READ 3 s2.0 B9780128191668001754 Main

From Groundwater to Drinking Water – Current Approaches for Microbial
Monitoring and Risk Assessment in Porous Aquifers

Julia Derxa,i, Rita Linkeb,i, Domenico Saviob,c,i, Monica Emelkod, Philip Schmidtd, Jack Schijvene,f, Liping Pangg, Regina
Sommerh,i, Margaret Stevensona,i, Harold van den Bergf, Saskia Rutjesf, Andreas H Farnleitnerb,c,i, and Alfred Paul Blaschkea,i,
a
TU Wien, Institute of Hydraulic Engineering and Water Resources Management, Vienna, Austria; bTU Wien, Institute of Chemical, Environmental
and Bioscience Engineering, Vienna, Austria; cKarl Landsteiner University of Health Sciences, Department of Pharmacology, Physiology and
Microbiology, Division Water Quality and Health, Krems, Austria; dUniversity of Waterloo, Department of Civil and Environmental Engineering,
Waterloo, ON, Canada; eUniversity of Utrecht, Department of Earth Sciences, Utrecht, The Netherlands; fNational Institute for Public Health and
the Environment (RIVM), Utrecht, The Netherlands; gInstitute of Environmental Science and Research Ltd., Christchurch, New Zealand; hMedical
University of Vienna, Institute for Hygiene and Applied Immunology, Vienna, Austria; iICC Water & Health, www.waterandhealth, Austria
© 2022 Elsevier Inc. All rights reserved.
This article was reviewed for the Encyclopedia of Inland Waters, Second Edition by Section Editor Christian Griebler.
The WHO approach for safe drinking water in the 21st century 581
How do microbial pathogens move through groundwater? 582
Observational methods in surface water and groundwater 582
Hydrological and physical-chemical parameters and monitoring design 582
Microbiological characterization of surface water 583
Fecal indicators and genetic fecal markers 583
Pathogens 584
Microbiological characterization of groundwater 585
Molecular tools for the characterization and vulnerability assessment of groundwater 585
Natural and artificial pathogen surrogates to characterize transport in groundwater 585
Observational data uncertainty 586
Modelling pathogen transport in surface water and groundwater 588
Quantitative microbial risk assessment (QMRA) and required treatment barriers 588
Conclusion 590
Current knowledge gaps 590
Further reading 590
References 590
Glossary
Aquifer An aquifer is an underground layer of permeable materials (gravel, sand or silt) or rock that can contain or transmit
groundwater.
Dose response model In the QMRA framework, dose response models are mathematical functions that describe the
relationship between the risk of infection, illness or death (response) given a known dose of a pathogen for specific pathogens,
transmission routes, and hosts (dose) (QMRA Wiki).
Epifluorescence microscopy (EFM) A specialized type of optical microscopy that uses fluorescence and allows to detect
fluorescently labeled cells or objects.
Fecal indicator A group of microorganisms that indicates the presence of fecal contamination such as E. coli. They indicate that
pathogens may be present.
First-order decay A decrease in quantity at a rate proportional to the current value. First-order microbial decay rates depend on
the microbial concentration and follow exponential decay (www.wikipedia.org).
Flow cytometry (FCM) A method that allows gathering statistical data on individual cell populations from a heterogenous cell
sample by optically analyzing single cells that have been separated prior to analysis.
Fluorescence-activated cell sorting (FACS) A special type of flow cytometry allowing the analysis and physical separation,
sorting and isolation of subpopulations of a heterogeneous cell sample. To do so, cells have to be stained with fluorescence
dyes prior to analysis and sorting.
Groundwater protection zones Zones around the drinking water abstraction sites in which any type of pollution must be
prevented and land use is not permitted. In these zones, groundwater sources are at a certain level of risk from contamination.
The closer the contamination source, the greater is the risk (Environment Agency, 2021).
580 Encyclopedia of Inland Waters, 2nd edition https://doi.org/10.1016/B978-0-12-819166-8.00175-4

STRUCTURES AND FUNCTIONS OF INLAND WATERS - GROUNDWATER | Microbial Monitoring and Risk Assessment 581
High-throughput DNA-sequencing (HTS) DNA sequencing technologies that are capable of processing massive amounts of
DNA sequences in parallel, enabling researchers to decipher full genomes or to resolve the composition of complex
microbiomes. HTS methods are often synonymously referred to as “Next-generation sequencing.”
Microbiome The microbiome is the sum of microbes and their genomic elements in a particular environment.
Natural isotopes Differing ratios of the oxygen and hydrogen isotopes exist that constitute the water molecule. Isotopes such
as deuterium, tritium or 18O are hydrogen or oxygen atoms that have a different number of neutrons in their nuclei. The water
molecules carry distinct fingerprints based on differing proportions of the hydrogen and oxygen isotopes.
Quantitative Microbial Risk Assessment (QMRA) A tool for estimating human health risks from exposure to pathogens via
any route such as food, water, air, and the environment.
Surrogate A particle or solute that serves as a substitute for pathogens for studying pathogen treatment and transport
characteristics, experimental tracer tests, analytical and numerical transport models and QMRA.
The WHO approach for safe drinking water in the 21st century
To achieve safety of drinking-water, the World Health Organization (WHO) recommend a risk assessment and risk management
approach called water safety plan (WSP) (Davison et al., 2005). This approach includes all steps in a water supply from catchment to
tap and includes water resource protection (Fig. 1). To implement such a plan is an interdisciplinary challenge, requiring the
expertise from many different fields. The national strategies for protecting drinking water serve as input to the WSP. In many
countries, groundwater protection in the vicinity of drinking water wells are based on a water travel time of 50–60 days from the
borders of the protection zones. These travel times were historically adopted based on die-off rates of E.coli (Knorr, 1937). The
parameter E.coli is an important representative of the standard fecal indicator bacteria (SFIB) used in water regulations worldwide
e.g. (EU, 2020). Many pathogens, especially viral and protozoan representatives, can survive considerably longer than bacteria.
However, information about their quantitative occurrence in water resources is very limited. Alternative water quality indicators and
methods to estimate the pathogen transport and required treatment have therefore been proposed for a long time (Gyllenberg et al.,
1960). The conceptual framework for implementing the WHO guidelines for safe drinking water include health-based targets, the
WSP, and a system of independent surveillance that verifies the proper operation (WHO, 2017). The health-based targets can be
defined either as maximum tolerable infection risk (maximum number of infections per person and year) or as maximum
tolerable illness risks which are often expressed as disability adjusted life years (DALYs). Models and planning tools are needed
for quantifying the potential sources and transport pathways of fecal-borne pathogens and the pathogen treatment reduction by soil
Fig. 1 Microbial fate and transport processes in a risk assessment for water supply according to water safety plans (WSP) (Davison et al., 2005).
582 STRUCTURES AND FUNCTIONS OF INLAND WATERS - GROUNDWATER | Microbial Monitoring and Risk Assessment
passage and subsequent disinfection steps. Such tools can be also useful for investigating the impact of future changes such as in
climate or wastewater management and thus support the sustainable water safety management (Demeter et al., 2021; Okaali et al.,
2021). Climate change, together with population growth, increasing urbanization and increased water demand, is expected to
present an additional burden to water supply. Increasing insight into impacts of global change phenomena and required adaptation
initiated some shift towards global and climate resilient water safety planning (WHO, 2017).
How do microbial pathogens move through groundwater?
When microbial pathogens enter subsurface environments from land discharges of human and animal fecal matter, how do they
move through the underground water systems called aquifers? Pathogens are bio-colloids ranging in size from nanometers to
micrometers. Once pathogens enter aquifers, they can be transported with the groundwater through pores in the aquifer media that
are larger than them and they disperse towards low concentration areas, they can attach to the surfaces of the aquifer matrix, to the
air-water interface, or they can be strained out by pores that are smaller than them. Some immobilized pathogens can remobilize if
the water flow or its chemistry changes. Pathogens become inactivated and non-infectious over time.
Depending on the degree of aquifer heterogeneity and the rates of attachment and detachment processes, the average speed of
pathogen transport in groundwater can be faster or slower than that of non-reactive solutes which do not adsorb or desorb (Sen,
2011; Ginn et al., 2002). In heterogeneous aquifers, pathogens tend to move through larger pores and cracks where water moves
more quickly, because they are excluded from small pores where water moves relatively slowly. Thus, if their attachment rate is slow
or their detachment rate is fast, the average speed of pathogen transport in heterogeneous aquifer media with large pores, for
example, alluvial gravel, karst and fractured rock aquifers, can be faster than that of non-reactive solutes (Schijven et al., 2017; Sen,
2011; Ginn et al., 2002). Such aquifers are therefore particularly vulnerable to contamination such as from fecal-borne pathogens
(Savio et al., 2021). Conversely, the average speed of pathogen transport in groundwater can be slower than that of non-reactive
solutes in uniform fine-grained aquifers, such as coastal and river sand, or if the pathogens are very strongly attached to the aquifer
matrix.
In summary, the attachment and detachment to and from soil particles, straining, detachment, and inactivation are the most
important transport mechanisms of colloids on top of the convection and dispersion (Sen, 2011; Ginn et al., 2002). The degree of
pathogen attenuation and transport in groundwater is largely determined by the properties of the aquifer media and pathogens,
groundwater chemistry (such as redox conditions, cf. Section “Modelling pathogen transport in surface water and groundwater”)
and flow conditions. Pathogen transport and survival in groundwater are favored by coarse-grained heterogeneous porous media
with preferential flow paths, highly negatively charged and hydrophilic pathogens, high flow velocities, high pH values, low ionic
strengths, high dissolved organic carbon levels, low dissolved oxygen levels and low groundwater temperatures (Schijven et al.,
2017; Sen, 2011; Ginn et al., 2002). These environmental factors enhance pathogen transport by increasing the quantities of
pathogens travelling through and/or by releasing pathogens previously retained by the aquifer media.
Pathogens can associate with other colloids (such as clays, metal oxides and organic matter) in effluent and environmental
waters, and, thus, they can hitch rides on other colloids when they travel through aquifers. Colloids play a dual role in pathogen
transport. Mobile colloids that travel through large pores and cracks increase the pathogen transport velocity and facilitate the
transport of colloid-bound pathogens. Conversely, immobile colloids with pathogens bound to them reduce the quantities of
pathogens travelling through the aquifer (Sen, 2011; Ginn et al., 2002). Which of these two processes dominate depends on the
number of large pores available for the transport of colloids and colloid-bound pathogens. Understanding how microbial
pathogens move through groundwater and the dual role of colloids in transporting pathogens have important implications for
risk analysis and remediating contaminated sites.
Observational methods in surface water and groundwater

Hydrological and physical-chemical parameters and monitoring design
The groundwater flow, and its recharge via precipitation or adjacent river water strongly control the microbial transport. It is
therefore important to monitor the levels and the geochemical status of the groundwater and river water from a network of
observation wells surrounding the area of interest. Loggers recording continuous datasets are immensely valuable in this respect, as
the conditions commonly vary along rivers. Standard physical and chemical parameters include e.g. the temperature, pH, electric
conductivity (EC), and oxygen content (Stuyfzand et al., 2006). The monitoring design is aligned to the type of drinking water
abstraction site. Managed aquifer recharge systems, such as riverbank filtration, are among the most widely used treatment systems
for drinking water production (Regnery et al., 2017). In case of riverbank filtration, the flow mechanisms are either driven by
pumping or are periodically changing such as when the flow direction reverses during receding flood events (Derx et al., 2010).
Suspended particles tend to infiltrate deep into the sediments of the gravelly river bed and riverbank. A clogging layer can drastically
reduce the groundwater flow velocity and strongly enhance the removal of pathogens (Blaschke et al., 2003). It is therefore
important to monitor the groundwater quantity and quality in wells that are situated within the first meters of soil passage from
the river towards the well. Fig. 2 shows an example of such a well setup at the Danube riverbank, where piezometer pipes are
installed underneath the river bed and in the riverbank.
Fig. 2 Groundwater piezometers located underneath the Danube river bed and bank in Vienna, Austria, to monitor the groundwater quantity and quality in wells
that are situated within the first meters of soil passage from the river towards the well.
Microbiological characterization of surface water

Fecal indicators and genetic fecal markers
The concept of fecal indicators was developed over a hundred years ago and is still successfully applied worldwide and are an
obligate part of guidelines and legislation (WHO, 2017; European Union, 2020). Required properties for a suitable fecal indicator
are that it is universally present in large numbers in the feces of humans and animals, readily detected by simple methods, not able
to proliferate in natural waters and soils as well as having a persistence in water and resistance to water treatment comparable to
other waterborne pathogens. However, there is no one single fecal indicator which can serve as a surrogate for all different
waterborne pathogens. Depending on the aim of the investigation, a set of indicators has to be selected. The most important
fecal indicator organisms (FIO) used as microbiological parameters are described below. Standard detection methods for these
indicators are based on cultivation, meaning that the organisms detected are capable of proliferation, which is the prerequisite of
pathogens to be infective.
Escherichia coli (E. coli) and enterococci bacteria normally live in the intestines of humans and animals. Most E. coli are harmless
and actually are an important part of a healthy human intestinal tract. However, some E. coli strains are pathogenic, meaning they
can cause illness, either diarrhea or illness outside of the intestinal tract. E. coli and enterococci have proven sensitive and reliable
general fecal indicators in alpine mountainous water resources (Farnleitner et al., 2010). E. coli is the most prominent member of
the coliform bacteria group. In contrast, coliform bacteria cannot be seen as reliable indicator for fecal contamination, as they
represent a very heterogeneous group, also including environmental species, which are even capable to proliferate in water
(Stevens, 2003).
Clostridium perfringens has been successfully used to detect fecal pollution in the aquatic environment in various geographic areas
(Fujioka and Shizumura, 1985). C. perfringens is able to form spores that allow prolonged detection of fecal contamination in the
environment. In contrast, the fecal indicators Escherichia coli and enterococci are considered to be short-lived in aquatic systems and
not representative for the behavior of the more resistant pathogens, including viruses and parasites. Monitoring of spores of
C. perfringens has shown to be useful to assess the water quality during the stages of natural and technical water treatment. The spores
can serve as surrogates for protozoan cysts and oocysts of Giardia lamblia and Cryptosporidium (Ryzinska-Paier et al., 2011; Vierheilig
et al., 2013; Payment, 1991).
(Bacterio)phages are viruses that infect prokaryotic cells. Phages are more persistent and resistant than fecal bacteria and can
serve as surrogates for human pathogenic viruses (Mesquita and Emelko, 2012). Coliphages (somatic and F-specific coliphages) are
fecal viral indicators using E. coli as host (Jofre et al., 2016). A European study on surface bathing water quality revealed the high
abundance of somatic coliphages and its usefulness as fecal viral indicator (Contreras-Coll et al., 2002). Regulatory authorities are
therefore beginning to implement coliphages as parameter of water quality (European Union, 2020).
The enumeration of cultivation-based fecal indicator bacteria described above still represents the gold standard for the
investigation of fecal pollution of water sources. However, due to their ubiquity in both human and animal fecal sources, source
determination without further analysis is not possible (Farnleitner et al., 2010; Ishii and Sadowsky, 2008; Ahmed et al., 2015).
Today, molecular techniques are available which enabled the development of methods for source identification (e.g. discrimination
between human and non-human sources), referred to as Microbial Source Tracking (MST) (Hagedorn et al., 2011). Many of these
approaches are library-independent DNA-based methods, most commonly targeting the 16S rRNA gene of highly abundant
host-associated intestinal bacteria (Hagedorn et al., 2011). These library-independent methods are opposed to library-dependent
methods which require the development of a so-called catchment-specific library, i.e. a dataset of characteristics of the target
microorganisms from relevant source hosts (Mott and Smith, 2011).
Detection of MST parameters usually involves enrichment of cells by filtration, followed by DNA extraction and finally detection
of the specific DNA target sequences by (quantitative) polymerase chain reaction (PCR). Currently, a plethora of methods targeting
fecal contamination of human and animal sources, including traditionally used fecal indicator bacteria, are available (e.g., Reischer
et al., 2013; Bernhard and Field, 2000; Boehm et al., 2013; Wuertz et al., 2011; Roslev and Bukh, 2011; Harwood et al., 2017).
Besides of methods targeting host-associated intestinal bacteria, also methods targeting viruses as MST tools become increasingly
available (e.g., Rusiñol et al., 2014; Rusiñol et al., 2016; Ahmed et al., 2020; Farkas et al., 2020). Finally, also molecular methods
targeting mitochondrial DNA of both humans and different animal species are emerging. These methods have the advantage of
enabling the assignment of a contamination source with certainty since the mitochondrial DNA sequence is unique to a species
(Schill and Mathes, 2008; Malla and Haramoto, 2020). However, there is contradictory information here if meat consumption,
either by humans or predators, can cause carryover of mitochondrial DNA from the consumed meat source possibly resulting in
false positive signals (Schill and Mathes, 2008; Malla and Haramoto, 2020).
The use of such molecular markers is a good complement to the use of traditional fecal indicators, especially when applied to
surface waters and waste- or stormwater impacted areas where genetic marker concentrations are high and sample volumes of
200–500 mL are sufficient for reliable detection and quantification. By the application of such MST methods, for example, the
dominance of human fecal impact in the River Danube could be proven (Kirschner et al., 2017). In ground water environments
however, much lower concentrations of these genetic fecal markers are observed. Here, tracer tests can aid in estimating the fate and
transport (cf. section “Natural and artificial pathogen surrogates to characterize transport in groundwater“).
No matter to which water source they are applied to, it is necessary to properly evaluate the methods used. Most of these
procedures have been developed and used in temperate latitudes. However, it could be shown that geographical differences in the
composition of the gut flora of humans and animal groups exist (Reischer et al., 2013). A direct transfer of such procedures to other
regions might therefore not always be straightforward and have to be evaluated (e.g. temperate zones versus tropics) (Reischer et al.,
2013; Boehm et al., 2013; Odagiri et al., 2015; Mayer et al., 2018).
Pathogens
Fecal-borne pathogens are pathogens that are introduced into aquatic systems by animal or human point or non-point fecal
pollution, e.g. by discharge of human sewage or washing in by rain events (GWPP, 2015), and transmitted by water to the
human (or animal) target. Infectious fecal-borne pathogens include bacteria and viruses as well as protozoa and helminths
(Leclerc et al., 2002; Ramírez-Castillo et al., 2015; Aw, 2018). Considering the large number of different fecal-borne pathogens
known today, it is practically impossible to target all of them to assess the microbiological water quality and estimate risks.
Unless there is no specific reason (e.g. epidemic occurring), pathogen detection is therefore focused to so-called reference
pathogens. These are representative members of each of the microbial groups mentioned (Ashbolt, 2015; Stalder et al.,
2011b). Ideally, a reference pathogen provides a conservative estimate of risk by representing a bad to worst-case combination
of high occurrence, high concentration and long survival, low removal and/or inactivation during treatment, and high
pathogenicity. Detection methods, preferably standardized cultivation-based or equivalent methods capable of testing for
viability and infectiousness, must be further available (Health Canada, 2019). Reference pathogens include norovirus,
rotavirus, adenovirus, Cryptosporidium sp., Giardia lamblia, Campylobacter spp., Salmonella enterica spp., and E. coli O157:H7
(USEPA, 2010; Soller et al., 2010; Stalder et al., 2011a,b). Sound water safety management measures have to be based on
carefully and appropriately chosen reference pathogens. The aim is to ensure sufficient protection from fecal-borne pathogen
infections and diseases based on conservative assumptions.
In contrast to host-associated intestinal bacteria used for MST applications, fecal pathogen concentrations are often very low in
water resources simply due to dilution effects. However, they show higher persistence and resistance to environmental conditions
which is why they may be transferred into groundwater systems and might be further distributed (cf. Section “Quantitative
microbial risk assessment (QMRA) and required treatment barriers”). For pathogen detection their very low abundance often
represents a great challenge. Large sample volumes and complex procedures for target enrichment and concentration might precede
detection (Ramírez-Castillo et al., 2015; Bofill-Mas and Rusiñol, 2020). Although culture-dependent detection methods are the gold
standard for detection and identification of many bacterial and viral pathogens (Haramoto et al., 2018; Maurya et al., 2020; McKee
and Cruz, 2021), molecular methods such as (quantitative) PCR methods are increasingly used. Culture-based methods are often
time consuming. However, they provide evidence of the presence of viable/infectious pathogens (Girones et al., 2010). Because
pathogens can also exist in the environment in a viable but nonculturable state, culture-dependent methods can lead to an
underestimation of risk (Girones et al., 2010; Law et al., 2015). Molecular methods, on the other hand, cannot distinguish between
viable and nonviable pathogens, so these methods tend to overestimate potential risk (Girones et al., 2010; Knight et al., 2013;
Farkas et al., 2020; McKee and Cruz, 2021). Therefore, a combination of different methods like integrated cell culture and molecular
detection can be useful, as for example for the detection of viruses (Farnleitner et al., 2018; Savio et al., 2018). Representative
estimates of the reference pathogen concentration in the considered water resource are an essential prerequisite to support QMRA
(cf. Section “Modelling pathogen transport in surface water and groundwater”).
Microbiological characterization of groundwater

Molecular tools for the characterization and vulnerability assessment of groundwater
As discussed in the above sections, the direct detection of pathogens in groundwater is challenging in many respects. The most
important limitations are related to low pathogen concentrations in the environment, insufficient sensitivity and high limits of
detection (LOD) of the respective methods – constraining their usefulness for health risk assessments. To overcome these
limitations and to address more specific issues related to water quality, efforts were made to discover additional indicators.
In this context, the natural water microbiome has been considered a valuable, but hidden treasure since many years.
Even the most pristine ground waters harbor an abundant and often highly diverse microbiome (Karwautz and Griebler, 2021).
These microbial communities comprise autochthonous and – depending on the vulnerability and type of the respective aquifer –
also allochthonous, transient microorganisms (Savio et al., 2018; Farnleitner et al., 2005; Pronk et al., 2009; Sinreich et al., 2014;
Smith et al., 2018). These microorganisms carry an intrinsic potential to serve as natural indicators (or ‘biomarkers’) for character-
izing aquifers or studying the water quality. For example, autochthonous surface water bacteria could serve as alternative tracers for
tracking infiltrated surface water through the aquifer (Harvey et al., 2018; Savio et al., 2019). On top of that, bacteria may serve as
indicators for the general ecological water quality and stability of the aquifer (Korbel et al., 2017; Fillinger et al., 2019). Recent
progress in molecular and optical methods such as High-throughput DNA-sequencing (HTS) or fluorescence-activated cell sorting
(Bonner et al., 1972) (from hereon abbreviated as FACS) will fuel the efforts towards discovering novel indicators (Tan et al., 2015;
Vartoukian et al., 2010; Kurm et al., 2019; Ben-Amor et al., 2005; Cichocki et al., 2020).
The analyses of the compositional community dynamics are advanced in terms of morphology and physiology. Epifluorescence
microscopy (EFM) and automated flow cytometry (FCM) are additionally used to study the dynamics of the bulk microbial
community abundance at high temporal resolution (Besmer et al., 2014, 2016). The total prokaryotic numbers – also referred to as total
cell counts (TCC) – for example can reveal valuable information on the stability and vulnerability of a (ground) water resource
(Sinreich et al., 2014; Fillinger et al., 2019; Retter et al., 2021). TCC have therefore potential as early warning parameter for water
quality deficiencies (Besmer et al., 2014, 2016).
Novel HTS methods have proven extremely valuable by opening the ‘black boxes’ of the diverse aquatic microbiomes and aiding
in identifying novel indicator microorganisms (Newton et al., 2013; Mclellan and Eren, 2014; Tan et al., 2015). In combination
with FACS, specific subpopulations can even be physically separated prior to genetic analyses based on morphological or
physiological characteristics or taxonomy (Müller and Nebe-von-Caron, 2010). The search for an alternative indicator generally
starts by looking for a specific taxonomic group (e.g. genus, species or strain) whose abundance (or physiology) is directly related to
temporary changes in hydrological conditions or physico-chemical water quality (Savio et al., 2019). Since the advent of HTS
methods, such correlations between the dynamics of specific taxonomic groups and environmental parameters can be identified
(Ramette, 2007; Buttigieg and Ramette, 2014; Savio et al., 2019; Tan et al., 2015). The causality of these correlations, however, is in
most cases not understood.
Once identified, novel alternative indicator organisms can be detected by a variety of molecular or optical methods, including
DNA-based methods such as qPCR and digital PCR (dPCR) or EFM and FCM in combination with fluorescence in situ hybridization
(FISH). Different taxonomic groups can inform about different water quality aspects such as potential (fecal) pollution-related
health risks, the deterioration of the ecosystem status (e.g. toxigenic algae/cyanobacteria), or the influence of surface water in an
aquifer (e.g. prokaryotic or eukaryotic photoautotrophs).
Natural and artificial pathogen surrogates to characterize transport in groundwater

Pathogen removal in groundwater can be investigated either by monitoring indicator organisms which are present in the
infiltrating water (as a consequence of fecal pollution, Section “Fecal indicators and genetic fecal markers”) or by conducting
classical injection tracer tests. Indicator organisms are part of the infiltrating water mass and often occur in moderate concentrations
(Table 2). Common surrogates include bacteriophages for viruses (e.g. enteroviruses in the Netherlands), E. coli for bacteria such as
Campylobacter, and anaerobic spores such as C. perfringens, which are commonly used as indicators for protozoa such as Cryptospo-
ridium and Giardia (Section “Fecal indicators and genetic fecal markers”). Thus far, MST markers have rarely been used for
groundwater protection studies. This is because, in groundwater environments, these genetic fecal markers occur in low concentra-
tions due to low persistence and resistance of the target organisms to environmental conditions and due to filtering effects of
soils (e.g. retention due to sorption on soil particles) (Krauss and Griebler, 2011). MST markers, however, have been used for the
characterization of karst springs (Diston et al., 2018; Reischer et al., 2011). Therefore, it could be demonstrated for the first time that
ruminants, rather than human sources, were the cause of fecal contamination (Reischer et al., 2008, 2011; Savio et al., 2018).
To study microbial transport in groundwater, tracers can be added on purpose in high concentrations (artificial surrogates or lab
strains of fecal indicators, Table 2). The classical tracer test in the field is with a conservative salt or dye tracer that is injected
upstream of a well or piezometer where samples are taken. Tracer tests can be performed with a natural gradient (between
piezometers) or with a forced gradient created by a pumping well. From the breakthrough curve of tracer concentration over
time, one can calculate different parameters analytically or numerically, such as the dispersivity, pore velocity and hydraulic
conductivity (cf. Section “How do microbial pathogens move through groundwater?”). A tracer can also be introduced into a
surface water body and monitored in groundwater, to determine the extent of surface-groundwater interaction.
Examples of common solute tracers are the dyes uranine (also known as fluorescein) (Buzady et al., 2006) and rhodamine WT
(Pang et al., 1998), or chloride and bromide salts (Harvey et al., 1989), as well as naturally occurring isotopes such as deuterium,
tritium or 18O (Mali et al., 2007). A disadvantage of the dye tracers is that transport may be retarded due to sorption, depending on
the dye and type of aquifer matrix (Kasnavia et al., 1999). High seasonal variation in the background concentration of chloride may
confuse results; conversely, low natural background concentrations of bromide make it a favorable tracer. Synthetic DNA tracers are
promising as a way to detect connections between surface water and groundwater (Ptak et al., 2004; Pang et al., 2017). These tracers
can be applied as free molecules or encapsulated in microparticles, and therefore acting like colloids, which usually travel faster than
a conservative tracer through heterogeneous aquifers (Pang et al., 2020).
Potential pathogen transport in groundwater can be estimated or predicted using tracer tests with pathogen surrogates and
numerical modelling (Schijven et al., 1999) (cf. Section “Modelling pathogen transport in surface water and groundwater”). A
pathogen surrogate is usually a synthetic colloid or another microorganism that is not dangerous to humans or the environment
and comparable in its behavior to pathogens. Aspects that need to be considered when searching for an appropriate surrogate are:
inactivation, adsorption to soil material, chemical reactions (geological chemistry), size, surface electrical charge, hydrophobicity,
and biological interactions.
Due to their small size, bacteriophages, such as MS2, PRD1 and jX174, are commonly used as surrogates for viruses (Schijven
and Hassanizadeh, 2000). Spores, such as Bacillus subtilis (Pang et al., 2005) and Clostridium perfringens (Hijnen et al., 2005), as well
as harmless Escherichia coli strains (Sinton et al., 1997), have acted as surrogates for pathogenic bacteria (Table 1). Examples of
synthetic colloids are silica or polystyrene nanoparticles or microspheres that have a similar density to a pathogenic microorganism.
These surrogate particles can have carboxyl surface functional groups (Bales et al., 1995) or other coatings to mimic the surface
charge of the pathogen in question. For example, it has been found that glycoprotein-coated microspheres imitated the transport of
the protozoa Cryptosporidium parvum better than carboxylated microspheres of the same size (Pang et al., 2012; Stevenson et al.,
2015). Likewise, DNA-labeled-protein-coated silica nanoparticles were found to have outperformed MS2 in mimicking rotavirus
attenuation in coastal sand (Pang et al., 2014), organosilane coated silica sand (Farkas et al., 2015) and silt loam over gravels dosed
with onsite wastewater (Clemens et al., 2020). The transport of pathogen surrogates in the field can be compared to column tests in
the laboratory, using the aquifer material taken from drilled boreholes. In this way, it is possible to compare the transport of the
surrogate and corresponding pathogenic microorganism in the column (usually 10–100 cm one-dimensional transport) and the
surrogate transport observed at the field site (Hijnen et al., 2005; Weaver et al., 2013).
Observational data uncertainty

A critical aspect of interpreting quantitative microbial data is that concentrations of pathogens or other target microorganisms in
water are not measured directly; they are estimated from raw data such as analyzed sample volumes (that may reflect only a portion
of a collected sample) and microscopic counts, colony counts, arrays of presence-absence data, or numbers of PCR cycles. This
estimation can be relatively straightforward for enumeration-based methods, and it may involve more elaborate statistical methods
such as most probable number (MPN) estimation or standard curves in detection-based methods and quantitative molecular
methods, respectively (Cochran, 1950; Rutledge and Côté, 2003). Microbial concentration values reported by commercial labora-
tories or in research investigation are often perceived as exact measurements. In fact, they are estimates and therefore reflect some
degree of uncertainty that may impact or even mislead interpretation or decision-making.
Quantitative microbial methods inherently feature random variation (i.e., error), as may be evidenced by repeated analyses
yielding variable results—even when samples and methods are controlled to be as identical as possible. When evaluating the
microorganism concentration in a source, random error adds variability to observed data and conversely adds uncertainty to
concentration estimates obtained from raw data. As a result, all microbial concentration estimates are uncertain, though some are
more precise than others. Selecting a sample size leading to observation of at least 10 microorganisms where possible is a useful
target for reducing this uncertainty (Emelko et al., 2008). Of course, lower counts remain valid but they do yield concentration
estimates with greater uncertainty. Ignoring the effect of random error in microbial concentrations presumes that it is trivially small
in relation to the spatial or temporal variability among samples (Emelko et al., 2019), which can be quite incorrect in some
environmental analyses.
Beyond basic uncertainty, concentration estimates also may be biased if losses in the analytical methodology are ignored or
incorrectly quantified. For some types of methods (e.g., plating, MPN) and/or microorganisms, it is common to implicitly presume
that there are no losses. For others (e.g., enumeration-based methods for waterborne protozoa), substantial losses are widely
recognized (Reynolds et al., 1999; U.S. EPA, 2005). Analytical recovery studies are designed to quantify these losses. Recovery is
evaluated by spiking samples with known quantities of microorganisms and may vary among water matrices and labs. Analyses in
matrix water must account for background quantities of target microorganisms using split samples or spiked microorganisms that
are distinguishable from native microorganisms. Correcting concentration estimates through division by recovered fractions is a
common practice, but this may introduce substantial bias when recovery values are small (Schmidt et al., 2013).
In settings with low numbers of target microorganisms, non-detects may be prevalent. It is common practice to regard
non-detects as concentrations below some limit of detection, but this practice is fundamentally flawed and is known to lead to
biased data analysis (Chik et al., 2018). Instead, the result may be reported as a concentration estimate of zero, recognizing that all
concentration estimates—not only non-detects—are uncertain. Availability of raw data is particularly critical to the interpretation of
non-detect data. Limits of detection in microbiology may have value in communicating the capacity of a method to detect low
concentrations, but they are not thresholds partitioning reliable and unreliable data.
Table 1 Overview of different groups of organisms and exemplary naming of explicit procedures (cultivation - and molecular based techniques) for assessing microbiological water quality.
Organism Target organism/ Fecal specificity Often used detection and example Additional characteristic Example referencesa
STRUCTURES AND FUNCTIONS OF INLAND WATERS - GROUNDWATER | Microbial Monitoring and Risk Assessment
group population methods
Bacteria Escherichia coli general Cultivation, qPCR methods (e.g., Indicates recent pollution event, not persistent in the environment, ISO (2001a) and Chern et al. (2011)
EC23S857, uidA405, rodA984) might occur autochthonous and replicate in the environment under
certain conditions
Enterococci general Cultivation, qPCR methods (e.g., ENT) Indicates recent pollution event, ENT more persistent than E. coli ISO (2000b) and Haugland et al. (2005)
Clostridium general Cultivation, qPCR methods Produces persistent spores, very conservative indicator ISO (2013), Chern et al. (2009), and
perfringens Maheux et al. (2013)
intestinal Human associated HF183/BacR287, BacHum, Lachno3, Detects human associated gut bacteria, more persistent compared to Green et al. (2014), Kildare et al. (2007),
Bacteroidetes, E. coli, detects DNA and cannot distinguish between living and dead and Feng et al. (2018)
Firmicutes organisms or free environmental DNA
intestinal Ruminant/cattle associated BacR, BacCow, BoBac Detects ruminant/cattle associated gut bacteria, more persistent Reischer et al. (2006), Kildare et al.
Bacteroidetes, compared to E. coli, detects DNA and cannot distinguish between (2007), and Layton et al. (2006)
Firmicutes living and dead organisms or free environmental DNA
intestinal Pig associated Pig2Bac Detects pig associated gut bacteria, more persistent compared to Mieszkin et al. (2009)
Bacteroidetes, E. coli, detects DNA and cannot distinguish between living and dead
Firmicutes organisms or free environmental DNA
Viruses Bacteriophages, general, mainly cultivation based methods, More persistent in the environment compared to bacteria, culture ISO (2000a, 2001b), USEPA (2001a, b),
somatic, some qPCR assays recently methods detect viable particles, qPCR assays detect DNA only and European Committee for
F-RNA, developed (e.g., CPQ056) cannot distinguish between living and dead particles or free Standardization (1995), Jebri et al.
Bacteroides environmental DNA (2017), and Stachler et al. (2018)
fragilis
Virus specific, human NoV, HAdV, HPyV, More persistent in the environment compared to bacteria, culture Rusiñol et al. (2014), Kirs et al. (2016),
methods detect viable/infectious particles, qPCR assays detect DNA Ahmed and Harwood (2017), and
only and cannot distinguish between infectious and dead particles or Farkas et al. (2020)
free environmental DNA
Specific, Ruminant/cattle BAdV, BPyV More persistent in the environment compared to bacteria, culture Wong and Xagoraraki (2010), Hundesa
methods detect viable/infectious particles, qPCR assays detect DNA et al. (2010), Ahmed and Harwood
only and cannot distinguish between infectious and dead particles or (2017), and Farkas et al. (2020)
Specific, pig PAdV More persistent in the environment compared to bacteria, culture Hundesa et al. (2009), Ahmed and
methods detect viable/infectious particles, qPCR assays detect DNA Harwood (2017), and Farkas et al.
only and cannot distinguish between infectious and dead particles or (2020)
Enteroviruses human Cell culture Mainly poliovirus and Coxsackievirus B serotypes are detected, they CEN (2005)
replicate and form plaques in BGM cells, only infectious viruses can
be analyzed
Protozoa Giardia lamblia general Direct laboratory methods mainly, Very persistent in the environment, form resistant cysts ISO (2006) and Guy et al. (2003)
some qPCR assays
Cryptosporidium general Direct laboratory methods mainly, Very persistent in the environment, form resistant cysts ISO (2006), USEPA (2005), Fayer et al.
sp. some qPCR assays (2000), and Hassan et al. (2020)
Abbreviations: BAdV: bovine adenovirus, BPyV: bovine polyomavirus, HAdV: human adenovirus, HPyV: human polyomavirus, NoV: norovirus, PAdV: pig adenvirus.
a
Please note that only a few examples of methods currently used are given here. Furthermore, MST procedures for sources other than humans, cattle, and swine also exist in the literature.
587
Table 2 Concentration ranges of representative culturable or synthetic microbial indicators naturally occurring in water and artificial pathogen surrogates that
can be injected into groundwater during tracer tests.
Fecal pollution indicators available in the environment Laboratory strains of indicators or synthetic surrogates
Escherichia coli n.d./100 ml (groundwater) – Phages, bacteria and spores (e.g. Bacillus subtilis), Total amount per tracer test (number per
108/100 ml (raw sewage) synthetic particles, synthetic DNA tracers amount injected): 1010–1012
Clostridium perfringens n.d./100 ml (groundwater) –
105/100 ml (raw sewage)
Somatic coliphages n.d./100 ml (groundwater) –
107/100 ml (raw sewage)
Modelling pathogen transport in surface water and groundwater
To encompass the full pathway of pathogens from the fecal sources towards the drinking water well, it requires models for the
microbial overland transport, infiltration via the unsaturated zone into groundwater, in-river transport, and transport via
river-groundwater exchange (Bradford et al., 2013; Drummond et al., 2018). Microbial transport models in rivers or lakes generally
account for mixing, dilution and microbial decay (Derx et al., 2016; Demeter et al., 2021). Flow velocity is described by the Manning
formula for open channel flow (Manning, 1891) or by hydrodynamic modelling (Tolouei et al., 2019). To validate such models,
data of fecal indicators, host-associated genetic fecal markers or pathogens in the wastewater source and surface water can be used
(Derx et al., 2016).
The actual pathogen treatment reduction by attenuation towards the abstraction wells can be estimated using groundwater flow
and microbial transport models. As for conservative tracers and solutes, the microbial transport in groundwater is described
numerically by the advection – dispersion equation (ADE). Advection refers to the movement of solutes as a function of
groundwater flow velocity. Diffusion is the result of the thermal motion of individual molecules or particles. Mechanical or
hydrodynamic dispersion results from solutes or microbial particles moving at different rates than the average velocity in
groundwater. This causes spreading of the contaminant plume in the porous media. Dispersion is a function of the dispersivity
coefficient and groundwater flow velocity. The dispersivity relates to the aquifer grain size distribution and generally increases with
the travel distance (Gelhar et al., 1992). Microbial subsurface transport is most commonly coupled with attachment – detachment
and first-order decay (Schijven et al., 2002). Attachment and detachment lead to immobilization and remobilization of microbial
particles to and from the solid matrix. In order to account for heterogeneity of the geochemical soil properties, two-site attachment –
detachment rate models can be used in groundwater (Schijven et al., 2002; Yang et al., 2019). Dual-porosity or dual-permeability
approaches can be used to account for the enhanced microbial transport in groundwater via preferential flow paths (Šimůnek
et al., 2003).
The attachment of microbial particles can be described by the colloid filtration theory (Yao et al., 1971), as the product of two
probabilities: the collector and the collision efficiency. The former is the probability that microbial particles collide with soil
particles when approaching the soil particle surface. The latter is the probability that they attach during the collision (Auckenthaler
and Huggenberger, 2003). The virus and microbial attachment and detachment rates are influenced by the soil geochemical
properties and changes in hydraulic flow conditions, as shown in soil laboratory column experiments (Sadeghi et al., 2011).
If applying the removal rates derived from laboratory experiments at the field scale, this can lead to strong underestimations of the
risks from groundwater contamination. It was therefore proposed to compare laboratory with field-site experiments (Stevenson
et al., 2021). Some of the uncertain parameters shown to affect microbial transport at pore scale may become irrelevant in complex
hydrological and porous systems at field scale. For instance, bacterial inactivation, straining, size of the bacterial cells, and the
permeability of the clogging layer with bed sediments (influencing the residence times) were found to determine the bacteria
transport during riverbank filtration at field scale (Knabe et al., 2021). The hydraulic and microbial transport parameters are
commonly obtained from fitting the models to the observed breakthrough concentrations at the observation wells. At a more
general level, studies relied on literature values, e.g. for providing recommendation guidelines. Blaschke et al. (2016) used a wide
range of reported hydraulic and virus transport parameters to model the virus removal by soil passage in the unsaturated and
saturated soil zone. They modelled safe setback distances for drinking water at on-site disposal sites of biologically treated
wastewater for different scenarios and types of aquifer media.
Quantitative microbial risk assessment (QMRA) and required treatment barriers
This chapter describes QMRA for drinking water, and specifically, how it is conducted in the Netherlands, because, since 2005, the
Dutch drinking water companies are obliged by law to demonstrate that less than 1 per 10,000 persons per year (health based target;
WHO (2011)) acquire an infection by consumption of unboiled drinking water (Schijven et al., 2011). To that aim, a guideline
document (Ilent, 2020) and computational tools (Schijven et al., 2010, 2011) have been developed. The drinking water
companies – producing drinking water from surface water and vulnerable groundwater sites – conduct QMRA of index pathogens
(enterovirus, Campylobacter, Cryptosporidium, Giardia, and occasionally other relevant pathogens) based on the reduction of indicator
organisms (bacteriophages for enterovirus, E. coli for Campylobacter and anaerobic spores for the (oo)cysts of Cryptosporidium and
Giardia) to determine treatment efficiency (Ilent, 2020). The risk of infection is estimated as follows:
Cdrw ¼ Craw =S ∗Z (1)

D ¼ Cdrw ∗V (2)
P inf ¼ f ðD, drparsÞ (3)
Where Cdrw is the drinking water concentration, Craw is the raw source water concentration of the index pathogen, S is the sensitivity
(or recovery efficiency) of the detection method and Z is the fraction of pathogens able to pass treatment. Log10Z is the log removal
by treatment. Commonly Z entails a sequence of treatment steps (multibarrier principle). Indicator organisms (cf. Section “Natural
and artificial pathogen surrogates to characterize transport in groundwater”) are used as proxy for removal of the corresponding
index pathogens. Dose D is the number of pathogens ingested by consuming V ml of unboiled drinking water. Pinf is the infection
risk for a given D as a function of dose response with parameters drpars.
The QMRA is repeated every 4 years. QMRAspot is the computational tool that is available to conduct the QMRA, entailing fitting
of the data to distributions, computing the risk using Monte Carlo simulations and dose response models including variability and
uncertainty (Schijven et al., 2011).
The drinking water companies - producing drinking water from groundwater - have to protect the groundwater source.
If adequately protected, additional treatment of the collected water to remove pathogens is not necessary. Basically, Z in Eq. (1)
has to be sufficiently small so that the probability of infection will not exceed the health based target. For a sufficiently small Z, the
setback distance between a fecal source of contamination and the groundwater production well must be large enough to enable
removal of pathogens by attachment to soil particles and/or inactivation and die-off of pathogens. The QMRA for groundwater is
mainly identical to the QMRA for surface waters, although limited to virus as index pathogen assuming that if virus removal is
sufficient, this applies to other pathogens too (Schijven et al., 2017). Even though this assumption is valid for homogeneous sandy
aquifers commonly found in the Netherlands, bacteria and protozoa may be likewise of concern in heterogeneous soil and
sediment. As described in the guideline document (Ilent, 2020), the risk analysis for a groundwater production site assumes no
problems with wellhead integrity. It also states that vulnerable groundwater aquifers require a QMRA to demonstrate compliance to
the legal infection risk of less than 1 in 10,000. To assess vulnerability, groundwater production sites are ranked from high to low in
terms of vulnerability, followed by QMRA of the most vulnerable sites. The vulnerability of a site (geohydrologic system) for
pathogens is determined by the intrinsic properties that affect survival of an index pathogen on its way from the contamination
source to the production well. A site is less vulnerable if confining (clay) layers are present, permeability is lower, the aquifer is
thicker and deeper, the well screen is deeper, the sand is finer, there is less heterogeneity and preferential flow paths are absent,
anisotropy is higher, the vadose zone is thicker and the groundwater has a higher ionic strength, lower pH, higher temperature, and
conditions are anoxic (Ilent, 2020; Schijven and Hassanizadeh, 2000).
Based on QMRA, the size of the protection zone is determined using a standard contamination scenario. A risk assessment is
needed if a contamination source is found within the protection zone. A monitoring program of 4 years is required if the
determined protection zone is larger than the protected zone. If fecal contamination is detected, the source needs to be found
and, if possible, to be removed. The QMRA required to determine the protection zone is based on a standard contamination
scenario of 100 enteroviruses per liter leak at Qleak ¼ 1 m3/day from a sewage pipe, and the following conditions of the aquifer: a
groundwater temperature of 10 C–12 C and under anoxic conditions virus inactivation is 0.01 per day on a log10-scale and
attachment of virus is conservative (sticking efficiency 10−5), resulting in a removal rate coefficient l ¼ 0.030/day (0.013 log10/day).
If (sub)oxic, then virus attachment is 100 times higher and l ¼ 1.1/day (0.49 log10/day). The virus source concentration must be
reduced by 8.5 log10 for an infection risk of <1 per 10,000 persons per year. Log10 reduction by dilution of the source by the
pumping at the production well, Qwell, and by dilution of the water from a single production well by the water from the complete
well field, Qtotal, are included. Virus removal in the unsaturated zone is set at 0.5 log10 per meter unsaturated zone. Following the
standard scenario, a protection zone can be computed using a numerical hydrologic model that includes first-order decay (l), or
using a hydrological model to compute groundwater travel times to determine the size of the zone that provides enough travel time
for the required virus removal, or by using computational model QMRAwell, with an empirical formula specifically developed for
this purpose (Schijven et al., 2010). The required travel time T can be computed as follows:
T ¼ ½8:5 − log 10 ðQwell =Qleak Þ − log 10 ðQtotal =Qwell Þ − log 10 unsat = log 10 l (4)
The computational model QMRAcatch (Schijven et al., 2015; Demeter et al., 2021; Derx et al., 2021) calculates the microbial
concentrations in rivers and the needed pathogen reduction treatment during drinking water production. It allows integrating data
of fecal indicators, MST markers and reference pathogens and to evaluate future scenarios to pro-actively determine robust and
sustainable water safety management options. Please also refer to the overview documents on QMRA by Teunis and Schijven (2019)
and WHO (2016).
Conclusion
A number of analytical and modelling methods exist to estimate suitable and required catchment protection and drinking water
treatment measures. The pathogen fate and transport processes in rivers and groundwater towards drinking water wells are subject of
ongoing research. A good understanding of these processes is needed to model the fecal microbial flow and transport and predict
the concentrations in raw water resources. Based on health-related water quality targets one can determine the status of drinking
water safety and for deriving required infection barriers.
Current knowledge gaps
• Interaction between microbial pathogens and diverse environmental surfaces, a multitude of physical, chemical, and microbi-
ological factors, and (emerging) contaminants in aquifers
• Upscaling tracer transport from the column scale to the field scale
• Tracer testing in heterogeneous material with preferential flow paths
• Identification of taxonomic groups from the water microbiome that can be used such as for the assessment of fecal pollution
sources, or as indicators for biostability, eutrophication, biofilm-formation, pipe corrosion, and surface water infiltration
• Dose response models for specific genotypes of pathogens such as protozoan cysts and oocysts of Giardia lamblia and
Cryptosporidium spp.
• Standardization of approaches for reporting non-detects and associated data needs to quantify uncertainty in microbial
count data
• Extension of quantitative description of uncertainty in microbiological data to next generation sequencing approaches
Further reading
• Schijven et al. (2017) provides an overview of tracers and surrogates for studying the transport of pathogenic microorganisms,
describes the major transport processes in terms of governing equations, lists typical removal rates of microorganisms (mainly
bacteriophages and fecal indicator bacteria) during soil passage.
• Sen (2011) and Ginn et al. (2002) reviewed the processes of pathogenic biocolloidal contaminant transport in saturated and
unsaturated porous media.
• Allaire et al. (2009) reviewed different tracer techniques to quantify preferential flow in soil.
• Chik et al. (2018) reviews approaches and provides guidance regarding approaches for reporting non-detects in microbial
count data.
• The Global Water Pathogen Project (GWPP) is a knowledge resource on pathogens supporting sanitation and safe water and
promoting quantitative information via monitoring of sewage, fecal sludges and freshwaters to inform public health measures
(www.waterpathogens.org).
• The QMRA Wiki (http://qmrawiki.org) is a community portal for current quantitative information and knowledge developed for
QMRA. It is an evolving knowledge repository intended to be the go to reference source for the microbial risk assessment
community.
References
Ahmed W and Harwood V (2017) Human and animal enteric viral markers for tracking the sources of faecal pollution. In: Rose JB and Jiménez-Cisneros B (eds.) Water and Sanitation
for the 21st Century: Health and Microbiological Aspects of Excreta and Wastewater Management. E. Lansing, Mi: Michigan State University. (Global Water Pathogen Project).
UNESCO.
Ahmed W, Gyawali P, and Toze S (2015) Quantitative PCR measurements of Escherichia coli including Shiga toxin-Producing E. coli (STEC) in animal feces and environmental waters.
Environmental Science & Technology 49: 3084–3090.
Ahmed W, Kitajima M, Tandukar S, and Haramoto E (2020) Recycled water safety: Current status of traditional and emerging viral indicators. Current Opinion in Environmental
Science & Health 16: 62–72.
Allaire SE, Roulier S, and Cessna AJ (2009) Quantifying preferential flow in soils: A review of different techniques. Journal of Hydrology 378: 179–204.
Ashbolt NJ (2015) Microbial contamination of drinking water and human health from community water systems. Current Environmental Health Reports 2: 95–106.
Auckenthaler A and Huggenberger P (2003) Pathogene Mikroorganismen im Grund-und Trinkwasser. Birkhäuser Basel.
Aw T (2018) Environmental Aspects and Features of Critical Pathogen Groups. In: Rose JB and Jiménez-Cisneros B (eds.) Water and Sanitation for the 21st Century: Health and
Microbiological Aspects of Excreta and Wastewater Management (Global Water Pathogen Project). Part 1: The Health Hazards of Excreta: Theory and Control. E. Lansing, Mi:
Michigan State University. UNESCO.
Bales RC, Li SM, Maguire KM, Yahya MT, Gerba CP, and Harvey RW (1995) Virus and Bacteria transport in a Sandy aquifer, Cape-Cod, Ma. Ground Water 33: 653–661.
Ben-Amor K, Heilig H, Smidt H, Vaughan EE, Abee T, and de Vos WM (2005) Genetic diversity of viable, injured, and dead fecal Bacteria assessed by fluorescence-activated cell sorting
and 16S rRNA gene analysis. Applied and Environmental Microbiology 71: 4679–4689.
Bernhard AE and Field KG (2000) Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes.
Applied and Environmental Microbiology 66: 1587–1594.
Besmer MD, Weissbrodt DG, Kratochvil BE, Sigrist JA, Weyland MS, and Hammes F (2014) The feasibility of automated online flow cytometry for in-situ monitoring of microbial
dynamics in aquatic ecosystems. Frontiers in Microbiology 5.
Besmer MD, Epting J, Page RM, Sigrist JA, Huggenberger P, and Hammes F (2016) Online flow cytometry reveals microbial dynamics influenced by concurrent natural and operational
events in groundwater used for drinking water treatment. Scientific Reports 6: 38462.
Blaschke AP, Steiner KH, Schmalfuss R, Gutknecht D, and Sengschmitt D (2003) Clogging processes in hyporheic interstices of an impounded river, the Danube at Vienna, Austria.
International Review of Hydrobiology 88: 397–413.
Blaschke AP, Derx J, Zessner M, Kirnbauer R, Kavka G, Strelec H, Farnleitner AH, and Pang L (2016) Setback distances between small biological wastewater treatment systems and
drinking water wells against virus contamination in alluvial aquifers. Science of the Total Environment 573: 278–289.
Boehm AB, van de Werfhorst LC, Griffith JF, Holden PA, Jay JA, Shanks OC, Wang D, and Weisberg SB (2013) Performance of forty-one microbial source tracking methods: A
twenty-seven lab evaluation study. Water Research 47: 6812–6828.
Bofill-Mas S and Rusiñol M (2020) Recent trends on methods for the concentration of viruses from water samples. Current Opinion in Environmental Science & Health 16: 7–13.
Bonner WA, Hulett HR, Sweet RG, and Herzenberg LA (1972) Fluorescence activated cell sorting. The Review of Scientific Instruments 43(3): 404–409.
Bradford SA, Morales VL, Zhang W, Harvey RW, Packman AI, Mohanram A, and Welty C (2013) Transport and fate of microbial pathogens in agricultural settings. Critical Reviews in
Environmental Science and Technology 43: 775–893.
Buttigieg PL and Ramette A (2014) A guide to statistical analysis in microbial ecology: A community-focused, living review of multivariate data analyses. FEMS Microbiology Ecology
90: 543–550.
Buzady A, Erostyak J, and Paal G (2006) Determination of uranine tracer dye from underground water of Mecsek Hill, Hungary. Journal of Biochemical and Biophysical Methods
69: 207–214.
Canada H (2019) Guidance on the use of quantitative microbial risk assessment in drinking water. In: Water and Air Quality Bureau, Healthy Environments and Consumer Safety
Branch, Health Canada, Ottawa, Ontario. (Catalogue No. H144–59/2019E-PDF).
CEN (2005) Water quality - Detection of human enteroviruses by monolayer plaque assay. (EN 14486:2005). Brussels, Belgium: European Standardization Organizations.
Chern EC, Brenner KP, Wymer L, and Haugland RA (2009) Comparison of fecal Indicator Bacteria densities in marine recreational waters by QPCR. Water Quality Exposure and Health
1: 203–214.
Chern EC, Siefring S, Paar J, Doolittle M, and Haugland RA (2011) Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes. Letters in
Applied Microbiology 52: 298–306.
Chik AHS, Schmidt PJ, and Emelko MB (2018) Learning something from nothing: The critical importance of rethinking microbial non-detects. Frontiers in Microbiology 9: 2304.
Cichocki N, Hübschmann T, Schattenberg F, Kerckhof F-M, Overmann J, and Müller S (2020) Bacterial mock communities as standards for reproducible cytometric microbiome
analysis. Nature Protocols 15: 2788–2812.
Clemens H, Pang L, Morgan LK, and Weaver L (2020) Attenuation of rotavirus, MS2 bacteriophage and biomolecule-modified silica nanoparticles in undisturbed silt loam over gravels
dosed with onsite wastewater. Water Research 169: 115272.
Cochran WG (1950) Estimation of bacterial densities by means of the “Most probable number” Biometrics 6: 105–116.
Contreras-Coll N, Lucena F, Mooijman K, Havelaar A, Pierzo V, Boque M, Gawler A, Höller C, Lambiri M, Mirolo G, Moreno B, Niemi M, Sommer R, Valentin B, Wiedenmann A, Young V,
and Jofre J (2002) Occurrence and levels of indicator bacteriophages in bathing waters throughout Europe. Water Research 36: 4963–4974.
Davison A, Howard G, Stevens M, Callan P, Fewtrell L, Deere D, and Bartram J (2005) Water Safety Plans - Managing Drinking Water Quality from Catchment to Consumer. World
Health Organization (WHO). WHO/SDE/WSH/05.06, 235.
Demeter K, Derx J, Komma J, Parajka J, Schijven J, Sommer R, Cervero-Arago S, Lindner G, Zoufal-Hruza CM, Linke R, Savio D, Ixenmaier SK, Kirschner AKT, Kromp H, Blaschke AP,
and Farnleitner AH (2021) Modelling the interplay of future changes and wastewater management measures on the microbiological river water quality considering safe drinking
water production. Science of the Total Environment 768: 144278.
Derx J, Blaschke AP, and Bloschl G (2010) Three-dimensional flow patterns at the river-aquifer interface - a case study at the Danube. Advances in Water Resources 33: 1375–1387.
Derx J, Schijven J, Sommer R, Zoufal-Hruza CM, van Driezum IH, Reischer G, Ixenmaier S, Kirschner A, Frick C, Husman AMD, Farnleitner AH, and Blaschke AP (2016) QMRAcatch:
Human-associated fecal pollution and infection risk modeling for a river/floodplain environment. Journal of Environmental Quality 45: 1205–1214.
Derx J, Demeter K, Linke R, Cervero-Aragó S, Lindner G, Stalder G, Schijven J, Sommer R, Walochnik J, Kirschner AKT, Komma J, Blaschke AP, and Farnleitner AH (2021) Genetic
microbial source Tracking support QMRA modeling for a riverine wetland drinking water resource. Frontiers in Microbiology 12.
Diston D, Robbi R, Baumgartner A, and Felleisen R (2018) Microbial source tracking in highly vulnerable karst drinking water resources. Journal of Water and Health 16(1): 138–149.
https://doi.org/10.2166/wh.2017.215.
Drummond JD, Boano F, Atwill ER, Li X, Harter T, and Packman AI (2018) Cryptosporidium oocyst persistence in agricultural streams -a mobile-immobile model framework
assessment. Scientific Reports 8.
Emelko MB, Schmidt PJ, and Roberson JA (2008) Quantification of uncertainty in microbial data—Reporting and regulatory implications. Journal AWWA 100: 94–104.
Emelko MB, Schmidt PJ, and Borchardt MA (2019) Confirming the need for virus disinfection in municipal subsurface drinking water supplies. Water Research 157: 356–364.
Environment Agency (2021) Guidance Groundwater source protection zones (SPZs). https://www.gov.uk/guidance/groundwater-source-protection-zones-spzs#how-we-defined-the-
zones. (accessed on 19.11.2021).
EU (2020) Directive (EU) 2020/2184 of the European Parliament and of the Council of 16 December 2020 on the quality of water intended for human consumption (recast) (Text with
EEA relevance).
European Committee for Standardization (1995) European Standard EN ISO 10705 - 1. Water Quality—Detection and Enumeration of Bacteriophages - Part 1: Enumeration of
F-specific RNA Bacteriophages. Brussels, Belgium: European Committee for Standardization.
European Union (2020) Directive (EU) 2020/2184 on the Quality of Water Intended for Human Consumption.
Farkas K, Varsani A, and Pang LP (2015) Adsorption of rotavirus, MS2 bacteriophage and surface-modified silica nanoparticles to hydrophobic matter. Food and Environmental Virology
7: 261–268.
Farkas K, Walker DI, Adriaenssens EM, Mcdonald JE, Hillary LS, Malham SK, and Jones DL (2020) Viral indicators for tracking domestic wastewater contamination in the aquatic
environment. Water Research 181: 115926.
Farnleitner AH, Wilhartitz I, Ryzinska G, Kirschner AKT, Stadler H, Burtscher MM, Hornek R, Szewzyk U, Herndl G, and Mach RL (2005) Bacterial dynamics in spring water of alpine
karst aquifers indicates the presence of stable autochthonous microbial endokarst communities. Environmental Microbiology 7: 1248–1259.
Farnleitner AH, Ryzinska-Paier G, Reischer GH, Burtscher MM, Knetsch S, Kirschner AKT, Dirnbock T, Kuschnig G, Mach RL, and Sommer R (2010) Escherichia coli and enterococci
are sensitive and reliable indicators for human, livestock and wildlife faecal pollution in alpine mountainous water resources. Journal of Applied Microbiology 109: 1599–1608.
Farnleitner AH, Savio D, Sommer R, Reischer G, Kirschner A, Zerobin W, and Stadler H (2018) Integrated Strategy to Guide Health-Related Microbial Quality Management at Alpine
Karstic Drinking Water Resources. Cham: Springer International Publishing185–192.
Fayer R, Morgan U, and Upton SJ (2000) Epidemiology of Cryptosporidium: Transmission, detection and identification. International Journal for Parasitology 30: 1305–1322.
Feng S, Bootsma M, Mclellan SL, and Elkins CA (2018) Human-associated Lachnospiraceae genetic markers improve detection of fecal pollution sources in urban waters. Applied and
Environmental Microbiology 84: e00309–e00318.
Fillinger L, Hug K, Trimbach AM, Wang H, Kellermann C, Meyer A, Bendinger B, and Griebler C (2019) The D-A-(C) index: A practical approach towards the microbiological-ecological
monitoring of groundwater ecosystems. Water Research 163.
Fujioka RS and Shizumura LK (1985) Clostridium perfringens, a reliable Indicator of stream water quality. Journal of the Water Pollution Control Federation 57: 986–992.
Gelhar LW, Welty C, and Rehfeldt KR (1992) A critical-review of data on Field-scale dispersion in aquifers. Water Resources Research 28: 1955–1974.
Ginn TR, Wood BD, Nelson KE, Scheibe TD, Murphy EM, and Clement TP (2002) Processes in microbial transport in the natural subsurface. Advances in Water Resources
25: 1017–1042.
Girones R, Ferrús MA, Alonso JL, Rodriguez-Manzano J, Calgua B, de Abreu Corrêa A, Hundesa A, Carratala A, and Bofill-Mas S (2010) Molecular detection of pathogens in water –
The pros and cons of molecular techniques. Water Research 44: 4325–4339.
Green HC, Haugland RA, Varma M, Millen HT, Borchardt MA, Field KG, Walters WA, Knight R, Sivaganesan M, Kelty CA, and Shanks OC (2014) Improved HF183 quantitative real-time
PCR assay for characterization of human fecal pollution in ambient surface water samples. Applied and Environmental Microbiology 80: 3086–3094.
Guy RA, Payment P, Krull UJ, and Horgen PA (2003) Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage. Applied and
Environmental Microbiology 69: 5178–5185.
GWPP (2015) Global Water Pathogens Project (GWPP). UNESCO.https://www.waterpathogens.org.
Gyllenberg H, Niemela S, and Sormunen T (1960) Survival of bifid Bacteria in water as compared with that of coliform Bacteria and enterococci. Applied Microbiology 8: 20–22.
Hagedorn C, Blanch AR, and Harwood VJE (2011) Microbial Source Tracking: Methods, Applications, and Case Studies. Springer Science & Business Media.
Haramoto E, Kitajima M, Hata A, Torrey JR, Masago Y, Sano D, and Katayama H (2018) A review on recent progress in the detection methods and prevalence of human enteric viruses
in water. Water Research 135: 168–186.
Harvey RW, George LH, Smith RL, and Leblanc DR (1989) Transport of microspheres and indigenous Bacteria through a Sandy aquifer - results of natural-gradient and forced-gradient
tracer experiments. Environmental Science & Technology 23: 51–56.
Harvey RW, Underwood JC, Bliznik PA, Leblanc DR, Hull RQ, Mccobb TD, and Bohlke JK (2018) In search of a microbial indicator for “groundwater under the direct influence of surface
water” (GWUDI): Results from studies of flow-through groundwater lakes (kettle ponds) in Cape Cod, Massachusetts. December 01, 2018. H22F-01.
Harwood V, Shanks O, Koraijkic A, Verbyla M, Ahmed W, and Iriate M (2017) General and host-associated bacterial indicators of faecal pollution. In: Rose JB and Jiménez-Cisneros B
(eds.) Water and Sanitation for the 21st Century: Health and Microbiological Aspects of Excreta and Wastewater Management. E. Lansing, Mi: Michigan State University. (Global
Water Pathogen Project). UNESCO.
Hassan EM, Örmeci B, Derosa MC, Dixon BR, Sattar SA, and Iqbal A (2020) A review of Cryptosporidium spp. and their detection in water. Water Science and Technology 83: 1–25.
Haugland RA, Siefring SC, Wymer LJ, Brenner KP, and Dufour AP (2005) Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative
polymerase chain reaction and membrane filter culture analysis. Water Research 39: 559–568.
Hijnen WAM, Brouwer-Hanzens AJ, Charles KJ, and Medema GJ (2005) Transport of MS2 phage, Escherichia coli, Clostridium perfringens, Cryptosporidium parvum and Giardia
intestinalis in a gravel and a sandy soil. Environmental Science & Technology 39: 7860–7868.
Hundesa A, Maluquer de Motes C, Albinana-Gimenez N, Rodriguez-Manzano J, Bofill-Mas S, Suñen E, and Rosina Girones R (2009) Development of a qPCR assay for the quantification
of porcine adenoviruses as an MST tool for swine fecal contamination in the environment. Journal of Virological Methods 158: 130–135.
Hundesa A, Bofill-Mas S, Maluquer de Motes C, Rodriguez-Manzano J, Bach A, Casas M, and Girones R (2010) Development of a quantitative PCR assay for the quantitation of bovine
polyomavirus as a microbial source-tracking tool. Journal of Virological Methods 163: 385–389.
Ilent (2020) Richtsnoer Analyse Microbiologische Veiligheid Drinkwater (AMVD). Inspectie Leefomgeving en Transport, Ministerie van Infrastructuur en Waterstaat.https://www.ilent.nl/
binaries/ilt/documenten/publicaties/2020/11/27/richtsnoer-analyse-microbiologische-veiligheid-drinkwater-amvd/ILT-+Richtsnoer+Analyse+Microbiologische+Veiligheid
+Drinkwater+%28AMVD%29+-.pdf.
Ishii S and Sadowsky MJ (2008) Escherichia coli in the environment: Implications for water quality and human health. Microbes and Environments 23: 101–108.
ISO (2000a) Water Quality - Detection and Enumeration of Bacteriophages - Part 2: Enumeration of Somatic Coliphages. (ISO 10705–2). Geneva, Switzerland: International
Organization of Standardization.
ISO (2000b) Water Quality - Detection and Enumeration of Intestinal Enterococci - Part 2: Membrane Filtration Method (ISO 7899e2: 2000). Geneva, Switzerland: International
Organization of Standardization.
ISO (2001a) Microbiology of Food and Animal Feeding Stuffs - Horizontal Method for the Enumeration of B-Glucoronidase-Positive Escherichia coli - Part 1: Colony-count Technique at
44oC Using Membranes and 5-Bromo-4-Chloro-3-Indolyl B-Glucuronide. (ISO 16649–1:2001 04). Geneva, Switzerland: International Organization of Standardization.
ISO (2001b) Water Quality - Detection and Enumeration of Bacteriophages - Part 4: Enumeration of Bacteriophages Infecting Bacteroides fragilis (ISO 10705e4:2001). Geneva,
Switzerland: International Organisation of Standardisation.
ISO (2006) Water quality — Isolation and identification of Cryptosporidium oocysts and Giardia cysts from water (15553:2006). Geneva, Switzerland: International Organisation of
Standardisation.
ISO (2013) Water Quality - Enumeration of Clostridium perfringens - Method Using Membrane Filtration (ISO 14189). Geneva, Switzerland: International Organisation of
Standardisation.
Jebri S, Muniesa M, and Jofre J (2017) General and host-associated bacteriophage indicators of faecal pollution. In: Rose JB and Jiménez-Cisneros B (eds.) Water and Sanitation for
the 21st Century: Health and Microbiological Aspects of Excreta and Wastewater Management. E. Lansing, Mi: Michigan State University. (Global Water Pathogen Project).
UNESCO.
Jofre J, Lucena F, Blanch AR, and Muniesa M (2016) Coliphages as model organisms in the characterization and management of water resources. Water 8: 199.
Karwautz D and Griebler C (2021) Section 4 Structures and Functions of Inland Waters – Groundwater. In: Tockner K and Mehner T (eds.) Inland Waters, 2nd edn. this issue.
Kasnavia T, Vu D, and Sabatini DA (1999) Fluorescent dye and media properties affecting sorption and tracer selection. Ground Water 37: 376–381.
Kildare BJ, Leutenegger CM, Mcswain BS, Bambic DG, Rajal VB, and Wuertz S (2007) 16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific
fecal Bacteroidales: A Bayesian approach. Water Research 41: 3701–3715.
Kirs M, Caffaro-Filho RA, Wong K, Harwood V, Moravcik P, Fujioka RS, and Elkins BV (2016) Human-associated Bacteroides spp. and human polyomaviruses as microbial source
Tracking markers in Hawaii. Applied and Environmental Microbiology 82: 6757–6767.
Kirschner AKT, Reischer GH, Jakwerth S, Savio D, Ixenmaier S, Toth E, Sommer R, Mach RL, Linke R, Eiler A, Kolarevic S, and Farnleitner AH (2017) Multiparametric monitoring of
microbial faecal pollution reveals the dominance of human contamination along the whole Danube River. Water Research 124: 543–555.
Knabe D, Guadagnini A, Riva M, and Engelhardt I (2021) Uncertainty analysis and identification of key parameters controlling Bacteria transport within a riverbank filtration scenario.
Water Resources Research 57: e2020WR027911.
Knight A, Li D, Uyttendaele M, and Jaykus L-A (2013) A critical review of methods for detecting human noroviruses and predicting their infectivity. Critical Reviews in Microbiology
39: 295–309.
Knorr M (1937) Die Schutzzonenfrage in der Trinkwasserhygiene. Das Gas- und Wasserfach 80(330–334): 350–355.
Korbel K, Chariton A, Stephenson S, Greenfield P, and Hose GC (2017) Wells provide a distorted view of life in the aquifer: Implications for sampling, monitoring and assessment of
groundwater ecosystems. Scientific Reports 7: 40702.
Krauss S and Griebler C (2011) Pathogenic microorganisms and viruses. In: Acatech Materialien Nr. 6, München 2011.
Kurm V, van der Putten WH, and Hol WHG (2019) Cultivation-success of rare soil bacteria is not influenced by incubation time and growth medium. PLoS One 14: e0210073.
Law JW-F, Ab Mutalib N-S, Chan K-G, and Lee L-H (2015) Rapid methods for the detection of foodborne bacterial pathogens: Principles, applications, advantages and limitations.
Frontiers in Microbiology 5.
Layton A, Mckay L, Williams D, Garrett V, Gentry R, and Sayler G (2006) Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of Total,
human, and bovine fecal pollution in water. Applied and Environmental Microbiology 72: 4214–4224.
Leclerc H, Schwartzbrod L, and Dei-Cas E (2002) Microbial agents associated with waterborne diseases. Critical Reviews in Microbiology 28: 371–409.
Maheux AF, Bérubé E, Boudreau DK, Villéger R, Cantin P, Boissinot M, Bissonnette L, and Bergeron MG (2013) Abilities of the mCP agar method and CRENAME alpha toxin-specific
real-time PCR assay to detect Clostridium perfringens spores in drinking water. Applied and Environmental Microbiology 79: 7654–7661.
Mali N, Urbanc J, and Leis A (2007) Tracing of water movement through the unsaturated zone of a coarse gravel aquifer by means of dye and deuterated water. Environmental Geology
51: 1401–1412.
Malla B and Haramoto E (2020) Host-specific mitochondrial DNA markers for tracking the sources of fecal pollution. Current Opinion in Environmental Science & Health 16: 34–46.
Manning R (1891) On the flow of water in open channels and pipes. Transaction of the Institution of Civil Engineers of Ireland 20: 161–207.
Maurya VK, Kumar S, and Saxena SK (2020) Novel Approaches for Detecting Water-Associated Pathogens. In: Saxena SK (ed.) Water-Associated Infectious Diseases. Springer
Singapore: Singapore.
Mayer RE, Reischer GH, Ixenmaier SK, Derx J, Blaschke AP, Ebdon JE, Linke R, Egle L, Ahmed W, Blanch AR, Byamukama D, Savill M, Mushi D, Cristobal HA, Edge TA, Schade MA,
Aslan A, Brooks YM, Sommer R, Masago Y, Sato MI, Taylor HD, Rose JB, Wuertz S, Shanks OC, Piringer H, Mach RL, Savio D, Zessner M, and Farnleitner AH (2018) Global
distribution of human-associated fecal genetic markers in reference samples from six continents. Environmental Science & Technology 52: 5076–5084.
McKee AM and Cruz MA (2021) Microbial and viral indicators of pathogens and human health risks from recreational exposure to waters impaired by fecal contamination. Journal of
Sustainable Water in the Built Environment 7: 03121001.
Mclellan SL and Eren AM (2014) Discovering new indicators of fecal pollution. Trends in Microbiology 22: 697–706.
Mesquita MMF and Emelko MB (2012) Bacteriophages as surrogates for the fate and transport of pathogens in source water and in drinking water treatment processes. In: Kurtboke I
(ed.) Bacteriophages. InTech: Croatia.
Mieszkin S, Furet JP, Corthier G, and Gourmelon M (2009) Estimation of pig fecal contamination in a river catchment by real-time PCR using two pig-specific Bacteroidales 16S rRNA
genetic markers. Applied and Environmental Microbiology 75: 3045–3054.
Mott JG and Smith AM (2011) Library-Dependent Source Tracking Methods. In: Hagedorn C, Blanch A, and Harwood V (eds.) Microbial Source Tracking: Methods, Applications, and
Case Studies. New York, NY: Springer. https://doi.org/10.1007/978-1-4419-9386-1_3,2011.
Müller S and Nebe-von-Caron G (2010) Functional single-cell analyses: Flow cytometry and cell sorting of microbial populations and communities. FEMS Microbiology Reviews
34: 554–587.
Newton RJ, Bootsma MJ, Morrison HG, Sogin ML, and Mclellan SL (2013) A microbial signature approach to identify fecal pollution in the waters off an urbanized coast of Lake
Michigan. Microbial Ecology 65: 1011–1023.
Odagiri M, Schriewer A, Hanley K, Wuertz S, Misra PR, Panigrahi P, and Jenkins MW (2015) Validation of Bacteroidales quantitative PCR assays targeting human and animal fecal
contamination in the public and domestic domains in India. Science of the Total Environment 502: 462–470.
Okaali DA, Kroeze C, Medema G, Burek P, Murphy H, Tumwebaze IK, Rose JB, Verbyla ME, Sewagudde S, and Hofstra N (2021) Modelling rotavirus concentrations in rivers: Assessing
Uganda’s present and future microbial water quality. Water Research 204: 117615.
Pang LP, Close M, and Noonan M (1998) Rhodamine WT and Bacillus subtilis transport through an alluvial gravel aquifer. Ground Water 36: 112–122.
Pang LP, Close M, Goltz M, Noonan M, and Sinton L (2005) Filtration and transport of Bacillus subtilis spores and the F-RNA phage MS2 in a coarse alluvial gravel aquifer: Implications
in the estimation of setback distances. Journal of Contaminant Hydrology 77: 165–194.
Pang LP, Nowostawska U, Weaver L, Hoffman G, Karmacharya A, Skinner A, and Karki N (2012) Biotin- and glycoprotein-coated microspheres: Potential surrogates for studying
filtration of Cryptosporidium parvum in porous media. Environmental Science & Technology 46: 11779–11787.
Pang LP, Farkas K, Bennett G, Varsani A, Easingwood R, Tilley R, Nowostawska U, and Lin SS (2014) Mimicking filtration and transport of rotavirus and adenovirus in sand media using
DNA-labeled, protein-coated silica nanoparticles. Water Research 62: 167–179.
Pang LP, Robson B, Farkas K, Mcgill E, Varsani A, Gillot L, Li JH, and Abraham P (2017) Tracking effluent discharges in undisturbed stony soil and alluvial gravel aquifer using synthetic
DNA tracers. Science of the Total Environment 592: 144–152.
Pang LP, Abeysekara G, Hanning K, Premaratne A, Robson B, Abraham P, Sutton R, Hanson C, Hadfield J, Heiligenthal L, Stone D, Mcbeth K, and Billington C (2020) Water tracking in
surface water, groundwater and soils using free and alginate-chitosan encapsulated synthetic DNA tracers. Water Research 184.
Payment P (1991) Fate of human enteric viruses, coliphages, and Clostridium perfringens during drinking-water treatment. Canadian Journal of Microbiology 37: 154–157.
Pronk M, Goldscheider N, and Zopfi J (2009) Microbial communities in karst groundwater and their potential use for biomonitoring. Hydrogeology Journal 17: 37–48.
Ptak T, Piepenbrink M, and Martac E (2004) Tracer tests for the investigation of heterogeneous porous media and stochastic modelling of flow and transport - A review of some recent
developments. Journal of Hydrology 294: 122–163.
Ramette A (2007) Multivariate analyses in microbial ecology. FEMS Microbiology Ecology 62: 142–160.
Ramírez-Castillo FY, Loera-Muro A, Jacques M, Garneau P, Avelar-González FJ, Harel J, and Guerrero-Barrera AL (2015) Waterborne pathogens: Detection methods and challenges.
Pathogens 4: 307–334.
Regnery J, Gerba CP, Dickenson ERV, and Drewes JE (2017) The importance of key attenuation factors for microbial and chemical contaminants during managed aquifer recharge: A
review. Critical Reviews in Environmental Science and Technology 47: 1409–1452.
Reischer GH, Kasper DC, Steinborn R, Mach RL, and Farnleitner AH (2006) Quantitative PCR method for sensitive detection of ruminant fecal pollution in freshwater and evaluation of
this method in alpine karstic regions. Applied and Environmental Microbiology 72: 5610–5614.
Reischer GH, Haider JM, Sommer R, Stadler H, Keiblinger KM, Hornek R, Zerobin W, Mach RL, and Farnleitner AH (2008) Quantitative microbial faecal source tracking with sampling
guided by hydrological catchment dynamics. Environmental Microbiology 10(10): 2598–2608. https://doi.org/10.1111/j.1462-2920.2008.01682.x.
Reischer GH, Kollanur D, Vierheilig J, Wehrspaun C, Mach RL, Sommer R, Stadler H, and Farnleitner AH (2011) Hypothesis-driven approach for the identification of fecal pollution
sources in water resources. Environmental Science & Technology 45(9): 4038–4045. https://doi.org/10.1021/es103659s.
Reischer GH, Ebdon JE, Bauer JM, Schuster N, Ahmed W, Astrom J, Blanch AR, Bloschl G, Byamukama D, Coakley T, Ferguson C, Goshu G, Ko G, Husman AMD, Mushi D, Poma R,
Pradhan B, Rajal V, Schade MA, Sommer R, Taylor H, Toth EM, Vrajmasu V, Wuertz S, Mach RL, and Farnleitner AH (2013) Performance characteristics of qPCR assays targeting
human- and ruminant-associated Bacteroidetes for microbial source Tracking across sixteen countries on six continents. Environmental Science & Technology 47: 8548–8556.
Retter A, Griebler C, Haas J, Birk S, Stumpp C, Brielmann H, and Fillinger L (2021) Application of the D-A-(C) Index as a Simple Tool for Microbial-Ecological Characterization and
Assessment of Groundwater Ecosystems—a Case Study of the Mur River Valley. Österreichische Wasser- und Abfallwirtschaft: Austria.
Reynolds DT, Slade RB, Sykes NJ, Jonas A, and Fricker CR (1999) Detection of Cryptosporidium oocysts in water: Techniques for generating precise recovery data. Journal of Applied
Microbiology 87: 804–813.
Roslev P and Bukh AS (2011) State of the art molecular markers for fecal pollution source tracking in water. Applied Microbiology and Biotechnology 89: 1341–1355.
Rusiñol M, Fernandez-Cassi X, Hundesa A, Vieira C, Kern A, Eriksson I, Ziros P, Kay D, Miagostovich M, Vargha M, Allard A, Vantarakis A, Wyn-Jones P, Bofill-Mas S, and Girones R
(2014) Application of human and animal viral microbial source tracking tools in fresh and marine waters from five different geographical areas. Water Research 59: 119–129.
Rusiñol M, Moriarty E, Lin S, Bofill-Mas S, and Gilpin B (2016) Human-, ovine-, and bovine-specific viral source Tracking tools to discriminate between the major fecal sources in
agricultural waters. Food and Environmental Virology 8: 34–45.
Rutledge RG and Côté C (2003) Mathematics of quantitative kinetic PCR and the application of standard curves. Nucleic Acids Research 31: e93.
Ryzinska-Paier G, Sommer R, Haider JM, Knetsch S, Frick C, Kirschner AKT, and Farnleitner AH (2011) Acid phosphatase test proves superior to standard phenotypic identification
procedure for Clostridium perfringens strains isolated from water. Journal of Microbiological Methods 87: 189–194.
Sadeghi G, Schijven JF, Behrends T, Hassanizadeh SM, Gerritse J, and Kleingeld PJ (2011) Systematic study of effects of pH and ionic strength on attachment of phage PRD1. Ground
Water 49: 12–19.
Savio D, Stadler P, Reischer GH, Kirschner AKT, Demeter K, Linke R, Blaschke AP, Sommer R, Szewzyk U, Wilhartitz IC, Mach RL, Stadler H, and Farnleitner AH (2018) Opening the
black box of spring water microbiology from alpine karst aquifers to support proactive drinking water resource management. Wiley Interdisciplinary Reviews Water 5.
Savio D, Stadler P, Reischer GH, Demeter K, Linke RB, Blaschke AP, Mach RL, Kirschner AKT, Stadler H, and Farnleitner AH (2019) Spring water of an alpine karst aquifer is dominated
by a taxonomically stable but discharge-responsive Bacteria community. Frontiers in Microbiology 10.
Savio D, Derx J, Lang R-P, Kirschner A, Sommer R, Küsel K, Blaschke AP, and Farnleitner AH (2021) From groundwater to drinking water – Microbiology of Karstic Water Resources.
In: Tockner K and Mehner T (eds.) Inland Waters, 2nd edn. this issue.
Schijven JF and Hassanizadeh SM (2000) Removal of viruses by soil passage: Overview of modeling, processes, and parameters. Critical Reviews in Environmental Science and
Technology 30: 49–127.
Schijven JF, Hoogenboezem W, Hassanizadeh SM, and Peters JH (1999) Modeling removal of bacteriophages MS2 and PRD1 by dune recharge at Castricum, Netherlands. Water
Resources Research 35: 1101–1111.
Schijven JF, Hassanizadeh SM, and de Bruin RHAM (2002) Two-site kinetic modeling of bacteriophages transport through columns of saturated dune sand. Journal of Contaminant
Hydrology 57: 259–279.
Schijven JF, Hassanizadeh SM, and de Roda Husman AM (2010) Vulnerability of unconfined aquifers to virus contamination. Water Research 44: 1170–1181.
Schijven JF, Teunis PPM, Rutjes SA, Bouwknegt M, and Husman AMD (2011) QMRAspot: A tool for quantitative microbial risk assessment from surface water to potable water. Water
Research 45: 5564–5576.
Schijven J, Derx J, Husman AMD, Blaschke AP, and Farnleitner AH (2015) QMRAcatch: Microbial quality simulation of water resources including infection risk assessment. Journal of
Environmental Quality 44: 1491–1502.
Schijven J, Pang L, and Ying GG (2017) Evaluation of subsurface microbial transport using microbial indicators, surrogates and tracers. In: Rose JB and Jiménez-Cisneros B (eds.) Part
2: Indicators and Microbial Source Tracking Markers. E. Lansing, Mi: Michigan State University. UNESCO.
Schill WB and Mathes MV (2008) Real-time PCR detection and quantification of mine potential sources of fecal contamination by analysis of mitochondrial cytochrome b targets.
Environmental Science & Technology 42: 5229–5234.
Schmidt PJ, Emelko MB, and Thompson ME (2013) Analytical recovery of protozoan enumeration methods: Have drinking water QMRA models corrected or created bias? Water
Research 47: 2399–2408.
Sen TK (2011) Processes in pathogenic biocolloidal contaminants transport in saturated and unsaturated porous media: A review. Water, Air, & Soil Pollution 216: 239–256.
Šimůnek J, Jarvis NJ, van Genuchten MT, and Gärdenäs A (2003) Review and comparison of models for describing non-equilibrium and preferential flow and transport in the vadose
zone. Journal of Hydrology 272: 14–35.
Sinreich M, Pronk M, and Kozel R (2014) Microbiological monitoring and classification of karst springs. Environmental Earth Sciences 71: 563–572.
Sinton LW, Finlay RK, Pang L, and Scott DM (1997) Transport of bacteria and bacteriophages in irrigated effluent into and through an alluvial gravel aquifer. Water, Air, and Soil
Pollution 98: 17–42.
Smith HJ, Zelaya AJ, de León KB, Chakraborty R, Elias DA, Hazen TC, Arkin AP, Cunningham AB, and Fields MW (2018) Impact of hydrologic boundaries on microbial planktonic and
biofilm communities in shallow terrestrial subsurface environments. FEMS Microbiology Ecology 94.
Soller JA, Bartrand T, Ashbolt NJ, Ravenscroft J, and Wade TJ (2010) Estimating the primary etiologic agents in recreational freshwaters impacted by human sources of faecal
contamination. Water Research 44: 4736–4747.
Stachler E, Akyon B, de Carvalho NA, Ference C, and Bibby K (2018) Correlation of crAssphage qPCR markers with Culturable and molecular indicators of human fecal pollution in an
impacted urban watershed. Environmental Science & Technology 52: 7505–7512.
Stalder GL, Farnleitner A, Sommer R, Beiglbock C, and Walzer C (2011a) Hazard- and risk based concepts for the assessment of microbiological water quality-part 2. Wiener
Tierärztliche Monatsschrift 98: 54–65.
Stalder GL, Sommer R, Walzer C, Mach RL, Beiglbock C, Blaschke AP, and Farnleitner AH (2011b) Hazard- and risk based concepts for the assessment of microbiological water
quality-part 1. Wiener Tierärztliche Monatsschrift 98: 9–24.
Stevens MA (2003) Recommendations to change the use of coliforms as microbial indicators of drinking water quality/Dr Melita Stevens, Dr Nicholas Ashbolt, Dr David Cunliffe.
Canberra: National Health and Medical Research Council.
Stevenson ME, Blaschke AP, Toze S, Sidhu JP, Ahmed W, Van Driezum IH, Sommer R, Kirschner AK, Cervero-Arago S, Farnleitner AH, and Pang L (2015) Biotin- and
glycoprotein-coated microspheres as surrogates for studying filtration removal of Cryptosporidium parvum in granular limestone aquifer media. Applied and Environmental
Microbiology 13: 4277–4283.
Stevenson M, Oudega T, Lindner G, Scheidl A, Eder A, Strauss P, and Blaschke AP (2021) Upscaling Subsurface Transport from the Column to the Field: A Focus on the Meso-Scale.
EGU General Assembly 2021, online, 19–30 Apr 2021, EGU21-16095.
Stuyfzand PJ, Juhàsz-Holterman MHA, and De Lange WJ (2006) Riverbank filtration in the Netherlands: well fields, clogging and geochemical reactions. In: Hubbs SA (ed.) Riverbank
Filtration Hydrology. The Netherlands: Springer.
Tan B, Ng C, Nshimyimana J, Loh L-L, Gin K, and Thompson J (2015) Next-generation sequencing (NGS) for assessment of microbial water quality: current progress, challenges, and
future opportunities. Frontiers in Microbiology 6.
Teunis P and Schijven J (2019) Generic Guidance to Quantitative Microbial Risk Assessment for Food and Water, Generieke richtlijn voor kwantitatieve microbiologische risicoschatting
voor voedsel en water, (in Dutch). Rijksinstituut voor Volksgezondheid en Milieu RIVM. https://doi.org/10.21945/rivm-2017-0188.
Tolouei S, Dewey R, Snodgrass WJ, Edge TA, Andrews RC, Taghipour M, Prevost M, and Dorner S (2019) Assessing microbial risk through event-based pathogen loading and
hydrodynamic modelling. Science of the Total Environment 693.
U.S. EPA (2005) United States Environmental Protection Agency (U.S. EPA). In: Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA; EPA 815-R-05-002. Office of
Water. Washington, DC.
USEPA (2001a) Method 1601: Detection of Male-specific (F +) and Somatic Coliphage in Water by Two-Step Enrichment Procedure. EPA 821-R-01-030. Washington, DC.
USEPA (2001b) Method 1602: Detection of Male-specific (F +) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure. EPA 821-R-01-029. Washington, DC.
USEPA (2005) Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. EPA 815-R-05-002 Office of Water. Washington, DC: Engineering and Analysis Division.
USEPA (2010) Quantitative Microbial Risk Assessment to Estimate Illness in Freshwater Impacted by Agricultural Animal Sources of Fecal Contamination. U.S. Environmental Agency.
Vartoukian SR, Palmer RM, and Wade WG (2010) Strategies for culture of ‘unculturable’ bacteria. FEMS Microbiology Letters 309: 1–7.
Vierheilig J, Frick C, Mayer RE, Kirschner AKT, Reischer GH, Derx J, Mach RL, Sommer R, and Farnleitner AH (2013) Clostridium perfringens is not suitable for the indication of fecal
pollution from ruminant wildlife but is associated with excreta from nonherbivorous animals and human sewage. Applied and Environmental Microbiology 79: 5089–5092.
Weaver L, Sinton LW, Pang LP, Dann R, and Close M (2013) Transport of microbial tracers in clean and organically contaminated silica sand in laboratory columns compared with their
transport in the field. Science of the Total Environment 443: 55–64.
WHO (2011) Guidelines for Drinking-Water Quality, 4th edn World Health Organization (WHO).
WHO (2016) Quantitative Microbial Risk Assessment: Application for Water Safety Management. World Health Organisation, Water, Sanitation, Hygiene and Health 204.
WHO (2017) Potable Reuse - Guidance for Producing Safe Drinking Water. World Health Organisation (WHO)1–138.
Wong K and Xagoraraki I (2010) Quantitative PCR assays to survey the bovine adenovirus levels in environmental samples. Journal of Applied Microbiology 109: 605–612.
Wuertz S, Wang D, Reischer GH, and Farnleitner AH (2011) Library-Independent Bacterial Source Tracking Methods. In: Hagedorn C, Blanch AR, and Harwood VJ (eds.) Microbial
Source Tracking: Methods, Applications, and Case Studies. New York, NY: Springer New York.
Yang L, Chen X, Zeng X, Radosevich M, Ripp S, Zhuang J, and Sayler GS (2019) Surface-adsorbed contaminants mediate the importance of chemotaxis and Haptotaxis for bacterial
transport through soils. Frontiers in Microbiology 10.
Yao KM, Habibian MM, and Omelia CR (1971) Water and waste water filtration - concepts and applications. Environmental Science & Technology 5: 1105.

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From Groundwater to Drinking Water – Current Approaches for Microbial

Monitoring and Risk Assessment in Porous Aquifers

580 Encyclopedia of Inland Waters, 2nd edition https://doi.org/10.1016/B978-0-12-819166-8.00175-4

How do microbial pathogens move through groundwater?

Observational methods in surface water and groundwater

Microbiological characterization of surface water

Microbiological characterization of groundwater

Natural and artificial pathogen surrogates to characterize transport in groundwater

Observational data uncertainty

Modelling pathogen transport in surface water and groundwater

Quantitative microbial risk assessment (QMRA) and required treatment barriers

Cdrw ¼ Craw =S ∗Z (1)

Current knowledge gaps

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