ISO 5667-16:1998

INTERNATIONAL ISO
STANDARD 5667-16
First edition
1998-10-01
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
A
Reference number
Contents Page
1 Scope . 1
2 Normative references. 1
3 Sampling. 1
4 Transport. 2
5 Preservation and storage. 3
6 Apparatus and equipment . 3
7 Pretreatment and preparation of samples . 4
8 Treatment of samples during the test . 10
9 General guidance regarding test design. 11
10 Special guidance regarding test performance . 13
11 Special biological assays . 16
12 Evaluation. 20
13 Presentation of results. 23
14 Test report. 24
15 Basic principles of quality assurance for biotesting . 25
Annex A Lowest Ineffective Dilution (LID) . 28
Annex B Bibliography . 29
©  ISO 1998
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced
or utilized in any form or by any means, electronic or mechanical, including photocopying and
microfilm, without permission in writing from the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet iso@iso.ch
Printed in Switzerland
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ISO ISO 5667-16:1998(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.
Draft International Standards adopted by the technical committees are
circulated to the member bodies for voting. Publication as an International
Standard requires approval by at least 75 % of the member bodies casting
a vote.
International Standard ISO 5667-16 was prepared by Technical Committee
ISO/TC 147, Water quality, Subcommittee SC 6, Sampling.
ISO 5667 consists of the following parts, under the general title Water
quality — Sampling:
 Part 1: Guidance on the design of sampling programmes
 Part 2: Guidance on sampling techniques
 Part 3: Guidance on the preservation and handling of samples
 Part 4: Guidance on sampling from lakes, natural and man-made
 Part 5: Guidance on sampling of drinking water and water used for
food and beverage processing
 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
 Part 10: Guidance on sampling of waste waters
 Part 11: Guidance on sampling of groundwaters
 Part 12: Guidance on sampling of bottom sediments
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 Part 13: Guidance on sampling of water, wastewater and related
sludges
 Part 14: Guidance on quality assurance of environmental water
sampling and handling
 Part 15: Guidance on preservation and handling of sludge and
sediment samples
 Part 16: Guidance on biotesting of samples.
Annexes A and B of this part of ISO 5667 are for information only.
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ISO ISO 5667-16:1998(E)
Introduction
Biological tests are suitable for determining the effect of chemical and
physical parameters on test organisms under specific experimental
conditions. In principle, the methods of chemical analysis are not suitable
for determining the biological effects. These effects can be enhancing or
inhibiting, and can be determined by the reaction of the organisms, e.g.
death, growth, proliferation, morphological, physiological and histological
changes. Inhibiting effects are triggered by toxic water constituents or by
other noxious influences.
Effects can refer to various levels, e.g. proceeding from (sub)cellular
structures or enzyme systems, concerning the whole organism, and
eventually the supra-organism or community level.
In the context of this part of ISO 5667, toxicity is the ability of a substance
to exert a deleterious effect on organisms or biocenoses due to its
chemical properties and its concentration.
The deleterious potential of a toxic substance can be counteracted by the
protective potential of the biological system, for instance by metabolic
detoxification and excretion. The apparent toxicity measurable in the
biological test is the result of the interaction between the substance and the
biological system.
Apart from the direct toxic effect of one or more water 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 medium 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. It also includes non-substance-related
effects such as impairment or damage due to extreme temperature.
Biological tests also include those tests which examine the effect of
organisms on substances, e.g. microbial degradation studies.
The results of the biological tests refer primarily to the organisms 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 biocenoses 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 sub-
organism systems, 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 and no biocenosis which can be used to
test all the effects on the ecosystem possible under the various
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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. Waters, in particular waste waters, 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).
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. It should be read in conjunction with the other
parts and in particular with ISO 5667-1, ISO 5667-2 and ISO 5667-3.
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INTERNATIONAL STANDARD  © ISO ISO 5667-16:1998(E)
Water quality — Sampling —
Part 16:
Guidance on biotesting of samples
1 Scope
This part of ISO 5667 gives practical guidance on sampling, pretreatment, performance and evaluation of waters in
the context of biotesting. Information is given on how to cope with the problems for biotesting arising from the nature
of the water 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, pretreatment and preparation of water
samples for biotesting and treatment of samples during the test, especially when performing tests with waters and
waste waters containing unstable or removable ingredients. Basic principles of quality assurance, evaluation of data
and presentation of results are outlined.
Special emphasis is laid on ecotoxicological testing with organisms ('single-species biotests'). Some features
addressed in this general guidance apply as well to biodegradation and/or bioaccumulation studies as far as
sampling and sample preparations is concerned. Preparation of poorly soluble substances and testing beyond the
water-solubility limit is also addressed.
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 standards contain provisions which, through reference in this text, constitute provisions of this part of
ISO 5667. At the time of publication, the editions indicated were valid. All standards are subject to revision, and
parties to agreements based on this part of ISO 5667 are encouraged to investigate the possibility applying the
most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid
International Standards.
ISO 5667-3 :1994, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples.
ISO 5667-10 :1992, Water quality — Sampling — Part 10: Guidance on sampling of waste waters.
3 Sampling
3.1 General
The choice of representative sampling points, frequency of sampling, type of samples taken, etc. is dependent on
the objective of the study. In general, the sampling approach for chemical analysis is compatible with the purpose of
biotesting.
© ISO
Some tests, however, require the water and waste water to be handled and kept in a particular way.
Depending on the type of investigation (e.g. toxicity or biodegradation tests) and the way the samples are to be
processed, it is necessary to divide a sample into different portions which are preserved and/or stored under
different conditions and processed in different ways.
If several samples have been taken (e.g. from different locations or at several times) they may be combined to
achieve greater representativity. These samples should be thoroughly mixed and, if necessary, divided into
subsamples. To obtain subsamples of equal quality, it should be ensured that the bulk sample maintains
homogeneity during the subsampling process, e.g. by continuous shaking or stirring. This holds particularly in the
case of two-phase mixtures, e.g. waters containing suspended particles, algal suspensions. It is recommended to
use cooling sampling apparatus when several samples taken at several times are combined.
3.2 Samplers/vessels/containers
The volume, shape and material of the vessels are dependent on the nature of the sample (e.g.
degradability/stability), the number of replicates, the volume required for these tests and the necessity of preserving
and storing the samples prior to further processing.
The time required for freezing and thawing should be minimized by reducing the sample volume, i.e. the size of the
vessel. In general it is appropriate to use one-litre vessels for freezing. For tests requiring larger volumes, the
sample should be divided into vessels holding not more than 10 l.
The total sample volume taken should be sufficient to cover any supplementary or repeated testing. Remaining
subsamples stored frozen separately should be saved until the final evaluation has been made.
The material of vessels should be chemically inert, easily cleaned and resistant to heating and freezing. Glassware,
polyethene or polytetrafluoroethene (PTFE) vessels are recommended.
3.3 Filling status of containers
It should be decided whether the containers should be filled completely to the brim or only partially, having an air
space, by taking into account the type of sample, the preservation mode and the biotest envisaged.
Problems related to partial filling can be
 enhanced agitation during transport, leading to breakdown of aggregated particles;
 interaction with gas phase, leading to stripping;
 oxidation of substances, leading e.g. to precipitation of compounds of heavy metals.
Problems related to complete filling can be
 oxygen depletion, with possible decomposition, leading to formation of toxic metabolites (e.g. nitrite, sulfide);
 impairment of homogenization by shaking or stirring the total volume.
Sample containers, when freezing is envisaged for preservation, should not be filled completely in order to allow
expansion of volume.
4 Transport
The samples collected should be protected from breakage, temperature increase and external contamination.
Misidentification of samples transported in melting ice should be avoided by using waterproof markers and/or labels.
© ISO
5 Preservation and storage
As stated in ISO 5667-3, it is impossible to give absolute rules for preservation, e.g. the duration of possible storage
and efficiency of various modes, because it depends primarily on the nature of the sample, especially its biological
activity.
Potable waters and ground waters are generally less susceptible to biological and chemical reactions than surface
waters, treated or raw waste waters. If the chemical composition can be approximately anticipated, reference
should be made to ISO 5667-3 for the purposes of biotesting. Some additional precautions, however, should be
considered as follows.
Samples for biotesting should be processed preferably without delay after collection to avoid changes in the original
composition as a result of physical and chemical reactions and/or biological processes. The maximum duration of
storage should not exceed 12 h at ambient temperature (maximum 25 °C). The samples should be kept in the dark
to prevent algal growth.
If testing almost immediately after sampling (or sample preparation) is not possible, e.g. when preparing composite
samples, cooling or freezing is recommended.
The most common and recommended way of preserving waste water samples is to cool to between 0 °C and 5 °C.
When cooled to this range and stored in the dark, most samples are normally stable for up to 24 h (see
ISO 5667-10). Cooling should commence as soon as possible after sampling, either in the field, for instance in cool
boxes with melting ice, or in a refrigerator in the transport vehicle.
Deep freezing below 218 °C in accordance with ISO 5667-10 allows in general an increase in conservation. A few
weeks up to 2 months, depending on the stability of samples, are generally the maximum storage periods.
Experience has shown that the quality of waste water can be affected during both freezing and thawing.
The use of biocidal preservatives should be excluded for the purpose of biotesting. The addition of highly
concentrated acids or bases to stabilize the samples, e.g. HCl or NaOH, is not recommended either.
It should be stressed that, if there is any doubt, the chemical analyst and the biotester should consult each other
before deciding on the method of handling and preserving the samples. If preservation techniques for the chemical
analysis and for biotesting are not compatible, separate subsamples should be provided for the different purposes.
6 Apparatus and equipment
6.1 Selection of apparatus
Type, shape and material of the technical equipment are dependent on the test and nature of the sample. All
materials which come into contact with the test sample should be such that interferences caused by sorption or
diffusion of the test material, by elution of foreign matter (e.g. plasticizers) or by growth of organisms, are kept to a
minimum. Inert materials are suitable, e.g. glass, PTFE. Tubing connections should be as short as possible and
replaced from time to time. Contamination of the test material, e.g. by grinding grease from stoppers or fittings,
should be avoided. Pipes made from copper, copper alloy or non-inert plastics are not suitable.
6.2 Silanization
In order to minimize adsorption of test material on containers, pipes, tubings, glassware or plasticsware can be
silanized (siliconized) by soaking or rinsing in a 5 % mass fraction solution of dichlorodimethylsilane in chloroform or
heptane. As the organic solvent evaporates, the silane is deposited on the surface, which should be rinsed many
times with water or heated at 180 °C for 2 h before use. Silanization should only be used if highly adsorbable
substances or water ingredients are to be tested and suitable inert material (e.g. PTFE) is not available.
© ISO
6.3 Cleaning of apparatus and equipment
Prior to use, the apparatus and equipment should be cleaned with suitable cleaning agents, e.g. hydrochloric acid,
sodium hydroxide, detergents, ethanol, sulfuric acid/ hydrogen peroxide and, where appropriate, sterilized, thermally
or chemically (e.g. with hypochlorite solution). Chromosulfuric acid should not be used.
Repeated rinsing of the apparatus with distilled water (or water with the same degree of purity), ensures that no
traces of cleaning or disinfection agent are left.
To efficiently remove traces of previous use, acid washing is recommended prior to final washing with distilled
water.
7 Pretreatment and preparation of samples
7.1 General
The flow diagram (figure 1) contains information on commonly (but sometimes differently) used terms in biotest
standards and guidelines.
Figure 1 — Preparation of samples for biotesting
© ISO
The sample, i.e. a chemical substance, is a preparation, solid or in solution, a mixture of various substances, water
or wastewater. The test sample is made from the sample by means of various preparatory steps specific to the
sample and the test, e.g. by dissolving, homogenizing, sedimenting, filtering, neutralizing or aerating. Dilution water
is added to prepare a series of defined dilutions. Following addition of the test-specific nutrient medium, the test
medium (including test sample) is obtained.
The final test batch is obtained by adding the test organisms – in the case of microorganisms called inoculum. The
control batch, or in several parallels, the controls are prepared from a mixture of dilution water and nutrient solution
with test organisms without the test sample.
When the effect or behaviour of a substance is known from previous tests ('reference substance') and when this
substance is examined within the framework of a test series as test sample, this is called the reference batch.
7.2 Thawing
Samples stored frozen should be thawed immediately before use. Running water or a warm water bath at a
temperature not exceeding 25 °C, together with gentle shaking, are recommended to avoid local overheating.
Complete thawing of the samples before use is essential, as the freezing process can have the effect of
concentrating some components in the inner part of the sample which freezes last. Microwave treatment involves
the risk of overheating.
7.3 Homogenization
An even distribution of all soluble and particulate components should be ensured. Gentle agitation, vigorous
shaking, ultrasonic treatment or high-speed mechanical dispersion may be applied, depending on the nature of the
samples. During this treatment step, attention should be given to the potential loss of volatile ingredients.
As a general rule, care should be taken that the original status of the sample be restored or at least be altered as
little as possible.
7.4 Separation of soluble and particulate matter
In general, biotests are carried out with the original sample. In some cases, however, large amounts of particulate
matter, sludge and sediment interfere with the behavioural requirements of test organisms (clogging of fish gills,
impairment of filter feeding of daphnids, light limitation of algae).
If these deleterious effects are not intended to be reflected by the test results, such interferences can be avoided or
overcome by various means.
Waters rich in particles can give rise to interferences, e.g. when quantifying by use of a particle counter.
Microscopic counting is strongly impaired as well. Continuous dosing is rendered unreliable by clogging and
blockage of tubing.
Filtration, centrifugation and other separation methods, however, involve the risk that active components, which are
bound to the particles, are removed prior to the test. Moreover, problems related to filtration, e.g. adsorption on and
leaching of filter materials, need to be taken into account. Sedimentation and centrifugation circumvent these
problems. When carrying out tests in the presence of particles causing severe problems, it is recommended that the
sample be allowed to settle for 30 min to 2 h or a coarse filtration (>50 μm) is carried out, thus removing only gross
particles. The separated particle mass may be examined separately.
Some test methods offer the possibility of determining a correction factor for parameters such as turbidity.
Waters rich in bacteria interfere in tests related to bacterial activity, e.g. respiration inhibition. The interference due
to the activity of bacteria in the sample can be accounted for, at least partially, by running suitable controls. When
testing certain algae, eggs and fry or cell cultures, interference can be caused by bacterial infections. Available
sterilization methods, such as thermal or UV-treatment or membrane filtration (0,2 μm), all involve a high risk of side
effects. Glass-fibre filtration is preferable when filtering is necessary. Centrifugation, e.g. 10 min at 4500 g + 1500 g,
is, in general, preferable to filtration.
© ISO
7.5 Preconcentration
Preconcentration of samples increases the concentration not only of harmful substances but of other water
constituents as well, which probably can be deleterious in higher concentrations.
Furthermore it is essential to take into consideration that in any case the preconcentration is selective depending on
the procedure applied. This alters the original composition pattern of water ingredients, e.g.
 liquid/liquid extraction with organic solvents and solid phase extraction by adsorption on solids (e.g. XAD-
resins) are particularly efficient for hydrophobic water constituents. Ionic strength and osmotic pressure can be
lowered. Toxic ions, polar chemicals and coefficient (e.g. masking) water ingredients, such as humic acids, can
be excluded;
 evaporation and freeze-drying can lead to a loss of volatile substances and enhance the ionic strength and
osmotic pressure;
 ultrafiltration can lead to a loss especially of small molecules penetrating the membrane.
The increase in concentration above the solubility threshold can lead to precipitation or flocculation of previously
dissolved substances.
Bioaccumulation cannot be simulated by preconcentration of samples, since bioconcentration factors (BCF) cannot
be related or extrapolated.
Certain ingredients of the water sample being concentrated can undergo chemical reactions at a higher rate than in
the original sample.
Appropriate blank values can be obtained only if unpolluted reference samples, e.g. upstream of the contamination
source, are available. The increase in salinity may be allowed for by preparing blanks with equal osmolarity and
similar ionic composition (e.g. Na:K ratio).
It is not possible to extrapolate from acute tests with preconcentrated samples to chronic effects of the original
sample.
Therefore it is preferable to choose a more sensitive test system or to prolong the exposure time rather than to
preconcentrate a sample. If there is no sensitive method available to test the original sample and a pre-
concentration procedure is applied, the result is the more contestable the higher the concentration factor.
For the above-mentioned reasons, tests for acute and chronic toxicity with pre-concentrated samples are generally
meaningless and not recommended. In all cases test results obtained with pre-concentrated water samples should
be interpreted with extreme caution. Preliminary investigations of this kind cannot be standardized and should be
validated by further extensive investigations.
7.6 pH adjustment
The selection of the pH value to which the sample is to be adjusted is governed by the objective of the test:
 adjustment to the pH of the receiving water will produce results more representative of the effect of toxicants
once in the environment;
 adjustment to a defined pH between 6 and 9 (which is usually tolerable for aquatic biota) will permit the
expression of ionizable toxicants that would otherwise be masked by pH conditions outside this range.
Usually samples with extreme pH values exceeding the tolerance limits of the test organisms are neutralized.
Neutralization 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. The concentration of the acid or base
required for neutralization should be such that the volume change is as small as possible. Passing the neutral point
should be avoided.
© ISO
The neutralizing agent should not undergo a reaction with the ingredients in the sample, which might, for example,
lead to precipitation or complexation. Also it should not influence the test organism by enhancement or inhibition.
Usually hydrochloric acid or sodium hydroxide solutions are recommended.
7.7 Preparation of stock solutions and test batches
7.7.1 Water-soluble substances
When preparing the stock solution, the weighed portion of the substance should not exceed the maximum amount
that will dissolve (< saturation concentration). By means of stirring and/or heating, the solution kinetics can be
enhanced. This should not lead, however, to substance loss or thermal decomposition of the test sample.
7.7.2 Emulsions and suspensions stable in water
In the case of emulsions (e.g. cutting oil emulsions) and suspensions (e.g. latex milk) that are stable in water, and
also with substances forming these stable entities with water, graduated dilutions should be prepared.
If a homogeneous distribution is not obtained in the stock liquor, the mixture should be stirred or shaken for up to
one day.
7.7.3 Poorly soluble substances
7.7.3.1 General
Substances with a solubility in water of less than approximately 100 mg/l should be considered as sparingly soluble.
When examining poorly soluble substances, ensure that no undissolved matter remains as sediment, as floating
particles or in dispersed form. Hence, in order to secure reproducible results, those methods are to be used that
ensure the best homogeneous distribution of the test compound in the test batch.
In the case of toxicity tests, it is advisable first to ascertain whether the substance has effects in the range of its
water solubility. It should be borne in mind that in the case of some pure substances there is sometimes overlapping
between molecular-dispersed and micellar- and colloid-dispersed up to coarsely dispersed systems (example:
isomer-pure surfactants). In the case of isomer mixtures, e.g. surfactants, there is no substance-specific solubility
limit. Simple optical methods (e.g. light-scattering measurements) do not permit any reliable determination of the
degree of dispersion.
It is impossible to recommend one single method for generating an optimum solution or distribution
of the substances in the medium, since the method selected should correspond to the physical properties of the
substance. For that reason it has to be left to the experience of the investigator and/or production information
of the manufacturer to select an appropriate method.
The following guidance is derived from practical experience and contains advice on ways and means which enable
the investigator, after weighing up the pros and cons, to select the most suitable method.
7.7.3.2 Testing in the water solubility range
For this purpose, a defined weighed portion of the substance (e.g. 100 mg) is mixed by stirring or shaking with 1 litre
of distilled water approximately 24 h, preferably in the dark. The weighed portion for preparing the stock liquor shall
be indicated. Following phase separation, the undissolved phase is fully separated by filtration (where necessary
using a membrane filter, pore size 0,2 μm) or by centrifuging. The dilution series is prepared with the aqueous
phase. Should it prove necessary, depending on the properties of the substance, (e.g. high viscosity or
decomposition in water), shorter or longer mixing times should be considered, possibly involving the use of auxiliary
agents.
NOTE Depending on the substances to be tested, centrifugation and filtration techniques can lead to different results.
If the solubility is reduced by adding media constituents (e.g. nutrient salts), it is advantageous to prepare a
saturated solution by mixing the substance with the test medium. In individual cases, consideration should be given
to replacing the salts (e.g. Ca or Mg ions) with others (e.g. Na or K ions), which do not precipitate.
© ISO
A fine distribution of highly viscous fluids may be achieved by embrittlement at low temperatures (e.g. using liquid
nitrogen) followed by mechanical crushing (e.g. in beater mills).
Filters for collecting of undissolved constituents shall be annealed (inorganic filters). Organic, e.g. polycarbonate,
filters require repeated treatment in boiling distilled water in order to ensure that no constituents of the filter material
are transferred into the test solution. Cellulose acetate filters are not recommended. Depending on the filter
material, not only can filtration capture undissolved constituents; but also dissolved test substance can be lost
through sorption. At concentrations <1 mg/l this can lead to considerable substance loss. The loss can be reduced
by discarding the first portion of the filtrate. The use of inorganic filter materials, in general, will lead to lower losses
than use of organic ones.
There is a variety of mechanical and chemical means to reach the saturation concentration. It should be borne in
mind, however, that preference should be given to mechanical aids for preparing saturated solutions. Recourse
should only be made to chemical aids (acids, bases, solvents) in exceptional cases.
Aids which can be used to reach the saturation concentration are:
a) Sonication/high speed grinder (advantage: higher dissolution speed; disadvantages: more difficult phase
separation, possible decomposition and heating through energy input. Further details are given in ISO 10634.
b) Temperature increase (advantage: usually higher solubility and higher dissolution speed; disadvantages:
enhanced volatilization, risk of decomposition and hydrolysis reactions, possibility of oversaturation with
ensuing delayed precipitation. Saturated solutions should not be cooled.
c) Use of acids/bases with ensuing neutralization (advantage: exploitation of higher solubility of a protonated or
deprotonated form; disadvantages: only applicable in exceptional cases, at extreme pH values risk of chemical
change in the test sample, e.g. through hydrolysis, sometimes perhaps very slow establishment of equilibrium
following neutralization, example: fatty acids/soaps).
d) Dissolution of the substance in a volatile, nonaqueous miscible solvent (e.g. n-hexane or petroleum ether)
which is stripped after mixing with water (advantage: rapid fine distribution of the test substance;
disadvantages: test substance can also be stripped off, low solvent residues in the test batch cannot be
precluded).
e) Dissolution of the substance in a water-miscible, nontoxic solvent (e.g. ethanol, acetone, acetonitrile, dimethyl
sulfoxide, dimethyl formamide). When selecting the solubilizer, its solubilizing capacity, toxicity, degradability
and its volatility should be considered. The solubilizing agents (concentration: < 0,1 g/l) initially remain for a
certain time in the test batch. It is unlikely that they lead to any major increase in the saturation concentration in
water; they lead rather to the rapid fine distribution of the test substance in the stock liquor. An additional
control batch with the maximum concentration of the solvent used in the test is necessary. Even if no damaging
effects have been observed, it cannot be excluded that the presence of the solvent affects the action of the test
substance on the test organisms, e.g. by aiding passage through the cell wall.
f) Sorption of the substance on to an inert carrier. A saturated aqueous solution can be prepared by applying the
substance, where necessary, using a volatile solvent such as n-hexane or petroleum ether, to an inert carrier
(e.g. glass beads, silica gel, chromatography resins). This is introduced into a flow-through system and rinsed
with water. The dissolving substances are frequently available as a genuine molecularly dispersed solution.
7.7.3.3 Testing above the solubility limit
In degradation studies, poorly soluble substances are eventually tested above the solubility limit. It is important to
achieve a high distribution rate for undissolved matter in order to ensure a large contact area between the
microorganisms and the substance. It is always necessary to shake or stir the test batches throughout the test in
order to guarantee a constant dissolution of the substance. The following methods are recommended.
a) Direct dosage
The substance is introduced directly into the test batch. Microscopic slides are suitable for introducing solid
substances or highly viscous liquids. Water-miscible, non-toxic, undegradable solvents, e.g. dimethyl sulfoxide,
are often used as solubilizer (see 7.7.3.2). This technique is restricted to non-volatile test substances.
© ISO
b) Ultrasonic treatment
Treatment with ultrasound (e.g. 20 kHz, 30 min) can frequently lead to a sufficiently stable mechanical
dispersion which, following approx. 15 min to 30 min settlement, can be distributed directly to the individual test
batches. Suitable analytic methods, e.g. Total Organic Carbon (TOC) or specific analysis, are necessary to
determine the initial concentration in the test batch.
c) Adsorption of the substance on to an inert carrier
This should be in line with the procedures outlined in 7.7.3.2 (adsorption of the substance on to an inert carrier).
The carrier with the homogeneously applied test substance is now a constituent in the test batch. This method
is not suitable for volatile substances, as they are eliminated during application and the subsequent drying of
the carrier.
d) Dispersion of the substance with an emulsifying agent
Emulsifying agents in the concentration used (<100 mg/l) should be non-toxic and stable throughout the test
period. The following substances may be used as emulsifying agents:
 polyethene sorbitol monolaurate;
 polyethene sorbitol trioleate;
 nonylphenol-20 EO-acetal (well suited for degradation tests);
 modified castor oil (less suited for degradation tests);
 ethene oxide/propene oxide copolymers (suited for degradation tests).
In order to prepare a chemically stable emulsion, it is recommended that the test substance be mixed with the
emulsifying agent prior to its introduction into water. Should an additional non water-miscible volatile solvent (e.g.
n-hexane or petroleum ether) be used for the preparation of an emulsion, the pre-treated test sample should be
stripped before the test organism is introduced.
Solid, poorly soluble substances are not usually tested above their solubility limit. Toxicity tests above the solubility
limit are advisable only for readily dispersible solid substances and solid substances which are marketed as
dispersions or come into contact with emulsifying agents when properly used.
Bioaccumulation studies and long term bioassays should not be carried out above the solubility limit.
The propensity of a substance to disperse in water is dependent above all on its density, its viscosity and its surface
tension. By increasing the energy input, a lower particle size can be achieved and thus often a higher stability of the
dispersion. There are, however, indications that the damaging effect of an emulsion is also influenced by the size of
the emulsified droplets. Different pathways are likely to be available to organisms for the uptake of emulsified
particles from that of dissolved substances.
Mechanical dispersions are often unstable. Phase separation can sometimes be avoided by constant stirring,
shaking or other mode of mixing the test sample during the test. Care should be taken to prevent any mechanical
damage to the test organisms.
NOTE Oil droplets and surface films can have deleterious effects on organisms, especially on daphnids. It is possible to
preclude the contact of daphnids with surface films by mechanical separation by means of nets/sieves or by darkening of the
surface.
The stability of an emulsion can be assessed by visual observation, chemical analysis (e.g. Dissolved Organic
Carbon (DOC)/TOC; and by turbidity measurements. In principle, priority is given to mechanically prepared
dispersions over dispersions with emulsifying agents.
© ISO
There are two ways of producing a dilution series from a stock dispersion with emulsifying agents:
—  maintaining a constant concentration of emulsifying agent;
—  maintaining a constant concentration ratio of test substance to emulsifying agent.
As a rule, preference should be given to maintaining the same concentration of emulsifying agent in all test batches
in order to ensure that the concentration-effect ratio depends only on the concentration of the test substance, thus
avoiding the breakdown of the emulsion at higher dilutions. An additional control batch with the highest
concentration of the emulsifier is always necessary.
When interpreting the results, it should be borne in mind that the effects observed are combined effects, even it the
emulsifier itself shows no effect in the control batch.
7.7.3.4 Special problems with mixtures of substance or technical products
When testing substances with readily soluble toxic sub-ingredients (e.g. tetrabutyl tin contaminated with tributyl tin
oxide) and poorly soluble substance mixtures (e.g. mineral oil products), the more readily soluble components can
be enriched in the aqueous extract. This is often indicated by a rise in the DOC with increasing mass portion in the
presence of an already existing undissolved phase. In order to record effects of this kind, it is recommended that an
additional saturated aqueous solution be prepared and tested with a higher mixture ratio (e.g. 0,1 g or 1 g of
substance per litre of water). Comparison with the findings obtained with various mixture ratios can indicate the
effects of readily soluble components of a heterogeneous mixture.
As an alternative to this procedure, it is advisable to test substance mixtures which are not readily soluble and
substances with readily soluble minor components by preparing a series of aqueous extracts in which the loading
rates decrease geometrically. The aqueous extracts should be tested in undiluted form and the result referred not to
the concentration in the aqueous phase but to the loading rates. It is important to note that the aqueous extracts
should not be diluted.
As the chemical composition of the dissolved fraction of mixtures often varies considerably from that of the original
product, it is advisable to examine also the effects of the original product in dispersed form (see 7.7.3.3).
8 Treatment of samples during the test
8.1 Aeration
The oxygen demand of test animals and heterotrophic microorganisms should be accounted for, e.g. by continuous
or intermittent aeration. In special cases, e.g. waste waters with an extremely high biochemical oxygen demand
(BOD), it is necessary to supply pure oxygen instead of air. Supersaturation (with respect to air) should be avoided.
Problems of stripping of volatiles and losses of substances by foaming can be avoided by special methods (see
10.1.3).
8.2 Suspension
Particulate matter contained in the water may be suspended during the test if no interference is anticipated (see
7.4). Planktonic microorganisms should be kept in suspension during the test by appropriate methods, e.g. by
shaking, stirring, rotation, aeration.
Care should be taken to avoid mechanical impairment of the test organisms.
8.3 pH adjustment and control
In certain circumstances (e.g. due to aeration) the pH will drift during the test even if the sample has been
neutralized previously. The adjustment and control of the pH during the test should be decided according to the
cause of pH drift and the objective of the test. Abiotic drift of pH can be distinguished by means of a batch without
organisms.
© ISO
From the analyst's and regulator's point of view it may be desirable to work with a substance in a clearly defined
constant chemical status. Increase in pH value due to practical problems, e.g. stripping of CO by aeration, should
be avoided or counteracted.
This is especially important for the pH-dependent change of fish toxicity with ionized and unionized ammonia. Other
examples for substances showing pH-dependent toxicity are sulfides, cyanides, amines, phenols and organic acids.
If, however, the change in pH during the test is an effect of fundamental vital processes such as photosynthesis in
algal growth, which is necessarily accompanied by CO consumption, it is reasonable not to adjust pH.
An allowed transition over a wide range would not only enhance the applicability and the indicative value of the test,
but also offer the chance to trigger the effect of a chemical compound which otherwise might have remained
undetected.
Increase in the pH value in algal assays can be reduced by various means, namely:
 addition of buffer;
 addition of carbonate;
 addition of gaseous CO ;
 increased gas exchange by aeration and/or shaking;
 limitation of light;
 limitation of nutrients;
 limitation of temperature;
 limitation of test duration;
 reduction of inoculum.
All of these options have undesirable side effects and consequences, e.g. reaction with a test compound,
precipitation, stripping of volatiles, complication of test design, limitation of growth, problems of measurability. In
general, optimized gas exchange and conducting tests at the lower limits of light and temperature will reduce the
problem.
Control of pH value in fish tests can be achieved by addition of carbon dioxid
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