Di-tert-butyl sec-butylidene diperoxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Date: 15/11/2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Denomination: 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE
Chemical name: 2,2-di(tertiobutylperoxy)butane 50% in isododecane
CAS number: 2167-23-9
Batch number: 41335 512306-109
Date of reception in the test facility: 7I10/2003
Expiration date of the test item: see § 3.5.
Attributed GRL number: 0099/03
Main characteristics:
- appearance: yellow liquid
- purity: 50%
- water solubility: < 1 mg/l at 20°C according to SDS
Conservation, storage and manipulation conditions: see manufacturer safety data sheet.

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

Preliminary test:
The test solutions nominal concentrations varying from 1 mg/l to 100 mg/l. Results of the preliminary test served to define the concentration range used in the definitive test.

Definitive test:
The test solutions nominal concentrations varying from 52.4 mg/l to 150 mg/l. The definitive test was performed to a concentration range forming a geometric progression with a factor of 1.3.

Vehicle:

no

Details on test solutions:

Solutions of the test item diluted in water were prepared 20 hours before the beginning of the test (time 0).
The test item is a mixture of 2 constituents with different solubilities and physicochemicals properties. Test solutions were prepared according to the WAF methodology (Water Accommodated Fraction).
6 WAF were prepared individually by adding respectively 150mg, 115.4mg, 88.8mg, 68.4mg and 52.4mg de 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE to 1 litre of dilution water. Solution were then vigorously stirred at 20°C during 20 hours.
After this duration period, solutions were separated by filtration (HAWP 0.45μm)

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna Straus (Cladocera, Crustacea), clone 5 or clone A (from IN. Animals come from a stock breading in our laboratory reared in VOLVIC water added with vitamin B12, Na2SeO3 5H2O, MgCl2.6H2O, Ca(NO3)2.4H2O, spiruline, unicellular green fresh-water algae Pseudokirchneriella subcapitata and Chlorela vulgaris. Selection of test organisms less than 24 hours old is made by filtration.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

19 ± 1°C

pH:

7.75-8.01

Dissolved oxygen:

7.8-8.9 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0, 52.4, 68.4, 88.8, 115.4, 150 mg/l
Measured concentrations: see the table under "Any other information on results".

Details on test conditions:

Principle
Daphnia are exposed over a time period of 48 hours to a range of concentration of the test item dissolved in dilution water. Solutions are not renewed during the test. Under identical test conditions and for adequate concentration range, different
concentrations of test item have different effects on mobility of Daphnia. Consequently, at the end of the test, to each concentration corresponds a different percentage of immobilisation of Daphnia. The test item concentrations causing 0 and 100 % of immobilisation are determined by observation, whereas EC50 - 24 h and EC50 - 48 h are estimated, if possible, by calculation.
The study includes:
- A range-finding test or an evaluation of existing data in order to obtain information about the range of concentrations to be used in the main test. The test solutions of the range-finding test are generally in a concentration range of 0.1 to 100 mg/I.
- A definitive or limit test. If no inhibition occurs in the range-finding test, a limit test may be performed at a nominal concentration of the test item of 100 mg/l if the item solubility allows it. Or, a definitive test may be carried out at concentrations higher than 100 mg/l if required by the Sponsor and/or Monitor. The definitive test is performed to a concentration range forming a geometric progression with a factor not exceeding 2.2. Each concentration is prepared in triplicates.

In the absence of analytical method described and provided by the Sponsor, a method is developed and validated after the preliminary test, which has allowed to specify the necessary quantification limit. At the beginning of the definitive test and prior to addition the organisms, test solutions are sampled and chemically analysed in order to verify that initial concentrations are equivalent to nominal concentrations (soluble, stable substances). Chemical analysis of test solution are also performed at the end of the test particularly if the item stability under the test conditions has not previously been shown (i.e. final concentrations are maintained within the designated limit of 80 % of the initial concentrations).

Materials
The study was performed in a thermostated room. The temperature, continuously recorded in one test flask, remained at l 9±1°C.
Physico-chemical parameters were measured using a METTLER TOLEDO 345 pHmeter for measures of pH, and with a WTW OXI 538 oxymeter for dissolved oxygen measurement.
The study was performed using 30 ml glass bottles stoppered with PTFE bungs and sealed with aluminium caps.

Product and reagents
- Water: ultra-pure water was used for the preparation of dilution water (resistivity > 17 MΩ).
- Chemical reagents used for the preparation of dilution water were of «analytical grade».

Preparation of test solutions
Solutions of the test item diluted in water were prepared 20 hours before the beginning of the test (time 0).
The test item is a mixture of 2 constituents with different solubilities and physicochemicals properties. Test solutions were prepared according to the WAF methodology (Water Accommodated Fraction).
6 WAF were prepared individually by adding respectively 150mg, 115.4mg, 88.8mg, 68.4mg and 52.4mg of 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE to 1 litre of dilution water. Solution were then vigorously stirred at
20°C during 20 hours.
After this duration period, solutions were separated by filtration (HAWP 0.45μm)

Preliminary test
The test solutions nominal concentrations varying from 1 mg/l to 100 mg/l.
A certain volume of the test solution was added into test flasks. 5 Daphnia aged from 24 hours were added into each test flask which were then totally filled with the test solution. 2 replicates were prepared for each concentration. 2 control flasks were prepared under the same conditions, with no test item. Test flasks were incubated in darkness at a temperature of 19±1°C.
After 48 hours of incubation, mobile Daphnia were counted.
Dissolved O2 and pH were measured at the highest tested concentration and in the control.
Results of the preliminary test served to define the concentration range used in the definitive test.

Chemical analysis
The chemical analysis of the test item was carried out by HPLC-MS-MS according to the method described in Annex 5.

Definitive test
The test solutions nominal concentrations varying from 52.4 mg/l to 150 mg/l. The definitive test was performed to a concentration range forming a geometric progression with a factor of 1.3.
A certain volume of the test solution was added into test flasks. 5 Daphnia aged from 24 hours were added into each test flask which were then totally filled with the test solution.
4 replicates were prepared for each concentration. 4 control flasks were prepared under the same conditions, with no test item. Test flasks were incubated in darkness at a temperature of 19 ± 1°C.
After 24 hours of incubation, mobile Daphnia were counted and flasks replaced under incubation. At time 48 hours mobile Daphnia were counted.
Dissolved O2 and pH were measured et the highest concentration and in the control at the beginning and at all concentration and in the control the end of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7), volumetric standard quality (Merck, purity> 99.95 %)

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

9.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 9.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear over the period of the test. No precipitation was observed at the end of the test.

Results with reference substance (positive control):

Immobilisation of Daphnia in the controls:
Percentage of immobilisation in the control was 0 % at the end of the test which did not exceed 10 %.

Aspect of Daphnia in the control vessels:
Test Daphnia in the control were not trapped at the surface of the water.

Dissolved oxygen and pH were measured in the test solutions at the beginning and at the end of the test:

Nominal concentration (WAF) (mg/l)	pH*		Dissolved O2 (mg/l)*
	T0	T48h	T0	T48h
0	7.81	7.61	8.9	8.2
52.4	7.93	7.62	8.3	7.7
68.4	7.75	7.54	8.2	7.7
88.8	7.79	7.55	7.9	7.6
115.4	7.95	7.64	8.2	7.8
150	8.01	7.64	7.8	7.4

*Measurements were carried out in non inoculated solutions atT0and in inoculated solutions after 48 hours of incubation.ND:not determined.

Percentage of inhibition:

The percentage inhibition of Daphnia mobility are presented in the following table:

Nominal concentration (WAF) (mg/l)	Immobilisation (%)
	24 h	48 h
0	0	0
52.4	0	0
68.4	0	10
88.8	10	10
115.4	0	10
150	0	40

NB:in the range finding test, immobilization rates were respectively 0% up to 50mg/l and 20% for 100 mg/l.

Test item concentrations:

The following table presents the concentrations (mg/l) of 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE measured in the test solutions at the beginning (without Daphnia) and at the end (with Daphnia) of the test.

Concentration of 2,2-DI(TERTIOBUTYLPEROXY)BUT ANE 50% IN ISODODECANE
Nominal (mg/l)	Measured in solutions
	Initial (mg/l)	Final (mg/l)	Final/Initial %
0	<LD	<LD
52.4	NA	8.02	-
68.4	7.70	7.58	98
88.8	7.88	6.96	88
115.4	8.56	8.08	94
150	9.22	8.82	96

<LQ: concentration lower than the Quantification Limit of the analytical method (0.08mg/l).

As shown in the previous table, the final concentrations of 2,2-Dl(TERTIOBUTYLPEROXY)BUT ANE 50% IN ISODODECANE were maintained within the designated limit of 80% of the initial concentrations in test solutions. Thus, the test item concentrations were maintained throughout the duration of the test.

Since the initial measured concentrations in non-inoculated flasks were not equal in value to the nominal concentrations, calculations of effective concentration EC₅₀ was performed using measured values.

Effective concentrations:

Annex 2 presents percentage of immobilisation after 24 and 48 hours of exposure. The first table shows raw data. Results used for calculations are shown in the second table.

The following table show results obtained by regression analysis using the Probit/log model:

Effective concentrations (mg/l)
	Value	95% CI
EC50-24h	>9.2	ND
EC50-48h	9.4	ND

CI:confident interval;ND:not determined

Quality criteria of the study:

Teneur en oxygene et pH:

The concentration of dissolved oxygen in the test vessels remained above 2 mg/l at the end of the test and pH did not vary by more than 1 unit.

Item concentrations:

The final concentrations (see § 6.3.) of 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE were maintained within the designated limit of 80% of the initial concentrations in test flasks.

Validity criteria fulfilled:

yes

Conclusions:

The determination of the inhibition of mobility (acute toxicity) of the freshwater Daphnia magna exposed to the test item 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE for a duration of 48 hours was performed following the method C2
described in Directive 92/69/EEC of the European Commission and in the guideline 202 (part I) of the OECD.
For each exposure concentration, the percentage of immobilisation after 24 hours and 48 hours was recorded. Endpoints were calculated by regression analysis using the Probit/log model. EL50-48h derived from loading rate of the WAF is reported.

The quality criteria of the study were all respected.

Executive summary:

The acute toxicity (inhibition of mobility) of the test item 2,2 -DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE to Daphnia magna was assessed according to the method C2 of the European Directive 92/69/CEE and in the guideline 202 (part I) of the OECD.

Daphnia were exposed to a range of concentrations of 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE dissolved in dilution water. The toxic effect measured during the assay was the inhibition of mobility after time periods of 24 hours and 48 hours.

Since 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE is sparingly soluble, the test was carried out with concentrations up to its water solubility. The test was performed using closed flasks as testing vessels, flasks were entirely filled with test solutions and closed with butyl rubber caps covered with PTFE. Daphnia were exposed in a static test to a concentration range of 52.5 to 150 mg/l, forming a geometric progression with a factor of 1.3. The test was performed with 5 Daphnia per vessel. Testing flasks were incubated in darkness at 19 ± 1 for 48 hours. For each exposure concentration, the percentage of immobilisation after 24 hours and 48 hours was recorded.

For each exposure concentration, the percentage of immobilisation after 24 hours and 48 hours was recorded. EC₅₀-24h and EC₅₀-48h were calculated by regression analysis using the Probit/log model. The results are presented in the following table:

Effective concentrations (mg/l)
	Value	95% CL
EC50-24h	>9.2	ND
EC50-48h	9.4	ND

Concentrations of 2,2-DI(TERTIOBUTYLPEROXY)BUTANE 50% IN ISODODECANE were measured by liquid chromatography coupled with mass spectrometry. Initial and final concentrations were not equivalent to nominal concentrations. Thus, effective concentrations shown in the previous table have been calculated using the initial measured concentrations of 2,2-DI(TERTIOBUTYLPEROXY)BUT ANE 50% IN ISODODECANE.

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear over the period of the test. No precipitation was observed at the end of the test.

The study was performed in compliance with its quality criteria: immobilisation in the control did not exceed 10 % at the end of the test; test Daphnia in the control were not trapped at the surface of the water; concentration of dissolved oxygen in the test vessels remained above 2 mg/l at the end of the test and pH did not vary by more than 1 unit; the concentrations have been maintained to within 80 % of the initial concentration throughout the duration of the test.

The study was performed according to the OECD principles of Good Laboratory Practice (GLP).

Description of key information

A valid acute Daphnia study according to OECD 202 performed under GLP is available.

50% effect on mobility was not reached, therefore EL50 (48h) may be expressed as >150 mg/L loading rate (the highest test concentration).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

150 mg/L