EN    EN 
 
 
 EUROPEAN  
COMMISSION   
Brussels, XXX   
SANTE/11249/2016 ANNEX 
(POOL/E2/2016/11249/11249 -EN 
ANNEX.doc) D047571/03  
[…](2016) XXX  draft 
ANNEX 1  
  
ANNEX  
to the  
COMMISSION REGULATION (EU) …/..  
amending Regulation (EC) No 152/2009 as regards the determination of the levels of 
dioxins and polychlorinated biphenyls    
EN 2   EN ANNEX  
In Annex V to Regulation (EC) No 152/2009, Part B 'DETERMINATION OF THE LEVELS 
OF DIOXINS (PCDD/ PCD F) AND PCBs' is replaced by the following:  
"B. DETERMINATION OF THE LEVELS OF DIOXINS (PCDD/ PCD F) 
AND PCBs  
CHAPTER I  
METHODS OF SAMPLING AND INTERPRETATION OF ANALYTICAL RESULTS   
1. SCOPE  AND DEFINITIONS  
The samples intended for the official control  of the levels of polychlorinated 
dibenzo -p-dioxins ( PCDDs), polychlorinated dibenzofurans (PCDFs), dioxin -like 
polychlorinated biphenyls  (PCBs)1 and non dioxin -like PCBs in feed shall be taken 
in accordance with the provisions of Annex I. The quantitative  requirements in 
relation to the control of substances or products uniformly distributed throughout the 
feed as provided for in point 5.1. of Annex I shall be applied. Aggregate samples 
thus obtained shall be considered representative for the lots or sublo ts from which 
they are taken. Compliance with maximum levels laid down by Directive 
2002/32/EC shall be established on the basis of the levels determined in the 
laboratory samples.  
                                                 
1 Table of TEF (= toxic equivalency factors) for PCDDs, PCDFs and dioxin -like PCBs:  WHO -TEFs for 
human risk assessment based on the conclusions of the World Health Organ ization (WHO) – 
International Programme on Chemical Safety (IPCS) expert meeting which was held in Geneva in June 
2005 (Martin van den Berg et al., The 2005 World Health Organization Re -evaluation of Human and 
Mammalian Toxic Equivalency Factors for Dioxin s and Dioxin -like Compounds. T oxicological 
Sciences 93(2), 223 –241 (2006)).   
 
Congener  TEF value  Congener  TEF value  
Dibenzo -p-dioxins 
(“PCDDs”) and 
Dibenzo -p-furans 
'("PCDFs")   “Dioxin -like” PCBs  
Non-ortho PCBs + Mono -ortho PCBs  
2,3,7,8 -TCDD  1   
1,2,3,7,8 -PeCDD  1 Non-ortho PCBs   
1,2,3,4,7,8 -HxCDD  0,1 PCB 77  0,0001  
1,2,3,6,7,8 -HxCDD  0,1 PCB 81  0,0003  
1,2,3,7,8,9 -HxCDD  0,1 PCB 126  0,1 
1,2,3,4,6,7,8 -HpCDD  0,01 PCB 169  0,03 
OCDD  0,0003   
Mono -ortho PCBs   
2,3,7,8 -TCDF  0,1 PCB 105  0,00003  
1,2,3,7,8 -PeCDF  0,03 PCB 114  0,00003  
2,3,4,7,8 -PeCDF  0,3 PCB 118  0,00003  
1,2,3,4,7,8 -HxCDF  0,1 PCB 123  0,00003  
1,2,3,6,7,8 -HxCDF  0,1 PCB 156  0,00003  
1,2,3,7,8,9 -HxCDF  0,1 PCB 157  0,00003  
2,3,4,6,7,8 -HxCDF  0,1 PCB 167  0,00003  
1,2,3,4,6,7,8 -HpCDF  0,01 PCB 189  0,00003  
1,2,3,4,7,8,9 -HpCDF  0,01   
OCDF  0,0003    
 
Abbreviations used: “T” = tetra; “Pe” = penta; “Hx” = hexa; “Hp” = hepta; “O” = octa; “CDD” = 
chlorodibenzodioxin; “CDF” = chlorodibenzofuran; “CB” = chlorobiphenyl.   
EN 3   EN For the purposes of this Part B, the definitions laid down in Annex I to C ommission 
Decision 2002/657/EC2 shall apply.  
In addition to those definitions, the following definitions shall apply for the purpose 
of this Part B:  
'Screening methods'means methods used for selection of those samples with levels of 
PCDD/Fs and dioxin -like PCBs that exceed the maximum levels or the action 
thresholds. They shall allow a cost -effective high sample -throughput, thus increasing 
the chance t o discover new incidents with high exposure and health risks to 
consumers. Screening methods shall be based on bioanalytical or GC -MS methods. 
Results from samples exceeding the cut -off value used to check compliance with the 
maximum level shall be verifie d by a full re -analysis from the original sample using 
a confirmatory method.  
'Confirmatory methods'  means methods that provide full or complementary 
information enabling the PCDD/Fs and dioxin -like PCBs to be identified and 
quantified unequivocally at the  maximum or in case of need at the action threshold. 
Such methods utilize gas chromatography/high resolution mass spectrometry (GC -
HRMS) or gas chromatography/tandem mass spectrometry (GC -MS/MS).  
2. COMPLIANCE OF THE LOT OR SUBLOT  WITH THE MAXIMUM LEV EL  
2.1. As regards non -dioxin -like PCBs  
The lot or sublot  complies with the maximum level if the analytical result for the sum 
of PCB 28, PCB 52, PCB 101, PCB 138, PCB 153 and PCB 180 (hereafter referred 
to as non -dioxin -like PCBs) does not exceed the maximum level l aid down by 
Directive 2002/32/EC, taking into account the expanded measurement uncertainty3 
The lot or sublot  does not comply with the maximum level as laid down by Directive 
2002/32/EC, if the mean of two upper -bound4 analytical results obtained from 
duplicate analysis5,taking into account the expanded measurement uncertainty, 
exceeds the maximum level beyond reasonable doubt , i.e. the analysed concentration 
after deduction of the expanded measurement uncertainty is used to assess 
compliance.   
                                                 
2 Commission Decision 2002/657/EC of 14 August 2002 implementing Council Directive 96/23/EC 
concerning the performance of analytical methods and interpretation of results (OJ L 221, 17.8.2002, 
p. 8). 
3 The principles described in the “Guidance Document on Measurement Uncertaint y for Laboratories 
performing PCDD/F and PCB Analysis using Isotope Dilution Mass Spectrometry” [ link to website ] 
shall be followed when applicable.  
4 The concept of ‘upper -bound’ requires using the limit of quantification for the contribution of each non -
quantified congener. The concept of ‘lower -bound’ requires using zero for the contribution of each non -
quantified congener. The concept of ‘medium -bound’ requires using half of the limit of quantification 
calculating the contribution of each non -quantified  congener.  
5 Duplicate analysis: Separate analysis of the analytes of interest using a second aliquot of the same 
homogenized sample.   
In general, the requirements for duplicate analysis as provided for in Annex II, Chapter C , point 3 
apply. However, for m ethods with the use of 13C-labelled internal standard for the relevant analytes, the 
duplicate analysis is only necessary if the result of the first determination is not compliant. The 
duplicate analysis is necessary to exclude the possibility of internal cross -contamination or an 
accidental mix -up of samples. In case the analysis is performed in the course of a contamination 
incident, confirmation by duplicate analysis may be omitted in case the samples selected for analysis 
are through traceability linked  to the contamination incident and the level found is significantly above 
the maximum level.   
EN 4   EN The expanded  measurement uncertainty is calculated using a coverage factor of 2 
which gives a level of confidence of approximately 95  %. A lot or sublot  is non -
compliant if the mean of the measured values minus the expanded uncertainty of the 
mean is above the maximum  level.  
The rules, mentioned in the paragraphs above under this point, shall apply for the 
analytical result obtained on the sample for official control. In case of analysis for 
defence or reference purposes, the national rules shall apply.  
2.2. As regards PCD D/Fs and dioxin -like PCBs  
The lot or sublot  complies with the maximum level if the result of a single analysis  
– performed by a screening method with a false -compliant rate below 5  %, 
indicates that the level does not exceed the respective maximum level of  
PCDD/Fs and the sum of PCDD/Fs and dioxin -like PCBs laid down by 
Directive 2002/32/EC ,  
– performed by a confirmatory method, does not exceed the respective maximum 
level of PCDD/Fs and the sum of PCDD/Fs and dioxin -like PCBs laid down by 
Directive 2002/32/ EC, taking into account the expanded measurement 
uncertainty . 
For screening assays a cut -off value shall be established for decisions on sample 
compliance with the respective maximum levels set for either PCDD/Fs, or for the 
sum of PCDD/Fs and dioxin -like PCBs.  
The lot or sublot  does not comply with the maximum level as laid down by Directive 
2002/32/EC if the mean of two upper -bound6 analytical result s obtained from  
duplicate analysis,7 using a confirmatory method, taking into account the expanded 
measurement uncertainty, exceeds the maximum level beyond reasonable doubt , i.e. 
the analysed concentration after deduction of the expanded measurement uncertainty 
is used to assess compliance.  
The expanded measurement uncertainty is calculated using a coverage factor of 2 
which gives a level of confidence of approximately 95  %. A lot or sublot  is non -
compliant if the mean of the measured values minus the expanded uncertainty of the 
mean is abov e the maximum level.  
The sum of the estimated expanded uncertainties of the separate analytical results of 
PCDD/Fs and dioxin -like PCBs shall be used for the sum of PCDD/Fs and dioxin -
like PCBs.  
                                                 
6 The concept of ‘upper -bound’ requires using the limit of quantification for the contribution of each non -
quantified congener to the Toxic Equivalent (TEQ). The c oncept of ‘lower -bound’ requires using zero 
for the contribution of each non -quantified congener to the TEQ. The concept of ‘medium -bound’ 
requires using half of the limit of quantification calculating the contribution of each non -quantified 
congener to th e TEQ.  
7 In general, the requirements for duplicate analysis as provided for in Annex II, Chapter C , point 2 
apply. However, for confirmatory methods with the use of 13C-labelled internal standard for the relevant 
analytes, the duplicate analysis is only n ecessary if the result of the first determination is not compliant. 
The duplicate analysis is necessary to exclude the possibility of internal cross -contamination or an 
accidental mix -up of samples. In case the analysis is performed in the course of a cont amination 
incident, confirmation by duplicate analysis may be omitted in case the samples selected for analysis 
are through traceability linked to the contamination incident and the level found is significantly above 
the maximum level.   
EN 5   EN The rules, mentioned in the paragraphs above under this point , shall apply for the 
analytical result obtained on the sample for official control. In case of analysis for 
defence or reference purposes, the national rules shall apply.  
3. RESULTS EXCEEDING AC TION THRESHOLDS AS L AID DOWN IN ANNEX II  TO 
DIRECTIVE 2002/32/ EC 
Action thresholds serve as a tool for the selection of samples in those cases where it 
is necessary to identify a source of contamination and to take measures for its 
reduction or elimination. Screening methods shall establish the appropriate cut -off 
values for selection of those samples. Where significant efforts are necessary to 
identify a source and to reduce or eliminate the contamination, it is appropriate to 
confirm exceedance of the action thresholds by duplicate analysis using a 
confirmatory metho d and taking into account the expanded measurement 
uncertainty8. 
CHAPTER II  
SAMPLE PREPARATION A ND REQUIREMENTS FOR METHODS OF ANALYSIS 
USED IN OFFICAL CONT ROL OF THE LEVELS OF  DIOXINS (PCDD/F s) AND 
DIOXIN -LIKE PCB s IN FEED  
1. FIELD OF APPLICATION  
The requirements set out in this Chapter shall be applied where feed is analysed for 
the official control of the levels of 2,3,7,8 -substituted PCDD/Fs and dioxin -like 
PCBs and as regards sample preparation and analytical requirements for other 
regulatory purposes,  which includes the controls performed by the feed business 
operator to ensure compliance with the provisions of Regulation (EC) No 183/2005 
of the European Parliament and of the Council9. 
Monitoring for the presence of PCDD/Fs and dioxin -like PCB s in feed may be 
performed with two different types of analytical methods:  
(a) Screening methods  
The goal of screening methods is to select those samples with levels of 
PCDD/Fs and dioxin -like PCBs that exceed the maximum levels or the action 
thresholds.  Screening methods sh all ensure  cost-effective high sample -
throughput, thus increasing the chance to discover new incidents with high 
exposure and health risks of consumers. Their application sh all aim to avoid  
false -compliant results. They may comprise bio analytical and GC/MS methods . 
Screening methods compare the analytical result with a cut -off value, 
providing a yes/no -decision over the possible exceedance of the maximum 
level or action threshold. The concentration of PCDD/Fs and the sum of 
PCDD/Fs and dioxin -like PCBs in samples suspected to be non -compliant with 
the maximum level shall  be determined  or confirmed by a confirmatory 
method . 
                                                 
8 Identical explanat ion and requirements for duplicate analysis for control of action thresholds as in 
footnote 7 for maximum levels.  
9 Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying 
down requirements for feed hygiene (OJ L 35, 8.2.2005, p. 1).   
EN 6   EN In addition, screening methods may give an indication of the levels of 
PCDD/Fs and dioxin -like PCBs present in the sample. In case of application of 
bioanalytical screening methods the result is expressed as Bioanalytical 
Equivalents (BEQ), whereas in ca se of application of physico -chemical GC -
MS methods it is expressed as Toxic Equivalents (TEQ). The numerically 
indicated results of screening methods are suitable for demonstrating 
compliance or suspected noncompliance or exceedance of action thresholds 
and give an indication of the range of levels in case of follow -up by 
confirmatory methods. They are not suitable for purposes such as evaluation of 
background levels, estimation of intake, following of time trends in levels or 
re-evaluation of action thres holds and maximum levels.  
(b) Confirmatory methods  
Confirmatory methods allow the unequivocal identification and quantification 
of PCDD/Fs and dioxin -like PCBs present in a sample and provide full 
information on congener level. Therefore, th ose methods all ow the control of 
maximum levels and action thresholds, including the confirmation of results 
obtained by screening methods. Furthermore, results  may be used for other 
purposes such as determination of low background levels in feed monitoring, 
following of  time trends, exposure assessment and building of a database for 
possible re -evaluation of action thresholds and maximum levels. They are also 
important for establishing congener patterns in order to identify the source of a 
possible contamination. Such me thods utili se GC -HRMS. For confirming 
compliance or non -compliance with the maximum level, also GC -MS/MS can 
be used.   
2. BACKGROUND  
For calculation of TEQ concentrations, the concentrations of the individual 
substances in a given sample shall be multipli ed by their respective Toxic 
Equivalency Factor (TEF) (see footnote 1 of Chapter I) and subsequently summed to 
give the total concentration of dioxin -like compounds expressed as TEQs.  
For the purposes of this Part B, the accepted specific limit of quantification of an 
individual congener means the lowest content of the analyte that can be measured 
with reasonable statistical certainty, fulfilling the identification criteria as described  
in internationally recognised standards, for example, in standard EN 16215:2012 
(Animal feed - Determination of dioxins and dioxin -like PCBs by GC/HRMS and of 
indicator PCBs by GC/HRMS) and/or in EPA methods 1613 and 1668 as revised.  
The limit of quantifi cation of an individual congener may be identified as  
(a) the concentration of an analyte in the extract of a sample which produces an 
instrumental response at two different ions to be monitored with a S/N 
(signal/noise) ratio of 3:1 for the less intensive  raw data signal; or  
(b) if for technical reasons the signal -to-noise calculation does not provide reliable 
results, the lowest concentration point on a calibration curve that gives an 
acceptable (≤ 30 %) and consistent (measured at least at the start and  at the end 
of an analytical series of samples) deviation to the average relative response 
factor calculated for all points on the calibration curve in each series of 
samples.  The limit of quantification ( LOQ ) is calculated from the lowest  
EN 7   EN concentration po int taking into account the recovery of internal standards and 
sample intake.  
Bioanalytical screening methods will not give results at the congener level but 
merely an indication10 of the TEQ level, expressed in BEQ to acknowledge the fact 
that not all comp ounds present in a sample extract that produce a response in the test 
may fulfill or meet all requirements of the TEQ -principle.  
Screening and confirmatory methods may only be applied for control of a certain 
matrix if the methods are sensitive enough to detect levels reliably at the action 
threshold or maximum level.   
3. QUALITY ASSURANCE REQ UIREMENTS  
3.1. Measures shall be taken to avoid cross -contamination at each stage of the sampling 
and analysis procedure.  
3.2. The samples shall be stored and transp orted in glass, aluminum, polypropylene or 
polyethylene containers suitable for storage without any influence on the levels of 
PCDD/Fs and dioxin -like PCBs in the samples. Traces of paper dust shall be 
removed from the sample container.  
3.3. The sample sto rage and transportation shall be performed in a way that maintains the 
integrity of the feed sample.  
3.4. Insofar as relevant, each laboratory sample shall be finely grinded and mixed 
thoroughly using a process that has been demonstrated to achieve complete 
homogeni sation (for example, ground to pass a 1 mm sieve). Samples shall be dried 
before grinding if the moisture content is too high.  
3.5. Control of reagents, glassware and equipment for possible influence of TEQ - or 
BEQ -based results shall be c arried out.  
3.6. A blank analysis shall be performed by carrying out the entire analytical procedure 
omitting only the sample.  
3.7. For bioanalytical methods, all glassware and solvents used in analysis shall be tested 
to be free of compounds that interfe re with the detection of target compounds in the 
working range. Glassware shall be rinsed with solvents or heated at temperatures 
suitable to remove traces of PCDD/Fs, dioxin -like compounds and interfering 
compounds from its surface.  
3.8. Sample quantity u sed for the extraction shall be sufficient to fulfill the requirements 
with respect to a sufficiently low working range including the concentrations of 
maximum levels or action threshold.  
3.9. The specific sample preparation procedures used for the products under 
consideration shall follow internationally accepted guidelines . 
                                                 
10 Bioanalytical methods are not specific to those congeners included in the TEF -scheme. Other 
structurally related AhR -active compounds may be present in the s ample extract which contribute to the 
overall response. Therefore, bioanalytical results cannot be an estimate but rather an indication of the 
TEQ level in the sample.    
EN 8   EN 4. REQUIREMENTS FOR LABO RATORIES  
4.1. In accordance with the provisions of Regulation (EC) No 882/2004, laboratories 
shall be accredited by a recognised body operating in accor dance with ISO Guide 58 
to ensure that they are applying analytical quality assurance. Laboratories shall be 
accredited following the EN ISO/IEC 17025 standard. The principles as described in 
the Technical Guidelines for the estimation of measurement uncer tainty and limits of 
quantification for PCDD/F and PCB analysis shall be followed when applicable11  
4.2. Laboratory proficiency shall be proven by the continuous successful participation in 
inter-laboratory studies for the determination of PCDD/Fs and diox in-like PCBs in 
relevant feed matrices and concentration ranges.  
4.3. Laboratories applying screening methods for the routine control of samples shall 
establish a close cooperation with laboratories applying the confirmatory method, 
both for quality contr ol and confirmation of the analytical result of suspected 
samples.  
5. BASIC REQUIREMENTS TO  BE MET BY ANALYTICAL  PROCEDURE FOR DIOXIN S 
(PCDD/F S) AND DIOXIN -LIKE PCB S 
5.1. Low working range and limits of quantification  
For PCDD/Fs, detectable quantities sha ll be in the upper femtogram (10-15g) range 
because of extreme toxicity of some of these compounds. For most PCB congeners a 
limit of quantification in the nanogram (10-9g) range is already sufficient. For the 
measurement of the more toxic dioxin -like PCB congeners (in particular non -ortho -
substituted congeners), the lower end of the working range shall reach the low 
picogram (10-12g) levels. For all other PCB congeners a limit of quantification in the 
nanogram (10-9g) range is sufficient.  
5.2. High selecti vity (specificity)  
5.2.1.  A distinction is required between PCDD/Fs and dioxin -like PCBs and a multitude of 
other, coextracted and possibly interfering compounds present at concentrations up to 
several orders of magnitude higher than those of the analytes of interest. For GC -MS 
methods, a differentiation among various congeners is required, such as between 
toxic (for example, the seventeen 2,3,7,8 -substituted PCDD/Fs, and twelve dioxin -
like PCBs) and other congeners.  
5.2.2.  Bioanalytical methods shall be a ble to detect the target compounds as the sum of 
PCDD/Fs, and/or dioxin -like PCBs. Sample clean -up shall aim at removing 
compounds causing false non -compliant results or compounds that may decrease the 
response, causing false compliant results.  
5.3. High a ccuracy (trueness and precision, bioassay apparent recovery)  
5.3.1.  For GC -MS methods, the determination shall provide a valid estimate of the true 
concentration in a sample. High accuracy is required to avoid the rejection of a 
sample analysis result on t he basis of poor reliability of the determined TEQ level. 
Accuracy is expressed as trueness  (difference between the mean value measured for 
an analyte in a certified material and its certified value, expressed as a percentage of 
                                                 
11 “Guidance Document on Measurement Uncertainty for Laboratories performing PCDD/F and PCB 
Analysis using Isotope Dilution Mass Spectrometry” [ link to website ], “Guidance Document on the 
Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food” [link to 
website] .  
EN 9   EN this value) and precision  (RSD R relative standard deviation calculated from results 
generated under reproducibility conditions).  
5.3.2.  For bioanalytical methods, the bioassay  apparent recovery shall be determined. 
Bioassay  apparent recovery means the BEQ level calculated from the TCDD or PCB 
126 calibration curve corrected for the blank and then divided by the TEQ level 
determined by the confirmatory method. It aims at correcting factors like the loss of 
PCDD/Fs and dioxin -like compounds during the extraction and clean -up steps, co -
extracted compounds increasing or decreasing the response (agonistic and 
antagonistic effects), the quality of the curve fit, or differences between the TEF 
values and the Relative Potency (REP) v alues. The bioassay apparent recovery is 
calculated from suitable reference samples with representative congener patterns 
around the level of interest.  
5.4. Validation in the range of maximum level and general quality control measures  
5.4.1.  Laboratories shall demonstrate the performance of a method in the range of the 
maximum level, for example, 0 ,5x, 1x and 2x the maximum level with an acceptable 
coefficient of variation for repeated analysis, during the validation procedure and 
during routine analysis.  
5.4.2.  Regular blank controls and spiking experiments or analysis of control samples 
(preferably, if available, certified reference material) shall be performed as internal 
quality control measures. Quality control charts for blank controls, spiking 
exper iments or analysis of control samples shall be recorded and checked to make 
sure the analytical performance is in accordance with the requirements.  
5.5. Limit of quantification  
5.5.1.  For a bioanalytical screening method, the establishment of the limit of  quantification 
(LOQ) is not an indispensable requirement but the method shall prove that it can 
differentiate between the blank and the cut -off value. When providing a BEQ level, a 
reporting level shall be established to deal with samples showing a respon se below 
this level. The reporting level shall be demonstrated to be different from procedure 
blank samples at least by a factor of three, with a response below the working range. 
It shall therefore be calculated from samples containing the target compound s around 
the required minimum level, and not from an S/N ratio or an assay blank.  
5.5.2.  The LOQ for a confirmatory method shall be about one fifth of the maximum level.  
5.6. Analytical criteria  
For reliable results from confirmatory or screening methods, the following criteria 
shall be met in the range of the maximum level for the TEQ or BEQ value, 
respectively, whether determined as total TEQ or total BEQ (as the sum of PCDD/Fs 
and dioxin -like P CBs) or separately for PCDD/Fs and dioxin -like PCBs:  
 Screening with bioanalytical 
or physico -chemical methods  Confirmatory methods  
False -compliant rate*  < 5 %  
Trueness   - 20 % to + 20  % 
Repeatability (RSD r) < 20 %  
Intermediate precision (RSD R)  < 25 % < 15 % 
* with respect to the maximum levels.   
EN 10   EN 5.7. Specific requirements for screening methods  
5.7.1.  Both GC -MS and bioanalytical methods may be used for screening. For GC -MS 
methods the requirements laid down in point 6 shall be met. For cell based 
bioanalytical methods specific requirements are laid down in point 7.  
5.7.2.  Laboratories applying screening methods for the routine control of samples shall 
establish a close cooperation with laboratories applying the confirmatory method.  
5.7.3.  Performan ce verification of the screening method is required during routine analysis, 
by analytical quality control and on -going method validation.  There shall be a 
continuous programme for the control of compliant results.  
5.7.4.  Check on possible suppression of t he cell response and cytotoxicity:  
20 % of the sample extracts shall be measured in routine screening without and with 
2,3,7,8 -TCDD added corresponding to the maximum level or action threshold, to 
check if the response is possibly suppressed by interfering  substances present in the 
sample extract. The measured concentration of the spiked sample shall be compared 
to the sum of the concentration of the unspiked extract plus the spiking 
concentration. If this measured concentration is more than 25 % lower than  the 
calculated (sum) concentration, this is an indication of potential signal suppression 
and the respective sample shall be submitted to GC/HRMS confirmatory analysis. 
Results shall be monitored in quality control charts.  
5.7.5.  Quality control on compli ant samples:  
Approximately 2 to 10  % of the compliant samples, depending on sample matrix and 
laboratory experience, shall be confirmed by GC/HRMS.  
5.7.6.  Determination of false -compliant rates from quality control data:  
The rate of false -compliant results  from screening  of samples below and above the 
maximum level or the action threshold shall be determined . Actual false -compliant 
rates shall be below 5  %. When a minimum of 20 confirmed results per 
matrix/matrix group is available from the quality control  of compliant samples, 
conclusions on the false compliant rate shall be drawn from this database. The results 
from samples analysed in ring trials or during contamination incidents, covering a 
concentration range up to for example 2x the maximum level (ML) , may also be 
included in the minimum of 20 results for evaluation of the false -compliant rate. The 
samples shall cover most frequent congener patterns, representing various sources.  
Although screening assays shall preferentially aim to detect samples exce eding the 
action threshold, the criterion for determining false -compliant rates is the maximum 
level, taking into account the expanded measurement uncertainty of the confirmatory 
method.  
5.7.7.  Potential non-compliant samples from screening shall always be verified by a full re -
analysis of the original sample by a confirmatory method of analysis. These samples 
may also be used to evaluate the rate of false non -compliant results. For screening 
methods, the rate of false non -compliant results shall be the fraction of results 
confirmed to be compliant from confirmatory analysis, while in previous screening 
the sample has been declared to be potentially non -compliant. E valuation of the 
advantage s of the screeni ng method shall be based on comparison of false -non-
compliant samples with the total number of samples checked. This rate shall be low 
enough to make the use of a screening tool advantageous.   
EN 11   EN 5.7.8.  Under validation conditions, bioanalytical methods shall provide a valid indication of 
the TEQ level, calculated and expressed as BEQ.  
Also for bioanalytical methods carried out under repeat ed conditions, the intra -
laboratory RSD r would typically be smalle r than under reproducibility  
conditions( RSD R)  
6. SPECIFIC REQUIREMENTS  FOR GC /MS METHODS TO BE COM PLIED WITH FOR 
SCREENING OR CONFIRM ATORY PURPOSES  
6.1. Acceptable differences between upper -bound and lower -bound WHO -TEQ 
results  
The difference between upper -bound level and lower -bound level shall not exceed 
20 % for confirmation of exceedance of maximum level or in case of need of action 
thresholds.  
6.2. Control of recoveries  
6.2.1.  Addition of 13C-labelled 2,3,7,8 -chlorine -substituted internal PCDD/F standards and 
of 13C-labelled internal dioxin -like PCB standards shall be carried out at the very 
beginning of the analytical method e.g. prior to extraction in order to validate the 
analytical procedure. At least one congener for each of the tetra - to oct a-chlorinated 
homologous groups for PCDD/Fs and at least one congener for each of the 
homologous groups for dioxin -like PCBs shall be added (alternatively, at least one 
congener for each mass spectrometric selected ion recording function used for 
monitorin g PCDD/Fs and dioxin -like PCBs). In the case of confirmatory methods, all 
17 13C-labelled 2,3,7,8 -substituted internal PCDD/F standards and all 12 13C-labelled 
internal dioxin -like PCB standards shall be used.  
6.2.2.  Relative response factors shall also be  determined for those congeners for which no 
13C-labelled analogue is added by using appropriate calibration solutions.  
6.2.3.  For feed of plant origin and feed of animal origin containing less than 10  % fat, the 
addition of the internal standards shall be  mandatory prior to extraction. For feed of 
animal origin containing more than 10  % fat, the internal standards shall be added 
either before or after fat extraction. An appropriate validation of the extraction 
efficiency shall be carried out, depending on the stage at which internal standards are 
introduced .  
6.2.4.  Prior to GC -MS analysis, 1 or 2 recovery (surrogate) standard(s) shall be added.  
6.2.5.  Control of recovery is required. For confirmatory methods, the recoveries of the 
individual internal stand ards shall be in the range of 60 to 120  %. Lower or higher 
recoveries for individual congeners, in particular for some hepta - and octa - 
chlorinated dibenzo -p-dioxins and dibenzofurans, shall be acceptable on the 
condition that their contribution to the TEQ  value does not exceed 10  % of the total 
TEQ value (based on sum of PCDD/F and dioxin -like PCBs). For GC/MS screening 
methods, the recoveries shall be in the range of 30 to 140  %. 
6.3. Removal of interfering substances  
– Separation of PCDD/Fs from interferin g chlorinated compounds such as non -
dioxin -like PCBs and chlorinated diphenyl ethers shall be carried out by 
suitable chromatographic techniques (preferably with a florisil, alumina and/or 
carbon column).   
EN 12   EN – Gas-chromatographic separation of isomers shall be < 25  % peak to peak 
between 1,2,3,4,7,8 -HxCDF and 1,2,3,6,7,8 -HxCDF.  
6.4. Calibration with standard curve  
The range of the calibration curve shall cover the relevant range of maximum level 
or action thresholds.  
6.5. Specific criteria for confirmatory meth ods  
– For GC -HRMS:  
In HRMS, the resolution shall typically be greater than or equal to 10 000 for 
the entire mass range at 10 % valley.  
Fulfilment of further identification and confirmation criteria as described in 
internationally recognised standards, for  example, in standard EN 16215:2012 
(Animal feed - Determination of dioxins and dioxin -like PCBs by GC -HRMS 
and of indicator PCBs by GC -HRMS) and/or in EPA methods 1613 and 1668 
as revised.  
– For GC -MS/MS:  
Monitoring of at least 2 specific precursor ions, ea ch with one specific 
corresponding transition product ion for all labelled and unlabelled analytes in 
the scope of analysis.  
Maximum permitted tolerance of relative ion intensities of ± 15  % for selected 
transition product ions in comparison to calculated or measured values 
(average from calibration standards), applying identical MS/MS conditions, in 
particular collision energy and collision gas pressure, for each transition of an 
analyte.  
Resolution for each quadrupole to be set equal to or better than uni t mass 
resolution (unit mass resolution: sufficient resolution to separate two peaks one 
mass unit apart) in order to minimi se possible interferences on the analytes of 
interest.  
Fulfilment of the further criteria as described in internationally recognised  
standards, for example, in standard EN 16215:2012 (Animal feed - 
Determination of dioxins and dioxin -like PCBs by GC -HRMS and of indicator 
PCBs by GC -HRMS) and/or in EPA methods 1613 and 1668 as revised, except 
the obligation to use GC -HRMS.  
7. SPECIFIC  REQUIREMENTS  FOR  BIOANALYTICAL  METHODS  
Bioanalytical methods are methods based on the use of biological principles like cell -
based assays, receptor -assays or immunoassays. This point 7 establishes 
requirements for bioanalytical methods in general.  
A screening method in principle classifies a sample as compliant or suspected to be 
non-compliant. For this, the calculated BEQ level is compared to the cut -off value 
(see point 7.3). Samples below the cut -off value are declared compliant, samples 
equal or  above the cut -off value are suspected to be non -compliant, requiring 
analysis by a confirmatory method. In practice, a BEQ level corresponding to two-
thirds  of the maximum level may serve as cut -off value provided that a false -
compliant rate below 5  % and  an acceptable rate for false non -compliant results are 
ensured. With separate maximum levels for PCDD/Fs and for the sum of PCDD/Fs  
EN 13   EN and dioxin -like PCBs, checking compliance of samples without fractionation 
requires appropriate bioassay cut -off values for  PCDD/Fs. For checking of samples 
exceeding the action thresholds, an appropriate percentage of the respective action 
threshold shall suit as cut -off value.  
If an indicative level is expressed in BEQs, sample results shall be in the working 
range and excee ding the reporting limit (see points  7.1.1 and 7.1.6).  
7.1. Evaluation of the test response  
7.1.1.  General requirements  
– When calculating the concentrations from a TCDD calibration curve, values at 
the higher end of the curve will show a high variation (hi gh coefficient of 
variation (CV)). The working range is the area where this CV is smaller than 
15 %. The lower end of the working range (reporting limit) shall be set at least 
by a factor of three above the procedure blanks. The upper end of the  working 
range is usually represented by the EC 70 value (70  % of maximal effective 
concentration), but lower if the CV is higher than 15  % in this range. The 
working range shall be established during validation. Cut -off values (see point 
7.3) shall be well within t he working range.  
– Standard solutions and sample extracts shall be tested in triplicate or at least in 
duplicate. When using duplicates, a standard solution or a control extract tested 
in four to six wells divided over the plate shall produce a response or 
concentration (only possible in the working range) based on a CV  < 15 %. 
7.1.2.  Calibration  
7.1.2.1.  Calibration with standard curve  
– Levels in samples shall be estimated by comparison of the test response with a 
calibration curve of TCDD (or PCB 126 or a P CDD/PCDF/dioxin -like PCB 
standard mixture) to calculate the BEQ level in the extract and subsequently in 
the sample.  
– Calibration curves shall contain 8 to 12 concentrations (at least in duplicates), 
with enough concentrations in the lower part of the curve  (working range). 
Special attention shall be paid to the quality of the curve -fit in the working 
range. As such, the R2 value is of little or no value in estimating the goodness 
of fit in non -linear regression. A better fit shall be achieved by minimising the 
difference between calculated and observed levels in the working range of the 
curve, for example by minimising the sum of squared residuals.  
– The estimated level in the sample extract shall be subsequently corrected for 
the BEQ level calculated for a ma trix or solvent blank sample (to account for 
impurities from solvents and chemicals used), and the apparent recovery 
(calculated from the BEQ level of suitable reference samples with 
representative congener patterns around the maximum level or action 
thres hold). To perform a recovery correction, the apparent recovery shall be 
within the required range (see point 7.1.4). Reference samples used for 
recovery correction shall comply with the requirements laid down in point 7.2.  
7.1.2.2.  Calibration with referen ce samples  
Alternatively, a calibration curve prepared from at least four reference samples (see 
point 7.2.4 ): one matrix blank, plus three reference samples at 0 ,5x, 1x and 2x the  
EN 14   EN maximum level or action threshold may be used, eliminating the need to corr ect for 
blank and recovery if matrix properties of the reference samples match those of the 
unknown samples.  In this case, the test response corresponding to two-thirds  of the 
maximum level (see point 7.3) may be calculated directly from these samples and 
used as cut -off value. For checking of samples exceeding the action thresholds, an 
appropriate percentage of these action thresholds shall  suit as cut -off value.  
7.1.3.  Separate determination of PCDD/Fs and dioxin -like PCBs  
Extracts may be split into frac tions containing PCDD/Fs and dioxin -like PCBs, 
allowing a separate indication of PCDD/Fs and dioxin -like PCB TEQ levels (in 
BEQ). A PCB 126 standard calibration curve shall preferentially be used to evaluate 
results for the fraction containing dioxin -like PCBs.  
7.1.4.  Bioassay apparent recoveries  
The “bioassay apparent recovery” shall be calculated from suitable reference samples 
with representative congener patterns around the maximum level or action threshold 
and expressed as percentage of the BEQ level  in comparison to the TEQ level. 
Depending on the type of assay and TEFs12 used, the differences between TEF and 
REP factors for dioxin -like PCBs can cause low apparent recoveries for dioxin -like 
PCBs in comparison to PCDD/Fs. Therefore, if a separate determination of PCDD/Fs 
and dioxin -like PCBs is performed, bioassay apparent recoveries shall be: for dioxin -
like PCBs 20  % to 60  %, for PCDD/Fs 50  % to 130  % (ranges apply for the TCDD 
calibration curve). As the contribution of dioxin -like PCBs to the su m of PCDD/Fs 
and dioxin -like PCBs can vary between different matrices and samples, bioassay 
apparent recoveries for the sum of PCDD/Fs and dioxin -like PCBs reflect these 
ranges and shall be between 30  % and 130  %. Any implication of substantially 
revised T EF values for the Union legislation for PCDD/Fs and dioxin -like PCBs 
requires the revision of these ranges.  
7.1.5.  Control of recoveries for clean -up 
The loss of compounds during the clean -up shall be checked during validation. A 
blank sample spiked with a  mixture of the different congeners shall be submitted to 
clean -up (at least n  = 3) and the recovery and variability checked by a confirmatory 
method. The recovery shall be within 60  % to 120  % especially for congeners 
contributing more than 10  % to the TE Q-level in various mixtures.  
7.1.6.  Reporting limit 
When reporting BEQ levels, a reporting limit shall be determined from relevant 
matrix samples involving typical congener patterns, but not from the calibration 
curve of the standards due to low precision  in the lower range of the curve. Effects 
from extraction and clean -up shall be taken into account. The reporting limit shall be 
set at least by a factor of three above the procedure blanks.  
7.2. Use of reference samples  
7.2.1.  Reference samples shall repr esent sample matrix, congener patterns and 
concentration ranges for PCDD/Fs and dioxin -like PCBs around the maximum level 
or action threshold.  
                                                 
12 Current requirements  are based on the TEFs pu blished in: M. Van den Berg et al, Toxicol Sci 93 (2), 
223–241 (2006).   
EN 15   EN 7.2.2.  A matrix blank, and where it is not possible, a procedure blank, and a reference 
sample at the maximum lev el or action threshold shall be included in each test series. 
These samples shall be extracted and tested at the same time under identical 
conditions. The reference sample shall show a clearly elevated response in 
comparison to the blank sample, thus ensur ing the suitability of the test. Th ose 
samples may be used for blank and recovery corrections.  
7.2.3.  Reference samples chosen to perform a recovery correction shall be representative 
for the test samples, meaning that congener patterns may not lead to an 
underestimation of levels.  
7.2.4.  Extra reference samples at e.g. 0 ,5x and 2x the maximum level or action threshold 
may be included to demonstrate the proper performance of the test in the range of 
interest for the control of the maximum level or action threshold. Combined, these 
samples may be used for calculating the BEQ levels in test samples (see point 
7.1.2.2).  
7.3. Determination of cut -off values  
The relationship between bioanalytical results in BEQ and results from the 
confirmatory method in TEQ sh all be established, for example by matrix -matched 
calibration experiments, involving reference samples spiked at 0, 0 ,5x, 1x and 2x the 
ML, with 6  repetitions on each level (n  = 24). Correction factors (blank and 
recovery) may be estimated from this relationship but shall be checked in accordance 
with point  7.2.2.  
Cut-off values shall be established for decisions over sample compliance with 
maximum levels or for the control of action thresholds, if relevant, with the 
respective maximum levels or actio n threshold set for either PCDD/Fs and dioxin -
like PCBs alone, or for the sum of PCDD/Fs and dioxin -like PCBs. They are 
represented by the lower  end-point of the distribution of bioanalytical results 
(corrected for blank and recovery) corresponding to the decision limit of the 
confirmatory method based on a 95  % level of confidence, implying a false -
compliant rate < 5  %, and on a RSD R < 25 %. The decision limit of the confirmatory 
method is the maximum level, taking into account the expanded measurement 
uncertainty.  
The cut -off value (in BEQ) may be calculated in accordance with one of the 
approaches set out in points 7.3.1, 7.3.2 and 7.3.3. (see Figure 1) . 
7.3.1.  Use of the lower  band of the 95  % prediction interval at the decision limit of the 
confirmatory  method  
Cut-off value = BEQ DL  sy,x * t α,f=m -2  
with:  
BEQ DL BEQ corresponding to the decision limit of the confirmatory method, 
being the maximum level taking into account the expanded 
measurement uncertainty  
sy,x residual standard deviation  
t α,f=m -2 student factor (α = 5  %, f = degrees of freedom, single -sided)  
xx i Qxxm n /) ( /1/12 
EN 16   EN m total number of calibration points (index j)  
n number of repetitions on each level  
xi sample concentration (in TEQ) of calibration point i determined by a 
confirmatory method  
 mean of the concentrations (in TEQ) of all calibration samples  
Qxx =  square sum parameter, i = index for calibration point i  
7.3.2.  Calculation from bioanalytical results (corrected for blank and recovery) of multiple 
analyses of samples (n > 6) contaminated at the decision limit of the confirmatory 
method, as the lower  endpoint of the data distribution at the corresponding mean 
BEQ value:  
Cut-off value = BEQ DL – 1,64xSD R 
with: 
SD R standard deviation of bioassay results at BEQ DL, measured under within -laboratory 
reproducibility conditions  
7.3.3.  Calculation as mean value of bioanalytical results (in BEQ, corrected for blank and 
recovery) from multiple analysis of samples (n  > 6) contaminated at two-thirds  of the 
maximum level or action threshold, based on the obse rvation that this level will be 
around the cut -off value determined under point 7.3.1 or point 7.3.2:   
Calculation of cut -off values based on a 95 % level of confidence implying a false -compliant rate 
< 5 %, and a RSD R < 25 %:  
1. from the lower  band of the 95 % prediction interval at the decision limit of the confirmatory 
method . 
2. from multiple analysis of samples (n > 6) contaminated at the decision limit of the 
confirmatory method as the lower  end-point of the data distribution (represented in the figure 
by a bell -shaped curve) at the corresponding mean BEQ value.  
x

m
jixx
12) ( 
EN 17   EN Figure 1   
 
7.3.4.  Restrictions to cut -off values  
BEQ -based cut -off values calculated from the RSD R achieved during validation 
using  a limited number of samples with different matrix/congener patterns may be 
higher than the TEQ -based maximum levels or action thresholds due to a better 
precision than attainable in routine when an unknown spectrum of possible congener 
patterns has to be controlled. In such cases, cut -off values shall be calculated from an 
RSD R = 25  %, or two -thirds of the maximum level or action threshold shall be 
preferred.  
7.4. Performance characteristics  
7.4.1.  Since no internal standards can be used in bioanalytical m ethods, tests on the 
repeatability of bioanalytical methods shall be carried out to obtain information on 
the standard deviation within and between test series. Repeatability shall be below 
20 % and intra-laboratory reproducibility shall be below 25  %. This shall be based on 
the calculated levels in BEQ after blank and recovery correction.  
7.4.2.  As part of the validation process, the test shall be shown to discriminate between a 
blank sample and a level at the cut -off value, allowing the identification of samples 
above the corresponding cut -off value (see point 7.1.2).  
7.4.3.  Target compounds, possible interferences and maximum tolerable blank levels shall 
be defined.  
7.4.4.  The percent standard deviation in the response or concentration calculated from  the 
response (only possible in working range) of a triplicate determination of a sample 
extract may not be above 15  %. 
 
EN 18   EN 7.4.5.  The uncorrected results of the reference sample(s) expressed in BEQ (blank and at 
the maximum level or action threshold) shall be  used for evaluation of the 
performance of the bioanalytical method over a constant time period.  
7.4.6.  Quality control charts for procedure blanks and each type of reference sample shall 
be recorded and checked to make sure the analytical performance is i n accordance 
with the requirements, in particular for the procedure blanks with regard to the 
requested minimum difference to the lower end of the working range and for the 
reference samples with regard to within -laboratory reproducibility. Procedure blank s 
shall be controlled in a manner to avoid false -compliant results when subtracted.  
7.4.7.  The results from the confirmatory methods of suspected samples and 2 to 10  % of the 
compliant samples (minimum of 20 samples per matrix) shall be collected and used 
to evaluate the performance of the screening method and the relationship between 
BEQ and TEQ. This database may be used for the re -evaluation of cut -off values 
applicable to routine samples for the validated matrices.  
7.4.8.  Successful method performance may also be demonstrated by participation in ring 
trials. The results from samples analysed in ring trials, covering a concentration 
range up to e.g. 2x maximum level, may be included in the evaluation of the false -
compliant rate, if a laboratory is able t o demonstrate its successful performance. The 
samples shall cover most frequent congener patterns, representing various sources.  
7.4.9.  During incidents, the cut -off values may be re -evaluated, reflecting the specific 
matrix and congener patterns of this s ingle incident.  
8. REPORTING  OF THE  RESULTS  
8.1. Confirmatory methods  
8.1.1.  The analytical results shall contain  the levels of the individual PCDD/F and dioxin -
like PCB congeners and TEQ -values shall be reported as lower -bound, upper -bound 
and medium -boun d in order to include a maximum of information in the reporting of 
the results and thereby enabling the interpretation of the results according to specific 
requirements.  
8.1.2.  The report shall include the method used for extraction of PCDD/Fs and dioxin -like 
PCBs.  
8.1.3.  The recoveries of the individual internal standards shall be made available in case the 
recoveries are outside the range referred to in point 6.2.5, in case the maximum level 
is exceeded (in this case, the recoveries for one of the two du plicate analysis) and in 
other cases upon request.  
8.1.4.  As the expanded measurement uncertainty is to be taken into account when deciding 
about the compliance of a sample, this parameter shall be made available. Thus, 
analytical results shall be reporte d as x +/ - U whereby x is the analytical result and U 
is the expanded measurement uncertainty using a coverage factor of 2 which gives a 
level of confidence of approximately 95  %. In the case of a separate determination of 
PCDD/Fs and dioxin -like-PCBs, the  sum of the estimated expanded uncertainty of 
the separate analytical results of PCDD/Fs and dioxin -like PCBs shall be used for the 
sum of PCDD/Fs and dioxin -like PCBs.  
8.1.5.  The results shall be expressed in the same units and with at least the same numb er of 
significant figures as the maximum levels laid down by Directive 2002/32/EC   
EN 19   EN 8.2. Bioanalytical screening methods  
8.2.1.  The result of the screening shall be expressed as "compliant" or "suspected to be 
non-compliant" ("suspected").  
8.2.2.  In addition, an indicative result for PCDD/Fs and/or dioxin -like PCBs expressed in 
BEQ, and not TEQ, may be given.  
8.2.3.  Samples with a response below the reporting limit shall be expressed as "lower than 
the reporting limit". Samples with a response abo ve the working range shall be 
reported as "exceeding the working range " and the level corresponding to the upper 
end of the working range shall be given in BEQ.  
8.2.4.  For each type of sample matrix, the report shall mention the maximum level or action 
threshold on which the evaluation is based.  
8.2.5.  The report shall mention the type of the test applied, the basic test principle and the 
kind of calibration.  
8.2.6.  The report shall include the method used for extraction of PCDD/Fs and dioxin -like 
PCBs.  
8.2.7.  In case of samples suspected to be non -compliant, the report needs to include a note 
on the action to be taken. The concentration of PCDD/Fs and the sum of PCDD/Fs 
and dioxin -like PCBs in those samples with elevated levels has to be 
determined/conf irmed by a confirmatory method.  
8.2.8.  Non-compliant results shall only be reported from confirmatory analysis.  
8.3. Physico -chemical screening  methods  
8.3.1.  The result of the screening shall be expressed as "compliant " or "suspected to be 
non-compliant " (“suspected”).  
8.3.2.  For each type of sample matrix, the report shall mention the maximum level or action 
threshold on which the evaluation is based.  
8.3.3.  In addition, levels for individual PCDD/F and/or dioxin -like PCB congeners and 
TEQ -values reported  as lower -bound, upper -bound and medium -bound may be 
given. The results shall be expressed in the same units and with at least the same 
number of significant figures as the maximum levels laid down by Directive 
2002/32/EC.  
8.3.4.  The recoveries of the indi vidual internal standards shall be made available in case the 
recoveries are outside the range referred to in point 6.2.5, in case the maximum level 
is exceeded (in this case, the recoveries for one of the two duplicate analysis) and in 
other cases upon re quest.  
8.3.5.  The report shall mention the GC -MS method applied.  
8.3.6.  The report shall include the method used for extraction of PCDD/Fs and dioxin -like 
PCBs.  
8.3.7.  In case of samples suspected to be non -compliant, the report needs to include a note 
on the action to be taken. The concentration of PCDD/Fs and the sum of PCDD/Fs 
and dioxin -like PCBs in those samples with elevated levels has to be 
determined/confirmed by a confirmatory method.  
8.3.8.  Non-compliance can only be decided after confirmatory an alysis .  
EN 20   EN CHAPTER III  
SAMPLE PREPARATION A ND REQUIREMENTS FOR METHODS OF ANALYSIS 
USED IN OFFICAL CONT ROL OF THE LEVELS OF  NON DIOXIN -LIKE PCB s IN 
FEED  
1. FIELD OF APPLICATION  
The requirements set out in this Chapter shall be applied where feed is analysed for 
the official control of the levels of non -dioxin -like PCBs and as regards sample 
preparation and analytical requirements for other regulatory purposes,  which 
includes t he controls performed by the feed business operator to ensure compliance 
with the provisions of Regulation (EC) No 183/2005.  
2. APPLICABLE DETECTION METHODS  
Gas chromatography / Electron Capture Detection (GC -ECD), GC -LRMS, GC-
MS/MS, GC -HRMS or equivalent methods.  
3. IDENTIFICATION AND CO NFIRMATION OF ANALYT ES OF INTEREST  
3.1. Relative retention time in relation to internal standards or reference standards 
(acceptable deviation of +/ - 0,25 %).  
3.2. Gas chromatographic separation of the non-dioxin -like PCBs from interfering 
substances, especially co -eluting PCBs, in particular if levels of samples are in the 
range of legal limits and non -compliance is to be confirmed13.  
3.3. Requirements for GC -MS tec hniques  
Monitoring of at least the following number of molecular ions or characteristic ions 
from the molecular cluster:  
(a) two specific ions for HRMS;  
(b) three specific ions for LRMS;  
(c) two specific precursor ions, each with one specific correspondin g transition 
product ion for for MS -MS. 
Maximum permitted tolerances for abundance ratios for selected mass fragments:  
Relative deviation of abundance ratio of selected mass fragments from theoretical 
abundance or calibration standard for target ion (most abundant ion monitored) and 
qualifier ion(s): ± 15 %   
3.4. Requirements for GC -ECD techniques  
Results exceeding the maximum level shall be confirmed with two GC columns  with 
stationary phases of different polarity.  
                                                 
13 Congeners often found to co -elute are for example PCB 28/31, PCB 52/69 and PCB 138/163/164. For 
GC-MS also possible interferences from fragments of higher chlorinated congeners shall be considered .  
EN 21   EN 4. DEMONSTRATION OF PERF ORMANCE OF METHOD  
The performance of the method shall be validated in the range of the maximum level 
(0,5 to 2 times the maximum level) with an acceptable coefficient of variation for 
repeated analysis (see requirements for intermediate precision in point 9).  
5. LIMIT OF QUANTIFICATION  
The sum of the LOQs14 of non-dioxin -like PCB s shall not be higher than one-third  of 
the maximum level15. 
6. QUALITY CONTROL  
Regular blank controls, analysis of spiked samples, quality control samples, 
participation in inter -laboratory studies on relevant matrices.  
7. CONTROL OF RECOVERIES  
7.1. Suitable internal standards with physico -chemical properties comparable to analytes 
of interest shall be used.  
7.2. Addition of internal standards:  
Addition to products (before extraction and clean -up pro cess).  
7.3. Requirements for methods using all six isotope -labelled non -dioxin -like PCB 
congeners  
(a) results shall be corrected for recoveries of internal standards;  
(b) recoveries of isotope -labelled internal standards shall be between 60 and 
120 %; 
(c) lower or higher recoveries for individual congeners with a contribution to the 
sum of non-dioxin -like PCBs  below 10 % are acceptable.  
7.4. Requirements for methods using not all six isotope -labelled internal standards or 
other internal standards:  
(a) recovery of internal standard(s) shall be controlled for every sample;  
(b) recoveries of internal standard(s) shall be between 60 and 120 %;  
(c) results shall be corrected for recoveries of internal standards.  
7.5. The recoveries of unlabelled congeners sh all be checked by spiked samples or 
quality control samples with concentrations in the range of the maximum level. 
Recoveries for these congeners shall be considered acceptable, if they are between 60 
and 120  %. 
                                                 
14 The principles as described in the “Guidance Document on the Estimation of LOD and LOQ for 
Measurements in the Field of Contaminants in Feed and Food” [ link to website ] shall be followed when 
applicable.  
15 It is highly recommendable to hav e a lower contribution of the reagent blank level to the level of a 
contaminant in a sample. It is in the responsibility of the laboratory to control the variation of blank 
levels, in particular, if the blank levels are subtracted.    
EN 22   EN 8. REQUIREMENTS FOR LABO RATORIES  
In accordan ce with the provisions of Regulation (EC) No 882/2004, laboratories 
shall be accredited by a recognised body operating in accordance with ISO Guide 58 
to ensure that they are applying analytical quality assurance. Laboratories shall be 
accredited following  the EN ISO/IEC 17025 standard. In addition, the principles as 
described in Technical Guidelines for the estimation of measurement uncertainty and 
limits of quantification for PCB analysis shall be followed when applicable16. 
9. PERFORMANCE CHARACTER ISTICS : CRITERIA FOR THE SUM  OF NON-DIOXIN -LIKE 
PCB S AT THE MAXIMUM EVEL  
 Isotope dilution 
mass spectrometry*  Other techniques  
Trueness  - 20 to + 20 %  - 30 to + 30 %  
Intermediate precision (RSD  %) ≤ 15 %  ≤ 20 %  
Difference between upper and 
lower -bound calculation  ≤ 20 %  ≤ 20 %  
* Use of all six 13C-labelled analogues as internal standards required  
10. REPORTING OF THE RESU LTS 
10.1.  The analytical results shall contain the levels of the individual non-dioxin -like PCBs 
and the sum of those PCB congeners reported as lower -bound, upper -bound and 
medium -bound in order to include a maximum of information in the reporting of the 
results and thereby enabling the interpretation of the results according to specific 
requirements.  
10.2.  The report shall include the method used for the extraction of PCBs.  
10.3.  The recoveries of the individual internal standards shall be made available in case the 
recoveries are outside the range referred to in point 7, in case the maximum level is 
exceed ed and in other cases upon request.  
10.4.  As the expanded measurement uncertainty is to be taken into account when deciding 
about the compliance of a sample, that parameter shall also be made available. Thus, 
analytical results shall be reported as x +/ - U whereby x is the analytical result and U 
is the expanded measurement uncertainty using a coverage factor of 2 which gives a 
level of confidence of approximately 95  %. 
10.5. The results shall be expressed in the same units and with at least the same numbe r of 
significant figures as the maximum levels laid down by Directive 2002/32/EC. " 
                                                 
16 See footnote 12.