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Administrative data

short-term toxicity to fish
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data from reliable source. Though no detailed test report is available, all tests from the Japanese Authorities are performed according to an OECD guideline and GLP incl. analytics.

Data source

Reference Type:
Information on Environmental Toxicity
NITE (National Institute of Technology and Evaluation) Japan
Bibliographic source:
NITE (National Institute of Technology and Evaluation) Japan, available at:

Materials and methods

Test guideline
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
not specified
probably yes

Test material

Constituent 1
Reference substance name:
EC Number:
EC Name:
Cas Number:
2,3,6-trichlorobenzoic acid
Details on test material:
Analogous: Assessment derived from a susbtance with similar chemical character:
- Name of test material (as cited in study report): 2,3,6-trichlorobenzoic acid

Sampling and analysis

Analytical monitoring:

Test solutions

not specified

Test organisms

Test organisms (species):
Oryzias latipes

Study design

Test type:
other: probably semi-static
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

Details on test conditions:
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.

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.

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.

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.

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.

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.

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.

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.

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.

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.
Reference substance (positive control):
not specified

Results and discussion

Effect concentrations
48 h
Dose descriptor:
Effect conc.:
417 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)

Applicant's summary and conclusion