The certification of the mass fraction of the total content and the aqua regia extractable content (ISO 12914 and ISO 11466) of As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn in sewage sludge, ERM®- CC144 : certification report.
Date: 2016-08-30
Institutions: Institute for Reference Materials and Measurements
Subjects: Community certification, analytical chemistry, chemical product, organic chemical, research report, research results, sewage sludge, wastewater, water treatment

CERTIFICATION REPORT 

The certification of the mass fraction of the total content 
and the aqua regia extractable content (ISO 12914 and  
ISO 11466) of As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn in 
sewage sludge: ERM®- CC144 

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

European Commission 
Joint Research Centre 
Directorate F – Health, Consumers and Reference Materials 

Contact information 
Reference materials sales 
Address: Retieseweg 111, 2440 Geel, Belgium 
E-mail: jrc-rm-distribution@ec.europa.eu 
Tel.: +32 (0)14 571 705 

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This publication is a Reference Materials Report by the Joint Research Centre, the European Commission’s in-house science service. It aims to provide 
evidence-based scientific support to the European policy-making process. The scientific output expressed does not imply a policy position of the 
European Commission. Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might 
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All images © European Union 2016 

JRC102360 

EUR 28012 EN 

ISBN 978-92-79-59857-9 (PDF) 
ISSN 1831-9424 (online) 
doi: 10.2787/264881 

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

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

Abstract 
This report describes the preparation and certification of ERM®-CC144, a sewage sludge material certified for total and aqua regia extractable content 
(ISO 12914 and ISO 11466) of As, Cd, Cr, Co, Cu, Fe, Hg, Mn, Ni, Pb and Zn. The material was produced by following ISO Guide 34: 2009. 
The whole certification process included checking of between unit-heterogeneity, stability during dispatch and best storage conditions which were 
assessed in accordance with ISO Guide 35:2006. Within-unit heterogeneity was also quantified to determine the minimum sample intake. The 
certified values were calculated through an inter-laboratory comparison study among laboratories of demonstrated competence and adhering to 
ISO/IEC 17025. Uncertainties of the certified values were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement 
(GUM), by including uncertainties related to possible heterogeneity, instability and characterisation. 
ERM-CC144 is available in glass bottles containing ca. 30 g of dried sewage sludge powder. 
The minimum amount of sample to be used is 300 mg for total content of As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn and 30 mg for total content of Hg. 
The minimum amount of sample to be used for aqua regia extractable content is 500 mg if using ISO 12914:2012 and 3 g if using ISO 11466:1995 
for all the certified elements. 
This material is intended for the quality control and assessment of method performance. As any reference material, it can also be used for control 
charts or validation studies.  
ERM-CC144 was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials 
consortium.

 
 
 
 
 
 
 
 
 
 
 
 
CERTIFICATION REPORT 

The certification of the mass fraction of the total content 
and the aqua regia extractable content (ISO 12914 and 
ISO 11466) of As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb and 
Zn in sewage sludge: ERM®- CC144 

Anna Santoro, Andres Perez, Toni Venelinov, 
Albert Oostra and Andrea Held 

European Commission, Joint Research Centre 
Directorate F – Health, Consumers and Reference Materials 
Geel, Belgium 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Disclaimer 

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

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Summary 

This  report  describes  the  preparation  and  certification  of  ERM®-CC144,  a  sewage  sludge 
material certified for total and aqua regia extractable content (ISO 12914 and ISO 11466) of 
As, Cd, Cr, Co, Cu, Fe, Hg, Mn, Ni, Pb and Zn. The material was produced by following ISO 
Guide 34: 2009 [1]. 
The  whole  certification  process  included  checking  of  between  unit-heterogeneity,  stability 
during  dispatch  and  best  storage  conditions  which  were  assessed  in  accordance  with  ISO 
Guide 35:2006 [2]. Within-unit heterogeneity was also quantified to determine the minimum 
sample  intake.  The  certified  values  were  calculated  through  an  inter-laboratory  comparison 
study among laboratories of demonstrated competence and adhering to ISO/IEC 17025 [3]. 
Uncertainties  of  the  certified  values  were  calculated  in  compliance  with  the  Guide  to  the 
Expression  of  Uncertainty  in  Measurement  (GUM)  [4],  by  including  uncertainties  related  to 
possible heterogeneity, instability and characterisation. 
ERM-CC144 is available in glass bottles containing ca. 30 g of dried sewage sludge powder. 
The minimum amount of sample to be used is 300 mg for total content of As, Cd, Co, Cr, Cu, 
Fe, Mn, Ni, Pb and Zn and 30 mg for total content of Hg. The minimum amount of sample to 
be used for aqua regia extractable content is 500 mg if using ISO 12914:2012 [5] and 3 g if 
using ISO 11466:1995 [6] for all the certified elements. 
This material is intended for the quality control and assessment of method performance. As 
any reference material, it can also be used for control charts or validation studies.  
ERM-CC144 was accepted as European Reference Material (ERM®) after peer evaluation by 
the partners of the European Reference Materials consortium. 
The following values were assigned: 

Certified Values 

Total content 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

Certified value 1) 
7.7 
14.5 
6.5 
168 
348 
32.9 
5.9 
352 
91 
157 
0.98 

Mass fraction based on dry mass 
Uncertainty 2) 
0.7 
1.4 
0.4 
14 
18 
1.6 
0.6 
14 
7 
9 
0.04 

Unit 
mg/kg 
mg/kg 
mg/kg 
mg/kg 
mg/kg 
g/kg 
mg/kg 
mg/kg 
mg/kg 
mg/kg 
g/kg 

1)  Certified values are expressed as unweighted mean value of the means of accepted sets of data, each set 
being obtained in a different laboratory and/or with a different method of determination. The certified value 
and its uncertainty are traceable to the International System of units (SI). 

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

1 

 
 
 
 
 
Aqua regia extractable 
content 1) 
As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

Unit 
mg/kg 
mg/kg 
mg/kg 
mg/kg 
mg/kg 
g/kg 
mg/kg 
mg/kg 
mg/kg 
mg/kg 
g/kg 
1)  Aqua regia extractable content is operationally defined by following ISO 11466:1995 or ISO 12914:2012  
2)  Certified values are expressed as unweighted mean value of the means of accepted sets of data each, set 
being obtained in a different laboratory and/or with a different method of determination. The certified value 
and its uncertainty are traceable to the International System of units (SI). 

Mass fraction based on dry mass 
Uncertainty 3) 
0.7 
0.9 
0.4 
11 
15 
1.5 
0.7 
13 
6 
7 
0.04 

Certified value 2) 
7.7 
13.3 
5.9 
150 
346 
32.8 
6.1 
340 
86 
156 
0.97 

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

Additional Material Information 

Additional Material Information 

Al 1)
Ca 1) 
Fe 1) 
K 1) 
Mg 1) 
Na 1) 
2) 
P2O5 
SiO2 
1) 
Ti 
TOC 3) 
TIC 3) 
Water content 4) 
Water activity 4) 
1)  Measured using k0-NAA 
2)  Analyses performed by gravimetry for SiO2 and spectrophotometry for P2O5 
3)  Total organic carbon (TOC) and total inorganic carbon (TIC) determined by using a combustion furnace 
4)  Water  content  determined  by  volumetric  Karl-Fischer  titration  and  water  activity  by  using  a  water  activity 

Unit 
g/kg 
g/kg 
g/kg 
g/kg 
g/kg 
g/kg 
g/kg 
g/kg 
g/kg 
g/100 g 
g/100 g 
g/100 g 
1 

Value  
19 
31 
34 
2.9 
3.8 
1.8 
38 
73 
1.5 
36 
0.1 
2.5 
0.15 

2) 

meter 

2 

 
 
 
 
 
 
 
 
Table of content 

Summary …………………………………………………………………………………...…………1 

Glossary ................................................................................................................................ 4 
Introduction ................................................................................................................ 7 
1. 
Participants ................................................................................................................ 7 
2. 
Project management and evaluation .......................................................................... 7 
2.1 
Processing ................................................................................................................. 7 
2.2 
Homogeneity study .................................................................................................... 8 
2.3 
Stability study (alphabetic order) ................................................................................ 8 
2.4 
Characterisation of total content (alphabetic order) .................................................... 8 
2.5 
Characterisation of aqua regia extractable content (alphabetic order) ........................ 8 
2.6 
Additional material information ................................................................................... 9 
2.7 
Material processing and process control .................................................................... 9 
3. 
Assessment of homogeneity .....................................................................................10 
4. 
4.1 
Between-unit homogeneity........................................................................................10 
4.2  Within-unit homogeneity and minimum sample intake...............................................13 
Stability .....................................................................................................................14 
5. 
Short-term stability study ..........................................................................................15 
5.1 
Long-term stability study ...........................................................................................16 
5.2 
Estimation of uncertainties ........................................................................................17 
5.3 
Characterisation study ..............................................................................................18 
6. 
6.1 
Selection of participants and study set-up .................................................................18 
6.2  Methods used ...........................................................................................................19 
Technical and statistical evaluation of results ...........................................................20 
6.3 
Value Assignment .....................................................................................................23 
7. 
Certified values and uncertainties .............................................................................23 
7.1 
7.2 
Additional material information ..................................................................................24 
Metrological traceability and commutability ...............................................................25 
8. 
8.1  Metrological traceability ............................................................................................25 
8.1.1 
Identity ......................................................................................................................25 
8.1.2  Quantity value ...........................................................................................................25 
8.1.3  Commutability ...........................................................................................................26 
Instructions for use ...................................................................................................26 
9. 
Storage conditions ....................................................................................................26 
9.1 
Safety and protection for the environment .................................................................26 
9.2 
9.3 
Preparation and use of the material ..........................................................................26 
9.4  Minimum sample intake ............................................................................................26 
Dry mass correction ..................................................................................................27 
9.5 
Use of the certified value ..........................................................................................27 
9.6 
Acknowledgments .....................................................................................................28 
10. 
References ...............................................................................................................29 
11. 
Annexes....................................................................................................................30 
12. 
Annex A: Methods used in the certification studies ...............................................................30 
Annex B: Homogeneity study ...............................................................................................38 
Annex C: Short term stability study .......................................................................................44 
Annex D: Long term stability study .......................................................................................56 
Annex E: Characterisation study ..........................................................................................60 

3 

 
 
 
Glossary 

AAS 
AFS  
ANOVA  
b 
BCR® 

CI 
CRM 
CV 
DMA  
ERM® 
ETAAS 
FAAS 
GUM 

ICP-OES 
ICP-MS 
ICP-SFMS 
ISO 
JRC 
k 
k0-NAA  
KFT 
MSbetween 
MSwithin  
n 
N 
n.a. 
n.c. 
PSA 
QC 
RSD 
s 
sbb
sbetween 
SI 
smeas 
swithin 
swb 
T 
t 
tsl 
u 
U 
u*

bb  

ubb 
ub 
uchar  
uCRM 

Atomic absorption spectrometry 
Atomic fluorescence spectrometry 
Analysis of variance 
Slope in the equation of linear regression y = a + bx 
Trademark of CRMs owned by the European Commission; formerly 
Community Bureau of Reference 
Confidence interval 
Certified reference material 
Cold vapour  
Direct Mercury Analyzer 
Trademark of European Reference Materials 
Electro thermal atomic absorption spectrometry 
Flame atomic absorption spectrometry 
Guide to the Expression of Uncertainty in Measurements 
[ISO/IEC Guide 98-3:2008] 
Inductively coupled plasma optical emission spectrometry 
Inductively coupled plasma mass spectrometry 
ICP sector field mass spectrometry 
International Organization for Standardization  
Joint Research Centre of the European Commission 
Coverage factor 
k0-Neutron Activation Analysis 
Karl Fischer titration 
Mean of squares between-unit from an ANOVA 
Mean of squares within-unit from an ANOVA 
Number of replicates per unit 
Number of samples (units) analysed 
Not applicable 
Not calculated 
Particle size analysis 
Quality control 
Relative standard deviation 
Standard deviation 
Between-unit standard deviation 
Standard deviation between groups as obtained from ANOVA 
International System of Units 
Standard deviation of measurement data 
Standard deviation within groups as obtained from ANOVA 
Within-unit standard deviation 
Temperature 
Time 
Proposed shelf life 
Standard uncertainty  
Expanded uncertainty 
Standard uncertainty related to a maximum between-unit heterogeneity 
that could be hidden by method repeatability 
Standard uncertainty related to a possible between-unit heterogeneity 
Error of the slope of the regression line  
Standard uncertainty of the material characterisation 
Combined standard uncertainty of the certified value; an additional index 
"rel" is added as appropriate 

4 

 
 
 
UCRM  
u∆ 

ults 
um 
urec  

usts 
ut 
ix  
x  
a  
D m 

n

MSwithin

Expanded uncertainty of the certified value 
Combined standard uncertainty of measurement result and certified 
value 
Standard uncertainty of the long-term stability 
Standard measurement uncertainty 
Standard uncertainty related to possible between-unit heterogeneity 
modelled as rectangular distribution 
Standard uncertainty of the short-term stability 
Standard uncertainty of trueness 
Time point for each replicate 

Mean results for all time points 

Significance level 
Absolute difference between mean measured value and the certified 
value 
Degrees of freedom of MSwithin 

5 

 
 
 
6 

 
1. 

Introduction  

The Certified Reference Material ERM-CC144 described in this report has been specifically 
developed to help laboratories that analyse sewage sludge, or similar matrices, in relation to 
environmental protection.  
The intended use of the Certified Reference Material ERM-CC144 is mainly for verification of 
the accuracy in determinations of trace element in sewage sludge or other similar matrices. 
Sewage  sludge  is  defined  as  a  'residual  sludge  from  sewage  plants  treating  domestic  and 
urban  waste  waters  and  from  other  sewage  plants  treating  waste  waters  of  a  composition 
similar to domestic and urban waste waters' [7]. 
In  recent  years,  treated  sewage  sludge,  either  of  industrial  or  domestic  origin,  has  been 
increasingly used on farmland, mostly for its high fertilising properties.  
Application  of  sewage  sludge  to  agricultural  land  partially  solves  the  problem  of  sewage 
sludge disposal after production, but also leads to contamination of soil and groundwater by 
inorganic (and organic) pollutants present in the sludge. 
The EU Directive 86/278/EEC (1986) [7] and its recent implementations, regulates the use of 
sewage  sludge  in  agriculture  disposal  so  as  to  prevent  harmful  effects  on  soil,  vegetation, 
animals and humans. Aiming at a correct management of the sludge from sewage treatment 
plants, the EU regulation prohibits farmland disposal of sludge unless specific requirements 
are  fulfilled,  including  compliance  with  limit  values  for  concentrations  of  a  range  of  trace 
elements.  
Mostly, laboratories prefer to determine the total content of trace elements; however, in some 
countries  (e.g.  Germany,  France,  Denmark  and  The  Netherlands),  national  regulations 
request evaluation of the aqua regia extractable content of the elements, rather than the total 
one.  
In this respect, certification of both total and aqua regia extractable content of As, Cd, Co, Cr, 
Cu, Fe, Hg, Mn, Ni, Pb and Zn was performed.  
In  this  specific  material  ERM-CC144,  the  results  obtained  for  total  content  and  aqua  regia 
extractable  content,  performed  either  following  ISO12914:2012  [5]  or  ISO  11466:1995  [6], 
were  similar  when  the  expanded  uncertainty  UCRM  is  taken  into  account. This  similarity  has 
already  been  observed  in  previous  studies  [8,  9],  however  it  cannot  be  considered  as  a 
general rule, but rather related to a specific element analysed and material processed [10]. 

ERM-CC144 was produced after careful consideration of the total and aqua regia extractable 
content of the certified elements, which were checked in a preliminary stage, so as to ensure 
concentrations below the limits imposed by the EU regulations [7]. 
Certification of this material included testing of the homogeneity and stability of the material, 
as well as the material characterisation using a laboratory inter-comparison approach. 

2. 

Participants 

2.1  Project management and evaluation 
Joint Research Centre Directorate F - Health, Consumers and Reference Materials Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No 268-RM) 

2.2  Processing  
Joint Research Centre Directorate F - Health, Consumers and Reference Materials Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No 268-RM) 

7 

 
 
 
 
 
 
 
2.3  Homogeneity study 
Vlaamse Instelling voor Technologische Onderzoek, Mol (BE)  
(measurements under the scope of ISO/IEC 17025 accreditation; BELAC, 045-TEST) 

2.4  Stability study (alphabetic order) 
ChemService S.r.l. Controlli e ricerche, Milano (IT)  
(measurements performed under the scope of ISO/IEC 17025 accreditation; SINAL-0004) 
The Macaulay Institute, Aberdeen (UK) 
(measurements performed under the scope of ISO/IEC 17025 accreditation; UKAS-1917) 
Umweltbundesamt, Umweltanalytik & Chemikalien, Wien (AT)  
(measurements performed under the scope of ISO/IEC 17025 accreditation, BMWA-92.714/0518-I) 

2.5  Characterisation of total content (alphabetic order) 
ALS Laboratory Group, ALS Scandinavia AB, Luleå (SE)  
(measurements performed under the scope of ISO/IEC 17025 accreditation; SWEDAC-1087) 
DSM Research, DSM Resolve Department, Geleen (NL)  
(measurements performed under the scope of ISO 9001 certification) 
Energieonderzoek Centrum Nederland (ECN), Petten (NL)  
(measurements performed under the scope of ISO/IEC 17025 accreditation; IEC-L135) 
Institut Jožef Stefan (IJS), Department of Environmental Sciences, Ljubljana (SI) 
(measurements performed ISO/IEC 17025 accreditation, Slovenka Akreditacija-LP090) 
Joint Research Centre Directorate F - Health, Consumers and Reference Materials Geel, BE 
(measurements performed under the scope of ISO/IEC 17025 accreditation; BELAC, 268-TEST) 
Minton, Treharne & Davies LTD, Cardiff (UK)  
(measurements performed under the scope of ISO/IEC 17025 accreditation; UKAS, 0024) 
Studiecentrum voor Kernenergie, SCK, Mol (BE)  
(measurements performed under ISO/IEC 17025 accreditation; BELAC, 015-TEST) 
Umweltbundesamt, Umweltanalytik & Chemikalien, Wien (AT)  
(measurements performed under the scope of ISO/IEC 17025 accreditation, BMWA-92.714/0518-I) 
VASYD, Dricksvattenavdelningen Vattenlaboratoriet, Malmö (SE)  
(measurements performed under the scope of ISO/IEC 17025 accreditation; SWEDAC 1056) 

2.6  Characterisation of aqua regia extractable content (alphabetic order) 
ALS Laboratory Group, ALS Scandinavia AB, Luleå (SE)  
(measurements performed under the scope of ISO/IEC 17025 accreditation; SWEDAC-1087) 
ALS Laboratory Group, ALS Czech Republic, s.r.o., Prague (CZ) 
(measurements performed under the scope of ISO/IEC 17025 accreditation; ČIA-521) 
ALS Laboratory Group, ALS Czech Republic, s.r.o., Harfe (CZ) 
(measurements performed under the scope of ISO/IEC 17025 accreditation; ČIA-521) 
DSM Research, DSM Resolve Department, Geelen (NL)  
(measurements performed under the scope of ISO 9001 certification) 
Energieonderzoek Centrum Nederland (ECN), Petten (NL)  
(measurements performed under the scope of ISO/IEC 17025 accreditation; IEC-L135) 
Minton, Treharne & Davies LTD, Cardiff (UK)  
(measurements performed under the scope of ISO/IEC 17025 accreditation; UKAS, 0024) 
Nacionalni laboratorij za zdravje, okolje in hrano (SI)  
(measurements performed under the scope of ISO/IEC 17025 accreditation, LP-014) 
Scientific Analytical Laboratories Ltd, Manchester (UK) 
(measurements performed under the scope of ISO/IEC 17025 accreditation; UKAS 1549) 

8 

 
 
 
 
Umweltbundesamt, Umweltanalytik & Chemikalien, Wien (AT)  
(measurements performed under the scope of ISO/IEC 17025 accreditation, BMWA-92.714/0518-I) 
Universitat de Barcelona, Facultat de Química, Barcelona (ES) 
Vlaamse Instelling voor Technologisch Onderzoek, Mol (BE)  
(measurements performed under the scope of ISO/IEC 17025 accreditation; BELAC, 045-TEST) 

2.7  Additional material information (alphabetic order) 
ALS Laboratory Group, ALS Czech Republic, s.r.o., Prague (CZ) 
(measurements performed under the scope of ISO/IEC 17025 accreditation; ČIA-521) 
Joint Research Centre Directorate F - Health, Consumers and Reference Materials Geel, BE 
(accredited to ISO Guide 34 for production of certified reference materials, BELAC No 268-TEST) 
Studiecentrum voor Kernenergie, SCK, Mol (BE)  
(measurements performed under ISO/IEC 17025 accreditation; BELAC, 015-TEST) 
Vlaamse Instelling voor Technologisch Onderzoek, Mol (BE)  
(measurements performed under the scope of ISO/IEC 17025 accreditation; BELAC, 045-TEST) 

3. 

Material processing and process control 

The base material for the preparation of ERM-CC144 consisted of a mixture of two types of 
sewage  sludge.  The  first  one  was  collected  by  the  Joint  Research  Centre  -  Environment 
Institute,  in  Ispra  (IT)  and  then  dried  at  the  Joint  Research  Centre  Directorate  F  -  Health, 
Consumers  and  Reference  Materials  Geel,  BE.  The  second  type  was  collected  by  the 
University of Amsterdam and dried at WEPAL, Wageningen (NL). The mixture was made of 
ca. 40 % of the first sewage sludge and ca. 60 % of WEPAL sludge. At the JRC-Geel the two 
materials  were  mixed  together  and  ten  sub-batches  of  ca.  10  kg  each  were  separately 
homogenised in a Turbula mixer for 30 minutes.  
The mixed sewage sludge was finely ground by using a Multi-Processing System from Alpine 
(100 AFG Jet Mill/ Ultrafine Classification system, Augsburg, DE). A total amount of ca. 94 kg 
of sewage sludge powder ground at < 250 µm was produced. Homogenisation and sampling 
were carried out in a multipurpose cone mixer of 250 L with semi-automatic filling equipment. 
A total of 2719 amber glass bottles were finally sampled with 30 g powder and closed with a 
polyethylene insert and a screw cap.  
The  particle  size  distribution  of  the  material  was  carried  out  on  three  randomly  selected 
samples by using a Helos laser light scattering instrument (Sympatec, DE) and as shown in 
Figure  1,  a  maximum  particle  size  of  ca.  250  µm  (average  particle  size  ca.  20  µm)  was 
obtained. 

9 

 
 
 
 
 
Figure 1: Typical particle size distribution for ERM-CC144 

The filled bottles were finally sterilised (Isotron Nederland B.V., 's-Hertogenbosch, NL) using 
gamma irradiation with a minimum radiation dose of 25 kGy in order to minimise the growth 
of bacteria and moulds which could damage the material. 

4. 

Assessment of homogeneity 

A key requirement for any reference material is the equivalence between the various units. In 
this respect, it is not relevant whether the variation between units is significant compared to 
the  analytical  variation,  but  whether  this  variation  is  significant  to  the  certified  uncertainty. 
Consequently,  ISO  Guide  34  requires  RM  producers  to  quantify  the  between  unit  variation 
via specific between-unit homogeneity studies [1]. 
Quantification  of  within-unit  heterogeneity  needs  also  to  be  evaluated  as  it  determines  the 
minimum sample intake, which is the minimum size of an aliquot that is representative for the 
whole  unit.  It  does  not  influence  the  uncertainty  of  the  certified  value  when  the  minimum 
sample intake is respected.  

4.1  Between-unit homogeneity 

The  between-unit  homogeneity  was  evaluated  to  ensure  that  the  values  of  the  CRM  were 
valid for all the bottles of the material produced, within the stated uncertainty.  
Fourteen  bottles  were  selected  for  this  study  using  a  randomly  stratified  sampling  scheme 
covering the whole batch. The whole batch was divided into 14 groups (with similar number 
of  bottles)  and  one  bottle  was  randomly  selected from  each group (the number  of  selected 
bottles corresponds to approximately the cubic root of the total number of bottles produced). 
From  each  bottle,  three  independent  aliquots  were  taken  and  analysed  for  total  content  by 
using HNO3, HCl and HF and three aliquots were analysed by following ISO 12914:2012 for 
aqua regia extractable content [5]. The quantification of all elements was performed by ICP-
OES,  with  the  exception  of  Hg  measured  by  CV-AFS  (Annex  A).  The  measurements  were 
performed under repeatability conditions, and in a randomised manner to be able to separate 
a potential analytical drift from a trend in the filling sequence. The results were corrected for 
the water content, which was determined for each bottle (see also section 9.5).  

10 

 
 
 
 
 
 
 
 
All  results,  reported  in  Annex  B,  were  first  checked  for  outliers  by  using  single  and  double 
Grubbs test. Two individual outliers were found using single Grubbs test at a 99 % CI in the 
analytical  sequence  for  Ni  total  content,  but  the  value  was  retained  for  the  final  statistical 
analysis as no specific technical reason for the outlier could be found. Regression analyses 
were also performed to evaluate potential trends in the analytical sequence as well as trends 
in the filling sequence.  
Some trends at a 95 % confidence level were observed in the analytical sequence for Cr and 
Hg total content and for Co, Cu, Mn and Zn aqua regia extractable content. These trends in 
the analytical sequence, not correlated to the unit numbers as the analyses were performed 
in  a  randomised  way,  were  corrected  if  the  trend  was  significant  on  at  least  a  95  % 
confidence  level,  in  order  to  improve  the  sensitivity  of  the  subsequent  statistical  analysis, 
through  a  reduction  in  analytical  variation,  without  anyhow  masking  potential  between-unit 
heterogeneities. The correction was performed using the formula:  

corrected

result

=

measured

result

ib

b = slope of the linear regression 
i = position of the result in the analytical sequence 
The  trend-corrected  datasets  were  tested  for  consistency  using  Grubbs  outlier  tests  on  a 
confidence level of 99 % on individual results and unit means. No outlying individual results 
and outlying unit means were detected (Table 1 and Table 2).  
Quantification  of  between-unit  heterogeneity  was  done  by  analysis  of  variance  (ANOVA), 
which could separate the between-unit variation (sbb) from the within-unit variation (swb). The 
latter is equivalent to the method repeatability if the individual samples are representative for 
the whole bottle.  
Evaluation  by  ANOVA  assumes  that  bottle  means  would  follow  at  least  a  unimodal 
distribution  and  results  for  each  unit  following  unimodal  distributions  having  approximately 
the same standard deviations. Considering that a small amount of data is available for each 
unit to make a clear statement of the distribution of individual results, distribution of the bottle 
averages  was  checked  by  using  histograms  and  normal  probability  plots.  Minor  deviations 
from unimodality of the individual values do not affect the estimate of between-unit standard 
deviations.  
The results of all statistical evaluations are given in Table 1 and Table 2. 

Table 1: Results of the statistical evaluation for homogeneity study, total content (for details 
see Annex B) 

Analyte 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

Significant trends 
(95% confidence) 
Filling 
sequence 
no 
yes 
no 
yes 
no 
yes 
no 
no 
no 
no 
no 

Analytical 
sequence 
no 
no 
no 
yes* 
no 
no 
yes* 
no 
no 
no 
no 

*no trends were observed after correction  

Outliers 

Distribution 

Bottle 
means 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 

Individual 
results 
normal 
normal 
normal 
normal 
normal 
normal 
normal 
unimodal 
normal 
unimodal 
normal 

Bottle 
means 
unimodal 
normal 
bimodal 
normal 
normal 
normal 
normal 
unimodal 
normal 
normal 
normal 

Individual 
results 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 

11 

 
 
 
 
(cid:215)
-
 
 
 
Table  2:  Results  of  the  statistical  evaluation  for  homogeneity  study,  aqua  regia  extractable 
content (ISO 12914:2012). Details in Annex B. 

Analyte 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

Significant trends 
(95% confidence) 
Filling 
sequence 
no 
no 
no 
no 
no 
no 
no 
no 
no 
no 
no 

Analytical 
sequence 
no 
no 
yes* 
no 
yes* 
no 
no 
yes* 
no 
no 
yes* 

Outliers 

Distribution 

Individual 
results 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 

Bottle 
means 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 

Individual 
results 
normal 
normal 
normal 
normal 
normal 
normal 
normal 
normal 
normal 
normal 
normal 

Bottle 
means 
normal 
normal 
normal 
unimodal 
normal 
normal 
bimodal 
normal 
unimodal 
normal 
normal 

*: no trends were observed after correction  

Method  repeatability  (swb,rel),  between-unit  standard  deviation  (sbb,rel)  and  u*
calculated as  

bb,rel  were 

s

,wb

rel

=

MSwithin 
y  

s

,bb

rel

=

MS

within

y 
mean square within a unit from an ANOVA  
average of all results of the homogeneity study 

MS

between

MS

within

n
y

MSbetween:   mean squares between-unit from an ANOVA 
n: 

average number of replicates per unit 

MS
within
n

4

ν

2

MSwithin

y

:  

degrees of freedom of MSwithin  

u

*
,bb

rel

=

ν

MSwithin

bb  is  comparable  to  the  limit  of  detection  of  an  analytical  method,  yielding 

The  parameter  u*
the maximum heterogeneity that might be undetected by the given study setup.  
Considering  that  sbb,rel  and  swb,rel  are  estimates  of  the  true  standard  deviations,  they  are 
subjected  to  random  fluctuations.  Therefore,  the  mean  square  between  groups  (MSbetween) 
can  be  smaller  than  the  mean  squares  within  groups  (MSwithin),  resulting  in  negative 
arguments  under  the  square  root  used  for  the  estimation  of  the  between-unit  variation, 
whereas  the  true  variation  cannot  be  lower  than  zero.  In  this  case,  u*
bb,  the  maximum 

12 

 
 
 
 
 
 
-
 
 
 
 
 
heterogeneity that could be hidden by method repeatability, was calculated as described by 
Linsinger et al. [11]. 

A  different  approach  was  adopted  for  Cd,  Cr  and  Fe  total  content,  for  which  a  trend  in  the 
filling sequence was significant at 95 % confidence level. The same approach was used for 
Co  total  content  and  aqua  regia  extractable  content  of  Cr,  Hg  and  Ni  for  which  the  bottle 
means  did  not  follow  a  perfect  normal  or  unimodal  distribution  but  apparently  consisted  of 
two populations with similar means.  

In these cases, urec was estimated using a rectangular distribution between the highest and 
lowest unit average: 

u rel
rec
,

=

highest re

sult - low

est result

2

y 3

The results of the evaluation of the between-unit variation are summarised in Table 3. 

Table 3: Results of the homogeneity study for total and aqua regia extractable content (n.c.: 
cannot be calculated as MSbetween < MSwithin) 

Element 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

swb,rel 
[%] 
3.4 
3.7 
2.9 
3.3 
1.9 
2.2 
3.4 
2.2 
4.3 
2.2 
2.2 

Total content 
u*
bb,rel 
[%] 
1.0 
1.1 
0.9 
1.0 
0.6 
0.7 
1.0 
0.7 
1.3 
0.7 
0.7 

urec,rel 
[%] 
- 
2.9 
1.9 
2.6 
- 
1.7 
- 
- 
- 
- 
- 

sbb,rel 
[%] 
2.8 
2.5 
1.9 
2.2 
n.c. 
1.1 
n.c. 
0.7 
2.1 
n.c. 
n.c. 

ubb,rel 
[%] 
2.8 
2.9 
1.9 
2.6 
0.6 
1.7 
1.0 
0.70 
2.1 
0.7 
0.7 

Aqua regia extractable content 
u*
bb,rel 
[%] 
0.8 
0.9 
0.5 
0.8 
0.4 
0.5 
1.1 
0.4 
0.7 
0.5 
0.3 

urec,rel 
[%] 
- 
- 
- 
2.1 
- 
- 
2.0 
- 
1.8 
- 
- 

sbb,rel 
[%] 
1.8 
2.0 
n.c. 
1.6 
n.c. 
0.9 
1.3 
0.7 
1.5 
n.c. 
0.2 

swb,rel 
[%] 
2.7 
3.0 
1.8 
2.6 
1.3 
1.8 
3.8 
1.5 
2.2 
1.5 
1.2 

ubb,rel 
[%] 
1.8 
2.0 
0.5 
2.1 
0.4 
0.9 
2.0 
0.7 
1.8 
0.5 
0.3 

The homogeneity study showed no outlying unit means or trends in the filling sequence for 
most  of  the  elements.  Therefore  the  between-unit  standard  deviation  could  be  used  as  an 
estimate of ubb. As u*
bb sets the limits for the detection power of the study, the larger value of 
sbb  and  u*
bb  was  adopted  as  an  uncertainty  contribution  to  account  for  potential 
heterogeneity. 

In  the  specific  case  of  Cd,  Co,  Cr  and  Fe  total  content  and  Cr,  Hg  and  Ni  aqua  regia 
extractable  content,  the  heterogeneity  was  quantified  as  urec,  which  was  sufficiently  small, 
demonstrating the feasibility of the material as a CRM. Eventually, urec was used as estimate 
of ubb. 

4.2  Within-unit homogeneity and minimum sample intake 

Concerning the micro-homogeneity of the material, individual aliquots of a material sampled 
from a single bottle may contain different amounts of analyte. In this respect, the within-unit 
heterogeneity needs to be evaluated as it is closely correlated to the minimum sample intake, 
which is the minimum amount of sample that is representative of the whole unit and thus can 

13 

 
(cid:215)
(cid:215)
 
 
 
 
 
 
be  used  in  an  analysis.  The  certified  values  and  stated  uncertainties  are  guaranteed  for 
sample sizes equal or above the minimum sample intake. 
In  this  study,  the  minimum  sample  intake  was  assessed  by  using  the  method  information 
supplied by the laboratories participating in the characterisation study (Annex A).  
Within-unit standard deviation (swb) per each set of results provided by each laboratory was 
calculated. The amount of sample used for the analysis and related to the smallest value of 
swb was considered as minimum sample intake.  
As reported in Annex B, sample intakes of 300 mg for all the elements represent the smallest 
values  of  swb.  A  few  remarks  are  however  needed  regarding  Cr,  Hg  and  Mn.  In  case  of  Cr 
and Mn, a similar swb was found for 300 mg and 100 mg sample intakes which indicates no 
difference in homogeneity between 100 mg and 300 mg. 
For  Hg,  a  30  mg  sample  intake  was  reported  by  analysing  the  samples  via  DMA 
instrumentation;  other  laboratories  using  different  techniques,  such  as  ICP-MS  or  CV-AAS, 
preferred higher sample intakes. The small swb found for 30 mg indicates that the material is 
homogeneous at a very small sample intake for this element (Annex B, Table B12). 

In summary, the overall minimum sample intake for total element content of As, Cd, Co, Cr, 
Cu, Fe, Mn, Ni, Pb and Zn is set at 300 mg. The minimum sample intake for Hg is 30 mg. 

The  sample  intake for  the  determination  of  the  aqua  regia  extractable  content  is  set  by  the 
ISO  guides  12914:2012  and  ISO  11466:1995  and  it  is  500  mg  and  3  g,  respectively.  One 
laboratory (L14) used a sample intake of 300 mg for the ISO guides 12914:2012 procedure. 
The results provided were retained for the final evaluation as they agreed with the required 
quality  control  parameters  and  were  comparable  to  all  the  other  ones  received  from  other 
laboratories. Nevertheless the sample intake for the ISO guides 12914:2012 procedure is set 
to 500 mg.  

5. 

Stability 

Stability testing is necessary to establish conditions for storage (long-term stability) as well as 
conditions  for  dispatch  to  the  customers  (short-term  stability).  Time,  temperature  and 
radiation were regarded as the most relevant influences on the stability of the materials. The 
influence  of  ultraviolet  or  visible  radiation  was  minimised  by  choosing  brown  glass  bottles 
which absorb most of the incoming light. In addition, materials are stored and dispatched in 
the dark, so as to reduce the possibility of degradation due to irradiation. Therefore, only the 
influences  of  time  and  temperature  were  investigated.  During  dispatch,  especially  in  the 
summer  period,  temperatures  up  to  60  °C  could  be  reached  and  stability  under  these 
conditions must be demonstrated if transport at ambient temperature should be applied. 

The  stability  studies  were  carried  out  using  an  isochronous  design  [12].  In  this  approach, 
samples  are  stored  for  a  certain  time  at  different  temperature  conditions.  Afterwards,  the 
samples are moved to conditions where further degradation can be assumed to be negligible 
("reference conditions"), effectively "freezing" the degradation status of the materials. At the 
end of the isochronous storage, the samples are analysed simultaneously under repeatability 
conditions.  Analysis  of  the  material  (after  various  exposure  times  and  temperatures)  under 
repeatability conditions greatly improves the sensitivity of the stability tests. 

14 

 
 
 
 
 
 
5.1  Short-term stability study 

Regarding the  short-term  stability  study,  selected  samples  were  stored at  18 °C  and  60 °C 
for 0, 1, 2 and 4 weeks. Two samples per each time/temperature combination were selected 
using a random stratified sampling scheme, for a total of 14 bottles (the two bottles for t = 0 
were used for both the 18 and 60 °C study). The reference temperature was set to -20 °C. 
From each bottle, three aliquots were digested by using HNO3 and HF for the total element 
content of As and Hg or using HNO3, HF and HCl for Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn 
total content. Three aliquots per bottle were also leached according to the ISO 11466:1995 
for aqua regia extractable content [6]. Quantification of the element mass fractions were then 
performed  by  FAAS  or  ICP-OES for  As,  FAAS for  Cd,  Co,  Cr,  Cu,  Fe, Mn,  Ni,  Pb  and  Zn; 
and  ETAAS  or  CV-AAS  for  Hg  (details  in  Annex  A).  The  measurements  were  performed 
under repeatability conditions, and in a randomised manner to be able to separate a potential 
trend  in  the  measurement  sequence from  a  trend  over  storage  time.  The  results  were  also 
corrected for the water content determined in each single bottle (see section 9.5).  
The  studies  were  evaluated  individually  for  each  temperature  by  screening  the  results  for 
outliers by using a Grubbs test on a 99 % confidence level. As reported in Table 4 and Table 
5,  some  outliers  were  detected  by  single  or  double  Grubbs  tests,  but  since  no  specific 
technical reasons were found; they were all retained in the final evaluation. 
Linear regressions were also performed and the slopes were tested for significance at 95 % 
CI (Annex C). In all cases the slope of the regression line was found to be not significantly 
different  from  zero  at  18 °C,  while  for  some  elements  the  slope  was  significantly  different 
from zero at 60 °C (Table 4). Specifically, the short term stability study showed instability of 
the total content of Cu and of the aqua regia extractable content of Cd, Cr, Cu, Fe, Pb and 
Zn at 60 °C (Table 4 and Table 5). 

Table  4:  Results  of  the  short  term  stability  test  for  total  element  content  (T = test 
temperature) 

Element 

Slope significantly different from zero at 
95 % confidence level 

Outlying value at 99 % CI 
(Grubbs test) 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

T = 18 °C 
no 
no 
no 
no 
no 
no 
no 
no 
no 
no 
no 

T = 60 °C 
no 
no 
no 
no 
yes 
no 
no 
no 
no 
no 
no 

T = 18 °C 
- 
1 
1 
- 
- 
- 
- 
- 
2 
2 
- 

T = 60 °C 
- 
- 
1 
2 
- 
- 
- 
- 
- 
- 
- 

15 

 
 
 
 
Table  5:  Results  of  the  short  term  stability  test  for  aqua  regia  leachable  content  (T = test 
temperature) 

Element 

Slope significantly different from zero at 
95 % confidence level 

Outlying value at 99 % CI 
(Grubbs test) 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

T = 18 °C 
no 
no 
no 
no 
no 
no 
no 
no 
no 
no 
no 

T = 60 °C 
no 
yes 
no 
yes 
yes 
yes 
no 
no 
no 
yes 
yes 

T = 18 °C 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 

T = 60 °C 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 
- 

The low uncertainty of stability found for one week transport at 60 °C shows however that the 
degradation  of  the  material  is  technically  insignificant  at  60  °C  for  periods  up  to  1  week. 
Therefore the material will be dispatched at ambient conditions. 

5.2  Long-term stability study 

Regarding the long-term stability study, samples were stored at 18 °C for 0, 4, 8, 12 and 0, 
12, 24 and 36 months. The reference temperature was set to -20 °C. Two samples per each 
time point were selected using a randomly stratified sampling scheme. From each bottle, 6 
aliquots  were  measured.  Only  the  aqua  regia  extractable  content  of  the  elements  was 
measured,  since this  parameter,  as  demonstrated  by  the  short  term  stability  study,  is  more 
affected by degradation rather than total content. The measurements were performed using 
ICP-MS for As, Cr, Co, Cd, Hg, Ni, Mn, Pb and Zn and by ICP-OES for Cu and Fe (Annex A), 
under repeatability conditions and in a randomised manner to be able to separate a potential 
analytical drift from a trend over storage time. The results were also corrected for the water 
content determined in each single bottle (see section 9.5). 
The  results  obtained  for  12  and  36  months  were  pooled  and  the  combined  study  was 
screened  for  outliers  using  the  single  and  double  Grubbs  test.  Some  outlying  individual 
results significant at 99 % CI were found for As, Co and Mn, but they were all kept in the final 
evaluation as no technical reasons for exclusion were found.  
All the results were also plotted against time (details in Annex D) and no significant trend for 
the slope was found at 95 % CI for all the elements.  The results are summarised in Table 6.  

16 

 
 
 
 
Table 6: Results of the 36 months long-term stability study for aqua regia leachable content 
(details in Annex C)  

Element 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

Slope significantly 
different from zero at 95 
% confidence level 
no 
no 
no 
no 
no 
no 
no 
no 
no 
no 
no 

Outlying value at 99 % 
CI (Grubbs test) 

2 
- 
2 
- 
- 
- 
- 
- 
2 
- 
- 

After the certification campaign, the material will be subjected to regular stability monitoring 
programme at the JRC-Geel to control its further stability. 

5.3  Estimation of uncertainties 

Uncertainties of stability during dispatch and storage were estimated as described in [13] for 
each  analyte.  In  this  approach,  the  uncertainty  of  the  linear  regression  line  with  a  slope  of 
zero  is  calculated.  The  uncertainty  contribution  (usts  and  ults)  are  then  calculated  as  the 
product of the chosen shelf life and the uncertainty of the regression lines as 

u

lts

,
rel

=

tu
b

sl

=

RSD
(

x
i

∑

t

sl

)

2

x

bu : 
RSD  
ix : 
x : 
tsl: 

error of the slope of the regression line  
relative standard deviation of all results of the stability study 
time point for each replicate 

mean results for all time points  
proposed shelf life 

In case of a slope significantly different from zero, the uncertainty is estimated by including 
the degradation according to the formula 

u

lts

,
rel

=

u

2
deg

+

u

2
b

t

sl

=

u

deg

b
3

b: 

slope of the regression line 

The following uncertainties were estimated: 

17 

 
 
 
 
 
(cid:215)
-
(cid:215)
 
 
 
 
(cid:215)
 
 
 
 
 
•  usts,rel,  the  uncertainty  of  degradation  during  dispatch.  This  was  estimated  from  the 
60 °C studies for a time of 0.25 months (1 week). The uncertainty therefore describes 
the possible change during a dispatch at 60 °C lasting for one week. 

•  ults,rel,  the  stability  during  storage.  This  uncertainty  contribution  was  estimated  from 
the  18 °C  study.  The  uncertainty  contribution  therefore  describes  the  possible 
degradation during 36 months at 18°C.  

The results of these evaluations are summarised in Table 7. 

Table  7:  Uncertainties  of  stability  during  dispatch  and  storage.  usts,rel  is  calculated  for  a 
temperature of 60 °C and 1 week; ults,rel is calculated for a storage temperature of 18 °C and 
3 years (36 months). Data for ults, rel total content derive from the aqua regia long term stability 
study 

Total content 

Aqua regia extractable 
content 

usts ,rel 
[%] 

0.63 
0.73 
0.64 
0.91 
0.73* 
0.42 
0.70 
0.49 
0.55 
0.60 
0.41 

ults, rel [%] 
for a shelf-life of 
36 months 
2.9 
1.9 
2.4 
2.6 
1.2 
1.5 
4.7 
1.4 
2.3 
1.4 
1.2 

usts ,rel 
[%] 

0.55 
1.0* 
0.53 
1.0* 
0.89* 
0.70* 
0.45 
0.38 
0.43 
0.92* 
0.81* 

ults, rel [%] 
for a shelf-life 
of 36 months 
2.9 
1.9 
2.4 
2.6 
1.2 
1.5 
4.7 
1.4 
2.3 
1.4 
1.2 

Element 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

* Degradation included in the calculation 

Summarising the stability studies: 

The material can be transported at ambient conditions without special precautions and stored 
at temperature not exceeding 18 °C –  5 °C. 
After the certification campaign, the material will be subjected to regular stability monitoring 
programme at the JRC-Geel to control its further stability. 

6. 

Characterisation study 

The characterisation of the material was based on an inter-comparison study involving expert 
laboratories.  The  mass  fractions  of  the  total  and  aqua  regia  extractable  content  of  As,  Cd, 
Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn were determined in different laboratories by applying 
different measurement procedures. This approach aims at randomisation of laboratory bias, 
which reduces the combined uncertainty. 

6.1  Selection of participants and study set-up 

Participants in the characterisation study were selected on the basis of a set of criteria that 
comprised 
in  previous 
technical  aspects:  quality  management  criteria,  experience 
certification exercises and successful previous participation in inter-comparison studies.  

18 

 
 
 
 
 
 
 
 
 
 
In  particular,  fulfilment  of  the  quality  management  requirements  ensured  that  the  technical 
standards were maintained from the beginning to the end of the characterisation study.  
Having a formal accreditation was not mandatory, but meeting the requirements of ISO/IEC 
17025 was obligatory. Where measurements are covered by the scope of accreditation, the 
accreditation number is stated in the list of participants (section 2). 

The  guiding  principle  of  the  characterisation  study  was  randomisation  of  laboratory  bias, 
which was achieved by taking the following measures: 

(cid:1)  A  minimum  of  six  laboratories  were  selected,  providing  independent  measurement 

results for each element. 

(cid:1)  Samples  for  the  characterisation  study  covered  the  whole  batch  and  were  selected 

(cid:1) 

(cid:1) 

using a random stratified sampling scheme [14]. 
In  case  of  total  content,  different  methodologies  were  employed  to  confirm  the 
absence of method specific bias. Laboratories were therefore free to select a method 
of their choice. Non-destructive methods, such as k0-NAA, were also used. 
In  case  of  aqua  regia  extractable  content,  two  ISO  methods  were  used:  ISO 
11466:1995 requiring aqua regia as reagent and an assembled system constituted of 
a  reaction  vessel,  reflux  condenser  and  absorption  vessel  [6]  or  ISO  12914:2012 
involving the use of aqua regia and a microwave-assisted extraction system [5]. 
(cid:1)  Six  independent  measurements  per  laboratory  and  method  were  performed.  These 
measurements  were  spread  over  two  days  to  ensure  within-laboratory  intermediate 
precision conditions. 
Independent calibrations were prepared for all days of the measurements.  

(cid:1) 
(cid:1)  All  laboratories  received  a  bottle  containing  BCR-144R  (sewage  sludge)  or  BCR-
146R (sewage sludge from industrial origin) as a blind sample to use as QC sample. 
The results for total content were compared to the certified ones of the material and 
used  to  check  the  absence  of  significant  bias,  with  the  exception  of  As  and  Fe  for 
which certified values were not reported. If the result deviated by 20 % (assumed as 
expanded  measurement  uncertainty  of  the  average  of  six  results)  or  more  from  the 
certified  value,  the  data  from  this  laboratory  for  this  specific  element  were  excluded 
from  the  final  characterisation  study  of  ERM-CC144.  Regarding  aqua  regia,  the  QC 
materials used  were certified according to a method different from the ones used in 
this  study  (DIN38414-S7  [15]);  therefore  all  results  received  from  the  laboratories 
were retained, except when the recoveries of the QC sample exceeded 120 %, as in 
any case aqua regia extractable content should be higher than the total one.  

(cid:1)  All  data  from  a  specific  laboratory  were  excluded  when  half  or  more  than  half  of  all 

delivered results deviated more than 20 % from the certified value.  

(cid:1)  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. 
(cid:1)  The  water  content  had  to  be  determined  on  separate  subsamples,  by  following  the 

procedure described in section 9.5. 

6.2  Methods used 

The  total  content  was  determined  through  an  inter-comparison  approach  using  different 
methods. 
The selected laboratories employed mainly closed microwave digestion of the samples using 
different  combination  of  digestion  media  (e.g.  HF,  HNO3,  HCl,  H2O2  and  HClO4).  The 
detection  techniques  used  for  measuring  the  elements  in  the  material  were  ICP-MS,  ICP-
OES,  FAAS  and  CV-AAS.  The  non-destructive  method  k0-NAA,  was  also  used.  The 
combination  of  results  from  different  methods  based  on  completely  different  principles 
virtually rules out undetected method bias.  

19 

 
 
 
 
All  methods  used  during  the  characterisation  study  are  summarised  in  Annex  A.  The 
laboratory code (e.g. L1, L2, etc.) is a random number and does not correspond to the order 
of laboratories in section 2.  

Concerning  the  aqua  regia  extractable  content,  the  certification  was  based  on  an  inter-
comparison study using the method ISO 11466:1995 or ISO 12914:2012.  
Data were first grouped and evaluated according to the method used, either ISO 11466:1995 
or ISO 12914:2012. As there was no difference between the results (p>0.05, t-Test) obtained 
with the two methods, all data were considered together in the final evaluation. The results of 
the t-Test are shown in Annex E (Table E1). 
All  methods  used  during  the  characterisation  study  are  summarised  in  Annex  A.  The 
laboratory code (e.g. L1, L2, etc.) is a random number and does not correspond to the order 
of laboratories in section 2. The same lab number for aqua regia and total content does not 
necessarily correspond to the same laboratory. 

6.3  Technical and statistical evaluation of results 

The  characterisation  campaign  resulted  in  a  minimum  of  seven  datasets  per  element.  All 
individual  results  of  the  participants,  grouped  per  element  are  displayed  in  tabular  and 
graphical form in Annex E.  
The  obtained  data  were  first  checked  for  compliance  with  the  requested  measurements 
protocol  and  for  their  validity  based  on  technical  reasons.  The  following  criteria  were 
considered during the evaluation:  

- 

compliance  with  the  analysis  protocol,  sample  preparations  and  measurements 
performed on minimum two days, randomisation of analysis order and water content 
determination. 
correctness of the measurements based on knowledge of the method 

- 
-  absence of values given as below limit of detection or below limit of quantification  
-  method  performance,  i.e.  agreement  of  the  measurement  results  with  the  assigned 

value of the QC sample, especially for total content 

Based on the abovementioned criteria some datasets were rejected as not technically valid.  
When  laboratories  provided  results  with  different  methods  using  the  same  sample  units 
and/or extracts, as for instance L6 and L10 for total content and L9 and L14 for aqua regia, 
these multiple results were considered as sample replicates of specific sample units. 
In Table  8 the  data  sets  that  were found to  be  not-valid,  together  with  the  action taken  are 
listed.  

20 

 
 
 
 
 
As 

Cd 

Hg 

Mn 

Pb 

Zn 

Aqua regia 
extractable 
content 

Cd 

Cr 

Table  8:  Datasets that showed  non-compliances  with  the  analysis  protocol  together  with  the 
technical  specifications,  and  action  taken.  A  list  of  the  laboratory  codes  together  with  the 
methods used is given in Annex A. 

Total content 

Laboratory 

Description of problem 

Action taken 

L1 (ICP-MS) 

Deviating results for QC sample 

L8 (ICP-OES) 
L9 (ICP-OES) 

Deviating results for QC sample 

L1 (ICP-MS) 

Deviating results for QC sample 

L6 (ICP-MS) 

Deviating results for QC sample 

L8 (ICP-OES) 

Deviating results for QC sample 

L1 (ICP-MS) 

Deviating results for QC sample 

Value not used for 
evaluation 
Value not used for 
evaluation 
Value not used for 
evaluation 
Value not used for 
evaluation 
Value not used for 
evaluation 
Value not used for 
evaluation 

Laboratory 

Description of problem 

Action taken 

L6 (ICP-MS) 
L9(k0-NAA) 

L1 (ICP-OES) 

Deviating results for QC sample 

Deviating results for QC sample 

All analytes 

L16 (ICP-MS) 
L17 (ICP-OES) 

Deviating results for more than 
50% of analytes in the QC sample 

Values not used for 
evaluation 

Values not used for 
evaluation 
Value not used for 
evaluation 

The  datasets  accepted  on  technical  grounds  were  tested  for  normality  of  dataset  means 
using  kurtosis/skewness  tests  and  normal  probability  plots,  as  well  as  for  outlying  means 
using the Nalimov test and for outlying standard deviations by using the Cochran test (both at 
a 95 % and 99 % confidence level). Standard deviation  within (swithin) and between (sbetween) 
laboratories  were  calculated  using  one-way  ANOVA.  The  results  of  these  evaluations  are 
shown in Table 9 and Table 10. 

21 

 
 
 
 
 
 
Table  9:  Statistical  evaluation  of  the  technically  accepted  datasets  for  total  content.  p: 
number of technically valid datasets 

Element  p 

Outlying mean 
values** 

Normally 
distributed 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

yes 
yes 
yes 
yes 
yes 
yes 
yes 
yes 
yes 
yes 
yes 
*Data before rounding for uncertainty calculation 
**Nalimov test at 95 % confidence level 

L2 (ICP-OES) 
- 
- 
L5 (ICP-SFMS) 
- 
- 
L6 (k0-NAA) 
- 
L8 (ICP-MS) 
L5 (ICP-SFMS) 
- 

7 
7 
8 
9 
9 
8 
8 
9 
7 
7 
8 

Statistical parameters* 

Average 
[mg/kg] 
7.7 
14.5 
6.5 
168 
348 
32936 
5.9 
352 
91 
157 
979 

s 
[mg/kg] 
0.44 
1.24 
0.17 
7.3 
21.6 
892.1 
0.40 
12.3 
5.5 
9.2 
39.0 

sbetween 
[mg/kg] 
0.21 
1.35 
0.07 
7.8 
24.4 
847.7 
0.54 
10.2 
5.2 
9.3 
37.8 

swithin 
[mg/kg] 
0.95 
0.95 
0.41 
9.2 
14.8 
1288.5 
0.32 
17.0 
4.6 
9.1 
49.6 

Table  10:  Statistical  evaluation  of  the  technically  accepted  datasets  for  aqua  regia 
extractable content. p: number of technically valid datasets 

Element 

p 

Outlying mean 
values** 

Normally 
distributed 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

15 
14 
15 
14 
15 
15 
14 
15 
15 
15 
15 

L3 (ICP-MS) 
L9 (FAAS) 
- 
L15 (ICP-OES) 
L2 (ICP-OES) 
L4 (ICP-SFMS) 
L9 (CV-AAS) 
L1 (ICP-MS) 
L8 (ICP-MS) 
L12 (ICP-OES) 
- 

yes 
yes 
yes 
yes 
yes 
yes 
yes 
yes 
yes 
yes 
yes 

*Data before rounding for uncertainty calculation 
**Nalimov test at 95 % confidence level 

Statistical parameters* 

Average 
[mg/kg] 
7.7 
13.3 
5.9 
150 
346 
32763 
6.1 
340 
86 
156 
970 

s 
[mg/kg] 
0.65 
0.87 
0.40 
6.7 
20.2 
1570.6 
0.37 
13.9 
4.1 
8.0 
43.1 

sbetween 
[mg/kg] 
0.58 
0.87 
0.40 
6.9 
20.0 

swithin 
[mg/k] 
0.73 
0.46 
0.20 
4.7 
7.9 

1533.7  1061.8 

0.41 
13.9 
4.0 
7.9 
44.6 

0.28 
7.1 
2.3 
3.7 
18.4 

In all cases, the laboratory averages follow normal distributions and only few outlying mean 
values  (Nalimov  test)  were  detected  at  95  %  CI.  Some  outlying  lab  variances  were  also 
detected  (data  not  shown).  As  different  laboratories  used  different  methods  and/or 
instrumentations,  the  outlying  variables  reflect  the  different  intrinsic  variability  that  different 
methods  may  have.  The  outlying  values  were  anyhow  within  the  certified  values  when  the 
uncertainty budget ulab provided by each laboratory is taken into account (Annex E). Thus, all 
data  for  measurement  methods  were  considered  technically  acceptable  and  retained.  The 
mean  of  laboratory  means  is  therefore  a  good  estimate  of  the  true  value  and  is  used  as 
certified value.  
The  contribution  of  the  characterisation  to  the  uncertainty  of  the  certified  value  uchar  was 
estimated  as  the  standard  deviation  of  laboratory  means  divided  by  √n,  with  n  number  of 
laboratories. 

22 

 
 
 
 
Individual  results  for  the  characterisation  study  together  with  the  uncertainty  stated  by  the 
laboratories (when provided) are reported in Annex E. 

7. 

Value Assignment 

For this material, certified values have been assigned. Additional material information on the 
material is also provided. 

Certified  values  are  values  that  fulfil  the  highest  standards  of  accuracy.  Procedures  at  the 
JRC-Geel  (Directorate  F  -  Health,  Consumers  and  Reference  Materials)  generally  require 
pooling  of  not  less  than  6  datasets  to  assign  certified  values.  Full  uncertainty  budgets  in 
accordance  with  the  Guide  to  the  expression  of  uncertainty  in  measurement  [4]  must  be 
established.  

Additional  material  information  refers  to  values  obtained  in  the  course  of  the  certification 
study.  For  example,  results  reported  by  only  one  or  two  laboratories  in  cases  where 
individual measurement uncertainty is high, would fall under this category.  

7.1  Certified values and uncertainties  

The  certified  values  reported  in  Table  11  and  12  have  been  calculated  as  the  unweighted 
mean of the means of the accepted datasets.  
uchar was estimated as the standard deviation of laboratory means, i.e. 
taken from Table 9 and Table 10. 
The  assigned  uncertainty  consists  of  uncertainties  related  to  characterisation,  uchar  (see 
section  6),  potential  between-unit  heterogeneity,  ubb  (see  section  4)  and  potential 
degradation during transport (usts) and long-term storage, ults (see section 5). These different 
contributions  were  combined  to  estimate  the  expanded,  relative  uncertainty  of  the  certified 
value (UCRM, rel) with a coverage factor k, as reported below 

 with s and p 

p=s

U

CRM,

rel

(cid:215)=
k

u

2
char,

rel

+

u

2
 bb,

rel

+

u

2
 sts,

rel

+

u

2
lts,

rel

. 

•  uchar was estimated as described in Table 9 and Table 10 
•  ubb was estimated as described in Table 3 
•  usts was estimated as described in Table 7  
•  ults was estimated as described in Table 7 

A coverage factor k of 2 corresponding to a level of confidence of approximately 95 % was 
applied. 

23 

 
 
 
 
 
 
 
 
 
 
 
 
Table 11: Summary of the certified values and uncertainties – total content (mass fraction) 

Analyte 

As 
Cd 
Co 
Cr 
Cu 
Fe [g/kg] 
Hg 
Mn 
Ni 
Pb 
Zn [g/kg] 

Analyte 

As 
Cd 
Co 
Cr 
Cu 
Fe [g/kg] 
Hg 
Mn 
Ni 
Pb 
Zn [g/kg] 

Certified 
value 
[mg/kg] 
7.7 
14.5 
6.5 
168 
348 
32.9 
5.9 
352 
91 
157 
0.98 

UCRM 
[mg/kg] 
k=2 
0.7 
1.4 
0.4 
14 
18 
1.6 
0.6 
14 
7 
9 
0.04 

2.8 
2.9 
1.9 
2.6 
0.6 
1.7 
1.0 
0.7 
2.1 
0.7 
0.7 

1) 

ubb,rel
[%] 

2) 

usts,rel
[%] 

2) 

ults,rel
[%] 

3) 

uchar,rel
[%] 

UCRM,rel 
[%] 

0.6 
0.7 
0.6 
0.9 
0.7 
0.4 
0.7 
0.5 
0.6 
0.6 
0.4 

2.9 
1.9 
2.4 
2.6 
1.2 
1.5 
4.7 
1.4 
2.3 
1.4 
1.2 

2.2 
3.2 
0.9 
1.4 
2.1 
1.0 
2.4 
1.2 
2.3 
2.2 
1.4 

9.2 
9.6 
6.5 
8.1 
5.1 
5.0 
10.8 
4.0 
7.8 
5.6 
4.0 

1) Values from homogeneity study (see Table 3) 
2) Values from short and long term stability study (see Table 7) 
3) Values from characterisation study (see Table 9) 

Table 12: Summary of the certified values and uncertainties – aqua regia extractable content 
(mass fraction) 

Certified 
value 
[mg/kg] 
7.7 
13.3 
5.9 
150 
346 
32.8 
6.1 
340 
86 
156 
0.97 

UCRM 
[mg/kg] 
k=2 
0.7 
0.9 
0.4 
11 
15 
1.5 
0.7 
13 
6 
7 
0.04 

1) 

ubb,rel
[%] 

2) 

usts,rel
[%] 

2) 

ults,rel
[%] 

3) 

uchar,rel
[%] 

UCRM,rel 
[%] 

1.8 
2.0 
0.5 
2.1 
0.4 
0.9 
2.0 
0.7 
1.8 
0.5 
0.3 

0.6 
1.0 
0.5 
1.0 
0.9 
0.7 
0.4 
0.4 
0.4 
0.9 
0.8 

2.9 
1.9 
2.4 
2.6 
1.2 
1.5 
4.7 
1.4 
2.3 
1.4 
1.2 

2.2 
1.8 
1.8 
1.2 
1.5 
1.2 
1.6 
1.1 
1.2 
1.3 
1.1 

8.2 
6.8 
6.1 
7.4 
4.3 
4.5 
10.8 
3.9 
6.4 
4.4 
3.7 

1) Values from homogeneity study (see Table 3) 
2) Values from short and long term stability study (see Table 7) 
3) Values from characterisation study (see Table 10) 

7.2  Additional material information 

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

The composition of ERM-CC144, together with TOC, TIC, water content and water activity is 
given in Table 13. 
The  determination  of  the  major  components  was  performed  mainly  by  k0-NAA,  with  the 
exception of SiO2 and P2O5 determined by gravimetry and spectrophotometry, respectively.  

24 

 
 
 
 
 
 
 
 
 
Table 13: Additional material information for ERM-CC144 

Parameter 
Al 
Ca 
Fe 
K 
Mg 
Na 
P2O5 
SiO2 
Ti 
TOC 
TIC 
Water content 
Water activity 

Value 
19 
31 
34 
2.9 
3.8 
1.8 
38 
73 
1.5 
36 
0.1 
2.5 
0.15 

Unit 
g/kg 
g/kg 
g/kg 
g/kg 
g/kg 
g/kg 
g/kg 
g/kg 
g/kg 
g/100 g 
g/100 g 
g/100 g 
1 

The  total  organic  carbon  (TOC)  and  total  inorganic  carbon  (TIC)  were  determined  in  two 
bottles selected in a randomly stratified way (3 replicates per bottle), according to ISO 10694 
procedure [16] by using a combustion furnace (Ströhleim C-mat 5500, DE).  
In  this  procedure  the  TOC  is  obtained  by  the  difference  between  the  results  of  the 
measurements of TC (total carbon) and TIC (total inorganic carbon). The TC present in the 
samples is converted to carbon dioxide by combustion in an oxygen-containing gas flow free 
of  carbon  dioxide  at  1200  °C,  while  the  TIC  is  determined  separately  from  another  sub-
sample  by  means  of  acidification  with  H3PO4,  purging  of  the  released  carbon  dioxide  and 
subsequent measurement of the released amount of CO2.  
The  water  content  was  determined  in  14  (one  replicate  per  bottle)  randomly  stratified 
selected bottles by volumetric Karl-Fisher titration, while the water activity was determined in 
1 bottle (3 replicates) by using a water activity meter (Aqualab CX3, Decagon, USA).  

8. 

Metrological traceability and commutability 

8.1  Metrological traceability 

8.1.1 

Identity 

The  total  content  of  As,  Cd,  Co,  Cr,  Cu,  Fe,  Hg,  Mn,  Ni,  Pb  and  Zn  in  ERM-CC144  is  a 
chemically  clearly  defined  parameter  and  therefore  independent  of  the  measurement 
method. 

Aqua  regia  extractable  content  of  As,  Cd,  Co,  Cr,  Cu,  Fe,  Hg,  Mn,  Ni,  Pb  and  Zn  in  ERM-
CC144 is a method-defined (i.e. operationally defined) measurand and can only be obtained 
by following the procedure specified in ISO 12914:2012 or ISO 11466:1995.  

8.1.2 

Quantity value 

The participants used different procedures for the sample preparation as well as for the final 
determination,  thus  minimising  the  possibility  of  a  measurement  bias.  Only  validated 
methods were used for the determination of the values assigned to total content of As, Cd, 
Co,  Cr,  Cu,  Fe,  Hg,  Mn,  Ni  and  Zn.  Different  calibrants  of  specified  traceability  of  their 
assigned values were used and all relevant input parameters were calibrated. The individual 
results  are  therefore  traceable  to  the  SI,  which  is  also  confirmed  by  the  agreement  among 

25 

 
 
 
 
 
 
 
the  technically  accepted  datasets.  Moreover  data  for  the  characterisation  study  were  only 
accepted if they agreed within 20% of the certified value of the QC material BCR-144R. As 
the assigned values are combinations of agreeing results individually traceable to the SI, the 
assigned quantity values are also traceable to the SI. 

Traceability of the obtained results for the aqua regia extractable content of As, Cd, Co, Cr, 
Cu,  Fe,  Hg,  Mn,  Ni  and  Zn  is  based  on  the  traceability  of  all  relevant  input  factors. 
Instruments  in  individual  laboratories  were  verified  and  calibrated  with  tools  ensuring 
traceability  to  the  SI.  Consistency  in  the  inter-comparison  study  demonstrates  that  all 
relevant input factors were covered. Moreover data for the characterisation study were only 
used if they were in reasonable agreement (≤120 %) with the certified values for BCR-144R 
or  BCR-146R.  As  the  assigned  values  are  combinations  of  agreeing  results  individually 
traceable to the SI, the assigned quantity values are also traceable to the SI. 

8.1.3   Commutability 
The commutability of a CRM defines its fitness for use and, thus, is a crucial characteristic in 
case of the application of different measurement methods. When commutability of a CRM is 
not  established,  the  results  from  routinely  used  methods  cannot  be  legitimately  compared 
with the certified value to determine if a bias exists.  

ERM-CC144  is  a  typical  sewage  sludge  material.  Regarding  the  total  content,  the 
laboratories  participating  in  the  certification  studies  were  all  selected  such  as  to  provide 
different  method  preparation,  calibration  and  detection.  Aqua  regia  extractable  content  was 
determined by following ISO 12914:2012 or ISO 11466:1995. The agreement between all the 
results obtained, shows that ERM-CC144 exhibit the same behaviour as a typical laboratory 
sample and therefore confirms its commutability. 

9. 

Instructions for use 

9.1  Storage conditions 
The  materials  shall  be  stored  at  18 °C  ±  5 °C  in  the  dark.  Care  shall  be  taken  to  avoid 
change  of  the  moisture  content  once  the  bottle  is  open,  as  the  material  could  pick  up 
moisture  from  the  air.  Elevated  moisture  content  could  favour  the  growth  of  mould  and 
enzyme activity, damaging the material. 

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

9.2  Safety and protection for the environment 
The usual laboratory safety measures apply. 

9.3  Preparation and use of the material 
The bottle should be shaken by turning it upside down for at least 2 min before opening to 
ensure re-homogenisation of the material. 

9.4  Minimum sample intake 
The minimum sample intake representative for all elements is 300 mg for the total content of 
As, Co, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn and 30 mg for Hg.  

26 

 
 
 
 
 
 
 
The minimum sample intake for aqua regia extractable content of all certified elements is 500 
mg  if  using  ISO  12914:2012  or  3  g  if  using  ISO  11466:1995  for  aqua  regia  extractable 
content. 

9.5  Dry mass correction 
Dry mass determination shall be carried out on a separate portion of at least 1 g, by drying in 
an oven at 105 °C ± 2 °C until constant mass (separate weighing should not differ by more 
than 5 mg) is attained. Weighing of the samples for dry mass determination and weighing for 
the analysis shall be done at the same time to avoid differences due to possible take up of 
moisture by the material. 

9.6  Use of the certified value 
The  main  purpose  of  these  materials  is  to  assess  method  performance,  i.e.  for  checking 
accuracy of analytical results. As with any reference material, it can also be used for control 
charts or validation studies. 

Comparing an analytical result with the certified value 

A result is unbiased if the combined standard uncertainty of measurement and certified value 
covers the difference between the certified value and the measurement result (see also ERM 
Application Note 1, www.erm-crm.org [17].  

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

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

value (D m). 

o  Combine  measurement  uncertainty 

(um)  with 

the  uncertainty  of 

the  

certified value (uCRM): 

u

=D

u

2

+
m u

2
CRM

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

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

o 

Use as a calibrant 

It  is  not  recommended  to  use  this  matrix  material  as  calibrant.  Nevertheless,  if  used,  the 
uncertainty  of  the  certified  value  shall  be  taken  into  account  in  the  estimation  of  the 
measurement uncertainty. 

Use in quality control charts 

The materials can be used for quality control charts. Different CRM-units will give the same 
result as heterogeneity was included in the uncertainties of the certified values.  

27 

 
 
 
 
 
10. 

Acknowledgments 

The  authors  would  like  to  acknowledge  the  support  received  from  some  colleagues  at  the 
Joint Research Centre (Belgium): Paul de Vos for his help during the processing of the CRM; 
Maria  Concepcion  Contreras-Lopez  and  Giovani  Kerckhove  for  setting  up  the  required 
isochronous  studies;  Mihaela  Caprioara-Buda,  Jens  Boertz,  Marco  Magini  and  Thomas 
Linsinger  for  reviewing  the  certification  report.  The  authors  would  also  like  to  thank  the 
experts of the Certification Advisory Panel ‘Elements and inorganic ions’ (alphabetic order): 
Steven  Balsley  (International  Atomic  Energy  Agency,  Austria),  Lars  Jorhem  (National  Food 
Administration, Sweden), Thomas Meisel (Unileoben, Austria), Thomas Prohaska (University 
of  Natural  Resources  and  Applied  Life  Sciences,  Austria),  Michel  van  Son  and  Peter 
Vermaecke (Studiecentrum voor Kernenergie, Belgium) for their constructive comments. 

28 

 
 
 
11. 

References 

1 

2 

3 

4 

5 

6 

ISO  Guide  34,  General  requirements  for  the  competence  of  reference  materials  producers, 
International Organization for Standardization, Geneva, Switzerland, 2009 
ISO  Guide  35,  Reference  materials  –  General  and  statistical  principles  for  certification, 
International Organization for Standardization, Geneva, Switzerland, 2006 
ISO 17025:2005, General requirements for the competence of testing and calibration laboratories, 
International Organization for Standardization, Geneva, Switzerland, Last revision 2010/ 
ISO/IEC  Guide  98,  Guide  to  the  Expression  of  Uncertainty  in  Measurement,  International 
Organization for Standardization, Geneva, Switzerland, 2008 
ISO 12914:2012, Soil quality – Microwave-assisted extraction of the aqua regia soluble fraction for 
the  determination  of  elements, 
for  Standardization,  Geneva, 
Switzerland, 2012 
ISO  11466,  Soil  quality  –  Extraction  of  trace  elements  soluble  in  aqua  regia,  International 
Organization for Standardization, Geneva, Switzerland, 1995 

International  Organization 

7  Council  Directive  86/278/EEC  of  12  June  1986  on  the  protection  of  the  environment,  and 

inparticular of the soil, when sewage sludge is used in agriculture 

8  J.  Ščančar,  R.  Milačič,  M.  Horvat  (2000)  Comparison  of  various  digestion  and  extraction 
procedures in analysis of heavy metals in sediments, Water, air and Soil Pollution, 118: 87-99 
9  J. Ščančar, R. Milačič, M. Stražar, O. Burica (2000) Total metal concentration and partitioning of 

Cd, Cr, Cu, Fe, Ni and Zn in sewage sludge, Sci. Tot. Environ., 250, 9-19 

10  K. J. Andersen, M. I. Kisser (2004) Digestion of solid matrices, Desk study –Horizontal, Eurofins 

A/S, Denmark. NÖ Umweltschutzanstalt, Austria 

11  T.P.J.  Linsinger,  J.  Pauwels  ,  A.M.H.  van  der  Veen,  H.  Schimmel,  A.  Lamberty  (2001) 

Homogeneity and stability of reference materials, Accred. Qual. Assur. 6: 20-25 

12  Lamberty,  H.  Schimmel,  J.  Pauwels,  (1998)  The  study  of  the  stability  of  reference  materials  by 

isochronous measurements, Fres. J. Anal. Chem. 360: 359-361 

13  T.P.J Linsinger, J. Pauwels, A. Lamberty, H. Schimmel, A.M.H. van der Veen, L. Siekmann (2001) 

Estimating the Uncertainty of Stability for Matrix CRMs, Fres. J. Anal. Chem. 370: 183-188 
14  Yates D S, Moore DS, McCabe G (2003) The Practice of Statistics. WH Freeman & Co, NY 
15  DIN  38414-17:  1989,  German  standard  methods  for  the  examination  of  water,  waste  water  and 

sludge — Sludge and sediments 

16  ISO  Guide  10694,  Soil  quality  –  Determination  of  organic  and  total  carbon  after  combustion 
(elementary analysis) International Organization for Standardization, Geneva, Switzerland, 1995 
17  T.P.J. Linsinger, ERM Application Note 1: Comparison of a measurement result with the certified 

value, www.erm-crm.org 

29 

 
 
 
 
12. 

Annexes 

Annex A: Methods used in the certification studies 

Table A1: Summary of the methods used for the homogeneity study of the total and aqua regia extractable content 

Parameter  Technique 

Sample 
mass (g) 

Sample preparation 

Calibration 

Instrumentation and 
measurement method 

Total 
content 

Aqua regia 
extractable 
content 

ICP-OES (As, Cd, 
Co, Cr, Cu, Fe, 
Mn, Ni, Pb and 
Zn); 
CV-AFS (Hg) 
ICP-OES (As, Cd, 
Co, Cr, Cu, Fe, 
Mn, Ni, Pb and 
Zn); 
CV-AFS (Hg) 

0.5 

0.5 

Closed microwave 
digestion with HF; HCl 
and HNO3 

2-point external calibration by 
using CPI, Merck or Spex 
Certiprep standards 

Perkin-Elmer Optima 3000 DV 
(ICP-OES)  

ISO 12914:2012 using a 
microwave heating rate 
of approx. 6 ºC/min 

2-point external calibration by 
using CPI. Merck or Spex 
Certiprep standards 

Perkin-Elmer Optima 3000 DV 
(ICP-OES) 
HYDRA AF Automated 
Mercury Analyser, Leeman 
Labs, Inc. (CV-AFS) 

30 

 
 
 
 
 
Table A2: Summary of the methods used for the short term stability study  

Parameter 

Technique 

Sample 
mass (g) 

Sample preparation 

Calibration 

Total 
content 

Aqua regia 
extractable 
content 

ICP-OES (As); 
CV-AAS (Hg) 

FAAS (As, Cd, 
Co, Cr, Cu, Fe, 
Mn, Ni, Pb and 
Zn) 
GF-AAS (Hg) 

ICP-OES (As); 
CV-AAS (Hg) 

FAAS (As, Cd, 
Co, Cr, Cu, Fe, 
Mn, Ni, Pb and 
Zn) 
GF-AAS (Hg) 

0.3 

0.3 

3 

3  

Closed microwave 
assisted digestion using 
6mL HCl+2mL 
HNO3+2mL HF 

Method EPA3052: HNO3 
and HF chemical 
dissolution 

According to ISO 
11466:1995 

According to ISO 
11466:1995 

Linear calibration using J.T.Baker 
standards solutions; 5-point 
external calibration curve 

Linear (As, Cd, Co) or quadratic 
(Cr, Cu, Fe, Hg, Mn, Ni, Pb and 
Zn) calibration using Merck or 
UltraScientific calibrants; 4-point 
calibration curve 

Linear calibration using J.T.Baker 
standards solutions; 7-point 
external calibration curve 

Linear (As, Cd, Co) or quadratic 
(Cr, Cu, Fe, Hg, Mn, Ni, Pb and 
Zn) calibration using Merck or 
UltraScientific calibrants; 4-point 
calibration curve 

Instrumentation and 
measurement method 
Perkin Elmer Optima 7300DV 
(ICP-OES) 
Perkin Elmer FIMS 400 (CV-
AAS) 

Varian AA240FS(AAS) and 
Varian AA240Z (GFAAS) 

Perkin Elmer Optima 7300DV 
(ICP-OES) 
Perkin Elmer FIMS 400 (CV-
AAS) 

Varian AA240FS(AAS) and 
Varian AA240Z (GFAAS) 

31 

 
 
Table A3: Summary of the methods used for the long term stability study (only aqua regia) 

Parameter 

Analytical 
technique 

Sample 
mass (g) 

Sample preparation 

Calibration 

Instrumentation and 
measurement method 

Aqua regia 
extractable 
content 

ICP-MS (As, Cr, 
Cd, Co, Hg, Mn, 
Ni, Pb, Zn) 

ICP-OES (Cu, Fe) 

3 

According to ISO 
11466:1995 

Linear (As, Cr, Cd, Co, Hg, Mn, 
Ni, Pb, Zn) calibration using 
Merck solutions; 

Linear (Cu, Fe) calibration using 
BDH solutions 

Thermo Iris Intrepid II XDL (ICP-
MS) 

Perkin Elmer Optima 3000 (ICP-
OES) 

Table A4: Summary of the methods used for the characterisation study of total element content 

Lab code 

Analytical 
technique 

Sample 
mass (g) 

Sample preparation 

Calibration 

L1 

L2 

L3 

ICP-MS (As, Cd, 
Co, Cr, Cu, Hg, Mn, 
Ni, Pb, Zn) 

ICP-OES (Fe) 

ICP-OES (As, Cd, 
Co, Cr, Cu, Fe, Mn, 
Ni, Pb, Zn) 

CV-AAS (Hg) 

ICP-MS (As, Cd, 
Co, Cr, Cu, Fe, Mn, 
Ni, Pb, Zn) 

CV-AAS (Hg) 

1.0 

0.3 

0.3 

Microwave digestion 
using 5 mL HNO3, 2 mL 
HCl, 3 mL HF 

Microwave digestion 
using 2 mL HNO3 + 6 mL 
HCl + 2 mL HF and 22 
mL 4% H3BO3 made up 
to 50 mL 

Microwave digestion 
using 2 mL HNO3 + 6 mL 
HCl + 2 mL HF and 22 
mL 4% H3BO3 made up 
to 50 mL 

32 

VWR, 3-point calibration 

Baker Instra Analyzed ICP 
Standard, 8-point external 
calibration 

Baker Instra Analyzed ICP 
Standard, 8-point external 
calibration 

Instrumentation and 
measurement method 

ICP-MS: Perkin Elmer Elan 6000 
DRC II 

ICP-OES: Thermo electron 
Atomscan 16 

ICP-OES: Perkin Elmer Optima 
3000 DV 

CV-AAS: Perkin Elmer FIMS 
400 

ICP-MS: Perkin Elmer Elan 
DRCII 

CV-AAS: Perkin Elmer FIMS 
400 

 
 
 
 
L5 

L6 

Lab code 

Analytical 
technique 

Sample 
mass (g) 

Sample preparation 

Calibration 

Instrumentation and 
measurement method 

L4 

k0-INAA 

ICP-SFMS (As, Cd, 
Co, Cr, Cu, Fe, Hg, 
Mn, Ni, Pb and Zn) 

k0-NAA (As, Cd, 
Co, Cr, Cu, Hg, Fe, 
Mn, Zn) 

0.4 

0.3 

- 

IRMM-530R 

k0-Neutron Activation Analyses 

Microwave digestion 
using 5 mL HNO3 + 3 mL 
HF mixture and diluted 
with 5% HNO3 prior to 
analysis. 

Ultra Scientific(US) ICP 
standard, 2-point external 
calibration after internal 
correction. 

Thermo Electron Corp, Cetac 

0.1 

N/A 

Validated k0-NAA by BCR 
certified reference materials 
(different matrices) 

HPGe coaxial detector (45 %) 
Flux 1.1E12 neutrons/(cm2 s) 

Merck ICP Multi Element 
Standard Solution XVI 
CertiPUR, 4-point external 
calibration 

Internal  stock  solution,  3-point 
external calibration 

FAAS: Varian Spectra AA 110 
(Mulgrave, Victoria, Australia); 

ICP-MS: Agilent 7500 ce 
Yokogawa Analytical Systems 
Inc. (Tokio, Japan) 

CV-AAS: Automatic Mercury 
Analyzer Model Hg-201, Sanso 
Seisakusho Co. LTD 

Internal stock solution, 8-point 
external calibration 

Direct Mercury Analyser (DMA-
80) Milestone S.r.l. 

FAAS (Cd, Cr, Cu, 
Fe, Mn, Ni, Pb, Zn) 

ICP-MS (As, Co) 

CV-AAS (Hg) 

0.3 

Microwave digestion 
using 4 mL HNO3 + 1 mL 
HCl + 0.2 mL HF + 5 mL 
H2O2 

DMA (Hg) 

0.03 

Sample dried and then 
thermally and chemically 
decomposed in a furnace 
at 650 °C. Absorbance 
measured at 254 nm as 
a function of mercury 
concentration. 

33 

 
 
Lab code 

Analytical 
technique 

Sample 
mass (g) 

Sample preparation 

Calibration 

Instrumentation and 
measurement method 

ICP-OES (As, Cd, 
Co, Cr, Cu, Fe, Mn, 
Ni, Pb, Zn) 

CV-AFS (Hg) 

0.25 

ICP-MS (As, Cd, 
Co) 

ICP-OES (Cr, Cu, 
Fe, Mn, Ni, Pb, Zn) 

0.3 

CV-AAS (Hg) 

Bomb digestion: 9.5g 
HNO3 + 0.5g HClO4, 
dilution to 50g 0.1 HNO3 
+ 0.5mL HF. 

Hg: the solution was 
diluted and subjected to 
a tin(II)chloride reduction 

Inorganic Ventures single 
element standards, 2-point 
external calibration 

ICP-OES: Spectroflame 
polychromator  

Microwave digestion 
using 15mL HNO3 
(suprapur 65%) + 1ml HF 
(suprapur) 

1000mg/L Merck solutions; 

5-point external calibration 

ICP-MS: Perkin Elmer DRC-e 

ICP-OES: Perkin Elmer Optima 
3000 

CV-AAS: Perkin Elmer AA200 

L7 

L8 

L9 

ICP-OES (Cr, Cu, 
Mn, Zn) 

L10 

ICP-MS (Hg) 

ICP-OES (Pb) 

0.1 

0.3 

Microwave digestion 
using a mixture of 
20%v/v HNO3 and 5% 
v/v HF 

1000mg/L Merck solutions; 

7-point external calibration 

IRIS Intrepid II XDL 

Digestion according to 
EPA 3052: 15 mL 
HNO3+1 mL H2O2+ 2 mL 
HF 

Spectrascan (ICP-MS) or 
Custom Grade (ICP-OES) 
standard supplies; 3-point 
external calibration 

ICP-MS: PerkiElmer ELAN 
DRCe 

ICP-OES: Optima 4300 DV 
Perkin-Elmer 

34 

 
 
 
Table A5: Summary of the methods used for the characterisation study of aqua regia extractable content 

Lab code  Method acronym 

Sample 
mass (g) 

Sample preparation 

Calibrants 

L1 

L2 

L3 

L4 

L5 

ICP-MS: Cd, Hg 

ICP-OES: As, Co, 
Cr, Cu, Fe, Mn, Ni, 
Pb, Zn 

ICP-OES (As, Cd, 
Co, Cr, Cu, Fe, Mn, 
Ni, Pb, Zn) 

CV-AAS (Hg) 

ICP-MS (As, Cd, 
Co, Cr, Cu, Fe, Mn, 
Ni, Pb, Zn) 

CV-AAS (Hg) 

ICP-SFMS (As, Cd, 
Co, Cr, Cu, Fe, Hg, 
Mn, Ni, Pb, Zn) 

ICP-OES (As, Cd, 
Co, Cr, Cu, Fe, Mn, 
Ni, Pb, Zn) 

CV-AFS (Hg) 

3  

3  

3  

3 

According to  

ISO 11466:1995 

VWR, 2-point calibration 

According to  

ISO 11466:1995 

Baker Instra Analyzed ICP 
Standard, 8-point external 
calibration 

According to ISO 
11466:1995 

Baker Instra Analyzed ICP 
Standard, 8-point external 
calibration 

Instrumentation and 
measurement method 

ICP-MS: Perkin Elmer Elan 
6000 DRC II 

ICP-OES: Thermo electron 
Atomscan 16 

ICP-OES: Perkin Elmer 
Optima 3000 DV 

CV-AAS: Perkin Elmer FIMS 
400 

ICP-MS: Perkin Elmer Elan 
DRCII 

CV-AAS: Perkin Elmer FIMS 
400 

Thermo Electron Corp. Cetac 

Ultra Scientific(US) ICP 
standard, 2-point external 
calibration after internal 
correction. 

Inorganic Ventures, single 
element standards. 2-point 
external calibration 

ICP-OES: Spectroflame 
polychromator 

According to  

ISO 12466:2012 

According to  

3  

ISO 11466:1995 

35 

 
 
 
 
Lab code  Method acronym 

Sample 
mass (g) 

Sample preparation 

Calibrants 

Instrumentation and 
measurement method 

ICP-MS: Perkin Elmer DRC-e 

ICP-OES: Perkin Elmer 
Optima 3000 

CV-AAS: Perkin Elmer AA200 

According to  

1000 mg/L Merck solutions; 

ISO 11466:1995 

5-point external calibration 

According to  

ISO 11466:1995 

High-purity standard solution, 5-
point external calibration curve 

ICP-MS Agilent 7500ce 

ICP-OES Perkin Elmer Optima 
3200RL 

According to  

ISO 11466:1995 

CPI or Merck high-purity 
standard solutions, 2-point 
external calibration curve 

ICP-OES: Perkin Elmer 
Optima 3000 DV 

3  

3 

3 

L6 

L7 

L8 

L9 

ICP-MS (As, Cd, 
Co) 

ICP-OES (Cr, Cu, 
Fe, Mn, Ni, Pb, Zn) 

CV-AAS (Hg) 

ICP-MS (As, Cd, 
Co, Hg, Ni, Pb) 

ICP-OES (Cr, Cu, 
Fe, Mn, Zn) 

ICP-OES (As, Cd, 
Co, Cr, Cu, Fe, Mn, 
Ni, Pb, Zn) 

CV-AFS (Hg) 

k0-NAA (As, Cd, 
Co, Cr, Cu, Hg, Fe, 
Mn, Zn) 

FAAS (Cd, Cr, Cu, 
Fe, Mn, Ni, Pb, Zn) 

CV-AAS (Hg) 

0.5 

According to  

ISO 11466:1995 

Validated k0-NAA by BCR 
certified reference materials  

Certipur or Merck standard 
solutions, minimum 3-point 
external calibration curve 

L10 

ICP-SFMS (all) 

0.5 

According to  

ISO 12914:2012 

UltraScientific standard 
solutions, 4-point external 
calibration curve 

L11 

ICP-OES (As, Cd, 
Co, Cr, Cu, Fe, Mn, 
Ni, Pb, Zn) 

AAS (Hg) 

0.5 

According to  

ISO 12914:2012 

Merck standard solutions, 8-
point external calibration curve 

36 

k0-NAA: HPGe coaxial 
detector (45 %), flux 1.1E12 
neutrons/(cm2 s) 

FAAS: Varian Spectra AA 110  

CV-AAS: AMA Model Hg-201, 
Sanso Seisakusho Co., LTD 

Thermo Scientific Element XR  
ICP-SFMS 

ICP-OES: Perkin Elmer, ICP-
OES OPTIMA 3000 XL 

AAS: AMA 254, Altec 

 
 
 
Lab code  Method acronym 

Sample 
mass (g) 

Sample preparation 

Calibrants 

Instrumentation and 
measurement method 

L12 

L13 

L14 

L15 

ICP-MS (As, Cd, 
Co, Pb) 

ICP-OES (Cr, Cu, 
Fe, Mn, Ni,  Zn) 

AFS (Hg) 

ICP-MS (As, Cd, 
Co, Hg, Pb, Ni) 

ICP-OES (Cr, Cu, 
Fe, Mn, Zn) 

ICP-MS (As, Cd, 
Co, Cr, Cu, Fe, Hg, 
Pb, Mn, Ni, Zn) 

ICP-OES (As, Cd, 
Co, Cr, Cu, Fe, Pb, 
Mn, Ni, Zn) 

CV-AFS (Hg) 

ICP-OES (As, Cd, 
Co, Cr, Cu, Fe, Pb, 
Mn, Ni, Zn) 

CV-AFS (Hg) 

1 

1 

0.3 

1 

According to  

ISO 12914:2012 

Analytika standard solutions, 3-
point external calibration curve 

ICP-MS: Agilent 7700 series 

ICP-OES: Varian 730-ES 

AFS: Millenium Merlin 

According to  

ISO 12914:2012 

Inorganic Ventures standards, 5-
point external calibration curve 

ICP-MS: Agilent 7500ce   

ICP-OES: Perkin Elmer 
Optima 3200RL 

According to  

ISO 12914:2012 

Inorganic Ventures standards, 
minimum 3-point external 
calibration curve 

According to  

ISO 12914:2012 

CPI or Merck standard solutions, 
minimum 2-point external 
calibration curve 

ICP-MS: Agilent 7700x ICP-
MS 

ICP-OES: Thermo iCAP 6300 

CV-AFS: Millennium System, 
PSA Analytical 

ICP-OES: IRIS Advantage 
Thermo Elemental 

CV-AFS: Hydra II AF Gold – 
Automated Mercury Analyzer 

37 

 
 
Annex B: Homogeneity study 

Table B1: Homogeneity data for As. Mass fraction in mg/kg on dry mass basis 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
7.7 
7.8 
7.3 
7.2 
7.3 
7.7 
7.7 
7.8 
7.7 
7.4 
7.9 
7.5 
7.6 
7.9 

Total content 
Replicate 2 
[mg/kg] 
7.9 
7.6 
7.0 
7.2 
7.6 
7.0 
7.8 
7.6 
8.0 
8.3 
8.1 
7.5 
7.7 
8.1 

Replicate 3 
[mg/kg] 
7.8 
8.3 
7.2 
7.1 
7.2 
7.8 
7.3 
7.1 
7.8 
7.6 
7.9 
7.5 
7.2 
7.7 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
6.9 
7.1 
7.0 
7.2 
7.2 
7.5 
6.8 
7.5 
7.1 
7.7 
7.6 
7.0 
7.1 
7.3 

Replicate 1 
[mg/kg] 
7.7 
7.3 
7.2 
7.2 
7.1 
7.1 
6.7 
7.4 
7.1 
7.6 
7.6 
7.4 
7.2 
7.4 

Replicate 3 
[mg/kg] 
7.4 
7.4 
7.1 
7.1 
7.4 
7.3 
7.1 
7.5 
7.2 
7.3 
7.3 
7.7 
7.5 
7.3 

Table B2: Homogeneity data for Cd. Mass fraction in mg/kg on dry mass basis 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
12.8 
14.1 
13.6 
13.1 
12.7 
12.6 
13.1 
13.3 
13.4 
13.1 
13.0 
13.4 
13.9 
12.7 

Replicate 1 
[mg/kg] 
12.7 
14.0 
13.5 
12.9 
13.4 
13.4 
13.5 
14.3 
13.2 
13.4 
14.2 
13.4 
13.4 
12.7 

Replicate 3 
[mg/kg] 
13.3 
13.7 
14.2 
12.6 
14.4 
14.0 
13.6 
13.5 
13.8 
13.5 
13.6 
13.3 
13.7 
12.9 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
14.5 
15.0 
13.9 
14.5 
13.3 
12.5 
13.8 
14.1 
13.3 
13.1 
13.7 
13.5 
13.9 
13.4 

Total content 
Replicate 2 
[mg/kg] 
14.1 
13.5 
14.2 
13.9 
12.3 
13.3 
13.9 
13.2 
13.7 
13.2 
13.3 
12.5 
14.2 
12.5 

Replicate 3 
[mg/kg] 
13.8 
13.5 
14.3 
13.5 
13.4 
13.1 
13.1 
14.0 
13.4 
13.4 
13.5 
13.7 
12.7 
12.4 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

38 

 
 
 
 
 
 
Table B3: Homogeneity data for Co. Mass fraction in mg/kg on dry mass basis 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
6.1 
6.0 
5.8 
5.6 
5.8 
6.0 
5.6 
5.9 
5.7 
6.0 
5.9 
5.8 
5.9 
5.5 

Total content 
Replicate 2 
[mg/kg] 
6.0 
5.6 
6.0 
5.9 
5.5 
6.3 
5.9 
5.8 
5.8 
6.1 
6.1 
5.7 
5.9 
5.6 

Replicate 3 
[mg/kg] 
6.1 
5.8 
5.8 
5.5 
5.7 
5.9 
5.7 
5.7 
6.4 
6.1 
6.0 
5.6 
6.1 
6.0 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
6.4 
6.3 
6.2 
6.0 
6.4 
6.2 
6.6 
6.1 
6.6 
6.3 
6.1 
6.1 
6.2 
6.0 

Replicate 1 
[mg/kg] 
6.1 
6.5 
6.0 
6.4 
6.1 
6.3 
6.5 
6.5 
6.2 
6.6 
6.6 
6.5 
6.6 
6.2 

Replicate 3 
[mg/kg] 
6.2 
5.9 
6.5 
6.5 
6.5 
6.4 
6.1 
6.2 
6.4 
6.1 
6.5 
6.1 
6.1 
6.4 

Table B4: Homogeneity data for Cr. Mass fraction in mg/kg on dry mass basis 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
154 
167 
162 
155 
152 
151 
157 
157 
159 
157 
154 
158 
163 
151 

Replicate 1 
[mg/kg] 
151 
164 
159 
153 
156 
161 
161 
167 
157 
158 
166 
159 
158 
151 

Replicate 3 
[mg/kg] 
158 
160 
167 
150 
167 
164 
160 
161 
163 
160 
161 
158 
165 
155 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
168 
176 
164 
170 
157 
147 
163 
168 
156 
157 
161 
163 
166 
157 

Total content 
Replicate 2 
[mg/kg] 
166 
157 
164 
163 
146 
158 
164 
156 
161 
156 
156 
149 
165 
149 

Replicate 3 
[mg/kg] 
163 
158 
168 
160 
159 
155 
155 
163 
163 
158 
163 
162 
152 
149 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

39 

 
 
 
 
 
Table B5: Homogeneity data for Cu. Mass fraction in mg/kg on dry mass basis 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
342 
344 
337 
347 
338 
350 
340 
348 
338 
339 
345 
333 
348 
344 

Total content 
Replicate 2 
[mg/kg] 
337 
349 
340 
338 
354 
346 
352 
338 
352 
350 
346 
339 
336 
342 

Replicate 3 
[mg/kg] 
349 
339 
341 
351 
349 
341 
344 
345 
333 
342 
329 
346 
343 
341 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
343 
345 
340 
327 
342 
338 
340 
333 
334 
337 
335 
332 
328 
328 

Replicate 1 
[mg/kg] 
335 
333 
331 
333 
327 
343 
338 
339 
340 
339 
335 
340 
341 
338 

Replicate 3 
[mg/kg] 
340 
328 
333 
335 
331 
334 
333 
339 
340 
334 
337 
338 
336 
336 

Table B6: Homogeneity data for Fe. Mass fraction in mg/kg on dry mass basis 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
36405 
34885 
34696 
34650 
36200 
35803 
35475 
34736 
34449 
35616 
34990 
34899 
33758 
35169 

Replicate 1 
[mg/kg] 
35676 
34175 
34131 
35047 
34180 
35424 
35126 
34416 
35517 
35685 
33884 
35862 
35596 
35578 

Replicate 3 
[mg/kg] 
35949 
33834 
34210 
35191 
33864 
34686 
34344 
34824 
35336 
34354 
34963 
35577 
34960 
35870 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
33747 
34111 
34110 
34101 
34514 
36214 
34000 
35307 
35038 
36569 
35351 
35024 
35752 
34480 

Total content 
Replicate 2 
[mg/kg] 
35746 
34355 
33142 
34177 
36000 
35487 
35756 
35503 
35168 
35649 
35113 
36270 
34141 
36315 

Replicate 3 
[mg/kg] 
35223 
34331 
34498 
35641 
36007 
36402 
35585 
33631 
35756 
34996 
35819 
35704 
35318 
36495 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

40 

 
 
 
 
 
 
Table B7: Homogeneity data for Hg. Mass fraction in mg/kg on dry mass basis 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
6.1 
6.0 
5.7 
5.9 
5.8 
5.6 
6.1 
6.0 
5.8 
5.7 
5.7 
6.1 
5.9 
5.8 

Total content 
Replicate 2 
[mg/kg] 
5.8 
5.6 
5.9 
5.7 
5.7 
5.9 
5.9 
5.5 
5.8 
5.7 
5.7 
5.5 
6.2 
5.2 

Replicate 3 
[mg/kg] 
5.9 
5.7 
6.1 
5.7 
5.8 
5.7 
5.5 
6.1 
5.8 
5.7 
5.8 
5.8 
5.6 
5.5 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
5.3 
6.1 
5.7 
5.5 
5.2 
5.3 
5.4 
5.6 
5.3 
5.2 
5.7 
5.3 
6.0 
5.2 

Replicate 1 
[mg/kg] 
5.6 
5.5 
5.4 
5.3 
5.4 
5.5 
5.4 
5.9 
5.6 
5.3 
6.1 
5.5 
5.6 
5.7 

Replicate 3 
[mg/kg] 
5.5 
5.6 
5.9 
5.4 
5.7 
5.5 
5.3 
5.5 
5.6 
5.5 
5.5 
5.5 
5.4 
5.2 

Table B8: Homogeneity data for Mn. Mass fraction in mg/kg on dry mass basis 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
372 
357 
352 
349 
364 
361 
360 
352 
350 
353 
355 
349 
348 
358 

Replicate 1 
[mg/kg] 
357 
352 
349 
355 
348 
361 
358 
355 
360 
360 
353 
363 
365 
365 

Replicate 3 
[mg/kg] 
360 
343 
349 
361 
348 
355 
348 
355 
359 
348 
354 
358 
355 
362 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
352 
353 
345 
359 
355 
377 
352 
366 
353 
355 
363 
351 
367 
363 

Total content 
Replicate 2 
[mg/kg] 
361 
367 
349 
350 
381 
365 
370 
360 
368 
379 
366 
362 
352 
368 

Replicate 3 
[mg/kg] 
365 
353 
354 
368 
372 
364 
364 
360 
353 
364 
353 
363 
366 
369 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

41 

 
 
 
 
 
Table B9: Homogeneity data for Ni. Mass fraction in mg/kg on dry mass basis. *Value was 
considered an outlier due to contamination during sample preparation and excluded. 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
94 
95 
90 
92 
87 
85 
89 
92 
87 
87 
89 
89 
91 
89 

Total content 
Replicate 2 
[mg/kg] 
91 
89 
92 
89 
84 
88 
92 
92 
93 
91 
90 
84 
94 
86 

Replicate 3 
[mg/kg] 
92 
89 
91 
88 
87 
86 
* 
94 
107 
99 
89 
89 
87 
83 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
82 
89 
88 
82 
86 
82 
86 
83 
86 
85 
82 
84 
86 
80 

Replicate 1 
[mg/kg] 
81 
86 
84 
82 
82 
85 
85 
89 
84 
84 
87 
84 
85 
81 

Replicate 3 
[mg/kg] 
86 
84 
88 
81 
87 
86 
86 
87 
87 
86 
85 
86 
87 
83 

Table B10: Homogeneity data for Pb. Mass fraction in mg/kg on dry mass basis 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
148 
154 
153 
151 
150 
150 
153 
151 
150 
148 
147 
153 
151 
151 

Replicate 1 
[mg/kg] 
148 
153 
148 
148 
149 
152 
147 
152 
155 
152 
152 
150 
153 
150 

Replicate 3 
[mg/kg] 
152 
147 
150 
146 
152 
154 
151 
150 
150 
149 
149 
154 
154 
146 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
154 
158 
155 
158 
154 
152 
152 
159 
155 
151 
157 
151 
158 
152 

Total content 
Replicate 2 
[mg/kg] 
157 
152 
151 
157 
152 
158 
159 
153 
160 
152 
153 
153 
155 
155 

Replicate 3 
[mg/kg] 
155 
155 
156 
158 
159 
160 
155 
152 
152 
157 
151 
163 
157 
151 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

42 

 
 
 
 
 
Table B11: Homogeneity data for Zn. Mass fraction in mg/kg on dry mass basis 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Replicate 1 
[mg/kg] 
981 
981 
953 
977 
966 
1001 
948 
975 
967 
948 
974 
944 
993 
995 

Total content 
Replicate 2 
[mg/kg] 
968 
991 
957 
952 
1020 
989 
998 
984 
995 
1013 
986 
976 
970 
991 

Replicate 3 
[mg/kg] 
1018 
962 
965 
988 
994 
974 
997 
990 
958 
991 
941 
992 
991 
948 

Bottle 
No 

72 
260 
289 
609 
885 
1033 
1274 
1396 
1730 
1897 
2093 
2321 
2452 
2659 

Aqua regia extractable content 
Replicate 2 
[mg/kg] 
994 
987 
969 
956 
973 
972 
966 
966 
945 
963 
962 
958 
956 
962 

Replicate 1 
[mg/kg] 
963 
963 
955 
952 
956 
989 
977 
979 
982 
978 
974 
989 
992 
986 

Replicate 3 
[mg/kg] 
979 
950 
964 
953 
964 
974 
963 
968 
981 
955 
972 
965 
978 
976 

Table B12: Relative within-unit standard deviation (swb) determined by statistically evaluating 
the  characterisation  data  (Annex  E).  The  technique  used  is  reported  below  each  sample 
intake. 

Element 

30 

DMA 

As 
Cd 
Co 
Cr 
Cu 
Fe 
Hg 
Mn 
Ni 
Pb 
Zn 

- 
- 
- 
- 
- 
- 
2.0 
- 
- 
- 
- 

Minimum sample intake [mg] 

100 
k0-
NAA 
2.3 
2.7 
- 
1.3 
9.4 
1.6 
2.5 
1.1 
- 
- 
0.7 

100 
ICP 
OES 
- 
- 
- 
5.0 
2.0 
- 
- 
3.4 
- 
- 
9.8 

250 
ICP 
OES 
- 
6.6 
3.4 
5.4 
1.6 
4.6 
3.5 
2.2 
4.0 
1.6 
1.9 

300 
ICP 
OES 
- 
4.4 
3.3 
5.1 
2.1 
2.8 
3.6 
3.3 
5.9 
6.5 
1.1 

300 
ICP 
MS 
5.9 
7.0 
3.1 
2.8 
1.1 
5.4 
6.5 
1.5 
5.3 
1.9 
2.1 

300 
ICP 
MS 
1.2 
2.0 
0.7 
3.7 
0.9 
1.2 
4.8 
4.0 
3.1 
1.7 
1.9 

300 
ICP 
MS 
3.6 
1.9 
3.6 
3.1 
2.2 
1.1 
1.9 
2.9 
1.5 
- 
0.5 

300 
ICP 
MS 
7.6 
- 
11.7 
11.5 
8.2 
5.1 
4.7 
7.8 
7.4 
- 
7.5 

400 

AFS 

- 
- 
- 
- 
- 
- 
3.1 
- 
- 
5.8 
- 

400 
k0-
NAA 
1.2 
3.3 
1.5 
1.3 
0.9 
1.3 
3.1 
2.5 
- 
- 
1.2 

1000 
ICP 
MS 
- 
9.0 
7.0 
5.9 
4.1 
1.8 
- 
7.2 
4.6 
12.6 
- 

43 

 
 
 
 
 
Annex C: Short term stability study 

TOTAL CONTENT 

Error bars are single standard deviations of 6 replicates per time point. 

18 °C

60 °C

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

18.0

16.0

14.0

12.0

10.0

8.0

As

8,0

7,5

7,0

6,5

6,0

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C1: Short term stability graph for total content of As at 18 °C and 60 °C. 

18 °C

60 °C

]
g
k
/

g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

15.0
14.0
13.0
12.0
11.0
10.0
9.0
8.0

Cd

13.0

12.5
12.0

11.5
11.0

10.5

10.0

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

44 

As

Cd

 
 
 
 
     
 
 
 
 
 
      
 
 
 
Figure C2: Short term stability graph for total content of Cd at 18 °C and 60 °C. 

18 °C

60 °C

7.0

6.5

6.0

5.5

5.0

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

Co

7.0

6.5

6.0

5.5

5.0

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Time [w eek]

Figure C3: Short term stability graph for total content of Co at 18 °C and 60 °C. 

18 °C

60 °C

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

150

140

130

120

110

Cr

150

140

130

120

110

100

/

]
g
k
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Co

Cr

Figure C4: Short term stability graph for total content of Cr at 18 °C and 60 °C. 

45 

 
 
     
 
 
 
 
 
 
     
 
 
 
 
18 °C

60 °C

350

330

310

290

270

250

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

Cu

350

340

330

320

310

300

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C5: Short term stability graph for total content of Cu at 18 °C and 60 °C. 

18 °C

60 °C

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

3.4E+04

3.2E+04

3.0E+04

2.8E+04

2.6E+04

Fe

3.1E+04

3.0E+04
2.9E+04

2.8E+04
2.7E+04

2.6E+04
2.5E+04

/

]
g
k
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C6: Short term stability graph for total content of Fe at 18 °C and 60 °C. 

46 

Cu

Fe

 
 
      
 
 
 
 
 
 
 
     
 
 
 
 
18 °C

60 °C

9.0

8.0

7.0

6.0

5.0

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

Hg

6,5

6,0

5,5

5,0

]
g
k
/

g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C7: Short term stability graph for total content of Hg at 18 °C and 60 °C. 

18 °C

60 °C

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

400

380

360

340

320

300

Mn

350

340

330

320

310

300

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C8: Short term stability graph for total content of Mn at 18 °C and 60 °C. 

47 

Hg

Mn

 
 
     
 
 
 
 
 
 
 
      
 
 
 
 
18 °C

60 °C

110

105

100

95

90

85

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

Ni

105

100

95

90

85

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f

s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C9: Short term stability graph for total content of Ni at 18 °C and 60 °C. 

/

]
g
k
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

18 °C

60 °C

160
155
150
145
140
135
130
125
120

Pb

160

150

140

130

120

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C10: Short term stability graph for total content of Pb at 18 °C and 60 °C. 

48 

Ni

Pb

 
 
 
      
 
 
 
 
 
 
 
     
 
 
 
1050

1000

950

900

850

800

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

18 °C

60 °C

Zn

1000

950

900

850

800

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

Zn

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C11: Short term stability graph for total content of Zn at 18 °C and 60 °C. 

49 

 
 
 
     
 
 
 
AQUA REGIA EXTRACTABLE CONTENT 

Error bars are single standard deviations of 6 replicates per time point. 

18 °C

60 °C

/

]
g
k
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

11.0

9.0

7.0

5.0

3.0

As

8,5

8,0

7,5

7,0

6,5

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Time [w eek]

Tim e [w eek]

Figure C12: Short term stability graph for aqua regia extractable content of As at 18 °C and 60 °C. 

18 °C

60 °C

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

15.0

14.5
14.0

13.5
13.0

12.5
12.0

Cd

15.0
14.5
14.0
13.5
13.0
12.5
12.0

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C13: Short term stability graph for aqua regia extractable content of Cd at 18 °C and 60 °C. 

50 

As

Cd

 
 
 
 
 
     
 
 
 
 
     
 
 
18 °C

60 °C

7.0

6.5

6.0

5.5

5.0

4.5

]
g
k
/

g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

Co

7.0

6.5
6.0
5.5

5.0
4.5

4.0

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Time [w eek]

Figure C14: Short term stability graph for aqua regia extractable content of Co at 18 °C and 60 °C. 

18 °C

60 °C

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

130

125

120

115

110

105

100

Cr

130

125

120

115

110

105

100

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C15: Short term stability graph for aqua regia extractable content of Cr at 18 °C and 60 °C. 

Co

Cr

51 

 
 
     
 
 
 
 
 
     
 
 
 
18 °C

60 °C

345

335

325

315

305

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

Cu

350

340

330

320

310

300

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C16: Short term stability graph for aqua regia extractable content of Cu at 18 °C and 60 °C. 

18 °C

60 °C

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

4.1E+04

4.0E+04

3.9E+04

3.8E+04

3.7E+04

3.6E+04

3.5E+04

Fe

4.1E+04

4.0E+04
3.9E+04

3.8E+04
3.7E+04

3.6E+04
3.5E+04

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w e ek]

Figure C17: Short term stability graph for aqua regia extractable content of Fe at 18 °C and 60 °C. 

Cu

Fe

52 

 
 
     
 
 
 
 
 
 
     
 
 
 
18 °C

60 °C

9.0

8.0

7.0

6.0

5.0

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

Hg

5.5

5.3

5.1

4.9

4.7

4.5

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C18: Short term stability graph for aqua regia extractable content of Hg at 18 °C and 60 °C. 

18 °C

60 °C

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

350

340

330

320

310

300

Mn

350

340

330

320

310

300

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Time [w eek]

Hg

Mn

Figure C19: Short term stability graph for aqua regia extractable content of Mn at 18 °C and 60 °C. 

53 

 
 
     
 
 
 
 
 
     
 
 
 
 
18 °C

60 °C

105

100

95

90

85

80

75

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

Ni

105

100

95

90

85

80

75

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f

s
s
a
M

0

1

2

3

4

0

1

2

3

4

Tim e [w eek]

Tim e [w eek]

Figure C20: Short term stability graph for aqua regia extractable content of Ni at 18 °C and 60 °C. 

18 °C

60 °C

]
g
k
/
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

170
165
160
155
150
145
140
135
130

Pb

160

155

150

145

140

135

130

/

]
g
k
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Time [w eek]

Tim e [w eek]

Figure C21: Short term stability graph for aqua regia extractable content of Pb at 18 °C and 60 °C. 

54 

Ni

Pb

 
 
 
     
 
 
 
 
 
 
 
      
 
 
 
18 °C

60 °C

1050

1000

950

900

850

800

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f
s
s
a
M

Zn

1000

950

900

850

800

Zn

/

]
g
k
g
m
[
n
o

i
t
c
a
r
f
s
s
a
M

0

1

2

3

4

0

1

2

3

4

Time [w eek]

Tim e [w eek]

Figure C22: Short term stability graph for aqua regia extractable content of Zn at 18 °C and 60 °C. 

55 

 
 
 
     
 
 
 
 
 
 
Annex D: Long term stability study 

Stability data for aqua regia extractable content measured by ICP-OES and ICP-MS. Reference samples stored at -20 °C. 

Error bars are single standard deviations of 12 replicates per time point 0 and 12 months and 6 replicates per time points 4, 8, 24 and 
36 months. 

As

Cd

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f

s
s
a
M

8.5

8.0

7.5

7.0

6.5

6.0

5.5

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f

s
s
a
M

13.5

13.0

12.5

12.0

11.5

11.0

0

4

8

12

16

24
20
Time [month]

28

32

36

0

4

8

12

16

20

24

28

32

36

Time [month]

Figure D1: Long term stability graph for aqua regia extractable As and Cd at 18 °C 

56 

 
 
 
 
 
 
          
 
 
 
Co

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f

s
s
a
M

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

Cr

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f

s
s
a
M

150

145

140

135

130

125

120

115

110

105

100

0

4

8

12

16

20

24

28

32

36

0

4

8

12

16

20

24

28

32

36

Time [month]

Time [month]

Figure D2: Long term stability graph for aqua regia extractable Co and Cr at 18 °C 

Cu

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f

s
s
a
M

330

325

320

315

310

305

300

295

290

Fe

3.1E+04

3.0E+04

2.9E+04

2.8E+04

2.7E+04

2.6E+04

]

g
k
/

g
m
n
o

[

i
t
c
a
r
f

s
s
a
M

0

4

8

12

16

20

24

28

32

36

0

4

8

12

16

20

24

28

32

36

Time [month]

Time [month]

Figure D3: Long term stability graph for aqua regia extractable Cu and Fe at 18 °C 

57 

 
 
          
 
 
 
 
          
 
 
Hg

Mn

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f

s
s
a
M

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

]

g
k
/

g
m
n
o

[

i
t
c
a
r
f

s
s
a
M

330

320

310

300

290

280

0

4

8

12

16

20

24

28

32

36

0

4

8

12

16

20

24

28

32

36

Time [month]

Time [month]

Figure D4: Long term stability graph for aqua regia extractable Hg and Mn at 18 °C 

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f

s
s
a
M

90

85

80

75

70

65

Ni

Pb

]
g
k
/

g
m
n
o

[

i
t
c
a
r
f

s
s
a
M

150

145

140

135

130

0

4

8

12

16

20

24

28

32

36

0

4

8

12

16

20

24

28

32

36

Time [month]

Time [month]

Figure D5: Long term stability graph for aqua regia extractable Ni and Pb at 18 °C 

58 

 
 
          
 
 
 
 
          
 
 
Zn

]
g
k
/
g
m
[
n
o
i
t
c
a
r
f

s
s
a
M

980

960

940

920

900

880

860

840

820

0

4

8

12

16

20

24

28

32

36

Time [month]

Figure D6: Long term stability graph for aqua regia extractable Zn at 18 °C 

59 

 
 
 
 
 
Annex E: Characterisation study 

t-Test results for aqua regia extractable content determined using ISO 11466:1995 and ISO 12914:2012 

Table E1: Results of the two-tail t-Test estimated assuming unequal variances. 

Element  p value (a =0.05)  Element 
0.74 
As 
0.19 
Cd 
0.50 
Co 
0.70 
Cr 
0.98 
Cu 
0.20 
Fe 

Hg 
Mn 
Ni 
Pb 
Zn 

p value (a =0.05) 
0.85 
0.95 
0.68 
0.43 
0.73 

TOTAL AND AQUA REGIA EXTRACTABLE CONTENT 

Tables - Individual results for each element are reported in mg/kg (with the exception of Fe and Zn reported in g/kg), together with the mean and 
uncertainty budget ulab when provided by the laboratory or with the calculated SD of the 6 replicates. Total content is reported on the left table 
and aqua regia extractable content on the right table. *: indicates results excluded because considered technically incorrect. 

Figures  –  Results for  each  element  are  presented  as  mean  value  (  ♦  ), with  related  uncertainty  budget  (when  provided  by  the  laboratory)  or 
twice the calculated SD of 6 replicates ( │ ); certified value ( __ ) and expanded uncertainty of the certified value (----). 

60 

 
 
 
 
 
 
 
 
 
 
 
 
Lab 

Method 

L2 
L3 
L4 
L5 
L6 
L6 
L7 
L8 

ICP-OES 
ICP-MS 
k0-NAA 
ICP-SFMS 
k0-NAA 
ICP-MS 
ICP-OES 
ICP-MS 

Total content 
Replicate 

3 
6.8 
7.3 
7.9 
7.8 
7.8 
6.9 

1 
2 
4 
9.5  3.7 
6.1 
8.0  7.9 
8.8 
8.1  8.1 
8.2 
7.8  8.0 
7.9 
7.6  8.0 
8.2 
7.1 
7.3  7.3 
8.2  7.3  2.6*  7.8 
6.2 
8.1  8.0 

7.7 

5 
9.3 
7.8 
8.0 
7.9 
8.3 
7.1 
7.6 
7.5 

6 
5.3 
8.0 
8.1 
7.9 
7.9 
7.6 
3.2* 
7.7 

Arsenic 

Mean 

u/SD 

Lab  Method 

6.8 
8.0 
8.1 
7.9 
8.0 
7.2 
7.7 
7.5 

1.3 
1.6 
0.4 
1.3 
0.7 
0.3 
0.4 
0.8 

L1 
L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L10 
L11 
L12 
L13 
L14 
L14 
L15 

ICP-MS 
ICP-OES 
ICP-MS 
ICP-SFMS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
k0-NAA 
ICP-SFMS 
ICP-MS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
ICP-OES 

Aqua regia extractable content 

1 
2 
9.8  9.5 
7.4  7.4 
6.3  6.3 
6.8  7.5 
9.1  9.1 
7.8  8.3 
7.8  7.4 
7.7  7.8 
7.4  7.7 
8.0  7.9 
7.1  7.2 
7.1  7.6 
8.1  7.8 
7.9  8.0 
7.9  7.4 
7.6  7.9 

Replicate 
5 
4 
3 
8.3 
9.3 
7.7 
7.8 
7.5 
7.8 
6.4 
6.4 
6.0 
7.9 
7.5 
7.3 
7.1  11.6  5.1 
8.2 
9.6 
7.8 
7.9 
7.4 
7.9 
8.0 
7.6 
8.1 
7.9 
7.6 
7.4 
7.0 
6.9 
8.0 
7.9 
8.1 
7.5 
7.8 
7.0 
7.3 
7.3 

6 
8.6 
7.3 
6.2 
7.6 
6.4 
8.9  10.2 
7.1 
7.3 
7.7 
7.3 
7.6 
7.7 
8.8 
8.1 
7.5 
7.9 
7.0 
6.2 
8.2 
8.2 
7.5 
8.1 
7.1 
7.7 
7.4 
8.0 

Mean  u/SD 

8.9 
7.5 
6.3 
7.4 
8.1 
8.8 
7.6 
7.6 
7.7 
8.2 
7.5 
7.0 
8.0 
7.8 
7.5 
7.6 

0.8 
1.5 
1.3 
1.7 
2.3 
1.0 
0.4 
0.5 
0.7 
0.8 
2.9 
1.5 
0.1 
0.6 
0.9 
0.6 

61 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lab 

Method 

L1 
L2 
L3 
L4 
L5 
L6 
L6 
L6 
L7 

ICP-MS 
ICP-OES 
ICP-MS 
k0-NAA 
ICP-SFMS 
k0-NAA 
FAAS 
ICPMS 
ICP-OES 

Total content 

Replicate 

1 

2 

3 

4 

5 

6 

15.1  15.2  16.5  15.1  18.1  18.8 
13.7  13.5  13.4  12.2  13.8  13.3 
14.1  14.0  15.3  12.3  14.2  14.5 
15.1  13.9  14.8  13.2  13.4  13.7 
15.0  14.7  15.2  14.5  15.2  14.9 
17.4  17.6  16.7  16.4  15.7  15.6 
16.1  15.3  15.6  15.0  14.8  14.8 
15.8  15.6  15.0  14.7  14.9  14.3 
13.8  13.3  11.9  13.5  12.2  13.4 

Cadmium 

Mean  u/SD  Lab 

Method 

Aqua regia extractable content 

1 

2 

Replicate 
3 

4 

5 

6 

Mean  u/SD 

16.5 
13.3 
14.1 
14.0 
14.9 
16.6 
15.3 
15.1 
13.0 

1.7 
1.6 
1.7 
1.0 
2.6 
1.9 
0.6 
0.7 
0.8 

L1 
L2 
L3 
L4 
L5 
L7 
L8 
L9 
L10 
L11 
L12 
L13 
L14 
L14 
L15 

ICP-MS 
ICP-OES 
ICP-MS 
ICP-SFMS 
ICP-OES 
ICP-MS 
ICP-OES 
FAAS 
ICP-SFMS 
ICP-MS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
ICP-OES 

13.4  13.4  14.9  13.3  13.3  13.2 
12.6  12.1  12.7  11.9  13.1  12.3 
12.1  12.4  13.2  12.7  11.6  11.9 
15.1  14.2  14.5  15.2  14.6  14.1 
12.8  12.8  11.9  12.7  12.7  13.0 
13.7  13.2  13.7  13.3  13.7  13.4 
13.2  13.2  13.3  13.4  12.9  13.0 
15.6  15.9  14.9  15.0  15.8  15.0 
12.6  13.1  13.5  12.9  13.3  12.6 
12.6  12.7  12.9  12.2  12.9  12.2 
12.6  12.7  12.9  12.2  12.9  12.2 
12.3  12.7  13.6  13.5  13.8  13.5 
13.5  13.2  13.8  13.4  12.3  13.3 
14.8  14.6  15.1  14.7  14.0  14.4 
13.4  13.2  12.6  13.9  12.8  12.6 

13.6 
12.5 
12.3 
14.6 
12.7 
13.5 
13.2 
15.4 
13.0 
12.6 
12.6 
13.2 
13.2 
14.6 
13.1 

1.4 
1.5 
1.5 
2.8 
0.4 
0.3 
0.8 
1.2 
0.9 
2.5 
5.6 
0.6 
1.3 
0.7 
1.0 

62 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lab  Method 

L1 
L2 
L3 
L4 
L5 
L6 
L6 
L7 
L8 

ICP-MS 
ICP-OES 
ICP-MS 
k0-NAA 
ICP-SFMS 
k0-NAA 
ICP-MS 
ICP-OES 
ICP-MS 

Total content 
Replicate 
3 
5.9 
6.9 
6.5 
6.5 
6.5 
6.8 
5.8 
6.4 
6.9 

4 
6.8 
5.4 
6.7 
6.4 
6.6 
6.7 
5.8 
6.7 
5.3 

2 

1 
6.6  7.0 
5.5  6.5 
6.9  6.5 
6.6  6.4 
6.4  6.6 
6.6  7.0 
6.3  6.2 
6.5  6.9 
6.7  7.0 

Cobalt 

Aqua regia extractable content 

Replicate 
3 
5.3 
5.3 
5.6 
5.9 
6.1 

4 
5.2 
5.2 
5.6 
5.7 
6.9 
7.3 
6.0 
6.1 
6.1 
5.5 
5.7 
5.7 
6.1 
5.8 
6.6 
6.6 

5 
5.3 
5.2 
5.4 
5.9 
6.2 
6.4 
5.8 
6.0 
6.2 
6.0 
5.7 
5.7 
6.2 
5.6 
6.2 
6.5 

6 
5.3 
5.0 
5.3 
5.9 
6.1 
6.4 
5.7 
6.1 
6.0 
5.9 
5.6 
5.6 
6.2 
5.9 
6.5 
6.4 

Mean  u/SD 

5.2 
5.2 
5.5 
5.9 
6.3 
6.4 
6.0 
6.2 
6.0 
5.9 
5.7 
5.7 
6.1 
5.8 
6.6 
6.5 

0.3 
0.5 
0.6 
1.1 
0.3 
0.6 
0.2 
0.4 
0.5 
0.5 
0.9 
1.1 
0.1 
0.2 
0.4 
0.6 

6.3 
6.3 
5.8 
6.3 
5.8 
5.8 
6.0 
5.7 
6.6 
6.5 

5 

6.2 
6.8 
7.1 
6.2 
6.6 
6.7 
6.0 
6.9 
6.9 

6 
6.3 
6.9 
7.2 
6.5 
6.6 
6.5 
5.7 
6.5 
7.5 

Mean  u/SD  Lab 

Method 

6.5 
6.3 
6.8 
6.4 
6.5 
6.7 
6.0 
6.6 
6.7 

0.6 
0.6 
0.7 
0.3 
1.1 
0.5 
0.2 
0.2 
1.1 

L1 
L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L10 
L11 
L12 
L13 
L14 
L14 
L15 

ICP-MS 
ICP-OES 
ICP-MS 
ICP-SFMS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
k0-NAA 
ICP-SFMS 
ICP-MS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
ICP-OES 

1 

2 
5.1  5.1 
5.3  5.2 
5.6  5.6 
5.8  6.1 
6.3  6.1 
6.1  6.0 
6.2  5.8 
6.4  6.1 
5.6  6.0 
5.8  5.9 
5.7  5.5 
5.7  5.5 
6.2  6.0 
5.9  5.9 
6.8  6.8 
6.7  6.5 

63 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lab  Method 

L1 
L2 
L3 
L4 
L5 
L6 
L6 
L6 
L7 
L8 
L9 

ICP-MS 
ICP-OES 
ICP-MS 
k0-NAA 
ICP-SFMS 
k0-NAA 
FAAS 
ICP-MS 
ICP-OES 
ICP-MS 
ICP-OES 

Total content 
Replicate 
3 

4 

1 

2 

167  174  151  170 
166  162  164  149 
178  179  168  156 
173  167  171  166 
194  182  177  183 
185  186  186  177 
183  182  179  175 
185  179  174  168 
173  164  152  164 
194  148  148  163 
158  170  181  172 

5 
156 
172 
161 
164 
176 
172 
169 
174 
154 
159 
167 

6 
164 
163 
162 
166 
183 
171 
184 
169 
167 
159 
169 

Chromium 

Mean  u/SD  Lab 

Method 

163 
163 
167 
168 
183 
180 
179 
175 
162 
162 
169 

23 
25 
26 
8 
36 
11 
7 
7 
8 
26 
8 

L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L9 
L10 
L11 
L12 
L13 
L14 
L14 
L15 

ICP-OES 
ICP-MS 
ICP-SFMS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
FAAS 
k0-NAA 
ICP-SFMS 
ICP-MS 
ICP-OES 
ICP-OES 
ICP-MS 
ICP-OES 
ICP-OES 

Aqua regia extractable content 

1 
2 
147  141 
152  151 
155  149 
153  153 
151  149 
152  154 
155  150 
159  163 
142  156 
148  148 
138  139 
138  139 
148  158 
159  160 
157  157 
168  166 

Replicate 
4 
3 
140 
150 
151 
149 
147 
143 
151 
142 
151 
145 
150 
153 
157 
149 
150 
153 
155 
153 
143 
150 
146 
138 
146 
138 
154 
159 
165 
160 
161 
162 
172 
165 

5 
152 
130 
142 
151 
151 
153 
151 
150 
165 
154 
142 
142 
154 
155 
149 
166 

6 
144 
132 
148 
154 
144 
151 
149 
153 
154 
145 
147 
147 
153 
159 
156 
162 

Mean 

u/SD 

146 
144 
147 
151 
149 
152 
152 
155 
154 
148 
142 
142 
154 
160 
157 
167 

22 
23 
28 
4 
4 
6 
35 
8 
16 
12 
29 
29 
6 
10 
12 
13 

64 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lab 

Method 

L1 
L2 
L3 
L4 
L5 
L6 
L6 
L6 
L7 
L8 
L9 

ICP-MS 
ICP-OES 
ICP-MS 
k0-NAA 
ICP-SFMS 
k0-NAA 
FAAS 
ICP-MS 
ICP-OES 
ICP-MS 
ICP-OES 

Total content 
Replicate 
4 
3 
376 
357 
315 
325 
333 
330 
335 
315 
374 
381 
414 
337 
374 
369 
380 
375 
334 
339 
325 
328 
350 
363 

2 
391 
325 
332 
316 
383 
379 
371 
376 
336 
336 
368 

1 
386 
310 
327 
322 
378 
392 
365 
388 
333 
401 
361 

5 
358 
323 
329 
335 
372 
334 
354 
384 
349 
333 
331 

6 
357 
321 
338 
337 
380 
381 
373 
385 
341 
332 
337 

Copper 

Mean  u/SD 

Lab 

371 
320 
332 
327 
378 
373 
368 
381 
339 
343 
352 

42 
38 
40 
50 
60 
109 
11 
11 
6 
40 
15 

L1 
L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L10 
L11 
L12 
L13 
L14 
L14 
L15 

Aqua regia extractable content 

Method 

1 
361 
ICP-MS 
309 
ICP-OES 
ICP-MS 
340 
ICP-SFMS  322 
343 
ICP-OES 
334 
ICP-MS 
349 
ICP-MS 
365 
ICP-OES 
FAAS 
360 
ICP-SFMS  323 
351 
ICP-MS 
361 
ICP-OES 
329 
ICP-OES 
358 
ICP-MS 
344 
ICP-OES 
371 
ICP-OES 

2 
375 
306 
332 
345 
339 
340 
345 
367 
367 
326 
355 
355 
336 
331 
341 
379 

Replicate 
3 
361 
309 
313 
333 
343 
333 
357 
374 
366 
327 
360 
377 
340 
349 
334 
373 

4 
367 
299 
321 
329 
340 
335 
352 
375 
358 
316 
361 
354 
330 
335 
339 
361 

5 
359 
313 
307 
345 
342 
334 
362 
394 
353 
336 
358 
376 
333 
350 
328 
366 

6 
378 
306 
317 
331 
349 
338 
360 
401 
364 
336 
358 
359 
333 
347 
327 
361 

Mean 

u/SD 

367 
307 
322 
334 
343 
336 
354 
379 
361 
327 
357 
364 
334 
345 
335 
369 

17 
37 
39 
62 
4 
4 
6 
35 
11 
23 
73 
72 
6 
28 
13 
29 

65 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Method 

Lab 

L1 
L2 
L3 
L4 
L5 
L6 
L6 
L6 
L7 
L8 

ICP-MS 
ICP-OES 
ICP-MS 
k0-NAA 
ICP-SFMS 
k0-NAA 
FAAS 
ICP-MS 
ICP-OES 
ICP-MS 

Total content 

Replicate 
4 
3 

5 

2 

6 

1 
34.3  33.4  34.0  32.1  33.4  32.2 
30.2  31.2  32.6  31.9  31.7  32.4 
34.3  33.2  31.3  36.6  32.9  33.2 
33.4  34.0  33.4  34.6  33.7  34.1 
31.5  31.8  32.5  31.6  31.4  31.5 
31.5  31.7  32.5  33.2  34.0  33.1 
31.2  32.0  31.7  33.4  33.1  33.0 
35.6  35.7  34.8  37.3  36.5  38.1 
30.3  32.0  33.8  32.3  34.1  32.1 
36.2  33.3  33.4  30.2  33.2  33.0 

Iron (mass fraction in g kg-1) 

Mean  u/SD 

Lab 

Method 

33.2 
31.7 
33.6 
33.9 
31.7 
32.6 
32.4 
36.3 
32.4 
33.2 

4.4 
3.1 
3.4 
1.6 
5.3 
1.9 
1.3 
2.4 
1.4 
1.7 

L1 
L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L9 
L10 
L11 
L12 
L13 
L14 
L14 

ICP-MS 
ICP-OES 
ICP-MS 
ICP-SFMS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
FAAS 
k0-NAA 
ICP-SFMS 
ICP-MS 
ICP-OES 
ICP-OES 
ICP-MS 
ICP-OES 

Aqua regia extractable content 

1 

2 

Replicate 
4 
3 

5 

6 

Mean  u/SD 

32.7  32.2  29.9  32.7  31.9  32.5 
33.0  33.0  34.0  33.5  32.4  32.0 
31.8  30.4  27.5  30.9  29.7  30.4 
28.7  31.4  31.3  27.6  28.1  30.9 
33.0  33.2  33.8  33.6  32.4  32.8 
32.2  32.9  32.7  32.2  32.4  32.9 
31.0  30.8  31.5  31.3  31.5  30.2 
33.9  32.5  35.4  34.5  33.8  35.2 
31.6  32.0  31.0  32.2  31.6  32.1 
30.1  31.6  32.0  33.5  33.4  33.0 
34.5  34.6  35.5  33.4  35.6  36.2 
33.3  35.5  34.4  34.9  35.0  34.6 
33.0  31.9  34.0  36.5  32.0  31.7 
33.8  32.3  32.9  33.1  33.9  33.8 
33.9  33.6  31.7  32.8  34.4  32.2 
34.4  33.0  32.5  34.6  34.6  33.8 

32.0 
33.0 
30.1 
29.7 
33.1 
32.5 
31.1 
34.2 
31.8 
32.3 
35.0 
34.6 
33.2 
33.3 
33.1 
33.8 

1.8 
3.3 
3.0 
6.1 
0.5 
0.4 
0.7 
3.2 
1.0 
2.5 
2.9 
6.7 
7.0 
0.7 
2.4 
2.0 

66 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Total content 

Aqua regia extractable content 

Mercury 

Method 

Lab 

ICP-OES 
L2 
ICP-MS 
L3 
k0-NAA 
L4 
ICP-SFMS 
L5 
k0-NAA 
L6 
DMA 
L6 
CV-AFS 
L6 
ICP-OES 
L7 
L8 
ICP-MS 
L10  CV-AFS 
ICP-MS 
L10 

1 
6.3 
6.2 
6.3 
5.9 
6.7 
6.8 
6.7 
5.1 
5.2 
5.7 
6.3 

2 
6.0 
6.5 
6.0 
5.5 
6.8 
6.8 
6.5 
5.3 
5.9 
5.7 
6.4 

Replicate 
4 
3 
5.6 
5.9 
5.4 
6.7 
6.0 
6.1 
5.3 
5.5 
6.4 
7.0 
6.9 
6.9 
6.5 
6.7 
5.3 
5.2 
6.1 
5.8 
5.5 
5.6 
5.3 
6.1 

5 
6.0 
6.2 
5.9 
5.9 
6.1 
6.6 
6.7 
5.5 
6.2 
5.2 
5.3 

6 
5.8 
6.4 
6.4 
5.8 
6.1 
6.6 
6.7 
5.8 
5.9 
5.6 
5.9 

Mean  u/SD 

Lab 

Method 

5.9 
6.2 
6.1 
5.6 
6.5 
6.8 
6.6 
5.4 
5.9 
5.6 
5.9 

1.1 
1.2 
0.3 
1.2 
0.5 
0.4 
0.3 
0.3 
0.4 
0.9 
1.3 

L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L9 
L10 
L11 
L12 
L13 
L14 
L14 
L15 

ICP-MS 
CV-AAS 
CV-AAS 
ICP-SFMS 
CV-AFS 
CV-AAS 
ICP-MS 
CV-AFS 
CV-AAS 
ICP-SFMS 
ICP-MS 
CV-AFS 
AAS 
ICP-MS 
ICP-OES 
ICP-OES 

1 
5.6 
5.6 
5.8 
6.2 
6.1 
5.9 
6.4 
6.7 
6.2 
6.1 
5.7 
5.7 
6.1 
6.3 
5.9 
6.4 

2 
5.5 
5.8 
5.6 
6.0 
6.2 
6.0 
6.1 
8.3 
7.6 
6.3 
5.7 
5.7 
7.0 
6.5 
5.9 
6.3 

Replicate 
4 
3 
5.4 
5.5 
5.9 
6.1 
5.7 
5.6 
6.1 
6.0 
6.2 
6.0 
6.1 
6.4 
6.1 
6.1 
6.8 
6.6 
6.7 
6.5 
6.3 
6.7 
5.5 
5.8 
5.5 
5.8 
6.9 
6.5 
6.2 
6.0 
6.1 
5.8 
6.6 
5.9 

5 
6.0 
5.8 
5.5 
6.2 
6.0 
5.7 
6.2 
6.5 
6.7 
6.4 
5.6 
5.6 
6.9 
6.1 
5.7 
6.0 

6 
5.6 
5.6 
5.7 
6.2 
5.9 
5.6 
6.1 
6.7 
6.5 
6.3 
5.7 
5.7 
6.5 
6.2 
5.8 
5.9 

Mean  u/SD 

5.6 
5.8 
5.7 
6.1 
6.1 
6.0 
6.2 
6.9 
6.7 
6.3 
5.7 
5.7 
6.6 
6.2 
5.9 
6.2 

1.0 
1.1 
1.0 
0.1 
0.1 
0.3 
0.3 
2.1 
1.5 
0.6 
1.2 
1.0 
0.4 
0.5 
0.4 
0.6 

67 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Method 

Lab 

L1 
L2 
L3 
L4 
L5 
L6 
L6 
L7 
L8 
L9 

ICP-MS 
ICP-OES 
ICP-MS 
k0-NAA 
ICP-SFMS 
k0-NAA 
FAAS 
ICP-OES 
ICP-MS 
ICP-OES 

1 
397 
312 
334 
348 
379 
342 
347 
335 
415 
367 

Total content 

5 

2 

6 

Replicate 
4 
3 
408  354  388  345  345 
333  342  340  339  343 
332  332  346  355  340 
334  342  334  348  354 
352  351  378  362  353 
344  341  369  361  359 
350  372  360  358  369 
337  349  342  354  339 
355  344  336  350  352 
375  358  354  329  334 

Manganese 

Mean  u/SD 

Lab 

Method 

Aqua regia extractable content 

1 

2 

Replicate 
4 
3 

5 

6 

Mean  u/SD 

373 
335 
340 
343 
363 
353 
359 
343 
359 
353 

43 
34 
35 
18 
70 
21 
11 
7 
39 
18 

L1 
L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L10 
L11 
L12 
L13 
L14 
L14 
L15 

ICP-MS 
ICP-OES 
ICP-MS 
ICP-SFMS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
FAAS 
ICP-SFMS 
ICP-MS 
ICP-OES 
ICP-OES 
ICP-MS 
ICP-OES 
ICP-OES 

306  305  294  311  303  314 
357  359  357  350  343  343 
338  335  324  318  328  333 
302  327  326  317  325  337 
328  336  343  345  335  341 
339  348  337  342  336  343 
347  341  352  348  349  337 
361  351  361  349  349  353 
349  350  349  351  353  351 
327  331  324  321  330  332 
323  338  332  336  334  335 
341  334  362  349  334  344 
362  345  358  353  358  356 
356  361  341  358  361  350 
356  355  339  347  344  338 
356  352  342  342  339  348 

306 
352 
329 
322 
338 
341 
346 
354 
351 
328 
333 
344 
355 
354 
346 
347 

31 
36 
33 
62 
6 
6 
7 
13 
10 
12 
69 
67 
9 
20 
19 
30 

68 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Total content 

Aqua regia extractable content 

Nickel 

Method 

Lab 

L1 
L2 
L3 
L5 
L6 
L6 
L7 
L8 

ICP-MS 
ICP-OES 
ICP-MS 
ICP-SFMS 
FAAS 
ICP-MS 
ICP-OES 
ICP-MS 

1 
94 
91 
91 
102 
87 
101 
90 
95 

2 
95 
90 
95 
97 
90 
97 
91 
86 

Replicate 
4 
3 
93 
84 
83 
94 
85 
98 
99 
98 
86 
89 
92 
95 
93 
84 
72 
79 

5 
90 
97 
92 
97 
88 
95 
86 
78 

6 
91 
94 
96 
104 
87 
94 
91 
78 

Mean  u/SD 

Lab 

Method 

91 
92 
93 
100 
88 
96 
89 
81 

18 
9 
9 
18 
3 
4 
3 
8 

L1 
L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L10 
L11 
L12 
L13 
L14 
L14 
L15 

ICP-MS 
ICP-OES 
ICP-MS 
ICP-SFMS 
ICP-OES 
ICP-MS 
ICPMS 
ICP-OES 
FAAS 
ICP-SFMS 
ICP-MS 
ICP-OES 
ICP-OES 
ICP-MS 
ICP-OES 
ICP-OES 

1 
93 
85 
83 
90 
90 
78 
92 
88 
93 
88 
81 
82 
82 
86 
92 
92 

2 
91 
82 
84 
90 
89 
78 
87 
86 
95 
87 
81 
76 
82 
84 
84 
89 

Replicate 
4 
3 
90 
94 
81 
86 
87 
88 
88 
86 
91 
84 
79 
79 
90 
91 
87 
85 
92 
92 
85 
90 
85 
80 
80 
83 
86 
84 
85 
84 
92 
90 
94 
89 

5 
88 
90 
79 
89 
88 
80 
91 
85 
93 
92 
83 
81 
87 
83 
85 
90 

6 
87 
84 
78 
89 
89 
77 
89 
85 
92 
86 
84 
80 
85 
85 
92 
89 

Mean  u/SD 

91 
85 
83 
89 
89 
79 
90 
86 
93 
88 
82 
80 
84 
85 
89 
91 

5 
9 
9 
16 
2 
3 
2 
13 
3 
7 
17 
16 
1 
3 
8 
6 

69 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lab 

L1 
L2 
L3 
L5 
L6 
L6 
L7 
L10 
L10 

Method 

ICP-MS 
ICP-MS 
ICP-OES 
ICP-SFMS 
FAAS 
ICP-MS 
ICP-OES 
ICP-OES 
ICP-MS 

1 
150 
143 
169 
173 
148 
164 
157 
129 
151 

Total content 

5 

2 

6 

Replicate 
4 
3 
135  169  133  169  172 
145  156  141  154  155 
171  152  171  151  155 
178  172  172  177  179 
153  154  151  150  153 
159  159  163  164  164 
152  152  156  155  159 
148  150  148  147  158 
151  157  139  135  157 

Lead 

Mean  u/SD 

Lab 

Method 

Aqua regia extractable content 

1 

2 

Replicate 
4 
3 

5 

6 

Mean  u/SD 

155 
149 
161 
175 
152 
162 
155 
147 
148 

20 
18 
20 
30 
4 
5 
3 
15 
31 

L1 
L2 
L3 
L4 
L5 
L6 
L7 
L8 
L9 
L10 
L11 
L12 
L13 
L14 
L14 
L15 

ICP-MS 
ICP-OES 
ICP-MS 
ICP-SFMS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
FAAS 
ICP-SFMS 
ICP-MS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
ICP-OES 

164  159  150  160  150  156 
152  151  153  151  153  149 
153  152  151  147  147  149 
158  151  157  149  155  166 
148  149  144  155  147  153 
158  161  155  157  157  157 
152  159  145  157  167  160 
155  155  156  152  152  155 
156  159  160  160  160  159 
160  163  157  162  162  163 
165  167  166  171  171  169 
138  137  137  136  137  134 
165  172  172  167  172  174 
156  153  153  153  153  150 
168  169  167  165  162  163 
158  159  153  153  151  151 

157 
152 
150 
156 
149 
158 
157 
154 
159 
161 
168 
137 
170 
153 
166 
154 

10 
18 
18 
32 
4 
3 
7 
4 
5 
6 
27 
34 
7 
4 
3 
7 

70 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Aqua regia extractable content 

1 

2 

Replicate 
4 
3 

5 

6 

Mean  u/SD 

0.98  0.96  0.95  0.96  0.95  0.97 
0.91  0.89  0.92  0.90  0.92  0.90 
0.96  0.95  0.92  0.93  0.91  0.93 
0.96  0.97  0.98  0.94  1.00  0.99 
0.91  0.91  0.90  0.94  0.90  0.91 
0.98  1.00  0.98  0.99  0.98  0.99 
1.04  1.04  1.05  1.02  1.04  1.01 
0.97  0.95  0.98  0.98  1.03  0.99 
1.02  1.03  1.02  1.00  1.04  1.03 
0.94  1.00  0.99  1.03  1.04  1.03 
0.90  0.92  0.91  0.89  0.93  0.92 
0.91  0.93  0.95  0.94  0.94  0.93 
0.99  0.97  0.98  0.98  0.97  0.97 
1.07  1.04  1.00  1.06  1.05  1.05 
1.01  1.00  0.98  1.01  1.00  0.99 
1.02  0.97  0.98  1.03  1.00  1.01 

0.97 
0.91 
0.93 
0.97 
0.91 
0.99 
1.03 
0.99 
1.02 
1.01 
0.91 
0.93 
0.98 
1.04 
1.00 
1.00 

0.05 
0.11 
0.11 
0.18 
0.03 
0.01 
0.03 
0.09 
0.03 
0.08 
0.05 
0.20 
0.19 
0.03 
0.03 
0.05 

Total content 

Zinc (mass fraction in g kg-1) 

Method 

Lab 

L2 
L3 
L4 
L5 
L6 
L6 
L7 
L8 
L9 

ICP-MS 
ICP-OES 
k0-NAA 
ICP-SFMS 
k0-NAA 
FAAS 
ICP-OES 
ICP-MS 
ICP-OES 

Replicate 
4 
3 

5 

2 

6 

1 
0.93  0.95  0.97  0.95  0.95  0.94 
0.91  0.90  0.94  0.92  0.93  0.96 
0.98  0.99  0.99  1.00  0.97  0.99 
1.02  0.99  0.99  0.99  1.03  1.01 
1.00  1.00  1.00  1.01  1.02  1.00 
1.04  1.05  1.05  1.05  1.05  1.05 
0.97  0.93  0.93  0.95  0.93  0.93 
1.18  1.01  0.99  0.98  1.00  1.00 
0.97  1.19  0.96  0.95  0.89  0.91 

Mean  u/SD 

Lab 

Method 

0.95 
0.93 
0.99 
1.00 
1.01 
1.05 
0.94 
1.03 
0.98 

0.11  L1 
0.11  L2 
0.05  L3 
0.17  L4 
0.06  L5 
0.03  L6 
0.02  L7 
0.11  L8 
0.11  L9 
L9 
L10 
L11 
L12 
L13 
L14 
L14 

ICP-MS 
ICP-OES 
ICP-MS 
ICP-SFMS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 
FAAS 
k0-NAA 
ICP-SFMS 
ICP-MS 
ICP-OES 
ICP-MS 
ICP-MS 
ICP-OES 

71 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
72 

 
 
European Commission 

EUR 28012 EN – Joint Research Centre – Directorate F – Health, Consumers and Reference Materials 
Title:  

The certification of the mass fraction of the total content and the aqua regia extractable content (ISO 
12914 and ISO 11466) of As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn in sewage sludge: ERM®-CC144 

Author(s): Anna Santoro, Andres Perez, Toni Venelinov, Albert Oostra and Andrea Held 
Luxembourg: Publications Office of the European Union 
2016 – 72 pp. – 21.0 x 29.7 cm 
EUR – Scientific and Technical Research series – ISSN 1831-9424 
ISBN 978-92-79-59857-9 
doi: 10.2787/264881 

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

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

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

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doi: 10.2787/264881 

ISBN: 978-92-79-59857-9