Certification of the taxon Gadus morhua (Atlantic cod) in fish powder : EURM®-023.
Date: 2025-10-09
Institutions: Joint Research Centre
Subjects: fish, genetics, organic certification, report, research method, sea fish, testing

Certification of the taxon Gadus morhua  
(Atlantic cod) in fish powder:  
EURM®-023 

A.M. Kortekaas, H. Leys, J. Seghers and S. Trapmann 

2025 

 
 
 
 
 
 
 
 
 
This  document  is  a  publication  by  the  Joint  Research  Centre  (JRC),  the  European  Commission’s  science  and  knowledge 
service. It aims to provide evidence-based scientific support to the European policymaking process. The contents of this 
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of its frontiers or boundaries. 

Contact information 
Name: EC, Joint Research Centre, Directorate F – Health and Food, Reference Materials Unit 
Address: Retieseweg 111, 2440 Geel, Belgium 
Email: JRC-RM-DISTRIBUTION@ec.europa.eu 
Tel.: 0032/14/571705 
Website : https://crm.jrc.ec.europa.eu  

EU Science Hub 
https://joint-research-centre.ec.europa.eu  

JRC143451 

PDF  

ISBN 978-92-68-31076-2  doi:10.2760/3032612 

KJ-01-25-420-EN-N 

Luxembourg: Publications Office of the European Union, 2025 
© European Union, 2025 

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How to cite this report: Kortekaas, A.M., Leys, H., Seghers, J. and Trapmann, S., Certification of the taxon Gadus morhua 
(Atlantic  cod) 
in  fish  powder:  EURM®-023,  Publications  Office  of  the  European  Union,  Luxembourg,  2025, 
https://data.europa.eu/doi/10.2760/3032612, JRC143451. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Contents  

Abstract ................................................................................................................................................................................................................................. 3 

Acknowledgements ...................................................................................................................................................................................................... 4 

1 

Introduction ................................................................................................................................................................................................................ 5 

1.1  Background ..................................................................................................................................................................................................... 5 

1.2  Choice of the material ........................................................................................................................................................................... 6 

1.3  Outline of the CRM project ................................................................................................................................................................ 7 

2  Participants ................................................................................................................................................................................................................. 8 

2.1  Project management, processing and data evaluation ............................................................................................. 8 

2.2  Homogeneity measurements .......................................................................................................................................................... 8 

2.3  Stability measurements ....................................................................................................................................................................... 8 

2.4  Characterisation measurements .................................................................................................................................................. 8 

3  Material processing and processing control ................................................................................................................................... 9 

3.1  Origin/Purity of the starting material ....................................................................................................................................... 9 

3.2  Processing ........................................................................................................................................................................................................ 9 

3.3  Processing control .................................................................................................................................................................................. 10 

3.4  Suitability, DNA extraction .............................................................................................................................................................. 11 

4  Homogeneity .......................................................................................................................................................................................................... 12 

4.1  Between-unit homogeneity ............................................................................................................................................................ 12 

4.2  Within-unit homogeneity and minimum sample size .............................................................................................. 13 

5  Stability ....................................................................................................................................................................................................................... 14 

5.1  Transport stability .................................................................................................................................................................................. 14 

5.2  Storage stability ...................................................................................................................................................................................... 15 

6  Characterisation .................................................................................................................................................................................................. 17 

6.1  Selection of participants ................................................................................................................................................................... 17 

6.2  Study setup .................................................................................................................................................................................................. 17 

6.3  Methods used ............................................................................................................................................................................................ 17 

6.4  Evaluation of results ............................................................................................................................................................................ 18 

6.4.1  Technical evaluation ............................................................................................................................................................. 18 

6.4.2  Evaluation of the genetic identification using the cytb barcode .................................................... 19 

6.4.3  Evaluation of the genetic identification using the COI barcode ...................................................... 22 

7  Value Assignment .............................................................................................................................................................................................. 25 

8  Metrological traceability and commutability .............................................................................................................................. 26 

8.1  Metrological traceability ................................................................................................................................................................... 26 

8.1.1 

Identity ............................................................................................................................................................................................. 26 

8.1.2  Qualitative property value ............................................................................................................................................... 26 

1 

 
 
8.2  Commutability ........................................................................................................................................................................................... 27 

9 

Instructions for use ........................................................................................................................................................................................... 28 

9.1  Safety information ................................................................................................................................................................................ 28 

9.2  Storage conditions ................................................................................................................................................................................. 28 

9.3  Use and reconstitution of the material ................................................................................................................................ 28 

9.4  Minimum sample size ......................................................................................................................................................................... 28 

9.5  Use of the certified value ................................................................................................................................................................ 28 

10 Conclusions .............................................................................................................................................................................................................. 29 

References ....................................................................................................................................................................................................................... 30 

List of abbreviations ................................................................................................................................................................................................ 36 

List of boxes ................................................................................................................................................................................................................... 38 

List of figures ................................................................................................................................................................................................................ 39 

List of tables .................................................................................................................................................................................................................. 40 

Annexes .............................................................................................................................................................................................................................. 41 

Annex 1. Summary of PCR methods used ..................................................................................................................................... 41 

Annex 2. Results of homogeneity measurements .................................................................................................................. 44 

Annex 3. Results of the short-term stability measurements ........................................................................................ 49 

Annex 4. Results of the long-term stability measurements .......................................................................................... 50 

Annex 5. Results of the characterisation measurements ................................................................................................ 51 

2 

 
 
 
 
 
 
Abstract 

EURM-023 is a certified reference material (CRM), consisting of a powder produced from fish fillet. It 
is certified for its taxon, the species Gadus morhua (Atlantic cod), in accordance with ISO 17034:2016. 
A certified qualitative property value was assigned as outlined in ISO 33406:2024. 

The material was produced at the European Commission’s (EC) Joint Research Centre (JRC) from one 
fillet originating from a single fish, which was diced, freeze-dried and milled. EURM-023 is available 
in glass vials, each containing at least 200 mg of fish powder and sealed under an atmosphere of 
argon. 

The between-unit homogeneity and stability during transport and storage were assessed following 
the recommendations in ISO 33406:2024. 

The  material  was  characterised  by  comparison  of  measurement  results  from  two  different 
mitochondrial DNA barcoding methods targeting the cytochrome b gene (cytb) and the cytochrome c 
oxidase  subunit  1  gene  (COI),  obtained  by  two  laboratories,  adhering  to  ISO/IEC  17025.  The 
uncertainty of the certified value is negligible. 

This material is intended  for use in method validation, specifically to assess the accuracy of DNA 
barcoding methods for the identification of the fish species Gadus morhua. 

Before release of the CRM, the certification project was subjected to peer-review involving JRC internal 
experts. 

3 

 
 
Acknowledgements 

The  authors  would  like  to  acknowledge  the  support  received  from  colleagues  of  JRC  for  the 
processing,  organising  of  stability  studies,  measuring,  reviewing  of  the  certification  project  and 
distribution of this CRM. 

Authors 

A.M. Kortekaas, H. Leys, J. Seghers and S. Trapmann  

European Commission, Joint Research Centre, Geel, BE 

4 

 
 
 
 
1 

Introduction 

1.1 Background 

To improve transparency in the fishery and aquaculture supply chain, the European Union (EU) has 
introduced Regulation (EU) No 1379/2013 [1] on the common organisation of markets for fishery and 
aquaculture  products.  Article  35  sets  out  the  rules  on  the  mandatory  marking  or  labelling  of  pre-
packed or non-prepacked fishery and aquaculture products. This includes indication of the commercial 
name of the species and its scientific name.   
Labelling of fish species may be wrongly applied, inadvertently or deliberately, e.g. when species of 
higher  value  are  substituted  with  lower  value  species.  Additionally,  species  from  unsustainable  or 
illegal  fisheries  may  enter  the  market  through  substitution  [2].  Furthermore,  correct  labelling  is 
important to avoid health risks, for instance, in relation to fish allergies [3]. 
Inspections  of  the  quality  and  authenticity  of  fresh  or  prepared  fish  products  on  the  market  are 
conducted to discourage operators from falsely labelling catches or products. These inspections are 
carried  out  following  European  regulations  such  as  Regulation  (EC)  No 1224/2009  [4]  which  was 
amended in (EU) 2023/2842 [5] and both set out a control regime, comprising the monitoring and 
inspection of the whole chain of fishery production and marketing while Regulation (EC) No 178/2002 
[6] covers the labelling of food products to avoid misleading consumers and the traceability of food 
products.  Regulation  (EC)  No  1005/2008  [7]  is  set  out  to  prevent,  deter  and  eliminate  illegal, 
unreported and unregulated fishing whereas Regulation (EU) No 2017/625 [8] is on official controls 
and other official activities performed to ensure the application of food and feed law.  
Morphological  identification  can  generally  only  be  applied  on  whole  fish.  Once  the  fish  has  been 
filleted or processed, fish species  are  identified mostly by using DNA-based  methods.  The current 
DNA-based  techniques  used  for  the  genetic  identification  of  fish  products  are  polymerase  chain 
reaction (PCR) and sequencing. PCR requires species-specific primers, while for sequencing, universal 
primers are used for specific genes/regions of fish mitochondrial DNA (mtDNA). The mutation rate for 
mtDNA  is  high  and  consequently,  significant  sequence  variation  can  be  observed  between  closely 
related  species  [9,  10].  The  specific  genes  or  parts  of  genes  on  the  mtDNA  that  are  found  to  be 
reliable for species identification are called DNA barcodes [10]. In addition, one cell contains many 
mitochondria and therefore mtDNA is present in cells at higher copy numbers compared to nuclear 
DNA.  The  variability  between  species  and  ease  of  amplification  makes  mtDNA  the  preferred  DNA 
template  together  with  the  availability  of  reference  databases  containing  sequences  of  different 
mtDNA barcodes from many species. 
The high mutation rate of mtDNA can also lead to sequence variations within one species, and the 
use of specific barcodes for species identification can only be reliable if the within-species variation 
is less than the between-species variation [10,  11] and sufficient data from closely related species 
are  available  [12].  For  the  identification  of  fish  species,  the  mitochondrial  cytochrome  b  gene 
(cytb) [13] and the mitochondrial cytochrome c oxidase subunit I gene (COI) [10] have been identified 
as  reliable  barcodes.  Although,  genetic  identification  using  a  single  barcode  might  lead  to 
misidentification because two different species share the sequence of the barcode, it is unlikely that 
there are two different distinctive species that share the sequences on several genes [14]. 
Publicly  available  species-specific  DNA  sequences,  used  as  reference  for  the  identification  of  fish 
samples  are  available  in  databases  such  as  Barcode  of  Life  Database  System  (BOLD)  [15,  16], 
GenBank® [17] and FishTrace [18]. They all contain data from “voucher specimens”, specimens from 
species identified via morphological and genetic methods, which are curated by museums or institutes 
in reference collections. Except for FishTrace, the databases also contain DNA sequences of samples 
that  are  connected  to  reviewed  publications  and  morphological  identification;  however,  they  also 
contain DNA sequences which may not all have a direct link to morphological identification. For the 
identification  of  species,  only  sequences  related  to  voucher  specimens  and  sequences  linked  to  a 
morphological identification connected to reviewed publications are preferred.   

5 

 
 
For the development and validation of DNA-based identification methods, laboratories can request 
tissue aliquots of voucher specimens from reference collections. As the access to these reference 
aliquots is often limited, the JRC decided to produce CRMs for commercially important fish species 
and species related to them. This CRM is intended for use in method validation, specifically to assess 
the accuracy of DNA barcoding methods for the identification of the fish species Gadus morhua.  

CRM EURM-023 is a freeze-dried fish powder prepared from a single fillet and certified for the taxon 
Gadus  mohua  (Linnaeus,  1758)1  with  AphiaID 126436  from  the  World  Register  of  Marine  Species 
(WoRMS) [19]. It is characterised based on its genetic identification by two DNA barcodes, cytb and 
COI.  

1.2 Choice of the material 

Gadus morhua is a species in the Gadidae family (cods and haddocks). The commercial name, Atlantic 
cod, follows the nomenclature of the Food and Agriculture Organization (FAO) of the United Nations 
[20], European Commission (EC) [21] and Fishbase [22].  

The head is relatively narrow with a protruding upper jaw, a barbel on the lower jaw, and light lateral 
line,  curved  above  the  pectoral  fins  [20,  23].  The  colour  is  variable,  brownish  to  greenish  or  grey 
dorsally and on the upper side, becoming pale ventrally and silvery at the tail. It is found in the North 
Atlantic Ocean along the North American coast, the east and west coasts of Greenland (extending for 
variable distances to the north, depending upon climate trends), around Iceland, the coasts of Europe 
from the Bay of Biscay to the Barents Sea, including the region around Bear Island. It is among the 
most important of all commercial fishes with a high food value. Although landings in 1983 reached 
their lowest level in the eastern Atlantic, the stock is recovering, and catches are again increasing 
[20]. Gadus morhua has been assessed in 1996 as vulnerable under criteria A1bd by the International 
Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species [22, 
24] and total allowable catches (TACs) or fishing opportunities are established. TACs are set annually 
for most stocks (every two years for deep-sea stocks) by the EU Council of fisheries ministers and 
agreed with non-EU countries for stocks that are shared and jointly managed [21].  

Substitution  of  fish  species  may  take  place  inadvertently  or  on  purpose  at  various  levels  in  the 
seafood supply chain. Due to overfishing, stock decline and the TACs to protect the stock, the prices 
for Gadus morhua increased and therefore it is prone to substitution with cheaper fish in all sections 
of the supply chain [25, 26], the reason to produce a CRM for Gadus morhua.  

The  mtDNA  copy  numbers  are  highest  in  high-energy  demanding  tissues  such  as  brain,  skeletal 
muscles and heart [27]. Hence, muscle tissue such as a fish fillet is ideal to produce a CRM from one 
fish. 

1 The taxon was first described in 1758 by C. Linnaeus as Gadus callarius, but later accepted as Gadus morhua Syst.Nat. ed.10:252 

6 

 
 
 
 
 
1.3 Outline of the CRM project 

Box 1. Reference material production 

Reference  material  (RM)  production  is  defined  in  ISO  17034  as  a  project  comprising  planning  and  processing  of  the 
material, followed by homogeneity and stability testing, characterisation and assigning of one or more property values. 
Depending on the intended use of the RM a commutability study is carried out. 

For certified reference materials (CRMs) a certificate is issued while for RMs a product information sheet is issued by the 
reference material producer (RMP). 

CRMs and RMs are distributed globally and the validity of their assigned values is monitored throughout the life-time of 
the material. 

The  certification  of  the  fish  species  in  EURM-023  is  based  on  genetic  analysis  performed  by  two 
independent  laboratories.  Both  laboratories  analysed  two  DNA  barcodes  that  are  reliable  for  fish 
identification (cytb and COI) by PCR amplification and subsequent bi-directional Sanger sequencing. 
The certification is based on the accepted sets of unambiguous data from these laboratories. The 
traceability of the certified value is established by comparing the mtDNA sequences of the cytb and 
COI barcodes with the mtDNA reference sequences from both voucher specimens and peer reviewed 
publications related to morphological identification, in the BOLD [15, 16], GenBank [17] and FishTrace 
[18] databases. 

The material is certified for its taxon, the species Gadus morhua. The material originates from a single 
fish and its genetic identity was verified by two genetic methods using different barcode loci. It is 
processed  into  a  dried  homogenous  powder  to  ensure  stable  storage,  easy  handling  and  efficient 
shipment. After reconstitution, the DNA can be extracted in the same manner as from fresh fish tissue. 

The uncertainty of the certified value was estimated in compliance with ISO 17034:2016 [28]. 

The CRM project, including the certification approach and the evaluation of the obtained measurement 
data, was subjected to peer-review involving internal experts. 

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

7 

 
 
 
 
2  Participants 

2.1  Project management, processing and data evaluation 

European Commission, Joint Research Centre, Directorate F – Health and Food, Geel, BE 

2.2  Homogeneity measurements 

Eurofins Genomics Europe Applied Genomics GmbH, Ebersberg, DE 

2.3  Stability measurements 

European Commission, Joint Research Centre, Directorate F – Health and Food, Geel, BE  

2.4  Characterisation measurements 

Eurofins Genomics Europe Applied Genomics GmbH, Ebersberg, DE 
SYNLAB Analytics & Services United Kingdom Ltd, Cramlington, UK 

The datasets are identified by a code (e.g. D01). The numbering is not related to the order of the 
laboratories presented above. 

8 

 
 
3  Material processing and processing control 

Box 2. Reference material processing 

RM processing covers the conversion of the raw material into a homogenous 
and stable material. It typically includes steps such as grinding, sieving and 
drying.  When  the  processed  material  fulfils  the  specifications,  the  final 
material is filled into individual containers, such as bottles or ampoules, and 
is labelled. These containers are referred to as RM units. 

3.1  Origin/Purity of the starting material 

The JRC purchased one fish of the species Gadus morhua (Atlantic cod) from a local fishmonger and 
used it for production of EURM-023. The fish was filleted, packed, labelled and stored at -20 °C until 
use. Genetic identification by bidirectional Sanger sequencing using two DNA barcodes (cytb and COI) 
confirmed the identity of the species Gadus morhua. The suitability of the raw material for PCR and 
sequencing  measurements  was  confirmed  by  the  visually  good  quality  of  the  sequencing 
electropherograms  generated  during  the  homogeneity  (Annex  2,  Figure  2.1  to  Figure  2.4)  and 
characterisation studies.  

3.2  Processing  

For the purpose of this report, the term ‘unit’ refers to one vial of EURM-023. 

One unit of EURM-023 is shown in Figure 1. 

Figure 1. EURM-023.  

Source: JRC 

During  processing,  contamination  of  the  EURM-023  material  with  foreign  DNA  was  avoided  by 
treating all contact surfaces prior to use with DNA degrading solution (DNA-Erase™, MP Biomedicals, 
Irvine,  US).  An  in-house  validation  study  had  previously  proven  that  the  solution  degraded  DNA 
effectively under the conditions used. Additionally, all staff involved in the processing of the material 
used clean laboratory clothing and powder-free gloves. 
The Gadus morhua single fillet was thawed and the skin and bones removed. The fillet was then cut 
into pieces of approximately 1 cm3 and about 2.2 kg of fillet cubes were freeze-dried (Epsilon 1-6D, 
Martin  Christ,  Osterode,  DE)  on  trays  using  a  programme  that  started  at  -40  °C  and  increased 
gradually to 25 °C. The total freeze-drying process lasted 55 h. The dried cubes were then stored in 
stainless  steel  drums  over  liquid  nitrogen  prior  to  being  cryogenically  milled  using  a  Palla  VM-KT 
cryogenic vibrating mill (KHD, Köln, DE). The resultant powder was then manually sieved over a 1000 
μm stainless steel mesh. The water content of the powder was further reduced by drying for 20 h 

9 

 
 
 
 
 
 
under vacuum in the freeze-dryer at 25 °C. The dried powder was homogenised in a DynaMIX CM200 
(WAB, Muttenz, CH) for 1 h. The final powder was packed in a plastic bag and stored in an airtight 
container at (4 ± 3) °C.  
A feeder, FD – SPAc 4A (MCPI, Meythet, FR), was used to fill 5 mL amber glass vials with at least 
200 mg of the fish fillet powder. Lyophilisation inserts were placed in the vials’ necks. The vials were 
placed in a freeze-dryer to provide an argon atmosphere, (Epsilon 1-6D, Martin Christ, Osterode, DE) 
and were closed inside the freeze-dryer. Capping was executed using an HV 100 B semi-automatic 
capping machine (Bausch & Ströbel, Ilshofen, DE) while labelling was completed manually. Each of 
the  units  processed  was  identified  by  a  numbered  label  indicating  the  EURM  code  and  a  number 
according to filling order. Following an inventory and the selection of units according to a random 
stratified sampling scheme for later analysis, the remaining units were stored in the dark at (4 ± 3) °C. 

3.3  Processing control 

The sample size per unit of EURM-023 is at least 200 mg. 

Particle  size  distribution  measurements  based  on  laser  diffraction  (Helos  KR,  Sympatec  GmbH, 
Clausthal-Zellerfeld,  DE)  and  water  mass  fraction  by  volumetric  Karl  Fischer  titration  (V-KFT,  841 
Titrando Karl Fischer titrator Metrohm, CH) took place after final processing of EURM-023.  

After  filling,  particle  size  analysis  was  performed  in  triplicate  on  five  units  of  the  final  material. 
Volume-weighted mean particle size of cumulative distributions for the final product are depicted in 
Figure 2. Cumulative volume distribution of the particles derived from laser diffraction data is based 
on their equivalent spherical diameters, i.e. the diameter of the particles derived from the volume 
occupied upon their rotation. For EURM-023, 99 % of the particles have an equivalent diameter below 
(480 ± 70) μm (U, k = 2) (Figure 2).  

Figure 2. Volume-based cumulative distribution of particle size in EURM-023. 

Source: JRC 

To determine the residual water mass fraction in EURM-023, 30 units were selected by a random 
stratified sampling scheme, covering the whole batch, and were measured with V-KFT. To reach the 
minimum required sample size for V-KFT measurements, the content from three units was used for 
one V-KFT measurement for a total of ten independent measurement results. The mean water mass 
fraction  of  EURM-023  was  (41.6 ± 2.4) g/kg  with  the  expanded  uncertainty  (U)  calculated  using  a 
coverage factor k = 2. The associated U has been estimated during validation of the V-KFT method 
on fish tissue. 

10 

 
 
 
 
3.4  Suitability, DNA extraction  

The suitability of EURM-023 depends on the quantity and quality of the DNA that can be extracted 
from the fish fillet powder in each unit. To evaluate this, in-house measurements were performed on 
the ten samples analysed in duplicate used for the homogeneity study representing the whole batch.  
The  DNA  extractions  were  performed  under  intermediate  precision  conditions  (two  DNA  extraction 
series) and in a randomised manner. The DNA extraction and the DNA quality checks were carried out 
at JRC (Geel, BE). Two independent replicates per unit of between 7 mg and 8 mg were weighed into 
2  mL  low-DNA-binding  micro-centrifuge  tubes.  The  DNA  extraction  is  described  in  the  ‘Standard 
operating  procedure  (SOP)  for  the  genetic  identification  of  fish  species  using  DNA  barcoding 
(mitochondrial cytochrome c oxidase subunit I sequencing)’ from the Labelfish Consortium [29]. The 
material was reconstituted in 160 μL nuclease-free water (Promega Benelux, Leiden, NL) and mixed 
for 10 s using a vortex mixer. Immediately, the DNA was extracted and purified following the in-house 
validated animal tissue protocol from the DNeasy Blood & Tissue Kit (QIAGEN Benelux BV, Antwerpen, 
BE).  The  incubation  time  was  30  min,  which  proved  to  extract  sufficient  DNA  during  the  method 
validation. At the end of the extraction, the DNA was eluted in 100 μL buffer.  
In checking the integrity of the DNA, gel electrophoresis was conducted using a gel-red pre-stained 1 
% agarose gel (PowerPac 3000 Electrophoresis Power Supply, Bio-Rad, NL). A significant degree of 
DNA fragmentation was observed for all the samples, mainly ranging between 300 bp and 1500 bp 
but also fragments up to about 8000 bp were visible for each sample. The quality of the DNA was 
sufficient for PCR amplification and Sanger sequencing of the targeted DNA barcodes of 413 bp and 
652 bp  (cytb  and  COI  genes,  respectively).  The  DNA  concentration  for  all  extracts  was  above  126 
ng/μL,  measured  by  UV  spectrophotometry  (ND  1000  Spectrophotometer,  NanoDrop  Technologies 
Inc.,  Wilmington,  US).  The  minimum  DNA  concentration  for  sequencing  should  be  above  10  ng/μL 
according to the SOP [29]. 
The results demonstrate that extracts from 7 mg powder yield enough DNA of sufficient quality and 
quantity for PCR amplification and sequencing.  

11 

 
 
 
 
 
 
 
 
4  Homogeneity 

Box 3. Homogeneity assessment 

A key requirement for any RM produced as a batch of units is equivalence 
between those units. It is important to know how much the variation between 
units contributes to the uncertainty of the certified value. Consequently, ISO 
17034  [28]  requires  RMPs  to  quantify  the  between-unit  variation  in 
homogeneity studies. 

The within-unit homogeneity is correlated to the minimum sample size, which 
is  the  minimum  amount  of  sample  that  is,  for  a  given  measurand, 
representative of the whole unit and that should be used in an analysis. Using 
sample intakes equal to or above the minimum sample size guarantees the 
assigned value within its stated uncertainty. 

The whole batch of EURM-023 was produced from a single fish fillet and it can therefore be concluded 
that  the  certified  value  is  valid  for  all  units  of  the  batch.  To  confirm  the  species  of  the  fish  fillet 
powder in EURM-023, samples were in the frame of a homogeneity study analysed by bidirectional 
Sanger sequencing of the mitochondrial cytb and COI genes. 

4.1  Between-unit homogeneity 

The between-unit homogeneity was evaluated to ensure that the certified value of the CRM is valid 
for all units of the material, within the stated uncertainty. 

The number of units selected corresponds to approximately the cube root of the total number of units 
produced.  Consequently,  ten  units  were  selected  for  the  between-unit  homogeneity  test  using  a 
random stratified sampling scheme covering the whole batch. Random stratified sampling involves 
dividing the batch into ten groups (with a similar number of units in each group) and selecting one 
unit randomly from each group. Two independent samples were taken from each selected unit and 
analysed.  The  measurements  were  performed  under  intermediate  precision  conditions  (two  DNA 
extraction  series)  and  in  a  randomised  manner  to  be  able  to  separate  a  potential  drift  in  the 
measurement results from a potential trend in the filling sequence. 

To investigate the homogeneity of the certified value, the species of the fish in EURM-023, the DNA 
was extracted as described in Section 3.4 and the samples were further prepared for sequencing by 
dilution in TE low buffer (pH 8.0, 1 mmol/L Tris(hydroxymethyl)aminomethane (TRIS) and 0.01 mmol/L 
Ethylenediamine-tetraacetic acid  (EDTA)) to the target DNA concentration (20 ng/μL) and shipped on 
dry ice to Eurofins Genomics, Ebersberg, DE, which performed the bidirectional Sanger sequencing of 
the mitochondrial cytb and COI genes after the PCR amplification and purification.  

Primers used for PCR amplification and Sanger sequencing of the cytb region were identical to the 
publication by Wolf et al. [13]. Primers used for the COI amplification and sequencing followed the 
SOP for the genetic identification of fish species using DNA barcoding (mitochondrial cytochrome c 
oxidase subunit I sequencing)’ from the Labelfish Consortium [29, 30, 31]. For PCR amplification of 
the COI barcode, two forward primers and two reverse primers were used. The M13-tail primers were 
used for the Sanger sequencing of the COI barcode (primers for cytb and COI are described in Annex 
1).  

All sequencing data were evaluated at JRC and sequencing results for the two barcodes (cytb and 
COI) were evaluated separately. Initially, the sequencing electropherograms were assessed for their 
quality using the SeqManPro application within the Lasergene 18.1 software (DNASTAR, Madison, US) 
[32]. Primer sequences and regions of low quality at either end of the sequencing electropherogram 
were removed and ambiguous bases and no-base calls were checked against the sense or anti-sense 
sequence and manually corrected, whenever justified. Forward and reverse sequences per extraction 
replicate were aligned to obtain an extraction consensus sequence. Subsequently, the two extraction 
consensus sequences per unit were aligned to obtain a unit consensus sequence. 

12 

 
 
 
The 20 cytb extraction consensus sequences were compared to a cytb barcode reference sequence 
for Gadus morhua (KX267041.1 [33], complete mitochondrial genome, downloaded from GenBank 
[17]),  using  the  SeqManPro  application  in  Lasergene  18.1  [32].  For  cytb,  12  of  the  20  sequences 
produced, covered the complete length (413 bp) of  the barcode sequence for Gadus  morhua. The 
subsequent eight sequences were shorter due to poorer quality base calls at the ends of the barcodes 
and ranged between 385 bp to 400 bp. One replicate had one ‘no base call’ in the sequence. No other 
sequence difference was observed between the 20 extraction consensus sequences and the reference 
sequence  KX267041.1.  Also,  no  base  differences  were  observed  between  the  ten  unit  consensus 
sequences (except for the different sequence lengths of 400 bp for one unit and 413 bp for the other 
units)  and  with  this  reference  sequence  (query  coverage,  the  percentage  of  the  sample  sequence 
length that is included in the aligned reference sequence, is 100 % for each sequence). 

The 20 COI extraction consensus sequences were also compared to the COI region of the reference 
sequence for Gadus morhua AM489716.1 [34] complete mitochondrial genome, downloaded from 
GenBank [17]) and similar with the record in BOLD with process ID number GBGC3821-07 [15, 16]. 
The COI barcode sequence has a length of 652 bp. Of the 20 sequences obtained, two covered the 
complete length of the COI barcode. The other sequences were some bases shorter due to poorer 
quality base calls at the ends of the barcodes but reached almost the full length and ranged between 
638 bp to 650 bp, of these 14 sequences were 650 bp long. Four replicate sequences had for one 
base position a ‘no base call’ close to the end of the sequence. No other sequence differences were 
observed between the 20 consensus sequences and the COI reference sequence. As a result of the 
‘no base calls’ in both the replicate consensus sequences, one unit consensus sequence had one ‘no 
base call’ on one sequence base position. No other base differences were observed, except for the 
different sequences lengths (647 bp and 652 bp), between the ten unit consensus sequences and 
this COI reference sequence (with a query coverage of 100 % for each sequence).  

The sequencing data produced were visually of good quality, indicating no interfering contamination 
with foreign DNA in any of the units sampled (see Annex 2, Figure 2.1 to Figure 2.4). The consensus 
sequences of the extraction replicates and the consensus sequences per unit have been identified as 
Gadus morhua at both barcode positions sequenced, cytb and COI.  

The  ten  units  analysed  had  identical  consensus  sequences,  except  for  the  small  differences  in 
sequence lengths and the ‘no base call’ in one of the unit sequences. The identity results were above 
99 % with 18 Gadus morhua GenBank [17] database records of the cytb barcode, using Basic Local 
Alignment Search Tool BLAST® software, (BLASTN 2.17.0+) [35, 36, 37, 38] (with a query coverage 
of 97 % to 100 %). Similarly for the COI barcode, identical consensus sequences were found for all 
units, except for small differences in sequence lengths. The sequence similarity results were above 
99 % with 41 Gadus morhua records selected of the COI barcode using the Barcode of Life Data 
Identification Systems v4 (BOLD) [15, 16] (with query coverage of 92 % to 100 %). For details about 
the  BLAST  and  BOLD  searches  see  Section  6.4.  The  results  of  the  BLAST  and  BOLD  searches  are 
summarised in Annex 2, Table 2.1 and Table 2.2. 

4.2  Within-unit homogeneity and minimum sample size 

EURM-023 was produced from a single fish fillet and no interfering contamination with foreign DNA 
was  detected.  Therefore,  the  powder  is  considered  homogenous  with  regard  to  its  certified  value, 
Gadus morhua. Homogeneity, stability and characterisation measurements were performed with a 
sample intake between 7 mg and 8 mg. These sample sizes ensured that sufficient DNA was extracted 
for PCR amplification and Sanger sequencing. Therefore, the minimum sample size is set at 7 mg.  

EURM-023 is a pure fish material consequently the CRM user may opt, at their own discretion and 
responsibility, for a smaller sample size provided that the method’s minimum DNA quantity specified 
by the applied DNA barcoding method is respected. 

13 

 
 
 
5  Stability 

Box 4. Stability assessment 

Stability testing is necessary to establish the conditions for storage as well as the 
transport conditions of the RMs to the customers. During transport, especially in 
summer,  temperatures  up  to  60 °C  can  be  reached,  and  stability  under  these 
conditions must be demonstrated if the RMs are to be transported without any 
additional cooling. 

Time, temperature, light (including ultraviolet radiation) and water content were regarded as the most 
relevant parameters on the stability of the materials. The influence of ultraviolet and visible light was 
minimised  by  storing  the  material  in  amber  glass  vials,  which  reduce  light  exposure.  In  addition, 
materials are stored in the dark and transported in boxes, thus removing any possibility of degradation 
by  light.  The  water  content  was  adjusted  to  an  optimum  during  processing.  Therefore,  only  the 
parameters time and temperature needed to be investigated. 

The stability studies were carried out using an isochronous design [39]. In this approach, units were 
stored for a particular length of time at different temperature conditions. Afterwards, the units were 
moved to conditions where further degradation can be assumed negligible (reference conditions). At 
the end of the isochronous storage, the samples were analysed simultaneously under intermediate 
precision conditions. Analysis of the material (after various exposure times and temperatures) under 
intermediate precision conditions greatly improves the sensitivity of the stability tests. 

5.1  Transport stability 

The conditions for the transport of the material to the customers were established in a short-term 
stability study. To this end, two units were stored at 4 °C, 18 °C and 60 °C for 0, 1, 2 and 4 weeks (at 
each temperature). The reference temperature was set to -70 °C. The two units per storage time were 
selected using a random stratified sampling scheme. From each unit, two samples were measured by 
quantifying the extracted DNA concentration using UV spectrophotometry and the DNA integrity was 
assessed  by  gel  electrophoresis,  both  described  in  Section  3.4.  The  amplicon  sizes  were  assessed 
through cytb and COI specific PCR (C1000 Touch Thermal Cycler, Bio-Rad, Nazareth, BE) using the 
methods described in Annex 1. The resulting PCR product sizes were estimated by comparison to a 
100 bp DNA ladder (Invitrogen, by ThermoFisher Scientific, Merelbeke, BE) using gel electrophoresis. 
These methods do not identify the fish species in EURM-023 but give an indication that the extracted 
DNA is of sufficient quantity and quality, as needed for PCR and sequencing methods to identify the 
species.  Additionally,  when  the  units  were  opened,  the  smell  and  colour  of  the  fish  powder  was 
described and compared with the samples stored at reference temperature. Different smell or colour 
may  indicate  degradation  of  the  fish  powder  and  further  fragmentation  of  the  DNA  at  the 
temperatures tested.  

The  measurements  were  performed  under  intermediate  precision  conditions  (four  DNA  extraction 
series), and a randomised sequence was used to differentiate any potential drift in the measurement 
results from a potential trend over storage time. The results of the extracted DNA concentration were 
recalculated to a sample size of 7 mg. These data were evaluated individually for each temperature. 
Significant differences between the extraction series means were checked using two-way analysis of 
variance at a 95 % confidence level. No statistically significant series effect was found for the DNA 
concentration  at  the  different  temperatures  tested.  The  data  for  the  4  °C,  18  °C  and  60  °C  study 
followed a normal distribution and were screened for outliers using the single and double Grubbs test 
on a confidence level of 99 %. No statistical outliers were found neither for the data over time nor 
for the analytical sequence. 

In addition, the data were evaluated against storage time, and regression analyses was performed to 
evaluate a potential trend at a 95 % confidence level in the measurement sequence as well as a 
trend in the storage time to test for potential increases or decreases of the DNA concentration due 

14 

 
 
 
 
to shipping conditions. None of the measurement sequence trends was statistically significant on a 
95 % confidence level for any of the temperatures. However, a statistically significant trend over time 
at a 95 % confidence level was found for 60 °C but not for the other temperatures tested.   

After opening, the samples from the 4°C and 18 °C studies did not smell differently in comparison to 
samples stored at the reference temperature -70 °C. The samples from the 60 °C study had a less 
profound fish smell than the samples from the reference temperature. The colour of the powder of 
the 60 °C samples was visually slightly different from the reference temperature samples and may 
indicate matrix degradation. A significant decrease was shown in the concentration of extracted DNA 
from samples stored at 60 °C for one or more weeks. Gel electrophoresis of the DNA extracts showed 
fragmented DNA for all samples at all temperatures (between 300 bp and 3000 bp) and all time 
points  but  significantly  increased  DNA  fragmentation  for  the  60  °C  samples.  However,  gel 
electrophoresis of the cytb and COI PCR products still showed bands at the expected sizes and there 
was  no  visual  difference  between  bands  from  samples  stored  at  60  °C  and  at  the  reference 
temperature. 

Extracted DNA concentration results measured by UV spectrophotometry for each temperature are 
shown in Figures 3.1 to 3.3 of Annex 3. The results of the DNA quality tests and gel electrophoresis 
are not presented. 

The results  of the short-term stability study showed significant  degradation  of the DNA  at 60 °C, 
giving a significant negative trend at 60 °C for the extracted DNA concentration. This indicates that 
the  temperature  condition  at  60  °C  has  a  negative  effect  on  the  stability  of  the  material.  As  no 
significant effect was observed at 18 °C and below, the material shall be shipped cooled to maintain 
matrix and DNA integrity. 

5.2  Storage stability 

Storage conditions and shelf life guaranteeing the stability of the material and the certified value 
were established in a long-term stability study. 

To  this  end,  two  units  were  stored  at  -20  °C  and  4  °C  for  0,  6,  12,  18  and  24  months  (at  each 
temperature).  The  reference  temperature  was  set  to  -70  °C.  The  two  units  per  storage  time  were 
selected using a random stratified sampling scheme. From each unit, two samples were measured as 
described in Section 5.1, except that the gel electrophoresis was performed by using the TapeStation 
4200 system, the genomic DNA ScreenTape kit for the DNA integrity and the D1000 ScreenTape kit 
for  the  PCR  products  (Agilent  Technologies  Belgium  N.V.,  Diegem,  BE).  The  measurements  were 
performed  under  intermediate  precision  conditions  (four  DNA  extraction  series)  and  in  a  random 
sequence to be able to separate any potential drift in the measurement results from a potential trend 
over storage time. The stability data evaluation is based on the extracted DNA concentration results 
that were recalculated to a sample intake of 7 mg. Statistically significant differences between the 
extraction series  means  were  found  by using two-way  analysis of variance at a 95  % confidence 
level. The data were first normalised to the respective series mean and then further evaluated, for 
each temperature individually as described in Section 5.1. The data of the isochronous study were 
normally distributed and screened for outliers using the single and double Grubbs test at a confidence 
level of 99 %. No statistical outliers were detected. Moreover, the data were also evaluated against 
the measurement sequence and no analytical trend was detected. In addition, the data were plotted 
against storage time and linear regression lines of the normalised data versus time were calculated. 
The  slopes  of  the  regression  lines  were  tested  for  statistical  significance  (loss/increase  due  to 
storage). None of the trends was statistically significant on a 95 % confidence level for any of the 
temperatures. 

The smell and the colour of the powder after opening of the units were similar for all temperatures 
tested and did not indicate matrix degradation. Although, for all samples the extracted DNA showed 
by  gel  electrophoresis  a  significant  degree  of  DNA  fragmentation  (200  bp  to  7000  bp),  the 

15 

 
 
 
 
fragmentation of the DNA is independent of the storage temperatures or time and did not result in 
deviating cytb and COI PCR product sizes. The quality and quantity of the extracted DNA (above 10 
ng/μL) were sufficient for PCR and sequencing. There was no significant difference with the material 
stored at reference temperature. Therefore, it can be concluded that long-term storage at -20˚C or 
4˚C has no detrimental impact on the material.  
Results of extracted DNA concentration measured by UV spectrophotometry are shown in Annex 4, 
Figure  4.1  and  Figure  4.2.  The  results  of  the  DNA  quality  tests  and  gel  electrophoresis  are  not 
presented.  

No statistical outliers were detected for the extracted DNA concentration. None of the trends was 
statistically significant on a 95 % confidence level for any of the temperatures of the isochronous 
study. The material can therefore be stored at 4 °C.  

The stability of the material after repeated opening and sampling has not been tested.  

The material is included in the JRC's regular stability monitoring programme, to control its further 
stability. 

Box 5. Stability monitoring 

RMs are produced as batches that, in most cases, should last for ten 
years or longer. This long lifetime means that a storage stability study 
of  limited  duration  cannot  provide  a  definite  “use  by”  date  for  the 
material.  It  therefore  needs  to  be  complemented  by  stability 
monitoring throughout the lifetime of the RM.  

Therefore, the stability of RMs whose assigned values might change is 
regularly  monitored.  The  monitoring  frequency  depends  on  the 
outcome of the storage stability assessment. 

If the tests confirm the stability of the assigned values, the material 
remains on sale. If not, possible actions include the retraction of the 
value in question, retraction of the complete material or a change of 
the certified value. Customers are notified if the change is larger than 
the uncertainty of the assigned value. 

16 

 
 
 
 
 
 
6  Characterisation  

Box 6. Reference material characterisation 

Material characterisation is the process of determining the property value(s) of a RM. 
While ISO 17034 allows to characterise an RM in various ways, quality management 
procedures  of  the  JRC  are  more  stringent  and  allow  characterisation  by  either 
interlaboratory comparison, the use of a primary method confirmed by independent 
analysis or value transfer. 

The material characterisation was based on a comparison between the results obtained by two expert 
laboratories, i.e. the DNA sequence of the material was determined in parallel in different laboratories 
to  demonstrate  the  absence  of  a  measurement  bias.  Due  to  the  nature  of  the  measurand,  both 
participants  used  PCR  amplification  and  bidirectional  Sanger  sequencing  methods  for  the 
measurements.   

6.1  Selection of participants  

Two  laboratories  were  selected  based  on  criteria  that  comprised  both  technical  competence  and 
quality management aspects. Each participating laboratory was required to operate a quality system 
and  to  deliver  documented  evidence  of  its  laboratory  proficiency  in  the  field  of  DNA  sequencing 
measurements in relevant matrices. Having a formal accreditation was not mandatory, but meeting 
the requirements of ISO/IEC 17025 [40] was obligatory. 

6.2  Study setup  

The sample preparations, DNA extraction and the quality and quantity checks of the extracted DNA, 
were all performed at JRC as described in Section 3.4. For one laboratory, five units were selected 
using a random stratified sampling scheme covering the whole batch of EURM-023. For the second 
laboratory,  as  the  homogeneity  and  characterisation  study  were  combined,  five  units  for  the 
characterisation  study  were  selected  from  the  homogeneity  units  and  likewise  covered  the  whole 
batch. Two DNA extracts per unit were sent to both laboratories that independently performed cytb 
and COI PCR amplification and Sanger sequencing (bidirectional).  

While DNA extraction was performed in two extraction series (intermediate precision), in a randomised 
order, DNA quality checks and Sanger sequencing measurements were completed under repeatability 
conditions, in a randomised order. 

6.3  Methods used 

From each unit two replicates of 7 mg to 8 mg fish powder were weighed into 2 mL low-DNA-binding 
microcentrifuge tubes. The DNA was extracted and the quality checked as described in Section 3.4. 
The DNA concentration was sufficient, and the DNA integrity was checked by gel electrophoresis and, 
despite a substantial degree of DNA fragmentation that was detected for all samples, the quality of 
the DNA was sufficient for PCR amplification and Sanger sequencing of the targeted DNA barcodes 
of 413 bp and 652 bp. The DNA extracts were diluted in TE low buffer (pH 8.0, 1 mmol/L TRIS and 
0.01 mmol/L EDTA) to the target DNA concentration for each lab and shipped on dry ice conditions to 
the laboratories listed in Section 2.4. 

17 

 
 
 
 
 
 
For the PCR amplification and sequencing, both laboratories used the cytb primers as described in 
Table  1.1  in  Annex  1  [13].  The  COI  primers  were  used  according  to  the  SOP  for  the  genetic 
identification of fish species by the Labelfish Consortium as described in Table 1.2 in Annex 1 [29, 
30,  31].  PCR  reagents  and  thermocycler  settings  for  both  barcodes  were  used  according  to  the 
laboratory’s  own  procedures.  For  the  COI  PCR  amplification,  the  laboratories  added  the  two  COI 
forward and two COI reverse primers to the PCR master-mix. For sequencing and according to the 
SOP one lab used the separate forward or reverse sequencing M13 tail primers. The second laboratory 
used the four single PCR COI primers for sequencing, resulting in four sequencing electropherograms.  
Successful  amplification  of  the  cytb  and  COI  barcode  regions  were  checked  by  either  gel 
electrophoresis or capillary electrophoresis of the PCR products. The purification of the PCR products, 
followed by cytb and COI bidirectional Sanger sequencing was executed by each laboratory using their 
own procedures according to ISO/IEC 17025 [40]. 
All sequencing data were evaluated at JRC using the SeqManPro application. The data for the two 
barcodes  (cytb  and  COI)  were  evaluated  separately.  The  resulting  extraction  consensus  sequences 
(per DNA extract) were uploaded to GenBank's BLASTN 2.17.0+ [35, 36, 37, 38] and the BOLD [15, 
16]  identification  tools  for  genetic  identification.  The  resulting  reference  sequences  for  the  cytb 
barcode  listed  in  the  alignment  hit  table  from  BLAST  were  checked  for  related  publications  and 
morphological  identification  or  for  voucher  reference.  Reference  sequences  that  had  suspicious 
genetic and morphological identification were not used for identification of the species in EURM-023. 
To obtain more results with reference sequences from different sources, valid reference sequences 
from Fish Trace [41] and GenBank were aligned with the extraction and the unit consensus sequences 
using the BLASTN tool. The reference sequences in the hit list from the BOLD identification tool for 
the COI barcode were checked similarly.  
For  each  barcode,  cytb  and  COI,  the  extraction  consensus  sequences  were  aligned  to  obtain  one 
sequence per unit and were also uploaded to GenBank's BLASTN and the BOLD identification tools 
for  genetic  identification.  A  consensus  sequence  per  laboratory  was  aligned  from  the  five  unit 
consensus sequences per barcode. A final consensus sequence for the cytb and the COI barcode was 
aligned with the sequences from both laboratories and are given in FASTA format in Tables 5.2 and 
5.3 of Annex 5.   

6.4  Evaluation of results 

The  characterisation  study  resulted  in  two  sets  of  sequencing  data  for  each  of  the  cytb  and  COI 
barcodes, thus four datasets in total. The identification results obtained by JRC by evaluating the data 
from  the  participants,  grouped  per  laboratory  and  barcode  method,  are  displayed  in  Table  5.1  of 
Annex 5. 

6.4.1  Technical evaluation 

The  sequencing  data  obtained  from  each  laboratory  were  first  checked  for  compliance  with  the 
requested instructions and for their validity based on technical reasons. The following criteria were 
considered during the evaluation: 

- Valid PCR sequencing procedure 
-  Compliance  with  the  analysis  protocol:  analytical  sequence  according  to  the  prepared 
scheme 
- PCR products showing a single band of about 464 bp and 738 bp in size for cytb and COI 

respectively (with primers) as visualised on a gel or by capillary electrophoresis 

- Quality of sequencing electropherograms: check for ambiguous bases in the forward and 
reverse trace sequences and in the aligned consensus sequence and, sequence length (at 
least 360 bp and 600 bp for cytb and COI respectively) to ensure an optimum length for 
differentiation between species. 

18 

 
 
 
The  PCR,  purification  of  PCR  products  and  sequencing  were  executed  meeting  the  requirement  of 
ISO/IEC 17025. The specific and requested primers as described in Annex 1 for the barcodes, cytb and 
COI,  were  used.  The  analyses  were  executed  according  to  the  requested  analytical  order.  The 
successful amplification of the DNA was confirmed by gel electrophoresis or capillary electrophoresis. 
The PCR products showed a single band consistent with the expected length of 464 bp and 738 bp 
for cytb and COI, respectively. The sequence electropherograms obtained were checked for quality. 
They were clean and reached suitable lengths for species identification, > 385 bp for cytb and > 638 
bp for COI. The two independent datasets per method (cytb and COI) gave identical sequence results. 
The  identification  resulted  in  the  same  species  for  all  characterisation  samples  for  both  barcodes 
(cytb  and  COI)  and  both  laboratories.  The  similarity  of  all  the  sequencing  results  between  the 
laboratories gave a good indication that the methods used were valid for the genetic identification of 
the species in this CRM. Based on the above, it was concluded that the datasets were technically valid. 

6.4.2  Evaluation of the genetic identification using the cytb barcode 

The two sequencing datasets for the cytb barcode, five units per laboratory and two DNA extracts per 
unit,  were  analysed  separately.  Alignment  was  performed  using  the  SeqManPro  application  within 
Lasergene 18.1 software (DNASTAR, Madison, US) [32]. Primer sequences and regions of low quality 
at  either  end  of  the  sequencing  electropherogram  were  removed  and,  where  possible,  ambiguous 
bases  and  no-base-calls  were  checked  against  the  forward  and  reverse  sequence  and  manually 
corrected,  whenever  justified.  Forward  and  reverse  sequences  were  checked  and  aligned  to  one 
consensus sequence per extraction replicate. 

The ten extraction consensus sequences per laboratory were compared to the reference sequence for 
Gadus morhua (KX267041.1 [33], complete mitochondrial genome, downloaded from GenBank [17]). 
From the cytb analysis of the two sets of ten extraction consensus sequences, 17 of the obtained 
extraction consensus sequences covered the complete length (413 bp) of the cytb barcode. The three 
remaining  sequences  were  shortened  due  to  lower  quality  base  calls  at  the  ends  of  the  barcodes 
(from  385  bp  to  398  bp).  No  other  sequence  differences  were  observed  between  the  extraction 
consensus sequences and additionally the reference sequence for Gadus morhua.  

Additionally,  all  extraction  consensus  sequences  were  aligned  to  a  cytb  reference  sequence 
downloaded  from  GenBank  of  the  two  nearest  species  identified,  Gadus  chalcogrammus  (Alaska 
pollock or walleye pollock, NC_004449.1 [42,  43], complete mitochondrial genome and identical to 
AB094061.1 [42, 17]) and with Gadus macrocephalus (Pacific cod, NC_036931.1 [44, 43] complete 
mitochondrial genome and identical to KY296294.1 [44, 17]). All the sequences deviated for the same 
19 bases with the Gadus chalcogrammus reference sequence and the same 18 bases with the Gadus 
macrocephalus reference sequence. 

The two extraction consensus sequences per unit were aligned to obtain a consensus sequence per 
unit and further used for species identification by using Megablast in the Nucleotide BLAST (blastn 
suite)  software      (BLASTN  2.17.0+)  [35,  36,  37,  38].  The  BLASTN  algorithm  compares  nucleotide 
sequences  pairwise;  searching  for  local  regions  of  similarity  in  nucleotide  sequences  against  the 
nucleotide database and calculates the statistical significance of matches [45]. Sequence similarity 
in BLAST refers to identity and means that the same nucleotide is found at the same position in the 
aligned  sequences  [46,  47,  48].  Identity  is  expressed  as  identity  percentage  (number  of  identical 
nucleotides divided by the length of the shortest of the compared sequences). 

The length of the consensus sequences for all units covered the whole length of the cytb barcode 
(413 bp). A Nucleotide BLAST alignment run was performed with 18 different reference sequences 
selected for Gadus morhua that were above 360 bp in size and varied in overall length between 464 
bp as a part of the cytb gene and the whole length of the mtDNA. These reference sequences were 
checked  for  morphological  identification  as  stated  in  a  related  publications  or  were  from  voucher 
specimens.  

19 

 
 
 
All consensus sequences of the units used for the characterisation study showed for the cytb barcode 
a very high similarity with the 18 Gadus morhua reference sequences selected, ranging from 100.0 
% to 99.3 % identity (with query coverage of between 100 % and 97 %), according to the Nucleotide 
BLAST algorithm. The found barcode sequences in the characterisation samples were identical to 10 
Gadus morhua reference sequences. They mismatched for two or three nucleotides with the eight 
other reference sequences. Consequently, they could not reach 100 % identity. All the mismatches 
with the reference sequences were at the amino acid third codon positions and thus without changes 
in  the  amino  acid  sequence  for  EURM-023.  A  summary  of  the  identity  percentage  results,  and 
reference sequences used is given in Table 1.  

A Nucleotide BLAST alignment search with the reference sequences of the two closest species showed 
that the similarity was between 95.9 % and 95.4 % identity for Gadus chalcogrammus and between 
95.6 % and 95.5 % identity for the Gadus macrocephalus. When using the SeqManPro application, all 
the  consensus  sequences  from  the  units  deviated  within  the  cytb  barcode  from  mainly  the  same 
minimum 17 bases when compared to three Gadus chalcogrammus reference sequences selected 
from GenBank [17, 43] (NC_004449.1 [42], DQ356946.1 [49], MW288643.1 [50] and minimum 17 
bases  when  compared  to  four  Gadus  macrocephalus  selected  from  GenBank  (NC_036931.1  [44], 
DQ356938.1 [49], AB248658.1 [51], EU729368.1 [52]). 

20 

 
 
Table  1. Average  DNA  sequence  alignment  identity  results of  EURM-023  compared with  18  Gadus  morhua 
reference sequences in GenBank and Fish Trace for the cytb barcode using BLASTN 2.17.0+. 

GenBank accession code 
of cytb reference 
sequence 

Voucher ID 

Species of 
cytb reference 
sequence 

Average 
identity [%] 1) 
of samples of 
EURM-023 
with cytb 
reference 
sequence 

Number of 
nucleotide 
mismatches 
of samples 
of EURM-
023 with 
reference 
sequence  

Sequence 
length [bp] 
samples of 
EURM-023 

Sample 
query 
coverage 
[%] with 
reference 
sequence 

KX267041.1 [33] 

KX267003.1 [33] 

KX266978.1 [33] 

EU877726.1 [53] 

EU877718.1 [53] 

AY946340.1 [54] 

AY946339.1 [54] 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

n.a. 

EU492304.1 [55] 

NRM 49416 

DQ174046.1 [56] 

DQ174045.1 [56] 

n.a. 

n.a. 

EU492303.1 [55] 

NRM 49423 

NC_002081.1 [57] 

DQ356939.1 [49] 

n.a. 

n.a. 

GadMor-EE-01 [58, 18] 

BMVP/1237 

GadMor-CS-02 [58, 18] 

BMVP/1193 

GadMor-CS-01 [58, 18] 

BMVP/1269 

EU492141.1 [59] 

MNHN 2005-1575 

EU492140.1 [59] 

MNHN 2005-1574 

Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 

1)  Identity [%] = Amount of identical nucleotides / minimum aligning length. 

Source: JRC

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

99.5 

99.5 

99.3 

99.3 

99.3 

99.3 

99.3 

99.3 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

2 

2 

3 

3 

3 

3 

3 

3 

413 

413 

413 

413 

413 

413 

413 

413 

413 

413 

413 

413 

413 

413 

413 

413 

413 

413 

100 

100 

100 

100 

100 

100 

100 

97 

97 

97 

97 

100 

100 

97 

97 

97 

97 

97 

21 

 
 
6.4.3  Evaluation of the genetic identification using the COI barcode 

The two datasets for the COI barcode were evaluated as described in Section 6.4.2. Five units per 
laboratory and sequencing results from two DNA extracts per unit were evaluated separately.  

The ten extraction consensus sequences per laboratory were compared to the reference sequence for 
Gadus morhua (AM489716.1 [34, 17], complete mitochondrial genome and with BOLD record number 
GBGC3821-07 [15, 16]). From the two sets of ten extraction consensus sequences for the COI analysis 
from each lab, the length of the sequences was found to be between 638 bp and 652 bp. Of those 
20 extraction consensus sequences, nine covered the complete length (652 bp) of the COI barcode 
and eight were 650 bp, while the three other sequences were relatively shorter in size (638 bp, 645 
bp or 647 bp). Five of the sequences had one ‘no base call’ in their sequence all due to lower quality 
base calls at the ends. The SeqManPro application reported no other sequence differences between 
the extraction consensus sequences and additionally with this reference sequence. 

The extraction consensus sequences were also aligned to reference sequences of the nearest species 
Gadus chalcogrammus and Gadus macrocephalus. From the 20 extraction consensus sequences 19 
deviated  for  the  same  14  bases  compared  to  the  reference  sequence  for  Gadus  chalcogrammus 
(NC_004449.1 [42, 43]). The other sequence deviated for 13 of the same bases as its sequence was 
shorter (638 bp).  From the 20 extraction consensus sequences, 19 deviated for the same 18 bases 
compared to the reference sequence for Gadus macrocephalus (NC_036931.1 [44, 43]). The other 
sequence was shorter and deviated 17 bases. The ‘no base call’ in some of the sequences caused in 
most of the cases an additional difference with both reference sequences. 

Identification of the species for the COI method was performed using the BOLD Identification Systems 
v4 [15, 16]. The search database for identification used, the COI Species Database, contains published 
COI records with a species level identification and a minimum sequence length of 500 bp. In BOLD, 
nucleotide  sequences  are  first  translated  into  amino  acids  sequences,  verified  that  they  originate 
from  the  COI  protein  and,  after  passing  some  additional  quality  checks,  a  linear  search  of  the 
reference library follows [15]. Sequence similarity in BOLD refers to the ratio to which nucleotide or 
protein sequences are related and takes substitutions into account; expressed as similarity percentage 
[47,  48].  For  sequences  showing  less  than  1  %  divergence  to  a  reference  sequence,  the  BOLD 
identification system delivers a species identification [15].  

The two extraction sequences attained per unit were aligned to obtain one consensus sequence per 
unit and further used for species identification. For six out of the ten consensus sequences, the entire 
region of the COI barcode (652 bp) was covered. For three of the four remaining consensus sequences, 
the lengths were 650 bp and the other sequence was 647 bp, there were no other differences between 
the unit sequences. All consensus sequences of the units used for the characterisation study for the 
COI barcode had comparable similarity results using the BOLD identification application. The sequence 
similarity with the 28 reference sequences selected for Gadus morhua was 100 % and the samples 
were all identified as Gadus morhua (Table 2). The query coverage was checked using BLAST and was 
between 92 % and 100 %. However, using BLAST, for four of these reference sequences (BCOLL162-
06  [60],  SCFAC022-05  [61],  SCFAC050-05  [61]  and  SCAFB692-07  [61])  the  identity  percentages 
differed from the BOLD similarity percentage (100 %) and were 99.9 %, 99.7 %, 99.7 % and 99.5 %, 
respectively. The different identity/similarity percentages may be caused by nucleotides codes within 
these sequences that have the code ‘N’ for all four nucleotides (A,T,G,C), code ‘W’ for the nucleotides 
A  or  T  and  code  ‘M’  for  the  nucleotides  A  or  C,  that  are  counted  as  difference  in  BLAST  but  not 
necessarily in BOLD. The similarity percentages are given in Table 2. 

The SeqManPro application confirmed that for the reference sequences BCOLL162-06, SCFAC022-
05, SCFAC050-05 and SCAFB692-07 there were deviating base calls within the COI barcode region 
of the characterisation samples. These four reference sequences had 1 to 3 unidentified nucleotides 
in their sequences. There was no difference with the 24 other Gadus morhua reference sequences 
selected from the hit list of the BOLD application (Table 2).  

22 

 
 
 
A BLAST search for the COI barcode gave comparable results for the unit consensus sequences, 100 
% identity with 99 of the 100 Gadus morhua records in the alignment hit list, and 99.8 % for one 
record.  

The samples were additionally compared to three Gadus chalcogrammus reference sequences with 
morphological  identification  (DQ356946.1  [49,  17],  MW288643.1  [50,  17]  and  NC_004449.1  [42, 
43]). The characterisation samples had at least 13 different bases within the COI barcode region of 
these reference sequences. Although, there were some differences between the reference sequences 
as only nine of the different bases covered all three reference sequences. Comparison with two Gadus 
macrocephalus  reference  sequences  with  morphological  identification  (NC_036931.1  [44,  43]  and 
DQ356938.1 [49, 17]), showed that the characterisation samples deviated within the COI barcode at 
least 18 different bases which were mainly the same for both reference sequences. 

Table 2. Average DNA sequence alignment similarity results of EURM-023 compared with 28 Gadus morhua 
reference sequences for the COI barcode using the BOLD system v4 identification tool (COI species Database). 

BOLD process ID of 
COI reference 
sequence 

GenBank 
accession 
code of COI 
reference 
sequence 

Voucher ID  

BCOLL157-06 [60] 

n.a. 

BCOLL163-06 [60] 

n.a. 

BCOLL164-06 [60] 

n.a. 

USNM-387534 -
AL200508-325 
USNM-387538-BBC 
2006-04-2 
USNM-387538-BBC 
2006-04-3 

DSFIB455-13 [62] 

n.a. 

ZMUB16088 

FCFUK252-06 [63] 

KJ205380.1  NTEN06-284 

FCFUK253-06 [63] 

KJ205379.1  NTEN06-285 

FCFUK058-06 [63] 

KJ205378.1  NTEN06-060 

UKFBI381-08 [64] 

KF929905.1  MCZ 155780, KUT2937 

SCFAC019-05 [61] 

KC015381.1 

SCFAC259-06 [61] 

KC015382.1 

ARC 25497, 05-313-
003a3 
ARC 25895, 06-095-
GeoB1-e1 

SCFAC839-06 [61] 

KC015377.1  ARC 26596, 06-906 

SCAFB081-07 [61] 

KC015375.1  ARC 24870, 05-828 

SCAFB023-07 [61] 

KC015376.1  ARC 25464, 05-389 

SCAFB377-07 [61] 

KC015384.1  ARC 25886, 06-363 

BNSF005-11 [63] 

KJ204885.1  MT00283 

BNSF007-11 [63] 

KJ204884.1  MT00285 

BNSF350-11 [63] 

KJ204875.1  MT01942 

Species 
of COI 
reference 
sequence 

Average 
similarity  
[%] 1)   of  
EURM-023 
with COI 
reference 
sequence 

Number of 
nucleotide 
mismatches 
of samples 
of EURM-
023 with 
reference 
sequence 

Sequence 
length 
[bp] 
samples 
of EURM-
023 

Sample 
query 
coverage 
[%] with 
reference 
sequence 

Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

100 

23 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

100 

100 

100 

100 

92 

99-100 

98-99 

100 

100 

100 

99-100 

100 

99-100 

99-100 

100 

100 

100 

 
 
BNSF102-11 [63] 

KJ204882.1  MT00671 

BNSF103-11 [63] 

KJ204881.1  MT00672 

BNSF592-12 [63] 

KJ204874.1  MT02994 

NOFIS088-10 [65] 
GBMNA16893-19 
[66] 

n.a. 

NHMO-DFH-541 

HG514359.1  n.a. 

GBGC3821-07 [34]  AM489716.1  n.a. 
ANGBF21909-19 
[53] 

EU877714.1  n.a. 

BCOLL162-06 [60] 

n.a. 

SCFAC050-05 [61] 

KC015383.1 

SCFAC022-05 [61] 

KC015380.1 

USNM-387538-BBC 
2006-04-1 
ARC 25464, 05-390-
003d12 
ARC 25486, 05-346-
003a7 

SCAFB692-07 [61] 

KC015372.1  ARC 26486, 07-080 

Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 
Gadus 
morhua 

100 

100 

100 

100 

100 

100 

100 

100 2) 

100 2) 

100 2) 

100 2) 

0 

0 

0 

0 

0 

0 

0 

1 

2 

2 

3 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

647-652 

100 

100 

100 

100 

100 

100 

100 

100 

99-100 

100 

100 

1)  The statistics related to the aligned sequences in BOLD, are expressed in similarity percentage. 
2)  Using BLAST, the identity percentages for BCOLL162-06, SCFAC022-05, SCFAC050-05, SCAFB692-07 were 99.9 %, 99.7 %, 99.7 % and 

99.5 %, respectively. 

Source: JRC 

24 

 
 
7  Value Assignment 

Box 7. Assignment of values to a reference material 

Based on the outcome of characterisation measurements three types 
of values can be assigned, namely certified, indicative or additional 
material information values. 

Certified values are values that fulfil the highest standards of accuracy. Procedures at JRC require a sufficient number 
of datasets to assign certified values. Full uncertainty budgets in accordance with ISO 17034 [28] and ISO 33405 [67] 
are required. Certified values of a CRM can be used for calibration and trueness controls as well as for quality control 
measures.  

Indicative  values  are  values  where  either  the  uncertainty  is  deemed  too  large  or  too  few  independent  datasets  are 
available to allow certification. Indicative values of an RM can be used for statistical quality control (homogeneity and 
stability  have  been  assessed)  but  not  for  calibration,  demonstration  of  method  or  laboratory  proficiency  or  method 
trueness.  

Additional material information values are values for which homogeneity and stability have usually not been assessed 
and only insufficient data for characterisation is available. Consequently, an estimate of the reliability of the values is 
not  possible  and  no  uncertainty  is  given.  Additional  material  information  values  cannot  be  used  for  calibration, 
demonstration  of  method  or  laboratory  proficiency  or  method  trueness.  They  can  be  used  to  e.g.  anticipate  possible 
interferences in measurement processes. 

A certified qualitative property value was assigned as outlined in ISO 33406:2024 [68, 69]. 
Four datasets were used to assign the certified value for EURM-023, the fish species Gadus morhua 
(AphiaID 126436 [19]). This is based on two genetic methods using different barcode loci, the mtDNA 
barcodes  cytb  and  COI.  These  barcodes  were  measured  by  two  laboratories  each,  applying 
bidirectional Sanger sequencing.  

The length of the resultant DNA sequence for the cytb barcode was 413 bp and for the COI barcode 
652 bp. The DNA sequences of both barcodes are given in FASTA format in the Tables 5.2 and 5.3 of 
Annex 5.  

The fish species identified for all characterisation samples from both datasets for the cytb barcode 
is Gadus morhua, based on the similarity scores of minimum 99 % identity, with a query coverage of 
97 % to 100 %, with the selected Gadus morhua reference sequences retrieved by the BLAST search 
tool (version BLASTN  2.17.0+, NCBI) [35, 36, 37, 38]. The resulting taxon, the fish species, is linked 
to the selected reference sequences in the GenBank [17] and FishTrace [18] databases (Table 1).  

The fish species identified for all characterisation samples from both datasets for the COI barcode is 
Gadus morhua, based on similarity scores of 100 % similarity, with a query coverage of 92 % to 100 
%, with the selected Gadus morhua reference sequences retrieved by the taxonomy browser of BOLD 
[15, 16]. The resulting taxon, the fish species, is linked to the selected reference sequences in the 
BOLD COI Species Database (Table 2). 

The certified value’s genetic identity  has a minimum of 99 % sequence similarity  with the Gadus 
morhua DNA sequences of the various selected specimens (voucher samples and/or specimens with 
morphological  identification),  deposited  in  the  GenBank  [17],  FishTrace  [18]  and  BOLD  [15,  16] 
databases.  It  is  unlikely  that  there  is  an  alternative  species  that  shares  the  sequences  of  the  two 
independent genes used in this study. Consequently, the uncertainty of the certified value is considered 
negligible. 

25 

 
 
 
 
 
 
 
8  Metrological traceability and commutability 

8.1  Metrological traceability 

Box 8. Metrological traceability 

Metrological traceability of measurement results is a key requirement for ensuring the comparability of data. As CRMs 
are used to make measurement results traceable to a common reference, metrological traceability of its certified values 
to a stated reference is essential.  

The certified value of a CRM is metrologically traceable if the measurement results used for establishing it can all be 
related to the same reference through an unbroken chain of calibrations.  

This requires that these measurements 

• refer to the same property (e.g. Pb content) and the same (kind of) quantity (e.g. Pb mass fraction),  
• result in a number and its uncertainty (e.g. 6 ± 2) expressed in the same measurement unit (e.g. μg/kg). 

The concept of metrological traceability rests in chemistry on several anchor points, namely identity, quantity value and 
measurement unit. The identity of a measurand can be defined by its chemical structure alone or can be operationally 
defined, the quantity value of the measurand can refer to the SI or to other appropriate references.   

8.1.1 

Identity 

The  fish  species  is  a  structurally  defined  qualitative  property,  independent  of  the  identification 
methods, as confirmed by two different mtDNA barcode loci, cytb and COI.  

8.1.2  Qualitative property value 

The certified value is traceable, via the mtDNA sequence of the cytb and COI barcodes, to the Gadus 
morhua reference sequences obtained from morphologically identified species in the GenBank [17], 
FishTrace [18] and BOLD [15, 16] databases.  

26 

 
 
 
8.2  Commutability  

Box 9. Commutability 

Commutability is a prerequisite for RMs intended to be used for calibration or quality control of different measurement 
procedures targeting the same measurand. The concept of commutability of a RM is defined by the VIM [12] as: 

“property  of  a  reference  material,  demonstrated  by  the  closeness  of  agreement  between  the  relation  among  the 
measurement results for a stated quantity in this material, obtained according to two given measurement procedures, 
and the relation obtained among the measurement results for other specified materials”  

Commutability  is  a  property  of  an  RM  indicating  how  well  an  RM  mimics  the 
characteristics of a typical routine sample in various measurement procedures for a 
stated measurand.  

The  same  RM  may  be  commutable  for  some  measurement  procedures  but  non-
commutable  for  others.  A  commutability  statement  is  therefore  only  valid  for  the 
mentioned measurement procedure(s). 

The CRM is prepared from fresh fish fillet that was freeze-dried and milled into a powder. 

PCR amplification and bidirectional Sanger sequencing of two different DNA barcodes, cytb and COI, 
works well for fresh or frozen tissue samples as well as for the dried fish fillet powder of EURM-023. 
It can therefore be concluded that the analytical behaviour is sufficiently similar to routine samples 
for PCR and DNA sequencing. 

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 ± 3) °C in the dark. Care should be taken to avoid any change of 
the moisture content once the units are open, as the material is hygroscopic. The user should close 
the unit immediately after taking a sample.  
For more information regarding the shelf life of reference materials please consult ERM Application 
Note 7 [70]. 

The  stability  of  samples  after  opening  has  not  been  tested.  Note  that  the  European  Commission 
cannot  be  held  responsible  for  changes  that  may  happen  to  samples  after  opening  or  when  the 
material is stored differently from the stated storage conditions at the customer's premises. 

9.3  Use and reconstitution of the material 

To  make  EURM-023  ready  for  use,  the  material  must  be  reconstituted  according  to  the  following 
procedure:  
Weigh 7 mg of the fish powder into a 2 mL low-DNA-binding microcentrifuge tube and add 160 μL 
nuclease-free  water,  mix  for  10  s  using  a  vortex  mixer  and  immediately  continue  with  the  DNA 
extraction. 
Repeated sampling or use has not been tested and occurs under the responsibility of the user. 

For general information on handling of reference materials, please consult ERM Application Note 6 
[71]. 

9.4  Minimum sample size 

The minimum sample size is 7 mg. 
EURM-023  is  a  pure  fish  material  consequently  the  user  may  opt,  at  their  own  discretion  and 
responsibility, for a smaller sample size provided that the method’s minimum DNA quantity specified 
by the applied DNA barcoding method is respected. 

9.5  Use of the certified value 

This material is intended  for use in method validation, specifically to assess the accuracy of DNA 
barcoding methods for the identification of the fish species Gadus morhua. 

28 

 
 
 
 
10 Conclusions 

EURM-023  is  a  fish  fillet  powder  material  certified  for  its  taxon,  the  species  Gadus  morhua.  This 
material was produced and certified in accordance with ISO 17034:2016 [28] and is certified for a 
qualitative property, as outlined in ISO 33406:2024 [68]. 
EURM-023 supports the correct labelling of fresh fish and fish products as it can be used to assess 
the  method  performance  for  fish  authenticity  measurements  that  are  executed  to  control  the 
compliance  with  the  rules  of  common  fisheries  policy  and  to  obtain  a  transparent  production, 
processing and marketing of fishery or aquaculture products (e.g. Regulation (EU) No 1379/2013 [1], 
(EC)  No  1224/2009  [4]  (last  amended  in  (EU) 2023/2842  [5],  (EC)  No 178/2002  [6],  (EC)  No 
1005/2008 [7] (EU) and (EU) No 2017/625 [8]). Analytical results of DNA-based authenticity tests are 
used to authorise import of products into the EU internal market from third countries and for official 
control of fisheries produced inside the EU. Moreover, these authenticity tests are used to control the 
correct labelling of products to avoid health risks in relation to fish allergies and to combat fish fraud: 
intentional mislabelling and species substitution. 
The following qualitative property value was assigned: 

Table 3. Value assigned to EURM-023. 

Certified value 

Fish species 

Uncertainty  

Taxon 1)  

Gadus morhua (Atlantic cod) 2) 

Negligible 3) 

1) According to the Linnaean Classification system 

2) Certified values are values that fulfil the highest standards of accuracy. Gadus morhua was first documented by Linnaeus in 1758 and 
is registered in the World Register of Marine Species as AphiaID 126436. The given value represents the consensus value of accepted 
datasets of two DNA barcodes, the mitochondrial cytochrome b gene (cytb) and the mitochondrial cytochrome c oxidase subunit I gene 
(COI), obtained in two laboratories by bidirectional Sanger sequencing. The certified value is traceable to the Gadus morhua reference DNA 
sequences deposited in the databases GenBank®, FishTrace and Barcode of Life Database System (BOLD). 

3) It is unlikely that there is an alternative species that shares the sequences of the cytb and COI barcodes, which showed to have at least 99 % 
similarity with the reference sequences with a query coverage of 92 % to 100 %. Consequently, the uncertainty of the certified value is negligible. 

Source: JRC 

This material is intended  for use in method validation, specifically to assess the accuracy of DNA 
barcoding methods for the identification of the fish species Gadus morhua. 

29 

 
 
 
 
 
 
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Note 

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Use 

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(last 

35 

 
 
 
 
List of abbreviations  

ANOVA 

AphiaID 

BLAST 

BLASTN 

BOLD 

bp 

CI 

COI 

CRM 

cytb 

DNA 

EC 

EDTA 

EU 

EURM® 

FAO 

FASTA 

GUM 

ID 

ISO 

IUCN 

JRC 

k 

mtDNA 

n 

N 

n.a. 

PCR 

RM 

RMP 

SOP 

T 

t 

Analysis of variance 

The data system behind WoRMS is called Aphia. All the names in 
WoRMS are tagged with a unique and persistent ID: the AphiaID 

Basic Local Alignment Search Tool 

Basic Local Alignment Search Tool Nucleotide 

Barcode of Life Database System 

Base pair 

Confidence interval 

Cytochrome c oxidase subunit I 

Certified reference material 

cytochrome b 

Deoxyribonucleic acid 

European Commission 

Ethylenediaminetetraacetic acid 

European Union 

Trademark owned by the European Commission; used by the JRC for 
reference materials 

Food and Agriculture Organization of the United Nations 

Fast-all 

Guide to the Expression of Uncertainty in Measurement 

Identity 

International Organization for Standardization 

International Union for Conservation of Nature and Natural Resources 

Joint Research Centre of the European Commission 

Coverage factor 

Mitochondrial DNA 

Number of replicate analysis per unit 

Number of units analysed 

Not applicable 

Polymerase chain reaction 

Reference material 

Reference material producer 

Standard operating procedure 

Temperature 

Time 

 
 
TACS 

TE 

TRIS 

U 

UV 

V-KFT 

WoRMS 

Total allowable catches 

Buffer containing TRIS and EDTA 

Tris(hydroxymethyl)aminomethane 

Expanded uncertainty 

Ultraviolet 

Volumetric Karl Fischer titration 

World Register of Marine Species 

 
 
List of boxes 

Box 1. Reference material production ...................................................................................................................................... 7 

Box 2. Reference material processing ...................................................................................................................................... 9 

Box 3. Homogeneity assessment ............................................................................................................................................. 12 

Box 4. Stability assessment ........................................................................................................................................................ 14 

Box 5. Stability monitoring ........................................................................................................................................................... 16 

Box 6. Reference material characterisation ....................................................................................................................... 17 

Box 7. Assignment of values to a reference material .................................................................................................. 25 

Box 8. Metrological traceability ................................................................................................................................................. 26 

Box 9. Commutability ...................................................................................................................................................................... 27 

 
 
 
 
 
 
 
List of figures 

Figure 1. EURM-023. .......................................................................................................................................................................... 9 

Figure 2. Volume-based cumulative distribution of particle size in EURM-023. .......................................... 10 

 
 
 
 
 
List of tables 

Table 1. Average DNA sequence alignment identity results of EURM-023, expressed as identity 
percentage, compared with 18 Gadus morhua reference sequences in GenBank and Fish Trace for 
the cytb barcode using BLASTN 2.17.0+. .............................................................................................................................. 21 

Table 2. Average DNA sequence alignment similarity results of EURM-023, expressed as similarity 
percentage, compared with 28 Gadus morhua reference sequences for the COI barcode using the 
BOLD system v4 identification tool (COI species Database). .................................................................................... 23 

Table 3. Value assigned to EURM-023. ................................................................................................................................ 29 

 
 
 
 
 
 
Annexes 

Annex 1. Summary of PCR methods used 

Primers for PCR-sequencing for the cytb and COI genes used for homogeneity, stability and 
characterisation studies: 

Table 1.1. PCR and sequencing primers cytb 1). 

Primer name 

Sequence 5' – 3' 

Forward primer 
L14735  

Reverse primer 
H15149ad  

AAA AAC CAC CGT TGT TAT TCA ACT A 

GCN CCT CAR AAT GAY ATT TGT CCT CA 

Reference 

[13] 

[13] 

1) The expected size of the PCR product is 464 bp. 
Source: C. Wolf, M. Burgener, P. Hübner, J. Lüthy, PCR-RFLP Analysis of Mitochondrial DNA: Differentiation of Fish Species, 
LWT - Food Science and Technology, 33(2) (2000) 144-150, doi:10.1006/fstl.2000.0630 

Table 1.2. PCR and sequencing primers COI 1). 

Primer name, 
PCR 
VF2_t1   

FishF2_t1  
FishR2_t1   
FR1d_t1   

Primer name, 
sequencing 

Sequence 5' – 3' 2)  

TGTAAAACGACGGCCAGTCAA CCA ACC ACA AAG ACA TTG GCA C 

TGTAAAACGACGGCCAGTCGA CTA ATC ATA AAG ATA TCG GCA C 

CAGGAAACAGCTATGACACT TCA GGG TGA CCG AAG AAT CAG AA 

CAGGAAACAGCTATGACACC TCA GGG TGT CCG AAR AAY CAR AA 

Sequence 5' – 3' 

M13_F 

TGTAAAACGACGGCCAGT 

Reference 

[29, 30, 31] 

[29, 30, 31] 

[29, 30, 31] 

[29, 30, 31] 

Reference 

[31] 

M13_R 

CAGGAAACAGCTATGAC 
1) The expected size of the PCR product is 738 bp. 
2) Primers with highlighted M13 tails.  
Source: Labelfish consortium, Standard operating procedure for the genetic identification of fish species using DNA 
barcoding (mitochondrial cytochrome I sequencing), https://www.seatraces.eu/sop/ 

[31] 

 
 
 
 
 
 
Method for cytb specific PCR used for stability measurements: 

Table 1.3. PCR conditions for cytb (C1000 Touch Thermal Cycler, Bio-Rad, NL). 

Section  

Hot start  

Denaturation 

Annealing 

Elongation 

Final elongation 

Cooling 

Source: JRC 

Temperature °C 

95 

95 

50 

72 

72 

10 

Time 

11 min 

45 s 

80 s 

90 s 

30 min 

Until manually stopped 

Cycles 

1 

35 

1 

1 

Table 1.4. Master-mix composition for cytb PCR reaction. 

Reagent  

Concentration 

Concentration 

Final solution 

μl per 
reaction 

Supplier 

Buffer  

Stock 

10 X 

1 x 

5.00 

AmpliTaq Gold PCR buffer, 
ThermoFisher scientific, Gent, 
BE 

Primer F: L14735 

7.5 μM 

Primer R: H15149ad  7.5 μM 

0.5 μM 

0.5 μM 

3.33 1) 
3.33 1) 

dNTPs 

10 mM 

250 μM 

1.25 

Polymerase 

5 U/μL 

0.05 U/μL 

0.50 

MgCl2 

25 mM 

3.0 mM 

6.00  

dNTP mix, 10 mMol each, 
ThermoFisher scientific, Gent, 
BE 

AmpliTaq Gold DNA 
polymerase, ThermoFisher 
scientific, Gent, BE 

MgCl2, AmpliTaq Gold DNA 
polymerase kit, ThermoFisher 
scientific, Gent, BE 

Water 
DNA 2) 

Total volume (μl) 

~20 ng/μL  

~60 ng/reaction  3.00 

50.00 

27.59 1) 

Promega Benelux, Leiden, NL 

1) For the stability study, the PCR reaction mix was prepared using the forward and reverse primer together in one master 
mix composition. Primers according Annex 1, Table 1.1. 
2) DNA concentration by UV spectrophotometry. 
Source: JRC 

 
 
 
 
  
  
  
 
  
  
 
 
  
  
 
Method for COI specific PCR according to the standard operating procedure from the Labelfish Consortium used 
for stability measurements: 

Table 1.5. PCR conditions for COI (C1000 Touch Thermal Cycler, Bio-Rad, NL). 

Section  

Hot start  

Denaturation 

Annealing 

Elongation 

Final elongation 

Cooling 

Temperature °C 

94 

94 

52 

72 

72 

10 

Time 

2 min 

30 s 

40 s 

60 s 

10 min 

Until manually stopped 

Cycles 

1 

35 

1 

1 

Source: Labelfish consortium, Standard operating procedure for the genetic identification of fish species using DNA 
barcoding (mitochondrial cytochrome I sequencing), https://www.seatraces.eu/sop/ 

Table 1.6. Master-mix composition for COI PCR reaction.  

Reagent 

Concentration 

Concentration  

Stock 

Final solution 

D(+)-Trehalose dihydrate  10 % 

Buffer  

10 X 

5 % 

1 x 

μl per 
reaction 

10.00 

2.00 

Supplier  

Alfa Aesar, VWR, Leuven, BE 

Reaction buffer, Bioline, 
GCbiotech, Waddinxveen, NL 

Forward  primers VF2_t1 
and FishF2_t1 

Reverse primers 
FishR2_t1 and FR1d_t1 

10 μM 

0.1 μM each 

0.20 each 1) 

10 μM 

0.1 μM each 

0.20 each 1) 

dNTPs 

10 mM 

200 μM 

0.40 

Polymerase 

5 U/μL 

0.025 U/μL 

0.10 

MgCl2 
Water 

DNA 2) 

50 mM 

2.5 mM 

1.00 
2.70 1) 

~20 ng/μL  

~60 ng/reaction  3.00 

dNTP mix, Bioline, GCbiotech, 
Waddinxveen, NL 

Biotaq DNA polymerase, 
Bioline, GCbiotech, 
Waddinxveen, NL 

MgCl2, Bioline 
Promega Benelux, Leiden, 
NL   

Total volume (μl) 

20.00 

1) For the stability study, the PCR reaction mix was prepared using a cocktail of the two forward and two reverse primers 
together in one master mix composition. Primers according to Annex 1, Table 1.2. 
2) DNA concentration by UV spectrophotometry. 
Source: Labelfish consortium, Standard operating procedure for the genetic identification of fish species using DNA 
barcoding (mitochondrial cytochrome I sequencing), https://www.seatraces.eu/sop/ 

 
 
 
 
  
  
  
  
 
 
  
  
 
  
  
 
Annex 2. Results of homogeneity measurements 

Table 2.1. Homogeneity results for EURM-023 of the species identification via the cytb barcode analysis 
using BLASTN 2.17.0+ (NCBI). Maximum and minimum identity results of the sequences of the two extraction 
replicates per unit, compared with 18 different Gadus morhua reference sequences in GenBank. 

Unit number- 
extraction 
replicate 

Species 
identification 

Maximum 
identity [%] 1), 
2) 

Minimum 
identity [%] 1), 
2) 

Sequence length 
replicate [bp] 

Query coverage 
[%] 

160-1 

160-2 

261-1 

261-2 

249-1 

249-2 

588-1 

588-2 

809-1 

809-2 

950-1 

950-1 

1167-1 

1167-2 

1317-1 

1317-2 

1431-1 

1431-2 

1602-1 

1602-2 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

Gadus morhua 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

100.0 

99.7 

100.0 

100.0 

100.0 

100.0 

99.3 

99.3 

99.3 

99.2 

99.2 

99.3 

99.3 

99.3 

99.3 

99.2 

99.3 

99.2 

99.3 

99.3 

99.3 

99.0 

99.2 

99.3 

99.3 

99.2 

413 

413 

413 

397 

398 

413 

413 

413 

413 

397 

413 

397 

413 

413 

400 

394 

385 

413 

413 

396 

97-100 

97-100 

97-100 

100 

100 

97-100 

97-100 

97-100 

97-100 

100 

97-100 

100 

97-100 

97-100 

100 

100 

100 

97-100 

97-100 

100 

1) Identity [%] = Amount of identical nucleotides / minimum aligning length. 
2)  Reference  sequences  GenBank  accession  number:  KX267041.1,  KX267003.1,  KX266978.1,  EU877726.1,  EU877718.1, 
AY946340.1,  AY946339.1,  NC_002081.1,  DQ356939.1,  EU492304.1,  DQ174046.1,  DQ174045.1,  EU492303.1, 
EU492141.1,  EU492140.1  and  Fish  Trace  voucher  codes  GadMor-EE-01a  BMVP/1237  ,  GadMor-CS-02  BMVP/1193, 
GadMor-CS-01 BMVP/1269. 

Source: JRC 

 
 
Table 2.2. Homogeneity results for EURM-023 of the species identification via the COI barcode analysis using 
the BOLD system v4 identification tool. Maximum and minimum similarity results of the sequences of the two 
extraction replicates per unit, compared with 41 different Gadus morhua reference sequences in BOLD. 

Unit number- 
extraction 
replicate 

Species 
identification 

Maximum 
similarity [%] 1), 
2) 

Minimum 
similarity [%] 1), 
2) 

Sequence length 
replicate [bp] 

Query coverage 
[%] 

160-1 
160-2 
261-1 
261-2 
349-1 
349-2 
588-1 
588-2 
809-1 
809-2 
950-1 
950-2 
1167-1 
1167-2 
1317-1 
1317-2 
1431-1 
1431-2 
1602-1 
1602-2 

Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 

100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 

100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 
100.0 

652 
650 
649 
650 
650 
650 
650 
650 
650 
638 
652 
650 
647 
645 
650 
650 
650 
650 
650 
650 

92-100 
92-100 
92-100 
92-100 
92-100 
92-100 
92-100 
92-100 
92-100 
94-100 
92-100 
92-100 
92-100 
93-100 
92-100 
92-100 
92-100 
92-100 
92-100 
92-100 

1) The statistics related to the aligned sequences in BOLD, are expressed in similarity percentage. 
2) Reference sequences BOLD process ID: ANGBF21909-19, ANGBF21963-19, BCGM136-08, BCGM137-08, BCOLL019-06, 
BCOLL157-06,  BCOLL162-06,  BCOLL163-06,  BCOLL164-06,  BNSF005-11,  BNSF006-11,  BNSF007-11,  BNSF008-11, 
BNSF102-11,  BNSF103-11,  BNSF350-11,  BNSF351-11,  BNSF352-11,  BNSF592-12,  DSFIB455-13,  FCFUK058-06, 
FCFUK252-06,  SFCFUK253-06,  FOAD165-05,  FOAD166-05,  FOAD167-05L,  FOAD168-05,  FOAD169-05,  GBGC3821-07, 
GBMNA16893-19,  NOFIS088-10,  SCAFB023-07,  SCAFB081-07,  SCAFB377-07,  SCAFB692-07,  SCFAC019-05, 
SCFAC022-05, SCFAC050-05, SCFAC259-06, SCFAC839-06, UKFBI381-08. 

Source: JRC 

 
 
 
Figure  2.1.  Sequence  electropherogram  (forward)  of  cytb  barcode  of  one  sample  for  homogeneity  and 
characterisation.  

Source: JRC 

Figure  2.2.  Sequence  electropherogram  (reverse)  of  cytb  barcode  of  one  sample  for  homogeneity  and 
characterisation. 

Source: JRC 

 
 
 
 
Figure 2.3. Sequence electropherogram (forward) of COI barcode of one sample for homogeneity and 
characterisation. 

Source: JRC 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2.4. Sequence electropherogram (reverse) of COI barcode of one sample for homogeneity and 
characterisation. 

Source: JRC 

 
 
 
 
 
 
 
 
  
Annex 3. Results of the short-term stability measurements 

Figure 3.1. Transport stability of EURM-023 assessed over four weeks at 4 °C, DNA concentration 
recalculated to a sample size of 7 mg. 

200

180

160

140

120

100

80

]
L
μ
/
g
n
[

n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

0

1

2

3

4

Storage time [week]

Source: JRC 

Figure 3.2. Transport stability of EURM-023 assessed over four weeks at 18 °C, DNA concentration 
recalculated to a sample size of 7 mg. 

200

180

160

140

120

100

80

]
L
μ
/
g
n
[

n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

0

1

2
Storage time [week]

3

4

Source: JRC 

Figure 3.3. Transport stability of EURM-023 assessed over four weeks at 60 °C, DNA concentration 
recalculated to a sample size of 7 mg. 

200

150

100

50

0

]
L
μ
/
g
n
[

n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

0

1

2
Storage time [week]

3

4

Source: JRC 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Annex 4. Results of the long-term stability measurements 

Figure 4.1. Storage stability of EURM-023 assessed over 24 months at -20 °C, DNA concentration 
recalculated to a sample size of 7 mg. 

180

160

140

120

100

80

60

]
L
μ
/
g
n
[

n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

0

6

Storage time [month]

12

18

24

Source: JRC 

Figure 4.2. Storage stability of EURM-023 assessed over 24 months at 4 °C, DNA concentration recalculated 
to a sample size of 7 mg. 

180

160

140

120

100

80

60

]
L
μ
/
g
n
[

n
o
i
t
a
r
t
n
e
c
n
o
c
A
N
D

0

6

12

18

24

Storage time [month]

Source: JRC 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
Annex 5. Results of the characterisation measurements 

Table 5.1. Fish species assignment for characterisation samples per laboratory and barcode.  
Unit 2 
Fish species 

Unit 4 
Fish species 

Unit 1 
Fish species 

Unit 3 
Fish species 

Unit 5 
Fish species 

Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 

Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 

Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 

Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 

Gadus morhua 
Gadus morhua 
Gadus morhua 
Gadus morhua 

Laboratory  
code/ 
barcode 
D01 / cytb 
D02 / cytb 
D01 / COI 
D02 / COI 

Source: JRC 

Table 5.2. FASTA format of the DNA sequence of the Gadus morhua fish fillet powder in EURM-023, cytb 
barcode.  

>EURM-023, Gadus morhua, cytb, 413 bp 
CAAGAACCTTAATGGCCAGCCTTCGGAAAACCCATCCAATCCTAAAAATTGCTAATAGCGCATTAGTTGATC
TCCCCGCCCCCTCCAATATCTCAGTATGATGAAATTTTGGCTCTCTTCTAGGCCTTTGCTTAATTACTCAAC
TTCTAACAGGACTATTTCTAGCCATACACTATACCTCAGACATCGAAACAGCCTTCTCATCCGTAGTCCACA
TCTGTCGTGATGTAAACTACGGCTGACTAATTCGGAATATACATGCTAATGGTGCCTCTTTCTTTTTCATTT
GTCTTTATATGCACATTGCCCGAGGTCTCTATTATGGTTCCTATCTTTTTGTAGAGACATGAAATATCGGGG
TTGTCCTTTTCCTTTTAGTAATAATAACCTCTTTCGTAGGTTATGTCCTCCCC 

Source: JRC 

Table 5.3. FASTA format of the DNA sequence of the Gadus morhua fish fillet powder in EURM-023, COI 
barcode.  

>EURM-023, Gadus morhua, COI, 652 bp 
CCTTTATCTCGTATTTGGTGCCTGAGCCGGCATAGTCGGAACAGCCCTAAGCCTGCTCATTCGAGCAGAGCT
AAGTCAACCTGGTGCACTTCTTGGTGATGATCAAATTTATAATGTGATCGTTACAGCGCACGCTTTCGTAAT
AATTTTCTTTATAGTAATACCACTAATAATTGGAGGCTTTGGGAACTGACTCATTCCTCTAATGATCGGTGC
ACCAGATATAGCTTTCCCTCGAATAAATAACATAAGCTTCTGACTTCTTCCTCCATCTTTCCTGCTCCTTTT
AGCATCCTCTGGTGTAGAAGCTGGGGCTGGAACAGGCTGAACTGTCTATCCACCTTTAGCCGGAAACCTCGC
TCATGCTGGGGCATCTGTTGATCTCACTATTTTTTCTCTTCATCTAGCAGGGATTTCATCAATTCTTGGGGC
AATTAATTTTATTACCACAATTATTAATATGAAACCTCCGGCAATTTCACAGTACCAAACACCCCTATTTGT
TTGAGCAGTACTAATTACAGCTGTGCTTCTACTATTATCTCTCCCCGTCTTAGCAGCTGGTATCACAATACT
TCTAACTGACCGTAATCTTAATACTTCTTTCTTTGACCCTGCTGGAGGAGGTGATCCCATTTTATACCAACA
CTTA 
Source: JRC 

 
 
 
 
 
 
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