Cyclohexylidenebis[tert-butyl] peroxide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 06, 2020 to March 29, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

adopted 02. Oct. 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

Regulation (EU) 2017/735 of 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Medium renewal was performed daily using freshly prepared test solutions. The content of the test item was measured weekly in the freshly prepared and in the used solutions. Samples were taken from the same batch of the solutions.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: semi-static test conditions, limit test
- Medium: Minerals were added to deionised water in order to derive ELENDT M4 medium in accordance with OECD-Guideline 211.
- Controls: a water control and a solvent control were included
- Chemical name of vehicle: Acetone, below 100 µL/L
Stock solutions containing 20 g/L in acetone (stock solution 1) were prepared. Stock solutions of 2 g/L and 1 g/L in acetone were prepared by dilution of stock solution 1 with acetone.
These stock solutions were used for preparation of the treatments levels. In the solvent control 100 μL/L acetone was mixed with test medium.
- Test concentration: Based on the results of a non-GLP pre-test a limit test was performed at the nominal concentration of 0.2 mg/L. For precautionary reasons, a lower concentration of nominal 0.1 mg/L was also tested.

Test organisms (species):

Daphnia magna

Details on test organisms:

SPECIFICATIONS:
- Species: Daphnia magna
- Variety: STRAUS
- Strain: Berlin
- Arrival of Strain at the test facility: 27. Sep. 2007
- Sex: female
- Age: between 0 and 24 hours

The animals which were used in the test did not belong to the first brood of the respective parent animals.

ANIMAL HUSBANDRY:
Daphnia magna was bred in-house at the test facility. The animals were originally obtained from the Umweltbundesamt in Berlin. They multiply by parthenogenesis, thus being genetically identical. The holding is performed similar to the method described in the OECD guideline 211.

HOLDING CONDITIONS:
- Vessels: glass beakers, nominal volume 2 litres
- Medium: M4-Medium (recipe of ELENDT, OECD 211), composition see Table below
- Food: green alga (Desmodesmus subspicatus)
- Medium renewal: twice a week
- Photo period: 16/8 hours light/dark
- Temperature: 20 ± 2 °C

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

Fresh medium: Control: 267 – 356 [mg CaCO3/L]; Test item: 267 – 356 [mg CaCO3/L]
Spent medium: Control: 267 – 356 [mg CaCO3/L]; Test item: 267 – 356 [mg CaCO3/L]

Test temperature:

19.6 – 21.3 °C

pH:

Fresh medium: Control: 7.6 – 7.8; Test item: 7.6 – 7.8
Spent medium: Control: 7.7 – 8.2; Test item: 7.7 – 8.2

Dissolved oxygen:

Fresh medium: Control: 8.5 – 9.1 [mg/L]; Test item: 8.6 – 9.1 [mg/L]
Spent medium: Control: 8.2 – 9.5 [mg/L]; Test item: 8.8 – 9.7 [mg/L]

Nominal and measured concentrations:

A limit test was performed at the nominal concentration of 0.2 mg/L. For precautionary reasons, a lower concentration of nominal 0.1 mg/L was also tested.
The measured concentrations were between 88 % and 123 % of the nominal concentration in the fresh medium and between 27 % and 39 % of the nominal concentration in spent medium.
Because the measured concentrations in the test solutions were not within the range of 80 % - 120 % of the nominal concentration, the time-weighted arithmetic mean values of 0.064 mg/L (64 % of nominal 0.1 mg/L) and 0.112 mg/L (56 % of nominal 0.2 mg/L) were used for determination of the biological results.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (nominal volume 100 mL) were used as test vessels and filled with 80 mL medium. Glass tubes and a stainless steel sieve were used to transfer the daphnia into the test vessels. Stock vessels as well as sampling or preparation materials (with the exception of the one-way tips of the pipettes) were made of glass or other inert material (e.g. PTFE).
- Aeration: none
- Renewal rate of test solution: Daily
- No. of organisms per vessel: 1 (animals were kept individually)
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- Biomass loading rate: 80 mL/animal

TEST MEDIUM / WATER PARAMETERS
De-ionised water was used and supplemented with specific ions to derive ELENDT M4 medium in-line with OECD 211.

OTHER TEST CONDITIONS
- Adjustment of pH: pH was not adjusted
- Photoperiod: 16/8 hours light/dark
- Light intensity: 1055 – 1092 Lux

EFFECT PARAMETERS MEASURED:
The test vessels were inspected for offspring every day. The newborn daphnids were counted and removed from the test vessel. The animals were fed on each day.
Abnormalities as ephippia, presence of males, presence of aborted eggs and dead offspring were recorded on each day of observation.

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.112 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.112 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

immobilisation

Details on results:

Immobile daphnids were observed in all treatments and the controls. In the control, the solvent control, the nominal 0.1 mg/L and 0.2 mg/L treatment groups, observed immobility was 10, 20, 20, and 10 %, respectively. There was no statistically significant difference between the treatments and the controls.

Total number of offspring was 951, 847, 866 and 868 in the control, the solvent control, the nominal 0.1 mg/L and 0.2 mg/L treatment groups, respectively. There was no statistically significant difference between the treatments and the controls. Based on mean offspring per surviving parent daphnid inhibition of reproduction compared to the controls was -2 and 9 % in the nominal 0.1 mg/L and 0.2 mg/L treatment groups, respectively. There was no statistically significant difference between the treatments and the controls.

Mean measured body length at study end was 5.1, 5.1, 5.3, and 5.2 mm in the control, the solvent control, the nominal 0.1 mg/L and 0.2 mg/L treatment groups, respectively.

No behavioural abnormalities were observed.

Reported statistics and error estimates:

For the parameter “Cumulative offspring per introduced parent (21 d)” and “Cumulative offspring per survived parent (0 - 21 d)” the data were tested for normal distribution using Shapiro-Wilk's Test (p > 0.01), followed by Levene's Test on variance homogeneity (with Residuals, p > 0.01). The linearity was tested using a trend analysis by Contrasts (Monotonicity of Concentration/Response, p > 0.05).
As the analysis of contrasts did not reveal a linear trend, a Dunnett's Multiple t-test procedure was finally performed (α = 0.05).
For the parameter “Immobility (0-21 d)” a qualitative trend analysis by Contrasts (Monotonicity of Concentration/Response, p > 0.05) was done. As the analysis of contrasts did not reveal a linear trend, the Fishers Exact Binomial Test with Bonferroni Correction was finally performed (α = 0.05)
Calculation of results was performed with the help of validated software (Microsoft Excel). The NOEC calculations of the biological results were accomplished using ToxRat Professional, version 3.3.0.
As no effects were observed, no ECx calculations could be provided.

Table 1: Summary of results

	Blank Control	Solvent Control	0.1 mg/L (nominal) (0.064 mg/L (TW mean))	0.2 mg/L (nominal) (0.112 mg/L (TW mean))
Total Offspring	951	847	866	868
Surviving Parent Animals	9	8	8	9
Mean length of survived animals (mm)	5.1	5.1	5.3	5.2

TW mean: time-weighted mean measured

 

Table 2: Offspring per surviving parent animal

Replicate	Blank Control	Solvent Control	0.1 mg/L (nominal) (0.064 mg/L (TW mean))	0.2 mg/L (nominal) (0.112 mg/L (TW mean))
1	91	117	M	84
2	98	M	106	89
3	100	M	99	127
4	142	111	M	108
5	98	95	126	79
6	M	109	108	123
7	68	87	107	96
8	114	91	110	63
9	100	108	100	99
10	140	129	110	M
Mean	106	106	108	96
SD	23	14	8	21
RSD	22	13	8	21

SD = standard deviation;

RSD = relative standard deviation = coefficient of variation

TW mean: time-weighted mean measured, M = mortality of parent animal

Validity criteria fulfilled:

yes

Remarks:

Mortality of parent animals in the control should be ≤ 20 % and was found to be 15 % (pooled controls). Mean surviving offspring in the control should be ≥ 60 and was 106 (mean offspring in the controls).

Conclusions:

No effects on daphnids were observed in the long-term toxicity test. Thus, the NOEC was determined to be >= 0.112 mg/L (time-weighted mean measured concentration). As no effects were observed, no ECx calculations could be provided.

Executive summary:

This study was performed in order to evaluate the effect of Cyclohexylidenebis[tert-butyl] peroxide on the reproduction of freshwater cladocera, using the species Daphnia magna  Straus. The aim of the study was to derive a NOEC (no observed effect concentration).

Young female Daphnia (the parent animals), aged less than 24 hours at the start of the test, were exposed to the test item added to ELENDT M4 medium under semi-static conditions. Medium renewal was performed daily. The test duration was 21 days. Ten daphnia each were exposed to the test item and the controls.

At the end of the test, total number of living offspring produced per introduced parent animal and per surviving parent animal was assessed and decrease in reproduction of the daphnia was determined. The offspring of the daphnids was counted each day.

Because of the limited water solubility of the test item, acetone was used to spike the test solutions. Based on the results of a non-GLP pre-test and under consideration that only a NOEC value has to be established, a limit test was performed at nominal 0.2 mg/L. For precautionary reasons, a lower concentration was also tested, i.e. 0.1 mg/L.

The water concentration of the test item was determined using Gas Chromatography (GC) with MS detection. The measured concentrations were between 88 % and 123 % of the nominal concentration in the new solutions and between 27 % and 39 % of the nominal concentration in the old solutions. Because the measured concentrations in the test solutions were not within the range of 80 % - 120 % of the nominal concentration, the time-weighted arithmetic mean of 0.064 and 0.112 mg/L were used for determination of the biological results

 

Biological effects

Immobile daphnids were observed in all treatments and the controls. In the control, the solvent control, the nominal 0.1 mg/L and 0.2 mg/L treatment groups, observed immobility was 10, 20, 20, and 10 %, respectively. There was no statistically significant difference between the treatments and the controls.

Total number of offspring was 951, 847, 866 and 868 in the control, the solvent control, the nominal 0.1 mg/L and 0.2 mg/L treatment groups, respectively. There was no statistically significant difference between the treatments and the controls. Based on mean offspring per surviving parent daphnid inhibition of reproduction compared to the controls was -2 and 9 % in the nominal 0.1 mg/L and 0.2 mg/L treatment groups, respectively. There was no statistically significant difference between the treatments and the controls.

Mean measured body length at study end was 5.1, 5.1, 5.3, and 5.2 mm in the control, the solvent control, the nominal 0.1 mg/L and 0.2 mg/L treatment groups, respectively.

No behavioural abnormalities were observed.

 

Conclusion

As no statistically significant effects were observed on immobility, reproduction based on introduced parents and reproduction based on surviving parents, the NOEC for long-term effects of the test item Cyclohexylidenebis[tert-butyl] peroxide on Daphnia magna was determined to be ≥ 0.112 mg/L (LOEC > 0.112 mg/L), the highest concentration tested.

Description of key information

No effects on daphnids were observed in the long-term toxicity test. Thus, the NOEC was determined to be >= 0.112 mg/L (time-weighted mean measured concentration).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 0.112 mg/L

Additional information

A study according to OECD 211 was performed in order to evaluate the effect of Cyclohexylidenebis[tert-butyl] peroxide on the reproduction of freshwater cladocera, using the species Daphnia magna  Straus. The aim of the study was to derive a NOEC (no observed effect concentration).

Young female Daphnia (the parent animals), aged less than 24 hours at the start of the test, were exposed to the test item added to ELENDT M4 medium under semi-static conditions. Medium renewal was performed daily. The test duration was 21 days. Ten daphnia each were exposed to the test item and the controls.

At the end of the test, total number of living offspring produced per introduced parent animal and per surviving parent animal was assessed and decrease in reproduction of the daphnia was determined. The offspring of the daphnids was counted each day.

Because of the limited water solubility of the test item, acetone was used to spike the test solutions. Based on the results of a non-GLP pre-test and under consideration that only a NOEC value has to be established, a limit test was performed at nominal 0.2 mg/L. For precautionary reasons, a lower concentration was also tested, i.e. 0.1 mg/L.

The water concentration of the test item was determined using Gas Chromatography (GC) with MS detection. The measured concentrations were between 88 % and 123 % of the nominal concentration in the new solutions and between 27 % and 39 % of the nominal concentration in the old solutions. Because the measured concentrations in the test solutions were not within the range of 80 % - 120 % of the nominal concentration, the time-weighted arithmetic mean of 0.064 and 0.112 mg/L were used for determination of the biological results

 

Biological effects

Immobile daphnids were observed in all treatments and the controls. In the control, the solvent control, the nominal 0.1 mg/L and 0.2 mg/L treatment groups, observed immobility was 10, 20, 20, and 10 %, respectively. There was no statistically significant difference between the treatments and the controls.

Total number of offspring was 951, 847, 866 and 868 in the control, the solvent control, the nominal 0.1 mg/L and 0.2 mg/L treatment groups, respectively. There was no statistically significant difference between the treatments and the controls. Based on mean offspring per surviving parent daphnid inhibition of reproduction compared to the controls was -2 and 9 % in the nominal 0.1 mg/L and 0.2 mg/L treatment groups, respectively. There was no statistically significant difference between the treatments and the controls.

Mean measured body length at study end was 5.1, 5.1, 5.3, and 5.2 mm in the control, the solvent control, the nominal 0.1 mg/L and 0.2 mg/L treatment groups, respectively.

No behavioural abnormalities were observed.

 

Conclusion

As no statistically significant effects were observed on immobility, reproduction based on introduced parents and reproduction based on surviving parents, the NOEC for long-term effects of the test item Cyclohexylidenebis[tert-butyl] peroxide on Daphnia magna was determined to be ≥ 0.112 mg/L (LOEC > 0.112 mg/L), the highest concentration tested.