Animal feeding stuffs: Methods of sampling and analysis - Screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in compound feed by a microbiological plate test

This European Standard presents a method describing the screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in complete feeding stuffs and milk replacers by a microbiological 3-plate test.
The limit of detection of the method is 1 mg/kg for avoparcin, tylosin, spiramycin and virginiamycin, and 5 mg/kg for zinc bacitracin. The presence of other (veterinary) antibiotics may interfere with the method.
Furthermore, high concentrations of metals (Cu, Zn) may interfere. The method should be used as a qualitative screening method. Positive results can be analysed further by TLC; for confirmatory purposes LC-MS is required [1].
A lower limit of detection for zinc bacitracin (3 mg/kg) is achievable (see Table 2), but should be established with an in house validation first.

Futtermittel - Probenahme- und Untersuchungsverfahren - Screening auf die Antibiotika Tylosin, Virginiamycin, Spiramycin, Bacitracin-Zink und Avoparcin in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln mittels mikrobiologischem Plattentest

Diese Europäische Norm stellt ein Verfahren dar, welches das Screening auf die Antibiotika Tylosin, Virginiamycin, Spiramycin, Bacitracin Zink und Avoparcin in Konzentrationen unterhalb von Zusatzstoffen in Alleinfuttermitteln und Milchaustausch Futtermitteln mittels mikrobiologischem 3 Platten Test (Allgemeiner Hemmstofftest) beschreibt. Die Nachweisgrenze des Verfahrens beträgt 1 mg/kg bei Avoparcin, Tylosin, Spiramycin und Virginiamycin sowie 5 mg/kg bei Bacitracin Zink. Das Vorhandensein weiterer Antibiotika (aus Tierarzneimitteln) kann das Verfahren beeinträchtigen. Weiterhin können hohe Konzentrationen von Metallen (Cu, Zn) stören. Das Verfahren sollte als ein qualitatives Screeningverfahren angewendet werden. Positive Ergebnisse können mittels Dünnschichtchromatographie (en: thin layer chromatography, TLC) weiter analysiert werden, für Bestätigungszwecke ist die Flüssigchromatographie mit Massenspektrometrie (en: liquid chromatography/mass spectrometry, LC MS) gefordert [1].
Eine niedrigere Nachweisgrenze bei Bacitracin Zink (3 mg/kg) ist erreichbar (siehe Tabelle 2), sie sollte jedoch erst durch eine laborinterne Validierung etabliert werden.

Aliments pour animaux : Méthodes d’échantillonnage et d’analyse - Dépistage des antibiotiques tylosine, virginiamycine, spiramycine, bacitracine-zinc et avoparcine à des niveaux sous-additifs dans les aliments composés par essai sur plaque microbiologique

La présente Norme européenne présente une méthode décrivant le dépistage des antibiotiques tylosine, virginiamycine, spiramycine, bacitracine-zinc et avoparcine à des niveaux sous-additifs dans les aliments complets pour animaux et les lactoremplaceurs par un essai microbiologique sur 3 plaques.
La limite de détection de la méthode est de 1 mg/kg pour l’avoparcine, la tylosine, la spiramycine, la virginiamycine et de 5 mg/kg pour la bacitracine-zinc. La présence d’autres antibiotiques (à usage vétérinaire) peut interférer avec la méthode.
En outre, des concentrations élevées en métaux (Cu, Zn) peuvent également causer des interférences. Il convient d’utiliser la méthode comme une méthode de dépistage qualitatif. Dans le cas de résultats positifs, l’analyse peut être approfondie par CCM (chromatographie sur couche mince). Il est nécessaire d’utiliser la CL-SM (chromatographie liquide couplée à la spectrométrie de masse) pour confirmer les résultats [1].
Il est possible d’atteindre une limite de détection inférieure pour la bacitracine-zinc (3 mg/kg) (voir le Tableau 2), mais il convient de l’établir préalablement par une validation en interne.

Krma: metode vzorčenja in analize - Presejalna analiza antibiotikov tilozina, virginiamicina, spiramicina, bacitracin-cinka in avoparcina pri koncentracijah pod vsebnostmi dodatkov v krmnih mešanicah s preskusom z mikrobiološko ploščo

Ta metoda opisuje presejalno analizo antibiotikov tilozin, virginiamicin, spiramicin, bacitracin-cink in avoparcin pri koncentracijah pod vsebnostmi dodatkov v popolnih krmnih mešanicah in mlečnih nadomestkih s preskusom s tremi mikrobiološkimi ploščami. Meja zaznavanja te metode je 1 mg/kg za avoparcin, tilozin, spiramicin in virginiamicin ter 5 mg/kg za bacitracin-cink. Metoda lahko ovira prisotnost drugih (veterinarskih) antibiotikov. Metodo lahko ovirajo tudi visoke vsebnosti kovin (Cu, Zn). Metodo je treba uporabiti kot kvalitativno presejalno metodo. Pozitivne rezultate je mogoče dodatno analizirati s TLC, za namen potrditve pa se priporoča metoda LCMS [1].
Spodnjo mejo detekcije za bacitracin-cink (3 mg/kg) je mogoče doseči (glej preglednico 2), vendar jo je treba najprej določiti z internim preskusom.

General Information

Status
Published
Publication Date
09-May-2017
Withdrawal Date
29-Nov-2017
ICS
65.120 - Animal feeding stuffs
Technical Committee
CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
Drafting Committee
CEN/TC 327/WG 3 - Feed additives and drugs
Current Stage
9093 - Decision to confirm - Review Enquiry
Start Date
01-Jan-2024
Completion Date
14-Apr-2025
Directive
882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
Mandate
M/521 - Mandate for standardisation addressed to CEN for methods of analysis in the field of animal nutrition - part 1
Ref Project
SIST EN 16936:2017 - Animal feeding stuffs: Methods of sampling and analysis - Screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in compound feed by a microbiological plate test

Overview

EN 16936:2017 (CEN) specifies a microbiological 3‑plate screening method for detecting sub‑additive levels of the antibiotics tylosin, virginiamycin, spiramycin, bacitracin‑zinc and avoparcin in complete animal feeding stuffs and milk replacers. The method is designed as a qualitative screening test using an agar plate inhibition assay: extracts of feed samples are loaded into wells on three different indicator plates and incubated; zones of inhibition indicate the likely presence of one of the target antibiotics.

Key facts:

  • Target analytes: tylosin, virginiamycin, spiramycin, bacitracin‑zinc, avoparcin
  • Screening principle: microbiological plate test (3 plates with different media/indicator strains)
  • Incubation: typically 16–18 h, followed by visual interpretation of inhibition zones
  • Confirmation: positive screens can be followed by TLC; LC‑MS is required for confirmatory identification and quantification

Key topics and technical requirements

  • Limit of detection (LOD): 1 mg/kg for avoparcin, tylosin, spiramycin and virginiamycin; 5 mg/kg for zinc bacitracin. A lower LOD of 3 mg/kg for zinc bacitracin is achievable but requires in‑house validation.
  • Test organisms and media: Kocuria rhizophila (ATCC 9341), Micrococcus luteus (ATCC 10240) and Bacillus megaterium (ATCC 10778) on specified culture media and pH settings to create differential inhibition patterns.
  • Sample extraction: acetone/hydrochloric acid/water mixture (475/25/500 v/v/v) is used for feed extraction prior to plate application.
  • Interferences: other veterinary antibiotics can interfere with interpretation; high concentrations of metals (Cu, Zn) may also affect results.
  • Scope of method: intended as a screening (qualitative) procedure - not a definitive quantitative assay. Use standard reference materials and positive controls as described; follow up positives with targeted chemical analysis (TLC → LC‑MS).

Applications and users

EN 16936:2017 is relevant to:

  • Feed testing and quality control laboratories performing routine surveillance for antibiotic residues in compound feed and milk replacers
  • Veterinary diagnostic labs screening for non‑authorized antibiotic inclusion at sub‑additive levels
  • Feed manufacturers and formulators implementing internal control and HACCP procedures
  • Regulatory authorities and enforcement laboratories conducting compliance monitoring and pre‑screening prior to confirmatory testing

Practical uses include screening suspect feed batches, monitoring supply chains, and prioritizing samples for LC‑MS confirmation.

Related standards

  • EN ISO 13969 - Guidelines for standardized description of microbial inhibitor tests (normative reference in EN 16936:2017)

Keywords: EN 16936:2017, animal feeding stuffs, microbiological plate test, antibiotic screening, tylosin, virginiamycin, spiramycin, bacitracin‑zinc, avoparcin, feed testing, LC‑MS, TLC, limit of detection.

Frequently Asked Questions

EN 16936:2017 is a standard published by the European Committee for Standardization (CEN). Its full title is "Animal feeding stuffs: Methods of sampling and analysis - Screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in compound feed by a microbiological plate test". This standard covers: This European Standard presents a method describing the screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in complete feeding stuffs and milk replacers by a microbiological 3-plate test. The limit of detection of the method is 1 mg/kg for avoparcin, tylosin, spiramycin and virginiamycin, and 5 mg/kg for zinc bacitracin. The presence of other (veterinary) antibiotics may interfere with the method. Furthermore, high concentrations of metals (Cu, Zn) may interfere. The method should be used as a qualitative screening method. Positive results can be analysed further by TLC; for confirmatory purposes LC-MS is required [1]. A lower limit of detection for zinc bacitracin (3 mg/kg) is achievable (see Table 2), but should be established with an in house validation first.

This European Standard presents a method describing the screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in complete feeding stuffs and milk replacers by a microbiological 3-plate test. The limit of detection of the method is 1 mg/kg for avoparcin, tylosin, spiramycin and virginiamycin, and 5 mg/kg for zinc bacitracin. The presence of other (veterinary) antibiotics may interfere with the method. Furthermore, high concentrations of metals (Cu, Zn) may interfere. The method should be used as a qualitative screening method. Positive results can be analysed further by TLC; for confirmatory purposes LC-MS is required [1]. A lower limit of detection for zinc bacitracin (3 mg/kg) is achievable (see Table 2), but should be established with an in house validation first.

EN 16936:2017 is classified under the following ICS (International Classification for Standards) categories: 65.120 - Animal feeding stuffs. The ICS classification helps identify the subject area and facilitates finding related standards.

EN 16936:2017 is associated with the following European legislation: EU Directives/Regulations: 882/2004; Standardization Mandates: M/521. When a standard is cited in the Official Journal of the European Union, products manufactured in conformity with it benefit from a presumption of conformity with the essential requirements of the corresponding EU directive or regulation.

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Standards Content (Sample)


2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Probenahme- und Untersuchungsverfahren - Screening auf die Antibiotika Tylosin, Virginiamycin, Spiramycin, Bacitracin-Zink und Avoparcin in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln mittels mikrobiologischem PlattentestAliments pour animaux : Méthodes d’échantillonnage et d’analyse - Dépistage des antibiotiques tylosine, virginiamycine, spiramycine, bacitracine-zinc et avoparcine à des niveaux sous-additifs dans les aliments composés par essai sur plaque microbiologiqueAnimal feeding stuffs: Methods of sampling and analysis - Screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in compound feed by a microbiological plate test65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16936:2017SIST EN 16936:2017en,fr,de01-september-2017SIST EN 16936:2017SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16936
May
t r s y ICS
x wä s t r English Version
Animal feeding stuffsã Methods of sampling and analysis æ Screening on the antibiotics tylosiná virginiamyciná spiramyciná bacitracinæzinc and avoparcin at subæadditive levels in compound feed by a microbiological plate test Aliments pour animaux ã Méthodes d 5échantillonnage et d 5analyse æ Dépistage des antibiotiques tylosineá virginiamycineá spiramycineá bacitracineæzinc et avoparcine à des niveaux sousæadditifs dans les aliments composés par essai sur plaque microbiologique
Futtermittel æ Probenahmeæ und Untersuchungsverfahren æ Screening auf die Antibiotika Tylosiná Virginiamyciná Spiramyciná BacitracinæZink und Avoparcin in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln mittels mikrobiologischem Plattentest This European Standard was approved by CEN on
x February
t r s yä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä
CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä
EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels
t r s y CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s x { u xã t r s y ESIST EN 16936:2017

Preparation of bacterial suspensions . 12 A.1 General . 12 A.2 Storage . 12 Annex B (informative)
Procedure for the additional identification of interfering antibiotics . 13 B.1 General . 13 B.2 Tetracyclines . 13 B.3 Quinolones/colistin . 13 B.4 Aminoglycosides . 13 B.5 Avilamycin . 14 B.6 Macrolides / ß-lactams . 14 B.7 Mixture of antibiotics . 14 Bibliography . 16
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본 기사에서는 유럽 표준 EN 16936:2017에 대해 다루고 있다. 이 표준은 복합 사료 및 우유 대체물에 포함되어 있는 항생제 타일로신, 비리지니아마이신, 스파이라마이신, 박락트라신-아연, 아보파르신의 저농도에서의 스크리닝 방법을 기술한다. 해당 방법의 검출 한계는 아보파르신, 타일로신, 스파이라마이신, 비리지니아마이신에 대해서는 1 mg/kg이며 박락트라신-아연의 경우 5 mg/kg이다. 다른 (수의) 항생제의 존재는 해당 방법에 방해가 될 수 있다. 또한, 고농도의 금속 (구리, 아연)도 방해 요인이 될 수 있다. 이 방법은 질적 스크리닝 방법으로 사용되어야 하며, 추가 확인이 필요한 양성 결과는 TLC로 분석할 수 있으며, 재확인을 위해서는 LC-MS가 필요하다. 박락트라신-아연의 낮은 검출 한계 (3 mg/kg)가 가능하지만, 이는 내부 검증을 통해 먼저 확립되어야 한다.

The article discusses a European Standard that outlines a method for screening antibiotics in animal feed and milk replacers. The method uses a microbiological plate test and has a limit of detection for certain antibiotics. The presence of other antibiotics and high concentrations of certain metals may interfere with the test. Positive results can be further analyzed using different methods. A lower limit of detection for one antibiotic can be achieved but requires validation.

この記事では、ヨーロッパ標準であるEN 16936:2017について説明しています。この標準では、微生物学的な3プレートテストを用いて、飼料やミルク代替品中の抗生物質であるタイロシン、バージニアマイシン、スピラマイシン、バシトラシン・亜鉛、アボパルシンの低濃度でのスクリーニング方法を示しています。この方法の検出限界は、アボパルシン、タイロシン、スピラマイシン、バージニアマイシンに対しては1 mg/kg、バシトラシン・亜鉛に対しては5 mg/kgです。他の（獣医用）抗生物質の存在は、この方法に干渉する可能性があります。さらに、高濃度の金属（銅、亜鉛）も干渉要因となる場合があります。この方法は、定性的なスクリーニング方法として使用することが推奨されており、陽性結果はTLCによってさらに分析することができ、確認のためにはLC-MSが必要です。バシトラシン・亜鉛の低い検出限界（3 mg/kg）は可能ですが、内部の検証が必要です。