the host to infection when it is

severe enough to evoke a recognisable pattern of clinical symptoms) is

necessary in that the factors controlling the occurrence of both may

be different and because infection without disease is a common

occurrence with many viruses. The most important determinants for

many common infections lie within the host itself with the factors

influencing the occurrence and severity of the response varying from

virus to virus and their mode of entry into the host.

The main characters of importance in the initiation of infection are:-

(a) factors promoting efficient transmission of the agent in the

environment;

(b) the ability of the agents to enter the host by more than one

route;

(c) the ability to successfully enter into and multiply in a wide

range of host cells;

(d) the effective release of viable particles into the environment;

(e) the means of developing alternative mechanisms of survival in the

face of host defence mechanisms (Evans, 1976).

The long term survival of a virus in a human population depends on its

ability to establish either a chronic infection without cell death or

in an effective method of virus release into the environment in a

manner ensuring its transport to a susceptible host. The external

environment exerts its influence on the agent itself, on the manner of

its spread and on the nature of the host responses to infection.

Viruses show much variation in their ability to survive the rigours of

environmental stress, but environmental factors also play an important

role in influencing the routes of transmission and the behavioural

patterns of the host. For example, the requirement of an insect

vector is obviously governed by environmental factors restricting the

occurrence of infection and disease to those areas which have the

correct climatic conditions, suitable vegetation and amplifying hosts

and other factors necessary for a successful insect population.

Another potent example of the significance of climate on the

transmission of many viral diseases is its effect on the social

behaviour of the host. Thus, in the tropics and during the warmer

summer months in temperate zones, the opportunity for the transmission

of gastrointestinal disease is greatly enhanced due to increased

contact with contaminated water both from swimming in and drinking

water from polluted areas. In contrast, in winter, people tend to

congregate in warm- buildings and thus promote the spread of airborne

and droplet infections such as colds and influenza.

TABLE 4 Transmission of Virus Infections

(Evans, 1976)

Exit Route Transmission Route Entry Route

Respiratory Bite SkinSalivary transfer MouthAerosol RespiratoryMouth to hand/object Oropharyng eal

Gastrointestinal tract Stool to hand/milk/water MouthThermometer Mouth

Skin Air RespiratorySkin to Skin Abraded skin

Blood Insects, needles, blood transfusion

Skin

Genital Cervix, semen GenitalPlacental Vertical to embryo BloodEye Tonometer Eye

The major routes of virus transmission are summarised in Table 4 and

the ways in which water, in particular, Can act as a vehicle for

disease dissemination are shown in Figure 4.

CROPS

EXCRETA

AEROSOLS

SEWAGE

SHELLFISH

MAN

RECREATION

IRRIGATION

WATER SUPPLY

R IVERS/LAKES

LAND RUN-O FF

GROUNDWATERSOCEANS/ESTUARIES

SOLID WASTE LANDFILL

FIGURE 4. WATER ROUTES FOR VIRUS TRANSMISSION(MELNICK, GERBA & WALLIS, 1978)

Minimal Infective Dose

One of the problems facing a viral epidemiologist is the question of

the minimal infective dose, that is, how much (or little) virus is

required to enter a susceptible host in order to initiate infection

and possibly cause disease.

In laboratory studies, using cell culture systems for example, it is

possible to determine the infectivity of viruses with some degree of

reproducibility, but this is less readily achieved in animal models.

In man only limited data have become available through vaccine trials

(Katz and Plotkin, 1967; Minor et a_l, 1981) . By the use of in vitro

systems and electron microscopy it has been possible to establish the

ratio of viable to non-viable viruses reaching the cells. Using such

models it has been demonstrated that a single viable virus particle is

capable of initiating infection and this concept has been extended to

apply to the initiation of infection in man (Berg et al, 1976).

However, this form of extrapolation is no longer measuring the

response of individuals to decreasing virus dosage, but is a

determination of the statistical probability that a single viable

virus particle will make effective contact with a susceptible host.

There have been few investigations of the minimal infective dose for

human viruses mainly because of the ethical aspects of administering

infectious virus to human volunteers. Studies have usually involved

the evaluation of vaccine-type polioviruses (Katz and Plotkin, 1967;

Minor et_ aJL, 1981) although recently Schiff et al (1984a) reported a

study using echovirus type 12. In a review of the human volunteer

work, Ward and Akin (1984) reinterpreted the data and found that each

virus type has its own particular infective dose (although this will

vary according to the host's susceptibility). They also realised that

the means of determining the initial virus dose administered was

important in that varying assay methods gave significantly different

results.

In the case of the echovirus 12 study, the initial report by Schiff et

al (1984a) used a plaque assay procedure with the monkey cell line,

LLC-MK2, as the host. Based on such a system the minimal infective

dose was calculated to be between one and two plaque forming units

(pfu) in order to induce a serological response in 1 % of the

volunteers. No illness was demonstrated in any of the subjects

although infection could be shown in 30% and 100% of the subjects

administered with 10 pfu and 300 pfu respectively. However,

retrospective titrations of the inoculum carried out by Schiff et al

(1984b) in human rhabdomyosarcoma cells by a soft agar plaque system,

indicated that the titre of the inoculum was 33-fold greater than had

been earlier reported (Schiff et al, 1984a). The implications of this

for the trial in human volunteers was that a dose of 919 pfu would

have been needed to infect 50% of the subjects and not the 1-2 pfu

which was thought to have been given. Ward and Akin (1984) concluded

"that the infectivity of echovirus 12 was much less in susceptible

healthy adults than in sensitive cultured cells. Furthermore, they

suggested that not only would other subjects, such as infants, be more

easily infected but that infectivity might have been more readily

achieved in individuals who had recently fed because of reduced

stomach acids and the protection afforded by the incorporation of the

viruses into the food during ingestion.

As enteric viruses are incapable of multiplying in the environment, it

is necessary to look for the consequences of viral pollution in subtle

ways. It is especially likely that persistent low-grade seeding of a

densely populated community will occur and, despite the development of

sbme immunity, this could maintain undetectable foci of endemic

infection without the production of definable outbreaks of disease.

More importantly, there would be persistent cycling of viruses in the

population. Under such conditions any factor which would lead to

increased host sensitivity could result in epidemic disease. Such

low-level seeding has been criticised on the grounds that there is

little evidence, epidemiological or clinical, to support the

hypothesis (Gamble, 1979).

Faecally Excreted Viruses

Viruses excreted in the faeces contribute most to the viral pollution

of water. Until recently, the viruses usually described as being

associated with the faecal-oral transmission route were the

enteroviruses, a sub-group of the Picornaviridae (Melnick, Wenner and

Phillips, 1979). They give rise to a wide range of clinical symptoms

ranging from headache and mild fever to severe muscular paralysis and

encephalitis, sometimes terminating in death (Table 5). Most of the

group can be readily grown in cell culture and most produce a marked,

and diagnostic, cytopathic effect (cpe).

TABLE 5 Human Enteric Viruses in the Water Cycle

Disease Caused

Meningitis, paralysis, fever Meningitis, diarrhoea, rash, fever, respiratory diseaseMeningitis, herpangina, fever, respiratory diseaseMyocarditis, congenital heart anomalies, pleurodynia, respiratory disease, fever, rash, meningitis Meningitis, encephalitis, acute haemorrhagic conjunctivitis, fever, respiratory disease Infectious hepatitis

Hepatic dysfunctionDiarrhoea, vomiting, feverGastroenteritisGastroenteritisGastroenteritisNot clearly establishedInfantile diarrhoeaGastroenteritis

(Goyal, 1984)

Virus

PolioEcho

Coxsackie A

Coxsackie B

No. Types

331

23

6

Entero 68-71

Hepatitis ANon-A, Non-BhepatitisNorwalkCaliciAstroEnteric coronaReoRotaFaecal adeno

1?3?1?2?1?322

In the last ten years other enteric viruses have been recognised,

typically associated with gastroenteritis rather than the syndromes

described for enteroviruses. Many of the gastrointestinal viruses are

highly contagious with an incubation period of as little as

twenty-four hours followed by gastroenteritis characterised by

diarrhoea, vomiting, headache, abdominal cramps and low-grade fever,

symptoms which together usually last only two to three days. However,

when an outbreak occurs, it moves very rapidly through a population,

apparently without giving rise to repeated infections. Even though

the disease tends to be epidemic, there is sufficient evidence to

postulate that the disease exists in an endemic form that is

responsible for sporadic cases or localised outbreaks, thus posing a

major recurring problem in public health (Blacklow et al, 1972;

Flewett, 1977).

The failure to isolate known pathogens from stools of patients with

diarrhoea led to the examination of faecal extracts by electron

microscopy. Apart from the known morphological groups, a range of

other virus types, previously unreported, was demonstrated. The major

virus group, associated with a large proportion of infantile

gastroenteritis cases, resembled the reoviruses with a diameter of

about 70nm. Similar viruses from diarrhoeal samples from other animal

species have been described and, because of certain highly

characteristic features, were named rotaviruses (Bishop et al, 1973;

Flewett, Bryden and Davies, 1973). To date, at least two distinct

serotypes have been described in man (Table 5).

Another group of viruses found in faecal extracts from

gastrointestinal cases has been referred to as Norwalk and

Norwalk-like agents. Hi is group probably contains at least three

serologically unrelated members (Kapikian et al, 1972; Thornhill et

al, 1975). These viruses are morphologically similar to the

polioviruses with the same pH tolerance. The disease caused by the

agents is characterised by a rapid onset of vomiting, with or without

diarrhoea, with peak virus excretion in the stools after twenty-four

hours. Little virus can be found in the faeces after seventy-two

hours. The disease is also unusual in that certain individuals

experience repeat infections which is taken to indicate that the

immune response is short-lived (Baron et al, 1984). It is thought

that the Norwalk-type viruses are the most significant cause of

waterborne outbreaks of gastroenteritis in certain parts of the world

(Holmes, 1979).

Other viruses described and associated with diarrhoea include the

astrovirus (Kurtz, Lee and Pickering, 1977; Ashley, Caul and Paver,

1978) which occurs in about 5% of cases, but is often detectable in

asymptomatic cases, calicivirus (Spratt et al, 1978; Oishi et al ,

1980; Cubbitt and McSwiggan, 1981) which is implicated in only a few

instances and coronavirus (Caul and Clarke, 1975; Maass and

Baumeister, 1983) which may only rarely give rise to gastroenteritis.

In addition, two newly-described adenovirus serotypes (types 41 and

42) have been shown to be faecally excreted and have been implicated

in outbreaks of gastroenteritis (Gary, Hierholzer and Black, 1979;

Johansson et al, 1980).

The virus causing hepatitis A is also found in faeces and is now

classified as a picornavirus (Skidmore and Tadros, 1976). Common

source outbreaks of this virus are frequent with spread of virus by

the faecal-oral route being most commonly by person-to-person

contact. Poor hygiene and low socio-economic status are major

contributory factors in the rapid spread of the virus. In common with

many gastroenteritis viruses, hepatitis A cannot readily be isolated

in cell culture systems although adaption of the virus to some cell

lines has been achieved (Flehmig, 1980; Crance et al, 1983). Recently

Binn &t al (1984) successfully isolated hepatitis A virus from liver

tissue from infected primates and from faecal extracts from human

cases using a range of cell lines, the most successful isolations

being made when primary green monkey kidney cells were used.

A second hepatitis-inducing virus (or viruses), currently termed

non-A, non-B hepatitis, has been implicated in several cases which had

been attributed to hepatitis A virus. The virus has been described as

being similar to hepatitis A virus in size (Balayan et al, 1983,

Sreenivasan et al (1984a) while Prince £t al (1984) demonstrated the

presence of a membrane enclosed particle of 85-90nm (with a core of

40-45nm) in cultures of chimpanzee liver cells inoculated with

material from patients showing typical non-A, non-B hepatitis. Ihis

possible multiple aetiology was further examined by Gitnick (1984) who

concluded that some of the types seemed to be related to hepatitis A

while others resembled hepatitis B virus. The picture is obviously

confused and it will probably be some time before it is known which

ones are of significance in the water context. However, it is certain

that some form of hepatitis due to one (or more) of these non-A, non-B

hepatitis agents can be traced to the consumption of contaminated

water as will be described later.

Faecally excreted viruses are usually present in a population at most

times of the year. However, many demonstrate marked seasonality in

terms of disease manifestation. For instance, rotavirus infections

tend to occur in late winter and early spring whereas

enterovirus-induced diseases tend to be dominant during the summer and

autumn months (Figure 5). It is arguable that in order to effectively

monitor the water cycle for the enteric viruses it is necessary to

take into account such seasonality.

9 0 0 — 1

3 0 0 —

mi1 9 8 0 1 9 8 1

□ ENTER0VIRAL INFECTIONS

R0TAVIRAL INFECTIONS

FIGURE 5. SEASONALITY OF VIRUS DISEASES(CDSC, 1 9 8 0 - 1 )

Waterborne Viral Diseases

A multiplicity of factors contribute to waterborne outbreaks of

disease (Committee 1979) the major ones being summarised in Table 6;

In general, to carry out such exhaustive surveillance programmes as

suggested by this review (Committee, 1979) is rarely practicable.

However, the value of intensive epidemiological investigations is

emphasised by the analysis of waterborne outbreaks in the US for the

'thirty-five year period 1946 to 1980 (Lippy and Waltrip, 1984). In

this time, 672 outbreaks were reported involving over 150,000 persons

and the authors found an underlying trend of increasing frequency of

outbreaks noting that in recent years that most of this increase could

be attributed to greater vigilance by a few areas. In 1980 over

20,000 cases of all types of waterborne disease were recorded in the

US with 14,000 being associated with only four outbreaks, but it was

concluded that these values would be much higher if surveillance

practices were more widely adopted.

In the UK little comparable epidemiological work has been done and

there is minimal co-ordination between the agencies concerned with the

public health aspects of water, namely the water authorities, the

public health laboratory services and the environmental health

departments. Indeed, the current attitude appears to be that

waterborne disease outbreaks in the UK are so uncommon as to be

effectively non-existent (Committee, 1978; Gamble, 1979). However,

this view must be questioned in light of the US data.

TABLE 6 Factors Contributing to Waterborne Disease

(Committee,

Surface water:

Ground water:

Inadequate treatment:

Storage deficiency:

Distribution deficiency

Water contact problems:

Other factors:

1979)

use of untreated surface water; contamination of water catchment area; use of unsafe source as supplementary supply; flooding, septage/sewage contamination; surface contamination eg. sewage sludge; contamination through fissures eg. chalk; flooding;improper construction of well/borehole.poor disinfection, if any;interruption of disinfectant;inadequate pre-treatment prior todisinfection.unprotected storage reservoirs;improper disinfection of new storagefacility.back-siphonage;cross connections;contamination of main during repairs; inadequate separation of water and sewage lines;improper disinfection of mains/plumbing, puncture injuries or wounds; recreation;inadequacy of water-holding facilities; improper pH control and disinfection, use of water not intended for drinking; contaminated water containers/drinking fountains;deliberate contamination; contaminated ice; aerosols.

Mosley (1959) concluded that in order to demonstrate satisfactorily

that water can be a route of transmission of viral diseases, it is

desirable to have unequivocal clinical criteria for the disease,

secure evidence that the causative viruses are present in the water

and to demonstrate a human source of the virus. It is quite clear

that viral infections may be transmitted by the ingestion of

contaminated drinking water and shellfish and also by swimming. What

is not so clearly established is that the water route is as important

as other routes, in particular person-to-person transmission. It is

evident, however, that the risks will be more or less eliminated by

vigorous application of good sanitary practice (Committee, 1970), but

that a serious departure from high sanitary standards could lead to

large outbreaks of disease. Even low grade transmission of infection

could, it is reasonable to assume, cause primary cases which would

probably result in a large number of secondary cases of disease.

Only a few virus diseases have been unequivocally shown to be

transmitted by the water route the best described being hepatitis A.

Outbreaks of this disease have been well documented by Mosley (1967).

Appendix A shows these data together with more recent reports. The

recognition of non-A, non-B hepatitis as an additional candidate for

waterborne transmission has, so far, only been supported by evidence

from India and Algeria. In particular, the 1955-6 outbreak of

hepatitis in India, originally attributed to hepatitis A virus, is now

thought to have been caused, in part, by the newer agent (Appendix A ) .

Incidents of acute gastroenteritis attributable to rotavirus and,

probably more significantly, Norwalk-type viruses have increased in

recent years (Appendix A).

Outbreaks of enterovirus disease caused by contaminated water are

apparently rare and those that have been reported have been regarded

with some scepticism (Appendix A). However, the range of symptoms

associated with the diseases caused by this group of viruses may well

make it almost impossible to recognise an outbreak when it occurs.

Records of waterborne viral diseases in the UK are infrequent.

However, some epidemiological and clinical data exist and it would

seem that many incidents of waterborne gastrointestinal illness can

probably be attributed to infection with viruses of the Norwalk group

(Appendix A ) . Since gastroenteritis is not a notifiable disease in

the UK, it is possible that there are many more outbreaks associated

with the drinking of polluted waters. Gamble (1979) has pointed out

that such outbreaks (as are reported) are typically associated with

water supplies that fail to meet accepted sanitary or treatment

standards. However, Akin (1984) noted that Gamble (1979) only

considered the enteroviruses and pointed out that the role of water as

a means of transmitting the other enteric viruses deserves further

investigation especially since viruses, albeit at low levels in most

cases, have now been shown to be present in drinking waters that

conform to accepted water quality standards (Akin, 1984; Slade, 1985).

Factors Affecting Virus Survival in Water

Before considering the levels and types of viruses which have been

found in the aquatic environment it is necessary to be aware of the

ability of viruses to survive in hostile surroundings.

TABLE 7 Survival Times o f •Enteroviruses in Various Environments

(Melnick and Gerba, 1980)

Environment Survival time (days)

2 - 1302 - 1885 - 168

25 - 1756 - 908 - 20

Melnick and Gerba (1980) summarised, in very broad terms, the ability

of viruses to withstand environmental pressures (Table 7) indicating

the variation that can be encountered because of the range of methods

used during investigations, the types of viruses and waters studied

and the influence of environmental factors. Those parameters

affecting virus survival are shown in Table 8. The more important of

these will be briefly considered.

Temperature is probably the single most important factor in

determining the survival of viruses in water. In general, as water

temperature rises, virus infectivity decreases. In temperate zones,

where water temperatures range from 10 - 20°C, virus numbers are

reduced only slowly. Typical of the published data are those of Lo,

Gilbert and Hetrick (1976) and Toranzo and Hetrick (1982). More

recent work describing the survival of calf rotavirus in water

Sea or estuary waterRiver waterTap waterSoilOystersMarine sediment

indicated that this group of viruses behaves in a similar fashion to

the enteroviruses. In one study, the virus was reduced by 90% after

80 days at 8°C (McDaniels et al, 1983) this being supported by the

findings of Sattar, Raphael and Springthorpe, (1984) who found that

the simian rotavirus, SA-11, was reduced by 50% after 64 days at 4°C.

TABLE 8 Factors Affecting Virus Survival in Water

(Sattar, 1981)

Hydrological^ Seasonal, Climatic:- Temperature- Turbulence, rate of flow, volume, depth

Nature and extent of suspended particulates and sediment- Rainfall and spring thaw- Sunshine

Chemical:pH

- Salinity- Heavy metals- Organics

Biological and Biochemical:- Bacteria and fungi- Protozoa- Shellfish- Other aquatic flora and fauna- Enzymes

Pollutants:- Domestic- Agricultural and farm

Industrial

The ability of viruses to adsorb to solid particulates influences the

inactivation of viruses in water. Viruses can be regarded as

collodial particles which tend to exhibit a nett negative charge at pH

levels around neutrality. Their adsorption to solids will depend upon

the characteristics of the material (including charge and nature of

the adsorbent) and the environment in which both are suspended (such

as ionic composition and pH; Valentine and Allison 1959). Under

optimal conditions, suspended particulates will have a nett positive

charge thus promoting interaction with viruses. A change in

conditions, for example elevation of pH, may lead to alteration of the

relative charges resulting in the desorption of viruses. This

phenomenon of adsorption and elution has been widely used in the

concentration of low levels of viruses from large volumes of water.

The range of materials which are capable adsorbents of viruses is

extensive (Table 9).

TABLE 9 Materials Capable of Adsorbing Viruses

(after Bitton, 1975)

Activated carbon Precipitable saltsGlass PolyelectrolytesDiatomaceous earths Other claysAlumina Silicates other than claysSand SoilIon exchange resins Iron oxidesMembrane filters

Adsorption has been shown to enhance virus survival in the

environment. LaBelle and Gerba (1982) demonstrated that poliovirus

type 1 adsorbed to estuarine sediments not only survived longer than

free virus but, to a certain extent, was also protected from the

effects of temperature. Adsorption of viruses, during water and

wastewater treatment processes, to chemical and biological floes

effectively removes many viruses from the aqueous phase. In a study

of the association of naturally-occurring enteroviruses with

wastewater solids, Hejkal et al (1981) showed that up to 47% of virus

was Solids-associated in the incoming sewage this being reduced to

about 4% after treatment (being related to the decrease in solids

levels of 94%). Adsorption of poliovirus type 1 and rotavirus SA-11

to aluminium hydroxide floes in tapwater indicated that adsorption of

the former was substantial (99.9%) whereas less than 90% of the

rotavirus was adsorbed. Similar findings were also found when

investigating the adsorption of the two viruses to activated sludge

floes (Farrah et: _al, 1978). The possibility that viruses behave

differently from one another in their ability to adsorb to surfaces

was investigated by Gerba et elL (1980). They successfully

demonstrated that viruses not only adsorbed to differing degrees, but

that strains of a single type also showed variation in their

behaviour, particularly when examining their interactions with sandy

loamy soils.

Aggregation of viruses in water may also play a role in protecting

viruses from treatment processes (particularly disinfection) and

environmental factors. Laboratory studies have shown that, given the

right conditions, viruses may aggregate into large clumps of several

hundreds of particles which may or may not dissociate when conditions

change (Floyd and Sharp, 1977). However, Young and Sharp (1977)

suggested that such aggregation may be due in part to complex

formation with insoluble material rather than virion to virion

interaction. In the water context, it is likely that the conditions

of variable pH, ionic strength, etc., together with the high

inter-virus distance because of dilution effects, result in only low

levels of aggregation, if any at all. Indeed, the low occurrence of

such aggregation can be monitored by the assay of effluents which

contain large numbers of viruses. These can be effectively separated

by the plaque detection system with no evidence of multiple serotypes

within the confines of a single plaque.

Photodynamic effects can also influence the infectivity of viruses in

water, especially those which are highly coloured. However, the

effect of light, usually taken to mean solar radiation, is marginal as

the degree of inactivation is dependent upon the ability of the

radiation to penetrate the water. Bitton, Fraxedas and Gifford (1979)

have shown that only the top few inches of water receive sufficient

energy to have an appreciable effect on virus levels. The same study

also demonstrated that virus adsorbed to clays was protected from

photodynamic inactivation. However, where wastewater can be treated

by long term lagooning, the addition of photosensitising dyes, such as

methylene blue, and the availability of long hours of intense

sunshine, can result in substantial reductions in virus numbers

(Gerba, Wallis and Melnick, 1977a,b; Hobbs et al, 1977; Bitton,

Fraxedas and Gifford, 1979).

Viruses in Water and Wastewater

Viruses occur in all types of water and wastewater, can be found in

association with particulate matter in soils, river sediments and

sludges, can be isolated from shellfish and may be present in

aerosols. The main source of virus contamination is sewage, levels of

viruses falling as the water cycle is followed through to the

consumption of drinking water. The occurrence of viruses in the

aquatic environment has recently been extensively reviewed by Bitton

et al (1985; Appendix B).

Drinking waters occasionally show evidence of virus contamination.

However, it is disturbing to note that viruses can be detected even

when residual disinfectant is present and the water meets current

standards of microbiological quality. Indeed, the finding of viruses

in water with chlorine residuals has resulted in the postulation that

such viruses may have attained a degree of chlorine resistance.

Shaffer, Metcalf and Sproul, (1980) found that two strains of

poliovirus type 1, isolated from conventionally treated drinking

water, were resistant to chlorine levels of up to i.35 mg 1

Inter-virus variation to the sensitivity to chlorine has been

demonstrated by Liu et al (1971) and Engelbrecht et al (1980) while

more recently Payment, Tremblay and Trudel (1985) have shown not only

inter-virus differences, but also inter-strain variation. In this

latter study, two strains of coxsackieviruses, types B4 and B5, could

be detected after sixteen hours exposure to 0.5 mg 1 ^ (initial

concentration) chlorine whereas little or no poliovirus could be

found. The finding of viruses in groundwater treated with chlorine to

1 mg 1 by Slade (1985) emphasises the potential risk to public

health despite satisfying microbiological standards.

Similarly, groundwaters are only infrequently contaminated with

viruses. The main factors which seem to influence the presence of

viruses in underground sources appear to be the depth at which water

is abstracted, the geological nature of the aquifer and the potential

for contamination from agricultural and industrial practices such as

sludge spreading on farm land. In one study, the presence of viruses

in a groundwater abstracted from a 60 metre borehole, could be shown

to be the probable cause of an outbreak of gastroenteritis and

hepatitis A in the recipient community (Hejkal e£ al, 1982). On this

occasion faecal contamination of the source was also demonstrated by

the presence of the conventional bacteriological indicators.

Surface waters inevitably reflect the influence of the sewage effluent

loading which they receive. The numbers and types will vary according

to the season as mentioned earlier. The main areas of concern about

viruses in surface waters are the potential risk of overloading water

treatment processes where such water is abstracted for potable supply

and where the water is used for recreational purposes. The frequency

of positive samples of river waters can be as high as 100% -depending

upon how much of the flow can be attributed to effluent discharges.

"However, the variety of methods available for the recovery and

detection of viruses in environmental samples, can result in levels as

low as 0.02 infective units being reported for the Ottawa river in

Canada (Sattar, 1978) and ->100 pfu 1 ^ in the River Thames in the

UK (Slade, 1977).

Other surface waters, lakes and reservoirs, tend to have lower levels

of virus, occasionally being apparently virus free. The factors

controlling the survival of viruses, obviously, are influential in

such waters.

Raw sewage may contain large numbers of viruses, again the levels

reported reflecting the techniques used for their detection. For

example, Schwartzbrod, Lucena and Finance (1979) used a concentration

system to recover viruses from sewage resulting in virus levels of up

to 400 MPN 1 \ In contrast, Buras (1974, 1976) did not use a

concentration method, but directly inoculated the sample onto the cell6 —Icultures. In these studies virus levels in excess of 10 pfu 1

could be detected. Wastewater effluents usually have slightly lower

levels than the incoming sewage because of the effects of solids

association in the formation of sludges and the effects of the various

treatment processes. However, virus levels can still be substantial

with Buras (1974, 1976) detecting < 10^ pfu 1

Disinfection of effluents is carried out in some parts of the world,

but even here there is evidence that many viruses escape

inactivation. The studies carried out in Hawaii by Fujioka and Loh

(1978) resulted in effluents being discharged into the sea after

disinfection still containing virus levels up to 750 pfu 1

Both estuarine and marine waters reflect the virological loading of

the rivers which flow into them and it is noticeable that many waters

at beaches used for bathing can be shown to be contaminated with

viruses. In the UK, studies on the virus contamination of coastal

waters, particularly in the vicinity of bathing areas, have been

carried out by Shoulder (1982) and Tyler (1982). Virus levels in sea

water have been found to be as high as 100 pfu 1 ^ with many

instances of virus being recovered from beach sand.

The presence of viruses associated with particulate matter has been

referred to several times. They may be found in conjunction with

water and wastewater sludges, adsorbed to river sediments and

associated with soils, in the latter case often as a result of the

application of wastewater products to land (Bitton 1975; Duboise et

al, 1979).

The accumulation of viruses by shellfish has been a cause for concern

in recent times as many outbreaks of foodborne gastroenteritis can be

associated with consumption of shellfish which have been harvested

from polluted waters, inefficiently depurated and inadequately

cooked. Figure 6 illustrates the outbreaks of shellfish-associated

gastroenteritis in the UK in the last few years.

FIGURE 6. VIRAL DISEASES FROM , CONSUMPTION OF SHELLFISH IN THE UK

(ADAPTED FROM SOCKE7T ET.AL, 1 985)

15 — I

10 — jt/5X.<111 <r

5 —

0 —

UA

PN

UA

HA

I 1 7q1 j 19 7 1-8 0 | 1981-3 j

ICOCKLES C3 MUSSELS E l OYSTERS □ MIXED/OTHER/UNSPECffTED

UAUJNKNOWN AETIOLOGY HA=HEPATITIS APN=PARVO/NORWALK VIRUSES

Aerosols from wastewater treatment plants and from areas where spray

irrigation using effluents is carried out may contain viruses. While

there is little evidence of disease outbreaks being attributable to

such aerial contamination there is sufficient evidence, based on

serological investigations, to suggest that infection does occur

(Iftimovici et al, 1980; Margalith et al, 1982; Morag et al, 1984).

Virus Removal by Water and Wastewater Treatment

Conventional water and wastewater treatment processes have been

developed in response to urbanisation, industrial development and

growing populations. In the present century, the physical and

biological processes developed during the previous hundred years have

been supplemented with chemical treatment including disinfection. The

efficiency of the various treatment procedures has been extensively

reviewed by Lloyd and Morris (1982) with particular reference to the

reduction of virus numbers.

Potable water treatment is governed by the raw water quality, in

particular the degree of microbiological contamination and the level

of turbidity. While other factors cannot be ignored, these determine

the regime of treatment in the production of good quality water.

Long term storage can be very effective in reducing virus levels and

factors influencing the survival of viruses have a major role to

play. Such stored water only occasionally shows evidence of virus

contamination and then usually only at very low levels (Slade, 1977).

The use of flocculation and coagulation processes, usually utilising

hydroxides of iron and aluminium, to reduce turbidity, colour and

micro-organisms is practiced in many areas with reductions of *<99.9%

being achieved (Chaudhuri and Engelbrecht, 1970; York and Drewry,

1974; Guy, Mclver and Lewis, 1977; Farrah et al, 1978).

Sand filtration is extensively used either as a rapid polishing filter

for the removal of floe or as a biological slow filter aimed at

removing micro-organisms. Virus removal by the former is ineffective

while the latter is capable of high efficiency. Poliovirus type 1 and

reoviruses have been reduced by as much as 99.99% (Poynter and Slade,

1977; McConnell, Sims and Barnett, 1984). The main disadvantage of

slow sand filtration is that large land areas are usually required in

order to process a raw potable water.

The use of activated carbon filtration to remove undesirable organic

material may also result in reduction of virus levels. However,

maintenance of the filters is essential because of saturation with

organic compounds and subsequent break-through of virus. Guy, Mclver

and Lewis (1977) found that 79% of poliovirus 1 could be removed, but

the efficiency of virus removal was adversely affected by the

frequency of back-washing with freshly disturbed material having

reduced adsorptive capacity. The same workers also noted that virus

removal was inversely related to the concentration of the organic

compounds present.

Disinfection of potable waters, primarily by chlorine, is probably the

single most effective barrier to the ingress of viruses into a water

supply. As already discussed, however, the presence of viruses in

finished waters which satisfy current microbiological criteria and

exhibiting residual disinfectant must be a major concern with regard

to the potential threat to public health.

The improvement of the quality of wastewaters using a variety of

physicochemical and biological treatment procedures has traditionally

been monitored by the reduction of non-biological parameters such as

biochemical oxygen demand and suspended solids. The treatments used

reduce the levels of such parameters substantially, but the vagaries

of the procedures, the effectiveness of which are influenced by the

quality of the incoming sewage, result in erratic reduction of virus

numbers.

Primary sedimentation is usually too short in duration to have a

noticeable effect, any virus removal being mainly associated with the

deposition of solids with attendant adsorbed viruses (Bloom et al ,

1959? Clarke et al, 1961; Kelly, Sanderson and Neidl, 1961; Mack et

al, 1962; Malherbe and Strickland-Cholmley, 1967). Purification of

wastewaters by percolating filtration through medium coated with

zoogloeal slime is similarly inefficient (Kelly and Sanderson, 1959;

Kelly, Sanderson and Neidl, 1961? Berg 1973) with Clarke and Chang

(1975) showing mean reductions of 94%, 83% and 85% for coxsackievirus

A9, echovirus 12 and poliovirus 1 respectively in an assessment of a

rotary filter. The use of activated sludge, an aerobic microbial

culture which rapidly assimilates organic and inorganic material

soluble in wastewater, can achieve substantial reductions of virus

levels with up to 99% removal being reached in some cases (England

et al, 1967; Lund, Hedstrom and Jantzen, 1969; Malina et al, 1974?

Slade, 1982). However, Slade (1982) pointed out that the performance

of the process varied according to the virus type being used in the

evaluations.

Lagooning (stabilisation ponds) is probably the most effective means

of removing viruses from wastewater, provided sufficient land is

available. Long term lagooning, under the influence of temperature

and light, can give major reductions in virus levels (>-99%) although

residual virus is often detectable (Sheladia, Ellender and Johnson,

1982). Disinfection of wastewaters is not normally carried out in the

UK whereas in some parts of the world, for example California and

Arizona, there is a legislative requirement to disinfect effluent

before discharge. As has been pointed out, viruses can still be

detected in disinfected effluent and the concern over the production

of undesirable halogenated organics precludes such treatment in many

parts of the world. It has been argued that disinfection of

wastewater effluents is unnecessary except as a terminal barrier prior

to water consumption, with other processes, when judiciously chosen

and operated correctly, reducing pollutant levels to satisfactory

levels before discharge to watercourses (Lloyd and Morris, 1982).

The Project

It is evident that much work has been carried out on the detection of

viruses in the hydrosphere. The methods used have almost always used

high levels of laboratory strains during evaluation studies, but often

the derived procedures have only been applied in a small way to the

examination of field samples. It is not unreasonable to postulate

that the behaviour of such highly adapted strains may, in fact, not

mirror what actually happens in the environment.

The methods chosen influence the numbers and types of viruses which

may be detected. Optimisation of procedures to detect the largest

frequency of positive samples and the highest numbers of viruses is of

paramount importance, particularly when monitoring large volumes of

drinking water for low levels of virus. Methods for such monitoring

have been examined and the influences of such factors as cell lines

and media constituents have been determined. A method has been

derived and applied to the 'real world' situation.

Enterovirus levels in waters and wastewaters have been measured, the\ .

data being used to determine the effectiveness of wastewater treatment*

procedures, to assess the relationships between viruses and

conventional microbiological indicators and to examine the potential

hazards associated with the use of polluted waters for potable supply.

SECTION 2 : MATERIALS AND METHODS

Viruses

Twenty-six enterovirus types were used in the evaluation of the virus

susceptibility of the ten cell lines investigated. Twenty-three, with

known passage histories in Vero cells, were obtained from the Wellcome

Research Laboratories and had been derived from the American Type

Culture Collection. The other three serotypes, echoviruses 13, 17 and

32 were isolates from environmental samples obtained by the Severn

Trent Water Authority and had been passaged in BGM cells only. Stocks

of each serotype were prepared in either Vero or BGM cells as2appropriate. Confluent monolayers (80cm flasks) were washed with

serum-free maintenance medium (Appendix C) and inoculated with 30.5cm of virus per flask. After adsorption at 37°C for 30 minutes,

330cm of fresh maintenance medium was added and. the cultures

incubated. They were examined daily for the development of cytopathic

effects and when this reached 100% the cultures were subjected to a

single cycle of freeze-thawing (-70°C) followed by clarification of

the fluid by centrifugation (3,000 xg, 10 minutes). The resultant

supernates were stored at -20°C as stock virus.

Monodispersion of Virus

Stock suspensions of viruses were filtered through cellulose nitrate

membranes (50nm porosity) to remove viral aggregates in order to

achieve standardised preparations regarded as being monodispersed.

Such preparations were thought to be more suitable for use in plague

tests because the presence of viral aggregates could lead to variable

infectivity titres. Monodispersed viruses were stored at -20°C.

Cell Lines

Ten cell lines were examined, five of human origin and five of simian

derivation. All were supplied by Flow Laboratories (Irvine, Scotland)

with the exception of MA-104 cells which were obtained from

Micro-biological Associates (Maryland, USA). The human cell lines

were: FL Amnion (FL; CCL 62), rhabdomyosarcoma (RD; embryonal; CCL

136), intestine 407 (1-407; CCL 6), Detroit-6 (D-6; sternal marrow

from lung carcinoma patient; CCL 3) and Chang conjunctiva (CC; clone

l-5c-4 from Chang conjunctival D cells; CCL 20.2). The simian cells

were: BGM , (African green monkey kidney), chimpanzee liver (CL),

LLC-MK2 (rhesus monkey kidney; CCL 7) , Vero (African green monkey

kidney; CCL 81) and MA-104 (foetal rhesus monkey kidney).

Propagation of Cell Lines

Stock cultures of each cell line were maintained by growth in2disposable tissue culture flasks (180cm , Nunc) using growth medium

based on either Parker's 199 (Morgan, Morton and Parker, 1950) or,

more usually, Eagle's minimal essential medium (EMEM; Eagle, 1959)

prepared in Earle's salts (Earle, 1943). These were supplemented with

foetal bovine serum (FBS; 10% v/v), non-essential amino acids,

vitamins, glutamine and antibiotics, all at levels recommended by the

supplier (Gibco-Europe). The growth medium was buffered with sodium

bicarbonate at a final concentration of l.lg 1 1 (Appendix C ) .

Cell lines were usually passaged on a weekly basis. The monolayers

were dispersed with a trypsin-versene mixture (Appendix C) after

removal of spent medium. After counting the cells obtained, they were

distributed into fresh flasks at approximately 2.5 million cells per 3flask m 100cm growth medium except MA-104 cells which were seeded

at 4 million per flask. All cultures were incubated at 37°C and after

formation of the monolayer (3-4 days) spent growth medium was replaced

with fresh maintenance medium.

Cell cultures for use in the studies were prepared in a variety of

ways:-21 virus stocks were prepared in cultures grown in 80cm flasks

seeded with one million cells which were inoculated with virus

after ,5-7 days growth;

2 growth of field isolates of viruses was in tube cultures seeded2with 0.5 million cells in 2cm growth medium and inoculated

after 2 days incubation;

3 for plaque assays, cells were grown in glass roller bottles2 . •(1,475cm , Bellco) which, after 7 days incubation, gave a cell

harvest of approximately 500 million having been planted out at350 million in 500cm growth medium. Excess cells were used for

assay by the suspended cell method (cell stock prepared as a 10-3million cm suspension) or for the microtitration assay system

-3at a concentration of 0.5 million cm

Adjuncts

A number of materials were examined for their effect on plaque

production by laboratory strains of viruses. Details of each are

given in Appendix C.

Assay of Viruses

Most" Probable Number Method — this is based on the method described

by Chang et alL (1958) . Dilutions of the test virus were prepared in

serum-free maintenance medium as four-fold steps. Tube cultures of

the appropriate cell line, 1-3 days post-initiation, were changed to3maintenance medium and 0.1cm of virus dilution added. Five tubes

were inoculated for each dilution. The infected cultures were

incubated at 37°C in the static position and the development of

cytopathic effect monitored daily until two consecutive days' readings

were the same. Using the tables of Chang et al (1958) the most

probable number of viruses present was calculated (MPN).

Microtitration Method - using a flat-bottomed microtitre plate, 25

^ul of virus dilution (prepared as logarithmic steps in serum-free

maintenance medium) were added to a well (five wells per dilution).4 ’Cells were added at a rate of 5 x 10 per well in 100 yul growth

medium and monolayers allowed to develop by incubation in a carbon

dioxide atmosphere (5% CC>2 in air, ^ 9 5 % relative humidity).

Cytopathic effect was monitored daily until two consecutive days'

readings were identical. Using the statistical method of Reed and

Muench (1938) the dilution required to infect 50% of the cultures was

calculated. The titre was expressed as the 50% tissue culture dose

(TCID50) cm"3 .

Plaque Assay - two methods were used: for the monolayer method2(Hsuing and Melnick, 1955) cultures of cells were grown in 80cm

flasks. The spent medium was removed, the monolayer washed once with3serum-free maintenance medium and the virus inoculated (<T2cm virus

per monolayer depending upon the type of sample being tested). After

adsorption at 37°C for 1-1.5 hours, the monolayer was overlaid with an3agar mixture (Appendix C; 25cm per flask) and the gel allowed to

set at room temperature in a shaded position. When the agar was set,

the flasks were inverted and the cultures incubated at 37°C. Plaque

development was monitored daily until cell cultures degenerated. The

suspended cell assay was based upon that described by Cooper (1967) as

modified by Slade (1978). Cells were prepared as a suspension of7 - 3 310 cm m growth medium. These were mixed (2cm ) with the

3sample (•< 2cm depending upon the type of sample) and added to 310cm agar mixture (Appendix C) . The mixture was gently agitated

and poured into a bacteriological grade plastic petri dish (90mm

diameter, Sterilin) before the agar set. Once the gel had hardened

the plate was inverted and incubated at 37°C in a carbon dioxide

atmosphere (5% CO2 in air with>95% relative humidity). Plaque

development was monitored daily until the cell culture degenerated.

Sample Collection

(a) River Waters - polypropylene containers of 10 litres capacity and

stainless steel vessels of 20 litres capacity were used. Water

was collected by dip sampling from the middle of the river flow

in order to avoid laminar effects at the bank which could

possibly result in unrepresentative samples. Normally samples

were processed the same day as sampling, but where this was not

possible they were stored overnight at 4°C.

(b) Tap waters and groundwaters - the same containers were filled

aseptically from taps at treatment works or at consumer

premises. The vessels contained sodium thiosulphate (final

concentration of 5mg 1 to remove any residual chlorine which

may have been present in the sample.

(c) Wastewaters were either taken as a dip sample or as aliquots from

a 24 hour composite sample. In either case the total sample3 :.volume was 20cm .

(d) Large volume treated water - samples of 100-1,000 litres of

treated water were processed in the field using a portable virus

concentrator (Figure 7) only the filters being returned to the

laboratory for further processing. Such an apparatus overcomes

the logistical problem of transporting large volumes over long

distances.

ACD/CAT10NS

''"^PROPORTIONATING PUMP

MAINS SUPPLY►-oWATER METER

7 H i H— D —

MIXINGCHAMBER

FILTERS WASTE

THIOSULPHATE

FIGURE 7. LARGE VOLUME FIELD CONCENTRATOR

Filters

Several types of filter were evaluated as virus adsorbents for the

concentration of viruses from water samples: cellulose nitrate

(Millipore, 293mm diameter, type HAWP-293-25, nominal porosity 0.4^u)

protected by a glass-fibre pre-filter (Millipore, type AP15),

epoxy-bound glass-fibre filter tube of nominal 8p porosity (Whatman,

type 80 in gamma-12 housing) and epoxy-bound glass-fibre filter tube

of nominal 8p porosity (Balston, either type 100-12-C in type 92

housing with a 6.3cm filter tube or type 100-25-C in type 45 housing

with a 19cm tube). All filters were sterilised in their respective

housings by autoclaving at 15 lb pressure for 15 minutes.

Concentration of Water Samples

The system employed was a two-step concentration procedure involving

an adsorption-elution stage, using electronegative filters, followed

by an organic flocculation step to further reduce the concentrate

volume. Using such a method it is possible to reduce a volume of31,000 litres to 20cm .

(a) Adsorption-elution step - the adsorptive matrix for all field

samples (except wastewaters) was the Balston glass-fibre filter

tube. Samples were conditioned to pH 3.5 by the addition of IN

hydrochloric acid and aluminium chloride added to a final-4concentration of 5 x 10 M. The treated water was passed

. through the filter (outside to inside flow to prevent filter

collapse) under pressure (-c.50 psi air line). Air was allowed

through the filter after all the water had been filtered.3Adsorbed viruses were recovered by passing through 400cm of

beef extract solution (3% w/v, pH 9.5, Oxoid Lablemco L29)

through the filter in the same direction. The eluted viruses

were then concentrated by flocculation.

Organic flocculation (Katzenelson, Fattal and Hostovesky, 1976) -

the beef extract eluate was flocculated by the addition of IN

hydrochloric acid to achieve a pH of 3.5 and the resultant

precipitate was allowed to coagulate at room temperature for 10

minutes. The floe was recovered by centrifugation (Sorvall RC-5B

centrifuge with type GS-3 rotor, 4,500 xg, 30 minutes, 4°C) and

the pellet was resuspended in sodium dihydrogen phosphate3solution (0.15M, pH 9.2) to a final volume of 10-20cm . The

floe usually required about 30 minutes to dissolve fully in the

buffer.

The concentration of water samples of greater than 100 litres

capacity was carried out using the field apparatus fitted with

the larger Balston filter, two such filters normally being used

in parallel. Using such an apparatus (based ,on that described by

Wallis, Homma and Melnick, 1972) it is possible to condition and

filter the water in the field and return filters with any

adsorbed viruses to the laboratory for further treatment as

described above. All concentrates were stored at -20°C.

Assay of Field Samples - Wastewaters

Wastewater samples were not concentrated prior to assay. The sample

was either tested on the day obtained or stored at -20°C. Once a

sample had been thawed it was not refrozen, any excess to the test

being discarded. All wastewaters were examined by the direct

inoculation method of Buras (1974) using the BGM suspended cell plaque3assay system with 2cm of untreated sample being included in the

assay medium (Appendix C). Plaque development was monitored daily and

subcultures of virus isolates made as necessary.

Assay of Field Samples - Other Waters

All other waters were assayed as concentrates using the BGM suspended

cell plaque assay method. All of the concentrate was assayed over

five plates. Subcultures of plaques were made as required.

Identification of Field Isolates

Plaques formed in the suspended cell assay system were subcultured by

removing a plug of agar (using a Pasteur pipette) from the edge of the

plaque and inoculating into a tube culture. Such subcultures were

incubated at 37°C, either in a static position or on a roller drum,

until the cell sheet had been destroyed by the virus isolate. If,

after 7 days incubation, no cytopathic effect was evident, the culture3was frozen-thawed once and an aliquot (0.5cm ) was inoculated into a

fresh culture. If after a further 7 days there was still no effect,

the isolate was scored as negative and discarded. All cultures

showing cytopathic effect were frozen and thawed once, clarified by

centrifugation at 3,000 xg for 10 minutes and the supernates stored at

- 20°C prior to identification of virus serotypes.

Identification of the isolate was carried out by mixing 25 yul of a-310 dilution of the virus (in serum-free medium) with 25 jil of a

1:25 dilution of a commercial preparation of specific antiserum

(Micro-biological Associates, anti-viral neutralising antisera

prepared in rabbits). Neutralisation of the virus was allowed to

occur by incubation at 37°C in a microtitre well.' At the end of this4time cells were added at the rate of 5 x 10 per well in 100 yil

growth medium. The monolayers were allowed to form at 37°C in a

carbon dioxide atmosphere (5% CO^ in air, >95% relative humidity)

and the . development of cytopathic effect monitored. Where

neutralisation occurred, no cytopathic effect developed and the virus

type was assumed to be that of the antiserum effecting neutralisation.

Bacteriological Analyses

These were conducted by the Bacteriology Laboratory at Finham Regional

Laboratory, STWA using the recommendations of Report 71 (HMSO, 1983).

Coliphage Determinations

Levels of coliphage infecting two strains of Escherichia coli

(E; coli) designated as W3110 and ED 391 strains (Seeley and Primrose,

1980) were determined using the method described by Balluz, Jones and3Butler (1977). Samples of up to 2cm per plate were examined and

levels per litre obtained by extrapolation.

Chemical Analyses

These were carried out by STWA laboratory staff using standard

Authority procedures (HMSO, 1980 onwards).

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SECTION 3A s LABORATORY INVESTIGATIONS - RESULTS

Efficiency of the Concentration Procedure

Waters used in the evaluation were a tap water derived from a river

source and subjected to full conventional treatment (Strensham water

works sampled at the Regional Laboratory, Coventry) and a river water

taken approximately 2km downstream of a sewage works discharge at

Coventry. The characteristics of the water samples are summarised in

Table 10.

TABLE 10 Physicochemical Characteristics of Test Waters

Electroconductivity us cm”3 pH 'Total Hardness mg 1“1 Ca Alkalinity mg I-1 Ca Total Organic Carbon mg 1”* C Chloride mg I”"-*- Cl Nitrate mg I--*- N Suspended Solids mg 1“ -. Temperature °C Adsorption pH * mean + std. dev.

Tap Water River Water

407 (62)* 1,197 (126)7.8 (0 .2 ) 7.4 (0 .1 )171 (34) 307 (33)103 (23) 915 (2 2 )2.4 (0.4) 9.4 (2.3)39 (7) 170 (29)

4.9 (0 .8 ) 13.9 (2.7)Not Done 7.2 (3.4)

11.0 (1.5) 14.6 (1 .8 )3.4 (0 .2 ) 3.4 (0 .2 )

Poliovirus type 2 was added to each water sample at 450 pfu in 20

litres of tap water and 1,750 pfu in five litres of river water. In

the latter case, the higher input level was used to overcome effects

associated with the indigenous viruses in the sample. All waters were3concentrated to 20cm and assayed by the monolayer plaque method

using Vero cells as the host system. Three filter types, in the

presence and absence of aluminium chloride, were assessed.

TABLE

11 The

Influence

of Differ

ent

Conc

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n Proc

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es

on the

Recovery

of Po

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Type

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Table 11 shows that viral recovery from both tap and river waters was

significantly improved if aluminium cations were present when using

Balston and Whatman filters although the cation did not influence

recovery by Millipore filtration. The variation in recoveries is

attributed, in part, to the different batches of filter materials used

in the study.

Based on cost and the ease of handling, the Balston glass-fibre filter

tube system was adopted as the adsorptive material for all

concentration procedures.

Evaluation of the Field - Concentrator

Two filters (Figure 7) were used in parallel to concentrate six

samples of tap water (840-1,000 litres; Table 10) seeded with

approximately 43 pfu of poliovirus type 2. Virus was recovered with

an efficiency of 43 + 19%, levels similar to those achieved in the

small scale filters. The procedure was evidently capable of detecting

1 pfu in 100 litres of tap water, making the method suitable for

processing large volumes (Table 25).

Evaluation of Flocculation

The organic flocculation stage of the concentration procedure was

examined by seeding monodispersed preparations of viruses into 200

cm volumes of beef extract solution (3% w/v, pH 9.5) and recovering

virus by flocculation as before. Assays were conducted using the Vero

monolayer plaque method. Table 12 shows that recoveries ranged widely

(7-98%) both between different virus types and between different

strains of the same virus. However, recovery values for one strain of

poliovirus type 2 were satisfactorily reproducible (starred entry,

Table 12) thus allowing the method to be adopted for routine use in

the recovery of indigenous viruses from environmental samples.

TABLE 12 The Value of Organic Flocculation (3% w/v Beef Extract

Solution, pH 3.5)

Virus

Polio 1 Polio 2* Polio 2 Polio 2 Polio 2 Polio 2 Cox B2 Cox B3 Cox B4 Cox B5 Echo 1 * ten Sources:

Source

LabLabRaw SludgeRiverRiverRiverLabLabLabLabLab

Input pfu

101171026

10555 64 5756 33 41

% Recovered

40;40 28+9 40;50 50 ;12 45; 40 35; 42 22; 6 98 ;98 7 ;11 9:3 7 ;3

replicates; all other tests as duplicates.Lab - strain subjected to many laboratory passages; River-sludge strains isolated from environmental samples with limited cell passage histories.

Evaluation of the Microtitration Assay

In order to assess the variability of the procedure, four viruses were

each titrated five times in the system using BGM as the host cell.

The results, summarised in Table 13, show that variation of *<^0.5 -3TCID_.cm (log..) can be encountered (mean 0.35 log.-). On5U XU XU

this basis, only titres differing by *“1 log.. TCID cm have1 U oUbeen considered as being significant. The dilution series and volumes

used in this test result in a minimum detection level of 1.6

-3TCID^cm and where virus material has been scored as NC after

titration this implies that levels were below this detection limit.

TABLE 13 Reproducibility of the Microtitration Assay

Virus Mean Titre (Std. dev.)*

Polio 1 Polio 2 Cox B3 Echo 11 -3

7.34 (0.42) 8.28 (0.18) 8.15 (0.51) 7.30 (0.27)

TCID_ cm 50 assayed in BGM cells.

Susceptibility of Selected Cell Lines

3Virus (0.1 cm ) was inoculated into tube cultures of each selected

cell line two days after culture initiation. Infected cells were

appearance of cytopathic effects (cpe). When the whole cell sheet had

unclarified medium inoculated into fresh tube cultures of the same

cell line. The procedure was repeated for a third time and the final

virus harvest was clarified by centrifugation before storage at

incubation, the culture was, nevertheless, treated in a like manner

and clarified medium from the third passage retained. All final

passage material, whether from cpe positive or negative cultures, was

assayed in the homologous cell line by the microtitration method

(Table 14).

incubated at 37°C in the static mode and monitored daily for the

3degenerated the cultures were freeze-thawed once and 0 .1cm of the

-20°C. In the event of no cpe being evident after seven days

TABLE

14 Infectious

Titres

of Cy

topa

thog

enic

Viruses

in Di

fferent

Cell

Line

s

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Cytopathic effect was noted for all viruses passaged in the simian

cell lines and also in the human FL line, but some failed to produce

cpe in other cell lines. For example, echovirus 16 did not cause cpe

in D-6 and CC cells, echovirus 30 was apparently negative in 1-407,

D-6 and CC cells while RD cells did not appear to support

cytopathogenic replication of some of the coxsackie B viruses.

However, some of these samples when titrated in BGM cells caused the

development of cytopathic effect (Table 15) which indicated that viral

replication had occurred in the original cell despite the absence of

cytopathic effect. It was also noted that the infectious titres of

some viruses, originally scored as non-cytopathogenic (NC) in certain

cells, were the same as the viruses originally passaged in BGM cells

in which they were cytopathogenic. Finally, some viruses which were

non-cytopathogenic were also, apparently, non-cytopathogenic when

tested in BGM cells. Whether or not such samples contained infectious

virus was not further tested.

TABLE 15 Titration of Viruses in BGM cells After Non-cytopathogenic

Replication in Certain Other Cells

1 2 Virus NC in NC in BGM BGM homologous

Cox B4 RD 7.1 3 7.8Cox B5 RD 7.4 8.2Cox B6 RD 6.8 7.9

CC 6.1 7.1Echo 17 RD 5.8 8.6Echo 24 CC 6.2 5.2Echo 30 1-407 7.8 7.4

CC 7.4 7.41 NC virus assayed in BGM2 BGM passaged virus assayed in BGM3 logio TCID5Qcm” 3

In general, virus titres tended to be lowest in CC and MA cells while

BGM, CL and RD cells tended to give the highest. Only in two cases

(echoviruses 24 and 28) did titres in BGM fall substantially lower

than those in other cell lines (Table 14) . In a few instances the

differences in titres were marked. For example, the titre of4coxsackie A7 virus was about 10 lower in D-6 cells than in BGM and4the echovirus 32 titre was 10 higher in BGM than in MA cells.

Polioviruses were similar in most cells, but titres in LLC and CC

"cells tended to be somewhat lower.

Coxsackie A viruses showed some variation with the titres in D-6 cells

being substantially lower than in other cells. Coxsackie B viruses

were similar in many of the cells, but RD failed to show cpe with

three types. In MA cells this group of viruses, with the exception of6 0_________3type B3, did not exceed titres of 10 * TCID^qCiii . Echoviruses

usually gave the highest titres in RD cells although in most cases

these levels were not too different from those with BGM cells. The

main exceptions were echoviruses 24 and 28 which were

<C10^*^TCID cm ^ lower in BGM.50

The effect of cell type on "the recovery of naturally occurring

enteroviruses by plaquing will be considered later.

Evaluation of Focal and Quantal Assays — Cell Presentation

Cell cultures in the form of tube cultures, plate monolayers and

suspension cultures were compared for the evaluation of naturally

occurring enteroviruses in river water concentrates. The waters were

from the rivers Avon and Sowe sampled downstream from major wastewater

effluent discharges (25 km and 2 km respectively). Five litre samples

were conditioned and concentrated by the Balston filter tube method3and organic, flocculation to a final volume of 25cm . Aliquots of

the concentrates were assayed by the three methods with three cell

lines.

Virus recovery was best in BGM cells especially when presented in the

suspended cell mode where the maximum level reached 620 pfu 1 ^

(Table 16). The MPN method detected viruses in all but two of the

samples, with the highest recovery being 585 MPN 1 Viruses were

also detected by monolayer plaquing, but the numbers were low, the

maximum level being 25 pfu 1

RD cells were generally less sensitive than BGM cells by all methods

tested and no one method always detected virus. The highest

sensitivity was by the suspended cell method, but even this system was

sometimes less sensitive than the other two procedures.

Virus titres in Vero cells were the lowest regardless of the mode of

cell presentation, in particular no sample was positive by the MPN

method (Table 16).

The suspended cell procedure was not apparently uniformly suitable

with other cell cultures when determining virus levels in effluents

(Table 17) . The highest frequency of positive samples was found in

BGM.cells and, in addition, the BGM system detected 79 plaques which

accounted for 50% of all plaques detected in the whole experiment.

Only once did BGM cells fail to detect viruses when present with

another cell line and levels in BGM were only lower than those i-n

TABLE

16 Titres

of In

fectious

Virus

in River

Water

Conc

entr

ates

uw Q Q c o r - o a Q L D i —i r - Z Z cm Z JZ H

s p i n c o c n p p p p i n 2 Z Z Z Z

p p p p p p p p p pz z z z z z z z z z

SI g

cm P lo o o Z oo H r -

iH cn rH

P P P p cn Z Z Z Z

p a ) M n p ( n p i n c o > Z CN Z Z

P P o o p c o P c o ^ c c o Z Z Z Z cri o

CM

£

ml

C M t n i n o o c o i n o t j ' C M^ ^ CM H H nr i in id cn in

CM CM CO CO IT) CM

p pz z cm H in co co h in oo h

H in

eCD

4Jcn>

cnw

urH cd CDrH cn >CD cn •HU <

O'! O'! *—• »—». 0\i t-" r-~ cr> n- i'- \ W r* i" W I—I H H \ \ CM CNrH rH rH CM CN rH rHW \ H H \ \ r - cn cn \ \ co orH CM CM t " r * H CM

CN PZ

CDCTi

<T> <T>r* n~ \ \ CM CM

\ \oCM CM

rHI>-lCD

rH X2 VD g rH D C

rHIrH

3rH UHI &

rH >1 3 cd kh cn a wcd> icd cn cn cd

CD3trcd

rHCXCD

rHX icd

— X3CTi O T- H \ & CMrH 4J\ cn r - O CM S

CD 3 c r rH cd rH

rH <D Pl, Ov-i >o CD CD > i ’V cd C

• aCD 4J O CD 4J CD n3

CDO j CD

C C CD C CD C0 0 * 0 5 0 > t> O > O >C < C Q < W < < W < C O

II II I! IIC CD C CDO * O * Zt> O > O Pj d O

S W

TABLE

17 Sensit

ivit

y of

Diff

erent

Cell

Lines

in the

Suspended

Cell

Plaque

Alssay

to

Infe

ctio

us

Viruses

in

Effl

uent

s

O C N H C O H O O O O O 03 ID

O O O H O O O O O H O C N C O

O O H O O O O C O O H H V O V D►Jl co

o o c o c o *i<h o h H o rrH CD

*<uc-H

U ^ H h W O N O O l C O ^ f O m r l ffl| H H H 03

rHa>u U O O H C O O O O C N H O O VX>U| CO

VDIQ ^ r o o o H o o o o o H v o rCN

r-OrrIH

o o r n o o o o o o o o n c T i

SI COCNVOCOOOOOHCNCNCOCOco r»

1-31 o o o o o o o o o o o o o Pul

<D4J(0o

&e(0w

r o r o n n M n f i n o o c o c o f f iID O VD N CO 'C ' f r - o

H H CN CN CN HCO CN H CN

CO(1)HCl£<0COCO

CDie

> u•ri o4-> H

(0 •HH CO D(0 O cw4->A Cl Cl

£ df> *

other cell lines on two occasions. RD cells detected 33 plaques in

total (21% of all plaques) with 8/11 samples being positive. FL cells

failed to detect any viruses in any samples while all other cell lines

gave only low plaque numbers and low frequency of positive samples;

On a single occasion the sample was negative by all cell lines.

Cell sensitivity may also be determined by the time taken for plaques

to appear and by plaque size. The maximum number of plaques detected

in BGM cells was after five days incubation, whereas in RD cells only

three days were required, although fewer were detected. All other

cell lines permitted maximum plaque counts between three and six days

incubation, but plaque definition was best in. BGM, CL and RD cells

respectively (Figure 8 ).

BGM80— iSIMIANHUMAN

aoo 40 —

RD

CL

MALLC

1-407FL

VERO0—1

DAYS

FIGURE 8. TIME OF APPEARANCE OF MAXIMUM PLAQUE COUNTS IN DIFFERENT CELL LINES.

Sensitivity of Cell Culture Types to Different Enterovirus Serotypes

The serotypes of the polio and coxsackie B virus groups detected in

the effluents are listed in Table 18. BGM cells detected six of the

nine serotypes, but failed to detect coxsackie B2, B5 and B6 which

were detected by LLC, MA and LLC cells respectively. On the single

occasion that BGM failed to reveal plaques (28.3), when the sample was

positive by other cells, the serotypes present were all polioviruses.

RD cells only detected polioviruses. Poliovirus type 2 was the most

commonly identified serotype accounting for one third of all

identifications. LLC cells detected several of the coxsackie B

viruses, but none of the polioviruses despite the presence of these

viruses in other cell lines. Overall, 101 isolates were identified,

6 8% being polioviruses and coxsackie B3 virus accounting for a further

18%.

The Sensitivity of Mixed Cell Cultures

7A sample of effluent was assayed in BGM and RD cells alone (2 x 107cells per plate) or as a mixture (1 x 10 cells of each type per

plate). All plaques formed were subcultured into tube cultures of

homologous cells as appropriate; the plaques from the mixed cell assay

were subcultured into both cell lines.

TABLE

18 Serotypes

in Effluent

Detected

by Ten

Cell

Line

s(0 cc <n ^ m ro h in m h +J CN i-t . o

rH

CDain rH rH

CQ(03u•rH><11•Ho<0inK8

ro

CN

<0inin<cd•H•acd4Jocd

4-1CD•OCDC

ro cn corH

a)a cn

rH co

oCN

H VO CN

•OCD•HUH•rl4-1CCDT3•H<Dd8 <T\

CN

in3u•H>o•H

rHoa

CDd•Hdl

CDO

CN n H n cn n inrH

rH CN CN

CN co CO

g g

r~o IDI IH D

mdo•H

4-1coO•HIH•rH4-1dCD•o•rlIHoUCD

X IE3d

TABLE 19 Mixed Cell Assay of Effluent

(a) Plaque N u m b e r s BGM assayRD assay BGM-RD assay

(b) Serotypes:

Assay System

BGM

RDBGM-RD

Isolate No.

12345 1 1

2

3

4

5

6 7

Subcultured in

BGMBGMBGMBGMBGMRDBGMRDBGMRDBGMRDBGMRDBGMRDBGMRDBGMRD

Serotype

Cox B5 Polio 3 Cox B2 Polio 2 Cox B5 Polio 3 Cox B5 NC*Cox B4 NCPolio 2 Polio 2 Cox B5 NCPolio 3 Polio 3 Cox B5 NCCox B5 NC

Table 19 shows that although virus levels were low, RD cells were less

sensitive than BGM. However, the mixed cell population had the same

sensitivity as BGM alone. Furthermore, all but two of the virus

isolates from the mixed cell assay only grew in BGM cells. The two

which grew in RD cells as well were both poliovirus serotypes. Where

cpe was detected after subculture in RD cells the serotype identified

was the same as in the BGM cells. The results also confirm the

failure to detect coxsackieviruses B4 and B5 in RD cells as previously

shown in Table 14.

Optimisation of the Suspended Cell Assay System

The effect of a range of test conditions was evaluated using BGM cells

in a suspended cell plaque assay.

a) Cell Concentration

Poliovirus type 1 was assayed using cells at concentrations7 7ranging from 1 x 10 to 5 x 10 per plate. Plaques were

counted and measured after three days incubation at 37°C.

7The highest number of plaques required at least 3 x 10 cells7(Figure 9). Below 2 x 10 cells per plate there were

significantly fewer plaques (P = 0.05) these plaques being more

diffuse and difficult to evaluate. All other concentrations of

cells produced well-defined plaques. Furthermore, variation in

the plaque size was not so great when the higher levels of cells

were used (Figure 10).

b) Adsorption Time

The optimum adsorption period before infected cells were mixed/

with‘ agar medium was determined. Cell suspensions were washed

three times in serum-free medium and allowed to interact with the

virus inoculum (poliovirus type 1) for up to one hour at 37°C

before being suspended in medium and incubated in the assay.

Plaques were counted after three days incubation at 37°C.

Adsorption time did not significantly influence the numbers or

quality of plaques formed (Figure 11).

<Uls

1 2 3 4 5

C ELL CO NCN.

X 1 0r 7

FIGURE 9. POLIOVIRUS 1 PLAQUING IN SUSPENDED CELL CULTURES AT DIFFERENT

CELL CONCENTRATIONS.

4 0 — ,

1 X 1 0

2 X 1 0

3 X 1 0

4 X 1 0

5 X 1 0

T W12| 18|

PLAQUE DIAMETER (MM)

FIGURE 10. EFFECT OF CELL CONCENTRATION ON POLIOVIRUS 1

PLAQUE SIZE

30— ,

PFU 15 — f

0 —

0 30 60ADSORPTION TIME (MIN)

FIGURE 11. EFFECT OF ADSORPTION TIME ON POLIOVIRUS 1 PLAQUING.

c) Time of Staining

In order to determine the effect of time of staining on the3numbers of plaques detected samples of effluent (2cm per

plate, five replicates per treatment) were assayed with the stain

incorporated at time of test initiation or applied as a second

overlay at 1, 2 or 3 days afterwards. Plaques were counted daily

on the tg test and 24 hours after application of the second

overlay. Table 20 indicates that although there may be a slight

advantage in applying stain later in the test the difference is

not significant when compared with the results obtained when dye

was added at the start of the test.

d) Gelling Agent ,

A range of agar-based gelling agents was used at a final

concentration of 1 .2 % to determine the effect on plaque formation

TABLE

20 Effect

of Time

of Staining

on Detection

of Viruses

in Ef

flue

nts

nto ^ ^• • • • • •

o o o o o o

ro

cr>CM

•O0■rlrHa§c•H(0•Pw> 10Q

CN

CNCN

CN

r-~CN

inCN

roCN

T}<in

n M D O H • • • • • •CN CN CN CN CN CN

O 00 O'! ID CTl CN • • • • • •n ro cn id oo

CN CO N 1 CN ro

> 10«acO

>0•CTD4JCOD«WP a

C00S

CQ0rHP aE0W

0rHPae0CO

by laboratory virus strains and field samples containing

indigenous viruses.

Plaque numbers, resulting from laboratory virus inocula, were

similar under most conditions (Table 21) although with some

viruses numbers could not be determined (UR) because of the poor

definition of the plaques. For example, coxsackievirus A7 when

assayed in the presence of Noble agar and agarose, and six

echoviruses when assayed with Number 1 agar.

TABLE 21 Effect of - Gelling Agents on Plaguing Efficiency

Virus Type Mean pfu Detected (Std. dev; )

Bacto Noble No. 1 No. 3 Agarose

Polio 1 40 (2 ) 41 (6 ) 42 (2 ) 45 (5) 41 (3)Polio 2 41 (3) 36 (3) 37 (4) 38 (2 ) 43 (6 )Polio 3 18 (2 ) 19 (4) 16 (6 ) 15 (6 ) 14 (3)Cox A7 34 (5) UR* 20 (5) 43 (13) URCox A9 53 (7) 54 (6 ) 54 (2 ) 51 (3) 50 (8 )Cox B3 23 (2 ) 19 (6 ) 18 (3) 21 (5) NT**Echo 1 10 (1 ) 11 (3) 8 (3) 11 (2 ) 11 (5)Echo 5 39 (6 ) 36 (4) UR 36 (8 ) NTEcho 7 167 (7) UR UR 141 (26) NTEcho 8 38 (9) 38 (9) UR 33 (5) NTEcho 14 35 (1) 31 (1) UR 30 (3) ' NTEcho 16 26 (8 ) 28 (3) UR 27 (6 ) NTEcho 20 25 (6 ) 23 (9) UR 21 (3) NT

UR unreadable due to poor plaque definition NT not tested

Plaque definition was best with Bacto, Number 3 and Noble agars while

plaques formed under agarose were the most difficult to read. The

effect of gelling agent choice on plaque size was marked with the

largest plaques being associated with the use of Number 3 agar and the

smallest with agarose (Figure 12).

POLIO 3 COXSACKIE B3 ECHO 120 — .

PLAQUE DIAMETER (MM)

FIGURE 12. EFFECT OF GELLING AGENTS ON PLAQUE SIZE • •• t(B=BACTO, N=NOBLE, 1 *N 0 .1 , 3 *N O ,3 , A*AGAROSE)

Examination of field samples (Table 22) . for the effect of gelling

agents on recovery of naturally occurring enteroviruses reflected the

above findings with only small differences being encountered in

numbers detected when using Bacto, Noble and Number 3 agars. In

general, however, plague numbers under Number 1 agar conditions tended

to be 2-3 times lower than with the other gelling agents. Agarose was

not used in this part of the evaluation because of the cost of the

material and of the poor results with laboratory strains in terms of

ease of test reading. Plaque definition tended to be best with Bacto

agar with the other gelling agents only being slightly more difficult

TABLE 22 Effect of Gelling Agents on Virus Detection , in Wastewaters

Sample Details Bacto Noble No. 1 n o . •:

Finham raw sewage*- 262 28 9 36Finham effluent 25 13 8 1 1Finham PAS influent 32 30 8 28Finham PF influent 26 33 17 30Finham PF effluent 26 33 17 30Balderton effluent 13 16 0 -16Crackley Point effluent 73 75 47 82Honiley effluent 24 34 22 23Balsall Common effluent 4 1 1 2 71 all Finham samples 4cm3 ; all others 8cm3 -

— 2 pfu detected

e) Evaluation of Serum

Bovine and equine sera at different concentrations were assessed

in the BGM plaque assay system using poliovirus type 1. Plaques

were read after three days incubation at 37°C.

60 — I FCS D -F C S

uj 60DCSNCS

-=>u.o. 60 — H I-H SHS

% SERUM

FIGURE 13. EFFECT OF SERUM ON POLIOVIRUS 1 PLAQUING.

Serum is a major influence on the plaquing efficiency of

poliovirus type 1 (Figure 13) . In all cases, as serum levels

increased there was a decrease in the numbers of plagues formed.

The sole exception was foetal bovine serum which had been

previously dialysed (D-PCS). However, the degree of effect

varied with the serum used. Donor calf serum (DCS) proved toxic

beyond the 3% level with the plaque numbers at that level only

being about half of those in the untreated control. The effect

with normal horse serum (HS) and after heat-inactivation (HI-HS)

was less marked, but still showed a substantial reduction in

plaque numbers. Newborn calf serum (NCS) was not toxic at the

higher levels, but did contain virus inhibiting materials.

Foetal bovine serum (FCS) had a slight effect, but a

concentration of 2% was not significantly different from the

dialysed serum (D-FCS).

Evaluation of a Range of Different Chemical Additives

(i) DEAE-Dextran was incorporated into the BGM suspended cell

assay (containing Bacto agar) at levels up to 250 p g -3cm , but was without obvious effect on the plaque

numbers and sizes formed by the different viruses (Figures

14 and 15).

-3 .(ii) Semicarbazide at levels of up to 250 jjg cm m the assay

medium was also without much effect on plaquing efficiency

(Figure 16). In two of the cases there was a slight

downward trend in the number of plaques formed as the

FREQ

UEN

CY

AT

EACH

D

IAM

ETE

R

40.POLIO 1

20.

0 —J-EE-3- J 31 ■ at "I I-I- x-±» 1

4 0 —,

20 —

PFU0 — >

COXSACKIE A9

I * I * J x I -1 I I- J

4 0 —.

20 —

0—J

ECHO 1

1100 200

-3pG CM DEAE-DEXTRAN

FIGURE 14. EFFECT OF DEAE-DEXTRAN ON PLAQUE NUMBERS

FIGURE 15. EFFECT OF DEAE-DEXTRAN ON COXSACKIEVIRUS A9 PLAQUE SIZE ( p c c M 3)

2 5 —

2 5 —

200

1 i L i _I I II I I LI "IT II I' IX 8 16

225

I I I I I I I I I I I I IT T 8 16

2 5 0

11111 m i nil 11 |i hi 111 LI 11111 U.o 16 16

DIAMETER (MM)

PFU

40—1

20H

6 0 —,

p<5LI0 1

— :— T . T ^ TrfibH-i

3 0 H

PFU

0 — 1

rtCOX A9

2 5ECHO 1

0

1 50

PG CM 3 SEMICARBAZIDE

FIGURE 16. EFFECT OF SEMICARBAZIDE ON PLAQUE NUMBERS

FIGURE 17. EFFECT OF SEMICARBAZIDE ON COXSACKIEVIRUS A9 PLAQUE SIZE.

20

2 0 —i

25 5 0 7 5

100 125 ■ 1 5 0 175

■J I JL I -fc.20—, 200 2 25 2 5 0

i in n r III II II iinnr I I I I I I I TiTrmr III III m i n i TITTTIT8 16 8 16 8 16 8 16

DIAMETER (MM)

concentration of the semicarbazide was increased, but no

effect on plaque size (Figure 17).

-3(lii) Low levels of magnesium chloride (500 jig cm ) had some

effect on the plaquing efficiency of poliovirus type 1 and

coxsackie virus A9 followed by a recovery to untreated

levels before the number of pfu was affected by increasing

concentration of the chemical (Figure 18) . The effect of

magnesium chloride on echovirus type 1 was more marked with

an initial enhancement to a maximum level of pfu at 2,500-3

jig cm followed by a rapid fall off. In the case of

this virus there was no initial depression of the plaque

numbers when low levels of the chemical was used.

The effect of magnesium chloride was also examined in the

assay of two wastewater effluent samples. Figure 19 shows

that despite an initial small increase in plaquing

efficiency high levels of magnesium chloride were

detrimental to plaque formation.

—3TABLE 23 Effect of Magnesium Chloride (2,000 jig cm ) on Plaque

Numbers and Serotypes in Wastewater Effluent

(a) Plaque Numbers pfu 10 cm~^- Mg Clp + Mg Cl?

Kilburn FE 331 350Curborough FE 153 147Whittington FE 131 124

(b) Serotypes (Whittington FE) No; when agar contained- Mg CIq + Mg Cl?

Cox B3 15 10Cox B4 2 7Cox B5 2 2

% OF

U

NTR

EATE

D

CO

NTR

OL

FIGURE 18. EFFECT OF MAGNESIUM CHLORIDE ON PLAQUING BY THREE VIRUSES.

150

100

5 0

COX A9POLIO 1 ECHO 1

105 5105 10PG C M 3 MGCL2 X1<5T3

120

Z 80

411

• BASLOW4 0u.OHATHERSAGE

5 ,0 0 0 10,000pG C M 3 MGCL2

FIGURE 19. EFFECT OF MAGNESIUM CHLORIDE ON VIRUS DETECTION

IN EFFLUENTS

-3Magnesium chloride, used at 2,000 ug cm . , also had no

appreciable effect on plaque numbers detected in three

not, the numbers of each serotype were slightly different.

In particular, more coxsackievirus B3 was identified when

plaques were subcultured from test plates with magnesium

chloride and conversely more coxsackievirus B4 if no cation

was present (Table 23).

(iv) The influence of protamine • sulphate (salmine) was assessed

(Figure 20) indicating that no enhancement of plaquing

efficiency could be detected although there was some

evidence that increasing levels of the compound may have

resulted in a slight decrease in plaque numbers of some

strains.

effluent samples. However, although the range of serotypes

detected was the same whether the cation was present or

4 0 —,p o l io i

o—>4 0 —,

ECHO 1

r0 5 0 0

JJG CM 3 PROTAMINE SULPHATE

1000

FIGURE 20. EFFECT OF PROTAMINE SULPHATE ON PLAQUING

(v) Incorporation of 5-iodo-2’-deoxyuridine (IDU) into the

medium during the microtitration assay of two laboratory

virus strains did not adversely affect the appearance of

cpe or the titres measured (Figure 21). However, when used

in the suspended cell plaque assay of naturally occurring

enteroviruses in wastewater effluents (Table 24),

increasing levels of IDU adversely affected plaque numbers

showing a direct relationship between numbers detected and

the concentration of the IDU (Figure 22).

TABLE 24 Effect of S-Iodo-^'—Deoxyuridine on Detection of Viruses in

Effluents

Sample Details Total pfu (% of untreated) at ug cm'-3

0 50 100 150

Ackleton 16 (1 0 0 ) 12 (75) 5 (31) 3 (19)Beckbury 23 (1 0 0 ) 13 (57) 14 (61) 2 (9)Stanley Downton (a) 56 (1 0 0 ) 44 (79) 17 (30) 3 (5)Stanley Downton (b) 75 (1 0 0 ) 61 (81) 26 (35) 15 (2 0 )Long Lawton 152 (1 0 0 ) 139 (92) 63 (42) 10 (7)Woolston 13 (1 0 0 ) 11 (85) 3 (23) 0 (0 )Brinklow 39 (1 0 0 ) 32 (82) 12 (31) 6 (15)Mean % 100 79 36 11Std. dev. 11 12 8

oinOOI-

8—,

4 —

0 —

COX B6 -£ r- i

■x~.— =COX A7

50

.-3100 150

fJG CM IODODEOXYURIDINE

FIGURE 21 EFFECT OF IDU ON VIRUS TITRES.

100

o

5 0

50 100 1 5 0

PG CM3 IODODEOXYURIDINE

FIGURE 22 . EFFECT OF IDU ON VIRUS DETECTION IN EFFLUENTS

SECTION 3B : LABORATORY INVESTIGATIONS - DISCUSSION

One of the main concerns when monitoring virus levels in the

environment has been the negative results obtained which may reflect

the inadequacies of the methods used rather than the actual absence of

viruses in the samples. Such findings may lead to a false sense of

security when attempting to detect low levels of virus in large

volumes of water, particularly drinking water, especially since the

true levels of viruses for the initiation of infection/disease have

not been satisfactorily established. It is obvious that if the

results obtained in monitoring programmes are to be credible, then the

methods used must be the most effective available. The present work

has derived a simple plaque assay for virus determination and has

examined the effect of a number of variations on the method efficiency.

Filter Evaluations

The observation that most viruses could be adsorbed to filter

materials under certain conditions opened up a means of rapidly

concentrating viruses from large volumes of water (Cliver, 1965).

Indeed, the procedure lends itself readily to large scale application

with flow-through devices enabling volumes of many thousands of litres

to be processed (Wallis, Homma and Melnick, 1972; Hill eit al, 1974;

Figure 7). The range of filter materials which can be used

successfully as virus adsorbents is extensive (Primrose, Seeley and

Logan, 1981) these being grouped into two classes depending upon their

surface charge, that is, whether they possess a nett negative or a

nett positive charge. However, many of the factors affecting both

virus adsorption to and elution from such filters are common to both

groups (Shields and Farrah, 1983;, Sobsey and Glass, 1984; Guttman-Bass

and Catalano-Sherman, 1985; Sobsey and Cromeans, 1985; Sobsey and

Hickey, 1985; Sobsey £t al, 1985).

Virus adsorption occurs most efficiently under pH and ionic conditions

where the nett charges of the virus and filter are either opposite in

sign or are small in magnitude, that is, near their isoelectric points

(Sobsey and Jones, 1979). Elution of adsorbed virus can be readily

achieved by a change in pH resulting in the nett charges of the virus

and filter becoming the same with subsequent repulsion. Until

recently only filter media classed as electronegative have been used

in virus concentration studies, particularly those based on cellulose

nitrate and epoxy-bound glass-fibre. The development of

electropositive filters based on charge-modified diatomaceous earth

materials has somewhat simplified the procedures necessary for

efficient virus recovery from large water volumes. The present

investigation, however, restricted itself to a consideration of

electronegative filter types.

The effectiveness of the different filter types available for virus

concentration and the role of cations in promoting enhanced virus

recovery is confused. For example, Wallis and Melnick (1967) and Rao

and Labzoffsky (1969) found that cation addition to the sample to be

concentrated using cellulose nitrate filters was beneficial. Low

recoveries from such filters in the absence of cations were also

reported by Jakubowski, Hill and Clarke, (1975) and Hill et al

(1976). However, Berg, Dahling and Berman (1971) recovered several

virus types with efficiencies ranging from 2 2% to 90% using the same

filter types without cations. The results obtained in the present

study support those of Berg, Dahling and Berman (1971) where addition

of aluminimum cations when using cellulose nitrate membranes had

little effect on virus recovery.

Glass-fibre filters, particularly the Balston type, have been examined

by several workers, again with conflicting findings. The results in

Table 11 conclusively show a positive requirement for aluminium

cations if the filter is to operate as efficiently as the cellulose

nitrate alternative. Guttman-Bass et al (1985) used the Balston

filter without cation addition and succeeded in recovering up to 94%

of virus. In contrast, Jakubowski et al (1974) could only detect up

to 50% of virus while Hill et a_l (1976) found a lower recovery of 21%,

not significantly different from the present findings. Low rates of

recovery with the Balston filter in the absence of cations were also

noted by Sobsey et al (1980a) who, in addition, also found low levels

when using cellulose nitrate without cations.

Why such disparity in results should exist could be explained in

several ways:-

(1) over a period of time, filter manufacturing processes may have

been modified thus altering the adsorption characteristics of the

materials;

(2 ) water quality may influence the adsorption and elution of viruses

with organics, mainly humic acids, exerting a blocking effect on

adsorption (Guttman-Bass and Catalano-Sherman, 1985);

(3) the degree of adsorption to filters by various virus serotypes

may differ widely as has been demonstrated with virus adsorption

to soils, sediments and sludges (Gerba et al, 1980);

(4) recovery efficiencies may have been affected by the aggregated

state of the test virus preparation with many of the earlier

studies failing to use monodispersed virus suspensions (e.g.

Wallis and Melnick, 1967; Rao and Labzoffsky, 1969) and even in

more recent reports (Guttman-Bass et al, 1985);

(5) some studies with cellulose nitrate filters have not used a

protective glass-fibre prefilter while others have. Rao and

Labzoffsky (1969) found that a prefilter was capable of adsorbing

65% of virus in the presence of calcium cations with 67% of the

adsorbed virus being recovered by elution. In contrast, Payment

and Trudel (1979) using aluminium cations found only 34% of virus

adsorbing to the prefilter, the prefilter and cellulose nitrate

membrane together adsorbing ^ 99% of the seeded virus.

However, as Payment and Trudel (1979) pointed out, virus adsorbency is

not the only useful property of filters. Their flow rates, cost and

resistance to clogging are major considerations in the choice of

appropriate filter. The present study only examined three filter

systems, the Balston method used in the presence of aluminium cations

having been shown to be reliable. The Balston system has the

advantages over the cellulose nitrate membrane system of relatively

low cost, ease of handling, capability of processing large volumes of

water by the judicious choice of filter size and good recovery

efficiency. Indeed, the system is so versatile that it has been used

to simultaneously concentrate enteroviruses and Salmonella bacteria

from surface waters (Rolland and Block, 1980).

Secondary Concentration

The present study used beef extract as the sole eluant for desorbing

viruses from filters. Other materials, usually used at elevated pH

levels, have also been found to be satisfactory eluants, for example,

skimmed milk (Bitton, Feldberg and Farrah, 1979), chaotropic solutions

such as sodium trichloracetate (Farrah, Shah and Ingram, 1981) and

glycine buffers (Fields and Metcalf, 1975; Sobsey et al, 1977).

The flocculation of beef extract solution at low pH enabled

Katzenelson, Fattal and Hostovesky (1976) to show that viruses eluted

from filters adsorbed to the precipitate resulting in efficient virus

recovery when the floe was dissolved in a small volume of phosphate

buffer. Using virus preparations which had not been monodispersed,

they reported a mean recovery of 74% for poliovirus type 1 compared

with 35% when using a glycine buffer system. Kedmi and Fattal (1981)

found good recoveries of echovirus type 7 and coxsackievirus A9 with

poliovirus 1 recoveries being slightly higher (94%) than those found

by Katzenelson, Fattal and Hostovesky, (1976). Again the recoveries

in excess of 1 0 0% pointed to the failure to remove virus aggregates in

the initial virus preparation by monodispersion. Using a

monodispersed suspension of poliovirus type 1 Landry et al (1978)

recovered about 8 6% by organic flocculation of beef extract solution.

Rotavirus could be recovered with an efficiency of only 61% in a study

reported by Guttman-Bass and Armon (1983).

In a comparison of several commercially available beef extracts, Hurst

et al (1984) showed that variation according to supplier (presumably

due to different manufacturing processes) can be expected such that

recoveries ranging from 6% to 6 8% could be found with poliovirus 1 .

Similar ranges were experienced with echovirus 7 and coxsackievirus

A9. The effect of several factors on the recovery of viruses by

organic flocculation of beef extract was investigated by Sobsey et al

(1980a) who demonstrated that variations of flocculating times and

beef extract concentrations were not important. However, when beef

extract was used below 0.5% w/v some slight decrease in recovery

"efficiency was observed. More recently, the same group of workers

(Sobsey, Oglesbee and Wait, 1985) demonstrated the ability of the

method to recover hepatitis A virus with efficiencies ranging from 8 %

to 62% the best recoveries being achieved when flocculation was

carried out at pH 3.3. Again variation was noted depending upon the

source of the beef extract. Changes in manufacturing practices have

resulted in an increased number of beef extract preparations failing,

or apparently failing, to form a floe on acidification. This has been

partially overcome by the supplementing of the organic flocculation

method with inorganic flocculation using ferric chloride (Payment,

Fortin and Trudel, 1984) recoveries of poliovirus being of the order

of 82%.

The results reported in the present study (Table 12) confirm that

organic flocculation of ‘ beef extract solution is capable of

concentrating a wide range of viruses. However, it is disappointing

to note that wide inter-serotypic variations were encountered and

possibly even intra-serotypic differences. Such variation was also

noted by Hurst et al (1984) where Oxoid beef extract (the same source

as the beef extract used in the study reported here) concentrated

poliovirus 1 with 52% efficiency, echovirus 7 with 12% efficiency and

coxsackievirus A9 with 59% efficiency. Interestingly, Jakubowski

(personal communication, 1978) could not recover the latter serotype

by beef extract flocculation.

Despite the limitations reported above, the flocculation of beef

extract solution as a convenient means of reducing the sample volume

further has received much use in recent years and as such has been

adopted as the secondary concentration method for all field studies

reported later in this study.

Cell Cultures

Having successfully concentrated low levels of viruses from large

volumes of water it is necessary to demonstrate their viability,

enumerate the viable units and identify the serotypes present. The

need to establish viability necessitates the recognition of virus

growth in cell cultures. This may take the form of the detection of

the products of intracellular virus multiplication by

immunocytochemical techniques or by the recognition of cytopathology

by plaque formation, the production of cytopathic effect or focus

formation. Whichever approach is adopted the cell host chosen must be

capable of supporting growth of a wide range of viruses and give the

maximum estimate of numbers of viable particles.

Many cell types have been used. Primary monkey cells tend to give the

highest titres and best isolation rates together with the broadest

spectrum of virus types (Schmidt et al, 1978; Grabow and Nupen,

1981) . However, the cost, general lack of availability and

possibility of contamination with latent simian viruses preclude use

by most laboratories. An alternative approach is the use of

continuous cell lines and, as no single cell line has yet been found

which satifies all requirements, a compromise has to be made. Several

factors will determine which cell line or lines would be most

appropriate. Firstly, the host cell must exhibit sensitivity to a

broad spectrum of viruses. Secondly, it must be capable of detecting

as many infectious virus units as possible. Lastly, it must be easily

handled, produce sufficient cell numbers to meet assay requirements

and not be fastidious in its media requirements.

The present report examined the susceptibility of ten cell lines to a

large number of laboratory virus strains. The evidence in Table 14

indicates that the cell lines are capable of supporting growth of the

majority of the serotypes available, in many cases producing high

titred virus. Such observations have been made by Lehmann-Grube

(1961), Hambling and Davis (1965), Davis and Phillpotts (1974), Harris

and Pindak (1975) and Wecker and Muelen (1977). However, in the water

context, the ability of the cell lines to detect naturally-occurring

viruses, that is, those which have not been adapted to growth in cell

cultures, with the maximum number of viable particles being detected,

is of major importance.

In recent years the BGM cell line has been the most favoured for the

detection of viruses in environmental samples, but has received little

attention from the clinical virologist. Dahling, Berg and Berman

(1974) originally compared the cell line with primary monkey kidney

cells and found that BGM gave higher detection rates and numbers in

water samples. The value of the BGM cell line has been seriously

questioned with reports that it was less sensitive than primary rhesus

monkey kidney cells, particularly when isolating echoviruses from

faeces and wastewater samples (Schmidt, Ho and Lennette, 1976; Schmidt

et al, 1978). Polioviruses and coxsackie B viruses were readily

detected, a situation which prompted Menegus and Hollick (1982) to

recommend the cell line for the rapid detection of these virus groups.

Despite such criticisms, the BGM cell line has been widely used for

environmental virological monitoring (Dahling and Saffirman, 1979;

Fujioka and Loh, 1978; Lucena et al, 1982; - for example). In a year

long survey of serotypes in wastewater effluent Irving and Smith

(1981) found that BGM did not isolate as many serotypes as HeLa cells

while Ridinger et al (1982) showed that LLC-MK2, BGM and 1-407 cells

were not as sensitive as the bovine kidney cell line, MDBK, for the

detection of naturally-occurring reoviruses. However, it should be

noted that 1-407 was efficient at detecting reovirus type 3. No

reoviruses were detected in the assay of effluents carried out in this

study.

Ridinger et al (1982) also noted that the BGM cell line gave the

greatest number of false plaques when determining the levels of

reoviruses. A similar situation was described by Bertucci et al.

(1983) who found that many plaques formed under BGM conditions could

not be confirmed as being of viral origin by subculturing. In this

case as many as 84% of plaques detected in wastewater failed to

confirm. The results reported in the present study indicate that the

majority of plaques confirmed when subcultured. Failure to confirm

viral aetiology , can usually be explained by the failure to subculture

plaques immediately into tube cultures or prolonged storage of agar

plugs from plaques usually resulting in poor virus growth when finally

subcultured.

Dahling, Safferman and Wright (1984) collated information from a large

number of laboratories worldwide concerning the BGM cell line with

particular references to the source of cells, passage history, media

and handling. Substantial differences were noted in the media used

for the propagation of the cell line and the ways in which it was used.

for the assay of viruses. One indication was that the higher the

passage level the less susceptible the cell line was. In the study

conducted here, BGM was used at passage levels between 75 and 100, a

range, according to Dahling, Safferman and Wright (1984), in which the

cell line had maximum sensitivity. It is not possible to obtain

passage levels lower than 70.

The present findings have demonstrated that BGM cells are efficient in

detecting the maximum - number of viable viruses in an environmental

sample and are capable of detecting most of the serotypes present in

such a sample. No other studies have been reported comparing the same

range of cell lines for the detection of enteroviruses in water

samples using the suspended cell technique, but the results presented

here support the contention that this system is probably as efficient,

if not more so, as any other assay technique. As work advances on the

development and assessment of other cell lines, BGM may no longer be

the host of choice. One such candidate is the human liver line,

designated PLC/PRF/5 described recently by Grabow et al (1983) which

has been used for the detection of polioviruses, coxsackieviruses

(groups A and B) , reoviruses, rotaviruses and hepatitis A virus

(Grabow, 1984).

Cell Presentation

Table 16 shows that the way in which cells are presented in a virus

assay can affect markedly the numbers of viable particles detected.

Plaque assays allow simultaneous enumeration and separation of viruses

while methods relying upon the recognition of cpe are statistical

estimations of the likely levels of viruses with inherent variability

(Table 13). However, the plaque assay does have limitations in that

some viruses may not form plaques despite producing cpe under liquid

overlay, they may not form plaques under solid overlays but may do so

under liquid or semi-solid overlays, toxic materials may give rise to

'false1 plaques and media components may affect plaquing efficiencies

(Schmidt et al., 1978).

Plaque techniques have been most favoured by water virologists

although some workers, for example Grabow and Nupen (1981), prefer to

use tube methods. Plaquing has conventionally been carried out using

monolayer cultures of the cell host, adsorbing the virus to the washed

cell sheet and applying a solid overlay medium. Plaques tend to

appear four to five days after inoculation of the monolayer (Hsuing

and Melnick, 1955). The system is inflexible in that preparation of

monolayers has to be carried out three to five days before the

initiation of the test. In addition, the washing and adsorption

procedures increase the amount of handling thus allowing a higher risk

of contamination by bacteria, fungi and, possibly, viruses.

Cooper (1967) described a suspended cell plaque assay based on methods

for determining levels of bacteriophages. The method was adapted by

Slade (1978) for the assay of viruses in slow sand-filtered waters and

has since been used successfully by other laboratories (Goddard and

Bates, 1981; Simmonds et a]., 1982; Simmonds, Loutit and Austin,

1983). The technique has the main advantages that, providing cell

stocks are adequate, assays can be initiated within minutes of a

sample being received, there is a minimal amount of handling (thus

minimising the risk of contamination), no washing or adsorption

periods are necessary and plaques form rapidly. Indeed, as Figure 8

indicates, the fastest growing viruses may form visible plaques within

two days of test initiation.

Much discussion has considered the relative merits of the different

assay systems, particularly with reference to detecting viruses in

water. Schmidt et al (1978) found that monolayer plaquing failed to

isolate some enteroviruses which had been detected by a tube

technique. Similar observations were reported by Mathews and Walters

(1983). In a comparison of monolayer plaquing and MPN determinations.

Rose et aJL, (1984) found the two methods comparable, the present study

supporting such results only in general terms. The results in Table

16 indicate that the mode of cell presentation is not the only factor

to be considered. The cell type used is of equal importance with the

BGM suspended cell plaque assay giving virus levels much higher than

other techniques and cells in many of the sanples tested. Only two

reports in the literature have compared the monolayer and suspended

cell plaquing methods. Slade, Chisholm and Harris (1984) found that

BGM cells used in the suspended mode were superior to other methods

for the detection of indigenous viruses in water samples, while Rao et

al (1984) recovered virus from samples apparently negative by

monolayer and tube tests with the same BGM system.

While it is important to detect the broadest spectrum of viruses

present in a water sample, this being determined largely by the cell

host chosen, the question arises as to whether this is the sole

requirement when monitoring the virological quality of waters.

Arguably, the need to detect the largest number of viable particles

assumes greater significance when assessing the health risk associated

with a water containing viruses. If this latter contention is

accepted, then the BGM suspended cell plaque assay goes a long way to

satisfying such requirements. In the present study the technique gave

the highest number of plaques, the highest incidence of positive

samples and the best recovery of serotypes (Table 17). Coupled with

its ease of application, the test has been adopted as the standard

approach in this laboratory for the routine examination of water

samples for enteroviruses.'

Optimisation of the BGM Suspended Cell Plaque Assay

Apart from cell host and cell presentation, many other factors can

affect the efficiency by which viruses are detected by plaquing.

Schmidt et al (1978) have suggested that some enteroviruses will only

plaque in monolayers if the vessel used for the test is closed, that

is there is no gas exchange system to ensure adequate buffering of the

assay medium. Adsorption times for virus-cell interactions using

monolayer systems indicate that prolonged adsorption can be beneficial

in some instances (Konowalchuck and Spiers, 1966; Schmidt and Wigand,

1980) with cultures which had been rocked gently over a four hour

period giving significantly higher plaquing rates (Richards and

Weinheimer, 1985). Such prolonged adsorption was found unnecessary in

the present study.

Cell concentration was not significantly critical in the suspended7cell plaque assay providing levels used were at least 2 x 10 cells

3 . .per 90mm (10cm medium) dish, similar findings having been described

by Slade, Chisholm and Harris, (1984) . Staining time was also not

critical but the convenience of incorporating the dye in the medium at

the initiation of the test reduced handling of the system. However,

it should be emphasised that neutral red stain may inactivate viruses

and also cause cell degeneration in visible light and it is advisable

to conduct tests in dim conditions followed by incubation in the dark

(Green and Ompton, 1959; Wilson and Cooper, 1965). Nevertheless, some

workers have found that the inclusion of the stain at the beginning of

the test can be detrimental to both cell survival and plaque formation

(Sellars and Stewart, 1960; Hatch and Marchetti, 1975) although this

may, in fact, have been due to photoinactivation mechanisms.

If cell cultures are to survive sufficiently long for use in the

plaque assay it is necessary to include a source of nutrients, usually

serum. Hull, Cherry and Tritch (1962) reported that cell sensitivity

to poliovirus was reduced when the cells were grown in the presence of

horse serum with similar findings with calf serum. Inhibition of

virus propagation by serum components was also noted by Allen,

Finkelstein and Sulkin (1958), Patty and Dougherty (1967), Hashimoto

(1975), Sairenji and Hinuma (1975) and Filczak and Korbecki (1976).

The present study has shown that the choice of serum for the inclusion

in the assay medium can seriously affect the numbers of virus plaques

formed and the ability of the assay to survive sufficiently long for

plaquing to occur. Foetal bovine serum was shown, unequivocally, to

support the best plaque formation with some other sera evidently

possessing marked inhibitory properties. In some cases other sera at

higher levels were cytotoxic to the test cell.

i

The gelling agent used to solidify the overlay medium has been shown

by many workers to be a critical component during virus plaquing. The

range of materials which can be used include starch (Maeyer and

Schonne, 1964; Wallis and Melnick, 1968), agars of differing purities

(Fiala and Kenny, 1966; Wallis and Melnick, 1968; Salo and Cliver,

1976; Matsuno, Inoue and Kono, 1977), agarose (Martinsen, 1970;

Sefcovicova, 1977) and methyl cellulose (Tytell and Neuman, 1963;

Dolan et al, 1968). The materials have affected plaque numbers,

plaque size and plaque definition. For example, Yoshii and Kono

(1978) showed that some echoviruses would only form plaques in the

presence of Bacto agar if DEAE-dextran had been included in the

medium. The inhibition of encephalomyocarditis virus by Noble agar

was demonstrated by Takemoto and Liebhaber (1961) while, in contrast,

Martinsen (1970) showed the superiority of Noble agar over agarose in

the assay of foot and mouth disease virus. Wallis, Melnick and

Bianchi (1962) found that Noble agar would allow the plaquing of some

enteroviruses but not others, these latter forming plaques if Bacto

agar was used. Yoshii and Kono (1978) established that many echovirus

types had specific requirements with respect to Bacto agar

concentration and levels of DEAE-dextran before plaquing could be

achieved. The results shown in Tables 21 and 22, to a certain extent,

confirm many of the observations reported in the literature although

much of the differences between agars was in terms of ease of test

reading with plaque definition varying with the agar type used.

However, it should be noted that only a few laboratory strains were

tested and the serotypes isolated from field samples did not include

any echoviruses.

Enhancement of plaquing by the inclusion of adjuncts in the test

medium aimed at off-setting inhibitory effects exerted by other medium

components or to stimulate greater virus replication, has been widely

reported although Wallis et al (1966) found that response to additives

may range from enhancement to reduction or have no effect on plaquing

of enteroviruses. Wallis and Melnick (1968) examined the role of

cations and polyions to reveal that many enterovirus serotypes had

enhanced plaquing efficiencies in the presence of magnesium cations,

protamine sulphate and DEAE-dextran. Such findings have been

supported by other reports (Feorino and Hannon, 1966; Fiala and Kenny,

1966; Conant and Barron, 1967; Martinsen, 1970). Pagano and Vaheri

(1965) suggested, however, that DEAE-dextran did not necessarily act

by overcoming the inhibitory properties of the agars and postulated

that in some cases enhanced plaquing could only be attained if the

chemical was present in the medium within three hours of addition of

virus to the cells. Similarly Sasaki, Furukawa and Plotkin (1981)

gained maximum enhancement when DEAE-dextran was applied immediately

after adsorption had occurred. The present results indicate that

enhancement was not achieved but, as with the agar investigation, only

a few strains of virus were tested.

Wallis et al (1966) noted that some additives had qualitative effects

that manifested themselves in improvement of plaque size, for example

echovirus type 12 with magnesium cations and echovirus type 14 with

cysteine. Studies with Rubella viru£ have shown that semicarbazide

improved plaque numbers and definition (Vaheri, Sedwick and Plotkin

(1967) . Furthermore, there was a suggestion that when it was used

with DEAE-dextran a synergistic effect occurred. The present results

showed that semicarbazide had little effect on either plaque numbers

or size although it is possible that there was a slight depression of

echovirus type 1 plaque numbers as the concentration of the chemical

increased.

The pretreatment of cells with 5-iodo-2'-deoxyuridine prior to

infection with virus has been reported to enhance levels of virus

(Jeor and Rapp, 1973; Jerkofsky and Rapp, 1975; Benton and Ward,

1982). In the present study pretreatment of cells with the chemical

was not done because it would have increased the complexity of the BGM

suspended cell plaque assay. However, when the compound was

incorporated in the assay medium at the time of test initiation

(Figure 21) no effect was detected. In contrast, the assay of

indigenous viruses in the presence of the chemical had a drastic

effect, showing a dose-related response.

The findings reported for the factors affecting the BGM suspended cell

plaque assay emphasise the importance of assessing recovery and

detection procedures using field samples. Reliance on results

obtained with laboratory virus strains can only be looked upon as

giving an indication of possible effects because it is possible that

after many cell passages, such cell-adapted strains may not

necessarily reflect the pattern of behaviour of viruses having just

been excreted by humans.

SECTION 4 ; FIELD INVESTIGATIONS - RESULTS AND DISCUSSION

Viruses in Drinking Waters

During the period 1980 to 1983 large volume samples (-cl,000 litres)

of most of the water supplies in the Authority were taken using the

field concentrator (Figure 7). Adsorbed viruses were further

concentrated by flocculation and assayed by the BGM suspended cell

plaquing method, a procedure shown to have a recovery efficiency of

about 43% (Table 25).

TABLE 25 Efficiency of Large Volume Concentrator for Recovery of

Poliovirus 2 from Tapwater

Sample volume (litres)

1,0001,0001,0001,0001,000

840

Input(pfu)

505520483846

Recovered(Pfu>

241314 12 13 26

% Recovered

4824 7025 34 57

Mean: 43.0- 18;5%

A total of 137,149 litres of treated potable water was examined

representing 284 individual samples taken at 265 locations throughout

the Authority, in which no cytopathic enteroviruses were detected.

Since the completion of the large volume survey, routine sampling (10

litre volumes) has been conducted and of 309 samples only one was

positive for virus, the single plaque detected being identified as a

strain of coxsackievirus B4. In this case the water was abstracted<

from the aquifer at depths of 70m and 94m.

Only a limited number of large volume surveys of drinking waters have

been conducted elsewhere. For instance, a report by the USA

Environmental Protection Agency (Report, 1978) on the examination of

over 140,000 litres of water from 40 communities (119 samples) failed

to demonstrate viral contamination and a comparable study in The

Netherlands also did not find viruses (102 samples, representing

51,000 litres; Olphen and Baan, 1982).

While such findings are reassuring in confirming the absence of gross

virus contamination of water supplies, it would be wrong to assume

that low levels do not occur from time to time. For example, Slade

(1985), who examined treated water « 10,000 litre samples) from a

chalk well in the south east of England, observed that 6/13 samples

were virus positive, all poliovirus serotypes. Untreated water from

the same well was positive on 12/15 occasions (polio and coxsackie B

viruses) . He suggested that the nature of the rock was such as to

allow seepage from sewers to penetrate the aquifer and he drew

attention to the disturbing fact that viruses were surviving

substantial disinfection treatment (-< 1.0 mg 1 free residual

chlorine). He also noted that no indicator bacteria were detectable

when one litre samples (a volume ten times the standard test volume)

of the raw water and treated water were examined.

The main drawback with large volume sampling is the restriction

imposed on the number of samples that can be processed because of the

time factor and geographical distribution of sources. Recently, Tyler

(1982) found that enteroviruses could occasionally be detected at some

locations in Wales using sample volumes of 20 litres taken as part of

a regular monitoring programme. .

It is probable that no gross contamination of the majority of water

supplies will persist where treatment is appropriate, adequate and

properly controlled. However, it is possible that any failure of

treatment, in particular terminal disinfection, may result in the

survival of viruses in the distribution system followed by outbreaks

of disease (Payment, 1981; Hejkal et al, 1982; Kaplan jet ajL, 1982c).

Viruses in Groundwaters

Most of the groundwater sources in the Authority have been monitored

and 399 samples were examined between 1978 and 1984, but only two

samples of relatively shallow aquifers proved to be contaminated with

viruses. The first, a 40 litre sample yielding 4 pfu all identified

as poliovirus type 2 , was a source deriving its supply from land

drainage pipes under a field regularly used for cattle grazing. The

use of this source has since been discontinued. The second

virus-positive groundwater was derived from a spring draining a

woodland. In this case 16 pfu were detected in a 40 litre sample

which, again, were all identified as poliovirus type 2. The result

was of some importance because the water was not treated but was being

supplied to a small hamlet for drinking purposes. Disinfection was

introduced and the source has been virus-free since. These two

results and the finding of coxsackievirus B4 in a treated groundwater

(referred to in the previous section) indicate that virus

contamination of groundwater is probably only sporadic , and when it

occurs only low levels of viruses are present.

Many reports of the presence of viruses in groundwaters have been

published (Appendix B, Table B2) but few workers have described the

nature of the aquifer or the overlying rock strata even if they were

aware of this and its potential significance. However, because of the

limited treatment usually applied to underground supplies it is not

surprising that the almost unavoidable contamination has resulted in

viruses entering supply and initiating disease outbreaks (Hejkal et

al, 1982). In some cases, contamination of the aquifer could be

traced to ingress of river water (Walter, Dobberkau and Durkop, 1982)

or to shallow wells being used in an area of wastewater recharge

(Wellings e_t al, 1975; Vaughn et al, 1978), while the example

discussed in the previous section shows that viruses can probably

penetrate to substantial depths.

Concern has grown about the production of mutagenic compounds during

the chemical treatment of river waters for potable supply. This has

directed increased attention to the use of groundwaters as sources

because these probably contain little or none of the necessary

precursors to form such harmful chemicals. The demands on some

aquifers have made their recharge necessary and recharge with

wastewater is one way of replenishing them. The passage of wastewater

through the soil into the underground sources relies upon the

filtration activity of the rock as a mechanism of purification.

However, care must be taken in how such recharge practices are carried

out. Experience in the Tucson area of Arizona (Gerba, personal

communication 1985) has shown that penetration of the aquifer by

viruses may be very rapid if pumping rates are too high resulting in

the wastewater entering the groundwater with only a minimal time in

the rock for purification.

Viruses in River Waters

a) General Survey

Monitoring for viruses in river waters has been concentratedi

mainly at points of abstraction with additional locations

providing extra data about the overall quality of the river

catchments. All the results are shown in Appendix D. Table 26

shows, in general terms, the virological quality of the main

river systems between 1979 and 1983. Samples (455) taken at 36

locations showed virus presence on 343 occasions with the maximum

level detected being 2,460 pfu 1 The range of serotypes

identified was limited to the polio and coxsackie B groups with

only two isolates of echoviruses (both type 32). The summary

data in this table show that the overall quality of the Trent

basin is somewhat lower than the Severn although the influence of

the river Tame (as will be seen later) may account for much of

this difference.

(b) Detailed Studies of the River Severn Basin

Monitoring of the river Severn was conducted between 1979 and

1982. Levels of up to 93 pfu 1 ^ were detected on occasions

and two-thirds of samples contained viruses.

TABLE

26 Range

and

Levels

of Viruses

in Rivers

in the

Severn

Trent

Water

Author

ity

1979

-198

3

(0 kin c ink k 0 k

in •H 'STk. 44 kCO in ro COk k k. o kCM •e*1 in CO •H CM ink k k. ■k VP k k

10 rH CO in CM •H rHd) CQ •. k. k CQ 4-> CQ ka k in CM ro •M- k C k co> i CO CQ CQ CQ k. ro in d> CO k44 k k. k. CO k k k CM in CM0 CM CO CO CO CQ CM •H CM CO CQ CQH k k k k k k CM k k w k k<U i—i CM CM CM CM rH CO in CO 0 rH k CM CMCO Al Pi Pi Pi Pi Pi W CQ CQ 2 Pi VO Pi Pi

3<pDiX<0s

O o o o O O O O o o• • • • • • • • • ■

in VD in VD r—1 CM O rH CMr-~ co VD *3* rH t " VD t " COm CM rH rH CM rr CM

vo oo a\ vd i"O rH (N H n 00 r l h CM rH rH

•sr in co

CO

^ ffl O CD CO VO VD CM CM rH CO CM i—IrH r- rH CM rH i—I

inin

00 rH rH rH O ' CO rH rH in VD rH rH IDCO

>1 -PU C c c P C(0 P P p ro 44 d) -P 44 4-> 4-13 <U d> <y 3 c £ C d) c C C to

-P > > > 44 <u P <0 > d> d> d) 310 d> o> co in P <U P 0 p 14 >4 14H CO CO CO H EH Q EH o Eh EH Eh •H

c14 p d) p 44d) Q) ip 3 C C> > 14 0 o fl>

-rH d) o 44 > 54Pi CO & CO < EH

44C0JSd)

44<L>J-i c O <U 14 Q)JD > 3 Ee o x : to< O U EH

d>10CO £ d) o S co

in >3 d) 01 14 -H 3 *H M >4 > U -HrH x .co o uf t U d)

II I! IIf t p s

cC >4 44

•H CD cin > <yro d) PCQ CO Eh

COhQ<EhOEH

inOJ44Ois

SHREWSBURY

PFU L*1

100—

ATCHAM

100 —

THE BURF

STRENSHAM

TEWKESBURY

HAW BRIDGE

I T 1 1 1 1 1 1 1 1 1 I I I I I I I I I I I T T T T r I ■1979 1980 1981FIGURE 23. VIRUS LEVELS IN THE R. SEVERN 1 9 7 9 - 1981.

Figure 23 details the virus levels at seven points, all being

abstraction points except Atcham, The Burf and Haw Bridge. Virus

levels from Shrewsbury to just above the Severn-Stour confluence

are low. The discharge from Monkmoor sewage works below

Shrewsbury elevated levels at Atcham on occasions. Below Atcham

the river Worfe contributes an extra 125 Mid, but virus levels at

Cosford (Figure 24) were very low. Below the Stour confluence

P F U L 1 O-

- 5

P F U L 1

O

6 0 0

P F U L 1 3 0 0

I I I I I I I I I IT i 11 i i 11 n r r I I I I T T1979 1980 1981

FIGURE 24. VIRUS LEVELS IN R. WORFE (COSFORD) 1979 - 1981.

1 I I II ITI 1 1 1ti n i 1 1 1 1 1iTI 1 N T1979 1980 1981

FIGURE 25. VIRUS LEVELS IN R. AVON (TEWKESBURY) 1979 - 1981.

TTTTT I I I I II TI I II I II I I I I II I I.1979 1980 1981

FIGURE 26. VIRUS LEVELS IN THE R. STOUR 1 9 7 9 - 1981

the virological quality of the Severn deteriorated despite

dilution from other rivers and occasionally high levels of virus

(compared with those at Shrewsbury) were detected at the

downstream abstractions. However, the further dilution of the

river by the 2,020 Mid flow from the Avon lowered the virus

levels substantially such that at Haw Bridge the quality was only

slightly less than that demonstrated at Shrewsbury 144 km

upstream. The Avon itself contributed very Tittle in the way of

virological contamination (Figure 25). However, the impact of

the Stour (Figure 26) was self-evident. Virus levels in this

tributary of the Severn were the highest found in the catchment.

Detailed Studies of the River Trent Basin

Sampling of the river Trent has only been carried out on a few

occasions (33 samples at seven locations, Table 26) and there was

evidence of much higher levels of virus than were found in the

river Severn. The Derwent tributary also showed fairly high

numbers of viruses (-<165 pfu 1 ^ in 25 samples) which were

probably derived from effluent discharges at Matlock, Belper and

Ambergate. In addition, effluents entering the upper reaches of

the river and its tributaries (for example Bakewell, Appendix D)

also, doubtless, contributed significant virus contamination.

The data for the intake at Little Eaton (Appendix D) shows that

this river abstraction was probably the most contaminated source

of all the surface water abstractions. Similarly, the Church

Wilne intake (Wilne) would also be expected to be heavily

contaminated, but here the water receives a substantial period of

storage prior to treatment.

The river Dove, which is abstracted for the Foremark and Staunton

Harold reservoirs, supports a game fishery but even here viruses

were readily detectable. The main source of contamination of

this river is the Churnet which, at best, is only diluted 1:2 on

entering the Dove. Samples taken from five points on the Churnet

suggest that it is heavily contaminated (Appendix D) . The major

sewage works discharging to this river is located at Leek, but

from the data available on the virological quality of the

effluent it would seem that the viruses found in the Churnet must

be derived from other sources. There is, obviously, a need to

further examine the catchment and identify the sources of viral

contamination.

Without doubt, the river Tame is the major contributor of viruses

to the Trent basin. Over a two year period 117 samples were

examined from six locations on a 38 km length of the river and

high levels of virus were detected (Appendix D) which confirmed

the gross contamination known to exist in the river. Indeed,

from this river have been obtained the highest reported virus

levels for a river water anywhere in the world (Appendix B) ,

2,460 pfu 1

TABLE 27 Effect of the Tame Lake on Physicochemical and

Microbiological Determinants

Effect of Lake (P=)

Total coliforms (12 samples)Escherichia coli (12 samples)Faecal streptococci (12 samples)Enteroviruses (13 samples)Suspended solids (13 samples)Biochemical oxygen demand (13 samples)

Insignificant (0.9) Insignificant (0.6) Insignificant (0.2) Insignificant (0.9) Very significant (0.02) Very significant (0.05)

CO

UN

TS

L"

As part of the 'clean-up' campaign for the river Tame, a large

settlement lake was constructed at Lea Marston in the early

1980's in order to reduce the loadings of suspended solids and

biochemical oxygen demand, much of which is derived from the

discharge of effluent from the Minworth sewage works (400 Mid) .

Table 27 shows that levels of solids and BOD were substantially

reduced.

FIGURE 27. MICROBIOLOGICAL QUALITY OF THE R. TAME 1982

4 —

LEA MARSTONWATER ORTON

8O

CHETW YND BD.KINGSBURY

JLY AIIGMAY

VIRUS (PFU),COLIFORMS E. COLI • — • F. STREP.

However, the influence of the lake on the levels of

microbiological determinants was, at best, marginal (Figure 27).

For instance, while bacterial levels were more or less stable,

the numbers of viruses during the test period apparently

increased. This may have largely reflected an increased amount

of enterovirus which can be expected during the summer months.

Another explanation for the increased virus levels may have been

the release of viruses associated with solids. However, the

latter probably did not occur to a significant degree because of

the slow progression of the water through the lake (seven hours

transit time) and the fact that levels in the water at Lea

Marston and Kingsbury, located at the inlet and outlet of the

lake, were similar.

It is interesting to note that virus levels at Water Orton above

the Minworth discharge of effluent were not reduced by the inflow

of wastewater indicating that the effluent has at least the same

level of contamination as the river at that point (Figure 27),

but, unfortunately, data was not available for the virological

quality of the Minworth effluent. Further downstream of

Kingsbury the Tame showed little improvement in quality with

levels of virus being fairly stable throughout until its

confluence with the Trent. Some slight reduction in

bacteriological levels may have occurred just before confluence

(Figure 27).

It is difficult to compare the results presented here with much

of the published data because of the differences in assay methods

used. For example, in a study of the Ottawa river, Sattar (1978)

examined concentrates of 10 litre water samples using BSC-1 cells

in a monolayer plaque technique. He found only low levels of

virus ( < 1 . 7 pfu 1 ■*■) even though 48% of the 59 samples

contained virus. Similarly, Olphen €5t al (1984) could only

detect < 5 pfu 1 ^ in the river Rhine and ^ 7.9 pfu 1 1 in the

river Drentse using a BGM monolayer plaque system. ^

Slade (1977), using methods similar to those reported in this

study, found 49 pfu 1 ^ in the river Thames and 12 pfu 1 ^

in the river Lee, although since then levels in excess of 100 pfu

1 ^ have been recorded (Slade, personal communication, 1985).

In Wales, Tyler (1982) has described levels in some rivers with a

maximum of 2.9 pfu 1 V again using the BGM suspended cell

plaque assay. The levels detected in the present study, in

particular with relation to the river Tame and river Stour are

the highest recorded for any surface water and similar levels

were occasionally recorded in the rivers Avon and Sowe (Table

16). Naturally, such levels reflect the degree of wastewater

contamination, but similar situations elsewhere in the world do

not give such high levels. This can only be interpreted as a

direct consequence of the improved detection systems used in the

present study.

The decrease of numbers of viruses in river waters throughout

their lengths from the point of contamination to the outflow into

their estuaries has rarely been studied. Wyn-Jones and Edwards

(1982) showed that there was substantial adsorption of virus to

river sediments and that this occurred within a short distance of

the discharge. Bitton, Chou and Farrah (1982) demonstrated

adsorption of about 40% of seeded poliovirus onto freshwater

sediments and such adsorbed viruses could be desorbed under

natural conditions. Table 16 demonstrates this phenomenon where

the peak levels of viruses in both the rivers Sowe and Avon were

detected under flood conditions when the turbidity of the waters

had increased, presumably due to disturbed sediments.

Viruses in Potable Waters from Storage Reservoirs

Table 28 examines levels of virus in a number of storage reservoirs

with detention times of up to 50+ days. Only one of the locations

(Blithfield) is an impoundment of a river, all other reservoirs

receive pumped river water. The Campion Hills site is prone to

short-circuiting (Kemsley, personal communication 1980) although in

theory the detention time for the reservoir is about seven days. In

general, the frequency with which viruses are detected in stored

waters, and the numbers detected, decreased as storage time

increased. In the case of Blithfield no viruses were ever encountered

over a period of two years.

In two instances there are data to compare virus levels in the

original river water and in the reservoirs after storage although no

allowance was made for temporal relationships. The river Severn

abstracted to Hampton Loade had the levels of viruses which would be

expected in the river at this point in its flow. In the stored water

viruses were, however, only infrequently detected although where found

the serotypes present matched those in the river water. Similarly,

the virus levels in the two other reservoirs (Staunton Harold and

Foremark), which receive water from the river Dove, were substantially

lower than in the river water (Table 29).

TABLE

28 Detection

of Viruses

in Stored

Wate

rs

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TABLE 29 Virus Levels in River and Stored Waters

(a) Hampton LoadeR Severn at Intake* Stored Water*

10.3.81 7.8 0.214.4.81 6.1 0.112.5.81 5.0 0.214.7.81 0.2 0.25.7.82 1.0 0.1

(b) MelbourneSample Date R D ove at Intakes* Foremark* Staunton Harold*

27.11.79 2.3 ND ND30.1.80 0.2 ND ND27.2.80 ND ND ND29.5.80 11.0 ND 0.124.6.80 5.2 ND ND23.7.80 7.8 ND ND29.10.80 ND ND ND25.11.80 ND ND ND

*pfu 1 ^ ND = none detected

Surprisingly, few studies of the effectiveness of potable water

storage have been carried out. Slade (1978), when examining the

efficiency of slow sand filtration of water, noted that stored water

(14+ days detention) had lower virus levels than the original river

waters (Thames and Lee) entering the reservoirs, but still managed to

detect 5.8 pfu 1 ^ and 84% positive samples. Tyler (1982) found

that two impoundments in Wales yielded viruses on 4/21 occasions with

a maximum level of 1.5 pfu 1 In a study in the Netherlands,

Olphen e_t al (1984) found that storage of the river Meuse for more

than 80 days still enabled viruses to be detected in 50% of the waters

while a second reservoir with storage in excess of 140 days only give

1/7 samples positive for viruses.

Open, raw water storage reservoirs vary enormously in area and depth.

Some are designed as an insurance against drought periods and dry

seasons, while others function primarily as sedimentation basins with

the resultant improvement in turbidity reducing the practical problems

of water treatment. The hygienic advantages of storage, as an initial

step in the treatment of potable supplies, are important but often

overlooked. The data presented here indicate that prolonged storage

is necessary if virus levels are to be markedly reduced, but even

after such treatment a number of samples will still contain low levels

of viruses.

Viruses in Wastewaters

Virus levels in wastewater (raw sewage, during treatment and final

effluents) have been measured in the present study in order to (a)

determine the efficiency of treatment processes and (b) determine the

role of effluent discharges in the viral pollution of receiving waters.

(a) Effectiveness of Wastewater Treatment

Samples were taken at two works in the period May 1982 to January

1983. The first works, Finham, serves a large conurbation of

about 350,000 inhabitants and receives a mixture of domestic and

industrial wastes. The second works, Monkmoor, receives sewage

from a major rural town of about 60,000 population and the nature

of this is mainly domestic. The treatment regimes for both works

are shown in Figures 28 and 29. The sampling at each stage of

the treatment did not allow for any temporal relationship, the

efficiency of individual processes thus being described in

general, rather than absolute, terms.

FIGU

RE

28.

FLOW

DI

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R FI

GURE

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(i) Incoming sewage: Virus levels in the incoming sewage at

Finham showed a degree of seasonality (Figure 30) with

highest levels being found in the summer months. This

would be expected with the enteroviruses (Figure 5) . The

peak levels of virus in raw sewage at the two works were

72,500 pfu 1 ^ at Finham and 57,500 pfu 1 ^ at Monkmoor.

FIGURE 30 . VIRUS LEVELS IN FINHAM SEWAGE 1982.

PFU L (LOGi0>

JUN JLY AUG SEP OCT NOV DEC

(ii) Sedimentation; Settling of raw sewage is usually only of

a few hours duration and, as was expected from the

experience with storage of river waters, such short

detention times had no significant effect on the levels of

virus detected (Table 30).. Similarly the use of short term

sedimentation (humus tanks) to remove solid materials after

percolating filtration and activated sludge treatment also

failed to have an impact on virus reduction (Table 30).

(iii) Percolating filtration; this process is aimed at the

reduction of biochemical oxygen demand by utilising

micro-organisms to oxidise the available nutrients in

TABLE

30 Ef

fect

of

Sedi

ment

atio

n on

Levels

of Virus

in Wa

stew

ater

a>o3COu*H

<4-4•Hccn•Hw

cs in > > • • • •o o o o

CM0)oc(0o•H

<4-4•Hccn

n oo n in • • • •o o o o

cn

i n O (T\ CMn cd in in

rn in co cn<Ti CN CN + + +. +

,-k o o O O-1 o o o O

CN o o Or-4 44 k k k k

3 rH CN co «sr3 CD t^- CO CO•44 3a r-4 1 I 1 1-— •44

•44 O o o oCQ w o o o oi—4 o VO CN0) k k k>&

1—1 CN VO

CQ o o o o3 o o o ou VO *3* in in

•H 44 k k k k> 3 r-4 co VO

CD CO VO in CO•44 30 r-4 1 1 1 1

•44<d c O o o ocn W o o o o3 CN o o CNCO k k k

rH rH CO

cd in ni cn CM CN H H

** * *CQ H U O Oe o. o

<o to e ejC XXc c c c•H *rl O OCn S S

(0c<0

4-1

(03e3.3**co•H4J(0

4430)E•rl<DIQ>1>-4COE•Hl-lCM

CO3M•H>•4-4oCQ

r-4<U>acoco•H4J(0M4JiH*Hfrccn 3 •H 4-> (0 r—4oo140)CM•4-4O4JOo•4-4>4-1W

a)cnj3(0Xio

CO CN CN ^ CD ^ CN

O ’sf CN ^ n N 1 04t i l l

O o O o-4 O o O oO o 00 inrH 44 k k k k3 CN CN 00 VO3 <u t"- t- in CO•w 3a rH 1 1 1 1-— •44•44 O o o oCQ H o o o o 3r-4 CN CN CN oCD \l k •H>3

V CO 44CO1444CQ o o o o r43 o o o o •H>4 00 o 00 CO •44•H 44 k k k k> 3 in CN CN CO 0)(D 00 rH•44 3 XX0 i—1U4 I l 1 1 3O(0 3 o o o o <ocn 4H o o o o3a ok

r-4

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oo in co cnCN CN CN H

** * *< CQ CQE E E OCO to CO g jc XX 43 JC3 3 3 3

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•44

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wastewaters and reduces virus levels only slightly (Table

31) . A variation of the process, alternating double

filtration (where wastewater is passed through a

percolating filter, a humus tank, a second filter and a

final humus tank) did not result in any greater removal of

virus.

(iv) Activated Sludge; this treatment is probably the most

important of the secondary treatments of wastewater. The

biological oxidation system is capable of high loadings,

but is susceptible to fluctuations in the quality of the

incoming wastewater. The detention times at the Finham

works are short (about 4 hours) and, not unexpectedly,

.virus removal was erratic (Table 32); the best reduction

achieved was 8 6 %. Even allowing for the lack of temporal

relationships, the activated sludge process at this works

was not effective in removing viruses.

(v) Tertiary Treatments; those examined were rapid sand

filtration and lagooning. The former is designed to remove

carried-over suspended solids and the only reduction of

virus which may occur is probably associated with the

adsorption of viruses to such floes (Table 33). Lagooning,

however, proved to be the most effective of all the

wastewater treatments examined (Table 33). At Monkmoor it

is only applied to about 1 0 % of the flow thus improving the

overall quality of the discharged effluent by only a small

margin. Reduction of virus by lagooning can be in excess

of 99% although 45% of the samples still showed evidence of

virus contamination.

TABLE

32 Effect

of Activated

Sludge

on Levels

of Virus

in Wa

stew

ater

iip

QJoctou•H

•HcCD•HCO

co oo • •

o o

IIPQ)Oc<0o

•HM-lcCl

•Hco

Or - oo o

V

(0o>c(0Xo

cm r~ co co

O CMco ro I +

in inCM CM

14(0*H4JU

SM-loXo<utwiww

O o O oiH O o to iH O o1 00 VO 3 1 O inrH 4-> w M iH 4J

c in in c3 <u t^ > 3 <D CNIH 3 44 3a rH l 1 44 & rH 1 I

144 0 *—* 4444 o o 44 O o

(0 W o o to to o inrH iH rH rH v0) Q) CD Y>3

>

a

>a

to o o 3 to O o3 o o 0 3 O o14 CM o 14 CM o•H -P hi . to •H jj *.> c rH CM 4J > c O «sr

<1) G <0 CO COU-J 3 <D U-4 30 rH 1 1 E o rH 1 1

44 •P 44(0 c O o to <U C o oc H • O o 0) C H o oc << 14 c rH os

E-is V

dP

,—.P 00to CO •CM o•---- ■»—■-

d)cn rHc •to CN cnX O cnu rH 1+

00 i-l CM rH

UlG00C>(0

(014Q)4J

< pae e (0 (0 x x c c

•H *Hfa ta

**U0OEc cos

•Hiw•cctotota•HcutoUl

Sedimentation has been shown to be ineffective for virus removal

unless long term storage is possible (compare short term primary

sedimentation with long term lagooning). Such findings have been

reported by many workers (for example: Bloom et al, 1959? Clarke

et al , 1961; Mack et al, 1962). More recently Rao et aJL (1981)

postulated that in developing countries sedimentation may be the

only cost effective way to reduce virus levels in wastewaters.

In industrialised nations, however, the cost of land to enable

long term storage to be carried out would almost certainly be

prohibitive.

Oxidation of wastewaters, either by percolating filtration or

activated sludge, is effective in improving the physicochemical

quality of wastewaters, but is not so useful for the reduction of

viruses. The results obtained here with percolating filtration

confirm the evidence of other workers where reductions were poor

under field conditions (Malherbe and Strickland-Cholmley, 1967).

Under laboratory conditions with careful control of the quality

of the incoming wastewater, an unreal situation, higher reduction

rates can be achieved (Clarke and Chang, 1975). Similarly,

activated sludge under ideal conditions can achieve 90%

reductions (Lund, Hedstrom and Jantzen, 1969; Malina et al, 1974;

Balluz, Jones and Butler, 1977) whereas under field situations

reductions tend to be lower (Kelly and Sanderson, 1959;

Schwartzbrod &t al, 1985). In an examination of a treatment

works employing primarily the activated sludge process Slade

(1982) found that virus levels could be reduced by as much as 99%

after an eight hour detention time. Such variability in

reduction rates emphasises the difficulties encountered when

using the 'real-world' situation for the measurement of process

efficiency.

Tertiary treatment of wastewaters is of no consequence unless it

takes the form of terminal disinfection of effluent prior to

discharge or involves the lagooning of the wastewater for

substantial periods of time. Lagooning can be very effective,

the above results confirming the experiences of Rao, Lakhe and

Waghmare (1981) and Sheladia, Ellender and Johnson (1982)

although the latter workers commented on the ability to detect

viruses in lagooned effluent even after 98 days detention.

TABLE,34 Overall Reduction of Naturally Occurring

Enteroviruses by Two Wastewater Treatment Works

% Reduction

Finham works Monkmoor works

without lagoons with lagoons

Neither of the treatment works examined consistently reduced

virus levels significantly (Table 34). The overall effect is a

reduction which may occasionally be significant in percentage

terms, but taken in the context of the numbers of viruses passing

through a sewage works reduction is, at best, marginal. For

example, at the Finham works (average daily flow of 100 Ml),

overall reduction is 63%. This represents a reduction of the

63

2633

12 11 daily virus loading from 1.4 x 10 pfu to 5.4 to 10 pfu,

an insignificant effect. Lagooning at Monkmoor substantially

improved the quality of the small amount of wastewater treated,

but it should be noted that when this is taken into account in

assessing the overall performance of the works, virus reduction

was still only 33%. Substantial reliance is thus set upon the

ability of the receiving waters to self-purify as well as an

adequate dilution of the discharged effluent.

Effluents

Figure 31 summarises the data relating to levels of virus in

wastewater effluents taken on 372 occasions at more than fifty

locations during 1980-1981. Levels of enteroviruses at numbers

greater than 100 pfu 1 1 (the detection limit of the assay

system) were detected in 46% of the samples. It is not possible

to indicate the pattern of virus discharge in the effluents using

this data, although it is obvious that the highest numbers of

viruses tend to be found in the summer months. However, data

from five wastewater treatment works (described in Table 35)

presented in Figure 32 shows that many variables can affect the

numbers of viruses present in the effluent. Only works A

consistently showed virus levels above the test limit,

occasionally giving high levels (maximum of 3,900 pfu 1 ^).

Both works B and C showed the effect of low domestic input and

high agricultural input with viruses only infrequently being

found. Works D, despite receiving substantial loadings of

domestic sewage, also showed low levels of virus in its effluent,

FIGURE 31. ENTEROVIRUS LEVELS IN EFFLUENTS 1980 - 1981.

100

-i<h-O»“ 50 u.O*

PFU L~1

... ..

.

\ _ A _ z V vl J i F | M | A | M j j | J | A | S | Q | N | D I

• <100 PFU L“ 1 + >100 PFU L* 1

FIGURE 32 . VIRUS LEVELS IN EFFLUENTS FROM FIVE WORKS 1980 .

__________________ NUMBER OF SAMPLES EACH MONTH26 I 26 125 I 101 5 I 24 I 41 I 34 I 30 I 24 I 31 I 19 I 10 I 10 I 16| 2 l 7 | 32

vVv»l

19811980

□ 0 - 99 ED 100 - 999 DU 1,000 - 9,999

Si 10,000 - 19,999 11 20,000 - 29,999 H 30,000+

TABLE

35 Incidence

of Virus

in Effl

uents

at Five

Works

1980

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possibly due to the intensive aeration of the incoming sewage in

order to prevent obnoxious odours from that part of the sewage

attributable to abbatoir waste. Works E similarly had only low

levels of virus which were possibly related to the discharge of

dye wastes into the tertiary effluent. It is conceivable that

such wastes may photoinactivate any viruses which may be present

(Gerba et al, 1977a, b) . On occasions the wastewater effluent

from this works was coloured red, blue or green.

The presence of a significant proportion of samples in which it

was not possible to show the presence of virus above the

detection limit of 100 pfu 1 ^ would seem to indicate that

either there was only a low level of virus excretion in the

contributory populations or that the treatment processes used

were effective in removing viruses.

Value of Indicator Systems for Predicting the Presence of

Enteroviruses

(a) Physico-chemical Determinants

No published data are available for the correlation of

enterovirus levels with such parameters. The present study

examined the relationship between levels of enteroviruses and two

physico-chemical determinants, suspended solids and total organic

carbon. A total of 285 samples were examined and the data showed

no correlation between the determinants. In order that any bias

attributable to the use of pooled data could be eliminated.

samples taken from two locations were also statistically analysed

with similar findings (Table 36) . It is obvious from such data

that the use of physico-chemical parameters as a means of

determining the risk from virus pollution is probably not valid.

TABLE 36 Relationship Between Levels of Cytopathic

Enteroviruses and Physico-chemical Determinants

Data Source No;-Samples Significance (P=)SS-V TOC-V

0.98 0.950.95 0.90

0.80 0.60

SS = suspended solids TOC = total organic carbon V = cytopathic enteroviruses

Bacteriological Determinants

In the present study the relationships between the enteroviruses

and the coliform group were determined in a large number of

samples, mainly wastewaters and river waters. Figure 33

summarises the data showing that as levels of coliforms increased

the proportion of samples likely to contain enteroviruses rose.

However, it should be noted that a small, but nevertheless

significant, number of samples contained viruses even when levels

of bacteria were below 10 1 Indeed, closer examination of

the data (Appendix D) shows that of four samples apparently free

of total coliforms one was contaminated with viruses and ten free

of E. coli had one virus positive. Bonde (1975) pooled data

All 285R Severn 13

(Tewkesbury)R Tame (Hopwas) 7

from many studies and found that where E. coli levels were less 4 -1than 10 1 , viruses may be expected to occur in about 18%

of samples with this level rising to 53% if E: • coli levels were4 -1 ' *above 10 1 . The present data, using more efficient virus

methodology than was available at the time the Bonde appraisal

was made, give virus positive sample frequencies as 30% and 70%

respectively. It is likely that as virus detection methods

further improve, such ratios will increase further.

TOTAL COLIFORMS E. COLI

>100PHAGE (ED391 HOST)PHAGE (W 3110 HOST)

LEVELS OF INDICATORS PER LITRE (LOGj J

FIGURE 33. RELATIONSHIPS BETWEEN LEVELSOF ENTEROVIRUSES AND INDICATORS.

The relationship between levels of enteroviruses and faecal

streptococci has received some attention lately. The bacteria

occur in the faeces of warm-blooded animals and are consistently

recovered from faecally-contaminated waters. ”Faecal streptococci

appear, in general, to be more persistent in the environment than

the coliforms and it may be that the group is perhaps a more

valid indicator of faecal pollution. However, the relationship

with enteroviruses has not been established (Sedita et al., 1977;

Marzouk, Goyal and Gerba, 1979; Berg, 1980). In contrast,

Cabelli (1980) has shown that the group correlates well with the

incidence of swimming-associated gastroenteritis.

The data presented in Figure 33 show no relationship between

levels of faecal streptococci and enteroviruses apart from the

increase in virus positive samples as levels of the bacterial

indicator increase.

Bacteriophage

In the present study a limited amount of data was generated on

the levels of coliphage in effluents using two E. coli strains as

host systems. Figure 33 shows that no virus positive samples2 -1could be found if coliphage levels were below 10 1 , but if

levels reached 1 0 1 ^ then virus presence could be expected

in the majority of the samples.

While coliphages do not at present satisfy the requirements of an

indicator of virus levels it is possible that more restrictive,

classification of the range and type of bacteriophage monitored

may enable a more suitable bacteriophage indicator system to be

developed.

Enteroviruses as Indicators of Virus Pollution

It is apparent that no single virus type or grouping can be

expected to act as an adequate indicator of virus pollution

despite the regular isolation of several serotypes (Figure 34).

On occasions it is possible to demonstrate that all plaques

detected in a sample were attributable to a single serotype. For

example, a sample from Hampton Loade (11.9.79) had 20 pfu of

virus in a 10 litre sample, all of which were coxsackie B4. Such

data reflect the findings of Katzenelson and Kedmi (1979) who

could not show sufficient evidence of poliovirus contamination to

warrant its use as an indicator since less than 50% of samples

contained the serotypes.

POLIO

cox B

i i i i i i i" i i r i i i r i i t i r r n . I I I I I I I I I I T1979 1 9 8 0 1981

FIGURE 34. SEROTYPES 1979 - 1981.

Reported data show that no currently used indicator system can be

expected to demonstrate the risk of contamination by viruses.

Despite the inadequacies of virological methods used in such

studies and the differences in volumes tested it has not been

possible to demonstrate a relationship between levels of

naturally-occurring viruses and indicators. The best the present

study achieved was that there was an increased likelihood of

virus being present as levels of bacteriological indicators rose

although, even here, there were exceptions. For instance, the

data in Appendix D shows the presence of low levels of virus in

the absence of one or more indicators similar to the findings of

Slade (1985) where no indicator bacteria could be found in well

water despite the presence of polioviruses. Conversely, viruses

have often been shown to be absent despite high levels of

bacteriological indicators. It is possible that newer indices of

faecal pollution may prove more useful, but it is quite clear, at

this time, that the most appropriate means of assessing the

potential risk posed by waterborne viruses is the direct

measurement of virus levels in the aquatic environment.

Nevertheless, the demonstration of heavy faecal pollution by

conventional indicators may be reliably taken as an indication of

potential presence of viruses. The failure, however, to

demonstrate such indicators must not and cannot be taken as

indicative of virus absence.

Enterovirus Serotypes Identified

The types of human viruses detected in the water cycle will,

obviously, reflect the types present in the excreting population;

Many reports have shown that a wide range of enteric viruses can be

found in sewage with a similar spectrum being demonstrated in river

waters receiving such wastes. However, it is apparent that the

methods used for the detection of viruses in the environment may

result in a bias towards the identification of specific types. For

instance, the earlier study of the concentration system showed a

recovery efficiency for poliovirus type 2 of about 40% (Table 11).

However, consideration of one part of the procedure, organic

flocculation (Table 12), suggested that some serotypes may not be

recovered and subsequently identified. This may be particularly true

for the echoviruses, although these are occasionally identified.

The present study examined a total of 1,907 isolates during the period

1979-1982 (Table 37). Polioviruses accounted for about 21% of all

identifications while three of the coxsackie B viruses accounted for

75% of the total.

Such a bias may be taken to indicate preferential recovery for such

serotypes, but examination of the frequency of isolation of these

types in waters and wastewaters shows that, in some cases, this merely

reflects the presence of the virus in the contributory population

(Figure 35). However, this is not always the case. In 1980 the

clinically dominant enterovirus was a strain of echovirus type 30.

Analysis of water samples at that time could not demonstrate this

serotype. Instead, the environmentally dominant virus was a coxsackie

B3 strain. ' . "

TABLE

37 En

tero

viru

s Serotypes

1979

- 19

82<#>rH(0JJ

in in in o o o

o j r o ^ o r o c r i i n o o o o o orH — ' w rH CN n — ' W W w W

r ' - r H c n i n ^ r c N i n c n c N ^ co oi id r- cd r-

cn cn in

H rH H CN t"oCJ\

CT> O rH O CO CO ID CTvi—I cn in h in corH rH CN

O rH O O COinID

cda

&

M CDCl) Cn

4J COCO 5

£ CDCQ

>X>COco

•H4J »oCO CDo S-l

-H 04-1 4->•H CQ4JCCDrOIH

•o!3 H

CD>

• r lPh

O r H O O l O r ' - O O O O O O rH CO

inin

o cnCN

n co co n 1 in in o rH o o o rHini—l

r i c r > c o m o o o i n o i H o o cn h co in co cn oo

rH rH Cn CNOo

43eooo

O C N O O O O O O O O O O O CN

CO] O O O O O O r H O O O O O O rH

<1>4J0UCL)cn

r H C N c n t H C N c n ^ r i n i o c n CQ rH.2 orH X Xo oU Ws

in cnr l CN cn CO

gioE-i

100COXSACKIE B4

Ui

<

u-100COXSACKIE B5

Eb!SZl

1979 1980 1981

— r i lN IC A L R E P O R T S — CL,N,CAL — ENVIRONMENTALCLINICAL REPORTS ECHOVIRUS 30 Q ISOLATES

FIGURE 35. COXSACKIE B VIRUSES IN WATERS AND WASTEWATERS 1979 - 1981.

POLIO 1

<100- i POLIO 2

- <

POLIO 3

n

JTJx = ^ l

1979 1980 1981

FIGURE 36. POLIOVIRUSES IN WATERS AND WASTEWATERS 1979 - 1981.

When the frequency of each serotype is examined over the three year

period (Figure 36) the changing pattern of dominance in the water

context can be seen. Coxsackie B4 was most frequently isolated in

1979, coxsackie B3 peaked in early 1980 with coxsackie B5 frequency

rising in mid 1980 and accounting for most isolates over the next

year. It is interesting to note that where the clinically dominant

virus is also the most prominent in the water cycle, evidence of

increased excretion rates, without reports of disease, can be detected

several months in advance. In addition, higher levels than normal can

still be detected after reports of disease have declined. In the case

of coxsackie B5 virus, the outbreak of disease occurred in 1981

whereas the virus was the most frequently isolated from water for

about a year before. As has already been commented upon, the

polioviruses accounted for less than a quarter of all isolates

identified. Figure 36 suggests that the isolation frequency of the

three types, which peak at the same time, may be correlated with

vaccination campaigns, but there are no data to substantiate this

hypothesis. It is also conceivable that the methods used allow for

interference between some virus serotypes such that one strain may be

detected while others may be suppressed by its presence.

Additionally, the large number of plaques detected in the assay of

river waters and wastewaters precluded the identification of all

isolates although it is reasonable to assume that the random

subculturing of such plaques reflects the actual occurrence of each

serotype.

SECTION 5 ; GENERAL DISCUSSION

The Need for Surveillance of Viruses in the Water Cycle

The Water Act of 1973 (HMSO, 1973) requires that water supply

undertakings provide drinking water which is 'wholesome'. This

nebulous term has been defined by Holden (1970) as:-

"water taken from a properly protected source and submitted to an

adequate system of purification can be rightly designated as puret

and wholesome if it is free from visible suspended matter, odour,

colour and taste, from all objectionable bacteria indicative of

mineral or organic origin which in quality or quantity would

render it dangerous to health."

The European Community directive on the quality of drinking water

(EEC, 1980) defines acceptable levels of indicator bacteria which must

not be compromised and additionally states that, as a rule, water

intended for human consumption should not contain pathogenic

organisms. Thus, no legislation on the permitted incidence of viruses

in drinking waters exists in Europe whereas in the USA a more positive

attitude has been adopted by the National Academy of Sciences (NAS,

1977) which declared that:-

"the presence of infective virus in drinking water is a potential

health hazard to the public health and there is no valid basis on

which a no-effect concentration of viral contamination in

finished water might be established."

This recognition of the importance of viruses in drinking water has

not yet lead to specific recommendations for virus surveillance and

the action necessary subsequent to their detection.

In contrast, monitoring bathing waters for viruses is a legislative

requirement (EEC, 1975) and such regulation is a tacit admission that

there is a risk to public health due to ingestion of polluted water.

Evidence that this is so was first collected in the USA by Cabelli

(1982) who demonstrated that only small amounts of virus-containing

water needs to be consumed, perhaps less than ten viable viruses being»

sufficient to induce infection/disease. That contaminated water can

be a source of viral diseases is also supported by the evidence from

shellfish-induced gastroenteritis outbreaks (Sockett, West and Jacob,

1985; figure 6) . In virtually all such cases, the shellfish

implicated were harvested from marine waters grossly contaminated with

wastewaters. At this time it is not known what levels of virus need

to be present in the shellfish flesh in order to induce illness but

the numbers are probably very low (West, 1986, personal communication).

Epidemiological evidence for the importance of viruses in drinking

water is, regrettably, much less well established. Only with

hepatitis A has transmission by the water route been conclusively

shown to be important in the spread of the disease (Appendix A) with

other viruses only recently receiving attention. Non-A, non-B

hepatitis is believed by some workers to be spread by the water route

(Appendix A) while rotavirus spread by water is apparently only of

minor importance. However, evidence is slowly accumulating indicating

that some viruses are capable of penetrating conventional water

treatment strategies (the 'multiple barrier' principle) and survive

disinfection to enter the distribution system. Such penetration has

been described by Payment (1981) and Brooker et a_l (1985) although in

neither of these examples was it possible to establish the

epidemiological significance of the contamination in terms of public

health.

It is apparent that present day UK attitudes towards the role of water

as a transmission route of viral diseases are no longer justifiable.

The case for examining water for viruses is irrefutable. It would be

foolhardy to suggest that water known to be contaminated with viruses,i

but satisfying other quality criteria, should be supplied for drinking

purposes. There is sufficient evidence to clearly demonstrate a risk

of waterborne viral infection associated with both water supplies (and

bathing waters) and that only by repeated failure over a long period

to demonstrate viruses in such waters, using techniques of maximum

sensitivity, can one hope to substantiate that confidence which

already exists in the virological wholesomeness of treated water

supplies.

The Practice of Virus Surveillance

If the rationale behind virus surveillance is accepted, two important

questions need to be considered. Firstly, should surveillance be

aimed at detecting all viruses present or should it be directed, as

with bacteriological analyses, to specific types or groups of

viruses? Secondly, should surveillance be conducted as a regular

monitoring programme, should it be based on a risk assessment of each

location or should effort only be directed to incidents which indicate

that viruses may be involved?

Which viruses should be screened for is determined by the methods

available for their detection. The need to assess the infectivity of

waterborne viruses restricts procedures to those which demonstrate

viability of the viruses. In all cases, this will involve isolation

and growth of viruses in cell cultures with visualisation of the

products of such propagation either by formation of cytopathic effect,

plaques or foci or by the immunocytochemical detection of

intracellular viral antigens. To date, easily applied methods only

exist for the cytopathic enteroviruses and the rotaviruses. The

former can be readily detected by plaquing while the latter can bel

demonstrated by immunofluorescent and immunoperoxidase techniques.

Unfortunately, at this time, no applicable methods are available to

demonstrate the presence of viable Norwalk-type viruses. Screening

for hepatitis A virus would be useful because the virus has been shown

to be waterborne, but the slow growth of the virus in cell culture (up

to 40 days) prevents current analytical procedures from having a

positive role to play in virological surveillance of water.

The techniques available for the cytopathic enteroviruses are fairly

broad spectrum although many factors, as reported in the present

study, can influence the numbers and types detected as well as

affecting the duration of the test. However, the more or less

consistent occurrence of these viruses in the water cycle serves as a

useful indication of virus presence provided that it is recognised

that others, such as rotavirus and Norwalk-type viruses, may be

present when the cytopathic enteroviruses are absent. It can be

argued that routine monitoring of waters should be mainly concerned

with such cytopathic enteroviruses with analysis for rotavirus, for

example, being limited to those seasons when the virus may most

reasonably be expected to occur.’ It is likely that, as reagents

become available and procedures are modified, it will become possible

to assay within a single test for total virus presence with or without

identification of the serotypes present. If viral monitoring is to

have a role to play in water quality assurance the move towards rapid

diagnostic procedures, reducing analysis times to a minimum, but still

demonstrating the infectiousness of the waters under scrutiny, must be

vigorously pursued.

I

The question of sampling frequency is more complex. Unless it is

possible to provide adequate laboratory facilities and manpower,

on-going regular surveillance of treated drinking waters, along with

associated raw potable waters and distribution samples, is not

practical. However, it can be argued that unless such data are

gathered it may not be possible to determine the health risk, in terms

of viruses, associated with each supply. The only option is to assess

risk based upon bacteriological, physicochemical and operational

information.

Another possible approach is to determine the health risk associated

with specific waters and then allocate a degree of priority. High

risk sources would be sampled frequently with low risk supplies only

being screened on an occasional basis. Such risk analysis may be

influenced by the size of the population served by the water supply,

the frequency of sampling being higher for a large urban population

than a smaller rural community as is normally applied in

bacteriological analyses (HMSO, 1983). However, such an approach can

be described as discriminatory with the UK water undertakings being

obliged to provide the same degree of quality to all its consumers who

do, after all, pay for such service.

A third approach, where only limited facilities are available, would

monitor those supplies most at risk, but allow sufficient time for the

monitoring of changing treatment regimes and the investigation of

incidents as and when they arise. Any water supply derived from a

surface water receiving effluents would automatically be monitored

with groundwaters known to be of unreliable quality by other criteriaI

falling within the same scope of surveillance. Other sources would be

allocated lower priority and screened as and when possible or

necessary. Wastewater surveillance, in terms of limited virological

capability, must be considered a luxury and should not normally be

pursued vigorously. However, where wastewater treatment practices

threaten the integrity of potable water sources, such as land disposal

of sludges in the vicinity of an aquifer, then surveillance is

necessary. Recreational waters are, to a certain extent, governed by

the EEC directive on bathing waters (EEC, 1975). It could be argued

that the present directive is too restrictive in that many waters used

for recreational purposes, with only occasional total immersion (for

example, canoeing), would not normally be considered bathing waters.

Such waters perhaps should be monitored at least in the warmer summer

months when there would be greater public usage.

Sample volumes will be determined by the type of water being examined

and the concentration-detection systems to be used. For routine

sampling as part of normal quality assurance surveillance, ten litres

is sufficient for most waters. Wastewaters do not often require prior

concentration to assay, thus only requiring small sample volumes. In3many cases 10-20 cm suffices. When investigating waterborne

outbreaks of possible viral aetiology, it may be advisable to sample

even larger volumes, 100-1,000 litres, the techniques available3(Figure 7) enabling concentrates of less than 20 cm to be obtained.

Is There a Need for Viral Standards?

Isolation of viruses from treated waters implies that potable waterl

treatment processes may not adequately insure the^ removal of viruses

from such waters. This is further exacerbated by the increasing

frequency of virus isolations from waters considered to satisfy other

water quality parameters, in particular the indicator bacteria. The

presence of low levels of residual disinfectant points to the

differences between the sensitivities of the various micro-organisms

to disinfection with many bacteria apparently being less resistant

than most viruses (Sproul, 1976; Engelbrecht etal, 1980; Grabow,

1982). The question arises as to whether there should be viral

standards for drinking waters as have already been applied to bathing

waters.

Legislation governing the permitted levels of viruses in drinking

waters has only been imposed on one occasion. In Arizona, a state

with an acute water shortage, drinking waters must not have

demonstrable virus in a forty litre sample. The European directive on

drinking water (EEC, 1980) makes the recommendation that viruses

should not normally be present in such water without laying down

specific guidelines let alone mandatory requirements, a situation

which has been approved by some UK workers (Gamble, 1979). The World

Health Organisation (WHO, 1984) while not able to impose mandatory

standards has endorsed the sentiments of the US National Academy of

Sciences (NAS, 1977). They recommend that where virological

facilities can be provided, it is desirable to examine waters for

viruses mainly as a means of gaining data in order to better assess

the risk to human health from the consumption of virus-contaminated

water. Rather than suggest a virus standard for drinking water, WHO

take the attitude that continued vigilant treatment of good qualityl

source waters, resulting in drinking waters of low turbidity and a

residual disinfectant level of 0.5 mgl ^ free chlorine after 30

minutes contact at pH 8.0, is adequate in most cases.

In the UK, several water authorities have recognised that the presence

of viruses in drinking water supplies may compromise their obligation

to provide 'wholesome' water. However, only one, the Thames Water

Authority, has seen fit to exert an internal standard as a policy

directive. In this case, water leaving a treatment works is required

to be free of virus using a 1,000 litres sample if viruses have been

detected previously in a ten litres sample of the distribution water.

An alternative to establishing a virus standard for drinking waters

would be to lay down minimum treatment requirements (WHO, 1984), aimed

specifically at removing the virus problem, coupled with

recommendations for remedial action as and when such treatment regimes

fail to remove viruses. Such an approach has its merits, but it would

still seem prudent to have at least a guideline value for permitted

virus levels in tapwater. The original suggestion by WHO (WHO, 1971)

of less than one viable virus in a ten litres sample would seem to be

the most reasonable approach. Such a volume can be easily handled.and

the water can be collected as part of a normal bacteriological

surveillance programme using similar sampling procedures.

Virological standards for wastewaters and wastewater products, such as

sludges, are probably unnecessary in the UK except where sources of

potable supply may be threatened. Where it is necessary to recycle

wastewater rapidly for the production of drinking water viralI

standards would seem to be appropriate, the degree of contamination

dictating the probable treatment the water would receive. In some

cases it may be necessary to disinfect wastewater prior to potable

treatment but even here, as has already been indicated, substantial

numbers of viruses may survive. Standards for wastewater sludges have

been applied by some countries. For example, in West Germany sludge

is irradiated to eliminate viruses prior to being used for

agricultural purposes (Koch, 1982, personal communication) as present

day sludge treatment processes give variable reduction of virus levels

(Berg and Berman, 1980).

Limited virological expertise and facilities precludes the ability to

carry out wide-ranging analyses in order to assess the degree of

conformation with any standards imposed and, where it is necessary to

satisfy such requirements, the surveillance of water intended for

human consumption must take the highest priority. There is no doubt

that, despite the lack of epidemiological evidence to support the

hypothesis that water is a significant route for virus transmission,

the presence of viruses in drinking waters is not only unacceptable

but inexcusable, but in cases where water is used for bathing such a

situation may be unavoidable.

Communications

In the UK three organisations are directly involved with water

quality? water supply undertakings, environmental health and the

public health laboratory service. Waterborne outbreaks of viral

disease probably do occur in the UK, but in many cases these arel

undetected or are not acted upon because of the lack of communication

within and between the concerned bodies. Three instances will

illustrate the difficulties encountered.

The first involves the failure to communicate the breakdown at a

potable water treatment plant processing river water. The floe

blanket had collapsed and it was nearly six weeks before it could be

re-established satisfactorily. In the meantime, highly chlorinated

river water, subjected only to rapid sand filtration, was being put

into supply (Kemsley, 1983, personal communication). Whether disease

outbreaks occurred is not known. In this case there was a total lack

of communication between operational and water quality staff either

through design or default. Such a situation should not happen.

A second example, the so-called Bramham incident, involved an outbreak

of gastroenteritis in a small Yorkshire village (CDSC, 1980) . For

several weeks before the disease was first reported, the lack of

disinfectant residual at the treatment plant was not made known to the

microbiological staff who were noting an increasing rate of failure on

bacteriological grounds (NWC, 1980). Ultimately it was found that the

chlorine dosing pump was defective resulting in unchlorinated water

entering supply. Such pumps are widely used in the water industry

where it is necessary to chlorinate waters with chloros solutions.

A third instance, involving the environmental health organisation,

resulted in samples of faeces and serum (from an outbreak of

apparently waterborne gastroenteritis; Edale incident, CDSC, 1982)

reaching the examining public health laboratory three weeks after

having been taken. By that time the samples had deteriorated.I

Such failure to co-operate with each other points to the many faults

within each organisation. In part it is evident that some sections of

the water industry are unwilling to lose face by admitting that

problems do occur. Similarly, the failure to appreciate the wide

range of agents which can be responsible for disease is a major

handicap to environmental health officers when dealing with

outbreaks. The PHLS is too overworked and under-staffed to deal with

material from such outbreaks quickly and effectively as and when such

incidents occur. In particular, there seems to be little sense of

urgency, perhaps related to the belief that waterborne disease does

not occur in the UK, a situation worsened by the fact that the

commonest waterborne outbreak, acute gastroenteritis, may not be

recognised because gastroenteritis is not a notifiable disease.

However, the apparent absence of waterborne disease in the UK can only

be attributed to lack of epidemiological surveillance. That outbreaks

of waterborne gastroenteritis do occur is highly likely but such

incidents go unrecognised. Increased surveillance as has already been

applied in the United States (Lippy and Waltrip, 1984) has shown how

common, relatively speaking, waterborne outbreaks of gastroenteritis

actually are. Such findings have now been supported by data from

Sweden where thirty-two outbreaks of waterborne disease occurred

between 1975 and 1984 involving nearly 12,000 people ranging from

single family outbreaks to community outbreaks affecting up to 3,000

people. About 60% of the cases could not be attributed to an

identifiable pathogen although it is likely that many may have been

caused by the Norwalk-type viruses (Andersson and Stenstrom, 1986).

\

Concluding Remarks

The present study, and indeed most of those which have been published,

has examined the incidence of cytopathic enteroviruses in water.

However, the lack of epidemiological evidence to support the

hypothesis that water is an important vehicle for transmission of such

viruses questions the validity of continued examination for these

pathogens. Certainly, the viruses which have been shown to be

unequivocally transmitted by water and give rise to disease outbreaks,

particularly rotaviruses, Norwalk-type viruses and hepatitis A virus,

are not detectable by the commonly used techniques. In addition, the

need to obtain results as rapidly as possible in order that

virological surveillance can have a role to play in operational

practices, indicates that current procedures for virus detection are

not satisfactory.

The advent of rapid techniques for the recognition of viral antigens

has major implications for present-day water virology and the use of

such procedures will permit water virology to become comparable to

current practices for bacteriological monitoring. However, only a few

of the current techniques are applicable to the water context because

the need to determine the infectiousness of any water is essential..

Two simple approaches are available, differing only in how the test is

presented for examination. Both rely upon the detection of

intracellular viral antigen, produced usually after only overnight

incubation of infected cells, as a result of a limited replication

cycle. By the use of indirect labelling techniques, the presence of

the antigen in the cell is amplified to a point where it becomesl

visible by low power microscopy. In one procedure the antigen is

visualised by fluorescence under ultra violet lighting conditions

while the other relies upon the production of a coloured product from

an enzyme-mediated reaction (peroxidase) . In either case, it is

possible to count individual or foci of labelled cells and determine

the numbers of viruses present, in essence a 'mini-plaque' technique.

Such immunocytochemical procedures (Lennette and Schmidt, 1979)

usually provide a result within 24 hours of test initiation. However,

such studies are essentially conducted with respect to specific virus

types, for example for rotavirus detection. Unfortunately, the

difficulty in cultivating such viruses as hepatitis A and the Norwalk

group, together with the lack of suitable reagents for their

detection, is a major handicap especially since these are, arguably,

the most important of the waterborne viruses.

The detection of hepatitis A virus has, traditionally been hampered by

the slow growth of the virus in cell cultures with incubation periods

of up to six weeks being necessary to obtain maximum titres (Provost

and Hilleman, 1979). However, the detection of specific hepatitis A

virus antigen by immunofluorescence within three days of test

initiation has recently been reported (Anderson ejt al, 1986) and may

provide the means of examining water concentrates for the virus when

immunological reagents are readily available.

A novel approach to the rapid detection of 'total' virus has been

proposed by Payment and . Trudel (1985) who have used commercially

available pooled human immune serum globulins for the detection of

viruses in water concentrates by immunoperoxidase. In three out ofl

seven drinking waters they were able to demonstrate low levels of

viruses which could not be detected by a cytopathic effect assay

system. The main drawback to the use of a system of pooled antibodies

for the detection of intracellular antigen (whether of an unknown

nature as in a commercial pool preparation or as a specially prepared

mixture of known antibodies) is that identification of specific

viruses is not possible, but in the water virology context, it may be

sufficient simply to detect and enumerate infectious virus. Another

disadvantage of a commercial preparation is that the antibodies

present may be of a wide range of titres, thus limiting the degree to

which dilution can be carried out. Too high a dilution and there

would be insufficient antibody of one kind or another to successfully

label intracellular antigens. The preparation of a standardised

mixture of antibodies (possibly produced using monoclonal technology)

would be a major step forward in providing a practical tool for the

water virologist which would elevate the discipline to a higher degree

of relevance even than current bacteriological analyses. The

prospects for sensitive, appropriate and reliable water virology are

now very promising.

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MORRIS,

MORRIS,

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BUTLER,

MORRIS,

LLOYD,

MORRIS,

R (1980). Viruses in water. Soc. gen. Microbiol. Quarterly,

2# 53-54.

R and WAITE, WM (1980). Evaluation of procedures for recovery

of viruses from water. 1. Concentration systems. Water

Res., 14, 791-793.

R and WAITE, WM (1980). Evaluation of procedures for recovery

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WALLIS, C & MELNICK, JL (1968). Mechanism of enhancement of virus plaques by cationic polymers. J. Virol., 2_, 267-274.

WALLIS, C, HOMMA, A & MELNICK, JL (1972). Apparatus for concentrating viruses from large volumes. J. Am. Wat. Wks. Assn., 64,189-196.

WALLIS, C, MELNICK, JL & BIANCHI, M (1962). Factors influencingenterovirus and reovirus growth and plaque formation. Texas Rep. Biol. Med., 20, 693-702.

WALLIS, C, MORALES, F, POWELL, J & MELNICK, JL (1966). Plaqueenhancement of enteroviruses by magnesium chloride, cysteine and pancreatin. J. Bacteriol., 91, 1932-1935.

WALTER, R & RUDIGER, S (1977). Untersuchungen zum virus vorkommen im grundwasser. Z. Ges; Hyg;, 23j, 461-463.

WALTER, R, DOBBERKAU, H-J & DURKOP, J (1982). A virological study of the health hazards associated with the direct reuse of water. In Butler, Medlen and Morris (1982) pp. 144-153.

WALTER, R, DOBBERKAU, H-J, DIENER, W & DURKOP, J (1982a).Experimentelle untersuchungen zur zirkulation von enteropathogen viren. Z. Ges. Hyg., 28, 391-395.

WALTER, R, DOBBERKAU, H-J, BARTELT, W, DIENER, W, HARTEL, I, HEINRICH,U, MULLER, U, RUDIGER, S & STETTNISCH, B (1982b). Long-term study of virus contamination of surface water in the German Democratic Republic. Bull. WHO., 60, 789-795.

WARD, RL & AKIN, EW (1984) . Minimum infective dose of animalviruses. Curr. Rev. Environ. Control, 14, 297-310.

WARREN, WR (1953). Epidemiology of infectious hepatitis. Armed Forces Med; J . , 4, 313-335.

WATSON, PG, INGLIS, JM & ANDERSON, KJ (1980). Viral content of a sewage polluted intertidal zone. J . Infect., 2_, 237-245.

WECKER, I & MUELEN, V ter (1977). RD cells in the laboratory diagnosis of enteroviruses. Med. Microbiol. Immunol., 163, 233-240.

WELLINGS, FM, LEWIS, AL & MOUNTAIN, CW (1976). Demonstration of solids associated virus in wastewater and sludge. Appl. Environ. Microbiol., 31, 354-358.

WELLINGS, FM, LEWIS, AL & MOUNTAIN, CW (1977). Survival of viruses in soil under natural conditions. In 'Wastewater Renovation and Reuse' edited by FM D'ltri. Marcel Dekker pp. 453-478.

WELLINGS, FM, LEWIS, AL, MOUNTAIN, CW & PIERCE, LV (1975).Demonstration of virus in groundwater after effluent discharge onto soil. Appl. Environ. Microbiol., 29,751-757.

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WHO (1973). Reuse of Effluents: Methods of Wastewater Treatment and Health Safeguards. World Health Organisation Technical Report Series'No. 517.

WHO (1984). Guidelines for Drinking Water Quality. 1. Recommendations. World Health Organisation.

WILCOX, KR (1961). An epidemic of infectious hepatitis in a ruralvillage attributable to widespread contamination of wells. Am. J. Hyg., 74, 249-258.

WILSON, JG (1957). An outbreak of infectious hepatitis. Med. J .Austr. , jL, 832-833.

WILSON, JN & COOPER, PD (1965). The effect of light on poliovirusgrown in neutral red. Virology, 26, 1-9.

WILTERDING, JB, WEILAND, HT & VERLIND, JD (1970). A longitudinal study on the significance of examination of sewage for the presence of poliovirus in the population. Arch, ges.Virusforsch., 32, 82-90.

WONG, DC, PURCELL, RH, SREENI VASAN, MA, PRASAD, SR & PAVRI, KM(1980). Epidemic and endemic hepatitis in India: evidence for a non-A, non-B hepatitis virus aetiology. Lancet, 2, 876-879.

WULLENWEBER, M & AGBALIKA, F (1984). Enterovirus types in samples of activated sewage sludge. Zbl. Bakt. Hyg. 1. Abt. Orig. B., 178, 522-526.

WYN-JONES, AP & EDWARDS, ER (1982). The adsorption of enteroviruses by river sediments. In Butlfer, Medlen and Morris (1982) pp. 77-83.

YORK, DW & DREWRY, WA (1974). Virus removal by chemical coagulation. J. Am. Wat. Wks. Assn., 6 6 , 711-715.

YOSHII, T & KONO, R (1978). Optimal conditions for plaque assay ofechoviruses in human embyronic lung fibroblast cells. Jap. J. Med. Sci. Biol., 31, 87-90.

YOUNG, DC & SHARP, DG (1977). Poliovirus aggregates and their survival in water. Appl. Environ. Microbiol., 33, 168-177.

ZAMOTIN, BA, et al (1981). Waterborne group infection of rotavirus aetiology. Zh. Mikrobiol. Epidemiol. Immunol. , (11) ,1 0 0 -1 0 2 .

ZAVATE, O, COTOR, F, IVAN, A, TIRON, S & AVRAM, G (1980).Investigations on the circulation of enteroviruses in achildren*s community. Rev. Roum. Med. Virol., 31, 289-293.

ZDRAZILEK, J, JADRNICK07A, N, JANDASEK, L, KASOVA, V, UVIZL, M & VALIH, J (1974). Presence of poliovirus and other enteroviruses in sewage: a survey of Czechoslavakia1962-72. Bull. WHO., 50, 562-563.

ZDRAZILEK, J et al (1982). Presence of polioviruses and other enteral viruses in sewage: a survey in the Czech Socialist Republic 1969-1976. J. Hyg. Epidemiol. Microbiol. Immunol., 26, 1-14.

ZEJDL, M, LITOV, M, HELEL, J & LHOTSKY, 0 (1965). Infectioushepatitis epidemic spread by water. J. Hyg. Epidemiol. Microbiol. Immunol. , _9, 374-386.

ZHANG, C et al (1983). Isolation and identification of viruses in the water of East Lake in Wuhan. J. Chin. Soc. Environ. Sci., j4, 55-58.

ZHANG, C et al (1984). A preliminary study of the virus contamination in tap water. J. Envirom. Sci., _5, 29-31.

ZHUMATOZ, KH & DARDIK, FG (1958). A waterborne outbreak of infective hepatitis. Probl. Virol., 3_, 37-41.

APPENDIX A: OUTBREAKS OF WATERBORNE DISEASES

Al Hepatitis A

A2 Non-A, non-B hepatitis

A3 Rotavirus

A4 Norwalk-type

A5 Enterovirus

A6 Possible Norwalk in UK

1194S

Table

Al Ou

tbreaks

of He

patitis

A At

trib

utab

le

to Co

ntam

inat

ed

Drinking

Wate

r(based

on the

review

by Mosley

(1967)

and

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ted)

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•H in •H D r - m <ts| CD E <Z i—i — ' 0 04 •H ID o ■—. CD ID - n >Cn in US cn CO w *—' CQ rH Ch to rH £ CTi U to

CD rH «. CTv k rH ■1G H co "—' rH o: ID AC rH tO cO >z N— <z AC rH co N — <d | to CD c p iZ <z 04 P —' X5 uG •H *— H i 0 •H N 0 •H CD •z rH 2 O <CD to c co c CD 4J C > i -tJ >4 C CO rH rH U co c —* X toV-i u 0) u CD to IH CO CO o CD to CD to H CD CD to u 0 rH k O DCD 0) in CD N •H •H in E in rH •H ■—1AC CD C 13 i—1 CD T5 CD H) rH O H i c

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Table

A2 Non-A, Non-B

Hepa

titis

Wate

rbor

ne

Outb

reak

s

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1194S

Table

A3 Rotavirus

Wate

rbor

ne

Outb

reak

s

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Table

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p ID e- CO CO 00 cn cn o o CN o G 0CO e- r- e'­ e r- r- r- CO CO CO 0 •H tpCD CTi cn en cn cn cn cn cn CTi cn p 0 c

rH rH rH rH rH rH rH i—l rH rH G p •H0 pU CO 0

0 Pi>i cn Sh

cn P prO 03 c Sh 0 0 1CD CD •H X P *0 cP P CD P 0 G p rH

CD G CO CO rH CD P 'V P 0 o 0cn 0 G c X CD 0 0 0 U X 2£co •H •H •H •H E P P £ 0 p p

C p p E E CO CO 0 CO 0 00 Pi (0 co CO CO p S c C r—1 p Z•rl CD G p P 0 CD •H o rH CN 0p CO•H G C Pi CD P Pi E •H 0 ID £ Oo E 0 0 cn CO CO 0 p S PCD X CO U U X CO £ p P o P tpIP p P cn P P p G C 0 'C 0 0 T3c •H c c G CD c CD•H o rH •V 0 C 0 X 0M o *H •H P •H P S X CO CO CD o G p p s P

o X CO CO Pi C T> P 0 0 p 0 3IP p p X s X 2E P •H o P COX p O c 3 •H X0 CD CD o CO CO CD CO •H CO c CO 0 •rH tp > •H

P cn rH •H t P •H p P P 'O •H •rH P CO U 0 PCD CO co o «P CDMH CD CO CO G E tp 0 CO o co p Pu S 2s rH > rH > 5 o CD CO COt—i & o rH 3 Pp CDip rH •H rH •H co P P P rH p X rH 0 03 Pi CO 0 CD P CD P Pi X O CO G 0 Pi u O Pi0 CO X X co CD 0 X 0 c c 0CO Et n* CO CO PS U CO E-i D * H X

1194S

Table

A5 En

tero

viru

s Wa

terb

orne

Outb

reaks

(after

Mosl

ey,

1967)

o r o r o c n r o o o u o in r H vc> in V D C O I ■—l

oroIroH'H'cooocriOjro^OVO'iffJOvO'iCOO'ir l r l H r l r l H H H

coj>o(-104c<D0JX!4->Oc

>V-I c c c 3 c c to CQ4J QJ a> QJ Q) 0) <D no cdG no no no no no no (0 JC3 a) Q) Q> QJ a) <u < c0 5 s £ 5 cn (0 ajU U0 cn cn cn cn cn D u G

uoJOuo4-1<0SCQcd

wMcd<DUJQ4J3OQJX

4-1

cnc•HJScn•HiHJOcd4-1CQQJ

HOu H vwa; Q)4J 4-1 CQno cd •HS S cn

cd0) H w QJ w JOO <u QJ U <ucd 4-1 c 4-1 cd C 4J OJvw cd o cd vw O cd JCw S •H s w •H 5 4-13 4-> 3 4-J

G no cn rH cd rH cn cd aj CQO c i—1 c rH c o (U•H o no 0) •H ai no •H cd CQ4J CU Q) E s QJ E vw CdU 4-1 cd 4-1 cd w O<u 1 cd & 4-1 04 cd 4-> 3

0)VH a> QJ c aj QJ G CQc rH w QJ o QJ u 0 CQH r-l

QJ4-> no o no 4-J O no

a>Q)JO

vw & no QJ no >1 Q> 4-1 4->0 rH Q) 4-1 .QJ i—1 4-> cd

0) rH 4-1 cd JW rH cd G iHo> 4-1 cd cd E cd cd E •H iHo (0 •r l QJ •H QJ •H •H U cdw > 4-1 w x W 4-1 X 03 •H W 4J 0 4J W 0 rH GO W cd C w C cd w X Hcn 04 Oi to P-i D P-4 Ph u '—

1194S

Table

A6 Waterborne

Outbreaks

of Possible Norwalk

Aetiology

in the UK

p<o0X

CNID«nrH

CO 030 tD 0

•iH tn jc04 rH 00 '—" -H0 . rHf—1 H P .—- .—- -•—.rH 0 3 o iH o o

0 •iH , 0* 00 00 00 00V U P C cn <n • cn cnc 0 | 3 rH rH rH rH0 US --- --- 'P G -0 0 0 < o U u UIP 0 0 & cn cn cn cn0 0 P fc4 Q Q D QPi GS o w u u u u

i—Iin

•VH

p0pto0o•r0P

o o o oO rH

oinro

ooo

oIDrH

tO Gs o•H1-1 4J 0 O

> QJ H C P Co

O tp

•H tO G5 tO 0

o *HP P 4-JQJ O <04-> CfO >i -H S XI P

o0 j rHto pE-J U

tpo

4-JcQJ Pe qj4-J 4-J tO tO QJ 51-4P ro

cQ» 10 4-J tH fO; 143 ’ 0 cn O QJ E 03 tO GH

14 QJ OJ 14jc 3 4-J rH QJ -Htn fO O 04 4-J

14QJ o Qj >1 O

4-J & (0 Or G 3 *H

GoH

14 4-Jcn rHto

QJ to

cnc•H

c•H14

C Ni—1

0 0 P p0 i—1 E 0 00 0 0 0 0p 0 •H P P PP rH rH E 0 0G 0 P 0 E E0 03 0 P O 0s w U CQ cn cn

cnc•H4*G•H14

03OJJCto-iH !—1 P 3CUc3

<

to

*c0•H >1

HeHe

>i >1 >1 >1P cn cn cn cn cn tn tn cn0 0 0 0 0 0 O o oo rH tH rH i—i i—i rH rH rH•rH o 0 O 0 o O o oIP •H •rH -H •H •H •rH •H •H

•iH E E E E E E E EP 0 0 0 0 0 0 0 0c 03 03 01 03 03 03 03 to0 •H •H -rH •rH -rH •rH •H •H03 04 a. 04 04 Ou 04 o. 04H 0 0 0 0 0 0 0 0

oin

QJI—Ito03W

o vd cn O o O O CNin vd r- 00 00 00 00 00tn cn cn cn cn cn cn <nrH rH tH tH tH tH tH i—!

•H 105 to o14 14QJ O 4->fO >iS.

OrtoEh

to

44 OJ O rH

Oto JC to QJOJ 14 14 Ocn PGrl

03 >i TOiH

-O ft'O QJ 04 QJ4-J 3 4-Jto to toC G•iH 14 -HE QJ EfO 4J fO4-J fO 4Jc £ co oo o

UHo4Jc0) 14E QJ

>i P P rH to fO Or 0 5 Or P 3 4-J 'O

CQJ fO

P 4J rH OJ tO P P 3 O fO C71 O 5 QJ E

03tOcH

to

P0)>OVQ>P

'O •H&top

03cto

to•rH-p■HPQJPCQJOpPtotocntpopQJtocoro•H04top

>1cnorHo•HEOJ03•H

topG<Ucnto

0)>•HPto to . 3too

QJ04 JC QJ P

03 QJ G P fO Q>

toE w O QJ P04 3

I Pto >

tHtOo•Hc•HrHoipo

to•Htoto-O

QJ > i JC rH P QJ

JsC C -H O rH

rO P 04 QJ JC O p cnO 3 jj0) O co P JC o

P sHD

„ to£ « • to •>

-GS •ID C « O•H QJ p -HO ^ QJ o ‘W CO G •H

rH0)cn•Hpcnop

jetoQJP

JOP3O

0-Cpc•H0 r—IoppGfOO*Hip•HGcn•Hto

to

03 0 -p o 3 ro JO

P °S oto 0QJ tOP 0 QJ tOS 3P0 > QJ O P P J2 0p ^ 3 CO o

0 0 JC JC P P

0 0 P 0X p 0 0 rH JC 0 4 P

to Pp 00 0 OJ rH0 P 0 i—1

•H0 > 0

h pp•H10 •» •Hs gJj J-i0 oP ^ G0

003O

JOP0Ecnc•H103

P P 0 0 0 £ cn

ip 'gO g

s »to <*>« Sa « 00EO C 0 -H

00 0 ID Gcn 0 rH cnoJCrH I P *1 «

o c

03 0 030 *rH

p0 0 0 0 o

1194S

APPENDIX B: ISOLATIONS OF VIRUSES FROM THE WATER CYCLE

B1 Drinking Waters

B2 Groundwaters

B3 Surface Waters

B4 Raw Wastewaters

B5 Marine Waters

B6 Wastewater Sludges

B7 Sediments

B 8 Soils

B9 Shellfish

BIO Aerosols

1194S

Table

B1 Isol

ations

of Viruses

from

Drinking

Waters

(after

Bitton

et al

f 19

85)

0)oc<1JX<DU-lQJPi

o00<Ti

JJ30XCQ)

o3m

c•HJxZO<0JJw

•s*CD<nr—I0JJJc (0 r—iCH

<urH HD CD 3 CTi X rH EH

JJ JJc cCD d)

(TJ <TJ CH PH

vr tnCD (1) CTv JJ rH C

oj \ 3rH Pm <0|QJ| O

xCTi 0J G X (TJ Xx qjtso m

co'O3(TJu

TOo1-1

XNJJ1-1(TJ

VD Xov OrH W

JJoj3 —• cn vd

•H VD r—I Ol O rH

*1QJ| CM00

O')

U Pm

o C i—1 _..00 CO 0 -—* oVD cn JJ rH CDcn rH JJ CH CO cn 00rH •— •H O cn rH cn

QJPQ

MlrH ' rH

* r—{ Uo

3 (TJ —e G Q 0) X rH

VD • n (TJ rH X Ml (0 *3’vo QJ C 00 CO M (U X n~ OO'! JS .‘•rH cn ncs (0 U> X cn 00rH Pm rH QJ 3 X (TJ rH cn

CO jj (0 co pM rH CD CDh. co w •H JrC CO cn cn

i—11 X rH I o Ml rH lS 1 11 rH rH(TJ | QJ (TJ <01 OJ QJ (0 <o| (TJ

JJ G X X W X •>JJ| JJ OJ JJI CD

cn■X (0 Q tn x | Ml i—11 ■Hi

<y| OJ O Qj| 0 e O (TJ QJ 1 QJ <ol <o|JS 3 D X CQ O

jj m3 JJ r l Cn k. i •H 4J I X IQJ QJ K. (TJ J»; c rH CO 0JI <u|3 c ■- 3 X & 3 (TJ i—ICn o tn cn >1 QJ O E •H X N X

•H rH 0) •H JJ in X •» N X Ml X X <0rH 3 v-i rH tn CD rH O Ml X X •H 0J Ml0 O (TJ 0 (L) cn (0 fts (TJ 3 OJ E QJ QJPm u PH Pm Pm rH £ Pi S o PH U i P O

QJX rH

QJ 3 (TJ> QJ rH 3

•H QJ c •H T3 0)0) X C •H (0 •H cc •H •H X X cn •H•H X 0 aj XX rH rH 0 o rH QJ X 0 rH X i—10 (TJ <0 CH rH X C rH <0 c (0

rH 3 3 X o •H i—I X 3 QJ 3X T3 ■O dP O X i o H3 E T3

•rl •H VD X O rH •H X •Htn tn X X rH X tn <0 tn

X QJ 0) w X •H X cn X QJ QJ OJX X X in •H £ o E •H X X X•H c XJ QJ tu QJ c •H QJ w CD QJ QJ QJc c > •H 0) (0 0J QJ > 0) • QJ > e rH CQ) •H •rl •rl (0 QJ > X > > •H > o > •H *H (TJ -H> U X X X > •rl X •H •H X •X •H X V-I C X

•H 0 O •H X •H X tn X X *H X c X •H 0 o Otn X i—ir—1 tn tn X •H •rl •H rH tn rH •H (0 QJ •H rH tn rH •H rHX •H X X O •H CO <u in in (0 o <0 tn X c W <0 O X X Xc tn o o ch — tn 0 d 0 0 3 CH 3 O X •H Q 3 CH o G oQJ o tj 0 CH •H CH CH tJ TO CH u CH 'O 0JE CH X X <*> rH CH o •H dP •H in 0 *H dP X > XE X X o •H <#> o dP dP tn r~ in dP tn r—1 dP tn t " X G X0 dP •H •H o 5 dP o <0 O rH aj • QJ i—1 QJ X ro 0) • •rl o •HU r- £ I—1 cn r- > ro rH X o X rH m3 o rr X CM £ O

CDG

•HXOi—Ixo

xj j•H

0J>•Hj j•H rH W (TJ

8.-3•H

dP inO 0) rH 1-1

'O QJ JJ O OJ JJ QJ rOinQJW3x

•H>

T3<

in

ro in -CQ CM

CO CQ rH in

(TJJJ

P-. pq pH U PH PP CQ

0 (0X X

OX

(0 VDX0 VDX LDro «•o o 0 0 r-~ 0 o in

CM X X X X Pm3 X X VD paQJ QJ QJ QJ 0) aj

0 X X X X •- X X *■0) G C C C rH c c CO rHPi W W w Px3 CH P>3 W m CH

X (0X T3G (TJ3 G0 (0O U

(TJo

•HPiQJ

(TJ (TJ OC JJ C

•H W (TJX O 1-1U U In

(0•HCd TD

Q C U M

OJ(TJ1-1WM

o>1 urH *H (TJ X JJ QJ H S

1194S

Table

Bl (C

onti

nued

)

Oro 00 0000 cn e'­cn rH eni—1 rHw VJ r"c 0 e'­ >10 JJ en CDJJ CQ t—I GX CD OCD £ k. 0cn G £•sr • W 0

00 iX 00 CQ . in HI CMcn e'­ c e~ e- 00i—l c 00 en CD cn e'­ Vj cn

c cn rH JO rH en CD rHCO iH *3* •H rH UJ

t-l E k. r" CD ». UJ k.CO rHl cn vQ rH ** CO rHl

0 rH CO rH CN in CO rH XZ C0|JJ VJ CO dts00 00 CO cn

CD CD U •. cn cn 4J 4JIU Jj CD 0 CO tH rH CD 4J K CD |G w CD XX > CD UJCD O >1 Vj xz 0 kk w 1 CD rH rH>-4 •H js CO 0 CQ xz VJ CD XX G CO COCD £ Oj JQ JJ •H CO CD 13 Vj XZ C3 J.C<4-1 cq VJ Vj CQ n i—1 rH CO CO CD JJ •roCD 0) 3 CD CD co CO rH i—1 O CD CDPi w £ O !S Pi £ EH cn U ffi £

CQCD CDG CD CQ

CD CD •H G CD 3C C VJ •H G Vj•H •H O VJ •H CD •H

kG U U rH O VJ G >JJ 0 0 XZ rH 0 •H•H rH rH o XZ tH VJ 13xz .G u XZ 0 o CD

u U rH o jj iH VjCD rH tH XZ 3> rH CO tH cn tH 13 o JJ•H CO 3 CO E cn CO CD Ojj 3 U 3 E 3 E tH 3

13 •H 13 o 13 Vj CO VjCQ r—I •H CQ •H . • cn •H O 3 I- JJ0 CO CQ CD CQ rH • CQ 4H 13 CQ CQO j 3 CD Vj CD \/ »H CD G •H 3

13 Vj Vj Y v Vj 0 CQ VJ 13dP •H rH Y O CD •H Cro CQ CD rH 1 CD H CD rH Vj > 3co CD > 1 rH > CD 1 > CD 1 CQ 0 O

VJ •H i—1 •H > rH •H > rH CD rH c VjC jj tn JJ •H jj •H > CQ 1 CD

CQ CO CD ■H cn E CD •H jj tn •H JJ tn 13 rH 13 rHJJ .G G CQ E G CQ •H E to •H E JJ Vj < rHC JJ •rl 0 in •H 0 CQ Q CQ *H CO tn COcd VJ Oj in cn U O j 0 cn O j O e~ CQ 13 E II Eg CQ 0 • • 0 Oj • O j • 0 G CQg CQ i—1 dP rH o rH dP o dP iH O j CO 00 130 CD x: cn V V xz dP v ro dP \/ JJ • < IIt_> tH o rH Y Y O i—1 ro Y *3* Y ro CQ o

>< cnPj

CD tnCQ •HVJ XX •k

fc. CD o CQ<0 CO XZ CO 3(I) JJ JJ CQ VjJJ 0 O X •Ho VJ 0 ><D CO + u 0JJ JJ w VJCD 0 in ng II CD13 VJ ». c JJt—i CO < GCQ jj CD0) 0 no 0 ro 0 0 0 kk.(0 VJ < w Vj Pr Vj VJ VJ 0 0 CQ II0 cd > CD CD CD •H •HVj jj - cn JJ JJ JJ tH rH CD O•H c rH Pi rH G G C O cn rH 0 cn -H Vj> H Pj cn Pi < W W W Pj Pj Pj Pj PQ XX CD

o JJCO CCQ wX0 Iku CQ

313 II VjC •HCO CQ >rH O coCQ ' o • «. jjH •H 0 o

Pi •H VJXX * CO rH

VJ 0 rH 0 •H 0 IIJJ o 0 JJ c PjC •H IH Vj co Pi CO3 X Vj CD E cn < II JJo a) 0 3 O cn « cn OU £ £ Pt Pi D D D Pj Vj

1194S

Table

B2 Isolations

of Viruses

from

Grou

ndwa

ters

(after

Bitton

et al,

1985

)

in cCD 0OJ 4-Ji—1 4-J

•rHinCD

k CN P CTiH CD HfO l

4-J 1OJrH

CTi o by

HiQJ|

£ CTiCDCTi ro

CDCTi

col

C 0 H H QJ H 4410 AC 4-J <u|4-J >4 k k •H in k4-J i" 3 (0 to o CD CO c•rH r - p X! X CTi X o

CTii—1 c8 J4

QJ>4OJ

L984 r-

H inr -

XQJ

XXX! 3

CTit-

CJ u CTiH

kCD

CTiH

CDn~

CNCD

U •Hp

14 (0 CTi <4! CO r- CTi CTi COno QJ AC rH •- CTi k H HQJ OJ X H H k H H Hi AC X

4-J •H QJ k (0 CO H 1 CO co! k k O*H no X Hi >i >i CO I k H 1 1—1J •H noo 3 X fO l Q Q in H 4-J |CO I col £ QJ

PJ 0 0 0 CD QJ CO QJ 1 in XQJ cn P 4-> 1 Qj|CTJ CTi r~ H 441 <u •HO QJ co Q)| k k rH •rH iH CTi in QJ 1 oj! ta u3 c k AC AC H H o>Q) •rH u X H 3 3 i—1 k H k c C H k XX Cn QJ QJ CO O O tO Hi •H QJ k •rH X CO H COQ) n3 4-J 4-J 4-J N N 4-J <0l 14 no AC rH OJ AC CO 1404 r-H r-H rH 4-J J4 X 4-J 4-J to O rH 3 •ro >i XQJ •rH (0 CO <0 CO CO CO 4-J QJ H to QJ CO QJ O COPJ > & £ pH s s P Q) Pc in 2 £ > « U Pc

inXc<1JEEOU

•y-toac10QJX£>4-J3O

IQ•HX H •H I>4 rqj4-» 5c o o>4 4-Ju in3 CO ro OJ

O'c

toOJXto tn XX •H X

3 X QJ k X 04in 0 X OJ OJ •H OJin X & X G c X no0) X cn aj CO •H 0 QJX X •H 3 no X X X EOJ 0) £ X O Oc in G CDc X •H E QJ QJ QJ QJ CN•H X QJ > CN X QJ E O

CO E 0 rH no •rH X XX O •rH c rH X toQJ c tn rH X ■H E tn> OJ 0 eO 'O’ c CO H-H > k & H in CN <0 •H OJ 1X QJ c <u Q> H H QJ H

o > — > QJ H 0 X tn X0 QJ •H •H ro •rH ? OJ X CO H o 3X H X X H X 5 H X

X to •H tO -H •rH 04 Oj QJ Q> AC 04X to c tn •H tn in cu £ 3 OJ 5 too X •H 0 X 0 O ' OJ O X QJQJ o X & OJ Pic c no i—1 in CO E X o

•ro QJ 0 X QJ •rH H H X X •X X H OP o E OP U E CO H o VD X o3 QJ X o to 0 [■'- & o X Q) OJ v 3 \/tn no o CN X in H in CO in S X 1 O Y

HDd)4-JO<D4-J0)roin<uin3x•H>

H inrH 3 >fct CN X < CN

o 0 r- 0 o 0 0 o 0 0 •H tn 0X X X X X X X X 0 X > X oQJ OJ 0 QJ OJ QJ Q) OJ QJ •H Q) 0 CO QJ •HX X X X X X X X X iH X X k X i—1G c o G G C G c c 0 C u CN G oW pa w w Pa P3 pa pa pa P4 pa pa P pa ft

> i ?X 0) HX O Q>G C CO3 to p ; XO X Q inU Px u H

(0 ri34-J WM D D

1194S

Table

B3 Isolations

of Viruses

from

Surface

Waters

(after

Bitton

et al,

1985

)

VO 44 k HVO 0 rHl 0CO o CP 0t " 00 (—! Gc r\ CP 0*1 GA 44 Or—1 rH 00 k uIJ 0 | 44 CM

CP G 4-* * 1 44 00k k 1—1 •rH 4-J •H CP ro

ID 4-1 td •HPQ c PQ rH 00

r- 3 k 0 0 CPcp 0 rH 3 44

> ik rH

pH xc0x s

COO' O >1S i

44•rH X I faO k

k 0 3 CP as PQ*30

r—Iin rH

xsIn 00

r~ O E-t x >1 3 CPcp 3 k k 0 0 X 4-J a rH VOrH PQ ro 0 VO In 44 •k •H

a-Ef f "

C > VO XI •H ro TD0as X 00 k CP

k o X 0 0 CP N 0 00 CM CP CP ■m * r - rH*3 00 C ro >4 rH 44 CP 3 00 rH rH •EJ1 00 r-0 CP 0 X 00 0 In

L9

82 rH •rl 00 0 CP 00 CP CP k

o rH 3 CP G k 0 uCP 1—1 k k CP rH rH G? c 0 rH rH rH 2 k r—1 in rH rH l rH •rH

X v •H •rH rH 0 | rC rHl ro 0 k 0 k k Xco CO E k. 0 u 0 | X rHl k rH l rH l CO0) r - o 0 rH 1£ CO 00 X 0 H x | rH l to! « l 0& CP 0 X 0 | 0| 441 CP 0 | 0 0 0| tel U

QJ rH 4-J CO k U3 01 rH Q X I X I HO OS CO fck 44 44 <u IT) X>| in 0] •rl 0 H <D| 01 asC k 44 0 I 0 0 0 c 00 10 k 0l 00 k rH lH 01QJ In S-l k 3 > 3 3 •H CP •rl G CP lH J-l rH 3 C In inin as to 0 0 CP 0 tP CP (7* H M 0 44 rH 0 0 •H 3 0 0 O O0 4-J 4-J rH E c 4*: •H •H (0 PQ CO in 0 X X 14 to X X X X

X 4-J 4-J J* 0 E rH rH rH k CO 00 In k r—1 r—1 X X 14 (fa to to0) 0 (0 0 0 x •H 0 0 •H rH 0 0 CP 0 rH 0 0 0 0 0 rH 0 0fa CO CO CO PH CO CO fa fa > 0 X ffi rH 0 0 & & fa 2 CD 0 52 2

>1iH&fa3100rHX0xainOOH

Viu In0 0 > > •H *rlJ-l In

SiasXOfaGJ >•H

X•HtOoChin0> >1*H i—IIn fa fadP 3co to

(1)>•HX•H10Ofa

inOun0>•H

x>1 >1 •H >1

In In in 0 rH rH 0 0 rH 0 00 0 0 0 fa fa > 0 fa > >

0 > X X rH fa & •rH fa fa •rl •rl4»£ •rl 0 0 fa 3 3 X 3 X X0 m 5 S >1 E to to •rl M 0 •rl •rlrH rH 0 0 0 0 0

0 in in c 0 in m 0 X in 0 0rH > 0 0 0 0 0 farH 0 0 fa fa0 •rl > > m > > 1 £ >G X •rl •H 0 0 •rl •H dP rH •rl dP dPO •rl U in rH > M in CP In m CD in•rl to 0 •rl in 3 0 rH rHX O 0 0 > M 0 0 X > 00 fa rH rH 0 ‘ rH 1—1 - fa •rl 0 1—1 k k0 X X 1—1 dP X X in in m X m inlH dP 0 0 CM 0 0 0 O 0 0 0 0O O X X 5 CP X X > CM dP X X > >0 O 0 0 0 \l 0 0

'u Y 00 G 0 •rl •rllH rH fa fa rH Y fa fa 0 fa 14 m

ocO CD 0 *0 In (0

0 0 0m m in<u a» a>X X Xc c c<p <d 0

<u qj4J Xc cQ) QJ

0 0 0 0G G G G 00 0 0 0 X 0

XS X CM X O 00 0 0 as in m

k k w 1—1 k k kO 0 O O 0 0 O O O O 0 Om in m m in In 0 in m m in In in0 0 0 0 0 0 •H 0 0 0 0 0 0X X X X X X rH X X X X X Xc c G c c G 0 G c G C G c0 0 0 0 0 0 & 0 0 0 0 0 0

HIn 0X 'OG 03 c0 0O u

(0•H(0>0

c•Hx

0 0 0 mX 0 >1 c O 00 G rH 0 •H X0 0 fa 0 fa X XN m O X 0 0 0u fa CD w X 2 Z

(0xc0 asi—I *rlc (0 e

3

l§

(0•rHc<0E3

s

1194S

river,

45%

positive

Cotor

et al,

1981

JQ

noCD44

CN00cn

•H H0

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1194S

Table

B4 Virus

Isol

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fro

m Raw

Wa

stew

ater

(after

Bitton

et al,

1985)

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1194S

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1194S

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1194S

Table

B5 Isolations

of Viruses

from' Marine

Waters

(after

Bitton

et al,

1985

)

0oc0u(1)U-l0

XI'O0X•Ho

ro00CTi

W3

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1194S

Table

B6

Isol

ations

of Viruses

from

Raw

and

Digested

Wast

ewat

er

Sludges

(after

Bitton

et al

f 19

85)

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1194S

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> i•H 00PQ cn X

c rH0 13E OS k- 0JH ro c X0 00 0 •H

X G cn id oX 0 i—1 rH C0cn

EJH

00cn fc.

X00 r—1 13 p 00

cniOS 3 JH0 0 os rl

cn X H £JH O 0 X k0 cn OS X G0 PQ 0 0o 0 l u 2 E in

G k. 0 JH000 c X X 0 w 0 cnJH 0 0 0 X cn 3 rH0 E JH JH iq •H 0

X JH JH Jl 0 X k.0 0 0 0 JH 0 O rHos PQ Cm Cm PQ p cn 0

0 o 0cn •H cn13 0 rH u 0 0 03 cn •H 3 cn cn cnrH 13 13 X rH 13 13 um 3 0 o, CQ 3 3 3rH X 0 iH rH rH13 in in IQ 13 CQ CQ CQ0 0 0 0X S cn E X S 130 •H CQ 0 0 00 JH 13 O 0 JH JH Xcn •H cn CQ•rl rH iH X •rH i—1 rH 013 1 1 o 13 1 1 cnrH rH JH rH iH •H

iH 0 rH 131 3 3 0 1 3 3cn x X G cn X X rH

O h Oh 0 O h Oh 1in 3 '— D cnX X O O H O VOG Q, O 0 o o O 30 o r- cn E-f o rH Xp r- 13 O hrH VO ro 3 o o Xs V Y V i—1CQ VOCN rH CN roin

u03C•HXcou

u0Xo0X013CQ0in3M•H>

oJH0Xc0

oJH0XG0

o o oJH JH JH0 0 0X X X G G C0 0 0

VOPQ

X0Eh

>iJHXc3Ou

<cnD

1194S

Table

B7 Isolat

ions

of

Viruses

from

Sediments

(after

Bitton

et al

f 1985

)

coLO 44r- 44<n CN •Hi—t VOr- 00cn PQr- cn iH >i•H cn rH44 rH w<0 t. CN (0 fOrH w G 00 -Q0 CN *H cn J4 QJ

ro 00 o CO rH QJ 44(0 cn •H 44 U •HCQ rH Gi—1 c3 G <45 o*0 ». OJ 0 CO TTG cn s ss o H *0 00CO •O CO Ul C cn*j-j c3 cS r- cn (0 0 rH•H CO f" i—I frj e 00N S CQ CQ cn TJ cnc ro •H •H rH •» <45 W rH 00<u W rH rH o cnJ-J rH QJ CO 3 w »- ■—lQJ <43 fO G rHl O J4 rHl inQ & cO ■•4-G QJ C0|,—100 -QJ IQ w QJ|U Cn QJ cn rHO a> CQ 441 J4 441Jg rH COC CO G 44 cn QJ QJ »■ QJ 041OJ J4 O rH c rH G XI 44J-i 0 D CO •H CO iH O •H rH rH QJ0) rH 1 O iH 42 0J 44 (0 CO CO44 Cu G 44 rH J4 PQ 44 -C >i o , O0) QJ >i OJ QJ QJ (0•H o Q CO "qj 1 P§Gj G £ s & O G PQ cn 0 G

CQ4-JG01 Ou

rHiH rl HOI I I inn QH 3

cn cn 3 3CJ U-l IH H-I

Eh Oi Oi OiCN J* 00 • • •

r H O OY Y Y

rHip i i—I rHI IO H ID

G 3

cn3 H IW U Oj Eh

rr ^ C N

IH 4-1 O j O j

H OV V Y Y

cn3

44Oj

OV

•CJ<1)4-JO(1)4-J(1)r0CO0cn3u

•H>

PQ

o 0 0 0 0 0 0 0 o uJ4 J-4 j-i J-i J-i J-i J-l j-i J-l 0OJ QJ OJ 0) Q> OJ 0 0 0 CO CQ4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Kc G c G G C G c G 0 0QJ 0) QJ Q> QJ 0 0 0 0 J-l u

tnJ-l4-Jc30U

CO <44 « cn H D D

1194S

Table

B8

Isol

ations

of Viruses

from

Soils

(after

Bitton

et al,

1985

)

J2

0)4->•HoincocniH oCOcni—IT3O inO CO e3 cn r—14J iH oCO jCQ)3: -Hi >i<0 i-Q

qj L» -Ml <43o o|c C(I) 14 c ou fti 0 coQJ 4J 4J iH4-1 4J 4-J 0J0J ro •H •HPd cn CQ 2

r-'cn

C•H ro<0 00 004-J cn cnC iH rH3Os <0 >-lJQ QJ14 •Hc8 0) Nu> •HDCO CO•H CO3 .scd) U iH•H CL)3 TDCfl •H0) oj10 « cncnc w•H rH OriH r- fO •rliH r~ >i r—1<D cn Q •H£ rH 0 Er

qjCO cn0) 43 0)

4-J 3 o(0 rH JSC ro 14<0 CO ro 44 OJOJ u 4-J -C

rH 3 44 4-J.sc QJ Cfl ro Orc 4-J QJ OJro 44 Or 3 rH 'O

4-J <C •H o ■HOr iH 0 c E

O CQ 0) CO o u•H £, 14 -O •H o4-J 4-J <L) > i 4-J rHOr C e E ro CNQ) 0 <u o ro cnCD e c CD CO o •H 4-J

0 iH rH 14 ro£1 14 •H 14 14(0 4-J 3 4-J ro 'a >i •H T3

4-J •rl 0 CO OJ oj 03 Q)C 3 44 o c 4-J G OJ 4-><y •rl o ro cn og rH rH rH iH 0) CO ro QJg •H •H Or -H 4-J 3 4-J0 0 0 Or 0 OJ 44 QJ OJO 10 CO to CO *0 0 CO 43

cnCQ fflcn

Q> OJ CN•rl •H \-SC -SC CNO o CN 0 Oro ro 14 14CO CO 0 QJ Q>X x jC 4-J 4-Jo o O c Go o QJ QJ QJ

>1C(0e14

H - V Q)14 ro 14 04-J •V roG ro E 4-J3 c G < CO0 ro . £ cn QJU u Q t> £

1194S

Table

B9 Virus

Isol

atio

ns

from

Shellfish

(after

Bitton

et al

f 1985

)CNCDcnr—1cn

CDcn 44 in COrH • r l4J COcn CDcn•» 3 t—i rH•H Q•sj* X P

r - CO CO COcn 3 ■43 0) a>rH PS C rH•H 1—i

•rH». os •H 4-1 4-1ro 4J cn cn cn

p - •H CO CDa) cn rH 3 cn <43 osu rH rH < r—1c Q) UH UH.oj i-. C P i—1j- i CO •H CO J4 (0 COai •rH 'o •rH qj u o

(44 c c £ rH 4-1 4-10JPS

qjp

a jcq

<UP

a;2

r- pcn CD■—1 in cn cn

p~ pfc. cn cn

MS P P wU X•rl ». JO 3c x MS * <0p o O <0 14<14 4-J •H o 4-12 <0 c CD cn

ffi. r-~ p cn o oOS cn ai P CD CD <43

os P 2 cn cnCO fe. p P 14•rl 14 •> <45 P | QJp <1J P | ml w *. cur—1 > col (0 P | P | o<0 •H X! 4-> 1 ml col o

iH 14 a>l uu a>l QJ 431K CP J4 ail Qj|UH UH QJ crH OJ P w'V c >i 0CO 4-> <0 P C X OJ COo <0 u co QJ cn CO c

4> cn 4-> t>i p 3 X! Xai 3 <u Q rH co O 02 2 O w > cn *3

0)cn QJ<0 4-J>4 COa> CO cCO QJC CO •rlEX E 14 CO4-1 CO O QJ 4-J CO c•H P JH 4-1 c 14 •rl <uQJCO UH C0

>i0u

QJ4-> CQ >•rlCO E O CO 14 4-JQJ 3 MS c >i QJ •rl CO4-1<0 E r - C•H •rl O 4-JCO CO

oEc0

C C QJ CO CO CO P >. rH Oi P•H •rl > 14 P 14 14 1 O CO i oQJ e P •H QJ 1 QJ QJ cn 14 cn CO> <0 P P 4-1 4-i cn 4-> 4-1 o QJ OJ o 14 p•rl 4-1 1 <0 QJ •rl CO o CO CO o > 4-1 o QJ 14-J C cn uh > CO >1 O >i p •rl <0 p 4-J cn•rl o 4-J •H Q O p o O 4-J s CO oCO u 3 3 4-1 & 3 •H 3 >1 oCO 0 UH O •H QJ 3 QJ 0) UH CO H3 UH O p4-1 Oi CO Oi CO CO P > UH > > 04 Q QJ 04

c p QJ 0 14 P •H Q| •H •H CO 04 4-1 UH 3OJ OP QJ cn cn Ol QJ <0 4-1 4-J 4-J >4 3 O O UHo CO <0 4-J UH •H VD •H •H QJ CN 4-1 P O Oi

j l p CO CN £ t#> C0 4-J CO CN CO in 4-J CN CO i—1CN OPou V

3E VQJCO ro

in> iO 3O & y a a <0£ V

oE av IDP

IDCN

'O<u+Jo4-1a)CQa>ca3>4>

CNO CQQ)14 CN O QJ•HP P 140 O O O O O O O O O QJ o14 14 14 14 14 14 0 14 14 14 O 14 44 COQJ 0) QJ QJ 0) QJ •H 0) QJ Q) -H QJ C CO4-J 4-J 4-1 4-> 4-1 44 rH 44 44 44 P 44 QJ XC c 3 C C C 0 C C C O C OQJ Q) QJ QJ QJ QJ Oi QJ QJ QJ Oi QJ P- U

cCOpH co 514 0) QJ44 o >11*C c p3 CO CO <o 14 44 QJ to cnU to H 2 D 0>

1194S

Table

BIO

Virus

Isolations

from

Aerosols

(after

Bitton

et al,

1985)

coX4JCQ JJ|CD

>1 GXI O

X C\00 1 1r0 •*-* r*ai <Xl CQ a\x rH >H•Ho wc X >44-1 0 >0

CDCD 01 O Xj_i 1—1 00 CD 14o CD CTi X o•d c

CDrH •rH

o 02

tS N <24-1CO

i—11C0| •“jl

M in K C0| •HCD CD •Pl cn

(D X CTl cS cd| x l coa cd rH CD | COc 5 X X0) c o u iH>4 CD H l 0) 01 CO CD<D rH cOl 4-1 X X in HU-l rH rH rH X 00 OCD 3 ■ 1 CD CD COCTv Oos £ CD EH EH lit iH 2

rO CDID CD 01 oC X 3 V-l•H CO >4 3£ cn •H 0c *H > 01s 14o 14 X B'D •H X

■rHOuB T3 X

•H o rHCO CD

•Hc0 B

roro T3 •H o 1B CD X X ro Bo X >4 >4 c COCM O CD CO CD cn 3iH CD rH CD 3 •H X

X X G iH 14 Xi02 C CD C X >4 'DX ■H to •H 01 X •H CDG U CD CD X «HCD 3 01 Xl i—1 >1 O OB X 3 01 Xi X CO CD •B cu 14 B X 14 X O0U ro

•H>

X0

CO01 -H5

Xl01 CDV

>1 <14 CD iH iH rHX o CD CD CDG G CO CO CO3 CO 14 >4 140 >4 01 01 01 COU PP M M H P

1194S

APPENDIX C: MATERIALS

1206S

CELL CULTURE MEDIA

Many components of the culture media used in this study were purchased

as sterile stock solutions from either Gibco-Europe Ltd or Flow

Laboratories Ltd.

% Stock Solution in MediumGrowth Maintenance Diluent Agar

Eagles MEM/199 (x 10c) 10 10 10 10Foetal calf serum 10 5 - 2Penicillin-streptomycin-*- 1 1 1 1Glutamine (200mM) 1 1 1Non-essential amino acids 1 1 - 1

(x 1 0 0 c)Vitamins (x 100c) 1 1 - 1Sodium bicarbonate^ 2.5 5 5 5Fungizone (250 ug cm”^) - - - 1Neomycin (10,000 u crrf^) _ _ _ q .5Kanamycin (10,000 u cm“ ) - - - 0.5Mycostatin (10,000 u cm"^) - - - 0.5Polymixin B sulphate - - - 0.5

(10, 0 0 0 u cm“^)Neutral Red^ - - - 3Magnesium chloride4 - . - 2Bacto agar^ - - - 33Deionised water** to 100 100 100 100

Notes:

. . . . . -31 Penicillm-streptomycin mixture; 10,000 ug cm and 10,000-3 .units cm respectively.

-32 Sodium bicarbonate stock of 44 mg cm gassed with carbon-3dioxide (phenol red as indicator 0.01 mg cm final) and

3autoclaved in sealed bottles of 25 cm capacity (15 minutes, 15

psi).

-33 Neutral red stock of 1 mg cm , autoclaved (15 minutes, 15 psi).

1206S

-34 Magnesium chloride at 200 mg cm , autoclaved (15 minutes, 15

psi) .

5 Bacto agar as 3.5%, autoclaved (20 minutes, 15 psi). Mixed with

rest of medium at 48°C in the ratios of one part agar two parts

medium.

6 Deionised water ex reverse osmosis plant, autoclaved (20 minutes,

15 psi) .

OTHER MEDIA COMPONENTS

Stock concentration -3Media component mg c m ...... Sterilised by

DEAE Dextran (Pharmacia)Protamine sulphate (Sigma)5-iodo-2'-deoxyuridine (Sigma)Semicarbazide (Sigma)

Note:

1 Filtration through sterile 0.2u cellulose nitrate membrane.

CHEMICALS USED DURING CONCENTRATION PROCEDURES

(a) Aluminium chloride (AlCl^.OH^O): stock prepared as 1M

solution. Not sterilised before use.

(b) Beef extract (Oxoid Lablemco L29) prepared as 3% solution and

sterilised by autoclaving at 15 psi for 20 minutes. pH adjusted

to 9.5 with 5N NaOH immediately before use as eluant.

10 f iltration-*-20 filtration10 filtration10 filtration

1206S

TRYPSINISATION OF CELL MONOLAYERS

-3Commercially available trypsin solution (250 mg cm ) was mixed with

sterile versene solution to give a final concentration of 5 mg-3cm . Monolayers were drained of medium and covered with the enzyme

mixture.

After 2-3 minutes at room temperature, most of the enzyme was removed

and the cultures incubated at 37°C until all the cells had detached

from the vessel surface. Cells were suspended in fresh growth medium 3(10-20 cm ) and counted by use of an improved Neubauer

haemocytometer.

Versene solution: per litre

8 . Og

0 .2g

0 .2g

1.15g

0 .2g

Sterilised by autoclaving for 20 minutes at 15 psi.

NaCl

kh2po4

KC1

Na2HP0 4

EDTA

1206S

APPENDIX D: VIROLOGICAL DATA

Wastewater effluents 1980-1

Microbiological levels in effluents from five

reclamation works

Wastewater treatment processes: virus levels

River Severn basin

River Trent basin

Stored surface waters

Notes: TC = total coliforms log^g 100 cm"^EC = Escherichia coli log^g 100 cm“^FS = Faecal streptococci log^g 100 cm”^V = Cytopathic enteroviruses pfu 1“^ND> = none detectedW3110 = coliphage assayed on E. coli strain W3110 pfu 1“-*-

(logio) ,ED391 = coliphage assayed on E. coli strain ED391 pfu 1 x

(log10)RS = raw sewage SS = settled sewageAS(I)/AS(E) = activated sludge influent/effluentPF(I)/PF(E) - percolating filtration influent/effluentADF(I)/ADF(E) = alternating double filtration influent/effluentSF(I)/SF(E) = rapid sand filtration influent/effluentHT(I)/HT(E) = humus tank influent/effluentL(I)/L(E) = lagooning influent/effluent

Serotypes: P = polioviruses, B = coxsackie viruses group B, E =echoviruses

D1

D2

D3

D4

D5

D6

1206S

TABLE D1 Virus Levels in Wastewater Effluents 1980-1981

Works Sample date pfu i i1 Serot

ALFRETON 24.6.80 1100 _23.7.80 1000 -

28.8.80 < 1 0 0 -

29.10.80 < 1 0 0 -

25.11.80 - < 1 0 0 -24.3.81 100 B329.5.81 6200 B3,525.6.81 17200 B3,5

APPERLEY 24 .6.80 < 1 0 0 -23.6.81 200 B5

ARLEY 12.6.80 - < 1 0 0 -_ ASHBOURNE 28.8.80 < 1 0 0 -

21.10.80 < 1 0 0 -AVENING 17.3.81 -<£100 -

1.6.81 < 100 -23.6.81 1100 P1,B3

BADGEWORTH 23.6.81 500 B5BAKEWELL 24.6.80 600 -

23.7.80 700 B328.8.80 -<100 -

29.10.80 -<100 -

25.11.80 < 1 0 0 -- 24.3.81 100 P3

29.5.81 2400 P2,B3,25.6.81 8200 P 2 rB5

BECKFORD 16.7.80 < 1 0 0 -BELPER 24.6.80 < 1 0 0 -

23.7.80 < 1 0 0 -28.8.80 < 1 0 0 - ■25.11.80 . < 1 0 0 -

BREDON 24.6.80 100 -

23.6.81 200 B5BROCKHAMPTON 15.9.80 200 B5(New) 17.2.81 < 1 0 0 - '

23.6.81 1300 B5BROCKHAMPTON 15.9.80 300 B5(Old) 17.2.81 < 1 0 0 -

23.6.81 300 -

BROMSBERROW 21.11.80 < 1 0 0 -BROOKTHORPE 23.6.81 < 1 0 0 -BROOKTHORPE 23.6.81 < 1 0 0 -(East)BUXTON 24.6.80 100

23.7.80 100 B328.8.80 < 1 0 0 -

29.10.80 < 1 0 0 -

25.11.80 < 100 -24.3.81 2800 B4,529.5.81 14400 B5, 625.6.81 31000 P2,B4,

CHEDDLETON 28.8.80 < 100 -

CHURCHAM 1.6.81 <.100 -

1206S

TABLE D1 (cont'd)

Works

COALEY

COLWALLCRUMPMEADOW

DERBY

DRAYCOTT

ENDONFILLONGLEYFRAMPTON

FROMES HILL

FURNACE ENDGOSCOTEHAYDEN

HUNTLEYKEMERTONLEDBURY

LEEK

LITTLEDEANLONGFORD

LONGHOPE

Sample date

16.7.8015.9.8021.11.80 17.2.8119.8.8024.6.8016.7.8023.6.8124.6.8023.7.8028.8.8025.11.8029.5.8125.6.8124.6.8023.7.8028.8.8025.6.8128.8.8012.6.8017.2.8117.3.811.6.8121.11.8017.3.8112.6.8014.10.8024.6.8019.8.8015.9.8021.11.8017.2.8117.3.811.6.8123.6.811.6.8116.7.8016.7.8021.11.8017.3.8123.6.8124.6.8028.8.8029.10.801.6.8116.7.8019.8.8015.9.8021.11.8017.2.8117.3.8124.6.8016.7.8023.6.81

pfu 1 1

<100 100

<100 400

<100 <100 1100 1500 1500 500

< 100 < 100 11600 2500 600 600

<100 6400 <100 2200

<100 1000 1100

<100 < 100 3400

< 100 1700 <100 <^100 <100 2667 <100 1800 1300 <100 <100 <100 <100 <100

400 <100 <100 <100 16600 <100 <100 <100 <100 167

<100 <100 500 200

Serotypes

B5

B3

B3,5

B4, 5 P2,B3,5

P2,B3

PI,3,B3,5

P2,B3 P2,3,B5

P2,B3,5

B2,5

B5 .

B5

P2,B3,4,5

1206S

TABLE Dl (cont'd)

Works Sample date pfu 1 1 Serotype!

LYDNEY 24.6.80 1000 _16.7.80 300 B3,523.6.81 1900 B5

MATLOCK 24.6.80 800 -

23.7.80 200 B328.8.80 <"100 -

29.10.80 < 1 0 0 -25.11.80 < 1 0 0 -24.3.81 1700 B529.5.81 7300 B525.6.81 17400 B3,5

_MONKMOOR 1.7.80 5800 -

(Humus) 5.8.80 1000 P2,B3,59.10.80 2800 P3,B32.12.80 < 1 0 0 -3.2.81 < 1 0 0 -3.3.81 < 1 0 0 ■ -7.4.81 2300 B58.6.81 21600 B5

MONKMOOR 1.7.80 100 -(Lagoon) 5.8.80 < 1 0 0 -

9.10.80 < 1 0 0 -2.12.80 < 1 0 0 -

3.2.81 < 1 0 0 -3.3.81 < 1 0 0 ■ -7.4.81 400 B58.6.81 < 1 0 0 -

NETHERIDGE 19.8.80 < 1 0 0 -17.3.81 9900 P2 ,B525.5.81 13100 B3, 527.5.81 8000 P2,B4 1523.6.81 7900 Pl f2,3,B5

NEWENT 21.11.80 < 1 0 0 -17.3.81 < 1 0 0 -

PITTS MILL 21.11.80 < 1 0 0 -QUEDGLEY 19.8.80 < 1 0 0 - .

17.3.81 80023.6.81 3500 P2,B4,5

RIDGE LANE 12.6.80 < 1 0 0 - ■'RIPLEY 24.6.80 900 -

23.7.80 100 B328.8.80 < 1 0 0 -25.11.80 < 1 0 0 -

SOUDLEY 24.6.80 100 -16.7.80 200 B315.9.80 400 B323.6.81 2300 B3,5

STANLEY 16.7.80 < 1 0 0 -DOWNTON 19.8.80 < 1 0 0 —

15.9.80 100 P221.11.80 < 1 0 0 -

17.2.81 200 B517.3.81 < 1 0 0 -

STOKE ORCHARD 24.6.80 < 1 0 0 -

1206S

TABLE D1 (cont!d)

Works

STRONGFORDTEWKESBURY

TIRLEYTWYNINGWESTBURYWINCHCOMBE

WOOLSTONE

Sample date pfu 1 Serotypes24.7.80 <10015.9.80 <10017.2.81 -^10021.11.80 < 1 0 024.6.80 2001.6.81 < 1 0 024.6.80 10023.6.81 300 B524.6.80 <100

1206S

TABLE D2 Microbiological Levels in Effluents from Five Reclamation Works

(a) Brinklow (virus levels log^n pfu 1 *)

ample date TC EC

21.1.80 7.1 5.924.1.80 5.2 4.828.1.80 5.9 5.531.1.80 6.0 5.34.2.80 5.4 4.811.2.80 5.4 5.114.2.80 6.0 6.218.2.80 6.6 5.525.2.80 6.3 5.23.3.80 6.8 5.810.3.80 7.1 6.217.3.80 6.7 5.824.3.80 6.3 5.731.3.80 6.6 5.714.4.80 6.7 5.721.4.8012.5.807.7.80 4.0 4.014.7.8021.7.80 5.1 4.028.7.80 4.8 3.011.8.8018.8.801.9.808.9.8015.9.8022.9.8013.10.8020.10.8027.10.803.11.8017.11.8024.11.801.12.8022.12.8029.12.805.1.8112.1.81

FS W3110 ED391 V

5.6 5.9 6.3 3.04.5 5.2 5.9 . 2.55.0 5.4 5.7 2.55.3 5.6 5.7 3.04.4 5.3 5.6 2.05.6 5.1 5.7 2.66.8 5.5 5.8 2.75.1 5.8 5.8 2.04.8 5.9 6.0 2.65.0 5.1 5.9 < 2.05.2 5.8 6.1 < 2.05.5 5.0 5.8 2.05.1 5.2 5.7 2.76.0 4.0 6.6 2.04.3 4.7 5.6 3.2

4.7 5.6 2.3< 2.0

3.0 4.0 5.3 < 2.0< 2.0

3.0 6.5 6.2 < 2.01.8 4.6 4.9 < 2.0

4.6 5.4 2.04.8 5.8 < 2.0

< 2.04.3 4.6 < 2.04.0 4.0 < 2.05.3 5.6 2.0

< 2.0< 2.0< 2.0< 2.0< 2.0< 2.0< 2.0< 2.0< 2.0< 2.0< 2.0

1206S

TABLE D2 (cont'd)

(b) Bubbenhall (virus levels log^p Pfu

Sample date TC EC W3110 ED391 V

2 1 .1 .80 5.5 5.1 4.8 5.4 5.5 < 2.024.1. 80 5.2 4.0 3.3 4.0 4.5 -<2.028.1. 80 5.2 4.3 4.2 4.0 4.7 2.131.1. 80 4.6 3.7 3.8 4.0 4.5 < 2.04.2.80 4.6 3.6 3.1 4.0 4.0 < 2.01 1 .2 .80 4.0 3.5 3.0 4.7 4.9 < 2.014.2. 80 5.1 5.0 4.7 4.1 4.5 2.518.2. 80 4.9 3.9 3.3 4.0 4.8 2.025.2. 80 5.7 3.7 3.3 4.0 4.0 < 2.03.3.80 5.5 4.4 4.0 5.6 5.6 < 2.010.3. 80 4.0 3.0 5.5 4.2 4.0 < 2.017.3. 80 5.6 4.6 4.5 5.3 5.5 2.024.3. 80 6.2 5.6 5.5 4.8 4.5 2.931.3. 80 5.1 4.3 4.4 3.3 3.3 < 2.014.4. 80 6.1 4.9 4.0 4.6 4.7 2.021.4. 80 5.3 5.4 2.312.5. 80 < 2.07.7.80 5.3 4.5 3.7 5.2 5.3 < 2.014.7. 80 2.021.7. 80 5.5 4.3 3.6 5.8 6.0 ^ 2.028.7. 80 6.0 5.0 4.0 5.9 5.9 < 2.04.8.80 5.7 4.8 3.7 5.1 5.4 < 2.01 1 .8 .80 4.9 5.4 < 2.018.8. 80 4.5 5.2 < 2.01.9.80 2.08.9.80 5.5 5.9 2.715.9. 80 6.0 6.2 < 2.022.9. 80 4.5 6.0 2.313.10 .80 < 2.020.10 .80 < 2.027.10 .80 2.53.11. 80 < 2.017.11 .80 < 2.024.11 .80 < 2.01 .1 2 .80 < 2.022.12 .80 < 2.029.12 .80 2.05.1.81 < 2.01 2 .1 .81 < 2.0

1206S

TABLE D2 (cont'd)

(c) Finham (virus levels log^Q P fu

Sample date TC EC

10.1.8017.1.80 5.7 5.021.1.80 6.3 5.924.1.80 5.4 5.028.1.80 6.2 5.631.1.80 6.3 5.84.2.80 6.2 5.511.2.80 5.7 5.314.2.80 5.3 4.918.2.80 5.5 5.225.2.80 6.0 5.33.3.80 6.8 5.310.3.80 6.0 5.517.3.80 6.4 5.724.3.80 5.9 5.331.3.80 6.2 5.614.4.80 6.1 5.221.4.8012.5.807.7.80 6.4 5.414.7.8021.7.80 6.0 4.928.7.80 5.0 4.84.8.80 5.9 5.011.8.8018.8.801.9.808.9.8015.9.8022.9.8029.9.8013.10.8020.10.8027.10.803.11.8017.11.8024.11.801.12.8015.12.8022.12.8029.12.805.1.8112.1.81

FS W3110 - ED391 V

3.64.6 5.6 5.9 3.25.3 6.2 6.3 3.14.4 5.7 5.9 3.05.0 5.7 6.1 . 3.15.6 6.4 5.9 3.05.6 5.4 5.6 3.04.9 5.4 5.8 2.64.6 5.8 5.5 3.04.6 5.8 5.9 2.74.9 5.7 5.8 2.94.8 5.9 6.2 3.05.0 5.4 5.9 3.15.5 6.2 6.0 3.35.1 5.5 5.7 3.25.0 5.8 5.9 3.14.5 5.7 5.8 2.8

6.0 6.2 3.23.0

4.2 5.6 6.0 3.12.0

3.3 5.6 5.9 2.53.6 5.6 5.8 2.83.9 5.5 5.6 3.2

5.1 5.8 3.25.8 6.3 2.9

3.06.0 6.3 2.05.8 6.2 2.95.9 6.3 3.5

3.6< 2.0 < 2.0 < 2.0 < 2.0 < 2.0 < 2.03.02.92.93.02.9

1206S

TABLE D2 (cont'd)

(d) Hinckley (virus levels log^p P fu

Sample date TC EC

10.1.8017.1.80 3.9 3.321.1.80 6.4 5.424.1.80 5.9 4.728.1.80 5.2 4.231.1.80 6.2 5.04.2.80 5.4 4.511.2.80 5.0 4.614.2.80 5.8 5.2

' 18.2.80 5.6 4.625.2.80 5.4 3.93.3.80 6.7 3.910.3.80 5.0 4.217.3.80 7.2 5.524.3.80 5.9 5.531.3.80 6.0 5.214.4.80 5.2 4.421.4.8012.5.807.7.80 6.7 5.914.7.8021.7.80 4.2 3.028.7.80 4.5 3.24.8.80 3.9 3.011.8.8018.8.801.9.808.9.8015.9.8022.9.8013.10.8020.10.8027.10.803.11.8017.11.801.12.8015.12.8022.12.8029.12.805.1.8112.1.81

FS W3110 ED391 V

< 2.02.2 4.0 5.0 <2.02.6 5.7 5.6 2.04.3 5.2 5.2 2.04.3 4.6 4.9 < 2 . 04.9 5.4 5.5 <2.04.3 4.8 4.9 < 2 . 04.3 5.7 5.1 <2. 04.7 5.0 5.1 <2.04.4 5.0 4.6 <2.03.5 4.5 4.9 2.03.8 4.6 4.9 2.64.4 4.6 <2.05.3 6.9 7.2 2.65.0 4.6 4.4 2.04.7 4.9 4.8 <2.04.0 4.5 4.0 2.0

3.0 4.0 < 2 . 0< 2.0

4.6 5.6 5.8 2.9< 2.0

2.0 4.0 4.5 <2.02.5 4.5 4.3 <2.04.2 3.0 4.5 <2.0

3.0 3.0 <2.04.0 4.0 <2.0

2.05.9 6.1 2.74.9 4.9 2.04.5 4.4 <2.0

< 2.0 < 2.0 < 2.0 < 2.0 < 2.0

2.7 2.03.0

< 2.02.02.9

1206S

TABLE D2 (cont'd)

(e) Ruqby (virus levels logio pfu I"1)

Sample date TC EC FS W3110 ED391 V

10.1.80 2.617.1.80 4.5 3.8 3.6 5.7 5.0 2.221.1.80 4.4 4.0 3.4 4.9 5.0 1.724.1.80 4.9 4.5 4.0 4.8 4.9 < 2.028.1.80 5.0 5.5 5.3 5.2 4.7 < 2.031.1.80 6.4 5.6 5.3 4.3 5.1 < 2.04.2.80 6.1 5.3 4.9 4.6 6.7 < 2.011.2.80 4.9 3.8 3.9 4.6 4.7 < 2.014.2.80 4.5 ' 3.0 3.0 4.3 4.5 < 2.018.2.80 4.7 3.4 2.7 4.8 4.5 < 2.025.2.80 4.5 3.7 3.3 4.0 4.5 < 2.03.3.80 5.0 4.6 4.0 4.8 4.8 < 2.010.3.80 5.8 3.8 3.3 4.0 4.0 < 2.017.3.80 5.2 4.1 3.6 5.4 5.5 2.024.3.80 5.4 .4.0 3.9 4.6 4.6 < 2.031.3.80 4.5 3.1 3.0 4.9 4.6 < 2.014.4.80 6.0 5.0 5.0 5.9 5.9 2.321.4.80 5.8 5.8 < 2.012.5.80 < 2.07.7.80 6.2 5.3 4.5 5.0 5.4 < 2.014.7.80 < 2.021.7.80 5.7 4.3 3.4 4.9 5.1 < 2.028.7.80 6.5 5.2 4.1 5.7 5.7 < 2.04.8.80 6.0 4.8 4.5 4.7 4.9 2.011.8.80 4.9 5.0 2.318.8.80 4.7 4.6 < 2.01.9.80 < 2.08.9.80 4.7 4.7 < 2.015.9.80 5.0 4.0 2.022.9.80 5.2 5.4 < 2.029.9.8013.10.8020.10.8027.10.803.11.8017.11.8024.11.801.12.8022.12.8029.12.805.1.8112.1.81

< 2.0< 2.0< -2.0< 2.0< 2.0< 2.0< 2.0< 2.0< 2.0< 2.0< 2.02.0

1206S

TABLE D3 Wastewater Treatment Processes: Virus Levels

(a) Finham A

Sample date RS SS/ADF(I)

21.5.82 3.7 4.027.5.82 4.5 4.521.6.82 4.5 4.929.6.82 4.8 4.87.7.82 4.1 4.315.7.82 4.6 4.923.7.82 4.7 4.62.8.82 4.4 4.610.8.82 4.4 4.516.8.82 4.0 3.98.9.82 3.9 3.914.9.82 3.8 3.620.9.82 3.9 3.927.9.82 3.6 4.15.10.82 3.9 4.021.10.82 3.9 4.129.10.82 4.0 3.98.11.82 3.7 3.715.11.82 3.6 3.822.11.82 3.4 3.529.11.82' 3.1 3.03.12.82 3.4 3.19.12.82 3.0 3.015.12.82 3.3 3.222.12.82 3.3 3.1

ADF(E)/AS(I) AS (E) PF(I) PF(E)

3.9 3.9 4.0 3.64.5 4.5 4.4 3.24.6 4.8 4.8 4.400* 4.8 4.6 4.74.1 4.3 4.2 3.94.9 4.9 4.9 4.84.7 4.5 4.6 4.34.5 4.5 . 4.4 4.14.4 4.5 4.5 4.14.2 3.4 4.0 3.23.7 4.0 3.7 2.33.8 3.6 4.1 3.64.1 3.8 3.6 2.63.4 3.7 4.2 4.14.0 3.9 4.2 3.93.9 3.8 3.8 3.53.9 3.7 3.9 3.63.5 3.6 3.7 3.23.7 3.6 3.7 3.83.5 3.5 3.3 2.93.2 3.7 3.3 2.63.2 3.5 3.5 3.22.6 3.2 3.1 2.93.2 3.2 3.2 3.13.2 3.5 2.3 2.6

1206S

TABLE D3 (cont'd)

(b) Finham B

Sample date RS SS/AS(I)

21.5.82 3.9 4.027.5.82 4.5 4.621.6.82 4.2 4.829.6.82 4.9 4.97.7.82 4.0 4.515.7.82 4.8 4.823.7.82 4.5 4.82.8.82 4.4 4.610.8.82 4.4 4.5

- 16.8.82 4.0 4.18.9.82 4.2 4.014.9.82 3.8 3.820.9.82 4.0 4.327.9.82 3.2 3.95.10.82 4.0 4.121.10.82 3.6 4.129.10.82 4.0 4.18.11.82 3.8 4.115.11.82 3.8 3.722.11.82 3.2 3.729 11.82 * 3.3 3.43.12.82 3.2 3.59.12.82 3.1 3.215.12.82 3.6 3.222.12.82 2.3 2.6

AS (E)/PF(I) PF(E) SF (I) SF (E)

4.0 3.7 3.4 3.24.4 4.2 3.8 3.94.7 4.4 4 .2 4.34.9 4.6 4.4 4.44.2 4.2 3.6 3.94.8 4.6 4.5 4.44.7 4.4 4.0 4.14.5 4.2 3.7 3.94.4 4.2 3.6 3.83.7 3.1 3.3 3.43.1 3.7 2.9 2.73.3 2.8 2.9 3.13.7 3.4 2.7 3.93.4 3.4 3.5 3.54.2 4.0 3.7 3.73.9 3.9 3.4 3.53.9 3.6 3.3 3.53.7 3.5 3.2 3.23.5 3.3 3.3 3.53.5 3.2 2.7 2.93.5 2.8 2.0 2.93.2 3.3 2.8 2.83.2 3.2 3.0 2.83.3 2.6 2.8 2.9

1 3.6 2.9 3.0 3.1

1206S

TABLE D3 (confd)

(c) Monkmoor

Sample date RS SS/PF<I)

10.6.82 4.5 4.515.6.82 4.5 4.322.6.82 3.9 4.029.6.82 4.1 4.28.7.82 3.6 3.713.7.82 4.8 4.422.7.82 4.5 4.527.7.82 3.4 4.45.8.82 4.6 4.4

“ 11.8.82 4.4 4.117.8.82 4.4 4.423.8.82 3.0 3.4

PF<E)/HT(I) HT{E)/L(I) L(E)

4.6 4.4 ^2.04.2 4.1 <2.03.7 4.14.0 4.1 <1.73.5 3.8 < 1 . 74.3 4.5 < 1 . 74.5 4.5 <1.74.2 4.4 1.74.3 4.1 1.73.9 4.0 2.04.1 4.2 2.33.5 3.6 < 2 . 0

1206S

1 —

34 —

6 —

78 — 1

9—

10

11

V

DWORFE

ISTOUR

S O W E

12□ AVON

-13

— 14 LEAM

Sampling Points in t h e .river Severn basin

1 Shrewsbury 8 Tewkesbury2 Atcham 9 Haw Bridge3 Hampton Loade res 10 Cosford4 Trimpley res 11 Stourport5 Burf 12 Stoneleigh6 Worcester 13 Campion Hills res7 Strensham 14 Draycote res

TABLE D4 River Severn Basin(a) Shrevsbury Water Abstraction Point

Sample date TC EC FS V

6.2.79 0.46.3.79 ND3.4.79 0.21.5.79 0.26.6.79 1.23.7.79 2.8 2.0 1.0 ND7.8.79 2.6 2.2 1.0 ND4.9.79 3.3 2.5 1.6 0.32.10.79 3.2 2.6 1.6 0.16.11.79 3.8 3.3 2.8 ND3.1.80 3.2 2.6 2.3 0.35.2.80 4.0 3.3 2.7 1.04.3.80 ND1.4.80 3.8 2.9 2.2 0.26.5.80 2.3 1.0 1.5 ND3.6.80 2.5 1.0 1.3 ND1.7.80 2.9 2.6 1.7 0.25.8.80 1.9 1.3 0.6 ND11.9.80 ND9.10.80 3.0 3.0 2.1 0.84.11.80 3.0 2.8 1.9 ND2.12.80 2.8 2.8 2.4 ND6.1.81 3.1 2.8 2.1 ND3.2.81 3.0 2.7 2.6 ND3.3.81 ND7.4.81 ND5.5.81 4.38.6.81 5.27.7.81 0.2

Serotypes

Pl,2,3,B2

P2,3B4

B3, 4 B2

B4

B3f 5

B5B5

1206S

TABLE D4 (cont*d)(b) Atcham below Monkrooor WRW Discharge

Sample date TC EC FS V

6.11.79 4.6 3.5 3.1 ND3.1.80 4.1 3.6 2.6 ND5.2.804.3.80

4.1 3.5 3.4 0.2ND

1.4.80 4.3 3.5 3.0 ND6.5.80 4.5 2.8 1.9 5.83.6.80 5.0 3.6 1.9 0.11.7.80 3.9 3.6 2.3 2.15.8.8011.9.80

4.0 3.1 2.0 0.53.3

9.10.80 3.0 ND ND 1.44.11.80 3.2 2.9 1.9 ND2.12.80 3.6 2.9 2.7 0.56.1.81 3.6 3.0 2.3 ND3.2.813.3.817.4.815.5.818.6.81 7.7.81

3.0 3.0 2.7 0.1ND0.51.6

28.393.0

Serotypes

Plf2 f3fB3f

P2 , B3

B5B5B5

1206S

TABLE D4 (cont'd)

(c) Hampton Loade Water Abstraction Point

Sample date V Serotypes

10.3.81 7.8 B514.4.81 6.1 B512.5.81 5.0 B514.7.81 0.25.7.82 1.0

1206S

TABLE D4 (cont'd)(d) Burf below Confluence with River Stour

Sample date TC EC

9.10.79 4.9 3.913.11.79 4.6 3.84.12.79 4.6 3.48.1.80 4.5 3.612.2.80 4.2 3.511.3.80 5.6 3.915.4.80 4.7 3.713.5.80 4.7 3.710.6.80 5.1 4.08.7.80 4.7 3.712.8.80 4.7 4.99.9.80 4.7 3.814.10.8011.11.80 5.6 3.813.1.81 4.7 3.910.2.81 5.3 4.410.3.8114.4.8112.5.8114.7.81

FS V Serotypes

2.7 9.4 P2,B2,42.5 3.0 P2,3,B42.8 0.2 B43.0 0.9 PifB52.9 ND2.6 ND2.8 0.12.1 5.82.5 38.02.5 36.02.0 6.0 P2 ,B3,4,52.0 ND

26.4 B52.7 ND2.7 ND3.0 ND

15.6 P2,B2,529.093.0 B2,568.0 B2,5

1206S

TABLE D4 (cont'd)

(e) Worcester Water Abstraction Point

Sample date TC EC FS V Serotypes

20.3.79 4.2 Pl,2f3 rB4,518.4.79 3.8 B415.5.79 2.8 B2,4,519.6.79 4.5 3.0 1.5 1.7 B417.7.79 3.7 2.8 1.8 0.121.8.79 4.6 3.3 1.9 ND25.10.79 .0 4.1 3.0 1.3 P2,B420.11.79 4.7 3.6 2.6 4.2 B422.1.80 5.1 4.1 4.0 1.019.2.80 ND18.3.80 5.0 3.8 3.4 ND22.4.80 5.1 3.7 2.6 ND19.5.80 9.6 P2,B3,417.6.80 4.2 3.9 2.6 0.216.7.80 4.3 3.4 2.2 7.219.8.80 4.4 3.9 2.4 ND16.9.80 3.7 3.5 1.7 1.5 B521.10.80 4.9 4.0 2.5 21.0 P2,B518.11.80 ND17.2.81 ND17.3.81 0.422.4.81 2.0 B519.5.81 2.616.6.81 ND

1206S

TABLE D4 (cont'd)(f) Strensham Water Abstraction Point

Sample date TC EC FS V Serotypes

20.3.79 4.4 P2f3,B4,518.4.79 4.2 P2,B1,4,515.5.79 1.3 P2,B419.6.79 4.5 3.0 1.5 3.717.7.79 3.9 2.9 1.5 0.7 B421.8.79 4.2 3.4 2.0 0.218.9.79 4.5 3.7 2.4 5.4 Pl,2,3rB425.10.79 4.8 3.9 2.5 3.7 P2,3,B420.11.79 4.8 3.8 2.8 0.722.1.80 5.2 4.0 3.9 9.3 B419.2.80 0.218.3.80 4.6 3.5 2.8 0.122.4.80 4.5 3.2 2.5 0.319.5.80 12.2 P2,B3,417.6.80 4.2 3.5 1.8 0.219.8.80 4.1 3.4 5.6 ND16.9.80 3.5 3.2 1.6 0.221.10.80 4.6 3.9 2.5 ND18.11.80 ND17.2.81 ND17.3.81 ND22.4.81 9.819.5.81 0.516.6.81 2.5

1206S

TABLE D4 (cont'd)(g) Tewkesbury Water Abstraction Point

Sample date TC EC FS V Serotypes

20.3.79 7.4 Pl,2,3,B4,518.4.79 2.4 P2,B1,415.5.79 0.5 B419.6.79 3.8 2.6 1.0 5.4 B417.7.79 3.0 2.1 ND 0.3 B421.8.79 4.2 2.0 1.7 ND25.10.79 4.7 3.5 2.3 1.8 B420.11.79 4.6 3.8 2.8 9.5 B3 r 422.1.80 5.3 4.0 4.0 3.019.2.80 ND18.3.80 4.5 3.8 3.2 0.422.4.80 4.7 2.7 2.0 ND19.5.80 4.0 P2,3,B317.6.80 3.7 3.4 2.3 1.6 B3,4,516.7.80 4.3 3.3 2.3 0.919.8.80 4.3 3.6 2.6 ND16.9.80 3.1 2.7 1.5 0.7 B521.10.80 4.2 3.3 2.0 ND18.11.80 ND17.2.81 ND17.3.81 4.022.4.81 1.719.5.81 0.416.6.81 1.3

1206S

TABLE D4 (cont’d)

(h) Haw Bridqe above Salt Water Flow

Sample date TC EC FS V Serotypes

25.10.79 4.5 3.6 2.2 0.6 B420.11.79 4.6 3.6 2.7 4.622.1.80 5.2 4.1 3.8 2.8 P3,B3,419.2.80 ND18.3.80 4.4 3.6 2.8 0.222.4.80 3.7 2.5 1.0 ND19.5.80 0.217.6.80 4.0 3.7 1.9 10.1 B3,416.7.80 5.6 3.6 2.3 4.719.8.80 4.1 3.3 2.3 ND16.9.80 3.2 2.0 1.7 ND21.10.80 4.9 4.3 2.6 25.2 P3,B2,3,518.11.80 ND17.2.81 ND17.3.81 ND22.4.81 0.519.5.81 0.216.6.81 6.5 B3,5

1206S

TABLE D4 (cont’d)(i) River Worfe Tributary at Cosford Water Abstraction Point

Sample date TC EC FS V

6.2.79 0.056.3.79 0.13.4.79 ND1.5.79 ND6.6.79 0.43.7.79 3.4 2.8 1.3 ND7.8.79 4.3 3.5 3.1 ND4.9.79 3.4 2.7 1.8 ND2.10.79 2.2 2.0 2.0 ND6.11.79 4.4 3.3 3.1 ND3.1.80 3.8 3.4 2.1 0.35.2.80 3.9 3.2 3.1 ND4.3.80 ND1.4.80 4.0 3.4 2.6 ND6.5.80 2.3 2.0 ND ND3.6.80 2.8 2.8 1.0 ND1.7.80 2.8 2.7 2.4 ND5.8.80 2.7 2.7 2.0 ND11.9.80 ND9.10.80 2.6 2.6 2.0 ND4.11.80 2.3 3.3 2.2 ND2.12.80 2.8 2.7 2.8 ND6.1.81 3.2 3.0 2.8 ND3.2.81 3.0 3.0 2.2 ND3.3.81 ND7.4.81 ND5.5.81 0.28.6.81 0.57.7.81 ND

1206S

TABLE D4 (cont'd)(j) River Stour Tributary at Stourport

Sample date TC EC FS V Serotypes

9.10.79 6.2 5.3 4.2 127.0 P3,B4,513.11.79 6.1 5.4 4.3 24.5 B34.12.79 6.3 5.1 3.5 19.38.1.80 6.0 5.2 4.8 3.5 P212.2.80 5.6 4.7 4.4 ND11.3.80 7.0 6.0 4.0 166.015.4.80 6.1 5.1 4.5 ND13.5.80 6.0 5.3 4.1 312.010.6.80 6.6 5.3 4 .2 133.0 B38.7.80 5.9 5.3 3.9 574.012.8.80 5.9 5.3 3.6 256.09.9.80 6.0 5.2 3.6 168.0 P2 ,B3,414.10.80 346.011.11.80 6.1 5.5 4.2 567.0 B3,513.1.81 6.0 5.3 4.4 10.0 B510.2.81 5.9 4.9 3.9 267.0 P2,B3,510.3.81 47.0 P2,3, B2,514.4.81 34.012.5.81 238.0 P2,B514.7.81 375.0 B5

1206S

TABLE D4 (cont'd)(k) River Avon Tributary at Tewkesbury

Sample date TC EC FS V Serotypes

25.10.79 3.6 2.8 1.7 0.320.11.79 4.2 2.9 1.6 1.9 P222.1.80 4.8 4.3 4.0 2.6 P2,3,B3,419.2.80 ND18.3.80 4.6 3.5 2.7 0.122.4.80 3.2 2.0 2.0 ND19.5.80 ND17.6.80 3.4 2.1 1.9 0.4 B316.7.80 4.6 3.8 2.7 5.019.8.80 3.9 3.4 2.3 ND16.9.80 3.1 2.7 1.6 ND21.10.80 4.9 4.4 2.8 ND18.11.80 ND17.2.81 5.0 P 2 fB517.3.81 ND22.4.81 3.2 B519.5.81 0.416.6.81 ND

1206S

2 —

DSOW3 — BOURNE/BLYTHE

1 6 _ f15

BLITHE4 — 1412 13

TAMEMEASE CHURNET

2 4^-23225 —

2120

DOVE

27 282 5 26

DERWENT7 — — 29

AMBER

V

Sampling Points in the river Trent basin

1 Tittensor 16 Whitacre res2 Salt 17 Harlaston3 Seven Springs 18 Clay Mills4 Yoxall bridge 19 Staunton Harold5 Walton Foremark res6 Willington 20 Rocester7 Sawley bridge 21 Quixhill8 Milford 22 Alton9 Blithfield res 23 Oakamoor

10 Chetwynd bridge 24 Froghall11 Elford 25 Church Wilne res12 Hopwas 26 Little Eaton13 Kingsbury 27 Ogston res14 Lea Marston 28 Whatstandwell15 Water Orton 29 Ambergate

TABLE D5 River Trent Basin(a) River Trent at Seven Locations

Location Sample date V Serotypes

TITTENSOR 30.10.79 4.526.3.81 ND23.4.81 58.0 B527.5.81 236.023.6.81 48.028.8.81 0.7

SEVEN SPRINGS 30.10.79 22.0 B3,426.3.81 ND23.4.81 51.0 B527.5.81 10.723.6.81 ND28.7.81 8.4 B3, 5

SALT 30.10.79 11.0 P2, B426.3.81 ND

- 23.4.81 51.027.5.81 141.023.6.81 27.028.7.81 22.0 B4,5

YOXALL BRIDGE 26.3.81 ND23.4.81 47.0 B527.5.81 173.023.6.81 12.028.7.81 10.3 B3,5

WALTON 26.3.81 ND9.4.81 57.014.5.81 ND10.6.81 2.016.7.81 0.7 B5

WILLINGTON 24.3.81 27.625.6.81 86.0 B5

SAWLEY BRIDGE 24.3.81 3.729.5.81 77.025.6.81 105.0

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TABLE D5 (cont'd)(b) River Derwent Tributary at Three Locations

Location

WHATSTANDWELL

LITTLE EATON (Abstraction)

WILNE(Abstraction)

Sample date TC EC FS V Serotypes

24.3.81 4.019.5.81 36.025.6.81 165.0 B528.3.79 ND24.4.79 3.6 PI,2,3,B2,422.5.79 9.4 Pl,2,3,B2,426.6.79 4.0 3.9 1.7 4.6 B2,430.8.79 4.7 3.7 2.5 ND26.9.79 4.4 3.0 2.0 19.3 P2,B424.10.79 4.6 3.7 2.0 11.4 P2,B2,3,427.11.79 2.8 2.0 1.5 0.530.1.80 4.4 3.8 3.6 ND27.2.80 4.9 4.8 3.7 ND29.5.80 5.8 4.3 2.9 5.1 PI,2 ,B3,E3224.6.80 5.1 4.5 3.3 5.223.7.80 4.1 3.4 1.3 4.728.8.80 4.7 4.3 2.7 0.129.10.80 ND19.11.80 ND25.11.80 5.0 3.8 2.6 ND24.3.81 ND29.5.81 74.025.6.81 49.0 P3,B524.3.81 ND29.5.81 62.025.6.81 95.0 B5

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TABLE D5 (cont'd)(c) River Churnet Tributary at Five Locations

Location Sample date TC EC V

FROGHALL 17.6.82 272.028.6.82 4.8 4.0 142.026.7.82 24.510.10.83 4.6 4.5 ND

OAKAMOOR 17.6.82 150.028.6.82 4.6 3.8 108.026.7.82 23.523.8.83 5.0 4.0 ND10.10.83 5.6 4.6 1.7

ALTON 17.6.82 189.028.6.82 4.6 3.6 78.026.7.82 16.323.8.83 4.6 3.4 0.210.10.83 5.5 4.7 ND

QUIXHILL 17.6.82 84.028.6.82 4.6 4.1 101. 026.7.82 24.323.8.83 4.6 3.5 2.410.10.83 5.7 4.8 1.1

ROCESTER 17.6.82 49.028.6.82 4.8 4.1 91.026.7.82 79.523.8.83 4.8 3.8 1. 610.10.83 5.3 4.5 2.2

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TABLE D5 (cont'd)

(d) River Tame Tributary at Water Orton

Sample date TC EC FS V

4.5. 82 6.2 4.6 3.8 16.711.5 .82 6.0 4.6 2.8 120.018.5 .82 6.5 4.9 3.8 97.025.5 .82 248.08.6. 82 6.7 5.6 4.1 418.015.6 .82 6.5 5.6 4.4 920.022.6 .82 7.0 5.5 4.5 220.06.7. 82 6.7 5.0 3.5 729.013.7 .82 6.4 5.3 3.7 1664.020.7 .82 6.0 4.9 3.5 1850.027.7 .82 6.0 5.3 3.5 1200.03.8. 82 6.8 5.9 4.3 1667.010.8 .82 6.4 5.6 4.1 2280.0

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Serotypes

Bl,4

TABLE D5 (cont'd)(e) River Tame Tributary at Lea Marston (above lake)

Sample date TC EC FS V Serotypes

5.3.81 94.0 B2 f 4,59.4.81 116.014.5.81 86.0 P2,B410.6.81 4.0 B516.7.81 659.04.8.81 122.0 B511.8.81 67.0 P2,B4,518.8.81 1.9 B325.8.81 8.0 P2,B3,47.9.81 14.8 P2 ,B215.9.81 102.0 P2,3,B3,4,522.9.81 273.0 P2,B2,3,429.9.81 71.0 Pi,3,B46.10.81 219.0 B3,413.10.81 100.0 P2,B4,620.10.81 400.0 B 2 ,3,4,527.10.81 110.0 Pl,2,B4,53.11.81 54.0 P1,B3,510.11.81 107.0 P2,B3,424.11.81 75.0 P3,B2,3,4,51.12.81 33.0 P3,B44.5.82 5.7 4.8 3.7 6.3 B 2 ,4,511.5.82 6.7 5.6 4.4 31.318.5.82 6.0 4.6 3.5 46.0 P2,B425.5.82 18.08.6.82 5.5 4.7 3.7 190.015.6.82 6.0 4.7 3.5 536.022.6.82 6.7 5.0 4.7 610.06.7.82 6.6 5.3 3.3 1376.013.7.82 6.0 5.1 3.4 1552.020.7.82 6.0 5.0 3.5 1770.027.7.82 5.8 4.3 3.4 860.03.8.82 5.8 5.5 3.5 1900.010.8.82 6.1 5.4 4.0 840.0

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TABLE D5 (confd)(f) River Tame Tributary at Kingsbury (below lake)

Sample date TC EC FS V Serotypes

5.3.81 69.0 B4,59.4.81 89.014.5.81 256.0 B4,510.6.81 7.7 B516.7.81 760.0 P2,B34.8.81 118.0 B2,4,511.8.81 58.0 P1,B3,4,518.8.81 130.0 P2,B2,3,525.8.81 47.0 P2,B57.9.81 10.0 B3,515.9.81 202.0 P2,B2,3,522.9.81 242.0 B4,529.9.81 106.0 B4,E326.10.81 185.0 B3,413.10.81 163.0 P2,B2,3,4,20.10.81 24.0 B2,4,527.10.81 70.0 B2 > 43.11.81 145.0 B3,410.11.81 198.0 P2 rB4,524.11.81 167.0 P2,B51.12.81 167.0 Pi,2,B3,44.5.82 4.7 4.6 3.5 9.411.5.82 5.6 4.8 3.4 1.518.5.82 5.5 4.5 3.0 83.025.5.82 90.08.6.82 5.7 4.2 2.8 346.015.6.82 5.5 4.2 3.4 756.022.6.82 6.7 5.7 4.0 900.06.7.82 7.0 5.2 3.4 776.013.7.82 5.6 4.8 3.6 1392.020.7.82 5.6 4.7 3.2 2460.027.7.82 5.8 5.4 3.4 1260.03.8.82 5.8 5.0 3.1 1520.010.8.82 5.5 5.0 3.4 640.0

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TABLE D5 (cont'd)(g) River Tame Tributary at Hopwas

Sample date TC EC FS V Serotypes

8.10.79 5.0 4.1 3.0 32.0 P2,B3,4,510.12.79 6.3 5.2 5.0 14.0 B47.1.80 5.3 4.7 4.4 8.0 Pl,2,B3,56.2.80 5.3 4.7 4.9 3.55.3.80 6.3 4.2 4.1 13.016.4.80 5.4 4.3 3.4 2.0 P27.5.80 5.5 4.3 3.7 88.011.6.80 5.3 4.6 3.0 337.0 P2,B3,52.7.80 5.2 4.4 2.7 146.013.8.80 5.5 4.1 2.0 221.016.10.80 223.0 B3,514.1.81 5.4 4.8 3.9 202.0 B519.2.81 110.0 B4, 55.3.81 107.0 P2,B2,4,59.4.81 75.014.5.81 197.0 B4,510.6.81 9.2 B416.7.81 647.0 B4

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TABLE D5 (cont'd)(h) River Tame Tributary at Elford and Chetwynd Bridge

Location

ELFORD

CHETWYND

ample date TC EC FS V

5.3.81 107.09.4.81 99.014.5.81 214.010.6.81 6.316.7.81 161.04.5.82 5.4 4.3 3.2 2.011.5.82 4.6 3.7 1.9 35.018.5.82 4.7 3.7 2.0 59.025.5.82 107.08.6.82 4.5 3.0 2.0 124.015.6.82 4.8 3.8 2.9 618.022.6.82 5.9 5.0 3.9 569.06.7.82 5.3 4.0 2.0 1248.013.7.82 5.0 4.0 3.0 792.020.7.82 5.3 4.2 2.5 990.027.7.92 4.6 4.2 2.5 580.03.8.82 5.5 4.4 2.5 820.010.8.82 5.2 4.4 3.2 260.0

Serotypes

B3,4,5

B2,4,5 B5B3,5

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TABLE D5 (cont'd)(i) Other Tributaries of the River Trent

Tributary(Location) Sample date TC EC FS V Serotypes

Amber 24.3.81 * 1.6(AMBERGATE) 29.5.81 146.0

25.6.81 135.0 B5Dove 24.10.79 4.1 2.8 2.7 5.9 B2 ,3,4,5(CLAY MILLS) 27.11.79 ,4.4 3.4 2.8 2.3 B4

30.1.80 4.7 3.9 3.1 0.227.2.80 5.2 4.5 3.5 ND29.5.80 4.8 4.5 2.7 11.0 B3,524.6.80 4.1 3.1 2.3 5.223.7.80 3.6 2.7 ND 7.829.10.80 5.1 4.0 3.7 ND25.11.80 5.0 3.8 3.4 ND24.3.81 0.325.6.81 9.6

Mease 5.3.81 ND(HARLASTON) 9.4.81 2.9

14.5.81 3.110.6.81 3.0 P2,B516.7.81 271.0

Sow 26.3.81 5.7(MILFORD) 23.4.81 32.0 P2,B3,5

27.5.81 0.223.6.81 3.028.7.81 0.2

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TABLE D6 Stored Waters

(a) River Blithe Impoundment at Blithfield Reservoir (50+ Days)

Sample date TC EC FS V

12.3.79 ND10.4.79 ND14.5.79 1.3 ND ND ND11.6.79 ND9.7.79 0.9 ND ND ND13.8.79 ND ND ND ND10.9.79 1.7 1.6 ND ND8.10.79 ND ND ND ND12.11.79 2.2 0.7 ND ND10.12.79 2.4 2.3 ND ND7.1.80 3.2 2.8 2.3 ND6.2.80 2.4 2.4 2.0 ND5.3.80 5.0 1.7 1.0 ND16.4.80 ND ND ND ND.7.5.80 ND ND ND ND11.6.80 ND2.7.80 ND13.8.80 ND10.9.80 ND6.10.80 ND12.11.80' ND14.1.81 ND19.2.81 ND

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TABLE D6 (cont'd)

(b) River Learn Impounded at Campion Hills Reservoir (Seven DaysShort Circuiting)

Sample date TC EC FS V Serotypes

9.1.79 6.3 PI,B2 ,4,E135.3.79 8.8 PI,2,3,B3,411.4.79 1.0 P2,B29.5.79 0.3 P2,B38.6.79 0.52.7.79 2.3 1.5 ND ND10.9.79 ND ND ND ND8.10.79 3.2 2.0 2.0 ND10.12.79 3.2 2.5 2.3 ND7.1.80 4.2 3.3 2.7 0.5 P26.2.80 3.7 3.2 2.9 ND5.3.80 3.8 2.1 1.6 ND16.4.80 3.0 1.6 ND ND7.5.80 2.4 1.0 ND ND2.6.80 ND3.6.80 ND4.6.80 ND5.6.80 ND6.6.80 ND9.6.80 ND10.6.80 ND11.6.80 ND12.6.80 0.113.6.80 0.2 B416.6.80 ND18.6.80 1.6 P2,B319.6.80 2.5 B320.6.80 1.1 B323.6.80 ND24.6.80 0.5 B326.6.80 0.227.6.80 0.62.7.80 0.757.7.80 0.98.7.80 0.29.7.80 0.310.7.80 0.511.7.80 0.114.7.80 0.2517.7.90 0.918.7.80 1.0 B321.7.80 ND22.7.80 0.124.7.80 0.8529.7.80 0.231.7.80 0.751.8.80 0.844.8.80 0.5

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TABLE D6 (cont'd)(b) River Leam Impounded at Campion Hills Reservoir (Seven Days with

Short Circuiting) (cont'd)

Sample date TC EC FS V Serotypes

6.8.807.8.808.8.80 11.8.80 12.8.8013.8.8014.8.8015.8.8018.8.801.9.808.9.8010.9.8015.9.8022.9.8029.9.806.10.8013.10.8020.10.8027.10.803.11.8012.11.8024.11.8015.12.8022.12.80 29.12.805.1.8114.1.81 19.2.91

NDNDNDND

0.30.1NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND

3.40.2

B5

P2, B5 B3

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TABLE D6 (cont'd)

(c) River Derwent Impoundment at Church Wilne Reservoir (50+ Days)

Sample date TC EC FS V Serotypes

28.3.79 ND24.4.79 0.1 P322.5.79 0.2 B426.6.79 2.5 ND ND 0.330.8.75 2.6 2.5 ND ND26.9.79 3.2 2.7 1.8 0.1 B324.10.79 2.5 2.2 0.3 ND27.11.79 3.0 2.9 2.4 0.130.1.80 3.1 2.6 2.6 ND27.2.80 2.6 ND 0.3 ND29.5.80 ND ND ND ND24.6.80 0.823.7.80 0.428.8.80 ND29.10.80 ND25.11.80 ND

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TABLE D6 (cont'd)(d) River Dove Impoundment at Foremark Reservoir (50+ Days)

Sample date TC EC

28.3.7926.6.79 ND ND30.8.79 0.3 ND26.9.79 0.3 ND24.10.79 0.9 ND27.11.79 2.1 0.330.1.80 2.4 2.227.2.80 4.0 0.629.5.8024.6.8023.7.8028.8.80 29.10.80

2.0 ND

FS V Serotypes

NDND NDND NDND 0.1 B4ND 0.10.3 ND2.0 NDND ND1.0 ND

ND ND ND ND

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TABLE D6 (cont'd)(e) River Severn Impounded at Hampton Loade Reservoir (50+ Days)

Sample date TC EC FS V

14.3.79 ND26.4.79 ND8.5.79 ND13.6.79 2.7 1.6 ND ND10.7.79 2.4 1.0 ND ND14.8.79 3.1 2.4 ND ND11.9.79 3.3 2.2 0.5 0.29.10.79 4.8 2.4 1.3 0.0813.11.79 3.5 2.9 1.5 ND4.12.79 3.2 2.2 2.1 ND8.1.80 2.8 2.6 2.2 0.0612.2.80 2.9 2.3 1.9 0.1211.3.80 3.0 2.5 2.0 ND15.4.80 3.2 2.0 1.5 ND13.5.80 2.7 1.0 1.0 ND10.6.80 3.3 1.0 1.0 ND8.7.80 1.0 ND 1.0 ND12.8.80 3.3 2.6 1.0 0.019.9.80 1.5 0.6 ND ND14.10.80 ND11.11.80-* 3.1 2.6 1.9 ND13.1.81 3.5 2.9 1.9 ND10.2.81 3.6 2.6 1.6 ND10.3.81 0.214.4.81 0.112.5.81 0.214.7.81 0.25.7.82 0.2

Serotypes

B4B4

B4

B5

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TABLE D6 (cont'd)(f) Rivers Derwent and Amber Impounded at Ogston Reservoir (50+

Days)

Sample date TC EC FS V

28.3.79 ND24.4.79 ND22.5.79 ND26.6.79 1.0 ND ND ND30.8.79 ND ND ND ND26.9.79 1.0 ND ND ND24.10.79 0.3 ND ND ND27.11.79 2.9 2.5 1.0 ND30.1.80 3.1 3.0 2.2 ND27.2.80 >5.0 2.7 2.3 ND29.5.80 2.6 1.0 1.0 ND24.6.80 0.123.7.80 ND28.8.80 ND29.10.80 ND25.11.80 ND17.6.81 ND

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TABLE D6 (cont'd)

(g) River Dove Impounded at Staunton Harold Reservoir (50+ Days)

Sample date TC EC FS V Serotypes

28.3.79 ND24.4.79 ND22.5.79 1.226.6.79 2.0 ND ND ND30.8.79 0.5 0.6 ND ND26.9.79 2.0 1.3 ND 0.3 B424.10.79 2.5 2.4 0.7 ND27.11.79 2.7 2.2 ND ND3.1.80 2.4 ND ND ND27.2.80 4.5 ND ND ND29.5.80 ND ND 1.3 0.124.6.80 ND23.7.80 ND28.8.80 ND29.10.80 ND25.11.80 ND

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TABLE D6 (cont'd)(h) River Severn Impounded at Trimpley Reservoir (50+ Days)

Sample date TC EC FS V

14.3.79 ND26.4.79 ND8.5.79 ND13.6.79 1.0 ND ND ND10.7.79 ND ND ND ND14.8.79 2.0 ND ND ND11.9.79 2.0 0.9 ND ND9.10.79 3.4 1.7 1.0 0.0213.11.79 1.0 ND ND ND4.12.79 ND ND ND ND8.1.80 1.0 ND ND ND12.2.80 ND ND ND ND11.3.80 3.1 1.5 ND 0.0215.4.80 ND ND ND 0.0113.5.80 1.0 ND ND ND10.6.80 2.0 1.0 ND ND8.7.80 0.6 ND ND ND12.8.80 1.6 0.5 ND ND9.9.80 1.2 0.3 ND ND14.10.80 ND11.11.80 * 0.9 0.8 ND ND13.1.80 ND ND ND ND10.2.81 1.1 0.5 ND ND

Serotypes

B4

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TABLE D6 (cont'd)(i) Rivers Bourne and Blythe Impounded at Whitacre Reservoir (50+

Days)

Sample date

8.1.7912.3.7910.4.7914.5.7911.6.799.7.7913.8.798.10.797.1.806.2.805.3.8016.4.807.5.8011.6.802.7.8013.8.8010.9.806.10.80 12.11.8014.1.8119.2.81

TC EC FS

1.5

0.90.8ND

3.02.6 3.5 2.4 1.7

ND

NDNDND

2.7 2.32.7 ND ND

ND

NDNDND

2.42.11.0NDND

V

2.80.05NDNDND0.1NDNDNDNDNDNDNDND0.20.5NDNDNDNDND

Serotypes

PI ,2,B2, 3,4

P2

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TABLE D6 (cont'd)

(j) River Learn Impounded at Draycote Reservoir (50+ Days)

Sample date V

16.7.79 ND29.7.80 ND4.8.80 ND14.8.80 ND1.9.80 ND8.9.80 ND15.9.80 ND22.9.80 ND29.9.80 ND13.10.80 ND20.10.80 ND27.10.80 ND3.11.80 ND1.12.80 ND15.12.80 0.122.12.80 ND29.12.80 ND5.1.81 ND

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TABLE D6 (cont'd)(k) River Wye Impounded at Mitcheldean (Overnight)

Sample date TC EC FS V Serotypes

20.3.79 0.5 Pl,2,B418.4.79 0.615.5.79 0.5 Bl,419.6.79 2.1 1.9 ND 0.117.7.79 2.8 1.8 ND 1.3 B421.8.79 3.4 2.3 1.0 ND18.9.79 3.6 2.0 1.3 0.4 PI,3 ,B425.10.79 3.6 2.5 1.0 1.4 P2 ,B3,420.11.79 3.8 3.1 1.8 3.2 B322.1.80 4.7 4.2 3.3 2.3 B319.2.80 ND18.3.80 3.6 2.5 1.9 ND22.4.80 3.2 2.0 1.3 ND19.5.80 4.3 B317.6.80 4.0 3.7 2.0 0.8 B316.7.80 3.8 2.8 2.1 3.619.8.80 3.6 3.2 2.3 ND16.9.80 2.7 2.3 1.2 ND21.10.80 3.2 3.3 1.0 1.95.11.80 ND17.2.81 ND17.3.81 ND22.4.81 0.219.5.81 1.516.6.81 ND

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