SIST EN 14735:2022
(Main)Characterization of waste - Preparation of waste samples for ecotoxicity tests
Characterization of waste - Preparation of waste samples for ecotoxicity tests
This European Standard describes the necessary steps to be performed before carrying out ecotoxicity tests on wastes. The purpose of this European Standard is to provide guidance on the taking of the sample, transport, storage of wastes and to define preparation, for the determination of ecotoxicological properties of wastes under the conditions specified in this European Standard by biological testing either as raw wastes or water extracts from wastes. Sample preparation for other applications (e.g. assessment of waste effects on aquatic and terrestrial organisms in a disposal scenario) is not considered.
Specifying a test battery to characterize ecotoxicological properties of wastes is not in the scope of this European Standard.
This European Standard is applicable to solid and liquid wastes.
Charakterisierung von Abfällen - Herstellung von Abfallproben für ökotoxikologische Untersuchungen
Dieses Dokument beschreibt die erforderlichen Arbeitsschritte, die durchzuführen sind, bevor Ökotoxizitäts¬prüfungen an Abfällen durchgeführt werden. Der Zweck dieses Dokuments ist, eine Anleitung für Probe¬nahme, Transport und Lagerung von Abfällen zu geben und die Vorbereitungen für die Bestimmung ökotoxischer Eigenschaften von Abfällen festzulegen; diese Bestimmung erfolgt unter den in diesem Dokument festgelegten Bedingungen durch biologische Prüfungen an Rohabfällen oder wässrigen Extrakten aus Abfällen. Die Probenvorbereitung für andere Anwendungsweisen (z. B. Beurteilung der Wirkungen von Abfällen auf Boden und Wasserorganismen im Rahmen einer Entsorgung) wird nicht berücksichtigt.
Die Festlegung einer Prüfbatterie zur Charakterisierung der ökotoxischen Eigenschaften von Abfällen liegt nicht im Aufgabenbereich dieses Dokumentes.
Dieses Dokument ist auf feste und flüssige Abfälle anwendbar.
Caractérisation des déchets - Caractérisation des déchets - Préparation des échantillons de déchets en vue d'essais écotoxicologiques
L’écotoxicité peut être estimée à l’aide de deux approches : une approche chimique spécifique et une approche fondée sur la toxicité. Dans le premier cas, des analyses chimiques sont comparées à des critères de qualité ou à des valeurs seuils pour évaluer l’écotoxicité. Dans le second cas, l’écotoxicité est mesurée directement à l’aide d’essais biologiques. Ces deux approches sont complémentaires. En effet, la détermination des polluants dans des mélanges complexes de composition inconnue, caractéristique d’un grand nombre de déchets, ne permet pas une évaluation pertinente de l’écotoxicité. Pour ce type d’échantillon, il est, en général, admis que l’approche toxicologique constitue une solution appropriée pour l’évaluation de la toxicité potentielle. En effet, les bioessais intègrent les effets de tous les polluants, y compris les effets additifs, synergiques et antagonistes. Ils sont uniquement sensibles à la fraction biodisponible des polluants. Enfin, ils intègrent les effets de tous les polluants, même ceux qui ne sont pas pris en compte ou détectés par des analyses chimiques.
Les essais d’écotoxicité peuvent être effectués sur des déchets afin d’identifier leurs propriétés présentant un danger potentiel pour l’environnement à des fins de classification ou d’évaluation du risque associé à un scénario d’exposition spécifique d’un site.
Karakterizacija odpadkov - Priprava vzorcev odpadka za ekotoksikološke preskuse
Ta evropski standard opisuje potrebne korake, ki jih je treba izvesti pred izvedbo ekotoksikoloških preskusov odpadkov. Namen tega evropskega standarda je podati smernice za odvzem vzorcev, prevoz in skladiščenje odpadkov ter opredeliti pripravo za določanje ekotoksikoloških lastnosti odpadkov pod pogoji, določenimi v tem evropskem standardu, z biološkim preskušanjem surovih odpadkov ali ekstraktov vode iz odpadkov. Priprava vzorcev za druge vrste uporabe (npr. ocenjevanje vplivov odpadkov na vodne in kopenske organizme v primeru odlaganja) ni obravnavana.
Določitev serije preskusov za karakterizacijo ekotoksikoloških lastnosti odpadkov ne spada na področje uporabe tega evropskega standarda.
Ta evropski standard se uporablja za trdne in tekoče odpadke.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 14735:2022
01-januar-2022
Nadomešča:
SIST EN 14735:2005
SIST EN 14735:2005/AC:2007
Karakterizacija odpadkov - Priprava vzorcev odpadka za ekotoksikološke preskuse
Characterization of waste - Preparation of waste samples for ecotoxicity tests
Charakterisierung von Abfällen - Herstellung von Abfallproben für ökotoxikologische
UntersuchungenCaractérisation des déchets - Caractérisation des déchets - Préparation des échantillons
de déchets en vue d'essais écotoxicologiquesTa slovenski standard je istoveten z: EN 14735:2021
ICS:
13.030.01 Odpadki na splošno Wastes in general
SIST EN 14735:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------SIST EN 14735:2022
---------------------- Page: 2 ----------------------
SIST EN 14735:2022
EN 14735
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 13.030.01 Supersedes EN 14735:2005
English Version
Characterization of waste - Preparation of waste samples
for ecotoxicity tests
Caractérisation des déchets - Préparation des Charakterisierung von Abfällen - Herstellung von
échantillons de déchets en vue d'essais Abfallproben für ökotoxikologische Untersuchungen
écotoxicologiquesThis European Standard was approved by CEN on 3 October 2021.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14735:2021 E
worldwide for CEN national Members.---------------------- Page: 3 ----------------------
SIST EN 14735:2022
EN 14735:2021 (E)
Contents Page
European foreword ....................................................................................................................................................... 4
Introduction .................................................................................................................................................................... 5
1 Scope .................................................................................................................................................................... 6
2 Normative references .................................................................................................................................... 6
3 Terms and definitions ................................................................................................................................... 7
4 Equipment and reagents............................................................................................................................... 9
5 Taking of laboratory sample ....................................................................................................................... 9
6 Transport ........................................................................................................................................................ 10
7 Storage ............................................................................................................................................................. 10
7.1 General ............................................................................................................................................................. 10
7.2 Waste sample ................................................................................................................................................. 10
7.3 Water extracts ............................................................................................................................................... 10
8 Waste characterization .............................................................................................................................. 11
9 Waste pre-treatment: particle size reduction (granular waste, monolithic waste,
paste-like waste and sludge) .................................................................................................................... 11
10 Tests performed on terrestrial organisms ......................................................................................... 12
10.1 General considerations .............................................................................................................................. 12
10.2 Dilution medium .......................................................................................................................................... 13
10.3 Introduction of waste into the dilution medium .............................................................................. 13
10.3.1 General ............................................................................................................................................................. 13
10.3.2 Monolithic waste, granular waste, paste-like waste and sludge ................................................. 13
10.3.3 Liquid sludge .................................................................................................................................................. 13
10.3.4 Liquid waste miscible with water .......................................................................................................... 14
10.3.5 Liquid waste non-miscible with water ................................................................................................. 14
10.4 Water extracts of waste ............................................................................................................................. 14
10.5 pH ....................................................................................................................................................................... 14
10.6 Addition of test organisms ........................................................................................................................ 15
11 Tests performed on aquatic organisms ............................................................................................... 15
11.1 General considerations .............................................................................................................................. 15
11.2 Monolithic waste, granular waste, paste-like waste and sludge ................................................. 15
11.2.1 Leaching procedure ..................................................................................................................................... 15
11.2.2 Liquid/solid separation procedure ....................................................................................................... 16
11.2.3 pH ....................................................................................................................................................................... 16
11.3 Liquid sludge .................................................................................................................................................. 17
11.3.1 Procedure........................................................................................................................................................ 17
11.3.2 pH ....................................................................................................................................................................... 17
11.4 Liquid waste miscible with water .......................................................................................................... 17
11.4.1 Procedure........................................................................................................................................................ 17
11.4.2 pH ....................................................................................................................................................................... 17
11.5 Liquid waste non-miscible with water ................................................................................................. 17
11.5.1 Procedure........................................................................................................................................................ 17
11.5.2 Liquid/liquid separation ........................................................................................................................... 18
11.5.3 pH ....................................................................................................................................................................... 18
---------------------- Page: 4 ----------------------SIST EN 14735:2022
EN 14735:2021 (E)
11.6 Preparation of test mixtures .................................................................................................................... 18
12 Test report ...................................................................................................................................................... 18
Annex A (normative) Preparation of test mixtures according to the ecotoxicity tests to be
performed ....................................................................................................................................................... 19
Annex B (informative) Ecotoxicity tests considered to establish this document ............................... 20
B.1 General ............................................................................................................................................................. 20
B.2 Terrestrial tests methods .......................................................................................................................... 21
B.2.1 Earthworms – Acute toxicity ..................................................................................................................... 21
B.2.2 Earthworms – Effects on reproduction ................................................................................................. 22
B.2.3 Earthworms – Avoidance ........................................................................................................................... 23
B.2.4 Collembola – Effects on reproduction ................................................................................................... 24
B.2.5 Coleoptera – Acute test ............................................................................................................................... 25
B.2.6 Enchytraeid – Reproduction test ............................................................................................................ 26
B.2.7 Nematoda – Effects on growth, fertility and reproduction ............................................................ 27
B.2.8 Juvenile land snails (Helix aspersa) ....................................................................................................... 28
B.2.9 Soil Flora – Inhibition of root growth .................................................................................................... 29
B.2.10 Effects on emergence and growth ........................................................................................................... 31
B.2.11 Ammonium oxidation – Rapid test ......................................................................................................... 33
B.2.12 Mineralization and nitrification .............................................................................................................. 34
B.2.13 Dehydrogenase activity of Arthrobacter globiformis....................................................................... 35
B.3 Aquatic tests methods ................................................................................................................................. 37
B.3.1 Daphnia magna – Inhibition of mobility .............................................................................................. 37
B.3.2 Daphnia magna – Inhibition of reproduction ..................................................................................... 38
B.3.3 Ceriodaphnia dubia reproduction test .................................................................................................. 39
B.3.4 Brachionus calyciflorus reproduction test ........................................................................................... 40
B.3.5 Aliivibrio fischeri – Luminescent bacteria test ................................................................................... 41
B.3.6 Pseudomonas putida growth inhibition test ....................................................................................... 42
B.3.7 Freshwater algal growth inhibition test ............................................................................................... 43
B.3.8 Lemna minor – Growth inhibition test .................................................................................................. 45
B.3.9 Fish egg test .................................................................................................................................................... 46
B.3.10 Freshwater fish acute toxicity test ......................................................................................................... 47
B.3.11 Marine copepods – Acute toxicity test ................................................................................................... 48
B.3.12 Marine algal growth inhibition test ....................................................................................................... 49
B.3.13 Salmonella / Microsome test .................................................................................................................... 51
B.3.14 UMU test ........................................................................................................................................................... 53
Bibliography ................................................................................................................................................................. 54
---------------------- Page: 5 ----------------------SIST EN 14735:2022
EN 14735:2021 (E)
European foreword
This document (EN 14735:2021) has been prepared by Technical Committee CEN/TC 444
“Environmental characterization of solid matrices”, the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2022, and conflicting national standards shall be
withdrawn at the latest by May 2022.Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 14735:2005.The main changes with respect to the previous edition are:
— the modification of Clause 12 “Test report” to specify whether ecotoxicity tests have been repeated
with pH adjustment of the test portion, allowing to clearly distinguish these results from those
obtained without pH adjustment;— the clarification of the notes of 10.5 and 11.2.3 and associated notes regarding the repetition of the
tests with pH adjustment;— the update of Annex B, describing the ecotoxicity tests considered to establish this document.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.---------------------- Page: 6 ----------------------
SIST EN 14735:2022
EN 14735:2021 (E)
Introduction
Ecotoxicity can be estimated using two approaches: a chemical-specific approach and a toxicity-based
approach. Chemical analyses are compared, in the first case, to quality criteria or threshold values to
estimate ecotoxicity. In the second one, ecotoxicity is measured directly using biological tests. These two
approaches complement each other; indeed, determination of pollutants in complex mixtures of
unknown composition, that is a characteristic of many wastes, does not allow a relevant estimation of
ecotoxicity. For such samples, the toxicity-based approach is usually recognized to be appropriate to
assess potential toxicity. Bioassays integrate, indeed, the effects of all contaminants including additive,
synergistic and antagonistic effects. They are sensitive to the bioavailable fraction of the contaminants
only. Finally, bioassays integrate the effects of all contaminants, including those, not considered or
detected by chemical analyses.Ecotoxicity tests can be applied to wastes to identify their potential hazardous properties with respect to
the environment for classification purposes or to assess the risk related to a site-specific exposure
scenario.Identification of properties potentially hazardous to the environment for classification purposes
A classification system, based on the assessment of intrinsic properties, should be independent of an
exposure scenario. The main requirement, in order to establish a relevant system for classifying wastes
and for assessment of hazard properties, is to obtain comparable test results. This can only be obtained
if the ecotoxicity tests on wastes are carried out according to a unique procedure describing more or less
conventional test conditions (an exclusive dilution medium for terrestrial tests, a unique L/S ratio for
preparation of water extracts, a unique liquid / solid separation step, etc.). This procedure should be
applicable to a very wide range of waste materials whatever their physical properties are.
Any strategy for the assessment of properties potentially hazardous to the environment used in a
classification system should include test organisms representing the terrestrial and the aquatic
compartment. Both types of tests should be considered because they expand the range of effect
expression due to differences in species sensitivity and exposure.For this specific purpose, the water extracts preparations for toxicity testing do not simulate leaching
from wastes under environmental conditions but measure the water available fraction of the toxic
components of the wastes.Site-specific exposure scenario
The second application of ecotoxicity tests to wastes refers to a risk assessment approach. In this
particular case, the test strategy should model site specific exposure conditions and should take into
account the transfer of contaminants via the food chain and to surface and ground water by run-off or
leaching. This application concerns firstly the definition of generic scenarios frequently encountered (e.g.
wastes deposit in stockpiles, re-use of wastes) and focus on the relevant way of exposure to terrestrial
and aquatic organisms.This document describes the necessary steps to be performed before carrying out ecotoxicity tests on
wastes within the context of assessment of ecotoxic properties for classification purposes.
It should be kept in mind that the use of this standard is currently not mandatory for assessing the
hazardous property HP14.---------------------- Page: 7 ----------------------
SIST EN 14735:2022
EN 14735:2021 (E)
1 Scope
This document describes the necessary steps to be performed before carrying out ecotoxicity tests on
wastes. The purpose of this document is to provide guidance on the taking of the sample, transport,
storage of wastes and to define preparation, for the determination of ecotoxicological properties of
wastes under the conditions specified in this document by biological testing either as raw wastes or water
extracts from wastes. Sample preparation for other applications (e.g. assessment of waste effects on
aquatic and terrestrial organisms in a disposal scenario) is not considered.Specifying a test battery to characterize ecotoxicological properties of wastes is not in the scope of this
document.This document is applicable to solid and liquid wastes.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 12457-2:2002, Characterisation of waste - Leaching - Compliance test for leaching of granular waste
materials and sludges - Part 2: One stage batch test at a liquid to solid ratio of 10 l/kg for materials with
particle size below 4 mm (without or with size reduction)EN 14899, Characterization of waste - Sampling of waste materials - Framework for the preparation and
application of a Sampling Plan)EN 16720-1, Characterization of sludges - Physical consistency - Part 1: Determination of flowability -
Method by extrusion tube apparatus)EN ISO 5667-3, Water quality - Sampling - Part 3: Preservation and handling of water samples (ISO 5667-
EN ISO 14238:2012, Soil quality - Biological methods - Determination of nitrogen mineralization and
nitrification in soils and the influence of chemicals on these processes (ISO 14238: 2012)
ISO 11465, Soil quality - Determination of dry matter and water content on a mass basis - Gravimetric
methodEN ISO 10390, Soil, sludge and treated biowaste - Determination of pH (ISO/FDIS 10390)
---------------------- Page: 8 ----------------------SIST EN 14735:2022
EN 14735:2021 (E)
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 https://www.electropedia.org/
3.1
dilution medium
liquid or solid used for the preparation of control vessels and the preparation of test mixtures
3.2ecotoxicological properties
potential adverse effects to biological systems which a waste has an inherent capacity to cause
3.3eluate
solution recovered from a leaching test
[SOURCE: EN 12457-2:2002, 3.3]
3.4
granular waste
waste not being monolithic, nor a liquid, a gas or a sludge
[SOURCE: adapted from EN 12457-2:2002, 3.10]
3.5
laboratory sample
sample or subsample(s) sent to or received by the laboratory (IUPAC definition)
Note 1 to entry: When the laboratory sample is further prepared (reduced) by subdividing, mixing, grinding or by
combinations of these operations, the result is the test sample. When no preparation of the laboratory sample is
required, the laboratory sample is the test sample. A test portion is removed from the test sample for the
performance of the test or for analysis. The laboratory sample is the final sample from the point of view of sample
collection, but it is the initial sample from the point of view of the laboratory.
Note 2 to entry: Several laboratory samples can be prepared and sent to different laboratories or to the same
laboratory for different purposes. When sent to the same laboratory, the set is generally considered as a single
laboratory sample and is documented as a single sample.3.6
leachant
liquid used in a leaching test
Note 1 to entry: For the purpose of this document the leachant is water as specified in Clause 4.
3.7leaching test
test during which a material is put into contact with a leachant and some constituents of the material are
extracted---------------------- Page: 9 ----------------------
SIST EN 14735:2022
EN 14735:2021 (E)
3.8
liquid sludge
sludge in a liquid physical state, determined according to EN 16720-1
3.9
liquid waste
waste that completely flows out of a calibrated opening, down to the upper level of the opening within a
limited period of time (see Annex B of EN 12457-2:2002)3.10
monolithic waste
waste prepared with a certain minimum dimensions and physical and mechanical properties that ensure
its integrity over a certain period of time in the considered scenario[SOURCE: CEN/TS 16675:2018, 3.7]
3.11
paste-like material
material of soft plastic or wet cement consistency – usually smooth
3.12
sludge
mixture of liquid and solid separated from various types of liquid as a result of natural or artificial
processes[SOURCE: adapted from EN 12457-2:2002, 3.8]
3.13
test mixture
mixture of the test portion (waste or water extract) with the dilution medium (3.1)
3.14test portion
amount or volume of the test sample taken for measurement of ecotoxicological properties by biological
testing and/or other properties of interest, usually of known weight or volume (adapted from IUPAC
definition)Note 1 to entry: The test portion can 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,
can be a test portion.3.15
test sample
sample, prepared from the laboratory sample, from which test portions are removed for biological testing
or analysis (adapted from IUPAC definition)Note 1 to entry: The preparation of the test sample can include particle size reduction, preparation of water
extract, etc.---------------------- Page: 10 ----------------------
SIST EN 14735:2022
EN 14735:2021 (E)
3.16
water extract
solution obtained from a leaching test, a liquid/liquid extraction and a liquid/solid separation
(centrifugation)4 Equipment and reagents
Usual laboratory equipment and the following.
4.1 Sieving equipment, e.g. with sieves of 4 mm square mesh.
NOTE Due to sieving, contamination of the sample can occur to an extent that affects the leaching of some
constituents of concern e.g. cobalt and tungsten from tungsten carbide equipment or chromium, nickel and
molybdenum from stainless steel equipment.4.2 Crushing equipment: jaw crusher or cutting device.
NOTE Due to crushing, contamination of the sample can occur to an extent that affects the leaching of some
constituents of concern e.g. cobalt and tungsten from tungsten carbide equipment or chromium, nickel and
molybdenum from stainless steel equipment.4.3 Balance of accuracy of at least 0,1 g.
4.4 End-over-end tumbler (5 rpm to 10 rpm) or rollertable rotating at about 10 rpm.
NOTE Other shaking or mixing devices can be used provided that they are proven to be equivalent.
4.5 Centrifuge that can attain a RCF between 2 000 g and 3 000 g.4.6 Filtering apparatus, either a vacuum filtration device (between 30 kPa and 70 kPa) (300 mbars
to 700 mbars) or a high-pressure filtration apparatus (<0,5 MPa) (5 bars).4.7 Large scale laboratory mixer.
4.8 pH meter.
4.9 Glass or high-density polyethylene (HDPE)/polypropylene (PP) bottles in accordance with
EN ISO 5667-3, glass bottles having caps of inert material, for example polytetrafluoroethylene. Rinsing
with sample material or water (4.10) is compulsory.NOTE For inorganic constituents HDPE/PP bottles are preferred, except for samples containing mercury.
4.10 Water, distilled water, demineralized water or deionized water with a conductivity < 5 µS/cm.
5 Taking of laboratory sampleObtain a representative laboratory sample of a quantity sufficient for the number of tests to be performed
and in accordance with the requirements of biological standardized methods to be used.
Special precautions should be taken to avoid any contamination of laboratory samples by material of
sampling devices and/or storage equipment, according to EN 14899.NOTE Sampling devices are described in the technical report CEN/TR 15310-2.
---------------------- Page: 11 ----------------------
SIST EN 14735:2022
EN 14735:2021 (E)
Some wastes are subject to chemical, physical and biological changes as soon as they are collected (e.g.
wastes that are fermentable, subject to oxidation or carbonation and wastes that contain volatile
substances). Possible changes shall be considered and sampling conditions shall be designed accordingly
in order to limit the effects of such changes on the results of ecotoxicity...
SLOVENSKI STANDARD
oSIST prEN 14735:2020
01-april-2020
Karakterizacija odpadkov - Priprava vzorcev odpadka za ekotoksikološke preskuse
Characterization of waste - Preparation of waste samples for ecotoxicity tests
Charakterisierung von Abfällen - Herstellung von Abfallproben für ökotoxikologische
UntersuchungenCaractérisation des déchets - Caractérisation des déchets - Préparation des échantillons
de déchets en vue d'essais écotoxicologiquesTa slovenski standard je istoveten z: prEN 14735
ICS:
13.030.01 Odpadki na splošno Wastes in general
oSIST prEN 14735:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------oSIST prEN 14735:2020
---------------------- Page: 2 ----------------------
oSIST prEN 14735:2020
DRAFT
EUROPEAN STANDARD
prEN 14735
NORME EUROPÉENNE
EUROPÄISCHE NORM
March 2020
ICS 13.030.01 Will supersede EN 14735:2005
English Version
Characterization of waste - Preparation of waste samples
for ecotoxicity tests
Caractérisation des déchets - Caractérisation des Charakterisierung von Abfällen - Herstellung von
déchets - Préparation des échantillons de déchets en Abfallproben für ökotoxikologische Untersuchungen
vue d'essais écotoxicologiquesThis draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 444.If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 14735:2020 E
worldwide for CEN national Members.---------------------- Page: 3 ----------------------
oSIST prEN 14735:2020
prEN 14735:2020 (E)
Contents Page
European foreword ............................................................................................................................... 4
Introduction ............................................................................................................................................. 5
1 Scope ............................................................................................................................................ 6
2 Normative references ............................................................................................................ 6
3 Terms and definitions ............................................................................................................ 7
4 Equipment and reagents ....................................................................................................... 9
5 Taking of laboratory sample ............................................................................................... 9
6 Transport ..................................................................................................................................10
7 Storage .......................................................................................................................................10
7.1 General ......................................................................................................................................10
7.2 Waste sample ..........................................................................................................................10
7.3 Water extracts ........................................................................................................................10
8 Waste characterization .......................................................................................................11
9 Waste pre-treatment: particle size reduction (granular waste, monolithicwaste, paste-like waste and sludge) ...............................................................................11
10 Tests performed on terrestrial organisms ...................................................................12
10.1 General considerations .......................................................................................................12
10.2 Dilution medium ....................................................................................................................13
10.3 Introduction of waste into the dilution medium ........................................................13
10.3.1 General ......................................................................................................................................13
10.3.2 Monolithic waste, granular waste, paste-like waste and sludge ..........................13
10.3.3 Liquid sludge ...........................................................................................................................13
10.3.4 Liquid waste miscible with water ....................................................................................13
10.3.5 Liquid waste non-miscible with water ...........................................................................14
10.4 Water extracts of waste .......................................................................................................14
10.5 pH ................................................................................................................................................14
10.6 Addition of test organisms .................................................................................................15
11 Tests performed on aquatic organisms .........................................................................15
11.1 General considerations .......................................................................................................15
11.2 Monolithic waste, granular waste, paste-like waste and sludge ..........................15
11.2.1 Leaching procedure ..............................................................................................................15
11.2.2 Liquid/solid separation procedure.................................................................................16
11.2.3 pH ................................................................................................................................................16
11.3 Liquid sludge ...........................................................................................................................17
11.3.1 Procedure .................................................................................................................................17
11.3.2 pH ................................................................................................................................................17
11.4 Liquid waste miscible with water ....................................................................................17
11.4.1 Procedure .................................................................................................................................17
11.4.2 pH ................................................................................................................................................17
11.5 Liquid waste non-miscible with water ...........................................................................17
11.5.1 Procedure .................................................................................................................................17
11.5.2 Liquid/liquid separation ....................................................................................................17
11.5.3 pH ................................................................................................................................................18
---------------------- Page: 4 ----------------------oSIST prEN 14735:2020
prEN 14735:2020 (E)
11.6 Preparation of test mixtures .............................................................................................18
12 Test report ...............................................................................................................................18
Annex A (normative) Preparation of test mixtures according to the ecotoxicity tests
to be performed......................................................................................................................19
Annex B (informative) Ecotoxicity tests considered to establish this document ........20
B.1 General ......................................................................................................................................20
B.2 Terrestrial tests methods ...................................................................................................20
B.2.1 Earthworms – Acute toxicity ..............................................................................................20
B.2.2 Earthworms – Effects on reproduction ..........................................................................22
B.2.3 Earthworms – Avoidance ....................................................................................................23
B.2.4 Collembola – Effects on reproduction ............................................................................24
B.2.5 Coleoptera – Acute test ........................................................................................................25
B.2.6 Enchytraeid – Reproduction test .....................................................................................27
B.2.7 Nematoda — Effects on growth, fertility and reproduction ...................................28
B.2.8 Juvenile land snails (Helix aspersa) ................................................................................29
B.2.9 Soil Flora – Inhibition of root growth .............................................................................30
B.2.10 Effects on emergence and growth ....................................................................................33
B.2.11 Ammonium oxidation – Rapid test ..................................................................................34
B.2.12 Mineralization and nitrification .......................................................................................35
B.2.13 36B.3 Aquatic tests methods ..........................................................................................................38
B.3.1 Daphnia magna – Inhibition of mobility .......................................................................38
B.3.2 Daphnia magna – Inhibition of reproduction ..............................................................39
B.3.3 Ceriodaphnia dubia reproduction test ...........................................................................40
B.3.4 Brachionus calicyflorus reproduction test ...................................................................41
B.3.5 Vibrio fischeri – Luminescent bacteria test ..................................................................42
B.3.6 Pseudomonas putida growth inhibition test ................................................................44
B.3.7 Freshwater algal growth inhibition test ........................................................................45
B.3.8 Lemna minor – Growth inhibition test ...........................................................................46
B.3.9 Fish egg test .............................................................................................................................47
B.3.10 Freshwater fish acute toxicity test ..................................................................................48
B.3.11 Marine copepods – Acute toxicity test ............................................................................49
B.3.12 Marine algal growth inhibition test ................................................................................50
B.3.13 Salmonella / Microsome test .............................................................................................51
B.3.14 UMU test ....................................................................................................................................53
Bibliography ..........................................................................................................................................54
---------------------- Page: 5 ----------------------oSIST prEN 14735:2020
prEN 14735:2020 (E)
European foreword
This document (prEN 14735:2020) has been prepared by Technical Committee CEN/TC 444 “Test
methods for environmental characterization of solid matrices”, the secretariat of which is held by
NEN.This document is currently submitted to the CEN Enquiry.
This document will supersede EN 14735:2005.
The main changes with respect to the previous edition are:
— the modification of Clause 12 “Test report” to specify when ecotoxicity tests have been
repeated with pH adjustment of the test portion, allowing to clearly distinguish these results
from those obtained without pH adjustment;— the clarification of the note of subclauses 10.5 and 11.2.3 regarding the repetition of the test
with pH adjustment;— the update of Annex B, describing the ecotoxicity tests considered to establish this document.
---------------------- Page: 6 ----------------------oSIST prEN 14735:2020
prEN 14735:2020 (E)
Introduction
Ecotoxicity can be estimated using two approaches: a chemical-specific approach and a toxicity-
based approach. Chemical analyses are compared, in the first case, to quality criteria or threshold
values to estimate ecotoxicity. In the second one, ecotoxicity is measured directly using biological
tests. These two approaches complement each other; however, determination of pollutants in
complex mixtures of unknown composition, that is a characteristic of many wastes, does not allow
a relevant estimation of ecotoxicity. For such samples, the toxicity-based approach is usually
recognized to be appropriate to assess potential toxicity. Bioassays integrate, indeed, the effects
of all contaminants including additive, synergistic and antagonistic effects. They are sensitive to
the bioavailable fraction of the contaminants only. Finally, bioassays integrate the effects of all
contaminants, including those, not considered or detected by chemical analyses.Ecotoxicity tests can be applied to wastes to identify their potential hazardous properties with
respect to the environment for classification purposes or to assess the risk related to a site-specific
exposure scenario.Identification of properties potentially hazardous to the environment for classification
purposesA classification system, based on the assessment of intrinsic properties, should be independent of
an exposure scenario. The main requirement, in order to establish a relevant system for classifying
wastes and for assessment of hazard properties, is to obtain comparable test results. This can only
be obtained if the ecotoxicity tests on wastes are carried out according to a unique procedure
describing more or less conventional test conditions (an exclusive dilution medium for terrestrial
tests, a unique L/S ratio for preparation of water extracts, a unique liquid / solid separation step
etc). This procedure should be applicable to a very wide range of waste materials whatever their
physical properties are.Any strategy for the assessment of properties potentially hazardous to the environment used in a
classification system should include test organisms representing the terrestrial and the aquatic
compartment. Both types of tests should be considered because they expand the range of effect
expression due to differences in species sensitivity and exposure.For this specific purpose, the water extracts preparations for toxicity testing do not simulate
leaching from wastes under environmental conditions but measure the water available fraction
of the toxic components of the wastes.Site-specific exposure scenario
The second application of ecotoxicity tests to wastes refers to a risk assessment approach. In this
particular case, the test strategy should model site specific exposure conditions and should take
into account the transfer of contaminants via the food chain and to surface and ground water by
run-off or leaching. This application concerns firstly the definition of generic scenarios frequently
encountered (e.g. wastes deposit in stockpiles, re-use of wastes) and focus on the relevant way of
exposure to terrestrial and aquatic organisms.This document describes the necessary steps to be performed before carrying out ecotoxicity tests
on wastes within the context of assessment of ecotoxic properties for classification purposes.
---------------------- Page: 7 ----------------------oSIST prEN 14735:2020
prEN 14735:2020 (E)
1 Scope
This document describes the necessary steps to be performed before carrying out ecotoxicity tests
on wastes. The purpose of this document is to provide guidance on the taking of the sample,
transport, storage of wastes and to define preparation, for the determination of ecotoxicological
properties of wastes under the conditions specified in this document by biological testing either
as raw wastes or water extracts from wastes. Sample preparation for other applications (e.g.
assessment of waste effects on aquatic and terrestrial organisms in a disposal scenario) is not
considered.Specifying a test battery to characterize ecotoxicological properties of wastes is not in the scope
of this document.This document is applicable to solid and liquid wastes.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies.
For undated references, the latest edition of the referenced document (including any
amendments) applies.EN 12457-2:2002, Characterisation of waste - Leaching - Compliance test for leaching of granular
waste materials and sludges - Part 2: One stage batch test at a liquid to solid ratio of 10 l/kg for
materials with particle size below 4 mm (without or with size reduction)EN 14899, Characterization of waste - Sampling of waste materials - Framework for the preparation
and application of a Sampling PlanEN 16720-1, Characterization of sludges - Physical consistency - Part 1: Determination of flowability
- Method by extrusion tube apparatusEN ISO 5667-3, Water quality - Sampling - Part 3: Preservation and handling of water samples (ISO
5667-3)EN ISO 14238:2012, Soil quality – Biological methods – Determination of nitrogen mineralization
and nitrification in soils and the influence of chemicals on these processesISO 11465, Soil quality — Determination of dry matter and water content on a mass basis —
Gravimetric methodISO 10390, Soil quality – Determination of pH
Under preparation. Stage at time of publication: ISO/DIS 10390.
---------------------- Page: 8 ----------------------
oSIST prEN 14735:2020
prEN 14735:2020 (E)
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
dilution medium
liquid or solid used for the preparation of control vessels and the preparation of test mixtures
3.2ecotoxicological properties
potential adverse effects to biological systems which a waste has an inherent capacity to cause
3.3eluate
solution recovered from a leaching test
[SOURCE: EN 12457-2:2002, 3.3]
3.4
granular waste
waste not being monolithic, nor a liquid, a gas or a sludge
[SOURCE: adapted from EN 12457-2:2002, 3.10]
3.5
laboratory sample
sample or subsample(s) sent to or received by the laboratory (IUPAC definition)
Note 1 to entry: When the laboratory sample is further prepared (reduced) by subdividing, mixing,
grinding or by combinations of these operations, the result is the test sample. When no preparation of the
laboratory sample is required, the laboratory sample is the test sample. A test portion is removed from the
test sample for the performance of the test or for analysis. The laboratory sample is the final sample from
the point of view of sample collection, but it is the initial sample from the point of view of the laboratory.
Note 2 to entry: Several laboratory samples can be prepared and sent to different laboratories or to the
same laboratory for different purposes. When sent to the same laboratory, the set is generally considered
as a single laboratory sample and is documented as a single sample.3.6
leachant
liquid used in a leaching test
Note 1 to entry: For the purpose of this document the leachant is water as specified in Clause 4.
3.7leaching test
test during which a material is put into contact with a leachant and some constituents of the
material are extracted---------------------- Page: 9 ----------------------
oSIST prEN 14735:2020
prEN 14735:2020 (E)
3.8
liquid sludge
sludge in a liquid physical state, determined according to EN 16720-1
3.9
liquid waste
waste that completely flows out of a calibrated opening, down to the upper level of the opening
within a limited period of time (see Annex B of EN 12457-2:2002)3.10
monolithic waste
material which has dimensional, physical and mechanical properties that comply with the criteria
defined in an on-going document3.11
paste-like material
material of soft plastic or wet cement consistency – usually smooth
3.12
sludge
mixture of liquid and solid separated from various types of liquid as a result of natural or artificial
processes[SOURCE: adapted from EN 12457-2:2002, 3.8]
3.13
test mixture
mixture of the test portion (waste or water extract) with the dilution medium
3.14
test portion
amount or volume of the test sample taken for measurement of ecotoxicological properties by
biological testing and/or other properties of interest, usually of known weight or volume (adapted
from IUPAC definition)Note 1 to entry: The test portion can 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, can be a test portion.3.15
test sample
sample, prepared from the laboratory sample, from which test portions are removed for biological
testing or analysis (adapted from IUPAC definition)Note 1 to entry: The preparation of the test sample can include particle size reduction, preparation of
water extract etc.3.16
water extract
solution obtained from a leaching test, a liquid/liquid extraction and a liquid/solid separation
(centrifugation)---------------------- Page: 10 ----------------------
oSIST prEN 14735:2020
prEN 14735:2020 (E)
4 Equipment and reagents
Usual laboratory equipment and the following.
4.1 Sieving equipment with sieves of 4 mm square mesh.
NOTE Due to sieving, contamination of the sample can occur to an extent that affects the leaching of
some constituents of concern e.g. cobalt and tungsten from tungsten carbide equipment or chromium, nickel
and molybdenum from stainless steel equipment.4.2 Crushing equipment: jaw crusher or cutting device.
NOTE Due to crushing, contamination of the sample can occur to an extent that affects the leaching of
some constituents of concern e.g. cobalt and tungsten from tungsten carbide equipment or chromium, nickel
and molybdenum from stainless steel equipment.4.3 Balance of accuracy of at least 0,1 g.
4.4 End-over tumbler (5 rpm to 10 rpm) or rollertable rotating at about 10 rpm.
NOTE Other shaking or mixing devices can be used provided that they are proven to be equivalent.
4.5 Centrifuge operating at 2 500 g.4.6 Filtering apparatus, either a vacuum filtration device (between 30 kPa and 70 kPa)
(300 mbars to 700 mbars) or a high-pressure filtration apparatus (<0,5 MPa) (5 bars).
4.7 Mixer.4.8 pH meter.
4.9 Glass or high-density polyethylene (HDPE)/polypropylene (PP) bottles in accordance
with EN ISO 5667-3, glass bottles having caps of inert material, for examplepolytetrafluoroethylene. Rinsing is compulsory.
NOTE For inorganic constituents HDPE/PP bottles are preferred, except for samples tested for
mercury.4.10 Distilled water, demineralized water or deionized water with a conductivity < 5 µS/cm.
5 Taking of laboratory sampleObtain a representative laboratory sample of a quantity sufficient for the number of tests to be
performed and in accordance with the requirements of biological standardized methods to be
used.Special precautions should be taken to avoid any contamination of laboratory samples by material
of sampling devices and/or storage equipment, according to EN 14899.NOTE Sampling devices are described in the technical report CEN/TR 15310-2.
Some wastes are subject to chemical, physical and biological changes as soon as they are collected
(e.g. wastes that are fermentable, subject to oxidation or carbonation and wastes that contain
volatile substances). Possible changes shall be considered and sampling conditions shall be
designed accordingly in order to limit the effects of such changes on the results of ecotoxicity tests.
However, addition of preservatives (e.g. acids, basic solutions, biocides) in order to retard
chemical and biological activity is prohibited.---------------------- Page: 11 ----------------------
oSIST prEN 14735:2020
prEN 14735:2020 (E)
6 Transport
Transport of laboratory samples shall be performed in the dark, in tight containers fully filled with
the waste to be tested. However, special precautions should be taken for transport and storage of
sludge or other microbial active wastes. Containers can become pressurized due to gas production
and explosions may occur. For such laboratory samples, containers should not be completely
filled. Nevertheless, headspace shall not exceed 10 % of the total capacity of the container. Manual
release of pressure during and after transport may be necessary.The container material shall be appropriate. The container, in which the waste laboratory sample
is transported, and the stopper shall not react with the constituents of the sample and shall not be
a cause of contamination. Wastes shall be stored in polyethylene, polypropylene,polytetrafluoroethene (PTFE) or glass containers. However, security aspects shall be considered,
including the risk of explosion due to gas generation (for example glass vessels are not suitable
for sludge samples).Transport of waste laboratory samples should be as short as possible. Possible changes shall be
considered and transport conditions shall be designed accordingly in order to limit the effects of
such changes on the results of ecotoxicity tests. Transport time shall be regarded as part of storage
time.A transport time of less than 48 h and/or a low temperature conditions (4 ± 2) °C are appropriate
to maintain the properties of laboratory samples.7 Storage
7.1 General
Storage should be carried out in the containers defined in Clause 6. Possible changes shall be
considered and storage conditions shall be designed accordingly in order to limit the effects of
such changes on the results of ecotoxicity tests.7.2 Waste sample
Storage time starting from reception of laboratory sample and ending with the start of definitive
tests should be as short as possible.A storage time of less than two months and low temperature conditions (4 ± 2) °C are appropriate
to maintain the properties of waste samples.NOTE Freezing can induce changes of characteristics of the waste sample.
7.3 Water extracts
Water extracts should be stored at (4 ± 2) °C in polyethylene, polypropylene,
polytetrafluoroethene (PTFE) or glass containers. Before testing, the containers shall be filled
with a headspace less than 5 %.NOTE 1 Freezing can induce changes of characteristics of water extracts.
It is recommended to minimize the time between the start of the different tests to be performed
on the same laboratory sample in order to minimize its changes.Ecotoxicological tests shall start immediately after production of water extract as specified in the
appl...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.