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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 March - 30 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Date of production: 28 July 2015
Expiry date: 28 July 2020
Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentration, samples were taken from the treatment group and the control at the start and at the end of each renewal period.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Method: As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). The following test item amounts were suspended in 5000 mL of the dilution water (ISO Medium): 0.5006 g in the first, 0.5005 g in the second, 0.5003 g in the third and 0.5008 g in the fourth renewal period. The loading rate of the test solutions was 100 mg test item/L at the start of each renewal period. The solution was handled by ultrasonic bath for 10 minutes thereafter stirred for a period of approximately 24 hours to achieve equilibrated concentration. The solution was filtrated through a membrane filter (0.45 μm2) to separate the possible nondissolved test material.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Akvárium Magazin Kft. (Pasaréti Gyula), 1222 Budapest, Dévény u. 36., Hungary
- Number of animals: 10 animals at each group (no replicates used)
- Age and size of animals: Juveniles; size within a range of 2.0 ± 1 cm
- Loading: The loading of the test aquaria did not exceed 1.0 g / L
- Sex: Both female and male (not separated)
- Animal health: Apparently healthy animals were used in the test

ACCLIMATION
Fish were held for at least 12 days before test initiation in the fish laboratory under the same conditions as used during the exposure period. During holding, fish were fed with appropriate, commercial diet for fish at least three times per week until 24 hours before the test started. The fish were not fed during the test. The health of the breeding was continuously monitored and any mortality or abnormal behaviour was recorded.
The mortalities were recorded for seven days immediately before the test and the following criteria were applied:
- Mortalities of greater than 10 per cent of population in seven days: Rejection of entire batch
- Mortalities of between 5 and 10 per cent of population: Acclimatisation continued for seven additional days
- Mortalities of less than 5 per cent of population: Acceptance of batch
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
The fish were observed at approximately 4, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality. Measurements of pH, dissolved oxygen concentration and temperature were carried out daily.
Post exposure observation period:
The body length of each fish were measured at the end of the test in order to check their compliance with the size range recommended for the species by the test guideline (OECD No. 203). The measured values were within the range of 2.10-2.60 cm.
Hardness:
The measured hardness of the ISO Medium used in the study was 242.08 mg/L (as CaCO3).
Test temperature:
The measured temperatures were within a range of 21.6 - 22.7°C in the test aquariums (the maximum deviation did not exceed ± 1°C / aquarium).
pH:
The test was carried out without adjustment of pH. The measured pH values were in the range of 7.18 - 7.80 during the test
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 75.8 - 97.2 % of the air saturation value at the temperature used.
Salinity:
Not applicable
Conductivity:
Not specified
Nominal and measured concentrations:
Limit test; using a single nominal concentration of 100 mg/L and a concurrent control. The measured concentrations deviated more than 20 % from the nominal concentration at the start and at the end of the renewal periods, therefore the geometric mean of the measured concentration was calculated to determine exposure concentration. The corresponding calculated concentration was 0.32 mg/L (based on total product).
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquaria
- Type: open
- Renewal rate of test solution: Daily renewal
- No. of organisms per vessel: 10
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: < 1.0 g fish / L test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Fish were held prior to the study in reconstituted water (ISO Medium, prepared according to recommendation of OECD 203 guideline). The test was performed using the same water. Separate stock solutions of individual substances were first prepared in deionised water. The ISO Medium was prepared by adding 25 mL from each of four stock solutions to one litre deionised water.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light: 8 h dark

EFFECT PARAMETERS MEASURED
The fish were observed at 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality. Fish were considered dead if there is no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction. Dead fish were removed when observed and mortalities were recorded. All sub-lethal effects were recorded. Measurements of pH, dissolved oxygen and temperature were carried out daily.


PRELIMINARY RANGE FINDING TEST
In order to select appropriate test concentrations for use in the definitive test, non-GLP preliminary tests were performed at the nominal concentration of 100 mg/L (limit concentration). The first pretest was a static test while in the second pre-test the test solutions were renewed daily. In both preliminary range-finding tests the test solution was prepared by dissolving an amount of 0.1 g test item in 1000 mL ISO Medium to obtain the 100 mg/L nominal test concentration. This solution was stirred rigorously for about 42 hours in the first pre-test. In the second pre-test stirring lasted about 67 hours in the first renewal period and about 23 hours in the following renewal periods, thereafter filtrated through a membrane filter (0.45 μm) to separate the possible non-dissolved test material. Three fish were exposed for 96 hours in the test item treated and control group. No replicates were included in the pre-tests. No mortality or any sub-lethal effects were detected in the treatment group or in the control group during the 96-h exposure period in both preliminary range finding tests.

- Test concentration: 100 mg/L (limit concentration)
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Details on results:
Validity of the Study
There was no mortality observed in control or in the treatment group during the study. The dissolved oxygen concentration in the test solutions did not fall below 60 % of air saturation value during the study: it ranged between 85.3 - 97.2 % for fresh solutions and between 75.8 - 84.6 % for old solutions. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Analytical Results
A saturated single concentration of 100 mg/L (limit test concentration) and a concurrent control was tested in the main test. The concentration of the test item was analytically determined based on total product at the start and at the end of each water renewal period. In the untreated control group the test item was not detected (i.e. signal intensities measured for the control samples were less than 20% of the analytical detection limit (LOD). The exposure concentration was calculated as the geometric mean of the start and end values of each renewal period and determined to be 0.32 mg/L. This concentration was considered as the saturation concentration in the test medium, based on water-accommodated fractions of the test item (equivalent to 100 mg/L nominal concentration). The temperature during the study was beyond the temperature range of validation in ISO Medium in some cases. As this deviation was not higher than 1 °C, it can be stated that it did not influenced the results of the study.

Biological Results
There was no mortality or sub-lethal effects observed in 10 fish in the control group or in 10 fish exposed to a mean measured test concentration of 0.32 mg/L. The 96-h No Observed Effect Concentration (NOEC) was determined to be 0.32 mg/L (based on total product).
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
Since a limit test was performed, no statistical analysis was necessary. The endpoints were determined directly from the raw data.
Validity criteria fulfilled:
yes
Conclusions:
In a 96-hour semi-static test according to EU Method C.1 and OECD Guideline 203 using the WAF method, the test item had no toxic effect on fish at aquatic saturation (i.e. limit test concentration). Accordingly, the 96-h NOEC was determined to be 0.32 mg/L and the LOEC > 0.32 mg/L (based on total product).
Executive summary:

The acute toxicity of the test item to Danio rerio was determined in a 96-hour semi-static test according to EU Method C.1 and OECD Guideline 203.

The 96 -h NOEC was determined to be 0.32 mg/L (equivalent to 100 mg/L nominal concentration) and the LOEC > 0.32 mg/L (equivalent to > 100 mg/L nominal concentration).

Description of key information

The acute toxicity of the structural analogue Blue Sema to Danio rerio was determined in a 96-hour semi-static test according to EU Method C.1 and OECD Guideline 203.The 96 -h NOEC was determined to be 0.32 mg/L (equivalent to 100 mg/L nominal concentration) and the LOEC > 0.32 mg/L (equivalent to > 100 mg/L nominal concentration).

Key value for chemical safety assessment

Additional information

No information on acute toxicity to fish is available for the submission substance itself. Therefore, read across to the structural analogue Blue Sema (CAS 1040873-93-5) was conducted.

The acute toxicity of the test item Blue Sema to Danio rerio was determined in a 96-hour semi-static test according to EU Method C.1 and OECD Guideline 203.The 96 -h NOEC was determined to be 0.32 mg/L (equivalent to 100 mg/L nominal concentration) and the LOEC > 0.32 mg/L (equivalent to > 100 mg/L nominal concentration).