Organic, organo-mineral and inorganic fertilizers - Detection of Enterocococaceae

This document specifies a method for the detection and enumeration of Enterococcaceae in fertilizers of the following Product Function Categories (PFCs) of EU fertilizing products, as described in Regulation (EU) 2019/1009 [1]:
•   PFC 1(A): Organic fertilizer;
•   PFC 1(B): Organo-mineral fertilizer;
•   PFC 1(C): Inorganic fertilizer, which contains more than 1 % by mass of organic carbon, other than organic carbon from chelating or complexing agents, nitrification inhibitors, denitrification inhibitors or urease inhibitors, coating agents, urea or calcium cyanamide. The present method was validated on products known as present on the market in April 2021 and conform to Regulation (EU) 2019/1009 [1] that are inorganic fertilizers with more than 1 % of organic carbon such as poultry manure and struvite with low level of organic matter. In case that other products would be developed having other physical and chemical characteristics, it might become necessary to develop different methods to correctly account for pathogens they might contain.
This document specifies a colony-count technique on selective media, Slanetz Bartley agar or Bile Esculin Azide agar, respectively. The method is based on EN ISO 7899 2:2000.

Organische, organisch-mineralische und anorganische Düngemittel - Nachweis von Enterococcaceae

Dieses Dokument legt ein Verfahren zum Nachweis von Enterococcaceae in Düngemitteln der folgenden Produktfunktionskategorien (PFCs, en: Product Function Categories) von EU-Düngeprodukten fest, wie in der Verordnung (EU) 2019/1009 [1] beschrieben:
— PFC 1(A): organisches Düngemittel;
— PFC 1(B): organisch-mineralisches Düngemittel;
— PFC 1(C): anorganisches Düngemittel, das mehr als 1 % Masseanteil an organischem Kohlenstoff enthält, ausgenommen organischer Kohlenstoff aus Chelatbildnern oder Komplexbildnern, Nitrifikations-hemmstoffen, Denitrifikationshemmstoffen oder Ureasehemmstoffen, Hüllstoffen, Harnstoff oder Kalkstickstoff. Das vorliegende Verfahren wurde an Produkten validiert, von denen bekannt ist, dass sie im April 2021 auf dem Markt waren und der Verordnung (EU) 2019/1009 [1] entsprechen und bei denen es sich um anorganische Düngemittel mit mehr als 1 % organischem Kohlenstoff handelt, wie z. B. Geflügelmist und Struvit mit geringem Gehalt an organischer Substanz. Für den Fall, dass andere Produkte mit anderen physikalischen und chemischen Eigenschaften entwickelt werden, könnte es notwendig werden, andere Verfahren zu entwickeln, um die darin möglicherweise enthaltenen Pathogene korrekt zu erfassen.
Dieses Dokument legt ein Koloniezählverfahren auf Selektivmedien fest, Slanetz-Bartley-Agar bzw. Galle-Äsculin-Azid-Agar. Dieses Verfahren basiert auf EN ISO 7899-2:2000.

Engrais organiques, organo-minéraux et inorganiques - Recherche des Enterococaceae

Le présent document spécifie une méthode pour la recherche et le dénombrement des Enterococcaceae dans les engrais des catégories fonctionnelles de produits (PFC) des fertilisants UE décrites dans le règlement (UE) 2019/1009 [1] :
•   PFC 1(A) : engrais organique ;
•   PFC 1(B) : engrais organo-minéral ;
•   PFC 1(C) : engrais inorganique, contenant plus de 1 % en masse de carbone organique, autre que le carbone organique des agents chélatants ou complexants, des inhibiteurs de nitrification, des inhibiteurs de dénitrification ou des inhibiteurs d’uréase, des agents d’enrobage, de l’urée ou du cyanamide calcique. La présente méthode a été validée sur des produits connus comme présents sur le marché en avril 2021 et conformes au règlement (UE) 2019/1009 [1] qui sont des engrais inorganiques contenant plus de 1 % de carbone organique, tel que la poulaitte et la struvite avec un niveau bas de matière organique. Dans le cas où d'autres produits seraient développés avec d'autres caractéristiques physiques et chimiques, il pourrait s'avérer nécessaire de développer différentes méthodes pour rendre compte correctement des pathogènes qu'ils pourraient contenir.
Le présent document spécifie une technique de comptage des colonies sur des milieux sélectifs, la gélose Slanetz Bartley ou la gélose Bile-Esculine-Azoture, respectivement. La méthode est basée sur l’EN ISO 7899 2:2000.

Organska, organsko-mineralna in anorganska gnojila - Ugotavljanje prisotnosti enterokokov (Enterococcaceae)

General Information

Status
Published
Publication Date
17-May-2022
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
18-May-2022
Due Date
04-Feb-2023
Completion Date
18-May-2022

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SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17804:2022
01-januar-2022
Organska, organsko-mineralna in anorganska gnojila - Ugotavljanje prisotnosti
enterokokov (Enterococcaceae)
Organic, organo-mineral and inorganic fertilizers - Detection of Enterococcaceae
Organische, organisch-mineralische und anorganische Düngemittel - Nachweis von
Enterococcaceae
Engrais organiques, organo-minéraux et inorganiques - Recherche des Enterococaceae
Ta slovenski standard je istoveten z: FprCEN/TS 17804
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17804:2022 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 17804:2022

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kSIST-TS FprCEN/TS 17804:2022


FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17804
SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION

December 2021
ICS 65.080
English Version

Organic, organo-mineral and inorganic fertilizers -
Detection of Enterococcaceae
Engrais organiques, organo-minéraux et inorganiques - Organische, organisch-mineralische und anorganische
Recherche des Enterococaceae Düngemittel - Nachweis von Enterococcaceae


This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee
CEN/TC 260.

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 17804:2021 E
worldwide for CEN national Members.

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kSIST-TS FprCEN/TS 17804:2022
FprCEN/TS 17804:2021(E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 7
5 Reagents . 8
5.1 General. 8
5.2 Diluents . 8
5.2.1 General. 8
5.2.2 Basic phosphate buffer . 8
5.2.3 Double-buffered phosphate buffer . 8
5.3 Selective media . 8
5.3.1 Slanetz Bartley agar . 8
5.3.2 Bile Esculin Azide agar . 8
6 Equipment and consumables . 9
6.1 General. 9
6.2 Equipment for dry sterilization (oven) and wet sterilization (autoclave) . 9
6.3 Incubator. 9
6.4 Blending equipment . 9
6.5 Mechanical stirrer . 9
6.6 Scale . 9
6.7 Water bath . 9
6.8 Cooling unit, adjustable at 5 °C ± 3 °C. . 9
6.9 pH meter, capable of reading to the nearest 0,1 pH unit at 20 °C to 25 °C. . 9
6.10 Sterile loops of approximate diameter, 3 mm (10 μl volume). . 9
6.11 Sterile tubes, bottles, or flasks with caps of appropriate capacity. . 9
6.12 Pipettes or pipettor and sterile tips of nominal capacities of 10 ml and 1 ml. . 9
6.13 Sterile Petri dishes with a diameter of approximately 90 mm and (optional) large size
(diameter approximately 140 mm). . 9
7 Sampling . 9
8 Preparation of test sample . 10
9 Procedure (see Figure A.1 in Annex A) . 10
9.1 Preparation of the initial suspension and decimal dilutions . 10
9.2 Inoculation and incubation . 10
9.2.1 General. 10
9.2.2 Procedure . 11
9.3 Enumeration of colonies . 11
9.4 Confirmation (optional) . 11
9.4.1 General. 11
9.4.2 Confirmation on the second selective medium . 11
9.4.3 Catalase Test . 12
2

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FprCEN/TS 17804:2021(E)
10 Expression of results . 12
11 Test report . 13
Annex A (normative) Diagram of the procedure . 14
Annex B (normative) Composition and preparation of culture media and reagents . 15
B.1 General . 15
B.2 Basic phosphate buffer . 15
B.2.1 Composition . 15
B.2.2 Preparation . 15
B.3 Double-buffered phosphate buffer . 16
B.3.1 Composition . 16
B.3.2 Preparation . 16
B.4 Slanetz Bartley agar . 16
B.4.1 Composition . 16
B.4.2 Preparation . 16
B.5 Bile Esculin Azide agar . 17
B.5.1 Composition . 17
B.5.2 Preparation . 17
Bibliography . 18

3

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kSIST-TS FprCEN/TS 17804:2022
FprCEN/TS 17804:2021 (E)
European foreword
This document (FprCEN/TS 17804:2021) has been prepared by the Technical Committee CEN/TC 260
“Fertilizers and liming materials”, the secretariat of which is held by DIN.
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.
4

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FprCEN/TS 17804:2021(E)
Introduction
This methodology has been developed to detect and enumerate Enterococcaceae in organic, organo-
mineral and inorganic fertilizers in order to be able to control certain hygienic requirements in
Regulation (EU) 2019/1009 [1].
Enterococcaceae in the sense of this document include several species of the genus Enterococcus (3.7, 3.8)
with a faecal origin. Consequently, it can be used as an indicator of faecal contamination. It can also be
used to monitor the effectiveness of pasteurization or disinfection treatments. Compared to E.coli, they
have a higher tenacity and can therefore better reflect the behaviour of all pathogens in fertilizers.
Because of the large variety of fertilizers, this method is not appropriate in every detail for certain
products. In this case, different methods which are specific to these products can be used if absolutely
necessary for justified technical reasons. Nevertheless, every attempt should be made to apply this
method as far as possible.
Mineral components in fertilizers can have a negative impact on the survivability of microorganisms
when they go into solution. In addition to an unfavourable shift in the pH value, the products can have a
strong osmotic effect or be toxic to cells themselves (e.g. copper). Therefore, it can be necessary to test
the inhibitory effect of the fertilizers to be investigated in a pre-test.
The method is validated in an interlaboratory study for the following products (Enterococcaceae were
investigated in both native and spiked test material):
Table 1 — Product groups and matrices for which the methods described in this method are
applicable and tested in a validation trial
Product group matrix
Organic fertilizers to be determined at an international ring trial
Organo-mineral fertilizers to be determined at an international ring trial
Inorganic fertilizers to be determined at an international ring trial
International ring trials will be conducted on the basis of this document.
5

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kSIST-TS FprCEN/TS 17804:2022
FprCEN/TS 17804:2021 (E)
1 Scope
This document specifies a method for the detection and enumeration of Enterococcaceae in fertilizers of
the following Product Function Categories (PFCs) of EU fertilizing products, as described in Regulation
(EU) 2019/1009 [1]:
• PFC 1(A): Organic fertilizer;
• PFC 1(B): Organo-mineral fertilizer;
• PFC 1(C): Inorganic fertilizer, which contains more than 1 % by mass of organic carbon, other than
organic carbon from chelating or complexing agents, nitrification inhibitors, denitrification
inhibitors or urease inhibitors, coating agents, urea or calcium cyanamide. The present method was
validated on products known as present on the market in April 2021 and conform to Regulation (EU)
2019/1009 [1] that are inorganic fertilizers with more than 1 % of organic carbon such as poultry
manure and struvite with low level of organic matter. In case that other products would be developed
having other physical and chemical characteristics, it might become necessary to develop different
methods to correctly account for pathogens they might contain.
This document specifies a colony-count technique on selective media, Slanetz Bartley agar or Bile Esculin
Azide agar, respectively. The method is based on EN ISO 7899-2:2000.
2 Normative references
There are no normative references in this document.
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
laboratory sample
sample intended for laboratory inspection or testing
3.2
test sample
sample prepared from the laboratory sample (3.1) and from which test portions (3.3) will be taken
3.3
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
3.4
initial suspension
primary dilution obtained after a weighed or measured quantity of the product under examination (or of
a test sample prepared from the product) has been mixed with, normally, a nine-fold quantity of diluent
6

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FprCEN/TS 17804:2021(E)
Note 1 to entry: A closer ratio between the diluent and the quantity of product is often not recommended because
of possible inhibiting influences of the matrix.
3.5
further dilution
suspension or solution obtained by mixing a measured volume of the initial suspension (3.4) with an x-
fold volume of diluent and by repeating this operation with further dilutions until a dilution series,
suitable for the inoculation of culture media, is obtained
Note 1 to entry: Ten-fold dilutions are normally used to produce a decimal dilution series, but other ratios can be
required for specific purposes.
3.6
Enterococcaceae
include among other genera and species several species of the genus Enterococcus (3.7, 3.8) with a faecal
origin
3.7
genus Enterococcus
gram-positive, catalase-negative facultative-anaerobic cocci of the family Enterococcaceae, which often
occur in pairs (diplococci) or short chains, unable of forming spores, tolerant of a wide range of
environmental conditions (extreme temperature (10 °C to 45 °C), pH (4,5 to 10,0), high sodium chloride
concentrations) and occur ubiquitously in the environment (water, soil), in animals and in humans (in
the normal intestinal flora) and for which they are considered indicator germs for faecal contamination
(intestinal enterococci)
3.8
enterococci
gram-positive, catalase-negative cocci, able to reduce 2,3,5-triphenyl tetrazolium chloride to formazan
on Slanetz Bartley agar and to hydrolyze esculin at 44 °C on Bile Esculin Azide agar
3.9
presumptive enterococci
gram-positive, catalase-negative cocci, able to reduce 2,3,5-triphenyl tetrazolium chloride to formazan
on Slanetz Bartley agar or to hydrolyze aesculin at 44 °C on Bile Esculin Azide agar
4 Principle
a) Preparation of Slanetz Bartley agar plates and/or Bile Esculin Azide agar.
b) Drawing a representative test sample under aseptic conditions.
c) Preparation of the initial suspension with a tempered diluent to obtain a homogeneous distribution
of bacterial cells from the test portion.
d) Preparation of further decimal dilutions of the initial suspension in order to reduce the number of
microorganisms per unit volume or to reduce the cell inhibitory properties of the initial suspension
to allow, after incubation, the counting of colonies.
e) Inoculation of plates with Slanetz Bartley agar or Bile Esculin Azide agar with an aliquot of the
optimum dilutions by plating method.
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f) In
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