Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data from reliablesource. Though no test report is available at present the data from this source are known to be well documented and the test are performed according to GLP and OECD guidelines.

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 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
not specified
probably: yes

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 2
Reference substance name:
Reference substance 001

Sampling and analysis

Analytical monitoring:

Test solutions

not specified

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
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.

Study design

Test type:
not specified
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

Details on test conditions:
The following test vessel and equipment are used for the study.

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.

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.

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.

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.

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.

Exposure period
Perform the exposure period for 48 hr.

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.

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.

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.

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.

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.
Reference substance (positive control):
not specified

Results and discussion

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

Applicant's summary and conclusion