2002L0069 — EN — 10.05.2004 — 001.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COMMISSIONDIRECTIVE2002/69/EC
of►C1 30July2002 ◄
laying downthe sampling methods and the methods of analysis for the official control of dioxins
andthedeterminationofdioxin-likePCBsinfoodstuffs
(TextwithEEArelevance)
(OJ L 209, 6.8.2002, p. 5)
Amended by:
Official Journal
No page date
►M1Commission Directive 2004/44/EC of 13 April 2004 L 113 17 20.4.2004
Corrected by:
►C1Corrigendum, OJ L 252, 20.9.2002, p. 40 (2002/69/EC)
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COMMISSIONDIRECTIVE2002/69/EC
of►C1 30July2002 ◄
layingdownthesamplingmethodsandthemethodsofanalysisfor
the official control of dioxins and the determination of dioxin-like
PCBsinfoodstuffs
(TextwithEEArelevance)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Community,
Having regard to Council Directive 85/591/EEC of 20 December 1985
concerning the introduction of Community methods of sampling andanalysis for the monitoring of foodstuffs intended for human consump-tion (
1), and►C1in particular Article 1 and 4 thereof, ◄
Whereas:
(1)Commission Regulation (EC) No 466/2001 (2), as last amended
by Regulation (EC) No 563/2002 (3), and amended by Council
Regulation (EC) No 2375/2001 (4) establishes maximum limits
for dioxins and furans in certain foodstuffs.
(2)Council Directive 89/397/EEC of 14 June 1989 on the official
control of foodstuffs(5) lays down the general principles for the
performance of control of foodstuffs. Council Directive 93/99/EEC of 29 October 1993 on the subject of additional measuresconcerning the official control of foodstuffs(
6) introduces a
system of quality standards for laboratories entrusted by theMember States withth e official control of foodstuffs.
(3)Directive 85/591/EEC has fixed general criteria for methods ofsampling and analysis. However, in certain cases it is necessaryto lay down more specific criteria and or requirements with
which the method of analysis should comply in order to ensure
that laboratories use methods of analysis with comparable levelsof performance.
(4)The provisions for sampling and methods of analysis have beendrawn up on the basis of present knowledge and they may beadapted to take account of advances in scientific and technolo-gical knowledge.
(5)The provisions laid down in this Directive relate only to thesampling and analysis of dioxins and dioxin-like PCBs for theimplementation of Regulation (EC) No 466/2001 and do notaffect the sampling strategy, sampling levels and frequency as
specified in Annexes III and IV to Council Directive 96/23/EC
of 29 April 1996 on measures to monitor certain substances andresidues thereof in live animals and animal products andrepealing Directives 85/358/EEC and 86/469/EEC and Decisions89/187/EEC and 91/664/EEC (
7). They do not affect the targeting
criteria for sampling as laid down in Commission Decision 98/
179/EC of 23 February 1998 laying down detailed rules on offi-
cial sampling for the monitoring of certain substances andresidues thereof in live animals and animal products (
8).
(6)An active approachsh ould be pursued in order to obtain compre-hensive reliable data on the presence of dioxin-like PCBs infoodstuffs. Requirements should therefore be laid down as2002L0069 — EN — 10.05.2004 — 001.001 — 2
(1) OJ L 372, 31.12.1985, p. 50.
(2) OJ L 77, 16.3.2001, p. 1.
(3) OJ L 86, 3.4.2002, p. 5.
(4) OJ L 321, 6.12.2001, p. 1.
(5) OJ L 186, 30.6.1989, p. 23.
(6) OJ L 290, 24.11.1993, p. 14.
(7) OJ L 125, 23.5.1996, p. 10.
(8) OJ L 65, 5.3.1998, p. 31.
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regards the methods of analysis to be used for the determination
of dioxin-like PCBs in foodstuffs.
(7)A screening method of analysis with proven, widely acceptablevalidation and high throughput could be used to select thesamples withsignificant levels of dioxins. Th e levels of dioxinsin these samples need to be determined by a confirmatory
method of analysis. It is therefore appropriate to establish strict
requirements for the confirmatory methods of analysis andminimum requirements for the screening method.
(8)The measures provided for in this Directive are in accordance
with the opinion of the Standing Committee on the Food chain
and Animal Health,
HAS ADOPTED THIS DIRECTIVE:
Article 1
The Member States shall ensure that the sampling for the official
control of the levels of dioxins and furans and the determination ofthe levels of dioxin-like PCBs in foodstuffs is carried out in accordancewith the methods described in Annex I.
Article 2
The Member States shall ensure that sample preparation and methods
of analyses used for the official control of the levels of dioxins andfurans and the determination of the levels of dioxin-like PCBs in food-
stuffs comply withth e criteria described in Annex II.
Article 3
Member States shall bring into force the laws, regulations and admin-
istrative provisions necessary to comply withth is Directive by 28
February 2003 at the latest. They shall forthwith inform the Commis-sion thereof.
When Member States adopt those provisions, they shall contain a refer-
ence to this Directive or be accompanied by such a reference on theoccasion of their official publication. Member States shall determine
how such reference is to be made.
Article 4
This Directive shall enter into force on the 20th day following that of
its publication in the Official Journal of the European Communities .
Article 5
This Directive is addressed to the Member States.2002L0069 — EN — 10.05.2004 — 001.001 — 3
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ANNEX I
METHODS OF SAMPLING FOR OFFICIAL CONTROL OF THE
LEVELS OF DIOXINS(PCDD/PCDF) AND THE DETERMINATIONOF
DIOXIN-LIKEPCBsINCERTAINFOODSTUFFS
1.Purposeandscope
Samples intended for the official control of the levels of dioxins (PCDD/
PCDF) content, as well for the determination of the content of dioxin-like
PCBs(1) in foodstuffs shall be taken according to the methods described
below. Aggregate samples thus obtained shall be considered as representa-
tive of the lots or sublots from which they are taken. Compliance with
maximum levels laid down in Commission Regulation (EC) No 466/2001
setting maximum levels for certain contaminants in foodstuffs shall be
established on the basis of the levels determined in the laboratory samples.
2.Definitions
Lot: an identifiable quantity of food delivered at one time and determined
by the official to have common characteristics, such as origin, variety, type2002L0069 — EN — 10.05.2004 — 001.001 — 4
(1) Table WHO TEFs for human risk assessment based on the conclusions of the World
HealthOrganisation meeting in Stockh olm, Sweden, 15-18 June 1997 (Van den Berget al., (1998) Toxic Equivalency Factors (TEFs) for PCBs, PCDDs, PCDFs for Humans
and for Wildlife. Environmental HealthPerspectives, 106(12), 775).
Congener TEF value
Dibenzo-p-dioxins(PCDDs)
2,3,7,8-TCDD 1
1,2,3,7,8-PeCDD 1
1,2,3,4,7,8-HxCDD 0,1
1,2,3,6,7,8-HxCDD 0,11,2,3,7,8,9-HxCDD 0,1
1,2,3,4,6,7,8-HpCDD 0,01
OCDD 0,0001
Dibenzofurans(PCDFs)
2,3,7,8-TCDF 0,1
1,2,3,7,8-PeCDF 0,05
2,3,4,7,8-PeCDF 0,5
1,2,3,4,7,8-HxCDF 0,1
1,2,3,6,7,8-HxCDF 0,1
1,2,3,7,8,9-HxCDF 0,1
2,3,4,6,7,8-HxCDF 0,11,2,3,4,6,7,8-HpCDF 0,01
1,2,3,4,7,8,9-HpCDF 0,01
OCDF 0,0001
‘Dioxin-like’ PCBs Non-ortho PCBs + Mono-ortho PCBs
Non-orthoPCBs
PCB 77 0,0001
PCB 81 0,0001
PCB 126 0,1
PCB 169 0,01
Mono-orthoPCBs
PCB 105 0,0001
PCB 114 0,0005
PCB 118 0,0001
PCB 123 0,0001
PCB 156 0,0005
PCB 157 0,0005PCB 167 0,00001
PCB 189 0,0001
Abbreviations used: T = tetra; Pe = penta; Hx = hexa; Hp = hepta; O = octa;
CDD = chlorodibenzodioxin; CDF = chlorodibenzofuran; CB = chlorobiphenyl.
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of packing, packer, consignor or markings. In the case of fish and fishery
products, also the size of fish shall be comparable.
Sublot: designated part of a large lot in order to apply the sampling method
on that designated part. Each sublot must be physically separated and iden-
tifiable.
Incremental sample: a quantity of material taken from a single place in the
lot or sublot.
Aggregate sample: the combined total of all the incremental samples taken
from the lot or sublot.
Laboratory sample: a representative part/quantity of the aggregate sample
intended for the laboratory
3.General provisions
3.1.Personnel
Sampling shall be performed by an authorised qualified person as specified
by the Member States.
3.2.Material to be sampled
Eachlot, wh ichis to be examined, must be sampled separately.
3.3.Precautions to be taken
In the course of sampling and preparation of laboratory samples precau-
tions must be taken to avoid any changes, which would affect the content
of dioxins and dioxin-like PCBs, adversely affect the analytical determina-
tion or make the aggregate samples unrepresentative.
3.4.Incremental samples
As far as practical incremental samples shall be taken at various places
distributed throughout the lot or sublot. Departure from this procedure
must be recorded in the record provided for under 3.8.
3.5.Preparation of the aggregate sample
The aggregate sample is made up by uniting all incremental samples. It
shall be at least 1 kg unless not practical, e.g. when a single package has
been sampled.
3.6.Subdivision of aggregate sample in laboratory samples for enforcement,
defence and referee purposes
The laboratory samples for enforcement, trade (defence) and referee
purposes shall be taken from the homogenised aggregate sample unless
this conflicts with Member States' regulations on sampling. The size of
the laboratory samples for enforcement shall be sufficient to allow at least
for duplicate analyses.
3.7.Packaging and transmission of aggregate and laboratory samples
Eachaggregate and laboratory sample sh all be placed in a clean, inert
container offering adequate protection from contamination, from loss of
analytes by adsorption to the internal wall of the container and against
damage in transit. All necessary precautions shall be taken to avoid change
of composition of the aggregate and laboratory samples, which might arise
during transportation or storage.
3.8.Sealing and labelling of aggregate and laboratory samples
Each sample taken for official use shall be sealed at the place of sampling
and identified following the Member States' regulations. A record must be
kept of eachsampling, permitting eachlot to be identified unambiguously
and giving the date and place of sampling together with any additional
information likely to be of assistance to the analyst.
4.Samplingplans
The sampling method applied shall ensure that the aggregate sample is
representative for the lot that is to be controlled.2002L0069 — EN — 10.05.2004 — 001.001 — 5
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Number of incremental samples
In the case of milk and oils, for which a homogeneous distribution of the
contaminants in question can be assumed within a given lot, it is sufficient
to take three incremental samples per lot which forms the aggregate
sample. Reference to the lot number shall be given. For other products,
the minimum number of incremental samples to be taken from the lot shall
be as given in Table 1.
The aggregate sample uniting all incremental samples shall be at least 1 kg
(see point 3.5). The incremental samples shall be of similar weight. The
weight of an incremental sample should be at least 100 grams. The weight
of the incremental sample is dependent on the size of the particles in the
lot. Departure from this procedure must be recorded in the record provided
for under 3.8. In accordance withth e provisions of Commission Decision
97/747/EC of 27 October 1997 fixing the levels and frequencies of
sampling provided for by Council Directive 96/23/EC for the monitoring
of certain substances and residues thereof in certain animal products (1),
the sample size for hen eggs is at least 12 eggs (for bulk lots as well for
lots consisting of individual packages, Tables 1 and 2).
TABLE 1
Minimumnumberofincrementalsamplestobetakenfromthelot
Weight of lot (in kg)Minimum number of incremental
samples to be taken
<5 0 3
50 to 500 5
> 500 10
If the lot consists of individual packages, then the number of packages,
which shall be taken to form the aggregate sample, is given in Table 2.
TABLE 2
Number of packages (incremental samples) which shall be taken to
formtheaggregate sampleifthelotconsistsofindividualpackages
Number of packages or units in the lotNumber of packages or units to be
taken
1 to 25 1 package or unit
26 to 100 about 5 %, at least 2 packages or
units
> 100 about 5 %, at maximum 10
packages or units
4.1.Specific provisions for the sampling of lots containing whole fishes
The number of incremental samples to be taken from the lot is defined in
Table 1. The aggregate sample uniting all incremental samples shall be at
least 1 kg (see point 3.5).
— In case the lot to be sampled contains small fish (individual fish
weighing < 1 kg), the whole fish is taken as incremental sample to
form the aggregate sample. In case the resulting aggregate sample
weighs more than 3 kg, the incremental samples can consist of the
middle part, weighing each at least 100 grams, of the fish forming the
aggregate sample. The whole part to which the maximum level is
applicable is used for homogenisation of the sample.
— In case the lot to be sampled contains larger fish (individual fish
weighing more than 1 kg), the incremental sample consists of the
middle part of the fish. Each incremental sample weighs at least 100
grams. In case the lot to be sampled consist of very large fish (e.g.2002L0069 — EN — 10.05.2004 — 001.001 — 6
(1) OJ L 303, 6.11.1997, p. 12.▼M1
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> 6 kg) and taking a piece of the middle part of the fish would result in
significant economic damage, taking three incremental samples of at
least 350 grams eachcan be considered sufficient, independently of
the size of the lot.
5.Complianceofthelotorsublotwiththespecification
The lot is accepted if the analytical result of a single analysis does not
exceed the respective maximum level as laid down in Regulation (EC) No
466/2001, taking into account the measurement uncertainty.
The lot is non-compliant with the maximum level as laid down in Regula-
tion (EC) No 466/2001, if the analytical result confirmed by duplicate
analysis and calculated as the mean of at least two separate determinations
exceeds the maximum level beyond reasonable doubt, taking into account
the measurement uncertainty.
The taking into account of the measurement uncertainty can be done
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 %,
— by establishing the decision limit (CC α)according to the provisions of
Commission Decision 2002/657/EC of 12 August 2002 implementing
Council Directive 96/23/EC concerning the performance of analytical
methods and the interpretation of results (1) (point 3.1.2.5 of the Annex
— the case of substances with established permitted levels).
The present interpretation rules apply for the analytical result obtained on
the sample for official control. In case of analysis for defence or referee
purposes, the national rules apply.2002L0069 — EN — 10.05.2004 — 001.001 — 7
(1) OJ L 221, 17.8.2002, p. 8. Decision as last amended by Decision 2004/25/EC (OJ L 6,
10.1.2004, p. 38).
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ANNEX II
SAMPLE PREPARATION ANDREQUIREMENTS FOR METHOD S OF
ANALYSIS USEDIN OFFICIAL CONTROL OF THE LEVELS OF
DIOXINS (PCDD/PCDF) AND THE DETERMINATION OF DIOXIN-
LIKEPCBsINCERTAINFOODSTUFFS
1.Objective andfieldofapplication
These requirements should be applied where foodstuffs are analysed for the
official control of the levels of dioxins (polychlorinated dibenzo-p-dioxins
(PCDD) and polychlorinated dibenzofurans (PCDF)) and the determination
of dioxin-like PCBs.
Monitoring for the presence of dioxins in foodstuffs can be performed by a
strategy involving a screening method in order to select those samples with
levels of dioxins and dioxin-like PCBs that are less than 30-40 % below or
exceed the level of interest. The concentration of dioxins in those samples
withsignificant levels needs to be determined/confirmed by a confirmatory
method.
Screening methods are methods that are used to detect the presence of
dioxins and dioxin-like PCBs at the level of interest. These methods have
a capacity for a high sample throughput and are used to sift large numbers
of samples for potential positives. They are specifically designed to avoid
false negatives.
Confirmatory methods are methods that provide full or complementary
information enabling the dioxins and dioxin-like PCBs to be identified
and quantified unequivocally at the level of interest.
2.Background
Because environmental and biological samples (including samples of food-
stuffs) in general contain complex mixtures of different dioxin congeners,
the concept of Toxic Equivalency Factors (TEFs) has been developed to
facilitate risk assessment. These TEFs have been established to express
concentrations of mixtures of 2,3,7,8-substituted PCDDs and PCDFs, and
more recently, some non-ortho and mono-ortho chlorine substituted PCBs
which possesses dioxin-like activity in toxic equivalents (TEQs) of
2,3,7,8-TCDD (see Annex I, footnote 1).
The concentrations of the individual substances in a given sample are
multiplied by their respective TEF and subsequently summed to give the
total concentration of dioxin-like compounds expressed as TEQs.
The concept of ‘upperbound’ requires using the limit of quantification for
the contribution of each non-quantified congener to the TEQ.
The concept of ‘lowerbound’ requires using zero for the contribution of
eachnon-quantified congener to th e TEQ.
The concept of ‘mediumbound’ requires using half of the limit of quantifi-
cation calculating the contribution of each non-quantified congener to the
TEQ.
For the purposes of this Directive only, the accepted specific limit of quan-
tification of an individual congener is the concentration of an analyte in the
extract of a sample which produces an instrumental response at two
different ions, to be monitored withan S/N (signal/noise) ratio of 3:1 for
the less sensitive signal and fulfilment of the basic requirements such as,
e.g., retention time, isotope ratio according to the determination procedure
as described in EPA method 1613 revision B.
3.Quality assurance requirements to be complied with for sample
preparation
— Measures must be taken to avoid cross-contamination at eachstage of
the sampling and analysis procedure.
— The samples must be stored and transported in glass, aluminium, poly-
propylene or polyethylene containers. Traces of paper dust must be
removed from the sample container. Glassware should be rinsed with
solvents previously controlled for the presence of dioxins.
— The sample storage and transportation has to be performed in a way
that maintains the integrity of the foodstuff sample.2002L0069 — EN — 10.05.2004 — 001.001 — 8
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— Insofar as relevant, finely grind and mix thoroughly each laboratory
sample using a process that has been demonstrated to achieve complete
homogenisation (e.g. ground to pass a 1 mm sieve); samples have to be
dried before grinding if moisture content is too high.
— Perform a blank analysis by carrying out the entire analytical procedure
omitting only the sample.
— Sample weight used for the extraction must be sufficient to fulfil the
requirements withrespect to sensitivity.
— There are many satisfactory specific sample preparation procedures,
which may be used for the products under consideration. The proce-
dures have to be validated according to internationally accepted
guidelines.
4.Requirementsforlaboratories
— Laboratories shall demonstrate the performance of a method in the
range of the level of interest, e.g. 0,5 ×, 1 × and 2 × the level of interest
withan acceptable coefficient of variation for repeated analysis. For
details of acceptance criteria, see point 5.
— Limit of quantification for a confirmatory method should be in the
range of about one fifth of the level of interest, to make sure that accep-
table coefficients of variations are met in the range of the level of
interest.
— Regular blank controls and spiking experiments or analysis of control
samples (preferably, if available, certified reference material) should
be performed as internal quality control measures.
— Successful participation in interlaboratory studies that assess laboratory
proficiency is the best way to prove the competence in specific
analyses. However successful participation in interlaboratory studies
for, e.g. soil or sewage samples, does not necessarily prove the compe-
tence also in the field of food or feedingstuff samples, which present
lower contamination levels. Therefore, the continuous participation in
interlaboratory studies for the determination of dioxins and dioxin-like
PCBs in the relevant feed/food matrices is mandatory.
— In accordance withth e provisions of Directive 93/99/EEC, laboratories
should be accredited by a recognised body operating in accordance with
ISO Guide 58 to ensure that they are applying analytical quality assur-
ance. Laboratories should be accredited following the ISO/IEC/
17025:1999 standard.
5.Requirementstobemetbyanalyticalprocedurefordioxinsanddioxin-
likePCBs
Basic requirements for acceptance of analytical procedures:
—High sensitivity and low limits of detection . For PCDDs and PCDFs,
detectable quantities have to be in the picogram TEQ (10-12g) range
because of extreme toxicity of some of these compounds. PCBs are
known to occur at higher levels than the PCDDs and PCDFs. For most
PCB congeners sensitivity in the nanogram (10-9g) range is already
sufficient. However, for the measurement of the more toxic dioxin-like
PCB congeners (in particular non-ortho substituted congeners), the
same sensitivity must be reached as for the PCDDs and PCDFs.
—High selectivity (specificity) . A distinction is required for PCDDs,
PCDFs and dioxin-like PCBs from a multitude of other, coextracted
and possibly interfering compounds present at concentrations up to
several orders of magnitude higher than those of the analytes of
interest. For gas chromatography/mass spectrometry (GC/MS) methods
a differentiation among various congeners is necessary, suchas between
toxic (e.g. the seventeen 2,3,7,8-substituted PCDDs and PCDFs and
dioxin-like PCBs) and other congeners. Bioassays should be able to
determine TEQ values selectively as the sum of PCDDs, PCDFs and
dioxin-like PCBs.
—High accuracy (trueness and precision) . The determination should
provide a valid estimate of the true concentration in a sample. 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 measurement) is necessary to avoid the rejection of a sample
analysis result on the basis of poor reliability of the estimate of TEQ.
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 (precision is
usually calculated as a standard deviation including repeatability and
reproducibility, and indicates the closeness of agreement between the
results obtained by applying the experimental procedure several times
under prescribed conditions).2002L0069 — EN — 10.05.2004 — 001.001 — 9
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Screening methods can comprise bioassays and GC/MS methods;
confirmatory methods are high-resolution gas chromatography/high
resolution mass spectrometry (HRGC/HRMS) methods. Following
criteria have to be complied with on total TEQ value:
Screening methods Confirmatory methods
False negative rate < 1 %
Trueness – 20 % to + 20 %
C V <3 0% <1 5%
6.Specific requirements for GC/MS methods to be complied with for
screeningorconfirmatorypurposes
— Addition of13C-labelled 2,3,7,8-chlorine substituted internal PCDD/F
standards (and of13C-labelled internal dioxin-like PCB standards, if
dioxin-like PCBs have to be determined) must be carried out at the
very beginning or start 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/F
(and at least one congener for each of the homologous groups for
dioxin-like PCBs, if dioxin-like PCBs have to be determined) must be
added (alternatively, at least one congener for eachmass spectrometric
selected ion recording function used for monitoring PCDD/F and
dioxin-like PCBs). There is a clear preference, certainly in case of
confirmatory methods, of using all 1713C-labelled 2,3,7,8-substituted
internal PCDD/F standards and all 1213C-labelled internal dioxin-like
PCB standard (if dioxin-like PCBs have to be determined).
Relative response factors should also be determined for those congeners
for which no13C-labelled analogue is added by using appropriate cali-
bration solutions.
— For foodstuffs of plant origin and foodstuffs of animal origin containing
less than 10 % fat, the addition of the internal standards is mandatory
prior to extraction. For foodstuffs of animal origin containing more than
10 % fat, the internal standards can be added either before extraction or
after fat extraction. An appropriate validation of the extraction effi-
ciency should be carried out, depending on the stage at which internal
standards are introduced and on whether results are reported on product
or fat basis.
— Prior to GC/MS analysis, 1 or 2 recovery (surrogate) standard(s) must
be added.
— Control of recovery is necessary. For confirmatory methods, the recov-
eries of the individual internal standards should be in the range of 60 %
to 120 %. Lower or higher recoveries for individual congeners, in parti-
cular for some hepta- and octa- chlorinated dibenzodioxins and
dibenzofurans, are acceptable on the condition that their contribution
to the TEQ value does not exceed 10 % of the total TEQ value (based
on PCDD/F only). For screening methods, the recoveries should be in
the range of 30 % to 140 %.
— Separation of dioxins from interfering chlorinated compounds such as
PCBs and chlorinated diphenyl ethers should be carried out by suitable
chromatographic techniques (preferably with a florisil, alumina and/or
carbon column).
— Gaschromatographic separation of isomers should be sufficient (< 25 %
peak to peak between 1,2,3,4,7,8-HxCDF and 1,2,3,6,7,8-HxCDF).
— Determination should be performed according to EPA Method 1613
revision B: Tetra- through octa-chlorinated dioxins and furans by
isotope dilution HRGC/HRMS or another with equivalent performance
criteria.
— The difference between upperbound level and lower bound level should
not exceed 20 % for foodstuffs witha dioxin contamination of about 1
pg WHO-TEQ/g fat (based on PCDD/PCDF only). For foodstuffs witha low fat content, the same requirements for contamination levels of
about 1 pg WHO-TEQ/g product have to be applied. For lower contam-
ination levels, for example 0,50 pg WHO-TEQ/g product, the difference
between upperbound and lowerbound level may be in the range of 25 to
40 %.2002L0069 — EN — 10.05.2004 — 001.001 — 10
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7.Screeningmethodsofanalysis
7.1.Introduction
Different analytical approaches can be performed using a screening
method: a pure screening approach and a quantitative approach.
Screening approachThe response of samples is compared to that of a reference sample at the
level of interest. Samples with a response less than the reference are
declared negative, those with a higher response are suspected positives.
Requirements:
— A blank and a reference sample(s) have to be included in each test
series, which is extracted and tested at the same time under identical
conditions. The reference sample must show a clearly elevated response
in comparison to a blank.
— Extra reference samples 0,5 × and 2 × the level of interest should be
included to demonstrate the proper performance of the test in the range
of interest for the control of the level of interest.
— When testing other matrices, the suitability of the reference sample(s)
has to be demonstrated, preferentially by including samples shown by
HRGC/HRMS to contain a TEQ level around that of the reference
sample or else a blank spiked at this level.
— Since no internal standards can be used in bioassays, tests on repeat-
ability are very important to obtain information on the standard
deviation within one test series. The coefficient of variation should be
below 30 %.
— For bioassays, the target compounds, possible interferences and
maximum tolerable blank levels should be defined.
Quantitative approachThe quantitative approach requires standard dilution series, duplicate or
triplicate clean up and measuring as well as blank and recovery controls.
The result may be expressed as TEQ, thereby assuming that the compounds
responsible for the signal correspond to the TEQ principle. This can be
performed by using TCDD (or a dioxin/furan standard mixture) to produce
a calibration curve to calculate the TEQ level in the extract and thus in the
sample. This is subsequently corrected for the TEQ level calculated for a
blank sample (to account for impurities from solvents and chemicals
used), and a recovery (calculated from the TEQ level in a quality control
sample around the level of interest). It is essential to note that part of the
apparent recovery loss may be due to matrix effects and/or differences
between the TEF values in the bioassays and the official TEF values set
by WHO.
7.2.Requirements for methods of analysis used for screening
— GC/MS methods of analysis and bioassays may be used for screening.
For GC/MS methods the requirements as laid down in point 6 are to be
used. For cell based bioassays specific requirements are laid down in
point 7.3 and for kit-based bioassays in point 7.4.
— Information on the number of false-positive and false-negative results
of a large set of samples below and above the maximum level or action
level is necessary, in comparison to the TEQ content as determined by
a confirmatory method of analysis. Actual false negative rates should be
under 1 %. The rate of false positive samples should be low enough to
make the use of a screening tool advantageous.
— Positive results have always to be confirmed by a confirmatory method
of analysis (HRGC/HRMS). In addition, samples from a wide TEQ-
range should be confirmed by HRGC/HRMS (approximately 2 % to
10 % of the negative samples). Information on correspondence between
bioassay and HRGC/HRMS results should be made available.
7.3.Specific requirements for cell-based bioassays
— When performing a bioassay, every test run requires a series of refer-
ence concentrations of TCDD or a dioxin/furan mixture (full dose-response curve witha R
2> 0,95). However, for screening purposes an
expanded low level curve for analysing low level samples could be
used.
— A TCDD reference concentration (about 3 × limit of quantification) on
a quality control sheet should be used for the outcome of the bioassay
over a constant time period. An alternative could be the relative
response of a reference sample in comparison to the TCDD calibration
line since the response of the cells may depend on many factors.2002L0069 — EN — 10.05.2004 — 001.001 — 11
▼B
— Quality control (QC) charts for each type of reference material should
be recorded and checked to make sure the outcome is in accordance
withth e stated guidelines.
— In particular for quantitative calculations, the induction of the sample
dilution used must be within the linear portion of the response curve.
Samples above the linear portion of the response curve must be diluted
and re-tested. Therefore, at least three or more dilutions at one time are
recommended to be tested.
— The percent standard deviation should not be above 15 % in a triplicate
determination for eachsample dilution and not above 30 % between
three independent experiments.
— The limit of detection may be set as 3 × the standard deviation of the
solvent blank or of the background response. Another approach is to
apply a response that is above the background (induction factor 5 ×
the solvent blank) calculated from the calibration curve of the day.
The limit of quantification may be set as 5 × to 6 × the standard devia-
tion of the solvent blank or of the background response or to apply a
response that is above the background (induction factor 10 × the solvent
blank) calculated from the calibration curve of the day.
7.4.Specific requirements for kit-based bioassays (1)
— Manufacturer's instructions for sample preparation and analyses have to
be followed.
— Test kits should not be used after the expiration date.— Materials or components designed for use with other kits should not be
used.
— Test kits should be kept within the specified range of storage tempera-
ture and used at the specified operating temperature.
— The limit of detection for immunoassays is determined as 3 × the stan-
dard deviation, based on 10 replicate analysis of the blank, to be
divided by the slope value of the linear regression equation.
— Reference standards should be used for tests at the laboratory to make
sure that the responsiveness to the standard is within an acceptable
range.
8.Reportingoftheresult
Insofar as the used analytical procedure makes it possible, the analytical
results should contain the levels of the individual PCDD/F and PCB conge-
ners and be reported as lowerbound, upperbound and mediumbound in
order to include a maximum of information in the reporting of the results
and thereby enabling the interpretation of the results according to specific
requirements.
The report should also include the lipid content of the sample as well the
method used for lipid extraction.
The recoveries of the individual internal standards must be made available
in case the recoveries are outside the range mentioned in point 6, in case
the maximum level is exceeded and in other cases upon request.2002L0069 — EN — 10.05.2004 — 001.001 — 12
(1) No evidence have yet been submitted of commercially available kit-based bioassays
having sufficient sensitivity and reliability to be used for screening for the presence of
dioxins at the required levels in samples of foodstuffs and feedingstuffs.