CEN/TS 17717:2022

Plant biostimulants - Detection of Salmonella spp.

This document describes a method for the detection of Salmonella spp. in biostimulants of the following Product Function Categories (PFCs) and Component Material Category (CMC) of EU fertilizing products, as described in the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1]:
—   PFC 6(A): Microbial plant biostimulant;
—   PFC 6(B): Non-microbial plant biostimulant;
—   CMC 7: Microorganisms.
It requires three successive steps: a selective enrichment, an isolation on a chromogenic agar, and if positive a confirmation with a serological test (and if required, a selective media).

Pflanzen-Biostimulanzien - Nachweis von Salmonella spp.

Dieses Dokument beschreibt ein Verfahren zum Nachweis von Salmonella spp. in Biostimulanzien der folgenden Produktfunktionskategorien (PFCs, en: Product Function Categories) und Komponenten-materialkategorien (CMC, en: Component Material Category) von EU Düngeprodukten, wie in der Verordnung (EU) 2019/1009 des Europäischen Parlaments und des Rates [1] beschrieben:
—   PFC 6(A): Mikrobielles Pflanzen-Biostimulans;
—   PFC 6(B): Nicht-mikrobielles Pflanzen-Biostimulans;
—   CMC 7: Mikroorganismen.
Es erfordert drei aufeinanderfolgende Schritte: eine selektive Anreicherung, eine Isolierung auf einem chromogenen Agar und bei positivem Befund eine Bestätigung mit einer serologischen Untersuchung (und, falls erforderlich, einem Selektivmedium).

Biostimulants des végétaux - Détection de Salmonella spp.

Le présent document décrit une méthode pour la recherche des Salmonella spp. dans les biostimulants des catégories fonctionnelles de produits (PFC) et de la catégorie de matières constitutives (CMC) des fertilisants UE décrites dans le règlement (EU) 2019/1009 du Parlement européen et du Conseil [1] :
- PFC 6(A) : Biostimulant microbien des végétaux ;
- PFC 6(B) : Biostimulant non-microbien des végétaux ;
- CMC 7 : Microorganismes.
Elle nécessite trois phases successives : un enrichissement sélectif, un isolement sur un milieu gélosé chromogène, et, en cas de résultat positif, une confirmation par un essai sérologique (et, si nécessaire, un milieu sélectif).

Rastlinski biostimulansi - Ugotavljanje prisotnosti salmonele (Salmonella spp.)

Ta dokument opisuje metodo za ugotavljanje prisotnosti Salmonella spp. v biostimulantih naslednjih funkcijskih kategorij proizvodov (PFC) in kategorij sestavnih materialov (CMC) v sredstvih za gnojenje EU, kot je opisano v Uredbi (EU) 2019/1009 Evropskega parlamenta in Sveta [1]:
–   PFC 6(A): mikrobni rastlinski biostimulanti;
–   PFC 6(B): nemikrobni rastlinski biostimulanti;
–   CMC 7: mikroorganizmi.
Metoda zahteva tri zaporedne korake: neselektivno obogatitev, izolacijo na kromogenem agarju in, če je rezultat pozitiven, potrditev s serološkim preskusom (in po potrebi s selektivnim gojiščem).

General Information

• Status: Withdrawn
• Publication Date: 22-Mar-2022
• Withdrawal Date: 13-Apr-2025
• ICS: 65.080 - Fertilizers
• Technical Committee: CEN/TC 455 - Plant Biostimulants and Agricultural Micro-Organisms
• Drafting Committee: CEN/TC 455/WG 3 - Pathogenic and non-pathogenic microorganisms
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Start Date: 27-Nov-2024
• Completion Date: 14-Apr-2025
• Directive: 2019/1009 - Regulation (EU) 2019/1009 of the European Parliament and of the Council of 5 June 2019 laying down rules on the making available on the market of EU fertilising products and amending Regulations (EC) No 1069/2009 and (EC) No 1107/2009 and repealing Regulation (EC) No 2003/2003
• Mandate: M/564 - Fertilising products in support of Regulation (EU) 2019/1009
• Ref Project: SIST-TS CEN/TS 17717:2023 - Plant biostimulants - Detection of Salmonella spp

Relations

• Replaced By: EN 17717:2024 - Plant biostimulants - Detection of Salmonella spp.
• Effective Date: 08-Feb-2023

Standards Content (Sample)

SLOVENSKI STANDARD
01-februar-2023
Rastlinski biostimulansi - Ugotavljanje prisotnosti salmonele (Salmonella spp.)
Plant biostimulants - Detection of Salmonella spp
Pflanzen-Biostimulanzien - Nachweis von Salmonella spp.
Biostimulants des végétaux - Détection de Salmonella spp.
Ta slovenski standard je istoveten z: CEN/TS 17717:2022
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TS 17717
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version
Plant biostimulants - Detection of Salmonella spp.
Biostimulants des végétaux - Détection de Salmonella Pflanzen-Biostimulanzien - Nachweis von Salmonella
spp. spp.
This Technical Specification (CEN/TS) was approved by CEN on 3 January 2022 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

EUROPÄISCHES KOMITEE FÜR NORMUN G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17717:2022 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
4.1  General . 6
4.2  Enrichment in selective liquid medium . 6
4.3  Plating out on selective solid media . 6
4.4  Confirmation . 6
5 Culture media, reagents, antisera . 6
5.1  General . 6
5.2  Isolation chromogenic agar . 7
5.3  Non-selective agar . 7
5.4  Confirmation selective agar . 7
6 Equipment and consumables . 7
7 Sampling . 8
8 Preparation of test sample . 8
9 Procedure . 8
9.1  Test portion and initial suspension . 8
9.2  Selective pre-enrichment . 9
9.3  Isolation . 9
9.4 Confirmation . 9
10 Expression of results . 11
11 Performance characteristics of the method . 11
11.1   Interlaboratory studies . 11
11.2   Sensitivity . 12
11.3   Specificity . 12
11.4   LOD . 12
12 Test report . 12
Annex A (normative) Diagram of the procedures . 13
Annex B (normative) Culture media and reagents . 14
Annex C (informative) Examples of selective plating-out media . 18
Bibliography . 19
European foreword
This document (CEN/TS 17717:2022) has been prepared by Technical Committee CEN/TC 455
“Plant Biostimulants”, the secretariat of which is held by AFNOR.
Attention is drawn to the possibility that some of the elements of this document may be the subject
of patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
Any feedback and questions on this document should be directed to the users’ national standards
body. A complete listing of these bodies can be found on the CEN website.
According to the CEN/CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to announce this Technical Specification: Austria, Belgium,
Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain,
Sweden, Switzerland, Turkey and the United Kingdom.
Introduction
This document was prepared by the experts of CEN/TC 455 “Plant Biostimulants”. The European
Committee for Standardization (CEN) was requested by the European Commission (EC) to draft
European standards or European standardization deliverables to support the implementation of
Regulation (EU) 2019/1009 of 5 June 2019 laying down rules on the making available on the
market of EU fertilizing products (“FPR” or “Fertilising Products Regulation”).
This standardization request, presented as M/564, also contributes to the Communication on
“Innovating for Sustainable Growth: A Bio economy for Europe”. The Working Group 5 “Labelling
and denominations”, was created to develop a work program as part of this request. The technical
committee CEN/TC 455 “Plant Biostimulants” was established to carry out the work program that
will prepare a series of standards. The interest in biostimulants has increased significantly in
Europe as a valuable tool to use in agriculture. Standardization was identified as having an
important role in order to promote the use of biostimulants. The work of CEN/TC 455 seeks to
improve the reliability of the supply chain, thereby improving the confidence of farmers, industry,
and consumers in biostimulants, and will promote and support commercialisation of the
European biostimulant industry.
Biostimulants used in agriculture can be applied in multiple ways: on soil, on plants, as seed
treatment, etc. A microbial plant biostimulant consists of a microorganism or a consortium of
microorganisms, as referred to in Component Material Category 7 of Annex II of the EU Fertilising
Products Regulation (EU) 2019/1009 [1].
This document is applicable to all microbial biostimulants in agriculture. The method is based on
the EN ISO 6579-1:2017 for the detection of Salmonella spp. in food.
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.
1 Scope
This document describes a method for the detection of Salmonella spp. in biostimulants of the
following Product Function Categories (PFCs) and Component Material Category (CMC) of EU
fertilizing products, as described in the Regulation (EU) 2019/1009 of the European Parliament
and of the Council [1]:
— PFC 6(A): Microbial plant biostimulant;
— PFC 6(B): Non-microbial plant biostimulant;
— CMC 7: Microorganisms.
It requires three successive steps: a selective enrichment, an isolation on a chromogenic agar, and
if positive a confirmation with a serological test (and if required, a selective media).
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies.
For undated references, the latest edition of the referenced document (including any
amendments) applies.
CEN/TS 17708, Plant biostimulants — Preparation of sample for microbial analysis
CEN/TS 17724, Plant Biostimulants — Terminology
EN ISO 7218:2007, Microbiology of food and animal feeding stuffs — General requirements and
guidance for microbiological examinations (ISO 7218:2007)
EN ISO 11133:2014, Microbiology of food, animal feed and water — Preparation, production,
storage and performance testing of culture media (ISO 11133:2014)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in CEN/TS 17724 and the
following apply.
3.1
Salmonella spp.
microorganisms which form typical colonies on solid selective media described and which display
the morphological, physiological and biochemical characteristics described when the analysis is
carried out in accordance with this document
3.2
detection of Salmonella spp.
determination of the detection or not detection of Salmonella spp. (3.1), in 25 g or 25 ml of
product, when tests are carried out in accordance with this document

As amended by EN ISO 7218:2007/A1:2013.
As amended by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020.
3.3
laboratory sample
sample intended for laboratory inspection or testing
3.4
test sample
sample prepared from the laboratory sample and from which test portions will be taken
3.5
test portion
quantity of material taken from the test sample (or if both are the same, from the laboratory
sample) and on which the test is carried out
4 Principle
4.1 General
The detection of Salmonella requires three successive steps as specified in Annex A. The three
steps are the selective enrichment, the isolation on a chromogenic agar, and the confirmation with
a serological test (and if required, a selective media).
NOTE Salmonella can be present in small numbers and are often accompanied by considerably larger
numbers of other bacteria, such as Enterobacteriaceae or of other families. Enrichment is used to allow the
detection of low numbers of Salmonella or stressed Salmonella.
Stressed microorganisms are defined here as those present in the environment that can be injured
or that can have developed in harsh environments. Such organisms can be difficult to detect
because they struggle to grow on selective media. However, under suitable conditions, they can
repair the cellular damages and recover their normal properties.
4.2 Enrichment in selective liquid medium
Buffered peptone water (BPW) containing 10 mg/l Novobiocin at room temperature is inoculated
with the test portion, then incubated between 34 °C and 38 °C for 18 h ± 2 h.
For large quantities (e.g. 1 l or more), it is recommended to pre-warm the BPW to 34 °C to 38 °C
before mixing it with the test portion.
4.3 Plating out on selective solid media
From the enrichment obtained in 4.2, the chromogenic solid media (5.2) is inoculated.
This selective agar is incubated between 34 °C and 38 °C for 24 h ± 3 h (or according to the
manufacturer’s instructions if explicitly recommended).
4.4 Confirmation
Colonies of presumptive Salmonella are confirmed by means of appropriate serological test. If the
serological test gives a negative result, the inoculation of a selective agar (B.5) is required.
If the test gives a negative result, up to 4 other presumptive colonies will be tested (if possible and
up to 5 colonies in total).
5 Culture media, reagents, antisera
5.1 General
1 2
For current laboratory practice, see EN ISO 7218:2007 and EN ISO 11133:2014 .
Composition of culture media and reagents and their preparation are described in Annex B.
5.2 Isolation chromogenic agar
This isolation medium is chosen by the testing laboratory and shall highlight the C8-esterase
enzymatic activity. For examples of isolation media, see Annex C, Table C.1.
5.3 Non-selective agar
General purpose agar supporting the growth of a wide range of non-fastidious strains. See B.4.
5.4 Confirmation selective agar
This isolation medium is chosen by the testing laboratory and shall highlight the production of
hydrogen sulphide (H S) by the strains (see B.5). For examples of isolation media, see Annex C,
Table C.2.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable
specifications.
Usual microbiological laboratory equipment (see EN ISO 7218:2007 ) and, in particular, the
following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave). As specified in
EN ISO 7218:2007 .
6.2 Drying cabinet or oven, capable of operating between 25 °C and 50 °C.
6.3 Incubator(s), capable of operating in the range 34 °C to 38 °C and at 37 °C ± 1 °C.
6.4 Incubator, capable of operating at 41,5 °C ± 1 °C or water bath capable of operating at
41,5 °C ± 1 °C.
6.5 Water bath, capable of operating at 47 °C to 50 °C.
6.6 Water bath, capable of operating at 37 °C ± 1 °C.
6.7 Water bath, capable of operating at 45 °C ± 1 °C. It is recommended to use a water bath (6.4
to 6.7) containing an antibacterial agent because of the low infective dose of Salmonella spp.
6.8 Cooling unit, adjustable at 5 °C ± 3 °C.
6.9 Freezer, capable of operating at − 20 °C ± 5 °C.
6.10 Sterile loops of approximate diameter, 3 mm (10 μl volume).
6.11 pH-meter having an accuracy of calibration of ± 0,1 pH unit from 20 °C to 25 °C.
6.12 Sterile tubes, bottles, or flasks with caps, of appropriate capacity.
6.13 Sterile graduated pipettes or automatic pipettes of nominal capacities of 25 ml, 10 ml,
1 ml, and 0,1 ml.
6.14 Sterile Petri dishes with a diameter of approximately 90 mm and (optional) large size
(diameter approximately 140 mm).
® 3
6.15 Peristaltic blender (stomacher ) with sterile bags.
6.16 Sterile filter with a 0,2 µm porosity.
7 Sampling
Sampling is not part of the method specified in this document (see CEN/TS 17702-1 dealing with
the product concerned). If there is no specific International or European Standard, it is
recommended that the parties concerned come to an agreement on this subject.
It is important that the laboratory receives a laboratory sample (3.3) which is representative and
has not been damaged or changed during transport or storage.
8 Preparation of test sample
Preparation of test sample (3.4) from the laboratory sample is not part of the method specified in
this document (see CEN/TS 17702-1).
For the microbiological examination, follow a specific standard appropriate to the product
concerned if no specific method is provided by the manufacturer. If necessary use one or more of
the apparatus on the basis of the nature of the product.
All the operations, before and after opening the products, shall be carried out aseptically to avoid
external contamination.
Sterile material and equipment shall be used.
Frozen products may be defrosted before testing, standing at 18 °C to 27 °C (laboratory ambient
temperature) for a maximum of 3 h, or at 5 °C ± 3 °C for a maximum of 24 h. After this, samples
shall be tested as quickly as possible.
Solid (powdered and granulated) products shall be thoroughly mixed in their container and weigh
out using aseptic techniques, taking the required test portion at random in small increments with
a spatula.
For dehydrated and other low-moisture products, it is important to weigh the diluent and then
add it gradually onto the test portion to reduce osmotic shock on any microorganism presen
...