HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN"L 91/8
EN
Official Journal of the European Union
29.3.2012
COMMISSION REGULATION (EU) No 278/2012
of 28 March 2012
amending Regulation (EC) No 152/2009 as regards the determination of the levels of dioxins and
polychlorinated biphenyls
(Text with EEA relevance)
THE EUROPEAN COMMISSION,
ground range. That is important for the follow-up of
time trends, exposure assessment and for the re-
Having regard to the Treaty on the Functioning of the European
evaluation of maximum and action thresholds.
Union,
Having regard to Regulation (EC) No 882/2004 of the European
(4) The
modification
of
the
maximum
levels
for
dioxins
and
Parliament and of the Council of 29 April 2004 on official
dioxin-like PCBs and the establishment of non-dioxin-like
controls performed to ensure the verification of compliance
PCBs in Directive 2002/32/EC and the need to update
with feed and food law, animal health and animal welfare
the criteria for screening methods makes it necessary to
rules (
amend the rules on the determination of dioxins and

1 ), and in particular Article 11(4) thereof,
PCBs in feed, as laid down by Part B of Annex V to
Whereas:
Regulation (EC) No 152/2009. For reasons of clarity
and comprehensibility, it is appropriate to replace Part
(1) Directive
2002/32/EC
of
the
European
Parliament
and
of
B of Annex V.
the Council of 7 May 2002 on undesirable substances in
animal feed ( 2 ) lays down maximum levels for dioxins,
(5) It
is
of
major
importance
that
analytical
results
are
furans and polychlorinated biphenyls (PCBs) in feed and
reported and interpreted in a uniform way in order to
action thresholds triggering investigations by Member
ensure a harmonised enforcement approach throughout
States to identify the sources of those substances.
the Union.
(2) Commission
Regulation
(EC)
No
152/2009
of
27
January (6) Annex
V
to
Regulation
(EC)
No
152/2009
should
2009 laying down the methods of sampling and analysis
therefore be amended accordingly.
for the official control of feed ( 3 ) includes methods for
the determination of the levels of polychlorinated
(7) The
measures
provided
for
in
this
Regulation
are
in
dibenzo-p-dioxins (PCDDs), polychlorinated dibenzo
accordance with the opinion of the Standing
furans (PCDFs) and dioxin-like polychlorinated
Committee on the Food Chain and Animal Health and
biphenyls (PCBs) in feed.
neither the European Parliament nor the Council have
(3) A
screening
method
of
analysis
with
widely
acceptable
opposed them,
validation and high throughput can be used to identify
HAS ADOPTED THIS REGULATION:
the samples with significant levels of PCDD/Fs and
dioxin-like PCBs (preferably selecting samples exceeding
Article 1
action thresholds and ensuring the selection of samples
exceeding maximum levels). The levels of PCDD/Fs and
Part B of Annex V to Regulation (EC) No 152/2009 is amended
dioxin-like PCBs in these samples need to be determined
in accordance with the Annex to this Regulation.
by a confirmatory method of analysis. It is therefore
appropriate to establish appropriate requirements for
Article 2
the screening method making sure that the false-
This Regulation shall enter into force on the twentieth day
compliant rate with respect to maximum levels is
following that of its publication in the Official Journal of the
below 5 % and strict requirements for the confirmatory
European Union.
methods of analysis. Furthermore, confirmatory methods
allow the determination of levels also in the low back
It shall apply from the date of entry into force.
This Regulation is binding in its entirety and directly applicable in all Member States.
Done at Brussels, 28 March 2012.
For the Commission
The President
José Manuel BARROSO
( 1 ) OJ L 165, 30.4.2004, p. 1.
( 2 ) OJ L 140, 30.5.2002, p. 10.
( 3 ) OJ L 54, 26.2.2009, p. 1.

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29.3.2012 Official
Journal
of
the
European
Union
L
91/9
ANNEX
In Annex V to Regulation (EC) No 152/2009, Part (B) ‘DETERMINATION OF THE LEVELS OF DIOXINS (PCDD/PCDF)
AND DIOXIN-LIKE PCBs’ is replaced by the following:
‘B. DETERMINATION OF THE LEVELS OF DIOXINS (PCDD/PCDF) AND PCBs
CHAPTER I
Methods of sampling and interpretation of analytical results
1. Purpose
and
scope
The samples intended for the official control of the levels of polychlorinated dibenzo-p-dioxins (PCDDs), poly
chlorinated dibenzofurans (PCDFs), dioxin-like polychlorinated biphenyls (PCBs) (*) and non-dioxin-like PCBs in
feed shall be taken in accordance with the provisions of Annex I. The quantitative requirements in relation to the
control of substances or products uniformly distributed throughout the feed as provided for in point 5.A of
Annex I shall be applied. Aggregate samples thus obtained shall be considered representative for the lots or sub-
lots from which they are taken. Compliance with maximum levels laid down in Directive 2002/32/EC shall be
established on the basis of the levels determined in the laboratory samples.
___________
(*) Table of TEF (= toxic equivalency factors) for dioxins, furans and dioxin-like PCBs:
WHO-TEFs for human risk assessment based on the conclusions of the World Health Organisation (WHO) –
International Programme on Chemical Safety (IPCS) expert meeting which was held in Geneva in June 2005
(Martin van den Berg et al., The 2005 World Health Organisation Re-evaluation of Human and Mammalian
Toxic Equivalency Factors for Dioxins and Dioxin-like Compounds. Toxicological Sciences 93(2), 223–241
(2006)).
Congener TEF
value
Congener TEF
value
Dibenzo-p-dioxins (PCDDs) and
“Dioxin-like” PCBs Non-ortho
Dibenzo-p-furans (PCDFs)
PCBs + Mono-ortho PCBs
2,3,7,8-TCDD
1
1,2,3,7,8-PeCDD
1
Non-ortho PCBs
1,2,3,4,7,8-HxCDD 0,1
PCB 77 0,0001
1,2,3,6,7,8-HxCDD 0,1
PCB 81 0,0003
1,2,3,7,8,9-HxCDD 0,1
PCB 126 0,1
1,2,3,4,6,7,8-HpCDD 0,01
PCB 169 0,03
OCDD 0,0003
Mono-ortho PCBs
2,3,7,8-TCDF 0,1
PCB 105 0,00003
1,2,3,7,8-PeCDF 0,03
PCB 114 0,00003
2,3,4,7,8-PeCDF 0,3
PCB 118 0,00003
1,2,3,4,7,8-HxCDF 0,1
PCB 123 0,00003
1,2,3,6,7,8-HxCDF 0,1
PCB 156 0,00003
1,2,3,7,8,9-HxCDF 0,1
PCB 157 0,00003
2,3,4,6,7,8-HxCDF 0,1
PCB 167 0,00003
1,2,3,4,6,7,8-HpCDF 0,01
PCB 189 0,00003
1,2,3,4,7,8,9-HpCDF 0,01
OCDF 0,0003
Abbreviations used: “T” = tetra; “Pe” = penta; “Hx” = hexa; “Hp” = hepta; “O” = octa; “CDD” = chlorodibenzodioxin; “CDF” =
chlorodibenzofuran; “CB” = chlorobiphenyl.
For the purposes of this Part of Annex V, the definitions laid down in Annex I to Commission Decision
2002/657/EC of 14 August 2002 implementing Council Directive 96/23/EC concerning the performance of
analytical methods and interpretation of results (**) shall apply.
___________
(**) OJ L 221, 17.8.2002, p. 8.

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Official Journal of the European Union
29.3.2012
2.
Compliance of the lot or sub-lot with the specification
2.1. As
regards
non-dioxin-like
PCBs
The lot complies with the specification if the analytical result does not exceed the maximum level of non-dioxin-
like PCBs laid down by Directive 2002/32/EC, taking into account the measurement uncertainty.
The lot does not comply with the specification if the upper-bound (***) analytical result confirmed by duplicate
analysis (****) exceeds the maximum level laid down by Directive 2002/32/EC, taking into account the
measurement uncertainty.
The measurement uncertainty shall be taken into account according to one of the following approaches:
— by calculating the expanded uncertainty, using a coverage factor of 2 which gives a level of confidence of
approximately 95 %. A lot or sub-lot is non-compliant if the measured value minus U is above the maximum
level,
— by establishing the decision limit (CCα) in accordance with point 3.1.2.5 of Annex I to Decision
2002/657/EC. A lot or sub-lot is non-compliant if the measured value is equal to or above the CCα.
These interpretation rules shall apply for the analytical result obtained on the sample for official control. In case
of analysis for defence or reference purposes, the national rules shall apply.
___________
(***) The concept of “upper-bound” requires using the limit of quantification for the contribution of each non-
quantified congener. The concept of “lower-bound” requires using zero for the contribution of each non-
quantified congener. The concept of “medium-bound” requires using half of the limit of quantification
calculating the contribution of each non-quantified congener.
(****) The duplicate analysis is necessary to exclude the possibility of internal cross-contamination or an
accidental mix-up of samples. The first analysis, taking into account the measurement uncertainty is
used for verification of compliance. In case the analysis is performed in the frame 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.
2.2. As
regards
PCDD/F
and
dioxin-like
PCBs
The lot complies with the specifications if the analytical 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/PCDFs and the sum of PCDD/PCDFs and dioxin-like PCBs laid
down by Directive 2002/32/EC,
— performed by a confirmatory method, does not exceed the respective maximum level of PCDD/PCDFs and
the sum of PCDD/PCDFs and dioxin-like PCBs laid down by Directive 2002/32/EC, taking into account the
measurement uncertainty.
For screening assays a cut-off value shall be established for decisions on sample compliance with the respective
levels of interest set for either PCDD/PCDFs, or for the sum of PCDD/PCDFs and dioxin-like PCBs.
The lot does not comply with the specification if the upper-bound (*****) analytical result obtained with a
confirmatory method and confirmed by duplicate analysis exceeds the maximum level laid down by Directive
2002/32/EC, taking into account the measurement uncertainty (******).
The measurement uncertainty shall be taken into account according to one of the following approaches:
— by calculating the expanded uncertainty, using a coverage factor of 2 which gives a level of confidence of
approximately 95 %. A lot or sub-lot is non-compliant if the measured value minus U is above the maximum
level. In case of a separate determination of PCDD/PCDFs and dioxin-like-PCBs, the sum of the estimated
expanded uncertainty of the separate analytical results of PCDD/PCDFs and dioxin-like PCBs shall be used for
the sum of PCDD/PCDFs and dioxin-like PCBs,
— by establishing the decision limit (CCα) in accordance with point 3.1.2.5 of the Annex I to Decision
2002/657/EC. A lot or sub-lot is non-compliant if the measured value is equal to or above the CCα.

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These interpretation rules shall apply for the analytical result obtained on the sample for official control. In case
of analysis for defence or reference purposes, the national rules shall apply.
___________
(*****) The concept of “upper-bound” requires using the limit of quantification for the contribution of each
non-quantified congener to the toxic equivalent (TEQ). The concept of “lower-bound” requires using
zero for the contribution of each non-quantified congener to the TEQ. The concept of “medium-bound”
requires using half of the limit of quantification calculating the contribution of each non-quantified
congener to the TEQ.
(******) The duplicate analysis is necessary to exclude the possibility of internal cross-contamination or an
accidental mix-up of samples. The first analysis, taking into account the measurement uncertainty is
used for verification of compliance. In case the analysis is performed in the frame 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.
3. Results
exceeding
action
thresholds
as
laid
down
in
Annex
II
to
Directive
2002/32/EC
Action thresholds serve as a tool for the selection of samples in those cases where it is necessary to identify a
source of contamination and to take measures to reduce or eliminate it. Screening methods shall establish
appropriate cut-off values for the selection of these samples. The efforts necessary to identify a source and to
reduce or eliminate the contamination shall be deployed only if exceedance of the action thresholds is confirmed
by duplicate analysis using a confirmatory method and taking into account the measurement uncer
tainty (*******).
___________
(*******) Identical explanation and requirements for duplicate analysis for control of action thresholds as in
footnote (*****) for maximum levels.
CHAPTER II
Sample preparation and requirements for methods of analysis used in official control of the levels of dioxins
(PCDD/PCDF) and dioxin-like PCBs in feed
1. Field
of
application
The requirements set out in this Annex shall be applied where feed is 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 polychlorinated biphenyls (dioxin-like PCBs) and for regulatory purposes.
Monitoring for the presence of PCDD/Fs and dioxin-like PCBs in feed may be performed with two different goals:
(a) Selection of those samples with levels of PCDD/Fs and dioxin-like PCBs that exceed the maximum levels, or
the action thresholds. This approach may involve a screening method allowing cost-effective high sample-
throughput, thus increasing the chance to discover new incidents with high exposure and health risks of
consumers. Screening methods may comprise bioanalytical methods and GC/MS methods. Their application
should aim at avoiding false-compliant results. The concentration of PCDD/Fs and the sum of PCDD/Fs and
dioxin-like PCBs in those samples with significant levels needs to be determined/confirmed by a confirmatory
method.
(b) Determination of the levels of PCDD/Fs and dioxin-like PCBs in feed samples in the range of low background
levels. This is important in order to follow time trends, exposure assessment of the population and to build a
database for possible re-evaluation of action and maximum levels. This goal is achieved by confirmatory
methods enabling the PCDD/Fs and dioxin-like PCBs to be identified and quantified unequivocally at the level
of interest. These methods can be used for confirmation of results obtained by screening methods and for
determination of low background levels in feed monitoring. They are also important for establishing
congener patterns in order to identify the source of a possible contamination. At present such methods
utilise high-resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS).
2.
Classification of methods by their degree of quantification (********)
___________
(********) Adapted to PCDD/Fs and dioxin-like compounds from “Guidelines for the validation of screening
methods for residues of veterinary medicines”, EU Reference Laboratories (EURLs) for residues of
veterinary medicines and contaminants in food of animal origin in Fougères, Berlin and Bilthoven,
20/1/2010, http://ec.europa.eu/food/food/chemicalsafety/residues/lab_analysis_en.htm

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2.1. Qualitative
methods give a yes/no response on the presence of analytes of interest, with no quantified indication of
the concentration of the putative analyte. Qualitative methods may have the potential for providing semi-
quantitative results but are used solely for report of a yes/no decision as indication of levels above or below
certain ranges, e.g. limit of detection, limit of quantification or cut-off values.
For the control of maximum levels and action thresholds for PCDD/PCDFs and dioxin-like PCBs in feed,
screening methods may be applied which are based on the comparison of the analytical result with a cut-off
value and give a yes/no decision for indication for the possible exceedance of the level of interest.
2.2. Semi-quantitative
methods are methods which give an approximate indication of the concentration of the putative
analyte, while the numerical result does not meet the requirements for quantitative methods. They may be used
to provide information on the range of the analyte concentration in order for the analyst to decide on the
calibration range for the confirmatory test subsequently to be performed and for quality control purposes. For
example, the following methods shall be considered as semi-quantitative methods:
(a) methods based on the use of biological principles like cell-based assays, receptor-assays or immunoassays,
hereinafter bioanalytical methods, which are able to detect the analytes of interest, include a calibration curve,
give a yes/no decision for indication for the possible exceedance of the level of interest and allow to report
the result as bioanalytical equivalents (BEQ), being an indication of the TEQ value in the sample;
(b) physicochemical test (e.g. Gas chromatography-Mass spectrometry/Mass spectrometry (GC-MS/MS) or Gas
chromatography/Low resolution mass spectrometry (GC/LRMS)) where the measured method precision char
acteristics do not meet the requirements for quantitative tests.
2.3. Quantitative
methods meet the same requirements for accuracy, dynamic range and precision as confirmatory
methods. When quantification is required, quantitative methods shall be validated as confirmatory methods.
3. Background
For calculation of toxic equivalent (TEQ) concentrations, the concentrations of the individual substances in a
given sample shall be multiplied by their respective toxic equivalency factor (TEF), as established by the World
Health Organisation and listed in the Appendix to this Annex, and subsequently summed to give the total
concentration of dioxin-like compounds expressed as TEQs.
For the purposes of this Part B of Annex V, the accepted specific limit of quantification of an individual congener
shall be the concentration of an analyte in the extract of a sample which produces an instrumental response at
two different ions to be monitored with an S/N (signal/noise) ratio of 3:1 for the less intensive signal and
fulfilment of identification criteria as described, for example, in standard prEN 16215 (Animal feed – Deter
mination of dioxins and dioxin-like PCBs by Gas chromatography/High resolution mass spectrometry
(GC/HRMS) and of indicator PCBs by GC/HRMS) and/or in EPA method 1613 revision B.
Bioanalytical screening methods will not give results at the congener level but merely an indication (*********) 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 obey all requirements of the TEQ-principle.
Screening and confirmatory methods can only be applied for control of a certain matrix if the methods are
sensitive enough to detect levels reliably at the level of interest (action threshold or maximum level).
___________
(*********) Bioanalytical methods are not specific to those congeners included in the TEF scheme. Other struc
turally 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.
4. Quality
assurance
requirements
4.1.
Measures shall be taken to avoid cross-contamination at each stage of the sampling and analysis procedure.
4.2. The
samples
shall
be
stored
and
transported
in
glass,
aluminium,
polypropylene
or
polyethylene
containers
suitable for storage without any influence on the levels of PCDD/PCDFs and dioxin-like PCBs in the samples.
Traces of paper dust shall be removed from the sample container.

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4.3.
The sample storage and transportation shall be performed in a way that maintains the integrity of the feed
sample.
4.4. In
so
far
as
relevant,
each
laboratory
sample
shall
be
finely
grinded
and
mixed
thoroughly
using
a
process
that
has been demonstrated to achieve complete homogenisation (for example, ground to pass a 1 mm sieve).
Samples shall be dried before grinding if the moisture content is too high.
4.5. Control
of
reagents,
glassware
and
equipment
for
possible
influence
of
TEQ-
or
BEQ-based
results
shall
be
carried out.
4.6.
A blank analysis shall be performed by carrying out the entire analytical procedure omitting only the sample.
4.7. For
bioanalytical
methods,
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 heated at temperatures suitable to remove traces of PCDD/PCDFs, dioxin-like compounds and
interfering compounds from its surface.
4.8.
Sample quantity used for the extraction shall be sufficient to fulfil the requirements with respect to a sufficiently
low working range including the concentrations of interest.
4.9. The
specific
sample
preparation
procedures
used
for
the
products
under
consideration
shall
follow
internationally
accepted guidelines.
5. Requirements
for
laboratories
5.1.
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.
5.2. Laboratory
proficiency
shall
be
proven
by
the
continuous
successful
participation
in
inter-laboratory
studies
for
the determination of PCDD/PCDFs and dioxin-like PCBs in relevant feed matrices and concentration ranges.
5.3.
Laboratories applying screening methods for the routine control of samples shall establish a close cooperation
with laboratories applying the confirmatory method, both for quality control and confirmation of the analytical
result of suspected samples.
6. Basic
requirements
to
be
met
by
analytical
procedure
for
dioxins
(PCDD/PCDFs)
and
dioxin-like
PCBs
6.1. Low
working
range
and
limits
of
quantification
For PCDD/PCDFs, detectable quantities shall be in the upper femtogram (10 –15 g) range because of extreme
toxicity of some of these compounds. For most PCB congeners limit of quantification in the nanogram (10 –9 g)
range is already sufficient. For the measurement of the more toxic dioxin-like PCB congeners (in particular non-
ortho substituted congeners) the lower end of the working range shall reach the low picogram (10 –12 g) levels.
For all other PCB congeners a limit of quantification in the nanogram (10 –9 g) range is sufficient.
6.2. High
selectivity
(specificity)
6.2.1. A
distinction
is
required
between
PCDD/PCDFs
and
dioxin-like
PCBs
and
a
multitude
of
other,
co-extracted
and
possibly interfering compounds present at concentrations up to several orders of magnitude higher than those of
the analytes of interest. For GC/MS methods, a differentiation among various congeners is required, such as
between toxic (for example, the seventeen 2,3,7,8-substituted PCDD/PCDFs, and twelve dioxin-like PCBs) and
other congeners.
6.2.2. Bioanalytical methods shall be able to detect the target compounds as the sum of PCDD/PCDFs, and/or dioxin-
like PCBs. Sample clean-up shall aim at removing compounds causing false-non-compliant results or compounds
that may decrease the response, causing false-compliant results.
6.3. High
accuracy
(trueness
and
precision,
bioassay
apparent
recovery)
6.3.1. For GC/MS methods, the determination shall provide a valid estimate of the true concentration in a sample. High
accuracy is required to avoid the rejection of a sample analysis result on the basis of poor reliability of the
determined TEQ level. Accuracy is expressed as trueness (difference between the mean value measured for an
analyte in a certified material and its certified value, expressed as percentage of this value) and precision (RSD R
relative standard deviation calculated from results generated under reproducibility conditions).

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6.3.2. For bioanalytical methods, the bioassay apparent recovery shall be determined. Bioassay apparent recovery means
the BEQ level calculated from the TCDD or PCB 126 calibration curve corrected for the blank and then divided
by the GC/HRMS determined TEQ level. It aims at correcting factors like the loss of PCDD/PCDFs and dioxin-like
compounds during the extraction and clean-up steps, co-extracted compounds increasing or decreasing the
response (agonistic and antagonistic effects), the quality of the curve fit, or differences between the toxic
equivalency factor (TEF) and the relative potency (REP) values. The bioassay apparent recovery is calculated
from suitable reference samples with representative congener patterns around the level of interest.
6.4. Validation
in
the
range
of
level
of
interest
and
general
quality
control
measures
6.4.1. Laboratories shall demonstrate the performance of a method in the range of the level of interest, for example,
0,5 ×, 1 × and 2 × the level of interest with an acceptable coefficient of variation for repeated analysis, during the
validation procedure and during routine analysis.
6.4.2. Regular
blank
controls
and
spiking
experiments
or
analysis
of
control
samples
(preferably,
if
available,
certified
reference material) shall be performed as internal quality control measures. Quality control charts for blank
controls, spiking experiments or analysis of control samples shall be recorded and checked to make sure the
analytical performance is in accordance with the requirements.
6.5. Limit
of
quantification
6.5.1. For a bioanalytical screening method, the establishment of the limit of quantification (LOQ) is not an indis
pensable 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 3, with 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 an S/N ratio
or an assay blank.
6.5.2. The
LOQ
for
a
confirmatory
method
shall
be
about
one
fifth
of
the
level
of
interest.
6.6. Analytical
criteria
For reliable results from confirmatory or screening methods, the following criteria shall be met for the TEQ or
BEQ value, respectively, whether determined as total TEQ (as sum of PCDD/PCDFs and dioxin-like PCBs) or
separately for PCDD/PCDFs and dioxin-like PCBs:
Screening with bioanalytical or
physico-chemical methods
Confirmatory methods
False-compliant rate (*) <
5
%
Trueness
– 20 % to + 20 %
Repeatability (RSD r ) <
20
%
Within-laboratory reproducibility
< 25 % <
15
%
(RSD R )
(*) With respect to the maximum levels.
6.7. Specific
requirements
for
screening
methods
6.7.1. Both
GC/MS
and
bioanalytical
methods
may
be
used
for
screening.
For
GC/MS
methods
the
requirements
laid
down in point 7 shall be met. For cell-based bioanalytical methods specific requirements are laid down in
point 8.
6.7.2. Laboratories
applying
screening
methods
for
the
routine
control
of
samples
shall
establish
a
close
cooperation
with laboratories applying the confirmatory method.
6.7.3. Performance
verification
of
the
screening
method
is
required
during
routine
analysis,
by
analytical
quality
control
and on-going method validation. There shall be a continuous programme for the control of compliant results.

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6.7.4. Check
on
possible
suppression
of
the
cell
response
and
cytotoxicity:
20 % of the sample extracts shall be measured in routine screening without and with 2,3,7,8-TCDD added
corresponding to the level of interest, to check if the response is possibly suppressed by interfering substances
present in the sample extract. The measured concentration of the spiked sample shall be compared to the sum of
the concentration of the unspiked extract plus the spiking concentration. If this measured concentration is more
than 25 % lower than the calculated (sum) concentration, this is an indication of potential signal suppression and
the respective sample shall be submitted to GC/HRMS confirmatory analysis. Results shall be monitored in
quality control charts.
6.7.5. Quality
control
on
compliant
samples:
Approximately 2 to 10 % of the compliant samples, depending on sample matrix and laboratory experience,
shall be confirmed by GC/HRMS.
6.7.6. Determination
of
false-compliant
rates
from
quality
control
data:
The rate of false-compliant results from screening of samples below and above the maximum level or the action
threshold shall be determined. Actual false-compliant rates shall be below 5 %. When a minimum of 20
confirmed results per matrix/matrix group is available from the quality control of compliant samples, conclusions
on the false-compliant rate shall be drawn from this database. The results from samples analysed in ring trials or
during contamination incidents, covering a concentration range up to for example 2 × maximum level (ML), may
also be included in the minimum of 20 results for evaluation of the false-compliant rate. The samples shall cover
most frequent congener patterns, representing various sources.
Although screening assays shall preferentially aim at detecting samples exceeding the action threshold, the
criterion for determining false-compliant rates is the maximum level, taking into account the measurement
uncertainty of the confirmatory method.
6.7.7. Suspected non-compliant samples from screening shall always be verified by a confirmatory method of analysis
(GC/HRMS). These samples may also be used to evaluate the rate of false-non-compliant results. For screening
methods, the rate of false-non-compliant results shall be the fraction of results confirmed to be compliant from
GC/HRMS confirmatory analysis, while in previous screening the sample has been declared to be suspected to be
non-compliant. Evaluation of the advantageousness of the screening method shall be based on comparison of
false-non-compliant samples with the total number of samples checked. This rate shall be low enough to make
the use of a screening tool advantageous.
6.7.8. 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 .
7. Specific
requirements
for
gc/ms
methods
to
be
complied
with
for
screening
or
confirmatory
purposes
7.1. General
requirements
The difference between upper-bound level and lower-bound level shall not exceed 20 % for feed with a
contamination of about 1 ng WHO-TEQ/kg product with 12 % moisture content (based on the sum of
PCDD/Fs and dioxin-like PCBs). For lower contamination levels, for example 0,5 ng WHO-TEQ/kg product,
the difference between upper-bound and lower-bound level may be in the range of 25 % to 40 %.
7.2. Control
of
recoveries
7.2.1. Addition
of 13 C-labelled 2,3,7,8-chlorine substituted internal PCDD/PCDF standards and of 13 C-labelled internal
dioxin-like PCB standards shall be carried out at the very beginning of the analytical method e.g. prior to
extraction in order to validate the analytical procedure. At least one congener for each of the tetra- to octa-
chlorinated homologous groups for PCDD/PCDFs and at least one congener for each of the homologous groups
for dioxin-like PCBs shall be added (alternatively, at least one congener for each mass spectrometric selected ion
recording function used for monitoring PCDD/PCDFs and dioxin-like PCBs). In the case of confirmatory
methods, all 17 13 C-labelled 2,3,7,8-substituted internal PCDD/PCDF standards and all 12 13 C-labelled
internal dioxin-like PCB standards shall be used.

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7.2.2. Relative response factors shall also be determined for those congeners for which no 13 C-labelled analogue is
added by using appropriate calibration solutions.
7.2.3. For feed of plant origin and feed of animal origin containing less than 10 % fat, the addition of the internal
standards shall be mandatory prior to extraction. For feed of animal origin containing more than 10 % fat, the
internal standards shall be added either before or after fat extraction. An appropriate validation of the extraction
efficiency shall be carried out, depending on the stage at which internal standards are introduced and on whether
results are reported on product or fat basis.
7.2.4. Prior to GC/MS analysis, 1 or 2 recovery (surrogate) standard(s) shall be added.
7.2.5. Control of recovery is required. 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, shall be acceptable on the condition that their
contribution to the TEQ value does not exceed 10 % of the total TEQ value (based on sum of PCDD/PCDF and
dioxin-like PCBs). For GC/MS screening methods, the recoveries shall be in the range of 30 to 140 %.
7.3. Removal
of
interfering
substances
— Separation of PCDD/PCDFs 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 < 25 % peak to peak between 1,2,3,4,7,8-HxCDF and
1,2,3,6,7,8-HxCDF.
7.4. Calibration
with
standard
curve
The range of the calibration curve shall cover the relevant range of levels of interest.
8. Specific
requirements
for
bioanalytical
methods
Bioanalytical methods are methods based on the use of biological principles like cell-based assays, receptor-assays
or immunoassays. This point 8 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 8.3). Samples below the cut-off value are declared
compliant, samples equal or above the cut-off value are suspected to be non-compliant, requiring analysis by a
confirmatory method. In practice, a BEQ level corresponding to 2/3 of the maximum level may serve as the most
suitable cut-off value ensuring a false-compliant rate below 5 % and an acceptable rate for false-non-compliant
results. 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 thresholds, an appropriate percentage of the respective level of
interest would suit as cut-off value.
Furthermore, in the case of certain bioanalytical methods, an indicative level expressed in BEQs may be given for
samples in the working range and exceeding the reporting limit (see 8.1.1 and 8.1.6).
8.1. Evaluation
of
the
test
response
8.1.1. G e n e r a l r e q u i r e m e n t s
— When calculating the concentrations from a TCDD calibration curve, values at the lower and 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) shall be set at least by a
factor of 3 above the procedure blanks. The upper end of the working range is usually represented by the
EC 70 value (70 % of maximal effective concentration), but lower if the CV is higher than 15 % in this range.
The working range shall be established during validation. Cut-off values (see point 8.3) shall be well within
the working range.
— Standard solutions and sample extracts shall be tested at least in duplicate. When using duplicates, a standard
solution or a control extract tested in 4 to 6 wells divided over the plate shall produce a response or
concentration (only possible in the working range) based on a CV < 15 %.
8.1.2. C a l i b r a t i o n
8.1.2.1. C a l i b r a t i o n w i t h s t a n d a r d c u r v e
— Levels in samples shall be estimated by comparison of the test response with a calibration curve of TCDD (or
PCB 126 or a PCDD/PCDF/dioxin-like PCB standard mixture) to calculate the BEQ level in the extract and
subsequently in the sample.

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Union L
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— 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 R 2 value is of little or no value in estimating the goodness of fit in nonlinear
regression. A better fit shall be achieved by minimising the difference between calculated and observed levels
in the working range of the curve, for example by minimising the sum of squared residuals.
— The estimated level in the sample extract shall be subsequently corrected for the BEQ level calculated for a
matrix/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 level of interest). To perform a recovery correction, the apparent recovery shall be within the
required range (see point 8.1.4). Reference samples used for recovery correction shall comply with the
requirements laid down in point 8.2.
8.1.2.2. C a l i b r a t i o n w i t h r e f e r e n c e s a m p l e s
Alternatively, a calibration curve prepared from at least four reference samples (see point 8.2.4: one matrix blank,
plus three reference samples at 0,5 ×, 1,0 × and 2,0 × the level of interest) around the level of interest may be
used, eliminating the need to correct for blank and recovery. In this case, the test response corresponding to 2/3
of the maximum level (see point 8.3) may be calculated directly from these samples and used as cut-off value.
For checking of samples exceeding the action thresholds, an appropriate percentage of these action thresholds
would suit as cut-off value.
8.1.3. S e p a r a t e d e t e r m i n a t i o n o f P C D D / P C D F s a n d d i o x i n - l i k e P C B s
Extracts may be split into fractions containing PCDD/PCDFs and dioxin-like PCBs, allowing a separate indication
of PCDD/PCDFs and dioxin-like PCB TEQ levels (in BEQ). A PCB 126 standard calibration curve shall prefer
entially be used to evaluate results for the fraction containing dioxin-like PCBs.
8.1.4. B i o a s s a y a p p a r e n t r e c o v e r i e s
The “bioassay apparent recovery” shall be calculated from suitable reference samples with representative congener
patterns around the level of interest and expressed as percentage of the BEQ level in comparison to the TEQ
level. Depending on the type of assay and TEFs (**********) used, the differences between TEF and REP factors for
dioxin-like PCBs can cause low apparent recoveries for dioxin-like PCBs in comparison to PCDD/PCDFs.
Therefore, if a separate determination of PCDD/PCDFs and dioxin-like PCBs is performed, bioassay apparent
recoveries shall be: for dioxin-like PCBs 25 % to 60 %, for PCDD/PCDFs 50 % to 130 % (ranges apply for the
TCDD calibration curve). As the contribution of dioxin-like PCBs to the sum of PCDD/PCDFs and dioxin-like
PCBs can vary between different matrices and samples, bioassay apparent recoveries for the sum of PCDD/PCDFs
and dioxin-like PCBs reflect these ranges and shall be between 30 % and 130 %. Any implication of substantially
revised TEF values for the Union legislation for PCDD/PCDFs and dioxin-like PCBs requires the revision of these
ranges.
___________
(**********) Current requirements are based on the TEFs published in: M. Van den Berg et al., Toxicol Sci 93 (2),
223–241 (2006).
8.1.5. C o n t r o l o f r e c o v e r i e s f o r c l e a n - u p
The loss of compounds during the clean-up shall be checked during validation. A blank sample spiked with a
mixture of the different congeners shall be submitted to clean-up (at least n = 3) and the recovery and variability
checked by GC/HRMS analysis. The recovery shall be within 60 % to 120 % especially for congeners contributing
more than 10 % to the TEQ-level in various mixtures.
8.1.6. R e p o r t i n g l i m i t
When reporting BEQ levels, a reporting limit shall be determined from relevant matrix samples involving typical
congener patterns, but not from the calibration curve of the standards due to low precision in the lower range of
the curve. Effects from extraction and clean-up shall be taken into account. The reporting limit shall be set at
least by a factor of 3 above the procedure blanks.
8.2. Use
of
reference
samples
8.2.1. Reference
samples
shall
represent
sample
matrix,
congener
patterns
and
concentration
ranges
for
PCDD/PCDFs
and dioxin-like PCBs around the level of interest.
8.2.2. A matrix blank, and where it is not possible, a procedure blank, and a reference sample at the level of interest
shall be included in each test series. These samples shall be extracted and tested at the same time under identical
conditions. The reference sample shall show a clearly elevated response in comparison to the blank sample, thus
ensuring the suitability of the test. These samples may be used for blank and recovery corrections.

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L 91/18
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Official Journal of the European Union
29.3.2012
8.2.3. Reference
samples
chosen
to
perform
a
recovery
correction
shall
be
representative
for
the
test
samples,
meaning
that congener patterns may not lead to an underestimation of levels.
8.2.4. Extra reference samples at e.g. 0,5 × and 2 × the level of interest may be included to demonstrate the proper
performance of the test in the range of interest for the control of the level of interest. Combined, these samples
may be used for calculating the BEQ levels in test samples (see point 8.1.2.2).
8.3. Determination
of
cut-off
values
The relationship between bioanalytical results in BEQ and GC/HRMS results in TEQ shall be established, for
example by matrix-matched calibration experiments, involving reference samples spiked at 0, 0,5 ×, 1 × and 2 ×
maximum level, with 6 repetitions on each level (n = 24). Correction factors (blank and recovery) may be
estimated from this relationship but shall be checked in accordance with point 8.2.2.
Cut-off values shall be established for decisions over sample compliance with maximum levels or for the control
of action thresholds, if relevant, with the respective levels of interest set for either PCDD/PCDFs and dioxin-like
PCBs alone, or for the sum of PCDD/PCDFs and dioxin-like PCBs. They are represented by the lower end-point of
the distribution of bioanalytical results (corrected for blank and recovery) corresponding to the GC/HRMS
decision limit based on a 95 % level of confidence, implying a false-compliant rate < 5 %, and on a RSD R
< 25 %. The GC/HRMS decision limit is the maximum level, taking into account the measurement uncertainty.
The cut-off value (in BEQ) may be calculated in accordance with one of the approaches set out in points 8.3.1,
8.3.2 and 8.3.3 (see Figure 1):
8.3.1. Use
of
the lower band of the 95 % prediction interval at the GC/HRMS decision limit:

Cut-off value BEQ
2

DL – s y;x t α
Q
;fm–2 q=
1=n 1=m x i – x xx
with:
BEQ DL
BEQ corresponding to the GC/HRMS decision limit, being the maximum level including measurement
uncertainty
s y,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
x i
GC/HRMS sample concentration (in TEQ) of calibration point i
x
mean of the concentrations (in TEQ) of all calibration samples
m
Q
2

xx
8.3.2.
X x i – x square sum parameter, i = index for calibration point i
j1
Calculation from bioanalytical results (corrected for blank and recovery) of multiple analyses of samples (n ≥ 6)
contaminated at the GC/HRMS decision limit, as the lower end-point of the data distribution at the corresponding
mean BEQ value:
Cut-off value = BEQ DL – 1,64 × SD R
With:
SD R
standard deviation of bioassay results at BEQ DL , measured under within-laboratory reproducibility
conditions
8.3.3. Calculation
as
mean
value
of
bioanalytical
results
(in
BEQ,
corrected
for
blank
and
recovery)
from
multiple
analysis of samples (n ≥ 6) contaminated at 2/3 the level of interest, based on the observation that this level will
be around the cut-off value determined under point 8.3.1 or point 8.3.2:

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29.3.2012 Official
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European
Union L
91/19
Figure 1
Calculation of cut-off values based on a 95 % level of confidence implying a false-compliant rate < 5 %, and a
RSD R < 25 %: 1. from the lower band of the 95 % prediction interval at the HRGC/HRMS decision limit; 2. from
multiple analysis of samples (n ≥ 6) contaminated at the HRGC/HRMS decision limit as the lower end-point of
the data distribution (represented in the figure by a bell-shaped curve) at the corresponding mean BEQ value.
8.3.4. 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 levels of interest 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 level
of interest shall be preferred.
8.4. Performance
characteristics
8.4.1. Tests on the repeatability of bioanalytical methods shall be carried out to obtain information on the standard
deviation within and between test series. Repeatability shall be below 20 %, intra-laboratory reproducibility below
25 %. This shall be based on the calculated levels in BEQ after blank and recovery correction.
8.4.2. As part of the validation process, the test shall be shown to discriminate between a blank sample and a level at
the cut-off value, allowing the identification of samples above the corresponding cut-off value (see point 8.1.2).
8.4.3. Target
compounds,
possible
interferences
and
maximum
tolerable
blank
levels
shall
be
defined.
8.4.4. The percent standard deviation in the response or concentration calculated from the response (only possible in
working range) of a triplicate determination of a sample extract may not be above 15 %.
8.4.5. The uncorrected results of the reference sample(s) expressed in BEQ (blank and level of interest) shall be used for
evaluation of the performance of the bioanalytical method over a constant time period.
8.4.6. Quality
control
charts
for
procedure
blanks
and
each
type
of
reference
sample
shall
be
recorded
and
checked
to
make sure the analytical performance is 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 shall be controlled in a
manner to avoid false-compliant results when subtracted.

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Official Journal of the European Union
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8.4.7. The results from the GC/HRMS analyses of suspected samples and 2 to 10 % of the compliant samples
(minimum of 20 samples per matrix) shall be collected and used to evaluate the performance of the
screening method and the relationship between BEQ and TEQ. This database may be used for the re-evaluation
of cut-off values applicable to routine samples for the validated matrices.
8.4.8. Successful
method
performance
may
also
be
demonstrated
by
participation
in
ring
trials.
The
results
from
samples analysed in ring trials, covering a concentration range up to e.g. 2 × maximum level, may be
included in the evaluation of the false-compliant rate, if a laboratory is able to demonstrate its successful
performance. The samples shall cover most frequent congener patterns, representing various sources.
8.4.9. During incidents, the cut-off values may be re-evaluated, reflecting the specific matrix and congener patterns of
this single incident.
9. Reporting
of
the
results
9.1. Confirmatory
methods
9.1.1. In so far as the used analytical procedure makes it possible, the analytical results shall contain the levels of the
individual PCDD/PCDF and dioxin-like PCB congeners and be 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.
9.1.2. The
report
shall
include
the
method
used
for
extraction
of
PCDD/PCDFs,
dioxin-like
PCBs
and
lipids.
9.1.3. The recoveries of the individual internal standards shall be made available in case the recoveries are outside the
range referred to in point 7.2.5, in case the maximum level is exceeded and in other cases upon request.
9.1.4. As the uncertainty of measurement is to be taken into account when deciding about the compliance of a sample,
this parameter shall be made available. Thus, analytical results shall be 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 the case of a separate determination of PCDD/PCDFs and dioxin-like-
PCBs, the sum of the estimated expanded uncertainty of the separate analytical results of PCDD/PCDFs and
dioxin-like PCBs shall be used for the sum of PCDD/Fs and dioxin-like PCBs.
9.1.5. If the uncertainty of measurement is taken into account by applying CCα (as described in point 2.2), this
parameter shall be reported.
9.1.6. 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 Directive 2002/32/EC.
9.2. Bioanalytical
screening
methods
9.2.1. The
result
of
the
screening
shall
be
expressed
as
“compliant”
or
“suspected
to
be
non-compliant”
(“suspected”).
9.2.2. In
addition,
a
result
for
PCDD/PCDF
and/or
dioxin-like
PCBs
expressed
in
BEQ,
and
not
TEQ,
may
be
given.
9.2.3. If
measurement
uncertainty
on
the
calculated
BEQ
level
is
given,
for
example
as
standard
deviation,
it
shall
be
based on at least a triplicate analysis of the sample, including extraction, clean up and determination of the test
response.
9.2.4. Samples
with
a
response
below
the
reporting
limit
shall
be
expressed
as
“lower
than
the
reporting
limit”.
9.2.5. For each type of sample matrix, the report shall mention the level of interest on which the evaluation is based.
9.2.6. The report shall mention the type of the test applied, the basic test principle and the kind of calibration.
9.2.7. The
report
shall
include
the
method
used
for
extraction
of
PCDD/PCDFs,
dioxin-like
PCBs
and
lipids.
CHAPTER III
Sample preparation and requirements for methods of analysis used in official control of the levels of non-dioxin-like
PCBs (PCB # 28, 52, 101, 138, 153, 180)
1. Applicable
detection
methods
Gas chromatography/Electron capture detection (GC/ECD), GC/LRMS, GC/MS-MS, GC/HRMS or equivalent
methods.

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European
Union L
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2.
Identification and confirmation of analytes of interest
2.1.
Relative retention time in relation to internal standards or reference standards (acceptable deviation of
+/– 0,25 %).
2.2.
Gas chromatographic separation of all six indicator PCBs (PCB 28, PCB 52, PCB 101, PCB 138, PCB 153 and
PCB 180) 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 confirmed.
Note: Congeners often found to co-elute are for example PCB 28/31, PCB 52/69 and PCB 138/163/164. For GC/MS also
possible interferences from fragments of higher chlorinated congeners shall be considered.
2.3. Requirements
for
GC/MS
techniques
Monitoring of at least:
(a) two specific ions for HRMS;
(b) two specific ions of m/z > 200 or three specific ions of m/z > 100 for LRMS;
(c) 1 precursor and 2 product ions for MS-MS.
Maximum permitted tolerances for abundance ratios for selected mass fragments:
Relative deviation of abundance ratio of selected mass fragments from theoretical abundance or calibration
standard for target ion (most abundant ion monitored) and qualifier ion(s):
Relative intensity of qualifier ion(s)
GC-EI-MS
GC-CI-MS, GC-MS n
compared to target ion
(relative deviation)
(relative deviation)
> 50 % ±
10
% ±
20
%
> 20 % to 50 % ±
15
% ±
25
%
> 10 % to 20 % ±
20
% ±
30
%
≤ 10 % ±
50
%
(*) ±
50
%
(*)
(*) Sufficient number of mass fragments with relative intensity > 10 % available, therefore not recommendable to use qualifier
ion(s) with a relative intensity of less than 10 % compared to the target ion.
2.4. Requirements
for
GC/ECD
techniques
Results exceeding the tolerance shall be confirmed with two GC columns with stationary phases of different
polarity.
3. Demonstration
of
performance
of
method
The performance of the method shall be validated in the range of the level of interest (0,5 to 2 times the level of
interest) with an acceptable coefficient of variation for repeated analysis (see requirements for intermediate
precision in point 8).
4. Limit
of
quantification
The blank values shall not be higher than 30 % of the level of contamination corresponding to the maximum
level (***********).
___________
(***********) 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 subtracted.
5. Quality
control
Regular blank controls, analysis of spiked samples, quality control samples, participation in inter-laboratory
studies on relevant matrices.

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Official Journal of the European Union
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6. Control
of
recoveries
6.1. Suitable
internal
standards
with
physico-chemical
properties
comparable
to
analytes
of
interest
shall
be
used.
6.2. Addition
of
internal
standards:
Addition to products (before extraction and clean-up process).
6.3. Requirements
for
methods
using
all
six
isotope-labelled
indicator
PCB
congeners:
(a) results shall be corrected for recoveries of internal standards;
(b) recoveries of isotope-labelled internal standards shall be between 50 and 120 %;
(c) lower or higher recoveries for individual congeners with a contribution to the sum of the six indicator PCBs
below 10 % are acceptable.
6.4.
Requirements for methods using not all six isotope-labelled internal standards or other internal standards:
(a) recovery of internal standard(s) shall be controlled for every sample;
(b) recoveries of internal standard(s) shall be between 60 and 120 %;
(c) results shall be corrected for recoveries of internal standards.
6.5.
The recoveries of unlabelled congeners shall be checked by spiked samples or quality control samples with
concentrations in the range of the level of interest. Recoveries for these congeners shall be considered acceptable,
if they are between 70 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.
8.
Performance characteristics: criteria for the sum of the six indicator PCBs at the level of interest
Trueness –
30
to
+
30
%
Intermediate precision (RSD%)
≤ 20 %
Difference between upper- and lower-bound calcu
≤ 20 %
lation
9. Reporting
of
the
results
9.1.
In so far as the used analytical procedure makes it possible, the analytical results shall contain the levels of the
individual PCB congeners and be 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.
9.2. The
report
shall
include
the
method
used
for
extraction
of
PCBs
and
lipids.
9.3.
The recoveries of the individual internal standards shall be made available in case the recoveries are outside the
range referred to in point 6, in case the maximum level is exceeded and in other cases upon request.
9.4.
As the uncertainty of measurement 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 %.
9.5.
If the uncertainty of measurement is taken into account by applying CCα (as described in point 2.1 of Chapter I),
this parameter shall be reported.
9.6.
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 Directive 2002/32/EC.’

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