CEN/TC 455 - Plant Biostimulants and Agricultural Micro-Organisms

This document specifies a method for the extraction and determination of phosphonates (P–PO3) in plant biostimulants using ion chromatography and conductivity detection (IC-CD).
This document is also applicable to the blends of fertilizing products where plant biostimulants are the main part of the blend. Otherwise, the Technical Specification for the main part of the blend applies.

This document specifies the terms and definitions referred to all the plant biostimulant field and it is constituted by 6 subclauses:
3.1   Claims
3.2   Terms relating to components
3.3   Terms relating to application method
3.4   Terms relating to sample preparation
3.5   Terms relating to physical form
3.6   Others terms relating to plant biostimulants

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].

This document specifies a method for determination of the content of mercury (Hg) in plant biostimulants using (cold) vapour generation apparatus coupled to an atomic absorption spectrophotometer and a method using a direct amalgamation technique. It is applicable to aqua regia digests prepared according to CEN/TS 17701-1.
NOTE    It is also possible to use other suitable methods for the determination of mercury described in Annex A if users prove that the method gives the same results as the methods described in this document.
This document is also applicable to the blends of fertilizing products where plant biostimulants are the main part of the blend. Otherwise, the Technical Specification for the main part of the blend applies.

This document specifies a horizontal method for the detection of enteropathogenic Vibrio spp., which causes human illness in or via the intestinal tract [1]. The species detectable by the methods specified include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
It is applicable to the following:
—   microbial plant biostimulants.
NOTE 1   The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and V. vulnificus are the major contaminants of Vibrio spp. [1].
NOTE 2   For confirmation, it is possible to use PCR tests; in this case the laboratory validates the procedure and data generated.

This document defines general rules for the aerobic preparation of the initial suspension and of dilutions for microbiological examinations of microbial plant biostimulants.
This horizontal method might not be appropriate in very detail for certain products. In this case, different methods which are specific to these products can be used if necessary, for justified technical reasons.

This document specifies methods for the reduction and preparation of samples of non-microbial plant biostimulants including those intended for determination of microbial pathogens and sets out the requirements for sample preparation reports. It specifies methods for the preparation of test samples and test portions from laboratory samples of plant biostimulants for subsequent chemical, biological or physical analysis.
It is also applicable to the sample preparation of blends of fertilizing products where plant biostimulants are main part of the blend. Otherwise, deliverables of sample preparation relevant for the main part of the blend apply.
This document does not include methods for the reduction and preparation of samples of microbial plant biostimulants, which will be covered by a different Technical Specification.
NOTE   This document is applicable to the category of EU fertilizing product (plant biostimulants) in the meaning of the Regulation (EU) 2019/1009.

This document was developed to provide a method for verifying that hexavalent chromium (CrVI) is not present in plant biostimulants in a concentration that exceeds the respective limits outlined in the EU Regulation on Fertilising Products [1].
This document is applicable to all types of plant biostimulants (solid and liquid ) used in agriculture.
The method described is suitable to quantify the chromium(VI) content in plant biostimulants down to 2 mg/kg.
The results obtained from this method are strictly dependent on the extraction conditions. Results obtained by using other extraction procedures (extraction solution, pH, extraction time, etc.) are not comparable with the results produced by the procedure described in this document.

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).

This document specifies a method for extraction, separation, and determination of inorganic arsenic (iAs) in plant biostimulants using anion-exchange high performance liquid chromatography (HPLC) or ion chromatography (IC) coupled to ICP-MS.
This document is also applicable to the blends of fertilizing products where plant biostimulants are the main part of the blend. Otherwise, the Technical Specification for the main part of the blend applies.

This document was developed to provide the methodology for the enumeration and determination of Azotobacter sp. in plant biostimulant products in accordance with the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1].

This document provides a method for verifying that the pathogen Shigella spp. is not present in microbial plant biostimulants in a concentration that exceeds the respective limits outlined in the EU Regulation on Fertilising Products.
The detection method for Shigella pathogens is not sensitive and quantification is rarely performed. Detection is usually performed using an enrichment medium followed by subculturing onto a variety of selective media.

This document specifies the procedure for the determination and calculation of the dry matter fraction of plant biostimulants for which the results of performed analysis are to be calculated to the dry matter basis.
This document is also applicable to the blends of fertilizing products where plant biostimulants are the main part of the blend. Otherwise, the Technical Specification for the main part of the blend applies.

This document specifies the method for the digestion of different plant biostimulants with aqua regia to enable a subsequent determination of arsenic (As), cadmium (Cd), copper (Cu), chromium (Cr), mercury (Hg), nickel (Ni), lead (Pb) and zinc (Zn). The method can be also applied for determination of other elements. The method is applicable for all solid and/or liquid plant biostimulants.
This document is also applicable to the blends of fertilizing products where plant biostimulants are the main part of the blend. Otherwise, the Technical Specification for the main part of the blend applies.
The extracts are suitable for analysis using CEN/TS 17701-2 (ICP-AES) and CEN/TS 17701-3 (Hg analysis).
NOTE   Alternatively, inductively coupled plasma mass spectrometry (ICP-MS) can be used for the measurement if the user proves that the method gives the same results.

This document specifies sampling plans and methods of representative sampling of plant biostimulants to obtain samples for physical, chemical and biological analysis.
It is applicable to the sampling of lots of plant biostimulants supplied or ready for supply to third parties, as such, or in smaller lots.
It is also applicable to the sampling of blends of fertilizing products where plant biostimulants are main part of the blend. Otherwise, deliverables of sampling relevant for the main part of the blend apply.
This document is intended to be used by manufacturers, buyers and competent authorities to obtain samples prior to transport and supply it to a laboratory for testing.
NOTE   This document is applicable to the category of EU fertilizing product (plant biostimulants) in the meaning of the Regulation (EU) 2019/1009.

This document specifies a method for laboratory measurement of the pH value in liquid microbial plant biostimulants, using pH electrodes with a glass membrane.
From the scope of this document plant biostimulants other than microbial plant biostimulants are excluded because there is no essential requirement in the Regulation (EU) 2019/1009 [1] for measuring the pH of non-microbial plant biostimulants.

This document specifies the general principles for justifying the product claims for plant biostimulants.
General principles consist of and define all general parameters, requirements and quality criteria, and are intended to be applied in order to assess the efficacy of trials used for claim(s) validation as a result of the use of a plant biostimulant.
This document is aimed primarily at manufacturers, laboratories, researchers, technical centres, companies that will put the products on the market, notifying authorities, notified bodies, and market surveillance authorities.

This document provides guidance for justifying quality traits claims of plant biostimulants used in agriculture.
This document is aimed primarily at manufacturers, laboratories, companies which will put the products on the market, notifying authorities, notified bodies, and market surveillance authorities.

This document provides the methodology for the enumeration and determination of Azospirillum spp. in plant biostimulant products in accordance to the Regulation of EU fertilising products [1].

This document was developed to provide a horizontal method for enumeration and genera/species determination [1], [2], [3] of mycorrhizal fungi in plant biostimulants products in accordance with the EU Fertilising Products Regulation.

The claim described in this document concerns the improvement of availability of confined nutrients in the soil or rhizosphere by a plant biostimulant.
This document is aimed primarily at manufacturers, laboratories, researchers, technical centres, companies that will put the products on the market, notifying authorities, notified bodies, and market surveillance authorities.

This document provides a horizontal method for enumeration of microorganisms that are able to grow and form colonies in a solid medium after anaerobic incubation at 30 °C.
The method is applicable to microbial plant biostimulants for verifying that the concentration of anaerobes does not exceed the respective limits outlined in the EU Regulation on Fertilising Products [1].
This method does not apply to the microbiological monitoring of the environment in which microbial plant biostimulants are manufactured.
No information about potential human pathogens can be inferred from anaerobic plate counts.

This document specifies the methods to be used for the determination of quantity of solid and liquid forms of plant biostimulants in packages, containers or in bulk.
This document is not applicable to the quantity determination of: soil improvers, growing media, organic and organo-mineral fertilizers and fertilizing product blends whose main constituent is a growing media or soil improver. The method for quantity determination for these products is given in EN 15761, EN 15238 and EN 12580.

This document specifies a method for the determination of arsenic (As), cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb), nickel (Ni) and zinc (Zn) in plant biostimulant digests using inductively coupled plasma-atomic emission spectrometry (ICP-AES).
This document is also applicable to the blends of fertilizing products where plant biostimulants are the main part of the blend. Otherwise, the Technical Specification for the main part of the blend applies.
This method is applicable to aqua regia digests prepared according to CEN/TS 17701-1. The method can be used for the determination of other elements, provided the user has verified the applicability.

This document provides guidance for justifying agronomic nutrient use efficiency claims of plant biostimulants used in agriculture.
This document is aimed primarily at manufacturers, laboratories, researchers, technical centres, companies that will put the products on the market, notifying authorities, notified bodies, and market surveillance authorities.

This document gives general guidelines for the detection and identification of the specified microorganism Escherichia coli in technical and formulated biostimulant products, both in liquid and solid state, and also the horizontal method for the enumeration of ß-glucuronidase-positive Escherichia coli in plant biostimulants products (both in liquid and solid state).
The qualitative method described in this document is based on the detection of Escherichia coli in a non-selective liquid medium (enrichment broth), followed by isolation on a selective agar medium. Other methods can be appropriate, depending on the level of detection required.
NOTE   For the detection of Escherichia coli, subcultures can be performed on non-selective culture media followed by suitable identification steps (e.g. using identification kits).
The quantitative method described in this document uses a colony-count technique at 44 °C on a solid medium containing a chromogenic ingredient for detection of the enzyme ß-glucuronidase.
WARNING — Strains of Escherichia coli which do not grow at 44 °C and, in particular, those that are ß glucuronidase negative, such as Escherichia coli O157, will not be detected.

This document provides the methodology for the enumeration and determination of Rhizobium sp., Mesorhizobium sp., Ensifer sp., or Bradyrhizobium sp. in plant biostimulant products in accordance with the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1].

This document provides a method for the detection of Listeria monocytogenes in microbial plant biostimulants for verifying that the concentration of this human pathogen does not exceed the respective limits outlined in the EU Regulation on Fertilising Products [1].

This document provides a method for verifying that the pathogen Staphylococcus aureus is present in microbial plant biostimulants according to the limits outlined in the EU Regulation on Fertilising Products [2].

This document specifies a horizontal method for the enumeration of yeasts and moulds present in plant biostimulants intended for use in agriculture, by means of the colony count technique after aerobic incubation at 25 °C ± 2,5 °C.
This document allows the enumeration of yeasts and moulds, in technical and formulated plant biostimulant, both in liquid and solid state. The method is applicable to microbial plant biostimulant except those composed of fungi or yeast to verify that the concentration of yeast and moulds does not exceed the respective limits described in the EU Fertilisers Regulation [1].
If necessary, yeast and mould enumerated can be identified using suitable identification tests.

This document specifies a potentiometric method for the determination of chloride (Cl-) content in the presence or in the absence of organic material. This method is applicable to plant biostimulants.

This document provides guidance for justifying abiotic stress tolerance claim of plant biostimulants used in agriculture.
This document is aimed primarily at manufacturers, laboratories, companies which will put the products on the market, notifying authorities, notified bodies, and market surveillance authorities.

This document specifies general rules to determine the concentration of microorganisms present in plant biostimulant products.
The method is applicable to microbial plant biostimulants for verifying that the concentration of microorganisms does not exceed the respective limits outlined in the EU Regulation on Fertilising Products [1].
This horizontal method might not be appropriate in every detail for certain products. In this case, it is necessary to refer to the methodology of specific determination and quantification of the microorganisms.

This document specifies a horizontal method for the detection of enteropathogenic Vibrio spp., which causes human illness in or via the intestinal tract [1]. The species detectable by the methods specified include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
It is applicable to the following:
-   microbial plant biostimulants.
NOTE 1   The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and V. vulnificus are the major contaminants of Vibrio spp. [1].
NOTE 2   For confirmation, it is possible to use PCR tests; in this case the laboratory must validate the procedure and data generated.

This document specifies a horizontal method for the enumeration of yeasts and moulds present in plant biostimulants intended for use in agriculture, by means of the colony count technique after aerobic incubation at 25 °C ± 2,5 °C.
This document allows the enumeration of yeasts and moulds, in technical and formulated plant biostimulant, both in liquid and solid state. The method is applicable to microbial plant biostimulant except those composed of fungi or yeast to verify that the concentration of yeast and moulds does not exceed the respective limits described in the EU Fertilisers Regulation [1].
If necessary, yeast and mould enumerated can be identified using suitable identification tests.

This document defines general rules for the aerobic preparation of the initial suspension and of dilutions for microbiological examinations of microbial plant biostimulants.
This horizontal method might not be appropriate in very detail for certain products. In this case, different methods which are specific to these products can be used if necessary, for justified technical reasons.

This document specifies the methods to be used for the determination of quantity of solid and liquid forms of plant biostimulants in packages, containers or in bulk.
This document is not applicable to the quantity determination of: soil improvers, growing media, organic and organo-mineral fertilizers and fertilizing product blends whose main constituent is a growing media or soil improver. The method for quantity determination for these products is given in EN 15761, EN 15238 and EN 12580.

This document specifies a method for extraction, separation, and determination of inorganic arsenic (iAs) in plant biostimulants using anion-exchange high performance liquid chromatography (HPLC) or ion chromatography (IC) coupled to ICP-MS. This document is applicable to the fertilizing product blends belonging to PFC 7 where the EU fertilising product plant biostimulant contained in the blend represents the highest % by mass in the blend. In case of equal shares, the user can apply either this or the standard(s) applicable to the other component product(s).

This document gives guidance on a consistent approach to justify the claims associated with the use of plant biostimulants.
This document is aimed primarily at manufacturers, laboratories, researchers, technical centres, and companies that intend to place plant biostimulants on the market, as well as notifying authorities, notified bodies, and market surveillance authorities.
To be in compliance with this standard, it is important to also follow the recommendations and quality criteria described in standard EN 17700-1:—1 on general principles to demonstrate plant biostimulant claims.

This document specifies a method for the extraction and determination of phosphonates (P-PO3) in plant biostimulants using ion chromatography and conductivity detection (IC-CD).
This document is applicable to the fertilizing product blends belonging to PFC 7 where the EU fertilising product plant biostimulant contained in the blend represents the highest % by mass in the blend. In case of equal shares, the user may apply either this or the standard(s) applicable to the other component product(s).

This document provides the methodology for the enumeration and determination of Rhizobiaceae (Rhizobium spp., Mesorhizobium spp., Ensifer spp., Bradyrhizobium spp.).
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilizing products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies a method for the determination of total contents of arsenic (As), cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb), nickel (Ni) and zinc (Zn) in aqua regia plant biostimulant digests using inductively coupled plasma-atomic emission spectrometry (ICP-AES).
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilizing products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Inhibitors, Plant Biostimulants, and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations, the user decides which standard to apply.
This method is applicable to aqua regia digests prepared according to EN 17701-1:—1. The method can be used for the determination of other elements, provided the user has verified the applicability.
NOTE   Alternatively, inductively coupled plasma mass spectrometry (ICP-MS) can be used for the determination of the elements in the aqua regia digests if the user proves that the method gives the same results.

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].

This document was developed to provide the methodology for the enumeration and determination of Azotobacter sp. [2] [3] in microbial plant biostimulants in accordance with the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1].

This document gives guidance on a consistent approach to justify the claims associated with the use of plant biostimulants.
This document is aimed primarily at manufacturers, laboratories, researchers, technical centres, and companies that intend to place plant biostimulants on the market, as well as notifying authorities, notified bodies, and market surveillance authorities.
To be in compliance with this standard, it is important to also follow the recommendations and quality criteria described in standard EN 17700-1:—1 on general principles to demonstrate plant biostimulant claims.

This document specifies the general principles for justifying the product claims for plant biostimulants. It is applicable to all claims and all types of application of plant biostimulants.
General principles define all general parameters, requirements and quality criteria to be applied in order to assess trials conducted to validate the claim(s) associated with the use of a plant biostimulant.
This document is aimed primarily at manufacturers, laboratories, researchers, technical centres and companies that intend to place plant biostimulants on the market, as well as notifying authorities, notified bodies, and market surveillance authorities.

This document specifies the procedure for the determination of dry residue and calculation of the dry matter fraction of plant biostimulants for which the results of performed analysis are to be calculated to the dry matter basis.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilizing products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Inhibitors, Plant Biostimulants, and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations, the user decides which standard to apply.

This document specifies methods for the reduction and preparation of samples of non-microbial plant biostimulants and sets out the requirements for sample preparation reports. It specifies methods for the preparation of test samples and test portions from laboratory samples of plant biostimulants for subsequent chemical, biological or physical analysis.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilizing products: Fertilizers/Liming Materials/Soil Improvers/Growing Media/Inhibitors/Plant Biostimulants, and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations1, the user decides which standard to apply.
NOTE   An example of such a blend is a product with two claimed functions consisting of a non-microbial plant biostimulant and an organic fertiliser composed of 1kg/kg of plant biostimulant from seaweed.
This document does not include methods for the reduction and preparation of samples of microbial plant biostimulants and samples intended for determination of microbial pathogens, which will be covered by a different European Standard.

This document specifies a horizontal method for the enumeration and genus/species determination [2], [3], [4] of mycorrhizal fungi in microbial plant biostimulants.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilizing products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document provides a method for verifying that the pathogen Shigella spp. is not present in microbial plant biostimulants.
The detection method for Shigella pathogens is not sensitive and quantification is rarely performed. Detection is usually performed using an enrichment medium followed by subculturing onto a variety of selective media.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilizing products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.