HORIZONTAL Factsheet
BACTERIOPHAGES
version: 3/6/2016
SUBJECT
Determination of BACTERIOPHAGES
HORIZONTAL number
4
Sludge, treated sludge (mesophilic digested /
dehydrated), compost and soil
ISO 10705-1 :1995 ; ISO 10705-2 : 2000 ; ISO
10705-4 : 2001
Matrices
Existing standards used as basis
Standardisation Committees
Work package
3
Project
STREP FP6 HORIZONTAL-HYG
Expert
Prof. Francisco Lucena
Organisation
University of Barcelona
Country
Spain
Email
flucena@ub.edu
Ongoing work
Status
Desk study First
Comments Phase I
Final version
Phase II Ruggedness
Hygienic parameters
Second consultation
Desk study due 31-06-05
Deadline 30-09-05
Due 31-07-06
Due 31-10-06
Validation
Summary of BACTERIOPHAGES procedure
This WP2/3 will consist in a pre-normative research on bacteriophages methods applied to
sludges, soils and treated biowastes.
The work will be organised in two main steps:
1- A 6 months preliminary critical review including an EU workshop;
2- A suitability study (INTRALABoratory study) based on a matrix-based approach with
3 partner laboratories.
1 - Critical review including a workshop
A 6 months critical review will be realised by partner 11 (UB). The aim of this work will be to
make a state of the art of the topic including a risk assessment and economical point of view.
Suitable target phages (somatic coliphages, F-specific RNA phages and/or phages infecting
Bacteroides fragilis) and methods for testing and extracting phages and viruses
(enteroviruses) will be identified and proposed. As well their fit for different sludges, soils and
treated biowastes matrices will be evaluated.
A 3 days workshop is included in this work in order that EU (including new EU countries)
experts could discuss on this topic. Partner 11 (UB) will be in charge of the scientific
preparation of the workshop on bacteriophages. Partner 11 will then have to prepare the
programme of the workshop and provide the WPL with a list of EU experts on this topic. The
WPL (partner 3, IPL) will be in charge of coordinating the whole workshop organisation.
A critical review report including the conclusions of the workshop will be produced. This
report would propose one to three method(s) identified to be tested for fit for purpose to
HORIZONTAL Factsheet
BACTERIOPHAGES
version: 3/6/2016
sludges, soils and biowastes matrices in a consecutive suitability study.
2 – Suitability study (INTRALABoratory study)
The aim of the suitability study is to evaluate the suitability of the bacteriophages extraction
and enumeration methods identified as relevant in the previous critical review, for the
matrices to be tested. The main organisation of this suitability study is the same than the one
described for the suitability study for E. coli and Salmonella spp methods. However, some
differences would however appear :
1) The work will be performed by partner 11 (including intralaboratory suitability study
organisation).
2) The suitability study will take into consideration the outputs of the critical review
(including the workshop) and will focus on bacteriophages (somatic coliphages, Fspecific RNA phages and/or phages infecting Bacteroides fragilis) enumeration
methods (1 to 4) including a preliminary extraction procedure that will be adapted
from ISO standard methods.
3) Only 3 partner laboratories have been identified as being able to be involved in this
study, due to their experience in routine bacteriophages analyses (partners 3, 9 and
11).
4) Four out of the 9 target matrices will be tested (raw sewage sludge, mesophilic
anaerobic digested sewage sludge, digested sewage sludge press cake, composted
sewage sludge). Those matrices have been chosen taking into consideration their
availability, the level of inactivation that the viruses may have suffered and the water
content of the sample.
5) Enteroviruses data will be compared to the phages data in a few selected samples.
Results of this comparison are expected to provide a first insight on the behaviour of
phages and human viruses in sludges, soils and biowastes.
6) No training session is planned for this suitability study, due to the pre-normative stage
of this study. However, a special care will be given to the description of methods and
corresponding materials and consumables and to the quality control traceability in
order to guaranty the harmonisation of protocols applied in all the participating partner
laboratories. Therefore, more time will be scheduled for the preparation and the
implementation of the laboratory work.
This study will so last for 15 months and will be organised in 4 months for the
preparation and organisation, 5 months for the laboratory work and 6 additional
months for the statistical analysis and reporting.
Results will be presented and discussed in a technical meeting between partners and the
statisticians. A report on the suitability study will be produced by partner 11 (UB) including
the issues of the discussion of the technical meeting. Recommendations will be made with
respect to each matrix for each method (fit for purpose and unfit for purpose). First draft CEN
standards will then be prepared by partner 11 (UB) for each of the methods tested and
proposed for CEN evaluation.
HORIZONTAL Factsheet
BACTERIOPHAGES
version: 3/6/2016
Placement of BACTERIOPHAGES method in overall structure HORIZONTAL
SLUDGE, SOIL, TREATED BIOWASTE AND RELATED
WASTES
SAMPLING
IN-SITU ,TRANSPORT, STORAGE
SAMPLE PRETREATMENT - INORG
INORGANIC
CONSTITUENTS
PHYSICAL
PROPERTIES
SAMPLE PRETREATMENT - ORG & BIOL
LEACHING
ORGANIC
PARAMETERS
BIOLOGICAL
PARAMETERS
SAMPLE PRETREATMENT
- HYG
HYGIENIC
PARAMETERS
PAH
pH
Bulk density
Characterisation
leaching tests
Impurities (e.g
glass, plastic)
E. Coli
Stability (bio)
Salmonella
Germinating
weed seeds
Clostridium
Phytotoxicity
Enterococci
AOX
Electrical
conductivity
Dry matter
Compliance
leaching tests
PCB
Organic matter*
Solidity
LAS, NP,NPE
Nutrients N, P,
K*
Thixotropic
behaviour
DEPH, DBP
Trace elements
solid *
Trace elements
determination*
Piling
behaviour
Helminth ova
BFR
Flowability
Rapid methods
PCCD/PCDF
Soluble P
Virusses and
bacteriophages
Pharmaceuticals
Plant
Pathogens
Position of BACTERIOPHAGES method in the sequence of steps from sampling to
reporting
Sampling Taking of
plan
sample
Transport & Preparation
storage
of test
portion
Not
Not defined. Not defined.
defined. To To be
To be
be
discussed discussed at
discussed at the
the Workshop
at the
Workshop
Workshop
Not defined.
To be
discussed at
the Workshop
Extraction/
Leaching
Pre-treatment
Detection &
Summary
Quantification, reports
Enumeration
Not defined. Not defined. To Not defined. To
To be
be discussed at be discussed at
discussed at the Workshop
the Workshop
the Workshop
1)Sample conservation, storage and transport are issues that generally are covered in as the final step in sampling standards
2) Preparation of the test portion from a laboratory sample is sometimes part of the next step (digestion or analysis preparation) or can
be covered in a separate standard
3) Summary of test results is a concise summary of the relevant data for the end-user, which can be merged in an overall test report
covering all aspects relevant for the given situation.
4) After defining sampling strategy based on the question to be answered either one of the sampling standards will generally apply. All
the following steps will be the same for either route.
5) It is at present not clear where extraction of organic parameters begins and pre-treatment ends. It seems that pre-treatment now includes
the extraction step.
Major issues of discussion
After the desk study (Critical Review), the elaboration of the results of the questionnaire
fulfilled by different laboratories and consultation of some experts, the present position,
pending of the discussions in the Lille Meeting, are the following.
Regarding sampling plan, sample collection, transport, storage and test portion, it seems that
the same decision taken for bacteria (E. coli) can be adopted. In any case since bacteriophages
HORIZONTAL Factsheet
BACTERIOPHAGES
version: 3/6/2016
are more persistent than bacteria the storage period can be extended.
The pre-treatment and the extraction stages had been identified as crucial steps for the
detection and enumeration of bacteriophages in biosolids and these will be the major issues of
discussion.
Regarding bacteriophages to test, all the information indicates that somatic coliphages are the
best suited for the purposes of testing bacteriophages in sludges. As well it seems to be a lot
of consensus on the methods for bacteriophage detection, since there is a feasible and well
accepted ISO method.