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September 2014

OECD GUIDELINE FOR TESTING OF CHEMICALS

Draft revised version

Fish, Acute Toxicity Test

Draft Update by H. Rufli 2.6.14, Track Changes 10.6.14, Comments 11.6.14, UK comments

9.-16.7.14, Update on OECD comments 9.-12.9.14

INTRODUCTION

1. OECD Guidelines for Testing of Chemicals are periodically reviewed to ensure that they reflect

the best available science. In the revision of this Guideline (originally adopted in 1981, updated

in 1984, 1992), special attention was given to possible improvements in relation to animal

welfare concerns in order to minimize unnecessary testing of laboratory animals (OECD 2010).

2. The main differences in comparison with the earlier versions are the reduction in group-size, the

introduction of the moribund stage as a surrogate of lethality/death, the possibility to use the

Fish Embryo Test as range-finder, and flexibility in the range of responses to cover (% mortality).

3. Definitions used in this Test Guideline are given in Annex 1.

PRINCIPLE OF THE TEST

4. The fish are exposed to the test chemical preferably for a period of 96 hours. Mortalities are recorded at 24, 48, 72 and 96 hours and the concentrations which kill (mortality/morbidity) 50% of the fish (LC50/LC50moribund) are determined where possible.

INFORMATION ON THE TEST CHEMICAL

5. Useful information about substance-specific properties include the structural formula, molecular

weight, purity, stability in water and light, pKa and Kow, water solubility (preferably in the test

medium) and vapour pressure as well as results of a test for ready biodegradability (OECD TG

301 (1) or TG 310 (2)). Solubility and vapour pressure can be used to calculate Henry's law

constant, which will indicate whether losses due to evaporation of the test chemical may occur.

6. A reliable analytical method for the quantification of the substance in the test solutions with

known and reported accuracy and limit of detection should be available.

7. If the Test Guideline is used for the testing of a mixture, its composition should, as far as

possible, be characterized, e.g. by the chemical identity of its constituents, their quantitative

occurrence and their substance-specific properties (see § 5). Before the use of the Test

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Guideline for regulatory testing of a mixture, it should be considered whether it will provide

acceptable results for the intended regulatory purpose.

VALIDITY OF THE TEST

8. For a test to be valid, the following conditions should be fulfilled:

- in the controls, the mortality should not exceed 10% (or one fish if less than ten are used) at

the end of the exposure and there should be no signs of stress of the remaining individuals.

- constant conditions should be maintained as far as possible throughout the exposure and, if

necessary, semi-static or flow-through procedures should be used (see Annex 1 for

definitions and OECD Guidance Document No. 23 for the use of semi-static and flow-through

procedures (3));

- the water temperature should not differ by more than ± 1.5o

C between test vessels or

between successive days at any time during the exposure, and should be within the

temperature ranges specified for the test species (Table 1);

- the dissolved oxygen concentration should be ≥60% of the air saturation throughout the

exposure;

- there should be evidence that the concentration of the test chemical has been satisfactorily

maintained, and preferably it should be at least 80% of the nominal concentration

throughout the exposure. If the deviation from the nominal concentration is >20% , results

should be based on the measured concentration (geometric mean in static and semi-static

tests, arithmetic mean in flow-through tests (3); time weighted average, where applicable).

DESCRIPTION OF THE METHOD

Apparatus

9. Normal laboratory equipment for the conduct of this assay include: (a) oxygen meter; (b) pH meter; (c) equipment for determination of hardness of water; (d) equipment for the determination of total organic carbon concentration (TOC); (e) equipment for the determination of chemical oxygen demand (COD); (f) adequate apparatus for temperature control; (g) tanks made of chemically inert material.

Test chambers

10. Any glass, stainless steel or other chemically inert vessels can be used. As silicone is known to

have a strong capacity to absorb lipophilic substances, the use of silicone tubing in flow-through

studies and use of silicone seals in contact with water should be minimized by the use of e.g.

monoblock glass aquaria. The dimensions of the vessels should be large enough to keep fish free of

stress in the control, maintenance of dissolved oxygen concentration (e.g. for small fish species, a 7 L

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tanks volume will achieve this) and compliance with loading rate criteria given in § 19. It is desirable

that test chambers be randomly positioned in the test area. The test chambers should be shielded

from unwanted disturbance. For difficult to test chemicals, the test system should preferably be pre-

conditioned with concentrations of the test chemical for a sufficient duration to demonstrate stable

exposure concentrations prior to the introduction of test organisms (3).

Selection of species

11. It is suggested that the species used be selected on the basis of such important practical criteria

as, for example, their ready availability throughout the year, ease of maintenance, convenience for

testing and any relevant economic, biological or ecological factors as well as historical use in safety

testing. The fish should be in good health (<5% mortality of population during the seven days

immediately preceding the exposure, see § 14) and free from any apparent malformations. Fish

previously treated against disease should not be used.

12. Examples of fish recommended for testing are given in the Table 1. The fish mentioned in Table 1

are easy to rear and/or widely available throughout the year. They can be bred and cultivated either

in fish farms or in the laboratory, under disease- and parasite-controlled conditions, so that the test

fish will be healthy and of known parentage. These fish are available in many parts of the world. If

other species fulfilling the above criteria are used, the test method should be adapted in such a way

as to provide suitable test conditions; such adaptations should be reported.

Holding of fish

13. All fish should be obtained and held in the laboratory for at least 12 days before they are used

for testing. They should be held in water of adequate and sufficient quality for use in the test (see

Annex 3 for relevant characteristics) for at least seven days immediately before testing and under the

following conditions:

Light: 12 to 16 hours photoperiod daily, 30 min transition period recommended;

Temperature: appropriate to the species (see Table 1);

Oxygen concentration: at least 80% of air saturation value;

Feeding: three times per week or daily until 24 hours before the exposure is started.

14. Following a 48 hour settling-in period, mortalities are recorded and the following criteria applied:

- mortalities >10% of population in seven days: rejection of entire batch;

- mortalities between 5 and 10% of population: acclimatization continued for seven

additional days;

- mortalities of <5% of population: acceptance of batch.

Water

15. Clean surface-, ground- sea- (for estuarine or marine species) or reconstituted water (see Annex

2) is preferred, although drinking water (dechlorinated, if necessary) may also be used. Any water

which conforms to the chemical characteristics of acceptable dilution water as listed in Annex 3 is

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suitable as a test water. It should be of constant quality during the period of the test. The water

quality is regarded as good, if fish will survive for the duration of the culturing, acclimatization and

testing without showing signs of stress. Waters with total hardness of 10 to 250 mg CaC03/L, and

with a pH 6.0 to 8.5 are preferable. The reagents used for the preparation of reconstituted water

should be of analytical grade and the deionised or distilled water should be of conductivity ≤10

μScm1. The dilution water is aerated prior to use for the test so that the dissolved oxygen

concentration has reached saturation.

16. If natural water (surface or ground water) is used, the quality parameters including conductivity

and total organic carbon (TOC) or chemical oxygen demand (COD) should be measured at least twice

a year or whenever it is suspected that these characteristics may have changed significantly (see § 15

and Annex 3). Chemical measurements should include heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni; note

that Cu-pipes may cause fish kills) and the substances and maximum concentrations shown in Annex

3. If dechlorinated tap water is used, daily chlorine analysis is desirable.

Test solutions

17. Test solutions of the selected concentrations can be prepared, e.g. by dilution of a stock solution.

The stock solutions should preferably be prepared by simply mixing or agitating the test chemical in

the dilution water by mechanical means (e.g. stirring and/or ultra-sonification). If the test chemical is

difficult to dissolve in water, procedures described in the OECD Guidance Document No. 23 for

handling of difficult substances should be followed (3). The use of solvents should be avoided, but

may be required in some cases in order to produce a suitably concentrated stock solution. Where a

solvent is used to assist in stock solution preparation, its final concentration should be minimized as

far as possible (not exceeding 100 mg/L and should be the same in all test vessels. When a solvent is

used, an additional solvent control is required.

18. The test should be carried out without adjustment of pH. Where the substance itself causes a

change of the pH of the test medium outside the range of pH 6.0-8.5, the procedure described in the

OECD Guidance Document No. 23 for handling difficult substances (3) should be followed.

PROCEDURE

Conditions of exposure

19. Duration: 96 hours. Prolongation may be necessary if the incipient LC50 is not reached

within 96 hours. This is best done by performing a test following TG 215, fish juvenile growth

test (4) or TG 210, fish early- life stage test (5).

Loading: maximum loading of 1.0 g fish/L for static and semi-static test is

recommended; for flow-through systems, maximum loading of 0.8 g fish/L passing through a

replicate in 24 hours.

Light: 12 to 16 hours photoperiod daily 30 min transition period recommended.

Temperature: appropriate to the species (see Table 1) and constant within a range of 2°C.

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Oxygen concentration: not less than 60% of the air saturation value. Aeration can be used

provided that it does not lead to a significant loss of test chemical as verified by analytical

measurements of test concentrations (see § 23).

Feeding: none.

Disturbance: disturbances that may change the behaviour of the fish should be avoided,

such as vibration or noise.

Number and handling of fish

20. At least 6 fish, randomly distributed among treatments, must be used at each test concentration

and in the control(s). Annex 4 provides details on the precision of the LC50 when using 6 fish per

concentration as compared to 7. Fish previously treated against disease should not be used in the

test.

Test concentrations

21. The threshold approach should be applied whenever possible (6). Alternatively, QSAR- methods

and other, non-animal alternatives such as the Fish Embryo Test (6) can be used as range-finding test

(see Annex 5 for range-finding procedure and in vivo fish confirmatory test), if no information on the

toxicity of the test chemical is available or if sufficient confidence cannot be gained from the use of in

silico/alternative methods.

A range-finding test is performed, for example starting at 100 (or the water solubility limit), 10 and

1.0 mg/L with three fish per concentration, no blank or solvent control and no replicate tanks.

Temperature, pH, test medium, preparation of test solution, test system (static or flow-through),

analytical determination of concentrations etc. should be kept the same as in the definitive test, as

far as possible, to ensure that results are comparable between the range-finding and the definitive

test.

For the definitive test with juvenile fish, at least five concentrations in a geometric series with a

factor preferably not exceeding 2.2 are used, although smaller separation factors of 1.6 to 1.8 should

be used whenever possible (see Annex 6). Fish should originate from the same source and

population. No test tank replication is required.

Controls

22. One water control and, if relevant, one solvent control are run in addition to the test series.

Frequency of analytical Determinations and Measurements

23. With difficult to test chemicals and when using flow-through systems, it is recommended to

perform chemical analysis of the test chemical concentration before initiation of the exposure to

check compliance with the acceptance criteria. All concentrations should be analyzed individually at

the beginning and termination of the exposure. If samples are stored to be analyzed at a later time,

the storage method of the samples should be previously validated. Samples should be filtered (e.g.

using a 0.45 μm pore size) or centrifuged to ensure that the determinations are made on the

chemical in true solution (3). For the analysis, a suitable analytical method is required with an

appropriate limit of quantification (LOQ).

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24. During the exposure, dissolved oxygen, pH, salinity (if relevant) and temperature should be

measured daily in each test vessel, hardness at the beginning of the exposure in the dilution water.

Temperature should preferably be monitored continuously in at least one test vessel.

Observations, humanely Killing and Measurement of Fish 25. The fish are inspected at least after 24, 48, 72 and 96 hours. Observations in the period 2 to 6

hours after the start of the exposure are desirable. Records are kept of all visible abnormalities in a

non-ambiguous way.

As far as possible, effects should be expressed by the most frequently occurring abnormalities

classified into 1) loss of equilibrium, 2) swimming behaviour, 3) respiratory function, 4) pigmentation

and 5) other clinical signs, together with specifications on the degree of the effects (see Annex 7 for

details on reporting sub-lethal effects).

1. Specifications on the type of visible abnormalities to be reported: Effects should be

expressed by the most frequently occurring abnormalities classified into 1) loss of

equilibrium, 2) swimming behaviour, 3) respiratory function, 4) pigmentation and 5)1

other clinical signs according to Table 2.

2. Specifications on the degree of the effects: Reporting is best done by stating the specific

number of fish exhibiting the specified abnormalities which increases the ability to

characterize the effects. If this is not possible, e.g. due to turbidity or colour of the

higher test concentrations, the degree of severity of the effects on the entire batch of

fish in each concentration needs to be classified by a scale from 0 (no effect) to 1

(slight), 2 (medium), and 3 (severe effects) for this study.

Fish are considered dead if there is no visible movement (e.g. gill movements) and if touching of the

caudal peduncle produces no reaction. Alternatively and preferably, the moribund state may be used

instead of death as an endpoint or other measures able to reliably predict mortality/morbidity. For

moribund fish, this is a premature discontinuation of the experiment to reduce the suffering (see

Annex 7 for the use of the moribund state). Dead/moribund fish are removed as soon as observed

and mortalities/morbidities are recorded. Moribund fish are humanely killed as soon as observed as

are the surviving fish at the end of the exposure. The individual size (wet weight, blotted dry and

total- or standard length, if fin rot or fin erosion occurs) of all the fish in the blank control is

measured.

LIMIT TEST

26. Using the procedures described in this Guideline, a limit test may be performed at 100 mg (active

ingredient)/L or at the limit of solubility in order to demonstrate that the LC50 is greater than this

concentration. The limit test should be performed using at least 7 fish, with the same number in the

1 Effects on the class on swimming behaviour include e.g. hyper-excitability, surfacing, sounding, erratic swimming, skittering, diving, spiraling, twitching, apathy, lethargy, weakness, immobility, quiescent as described in Table 2. Similarly, effects on the other classes of sub-lethal effects include the clinical signs shown in the Table for this class. Tumbling fish may be classified as showing both a change in swimming behaviour and loss of equilibrium. Lying on the bottom may be allocated to altered swimming behaviour, but may also show loss of equilibrium.

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control(s)2. If any mortalities occur, a full study should be conducted. If sub-lethal effects are

observed, these should be recorded (see Annex 7).

DATA AND REPORTING

Treatment of results

27. In this test, replicates are defined as test vessels which are the unit of comparison. Data should

be summarized in tabular form, showing the number of fish used, mortality/morbidity and sub-lethal

effects (see Annex 7) for each treatment group and control(s) at each observation. If a limit test is

performed, no graphical representation of responses or statistical calculations are needed.

Otherwise, the cumulative percentage mortality/morbidity for each exposure period, preferably in

probit or probability scale in order to produce a straight line, is plotted against concentration in

logarithmic scale. When an experiment results in at least two concentrations with partial

mortalities/morbidities (mortality >0 and <100%), the LC50/LC50moribund, the confidence limits (95%)

and the slope of the curve should be estimated. These estimates should be obtained using

appropriate statistical methods such as the classical maximum likelihood methods for fitting probit or

logit models (8, 9 and 10). When an experiment results in only one concentration with partial

mortality/morbidity or none, classical maximum likelihood methods cannot be used to estimate the

LC50/LC50moribund, the slope of the concentration-response curve cannot be estimated, and a

confidence interval for the LC50/LC50moribund may not be estimable. In such cases, estimates of the

LC50/LC50moribund can be made using various techniques such as the Spearman-Kaerber method (10),

the binomial method (11), the moving average method (11), or as a last resort, the graphical method

(12). These non-classical methods can give precise LC50/LC50moribund estimates for well designed

studies (11), and are extremely useful, as up to 75% of acute fish studies yield results that cannot be

analyzed using classical probit maximum likelihood techniques (13).

Test Report

28. The test report should include the following information:

Test chemical:

Mono-constituent substance

- physical appearance, water solubility, and additional relevant physicochemical properties;

- chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code,

structural formula, purity, chemical identity of impurities as appropriate and practically

feasible, etc. (including the organic carbon content, if appropriate).

Multi-constituent substance, UVBCs and mixtures:

- characterized as far as possible by chemical identity (see above), quantitative occurrence and

relevant physicochemical properties of the constituents.

Test fish:

2 Binomial theory dictates that when 10 fish are used with zero mortality, there is a 99.9% confidence that the

LC50 is greater than the concentration used in the limit test. With 7, 8 or 9 fish, the absence of mortality provides at least 99% confidence that the LC50 is greater than the concentration used in the limit test.

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- scientific name, strain, size (wet weight, blotted dry, and total- or standard length), supplier,

any pretreatment, etc.

Test conditions:

- test procedure used (e.g. static, semi-static, flow-through; aeration; fish loading; etc.);

- water quality characteristics (pH, hardness, temperature; TOC, COD for surface or ground

water) and adaptations made to suite fish species used other than those in Table 1;

- dissolved oxygen concentration, pH values and temperature of the test solutions at 24 hour

intervals in each tank and continuous in one tank (in semi-static systems, the pH should be

measured prior to and after water renewal);

- methods of preparation of stock and test solutions;

- concentrations used;

- information on concentrations of the test chemical in the test solutions;

number of fish in each test solution.

Results:

- maximum concentration causing no mortality/morbidity within the period of the exposure

(no mandatory requirement to get a maximum concentration causing 0%

mortality/morbidity with one of the 5 concentrations);

- minimum concentration causing 100% mortality/morbidity within the period of the exposure

(no mandatory requirement to get a minimum concentration causing 100%

mortality/morbidity with one of the 5 concentrations);

- cumulative mortality/morbidity at each concentration at the recommended observation

times;

- the LC50/LC50moribund values at 24, 48, 72 and 96 hours with 95% confidence limits, if

possible;

- the slope of the concentration-response curve, if possible;

- graph of the concentration-mortality/morbidity curve at the end of the exposure preferably

on probit or probability scale versus concentration in log scale; (note that the control group

cannot be plotted on log scale axes. Likewise, neither 0 nor 100% mortality can be plotted on

a probit scale (undefined values), and the slope cannot be meaningfully represented for

experiments with less than two partial mortalities/morbidities or if the 50% response is

between the control and lowest test concentration. Therefore, graphs are not required

under such circumstances);

- mortality/morbidity in the controls;

- incidence and description of visible abnormalities such as loss of equilibrium, swimming

behaviour, respiratory function, pigmentation and other clinical signs including degree of the

effects (according to Annex 7);

- incidents in the course of the test which might have influenced the results;

- description of the statistical methods used and treatment of data (e.g. probit analysis, logistic

regression model, arithmetic or geometric mean for LC50/LC50moribund values, time weighted

average)

Any deviation from the Guideline and relevant explanations

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TABLE 1A: FRESHWATER FISH SPECIES RECOMMENDED FOR TESTING

Recommended species Recommended test temperature range

(°C)

Recommended total length of test fish

(cm)*

Danio rerio Zebrafish

21-25

2.0 ± 1.0

Pimephales promelas Fathead minnow

21-25

2.0 ± 1.0

Cyprinus carpio Carp

20-24

3.0 ± 1.0

Oryzias latipes Japanese Medaka

21-25

2.0 ± 1.0

Poecilia reticulate Guppy

21-25

2.0 ± 1.0

Lepomis macrochirus Bluegill

21-25

2.0 ± 1.0

Oncorhynchus mykiss Rainbow trout

13-17

5.0 ± 1.0

TABLE 1B: ESTUARINE and MARINE FISH SPECIES RECOMMENDED FOR TESTING

Recommended species Recommended test temperature range

(°C)

Recommended total length of test fish

(cm)

Recommended salinity range

(ppt)*

Cyprinodon variegates Sheepshead minnow

23-27

2.0 ± 1.0

15-35**

Menidia sp. Silverside

22-25

3.0 ± 1.0

15-35**

Gasterosteus aculeatus Three-spined stickleback

18-20

3.0 ± 1.0

20 ± 5

* If fish of sizes other than those recommended are used, this should be reported together with the

rationale.

** For any given test this shall be performed to ± 2‰.

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LITERATURE

(1) (2)

OECD (1992) Ready Biodegradability, Test Guideline No. 301, Guidelines for the Testing of Chemicals, OECD, Paris. OECD (2006) Ready Biodegradability, CO2 in sealed vessels, Test Guideline No. 310, Guidelines for the Testing of Chemicals, OECD, Paris.

(3) OECD (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Series on Testing and Assessment No. 23, OECD, Paris.

(4) OECD (2000) Fish, Juvenile Growth Test, Test Guideline No. 215, Guidelines for the Testing of Chemicals, OECD, Paris.

(5) OECD (2013) Fish, Early-life Stage Toxicity Test, Test Guideline No. 210, Guidelines for the Testing of Chemicals, OECD, Paris.

(6) OECD (2013) Fish Embryo Acute Toxicity (FET) Test, Test Guideline No. 236, Guidelines for the Testing of Chemicals, OECD, Paris.

(7) OECD (2010) SHORT GUIDANCE ON THE THRESHOLD APPROACH FOR ACUTE FISH TOXICITY. Series on Testing and Assessment No. 126, OECD, Paris.

(8) ISO (2006) International Standard. Water quality – Guidance on statistical interpretation of

ecotoxicity data ISO TS 20281. Available: [http://www.iso.org].

(9) OECD (2006) Guidance Document on Current Approaches in the Statistical Analysis of Ecotoxicity Data: a Guidance to Application: Series on Testing and Assessment No. 54, OECD, Paris.

(10) Finney, DJ (1978) Statistical Methods in Biological Assays. Griffin, Weycombe, U.K.

(11) Stephan, CE (1977) Methods for calculating an LC50. In Aquatic toxicology and hazard

evaluation ASTM STP 634, ed. F.L Mayer and J. L Hamelink. Philadelphia: American Society for

Testing and Materials.

(12) USEPA (2002) Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms. Fourth edition. US Environmental Protection Agency, Office of Water, Washington, DC. EPA-821-R-02-013. October 2002.

(13)

Rufli H, Springer TA (2011) Can we reduce the number of fish in the OECD acute fish toxicity test? Environ Toxicol Chem 30: 1006-1011.

ANNEX 1

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DEFINITIONS

Flow-through test is a test with continued flow of test solutions through the test system during the

duration of exposure.

IUPAC: International Union of Pure and Applied Chemistry.

Median Lethal Concentration (LC50) is the concentration of a test chemical that is estimated to be

lethal to 50% of the test organisms within the test duration.

Median Lethal Concentration (LC50moribund) is the concentration of a test chemical that is estimated

to be lethal to 50% of the test organisms within the test duration based on the endpoint moribund.

Moribund: Dictionary definitions: “dying”, “at the point of death”, “in the state of dying” or

“approaching death”.

Semi-static renewal test is a test with regular renewal of the test solutions after defined periods (e.g.

every 24 hours).

SMILES: Simplified Molecular Input Line Entry Specification.

Static test is a test in which test solutions remain unchanged throughout the duration of the test.

TC: The lowest EC50-value of existing and reliable algae or acute invertebrate (e.g. daphnia) toxicity

data is set as threshold concentration (TC).

UVCB: Substances of unknown or variable composition, complex reaction products or biological

materials.

ANNEX 2

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EXAMPLE OF A SUITABLE RECONSTITUTED WATER (ISO 6341 -1982)

(a) Calcium chloride solution

Dissolve 11.76 g CaCl2 · 2H2O in deionised water; make up to 1 litre with deionised water

(b) Magnesium sulphate solution

Dissolve 4.93 g MgSO4 · 7H2O in deionised water; make up to 1 litre with deionised water

(c) Sodium bicarbonate solution

Dissolve 2.59 g NaHCO3 in deionised water; make up to 1 litre with deionised water

(d) Potassium chloride solution

Dissolve 0.23 g KCl in deionised water; make up to 1 litre with deionised water

All chemicals must be of analytical grade.

The conductivity of the distilled or deionised water should not exceed 10µScm-1

25 ml each of (a) to (d) are mixed and the total volume made up to 1 litre with deionised

water. The sum of the calcium and magnesium ions in this solution is 2.5 mmol/L.

The proportion Ca:Mg ions is 4:1 and Na:K ions 10:1. The acid capacity KS4 3 of this solution is

0.8 mmol/L.

Aerate the dilution water until oxygen saturation is achieved, then store it for about two days

without further aeration before use.

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ANNEX 3

SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION/TEST WATER

Substance Limit concentration

Particulate matter 5 mg/L

Total organic carbon 2 mg/L

Un-ionised ammonia 1 µg/L

Residual chlorine 10 µg/L

Total organophosphorous pesticides 50 ng/L

Total organochlorine pesticides plus polychlorinated biphenyls 50 ng/L

Total organic chlorine 25 ng/L

Aluminium 1 µg/L

Arsenic 1 µg/L

Chromium 1 µg/L

Cobalt 1 µg/L

Copper 1 µg/L

Iron 1 µg/L

Lead 1 µg/L

Nickel 1 µg/L

Zinc 1 µg/L

Cadmium 100 ng/L

Mercury 100 ng/L

Silver 100 ng/L

Chemical oxygen demand ≤5 mg/L

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ANNEX 4

PRECISION OF THE LC50 WHEN USING SIX FISH

Monte Carlo Simulations representing different experimental scenarios with five test concentrations (plus control) and six or seven fish per concentration, following a range- finding test of three concentration groups of four fish each were performed. Concentration-response slopes used in the simulations were selected based on data from the Industry Laboratory Database (523 acute fish studies) and U.S. EPA Office of Pesticide Programs (OPP) Ecotoxicity Oneliner Database (http://www.ipmcenters.org/Ecotox/index.cfm) (4010 studies). The simulations showed that for about 75% of the studies, six fish per concentration yield the same quality of the LC50-value as does a minimum of seven fish (Fig. 1) (1). For about 25% of the studies, with concentration-response curve slopes of less than four, six fish did not yield LC50 estimates of similar quality as when using seven fish. Fig. 1. Values of R95/5 (ratio of 95 to 5th centile of LC50 estimates) as a measure of precision of the

LC50 distributions obtained in the simulations (lower values of R95/5 correspond to a higher

precision, lower variability). Each slope is represented by three points representing sets of simulations that assumed different true LC50-values. y Axis: R95/5 of scenario 1 (47 + 7 fish: 47 fish in range-finder and definitive test plus 7 in control):

range-finder with three concentrations containing four fish each; definitive test consisting of five concentrations with seven fish each. x Axis: R95/5 of scenario 2 (42 + 7 fish): same as scenario 1, but definitive test with six instead of

seven fish per concentration.

(1) Rufli H, Springer TA (2011) Can we reduce the number of fish in the OECD acute fish toxicity

test? Environ Toxicol Chem 30: 1006-1011.

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ANNEX 5

FISH EMBRYO TEST AS RANGE-FINDER

When a range-finding test is required, the fish use can be reduced by using fish embryos instead of

juvenile fish. The 96 hours fish embryo toxicity test (1) is classified as a non-animal test according to

the definitions of the European Union animal protection directive (2) because the embryo does not

begin to feed freely until after this period (3). The range-finding-test is started with embryos at the

Threshold Concentration (TC) corresponding to the lowest 50% effective concentration (EC50) value

from algae and acute invertebrate (e.g. daphnia) tests with the test chemical (4), if available, or

another reasonable starting concentration ≤100 mg/L based on the information available3. If the

embryo test shows no toxicity at this concentration, it is followed by an in vivo fish confirmatory test

performed at the concentration of the fish embryo test as a limit test, or as a full test if a

concentration-effect relationship is required. If the concentration is toxic, the embryo test is

repeated, stepping down from the previous test concentration until there is no toxicity, followed by

the in vivo confirmatory limit or full test with fish as above (five fish in a single concentration and

control for hazard classification (6) and at least seven for risk assessment with concentration-

response curve according to OECD TG 203); end testing if there is no mortality.

Instead of the 96 hours fish embryo test, a 48 hours test might be used. However, for some

substances like quaternary ammoniums, a 96 hours test is required to give better correlation to the

OECD 203 test as embryos are protected by the selective permeability of the chorion until hatching

(zebrafish hatching: after 2-3 days, fathead minnow: after 4-5 days, medaka: after 9-14 d) (7).

FLOW-CHART

1 Fish embryo test at TC (or other reasonable starting concentration ≤100 mg/L)

No toxicity Proceed to step 2, or step 3 for a concentration-response curve

↓

2 Limit test according to OECD TG 203 at the starting concentration

No toxicity Toxicity

LC50 fish > starting concentration Repeat step 1 at a lower concentration

↓

3 Performance of a full study according to OECD TG 203

Concentration-response curve

LC50 fish

3 An evaluation of 694 acute algae, daphnia and fish tests revealed that fish were the most sensitive in only

15.6% of these tests whereas in 84.4%, the fish LC50 was ≤TC (5).

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(1) OECD (2013) Fish Embryo Acute Toxicity (FET) Test, Test Guideline No. 236, Guidelines for the Testing of Chemicals, OECD, Paris.

(2) European Commission (2010) Directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union L 276, 20.10. pp 33-79.

(3) Belanger SE, Balon EK, Rawlings JM (2010) Saltatory ontogeny of fishes and sensitive early life stages for ecotoxicology tests. Aquat Toxicol 97:88-95.

(4) OECD (2010) Short Guidance on the Threshold Approach for Acute Fish Toxicity. Series on Testing and Assessment No. 126, OECD, Paris.

(5) Weyers A, Sokull-Klüttgen B, Baraibar-Fentanes J, Vollmer G (2000) Acute toxicity data: a comprehensive comparison of results of fish, Daphnia and algae tests with new substances notified in the EU. Environ Toxicol Chem 19:1931-1933.

(6) Jeram S, Riego Sintes JM, Halder M, Baraibar Fentanes J, Sokull-Klüttgen B, Hutchinson TH (2005) A strategy to reduce the use of fish in acute ecotoxicity testing of new chemical substances notified in the European Union. Regul Toxicol Pharmacol 42:218-224.

(7) Braunbeck T, Lammer E (2006) Background on Fish Embryo Toxicity Assays. UBA Contract Number 203 85 422. Prepared for German Federal Environment Agency, D-06813 Dessau.

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ANNEX 6

FACTOR BETWEEN CONCENTRATIONS

Although a factor between concentrations (separation factor) of 2.2 is permissible, when the LC50

can be estimated with sufficient confidence during the design of the test, separation factors of 1.6 to

1.8 are preferred for the following reasons:

1. It is not uncommon in fish tests to find none or just one concentration with a partial

mortality (>0 and <100% mortality). In the Industry laboratory Database, these studies

amounted to 75% (1)4 for which classical statistical methods cannot be used5.

2. Little information is gained from the multiple test concentrations with no or complete

mortality. In such cases, fish in 3 of 5 concentrations do not contribute to the determination

of the LC50 and are, thus, wasted.

3. The plot of the factor between concentrations for two partial mortalities (for 13 and 87%

mortality) versus the slope of the probit transformed concentration-response curve shows

that separation factors of 1.2 to 2.0 would produce two partial mortalities for slopes

between 7 and 30 (Fig. 1, dotted lines). To get two partial mortalities for 75% of the studies

would require a ´Factor for 2PM´ of approximately ≤1.4 for slopes up to 18.8 (75th centile)

according to the Industry Database (Fig. 2), whereas a ´Factor for 2PM´ of ≤1.8 would be

sufficient for slopes up to 8.8 (75th centile) according to the U.S. EPA Oneliner Database.

Thus, separation factors of ≤1.4 would produce two partial mortalities if the distribution

follows that of the Industry Database (slopes ≤18.8), and separation factors of ≤1.8 if the

distribution follows that of the U.S. EPA Oneliner Database (slopes ≤8.8). Even if two partial

mortalities are not obtained so that mortality goes from 0 to 100% in adjacent

concentrations, there is still an advantage in keeping the separation factor small, because

the region of the transition from survival to mortality is more narrowly defined. So for the

Industry Database, one would run into the practical lower limit for the separation factor. The

only reason to use broader spacing is to increase the probability that the test concentrations

selected during study design will encompass the unknown LC50.

As a consequence, separation factors should be selected as a compromise between the need to

bracket the true LC50 and the desire to minimize fish waste. A reasonable compromise appears to be

using separation factors of 1.6-1.8, e.g. concentrations of 1.0, 1.8, 3.2, 5.6, 10 mg/L when using factor

1.8.

4 Because the goal of performing a test according to OECD 203 is to estimate the 50% lethal

concentration (LC50), the results of many fish acute tests performed for regulatory submission with low-toxicity chemicals must be expressed as a one-sided interval, such as LC50 >100 mg/L (37% in Industry Laboratory Database: 194 of 523 studies; 8% in U.S. EPA Oneliner Database: 326 of 4010 studies). 5 For the use of classical Probit maximum likelihood techniques, and to obtain an estimate of the

slope, at least 2 partial mortalities are required.

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Figure 1. Plot of maximum ratios of adjacent test concentrations (Factor for 2PM) that would result in the expectation of two adjacent concentrations with 13 and 87% mortality if centered on the true LC50 (D-optimal dose placement). ‘Factor for 2PM’ is plotted as a function of concentration-response curve slopes. The 13 and 87% mortality rates correspond to one mortality in the test concentration below the LC50, and one survivor in the test concentration above the LC50. The plot shows that factors between concentrations of 1.2 to 2.0 would produce two partial mortalities for slopes between 7 and 30 (dotted lines). Concentrations for use in a study should be selected using a step size less than the ‘Factor for 2PM’ obtained assuming some likely slope of the concentration-response curve. PM: partial mortality (mortality in concentration >0 and <100%)

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Figure 2: Distribution of the concentration-response slopes in two databases of results from tests performed according to regulatory test guidelines (Industry Laboratory and U.S. EPA Oneliner Database). The reasons for the different distributions in the two databases (Industry Laboratory Database: bimodal, median of slopes 13.1 versus U.S. EPA Oneliner Database: unimodal, median of slopes 6.5) are not known, but might include different distributions of modes of action or artifacts such as from selection criteria for inclusion of tests in the databases.

SD: standard deviation

N: total number of studies

(1) Rufli H, Springer TA (2011) Can we reduce the number of fish in the OECD acute fish toxicity test? Environ Toxicol Chem 30: 1006-1011.

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ANNEX 7

MORIBUND AND REPORTING OF SUB-LETHAL EFFECTS

It has become common practice in many laboratories in Europe to introduce the criterion of “moribund” in the acute fish test as it reduces the terminal suffering of the fish. The definitions of “moribund” are severely limited to laboratory animal research because they do not describe the moribund state in behavioral or physiological terms. Developing a sound approach to identifying the moribund state is crucial to its effective use as an experimental endpoint (1). The LC50moribund is already used for the hazard and risk assessment of chemicals, for example in the UK. To produce comparable results between laboratories, it requires reducing the subjectivity of the criterion moribund (2). This is best done by:

3. A unique definition of the moribund state6: Observation of both “impaired swimming behaviour” and “loss of equilibrium” at two successional observation times 24 hours apart from each other7 as an approximation of ecological death8.

4. Specifications on the type of visible abnormalities to be reported: Effects should be expressed by the most frequently occurring abnormalities classified into 1) loss of equilibrium, 2) swimming behaviour, 3) respiratory function, 4) pigmentation and 5)9 other clinical signs according to Table 2.

5. Specifications on the degree of the effects: Reporting is best done by stating the specific number of fish exhibiting the specified abnormalities which increases the ability to characterize the effects. If this is not possible, e.g. due to turbidity or colour of the higher test concentrations, the degree of severity of the effects on the entire batch of fish in each concentration needs to be classified by a scale from 0 (no effect) to 1 (slight), 2 (medium), and 3 (severe effects) for this study.

6 In the retrospective analysis of 328 fish acute toxicity tests of an industry laboratory (IL) in Europe and 111 tests of other laboratories (OL) from Europe and the United States, different definitions of the moribund stage lead to a different number of fish declared as moribund. Furthermore, it was shown that different definitions may affect the difference between the LC50moribund and the LC50, because this depends on the number of concentrations with fish declared as moribund. Therefore, a unique definition of the moribund stage needs to be given in the Guideline. 7 This definition has been selected out of five definitions analyzed (1) and appears as the most suitable because: a) it is the easiest to be applied, b) it resulted in a reasonable amount of studies (45% IL; 49% OL) and fish being declared as moribund (13% IL; 10% OL) reducing the suffering up to 72 (IL) and 92 hours (OL), c) it included fewer studies with lower values of LC50moribund versus LC50 (36%), whilst other definitions lead to an increase of this number (36 to 40% IL; 36 to 52% OL) with more fish declared as moribund while they actually survived. No other studies on the moribund state in fish are presently known. 8 Any fish that are exhibiting serious sub-lethal effects are likely to be compromised and hardly ever recover. In the wild, such fish would fall victim to predation; for this reason, and together with the animal welfare concerns, the precautionary approach of using the LC50moribund is justified in chemical risk assessments, although it must be tightly defined. 9 Effects on the class on swimming behaviour include e.g. hyper-excitability, surfacing, sounding, erratic swimming, skittering, diving, spiraling, twitching, apathy, lethargy, weakness, immobility, quiescent as described in Table 2. Similarly, effects on the other classes of sub-lethal effects include the clinical signs shown in the Table for this class. Tumbling fish may be classified as showing both a change in swimming behaviour and loss of equilibrium. Lying on the bottom may be allocated to altered swimming behaviour, but may also show loss of equilibrium.

21

Table 2: Sub-lethal effects classified into 1) loss of equilibrium, 2) swimming behaviour, 3) respiratory function, 4) pigmentation and 5) other clinical signs.

(1) Toth LA (2000) Defining the moribund condition as an experimental endpoint for animal research. ILAR J 41:72-79.

(2) Rufli H (2012) Introduction of moribund category to OECD fish acute test and its effect on suffering and LC50-values. Environ. Toxicol. Chem. 31, 2012.

Swimming Behaviour* Loss of Equilibrium* Respiratory Function* Pigmentation* Other Clinical Signs

Tumbling** Tumbling** Rapid respiration, Strong ventilation,

Hyperventilation

Dark discoloured, Darkended

pigmentation, Increased

pigmentation

Strongly extended gills

Hyperexcitability Partial loss of balance Slow respiration Discouloured Distended abdomen, Abdominal

distension, Bloated, Swollen

abdomenAt surface, Surfacing Complete loss of equilibrium,

Keeling

Laboured respiration Changed colour Convulsions

Lying on the bottom***, Sounding Irregular respiration Mottled Mucus secretion

Erractic swimming Gasping respiration Haemorrhaging

Skittering Gulping respiration Exophthalmus

Diving Coughing Dilated pupils

Spiralling Aggression

Twitching Mouth open

Apathy , Lethargic, Weak

Immobility, Ceased swimming,

Quiescent

* Abnormalities given in the OECD Guideline 203, fish acute toxicity test.

** Tumbling fish may be classified as showing both a change in swimming behaviour and partial loss of equilibrium

*** Lying on the bottom may be allocated to altered swimming behaviour, but may also show a loss of equilibrium