SIST EN ISO 5667-16:2017
(Main)Water quality - Sampling - Part 16: Guidance on biotesting of samples (ISO 5667-16:2017)
Water quality - Sampling - Part 16: Guidance on biotesting of samples (ISO 5667-16:2017)
This document gives practical guidance on sampling, pre-treatment, performance and evaluation of
environmental samples in the context of performing biological tests. Information is given on how to
cope with the problems of biotesting arising from the sample and the suitability of the test design.
It is intended to convey practical experience concerning precautions to be taken by describing methods
successfully proven to solve or to circumvent some of the experimental problems of biotesting of, for
example, waters.
Primarily dealt with are substance-related problems concerning sampling and pre-treatment of
environmental samples (e.g. waste water samples) for the performance of biotests.
This guidance is on ecotoxicological testing with organisms (single-species biotests; in vivo and in
vitro). Some features addressed in this document also apply to biotests using single-cell systems (in
vitro bioassays) and biodegradation studies as far as sampling and sample preparations are concerned.
Testing of substances in the water solubility range is also addressed.
Reference has been made as far as possible to existing International Standards and guidelines.
Information taken from published papers or oral communication has been utilized as well.
This document is applicable to biological tests for determining the effect of environmental samples
like treated communal and industrial waste water, groundwater, fresh water, aqueous extracts (e.g.
leachates, eluates), pore water of sediments and whole sediments. This document is also applicable to
chemical substances.
This document is not applicable to bacteriological examination of water. Appropriate methods for
bacteriological examination are described in other documents (see ISO 19458[17]).
Wasserbeschaffenheit - Probenahme - Teil 16: Anleitung zur Probenahme und Durchführung biologischer Testverfahren (ISO 5667-16:2017)
Dieses Dokument gibt praktische Hinweise zur Probenahme, Vorbehandlung, Durchführung und Bewertung von Umweltproben in Verbindung mit der Durchführung von biologischen Testverfahren. Es werden Informationen gegeben, wie bei biologischen Testverfahren mit Problemen umzugehen ist, die das Probenmaterial und das Testdesign mit sich bringen.
Es ist das Ziel, praktische Erfahrung hinsichtlich zu treffender Vorkehrungen zu vermitteln, indem erfolgreich bewährte Verfahren beschrieben werden, um einige der experimentellen Probleme bei der Untersuchung von z. B. Wässern mit biologischen Testverfahren zu lösen oder zu umgehen.
Vor allem werden substanzbezogene Probleme in Bezug auf die Probenahme und Vorbehandlung von Umweltproben (z. B. Abwasserproben) bei der Durchführung von Biotests berücksichtigt.
Diese Anleitung behandelt ökotoxikologische Testverfahren mit Organismen (Tests mit jeweils einer Art; in vivo und in vitro). Einige Ausführungen in diesem Dokument beziehen sich auch auf Biotests, die einzellige Mikroorganismen (in-vitro Biotests) verwenden, und auf Untersuchungen zum biologischen Abbau, sofern die Probenahme und die Probenvorbereitung betroffen sind. Ebenso wird auf die Untersuchung von Substanzen im Bereich ihrer Wasserlöslichkeit eingegangen.
Soweit möglich, wird auf bestehende Internationale Normen und Anleitungen verwiesen. Die Informationen aus Veröffentlichungen und mündliche Mitteilungen sind ebenfalls berücksichtigt worden.
Dieses Dokument gilt für biologische Testverfahren zur Bestimmung der Wirkung von Umweltproben, wie behandeltes kommunales und Industrieabwasser, Grundwasser, Süßwasser, wässrige Extrakte (z. B. Sickerwasser, Eluate), Porenwasser von Sedimenten und Gesamtsedimente. Dieses Dokument ist auch auf chemische Substanzen übertragbar.
Dieses Dokument ist nicht anwendbar auf die bakteriologische Untersuchung von Wasser. Entsprechende Verfahren sind in anderen Dokumenten beschrieben (siehe ISO 19458) [17].
Qualité de l'eau - Échantillonnage - Partie 16: Lignes directrices pour les essais biologiques des échantillons (ISO 5667-16:2017)
ISO 5667-16:2017 fournit des lignes directrices pour l'échantillonnage, le prétraitement, la mise en ?uvre et l'évaluation des échantillons environnementaux dans le cadre de la réalisation d'essais biologiques. Des informations sont données sur la manière de traiter les problèmes, pour les essais biologiques, liés à l'échantillon et à l'adaptation du dispositif expérimental.
Elles sont destinées à diffuser une expérience pratique concernant les précautions à prendre en décrivant des méthodes qui se sont révélées satisfaisantes pour résoudre ou éliminer certains problèmes expérimentaux au cours des essais biologiques des eaux, par exemple.
ISO 5667-16:2017 traite en premier lieu des problèmes liés aux substances en ce qui concerne l'échantillonnage et le prétraitement des échantillons environnementaux (par exemple échantillons d'eaux résiduaires) pour la réalisation d'essais biologiques.
Ces lignes directrices portent sur les essais écotoxicologiques réalisés avec des organismes (essais biologiques monospécifiques; in vivo et in vitro). Certaines caractéristiques traitées dans le présent document s'appliquent également à l'échantillonnage et la préparation d'échantillons pour les essais biologiques utilisant des systèmes unicellulaires (essais biologiques in vitro) et les études de biodégradation. Les essais sur substances dans la gamme de solubilité dans l'eau sont également traités.
Il a été fait référence aux Normes internationales et aux directives existantes dans toute la mesure du possible. Des informations provenant de publications ou de communications orales ont également été utilisées.
ISO 5667-16:2017 s'applique aux essais biologiques destinés à déterminer l'effet des échantillons environnementaux tels que les eaux résiduaires communales et industrielles traitées, les eaux souterraines, les eaux douces, les extraits aqueux (par exemple lixiviats et éluats), l'eau interstitielle des sédiments et les sédiments entiers. Il s'applique également aux substances chimiques.
ISO 5667-16:2017 n'est pas applicable à l'examen bactériologique des eaux. Des méthodes appropriées pour l'examen bactériologique sont décrites dans d'autres documents (voir l'ISO 19458[17]).
Kakovost vode - Vzorčenje - 16. del: Navodilo za biološko preskušanje vzorcev (ISO 5667-16:2017)
Ta dokument podaja praktične smernice glede vzorčenja, predhodne obdelave, zmogljivosti in ocenjevanja okoljskih vzorcev za biološke preskuse. Vsebuje informacije o tem, kako se soočiti s težavami biološkega preskušanja, ki so posledica vzorca, in o primernosti zasnove preskusa.
Namenjen je podajanju praktičnih izkušenj v zvezi s previdnostnimi ukrepi, ki jih je treba izvesti, in sicer z opisovanjem metod, ki so se izkazale za uspešne pri reševanju ali preprečevanju nekaterih poskusnih težav, na primer pri biološkem preskušanju vod.
Zajema predvsem težave zaradi snovi, ki so povezane z vzorčenjem in predhodno obdelavo okoljskih vzorcev (npr. vzorci odpadne vode) za biološko preskušanje.
Smernice podajajo informacije o ekotoksikološkem preskušanju z organizmi (biološko preskušanje posamezne vrste; in vivo in in vitro). Nekatere značilnosti, obravnavane v tem dokumentu, veljajo tudi za biološke preskuse, ki uporabljajo enocelične sisteme (biološki preskusi in vitro) in študije biorazgradljivosti v povezavi z vzorčenjem in pripravo vzorcev.
Obravnavano je tudi preskušanje topnosti snovi v vodi.
Dodani so bili sklici na obstoječe mednarodne standarde in smernice, in sicer v največjem možnem obsegu.
Uporabljene so bile tudi informacije iz objavljenih študij ali ustne komunikacije. Ta dokument se uporablja za biološko preskušanje za določanje učinka okoljskih vzorcev, kot so obdelana javna in industrijska odpadna voda, podtalnica, sveža voda, vodni ekstrakti (npr. izcedne vode, izlužki), voda v porah sedimentov in celotnih sedimentov. Ta dokument se uporablja tudi za kemične snovi.
Ta dokument se ne uporablja za bakteriološko preskušanje vode. Metode, ki so primerne za bakteriološko preskušanje, so opisane v drugih dokumentih (glej ISO 19458[17]).
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 5667-16:2017
01-julij-2017
1DGRPHãþD
SIST EN ISO 5667-16:2000
.DNRYRVWYRGH9]RUþHQMHGHO 1DYRGLOR]DELRORãNRSUHVNXãDQMHY]RUFHY
,62
Water quality - Sampling - Part 16: Guidance on biotesting of samples (ISO 5667-
16:2017)
Wasserbeschaffenheit - Probenahme - Teil 16: Anleitung zur Probenahme und
Durchführung biologischer Testverfahren (ISO 5667-16:2017)
Qualité de l'eau - Échantillonnage - Partie 16: Lignes directrices pour les essais
biologiques des échantillons (ISO 5667-16:2017)
Ta slovenski standard je istoveten z: EN ISO 5667-16:2017
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 5667-16:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 5667-16:2017
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SIST EN ISO 5667-16:2017
EN ISO 5667-16
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2017
EUROPÄISCHE NORM
ICS 13.060.45 Supersedes EN ISO 5667-16:1998
English Version
Water quality - Sampling - Part 16: Guidance on biotesting
of samples (ISO 5667-16:2017)
Qualité de l'eau - Échantillonnage - Partie 16: Lignes Wasserbeschaffenheit - Probenahme - Teil 16:
directrices pour les essais biologiques des échantillons Anleitung zur Probenahme und Durchführung
(ISO 5667-16:2017) biologischer Testverfahren (ISO 5667-16:2017)
This European Standard was approved by CEN on 9 February 2017.
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, 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. EN ISO 5667-16:2017 E
worldwide for CEN national Members.
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SIST EN ISO 5667-16:2017
EN ISO 5667-16:2017 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 5667-16:2017
EN ISO 5667-16:2017 (E)
European foreword
This document (EN ISO 5667-16:2017) has been prepared by Technical Committee ISO/TC 147 “Water
quality” in collaboration with Technical Committee CEN/TC 230 “Water analysis” the secretariat of
which is held by DIN.
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 November 2017, and conflicting national standards
shall be withdrawn at the latest by November 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document supersedes EN ISO 5667-16:1998.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: 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 the United Kingdom.
Endorsement notice
The text of ISO 5667-16:2017 has been approved by CEN as EN ISO 5667-16:2017 without any
modification.
3
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SIST EN ISO 5667-16:2017
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SIST EN ISO 5667-16:2017
INTERNATIONAL ISO
STANDARD 5667-16
Second edition
2017-04
Water quality — Sampling —
Part 16:
Guidance on biotesting of samples
Qualité de l’eau — Échantillonnage —
Partie 16: Lignes directrices pour les essais biologiques des
échantillons
Reference number
ISO 5667-16:2017(E)
©
ISO 2017
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SIST EN ISO 5667-16:2017
ISO 5667-16:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
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SIST EN ISO 5667-16:2017
ISO 5667-16:2017(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General guidance regarding test design . 5
4.1 General . 5
4.2 Replicates . 5
4.2.1 General. 5
4.2.2 Lowest ineffective dilution (LID) . 5
4.2.3 Hypothesis testing — two-sample comparisons . 6
4.2.4 Concentration and dilution response relationship . 6
5 Evaluation . 7
5.1 General . 7
5.2 Statistical analysis . 7
6 Sampling and transportation . 7
6.1 General . 7
6.2 Sampling equipment . 8
6.2.1 General. 8
6.2.2 Sample container . 8
6.3 Filling status of sample containers . 9
6.4 Sample identification and records . 9
6.5 Sub-sampling . 9
6.6 Transportation .10
6.7 Contamination during sampling .10
6.8 Sampling quality control techniques .10
7 Pre-treatment .11
7.1 General .11
7.2 Preservation and storage .11
7.3 Thawing .12
7.4 Homogenization .12
7.5 Separation of soluble and particulate matter .12
7.6 Preconcentration .13
7.6.1 General.13
7.6.2 Extraction methods .14
7.7 pH adjustment .14
8 Apparatus and equipment .14
8.1 Selection of apparatus .14
8.2 Cleaning of apparatus and equipment .15
9 Impairment of test performance .15
9.1 Problems and preventive measures for samples containing removable ingredients .15
9.1.1 General.15
9.1.2 Volatilization . .16
9.1.3 Foaming .16
9.1.4 Adsorption . . .16
9.1.5 Precipitation/flocculation .16
9.1.6 Degradation .16
9.2 Problems and preventive measures concerning coloured and/or turbid samples .17
10 Preparation of stock solutions and test batches .17
10.1 Water-soluble substances .17
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SIST EN ISO 5667-16:2017
ISO 5667-16:2017(E)
10.2 Poorly soluble substances .17
10.2.1 General.17
10.2.2 Testing in the water solubility range .17
10.2.3 Dispersions and emulsions .18
10.2.4 Special problems with mixtures of substances or technical products .18
10.2.5 Limit test .19
11 Quality assurance for biotesting .19
11.1 General .19
11.2 Quality assurance in the context of the investigation of environmental samples .19
12 Reporting .20
Bibliography .23
iv © ISO 2017 – All rights reserved
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SIST EN ISO 5667-16:2017
ISO 5667-16:2017(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by ISO/TC 147, Water quality, Subcommittee SC 6, Sampling (general
methods).
This second edition cancels and replaces the first edition (ISO 5667-16:1998), which has been technically
revised.
A list of all parts in the ISO 5667 series can be found on the ISO website.
© ISO 2017 – All rights reserved v
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SIST EN ISO 5667-16:2017
ISO 5667-16:2017(E)
Introduction
Biological tests are suitable for determining the effect of environmental samples or chemical substances
on the respective test organism under the specific standardized test conditions. Environmental samples
are e.g. treated communal and industrial waste water, fresh water, aqueous extracts of solid material
(e.g. leachates, eluates), pore water of sediments. The effect can be stimulative or inhibiting, and can be
determined by the reaction of the test organism (e.g. death, growth, morphological and physiological
changes or generally, changes in molecular mechanisms of action). Inhibiting effects can be triggered
by toxic water constituents or by other noxious influences.
The toxicity measurable in the biological test is the result of the interaction between a single toxic
substance, a mixture of substances or the constituents of an environmental sample and the test
organism. The protective potential of the biological system, i.e. the test organism, for instance by
metabolic detoxification and excretion, is an integral part of the biological test.
Apart from the direct toxic effect of one or more sample constituents, biological effects can be exerted
by the combined action of all constituents of a sample. Such a combined effect includes the impact of, for
example, substances which are not toxic per se but affect the chemical or physical properties of the test
batches by interfering with the test specific additives (e.g. nutrients, salts) and, consequently, the living
conditions for the test organisms. This applies for instance to oxygen-depleting substances, coloured
substances or turbid matter which reduce light exposure.
Biological tests also include those tests which examine the effect of organisms on substances (e.g.
microbial degradation studies).
The results of the biological test refer primarily to the organism used in the test and the defined
conditions stipulated for the test procedure. A harmful effect stated by means of standardized biological
tests can justify concern that aquatic organisms and biocoenosis might be endangered. The results,
however, do not permit direct or extrapolative conclusions as to the occurrence of similar effects in
the aquatic environment. This applies in particular to suborganismic tests, as important properties
and physiological functions of intact organisms (e.g. protective integuments, repair mechanisms) are
removed or deactivated.
In principle there is no test organism which can be used to test all the effects on the biocoenosis or
the ecosystem possible under the various combinations of abiotic and biotic conditions. Only a few
(“model”) species representing relevant ecological functions can be tested in practice.
Besides these fundamental and practical limitations in the selection of test organisms some issues
should be taken into account during sampling and sample treatment in order to avoid a change in the
sample properties. This applies to the method of sampling, including the sampling equipment and
sample container as well as the transport to the laboratory. The method of sample pre-treatment and
storage, as well as the preparation of, for example, stock solutions, may have an influence on the test
result as well.
Furthermore, the sample to be tested can pose experimental problems on biotesting. Environmental
samples (e.g. waste water, eluates) are complex mixtures and may contain, for example, sparingly
soluble, volatile, unstable, coloured substances or suspended, sometimes colloidal, particles. The
complexity and heterogeneity of materials give rise to a variety of experimental problems when
performing biotests.
Special problems are related to the instability of the test material due to reactions and processes such as
— physical (e.g. phase separation, sedimentation, volatilization),
— chemical (e.g. hydrolysis, photodegradation, precipitation), and/or
— biological (e.g. biodegradation, biotransformation, biological uptake in organisms).
Other problems, especially if spectrometric measurements are applied, relate to turbidity and colour of
the test batch.
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SIST EN ISO 5667-16:2017
ISO 5667-16:2017(E)
The statistical analysis of the data from biological testing of environmental samples should be
conducted according to the current state of the art if not stipulated by the specific biotest standard.
Finally, it is recommended to implement and maintain a quality management system regardless if a
laboratory is involved in testing of substances or environmental samples.
This document is one of a group of International Standards dealing with the sampling of waters and
sediments and is intended to be read in conjunction with the other parts of the ISO 5667 series, in
particular with ISO 5667-1, ISO 5667-3 and ISO 5667-15.
© ISO 2017 – All rights reserved vii
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SIST EN ISO 5667-16:2017
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SIST EN ISO 5667-16:2017
INTERNATIONAL STANDARD ISO 5667-16:2017(E)
Water quality — Sampling —
Part 16:
Guidance on biotesting of samples
1 Scope
This document gives practical guidance on sampling, pre-treatment, performance and evaluation of
environmental samples in the context of performing biological tests. Information is given on how to
cope with the problems of biotesting arising from the sample and the suitability of the test design.
It is intended to convey practical experience concerning precautions to be taken by describing methods
successfully proven to solve or to circumvent some of the experimental problems of biotesting of, for
example, waters.
Primarily dealt with are substance-related problems concerning sampling and pre-treatment of
environmental samples (e.g. waste water samples) for the performance of biotests.
This guidance is on ecotoxicological testing with organisms (single-species biotests; in vivo and in
vitro). Some features addressed in this document also apply to biotests using single-cell systems (in
vitro bioassays) and biodegradation studies as far as sampling and sample preparations are concerned.
Testing of substances in the water solubility range is also addressed.
Reference has been made as far as possible to existing International Standards and guidelines.
Information taken from published papers or oral communication has been utilized as well.
This document is applicable to biological tests for determining the effect of environmental samples
like treated communal and industrial waste water, groundwater, fresh water, aqueous extracts (e.g.
leachates, eluates), pore water of sediments and whole sediments. This document is also applicable to
chemical substances.
This document is not applicable to bacteriological examination of water. Appropriate methods for
[17]
bacteriological examination are described in other documents (see ISO 19458 ).
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
blank
mixture of water and nutrients without test organism
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SIST EN ISO 5667-16:2017
ISO 5667-16:2017(E)
3.2
cell density
x
number of cells per unit volume of medium
Note 1 to entry: Cell density is expressed in cells per millilitre.
[6]
[SOURCE: ISO 10253:2016, 3.1]
3.3
control
control medium (3.4), or control sediment (3.5), including organisms used in the test, without test sample
3.4
control medium
combination of dilution water and/or nutrient medium used in the test
[18]
[SOURCE: ISO 20079:2005, 3.6]
3.5
control sediment
defined artificial or natural sediment used in the test
3.6
dilution level
D
reciprocal value of the volume fraction of test sample in dilution water (3.7) in which the test is
conducted
EXAMPLE 250 ml of waste water in a total volume of 1 000 ml (volume fraction of 25 %) represents dilution
level D = 4.
[13]
[SOURCE: ISO 15088:2007, 3.2, modified — “waste water” replaced by “test sample”]
3.7
dilution water
water added to the test sample to prepare a series of defined dilutions
Note 1 to entry: The composition of the water is specified in the respective standard.
[18]
[SOURCE: ISO 20079:2005, 3.7, modified — “Note 1 to entry” has been added]
3.8
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, 3.8.1, modified — “quantal” has been removed from the term and
abbreviated term; “soil” and “(e.g. immobility)” have been removed from the definition; the EXAMPLE
[20]
and Notes 1 and 2 to entry are not included]
3.9
field blank
container prepared in the laboratory using reagent water or other blank matrix and sent with the
sampling personnel for exposure to the sampling environment to verify possible contamination during
sampling
[9]
[SOURCE: ISO 11074:2015, 4.5.3]
2 © ISO 2017 – All rights reserved
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SIST EN ISO 5667-16:2017
ISO 5667-16:2017(E)
3.10
growth rate
proportional rate of increase in biomass per unit of time: (1/day)
[SOURCE: ISO 10253:2016, 3.2, modified — “specific grow rate” replaced by “growth rate
...
SLOVENSKI STANDARD
oSIST prEN ISO 5667-16:2016
01-marec-2016
.DNRYRVWYRGH9]RUþHQMHGHO 1DYRGLOR]DELRORãNHSUHVNXVHY]RUFHY
,62',6
Water quality - Sampling - Part 16: Guidance on biotesting of samples (ISO/DIS 5667-
16:2016)
Wasserbeschaffenheit - Probenahme - Teil 16: Anleitung zur Probenahme und
Durchführung biologischer Testverfahren
Qualité de l'eau - Échantillonnage - Partie 16: Lignes directrices pour les essais
biologiques des échantillons (ISO/DIS 5667-16:2016)
Ta slovenski standard je istoveten z: prEN ISO 5667-16
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST prEN ISO 5667-16:2016 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 5667-16:2016
DRAFT INTERNATIONAL STANDARD
ISO/DIS 5667-16
ISO/TC 147/SC 6 Secretariat: BSI
Voting begins on: Voting terminates on:
2016-01-28 2016-04-28
Water quality — Sampling —
Part 16:
Guidance on biotesting of samples
Qualité de l’eau — Échantillonnage —
Partie 16: Lignes directrices pour les essais biologiques des échantillons
ICS: 13.060.45
ISO/CEN PARALLEL PROCESSING
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bodies for a parallel five month enquiry.
Should this draft be accepted, a final draft, established on the basis of comments
received, will be submitted to a parallel two-month approval vote in ISO and
THIS DOCUMENT IS A DRAFT CIRCULATED
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THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
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ISO/DIS 5667-16:2015(E)
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PROVIDE SUPPORTING DOCUMENTATION. ISO 2015
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ISO/DIS 5667-16:2015(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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Contents Page
Foreword . v
Introduction . vii
1 Scope .1
2 Normative references .1
3 Terms and definitions .2
4 General guidance regarding test design .6
4.1 General .6
4.2 Replicates .6
5 Evaluation .8
5.1 General .8
5.2 Statistical analysis .8
6 Sampling and transport .8
6.1 General .8
6.2 Sampling equipment .9
6.3 Filling status of sample containers . 10
6.4 Sample identification and records . 11
6.5 Sub-sampling . 11
6.6 Transport . 11
6.7 Contamination during sampling . 12
6.8 Sampling quality control techniques . 12
7 Pre-treatment . 12
7.1 Preservation and storage. 12
7.2 Thawing . 13
7.3 Homogenization . 13
7.4 Separation of soluble and particulate matter . 13
7.5 Preconcentration . 14
7.6 pH adjustment. 15
8 Apparatus and equipment . 16
8.1 Selection of apparatus. 16
8.2 Cleaning of apparatus and equipment . 16
9 Impairment of test performance . 16
9.1 Problems and preventive measures for samples containing removable ingredients . 16
9.2 Problems and preventive measures concerning coloured and/or turbid samples . 18
10 Preparation of stock solutions and test batches . 19
10.1 Water-soluble substances . 19
10.2 Poorly soluble substances . 19
11 Quality assurance for biotesting . 21
11.1 General . 21
11.2 Quality assurance in the context of the investigation of environmental samples . 22
12 Test report . 22
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Bibliography . 26
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Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national
standards bodies (ISO member bodies). The work of preparing International Standards is normally
carried out through ISO technical committees. Each member body interested in a subject for which a
technical committee has been established has the right to be represented on that committee.
International organizations, governmental and non-governmental, in liaison with ISO, also take part in
the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all
matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO's adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committees responsible for this document are ISO/TC 147, Water quality, Subcommittee SC 6,
Sampling (general methods) and, under the Vienna Agreement, CEN/TC 230, Water analysis.
This second edition cancels and replaces the first edition (ISO 5667-16:1998), which has been
technically revised.
ISO 5667 consists of the following parts, under the general title Water quality — Sampling:
Part 1: Guidance on the design of sampling programmes and sampling techniques
Part 3: Preservation and handling of water samples
Part 4: Guidance on sampling from lakes, natural and man-made
Part 5: Guidance on sampling of drinking water from treatment works and piped distribution systems
Part 6: Guidance on sampling of rivers and streams
Part 7: Guidance on sampling of water and steam in boiler plants
Part 8: Guidance on the sampling of wet deposition
Part 9: Guidance on sampling from marine waters
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Part 10: Guidance on sampling of waste waters
Part 11: Guidance on sampling of groundwaters
Part 12: Guidance on sampling of bottom sediments
Part 13: Guidance on sampling of sludges
Part 14: Guidance on quality assurance and quality control of environmental water sampling and
handling
Part 15: Guidance on the preservation and handling of sludge and sediment samples
Part 16: Guidance on biotesting of samples
Part 17: Guidance on sampling of bulk suspended solids
Part 19: Guidance on sampling of marine sediments
Part 20: Guidance on the use of sampling data for decision making - Compliance with thresholds and
classification systems
Part 21: Guidance on sampling of drinking water distributed by tankers or means other than
distribution pipes
Part 22: Guidance on design and installation of groundwater monitoring points
Part 23: Guidance on passive sampling in surface waters
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Introduction
Biological tests are suitable for determining the effect of environmental samples like treated communal
and industrial waste water, fresh water, aqueous extracts (e.g. leachates, eluates) of solid material, pore
water of sediments, whole sediments or chemical substances 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).
Inhibiting effects are triggered by toxic water constituents or by other noxious influences.
The apparent toxicity measurable in the biological test is the result of the interaction between the
constituents of an environmental sample or a toxic substance 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.
Apart from the direct toxic effect of one or more sample constituents, damaging biological effects can be
exerted by the combined action of all noxious substances (e.g. by substances which are not toxic per se
but affect the chemical or physical properties of the test batches by interfering with the test specific
additives (e.g. nutrients, salts) and, consequently, the living conditions for the organisms. This applies
for instance to oxygen-depleting substances, coloured substances or turbid matter which reduce light
exposure.
Biological tests also include those tests which examine the effect of organisms on substances (e.g.
microbial degradation studies).
The result of the biological test refer primarily to the organism used in the test and the conditions
stipulated in the test procedure. A harmful effect stated by means of standardized tests can justify
concern that aquatic organisms and biocoenosis might be endangered. The results, however, do not
permit direct or extrapolative conclusions as to the occurrence of similar effects in the aquatic
environment. This applies in particular to suborganismic tests, as important properties and
physiological functions of intact organisms (e.g. protective integuments, repair mechanisms) are
removed or deactivated.
In principle there is no organism which can be used to test all the effects on the biocoenosis or the
ecosystem possible under the various constellations of abiotic and biotic conditions. Only a few
("model") species representing relevant ecological functions can be tested in practice.
Besides these fundamental and practical limitations in the selection of test organisms, the sample to be
tested can also pose experimental problems on biotesting. Environmental samples (e.g. waste water,
whole sediment) are complex mixtures and often contain sparingly soluble, volatile, unstable, coloured
substances and/or suspended, sometimes colloidal, particles. The complexity and heterogeneity of
materials give rise to a variety of experimental problems when performing biotests.
Special problems are related to the instability of the test material due to reactions and processes such
as:
physical (e.g. phase separation, sedimentation, volatilization);
chemical (e.g. hydrolysis, photodegradation, precipitation); and/or
biological (e.g. biodegradation, biotransformation, biological uptake in organisms).
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Other problems, especially if spectrometric measurements are applied, relate to turbidity and colour.
This part of ISO 5667 is one of a group of International Standards dealing with the sampling of waters
and should be read in conjunction with the other parts of ISO 5667 and in particular with parts 1, 3 and
15.
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DRAFT INTERNATIONAL STANDARD ISO/DIS 5667-16
Water quality — Sampling — Part 16: Guidance on biotesting
of samples
1 Scope
This part of ISO 5667 gives practical guidance on sampling, pre-treatment, performance and evaluation
of environmental samples in the context of performing biological tests. Information is given on how to
cope with the problems for biotesting arising from the sample and the suitability of the test design.
It is intended to convey practical experience concerning precautions to be taken by describing methods
successfully proven to solve or to circumvent some of the experimental problems of biotesting of
waters.
Reference has been made as far as possible to existing International Standards and guidelines.
Information taken from published papers or oral communication is utilized as well.
Primarily dealt with are substance-related problems concerning sampling and pre-treatment of
environmental samples (e.g. waste water samples) for the performance of biotests.
Special emphasis is laid on ecotoxicological testing with organisms (single-species biotests). Some
features addressed in this general guidance apply as well to biotests using single-cell systems (in vitro
biotest) and biodegradation studies as far as sampling and sample preparations are concerned. Testing
of substances in the water solubility range is also addressed.
This part of ISO 5667 is applicable to biological tests for determining the effect of environmental
samples like treated communal and industrial waste water, fresh water, aqueous extracts (e.g.
leachates, eluates), pore water of sediments and whole sediments. This part of ISO 5667 is also
applicable to chemical substances.
This part of ISO 5667 is not applicable to bacteriological examination of water. Appropriate methods
are described in other International Standards.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes and
sampling techniques
ISO 5667-10, Water quality — Sampling — Part 10: Guidance on sampling of waste waters
ISO 5667-15, Water quality — Sampling — Part 15: Guidance on the preservation and handling of sludge
and sediment samples
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3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
blank
mixture of water and nutrients without test organism
3.2
cell density
x
number of cells per unit volume of medium
[1]
[SOURCE: ISO 10253:2006 , 3.1]
Note 1 to entry: Cell density is expressed in cells per millilitre.
3.3
control
control medium (3.4), or control sediment (3.5), including organisms used in the test, without test
sample
3.4
control medium
combination of water and additives (e.g. nutrients, salt) used in the test
3.5
control sediment
defined artificial or natural sediment used in the test
[2]
[SOURCE: ISO 10872:2010 , 3.7, modified]
3.6
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 waste water in a total volume of 1 000 ml (volume fraction of 25 %) represents dilution
level D = 4.
[3]
[SOURCE: ISO 15088:2007 , 3.2]
3.7
dilution water
water added to the test sample to prepare a series of defined dilutions
[4]
[SOURCE: ISO 20079:2005 , 3.7]
Note 1 to entry: The composition of the water is specified in the specific test.
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3.8
effective concentration
EC
x
concentration of the test material in water or sediment that causes x % change in response during a
specified time interval
[4]
[SOURCE: ISO/TS 20281:2006 , 3.8.1]
3.9
field blank
container prepared in the laboratory, using reagent water or other blank matrix, and sent with the
sampling personnel for exposure to the sampling environment to verify possible contamination during
sampling
[5]
[SOURCE: ISO 11074:2005 , 4.5.3]
3.10
growth rate
proportional rate of increase in biomass per unit of time: (1/day)
[1]
[SOURCE: ISO 10253:2006 , 3.2]
3.11
lowest ineffective dilution / dilution factor
LID
lowest ineffective dilution tested, expressed as dilution level D (3.6), at which no inhibition, or only
effects not exceeding the test-specific variability, are observed
[3]
[SOURCE: ISO 15088:2007 , 3.5]
3.12
nutrient medium
solution of nutrients and micronutrients in water which are essential for the growth of the test
organism
[4]
[SOURCE: ISO 20079:2005 , 3.17, modified]
3.13
positive control
any well-characterized reference substance that, when evaluated by a specific test method,
demonstrates the suitability of the test system to yield a reproducible, appropriately positive or
reactive response in the test system.
[7]
[SOURCE: ISO 10993-10:2010 , 3.14, modified]
3.14
reference substance
known substance to verify the sensitivity of the method
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3.15
reference batch
effect or behaviour of a substance known from previous tests (reference substance) and examined
within the framework of a test series as test sample
[8]
[SOURCE: ISO 21338:2010 , 3.5]
3.16
replicate
single test chamber containing a prescribed number of organisms, either in one concentration
(treatment) of test sample, or in a control
[21]
[SOURCE: Reference Environment Canada 2005 ]
3.17
sample
portion of material selected from a larger quantity of material collected and taken away for testing
[20]
[SOURCE: ISO 18589-1:2005 , 3.3.1]
NOTE 1 to entry: The method of sample selection can be described in the sampling plan.
Note 2 to entry: The material is from the environment (e.g. waste water, sediment or an eluate), a chemical
substance or preparation or related material.
3.18
sample pre-treatment
collective noun for all procedures used for conditioning a sample to a defined state which allows
subsequent examination
Note 1 to entry: Depending on the requirements of the method sample pre-treatment includes for example
preservation and storage, centrifugation, filtration, homogenization, preconcentration, pH adjustment.
3.19
sample storage
process, and the result, of keeping a sample available under predefined conditions for a (usually)
specified time interval between collection and further treatment of a sample (storage time)
[10]
[SOURCE: ISO 5667-3:2012 , 3.3]
Note 1 to entry: Specified time is the maximum time interval.
3.20
stock culture
culture of a single species to conserve the original defined species in the laboratory
[4]
[SOURCE: ISO 20079:2005 , 3.21, modified]
3.21
stock solution
solution with known analyte concentration(s), prepared from chemicals with an appropriate purity
[9]
[SOURCE: ISO 11885:2007 , 3.23]
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3.22
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
[10]
[SOURCE: ISO 5667-3:2012 , 3.4]
Note 1 to entry: Sampling finishes as soon as the sample container has been filled with the sample. Storage time
ends when the sample is taken by the analyst to start sample preparation prior to analysis.
3.23
sub-sample
representative portion removed from a sample
[11]
[SOURCE: ISO 5667-19:2004 , 3.7]
3.24
test batch
mixture of test medium, undiluted or diluted with water, including test organisms
[13]
[SOURCE: ISO 8692:2004 , 3.6, modified]
3.25
test medium
mixture of dilution water, test specific additives and test sample
[13]
[SOURCE: ISO 8692:2004 , 3.5]
3.26
test sample
undiluted, diluted or otherwise prepared portion of a sample to be tested, after completion of all pre-
treatment (x.y) steps
[6]
[SOURCE: ISO 6107-6:2004 , 92, modified]
3.27
test substance
chemical substance under investigation added to the test system
[12]
[SOURCE: ISO 15473:2002 , 3.10]
3.28
test material
material to be tested
[14]
[SOURCE: ISO 17126:2005 , 3.3]
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4 General guidance regarding test design
4.1 General
For each test several replicates of the control and the treatment groups should be examined. The
minimum number of replicates is usually prescribed in the respective standard. An example of how the
required number of observations (replicates) can be calculated is given in 4.2.3.
It is recommended to minimize the influence of differences in testing conditions (e.g. light,
temperature) for example by randomizing the design for location of the test vessels in the test chamber.
To assure that the laboratory test conditions (including the condition and sensitivity of the test
organisms) are adequate and have not changed significantly, a reference substance should be tested as
a positive control.
4.2 Replicates
4.2.1 General
Mainly three statistical approaches are performed in statistical analysis of the results from ecotoxicity
tests with environmental samples: (1) determining the lowest ineffective dilution (LID) in testing of
waste water, (2) two-sample comparisons between the control (or reference batch) and either test or
positive control batches (toxic standard) and (3) computing point estimates (e.g. EC20, LC50) from
modelled concentration/dilution/response relationships.
In ecotoxicity testing of environmental samples the determination of a NOEC (no observed effect
concentration) and LOEC (lowest observed effect concentration) is normally not intended and thus is
not considered further here. If the assessment of the NOEC has to be exceptionally performed consider
[15]
ISO/TS 20281 .
The number of replicates is mainly critical in hypothesis testing (e.g. two-sample comparisons) and is
dependent on the variability of the respective endpoint to be evaluated, the minimum effect size which
has to be detected by a statistical test and the statistical power. The estimation of an ECx puts different
[15]
demands on the study design than do the two-sample comparison (see ISO/TS 20281 ).
4.2.2 Lowest ineffective dilution (LID)
In toxicity testing of waste water the water sample is diluted according to a defined scheme of dilutions
(D). The lowest ineffective dilution (LID) denotes the most concentrated test batch at which no
[4]
inhibition or mortality, or only effects not exceeding the test-specific limit occur (e.g. ISO/TS 20281 ]:
10 % inhibition of the growth rate of Lemna spec.). D is expressed as the reciprocal value of the volume
fraction of waste water in the test batch.
This approach does not require further statistical analysis (e.g. hypothesis testing).
4.2.3 Hypothesis testing – two-sample comparisons
Statistical two-sample comparisons play an important role in testing of environmental samples. They
are performed in order to compare samples from various sample sites with a reference site (e.g.
samples from a polluted area of running waters with a sample from an unp
...
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