SAMPLING METHODS AND METHODS OF ANALYSIS FOR THE OFFICIAL CONTROL OF DIOXINS AND THE DETERMINATION OF

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

SUBSIDIARY LEGISLATION 437.75

SAMPLING METHODS AND METHODS

OF ANALYSIS FOR THE OFFICIAL CONTROL

OF DIOXINS AND THE DETERMINATION OF

DIOXIN-LIKE PCBs IN FOODSTUFFS RULES *

5th July, 2005

LEGAL NOTICE 241 of 2005.

(1) The title of these rules is the Sampling Methods and

Methods of Analysis for the Official Control of Dioxins and the

Determination of Dioxin-like PCBs in Foodstuffs Rules.

(2) The scope of these rules is to implement the rules found

under European Union Council Directive 2002/69/EC on the

sampling methods and the methods of analysis for the official

control of dioxins and the determination of dioxin-like PCBs in

foodstuffs.

METHODS OF SAMPLING FOR OFFICIAL CONTROL OF THE LEVELS OF

DIOXINS (PCDD/PCDF) AND THE DETERMINATION OF DIOXIN-LIKE PCBs

IN CERTAIN FOODSTUFFS

1. Purpose and scope

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 in

foodstuffs shall be taken according to the methods described below. Aggregate

samples thus obtained shall be considered as representative of the lots or sublots

from which they are taken. Compliance with maximum levels laid down in

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.

Congener

TEF value

Congener

TEF value

Dibenzo-p-dioxins (PCDDs)

‘Dioxin-like’ PCBsNon-ortho PCBs +

Mono-ortho PCBs

2,3,7,8-TCDD

Non-ortho PCBs

1,2,3,7,8-PeCDD

PCB 77

0,0001

1,2,3,4,7,8-HxCDD

0,1

PCB 81

0,0001

1,2,3,6,7,8-HxCDD

0,1

PCB 126

0,1

1,2,3,7,8,9-HxCDD

0,1

PCB 169

0,01

1,2,3,4,6,7,8-HpCDD 0,01

OCDD

0,0001

Dibenzofurans (PCDFs)

Mono-ortho PCBs

2,3,7,8-TCDF

0,1

PCB 105

0,0001

1,2,3,7,8-PeCDF

0,05

PCB 114

0,0005

2,3,4,7,8-PeCDF

0,5

PCB 118

0,0001

1,2,3,4,7,8-HxCDF

0,1

PCB 123

0,0001

1,2,3,6,7,8-HxCDF

0,1

PCB 156

0,0005

1,2,3,7,8,9-HxCDF

0,1

PCB 157

0,0005

2,3,4,6,7,8-HxCDF

0,1

PCB 167

0,00001

1,2,3,4,6,7,8-HpCDF 0,01

PCB 189

0,0001

1,2,3,4, 7,8,9-HpCDF 0,0001

Abbreviations used: T = tetra; Pe = penta; Hx = hexa; Hp = hepta; O = octa; CDD

= chlorodibenzodioxin; CDF = chlorodibenzofuran; CB = chlorobiphenyl.

2. General provisions

2.1. Personnel

Sampling shall be performed by an authorised qualified person as specified by the

Veterinary Services.

2.2. Material to be sampled

Each lot, which is to be examined, must be sampled separately.

2.3. Precautions to be taken

In the course of sampling and preparation of laboratory samples precautions must

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

be taken to avoid any changes, which would affect the content of dioxins and dioxin-

like PCBs, adversely affect the analytical determination or make the aggregate

samples unrepresentative.

2.4. 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 2.8.

2.5. 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.

2.6. 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 homogenized 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.

2.7. Packaging and transmission of aggregate and laboratory samples

Each aggregate and laboratory sample shall be placed in a clean, inert container

offering adequate protection 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.

2.8. 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 Veterinary Services’ regulations. A record must be kept of

each sampling, permitting each lot to be identified unambiguously and giving the

date and place of sampling together with any additional information likely to be of

assistance to the analyst.

3. Sampling plans

T h e s a m p l i ng m e t h o d ap p l ie d s h a ll e n s u r e t ha t t h e a g g r e g a t e s a mp le is

representative for the lot that is to be controlled.

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 2.5). 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 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,

the sample size for hen eggs is at least 12 eggs (for bulk lots as well for lots

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

consisting of individual packages, Tables 1 and 2).

TABLE 1

Minimum number of incremental samples to be taken from the lot

Weight of lot (in kg)

Minimum number of incremental samples

to be taken

< 50

50 to 500

> 500

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 form the

aggregate sample if the lot consists of individual packages

Number of packages or units in the lot Number 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

For the purposes of these rules, and unless context

otherwise requires -

"aggregate sample" means the combined total of all the

incremental samples taken from the lot or sublot;

"the Commission" means the European Commission;

"the Community" means the European Community as established

under the Treaty establishing the European Community;

"the competent authority" means the Veterinary Services within

the territory of Malta as provided under article 2 of the Veterinary

Services Act;

"incremental sample" means a quantity of material taken from a

single place in the lot or sublot;

"laboratory sample" means a representative part/quantity of the

aggregate sample intended for the laboratory;

"lot" means an identifiable quantity of food delivered at one time

and determined by the official to have common characteristics,

such as origin, variety, type of packing, packer, consignor or

markings. In the case of fish and fishery products, also the size of

fish shall be comparable;

"Member State" means a State which is a Member within the

European Community;

"sublot" means the designated part of a large lot in order to apply

the sampling method on that designated part. Each sublot must be

physically separated and identifiable;

*These rules have been repealed by Legal Notice 94 of 2025.

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

"third country" means a State which is not a Member within the

European Community;

"Veterinary Services" means the competent authority within the

territory of Malta as established under article 2 of the Veterinary

Services Act<

SAMPLE PREPARATION AND REQUIREMENTS FOR METHODS OF

ANALYSIS USED IN OFFICIAL CONTROL OF THE LEVELS OF DIOXINS

(PCDD/PCDF) AND THE DETERMINATION OF DIOXIN-LIKE PCBs IN

CERTAIN FOODSTUFFS

1. Objective and field of application

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 with significant 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

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

sample throughput and are used to sift large numbers of samples for potential

positives. They are specifically designed to avoid false negatives.

C o n f i r m a t o r y m e t h o d s a r e m e t h o d s t h a t p r o v i d e f u l l o r c om p l e m e n t a r y

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 foodstuffs)

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 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 each non-

quantified congener to the TEQ.

The concept of ‘mediumbound’ requires using half of the limit of quantification

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

the sampling and analysis procedure.

The samples must be stored and transported in glass, aluminium,

polypropylene 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.

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 with respect to sensitivity.

There are many satisfactory specific sample preparation procedures,

which may be used for the products under consideration. The

procedures have to be validated according to internationally accepted

guidelines.

4. Requirementsfor laboratories

Laboratories shall demonstrate the performance of a method in the

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

range of the level of interest, e.g. 0,5 ×, 1 × and 2 × the level of interest

with an 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 acceptable

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

competence 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 with the 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

assurance. Laboratories should be accredited following the ISO/IEC/

17025:1999 standard.

5. Requirementsto be met by analytical procedure for dioxinsand dioxin-like

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 pictogram TEQ (10-12 g) 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 (10-9 g) 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, such as 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

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

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).

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

False negative rate

< 1%

Trueness

– 20% to + 20%

< 30%

< 15%

Malta shall ensure that the sampling for the official control

of the levels of dioxins and furans and the determination of the

l e v e l s o f d i o x i n - l i k e P C B s i n f o o d s t u ff s i s c a r r i e d o u t i n

accordance with the methods described in Schedule I.

Malta shall ensure that sample preparation and methods of

analyses used for the official control of the levels of dioxins and

furans and the determination of the levels of dioxin-like PCBs in

foodstuffs comply with the criteria described in Schedule II.

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

Compliance of the lot or sublot with the specification

The control laboratory shall analyse the laboratory sample for enforcement in

duplicate analysis in case the obtained result of the first analysis is less than 20%

below or above the maximum level, and calculate the mean of the results. The lot is

accepted if the result of the first analysis is more than 20% below the maximum level

or, where duplicate analysis is necessary, if the mean conforms to the respective

maximum level as laid down in Regulation (EC) No 466/2001.

Specific requirements for GC/MS methods to be complied with for

screening or confirmatory purposes

Addition of 13C-labelled 2,3,7,8-chlorine substituted internal PCDD/F

standards (and of 13C-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 t he t etra to octa- chlorinated

homologous groups for PCDD/F (and at least one congener for each of the

ho m o l o g o us g r ou p s f o r d i o xi n - l i k e P C B s , i f di o x i n - li k e P C B s h a ve t o b e

determined) must be added (alternatively, at least one congener for each mass

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 17 13C-labelled 2,3,7,8-substituted internal PCDD/F standards

and all 12 13C-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

no 13C-labelled analogue is added by using appropriate calibration 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

efficiency 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

Control of recovery is necessary. For confirmatory methods, the

recoveries of the individual internal standards should be in the range of

60% to 120%. Lower or higher recoveries for individual congeners, in

particular 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

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

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 with a dioxin contamination of about 1 pg WHO-TEQ/g

fat (based on PCDD/PCDF only). For foodstuffs with a low fat content, the same

requirements for contamination levels of about 1 pg WHO-TEQ/g product have to be

applied. For lower contamination levels, for example 0,50 pg WHO-TEQ/g product,

the difference between upperbound and lowerbound level may be in the range of 25

Screening methodsof analysis

7.1. Introduction

Different analytical approaches can be performed using a screening method: a

pure screening approach and a quantitative approach.

Screening approach

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 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

Since no internal standards can be used in bioassays, tests on

repeatability 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 approach

The quantitative approach requires standard dilution series, duplicate or triplicate

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

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

reference concentrations of TCDD or a dioxin/furan mixture (full dose-

response curve with a R2 > 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.

Quality control (QC) charts for each type of reference material should

be recorded and checked to make sure the outcome is in accordance

with the 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 each sample dilution and not above 30% between

three independent experiments.

DETERMINATION OF DIOXIN-LIKE PCBs IN FOODSTUFFS

-

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 deviation 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

temperature and used at the specified operating temperature.

-

The limit of detection for immunoassays is determined as 3 × the

standard 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.

Reporting of the result

Insofar as the used analytical procedure makes it possible, the analytical results

should contain the levels of the individual PCDD/F and PCB congeners 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.