2002L0069—EN—26.08.2002—000.001—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)
(OJL209,6.8.2002,p.5)
Correctedby:
►C1Corrigendum,OJL252,20.9.2002,p.40(2002/69/EC)
▼B
COMMISSIONDIRECTIVE2002/69/EC
of►C1 30July2002 ◄
layingdownthesamplingmethodsandthemethodsofanalysisfor
the official control of dioxins and the determination of dioxin-like
PCBsinfoodstuffs
(TextwithEEArelevance)
THECOMMISSIONOFTHEEUROPEANCOMMUNITIES,
HavingregardtotheTreatyestablishingtheEuropeanCommunity,
HavingregardtoCouncilDirective85/591/EECof20December1985
concerning the introduction of Community methods of sampling andanalysisforthemonitoringoffoodstuffsintendedforhumanconsump-tion(
1),and►C1inparticularArticle1and4thereof, ◄
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
fordioxinsandfuransincertainfoodstuffs.
(2)Council Directive 89/397/EEC of 14 June 1989 on the official
control offoodstuffs(5)laysdownthe general principles forthe
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
systemof quality standards for laboratories entrusted by theMemberStateswiththeofficialcontroloffoodstuffs.
(3)Directive 85/591/EEC has fixed general criteria for methods ofsamplingandanalysis.However,incertain casesitisnecessaryto lay down more specific criteria and or requirements with
which the method of analysis should comply in order to ensure
thatlaboratoriesusemethodsofanalysiswithcomparablelevelsofperformance.
(4)Theprovisionsforsampling andmethodsofanalysishavebeendrawn up on the basis of present knowledge and they may beadapted to take account of advances in scientific and technolo-gicalknowledge.
(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
of29April1996onmeasurestomonitorcertainsubstancesandresidues thereof in live animals and animal products andrepealingDirectives85/358/EECand86/469/EECandDecisions89/187/EECand91/664/EEC(
7).Theydonotaffectthetargeting
criteria for sampling as laid down in Commission Decision 98/
179/ECof23February1998layingdowndetailedrulesonoffi-
cial sampling for the monitoring of certain substances andresiduesthereofinliveanimalsandanimalproducts(
8).
(6)Anactiveapproachshouldbepursuedinordertoobtaincompre-hensive reliable data on the presence of dioxin-like PCBs infoodstuffs. Requirements should therefore be laid down as2002L0069—EN—26.08.2002—000.001—2
(1) OJL372,31.12.1985,p.50.
(2) OJL77,16.3.2001,p.1.
(3) OJL86,3.4.2002,p.5.
(4) OJL321,6.12.2001,p.1.
(5) OJL186,30.6.1989,p.23.
(6) OJL290,24.11.1993,p.14.
(7) OJL125,23.5.1996,p.10.
(8) OJL65,5.3.1998,p.31.
▼B
regardsthemethodsofanalysistobeusedforthedetermination
ofdioxin-likePCBsinfoodstuffs.
(7)A screening method of analysis with proven, widely acceptablevalidation and high throughput could be used to select thesampleswithsignificantlevelsofdioxins.Thelevelsofdioxinsin 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 andminimumrequirementsforthescreeningmethod.
(8)The measures provided for in this Directive are in accordance
with the opinion of the Standing Committee on the Food chain
andAnimalHealth,
HASADOPTEDTHISDIRECTIVE:
Article 1
The Member States shall ensure that the sampling for the official
control of the levels of dioxins and furans and the determination ofthelevelsofdioxin-likePCBsinfoodstuffsiscarriedoutinaccordancewiththemethodsdescribedinAnnexI.
Article 2
The Member States shall ensure that sample preparation and methods
of analyses used for the official control of the levels of dioxins andfuransandthedeterminationofthelevelsofdioxin-likePCBsinfood-
stuffscomplywiththecriteriadescribedinAnnexII.
Article 3
Member States shall bringinto force the laws,regulations andadmin-
istrative provisions necessary to comply with this Directive by 28
February 2003 at the latest. They shall forthwith inform the Commis-sionthereof.
WhenMemberStatesadoptthoseprovisions,theyshallcontainarefer-
ence to this Directive or be accompanied by such a reference on theoccasion of their official publication. Member States shall determine
howsuchreferenceistobemade.
Article 4
ThisDirective shall enterinto forceonthe 20thdayfollowingthat of
itspublicationinthe Official Journal of the European Communities .
Article 5
ThisDirectiveisaddressedtotheMemberStates.2002L0069—EN—26.08.2002—000.001—3
▼B
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,aswellforthedetermination ofthecontent ofdioxin-like
PCBs(1) in foodstuffs shall be taken according to the methods described
below.Aggregatesamplesthusobtainedshallbeconsideredasrepresenta-
tive of the lots or sublots fromwhich they are taken. Com pliance with
maximum levels laid down in Commission Regulation (EC) No 466/2001
setting maximum levels for certain contaminants in foodstuffs shall be
establishedonthebasisofthelevelsdeterminedinthelaboratorysamples.
2.Definitions
Lot:anidentifiable quantity offooddelivered atonetimeanddetermined
bytheofficialtohavecommoncharacteristics,suchasorigin,variety,type2002L0069—EN—26.08.2002—000.001—4
(1) Table WHO TEFs for human risk assessment based on the conclusions of the World
Health Organisation meeting in Stockholm, Sweden, 15-18 June 1997 (Van den Berget al.,(1998)ToxicEquivalencyFactors(TEFs)forPCBs,PCDDs,PCDFsforHumans
andforWildlife.EnvironmentalHealthPerspectives,106(12),775).
Congener TEFvalue
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
PCB77 0,0001
PCB81 0,0001
PCB126 0,1
PCB169 0,01
Mono-orthoPCBs
PCB105 0,0001
PCB114 0,0005
PCB118 0,0001
PCB123 0,0001
PCB156 0,0005
PCB157 0,0005PCB167 0,00001
PCB189 0,0001
Abbreviations used: T = tetra; Pe = penta; Hx = hexa; Hp = hepta; O = octa;
CDD=chlorodibenzodioxin;CDF=chlorodibenzofuran;CB=chlorobiphenyl.
▼B
of packing, packer, consignor or markings. In the case of fish and fishery
products,alsothesizeoffishshallbecomparable.
Sublot:designatedpartofalargelotinordertoapplythesamplingmethod
onthatdesignatedpart.Eachsublotmustbephysicallyseparatedandiden-
tifiable.
Incrementalsample:aquantityofmaterialtakenfromasingleplaceinthe
lotorsublot.
Aggregatesample:thecombinedtotalofalltheincrementalsamplestaken
fromthelotorsublot.
Laboratory sample: a representative part/quantity of the aggregate sample
intendedforthelaboratory
3.General provisions
3.1.Personnel
Samplingshallbeperformedbyanauthorisedqualifiedpersonasspecified
bytheMemberStates.
3.2.Material to be sampled
Eachlot,whichistobeexamined,mustbesampledseparately.
3.3.Precautions to be taken
In the course of sampling and preparation of laboratory samples precau-
tionsmustbe taken toavoid anychanges, whichwouldaffect the content
ofdioxinsanddioxin-likePCBs,adverselyaffecttheanalyticaldetermina-
tionormaketheaggregatesamplesunrepresentative.
3.4.Incremental samples
As far as practical incremental samples shall be taken at various places
distributed throughout the lot or sublot. Departure fromthis procedure
mustberecordedintherecordprovidedforunder3.8.
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
beensampled.
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
thelaboratorysamplesforenforcementshallbesufficienttoallowatleast
forduplicateanalyses.
3.7.Packaging and transmission of aggregate and laboratory samples
Each aggregate and laboratory sample shall be placed in a clean, inert
container offering adequate protection fromcontam ination, fromloss of
analytes by adsorption to the internal wall of the container and against
damageintransit.Allnecessaryprecautionsshallbetakentoavoidchange
ofcompositionoftheaggregateandlaboratorysamples,whichmightarise
duringtransportationorstorage.
3.8.Sealing and labelling of aggregate and laboratory samples
Eachsampletakenforofficialuseshallbesealedattheplaceofsampling
andidentified followingtheMemberStates'regulations.Arecordmustbe
kept ofeachsampling, permitting each lottobeidentified unambiguously
and giving the date and place of sampling together with any additional
informationlikelytobeofassistancetotheanalyst.
4.Samplingplans
The sampling method applied shall ensure that the aggregate sample is
representativeforthelotthatistobecontrolled.2002L0069—EN—26.08.2002—000.001—5
▼B
Number of incremental samples
In the case of milk and oils, for which a homogeneous distribution of the
contaminantsinquestioncanbeassumedwithinagivenlot,itissufficient
to take three incremental samples per lot which forms the aggregate
sample. Reference to the lot number shall be given. For other products,
theminimumnumberofincrementalsamplestobetakenfromthelotshall
beasgiveninTable1.
Theaggregatesampleunitingallincrementalsamplesshallbeatleast1kg
(see point 3.5). The incremental samples shall be of similar weight. The
weightofanincrementalsampleshouldbeatleast100grams.Theweight
of the incremental sample is dependent on the size of the particles in the
lot.Departurefromthisproceduremustberecordedintherecordprovided
for under 3.8. In accordance with the 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
lotsconsistingofindividualpackages,Tables1and2).
TABLE1
Minimumnumberofincrementalsamplestobetakenfromthelot
Weightoflot(inkg)Minimumnumberofincremental
samplestobetaken
<50 3
50to500 5
>500 10
If the lot consists of individual packages, then the number of packages,
whichshallbetakentoformtheaggregatesample,isgiveninTable2.
TABLE2
Number of packages (incremental samples) which shall be taken to
formtheaggregate sampleifthelotconsistsofindividualpackages
NumberofpackagesorunitsinthelotNumberofpackagesorunitstobe
taken
1to25 1packageorunit
26to100 about5%,atleast2packagesor
units
>100 about5%,atmaximum10
packagesorunits
5.Complianceofthelotorsublotwiththespecification
Thecontrollaboratoryshallanalysethelaboratorysampleforenforcement
induplicate analysisincasetheobtainedresultofthefirstanalysisisless
than 20%beloworabove the maximum level, andcalculate the mean of
theresults.Thelotisacceptediftheresultofthefirstanalysisismorethan
20%belowthemaximum level or,whereduplicate analysisisnecessary,
if the mean conforms to the respective maximum level as laid down in
Regulation(EC)No466/2001.2002L0069—EN—26.08.2002—000.001—6
(1) OJL303,6.11.1997,p.12.
▼B
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
Theserequirementsshouldbeappliedwherefoodstuffsareanalysedforthe
official control of the levels of dioxins (polychlorinated dibenzo-p-dioxins
(PCDD)andpolychlorinateddibenzofurans(PCDF))andthedetermination
ofdioxin-likePCBs.
Monitoringforthepresenceofdioxinsinfoodstuffscanbeperformedbya
strategyinvolvingascreeningmethodinordertoselectthosesampleswith
levelsofdioxinsanddioxin-likePCBsthatarelessthan30-40%belowor
exceedthelevelofinterest.Theconcentration ofdioxinsinthosesamples
withsignificantlevelsneedstobedetermined/confirmedbyaconfirmatory
method.
Screening methods are methods that are used to detect the presence of
dioxins and dioxin-like PCBsat the level of interest. These methods have
acapacityforahighsamplethroughputandareusedtosiftlargenumbers
of samples for potential positives. They are specifically designed to avoid
falsenegatives.
Confirmatory methods are methods that provide full or complementary
information enabling the dioxins and dioxin-like PCBs to be identified
andquantifiedunequivocallyatthelevelofinterest.
2.Background
Becauseenvironmentalandbiologicalsamples(includingsamplesoffood-
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(seeAnnexI,footnote1).
The concentrations of the individual substances in a given sample are
multiplied by their respective TEF and subsequently summed to give the
totalconcentrationofdioxin-likecompoundsexpressedasTEQs.
The concept of ‘upperbound’ requires using the limit of quantification for
thecontributionofeachnon-quantifiedcongenertotheTEQ.
The concept of ‘lowerbound’ requires using zero for the contribution of
eachnon-quantifiedcongenertotheTEQ.
Theconceptof‘mediumbound’requiresusinghalfofthelimitofquantifi-
cation calculating the contribution of each non-quantified congener to the
TEQ.
3.Quality assurance requirements to be complied with for sample
preparation
— Measures must be taken to avoid cross-contamination at each stage of
thesamplingandanalysisprocedure.
— Thesamplesmustbestoredandtransportedinglass,aluminium,poly-
propylene or polyethylene containers. Traces of paper dust must be
removed from the sample container. Glassware should be rinsed with
solventspreviouslycontrolledforthepresenceofdioxins.
— The sample storage and transportation has to be performed in a way
thatmaintainstheintegrityofthefoodstuffsample.
— Insofar as relevant, finely grind and mix thoroughly each laboratory
sampleusingaprocessthathasbeendemonstratedtoachievecomplete
homogenisation(e.g.groundtopassa1mmsieve);sampleshavetobe
driedbeforegrindingifmoisturecontentistoohigh.
— Performablankanalysisbycarryingouttheentireanalyticalprocedure
omittingonlythesample.
— Sample weight used for the extraction must be sufficient to fulfil the
requirementswithrespecttosensitivity.
— There are many satisfactory specific sample preparation procedures,
which may be used for the products under consideration. The proce-2002L0069—EN—26.08.2002—000.001—7
▼B
dureshavetobevalidatedaccordingtointernationally accepted guide-
lines.
4.Requirementsforlaboratories
— Laboratories shall demonstrate the performance of a method in the
rangeofthelevelofinterest,e.g.0,5×,1×and2×thelevelofinterest
with an acceptable coefficient of variation for repeated analysis. For
detailsofacceptancecriteria,seepoint5.
— Limit of quantification for a confirmatory method should be in the
rangeofaboutonefifthofthelevelofinterest,tomakesurethataccep-
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
beperformedasinternalqualitycontrolmeasures.
— Successfulparticipationininterlaboratorystudiesthatassesslaboratory
proficiency is the best way to prove the competence in specific
analyses. However successful participation in interlaboratory studies
for,e.g.soilorsewagesamples,doesnotnecessarilyprovethecompe-
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
PCBsintherelevantfeed/foodmatricesismandatory.
— InaccordancewiththeprovisionsofDirective93/99/EEC,laboratories
shouldbeaccreditedbyarecognisedbodyoperatinginaccordancewith
ISOGuide58toensurethattheyareapplyinganalytical qualityassur-
ance. Laboratories should be accredited following the ISO/IEC/
17025:1999standard.
5.Requirementstobemetbyanalyticalprocedurefordioxinsanddioxin-
likePCBs
Basicrequirementsforacceptanceofanalyticalprocedures:
—High sensitivity and low limits of detection . For PCDDs and PCDFs,
detectable quantities have to be in the picogramTEQ (10-12g) range
because of extreme toxicity of some of these compounds. PCBs are
knowntooccurathigherlevelsthanthePCDDsandPCDFs.Formost
PCB congeners sensitivity in the nanogram(10-9g) range is already
sufficient.However,forthemeasurementofthemoretoxicdioxin-like
PCB congeners (in particular non-ortho substituted congeners), the
samesensitivitymustbereachedasforthePCDDsandPCDFs.
—High selectivity (specificity) . A distinction is required for PCDDs,
PCDFs and dioxin-like PCBs froma m ultitude of other, coextracted
and possibly interfering compounds present at concentrations up to
several orders of magnitude higher than those of the analytes of
interest. Forgaschromatography/mass spectrometry (GC/MS)methods
adifferentiationamongvariouscongenersisnecessary,suchasbetween
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-likePCBs.
—High accuracy (trueness and precision) . The determination should
provide a valid estimate of the true concentration in a sample. High
accuracy(accuracyofthemeasurement:theclosenessoftheagreementbetweentheresultofameasurementwiththetrueorassignedvalueof
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
underprescribedconditions).
Screening methods can comprise bioassays and GC/MS methods;
confirmatory methods are high-resolution gas chromatography/high
resolution mass spectrometry (HRGC/HRMS) methods. Following
criteriahavetobecompliedwithontotalTEQvalue:
Screeningmethods Confirmatorymethods
Falsenegativerate <1%
Trueness –20%to+20%2002L0069—EN—26.08.2002—000.001—8
▼B
Screeningmethods Confirmatorymethods
CV <30% <15%
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
verybeginningorstartoftheanalyticalmethode.g.priortoextraction
inordertovalidate theanalytical procedure.Atleastonecongenerfor
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 PCBshave to be determined) must be
added(alternatively, atleastonecongenerforeachmassspectrometric
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
PCBstandard(ifdioxin-likePCBshavetobedetermined).
Relativeresponsefactorsshouldalsobedeterminedforthosecongeners
for which no13C-labelled analogue is added by using appropriate cali-
brationsolutions.
— Forfoodstuffsofplantoriginandfoodstuffsofanimalorigincontaining
less than 10 % fat, the addition of the internal standards is mandatory
priortoextraction.Forfoodstuffsofanimalorigincontainingmorethan
10%fat,theinternalstandardscanbeaddedeitherbeforeextractionor
after fat extraction. An appropriate validation of the extraction effi-
ciency shouldbe carried out, dependingon thestage at whichinternal
standardsareintroducedandonwhetherresultsarereportedonproduct
orfatbasis.
— Prior to GC/MS analysis, 1 or 2 recovery (surrogate) standard(s) must
beadded.
— Controlofrecoveryisnecessary.Forconfirmatorymethods,therecov-
eriesoftheindividualinternalstandardsshouldbeintherangeof60%
to120%.Lowerorhigherrecoveriesforindividualcongeners,inparti-
cular for some hepta- and octa- chlorinated dibenzodioxins and
dibenzofurans, are acceptable on the condition that their contribution
totheTEQvaluedoesnotexceed10%ofthetotalTEQvalue(based
on PCDD/Fonly). For screening methods, the recoveries should be in
therangeof30%to140%.
— Separation of dioxins frominterfering chlorinated com pounds such as
PCBsandchlorinateddiphenylethersshouldbecarriedoutbysuitable
chromatographic techniques (preferably with a florisil, alumina and/or
carboncolumn).
— Gaschromatographicseparationofisomersshouldbesufficient(<25%
peaktopeakbetween1,2,3,4,7,8-HxCDFand1,2,3,6,7,8-HxCDF).
— Determination should be performed according to EPA Method 1613
revision B: Tetra- through octa-chlorinated dioxins and furans by
isotope dilution HRGC/HRMSor another with equivalent performance
criteria.
— Thedifferencebetweenupperboundlevelandlowerboundlevelshould
notexceed20%forfoodstuffswithadioxincontaminationofabout1
pgWHO-TEQ/gfat(basedonPCDD/PCDFonly).Forfoodstuffswith
a low fat content, the same requirements for contamination levels of
about1pgWHO-TEQ/gproducthavetobeapplied.Forlowercontam-
inationlevels,forexample0,50pgWHO-TEQ/gproduct,thedifference
betweenupperboundandlowerboundlevelmaybeintherangeof25to
40%.
7.Screeningmethodsofanalysis
7.1.Introduction
Different analytical approaches can be performed using a screening
method:apurescreeningapproachandaquantitativeapproach.
Screeningapproach
The response of samples is compared to that of a reference sample at the
level of interest. Samples with a response less than the reference are2002L0069—EN—26.08.2002—000.001—9
▼B
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.Thereferencesamplemustshowaclearlyelevatedresponse
incomparisontoablank.
— Extra reference samples 0,5 × and 2 × the level of interest should be
includedtodemonstratetheproperperformanceofthetestintherange
ofinterestforthecontrolofthelevelofinterest.
— 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
sampleorelseablankspikedatthislevel.
— 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
below30%.
— For bioassays, the target compounds, possible interferences and
maximumtolerableblanklevelsshouldbedefined.
QuantitativeapproachThe quantitative approach requires standard dilution series, duplicate or
triplicate clean up and measuring as well as blank and recovery controls.
TheresultmaybeexpressedasTEQ,therebyassumingthatthecompounds
responsible for the signal correspond to the TEQ principle. This can be
performedbyusingTCDD(oradioxin/furanstandardmixture)toproduce
acalibrationcurvetocalculatetheTEQlevelintheextractandthusinthe
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 fromthe TEQ level in a quality control
sample around the level of interest). It isessential 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
byWHO.
7.2.Requirements for methods of analysis used for screening
— GC/MSmethods ofanalysis and bioassaysmay be usedfor screening.
ForGC/MSmethodstherequirementsaslaiddowninpoint6aretobe
used. For cell based bioassays specific requirements are laid down in
point7.3andforkit-basedbioassaysinpoint7.4.
— Information on the number of false-positive and false-negative results
ofalargesetofsamplesbelowandabovethemaximumleveloraction
levelisnecessary,incomparisontotheTEQcontentasdeterminedby
aconfirmatorymethodofanalysis.Actualfalsenegativeratesshouldbe
under1%.Therateoffalsepositivesamplesshouldbelowenoughto
maketheuseofascreeningtooladvantageous.
— Positiveresultshavealwaystobeconfirmedbyaconfirmatorymethod
of analysis (HRGC/HRMS). In addition, samples from a wide TEQ-
range should be confirmed by HRGC/HRMS (approximately 2 % to
10%ofthenegativesamples).Informationoncorrespondencebetween
bioassayandHRGC/HRMSresultsshouldbemadeavailable.
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 purposesan
expanded low level curve for analysing low level samples could be
used.
— ATCDDreferenceconcentration(about3×limitofquantification)on
aquality control sheetshouldbeusedfortheoutcome ofthebioassay
over a constant time period. An alternative could be the relative
responseofareferencesampleincomparisontotheTCDDcalibration
linesincetheresponseofthecellsmaydependonmanyfactors.
— Quality control (QC) charts for each type of reference material should
be recorded and checked to make sure the outcome is in accordance
withthestatedguidelines.
— In particular for quantitative calculations, the induction of the sample
dilution used must be within the linear portion of the response curve.
Samplesabovethelinearportionoftheresponsecurvemustbediluted
andre-tested.Therefore,atleastthreeormoredilutionsatonetimeare
recommendedtobetested.2002L0069—EN—26.08.2002—000.001—10
▼B
— Thepercentstandarddeviationshouldnotbeabove15%inatriplicate
determination for each sample dilution and not above 30 % between
threeindependentexperiments.
— The limit ofdetection may beset as 3×the standarddeviation ofthe
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 fromthe calibration curve of the day.
Thelimitofquantificationmaybesetas5×to6×thestandarddevia-
tion of the solvent blank or of the background response or to apply a
responsethatisabovethebackground(inductionfactor10×thesolvent
blank)calculatedfromthecalibrationcurveoftheday.
7.4.Specific requirements for kit-based bioassays (1)
— Manufacturer'sinstructionsforsamplepreparationandanalyseshaveto
befollowed.
— Testkitsshouldnotbeusedaftertheexpirationdate.— Materialsorcomponentsdesignedforusewithotherkitsshouldnotbe
used.
— Testkitsshouldbekeptwithinthespecifiedrangeofstoragetempera-
tureandusedatthespecifiedoperatingtemperature.
— Thelimitofdetectionforimmunoassaysisdeterminedas3×thestan-
dard deviation, based on 10 replicate analysis of the blank, to be
dividedbytheslopevalueofthelinearregressionequation.
— Reference standardsshouldbeusedfortestsat thelaboratory 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
resultsshouldcontainthelevelsoftheindividualPCDD/FandPCBconge-
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
methodusedforlipidextraction.
Therecoveriesoftheindividualinternalstandardsmustbemadeavailable
in case the recoveries are outside the range mentioned in point 6, in case
themaximumlevelisexceededandinothercasesuponrequest.2002L0069—EN—26.08.2002—000.001—11
(1) No evidence have yet been submitted of commercially available kit-based bioassays
havingsufficientsensitivityandreliabilitytobeusedforscreeningforthepresenceof
dioxinsattherequiredlevelsinsamplesoffoodstuffsandfeedingstuffs.