COMMISSION REGUL ATION (EU) 2017/644 
of 5 Apr il 2017 
laying down methods of sampling and analysis for the control of levels of dioxins, dioxin-like 
PCBs and non-dio xin-like PCBs in certain foodst uffs and repealing Regulation (EU) No 589/2014 
(Text with EEA relevance) 
THE EUR OPEAN COMMISSION, 
Having regar d to the Treaty on the Functioning of the European Union, 
Having regard to Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 Apr il 2004 on 
official controls perf ormed to ensure the verification of compliance with feed and food law, animal health and animal 
welf are rules (1), and in particular Article 11(4) thereof, 
Whereas: 
(1)  Commission Regulation (EC) No 1881/2006 (2) sets out the maximum levels for non-dio xin-like poly chlor inated 
biphen yls (PCBs) dioxins and furans and for the sum of dioxins, furans and dioxin-like PCBs in certain foodstuffs. 
(2)  Commission Recommendation 2013/711/EU (3) sets out action levels in order to stimulate a proactive approach 
to reduce the presence of poly chlor inated dibenzo-para-dio xins and poly chlor inated dibenzofurans (PCDD/Fs) and 
dioxin-like PCBs in food. Those action levels are a tool used by compet ent author ities and operators to highlight 
those cases where it is appropr iate to identify a source of contamination and to take the necessar y measures in 
order to reduce or eliminate it. 
(3)  Commission Regulation (EC) No 589/2014 (4) establishes specifi c provisions concer ning the samp ling procedure 
and the methods of analysis to be applied for the official control of levels of dioxins, dioxin-lik e PCBs and non- 
dioxin-like PCBs. 
(4)  The provisions laid down in this Regulation relat e only to the sampling and analysis of dioxins, dioxin-like PCBs 
and non-dio xin-like PCBs for the implementation of Regulation (EC) No 1881/2006 and Recommendation 
2013/711/EU. They do not affect the sam pling strateg y, sam pling levels and frequency as set out in Annex es III 
and IV to Council Directive 96/23/EC (5). They do not affect the targeting criteria for sampling as laid down in 
Commission Decision 98/179/EC (6). 
(5)  It is appropr iate to ensure that food business operat ors applying the controls perf ormed within the framework of 
Article 4 of Regulation (EC) No 852/2004 of the European Parliament and of the Council (7) apply sam pling 
procedures equivalent to the sampli ng procedures provided for by this Regulation in order to ensure that samples 
take n for those controls are representative samples. Further more, the European Union Reference Laboratory for 
Dioxins and PCBs has provided evidence that analytical results in certain cases are not reliable when the 
performa nce criteria as provided in this Regulation are not applied by laborat ories perf orming the analysis of 
samp les taken by food business operators within the framew ork of Article 4 of Regulation (EC) No 852/2004. It 
is theref ore appropr iate to make the application of the perf ormance criteria also obligat ory for the analysis of 
those sample s. 6.4.2017 L 92/9 Official Jour nal of the European Union EN     
(1)OJ L 165, 30.4.2004, p. 1. 
(2)Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs 
(OJ L 364, 20.12.2006, p. 5). 
(3)Commission Recommendation 2013/711/EU of 3 December 2013 on the reduction of the presence of dioxins, furans and PCBs in feed 
and food (OJ L 323, 4.12.2013, p. 37). 
(4)Commission Regulation (EU) No 589/2014 of 2 June 2014 laying down methods of sampli ng and analysis for the official control of 
levels of dioxins, dioxin-like PCBs and non-dio xin-like PCBs in certain foodstuffs and repealing Regulation (EU) No 252/2012 (OJ L 164, 
3.6.2014, p. 18). 
(5)Council Directive 96/23/EC of 29 Apr il 1996 on measures to monit or certain substances and residues thereof in live animals and animal 
products and repealing Directives 85/358/EEC and 86/469/EEC and Decisions 89/187/EEC and 91/664/EEC (OJ L 125, 23.5.1996, 
p. 10). 
(6)Commission Decision 98/179/EC of 23 Febr uary 1998 laying down detailed rules on official samplin g for the monit oring of certain 
substances and residues thereof in live animals and animal products (OJ L 65, 5.3.1998, p. 31). 
(7)Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 Apr il 2004 on the hygiene of foodstuffs (OJ L 139, 
30.4.2004, p. 1). (6)  Given that the approach of the use of a decision limit to ensure that an analytical result is above the maximum 
level with a certain probability , as provided for in Commission Decision 2002/657/EC (1), is no long er applied 
for the analysis of dioxins and PCBs in food, it is appropr iate to delete this approach and to keep only the 
approac h of the expanded uncer tainty using the coverag e factor of 2, giving a level conf idence of appro ximatel y 
95 %. 
(7)  In line with the repor ting requirements for bioanalytical screening methods, it is appropr iate to also provide for 
physico-chem ical methods used for screening specifi c repor ting requirements. 
(8)  Given that the analysis of dioxins, dioxin-like PCBs and non-dio xin-like PCBs are in most cases deter mined 
together it is appropr iate to align the perf ormance criteria for the non-dio xin-like PCBs to the performa nce 
criteria for dioxins and dioxin-like PCBs. This is a simplification, without substantial change s in practice as in the 
case of non-dio xin-like PCBs the relative intensity of qualifi er ions compared to target ions is > 50 %. 
(9)  Further more there are several other minor modifi cations proposed to the current provisions, requir ing the repeal 
of Regulation (EU) No 589/2014 and its replacing by a new Regulation to maintain the readability of the text. 
(10)  The measures provided for in this Regulation are in accordance with the opinion of the Standing Committ ee on 
Plants, Animals, Food and Feed, 
HAS ADOPTED THIS REGUL ATION: 
Article 1 
For the purposes of this Regulation, the definitions and abbreviations set out in Annex I shall apply . 
Article 2 
Samp ling for the official control of the levels of dioxins, furans, dioxin-like PCBs and non-dio xin-like PCBs in foodstuffs 
listed in Section 5 of the Annex to Regulation (EC) No 1881/2006 shall be carried out in accordance with the methods 
set out in Annex II to this Regulation. 
Article 3 
Samp le preparation and analyses for the control of the levels of dioxins, furans and dioxin-like PCBs in foodstuffs listed 
in Section 5 of the Annex to Regulation (EC) No 1881/2006 shall be carried out in accordance with the methods set 
out in Annex III to this Regulation. 
Article 4 
Analyses for the control of the levels of non-dio xin-like PCBs in foodstuffs listed in Section 5 of the Annex to 
Regulation (EC) No 1881/2006 shall be carried out in accordance with the requirements for analytical procedures set 
out in Annex IV to this Regulation. 
Article 5 
Regulation (EU) No 589/2014 is repealed. 
References to the repealed Regulation shall be constr ued as references to this Regulation. 6.4.2017 L 92/10 Official Jour nal of the European Union EN     
(1)Commission Decision 2002/657/EC of 14 Aug ust 2002 implementing Council Directive 96/23/EC concer ning the performance of 
analytical methods and the interpretation of results (OJ L 221, 17.8.2002, p. 8). Article 6 
This Regulation shall enter into force on the twentieth day following that of its publication in the Official Journal of the 
European Union. 
This Regulation shall be binding in its entirety and directly applicable in all Member State s. 
Done at Brussels, 5 Apr il 2017. 
For the Commission 
The Presid ent 
Jean-Claude JUNCKER  6.4.2017 L 92/11 Official Jour nal of the European Union EN     ANNEX I 
DEFINITIONS AND ABBREVIA TIONS  
I. DEFINITIONS 
For the purposes of this Regulation the definit ions laid down in Annex I to Decision 2002/657/EC 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 detect ed.  
1.2. ‘Screening methods ’ means methods used for the selection of those sam ples with levels of PCDD/Fs and 
dioxin-like PCBs that exceed the maximum levels or the action levels. They shall allow for a cost-effective high 
sample-throughput, thus increasing the chance of disco vering 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 sam ples exce eding the cut-off value established to check compl iance with the maximum level shall be 
verified by a full re-analysis from the original sam ple using a conf irmatory method.  
1.3. ‘Conf irmatory methods ’ means methods that provide full or compl ementar y information enabling the PCDD/Fs 
and dioxin-like PCBs to be identif ied and quantified unequivocally at the maximum or, in case of need, at the 
action level. Such methods utilise gas chromatograph y/high resolution mass spectrometr y (GC-HRMS) or gas 
chromat ography /tandem mass spectrometr y (GC-MS/MS).  
1.4. ‘Bioanalytical methods’ means methods based on the use of biological principles such as cell-based assa ys, 
recept or-assa ys or immunoassa ys. They do not give results at the congener level but merely an indication (1) of 
the TEQ level, expressed in Bioanalytical Equivalents (BEQ) to ackn owledge the fact that not all compo unds 
present in a sam ple extract that produce a response in the test may meet all requirements of the 
TEQ-pr inciple.  
1.5. ‘Bioassa y apparent reco very’ 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 confir matory method. It attemp ts 
to correct factors like the loss of PCDD/Fs and dioxin-like compo unds during the extraction and clean-up 
steps, co-extract ed compounds increasing or decreasing the response (agonistic and antagonistic effects), the 
quality of the curve fit, or diffe rences between the TEF and the REP values. The bioassa y apparent reco very is 
calculated from suitable reference samp les with representative cong ener patter ns around the maximum or 
action level.  
1.6. ‘Duplicate analysis ’ means separate analysis of the analyt es of interest using a second aliquot of the same 
homogenised sample.  
1.7. ‘Accept ed specific limit of quantification (2) of an individual cong ener in a sample’ means the lowest content of 
the analyte that can be measured with reasonable statistical certainty , fulfilling the identif ication criteria as 
descr ibed in internatio nally recognised standards, for exam ple, in standard EN 16215:2012 ('Animal feed — 
Determi nation 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 cong ener may be identified as 
(a)  the concentration of an analyte in the extract of a sample whic h produces an instr umental response at two 
different ions to be monit ored with a S/N (signal/noise) ratio of 3:1 for the less intensive raw data signal; 6.4.2017 L 92/12 Official Jour nal of the European Union EN     
(1)Bioanalytical methods are not specific to those cong eners included in the TEF-scheme. Other structurally relate d AhR -active compounds 
may be present in the sample extract whic h contr ibut e to the overall response. Theref ore, bioanalytical results cannot be an estimat e but 
rather an indication of the TEQ level in the sample. 
(2)The principles as descr ibed 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. or, if for technical reasons the signal-t o-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 sam ples) deviation to the average 
relative response factor calculated for all points on the calibration curve in each series of samples (1).  
1.8. ‘Upper -bound’ means the concept which requires using the limit of quantifi cation for the contr ibution of each 
non-quantif ied congener .  
1.9. ‘Lower-bound’ means the concep t whic h requires using zero for the contr ibution of each non-quantified 
congener .  
1.10. ‘Medium-bound’ means the concept which requires using half of the limit of quantifi cation calculating the 
contr ibution of each non-quantified cong ener .  
1.11. ‘Lot’ means an identifiable quantity of food delivered at one time and deter mined by the official to have 
common charact eristics, such as origin, variety, type of packing, pack er, consignor or markings. In the case of 
fish and fisher y products, also the size of fish shall be comparable. In case the size and/or weight of the fish is 
not compar able within a consignment, the consignment may still be considered as a lot but a specific sampling 
procedure has to be applied.  
1.12. ‘Sublot’ means designate d part of a large lot in order to apply the sampli ng method on that designated part. 
Each sublot must be physically separated and identif iable.  
1.13. ‘Incremental sam ple’ means a quantity of mater ial taken from a sing le place in the lot or sublot.  
1.14. ‘Aggreg ate sample’ means the combined total of all the incremental samples taken from the lot or sublot.  
1.15. ‘Laborator y sample’ means a representative part/quantity of the aggregat e sample intended for the laboratory .  
II. ABBREVIA TIONS USED 
BEQ  Bioanalytical Equivalents 
GC  Gas chromatograph y 
HRMS  High resolution mass spectrometr y 
LRMS  Low resolution mass spectrometr y 
MS/MS  Tandem mass spectrometr y 
PCB  Polychlor inated biphenyl 
Non-dio xin-like PCBs  PCB 28, PCB 52, PCB 101, PCB 138, PCB 153 and PCB 180 
PCDD  Polychlor inated dibenzo-p-dio xins 
PCDF  Polychlor inated dibenzofurans 
QC  Quality control 
REP  Relative poten cy 
TEF  Toxic Equivalency Fact or 
TEQ  Toxic Equivalents 
TCDD  2,3,7,8- Tetrac hlorodibenzo-p-dio xin 
U  Expanded measurement uncer tainty  6.4.2017 L 92/13 Official Jour nal of the European Union EN     
(1)The LOQ is calculated from the lowest concentration point taking into account the reco very of inter nal standards and sample intake. ANNEX II 
METHODS OF SAM PLING FOR OFFICIAL CONTR OL OF LEVELS OF DIO XINS (PCDD/PCDF), DIO XIN-LIKE 
PCBs AND NON-DIO XIN-LIKE PCBs IN CERT AIN FOODSTUFFS  
I. SCOPE 
Sam ples intended for the official control of the levels of dioxins (PCDD/Fs), dioxin-like PCBs and non-dio xin-like 
PCBs in foodstuffs shall be taken according to the methods descr ibed in this Annex. Aggreg ate 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 laborat ory samp les. 
To ensure compliance with the provisions in Article 4 of Regulation (EC) No 852/2004, food business operat or 
shall, when sample s are taken to control the levels of dioxins (PCDD/Fs), dioxin-like PCBs and non-dio xin-like PCBs, 
take the samples according to the methods descr ibed in Chapt er III of this Annex or apply an equivalent sampli ng 
procedure which is demonstrated to have a same level of representation as the sampli ng procedure descr ibed in 
Chapt er III of this Annex.  
II. GENERAL PROVISIONS 
1.Personnel 
Offcial sam pling shall be perf ormed by an author ised person as designate d by the Member State. 
2.Mater ial to be sampled 
Each lot or sublot whic h is to be examined shall be sample d separately . 
3.Precautions to be taken 
In the course of sam pling 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 analytical determination or make the aggregat e 
samp les unrepresentative. 
4.Incrementa l samples 
As far as possible, incremental samples shall be taken at various places distr ibut ed throughout the lot or sublot. 
Depar ture from such a procedure shall be recorded in the record provided for under point II.8. 
5.Preparation of the aggregate sample 
The aggregat e samp le shall be made up by combining the incremental samples . It shall be at least 1 kg unless not 
practical, e.g. when a sing le packag e has been sampled or when the product has a very high commercial value. 
6.Replicate samples 
The replicate sam ples for enforcement, defence and referen ce purposes shall be take n from the homogenised 
aggrega te sample, unless such procedure conf licts with a Member State 's rules as regard the rights of the food 
business operat or. The size of the laboratory samples for enforcement shall be suffi cient to allow at least for 
duplicat e analyses. 
7.Packa ging and transmission of samples 
Each sample shall be placed in a clean, iner t container offering adequate protection from contamination, from loss 
of analytes by adsor ption to the internal wall of the container and against damage in transit. All necessar y 
precautions shall be taken to avoid any change in compo sition of the sam ple which might arise during transpor ­
tation or storage. 6.4.2017 L 92/14 Official Jour nal of the European Union EN     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 State s. 
A record shall be kept of each sampling, permitting each lot to be identif ied unambiguously and giving the date and 
place of sam pling together with any additional information likely to be of assistance to the analyst.  
III. SAM PLING PLAN 
The sampling method applied shall ensure that the aggregat e sample is representative of the (sub)lot that is to be 
controlled. 
1.Division of lots into sublots 
Larg e lots shall be divided into sublots on condition that the sublot can be separated physically . 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 alwa ys 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 
≥ 1 500 500 tonnes 
> 300 and < 1 500 3 sublots 
≥ 50 and ≤ 300 100 tonnes 
< 50 —  
Table 2 
Subdivision of lots into sublots for other products 
Lot weight (ton) Weight or number of sublots 
≥ 15 15-30 tonnes 
< 15 —  
2.Number of incremental samples 
The aggregat e sam ple 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 mech anical means immediately prior to sampling. In 
that case, a homog eneous distr ibution of contaminants is assumed within a given lot or sublot. It is theref ore 
sufficient to take three incremental samples from a lot or sublot to form the aggreg ate sample. 
The incremental samples shall be of similar weight. The weight of an incremental sample shall be at least 
100 grams. 6.4.2017 L 92/15 Official Jour nal of the European Union EN     Depar ture 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/EC (1), the aggregat e sample size for hens' eggs is at 
least 12 eggs (for bulk lots as well as for lots consisting of individual packag es, Tables 3 and 4 shall apply). 
Table 3 
Minimum number of incrementa l samples to be taken from the lot or sublot 
Weight or volum e 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 packag es or units, then the number of packag es or units whic h shall be 
take n to form the aggregat e sample is given in Table 4. 
Table 4 
Number of packa ges or units (increment al samples) which shall be taken to form the aggregate sample if 
the lot or sublot consis ts of individual packages or units 
Number of pack ages or units in the lot/sublot Number of packag es or units to be taken 
1 to 25 at least 1 packag e or unit 
26 to 100 about 5 %, at least 2 packag es or units 
> 100 about 5 %, at maximum 10 packag es or units  
3.Specif ic provisions for the sampling of lots containing whole fishes of comparable size and weight 
Fishes are considered to be of compar able size and weight where the differ ence in size and weight does not exceed 
about 50 %. 
The number of incremental sample s to be taken from the lot are defined in Table 3. The aggreg ate sample uniting all 
incremental samp les shall be at least 1 kg (see point II.5). 
—  Where the lot to be sam pled contains small fishes (individual fishes weighing < about 1 kg), the whole fish is 
taken as incremental samp le to form the aggregat e sample. Where the resulting aggregat e 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 aggregat e sample . The whole part to which the maximum level is applicable is used for 
homogenisation of the sam ple. 
The middle part of the fish is where the centre of gravity is. This is locat ed in most cases at the dorsal fin (in 
case the fish has a dorsal fin) or halfwa y between the gill opening and the anus. 
—  Where the lot to be sam pled contains larger fishes (individual fishes weighing more than about 1 kg), the 
incremental sample consists of the middle part of the fish. Each incremental sam ple weighs at least 100 grams. 
For fishes of interm ediat e 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. 6.4.2017 L 92/16 Official Jour nal of the European Union EN     
(1)Commission Decision 97/747/EC of 27 October 1997 fixing the levels and frequencies of samplin g provided for by Council Directive 
96/23/EC for the monitoring of certain substances and residues thereof in certain animal products (OJ L 303, 6.11.1997, p. 12). For very large fishes (e.g. > about 6 kg), the incremental part is taken from the right side (frontal 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 signif icant economic damage, the taking of three incremental sam ples of at least 350 grams each 
may be considered 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 take n to form 
the incremental sam ple being representative for 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 regar ds sam ple constitution shall apply . 
—  Where a size or weight class/cat egor y 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/cat egor y predominates, then it must be ensured that the fishes select ed 
for the sam ple are representative for the lot. Specifi c guidance for such cases is provided in ‘Guidance document 
on samp ling of whole fishes of differ ent size and/or weight’ (2). 
5.Sampling at retail stage 
Sam pling of foodstuffs 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 altern ative method of sam pling at retail stage may be used provided that it ensures 
sufficient representativeness for the sample d lot or sublot.  
IV. COMP LIANCE OF THE LOT WITH SPECIFICA TION 
1.As regards non-dio xin-like PCBs 
The lot is compl iant if the analytical result for the sum of non-dio xin-like PCBs does not exce ed the respective 
maximum level, as laid down in Regulation (EC) No 1881/2006 taking into account the expanded measurement 
uncer tainty (3). 
The lot is non-com pliant with the maximum level as laid down in Regulation (EC) No 1881/2006 if the mean of 
two upperbound analytical results obtained from duplicat e analysis (4), taking into account the expanded 
measurement uncer tainty , excee ds the maximum level beyond reasonable doubt. 
The expanded measurement uncer tainty is calculat ed using a coverag e factor of 2 which gives a level of conf idence 
of appro ximately 95 %. A lot is non-compliant if the mean of the measured values minus the expanded uncer tainty 
of the mean is above the established maximum level. 
The rules, mentioned in the paragraphs above under this point, shall apply for the analytical result obtained on the 
samp le for official control. In case of analysis for defe nce or reference purposes, the national rules apply . 
2.As regards dioxins (PCDD/Fs) and dioxin-lik e PCBs 
The lot is compliant if the result of a sing le analysis 
—  performed by a screening method with a false-compliant rate belo w 5 % indicate s that the level 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, 6.4.2017 L 92/17 Official Jour nal of the European Union EN     
(2)https://ec.europa.eu/f ood/sites/f ood/fil es/safety/d ocs/cs_contaminants_catalogue_dio xins_guidance-samplin g_exem ples-dec2006_en. 
pdf 
(3)The principles as descr ibed in the ‘Guidance Document on Measurement Uncer tainty for Laboratories perf orming PCDD/F and PCB 
Analysis using Isotope Dilution Mass Spectrometr y’ [link to website] shall be followed when applicable. 
(4)The duplicate analysis is necessar y if the result of the first deter mination is non-compli ant. The duplicate analysis is necessar y to exclude 
the possibility of inter nal cross-contamination or an accidental mix-up of samples. In case the analysis is performed in the course of a con­
tamination incident, confir mation 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. —  performed by a conf irmatory method does not excee d 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 uncer tainty (5). 
For screening assa ys a cut-off value shall be established for the decision on the compl iance 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-com pliant with the maximum level as laid down in Regulation (EC) No 1881/2006 if the mean of 
two upperbound analytical results (duplicat e analysis (6)) obtained using a conf irmatory method, taking into account 
the expanded measurement uncer tainty , excee ds the maximum level beyond reasonable doubt. 
The expanded measurement uncer tainty is calculat ed using a coverag e factor of 2 which gives a level of conf idence 
of appro ximately 95 %. A lot is non-compliant if the mean of the measured values minus the expanded uncer tainty 
of the mean is above the established maximum level. 
The sum of the estimated expanded uncer tainties of the separate analytical results of PCDD/Fs and dioxin-like PCBs 
has to be used for the estimate d expanded uncer tainty 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 
samp le for official control. In case of analysis for defe nce or reference purposes, the national rules apply .  
V. EXCEED ANCE OF ACTION LEVELS 
Action levels serve as a tool for the selection of sample s in those cases where it is appropr iate to identify a source of 
contamination and to take measures for its reduction or elimination. Screening methods shall establish the 
appropr iate cut-off values for selection of those sam ples. Where signif icant efforts are necessar y to identify a source 
and to reduce or eliminate the contamination, it might be appropr iate to confir m excee dance of the action level by 
duplicat e analysis using a conf irmatory method and taking into account the expanded measurement uncer tainty (7).  6.4.2017 L 92/18 Official Jour nal of the European Union EN     
(5)Guidance Document on Measurement Uncer tainty for Laboratories perf orming PCDD/F and PCB Analysis using Isotope Dilution Mass 
Spectrometr y [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] . 
(6)The duplicate analysis is necessar y if the result of the first determination applying confir matory methods with the use of 13C-labelled 
internal standard for the relevant analyt es is non-compliant. The duplicate analysis is necessar y to exclude the possibility of internal 
cross-contamination or an accidental mix-up of samples. In case the analysis is perfor med in the course of a contamination incident, 
confir mation by duplicate analysis might be omitted in case the samples selected for analysis are through traceability linked to the con­
tamination incident and the level found is signifi cantly above the maximum level. 
(7)Identical explanation and requirements for duplicate analysis for control of action levels as in footnote 6 for maximum levels. ANNEX III 
SAMPLE PREP ARA TION AND REQUIREMENTS FOR METHODS OF ANAL YSIS USED IN CONTR OL OF THE 
LEVELS OF DIO XINS (PCDD/FS) AND DIO XIN-LIKE PCBs IN CERT AIN FOODSTUFFS 
1.  FIELD OF APPLICA TION 
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-substitute d poly chlor inate d dibenzo-p-dio xins and poly chlor inated dibenzofurans (PCDD/ 
Fs) and dioxin-like poly chlor inated biphenyls (dioxin-lik e PCBs) and as regar ds sample preparation and 
analytical requirements for other regulatory purposes, including the controls performe d by the food business 
operat or to ensure compl iance 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 perf ormed with two 
different types of analytical methods:  
(a) Screening methods 
The goal of screening methods is to select those sample s with levels of PCDD/Fs and dioxin-like PCBs that 
excee d the maximum levels or the action levels. Screening methods shall ensure cost-eff ective high sample- 
throughput, thus increasing the chance to disco ver new incidents where high exposure may lead to health 
risks for consumers. Their application shall aim to avoid false-com pliant results. They may compris e bioana ­
lytical and GC/MS methods. 
Screening methods compar e the analytical result with a cut-off value, providing a yes/no-decision over the 
possible excee dance of the maximum or action level. The concentration of PCDD/Fs and the sum of PCDD/ 
Fs and dioxin-like PCBs in sam ples suspecte d to be non-compliant with the maximum level must be 
determined or conf irmed by a conf irmatory method. 
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 case of application of physico-c hemical GC-MS methods it is 
expressed as Toxic Equivalents (TEQ). The numer ically indicated results of screening methods are suitable 
for demonstrating compliance or suspect ed non-com pliance or excee dance of action levels and give an 
indication of the range of levels in case of follow-up by conf irmatory methods. They are not suitable for 
purposes such as evaluation of back ground levels, estimation of intake , following of time trends in levels or 
re-evaluation of action and maximum levels.  
(b) Confir mator y methods 
Confir matory methods allow the unequivocal identif ication and quantification of PCDD/Fs and dioxin-like 
PCBs present in a sample and provide full information on cong ener basis. Theref ore, those methods allow 
the control of maximum and action levels, including the confir mation of results obtained by screening 
methods. Further more, results may be used for other purposes such as determination of low backg round 
levels in food monitori ng, 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 important for 
establishing congener patter ns in order to identify the source of a possible contamination. Such methods 
utilise GC-HRMS. For confir ming compl iance 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 sam ple shall 
be multiplied by their respective TEF, as established by the World Health Org anisation and listed in the 
Appendix to this Annex, and subsequently summed to give the tota l concentration of dioxin-like compo unds 
expressed as TEQs. 
Screening and conf irmat ory methods may only be applied for control of a certain matr ix if the methods are 
sensitive enough to detect levels reliably at the maximum or action level. 6.4.2017 L 92/19 Official Jour nal of the European Union EN     3.  QUALIT Y ASSURANCE REQUIREMENTS 
—  Measures must be taken to avoid cross-contamination at each stage of the sam pling and analysis procedure. 
—  The samples must be stored and transpor ted in glass, aluminum, polyprop ylene or polyeth ylene containers 
suitable for storage without any influence on the levels of PCDD/Fs and dioxin-like PCBs in the sam ples. 
Traces of paper dust must be remo ved from the sample container . 
—  The sample storag e and transpor tation has to be perf ormed in a way that maintains the integrity of the 
foodstuff samp le. 
—  Insofar as relevant, finely grind and mix thoroughly each laborat ory sample using a process that has been 
demonstrated to achi eve compl ete homog enisation (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 impor tance. 
—  A blank analysis shall be performed by carrying out the entire analytical procedure omitting only the 
sample. 
—  For bioanalytical methods, it is of great impor tance that all glassware and solvents used in analysis shall be 
tested to be free of compo unds that interf ere with the detec tion of target compo unds in the working rang e. 
Glassware shall be rinsed with solvents or/and heat ed at temperatures suitable to remo ve traces of PCDD/Fs, 
dioxin-like compo unds and interfer ing compo unds from its surface. 
—  Sample quantity used for the extraction must be sufficient to fulfill the requirements with respect to 
a suffi ciently low working rang e including the concentrations of maximum or action levels. 
—The specifi c sample preparation procedures used for the products under consideration shall follow internat ­
ionally accept ed guidelines. 
—  In the case of fish, the skin has to be remo ved as the maximum level applies to muscle meat without skin. 
However it is necessar y that all remaining muscle meat and fat tissue on the inner side of the skin are 
carefully and compl etely scraped off from the skin and added to the sample to be analysed. 
4.  REQUIREMENTS FOR LABORA TORIES 
—  In accordance with the provisions of Regulation (EC) No 882/2004, laboratori es shall be accredite d by 
a recognised body operating in accordance with ISO Guide 58 to ensure that they are applying analytical 
quality assurance. Laboratori es shall be accredited following the EN ISO/IEC 17025 standard. The principles 
as descr ibed in the Techn ical Guidelines for the estimation of measurement uncer tainty and limits of quanti ­
fication for PCDD/F and PCB analysis shall be followed when applicable (1). 
—  Laboratory proficiency shall be proven by the continuous successful participation in inter laboratory studies 
for the determination of PCDD/Fs and dioxin-like PCBs in relevant food matr ices and concentration rang es. 
—  Laboratori es applying screening methods for routine control of samples shall establish a close cooperation 
with laboratories applying the conf irmat ory method, both for quality control and conf irmation of the 
analytical result of suspect ed samples. 
5.  BAS IC REQUIREMENTS TO BE MET BY ANAL YTICAL PROCEDURE FOR DIO XINS (PCDD/FS) AND DIO XIN-LIKE PCBS 
5.1.  Low working range and limits of quantif ication 
—  For PCDD/Fs, detectable quantities have to be in the upper femtogram (10– 15 g) range because of extreme 
toxicity of some of these compo unds. For most PCB cong eners limit of quantification in the nanogram 
(10– 9 g) range is already suffi cient. However , for the measurement of the more toxic dioxin-like PCB 
cong eners (in particular non-or tho-substitut ed congeners) the lower end of the working range must reach 
the low picogram (10– 12 g) levels. 6.4.2017 L 92/20 Official Jour nal of the European Union EN     
(1)Guidance Document on Measurement Uncer tainty for Laboratories perf orming PCDD/F and PCB Analysis using Isotope Dilution Mass 
Spectrometr y [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] . 5.2.  High selectivity (specif icity) 
—  A distinction is required between PCDD/Fs and dioxin-like PCBs and a multitude of other , coextracte d and 
possibly interfe ring compo unds present at concentrations up to several orders of magnitude higher than 
those of the analyte s of interest. For gas chromat ography/mass spectrometr y (GC-MS) methods, a differ en­
tiation among various cong eners is necessar y, such as between toxic (e.g. the seventeen 2,3,7,8-substitute d 
PCDD/Fs, and twelve dioxin-like PCBs) and other cong eners. 
—  Bioanalytical methods shall be able to detect the targe t compo unds as the sum of PCDD/Fs, and/or dioxin- 
like PCBs. Samp le clean-up shall aim at remo ving compounds causing false non-com pliant results or 
compo unds that may decrease the response, causing false-com pliant results. 
5.3.  High accuracy (trueness and precision, bioassay apparent reco very) 
—  For GC-MS methods, the determination shall provide a valid estimate of the true concentration in a sam ple. 
High accuracy (accuracy of the measurement: the closeness of the agreement between the result of 
a measurement with the true or assigned value of the measurand) is necessar y 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 (differ ence between the mean value measured for an analyte in a certified mater ial and its 
certified value, expressed as percentag e of this value) and precision (RSDR relative standard deviation 
calculated from results generated under reproducibility conditions). 
—  For bioanalytical methods, the bioassa y apparent reco very shall be deter mined. 
5.4.  Validation in the range of maximum level and general quality control measures 
—  Laboratori es shall demonstrate the performa nce of a method in the range of the maximum level, e.g. 0,5×, 
1× and 2× the maximum level with an accepta ble coeffi cient of variation for repeate d analysis, during the 
validation procedure and/or during routine analysis. 
—  Regular blank controls and spiking exper iments or analysis of control sam ples (prefe rably , if available, 
certified reference mat erial) shall be performe d as intern al quality control measures. Quality control (QC) 
char ts for blank controls, spiking exper iments or analysis of control samples shall be recorded and check ed 
to make sure the analytical perf ormance is in accordance with the requirements. 
5.5.  Limit of quantif ication 
—  For a bioanalytical screening method, establishment of the LOQ is not an indispensable requirement but the 
method shall prove that it can differentiat e between the blank and the cut-off value. When providing a BEQ- 
level, a repor ting level shall be established to deal with samples showing a response belo w this level. The 
repor ting level shall be demonstrat ed to be diffe rent from procedure blank samples at least by a factor of 
three, with a response belo w the working range . It shall theref ore be calculated from samples containing the 
target compo unds around the required minimum level, and not from a S/N ratio or an assa y blank. 
—  Limit of quantif ication (LOQ) for a conf irmat ory method shall be about one fifth of the maximum level. 
5.6.  Analytical criter ia 
—  For reliable results from conf irmat ory 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-lik e PCBs) or separate ly for PCDD/Fs and dioxin-like PCBs.  
Screening with bioanalytical or 
physico-c hemical methods Confir matory methods 
False-compli ant rate (*) < 5 %  
Trueness  – 20 % to + 20 % 6.4.2017 L 92/21 Official Jour nal of the European Union EN     Screening with bioanalytical or 
physico-c hemical methods Confir matory methods 
Repeatability (RSDr) < 20 %  
Intermediate precision (RSDR) < 25 % < 15 % 
(*)  With respect to the maximum levels  
5.7.  Specif ic 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 specifi c requirements are laid 
down in point 7. 
—  Laboratori es applying screening methods for routine control of samples shall establish a close cooperation 
with laboratories applying the conf irmat ory 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 compl iant 
results. 
—  Check on possible suppression of the cell response and cytotoxicity . 
20 % of the sam ple extracts shall be measured in routine screening without and with TCDD added 
corresponding to the maximum or action level, to chec k if the response is possibly suppressed by 
interfer ing substances present in the sample extract. The measured concentration of the spik ed sample is 
compared to the sum of the concentration of the unspike d extract plus the spiking concentration. If this 
measured concentration is more than 25 % lower than the calculat ed (sum) concentration, this is an 
indication of a poten tial signal suppression and the respective sample must be submitted to conf irmatory 
analysis. Results shall be monitor ed in quality control charts. 
—  Quality control on compl iant samples 
Appro ximat ely 2 % to 10 % of the compliant samples , depending on sample matr ix and laborat ory 
exper ience, shall be confir med. 
—  Determination of false-compliant rates from QC data 
The rate of false-compliant results from screening of samples belo w and above the maximum level or the 
action level shall be determined. Actual false-com pliant rates shall be belo w 5 %. 
After a minimum of 20 conf irmed results per matr ix/matr ix group is available from the quality control of 
compliant sample s, 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 rang e up 
to, e.g. 2× the maximum level (ML), may also be included in the minimum of 20 results for evaluation of 
the false-compliant rate. The sam ples shall cover most frequent congener patter ns, representing various 
sources. 
Although screening assa ys shall preferen tially aim to detect samples exceed ing the action level, the criterion 
for determining false- compliant rates is the maximum level, taking into account the expanded measurement 
uncer tainty of the conf irmat ory method. 
—  Poten tial non-com pliant results from screening shall alwa ys be verified by a full re-analysis of the original 
sample by a conf irmat ory method. These samples may also be used to evaluate the rate of false non- 
compliant results. For screening methods, the rate of false non-com pliant results is the fraction of results 
conf irmed to be compliant from confir matory analysis, while in previous screening the sam ple had been 
declared to be suspect ed to be non-compliant. However , evaluation of the advantage ousness of the 
screening method shall be based on comparison of false non-com pliant samp les with the total number of 
samples chec ked. This rate shall be low enough to make the use of a screening tool advantage ous. 6.4.2017 L 92/22 Official Jour nal of the European Union EN     —  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 RSDr would 
typically be smaller than the reproducibility RSDR. 
6.  SPECIFIC REQUIREMENTS FOR GC-MS METHODS TO BE COMP LIED WITH FOR SCREENING OR CON FIRMA TOR Y 
PURPOSES 
6.1.  Accepta ble differences betw een upperbound and lowerbound WHO- TEQ levels 
—  The differ ence between upperbound level and lowerbound level shall not exceed 20 % for conf irmation of 
the exceedance of maximum or in case of need of action levels. 
6.2.  Control of reco veries 
—  Addition of 13C-labelled 2,3,7,8-chlor ine-substitute d intern al 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 validat e the analytical procedure. At least one congener for each of the tetra- to octa- 
chlorinate d homologous groups for PCDD/Fs and at least one cong ener for each of the homologous groups 
for dioxin-lik e PCBs must be added (alternatively , at least one cong ener for each mass spectrometr ic select ed 
ion recording function used for monitori ng PCDD/Fs and dioxin-like PCBs). In case of conf irmat ory 
methods, all seventeen 13C-labelled 2,3,7,8-substituted intern al PCDD/F standards and all twelve 13C-labelled 
internal dioxin-like PCB standards shall be used. 
—  Relative response factors shall also be deter mined for those cong eners for which no 13C-labelled analogue is 
added by using appropr iate calibration solutions. 
—  For foodstuffs of plant origin and foodstuffs of animal origin containing less than 10 % fat, the addition of 
the intern al standards is mandat ory 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 appropr iate 
validation of the extraction efficiency shall be carried out, depending on the stage at which intern al 
standards are introduced and on whether results are repor ted on product or fat basis. 
—  Prior to GC-MS analysis, one or two reco very (surrogate) standard(s) must be added. 
—  Control of reco very is necessar y. For confir matory methods, the reco veries of the individual internal 
standards shall be in the range of 60 to 120 %. Lower or higher reco veries for individual cong eners, in 
particular for some hepta- and octa- chlor inate d dibenzo-p-dio xins and dibenzofurans, are accepta ble on the 
condition that their contr ibution 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 reco veries shall be in the range of 
30 to 140 %. 
6.3.  Remo val of inter fering subst ances 
—  Separation of PCDD/Fs from interfe ring chlor inated compo unds such as non-dio xin-like PCBs and 
chlorinate d diphen yl ethers shall be carried out by suitable chromat ographic techniques (prefe rably with 
a florisil, alumina and/or carbon column). 
—  Gas-chro matographic separation of isomers shall be suffic ient (< 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 rang e of the calibration curve shall cover the relevant range of maximum or action levels. 
6.5.  Specif ic criter ia for conf irmat ory methods 
—  For GC-HRMS: 
In HRMS, the resolution shall typically be greate r than or equal to 10 000 for the entire mass range at 
10 % valley . 6.4.2017 L 92/23 Official Jour nal of the European Union EN     Fulfi lment of further identif ication and conf irmation criteria as descr ibed in intern ationally 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: 
Monitor ing of at least two specific precursor ions, each with one specific corresponding transition product 
ion for all labelled and unlabelled analyte s in the scope of analysis. 
Maximum permitted tolerance of relative ion intensi ties of ± 15 % for selecte d transition product ions in 
compari son to calculated or measured values (averag e from calibration standards), applying identical MS/MS 
conditions, in particular collision energy and collision gas pressure, for each transition of an analyt e. 
Resolution for each quadr upole to be set equal to or better than unit mass resolution (unit mass resolution: 
suffi cient resolution to separate two peaks one mass unit apar t) in order to minimise possible interf erences 
on the analyt es of interest. 
Fulfi lment of the further criteria as descr ibed 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, excep t the oblig ation to 
use GC-HRMS. 
7.  SPECIFIC REQUIREMENTS FOR BIO ANAL YTICAL METHODS 
Bioanalytical methods are methods based on the use of biological principles like cell-based assa ys, recep tor- 
assa ys or immunoassa ys. This point establishes requirements for bioanalytical methods in general. 
A screening method in principle classif ies a sample as compl iant or suspected to be non-com pliant. For this, 
the calculat ed BEQ level is compar ed to the cut-off value (see point 7.3). Sam ples belo w the cut-off value are 
declared compliant, sam ples equal or above the cut-off value as suspect ed to be non-com pliant, requir ing 
analysis by a conf irmat ory 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 belo w 5 % and an accept able rate for false non- 
compl iant results are ensured. With separate maximum levels for PCDD/Fs and for the sum of PCDD/Fs and 
dioxin-like PCBs, chec king compl iance of sam ples without fractionation requires appropr iate bioassa y cut-off 
values for PCDD/Fs. For chec king of samples excee ding the action levels, an appropr iate 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 repor ting limit (see points 7.1.1 and 7.1.6). 
7.1.  Evaluation of the test response 
7.1.1.  Gener al requirements 
—  When calculating the concentrations from a TCDD calibration curve, values at the higher end of the curve 
will show a high variation (high coeffi cient of variation (CV)). The working range is the area where this CV 
is smaller than 15 %. The lower end of the working range (repor ting limit) must further be set signif icantly 
(at least by a factor of three) above the procedure blanks. The upper end of the working range is usually 
represented by the EC70 value (70 % of maximal effective concentration), but lower if the CV is higher than 
15 % in this range. The working rang e shall be established during validation. Cut-off values (see point 7.3) 
must be within the working rang e. 
—  Standard solutions and sample extracts shall be tested in triplicate or at least in duplicate. When using 
duplicat es, 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 rang e) based on a CV < 15 %. 6.4.2017 L 92/24 Official Jour nal of the European Union EN     7.1.2.  Calibr ation 
7.1.2.1.  Calibration with standard curve 
—  Levels in samples may be estimate d by comparison of the test response with a calibration curve of TCDD 
(or PCB 126 or a PCDD/F/dio xin-like PCB standard mixture) to calculat e 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 
nonlinear regression. A bette r fit will be achi eved by minimising the difference between calculated and 
obser ved levels in the working rang e of the curve (e.g. by minimising the sum of squared residuals). 
—  The estimat ed level in the sample extract is subsequently correcte d for the BEQ level calculat ed for a matr ix 
or solvent blank sample (to account for impurities from solvents and chemicals used), and the apparent 
reco very (calculat ed from the BEQ level of suitable reference samples with representative congener patt erns 
around the maximum or action level). For performin g a reco very correction, the apparent reco very must 
alwa ys be within the required rang e (see point 7.1.4). Reference sam ples used for reco very correction must 
comply with requirements as given in point 7.2. 
7.1.2.2.  Calibration with reference sam ples 
Alternatively , a calibration curve prepared from at least four reference samples (see point 7.2: one matr ix blank, 
plus three reference samples at 0,5×, 1,0× and 2,0× the maximum or action level may be used, eliminating the 
need to correct for blank and reco very if matr ix proper ties of the reference samp les match those of the 
unkno wn samples. In this case, the test response corresponding to two- thirds of the maximum level (see 
point 7.3) may be calculat ed directly from these sam ples and used as cut-off value. For checking of samples 
exce eding the action levels, an appropr iate percentage of these action levels would suit as cut-off value. 
7.1.3.  Separ ate deter mination 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 calibration curve shall prefe rentially be 
used to evaluate results for the fraction containing dioxin-like PCBs. 
7.1.4.  Bioassay apparen t recoveries 
The ‘bioassa y apparent reco very’ shall be calculat ed from suitable reference sam ples with representative 
cong ener patter ns 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 assa y and TEFs (1) used, the differences between TEF 
and REP factors for dioxin-lik e PCBs may cause low apparent reco veries for dioxin-lik e PCBs in comparison to 
PCDD/Fs. Theref ore, if a separate determi nation of PCDD/Fs and dioxin-like PCBs is performed, bioassa y 
apparent reco veries shall be: for dioxin-lik e PCBs 20 % to 60 %, for PCDD/Fs 50 % to 130 % (range s apply for 
TCDD calibration curve). As the contr ibution of dioxin-like PCBs to the sum of PCDD/Fs and dioxin-like PCBs 
may vary between differ ent matr ices and sample s, bioassa y apparent reco veries for the sum paramete r reflect 
these ranges and shall be between 30 % to 130 %. 
7.1.5.  Contro l of recoveries for clean-up 
The loss of compo unds during the clean-up shall be chec ked 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 reco very and 
variability chec ked by a conf irmatory method. The reco very shall be within 60 to 120 % especially for 
cong eners contr ibuting more than 10 % to the TEQ-level in various mixtures. 6.4.2017 L 92/25 Official Jour nal of the European Union EN     
(1)Current requirements are based on the TEFs published in: M. Van den Berg et al, Toxicol Sci 93 (2), 223-241 (2006). 7.1.6.  Repor ting Limit 
When repor ting BEQ levels, a repor ting limit shall be determi ned from relevant matr ix samples involving 
typical cong ener patt erns, but not from the calibration curve of the standards due to low precision in the lower 
rang e of the curve. Effects from extraction and clean-up must be take n into account. The repor ting limit must 
be set signif icantly (at least by a factor of three) above the procedure blanks. 
7.2.  Use of reference samples 
—  Reference samples shall represent samp le matr ix, congener patter ns and concentration range s for PCDD/Fs 
and dioxin-like PCBs around the maximum or action level. 
—  A procedure blank, or preferably a matr ix blank, and a reference sample at the maximum or action level 
have to be included in each test series. These samples must be extract ed and tested at the same time under 
identical conditions. The referen ce sample must show a clearly elevate d response in compar ison to the 
blank samp le, thus ensur ing the suitability of the test. Those samp les may be used for blank and reco very 
corrections. 
—  Reference samples chosen for performi ng a reco very correction shall be representative for the test sam ples, 
meaning that congener patter ns shall not lead to an underestimation of levels. 
—  Extra reference samples at, e.g. 0,5× and 2× the maximum or action level may be included to demonstrate 
the proper performa nce of the test in the range of inter est for the control of the maximum or action level. 
Combined, these samp les may be used for calculating the BEQ-levels in test samples (see point 7.1.2.2). 
7.3.  Deter mination of cut-of f values 
The relationship between bioanalytical results in BEQ and results from conf irmatory methods in TEQ shall be 
established (e.g. by matr ix-match ed calibration exper iments, involving reference sam ples spik ed at 0, 0,5×, 1× 
and 2× the maximum level (ML), with six repetitions on each level (n = 24)). Correction factors (blank and 
reco very) may be estimat ed from this relationship but shall be chec ked in each test series by including 
procedure/matr ix blanks and reco very 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 PCBs. They are represented by the lower endpoint of the 
distr ibution of bioanalytical results (cor rected for blank and reco very) corresponding to the decision limit of the 
confir matory method based on a 95 % level of conf idence, implying a false-compliant rate < 5 %, and on 
a RSDR < 25 %. The decision limit of the confir matory method is the maximum level, taking into account the 
expanded measurement uncer tainty . 
In practice, the cut-off value (in BEQ) may be calculat ed from the following approach es (see Figure 1): 
7.3.1.  Use of the lower band of the 95 % prediction inter val at the decision limit of the confirmator y method 
Cut-off value ¼BEQ DL − s y,x�tα,f¼m − 2ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi
1=nþ1=m þðxi − xÞ2=Q xxq
with: 
BEQDL  BEQ corresponding to the decision limit of the conf irmatory method, being the ML taking into 
account the expanded measurement uncer tainty 
sy,x  residual standard deviation 
t α,f = m – 2  student factor (α = 5 %, f = degrees of freedom, sing le-sided) 
m  total number of calibration points (index j) 
n  number of repetitions on each level 6.4.2017 L 92/26 Official Jour nal of the European Union EN     xi  sam ple concentration (in TEQ) of calibration point I determined by a conf irmatory method 
x  mean of the concentrations (in TEQ) of all calibration samples 
Qxx¼Xm
j¼1ðxi − xÞ2 square sum parameter 
i  =index for calibration point i 
7.3.2.  Calculation from bioanalytical results (cor rected for blank and reco very) of multiple analyses of samples (n ≥ 6) 
contaminate d at the decision limit of the conf irmatory method, as the lower endpoint of the data distr ibution at 
the corresponding mean BEQ value: 
Cut-off value = BEQDL – 1,64 × SDR 
with 
SDR  standard deviation of bioassa y results at BEQDL, measured under within-laboratory reproducibility 
conditions 
7.3.3.  Calculation as mean value of bioanalytical results (in BEQ, corrected for blank and reco very) from multiple 
analysis of samples (n ≥ 6) contaminate d at two- thirds of the maximum or action level. This is based on the 
obser vation 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 conf idence implying a false-compliant rate < 5 %, and 
a RSDR < 25 %:  
1. from the lower band of the 95 % prediction inter val at the decision limit of the conf irmatory method,  
2. from multiple analysis of sam ples (n ≥ 6) contaminate d at the decision limit of the confir matory method as 
the lower endpoint of the data distr ibution (represent ed in the figure by a bell-shaped curve) at the 
corresponding mean BEQ value. 
Figure 1 
6.4.2017 L 92/27 Official Jour nal of the European Union EN     7.3.4.  Restr ictions to cut-off values 
BEQ-based cut-off values calculat ed from the RSDR achi eved during validation using a limited number of 
sam ples with differ ent matr ix/congener patter ns may be higher than the TEQ-based maximum or action levels 
due to a better precision than attainable in routine when an unkno wn spectr um of possible congener patte rns 
has to be controlled. In such cases, cut-off values shall be calculated from an RSDR = 25 %, or two- thirds of the 
maximum or action level shall be prefer red. 
7.4.  Perfor mance character istics 
—  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 belo w 
20 % and intra-laboratory reproducibility shall be belo w 25 %. This shall be based on the calculat ed levels 
in BEQs after blank and reco very correction. 
—  As part of the validation process, the test must be shown to discr iminate between a blank sam ple and 
a level at the cut-off value, allowing the identification of sample s above the corresponding cut-off value (see 
point 7.1.2). 
—  Target compounds, possible interf erences and maximum tolerable blank levels shall be defined. 
—  The per cent standard deviation in the response or concentration calculated from the response (only 
possible in working rang e) of a triplicate determination of a sample extract shall not be above 15 %. 
—  The uncor rected results of the reference samp le(s) expressed in BEQs (blank and at the maximum or action 
level) shall be used for evaluation of the performa nce of the bioanalytical method over a constant time 
period. 
—  QCchar ts for procedure blanks and each type of reference sample shall be recorded and check ed to make 
sure the analytical performa nce is in accordance with the requirements, in particular for the procedure 
blanks with regar d to the request ed minimum differ ence to the lower end of the working range and for the 
reference sam ples with regar d to within-laboratory reproducibility . Procedure blanks must be well controlled 
in order to avoid false-com pliant results when subtracted . 
—  The results from the conf irmat ory methods of suspected samples and 2 to 10 % of the compl iant sam ples 
(minimum of 20 samples per matr ix) shall be collect ed and used to evaluate the performa nce 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 validate d matr ices. 
—  Successful method performa nce may also be demonstrated by participation in ring trials. The results from 
samples analysed in ring trials, covering a concentration rang e up to, e.g. 2× ML, may also be included in 
the evaluation of the false-compliant rate, if a laboratory is able to demonstrate its successful performa nce. 
The sample s shall cover most frequent cong ener patt erns, representing various sources. 
—  Dur ing incidents, the cut-off values may be re-evaluate d, reflecting the specific matr ix and cong ener 
patter ns of this sing le incident. 
8.  REPOR TING OF THE RESUL T 
Confir mator y methods 
—  The analytical results shall contain the levels of the individual PCDD/F and dioxin-like PCB cong eners and 
TEQ-values shall be repor ted as lower-bound, upper -bound and medium-bound in order to include 
a maximum of information in the repor ting of the results and thereby enabling the interpretation of the 
results according to specif ic requirements. 
—  The repor t 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 matr ices with maximum levels 
expressed on fat basis and with an expecte d fat concentration in the range of 0-2 % (in correspondence to 
existing legislation). For other samples, the deter mination of the lipid content is optional. 6.4.2017 L 92/28 Official Jour nal of the European Union EN     —  The reco veries of the individual intern al standards must be made available in case the reco veries are outside 
the range mentioned in point 6.2 where the maximum level is excee ded (in this case, the reco veries for one 
of the two duplicat e analysis) and in other cases upon request. 
—  As the expanded measurement uncer tainty is to be taken into account when deciding about the compl iance 
of a sample, this parameter shall also be made available. Thus, analytical results shall be repor ted as 
x +/– U whereby x is the analytical result and U is the expanded measurement uncer tainty using a coverag e 
factor of 2 whic h gives a level of conf idence of appro ximat ely 95 %. In case of a separate determination of 
PCDD/Fs and dioxin-like-PCBs the sum of the estimated expanded uncer tainty 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. 
—  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 screeni ng methods 
—  The result of the screening shall be expressed as compl iant 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 belo w the repor ting limit shall be expressed as lower than the 
repor ting limit. Sam ples with a response above the working rang e shall be repor ted as exceeding the 
working range and the level corresponding to the upper end of the working range shall be given in BEQ. 
—  For each type of sam ple matr ix, the repor t shall mention the maximum or action level on which the 
evaluation is based. 
—  The repor t shall mention the type of test applied, the basic test principle and kind of calibration. 
—  The repor t 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 matr ices with maximum levels 
expressed on fat basis and with an expecte d fat concentration in the range of 0-2 % (in correspondence to 
existing legislation). For other samples, the deter mination of the lipid content is optional. 
—  In the case of samples suspected to be non-compliant, the repor t 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/confir med by a conf irmatory method. 
—  Non-compliant results shall only be repor ted from conf irmat ory analysis. 
Physico-chemical screening methods 
—  The result of the screening shall be expressed as compl iant or suspected to be non-compliant (‘suspected’). 
—  For each type of sam ple matr ix, the repor t 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 cong eners and TEQ-values repor ted 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 in 
Regulation (EC) No 1881/2006. 
—  The reco veries of the individual intern al standards must be made available in case the reco veries are outside 
the range mentioned in point 6.2 and in other cases upon request. 
—  The repor t shall mention the GC-MS method applied. 
—  The repor t 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 matr ices with maximum levels 
expressed on fat basis and with an expecte d fat concentration in the range of 0-2 % (in correspondence to 
existing legislation). For other samples, the deter mination of the lipid content is optional. 6.4.2017 L 92/29 Official Jour nal of the European Union EN     —  In case of sam ples suspected to be non-com pliant, the repor t 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 sam ples with 
elevated levels has to be determined/conf irmed by a conf irmatory method. 
—  Non-compliance can only be decided after confir matory analysis.  6.4.2017 L 92/30 Official Jour nal of the European Union EN     Appendix 
WHO- TEFs for human risk assessment based on the conclusions of the World Health Org anisation (WHO) 0151 Inter­
national Programme on Chemical Safety (IPCS) exper t meeting which was held in Geneva in June 2005 (1) 
Congener TEF value Cong ener TEF value 
Dibenzo-p-dioxins (‘PCDDs’) ‘Dioxin-like ’ PCBs 
Non-or tho PCBs + Mono-or tho PCBs 
2,3,7,8- TCDD 1   
1,2,3,7,8-P eCDD 1 Non-or tho 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   
Dibenzofur ans (‘PCDFs’) Mono-or tho PCBs 
2,3,7,8- TCDF 0,1 PCB 105 0,00003 
1,2,3,7,8-P eCDF 0,03 PCB 114 0,00003 
2,3,4,7,8-P eCDF 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’ = chlorodibenzo dioxin; ‘CDF’ = chlorodi benzo ­
furan; ‘CB’ = chlorobiphen yl.   6.4.2017 L 92/31 Official Jour nal of the European Union EN     
(1)Mar tin van den Berg et al., The 2005 World Health Organisation Re-evaluation of Human and Mammalian Toxic Equivalency Fact ors for 
Dioxins and Dioxin-like Compounds. Toxicological Sciences 93(2), 223–241 (2006). ANNEX IV 
SAMPLE PREP ARA TION AND REQUIREMENTS FOR METHODS OF ANAL YSIS USED IN CONTR OL OF THE LEVELS 
OF NON-DIO XIN-LIKE PCBS IN CERT AIN FOODSTUFFS 
The requirements set out in this Annex shall be applied where foodstuffs are analysed for the official control of the 
levels of non-dio xin-like PCBs and as regards sample preparation and analytical requirements for other regulator y 
purposes, including the controls performe d by the food business operat or to ensure compliance with the provisions in 
Article 4 of Regulation (EC) No 852/2004. 
The provisions on sam ple preparation provided for in point 3 of Annex III of this Regulation shall also be applicable for 
the control of the levels of non-dio xin-like PCBs in food.  
1. Applicable detection methods 
Gas Chromatograp hy/Electron Cap ture Detection (GC-ECD), GC-LRMS, GC-MS/MS, GC-HRMS or equivalent 
methods.  
2. Identif ication and conf irmation of analytes of interest : 
—  Relative retention time in relation to internal standards or reference standards (acceptable deviation of 
+/– 0,25 %). 
—  Gas chromat ographic separation of the non-dio xin-like PCBs (from interfer ing substances, especially co-eluting 
PCBs, in particular if levels of samples are in the range of legal limits and non-compliance is to be confir med (1)). 
—  For GC-MS techniques: 
—  Monitoring of at least the following number of molecular ions or charact eristic ions from the molecular 
cluster: 
—  two specific ions for HRMS, 
—  three specifi c 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 select ed mass fragments: 
Relative deviation of abundance ratio of select ed mass fragments from theoretical abundance or calibration 
standard for target ion (most abundant ion monitored) and qualifi er ion(s): ± 15 %. 
—  For GC-ECD: 
Confi rmation of results excee ding the maximum level with two GC columns with stationar y phases of diffe rent 
polar ity.  
3. Demonstration of perfor mance of method: 
Validation in the rang e of the maximum level (0,5 to 2 times the maximum level) with an acceptable coeff icient of 
variation for repeated analysis (see requirements for intermediate precision in point 8).  
4. Limit of quantif ication: 
The sum of the LOQs (2) of non-dio xin-like PCBs shall not be higher than one-third of the maximum level (3).  
5. Quality control: 
Regular blank controls, analysis of spiked samples , quality control samples, participation in inter laboratory studies on 
relevant matr ices. 6.4.2017 L 92/32 Official Jour nal of the European Union EN     
(1)Cong eners 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 chlor inated congeners have to be considered. 
(2)The principles as descr ibed 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. 
(3)It is highly recommendable to have a lower contr ibution of the reagent blank level to the level of a contaminant in a sample. It is in the re­
sponsibility of the laborato ry to control the variation of blank levels, in particular , if the blank levels are subtracted. 6. Control of reco veries: 
—  Use of suitable internal standards with physico-chemical proper ties comparab le to analytes of inter est. 
—  Addition of intern al standards: 
—  Addition to products (before extraction and clean-up process), 
—  Addition also possible to extract ed fat (before clean-up process), if maximum level is expressed on fat basis. 
—  Requirements for methods using all six isotope-labelled non-dio xin-like PCB cong eners: 
—  Correction of results for reco veries of intern al standards, 
—  Generally accep table reco veries of isotope-labelled intern al standards are between 60 and 120 %, 
—  Lower or higher reco veries for individual congeners with a contr ibution to the sum of non-dio xin-like PCBs 
belo w 10 % are accept able. 
—  Requirements for methods using not all six isotope-labelled internal standards or other intern al standards: 
—  Control of reco very of intern al standard(s) for ever y sam ple, 
—  Acceptable reco veries of intern al standard(s) between 60 and 120 %, 
—  Correction of results for reco veries of intern al standards. 
—  The reco veries of unlabelled cong eners shall be chec ked by spik ed samples or quality control sample s with 
concentrations in the range of the maximum level. Acceptable reco veries for these cong eners are between 60 
and 120 %.  
7. Requirements for laboratories 
In accordance with the provisions of Regulation (EC) No 882/2004, laborat ories 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 accredit ed following the EN ISO/IEC 17025 standard. In addition, the principles as descr ibed in 
Technical Guidelines for the estimation of measurement uncer tainty and limits of quantification for PCB analysis shall 
be followed when applicable (1).  
8. Perfor mance character istics: Criter ia for the sum of non-dio xin-like PCBs at the maximum level  
Isotope dilution mass spectrometr y(*) Other techniques 
Trueness – 20 to + 20 % – 30 to + 30 % 
Intermediat e precision (RSDR) ≤ 15 % ≤ 20 % 
Difference between upper and lower 
bound calculation ≤ 20 % ≤ 20 % 
(*)  Use of all six 13C-labelled analogues as internal standards required   
9. Repor ting of results 
—  The analytical results shall contain the levels of the individual non-dio xin-like PCB congeners and the sum of 
non-dio xin-like PCBs, repor ted as lower-bound, upper -bound and medium-bound, in order to include 
a maximum of information in the repor ting of the results and thereby enabling the interpretat ion of the results 
according to specific requirements. 6.4.2017 L 92/33 Official Jour nal of the European Union EN     
(1)‘Guidance Document on Measurement Uncer tainty for Laborato ries perfor ming PCDD/F and PCB Analysis using Isotope Dilution Mass 
Spectrometr y’ [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]. —  The repor t shall also include the method used for the extraction of PCBs and lipids. The lipid conte nt of the 
sample shall be determined and repor ted for food matr ices with maximum levels expressed on fat basis and with 
an expect ed fat concentration in the rang e of 0-2 % (in correspondence to existing legislation). For other samp les, 
the determination of the lipid content is optional. 
—  The reco veries of the individual internal standards must be made available in case the reco veries are outside the 
range mentioned in point 6, in case the maximum level is exce eded and in other cases upon request. 
—  As the expanded measurement uncer tainty is to be taken into account when deciding about the compl iance of 
a sample, that parameter shall also be made available. Thus, analytical results shall be repor ted as 
x +/– U whereby x is the analytical result and U is the expanded measurement uncer tainty using a coverag e factor 
of 2 whic h gives a level of conf idence of appro ximatel y 95 %. 
—  The results shall be expressed in the same units and with the same number of signif icant figures as the maximum 
levels laid down in Regulation (EC) No 1881/2006.  6.4.2017 L 92/34 Official Jour nal of the European Union EN