SIST-TS CEN/TS 17720:2023
(Main)Plant biostimulants - Determination of Enterococcaceae
Plant biostimulants - Determination of Enterococcaceae
This methodology has been developed to determine enterococci in biostimulants as a single microorganism component or in a mixture with other microorganisms. This document is not applicable to mineral fertilizers that are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40 % crude ash (Council Directive 79/373/EEC) [3].
Pflanzen-Biostimulanzien - Bestimmung von Enterococcaceae
Dieses Verfahren wurde zur Bestimmung von Enterokokken in Biostimulanzien als einzelnen Mikroorganismus-Bestandteil oder in einem Gemisch mit anderen Mikroorganismen entwickelt. Dieses Dokument ist nicht anwendbar auf mineralische Düngemittel, die als Ergänzungsfuttermittel definiert sind, hauptsächlich aus Mineralstoffen zusammengesetzt sind und mindestens 40 % Rohasche enthalten (Richtlinie des Rates 79/373/EWG) [3].
Biostimulants des végétaux - Détermination des Enterococcaceae
Cette méthodologie a été développée pour déterminer les entérocoques dans les biostimulants en tant que composant unique d'un microorganisme ou dans un mélange avec d'autres microorganismes. Le présent document ne s’applique pas aux engrais minéraux utilisés comme des aliments complémentaires principalement composés de minéraux et contenant au moins 40 % de cendres brutes (Règlement (CE) N° 767/2009)) [3].
Rastlinski biostimulanti - Določanje Enterococcaceae
Ta metodologija je bila razvita za določanje enterokokov v biostimulantih kot posamezne komponente mikroorganizmov ali v mešanici z drugimi mikroorganizmi. Dokument se ne uporablja za mineralna gnojila, ki so opredeljena kot dopolnilna krmna mešanica, sestavljena predvsem iz mineralov, in vsebujejo najmanj 40 % surovega pepela (Direktiva Sveta 79/373/EGS) [3].
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 17720:2023
01-februar-2023
Rastlinski biostimulanti - Določanje Enterococcaceae
Plant biostimulants - Determination of Enterococcaceae
Pflanzen-Biostimulanzien - Bestimmung von Enterococcaceae
Biostimulants des végétaux - Détermination des Enterococcaceae
Ta slovenski standard je istoveten z: CEN/TS 17720:2022
ICS:
65.080 Gnojila Fertilizers
SIST-TS CEN/TS 17720:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN/TS 17720:2023
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SIST-TS CEN/TS 17720:2023
CEN/TS 17720
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version
Plant biostimulants - Determination of Enterococcaceae
Biostimulants des végétaux - Détermination des Biostimulanzien für die pflanzliche Anwendung -
Enterococcaceae Bestimmung von Enterococcaceae
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
E UR OPÄISCHES 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 17720:2022 E
worldwide for CEN national Members.
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SIST-TS CEN/TS 17720:2023
CEN/TS 17720:2022 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 7
5 Diluents, selective media and test kit for phenotypic characterization . 7
5.1 General . 7
5.2 Diluent for initial suspension and for serial dilutions . 7
5.3 Selective media . 7
5.4 Phenotypic characterization and confirmation . 7
6 Apparatus and glassware . 8
7 Sampling . 8
8 Preparation of test sample . 8
8.1 General . 8
8.2 Liquid – water-based formulations . 8
8.3 Liquid – oil-based (emulsifiable concentrate - EC) formulations . 9
8.4 Solid – wettable powder (WP) formulations . 9
8.5 Solid – water dispersible granules (WDG) formulations . 9
8.6 Solid – pellets, granules, microgranules (slow release) formulations . 9
– substrate . 9
8.7 Solid
9 Procedure. 9
9.1 Inoculation and incubation of the plates . 9
9.2 Evaluation of results . 10
9.3 Confirmation step on BEA . 10
9.4 Catalase test . 10
10 Expression of results . 10
11 Precision . 10
11.1 General . 10
11.2 Interlaboratory study . 10
11.3 Repeatability . 10
11.4 Reproducibility . 11
12 Test report . 11
Annex A (normative) Solutions, media . 12
A.1 Diluents for initial suspension and serial dilutions . 12
A.2 Elective media . 13
Bibliography . 14
2
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SIST-TS CEN/TS 17720:2023
CEN/TS 17720:2022 (E)
European foreword
This document (CEN/TS 17720:2022) 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.
3
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SIST-TS CEN/TS 17720:2023
CEN/TS 17720:2022 (E)
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 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 (“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”. Working Group 5 “Labelling and
denominations” was created to develop a work program as part of this standardization request.
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 commercialization of the European biostimulant industry.
Biostimulants used in agriculture can be applied in multiple ways: to the soil, to plants, as seed
treatments, 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.
This document is applicable to all microbial biostimulants in agriculture.
Table 1 summarizes many of the agro-ecological principles and the role played by biostimulants.
Table 1 — Agro-ecological principles and the role played by biostimulants [1]
Increase biodiversity
By improving soil microorganism quality/quantity
Reinforce biological regulation and interactions
By reinforcing plant- microorganism interactions
— symbiotic exchanges i.e. Mycorrhizae
— symbiotic exchanges i.e. Rhizobiaceae/Fava
— secretions mimicking plant hormones (i.e. Trichoderma)
By regulating plant physiological processes
— e.g. growth, metabolism, plant development…
Improve biogeochemical cycles
— improve absorption of nutritional elements
— improve bioavailability of nutritional elements in the soil
— stimulate degradation of organic matter
4
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SIST-TS CEN/TS 17720:2023
CEN/TS 17720:2022 (E)
This methodology has been developed to enumerate enterococci and enable the European Commission
to control proper labelling of plant biostimulant products. It is mainly based on EN 15788:2009, Animal
feeding stuffs - Isolation and enumeration of Enterococcus (E. faecium) spp. The method is based on an
extensive screening of 12 pre-selected, commercially available media for the detection and enumeration
of enterococci. The described methodology was validated in an interlaboratory study [2].
This method can be applied to enumerate enterococci in additives, premixtures and biostimulants.
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.
5
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SIST-TS CEN/TS 17720:2023
CEN/TS 17720:2022 (E)
1 Scope
This methodology has been developed to determine enterococci in biostimulants as a single
microorganism component or in a mixture with other microorganisms. This document is not applicable
to mineral fertilizers that are defined as complementary feeding stuffs composed mainly of minerals and
containing at least 40 % crude ash (Regulation (EC) No 767/2009) [3].
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.
1
EN ISO 7218:2007, Microbiology of food and animal feeding stuffs - General requirements and guidance
for microbiological examinations (ISO 7218:2007)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
Enterococcus spp.
bacteria forming colonies on the specified selective medium Slanetz Bartley agar after incubation of
spread plates for 44 h at a temperature of 36 °C under aerobic conditions
Note 1 to entry: Colony description:
— circular;
— convex to dome-shaped;
— entire;
— glistening surface;
— pink, red, brown colour;
— opaque.
Note 2 to entry: Colony size varies between 0,5 mm and 2 mm in diameter.
Note 3 to entry: When using Bile Esculin Azide agar (BEA) the medium surrounding the colonies shows a dark
brown to black coloration, due to the hydrolysis of esculin.
Note 4 to entry: Microscopic examination of selected colonies typically shows diplococci or short chains of cocci.
1
As impacted by EN ISO 7218:2007/A1:2013.
6
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SIST-TS CEN/TS 17720:2023
CEN/TS 17720:2022 (E)
4 Principle
An initial suspension of the sample is prepared in a diluent with suitable buffer capacity using a suitable
homogenizer. Dilutions of the initial suspension (serial ten-fold dilutions) shall be immediately prepared
before the suspension settles. Subsequently plate 0,1 ml of each dilutio
...
SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17720:2021
01-november-2021
[Not translated]
Plant biostimulants - Determination of Enterococcaceae
Biostimulanzien für die pflanzliche Anwendung - Bestimmung von Enterococcaceae
Ta slovenski standard je istoveten z: FprCEN/TS 17720
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17720:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST-TS FprCEN/TS 17720:2021
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kSIST-TS FprCEN/TS 17720:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17720
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
September 2021
ICS 65.080
English Version
Plant biostimulants - Determination of Enterococcaceae
Biostimulanzien für die pflanzliche Anwendung -
Bestimmung von Enterococcaceae
This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee
CEN/TC 455.
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.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change
without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17720:2021 E
worldwide for CEN national Members.
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kSIST-TS FprCEN/TS 17720:2021
FprEN 17720:2021 (E)
Contents
European foreword . 3
Introduction . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 7
5 Diluents, selective media and test kit for phenotypic characterization . 7
5.1 General . 7
5.2 Diluents . 7
5.2.1 Diluent for initial suspension and for serial dilutions. 7
5.3 Selective media . 7
5.4 Phenotypic characterization and confirmation . 8
6 Apparatus and glassware . 8
7 Sampling . 8
8 Preparation of test sample . 8
8.1 General . 8
8.2 Liquid - water based- formulations . 8
8.3 Liquid – oil based (emulsifiable concentrate - EC) formulations . 9
8.4 Solid - Wettable Powder (WP) formulations . 9
8.5 Solid - Water dispersible granules (WDG) formulations . 9
8.6 Solid – Pellets, granules, microgranules (slow release) formulations . 9
8.7 Solid - substrate . 9
9 Procedure . 9
9.1 Inoculation and incubation of the plates . 9
9.2 Evaluation results . 10
9.3 Confirmation step on BEA . 10
9.4 Catalase test. 10
10 Expression of results . 10
11 Precision . 10
11.1 General . 10
11.2 Interlaboratory study . 10
11.3 Repeatability . 10
11.4 Reproducibility . 10
12 Test report . 11
Annex A (normative) Solutions, media . 12
Bibliography . 14
2
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kSIST-TS FprCEN/TS 17720:2021
FprEN 17720:2021 (E)
European foreword
This document (FprCEN/TS 17720:2021) has been prepared by Technical Committee CEN/TC
455 “Plant Biostimulants”, the secretariat of which is held by AFNOR.
This document is currently submitted to the Vote on TS.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
3
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kSIST-TS FprCEN/TS 17720:2021
FprEN 17720:2021 (E)
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 fertilising products (“FPR” or “Fertilising Products Regulation”). This request,
presented as SR 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 commercialization of the
European biostimulant industry.
Biostimulants used in agriculture can be applied in multiple ways: to the soil, to plants, as seed
treatments, 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 Fertilizing
Products Regulation.
This document is applicable to all microbial biostimulants in agriculture.
Table 1 summarizes many of the agro-ecological principles and the role played by biostimulants.
Table 1 — Agro-ecological principles and the role played by biostimulants [1]
Increase biodiversity
By improving soil microorganism quality/quantity
Reinforce biological regulation and interactions
By reinforcing plant- microorganism interactions
— symbiotic exchanges i.e. mycorrhize
— symbiotic exchanges i.e. rhizobiaciae/fava
— secretions mimicking plant hormones (i.e. trichoderma)
By regulating plant physiological processes
— for ex growth, metabolism, plant development…
Improve biogeochemical cycles
— improve absorption of nutritional elements
— improve bioavailability of nutritional elements in the soil
— stimulate degradation of organic matter
4
---------------------- Page: 6 ----------------------
kSIST-TS FprCEN/TS 17720:2021
FprEN 17720:2021 (E)
This methodology has been developed to enumerate enterococci and enable the European
Commission to control proper labelling of plant biostimulant products. It is mainly based on
EN 15788:2009, Animal feeding stuffs - Isolation and enumeration of Enterococcus (E. faecium) spp.
The method is based on an extensive screening of 12 pre-selected, commercially available media
for the detection and enumeration of enterococci. The described methodology was validated in an
interlaboratory study [2].
This method can be applied to enumerate enterococci in additives, premixtures and biostimulants.
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.
5
---------------------- Page: 7 ----------------------
kSIST-TS FprCEN/TS 17720:2021
FprEN 17720:2021 (E)
1 Scope
This methodology has been developed to determine enterococci in biostimulants as a single
microorganism component or in a mixture with other microorganisms. This document is not
applicable to mineral fertilizers that are defined as complementary feeding stuffs composed
mainly of minerals and containing at least 40 % crude ash (Council Directive 79/373/EEC) [3].
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.
1
EN ISO 7218:2007, Microbiology of food and animal feeding stuffs - General requirements and
guidance for microbiological examinations (ISO 7218:2007)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following
addresses:
• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
3.1
enterococcus spp.
enterococcus spp. form colonies on the specified selective medium Slanetz Bartley agar after
incubation of spread plates for 44 h at a temperature of 36 °C under aerobic conditions
Note 1 to entry: Described by their characteristics.
Note 2 to entry: Colony description:
• circular;
• convex to dome-shaped;
• entire;
• glistening surface;
• pink, red, brown colour;
• opaque.
Note 3 to entry: Colony size varies between 0,5 mm and 2 mm in diameter.
1
As impacted by EN ISO 7218:2007/A1:2013.
6
---------------------- Page: 8 ----------------------
kSIST-TS FprCEN/TS 17720:2021
FprEN 17720:2021 (E)
Note 4 to entry: When using Bile Esculin Azide agar (BEA) the medium surrounding the colonies shows a
dark brown to black coloration, due to the hydrolysis of esculin.
Note 5 to entry: Microscopic examination of selected colonies typically shows diplococci or short chains of
cocci.
4 Principle
An initial suspension of the sample is prepared in a diluent with suitable buffer capacity using a
suitable homogenizer. Dilutions of the initial suspension (serial ten-fold dilutions) shall be
immediately prepared before the suspension settles. Subsequently plate 0,1 ml of each dilution
onto Slanetz-Bartley agar. This selective medium (as reported in EN ISO 7899-2:2000) is a
selective media that inhibits the growth of Gram-negative bacteria and Staphylococci. Incubate at
36 ± 1 °C for 44 h. The growing colonies show a pink-red or red-brown colour and should be
further confirmed by subsequent inoculation onto BEA pre-heated plates (44 °C for 2 h).
If reaching a lower detection limit is required 1 ml of the undiluted biostimulant product or 1 ml
−1
of the first dilution (10 ) can be spread over the surface of agar plates (140 mm diameter or 3
smaller plates having a diameter of 90 mm).
BEA plates are inoculated with 0,1 ml of the chosen dilutions. The plates are incubated aerobically
for 24 h ± 2 h at 44 °C ± 1 °C.
Presumptive enterococci colonies are counted and the number of colony-forming units (CFU) per
g or ml i
...
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