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

Biodegradation in water and sediment: simulation tests

Administrative data

Endpoint:

biodegradation in water: sewage treatment simulation testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was performed according to modified OECD Test Guidelines to permit prolonged measurements with no GLP statement. This study was reliable with restrictions because all validity criteria could not be verified. No information on oxygen depletion in the inoculum blank is available

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,5-dimethyl-2,5-di(tert-butylperoxy)hexane

IUPAC Name:

2,5-dimethyl-2,5-di(tert-butylperoxy)hexane

Constituent 2

Reference substance name:

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

EC Number:

201-128-1

EC Name:

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

Cas Number:

78-63-7

Molecular formula:

C16H34O4

IUPAC Name:

2,5-bis(tert-butylperoxy)-2,5-dimethylhexane

Test material form:

other: liquid

Details on test material:

Name of test material (as cited in study report): Trigonox 101 (2,5-dimethyl-2,5-di(tert-butylperoxy)hexane).

Radiolabelling:

no

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge to inoculate the test at the start was collected from the wastewater treatment plant (WWTP) Nieuwgraaf in Duiven, The Netherlands.
The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic sewage. 0.35 liter of secondary activated sludge containing approximately 3 g/L dry weight was used as an inoculum for each CAS unit. This dry weight was obtained by diluting the sludge obtained from the treatment plant.
To maintain an activated sludge concentration between 2.0 and 3.0 g/L, secondary activated sludge containing approximately 3 g/L dry weight was collected weekly. The primary settled sewage was collected from the same plant weekly and stored frozen until required. Before use 10 mL/L of a NaHCO3 (10 g/L) solution was added.

Duration of test (contact time):

59 d

Initial test substance concentration

Initial conc.:

10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Details on study design:

The CAS unit was started with activated sludge. The aeration was achieved by operating an air-lift. The aeration rate was regulated so that the activated sludge was kept in suspension and the dissolved oxygen concentration was at least 2 mg/L. This oxygen concentration in the aeration vessel was measured at least two times a week. The domestic sewage was supplied at a rate of approximately 1.4 L/day to give a hydraulic retention time of 6 hours. The flow was checked by measuring the total volume of effluent over a 24-hour period. After brushing, 35 mL of sludge was daily removed from the aeration tank to maintain a sludge retention time of 10 days. To maintain an activated sludge concentration between 2 and 3 g/L dry weight in the CAS unit, 20 mL of unacclimated secondary activated sludge containing approximately 3 g/L dry weight was added daily. The effluent samples (50 mL) were taken from the settler.
2,5-dimethyl-2,5-di(tert-butylperoxy)hexane was directly added to the test unit using a syringe pump. To prevent precipitation of the stock suspension in the syringe the stock suspension was stirred by using a small magnetic stirrer bar inside the syringe. The flow rate of the syringe pump was 9.6 mL/day giving a nominal concentration of the test substance in the influent of the unit of 10 mg/L at a sewage supply rate of 1.4 L/day.
Specific analyses of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane were used to determine the primary removal of the test substance.

Results and discussion

% Degradation

% Degradation 1

Parent/product:

parent

% Degr.:

99.95

Parameter:

test mat. analysis

Sampling time:

58 d

Remarks on result:

other: removal from effluent

% Degradation 2

Parent/product:

parent

% Degr.:

99.89

Parameter:

test mat. analysis

Sampling time:

59 d

Remarks on result:

other: removal from effluent

Transformation products:

no

Details on results:

The incubation temperature of the CAS units ranged from 19.5 to 20.2°C. The pH of the effluent of the CAS units varied from 6.9 to 7.4. The oxygen concentrations measured in both units were always ≥4.0 mg/L. These test conditions are believed to allow biodegradation by micro-organisms present in activated sludge.
The CAS test was started with a high concentration of aerobic micro-organisms (3.0-3.1 g/L dry weight) maintained by the daily addition of primary settled sewage and sludge from a full-scale treatment plant. The daily removal of 35 mL of activated sludge from the aeration vessel resulted in a sludge retention time of 10 days. The dry weight in the CAS units ranged from 2.6 to 3.2 g/L.
Aqueous and sludge samples spiked with 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane were analyzed by LC-MS/MS to determine the recovery.
Fortified aqueous samples were prepared at a concentration of 10 mg/L 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane and fortified sludge samples were prepared at a concentration of 400 mg/L 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane. Recoveries were higher than the general prescribed recovery range from 70-110% of the nominal concentration. Higher recoveries might be caused due to the spiking of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane in an suspension with Tween 80. Measured concentrations of the effluent and mixed liquid suspended solids samples might therefore be overestimated.
The concentration of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane in the effluent of the CAS unit on day 58 and 59 were < 5 and 11 μg/L. These concentrations correspond with a removal of 99.95 and 99.89%. The average concentrations of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane in the mixed liquid suspended solids (activated sludge) of the reactor were 2.6 and 3.0 mg/L,
respectively. Based on these concentrations in the mixed liquid suspended solids 0.65% and 0.75% removal by adsorption was calculated.

Any other information on results incl. tables

Removal of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane in a properly operating conventional biological wastewater treatment plants is assumed to be primarily achieved through biodegradation. Formation of intermediates is not detected by the specific analysis therefore the results found in the CAS test are only prove of primary biodegradation.
Based on results found in closed bottle tests, inoculated with unacclimated and acclimated activated sludge, it is concluded that it is unlikely that 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is degraded by microorganisms growing on this substance under the conditions of the ready biodegradability tests. Addition of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane (9 mg/l organic carbon) in a semi continuously-fed activated sludge (SCAS) test did not result in an increase of the organic carbon concentration demonstrating that water soluble persistent substances were not formed (van Ginkel, 2011). It is also hypothized by van Ginkel (2011) that the peroxide bond of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is co-metabolically reduced by microorganisms possessing reductases. Reduction of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane results in the release of the intermediates 2,5-dimethyl-2,5-hexanediol and tert-butanol which both degrade >80% within 56 days in standard closed bottle tests.
Biodegradation of 2,5-dimethyl-2,5-hexanediol and tert-butanol in the closed bottle tests using unacclimated sludge as inoculum starts with a lag period of 2-4 weeks, whereas the biodegradation starts immediately using sludge from a SCAS test fed with 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane. This suggests that the intermediates 2,5-dimethyl-2,5-hexanediol and tert-butanol are formed in the SCAS test and subsequently are biodegraded. The same mechanism of co-metabolic reduction and subsequent biodegradation may also apply for removal of 2,5-dimethyl-2,5-di(tert-butyperoxy)hexane in the CAS test. Formation of other adsorbing intermediates by different (bio)degradation mechanisms could also explain the high removals but are expected very unlikely.
Primary biodegradation of the test substance was shown in a continuously-fed activated sludge unit treating domestic wastewater. Based on biodegradation results found in SCAS and CBT tests 2,5-dimethyl-2,5-di(tert-butyperoxy)hexane is probably biodegraded ultimately (complete) in the CAS test.

Applicant's summary and conclusion

Conclusions:

Since very low concentrations of the test substance were found in effluent, the CAS test demonstrates that 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane, is nearly completely removed from the wastewater in conventional biological wastewater treatment plants.

Executive summary:

The continuous activated sludge (CAS) test was performed according to ISO (1995), EC (1988) and OECD (1981) Guidelines. 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane was exposed to micro-organisms maintained by addition of domestic wastewater in the CAS test. 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane was spiked at a nominal influent concentration of 10.0 mg/L test substance for a period of 59 days. An accurate assessment of the removal of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane was established with specific analyses using LC-MS/MS.
The average removal percentage in the test unit was quantified with the specific analysis on day 58 and 59 was 99.95% and 99.89%, respectively. These analyses demonstrate that the removal of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane from the wastewater is nearly complete. Average concentrations of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane in the sludge of the reactor sampled on days 58 and 59 were 2.6 and 3.0 mg/L, respectively. The mean removal percentage of 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane from the influent through adsorption onto sludge assessed is therefore assumed to be only 0.65% and 0.75%. The low removal percentage by adsorption suggests that 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is primarily removed by (bio)degradation.
In conclusion, the CAS test demonstrates that 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane, is removed from the wastewater in conventional biological wastewater treatment plants. 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane is probably removed by biodegradation. Formation of intermediates is not detected by the specific analysis and therefore the results found in the CAS test are only prove of primary (bio)degradation. Ultimate (complete) biodegradation is
suggested in combination with results found in SCAS and CBT tests.