Di-tert-butyl 1,1,4,4-tetramethylbut-2-yn-1,4-ylene diperoxide

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 2017-08-02 to 2017-09-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-UV

Details on sampling:

- Concentrations: control and treatment group
- Sampling method: For determination of the test item concentration five replicate samples were taken from the test solution and from the control at the start and at the end of each renewal period of the test.

Vehicle:

no

Details on test solutions:

The test solution used in the test was prepared by mechanical dispersion. An amount of 0.5 g test item was dissolved in 5000 mL dilution water (ISO Medium) in order to obtain the concentration of 100 mg/L at the start of each renewal period (always one day before treatment). This suspension was handled by ultrasonic bath for 10 minutes, rigorously stirred for one day, and thereafter, filtrated through a 0.45 μm pore size membrane filter. The resulted solution was the test concentration.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Akvárium Magazin Kft., 1222-Budapest, Dévény u. 36., Hungary
- Age at study initiation: juveniles
- Length at study initiation: 2.20 - 2.50 cm
- Weight at study initiation: 0.27 - 0.30 g/fish

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions: same as test
- Type and amount of food during acclimation: appropriate, commercial diet for fish
- Feeding frequency during acclimation: 3 times per week until one day before the test
- Health during acclimation: No mortality occurred in seven days before the start of the experiment.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

249 mg/L CaCO3

Test temperature:

20.7 - 22.4 °C

pH:

7.36 - 7.77

Dissolved oxygen:

74.9 - 93.1 % of the air saturation value at the tested temperature

Salinity:

NA

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentrations: 1.2 mg/L at the start of the renewal period, 0.6 mg/L at the end of the renewal period. Measured geometric mean concentration: 0.9 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquariums with approx. 6 litres capacity
- Renewal rate of test solution: every 24 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.54 g/L in the control, 0.60 g/L in the test group

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Medium prepared according to OECD 203

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

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.1, 1, 10, 50, 100 mg/L
- Results used to determine the conditions for the definitive study: No mortality and any significant toxic effects were observed after 96 hours of the preliminary test.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

No mortality occurred in the control group and in the test item group of 100 mg test item/L medium (nominal).

Sublethal observations / clinical signs:

Pre-experiment

No mortality or any sub-lethal effects were detected neither in the treatment groups nor in the control group during the 96-h exposure period.

 

Validity of the study

- No mortality was observed in the control group during the test.

- The dissolved oxygen concentration in the test solutions did not fall below 60 % of air saturation value during the study. The dissolved oxygen concentration was in the range of 74.9 – 93.1 % of the air saturation value.

- No significant change (more than one unit) in the pH value was observed during the test. The pH was in the range of 7.36 – 7.77.

All acceptance criteria were met, therefore, the study was considered to be valid.

 

Analytical Results

The measured concentration at the end of each renewal period was lower than 80 % of the mean measured concentration at the start of the test.

Table 1. Results of the Analytical Measurements during the Study

Renewal Periods	Mean Measured Test Item Concentration at the Start of the Renewal Period (mg/L)	Mean Measured Test Item Concentration at the End of the Renewal Period (mg/L)	Measured Concentration in Percentage of the Start (%)
1.	1.20	0.71	59
2.	1.24	0.61	49
3.	1.19	0.61	52
4.	1.23	0.59	48

 

Biological results

No mortality occurred in the control group and in the test item group of 100 mg test item/L medium (nominal). No sub-lethal effect was observed in the control group, while in the test item group a slight inactivity and orientation on the bottom of the aquarium was shown in case of 5 fish 3 hours after treatment and by 3 fish 6 hours after treatment on day 0. No sub-lethal effect occurred later during the study in this test group. Accordingly, the 96-h NOEC was determined as 100 mg/L and the LOEC and the LC50 values were determined to be higher than 100 mg/L.

 

Table 2. Summary of the biological endpoints
Parameter	Nominal concentration [mg/L]
96-h LC50	> 0.9 (nominal 100)
96-h NOEC	0.9 (nominal 100)
96-h LC0	0.9 (nominal 100)
96-h LOEC	> 0.9 (nominal 100)
96-h LC100	> 0.9 (nominal 100)

Validity criteria fulfilled:

yes

Conclusions:

The test item had no toxic effect up to at least the limit concentration of 0.9 (nominal 100) mg/L on Zebrafish. Accordingly, the 96-h LC50 value was determined to be >0.9 (nominal >100) mg/L. The 96-h NOEC was determined to be 0.9 (nominal 100) mg/L.

Executive summary:

The acute toxicity of the test item to fish (zebrafish) was determined according to the OECD Guideline 203, EU Council Regulation No. 440/2008 method C.1 and EPA OPPTS 850.1075. A semi-static test was performed as a limit test with a concentration of 100 mg/L. The test item solution was prepared by mechanical dispersion – 0.5 g test item were dissolved in 5000 mL ISO Medium. Ten test organisms were exposed to the test concentration and the control, respectively, for 96 hours. The measured concentration of the test item at the start of the renewal periods was 1.2 mg/L and at the end of the renewal periods was 0.6 mg/L, respectively. The limit concentration of the test item was found to have no effect on the vitality of zebrafish. In conclusion, the NOEC corresponds to the nominal concentration 100 mg/L (0.9 mg/L measured geometric mean concentration). The LC50 and the LC100 correspond to the nominal concentration >100 mg/L (>0.9 mg/L measured geometric mean concentration).

Description of key information

The test item had no toxic effect up to at least the limit concentration of 0.9 (nominal 100) mg/L on Zebrafish. Accordingly, the 96-h LC50 value was determined to be >0.9 (nominal >100) mg/L. The 96-h NOEC was determined to be 0.9 (nominal 100) mg/L.

Additional information

The acute toxicity of the test item to fish (zebrafish) was determined according to the OECD Guideline 203, EU Council Regulation No. 440/2008 method C.1 and EPA OPPTS 850.1075. A semi-static test was performed as a limit test with a concentration of 100 mg/L. The test item solution was prepared by mechanical dispersion – 0.5 g test item were dissolved in 5000 mL ISO Medium. Ten test organisms were exposed to the test concentration and the control, respectively, for 96 hours. The measured concentration of the test item at the start of the renewal periods was 1.2 mg/L and at the end of the renewal periods was 0.6 mg/L, respectively. The limit concentration of the test item was found to have no effect on the vitality of zebrafish. In conclusion, the NOEC corresponds to the nominal concentration 100 mg/L (0.9 mg/L measured geometric mean concentration). The LC50 and the LC100 correspond to the nominal concentration >100 mg/L (>0.9 mg/L measured geometric mean concentration).