Di-tert-butyl sec-butylidene diperoxide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 10 August 2000 Experimental completion date: 8 September 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

yes

Remarks:

The temperature in the culture room dropped several times below 20°C during the test (minimum of 19°C each time). This minor deviation was not considered to have compromised the validity or integrity of the study.

Qualifier:

according to guideline

Guideline:

other: Commission Directive 92/96/EEC

Deviations:

yes

Remarks:

The temperature in the culture room dropped several times below 20°C during the test (minimum of 19°C each time). This minor deviation was not considered to have compromised the validity or integrity of the study.

Qualifier:

according to guideline

Guideline:

other: Commission Directive 93/21/EEC

Deviations:

yes

Remarks:

The temperature in the culture room dropped several times below 20°C during the test (minimum of 19°C each time). This minor deviation was not considered to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test substance 2,2-DI-(T-BUTYLPEROXY)BUTANE IN ISODODECANE was supplied by
ATOHNA.
It was identified as follows:
name:
- protocol: 2,2-DI-(T-BUTYLPEROXY)BUTANE IN ISODODECANE
- labelling: LUPEROX 220EM50
- ATOHNA trade name: LUPEROX 220 M50
All names correspond to the same test substance.
batch number:
- protocol and labelling: 11 579-99 10- 1 02
date of receipt: 1 December 1 999
container: one plastic flask
description: colourless liquid
storage conditions: at room temperature
composition
- peroxide content: approximately 50%
- ISODODECANE content: approximately 50%
carbon content:
- test substance: 72.7% (weight/weight)
- peroxide: 6 1 .5% (weight/weight).

The test substance stabilizing agent was s upplied by A TOHNA.
It was identified as follows:
name:
- protocol: ISODODECANE
- labelling: ISODODECAN
B oth names correspond to the same substance.
batch number:
- protocol and labelling: 08. 1 1 .99
date of receipt: 1 December 1 999
container: one plastic flask
description: colourless liquid
storage conditions: at room temperature
carbon content: 84.6% (weight/weight).

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Type: inoculum was collected from a water treatment plant containing effluent from a predominantly domestic origin.
Collected from the water treatment plant Emeraude (SIARR) (761 41 Petit-Quevilly, France).

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparation of the solutions
The test substance and the test substance stabilizing agent were weighed on two superposed glass microfibre filters and these filters were placed directly into test vessels containing 3 litres of mineral medium.
The quantity of test substance (2,2-DI-(T-BUTYLPEROXY)BUTANE IN ISODODECANE) added per flask was 94.9 mg, corresponding to a peroxide (2,2-DI-(T-BUTYLPEROXY) BUTANE) concentration of 10.0 mg/L of TOC.
The quantity of stabilizing agent (ISODODECANE) added per flask was 47 .6 mg, corresponding to that added to the test solution flasks of the CIT/Study No. 19578 ECS via the test substance (and also to 13.4 mg/L of TOC).
The reference substance (sodium acetate) was placed directly into the test vessel containing 3 litres of mineral medium. The quantity added was 102.4 mg, corresponding t o 10.0 mg/L of TOC.
When test, stabilizing and reference substances were placed into the appropriate test vessels, two superposed glass microfibre filters were also placed into the two blank test vessels and the test vessel containing the reference substance.

TEST SYSTEM
Conditions during preconditioning
During the preconditioning period, the conditions were set as follows:
Temperature: 22 ± 2°C. The temperature in the culture room was recorded continuously
and records retained.
Aeration: air was bubbled through the inoculum until use.
Mineral medium:reconstituted water (OECD and EEC recommended) was prepared using
deionised water which contained no more than 5 % of the organic carbon
content introduced by addition of the test or reference substance and
analytical grade reagents (see appendix 2 for details of fabrication).
The mineral medium is checked periodically for carbon content by Total
Organic Carbon analysis (analysis undertaken by the Laboratoire
Departemental d'Analyses, 27000 Evreux, France).
Conditions during the test
The test conditions were set as follows:
Temperature: between 1 9 °C and 24°C.
Illumination: the test was carried out in dark glass bottles fitted with dark glass
stoppers and aeration tubes to reduce the quantity of light reaching the
test suspensions.
Culture homogeneity: in order to homogenize the solution, all test suspensions were agitated
using magnetic stirrers.
Aeration: air was bubbled through each parallel at the rate of 30 - 1 00 mUmin
(checked daily and reset if necessary) during the test.
pH: was measured before the beginning of the test in the mineral medium and
at the end of the test in all test suspensions.
Loading: test vessels were loaded at 3 litres of suspension per flask.

TREATMENT
Test system
Seven flasks were used to determine the quantity of carbon dioxide evolved by the degradation of the peroxide 2,2-DI-(T-BUTYLPEROXY)BUTANE: two flasks containing the inoculum (inoculum blanks), two flasks containing the test substance (to obtain a peroxide concentration of 1 0.0 mg/L of TOC) and inoculum (test solutions), two flasks containing a known quantity of ISODODECANE and inoculum (stabilizing agent solutions), one flask containing the reference substance sodium acetate (to obtain a concentration of 10.0 mg/L of TOC) and inoculum (procedure control).
The three control flasks (inoculum blanks and procedure control) and the two ISODODECANE flasks (stabilizing agent solutions) were those of the CIT/Study No. 19578 ECS.
Parallel groups were prepared by adding 2.4 litres of mineral medium to each of the test flasks.
Inoculum was added to provide a final concentration of 12.0 mg/L of suspended solids (dry weight) in 3 litres of suspension.
The test flasks were aerated with C02-scrubbed air overnight to purge the system of carbon dioxide and then attached in parallel to a series of three wash bottles filled with 100 mL of 0.0125 M barium hydroxide solution (to trap any CO2 released from the test vessels).
The test and reference substances were added, as appropriate, to the flasks to give a peroxide and reference substance concentrations of 10.0 mg of TOC per litre (94.9 mg of 2,2-DI-(T-BUTYLPEROXY)BUTANE IN ISODODECANE and 102.4 mg of sodium acetate) .
The quantity of ISODODECANE added to the stabilizing agent solution was 47.6 mg, corresponding to that added to the test solution flasks of the CIT/Study No. 19578 ECS via the test substance (and also to 13.4 mg/L of TOC).
When all the substances had been added, the volume of the suspensions was made up to 3 litres.
Carbon dioxide-scrubbed air was bubbled through the suspensions at the same rate for all preparations.
Measurements of C02 were made at the following times: days 1, 4, 6, 8, 11, 14, 18, 22, 25 and 29.
For each measurement, the first wash bottle nearest to the test flask was disconnected and titrated with 0.05 M HCl, using phenolphthalein as an indicator.
For calculation purpose, it was assumed that the volume necessary to titrate untitrated wash bottle would be the same as the volume needed to titrate 100 rnL of the B a(OH)2 s tock solution.
The remaining C02 absorber bottles were connected to the test flasks so that the second wash bottle replaced the first one and an extra bottle containing fresh barium hydroxide solution was added to the far end of the series.
Each time C02 was analyzed, 100 mL of the barium hydroxide stock solution (used to fill the wash bottles) was titrated with the HCl solution in order to determine the maximum amount of acid required to titrate the wash bottles.
Barium hydroxide (from analytical grade B a(OH)z crystals) arid hydrochloric acid solutions (from IM analytical grade HCl) were made up as necessary.
On the 28th day, 1 mL of concentrated hydrochloric acid was added to each flask to stop the biodegradation and aerated overnight to drive off the remaining carbon dioxide. A final CO2 analysis of all wash bottles was made on the 29th day, this final analysis being representative of biodegradation of the 28th day.

Reference substance:

other: Sodium Acetate

Key result

Parameter:

% degradation (CO2 evolution)

Value:

48

Sampling time:

28 d

Details on results:

VALIDITY CRITERIA
All validity criteria were respected:
biodegradation values of peroxide contained in test substance replicates did not deviate by more than 20% at the end of the 10-day window (the 10 days immediately following the attainment of 10% biodegradation) and at the end of the test, biodegradation in the reference test was 6 1% after 14 days then it was at least 60% within this period, the blank value (average of the two controls) was less or equal to 70 mg of C02/L at the end of the test (32.6 mg/L).

WATER QUALITY
The minimum and maximum values of parameters measured during the test were:
temperature: 19°C and 24°C in the culture room, throughout the test,
pH: 7.58 measured in the mineral medium before the start of the test; 8.01 to 8.45 measured in the test flasks at the end of the test.

BIODEGRADATION
The 10-day window of the peroxide started on the 8th day. Biodegradation of 2,2-DI-(TBUTYLPEROXY) BUTANE totalled 29% (mean of the two flasks) at the end of this 10-day window on the 18th day and 48% over the test period.

Results with reference substance:

The reference compound degraded normally under the test conditions.

Cumulative percentage of biodegradation

	Isododecane			Peroxide			Reference Substance
day	No. 1	No. 2	Average	No. 1	No. 2	Average
1	0	0	0	0	0	0	1
4	0	0	0	0	2.1	1.05	16.68
6	0.41	1.15	0.78	2.83	5.53	4.18	36.72
8	0.41	1.6	1	7.74	10.43	9.09	46.51
11	1.12	2.23	1.67	14.71	17.91	16.31	54.25
14	1.12	2.23	1.67	20.55	22.95	21.75	61.49
18	1.12	2.23	1.67	27.7	29.8	28.75	65.74
22	1.45	2.23	1.84	34.12	34.62	34.37	70.09
25	1.45	2.23	1.84	39.17	38.17	38.67	73.53
28	2.71	3.94	3.33	47.73	48.63	48.18	80.53

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The biodegradation of 2,2-DI-(T-BUTYLPEROXY)BUTANE reached 29% at the end of the 10-day window (the 10 days immediately following the attainment of 10% biodegradation) and 48% at the end of the test.

Under our experimental conditions, the peroxide 2 ,2-DI-(T-BUTYLPEROXY)BUTANE was therefore non readily biodegradable in the 28-day modified Sturm test.

Executive summary:

At the request of ATOHNA, Paris-la-Defence, France, the ready biodegradability of the peroxide 2,2-DI-(T-BUTYLPEROXY)BUTANE was evaluated using a 28-day modified Sturm test according to Commission Directive 92/69/EEC (C4-C., 31 st July, 1992), Commission Directive 93/2 1/EEC (27th April 1993) and OECD guideline No. 301 B (17th July 1992).

In order to classify a test substance as readily biodegradable: the biodegradation value of 60% has to be reached in the 10-day window (the 10 days immediately following the attainment of 10% biodegradation) within 28 days according to Commission Directive 93/21 /EEC and OECD guideline No. 301 B, or the biodegradation must exceed 70% after 28 days according to Commission Directive 93/21/EEC.

Methods

The test substance contained approximately 50% of peroxide (2,2-DI-(T-BUTYLPEROXY) BUTANE) and 50% of stabilizing agent (ISODODECANE).

The test substance was dispersed in reconstituted. water (OECD mineral medium) prepared from deionised water with a conductivity < 10 μS/cm.

Seven flasks were used to determine the quantity of carbon dioxide evolved by the degradation of the peroxide:

two flasks containing the inoculum (inoculum blanks), two flasks containing the test substance (to obtain a peroxide concentration of 10.0 mg/L of TOC) and inoculum (test solutions), two flasks containing a known quantity of ISODODECANE and inoculum (stabilizing agent solutions), one flask containing the reference substance sodium acetate (to obtain a concentration of 10.0 mg/L of TOC) and inoculum (procedure control).

The inoculum consisted of sewage sludge sampled from the aeration tank of a sewage treatment plant and then aerated for 7 days. Inoculum concentration was 12.0 mg/L (dry weight) in all test vessels.

CO2 scrubbed air was bubbled through the flasks for the 28-day test period. Environmental parameters were recorded as:

pH: 7.58 to 8.45,

temperature: l 9 °C to 24°C.

Results

Validity criteria

All validity criteria were respected:

biodegradation values of peroxide contained in test substance replicates did not deviate by more than 20% at the end of the 10 -day window (the 10 days immediately following the attainment of 10% biodegradation) and at the end of the test, biodegradation in the reference test was at least 60% within 14 days, the blank value (a measurement of CO2 evolved uniquely from the breakdown of organic

matter in the inoculum, mineral medium, etc ... ) was less or equal to 70 mg of CO2 evolved per litre of suspension at the end of the test (average of the two controls).

Peroxide biodegradation

The 10-day window of the peroxide started on the 8th day. Biodegradation of 2,2-DI-(T BUTYLPEROXY)BUTANE totalled 29% at the end of this 10-day window (18th day) and 48% at the end of the test.

Conclusion

Under our experimental conditions, the p eroxide 2,2-DI-(T-BUTYLPEROXY)BUTANE was non readily biodegradable in the 28-day modified Sturm test.

Description of key information

The biodegradation of 2,2-DI-(T-BUTYLPEROXY)BUTANE reached 29% at the end of the 10-day window and 48% at day 28, the end of the test.

Under experimental conditions, the peroxide 2 ,2-DI-(T-BUTYLPEROXY)BUTANE was therefore non readily biodegradable in the 28-day modified Sturm test.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria