ISO 54321:2020

INTERNATIONAL ISO
STANDARD 54321
First edition
2020-08
Soil, treated biowaste, sludge and
waste — Digestion of aqua regia
soluble fractions of elements
Sols, biodéchets traités, boues et déchets — Digestion des éléments
solubles dans l’eau régale
Reference number
©
ISO 2020
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Interferences and sources of errors . 2
6 Reagents . 3
7 Apparatus . 3
7.1 General . 3
7.2 Method A — Apparatus for thermal heating under atmospheric conditions . 3
7.2.1 Method A1 — Thermal heating under reflux conditions . 3
7.2.2 Method A2 — Thermal heating with a heating block with containers . 4
7.3 Method B — Microwave digestion with temperature control, closed vessels . 4
8 Procedure. 5
8.1 General . 5
8.2 Blank test . 5
8.3 Method A — thermal heating under atmospheric conditions . 5
8.3.1 Method A1 — Thermal heating under reflux conditions . 5
8.3.2 Method A2 — Thermal heating with a heating block with containers . 6
8.4 Method B — Microwave digestion with temperature control, closed vessels . 7
9 Test report . 7
Annex A (informative) Repeatability and reproducibility data for soil, biowaste and sludge
samples . 9
Annex B (informative) Repeatability and reproducibility data for waste samples .10
Bibliography .37
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee
SC 3, Chemical and physical characterization, in collaboration with the European Committee for
Standardization (CEN) Technical Committee CEN/TC 444, Environmental characterization of solid
matrices, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna
Agreement).
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved

Introduction
Regarding the comparability of the procedure described in this document with those of the other
standards mentioned above the next remarks can be made:
— This document describes the digestion of solid samples with aqua regia.
— Differences in the procedures of the different standards are small. An important difference between
the reflux procedures as described in ISO 11466 and EN 13657 and EN 16174 concerns the waiting
time after addition of the acid to the sample, before the digestion starts. ISO 11466 specifies a
waiting time of 16 h, both European standards state that the digestion can start after the first strong
reactions have ceased. In validation work it was proven that the difference between 2 h and 16 h of
waiting was negligible, therefore this document follows the approach of EN 13657 and EN 16174.
— The heating block procedure was added to the reflux and microwave digestion procedures. The
procedure was adopted from the Dutch standard NEN 6961, which specifies a boiling time of 2 h to
4 h. This document specifies a boiling time of 2 h.
The methods specified in this document are providing multi-element aqua regia digestion techniques
for soil, treated biowaste, sludge and waste prior to analysis. It is known that the digestion of
environmental samples with aqua regia will not necessarily lead to complete element recoveries, and
that the extract from a test sample may not reflect the total concentrations of the target analytes.
However, for most environmental applications the result obtained based upon digestion methods
specified in this document are considered to be fit for the intended purpose.
This document is validated for several types of matrices as indicated in Table 1.
Table 1 — Matrices for which this document is validated
Matrix Materials used in the validation test
Municipal sludge
Industrial sludge
Sludge Sludge from electronic industry
Ink waste sludge
Sewage sludge
Compost
Biowaste (Meth-
od A)
Composted sludge
Agricultural soil
Soil
Sludge amended soils
City waste incineration fly ash (“oxidised” matrix)
City waste incineration bottom ash (“silicate” matrix)
Ink waste sludge (organic matrix)
Waste
Electronic industry sludge (“metallic” matrix)
BCR 146R (sewage sludge)
BCR 176 (city waste incineration ash)
WARNING — Persons using this document should be familiar with usual laboratory practice.
Some of the reagents used in this document are highly corrosive and very toxic. Safety
precautions are absolutely necessary, not only due to the strong corrosive reagents, but also to
the high temperature and high pressure.
The use of laboratory-grade microwave equipment with isolated and corrosion resistant safety
devices is required. Domestic (kitchen) type microwave ovens shall not be used, as corrosion
by acid vapours may compromise the function of the safety devices and prevent the microwave
magnetron from shutting off when the door is open, which could result in operator exposure to
hazardous levels of microwave energy.
All procedures should be performed in a fume hood or in closed force-ventilated equipment. By
the use of strong oxidising reagents, the formation of explosive organic intermediates is possible,
especially when dealing with samples with a high organic content. Do not open pressurized
vessels before they have cooled down. Avoid contact with the chemicals and the gaseous reaction
products.
IMPORTANT — It is absolutely essential that tests conducted according to this document be
carried out by suitably trained staff.
vi © ISO 2020 – All rights reserved

INTERNATIONAL STANDARD ISO 54321:2020(E)
Soil, treated biowaste, sludge and waste — Digestion of
aqua regia soluble fractions of elements
1 Scope
This document specifies two methods for digestion of soil, treated biowaste, sludge and waste by the
use of an aqua regia digestion.
Digestion with aqua regia will not necessarily accomplish total decomposition of the sample. The
extracted analyte concentrations may not necessarily reflect the total content in the sample but
represent the aqua regia soluble metals under the condition of this test procedure. It is generally agreed
that for environmental analysis purposes, the results are fit for the intended purpose to protect the
environment.
This document is applicable for the following elements:
Aluminium (Al), antimony (Sb), arsenic (As), barium (Ba), beryllium (Be), boron (B), cadmium (Cd),
calcium (Ca), chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), magnesium (Mg),
manganese (Mn), mercury (Hg), molybdenum (Mo), nickel (Ni), phosphorus (P), potassium (K),
selenium (Se), silver (Ag), sodium (Na), strontium (Sr), sulfur (S), tellurium (Te), thallium (Tl), tin (Sn),
titanium (Ti), vanadium (V), and zinc (Zn).
This document can also be applied for the digestion of other elements, provided the user has verified
the applicability.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
aqua regia
digestion (3.2) solution obtained by mixing 1 volume of nitric acid (mass fraction of 65 % to 70 %) and
3 volumes of hydrochloric acid (mass fraction of 35 % to 37 %)
Note 1 to entry: These mass percentages agree with the concentrations of 6.2 and 6.3.
3.2
digestion
mineralization of the organic matter of a sample and dissolution of its mineral part, more or less
completely, when reacting with a reagent mixture
3.3
dry residue
dry matter expressed as a percentage by mass after drying at 105 °C ± 5 °C to the constancy of weight
3.4
laboratory sample
sample (3.5) intended for laboratory inspection of testing
[SOURCE: ISO 11074:2015, 4.3.7]
3.5
sample
portion of material selected from a larger quantity of material
[SOURCE: ISO 11074:2015, 4.1.17]
3.6
test portion
analytical portion
quantity of material of proper size for measurement of the concentration or other properties of interest,
removed from the test sample (3.7)
Note 1 to entry: The test portion may be taken from the laboratory sample directly if no preparation of sample is
required (e. g. with liquids), but usually it is taken from the prepared test sample.
Note 2 to entry: A unit or increment of proper homogeneity, size and fineness, needing no further preparation,
may be a test portion.
[SOURCE: ISO 11074:2015, 4.3.15]
3.7
test sample
analytical sample
portion of material resulting from the laboratory sample (3.4) by means of an appropriate method of
sample pre-treatment and having the size (volume/mass) necessary for the desired testing or analysis
[SOURCE: ISO 11074:2015, 4.1.3]
4 Principle
A test portion is digested with aqua regia according to one of the following heating procedures:
— Method A: procedure under atmospheric conditions
— A1: reflux for (120 ± 10) min, followed by filtration/centrifugation;
— A2: heating block at (105 ± 5) °C for (120 + 10) min, followed by filtration/centrifugation.
— Method B: microwave digestion
— B1: Temperature controlled procedure: at (175 ± 5) °C for (10 ± 1) min in a closed vessel followed
by filtration/centrifugation.
5 Interferences and sources of errors
The container in which the sample is delivered and stored can be a source of errors. Its material shall
be chosen according to the elements to be determined (e.g. elemental Hg can penetrate polyethylene
walls very fast in both directions. Glass can contaminate samples with its major elements: e.g. B, Na, K,
Si and Al).
Grinding or milling samples includes a risk of contamination of the sample by the environment (air, dust,
wear of milling equipment). Due to elevated temperature losses of volatile compounds are possible.
For the determination of elements forming volatile compounds (e.g. Hg, As) special care has to be taken
during sample pre-treatment.
2 © ISO 2020 – All rights reserved

All glassware and plastics ware shall be adequately cleaned and stored in order to avoid any
contamination.
In the case of filtration of the digested solution it is necessary to take care that the filtration procedure
does not introduce contaminants.
Ensure that all of the test portion is brought into contact with the acid mixture in the digestion vessel.
Some elements of interest can be lost due to precipitation with ions present in the final digest solution,
e.g. low soluble chlorides, fluorides and sulfates.
6 Reagents
Use only acids and reagents of recognized analytical grade to avoid high blank values for subsequent
analytical measurements. Use a test blank solution throughout the procedure applying all steps with
the same amount of acids, but without a sample.
6.1 Water, e.g. deionized.
6.2 Hydrochloric acid, c(HCl) ≈ 12 mol/l.
6.3 Nitric acid, c(HNO ) ≈ 15 mol/l.
6.4 Nitric acid, c(HNO ) ≈ 0,5 mol/l.
Dilute 35 ml nitric acid (6.3) to 1 l with water (6.1).
6.5 Antifoaming agent, e.g. n-dodecane (C H ) or n-octanol (C H O) are suitable.
12 26 8 18
7 Apparatus
7.1 General
Usual laboratory apparatus. All glassware and plastics ware shall be adequately cleaned and stored in
order to avoid any contamination.
Depending upon the concentration of the element of interest, particular care should be exercised with
respect to the effective cleaning of the vessels.
7.2 Method A — Apparatus for thermal heating under atmospheric conditions
7.2.1 Method A1 — Thermal heating under reflux conditions
7.2.1.1 Digestion vessel, temperature- and pressure-resistant and capable of containing the mixture
of sample and digest solution, for example a quartz vessel. The digestion vessel shall have a volume of at
least 5 times of the volume of the aqua regia used. The inner wall of the vessel shall be inert and shall not
release substances to the digest in excess of the purity requirements of the subsequent analysis.
NOTE 1 Silica or borosilicate glass vessels can be used instead of quartz vessels.
NOTE 2 It can be necessary to periodically clean the digestion vessels with a suitable surfactant to remove
persistent deposits.
7.2.1.2 Reflux condenser, adaptable to the digestion vessel (7.2.1.1).
7.2.1.3 Absorption vessel, volatile species trap, in an open digestion system capable of trapping one
or more volatile measurement species, adaptable to the reflux condenser (7.2.1.2).
7.2.1.4 Heating device, for example a heating mantle, thermostatic controlled, or an aluminium block
thermostat.
7.2.2 Method A2 — Thermal heating with a heating block with containers
7.2.2.1 Digestion tube, 50 ml propylene tube with a screw cap from polypropylene.
The part of the tube not being heated and the screw cap function as a condenser, but are not really
a reflux system. The material of the tube and screw cap need to be tested in order to be sure that
release of elements of interest does not take place. Other materials and vessels with other volumes than
mentioned above are allowed to be used if suitability has been proven.
7.2.2.2 Temperature controlled heating block, heating block able to heat the tube(s) to a temperature
of (105 ± 5) °C.
7.3 Method B — Microwave digestion with temperature control, closed vessels
7.3.1 Digestion vessel, for pressurized microwave digestion, typically 100 ml volume, reagent-,
temperature- and pressure-resistant and capable of containing the mixture of sample and digest solution.
The vessel shall be suitable for the safe application in the temperature and pressure range applied,
capable of withstanding pressures of at least 3 000 kPa.
Digestion vessels made of perfluoroalkoxylalkane (PFA), modified polytetrafluoroethylene (PTFE) or
quartz, and equipped with a safety pressure releasing system to avoid explosion of the vessel, shall
be used. The inner wall of the vessel shall be inert and shall not release contaminations to the digest
solution.
It can be necessary to periodically clean the digestion vessels with a suitable surfactant to remove
persistent deposits.
7.3.2 Microwave digestion system, corrosion resistant and well ventilated. All electronics shall be
protected against corrosion for safe operation.
Use a laboratory-grade microwave oven with temperature feedback control mechanisms.
The microwave digestion system should be able to control the temperature with an accuracy of ±5 °C
and automatically adjust the microwave field output power within 2 s of sensing. Temperature sensors
shall be accurate to ±2 °C, including the final reaction temperature of (175 ± 5) °C. Temperature
feedback control provides the primary performance mechanism for the method. Due to the variability
in sample matrix types and microwave digestion equipment (i.e. different vessel types and microwave
designs), control of the temperature during digestion is important for reproducible microwave heating
and comparable data. Manufacturer specifications of the microwave digestion system must fit these
specifications. The accuracy of the temperature measurement system should be periodically tested
on blank samples at an elevated temperature according to the manufactures instructions. If the
temperature deviates by more than 2 °C from the temperature measured by an external, calibrated
temperature measurement system, the microwave temperature measurement system should be re-
calibrated.
7.4 Sample containers, plastics and glass containers are both suitable.
7.5 Filter paper, usually with a pore size of 0,45 µm and resistant to the diluted aqua regia final
digestion solution.
4 © ISO 2020 – All rights reserved

7.6 Volumetric flasks, usually of nominal capacity of 50 ml or 100 ml.
7.7 Analytical balance, with an accuracy of 1 mg or better.
7.8 Boiling aids, anti-bumping granules or glass beads, diameter 2 mm to 3 mm, acid washed.
8 Procedure
8.1 General
Pre-treat, soil, sludge and biowaste samples according to e.g. EN 16179 or ISO 11464 and waste samples
according to e.g. EN 15002.
Determine the dry matter content, depending on the matrix of the sample, e.g. according to EN 15934.
For waste samples the next remarks apply:
— Pre-treatment should include drying or grain size reduction below a particle size of 250 µm for solid
waste or homogenizing by use of a high speed mixer or sonification for liquid waste samples.
— The mass of test portion for a single digestion has to be selected in a way, that:
— it is representative for the laboratory sample;
— it complies with the specifications of manufacturer of the digestion unit.
Referring to the manufacturer’s instructions, the upper limits of mass of the test portion shall be taken
into account.
— For representativeness reasons a mass above 200 mg is to be preferred for the test portion. Follow,
for safety reasons, the manufacturer’s instructions regarding the maximum amount of organic
carbon in the sample.
8.2 Blank test
Carry out a reagent blank test digestion in parallel with the determination, using the same procedure
and the same quantities of all the reagents as in the determination, but omitting the test portion. The
laboratory shall define acceptable limits.
NOTE The measurement of a blank is introduced to determine the contribution of the extracting solution,
glassware, digestion tube and filter paper used to the measured value.
8.3 Method A — thermal heating under atmospheric conditions
8.3.1 Method A1 — Thermal heating under reflux conditions
Weigh approximately 3 g of the test sample (waste samples 1 g to 10 g), with an accuracy of 0,001 g (or
at least three significant figures), and transfer to the digestion vessel (7.2.1.1).
In case of dry samples moisten the test portion with about 0,5 ml to 1,0 ml of water (6.1) and add,
dropwise, if necessary, to reduce foaming, with mixing, (21,0 ± 0,1) ml of hydrochloric acid (6.2)
followed by (7,0 ± 0,1) ml of nitric acid (6.3). Connect the reflux condenser (7.2.1.2) to the digestion
vessel (7.2.1.1). Fill the absorption vessel (7.2.1.3) with approximately 15 ml nitric acid (6.4). Connect
the absorption vessel to the reflux condenser, and let stand at room temperature until any effervescence
almost ceases to allow for slow oxidation of the organic matter in the sample.
The time of standing at room temperature can have an influence on the digestion rate of aqua regia.
For consistency, it is recommended to start heating as soon as possible after the first strong reaction
has ceased.
30 ml of aqua regia is only sufficient for the oxidation of about 0,5 g organic carbon. If there is any
doubt of the amount of carbon present, estimate the amount of carbon in the sample or carry out a
determination of TOC. If there is more than 0,5 g of organic carbon in the test portion, proceed as
follows.
Allow first reaction with the aqua regia to subside. Then add an extra 1 ml of nitric acid (6.3) only to
every 0,1 g of organic carbon above 0,5 g. Do not add more than 10 ml of nitric acid at any given time,
and allow any reaction to subside before proceeding further.
Connect the digestion vessel (7.2.1.1) to the heating device (7.2.1.4) and raise the temperature of the
reaction mixture to reflux conditions and maintain for 2 h ensuring that the condensation zone is lower
than 1/3 of the height of the reflux condenser, then allow to cool. Add the content of the absorption
vessel to the reaction vessel via the reflux condenser, rinsing both the absorption vessel and condenser
with further 10 ml of diluted nitric acid (6.4).
Transfer quantitatively the solution content of each vessel into a suitable sized volumetric flask and add
water (6.1) to the volume mark.
Alternatively, another procedure can be applied, such that the adjustment to volume with the solid
residue still present shall be carried out immediately after digestion.
If the measurement solution contains particles due to precipitation which may clog nebulizers or
interfere with an injection of the sample into the instrument, the sample may be centrifuged, allowed to
settle, or filtered (7.5).
The measurement solution is now ready for analysis for elements of interest using appropriate
elemental analysis techniques.
8.3.2 Method A2 — Thermal heating with a heating block with containers
Weigh an amount of not more than 2 g of the test portion (typically 0,5 g to 1 g of dry sample) containing
not more than 0,15 g of organic carbon with an accuracy of 0,001 g (or at least three significant figures)
and transfer it into the digestion vessel (7.2.2.1).
The amount of the test sample depends on the amount of organic matter. The maximum amount of
organic carbon shall not exceed 0,15 g when 8 ml of aqua regia is used. Per additional 0,1 g organic
carbon (more than this 0,15 g), 1 ml of additional concentrated HNO (6.3) shall be added before the
digestion process is started.
N.B.: For some elements, e.g. barium and chromium, the additional volume of HNO is essential in order
to have a sufficient recovery upon digestion.
In case of dry samples moisten the test portion with a few drops of water (6.1). Add (6,0 + 0,1) ml
hydrochloric acid (6.2) followed by (2,0 + 0,1) ml nitric acid (6.3). Let stand at room temperature until
any effervescence almost ceases to allow for slow oxidation of the organic matter in the sample.
The time of standing at room temperature can have an influence on the digestion rate of aqua regia.
For consistency, it is recommended to start heating as soon as possible after the first strong reaction
has ceased.
Loosely screw on the tube cap (not very tight!) and place the digestion vessel on the heating block
(7.2.2.2) and slowly increase the temperature to the boiling point. Keep the temperature on the boiling
point during (120 + 10) min.
Let the vessel cool down to room temperature and fill up with water (6.1) to the volume mark.
If a non-graduated digestion tube is used, transfer quantitatively the solution into a suitable sized
volumetric flask and add water (6.1) to the volume mark. Alternatively, another procedure can be
applied, such that the adjustment to volume with the solid residue still present shall be carried out
immediately after digestion.
6 © ISO 2020 – All rights reserved

If the measurement solution contains particles due to precipitation which may clog nebulizers or interfere
with an injection of the sample into the instrument, the sample may be centrifuged or filtered (7.5).
The measurement solution is now ready for analysis for elements of interest using appropriate
elemental analysis techniques.
8.4 Method B — Microwave digestion with temperature control, closed vessels
Weigh an amount of not more than 2 g of the test portion (typically 0,5 g to 1 g of dry sample) containing
not more than 0,15 g of organic carbon with an accuracy of 0,001 g (or at least three significant figures)
and transfer it into the digestion vessel (7.3.1).
Add for each additional intake of 0,1 g organic carbon 1 ml of concentrated HNO (6.3) before the
digestion process is started.
Referring to the manufacturer’s instructions, the upper limits of mass of the test portion shall be taken
into account.
In case of dry samples moisten the test portion with a few drops of water (6.1). Add separately
(6 ± 0,1) ml of hydrochloric acid (6.2) and (2 ± 0,1) ml of nitric acid (6.3) and mix well.
If a vigorous reaction occurs, allow the reaction cease before capping the vessel. If excessive foaming
occurs, add a drop of anti-foaming agent (6.5).
This method is an operationally defined method, designed to achieve consistent digestion of samples
by specific reaction conditions. The temperature of the digestion mixture in each vessel shall be raised
with a heating rate of approximately 10 °C/min to 15 °C/min to (175 ± 5) °C and remain at (175 ± 5) °C
for (10 ± 1) min. Cool down to room temperature.
Check this procedure regularly with a blank sample of aqua regia.
WARNING — Too high a temperature increase may cause a vigorous, exothermic reaction in
the digestion solution with high pressure increase and blow-off of the security valve. Losses of
analytes are possible.
At the end of the microwave programme, allow the vessels to cool according to the manufacturer’s
instructions before removing them from the microwave system. Cooling of the vessels may be
accelerated by internal or external cooling devices.
After reaching room temperature, check if the microwave vessels maintained their seal throughout the
digestion. Due to the wide variety of vessel designs, a single procedure is not appropriate. Carefully
uncap and vent each vessel in a well-ventilated fume hood according to the manufacturer’s instructions.
Transfer quantitatively the solution content of each vessel into a suitable sized volumetric flask and add
water (6.1) to the volume mark.
Alternatively, another procedure can be applied, such that the adjustment to volume with the solid
residue still present shall be carried out immediately after digestion. If the measurement solution
contains particles due to precipitation which may clog nebulizers or interfere with an injection of the
sample into the instrument, the sample may be centrifuged, allowed to settle, or filtered.
The measurement solution is now ready for analysis for elements of interest using appropriate
elemental analysis techniques.
9 Test report
This test report shall contain at least the following information:
a) the digestion method used, together with a reference to this document, i.e. ISO 54321;
b) identity of the sample;
c) the results;
d) the date of the test;
e) any deviation from this method and report of circumstances that can have affected the results.
8 © ISO 2020 – All rights reserved

Annex A
(informative)
Repeatability and reproducibility data for soil, biowaste and
sludge samples
The interlaboratory comparison of digestion of aqua regia soluble fractions of trace elements in sludge,
treated biowaste and soil was carried out with contributions by 20 to 23 laboratories from European
countries on five materials. Detailed information can be found in Reference [27].
Table A.1 lists the types of materials tested.
Table A.1 — Materials tested and parameters analysed in the interlaboratory comparison of
the digestion for the extraction of aqua regia soluble fractions of trace elements in soil, treated
biowaste and sludge
Grain size Sample Material Parameters
Mix 1 of municipal waste water treatment
Sludge 1 plant sludges from North Rhine As, Cd, Cr, Cu, Fe, Mn, Ni, P, Pb, Zn
Sludge
Westphalia, Germany
Mix 2 of municipal waste water treatment
(<0,5 mm)
Sludge 2 plant sludges from North Rhine As, Cd, Cr, Cu, Fe, Mn, Ni, P, Pb, Zn
Westphalia, Germany
Compost 2 Compost from Germany As, Cd, Cr, Cu, Fe, Mn, Ni, P, Pb, Zn
Fine grained
Soil 1 A sludge amended soil from Pavia, Italy As, Cd, Cr, Cu, Fe, Mn, Ni, P, Pb, Zn
(<2,0 mm)
A sludge amended soil from Düsseldorf,
Soil 2 As, Cd, Cr, Cu, Fe, Mn, Ni, P, Pb, Zn
Germany
Annex B
(informative)
Repeatability and reproducibility data for waste samples
B.1 Inter-laboratory study (methods A1 and B)
B.1.1 General
During 1998 to 1999 a project for validation of EN 13657 has been organised and carried out. The
validation included an inter-laboratory study for evaluation of performance characteristics of methods
included in the standard (reproducibility, repeatability, accuracy where applicable), and a robustness
study (i.e. the evaluation of the influence of some defined operational parameters on the methods).
The validation included method A1 (Digestion by thermal heating, with aqua regia in reflux systems)
and method B (Microwave assisted digestion with aqua regia in closed vessels).
B.1.2 Selection of laboratories
A questionnaire has been circulated by all CEN/TC 292 ‘Characterization of waste’ members to
collect a list of interested European laboratories. About seventy laboratories gave their availability to
participate to the inter-laboratory trial. All of them were asked to declare that they fulfil the minimum
requirements to carry out digestion and analyses according to this standard. According to the ISO 5725
series no selection has been made in advance on the basis of the supposed “ability” of laboratories, their
certifications, etc: it’s therefore possible to assume that participating laboratories are a rather good
“sample” of “normal” European laboratories.
B.1.3 Selection of samples
The materials to be used in the inter-laboratory study had to satisfy all the following requisites:
— representative of a wide range of matrices, as much as possible;
— available in a homogeneous form or, alternatively, not too difficult to grind, sieve and homogenise;
— available in a sufficient quantity.
After a survey, the following materials have been found:
— city waste incineration fly ash (“oxidised” matrix) (CEN6/99 FLY ASH CW6 POWDER);
— city waste incineration bottom ash (“silicate” matrix) (CEN7/99 “ASH CW4 POWDER”);
— ink waste sludge (organic matrix) (CEN8/99 “INK WASTE CW12 POWDER”);
— electronic industry sludge (“metallic” matrix) (CEN9/99 “SEWAGE SLUDGE SL11 POWDER”);
For the evaluation of performances of digestion procedures, independently from the subsequent
analyses performed on digested samples, all laboratories have been asked to analyse some already-
prepared aqueous solutions with different degrees of difficulty (clean synthetic solutions, acid digested
solutions of the above four materials). This has been used as a tool for discarding from the evaluation
laboratories that did not prove their analytical ability for some matrices/elements.
For accuracy evaluation, two certified reference material (CRM) have been also included:
— BCR 146 R (sewage sludge);
10 © ISO 2020 – All rights reserved

— BCR 176 (city waste incineration ash).
All samples, including the two CRMs, have been delivered to laboratories in anonymous form.
B.1.4 Experimental
Preparation and homogenisation of samples, packaging, delivering, collection and evaluation of results
have been carried out by Environmental Monitoring Sector of European Commission Joint Research
Centre in Ispra (Italy).
B.1.5 Results
About fifty laboratories have actually returned results for the inter-laboratory study. The evaluation of
results has been performed by following these steps:
— removing of “obviously erroneous data”, both means and single data according to ISO 5725-2:1994,
7.2.6;
— results from laboratories failing to correctly measure some elements in “clean metals” solution
were removed from the whole data set (for the failed elements only);
— results from laboratories failing to correctly measure some elements in digested aqueous solutions
were removed from the whole data set (for the failed elements only);
— the remaining data sets were evaluated according to ISO 5725 series, with calculation of repeatability,
reproducibility and, where a “conventional true value” was available, accuracy (recovery); results of
this evaluation are reported in the tables below.
The inter-laboratory study involved a large number of laboratories, performing analyses in four
replicates on several samples (five aqueous, six powders), for the determination of a large number of
elements (up to 31), by using one to three digestion methods: this led to a very large data set. For some
digestion methods and for some elements determination, only few data were available (a minimum
of 24 outlier-free results is generally required); anyway, even for these methods and elements, useful
information on performance has been obtained.
B.1.6 Conclusions
The performances of the three methods should be compared on an element-by-element, matrix-by-
matrix basis, in the tables below. In general, performances are considered to be acceptably consistent,
especially for most environmentally-sensitive (toxic) elements.
Recovery rates for CRM: sewage sludge (BCR 146 R, non-refractory matrix) are in generally high, for
CRM: city waste incineration ash (BCR 176, refractory matrix) in many cases low. Digestion with aqua
regia will not necessarily release elements completely from many geological matrices.
B.2 Inter-laboratory study (method A2)
B.2.1 General
Between October 2018 and February 2019, a project for validation of method A2 (Digestion by thermal
heating, with aqua regia, with a heating block with containers) was organised and carried out. The
validation included an inter-laboratory study for evaluation of performance characteristics of the
method (reproducibility, repeatability, accuracy where applicable).
B.2.2 Selection of laboratories
Several CEN/TC 444 members were requested to circulate information about the inter-laboratory trial
and collect a list of interested European laboratories. Twelve laboratories gave their availability to
participate to the inter-laboratory trial. All laboratories carried out the digestion with a heating block
at regular basis. The laboratories were informed about the necessity to carry out digestion and analysis
according to this document. According to ISO 5725 series no selection has been made in advance on the
basis of the supposed “ability” of laboratories, their certifications, etc. it’s therefore possible to assume
that participating laboratories are a rather good “sample” of “normal” European laboratories.
B.2.3 Selection of samples
Two samples were taken from the interlaboratory trial for the validation of EN 13657:
— ink waste sludge (organic matrix) (CEN 8/99 “INK WASTE CW12 POWDER”);
— electronic industry sludge (“metallic” matrix) (CEN 9/99 “SEWAGE SLUDGE SL11 POWDER”);
For accuracy evaluation, two certified reference materials (CRM) have been included, as well as two
[27] [28]
materials used in robustness validation tests for CEN/TC 351 :
— ISE 859 (sediment);
— BCR 176R (city waste incineration ash, with identical matrix as BCR 176, used in the inter laboratory
trial for the evaluation of EN 13657);
— CFA Coal fly ash;
— GSS Steel slag;
A sample material “Bottom ash” was added for the interlaboratory trial.
All samples, including the two CRMs, have been delivered to laboratories in anonymous form.
B.2.4 Experimental
Preparation and homogenisation of samples, packaging, delivering, collection and evaluation of results
have been carried out by VITO NV in Mol (Belgium) and Synlab Analytics & Services B.V. in Rotterdam
(The Netherlands).
B.2.5 Results
All twelve laboratories have returned results for the inter-laboratory study. The means and standard
deviations of reproducibility have been evaluated according to ISO 13528:2015, C.1, following these steps:
— removing of data with a value below the detection limit (“<”-values);
— data have been evaluated if the mean measured concentration is above 1 mg/kg dm (for Hg
0,1 mg/kg dm);
— data have been evaluated only if more than six results remain;
— for the remaining data sets, means and standard deviations were calculated as Huber-M estimates
with Winsorising at 1,5 standard deviations;
— where a “conventional true value” was available, accuracy (recovery) has been calculated.
The inter-laboratory study involved twelve laboratories, performing analyses in two replicates on seven
samples, for the determination of a large number of elements (up to 28): this led to a large data set. For
some elements, only few data were available, anyway, even for these elements, useful information on
performance has been obtained.
B.2.6 Conclusions
The performances of the method should be compared on an element-by-element, matrix-by-matrix
basis, in the tables below. In general, the performance of method A2 is considered to be acceptably
consistent with methods A1 and B, especially for most environmentally-sensitive (toxic) elements.
12 © ISO 2020 – All rights reserved

Recovery rates for CRM: sediment (ISE 859) and for samples used in former robustness validation
tests: CFA Coal fly ash and GSS Steel slag, are in generally high. For CRM: city waste incineration ash
(BCR 176R, refractory matrix) in many cases low, which agrees with conclusions of B.1.5. Digestion
with aqua regia will not necessarily release elements completely from many geological matrices.
14 © ISO 2020 – All rights reserved
Table B.1 — Analytical results, SAMPLE CEN 6/99 “FLY ASH CW6 POWDER”
SAMPLE CEN 6/99 “FLY ASH CW6 POWDER”
Method A1: Thermal heating, with aqua regia in reflux systems Method B: Microwave assisted, with aqua regia in closed vessels
XREF Mean Recov Reprod Repeat XREF Mean Recov Reprod Repeat
N L NA N L NA
mg/kg mg/kg % % % mg/kg mg/kg % % %
Al 38 9 0 41 811 20,2 2,8 71 18 0 40 382 25,4 4,6
Sb 18 4 0 800,1 21,2 2 52 12 0 980,8 24,4 4,6
As 36 8 0 35,22 26,5 7,7 57 14 0 41,67 30,6 8,5
B 19 4 0 269,6 25,1 6,8 37 9 1 210,2 16,2 3,7
Ba 19 4 5 196,6 29,5 14,7 58 14 1 844,3 36,8 11,3
Be 9 2 0 0,91 9,6 2,6 26 6 5 0,92 11,7
...