The certification of the mass concentrations of Cd, Cr, Hg, Ni, Pb and Tl in human blood : ERM-DA634, ERM-DA635 and ERM-DA636.
Date: 2025-02-11
Institutions: Joint Research Centre
Subjects: approval, medical research, report, research method, scientific research, testing

The certification of the mass concentrations of  
Cd, Cr, Hg, Ni, Pb and Tl in human blood. 
ERM®-DA634, ERM®-DA635 and ERM®-DA636 

T.P.J. Linsinger, L. Deprez, G. Auclair 

2025 

 
 
 
 
 
 
This  publication  is  a  Reference  Materials  report  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  publication  do  not  necessarily  reflect  the  position  or 
opinion of the European Commission. Neither the European Commission nor any  person acting on behalf of 
the  Commission  is  responsible  for  the  use  that  might  be  made  of  this  publication.  For  information  on  the 
methodology and quality underlying the data used in this publication for which the source is neither Eurostat 
nor other Commission services, users should contact the referenced source. The designations employed and 
the presentation of material on the maps do not imply the expression of any opinion whatsoever on the part 
of the European Union concerning the legal status of any country, territory, city or area or of its authorities, or 
concerning the delimitation 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  

JRC138775 

PDF  

ISBN 978-92-68-21621-7   doi:10.2760/4057065  

KJ-01-24-123-EN-N 

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

The  reuse  policy  of  the  European  Commission  documents  is  implemented  by  the  Commission  Decision  2011/833/EU  of  12  December 
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How  to  cite  this  report:  European  Commission:  Joint  Research  Centre,  LINSINGER,  T.P.J.,  DEPREZ,  L.,  AUCLAIR,  G., 
Certification of the mass concentrations of Cd, Cr, Hg, Ni, Pb and Tl in human blood ERM-DA634, ERM-DA635 and ERM-
DA636, Publications Office of the European Union, Luxembourg, 2025, https://data.europa.eu/doi/:10.2760/4057065, JRC138775. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Contents  

Abstract .......................................................................................................................................................................................................................................................................... 1 

Acknowledgements ................................................................................................................................................................................................................................................. 2 

1 

Introduction..................................................................................................................................................................................................................................................... 3 

1.1.  Background ........................................................................................................................................................................................................................................ 3 

1.2.  Choice of the material ............................................................................................................................................................................................................ 4 

1.3.  Outline of the CRM project ................................................................................................................................................................................................. 4 

2  Participants ..................................................................................................................................................................................................................................................... 5 

2.1  Project management and data evaluation ......................................................................................................................................................... 5 

2.2  Processing .......................................................................................................................................................................................................................................... 5 

2.3  Homogeneity and stability measurements......................................................................................................................................................... 5 

2.4  Characterisation measurements .................................................................................................................................................................................. 5 

3  Material processing and processing control .................................................................................................................................................................... 6 

3.1  Origin of the starting material ........................................................................................................................................................................................ 6 

3.2  Processing .......................................................................................................................................................................................................................................... 6 

3.3  Processing control ....................................................................................................................................................................................................................... 7 

4  Homogeneity ................................................................................................................................................................................................................................................. 8 

4.1  Between-unit homogeneity ............................................................................................................................................................................................... 8 

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

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

5.1  General ............................................................................................................................................................................................................................................... 14 

5.2  Transport stability .................................................................................................................................................................................................................... 14 

5.3  Storage stability ........................................................................................................................................................................................................................ 16 

5.3.1 

General considerations ..................................................................................................................................................................................... 16 

5.3.2 

Stability data on BCR-634, -635 and -636 .................................................................................................................................. 16 

5.3.3 

12-months stability study ............................................................................................................................................................................. 17 

5.4  Estimation of uncertainties............................................................................................................................................................................................. 19 

5.5  Stability of reconstituted samples ........................................................................................................................................................................... 21 

6  Characterisation ...................................................................................................................................................................................................................................... 22 

6.1  Characterisation principle ................................................................................................................................................................................................. 22 

6.2  Selection of participants .................................................................................................................................................................................................... 22 

6.3  Study setup .................................................................................................................................................................................................................................... 22 

6.4  Measurement procedures used .................................................................................................................................................................................. 23 

6.5  Evaluation of results ............................................................................................................................................................................................................. 23 

6.5.1 

Technical evaluation ............................................................................................................................................................................................ 23 

6.5.2 

Statistical evaluation .......................................................................................................................................................................................... 25 

7  Value Assignment .................................................................................................................................................................................................................................. 28 

7.1  Certified values and their uncertainties ............................................................................................................................................................. 28 

i 

 
7.2  Additional material information ................................................................................................................................................................................. 30 

8  Metrological traceability and commutability ............................................................................................................................................................... 31 

8.1  Metrological traceability .................................................................................................................................................................................................... 31 

8.1.1 

Identity ............................................................................................................................................................................................................................. 31 

8.1.2  Quantity value ........................................................................................................................................................................................................... 31 

8.2  Commutability ............................................................................................................................................................................................................................. 32 

9 

Instructions for use .............................................................................................................................................................................................................................. 33 

9.1  Safety information .................................................................................................................................................................................................................. 33 

9.2  Storage conditions ................................................................................................................................................................................................................... 33 

9.3  Use and reconstitution of the material ............................................................................................................................................................... 33 

9.4  Minimum sample size .......................................................................................................................................................................................................... 33 

9.5  Use of the certified values .............................................................................................................................................................................................. 33 

10 Conclusions .................................................................................................................................................................................................................................................. 35 

References ............................................................................................................................................................................................................................................................. 36 

List of abbreviations .................................................................................................................................................................................................................................... 38 

List of boxes......................................................................................................................................................................................................................................................... 41 

List of figures ..................................................................................................................................................................................................................................................... 42 

List of tables ........................................................................................................................................................................................................................................................ 43 

Annexes .................................................................................................................................................................................................................................................................... 44 

Annex 1. Results of the homogeneity measurements ....................................................................................................................................... 45 

Annex 2. Results of the transport stability measurements ........................................................................................................................... 48 

Annex 3. Results of the storage stability measurements ............................................................................................................................... 51 

Annex 5. Summary of methods used in the characterisation study ..................................................................................................... 56 

Annex 6. Results of the Characterisation study ........................................................................................................................................................... 0 

Annex 7. Results of the commutability study ............................................................................................................................................................. 18 

ii 

 
Abstract 

ERM-DA634,  ERM-DA635  and  ERM-DA636  are  human  blood  reference  materials  certified  for  their  mass 
concentrations  of  cadmium,  chromium,  mercury,  nickel,  lead  and  thallium.  They  were  produced  within  the 
scope of ISO 17034 accreditation [1]. 

Human  blood  was  lysed,  spiked  with  the  six  elements,  dispensed  into  vials  and  lyophilised.  The  CRMs  are 
available in sets of three glass vials, each containing 1 mL freeze-dried blood.  

Between-unit  homogeneity  was  quantified  and  stability  during  transport  and  storage  was  assessed  in 
accordance with ISO 33405:2024 [2]. The minimum sample size for one measurement is 150 mg for Hg and 
250 mg for all other elements. 

The  material  was  characterised  by  an  interlaboratory  comparison  of  laboratories  of  demonstrated 
competence and adhering to ISO/IEC 17025:2017 [3] or ISO 15189 [4].  

Uncertainties  of  the  certified  values  were  calculated  in  accordance  with  ISO  17034:2016  [1]  and  include 
uncertainties related to possible inhomogeneity, instability and characterisation. 

The materials are intended for the assessment of method performance, calibration and quality control. 

Before release of the CRMs, the certification project was subjected to peer-review involving both internal and 
external experts. 

1 

 
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 these CRMs. 

Furthermore,  the  authors  would  like  to  thank  the  experts  of  the  Reference  Material  Review  Panel  Dr  Paola 
Fisicaro  (Laboratorie  National  de  Métrologie  et  d’Essays,  FR)  and  Dr  Jens  Sloth  (Danmarks  Tekniske 
Universitet, DK) for their constructive comments and the external review. 

Authors 

Thomas P.J. Linsinger 1), Liesbet Deprez 1), Guy Auclair 1) 

1) European Commission, Joint Research Centre, Geel, BE 

2 

 
 
 
 
1 

Introduction 

1.1. Background 

Metals, pervasive in our environment, can have significant implications on human health.  

Acute  cadmium  (Cd)  poisoning  is  relatively  rare,  but  chronic  exposure  can  result  in  kidney  disease  and  lung 
cancer [5]. Additionally, heightened Cd exposure is associated with an increased risk of breast cancer [6]. Cd 
enters  the  environment  mainly  through  the  atmosphere  from  processes  like  Cd  production  and  processing; 
combustion of fossil fuels, garbage and sewage sludge. After deposition it is readily resorbed by plants. The 
main exposure routes for human is through food [7], but also smoking [8] is a major exposure route. 

Chromium (Cr) exists in a trivalent and hexavalent form and its toxicity is largely dependent on its oxidation 
state.  Cr(III)  at  low  doses  is  an  essential  dietary  nutrient  whereas  Cr(VI)  is  more  toxic  than  Cr(III)  and  is 
cancerogenic [9]. Exposure to Cr is mainly through ingestion (food, water) but inhalation can form a significant 
route in occupational settings. Acute poisoning is rare, but the practice of adding lead chromate to turmeric to 
enhance its colour is a public health issue in Bangladesh [10]. 

The toxic effects of mercury (Hg) are well-documented, with the infamous Minamata disease being the most 
notable  case.  However,  even  low  levels  of  Hg  exposure  can  result  in  neurological,  renal,  and  cardiovascular 
complications  [11].  Exposure  to  Hg  is  primarily  through  consumption  of  seafood  containing  methylmercury 
and  amalgam  dental  fillings  [7].  Industrial  exposure  to  inorganic  Hg  is  typically  confined  to  workers  in  the 
dental products, fluorescent lighting, and chloralkali electrolysis industries [11]. 

Exposure no nickel (Ni) can harm the lungs, stomach, and kidneys, and may also increase the risk of cancer 
[12].  In  addition,  Ni  can  cause  skin  eczema  in  sensitized  persons  [13].  Harmful  exposure  to  Ni  is  largely 
confined to workers in Ni processing plants and the jewellery industry, but exposure through food can cause 
effects in sensitized humans [12, 13]  

Lead  (Pb)  toxicity  is  also  well-established  and  includes  inhibition  of  the  synthesis  of  the  haem-complex  of 
haemoglobin,  obstruction  of  motoric  nerves  as  well  as  fatigue,  headache  and  tremor  [7].  Pb  is  particularly 
detrimental to children and unborn babies, leading to low birth weight, miscarriage, and cognitive impairment 
[14]. Alarmingly, one-third of children worldwide have blood Pb levels above 50 µg/L [15], which can result in 
stunting and cognitive impairment. The major exposure route for the general population is through food [16] 
but  exposure  can  also  occur  in  occupational  settings,  and  through  inhalation.  Residing  near  industrial 
processes can lead to increased blood Pb levels, as was observed in children living near an industrial site in 
Hoboken, Belgium [17]. 
To  protect  workers  from  these  chemical  risks,  Council  Directive  98/24/EC  [18]  has  been  introduced.  This 
directive  mandates  the  monitoring  of  workers'  blood  Pb  levels  and  necessitates  medical  surveillance  of 
workers with a blood Pb concentration exceeding 400 µg/L. 

Chronic exposure to thallium (Tl) can affect kidney function, lead to preterm births and there are reports that 
Tl is associated with autism spectrum disorder [19. While Tl was used as rat and ant poison in the past, its 
use  for  this  purposed  has  been  banned.  Exposure  today  is  mainly  occupational,  for  example,  cement 
production, coal-burning industries or smelting of sulphide ores. Exposure can also come from food grown in 
the vicinity of such sources [20]. 

Within the context of “One Health”, monitoring of exposure routes (e.g. air, food, water etc.) contributes greatly 
to  limit  the  exposure  of  the  general  population  to  harmful  concentrations  of  metals.  However,  biological 
monitoring  remains  a  very  important  tool  to  assess  actual  exposure  to  metals  where  monitoring  of 
environmental contamination may not be a reliable proxy for exposure. 

To make this monitoring meaningful and reliable, its results must be accurate and traceable To support the 
quality of monitoring results, the European Commission has funded projects for the development of certified 
reference materials (CRMs) for Cd and Pb in blood: BCR-194, BCR-195 and BCR-196, three lysed bovine blood 
materials were released in 1985 [21].  A set of lysed  human blood CRMs (BCR-634,  BCR-635 and  BCR-636) 
was released in 2004 [22]. These materials were exhausted. To ensure the constant quality of the biological 
monitoring for metals, the JRC decided to replace these materials. 

3 

 
 
 
 
1.2. Choice of the material 

Given the popularity of the predecessor materials, it was decided that the new materials should resemble the 
previously  produced  ones  as  closely  as  possible.  The  material  of  choice  was  therefore  also  a  lysed  human 
blood. The Cd and Pb concentrations were chosen to closely match those of BCR-634, BCR-635 and BCR-636. 
The Cr, Hg. Ni and Tl concentrations were chosen after checking the ranges used in human blood proficiency 
testing and  discussion  with  clinical laboratories. All element concentrations  cover the relevant concentration 
range for biological monitoring. 

Several  clinical  laboratories  were  contacted  regarding  the  format  (frozen  or  lyophilised)  and  the  volume. 
These discussions confirmed that a lyophilised material was preferred, as it guarantees long-term stability. 

Sourcing naturally incurred  material with the  desired  metal levels was deemed impossible. It was therefore 
decided to spike the starting materials for all three concentrations. 

1.3. Outline of the CRM project 

Box 1. Reference material production 

Reference  material  (RM)  production  is  defined  in  ISO  17034  [1]  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 stability of their assigned values is monitored throughout the 
life-time of the material. 

Human blood from healthy donors without risk factors for elevated metal concentrations was acquired. The 
blood  donations were lysed,  spiked to achieve the intended element concentrations,  filtered and lyophilised. 
Homogeneity was checked and the variation between units was quantified. The stability during transport was 
assessed using a 4-weeks stability study. Stability during storage for Pb and Cd was assessed using the data 
from  the  long-term  stability  monitoring  of  the  previous  certified  reference  materials.  The  findings  of  this 
assessment  were  confirmed  by  a  6-months  stability  study.  Finally,  the  materials  were  characterised  in  an 
interlaboratory  comparison  among  laboratories  of  demonstrated  competence  using  different  methods  of 
sample preparation and quantification. 

Uncertainties of certified values were assigned in compliance with ISO 17034 [1], which implements the basic 
principles of ISO/IEC Guide 98 (GUM) [23]. 

The CRM project, including the certification approach and the evaluation of the obtained measurement data, 
was subjected to peer-review involving both internal and external 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 
European Commission, nor does it imply that the material or equipment is necessarily the best available for 
the purpose. 

4 

 
 
2  Participants  

2.1  Project management and data evaluation 

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

(accredited to ISO 17034:2016 for production of certified reference materials, BELAC No. 268-RM) 

2.2  Processing 

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

2.3  Homogeneity and stability measurements 

ALS Scandinavia, Luleå, SE  
(measurements performed under the scope of ISO/IEC 17025:2017 accreditation SWEDAC 2030) 

2.4  Characterisation measurements 

ALS Scandinavia, Luleå, SE  
(measurements performed under the scope of ISO/IEC 17025:2017 accreditation SWEDAC 2030) 

Institut za medicinska istraživanja i medicinu rada, Zagreb, HR 

Institut Jožef Stefan, Ljubljana, SI   

Laboratoire National de Métrologie et d'Essais, Trappes Cedex, FR 
(measurements for Cd and Pb performed under the scope of ISO/IEC 17025:2017 accreditation COFRAC 2-54) 

Medizinisches Labor Ostsachsen MVZ GbR, Görlitz, DE 
(measurements performed under the scope of ISO 15189 accreditation DAkkS D-ML-13288-01-00) 

Sciensano, Tervuren, BE 

Työterveyslaitos, Helsinki, FI 
(measurements for Cd, Cr, Hg and Pb performed under the scope of ISO/IEC 17025:2017 accreditation FINAS 
T013) 

Umweltbundesamt GmbH, Wien, AT 
(measurements under the scope of ISO/IEC 17025:2017 accreditation Akkreditierung Austria 0200) 

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

5 

 
 
 
 
 
 
 
3  Material processing and processing control   

Box 2. Reference material processing 

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

3.1  Origin of the starting material 

A  total  of  28  frozen  donations  of  206  mL  to  311  mL  from  healthy  human  donors  (19  male,  9  female)  of 
human  blood  were  delivered  by  In.Vent  Diagnostica  GmbH  (Hennigsdorf,  DE).  Each  donation  contained 
2.21 mmol K3EDTA as anticoagulant.  

Each individual blood donation has been tested for HIV-1/2 antibodies, HCV antibodies and HBs antigen with 
methods cleared in compliance with the European Directive 98/79/EC, Annex II, List A and found negative or 
non-reactive. 

The samples were collected according to the principles of the Declaration of Helsinki (DoH) and Taipei (DoT), 
processed and verified in a way that is ethically and legally compliant for the purposes of diagnostic research 
and development, production and quality assurance. 

Furthermore,  the  samples  were  collected  under  ethic’s  approval.  The  ethic’s  approval  was  granted  by  the 
ethics commission review board of the Freiburger Ethik Kommission international under feki Code: 011/1763. 

3.2  Processing 

After thawing, the contents of each blood bag were transferred into a beaker and 2.21 mmol CaCl2 (99.99 % 
on metal basis; Thermo Scientific, Kandel, Germany) was added to each donation to bind the EDTA. The beaker 
was  placed  on  ice  and  the  blood  was  lysed  for  10  minutes  using  an  ultrasonic  probe  (Q700,  Qsonica, 
Newtown,  US)  with  a  total  energy  input  of  50  -  65  J.  The  sonication  was  performed  in  two  stages  of  5 
minutes to avoid overheating the blood. After lysing, the donations were placed in a 37 °C warm water bath 
for  2-3  h  for  clotting.  Then  lysates  were  pooled  into  1-L  bottles  and  were  centrifuged  at  4  °C at  12000  G 
(Sorvall  RC6+,  Thermo  Fisher  Scientific,  Waltham,  US)  and  filtered  using  a  0.45 µm  polyethersulfone  filter 
(Thermo Fisher Scientific, Waltham, US) followed by a second filtration through a 0.22 µm polyethersulfone 
filter (VWR, Haasrode, BE). At that stage, the lysate for each of the materials was pooled and spiked to obtain 
the desired metal levels. Afterward the pools were diluted by adding two times the volume of ultrapure water 
(resistivity 18.2 MΩ.cm at 25 °C) and stirred overnight at 4 °C. The purpose of the dilution was to reduce the 
viscosity of the lysate and to increase the repeatability of the filling volume by filling a larger volume. 

Five-mL  amber  glass  vials  were  rinsed  with  5  %  nitric  acid  and  rinsed  with  ultrapure  water  (resistivity 
18.2 MΩ·cm  at  25  °C).  Three  mL  of  the  spiked  and  diluted  lysate  were  dispensed  into  each  vial  using  a 
peristaltic pump. The lysate was stirred during the filling process. The lysate was then freeze-dried (Epsilon2-
100D, Martin Christ, Osterode am Harz, DE). After freeze drying, the vacuum was broken with Ar and the vials 
were closed with silicone stoppers and capped. The dry matter content of each vial was about 200 mg. The 
number  of  vials  retained  of  ERM-DA634,  ERM-DA635  and  ERM-DA636  was  2454,  1935,  and  1314, 
respectively. 

For the purpose of this report, the term ‘unit’ refers to one vial of ERM-DA634, ERM-DA635 and ERM-DA636. 
In this way it is ensured that the certified values with their uncertainty are valid for the individual unit. The 
materials were assembled into sets of three units, with the code xa, xb and xc with x being the set number. 
The  labelling  was  done  in  a  way  that  each  set  contains  units  from  the  complete  filling  sequence.  If,  for 
example, 2400 units were filled, labelling was done as follows: Unit 1 to 800 were labelled as “a”, unit 801 to 
1600 were labelled as “b” and units 1601 to 2400 were labelled as “c”. The labelling within a, b and c was 
done in order of the filling sequence. 

6 

 
 
 
One set of CRM ERM-DA635 is shown in Figure 1 as an example for the three CRMs. 

Figure 1. CRM ERM-DA635.  

Source: JRC 

3.3  Processing control  

16 pre-weighed empty vials were entered at approximately every 100th position of the filling sequence. After 
filling, these units were removed and weighed. The relative standard deviations of the masses of ERM-DA634 
and  ERM-DA635  were  0.9  %  and  no  trend  was  visible  in  the  masses,  showing  that  the  filling  was  under 
control. A significant start-up effect was visible for the masses for ERM-DA636, where the first unit had a 5 % 
lower filling mass than later units. As the blood was subsequently lyophilised and reconstituted with the same 
volume  of  water  regardless  of  the  initial  filling  volume,  this  was  expected  to  result  in  lower  metal 
concentrations for these vials. This effect was confirmed by measurement of the metal concentration of six 
samples from unit number 3 to 126. Therefore, the first 50 sets were removed from the batch. The relative 
standard  deviation  of  the  filling  masses  of  the  remaining  tested  unit  masses  was  0.4 %,  demonstrating 
sufficient control after the problems at the start. 

7 

 
 
 
 
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 [1] 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 
its  stated 
sample  size  guarantees  the  assigned  value  within 
uncertainty. 

4.1  Between-unit homogeneity 

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

The  number  of  units  selected  corresponds  to  approximately  the  cube  root  of  the  total  number  of  units 
produced  for  ERM-DA634,  ERM-DA635  and  ERM-DA636  (2454,  1935,  and  1524,  respectively).  In  total  15 
units  were  selected  using  a  random  stratified  sampling  scheme  covering  the  whole  batch  for  the  between-
unit homogeneity test. This random stratified sampling involves dividing the total number of set numbers into 
five  equal  groups  and  randomly  choosing  one  set  from  each  group.  As  each  set  contains  three  units  from 
different thirds of the batch, this setup ensures coverage of the whole batch in a random-stratified manner. 
The  samples  were  reconstituted  and  three  independent  samples  were  taken  from  each  selected  unit  and 
analysed  by  acid-digestion  inductively  coupled  plasma  sector  field  mass  spectrometry  (ICP-SFMS).  The 
measurements  were  performed  under  repeatability  conditions  and  in  a  randomised  manner  to  separate  a 
potential drift in the measurement results from a potential trend in the filling sequence.  

Regression analyses were performed to evaluate potential trends in the measurement sequence. For several 
elements (see Table 1, Table 2 and Table 3) trends in the measurement sequence were visible, pointing at a 
changing parameter, e.g. a signal drift in the measurement system. Research has shown that the correction of 
even non-significant biases is beneficial. Such correction of non-significant biases has two advantages: on the 
one  hand,  the  resulting  uncertainties  are  smaller  than  for  non-corrected  biases.  On  the  other  hand,  after 
correction the  uncertainty interval  has the  highest probability of including the true value [24]. Correction of 
trends is therefore expected to improve the sensitivity of subsequent statistical analysis through a reduction 
in  the  variation  of  measurement  results  without  masking  potential  between-unit  heterogeneities.  As  the 
measurement  sequence  and  the  unit  numbers  were  not  correlated,  trends  significant  on  at  least  a  95  % 
confidence level were corrected as shown below: 

xi_corr = xi

- b ∙ i  

Equation 1 

b 
i 

slope of the linear regression 
position of the result in the measurement sequence 

Regression analyses were performed on the (if necessary) trend-corrected data to evaluate potential trends in 
the  filling  sequence.  For  Pb  in  ERM-DA634  a  trend  in  the  filling  sequence  that  was  significant  on  a  95  % 
confidence level was detected. This is about what is expected by chance (5 % of 18 sequences = 0.9) and, as 
this  trend  is  not  visible  for  any  other  of  the  elements  in  the  same  material,  might  be  a  statistical  artefact. 
Nevertheless, this trend was taken into consideration in the evaluation of ubb of Pb.  

The (if necessary) trend-corrected dataset was assessed for consistency using the single and double Grubbs 
outlier tests at a confidence level of 99 % on the individual results and on the unit means. 

8 

 
 
 
 
 
 
 
 
For ERM-DA634, two individual outliers were detected for Cr. These outliers only concerned one of the three 
individual replicates of one unit and were removed.  

For ERM-DA635, two Individual outliers were detected for Cr. These outliers only concerned one of the three 
individual  replicates  of  one  unit  and  were  removed.  For  none  of  the  materials  outlying  unit  means  were 
detected.  

For ERM-DA636, two outlying unit means were observed for Pb. These were flagged as outliers, although the 
results agreed within their confidence intervals with the other results. These two means were retained. 

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

Evaluation by ANOVA requires mean values per unit, which follow at least a unimodal distribution and results 
for  each  unit  that  follow  unimodal  distributions  with  approximately  the  same  standard  deviations.  The 
distribution  of  the  mean  values  per  unit  was  visually  tested  using  histograms  and  normal  probability  plots. 
Too few data are available for the unit means to make a clear statement of the distribution. Therefore, it was 
checked  visually  whether  all  individual  data  follow  a  unimodal  distribution  using  histograms  and  normal 
probability  plots.  Minor  deviations  from  unimodality  of  the  individual  values  do  not  significantly  affect  the 
estimate of between-unit standard  deviations. The results of all statistical evaluations are given in Table 1, 
Table 2 and Table 3; the results of the homogeneity tests are shown in graphical form in Annex 1. 

The unit means for NI do not follow normal distributions. The distributions are broad with a hint of bimodality. 
Evaluation by ANOVA is therefore not applicable. 

Table 1. Results of the statistical evaluation of the homogeneity study of ERM-DA634. Trends in the measurement 
sequence were corrected before further data treatment.  

ERM-DA634 

Trends 1) 

Outliers 2) 

Distribution 

Measure
ment 
sequence 

yes 

yes 

no 

no 

yes 

yes 

Filling 
sequence 

Individual 
results 

Unit means 

Individual 
results 

Unit means 

no 

no 

no 

no 

yes 

no 

no 

2 (removed) 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

normal 

normal 

normal 

normal 

normal 

normal 

normal 

not normal 

unimodal 

normal 

normal 

normal 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

95 % confidence level. 
99 % confidence level. 

1) 
2) 
Source: JRC 

9 

 
 
 
Table 2. Results of the statistical evaluation of the homogeneity study of ERM-DA635: Trends in the measurement 
sequence were corrected before further data treatment.  

ERM-DA635 

Trends 1) 

Outliers 2) 

Distribution 

Measure
ment 
sequence 

Filling 
sequence 

Individual 
results 

Unit means 

yes 

yes 

yes 

no 

no 

yes 

no 

no 

no 

no 

no 

no 

no 

2 (removed) 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

Individual 
results 

unimodal 

normal 

normal 

Unit means 

normal 

normal 

normal 

normal 

not normal 

normal 

normal 

normal 

normal 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

95 % confidence level. 
99 % confidence level. 

1) 
2) 
Source: JRC 

Table 3. Results of the statistical evaluation of the homogeneity study of ERM-DA636. Trends in the measurement 
sequence were corrected before further data treatment.  

ERM-DA636 

Trends 1) 

Outliers 2) 

Distribution 

Measure
ment 
sequence 

yes 

no 

no 

no 

no 

yes 

Filling 
sequence 

Individual 
results 

Unit means 

Individual 
results 

Unit means 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

normal 

normal 

normal 

normal 

normal 

normal 

normal 

not normal 

2 (retained) 

normal 

unimodal 

no 

normal 

normal 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

95 % confidence level. 
99 % confidence level. 

1) 
2) 
Source: JRC 

It should be noted that sbb, rel and swb, rel are estimates of the standard deviations and are therefore subject to 
random  fluctuations.  Therefore,  the  mean  square  between  groups  (MSbetween)  can  be  smaller  than  the  mean 
squares within groups (MSwithin), resulting in a negative number under the square root used for the estimation 
of  the  between-unit  variation,  whereas  the  true  variation  cannot  be  less  than  zero.  In  this  case,  u*
bb,  the 
maximum  inhomogeneity  that  could  be  hidden  by  method  repeatability,  was  calculated  as  described  by 
Linsinger et al. [25]. u*
bb is comparable to the limit of detection (LOD) of a measurement method yielding the 
maximum degree of inhomogeneity that might be undetected by the given study setup. 

Method  repeatability  (swb,  rel)  (equivalent  to  the  within-unit  standard  deviation),  between–unit  standard 
deviation (sbb, rel) and u*

bb, rel were calculated as:  

swb, rel = 

MSwithin
y

�

�

Equation 2 

10 

 
 
 
 
 
 
 
 
 
sbb, rel = 

MSbetween
n
y

�

�

4

MSwithin
n

- MSwithin

2
νMSwithin

*
ubb, rel

 =  �

�
y

�

Equation 3 

Equation 4 

MSwithin  mean of squares within-unit from ANOVA  
MSbetween  mean of squares between-unit from ANOVA 
mean of all results of the homogeneity study 
y
mean number of replicate analysis per unit 
n 
�
νMSwithin 
degrees of freedom of MSwithin 

For Pb in ERM-DA634, the trend in the filling sequence was significant on a 95 % confidence level. Therefore 
the  uncertainty  was  assessed  in  a  different  way.  Here,  urec  was  estimated  using  a  rectangular  distribution 
between the highest and lowest unit mean [23] as given in Equation 5. The same approach was adopted for 
Ni (all materials), where the unit means do not follow normal distributions.  

The  Pb  mass  concentrations  in  ERM-DA636  are  a  special  case:  The  unit  means  show  two  outliers  with 
concentrations  above  the  mean  value  (451  µg/L  and  445  µg/L  compared  to  the  average  of  432  µg/L). 
Removal  of  these  two  outliers  reveals  another  outlier  with  a  lower  concentration  (421  µg/L).  Despite  these 
outliers, the homogeneity of the Pb mass concentration is still sufficient for use. Throughout the other studies 
(stability, characterisation) no other such outliers were found  supporting the assumption that the outliers in 
the homogeneity study indeed cover the highest and lowest concentrations that can be expected. Therefore, 
the uncertainty of homogeneity was also estimated using a rectangular distribution between the highest and 
lowest unit mean, hence taking the outliers into consideration (Equation 5). 

urec= 

max - x
x
2 ∙ 
|
�
mean of all results of the homogeneity study 
highest unit mean of the homogeneity study 
lowest unit mean of the homogeneity study 

min
3 ∙ y
�

�

|

√

y
max 
x
�
min 
x
�
The results of the evaluation of the between-unit variation are summarised in Table 4, Table 5 and Table 6. In 
�
half of the cases, the uncertainty contribution for homogeneity was determined by the method repeatability. 

Equation 5 

Table 4. Results of the homogeneity study of ERM-DA634. 

ERM-DA634 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

swb, rel 

[%] 

6.64 

11.86 

9.91 

13.53 

6.30 

6.05 

n.a.: not applicable as not evaluated by ANOVA.  

1) 
2) n.c.: not calculated, as MSbetween < MSwithin 

Source: JRC 

sbb, rel 

[%] 

1.84 

n.c. 2) 

n.c. 2) 

n.a.1) 

n.a.1) 

3.48 

11 

u*

bb, rel 

[%] 

1.95 

3.62 

2.91 

3.97 

1.85 

1.78 

urec, rel 

[%] 

n.a.1) 

n.a.1) 

n.a.1) 

9.55 

3.67 

n.a.1) 

ubb, rel 

[%] 

1.95 

3.62 

2.91 

9.55 

3.67 

3.48 

 
 
  
 
 
  
 
 
 
 
   
 
 
 
 
 
Table 5. Results of the homogeneity study of ERM-DA635. 

ERM-DA635 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

swb, rel 

[%] 

2.30 

3.15 

3.56 

3.73 

5.27 

3.92 

sbb, rel 

[%] 

1.30 

1.18 

1.20 

n.a.1) 

n.c.2) 

n.c.2) 

n.a.: not applicable. As not evaluated by ANOVA 
n.c.: cannot be calculated as MSbetween < MSwithin. 

1) 
2) 
Source: JRC 

Table 6. Results of the homogeneity study of ERM-DA636. 

ERM-DA636 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

swb, rel 

[%] 

3.16 

4.00 

5.07 

4.07 

4.94 

4.92 

sbb, rel 

[%] 

1.17 

2.38 

n.c.2) 

n.a.1) 

n.a.1) 

n.c.2) 

u*

bb, rel 

[%] 

0.68 

0.96 

1.04 

1.09 

1..55 

1.15 

u*

bb, rel 

[%] 

0.94 

1.19 

1.51 

1.21 

1.48 

1.47 

urec, rel 

[%] 

n.a.1) 

n.a.1) 

n.a.1) 

2.05 

n.a.1) 

n.a.1) 

urec, rel 

[%] 

n.a.1) 

n.a.1) 

n.a.1) 

2.65 

2.31 

n.a.1) 

ubb, rel 

[%] 

1.30 

1.18 

1.20 

2.05 

1.55 

1.15 

ubb, rel 

[%] 

1.17 

2.38 

1.51 

2.65 

2.31 

1.47 

n.a.: not applicable. As not evaluated by ANOVA 
n.c.: cannot be calculated as MSbetween < MSwithin.   

1) 
2) 
Source: JRC 

The homogeneity studies showed outlying unit means for Pb in ERM-DA636, but the deviation was within the 
confidence  intervals  of  the  various  means.  Except  for  Pb  in  ERM-DA634,  none  of  the  trends  in  the  filling 
sequence were significant on a 95 % confidence level. Therefore, the between-unit standard deviation sbb or 
u*
bb can be used as an estimate of ubb. This holds for all elements except Pb in ERM-DA634 and Ni: these data 
do  not  follow  normal  distributions  and  hence  a  calculation  of  a  standard  deviation  would  be  not  valid. 
Therefore,  ubb  was  estimated  from  a  rectangular  distribution.  As  u*
bb  sets  the  limits  of  the  study  to  detect 
inhomogeneity, the larger value of sbb and u*
bb is adopted as uncertainty contribution to account for potential 
inhomogeneity. 

12 

 
 
 
4.2  Within-unit homogeneity and minimum sample size 

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

The minimum sample size was established based on the technically valid results of the characterisation and 
stability studies, using the method information supplied by the participants. The smallest sample intake that 
still yielded results with acceptable precision to be included in the respective studies was taken as minimum 
sample size. The following minimum sample sizes of reconstituted material are derived: 

Hg: 150 mg which is for practical purposes equivalent to 150 µL. 

Cd, Cr, Ni, Pb, Tl: 200 mg which is for practical purposes equivalent to 200 µL. 

13 

 
5  Stability 

5.1  General 

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 and light (including ultraviolet radiation) were regarded as the most relevant influences on 
the stability of the materials. The influence of ultraviolet and visible light is minimised by storing the material 
in amber glass units, which reduce light exposure. In addition, materials are stored in the dark and transported 
in  boxes,  thus  removing  any  possibility  of  degradation  by  light.  Therefore,  only  the  influences  of  time  and 
temperature needed to be investigated. 

The stability studies were carried out using an isochronous design [26]. In this approach, units are stored for a 
particular  length  of  time  in  different  temperature  conditions.  Afterwards,  the  units  are  moved  to  conditions 
where  further  degradation  can  be  assumed  negligible  (reference  conditions).  At  the  end  of  the  isochronous 
storage,  the  samples  are  analysed  simultaneously  under  repeatability/intermediate  precision  conditions. 
Analysis  of  the  material  (after  various  exposure  times  and  temperatures)  under  repeatability/intermediate 
precision conditions greatly improves the sensitivity of the stability tests. 

5.2  Transport stability 

The conditions for the transport of the material to the  customers  were established in  a short-term stability 
study. To this end, units were stored at 18 °C and 60 °C for 0, 1, 2 and 4 weeks (at each temperature). The 
reference temperature was set to -20 °C. Two units per storage time were selected using a random stratified 
sampling  scheme.  From  each  unit,  three  samples  were  measured  by  acid-digestion  ICP-SFMS.  The 
measurements  were  performed  under  repeatability  conditions  and  a  randomised  sequence  was  used  to 
differentiate  any  potential  drift  in  the  measurement  results  from  a  potential  trend  over  storage  time.  The 
data were evaluated individually for each temperature. For ERM-DA636, only units with numbers > 70 were 
used because of the run-in effects found in the processing. 

The  results  were  screened  for  trends  in  the  analytical  sequence  and  trends  were  corrected  if  they  were 
significant on a 95 % confidence level as described in section 4.1. The results were screened for outliers using 
the single and double Grubbs test on a confidence level of 99 %. 

In addition, the data were evaluated against storage time, and regression lines of mass concentration versus 
time were calculated, to test for potential increases or decreases of the mass concentration due to shipping 
conditions. The slopes of the regression lines were tested for statistical significance. 

The results of the measurements are shown in Annex 2. The results of the statistical evaluation of the short-
term stability are summarised in Table 7, Table 8 and Table 9.  

14 

 
 
 
 
 
Table 7. Results of the short-term stability tests of ERM-DA634. 

ERM-DA634 

Number of individual outlying results 1) 

Significance of the trend 2) 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

18 °C 

none 

none 

none 

none 

none 

none 

60 °C 

none 

none 

none 

none 

none 

none 

18 °C 

60 °C 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

yes (95 %) 

no 

1) 
99 % confidence level.  
2)   95 % confidence level. 
Source: JRC 

Table 8. Results of the short-term stability tests of ERM-DA635. 

ERM-DA635 

Number of individual outlying results 1) 

Significance of the trend 2) 

18 °C 

none 

none 

none 

none 

none 

none 

60 °C 

none 

none 

none 

none 

none 

none 

18 °C 

60 °C 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

no 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

1) 
99 % confidence level.  
2)   95 % confidence level. 
Source: JRC 

Table 9. Results of the short-term stability tests of ERM-DA636. 

ERM-DA636 

Number of individual outlying results 1) 

Significance of the trend 2) 

18 °C 

none 

none 

none 

none 

none 

none 

Cd 

Cr 

Hg 

Ni 

Pb 

Hg 

1) 
99 % confidence level.  
2)   95 % confidence level. 
Source: JRC 

60 °C 

none 

none 

none 

none 

none 

none 

15 

18 °C 

60 °C 

no 

no 

no 

no 

no 

no 

no 

no 

no 

yes (95 %) 

no 

no 

 
 
The trends of mass concentration versus time at 60 °C were significant at a 95 % confidence level for Pb in 
ERM-DA634 and Ni in ERM-DA636. Two of 36 positive results is about the expected rate of false positives on 
a 95 % confidence level. The elements themselves are stable and any trend can only be caused by changes in 
the  matrix,  which  should  affect  all  elements.  As  none  of  the  other  elements  showed  a  trend  for  these  two 
materials, these trends are probably statistical artefacts. Nevertheless, they will be taken into consideration 
when assessing the uncertainties. 

The material can be dispatched without further precautions under ambient conditions. 

5.3  Storage stability 

5.3.1  General considerations 

Pb and Cd are stable elements that cannot degrade. However, degradation of the blood matrix is possible and 
therefore stability during storage must be assessed. 

Storage  conditions  and  shelf  life  guaranteeing  the  stability  of  the  material  and  the  certified  values  were 
established in a long-term stability study. Stability of the Pb and Cd mass concentrations was assessed using 
the  stability  data  of  BCR-634,  BCR-635  and  BCR-636.  To  verify  that  the  data  obtained  from  the  stability 
monitoring  of  these  CRMs  produced  and  stored  in  the  same  way  could  be  used  to  estimate  the  stability 
uncertainty contribution for ERM-DA634, ERM-DA635 and ERM-DA6365, the data of the a 12-months stability 
study were compared to the stability monitoring data. The data of the 12-months stability study supports the 
assumption  that  the  Cd  and  Pb  mass  concentrations  in  ERM-DA634,  ERM-DA635  and  ERM-DA636  are  as 
stable  as  in  BCR-634,  BCR-635  and  BCR-636  and  therefore  uncertainty  estimations  based  on  stability 
monitoring data can be used to estimate the uncertainty contribution relating to the storage of the CRM. 

5.3.2  Stability data on BCR-634, -635 and -636 

The CRMs BCR-194, BCR-195 and BCR-196 with certified values for Pb and Cd in bovine blood, have been for 
sale  for  22  years  (BCR-194)  and  35  years  (BCR-195,  BCR-196),  respectively.  During  this  time,  they  were 
tested 15 times for stability with no indication of degradation.  

BCR-634, BCR-635 and BCR-636 were certified in 2003. A 24-months stability study with time points of 0, 6, 
12 and 24 months and a 48-months stability study with time points of 0, 12, 24 and 48 months were started 
after the release. In each of the studies, 6 independent measurements per time point were performed under 
repeatability conditions. 

In addition, units were moved from the normal storage temperature at -20 °C to the reference temperature of 
-70 °C in October 2002. These reference samples were used to assess potential changes in the materials in 
the  stability tests of  2009, 2011, 2013, 2015, 2017, 2019,  2021 and 2023 (82-222  months). For each of 
these  tests,  six  independent  measurements  of  samples  stored  at  the  storage  temperature  and  six 
independent measurements of units stored at the reference temperature were performed. 

A quantitative assessment for the possible degradation of the materials was made separately for Cd and Pb 
on the basis of the human materials (BCR-634, -635 and -636) as follows: 

For each time point of the isochronous studies and the follow-up stability monitoring studies, the ratio of the 
mean result from normal stock to mean result from reference stock was calculated. These data are shown in 
a graph in Annex 3. 

The data of BCR-634, -635 and -636 were pooled. This is justified as the three materials were processed in 
the same way and as the analytes under investigation are the same. 

The  data  were  checked  for  outliers  and  significance  of  the  trends.  The  results  of  these  investigations  are 
shown in Table 10 

16 

 
 
 
Table 10. Evaluation of the pooled data of the stability monitoring of BCR-634, BCR-635 and BCR-636. 

Number  of 
results 1) 

individual  outlying 

Significance of the trend 2) 

1 (removed) 

none 

yes 

no 

Cd 

Pb 

99 % confidence level. 
95 % confidence level. 

1) 
2) 
Source: JRC 

The dataset for Cd contained one outlying ratio where the result at reference temperature was 16 % lower 
than at the storage temperature, indicating a more severe degradation at -70 °C than at -20 °C. The reason 
for this is that this result (time point 130 months) was close to the limit of quantification of the method. As 
this finding is unlikely and was also not confirmed by the results of BCR-635 and BCR-636 at this time point 
and neither from any other time, the outlier was removed. 

The  trend  for  Cd  is  statistically  significant.  This  significance  hinges  at  the  results  at  221  and  248  months. 
Removing one of these two time points means that the trend is no longer significant, which indicates a lack of 
robustness  of  the  finding.  In  addition,  the  trend  is  small:  at  a  rate  of  0.11  %  per  year,  it  is  technically  not 
relevant. This means that despite the apparent trend, the Cd mass concentration is sufficiently stable even if 
this apparent trend is real. As a precautionary measure, the apparent degradation is included in the estimation 
of the uncertainty. 

The data for Pb showed neither outliers nor a significant trend. 

The uncertainty evaluation of the data is described in section .5.4. 

5.3.3  12-months stability study  

Units were stored at -20 °C for 0, 4, 8 and 12 months. The reference temperature was set to -70 °C. Two units 
per  storage  time  were  selected  using  a  random  stratified  sampling  scheme.  From  each  unit,  three  samples 
were digested and measured by ICP-SFMS. The measurements were performed under repeatability conditions 
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  results  were  screened  for  trends  in  the  analytical  sequence  and  trends  were  corrected  if  they  were 
significant on a 95 % confidence level as described in section 4.1. Single and double Grubbs outliers tests at a 
confidence level of 99 % were performed.  

Finally, the data were plotted against storage time and linear regression lines of mass concentration versus 
time were calculated. The slopes of the regression lines were tested for statistical significance (loss/increase 
due to storage). 

The results of the statistical evaluation of the long-term stability study are summarised in Table 11 to Table 
14.  

17 

 
 
 
 
 
Table 11. Results of the 12-months study of ERM-DA634. 

Element 

Number  of  individual 
outlying results 1) 

Significance  of  the  trend  in  the 
analytical sequence 2) 

Significance of the trend in time 2) 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

none 

none 

none 

none 

none 

none 

no 

no 

no 

no 

no 

yes 

99 % confidence level.   

1) 
Source: JRC 

2) 95 % confidence level. 

Table 12. Results of the 12-months study of ERM-DA635  

no 

no 

no 

no 

no 

no 

Element 

Number  of  individual 
outlying results 1) 

Significance  of  the  trend  in  the 
analytical sequence 2) 

Significance of the trend in time 2) 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

2 (removed) 

none 

none 

none 

1 (removed) 

none 

99 % confidence level. 
95 % confidence level. 

1) 
2) 
Source: JRC 

yes 

no 

no 

no 

no 

no 

no 

no 

no 

yes 

no 

no 

Table 13. Results of the 12-months study of ERM-DA636. 

Element 

Number  of  individual 
outlying results 1) 

Significance  of  the  trend  in  the 
analytical sequence 2) 

Significance of the trend in time 
2) 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

none 

none 

none 

none 

none 

none 

99 % confidence level. 
95 % confidence level. 

1) 
2) 
Source: JRC 

no 

no 

no 

yes 

no 

no 

no 

no 

no 

yes 

no 

no 

18 

 
 
 
 
 
 
 
5.4  Estimation of uncertainties 

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

The  uncertainties  of  stability  during  transport  and  storage  were  estimated,  as  described  in  [27]  for  each 
element. In this approach, the uncertainty of the linear regression line with a slope of zero was calculated. The 
uncertainty  contributions  usts  and  ults  were  calculated  as  the  product  of  the  chosen  transport  time/shelf  life 
and the uncertainty of the regression lines as: 

usts, rel = 

srel

2
)

- t

(ti

 ∙ ttt 

�∑

�

ults, rel = 

srel

2
)
- t

(ti

 ∙ tsl 

�∑

Equation 6 

Equation 7 

srel 
ti 
t
ttt 
tsl 

relative standard deviation of all results of the stability study 
time elapsed at time point i 
mean of all ti 
chosen transport time (1 week at 60 °C) 
chosen shelf life (18 months at 18 °C) 

For Pb in ERM-DA634 (short-term), Ni in ERM-DA635 (1 year) and Ni in ERM-DA636 (1 year and short-term) 
where  the  slope  b  of  the  regression  line  was  significant  on  a  95  %  confidence  level  as  well  as  for  the 
assessment of the long-term stability for Cd, the potential effect of change was included in the uncertainty 
estimation.  This  effect  was  modelled  as  a  rectangular  distribution  and  added  to  the  uncertainty  of  the 
regression line itself as shown below: 

Equation 8 

usts, rel = 

 ∙ ttt 

2
𝑠𝑠𝑦𝑦𝑦𝑦
∑(𝑡𝑡𝑖𝑖 − 𝑡𝑡�)

�

2

2
𝑏𝑏𝑟𝑟𝑟𝑟𝑟𝑟

+

3

b 
syx 
ti 
t
ttt 

slope of the regression line 
relative standard error of the regression 
time elapsed at time point i 
mean of all ti 
chosen transport time (1 week at 60 °C) 

The following uncertainties were estimated: 

- 

- 

usts,rel, the uncertainty of stability during transport. This was estimated from the 60 °C study. The 
uncertainty describes the possible change during a transport at 60 °C lasting for one week. 

ults,rel,  the  uncertainty  of  stability  during  storage.  This  uncertainty  contribution  was  estimated 
from stability monitoring for Pb and Cd of the CRMs BCR-634, BCR-635 and BCR-636 and was 
estimated  for  3  years  of  storage.  For  all  other  elements,  the  stability  was  estimated  for  18 
months of storage based on the results of the 1-year study. 

The results of these evaluations are summarised in Table 15. 

19 

 
 
 
 
 
 
 
̅
 
 
 
 
̅
 
 
 
 
 
 
̅
 
 
Table 14. Uncertainties of stability during transport and storage for ERM-DA634. usts,rel was calculated for a temperature 

of 60 °C and 1 week; ults,rel was calculated for a storage temperature of -20 °C and 18 months storage time. 

ERM-DA634 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

Source: JRC 

usts,rel 

[%] 

0.48 

1.39 

0.60 

1.72 

0.67 (degradation included) 

0.57 

ults,rel 

[%] 

0.12 

8.00 

2.73 

4.65 

0.10 

1.51 

Table 15. Uncertainties of stability during transport and storage for ERM-DA635. usts,rel was calculated for a temperature 

of 60 °C and 1 week; ults,rel was calculated for a storage temperature of -20 °C and 18 months storage time. . 

ERM-DA635 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

Source: JRC 

usts,rel 

[%] 

0.32 

0.54 

0.38 

0.70 

0.33 

0.54 

ults,rel 

[%] 

0.12 

4.55 

2.49 

3.53 (degradation included) 

0.10 

2.08 

Table 16. Uncertainties of stability during transport and storage for ERM-636. usts,rel was calculated for a temperature of 

60 °C and 1 week; ults,rel was calculated for a storage temperature of -20 18°C and 18 months storage time. 

ERM-DA636 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

Source: JRC 

usts,rel 

[%] 

0.54 

0.43 

0.42 

ults,rel 

[%] 

0.12 

2.90 

2.02 

1.1 (degradation included) 

4.06 (degradation included) 

0.10 

1.70 

0.33 

0.51 

20 

 
The  calculation  of  the  uncertainties  shows  that  the  statistically  significant  trends  in  the  60  °C  study  for  Pb 
(ERM-DA634)  and  Ni  (ERM-DA636)  are  technically  insignificant.  Therefore,  the  material  can  be  shipped  at 
ambient conditions without special precautions. 

An increasing trend was found in the  1-year study  for  Ni in ERM-DA635. This  would  only be possible if the 
matrix  degraded,  which  would  be  visible  also  for  the  other  elements.  The  stability  monitoring  will  show 
whether this trend is real. As a precautionary measure, the extent of the trend was included in the uncertainty 
estimation. 

A decreasing trend was found in the 1-year study for Ni in ERM-DA636. This is very unlikely to be real as Ni 
itself  is  a  stable  element.  A  degradation  could  be  due  to  changes  in  the  matrix  that  result  in  incomplete 
reconstitution. However, this effect should be visible for the other elements as well, which is not the case. The 
stability monitoring will show whether this trend is real. As a precautionary measure, the extent of the trend 
was included in the uncertainty estimation. 

The  materials  are  included  in  the  JRC's  regular  stability  monitoring  programme,  to  control  their  further 
stability. 

Box 5. Stability monitoring 

RMs  are  produced  as  batches  that  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. 

5.5  Stability of reconstituted samples 

One set of each material was used to test the stability of reconstituted samples. On day one of the study, the 
first  unit  of  each  set  was  reconstituted  and  stored  at  4  °C.  On  day  3  of  the  study,  the  second  unit  was 
reconstituted and stored at 4 °C. On day 7, the third unit of the set was reconstituted. All reconstituted units 
were  re-homogenised  on  day  8.  Three  separate  digestions  were  performed  on  each  of  the  vials  and  each 
digest was measured once. 

The results were screened for individual outlying results using a Grubbs test on a 99 % confidence level. The 
results were evaluated using one-way ANOVA with the day of the reconstitution as the grouping variable. In 
addition, the trend of the results over the reconstitution day was tested for significance. 

No  outlier  was  detected  for  ERM-DA634.  The  variation  between  days  was  not  significant  on  a  95 % 
confidence level for Cd, Cr, Hg, Pb and Tl. It was significant for Ni on a 95 %, but not on a 99 % confidence 
level. None of the trends was significant on a 95 % confidence level.  

For ERM-DA635, one individual statistical outlier for Cd  was removed. None of the  variations between days 
was significant on a 95 % confidence level. None of the trends was significant on a 95 % confidence level.  

No  outlier  was  detected  for  ERM-DA636.  None  of  the  variations  between  days  was  significant  on  a  95  % 
confidence level. None of the trends was significant on a 95 % confidence level.  

All the data are shown in graphical form in Annex 4. 

The results show that reconstituted samples can be stored for 1 week at 4 °C.  

21 

 
 
 
 
6  Characterisation  

6.1  Characterisation principle 

The  material  characterisation  was  based  on  an  interlaboratory  comparison,  i.e.  the  element  mass 
concentrations of the material were determined by combining independent datasets obtained in laboratories 
applying  different  measurement  procedures  to  demonstrate  the  absence  of  a  measurement  bias.  This 
approach converts the systematic bias of each dataset into a random variable, the combined effect of which 
is reduced by averaging over several datasets. 

Box 6. Reference material characterisation 

Material characterisation is the process of  determining the property value(s) 
of an RM. While ISO 17034 [1] allows to characterise a RM in various ways.. 
Quality  management  procedures  of  the  JRC  are  more  stringent  and  allow 
characterisation  only  by  either  interlaboratory  comparison  or  the  use  of  a 
primary method confirmed by independent analysis. 

6.2  Selection of participants 

Eight  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  determination  of  elements  in  biological  matrices, 
preferably  in  blood  by  providing  results  for  interlaboratory  comparison  exercises.  Having  a  formal 
accreditation was not mandatory, but meeting the requirements of ISO/IEC 17025 [3] or ISO 15189 [4] was 
obligatory.  All  laboratories  are  accredited,  but  not  all  measurements  were  performed  under  the  scope  of 
accreditation.  Where  measurements  were  performed  under  the  scope  of  accreditation,  the  accreditation 
number is stated in the list of participants (Section 2). 

6.3  Study setup 

Each laboratory provided one dataset. Not all laboratories measured all elements. 

Laboratories  received  several  units  of  ERM-DA634,  ERM-DA635  and  ERM-DA636  and  were  requested  to 
provide for each material six independent results. The number of units per laboratory varied depending on the 
sample  intake: laboratories  with  a  sufficiently  small  sample  intake  received  three units  and  performed  two 
independent measurements per unit; laboratories with a sample intake above 1 mL pooled two or three units 
to obtain  sufficient  material  for the analyses. The  units for  material characterisation  were selected  using a 
random  stratified  sampling  scheme  and  covered  the  whole  batch.  The  sample  preparations  and 
measurements  had  to  be  done  on  at  least  three  days  to  ensure  intermediate  precision  conditions.  An 
independent calibration was performed for each day.  

Each  laboratory  also  received  one  unit  each  of  the  certified  reference  materials  BCR-634  and  BCR-636  as 
quality  control  for  the  Pb  and  Cd  measurements.  In  addition,  laboratories  were  instructed  to  perform  one 
standard  addition  experiment  for  all  elements  on  an  independent  preparation  of  ERM-DA635  as  a  quality 
control  for  all  elements.  The  results  of  these  measurements  were  used  to  support  the  evaluation  of  the 
characterisation results. 

Laboratories were also requested to give estimations of the expanded uncertainties of the mean value of the 
six results. No approach for the estimation was prescribed, i.e. top-down and bottom-up [23] were regarded as 
equally valid procedures. 

22 

 
 
 
 
 
6.4  Measurement procedures used 

A  variety  of  digestion  and  dilution  methods  using  pressurised  digestion  HNO3  or  HNO3-H2O2,  digestion  at 
ambient pressure with HNO3, dilution with various  media  with different  quantification steps (ICP-SFMS, ICP-
QMS, ICP-Q3MS, ICP-IDCQMS, AAS) were used to characterise the material. The combination of results from 
methods  all  targeting  the  same  measurand  but  based  on  completely  different  measurement  principles 
mitigates undetected method bias. 

All methods used during the characterisation study are summarised in Annex 5. The dataset code (e.g. D01-
ICP-SFMS) is a random number and does not correspond to the order of laboratories in Section 2. The dataset 
code consists of a number assigned to each dataset (e.g. D01) and abbreviation of the measurement method 
used (e.g. ICP-SFMS). 

6.5  Evaluation of results 

The  characterisation  study  resulted  in  6-8  datasets  per  element.  All  individual  datasets  of  the  participating 
laboratories, grouped per element, are displayed in tabular and graphical form in Annex 5. 

6.5.1  Technical evaluation 

The  obtained  data  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: 

- 

- 

compliance with the instructions given: sample preparations and measurements performed on three 
days,  

absence of values given as below limit of detection (LOD) or below limit of quantification (LOQ) 

-  method performance, i.e. agreement of the measurement results with the certified values of the QC 

samples (BCR-634 and BCR-636) and the results of the standard addition experiments 

Based on the above criteria, the following data or datasets were rejected as not technically valid (Table 18).  

Cd:  The  result  for  Cd  for  the  quality  control  sample  BCR-634  of  Dataset  06  (measured  on  day  1)  were 
significantly  above  the  certified  value.  The  laboratory  suspected  a  contamination  of  the  blank  and  re-
measured  ERM-DA634,  ERM-DA635  and  ERM-DA636  (but  not  BCR-634)  on  day  2.  The  results  of  the  re-
measured  samples  are  significantly  lower  than  those  obtained  on  day  1  and  are  in  line  with  the  results 
obtained on days 2 and 3. This confirms the suspicion of a contamination of day 1. The results of day 1 were 
therefore ignored and the data obtained from the re-measurement on day 2 were used. 
The result of the standard addition of Dataset 03 disagreed with the added concentration on a 95 %, but not 
on  a  99  %  confidence  level.  This  disagreement  was  therefore  attributed  to  a  statistical  artefact  and  the 
results were retained. 

Cr: One digest for ERM-DA635 of Dataset 03 showed markedly higher results than the other digests (36 µg/L 
compared  to  21  µg/L).  This  was  the  same  digest  that  also  showed  higher  results  for  Ni  (see  below).  As 
contamination  comes  most  likely  through  stainless  steel,  also  this  deviating  result  was  attributed  to  a 
contamination of the digest and the results of this digest were not used for evaluation.  
Dataset 08 also included results for Cr. The laboratory confirmed that the method was not yet validated for 
Cr.  The  results  were  obtained  in  the  same  measurement  run  as  the  other  elements  and  the  sample 
preparation  was  therefore  the  same;  the  results  also  agreed  with  the  assigned  value  within  the  stated 
uncertainty. However, as the method was not validated, the results were not used for the calculation of the 
certified value. 

Ni: The result of the standard addition of Dataset 03 disagreed with the added concentration on a 95 %, but 
not on a 99 % confidence level. This disagreement was therefore attributed to a statistical artefact and the 
results were retained. In addition, one digest for ERM-DA635 of Dataset 03 showed markedly higher results 
than the other digests (32 µg/L compared to 18 µg/L). Re-measurement of the same digests gave the same 
results,  as  did  a  measurement  of  diluted  digests.  The  laboratory  attributed  this  deviating  result  to 
contamination and the result of this digest was therefore not used for the evaluation. 

Pb:  Dataset  02  showed  low  results  for  BCR-636.  The  laboratory  reported  that  BCR-636  was  not  a 
homogeneous  solution  after  reconstitution,  which  could  explain  the  deviation.  However,  also  the  result  for 
BCR-634  was  low.  Contrary  to  these  low  findings,  the  result  of  the  standard  addition  experiment  on  ERM-
DA635  was  indeed  the  measured  concentration  of  ERM-DA635  plus  the  added  concentration  of  the  spike. 

23 

 
 
Nevertheless, the low findings on the two BCR-materials cast doubt on the result and the complete dataset 
for Pb was not used for evaluation. 
For  ERM-DA634,  Dataset  07  showed  a  markedly  lower  results  for  day  1  than  on  day  2  and  day  3.  The 
laboratory  confirmed  that  the  concentration  level  is  close  to  the  LOQ  and  quite  far  below  the  lowest 
calibration point of 85 µg/L. In addition, day 2 and 3 had been measured with a fresh graphite furnace, which 
tends to have better sensitivity for Pb. For this reason, the values for Pb for ERM-DA634 were not included in 
the calculation of the certified value, although the average is in agreement with all other results. 

Table 17. Datasets that showed non-compliance with the instructions given and technical specifications, and action taken.  

Dataset 
code 

Description of problem 

Action taken 

Cd, all CRMs 

D06 

Contamination of the blank on 
measurement day 1 

Cd, standard 
addition 

D03 

Results for the standard addition deviated 
from the added value on a 95 %, but not 
on a 99 % confidence level. 

Cr, ERM-DA635 

D03 

One digest gave markedly higher results. 

Data of the re-measured 
samples on day 2 were 
used. 

Results retained 

Results of this digest not 
used for evaluation 

D08 

D03 

Cr, all CRMs 

Ni, standard 
addition 

Ni, ERM-DA635 

Method not validated for Cr 

Not used for evaluation 

One digest gave markedly higher results. 

Results for the standard addition deviated 
from the added value on a 95 %, but not 
on a 99 % confidence level. 

Results of this digest not 
used for evaluation 

Results retained 

Pb, all materials 

D02 

Results for BCR-634 and BCR-636 do not 
agree with the certified values. 

Data not used for 
evaluation 

Pb, ERM-DA634 

D07 

Result close to LOQ and far below lowest 
calibration point 

Data not used for 
evaluation 

Source: JRC 

24 

 
 
 
 
 
 
6.5.2  Statistical evaluation 

The datasets accepted based on technical reasons were tested for normality of dataset means using normal 
probability  plots  and  were  tested  for  outlying  means  using  the  Grubbs  test  and  using  the  Cochran  test  for 
outlying standard deviations (both at a 99 % confidence level). Standard deviations within (swithin) and between 
(sbetween)  laboratories  were  calculated  using  one-way  ANOVA.  The  results  of  these  evaluations  are  shown  in 
Table 19 to Table 21. The individual data are shown as table and as charts in Annex 6. 

Table 18. Statistical evaluation of the technically accepted datasets for ERM-DA634. p: number of technically valid 
datasets. n.a. not applicable, as the data do not follow a normal distribution 

Element 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

Source: JRC 

p 

8 

6 

7 

6 

6 

6 

Outliers 

Means 

Variances 

Normally 
distributed 

no 

no 

no 

no 

no 

no 

no 

D02, D07 

D06 

D02, D03, D08 

no 

D06, D08, D02 

yes 

yes 

yes 

no 

yes 

yes 

Statistical parameters 

s 

sbetween 

swithin 

[µg/L] 

0.088 

0.306 

0.274 

0.831 

1.15 

[µg/L] 

0.079 

0.285 

0.252 

n.a. 

1.13 

[µg/L] 

0.095 

0.267 

0.259 

0.617 

0.54 

0.062 

0.060 

0.037 

Mean 

[µg/L] 

1.289 

1.462 

1.614 

3.069 

18.62 

0.809 

Table 19. Statistical evaluation of the technically accepted datasets for ERM-DA635. p: number of technically valid 
datasets.  

Element 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

Source: JRC 

p 

8 

6 

7 

6 

7 

6 

Outliers 

Means 

Variances 

Normally 
distributed 

no 

no 

no 

no 

no 

no 

D05 

no 

D06 

D8 

no 

no 

yes 

yes 

yes 

yes 

yes 

yes 

Statistical parameters 

s 

sbetween 

swithin 

[µg/L] 

0.377 

[µg/L] 

0.369 

1.33 

3.31 

0.50 

11.2 

1.27 

3.27 

0.30 

11.0 

[µg/L] 

0.183 

0.93 

1.31 

0.96 

4.5 

0.601 

0.593 

0.247 

Mean 

[µg/L] 

5.655 

22.03 

25.52 

18.27 

182.0 

8.311 

Table 20. Statistical evaluation of the technically accepted datasets for ERM-DA636. p: number of technically valid 
datasets. 

Element 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

Source: JRC 

p 

8 

6 

7 

6 

7 

6 

Outliers 

Means 

Variances 

Normally 
distributed 

no 

no 

no 

no 

no 

no 

D05 

D07 

no 

D08 

D07 

no 

yes 

yes 

yes 

yes 

yes 

yes 

25 

Statistical parameters 

s 

sbetween 

swithin 

[µg/L] 

[µg/L] 

[µg/L] 

0.65 

2.99 

6.00 

2.87 

34.7 

1.44 

0.63 

2.95 

5.85 

2.63 

33.9 

1.42 

0.35 

1.86 

2.39 

2.78 

9.0 

0.46 

Mean 

[µg/L] 

10.85 

41.93 

56.36 

35.96 

438.8 

16.57 

 
The results for Ni in ERM-DA634 show three groups of results: one at 2.0 µg/L, three at (2.7-2.9) µg/L and two 
at 4.0 - 4.2 µg/L. The lowest dataset (2.0 µg/L) was the only one obtained by a sector field instrument at a 
higher mass resolution (4000) than the other data; these results were also obtained using a different isotope 
(62Ni rather than 60Ni). The Ni-level of this material is also rather close to the LOQ for other methods and how 
the  intercept  of  the  calibration  curve  is  drawn  becomes  increasingly  important.  In  addition,  Ni  is  prone  to 
contamination.  There  is  therefore  significant  doubt  on  what  the  correct  value  for  Ni  for  ERM-DA634  is  and 
consequently no certified value is assigned. The range is given as information value. 

All other dataset means follow normal distributions. None of the data contains outlying means. The statistical 
evaluation  flagged  several  datasets  as  outlying  variances.  This  merely  reflects  the  fact  that  different 
methods  have  different  intrinsic  variability.  As  all  measurement  methods  were  found  technically  sound,  all 
results were retained.  
The datasets are therefore consistent and the mean of means is a good estimate of the true value. Most of 
the  standard  deviations  between  dataset  means  are  considerably  larger  than  the  standard  deviation  within 
datasets, showing that confidence intervals of replicate measurement results are unsuitable as estimates of 
measurement uncertainty. 

The  uncertainty  related  to  the  characterisation  is  estimated  as  the  standard  error  of  the  mean  of  means 
(s/

p) (Table 22 to Table 24).  

Table 21. Uncertainty of characterisation for ERM-DA634. 

√

s 

[µg/L] 

0.088 

0.306 

0.274 

1.15 

0.062 

s 

[µg/L] 

0.377 

1.33 

3.31 

0.50 

11.2 

0.60 

uchar 

[%] 

2.41 

8.53 

6.42 

2.53 

3.13 

uchar 

[%] 

2.36 

2.46 

4.90 

1.12 

2.32 

2.95 

Element 

Cd 

Cr 

Hg 

Pb 

Tl 

Source: JRC 

p 

8 

6 

7 

6 

6 

Mean 

[µg/L] 

1.289 

1.462 

1.614 

18.62 

0.809 

Table 22. Uncertainty of characterisation for ERM-DA635. 

Element 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

Source: JRC 

p 

8 

6 

7 

6 

7 

6 

Mean 

[µg/L] 

5.655 

22.03 

25.52 

18.27 

182.0 

8.311 

26 

 
 
 
 
Table 23. Uncertainty of characterisation for ERM-DA636. 

Element 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

Source: JRC 

p 

8 

6 

7 

6 

7 

6 

Mean 

[µg/L] 

10.85 

41.93 

56.36 

35.96 

438.8 

16.57 

s 

[µg/L] 

0.65 

2.99 

6.00 

2.87 

34.7 

1.44 

uchar 

[%] 

2.11 

2.91 

4.02 

3.26 

2.99 

3.54 

27 

 
 
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  Directorate  F 
require a sufficient number of datasets to assign certified values. Full uncertainty budgets in accordance with 
ISO 17034 [1] and ISO Guide 35 [2] are required. Certified values of a CRM can be used for calibration and 
trueness controls.  

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 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. 

7.1  Certified values and their uncertainties 

The unweighted mean of the means of the accepted datasets as shown in Table 19 to Table 21 was assigned 
as certified value for each parameter. 

The  assigned  uncertainty  consists  of  uncertainties  relating  to  characterisation  (uchar),  potential  between-unit 
inhomogeneity  (ubb),  and  potential  degradation  during  transport  (usts),  and  storage  (ults).  These  different 
contributions were combined to estimate the relative expanded uncertainty of the certified value (UCRM, rel) with 
a coverage factor k given as: 

UCRM, rel = k 

2
ubb, rel

2
+usts, rel

2
+ults, rel

2
+uchar, rel

Equation 9 

- 

- 

- 

�
uchar was estimated as described in Section 6.5  

∙

ubb was estimated as described in Section 4.1 

usts and ults were estimated as described in Section 5 

The  mass  concentration  is  in  principle  dependent  on  the  temperature  because  of  the  thermal  expansion  of 
water.  However,  in  the  range  of  from  4  °C  to  25  °C,  the  change  of  volume  compared  to  20  °C  caused  by 
thermal  expansion  amounts  to  0.17  %  and  0.12  %,  respectively,  which  is  negligible  compared  to  the  other 
uncertainty contributions. 

The number of effective degrees of freedom were calculated using the Welch-Satterthwaite equation [23] and 
are shown in Table  25 to Table 27. The number of degrees of freedom are sufficient to justify a coverage 
factor  (k) of k=2 for all elements. 

28 

 
 
 
 
  
 
 
 
 
 
Table 24. Certified values and their uncertainties for ERM-DA634. 

CRM 

Element 

Certified value 

uchar, rel 

ubb, rel 

usts, rel 

ults, rel 

νUCRM 

UCRM, rel 

[µg/L] 

[%] 

[%] 

[%] 

[%] 

ERM-DA634 

Cd 

Cr 

Hg 

Pb 

1.29 

1.5 

1.6 

18.6 

2.41 

8.53 

6.42 

2.53 

1.95 

3.62 

2.91 

3.67 

0.48 

1.39 

0.60 

0.67 

0.12 

8.00 

2.73 

0.10 

18 

18 

11 

34 

[%] 

6.3 

24.6 

15.2 

9.0 

UCRM 

1) 

[µg/L] 

0.09 

0.4 

0.3 

1.7 

1) 

Tl 

3.48 
Expanded  (k = 2)  and  rounded  uncertainty;  uncertainties  are  always  rounded  up  [28]  and  in  a  way  that  the  rounding  error 
corresponds to 3 % to 30 % of the uncertainty. 

0.81 

3.13 

0.57 

1.51 

0.08 

9.9 

20 

Source: JRC 

Table 25. Certified values and their uncertainties for ERM-DA635. 

CRM 

Element 

Certified value 

uchar, rel 

ubb, rel 

usts, rel 

ults, rel 

νUCRM 

UCRM, rel 

ERM-DA635 

[µg/L] 

5.7 

22.0 

25.5 

18.3 

182 

[%] 

2.36 

2.46 

4.90 

1.12 

2.32 

[%] 

1.30 

1.18 

1.20 

2.05 

1.54 

[%] 

0.32 

0.54 

0.38 

0.70 

0.33 

[%] 

0.12 

4.55 

2.49 

3.53 

0.10 

11 

30 

10 

44 

12 

[%] 

5.5 

10.7 

11.3 

8.6 

5.6 

Cd 

Cr 

Hg 

Ni 

Pb 

UCRM 

1) 

[µg/L] 

0.4 

2.4 

2.9 

1.6 

11 

1) 

Tl 

1.15 
Expanded  (k = 2)  and  rounded  uncertainty;  uncertainties  are  always  rounded  up  [28]  and  in  a  way  that  the  rounding  error 
corresponds to 3 % to 30 % of the uncertainty. 

0.54 

2.95 

2.08 

0.7 

7.7 

8.3 

13 

Source: JRC 

Table 26. Certified values and their uncertainties for ERM-DA636. 

CRM 

Element 

Certified value 

uchar, rel 

ubb, rel 

usts, rel 

ults, rel 

νUCRM 

UCRM, rel 

ERM-DA636 

[µg/L] 

10.9 

42 

56 

36 
0.44*103 

[%] 

2.11 

2.91 

4.02 

3.26 

2.99 

[%] 

1.17 

2.38 

1.51 

2.65 

2.31 

[%] 

0.54 

0.43 

0.42 

1.1 

0.33 

[%] 

0.12 

2.90 

2.02 

4.06 

0.10 

16.6 

3.54 

1.47 

0.51 

1.70 

12 

26 

11 

30 

15 

9 

[%] 

5.0 

9.5 

9.5 

11.9 

7.6 

8.4 

Cd 

Cr 

Hg 

Ni 

Pb 

Tl 

UCRM 

1) 

[µg/L] 

0.6 

4 

6 

5 
0.04*103 

1.4 

1) 

Expanded  (k = 2)  and  rounded  uncertainty;  uncertainties  are  always  rounded  up  [28]  and  in  a  way  that  the  rounding  error 
corresponds to 3 % to 30 % of the uncertainty. 

Source: JRC 

29 

 
 
 
7.2  Additional material information 

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

The  range  of  the  Ni  mass  concentrations  in  ERM-DA634  is  given  as  information  value  only  because  of  the 
disagreement of results in the characterisation study. 

Table 27. Additional material information for ERM-DA634. 

CRM 

Element 

Additional material information 

ERM-DA634 
Source: JRC 

Ni 

[µg/L] 

2.0 - 4.2 

30 

 
 
 
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, metrological traceability of its certified values to a 
stated reference is essential.  

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

This requires that these measurements 

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

The concept of traceability rests on several anchor points, namely identity, quantity value and measurement 
unit. The identity of a measurand can be defined by its 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 

Cd,  Cr,  Hg  and  Pb  are  chemically  clearly  defined  analytes.  The  mass  concentration  data  were  obtained  by 
applying different measurement principles for the sample preparation as well as for the final determination, 
demonstrating  absence  of  measurement  bias.  The  measurands  are  therefore  structurally  defined  and 
independent of the measurement methods. 

Also  Ni  and  Tl  are  clearly  defined  analytes.  All  determinations  are  based  on  ICP-MS  techniques.  The  MS 
techniques vary widely, from sector field-, single- and triple quadrupole- MS, which enhances the confidence 
in the absence of method bias. However, as all mass concentration data were obtained by ICP-MS, the identity 
for Ni and Tl is therefore operationally defined by ICP-MS. 

8.1.2  Quantity value 

Only  validated  methods  were  used  during  the  characterisation  study.  Different  calibrants  of  known 
concentration and specified traceability of their assigned values were used. Investigation of the method and 
measurement  details  of  the  individual  results  show  that  all  relevant  input  parameters  of  each  technically 
accepted dataset have been properly calibrated. All technically accepted datasets are therefore traceable to 
the  same  reference,  namely  the  SI.  This  traceability  to  the  same  reference  is  also  confirmed  by  the 
agreement  of  results  within  their  respective  uncertainties.  As  the  assigned  values  are  combinations  of 
agreeing results individually traceable to the International System of Units (SI), the assigned quantity values 
themselves are traceable to the SI as well. 

31 

 
 
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 an RM is defined 
by the VIM [29] 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). 

Sample preparation in  routine laboratories consists often only of a dilution step, compared to the complete 
digestion  applied  in  the  characterisation  study  of  these  materials.  In  addition,  routine  samples  consist  of 
whole blood whereas these CRMs are lysed, which can also lead to a lack of commutability.  

The  commutability  study  was  carried  out  according  to  the  guidelines  of  the  IFCC  working  group  on 
Commutability  [30]  using  the  ‘difference  in  bias  analysis’.  In  this  commutability  study  the  CRMs  were 
compared to 20 fresh, single donation patient samples of persons with a higher risk profile for elevated metal 
concentrations in blood. In addition, 22 spiked, fresh blood samples were included in the study to ensure that 
the  whole  concentration  range  was  covered.  The  following  methods  were  tested:  ICP-MS  after  dilution  (all 
elements), ET-AAS after dilution (Cd and Pb only) and ICP-MS after digestion (all elements). Commutability of 
the CRM was assessed for each combination of digestion-ICP-MS with one routine method (dilution ICP-MS or 
ET-AAS).  The  commutability  criteria  were  set  on  the  basis  of  the  performance  criteria  of  external  quality 
assessment schemes and ranged from 3.7 % to 17.9 %. The results are shown in Annex 7. 

Due to the narrow criteria, the results were mostly inconclusive, as shown in Table 28.  

Table 28. Result of the commutability study. 

Material 

Method 

Cd 

ERM-DA634  Dilution-ICP-MS 

Dilution-ET-AAS 

ERM-DA635  Dilution-ICP-MS 

Dilution-ET-AAS 

ERM-DA636  Dilution-ICP-MS 

Dilution-ET-AAS 

I 

I 

I 

I 

I 

I 

Cr 

NC 

n.a. 

I 

n.a. 

I 

n.a. 

Hg 

C 

n.a. 

I 

n.a. 

I 

n.a. 

Ni 

NC 

n.a. 

I 

n.a. 

I 

n.a. 

Pb 

C 

I 

I 

NC 

I 

I 

Tl 

I 

n.a. 

I 

n.a. 

I 

n.a. 

C: Commutable. I: Inconclusive; NC: Not commutable; n.a. not applicable, as it was not measured) 

Source: JRC 

Although the results are mainly inconclusive, the graphs in Annex 5 show that normalising the results to the 
result  of  the  CRMs  would  in  all  cases  have  reduced  the  bias  for  routine  sample  between  the  methods.  The 
commutability study therefore shows that these three CRMs are an effective way to reduce the bias between 
laboratories. A detailed description of the evaluation is given in [31].  

32 

 
 
 
 
9 

Instructions for use 

9.1  Safety information 

Each individual blood donation has been tested for HIV-1/2 antibodies, HCV antibodies and HBs antigen with 
methods cleared in compliance with the European Directive 98/79/EC, Annex II, List A and found negative or 
non-reactive.  Nevertheless,  biological  specimens  should  be  treated  as  potentially  infectious.  Therefore, 
standard precautions for a safe handling and disposal are advised. 

9.2  Storage conditions 

The materials should be stored at (-20 ± 5) °C in the dark.  

Reconstituted samples stored at 4 °C were found to be stable for 7 days, i.e. certified values are valid over 
this period. 

For more information regarding the  shelf life of  reference materials please consult ERM  Application Note  7 
[32]. 

The  European  Commission  cannot  be  held  responsible  for  changes  that  happen  to  samples  after 
reconstitution  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 it ready for use, the material has to be reconstituted according to the following procedure:  

•  Allow the unit to reach ambient temperature before opening.  

• 

Tap  the  bottom  of  the  unit  to  loosen  any  blood  material  adhering  to  the  stopper  and  carefully 
remove the rubber stopper. 

•  Gently add 1.00 mL water (20 ± 4) °C and swirl by hand or vortex for 2 minutes 

Samples need to be reconstituted on the day before and should be re-homogenised on the day of the 
measurement. Subsampling of dry material of one vial is not acceptable. 

Dispose in accordance with good laboratory practice. 

For general information on handling of reference materials, please consult ERM Application Note 6 [33]. 

9.4  Minimum sample size 

The minimum sample size representative for Hg is 150 µL and 200 µL of the reconstituted material for all 
other elements.  

9.5  Use of the certified values 

The  intended  use  of  these  materials  is  to  assess  method  performance,  i.e.  for  checking  accuracy  of 
measurement  results/calibration.  It  can  also  be  used  for  the  calibration  of  methods,  thereby  making 
measurements results metrologically traceable.  

The material is intended for in vitro analysis only. 

Use as a calibrant 

If the materials are used as calibrants, the uncertainty of the certified value shall be taken into account in the 
estimation of the measurement uncertainty. 

Comparing a measurement result with the certified value 

A  result  is  unbiased  if  the  combined  standard  uncertainty  of  measurement  and  certified  value  covers  the 
difference between the certified value and the measurement result (see also ERM Application Note 1 [34]). 

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

33 

 
—  Calculate the absolute difference between mean measured value and the certified value (Δmeas). 

—  Combine the measurement uncertainty (umeas) with the uncertainty of the certified value (uCRM): u∆ = 

2
umeas

2
 + uCRM

—  Calculate  the  expanded  uncertainty  (U∆)  from  the  combined  uncertainty  (u∆)  using  an  appropriate 

�
coverage factor, corresponding to a level of confidence of approximately 95 %. 

—  If Δmeas ≤ U∆ then no significant difference exists between the measurement result and the certified 

value, at a confidence level of approximately 95 %. 

Use in quality control charts 

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

34 

 
 
10  Conclusions 

ERM-DA634,  ERM-DA635  and  ERM-DA636  are  human  blood  reference  materials  certified  for  their  mass 
concentrations of Cd , Cr, Hg, Ni, Pb and Tl. These materials were produced and certified in accordance with 
ISO  17034:2016  [1]  and  ISO  33405:2024  [2].  They  were  produced  within  the  scope  of  ISO  17034 
accreditation. 

The  material  helps  implementing  of  biomonitoring  schemes  for  the  exposure  of  humans  to  metals  and 
ensures that data collected as mandated by Council Directive 98/24/EC are reliable. 

The following values were assigned: 

Table 29. Certified values assigned to ERM-DA634, ERM-DA635 and ERM-DA636.  

Material 

Element 

Certified value 3) 

Uncertainty 4) 

Mass concentration in the reconstituted material 

ERM-DA634 

ERM-DA635 

ERM-DA636 

Cd 1) 

Cr 1) 

Hg 1) 

Pb 1) 

Tl 2) 

Cd 1) 

Cr 1) 

Hg 1) 

Ni 2) 

Pb 1) 

Tl 2) 

Cd 1) 

Cr 1) 

Hg 1) 

Ni 2) 

Pb 1) 

Tl 2) 

1.29 

1.5 

1.6 

18.6 

0.81 

5.7 

22.0 

25.5 

18.3 

182 

8.3 

10.9 

42 

56 

36 

0.44 

16.6 

0.09 

0.4 

0.3 

1.7 

0.08 

0.4 

2.4 

2.9 

1.6 

11 

0.7 

0.6 

4 

6 

5 

0.04 

1.4 

Unit 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

µg/L 

mg/L 

µg/L 

1) Independent of the measurement method 

2) As obtained by ICP-MS 

3) Certified values are values that fulfil the highest standards of accuracy. The given values represent the unweighted mean values of 
the  means  of  accepted  sets  of  data,  each  set  being  obtained  in  a  different  laboratory  and/or  with  a  different  method  of 
determination. The certified value and its uncertainty are traceable to the International System of Units (SI). 
The values are valid in the temperature range from 4 ° to 25 °C. 

4) The uncertainty of the certified value is the expanded uncertainty with a coverage factor k = 2 corresponding to a level of confidence 

of 95 %, estimated in accordance with ISO 17034:2016 and ISO 33405:2024  

Source: JRC 

The  materials  are  intended  for  the  assessment  of  method  performance  calibration  of  methods  and  quality 
control. 

35 

 
 
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[2]  

[3]  

[4]  

[5] 

[6] 

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homogeneity and stability, International Organization for Standardization, Geneva, Switzerland 

ISO/IEC 17025:2017, General requirements for the competence of testing and calibration laboratories, 
International Organization for Standardization, Geneva, Switzerland 

ISO  15189:2022,  Medical  laboratories  Requirements  for  quality  and  competence,  International 
Organization for Standardization, Geneva, Switzerland 

Agency  for  Toxic  Substances  and  Disease  Registry,  Case  Studies  in  Environmental  Medicine  (CSEM) 
Cadmium Toxicity, https://www.atsdr.cdc.gov/csem/cadmium/docs/cadmium.pdf ; accessed 19.4.2024 

VA. Florez-Garcia, EC. Guevara-Romero, MM. Hawkins LE. Bautista TE. Jenson J. Yu and AE. Kalkbrenner , 
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115109.  

[7] 

H. Greim, E. Demel, Toxikologie,VCH, Weinheim, 1996 

[8]  

[9] 

[10] 

Järup,  L.,  &  Åkesson,  A.  (2009).  Current  status  of  cadmium  as  an  environmental  health  problem. 
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Agency  for  Toxic  Substances  and  Disease  Registry,  Case  Studies  in  Environmental  Medicine  (CSEM) 
,  accessed 
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19.4.2024 

,  https://www.atsdr.cdc.gov/csem/chromium/docs/chromium.pdf 

J.R.  Forsyth  et  al,  Turmeric  means  “yellow”  in  Bengali:  Lead  chromate  pigments  added  to  turmeric 
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food , and drinking water, EFSA Journal 18 (2020), doi: 10.2903/j.efsa.2020.6268 

[14]  Lead, European Chemical Agency, https://echa.europa.eu/hot-topics/lead , accessed 19.4.2024  

[15]  Eén  kind  op  drie  wereldwijd  vergiftigd  met  lood,  ook  400.000  Belgische  kinderen,    De  Standaard, 

30.6.2020, https://www.standaard.be/cnt/dmf20200730_91662519  

[16]   EFSA,  Lead  dietary  exposure  in  the  European  population,  EFSA  Journal  10  (2012),  2831, 

https://doi.org/10.2903/j.efsa.2012.2831 

[17]  Dalende 

in 
https://www.standaard.be/cnt/dmf20211205_98029601  

loodwaarden 

Hobokense 

bloed 

kinderen, 

De 

Standaard, 

6.12.2021 

[18]   Council Directive 98/24/EC of 7 April 1998 on the protection of the health and safety of workers from 

the risks related to chemical agents at work 

[19]   Y.  Chang  1  and  C.-K.  Chiang,  The  Impact  of  Thallium  Exposure  in  Public  Health  and  Molecular 
4750, 

Comprehensive 

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(2024), 

Sci., 

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25 

Toxicology: 
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[21]   W.B. Yeoman, E. Colinet, B. Grieping, The certification of lead and  cadmium in three lyophilized blood 
materials  BCR-194,  BCR-195,  BCR-196,  EU  Report  EUR  10380,  Commission  of  the  European 
Communities, Luxembourg, 1985, ISBN 92-894-7112-3 

[22]   K. Byrialsen, J.M. Christensen, J. Kristiansen, The certification of the  mass concentrations of lead and 
cadmium in reconstituted human blood BCR-634, BCR-635, BCR-636, EU Report EUR 21066, Office for 
Official Publications of the European Communities, Luxembourg, 2004, ISBN 92-894-7112-3 

[23]  

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in Measurement (GUM 1995), International Organization for Standardization, Geneva, Switzerland 

[24]   G.E.  O’Donnell,  D.B.  Hibbert,  Treatment  of  bias  in  estimating  measurement  uncertainty,  Analyst  130 

(2005) 721-729 

[25]   T.P.J. Linsinger, J. Pauwels, A.M.H. van der Veen, H. Schimmel, A. Lamberty, Homogeneity and stability of 

reference materials, Accred. Qual. Assur. 6 (2001) 20-25 

[26]   A. Lamberty, H. Schimmel, J. Pauwels, The study of the stability of reference materials by isochronous 

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

[27]  T.P.J. Linsinger, J. Pauwels,  A. Lamberty, H.G. Schimmel,  A.M.H.  van  der Veen,  L. Siekmann, Estimating 

the uncertainty of stability for matrix CRMs, Fres. J. Anal. Chem. 370 (2001) 183-188 

[28]   DIN 1333:1992-02, Zahlenangaben, Deutsches Institut für Normung e.V., Berlin, Germany 

[29]  

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associated terms (VIM), International Organization for Standardization, Geneva, Switzerland 

[30]   W.G. Miller, H. Schimmel, R. Rej, N. Greenberg, F. Ceriotti, C. Burns, J.R. Budd, C. Weykamp, V. Delatour, G. 
Nilsson, F. MacKenzie, M. Panteghini, T. Keller, J.E. Camara, I. Zegers, H.W. Vesper, IFCC Working Group 
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(2018) 447–454 

[31]   T.P.J. Linsinger, G. Auclair, L. Deprez, Commutability study on three CRMs evaluating their suitability for 
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in  human  blood,  Analytical  and  Bioanalytical  Chemistry,  2025 

[32]   ERM  Application  Note  7:  Prolonging 

the 

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support-Application-Notes, accessed on 3 June 2024 

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https://crm.jrc.ec.europa.eu/e/132/User-support-Application-Notes, accessed on 3 June 2024 

37 

 
                                                                                                                                                                             
 
List of abbreviations  

AAS (DMA) 

ANOVA 

b 

BCR® 

c 

CI 

CRM 

CV-AAS 

EC 

EDTA 

ERM® 

EU 

ET-AAS 

GUM 

ICP 

Atomic absorption spectrometry (direct mercury analyser) 

Analysis of variance 

Slope in the equation of linear regression y = a + bx 

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

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

Confidence interval 

Certified reference material 

Cold vapour- atomic absorption spectrometry 

European Commission 

Ethylenediaminetetraacetic acid  

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

European Union 

Electrothermal atomic absorption spectrometry 

Guide to the Expression of Uncertainty in Measurement 

Inductively coupled plasma 

ICP-IDCQMS 

ICP isotope dilution collision cell quadrupole mass spectrometry  

ICP-QMS 

ICP-Q3MS 

ICP-SFMS 

IFCC 

ISO 

JRC 

k 

LOD 

LOQ 

MSbetween 

MSwithin 

n 

N 

n.a. 

n.c. 

p 

QC 

rel 

RM 

RMP 

ICP-quadrupole mass spectrometry  

ICP-triple quadrupole mass spectrometry 

ICP-sector field mass spectrometry  

International Federation of Clinical Chemistry and Laboratory Medicine 

International Organization for Standardization 

Joint Research Centre of the European Commission 

Coverage factor 

Limit of detection 

Limit of quantification 

Mean of squares between-unit from an ANOVA 

Mean of squares within-unit from an ANOVA 

Number of replicate analysis per unit 

Number of units analysed 

Not applicable 

Not calculated 

Number of technically valid datasets 

Quality control 

Index denoting relative figures (uncertainties etc.) 

Reference material 

Reference material producer 

38 

 
 
s 

sbb

sbetween 

SI 

smeas 

swb 

swithin 

T 

t 

ti 

tα, df 

tsl 

ttt 

u 

U 

u*

bb 

ubb 

uc 

uchar 

uCRM 

UCRM 

u∆ 

ults 

umeas 

Umeas 

urec 

usts 

VIM 

Standard deviation 

Between-unit standard deviation; an additional index "rel" is added when 
appropriate; this parameter is linked to the homogeneity of the material 

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

International System of Units 

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

Within-unit standard deviation; this parameter is linked to the homogeneity of 
the material 

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

Temperature 

Time 

Time point for each replicate 

Critical t-value for a t-test, with a level of confidence of 1-α and df degrees of 
freedom 

Proposed shelf life 

Proposed transport time 

Standard uncertainty 

Expanded uncertainty 

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

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

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

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

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

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

Combined standard uncertainty of measurement result and certified value 

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

Standard measurement uncertainty 

Expanded measurement uncertainty 

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

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

International Vocabulary of Metrology – Basic and General Concepts and 
Associated Terms 

39 

 
 
 
x

Arithmetic mean 

min 

max 

x
�
x
�
∆meas 
�
νMSwithin 

νUCRM 

y

�

Highest unit mean of the homogeneity study 

Lowest unit mean of the homogeneity study 

Absolute difference between mean measured value and the certified value 

Degrees of freedom of MSwithin 

Degrees of freedom of UCRM 

Mean of all results of the homogeneity study 

40 

 
 
 
 
 
 
 
 
 
List of boxes 

Box 1. Reference material production ..................................................................................................................................................................4 

Box 2. Reference material processing ..................................................................................................................................................................6 

Box 3. Homogeneity assessment ............................................................................................................................................................................8 

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

Box 5. Stability monitoring ...................................................................................................................................................................................... 21 

Box 6. Reference material characterisation ................................................................................................................................................. 22 

Box 7. Assignment of values to a reference material ............................................................................................................................ 28 

Box 8. Metrological traceability ............................................................................................................................................................................ 31 

Box 9. Commutability .................................................................................................................................................................................................. 32 

41 

 
 
 
 
 
List of figures 

Figure 1. CRM ERM-DA635  ........................................................................................................................................................................................7 

42 

 
 
 
 
 
List of tables 

Table 1. Results of the statistical evaluation of the homogeneity study of ERM-DA634. Trends in the 
measurement sequence were corrected before further data treatment. .......................................................................................9 

Table 2. Results of the statistical evaluation of the homogeneity study of ERM-DA635: Trends in the 
measurement sequence were corrected before further data treatment. ................................................................................... 10 

Table 3. Results of the statistical evaluation of the homogeneity study of ERM-DA636. Trends in the 
measurement sequence were corrected before further data treatment. ................................................................................... 10 

Table 4. Results of the homogeneity study of ERM-DA634............................................................................................................... 11 

Table 5. Results of the homogeneity study of ERM-DA635............................................................................................................... 12 

Table 6. Results of the homogeneity study of ERM-DA636............................................................................................................... 12 

Table 7. Results of the short-term stability tests of ERM-DA634. ................................................................................................ 15 

Table 8. Results of the short-term stability tests of ERM-DA635. ................................................................................................ 15 

Table 9. Results of the short-term stability tests of ERM-DA636. ................................................................................................ 15 

Table 10. Evaluation of the pooled data of the stability monitoring of BCR-634, BCR-635 and BCR-636. ....... 17 

Table 11. Results of the 12-months study of ERM-DA634. .............................................................................................................. 18 

Table 12. Results of the 12-months study of ERM-DA635 ............................................................................................................... 18 

Table 13. Results of the 12-months study of ERM-DA636. .............................................................................................................. 18 

Table 15. Uncertainties of stability during transport and storage for ERM-DA634. .......................................................... 20 

Table 16. Uncertainties of stability during transport and storage for ERM-DA635.. ......................................................... 20 

Table 17. Uncertainties of stability during transport and storage for ERM-636. ................................................................. 20 

Table 18. Datasets that showed non-compliance with the instructions given and technical specifications,  
                 and action taken. .................................................................................................................................................................................... 24 

Table 19. Statistical evaluation of the technically accepted datasets for ERM-DA634. ................................................. 25 

Table 20. Statistical evaluation of the technically accepted datasets for ERM-DA635. ................................................. 25 

Table 21. Statistical evaluation of the technically accepted datasets for ERM-DA636.. ................................................ 25 

Table 22. Uncertainty of characterisation for ERM-DA634................................................................................................................ 26 

Table 23. Uncertainty of characterisation for ERM-DA635................................................................................................................ 26 

Table 24. Uncertainty of characterisation for ERM-DA636................................................................................................................ 27 

Table 25. Certified values and their uncertainties for ERM-DA634. ............................................................................................. 29 

Table 26. Certified values and their uncertainties for ERM-DA635. ............................................................................................. 29 

Table 27. Certified values and their uncertainties for ERM-DA636. ............................................................................................. 29 

Table 28. Additional material information for ERM-DA634............................................................................................................... 30 

Table 28. Result of the commutability study. ............................................................................................................................................. 32 

Table 29. Certified values assigned to ERM-DA634, ERM-DA635 and ERM-DA636.......................................................... 35 

43 

 
 
 
Annexes 

44 

 
 
 
 
Annex 1. Results of the homogeneity measurements 

Shown are the averages per unit. The error bars correspond to the 95 % confidence interval of the average 
per unit and are based on the within-unit standard deviation as calculated by one-way ANOVA 

Source: JRC 

45 

 
 
 
 
 
 
 
 
 
Source: JRC 

46 

 
 
 
 
 
 
 
Source: JRC 

47 

 
 
 
 
 
 
 
 
Annex 2. Results of the transport stability measurements 

Shown  are  the  averages  per  time  point.  Error  bars  correspond  to  the  confidence  interval  of  the  means  per 
time point, calculated using the standard deviation of all measurements. The data for 18 °C and 60 °C were 
separated graphically. 

Source: JRC 

48 

 
 
 
 
 
 
 
Source: JRC 

49 

 
 
 
 
 
 
 
Source: JRC 

50 

 
 
 
 
 
 
 
 
 
Annex 3. Results of the storage stability measurements 

Data  of  the  stability  monitoring  of  BCR-634,  BCR-635  and  BCR-636.  Shown  are  the  ratios  of  results  from 
units stored at -20 °C to samples stored at -70 °C. Each data point is usually an average of six results. 

Source: JRC 

51 

 
 
 
 
 
 
 
Data  of  the  1-year  study  of  ERM-DA634,  ERM-DA635  and  ERM-DA636.  Shown  are  the  averages  per  time 
point.  Error  bars  correspond  to  the  confidence  interval  of  the  means  per  time  point,  calculated  using  the 
standard deviation of all measurements. 

Source: JRC 

52 

 
 
 
 
 
 
 
 
Source: JRC 

53 

 
 
 
 
 
 
 
 
 
Source: JRC 

54 

 
 
 
 
 
 
 
 
 
Annex 4: Results of the measurements of the repeated use study.  

Shown are the averages of the three results per day as  percentage of the average over all  days. The error 
bars  correspond  to  the  95  %  confidence  interval  using  the  within-group  standard  deviation  as  obtained  by 
one-way ANOVA. 

Source: JRC 

55 

 
 
 
 
 
Annex 5. Summary of methods used in the characterisation study 

Method information is reported as given by the laboratories 

Dataset  Sample preparation 

Elements 

Quantification 

D01  

Sample intake 500 mg 

Closed  vessel,  microwave-assisted  digestion 
with HNO3 

Cd,  Cr,  Hg,  Ni, 
Pb, Tl 

Sector  field 
resolution >300 (Cd, Hg, Pb, Tl) and >4000 (Cr, Ni)  

ICP-MS,  ThermoScientific 

  ELEMENT  XR; 

Masses  used:  111,  114  (Cd),  52,  53  (Cr),  199,  200,  201 
(Hg), 60, 62 (Ni), 206, 207, 208 ({b), 203, 205 (Tl) 

Calibration 

External calibration 

Single  element  standard  solutions  from 
Agilent;    traceable  to  the  SI  via  NIST 
CRMs 

D02  

Sample intake 300 mg 

Microwave  pressure  digestion:  0.3  g  sample 
+  0.5  mL  65  %  HNO3;  keep  for  20  min  at 
240 °C; dilution to 5 mL 

Cd,  Cr,  Hg,  Ni, 
Pb, Tl 

ICP-triple  quadrupole  MS  Agilent  8800;  resolution  approx.. 
0.7 amu for both quadrupoles 

External calibration 

Hg: NIST SRM 3133  

Masses measured: 114 (Cd), 53 (Cr), 202 (Hg), 60 (Ni), 208 
(Pb), 205 (Tl) 

Multi-element  standard  solution  Supelco 
TraceCert  Mix  1;  traceable  to  the  SI  via 
NIST CRMs 

solution 

External  calibration  with  multi-element 
from 
standard 
ThermoScientific 
element 
solutions;  each  traceable  to  the  SI  via 
NIST CRMs 

Single 

made 

D03  

Sample intake: 2.5 mL 

Pooling of 3 reconstituted samples.  

Cd,  Cr,  Ni,  Pb, 
Tl 

Microwave  pressure  digestion  according  to 
EN 13805:2014 with 2.5 mL sample + 2 mL 
67-69 % HNO3 + 2 mL 30 % H2O2 

Hg 

ICP-single  quadrupole  MS  Perkin  Elmer  NexION  350D; 
resolution 0.7 amu 
;  
Masses  measured:  114 
208+206+207(Pb), 205 (Tl) 

(Cd),  52 

(Cr),  60 

(Ni), 

AAS  (DMA)  Analytik  Jena  Mercur  Duo  Plus;  wavelength 
253.7 nm 

56 

 
 
 
D04  

Cd, Pb:  Sample intake 800 mg 

Digestion 
in  a  closed  microwave  oven  
(Multiwave5000,  ANTON  PAAR)  with  4  mL 
HNO3  (69  %,  V/)  (25  min  ramp  time  to  190 
°C, 20 min at 190 °C). 

Cd, Pb 

ICP-QMS  with  collision  cell  (Thermo  Fisher  iCAP  Q  ICP-MS) 
in  kinetic  energy  discrimination  mode  at  a  He  flow  of  5 
mL/min 

Masses  measured:  110,  111  (Cd),  206,  208  (Pb)  in  low 
resolution mode 

Hg: Sample intake 150 µL.  
Aliquots introduced directly into an AMA254 
mercury  analyser:  combustion  at  750°C 
under oxygen, followed by atomic absorption 
analysis  (DMA)  after  amalgamation  on  a 
gold amalgam. 

Hg 

AAS (DMA) Altac AMA 254, wavelength 253.7 nm 

D05 

Samples  diluted  1:50 
  with  a  solution 
containing    0.7  mmol/l  NH4OH,  0.01  mmol/l 
EDTA, 0.07 % (v/v) Triton X-100 in ultrapure 
water. Addition of internal standard 

Cd,  Cr,  Hg,  Ni, 
Pb, Tl 

ICP- triple quadrupole MS, Agilent 8900MS Agilent 8900 

Masses measured:  114 (Cd), 52 (Cr), 60 (Ni), 202 (Hg), 205 
(Tl), 208 (Pb) 

Double 
mass 
isotope-dilution 
spectrometry.  Two  CRMs  are  used  to 
determine  the 
  spike  concentrations: 
Supelco Tracecert CRMs (traceable to the 
SI  via  NIST  CRM)  and  digestion  high 
purity  metal  (99.999  %  and  99.9999  % 
purity, respectively). 

standard  additions  per 

3 
sample 
corresponding  to  0.5,  1  and  2  times  the 
measured concentration.   
Calibration  with  standard  from  SCP 
Science,  directly  traceable  to  NIST  SRM 
3133 

Standard  addition  with  single  element 
standards  from  Agilent,  SCP  Science, 
Inorganic Ventures produced by digestion 
of pure elements. Values traceable to the 
SI  by 
the  gravimetric  preparation 
confirmed by comparison with NIST CRMs  
(Cd,  Ni,  Pb,  Tl),  direct  comparison  with 
NIST CRM (Cr) or a combination of EDTA 
assay/direct comparison with NIST CRM. 

Correctness  of  the  calibration  confirmed 
by  analysing 
"SeronormTM  Trace 
Elements  Whole  Blood"  and  "ClinChek® 
Whole Blood Controls" 

57 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
D06  

Sample  intake  2000  mg  (Hg),  250  mg  (all 
others) 

Cd,  Cr,  Ni,  Pb, 
Tl 

ICP- triple quadrupole  MS Agilent 8800;  resolution approx.. 
0.7 amu for both quadrupoles 
Sc, Ge, Y, In and Bi used as internal standards 
Masses used: 111 (Cd), 52, 53 (Cr), 60 (Ni), 206, 207, 208 
(Pb), 203, 205 (Tl) 

Hg 

CV-AAS Perkin-Elmer AAnalyst 800; 0.7 nm slit width 

Cd,  Cr,  Ni,  Pb,  Tl:  samples  diluted  20-fold 
with a solution containing 2 % v/v 1-butanol, 
0.5  g/L  EDTA,  0.05  %  v/v  Triton  X-100  and 
1 % v/v NH4OH. 

Hg:  1 % L-Cysteine, 1 % NaCl, 45 % NaOH 
and  octanol  added.  Inorganic  Hg  reduced 
with  SnCl;  then  organic  Hg  reduced  with 
mixture  of  SnCl/CdCl2.  Total  Hg  calculated 
sum of inorganic and organic mercury. 

D07  

Sample  intake  400   µL  (Cd,  Pb)  or  200   µL 
(Cr) 

Addition  of  700  µL  HNO3  and  1  mL  0.1  % 
The 
Triton-X-100; 
supernatant is measured. 

centrifugation. 

Cd, Cr, Pb 

ET-AAS (Perkin Elmer PinAAcle 900T) 

Wavelengths used: 228.80 nm (Cd), 357.87 nm (Cr), 283.31 
(Pb) 

Slit width 0.7 nm 

External calibration 

Cd,  Cr,  Ni.  Pb,  Tl:  multielement  or  single 
element (Tl) standards from Romil Ltd or 
Inorganic Ventures;  

Traceability  for  Ni  via  NIST  CRMs  SRM 
3136, 928 

Hg: 
Inorganic  Hg  from  SigmaAldrich; 
organic  Hg  from Alfa  Aesar;  traceable to 
the SI via NIST CRMs SRM 728 

External  calibration  with  matrix-matched 
multi-element  standard  solution  from 
Recipe  (lyophilised  whole  blood).  The 
values  are  traceable  to  the  SI  through  a 
combination of results from at least two 
different  methods 
from 
laboratories accredited to ISO/IEC 17025 
or ISO 15189 

derived 

Checked  with  control  samples  from 
Recipe 

Cd,  Cr,  Ni,  Pb, 
Tl: 

Hg 

D08  

Sample  intake  150  mg  (Hg),  500  mg  (all 
other  elements  ERM-DA634)  200  mg  (all 
other  elements  ERM-DA635  and  ERM-
DA636)) 

Cd,  Cr,  Ni,  Pb,  Tl:  Digestion  with    Digestion 
with  HNO3:H2O2:H2O  2:1:3  at  ambient 
pressure (Digiprep blok digestion) 

Hg:  Direct  measurement;  sample 
150 mg 

intake 

Source: JRC 

ICP- single quadrupole MS (Thermo iCAP) 

External calibration  

Masses measured: 114 (Cd), 52 (Cr), 60 (Ni), 206,207, 208 
(Pb), 203 (Tl), 

AAS (DMA) Milestone DMA-80); wavelength 253.7 nm 

58 

Cd,  Cr,  Ni,  Pb,  Tl:  Multi  element  standard 
solution 
Analytica. 
Traceable to the SI via NIST CRMs, 3108, 
3112a, 3136, 928, 3128 ands 3158 

Astasol 

from 

Hg:  Mono  element  standard  from  CPA 
Chem: Production from high purity metal 
and checked using NIST and BAM CRMs. 

 
 
 
 
 
 
 
 
 
 
 
Annex 6. Results of the Characterisation study 

Cadmium 

Table 6.1. Data from the characterisation for Cd, ERM-DA634. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

D01-ICP-SFMS 

1.4 

1.28 

1.29 

D02-ICP-Q3MS 

1.316 

1.345 

1.273 

D03-ICP-QMS 

1.29 

D04-ICP-IDCQMS 

1.27 

D05-ICP-Q3MS 

D06-ICP-Q3MS 

D07-ET-AAS 

D08-ICP-QMS 
Source: JRC 

1 

1.07 

1.42 

1.24 

1.28 

1.28 

1.1 

1.15 

1.41 

1.42 

1.25 

1.26 

1.3 

1.41 

1.48 

1.44 

1.38 

1.27 

1.22 

1.33 

1.3 

1.44 

1.39 

1.37 

1.32 

1.34 

1.261 

1.315 

1.29 

1.23 

1.1 

1.13 

1.23 

1.37 

1.25 

1.22 

1 

1.18 

1.33 

1.63 

mean 

[µg/L] 

1.34 

1.30 

1.26 

1.27 

1.13 

1.23 

1.38 

1.41 

Expanded  
uncertainty 

0.12 µg/L 

0.14 µg/L 

0.13 µg/L 

0.06 µg/L 

47 % 

38 % 

20 % 

0.4 µg/L 

Figure 6.1. Characterisation data for Cd, ERM-DA634, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively. 

Source: JRC 

0 

 
 
 
 
 
 
 
 
 
 
Table 6.2. Data from the characterisation for Cd, ERM-DA635. The data are shown as provided by the laboratories. 

Dataset code 

and method 

D01-ICP-SFMS 

D02-ICP-Q3MS 

D03-ICP-QMS 

5.68 

5.81 

5.79 

D04-ICP-IDCQMS 

5.74 

D05-ICP-Q3MS 

D06-ICP-Q3MS 

D07-ET-AAS 

D08-ICP-QMS 
Source: JRC 

5.0 

5.26 

6.31 

6.13 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

5.67 

5.9 

5.65 

5.61 

4.8 

5.18 

6.28 

5.78 

5.63 

5.81 

5.8 

5.68 

5.5 

5.26 

6.13 

6.07 

5.57 

5.76 

5.68 

5.65 

5.6 

5.18 

6.4 

5.9 

5.73 

5.81 

5.7 

5.64 

4.7 

5.39 

6.06 

6.17 

5.81 

5.54 

5.61 

5.42 

4.7 

4.99 

5.98 

5.98 

mean 

[µg/L] 

5.68 

5.77 

5.71 

5.62 

5.05 

5.21 

6.19 

6.01 

Expanded  
uncertainty 

0.38 µg/L 

0.49 µg/L 

0.57 µg/L 

0.19 µg/L 

25 % 

38 % 

15 % 

1.68 µg/L 

Figure 6.2. Characterisation data for Cd, ERM-DA635, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively. 

Source: JRC 

1 

 
 
 
 
 
 
 
 
Table 6.3. Data from the characterisation for Cd, ERM-DA636. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

D01-ICP-SFMS 

11.4 

11.5 

11.2 

11.4 

D02-ICP-Q3MS 

11.22* 

10.56 

11.41 

10.29 

D03-ICP-QMS 

10.5 

D04-ICP-IDCQMS 

10.9 

D05-ICP-Q3MS 

D06-ICP-Q3MS 

9.8 

10.1 

D07-ET-AAS 

11.01 

10.7 

11.1 

9.7 

9.7 

11 

10.1 

11.1 

10.6 

10.15 

10.5 

11.0 

10.9 

9.94 

11.9 

11.2 

10.8 

11.2 

9.5 

11.6 

11.17 

10.6 

11.0 

9.1 

10.21* 

10.17 

11.14 

11.13 

10.97 

11.31 

11.50 

0.64 µg/L 

10.93 

0.72 µg/L 

10.53 

1.1 µg/L 

11.05 

0.4 µg/L 

9.93 

10.01 

11.10 

11 % 

38 % 

10 % 

D08-ICP-QMS 

11.34 

11.35 

11.63 

12.01 

12.17 

11.81 

11.72 

3.28 µg/L 

*: result not used as the unit was in the part of a rising trend in the filling volume 

Source: JRC 

Figure 6.3. Characterisation data for Cd, ERM-DA636, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively. 

Source: JRC 

2 

 
 
 
 
 
 
 
 
 
Chromium 

Table 6.4. Data from the characterisation for Cr, ERM-DA634. The data are shown as provided by the laboratories. 

Dataset code 

and method 

D01-ICP-SFMS 

D02-ICP-Q3MS 

D03-ICP-QMS 

D05-ICP-Q3MS 

D06-ICP-Q3MS 

D07-ET-AAS 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

1.29 

1.7 

1.78 

1.2 

1.61 

1.14 

1.24 

1.75 

1.9 

0.95 

1.9 

2.32 

1.26 

1.1 

1.91 

1.1 

1.72 

1.2 

1.33 

1.93 

1.94 

1.2 

1.62 

1.13 

1.3 

1.08 

2.01 

1 

1.41 

1.18 

1.33 

1.29 

2.08 

1 

1.76 

0.97 

mean 

[µg/L] 

1.29 

1.48 

1.94 

1.08 

1.67 

1.32 

Expanded  
uncertainty 

0.14 µg/L 

0.3 µg/L 

0.29 µg/L 

44 % 

26 % 

20 % 

Results not used for value assignment  

D08-ICP-QMS 
Source: JRC 

1.09 

2.73 

1.27 

0.3 

3.01 

2.25 

1.78 

0.53 µg/L 

Figure 6.4. Characterisation data for Cr, DA634 bold red lines and dashed red lines represent the certified value and its  
expanded uncertainty, respectively. Results in the shaded area were no used for the calculation of the certified value and 
uncertainty.  

Source: JRC 

3 

 
 
 
 
 
 
 
 
 
 
Table 6.5. Data from the characterisation for Cr, ERM-DA635. The data are shown as provided by the laboratories. 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

Dataset code 

and method 

D01-ICP-SFMS 

D02-ICP-Q3MS 

D03-ICP-QMS 

D05-ICP-Q3MS 

24.1 

22.7 

20.6 

21.3 

22.6 

22.9 

20.4 

21 

24.9 

20.8 

19.2 

20.1 

24 

21.7 

21.3 

20.4 

D06-ICP-Q3MS 

23.63 

23.12 

21.84 

22.24 

D07-ET-AAS 

23.7 

23.6 

21.5 

22.3 

Results not used for value assignment  

24.7 

20.1 

22.3 

21 

22.9 

21.8 

24.5 

19.7 

24.13 

2.1 µg/L 

21.32 

2.2 µg/L 

36.2* 

20.76 

3.5 µg/L 

20.8 

20.77 

22.68 

22.74 

21.9 

22.47 

31 % 

26 % 

15 % 

D08-ICP-QMS 

27.24 

30.5 

26.38 

28.28 

27.47 

15.78 

25.94 

7.78 µg/L 

*: Result not used (contamination) 

Source: JRC 

Figure 6.4. Characterisation data for Cr, DA635, bold red lines and dashed red lines represent the certified value and its 
expanded uncertainty, respectively. Results in the shaded area were no used for the calculation of the certified value and 
uncertainty.  

Source: JRC 

4 

 
 
 
 
 
 
 
 
 
 
Table 6.6. Data from the characterisation for Cr, ERM-DA636. The data are shown as provided by the laboratories. 

Dataset code 

and method 

D01-ICP-SFMS 

D02-ICP-Q3MS 

D03-ICP-QMS 

D05-ICP-Q3MS 

D06-ICP-Q3MS 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

45.5 

42* 

36.6 

41 

43 

44.8 

40 

38.3 

41.3 

44.4 

39.7 

39.7 

38.5 

43.2 

42.1 

38.8 

39 

46.1 

39.5 

37.8 

40.4 

44 

37.9 

39.2 

41.2 

44.67 

3.4 µg/L 

39.84 

4 µg/L 

38.40 

5.8 µg/L 

40.23 

42.29 

41.22 

42.46 

43.14* 

42.81 

42.36 

D07-ET-AAS 

50.2 

48.1 

48.7 

46.2 

41.4 

41.9 

46.08 

Results not used for value assignment  

20 % 

26 % 

10 % 

D08-ICP-QMS 

48.78 

43.11 

56.37 

57.49 

41.56 

44.94 

48.71 

14.61 µg/L 

*: result not used as the unit was in the part of a rising trend in the filling volume 

Source: JRC 

Figure 6.6. Characterisation data for Cr, ERM-DA636, bold red lines and dashed red lines represent the certified value and 
its expanded uncertainty, respectively. Results in the shaded area were no used for the calculation of the certified value 
and uncertainty 

Source: JRC 

5 

 
 
 
 
 
 
 
 
 
Mercury 

Table 6.7. Data from the characterisation for Hg, ERM-DA634. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

D01-ICP-SFMS 

D02-ICP-Q3MS 

D03- AAS (DMA) 

D04- AAS (DMA) 

D05-ICP-Q3MS 

D06- CV-AAS 

D08- AAS (DMA) 
Source: JRC 

1.54 

1.59 

1.84 

1.73 

1.4 

1.76 

1.22 

1.61 

1.56 

1.85 

1.59 

1.4 

1.47 

1.15 

1.65 

1.46 

1.85 

1.55 

1.3 

3.12 

1.14 

1.59 

1.43 

1.91 

1.67 

1.2 

2.5 

1.2 

1.58 

1.49 

1.67 

1.81 

1.6 

1.56 

1.29 

1.7 

1.49 

1.63 

1.76 

1.7 

1.98 

1.25 

mean 

[µg/L] 

1.61 

1.50 

1.79 

1.69 

1.43 

2.07 

1.21 

Expanded  
uncertainty 

0.15 µg/L 

0.24 µg/L 

0.27 µg/L 

0.30 µg/L 

57 % 

24 % 

0.22 µg/L 

Figure 6.7. Characterisation data for Hg, ERM-DA634, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively. 

Source: JRC 

6 

 
 
 
 
 
 
 
 
 
 
Table 6.8. Data from the characterisation for Hg, ERM-DA635. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

D01-ICP-SFMS 

D02-ICP-Q3MS 

D03- AAS (DMA) 

D04- AAS (DMA) 

D05-ICP-Q3MS 

D06-CV-AAS 

D08- AAS (DMA) 
Source: JRC 

26.4 

23.5 

30.6 

27.9 

27.9 

18.7 

25.6 

24.1 

31 

26.5 

27.7 

26.0 

23.6 

30.9 

27.5 

26.1 

25.9 

23.6 

31.1 

26.7 

26.1 

16.36 

18.58 

20.04 

26.3 

23.7 

29.4 

26.2 

29.3 

24.1 

26.3 

23.7 

28.9 

26.4 

29.2 

26.08 

2.2 µg/L 

23.70 

1.5 µg/L 

30.32 

4.5 µg/L 

26.87 

4.5 µg/L 

27.72 

22.54 

20.05 

30 % 

24 % 

24.65 

23.3 

23.06 

23.71 

24.59 

24.25 

23.93 

4.31 µg/L 

Figure 6.8. Characterisation data for Hg, ERM-DA635, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively. 

Source: JRC 

7 

 
 
 
 
 
 
 
 
 
 
Table 6.9. Data from the characterisation for Hg, ERM-DA636. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

D01-ICP-SFMS 

D02-ICP-Q3MS 

D03- AAS (DMA) 

D04- AAS (DMA) 

D05-ICP-Q3MS 

54.9 

51.5* 

59.7 

66.9 

59.6 

54.1 

47.4 

60.9 

62.0 

58.6 

55.3 

48.8 

66 

63.9 

54.5 

53.8 

54 

65 

65.5 

54.2 

53.9 

50.4 

59.6 

65.3 

61.7 

54.7 

49.9 

65.8 

66.6 

62.9 

54.45 

4.7 µg/L 

50.10 

2.6 µg/L 

62.83 

9.4 µg/L 

65.03 

15 µg/L 

58.58 

20 % 

24 % 

D06-CV-AAS 

49.05 

46.42 

51.79 

48.43 

49.76* 

52.68 

49.67 

D08- AAS (DMA) 

54.86 

55.02 

53.86 

51.77 

54.19 

53.52 

53.87 

9.7 µg/L 

*: result not used as the unit was in the part of a rising trend in the filling volume 

Source: JRC 

Figure 6.9. Characterisation data for Hg,  ERM-DA636, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively. 

ERM-DA636: Hg

]
l
L
g
µ
[
n
o
i
t
a
r
t
n
e
c
n
o
c

s
s
a
M

85

80

75

70

65

60

55

50

45

40

35

S
M
F
S
P
C

-

I
-
1
0
D

S
M
3
Q
P
C

-

I
-
2
0
D

S
M
3
Q
P
C

-

I
-
5
0
D

-

S
A
A
V
C
-
6
0
D

)

A
M
D

(

S
A
A

-
8
0
D

)

A
M
D

(

)

A
M
D

(

S
A
A

-
3
0
D

S
A
A

-
4
0
D

Source: JRC 

8 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Nickel 

Table 6.10. Data from the characterisation for Ni, ERM-DA634. The Ni mass concentration is not certified in ERM-DA634. 
The data are shown as provided by the laboratories. 

Dataset code 

and method 

D01-ICP-SFMS 

D02-ICP-Q3MS 

D03-ICP-QMS 

D05-ICP-Q3MS 

D06-ICP-Q3MS 

D08-ICP-QMS 
Source: JRC 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

2.17 

3.35 

3.28 

2.8 

2.96 

3.57 

1.82 

3.06 

3.29 

2.4 

3.07 

2.15 

1.98 

3.77 

3.51 

2.6 

2.45 

3.33 

2.03 

5.33 

4.55 

3 

2.49 

2.31 

1.72 

2.01 

3.7 

4.4 

3.3 

2.7 

4.5 

5.9 

3.2 

2.8 

2.33 

2.67 

mean 

[µg/L] 

1.96 

3.95 

4.16 

2.88 

2.75 

2.73 

Expanded  
uncertainty 

0.27 µg/L 

0.69 µg/L 

0.46 µg/L 

25 % 

27 % 

0.82 µg/L 

Figure 6.10. Characterisation data for Ni, ERM-DA634. The Ni mass concentration is not certified in ERM-DA634.  

Source: JRC 

9 

 
 
 
 
 
 
 
 
 
Table 6.11. Data from the characterisation for Ni, ERM-DA635. The data are shown as provided by the laboratories. 

31 % 

27 % 

Dataset code 

and method 

D01-ICP-SFMS 

D02-ICP-Q3MS 

D03-ICP-QMS 

D05-ICP-Q3MS 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

18.5 

17.7 

17.4 

19.0 

18.9 

18.2 

17.5 

17.7 

18.2 

17.7 

17.9 

17.5 

18.6 

17.5 

18.1 

17.8 

18.9 

18.1 

17.7 

18.7 

19 

18.68 

1.6 µg/L 

16.8 

17.67 

1.6 µg/L 

31.9* 

17.72 

2.2 µg/L 

18.3 

18.17 

D06-ICP-Q3MS 

17.96 

18.25 

19.59 

17.92 

18.52 

19.34 

18.60 

D08-ICP-QMS 

20.95 

19.55 

15.31 

18.88 

20.3 

17.88 

18.81 

5.64 µg/L 

*: Result not used (contamination) 

Source: JRC 

Figure 6.11. Characterisation data for Ni, ERM-DA635, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively.  

Source: JRC 

10 

 
 
 
 
 
 
 
 
 
Table 6.12. Data from the characterisation for Ni, ERM-DA636. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

D01-ICP-SFMS 

36.4 

D02-ICP-Q3MS 

35.2* 

D03-ICP-QMS 

D05-ICP-Q3MS 

32.5 

35.2 

36.9 

32.9 

33.8 

35.7 

36.6 

33.9 

31 

35.8 

36.2 

29.5 

33.4 

34.4 

35.9 

33 

34.8 

36.8 

36.8 

34.4 

31 

39.2 

D06-ICP-Q3MS 

37.56 

35.94 

37.69 

39.15 

38.06* 

36.89 

D08-ICP-QMS 

51.23 

36.82 

40.76 

38.46 

34.56 

39.33 

*: result not used as the unit was in the part of a rising trend in the filling volume 

44.67 

2.9 µg/L 

39.84 

3 µg/L 

38.40 

3.6 µg/L 

36.18 

42.36 

46.08 

20 % 

27 % 

12.06 

Source: JRC 

Figure 6.12. Characterisation data for Ni, ERM-DA636, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively.  

Source: JRC 

11 

 
 
 
 
 
 
 
 
 
Lead 

Table 6.13. Data from the characterisation for Pb, ERM-DA634. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

D01-ICP-SFMS 

D03-ICP-QMS 

D04-ICP-IDCQMS 

D05-ICP-Q3MS 

17.6 

17.9 

19.6 

19.4 

16.8 

17.5 

20.3 

19.3 

18.8 

18 

19.2 

19.9 

18.6 

17.8 

18.9 

19.6 

18.3 

17.9 

19.3 

19.6 

17.4 

17.9 

19.8 

19.2 

17.92 

1.5 µg/L 

17.83 

2.3 µg/L 

19.52 

0.8 µg/L 

19.50 

20 % 

23 % 

D06-ICP-Q3MS 

16.48 

16.36 

17.22 

17.12 

17.78 

17.48 

17.07 

D08-ICP-QMS 

19.3 

19.93 

20.37 

18.71 

20.59 

20.28 

19.86 

5.96 µg/L 

Results not used for value assignment  

D02-ICP-Q3MS 

18.27 

17.49 

17.44 

D07-ET-AAS 
Source: JRC 

11.65 

11.25 

16.6 

17.3 

19.4 

17.3 

16.82 

21.76 

19.59 

17.4 

16.7 

0.81 µg/L 

20 % 

Figure 6.13. Characterisation data for Pb, ERM-DA634, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively. Results in the shaded area were no used for the calculation of the certified 
value and uncertainty. 

Source: JRC 

12 

 
 
 
 
 
 
 
 
 
Table 6.14. Data from the characterisation for Pb, ERM-DA635. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

D01-ICP-SFMS 

D03-ICP-QMS 

D04-ICP-IDCQMS 

D05-ICP-Q3MS 

178 

178 

188 

188 

181 

181 

182 

186 

177 

177 

184 

189 

173 

173 

184 

190 

176 

176 

181 

185 

172 

172 

174 

183 

176.2 

15 µg/L 

176.2 

23 µg/L 

182.2 

7 µg/L 

186.8 

D06-ICP-Q3MS 

157.5 

156.9 

165.5 

165.3 

171.5 

170.2 

164.5 

D07-ET-AAS 

192.7 

192.1 

190.4 

193.2 

179.4 

184.7 

188.8 

20 % 

23 % 

15 % 

D08-ICP-QMS 

200.94 

202.24 

195.23 

193.17 

204.71 

200.28 

199.4 

59.83 µg/L 

Results not used for value assignment  

D02-ICP-Q3MS 
Source: JRC 

170.6 

168.2 

166 

166.8 

163.3 

160.0 

165.8 

5.6 µg/L 

Figure 6.14. Characterisation data for Pb, ERM-DA635 bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively. Results in the shaded area were no used for the calculation of the certified 
value and uncertainty. 

Source: JRC 

13 

 
 
 
 
 
 
 
 
 
Table 6.15. Data from the characterisation for Pb, ERM-DA636. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

D01-ICP-SFMS 

D03-ICP-QMS 

D04-ICP-IDCQMS 

D05-ICP-Q3MS 

421 

404 

445 

448 

427 

404 

449 

447 

D06-ICP-Q3MS 

367.3 

377.3 

426 

413 

444 

449 

398 

417 

413 

446 

452 

412 

404 

448 

451 

422 

412 

445 

448 

420.8 

35 µg/L 

408.3 

53 µg/L 

446.2 

15 µg/L 

449.2 

392.9 

407.7* 

407.5 

388.6 

D07-ET-AAS 

474.4 

480.4 

465.2 

471.8 

489.6 

507.6 

481.5 

10 % 

23 % 

10 % 

D08-ICP-QMS 

479.49 

470.79 

473.52 

471.02 

488.82 

480.22 

477.3 

143.19 µg/L 

Results not used for value assignment  

D02-ICP-Q3MS 

390* 

387 

395 

375 

387 

388 

386.4 

12 µg/L 

*: result not used as the unit was in the part of a rising trend in the filling volume 

Source: JRC 

Figure 6.15. Characterisation data for Pb, ERM-DA636, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively. Results in the shaded area were no used for the calculation of the certified 
value and uncertainty. 

Source: JRC 

14 

 
 
 
 
 
 
 
 
 
Thallium 

Table 6.16. Data from the characterisation for Tl, ERM-DA634. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

D01-ICP-SFMS 

0.774 

0.778 

0.751 

0.744 

0.759 

0.739 

D02-ICP-Q3MS 

0.811 

0.778 

0.736 

0.752 

0.761 

0.749 

D03-ICP-QMS 

0.789 

0.77 

0.744 

0.752 

0.775 

0.762 

D05-ICP-Q3MS 

D06-ICP-Q3MS 

D08-ICP-QMS 
Source: JRC 

0.8 

0.85 

0.88 

0.8 

0.83 

0.88 

0.8 

0.91 

0.93 

0.8 

0.9 

0.8 

0.8 

1.01 

0.8 

0.8 

0.96 

0.86 

mean 

[µg/L] 

0.76 

0.76 

0.77 

0.80 

0.91 

0.86 

Expanded  
uncertainty 

0.06 µg/L 

0.13 µg/L 

0.11 µg/L 

110 % 

35 % 

0.26 µg/L 

Figure 6.16. Characterisation data for Tl, ERM-DA634 bold lines bold red lines and dashed red lines represent the 
certified value and its expanded uncertainty, respectively. Results in the shaded area were no used for the calculation of 
the certified value and uncertainty.  

Source: JRC 

15 

 
 
 
 
 
 
 
 
 
 
Table 6.17. Data from the characterisation for Tl, ERM-DA635. The data are shown as provided by the laboratories. 

Dataset code 

and method 

D01-ICP-SFMS 

D02-ICP-Q3MS 

D03-ICP-QMS 

D05-ICP-Q3MS 

D06-ICP-Q3MS 

D08-ICP-QMS 
Source: JRC 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

8.13 

7.76 

8.13 

8.3 

8.38 

9.5 

8.23 

7.71 

8.23 

8.2 

8.43 

9.53 

7.79 

7.73 

7.79 

8.2 

9.2 

9.06 

7.65 

7.96 

7.91 

8.2 

8.75 

8.89 

7.91 

7.51 

7.94 

8.3 

9.1 

7.94 

7.43 

7.65 

8.1 

9.3 

9.34 

9.01 

mean 

[µg/L] 

7.94 

7.68 

7.94 

8.22 

8.86 

9.22 

Expanded  
uncertainty 

0.65 µg/L 

0.5 µg/L 

1.2 µg/L 

20 % 

35 % 

2.77 µg/L 

Figure 6.17. Characterisation data for Tl, ERM-DA635, bold red lines and dashed red lines represent the certified value 
and its expanded uncertainty, respectively.  

Source: JRC 

16 

 
 
 
 
 
 
 
 
 
 
Table 6.18. Data from the characterisation for Tl, ERM-DA636. The data are shown as provided by the laboratories. 

Dataset code 

and method 

replicate 
1 
[µg/L] 

replicate 
2 
[µg/L] 

replicate 
3 
[µg/L] 

replicate 
4 
[µg/L] 

replicate 
5 
[µg/L] 

replicate 
6 
[µg/L] 

mean 

[µg/L] 

Expanded  
uncertainty 

D01-ICP-SFMS 

16.2 

15.8 

15.7 

16.1 

15.7 

D02-ICP-Q3MS 

15.15* 

14.81 

15.58 

14.39 

15.47 

D03-ICP-QMS 

D05-ICP-Q3MS 

15.5 

16.7 

15.7 

16.5 

15.4 

16.5 

15.5 

16.7 

15.5 

16.9 

14.5 

15.2 

15.4 

16.7 

15.67 

1.3 µg/L 

15.09 

0.96 µg/L 

15.50 

2.3 µg/L 

16.67 

20 % 

35 % 

D06-ICP-Q3MS 

17.03 

16.94 

18.03 

18.34 

18.85* 

18.51 

17.77 

D08-ICP-QMS 

18.79 

18.51 

19 

18.05 

19.05 

19.05 

18.74 

5.62 µg/L 

*: result not used as the unit was in the part of a rising trend in the filling volume 

Source: JRC 

Figure 6.18. Characterisation data for Tl, bold red lines and dashed red lines represent the certified value and its 
expanded uncertainty, respectively.  

Source: JRC 

17 

 
 
 
 
 
 
 
 
 
 
Annex 7. Results of the commutability study 

The  graphs  show  the  differences  of  the  natural  logarithms  of  the  averages  of  the  two  methods  of  each 
sample versus the average concentration in µg/l. Black diamonds are the fresh samples (spiked and unspiked), 
red squares correspond to the CRMs. The error bars on the CRMs correspond to the expanded uncertainty of 
the difference. The dashed red line correspond to the chosen commutability limit. 

18 

 
 
 
 
 
 
 
  
 
 
 
Source: JRC 

19 

 
 
  
 
 
 
 
 
 
 
 
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