Cyclohexylidenebis[tert-butyl] peroxide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: screening test, other

Remarks:

A Semi-Continuous fed Activated Sludge (SCAS) test was performed according to modified OECD Test Guidelines 302A and exposed sludge was used in a consequently OECD test guideline 301D

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No GLP certificate

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Oxygen conditions:

aerobic/anaerobic

Remarks:

aerobic condition for SCAS, anaerobic conditions for Closed Bottel test.

Inoculum or test system:

other: activated sludge, domestic, not adapted for SCAS, adapted for Closed Bottle test.

Remarks:

Secondary activated sludge and primary settled sewage were obtained from a wastewater treatment plant. Pre-exposed activated sludge from the SCAS test unit were used as inoculum in the Closed Bottle test.

Details on inoculum:

The primary settled sewage was collected weekly and stored in a refrigerator until required. A volume of 150 mL of secondary activated sludge containing approximately 2.0 g DW (dry weight)/L of mixed liquid suspended solids (MLSS) was used as inoculum for SCAS units.

Pre-exposed activated sludge from both the isododecane control and the test SCAS unit were used as inoculum in the Closed Bottle test. Pre-exposed activated sludge from the test SCAS unit was sampled after 11 and 30 weeks of operation. Pre-exposed activated sludge from the isododecane control SCAS unit was sampled after 25 weeks of operation.

Duration of test (contact time):

30 wk

Initial conc.:

16 mg/L

Based on:

test mat.

Remarks:

SCAS test concentration

Initial conc.:

2 mg/L

Based on:

test mat.

Remarks:

Closed Bottle test concentration

Parameter followed for biodegradation estimation:

other: NPOC (non-purgeable organic carbon) for SCAS test

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

for closed bottle test

Details on study design:

Secondary activated sludge and primary settled sewage were obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands, treating predominantly domestic wastewater. The primary settled sewage was collected weekly and stored in a refrigerator until required. A volume of 150 mL of secondary activated sludge containing approximately 2.0 g DW (dry weight)/L of mixed liquid suspended solids (MLSS) was used as inoculum for SCAS units.

The SCAS unit were 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 one hour. 100 mL of the supernatant liquor was withdrawn from the tap of the unit. Subsequently, 100 mL of primary settled sewage was added, and the aeration was started. This fill and draw cycle was repeated daily for one week. After one week 100 mL of the settled supernatant liquor was withdrawn from the tap and subsequent 100 mL of primary settled sewage and 20 µL of a ~100 g/L stock solution of the test substance in methanol ( ~80 g/L 1,1-di(tert-butylperoxy)cyclohexane) was administrated to the test SCAS unit.

This resulted in an 1,1-di(tert-butylperoxy)cyclohexane and methanol influent concentration of 16.0 mg/L and 158 mg/L, respectively. Aeration was started again and the above fill and draw procedure for dosing the test substance was repeated five times per week throughout the test.

The performance of the SCAS units was followed by measuring the Non-Purgeable Organic Carbon (NPOC) content in the effluent of the test unit and to compare this with the NPOC content in a control SCAS unit and an Isododecane control SCAS unit. Both control units were operated identically to the test SCAS unit. The control SCAS unit was dosed only with primary settled sewage, and the isododecane control SCAS unit was dosed with primary settled sewage that was supplemented with isododecane and methanol. The isododecane and methanol concentration in the influent of the isododecane control SCAS unit was 4.3 mg/L and 158 mg/L, respectively. The isododecane was dosed to the isododecane control SCAS unit from a ~21 g/L isododecane stock solution in methanol (20µL / 100 ml primary settled sewage).

To determine the NPOC in the effluent of the SCAS units, the samples were filtered using cellulose nitrate filters (8 μm pore size) to remove sludge particles from the effluent. The filtered samples were acidified prior to injecting in TOC apparatus.

The total removal of 1,1-di(tert-butylperoxy)cyclohexane in the test SCAS unit from the wastewater, the removal by adsorption to the activated sludge, and the removal by evaporation from the test SCAS unit was determined by using specific analysis (please see details on analytical methods). The 1,1-di(tert-butylperoxy)cyclohexane content was measured in the effluent, in the mixed liquid suspended solids (MLSS) and in the air of the SCAS test unit. Samples for the specific analyzes in effluent and MLSS were taken after 7 to 13 weeks of operation of the test SCAS unit. To detect the formation of degradation products, MLSS samples were taken at the start of the cycle and after 15 min, 30 min, 1 hour, 3 hours, 5 hours and 24 hours.

After 30 weeks of operation the air from the test SCAS unit was sampled by closing the top of the SCAS unit and directing the air from the SCAS unit for two hours through two tenax tubes placed in series ana analyzed by thermal desorption.

Pre-exposed activated sludge from both the isododecane control and the test SCAS unit were used as inoculum in a Closed Bottle test (OECD test guideline 301).

The nutrient medium of the Closed Bottle test contained per liter of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification not related to the biodegradation of the test substance. The test substance was dosed on silica gel in a 50-mL serum flask to obtain a concentration of 3.0 mg / g silica gel. Only the upper layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw cap and mixed vigorously. Subsequently, 0.20 g of silica gel was administrated to each test bottle. The final concentration in the bottles was 2.0 mg/L. Closed Bottle tests with isododecane (0.46 mg/L) were performed to correct for the biodegradation of the isododecane in the test with the test substance (= isododecane control bottles). To assess the biodegradability of the isododecane another series of tests were performed using isododecane as the test substance. In these tests an isododecane concentration of 1.5 mg/L was used in the bottles.
Isododecane was dosed to the Closed Bottles using a silica gel stock of 3.0 mg / g silica gel which was prepared using the same procedure to prepare the test substance silica gel stock. The tests were performed in 0.3 L BOD bottles with glass stoppers. Activated sludge pre-exposed in SCAS units to the test substance and to isododecane were used as inocula in the Closed Bottle tests. Use was made of 3 control bottles containing respective inoculum and 0.2 g of (clean) silica gel, 3 test bottles with the test substance and respective inoculum, and for the peroxide test substance also 3 isododecane control bottles were included
containing isododecane and the respective inoculum. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were filled without air bubbles.

The bottles were closed and incubated in the dark at temperatures ranging from 22 to 24°C. The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle, when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium. Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle.

Key result

Parameter:

% degradation (test mat. analysis)

Value:

98.8

Sampling time:

24 h

Details on results:

The calculated carbon content from stock solutions daily dosed to the test and isododecane SCAS unit were 71 mg/L and 63 mg/L for the test substance stock solution in methanol and the isododecane stock solution in methanol, respectively. NPOC measurements in filtered effluent samples of both units and the control unit showed that there was no increase in organic carbon (NPOC) in the effluent of the test and isododecane SCAS unit compared to the control SCAS unit.

NPOC concentrations in the effluent of the test substance SCAS unit were lower compared to the concentrations in the control and isododecane control SCAS unit and hence calculated biodegradation % are >100%. The high NPOC removals in both units may be the result of adsorption however this does not explain organic carbon removals >100%. Based on the measured organic content it can however be concluded that the dosed methanol was completely removed and the formation of water-soluble products during the (bio)degradation of the test substance and the isododecane can be excluded.

Specific analysis by GC-MS show that the primary removal of 1,1-di(tert-butylperoxy)cyclohexane was relatively fast and >80% was already removed within the first five hours of the SCAS test. The lack of removal in the heat-killed activated sludge indicates that this removal was the result of a primary biodegradation step and not a physical / chemical degradation step. The total primary removal of 1,1-di(tert-butylperoxy)cyclohexane measured in the effluent of the SCAS unit after 24 hours was 98.8%. This primary removal was not the result of adsorption because only 0.2 – 0.3 % of the 1,1-di(tert-butylperoxy)cyclohexane was recovered from the MLSS after 24 hours. A large removal of 1,1-di(tert-butylperoxy)cyclohexane by volatilization from the SCAS unit was also excluded because no relevant and no significant amounts of organics were detected in the air of the test SCAS unit. Specific analyses were also used to search for the formation of (bio)degradation products. No (bio)degradation products were found during the SCAS cycle.

Next, Closed Bottle tests with the pre-exposed activated sludge from the SCAS test were performed to confirm that the removal in the SCAS test was the result of a biological step (biodegradation) and to determine the extent of this biodegradation (partial or ultimate).

Biodegradation percentages ≥60 in the Closed Bottle test suggest a complete mineralization of a test substance. The ThODNH3 used to calculate the biodegradation of the test substance was 2.50 mg oxygen/mg test substance, the ThODNH3 of 1,1-di(tert-butylperoxy)cyclohexane was 2.34 mg oxygen/mg test substance, and the ThOD NH3 of the isododecane was 3.48 mg oxygen/mg test substance. Biodegradation percentages for the test substance leveled off from day 42 between 32 and 45% and suggest partial degradation. The partial biodegradation was reproducible between the tests performed with 11 and 30 weeks pre-exposed activated sludge. Biodegradation percentages of the isododecane control bottles and in the biodegradation tests with isododecane as test substance all gave negative biodegradation percentages. Negative percentages may be the result of toxicity to the inoculum and certainly demonstrate a lack of biodegradation. The high organic carbon removal measured in the SCAS test was therefore most likely the result of removal by adsorption to the activated sludge. The observed partial biodegradation of the test substance can be explained by the lack of the biodegradation of the isododecane. It is however not understood why isododecane (2,2,4,6,6-pentamethylheptane) is reported as ready biodegradable (ECHA, 2010) and in this study no biodegradation was achieved. Because of the lack of biodegradation in this study the biodegradation percentages for the test substance were calculated using the oxygen consumption in the control bottles (Mc) and not the oxygen consumption in the isododecane control bottles (Mci). The biodegradation of the isododecane would account for approximately 28% of the total ThOD. This means that without the biodegradation of the isododecane the maximum oxygen consumption in the Closed Bottle test for the test substance would be 43% (= 60% x the biodegradable ThOD fraction of 72%). The achieved ready biodegradation results for the test substance in this study therefore indicate mineralization by growth linked biodegradation of the 1,1-di(tert-butylperoxy)cyclohexane fraction in the test substance. The potential of 1,1-di(tert-butylperoxy)cyclohexane being biodegraded through a growth-linked metabolism increases the chance of finding (ultimate) biodegradation of 1,1-di(tert-butylperoxy)cyclohexane in other environmental matrices.

Validity criteria fulfilled:

not specified

Interpretation of results:

readily biodegradable

Remarks:

The total primary removal of the test item measured in the effluent of the SCAS unit after 24 hours was 98.8%

Conclusions:

Microorganisms were exposed to 1,1-di(tert-butylperoxy)cyclohexane in the SCAS test. To this end 1,1-di(tert-butylperoxy)cyclohexane was spiked to the influent of the SCAS test as at a nominal influent concentration of 16 mg/L for a period of 30 weeks. The total removal percentage from the influent measured after ~14 weeks was 98.8%. The removal from the influent through adsorption onto the sludge was only 0.2 – 0.3% and no (significant) removal of the 1,1-di(tert-butylperoxy)cyclohexane by volatilization from the SCAS was measured. A rapid primary biodegradation of 1,1-di(tert-butylperoxy)cyclohexane was demonstrated in the SCAS test, however formation of (bio)degradation products was notdetected. The removal of 1,1-di(tert-butylperoxy)cyclohexane is most likely the result of biodegradation because 1,1-di(tert-butylperoxy)cyclohexane was found stable (persistent) in heat-killed activated sludge. More important the biodegradation of the test substance achieved in the OECD 301D test with pre-exposed (11 weeks and 30 weeks) sludge from the SCAS tests demonstrated the growth linked (ultimate) biodegradation of the 1,1-di(tert-butylperoxy)cyclohexane fraction in the test substance.

Executive summary:

Secondary activated sludge and primary settled sewage were obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands, treating predominantly domestic wastewater. The primary settled sewage was collected weekly and stored in a refrigerator until required. A volume of 150 mL of secondary activated sludge containing approximately 2.0 g DW (dry weight)/L of mixed liquid suspended solids (MLSS) was used as inoculum for SCAS units.

The performance of the SCAS units was followed by measuring the Non-Purgeable Organic Carbon (NPOC) content in the effluent of the test unit and to compare this with the NPOC content in a control SCAS unit and an Isododecane control SCAS unit. 

The 1,1-di(tert-butylperoxy)cyclohexane content was measured in the effluent, in the mixed liquid suspended solids (MLSS) and in the air of the SCAS test unit. Samples for the specific analyzes in effluent and MLSS were taken after 7 to 13 weeks of operation of the test SCAS unit. To detect the formation of degradation products, MLSS samples were taken at the start of the cycle and after 15 min, 30 min, 1 hour, 3 hours, 5 hours and 24 hours.

After 30 weeks of operation the air from the test SCAS unit was sampled by closing the top of the SCAS unit and directing the air from the SCAS unit for two hours through two tenax tubes placed in series ana analyzed by thermal desorption.

Pre-exposed activated sludge from both the isododecane control and the test SCAS unit were used as inoculum in a Closed Bottle test.

The total removal percentage from the influent measured after ~14 weeks was 98.8%. The removal from the influent through adsorption onto the sludge was only 0.2 – 0.3% and no (significant) removal of the 1,1-di(tert-butylperoxy)cyclohexane by volatilization from the SCAS was measured. A rapid primary biodegradation of 1,1-di(tert-butylperoxy)cyclohexane was demonstrated in the SCAS test, however formation of (bio)degradation products was notdetected. The removal of 1,1-di(tert-butylperoxy)cyclohexane is most likely the result of biodegradation because 1,1-di(tert-butylperoxy)cyclohexane was found stable (persistent) in heat-killed activated sludge. More important the biodegradation of the test substance achieved in the OECD 301D test with pre-exposed (11 weeks and 30 weeks) sludge from the SCAS tests demonstrated the growth linked (ultimate) biodegradation of the 1,1-di(tert-butylperoxy)cyclohexane fraction in the test substance.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2000-08-10 to 2000-09-08

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)

Version / remarks:

17th July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

31st July 1992

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: the water treatment plant Emeraude (SIARR); 76141 Petit-Quevilly; France)
- Laboratory culture: no
- Preparation of inoculum for exposure: The inoculum was prepared by initially sieving sewage sludge. The sludge was then centrifuged for 5 minutes, the supernatant was rejected and the pellet was re-dispersed in the mineral medium. In order to wash out the dissolved organic carbon (DOC) and to lower the carbon organic content, the inoculum was preconditioned for 7 days before use. Air was bubbled through the inoculum during this preconditioning period.
- Concentration of sludge: 12 mg/L
- Water filtered: no

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:

TEST CONDITIONS
- Composition of medium: According to OECD guideline
- Additional substrate: no
- Test temperature: between 19 °C and 24 °C
- pH: 7.58 (before the start of the test); 8.04 to 8.45 (at the end of the test)
- Aeration of dilution water: air was bubbled through each parallel at the rate of 30 - 100 mL/min during the test

TEST SYSTEM
- Culturing apparatus: test vessels were loaded at 3 litres of suspension per flask
- Number of culture flasks/concentration: 2

SAMPLING
- Sampling frequency: on day 1, 4, 6, 8, 11, 14, 18, 22, 25, 29
- Sampling method: 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 volume needed to titrate 100 mL of the Ba(OH)2 stock solution. The remaining CO2 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.
- Sample storage before analysis: none

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

5

Sampling time:

28 d

Details on results:

Biodegradation of the peroxide totalled 5 % (mean of the two flasks) over the test period.

Results with reference substance:

The biodegradation in the reference test was 61 % after 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The biodegradation of cyclohexylidenebis[tert-butyl] peroxide totalled 5 % at the end of the test. Cyclohexylidenebis[tert-butyl] peroxide was not readily biodegradable in the 28-day modified Sturm test.

Executive summary:

The ready biodegradability of cyclohexylidenebis[tert-butyl] peroxide was evaluated using a 28-day modified Sturm test according to EU method C.4 -C and OECD guideline no. 301 B.

The flasks of the test item obtained a peroxide concentration of 10 mg/L of TOC and inoculum. The inoculum consisted of sewage sludge sampled from the aeration tank of a sewage treatment plant and was 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.

The biodegradation of cyclohexylidenebis[tert-butyl] peroxide totalled 5 % at the end of the test. Cyclohexylidenebis[tert-butyl] peroxide was not readily biodegradable in the 28-day modified Sturm test.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

yes

Principles of method if other than guideline:

The goal of this study is to assess the inoculum giving the most advantageous classification of 1,1-di(tert-butylperoxy)cyclohexane in the Closed Bottle test. The Closed Bottle tests are performed according to slightly modified OECD Test Guidelines (OECD, 1992).

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: Please see below

Details on inoculum:

Inocula
Activated sludge was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to 2.0 mg DW/L in the biological oxygen demand (BOD) bottles (van Ginkel and Stroo, 1992).
River water was sampled from the Rhine near Heveadorp, The Netherlands. The river water was aerated for 7 days to reduce the endogenous respiration. Particles were removed by sedimentation after one day while moderately aerating. Particle free river water was used as inoculum. The river water spiked with mineral salts of the nutrient medium was used undiluted.

Duration of test (contact time):

196 d

Details on study design:

Test procedures of Closed Bottle test
The Closed Bottle test was conducted according to Test Guidelines (OECD 1992). The nutrient medium of the Closed Bottle test contained per liter of deionised water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, and 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. The test substance and isododecane were dosed on silica gel in a 50-mL serum flask to obtain a concentration of 3.0 mg / g silica gel. Only the upper layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw cap and mixed vigorously. Subsequently, silica gel with test substance (0.2 g) and isododecane (0.1 g) were administrated to each bottle. The tests were performed in 0.3 L BOD bottles with glass stoppers. Use was made of 3 bottles containing only respective inoculum silica gel and isododecane, 3 bottles with test substance and the respective inoculum. The final concentrations of the test substance and mineral spirits in the bottles were 2.0 and 1.0 mg/L, respectively. 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 bottles were closed and incubated in the dark at 23 °C (± 1 °C). The biodegradation was measured by following the course of the oxygen decrease in the bottles using a special funnel and an oxygen electrode. This funnel fitted exactly in the BOD bottle and when the oxygen electrode was inserted in the BOD bottle the funnel collected the dissipated medium. Upon the removal of the oxygen electrode the collected medium flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992).

Key result

Parameter:

% degradation (O2 consumption)

Value:

64

Sampling time:

112 d

Remarks on result:

other: sludge

Key result

Parameter:

% degradation (O2 consumption)

Value:

24

Sampling time:

112 d

Remarks on result:

other: sludge from the standard Closed Bottle test

Key result

Parameter:

% degradation (O2 consumption)

Value:

14

Sampling time:

196 d

Remarks on result:

other: River water

Test conditions

The validity of the test is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period. The pH of the media was 7.3 (activated sludge) and 8.0 (river water) at the start of the test. The pH was 7.2 ± 0.1 (activated sludge) and 7.9 ± 0.1 (river water) at day 28. The temperature was 23 °C (± 1 °C). The inhibition of biodegradation by the test substances is usually detected prior to the onset of the biodegradation through suppression of the endogenous oxygen consumption. Inhibition of the endogenous respiration of the inoculum was not detected with 1,1-di(tert-butylperoxy)cyclohexane.

 

The Closed Bottle test results

The ThOD used to calculate the biodegradation percentages was 2.3 g O2/g test substance. Only little biodegradation was found after 28 and 60 days with both activated sludge and river water (Table). The test substance should therefore not be classified as readily biodegradable nor as not persistent.

However, after 84 days biodegradation in excess of 60 % was found with activated sludge as inoculum. The long period (lag) before oxygen consumption was observed indicates that degradation of 1,1-di(tert-butylperoxy)cyclohexane is most likely initiated by a biochemical reaction (hydrolysis). Hydrolysis results in the formation of tert-butanol and cyclohexanone. Cyclohexanone is readily biodegradable. Tert-Butanol degrades within 4 to 6 weeks in Closed Bottle tests (AkzoNobel 2017).

The content of the bottles (day 112) showing complete degradation were used to inoculate a follow-up test. In this test with microorganisms adapted to the test item an immediate degradation was expected. However, only partial degradation was observed in this test. Degradation did start within 14 days (Table 1). The results do show that biodegradation is possible. The results are however inconsistent with respect to the extent of the biodegradation (final percentage) and the chance of occurring (no degradation in river water).

 

Table 1 Percentage biodegradation of the registered substance in modified bottle test

 

Inoculum	Biodegradation percentage at day (based on oxygen consumption)
	7	14	21	28	42	56	84	112	140	196
Sludge	0	-4	-3	-1	1	4	63	64
Sludge*	1	14	14	14	19	19	24	24
River water	1	-3	3	3	3	4	9	11	14	14

* The Closed Bottle test was inoculated with sludge from the standard Closed Bottle test.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Description of key information

In a weight of evidence approach, the test item is considered to be degradable, but not fullfilling the readily biodegradable criteria.

In a further test, microorganisms were exposed to 1,1-di(tert-butylperoxy)cyclohexane in the SCAS test. To this end 1,1-di(tert-butylperoxy)cyclohexane was spiked to the influent of the SCAS test as at a nominal influent concentration of 16 mg/L for a period of 30 weeks. The total removal percentage from the influent measured after ~14 weeks was 98.8%. The removal from the influent through adsorption onto the sludge was only 0.2 – 0.3% and no (significant) removal of the 1,1-di(tert-butylperoxy)cyclohexane by volatilization from the SCAS was measured. A rapid primary biodegradation of 1,1-di(tert-butylperoxy)cyclohexane was demonstrated in the SCAS test, however formation of (bio)degradation products was notdetected. The removal of 1,1-di(tert-butylperoxy)cyclohexane is most likely the result of biodegradation because 1,1-di(tert-butylperoxy)cyclohexane was found stable (persistent) in heat-killed activated sludge. More important the biodegradation of the test substance achieved in the OECD 301D test with pre-exposed (11 weeks and 30 weeks) sludge from the SCAS tests demonstrated the growth linked (ultimate) biodegradation of the 1,1-di(tert-butylperoxy)cyclohexane fraction in the test substance.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

In a modified Sturm Test (Key, 2001) according to OECD 301B, no biodegradation was observed - 5 %.

In a modified Closed Bottle Test (Supporting, 2018) according to OECD 301D, mineralisation of the test item up to 64 % was observed after 114 days of incubation with activated sludge. This test shows that the test item is being consumed to some level after a lag period, which shows that the test item is probably hydrolysed and degraded. Still the obtained mineralisation level is not fulfilling the criteria for ready biodegradability.

In a modified test, microorganisms were exposed to 1,1-di(tert-butylperoxy)cyclohexane in the SCAS test. To this end 1,1-di(tert-butylperoxy)cyclohexane was spiked to the influent of the SCAS test as at a nominal influent concentration of 16 mg/L for a period of 30 weeks. The total removal percentage from the influent measured after ~14 weeks was 98.8%. The removal from the influent through adsorption onto the sludge was only 0.2 – 0.3% and no (significant) removal of the 1,1-di(tert-butylperoxy)cyclohexane by volatilization from the SCAS was measured. A rapid primary biodegradation of 1,1-di(tert-butylperoxy)cyclohexane was demonstrated in the SCAS test, however formation of (bio)degradation products was notdetected. The removal of 1,1-di(tert-butylperoxy)cyclohexane is most likely the result of biodegradation because 1,1-di(tert-butylperoxy)cyclohexane was found stable (persistent) in heat-killed activated sludge. More important the biodegradation of the test substance achieved in the OECD 301D test with pre-exposed (11 weeks and 30 weeks) sludge from the SCAS tests demonstrated the growth linked (ultimate) biodegradation of the 1,1-di(tert-butylperoxy)cyclohexane fraction in the test substance.

In a weight of evidence approach, the test item is considered to be degradable, but not fullfilling the readily biodegradable criteria.