Dioxin-like PCBs in Feedingstuffs Rules, 2005

L.N. 71 of 2005

VETERINARY SERVICES ACT

(CAP. 437)

Requirements for the Determination of Levels of Dioxins and

Dioxin-like PCBs in Feedingstuffs Rules, 2005

IN exercise of the powers conferred by article 5(2) of the Veterinary

Services Act, 2001, the Minister for Rural Affairs and the Environment

has made the following rules>–

(1) The title to these rules is the Requirements for the Title and scope.

Determination of Levels of Dioxins and Dioxin-like PCBs in

Feedingstuffs Rules, 2005.

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

under European Union Council Directive 2002#70#EC establishing

requirements for the determination of dioxins and dioxin-like PCBs in

feedingstuffs.

For the purpose of these regulations –

METHODS OF SAMPLING FOR OFFICIAL CONTROL OF THE LEVELS OF DIOXINS

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

FEEDINGSTUFFS

1. Purpose and scope

The 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 feedingstuffs, shall be taken

according to the provisions of European Union Commission Directive 76/371/EEC of 1 March

1976 establishing Community methods of sampling for the official control of feedingstuffs.

Aggregate samples thus obtained shall be considered as representative for the lots or sublots from

which they are taken. Compliance with maximum levels laid down in European Union Council

Directive 1999/29/EC shall be established on the basis of the levels determined in the laboratory

samples.

2. 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 European Union Council Directive 1999/29/EC.

Table WHO TEFs for human risk assessment based on the conclusions of the World Health Organisation

meeting in Stockholm, Sweden, 15 to 18 June 1997 (Van den Berg et al., (1998) Toxic Equivalency Factors

(TEFs) for PCBs, PCDDs, PCDFs for Humans and for Wildlife. Environmental Health Perspectives,

106(12), 775).

Congener

TEF value

Congener

TEF value

Dibenzo-p-dioxins (PCDDs)

‘Dioxin-like’ PCBs: Non-ortho PCBs + Mono-ortho

2,3,7,8-TCDD

PCBs

1,2,3,7,8-PeCDD

Non-ortho PCBs

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

0,1

PCB 77

0,0001

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

0,1

PCB 81

0,0001

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

0,1

PCB 126

0,1

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

0,01

PCB 169

0,01

OCDD

0,0001

Mono-ortho PCBs

Dibenzofurans (PCDFs)

PCB 105

0,0001

2,3,7,8-TCDF

0,1

PCB 114

0,0005

1,2,3,7,8-PeCDF

0,05

PCB 118

0,0001

2,3,4,7,8-PeCDF

0,5

PCB 123

0,0001

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

0,1

PCB 156

0,0005

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

0,1

PCB 157

0,0005

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

0,1

PCB 167

0,00001

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

0,1

PCB 189

0,0001

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

0,01

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

0,01

OCDF

0,0001

Abbreviations used: T = tetra; Pe = penta; Hx = hexa; Hp = hepta; O = octa; CDD = chlorodibenzo-p-dioxin; CDF =

chlorodibenzofuran; CB = chlorobiphenyl.

Definitions.

“the competent authority” means the Veterinary Services

within Malta as provided under article 2 of the Veterinary Services

Act, or any other authority to which such responsibility has been

delegated by the Veterinary Services.

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 FEEDINGSTUFFS

1. Objective and field of application

These requirements should be applied where feed materials and feedingstuffs are analysed for the

determination of the dioxins (polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated

dibenzofurans (PCDF)) and dioxin-like polychlorinated biphenyls (PCBs).

Monitoring for the presence of dioxins in feedingstuffs 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 % to 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 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 feed materials/feedingstuffs) 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 possess dioxin -like activity in toxic

equivalents (TEQs) of 2,3,7,8-TCDD (see Schedule 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 in

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

The general provisions on the preparation of samples for analysis as laid down in the Annex to

European Union Commission Directive 81/680/EEC of 30 July 1981 amending European Union

Directives 71/250/EEC, 71/393/EEC, 72/199/EEC, 73/46/EEC, 74/203/EEC, 75/84/EEC, 76/372/EEC

and 78/633/EEC establishing Community methods of analysis for the official control of feedingstuffs

are applicable.

In addition the following requirements have to be complied with

— 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,

— 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.

4. Requirements for laboratories

— Laboratories shall demonstrate the performance of a method in the range of the level of interest,

for example, 0,5 ×, 1 × and 2 × the level of interest with an acceptable coefficient of variation for

repeated analysis. For details of acceptance criteria, see point 5.

— Limit of quantification for a confirmatory method shall 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) shall 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, for example, 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.

— Laboratories shall be accredited by a recognised body operating in accordance with ISO Guide 58

to ensure that they are applying analytical quality assurance. Laboratories should be accredited

following the ISO/IEC/17025:1999 standard.

The competent authority shall ensure that the sampling for Sampling for the

the official control of the levels of dioxins and furans and the official

the levels of dioxins

determination of the levels of dioxin-like PCBs in feedingstuffs is carried and furans.

out in accordance with the methods described in Schedule I.

The competent authority shall ensure that sample preparation Sample preparation

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

analyses.

dioxins and furans and the determination of the levels of dioxin-like

PCBs in feedingstuffs comply with the criteria described in Schedule

II.

Requirements for the analytical procedures for dioxins and dioxin-like PCBs

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- 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 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 and reliable

estimate of the true concentration in a sample. High accuracy (accuracy of the measurement: the

closeness of 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).

Screening methods can comprise bioassays and GC/MS methods; confirmatory methods are high-

resolution gas -chromatography/high-resolution mass-spectrometry (HRGC/HRMS) methods. The

following criteria have to be complied with on total TEQ value

Screening methods

Confirmatory Levels

False negative rate

< 1 %

Trueness

- 20 % to + 20 %

CV

< 30 %

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, for example, 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 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 feedingstuffs of plant origin and feedingstuffs of animal origin containing less than 10 % fat,

the addition of the internal standards is mandatory prior to extraction. For feedingstuffs 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 a product or fat basis.

— Prior to GC/MS analysis, one or two recovery (surrogate) standard(s) must be added.

— 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 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

feedingstuffs with a dioxin contamination in the range or above the maximum level. For

feedingstuffs with contamination levels well below the maximum level, the difference may be in

the range of 25 % to 40 %.

Screening methods of 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,

— additional reference samples at 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 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 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 limit 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 concentration 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 three times the 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.

— The limit of detection may be set as three times 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 five times the solvent blank) calculated from the calibration curve of the day. The

limit of quantification may be set as five to six times the standard deviation of the solvent blank or

of the background response or to apply a response that is clearly above the background (induction

factor 10 times 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 the sum of the mean and three times 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 results

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 the other

cases upon request.

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.