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COMPARATIVE ANALYSIS OF PATHOGEN
OCCURRENCE IN WASTEWATER – MANAGEMENT
STRATEGIES FOR BARRIER FUNCTION AND MICROBIAL
CONTROL
JAKOB OTTOSON
APRIL 2004
TRITA-LWR PhD Thesis: 1021
ISSN 1650-8602
© Jakob Ottoson 2005
PhD Thesis
Department of Land and Water Resources Engineering
Royal Institute of Technology (KTH)
SE-100 44 Stockholm
www.kth.se
Department of Parasitology, Mycology, Water and Environmental Microbiology
Swedish Institute for Infectious Disease Control (SMI)
SE-171 82 Solna
www.smittskyddsinstitutet.se
Front cover photography: Faecal Bacteria by Kjell-Olof Hedlund
ISBN 91-7178-059-9
Universitetsservice US AB, Stockholm 2005
TRITA-LWR PhD Thesis: 1021
ISSN 1650-8602
Comparative occurrence of pathogens in wastewater
SUMMARY
This project was initiated to fill knowledge gaps on the occurrence of pathogens in different
streams of wastewater, e.g. greywater and domestic wastewater. The aims were also to measure
the removal of pathogens in different treatment processes, conventional and innovative, and
correlate the removal to that of common microbial process indicators, such as faecal coliforms,
enterococci, Cl. perfringens spores and bacteriophages. One study also assessed the correlation
between the removal of microorganisms and some commonly measured physico-chemical
process indicators. The results can be applied in microbial risk assessments (MRAs) of urban
wastewater systems.
Indicators and parasitic (oo)cysts were enumerated with standard methods and viruses with
rtPCR. High levels of Giardia cysts and enteroviruses were found in untreated wastewater (103.2
and 104.2 L-1 respectively) indicating high incidences in the society. Noroviruses were also often
found in high numbers (103.3 L-1) during winter, but less frequent and in lower numbers (102.3 L-1)
during the rest of the year. This temporal variation correlated to the clinical laboratory reporting
of noroviruses. A temporal variation was also shown for Giardia with significantly lower cyst
counts in untreated wastewater during spring. Cryptosporidium oocysts were not as numerous in
untreated wastewater (5 L-1) reflecting a lower incidence in the society than for the other
pathogens during the time of the study. Since temporal variation had a larger impact than spatial,
site-specific measurements may not be necessary to perform screening level MRAs of wastewater
discharge and reuse. Good data can be found in the literature and corrected for by recovery of
the detection method, flow and incidence in the society.
Removal of microorganisms in wastewater treatments varied from 0 to >5.8 log due to process
combination and organism in question. Treatment in integrated hydroponics removed
microorganism more efficiently than did secondary conventional treatment, though having longer
hydraulic retention time. Tertiary treatment and treatment in a membrane bioreactor (MBR)
showed better removal potential than treatment in upflow anaerobic sludge blankets (UASB) in a
pilot plant. Human virus genomes were less removed and Giardia cysts more removed than all of
the studied indicators. Enumeration with PCR, however, may underestimate infectious virion
removal. Spores of sulphite-reducing anaerobes and somatic coliphages were significantly less
removed than E. coli and enterococci in all the studied processes. Bacterial indicator and spore
removals correlated to enterovirus genome removal (p<0.05), but the predictive values were low
(R<0.4). Removals between microbial indicators and NH4-N, Kjeldal-N, COD and TOC
correlated stronger (10-18<p<0.02; 0.43<R<0.90).
To manage the risk with reuse and discharge of wastewater, treatment performance targets have
been calculated as a step in a hazard analysis and critical control point (HACCP) approach. These
targets varied from 0 to 10.4 log removal due to water (grey or wastewater), organism (rotavirus,
Campylobacter or parasitic (oo)cysts) and exposure (drinking water, surface water, aerosols,
irrigation of crops or public parks). Faecal contamination in greywater was measured by
coprostanol and was shown to be 980 times lower than in wastewater, corresponding to 2.9 log
removal in treatment. Somatic coliphages were suggested to function as an index of virus removal
in wastewater treatment processes as well as to be included in the monitoring of bathing water.
The guideline level was suggested to be 300 PFU 100 mL-1 based on MRA of enteroviruses. This
level in a water sample would equal a probability of infection of 0.3% (95th percentile 4%). The
risk is overestimated if animal sources dominate the faecal pollution. Development in methods to
track sources of faecal pollution showed that if somatic coliphages are enumerated together with
phages infecting Bacteroides strain GA17, discriminating human from animal faecal pollution is
possible based on the ratio between the phages.
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Comparative occurrence of pathogens in wastewater
TABLE OF CONTENTS
SUMMARY..............................................................................................................................iii
TABLE OF CONTENTS......................................................................................................... v
LIST OF PAPERS.................................................................................................................. vii
ABBREVIATIONS ................................................................................................................. ix
1. INTRODUCTION............................................................................................................ 1
1.1.
Background .................................................................................................................... 1
1.2.
Health and hygiene......................................................................................................... 2
1.3.
Wastewater ..................................................................................................................... 2
1.3.1. Greywater ............................................................................................................... 4
1.4.
Wastewater treatment..................................................................................................... 4
1.4.1. Conventional treatment .......................................................................................... 4
1.4.2. Alternative treatments ............................................................................................ 5
1.5.
Public exposure to wastewater ....................................................................................... 8
1.5.1. Water discharge...................................................................................................... 8
1.5.2. Water reuse............................................................................................................. 9
1.6.
Water management....................................................................................................... 10
1.6.1. Indicators of faecal pollution................................................................................ 10
1.6.2. Microbial Source-Tracking .................................................................................. 12
1.6.3. Microbial Risk Assessment (MRA) ..................................................................... 12
1.6.4. Hazard Analysis and Critical Control Point (HACCP)........................................ 18
2. OBJECTIVES AND SCOPE......................................................................................... 21
3. MATERIALS AND METHODS................................................................................... 23
3.1.
Study Sites.................................................................................................................... 23
3.2.
Microbiological Methods ............................................................................................. 23
3.2.1. Human virus detection ......................................................................................... 23
3.2.2. Protozoan analyses ............................................................................................... 24
3.3.
Microbial Risk Assessment Methods........................................................................... 25
3.3.1. Pathogens assessed and data used ........................................................................ 25
3.3.2. Exposures assessed............................................................................................... 27
4. SUMMARY OF RESULTS........................................................................................... 31
4.1.
Occurrence ................................................................................................................... 31
4.1.1. Faecal indicators in wastewater............................................................................ 31
4.1.2. Pathogens in wastewater ...................................................................................... 32
4.2.
Removal ....................................................................................................................... 34
4.2.1. Correlation with process indicators...................................................................... 36
4.3.
Risk............................................................................................................................... 37
4.3.1. Exposure to surface water .................................................................................... 38
4.4.
Microbial source-tracking ............................................................................................ 38
5. DISCUSSION ................................................................................................................. 43
5.1.
Do we need site-specific data? ..................................................................................... 43
5.2.
How do we measure the microbial removal in treatment?........................................... 43
5.3.
How can we manage the risks? .................................................................................... 45
5.3.1. Surface water management .................................................................................. 47
6. CONCLUSIONS............................................................................................................. 49
7. ACKNOWLEDGEMENTS........................................................................................... 51
8. REFERENCES ............................................................................................................... 53
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Comparative occurrence of pathogens in wastewater
LIST OF PAPERS
This thesis is based on the following papers, which have been referred to in the text by their
roman numerals. Published papers were reproduced by the kind permission from the publishers.
I. Ottoson, J. & Stenström, T.A. (2003) Faecal contamination of greywater and associated
microbial risks. Water Res 37(3), 645-655
II. Ottoson, J., Hansen, A., Westrell, T., Johansen, K., Norder, H. & Stenström, T. A. (2004).
Removal of noro- and enteroviruses, Giardia cysts, Cryptosporidium oocysts and faecal
indicators at four secondary wastewater treatment plants in Sweden. Submitted to Water
Environ Res.
III. Ottoson, J., Hansen, A., Björlenius, B., Norder, H. & Stenström, T. A. (2005). Removal of
viruses, parasitic protozoa microbial indicators and correlation with process indicators in
conventional and membrane processes in a wastewater pilot plant. (Manuscript).
IV. Ottoson, J., Norström, A. & Dalhammar, G. (2005). Removal of microorganisms in a
small scale hydroponics wastewater treatment system. Letters Appl Microbiol, In Press
(Published on-line early).
V. Ottoson, J., Stenström, T. A. & Ashbolt, N. J. (2005). Proposed guidelines for bathing
water based on the occurrence of somatic coliphages. (Submitted to Lett Appl Microbiol).
VI. Blanch, A. R., Belanche-Muñoz, L., Bonjoch, X., Ebdon, J., Gantzer, C., Lucena, F.,
Ottoson, J., Kourtis, C., Iversen, A., Kühn, I., Moce, L., Muniesa, M., Schwartzbrod, J.,
Skraber, S., Papageorgiou, G., Taylor, H. D., Wallis, J. & Jofre, J. (2004). Tracking the
origin of faecal pollution in surface water. An ongoing project within the European Union
research programme. J Water Health, 4(2), 249 – 260.
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Comparative occurrence of pathogens in wastewater
ABBREVIATIONS
Bact.
Bif.
BOD
C.
Camp.
CFU
Cl.
COD
DALY
E.
EPA
EHEC
Ent.
FITC
G.
GC-MS
HACCP
HMU
IMS
MBR
MPN
MRA
PDF
PE
PFU
RNA
RO
rtPCR
SD
SS
TOC
UV
UASB
VBNC
WWTP
XOC
Bacteroides
Bifidobacterium
Biological oxygen demand
Cryptosporidium
Campylobacter
Colony forming unit
Clostridium
Chemical oxygen demand
Disability adjusted life years
Escherichia
Environmental protection agency
Enterohaemorrhagic E. coli
Enterococcus
Fluorescein isothiocyanate
Giardia
Gas chromatography mass spectrometry
Hazard analysis and critical control point
Health-related modelling unit
Immunomagnetic separation
Membrane bioreactor
Most probable number
Microbial risk assessment
Probability density function
Person equivalents
Plack forming unit
Ribonucleic acid
Reverse osmosis
Reverse transcriptase polymerase chain reaction
Standard deviation
Suspended solids
Total organic carbon
Ultraviolet (light)
Upflow anaerobic sludge blanket
Viable but non-culturable
Wastewater treatment plant
Xenobiotic organic compound
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Comparative occurrence of pathogens in wastewater
1.
INTRODUCTION
1.1.
Background
water. Diseases can be transmitted to humans
via water due to activities such as swimming
in or drinking contaminated water and food
production from irrigation with contaminated
water. Run-off from agriculture land will
reintroduce pathogens from sludge and
manure to surface waters, etc. The circulation
of pathogens in the society is to a large extent
dependent on the water and wastewater
systems that we build. The design of the
systems will also affect the possibilities to
restrict environmental transmission of
pathogens.
Poor sanitation and lack of drinking water of
high quality is estimated to be the main
global cause of approximately 4,000 deaths
per day (20). To be able to fulfil the
millennium development goals, water and
sanitation are of highest international priority.
The global perspective on water and
sanitation is very different from the Swedish
situation with abundance of clean water and
possibilities of surface water discharge.
However, water shortage may temporarily
occur as well as infections due to
contaminated water (220). Still, when water
management is discussed the starting point is
sustainability rather than acute need of fresh
water sources.
A Swedish research programme "Sustainable
Urban Water Management” was started in
1999. The programme aimed to develop tools
and knowledge to guide the planning and
management of the water and wastewater
systems in the future sustainable Sweden
(238). The water and wastewater system
consists of its water users, technical
infrastructure and the institutions involved
and it can be judged by how well it meets
chosen aspects of sustainability: health,
environment, economy, socio-culture and
technical function (Fig. 1). Sustainability
criteria for these five aspects have been
defined and a toolbox intended for urban
planners, water utilities and other actors
involved in the planning of future systems is
now in its end stages (www.urbanwater.org).
This thesis is linked to the health criterion
and hygiene risks with reuse and discharge of
wastewater.
For the water sector, sustainability can be
defined as the ability to plan and manage
resources so they can be used for future
generations. Within the urban water cycle this
means that: water that is used, should be able
to be reused, and returned to nature. Plant
nutrients in the wastewater - nitrogen N,
phosphorous, P and potassium, K - should
be returned to productive land as fertilisers
and finally, harmful substances should be
removed from the water and nutrient cycles
(238). Further, all these activities must be
performed in a way to minimise adverse
human health risks. In Sweden and other
developed areas of the world, wastewater
from cities is most often treated at municipal
treatment plants. All collected water –
stormwater from roofs, streets and other
hard surfaces, blackwater from toilets,
greywater from kitchen sinks, bathrooms and
washing facilities and industrial wastewater is transported to the treatment plant.
Potentially more than 100 different types of
pathogenic viruses, bacteria and parasites that
may cause an immense range of diseases and
clinical symptoms in humans such as
diarrhoea, meningitis, myocarditis and
hepatitis, can be present in faecally-polluted
Fig. 1. The urban water conceptual framework. At the
top is the system with its subsystems and below are the
aspects of sustainability (238).
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Jakob Ottoson
Trita LWR PHD
1.2. Health and hygiene
pathogenic agents. Epidemiological studies
have broadened our understanding of the
importance of transmission of infectious
diseases from the environment, for example
by identifying key pathogens from
waterborne outbreak data. Epidemiological
background data have also been used within
the framework of MRA to enable the
estimation of pathogen loads in wastewater
when direct pathogen counts may be
unavailable.
The
problem
with
epidemiological studies in this context is their
lack of sensitivity (51). The detection limit for
exposure to bathing water has been estimated
to be an increase in disease incidence in the
range of 1 – 5% (55), which is 100 – 500
times higher than the 0.01% suggested by
Regli et al. (1991) (182). Higher sensitivity in
risk estimates is therefore needed to meet the
future demands on security of water and
sanitation systems in many parts of the
world. Further, epidemiological studies are
time-consuming and costly and the
possibilities to do prospective studies are
limited. Thus, innovative full-scale systems
cannot be properly evaluated.
The overall criterion for the water and
wastewater systems from a hygiene point of
view is that the risk of infection from
environmental sources should never exceed a
background level. This background level may
however differ between various regions of
the world and over time (239). Microbial risk
assessment, MRA (1.6.3), has been developed
as a tool to predict the consequences of
potential or actual exposure to infectious
microorganisms (85) and has been applied
within the Urban Water programme as a tool
to compare water and wastewater systems
within a systems´ analytical framework. In
the MRA tool, the first criterion assessed is
the ability to maintain an acceptably low
infection level, currently considered to be <1
per 10,000 people per annum, as proposed by
the US EPA and Dutch regulation for
microbial risks from drinking water (6, 182).
This criterion implies that infections in the
receiving population can be predicted or
measured (239). The second criterion
addressed is system robustness. Multiple
barriers provide pathogen reduction with a
high degree of redundancy. The MRA Tool is
set up with the potential to characterise the
performance of each barrier by its reliability
and variability. Hence, the second criterion
implies a number of barriers as well as a
stochastic factor (probability of performance)
for each major pathogen group by exposure
points (239).
1.3. Wastewater
The pathogens of main concern in the
wastewater are those from human faeces
transmitted by the faecal-oral route, thus
emanating from the toilet. The other urban
wastewater flows are greywater (bath, dish,
laundrywater), industrial wastewater and
stormwater from hard surfaces (Table 1). The
main focus in this thesis is the domestic
wastewater (household wastewater) including
a specific study of the greywater flow. In
some of the investigated treatment plants,
treatment of storm- and industrial wastewater
also took place. These were, however, not
explicitly examined.
Traditionally, epidemiological studies have
been the main tool to measure health effects
within a population. Epidemiology is defined
as “the study of the distribution and
determinants of health-related states or
events in specified populations, and the
application to control health problems” (22),
for example how a population react to
2
Comparative occurrence of pathogens in wastewater
Table 1. Urban wastewater streams and their approximate volumes [L] (116, 157).
Flow
Domestic wastewater
Stormwater
Vol
200
Flow
Blackwater
Vol
50
Greywater
150
Rain run-off from hard surfaces
Industrial wastewater
Flow
Urine
Faeces
Flushwater
Bath, Showers, Handbasins
Laundry
Dishwasher, Kitchen sink
Roads
Roofs
Wastewater from industries
wastewater is normally connected with a
health risk. In Table 2, some international
data on pathogen occurrence in wastewater
are reported.
Before this study, the knowledge of pathogen
occurrence in different wastewater streams in
Sweden was limited and scattered. Risk
assessments would thus have to rely on
epidemiological
approaches
and/or
international data, resulting in some
limitations. Within the epidemiological
approaches the asymptomatic carriers are not
accounted for since only a fraction seeks
medical care and many cases are not
reported. Numerical adjustments can be
made (as in paper I) but a high degree of
uncertainty is then integrated into the
models. Using international data is another
option, however, the endemic rates may
differ between regions. Furthermore, the
microbial survival is longer and removal in
treatment may differ due to the colder water
in the Nordic countries than for example in
tropical and sub-tropical climates. All these
factors stress the need for Swedish
investigations. In a larger population, some
people are most certainly infected with a
gastro-intestinal
pathogen
and
thus
Protozoa and enteric viruses are of specific
concern in wastewater reuse as they generally
pose a greater challenge than bacteria in
wastewater treatment, and the risk of
infection can be 10 to 1,000 times higher
than for bacteria at a similar level of exposure
(189). Highly infectious bacteria like
Campylobacter (231) and enterohaemorrhagic
E. coli, EHEC (229), are notable exceptions.
Enteric viruses are of major health concern
for waters in industrialised countries with
high health standards, where immunity is
low. Illness may here be detectable
epidemiologically at relatively low incidences
(7). Norovirus gastroenteritis is probably the
most common single agent responsible for
diarrhoea in developed regions of the world
(133).
Table 2. Reported numbers of pathogens in wastewater [L-1]
Pathogen
Bacteria
Salmonella spp.
Campylobacter spp.
Enteric viruses
Enteroviruses
Rotavirus
Norovirus
Adenovirus
Protozoa
Giardia cysts
Cryptosporidium oocysts
Vol
1.5
0.1
48.4
120
20
10
Range
Country
Reference
930 – 110,000
8,900 – 290,000*
500 – 4.4·106
16,300
Finland
Germany
Germany
Italy
(121)
(42)
(102)
(214)
100 – 10,000
< 1 – 10,000
6
< 1,000 – 1,6·10
250 – 250,000
Italy
Netherlands
Germany
Spain
(15)
(132)
(180)
(28)
1,100 – 52,000
100 – 9,200
< 20 - 400
1 – 560
Scotland
Canada
Scotland
Canada
(185)
(175)
(185)
(175)
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Jakob Ottoson
Trita LWR PHD
Table 3. Reported numbers of faecal indicator bacteria in grey wastewater [log10 100mL-1]
Wastewater origin
Bath, hand basin
Laundry
Shower, handbasin
Greywater
Shower, bath
Laundry, wash
Laundry, rinse
Greywater
Hand basin, kitchen sink
Greywater
Greywater, 79% shower
Kitchen sink
Greywater
Total
Coliforms
3.4-5.5
2.7-7.4
7.9
1.8-3.9
1.9-5.9
2.3-5.2
7.2-8.8
Thermotolerant
Coliforms E. coli
4.4
2.0-3.0
2.2-3.5
5.8
0-3.7
1.0-4.2
0-5.4
5.0
5.2-7.0
4.3-6.9
7.6
5.8
7.4
Enterococci
1.0-5.4
1.4-3.4
1.9-3.4
2.4
0-4.8
1.5-3.9
0-6.1
3.2-5.1
7.4
5.4
Reference
(2)
(38)
(38)
(37)
(56)
(56)
(56)
(74)
4.6 (83)
(131)
(190)
7.7 (157)
4.6 (157)
(Table 3). Greywater may, however, have an
elevated load of easily degradable organic
compounds, which may favour growth of
enteric bacteria such as the faecal indicators.
Such growth has been reported in wastewater
systems (137). Hence, a focus on bacterial
indicator numbers may lead to an
overestimation of faecal loads and thus risk.
Occasionally enteric pathogenic bacteria,
such as Salmonella and Campylobacter, can be
introduced by food handling in the kitchen
(39).
1.3.1. Greywater
Greywater is defined as household wastewater
excluding toilet waste, i.e. wastewater from
the sinks, shower, washing machine and
dishwasher. The average flow is 150 L p·d-1
(157). The exclusion of blackwater (toilet
waste) results in greywater with low faecal
contamination and with less eutrophying
substances, thus simplifying local treatment
and reuse. The load of adverse chemical
compounds is also significant. Eriksson et al.,
(2002) (54) identified 900 xenobiotic organic
compounds (XOCs) that may be present in
greywater. Still, the interest in reusing
greywater has increased dramatically in recent
years, especially in arid areas. In some densely
populated areas such as Singapore and
Tokyo, greywater reuse is already a common
practice (9). The main hazard of greywater
emanates
from
the
faecal
crosscontamination. Since toilet waste is not
included in greywater, faecal contamination is
limited to activities such as washing faecally
contaminated laundry (i.e. diapers), childcare,
anal cleansing and showering. Faecal
contamination has historically been measured
by the use of the common indicator
organisms such as coliforms and enterococci
(1.6.1). These have also been applied for
assessing faecal contamination of greywater.
Some studies have reported high numbers of
these organism groups, which would indicate
substantial input of faeces to the greywater
1.4. Wastewater treatment
Most of the treatment plants in Sweden have
both biological treatment (activated sludge,
biofilters, rotating discs etc.) and chemical
precipitation (flocculation followed by
sedimentation, flotationat or filtration). An
investigation on the removal of indicators in
Swedish secondary wastewater treatment
plants (WWTP) reported removals from 80
to 99.9% depending on process combination
and organism in question (218). Somatic
coliphages as viral indicators were, however,
removed to a significantly lower degree than
bacterial indicators.
1.4.1. Conventional treatment
Primary treatment refers to the initial
processing of wastewater for removal of
4
Comparative occurrence of pathogens in wastewater
particulate matter.
In conventional
wastewater treatment facilities, primary
treatment includes screening for removal of
large solids, grit removal, and sedimentation.
Incorporating
coagulation/flocculation
upstream of sedimentation, which is a
common practise in Swedish wastewater
treatment plants, increases the removal
efficiency of primary treatment processes.
The flocs that form in the chemical
precipitation capture organic matter, other
particulate material and microorganisms,
which can be removed efficiently by
sedimentation. The temperature, dose of
coagulant, pH and characteristics of the
organic matter governs the overall efficiency
of chemical treatment (43). The reduction
was 93-99.6% for indicator organisms in
Swedish wastewater treatment plants in the
chemical-mechanical process (218). Chemical
precipitation has been in use in the
investigated treatment plants reported in
papers II and III.
investigated treatment plants in papers I, II,
III and IV.
In general, tertiary treatment refers to
additional removal of colloidal and
suspended solids by chemical coagulation
and/or granular media filtration. Filtration is
an effective method for the removal of
microorganisms present in wastewater. The
mechanisms of removal are a combination of
size exclusion and adsorption (201). Further,
microorganisms are removed by protozoan
predation and degradation by autochthonous
microbiota in biological treatments such as
slow sand filtration (99). Thus, slow sand
filtration is a barrier in itself and herein seen
as tertiary treatment whereas rapid filtration
is considered a means to capture flocs from
earlier steps of the treatment. At Klagshamn,
Malmö, rapid sand filtration is considered as
a complement to secondary treatment (II)
whereas in Henriksdal, Stockholm, a
coagulant is added before filtration. Further
the retention time in the sand-filter is longer
and therefore considered to be tertiary
treatment (III).
Secondary treatment systems include an array
of biological treatment processes coupled
with solid/liquid separation.
Biological
processes are engineered to provide effective
bio-oxidation of organic substrates dissolved
or suspended in the wastewater.
The
microbial biomass interacts with the
compounds in the wastewater using
suspended-growth or fixed-film processes.
Examples of suspended-growth processes
include activated sludge, aerated lagoons and
waste stabilization ponds. Examples of
fixed-film processes are trickling filters,
rotating biological contactors, and other
biofilm bioreactors. In the biological
treatment, bacteria digest organic matter in
the wastewater, reducing the organic material,
measured as BOD and COD, significantly. In
the process, nitrogen, phosphorus, inorganic
substances and pathogens are also reduced.
The pathogen reduction is due to
competition, digestion and sedimentation. In
Swedish wastewater treatment plants, a 5398% reduction of different indicator
organisms was observed in the activated
sludge treatment process (218). Biological
treatment in any form is included in the
1.4.2. Alternative treatments
As a response to waste of resources, poor
sludge quality, small-scale solutions and
higher demands on water quality, different
alternative techniques to treat wastewater are
used. Some alternative treatment methods are
briefly exemplified below, among which
some are common small-scale solutions in
households and are not connected to the
municipal sewers.
Infiltration is the most common way to treat
greywater in Sweden and is normally used in
small-scale (single households) treatment of
wastewater, but may sometimes be used for
municipal wastewater treatment (219).
Wastewater is distributed evenly over a
subsurface disposal system. Filters, consisting
of 60-150 cm of sand (or other material) in a
bed, improve the quality further. Intermittent
dosing of the wastewater is mainly applied
(224). Infiltration systems may affect
5
Jakob Ottoson
Trita LWR PHD
groundwater quality due to hydrological prerequisites. Guidance for suitable sites is
provided in Naturvårdsverket, (1985) (156).
and protozoa (222, 245, 251), UV and the
retention-time are important factors for virus
reduction (67). The system investigated in
paper I includes a pond treatment of
greywater.
If the water from the infiltration unit is
collected for further treatment, reused or
released into a receiving water body, the
terms sand filter trenches, biofilters or constructed
wetlands are used. The latter are planted,
forming a root-zone for direct uptake of
water and plant nutrients (Fig. 2). Biofilters
and constructed wetlands using light-weight
aggregates were pioneered in Norway (114).
More than 5 log removal of E. coli have been
observed during intermittent filtration with
hydraulic loading rate, media grain size and
retention time being the most important
factors (16, 223, 224). Pre-treatment with
aeration in a biofilter reduces BOD and
bacteria, so higher loading rates can be
applied for the subsequent wetland or
infiltration system (114).
Hydroponics integrated with conventional
systems has been developed as a means to
increase nutrient recirculation (233). Plants
assimilate nitrogen and phosphorous directly
from the wastewater, thereby minimising
sludge production. To cope with the Swedish
climate, the investigated hydroponics was
situated in a greenhouse (IV).
Membranes of different pore sizes can be used
to produce high quality water from a hygienic
point of view. Processes use osmotic or other
pressure to force water through a
semipermeable membrane as permeate with
dissolved solids and other constituents
captured as retentate or concentrate. The
membranes were commonly made of organic
polymers, but new types of inorganic
polymers as well as ceramic and metallic
membranes are now also in use. The basic
membrane systems include microfiltration,
ultrafiltration, nanofiltration and reverse
osmosis (RO), retaining different size ranges
of particles (Fig. 3) (241).
Wetlands and ponds have become a more
frequent way to polish wastewater in order to
remove nitrogen and microorganisms. The
reduction of pathogens is dependent on
system design, retention time and dilution
(146, 181). In Sweden, the cold climate is not
favourable for biological pond treatment.
Since virions do not settle as easily as bacteria
Fig. 2. A subsurface flow wetland with an integrated biofilter (114).
6
Comparative occurrence of pathogens in wastewater
Fig. 3. Membrane process classification (241).
A membrane bioreactor (MBR) is a
modification of the activated sludge process
in which solids separation is achieved without
the requirement of a secondary clarifier.
Instead this function is carried out by a
membrane, which retains the particulate
phase within the reactor and allows the
treated, clarified effluent to pass to the next
process/effluent. The small pore size of the
membranes employed for solids separation
results in the removal of a wide range of
microorganisms. MBR has several potential
advantages over conventional wastewater
treatment technologies, including lower
energy use, smaller space requirements and
better reduction of microorganisms and
organic matter (113). Studies have reported 5
– 7 log removal of indicator bacteria in MBR
processes (66, 236). Viruses however, are
smaller than the pore sizes used in
microfiltration processes. High removals of
viruses have been reported anyway, but first
after the build-up of a biofilm on the
membrane (59, 236). Except for pore-size
and biofilm build-up, water chemistry
(conductivity and pH), feed concentration
and particulate matter in the feed water
contributes to the removal of viruses (92,
110, 247). In the pilot plant at Hammarby
Sjöstad (3.1), MBR treatment with
microfiltration is in operation and has been
evaluated (III).
In the pilot plant at Hammarby Sjöstad (3.1),
trials with anaerobic treatment of wastewater are
made. The advantages with anaerobic
compared to conventional treatment are
lower energy use, less sludge production and
production of biogas (225). The anaerobic
treatment line includes upflow anaerobic sludge
blankets, (UASB). In the UASB, biological
treatment is performed on granules, formed
by existing bacteria under the right hydraulic
conditions (225). The mechanisms by which
these granules are formed are not well
understood (196). A study on indicators in a
UASB process in Colombia reported
removals from 0.3 to 0.7 log (134). The
microbial removal in anaerobic treatment in
UASB was investigated in paper III.
Disinfection with chlorine has traditionally
been the most common method. It will
reduce the number of many, but not all,
pathogens. The protozoan oocysts have
shown to be resistant to disinfection by
chlorine (188). Chlorinated byproducts may
function as endocrine disruptors and can be
toxic to aquatic life. Ozonation is an
alternative disinfection method that will not
create chlorinated organic compounds.
Ozonation is also more effective in
inactivating protozoan (oo)cysts than
chlorine compounds (186). Chemical
7
Jakob Ottoson
Trita LWR PHD
disinfection is, however, not a method
practised to treat wastewater in Sweden, but
ultraviolet light, UV, disinfection is
sometimes applied in low-turbid wastewater
streams. It is for example used in water
recycling applications, where pathogenic
contamination is of concern. An example is
the AquatronTM that separates the liquid from
the solid part of the toilet waste by
centrifugal forces. The liquid flow then enters
the greywater stream after UV disinfection.
This system is used in an eco-village,
Ekoporten, Norrköping, with subsequent
treatment in a settling tank and a constructed
wetland (131). At this location, the UV unit
did, however, not disinfect the toilet
flushwater properly due to the amount of
suspended solids (131). Similar problems
with UV-disinfection have been reported
from other sites (251).
products is poorly documented (221), but
evidence for higher disease incidences in
population exposed to reused wastewater
than unexposed have been documented (144,
221).
1.5.1. Water discharge
Wastewater is mainly discharged to surface
water recipients. Besides the health risks,
faecal contamination will also degrade water
environments that are important for
recreation, fishing and drinking water
production. Recreational water is an
important exposure route for transmission of
disease. Several studies have shown a relation
between gastroenteritis in populations
swimming in water polluted by faecal
indicator bacteria. When comparing the
groups “swimmers” and “non-swimmers” it
was found that swimming in even marginally
polluted water was a significant route of
transmission (33, 61, 118, 240, 258). Also an
increase in non-enteric disease has been
registered from waters with elevated
microbial contamination (62). Performance
guidelines for wastewater treatment have
been developed for reclamation and reuse
(Table 5). These refer to both levels of
organisms and degree of treatment (126),
while risk management of receiving waters
usually relies on the monitoring of faecal
indicator bacteria, i.e. faecal coliforms and in
marine waters enterococci, at bathing sites
(55). There are, however, limitations on how
well these indicator organisms can predict
microbial risks. Their removal and
environmental decay have proven to differ
from pathogen removal and decay, especially
for viruses and (oo)cysts (15, 29, 111, 155,
188, 200, 244). Alternative indicators
suggested are bacteriophages and spores of
sulphite-reducing anaerobic bacteria (1.6.1).
While these are supposed to be better
indicators of enteric viruses and (oo)cyst
occurrence of wastewater origin (41, 88, 174),
they should be used with precaution. Spores
are to a large extent absent in herbivore
faeces at the same time as cattle are an
important source of oocyst contamination
1.5. Public exposure to wastewater
The public can be exposed to wastewater by
several routes. The most common ones are
through recreational activities, such as
bathing, and drinking water produced from
surface water sources. An indirect exposure
route is from shellfish production. Filter
feeders such as molluscs concentrate
microorganisms occurring in contaminated
water by filtration that may infect the
consumers (226). At a mussel-farm used for
bioremediation, enteric viruses were found in
50 – 60 % of the mussel samples (93). In all
the above-mentioned exposure scenarios
wastewater has been discharged into the
receiving water. Shuval, (2003) (203)
estimated the total cost of human infectious
diseases from swimming/bathing in coastal
waters polluted by wastewater and eating
filter-feeders harvested from such waters to
some 12 billion dollars yearly. Direct use of
wastewater for irrigation may expose people
to aerosols during irrigation as well as
through contact with the irrigated area or
ingestion of irrigated crops. Other uses
exposing people to wastewater are toilet
flushing and groundwater production (Table
4). Increase in disease due to use of waste
8
Comparative occurrence of pathogens in wastewater
(123). Only humans excrete enteric viruses of
health significance for a human population,
while most warm-blooded animals excrete
indicators. Methods for tracking the sources
of faecal pollution are thus very important in
order to assess the risk in a correct way as
well as for an effective water management
where the pollution can be counteracted at its
source (127, 136).
Wastewater reuse is a common practise in
many parts of the world even though the
pathogen load is higher than in greywater. It
is for example used for toilet flushing and
irrigation of gardens in Japan (164) and
Australia (4). In Sweden, secondary treated
wastewater is used for irrigation of energy
crops (36). In Singapore, treated wastewater
is returned to a raw water reservoir to be
used in process industries (82). Reuse of
wastewater need more treatment, due to the
higher pathogen content, than greywater and
urine. However, all flows are possibly
contaminated to some extent and safety
measures have to be taken into consideration
before building new systems, potentially
leading to new transmission routes of
pathogens to humans. In this context, MRA
(1.6.3) can serve as a very useful tool. The
new WHO drinking water guidelines (257), as
well as the forthcoming wastewater use
guidelines, are based on MRA, where
treatment targets are decided based on the
occurrence of pathogens in the raw water
(Fig. 4). This approach can also be useful as a
means to harmonise and communicate the
risks connected with the reuse of different
wastewater streams. But what should the
targets be and how do we best measure that
they are reached?
1.5.2. Water reuse
Wastewater reuse is driven by economical
reasons, a demand for nutrients to be
recirculated as fertilisers or to account for
water shortages. In the latter case, wastewater
and greywater have been used for irrigation,
but also for indoor house activities as toilet
flushing and even for drinking water
production (3, 9, 25, 38, 44, 95, 115, 160, 165,
192, 235). In new Australian settlements, reuse of greywater is a common practise and a
third pipe included in many buildings (10).
To meet proposed guidelines for wastewater
reuse (Table 5), extensive treatment is most
often needed, also for greywater that may
have an elevated load of coliforms bacteria.
The treatment performance based on the
removal of coliform bacteria may however be
overestimated due to the possible growth of
these of bacteria within greywater system.
Table 4. Examples of end-uses of treated wastewater, types of human exposure and approximate
exposure volumes.
Exposure route
Exposure type
Toilet flushing
Crop irrigation
Pond
(As part of treatment)
Receiving water
Ground water
Drinking water
Inhalation of aerosol
Ingestion of crop
Inhalation of aerosol
Accidental ingestion
Approx.
exposure volume
0.01 mL
10 mL
0.05 mL
1 mL
(45)
(8, 204)
(45, 120)
(13)
Ingestion
Ingestion
Ingestion
50 mL
1L
1L
(12)
(191, 253)
(191, 253)
9
References
Jakob Ottoson
Trita LWR PHD
Table 5. Reported microbial guidelines on reuse of wastewater.
Guidelines
Total coliforms
< 10
< 10
< 100
< 1,000
Volume Specified use
Reference
100 mL
100 mL
(126)
(119)
Unrestricted irrigation
Primary contact recreation, toilet flushing, car washing, food
a
aquaculture, unrestricted public access
Secondary contact recreation, ponds parks and gardens with access,
a
food crops
Passive recreation, parks and gardens with no public access during
a
irrigation
Irrigation of pasture and fodder, non food crops
Food crops
Non-food crops
Unrestrictrd recreational reuse
< 10,000
< 23 avg 2.2
100 mL
< 240 avg 23
< 240 avg 23
Faecal
coliforms
< 1,000
100 mL Unrestricted irrigation
< 400 avg 200
100 mL Food crops
< 400 avg 200
Non-food crops
< 23 avg 2.2
Unrestrictrd recreational reuse
<1
100 mL Unrestricted irrigation
Enteric viruses
<2
50 L
Unrestricted irrigation
Parasitic
(oo)cysts
<1
50 L
Unrestricted irrigation
Nematode eggs
<2
1L
Unrestricted irrigation
a
Intestinal parasites and viruses need to be considered, b California, c WHO Guideline, dNevada
(243)
b
(256)c
(243)d
(126)
(126)
(126)
(256)
c
density in the wastewater and wastewater
systems is dependent on the number of
infected people in the population
contributing to the wastewater flow. Since
the enumeration of pathogens is often
difficult, indicators of faecal pollution play an
important role in water guidelines and
management (216). A good indicator of
faecal pollution should be a common part of
the intestinal microbiota and occur naturally
in relatively high numbers in faeces from
humans and warm-blooded animals, but not
elsewhere. It should have the same
growth/reduction pattern in the environment
and different water treatment processes as
the pathogenic microorganisms that are
indicated. Finally, it should be easy to analyse
(216). The most widely used indicators are
members of the coliform group, but
enterococci, bacteriophages and spores of
sulphite reducing anaerobic bacteria are
becoming more frequently used as
alternatives or supplementary indicators to
the coliforms. Most bacteria in human and
animal faeces are anaerobic, such as Bacteroides
fragilis and bifidobacteria. Their rapid die-off
in the environment, however, makes them
Fig. 4. Treatment performance targets for
selected viral, bacterial and protozoan
pathogens in relation to raw water quality
(257).
1.6. Water management
1.6.1. Indicators of faecal pollution
Gastrointestinal pathogenic microorganisms
do not occur as a natural part of the normal
intestinal microbiota. Their presence and
10
Comparative occurrence of pathogens in wastewater
unsuitable as indicators of faecal pollution
(216), but their phages can be used. There are
also some chemical substances that can be
used to track faecal contamination, of which
faecal sterols are the most frequently used.
resistant than vegetative bacteria to chemical
and physical stress and may give an indication
of remote or intermittent faecal pollution
(104). They have been suggested as index
organisms for parasitic protozoan reduction
in water treatment processes (94, 174).
Coliform bacteria
Coliform bacteria are the most widely used
faecal indicators. They are a heterogeneous
group that can originate from several
environments. Thermotolerant coliform
bacteria have a direct connection to sewage
pollution but some Klebsiella spp. can also
originate from plant degradation. E. coli is
almost exclusively of faecal origin and thus a
more reliable faecal indicator than the other
coliform groups (216). E coli is excreted in
densities of 105 – 108 CFU g-1 (72). However,
regrowth in the environment may occur.
Coliforms may also differ in sensitivity to
treatment processes or environmental stress
compared to many pathogens (15, 29, 111,
155, 188, 200, 244), which restrict their
suitability as a sole indicator of the hygiene
quality of reuse products (27).
Bacteriophages
Bacteriophages are viruses that infect
bacterial host cells but are harmless to
humans. Many enteric viruses are more
resistant in the environment, as well as to
different treatments, than bacteria. They are
also smaller, which assigns them different
transport features. Therefore bacteriophages
have been suggested as indicator organisms
to predict the presence and behaviour of
enteric viruses in the environment (88).
Bacteriophages used as indicators for faecal
pollution mainly belong to three groups: 1)
Somatic coliphages, infecting various E. coli
and related strains by attachment to the cell
wall as the first step in the infection process.
2) F-specific RNA bacteriophages are capable
of infecting bacteria possessing the F-plasmid
(sex plasmid) by adsorbtion to the F-pili as
the first step in the infection process. Their
presence indicates pollution by wastewater
contaminated by human or animal faeces
(89). 3) Phages infecting Bacteroides fragilis
attach to molecules in the cell wall of host
bacteria as the first step in the infection
process. Phages infecting Bact. fragilis
RYC2056 in a water sample indicate human
or animal faecal pollution, while phages
infecting Bact. fragilis HSP40 in a sample
preferably indicate faecal pollution of human
origin (178, 179).
Enterococci
Enterococci (faecal streptococci) are present
in faeces in densities between 105 – 107 CFU
g-1 (72). They are considered to be an
alternative to coliform bacteria since they are
more tolerant to environmental stress.
Enterococci have also been suggested as an
indicator for the die-off and potential
occurrence of enteric viruses, particularly in
sludge and seawater (27). Health risks from
exposures to recreational water have been
reported to correlate to enterococci densities
(118). Enterococci may however adsorb
more strongly to soil particles and are more
sensitive to detergents than many pathogens
(216), which may restrict their use as
indicator in some greywater applications and
groundwater recharge.
Chemical indicators
Faecal sterols are the collective term for the
sterols and stanols excreted in faeces. The
composition of faecal sterols in faeces
depends on diet, age and endogenous
synthesis and biohydrogenation in the
digestive tract (129, 147, 148). Coprostanol
(5β-cholestan-3β-ol) is the principal faecal
sterol in human faeces, constituting about 4060% of the total sterol content (246). It is
produced from cholesterol by anaerobic
bacteria in the digestive tract (149). Analyses
Spores of sulphite reducing anaerobic bacteria
Spores of sulphite reducing anaerobic
bacteria are dominated by Clostridium
perfringens spores, which are present in human
and animal faecal matter. The spores survive
for long periods in water and are more
11
Jakob Ottoson
Trita LWR PHD
of faecal sterols, especially coprostanol, have
been used as an alternative to indicator
bacteria to determine faecal contamination
(127, 246). Coprostanol can also be used to
quantify the faecal load under specific
circumstances (198).
biochemical tests, performed in
microplates to type common
indicator bacteria isolates from
surface water (124, 125).
4. Genotypic identification of faecal
organisms. Genotyping has been
more
time-consuming
than
phenotyping, but species-specific
genes may be targeted giving a
better discrimination, although
losing in sensitivity.
5. Detection and ratio of faecal sterols
and stanols. Depending on diet and
intestinal microbiota different
species excrete different sterols and
stanols. Coprostanol has been used
as a marker of human faecal
pollution (129). Ratios of sterols
and
stanols
together
with
information from occurrence of
indicator bacteria has been used to
distinguish sources of faecal
pollution successfully also giving
information of the age of the
pollution (128).
1.6.2. Microbial Source-Tracking
Several methods to discriminate human from
animal faecal pollution have been reported
(206). The approaches can be grouped into
five different areas (228), all with their pros
and cons:
1. Isolation or identification of species
that may be host specific. Host
specific species will give a
distinction between human and
animal faecal pollution. The
problem is that in the environment
they are usually occurring in low
numbers giving a low sensitivity for
the methods based on this
approach.
2. Ratios between various faecal
groups or species. Indicator groups
are easily found in the environment
and the methods are cheap and easy
to perform. However, methods
based on various faecal groups or
species
are
usually
not
discriminative enough. Further,
ratios at the source may differ due
to different die-off and dispersal
patterns in the environment.
3. Phenotypic analysis of mixed
microbial
communities
and
comparison with those found in the
faeces of humans and various
animals. If host specificity of
individual species may be limited,
certain subspecies or biotypes often
predominate in the faeces of
particular hosts. Full discrimination
is difficult to achieve and
comparing
mixed
microbial
populations is potentially time
consuming. Methods have been
developed based on the kinetics of
Taylor, (2003) (228) concluded that no single
approach to track faecal pollution is useful in
all situations but multivariate analysis of
appropriate baskets of determinants may
offer the possibility of identifying and
apportioning human and animal faecal inputs
to surface water.
1.6.3. Microbial Risk Assessment (MRA)
Risk assessment is a tool that has been used
to assess chemical risks and is becoming
more frequently used as a tool to measure
microbial health effects on a population.
MRAs were first developed for drinking
water (182) but have lately been applied to
other practices such as reuse of human urine
(101) and discharge to recreational waters
(12). At a screening level, MRA is a valuable
tool to initially estimate risks even when the
set of data is poor, giving the potential to
make rational decisions at far less cost than
epidemiological studies. Sensitivity analyses
are made in order to obtain information on
12
Comparative occurrence of pathogens in wastewater
knowledge gaps and critical control points.
Besides giving information allowing for a
prioritisation of preventive measures within
the risk management strategies, they also help
to direct research in order to fill identified
information gaps (13). Whether the risk
assessment is of a chemical or
microbiological nature, the procedure
contains four primary elements: 1) Hazard
identification, 2) Exposure assessment, 3)
Dose-response assessment and 4) Risk
characterisation (84).
jejuni, Camp. coli, Camp. lari, Camp. upsaliensis
and Camp. helveticus form a genetically close
group, which are the most commonly
isolated ones from human and animal
diarrhoea (166). Camp. jejuni alone stand for
approximately 85% of the nationally acquired
cases, followed by Camp. coli and Camp.
upsaliensis. Clinical symptoms are: severe and
often bloody diarrhoea, stomach pain and
fever. Malaise and vomiting can also occur
(211). The disease is, however, often selflimiting but complications such as arthritis
and Guillain-Barré syndrome have been
documented (213). In a survey by the
Swedish Food Administration (211) on the
occurrence of Campylobacter in Sweden, 9.5%
of the chickens tested were positive.
Furthermore, Campylobacter could be detected
in the raw water used in 38% (n = 102) of
surface water treatment plants.
Hazard identification
In the hazard identification step, background
information on the pathogens in a specific
system is described. It also includes the
spectra of human illness and disease
associated with the identified microorganisms
(85). Since this thesis focused on water as a
transmission
route,
the
faecal-oral
transmission of gastrointestinal pathogens is
the main hazard emanating from faecal
contamination of the water. In Table 6, some
of the most important waterborne agents and
the disease they cause are listed. While faecal
contamination
and
gastro-intestinal
pathogens are the primary source of risk,
environmental microorganisms such as
Legionella spp. among others, are present in
sewage in low numbers but can multiply
within a recycled water system (11). The
focus lies on Campylobacter, enteric viruses,
Giardia and Cryptosporidium whilst different
opportunistic pathogens are not considered
in the risk models of this thesis.
It is believed that a major part of the
waterborne outbreaks with unknown
etiological agent is viral (197). The main
reason why they are not diagnosed is due to
methodological problems and the fact that
gastrointestinal viral diseases usually have a
shorter duration than bacterial and parasitic
ones (91). Not all viruses are culturable, but
new and improved detection methods will
improve surveillance. Viruses excreted in the
faeces are more host-specific than enteric
bacteria and parasites, thus having a low
zoonotic potential (17).
Rotaviruses are the most common cause of
diarrhoea in children but can also infect
adults (17). They were the major cause of
gastrointestinal illness in Swedish children
(237). The incubation period is less than 48
hours, with duration of illness for 5 to 8 days.
Symptoms
usually
include
vomiting,
diarrhoea and dehydration. Fever and
respiratory problems can also occur (17). The
virus has the highest infectivity of the
waterborne viruses (73). The shedding at its
maximum coincides with the third to fourth
day of disease with excretion of as much as
1011 virions g-1 faeces (56).
Campylobacter was chosen as an index
organism for the bacteria group due to their
high endemic infection level and low
infectious dose. Campylobacter is the most
common cause of bacterial gastroenteritis in
Sweden with more than 7,000 reported cases
in 2003, of which one third were acquired in
Sweden (212). Campylobacter is harboured in
the intestines of a wide range of domestic
and wild animals including almost all bird
species examined. They are particularly
prevalent in poultry, which is a likely source
of human infection (17). At present the genus
contains 16 species and six subspecies. Camp.
13
Jakob Ottoson
Trita LWR PHD
Table 6. Example of pathogens that may be excreted in faeces (can be transmitted through water
and improper sanitation) and related diseases, including examples of symptoms they may cause
(199).
Group
Pathogen
Disease - Symptoms
Bacteria
Aeromonas spp
Enteritis
Campylobacter jejuni/coli
Campylobacteriosis - diarrhoea, cramping, abdominal pain,
fever, nausea; arthritis; Guillain-Barré syndrome
Escherichia coli (EIEC, EPEC,
ETEC, EHEC)
Enteritis
Plesiomonas shigelloides
Enteritis
Salmonella typhi/paratyphi
Typhoid/paratyphoid fever - headache, fever, malaise, anorexia,
bradycardia, splenomegaly, cough
Salmonella spp.
Salmonellosis - diarrhoea, fever, abdominal cramps
Shigella spp.
Shigellosis - dysentery (bloody diarrhoea), vomiting, cramps,
fever; Reiter’s syndrome
Vibrio cholerae
Cholera - watery diarrhoea, lethal if severe and untreated
Yersinia spp.
Yersiniosis – fever, abdominal pain, diarrhoea, joint pains, rash
Enteric adenovirus 40 and 41
Enteritis
Astrovirus
Enteritis
Calicivirus (incl. Noroviruses)
Enteritis
Coxsackievirus
Various; respiratory illness; enteritis; viral myocarditis; viral
meningitis
Echovirus
Aseptic meningitis; encephalitis; often asymptomatic
Enterovirus types 68-71
Meningitis; encephalitis; paralysis
Hepatitis A
Hepatitis - fever, malaise, anorexia, nausea, abdominal
discomfort, jaundice
Hepatitis E
Hepatitis
Poliovirus
Poliomyelitis – often asymptomatic, fever, nausea, vomiting,
headache, paralysis
Rotavirus
Enteritis
Virus
Parasitic
protozoa
Helminths
Cryptosporidium parvum and Cryptosporidiosis - watery diarrhoea, abdominal cramps and
C. hominis
pain
Entamoeba histolytica
Amoebiasis - Often asymptomatic, dysentery, abdominal
discomfort, fever, chills
Giardia intestinalis
Giardiasis – diarrhoea, abdominal cramps, malaise, weight loss
Ascaris lumbricoides
Generally no or few symptoms; wheezing; coughing; fever;
enteritis; pulmonary eosinophilia
14
Comparative occurrence of pathogens in wastewater
Noroviruses form together with sapoviruses
the group Caliciviruses, former NorwalkLike Viruses (NLV) or Small Round
Structured Viruses (SRSV), known as the
causative agents of winter vomiting disease.
Noroviruses are the most common cause of
gastroenteritis in the western world (122)
and, next to rotavirus, the most important
cause of pediatric acute gastroenteritis (151).
Symptoms usually include diarrhoea,
vomiting, nausea, abdominal cramps, fever
and malais after an incubation period of 24 –
48 hours. The duration of illness is normally
three to four days and is selflimited in
immunocompetent patients (151). Excretion
of virions can however continue for up to
two weeks (80). The main transmission is
from person to person, but noroviruses can
also be transmitted via contaminated water
or food. The number of reported outbreaks
caused by noroviruses has steadily increased
in Sweden as well as internationally (5).
causing disease in humans and is considered
to be the most common intestinal parasite in
the world (139). With 1,360 human cases,
Giardia was the third most reported cause of
gastrointestinal infection in Sweden in 2003,
(212). The infectious life stage of Giardia is
the cyst, which is a 10-12 µm long and 5-8
µm wide oval highly resistant stage of its
life-cycle that can survive and remain
infectious for several months in water (261).
The manifestation of giardiasis in humans
varies from asymptomatic to chronic
diarrhoea and is very much dependent on
the immunological status of the host. First
symptoms are often nausea, anorexia,
malaise, fever and chills, followed by
explosive,
watery
and
foul-smelling
diarrhoea (139).
Cryptosporidium is divided into 24 species, of
which 4 have been shown to be able to
infect humans (123). It is however only C.
parvum (former bovine strain or C. parvum
genotype 2) and C. hominis (former C. parvum
genotype 1 or human strain) that seem to be
of any human health significance (152).
Cryptosporidium is present in the environment
as an oocyst, which is a 4.5-6 µm in diameter
spherical, thick-walled stage in its life-cycle.
In aqueous solutions, oocysts remain
infectious for up to 6 months and are viable
for 9 months (143). Oocysts are also tolerant
to high doses of disinfectants (186).
Cryptosporidium caused the largest waterborne
outbreak in the industrialised world during
the past decade in Milwaukee, where more
than 400,000 people were assumed to be
infected by contaminated municipal drinking
water (1). A Cryptosporidium infection can
give
cholera-like
diarrhoea
in
immunocompromised individuals, with up
to 71 stools and 17 L water loss per day
having been reported as being the ultimate
cause of death for AIDS patients.
Immunocompetent people usually have selflimiting watery diarrhoea with a flu-like lowgrade fever. There is still no medication for
cryptosporidiosis but paromomycin has
been shown to decrease stool frequency and
oocyst excretion in humans (123). C. parvum
is a notifiable disease since June 2004 within
Pathogenic enteroviruses are divided into four
main groups, Coxsackie, Echo, Polio and
Enterovirus type 68 – 71 that can cause a
wide variety of illnesses (Table 6). The
incubation period varies greatly and may
range from 1 to 35 days. Shorter periods are
typical for respiratory tract syndromes (17).
Enterovirus infections are very common in
young children, 10 % of faecal soiled diapers
contained enteroviruses (176). Viruses have
also been isolated from a range of animals
(81) although serologically and genetically
distinct from human strains (17, 162).
Protozoa are single or unicellular, eucaryotic
organisms divided into four main groups:
flagellated,
amoeboid,
ciliated
and
sporozoids. All four groups contain
intestinal parasites, of which many are
zoonotic, i.e. can be transmitted directly
from animals to humans. This is the case for
both Giardia and Cryptosporidium (139).
Giardia is a flagellate belonging to the order
Diplomanidida. The genus comprises several
species, all intestinal parasites, distinguished
from each other on morphological criteria
and host range. G. intestinalis (lamblia,
duodenalis) is probably the only species
15
Jakob Ottoson
Trita LWR PHD
in the Swedish surveillance system. Together
with viral agents, Cryptosporidium were
thought to be a main cause of outbreaks
with unknown etiological agents (217).
organism or the exposed population, two
parameters, α and β, describe the relation as:
Exposure assessment
In the exposure assessment step, the size
and nature of the population exposed and
the routes, concentrations and distribution
of the microorganisms are determined. If
greywater is used for groundwater recharge,
drinking water from the tap will be the
primary transmission route and the exposure
about 1 L p·d-1 (191). The dose of a
pathogen is calculated from the density of
the organism in the water times the volume
ingested. Densities are preferably based on
occurrence data from direct measurements,
but also on index organisms* or through
indirect estimation (density in untreated
wastewater – expected removal). Reuse
options and approximate volumes were
given in Table 4.
The Beta-Poisson model fits well with many
dose-response datasets, adds plausibility to
the assumption that ingestion of a single
organism is sufficient to cause infection and
is conservative when extrapolating to low
doses (231). It can however be misused.
Equation (2) is a simplified relation that is
only valid for certain parameter values, e.g. β
>> α, β >> 1 (64). Furthermore, the upper
95% confidence region of the Beta-Poisson
model exceeds the limiting exponential
curve at low doses for some organisms since
β is a scale parameter just moving the doseresponse curve across the dose axis without
limits in low doses (230). The suggestion is
to use a hypergeometric model which has a
good fit of data and for which the risk of
infection is never higher than the probability
of exposure to at least one organism (230).
However, the constants for different
pathogens have yet to be published.
Pinf ~ 1- (1 + Dose/β)-α
Dose-response assessment
To establish a relationship between the dose
of a microbial agent and the rate of infection
in a population, human volunteer studies
have been performed. The resulting infected
and unaffected individuals were used to
create a mathematical relationship between
the dose administered and the probability of
infection in the exposed population. The
current baseline information from such
studies has been compiled by Haas et al.
(1999) (87) and Teunis et al. (1996) (231).
Two main equations have been used to
describe the relationship; exponential (1) and
Beta-Poisson (2):
Another approach, applicable when dealing
with pathogens for which no dose-response
studies have been made, when vulnerable
populations are exposed and in worst-case
scenarios, is to use the exponential
relationship with r = 1. This is a generic
single hit model where the ingestion,
inhalation or contact with one organism will
lead to Pinf = 0.63.
Drinking water is something people do every
day. To assess repeated exposures, equation
(3) is used so that the risk can be measured
on a yearly basis (n = 365).
When organisms are distributed randomly
(Poisson) and the probability of infection for
any organism equals r, then:
-rDose
Pinf = 1 – e
(2)
Pn(inf) = 1 – (1 – Pinf)n
(1)
(3)
Equation (3) can be simplified at low Pinf to:
When the r is not constant, but has a
probability distribution in itself (βdistribution) due to either the nature of the
Pn(inf) = n · Pinf
An index organism is a group or species indicative
of pathogen presence.
*
16
(4)
Comparative occurrence of pathogens in wastewater
Risk characterisation
The information from the hazard
identification, exposure assessment and
dose-response
relationship
steps
is
integrated in the risk characterisation in
order to estimate the magnitude of the
public health problem. Since the information
is often incomplete and since the densities
of pathogens fluctuate, probability density
functions (PDFs) are often used instead of
point estimates or constant values. MonteCarlo simulations are then used to sample
the PDFs in risk calculations (100). Most
often the microbial risk is presented as the
quotient infected/exposed number of
people = probability of infection, Pinf. The
risk can also be presented as total number of
infections per annum or system lifetime (58).
Another metric is the Disability Adjusted
Life Years (154), DALYs, where the severity
of an infection is taken into account. In the
3rd WHO Drinking Water Guidelines a
reference level of risk of 1 µDALY is
suggested (257). This level is equivalent to
1:1,000 risk of disease from a pathogen
causing watery diarrhoea (252). In the
overall
“Sustainable
Urban
Water
Management” programme, MRAs are used
to compare different systems to each other
and from a management point of view, the
performance and reliability of a system
might be more important than the absolute
number of infections. Another approach,
used by Westrell et al., (2004) (254) is to link
the number of infections to the
epidemiological situation in the society
(Table 7). This is in line with other efforts to
weigh different risks in society to each other
and to other parameters such as cost or
environmental impact (239).
Acceptable Microbial Risk
Acceptable risk is important to define, as
well as to know when to take preventive
measures as when integrating information
from the different aspects of sustainability.
In Urban Water (2005) (239) an acceptably
low infection level was considered to be <1
per 10,000 people per annum, as proposed
by the US EPA and Dutch regulation for
microbial risks from drinking water (6, 182).
Haas, (1996) (84) argued that it should be
lowered to 1:1,000. In this study, 1:1,000 was
the level used for proposing wastewater
treatment targets, comparable to one
µDALY, which WHO uses in the 3rd edition
guidelines for drinking water quality (257).
Table 7. Suggested definitions of severity of consequences of hazards based on increase of
disease in the community (254).
Item
Definition
Catastrophic Major increase in diarrhoeal disease (> 25%) or > 5% increase in more severe
diseases, for example EHEC , or larger community outbreak (> 100 cases) or death.
Major
Increase in more severe diseases (0.1 – 5%) or large increase in diarrhoeal disease (5 < 25%).
Moderate
Increase in diarrhoeal disease (1 - < 5%)
Minor
Slight increase in diarrhoeal disease (0.1 - < 1%)
Insignificant No increase in disease incidence (< 0.1% (= 1/1,000))
17
Jakob Ottoson
Trita LWR PHD
4. Establish a system to monitor
control of the CCP by scheduled
testing or observations.
5. Establish corrective actions to be
taken when monitoring indicates that
a particular Critical Control Point is
not under control.
6. Establish procedures for verification,
which include supplementary tests,
and procedures to confirm that the
HACCP
system
is
working
effectively.
7. Establish a system of documentation
concerning all procedures, and a
record-keeping system appropriate
to these principles and their
application.
1.6.4. Hazard Analysis and Critical Control Point
(HACCP)
Hazard Analysis and Critical Control Point
(HACCP) is a systematic approach to the
identification, assessment, and control of
hazards during production, processing,
manufacturing, preparation, and use of food,
water, or other substances to ensure the
safety of the product when consumed or
used. The HACCP system incorporates
safety control into the design of the whole
process rather than relying solely on endproduct testing (34). HACCP has been
applied to a number of different processes,
including drinking-water treatment (87),
aquaculture
production
(70),
and
management of the wastewater and sludge
from a medium Swedish city wastewater
treatment plant (254). The HACCP system
consists of the following seven principles
(34):
The HACCP system contains some of the
elements of MRA such as the hazard
identification and exposure assessments.
MRA can also be of help to determine
critical limits based on the acceptable level
decided under point 2, which has been one
of the main objectives of this thesis. In Fig.
5 different barriers, which can function as
control points between wastewater and
exposure to humans are shown. This study
investigates different wastewater treatment
processes, but also discusses some of the
other possible barriers to human exposure.
For example, successful vaccination
campaignes have helped to eradicate polio
from many countries in the world. Other
barriers can be crop restriction, choice of
recipients and different exposure controls
such as workers personal protection,
location of beaches and time between
irrigation and public access of wastewaterirrigated areas (Fig. 5).
1. Conduct a hazard analysis by
identifying and evaluating the
potential hazards associated with
each stage of the production; assess
the likelihood of occurrence of the
hazards and identify measures for
their control.
2. Determine Critical Control Points
(CCP). A CCP is a step where
control can be applied and is
essential to prevent or eliminate a
safety hazard or reduce it to an
acceptable level.
3. Establish critical limits that must be
met to ensure that the CCP is under
control.
18
Comparative occurrence of pathogens in wastewater
Fig. 5. Control points or barriers for the prevention of transmission of wastewater pathogens to
humans.
19
Comparative occurrence of pathogens in wastewater
2.
OBJECTIVES AND SCOPE
The overall aims of this study have been to investigate the occurrence of pathogens in
wastewater, their removal in wastewater treatment and the risk outcome of different wastewater
system structures. The main questions have been:
•
•
•
What is the pathogen load and how important is it to use local data?
How can we estimate the removal of pathogens, especially of viruses and (oo)cysts, with
simple process indicators?
How can we manage the risks of different exposures to wastewater?
More specifically, the objectives of the respective investigations were to:
Quantify the faecal contamination in the
greywater flow. Faecal contamination has
traditionally been measured by indicator
bacteria of the coliforms group. Regrowth in
wastewater systems however leads to an
overestimation of the faecal load. Cholesterol
and coprostanol were therefore used in
paper I as an alternative assessment of the
faecal load in a greywater treatment system.
Correlate the removal of pathogens, i.e.
enteric viruses and parasitic (oo)cysts to
that
of
process
indicators,
microbiological (E. coli, enterococci,
phages and spores of sulphite-reducing
anaerobes)
and
physico-chemical
parameters as a means to measure the
treatment
targets.
Removal
of
microorganisms in treatment and correlation
between removals were calculated from
paired samples, i.e. samples from untreated
and treated wastewater were collected the
same day and analysed for a large number of
parameters.
Statistical
analyses
were
performed to find correlation between
pathogens and possible process indicators
(papers II, III).
Measure the occurrence of pathogens in
wastewater and to compare that to
international data as well as to
epidemiological data. Handling wastewater
is connected with a risk. This risk is
dependent on the pathogen occurrence in the
wastewater as well as the removal in
treatment and other barriers in the
environment. Information on the occurrence
of microorganisms and their removal in
treatment has been collected from five
different cities and eight different treatment
plants in Sweden (papers I, II, III, IV).
Suggest guidelines for bathing waters
based on the enumeration of somatic
coliphages. For some exposures, for
example groundwater, drinking water
production and recreational water, the local
context is very important and general risk
models are not feasible. That is why the
indicators of faecal pollution will still play an
important roll in risk management of urban
water systems. The problem is the relevance
of the indicators commonly used today.
Alternatives to indicate the occurrence of
viruses and parasitic (oo)cysts have been
investigated and suggested guidelines for
bathing water presented in paper V.
Based on occurrence data in different
flows, suggest treatment targets for the
removal of pathogens depending on
water use. Based on an acceptable level of
risk (10-3), MRA was used to calculate the
performance targets for different key
pathogens taking into account the variation
in their occurrence in waste/grey water
(papers I, II, III), environmental barriers
and the end use of the water.
21
Jakob Ottoson
Trita LWR PHD
Together with European partners,
provide a model for the tracking of
sources of faecal pollution in surface
waters. The indicators detect faecal
contamination
from
warm-blooded
animals. Human wastewater is however
connected with a larger risk than for example
exposure
to
wastewater
from
a
slaughterhouse, due to the risk of infection of
human viruses that are not transmitted from
animals. It is also of highest priority to locate
point pollution to be able to manage faecal
contamination that degrades surface waters.
Thus, methods discriminating human from
animal faecal pollution were developed
within a group of five European partners and
results presented in paper VI and (234). This
was possible by the collection of wastewater
samples of human and animal origin, from
five different regions in Europe, which were
analysed for 38 different sets of parameters.
Multivariate analyses were used to distinguish
animal from human wastes based on the
lowest number of parameters.
22
Comparative occurrence of pathogens in wastewater
3.
MATERIALS AND METHODS
3.1. Study Sites
An integrated hydroponics treatment system
has been built in a greenhouse, to cope with
the Swedish climate. Information on the
treatment processes are provided in
Norström et al., (2003) (163) and paper IV.
Removal of microbial indicators was
assessed during the autumn 2002 and results
presented in paper IV. The greenhouse is
situated in Ulriksdal, Solna Stad, just north
of Stockholm.
Information on the occurrence of
pathogens, faecal contamination and
removal in treatment has been collected
from various sites, described briefly below
and in the papers.
Vibyåsen is situated in Sollentuna, north of
Stockholm. The wastewater is separated into
blackwater, treated in the municpal
treatment plant, and greywater, which is
treated locally in the respective area. Details
of treatment are given in paper I and in
Palmquist, (2004) (171). Separation of
greywater is common in cottages where dry
sanitation is a practise in Sweden. Most
often the greywater stream is based on single
household treatment. In Vibyåsen, however,
greywater from one system was collected
from 85 households and 212 inhabitants of
whom 17 (8%) were children (I).
3.2. Microbiological Methods
For the detection and enumeration of
microorganisms, different methods have
been used (Table 8). Indicators and parasitic
(oo)cysts were enumerated with standard
methods and viruses with rtPCR.
3.2.1. Human virus detection
To be able to reach a reasonably low limit of
detection for viruses, some concentration of
the water, especially the treated wastewater,
is necessary. Virus concentration methods
with adsorption to glassfiber (69, 71)
negatively charged filters (24, 52, 117, 172)
positively charged filters (52, 75, 98), PEGprecipitation (14), two-phase separation
(132) and ultracentrifugation (65, 77) have
been developed. The method used here
captured virions on positively charges filters
as described by Gilgen et al. (1997) (75).
Detection of infectious virions are made on
cell-culture, however, many viruses are not
possible to culture on existing cell lines.
Polymerase chain reaction, PCR, is a very
sensitive method that enables detection of a
lot of unculturable viruses. There are,
however, implications on the viability of
virions detected by the replication of part of
their nucleic acid content (46). In this study,
the occurrence and removal of entero- and
noroviruses in wastewater were measured
Secondary treatment with coagulation and
active sludge treatment is conventional in
Sweden. Four different plants situated in
three different cities (Malmö, Göteborg and
Umeå) were sampled monthly during the
winter - spring 2003 – 2004 looking at
spatial and temporal differences in the
occurrence and removal of microorganisms
(II).
In Henriksdal WWTP pilot plant, three
different treatment processes have been
evaluated (III). The existing process in
Henriksdal (www.stockholmvatten.se) was
used as a reference, but in pilot scale (150
PE). The other processes have been
treatment in a membrane bioreactor, MBR,
after primary treatment in a drum filter and
anaerobic treatment in UASB (III). Samples
from the pilot plant study have been sent for
analysis of physico-chemical parameters and
correlation
studies
with
microbial
parameters performed.
23
Jakob Ottoson
Trita LWR PHD
with PCR, and enumerated with serial
dilutions, as described in paper II and III.
distinction of a specific strain is wanted,
more diverse parts of the genome, such as
the VP1 gene, can be targeted (161, 162).
Noroviruses were detected by amplification
of part of the polymerase gene with primers
described by Vinje and Koopmans, (1996)
(260).
Enteroviruses were detected with nested
PCR. The outer primer pair attached to a
site in the 5´non-coding part. This is a
conserved part of the genome making it
possible to detect all enteroviruses. If a
Table 8. Substrates and methods used for the detection of microorganisms in the respective
papers.
Organism
Substrate
Indicator Bacteria
Coliform bacteria
m-endoagar LES (Difco,
Franklin Lakes, New Jersey)
Colilert 18 (IDEXX,
Westbrook, Maine)
Mfc agar (Difco)
E. coli
Enterococci
Spores of sulphitereducing
anaerobes
Phages
Somatic
coliphages
F-specific phages
Parasites
Giardia cysts
Cryptosporidium
oocysts
Human Viruses
Enteroviruses
Noroviruses
Incubation
Verification Paper Reference
time and temp.
21 ± 3 h, 35°C
Oxidase
I
(106)
18 ± 2 h, 35°C
21 ± 3 h, 44°C
Colilert 18 (IDEXX)
18 ± 2 h, 35°C
Enterococcus agar (Difco)
44 ± 3 h, 35°C
Enterolert (IDEXX)
24 ± 2 h, 41°C
Perfringens agar (Difco)
24 ± 3 h, 37°C
Host strain
ATCC 13706
21 ± 3 h, 37°C
WG49
Concentration
Centrifugation
Centrifugation
Positively charged filters
(Zetapore; Cuno, Meriden,
Conneticut), gradient
centrifugation (Centricon;
Millipore, Bedford, Maine)
Positively charged filters
(Zetapore), gradient
centrifugation (Centricon)
LTLSB
Bile
Eskuline
II, III,
IV
I
(48, 49)
II, III,
IV
I
(48, 49)
II, III,
IV
I, II,
III, IV
21 ± 3 h, 37°C
Purification
IMS (Dynal,
Oslo, Norway)
IMS (Dynal)
Extraction
QiAMP Viral
RNA (Qiagen,
Hilden,
Germany)
Primers
Ent 5/6
Nest Ent
1/2
QiAMP Viral
RNA (Qiagen)
Jv 12/13
(106)
(105)
(104)
I, II,
III, IV
II, III
(108)
II, III
(242)
II, III
(242)
II, III
(75, 162)
II, III
(75, 260)
(107)
centrifugation (185). After concentration
and elution, (oo)cysts are usually separated
from background microbiota and debris for
example with the help of ether clarification
(202), gradient centrifugation (168, 184,
202), immunomagnetic separation, IMS, (31,
35) or flow cytometry (249). The method
most widely used today is to concentrate
3.2.2. Protozoan analyses
Different methods to concentrate (oo)cysts
from water samples have been reported.
These have been based on the filtration of
the water through different types of filters
(60, 150, 167, 205), flocculation (250) and
24
Comparative occurrence of pathogens in wastewater
water by membrane filtration, separate
(oo)cysts
from
debris
and
other
microorganisms with IMS and finally
enumerate them with direct microscopy
after
staining
with
fluorescein
isothiocyanate, FITC (242). Also PCR
methods have been developed for the
detection of Giardia and Cryptosporidium in
environmental samples (103, 140, 262). In
this study, concentration was performed
with centrifugation before separation of
(oo)cysts with IMS and staining. Recovery
rates between 56-89% have been reported in
the clarification step (141) and 87±15% in
the centrifugation (185). To know the
recovery in a specific sample, (oo)cysts
stained with Texas Red fluorochrome are
commercially available. One hundred
(oo)cysts are added to the sample and
enumerated together with the FITC-stained
oocysts. The percentage recovery is then
equal to the number of Texas Red stained
(oo)cysts enumerated. This was done for
some samples with Color Seed (Biotech
Frontiers Decisive, North Ryde, Australia).
The recoveries were 22 ± 1.5% for Giardia
cysts in untreated wastewater and 25 ± 12%
in treated wastewater (n = 3). For
Cryptosporidium oocysts the recoveries were
15 ± 4.6% and 39 ± 13% respectively (II,
III).
3.3. Microbial
Methods
Risk
rotaviruses may be 100 times lower than for
noro
and
picornaviruses,
due
to
uncompletely denaturation of the doublestranded RNA. Data on rotavirus
occurrence in wastewater were therefore
based on an epidemiological approach
(Table 9). Mead et al. (1999) (142) estimated
the incidence of disease to be 0.71% in the
U.S. With a disease rate of 75% (73), the
incidence of infection is thus 0.95%. A mean
of 10-9 virions g-1 were supposed to be
excreted for 10 days by infected individuals
(Table 9). Background data for the
calculations of the other pathogens are
presented in Table 9. For reportable
diseases, giardiasis and campylobacteriosis,
the number of infected individuals were
estimated from reported cases by the
national surveillance system (212) and
corrected for underreporting (142, 255) and
unsymptomatic carriers (infections leading
to disease, Table 9). For norovirus and
Cryptosporidium infections, international data
were used (142, 252).
The constants for the dose-response models
that have been used in this study are
presented in Table 10 and further illustrated
in Fig. 6.
Variability and uncertainty of the data
The variability stands for the variation in the
data, which cannot be reduced by increased
sampling. This variation can be either spatial
or temporal. Connecting this to the data-set,
the variability is dependent on variation in
disease incidences between regions and over
time. The uncertainty, on the other hand,
reflects flaws in the data collection and can
thus be reduced by increased investigations.
In the measured data, uncertainty is seen in
the recovery of the detection methods used,
where no or only a limited number of
recovery trials have been performed.
Assessment
3.3.1. Pathogens assessed and data used
A rotavirus PCR developed for faecal
samples, picking up all the four types, was
tested. It turned out to be too unsensitive
for the detection of rotaviruses in
wastewater. Metcalf et al., (1995) (145)
reported that the sensitivity for rtPCR of
25
Jakob Ottoson
Trita LWR PHD
Table 9. Literature data and PDFs that were used to calculate pathogen occurrence in
wastewater and greywater. The faecal contamination and flow were 0.1 g p·d-1 and 150 L p·d-1 in
greywater and 130 g p·d-1 and 200 L p·d-1 in wastewater (Table 12, 116, 157).
Parameter
Giardia
Reported disease incidence [%] 0.0152(212)
Infections leading to disease
39(85)
[%]
Underreporting
18.7(142)
Excretion time [d]
101.3 ± 0.3
Cryptosporidium
0.0027(142)
39(85)
Norovirus
0.012(252)
75(73)
45.2(142)
1.4 ± 0.2 (215)
10
1500(252)
101.16 ± 0.3
(112)
-1
Excretion density [g ]
107.0 ± 1.0
7.0 ± 1.0 (76)
10
108.0 ± 1.0
Campylobacter
0.078(212)
23(85)
7.6(255)
1.0 ± 0.3 (142,
10
(252)
(112)
Σ pathogens in wastewater
[L-1]
Σ pathogens in greywater
[L-1]
Rotavirus
0.71(142)
75(73)
1.18 ± 0.3 (56)
10
56)
9.0 ± 1.0 (56,
10
(252)
73, 248)
8.0 ± 1.0 (56)
10
3.41 ± 1.22
103.15 ± 1.01
104.79 ± 1.06
105.23 ± 1.05
104.84 ± 1.16
0.60 ± 1.23
100.16 ± 1.04
101.80 ± 1.07
102.24 ± 1.08
101.85 ± 1.20
10
10
Table 10. Dose-response models and constants used in the risk calculations.
Organism
Rotavirus
Campylobacter jejuni
Enteroviruses (Coxsackie B)
Giardia intestinalis
Cryptosporidium parvum
Model
Beta-Poisson
Beta-Poisson
Exponential
Exponential
Exponential
Constants
α = 0.253; β = 0.422
α = 0.145; β = 7.589
r = 0.0145
r = 0.0199
r = 0.00405
Reference
(231)
(231)
(85)
(231)
(231)
Fig. 6. The probability of infection from the ingestion of pathogenic cells in different doseresponse relationships: Exponential models for (a) Giardia, (b) Cryptosporidium and BetaPoisson models for (c) rotavirus and (d) Campylobacter.
26
Comparative occurrence of pathogens in wastewater
conservative 1 mL event-1 (13). No dilution
of the treated wastewater was considered.
3.3.2. Exposures assessed
If the water was used for irrigation of a
public park (Exp I, Fig. 7), the irrigation was
assumed to take place the day before access.
The water intake was a conservative 1 mL 26
times per year based on weekly activities
during the summer season in Sweden.
During irrigation, people can be exposed to
aerosols (Exp. A, Fig. 7). The water intake
from inhalation of aerosols is time
dependent based on average aerosol
ingestion (45) and droplet size distribution
(120), giving a log normal distribution (Fig.
8). This exposure can, with some
modifications, be applied to toilet flushing.
The water intake is however smaller since
the time a person is exposed to aerosols is
shorter when flushing the toilet than during
irrigation. To assess the treatment need for
crop irrigation, several assumptions have
been made. Shuval et al., (1997) (204)
measured 10.8 mL wastewater to attach to
100 g of lettuce. Asano et al., (1992) (8) have
assumed 10 mL to be ingested by
consuming crops irrigated with wastewater.
Die-off of viruses on crops have been
reported by Asano et al. (1992) (8) and
Petterson et al. (2001) (177) to be 0.69 and
0.45 log10 d-1 respectively. Due to the colder
climate in Sweden, the lower value was used
for all microorganisms and with a
withholding period of 1 week as in Höglund
et al., (2002) (101).
In the modelling of microbial risks, a system
has been built up of several so-called healthrelated modelling units (HMUs), defined as
physical units that in some ways alter the
concentration of pathogens (57). They are
most often some kind of microbiological
barrier, such as a treatment unit, but also
include reduction and/or growth in the
environment. The most important HMUs in
water and wastewater management are in
most cases different steps in the water
treatment. The focus in the present work
was on wastewater treatment efficiency and
reliability. The pathways assessed in paper I
were: accidental ingestion of greywater,
consumption of recharged groundwater with
varying retention times and direct exposure
at a sports field irrigated with greywater.
This study was later extended to include
more pathways (Table 11, Fig. 7), looking at
the treatment efficiency needed to be inside
an acceptable level of infection (10-3).
For exposure to artificially produced
groundwater (exp. G, Fig. 7), the depth of
the unsaturated zone was assumed to be
three metres and the retention time in the
saturated zone two months, based on the
recommendation in the report “Wastewater
Infiltration:
Conditions,
Function,
Environmental Consequences” (156). The
dilution factor was two as in Asano et al.,
(1992) (8), i.e. half of the water from the tap
will be reclaimed wastewater (Table 11). The
water intake was log normally distributed
with a medium daily intake of 860 mL p·d-1
(253), exemplified in Fig. 8.
The common practice for exposure to
recreational water (Exp. R, Fig. 7) has been a
conservative 100 mL water intake (50 mL h1
, mean bathing time 2 h d-1) (12). The
ingested volume was changed for the
present simulation to a time dependent
distribution with the mean time of bathing
being 1 hour (Fig. 8). The dilution in the
receiving water was 1,000 times.
If the treated wastewater was used to create
blue-green areas (259) (Exp. P, Fig. 7), there
is a risk for accidental contact with the water
and the assumed water intake is a
27
Jakob Ottoson
Trita LWR PHD
Blackwater
Greywater
Exp. A
Exp. D
Waste/greywater treatment
Crop/Grass
Exp. I/C
Exp. P
Pond
Percolation
Lake
Exp. R
Saturated zone
Exp. G
Figure 7. Exposure pathways and HMUs involved for studied uses of treated grey and
wastewater. D = Drinking water production, P = accidental ingestion to treated
grey/wastewater, G = artificial groundwater production, R = Recreational water, I = Irrigation
of parks, C = irrigation of crops and A = exposure to aerosols during irrigation.
Table 11. Transmission pathways for exposures to reused or discharged grey/waste-water and
health-related modelling units (HMUs), except treatment, involved.
Exposure
HMUs involved
G. Drinking recharged groundwater (yearly risk Dilution(8), unsaturated
from 365 exposures).
zone(8) and saturated
zone(143, 187, 232, 263)
P. Accidental ingestion to treated wastewater
I. Ingestion from a field irrigated with treated
Survival on grass(18)
wastewater
A. Ingestion/inhalation of aerosols
R. Swimming in recreational water receiving
Dilution
treated wastewater.
D. Drinking water produced from wastewater,
(yearly risk from 365 exposures).
C. Eating crops fertilised with treated
Survival on crop(177)
wastewater
28
Volume ingested
e(6.61 ± 0.57) mL day-1 (253)
1 mL exposure-1 (13)
1 mL exposure-1 (13)
e(-4.2 ± 2.2) mL (45, 120)
e(3.9 ± 0.3) mL(12)
e(6.61 ± 0.57) mL day-1 (253)
10 mL(8, 204)
Comparative occurrence of pathogens in wastewater
a
Figure 8. Cumulative distributions for the water intake from inhalation of aerosols (left) and
ingestion of recreational (middle) and drinking water (right) used in the quantitative microbial
risk assessment.
29
Comparative occurrence of pathogens in wastewater
4.
SUMMARY OF RESULTS
also used in paper I as an alternative to
measure the faecal input in greywater, since
growth of indicator bacteria in the system led
to an overestimation of the faecal
contamination and thus risk (Table 12).
Bacterial indicators can also grow to a lesser
degree in wastewater treatment systems. This
growth does not lead to the same relative
overestimations as in greywater. The ratio
between indicators and coprostanol was 12,
4.8 and 15 for E. coli, enterococci and Cl.
perfringens spores in wastewater respectively.
The corresponding ratios in greywater were
1,400, 110 and 180. The measured faecal
contaminations from coprostanol values were
further used in the risk calculations.
4.1. Occurrence
4.1.1. Faecal indicators in wastewater
Wastewater from a larger population usually
contains a consistent range of the most
commonly-used indicators such as faecal
coliforms, enterococci, spores of Cl.
perfringens and bacteriophages (Table 12).
Alternative measures of faecal contamination
such as faecal sterols and stanols have been
widely used to distinguish human from
animal faecal contamination in surface water
and sea sediment (130). Faecal sterols were
Table 12. Data from municipal or hospital wastewater (VI) and faecal indicators in Vibyåsen
greywater system (I) and the corresponding faecal loads [g p·d-1] with an average flow of 200 and
150 L p·d-1 (157). Daily excretion values on E. coli and enterococci were taken from Geldreich,
(1978) (72) and Cl. perfringens spores, coprostanol and cholesterol from Leeming et al., (1998)
(130). Results are the average of log CFU/100 ml or log PFU/100 ml for E. coli (EC), enterococci
(FE), clostridia (CL) and somatic coliphages (SOMCPH). Faecal sterols are in mg/L:
coprostanol (COP) and cholesterol (CHOL).
Wastewater
Average
SD
EC
6.91*
-
FE
6.00
CL
Greywater
Average
SD
N
Mean faecal load
1,600
5.98
0.60
24
140
0.81 89
630
4.38
0.48
24
11
5.20
0.69 89
1,900
3.31
0.61
24
18
SOMCPH
6.07
0.93 94
-
3.32
0.63
24
-
COP
8.42
-
13
130
0.0086
0.0044 10
0.1
-
-
-
-
0.0173
0.0084 10
0.3
CHOL
N Mean faecal load
89
* Based on faecal coliforms value and percentage of confirmed E. coli, Table 16.
31
Jakob Ottoson
Trita LWR PHD
differences could be detected between the
epidemiological and measured data (Fig. 9).
4.1.2. Pathogens in wastewater
In Sweden, the variation in numbers was
higher for Giardia in Umeå than in the other
cities. In general, variations were larger for
the epidemiological approaches than for the
measured data, with oocyst occurrence in
Australia as a striking exception (Fig. 9).
Pathogen levels in greywater were lower
reflecting a difference in the faecal
contamination, 0.1 g p·d-1 compared to 130 g
p·d-1 in wastewater (Table 12).
There was no significant difference (p>0.05)
in densities of the measured pathogens
between the Swedish wastewater treatment
plants, indicating a similar incidence for all
organisms in the examined cities. Neither
were there significant differences between
Swedish
and
presented
international
(developed temperate regions) data, with the
exception of noroviruses being more
numerous in the Netherlands and Germany
than in Sweden (p<0.05). Giardia cyst
numbers were significantly more numerous
in Scottish (Strathclyde region) than
Australian (Sydney) wastewater (Fig. 9).
Norovirus outbreaks are connected with
winter in most parts of Europe and thus the
pathogen is more likely to be found in high
numbers in the wastewater during that
season. This was also the case in the studies
(papers II and III), where noroviruses were
detected in 81% (n = 16) of the untreated
wastewater samples during the period Nov –
Feb, but only in 8% (n = 25) throughout the
rest of the year. A temporal variation in
Giardia cyst occurrence was also detected
with significantly lower cyst numbers
between mid February – mid April in 2004
(Fig. 10). A similar variation could not be
verified for enteroviruses.
Comparison of measured to epidemiological
data showed a resemblance between them.
Cyst numbers were slightly underestimated in
the epidemiological approach, however, only
significantly lower than Scottish cyst numbers
(Fig. 9). For Cryptosporidium on the other
hand, the epidemiological approach leads to
an overestimation of oocysts in wastewater
(Fig. 9). Comparing the two sets of rotavirus
data (Fig. 9), Dutch and epidemiological, the
epidemiological data were significantly
higher, whereas for noroviruses no significant
32
Comparative occurrence of pathogens in wastewater
8
8
6
6
4
4
2
2
0
0
-2
Malmö
Sjöstad
Umeå
Göteborg
-2
Scotland
Australia
Epi
Greywater
Sjöstad
8
8
6
6
4
4
2
2
0
0
-2
Sjöstad
Malmö
Göteborg
Umeå
Sjöstad
Australia
Göteborg
-2
Rota-Epi
Italy
Netherlands (rota) Rota-greywater
Umeå
Malmö
Malmö
Göteborg
Umeå
Scotland
Epi
Epi
Netherlands
Germany
Greywater
Greywater
Fig. 9. Box-plots showing the range, 25th, 50th and 75th percentiles on the occurrence of: Giardia
cysts (upper left), Cryptosporidium oocysts (upper right), enteroviruses (lower left) and
noroviruses (lower right) [log10 L-1] in wastewater from different studies at different places and by
an epidemiological approach based on reported incidence and excretion data (Table 9). Data
from Sjöstad were taken from paper III, from Malmö, Göteborg and Umeå (II), Australia (10),
Scotland (185), Italy (15), Netherlands (132) and Germany (180).
Fig. 10. Giardia cyst and enterovirus genome numbers [L-1] in untreated wastewater (II, III).
33
Jakob Ottoson
Trita LWR PHD
for
enteroviruses,
noroviruses
and
Cryptosporidium oocysts (Table 13). For all the
indicators, most of the removal took place in
primary chemical treatment whereas activated
sludge treatment was not as efficient in
removing microorganisms, especially not
phages. In paper III, tertiary filtration was
shown to remove indicators further, but not
human viruses as efficiently (Table 13). Also
in MBR treatment, a relatively higher
reduction of indicators than virus genomes
could be detected.
4.2. Removal
Microorganism removal have been measured
at eight different treatment plants and results
are presented in papers I, II, III and IV. In
Table 13, these removals have been
summarised Noroviruses were the least
removed of the pathogens, followed by
enteroviruses. Cysts were removed more
efficiently than oocysts. Somatic coliphages
and spores of sulphite-reducing anaerobes
were significantly less removed than the
bacterial indicators in most treatment
processes (I, II, III, IV).
Removal in the anaerobic line with UASB
treatment was significantly lower than in the
aerobic treatment lines (III). All the three
lines described in paper III are compact
systems with <24 h retention times.
Alternatives such as the integrated
hydroponics (IV) and the greywater
treatment system in Vibyåsen (I) have
significantly longer retention times (12.7 and
10 days respectively).
Secondary
treatment
efficiency
was
investigated in paper II. Coliphages showed
a significantly lower removal than all the
other indicators (1.0 log), followed by spores
(1.5 log), F-specific phages (1.7 log),
enterococci (2.1 log) and E. coli (2.4 log).
Giardia cysts removal was >1 log higher than
Table 13. Removal, expressed as log10 (mean (SD)), of microorganisms in different types of
wastewater treatments. Secondary, and tertiary, treatments are the cumulative removals from
chemical, biological treatment and for tertiary filtration. The removals in the membrane
bioreactor (MBR) and upflow anaerobic sludge blanket (UASB) include the whole treatment lines
2 and 3 in paper III.
Primary
Secondary
Tertiary
MBR
UASB
Pond
Integrated
hydroponics
E.
coli
Enterococci
Spores
Somatic
coliphages
Fspecific
phages
Giardia
Cysts
Crypto
Oocysts
Enteroviruses
Noroviruses
1.8
(0.5)
2.4
(0.6)
3.2
(0.8)
5.0
(0.9)
1.9
(0.8)
3.0
(0.8)
>
5.8
1.5 (0.3)
1.1 (0.4)
0.8 (0.4)
1.3 (0.7)
-
-
-
-
2.1 (0.5)
1.5 (0.4)
1.0 (0.3)
1.7 (0.6) 2.7 (0.6)
1.2 (0.3)
1.3 (0.7)
3.2 (0.9)
2.4 (0.6)
2.3 (0.6)
3.5 (0.8) 3.5 (0.9)
1.6 (1.3)
1.7 (0.5)
4.5 (1.1)
3.0 (0.4)
3.1 (0.7)
3.8 (0.9)
> 3.9
> 1.5
1.8 (0.6)
1.8 (0.8)
0.7 (0.2)
0.8 (0.7)
2.4 (0.2)
-
-
0.5 (0.6)
0.9
(0.3)
1.0
(0.8)
1.1
(0.9)
0
2.3 (0.6)
1.0 (0.6)
1.2 (0.7)
-
-
-
-
-
4.5 (0.4)
2.3 (0.4)
2.5 (0.4)
-
-
-
-
-
34
Comparative occurrence of pathogens in wastewater
8
7
6
Total Coliforms
E. coli
Enterococci
Somatic Coliphages
Spores of sulphite-reducing anaerobic bacteria
7
anaerobic bacteria
Log cfu/pfu 100 mL-1
log10 cfu/pfu 100 mL-1
8
Total Coliforms
E. coli
Enterococci
Somatic Coliphages
Spores of sulphite-reducing
5
4
3
6
5
4
3
2
2
1
1
0
0
0
In
1
2
2
4
6
Out
8
Days
10
12
Figure 11. Removal of indicators in an integrated hydroponics treatment plant as a function of
treatment systems, Closed tanks (in-1) hydroponics (1-2) and algal tanks and sandfilters (2-out)
(Errorbars indicate 1 SD) (left), and hydraulic retention time (right)(IV).
Microbial removal in the hydroponics ranged
from 2.3 to >5.8 log (Table 13). The three
different systems – closed tanks, hydroponics
and algal tanks and sand filters - removed
microorganisms with approximately the same
efficiency (Fig. 11). However, looking at the
different time the water spent in every system
it is clear that the hydroponics did not
remove microorganisms as efficient as the
closed tank, algal tanks and sandfilters (Fig.
11). In the hydroponics, the mean removal
was almost similar to what Gantzer et al.,
(2001) (68) found in wastewater at 22° C.
These data can be applied to wetlands, which
probably is the most appropriate process to
compare hydroponics treatment with. In
wetlands, dilution of the water as well as
microbial input from wild animals may occur.
In paper I, a greywater treatment system was
evaluated. As for all the other types of
treatments, E. coli was removed the most (4.1
log), followed by enterococci (2.9 log), which
were both significantly more reduced than
spores (1.2 log) and somatic coliphages (1.5
log). The treatment includes a pond where
approximately 80% of the total removal on a
log basis took place (I) (Fig. 12). As for the
hydroponics, removals in the pond could
almost exclusively be explained by the
retention time.
9
9
8
Log CFU/PFU 100 mL-1
7
6
5
4
3
Total coliforms
E. coli
Enterococci
Spores of Cl. perfringens
Somatic coliphages
8
Log CFU/PFU 100 mL-1
Total coliforms
E. coli
Enterococci
Spores of Cl. perfringens
Somatic coliphages
2
7
6
5
4
3
2
1
1
0
0
In
Before pond
2
4
6
8
10
Days
After pond
Fig. 12. Removal of indicators in Vibyåsen greywater treatment plant as a function of treatment
systems (Errorbars indicate 1 SD) (left) and hydraulic retention time (right)(I).
35
Jakob Ottoson
Trita LWR PHD
formed a closer group, especially E. coli,
enterococci, Cl. perfringens spores and somatic
coliphages showed correlated removals.
These indicators also correlated positively to
N- and C-parameters, such as ammoniumnitrogen and total organic carbon, TOC. The
removal in turbidity, measured as suspended
solids (SS), resembled that of the indicators
and correlated positively with enterococci. SS
was, however, too insensitive, i.e. all the
MBR effluents were below the detection
value 0.5 mg L-1 (III). The indicator removals
reported in papers I and II are not included
in Table 14.
4.2.1. Correlation with process indicators
Enteroviruses have been detected in almost
all samples positive for any other pathogen
(II, III) and have therefore been suggested as
an indicator for virus removal/risk.
Correlation between enteroviruses and other
parameters are shown in Table 14. Whilst
there is a significant correlation (p<0.5)
between enterovirus removal and E. coli,
enterococci, Cl. perfringens spores and Fspecific phages, the predictive value is low
with the correlation coefficients between 0.33
and 0.37 (Table 14). Instead, the indicators
Table 14. Pearson Product Moment correlation for the log10 removal of microorganisms and physchem parameters in wastewater treatment. The values stand for correlation coefficient (= R), pvalue and number of replicates (bold when significant correlation, α = 95%), EV = enterovirus,
FE = enterococci, CL = Cl. perfringens spores, SCP = somatic coliphages and F-SP = F-specific
phages.
EV
E. coli
FE
CL
SCP
F-SP
E. coli
0.334
0.016
52
Microbial parameters
FE
CL
SCP
0.33
0.37
0.24
0.02
0.007 0.08
48
52
52
0.90
0.80
0.72
1E-18 8E-13 2E-9
48
52
52
0.76
0.65
8E-10 6E-7
48
48
0.76
1E-12
52
F-SP
0.34
0.04
36
0.62
6E-5
36
0.80
4E-8
32
0.59
1E-4
36
0.67
7E-6
36
PO4-P
0.17
0.42
24
-0.21
0.93
24
-0.02
0.93
23
0.03
0.89
24
0.18
0.59
24
0.54
0.05
13
Tot-P
0.31
0.15
23
-0.08
0.72
23
0.14
0.54
22
0.11
0.61
23
0.11
0.61
23
0.47
0.10
13
36
Physico-chemical parameters
Alk. NH4-N Kjel-N SS BOD7
-0.34 0.08
0.05
0.36 0.09
0.13 0.72
0.82
0.09 0.70
21
23
23
25
23
0.46
0.16 0.34
0.22 -0.02
0.03
0.49 0.11
0.35 0.92
23
21
23
23
22
0.50
0.50 0.35
0.37 0.43
0.02
0.02 0.12
0.11 0.05
22
22
22
20
21
0.65
0.37 0.55
0.41 0.16
0.09 0.007 8E-4 0.06 0.47
23
23
21
23
22
0.81
0.42 0.66
0.31 0.15
0.06 5.5E-4 2E-6 0.16 0.50
23
23
21
23
22
0.09 -0.52 0.11
0.46 0.44
0.78 0.86
0.72
0.10 0.12
12
13
13
14
14
COD
0.24
0.27
23
0.54
0.008
23
0.58
0.005
22
0.63
0.001
23
0.69
3.3E-4
23
0.35
0.24
13
TOC
0.18
0.42
23
0.48
0.02
23
0.54
0.009
22
0.67
4.3E-4
23
0.75
3.4E-5
23
0.34
0.26
13
Comparative occurrence of pathogens in wastewater
4.3. Risk
from 365 repeated exposures. For exposure
to irrigated parks or golf courses this was
based on 26 repeated exposures, whereas in
the other scenarios 1:1,000 were accepted
risks per exposure. Results are presented in
Fig. 13.a-c. Excluded from the figures are the
uncertainties in the performance targets.
Adding an extra log would be equal to one
SD for all the scenarios except exposure to
ground water where one log security margin
is equal to 0.5 SD. Due to more HMUs
included in exposure to ground water, more
uncertainty is added in that scenario.
Depending on the contamination of the
water and type of exposure, different levels
of treatment is needed to achieve an
acceptable level of risk. This level, “the
performance target”, has been calculated for
wastewater and greywater for the exposure
scenarios listed in Table 11 with the
acceptable risk of infection equal to 1:1,000
as suggested by Haas et al. (1996) (84). For
exposure to drinking tap water or artificially
produced groundwater, this was a yearly risk
10
Wastewater, rotavirus
Greywater, rotavirus
Performance target [Log10]
10
Performance target
8
6
4
6
4
2
2
0
0
Crop
Pond/Park
Bathing/Aerosol
Groundwater
Tap Water
a
Crop
Pond/Park
Bathing/Aerosol
Groundwater
Tap water
b
c
10
Tap Water
Groundwater
8
Performance target
8
Rotavirus, 130,000 L-1
Campylobacter, 53,000 L-1
Giardia cysts, 3,000 L-1
Cryptosporidium oocysts, 1,100 L-1
Pond treatment, irrigation of parks
Bathing and exposure to aerosols
Crop irrigation
6
4
2
0
Giardia
Cryptosporidium
Campylobacter
rotavirus
Fig. 13.a-c. Performance targets for the removal
of rotavirus, Campylobacter, Giardia cysts and
Cryptosporidium oocysts from greywater and
wastewater via different exposure-routes: Tap
(drinking)
water
production,
artificial
groundwater production, accidental ingestion
to pond water, contact with irrigated areas,
bathing (receiving) water and aerosols during
irrigation. Figures represent median number in
wastewater [L-1] based on the incidence status
in the society, excretion (130 g p·d-1) and flow
(200 L p·d-1) (Table 9).
37
Jakob Ottoson
Trita LWR PHD
A mean 2.9 log higher reduction is necessary
for wastewater compared to greywater for all
exposure scenarios, reflecting the difference
in the faecal contamination of the different
flows, 130 and 0.1 g p·d-1, respectively (Fig.
13.a). Based on the average pathogen
numbers in wastewater, treatment should be
focused on removing viruses (Fig. 13.b). If
viruses are removed, other organisms are also
likely to be removed, most of all due to their
larger size. A mean 1.8, 3.4 and 4,2 log lower
removals are needed for Campylobacter, Giardia
cysts and Cryptosporidium oocysts, respectively,
than for viruses (Fig. 13.b). Producing
drinking water from wastewater would need a
mean 10.4 log removal (greywater 7.5) of
rotaviruses. The performance targets of
rotaviruses for the other exposures are:
groundwater 6.2 (3.3), pond treatment and
irrigation of parks 4.9 (2.0), aerosols and
bathing water 3.7 (0.8) and crop irrigation 2.7
(0) log respectively (Fig. 13.c).
Fig. 14. The probability of infection (Pinf),
with 90% confidence interval, as a function
of coliphage densities [PFU 100 mL-1], after
exposure to 50 mL surface water. This was
calculated from the ratio somatic
coliphages:enteroviruses in wastewater
assuming a stable relation in the
environment.
Table 15. Somatic coliphage (SOMCPH)
densities (95th percentile) [PFU 100 mL-1] in
surface water corresponding to different
probability of infections [%], Pinf (95th
percentiles).
4.3.1. Exposure to surface water
The treatment targets presented above used
1,000 times dilution of treated wastewater in
the bathing water scenario, but no microbial
die-off in the environment. Thus, the model
is a generalision intended for screening
microbial risks. Exposures to environmental
sources will in the nearest future still rely on
indicators. The inadequacy of the bacterial
indicators to predict the risks with exposure
to human viruses has been explained.
Therefore a guideline based on the
enumeration of somatic coliphages was
suggested in paper V. The risk of infection
from ingesting 50 mL surface water with a
known coliphage density is shown in Fig. 14,
with 90% confidence interval. In Table 15,
key levels of probability of infections and
their corresponding coliphage densities (95th
percentiles) in the water sample are given
together with suggested guidelines, 200 or
300 somatic coliphages, and the following Pinf
(95th percentiles). At the proposed level, 300
PFU 100 mL-1, the risk of infection from
bathing was 0.3% (4%, 95th percentile).
Pinf
SOMCPH
0.1
89 (7.0)
0.2 (3.0)
200
0.3 (4.0)
300
1.0
900 (71)
5.0
4,600 (360)
4.4. Microbial source-tracking
No single microorganism or chemical
biomarker has so far reliably distinguished
between human and animal faecal
contamination in water (206, 207). A
comparative evaluation between different
methods, based on a wide variety of samples
from different European regions, was made
and reported in paper VI. The results on the
occurrence of the selected parameters are
presented in Table 16. The parameters can be
divided into four classes, normal faecal
indicators, genotypes of F-specific RNA
38
Comparative occurrence of pathogens in wastewater
input in these abattoir wastewaters from staff
toilets. Different tolerance to environmental
stress between the genotypes must also be
taken into consideration if genotyping should
be used for source-tracking purposes (193).
Further, genotyping with the method used
(194) is a cumbersome procedure and not
proposed for routine purposes.
bacteriophages, faecal sterols and phenotypes
of faecal coliforms and enterococci.
Faecal indicators
The numbers of the respective faecal
indicators (Table 16) were similar to those
described in the literature for wastewater of
human (79, 135) as well as animal origin (89,
90, 227). No significant differences were
observed between the wastewater samples
from the different geographical areas. The
indicators and ratios between them do not
provide a distinguishing tool. The faecal
coliforms to enterococci ratio was proposed
to indicate the origin of pollution, where a
ratio of > 4 would indicate contamination of
human origin and a ratio < 0.7 is indicative
of animal pollution (72). This ratio is
however only valid for recent (24 hours)
faecal pollution due to different die-off
kinetics (27). The data indicate that the ratios
does not differ very much between animal
and human wastewaters (Table 16). Bacteroides
fragilis phages detected by strain 2056 were
found in all samples of human origin but to a
lesser degree and in lower numbers in animal
samples. The ratio between any of the other
indicators and phages infecting RYC2053
were 10 times as high in human compared to
animal samples. These differences were
however not enough to track the origin of
pollution (VI).
Sterols
The concentrations of four sterols and
stanols were determined: coprostanol,
stigmastanol, epicoprostanol and cholestanol.
Higher concentrations of coprostanol (COP)
were found in human than animal samples.
Epicoprostanol (EPICOP) was more
abundant in animal samples. None of the
studied faecal sterols are exclusively related to
humans or a specific animal species, but the
ratio COP:COP + EPICOP, as proposed in
Leeming et al., (1998) (130), proved to have a
high grade of discriminative power. The
exclusive use of sterols was however not
enough to ascertain the origin of pollution.
To do the analysis, an organic chemistry
laboratory and GC-MS facilities are needed.
The method is costly and time-consuming
and the application of faecal sterols for
routine purposes may be unreliable.
Phenotyping of faecal coliforms and enterococci
High diversities were generally associated
with urban and slaughterhouse wastewater,
whereas lower diversities were obtained from
hospital wastewater and animal faeces. These
findings reflect the number of individuals
contributing to the pollution rather than
being a discriminative parameter itself. The
percentage Enterococcus faecalis + Ent. faecium
(FM-FS), E. coli phene plate system pattern
and confirmed E. coli (Table 16)
differentiated the most between human and
animal pollution. Phenotyping of faecal
coliforms and enterococci is not very
discriminative but can provide some
complementary information (VI).
Genotyping
Genotyping of F-specific RNA phages have
been proposed as a method to distinguish
between animal and human faecal pollution
(194). Of the genotypes, II and III are
associated with human faecal pollution,
whereas I and IV are associated with animal
wastewater (195). These results were
confirmed in the European study (Table 16),
but 27% of animal samples failed to fulfil the
assumption that genotypes I and IV
predominate in animal faeces (VI). It is,
however, unclear if there could be human
39
Jakob Ottoson
Trita LWR PHD
Table 16. Data from municipal or hospital sewage (human) and slaughterhouse wastewater
(animal). Results are the average of log CFU/100 ml or log PFU/100 ml for faecal coliforms (FC),
enterococci (FE), clostridia (CL), somatic coliphages (SOMCPH), total F bacteriophages
(FTOTAL), F-RNA bacteriophages (FRNAPH), Bact. fragilis bacteriophages (RYC2056). Faecal
sterols are in µg/g: coprostanol (COP), stigmastanol or 24-ethylcoprostanol (ETHYLCOP),
epicoprostanol (EPICOP) and cholestanol (CHOL). The percentage of the rest of the values is
also the average of samples: diversity of enterococci (DiE), Ent. faecium plus Ent. faecalis (FMFS), Ent. hirae (Hir), E. coli Phene System patterns (ECP), E. coli confirmed (ECT). N: number
of samples (adapted from paper VI).
Class
Human
Animals
Average
SD
N
Average
SD
N
FC
7.05
0.58
89
7.60
0.94
95
FE
6.00
0.81
89
6.52
0.83
95
CL
5.20
0.69
89
5.29
1.26
95
SOMCPH
6.07
0.93
94
6.77
1.10
94
RYC2056
3.82
1.01
85
3.76
1.23
85
FTOTAL
5.48
0.76
87
5.43
1.52
85
FRNAPH
5.23
1.01
87
5.31
1.58
81
Percentage of genotypes
of F-RNA phages
I
7
–
83
34
–
71
II
40
–
83
12
–
71
III
48
–
83
28
–
71
IV
5
–
83
26
–
71
COP
1042.73
3436.66
70
252.92
1147.86
69
ETHYLCOP
657.96
3151.15
70
2840.69
10210.25
69
EPICOP
643.79
3316.43
70
2033.00
10347.60
69
CHOL
1361.60
6351.94
70
441.26
1351.80
69
Phenotypes of coliforms and
enterococci
DiE
0.85
0.19
84
0.76
0.25
95
FM-FS
66.10
20.69
84
49.03
30.63
94
Hir
17.15
18.48
84
28.69
31.12
94
DiC
0.89
0.16
88
0.88
0.09
95
ECP
79.10
22.65
77
97.11
5.37
84
ECT
72.13
24.29
77
93.30
9.10
84
Faecal indicators
Faecal sterols
40
Comparative occurrence of pathogens in wastewater
the size of colonies that should be counted.
Usually there are a lot of small colonies of
non-sorbitol fermenters. These are probably
not bifidobacteria, or even anaerobic bacteria,
but may grow slowly due to less strict
anaerobic conditions (30). Only colonies > 1
mm in diameter has been counted in Swedish
wastewater samples giving the ratio < 10.
Further, the pollution must be of recent time
since detection of bifidobacteria have been
reported to be difficult, especially during
summer (183). To overcome problems with
culture-based methods, probes were
developed for hybridisation to the genome of
Bifidobacterium adolescentis and Bif. dentium,
which were found exclusively in wastewaters
of human origin. This multiplex PCR can be
useful to discriminate between human and
animal faecal pollution in waters with high
concentrations of bifidobacteria (30).
Multivariate analysis
Numerical analyses during the first sampling
campaign showed that no single parameter
alone could discriminate animal from human
pollution satisfactorily. However, baskets of
four to five indicators would offer the
possibility of identifying human and animal
faecal inputs to surface waters with >60%
success. Not surprisingly F-RNA phage
genotype II together with the ratio
COP:COP + EPICOP were those
parameters with the greater discriminative
power (VI). A total discrimination was,
however, not possible based on the abovepresented parameters. Thus, in a second
sampling campaign, new parameters were
included. These were phages infecting
Bacteroides thetaiotaomicron, strain GA17,
hereafter called GA17, detection and ratio of
total:sorbitol-fermenting bifidobacteria (138,
183), detection of human specific
bifidobacteria with PCR (30) and antibiotic
resistance patterns of enterococci (VI).
Phages infecting Bacteroides spp.
Phages infecting Bacteroides, strain GA17, are
as human specific as HSP 40, suggested as
source-tracking
parameter
in
the
Mediterranean area (179), but occur in higher
titres, from 103 in UK to 105 in Spain (173).
The enumeration method is the same as for
the detection of phages infecting other
Bacteroides strains (109), making it easy to
implement for routine purposes. The use of
the enumeration of somatic coliphages and
the ratio SOMCPH:GA17 provided a 100%
correct classification with any of the four
learning machines used; 1-nearest neighbour
classifier, linear Bayesian classifier, quadratic
Bayesian classifier and support vector
machine (234).
Bifidobacteria
Bifidobacteria are excreted in high amounts
by humans and animals (109 g-1, (72)). Their
rapid die-off in the environment has limited
their use as indicators (216). Methods to
increase recovery (159) and detection of
humanspecific bifidobacteria by PCR (158)
can however increase the usefulness of
bifidobacteria as indicators of faecal
pollution. Culture of bacteria anaerobically
on HBSA media (138) gives the opportunity
to separate sorbitol-fermenting from total
bifidobacteria. The ratio of total to
sorbitolfermenters in Swedish wastewater
was typically less than 10, whereas it in
animal wastes was > 100. If a similar die-off
pattern of sorbitol as non-sorbitolfermentors
in the environment can be assumed, a cheap
and simple model for apportioning faecal
pollution is available where a ratio of less
than 10 would indicate human pollution, 10 –
100 mixed pollution and for ratios > 100 a
major impact of animal faecal pollution. Dieoff studies of the different phenotypes are
thus warranted. Definition of the laboratory
protocol is also very important, for example
Antibiotic resistance patterns of enterococci
Twenty-one
antibiotics
in
four
concentrations were chosen to represent
classes of broad-spectrum and narrowspectrum antibiotics used for treatment of
humans and animals. Resistance testing of
enterococci isolates were performed in
microplates (47). Studies were carried out in
the UK and the average rate of correct
classification was 72% (VI). Further
improvements based on initial results have
41
Jakob Ottoson
Trita LWR PHD
reduced the number of tests at the same time
as the discriminative power was increased to
86% (47). The problem is that antibiotic
databases work on a regional scale and new
databases may have to be created for every
region, which is costly and time-consuming
(234).
42
Comparative occurrence of pathogens in wastewater
5.
DISCUSSION
not significantly. Usually, the enterovirus
season starts during summer and peaks at fall
(19, 50, 97), a pattern that could not be
verified by studies in Swedish wastewater.
5.1. Do we need site-specific data?
In the introduction, the need for Swedish
studies on the occurrence of pathogens was
stressed. There is a wide range of possible
target organisms considering that more than
100 different disease-causing agents may
occur in wastewater. Four of these were
chosen due to their importance as pathogens
as well as their tolerance to treatment and
environmental stress. Comparing the results
from paper II and III with international data
(developed areas) does not show striking
spatial differences. The levels of noroviruses
were however significantly higher in the
Netherlands and Germany than in Sweden.
The study from Germany (180) was
conducted from October 2002 to September
2003. This was a year with a striking increase
in norovirus outbreaks in Europe that
coincided with the detection and emergence
of a new predominant norovirus variant
(133). Thus, this difference was temporal
rather then spatial. Also the epidemiological
approach
showed
resemblance
with
measured data and differences could be
explained and corrected in a rational manner,
for example by lower excretion values,
underreporting and disease incidences. The
epidemiological approach is, however,
connected with higher uncertainty leading to
less exact risk assessments, which may be
more difficult to communicate. So, do we
need site-specific data? It is partly a question
of cost/money. Site-specific data is valuable
and rectified. With a limited budget, however,
a lot of information can be provided from
reported data or by using epidemiological
approaches as demonstrated in this study.
Variations showed to be temporal rather than
spatial, with noroviruses as an obvious
example. Occurrence of Giardia was
significantly lower during two winter–spring
months in 2004. At the same period of time,
enterovirus occurrence was lower, though
Pathogens have been measured in larger
wastewater treatment plants with a large
contributing population. Someone in this
population is presumably infected with the
most common infectious agents, making it
possible to detect them in rather stable
numbers in the wastewater. In an area as
Vibyåsen on the other hand, with a
population of 212 people, statistically two
rotavirus infections will occur yearly
(incidence 0.95%) with a mean duration of 10
days each. Hence, most of the year, no
rotaviruses will be found, whereas during the
20 days of rotavirus occurrence, titres will be
very high leading to high risk of infection and
subsequently high performance targets. Scale
may therefore contribute more to the
variability than geography and or time.
However, integrating results from a number
of small systems to compare with a large one
may overcome scale-biases.
5.2. How do we measure the microbial
removal in treatment?
With unlimited time, space or energy, water
could be treated more or less as efficiently as
one would want. In a sustainable urban
environment, however, all these three
variables are limited. In this study compact
traditional treatments have been compared
with compact (MBR, UASB) as well as
extensive
(ponds
and
hydroponics)
alternatives. E. coli and enterococci were
more sensitive in all treatments than spores
and coliphages, with the F-specific phages
somewhere intermediate. This is in line with
earlier reported data (111, 218). Therefore,
bacterial indicators are not considered to be
suitable as process indicators of more
resistant organisms, such as viruses and
(oo)cysts, unless in mild treatment processes
43
Jakob Ottoson
Trita LWR PHD
such as primary sedimentation without the
addition of coagulants (134). Cl. perfringens
spores and somatic coliphages were tolerant
to treatment. Further, the analyses of these
organisms are cheap and easy to perform and
therefore Lucena et al. (2004) (134) suggested
that they should be included as additional
parameters to measure the removal efficiency
in wastewater treatment processes.
Enteroviruses were shown to be a good
index of virus occurrence and formed the
basis for the search for possible process
indicators (II, III). Bacteriophages are
structurally the most similar to enteroviruses
amongst the indicators and logically the best
predictor of virus removal in treatment
processes. This was also the case in
secondary treatment (II) where somatic
coliphage removal resembled enterovirus
genome removal. F-specific phages have
shown to be more sensitive to treatment (II,
III) and in the environment (40) than viruses
and coliphages and for that reason not
further discussed. In paper III, however,
coliphage removal did not correlate with
enteroviruses. Instead spores of Cl. perfringens
were the better predictor of virus removal
amongst the indicators. None of the tested
microorganisms did provide a satisfactory
model for virus removal (R-values around
0.4) (III). On the other hand, indicator
removals correlated to each others and to
other commonly-used parameters such as
NH4-N, COD and TOC, indicating that it
can be the enterovirus detection method that
is inappropriate to use. When culture-based
methods are used, phages have proved to be
infectious and bacteria the ability to grow.
Viruses on the other hand were detected by
rtPCR. Naked RNA is rapidly degraded in a
hostile environment such as wastewater, so
the RNA detected in the virus assays are
probably coming from intact virions, but are
they infectious? Duizier et al., (2004) (46),
studied the inactivation of the enteric canine
calicivirus and the respiratory feline
calicivirus and correlated inactivation to
reduction in PCR units of feline calicivirus,
canine calicivirus and a norovirus. For all
treatments, detection of viral RNA
underestimated the reduction in viral
infectivity. Further, positive/negative samples
from serial dilutions, as viruses were
enumerated in paper II and III, render a
discrete variable whereas indicator methods
render a continuous variable. Development
in quantitative PCR will hopefully eliminate
this difference in the future. The reason to
the poor correlation between coliphages and
human viruses may therefore be attributed to
Besides measuring the treatment efficiency
the objective has been to correlate removal of
indicators to those of pathogens. The focus
has been removal of enteric viruses and
parasitic (oo)cysts. Bacterial indicators, where
a lot of data are available, have been assumed
to be relevant models of bacterial pathogens.
The removal of Salmonella is likely to correlate
with that of E. coli, but if a more conservative
model is wanted enterococci can be used.
Campylobacter are very sensitive to treatment
and E. coli can be seen as a conservative
model. There are, however, studies claiming
that Campylobacter enters a coccoid, viable but
non-culturable (VBNC), form due to
environmental stress (23, 32). For this form,
not competing for nutrients, removal is
probably more appropriately predicted with
spores of sulphite-reducing anaerobes than
coliforms or enterococci. Cl. perfringens spores
have also been suggested as indicators of
parasitic (oo)cyst removal in drinking water
treatment processes (94, 174). Giardia cysts
were detected frequently in high numbers but
significantly more removed than spores in all
types of treatments (II, III). The use of
spores as process indicators will therefore
overestimate the risk of giardiasis. The
removal of Cryptosporidium oocysts was not
significantly different from spore removal,
however, the data on oocyst removal are
uncertain due to the low number of positive
samples. Oocyst occurrence was of little
relative importance when considering the risk
with reuse of wastewater. Instead the risks
were dictated by exposure to human viruses
and removal of these should be the focus for
treatment processes. Thus the further
discussion has its starting-point in virus
removal.
44
Comparative occurrence of pathogens in wastewater
the different detection methods used. The
advantages of PCR detection of viruses,
however, overweighed this disadvantage.
Moreover, culture-based methods for some
viruses (i.e. noroviruses) are not yet available.
More studies on the relations between PCR
and culture-based methods for microbial
enumeration, indicator as well as pathogen,
are though warranted.
on results from MRA (257). This should be
done in an early phase of the planning
process to give an indication of the
performance need of the WWTP for the
intended use of the wastewater. Based on
this, different systems can be suggested for
the future process of planning, weighing in
parameters from the other aspects of
sustainability (Fig. 1). On the management
level, measuring the performance targets
(discussed above) should be prioritised. In
the introduction a multiple microbial barrier
approach (HMUs) was stressed. The most
important HMUs are probably the different
treatment processes, and several of them, or
whole treatment lines, could be used as
critical control points in a HACCP approach.
None of the physical-chemical parameters
tested correlated to enterovirus removal, but
Kjeldal-N, ammonium-N, COD and TOC
removals correlated positively to indicator
organism removal (III). These parameters
were not measured on-line. Turbidity can be
measured on-line but was in this study
measured as mg suspended solids L-1, which
was too insensitive as a method, especially
for effluents from MBR-treated water.
Phosphorous removal did neither correlate to
enterovirus nor indicator removal, with the
exception of F-specific phages. The reason to
the different behaviour of phosphorous in
the treatment processes could be that the
majority of replicates came from the tertiary
treatment line with chemical precipitation as
a treatment process. In this step
phosphorous may be more efficiently
removed than the other process indicators
(III).
Removal varied greatly in treatment due to
process combination and organism in
question. Human viruses were in general less
removed than the indicators, however, viral
genome removal may underestimate
infectious virion removal (46). Secondary
treatment
systems
removed
somatic
coliphages, suggested as an indicator of
human viruses, with 1 log, i.e. 90%. Tertiary
filtration removed phages 1.3 log further, but
the treatment efficiency is still below the
suggested performance target of 3.7 log for
rotaviruses for discharge to surface water
recipients (Fig. 13). Alternative treatments
could remove microorganisms at least as
efficiently as tertiary treatment. The
integrated hydroponics (IV) and the
greywater treatment system in Vibyåsen (I),
however, needed longer hydraulic retention
times for their proper removal of
microorganisms. Systems like these may
therefore be unreliable in a dense urban
environment. MBR treatment is compact and
has shown high removals of the indicators.
UASB is also compact but with limited
removal efficiency (III).
The answer to the question: How do we
measure the removal of pathogens?” is that
we need more studies and better
understanding if we want easy measures on
virus removal. Potentially, somatic coliphages
can be used as an adequate model and
turbidity as an early warning, but should be
interpreted with care. Turbidity is a measure
ensuring the proper function of the treatment
process, not the absence of microorganisms.
Development of particle counters may be a
useful tool to ensure microbial safety on-line
as a control in a HACCP approach.
Apart from the actual treatment processes,
other barriers may be linked to sourceseparation and limitations in exposure.
Separation of greywater from toilet waste at
the source is beneficial, especially if the water
resource is the main interest and not the
5.3. How can we manage the risks?
The suggested approach is in line with WHO
guidelines, setting up treatment targets based
45
Jakob Ottoson
Trita LWR PHD
plant nutrients. The need for extensive
treatment is significantly reduced (99.9%).
This level of removal was not achieved for
viruses, even with tertiary treatment. If
viruses are removed, other organisms are also
likely to be removed, most of all due to their
larger size. Further, viruses are more tolerant
to different types of treatments as well as in
the environment, especially than vegetative
bacteria competing for nutrients.
diluted (1,000 times) wastewater. For
wastewater, the performance target was 3.7
log removal for rotaviruses which is not
achieved with secondary treatment. Thus, the
wastewater treatment plants of today do not
fulfil the safety levels suggested in this study.
Environmental die-off was however not
included in the receiving water scenario,
therefore making the model conservative.
The risks for exposures to aerosols are lower
than for the exposure to the irrigated areas.
Bacteriophage transport over 500 m in
aerosols have, however, been shown to occur
downwind (21). An easy control measure is
to ensure that irrigation takes place when the
wind and time of day minimises exposure to
humans. For the last exposure scenario,
ingestion of irrigated crops, the performance
target was the lowest of all, 2.7 log for
rotaviruses in wastewater, none in greywater.
On the other hand, this was measured for a
single exposure, not repeated exposures such
as in the irrigation of parks scenario. Further,
public acceptance to consume unprocessed
crops irrigated with wastewater is probably
lower than the risk of infection, highlighting
another issue, the difference between
measured and perceived risk, discussed for
example by Slovic, (1987) (209).
If bottled drinking water is produced from
wastewater the simulations resulted in a 10.4
log removal for rotavirus to be below an
acceptable yearly risk of 1:1000. The
corresponding performance target for
greywater was 7.5 log, which is probably not
achievable unless disinfection and RO
treatment are applied. In the pilot-plant in
Hammarby Sjöstad, the aim is to produce
effluent water of drinking water quality with
the inclusion of RO. Better pre-treatment
than in the UASB–line is, however, needed.
For exposure to artificial groundwater the
performance targets are also high since the
exposure is based on a yearly risk of 365
exposures to about 1 L of water (0.5 L
wastewater considering the dilution factor).
Increasing the retention time in the aquifer
will significantly reduce the performance
target as shown in paper I. Exposure to
irrigated areas and pond had performance
targets of 4.9 and 2.0 log for rotaviruses in
wastewater and greywater respectively. Some
dilution of the pond water is possible but not
accounted for in the risk assessment.
Exposure restriction like fencing the pond
also functions as a barrier. For irrigation of
public parks and golf courses, performance
targets equal to exposure to ponds would be
necessary.
Exposure restrictions like
prolonging the time between irrigation and
access to the irrigated area is also a barrier
that decreases the need of treatment. This
has been proposed for reuse of human urine
(101) and greywater irrigation (170).
Performance targets for exposure scenarios
aerosols and receiving water are lower than the
above-presented due to the lower water
intake, mean 0.05 mL wastewater either
through inhalation of aerosols or ingestion of
Campylobacter is the most common gastrointestinal pathogen in Sweden and not
surprisingly very important to control when
reuse is considered. Controlling viruses will
probably be enough to control Campylobacter
as well. The zoonotic potential is, however,
higher and Campylobacter may be the pathogen
of main concern if animal sources are likely
to dominate the faecal contamination. Giardia
is also common in the population and high
and steady numbers have been detected in
wastewater (II, III). The performance target
was anyway 3.4 log lower than for rotaviruses
reflecting a difference both in the occurrence
in wastewater as well as a lower risk of
infection to single cysts than to rotavirions
(Fig. 6). Unlike for the other organisms, cyst
levels in the risk calculations were probably
underestimated and Giardia will be one of the
most important organisms to control in reuse
systems, especially during failure events. In
46
Comparative occurrence of pathogens in wastewater
one of the largest waterborne outbreaks in
Sweden, when untreated wastewater
contaminted the tap-water, most of the
clinical isolates were positive for Giardia
(220). Looking at the dose-response curves
(Fig. 6), it could be seen that Giardia is the
most infective of the studied organisms at
doses above 70 infectious particles (virions,
bacteria or (oo)cysts). Cryptosporidium are not
as numerous in the wastewater, with low risk
of infection for exposure to a single oocyst,
and is consequently not as an important
organism for most of these exposure
scenarios. If an exposure involves systems
where chlorination is an important barrier,
for example drinking water production or
exposure to water in chlorinated swimmingpools or systems relying on long retention
times before reuse, oocysts can be an
important agent due to their chlorine
resistance and ability to survive for extended
periods in water (143). Agriculture run-off is
also a risk factor for oocyst occurrence since
cattle is supposed to be a reservoir for
Cryptosporidium (parvum) (123). Agricultural
run-off was supposed to be the reason for
the Milwaukee outbreak of cryptosporidiosis
(1). The main input of Gothenburg raw water
intake, frequently contaminated with oocysts
(86), was also supposed to be agricultural
(169).
occurrence in water. Correlation with virus
occurrence in surface water has been
documented in temperate regions (135, 208)
and somatic coliphages as well as phages
infecting Bacteroides are useful to predict the
presence of pathogens with higher survival
rates than the bacterial indicators (40).
Bacteroides phages were not as numerous in
wastewater as somatic coliphages (VI).
Coliphages on the other hand have been
reported to be able to multiply in the
environment, thereby overestimating the
faecal contamination (88). Environmental
multiplication is, however, unlikely to happen
under the circumstances prevailing in a
surface water environment (153), especially
not in temperate regions (135). Spores of
sulphite-reducing anaerobes or Cl. perfringens
spores have also been suggested as indicators
of virus occurrence in surface waters (143).
The former is a cheap and easy assay to
perform. Sulphite-reducers are, however, not
exclusively of faecal origin (104). To detect
Cl. perfringens spores a battery of biochemical
tests have to be included making the analysis
more cumbersome than the enumeration of
somatic coliphages.
In receiving waters, viruses stood out as the
largest risk in terms of probability of
infection. It is, however, believed that gastrointestinal viruses infecting humans are
exclusively of human origin and cannot
emanate from farmland run-off. Thus it is
important to know the source of pollution.
Not only to be able to calculate the risk but
also to be able to manage the pollution in an
effective way. In a European study, a model
to discriminate human from animal faecal
pollution to 100% including just two
microbial parameters was developed (234).
Only one additional parameter to somatic
coliphages, phages infecting Bacteroides
tethaiotaomicron, strain GA17, was needed.
These bacteriophages have so far turned out
to be human specific (173). The method
seems to be working in the Mediterranean
region, and to some degree in Sweden. The
possibility to search for other more suitable
regional host strains is described (173). The
5.3.1. Surface water management
In the risk models, exposure to bathing water
had performance targets between 0 and 3.7
log removal due to water and organism. The
model was, however, a generalisation based
on a fixed dilution and no environmental dieoff. To be able to quantify the risk at specific
sites, indicators of faecal pollution are used.
The lack of accuracy in determining the risk
based on these has been explained. In paper
V, guidelines for the management of bathing
water were proposed. These were based on
MRA instead of epidemiological studies, such
as the existing EU bathing water directive.
Further, somatic coliphages were suggested
as a supplementary parameter due to its
better prerequisites to predict virus
47
Jakob Ottoson
Trita LWR PHD
cost both to develop the method, implement
it and the cost for analysis are low. In less
contaminated waters there may be a need to
concentrate phages and method development
is
warranted.
Method
for
phage
concentration needs also to be standardised
for the sake of including somatic coliphages
in bathing water management.
As detection methods of human viruses have
improved, many studies have suggested the
use of direct detection of these, for example
enteroviruses (96), adenoviruses (63) and
polyomaviruses (28) as a means to manage
recreational waters. Enteroviruses were
present in almost all of the treated
wastewaters containing any other pathogen
(II, III). They are frequently detected in
surface waters (75) and may serve as an
indicator (96) as well as being a parameter for
the control of heavily contaminated bathing
waters (210). Most animals also excrete
enteroviruses. A distinction between animal
and human faecal pollution based on
enterovirus occurrence is possible, but not
with the set of primers used in papers I and
II. Adenoviruses are also numerous (78),
tolerant (53) and indicates human faecal
pollution (234), but still none of the human
virus parameters are as easy and cheap to
perform as the phage assays and somatic
coliphages are strongly advocated to be used
in future guidelines for the safe use of or
exposure to water.
48
Comparative occurrence of pathogens in wastewater
6.
CONCLUSIONS
•
High levels of Giardia cysts and
enteroviruses were found in the
wastewater indicating high incidences
in the society. Noroviruses were also
found in high numbers, mostly
during winter, correlating to the
reporting of winter vomiting disease.
Cryptosporidium oocysts were not as
numerous
in
the
wastewater
indicating lower prevalence in the
society than for the other pathogens
during the time of the study.
•
Measured data minimises uncertainty
in risk assessments. Site-specific data
are, however, not necessary in every
case (e.g. occurrence in wastewater).
Good data could be found in the
literature and corrected for by
recovery of the detection method,
flow and incidence in the society.
Variation proved to be temporal
rather than spatial, with noroviruses
as an obvious example.
•
The
faecal
contamination
in
greywater,
measured
with
coprostanol, is 1,300 times lower than
in domestic wastewater. Adjusting for
the different dilutions gives a
pathogen density 980 times lower in
greywater than in wastewater, equal
to 2.9 log removal in treatment.
•
suggested to be included
supplementary
indicators
wastewater treatment processes.
as
in
•
Bacterial indicator and spore
removals correlated to enterovirus
genome removal (p<0.05), but the
predictive values were low (R<0.4).
Removal
between
microbial
indicators and nitrogen, COD and
TOC correlated stronger. Somatic
coliphages are suggested as an index
organism of virus removal.
•
To manage the risk with reuse and
discharge of wastewater, performance
targets for wastewater treatment have
been calculated as a step in a HACCP
system. These targets varied from 0
to 10.4 log removal due to water
(grey or wastewater), organism
(rotavirus, Campylobacter or parasitic
cysts) and exposure (drinking water,
surface water, aerosols, irrigation of
crops or public parks) and were:
2.9 log removal higher for wastethan greywater.
1.8, 3.4 and 4.2 log higher for
viruses than Campylobacter, Giardia
cysts and Cryptosporidium oocysts
respectively.
The highest for drinking water
production followed by ground
water production, open pond
treatment, irrigation of parks,
aerosols, bathing water and
ingestion of irrigated crops.
Removal of microorganisms in
treatment varied from 0 to >5.8 log
due to process combination and
organism in question. Human virus
genomes were less removed than all
the indicators whilst Giardia cysts
more removed than the indicators.
Enumeration with PCR, however,
may underestimate infectious virion
removal. Spores of sulphite-reducing
anaerobes and somatic coliphages
were significantly less removed than
E. coli and enterococci and are
Needing one log extra treatment
for a safety margin equal to one
standard deviation.
49
Jakob Ottoson
•
Trita LWR PHD
Somatic coliphages are suggested as a
supplementary
parameter
in
management of bathing waters as an
indication of virus occurrence. The
guideline level is proposed to be 300
PFU 100 mL-1 based on a MRA of
enteroviruses. If somatic coliphages
are enumerated together with phages
infecting a regionally adequate
Bacteroides spp. strain, tracking the
source of faecal pollution can be
possible.
50
Comparative occurrence of pathogens in wastewater
7.
ACKNOWLEDGEMENTS
Thor Axel Stenström, Bengt Hultman and Rhys Leeming are acknowledged for their supervision. Nick
Ashbolt for coming to Sweden from time to time, functioning as an informal supervisor, sharing
his MRA knowledge and Australian(?) humour.
All the members of our group “Vattenskallarna”, Water and Environmental Microbiology, at
SMI for making it such a great place. Görel, I guess that I have learnt more from you than any
other teacher during my whole life of education; Caroline always wanting to have a talk over
coffee (preferably with a cake), sharing recipies and your experience from children (and television
shows); Anette, presumably my new tennispartner, doing the hard work with the parasites; Therese
thanks for all the support and nice words during hard times; Jonas, always being so kind and
helpful; Johan, I have enjoyed our discussions, scientific as well as non-scientific; Anneli, I have
missed you during your maternity leave; Anna, the nice girl next door; Emma and Sabina, keeping
the lab in perfect shape, just for me (?) and finally Michael (sorry, Dr. Storey), my former
tennispartner, unfortunately on the wrong side of the globe today.
The Foundation for Strategic Environmental Research, MISTRA, is acknowledged for its
financial support through the reseach programme “Sustainable Urban Water Management”. A
special thank to all the members of the programme; Per-Arne and Henriette at the HQ; Jan-Olof
running the research school; all the PhD-students (I really enjoyed your company during our
courses and meetings), Anna and Gunnel, co-authors of paper IV; Daniel and Håkan, coordinators
of the model-cities and everyone else.
Employees at the Department of Virology at SMI; Helene, co-author of paper II and III and Lena
for invaluable help with enterovirus assays; Kari, co-author of paper II, Margaretha and Klas for
introducing me to norovirus diagnostics and Kjell-Olof, providing me with EM-positive controls
and a nice cover photo.
All the members of PMV, the best Department at SMI and the rest of SMI, especially those who
fikar at level 4.
People at KTH, Department of Land and Water Resources Engineering, especially Aira, for their
administrative help around my theses presentations.
Tore Midtvedt, Lizza and Anna-Karin for nice discussions and help with faecal sterol extractions
and analyses for paper I. Elisabet Börjesson, SLU, is also acknowledged for her invaluable help,
running the GC-MS.
Björn Olsen and Per-Erik Jarnheimer of the Kalmar County Hospital are acknowledged for nice
coorperation, however, our papers are saved for a better occasion.
Members of the TOFPSW consortium; Anicet, Joan, Maite, and all the girls and Xavis at the lab, in
Barcelona, Sylvain and Christophe in Nancy, Inger and Aina at MTC, James, Jess and Huw in Brighton
and Georgios and Christos in Nicosia. Thanks for including me in your project; it was great to work
with you all.
Karsten, COWI, Denmark for nice meetings, preferably with a Tuborg. Sorry for my poor
engagemnet in the LOT-project.
51
Jakob Ottoson
Trita LWR PHD
Per-Erik, Sollentuna Vatten, for helping me with sampling at Vibyåsen; Inger, Per and Karin for
sampling and sending wastewater from Göteborg, Umeå and Malmö. Thanks also to Berndt, coauthor of paper III, and all the other helpful people at the pilot-plant.
Britt-Marie, for lending me your car, time and son, so I finally was able to take my driver´s license.
All the ones that I should thank but forgot.
Finallly, thanks to my family and my friends for all their love and support. Pappa och Mamma,
presumably my greatest fans; Amanda, Klara, Figo, Teo and Lotta, Life wouldn´t be anything
without You, I love You.
52
Comparative occurrence of pathogens in wastewater
8.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
REFERENCES
Addiss, D., W. McKenzie, N. Hoxie, M. Gradus, K. Blair, M. Proctor, J.
Kazmierczak, W. Schell, P. Osewe, H. Frisby, H. Cicerello, R. Cordell, J. Rose, and
J. Davis. 1995. Epidemiologic features and implications of the Milwaukee
cryptosporidiosis outbreak. pp 19-25. In W. Betts, D. Casemore, C. Fricker, H.
Smith, and J. Watkins (ed.), Protozoans Parasites and Water. The Royal Society of
Chemistry, Cambridge.
Albrechtsen, H.-J. 1998. Water consumption in residences. Microbiological
investigations of rain water and greywater reuse systems. Miljöstyrelsen och
Boligministeriet.
Al Jayyousi, O. 2002. Focused environmental assessment of greywater reuse in
Jordan. Environ Eng Policy 3:67-73.
Anderson, J. M. 1996. Current water recycling initiatives in Australia: Scenarios for
the 21st century. Water Sci Technol 33:37-43.
Andersson, Y., and P. Bohan. 2001. Disease surveillance and waterborne outbreaks.
pp 115-133. In Fewtrell, L. and J. Bartram (ed.), Water Quality: Guidelines,
standards and health. IWA Publishing, London.
Anonymous. 2001. Waterleidingbesluit 2001. Staatsblad van het Koninkrijk der
Nederlanden 31:1-53.
Asano, T. and A. Levine. 1998. Wastewater reclamation, recycling and reuse: an
introduction. pp 1-56. In T. Asano (ed.), Wastewater reclamation and reuse.
Technomic Publishing Company Inc., Lancaster, Pennsylvania.
Asano, T., L. Y. C. Leong, M. B. Rigby, and R. H. Sakaji. 1992. Evaluation of the
California wastewater reclamation criteria using enteric virus monitoring data. Water
Sci Technol 26:1513-1524.
Asano, T., and A. D. Levine. 1996. Wastewater reclamation, recycling and reuse:
past, present and future. Water Sci Technol 33:1-14.
Ashbolt, N. 2005. Personal Communication.
Ashbolt, N., S. Petterson, D. Roser, T. Westrell, J. Ottoson, C. Schönning, and T.
Stenström. 2004. Microbial risk assessment tool to aid in the selection of sustainable
urban water systems. Presented at the Proceedings from 2nd IWA Leading-Edge
Conference on Sustainability in Water Limited Environments., Sydney, 8 - 10
November.
Ashbolt, N., C. Riedy, and C. N. Haas. 1997. Microbial health risk at Sidney´s
coastal bathing beaches. Advances in risk assessment approaches to waterborne
disease: outbreak and occurrence. Presented at the Proc. 17th Federal Convention
of AWWA, Melbourne, 16-21 March.
Ashbolt, N. J. 1999. Presented at the 2nd International Conference on the Safety of
Water Disinfection: Balancing Chemical and Microbial Risks, Miami, FL, USA,
November 15-17.
Atmar, R. L., F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G.
Metcalf, and M. K. Estes. 1995. Detection of Norwalk virus and hepatitis A virus in
shellfish tissues with the PCR. Appl Environ Microbiol 61:3014-3018.
Aulicino, F. A., A. Mastrantonio, P. Orsini, C. Bellucci, M. Muscillo, and G. Larosa.
1996. Enteric viruses in a wastewater treatment plant in Rome. Water Air Soil Poll
91:327-334.
Ausland, G., T. K. Stevik, J. F. Hanssen, J. C. Kohler, and P. D. Jenssen. 2002.
Intermittent filtration of wastewater-removal of fecal coliforms and fecal
streptococci. Water Res 36:3507-3516.
53
Jakob Ottoson
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
Trita LWR PHD
AWWA. 1999. Waterborne Pathogens, First Edition ed. AWWA, Denver,
Colorado.
Badawy, A. S., J. B. Rose, and C. P. Gerba. 1990. Comparative survival of enteric
viruses and coliphage on sewage irrigated grass. J Environ Sci Health Part A Tox
Hazard Subst Environ Eng 25:937-952.
Bahri, O., D. Rezig, B. B. Nejma-Oueslati, A. B. Yahia, J. B. Sassi, N. Hogga, A.
Sadraoui, and H. Triki. 2005. Enteroviruses in Tunisia: virological surveillance over
12 years (1992-2003). J Med Microbiol 54:63-69.
Bartram, J., K. Lewis, R. Lenton, and A. Wright. 2005. Focusing on improved water
and sanitation for health. Lancet 365:810-812.
Bausum, H. T., S. A. Schaub, K. F. Kenyon, and M. J. Small. 1982. Comparison of
coliphage and bacterial aerosols at a wastewater spray irrigation site. Appl Environ
Microbiol 43:28-38.
Beaglehole, R., R. Bonita, and T. Kjellström. 1993. Basic epidemiology. WHO,
Geneva. 175 p.
Beumer, R. R., J. de-Vries, and F. M. Rombouts. 1992. Campylobacter jejuni nonculturable coccoid cells. Int J Food Microbiol 15:153-163.
Beuret, C. 2003. A simple method for isolation of enteric viruses (noroviruses and
enteroviruses) in water. J Virol Meth 107:1-8.
Bingley, E. B. 1996. Greywater reuse proposal in relation to the Palmyra Project.
Desal 106:371-375.
Bishop, R. F. 1996. Natural history of human rotavirus infection. Arch Virol Suppl
12:119-128.
Bitton, G. 1994. Wastewater microbiology. Wiley-Liss Inc., New York. 478 p.
Bofill-Mas, S., S. Pina, and R. Girones. 2000. Documenting the epidemiologic
patterns of polymaviruses in human populations by studying their presence in urban
sewage. Appl Environ Micrbiol 66:238-245.
Bonadonna, L., R. Briancesco, C. Cataldo, M. Divizia, D. Donia, and A. Pana. 2002.
Fate of bacterial indicators, viruses and protozoan parasites in a wastewater multicomponent treatment system. New Microbiol 25:413-420.
Bonjoch, X., E. Balleste, and A. R. Blanch. 2004. Multiplex PCR with 16S rRNA
gene-targeted primers of bifidobacterium spp. to identify sources of fecal pollution.
Appl Environ Microbiol 70:3171-3175.
Bukhari, Z., R. M. McCuin, C. R. Fricker, and J. L. Clancy. 1997. Immunomagnetic
separation Cryptosporidium parvum from source water samples of various turbidities.
Appl Environ Microbiol 64:4495-4499.
Buswell, C. M., Y. M. Herlihy, L. M. Lawrence, J. T. M. McGuiggan, P. D. Marsch,
C. W. Keevil, and S. A. Leach. 1998. Extended survival and persistance of
Campylobacter spp. in water and aquatic biofilms and their detection by
immunofluorescent-antibody and -rRNA staining. Appl Environ Microbiol 64:733741.
Cabelli, V. J., A. P. Dufour, L. J. McCabe, and M. A. Levin. 1982. Swimming
associated gastroenteritis and water quality. Am J Epidemiol 115:606-616.
CAC. 1997. Hazard Analysis and Critical Control Point (HACCP) system and
guidelines for its application Annex to CAC/RCP 1-1969. CODEX Alimentarius
Commission, Rome.
Cambell, A., and H. Smith. 1997. Immunomagnetic separation of Cryptosporidium
oocysts from water samples: Round Robin comparison of techniques. Water Sci
Technol 35:397-401.
54
Comparative occurrence of pathogens in wastewater
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
Carlander, A., T. Stenström, A. Albihn, and K. Hasselgren. 2002. Hygieniska
aspekter vid avloppsbevattning av Salixodlingar - undersökningar vid tre
fullskaleanläggningar 2002-1. VA-Forsk.
Casanova, L. M., C. P. Gerba, and M. Karpiscak. 2001. Chemical and microbial
characterization of graywater. J Environ Sci Health A36:395-401.
Christova-Boal, D., R. E. Eden, and S. McFarlane. 1996. An investigation into
greywater reuse for urban residential properties. Desal 106:391-397.
Cogan, T. A., S. F. Bloomfield, and T. J. Humphrey. 1999. The effectiveness of
hygiene procedures for prevention of cross-contamination from chicken carcasses
in the domestic kitchen. Lett Appl Microbiol 29:354-358.
Contreras-Coll, N., F. Lucena, K. Mooijman, A. Havelaar, V. Pierzo, M. Boque, A.
Gawler, C. Holler, M. Lambiri, G. Mirolo, B. Moreno, M. Niemi, R. Sommer, B.
Valentin, A. Wiedenmann, V. Young, and J. Jofre. 2002. Occurrence and levels of
indicator bacteriophages in bathing waters throughout Europe. Water Res 36:49634974.
Davies, C. M., J. A. Long, M. Donald, and N. J. Ashbolt. 1995. Survival of fecal
microorganisms in marine and freshwater sediments. Appl Environ Microbiol
61:1888-1896.
de Zutter, L., and J. van Hoof. 1984. Occurrence of Salmonella in a chemical
wastewater treatment plant. Zentralbl Bakteriol Mikrobiol Hyg [B] 179:440-448.
Dennett, K. E., A. Amirtharajah, T. F. Moran, and J. P. Gould. 1996. Coagulation:
Its effect on organic matter. J AWWA 88:129-142.
Dixon, A. M., D. Butler, and A. Fewkes. 1999. Guidelines for greywater re-use:
Health issues. J Inst Water Environ Manage 13:322-326.
Dowd, S. E., C. P. Gerba, I. L. Pepper, and S. D. Pillai. 2000. Bioaerosol transport
modeling and risk assessment in relation to biosolid placement. J Environ Qual
29:343-348.
Duizer, E., P. Bijkerk, B. Rockx, A. De Groot, F. Twisk, and M. Koopmans. 2004.
Inactivation of caliciviruses. Appl Environ Microbiol 70:4538-4543.
Ebdon, J. E., J. L. Wallis, and H. D. Taylor. 2004. A simplified low-cost approach
to antibiotic resistance profiling for faecal source tracking. Water Sci Technol
50:185-191.
Eckner, K. F. 1998. Comparison of membrane filtration and multiple-tube
fermentation by the colilert and enterolert methods for detection of waterborne
coliform bacteria, Escherichia coli, and enterococci used in drinking and bathing
water quality monitoring in southern Sweden. Appl Environ Microbiol 64:3079-83.
Edberg, S., M. Allen, and D. Smith. 1994. Comparison of the Colilert method and
standard fecal coliform methods. AWWA research foundation, Denver Colorado.
Ehrnst, A., and M. Eriksson. 1993. Epidemiological features of type 22 echovirus
infection. Scand J Infect Dis 25:275-281.
Eisenberg, J. N., J. A. Soller, J. Scott, D. M. Eisenberg, and J. M. Colford, Jr. 2004.
A dynamic model to assess microbial health risks associated with beneficial uses of
biosolids. Risk Anal 24:221-236.
Enriquez, C. E., and C. P. Gerba. 1995. Concentration of enteric adenovirus 40
from tap, sea and wastewater. Water Res 29:2554-2560.
Enriquez, C. E., C. J. Hurst, and C. P. Gerba. 1995. Survival of the enteric
adenoviruses 40 and 41 in tap, sea and wastewater. Water Res 29:2548-2553.
Eriksson, E., K. Auffart, M. Henze, and A. Ledin. 2002. Characteristsics of grey
wastewater. Urban Water 4:85-104.
EU. 2002. Directive of the European Parliament and of the Council concerning the
quality of bathing water. Report 2002/0254 (available at http://europa.eu.int/eur-
55
Jakob Ottoson
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
Trita LWR PHD
lex/en/com/pdf/2002/com2002_0581en01.pdf). Commission of the European
Communities.
Faechem, R. G., D. J. Bradley, H. Garelick, and D. D. Mara. 1983. Sanitation and
disease: health aspects of excreta and wastewater management. John Wiley & Sons,
Washington. 501 p.
Fane, S. A., and N. J. Ashbolt. 2000. A methodology for assessing comparative
pathogen impact from novel wastewater systems. Presented at the Water recycling
Australia 2000, Adelaide, 19-20 Oct.
Fane, S. A., N. J. Ashbolt, and S. B. White. 2002. Decentralised urban water reuse:
the implications of system scale for cost and pathogen risk. Water Sci Technol
46:281-288.
Farahbakhsh, K., and D. W. Smith. 2004. Removal of coliphages in secondary
effluent by microfiltration-mechanisms of removal and impact of operating
parameters. Water Res 38:585-592.
Fayer, R., T. K. Graczyk, E. J. Lewis, J. M. Trout, and C. A. Farley. 1998. Survival
of infectious Cryptosporidium parvum oocysts in seawater and Eastern oysters
(Crassostrea virginica) in the Chesapeake Bay. Appl Environ Microbiol 64:10701074.
Ferley, J. P., D. Zmirou, F. Balducci, B. Baleux, P. Fera, G. Larbaigt, E. Jacq, B.
Moissonnier, A. Blineau, and J. Boudot. 1989. Epidemiological significance of
microbiological pollution criteria for river recreational waters. Int J Epidemiol
18:198-205.
Fleisher, J. M., D. Kay, R. L. Salmon, F. Jones, M. D. Wyer, and A. F. Godfree.
1996. Marine waters contaminated with domestic sewage: nonenteric illnesses
associated with bather exposure in the United Kingdom. Am J Public Health
86:1228-1234.
Formiga-Cruz, M., G. Tofino-Quesada, S. Bofill-Mas, D. N. Lees, K. Henshilwood,
A. K. Allard, A. C. Conden-Hansson, B. E. Hernroth, A. Vantarakis, A. Tsibouxi,
M. Papapetropoulou, M. D. Furones, and R. Girones. 2002. Distribution of human
virus contamination in shellfish from different growing areas in Greece, Spain,
Sweden, and the United Kingdom. Appl Environ Microbiol 68:5990-5998.
Furumoto, W. A., and R. Mickey. 1967. A mathematical model for the infectivitydilution curve of tobacco mosaic virus: theoretical considerations. Virol 32:216-223.
Gale, P. 2003. Using event trees to quantify pathogen levels on root crops from
land application of treated sewage sludge. J Appl Microbiol 94:35-47.
Gander, M. A., B. Jefferson, and S. J. Judd. 2000. Membrane bioreactors for use in
small wastewater treatment plants: membrane materials and effluent quality. Water
Sci Technol 41:205-211.
Gantzer, C., E. Dubois, J.-M. Crance, S. Billaudel, H. Kopecka, L. Schwartzbrod,
M. Pommepuy, and F. Le-Guyader. 1998. Devenir des virus enteriques en mer et
influence des facteurs environnementaux. Oceanol. Acta 21:983-992.
Gantzer, C., L. Gillerman, M. Kuznetzov, and G. Oron. 2001. Adsorption and
survival of faecal coliforms, somatic coliphages and F-specific RNA phages in soil
irrigated with wastewater. Water Sci Technol 43:117-124.
Gantzer, C., S. Senouci, A. Maul, Y. Levi, and L. Schwartzbrod. 1997. Enterovirus
genomes in wastewater: concentration on glass wool and glass powder and
detection by RT-PCR. J Virol Methods 65:265-271.
Garrett, E. S., C. L. dosSantos, and M. L. Jahncke. 1997. Public, animal, and
environmental health implications of aquaculture. Emerg Inf Dis 3:453-457.
56
Comparative occurrence of pathogens in wastewater
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
Gassilloud, B., M. Duval, L. Schwartzbrod, and C. Gantzer. 2003. Recovery of
feline calicivirus infectious particles and genome from water: comparison of two
concentration techniques. Water Sci Technol 47:97-101.
Geldreich, E. E. 1978. Bacterial populations and indicator concepts in feces,
sewage, stormwater and solid wastes. pp 51-97. In G. Berg (ed.), Indicators of
viruses in waters. Ann Arbor Science Publishers Inc., Ann Arbor, Michigan.
Gerba, C. P., J. B. Rose, C. N. Haas, and K. D. Crabtree. 1996. Waterborne
rotavirus: a risk assessment. Water Res 30:2929-2940.
Gerba, C. P., T. M. Straub, J. B. Rose, M. M. Karpisack, and K. E. Foster. 1995.
Water quality of graywater treatment system. Water Res Bull 31:109-116.
Gilgen, M., D. Germann, J. Lüthy, and P. Hübner. 1997. Three-step isolation
method for sensitive detection of enterovirus, rotavirus, hepatitis A virus and small
round structured viruses in water samples. Int J Food Microbiol 37:189-199.
Girdwood, R. V. A., and H. V. Smith. 1999. Cryptosporidia, pp 487-497. In R.
Robertson (ed.), Encyclopaedia of Food Microbiology. Academic Press, London.
Girones, R., A. Allard, G. Wadell, and J. Jofre. 1993. Application of PCR to the
detection of adenoviruses in polluted waters. Water Sci Technol 27:235-241.
Girones, R., M. Puig, A. Allard, F. Lucena, G. Wadell, and J. Jofre. 1995. Detection
of adenovirus and enterovirus by PCR amplification in polluted waters. Water Sci
Technol 31:351-357.
Grabow, W. O. K., T. E. Neubrech, C. S. Holtzhausen, and J. Jofre. 1995.
Bacteroides-Fragilis and Escherichia-Coli Bacteriophages - Excretion by Humans
and Animals. Water Sci Technol 31:223-230.
Graham, D. Y., X. Jiang, T. Tanaka, A. R. Opekun, H. P. Madore, and M. K. Estes.
1994. Norwalk virus infection of volunteers: new insights based on improved
assays. J Infect Dis 170:34-43.
Grew, N., R. S. Gohd, J. Arguedas, and J. I. Kato. 1970. Enteroviruses in rural
families and their domestic animals. Am J Epidemiol 91:518-526.
Guendert, D. 2004. Singapore success. Water 21:23(June).
Gunther, F. 2000. Wastewater treatment by greywater separation: Outline for a
biologically based greywater purification plant in Sweden. Ecol Eng 15:139-146.
Haas, C. N. 1996. Acceptable microbial risk. J AWWA 88:8(12).
Haas, C. N., J. B. Rose, and C. P. Gerba. 1999. Quantitative Microbial Risk
Assessment. John Wiley and Sons Inc., New York. 450 p.
Hansen, A., and T.-A. Stenström. 1998. Kartläggning av Giardia och Cryptosporidium
i svenska ytvattentäkter SMI-tryck 118-1998. Solna.
Havelaar, A. H. 1994. Application of HACCP to drinking-water supply. Food
Control 5:145-152.
Havelaar, A. H., S. R. Farrah, J. Jofre, E. Marques, A. Ketratanakul, M. T. Martins,
S. Ohgaki, M. D. Sobsey, and U. Zaiss. 1991. Bacteriophages as model viruses in
water quality control. Water Res 25:529-545.
Havelaar, A. H., K. Furuse, and W. M. Hogeboom. 1986. Bacteriophages and
indicator bacteria in human and animal faeces. J Appl Bacteriol 60:255-262.
Havelaar, A. H., W. M. Pothogeboom, K. Furuse, R. Pot, and M. P. Hormann.
1990. F-specific RNA bacteriophages and sensitive host strains in feces and
wastewater of human and animal origin. J Appl Bacteriol 69:30-37.
Hedberg, C. W., and M. T. Osterholm. 1993. Outbreaks of food-borne and
waterborne viral gastroenteritis. Clin Microbiol Rev 6:199-210.
Herath, G., K. Yamamoto, and T. Urase. 1998. Mechanism of bacterial and viral
transport through microfiltration membranes. Water Sci Technol 38:489-496.
57
Jakob Ottoson
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
Trita LWR PHD
Hernroth, B. 2002. Uptake and fate of pathogenic microbes in the blue mussel,
Mytilis edulis. PhD thesis. Göteborg University, Göteborg.
Hijnen, W. A. M., J. Willemsen-Zwaagstra, P. Hiemstra, G. J. Medema, and D. van
der Kooij. 2000. Removal of sulphite-reducing clostridia spores by full-scale water
treatment processes as a surrogate for protozoan (oo)cyst removal. Water Sci
Technol 44:165-171.
Hills, S., A. Smith, P. Hardy, and R. Birks. 2001. Water recycling at the Millennium
Dome. Water Sci Technol 43:287-294.
Hot, D., O. Legeay, J. Jacques, C. Gantzer, Y. Caudrelier, K. Guyard, M. Lange, and
L. Andreoletti. 2003. Detection of somatic phages, infectious enteroviruses and
enterovirus genomes as indicators of human enteric viral pollution in surface water.
Water Res 37:4703-4710.
Hovi, T., M. Stenvik, and M. Rosenlew. 1996. Relative abundance of enterovirus
serotypes in sewage differs from that in patients: clinical and epidemiological
implications. Epidemiol Infect 116:91-97.
Huang, P., D. Laborde, V. Land, D. Matson, and X. Jiang. 1999. Detection of
human Caliciviruses in seeded water samples by reverse transcription-polymerase
chain reaction (rtPCR). Presented at the International workshop on human
Caliciviruses, Atlanta, GA.
Hutchinson, M., and J. Ridgway. 1977. Microbial aspects of drinking water supplies.
Aquatic microbiology. Academic Press, London. 180 p.
Höglund, C. 2001. Evaluation of microbial health risks associated with the reuse of
source-separated human urine. PhD thesis. Royal Institute of Technology (KTH),
Stockholm.
Höglund, C., N. Ashbolt, and T. A. Stenström. 2002. Microbial risk assessment of
source-separated urine used for reuse in agriculture. Waste Manage Res 20:150-161.
Höller, C. 1988. Quantitative and qualitative studies of Campylobacter in a sewage
treatment plant. Zentralbl Bakteriol Mikrobiol Hyg 185:326-339.
Hörnman, A., R. Rimhanen-Finne, L. Maunula, C. H. von Bonsdorff, N. Torvela,
A. Heikinheimo, and M. L. Hanninen. 2004. Campylobacter spp., Giardia spp.,
Cryptosporidium spp., noroviruses, and indicator organisms in surface water in
southwestern Finland, 2000-2001. Appl Environ Microbiol 70:87-95.
ISO. 1986. ISO 6461/2, Water quality - Detection and enumeration of the spores
of sulphite-reducing anaerobes (clostridia) - Part 2: Method by membrane filtration.
International Standard Organisation, Geneva.
ISO. 2000. ISO 7899-2, Water quality - Detection and enumeration of intestinal
enterococci - Part 2: Membrane filtration method. International Standardisation
Organisation, Geneva.
ISO. 2000. ISO 9308-1, Water quality - Detection and enumeration of coliform
organisms, thermotolerant coliform organisms and presumptive Escherichia coli - Part
1: Membrane filtration method. International Standardisation Organisation,
Geneva.
ISO. 2000. ISO 10705-2, Water quality - Detection and enumeration of
bacteriophages - Part 1: Enumeration of somatic coliphages. International
Standardisation Organisation, Geneva.
ISO. 2000. ISO 10705-2, Water quality - Detection and enumeration of
bacteriophages - Part 2: Enumeration of somatic coliphages. International
Standardisation Organisation, Geneva.
ISO. 2001. ISO 10705-4, Water quality - Detection and enumeration of
bacteriophages - Part 1: Enumeration of bacteriophages infecting Bacteroides fragilis.
International Standardisation Organisation, Geneva.
58
Comparative occurrence of pathogens in wastewater
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
Jacangelo, J., S. Adham, and J. Laine. 1995. Mechanism of Cryptosporidium,
Giardia and MS2 virus removal by MF and UF. J AWWA 87:107-121.
Jacangelo, J. G., P. Loughran, B. Petrik, D. Simpson, and C. McIlroy. 2003.
Removal of enteric viruses and selected microbial indicators by UV irradiation of
secondary effluent. Water Sci Technol 47:193-198.
Jakubowski, W., J. L. Sykora, C. A. Sorber, L. W. Casson, and P. D. Gavaghan.
1991. Determining Giardiasis Prevalence by Examination of Sewage. Water Sci
Technol 24:173-178.
Jefferson, B., A. L. Laine, T. Stephenson, and S. J. Judd. 2001. Advanced biological
unit process for domestic water recycling. Water Sci Technol 43:211-218.
Jenssen, P. D. 2001. Design and performance of ecological sanitation systems in
Norway. Presented at the First International Conference on Ecological Sanitation.,
Nanning, China, November 5-7.
Jeppesen, B. 1996. Domestic greywater re-use: Australia's challenge for the future.
Desal 106:311-315.
Jönsson, H., B. Vinnerås, C. Höglund, T. Stenström, G. Dalhammar, and H.
Kirchman. 2000. Källsorterad humanurin (Recycling source-separated human urine)
2000-1. VA-Forsk.
Katayama, H., A. Shimasaki, and S. Ohgaki. 2002. Development of a virus
concentration method and its application to detection of enterovirus and norwalk
virus from coastal seawater. Appl Environ Microbiol 68:1033-1039.
Kay, D., J. M. Fleisher, R. L. Salomon, F. Jones, M. D. Wyer, A. F. Godfree, Z.
Zelenauch-Jacquotte, and R. Shore. 1994. Predicting likelihood of gastroenteritis
from seabathing: results from randomised exposure. Lancet 344:905-909.
Kayaalp, N. M. 1996. Regulatory framework in South Australia and reclaimed water
reuse options and possibilities. Desal 106:317-322.
Kincaid, D., K. Solomon, and J. Oliphant. 1996. Drop size distributions for
irrigation sprinklers. Transact ASAE 39:839-845.
Koivunen, J., A. Siitonen, and H. Heinonen-Tanski. 2003. Elimination of enteric
bacteria in biological-chemical wastewater treatment and tertiary filtration units.
Water Res 37:690-698.
Koopmans, M., and E. Duizer. 2004. Foodborne viruses: an emerging problem. Int
J Food Microbiol 90:23-41.
Kosek, M., C. Alcantara, A. A. Lima, and R. L. Guerrant. 2001. Cryptosporidiosis:
an update. Lancet Infect Dis 1:262-269.
Kühn, I., G. Allestam, M. Engdahl, and T. A. Stenström. 1997. Biochemical
fingerprinting of coliform bacterial populations - comparisons between polluted
river water and factory effluents. Water Sci Technol 35:343-350.
Kühn, I., L. G. Burman, S. Haeggman, K. Tullus, and B. E. Murray. 1995.
Biochemical fingerprinting compared with ribotyping and pulse-field gel
electrophoresis of DNA for epidemiological typing of enterococci. J Clin Microbiol
33:2812-2817.
Law, I. B. 1996. Rouse Hill - Australia's first full scale domestic non-potable reuse
application. Water Sci Technol 33:71-78.
Leeming, R., A. Ball, N. Ashbolt, and P. Nichols. 1996. Using faecal sterols from
human and animals to distinguish faecal pollution in receiving waters. Water Res
30:2893-2900.
Leeming, R., N. Bate, R. Hewlett, and P. D. Nichols. 1998. Discriminating faecal
pollution: A case study of stormwater entering Port Phillip Bay, Australia. Water Sci
Technol 38:15-22.
59
Jakob Ottoson
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
Trita LWR PHD
Leeming, R., and P. D. Nichols. 1996. Concentrations of coprostanol that
correspond to existing bacterial indicator guideline limits. Water Res 30:2997-3006.
Leeming, R., P. D. Nichols, and N. J. Ashbolt. 1998. Distinguishing sources of
faecal pollution in Australian inland and coastal waters using sterol biomarkers and
microbial faecal indicators. Report 204. Urban Water Research Association of
Australia. Sydney.
Lindgren, S., and S. Grette. 1998. Vatten- och avloppssystem. Ekoporten
Norrköping. (Water and sewerage system. Ekoporten in Norrköping). Trycksak
13303/1998-06.500. SABO Utveckling. Stockholm.
Lodder, W. J., J. Vinjé, R. van de Heide, A. M. de Roda Husman, E. J. T. M.
Leenen, and M. P. G. Koopmans. 1999. Molecular detection of Norwalk-like
caliciviruses in sewage. Appl Environ Microbiol 65:5624-5627.
Lopman, B., H. Vennema, E. Kohli, P. Pothier, A. Sanchez, A. Negredo, J. Buesa,
E. Schreier, M. Reacher, D. Brown, J. Gray, M. Iturriza, C. Gallimore, B. Bottiger,
K. O. Hedlund, M. Torven, C. H. von Bonsdorff, L. Maunula, M. Poljsak-Prijatelj,
J. Zimsek, G. Reuter, G. Szucs, B. Melegh, L. Svennson, Y. van Duijnhoven, and
M. Koopmans. 2004. Increase in viral gastroenteritis outbreaks in Europe and
epidemic spread of new norovirus variant. Lancet 363:682-688.
Lucena, F., A. E. Duran, A. Moron, E. Calderon, C. Campos, C. Gantzer, S.
Skraber, and J. Jofre. 2004. Reduction of bacterial indicators and bacteriophages
infecting faecal bacteria in primary and secondary wastewater treatments. J Appl
Microbiol 97:1069-1076.
Lucena, F., X. Mendez, A. Moron, E. Calderon, C. Campos, A. Guerrero, M.
Cardenas, C. Gantzer, L. Shwartzbrood, S. Skraber, and J. Jofre. 2003. Occurrence
and densities of bacteriophages proposed as indicators and bacterial indicators in
river waters from Europe and South America. J Appl Microbiol 94:808-815.
Malakoff, D. 2002. Water quality. Microbiologists on the trail of polluting bacteria.
Science 295:2352-2353.
Manville, D., E. J. Kleintop, B. J. Miller, E. M. Davis, J. J. Mathewson, and T. D.
Downs. 2001. Significance of indicator bacteria in a regionalized wastewater
treatment plant and receiving waters. Int J Environ Pollut 15:461-466.
Mara, D. D., and J. I. Oragui. 1983. Sorbitol-fermenting bifidobacteria as specific
indicators of human faecal pollution. J Appl Bacteriol 55:349-357.
Marshall, M. M., D. Naumovitz, Y. Ortega, and C. R. Sterling. 1997. Waterborne
protozoan pathogens. Clin Microbiol Rev 10:67-85.
Mayer, C. L., and C. J. Palmer. 1996. Evaluation of PCR, nested PCR, and
fluorescent antibodies for detection of Giardia and Cryptosporidium species in
wastewater. Appl Environ Microbiol 62:2081-2085.
McCuin, R. M., Z. Bukhari, J. Sobrinho, and J. L. Clancy. 2001. Recovery of
Cryptosporidium oocysts and Giardia cysts from source water concentrates using
immunomagnetic separation. J Microbiol Methods 45:69-76.
Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M.
Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States.
Emerg Inf Dis 5:607-625.
Medema, G. J., M. Bahar, and F. M. Schets. 1997. Survival of Cryptosporidium parvum,
Escherichia coli, faecal enterococci and Clostridium perfringens in river water: influence
of temperature and autochthonous microorganism. Water Sci Technol 35:249-252.
Melloul, A., O. Amahmid, L. Hassani, and K. Bouhoum. 2002. Health effect of
human wastes use in agriculture in El Azzouzia (the wastewater spreading area of
Marrakesh city, Morocco). Int J Environ Health Res 12:17-23.
60
Comparative occurrence of pathogens in wastewater
145.
146.
147.
148.
149.
150.
151.
152.
153.
154.
155.
156.
157.
158.
159.
160.
161.
Metcalf, T. G., J. L. Melnick, and M. K. Estes. 1995. Environmental virology: from
detection of virus in sewage and water by isolation to identification by molecular
biology--a trip of over 50 years. Annu Rev Microbiol 49:461-487.
Mezrioui, N., K. Oufdou, and B. Baleux. 1995. Dynamics of non-O1 Vibrio
cholerae and fecal coliforms in experimental stabilization ponds in the arid region
of Marrakesh, Morocco, and the effect of pH, temperature, and sunlight on their
experimental survival. Can J Microbiol 41:489-498.
Midtvedt, A. C., B. Carlstedt-Duke, K. E. Norin, H. Saxerholt, and T. Midtvedt.
1988. Development of five metabolic activities associated with the intestinal
microflora of healthy infants. J Pediatr Gastroenterol Nutr 7:559-567.
Midtvedt, A. C., and T. Midtvedt. 1993. Conversion of cholesterol to coprostanol
by the intestinal microflora during the first two years of human life. J Pediatr
Gastroenterol Nutr 17:161-168.
Midtvedt, T. 1999. Microbial functional activities. pp 79-96. In L. Hansson and R.
Yolken (ed.), Probiotics, other nutritional factors, and intestinal microflora, Nestle
Nutrition Workshop Series, Vol 42. Vevey/Lippincott-Raven Publishers,
Philadelphia.
Morales-Morales, H. A., G. Vidal, J. Olszewski, C. M. Rock, D. Dasgupta, K. H.
Oshima, and G. B. Smith. 2003. Optimization of a reusable hollow-fiber ultrafilter
for simultaneous concentration of enteric bacteria, protozoa, and viruses from
water. Appl Environ Microbiol 69:4098-4102.
Moreno-Espinosa, S., T. Farkas, and X. Jiang. 2004. Human caliciviruses and
pediatric gastroenteritis. Semin Pediatr Infect Dis 15:237-245.
Morgan-Ryan, U. M., A. Fall, L. A. Ward, N. Hijjawi, I. Sulaiman, R. Fayer, R. C.
Thompson, M. Olson, A. Lal, and L. Xiao. 2002. Cryptosporidium hominis n. sp.
(Apicomplexa: Cryptosporidiidae) from Homo sapiens. J Eukaryot Microbiol
49:433-440.
Muniesa, M., and J. Jofre. 2004. Factors influencing the replication of somatic
coliphages in the water environment. Antonie Van Leeuwenhoek 86:65-76.
Murray, C. J., A. D. Lopez, and D. Tamison. 1994. The global burden of disease in
1990: summary results, sensitivity analysis and future directions. Bull World Health
Organ 72:495-509.
Nasser, A. M., N. Zaruk, L. Tenenbaum, and Y. Netzan. 2003. Comparative
survival of Cryptosporidium, coxsackievirus A9 and Escherichia coli in stream,
brackish and sea waters. Water Sci Technol 47:91-96.
Naturvårdsverket. 1985. Avloppsvatteninfiltration, förutsättningar, funktion,
miljökonsekvenser. ISBN 91-7590-221-4. Statens naturvårdsverk, Swedish EPA.
Stockholm.
Naturvårdsverket. 1995. Vad innehåller avlopp från hushåll? Näring och metaller i
urin och fekalier samt i disk-, tvätt-, bad- & duschvatten. (What does household
wastewater contain? Nutrients and metals in urine, faeces and dish-, laundry and
showerwater). Rapport 4425. Naturvårdsverket (Swedish EPA). Stockholm.
Nebra, Y., X. Bonjoch, and A. R. Blanch. 2003. Use of Bifidobacterium dentium as
an indicator of the origin of fecal water pollution. Appl Environ Microbiol 69:26512656.
Nebra, Y., J. Jofre, and A. R. Blanch. 2002. The effect of reducing agents on the
recovery of injured Bifidobacterium cells. J Microbiol Meth 49:247-254.
Nolde, E. 1999. Greywater reuse systems for toilet flushing in multi-storey
buildings - over ten years experience in Berlin. Urban Water 1:275-284.
Norder, H., L. Bjerregaard, L. Magnius, B. Lina, M. Aymard, and J. J. Chomel.
2003. Sequencing of 'untypable' enteroviruses reveals two new types, EV-77 and
61
Jakob Ottoson
162.
163.
164.
165.
166.
167.
168.
169.
170.
171.
172.
173.
174.
175.
176.
177.
178.
179.
Trita LWR PHD
EV-78, within human enterovirus type B and substitutions in the BC loop of the
VP1 protein for known types. J Gen Virol 84:827-836.
Norder, H., L. Bjerregaard, and L. O. Magnius. 2001. Homotypic echoviruses share
aminoterminal VP1 sequence homology applicable for typing. J Med Virol 63:35-44.
Norström, A., K. Larsdotter, L. Gumaelius, J. la Cour Jansen, and G. Dalhammar.
2003. A small scale hydroponics wastewater treatment system under Swedish
conditions. Water Sci Technol 48:161-167.
Ogoshi, M., Y. Suzuki, and T. Asano. 2001. Water reuse in Japan. Water Sci
Technol 43:17-23.
Okun, D. A. 1997. Distributing reclaimed water through dual systems. J AWWA
89:52-64.
On, S. L. 2001. Taxonomy of Campylobacter, Arcobacter, Helicobacter and related
bacteria: current status, future prospects and immediate concerns. Symp Ser Soc
Appl Microbiol:1S-15S.
Ongert, J. E., and H. H. Stibbs. 1986. Isolation and identification of Cryptosporidium
oocysts in river water. Appl Environ Microbiol 53:672-676.
Ottoson, J. 1999. Giardia and Cryptosporidium in Swedish wastewater treatment
plants: a report including occurrence in raw water, risk assessment analysis and
validation of methods. Master Thesis. Stockholm University and SMI, Stockholm.
Ottoson, J. 2001. Giardia and Cryptosporidium in Swedish wastewater treatment
plants. Vatten 57:283-289.
Ottosson, J. 2003. Hygiene aspects of greywater and greywater reuse. Licentiate
Thesis. Royal Institute of Technology, Stockholm.
Palmqvist, H. 2004. Hazardous substances in wastewater management. PhD Thesis.
Luleå University of Technology, Luleå.
Papageorgiou, G. T., L. Moce´-Llivina, C. G. Christodoulou, F. Lucena, D.
Akkelidou, E. Ioannou, and J. Jofre. 1999. A simple methodological approach for
counting and identifying culturable viruses adsorbed to cellulose nitrate membrane
filters. Appl Environ Microbiol 66:194-198.
Payan, A., J. Ebdon, H. Taylor, C. Gantzer, J. Ottoson, G. Papageorgiou, A.
Blanche, F. Lucena, J. Jofre, and M. Muniesa. 2005. A method for the isolation of
suitable bacteriophage host strains of Bacteroides for tracking sources of fecal
pollution in water. Accepted for publication in Appl Environ Microbiol.
Payment, P., and E. Franco. 1993. Clostridium perfringens and somatic coliphages as
indicators of the efficiency of drinking water treatment for viruses and protozoan
cysts. Appl Environ Microbiol 59:2418-2424.
Payment, P., R. Plante, and P. Cejka. 2001. Removal of indicator bacteria, human
enteric viruses, Giardia cysts, and Cryptosporidium oocysts at a large wastewater
primary treatment facility. Can J Microbiol 47:188-193.
Peterson, M. L. 1974. Soiled disposable diapers: a potential source of viruses. Am J
Public Health 64:912-914.
Petterson, S. R., P. F. M. Teunis, and N. J. Ashbolt. 2001. Modeling virus
inactivation on salad crops using microbial count data. Risk Anal 21:1097-1108.
Puig, A., J. Jofre, and R. Araujo. 1997. Bacteriophages infecting various Bacteroides
fragilis strains differ in their capacity to distinguish human from animal faecal
pollution. Water Sci Technol 35:359-362.
Puig, A., N. Queralt, J. Jofre, and R. Araujo. 1999. Diversity of Bacteroides fragilis
strains in their capacity to recover phages from human and animal wastes and from
fecally polluted wastewater. Appl Environ Microbiol 65:1772-1776.
62
Comparative occurrence of pathogens in wastewater
180.
181.
182.
183.
184.
185.
186.
187.
188.
189.
190.
191.
192.
193.
194.
195.
196.
Pusch, D., D. Y. Oh, S. Wolf, R. Dumke, U. Schroter-Bobsin, M. Hohne, I. Roske,
and E. Schreier. 2005. Detection of enteric viruses and bacterial indicators in
German environmental waters. Arch Virol. 150:929-947.
Raangeby, M., P. Johansson, and M. Pernrup. 1996. Removal of faecal coliforms in
a wastewater stabilization pond system in Mindelo, Cape Verde. Water Sci Technol
34:149-57.
Regli, S., J. B. Rose, C. N. Haas, and C. P. Gerba. 1991. Modelling the risk from
Giardia and viruses in drinking water. J AWWA 83:76-84.
Rhodes, M. W., and H. Kator. 1999. Sorbitol-fermenting bifidobacteria as indicators
of diffuse human faecal pollution in estuarine watersheds. J Appl Microbiol 87:528535.
Robertson, L. J., and B. Gjerde. 2001. Factors affecting recovery efficiency in
isolation of Cryptosporidium oocysts and Giardia cysts from vegetables for
standard method development. J Food Prot 64:1799-1805.
Robertson, L. J., C. A. Paton, A. T. Cambell, P. G. Smith, M. H. Jackson, R. A.
Gilmour, S. E. Black, D. A. Stevenson, and H. V. Smith. 2000. Giardia cysts and
Cryptosporidium oocysts at sewage treatment works in Scotland, UK. Water Res
34:2310-2322.
Robertson, L. J., H. V. Smith, and J. E. Ongerth. 1994. Cryptosporidium and
cryptosporidiosis. Part III: Development of water treatment technologies to remove
and inactivate oocysts. Microbiol Europe(Jan/Feb).
Romig, R. R. 1990. The effect of temperature and deposition in reservoir sediments
or water on the viability of cultured Giardia lamblia cysts, as determined by
fluorogenic dyes. Diss Abst Int PtB - Sci Eng 51:82-88.
Rose, J. B., L. J. Dickson, S. R. Farrah, and R. P. Carnahan. 1996. Removal of
pathogenic and indicator microorganisms by a full-scale water reclamation facility.
Water Res 30:2785-2797.
Rose, J. B., and C. P. Gerba. 1991. Assessing potential health risks from viruses and
parasites in reclaimed water in Arizona and Florida, USA. Water Sci Technol
23:2091-2098.
Rose, J. B., G. Sun, C. P. Gerba, and N. A. Sinclair. 1991. Microbial quality and
persistence of of enteric pathogens in graywater from various household sources.
Water Res 25:37-42.
Roseberry, A. M., and D. E. Burmaster. 1992. Lognormal distribution for water
intake by children and adults. Risk Anal 12:99-104.
Santala, E., J. Uotila, G. Zaitsev, R. Alasiurua, R. Tikka, and J. Tengvall. 1998.
Microbial greywater treatment and recycling in an apartment building. Presented at
the AWT-98, Advanced Wastewater Treatment, Recycling and Reuse, Milan, 14-16
September.
Schaper, M., A. E. Duran, and J. Jofre. 2002. Comparative resistance of phage
isolates of four genotypes of F-specific RNA bacteriophages to various inactivation
processes. Appl Environ Microbiol 68:3702-3707.
Schaper, M., and J. Jofre. 2000. Comparison of methods for detecting genotypes of
F-specific RNA bacteriophages and fingerprinting the origin of faecal pollution in
water samples. J Virol Methods 89:1-10.
Schaper, M., J. Jofre, M. Uys, and W. O. Grabow. 2002. Distribution of genotypes
of F-specific RNA bacteriophages in human and non-human sources of faecal
pollution in South Africa and Spain. J Appl Microbiol 92:657-667.
Schmidt, J. E., and B. K. Ahring. 1996. Granular sludge formation in upflow
anaerobic sludge blanket (UASB) reactors. Biotechnol Bioeng 49:229-246.
63
Jakob Ottoson
197.
198.
199.
200.
201.
202.
203.
204.
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
Trita LWR PHD
Schwartzbrod, L. 1995. Effect of human viruses on public health associated with
the use of wastewater and sewage sludge in agriculture and aquaculture. World
Health Organization, Geneva.
Schönning, C., R. Leeming, and T. A. Stenström. 2002. Faecal contamination of
source-separated human urine based on the content of faecal sterols. Water Res
36:1965-1972.
Schönning, C., and T. Stenström. 2004. Guidelines for the safe use of urine and
faeces in ecological sanitation systems. Report 2004-1. Swedish Environmental
Institute. Stockholm.
Scott, T., M. McLaughlin, V. Harwood, N. Chivukula, A. Levine, A. Gennacaro, J.
Lukasik, S. Farrah, and J. Rose. 2002. Reduction of pathogens, indicator bacteria
and alternative indicators by wastewater treatment and reclamation processes.
Water Sci Technol. Water Supply 3:247-252.
Sharma, P., and M. McInerney. 1994. Effect of grain size on bacterial penetration,
reproduction and metabolic activity in porous glass bead chambers. Appl Environ
Microbiol 60:1481-1486.
Shepherd, K. M., and A. P. Wyn-Jones. 1996. An evaluation of methods for the
simultaneous detection of Cryptosporidium oocysts and Giardia cysts from water.
Appl Environ Microbiol 62:1317-1322.
Shuval, H. 2003. Estimating the global burden of thalassogenic diseases: human
infectious diseases caused by wastewater pollution of the marine environment. J
Water Health 1:53-64.
Shuval, H., Y. Lampert, and B. Fattal. 1997. Development of a risk assessment
approach for evaluating wastewater reuse standards for agriculture. Water Sci
Technol 35:15-20.
Simmons, O. D., 3rd, M. D. Sobsey, C. D. Heaney, F. W. Schaefer, 3rd, and D. S.
Francy. 2001. Concentration and detection of Cryptosporidium oocysts in surface
water samples by method 1622 using ultrafiltration and capsule filtration. Appl
Environ Microbiol 67:1123-1127.
Simpson, J. M., J. W. Santo Domingo, and D. J. Reasoner. 2002. Microbial source
tracking: state of the science. Environ Sci Technol 36:5279-5288.
Sinton, L., R. Finlay, and D. Hannah. 1998. Distinguishing human from animal
faecal contamination in water: a review. NZJ Mar Freshwater Res 32:323-348.
Skraber, S., B. Gassilloud, and C. Gantzer. 2004. Comparison of coliforms and
coliphages as tools for assessment of viral contamination in river water. Appl
Environ Microbiol 70:3644-3649.
Slovic, P. 1987. Perception of risk. Science 236:280-285.
SLV. 1993. Livsmedelsverkets kungörelse om dricksvatten SLV FS 1993:35.
Uppsala.
SLV. 2002. Campylobacter i kött och vatten (Campylobacter in meat and water).
Rapport 10 - 2002. Livsmedelsverket (National Food Administration). Uppsala.
SMI. 2004. Verksamhetsberättelse och epidemiologisk årsrapport 2003 SMI-tryck
150-2004. Swedish Institute for Infectious Disease Control. Solna.
Smith, J. L. 2002. Campylobacter jejuni infection during pregnancy: long-term
consequences of associated bacteremia, Guillain-Barre syndrome, and reactive
arthritist. J Food Prot 65:696-708.
Stampi, S., O. Varoli, G. de Luca, and F. Zanetti. 1992. Occurrence, removal and
seasonal variation of "thermophilic" campylobacters in a sewage treatment plant in
Italy. Zentralbl Hyg Umweltmed 193:199-210.
64
Comparative occurrence of pathogens in wastewater
215.
216.
217.
218.
219.
220.
221.
222.
223.
224.
225.
226.
227.
228.
229.
230.
231.
232.
233.
Stehr-Green, J. K., L. McCaig, H. M. Remsen, C. S. Rains, M. Fox, and D. D.
Juranek. 1987. Shedding of oocysts in immunocompetent individuals infected with
Cryptosporidium. Am J Trop Med Hyg 36:338-342.
Stenström, T. A. 1985. Infiltration i mark, mikroorganismers transport och
överlevnad. SNV PM 3051. Svenska Naturvårdsverket. Stockholm.
Stenström, T. A. 1994. A review of water-borne outbreaks of gastroenteritis in
Scandinavia. pp 137-143. In A. Golding, N. Noah, and R. Stanwell-Smith (ed.),
Water public and health. Smith-Gordon, London.
Stenström, T.A. 1986. Kommunalt avloppsvatten från hygienisk synpunkt.
(Municipal wastewater from a hygienic point of view). PM 1956. Statens
Naturvårdsverk. Stockholm.
Stenström, T. A., G. Allestam, and K. E. Kihlmark. 1982. Spårorganismförsök i
markbäddsanläggningar, Abisko Reningsverk. Delrapport 6: 1982. Statens
Naturvårdsverk. Stockholm.
Stenström, T. A., F. Boisen, F. Georgsson, K. Lahti, V. Lundh, Y. Andersson, and
K. Ormerod. 1994. Vattenburna infektioner i Norden (Waterborne infections in the
Nordic countries). 1994:585. TemaNord. Köpenhamn.
Stenström, T. A., and A. Carlander. 1999. Mikrobiella risker för smittspridning och
sjukdomsfall - slamspridning och behandling. Rapport 5039. Naturvårdsverket.
Stockholm.
Stenström, T. A., and A. Carlander. 2001. Occurrence and die-off of indicator
organisms in the sediment in two constructed wetlands. Water Sci Technol 44:223230.
Stevik, T. K., G. Ausland, J. F. Hanssen, and P. D. Jenssen. 1999. The influence of
physical and chemical factors on the transport of E. coli through biological filters
for wastewater purification. Water Res 33:3701-3706.
Stevik, T. K., G. Ausland, P. D. Jenssen, and R. L. Siegrist. 1999. Removal of E. coli
during intermittent filtration of wastewater effluent as affected by dosing rate and
media type. Water Res 33:2088-2098.
Stockholm Vatten. 2004. Hammarby Sjöstads reningsverk (The treatment plant of
Hammarby Sjöstad). Stockholm Vatten, Stockholm.
Tamburrini, A., and E. Pozio. 1999. Long-term survival of Cryptosporidium parvum
oocysts in seawater and in experimentally infected mussels (Mytilus galloprovincialis).
Int J Parasitol 29:711-715.
Tartera, C., F. Lucena, and J. Jofre. 1989. Human origin of Bacteroides fragilis
bacteriophages present in the environment. Appl Environ Microbiol 55:2696-2701.
Taylor, H. D. 2003. Surface waters. pp 611-626. In D. D. Mara and N. J. Horan
(ed.), Water and wastewater microbiology. Academic press, London.
Teunis, P., K. Takumi, and K. Shinagawa. 2004. Dose response for infection by
Escherichia coli O157:H7 from outbreak data. Risk Anal 24:401-407.
Teunis, P. F. M., and A. H. Havelaar. 2000. The Beta Poisson dose-response model
is not a single hit model. Risk Anal 20:513-520.
Teunis, P. F. M., O. G. van der Heijden, J. W. B. van der Giessen, and A. H.
Havelaar. 1996. The dose-response relation in human volunteers for gastrointestinal pathogens 28450002. RIVM. Bilthoven.
Thomas, C., D. J. Hill, and M. Mabey. 1999. Evaluation of the effect of temperature
and nutrients on the survival of Campylobacter spp. in water microcosms. J Appl
Microbiol 86:1024-1032.
Todd, J., and B. Josephson. 1996. The design of living technologies for waste
treatment. Ecol Eng 6:109-136.
65
Jakob Ottoson
234.
235.
236.
237.
238.
239.
240.
241.
242.
243.
244.
245.
246.
247.
248.
249.
250.
251.
252.
253.
Trita LWR PHD
TOFPSW 2005, 25/04/2005. Tracking the origin of faecal pollution in surface
water.
Anicet
Blanche
http://www.ub.es/microbiologia/TOFPSW_archives/TOFPSW1.html. [Online.]
Trujillo, S., A. A Hanson, W. Zachritz, and R. Chacey. 1998. Potential for greywater
recycle and reuse in New Mexico. New Mexico J Sci 38:293-313.
Ueda, T., and N. J. Horan. 2000. Fate of indigenous bacteriophage in a membrane
bioreactor. Water Res 34:2151-2159.
Uhnoo, I., G. Wadell, L. Svensson, E. Olding-Stenkvist, E. Ekwall, and R. Mölby.
1986. Aetiology and epidemiology of acute gastro-enteritis in Swedish children. J
Infect 13:73-89.
Urban Water. 2001. Progress Report ISSN 1650-3791. Chalmers University of
Technology. Gothenburg.
Urban Water. 2005. Microbial Risk Assessment (MRA) Tool. Report 2005:7.
Chalmers University of Technology. Gothenburg.
US EPA. 1984. Health effects criteria for fresh recreational waters. EPA 600/1-84004. US EPA. Washington DC.
US EPA. 2001. Low-pressure membrane filtration for pathogen removal:
Application, implementation, and regulatory issues 815-C-01-001. United States
Environmental Protection Agency, Office of ground water and drinking water,
standards and risk management division, techical support center. US EPA.
Washington DC.
US EPA. 2001. Method 1623. Cryptosporidium and Giardia in Water by
Filtration/IMS/IFA. US EPA. Washington DC.
US EPA. 2004. Guidelines for water reuse. US EPA. Washington DC.
Wait, D. A., and M. D. Sobsey. 2001. Comparative survival of enteric viruses and
bacteria in Atlantic Ocean seawater. Water Sci Technol 43:139-142.
Walker, J., H. Leclerc, and J. M. Foliguet. 1980. Experimental study of
bacteriophage removal from the lagoon basin. Can J Microbiol 26:27-32.
Walker, R. W., C. K. Wun, and W. Litsky. 1982. Coprostanol as an indicator of fecal
pollution. CRC Crit. Rev. Environ. Contr. 10:91-112.
van Voorthuizen, E., N. Ashbolt, and A. Schäfer. 2001. Role of hydrophobic and
electrostatic interactions for initial enteric virus retention by MF membranes. J
Membr Sci 194:69-79.
Ward, R. L., D. I. Bernstein, E. C. Young, J. R. Sherwood, D. R. Knowlton, and G.
M. Schiff. 1986. Human rotavirus in volunteers: Determination of infectious dose
and serological response to infection. J Infect Dis 154:871-880.
Vesey, G., J. S. Slade, M. Byrne, K. Shepherd, P. J. Dennis, and C. R. Fricker. 1993.
Routine monitoring of Cryptosporidium oocysts in water using flow cytometry. J Appl
Bacteriol 75:87-90.
Vesey, G., J. S. Slade, M. Byrne, K. Shepherd, and C. R. Fricker. 1993. A new
method for the concentration of Cryptosporidum oocysts from water. J Appl
Bacteriol 75:82-86.
Westrell, T. 1997. Microbiological reduction efficiency of the sewage treatment
system in the ecovillage Understenshöjden, with special examination of the UV
unit. Master Thesis. Stockholm University and SMI.
Westrell, T. 2004. Microbial risk assessment and its implications for risk
management in urban water systems. PhD Thesis. University of Linköping,
Linköping.
Westrell, T., Y. Andersson, and T. Stenström. 2005. Drinking water consumption
patterns in Sweden. Accepted for publication in J Water Health.
66
Comparative occurrence of pathogens in wastewater
254.
255.
256.
257.
258.
259.
260.
261.
262.
263.
Westrell, T., C. Schönning, T. A. Stenström, and N. J. Ashbolt. 2004. QMRA
(quantitative microbial risk assessment) and HACCP (hazard analysis and critical
control points) for management of pathogens in wastewater and sewage sludge
treatment and reuse. Water Sci Technol 50:23-30.
Wheeler, J., D. Sethi, J. Cowden, P. Wall, L. Rodrigues, D. Tompkins, M. Hudson,
and P. Roderick. 1999. Study of infectious intestinal disease in England: rates in the
community, presenting to general practise, and reported to national surveillance.
Brit Med J 318:1046-1050.
WHO. 1989. Guidlines for the safe use of wastewater and excreta in agriculture and
aquaculture Technical report series 778. World Health Organization. Geneva.
WHO. 2004. Guidelines for drinking water quality, 3rd edition. World Health
Organization. Geneva.
Wiedenmann, A. 2003. Control of recreational water quality in the European union:
From epidemiology to legislation. Epidemiol 14:S122-S122.
Villareal, E. 2005. Beneficial use of stormwater. PhD Thesis. Lund Institute of
Technology, Lund.
Vinje, J., and M. P. Koopmans. 1996. Molecular detection and epidemiology of
small round-structured viruses in outbreaks of gastroenteritis in the Netherlands. J
Infect Dis 174:610-615.
Wolfe, M. S. 1992. Giardiasis. Clin Microbiol Rev 5:93-100.
Xiao, L., K. Alderisio, J. Limor, M. Royer, and A. A. Lal. 2000. Identification of
species and sources of Cryptosporidium oocysts in storm waters with a smallsubunit rRNA-based diagnostic and genotyping tool. Appl Environ Microbiol
66:5492-5498.
Yates, M. V., C. P. Gerba, and L. M. Kelley. 1985. Virus persistence in
groundwater. Appl Environ Microbiol 49:778-781.
67