EN 14735:2005

SLOVENSKI STANDARD
01-oktober-2005
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 14735:2005
ICS:
13.030.01 Odpadki na splošno Wastes in general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN 14735
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2005
ICS 13.030.01
English Version
Characterization of waste - Preparation of waste samples for
ecotoxicity tests
Caractérisation des déchets - Préparation des échantillons Charakterisierung von Abfällen - Herstellung von
de déchets en vue d'essais écotoxicologiques Abfallproben für ökotoxikologische Untersuchungen
This European Standard was approved by CEN on 27 June 2005.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2005 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14735:2005: E
worldwide for CEN national Members.

Contents page
Foreword .3
0 Introduction.4
1 Scope.5
2 Normative references .5
3 Terms and definitions.6
4 Equipment and reagents .7
5 Taking of laboratory sample .8
6 Transport .8
7 Storage .9
8 Waste characterization.10
9 Waste pre-treatment : particle size reduction (granular waste, monolithic waste,
paste-like waste and sludge).10
10 Tests performed on terrestrial organisms .11
11 Tests performed on aquatic organisms .14
12 Test report .17
Annex A (normative) Preparation of test mixtures according to the ecotoxicity tests to be
performed .18
Annex B (informative) Ecotoxicity tests considered to establish this European Standard.20
Bibliography.44

Foreword
This European Standard (EN 14735:2005) has been prepared by Technical Committee CEN/TC 292
“Characterization of waste”, 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 February 2006, and conflicting national standards
shall be withdrawn at the latest by February 2006.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy,
Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia,
Spain, Sweden, Switzerland and United Kingdom.

0 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 recognised 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.
0.1 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.
0.2 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 European Standard describes the necessary steps to be performed before carrying out
ecotoxicity tests on wastes within the context of assessment of ecotoxic properties used in a
classification system.
1 Scope
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.
2 Normative references
The following referenced documents are indispensable for the application of this European Standard.
For dated references, only the edition cited applies. For undated references, the latest edition of the
referenced document (including any amendments) applies.
prEN 14899, Characterization of waste - Sampling of waste materials - Framework for the preparation
and application of a Sampling Plan
EN 12457-2:2002, Characterization 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 ISO 5667-3, Water quality - Sampling - Part 3: Guidance on the preservation and handling of water
samples (ISO 5667-3:2003)
ISO 10390, Soil quality – Determination of pH
ISO 11268-1, Soil quality – Effects of pollutants on earthworms (Eisenia fetida) – Part 1: Determination
1)
of acute toxicity using artificial soil substrate
ISO 11465, Soil quality – Determination of dry matter and water content on a mass basis – Gravimetric
method
ISO 14238:1997, Soil quality – Biological methods – Determination of nitrogen mineralization and
nitrification in soils and the influence of chemicals on these processes

1) Definition of soil substrate.
3 Terms and definitions
For the purposes of this European Standard, the following terms and definitions apply.
3.1
dilution medium
liquid or solid used for the preparation of control vessels and the preparation of test mixture
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
[EN 12457-2:2002]
3.4
granular waste
waste not being monolithic, nor a liquid, a gas or a sludge
[EN 12457-2:2002]
3.5
laboratory sample
sample or subsample(s) sent to or received by the laboratory (IUPAC definition)
NOTE 1 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 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 For the purpose of this European Standard 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
3.8
liquid sludge
sludge that has the characteristic of a liquid as specified in the definition of a liquid waste
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
[EN 12457-2:2002]
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 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 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 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)
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 may 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 may 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 laboratory sample of a quantity sufficient for the number of tests to be performed and in
accordance with the requirements of biological standardised methods to be used.
The laboratory sample should be obtained according to the guide to the preparation of a sampling plan
for liquid and granular waste materials including paste-like materials and sludge, under development
by CEN/TC 292/WG 1.
Special precautions should be taken to avoid any contamination of laboratory samples by material of
sampling devices and/or storage equipment, according to prEN 14899.
NOTE Sampling devices are described in a standard under development by prCEN/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.
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 pressurised 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 shall be (4 ± 2) °C in order to
appropriately 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 shall be (4 ± 2) °C in order to
appropriately to maintain the properties of waste samples.
NOTE Freezing may 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 may induce changes of characteristics of water extracts.
It is recommended to minimise the time between the start of the different tests to be performed on the
same laboratory sample in order to minimise its changes.
Ecotoxicological tests shall start immediately after production of water extract as specified in the
applicable standard for the considered ecotoxicity test and in no case later than 72 h after production
of water extract. If a range-finding test and a definitive test are performed, the definitive test shall be
completed within 10 days after production of the water extract.
For longer tests (e.g. semi-static chronic tests), several water extracts shall be produced and used
within 10 days after production.
If definitive test results are not in accordance with the range-finding test, water extraction shall be
repeated and the test shall be performed on the new water extract.
NOTE 2 It could be possible to extend the duration of storage if it has been proved that no modification of
toxicity occurred within the storage period (e.g. carry out the same ecotoxicity test immediately after extraction
period and at the end of storage period).
8 Waste characterization
The following characteristics shall be determined prior to the performance of ecotoxicity tests:
 pH, according to ISO 10390 for granular waste, monolithic waste, paste-like waste and sludge;
 dry matter content, according to ISO 11465 for granular waste, monolithic waste, paste-like waste
and sludge (see Clause 9);
 water holding capacity, according to Annex A of ISO 14238:1997 for granular waste, monolithic
waste, paste-like waste and sludge (see note).
NOTE The method described in Annex A of ISO 14238:1997 has been found to be appropriate for most of
the different kinds of waste.
9 Waste pre-treatment : particle size reduction (granular waste, monolithic
waste, paste-like waste and sludge)
Both ecotoxicological and leaching tests are performed on material which originally and after
pretreatment has a particle size less than 4 mm.
The tests shall be made on material with a grain size of at least 95 % (mass) less than 4 mm.
Therefore, the laboratory sample shall be sieved (4.1). If oversized material exceeds 5 % (mass) the
entire oversized fraction shall be crushed with crushing equipment (4.2). On no account shall the
material be finely ground. Non-crushable material (e.g. metallic parts such as nuts, bolts, scrap) in the
sample shall be separated and the weight and nature of the material shall be recorded. The method of
size-reduction applied shall be documented and recorded in the test report. Irrespective of any
necessary size reduction, the separate fractions with the exception of the non-crushable material shall
be mixed to constitute the test sample. If the laboratory sample cannot be crushed or sieved because
of its moisture content, it is allowed, only in this case, to dry the laboratory sample. The drying
temperature shall not exceed 40 °C.
NOTE 1 Fibrous materials, paste-like waste and plastics can often be size-reduced after cryogenic treatment.
The sample is usually plunged into liquid nitrogen (- 196 °C) just before crushing to make it fragile and brittle. It
also limits the overheating during crushing. As a result, the sample obtained is fine and perfectly homogeneous.
NOTE 2 In order to minimise the possible contamination during the sieving, fragmentation and splitting, it is
recommended, before preparing the test sample, to process a portion of the laboratory sample through the
devices for sieving, fragmentation and splitting and to discard such material thereafter. This recommendation
does not cover the situation described in the notes under 4.1 and 4.2.
For this European Standard, any other waste pre-treatment is excluded ; especially, the test sample
which shall not be further dried. The determination of the dry matter content ratio and the moisture
content ratio shall be determined on a dedicated test portion. The moisture content of the test sample
shall be determined at (105 ± 5) °C. It shall be taken into account when adjusting the L/S ratio in
leaching test. The dry mass of the sample shall be determined at (105 ± 5) °C according to ISO 11465
and the dry matter content ratio is calculated as follows:
M
D
DR = 100 × (1)
M
W
where
DR is the dry matter content ratio (%);
M is the mass of the dried test portion (kg);
D
M is the mass of undried test portion (kg).
W
The moisture content ratio is calculated as follows:
(M − M )
W D
MC = 100 ×        (2)
M
D
where
MC is the moisture content ratio (%).
NOTE 3 The basis for the calculation of the moisture content is the mass of the moisture content of the dry
residue in this European Standard, as specified in ISO 11465 (for the determination of the water content of soil). It
should be noted that in EN 12880 (for the determination of water content of sludge), the water content is
calculated on the basis of the raw mass.
NOTE 4 The above moisture content determination could be not accurate enough in some cases (e.g. large
amount of volatile or unstable compounds). In such cases a direct determination of the true water content should
be performed and the moisture content calculated accordingly.
10 Tests performed on terrestrial organisms
10.1 General considerations
The determination of ecotoxicological properties of wastes under conventional conditions requires
using a dilution medium as inert as possible. This dilution medium shall allow the survival and the
good development of organisms during the test period. Both requirements may be difficult to reconcile
particularly considering plant growth inhibition tests and microbial tests (tests that required indigenous
population of micro-organisms).
In order to fulfil theses requirements, the dilution medium called "artificial soil" shall be used unless
otherwise specified in the standardized terrestrial test methods. The same medium shall be used for
both control and dilution.
Moreover, preparation of a medium should be reproducible to allow comparison of ecotoxicity tests
results.
Several standardized ecotoxicity tests were considered to establish the following conditions for testing
wastes on terrestrial organisms. This compilation of tests is given in Annex B.
Preparation of test mixtures may differ according to the type of waste and according to the ecotoxicity
tests to be performed. Preparation of the different test mixtures is summarized in Annex A.
10.2 Dilution medium
The dilution medium shall have the following composition (such as defined in ISO 11268-1):
 sphagnum peat finely ground and with no visible plant remains: 10 % (percentage expressed on
dry mass basis);
 kaolinite clay containing not less than 30 % kaolinite: 20 % (percentage expressed on dry mass
basis);
 industrial quartz sand (more than 50 % of particle size from 0,05 mm to 0,20 mm): 70 %
(percentage expressed on dry mass basis).
Calcium carbonate (CaCO ), pulverised and of recognised analytical grade is added to bring the pH of
the wetted substrate to 6,0 ± 0,5 (generally between 0,5 % and 1 % of the mass of the dry ingredients).
Water (4.10) or mixture of water extract with water is added to the dilution medium to reach the
percentage of the total water holding capacity recommended for each test organism.
10.3 Introduction of waste into the dilution medium
10.3.1 General
Preparation of test mixtures differs according to the waste to be tested (see Annex A). The different
methods of preparation are described below.
10.3.2 Monolithic waste, granular waste, paste-like waste and sludge
Different methods can be applied to introduce the test portion into the dilution medium. Several
parameters influence the selection of introduction method such as physical properties of waste or
amounts to be tested. The following methods are recommended:
 for small amounts, introduce the test portion in the water (or in part of it) necessary to wet the
dilution medium, then mix this suspension thoroughly with the dilution medium;
 for large amounts, mix the test portion thoroughly with the already hydrated dilution medium;
 for hydrophobic waste, mix the test portion thoroughly with the dilution medium, then add the
water necessary to wet this mixture.
Test mixtures are expressed in percentages (dry mass of waste per total dry mass of test mixture).
10.3.3 Liquid sludge
Introduce the test portion in the water (or in part of it) necessary to wet the dilution medium, then mix
this suspension thoroughly with the dilution medium in order to obtain the test mixture to perform the
considered ecotoxicological test. Test mixtures are expressed in percentages (mass of waste per total
dry mass of test mixture).
The volume of the liquid sludge to be added is limited by the percentage of water holding capacity of
the test mixture recommended in the test methods.
10.3.4 Liquid waste miscible with water
Introduce the test portion in the water (or in part of it) necessary to wet the dilution medium, then mix
this suspension thoroughly with the dilution medium.
Some liquid waste may not contain any water and does not hydrate the test mixture, in which case it is
necessary to add water to allow survival of test organisms within the given test period.
The maximum quantity of liquid waste and water to be added shall correspond to the water holding
capacity of the dilution medium recommended for each test method. The structure achieved after
adding the liquid waste should meet the requirements of the test organisms.
10.3.5 Liquid waste non miscible with water
The following methods are recommended.
a) For small amounts:
 introduce by ultrasonic dispersion the test portion in the water (or in part of it) necessary to wet
the dilution medium, then mix this suspension thoroughly with the dilution medium; or
 prepare a mixture of quartz sand (see 10.2) and the quantity of test portion required to obtain the
desired amount (a ratio of 10 g of sand per kilogram of soil is usually recommended). Mix with the
dilution medium thoroughly, then add the water necessary to wet this mixture.
b) For large amounts:
 mix the test portion thoroughly with the dilution medium already hydrated; or
 mix the test portion thoroughly with the dilution medium, then add the water necessary to wet this
mixture.
Test mixtures are expressed in percentages (mass of waste per total dry mass of test mixture).
10.4 Water extracts of waste
Introduce the water extract (see Clause 11) in the water (or in part of it) necessary to wet the dilution
medium, then mix this suspension thoroughly with the dilution medium.
The maximum quantity of water extract to be added shall correspond to the water holding capacity of
the dilution medium recommended for each test method.
Test mixtures are expressed in percentages (mass of water extract per total dry mass of test mixture).
10.5 pH
Tests shall be carried out without pH adjustment of the test portion.
pH of all test mixtures is measured at the beginning and at the end of the test and reported.
NOTE 1 pH of test mixtures may significantly differ from pH of test portion according to the selected dilution
range and according to buffer capacity of test medium or test portion.
NOTE 2 If toxic effects are observed in the dilutions where pH is not compatible with the survival of the
organisms, the test(s) can be repeated with pH adjustment of the test portion.
10.6 Addition of test organisms
The test procedure is selected according to the aim of the study: determination of a dose-effect
relationship or limit test at a given dilution.
Test organisms shall be added as soon as possible after test mixture preparation and on no account
later than 24 h after preparation.
11 Tests performed on aquatic organisms
11.1 General considerations
Ecotoxicological properties of wastes are assessed under conventional conditions (and not according
to specific scenarios) and a unique procedure is specified, for each material status, to produce the
water extract in order to obtain comparable ecotoxicity tests results performed on different wastes.
Water extract preparation is influenced by several parameters such as physical properties of waste
and required volume of water extract. This last parameter may become crucial when chronic
ecotoxicity tests are performed.
The L/S = 10 ratio is recommended, since it is applicable to a very wide range of wastes. The volume
of leachant provided is suitable to obtain a water extract even for wastes with high water holding
capacities (e.g. sludge). The volume of water extract obtained is consistent with the implementation of
chronic ecotoxicity tests.
NOTE Deviation from an L/S ratio of 10 leads to test results not comparable with test results obtained with a
ratio of 10.
Several standardized ecotoxicity tests were considered to establish the following conditions for testing
wastes on aquatic organisms. This compilation of tests is given in Annex B.
Preparation of test mixtures may differ according to the type of waste and according to the ecotoxicity
tests to be performed. Preparation of the different test mixtures is summarized in Annex A.
11.2 Monolithic waste, granular waste, paste-like waste and sludge
11.2.1 Leaching procedure
The following procedure is based on EN 12457-2. The following text includes the modifications
required by the tests listed in Annex B.
Place the test portion with the total mass M corresponding to (90 ± 5) g of dry mass M in a bottle (4.9)
D
with a nominal volume of 1 l to minimise headspace.
Ecotoxicological tests usually require several litres of eluate leading to an adjustment of mass of the
test portion. When a large volume of eluate is required, it is possible to perform the leaching test in a
vessel of appropriate capacity into which the corresponding number of test portions (a dry mass of
(95 ± 5) g) are introduced or to perform parallel leaching tests at the same time with an appropriate
number of vessels of 1 l capacity each (see above). In that case, all the individual eluates shall be
mixed in order to obtain the test sample. When the leaching test is performed in one vessel, the size of
the vessel shall be appropriate to minimise the headspace (≤ 5 % of the total capacity of the vessel).
Add an amount of leachant (L) establishing a liquid to solid ratio (L/S) = 10 l/kg ± 2 % during the
extraction. Care should be taken to obtain good mixing of solid and liquid.
 MC
L =10 −  × M (3)
D
 
where
L is the volume of leachant used (in l);
M is the dry mass of the test portion (in kg);
D
MC is the moisture content ratio (in %).
Place the capped bottle in an agitation device (an end-over-end tumbler 5 rpm to 10 rpm or rollertable
rotating at about 10 rpm). Agitate for (24 ± 0,5) h at a temperature within the range 15 °C to 25 °C.
During the extraction care should be taken to prevent settlement of solids in the bottle.
Excessive abrasion leading to significant particle size reduction shall be avoided.
NOTE Some wastes generate gas when they are wetted. Examples are waste incineration fly ash and sand
blasting waste that may contain metallic particles. If gas emission occurs, careful opening of the bottle a few times
during the leaching can prevent too high pressure. Such opening should be documented in the test report.
11.2.2 Liquid/solid separation procedure
As ecotoxicological properties of wastes are assessed under conventional conditions, the eluates shall
be filtrated through 0,45 µm membrane filter. It is recommended to use PTFE or nylon filters instead of
acetate or nitrate cellulose filters. This filtration has the advantage that several bioassays, which may
be influenced by suspended materials in the test mixture (determination of inhibitory effects on the
light emission of Vibrio fischeri, algal growth inhibition test etc), may be performed.
The separation of the solid and the liquid phases is carried out as follows in the same temperature
conditions as defined above.
Allow the suspended solids to settle for (15 ± 5) min. When the separation of the solid and liquid
phases is not achieved within this time period, the mixture is centrifuged for 30 min at 2 500 g.
Filter the eluate over a 0,45 µm membrane filter using a vacuum or pressure filtration device (4.6).
Rinsing of the filter with water or another solvent is not allowed after filtration.
NOTE When volatile components are analysed, vacuum filtration should not be used.
Determine the volume of eluate VE.
Measure immediately conductivity (in µS/cm), pH (and optionally redox potential E in mV and
h
temperature) of the eluate.
11.2.3 pH
Tests shall be carried out without pH adjustment of the test portion.
pH of all test mixtures is measured at the beginning and at the end of the test and reported.
NOTE 1 pH of test mixtures may significantly differ from pH of test portion according to the selected dilution
range and according to buffer capacity of test medium or test portion.
NOTE 2 If toxic effects are observed in the dilutions where pH is not compatible with the survival of the
organisms, the test(s) can be repeated with pH adjustment of the test portion.
11.3 Liquid sludge
11.3.1 Procedure
Large particles should be removed from sludge by centrifugation (for 30 min at 2 500 g) and the water
extract is filtrated through a 0,45 µm membrane filter. It is recommended to use PTFE or nylon filters
instead of acetate or nitrate cellulose filters.
11.3.2 pH
See 11.2.3.
11.4 Liquid waste miscible with water
11.4.1 Procedure
No specific procedure is needed, the liquid waste miscible with water should be used for aquatic
toxicity testing after filtration (see 11.2.2).
Measure conductivity (in µS/cm), pH (and optionally redox potential E in mV and temperature) of the
h
liquid waste.
11.4.2 pH
See 11.2.3.
11.5 Liquid waste non miscible with water
11.5.1 Procedure
A liquid/liquid extraction is performed by bringing water in contact with the test portion. In order to
compare liquid waste with the other waste materials, the parameters used for this procedure should be
the same as those defined in other extraction procedures. The liquid/liquid extraction is performed as
follows.
Place the test portion with the total mass M corresponding to (90 ± 5) g of dry mass M in a bottle (4.9)
D
with a nominal volume of 1 l to minimise headspace.
Ecotoxicological tests usually require several litres of water extract leading to an adjustment of mass
of the test portion. When a large volume of water extract is required, it is possible to perform the
liquid/liquid extraction in a vessel of appropriate capacity into which the corresponding number of test
portions (a mass of (90 ± 5) g) are introduced or to perform parallel liquid/liquid extraction at the same
time with an appropriate number of vessels of 1 l capacity each (see above). In that case, all the
individual water extracts shall be mixed in order to obtain the test sample. When the liquid/liquid
extraction is performed in one vessel, the size of the vessel shall be appropriate to minimise the
headspace (≤ 5 % of the total capacity of the vessel).
Add an amount of leachant establishing a ratio of 10 during the extraction.
Place the capped bottle in an agitation device (an end-over-end tumbler 5 rpm to 10 rpm or rollertable
rotating at about 10 rpm). Agitate for (24 ± 0,5) h at a temperature within the range 15 °C to 25 °C.
11.5.2 Liquid/liquid separation
Allow the two phases to separate for (15 ± 5) min in the same temperature conditions as defined
above. When separation of the two liquid phases is not achieved within this time period, the mixture is
centrifuged for 30 min at 2 500 g.
Filter the water extract over a 0,45 µm membrane filter using a vacuum or pressure filtration device
(4.6). Rinsing of the filter with water or another solvent is not allowed after filtration.
11.5.3 pH
See 11.2.3.
11.6 Preparation of test mixtures
The water extract is mixed thoroughly with the dilution medium in order to obtain homogenous test
mixtures. The water extract is diluted with the dilution medium specified in the selected ecotoxicity test
method.
Test mixtures are expressed in percentages (volume of water extract per total volume of test mixture).
The test procedure is selected according to the aim of the study: determination of a dose-effect
relationship or limit test at a given dilution.
12 Test report
The test report shall include the following information:
a) reference to this European Standard;
b) waste characterization (pH, dry matter content, water holding capacity, granularity etc);
c) complete description of sampling conditions;
d) complete description of transport and storage conditions (temperature, duration, containers etc);
e) complete description of waste pre-treatment, including method of size reduction, fraction above
4 mm, fraction of non crushable material (if relevant information);
f) complete description of water extraction procedure including type, size and material of the vessel
being used; description of the liquid-solid separation procedure; volume of water extract;
g) description of storage conditions of water extract (if relevant information);
h) presentation of the results and the test conditions according to the ecotoxicity test standards.
Annex A
(normative)
Preparation of test mixtures according to the ecotoxicity tests to be
performed
Table A.1 — Preparation of test mixtures (3.13) according to the ecotoxicity tests to be performed
Category of waste Preparation of water extract Test mixtures for tests performed on Test mixtures for tests performed on
aquatic organisms terrestrial organisms
Waste with particle size below 4 mm Leaching test according to Eluate is diluted with the dilution - Waste is mixed with the dilution

a
(originally or after pre-treatment) EN 12457-2 medium of the selected ecotoxicity medium
.
tests. - Eluate is added to the water used to
wet the dilution medium.
Sludge Leaching test according to Eluate is diluted with the dilution - Waste is mixed with the dilution

b
EN 12457-2 medium of the selected ecotoxicity medium .
tests. - Eluate is added to the water used to
wet the dilution medium.
Liquid sludge Separation of liquid and The water extract is diluted with the The water extract is added to the water
particulate matter by dilution medium of the selected use
...