Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-01-24 to 2008-01-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
For the analysis of the test item concentrations, samples from the test media of the test concentration and the control were taken at the start and end of the study. All test medium samples were taken from the approximate centre of the aquaria without mixing of the test media.
- Sample storage conditions before analysis:
All samples were analysed directly after sampling. The samples are kept stored in deep-freezer up to the date of the final report.

Vehicle:

no

Details on test solutions:

Dosage of Test Item:
A supersaturated test item solution was prepared by dispersing/dissolving the test item without the use of any organic solvent into the Test Medium two days before the start of the study (on day -2). This solution was treated in ultrasonic bath 3 times for 10 minutes, shaken for 24 hours at 30 ºC, than for 4 hours at 20 ºC and thereafter filtrated through a 0.22 μm-MILLIPORE filter. The concentration of this stock solution was 26 mg/L and the test solutions were prepared by the appropriate diluting of the stock solution on day 0, just before the start of the study (= introduction of fish) according to the following table:

Concentration [mg/L] Stock solution (mL) Aquarium water (mL)
nominal mean measured
Control – 4000
0.625 0.477 96 ad 4000
1.25 0.964 193 ad 4000
2.5 2.64 385 ad 4000
5.0 5.34 770 ad 4000
10.0 9.81 1540 ad 4000

Control:
In the control, aquarium water (filtered tap water) was used without addition of the test item.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Zebrafish (Danio rerio)
Age and Size: Juveniles; The mean body lengths of the fish were 2.8 - 3.3 cm. The body length of 10 test animals from the test fish batch were measured prior to the start of the test.
The mean body weight of the fish in the test groups and control ranged between 0.299 – 0.363 g. The body weight of fish was recorded on the day 0 (at the start of the main study). First the weight of a vessel filled with water was determined. Then 7 fish were placed into this vessel. The weighting of all fish were registered and the average body weight was calculated for 1 fish. On the basis of the above, the loading of the testing liquid in the test-aquaria was calculated (g fish/litre testing liquid). The maximum loading was 0.598 g /L in the test.
Sex: male and female
Origin: The test fish were obtained from “Black Molly” Fish Farm, Veszprém, Hungary.
Holding Conditions: In accordance with the test guidelines the fish were held in test water in the laboratories for at least 12 days prior to the start of the test without any medication. During holding until one day before test start the fish were fed with a commercial fish diet (“Aquafood”) for Zebrafish. During the 12 days prior to the test no fish died in the test fish batch and all fish were healthy.
Acclimatisation: For approx. 16 weeks before the start of the test the fish were held in the test water at test temperature.
Feeding: None

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

169.5 mg/L

Test temperature:

21.4 - 21.9 °C

pH:

8.32 - 8.47

Dissolved oxygen:

at least 89 % or higher

Nominal and measured concentrations:

nominal: 0.625; 1.25, 2.5;5.0; 10.0 mg/L
measured: 0.477; 0.964; 2.64; 5.34; 9.81 mg/L

Details on test conditions:

Holding Conditions: In accordance with the test guidelines the fish were held in test water in the laboratories for at least 12 days prior to the start of the test without any medication. During holding until one day before test start the fish were fed with a commercial fish diet (“Aquafood”) for Zebrafish. During the 12 days prior to the test no fish died in the test fish batch and all fish were healthy.
Test Environment: The aquaria were in a temperature-controlled tray
Light period: 16 h light : 8 h dark
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.
Test water: Similar holding water (tap water biological and mechanical filtrated by a submersible internal filter Type SP-1800B) was used in the test as it was used during the holding. Before the start of the study, chemical parameters of this water were determined. The hardness (CaCO3) of water was: 168.5 mg/L.

Reference substance (positive control):

yes

Remarks:

aquarium water (filtered tap water)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

1.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

3.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

1.79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

3.06 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.964 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.964 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.964 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

0.964 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

2.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

2.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

2.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: significant effect

Details on results:

Analytical Results
The analytically determined mean test item concentrations in the analysed test media ranged from 90 % to 117 % at the start and from 62 % to 108 % at the end of the study. The deviation of the measured concentrations from the nominal values was higher than 20 % in some cases at the end of the study. Therefore, all reported biological results are related to the geometric mean measured concentrations of the test item calculated by EXCEL Software Program.

Biological Results and Conclusion
Mortality: At the test item concentrations of 2.65 (nominal 2.5); 5.34 (nominal 5.0) and 9.81 (nominal 10.0 mg/L) occurred mortality.
Symptoms: At the test item concentration of 2.65 (nominal 2.5) mg/L fast motility of operculum of the fish was observed at the 24-hour and 48-hour observations. At the test item concentration of 5.34 (nominal 5.0) mg/L slow swimming was observed and the fish settled on the ground of the aquarium.
Results: The 96-hour LC50 was calculated to be 1.62 mg/L (95 % confidence intervals: 0.67 – 2.97 mg/L, SPSS+ statistical software program). The 96-hour LC50 nominal value was calculated to be 1.79 mg/L (95 % confidence intervals: 1.20 – 2.48 mg/L, SPSS+ statistical software program). The 96-hour LC100 was 2.64 (nominal 2.5) mg/L. The 96-hour NOEC (highest concentration tested without any toxic effect after the exposure period of 96 hours) and the 96-hour LC0 of the test item was determined to be 0.964 (nominal 1.25) mg/L. The 96-hour LOEC was 2.64 (nominal 2.5) mg/L.

Reported statistics and error estimates:

The NOEC, LOEC, LC0 and the LC100 were determined directly from the raw data. The LC50 was calculated by Probit analysis (SPSS+ statistical software program).

Sublethal observations / clinical signs:

Calculation based on nominal concentration

Parameter	24 h (mg/L)	48 h (mg/L)	72 h (mg/L)	96 h (mg/L)
LOEC	2.5	2.5	2.5	2.5

Validity criteria fulfilled:

yes

Conclusions:

In the 96-hour static acute toxicity test to Zebrafish (Danio rerio) with the test item the LC50 values were in the range of 1 - 10 mg/L (Probit analysis). The overall NOEC was determined to be 0.964 mg/L.

Executive summary:

The purpose of this study was to evaluate the acute toxicity of the test item to fish. Young fish were exposed to aqueous test media containing the test item at the nominal concentrations of 0.625; 1.25; 2.5; 5.0 and 10.0 mg/L under defined conditions. The corresponding mean concentrations measured were 0.477; 0.964; 2.64; 5.34 and 9.81 mg test item/L.

For the analysis of the test item concentrations, samples from the treatment groups and the untreated control were taken at the start and end of the study. The analytically determined mean test item concentrations in the analysed test media ranged from 90 % to 117 % at the start and from 62 % to 108 % at the end of the study. The deviation of the measured concentrations from the nominal values was higher than 20 % in some cases at the end of the study. Therefore, all reported biological results are related to the geometric mean measured concentrations of the test item calculated by EXCEL Software Program.

The 96-hour LC50 was calculated to be 1.62 mg/L (95 % confidence intervals: 0.67 – 2.97 mg/L, SPSS+ statistical software program). The 96-hour LC50 nominal value was calculated to be 1.79 mg/L (95 % confidence intervals: 1.20 – 2.48 mg/L, SPSS+ statistical software program). The 96-hour NOEC (highest concentration tested without any toxic effect after the exposure period of 96 hours) and the 96-hour LC0 of the test item was determined to be 0.964 (nominal 1.25) mg/L. The 96-hour LOEC was 2.64 (nominal 2.5) mg/L.

Description of key information

In a 96-hour static acute toxicity test to Zebrafish (Danio rerio) with the test item according to EU Method C.1 (Acute toxicity to fish), the 96-h LC50 value was determined to be 1.62 mg/L. The overall NOEC was determined to be 0.964 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.62 mg/L

Additional information

The purpose of this study was to evaluate the acute toxicity of the test item to fish. Young fish were exposed to aqueous test media containing the test item at the nominal concentrations of 0.625; 1.25; 2.5; 5.0 and 10.0 mg/L under defined conditions. The corresponding mean concentrations measured were 0.477; 0.964; 2.64; 5.34 and 9.81 mg test item/L.

For the analysis of the test item concentrations, samples from the treatment groups and the untreated control were taken at the start and end of the study. The analytically determined mean test item concentrations in the analysed test media ranged from 90 % to 117 % at the start and from 62 % to 108 % at the end of the study. The deviation of the measured concentrations from the nominal values was higher than 20 % in some cases at the end of the study. Therefore, all reported biological results are related to the geometric mean measured concentrations of the test item concentration.

The 96-hour LC50 was calculated to be 1.62 mg/L (95 % confidence intervals: 0.67 – 2.97 mg/L, SPSS+ statistical software program). The 96-hour LC50 nominal value was calculated to be 1.79 mg/L (95 % confidence intervals: 1.20 – 2.48 mg/L, SPSS+statistical software program).

The 96-hour NOEC (highest concentration tested without any toxic effect after the exposure period of 96 hours) and the 96-hour LC0 of the test item was determined to be 0.964 (nominal 1.25) mg/L. The 96-hour LOEC was 2.64 (nominal 2.5) mg/L.