Recast Drinking Water Directive, state of play : guidance note for the analysis of microbiological parameters.
Date: 2022-06-29
Institutions: Joint Research Centre
Subjects: consumer protection, directive (EU), drinking water, public health, quality standard, research report, water pollution, water quality

Recast Drinking Water Directive  
State of play: Guidance Note for the 
analysis of microbiological parameters 

Magdalena Niegowska, Tarja Pitkänen, Regina 
Sommer, João Brandão, Lucia Bonadonna, Dana 
Baudišová, Nadine Burlion, Benoît Gassilloud, 
Nicholas Pissarides, Miroslava Prokšová, Tatjana 
Rupel, Benedikt Schaefer, Ciska Schets, Tommy 
Šlapokas, Marta Vargha, Teresa Lettieri 

June 2022 

1 

EUR 31130 EN 

 
 
 
 
This publication is a Science for Policy report by the Joint Research Centre (JRC), the European Commission’s science and knowledge service. It 
aims to provide evidence-based scientific support to the European policymaking process. The scientific output expressed does not imply a policy 
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Contact information  
Name: Teresa Lettieri 
Address:  
European Commission 
Joint Research Centre 
Directorate D-Sustainable Resources  
Unit D.02 Water and Marine Resources TP 121  
Via Enrico Fermi, 2749 
I-21027 Ispra (VA), Italy  
Email: teresa.lettieri@ec.europa.eu 
Tel.: +39 0332 78 9868 

EU Science Hub 
https://ec.europa.eu/jrc 

JRC129755 

EUR 31130 EN  

PDF 

ISBN 978-92-76-53688-8 

ISSN 1831-9424 

doi:10.2760/14494 

Luxembourg: Publications Office of the European Union, 2022  

© European Union, 2022  

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How to cite this report:  Magdalena Niegowska, Tarja Pitkänen, Regina Sommer, João Brandão, Lucia Bonadonna, Dana Baudišová, Nadine Burlion, 
Benoît Gassilloud, Nicholas Pissarides, Miroslava Prokšová, Tatjana Rupel, Benedikt Schaefer, Ciska Schets, Tommy Šlapokas, Marta Vargha, 
Teresa Lettieri, - Recast Drinking Water Directive - State of play: Guidance Note for the analysis of microbiological parameters, EUR 31130 EN, 
Publications Office of the European Union, Luxembourg, 2022, ISBN 978-92-79-76-53688-8, doi:10.2760/14494, JRC129755. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Contents 

Abstract ...................................................................................................................................... 1 

Foreword .................................................................................................................................... 2 

Acknowledgements .................................................................................................................... 3 

Executive summary .................................................................................................................... 4 

Rationale of the Guidance Note ................................................................................................. 5 

New microbiological parameters included in DWD 2020/2184 ................................................ 6 

Somatic coliphages ................................................................................................................ 6 

Legionella .............................................................................................................................. 9 

Method specifications for the new microbiological parameters (Annex III) ........................... 11 

Somatic coliphages .............................................................................................................. 11 

Legionella ............................................................................................................................ 11 

EMEG proposals for the next steps in implementing the new microbiological parameters .... 13 

Other microbiological parameters in DWD 2020/2184 established by DWD 98/83/EC  ....... 15 

Escherichia coli ................................................................................................................... 15 

Intestinal enterococci ........................................................................................................... 15 

Clostridium perfringens including spores ........................................................................... 16 

Colony count 22°C .............................................................................................................. 16 

Coliform bacteria ................................................................................................................. 16 

Other parameters ................................................................................................................. 17 

Conclusions .............................................................................................................................. 18 

References ................................................................................................................................ 19 

List of tables ............................................................................................................................. 22 

Supplementary Information ..................................................................................................... 23 

i 

 
 
 
 
Abstract 

The quality of drinking water in the European Union is verified by measuring parameters established 
in the recast of the Drinking Water Directive (DWD) 2020/2184. Compared to the former Directive 
98/93/EC, microbiological parameters have been extended to additionally cover somatic coliphages 
as an indicator of removal efficiency following water treatment and Legionella as a parameter for the 
risk assessment in buildings.  

Implementation of these parameters and their transposition is of utmost importance for the Member 
States  to  ensure  an  adequate  level  of  protection  of  human  health.  However,  existing  standardised 
methods  used  to  measure  the  new  microbiological  parameters  present  technical  drawbacks.  For 
somatic coliphages, large sample volumes require a supplementary concentration step extending time 
of analysis, while for Legionella, the reliability of results substantially depends on the selection of a 
proper protocol variant, restricted choice of variants for certain drinking water types and detection 
limit higher than the parametric value.  

Alternative  methods  provide  a  solution  to  overcome  the  mentioned  technical  limitations  and 
significantly  reduce  time  to  results  allowing  an  early  action  in  case  of  detected  contamination. 
According to DWD 2020/2184, such methods can be implemented for analysing somatic coliphages 
and Legionella if their reliability is at least equal to that of the standard method.  

The  Guidance  Note  presents  an  overview  of  current  and  novel  methods  for  testing  the  new 
microbiological parameters and provides recommendations for harmonising the choice of alternative 
methods  used  across  the  Member  States  with  the  aim  to  support  the  European  Commission  and 
Member  States  in  the  implementation  and  transposition  of  the  mentioned  parameters,  taking  into 
account the parameters formerly established in Directive 98/93/EC.  

1 

 
 
Foreword 

In 2002, the European Commission  (EC) established the European Microbiology Advisory Group 
(EMAG) as  a sub-group of experts with the mandate to  advise the EC  and the art. 12 Committee 
(from 2013 replaced by Expert Group) on different microbiological scientific and technical issues 
under the Drinking Water Directive (DWD). In 2004, EMAG was named European Microbiology 
Group  (EMG)  and  the  mission  extended  to  advice  other  Directive  and  Committees  on  microbial 
quality of water. The name European Microbiology Expert Group (EMEG) was established in 2012 
along with the new mandate towards a horizontal support of the EC across water-related European 
directives: DWD, Bathing Water Directive (BWD), Water Framework Directive (WFD) and Urban 
Waste Water Directive (UWWD). 

At present, EMEG is composed of scientific experts in the field of water microbiology nominated by 
the respective Member States. Its role is to provide scientific input on novel methods for monitoring 
the microbiological quality of drinking water, including molecular and innovative methods, and on 
the  research  needs  for  the  implementation  of  new  methods.  Provided  advice  may  also  include 
emerging pathogens and aspects related to water quality management, such as water disinfection or 
prevention of contamination, with reference to those highlighted by the World Health Organisation 
(WHO) as relevant at European dimension and in close collaboration with WHO. 

For testing microbiological parameters in drinking water, the European DWD 2020/2184 allows the 
use of alternative methods if their reliability is at least equivalent to the reference methods established 
by the Directive. To support the Member States in the submission of alternative methods, EMEG is 
in charge of drafting guidelines for the submission of such methods and of checking the fulfilment of 
criteria for acceptance according to ISO 17994 or ISO 16140. In the present Guidance Note, these 
activities  specifically  aim  at  facilitating  the  practical  implementation  of  new  microbiological 
parameters and the harmonisation of methods among the Member States. 

2 

 
 
Acknowledgements 

We acknowledge the support given by Isabella Sanseverino to the final editing of the document. We 
also thank Ramón Pérez de Lara for creating the cover image. 

Authors 

Magdalena Niegowska 

Tarja Pitkänen  

Regina Sommer 

João Brandão  

Lucia Bonadonna 

Dana Baudišová 

Nadine Burlion 

Benoît Gassilloud 

Nicholas Pissarides 

Miroslava Prokšová 

Tatjana Rupel 

Benedikt Schaefer 

Ciska Schets 

Tommy Šlapokas 

Marta Vargha 

Teresa Lettieri 

3 

 
 
 
 
 
 
 
 
 
Executive summary 

Policy context 
The recast of the European Drinking Water Directive (DWD 2020/2184) introduced two additional 
microbiological  parameters  for  evaluating  the  quality  of  water  intended  for  human  consumption: 
somatic coliphages as an indicator of removal efficiency along the train of water treatment barriers at 
the  waterworks,  and  Legionella  as  a  parameter  for  the  risk  assessment  in  buildings.  The  new 
parameters should be implemented by the Member States along with standardised methods or equally 
reliable alternative methods for their detection and enumeration.  

Purpose of the Guidance Note 
The document provides an overview of current and new methods for analysing the microbiological 
parameters introduced by DWD 2020/2184 and to support the European Commission and Member 
States  in  the  implementation  and  transposition  of  the  mentioned  parameters  taking  into  account 
parameters formerly set in Directive 98/93/EC. The aim is to give advice and recommendation for 
harmonisation  of  methodological  choices  in  order  to  minimise  the  number  of  alternative 
microbiological methods used across the Member States. 

Key points 
The Guidance Note should be considered as  a document  increasing the awareness of the Member 
States  about  the  new  microbiological  parameters  introduced  by  DWD  2020/2184  and  possible 
challenges in their measurement.  

Recommendations 
In order to facilitate determination of Legionella in drinking water (ISO 11731) and the analysis of 
somatic coliphages in large sample volumes (ISO 10705-3), respective reference methods should be 
revised according to standardisation rules with the aim to agree on a single protocol for testing these 
parameters. It is necessary to define methods applicable by the Member States including distinction 
between  alternative  and  established  methods, and indicating those methods and circumstances for 
which validation or equivalency testing are required. 

Recommended actions 

-  To increase the knowledge of available protocols for the detection and 

enumeration of Legionella and somatic coliphages in water distribution systems 

-  To increase the laboratory capability through practical training on reference and 

available methods 

Related and future work 
Support  in  the  increase  of  laboratory  capability  in  the  Member  States  through  training  of  the 
laboratory staff involved in the analysis of new microbiological parameters is a key action to achieve 
a high-quality assessment of drinking water. Meetings of the EMEG members and experts from the 
Member States are expected to facilitate this process and to enable sharing of experiences and best 
practices. Discussions will include indications on how to interpret and transpose other methods and 
will clarify the steps confirming the procedure  which should be followed by the Member State to 
allow the use of alternative method at national level. Additional discussions with the participation of 
science  and  public  health  professionals  and  other  stakeholders  will  help  in  identifying  possible 
concerns and questions. 

4 

 
 
 
 
 
 
 
 
 
 
Rationale of the Guidance Note 

The  European  Microbiology  Expert  Group  (EMEG)  is  a  group  of  experts  in  the  field  of  water 
microbiology of the European Union nominated by their respective Members States. As a European 
(JRC)  Science  Hub  Community,  EMEG  mandate 
Commission  Joint  Research  Centre 
(https://ec.europa.eu/jrc/communities/en/node/279/page/drinking-water-emeg-mandate) includes the 
provision  of  support  and  input  on  microbiological  related  aspects  of  relevant  Directives,  in  co-
operation  with  the  respective  Committees  or  Expert  Groups  established  under  the  scope  of  the 
Directives.  

Regarding the microbiological parameters, EMEG members often have a central role in the practical 
implementation of the Directives in their respective Member State. In this Guidance Note, EMEG 
members provide a consensus statement towards a common European understanding of the practical 
implementation aspects related to the microbiological analysis methods included in DWD 2020/2184. 
The intention of this document is to provide a compilation of the information needs identified by MS 
during the transposition period, and to display the boundary limits for harmonisation of the analysis 
methods. 

The objective of this Guidance Note is to provide an overview on the scientific and informal expert 
discussions of EMEG, related to microbiological parameters in DWD 2020/2184. The specific aims 
are: 

•  To ease the implementation of new parameters Legionella and somatic coliphages 

•  To harmonise methodological choices in MS as much as possible 

•  To minimise the number of various alternative microbiological methods to be evaluated by 

EMEG in the coming years 

5 

 
 
 
New microbiological parameters included in DWD 2020/2184 

In  the  recast  of  the  European  Directive  for  the  drinking  water  (DWD  2020/2184),  two  additional 
microbiological parameters were included: somatic coliphages as an indicator of removal efficiency 
along the train of water treatment barriers at the waterworks, and Legionella as a parameter for the 
risk assessment in buildings.  

Somatic coliphages 
ANNEX II, Part A (Monitoring; the operational monitoring programme shall include the monitoring 
of  somatic  coliphages  in  raw  water  to  control  the  efficacy  of  the  treatment  processes  against 
microbiological risks) 
-  Reference  value  50  (for  raw  water)  (Unit:  Plaque  Forming  Units  (PFU)/100  mL;  notes:  this 
parameter shall be measured if the risk assessment indicates that it is appropriate to do so. If it is 
found in raw water at concentrations > 50 PFU/100 mL, it should be analysed after steps of the 
treatment train in order to determine log removal by the barriers in place and to assess whether the 
risk of a breakthrough of pathogenic viruses is sufficiently under control). 

ANNEX III (Analysis method) 
-  For operational monitoring, Part A of Annex II, EN ISO 10705-2, and EN ISO 10705-3 can be 

used. 

The  parameter  somatic  coliphages  was  introduced  for  the  first  time  in  the  DWD  2020/2184.  Its 
monitoring requirement is based on risk assessment. It is thus a useful parameter in the identification 
of  hazardous  pollution  events  and  in  the  verification  of  the  success  of  risk  management  of  the 
catchment areas, for abstraction points (Article 8 of DWD 2020/2184) as well as the efficacy of water 
treatment  and  disinfection,  i.e.,  in  risk  assessment  and  risk  management  of  water  supply  systems 
(Article 9 of DWD 2020/2184). 

The  information  regarding  the  numbers  of  somatic  coliphages  in  European  raw  waters  is  not 
exhaustive. Most of the analysis methods provided are out-dated and not in active use in most MS. 
The  method  EN  ISO  10705-2  alone  is  not  sufficient  for  analysing  100  mL  volumes  of  water. 
Furthermore, much higher volumes than 100 mL are required in order to determine log removal after 
steps of the treatment train. Therefore, sample concentration method verification, according to EN 
ISO 10705-3, is essential. Analysis method development and laboratory training for this parameter is 
necessary.  

Table  1  summarises  the  available  standardised  methods  for  somatic  coliphages.  In  supplementary 
information (Tables S1 and S2), other promising methods, culture-based and not requiring culture, 
are listed. 

6 

 
 
 
 
Table 1. Standardised methods for detection, enumeration and concentration of somatic coliphages. Adapted from Lamy et al. (2020). 

Method 

Purpose 

ISO10705-2 
(2000)  

Detection and 
enumeration of 
bacteriophages.  
Enumeration of 
somatic 
coliphages  

Control 
coliphage* 

Somatic 
coliphage 
ΦX174  

ISO10705-3 
(2003)  

Validation of 
methods for 
concentration of 
bacteriophage 
from water  

F-specific 
RNA and 
somatic 
coliphages 
used with 
other parts 
of ISO10705  

E. coli strain C 
(drinking 
water, 
unpolluted 
natural waters)  
E. coli strain 
CN, also 
known as WG5 
(polluted 
natural waters 
or 
wastewaters)  

To define 
according to 
the detection 
method  

Host cell  Water type 

Required 
sample 
volume 

Output 

Time to 
results 

Sensitivity 

Advantages 

Disadvantages 

All kinds of 
water 
drinking 
water, 
bathing 
water, 
sediments, 
sludge, 
shellfish  

1 mL (or 5mL 
when expected 
low counts)  

PFU/ 
volume  

24-48h  

1 PFU per 
sample  

- In case of samples 
with high bacterial 
background flora, 1 
mL of samples or 
dilutions is sufficient 
(standard 
procedure)  

- In case of samples 
with low phage 
counts, the volume of 
sample is modified to 
5 mL (instead of 1 
mL)  

Water 
samples 
expected to 
contain < 3 
PFU/mL  

From 100 mL, 
up to 10 L, the 
sample is 
concentrated 
in 5-30 mL  

PFU/ 
volume  

A few hours, 
depending 
on the 
detection 
method  

Depends on 
the method for 
concentration  

- Enables the 
concentration prior 
to further 
enumeration of 
somatic coliphages 
using ISO 10705-
2:2000.  

- 100 mL samples 
can be rapidly 
filtrated (a few 
seconds or minutes)  

- Annex A 
recommends 
methods for 
concentration of 
bacteriophages from 
water depending on 
the volume, 
turbidity and particle 
content.  

- After an 
enrichment step, 
samples are spotted 
onto a lawn of 
specific host bacteria 
cultured on Double 
Agar Layer (DAL) 
and incubated for 
18-24 hours.  

- Filtration of samples 
with volumes greater 
than 1 L can take a 
few minutes to a 
longer time  

- Suspension solids or 
dissolved matter can 
interfere with the 
concentration 
procedure, and 
filtration may take up 
to several hours. 

- Quantitative (for 1 
mL sample, as all the 
sample can be tested)  

- Qualitative 
(presence/absence) 
for larger volumes (for 
100 mL, 1 L samples)  

US EPA 
1601 
(2001)  

Detection and 
quantification of 
coliphages by a 
two-step 
enrichment 
procedure  

Male-specific 
coliphage 
(MS2) and 
somatic 
coliphage 
(ΦX174)  

E. coli Famp 
(for male-
specific 
coliphage and 
E. coli CN-13 
for somatic 
coliphage  

Ground 
water (only 
validated for 
ground 
water) and 
other waters  

100 mL, 1 L  

PFU/ 
volume  

24-48 h  

1 PFU per 
sample  

7 

 
 
US EPA 
1602 
(2001)  

Detection and 
quantification of 
coliphages by 
Single Agar Layer 
(SAL) procedure  

Male-specific 
coliphage 
(MS2) and 
somatic 
coliphage 
(ΦX174)  

E. coli Famp (for 
male-specific 
coliphage and 
E. coli CN-13 
for somatic 
coliphage  

Ground 
water (only 
validated for 
ground 
water)  

*Recommended. PFU: Plaque Forming Unit

- Circular lysis zones 
indicate the 
presence of 
coliphages 
detectable even at 1 
PFU/sample. 

100 mL  

PFU/ 
volume  

24-48h  

1 PFU per 
sample  

- Quantitative 
method (PFU/vol)  

- It can be easily 
applied to 100 mL 
water samples. After 
preparation of the 
material, the mix is 
poured on plates, 
and the plates 
incubated 24 h). 
Lysis plaques are 
counted and 
summed for all 
plates corresponding 
to a single sample of 
100 mL. 

8 

 
 
Legionella 
ANNEX I, Part D (Risk assessment of domestic distribution systems) 
-  Parametric value < 1 000 (Unit: CFU/L; note: this parametric value is set for the purposes of 
Articles 10 and 14. Actions provided in those Articles could be considered even when the value 
is below the parametric value, e.g., in cases of infections and outbreaks. In such cases, the source 
of infection should be confirmed and the species of Legionella should be identified). 

ANNEX II, Part D (Monitoring) 
-  Samples  for  Legionella  in  domestic  distribution  systems  shall  be  taken  at  risk  points  for 
proliferation of Legionella, points representative for systemic exposure to Legionella, or both. 
MS shall establish guidelines for sampling methods for Legionella. 

ANNEX III (Analysis method) 
-  EN ISO 11731 (for compliance with the value in Part D of Annex I); for risk-based verification 
monitoring and to complement culture methods, in addition to methods such as ISO/TS 12869, 
rapid culture methods, non-culture-based methods, and molecular-based methods, in particular 
qPCR, can be used. 

The parameter Legionella was introduced for the first time in the DWD 2020/2184. Its monitoring 
requirement is based on risk assessment of domestic distribution systems. The analysis method EN 
ISO 11731 is intended for the detection of all culturable species included in the Legionella genus and 
should  be  used  for  compliance  monitoring  (Annex  I,  Part  D).  However,  EN  ISO  11731  includes 
several method variants and only some are reliable for testing of domestic water systems. In many 
MS, laboratory training for these analysis method variants is needed. 

For risk-based verification monitoring and to complement culture methods, alternative methods can 
be used. For example, ISO/TS 12869 is intended for detection and quantification of Legionella spp. 
and/or  Legionella  pneumophila  by  concentration  and  gene  amplification  through  quantitative 
polymerase chain reaction (qPCR). Standard methods for detection of Legionella spp. are listed in 
Table 2 while alternative methods are described in the supplementary information (Table S3). 

9 

 
 
Table 2. Standardised methods for detection and enumeration of Legionella. Adapted from the JRC report Lamy et al. (2020). 

Method 

Purpose 

Water type 

ISO 
11731:2017  

Enumeration of 
Legionella  

All kinds of 
water samples 
(potable, 
industrial, 
waste and 
natural 
waters) and 
water related 
matrices 
(biofilms, 
sediments)  

ISO/TS 
12869:2019  

All types of 
water (hot or 
cold water, 
cooling tower, 
etc.)  

Detection and 
quantification of 
Legionella spp. 
and/or Legionella 
pneumophila by 
concentration and 
genic 
amplification by 
quantitative 
polymerase chain 
reaction (qPCR)  

CFU: Colony-Forming Unit

Sample 
volume 

Litres 

Advantages 

Disadvantages 

- Concentration by membrane filtration or dilution 
can be used prior to plating  

- Not all Legionellae are culturable, so the 
methods do not recover all species of Legionella. 

- In case of interfering microbiota the most reliable 
method is concentrating the bacteria via 
polycarbonate membrane filtration, resuspension in 
5-10 mL Ringer solution and plating. 

- Some Legionella species form colonies slowly. 

- Risk of not correctly applying the decision 
matrix and need of highly qualified staff. 

- ISO 11731:2017 procedure comprises 2 steps 
(incubation of plates containing specific media for 
7 to 10 days - recommended to check colonies 
regularly during the incubation, with microscope, 
presumptive colonies are then confirmed after 2 
additional days of incubation on selective agar 
plates). Laborious and time-consuming for 
certain analysis protocols. 

- A total of 10 days to results, after which 
confirmation is needed, requiring long time 
incompatible with early warning. 

- Initial cost of the real-time PCR instrument and 
maintenance and probe, but this will cost will be 
reduced if the instrument is also used for the 
detection and quantification of other pathogens 
by qPCR.  

- Selective culture media give presumptive 
Legionella (CFU Legionella/L)  

- Possible to obtain simultaneously Legionella spp. 
and Legionella pneumophila results 

- Possible determination of Legionella serogroup 
with the use of serum in a subsequent step 

Litres 

- Enables the detection and the quantification of 
Legionella pneumophila but also of Legionella spp. 
(detection of the mip gene)  

- Suitable for high volumes as a concentration step 
is included prior to DNA extraction  

- Rapid (a few hours compared to a few or 10- 13 
days for the reference method): from 
concentration of water sample by filtration, DNA 
extraction from the filter, amplification of the 
specific DNA sequence (s) by real-time qPCR  

- Expressed as genome unit (GU) or genome 
equivalent (GE) Legionella spp. or Legionella 
pneumophila /L, enables the quantification of 
viable but non culturable Legionella spp.  

10 

 
 
 
 
 
 
 
 
 
Method specifications for the new microbiological parameters (Annex III) 

The specifications of the microbiological methods to be used for microbiological parameters are set 
out  in  Annex  III  of  DWD  2020/2184.  The  specifications  include  that  the  laboratories  or  parties 
contracted by the laboratories apply quality management system practices, and that the methods of 
analysis  used  for  the  purposes  of  monitoring  and  demonstrating  compliance,  are  validated  and 
documented  in  accordance  with  EN  ISO/IEC  17025  or  other  equivalent  standards  accepted  at 
international level. 

Alternative methods may be used, provided that it is demonstrated that the results obtained are at least 
as  reliable  as  those  produced  by  the  methods  specified.  The  demonstration  should  be  done  by 
providing the Commission with all the relevant information concerning the equivalence. In practice, 
the MS are expected to prepare and submit an equivalency report based on the procedure stablished 
in standards EN ISO 17994 or EN ISO 16140. Further guidance on the submitting of equivalency 
reports can be found at the EMEG webpage: 

https://ec.europa.eu/jrc/communities/en/community/emeg. 

Regarding the new microbiological parameters, it is obvious that the implementation of Annex III is 
unclear  as  discussed  below.  To  highlight  the  current  status  and  some  challenges,  Table  S4  in  the 
supplementary information shows the methods that the MS have so far in use for the detection of 
somatic coliphages and Legionella.   

Somatic coliphages 
For the first time, Annex III of the DWD 2020/2184 provides a reference method for an operational 
parameter  (somatic  coliphages).  However,  for  this  parameter,  Annex  III  gives  two  methods, 
specifically: “for operational monitoring, Part A of Annex II, EN ISO 10705-2, and EN ISO 10705-
3 can be used.” The phrasing “can be used” is interpreted that it is possible for the MS to use also 
available alternative methods for this parameter without the need to assess the equivalence against 
the given reference methods. I.e., the need for comparative performance study by using ISO 17994 
or  ISO  16140  would  be  lifted  for  this  operational  monitoring  parameter.  However,  to  ensure  the 
sufficient  recovery  of  the  somatic  coliphages  from  both  source  waters  and  the  sufficient  removal 
during the drinking water purification process, the requirement to validate and document the method 
in accordance with EN ISO/IEC 17025 is recommended.  

The method EN ISO 10705-2 is not suitable for the quantitative enumeration of somatic coliphages 
directly in volumes of 100 mL water. The maximum sample inoculation volume on diameter 90 mm 
Petri dishes is only 1 mL. For analysing a 100 mL drinking water sample with this method, twenty 
(20) Petri dishes with diameter 140 mm would be needed, which is not practical nor resource efficient. 
Therefore, prior to using the EN ISO 10705-2 method, an additional concentration step is necessary. 
The standard EN ISO 10705-3 specifies the general principles for assessing the performance of the 
methods for the concentration of bacteriophages in water. It gives performance criteria for various 
concentration  method  testing  purposes.  Indeed,  very  large  volumes  (up  to  1000  litres)  might  be 
required when the logarithmic removal efficiency in the treatment train is evaluated.  

Legionella 
For compliance monitoring of Legionella with the parametric value specified in Part D of Annex I, 
the use of EN ISO 11731 for priority buildings is the reference method and the alternative methods 
need to be equivalent, according to the EN ISO 17994 or EN ISO 16140 for monitoring Legionella 
species.  

11 

 
 
 
 
Alternative methods can however be used without the need for equivalency assessment in specific 
cases of risk-based verification monitoring. In these cases, the requirement to validate and document 
the  method  in  accordance  with  EN  ISO/IEC  17025  is  valid.  The  use  of  methods  intended  for 
enumeration of only a single species, such as Legionella pneumophila, is not a choice of preference, 
because  the  risk  of  other  Legionella  species  should  also  be  covered.  Specifically,  Legionella 
pneumophila  testing  alone  does  not  provide  information  on  the  potential  proliferation  of  other 
Legionella species in the distribution system of a building. 

EN  ISO  11731  specifies  culture  methods  for  the  isolation  of  Legionella  and  estimation  of  their 
numbers in water samples. This standard method is applicable to all kinds of water samples including 
potable, industrial, waste and natural waters. Thus, the 2017 version of ISO 11731 introduced the 
notion of a decision matrix which offers different analytical paths depending on the type of water 
under scrutiny. The analysis protocol will vary depending on the type of water defined according to 
the presumed numbers of Legionella and according to the quantity of interfering bacteria. 

Depending on the type of water, the analyst will have the choice between 4 protocols and 4 culture 
media.  Waters  for  which  the  concentration  of  Legionella  and  interfering  microbiota  is  unknown, 
which is usually the case, the method with the highest reliability is 8.2.3 Membrane filtration followed 
by washing procedure. In this protocol, 1 L sample volume is concentrated to a volume of 5-10 mL. 
The  concentrate  is  divided  into  three  portions,  which  are  directly  plated  on  Glycine  Vancomycin 
Polymyxin Cycloheximide (GVPC) agar plates untreated, heat and acid treated. The choice of path 
in the decision matrix is very important as the reliability of the results depends on it. Some of the 
method variants described in the EN ISO 11731 are not suitable at all for drinking water samples as 
their detection limit is higher than the parametric value (1000 CFU/L). 

The major drawbacks of ISO 11731 are: 

- 

- 

- 

- 

the risk of not applying the decision matrix correctly; 

the need to have highly qualified staff; 

the laborious and time-consuming analysis protocols; 

the long time taken to obtain results (up to 10 days). 

The  advantage  of  ISO  11731  is  that  it  is  possible  to  obtain  simultaneously  Legionella  spp.  and 
Legionella pneumophila results. In addition, the use of serum makes it possible, in a subsequent step, 
to determine the serogroup to which the strain of Legionella belongs. 

12 

 
 
 
EMEG proposals for the next steps in implementing the new microbiological 
parameters 

Implementation of the new microbiological parameters, somatic coliphages and Legionella, and their 
transposition is of utmost importance for the MS to ensure the level of protection at EU level. Table 
3  summarises  the  requirements  set  in  DWD  2020/2184  for  these  new  parameters.  EMEG  also 
identified the key steps and the needs for action which may contribute to the smooth and successful 
implementation of the DWD, shown in Table 4.  

Table 3. Overview of new microbiological parameters in the Drinking Water Directive 2020/2184. 

Parameter 

Data to be set 
accessible to 
Commission; 
Article 18.1 (b) 

Obligatory use of the 
analytical method specified; 
Annex III 

Equivalent performance of the 
alternative methods required; 
Article 13.4 

Somatic 
coliphages 

Yes 

Legionella 

Yes 

No. EN ISO 10705-2 and EN 
ISO 10705-3 can be used for 
operational monitoring 

No 

Yes, at the point of compliance 
for priority premises;  
EN ISO 11731. 

Yes, for monitoring the parametric 
value at the point of compliance; an 
equivalency report to be prepared 
and submitted to EMEG for 
evaluation 

Other methods can be used for 
risk-based verification 
monitoring and to complement 
culture methods 

No 

13 

 
 
 
 
 
 
 
 
 
 
Table 4: Key steps and actions proposed by EMEG for the implementation of the Drinking Water Directive 2020/2184. 

Proposal 

Actions 

Questionnaire about methodological 
details 

-  Define methods which can be used by the MS including 

distinction between alternative methods and methods in place 

A forum to share plans and raise 
concerns/questions  

-  Share a discussion on how to interpret and transpose 

other methods  

-  Indicate methods and circumstances for which 
validation/equivalency testing is requested 

-  Arrangement of informal video-meetings to enable sharing of 

experiences and best practices between the European 
microbiologists 

-  Regular EMEG meetings every six months, in particular for the 

compilation of the DWD file 

-  EMEG sub-group could organise discussions independently 

(scientific/public health interest) 

-  A connection to the Expert Group is essential for success of 

communications 

Support of practical implementation 
of the new DWD by the Member 
States in terms of microbiological 
methods - new variables (possibly 
ring trials) 

-  Harmonise a protocol for Legionella determinations in DW 
including sampling methods for the check of compliance 
-  Harmonise a protocol for analysing somatic coliphages from 

large volumes of DW 

-  Support creation of laboratory capacity in the MS 

Revision of reference methods:  
ISO 11731 (Legionella spp.) and 
ISO 10705-2 and ISO 10705-3 
(somatic coliphages) 

-  Revise as soon as possible according to the ISO rules with aim 
to agree on a single standardised protocol available for drinking 
water testing of these parameters 

14 

 
Other microbiological parameters in DWD 2020/2184 established by DWD 98/83/EC  

For determination of microbiological quality of the water supplied for human consumption, DWD 
98/83/EC required the check monitoring for parameters listed below which have been maintained in 
the  current  DWD  2020/2184.  The  former  Directive  also  specified  methods  of  analysis  for  these 
parameters. 

Escherichia coli (E. coli) 
ANNEX I, Part A (Minimum requirement) 
-  Parametric value 0 (Unit: number/100 mL; note: for water put into bottles or containers, the unit 

is number/250 mL) 

ANNEX II, Part B (Monitoring) 
-  Parameter of group A + considered as “core parameter” 

ANNEX III (Analysis method) 
-  EN ISO 9308–1 or EN ISO 9308–2 

There are no changes in E. coli parameter and microbiological method specifications. However, the 
number of E. coli analysis is suspected to increase due to the monitoring in the catchment areas for 
abstraction points, or in raw water (Article 8 of DWD 2020/2184). It is a useful parameter also in the 
identification of hazardous events and verification of the success of risk management of the catchment 
areas  for  abstraction  points  (Article  8  of  DWD  2020/2184),  and  in  risk  assessment  and  risk 
management  of  the  supply  system  (Article  9  of  DWD  2020/2184),  especially  in  non-disinfected 
systems.  Microorganisms  more  persistent  against  disinfection  should  be  used  for  validation  of 
disinfection efficiency. 

Intestinal enterococci 
ANNEX I, Part A (Minimum requirement) 
-  Parametric value 0 (Unit: number/100 mL; note: for water put into bottles or containers, the unit 

is number/250 mL) 

ANNEX II, Part B (Monitoring) 
-  Parameter of group A + considered as “core parameter” 

ANNEX III (Analysis method) 
-  EN ISO 7899–2 

The parameter name is changed from Enterococci to Intestinal enterococci. However, there are no 
changes  in  the  microbiological  method  specification.  It  is  noteworthy  that  the  bacterial  genera 
Enterococcus includes a wide variety of species. The monitoring of water is focused on detecting 
faecal contamination and thus the analysis method (EN ISO 7899-2) is targeted on species known to 
have an intestinal origin. Some other methods (proposed as alternative methods) might target on a 
wider group of bacteria within Enterococcus genera.  

According  to  DWD  2020/2184,  the  monitoring  frequency  of  intestinal  enterococci  increases 
(inclusion as a Group A parameter). Further, intestinal enterococci is recommended to be included 
into the monitoring in the catchment areas for abstraction points, or in raw water (Article 8 of DWD 
2020/2184). It is a useful parameter also in the identification of hazardous events and verification of 
the  success  of  risk  management  of  the  catchment  areas  for  abstraction  points  (Article  8  of  DWD 
2020/2184) and in risk assessment and risk management of the supply system (Article 9 of DWD 
2020/2184),  especially  in  non-disinfected  systems.  Microorganisms  more  persistent  against 
disinfection should be used for validation of the disinfection efficiency. 

15 

 
 
 
 
 
Clostridium perfringens including spores 
ANNEX I, Part C (Indicator parameters) 
-  Parametric value 0 (Unit: number/100 mL; note: this parameter shall be measured if the risk 

assessment indicates that it is appropriate to do so) 

ANNEX III (Analysis method) 
-  EN ISO 14189 

According to the DWD 2020/2184, Clostridium perfringens (C. perfringens) monitoring requirement 
is based on risk assessment. For example, the C. perfringens monitoring can be useful at any time 
when surface water might enter groundwater (where preferably the analysed water volume should be 
1000 mL or more). Due to extended environmental survival of Clostridium spores, this parameter is 
also useful in identification of past faecal contamination events (Articles 8 and 9 of DWD 2020/2184). 
Furthermore,  C.  perfringens  spores  are  especially  useful  in  validation  of  disinfection  efficiency. 
However,  C.  perfringens  occurs  in  faeces  of  humans  and  warm-blooded  animals  in  1-2  orders  of 
magnitude lower numbers than E. coli or intestinal enterococci. Thus, large enough water volumes 
should be analysed. 

Colony count 22°C 
ANNEX I, Part C (Indicator parameters) 
-  Parametric value ‘No abnormal change’ 

ANNEX II, Part B (Monitoring) 
-  Parameter of group A 

ANNEX III (Analysis method) 
-  EN ISO 6222 

According to the DWD 2020/2184, the monitoring frequency of colony count increases (inclusion as 
a Group A parameter). The changes in the pattern of colony counts from a given sampling point are 
usually  more  significant  than  the  actual  numerical  count  of  any  particular  sample.  It  is  a  useful 
parameter  in  hazard  evaluation  and  risk  management  of  the  supply  system  (Article  9  of  DWD 
2020/2184), and it is affected if the materials in contact with water enhance microbial proliferation 
(Article 11 of DWD 2020/2184). In oligotrophic drinking water distribution systems, the method EN 
ISO  6222  might  produce  no  colonies.  For  operational  monitoring  of  such  systems,  an  alternative 
method, such as the colony count method by spread plate inoculation on R2A medium might be useful 
(international standard method in preparation). 

Coliform bacteria 
ANNEX I, Part C (Indicator parameters) 
-  Parametric value 0 (Unit: number/100 mL; note: for water put into bottles or containers, the 

unit is number/250 mL) 

ANNEX II, Part B (Monitoring) 
-  Parameter of group A 

ANNEX III (Analysis method) 
-  EN ISO 9308–1 or EN ISO 9308–2 

Coliform bacteria are useful indicators in the identification of hazardous events and for verifying the 
success  of  risk  management  of  the  catchment  areas  for  abstraction  points  (Article  8  of  DWD 
2020/2184) and in risk assessment and risk management of the supply system (Article 9 of DWD 

16 

 
 
 
2020/2184). However, many species included in coliform group are environmental bacteria without 
direct health significance. 

Other parameters 
A chemical parameter microcystin-LR, which is a toxin produced by cyanobacteria, is included in the 
Annex I part B with reference value 1.0 μg/L. This parameter shall be measured only in the event of 
potential blooms in source water (increasing cyanobacterial cell density or bloom forming potential). 

According to DWD 2020/2184, MS shall ensure that additional monitoring is carried out, if there is 
reason to suspect a potential danger to human health. Such risk-based parameters need to be addressed 
on  a  case-by-case  basis  and  fall  outside  the  scope  of  this  document.  Each  member  state  will  be 
expected  to  assess  additional  parameters  and  values  on  the  basis  of  territorial,  climatic  and 
epidemiological conditions which vary geographically, without specifying further details. This choice 
is part of the application of water safety plans adopted by each water utility. 

Note  1:  The  DWD  2020/2184  does  not  include  the  microbiological  parameters  Pseudomonas 
aeruginosa  and  colony  count  36°C  used  previously  for  monitoring  water  put  into  bottles  and 
containers. These parameters can be additionally considered based on the assessment. 

Note 2: The DWD 2020/2184 does not yet include microfungi, although there are currently MS which 
perform the analysis of this parameter and there is scientific evidence and recommendation to do so 
(Caggiano  et  al.  2020;  Novak  Babič  et  al.  2017;).  At  this  stage,  they  should  be  regarded  in  risk 
assessment  and  recommended  to  be  monitored  at  end-points  in  hospitals  and  buildings  where 
immune-compromised users may congregate.  

17 

 
 
 
Conclusions 

Alternative methods for analysing somatic coliphages and Legionella according to DWD 2020/2184 
provide a solution in situations when single standard methods are not suitable for the analysis of large 
sample volumes, and testing of domestic water systems, or require high-level expertise in selecting 
proper  method  variant.  The  average  time  to  results  of  alternative  methods  is  shorter  compared  to 
standard methods which helps to undertake early actions when the parametric value is exceeded or in 
case  of  outbreaks.  While  some  promising  methods  are  described  in  scientific  literature  and  need 
further  development,  several  alternative  methods  are  already  validated  but  may,  however,  require 
additional laboratory training.  

Several Member States have already adopted alternative methods, however, they do not substitute 
reference  methods  for  regulatory  monitoring  of  Legionella  and  somatic  coliphages  in  distributed 
water systems. Rapid methods involving molecular technologies, such as those relying on polymerase 
chain reaction (PCR), are at present employed for environmental monitoring, research projects or in 
case of outbreak investigation in addition to reference methods.  

To  facilitate  the  implementation  of  the  new  microbiological  parameters,  a  single  protocol  for 
reference  methods  should  be  adopted  by  the  Member  States  with  adequate  revision  according  to 
standardisation rules. A harmonised selection of methods that can be applied by the Member States 
should clearly distinguish between the application of alternative and established methods, and should 
also provide an indication of situations in which validation or equivalency testing are required. 

18 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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21 

 
 
 
 
 
 
 
 
List of Tables 

Table 1. Standardised methods for detection, enumeration and concentration of somatic coliphages. 
Adapted from Lamy et al. (2020)………………………………………………………………….....7 

Table 2. Standardised methods for detection and enumeration of Legionella. Adapted from the JRC 
report Lamy et al. (2020)……………………………………………………………………………10 

Table  3.  Overview  of  new  microbiological  parameters  in  the  Drinking  Water  Directive 
2020/2184…………………………………………………………………………………………...13 

Table 4. Key steps and actions proposed by EMEG for the implementation of the Drinking Water 
Directive 2020/2184……………………………………………………..………………………….14 

Table S 1. Culture-based methods for the detection of somatic and F-specific coliphages. Adapted 
from the JRC report Lamy et al. (2020)…………………………………………………..………...23 

Table  S  2.  Non-culture-based  methods  for  the  detection  of  somatic  and  F-specific  coliphages. 
Adapted from the JRC report Lamy et al. (2020)…………………….………………………….….25 

Table S 3. Alternative methods for the detection and enumeration/quantification of Legionella spp. 
and Legionella pneumophila………………………………….………………………………….….26 

Table S 4. Methods applied currently in the MS for detection of somatic coliphages and Legionella. 
The information was provided by national experts nominated as members of EMEG and from DWD 
Expert Group………………………………………………………………………………….……..28 

22 

 
 
 
 
 
 
Supplementary Information  

Table S1. Culture-based methods for the detection of somatic and F-specific coliphages. Adapted from the JRC report Lamy et al. (2020) 

Method 

Purpose/ 
Type 

Target 
coliphage 

Host cell 

Sample 
type tested 

FastPhage  
qualitative 
method 

Presence/ 
absence  

Somatic or F-
specific 
coliphages  

E. coli CN-
13 for 
somatic Famp 
for male-
specific 
coliphage  

Ground 
water 
(validated 
by US EPA), 
drinking 
water  

Required 
sample 
volume 

100 mL  

Sensitivity 

Time to 
results 

Output 

Advantages/ 
Disadvantages 

PFU/100mL   Advantages:  

1.5 PFU/100 
mL  

Prediction 
(visual 
fluorescence 
test: 8 h) 
followed by 
confirmation 
(plaque test: 
16 h)  
Total:24 h  

- no need of overnight 
preculture for host cell 
(ready-to use tablet)  
- fluorescence-based 
prediction enables early 
warning.  
Disadvantage:  
- not quantitative  
- time to result comparable 
to classic culture-based 
methods. 

Advantages:  
- quantitative. 
Disadvantages:  
- initial investment for the 
devices. 

5.5-6 h 

PFU/4mL 
(TEMPO 
format)  
or  

3.5 h, plus 
time 
necessary to 
pre-grow host 

PFU/100 mL  Advantages: 

- applicable to a wide range 
of volumes and sample 
types 
- can be adapted to the 
MPN format for 
quantification 
- allows a simultaneous 
detection of somatic and F-
specific coliphages. 
Disadvantages: 
- preculture required. 

Reference 

Salter et al. 
2010 

Salter et al. 
2010 

Muniesa et 
al. 2018, 
Toribio-
Avedillo et 
al. 2019 

FastPhage 
quantitative 
method 

Detection 
and 
enumeration 

Somatic or F-
specific 
coliphages 

Drinking 
water 

E. coli  
no other 
information 
on the 
strain 

BluePhage 
qualitative 
method 

Presence/ 
absence 

E. coli CB10 
and CB12 

Somatic and 
F-specific 
coliphages, 
(simultaneous 
detection with 
CB12) 

Surface 
water, 
wastewater, 
drinking 
water, 
reclaimed 
water, 
sediments, 
sludge 

TEMPO 
format:  
4 mL  
or  
MPN format: 
100 mL 

100 mL 

<0.25 PFU/ 
4 mL 
(TEMPO 
format)  
or  
<1 PFU/100 
mL (MPN 
format) 

1 PFU/100 
mL 

BluePhage 
quantitative 
method 

Detection 
and 
enumeration  

Somatic or F-
specific RNA 
coliphages  

E. coli CB10 
and CB12 

1 mL 
(dilution if 
necessary) 

Raw or 
treated 
wastewater, 
surface 
water, 

1 PFU/mL 

18-24 h 

PFU/mL 

Advantages:  
- no need of overnight 
culture but 2 h of pre-
growth of the host strain. 
Disadvantage: 

Muniesa et 
al. 2018, 
Toribio-
Avedillo et 
al. 2019 

23 

 
 
 
QuantiPhage  Detection 

and 
enumeration 

Somatic and 
F-specific RNA 
coliphages 

E. coli CN- 
13 for 
somatic, Famp 
for male- 
specific 
coliphage 

EasyPhage 

Detection 
and 
enumeration 

Somatic and 
F-specific RNA 
coliphages 

E. coli Famp 

recreational 
water, 
shellfish 
extracts, 
sediments, 
sludge 
extracts  

Surface 
water, 
drinking 
water, 
recreational 
water, 
wastewater 

Marine 
swimming 
beaches, 
streams, 
non 
swimming 
coastal 
waters and 
raw sewage 

- results not available in the 
same working day. 

1 mL, 10 
mL, 100 mL 

1 PFU/mL 
(when 1 mL 
of water is 
analysed), 1 
PFU/10 mL 

1.5-2 h for 
somatic 
coliphages,  
2.5-3 h for F-
specific 
coliphages 

PFU/volume  Advantages:  

- visual. 
Disadvantage:  
- preparation of host cells 
overnight culture is needed. 

10 mL 

1 PFU/10 
mL 

16 h 

PFU/10mL 

Antibody-
coated 
polymeric 
bead 
agglutination 
assay 
(CLAT)*, ** 

Detection 
and 
classification 
of F-
coliphages 
in a variety 
of samples 

F-specific RNA 
and DNA 
coliphages 

E. coli Famp 

Estuary 
water, bird 
faeces, 
shellfish 
tissue 

2.5 l of 
coliphage 
enrichment 
culture 

5x103 
PFU/2.5 l 
or 1 PFU/mL 
with phage 
enrichment 
culture 

3 h, plus 2 h 
preincubation 

PFU/mL 

Rames and 
Macdonald 
2019 

Fung et al. 
2007 

Love and 
Sobsey 
2007 

Advantages: 
- rapid formation of the 
growth gel without the use 
of heat 
- kit containing all the 
components to complete 
the assay 
- possibility to remove 
background bacteria 
through antibiotic addition. 

Advantages:  
- faster than classic culture-
based methods  
- can be performed in the 
field. 
Disadvantages: 
- high limit of detection for 
regulatory purposes (i.e. 
drinking and bathing water) 
- need of phage-enrichment 
culture in matrices with low 
bacteriophage load 
- not adapted for all F-
specific coliphages 
- specific antisera not 
readily available. 

*Method not requiring culture for samples with a high bacteriophage load. **Methods available non-commercially and described in scientific literature. 

24 

 
 
 
 
Table S2. Non-culture-based methods for the detection of somatic and F-specific coliphages. Adapted from the JRC report Lamy et al. (2020) 

Target 
coliphage 

Sample 
type tested 

Required 
sample 
volume 

Sensitivity 

Time to 
results 

Output 

Advantages/ 
Disadvantages 

Reference 

Method 

Purpose 

Reverse 
Transcription 
quantitative 
PCR (RT-
qPCR)  

Detection and 
quantification of 
bacteriophages 
in a variety of 
samples  

Quantitative 
PCR (qPCR)  

Multiplex 
quantitative 
PCR  

Various 
kinds of 
samples  

1, 100 mL 
(after 
concentration)  

Increased 
respect to 
the culture  

Developed for 
F-specific 
coliphages,  
can be used 
for somatic 
coliphage and 
other 
bacteriophage  

Total time: 4-5 
h for F-specific 
RNA coliphages 
(RNA extraction, 
retro-
transcription 
and real-time 
PCR)  

DNA copy 
number or 
genome 
unit (gu) 
or genome 
copy 
number 
(gc)  

Digital PCR  

Detection and 
quantification of 
bacteriophages 
in a variety of 
samples  

In-gel loop-
mediated 
isothermal 
amplification 
(gLAMP) 
system  

Detection and 
quantification of 
bacteriophages 
in a variety of 
samples  

Developed for 
F-specific RNA 
coliphage 
(MS2), can be 
used for 
somatic 
coliphage and 
other 
bacteriophage  

Developed for 
F-specific RNA 
coliphage 
(MS2), can be 
used for 
somatic 
coliphage and 
other 
bacteriophage  

Mostly 
wastewater  

Not reported  

Increased 
respect to 
the culture  

4 h  

Environment
al waters, 
wastewater  

20 mL  

0.7 PFU per 
reaction or 
10 DNA 
copies/μL  

RNA extraction: 
approx. 2 h  
gLAMP:30 min  
Total time: 2 h  

DNA copy 
number or 
genome 
unit (gu) 
or genome 
copy 
number 
(gc)  

Amplicon  

25 

Ogorzaly 
and 
Gantzer 
2006 

Leeds 
2010 

Wolf et al. 
2010, Kirs 
and Smith 
2007 

Morella et 
al. 2018 

Huang et 
al. 2018, 
Lin et al. 
2019 

Advantages:  
- faster than 
traditional USEPA or 
ISO culture based- 
method (4 h vs 24-
48 h), but as fast as 
the new culture 
based-methods  
- multiple assays 
could be used to 
target more than one 
family of 
bacteriophages. 
Disadvantages:  
- does not provide 
the infectivity status 
of the detected 
genome  
- it should be coupled 
to other methods.  

Advantages:  
- rapid  
- ∼10 $/ sample.  
Disadvantage:  
- initial investment 
(instrument).  

Advantages:  
- rapid, visual (dye, 
fluorescence)  
- ∼0.1$/ sample. 
Disadvantages:  
- initial investment 
(instrument)  
- fluorescence 
microscope.  

 
 
 
 
 
 
 
Table S3. Alternative methods for the detection and enumeration/quantification of Legionella spp. and Legionella pneumophila.  

Method 

Purpose/ 
Type 

Sample type 
tested 

Required 
sample 
volume 

Sensitivity 

Time to 
results 

Output 

Advantages/ Disadvantages 

Reference 

Legiolert/Quanti-
Tray  

Enumeration of 
L. pneumophila 

Drinking water and 
other water 
samples 

100 mL  

1 MPN /100 mL 

7 days   MPN /100 

mL 

ICGene LAMP 
detection kit 

Detection of 
Legionella spp. 
and L. 
pneumophila 

Drinking water, 
hot water, cooling 
towers, mineral 
waters 

~1 h 

Ct values 
or DNA 
copy 
numbers 

Advantages:  
- high yields of L. pneumophila counts 
- single condition run (low complexity) 
- faster to perform than the ISO 11731. 
Disadvantage:  
- time to result moderately shorter than the 
classic culture-based methods (7 vs 10 days). 

Advantages: 
- rapid DNA extraction (15 min) 
- adequate for field application 
- no need to process data post run. 
Disadvantages: 
- investment for the LAMP instrument 
- not able to distinguish dead/viable cells. 

Sartory et al. 
2017 

Lu et al. 
2011 

AquaScreen qPCR 
detection kit 

Detection and 
quantification of 
Legionella spp. 
and L. 
pneumophila 

Drinking water, 
bathing water, 
wastewater, 
surface waters 

100 mL to 
1 L 

LOQ=20 
copies/assay 

<6 h 

Ct values 
or DNA 
copy 
numbers 

Advantages: 
- no need to dilute samples (linear detection up to 
1 x 106 particles per sample). 
Disadvantages: 
- cross-reactivity of the target gene (mip) to other 
waterborne microorganisms is not known 
- not able to distinguish dead/viable cells. 

Yaradou et 
al. 2007 

Spartan 
Legionella 
detection system 

Detection and 
quantification of 
L. pneumophila 

Water systems of 
buildings 

45 min  

LOD=2 genomic 
units/reaction 
LOQ=20 genomic 
units/reaction 

Ct values 
or DNA 
copy 
numbers 

Advantages: 
- detects live and VBNC bacteria 
- calibrated to culture so that 1 genomic unit/mL 
= 1 CFU/mL 
- on-site testing possible. 
Disadvantages: 
- investment for the Spartan Cube instrument. 

Ahmed et al. 
2019 

Bio-Rad iQ-Check 
PCR kits 

Detection and 
quantification of 
Legionella spp. 
or L. 
pneumophila 

Water supply 
systems, water for 
industrial use, 
thermal and 
swimming pools, 
other bathing 
water, biofilm, 
aerosol 

<4 h 

LOD=5 genomic 
units/reaction 
LOQ=15 genomic 
units/reaction 

Ct values 
or DNA 
copy 
numbers 

Advantages: 
- different assays can be run in parallel on the 
same plate. 

Yaradou et 
al. 2007 

26 

 
 
 
 
 
 
 
GeneDisc 
Legionella kit 

Mericon Quant 
Legionella spp. kit 

Diateva DI-Check 
Legionella 
pneumophila kit 

Detection and 
quantification of 
Legionella spp. 
and L. 
pneumophila 

Detection and 
quantification of 
Legionella spp. 

Detection and 
quantification of 
Legionella spp. 
and L. 
pneumophila 

Various water 
types 

250 mL to 
1 L 

Various water 
types 

1 L 

Various water 
types 

up to 1 L 

<3 h 

LOD=5 genomic 
units/reaction 
LOQ=25 genomic 
units/reaction 

<3 h 

<4 h 

LOD=5 
copies/reaction 
LOQ= 10 
copies/reaction 

LOD=5 genomic 
units/5 µL 
LOQ=25 genomic 
units/5 µL 

Multiplex real-
time PCR* 

Enumeration of 
Legionella spp. 

Sputum, 
environmental 
(type non 
reported)  

Non 
reported 

10 fg per reaction 

~5 h 

Reverse 
transcription PCR 
(RT-PCR)* 

Detection of 
viable L. 
pneumophila 

Drinking water 

1 L 

103 cells/L 

8 h 

Membrane 
filtration and 
immunodetection
* 

Detection and 
enumeration of 
L. pneumophila 

Drinking water, 
various water 
types 

200 mL up 
to 1 L 

70 CFU per filter 

2 h 

Ct values 
or DNA 
copy 
numbers 

Ct values 
or DNA 
copy 
numbers 

Ct values 
or DNA 
copy 
numbers 

Ct values 
or DNA 
copy 
numbers 

Ct values 
or DNA 
copy 
numbers 

CFU/200 
mL or per 
filtered 
volume  

Advantages: 
- shorter time to results for clear water samples 
(<2 h). 

Yaradou et 
al. 2007 

Advantages: 
- adapted for the multiplex real-time PCR 
approach. 

Ballard et al. 
2000 

Kim et al. 
2015 

Boss et al. 
2018 

Párraga-Niño 
et al. 2018 

Advantages:  
- quantitative method 
- simultaneous detection of multiple non-
pneumophila species 
- adaptable to other Legionella species 
Disadvantage:  
- not able to distinguish dead/viable cells 

Advantages: 
- discriminates between VBNC and dead cells. 
Disadvantages: 
- complex due to additional extraction step 
- more expensive than ISO 11731. 

Advantages:  
- low level of unspecific binding to filter 
membranes 
- rapid, simple 
- volume magnitude adaptable to ISO 11731 
- discriminates between VBNC and dead cells. 
Disadvantages: 
- LOD not determined for natural water samples 
- cross-reactivity and false positive rates higher 
than for PCR-based methods. 

*Methods available non-commercially and described in scientific literature. 

27 

 
 
 
 
 
 
 
 
Table S4. Methods applied currently in the MS for detection of somatic coliphages and Legionella. The information was provided by national experts nominated as members 
of EMEG and from DWD Expert Group. 

Country 

Somatic coliphages 

Legionella 

Method 

Purpose and notes 

Method 

Purpose and notes 

AT 

ISO 10705-2  

For surface water and 
wastewater. 

ISO 111731  

For official monitoring (on a 3-6 month basis, or even 
yearly basis). 

ISO 10705-3 cellulose acetate 
membrane/beef extract method.   

50% mean recovery for surface 
water. 
5 mL concentrate is processed in 
portions: 3 x 1 mL, 2 x 0.5 mL 
and 2 x 0.1 mL (5 plates).   

"Concentrating and wash" method; 1 L 
(clause 8.2.3; [B (8/9/10) GVPC]) 

Sample processing. 

Additional methods (e.g., qPCR) 

For outbreak investigations to reduce time to results 
(from 7-10 days required for regular monitoring). 

No legal regulation for drinking water. 

Legionella will be introduced as a new parameter in 
the legislation. 

BE 

Legiolert covers L. pneumophila only; the 
sample volume processed is 100 mL and 
not 1 L. 

ISO 11731  

Not yet legal regulation for drinking water (Wallonia). 
But present in swimming pool legislation. Regulatory 
oversight (swimming pools, public buildings, etc.) and 
to investigate epidemics. 

NF T-90 or ISO 12869 

Used for preliminary data, but not used for regulatory 
compliance monitoring (under the current regulation). 

CY 

ISO 10705-2 without 
concentration method. 

Surface and ground water. Used 
2 x 5 mL samples in 2140 mm 
Petri dishes. 

ISO 11731, latex kits to determine 
serotypes. 

For monitoring Public Hospitals and to investigate 
outbreaks. 

CZ 

Various techniques are currently 
being tested and any experience 
are welcome. 

Not much practical experience of 
somatic coliphage detection in 
drinking and raw waters so far. 

ISO 11731  

For regular monitoring of warm water in "important 
buildings" and swimming pools.  

Plans to use also the PCR method. 

Only for risk analyses dealing with domestic 
monitoring. 

DE 

 ISO 10705-2 

Used for surface waters and from 
reservoirs to produce drinking 
water on a voluntary basis. Few 
laboratories are accredited for 
this method. 

Combination of ISO 11731 annex J method 
1 and method 7. 

For routine monitoring of Legionella species in 
drinking water (drinking water distribution systems 
exceeding 400 L hot water storage or 3 L contents of 
warm water in the longest pipes): every year for 
systems providing water to the public and every 3 
years for commercially operated systems. 
Combination of methods allows to overcome the 

28 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
EE 

Little experience with ISO 
10705-2 and ISO 10705-3. 

Need to be implemented.   

ISO 11731  

ISO/TS 12869 and Legiolert  

FI 

US EPA 1602 method (Single 
agar layer procedure where the 
total analysed sample volume is 
100 mL). 

ISO 12869 

In use for research purposes. 

ISO 11731  

Membrane filtration (MF), dead-
end ultrafiltration (DEUF), PEG 
precipitation. 

Various techniques are currently 
being tested for sample 
concentration. 

No prior experience by using 
ISO 10705-2 or ISO 10705-3 
method.  

Preliminary results indicate 
superior recovery of ISO method 
as compared to US EPA method. 

problem of the quite high lower limit of detection for 
direct plating alone. 

For operational purposes for L. pneumophila in 
addition to ISO 11731. 

Accredited by Estonian Accreditation Centre (EAK), 
routinely in use. 

Needs to be implemented as alternative to ISO 11731 
with isolation procedure according to ISO 11731. 

Legionella will be introduced as a new parameter in 
the legislation. 

Alternative methods not included in ISO 
11731. 

Require acceptance based on equivalence. The 
exception is the outbreak investigation, where also 
rapid methods may be used. 

Not for regulatory monitoring but 
used for research purposes 
concerning characterisation of 
the faecal contamination (surface 
water).  

NF T 90-431 for detection and 
enumeration of Legionella spp. and L. 
pneumophila (corresponding to ISO 
11731 variant using GVPC media). 

Ground water: parameter is used 
for tracing possible faecal 
contamination. 

New version of the French standard, closer 
to the ISO standard, was published in 
2014.  

FR 

ISO 10705-2 without 
concentration method.  

Various techniques are currently 
being tested for concentration of 
surface water and drinking water 
samples. One of the evaluated 
concentration methods is a 
based-filtration method using a 
binding and elution buffers. The 
concentrate is analysed in 
accordance with the 
recommendations of the 
standard ISO 10705-2. 

NF T90-471 (corresponding to ISO 12869) 
and real-time PCR. 

29 

For regulatory monitoring and to investigate 
outbreaks. 

Note concerning NF T 90-431 standard: Each clean 
water samples are analysed by three ways: (i) 0.2 mL 
are plated on GVPC agar; 10 mL and 100 mL are 
filtered through cellulose nitrate or mixed cellulose 
ester membranes filters direct placing on GVPC agar. 
Previously, acid treatment is done directly on the 
membrane filter in the funnel.  Where concentration 
by filtration is not possible the sample can be 
concentrated by centrifugation or by filtration through 
polycarbonate membrane filter followed by an elution 
step (vortex or sonication). Latex kits are used to 
discriminate between Legionella spp. and 
L. pneumophila and to identify specifically 
L. pneumophila serogroup 1 from the other 
serogroups (2-15). 

Real-time PCR for Legionella not used for regulatory 
monitoring but as a complementary method to the 
method described by the NF T90-431. PCR is used to 
investigate Legionella proliferation in sanitary water 
networks and outbreaks. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HU 

No previous experience with the 
detection of somatic coliphage 
from drinking water. 

Need for expert advice from the 
group acknowledged. 

ISO 11731  

Legiolert  

Monitoring in relevant sites such as public buildings. 

Accepted as an alternative method (under the current 
regulation). 

Support for direct use of 
alternatives. 

IT 

5 mL using the lysis plate 
method. For the same samples 
we sometimes filtered through 
electropositive filters in parallel, 
but we obtained much lower 
yields. 

Filter/concentrate 1500 - 2500 L 
through electropositive filters 
(Nanoceram equivalent to the 
Zetaplus according to USEPA). 
We obtain 1-2 mL of concentrate 
that is analyse by the lysis plate 
method. The final counts 
consider the various 
concentration steps. 

NL 

ISO EN-ISO 10705-2, using 
Scholtens Agar.  

qPCR method (preliminary data)  

Not used for regulatory compliance monitoring (under 
the current regulation). 

A few rapid methods tested. 

Surface water. 

ISO 11731 

None of them was found to be sufficiently reliable to 
be adopted. 

European Technical Guidelines for the Prevention, 
Control and Investigation of Infections Caused by 
Legionella species (ECDC 2017) and the Italian 
Guidelines on legionellosis (Ministry of Health 2015). 

Legiolert 

For L. pneumophila. 

Clean samples (e.g., at the exit 
of drinking water treatment 
plants). 

Faster methods can probably be used as 
screening methods. 

Real-time PCR 

ISO 11731 

The parameter is used for tracing 
possible faecal contamination in 
wells (where they analyse 100 – 
1000 L samples), but not in 
further drinking water 
purification steps.  

With all the steps that are required for the different 
types of water. 

PT 

SI 

SK 

Concentration steps are applied 
such as negative membrane 
filtration or hemoflow filtration. 

To determine the recovery of the 
concentration method that is 
used in our QMRA approach. 

ISO 10705-2 without 
concentration method. 

Surface water and wastewater. 

ISO 11731, latex kits to determine 
serotypes. 

For regulatory monitoring and to investigate 
outbreaks. 

ISO 11731 and ISO 12869 

Legionella will be introduced as a new parameter in 
the legislation. 

PCR method  

For private requirements and research. 

ISO 11731, latex kits to determine 
serotypes too. 

Legionella will be introduced as a new parameter in 
the legislation.  

30 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ISO 12869 

Settlement of water supply systems by Legionella 
(drinking water, warm water, pools, air conditioning 
units). 

Real-time PCR 

For Legionella in the environment. 

31 

 
 
 
 
 
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doi:10.2760/14494 
ISBN 978-92-76-53688-8