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Ecotoxicological information

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:
January - March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
TEST MATERIAL
- Molecular formula: C21H30N2
- Analysis date: August 12, 2014
- Date of production: August 11, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25ºC, <70 RH%), protected from light and humidity.
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Expiry date: August 10, 2017
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentrations, samples were taken from each testing concentrations and from the control at the start and at the end of each water renewal period (daily renewal period). Three replicate samples were taken from the test solutions and two control samples on each analytical occasion.
Vehicle:
no
Details on test solutions:
The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. The test item is poorly water soluble material. In order to achieve maximum dissolved concentration, a supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving an amount of 0.4 g test item into 4000 mL test medium (ISO medium) at least one day before the start of the test. This solution was agitated by orbital shaker (~350 rpm) for a period of at least one day and then the non-dissolved test material was separated by filtration through a fine (0.22 μm) filter in order to obtain the saturated test solution (i.e. 100 % v/v saturated solution). The test solutions of the chosen test concentrations were prepared by appropriate diluting of this stock solution. The test solutions were prepared just before introduction of the test animals (start of the water renewal periods).
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Species: Zebrafish (Brachydanio rerio)
- Origin: Black Molly Bt. 8200 Veszprém, Jutasi út 29, Hungary
- Number of animals: There were 7 animals in the test item concentrations and in the control group respectively (no parallels were used).
- Age and size of animals: Juveniles; size within a range of 2.0 ± 1 cm. The loading of the test aquaria did not exceed 1.0 g / L.
- Sex: Both female and male (were not separated)
- Animal health: Apparently healthy animals were used in the test
- Acclimatization, holding of fish prior to the study:
Fish were held for at least 12 days before test initiation in the fish laboratory of the CRO under the same conditions as used during the exposure period.
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
There was no mortality occurred in seven days before the start of experiment, therefore the batch was considered to be acceptable for testing.
- Feeding: The fish were not fed during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
The fish were observed at approximately 3, 6, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality.
Post exposure observation period:
None.
Hardness:
The reconstituted water (ISO medium) has an approximate theoretical total hardness of 249 mg/L (as CaCO3).
Test temperature:
The aquariums were kept in a temperature-controlled tray. The measured temperatures were within a range of 21.5 – 22.3°C in the aquariums (the maximum deviation did not exceed ± 1°C).
pH:
The test was carried out without adjustment of pH. The measured pH values were in the range of 7.27 – 7.86 during the test.
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 62.8 – 92.1 % of the air saturation value at the temperature used.
Salinity:
Freshwater used
Conductivity:
No data
Nominal and measured concentrations:
Based on the results of the preliminary experiment, nominal concentrations of 19.8, 29.6, 44.4, 66.7 and 100 % v/v saturated solution were tested in the main study. The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were 0.24, 0.36, 0.55, 0.87 and 1.36 mg/L. All biological results are based on the geometric mean of the measured concentrations.
Details on test conditions:
- Light: 12 to 16 hours photoperiod daily;
- Temperature: 21 – 25°C, constant within ± 1°C.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % conf. limits: 1.02 – 2.65 mg/L
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.55 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
behaviour
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
0.87 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.87 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
behaviour
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 1.36 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
MORTALITY:
There was no mortality observed in the control group and in the concentrations of 0.24, 0.36, 0.55 and 0.87 mg/L during the whole test period. In the highest concentration of 1.36 mg/L four fish died during the 96 h exposure period.

TOXIC EFFECTS:
No signs abnormalities were observed in the control and in the concentrations of 0.24, 0.36 and 0.55 mg/L. Slight to severe decreased swimming activity, staying nearby the bottom of aquarium, furthermore in some cases lying at the bottom of aquarium in lateral position with periodic gill movements were observed in the two highest concentrations of 0.87 and 1.36 mg/L.
Results with reference substance (positive control):
No reference substance tested.
Reported statistics and error estimates:
The LC50 value of the test item was calculated by Probit analysis using SPSS PC+ software. The LC0, the NOEC, the LOEC and the LC100 values of the test item were determined directly from the raw data.
Validity criteria fulfilled:
yes
Conclusions:
The 96-h LC50 value was determined to be 1.32 mg/L. The highest test concentration causing no mortality within the test period (96-h LC0) was determined to be 0.87 mg/L and the lowest test concentration causing 100 % mortality within the test period (96-h LC100) was determined to be higher than 1.36 mg/L. The results are based on the measured concentrations.
Executive summary:

The purpose of this study was to evaluate the acute toxicity of the test item on Zebrafish (Brachydanio rerio). The study was performed according to EU testing method C.1 and OECD no. 203. Young fish were exposed in an acute toxicity test to aqueous test media containing the test item for 96 hours at a range of concentration. Based on the analytical results obtained during the previously performed acute toxicity test, the test item was not expected to be stable for the duration of 96 hours in ISO Medium, therefore a semi-static test was performed using daily water renewal periods. Based on the results of the preliminary experiment, nominal concentrations of 19.8, 29.6, 44.4, 66.7 and 100 % v/v saturated solution were tested in the main study. The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were 0.24, 0.36, 0.55, 0.87 and 1.36 mg/L. All biological results are based on the geometric mean of the measured concentrations. The fish were observed at approximately 3, 6, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality. There was no mortality observed in the control group and in the concentrations of 0.24, 0.36, 0.55 and 0.87 mg/L during the whole test period. In the highest concentration of 1.36 mg/L four fish died during the 96 h exposure period. Sub-lethal effects were observed in the two highest concentrations of 0.87 and 1.36 mg/L.

In conclusion, the test item had significant toxic effects on fish. The 96-h LC50 value was determined to be 1.32 mg/L. The highest test concentration causing no mortality within the test period (96 -h LC0) was determined to be 0.87 mg/L and the lowest test concentration causing 100 % mortality within the test period (96-h LC100) was determined to be higher than 1.36 mg/L. The results are based on the measured concentrations.

Description of key information

The purpose of this study was to evaluate the acute toxicity of the test item on Zebrafish (Brachydanio rerio). The study was perfored according to EU testing method C.1 and OECD no. 203 as semi-static test. The test item had significant toxic effects on fish. The 96-h LC50 value was determined to be 1.32 mg/L. The highest test concentration causing no mortality within the test period (96-h LC0) was determined to be 0.87 mg/L and the lowest test concentration causing 100 % mortality within the test period (96-h LC100) was determined to be higher than 1.36 mg/L. The results are based on the measured concentrations.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.32 mg/L

Additional information

Source: GLP-report