Animal feeding stuffs: Methods of sampling and analysis - Determination of OCPs by GC-ECD

This document specifies a gas chromatographic method with electron capture detection (ECD) for the determination of organochlorine pesticides (OCPs) in compound feeds and oil and fats.
The method is applicable to animal compound feed, oils and fats and fish meals with a water content up to about 20 % by weight and oil/fatty samples containing residues of one or more of the following OCPs, toxaphene and some of their isomers and degradation products:
—   aldrin;
—   dieldrin;
—   dichlorodiphenyltrichloroethane (DDT) (the isomers op'-DDT, pp'-DDT, pp'-TDE (pp'-DDD), and pp'-DDE);
—   endosulfan (as the sum of α-/β-isomers);
—   endrin;
—   hexachlorobenzene (HCB);
—   hexachlorocyclohexane isomers α-HCH (α-BHC), β-HCH (β-BHC), γ-HCH (γ-BHC or lindane);
For the following OCPs, the method is considered a screening method. Additional in-house validation is required for reporting validated data.
—   chlordane (as the sum of chlordane isomers and oxychlordane);
—   endosulfan-sulphate;
—   delta-keto-endrin;
—   heptachlor (as the sum of heptachlor and heptachlor epoxide);
—   photo-heptachlor;
—   cis
—   and trans-nonachlor.
A limit of quantification (LOQ) for the mentioned OCPs of 5 µg/kg is intended to be obtained. However, 10 µg/kg applies for heptachlor, aldrin, endrin, dieldrin, and endosulfan (α-/β– and sulphate). Individual laboratories are responsible for ensuring that the equipment that they use, achieves these limits of quantifications. The LOQs apply to the individual OCPs.

Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von OCP mittels GC-ECD

Dieses Dokument legt ein gaschromatographisches Verfahren mit Elektroneneinfangdetektor (ECD, en: electron capture detection) für die Bestimmung von Organochlorpestiziden (OCP) in Mischfuttermitteln und Ölen und Fetten fest.
Das Verfahren ist auf Mischfuttermittel, Öle und Fette und Fischmehl mit einem Wassergehalt bis zu etwa 20 % (Massenanteil) und für öl-/fetthaltige Proben anwendbar, die Rückstände von einem oder mehreren der folgenden OCP, Toxaphen und einige von deren Isomeren und Abbauprodukten enthalten::
-   Aldrin;
-   Dieldrin;
-   Dichlordiphenyltrichlorethan (DDT) (die Isomere 'op'-DDT', 'pp'-DDT', 'pp'-TDE' ('pp‘-DDD') und 'pp'-DDE');
-   Endosulfan (als Summe von α-/β-Isomeren);
-   Endrin;
-   Hexachlorbenzol (HCB);
-   Hexachlorcyclohexan-Isomere α-HCH (α-BHC), β-HCH (β-BHC), γ-HCH (γ-BHC oder Lindan);
Für die folgenden OCP wird das Verfahren als Screening-Verfahren angesehen. Um validierte Daten angeben zu können, ist eine zusätzliche laborinterne Validierung erforderlich.
-   Chlordan (als Summe von Chlordanisomeren und Oxychlordan);
-   Endosulfan-Sulfat;
-   Delta-Ketoendrin;
-   Heptachlor (als Summe von Heptachlor und Heptachlorepoxid);
-   Photoheptachlor;
-   cis-  und trans-Nonachlor.
Für die genannten OCP soll eine Bestimmungsgrenze (LOQ) von 5 μg/g erreicht werden. Jedoch gilt bei Heptachlor, Aldrin, Endrin, Dieldrin und Endosulfan (α-, β- und Sulfat) eine Bestimmungsgrenze von 10 μg/g. Die einzelnen Labore sind dafür verantwortlich, dass die von ihnen verwendeten Geräte diese Bestimmungsgrenzen erreichen. Die Bestimmungsgrenzen gelten für die einzelnen OCP.

Aliments des animaux: Méthodes d'échantillonnage et d'analyse - Dosage des pesticides organochlorés (POC) par CPG/ECD

Le présent document spécifie une méthode de chromatographie en phase gazeuse (CPG) avec détection par capture d’électrons (ECD) pour le dosage des pesticides organochlorés (POC) dans les aliments pour animaux.
La méthode est applicable aux aliments composés pour animaux, aux huiles et graisses ainsi qu’aux farines de poisson dont la teneur en eau s’élève jusqu’à environ 20 % en masse et aux échantillons d’huiles/de graisses contenant des résidus d’un ou de plusieurs des POC, du toxaphène et de certains de leurs isomères et produits de dégradation suivants :
- aldrine ;
- dieldrine ;
- dichlorodiphényltrichloroéthane (DDT) (isomères de 'op'-DDT', 'pp'-DDT', 'pp'-TDE' ('pp'-DDD') et 'pp'-DDE') ;
- endosulfan (sous forme de somme d’isomères α-/β) ;
- endrine ;
- hexachlorobenzène (HCB) ;
- isomères d’hexachlorocyclohexane α-HCH (α-BHC), β-HCH (β-BHC), γ-HCH (γ-BHC ou lindane) ;
Pour les POC suivants, la méthode considérée est une méthode de dépistage. Une validation interne supplémentaire est requise pour rendre des résultats valides.
- chlordane (sous forme de somme d’isomères de chlordane et d’oxychlordane) ;
- endosulfan-sulfate ;
- delta-kéto-endrine ;
- heptachlor (sous forme de somme d’heptachlore et d’heptachlore époxyde) ;
- photo-heptachlore ;
- cis
- et trans-nonachlore.
Il est prévu d’obtenir une limite de quantification (LQ) de 5 μg/kg pour les POC mentionnés. Cependant, une limite de 10 μg/kg s’applique à l’heptachlore, l’aldrine, l’endrine, la dieldrine et l’endosulfan (α-/β- et sulfate). Il incombe aux laboratoires individuels de s’assurer que les appareils qu’ils utilisent atteignent ces limites de quantification. Les LQ s’appliquent aux POC individuels.

Krma: metode vzorčenja in analize - Določevanje OCP z GC/ECD

General Information

Status
Published
Publication Date
24-Mar-2020
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
25-Mar-2020
Completion Date
25-Mar-2020

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SLOVENSKI STANDARD
SIST EN 15742:2020
01-maj-2020
Nadomešča:
SIST EN 15742:2009
Krma: metode vzorčenja in analize - Določevanje OCP z GC/ECD

Animal feeding stuffs: Methods of sampling and analysis - Determination of OCPs by

GC/ECD

Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von OCP mittels

GC-ECD

Aliments des animaux: Méthodes d'échantillonnage et d'analyse - Dosage des pesticides

organochlorés (POC) par CPG/ECD
Ta slovenski standard je istoveten z: EN 15742:2020
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN 15742:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 15742:2020
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SIST EN 15742:2020
EN 15742
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2020
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN 15742:2009
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of OCPs by GC-ECD

Aliments des animaux: Méthodes d'échantillonnage et Futtermittel - Probenahme- und

d'analyse - Dosage des pesticides organochlorés (POC) Untersuchungsverfahren - Bestimmung von OCP

par CPG/ECD mittels GC-ECD
This European Standard was approved by CEN on 6 January 2020.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

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 NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 15742:2020 E

worldwide for CEN national Members.
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SIST EN 15742:2020
EN 15742:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 6

4 Principle ............................................................................................................................................................. 6

5 Reagents and materials ................................................................................................................................. 6

6 Apparatus ........................................................................................................................................................ 12

7 Sampling .......................................................................................................................................................... 14

8 Preparation of test sample ....................................................................................................................... 14

9 Procedure........................................................................................................................................................ 14

9.1 General ............................................................................................................................................................. 14

9.2 Extraction ........................................................................................................................................................ 14

9.3 Clean-up procedure ..................................................................................................................................... 15

9.4 Gas chromatography ................................................................................................................................... 15

10 Calculation and expression of results ................................................................................................... 16

10.1 Calibration criteria ...................................................................................................................................... 16

10.2 Identification and confirmation.............................................................................................................. 16

10.3 Calculation ...................................................................................................................................................... 16

10.4 Recovery .......................................................................................................................................................... 18

10.5 Identification and confirmation.............................................................................................................. 18

11 Precision .......................................................................................................................................................... 18

11.1 Interlaboratory test ..................................................................................................................................... 18

11.2 Repeatability and precision within participating laboratories .................................................. 19

11.3 Reproducibility and precision between participating laboratories .......................................... 19

12 Test report ...................................................................................................................................................... 20

13 Important considerations for this method ......................................................................................... 21

13.1 Alternative extraction techniques ......................................................................................................... 21

13.2 Amount of silica ............................................................................................................................................ 21

13.3 Internal standards ....................................................................................................................................... 21

Annex A (informative) Results of interlaboratory tests .............................................................................. 22

Bibliography ................................................................................................................................................................. 23

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SIST EN 15742:2020
EN 15742:2020 (E)
European foreword

This document (EN 15742:2020) has been prepared by Technical Committee CEN/TC 327 “Animal

feeding stuffs: Methods of sampling and analysis”, the secretariat of which is held by NEN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by September 2020, and conflicting national standards shall

be withdrawn at the latest by September 2020.

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 supersedes EN 15742:2009.

In comparison with the previous edition, the following technical modifications have been made:

The analysis of polychlorinated biphenyls (PCBs) has been removed from this standard as current

legislation on maximum limits requires sensitivity that cannot be provided by GC-ECD. Additionally,

editorial changes were made.

This document has been prepared under a standardization request given to CEN by the European

Commission and the European Free Trade Association.

According to the CEN-CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to implement this European Standard: 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.
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Introduction

This document was developed in response to Directive 2002/32/EC of the European Parliament and the

Council of 7 May 2002 on undesirable substances in animal feed.

The previous edition of this document (EN 15742:2009) was fully validated by means of a collaborative

study for aldrin, dieldrin, endrin, p,p’-DDT, o,p’-DDT, p,p’-TDE, pp-DDE, alpha-endosulfan, beta-

endosulfan, HCB, alpha-HCH, beta-HCH and gamma-HCH. Attempts in the framework of the third Mandate

from the European Commission to CEN/TC 327 to perform additional validation of the method through

a full collaborative study (2017) for photo heptachlor, cis/trans nonachlor and keto-endrin were

unsuccessful as no more than three laboratories volunteered to send in results [1].

WARNING — The use of this document can involve hazardous materials, operations and

equipment. This standard does not purport to address all the safety problems associated with its

use. It is the responsibility of the user of this European Standard to establish appropriate safety

and health practices and determine the applicability of regulatory limitations prior to use.

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EN 15742:2020 (E)
1 Scope

This document specifies a gas chromatographic method with electron capture detection (ECD) for the

determination of organochlorine pesticides (OCPs) in compound feeds and oil and fats.

The method is applicable to animal compound feed, oils and fats and fish meals with a water content up

to about 20 % by weight and oil/fatty samples containing residues of one or more of the following OCPs,

toxaphene and some of their isomers and degradation products:
— aldrin;
— dieldrin;

— dichlorodiphenyltrichloroethane (DDT) (the isomers 'op'-DDT', 'pp'-DDT', 'pp'-TDE' ('pp'-DDD'), and

'pp'-DDE');
— endosulfan (as the sum of α-/β-isomers);
— endrin;
— hexachlorobenzene (HCB);

— hexachlorocyclohexane isomers α-HCH (α-BHC), β-HCH (β-BHC), γ-HCH (γ-BHC or lindane);

For the following OCPs, the method is considered a screening method. Additional in-house validation is

required for reporting validated data.
— chlordane (as the sum of chlordane isomers and oxychlordane);
— endosulfan-sulphate;
— delta-keto-endrin;
— heptachlor (as the sum of heptachlor and heptachlor epoxide);
— photo-heptachlor;
— cis- and trans-nonachlor.

A limit of quantification (LOQ) for the mentioned OCPs of 5 µg/kg is intended to be obtained. However,

10 µg/kg applies for heptachlor, aldrin, endrin, dieldrin, and endosulfan (α-/β– and sulphate). Individual

laboratories are responsible for ensuring that the equipment that they use, achieves these limits of

quantifications. The LOQs apply to the individual OCPs.
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.

EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)

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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 http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
3.1
limit of detection

smallest measured content, from which it is possible to deduce the presence of the analyte with

reasonable statistical certainty

Note 1 to entry: The limit of detection is numerically equal to three times the standard deviation of the mean of

blanc determinations (n > 10).
3.2
limit of quantification

lowest content of the analyte which can be measured with reasonable statistical certainty

Note 1 to entry: If both accuracy and precision are constant over a concentration range around the limit of

detection, then the limit of quantification is numerically equal to 6 times the standard deviation of the mean of blanc

determinations (n > 10).
4 Principle

In order to check for the presence of OCPs, a test portion of animal feeding stuff is fortified with internal

standard (PCB 198), and is extracted with ethyl acetate. The extract is concentrated and subsequently

purified by:

— gel permeation chromatography (GPC), with cyclohexane or ethyl acetate as eluting solvent;

— chromatography on partially deactivated silica gel.

The collected fraction containing the compounds of interest is concentrated and re-dissolved in a solution

containing another internal standard (PCB 209) as a reference standard. After clean up, the analytes are

measured using GC-ECD. Identification is done on the basis of comparing retention times on capillary

columns of different polarity. Quantification is done using the internal standard method.

5 Reagents and materials
5.1 General

Use only reagents of recognized analytical grade and with a purity suitable for OCP and PCB residue

analysis. Check the purity of the reagents by performing a blanc test under the same conditions as used

in the method. The chromatogram should not show any interfering impurity at the retention time of

compounds of interest.
5.2 Chemicals
5.2.1 Cyclohexane
5.2.2 Ethyl acetate
5.2.3 Hexane
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EN 15742:2020 (E)
5.2.4 Dichloromethane
5.2.5 Iso-octane
5.2.6 Toluene
5.2.7 Hexane/toluene = 3+7, parts by volume

Mix 30 ml of hexane (5.2.3) with 70 ml of toluene (5.2.6) thoroughly. Store at room temperature in a

tightly closed glass bottle.
5.2.8 Sodium sulphate, anhydrous
Heated to 160 °C to 200 °C during at least 24 h.
5.2.9 Ethyl acetate/cyclohexane = 1+1, parts by volume

Mix 500 ml of ethyl acetate (5.2.2) with 500 ml of cyclohexane (5.2.1) thoroughly. Store at room

temperature in a tightly closed glass bottle.
5.2.10 Silica gel, deactivated with 3,5 % water

Heat silica gel 60 (63 µm to 200 µm = 70 mesh to 230 mesh), at 130 °C for at least 5 h, allow to cool in a

desiccator, and store in a tightly stopped container in the desiccator.

Add 3,5 ml water dropwise from a burette, with continuous swirling, to 96,5 g dried silica gel in a 300 ml

Erlenmeyer flask with a ground joint.

Immediately stopper the flask with a ground stopper and shake vigorously for 5 min until all lumps have

disappeared.

Next, shake for 2 h on a mechanical shaker, and then store in a tightly stoppered container.

Deactivated silica gel is tenable during approximately 2 weeks if carefully stored.

5.2.11 Internal standard (PCB 198)
5.2.12 Internal Standard (PCB 209)
5.2.13 OCP reference standards, each with a purity not less than 99 %:
Aldrin
(1R,4S,4aS,5S,8R,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-1,4:5,8-
dimethanonaphthalene; CAS Number: 309-00-2.
Dieldrin

(1R,4S,4aS,5R,6R,7S,8S,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,6,7,8,8a-octahydro-6,7epoxy-1,4:5,8-

dimethanonaphthalene; CAS Number: 60-57-1.
Delta-keto-endrin
CAS Number 53494-70-5.
Chlordane, α isomer

|1,2,4,5,6,7,8,8 octachloro-2,3,3a,4,7,7a-hexahydro-4,7-ethano-1H-indene; α isomer; CAS Number: 5103-

71-9.
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EN 15742:2020 (E)
Chlordane, β isomer

1,2,4,5,6,7,8,8-octachloro-2,3,3a,4,7,7a-hexahydro-4,7-ethano-1H-indene; β isomer; CAS Number: 5103-

74-2.
Oxychlordane

4,7-Methanoindan, 1,2,4,5,6,7,8,8-octachloro-2,3-epoxy-3a,4,7,7a-tetrahydro-, exo, endo-; CAS Number:

27304-13-8.

'op'-DDT' 1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane ; CAS Number: 789-02-6.

'pp'-DDT' 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane ; CAS Number: 50-29-3.
'pp'-TDE'
('p,p'-DDD')1,1-dichloro-2,2-bis(4-chlorophenyl) ethane; CAS Number: 72-54-8.
'pp'-DDE' 1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene; CAS Number: 72-55-9.
Endosulfan, α stereoisomer

6,9-Methano-2,4,3-benzodioxathiepin, 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-, 3-oxide, (3α,

5aβ, 6α, 9α, 9aβ); CAS Number: 959-98-8.
Endosulfan, β stereoisomer

6,9-Methano-2,4,3-benzodioxathiepin, 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-, 3-oxide,

(3α,5aα,6β,9β,9aα); CAS Number: 33213-65-9;
Endosulfan sulphate

6,9-Methano-2,4,3-benzodioxathiepin, 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-, 3,3-dioxide;

CAS Number: 1031-07-8.
Endrin

(1R,4S,4aS,5S,6S,7R,8R,8aR)-1,2,3,4,10,10-hexachloro-1,4,4a,5,6,7,8,8a-octahydro-6,7-epoxy-1,4:5,8-

dimethanonaphthalene; CAS Number: 72-20-8;
Delta-keto endrin

2,5,7-Metheno-3H-cyclopenta[a]pentalen-3-one,3b,4,5,6,6,6a-hexachlorodecahydro-, (2α,

3aβ,3bβ,4β,5β,6aβ,7α7aβ,8R); CAS 53494-70-5;
Heptachlor

1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene; CAS Number: 76-44-8.

β-Heptachlor epoxide

1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene(exo); CAS Number: 1024-57-3.

HCB
hexachlorobenzene; CAS Number: 118-74-1.
α-HCH (α-BHC)
α-1,2,3,4,5,6-hexachlorocyclohexane; CAS Number: 319-84-6.
β-HCH (β-BHC)
β-1,2,3,4,5,6-hexachlorocyclohexane; CAS Number: 319-85-7.
γ-HCH (γ-BHC; lindane)
γ-1,2,3,4,5,6-hexachlorocyclohexane; CAS Number: 58-89-9.
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Photoheptachlor
CAS Number 33442-83-0.
Cis-nonachlor
CAS Number 5103-73-1.
Trans-nonachlor
CAS Number 39765-80-5.

Alternatively to each individual OCP reference standard listed above, use a certified mixture at a

concentration of 10 µg/ml.
5.2.14 Chlorocamphene (toxaphene)
5.3 Stock solutions, 100 µg/ml

Weigh 5 mg−10 mg (±0,01 mg) of each compound (5.2.11, 5.2.12, 5.2.13 and 5.2.14) in separate brown

medicine glass bottles of 100 ml and add iso-octane (5.2.5.) to achieve a concentration of 100 μg/ml.

Store the solutions in a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during

at least 5 years if the weight is carefully controlled.

Dissolve ß-HCH in 10 ml toluene (5.3), to achieve complete solvability and dilute further with iso-octane

(5.2.5) to achieve a concentration of 100 μg/ml.
5.4 Mixed stock solutions
5.4.1 Mixed stock solution OCP (without endosulfan and toxaphene)

Pipet of each OCP-stock solution (5.3) the indicated volume (Table 1) in a volumetric flask of 100 ml. Fill

up to 100 ml with iso-octane (5.2.5) and mix. The achieved concentration is given in Table 1. Transport

this solution to a brown medicine glass bottle of 100 ml and store it in a refrigerator at 4 °C (±3 °C). The

solution is tenable under these conditions during at least 5 years if the weight is carefully controlled.

5.4.2 Mixed stock solution endosulfan

Pipet of each endosulfan-stock solution (5.3) the indicated volume (Table 1) in a volumetric flask of

100 ml. Fill up to 100 ml with iso-octane (5.2.5) and mix. The achieved concentration is given in Table 1.

Transport this solution to a brown medicine glass bottle of 100 ml and store it in a refrigerator at 4 °C

(±3 °C). The solution is tenable under these conditions during at least 5 years if the weight is carefully

controlled.

Table 1 — Concentration of OCPs in mixed stock solution (5.4) and mixed standard solution (5.5)

Compound Pipet Mixed stock solution Mixed standard
volume (5.4.1 and 5.4.2) solution (5.5.1 and
5.5.2)
(ml) (μg/ml)
(µg/ml)
aldrin 2,0 2,0 0,10
dieldrin 2,0 2,0 0,10
α-chlordane 1,0 1,0 0,05
γ-chlordane 1,0 1,0 0,05
oxychlordane 1,0 1,0 0,05
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Compound Pipet Mixed stock solution Mixed standard
volume (5.4.1 and 5.4.2) solution (5.5.1 and
5.5.2)
(ml) (μg/ml)
(µg/ml)
o,p'-DDT 4,0 4,0 0,20
p,p'-DDT 4,0 4,0 0,20
p,p'-TDE 4,0 4,0 0,20
p,p'-DDE 4,0 4,0 0,20
α-endosulfan 2,0 2,0 0,10
β-endosulfan 2,0 2,0 0,10
endosulfan-sulphate 1,0 1,0 0,05
endrin 1,0 1,0 0,05
heptachlor 2,0 2,0 0,10
heptachlor epoxide 2,0 2,0 0,10
HCB 1,0 1,0 0,05
α-HCH 2,0 2,0 0,10
ß-HCH 1,0 1,0 0,05
γ-HCH (lindane) 1,0 1,0 0,05
photo heptachlor 2,0 2,0 0,10
cis-nonachlor 2,0 2,0 0,10
trans-nonachlor 2,0 2,0 0,10
5.4.3 Mixed stock solution toxaphene, 10,0 μg/ml

Pipet 10,0 ml of the toxaphene-stock solution (5.3) in a volumetric flask of 100 ml. Fill up to 100 ml with

iso-octane (5.2.5) and mix. Transport this solution to a brown medicine glass bottle of 100 ml and store

it in a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during at least 5 years if

the weight is carefully controlled.
5.4.4 Internal standard stock solution PCB 198, 2,0 µg/ml

Pipet 2,0 ml of the PCB 198-stock solution (5.3) in a volumetric flask of 100 ml. Fill up to 100 ml with iso-

octane (5.2.5) and mix. Transport this solution to a brown medicine glass bottle of 100 ml and store it in

a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during at least 5 years if the

weight is carefully controlled.
5.4.5 Internal standard stock solution PCB 209, 2,0 µg/ml

Pipet 2,0 ml of the PCB 209-stock solution (5.3) in a volumetric flask of 100 ml. Fill up to 100 ml with iso-

octane (5.2.5) and mix. Transport this solution to a brown medicine glass bottle of 100 ml and store it in

a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during at least 5 years if the

weight is carefully controlled.
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5.5 Mixed standard solution
5.5.1 Mixed standard solution OCPs, 0,05-0,20 µg/ml

Pipet 5,0 ml of the mixed stock solution OCPs (5.4.1) in a volumetric flask of 100 ml. Fill up to 100 ml with

iso-octane (5.2.5) and mix. The achieved concentration is given in Table 1. Transport this solution to a

brown medicine glass bottle of 100 ml and store it in a refrigerator at 4 °C (±3 °C). The solution is tenable

under these conditions during at least 5 years if the weight is carefully controlled. The calibration

solutions can also be prepared on the basis of a certified mixed standard solution of for example 10 µg/ml.

In that case, the solutions shall cover the concentration range as shown in Table 2.

5.5.2 Mixed standard solution endosulfan, 0,05-0,10 µg/ml

Pipet 5,0 ml of the mixed stock solution endosulfan (5.4.2) in a volumetric flask of 100 ml. Fill up to

100 ml with iso-octane (5.2.5) and mix. The achieved concentration is given in Table 1. Transport this

solution to a brown medicine glass bottle of 100 ml and store it in a refrigerator at 4 °C (±3 °C). The

solution is tenable under these conditions during at least 5 years if the weight is carefully controlled.

5.5.3 Mixed standard solution toxaphene, 0,5 μg/ml

Pipet 5,0 ml of the mixed stock solution toxaphene (5.4.3) in a volumetric flask of 100 ml. Fill up to 100 ml

with iso-octane (5.2.5) and mix. Transport this solution to a brown medicine glass bottle of 100 ml and

store it in a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during at least

5 years if the weight is carefully controlled.
5.6 Internal standard solutions
5.6.1 PCB 198, 0,1 µg/ml

Pipet 5,0 ml PCB 198 standard solution (5.4.4) in a volumetric flask of 100 ml. Fill up with ethyl

acetate/cyclohexane (5.2.9) and mix. Transport this solution to a brown medicine glass bottle of 100 ml

and store it in a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during at least

5 years.
5.6.2 PCB 209, 0,1 µg/ml

Pipet 5,0 ml PCB 209 standard solution (5.4.5) in a volumetric flask of 100 ml. Fill up with ethyl

acetate/cyclohexane (5.2.9) and mix. Transport this solution to a brown medicine glass bottle of 100 ml

and store it in a refrigerator at 4 °C (±3 °C). The solution is tenable under these conditions during at least

5 years.
5.7 GC-standard solutions – GC-standard OCPs, endosulfan, toxaphene

Prepare calibration mixtures according to Table 2 for OCPs, endosulfan and toxaphene each in a final

volume of 5,0 ml ethyl acetate/cyclohexane (5.2.9). Store in a refrigerator at 4 °C (±3 °C). The solution is

tenable under these conditions during at least 1 year.

Depending on the expected mass fractions in the samples, the highest levels (6 and 7) depicted in Table 2

may be ignored. However, the response in the sample extract should lie within the range of the calibration

solutions.
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Table 2 — Calibration mixtures for OCPs, endosulfan, toxaphene and PCB, each in a final volume

of 5,0 ml ethyl acetate/cyclohexane
PCB 198 PCB 209 Pipet from Concentration Concentration Concentration
Level
(5.6.1) (5.6.2) OCP (5.5.1) in calibration in calibration in calibration
or solution OCP solution solution
[ml] [ml]
endosulfan endosulfan toxaphene
[ng/ml]
(5.5.2) or
[ng/ml] [ng/ml]
toxaphene
(5.5.3)
solution
[ml]
1 0,25 0,25 0 0 0 0
2 0,25 0,25 0,05 0,5 – 2,0 0,5 – 2,0 5
3 0,25 0,25 0,10 1,0 – 4,0 1,0 – 2,0 10
4 0,25 0,25 0,20 2,0 – 8,0 2,0 – 4,0 20
5 0,25 0,25 0,40 4,0 – 16,0 4,0 – 8,0 40
6 0,25 0,25 0,80 8,0 – 32,0 8,0 – 16,0 80
7 0,25 0,25 1,60 16,0 – 64,0 16,0 – 32,0 160
5.8 Blanc animal feeding stuff
The sample should be free of residues of OCPs and interfering impurities.
5.9 Glass wool
6 Apparatus
6.1 General
Usual laboratory glassware and equipment and, in particular, the following:

NOTE All technical descriptions below are examples of possible system setups and parameters that are scaled

or adopted to the user's equipment.
6.2 Analytical balance, accuracy 0,01 mg
6.3 Analytical balance, accuracy 10 mg
6.4 Tubes 50 ml
For example polypropylene or glass tubes.
6.5 Mechanical shaker

6.6 Evaporation system, equipped with 10 ml graduated glass tubes and nitrogen gas

EXAMPLE A turbovab evaporator can be used.
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6.7 GPC system

WARNING — In case the recovery of β-HCH and γ-chlordane in the GPC control with a standard solution

is too low, the start of the collection time of the OCP fraction is too late. In case the recovery of HCB is too

low, the end time of the OCP fraction is too early.

Equilibrate the GPC-system (6.7) under the recommended operating conditions and check the GPC

column performance as subscribed in EPA method 3640 [2].
The GPC clean up system shall consist of the components below:
6.7.1 HPLC-pump

The HPLC pump shall be capable of maintaining a flow-rate of 1,0 ml per min ethyl

acetate/cyclohexane = 1+1, parts by volume (5.2.9).
6.7.2 Automated injection system

The automated injection system shall be capable of performing a series of unattended injections of a

volume of 500 μl.
6.7.3 GPC-column

The GPC-column shall be capable of performing a separation as specified by criteria in EPA Method 3640

[2].

EXAMPLE A column with length of 45 cm, and 10 mm internal diameter, packed with stationary phase Bio

Beads SX-3 can be used for this purpose. In this example, the OCP/PCB containing fraction elutes between 16 min –

26 min. This can be tested before use.
6.7.4 Fraction collector
6.8 GC-ECD

The GC-ECD consists of an automatic injection system, a gas chromatograph equipped with a splitless

injector, a capillary column, an electron capture detector (ECD) and a computer with appropriate

software. The total chromatographic system should be adjusted and optimized according to the

manufacturer's instructions.
6.8.1 Automated Injection System
The autosampler shall be capable of injecting 2 µl.
6.8.2 Gas chromatograph

The gas chromatograph shall be capable of working with capillary columns. The use of two capillary

columns coated with non-polarity and a mid-range polarity stationary phase (dimensions: 25 m –

30 m × 0,20 – 0,40 mm, film thic
...

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