Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 A (Inherent Biodegradability: Modified SCAS Test)

GLP compliance:

no

Specific details on test material used for the study:

The potential decomposition products tert-butanol and 2,5-dimethyl-2,5-hexanediol were purchased from Acros, Geel, Belgium.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge and primary settled sewage were collected from the WWTP Nieuwgraaf in Duiven, The Netherlands. The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic sewage. The primary settled sewage was collected weekly and stored at -20°C until required. 150 ML of secondary activated sludge containing approximately 2 g DW/L of suspended solids was used as an inoculum for each unit.

Duration of test (contact time):

8 wk

Initial conc.:

1 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

other: NPOC

Details on study design:

The SCAS test was performed according to Test Guidelines (OECD, 1981). The test was performed in 150 ml SCAS units. At the start each SCAS unit was filled with 150 ml of activated sludge and the aeration was started. After 23 hours the aeration was stopped and the sludge was allowed to settle for 45 minutes. After settling 100 ml of the supernatant liquor was withdrawn from the tap. Subsequently, 100 mL of primary settled sewage was added to the control unit, and
100 ml of primary settled sewage and 1 µl of the test substance were added to the test unit. The reason for the direct administration of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is the insolubility of the substance in water. Aeration was started again and continued for 23 hours. The unit was operated at a sludge retention time of 30 days by daily removing 5 mL of activated sludge before the settling period. The above fill and draw procedure was repeated daily.
Supernatant drawn off was analyzed for non-purgeable organic carbon (NPOC).
The NPOC values were used to follow the removal of the test substance.

Details on results:

The calculated organic carbon concentration of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane in the influent of the SCAS test was 9 mg/L. The incubation temperature of the SCAS units ranged from 18 to 22°C. The pH of the effluent of both SCAS units varied from 6.5 to 7.3. The SCAS test was started with a high concentration of activated sludge (2 g DW/L) maintained by the daily additions of sewage. The test substance caused no reduction of the removal of organic carbon present in the sewage. Therefore 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is considered to be non-inhibitory to the activated sludge.
Before the additions of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane, the effluent NPOC values obtained from the test compound unit and the control unit are comparable and constant. Additions of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane did not result in an increase of the organic carbon concentration. An increase due to the presence of the organic peroxide in the aqueous phase was not expected due to the poor water solubility of the test substance.
The comparable NPOC values during the test period demonstrate that water soluble persistent substances were not formed.

Executive summary:

In the SCAS test, 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane was exposed to activated sludge maintained by daily addition of sewage for a period of 8 weeks.
The test substance caused no reduction of the biodegradation of the organic compounds present in primary settled sewage.
2,5-Dimethyl-2,5-di(tert-butylperoxy)hexane was not degraded in prolonged Closed Bottle tests inoculated with both unacclimated and acclimated activated sludge from SCAS units. This result demonstrates that microorganisms do not have the ability to grow on this organic peroxide prevailing in ready biodegradability tests. Potential decomposition products of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane i.e. tert butanol and 2,5-dimethyl-2,5-hexanediol were degraded in Closed Bottle tests with both acclimated and unacclimated sludge. The length of the lag period found with these biodegradation products with acclimated sludge was short compared to the period obtained with unacclimated sludge. This result strongly suggests that the peroxide bond of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is cleaved in the SCAS  unit.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

Also adapted sludge from a SCAS unit was used.

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: Inocula were derived from SCAS units (see details on inoculum)

Details on inoculum:

The inocula were derived from the control unit (unacclimatized) and the SCAS unit (acclimatized). See RSS 78-63-7, Biodegradation in water: SCAS, van Ginkel, 2011, SS.
Concentration of sludge: approximately 2 g DW/L of suspended solids

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Use was made of bottles containing only inoculum, and bottles containing the test substance. The concentration of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane, tert butanol and 2,5-dimethyl-2,5-hexanediol (the potential decomposition products) in the bottles was 2 mg/L. The inoculum was diluted to 2 mg DW/L in the closed bottles. The inocula were derived from the communal wastewater treatment plant (unacclimatized) and the SCAS unit (acclimatized). Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. Subsequently the bottles were closed and incubated in the dark. Bottles of all series were withdrawn for analyses of the dissolved oxygen concentration. The course of the oxygen decrease in the bottles was measured using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

Test performance:

Inhibition of the endogenous respiration of the inocula by the test substances was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentrations of the test substances is expected. The pH of the media was 7.0 at the start of the test. The pH of the medium at day 28 was 6.8 to 7.0. Temperatures ranged from 19 to 21°C. The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Parameter:

% degradation (O2 consumption)

Value:

ca. 0

Remarks on result:

other: after average 60 days

Details on results:

The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period.

See Figure in the "Attached background material".
2,5-Dimethyl-2,5-di(tert-butylperoxy)hexane is not biodegraded in the prolonged Closed Bottle test inoculated with unacclimated sludge.
Biodegradation of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane was also not noted in the prolonged Closed Bottle test inoculated with sludge from the SCAS test. The sludge from the SCAS test was exposed to the test substance for 8 weeks allowing acclimatization. Based on these results it is concluded that it is very unlikely that 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is biodegraded by microorganisms capable of growing on this test substance under completely aerobic conditions.

Results with reference substance:

Not applicable

2,5-Dimethyl-2,5-hexanediol and tert butanol are potential intermediates in the biodegradation process of the organic peroxide. 2,5-Dimethyl-2,5-hexanediol and tert butanol are degraded >80% within 56 days in the Closed Bottle test (See Figure in the "Attached background material"). Both potential intermediates were also as expected degraded in Closed Bottle tests inoculated with sludge exposed to 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane. The degradation of 2,5-dimethyl-2,5-hexanediol with sludge exposed to the peroxide started almost immediately whereas with unacclimated sludge, lag periods of 2 to 4 weeks were noted. The absence of a lag period indicates that 2,5-dimethyl-2,5-hexanediol is formed in the SCAS unit fed with 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane. This strongly indicates that 2,5-dimethyl-2,5-hexanediol was produced from 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane in the SCAS unit. It is hypothized that 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is converted chemically in the SCAS unit during the settle period or with the help of chemicals excreted by the activated sludge present in the unit. During the settle period anaerobic conditions (reducing conditions) are created because aeration is stopped. Conversion of highly oxidized peroxide bonds is more likely under reducing anaerobic conditions. It is also possible that the peroxide bond of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane-2,5-di(tert-butylperoxy)hexane is reduced by microorganisms possessing reductases (enzymes) capable of inactivating toxic lipid peroxides. Microorganisms are present at very low concentrations in ready biodegradability tests. At these low concentrations, activity of these reductases may be insufficient to reduce the organic peroxides administered in the Closed Bottle test.

Validity criteria fulfilled:

not specified

Remarks:

all validity criteria could not be verified.

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

2,5-Dimethyl-2,5-di(tert-butylperoxy)hexane was not degraded in prolonged Closed Bottle tests inoculated with unacclimated and acclimated sludge from the SCAS unit. However, potential decomposition products of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane i.e. tert butanol and 2,5-dimethyl-2,5-hexanediol were degraded in Closed Bottle tests with both acclimated and unacclimated sludge.

Executive summary:

This study was performed according to modified OECD Test Guidelines to permit prolonged measurements with no GLP statement.

2,5-Dimethyl-2,5-di(tert-butylperoxy)hexane was not degraded in prolonged Closed Bottle tests inoculated with unacclimated and acclimated sludge from the SCAS unit. This result demonstrates that microorganisms do not have the ability to grow on this organic peroxide under aerobic conditions. Potential decomposition products of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane i.e. tert butanol and 2,5-dimethyl-2,5-hexanediol were degraded in Closed Bottle tests with both acclimated and unacclimated sludge. The length of the lag period found with 2,5-dimethyl-2,5-hexanediol with acclimated sludge was short compared to the period obtained with unacclimated sludge. This result suggests that the peroxide bond of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is cleaved in the SCAS unit.

Description of key information

One key study was available on the biodegradation of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane. The substance was not degraded in prolonged Closed Bottle tests inoculated with unacclimated and acclimated sludge from the SCAS unit. However, potential decomposition products of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane i.e. tert-butanol and 2,5-dimethyl-2,5-hexanediol were degraded in Closed Bottle tests with both acclimated and unacclimated sludge (>80% within 56 days).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

One key study was available on the biodegradation of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane. This study was performed according to modified OECD Test Guidelines to permit prolonged measurements with no GLP statement. In the SCAS test, 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane was exposed to activated sludge maintained by daily addition of primary settled sewage for a period of 8 weeks. The test substance caused no reduction of the biodegradation of the organic compounds present in primary settled sewage. Therefore the test substance is considered to be non-inhibitory to the activated sludge.

2,5-dimethyl-2,5-di(tert-butylperoxy)hexane was not degraded in prolonged Closed Bottle tests inoculated with unacclimated and acclimated sludge from the SCAS unit. This result demonstrates that microorganisms do not have the ability to grow on this organic peroxide under aerobic conditions. Potential decomposition products of the test substance i.e. tert butanol and 2,5-dimethyl-2,5-hexanediol were degraded in Closed Bottle tests with both acclimated and unacclimated sludge (> 80% within 56 days).The length of the lag period found with 2,5-dimethyl-2,5-hexanediol with acclimated sludge was short compared to the period obtained with unacclimated sludge. This result suggests that the peroxide bond of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is cleaved in the SCAS unit.