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

General Information

Status
Published
Public Enquiry End Date
03-May-2020
Publication Date
13-Dec-2021
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
26-Nov-2021
Due Date
31-Jan-2022
Completion Date
14-Dec-2021

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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

Untersuchungen

Caractérisation des déchets - Caractérisation des déchets - Préparation des échantillons

de déchets en vue d'essais écotoxicologiques
Ta 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
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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

écotoxicologiques
This 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

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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

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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.
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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.
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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

method

EN ISO 10390, Soil, sludge and treated biowaste - Determination of pH (ISO/FDIS 10390)

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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

3.2
ecotoxicological properties

potential adverse effects to biological systems which a waste has an inherent capacity to cause

3.3
eluate
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.

3.7
leaching test

test during which a material is put into contact with a leachant and some constituents of the material are

extracted
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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)

3.14
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.
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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 sample

Obtain 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.
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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

Untersuchungen

Caractérisation des déchets - Caractérisation des déchets - Préparation des échantillons

de déchets en vue d'essais écotoxicologiques
Ta 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 écotoxicologiques

This 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, 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 ....................................................................................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

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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 36

B.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

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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.

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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

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.

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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

5667-3)

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 11465, Soil quality — Determination of dry matter and water content on a mass basis —

Gravimetric method
ISO 10390, Soil quality – Determination of pH
Under preparation. Stage at time of publication: ISO/DIS 10390.
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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

3.2
ecotoxicological properties

potential adverse effects to biological systems which a waste has an inherent capacity to cause

3.3
eluate
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.

3.7
leaching test

test during which a material is put into contact with a leachant and some constituents of the

material are extracted
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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 document
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
3.14
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)
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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 example
polytetrafluoroethylene. 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 sample

Obtain 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.
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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
...

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