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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:
01-Nov-2010 to 30-Nov-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Conclusive valid guideline study under GLP conditions.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), C.
Deviations:
no
Principles of method if other than guideline:
At the request of the Sponsor, the test consisted of two parts. A single test concentration was tested in each test part under a different test design (semi-static or flow-through). Thus, both test parts were performed as limit tests based on available toxicity data. The preparation of both test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
GLP compliance:
yes (incl. certificate)
Remarks:
swissmedic 19-Nov-2010
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
yes
Details on sampling:
In both tests, all samples were analyzed immediately after sampling. The concentrations of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide and the potential degradation product 3,3,5-Trimethylcyclo-hexanone were determined in all duplicate test medium, control and solvent control samples.

Part 1 of the test (semi-static):
For measurement of the actual concentrations of the test item, duplicate samples were taken daily from the freshly prepared and aged test medium. From the sampling times at the start and end of the test, one sample was also taken from the control.

Part 2 of the test (flow-through):
Duplicate samples were taken before the start of the test, after 48 hours and at the end of the test after 96 hours. One sample was taken from the control and from the solvent control at the end of the test. Additionally, duplicate samples were taken from the application solution at the start and end of the test. These samples have not been analyzed.

The analytical procedure and results are described in the attached Appendix I - Analytical Investigations.
Vehicle:
no
Details on test solutions:
The preparation of the test medium in both test parts was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Part 1 of the test (semi-static):
At the start of the test and before each test medium renewal, the test concentration was freshly prepared as follows:

First, a dispersion of the test item with the loading rate of 1.0 mg/L was prepared by homogeneously dispersing 11.3 mg (dosing range: 11.3-11.4 mg) in 11.3 liters of test water and intensely stirring for 4 hours at room temperature in the dark in a completely filled and tightly closed stirring vessel to avoid test item losses due to evaporation. After stirring, the dispersion was left to settle for about 10 minutes to separate undissolved test material from the test water (as far as possible), and thereafter, the supersaturated solution was drawn off from the middle layer of the water column (with a teflon tube). The test item could not be filtered due to potential absorption of the test item to filter materials. The so obtained supersaturated solution was diluted 2-fold and used as the test medium.

Part 2 of the test (flow-through):
The concentration of the test item in the test medium was maintained by dosing the application solution (concentrated solution of the test item in organic solvent) into test water using an automatic dosing system. N,N-dimethylformamide p.a. (DMF) was chosen based on its solubilizing properties and its relative non-toxicity to fish. The DMF concentration in the test media and solvent control was 100 µL/L.

The application solution of 10 g/L, used for the dosage of the single test concentration, was freshly prepared before the test start by completely diluting 1104.4 mg of the test item in 110 mL of DMF using intense stirring for 5 minutes.

The application solution was protected from light and was continuously stirred using magnetic stirrers. It was dosed automatically by a timer controlled HAMILTON digital dispenser unit (HAMILTON, Reno, Nevada, USA) into a mixing vessel with a volume of about 2 liters (dosage: 0.60 mL application solution per hour; 15 dosages per hour; 0.040 mL application solution per dosage). Test water flowed at a constant rate of 6.0 liter/hour (controlled by a flow-meter) into the mixing vessels, where test water and application solutions were mixed together continuously by intensive stirring with a magnetic stirrer.
From the mixing vessel, the test media flowed into 48-liter flow-through aquaria (containing 22 liters test medium) at a rate of 144 liters per 24 hours. Thus, the flow rate of the test media through the aquaria corresponded to an at least 6-fold theoretical volume exchange per day.

At the start of the test, 2.2 ml of the application solution (10 g/L) were added to the aquarium. Then the dosing system was started.

Test water without any additions flowed at the same rate into the replicates of the control. For the solvent control, DMF was dosed in the same way as described above.

Water-flow and dosing systems were calibrated before the start of the dosage. The whole dosing system was controlled at least once per day and adjusted if necessary.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The study was performed with zebra fish (Brachydanio rerio). The fish were obtained from a breeding culture at Harlan Laboratories. No medication was applied during holding and acclimatization. Prior to test start, the test fish were acclimated for one week to the test water and temperature. During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany). During holding and acclimatization, no fish died in the test fish batch and all fish were healthy.

From the acclimated test fish batch, 10 fish were measured at the start of the test. The mean body length of the fish was 3.1 ± 0.14 cm (Mean ± SD), the mean body wet weight was 0.23 ± 0.03 g (Mean ± SD).

The test method and test species are recommended by the international test guidelines.
Test type:
other: semi-static and flow through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
not applicable
Hardness:
1.25 mmol/L
Test temperature:
The water temperature was 22-23 °C during the test (see attached Table 4).
pH:
The pH values in all treatments ranged from 7.0 to 7.2 (see attached Table 2).
Dissolved oxygen:
The oxygen concentration was always 6.3 mg/L or higher (see attached Table 3), and thus higher than 60% oxygen saturation.
Salinity:
according to OECD guideline.
Nominal and measured concentrations:
The nominal test item concentrations of both test parts were: 500µg/L.
At the start of the test medium renewal periods, the concentration in the test item treatment ranged between 35 to 132 µg/L . During the test medium renewal periods of 24 hours, the test item concentration decreased to 11 - 26 µg/L.
Details on test conditions:
Reconstituted test water was used in the study. For further information on test water please see section " any other information on materilas and methods incl. tables" below.

The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The test water was aerated prior to the preparation of the test media until oxygen saturation was reached.

In both test parts, the test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

Part 1 of the test (semi-static):
Since the test item was supposed to be volatile (information provided by the Sponsor), the test was performed in a closed system. One 5-liter Erlenmeyer flask was used for each treatment. The Erlenmeyer flasks were completely filled to keep the air-space in the test flasks as small as possible and were tightly sealed.

Part 2 of the test (flow-through):
One glass test vessel with 22 liters of test medium was used for each treatment.

In both test parts, a 16 hour light to 8-hour dark photoperiod, with a 30-minute transition period was used (light intensity during the light period was approximately within the range of 140 to 480 Lux). The test duration was 96 hours and the fish were not fed during the test.

Part 1 of the test (semi-static):
The water temperature in the test vessels was maintained at 22-23 °C (see attached Table 4). The test vessels were not aerated during the test period.

Part 2 of the test (flow-through):
The water temperature in the test vessels was maintained at 23 °C (see attached Table 8). The test vessels were not aerated during the test period. Since a closed system was used, the air from the small air-space above the test media was siphoned off by means of a pump and was slowly bubbled back into the test medium at the bottom of the test flask. Thus, the dissolved oxygen concentrations in the test media were kept sufficiently high, and additionally evaporated test item was dosed back into the test medium.

At the request of the Sponsor, the test consisted of two parts. A single test concentration was tested in each test part under a different test design (semi-static or flow-through). Thus, both test parts were performed as limit tests. In both test parts, 7 fish were introduced at the start of the exposure into each test vessel in a random order. The loading rate was 0.32 and 0.07 g fish wet weight per liter test medium in parts 1 and 2 of the test, respectively. Thus, the requirement of a loading rate not exceeding 1 g fish/L was fulfilled.

Part 1 of the test (semi-static):
The test was performed semi-statically in a closed system to keep the test item concentration as stable as possible. During the semi-static part of the test, the test fish were daily placed into a new test vessel with freshly prepared test medium of the corresponding treatment. Additionally, a control was tested in parallel.

Part 2 of the test (flow-through):
The test was performed in a flow-through system to keep the test item concentration as stable as possible. The test concentration was dosed with DMF. Additionally, a control and a solvent control group were tested in parallel.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 43 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: test part 1
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 250 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: test part 2
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
>= 43 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: test part 1
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
>= 250 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: test part 2
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 43 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: test part 1
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 250 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: test part 2
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 43 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: test part 1
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 43 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: test part 1
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 250 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: test part 2
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 250 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: test part 2
Details on results:
Influence of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide on zebra fish (test part 1, semi-static conditions)
At the start of the test medium renewal periods, the concentration in the test item treatment ranged between 35 to 132 µg/L. During the test medium renewal periods of 24 hours, the test item concentration decreased to 11 - 26 µg/L. Therefore the biological results were based on mean measured concentration of 43 µg/L, calculated as arithmetic mean of the geometric means of the concentrations measured at the start and at the end of each test medium renewal period.

The analytically determined concentrations of the potential degradation product 3,3,5 trimethylcyclohexanone had not increased over the test period. Concentrations ranged between 12 and 23 µg/L at the start of the test medium renewal periods and between 12 and 21 µg/L at the end of the test medium renewal periods.

The biological results (visible abnormalities observed at the test fish, mortalities and the LC50 values) are listed in Table 1. In the control and test medium with the mean measured concentration of 43 µg/L, all fish survived until the end of the test and no visible abnormalities were observed at the test fish. Therefore, the 96 hour NOEC and LC0 of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide to zebra fish were both determined to be at least 43 µg/L. The 96 hour LOEC, LC50 and LC100 were clearly higher than 43 µg/L. These values could not be quantified due to the absence of toxicity of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide at the tested concentration.

In conclusion, the test item Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide had no acute toxic effects on zebra fish up to mean measured concentration of 43 µg/L under the present test conditions.

No remarkable observations were made concerning the appearance of the test medium. It was a clear solution throughout the entire test duration.

The pH values in all treatments ranged from 7.0 to 7.2 (see attached Table 2). The oxygen concentration was always 6.3 mg/L or higher (see attached Table 3), and thus higher than 60% oxygen saturation. The water temperature was 22-23 °C during the test (see attached Table 4).


Influence of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide on zebra fish (test part 2, flow-through conditions)
This test was performed in a flow-through system. The nominal concentration of 1000 µg/L was obtained by dosing 1000 µg/L with the solvent DMF. Additionally, a control and a solvent control group were tested in parallel.

The average test item concentrations in the test medium of nominal 1000 µg/L varied in the range from 210 to 270 µg/L. The mean measured concentration of the test item was calculated as arithmetic mean over all measurements during the test period and was 250 µg/L. The biological results were based on the mean measured concentration of di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide.

The analytically determined concentration of the potential degradation product 3,3,5 trimethylcyclohexanone had not increased over the test period. The concentration was 31 µg/L at the start and 19 µg/L at the end of the test.

In the control and test medium with the mean measured concentration of 250 µg/L (nominal 1000 µg/L), all fish survived until the end of the test and no visible abnormalities were observed at the test fish. Therefore, the 96 hour NOEC and LC0 of Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide to zebra fish were both determined to be at least 250 µg/L. The 96 hour LOEC, LC50 and LC100 were clearly higher than 250 µg/L. These values could not be quantified due to the absence of toxicity of Di-tert-butyl 3,3,5 trimethylcyclohexylidene diperoxide at the solubility limit of the test item.

In conclusion, the test item Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide had no acute toxic effects on zebra fish up to the solubility limit of the test item under the present test conditions.

No remarkable observations were made concerning the appearance of the test medium. It was a clear solution throughout the entire test duration.

The pH values in all treatments were between 7.1 and 7.2 (see attached Table 6). The oxygen concentration was always 7.7 mg/L or higher (see attached Table 7), and thus higher than 60% oxygen saturation. The water temperature was 23 °C during the test (see attached Table 8).
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The NOEC and LC0 were determined directly from the raw data. The LOEC, LC100 and LC50 at the observation times could not be quantified due to the absence of a toxic effect of the test item at the tested concentrations.

   REFERENCES

            1.         OECD Series on Testing and Assessment No. 23 (2000):
Guidance Document on Aquatic Toxicity Testing
of Difficult Substances and Mixtures

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the test item Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide had no acute toxic effects on zebra fish up to the its solubility limit in test water under the present test conditions.
Executive summary:

The acute toxicity of the test item Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide to zebra fish (Brachydanio rerio) was determined in a 96-hour test according to the EU Commission Directive 92/69/EEC, Part C.1, the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992)).

 

At the request of the Sponsor, the test consisted of two parts. A single test concentration was tested in each test part under a different test design (semi-static or flow-through). Thus, both test parts were performed as limit tests based on available toxicity data. The preparation of both test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

Test part 1 (semi-static):

The test was performed semi-statically in a closed system to keep the test item concentration as stable as possible. A dispersion of the test item with the loading rate of 1000 µg/L was prepared. After stirring and separation of undissolved test item the test water was diluted 2-fold and used as test medium for test part 1 (nominal test concentration of 500 µg/L). Additionally, a control group was tested in parallel.

 

At the start of the test medium renewal periods, the concentration in the test item treatment ranged between 35 to 132 µg/L. During the test medium renewal periods of 24 hours, the test item concentration decreased to 11 - 26 µg/L. Therefore the biological results were based on mean measured concentration of 43 µg/L, calculated as arithmetic mean of the geometric means of the concentrations measured at the start and at the end of each test medium renewal period.

 

Test part 2 (flow-through):

This test was performed in a flow-through system. The nominal concentration of 1000 µg/L was obtained by dosing 1000 µg/L with the solvent DMF. Additionally, a control and a solvent control group were tested in parallel.

The average test item concentrations in the test medium of nominal 1.0 mg/L varied in the range from 210 to 270 µg/L. The mean measured concentration of the test item was calculated as arithmetic mean over all measurements during the test period and was 250 µg/L.

 

 

Summary

The following biological results were based on the mean measured concentration of di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide:

 

 

Test Part 1

Test Part 2

 

– 96-hour LC50:

>43

>250

µg/L

 

 

 

 

– 96-hour LC0:

≥43

≥250

µg/L

 

 

 

 

– 96-hour LC100:

>43

>250

µg/L

 

 

 

 

– 96-hour NOEC:

≥43

≥250

µg/L

 

 

 

 

– 96-hour LOEC:

>43

>250

µg/L

 

In conclusion, the test item Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide had no acute toxic effects on zebra fish up to the its solubility limit in test water under the present test conditions.

Description of key information

There is one valid key study to assess the acute toxicity of the test substance to fish. There were no acute toxic effects on zebra fish up to the solubility limit o the test item (Hoger, 20122).

Key value for chemical safety assessment

Additional information

Influence of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide on zebra fish (test part 1, semi-static conditions) At the start of the test medium renewal periods, the concentration in the test item treatment ranged between 35 to 132 µg/L. During the test medium renewal periods of 24 hours, the test item concentration decreased to 11 - 26 µg/L. Therefore the biological results were based on mean measured concentration of 43 µg/L, calculated as arithmetic mean of the geometric means of the concentrations measured at the start and at the end of each test medium renewal period.

In conclusion, the test item Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide had no acute toxic effects on zebra fish up to mean measured concentration of 43 µg/L under the present test conditions.

Influence of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide on zebra fish (test part 2, flow-through conditions) This test was performed in a flow-through system. The nominal concentration of 1000 µg/L was obtained by dosing 1000 µg/L with the solvent DMF. Additionally, a control and a solvent control group were tested in parallel. The average test item concentrations in the test medium of nominal 1000 µg/L varied in the range from 210 to 270 µg/L. The mean measured concentration of the test item was calculated as arithmetic mean over all measurements during the test period and was 250 µg/L. The biological results were based on the mean measured concentration of di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide.

In conclusion, the test item Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide had no acute toxic effects on zebra fish up to the solubility limit of the test item under the present test conditions.