The certification of the mass fractions of hexachlorobenzene (HCB) and hexachlorobutadiene (HCBD) in fish tissue : ERM®-CE100 : certification report.
Date: 2016-06-27
Institutions: Institute for Reference Materials and Measurements
Subjects: EU research policy, fish, research method, research report, sampling, scientific research

CERTIFICATION REPORT 

The certification of the mass fractions of 
hexachlorobenzene (HCB) and hexachlorobutadiene (HCBD) 
in fish tissue: ERM®-CE100

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JRC-IRMM promotes a common and reliable European measurement  
provides reference measurements. 

European Commission 
Joint Research Centre 
Institute for Reference Materials and Measurements (IRMM)  

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JRC102041 

EUR 27965 EN 

ISBN 978-92-79-59111-2 (PDF) 
ISSN 1831-9424 (online) 
doi: 10.2787/359451 

Luxembourg: Publications Office of the European Union, 2016 
© European Union, 2016 

Reproduction is authorised provided the source is acknowledged. 
Printed in Belgium 

Abstract 
This report describes the production of ERM®-CE100, a fish (biota) material certified for the mass fractions of hexachlorobenzene and 
hexachlorobutadiene. This material was produced following ISO Guide 34:2009 and is certified in accordance to ISO Guide 35:2006. 
The starting material was wild Wels catfish (Silurus glanis) caught in the area of the Flix reservoir at the Ebro River, Spain. The filleted fish was cut 
and cryogenically milled before mixing. Pre-cooking followed and after further mixing, the material was filled in jars, sterilised in an autoclave and 
finally labelled as ERM-CE100. 
Between-unit homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. Within-unit 
homogeneity was quantified to determine the minimum sample intake. 
The material was characterised by an interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025. 
Technically invalid results were removed but no outlier was eliminated on statistical grounds only.  
Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include 
uncertainties related to possible inhomogeneity, instability and characterisation. 
The material is intended for the quality control and assessment of method performance. As with any reference material, it can be used for 
establishing quality control charts or validation studies. The CRM is available in glass jars closed with twist-off lids containing at least 40 g of 
sterilised fish paste. The minimum amount of sample to be used is 1 g (wet mass). 
The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.

 
 
 
 
 
 
 
 
 
 
 
 
CERTIFICATION REPORT 

The certification of the mass fractions of 
hexachlorobenzene (HCB) and hexachlorobutadiene 
(HCBD) in fish tissue: 
ERM®-CE100 

I. Dosis, M. Ricci, L. I. Majoros, R. Lava, J. Seghers, H. Emteborg, 
A. Held, H. Emons 

European Commission, Joint Research Centre 
Institute for Reference Materials and Measurements (IRMM) 
Geel, Belgium 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Disclaimer 

Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the 
experimental  procedure.  In  no  case  does  such  identification  imply  recommendation  or  endorsement  by  the 
European Commission, nor does it imply that the material or equipment is necessarily the best available for the 
purpose.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Summary 
This report describes the production of ERM®-CE100, a fish (biota) material certified for the 
mass fractions of hexachlorobenzene and hexachlorobutadiene. This material was produced 
following ISO Guide 34:2009 [1] and is certified in accordance to ISO Guide 35:2006 [2]. 

The  starting  material  was  wild  Wels  catfish  (Silurus  glanis)  caught  in  the  area  of  the  Flix 
reservoir  at  the  Ebro  River,  Spain.  The filleted fish  was  cut  and  cryogenically  milled  before 
mixing. Pre-cooking followed and after further mixing, the material was filled in jars, sterilised 
in an autoclave and finally labelled as ERM-CE100. 

Between-unit  homogeneity  was  quantified  and  stability  during  dispatch  and  storage  were 
assessed in accordance with ISO Guide 35:2006 [2]. Within-unit homogeneity was quantified 
to determine the minimum sample intake. 

The  material  was  characterised  by  an  interlaboratory  comparison  of  laboratories  of 
demonstrated  competence  and  adhering  to  ISO/IEC  17025  [3].  Technically  invalid  results 
were removed but no outlier was eliminated on statistical grounds only.  

Uncertainties  of  the  certified  values  were  calculated  in  accordance  with  the  Guide  to  the 
Expression  of  Uncertainty  in  Measurement  (GUM)  [4]  and  include  uncertainties  related  to 
possible inhomogeneity, instability and characterisation. 

The material is intended for the quality control and assessment of method performance. As 
with any reference material, it can be used for establishing quality control charts or validation 
studies. The CRM is available in glass jars closed with twist-off lids containing at least 40 g of 
sterilised fish paste. The minimum amount of sample to be used is 1 g (wet mass). 

The  CRM  was  accepted  as  European  Reference  Material  (ERM®)  after peer  evaluation  by 
the partners of the European Reference Materials consortium. 

The following values were assigned: 

Mass fraction (relative to wet weight) 

Certified value 2) 
[µg/kg] 

120  

36  

Uncertainty 3) 
[µg/kg] 

8  

4  

Hexachlorobenzene 1) 
Hexachlorobutadiene 1) 

1) as obtained by using gas chromatography 

2) Unweighted mean value of the means of accepted sets of data, each set being obtained in a different laboratory 
and/or  with  a  different  method  of  determination.  The  certified  values  and  their  uncertainties  are  traceable  to  the 
International System of units (SI). 

3) The uncertainty of the certified value is the expanded  uncertainty  with  a  coverage  factor  k  =  2  corresponding  to a 
level  of  confidence  of  about  95  %  estimated  in  accordance  with  ISO/IEC  Guide  98-3,  Guide  to  the  Expression  of 
Uncertainty in Measurement (GUM:1995), ISO, 2008. 

 
 
 
 
 
 
 
 
 
Table of contents 
Summary .............................................................................................................................. 1 
Table of contents ................................................................................................................. 3 
Glossary ............................................................................................................................... 4 
Introduction .............................................................................................................. 7 
1 
Background ............................................................................................................... 7 
1.1 
Choice of the material ................................................................................................ 7 
1.2 
Design of the project .................................................................................................. 7 
1.3 
Participants .............................................................................................................. 8 
2 
Project management and evaluation .......................................................................... 8 
2.1 
Processing ................................................................................................................. 8 
2.2 
Homogeneity study .................................................................................................... 8 
2.3 
Stability study ............................................................................................................ 8 
2.4 
Characterisation ......................................................................................................... 8 
2.5 
Material processing and process control .............................................................10 
3 
Origin of the starting material ....................................................................................10 
3.1 
Processing ................................................................................................................10 
3.2 
3.3 
Process control .........................................................................................................13 
3.3.1  Particle size analysis .................................................................................................13 
3.3.2  Water content by oven drying method .......................................................................14 
Homogeneity ...........................................................................................................15 
4 
Between-unit homogeneity........................................................................................15 
4.1 
4.2  Within-unit homogeneity and minimum sample intake...............................................17 
Stability....................................................................................................................19 
5 
Short-term stability study ..........................................................................................19 
5.1 
Long-term stability study ...........................................................................................20 
5.2 
Estimation of uncertainties ........................................................................................20 
5.3 
Characterisation .....................................................................................................23 
6 
Selection of participants ............................................................................................23 
6.1 
6.2 
Study setup ...............................................................................................................23 
6.3  Methods used ...........................................................................................................23 
6.4 
Evaluation of results .................................................................................................24 
6.4.1  Technical evaluation .................................................................................................24 
6.4.2  Statistical evaluation .................................................................................................25 
Value Assignment ...................................................................................................26 
7 
Certified values and their uncertainties .....................................................................26 
7.1 
Additional material information ..................................................................................26 
7.2 
8 
Metrological traceability and commutability .........................................................27 
8.1  Metrological traceability ............................................................................................27 
Commutability ...........................................................................................................27 
8.2 
Instructions for use ................................................................................................28 
9 
Safety information .....................................................................................................28 
9.1 
Storage conditions ....................................................................................................28 
9.2 
Preparation and use of the material ..........................................................................28 
9.3 
9.4  Minimum sample intake ............................................................................................28 
Use of the certified value ..........................................................................................28 
9.5 
Acknowledgements ................................................................................................29 
10 
References ..............................................................................................................30 
11 

3 

 
Glossary 

ANOVA  

Analysis of variance 

ASE 

c 

C-QC 

CI 

CLSI 

CRM 

EC 

EI 

EQS 
ERM® 

EU 

Accelerated solvent extraction 

Mass concentration c = m / V (mass / volume) 

Calibration quality control 

Confidence interval 

Clinical and Laboratory Standards Institute 

Certified reference material 

European Commission 

Electron ionisation 

Environmental Quality Standards 

Trademark of European Reference Materials 

European Union 

GC-ECD 

Gas chromatography-electron capture detection 

GC-(HR)MS 

Gas chromatography-(high resolution)mass spectrometry 

GC-MS/MS 

Gas chromatography- tandem mass spectrometry 

GC-IDMS 

Gas chromatography isotope dilution-mass spectrometry 

GPC 

GUM 

HCB 

HCBD 

IEC 

IRMM 

ISO 

JRC 

k 

LLE 

LOD 

LOQ 

MSbetween 

MSwithin  

M-QC 

n 

n.c. 

NIST 

PS 

Gel permeation chromatography 

Guide to the Expression of Uncertainty in Measurements 

Hexachlorobenzene 

Hexachlorobutadiene 

International Electrotechnical Commission 

Institute for Reference Materials and Measurements of the JRC  

International Organization for Standardization  

Joint Research Centre of the European Commission 

Coverage factor 

Liquid liquid extraction 

Limit of detection 

Limit of quantification 

Mean of squares between-unit from an ANOVA 

Mean of squares within-unit from an ANOVA 

Method quality control 

Number of replicates per unit 

Not calculated 

National Institute of Standards and Technology (US) 

Priority substances 

4 

 
QA 

QC 

rel 

RM 

RSD 
r2 

s 

sbb

sbetween 

SI 

smeas 

SPE 

swithin 

swb 

T 

t 

ti 

ta

, df 

tsl 

ttt 

u 

U 
u*

bb  

ubb 

uc 

ucal 

uchar  

uCRM 

UCRM  

Quality assurance 

Quality control 

Index denoting relative figures (uncertainties etc.) 

Reference material 

Relative standard deviation 

Coefficient of determination of the linear regression 

Standard deviation 

Between-unit standard deviation; an additional index "rel" is added when 
appropriate 

Standard deviation between groups as obtained from ANOVA; an 
additional index "rel" is added as appropriate 

International System of units 

Standard deviation of measurement data; an additional index "rel" is 
added as appropriate 

Solid phase extraction 

Standard deviation within groups as obtained from ANOVA; an additional 
index "rel" is added as appropriate 

Within-unit standard deviation 

Temperature 

Time 

time elapsed at time point i of the short-term stability study (for usts,rel) or 
long-term stability study (for ults,rel) 
Critical t-value for a t-test, with a level of confidence of 1-a
degrees of freedom 

 and df 

Proposed shelf life 

Transport time chosen 

Standard uncertainty  

Expanded uncertainty 

Standard uncertainty related to a maximum between-unit inhomogeneity 
that could be hidden by method repeatability; an additional index "rel" is 
added as appropriate 

Standard uncertainty related to a possible between-unit inhomogeneity;  
an additional index "rel" is added as appropriate 

Combined standard uncertainty; an additional index "rel" is added as 
appropriate 

Standard uncertainty of calibration 

Standard uncertainty of the material characterisation; an additional index 
"rel" is added as appropriate 

Combined standard uncertainty of the certified value; an additional index 
"rel" is added as appropriate 

Expanded uncertainty of the certified value; an additional index "rel" is 

5 

 
 
u∆ 

ults 

umeas 

Umeas 

urec  

usts 

ut 

WFD 
a  
D meas 

n s,meas 
n

MSwithin

added as appropriate 

Combined standard uncertainty of measurement result and certified 
value 

Standard uncertainty of the long-term stability; an additional index "rel" is 
added as appropriate 

Standard measurement uncertainty 

Expanded measurement uncertainty 

Standard uncertainty related to possible between-unit inhomogeneity 
modelled as rectangular distribution; an additional index "rel" is added as 
appropriate 

Standard uncertainty of the short-term stability; an additional index "rel" 
is added as appropriate 

Standard uncertainty of trueness 

Water Framework Directive 

Significance level 

Absolute difference between mean measured value and the certified 
value 

Degrees of freedom for the determination of the standard deviation smeas 

Degrees of freedom of MSwithin 

6 

 
 
1  Introduction  

1.1  Background 

Hexachlorobenzene (HCB) and hexachlorobutadiene (HCBD) are two substances which are 
considered  as  global  environmental  pollutants,  especially  of  aquatic  ecosystems.  They  are 
used  in  agricultural  and  industrial  applications  [5-6]  and  are  listed  as  Persistent  Organic 
Pollutants (POP) of the Stockholm convention. During the last decades both pollutants were 
detected in surface waters and related matrices (sediments, biota) [7]. Bioaccumulation and 
biomagnification effects have been shown for both chemicals, most pronounced for HCB [8-
9]. 

Since 2000, Directive 2000/60/EC, known as the Water Framework Directive (WFD) [10] is in 
force in the European Union (EU) for developing a pollution control strategy of all EU water 
bodies.  Subsequently,  Directive  2008/105/EC  [11]  set  Environmental  Quality  Standards 
(EQS) for the established list of Priority Substances (PS). HCB and HCBD are among the PS 
that Member States are expected to assess, monitor and control in EU water resources. As 
foreseen in Article 3 of this Directive, EU Member States that opt to apply EQS for sediment 
and biota, shall apply an EQS of 10 µg/kg for HCB and 55 µg/kg for HCBD. The most recent 
Directive  2013/39/EU  [12]  re-evaluated  the  EQS  for  some  of  the  PS  and  biota  EQS  were 
introduced for more PS, with the specification that, wherever a biota EQS is given, biota shall 
be the "default" monitoring matrix. 

To ensure the quality and comparability of analytical results reported by the Member States, 
the  EU  issued  Directive  2009/90/EC  [13]  in  2009,  setting  minimum  analytical  performance 
criteria for monitoring water quality. The competence of EU laboratories selected for this task 
must be guaranteed by the use of certified reference materials (CRMs) of appropriate matrix 
and with corresponding levels to the established EQS. 

1.2  Choice of the material 

In  support  of  the  EU  legislation,  ERM-CE100  was  developed  as  a  fresh-like  biota  matrix 
CRM,  addressing  the  legislative  trend  of  the  Directive  2013/39/EU  which  introduced  biota 
EQS  for  more  PS,  and  declared  that,  for  those  substances,  biota  shall  be  the  "default" 
monitoring matrix. The fish selected for the production of ERM-CE100 was wild Wels catfish 
(Silurus glanis). The choice of the species Silurus glanis for the production of a biota CRM for 
the analysis of HCB and HCBD was made for several reasons. This fish can reach very large 
sizes  and  is  a  predator,  i.e.  positioned  high  in  the  trophic  chain,  which  potentially  leads  to 
large 
bioaccumulation  and  biomagnification  of  organic  pollutants.  The  presence  of 
specimens should lead to a reduced variation of pollutant levels between pooled individuals, 
introducing  a  clear  advantage  to  the  CRM  preparation.  The  overall  purpose  of  the  project 
was to develop a naturally rather than artificially contaminated (spiked) fresh-like biota matrix 
material. 

1.3  Design of the project 

The  CRM  consists  of  units  containing  approximately  40  g  of  sterilised  fish  paste.  The 
material  was  not  freeze-dried  in  order  to  resemble  the  routine  environmental  samples  as 
close as possible and in consideration of the fact that EQS are expressed as mass fraction 
relative to wet weight. Homogeneity and stability were assessed for both analytes in the final 
CRM.  The  certification  was  performed  by  interlaboratory  comparison  using  analytical 
methods based on different analytical principles to reduce bias in the analytical result.  

The HCB and HCBD levels in the designed CRM were targeted to be as close as possible to 
the  EQS  of  these  pollutants  [12].  On  the  other  hand,  the  natural  level  of  the  analytes  of 
interest in the individual fish collected had to be taken into consideration [7] and determined 
the final certified value obtained.   

7 

 
 
2  Participants 

2.1  Project management and evaluation 

European  Commission,  Joint  Research  Centre,  Institute  for  Reference  Materials  and 
Measurements (IRMM), Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No. 268-RM) 

2.2  Processing  

European  Commission,  Joint  Research  Centre,  Institute  for  Reference  Materials  and 
Measurements (IRMM), Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No. 268-RM) 

2.3  Homogeneity study 

European  Commission,  Joint  Research  Centre,  Institute  for  Reference  Materials  and 
Measurements (IRMM), Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No. 268-RM; measurements 
under the scope of ISO/IEC 17025 accreditation BELAC No. 268-TEST) 

Helmholtz Zentrum München, Deutsches Forschungzentrum für Gesundheit und Umwelt 
(GmbH), Neuherberg, DE 
(measurements under the scope of ISO/IEC 17025 accreditation DAkkS; D-PL-14138-02-00) 

2.4  Stability study 

European  Commission,  Joint  Research  Centre,  Institute  for  Reference  Materials  and 
Measurements (IRMM), Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No. 268-RM; measurements 
under the scope of ISO/IEC 17025 accreditation BELAC No. 268-TEST) 

2.5  Characterisation 

BAM, Bundesanstalt für Materialforschung und –prüfung, Berlin, DE 
(measurements partially under the scope of ISO/IEC 17025 accreditation DAkkS; D-PL-11075-14-00) 

European  Commission,  Joint  Research  Centre,  Institute  for  Reference  Materials  and 
Measurements (IRMM), Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No. 268-RM; measurements 
under the scope of ISO/IEC 17025 accreditation BELAC No. 268-TEST) 

GBA, Gesellschaft für Bioanalytik mbH, Pinneberg, DE 
(measurements under the scope of ISO/IEC 17025 accreditation DAkkS; D-PL-14170-01-00) 

Helmholtz Zentrum München, Deutsches Forschungzentrum für Gesundheit und Umwelt 
(GmbH), Neuherberg, DE 
(measurements under the scope of ISO/IEC 17025 accreditation DAkkS; D-PL-14138-02-00) 

IDAEA-CSIC, Institut de Diagnosi Ambiental i Estudis de l'Aigua, Consejo Superior de 
Investigaciones Científicas, Departamento de Química Ambiental, Barcelona, ES 

IMARES, Institute for Marine Resources and Ecosystem Studies, Wageningen UR, 
IJmuiden, NL 
(measurements partially under the scope of ISO/IEC 17025 accreditation Raad voor Accreditatie/Dutch 
Accreditation Council; L097) 

Instituto Hidrográfico, Divisão de Química e Poluição do Meio Marinho, Lisboa, PT 

IVM, Institute for Environmental Studies, Vrije Universiteit, Amsterdam, NL 

LABERCA – ONIRIS, Laboratoire d' Etude des Résidus et Contaminants dans les Aliments – 
École Nationale Vétérinaire, Agroalimentaire et de l'Alimentation Nantes Atlantique, Nantes, 
FR  
(measurements under the scope of ISO/IEC 17025 accreditation COFRAC; 1-0549) 

8 

 
 
 
 
 
RIKILT, Institute of Food Safety, Wageningen UR, Wageningen, NL 
(measurements under the scope of ISO/IEC 17025 accreditation Raad voor Accreditatie/Dutch Accreditation 
Council; L014) 

University of Barcelona, Department of Analytical Chemistry, Group of Chromatography, 
Capillary Electrophoresis and Mass Spectrometry, Barcelona, ES  

9 

 
 
3  Material processing and process control 

3.1  Origin of the starting material 

The  starting  material  used  for  the  production  of  ERM-CE100  was  approximately  110  kg  of 
filleted Wels catfish (Silurus glanis) originating from the area of the Flix reservoir at the Ebro 
River,  Spain.  Twenty-nine  specimens  were  caught  by  angling  during  the  period  December 
2010 – June 2011. The fish was filleted (excluding skin, inner part and bones but including 
the  fatty  part  of  the  fish  tissue),  separated  into  head  and  tail,  sealed  into  plastic  bags  and 
labelled  to  unambiguously  identify  the  origin. The  material  was  transported frozen  to  IRMM 
and kept at -20 °C until further treatment. 

3.2  Processing 

Flowchart and scheme of the complete production process are presented below in Figures 1 
and 3. 

Aliquotes were analysed from all heads and tails to screen the content of HCB and HCBD. 
Sampling was performed using a steel drill on the still frozen parts. 

In total, 60 kg of head parts were thereafter thawed and manually cut into pieces. After flash-
cooling  in  liquid  nitrogen  the  manually  cut  pieces  were  cryogenically  milled  in  a  vibrating 
cryogenic  mill,  Palla  VM-KT  (Janke  Kunkel,  Köln,  DE).  Thereafter  50  kg  of  fish  tails  were 
cryogenically  milled  and  processed  separately  in  the  same  way.  Each  specimen  was 
processed separately and could be traced back to the original part and fish (part being head 
or  tail). The  cryogenic mill  was  cleaned  with  about  5 kg  of  crushed  ice  between  the  milling 
events to prevent carryover from one piece of fish to another. Each piece of milled fish was 
thereafter homogenised in a Stephan UM12 mixer (Hameln, DE) and samples were taken for 
additional checks of the contaminants' levels. The milled fish was stored at -20 °C until the 
analysis results for HCB and HCBD became available. 

According  to  the  analysis  results,  different  head  and  tail  parts  were  selected  in  a  mixing 
scheme to target the EQS levels of HCB and HCBD as close as possible (Batch 4, Figure 3). 
For  the  mixing  to  obtain  the  different  batches  (Figure  3)  a  Stephan  UM12  mixer  (Hameln, 
DE) and a larger Stephan UM80 mixer were used. During the in-between batches analysis, 
the  paste  material  was  stored  at  4  °C  overnight.  The  final  control  analysis  on  batch  4 
confirmed values close to the targeted mass fraction levels for the two analytes. The paste 
was  thereafter  transferred  into  a  Firex  cucimix  mixer  (Sedico,  IT)  with  a  70  L  capacity  that 
was  used  to  gradually  increase  the  temperature  of  the  fish  paste  from  19  to  82  °C  in  one 
hour under constant mixing and to finally maintain it for two minutes. 

For rapid cool-down the still warm material was thereafter transferred to trays and placed in 
an Martin Christ Epsilon 2-100D freeze dryer (Osterode, DE) where the shelves were kept at 
1  °C.  The  cooled  fish  paste  was  thereafter  placed  at  4  °C  overnight  and  the  following  day 
transferred  to  the  Stephan  UM  80  mixer  where  it  was  mixed  3  x  3  minutes  to  achieve  a 
homogeneous viscous paste, see Figure 2. 

Approximately 40 g of the fish paste was filled into 65 mL glass jars using a PSV Villa SP25 
piston filling machine (Genainville, FR), see Figure 2. Twist-off lids of 66 mm diameter were 
placed on the jars using a Lenssen twist-off machine (Sevenum, NL). 

The filled jars were placed according to fill order in a basket and sterilised at 121 °C (peak 
temperature  maintained  for  about  10  minutes)  using  a  JBTC  autoclave  (Sint-Niklaas,  BE). 
The temperature inside the material during autoclavation was monitored online with a probe 
and additionally using autoclavation tape. 

Labelling was performed manually according to the filling order. A label was placed both on 
the lid and at the underside of each glass jar. A total of 1023 CRM units were produced. 

10 

110 kg of filleted Silurus 
glanis tissue split in 
heads and tails 

Manual cutting of heads and 
tails 

Separate cryogenic milling 
of heads and tails 

in-house measurements of 
HCB & HCBD by GC-MS* 

Heads/tails separately 
homogenised and stored 
at - 20 °C 

Mixing of selected heads and 
tails based on analysis results* 
and storage at 4 °C 

in-house measurements of 
HCB & HCBD by GC-MS for 
confirming target levels* 

Mixing of final batch 

Pre-cooking to 82 °C under 
constant mixing 

Filling and closing jars 

Thermal sterilisation at 121 °C 

Additional analysis [HCB & HCBD 
by GC-MS, particle size, water 
content] 

Manual labeling of the 
1023 units produced 

Figure 1: Flow chart of main steps in the preparation of ERM-CE100. 

* the steps concerning the in-house checks of the analytes' levels and the mixing are 
reported in more details in Figure 3.                           

11 

 
 
 
Figure 2: Pre-cooking and filling of ERM-CE100 

Figure 3: Processing of ERM-CE100: "mix and match" scheme (in blue the theoretical fat content [7,14] and mass fractions of HCB and HCBD, 
in  green  the  mass  fractions  measured  in-house,  in  purple  the  fat  content  and  mass  fractions  of  HCB  and  HCBD  measured  in-house  on  the 
finally produced material; mass fractions are expressed relative to wet weight) 

12 

 
 
 
 
An example of a final unit produced can be seen in Figure 4 below:  

Figure 4: Example of ERM-CE100 unit 

3.3  Process control 

3.3.1  Particle size analysis 
The average particle size distribution of ERM-CE100 is displayed in Fig. 5 and relevant data 
are reported in Table 1. Five units were measured in duplicate using a Sympatec Helos laser 
light diffraction instrument (Clausthal Zellerfeld, DE). 

Figure 5: Average particle size distribution in ERM-CE100 using water as dispersant 

13 

 
 
 
 
 
 
 
Table 1: Particle size data for ERM-CE100 (average of ten measurements) 

Upper band limit 

Average particle size (µm)  

s (µm) 

RSD (%) 

X10 

X50 

X90 

2.76 

28.14 

118.97 

0.21 

1.17 

3.47 

4.78 

3.09 

1.52 

As  an  overall  assessment  of  comparability  of  the  particle  size  distribution  between  the 
different units, the average of the deviation for X10, X50 and X90 from their respective average 
values is calculated. Results with an average deviation for X10, X50 and X90 below 20 % are 
considered as acceptable. 

For the ten replicates, the average deviations are all below 10 %. Consequently the material 
is homogeneous and uniform over the whole batch with respect to particle size distribution. 

3.3.2  Water content by oven drying method 
A  ventilated  oven  method  at  102  °C  provided  the  best  option for  measuring the  high  water 
content in ERM-CE100, typical of a fresh tissue material. Volatile components are also lost 
but  these  are  assumed  to  constitute  a  very  small  fraction  of the  total  mass  loss.  Five  units 
randomly  selected  over  the  whole  batch  were  analysed  in  duplicate.  An  analytical  balance 
was  used  to  record  the  mass  loss  after  one  and  two  hours,  respectively.  Two  hours  were 
deemed  sufficient  to  obtain  reliable  and  relatively  uniform  mass  loss  data.  The  sample 
amounts placed in the drying oven varied between 4 to 12 g of wet material and the recorded 
mass  loss  was  found  to  be  independent  from the  sample  mass.  Based  on  the  variability  in 
the  data  from  the  same  unit,  no  trend  over  the  filling  sequence  could  be  observed  for  the 
water content in this material. An average water content of 74.4 % (m/m) was measured.  

14 

 
 
 
4  Homogeneity 
A  key  requirement  for  any  reference  material  distributed  into  units  is  the  equivalence 
between  those  units.  In  this  respect,  it  is  relevant  whether  the  variation  between  units  is 
significant  compared  to  the  uncertainty  of  the  certified  value,  but  it  is  not  relevant  if  this 
variation between units is significant compared to the analytical variation. Consequently, ISO 
Guide  34  [1]  requires  RM  producers  to  quantify  the  between  unit  variation.  This  aspect  is 
covered in between-unit homogeneity studies. 

The within-unit inhomogeneity does not influence the uncertainty of the certified value when 
the minimum sample intake is respected, but determines the minimum size of an aliquot that 
is  representative  for  the  whole  unit.  Quantification  of  within-unit  inhomogeneity  is  therefore 
necessary to determine the minimum sample intake. 

4.1  Between-unit homogeneity 

The between-unit homogeneity was evaluated to ensure that the certified values of the CRM 
are valid for all units of the material, within the stated uncertainties. 

The number of units selected corresponds to approximately the cube root of the total number 
of  units  produced.  Twelve  units  were  selected  using  a  random  stratified  sampling  scheme 
covering  the  whole  batch  for  the  between-unit  homogeneity  test.  For  this,  the  batch  was 
divided  into  twelve  groups  (with  a  similar  number  of  units)  and  one  unit  was  selected 
randomly  from  each  group.  Two  independent  samples  of  2  g  each  (wet  mass)  were  taken 
from  each  selected  unit  and  in-house  analysed  by  accelerated  solvent  extraction  (ASE), 
solid-phase extraction (SPE) clean-up and GC-IDMS [7,14]. For each independent replicate, 
sample preparation and analytical determination were carried out within one day. Due to the 
high  number  of  samples  (twenty-four), 
the  measurements  were  performed  under 
intermediate precision conditions (spread over four days). Consequently, day-to-day effects 
could  occur  that  could  mask  the  between-bottle  variation.  Therefore,  it  was  first  checked  if 
there was a significant difference between the day means using ANOVA. Indeed, significant 
day-to-day effect was present for HCBD content and a correction was applied by normalising 
i.e., dividing the data points by the respective day mean. The results are shown as graphs in 
Annex A.  

Regression analyses were performed to evaluate potential trends in the analytical sequence 
as  well  as  trends  in  the  filling  sequence.  No  trends  in  the  filling  sequence  or  the  analytical 
sequence were observed at a 95 % confidence level.  

The datasets were assessed for consistency using Grubbs outlier tests at a confidence level 
of 99 % on the individual results and on the unit means. One outlying result was detected for 
HCBD. Since no technical reason for the outliers could be found, all data were retained for 
statistical analysis.  

Quantification  of  between-unit  inhomogeneity  was  undertaken  by  analysis  of  variance 
(ANOVA), which separates the between-unit variation (sbb) from the within-unit variation (swb). 
The  latter  is  equivalent  to  the  method  repeatability  if  the  individual  samples  of  2  g  were 
representative for the whole unit.  

Evaluation  by  ANOVA  requires  mean  values  per  unit,  which  follow  at  least  a  unimodal 
distribution and results for each unit that follow unimodal distributions with approximately the 
same  standard  deviations.  The  distribution  of  the  mean  values  per  unit  was  visually  tested 
using histograms and normal probability plots. Too few data are available for the unit means 
to make a clear statement of the distribution. Therefore, it was checked visually whether all 
individual  data follow  a  unimodal  distribution  using  histograms  and  normal  probability  plots. 
Minor  deviations  from  unimodality  of  the  individual  values  do  not  significantly  affect  the 
estimate  of  between-unit  standard  deviations.  The  results  of  all  statistical  evaluations  are 
given in Table 2. 

15 

 
Table 2: Results of the statistical evaluation of the homogeneity studies 

Analyte 

Trends 
(before correction)* 

Outliers** 

Distribution 

HCB 

HCBD 

Analytical 
sequence 

Filling 
sequence 

Individual results 

No 

No 

No 

No 

None 

1-statistical 
reason (retained) 

Unit 
means 

None 

None 

Individual 
results 
normal/ 
unimodal 
normal/ 
unimodal 

Unit 
means 
normal/ 
unimodal 
normal/ 
unimodal 

* 95 % confidence level 
** 99 % confidence level 

It should be noted that sbb,rel and swb,rel are estimates of the true standard deviations and are 
therefore  subject  to  random  fluctuations.  Therefore,  the  mean  square  between  groups 
(MSbetween)  can  be  smaller  than  the  mean  squares  within  groups  (MSwithin),  resulting  in 
negative  arguments  under  the  square  root  used  for  the  estimation  of  the  between-unit 
variation,  whereas  the  true  variation  cannot  be  lower  than  zero.  In  this  case,  u*
bb,  the 
maximum  inhomogeneity  that  could  be  hidden  by  method  repeatability,  was  calculated  as 
described  by  Linsinger  et  al.  [15].  u*
bb  is  comparable  to  the  LOD  of  an  analytical  method, 
yielding the maximum inhomogeneity that might be undetected by the given study setup.  
Method  repeatability  (swb,rel),  between–unit  standard  deviation  (sbb,rel)  and  u*
calculated as:  

bb,rel  were 

s

wb

,

rel

=

MS

within
y

MS

between

MS

within

s

bb

,

rel

=

n
y

MS
within
n

4

ν

2

MSwithin

y

u

*
,bb

rel

=

Equation 1 

Equation 2 

Equation 3 

MSwithin 

mean of squares within-unit from an ANOVA  

MSbetween  mean of squares between-unit from an ANOVA 

y  

mean of all results of the homogeneity study 

n 
ν

MSwithin

number of replicates per unit 

degrees of freedom of MSwithin  

The  results  of  the  evaluation  of  the  between-unit  variation  are  summarised  in  Table  3.  For 
HCBD, the uncertainty contribution was determined by the method repeatability. 

16 

 
 
 
 
-
 
 
 
 
 
 
 
Table 3: Outcome of the homogeneity study 

swb,rel  

sbb,rel  

u*

bb,rel 

ubb,rel 

[%] 

2.6  

3.0 

[%] 

2.0  

n.c. 1) 

[%] 

1.2  

1.3  

[%] 

2.0  

1.3 

Analyte 

HCB 

HCBD 

1) n.c.: cannot be calculated as MSbetween < MSwithin 

The  homogeneity  study  showed  no  outlying  unit  means  or  trend  in  the  filling  sequence  for 
both  analytes.  Therefore,  the  between-unit  standard  deviation  can  be  used  as  estimate  of 
ubb.  As  u*
bb  sets  the  limits  of  the  study  to  detect inhomogeneity,  the  larger  value  of  sbb  and 
u*

bb is adopted as uncertainty contribution to account for potential inhomogeneity. 

4.2  Within-unit homogeneity and minimum sample intake 

The within-unit homogeneity is closely correlated to the minimum sample intake. Due to this 
correlation, individual aliquots of a material will not contain the same amount of analyte. The 
minimum  sample  intake  is  the  minimum  amount  of  sample  that  is  representative  for  the 
whole  unit  and  thus  can  be  used  in  an  analysis.  Using  sample  sizes  equal  or  above  the 
minimum sample intake guarantees the certified value within its stated uncertainty. 

Study of decreasing sample intakes 

To estimate the minimum sample intake, a series of measurements with decreasing amounts 
of sample on six randomly selected units were performed. The following sample intakes were 
tested:  0.5,  1  and  2  g  (wet  mass).  For  each  sample  intake,  2  units  were  measured  in 
triplicate each by ASE, silica column clean-up and GC-MS quantification under repeatability 
conditions (for the 0.5 and 1 g; the 2 g sample intake measurements were carried out for the 
scope of the characterisation campaign few days before), and in a randomised manner. The 
measurement method was robust over the whole range of the sample intake tested and its 
repeatability  was  in  the  same  range  or  better  than  the  repeatability  achieved  during  the 
material characterisation (Section 6). 

The obtained datasets (for the sample intakes 0.5 and 1 g taken together) were first tested 
whether  they  follow  a  normal,  or  at  least  unimodal  distribution.  This  was  done  by  visual 
inspection  of  normal  probability  plots  and  histograms  (if  the  data  do  not  follow  at  least  a 
unimodal  distribution,  the  calculation  of  standard  deviations  is  doubtful  or  impossible).  All 
results were normally and unimodally distributed. 

Furthermore,  the  results  (corresponding  to  the  sample  intakes  0.5  and  1  g  taken  together) 
were  scrutinised  for  outliers  using  the  single  Grubbs-test  at  a  99  %  confidence  level.  No 
outliers were detected. 

The  minimum  sample  intake  was  established  by  comparison  of  the  variances  obtained  for 
0.5  and  1  g  sample  intakes  with  the  variance  obtained  for  2  g  sample  intake.  It  was  done 
using the  F-test for  equality  of two  samples for variances  with  5  degrees  of freedom  and a 
confidence level of 95 %. 

The  study  results  are  presented  in  Annex  B  and  the  minimum  sample  intakes  are 
summarised in Table 4.  

17 

 
 
 
 
 
 
Table 4: Outcome of the minimum sample intake determination 

Analyte 

HCB 

HBCD 

Minimum sample intake 
(wet mass) [g] 
1 

0.5 

As  shown  above,  the  minimum  sample  intake  representative  for  both  analytes  is  1  g  (wet 
mass). 

18 

 
 
 
 
5  Stability 
Time  and  temperature  were  regarded  as  the  most  relevant  influences  on  stability  of  the 
material.  Additionally  the  material  was  sterilised  by  heat  treatment  (in  an  autoclave)  to 
eliminate microbial growth. Therefore, only the influences of time and temperature needed to 
be investigated. 

Stability  testing  is  necessary  to  establish  the  conditions  for  storage  (long-term  stability)  as 
well  as  the  conditions  for  dispatch  of  the  materials  to  the  customers  (short-term  stability). 
During transport, especially in summer time, temperatures up to 60 °C can be reached and 
stability  under  these  conditions  must  be  demonstrated  if  the  samples  are  to  be transported 
without any additional cooling. 

The  stability  studies  were  carried  out  using  an  isochronous  design  [16].  In  this  approach, 
samples  were  stored  for  a  particular  length  of  time  at  different  temperature  conditions. 
Afterwards,  the  samples  were  moved  to  conditions  where  further  degradation  can  be 
assumed to be negligible (reference conditions). At the end of the isochronous storage, the 
samples  were  analysed  simultaneously  under  repeatability  conditions.  Analysis  of  the 
material  (after  various  exposure  times  and  temperatures)  under  repeatability  conditions 
greatly improves the sensitivity of the stability tests.  

5.1  Short-term stability study 

For the short-term stability study, samples were stored at 18 °C and 60 °C for 0, 1, 2 and 4 
weeks  (at  each  temperature).  The  reference  temperature  was  set  to  4  °C.  Two  units  per 
storage time were selected using a random stratified sampling scheme. From each unit, two 
samples  of  2 g  each  (wet mass)  were  in-house measured  by  ASE,  SPE  clean-up  and  GC-
IDMS  [7,14].  The  measurements  were  performed  under  intermediate  precision  conditions 
(different  days)  and  a  randomised  sequence  was  applied  to  differentiate  any  potential 
analytical drift from a trend over storage time. It was first checked if there was a significant 
difference  between  the  day  means  using  ANOVA  (measurements  spread  over  three  days). 
Significant  day-to-day  effect  was  present  for  HCB  content  and  a  correction  was  applied  by 
normalising i.e. dividing the data points by the respective day mean. 

The  data  were  evaluated  individually  for  each  temperature.  The  results  were  screened  for 
outliers using the single and double Grubbs test on a  confidence level of 99 %. No outliers 
were found (Table 5). 

In  addition,  the  data  were  evaluated  against  storage  time,  and  regression  lines  of  mass 
fraction  versus  time  were  calculated,  to  test  for  potential  increases/decreases  of  the 
measurands  due  to  shipping  conditions.  The  slopes  of  the  regression  lines  were  tested  for 
statistical  significance.  None  of the  trends  was  statistically  significant  at  a  95  %  confidence 
level for any of the temperatures for HCB, while a significant trend at a 95 % confidence level 
was evidenced for HCBD at 60 °C.  

The  results  of  the  measurements  are  shown  in  Annex  C.  The  results  of  the  statistical 
evaluation of the short-term stability are summarised in Table 5. 

Table 5: Results of the statistical evaluation of the short-term stability tests 

Analyte 

HCB 

HCBD 

* 99 % confidence level 
** 95 % confidence level 

Number  of  individual 
outlying results* 

Significance of the trend** 

18 ºC 

None 

None 

60 ºC 

None 

None 

18 ºC 

60 ºC 

No 

No 

No 

Yes 

19 

 
Neither  technically  unexplained  nor  statistical  outliers  were  detected  for  both  analytes.  A 
significant  trend  at  60  °C  was  found  for  HCBD,  but  the  material  appeared  to  be  stable  at 
18 °C. 

The material shall be shipped under cooled conditions. 

5.2  Long-term stability study 

For the long-term stability study, data from two isochronous studies have been combined to 
assess the stability of the CRM. 

For the first isochronous study, four units were stored at 4 °C for 0, 4, 8 and 12 months. For 
the second isochronous study, four units were stored at 4 °C for 0, 8, 16 and 24 months. The 
reference  temperature  was  set  to  -20  °C  in  both  studies.  Two  units  per  storage  time  were 
selected using a random stratified sampling scheme. From each unit within each study, two 
samples  of  2 g  each  (wet mass)  were  measured  in-house  by  ASE,  SPE  clean-up  and  GC-
IDMS  [7,14].  The  measurements  were  performed  under  intermediate  precision  conditions 
(different  days)  in  a  random  sequence  to  be  able  to  separate  any  potential  analytical  drift 
from  a  trend  over  storage  time.  It  was  first  checked  if  there  was  a  significant  difference 
between  the  day  means  using  a  t-test  at  a  95  %  confidence  level  (measurements  spread 
over  two  days).  Significant  day-to-day  effect  was  present  for  HCBD  content  in  the  second 
isochronous  study  and  a  correction  was  applied  by  normalising  i.e.  dividing the  data  points 
by the respective day mean.  

To combine the two studies, the data of the first isochronous study was also normalised by 
dividing each data point by the mean of the study. 

The  results  were  screened  for  outliers  using  the  single  and  double  Grubbs  test  at  a 
confidence level of 99 %. One outlying individual result for HCBD was found. As no technical 
reason  for  this  outlier  could  be  found  all  data  were  retained  for  statistical  analysis.  No 
outlying individual results were found for HCB (Table 6).  

In  addition,  the  data  were  plotted  against  storage  time  and  linear  regression  lines  of  mass 
fraction  versus  time  were  calculated.  The  slopes  of  the  regression  lines  were  tested  for 
statistical  significance  (loss/increase  due  to  storage).  No  significant  trend  was  detected  for 
both analytes at a 95 % confidence level.  

The results of the long term stability measurements are shown in Annex D. The results of the 
statistical evaluation of the long-term stability study are summarised in Table 6. 

Table 6: Results of the statistical evaluation of the long-term stability tests (4 ºC) 

Analyte 

HCB 

HCBD 

* 99 % confidence level 
** 95 % confidence level 

Number  of 
outlying results* 

individual 

Significance of the trend**  

None 

One 

No 

No 

One  statistical  outlier  was  observed  for  HCBD  and  none  for  HCB.  None  of  the  trends  was 
statistically significant on a 95 % confidence level. The material can therefore be stored at 4 
°C.  

5.3  Estimation of uncertainties 

Due  to  the  intrinsic  variation  of  measurement  results,  no  study  can  entirely  rule  out 
degradation of materials, even in the absence of statistically significant trends. It is therefore 
necessary  to  quantify  the  potential  degradation  that  could  be  hidden  by  the  method 
repeatability,  i.e.  to  estimate  the  uncertainty  of  stability.  This  means  that,  even  under  ideal 

20 

 
conditions,  the  outcome  of  a  stability  study  can  only  be  that  there  is  no  detectable 
degradation within an uncertainty to be estimated.  

The uncertainties of stability during dispatch and storage were estimated as described in [17] 
for  HCB  and  HCBD.  In  this  approach,  the  uncertainty  of  the  linear  regression  line  with  a 
slope  of  zero  was  calculated.  The  uncertainty  contributions  usts  and  ults  were  calculated  as 
the product of the chosen transport time/shelf life and the uncertainty of the regression lines 
as: 

u

sts

,

rel

=

u

lts

,

rel

=

sts

RSD
(
∑
t

i

)

2

t

lts

RSD
(
∑
t

i

)

2

t

t

tt

t

sl

Equation 4 

Equation 5 

RSDsts 

RSDlts 

ti 

t  

ttt 

tsl 

relative standard deviation of all results of the short-term stability study 

relative standard deviation of all results of the long-term stability study 

time elapsed at time point i of the short-term stability study (for usts,rel) or long-
term stability study (for ults,rel) 

mean  of  all  ti  of  the  short-term  stability  study  (for  usts,rel)  or  long-term  stability 
study (ults,rel) 

chosen transport time (1 week at 18 ºC) 

chosen shelf life (36 months at 4 ºC) 

The following uncertainties were estimated: 

-  usts,rel,  the  uncertainty  of  degradation  during  dispatch.  This  was  estimated  from  the 
18 °C  studies.  The  uncertainty  describes  the  possible  change  during  a  dispatch  at 
18 °C lasting for one week. 

-  ults,rel,  the  stability  during  storage.  This  uncertainty  contribution  was  estimated  from 
the  4 °C  studies.  The  uncertainty  contribution  describes  the  possible  degradation 
during 36 months storage at 4 °C.  

The results of these evaluations are summarised in Table 7. 

Table 7: Uncertainties of stability during dispatch and storage. usts,rel was calculated for 
a temperature of 18 °C and 1 week; ults,rel was calculated for a storage temperature of 4 
°C and 36 months 

Measurand 

HCB 

HCBD 

usts ,rel 
[%] 
18 °C 

0.4 

0.5 

ults,rel 
[%] 
4 °C 

0.9 

2.6 

The  material  showed  significant  degradation  at  60  °C  but  no  significant  degradation  was 
observed for transport at or below 18 °C. Cooled shipment is therefore necessary. 

No trend was statistically significant on a 95 % confidence level. The material can therefore 
be stored at 4 °C.  

21 

 
(cid:215)
-
 
(cid:215)
-
 
 
 
After  the  certification  study,  the  material  will  be  included  in  the  IRMM's  regular  stability 
monitoring programme, to control its further stability. 

22 

 
 
 
6  Characterisation 
The material characterisation is the process of determining the property values of a reference 
material. 

The  material  characterisation  was  based  on  an  interlaboratory  comparison  of  expert 
laboratories,  i.e.  the  analytes  in  the  material  were  determined  in  different  laboratories  that 
applied  different  measurement  procedures  to  demonstrate  the  absence  of  a  measurement 
bias.  This  approach  aims  at  randomisation  of  laboratory  bias,  which  reduces  the  combined 
uncertainty. 

6.1  Selection of participants 

Eleven  laboratories  were  selected  based  on  criteria  that  comprised  both  technical 
competence  and  quality  management  aspects.  Each  participant  was  required  to  operate  a 
quality system and to deliver documented evidence of its laboratory proficiency in the field of 
HCB  and  HCBD  measurements  in  biota  matrices  by  submitting  results  for  intercomparison 
exercises or method validation reports. Having a formal accreditation was not mandatory, but 
meeting  the  requirements  of  ISO/IEC  17025  [3]  was  obligatory.  Where  measurements  are 
covered  by  the  scope  of  accreditation,  the  accreditation  number  is  stated  in  the  list  of 
participants (Section 0). 

6.2  Study setup 

Each  laboratory  received  two  units  of  ERM-CE100  and  was  requested  to  provide  six 
independent results reported relative to wet weight, three per unit. The units for the material 
characterisation were selected using a random stratified sampling scheme and covered the 
whole  batch.  For  each  unit,  the  sample  preparations  and  measurements  had  to  be  spread 
over  at  least  two  days  to  ensure  intermediate  precision  conditions.  An  independent 
calibration was performed for each day. Results are reported as µg/kg wet weight. 

Each participant received a sample of NIST SRM1947 Lake Michigan Fish Tissue, certified 
for  HCB  mass  fraction,  as  blind  method  quality  control  (M-QC)  and  an  iso-octane  solution 
containing  both  HCB  and  HCBD  as  blind  calibration  quality  control  (C-QC). The  C-QC  was 
gravimetrically  prepared  in-house  targeting  concentrations  of  HCB  and  HCBD  lying 
approximately in the middle of the envisaged calibration curve. The necessity of having also 
the  C-QC  was  due  to  the  absence  of  matrix  reference  materials  certified  for  HCBD.  The 
results for these samples were used to support the evaluation of the characterisation results. 

Laboratories  were  also  requested  to  give  estimations  of  the  expanded  uncertainties  of  the 
mean value of the six results. No approach for the estimation was prescribed, i.e. top-down 
and bottom-up were regarded as equally valid procedures. 

6.3  Methods used 

A  variety  of  extraction  [ASE,  Soxhlet,  ultrasonic  extraction  after  freeze  drying,  liquid  liquid 
extraction (LLE), organic solvent extraction] and clean-up methods [Florisil column, silica gel 
column, gel permeation chromatography (GPC)] with different quantification techniques (GC-
ECD,  GC-MS,  GC-HRMS,  GC-MS/MS)  were  used  to  characterise  the  material.  The 
combination  of  results  from  methods  based  on  completely  different  principles  mitigates 
undetected  method  bias.  Two  of  the  expert  laboratories  offered  two  different  analytical 
methods and these were handled as separate data sets. 

All  methods  used  during  the  characterisation  study  are  summarised  in  Annex  E.  The 
laboratory  code  (e.g.  L01)  is  a  random  number  and  does  not  correspond  to  the  order  of 
laboratories  in  Section  0.  For  the  laboratories  offering  two  different  analytical  methods,  the 
letter A and B was added to the laboratory code. The lab-method code consists of a number 
assigned  to  each  laboratory  and  abbreviation  of  the  measurement  method  used  (e.g.  L01-
GC-MS). 

23 

 
6.4  Evaluation of results 

The characterisation campaign resulted in thirteen datasets per analyte. All individual results 
of the participants, grouped per analyte, are displayed in tabular form in Annex F.  

6.4.1  Technical evaluation 
The  obtained  data  were  first  checked  for  compliance  with  the  requested  analysis  protocol 
and  for  their  validity  based  on  technical  reasons.  The  following  criteria  were  considered 
during the evaluation:  

-  appropriate validation of the measurement procedure 

- 

compliance  with  the  analysis  protocol:  sample  preparations  and  measurements,  as 
well as analytical sequence determination performed on two days. 

-  absence of values given as below limit of detection or below limit of quantification 

-  method performance, i.e.  

agreement of the measurement result with the assigned value of the C-QC sample. 
Values  reported  were  expected  to  remain  within  a  15  %  offset  from  the  assigned 
value (median value of measurement results from all laboratories) 

and 

agreement  of  the measurement  result  with  the  certified  value  of  HCB  in  SRM  1947 
(7.44 ± 0.66 µg/kg), according to ERM Application Note 1 [18]. 

Based  on  the  above  criteria,  the  following  datasets  were  rejected  as  not  technically  valid 
(Table 8).  

L04: the results for both HCB and HCBD were rejected because the measurement result of 
the  C-QC  sample  was  deviating  more  than  15  %  from  the  assigned  value;  in  addition  the 
measurement result for the M-QC sample did not agree with the certified value.  

L05: the results for HCB was rejected because the measurement result of the C-QC sample 
was deviating more than 15 % from the assigned value. 

L07:  the  results  for  HCBD  were  rejected  because  the  measurement  result  of  the  C-QC 
sample was deviating more than 15 % from the assigned value. 

L09: the results for both HCB and HCBD were rejected because the measurement result of 
the C-QC sample was deviating more than 15 % from the assigned value. 

L10: the results for both HCB and HCBD were rejected because the measurement result of 
the  C-QC  sample  was  deviating  more  than  15  %  from  the  assigned  value;  in  addition  the 
measurement result for the M-QC sample did not agree with the certified value. 

24 

 
 
 
 
 
Table 8: Datasets that showed non-compliances with the analysis' protocol and 
technical specifications, and action taken 

Analyte 

Lab code 

Description of problem 

Action taken 

HCB 

HCBD 

L04, L05, 
L09, L10 

Failure to measure C-QC 
sample (and M-QC sample 
for L04 and L10) 

L04, L07, 
L09, L10 

Failure to measure C-QC 
sample 

not used for 
evaluation 

not used for 
evaluation 

6.4.2  Statistical evaluation 
The  datasets  accepted  based  on  technical  reasons  were  tested  for  normality  of  dataset 
means  using  kurtosis/skewness  tests  and  normal  probability  plots  and  were  tested  for 
outlying  means  using  the  Grubbs  test  and  using  the  Cochran  test  for  outlying  standard 
deviations  (at  a  99  %  confidence  level).  Standard  deviations  within  (swithin)  and  between 
(sbetween)  laboratories  were  calculated  using  one-way  ANOVA.  The  results  of  these 
evaluations are shown in Table 9. 

Table 9: Statistical evaluation of the technically accepted datasets for ERM-CE100. p: 
number of technically valid datasets 

Analyte 

HCB 

HCBD 

Outliers 

Means  Variances 

No 

No 

Yes 
(L03B) 

No 

p 

9 

9 

Normally 
distributed 

Yes 

Yes 

Statistical parameters 

Mean 
[µg/kg] 

s 
[µg/kg] 

sbetween 
[µg/kg] 

swithin 
[µg/kg] 

120.211 

8.546 

8.249 

5.469 

36.365 

4.378 

4.229 

2.777 

The laboratory means follow normal distributions (at a 99 % confidence level). 

The  statistical  evaluation  flags  laboratory  L03B  as  outlying  variance  for  HCB.  This  merely 
reflects the fact that different methods have different intrinsic variability. As all measurement 
methods were found technically sound, all results were retained. 

The uncertainty related to the characterisation is estimated as the standard error of the mean 
of laboratory means (Table 10). 

Table 10: Uncertainty of characterisation for ERM-CE100 

Analyte 

HCB 

HCBD 

p 

9 

9 

Mean 
[µg/kg] 

s 
[µg/kg] 

uchar 
[µg/kg] 

120.211 

8.546 

2.844 

36.365 

4.378 

1.459 

uchar,rel 
[%] 

2.4 

4.0 

25 

 
 
 
 
 
 
7  Value Assignment 
Certified values were assigned. 

Certified values are values that fulfil the highest standards of accuracy. Procedures at IRMM 
require  generally  pooling  of  not  less  than  6  datasets  to  assign  certified  values.  Full 
uncertainty budgets in accordance with the ISO/IEC Guide 98-3 'Guide to the Expression of 
Uncertainty in Measurement' [4] were established.  

7.1  Certified values and their uncertainties 

The  unweighted  mean  of  the  means  of  the  accepted  datasets  as  shown  in  Table  10  was 
assigned as certified value for each parameter.  

The  assigned  uncertainty  consists  of  uncertainties  related to  characterisation,  uchar  (Section 
0), potential between-unit inhomogeneity, ubb (Section 4.1) and potential degradation during 
transport (usts) and long-term storage, ults (Section 0). The uncertainty related to degradation 
during  transport  (usts)  was  found  to  be  negligible.  These  different  contributions  were 
combined to estimate the expanded, relative uncertainty of the certified value (UCRM, rel) with a 
coverage factor k as:  

U

CRM,

rel

(cid:215)=
k

u

2
char,

rel

+

u

2
 bb,

rel

+

u

2
lts,

rel

-  uchar was estimated as described in Section 6 

-  ubb was estimated as described in Section 4.1. 

-  ults was estimated as described in Section 5.3.  

Equation 6 

Because  of  the  sufficient  number  of  the  degrees  of  freedom  of  the  different  uncertainty 
contributions, a coverage factor k of 2 was applied, to obtain the expanded uncertainties.  

The certified values and their uncertainties are summarised in Table 11. 

Analyte 

Table 11: Certified values and their uncertainties for ERM-CE100 
Certified value 1) 
[µg/kg]  

UCRM, rel 
[%] 

uchar, rel 
[%] 

ubb, rel 

ults, rel 

UCRM 

[%] 

[%] 

[µg/kg] 

2) 

HCB 

120 

2.4 

2.0 

0.9 

6.5 

8 

HCBD 
1.3 
1) The certified values and their uncertainties are expressed as mass fractions relative to wet weight 
2) Expanded (k = 2) and rounded uncertainty 

9.9 

4.0 

2.6 

36 

4 

7.2  Additional material information 

The  data  provided  in  this  section  should  be  regarded  as  informative  only  on  the  general 
composition of the material and cannot be, in any case, used as certified or indicative value. 

An  additional  material  information  value  was  assigned  for  the  fat  content  of  ERM-CE100. 
Three CRM units were randomly selected over the whole batch and for each unit a sample of 
1  g  was  processed  in-house  according  to  the  following  procedure  [7,14]:  after  ASE,  the 
extract was concentrated under nitrogen stream and finally placed in an oven at 105 °C until 
constant  mass  was  reached.  In  Table  12,  the  extractable  fat  content  is  expressed  as  the 
mean of the three replicates and given in mass fraction % (equivalent to g/100 g) relative to 
wet weight of ERM-CE100. 

Table 12: Fat content of ERM-CA100 (as additional material information) 

Extractable fat  

Value [% m/m] 

5.9 

26 

 
 
 
 
 
8  Metrological traceability and commutability 

8.1  Metrological traceability  

Identity 

HCB  and  HCBD  are  chemically  clearly  defined  analytes.  The  participants  used  different 
methods for the sample preparation as well as for the final detection, demonstrating absence 
of  measurement  bias.  All  participants  used  GC-based  analytical  methods,  therefore  the 
measurands are operationally defined by GC. 

Quantity value 

Only  validated  methods  were  used  for  the  determination  of  the  assigned  values.  Different 
calibrants of known purity and specified traceability of their assigned values were used and 
all relevant input parameters were calibrated. The individual results are therefore traceable to 
the  International  System  of  units  (SI),  as  it  is  also  confirmed  by  the  agreement  among  the 
technically accepted datasets. As the assigned values are combinations of agreeing results 
individually traceable to the SI, the assigned quantity values themselves are traceable to the 
SI as well. 

8.2  Commutability 

Many measurement procedures include one or more steps, which are selecting specific (or 
specific  groups  of)  analytes  from  the  sample  for  the  subsequent  whole  measurement 
process.  Often  the  complete  identity  of  these  'intermediate  analytes'  is  not  fully  known  or 
taken into account. Therefore, it is difficult to mimic all the analytically relevant properties of 
real  samples  within  a  CRM.  The  degree  of  equivalence  in  the  analytical  behaviour  of  real 
samples  and  a  CRM  with  respect  to  various  measurement  procedures  (methods)  is 
summarised  in  a  concept  called  'commutability  of  a  reference  material'.  There  are  various 
definitions that define this concept. For instance, the CLSI Guideline C-53A [19] recommends 
the use of the following definition for the term commutability: 

"The  equivalence  of  the  mathematical  relationships  among  the  results  of  different 
measurement procedures for an RM and for representative samples of the type intended 
to be measured." 

The  commutability  of  a  CRM  defines  its  fitness  for  use  and  is  therefore  a  crucial 
characteristic  when  applying  different  measurement  methods. When  the  commutability  of  a 
CRM  is  not  established,  the  results  from  routinely  used  methods  cannot  be  legitimately 
compared with the certified value to determine whether a bias does not  exist in calibration, 
nor can the CRM be used as a calibrant. 

It should be borne in mind that the methods used in the characterisation of ERM-CE100 are 
methods routinely applied for measuring HCB and HCBD in biota matrices. The agreement 
of results from different methods demonstrates that ERM-CE100 behaves like a real sample. 

ERM-CE100  was  produced  from  naturally  contaminated  wild  fish  specimens  by  cryogenic 
milling, mixing and cooking to produce a sterilised paste, enhancing the commutability of the 
material  by  avoiding  any  freeze  drying  process.  The  analytical  behaviour  should  match  as 
close as possible a routine biota sample.  

27 

 
 
 
9  Instructions for use 

9.1  Safety information 

The usual laboratory safety measures apply. 

9.2  Storage conditions 

The material should be stored at 4 °C ± 3 °C in the dark. 

Please  note  that  the  European  Commission  cannot  be  held  responsible  for  changes  that 
happen during storage of the material at the customer's premises, especially of opened units. 

9.3  Preparation and use of the material 

Before analysis, ERM-CE100 units should be left to equilibrate to room temperature. 

To  make  it  ready  for  use  and  before  sub-sampling,  the  material  has  to  be  thoroughly  and 
manually re-homogenised with the help of a spatula. In case that a small quantity of oxidised 
material is observed attached to the lid, it is advisable not to include it. 

9.4  Minimum sample intake 

The minimum sample intake representative for both analytes is 1 g (wet mass).  

9.5  Use of the certified value 

The  main  purpose  of  this  material  is  to  assess  method  performance,  i.e.  for  checking 
accuracy  of  analytical  results.  As  any  reference  material,  it  can  be  used  for  establishing 
quality control charts or validation studies. 

Use as a calibrant 

It is not recommended to use this matrix material as calibrant.  

Comparing an analytical result with the certified value 

A result is unbiased if the combined standard uncertainty of measurement and certified value 
covers the difference between the certified value and the measurement result (see also ERM 
Application Note 1, www.erm-crm.org [19].  

When assessing the method performance, the measured values of the CRM are compared 
with the certified values. The procedure is summarised here:  

-  Calculate  the  absolute  difference  between  mean  measured  value  and  the  certified 

value (D meas). 

-  Combine 

the  measurement  uncertainty  (umeas)  with 

the  uncertainty  of 

the  

certified value (uCRM): 

=D
u

+
2
meas u
u

2
CRM

-  Calculate the expanded uncertainty (UD ) from the combined uncertainty (uD

,) using an 
appropriate coverage factor, corresponding to a level of confidence of approximately 
95 % 
If D meas £  UD  no significant difference exists between the measurement result and the 
certified value, at a confidence level of about 95 %. 

- 

Use in quality control charts 

The  materials  can  be  used  for quality  control  charts.  Using  CRMs  for quality  control  charts 
has the added value that a trueness assessment is built into the chart.  

28 

 
 
10  Acknowledgements 
The  authors  would  like  to  acknowledge  the  support  received  from  Hanne  Leys,  Jean 
Charoud-Got,  Katharina  Teipel,  Marie-France  Tumba-Tshilumba,  Albert  Oostra  and  Patrick 
Conneely  from  IRMM  relating  to  the  processing  of  this  CRM  and  from  Maria  Concepción 
Contreras  Lopez  concerning  the  set-up  of  the  required  isochronous  studies.  B.  Binici 
(TÜBITAK UME, Turkey) is acknowledged for laboratory work in connection with the project. 

Furthermore, the authors would like to thank Saioa Elordui-Zapatarietxe and Yannic Ramaye 
(IRMM) for the reviewing of the certification report, as well as the experts of the Certification 
Advisory  Panel  "small  organic  molecules  and  operationally  defined  parameters  in  food 
analysis",  S.  De  Saeger  (Ghent  University,  BE),  L.  Alder  (formerly  Bundesinstitut  für 
Risikobewertung,  DE)  and  S.  van  Leeuwen  (RIKILT,  Wageningen  UR,  NL)  for  their 
constructive comments. 

29 

 
11 References 

1  

2  

3  

4  

5  

6  

7  

8 

9 

ISO  Guide  34,  General  requirements  for  the  competence  of  reference  materials 
producers, International Organization for Standardization, Geneva, Switzerland, 2009. 

ISO Guide 35, Reference materials – General and statistical principles for certification, 
International Organization for Standardization, Geneva, Switzerland, 2006. 

ISO/IEC  17025:2005,  General  requirements  for  the  competence  of  testing  and 
for  Standardization,  Geneva, 
calibration 
Switzerland, 2005. 

International  Organization 

laboratories, 

ISO/IEC  Guide  98-3,  Guide  to  the  Expression  of  Uncertainty  in  Measurement,  (GUM 
1995), International Organization for Standardization, Geneva, Switzerland, 2008. 

C.F.  Mumma,  E.W.  Lawless,  Survey  of 
I-
hexachlorobenzene  and  hexachlorobutadiene  pollution  for  chlorocarbon  processes 
(EPA 560/3-75-003), US EPA, Washington, DC7, 1975. 

industrial  processing  data.  Task 

Canadian  Environmental  Protection  Act,  Priority  Substances  List  Assessment  Report, 
Environment Canada, 1999. 

R. Lava, L.I. Majoros, I. Dosis, M. Ricci, A practical example of the challenges of biota 
monitoring under the Water Framework Directive, TrAC, 59 (2014) 103-111. 

A.P  van  Birgelen,  Hexachlorobenzene  as  a  possible  major  contributor  to  the  dioxin 
activity of human milk, Environ. Health Perspect. 106 (1998) 683-688. 

K.  Weber,  H.  Goerke,  Persistent  Organic  Pollutants  (POPs)  in  Antarctic  fish:  levels, 
patterns, changes, Chemosphere 53 (2003) 667-678. 

10  Directive  2000/60/EC  of  the  European  Parliament  and  of  the  Council  of  23  October 
2000 establishing a framework for Community action in the field of water policy, Off. J. 
Eur. Comm. L 327 (2000) 1. 

11  Directive 2008/105/EC of the European Parliament and of the Council of 16 December 
2008 concerning environmental quality standards in the field of water policy, Off. J. Eur. 
Union L 348 (2008) 84. 

12  Directive 2013/39/EU of the European Parliament and of the Council of 12 August 2013 
amending  Directives  2000/60/EC  and  2008/105/EC  as  regards  priority  substances  in 
the field of water policy, Off. J. Eur. Union L 226 (2013) 119. 

13  Commission Directive 2009/90/EC of 31 July 2009, laying down, pursuant to Directive 
2000/60/EC of the European Parliament and of the Council, technical specifications for 
chemical analysis and monitoring of water status, Off. J. Eur. Union L 201, (2009) 36. 

14 

L.I.  Majoros,  R.  Lava,  M.  Ricci,  B.  Binici,  F.  Sandor,  A.  Held,  H.  Emons,  Full  method 
validation for the determination of hexachlorobenzene and hexachlorobutadiene in fish 
tissue by GC-IDMS, Talanta 116 (2013) 251-258. 

15  T.P.J.  Linsinger,  J.  Pauwels,  A.M.H.  van  der  Veen,  H.  Schimmel,  A.  Lamberty, 
Homogeneity and stability of reference materials, Accred. Qual. Assur. 6 (2001) 20-25. 

16  A. Lamberty, H. Schimmel, J. Pauwels, The study of the stability of reference materials 

by isochronous measurements, Fres. J. Anal. Chem. 360 (1998) 359-361. 

17  T.P.J.  Linsinger,  J.  Pauwels,  A.  Lamberty,  H.  Schimmel,  A.M.H.  van  der  Veen,  L. 
Siekmann, Estimating the uncertainty of stability for matrix CRMs, Fres. J. Anal. Chem. 
370 (2001) 183-188. 

18  T.P.J.  Linsinger,  ERM  Application  Note  1:  Comparison  of  a  measurement  result  with 

the certified value, www.erm-crm.org (last accessed on 19/12/2014). 

30 

 
 
19  H.  Vesper,  H.  Emons,  M.  Gnezda,  C.  P.  Jain,  W.  G.  Miller,  R.  Rej,  G.  Schumann,  J. 
Tate,  L.  Thienpont,  J.  E.  Vaks,  Characterization  and  Qualification  of  Commutable 
Reference  Materials  for  Laboratory  Medicine;  Approved  Guideline,  CLSI  document 
EP30-A, Clinical and Laboratory Standards Institute, Wayne, PA, USA, 2010. 

31 

 
 
 
Annexes 

Annex A: Results of the homogeneity measurements 

•  The  graphs  report  mass  fraction  unit  means  (normalised  for  HCBD)  ±  95  % 

confidence interval (CI) of the means. 

32 

 
 
 
 
 
Annex B: Measurement results of the minimum sample intake study 

Hexachlorobenzene (HCB) µg/kg (relative to wet weight) 

2 g 
Unit 282 
118.00 
117.00 
124.00 

Unit 959 
115.00 
127.00 
124.00 

1 g 
Unit 287 
114.00 
94.00 
84.10 

Unit 100 
99.90 
104.00 
97.00 

0.5 g 
Unit 851 
105.00 
116.00 
98.50 

Unit 667 
82.80 
87.00 
99.80 

RSD 

4.0 % 

10.1 % 

12.3 % 

Hexachlorobutadiene (HCBD) µg/kg (relative to wet weight) 

2 g 
Unit 282 
36.90 
38.80 
40.00 

Unit 959 
37.20 
38.10 
41.50 

1 g 
Unit 287 
34.20 
33.50 
31.50 

Unit 100 
35.10 
34.90 
35.80 

0.5 g 
Unit 851 
35.80 
39.40 
34.00 

Unit 667 
35.20 
33.60 
38.80 

RSD 

4.5 % 

4.5 % 

6.7 % 

33 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Annex C: Results of the short-term stability measurements 

•  Data  for  the  short-term  stability  study  at  18  °C.  The  graphs  report  mass  fraction 

means per time point (normalised for HCB) ± 95 % CI of the means. 

34 

 
 
 
 
 
•  Data  for  the  short-term  stability  study  at  60  °C.  The  graphs  report  mass  fraction 

means per time point (normalised for HCB) ± 95 % CI of the means. 

35 

 
 
 
 
 
 
Annex D: Results of the long-term stability measurements 

•  Data  for  the  long-term  stability  study  at  4  °C.  The  graphs  report  normalised  mass 

fraction means per time point ± 95 % CI of the means. 

36 

 
 
 
 
 
Annex E: Summary of methods used in the characterisation study 

•  Method information is reported as given by laboratories 

Laboratory 
code–method 

Sample pre-
treatment 

Detection method 

Type of calibration 

LOQ  

Calibrants’ details (supplier 
and purity) 

[µg/kg wet 
weight] 

L00-GC-MS 

accelerated solvent 
extraction (ASE), 
clean-up by SPE 
(solid phase 
extraction) column 

GC-IDMS  

13C6-HCB and 13C4-
HCBD as internal 
standards 

L01A-GC-ECD 

Soxhlet extraction, 
clean-up with alumina 
and silica columns 

GC-ECD 

PCB 112 as internal 
standard 

L01B-GC-MS 

ASE (inline fat 
removal with Florisil), 
clean-up with alumina 
and silica columns 

GC-MS  

PCB 112 as internal 
standard 

L02-GC-HRMS 

ASE, clean-up with 1) 
silica and alumina 
column, 2) C18-
modified silica column 

GC-ID-HRMS 

13C6-HCB and 13C4-
HCBD as internal 
standards 

L03A-GC-ECD 

ASE, clean-up with 
alumina 

L03B-GC-MS 

ASE, clean-up with 
alumina 

GC-ECD 

PCB 103 as internal 
standard (used for 
recovery) 

GC-MS 

13C6-HCB and 13C4-
HCBD as internal 
standards (used for 
recovery) 

5 points internal standard 
calibration 

HCB (purity 99.5 %) and 
HCBD (purity 99 %) by Dr. 
Ehrenstorfer. 

Quadratic calibration curve 

Pesticide standard in iso-
octane by Accustandard, Inc. 
Purity: HCBD 98.1 %, HCB: 
100 % 

Quadratic calibration curve 

Pesticide standard in iso-
octane by Accustandard, Inc. 
Purity: HCBD 98.1 %, HCB: 
100 % 

Single point 

HCB solution by Wellington 
Laboratories, > 98 % 

HCBD solution by Dr. 
Ehrenstorfer, 99 %. 

8 points external standard 
calibration curve  

Pesticide standard in iso-
octane by Accustandard, Inc. 
Purity: HCBD 98.1 %, HCB: 
100 % 

8 points external standard 
calibration curve  

Pesticide standard in iso-
octane by Accustandard, Inc. 
Purity: HCBD 98.1 %, HCB: 
100 % 

L05-GC-MS/MS 

Extraction with 
ethylacetate and 
centrifugation, clean-
up by gel permeation 
chromatography 
(GPC)  

GC-MS/MS 

PCB 137 as internal 
standard 

6 points calibration curve 

HCB by Dr. Ehrenstorfer, 
purity 99.5 % 

HCBD by Sigma-Aldrich, purity 
97.7 % 

HCB: 3.7 

HCBD:15.7 

HCB: 1.35 

HCBD:1.35 

HCB:  1.6 

HCBD:  1.6 

HCB: 0.02 

HCBD:0.1 

HCB: 0.7 

HCBD:0.7 

HCB: 0.3 
(LOD) 

HCBD:0.3 
(LOD)  

HCB: 7 

HCBD: 7 

37 

 
 
L06-GC-MS 

ASE, GPC clean-up  

L07-GC-HRMS 

Acid digestion, liquid 
liquid extraction, 
clean-up with Florisil  

GC-IDMS 

13C6-HCB and 13C4-
HCBD as internal 
standards 

GC-ID-HRMS  

13C6-HCB and 13C4-
HCBD as internal 
standards 

6-7 points internal standard 
calibration 

HCB (purity 99.5 %) and 
HCBD (purity 99.0 %) by Dr. 
Ehrenstorfer 

7 points internal standard 
calibration 

HCB and HCBD from 
Cambridge Isotope 
Laboratories, ≥ 98 % 

HCB: 1.4 

HCBD: 3.7 

HCB: 0.004 

HCBD: 0.015 

L08-GC- 
MS/MS 

Extraction with 
organic solvent, 
clean-up with 
Kieselguhr/H2SO4 

GC- MS/MS  

13C6-HCB and 13C4-
HCBD as internal 
standards 

5 points external standard 
calibration curve 

HCB and HCBD solutions from 
Neochema, 99 % 

HCB: 1 

HCBD: 1 

Not used in certification 

L04-GC-MS 

Freeze-drying of the 
sample; ultrasonic 
extraction and clean-
up with Florisil SPE 

GC-MS  

13C6-HCB as internal 
standard for both 
analytes 

L09-GC-ECD 

Soxhlet, clean-up with 
Florisil column  

GC-ECD 

no internal standard 

L10-GC-MS 

Pressurised liquid 
extraction, clean-up 
with Florisil column  

GC-MS  

13C6-HCB as internal 
standard for both 
analytes 

8 points internal standard 
calibration 

HCB from Supelco,  99.6 % 

HCBD solution from Supelco, 
99 % 

5 points external calibration 
curve 

Mix organochlorine pesticides 
solution from ULTRA 
SCIENTIFIC (purity not 
specified) 

7 points internal standard 
calibration 

HCB from Fluka/ Riedel-de-
Haën, > 99.6 % 

HCBD solution from Supelco 
(purity not specified) 

HCB: 0.01 

HCBD: 0.02 

HCB: 0.02 

HCBD: 0.12 

HCB: 0.4 

HCBD: 1.5 

N.B. the purity values refer to the neat substances, unless otherwise specified 

38 

 
 
 
Annex F: Results of the characterisation measurements 

 Note: values as reported by the laboratory and expressed relative to wet weight 

Hexachlorobenzene 

Laboratory  
code - Method 

replicate 
1 
[µg/kg] 

replicate 
2 
[µg/kg] 

replicate 
3 
[µg/kg] 

replicate 
4 
[µg/kg] 

replicate 
5 
[µg/kg] 

replicate 
6 
[µg/kg] 

mean 

[µg/kg] 

Expanded  
uncertainty 
[µg/kg] 

L00-GC-MS 

120.84 

122.10 

121.02 

118.73 

120.04 

120.17 

120.48 

12.65 

L01A-GC-ECD 

131.82 

124.66 

118.65 

120.03 

128.29 

129.88 

125.56 

49.09 

L01B-GC-MS 

119.33 

118.54 

113.28 

115.33 

108.41 

109.98 

114.15 

42.46 

L02-GC-HRMS 

118.00 

117.00 

124.00 

115.00 

127.00 

124.00 

121.00 

35.00 

L03A-GC-ECD 

130.09 

139.82 

140.46 

130.50 

137.69 

139.07 

136.27 

54.51 

L03B-GC-MS 

116.73 

143.40 

113.43 

112.07 

112.53 

118.15 

119.39 

23.88 

L06-GC-MS 

105 

99 

104 

109 

106 

102 

104 

5 

L07-GC-HRMS 

120.70 

124.60 

119.80 

117.18 

118.09 

116.48 

119.48 

18.40 

L08-GC-MS 

119.73 

121.27 

125.14 

117.38 

125.00 

121.00 

121.59 

21.89 

Results not used for certification 

L04-GC-MS 

77.32 

85.79 

81.87 

88.89 

87.90 

90.72 

85.42 

6.58* 

L05-GC-MS/MS 

154.00 

142.50 

152.30 

141.90 

153.60 

157.70 

150.33 

60.13  

L09-GC-ECD 

95.58 

97.86 

88.03 

93.70 

83.06 

86.19 

90.74 

26.32 

L10- GC-MS 

90.46 

82.68 

90.65 

79.01 

86.16 

78.58 

84.59 

23.69 

* inter-day variability 

Hexachlorobenzene

]
g
k
/
g
µ
[
n
o
i
t
c
a
r
f

s
s
a
M

250

200

150

100

50

0

L00

L01A

L01B

L02

L03A

L03B

L06

L07

L08

N.B. error bars represent expanded uncertainties 

39 

laboratory mean

Certified value

+ U CRM

- U CRM

 
 
 
 
 
 
 
 
 
 
 
 
 
Hexachlorobutadiene 

Laboratory  
code - Method 

replicate 
1 
[µg/kg] 

replicate 
2 
[µg/kg] 

replicate 
3 
[µg/kg] 

replicate 
4 
[µg/kg] 

replicate 
5 
[µg/kg] 

replicate 
6 
[µg/kg] 

mean 

[µg/kg] 

Expanded  
uncertainty 
[µg/kg] 

L00-GC-MS 

34.60 

34.88 

35.80 

35.15 

34.42 

35.83 

36.61 

4.39 

L01A-GC-ECD 

31.82 

29.94 

32.36 

32.06 

33.57 

32.40 

32.03 

4.16** 

L01B- GC-MS 

36.24 

39.55 

39.30 

32.58 

43.25 

42.48 

38.90 

3.19** 

L02-GC-HRMS 

36.90 

38.80 

40.00 

37.20 

38.10 

41.50 

38.75 

11.24 

L03A-GC- ECD 

39.68 

39.75 

43.38 

41.42 

42.65 

42.89 

41.63 

16.65 

L03B-GC-MS 

33.97 

33.40 

34.83 

27.05 

23.12 

28.77 

30.19 

9.36 

L05-GC-MS/MS 

32.60 

31.90 

32.40 

31.10 

25.10 

33.40 

31.08 

12.43 

L06-GC-MS 

37.3 

35.8 

36.5 

37.5 

36.3 

34.9 

36.4 

2.9 

L08-GC-MS 

41.06 

41.75 

43.79 

40.61 

42.65 

40.41 

41.71 

7.51 

Results not used for certification 

L04-GC-MS 

5.47 

6.17 

5.73 

5.05 

5.17 

4.76 

5.39 

0.43* 

L07-GC-HRMS 

82.75 

83.37 

84.92 

86.30 

86.06 

87.09 

85.08 

not given 

L09-GC-ECD 

23.47 

25.58 

25.43 

21.85 

28.46 

27.59 

25.40 

L10- GC-MS 

5.38 

5.32 

5.48 

6.77 

5.88 

6.85 

5.95 

8.13 

1.67 

* inter-day variability 

** within-laboratory reproducibility 

Hexachlorobutadiene

]
g
k
/
g
µ
[
n
o
i
t
c
a
r
f

s
s
a
M

70

60

50

40

30

20

10

0

L00

L01A

L01B

L02

L03A

L03B

L05

L06

L08

N.B. error bars represent expanded uncertainties 

40 

laboratory mean

Certified value

+ U CRM

- U CRM

 
 
 
 
 
 
 
 
 
 
 
 
 
 
European Commission 

EUR 27965 EN – Joint Research Centre – Institute for Reference Materials and Measurements 
Title:  

The certification of the mass fractions of hexachlorobenzene (HCB) and hexachlorobutadiene (HCBD) in 
fish tissue: ERM®-CE100  

Author(s): I. Dosis, M. Ricci, L. I. Majoros, R. Lava, J. Seghers, H. Emteborg, A. Held, H. Emons 
Luxembourg: Publications Office of the European Union 
2016 – 40 pp. – 21.0 x 29.7 cm 
EUR – Scientific and Technical Research series – ISSN 1831-9424 
ISBN 978-92-79-59111-2 
doi: 10.2787/359451 

 
 
 
 
 
 
 
As the Commission's in-house science service, the Joint Research Centre's mission is to provide EU policies 
with independent, evidence-based scientific and technical support throughout the whole policy cycle. 

Working in close cooperation with policy Directorates-General, the JRC addresses key societal challenges 
while stimulating innovation through developing new methods, tools and standards, and sharing its know-
how with the Member States, the scientific community and international partners. 

Key policy areas include: environment and climate change; energy and transport; agriculture and food 
security; health and consumer protection; information society and digital agenda; safety and security, 
including nuclear; all supported through a cross-cutting and multi-disciplinary approach. 

L
A
-
N
A
-
2
7
9
6
5
-
E
N
-
N

doi: 10.2787/359451 

ISBN: 978-92-79-59111-2