CEN/TR 17105:2017

SLOVENSKI STANDARD
01-september-2017
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Construction products - Assessment of release of dangerous substances - Guidance on
the use of ecotoxicity tests applied to construction products
Bauprodukte - Bewertung der Freisetzung von gefährlichen Stoffen - Leitfaden für die
Anwendung von ökotoxikologischen Untersuchungen auf Bauprodukte
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Ta slovenski standard je istoveten z: CEN/TR 17105:2017
ICS:
13.020.99 Drugi standardi v zvezi z Other standards related to
varstvom okolja environmental protection
91.100.01 Gradbeni materiali na Construction materials in
splošno general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TR 17105
TECHNICAL REPORT
RAPPORT TECHNIQUE
June 2017
TECHNISCHER BERICHT
ICS 91.100.01
English Version
Construction products - Assessment of release of
dangerous substances - Guidance on the use of ecotoxicity
tests applied to construction products
Produits de construction - Evaluation de l'émission de Bauprodukte - Bewertung der Freisetzung von
substances dangereuses ¿ Préconisations concernant gefährlichen Stoffen - Leitfaden für die Anwendung von
l'utilisation des essais visant à évaluer l'écotoxicité des ökotoxikologischen Untersuchungen auf Bauprodukte
produits de construction
This Technical Report was approved by CEN on 14 May 2017. It has been drawn up by the Technical Committee CEN/TC 351.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TR 17105:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Terms and definitions . 6
3 General information on ecotoxicity assessment . 8
3.1 Basic approaches for ecotoxicity estimation . 8
3.2 Principles for ecotoxicity testing . 8
3.3 Information on the biological test battery . 9
4 Sampling and transport of construction products . 10
5 Leaching procedures for ecotoxicological testing . 10
5.1 Suitable leaching tests and selection of fractions from leaching tests . 10
5.1.1 General . 10
5.1.2 Dynamic Surface Leaching Test (DSLT) CEN/TS 16637-2 . 10
5.1.3 Horizontal up-flow percolation test (CEN/TS 16637-3) . 11
5.1.4 Further leaching tests . 11
5.2 Sampling, transport and storage of eluates . 11
5.3 Pre-treatment of eluates. 12
6 Aquatic ecotoxicological testing . 12
6.1 Purpose of aquatic ecotoxicity testing . 12
6.2 Selection of ecotoxicity tests and minimum test battery . 13
6.3 Testing strategy and conditions . 13
6.3.1 General . 13
6.3.2 Consideration of additional blank samples . 14
6.3.3 pH adjustment . 14
6.3.4 Test concentrations . 14
6.3.5 Colour and turbidity . 14
6.3.6 Unstable substances . 15
6.4 Limitations of aquatic ecotoxicity tests . 15
7 Assessment of biodegradability . 15
8 Terrestrial ecotoxicological testing . 15
8.1 Purpose of terrestrial ecotoxicity testing. 15
8.2 Pre-treatment of construction products for terrestrial tests . 16
8.3 Selection of ecotoxicity tests and minimum test battery . 17
8.4 Test report and quality assurance procedure . 17
Annex A (informative) CEN/TC 351 workshop on Ecotoxicity, biodegradability and
construction products on 10 April 2014 in Brussels – Conclusions and
recommendations . 19
Annex B (informative) Interface with information derived from REACH . 21
Annex C (informative) Information on regulations and guidelines with relevance for
ecotoxicological assessment of construction works with implications on
construction products . 22
Annex D (informative) Results from a European round robin test “Ecotoxicological
characterisation of eluates from construction products” . 24
Annex E (informative) Evaluation of test results . 27
E.1 Aquatic tests . 27
E.2 Terrestrial tests . 28
E.3 Example: LID approach in German regulation . 28
Bibliography . 29

European foreword
This document (CEN/TR 17105:2017) has been prepared by Technical Committee CEN/TC 351
“Construction Products - Assessment of release of dangerous substances”, the secretariat of which is
held by NEN.
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 has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
This Technical Report gives guidance for the combination of the recommended horizontal leaching tests
harmonized by CEN/TC 351 with existing biological test methods for the assessment of ecotoxicological
properties of eluates of construction products.
Guidance regarding biological tests for the effects of construction products on soil organisms is also
included. This document takes into account relevant information that had become available by
March 2016. This document is intended as easy-to-use guidance especially for the Group of Notified
Bodies, test laboratories and EOTA. Technical committees for construction products (product TCs) are
expected to benefit from the information given in the report, if they have been mandated to address
ecotoxicity in their product standards or if they are interested to include ecotoxicity in a dossier
prepared in the context of qualifications for a “without testing” status.
Introduction
Ecotoxicological analysis of construction products and their eluates and biodegradability of the organic
substances in eluates belong to the essential characteristics covered by the basic requirement for
construction works “hygiene, health and the environment” from Annex 1 of the Construction Products
Regulation [1]. Under the European Commission’s mandate M/366 (see mandate database at
http://ec.europa.eu/growth/tools-databases/mandates) and according to the Indicative List (see
https://www.nen.nl/ under search term CEN/TC 351) which specifies the mandated parameters,
CEN/TC 351 has been assigned to deal with these essential characteristics. Now that the mandated
leaching tests from CEN/TC 351 (CEN/TS 16637-2, CEN/TS 16637-3) [2], [3] are available and also
work on the methods for the chemical analysis of eluates has progressed, CEN/TC 351 has included a
Technical Report on ecotoxicity / biodegradability in its active programme of work. The background for
the decision to cover this topic was presented in an open expert workshop in Brussels in April 2014.
The conclusions and recommendations of the workshop are presented in Annex A.
The regulatory background for the work is explained in Annex B and information on its possible
interface with data generated under the REACH Regulation is given in Annex C. In the context of
harmonized specifications for construction products currently only Germany requests performance
data on ecotoxicity/biodegradability in certain cases, i.e. when and where a chemical analysis and
assessment of the eluates of construction products is considered to be too onerous or not possible due
to the lack of analytical methods/data. Examples of products addressed are fire protective products and
fire stopping and fire sealing products depending on their ingredients. Ecotoxicity assessment is
considered to be especially relevant for the building and for the demolition phase in the life cycle of
construction works. These life cycle phases have been covered by the framework of the Construction
Products Regulation since 2013.
The majority of existing internationally harmonized ecotoxicity tests were developed firstly to assess
the ecotoxic potential of chemicals, waste water or contaminated soils. More recently, these tests have
been successfully applied to waste and waste eluates [4] to [7]. These methods can be applied with
some modifications for the ecotoxicological characterization of construction products and their eluates.
Several studies as well as an international round robin test have been conducted to validate some test
methods for construction product eluates and the results have been used as background information
[8] to [13]. The validation of the methods for construction products is not yet complete. Further
validation of the recommended test procedure is needed, if this TR is intended to be further developed
into a CEN/TS or EN (to be decided later).
1 Scope
This Technical Report gives information on existing methods to test ecotoxicity of construction
products. Information is given on how to combine recommended leaching tests with biological tests for
the aquatic environment and how to avoid possible problems, when performing biological tests. Also
suitable terrestrial tests on granular construction products diluted with artificial soil are proposed for a
minimum test battery.
Reference has been made as far as possible to existing International and European Standards and
guidelines.
The test procedure described in this Technical Report is technically suitable for all construction product
eluates and for terrestrial tests on granular or paste-like construction products. However, from the
point of view of test efficiency it is recommended mainly for products containing organics or polymers
in case chemical analysis alone is not deemed to be sufficient. For inorganic products the chemical
analysis is seen as straightforward in construction product eluates and therefore the added value of
data received through ecotoxicity tests is seen as limited.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
biodegradation
mineralization of organic compounds by bacteria and fungi to carbon dioxide, water and inorganic
compounds
2.2
control
mixture of control medium and organisms used in the test without test sample
2.3
control medium
combination of water and additives (e.g. nutrients, salts) used in the test
2.4
dilution level
D
reciprocal value of the volume fraction of test sample in dilution water in which the test is conducted
EXAMPLE 250 ml of test sample in a total volume of 1 000 ml (volume fraction of 25 %) represents dilution
level D = 4.
[SOURCE: EN ISO 15088:2008 [14], 3.2, modified - "waste water" replaced by "test sample"]
2.5
dilution soil
soil added to the test sample to prepare a series of defined dilutions
Note 1 to entry: The origin and composition of the soil is specified in the specific test.
2.6
dilution water
water added to the test sample to prepare a series of defined dilutions
[SOURCE: EN ISO 20079:2006 [15], 3.7]
Note 1 to entry: The composition of the water is specified in the specific test.
2.7
effective concentration
EC
x
concentration of the test material in water or sediment that causes x % change in response during a
specified time interval
[SOURCE: ISO/TS 20281:2006 [16], 3.8.1]
2.8
eluate
aqueous solution recovered from a leaching test
[SOURCE: CEN/TR 16110:2010 [4], 3.2]
2.9
leaching test
laboratory test during which a construction product is put into contact with a leachant under strictly
defined conditions for the determination of the release of substances into water
2.10
lowest ineffective dilution
dilution factor
LID
lowest ineffective dilution tested, expressed as dilution level D (2.4), at which no inhibition, or only
effects not exceeding the test-specific variability, are observed
[SOURCE: EN ISO 15088:2008 [14], 3.5]
2.11
storage time
period of time between filling of the sample container and further treatment of the sample in the
laboratory, if stored under predefined conditions
[SOURCE: EN ISO 5667-3:2012 [17], 3.4]
2.12
test material
material to be tested
[SOURCE: ISO 17126:2005 [18], 3.3]
3 General information on ecotoxicity assessment
3.1 Basic approaches for ecotoxicity estimation
Ecotoxicity can be estimated using two approaches: a chemical-specific approach and a toxicity-based
approach. In the first situation, results of chemical analyses are compared to quality criteria or
threshold values to estimate toxicity. This approach is used, e.g. for the evaluation of biocides and is not
covered by this report. In the second case, toxicity is measured directly using biological toxicity tests.
These two approaches complement each other. However, determination of individual target substances
in complex mixtures of unknown or undisclosed composition does not allow a relevant estimation of
toxicity via the chemical-specific approach. For such samples, the toxicity based approach is recognised
to be practicable to estimate ecotoxicity.
Ecotoxicity tests integrate the effects of all dangerous or hazardous substances including additive,
synergistic and antagonistic effects. They are sensitive to the bioavailable fraction of the substances
only and integrate the effects of all dangerous substances, including those, not directly addressed by
chemical analyses. In principle there is no organism which can be used to test all possible effects on
ecosystems. Only a few ("model" or reference) species representing relevant ecological functions can be
tested in practice.
3.2 Principles for ecotoxicity testing
Biological tests are suitable for determining the effect of e.g. eluates or of solid material on test
organisms under specific experimental conditions. These effects can be enhancing or inhibiting, and can
be determined by the reaction of the organisms (e.g. death, growth, proliferation, morphological and
physiological changes). The apparent toxicity measurable in the biological test is the result of the
interaction between the constituents of the tested sample and the test organism. The protective
potential of the biological system, for instance by metabolic detoxification and excretion, is an integral
part of the biological test. Biological tests also include those tests which examine the effect of organisms
on substances (e.g. microbial degradation studies).
However, the sample to be tested can pose experimental challenges on biotesting. These challenges
should be paid attention to, when testing samples that contain sparingly soluble, volatile, unstable,
coloured substances and/or suspended, sometimes colloidal, particles. The complexity and
heterogeneity of materials should be taken into account when performing biotests. Special care should
be taken, if the test material is instable due to reactions and processes such as photo-degradation or
biodegradation. If spectrometric measurements are applied, turbidity and colour of the eluate should be
considered.
Ecotoxicity tests can be applied to construction product eluates (aquatic tests) or to test samples of
construction products mixed with artificial soil (terrestrial tests) to identify their potential hazardous
intrinsic properties with respect to the environment, if the construction product comes into contact
with soil or water in its intended use. In general aquatic test are performed with construction product
eluates mixed with dilution water but in a few cases liquid construction products could directly be
mixed with dilution water. The result of the biological test refers primarily to the organism used in the
test and the conditions stipulated in the test procedure. A harmful effect stated by means of
standardised tests can justify concern that aquatic or terrestrial organisms and ecosystems might be
endangered. The results, however, do not permit direct or extrapolative conclusions as to the
occurrence of similar effects in the aquatic or terrestrial environment.
The interpretation of the ecotoxicological testing is related to the purpose of testing and the intended
use scenario for the tested product and further criteria when defined in regulations or guidelines.
Depending upon the purpose of testing, technical choices can be made to enhance the reliability of the
results.
Any construction product that falls under the scope of the leaching tests in the CEN/TS 16637 series
may also be assessed with biological tests, when and where desired. Currently biological tests are only
requested and applied in certain cases, if a chemical assessment of eluates from the CEN/TS 16637 tests
is not considered to cover all organic substances with hazardous properties that may be released from a
product [19], [20]. For construction products in direct contact with soil in their intended use also
terrestrial tests are a relevant assessment tool.
3.3 Information on the biological test battery
Sensitivity of animal and plant communities to toxicants may vary significantly from one species to
another. If testing is performed on one species or function only, the high diversity in the sensitivity
between species results in a high level of uncertainty. Only a combination of several ecotoxicity tests
can give a clear view of the toxic effects of product samples. The recommended approach for the
ecotoxicological characterisation of the toxicity of construction products and eluates is therefore to use
a battery of tests with several species belonging to different taxonomic groups and trophic levels.
Two approaches can be applied for selecting bioassays in order to establish a test battery: (i) an “a
priori” method, in which the selection is made, independently of the results, according to decision
criteria such as standardization of the method, ecological relevance of test organisms, or cost, (ii) an “a
posteriori” method, in which the selection is made after analysing test results obtained on a large series
of bioassays. Regarding the “a priori” approach, there is a good overall agreement on the criteria to be
considered to establish a test battery. These are: robustness, relevance, reproducibility, sensitivity,
endpoints (chronic, acute), standardisation status, discriminative power, cost, ease of use. The
importance of each individual criterion is clearly related to the aim of the ecotoxicological assessment.
These criteria fall into three main categories (see Table 1 below).
Table 1 — Relevant criteria for establishing a test battery for construction products
Decision Criteria
categories
General criteria — Good ratio cost efficiency of selected tests
— Lack of test results redundancy
— Ability of discrimination between samples
Test battery — Combination of different trophic levels
organization
— Combination of assessment endpoints (e.g. acute, chronic, genotox)
— Are the most important exposure pathways related to construction
products (soil, groundwater, surface water) covered?
Test methods — Ecological relevance: e.g. the species dominance in terms of abundance or
included in the biomass, representation of trophic level
test battery
— Ecological tolerance: degree of sensitivity of the species to changes in
environmental conditions
— Keeping/breeding: the easiness and quickness of the species to be bred in
the laboratory
— Practicability: Are a plenty of or few resources (costs, staff, and time)
required to perform the test?
— Reproducibility of the results obtained
— Sensitivity of the species
— Standardization: Is the method published as a validated (international)
guideline, preferably ISO?
In ecotoxicology the measured endpoints are intended to be indicative for potential negative effects on
the survival of the populations (not of the individuals). Relevant endpoints therefore are mortality and
reproduction impairment. Next to these direct endpoints also the presence of genotoxic substances
(inducing DNA damage) and hormone disturbing compounds (inducing reproductive impairment) are
considered as relevant for interfering with normal population survival.
Due to general reasons of practicability, an ecotoxicological test battery usually consists of three tests
for each medium considered (aquatic, terrestrial), often each divided into three trophic/taxonomical
groups. The current approaches for the evaluation of test results are described in Annex E.
4 Sampling and transport of construction products
In addition to the relevant product standards guidance on sampling of construction products is given in
CEN/TR 16220 [21]. Also guidance given in EN 15002 [22] on how to produce representative test
portions from laboratory samples can be applied to construction products before biological tests are
carried out.
In the case of aquatic tests and in order to generate suitable eluates for ecotoxicological testing, it is
recommended additionally to use the guidance for taking laboratory samples of products and for
transport, storage and product sample pre-treatment for leaching tests given in CEN/TS 16637 [23], [2]
and [3], EN 14735 [24] or EN 16105 [25] and in relevant product standards or specifications.
5 Leaching procedures for ecotoxicological testing
5.1 Suitable leaching tests and selection of fractions from leaching tests
5.1.1 General
A leaching method should be selected using guidance given in CEN/TS 16637-1 [21]. Eluates from the
DSLT (CEN/TS 16637-2) and column test (CEN/TS 16637-3) are considered to be basically suitable for
ecotoxicological tests on the basis of current experience.
Products that come into contact with water only occasionally in their intended use may not be stable in
a test set up with long time immersion in water. For these products a leaching test with short
immersion phases ─ EN 16105 Laboratory method for determination of release of substances from
coatings in intermittent contact with water [25] ─ is recommended instead.
The results of biological tests with the same product, but using a different leaching test are not
comparable due to the different test conditions.
The selection of fractions from leaching tests for biological tests depends on the purpose of the testing
approach. Generally, for many construction products the maximum effect is expected to occur with the
first elution steps. It is often also practicable to test the first two fractions, because organic substances
are not always stable during long immersion periods.
An alternative option for the selection of fractions is to define an indicative parameter (e.g. TOC or
substances of interest) which is analysed in each eluate fraction. The two fractions with the highest
amount of the indicative parameter are combined and tested.
For construction products with continuously releasing compounds, those fractions with the longest
contact time may exhibit higher effects and the fractions should be taken from an advanced stage of the
leaching procedure. By comparison of effects from different fractions, the longer term behaviour of the
construction products may be assessed. The use of this option should be justified with an indicative
parameter.
5.1.2 Dynamic Surface Leaching Test (DSLT) CEN/TS 16637-2
The test describes a tank test for monolithic construction products of > 40 mm edge length in all
directions and for plates or sheets with a surface area exposed to the eluate of > 100 cm2. In the
standard procedure, this test is carried out for 64 days, while the eluate water is replaced at distinct
time intervals (after 6 h, 24 h, 2,25 d, 4 d, 9 d, 16 d, 36 d and 64 d).
Because of the numerous eluates from DSLT and the effort required for ecotoxicity testing, it is
recommended to consider the following adjustments:
— Only the first two elution steps after 6 h and additional 18 h are carried out and both eluates are
unified for ecotoxicity testing.
2 2
— The lower limit of the liquid / surface area relation (L/A), corresponding to 20 l/m (2 ml/cm ), is
applied in order to maximize the concentration level in the eluates (smallest liquid to surface area).
These adjustments have been applied in the European round robin test for the ecotoxicological
characterisation of construction products [8]. It can be useful to investigate eluates that represent other
elution steps, if information on later stages of leaching processes is desired.
5.1.3 Horizontal up-flow percolation test (CEN/TS 16637-3)
The horizontal up-flow percolation test is a method to determine the leaching behaviour of non-volatile
inorganic and organic substances from granular construction products (without or with size reduction).
The construction products are subjected to percolation with water as a function of liquid to solid ratio
under specified percolation conditions. The method is a once-through column leaching test.
Eluates from up-flow percolation tests are collected until an L/S ratio of 2 l/kg is reached. Mass of test
material and dimension of the applied columns have to be adapted to obtain sufficient eluate volume for
testing. Furthermore the relationship between maximum grain size and minimal column dimension as
specified in CEN/TS 16637-3 has to be fulfilled. It can be useful to investigate eluates that represent
higher L/S ratios if information on later stages of leaching processes is desired.
5.1.4 Further leaching tests
CEN/TS 16637-1 provides guidance for the determination of leaching tests and refers, among others, to
EN 12457, all parts.
EN 12457-1 [26] has been used for granular products in the European round robin test for the
ecotoxicological characterisation of construction products [8]. The one stage batch test has a liquid to
solid ratio of 2 l/kg for materials with high solid content and with particle size below 4 mm (without or
with size reduction). EN 12457-2 [27] with liquid to solid ratio of 10, which is applicable to a wide
range of wastes, has been recommended and used for ecotoxicological testing of waste [5], [24], [28].
The test in EN 16105 [25] has been designed to determine the release of substances from paints and
varnishes in intermittent contact with water. Under natural weather conditions, the construction
products are exposed to intermittent cycles of wetting (rainfall, thaw, and condensate) and drying.
5.2 Sampling, transport and storage of eluates
The goal of sampling is to obtain a representative eluate and to supply it to the laboratory in the correct
manner. The eluates may be susceptible to changes as a result of physical, chemical or biological
reactions. The eluates should be preferably tested immediately after sampling to avoid changes in the
original composition as a result of physical and chemical reactions and/or biological processes. If this is
not possible, e.g. because several biotests are intended to be applied, samples should be cooled down
for transport and storage in closed, dark glass bottles to temperatures between 2 °C and 8 °C and kept
in the dark or should be frozen down to ≤ -18 °C. The transport should be realised in cool boxes with
ice, frozen gel packs or cooling elements. The time span between sampling and ecotoxicity tests
(storage time) should remain within the storage time set of 48 h (cooled samples) or 2 months (frozen
samples). For further information see EN ISO 5667-16 [29] and related standards referred to there.
NOTE 1 Eluates are not considered suitable for biological tests, if they have been treated to prevent
biodegradation processes during the leaching test. The addition of preservatives such as biocides, acids or basic
solutions in order to retard chemical and biological activity modifies the test sample in an unacceptable way.
The amount of eluates and number of replicate vessels depends on the test battery chosen and the test
design and should be decided beforehand. It is practicable to split the total volume of the eluate into
appropriate flasks. In this way, the different tests can be carried out independently from each other and
suitable test portions are available for potential repetitions. As a rough estimate about 100 mL to 200
mL are needed for one test with one organism. For the algae test up to about 500 ml are required
depending on the size of the incubation vessels. It is recommended to consider additional replicate
samples for retesting the eluates in cases, where the toxicity range has not been met.
Frozen samples are thawed on the day of testing before use. A warm water bath at a temperature not
exceeding 25 °C, together with gentle shaking, are recommended to avoid local overheating.
Alternatively, the sample may be thawed in the dark at a temperature between 2 °C and 8 °C over night.
A once thawed sample may not be frozen again for subsequent testing, but a replicate sample should be
used. An even distribution of all soluble and particulate components should be ensured (e.g. by gentle
agitation or vigorous shaking) [29].
NOTE 2 Samples thawed with a microwave treatment are not considered suitable for biological tests as
thawing in a microwave oven modifies the test sample in an unacceptable way.
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).
5.3 Pre-treatment of eluates
It is recommended that any pre-treatment of the eluates should be limited to such cases, where
suspended solids resulting from construction products disturb the performance of the bioassays. This is
in accordance with EN ISO 5667-16. In general, biotests are carried out with the original sample. Only
when large amounts of particulate matter interfere with the requirements of test organisms these
should be removed by centrifugation or filtration. Centrifugation (e.g. 10 min at 5000 g) in general is
preferred to filtration.
Alternatively coarse particles can be separated by filtration (> 50 µm). The filter material should be
made of inert material (e.g. glass-fiber) and the filters should be rinsed with high-purity water prior to
use to reduce the risk of contamination of the eluates. Filtration can be carried out under pressure or
with vacuum. Any method used for separation of particles should be reported in the test report [29].
When phase separation is restricted, e.g. due to the specific weight of the material, it may also be
supported by sieving the eluate through a (0,5 ± 0,2) mm stainless steel sieve and subsequent
centrifugation [8].
It is recommended that the eluate should be allowed to settle for 30 min to 2 h after treatment. The
required quantity of supernatant can be sampled using a pipette.
6 Aquatic ecotoxicological testing
6.1 Purpose of aquatic ecotoxicity testing
Aquatic ecotoxicity tests measure the water available fraction of the toxic components of the eluate.
When carrying out aquatic tests, attention should be focussed on possible confounding factors which
might interfere with the test results and make adaptations in the test schemes advisable. Differences in
the ionic composition might interfere with many of the test organisms; coloured or turbid samples
might interfere with algae as well as in the luminescent bacteria test and pH values might be outside the
normal range for many test organisms. In those cases tests can best be carried out twice: once according
to the standardized method to provide comparable results and once with an adaptation in the method
to provide more insight for the evaluation of results.
6.2 Selection of ecotoxicity tests and minimum test battery
It is generally accepted in environmental hazard assessment, that a test battery with organisms from
different trophic levels and combining different assessment endpoints, is required.
A suitable test battery is detailed in the following table (Table 2).
Table 2 — Recommended minimum test battery for aquatic ecotoxicity tests on construction
products
Recommended Expression of
Test species Source Endpoint
test battery results
Luminescent bacteria EN ISO 11348-3 Luminescence EC (30 min)
(Vibrio fischeri) [30]
or LID
Algae EN ISO 8692 Growth EC (72 h) or
[31]
Minimum Pseudokirchneriella LID
battery subcapitata or
Desmodesmus subspicatus
Crustacea EN ISO 6341 Mobility EC (48 h) or
[32]
Daphnia magna LID
Danio rerio (zebrafish EN ISO 15088 Mortality EC (48 h) or
eggs) [14]
LID
umu-assay with ISO 13829 [33] Gene induction Dmin ≥ 1,5
Salmonella typhimurium (24 h)
TA 1535/pSK1002
or,
Ames fluctuation test ISO 11350 [34] Increase in the Significant
number of concentration-
mutant colonies related increase
Additional
of revertant
tests
wells over the
concentration
range tested
and/or a
reproducible
increase at one
or more
concentrations
in the number
of revertant
wells
6.3 Testing strategy and conditions
6.3.1 General
As a general guidance for testing strategy and conditions the guidance given in EN 14735 [24] is
recommended.
6.3.2 Consideration of additional blank samples
It is strongly recommended to consider additional blank vessels and eluates to be tested in parallel,
which include all experimental steps (from dilution water over the leaching vessels to the sampling
vessels). These additional blanks are also examined in the ecotoxicity tests, in order to detect possible
influence from the testing device such as the quality of the eluent water, contamination of the leaching
vessels or the surrounding environment, the storage conditions etc. Normally no ecotoxicity should be
detectable in these additional blanks at a dilution level of 2 (50 % by volume of the eluate and 50 %
dilution water).
6.3.3 pH adjustment
PH of test mixtures (i.e. test sample diluted with control/dilution medium) may significantly differ from
pH of test sample according to the selected dilution range and according to buffer capacity of
control/dilution medium or test sample. Therefore, different approaches have been developed and are
available in guidance documents referring to ecotoxicity testing according to the type of sample under
test. For instance, EN 14735 [24] recommends to perform ecotoxicity tests on waste eluates without
any pH adjustment of the eluate and to measure the pH of all test mixtures at the beginning and at the
end of the test. 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 eluate. Conversely,
EN ISO 5667-16 includes the following statements:
— Samples with extreme pH values exceeding the tolerance limits of most test organisms (usually pH
<6 or >9, depending on the organism) should be adjusted to pH 7,0 ± 0,2 e.g. by adding sodium
hydroxide NaOH (1 mol/l) or hydrogen chloride HCl (1 mol/l). However, the volume should
generally not exceed 5% of the total volume (see EN ISO 11348-3) [30]. The physiological limits of
the organisms should be defined in the respective standards. Exceeding the neutral pH value should
be avoided.
— Adjustment should be omitted if the effect of the pH is to be reflected in the test result or if physical
modification or chemical reactions (e.g. precipitation) are observed due to pH adjustment [29].
EXAMPLE The 5 % limit may be considered as suitable for instance for the test with Daphnia magna with an
eluate from a construction product: Vleachate = 200 ml; pH initial = 4,7. By adding 270 μl of NaOH (1 mol/l), the
pH level reaches 6,8 (which is inside the pH range for Daphnia magna test: 6,7 to 8,5). 270 μl is far below the limit
of 5 %.
6.3.4 Test concentrations
The results of ecotoxicity tests can be expressed as ECx (= effective concentrations) values or as LID (=
lowest ineffective dilution) values in order to fulfill the two main ways of expressing results of
ecotoxicity tests in Europe. In order to determine these values, the eluate is diluted according to a
defined scheme of dilutions to establish an exposure-effect relationship.
The EC is the concentration of eluate or percent dilution of eluate estimated to reduce the selected
endpoint at the end of the test to 50 % compared to the control within the test period.
The lowest ineffective dilution (LID) denotes the most concentrated tested dilution at which no
inhibition or mortality occurs, or only effects not exceeding the test-specific limit occur. The dilution
level D is expressed as the reciprocal value of the volume fraction of eluate in the respective test
medium.
6.3.5 Colour and turbidity
Highly coloured eluates might show ecotoxicological effects without being toxic, since UV radiation is
essential for some organisms. If eluates are turbid, they might show ecotoxicological effects without
being toxic for the same reasons. In such cases, duckweed (EN ISO 20079) can be used as test species
instead of algae [15].
6.3.6 Unstable substances
Furthermore, organic substances in eluates can be unstable during the performance of the leaching test
and/or the storage time before ecotoxicological testing starts. Guidance given on biotesting of samples
provided in EN ISO 5667-16 [29] should be considered when testing unstable eluates.
6.4 Limitations of aquatic ecotoxicity tests
A general limitation of all ecotoxicity tests is that part of the components of a leachate can be lost from
the test system because of evaporation of volatile substances, foaming of surface-active agents, sorption
to the test vessels or filters, biodegradation or abiotic degradation (e.g. hydrolysis, photolysis). In some
aquatic biological tests, the endpoint determination is based on a spectrometric measurement
(photometry, fluorometry) which is disturbed by highly coloured or turbid samples. For details
reference is given to EN ISO 5667-16 [29].
7 Assessment of biodegradability
Those eluates which exhibit higher ecotoxic effects and contain a significant amount of organic
substances may be tested for biodegradability for assessing, whether the organics are resistant to
biodegradation, thus causing longer-term concerns. Eluates with a signifi
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