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Ecotoxicological information

Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-11-04 - 2017-04-17
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Also conducted according to GLP.
Justification for type of information:
Study run in 2017 for the purpose of a Chinese registration which requires an acute toxicity test specifically on the fish species Gobiocypris rarus.
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Principles of method if other than guideline:
In addition, the following guidance document was followed:
Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000
GLP compliance:
Analytical monitoring:
Details on sampling:
For the analysis of the actual test substance concentration, 2mL of test solution was taken from each test vessel at 0, 24, 48, 72 and 96 hours after test initiation.
The samples were analyzed directly without storage.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A nominal concentration at 100mg/L of the test substance was prepared and stirred (via magnetic stirrer) in the dark for 72h followed by filtration using a 0.45 µm nitrocellulose membrane to make a saturated solution of the test substance in the test water (referred to as "saturated solution").
Test organisms (species):
other: Gobiocypris rarus
Details on test organisms:
- Common name: Chinese rare minnow
- Source: In-house culture
- Length at study initiation (length definition, mean, range and SD): 2.3 +/- 0.2 cm
- Weight at study initiation (mean and range, SD): 0.23 +/- 0.06 g

- Acclimation period: at least 14 days
- Acclimation conditions (same as test or not): yes
- Type and amount of food during acclimation: Fed brine shrimp flakes
- Health during acclimation (any mortality observed): No diseases or mortality observed

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
Test temperature:
21.5 - 22.1 °C
7.87 - 8.36
Dissolved oxygen:
70.2 - 94.1 %ASV
Nominal and measured concentrations:
Nominal: 100 mg/L
Measured: 0.06 mg/L (time-weighted mean measured concentration)
Details on test conditions:
- Test vessel: 5L glass tanks
- Material, size, headspace, fill volume: Fill volume was 4L
- Renewal rate of test solution: 24h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.6 g fish/L

- Source/preparation of dilution water: Dechlorinated tap water (by activated carbon and sterilized by UV lamp)

- Adjustment of pH: No
- Photoperiod: 12/12

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality and behavioural abnormalities were assessed at 0, 3, 24, 48, 72 and 96h.

- Spacing factor for test concentrations: Not applicable, limit test
- Justification for using less concentrations than requested by guideline: Limit test
Range finding study
- Test concentrations: 100 mg/L loading rate
- Results used to determine the conditions for the definitive study: The measured concentration of the test substance was not stable in the test system, with a decreasing concentration over time up to no substance detected after 96 hours. Therefore a semi-static method (renewal every 24h) was used for the definitive test. No mortalities or abnormalities observed.
Reference substance (positive control):
Potassium dichromate
96 h
Dose descriptor:
Effect conc.:
> 0.06 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: No toxicity at the limit of solubility.
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
There were no mortalities and abnormalities observed in the fish of the control group and the treatment group during the test.
Results with reference substance (positive control):
96h-LC50 243 mg/L with 95% confidence limits of 194 and 297 mg/L.
This is within the ± 2SD range of the QC chart (81.6-307mg/L).
Sublethal observations / clinical signs:

Measured concentrations of the test substance in the limit test

Group Blank Control Treatment group (Saturated solution) Before vs After renewal
Measured concentration 0h Not detected 0.0934 -52.5
24h Before renewal Not detected 0.0444
After renewal Not detected 0.0802 -48.1
48h Before renewal Not detected 0.0416
After renewal Not detected 0.0886 -40.6
72h Before renewal Not detected 0.0526
After renewal Not detected 0.0894 -53.7
96h Not detected 0.0414
Time-weight mean (mg/L) - 0.064
Variation of the new solutions (%) -  -8.76 to 6.26

Mortalities and abnormalities in the limit test (10 fish in each concentration)

Group Duration (h) Mortalities Cumulative mortalities Abnormalities
Control 0 0 0 None
3 0 0 None
24 0 0 None
48 0 0 None
72 0 0 None
96 0 0 None
Treatment Group 0 0 0 None
3 0 0 None
24 0 0 None
48 0 0 None
72 0 0 None
96 0 0 None
Validity criteria fulfilled:
There was no acute toxicity of the saturated solution in the limit test. The test results showed that the 96h LC50 for the test substance 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) to Gobiocypris rarus was higher than the concentration of the saturated solution (the time-weighted mean measured concentration was 0.0640 mg/L) under the tested conditions. Therefore, 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) was not harmful to the tested fish species under the present experimental conditions.
Executive summary:

A study was performed to assess the acute toxicity of the test item to fish using the species Gobiocypris rarus. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 203 also the Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000 under GLP conditions.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF).Ten fish were exposed to a Water Accommodated Fraction (WAF) of the test item at a single nominal loading rate of 100 mg/L for 96 hours at a temperature of ca. 22 °C under semi-static test conditions. The number of dead fish and any adverse reactions to exposure were recorded after 3, 24, 48, 72 and 96 hours.

The 96 -Hour LL50 for fish based on nominal loading rates was greater than 100 mg/L loading rate, equivalent to greater than 0.06 mg/L (Time-weighted average) based on measured concentrations (LC50). This indicates that, in the range of its water solubility, the test item was not toxic under the test conditions.

Description of key information

LC50 (96h) > 0.06 mg/L (measured) for Chinese rare minnow (OECD 203).

No toxicity observed at the limit of solubility of the test substance.

The same observation was done in a 1991 study on Oncorhynchus mykiss (OECD 203) although a solubilizer was used and so the fish were exposed to concentrations above the limit of solubility of the test substance.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

Two studies are available on the short-term toxicity of 4,4´-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS No. 107934-68-9) to fish. Both studies were performed according to OECD guideline 203 and GLP, with Chinese rare minnow or Rainbow trout as test organism. The fish were exposed to the substance for 96 hours under semi-static conditions. Both tests were conducted as limit tests with a measured test substance concentration of 0.06 mg/L (Chinese rare minnow, no solubilizer, loading rate 100 mg/L) and 6.5 mg/L (Rainbow trout, solubilizer, loading rate 10 mg/L). During the 96 hours test duration no effects on the test fish were observed.