1
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
2
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
3
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
4
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.
5
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).
6
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.
7
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.
8
- 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
9
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‰.
10
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
11
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
12
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.
13
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
14
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.
15
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).
16
(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.
17
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.
18
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%)
19
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.
20
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
Top Related