EN    EN 
 
 
 EUROPEAN  
COMMISSION   
Brussels, XXX   
SANTE/10521/2016 ANNEX Rev. 1 
(POOL/E2/2016/10521/10521R1 -EN 
ANNEX.doc) D045998/04  
[…](2016) XXX  draft  
ANNEXES 1 to 4  
  
ANNEXES  
to the  
COMMISSION REGULATION (EU) …/…  
laying down methods of sampling and analysis for the control of levels of dioxins, dioxin -
like PCBs and non -dioxin -like PCBs in certain foodstuffs and repealing Regulation (EU) 
No 589/2014    
EN 2   EN ANNEX I  
DEFINITIONS AND ABBREVIATIONS  
I. DEFINITIONS  
For the purposes of this Regulation the definitions laid down in Annex I to 
Commission Decision 2002/657/EC1 shall apply.  
Further to those definitions, the following definitions shall apply for the purposes of 
this Regulation:  
1.1. 'Action level 'means the level of a given substance, as laid down in the Annex to 
Recommendation 2013/711/EU, which triggers investigations to identify the source 
of that substance in cases where increased levels of the substance are detected.  
1.2. 'Screening methods'  means methods used for the selection of those samples with 
levels of PCDD/Fs and dioxin -like PCBs that exceed the maximum levels or the 
action level s. They shall allow for a cost -effective high sample -throughput, thus 
increasing the chance of discover ing new cases where high exposure may lead to  
health risks for consumers . Screening methods shall be based on bioanalytical or GC -
MS methods. Results from samples exceeding the cut -off value established to check 
compliance with the maximum level shall be verified by a full re -analysis from the 
origin al sample using a confirmatory method.  
1.3. '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 level . 
Such methods utilise gas chromatography/high resolution mass spectrometry (GC -
HRMS) or gas chromatography/tandem mass spectrometry (GC -MS/MS).  
1.4. 'Bioanalytical methods'  means methods based on the use of biological principles such 
as cell-based assays, receptor -assays or immunoassays. They do not give results at 
the congener level but merely an indication2 of the TEQ level, expressed in 
Bioanalytical Equivalents (BEQ) to acknowledge the fact that not all compounds 
present in a sample extract that produce a response in the test may meet  all 
requirements of the TEQ -principle.  
1.5. '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 attempts to correct factors like the loss of 
PCDD/Fs and dioxin -like compounds du ring 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 and 
the REP values. The bioassay apparent recovery is ca lculated from suitable reference 
samples with representative congener patterns around the maximum or action level .  
1.6. 'Duplicate analysis ' means  separate analysis of the analytes of interest using a second 
aliquot of the same homogeni sed sample.  
                                                 
1 Commi ssion Decision 2002/65 7/EC of 1 4 August 2002 implementing Council Directive 96/23/EC 
concerning the performance of analytical methods and the interpretation of results  (OJ L 221, 
17.8.2002, p. 8 ).  
2 Bioanal ytical methods are not specific to those congeners included in the TEF -scheme. Other 
structurally related AhR -active compounds may be present in the sample 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 3   EN 1.7. 'Accepted specific limit of quantification3 of an individual congener in a sample'  
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 quantification of an individu al 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, if for technical reasons the signal -to-noise calculation does not provide reliable 
results,  
(b) 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 samples4. 
1.8. 'Upper-bound'  means the concept which requires using the limit of quantification for 
the contribution of each non -quantified congener.  
1.9. 'Lower -bound'  means the concept which requires using zero for the contribution of 
each non -quantified congener.  
1.10.  'Medium -bound'  means the concept which requires using half of the limit of 
quantification calculating the contribution of each non -quantified congener.  
1.11.  'Lot' means an identifiable quantity of food delivered at one time and determined by 
the official to have c ommon characteristics, such as origin, variety, type of packing, 
packer, consignor or markings. In the case of fish and fishery products, also the size 
of fish shall be comparable. In case the size and/or weight of the fish is not 
comparable within a consi gnment, the consignment may still be considered as a lot 
but a specific sampling procedure has to be applied.  
1.12.  'Sublot' means designated part of a large lot in order to apply the sampling method on 
that designated part. Each sublot must be physically  separated and identifiable.  
1.13.  'Incremental sample'  means a quantity of material taken from a single place in the lot 
or sublot.  
1.14.  'Aggregate sample'  means the combined total of all the incremental samples taken 
from the lot or sublot.  
1.15.  'Labor atory sample'  means a representative part/quantity of the aggregate sample 
intended for the laboratory.  
                                                 
3 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.  
4 The LOQ is calculated from the lowest concentratio n point taking into account the recovery of internal 
standards and sample intake.   
EN 4   EN II. ABBREVIATIONS USED  
BEQ  Bioanalytical Equivalents  
GC Gas chromatography  
HRMS  High resolution mass spectrometry  
LRMS  Low resolution mass spectrometry  
MS/MS  Tandem mass spectrometry  
PCB  Polychlorinated biphenyl  
Non-dioxin -like PCBs  PCB 28, PCB 52, PCB 101, PCB 138, PCB 153 and 
PCB 180  
PCDD  Polychlorinated dibenzo -p-dioxins  
PCDF  Polychlorinated dibenzofurans  
QC Quality control  
REP Relative potency  
TEF Toxic Equivalency Factor  
TEQ  Toxic Equivalents  
TCDD  2,3,7,8 -Tetrachlorodibenzo -p-dioxin  
U Expanded measurement uncertainty   
EN 5   EN ANNEX II  
METHODS OF SAMPLING  FOR OFFICIAL CONTROL OF LEVELS OF DIOXINS  
(PCDD/PCDF), DIOXIN -LIKE PCBs  AND NON -DIOXIN -LIKE PCBs IN CERTAIN 
FOODSTUFFS  
I. SCOPE  
Samples intended for the official control of the levels of dioxins (PCDD/Fs), dioxin -
like PCBs and non -dioxin -like PCBs  in foodstuffs shall be taken according to the 
methods described in this Annex. Aggregate samples thus obtained shall be 
considered as representative of the lots or sublots from which they are taken. 
Compliance with maximum levels laid down in Regulation (EC) No 1881/2006 shall 
be established on the basis of the levels determined in the laboratory samples.  
To ensure compliance with the provisions in Arti cle 4 of Regulation (EC) 
No 852/2004 , food business operator shall , when samples are taken to control the 
levels of dioxins  (PCDD/Fs) , dioxin -like PCBs and non -dioxin -like PCBs , take the 
samples according to the methods described in Chapter III of this Annex or apply an 
equivalent sampling procedure which is demonstrated to have a same level of 
representation  as the sampling procedure described in  Chapter III of  this Annex.  
II. GENERAL PROVISIONS  
1. Personnel  
Offcial s ampling shall be performed by an authorised person as designated by the 
Member State.  
2. Material to be sampled  
Each lot or sublot which is to be examined shall be sampled separately.  
3. Precautions to be taken  
In the course of sampling and the preparation of the samples, precautions shall be 
taken to avoid any changes which would affect the content of dioxins and PCBs, 
adversely affect the analytic al determination or make the aggregate samples 
unrepresentative.  
4. Incremental samples  
As far as possible , incremental samples shall be taken at various places distributed 
throughout the lot or sublot. Departure from such a procedure shall be recorded in the 
record provided for under point II.8.  
5. Preparation of the aggregate sample  
The aggregate sample shall be made up by combining the incremental samples. It 
shall be at least 1 kg unless not practical, e.g. when a single package has been 
sampled or when  the product has a very high commercial value.  
6. Replicate samples  
The replicate samples for enforcement, defence and reference purposes shall be taken 
from the homogenised aggregate sample, unless such procedure conflicts with a 
Member State's  rules as r egard the rights of the food business operator. The size of 
the laboratory samples for enforcement shall be sufficient to allow at least for 
duplicate analyses.   
EN 6   EN 7. Packaging and transmission of samples  
Each sample shall be placed in a clean, inert containe r offering adequate protection 
from contamination, from loss of analytes by adsorption to the internal wall of the 
container and against damage in transit. All necessary precautions shall be taken to 
avoid any change in composition of the sample which migh t arise during 
transportation or storage.  
8. Sealing and labelling of samples  
Each sample taken for official use shall be sealed at the place of sampling and 
identified in accordance with  the rules of the Member States.  
A record shall be kept of each samp ling, permitting each lot to be identified 
unambiguously and giving the date and place of sampling together with any 
additional information likely to be of assistance to the analyst.  
III. SAMPLING PLAN  
The sampling method applied shall ensure that the aggregate sample is representative 
of the (sub)lot that is to be controlled.  
1. Division of lots into sublots  
Large lots shall be divided into sublots on condition that the sublot can be separated 
physicall y. For products traded in large bulk consignments (e.g. vegetable oils) Table 
1 shall apply. For other products Table 2 shall apply. Taking into account that the 
weight of the lot is not always an exact multiple of the weight of the sublots, the 
weight of the sublot may exceed the mentioned weight by a maximum of 20%.  
Table 1   
Subdivision of lots into sublots for products traded in bulk consignments  
Lot weight (ton)  Weight or number of sublots  
1 500  
> 300 and < 1 500  
 50 and  300 
< 50 500 tonnes  
3 sublots  
100 tonnes  
-- 
Table 2: Subdivision of lots into sublots for other products  
Lot weight (ton)  Weight or number of sublots  
 15 
<15 15-30 tonnes  
- 
2. Number of incremental samples  
The aggregate sample uniting all incremental samples shall be at least 1 kg (see 
point  II.5).  
The minimum number of incremental samples to be taken from the lot or sublot shall 
be as given in Tables 3 and 4 . 
In the case of bulk liquid products , the lot or  sublot shall be thoroughly mixed insofar 
as possible and insofar as it does not affect the quality of the product by either 
manual or mechanical means immediately prior to sampling. In that case, a 
homogeneous distribution of contaminants is assumed withi n a given lot or sublot. It  
EN 7   EN is therefore sufficient to take three incremental samples from a lot or sublot to form 
the aggregate sample.  
The incremental samples shall be of similar weight. The weight of an incremental 
sample shall be at least 100 grams.  
Departure from this procedure must be recorded in the record provided for under 
point II.8 of this Annex. In accordance with the provisions of Commission Decision 
97/747/EC5, the aggregate sample size for hen eggs is at least 12 eggs (for bulk lots 
as well as for lots consisting of individual packages, Tables 3 and 4 shall apply).  
Table 3: Minimum number of incremental samples to be taken from the lot or 
sublot  
Weight or volume of lot/sublot (in kg 
or litre)  Minimum number of incremental 
samples to be taken  
< 50 3 
50 to 500  5 
> 500  10 
If the lot or sublot consists of individual packages or units, then the number of 
packages or units which shall be taken to form the aggregate sample is given in 
Table  4. 
Table 4: Number of packages or units (incremental samples) which shall be 
taken to form the aggregate sample if the lot or sublot consists of individual 
packages or units  
Number of packages or units in the 
lot/sublot  Number of packages or units to be 
taken  
1 to 25  at least 1 package or unit  
26 to 100  about 5 %, at least 2 packages or units  
> 100  about 5 %, at maximum 10 packages or 
units  
3. Specific provisions for the sampling of lots containing whole fishes of 
comparable size and weight  
Fishes are considered to be  of comparable size and weight wher e the difference in 
size and weight does not exceed about 50 %.  
The number of incremental samples to be taken from the lot are defined in Table 3. 
The aggregate sample uniting all incremental samples shall be at least 1 kg (see 
point  II.5).  
– Where  the lot t o be sampled contains small fishes (individual fishes weighing 
< about  1 kg), the whole fish is taken as incremental sample to form the 
aggregate sample. Where  the resulting aggregate sample weighs more than 
3 kg, the incremental samples may consist of the middle part, weighing each at 
least 100  grams, of the fishes forming the aggregate sample. The whole part to 
                                                 
5 Commission Decision 97/747/EC of 27 October 1997 fixing the levels and frequencies of sampling 
provided for by Council Directive 96/23/EC for the monitoring of certain subs tances and residues 
thereof in certain animal products  (OJ L 303, 6.11.1997, p. 12 ).  
EN 8   EN which the maximum level is applicable is used for  homogenisation of the 
sample.  
The middle part of the fish is where the centre of gravity is. This is located in 
most cases at the dorsal fin (in case the fish has a dorsal fin) or halfway 
between the gill opening and the anus.  
– Where  the lot to be sampled  contains larger fishes (individual fishes weighing 
more than about 1 kg), the incremental sample consists of the middle part of 
the fish. Each incremental sample weighs at least 100 grams.  
For fishes of intermediate size (about 1 -6 kg) the incremental sample is taken 
as a slice of the fish from backbone to belly in the middle part of the fish.  
For very large fishes (e.g. > about 6 kg), the incremental part is taken from the 
right side (fron tal view) dorso -lateral muscle meat in the middle part of the 
fish. Where  the taking of such a piece of the middle part of the fish would 
result in significant economic damage, the taking of three incremental samples 
of at least 350 grams each may be consi dered as being sufficient independent 
of the size of the lot or alternatively an equal part of the muscled meat close to 
the tail part and the muscle meat close to the head part of one fish may be taken 
to form the incremental sample being representative f or the level of dioxins in 
the whole fish.  
4. Sampling of lots of fish containing whole fishes of different size and/or weight  
– The provisions of point III.3 as regards sample constitution shall apply.  
– Where  a size or weight class/category is predominant ( about 80 % or more of 
the lot), the sample is taken from fishes with the predominant size or weight. 
This sample is to be considered as being representative for the whole lot.  
– Where  no particular size or weight class/category predominates, then it must be 
ensured that the fishes selected for the sample are representative for the lot. 
Specific guidance for such cases is provided in “Guidance document on 
sampling of whole fishes of different size and/or weight"6. 
5. Sampling at retail stage  
Sampling of foodst uffs at the retail stage shall be done where possible in accordance 
with the sampling provisions set out in point III.2.  
Where this is not possible, an alternative method of sampling at retail stage may be 
used provided that it ensures sufficient represen tativeness for the sampled lot or 
sublot.  
IV. COMPLIANCE OF THE LOT WITH SPECIFICATION  
1. AS REGARDS NON -DIOXIN -LIKE PCB S  
The lot is compliant if the analytical result for the sum of non-dioxin -like PCB s does 
not exceed the respective maximum level, as laid down in Regulation (EC) 
No 1881/2006 taking into account the expanded measurement uncertainty7. 
                                                 
6 https://ec.europa.eu/food/sites/food/files/safety/docs/cs_contaminants_catalogue_dioxins_guidance -
sampling_exemples -dec2006_en.pdf   
7 The principles as described in the “Guidance Document on Measurement Uncertainty for Laboratories 
performing PCDD/F and PCB Analysis using Isotope Dilution Mass Spectrometry” [link to website] 
shall be followed when applicable.   
EN 9   EN The lot is non -compliant with the maximum level as laid down in Regulation (EC) 
No 1881/2006 if the mean of two upperbound analytical results obtained from 
duplicate analysis8, taking into account the expanded measurement uncertainty, 
exceeds the maximum level beyond reasonable doubt.  
The expanded measurement uncertainty is calculated using a coverage factor  of 2 
which gives a level of confidence of approximately 95  %. A lot is non -compliant if 
the mean of the measured values minus the expanded uncertainty of the mean is 
above the established maximum level.  
The rules , mentioned in the paragraphs above under t his 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 apply.  
2. AS REGARDS DIOXINS (PCDD/F S) AND DIOXIN -LIKE PCB S 
The lot is compliant 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 as l aid down in 
Regulation (EC) No 1881/2006 ,  
– 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 as laid down 
in Regulation (EC) No 1881/2006 taking into account the expanded 
measurement uncertainty9. 
For screening assays a cut -off value shall be established for the decision on the 
compliance with the respective maximum levels set for either PCDD/Fs or for the 
sum of PCDD/Fs and dioxin -like PCBs.  
The lot is non -compliant with t he maximum level as laid down in Regulation (EC) 
No 1881/2006 if the mean of two upperbound analytical results (duplicate analysis10) 
obtained using a confirmatory method, taking into account the expanded 
measurement uncertainty, exceeds the maximum level b eyond reasonable doubt.  
The expanded measurement uncertainty is calculated using a coverage factor of 2 
which gives a level of confidence of approximately 95  %. A lot is non -compliant if 
the mean of the measured values minus the expanded uncertainty of the mean is 
above the established maximum level.  
                                                 
8 The duplicate analysis is necessary if the result of the firs t determination is noncompliant.  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 might 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.  
9 see footnote 12  
10 The duplicate analysis is necessary if the result of the first determination applying confirmatory 
methods with the use of 13C-labelled internal standard for the relevant analytes is non -compliant. The 
duplicate analysis is necessary to exclude the possibi lity 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 might 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 10   EN The sum of the estimated expanded uncertainties of the separate analytical results of 
PCDD/Fs and dioxin -like PCBs  has to be used for the estimated expanded 
uncertainty of the sum of PCDD/Fs and dioxin -like PCBs,  
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 apply.  
V. EXCEEDANCE OF ACTION LEVEL S  
Action level s serve as  a tool for the selection of samples in those cases where it is 
appropriate 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 might be 
appropriate to confirm exceedance of the action level  by duplicate analysis using a 
confirmatory method and taking into account the expanded me asurement 
uncertainty11.  
                                                 
11 Identical explanation and requirements for duplicate analysis for control of action level s as in footnote 
10 for maximum levels.   
EN 11   EN ANNEX III  
SAMPLE PREPARATION A ND REQUIREMENTS FOR METHODS OF ANALYSIS 
USED IN CONTROL OF T HE LEVELS OF DIOXINS  (PCDD/FS) AND DIOXIN -
LIKE PCBS IN CERTAIN  FOODSTUFFS  
1. FIELD OF APPLICATION  
The requirements set out in this Annex shall be applied where foodstuffs are 
analysed for the official control of the levels of 2,3,7,8 -substituted polychlorinated 
dibenzo -p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) and dioxin -like 
polychlorinate d biphenyls (dioxin -like PCBs) and as regards sample preparation and 
analyti cal requirements for other regulatory purposes, including the controls 
performed by the food business operator to ensure compliance with provisions in 
Article  4 of Regulation (EC) No 852/2004 . 
Monitoring for the presence of PCDD/Fs and dioxin -like PCBs in foodstuffs may be 
performed with two different types of analytical methods:  
(a) Screening methods  
The goal of screening methods  is to select those samples with le vels of 
PCDD/Fs and dioxin -like PCBs that exceed the maximum levels or the action 
level s. Screening methods shall ensure  cost-effective high sample -throughput, 
thus increasing the chance to discover new incidents where high exposure may 
lead to health risk s for consumers  . Their application shall aim to avoid false -
compliant results. They may comprise bioanalytical 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 or 
action level . 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 must be determined  or confirmed by a confirmatory method.  
In additi on, 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 case of appli cation 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 non -compliance or exceedance of action level s and 
give an ind ication 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 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 basis. Therefore, those methods allow the control of 
maximum  and action level s, 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 food monitoring, following of 
time trends, exposure assessment of the population and building of a database 
for possible re -evaluation of action and maximum levels. They are also  
EN 12   EN important for establishing congener patterns in order to identify the source of a 
possible con tamination. Such methods utilise 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 multiplied by their respective TEF, as 
established by the World Health Organization and listed in the Appendix to this 
Annex, and subsequently summed to give the total concentration of dioxin -like 
compounds expressed as TEQs.  
Screening and confirm atory methods may only be applied for control of a certain 
matrix if the methods are sensitive enough to detect levels reliably at the maximum 
or action level . 
3. QUALITY ASSURANCE RE QUIREMENTS  
– Measures must be taken to avoid cross -contamination at each s tage of the 
sampling and analysis procedure.  
– The samples must be stored and transported 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. Tr aces of paper dust 
must be removed from the sample container.  
– The sample storage and transportation has to be performed in a way that 
maintains the integrity of the foodstuff sample.  
– Insofar as relevant, finely grind and mix thoroughly each laboratory samp le 
using a process that has been demonstrated to achieve complete 
homogeni sation (e.g. ground to pass a 1 mm sieve); samples have to be dried 
before grinding if moisture content is too high.  
– Control of reagents, glassware and equipment for possible influence of TEQ - 
or BEQ -based results is of general importance.  
– A blank analysis shall be performed by carrying out the entire analytical 
procedure omitting only the sample.  
– For bioanalytical methods, it is of great importance that all glassware and 
solvents used in analysis shall be tested to be free of compounds that interfere 
with the detection of target compounds in the working range. Glassware shall 
be rinsed with solvents or/and heated at  temperatures suitable to remove traces 
of PCDD/Fs, dioxin -like compounds and interfering compounds from its 
surface.  
– Sample quantity used for the extraction must be sufficient to fulfill the 
requirements with respect to a sufficiently low working range in cluding the 
concentrations of maximum or action levels.  
– The specific sample preparation procedures used for the products under 
consideration shall follow internationally accepted guidelines.  
– In the case of fish, the skin has to be removed as the maximum le vel applies to 
muscle meat without skin. However it is necessary that all remaining muscle  
EN 13   EN meat and fat tissue on the inner side of the skin are carefully and completely 
scraped off from the skin and added to the sample to be analysed.  
4. REQUIREMENTS FOR LABORATORIES  
– In accordance with the provisions of Regulation (EC) No 882/2004, 
laboratories shall be accredited by a recogni sed body operating in accordance 
with ISO Guide 58 to ensure that they are applying analytical quality 
assurance. Laboratories shal l be accredited following the EN ISO/IEC 17025 
standard. The principles as described in the Technical Guidelines for the 
estimation of measurement uncertainty and limits of quantification for PCDD/F 
and PCB analysis shall be followed when applicable12. 
– Labo ratory proficiency shall be proven by the continuous successful 
participation in interlaboratory studies for the determination of PCDD/Fs and 
dioxin -like PCBs in relevant food matrices and concentration ranges.  
– Laboratories applying screening methods for r outine control of samples shall 
establish a close cooperation with laboratories applying the confirmatory 
method, both for quality control and confirmation of the analytical result of 
suspected samples.  
5. BASIC REQUIREMENTS T O BE MET BY ANALYTIC AL PROCEDU RE 
FOR DIOXINS (PCDD/FS ) AND DIOXIN -LIKE PCBS  
5.1. Low working range and limits of quantification  
– For PCDD/Fs, detectable quantities have to be in the upper femtogram (10-15g) 
range because of extreme toxicity of some of these compounds. For most PCB 
conge ners limit of quantification in the nanogram (10-9g) range is already 
sufficient. However, for the measurement of the more toxic dioxin -like PCB 
congeners (in particular non -ortho -substituted congeners) the lower end of the 
working range must reach the low  picogram (10-12g) levels.  
5.2. High selectivity (specificity)  
– 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 gas chrom atography/mass spectrometry (GC -MS) 
methods, a differentiation among various congeners is necessary, such as 
between toxic (e.g. the seventeen 2,3,7,8 -substituted PCDD/Fs, and twelve 
dioxin -like PCBs) and other congeners.  
– Bioanalytical methods shall be ab le 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 -noncompliant results or compounds that may 
decrease the response, causing false -compliant results.  
5.3. High acc uracy (trueness and precision, bioassay apparent recovery)  
– For GC -MS methods, the determination shall provide a valid estimate of the 
true concentration in a sample. High accuracy (accuracy of the measurement: 
                                                 
12 Guidance Do cument 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 F ood [link to 
website]   
EN 14   EN the closeness of the agreement between the res ult of a measurement with the 
true or assigned value of the measurand) is necessary to avoid the rejection of a 
sample analysis result on the basis of poor reliability of the determined TEQ 
level. Accuracy is expressed as trueness (difference between the m ean value 
measured for an analyte in a certified material and its certified value, expressed 
as percentage of this value) and precision  (RSD R relative standard deviation 
calculated from results generated under reproducibility conditions).  
– For bioanalytical  methods, the bioassay  apparent recovery shall be determined.  
5.4. Validation in the range of maximum level and general quality control measures  
– Laboratories shall demonstrate the performance of a method in the range of the 
maximum level, e.g. 0 ,5x, 1x an d 2x the maximum level with an acceptable 
coefficient of variation for repeated analysis, during  the validation procedure 
and/or during routine analysis.  
– Regular blank controls and spiking experiments or analysis of control samples 
(preferably, if availab le, certified reference material) shall be performed as 
internal quality control measures. Quality control (QC) charts for blank 
controls, spiking experiments or analysis of control samples shall be recorded 
and checked to make sure the analytical performa nce is in accordance with the 
requirements.  
5.5. Limit of quantification  
– For a bioanalytical screening method, establishment of the 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 response 
below this level. The reporting level shall be demonstrated to be different from 
procedure blank samples at least by a factor of three, wit h a response below the 
working range. It shall therefore be calculated from samples containing the 
target compounds around the required minimum level, and not from a S/N ratio 
or an assay blank.  
– Limit of quantification (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 must be met in the range of the maximum level for the TEQ value 
respectively the BEQ value, whether determined as total TEQ or total BEQ (as 
sum of PCDD/F and dioxin -like PCBs) or separately for PCDD/Fs and dioxin -
like PCBs.   
EN 15   EN  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  
5.7. Specific requirements for screening methods  
– Both GC -MS and bioanalytical methods may be used for screening. For GC -
MS methods the requirements as laid down in point 6 are to be used. For cell 
based bioanalytical methods specific requirements are laid down in point 7.  
– Laboratories applying screening methods for routine control of samples shall 
establish a close cooperation with labora tories applying the confirmatory 
method.  
– Performance verification  of the screening method is required during routine 
analysis, by analytical quality control and ongoing method validation.  There 
must be a continuous programme for control of compliant result s. 
– Check on possible suppression  of the cell response and cytotoxicity . 
20 % of the sample extracts shall be measured in routine screening without and 
with TCDD added corresponding to the maximum or action level , to check if 
the response is possibly suppre ssed by interfering substances present in the 
sample extract. The measured concentration of the spiked sample is 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 a potential signal 
suppression and the respective sample must be submitted to confirmatory 
analysis. Results shall be monitored in quality control charts.  
– Quality control  on complia nt samples  
Approximately 2 % to 10  % of the compliant samples, depending on sample 
matrix and laboratory experience, shall be confirmed.  
– Determination of false -compliant rates from QC data  
The rate of false -compliant results from screening  of samples below and above 
the maximum level or the action level  shall be determined. Actual false -
compliant rates shall be below 5  %. 
After a minimum of 20 confirmed results per matrix/matrix group is available 
from the quality control of compliant sampl es, conclusions on the false -
compliant rate shall be drawn from this database. The results from samples 
analy sed in ring trials or during contamination incidents, covering a 
concentration range up to e.g. 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.   
EN 16   EN Although screening assays shall preferentially aim to detect samples exceeding 
the action level , the criterion for determining false -compliant rates is the 
maximum  level, taking into account the expanded measurement uncertainty of 
the confirmatory method.  
– Potential non -compliant results from screening  shall always be verified by a 
full re -analysis of th e original sample by a confirmatory method. These 
samples may also be used to evaluate the rate of false -noncompliant results. 
For screening methods, the rate of false -noncompliant results is the fraction of 
results confirmed to be compliant from confirmat ory analysis, while in 
previous screening the sample had been declared to be suspected to be non -
compliant. However, evaluation of the advantageousness  of the screening 
method shall be based on comparison of false -non compliant samples with the 
total numbe r of samples checked. This rate shall be low enough to make the 
use of a screening tool advantageous.  
– At least 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 repeatability conditions, the 
intra-laboratory RSD r would typically be smaller than the reproducibility 
RSD R. 
6. SPECIFIC REQUIREMENT S FOR GC -MS METHODS TO BE COM PLIED 
WITH FOR SCREENING O R CONFIRMATORY PURPO SES 
6.1. Acceptable differences between upperbound and lowerbound WHO -TEQ levels  
– The difference between upperbound level and lowerbound level shall not 
exceed 20  % for confirmation of the exceedance of maximum or in case of 
need of action levels.  
6.2. Control of recoveries  
– Addition of 13C-labelled 2,3,7,8 -chlorine -substituted internal PCDD/F 
standards and of 13C-labelled internal dioxin -like PCB standards must be 
carried out at the very beginning of the analytical method e.g. prior to 
extraction in order to valida te the analytical procedure. At least one congener 
for each of the tetra - to octa -chlorinated homologous groups for PCDD/Fs and 
at least one congener for each of the homologous groups for dioxin -like PCBs 
must be added (alternatively, at least one congener  for each mass spectrometric 
selected ion recording function used for monitoring PCDD/Fs and dioxin -like 
PCBs). In 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.  
– Relative response factors shall also be determined for those congeners for 
which no 13C-labelled analogue is added by using appropriate calibration 
solutions.  
– For foodstuffs of plant origin and foodstuffs of animal origin conta ining less 
than 10  % fat, the addition of the internal standards is mandatory prior to 
extraction. For foodstuffs of animal origin containing more than 10  % fat, the 
internal standards may be added either before or after fat extraction. An 
appropriate vali dation of the extraction efficiency shall be carried out,  
EN 17   EN depending on the stage at which internal standards are introduced and on 
whether results are reported on product or fat basis.  
– Prior to GC -MS analysis, 1 or 2 recovery (surrogate) standard(s) must b e 
added.  
– Control of recovery is necessary. For confirmatory methods, the recoveries of 
the individual internal standards 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, are 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 sha ll be in the range of 30 to 140  %. 
6.3. Removal of interfering substances  
– Separation of PCDD/Fs from interfering 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).  
– Gas-chromatographic separation of isomers shall be sufficient (< 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 calibrati on curve shall cover the relevant range of maximum 
or action levels.  
6.5. Specific criteria for confirmatory methods  
– 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.  
Fulfilme nt 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) an d/or in EPA methods 1613 and 1668 
as revised.  
– For GC -MS/MS:  
Monitoring of at least 2 specific precursor ions, each with one specific 
corresponding transition product ion for all labelled and unlabelled analytes in 
the scope of analysis.  
Maximum permitted t olerance 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 p ressure, for each transition of an 
analyte.  
Resolution for each quadrupole to be set equal to or better than unit mass 
resolution (unit mass resolution: sufficient resolution to separate two peaks one 
mass unit apart) in order to minimi se possible interfer ences on the analytes of 
interest.   
EN 18   EN 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 REQUIREMENT S FOR BIOANALYTICAL METHODS  
Bioanalytical methods are methods based on the use of biological principles like cell -
based assays, receptor -assays or immunoassays. This point 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 as 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 -noncompliant results are ensured. With 
separate maximum levels for PCDD/Fs and for the sum of PCDD/Fs 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 
level s, an appropriate percentage of the respective action level  would suit as cut -off 
value.  
If an indicative level is expressed in BEQs, the results from the the sample must be 
given in the working range and exceeding 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 (high 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) must further be set 
significantly (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 hi gher than 15  % in 
this range. The working range shall be established during validation. Cut -off 
values ( see point 7.3) must be within the working range.  
– Standard solutions and sample extracts shall be tested in triplicate or at least in 
duplicate. When usi ng 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 sta ndard curve  
– Levels in samples may be estimated by comparison of the test response with a 
calibration curve of TCDD (or PCB 126 or a PCDD/F/dioxin -like PCB 
standard mixture) to calculate the BEQ level in the extract and subsequently in 
the sample.   
EN 19   EN – Calibrati on curves shall contain eight  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 nonlinear regression. A better fit will be achieved by 
minimi sing the difference between calculated and observed levels in the 
working range of the curve (e.g. by minimi sing the sum of sq uared residuals).  
– The estimated level in the sample extract is subsequently corrected for the BEQ 
level calculated for a matrix 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 or action level). For performing a recovery 
correction, the apparent recovery must always be within the required range (see 
point 7.1.4.). Reference sampl es used for recovery correction must comply 
with requirements as given in point 7.2.  
7.1.2.2.  Calibration with reference samples  
Alternatively, a calibration curve prepared from at least 4 reference samples (see 
point 7.2: one matrix blank, plus three refe rence samples at 0 ,5x, 1 ,0x and 2 ,0x the 
maximum or action level may be used, eliminating the need to correct for blank and 
recovery if matrix properties of the reference samples match those of the unknown 
samples. In this case, the test response correspon ding 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 level s, an appropriate 
percentage of these action level s would suit as cut -off val ue.  
7.1.3.  Separate determination of PCDD/Fs and dioxin -like PCBs  
Extracts may be split into fractions containing PCDD/Fs and dioxin -like PCBs, 
allowing a separate indication of PCDD/Fs and dioxin -like PCB TEQ levels (in 
BEQs). A PCB 126 standard calibrat ion 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 or action level and 
expressed as percentage of the BEQ level in comparison to the TEQ level. 
Depending on the type of assay and TEFs13 used, the differences between TEF and 
REP factors for dioxin -like PCBs may 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 TCDD 
calibration curve). As the contribution of dioxin -like PCBs to the sum of PCDD/Fs 
and dioxin -like PCBs may vary between different matrices and samples, bioassay 
apparent recoveries for the sum parameter reflect these ranges and shall be betw een 
30 % to 130  %.  
                                                 
13 Current requirements are based  on the TEFs published in: M. Van den Berg et al, Toxicol Sci 93 (2), 
223–241 (2006).   
EN 20   EN 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 th e 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 TEQ -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 must be taken into ac count. The reporting limit must be 
set significantly (at least by a factor of three) above the procedure blanks.  
7.2. Use of reference samples  
– Reference samples shall represent sample matrix, congener patterns and 
concentration ranges for PCDD/Fs and dioxi n-like PCBs around the maximum 
or action level.  
– A procedure blank, or preferably a matrix blank, and a reference sample at the 
maximum or action level have to be included in each test series. These samples 
must be extracted and tested at the same time unde r identical conditions. The 
reference sample must show a clearly elevated response in comparison to the 
blank sample, thus ensuring the suitability of the test. Those samples may be 
used for blank and recovery corrections.  
– Reference samples chosen for perf orming a recovery correction shall be 
representative for the test samples, meaning that congener patterns shall not 
lead to an underestimation of levels.  
– Extra reference samples at e.g. 0 ,5x and 2x the maximum or action level may 
be included to demonstrat e the proper performance of the test in the range of 
interest for the control of the maximum or action level. 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 confirmatory 
methods in TEQ shall be established (e.g. by matrix -matched calibration 
experiments, involving reference samples spiked at 0, 0 ,5x, 1x and 2x the maximum 
level (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 each test 
series by including procedure/matrix blanks and recovery samples ( see point 7.2). 
Cut-off values shall be established for decision over sample compliance with 
maximum levels or for control of action levels, if of interest, with the respective 
maximum or action levels set for either PCDD/Fs and dioxin -like PCBs alone, or for 
the sum of PCDD/Fs and dioxin -like P CBs. They are represented by the lower  
endpoint 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 r ate < 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.   
EN 21   EN In practice, the cut -off value (in BEQ) may be calculated from the following 
approaches (see Figu re 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 x t α,f=m -2  
with:  
BEQ DL BEQ corresponding to the decision limit of the confirmatory method, 
being the ML 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)  
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 or action level. This is based on the observation that this level will be 
around the cut -off determined under point 7.3.1 or 7.3.2.   
Calculation of cut -off values based on a 95% level of confidence im plying 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  endpoint of the data distribution 
(represented in the figure by a bell -shaped curve) at the corresponding mean 
BEQ value.   
xx i Qxxm n /) ( /1/12
x

m
jixx
12) ( 
EN 22   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 or action level s 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 or action level  shall be preferred.  
7.4. Performance characteristics  
– Since no internal standards can be used in bioanalytical methods, tests on 
repeatability 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 BEQs after blank and recovery correction.  
– As part of the validation process, the test must be shown to discriminate 
between a blank sample and a level at the cut -off value, allowing the 
ident ification of samples above the corresponding cut -off value (see point 
7.1.2).  
– Target compounds, possible interferences and maximum tolerable blank levels 
shall be defined.  
– 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 shall not be above 15  %. 
– The uncorrected results of the reference sample(s) expressed in BEQs (blank 
and at the maximum or action level) shall be used for evaluati on of the 
performance of the bioanalytical method over a constant time period.  
 
EN 23   EN – QCcharts for procedure blanks and each type of reference sample shall be 
recorded and checked to make sure the analytical performance is in 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 blanks must be well controlled in order to a void false -compliant 
results when subtracted.  
– 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 BEQs and TEQs. This database might be used for re -
evaluation of cut -off values applicable to routine samples for the validated 
matrices.  
– Successful method performance may also be demonstrated by participation in 
ring tr ials. The results from samples analy sed in ring trials, covering a 
concentration range up to e.g. 2x ML, may also be included in the evaluation of 
the false -compliant rate, if a laboratory is able to demonstrate its successful 
performance. The samples shal l cover most frequent congener patterns, 
representing various sources.  
– During incidents, the cut -off values may be re -evaluated, reflecting the specific 
matrix and congener patterns of this single incident.  
8. REPORTING OF THE RES ULT  
Confirmatory methods  
– 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 -bound in order to include a maximum of information 
in the reporting of the result s and thereby enabling the interpretation of the 
results according to specific requirements.  
– The report shall also include the method used for extraction of PCDD/Fs, 
dioxin -like PCBs and lipids. The lipid content of the sample shall be 
determined and repor ted for food matrices with maximum levels expressed on 
fat basis and with an expected fat concentration in the range of 0 - 2 % (in 
correspondence to existing legislation) . For other samples , the determination of 
the lipid content is optional.  
– The recoveri es of the individual internal standards must be made available in 
case the recoveries are outside the range mentioned in point 6.2 where  the 
maximum level is exceeded (in this case, the recoveries for one of the two 
duplicate analysis) and in other cases u pon request.  
– As the expanded measurement uncertainty is to be taken into account when 
deciding about the compliance of a sample, this 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  %. 
In 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 has to be used for the sum of PCDD/Fs and 
dioxin -like PCBs.   
EN 24   EN – The results shall be expressed in the same units and with the same number of 
significant figures as the maximum levels laid down in Regulation (EC) No 
1881/2006.  
Bioanalytical screening methods  
– The result of the screening shall be expressed as compliant or suspected to be 
non-compliant (“suspected”).  
– In addition, an indicative result for PCDD/F and/or dioxin -like PCBs expressed 
in BEQ  (not TEQ) may be given (see point  1). Samples with a response below 
the reporting limit shall be expressed as lower than the reporting limit. Samples 
with a response above the working range shall be reported as exceeding the 
working range and the level co rresponding to the upper end of the working 
range shall be given in BEQ.  
– For each type of sample matrix, the report shall mention the maximum or 
action level on which the evaluation is based.  
– The report shall mention the type of test applied, the basic tes t principle and 
kind of calibration.  
– The report shall also include the method used for extraction of PCDD/Fs, 
dioxin -like PCBs and lipids. The lipid content of the sample shall be 
determined and reported for food matrices with maximum levels expressed on 
fat basis and with an expected fat concentration in the range of 0 - 2 % (in 
correspondence to existing legislation) . For other samples , the determination of 
the lipid content is optional.  
– In the 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.  
– Non-compliant results shall only be reported  from confirmatory analysis . 
Physico -chemical screening methods  
– The result of the screening  shall be expressed as compliant or suspected to be 
non-compliant (“suspected”).  
– For each type of sample  matrix, the report shall mention the maximum or action 
level on which the evaluation is based.  
– 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 giv en. 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 
in Regulation (EC) No 1881/2006.  
– The recoveries of the individual internal standards must be made available in 
case the recoveries are outside the range mentioned in point 6.2 and in other 
cases upon request.  
– The report shall mention the GC -MS method applied.  
– The report shall also include the method used for extraction of PCDD/Fs, 
dioxin -like PCBs and lipids. The li pid content of the sample shall be 
determined and reported for food matrices with maximum levels expressed on  
EN 25   EN fat basis and with an expected fat concentration in the range of 0 - 2 % (in 
correspo ndence to existing legislation). F or other samples , the deter mination of 
the lipid content is optional.  
– 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.  
– Non-compliance can only be decided after confirmatory analysis.   
EN 26   EN Appendix  
WHO -TEFs  for human risk assessment based on the conclusions of the World Health 
Organization (WHO) – International Programme on Chemical Safety (IPCS) expert meeting 
which was held in Geneva in June 200514  
Congener  TEF value  Congener  TEF value  
Dibenzo -p-dioxins (“PCDDs”)   “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    
 
Dibenzofurans ("PCDFs")   
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.  
                                                 
14 Martin van den Berg et al. , The 2005 World Health Organization Re -evaluation of Human and 
Mammalian Toxic Equivale ncy Factors for Dioxins and Dioxin -like Compounds. T oxicological 
Sciences 93(2), 223 –241 (2006) .  
EN 27   EN ANNEX IV  
SAMPLE PREPARATION A ND REQUIREMENTS FOR METHODS OF ANALYSIS 
USED IN CONTROL OF T HE LEVELS OF NON -DIOXIN -LIKE PCBS IN CERTAIN  
FOODSTUFFS  
The requirements set out in this Annex shall be applied where foodstuffs  are 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 , including the controls performed by the 
food business operator to ensure compliance with the provisions in Ar ticle 4 of Regulation 
(EC) No 852/2004.   
The provisions on sample preparation provided for in point 3  of Annex III of this Regula tion 
shall also be applicable for the control of the levels of non -dioxin -like PCBs in food.  
1. Applicable detection methods:  
Gas Chromatography / Electron Capture Detection (GC -ECD), GC -LRMS, GC -
MS/MS, GC -HRMS or equivalent methods.  
2. Identification and confirmation of analytes of interest:  
– Relative retention time in relation to internal standards or reference standards 
(acceptable deviation of +/ - 0,25 %).  
– 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 confirmed15). 
– For GC -MS techniques:  
– Monitoring of at least the following number of molecular ions or 
characteristic ions from the molecular cluster:  
– two specific ions for HRMS,  
– three specific ions for LRMS,  
– two specific precursor ions, each with one specific corresponding 
transition product ion for MS -MS. 
– Maximum permitted tolerances for abundance ratios for selected mass 
fragments:  
Relative deviation of a bundance ratio of selected mass fragments from 
theoretical abundance or calibration standard for target ion (most 
abundant ion monitored) and qualifier ion(s): ± 15 % . 
– For GC -ECD:  
Confirmation of results exceeding the maximum level with two GC columns 
with stationary phases of different polarity.  
3. Demonstration of performance of method:  
Validation 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 8).  
                                                 
15 Congeners often found to co -elute are e.g. PCB 28/31, PCB 52/69 and PCB 138/163/164. For GC -MS 
also possible interferences from fragments of higher chlorinated c ongeners have to be considered.   
EN 28   EN 4. Limit of quantification:  
The sum of the LOQs16 of non-dioxin -like PCB s shall not be higher than one-third  of 
the maximum level17. 
5. Quality control:  
Regular blank controls, analysis of spiked samples, quality control samples, 
participation in interlaboratory studies on relevant matrices.  
6. Control of recoveries:  
– Use of suitable internal standards with physico -chemical properties comparable 
to analytes of interest.  
– Addition of internal standards:  
– Addition to products ( before extraction and clean -up process) , 
– Addition also possible to extracted fat (before clean -up process), if 
maximum level is expressed on fat basis.  
– Requirements for methods using all six isotope -labelled non -dioxin -like PCB 
congeners:  
– Correction of res ults for recoveries of internal standards,  
– Generally acceptable recoveries of isotope -labelled internal standards are 
between 60 and 120 % , 
– Lower or higher recoveries for individual congeners with a contribution 
to the sum of non-dioxin -like PCB s below 10 % are acceptable.  
– Requirements for methods using not all six isotope -labelled internal standards 
or other internal standards:  
– Control of recovery of internal standard(s) for every sample,  
– Acceptable recoveries of internal standard(s) between 60 and 120 %,  
– Correction of results for recoveries of internal standards.  
– The recoveries of unlabelled congeners shall be checked by spiked samples or 
quality control samples with concentrations in the range of the maximum level. 
Acceptable recoveries for these congener s are between 60 and 120 %.  
7. Requirements for laboratories:  
In accordance 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 
                                                 
16 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.  
17 It is highly recommendable  to have 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 subtra cted.   
EN 29   EN described in Technical Guidelines for the estimation of measurement uncertainty and 
limits of quantification for PCB ana lysis shall be followed when applicable18. 
8. Performance characteristics: Criteria for the sum of non-dioxin -like PCB s at 
the maximum level:  
 Isotope dilution 
mass spectrometry*  Other techniques  
Trueness  - 20 to + 20 %  - 30 to + 30 %  
Intermediate precision (RSD R) ≤ 15 %  ≤ 20 %  
Difference between upper and lower 
bound calculation  ≤ 20 %  ≤ 20 %  
* Use of all six 13C-labelled analogues as internal standards required  
9. Reporting of results  
– The analytical results shall contain the levels of the individual non-dioxin -like 
PCB congeners and the sum of non-dioxin -like PCB s, 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.  
– The report shall also include the method used for the extraction of PCBs and 
lipids. The lipid content of the sample shall be determined and reported for 
food matrices with maximum levels expressed on fat basis and with an 
expected fat concentration in the range of 0 - 2 % (in correspondence to 
existing legislation) . For other samples , the determination of the lipid content is 
optional.  
– The recoveries of the individual internal standards must be made available in 
case the recoveries are outside the range mentioned in point 6, in case the 
maximum level is exceeded and in other cases upon request.  
– As the expanded measurement uncertainty is to be taken into accou nt 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 o f 2 which gives a level of confidence of approximately 95  %. 
– The results shall be expressed in the same units and with the same number of 
significant figures as the maximum levels laid down in Regulation (EC) 
No 1881/2006.  
                                                 
18 “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 Cont aminants in Feed and Food” [link to 
website] .