Algal Growth Inhibition Test, Daphnia Acute Immobilization Test,
and Fish Acute Toxicity Test

I. Scope of application
A standard method for the algal growth inhibition test, Daphnia acute immobilization test or fish acute toxicity test of chemicals is described here.

II. Definitions

Objective
The definitions of the terms used in this test method are given below.

1. Test system
   

• Static test: A test performed without replacing the test solution in the test vessel throughout the exposure period.

• Semi-static test: A test performed by replacing the test solution in the test vessel with a new test solution like a batch system at certain intervals (e.g., 24 hr).
• Flow-through test: A test performed by constantly and automatically replacing the test solution in the test vessel with a new test solution while draining the old solutions.

2. End-points
   

• LC₅₀ in this Test Guideline is the median lethal concentration, i.e. that concentration of the test substance in water which kills 50 per cent of a test batch of fish within a particular period of exposure (which must be stated).

• EC_x is the concentration of the test substance dissolved in test medium that results in an x % (e.g. 50%) reduction growth, mobility, reproduction, etc., of test organism within a stated exposure period.
• LOEC is the lowest tested concentration at which the substance is observed to have a statistically significant (at p < 0.05) when compared with the control, within a given exposure time. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. When these two conditions cannot be satisfied, a full explanation must be given for how the LOEC (and hence the NOEC) has been selected.
 
• NOEC is the test concentration immediately below the LOEC and the highest tested concentration at which the substance is not observed to have a statistically significant harmful effect (at p < 0.05) when compared with the control, within a given exposure time.

III. General rules

1. Basic idea of the test
   Toxicity tests using algae, Daphnia sp. or fish are intended for determining the toxicity of test substances by exposing the test organisms to the test substances dissolved in medium or dilution water (hereinafter referred to as "medium, etc."). Therefore, the solubility of the test substance to the medium, etc. under the test condition must be checked before conducting the test. Moreover, a reliable method for quantifying the test substance in the test solution must be available.
   Wherever possible, the test conditions should be kept constant throughout the exposure period. For example, the test substance concentration is preferably maintained at least 80% of the initial concentration (i.e., the nominal concentration or measured concentration at the beginning of the exposure). Information on the test substance which may be useful in establishing the test conditions includes structural formula, purity, stability in light stability under the conditions of the test pKa, octanol water partition coefficient (Pow), vapor pressure, and results of transformation including biodegradability in water. Test substances with large vapor pressures may be lost during the exposure period, so it is suggested to obtain the Henry constant that serves as the index of the loss. The Henry constant can be calculated from the solubility and the vapor pressure.

   　

2. Preparation of the test solution
   To prepare a test solution of each concentration, directly dissolve the required amount of the test substance in the medium, etc., or prepare a stock solution of the test substance at an appropriate concentration and dilute it with the medium, etc. Prepare the stock solution without using any vehicle, but if it is difficult to dissolve the test substance in water or the medium, etc., perform mechanical dispersion by means of ultrasonic wave, etc., or use a low-toxicity vehicle (which hereinafter refers to a solvent or dispersant) such as an organic solvent. However, as a rule, do not use any surfactant. Set the test concentrations so that they do not exceed the solubility (hereinafter referred to as "solubility limit") of the test substance to the medium, etc. under the test conditions.
   When using a vehicle, additionally establish a vehicle control containing the vehicle at the same concentration as that used for preparing the test concentrations. In principle, the concentration of the vehicle must not exceed 100 mg/L and must be constant among all the test concentrations. Note that the obtained test results may be attributed to the test substance itself or its interactive effect with the vehicle.

   　

3. Handling of water-insoluble substances
   Even if the test substance is insoluble in water, basically avoid using any dispersant. Set the test concentrations so that they do not exceed the solubility limit of the test substance. However, if the test substance has an extremely low solubility in the medium, etc. and its solubility limit cannot be determined by usual methods, and if test concentrations above the solubility limit are inevitable for determining the toxicity values such as LC50, perform the test using a dispersed system. If such test substances are intended to be used with dispersants or emulsifiers, perform the test using a dispersant.
   Before concluding that the toxicity values such as LC50 cannot be determined at concentrations within the soluble or dispersible limit of the test substance in the medium, etc., take every possible measure for dissolving or dispersing the test substance in the medium, etc. and determine the upper limit of the concentration at which the test substance can be dissolved or dispersed in the medium, etc.

IV. Algal growth inhibition test

Objective
The purpose of the present study is to determine the toxicity of the test substance against algal growth by exposing algae at the exponential growth phase to the test substance and measuring the growth inhibition rate against the control. Growth is quantified from measurements of the algal biomass as a function of time.

1. Test organism
   The recommended species is Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), but other species such as Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus) may also be used. If species other than these two are used, it must be confirmed that their exponential growth can be maintained throughout the exposure phase.

2. Test vessel and equipment
   The following test vessel and apparatus are used for the study.

   2-1. Test vessel
   Test vessels and other apparatus which will come into contact with the test solutions should be made entirely of glass or other chemically inert material. The test vessel has a sufficiently large surface exposed to air. For example, a 250 mL conical flask is suitable for holding a 100 mL test solution. If the test substance is volatile, take appropriate measures such as using sealable flasks.

   2-2. Culture apparatus
   Culture the test organisms in an incubator or incubation chamber where constant temperature and lighting conditions can be maintained.

   2-3. Cell counter
   Cell count, which is the most frequently used surrogate parameter for algal biomass, may be made using an electronic particle counter, a microscope with counting chamber. Other biomass surrogates can be measured using a fluorimeter, spectrophotometer or colorimeter. In order to provide useful measurements at low biomass concentrations when using a spectrophotometer, it may be necessary to use cuvettes with a light path of at least 4 cm.

3. Growth media
   The recommended composition of the medium is as follows.

• Ammonium chloride 15 mg/L

• Magnesium chloride hexahydrate 12 mg/L
• Calcium chloride dehydrate 18 mg/L
• Magnesium sulfate heptahydrate 15 mg/L
• Potassium dihydrogen phosphate 1.6 mg/L
• Ferric chloride (III) hexahydrate 0.064 mg/L
• Disodium ethylene diamine tetraacetiate dihydrate 0.1 mg/L
• Boric acid 0.185 mg/L
• Manganese chloride tetrahydrate 0.415 mg/L
• Zinc chloride 0.003 mg/L
• Cobalt chloride hexahydrate 0.0015 mg/L
• Copper chloride dihydrate 0.00001 mg/L
• Disodium molybdate dihydrate 0.007 mg/L
• Sodium hydrogen carbonate 50 mg/L

The pH of this Media will be 8.1 in the equilibrium condition with atmosphere.

4. Preculture
   In order to adapt the test alga to the test conditions and ensure that the algae are in the exponential growth phase when used to inoculate the test solutions, an inoculum culture in the test medium is prepared 2-4 days before start of the test. The algal biomass should be adjusted in order to allow exponential growth to prevail in the inoculum culture until the test starts.
   　
5. Test solution
   To prepare a test solution of each concentration, directly dissolve the required amount of the test substance in the medium, or prepare a stock solution of the test substance at an appropriate concentration and dilute it with the medium. Follow the descriptions in "Preparation of the test solution" under "III. General rules."
   　
6. Test condition
   6-1. Exposure period
   Exposure period is normally 72 hours.

   6-2. Initial biomass concentration
   The initial biomass in the test cultures must be the same in all test cultures and sufficiently low to allow exponential growth throughout the incubation period. The initial biomass should not exceed 0.5 mg/L as dry weight. The following initial cell concentrations are recommended:
   Pseudokirchneriella subcapitata: 5 x 10³ - 10⁴ cells/mL
   Desmodesmus subspicatus 2-5 x 10³ cells/mL
   For other species, adjust the initial biomass concentration to achieve an equivalent dry weight.

   6-3. Test concentration
   For the final definitive test at least five concentrations, arranged in a geometric series, should be selected. The concentration series should preferably cover the range causing 0-75 % inhibition of algal growth rate. Concentrations of 100 mg/L or higher do not need to be tested. Perform a control, and additionally a vehicle control if using any vehicle.

   6-4. Number of replicates
   Perform a test with at least three replicates at each test concentration including the control(s). The number of control replicates of six is recommended (e.g. three control replicates and vehicle control in six replicates when using any vehicle). each test concentration in 3 replicates. It is recommended to perform the control in 6 replicates.

   6-5. Incubation
   

   • Temperature: The cultures should be maintained at a tempareture in the range of 21 to 24°C, controlled at ± 2°C.
   • Illumination: 60 - 120μE/m²/s (The surface where the cultures are incubated should receive continuous, uniform fluorescent illumination e.g. of <<cool-white>> or <<daylight>> type.)
   • Incubation: Cap the test vessels with air-permeable stoppers. The vessels are shaken and placed in the culturing apparatus. During the test it is necessary to keep the algae in suspension and to facilitate transfer of CO₂. To this end constant shaking or stirring should be used.

7. Beginning of the exposure to the test substance
   Start the exposure by inoculating each test vessel with the algae that have been precultured to the initial biomass concentration established according to 6-2.
   　
8. Measurement of biomassCell density measurement
   Measure the biomass in each test vessels at least at 24, 48 and 72 hr after the beginning of the exposure. Use sterilized medium as a background of the particle counter or a blank of the spectrophotometer, etc.
   　

9. Measurements of test substance concentration
   9-1. Analysis of the concentration of the test substance
   At the beginning and end of the test, analyze the concentrations of the test substance at least in the lowest and highest test concentration and the concentration around the predicted EC50. If it is predicted that the concentrations of the test substance decreases from the nominal concentration by 20% or more during the exposure period, it is recommended to analyze all test concentration at the beginning and end of the test. Furthermore, for volatile or adsorbing test substances or those that are likely to be greatly decreased during the exposure period, additional sampling for analysis at 24 hr intervals during the exposure period are recommended.

   9-2. Measurement of the test condition
   Measure the pH of the test solution at the beginning and end of the test. Generally, the variation of the pH in the control must not exceed 1.5 units during the test.

10. Limit test
    Under some circumstances, e.g. when a preliminary test indicates that the test substance has no toxic effects at concentrations up to 100 mg/L or up to its limit of solubility in water (whichever is the lower), a limit test can be performed at this concentration to demonstrate that NOEC, etc., is higher than this concentration. All previously described test conditions and validity criteria apply to a limit test, with the exception that the number of treatment replicates must be at least six. The average specific growth rate in the control (in vehicle control if using any vehicle) and treatment group may be analyzed using a statistical test to compare means, e.g. a Student's t-test.
    　

11. Validity of the test
    For the test to be valid, the following performance criteria should be met for Pseudokirchneriella subcapitata and Desmodesmus subspicatus.

    • The biomass in the control (also in the vehicle control) cultures should have increased exponentially by a factor of at least 16 within the exposure period.
    • The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control (also in the vehicle control) cultures must not exceed 35%.
    • The coefficient of variation of average specific growth rates during the whole test period in replicate control (also in vehicle control) cultures must not exceed 7% in tests.

12. Analysis of the results
    12-1. Processing of the results
    In principle, analysis of the results must be based on appropriate averages of measured values. If there is evidence that the concentration of the substance being tested has been satisfactorily maintained within ± 20 % of the nominal or measured initial concentration throughout the test, analysis of the results can be based on nominal or measured initial values.
    Tabulate the estimated biomass concentration in test cultures and controls (including vehicle control) together with the concentrations of test material and the times of measurement to produce plots of growth curves. Using the plots, examine whether control (including vehicle control) cultures grow exponentially at the expected rate throughout the test.
    It is recommended to obtain a quantitative concentration-response relationship of the test substance using the methods described in 12-2.

    12-2. Comparison of growth rates
    The average specific growth rate for a period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:

    
    

    where,
    μ_i-j is the average specific growth rate between time i to j;
    X_i is the biomass at time i;
    X_j is the biomass at time j;
    t_i is the time (d) of i^th biomass measurement after beginning the exposure;
    t_j is the time (d) of j^th biomass measurement after beginning the exposure

    
    For calculating EC₅₀, determine the average specific growth rate over the entire test duration (normally days 0-3).
    The growth rate can also be obtained from the slope of the regression line drawn by plotting the logarithmic values of the biomass measurements.
    Calculate the percent inhibition of growth rate for each treatment replicate from equation:

    
    　

    where:

    I_μ is the percent inhibition in average specific growth rate;
    μ_C is the mean value for average specific growth rate (μ) in the control (in vehicle control if using any vehicle) group;
    μ_T is the average specific growth rate for the treatment replicate

    12-3. Calculation of the EC₅₀ and NOEC
    Plot the values for I_μ against the logarithmic value of the test substance concentration. Using the regression formula, etc., determine the 50% inhibition concentration. The EC₅₀ values obtained using I_μ is referred to as ErC₅₀.
    The values μ_0-3d of the control (vehicle control if using any vehicle) and each test concentration are further subjected to an appropriate method for analysis of variance (ANOVA) and a multiple comparison method to determine the NOEC.

13. Summary of the results
    Summarize the test results in Form 7 and attach it to the final report.

    　

V. Daphnia Acute Immobilization Test

Objective
The purpose of the present study is to determine the toxicity of the test substance against the mobility of Daphnia spp. by exposing them to the test substance for 48 hr and measuring the immobilization rate against the control. The present study defines an organism as being immobilized when it does not move for 15 sec after the test vessel is gently shaken.

1. Test organism
   The recommended species is Daphnia magna, but other Daphnia species such as Daphnia pulexs may also be used.
   Use organisms that are 24 hr old or younger at the beginning of the exposure. To reduce variation, do not use the first offsprings of the parents. The test organisms must be obtained from healthy parents (i.e., showing no sign of stress under the culture conditions such as a high mortality, the appearance of males or oostegites, a prolonged period before the first offsprings and discoloration) of the same strain.
   The parent organisms must be cultured under conditions (light, temperature and water) as same as those employed in the test. When performing the test using water different from that usually used for culturing Daphnia spp., establish an acclimatization period before beginning the exposure. The acclimatization can be performed by culturing the organisms in the material water at the test temperature for at least 48 hr before beginning the exposure. Use the offsprings obtained from acclimatized parents for the test.

2. Test vessel and equipment
   The following test vessel and equipment are used for the study.

   2-1. Test vessel
   The test vessel or other instrument that contacts the test solution must be made of glass or other chemically inert materials. Cap the vessel loosely to prevent evaporation and dust contamination.
   If the test substance is volatile, perform the test in a sealed system. Use a sufficiently large vessel to prevent shortage of dissolved oxygen.

   2-2. Instrument
   Use a dissolved oxygen meter (a microelectrode or other instrument suitable for measuring the dissolved oxygen concentration with a small amount of sample), a pH meter, an appropriate instrument for controlling temperature, etc., for the test.

3. Material water
   Use water suitable for culturing and testing Daphnia spp. It can be natural water (surface water or groundwater), dechlorinated tap water or artificially prepared water (e.g., Appendix Table 1), but must satisfy the conditions listed in Appendix Table 2.
   Do not use Elendt M4 or M7 media or water containing chelating agents for testing metal-containing substances. The water hardness should be 250 mg/L or smaller in terms of calcium carbonate concentration, and the pH should be 6-9.
   Aerate the material water before using it for the test.

4. Test solution
   To prepare a test solution of each concentration, directly dissolve the required amount of the test substance in the material water, or prepare a stock solution of the test substance at an appropriate concentration and dilute it with the material water. Follow the descriptions in "Preparation of the test solution" under "III. General rules".
   Perform the test without adjusting the pH. If the pH of the material water is not within the range of 6-9, it is recommended to perform an additional test after adjusting the pH to that observed prior to the addition of the test substance. Perform the pH adjustment through a method causing no change in the concentration, chemical reaction or precipitation of the test substance. Preferably, use HCl or NaOH for the pH adjustment.

5. Test conditions
   5-1. Test method
   The test can be performed under a static, semi-static or flow-through condition. If the test substance concentration is unstable, a semi-static or flow-through test is recommended.

   5-2. Exposure period
   Perform the exposure period for 48 hr.

   5-3. Volume and number of test organisms

   • Volume: Use at least 2 mL or the test solution per organism.
   • Number of test organisms: Use at least 20 organisms for each of the test concentrations and the control. Preferably, divide the organisms into 4 groups of 5 organisms.

   5-4. Test concentrations
   Adopt a concentration range comprising at least 5 concentrations that are setup in a geometric progression, preferably at a geometric ratio of within 2.2. The highest test concentration preferably induces 100% immobilization, but concentrations of 100 mg/L or higher do not need to be tested. Preferably, no effect is observed at the lowest concentration.
   Perform a control, and additionally an auxiliary control if using any auxiliary.

   5-5. Culture method

   • Illumination: The photoperiod is preferably set to 16 hr light and 8 hr dark. The test can be conducted in dark if the test substance is unstable against light.
   • Temperature: The temperature is set within the range of 18°C - 22°C, with variations among the test vessels of ± 1.0°C.
   • Dissolved oxygen concentration: It must be kept at 3 mg/L or higher. In principle, do not perform aeration during the exposure period.
   • Feeding: Do not feed the organisms.

6. Beginning of the exposure to the test substance
   Start the exposure by transferring a specified number of organisms established in 5-3. to each test vessel.

7. Observation
   Observe the mobility of the organisms at least twice, i.e., at 24 and 48 hr after the beginning of the exposure. The organisms are considered as being immobilized when they do not move for 15 sec after the test vessel is gently shaken. During observation, record any anomaly in behavior or appearance besides immobilization.

8. Measurements of test substance concentration, etc.
   8-1. Test substance concentration measurement
   At the beginning and end of the exposure, measure the test substance concentrations at least in the lowest and highest test concentration groups. If it has been predicted that the test substance concentration decreases from the initial concentration by 20% or more during the exposure period, it is recommended to take measurements for all test concentration groups at the beginning and end of the exposure. Furthermore, for volatile or adsorptive substances or those that are likely to be greatly decreased during the exposure period, additional measurements are recommended at 24 hr intervals during the exposure period.
   In a semi-static test, perform at least two sets of measurements, provided that the measurements taken immediately after the water renewal and immediately before the next renewal are counted as one set.

   8-2. Test condition measurement
   Measure the dissolved oxygen concentration and the pH in the control and at the highest test concentration at the beginning and end of the exposure period. The water temperature in the control should be measured at least twice, i.e., at the beginning and end of the exposure period, but to monitor the variation in the water temperature, it is recommended to continuously measure the atmospheric temperatures in the control or in the surrounding atmosphere, etc., and record their variations. Generally, the variation of the pH must not exceed 1.5 during the exposure period.

9. Limiting test
   If the test substance concentration of 100 mg/L or at the water solubility limit, whichever is lower, is predicted to show no toxicity, a limiting test can be performed at this concentration to demonstrate that EC₅₀ is higher than this concentration. Use 20 organisms (preferably divided into 4 groups of 5 organisms) for the limiting test and also for the control. If the immobilization rate has exceeded 10% at the end of the exposure, perform a regular test. Record any abnormal behavior observed.

10. Validity of the test
    The test is considered valid if it satisfies the following conditions.
    • The control group shows an immobilization rate of 10% or less and no organism floating on the surface.
    • The dissolved oxygen concentration is 3 mg/L or higher at the end of the exposure.

11. Calculation of the results
    In principle, perform the calculation of the results based on appropriate averages of the test substance concentration measurements. If it can be demonstrated that the test substance concentration was kept within ± 20% of the initial concentration throughout the exposure period, the calculation of the results can be performed based on the initial concentration.
    Tabulate the immobilization rate for the individual test concentrations and the control together with the exposure period and test substance concentrations. Plot the immobilization rate at 24 hr and 48 hr against the test concentrations. Then, using the probit method or other appropriate statistical method, determine the slope of the regression line at 95% confidence limit and the EC₅₀ for 48 hr exposure.
    If the obtained data are insufficient for performing statistical calculations, estimate EC₅₀ as the geometric average of the highest test concentration inducing no immobilization and the lowest test concentration inducing 100% immobilization.

12. Summary of the results
    Summarize the test results in Form 8 and attach it to the final report.

    　

Appendix Table 1. Artificially prepared water

(1) ISO test water

(a) Calcium chloride solution
Dissolve 11.76 g calcium chloride dihydrate in dilution water and add dilution water up to 1 L.

(b) Magnesium sulfate solution
Dissolve 4.93 g magnesium sulfate heptahydrate in dilution water and add dilution water up to 1 L.

(c) Sodium hydrogencarbonate solution
Dissolve 2.59 g sodium hydrogencarbonate in dilution water and add dilution water up to 1 L.

(d) Potassium chloride solution
Dissolve 0.23 g potassium chloride in dilution water and add dilution water up to 1 L.

Mix 25 mL each of solutions (a)-(d) and make up to 1 L with dilution water.
Use appropriate purified water (e.g., ion-exchanged water, distilled water or reverse osmosis water) with an electric conductivity of 10 μS/cm or lower as the dilution water. All reagents must be of analytical grade.

(2) Elendt M4 and M7 media
Prepare each medium by adding the medium stock solutions I (microelements) and medium stock solutions II (principal components) to dilution water (use appropriate purified water, e.g., deionized water, distilled water or reverse osmosis water).

(a) Preparation of the medium stock solutions I
Prepare the medium stock solution I of each substance shown in the left column in Table 1 by dissolving the amount shown in the second column in Table 1 in 1 L of dilution water. Prepare the ferrous ethylene diamine tetraacetate solution (II) by mixing the sodium ethylene diamine tetraacetate dihydrate and ferrous sulfate heptahydrate, which have been separately prepared, and immediately autoclaving them.
After preparing the medium stock solution I of each substance, take the amount shown in the third column in Table 1 from each solution, mix them and add dilution water up to 1 L to achieve "medium stock solution I mixture."

Table 1. Components of the medium stock solutions I and their amounts, etc.

*Final dilution ratio: The final dilution ratio of the medium stock solution I in Elendt M4 or M7 medium.

(b) Preparation of the medium stock solutions II
Except for the medium stock solution I mixture, prepare the medium stock solution II of each substance shown in the left column in Table 2 by dissolving the amount shown in the second column in Table 2 in 1 L of dilution water. After preparing the vitamin mixture stock solution, freeze them in small portions and add them to the medium immediately before use.

(c) Preparation of each medium
Prepare each medium by taking the amount shown in the third column in Table 2 from each medium stock solution II, mixing them and adding dilution water up to 1 L. When preparing each medium, add these amounts of medium stock solutions to about 500 - 800 mL dilution water and further add dilution water up to 1 L to avoid precipitation of salts.

Table 2. Components of the medium stock solutions II and their amounts (For both Elendt M4 and M7)

Appendix Table 2. Chemical conditions of the material water

VI. Fish acute toxicity test

Objective
The purpose of the present study is to determine the toxicity of the test substance against fish by exposing them to the test substance for 96 hr and measuring the mortality.

1. Test organism
   The recommended species is Ricefish (Oryzias latipes), but other fish species listed in Appendix Table 1 may also be used. Use healthy fish showing no abnormal appearance. In each test, the size of the organisms should be as uniform as possible.

Appendix Table 1.

Fish species	Recommended test temperature (°C)	Recommended length of test fish (cm)
Danio rerio Zebra-fish	21 - 25	2.0 ± 1.0
Pimephales promelas Fathead minnow	21 - 25	2.0 ± 1.0
Cyprinus carpio Common carp	20 - 24	4.0 ± 2.0
Oryzias latipes Ricefish	21 - 25	2.3 ± 1.2
Poecilia reticulata 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

2. Test vessel and equipment
   The following test vessel and equipment are used for the study.

   2-1. Test vessel
   The test vessel or other instrument that contacts the test solution must be made of glass or other chemically inert materials. Use a test vessel of an appropriate size in regard to the recommended volume. Cap the vessel loosely to prevent evaporation and dust contamination.
   If the test substance is volatile, perform the test in a sealed system. Use a sufficiently large vessel to prevent shortage of dissolved oxygen.

   2-2. Instrument
   Use a dissolved oxygen meter and an appropriate instrument or apparatus for controlling the temperature for the test.

3. Material water
   Use water suitable for culturing and testing the fish. It can be natural water (surface water or groundwater), dechlorinated tap water or artificially prepared water (see the note). The recommended total hardness is 10 - 250 mg/L in terms of calcium carbonate concentration, and the recommended pH is 6.0 - 8.5. Prepare the artificially prepared water using reagents of analytical grade and deionized or distilled water having an electric conductivity of 10 μS/cm or lower.

4. Acclimatization
   All test fish must be acquired at least 12 days before the test and acclimatized. After monitoring the fish for 48 hr, acclimatize them in water used for the test under the following conditions for at least 7 days before beginning the exposure. If possible, avoid using medicated bath after the monitoring period.
   • Illumination: 12 - 16 hr light per day
   • Temperature: Appropriate temperature for the species tested (see Appendix Table 1 under VI.)
   • Oxygen concentration: At least 80% of the saturated oxygen concentration
   • Feeding: Daily or three times per week until 24 hr before beginning the exposure.

   Record the mortality during the acclimatization period and apply the following conditions to the test fish.

   • If the overall mortality for seven consecutive days during the acclimatization period exceeds 10%, do not use them for the test.
   • If the overall mortality for seven consecutive days during the acclimatization period falls within 5 - 10%, extend the acclimatization period for 7 days.
   • If the overall mortality for seven consecutive days during the acclimatization period is below 5%, use them for the test.

5. Test solution
   To prepare a test solution of each concentration, directly dissolve the required amount of the test substance in the material water, or prepare a stock solution of the test substance at an appropriate concentration and dilute it with the material water. Follow the descriptions in "Preparation of the test solution" under "III. General rules."
   Perform the test without adjusting the pH. If the pH of the material water is greatly changed by the addition of the test substance, it is recommended to perform an additional test after adjusting the pH to that observed prior to the addition of the test substance. Perform the pH adjustment through a method causing no change in the concentration, chemical reaction or precipitation of the test substance. Preferably, use HCl or NaOH for the pH adjustment.

6. Test conditions
   6-1. Test method
   The test should be performed under a flow-through or semi-static condition. If the test substance concentration is unstable, a flow-through test is recommended.

   6-2. Exposure period
   Perform the exposure period for 96 hr.

   6-3. Volume and number of test fish
   

   • Volume: A maximum density of 1.0 g fish weight/L is recommended for a semi-static test. The volume can be larger in a flow-through test.
   • Number of test fish: Use at least 7 fish for each of the test concentrations and the control.

   6-4. Test concentrations
   Adopt a concentration range comprising at least 5 concentrations that are setup in a geometric progression, preferably at a geometric ratio of within 2.2. The highest test concentration preferably gives fatal effects on all individuals tested, but concentrations of 100 mg/L or higher do not need to be tested. Preferably, no effect is observed at the lowest concentration.
   Perform a control, and additionally an auxiliary control if using any auxiliary.

   6-5. Culture method
   

   • Temperature: The temperature is set within the appropriate range for the species tested (see Appendix Table 1 under VI.), and kept constant with a variation of ± 2°C.
   • Illumination: The photoperiod is set to 12 - 16 hr light per day.
   • Dissolved oxygen concentration: It must be kept at 60% of the saturated oxygen concentration or higher. Aeration may be performed unless it causes a great loss of the test substance.
   • Feeding: Do not feed the organisms.
   • Disturbing factor: Avoid any factor that should disturb the fish behavior.

7. Beginning of the exposure to the test substance
   Start the exposure by transferring a specified number of acclimatized fish established in 6-3 to each test vessel.

8. Observation
   Observe the appearance and behavior of the fish at least at 24, 48, 72 and 96 hr after the beginning of the exposure. Organisms showing no motion (e.g., opercular movements) and no response when touched at their peduncles are considered as being dead. Remove dead organisms and record the mortality. Additional observations at 3 and 6 hr after the beginning of the exposure are also recommended. Record any anomaly in balance, swimming behavior, respiratory function or body color.

9. Measurements of test substance concentration, etc.
   9-1. Test substance concentration measurement
   At the beginning and end of the exposure, measure the test substance concentrations at least in the lowest and highest test concentration groups. If it has been predicted that the test substance concentration decreases from the initial concentration by 20% or more during the exposure period, it is recommended to take measurements for all test concentration groups at the beginning and end of the exposure. Furthermore, for volatile or adsorptive substances or those that are likely to be greatly decreased during the exposure period, additional measurements are recommended at 24 hr intervals during the exposure period.
   In a semi-static test, perform at least two sets of measurements, provided that the measurements taken immediately after the water renewal and immediately before the next renewal are counted as one set.

   9-2. Test condition measurement
   Measure the pH, dissolved oxygen concentration and water temperature at least once daily.

10. Limiting test
    If the test substance concentration of 100 mg/L or at the water solubility limit, whichever is lower, is predicted to show no fatality, etc., a limiting test can be performed at this concentration to demonstrate that LC₅₀ is higher than this concentration. Use at least 7 organisms for the limiting test and also for the control. If any dead fish is observed by the end of the exposure, perform a regular test. Record any sublethal effects observed.

11. Validity of the test
    The test is considered valid if it satisfies the following conditions.
    
    • The mortality in the control group does not exceed 10% (no more than one fish dead when using less than 10 individuals).
    • The dissolved oxygen concentration is kept at 60% of the saturated oxygen concentration or higher throughout the exposure period.
    • A sufficient level of the test substance concentration is kept throughout the exposure period.

12. Calculation of the results
    In principle, perform the calculation of the results based on appropriate averages of the test substance concentration measurements. If it can be demonstrated that the test substance concentration was kept within ± 20% of the initial concentration throughout the exposure period, the calculation of the results can be performed based on the initial concentration.
    Tabulate the cumulative mortalities for the individual test concentrations and the control together with the exposure period and test substance concentrations. On a logarithmic-normal probability paper, plot the cumulative mortality during the exposure period against the test concentrations. Then, using the probit method or other appropriate statistical method, determine the slope of the regression line at 95% confidence limit and the LC₅₀ for 96 hr exposure.
    If the obtained data are insufficient for performing statistical calculations, estimate LC₅₀ as the geometric average of the highest test concentration inducing no mortality and the lowest test concentration inducing 100% mortality.

13. Summary of the results
    Summarize the test results in Form 9 and attach it to the final report.

Note: Artificially prepared water
Composition designated in OECD (ISO6341-1982)

(a) Calcium chloride solution

Dissolve 11.76 g calcium chloride dihydrate in deionized water and add deionized water up to 1 L.

(b) Magnesium sulfate solution

Dissolve 4.93 g magnesium sulfate heptahydrate in deionized water and add deionized water up to 1 L.

(c) Sodium hydrogencarbonate solution

Dissolve 2.59 g sodium hydrogencarbonate in deionized water and add deionized water up to 1 L.

(d) Potassium chloride solution

Dissolve 0.23 g potassium chloride in deionized water and add deionized water up to 1 L.
Mix 25 mL each of solutions (a)-(d) and add deionized water up to 1 L. The total amount of calcium and magnesium ions in this solution is 2.5 mmol/L. The ionic ratio of calcium to magnesium is 4:1, and the ionic ratio of sodium to potassium is 10:1.
The electric conductivity of the deionized water must not exceed 10 μS/cm. All reagents must be of analytical grade.
Aerate the artificially prepared water until the dissolved oxygen concentration reaches saturation. Store the water without aeration for about 2 days until use.