Unclassified ENV/JM/MONO(2000)6

Organisation de Coopération et de Développement Economiques OLIS : 14-Dec-2000

Organisation for Economic Co-operation and Development Dist. : 15-Dec-2000

__________________________________________________________________________________________

Or. Eng.

ENVIRONMENT DIRECTORATE

JOINT MEETING OF THE CHEMICALS COMMITTEE AND

THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGY

OECD SERIES ON TESTING AND ASSESSMENT

Number 23

GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF

DIFFICULT SUBSTANCES AND MIXTURES

UnclassifiedENV/JM/MONO(2000)6 Or. Eng.

98702

Document complet disponible sur OLIS dans son format d’origine

Complete document available on OLIS in its original format
ENV/JM/MONO(2000)6

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ENV/JM/MONO(2000)6

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OECD Environmental Health and Safety Publications

Series on Testing and Assessment

No. 23

GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF

DIFFICULT SUBSTANCES AND MIXTURES

Environment Directorate

ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT

Paris

September 2000
ENV/JM/MONO(2000)6

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Also published in the Series on Testing and Assessment

Also Published in the Series Testing and Assessment:

No. 1, Guidance Document for the Development of OECD

Guidelines for Testing of Chemicals (1993; reformatted 1995)

No. 2, Detailed Review Paper on Biodegradability Testing (1995)

No. 3, Guidance Document for Aquatic Effects Assessment

(1995)

No. 4, Report of the OECD Workshop on Environmental

Hazard/Risk Assessment (1995)

No. 5, Report of the SETAC/OECD Workshop on Avian

Toxicity Testing (1996)

No. 6, Report of the Final Ring-test of the Daphnia magna

Reproduction Test (1997)

No. 7, Guidance Document on Direct Phototransformation of

Chemicals in Water (1997)

No. 8, Report of the OECD Workshop on Sharing Information

about New Industrial Chemicals Assessment (1997)

No. 9, Guidance Document for the Conduct of Studies of

Occupational Exposure to Pesticides During Agricultural

Application (1997)

No. 10, Report of the OECD Workshop on Statistical Analysis of

Aquatic Toxicity Data (1998)

No. 11, Detailed Review Paper on Aquatic Testing Methods for

Pesticides and industrial Chemicals (1998)

No. 12, Detailed Review Document on Classification Systems for

Germ Cell Mutagenicity in OECD Member Countries (1998)

No. 13, Detailed Review Document on Classification Systems for

Sensitising Substances in OECD Member Countries (1998)

No. 14, Detailed Review Document on Classification Systems

for Eye Irritation/Corrosion in OECD Member Countries (1998)

No. 15, Detailed Review Document on Classification Systems

for Reproductive Toxicity in OECD Member Countries (1998)
ENV/JM/MONO(2000)6

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No. 16, Detailed Review Document on Classification Systems

for Skin Irritation/Corrosion in OECD Member Countries (1998)

No. 17, Environmental Exposure Assessment Strategies for

Existing Industrial Chemicals in OECD Member Countries

(1999)

No. 18, Report of the OECD Workshop on Improving the Use of

Monitoring Data in the Exposure Assessment of Industrial

Chemicals (2000)

No. 19, Guidance Document on the Recognition, Assessment and

Use of Clinical Signs as Humane Endpoints for Experimental Animals

used in Safety Evaluation (2000)

No. 20, Revised Draft Guidance Document for Neurotoxicity

Testing (in preparation.

No. 21, Detailed Review Paper: Appraisal of Test Methods for Sex

Hormone Disrupting Chemicals (2000)

No. 22, Guidance Document for the Performance of Out-door

Monolith Lysimeter Studies (2000)
ENV/JM/MONO(2000)6

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About the OECD

The Organisation for Economic Co-operation and Development (OECD) is an intergovernmental

organisation in which representatives of 29 industrialised countries in North America, Europe and the

Pacific, as well as the European Commission, meet to co-ordinate and harmonise policies, discuss issues of

mutual concern, and work together to respond to international problems. Most of the OECD’s work is

carried out by more than 200 specialised Committees and subsidiary groups composed of Member country

delegates. Observers from several countries with special status at the OECD, and from interested

international organisations, attend many of the OECD’s Workshops and other meetings. Committees and

subsidiary groups are served by the OECD Secretariat, located in Paris, France, which is organised into

Directorates and Divisions.

The work of the OECD related to chemical safety is carried out in the Environmental Health

and Safety Programme. As part of its work on chemical testing, the OECD has issued several Council

Decisions and Recommendations (the former legally binding on Member countries), as well as numerous

Guidance Documents and technical reports. The best known of these publications, the OECD Test

Guidelines, is a collection of methods used to assess the hazards of chemicals and chemical preparations.

These methods cover tests for physical and chemical properties, effects on human health and wildlife, and

accumulation and degradation in the environment. The OECD Test Guidelines are recognised world-wide

as the standard reference tool for chemical testing.

More information about the Environmental Health and Safety Programme and its publications

(including the Test Guidelines) is available on the OECD’s World Wide Web site (see page 8).

The Environmental Health and Safety Programme co-operates closely with other international

organisations. This document was produced within the framework of the Inter-Organisation Programme for

the Sound Management of Chemicals (IOMC).

The Inter-Organization Programme for the Sound Management of Chemicals (IOMC)

was established in 1995 by UNEP, ILO, FAO, WHO, UNIDO and the OECD (the

Participating Organisations), following recommendations made by the 1992 UN

Conference on Environment and Development to strengthen co-operation and increase

international co-ordination in the field of chemical safety. UNITAR joined the IOMC in

1997 to become the seventh Participating Organisation. The purpose of the IOMC is to

promote co-ordination of the policies and activities pursued by the Participating

Organisations, jointly or separately, to achieve the sound management of chemicals in

relation to human health and the environment.
ENV/JM/MONO(2000)6

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This publication is available electronically, at no charge.

For the complete text of this and many other Environmental

Health and Safety publications, consult the OECD’s

World Wide Web site (http://www.oecd.org/ehs/)

or contact:

OECD Environment Directorate,

Environmental Health and Safety Division

2 rue André-Pascal

 75775 Paris Cedex 16

France

Fax: (33-1) 45 24 16 75

E-mail: ehscont@oecd.org
ENV/JM/MONO(2000)6

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FOREWORD

Guidance for the aquatic toxicity testing and assessment of difficult substances and mixtures was

identified as a high priority requirement by the National Co-ordinators of the Test Guidelines Programme

(TGP) and the Risk Assessment Advisory Body (RAAB) of the OECD. The OECD Task Force on

Ecotoxicology for priority setting in development and revision of Test Guidelines and Guidance documents

appropriate for pesticides testing also gave high priority to the development of a guidance document for the

testing of difficult substances.

The first draft proposal for an OECD guidance document on the aquatic toxicity testing of

difficult substances and mixtures was prepared following a meeting of the Expert Panel on the 2nd and 3rd

of April 1998 in Paris, France. It was circulated to National Experts for review in March 1999 through the

National Co-ordinators of the Test Guidelines Programme. Comments were received from twelve countries

(Australia, Belgium, France, Germany, Hungary, Italy, Japan, the Netherlands, Sweden, Switzerland, The

United-Kingdom and the United States) and industry. A revised draft was circulated in January 2000 to

National Co-ordinators for approval.

The revised proposal was well appreciated and the proposed guidance document was generally

fully accepted. However, Denmark, Germany, the Netherlands, the United-Kingdom and the United-States

made several additional comments. The document was again revised taking into account the amendments

suggested and then submitted to National Co-ordinators of TGP for final approval. In May 2000, the 12th

National Co-ordinators Meeting approved this last version and agreed that the draft Guidance Document be

submitted to the Joint Meeting for declassification.

The Joint Meeting of the Chemicals Committee and the Working Party on chemicals, Pesticides

and Biotechnology recommended that this document be made public. It is being published on the

responsibility of the Secretary General of the OECD.
ENV/JM/MONO(2000)6

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TABLE OF CONTENTS

Glossary of important terms used in the Guidance Document........................................................ 11

Introduction ................................................................................................................................... 12

Scope ................................................................................................................................... 13

1. Preliminary assessment of stability of test substance........................................................ 15

1.1 Review of existing data on the test substance............................................................ 15

1.2 Preliminary stability study ......................................................................................... 16

2. General considerations on selection of exposure regimes ................................................ 19

3. Media preparation and exposure systems for difficult test substances........................... 20

3.1 Poorly water-soluble substances ................................................................................ 20

3.1.1 Preliminary experiment.................................................................................... 20

3.1.2 Media preparation methods.............................................................................. 21

 Direct addition ................................................................................................. 21

 Solvents............................................................................................................ 22

 Generator systems............................................................................................ 23

 Dispersions and emulsions............................................................................... 23

3.1.3 Flow-through exposure systems....................................................................... 24

3.1.4 Culture and test media considerations when testing

 metals and metal compounds ........................................................................... 25

3.2 Substances that are not acutely toxic at the limit of their water solubility................. 25

3.3 Substances toxic at low concentrations...................................................................... 25

3.4 Volatile substances..................................................................................................... 26

3.5 Substances that degrade in the test system................................................................. 28

3.5.1 Photolysis......................................................................................................... 29

3.5.2 Hydrolysis........................................................................................................ 30

3.5.3 Oxidation ......................................................................................................... 30

3.5.4 Biodegradation................................................................................................. 31

3.6 Adsorbing substances................................................................................................. 31

3.7 Complexing substances.............................................................................................. 32

3.8 Coloured substances................................................................................................... 33

 Algal tests ............................................................................................................... 33

 Plant tests ................................................................................................................ 34

 Invertebrate tests ..................................................................................................... 34

3.9 Hydrophobic substances ............................................................................................ 34

3.10 Ionised substances...................................................................................................... 35

3.11 Multi-component mixtures......................................................................................... 36

 Fully soluble .......................................................................................................... 36

 Partially soluble ..................................................................................................... 36

3.12 Preparations ............................................................................................................... 37
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4. Sampling of test media for analysis of exposure concentrations...................................... 39

4.1 Sampling schedules for analysis of test media........................................................... 39

4.1.1 Algal growth inhibition test ............................................................................. 39

4.1.2 Lemna growth inhibition test .......................................................................... 39

 Static .............................................................................................................. 40

 Semi-static ...................................................................................................... 40

 Continuous flow-through ................................................................................ 40

4.1.3 Daphnia acute immobilisation test .................................................................. 40

4.1.4 Daphnia reproduction test................................................................................ 40

 Semi-static ....................................................................................................... 40

 Continuous flow-through ................................................................................. 41

4.1.5 Fish acute toxicity test ..................................................................................... 41

 Static ................................................................................................................ 41

 Semi-static ....................................................................................................... 41

 Continuous flow-through ................................................................................. 41

4.1.6 Fish prolonged toxicity test, fish early-life stage toxicity test, fish juvenile

 growth toxicity and fish short-term toxicity on embryo and sac-fry stages .....41

 Semi-static ....................................................................................................... 41

 Continuous flow-through................................................................................. 42

4.2 Taking of media samples for chemical analysis ........................................................ 42

5. Calculation and expression of test results.......................................................................... 43

6. References............................................................................................................................. 44

Annex 1 Computer programs for predicting physical/chemical properties, and

fate, transport and aquatic toxicity of substances............................................................. 48

Annex 2 Formula for calculating geometric mean exposure concentration ................................... 50

Annex 3 Toxicity mitigation testing for cationic substances.......................................................... 51

Annex 4 Toxicity mitigation testing with algae for chemicals which form complexes with

and/or chelate polyvalent metals...................................................................................... 52

Annex 5 Expert panel on aquatic toxicity testing of difficult substances /

Peer review committee..................................................................................................... 53
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Glossary of important terms used in the Guidance Document(1)

Substance (2) Chemical elements and their compounds in the natural state or obtained by any

production process, including any additive necessary to preserve the stability of

the product and any impurities deriving from the process used, but excluding

any solvent which may be separated without affecting the stability of the

substances or changing its composition.

Mixture (2) Mixtures or solutions composed of two or more substances in which they do

not react.

Preparation Deliberate physical mixture of substances.

Multi-component

substances (3)

Mixtures comprising a complex mix of individual substances with different

solubilities and physical-chemical properties. In most cases, they can be

characterised as a homologous series of substances with a certain range of

carbon chain length/number or degree of substitution. These materials are

frequently referred to as “complex mixtures”. But, in this Guidance Document,

these are referred to as “multi-component substances.

Water solubility The maximum attainable concentration or concentration at thermodynamic

equilibrium between aqueous phase and solid (or liquid or gaseous) pure phase.

Concentration in pure water is meant.

Saturation

concentration

The maximum dissolved concentration of a test substance that can be achieved

under the test conditions.

Poorly (or sparingly)

water-soluble substance

Substance with a limit of water solubility of <100 mg/l.

Water-miscible solvent A solvent in which a test substance can be dissolved to facilitate preparation of

stock solutions and/or treatment of test media.

Suspension A stable dispersion of solid particles in a liquid

Emulsion A stable dispersion of liquid droplets in a liquid.

(Note: both liquids are no miscible)

Chemical dispersant /

emulsifying agent

A chemical reagent (e.g. surfactant) which aids in the production of a

suspension / emulsion of a substance

Critical micelle

concentration

A maximum concentration of the freely solubilised surfactant in water.

Stable exposure

concentration

A condition in which the exposure concentration remains within 80-120% of

nominal over the entire exposure period.

Water-accommodated

fraction (WAF)

An aqueous fraction containing the dissolved and/or suspended and/or

emulsified fraction of a multi-component substances or a mixture.

Loading rate The ratio of test material to water (in mg/l) used in the preparation of a WAF.

1. All terms and their descriptions should be considered as working definitions for the purpose of this Guidance

Document only.

2. The definition is cited from a paper (ENV/JM/HCL(99)11), entitled “Step 2 Proposal for Harmonised

Classification Criteria for Mixtures” and therefore considered as a provisional definition.

3. Consideration is given to the consistency with the definition of “multi-component substances” (or “complex

substances”) in Chapter 1 of a draft “Guidance Document on the Use of the Harmonized System for the

Classification of Chemicals which are Hazardous for the Aquatic Environment” (ENV/JM/HCL(2000)11).
ENV/JM/MONO(2000)6

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Introduction

Guidance for the aquatic toxicity testing and assessment of difficult substances and mixtures was

identified as a high priority requirement by the National Co-ordinators of the Test Guidelines Programme

and the Risk Assessment Advisory Body of the OECD. An Expert Panel was therefore established to

review relevant literature and develop a guidance document.

The first draft guidance document was prepared following a meeting of the Expert Panel on the

2nd and 3rd of April 1998 in Paris, France (see Annex 5). The group considered relevant documents

published by the United Kingdom Department of the Environment (DoE, 1996), the United States

Environmental Protection Agency (US EPA, 1996a), the International Organization for Standardization

(ISO, 1997), the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC, 1996), the

Oil Companies European Organisation for Environment, Health and Safety (CONCAWE, 1992) and the

Danish Water Quality Institute (VKI, 1998).

The members of the Expert Panel agreed that the document “Guidance on the Aquatic Toxicity

Testing of Difficult Substances” published by the UK Department of the Environment should form the

basis for the OECD guidance document. The text of the Guidance Document is therefore based, to a large

extent, on the UK document but with amendments and additions as considered appropriate by the Expert

Panel and consequent review by national experts.

The first draft was circulated for review in March 1999 to national experts through the OECD

National Co-ordinators of the Test Guidelines Programme. A revised draft was submitted to National Coordinators for approval in January 2000. Additional comments were received, therefore a revised version

was prepared and again submitted to National Co-ordinators for approval in April 2000. It was approved at

the 12th National Co-ordinators Meeting in May 2000.
ENV/JM/MONO(2000)6

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Scope

This document provides guidance for the testing of substances, and mixtures, classed as “difficult

to test” for the purposes of determining their aquatic toxicity. Whilst the guidance given is targeted

towards tests described by the OECD Guidelines identified in Table 1 it may also be relevant to other tests.

The guidance relates to the practical aspects of carrying out valid tests with “difficult” test

substances and mixtures and presenting the results. As such it considers those substances and mixtures

with the properties described in Table 2 that require modifications or additions to standard testing

procedures. Guidance herein does not extend to interpretation of results, after testing of these substances,

or their use in classification and risk assessment.

The guidance is considered to reflect good practice but it is important to recognise that some test

substances and mixtures will present specific scientific and technical issues that may fall outside its scope.

It is also important to acknowledge that the guidance may not be applicable or appropriate where the test

results are to be used for applications which fall outside the scope of the guidelines. Under such

circumstances it is important to consult with the relevant end user of the data to ensure that appropriate

testing procedures are agreed upon and followed.

This document is divided into five substantive sections covering:

• guidance for carrying out a preliminary assessment of test substance stability;

• general considerations on selection of exposure regimes;

• development of media preparation and exposure systems;

• sampling of test media for analysis, and

• calculating and reporting test results.

Specific guidance on testing of metals and metal compounds can be found in a draft report of an

OECD Workshop on Aquatic Toxicity Testing of Sparingly Soluble Metals, Inorganic Metal Compounds

and Minerals (OECD, 1995). Where appropriate, the recommendations made at the workshop have been

incorporated into this document.

It should also be mentioned that revision of the OECD Test Guideline 201 (Micro-Algae, Growth

Inhibition Test) has been underway. Since algal tests represent a special testing field and are different in

many ways from the traditional ecotoxicological test methods for aquatic organisms, the updated OECD

Test Guideline 201 should be consulted, once available, when specific guidance is needed for handling of

difficult substances in algal tests.
ENV/JM/MONO(2000)6

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Table 1. Relevant OECD Ecotoxicity Test Guidelines

Guideline number Description of test

201 Alga, growth inhibition test

202 (updated draft) Daphnia sp. acute immobilisation test

203 Fish acute toxicity test

204 Fish prolonged toxicity test

210 Fish early life-stage toxicity test

211 Daphnia magna reproduction test

212 Fish, short-term toxicity test on embryo and sac-fry stages

215 Fish juvenile growth test

219 (draft) Sediment-Water Chironomid Toxicity Test Using Spiked Water

Draft Lemna sp. growth inhibition test

Table 2. Properties of “difficult” substances and mixtures

Property Nature of difficulty

Poorly water-soluble • Achieving/maintaining required exposure concentrations

• Analysing exposure

Toxic at low concentrations • Achieving/maintaining required exposure concentrations

• Analysing exposure

Volatile • Maintaining exposure concentrations

Photo-degradable • Maintaining exposure concentrations

• Toxicity of breakdown products

Hydrolytically unstable • Maintaining exposure concentrations

• Toxicity of breakdown products

Oxidizable • Achieving, maintaining and measuring exposure concentrations

• Toxicity of modified chemical structures or breakdown products

Subject to corrosion/

transformation

• Achieving, maintaining and measuring exposure concentrations

• Toxicity of breakdown products

Colloids • Achieving, maintaining and measuring exposure concentrations

• Light attenuation/scatter

Biodegradable • Maintaining exposure concentrations

• Toxicity of breakdown products

Adsorbing • Maintaining exposure concentrations

• Analysing exposure

Complexing • Distinguishing complexed and non-complexed fractions in media

• Depletion of nutrients in media

Coloured • Light attenuation

Hydrophobic • Maintaining exposure concentrations

Ionised • Distinguishing ionised and non-ionised fractions in media

• Defining exact exposure concentrations

Multi-component substances and

preparations

• Preparing representative test media

• Defining exact exposure concentrations
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1. Preliminary assessment of stability of test substance

Modifications to media preparation and exposure systems may be required where exposure

concentrations of a test substance are likely to decline significantly over the test period. In OECD

guidelines for aquatic toxicity testing a decline in concentration of 20% is considered sufficient to warrant

consideration of measures to reduce the decline. Data providing an indication of the stability of the test

substance under the test conditions should therefore be obtained before commencing testing. Two potential

sources of these data are:

• review of existing data on the physical and chemical properties of the substance; and

• data determined from a preliminary stability study carried out under test conditions.

1.1 Review of existing data on the test substance

Data relating to the physical/chemical properties, fate, transport and environmental toxicity of a

test substance will be of assistance in selecting a media preparation regime and exposure system which will

control the decline in exposure concentration. An example of a data profile which might be useful to assess

these parameters is given in Table 3. The table includes values for some of the properties which are likely

to be indicative of presenting technical difficulties for testing.

Existing reference sources or predictive methods (see Annex 1 for examples of computer-based

predictive methods) should be used to construct the profile. The profile can then be reviewed with respect

to standard media preparation and testing procedures and modifications identified and implemented prior

to commencement of testing.
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Table 3. Data profile for review of test substance properties and indicator values of difficulties

for media preparation and testing

Property Indicator value (1)

Physical state: liquid, solid, gas -

Compositional information and chemical structures -

Molecular weight(s) >300 g/mol (2)

Is the substance ionisable? If yes, dissociation constant (pKa)? -

n-Octanol/water partition coefficient (log Kow) >4

Water solubility in test media under test conditions < 100 mg/l

Water solubility in pure water at 25°C < 100 mg/l

Critical micelle concentration -

Vapour pressure at 25°C -

Henry’s Law Constant (H) >0.1 Pa.m3/mol

Boiling point -

Melting point -

Solubility in organic solvents -

Water dispersibility for surfactants/detergents -

Complexation constants -

Time for ultimate aerobic biodegradation <10 days (3)

Time for primary aerobic biodegradation <5 days (3)

Soil adsorption coefficient (log Koc) -

Hydrolysis half-life at 25°C and pH=7 <24 hours (4)

Fish bioconcentration factor (BCF) > 500

Volatilisation (half-lives) from water: model river and lake -

Sewage (wastewater) treatment plant removal percentage -

Photodegradability Absorbs light at wavelengths 290 - 600 nm

Toxicity to aquatic species <1 mg/l

Dissolution rate and extent for metal compounds -

1. Indicator values are not criteria of difficult-to-test substances. They indicate whether these substances are likely

to present technical difficulties for testing.

2. Experience indicates that many difficult-to-test substances appear to have molecular weights > 300 g/mol.

3. This test is not routinely performed for many chemicals. The value depends on test duration.

4. The value depends on test duration.

1.2 Preliminary stability study

A number of physical, chemical and biological processes can result in significant declines in

actual exposure concentrations of a test substance in aqueous media over time (see Table 2 and later

sections). Where data are absent or insufficient to identify the process responsible for the decline it may be

appropriate to carry out a preliminary study for assessing the stability of the test substance.

An example of a design for such an experiment is given in Figure 1. Solutions of the test

substance are prepared under conditions equivalent to those to be used in the toxicity test (including

aeration if that is envisaged), but excluding test organisms. Stable dispersion and emulsions of the test

substance can only be used in exceptional cases (see Section 3.1.2 on “Dispersions and emulsions”).

Samples of the test media are analysed at 0, 24 hours and at the end of the test or at a shorter period for

unstable and adsorbing substances. A saturated solution need not be used for this test but it should contain

a detectable concentration of the test substance. A sufficiently sensitive analytical method is necessary for

the analysis of the test substance in the test medium. The possibility of losses during sampling, sample
ENV/JM/MONO(2000)6

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treatment and analysis must be considered. Where samples are stored before analysis, losses during

sample storage should also be assessed. If the test substance is so sparingly soluble that it cannot be

detected using reasonable analytical techniques it will normally be necessary for the experimenter to use

whatever physical-chemical data are available in conjunction with expert judgement in order to assess

stability. Moreover, if a substance is so sparingly soluble that it cannot be detected, it is unlikely to be

used in an aquatic toxicity test without a dispersant/solvent, and there would be no need to establish its

stability without these. In exceptional circumstance it may be justified to use specialised techniques (e.g.

radiolabelled test substance) in order to achieve quantitative assessments. The 14C identity of the

radiolabelled substance should be verified at the beginning and during the study. It may be noteworthy to

mention that even if the radioactivity level is maintained, test substance can still be lost from solution.

The significance of volatility can be assessed by comparing concentrations in open and closed

vessels. Similarly, the significance of adsorption onto test vessel surfaces can be assessed by a comparative

study of concentrations determined in vessels conditioned to reduce adsorption and in unconditioned

vessels. Methods to reduce volatile losses and partitioning onto test vessels are described in Sections 3.4

and 3.6.

The significance of adsorption onto test vessel surfaces can be assessed by a comparative study

of concentrations determined in closed vessels conditioned to reduce adsorption (see Part B of Figure 1)

and in unconditioned closed vessels (see Part A of Figure 1). Methods to reduce partitioning onto test

vessels are described in Sections 3.6.

If conditioning of test vessels has no appreciable impact on maintaining measured chemical

concentrations, then it is most likely that the loss process is either photolysis, biodegradation or hydrolysis.

However, it may also be due to oxidation. Photodegradation can be distinguished from a comparison of

analysis of media held under conditions of light and darkness. Greater losses in the light point to photolysis

whilst similar losses in both vessels suggest that hydrolysis or biodegradation are the more important loss

processes. Methods to reduce losses by these routes are outlined in Section 3.5.

The design of the preliminary stability study may need to be tailored on a case-by-case basis to

address other mechanisms responsible for reductions in exposure concentration, e.g. sorption onto organic

food substances. Further guidance on the design and conduct of preliminary assessment studies can be

found in documents published by the EC (EC, 1996b) and the US EPA (US EPA, 1996a); Nabholz (1991);

Nabholz et al (1993); Auer et al. (1990); Lynch et al. (1994); Boethling and Nabholz (1997); and

Newsome et al. (1996). The preliminary stability studies were not intended to be limited to laboratory

studies.

It may be necessary to run the preliminary stability study alongside a preliminary assessment of

toxicity, if stability is only of concern at concentrations that will be used in the toxicity test. Those

concentrations may not be known beforehand and they may affect the stability of test substances in the test

media. For example, complexation may occur at higher concentrations and losses may be greater

(proportionally) at low concentrations than at high concentrations. It may be necessary to carry out the

preliminary study at least with two concentrations to identify these problems.
ENV/JM/MONO(2000)6

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Figure 1. Design of a preliminary experiment for identifying process(es) responsible for

loss of test substance from aquatic toxicity test media

Part A

Yes Select suitable

test system

Prepare aqueous solution/suspension of test substance

(preferably assess as solution but if inappropriate consider testing as dispersion)

Open vessels

Analyse at 0, 24 h and end of acute test (or

maximum period before renewal if chronic

Physical-chemical data available, QSAR analysis

Substance lost only from open vessel?

No

Concentration > 80 % of initial concentration at end of renewal period

Closed vessels

Analyse at 0, 24 h and end of acute test (or

maximum period before renewal if chronic

Aqueous solution/suspension of test substance

“Conditioned” vessels in light "Conditioned" vessels in dark

Analyse at 0, 24 h and end of acute test (or

maximum period before renewal if chronic

Analyse at 0, 24 h and end of acute test (or

maximum period before renewal if chronic

Losses in A > B

No

Losses in light > dark

No

Hydrolytically unstable, oxidizable or biodegradable

Yes Stable (consider

influence of pH)

Yes Volatile

Yes Sorbed onto glass

Yes Photodegraded

No

No

Part B
ENV/JM/MONO(2000)6

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2. General considerations on selection of exposure regimes

The selection of the exposure regime to be used in aquatic toxicity tests should be guided by the

time course of the experiment, the test species used, and the characteristics (e.g. physico-chemical

properties) of the test substance and/or the results of a preliminary stability study. The latter is conducted

to determine the likely fate and behaviour of the substance under very similar test conditions as those

prevalent in the actual aquatic toxicity test. The exposure regime could be:

• Static, no replacement - the test medium is not replaced for the duration of the study;

• Semi-static replacement - the test medium is periodically replaced on a batch basis;

• Intermittent flow-through replacement - the test medium is replaced over set periods during the

exposure; and

• Continuous flow-through replacement - the test medium is continually replaced.

Note: For some chemicals, especially crop protection products, pulsed or time-varying exposure

designs have been developed.

It is not possible to give definitive guidance on criteria for deciding on exposure regime since

requirements may vary between different regulatory authorities. However, a static exposure regime is

likely to be appropriate if exposure concentrations can be expected to remain within 80-120% of nominal

over the whole test period without renewal of the test media. Likewise, a semi-static or intermittent or

continuous flow-through regime will probably be required where concentrations are unlikely to remain

within 80-120% of nominal. Where uncertainty exists over the suitability of an exposure regime it is good

practice to consult with the appropriate regulatory authority prior to commencement of the test.

Renewal of test solutions after 24 hours exposure will frequently allow exposure concentrations

to be maintained and is a relatively simple modification to the test procedure. More frequent semi-static

renewal is possible but care must be exercised not to excessively stress fragile test organisms, e.g.

daphnids, by excessive handling. An intermittent flow-through regime may reduce the stress imposed on

test organisms compared with a semi-static regime and systems for use in acute and chronic tests with

daphnids and early life-stage tests with fish have been referred to by Van Leeuwen et al. (1986). A

continuous flow-through regime should generally be considered when a semi-static regime with renewal

following each successive 24 hours exposure period is incapable of maintaining exposure concentrations.

Flow-through test systems are discussed further in Section 3.1.3 and 3.4 in particular.

Semi-static renewal and flow-through regimes are likely to require frequent cleaning of the

exposure systems to prevent the accumulation of organic debris and the development of excessive

microbial populations. Care should be taken to minimize stress on test organisms caused by cleaning.

It should be stressed that flow-through systems cannot be used for algal testing.
ENV/JM/MONO(2000)6

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3. Media preparation and exposure systems for difficult test substances

The objective in preparing test media should be to achieve the required exposure concentrations

of dissolved test substance at the start of the test. These concentrations should then be maintained, where

technically possible, throughout the test using either static, semi-static renewal or flow-through exposure

regimes.

The method used for preparing aqueous stock or test solutions is dictated by factors such as the

physical state of a test substance, its solubility in the test dilution water and the desired range of test

concentrations relative to the solubility of the substance. All reasonable efforts should be made to achieve

true test solutions containing dissolved concentrations of the test substance that are stable and close to

target. However, it has to be recognised that actual exposure concentrations can be substantially lower

than nominal concentrations as a consequence of a number of loss processes. Hence, while the target

should be to achieve exposure concentrations up to the aqueous solubility of the test substance, it may be

necessary to utilise higher nominal concentrations to achieve it. Visible non-solubilized test substance in

the exposure vessels is generally to be avoided or minimized. Exceptions are possible when suitably

justified (e.g. see two exceptions in Section 3.1.2 on “Dispersions and emulsions”).

The media preparation and exposure methods should be evaluated prior to commencing testing to

ensure that the required quantities of test medium can be produced and the required concentrations can be

achieved and maintained. Consideration should also be given to testing a reference substance in order to

establish that the methods achieve results comparable to those obtained using standard methods. Approval

for non-standard methods should be sought from the appropriate regulatory authority and the methods

should be fully described in the test report.

It may be noteworthy that for assisting classification of metals and metal compounds, an OECD

protocol on transformation/dissolution of metals and metal compounds is currently being developed.

3.1 Poorly water-soluble substances

3.1.1 Preliminary experiment

A preliminary experiment should be considered for all studies with poorly water-soluble

substances to determine the maximum dissolved concentration that can be achieved under test conditions,

which is defined as the saturation concentration, and the preparation conditions that are required to achieve

it. The results of the preliminary experiment will form a basis of and justification for the media preparation

procedures adopted for the toxicity tests and a reference point (i.e. water solubility under test conditions)

against which the test results can be evaluated. In view of its importance the preliminary experiment should

be fully reported.

Solutions of test substance could be prepared at higher nominal concentrations than the

theoretical water solubility limit to achieve the maximum dissolved concentration provided any nondissolved substance is separated before testing. This method is also useful to take into account soluble

impurities.

The design of the preliminary experiment should take into account the possible need to remove

non-dissolved test substance from the test medium. It should not be assumed that a clear test medium
ENV/JM/MONO(2000)6

21

indicates a true solution since crystals, aggregates, micelles etc. cannot easily be detected by visual

observation. Possible separation techniques include:

• Centrifugation - the preferred separation method but there may be practical difficulties in applying the

technique to large volumes of test medium. As a guide, centrifugation at 100,000 to 400,000 m.s-2 for

30 minutes may achieve adequate separation. It should be noted that most centrifuge containers are

made of various sorts of plastics which may adsorb the test substance, and that glass containers are

more likely to break.

• Filtration through a membrane filter - less widely advocated because of the potential for losses due to

adsorption onto the filter matrix. Filtration may represent the only practical option where large

volumes of test media are required. Filter sizes of 0.22 to 0.45 μm may be suitable for achieving

adequate separation. Filter matrix should be made of inert materials (i.e. chemically and physically

non-reactive with testing compounds). Filters should be rinsed with high-purity water prior to use to

reduce the risk of contamination of test solutions with toxic residues. Adsorption of the test substance

may be reduced to insignificant levels by preconditioning filters with solutions of the test substance

prepared at the appropriate test concentrations. Filtration under pressure is preferable to vacuum

filtration due to potential losses by evaporation.

A justification for, or validation of, the separation technique should always be provided in the test

report. Separation techniques should not be applied to surfactant solutions.

It should be noted that a high level of uncertainty is often associated with solubility estimates for

poorly water-soluble substances. It is therefore often not possible to specify an upper exposure limit with a

high degree of confidence. Exposure concentrations which exceed a “reported” water solubility should

therefore not necessarily be considered invalid but reviewed on a case-by-case basis.

3.1.2 Media preparation methods

Four methods of test media preparation have been applied to poorly water-soluble substances.

These methods are referred to here by the generic terms: direct addition, water-miscible solvents, generator

systems and dispersions and emulsions. Care should be taken due to the fact that stability of a substance

measured in the laboratory can differ greatly from that observed in the test system.

Direct addition

Direct addition, as the term implies, involves the bulk addition of a test substance to the water. It

will usually be necessary to mix the substance with the water to ensure its dispersion through and solution

in the water column. The effectiveness of direct addition is therefore very dependent upon using a mixing

regime appropriate to properties of the substance such as viscosity and density. The following mixing

techniques are identified below in increasing order of severity:

• low energy stirring;

• vigorous shaking;

• blending;

• homogenisation/high-shear mixing;

• ultrasonication.

For stirring, a 48-hour period might be considered reasonable to achieve maximum solubility,

although a slightly higher concentration might be achieved by stirring for 6 weeks. A period of 30 minutes

has been suggested for ultrasonication, but this could lead to considerable heating, particularly of small

volumes, and potential consequences for stability.
ENV/JM/MONO(2000)6

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Mild heating of the substance and water may also help to achieve more rapid solution of a test

substance but care must be taken to ensure that this does not result in changes to either the test substance or

the water which could compromise the outcome of the test. Attention should also be given to potential

decrease of dissolved oxygen concentration.

It is recommended that use of direct addition should be limited to the cases when the

concentrations to be reached are lower than 50% of the water solubility. However, direct addition by

weighting may be difficult when testing a substance with very low solubility at low concentrations,

because making a large volume of a very diluted solution may inherently be uncertain as regards whether

or not microparticles may be present and thus included or not when using a small fraction of the solution.

Although direct addition of a suitable volume of a poorly soluble test chemical is the preferred method, in

practice it may be impossible for very toxic chemicals. In general, it is not possible to weigh out an amount

less than 5-10 mg with a sufficient precision. The preparation of a test solution with a nominal

concentration of less than 1 mg/litre will thus require more than 10 litres of the test medium for dispersion,

and a test concentration of 0.1 mg/litre will similarly require more than 100 litres. It is not possible to

handle such large volumes in practice. Therefore, serial dilution of the Water Accommodated Fraction

(WAF, see Section 3.11) of a properly prepared stock solution may be the only applicable method for such

very toxic chemicals.

Solvents

Water-miscible solvents provide a vehicle in which some poorly soluble substances can be

dissolved to produce a stock solution which is more amenable to adding to, and mixing with, the test

media. In particular, solvents could be helpful for hydrolytically unstable and highly viscous substances.

However, because of the potential for interaction with the test substance resulting in an altered response in

the test, their use should be restricted to situations where no other acceptable method of media preparation

is available. If solvents are used, their effects on the test results, if any, need to be determined. It should be

emphasised that solvents are not appropriate for mixtures where the use of the solvent can give preferential

dissolution of one or more components and thereby affect the toxicity. This could also be true where the

technical grade of a substance is tested and a toxic impurity dissolves preferentially.

The choice of solvent will be determined by the chemical properties of the substance. Solvents

which have been found to be effective for aquatic toxicity testing include acetone, ethanol, methanol,

tertiary-butyl alcohol, acetonitrile, dimethyl formamide and triethylene glycol. The physico-chemical

properties and aquatic toxicity of a range of solvents have been reviewed by Tarr and Hutchinson (1992)

and ECETOC (1996). It should be noted that the use of solvents such as acetone, ethanol and methanol

can be problematic for static and semi-static systems due to substantial growth of bacteria in aquatic test

systems and resulting depletion of oxygen. Caution should be exercised to avoid critical oxygen depletion

in long term tests with intermittent or continuous flow-through regimes.

The recommended approach to media preparation is to prepare a concentrated stock solution of

the test substance in the solvent. Measured quantities of the stock solution are then added gradually to the

test dilution water whilst continuously mixing using an appropriate technique. The concentration of the

solvent in the final test medium should not exceed the corresponding toxicity thresholds determined for the

solvent under the test conditions. The suggested level is at least one order of magnitude below the

appropriate no-observed effect concentration (NOEC) depending on the test species and the length/type of

toxicity test or in any case below 100 mg/l (or 0.1ml/l). In practice, a concentration of 100 mg/l (or 0.1

ml/l) is a reasonable working maximum concentration for most of the commonly used solvents. It is

unlikely that a solvent concentration of 100 mg/l will significantly alter the maximum dissolved

concentration of the test substance which can be achieved in the medium.
ENV/JM/MONO(2000)6

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The use of volatile solvents (n-hexane and petroleum ether) which can be stripped from the

medium after mixing has also been referred to in ISO (1997). The potential for co-stripping of volatile test

substance from, and the retention of toxic solvent residues in, the test medium are likely to restrict the

applicability of this approach.

OECD guidelines for aquatic toxicity tests require that the concentration of the solvent must be

the same in all treatments, where technically feasible in flow-through systems, and that the test design

includes a control group of organisms which are exposed to this concentration.

Generator systems

Aqueous solutions of a poorly soluble pure substance can be produced by bringing water into

contact with a chemically inert matrix which has been coated with the substance. A matrix with a high

surface area to volume ratio (such as pumice or glass beads) allows a relatively large amount of test

substance to be coated on the surface and provides a large area over which dissolving of the test substance

can take place. Systems utilising columns packed with a suitable matrix have been described by Veith and

Comstock (1975), Gingerich et al. (1979) and Phipps et al. (1982). The aqueous solution drawn off from

the column can be used either directly for testing or diluted to produce a concentration series.

Generator systems based on semi-permeable membrane disks (e.g. EmporeTM) offer an

alternative to column-based systems and have the advantage of ensuring that only dissolved material is

present in the resulting test medium. A system which utilises a disk placed in the headspace has been

described by Urrestarazu Ramos et al. (1997).

It may be necessary to re-circulate a test medium repeatedly through a column or disk in order to

achieve a saturated solution. Where the test medium is recycled it may also be necessary to re-circulate in

order to replenish losses due to sorption or bioaccumulation. The latter practice may result in

complexation of the test substance with dissolved organic matter and measured total concentrations of the

test substance exceeding the saturation concentration (Billington, 1988).

Generator systems are not considered appropriate for substances containing components or

impurities which differ in their water solubility. Differences in water solubility will result in selective

depletion of the more water soluble components from the column or disk matrix and their relative

concentration in the water phase. Generator systems are also not suited to substances which are

hydrolytically unstable since the composition of the derived test medium is likely to change over time.

Another disadvantage of the generator column method is the difficulty in obtaining large volumes of

solution which makes their use in flow-through systems problematic.

Dispersions and emulsions

The testing of aqueous dispersions and emulsions is not generally advocated for the following

reasons:

• Effects observed in toxicity tests are generally best explained when considered in relation to exposure

concentrations of dissolved test substance;

• The presence of non-dissolved material presents significant difficulties for the determination of

exposure concentrations’; and

• Non dissolved material present in test media has the potential to exert physical effects on test

organisms which are unrelated to toxicity.
ENV/JM/MONO(2000)6

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There are two exceptions to this rule:

• Where there is a regulatory requirement, such as assessing oil dispersing agents, pesticides which are

formulated for use as a dispersion or emulsion, and industrial chemicals which are emulsified for use

and released as an emulsion, such as some polycationic polymers with a silicone polymer backbone.

Guidance on the testing of substances in the presence of chemical dispersing or emulsifying agents is

best sought from the appropriate regulatory authority;

• Where the test substance has an inherent tendency to form an aqueous dispersion or emulsion such as

surfactants and detergents. The highest test concentration should either be 1000 mg active

ingredients/litre or the dispersibility limit (i.e., the limit at which phase separation takes place),

whichever is lower.

Stable dispersions or emulsions can sometimes be produced by the simple expedient of

physically mixing the test substance with the aqueous phase. Appropriate solvents used in conjunction

with physical mixing may also be effective, particularly where the test substance is a solid. The use of

chemical dispersants or emulsifying agents is not generally advocated because of the potential for physicalchemical interactions influencing the apparent toxicity of the test substance. The potential for any method

of dispersion or emulsification causing breakdown of the test substance should be checked before using it

in the toxicity test.

A dispersion or emulsion should be stable before the test organisms are introduced. Any excess

of test substance not uniformly distributed throughout the test medium should be removed. No attempt

should be made to maintain test material in suspension, by for example stirring, except where this is

required by specific test protocols. A semi-static replacement regime should be considered if a stable

dispersion or emulsion cannot be maintained for the duration of the test.

The potential for non-dissolved test material to cause physical effects on the test organisms

should be estimated during the test. Physical effects, such as the blocking of fish gill membranes, the

encapsulation/entrapment of daphnids or the reduction of light intensity in the algal test, can lead to an

overestimation of toxicity. Techniques for physically separating the test organisms from non-dissolved

material, whilst maintaining contact with the water column, should be considered where physical effects

are likely to be significant (see Section 3.9 on “Hydrophobic substances”).

In tests with dispersions or emulsions of pure single substances, exposure concentrations should

be expressed in terms of the concentration of the test substance in solution, otherwise the toxicity may be

underestimated. However, there are some exceptional cases. For example, where the pure substances are

self-dispersing in water forming micelles, micro-dispersions and macro-dispersions such as certain

surfactants, some charged polymers, many aliphatic amines, exposure concentrations should be expressed

in terms of the whole test substance dispersed in dilution water. It is therefore recommended to consult the

regulatory authority to decide how exposure concentrations are to be expressed. Dissolved concentrations

can be approximated by measurement following treatment to separate non-dissolved material from the

aqueous phase (see Section 3.1.1).

Toxic effect concentrations for dispersions and emulsions should be compared with the

dispersibility limit or the critical micelle concentration for a substance in water rather than with its water

solubility limit.

3.1.3 Flow-through exposure systems

Substances which are solids at room temperature may take a significant amount of time to reach

their solubility limit, even when added as a solution in a water-miscible solvent and subject to vigorous
ENV/JM/MONO(2000)6

25

mixing. Consequently a flow-through system, using a solvent stock solution of the substance for dosing

the medium, may not give sufficient time for the substance concentration to equilibrate. Under such

circumstances it may be appropriate to prepare, in advance, sequential batches of test solution sufficient for

a period of e.g. 24 hours or longer as necessary, which can be pumped directly into the test vessels. It

should be noted that such solvent systems are not appropriate for metals.

3.1.4 Culture and test media - considerations when testing metals and metal compounds

Media specified for culturing and testing with invertebrates, plants and algae may contain

concentrations of some essential elements, such as copper, zinc and nickel, that are only just sufficient to

meet nutritional requirements. As nutritional requirements depend on the acclimatisation history of the

organisms, ecologically relevant culture conditions need to be used. The composition of culture and test

media may therefore require special consideration to ensure that test results correctly reflect the toxicity of

metals and metal compounds.

3.2 Substances that are not acutely toxic at the limit of their water solubility

It is important to recognise that the maximum achievable dissolved concentration of a substance

in the test medium, i.e. saturation concentration, may not be the same as the water solubility of the

substance as determined by, for example, OECD Guideline 105. Typically, the concentration will be less.

It is also important to note that water solubility measurements made for regulatory purposes are usually

made in distilled water (pH=6-9) and not test media (pH=7-8) and that differences in pH of the test media

and distilled water may significantly affect the solubility, especially of ionised substances with a pKa

between 5 and 9.

If it is necessary to assess the toxicity of a substance at the saturation concentration, it is

important to provide evidence that all reasonable efforts have been taken to achieve this. The evidence

might include:

• the report of a preliminary experiment demonstrating that the media preparation regime is sufficient to

maximise the concentration of the test substance in solution (see Section 3.1 “Poorly water-soluble

substances”);

• a description of the method of media preparation regime used for the test;

• a statement of the water solubility; and

• an assessment of saturation concentration.

An effect concentration can be measured only if it is equal to, or less than, the saturation

concentration. If an effect cannot be detected in a test with a saturated solution the result should be

reported as no toxic effects at saturation. It is important to note that an absence of acute toxic effects at the

saturation concentration cannot be used as the basis for predicting no chronic toxicity at saturation or at

lower concentrations. Where chemicals are predicted to have no acute toxic effects at saturation, it is

recommended to consult the regulatory agency. Some regulatory authorities may prefer to omit acute

toxicity tests and proceed straight to chronic toxicity testing.

3.3 Substances toxic at low concentrations

Serial dilution of stock solutions provides a mechanism for adding small quantities of test

substance. Stock solutions may be prepared in water or a water-miscible solvent as appropriate (see
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26

Section 3.1). Effective mixing is essential to ensure that the test substance is uniformly dissolved

throughout the test medium.

Exposure concentrations should ideally be confirmed and their stability demonstrated by analysis.

The use of a radio-labelled test substance may enable very low concentrations to be analysed but this may

lead to significant additional costs associated with its synthesis, handling and analysis. In the absence of a

suitable analytical method for quantifying exposure, a semi-static renewal or flow-through regime may be

necessary to ensure that exposure concentrations are in line with target values.

Where measured concentrations decline significantly over the exposure period, the geometric

mean concentration should be used as the basis for assessing effects. However, it should be noted that

several types of loss processes (e.g. precipitation) can occur very fast and new equilibrium conditions can

be attained relatively quickly during the exposure. If this was the case, the median of the concentrations

which are measured after the decline would be more appropriate as a surrogate for the mean exposure

concentration. Where a measured concentration at the end of the exposure period is absent or where it

indicates that the substance is not detected, the validity of the test should be reconfirmed. In order to

calculate a mean exposure concentration, the final concentration may be taken as the limit of detection for

the method if the substance is not detected. When the substance is detected but not quantified, it may be a

good practice to use half of the limit of quantification. Since there may be various methods for determining

that, the method selected to determine mean measured concentrations should be made explicit in the

reporting of test results.

3.4 Volatile substances

The vapour pressure (vp) and Henry’s law constant (H) of a substance are important parameters

determining the potential for a substance to be lost to the atmosphere by evaporation.

Vapour pressure is a measure of the equilibrium between the condensed and vapour phases of a

substance. Vapour pressure is relevant to aquatic toxicity testing where non-dissolved test substance on

the surface of the medium is in contact with the atmosphere. Under this condition transfer to the vapour

phase alters the equilibrium between the dissolved and non-dissolved phases and changes the composition

of the medium. The significance of losses from the medium via this route can be calculated assuming ideal

gas behaviour and the volume of a mole of substance at the temperature and pressure of the system.

The Henry’s law constant for a substance is a measure of its equilibrium between an ideal

solution phase and the vapour phase. As such it is a measure of the potential for a substance to be lost

from solution by evaporation. Molecular weight (MWt, g/mol), water solubility (S, mg/l) and vapour

pressure (vp, Pa) can be used to estimate Henry’s law constant (H, Pa.m3

/mol) from the relationship:

H vp MWt

S = ×

As an approximation, if H is greater than 100 Pa.m3/mol, more than 50% of the substance could

be lost from the water phase in 3-4 hours (Mackay, 1992). However, other factors in the test system may

affect the rate of loss, principally test vessel size and shape, depth and temperature of the medium and rate

of aeration. The losses due to volatilisation may become significant for substances with Henry’s law

constants of 1-10 Pa.m3

/mol under vigorous mixing conditions where the opportunity for water/air

exchange is high.
ENV/JM/MONO(2000)6

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Losses of volatile substances from test media during preparation and exposure can be minimised

using relatively straightforward modifications to procedures. As a general rule vessels should be sealed

during preparation and exposure and the headspace kept to a minimum. Likewise, test concentrations

should, where possible, be prepared individually by addition of test substance directly to the test vessels

rather than by dilution of a stock solution. Systems with zero headspace should be used where it is not

possible to analyse exposure concentrations. Syringe pumps can be used to dispense concentrated

solutions of volatile substances (in water miscible carrier solvents, if required) into sealed vessels.

Samples collected for analysis should be placed in zero headspace vials.

A tiered approach, along the lines of the following, is suggested for selecting an appropriate

exposure system:

1. Open system, no test medium renewal, analytically determined exposure concentrations;

2. Open system, static renewal of test media, analytically determined exposure concentrations;

3. Open system, flow-through medium replacement, analytically determined exposure concentrations;

4. Closed semi-static renewal or continuous flow-through system, with or without headspace, analytically

determined exposure concentrations; and

5. Closed semi-static renewal or continuous flow-through system, no headspace, nominal exposure

concentrations.

Selection of the appropriate system should be dictated, where possible, by the goal of maintaining

analytically quantifiable test substance concentrations throughout the test.

Ideally, the maximum exposure concentration should not exceed the water solubility of the

substance. If it is not the case, the approaches adopted should be consistent with those described in

Sections 3.1 and 3.11 dealing with “Dispersions and emulsions” and “Multi-component substances”,

respectively.

The following comments relate to specific tests:

• Fish tests: Depletion of dissolved oxygen from media in sealed vessels may be significant in fish tests.

Consideration should therefore be given to using fish at the smaller end of the preferred size range,

larger test volumes, aeration with pure oxygen or more frequent medium renewal (semi-static or flowthrough) in order to maintain oxygen concentrations within guideline values. When using pure oxygen,

the pH of the test solutions should be monitored. Dosing systems for flow-through tests have been

described by Mount and Brungs (1967) and Benville and Korn (1974).

• Daphnia reproduction test: Removal of progeny and renewal of the test media should be undertaken at

the same time in the 21-day Daphnia reproduction test to minimise the number of occasions when the

test vessels are unsealed. Depletion of dissolved oxygen is not generally a factor in Daphnia tests

providing test volumes are not too small and the test media are changed relatively frequently e.g. daily.

Flow-through exposure systems for Daphnia chronic tests have been described in ASTM (1997),

Diamantino et al. (1997), Sousa et al. (1995) and US EPA (1996b).

• Algal test: Algal tests with very volatile substances are technically very difficult to perform

satisfactorily and may as a consequence yield results that are difficult to interpret and of limited

relevance to real-world conditions. Guidance for algal growth inhibition tests has been given in ISO

(1998). The use of a sealed exposure system in the algal growth inhibition test will result in culture

growth being limited by CO2 depletion and increasing pH. Consideration should therefore be given to

reducing the inoculum cell density and adding additional sodium bicarbonate to the medium.

Alternatively, a flow-through system capable of maintaining the supply of CO2 whilst avoiding a
ENV/JM/MONO(2000)6

28

headspace could be considered. Such a system has been developed by Tjeerdema and Singer (1991)

but has yet to be described in an internationally or nationally agreed test guideline. Halling-Soerensen

et al. (1996) have described a system for testing volatile substances which utilises a CO2 enriched

headspace and Mayer et al. (2000) described a system using completely closed flasks with no gas

phase in which gaseous CO2 is simply replaced with NaHCO3 and pH is adjusted with HCL. Whatever

system is adopted it is advisable to establish that acceptable control culture growth can be achieved

before starting the test. A test with a reference substance should also be performed to confirm that the

result is consistent with that obtained in a conventional test system. The test result should be reported

with the reference substance.

3.5 Substances that degrade in the test system

The preliminary stability assessment study outlined in Section 1.2 should consider the stability of

the test substance in water. If the substance is likely to be unstable, a decision to test the parent substance

and/or its degradation products, if identified, should be based on a consideration of its half-life under test

and real-world conditions. The following decision criteria are suggested only as a guide for static and

semi-static tests with medium renewal times of 24 hours:

• Half-life >3 days: test parent substance;

• Half-life <3 days and >1 hour: consider on a case-by-case basis, and include possible testing of

degradation products;

• Half life <1 hour: test degradation products.

These criteria are based on the assumption that problems associated with maintaining exposure

concentrations are often more noticeable in static and semi-static renewal tests compared with flowthrough tests. Loss of 20% of the initial concentration of a substance over a 24-hour period corresponds

with a half-life of approximately 3 days. A half-life of <3 days is therefore likely to result in exposure

concentrations decreasing to below target values and a possible build-up of degradation products over 24

hours.

Testing of both degradation products and parent compound depends on the objectives and

regulatory requirements.

The results of a preliminary toxicity test in which test organisms are initially affected and then

recover may indicate that the parent substance is more toxic than its degradation products. In this case the

toxicity of the parent substance should be determined, even if exposure levels cannot be maintained to the

extent necessary to comply absolutely with test guidelines. It should be noted that some regulations also

require the toxicity of the parent substance to be determined.

Testing of degradation products will normally be required where the results of a preliminary

range-finding experiment or a (Q)SAR analysis indicates that they have significant toxicities or other

relevant properties (e.g. low or no degradability). The aquatic toxicity of degradation products may be

determined by allowing the parent compound to degrade and then exposing the test organisms to the

resulting media. Leaving a stock or test solution of the parent substance for a period equal to 6 half-lives

of the substance will generally be sufficient to ensure that the medium contains only degradation products.

The pH of the degraded solution should be neutralised to that of the control medium prior to testing.

Test results obtained for non-degraded and degraded solutions should initially be compared on

the basis of the concentrations of the parent substance used in preparing the solutions. In this way the
ENV/JM/MONO(2000)6

29

relative toxicity of the parent substance and degradation products can be assessed. Identification and

quantification of the degradation products may subsequently be necessary to aid in interpreting test results.

The choice of medium replacement regime in fish tests with degradation products needs to take

into account water quality considerations as well as chemical and concentration stability. Where the

degradation products have been clearly identified it may be preferable to determine their toxicity

separately. The testing of degradation products imposes a requirement for an analytical method to

determine their concentration. This will be additional to the method required to determine the parent

substance. Samples taken for analysis should be treated and/or stored in an appropriate manner to prevent

further degradation prior to and during analysis.

A useful bench-mark when designing flow-through exposure systems is that, for a substance with

a half-life of 4 hours, approximately 50% of the nominal concentration should be able to be maintained

using a system with 6 volume renewals in 24 hours. A flow-through system which delivers more than 6

volume replacements in 24 hours will therefore be required when it is necessary to achieve concentrations

which are closer to nominal.

It should be noted that when selecting exposure regime and test vessels for unstable substances,

the tiered approach suggested for volatile substances (see Section 3.4) is applicable. Selection of the

appropriate systems should be dictated by the goal of maintaining analytically quantifiable test substance

concentrations throughout the test.

3.5.1 Photolysis

In short-term acute fish and Daphnia tests breakdown of chemical structures by photolysis may

be reduced or prevented by working in a darkened environment and using red light where necessary (ISO,

1997). It may also be possible to identify and selectively eliminate the light wavelength(s) responsible for

photolysis. For longer-term chronic tests it is not advisable to carry out tests in complete darkness because

of the risk of imposing additional stress by disrupting normal behaviour.

In algal tests it may be possible to determine the toxicity of the parent substance using one of two

approaches:

• The first approach is based on selective removal from the illumination source of light wavelengths

responsible for photolysis whilst retaining those wavelengths necessary for photosynthesis. However,

this is not often workable since in most cases information on the wavelengths that are responsible for

photolysis is not available. Although the UV spectrum may be available, the particular wavelengths

causing degradation are unlikely to have been identified. It is also worth considering that, in the

majority of cases, it is high energy wavelengths that give rise to photodegradation and these would not

pass through borosilicate glass flasks. When considering such an approach it is important to recognise

that algal tests should be performed at light saturation (about 100 μE/m2

/s for species of green algae).

Increased illumination may be required to compensate for the reduction in photosynthetic light when

filters are used to remove light wavelengths responsible for photolysis. Appropriate controls should be

included in the experiment design to demonstrate that illumination conditions are capable of

sustaining acceptable algal culture growth.

• The second approach involves carrying out the test using a dark exposure phase followed by an

illuminated phase. In this case interpretation of the test result will only be straightforward if the parent

substance is algicidal, since once illuminated the potential exists for both the parent substance and the

degradation intermediates to exert their effect(s).
ENV/JM/MONO(2000)6

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However, it should also be mentioned that these approaches have been questioned. Instead,

performing an abiotic experiment and subsequently doing an algal test on the control, simply inoculating

the abiotic control with algae as appropriate (or as in the test) has been proposed.

The use of flow-through exposure systems should also be considered for photo-degradable

substances. Key considerations in their design include eliminating the potential for photo-degradation of

stock solutions and selection of appropriate flow-through rates to limit the extent of degradation in the

exposure vessels.

3.5.2 Hydrolysis

Exposure concentrations of the parent substance should be maximised by keeping duration of the

media preparation stage to a minimum. Direct addition of the substance to the test medium combined with

methods to achieve rapid dissolution are preferred. Where stock solutions are required, consideration

should be given to preparing these using non-reactive water-miscible solvents to minimise hydrolysis prior

to dosing test vessels (see Section 3.1.2).

Flow-through exposure systems are not considered appropriate for the small number of

substances which hydrolyse at high concentrations to form polymers (e.g. alkyloxysiloxanes and

isocyanates) because of the potential for fouling of the test organisms and apparatus. These substances

should be added directly to the exposure vessels, vigorously mixed. All test organisms need to be added as

quickly and safely as possible after test substance addition within the time-scale required to maintain the

health of the control organisms, for example, 10 minutes. If the substance is too insoluble to achieve a

homogeneous dispersion after vigorous mixing, it should first be diluted in a non-reactive solvent and then

added directly to the exposure vessels and mixed. Once again the organisms should be added as quickly

and safely as possible.

Solutions of hydrolysis products of substances which polymerise should be prepared by adding

the substance very slowly to a vessel which is part-filled with water and being stirred rapidly. Once the

substance has been added, the vessel should be topped up with water to the required volume and stirred

continuously for a period sufficient to ensure complete hydrolysis. This procedure should enable a

solution of the hydrolysis products to be produced without the formation of polymers.

Temperature and pH can influence the rate of hydrolysis of some substances. Adjustment of

these parameters, within the range permitted for the test, may therefore be appropriate in order to optimise

exposure concentrations of the parent substance. The significance of pH for the rate of hydrolysis should

be determined and the result used (if required) to identify conditions for producing hydrolysis products for

testing. Testing of hydrolysis products should be performed at the normal test medium pH.

3.5.3 Oxidation

Oxidation is an abiotic transformation process which can result in breakdown of chemical

structure and consequent effects on the toxicity of a substance. In aquatic systems oxidation also reduces

the amount of dissolved oxygen which is available for respiration by aquatic species. The term “reducing

agents” is applicable to substances which are subject to oxidation.

Oxidation cannot be prevented in oxygenated aqueous media. Maintenance of dissolved oxygen

concentration is therefore a key consideration for fish and invertebrate testing. Selection of a static, semistatic renewal or flow-through exposure regime should be guided by the need to maintain the dissolved

oxygen concentration within the range permitted for the test. Maintenance of oxygen concentration may

be facilitated by aeration of the medium, increasing the volume of test medium and/or reducing the test
ENV/JM/MONO(2000)6

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organism loading or increasing the rate/frequency of test medium renewal. Flow-through test systems may

often be appropriate. Stock solutions of the test substance should be kept under anoxic conditions (e.g.

under nitrogen) until introduced to the test medium.

3.5.4 Biodegradation

Readily biodegradable test substances are likely to be degraded in aquatic test systems once

competent bacterial populations become established. Maintenance of exposure concentrations is therefore

dependent upon preventing the development of significant microbial populations.

Strict test vessel hygiene, at the start and during the course of the test, will delay and limit but not

prevent the development of populations of bacteria capable of degrading the test substance. It is also

important that carryover of old test medium is kept to a minimum and that test vessels are thoroughly

cleaned and, where possible, sterilised at medium renewals. Antibiotic use is to be avoided. When they

must be used, this should be defended by the sponsor and the testing laboratory. An antibiotic control will

also need to be added to the experimental design.

A flow-through exposure regime with sufficient volume renewal and a high concentration stock

solution maintained under nitrogen has been shown to: prevent aerobic biodegradation, minimise the

concentration of breakdown products and maintain exposure concentration of the parent substance (Tolls et

al., 1997). Good hygiene procedures will also help to prevent the development of high bacterial

populations on surfaces in the exposure system.

Care should also be taken to maintain dissolved oxygen concentrations, particularly in fish tests.

3.6 Adsorbing substances

In most cases adsorption onto surfaces of exposure systems and onto organic material will only

be important where relatively low concentrations of a substance are being tested (e.g. <1 mg/l). The most

common adsorption mechanisms responsible for losses are as follows:

• adsorption to test vessels (typically made of glass) via hydrogen or ionic bonding between the

substance and hydroxyl groups on the vessel surfaces. Glass surfaces offer negatively charged

hydroxyl groups which can bind with cationic substances such as surfactants. Salt bridges, formed by

divalent cationic substances, such as Ca2+ and Mg2+, may also be responsible for binding anionic

substances;

• adsorption to negatively charged biological material, such as algal cells, by the same mechanisms

described above.

The following approaches to reducing adsorption should be considered where losses of the

substance are likely to compromise compliance with test guidelines:

• reducing the surface area to test medium volume ratio;

• increasing the frequency or rate of test medium renewal in semi-static and flow-through tests;

• construction of exposure systems using non-adsorptive materials such as polytetrafluoroethylene

(PTFE);

• avoiding using highly adsorptive materials in the construction of exposure systems (in particular,

rubber and polyethylene should never be used as part of exposure systems in any situation);

• pre-conditioning of test vessels using solutions of the test substance (N.B.: The concentration of the

test substance used to condition a vessel should not exceed the test concentration appropriate to the
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vessel otherwise the substance may desorb during the test and increase the exposure concentration. It

should be noted that any flow through regime should always be equilibrated with the test substance,

regardless of adsorptive substance testing or not, and confirmed by the pre-test samples on e.g. two

days at least.);

• pre-conditioning of test vessels using silanising agents (e.g. a 5% solution of dichlorodimethylsilane in

chloroform or heptane) [N.B.: many silanising agents are highly toxic and great care should be taken to

ensure that residues are removed from the vessels before they are used in a test. A regime of repeated

rinsing with water or baking at 180o

C for 2 hours has been recommended (ISO, 1997). Organic

solvents such as methanol could also be used in the rinsing steps];

• maintaining dissolved total organic carbon concentrations (other than that due to the test substance) in

all tests at or below 2 mg/l, unless special studies are being conducted;

• removing excess food from test vessels after feeding has finished;

• feeding of fish a few hours before test medium renewal in semi-static tests; and

• using semi-static or flow-through exposure regimes instead of a static one.

Substances which sorb onto glass are usually very hydrophobic. Adsorption losses of test

substance onto the surfaces of vessels used for analysis sampling can be prevented by priming the vessel

with a small volume (usually 10-20 ml when a 50 ml sample is taken) of a water-non miscible solvent,

such as hexane. Partitioning into the solvent phase is favoured at the expense of sorption onto the glass

surfaces of the sampling vessel.

The influence of sorption on algal biomass in algal toxicity tests may be mitigated by reducing

the inoculated algae concentration and/or the test duration in order to reduce the final algal biomass.

Test results should generally be expressed relative to measured exposure concentrations.

Although analysis methods for cationic substances are incapable of distinguishing between bound and free

test substance results should, nonetheless, be expressed in terms of measured exposures. For some

regulatory applications it may be necessary to determine the extent to which the toxicity of a cationic

substance is mitigated by adsorption to dissolved organic carbon. This topic is briefly addressed in

Annex 3.

3.7 Complexing substances

Complexation may significantly affect the bio-availability and toxicity of a test substance. It may

also reduce the availability in the test medium of salts (such as calcium and magnesium) and trace elements

which are essential for supporting healthy test organisms. The following are examples of substances which

may be involved in complexation:

• EDTA;

• Polyanionic polymers with carboxylic and phosphoric acids;

• Phosphonates; and

• Metals.

The extent to which a substance is complexed will depend upon the availability of co-complexing

agents and other properties of the medium such as pH. Speciation models may be used to calculate the

concentrations of dissolved and complexed test substance from the total nominal concentration added.

Data from tests in which complexation has been judged to have had a significant bearing on the

result are likely to be of questionable value for classifying substances and for extrapolating to a predicted
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33

no effect concentration for risk assessment. The extent to which effects are a direct consequence of

chemical toxicity or a secondary effect, resulting from for example complexation induced nutrient

limitation, should be determined where possible. Compensatory adjustment to water quality parameters or

the testing of an appropriate salt of the test substance help to achieve a valid test result but protocols

incorporating modifications to standard procedures should be validated and approved for use by the

appropriate regulatory authority.

The effects of metal complexing substances in algal growth inhibition tests are mainly caused

by chelation of essential cations, which leads to growth limiting reductions in the concentration of

uncomplexed physiologically active ions. Inhibition of algal growth by metal complexing agents is

therefore a secondary effect, which cannot be attributed to substance specific inherent toxic properties. The

secondary effects can be eliminated by compensating for the deficit in the concentration of the essential

ion(s). Guidance on toxicity mitigation testing with algae for chemicals which form complexes with and/or

chelate polyvalent metals is given in Annex 4.

Analysis methods for quantifying exposure concentrations, which are capable of distinguishing

between the complexed and non-complexed fractions of a test substance, may not always be available or

economic. Where this is the case approval should be sought from the regulatory authority for expressing

the test result in terms of nominal concentrations.

Complexation of metals to organic and inorganic ligands in test media and natural environments

can be estimated from metal speciation models. Speciation models for metals, including pH, hardness,

DOC, and inorganic substances such as MINTEQ (Brown and Allison, 1987), WHAM (Tipping, 1994) and

CHESS (Santore and Driscoll, 1995) can be used to calculate the non-complexed and complexed fractions

of the metal ions. Alternatively, the Biotic Ligand Model (BLM), allows for the calculation of the

concentration of metal ion responsible for the toxic effect at the level of the organism. The BLM model

has at present only been validated for a limited number of metals, organisms, and end-points (Santore and

Di Toro, 1999). The models and formulae used for the characterisation of metal complexation in the media

should always be clearly reported, allowing for their translation back to natural environments.

3.8 Coloured substances

The prime objective of aquatic toxicity tests is to determine the inherent toxicity of a substance.

Coloured substances can present particular problems for determining true toxicity in algal tests and in tests

with Daphnia sp. In fish tests, observation of behaviour and mortality of fish could also be difficult.

Algal tests

Coloured substances can absorb photosynthetically active light and hence limit growth of algal

cultures. Absorption will be proportional to test substance concentration and as a consequence it can result

in growth inhibition which is difficult to distinguish from inherent toxicity.

The need for clear guidance on how to determine the inherent toxicity of coloured substances to

algae is widely recognised (EC, 1996a) and work is ongoing in this area (Justesen and Nyholm, 1998).

The International Organization for Standardization (ISO, 1997) has identified the following general

strategies for discriminating between effects resulting from toxicity and light absorption:

• testing at different light intensities, since the growth rate above light saturation is almost independent

from the light intensity;

• reducing the light path by reducing the depth or the volume;
ENV/JM/MONO(2000)6

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• measuring the reduction of cell proliferation in a control batch when the light previously passes

through a liquid light transmission filter, e.g. a flat dish containing corresponding dilutions of the

sample with the corresponding colour and layer thickness; and

• utilising a light transmission filter by preparing reciprocal dilutions to the exposure concentrations thus

maintaining a common light path length (constant depth) and the spectral absorbance characteristics of

the sample.

Note: Care should be taken with the approaches described in the last two bullets. Utilising external

filters is questionable because the light conditions in the test flasks are affected by a coloured

substance in a much more complicated way than with the external absorption method. Instead, it

is better to change the incubation system to compensate for the internal light absorption, thereby

minimising its effect. In practice, this can be done by shortening the light path, increasing the

light intensity, and maintaining a good turbulence (Justesen and Nyholm, 1998).

Specific methods under development which incorporate some of the above features have been

described by Memmert (1994) and Comber et al.(1995). There is, however, limited experience of their

application and as a consequence they cannot be endorsed at this stage.

Plant tests

Testing with the floating plant, Lemna sp., may provide a further alternative for coloured

substances since it is unaffected by the optical properties of the test medium. A draft OECD Test Guideline

for a growth inhibition test with this species has been developed but it still has to be established whether

the test can be considered equivalent to the algal growth inhibition test.

Invertebrate tests

Observation of small invertebrate test organisms, such as Daphnia sp., can be difficult in highly

coloured test media. Observations may be made easier by placing test vessels on a light box or transferring

the contents of the test vessels to shallow containers for scoring.

3.9 Hydrophobic substances

Partitioning of test substances into or onto test organism biomass and onto food or other organic

detritus in the test system is a potentially important loss mechanism for hydrophobic substances with high

octanol/water partition coefficient (log Kow >4) or bioconcentration factor (BCF >500). High octanol/water

partition coefficients are generally associated with low water solubility and hence losses due to partitioning

are likely to be more significant for hydrophobic substances.

Methods of preparing solutions of poorly water-soluble substances have been discussed in

Section 3.1. Strategies for maintaining exposure concentrations of hydrophobic substances in aquatic

toxicity test media include:

• reducing the ratio of test organism biomass to test medium volume (i.e. the use of a ratio of <1 g/l in

fish tests);

• using a semi-static renewal or flow-through exposure regime;

• increasing the frequency or rate of test medium renewal in semi-static and flow-through tests;

• removing excess food and detritus;
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• maintaining dissolved total organic carbon concentrations (other than that due to the test substance) in

acute tests at or below 2 mg/l;

• feeding (e.g. of fish) a few hours before test medium renewal in semi-static tests; and

• making the system surfaces saturated with the compound so that it does not ‘plate’ out.

Analyses of exposure concentrations in algal tests with hydrophobic substances may need to take

account of the potential for partitioning of the test substance onto the algal biomass. Where partitioning is

likely to be significant it may be appropriate to quantify exposure following separation of algal cells from

the test medium. Separation techniques include centrifugation and filtration and both of these are subject

to the comments made in Section 3.1.1.

A film of hydrophobic test substance floating on the surface of test media may physically trap

small aquatic invertebrates, such as daphnids. It should therefore either be removed before introducing the

test organisms or the organisms should be prevented from coming into contact with it using screens, cages

or other suitable devices. A device for preventing surface trapping of daphnids has been described by Dean

and De Graeve (1986).

It is advisable to consult Section 3.6, since there is much overlap between hydrophobic

substances and adsorbing substances.

3.10 Ionised substances

Relatively small changes in pH can significantly alter the balance between the dissociated and

non-dissociated forms of some organic acids and bases. Altered dissociation equilibrium may in turn

significantly affect the water solubility and partition coefficient of the substance, and thus also its

bioavailability and measurable toxicity. It is, therefore, essential that the relevant dissociation constants

(pKa values) are known prior to commencement of testing.

Design of the toxicity test should take into account the effects on dissociation equilibrium that

adjustments to the pH may cause. A preliminary test, to determine the potential for differing toxicity of the

two or more forms of the substance, should be considered where the pKa for the test substance falls within

the normal pH range of a test. The definitive test should be conducted at a pH consistent with the more

toxic form of the substance whilst remaining within the range required to maintain the health of the control

organisms.

Where the substance itself causes a change to the pH of the test medium, the pH should be

adjusted to lie within the specified range for the test using acid, alkali or other suitable buffer. It should be

noted that the use of buffers can affect the result of the test - particularly for algae. Furthermore, this can

cause sedimentation and/or degradation of the test substance. The suitability of any proposed buffer system

should therefore be assessed prior to use in the definitive test. Adjustments to pH may be carried out in the

stock solutions used to prepare the test media or in the media itself, as judged appropriate. In either case

the procedure should be applied to all treatments, including the controls.

Growth of algal test cultures can cause increase of pH due to consumption of HCO3 ions.

Maintenance of stable pH when testing an ionised substance is therefore important to ensure that the

balance between dissociated and non-dissociated forms of the substance is maintained. Strategies for

maintaining the concentration of HCO3 ions and therefore reducing pH shifts have been discussed in

Section 3.4.
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3.11 Multi-component substances

Mixtures comprising a complex mix of individual substances with different solubility and

physical-chemical properties are frequently referred to as “complex mixtures”. In most cases, they can be

characterised as a homologous series of substances with a certain range of carbon chain length/number or

degree of substitution (e.g. petroleum). In this Guidance Document, these are referred to as “multicomponent substances”. Different approaches to media preparation and/or testing are required for multicomponent substances depending upon whether they are fully or partially soluble in the test media across a

proposed range of test concentrations.

Fully soluble

Test methods consistent with those described for water-soluble substances are appropriate for

mixtures comprising components which dissolve fully within the proposed range of test concentrations.

Mixture components may, however, have individual properties (e.g. degradability, volatility etc.) that

require steps to be taken to control losses.

Partially soluble

When multi-component substances is not fully soluble in water, it is important to seek to identify

its components as far as practically possible and to examine the possibility of determining its toxicity using

available information on its components. Only when the toxicity cannot be calculated, testing of the multicomponent substances is considered.

The toxicity of complex multi-component substances, which are only partially soluble in water,

can be determined by preparing water-accommodated fractions (WAFs) of them. The term wateraccommodated fraction is applied to aqueous media containing only the fraction of multi-component

substances that is dissolved and/or present as a stable dispersion or emulsion. Test data obtained with

WAFs apply to the multi-component substances as an entity. Using the data derived from the testing of

WAFs for classifying for aquatic toxicity is referred to in the draft Guidance Document on Use of the

Harmonized System for the Classification of Chemicals which are Hazardous for the Aquatic Environment

(OECD, 2000). It should be noted that applicability of WAFs depends on the objectives of regulatory

authorities. When doing WAF testing, the appropriate regulatory authority should be consulted so that the

data generated on WAFs are deemed appropriate by the regulatory authority receiving the data. For

example, in the cases where poorly soluble components in the multi-component substances are of concern,

the WAF method is not adequate to determine the toxicity of such multi-component substances.

WAFs are prepared individually and not by serial dilution of a single stock WAF. Measured

amounts of multi-component substances are added directly to water and mixed for a period of time

sufficient to achieve an equilibrated concentration of dissolved and dispersed or emulsified components in

the aqueous phase. Following cessation of mixing and a period of settling (to allow phase separation) the

aqueous phase, i.e. the WAF, is drawn off for testing. The duration of the mixing and settling phases

should normally be determined by carrying out a preliminary study. Techniques such as turbidimetry and

total organic carbon analysis may provide a useful indication of the progress of mixing and phase

separation. Procedures for preparing WAFs of oil products and products such as creosote, have been

described by Girling (1989) and Tadokoro et al. (1991) respectively.

WAFs should be prepared by adding carefully measured amounts of the substance to the test

medium and then applying appropriate mixing techniques. The presence of water-miscible solvents can

modify the composition of a WAF and as a consequence they should not be used during preparation. The

severity of the mixing regime should be guided by the desire to attain an equilibrated test medium as
ENV/JM/MONO(2000)6

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quickly as possible whilst avoiding, where possible, the formation of emulsions. Care should be taken due

to the fact that some multi-component substances will have an innate tendency to form an emulsion or

dispersion and will require to be tested as such (see Section 3.1.2 on “Dispersions and emulsions”). It is

important to recognise that the duration of mixing and energy input can have a marked influence on the

composition, particle size and proportion of dispersed and non-dispersed test material in the WAF.

Generator systems described in Section 3.1.2 are not appropriate media preparation methods for multicomponent substances. The method used to prepare the WAF should therefore be fully described in the

test report and evidence of its compositional stability over time provided.

Generally, any non-dissolved test material which has sedimented or precipitated out in the test

vessels should be removed from the test media using, for example, a separating funnel. Similarly any

fraction of the mixture forming a surface film should be decanted to prevent surface trapping. Where there

is a need to retain an excess of the multi-component substances in the test vessels, steps should be taken to

prevent trapping or fouling of test organisms (see Section 3.9 on “Hydrophobic substances”).

Only a fraction of the total mass of multi-component substances responsible for the composition

of a WAF may be present in the WAF. The “loading rate” has therefore been advocated for expressing

exposures of mixtures that neither wholly dissolve nor completely form a stable dispersion or emulsion

over the required test range (Girling et al., 1992). The loading rate is the mass to volume ratio of the

mixture to medium used in the preparation of a WAF. WAFs may be thus considered analogous to the

term “nominal concentration” used for typical test substances, with all the limitations inherent to that term.

Chemical specific analysis is required to demonstrate attainment of equilibrium in WAF

preparation and stability during the test. Methods capable of identifying gross changes in the composition

of WAFs with time are required. Infra-red or ultra-violet spectroscopy or total peak area have been used

successfully for this purpose. Total organic carbon analysis may also be useful in monitoring any losses of

test material from test media but the low sensitivity of the method (approximately 1 mg/l) may limit its

applicability.

Effects concentrations in tests based upon WAFs can be calculated from (1) the loading rates and

are identified as either LL50 or EL50 values and/or (2) the measured mass of test substance in the WAF and

are identified as either LC50 or EC50 values. For example, some regulatory authorities in the UnitedStates require the latter approach so that an adequate risk assessment can be done for the test substance.

LL50 or EL50 values are comparable to LC50 or EC50 values determined for pure substances tested within

their solubility range. Similarly the NOEC (No Observable Effect Concentration) becomes the NOELR

(No Observable Effect Loading Rate). The statistical methods used to determine LL50, EL50 and NOELR

values are the same as those used to determine LC50, EC50 and NOEC values.

3.12 Preparations

Preparations are defined as a deliberate physical mixture of substances. Testing of preparations

is only advocated in the following instances:

• where the toxicity of the preparation cannot be calculated from the toxicity of its component

substances (N.B.: In this instance, consideration should first be given to determining the toxicity for

any of the component substances for which data are lacking, so that the toxicity of the preparation can

then be calculated);

• where data confirming the calculated toxicity are specifically requested; and

• where there is a regulatory requirement which is commonly the case for registration of pesticides and

biocides.
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Where testing of the preparation as a whole is required, the approaches identified above for

multi-component substances should be considered.

In general, alloys can be considered separately from other preparations due to their unique

physical and chemical properties that differentiate them from simple mixtures of constituent elements. An

alloy can be defined as a metallic material, homogeneous on a macroscopic scale, consisting of two or

more elements so combined that they cannot be readily separated by mechanical means. Due to the

decreased solubility or lack of solubility of many alloys, the approaches identified for multi-component

substances are not appropriate. However, care should be taken due to the fact that some alloys such as

powered brass show toxicity to aquatic organisms.
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4. Sampling of test media for analysis of exposure concentrations

A detailed consideration of analysis techniques for use in aquatic toxicity tests is outside the

scope of this document. Sections 4.1 and 4.2 are therefore limited to providing guidance on the design of

sampling schedules and methods of sampling.

4.1 Sampling schedules for analysis of test media

Schedules for collecting samples of test media for analysis are often included in testing

guidelines. The following recommendations are designed to supplement the information given in the

guidelines and provide a lead where guidance is lacking.

Sampling schedules for analytical support to tests with algae, plants, daphnids and fish are

outlined below. These schedules only apply to tests carried out within the solubility range of pure

substances. It should be noted that for difficult substances there is a risk in analysing only the minimum

number of samples that the exposure concentrations will be inadequately described. As a general rule it is

therefore advisable, to schedule for sampling all concentrations at a higher frequency. Under some

circumstances it may be possible to take additional samples and preserve them using fully validated

methods. Analysis of the additional samples is only carried out if results from the minimum sample set

provide insufficient data to adequately quantify exposure. It is recommended that mean measured

concentrations be taken at the beginning of the test, and 24-hour intervals throughout the test in order to

obtain the mean measured concentrations, where losses due to e.g. volatility are anticipated. It should also

be noted that where the flow-through exposure system is selected, analytical samples should be taken at the

beginning of the test to verify that the system is stable and operating correctly.

4.1.1 Algal growth inhibition test

Test media prepared specifically for analysis of exposure concentrations should be treated the

same as those used for testing i.e. they should be inoculated with algae and incubated under test conditions.

It may be necessary to subsequently separate algae from the medium or to analyse a series of test solutions

incubated under test conditions but with no algae in order to obtain a correct determination of the dissolved

test substance concentration (see Section 3.9). Since requirements may vary between different regulatory

authorities, it is good practice to consult with the appropriate authority.

Analysis at the start and end of the test of a low and high test concentration and a concentration

around the expected EC50 may be sufficient where it is likely that exposure concentrations will remain

within 20% of nominal values. Analysis of all test concentrations at the start and end of the test is required

where concentrations are unlikely to remain within 80-120% of nominal.

4.1.2 Lemna growth inhibition test

This test may be carried out using static, semi-static renewal or continuous flow-through

exposure regimes.
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Static

A static test is appropriate where exposure concentrations are expected to remain within 80-120%

of nominal over the 7-day exposure period. Analysis of the highest and lowest test concentration and a

concentration around the expected LC50 at the start and end of the exposure period is considered the

minimum requirement.

Semi-static

A semi-static renewal regime is recommended where exposure can not be maintained within 80-

120% of the nominal values over the test duration (7 days). Analysis of the highest and lowest test

concentrations and a concentration around the expected LC50 at the start and end of each renewal period is

recommended. Renewing the test media on, at least, two occasions during the test (e.g. days 3 and 5), is

considered the minimum requirement.

Continuous flow-through

A continuous flow-through regime may be preferred in some circumstances, e.g. where

concentrations are expected to decline from nominal by more than 20% over 24 hours.

If, under flow-through conditions, measured concentrations are likely to remain within 80-120%

of nominal then the highest and lowest test concentrations and a concentration around the expected LC50

should be analysed at the start, mid-way through and at the end of the test. If the concentrations are

expected to decline by more than 20% then all the concentrations should be analysed at the start, mid-way

through and at the end of the test.

4.1.3 Daphnia acute immobilisation test

Analysis of highest and lowest test concentrations and a concentration around the expected EC50

at the start and end (typically after 48 hours) of the exposure period may be sufficient where it is likely that

exposure concentrations will remain within 80-120% of nominal values. Analysis of all test concentrations

is required where concentrations are unlikely to remain within 80-120% of nominal.

Daphnia acute immobilisation test might be carried out using semi-static renewal or flow-through

regimes, but these approaches are not prevalent due to practical and cost reasons. Renewal systems are not

recommended in the OECD Test Guideline.

4.1.4 Daphnia reproduction test

This test may be carried out using semi-static renewal or continuous flow-through exposure

regimes.

Semi-static

Where the concentration of the test substance is expected to remain within 80-120% of nominal,

it is recommended that the highest and lowest concentrations and a concentration around the expected

NOEC/ECx are analysed when freshly prepared and immediately prior to renewal on one occasion during

the first week and thereafter weekly during the test. Where the test concentrations are not expected to

remain within 80-120% of nominal, it is necessary to either analyse all concentrations or consider a

continuous flow- through test.
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Continuous flow-through

Where the test substance concentrations are expected to remain within 80-120% of nominal, it is

recommended that the highest and lowest test concentrations and a concentration around the expected

NOEC/ECx are analysed three times during the first week and thereafter weekly during the test. Where the

test concentrations are not expected to remain within 80-120% of nominal, all the concentrations should be

analysed three times during the first week and thereafter weekly during the test.

4.1.5 Fish acute toxicity test

The test may be carried out using static, semi-static renewal or continuous flow-through exposure

regimes.

Static

A static test is appropriate where exposure concentrations are expected to remain within 80-120%

of nominal over the 96 hour exposure period. Analysis of the highest and lowest test concentration and a

concentration around the expected LC50 at the start and end of the exposure period is considered the

minimum requirement.

Semi-static

A semi-static renewal regime is recommended where exposure can be maintained within 80-

120% of the nominal values by renewing the test media at 24 or 48 hour intervals. Analysis of the highest

and lowest test concentrations and a concentration around the expected LC50 at the start and end of the first

24 or 48 hour renewal period is considered the minimum requirement.

Continuous flow-through

A continuous flow-through regime is recommended where concentrations are expected to decline

from nominal by more than 20% over 24 hours.

If, under flow-through conditions, measured concentrations are likely to remain within 80-120%

of nominal then the highest and lowest test concentrations and a concentration around the expected LC50

should be analysed at the start, mid-way through and at the end of the test. If the concentrations are

expected to decline by more than 20% then all the concentrations should be analysed at the start, mid-way

through and at the end of the test.

4.1.6 Fish prolonged toxicity test, fish early-life stage toxicity test, fish juvenile growth

toxicity and fish short-term toxicity on embryo and sac-fry stages

Fish prolonged toxicity test, fish early-life stage toxicity test, fish juvenile growth toxicity and

fish short-term toxicity on embryo and sac-fry stages are classified into this category. These tests may be

carried out using semi-static renewal or continuous flow-through exposure regimes.

Semi-static

A semi-static renewal regime is recommended where exposure can be kept to within 80-120% of

the nominal values by renewing the test media at intervals. Analysis of the highest and lowest test

concentrations and a concentration around the expected NOEC/ECx at the start and end of the first renewal

period and thereafter at least weekly is considered the minimum requirement.
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Continuous flow-through

A continuous flow-through regime is recommended where concentrations are expected to decline

from nominal by more than 20% over 24 hours.

If, under flow-through conditions, measured concentrations are likely to remain within 80-120%

of nominal then the highest and lowest test concentrations and a concentration around the expected

NOEC/ECx should be analysed three times during the first week and thereafter at least at weekly intervals.

If the concentrations are expected to decline by more than 20% then all the concentrations should be

analysed three times during the first week and thereafter at least at weekly intervals.

4.2 Taking of media samples for chemical analysis

Sampling of test media for chemical analysis will be case specific and it is therefore not possible

to give guidance which will be applicable in all cases. However, it is likely that it will be important to

consider the following when developing a suitable method:

• Does the test substance have physical-chemical properties which warrant special consideration. The

properties of difficult test substances discussed earlier in the context of testing may also be relevant to

sampling and analysis?

• What sample volume is required in order to measure exposure to the required level of accuracy and

precision?

• Will it be necessary to set up additional test vessels in order to obtain an adequate sample volume?

• What method of sampling should be employed and where in the test vessels should samples be taken?

• What time interval is acceptable between taking and analysing samples?

• Do samples require immediate fixing or extraction into an organic solvent?

• Is sample storage acceptable and if so are there special requirements?

 Sample collection and storage methods should be validated before they are applied to a definitive test.
ENV/JM/MONO(2000)6

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5. Calculation and expression of test results

The procedures described in Section 4 provide either nominal or measured exposure

concentrations of pure substances which can be used to determine effects concentrations. The following

general principles apply with respect to how these concentrations are to be used when calculating effect

concentrations in accordance with test guidelines:

• For static and semi-static and flow-through tests, where the concentrations remain within 80-120% of

nominal, the effect concentrations can be expressed relative to nominal or measured concentrations;

• For static and semi-static tests, where the concentrations do not remain within 80-120% of nominal,

the effect concentrations could be determined and expressed relative to the geometric mean of the

measured concentrations. A formula for calculating the geometric mean is given in Annex 2;

• For flow-through tests, where the concentrations do not remain within 80-120% of nominal, the effects

concentrations should be determined and expressed relative to the arithmetic mean concentration; and

• For tests with chemicals that cannot be quantified by analytical methods at the concentrations causing

effects, the effect concentration can be expressed based on the nominal concentrations.

It should be noted that several types of loss processes can occur very fast and new equilibrium

conditions can be attained relatively quickly during the exposure. If this is the case, the median of the

concentrations which are measured after the decline would be more appropriate as a surrogate for the mean

exposure concentration. Since there may be various methods for determining that, the method to determine

mean measured concentrations should be made explicit in the reporting of test results (see Section 3.3).

It is generally recommended that all test results be expressed in terms of mean measured

concentrations as far as possible. Some regulatory authorities invalidate, in certain regulatory context,

toxicity data from tests conducted above the aqueous water solubility limits using nominal concentrations.

It should also be noted that it is often useful to have both measured and nominal effect concentrations

quoted.

Guidance on the conventions used for expressing exposure and determining effect concentrations

in tests with multi-component substances is given in Section 3.11.
ENV/JM/MONO(2000)6

44

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ENV/JM/MONO(2000)6

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Annex 1

Computer programs for predicting physical/chemical properties, and fate,

transport and aquatic toxicity of substances

The following computer programs are used by the Office of Pollution Prevention and Toxics

(OPPT) of the United States Environmental Protection Agency for estimating physical/chemical properties,

and fate, transport and toxicity of substances.

1. Physical/chemical properties, fate and transport.

“EPI” (Estimation Programs Interface)

Available from:

Syracuse Research Corp, 6225 Running Ridge Rd, N. Syracuse, NY 13210, U.S.A., Tel: 315-452-3350,

Fax: 315-426-3429, Email: howardp@syrres.com, Internet: http://esc.syrres.com.

and

Peter Fisk Associates, 9 St. Swithins Road, Tankerton, Whitstable, Kent, CT5 2HT, U.K., Tel: 01227

273797, Fax: 01227 794982, Email: pfa@enterprise.net, Internet: http://www.ecotoxchem.co.uk

2. Aquatic toxicity

“ECOSAR”

Available as a computer program with a user’s manual and a technical support manual from:

J. V. Nabholz, RAD(7403), USEPA, 401 M St, SW, Washington, DC 20460-0001, U.S.A., Tel: 202-260-

1271, Email: nabholz.joe@epa.gov, Internet: http://www.epa.gov/opptintr/newchms/

and

P. H. Howard, Syracuse Research Corp. 6225 Running Ridge Rd, N. Syracuse, NY 13210, U.S.A., Tel:

315-452-8417, Email: howardp@syrres.com).

and

Peter Fisk Associates, 9 St. Swithins Road, Tankerton, Whitstable, Kent, CT5 2HT, U.K., Tel: 01227

273797, Fax: 01227 794982, Email: pfa@enterprise.net, Internet: http://www.ecotoxchem.co.uk

or as a manual - Clements, R.G. (editor), Nabholz, J.V., and Zeeman, M. (1996). Estimating toxicity of

industrial chemicals to aquatic organisms using structure activity relationships. 2nd edition. 402 p.

from:
ENV/JM/MONO(2000)6

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New Chemicals Screening Branch, Risk Assessment Division (7403), Office of Pollution Prevention and

Toxics, United States Environmental Protection Agency. EPA Report No. EPA-748-R-93-001.

Washington, DC 20460-0001.

3. n-Octanol-water partition coefficient (log Kow)

“ClogP”

Available from:

BioByte Corporation, 201 W. Fourth St., Suite 204, Claremont, CA 91711-4707, U.S.A., Tel: 909-624-

5992, Fax: 909-624-1398, Email: clogp@biobyte.com, Internet: http://www.biobyte.com/~clogp/

“KOWWIN”

Available from:

Syracuse Research Corporation Corp, 6225 Running Ridge Rd, N. Syracuse, NY 13210, U.S.A., Tel: 315-

452-3350, Fax: 315-426-3429, Email: howardp@syrres.com, Internet: http://esc.syrres.com.

and

Peter Fisk Associates, 9 St. Swithins Road, Tankerton, Whitstable, Kent, CT5 2HT, U.K., Tel: 01227

273797, Fax: 01227 794982, Email: pfa@enterprise.net, Internet: http://www.ecotoxchem.co.uk

“AUTOLOGP”

Described by:

Devillers, J., Domine, D. and Karcher, W. (1995). SAR QSAR. Environ. Res., 3, 301-306.
ENV/JM/MONO(2000)6

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Annex 2

Formula for calculating geometric mean exposure concentration

The following formula allows calculation of a geometric mean exposure concentration where

concentrations have been determined on more than two occasions during a test.

Mean concentration = antilog ( ) [ ] ( ) ()( ) ( ) 1

2 1

1 1

1

1

t t

conc conc t t

n

i i ii

i

n

− + ⋅−













 + +  =

−

∑ log log

Where: t1 = initial time < t2 <..... tn = final time

conc1 = initial concentration, conc2, ....., concn = final concentration
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Annex 3

Toxicity mitigation testing for cationic substances

Under some circumstances it may be required to determine the extent to which the toxicity of a

substance is mitigated by adsorption to dissolved organic carbon. For example, where cationic substances

show moderate to high aquatic toxicity in dilution water containing <2 mg/l total organic carbon. Toxicity

mitigation testing is usually carried out with fish. Fish are least susceptible to physical effects resulting

from clogging and/or coating of gills by precipitate formed by the reaction of the cationic substance with

the anionically charged dissolved organic carbon. Mitigation testing has, however, also been successfully

carried out with daphnids and green algae.

Once the intrinsic toxicity of a cationic substance has been determined in dilution water

containing less than 2.0 mg/l total organic carbon, the substance should be tested at least two more times

with two different concentrations of dissolved humic acid. The first of these tests should be carried out at a

humic acid concentration of 20 mg /l (or lower if a floc, precipitate, or a viscous mixture forms). The

second test should be carried out with a reduced humic acid concentration, for example, 10 mg/l.

The concentration of total organic carbon should be measured in the controls of the test without

added humic acid, the test with 20 mg/l humic acid and the test with 10 mg/l humic acid. TOC samples

should be taken from the controls at the beginning of the toxicity tests and it is recommended that total

organic carbon be measured three times in each of the three tests. The three measurements should be

reported separately and with the mean value for each test.

Toxicity mitigation is determined by regressing the effect concentrations determined in the test

(EC50, LC50 values etc.) against the total organic carbon concentration.
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Annex 4

Toxicity mitigation testing with algae for chemicals which form complexes with

and/or chelate polyvalent metals

The requirements for algal toxicity testing in relation to risk assessment for substances which

complex metals depend upon the water quality which is envisaged in the release scenario. The following

testing scheme has been suggested by the US EPA:

1. test chemical as it is in standard algal growth medium with a hardness in the range 15 to 24 mg/l as

CaCO3;

2. test chemical as it is in modified algal growth medium with a hardness of approximately 150 mg/l as

CaCO3;

3. test the chemical as the Ca salt in standard algal medium by adding an equivalent amount of Ca2+ to the

stock solution; and

4. test the chemical as the Ca salt in modified algal medium as for (2) above.

Testing a substance as the Ca salt requires the addition of an equivalent of Ca2+ to the stock

solution. A suitable procedure for preparing a calcium salt of a substance might be to add 1 g active

ingredient of the substance to a 1 litre volumetric flask which is partly filled with water and being stirred

continuously. An equivalent of Ca2+ is then added and stirred for at least one hour. The flask is then

topped up to 1 litre with water and used to prepare test media. Precipitate and/or flocculant which forms in

the stock solution should be maintained, to the extent possible, as a homogeneous dispersion during

preparation of the test media and should not be removed by filtration or centrifugation.
ENV/JM/MONO(2000)6

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Annex 5

Expert Panel on Aquatic Toxicity Testing of Difficult Substances

Peer Review Committee

Estelle Bjornestad VKI, Denmark

Norbert Bornatowicz Seibersdorf Research Centre, Austria

Elaine Dorward-King Rio Tinto plc, United Kingdom

Andrew Girling Environmental consultant, United Kingdom

Marie-Hélène Lamy INERIS, France

Stuart Marshall Unilever, United Kingdom

Vincent Nabholz EPA/OPPT, United States

Margrethe Winther-Nielsen VKI, Denmark

Joanne Parrot Environment Canada, Canada

Erik van de Plassche RIVM, the Netherlands

Hans-Juerguen Pluta UBA, Germany

Helena Parkman KemI, Sweden

Hans Rufli Novartis, Switzerland

Leslie Touart EPA/OPP, United States

Sabine Zok Hoechst, Germany