Bis(α,α-dimethylbenzyl) peroxide

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-11-12 to 2020-08-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Version / remarks:

October 2004

Deviations:

yes

Remarks:

Acetonitrile was used as a solvent, to ensure stability, dissolution and minimisation of volatilisation of the initial test concentration; test duration was up to 97 days (instead of 90 days) for the low concentration (mineralisation only testing).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Bis(α,α-dimethyl[ring-14C]benzyl)peroxide (14C-Dicumylperoxide) (numbering CFQ43623), Pharmaron UK Ltd

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 98.4%
- Specific activity: 52 mCi/mmol, corresponding to 1.92 GBq/mmol
- Locations of the label: One carbon atom in one of the two benzyl rings; the exact C position within benzyl not indicated
- Expiration date of radiochemical substance: Not specified, radiochemical purity was verified prior to start of the study

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: -20 ± 2 °C, dark, in tightly closed original container (borosilicate multidose vial with screw cap)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: the material was supplied as a solution in toluene
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: No data
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: No data
- Reactivity of the test material with the incubation material used (e.g. plastic ware): No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): None
- Preliminary purification step (if any): By HPLC at the manufacturing site
- Final concentration of a dissolved solid, stock liquid or gel: application solution: 6.72 MBq in 1.15 mL acetonitrile; nominal test item concentration: 50 µg/L
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): Not applicable

Radiolabelling:

yes

Oxygen conditions:

aerobic (low dissolved oxygen)

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

SAMPLING:
Natural surface water (field fresh sampled) of the river ‘Leine’ was collected from the surface of the river (approx. upper 20 cm water) at an undisturbed recess. The sampling site was selected with respect to the regional biological and chemical water quality maps (interactive online version) of the Lower Saxony Water Management, Coastal Defence and Nature Conservation Agency (German: NLWKN). Water temperature, pH value and O2 concentration of the water were measured at field sampling.
- Test on mineralisation and transformation: 9.6 °C; pH 8.04; 9.93 mg O2/L
- Test on mineralisation at low concentration: 12.2 °C; pH 7.64; 9.43 mg O2/L
HANDLING:
The water was clear and colourless. Further pre-treatment like filtration was based on visual assessment considered not being necessary.
ACCLIMATISATION:
Until test start within 8 days after collection of the surface water, the water was acclimated under test conditions at 12 ± 2 °C in the dark with aeration.

Details on inoculum:

- Source of inoculum: surface water; coordinates: 52˚ 11’ 39.419'' N, 9˚ 47’ 7.49'' E
- Storage conditions: 12 ± 2 °C in the dark with aeration
- Storage length: within 8 days after collection of the water
- Temperature (°C) at time of collection: water temperature: 9.6 °C for first sampling for test conc of 50 µg/L (2019-11-04); 12.2 °C for second sampling for test conc 0.5 µg/L (2020-05-04)
- pH at time of collection: 8.04 (first sampling), 7.64 (second sampling)
- Electrical conductivity: not measured
- Redox potential (mV): not measured
- Oxygen concentration (mg/l): 9.93 (first sampling), 9.43 (second sampling)
- Dissolved organic carbon (%): 6.04 (first sampling), 5.21 (second sampling)
- Biomass (e.g. in mg microbial C/100 mg, CFU): not measured (surface water)
- Biomass concentration (mg/L) used in test: not measured (surface water)
- Chemical oxygen demand (COD) - water filtered: not measured (surface water)

Duration of test (contact time):

>= 90 - <= 97 d

Initial conc.:

0.5 µg/L

Based on:

test mat.

Initial conc.:

50 µg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

test mat. analysis

Details on study design:

TEST CONDITIONS
- Test vessel: 120 mL glass bottles and CO2 traps, see below
- Solubilising agent (type and concentration if used): 0.0063 % acetonitrile
- Test temperature: 12 ± 2 °C in the dark
- pH: measured at day 0, 7, 31, 56, 92: pH 6.76 to 8.44
- pH adjusted: no
- O2 measured at day 0, 39, 62, 78, 97: 6.97 to 9.66 mg/L
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: Yes, in the preliminary studies. In response, the test concentrations were lowered

TEST SYSTEM
- Test performed in closed vessels due to certain volatility of test substance: 120 mL gastight glass bottles for testing of mineralization and transformation of the test item. 120 mL gastight glass bottles for testing of mineralization of the reference control. 150 mL gastight glass bottles with internal 14CO2 trap for testing the mineralization of test item and reference control at selected time points and the mineralization of test item at the low concentration. 150 mL gastight glass bottles for the controls.
- Details of trap for CO2: 14 CO2 (internal): Internal traps containing 2 g soda lime or 1.5 mL 5 mol/L NaOH solution were used for trapping 14CO2; 14CO2 (external): Crimped bottles containing 10 mL 1 mol/L aqueous sodium hydroxide
- Culturing apparatus: 120 mL glass bottles, 14CO2 traps,
- Number of culture flasks/concentration: 2
Two replicates per test item concentration were harvested at each sampling time for determination of transformation and mineralisation.

SAMPLING
- Sampling frequency for transformation: day 0, 3, 7, 14, 20, 24, 29, 34, 37, 48, 55, 62, 70, 77, 83, 90
- Sampling frequency for mineralisation: day 14, 29, 37, 48, 55, 62, 70, 77, 83 and 90
- Sampling frequency for mineralisation at low concentration: 0, 5, 11, 20, 26, 33, 41, 46, 50, 54, 62, 78 and 97
- The volatile traps were connected to syringe needles and the needles connected to the headspace of the test vessels.

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION CONTROL AND BLANK SYSTEM
- Inoculum blank: Surface water with the respective amount solvent but without test and/or reference item
- Abiotic sterile control: Surface water with the respective amount solvent but without test and/or reference item
- Toxicity control: none

STATISTICAL METHODS: Calculations were carried out using the following software:
- Excel, MICROSOFT CORPORATION
- CAKE (COMPUTER ASSISTED KINETIC EVALUATION, Version 3.3), Tessella Ltd.

Reference substance:

other: Aniline sulfate, [14C (U)]-

Remarks:

Batch number: 373-002-110-A-20141024-PVA; Radiochemical purity: 99.2%; Specific activity: 110 mCi/mmol; Test concentration: 10 µg/L

Test performance:

Mineralisation and transformation: The results of sampling on day 24 indicate losses due to volatilisation. Therefore the results were not included in the evaluation and the headspace sampling was included in the further samplings. Results of sampling on day 34 were not plausible (mass balance < 80 %) and therefore not included in the evaluation. A new sampling was done on day 37.
Mineralisation at low concentration: Results of sampling on day 26 were not plausible (interference with background) and therefore not included in the evaluation. The measurement procedure was modified and a new sampling was done on day 33.

Compartment:

natural water: freshwater

Sampling date:

2020

% Total extractable:

ca. 83.5

% CO2:

ca. 12.9

% Other volatiles:

ca. 11.4

Remarks on result:

other: 50 µg/L; mean recovery for sampling day 90 (start of experiment (2019-11-12) + 90 days).

Remarks:

Recovery rates for day 0–90 are attached as table under overall remarks.

Compartment:

natural water: freshwater

Sampling date:

2020

% Total extractable:

26.1

% CO2:

60.21

Remarks on result:

other: 0.5 µg/L, dicumylperoxide not determined, only measurements on CO2; "total extractable" refers to CO2 in solution; indications for sampling day 97 (start of experiment (2019-11-12) + 97 days), also see attachments

Remarks:

Recovery rates for day 0–97 are attached as table under overall remarks.

Parent/product:

parent

Compartment:

water

Key result

% Degr.:

>= 15.7 - <= 25.5

Parameter:

radiochem. meas.

Sampling time:

90 d

Remarks on result:

other: starting conc. 50 µg/L; degradation calculated based on measured parent compound as % of applied radioactivity

Key result

Compartment:

natural water: freshwater

DT50:

ca. 142

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: primary transformation with 50 µg/L initial conc of dicumylperoxide

Compartment:

natural water: freshwater

DT50:

ca. 164

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Remarks on result:

other: mineralisation at low initial conc of 0.5 µg/L of dicumylperoxide, but first order kinetics gave poor fit

Compartment:

natural water: freshwater

DT50:

ca. 92.2

Type:

other: Hockey Stick

Temp.:

12 °C

Remarks on result:

other: Mineralisation at low initial con 0.5 µg/L of dicumylperoxide

Mineralization rate (in CO2):

12.9 other: % until day 90

Transformation products:

yes

No.:

#1

Details on transformation products:

The metabolite 2-Phenyl-2-propanol was determined from day 29 on. The mean maximum concentration of 2-Phenyl-2-propanol was 9.8 % applied radioactivity (AR),day 37 and day 48. Thereafter the concentration decreased continuously and was < 1 % from day 62 on until test end.

Evaporation of parent compound:

yes

Remarks:

To account for evaporation of the parent compound, the volatile fraction was measured in the headspace of the vessels. At test end 84.3 % and 74.5 % of Dicumylperoxide were determined in the water phase and 14.3% and 8.4% were determined in the headspace.

Volatile metabolites:

no

Residues:

no

Results with reference substance:

Mineralisation of the reference item Aniline sulphate, [14C(U)]- in the reference control was ≥ 60 % after 7 days and 12 days, respectively. The high mineralization (formation of 14CO2) indicates that the surface water contained an active microbial population.

Validity criteria:

According to the OECD 309 guideline, aniline should degrade within less than two weeks. In addition, the total recovery at the end of the experiment should be 90% to 110 % for radiolabelled substances.

Observed value:

Aniline was degraded (> 50%) within 5 and 7 days. Mass balance: 84.7–120.3 % (mean values, 50 μg/L for determination of transformation). Mass balance: 80.4–100.8 % (mean values, 0.5 μg/L for determination of mineralisation).

Validity criteria fulfilled:

yes

Conclusions:

Transformation and mineralisation at 50 µg/L and 0.5 µg/L (the latter being a potentially environmentally relevant concentration) of dicumylperoxide was slow during the whole test period of 90 or 97 days. The metabolite 2-Phenyl-2-propanol was determined from day 29 on achieving 9.8% applied radioactivity at maximum (at day 37 and day 48).

Executive summary:

The biotransformation of radiolabelled dicumyl peroxide was studied in unfiltered river water (pH 7.64 to 8.04, dissolved organic carbon 5.21 to 6.04 mg/L) from Germany for 90 and 97 days under aerobic conditions in the dark at 12 °C. Dicumyl peroxide was applied at concentrations of 50 and 0.5 µg /L. The experiment was conducted in accordance with the OECD guideline 309, and in compliance with the OECD-GLP standards. The test system consisted of gastight glass bottles attached with traps for the collection of CO₂ and organic volatiles. Samples were analysed at 0, 3, 7, 14, 20, 24, 29, 34, 37, 48, 55, 62, 70, 77, 83, 90 days of incubation for primary transformation and mineralisation (50 µg/L dicumyl peroxide), and at 0, 5, 11, 20, 26, 33, 41, 46, 50, 54, 62, 78 and 97 days of incubation (0.5 µg/L dicumyl peroxide). The water samples were not extracted, and dicumyl peroxide and the main transformation products were analysed by HPLC with fraction collector and offline LSC. Mineralisation was determined by LSC counting of CO₂ traps.

Material balance ranged from 84 to 94 % of applied radioactivity. The concentration of parent compound decreased from 93.3 % at day 0 to 84.3 % and 74.5 % of the applied amount at the end of study period. The half-life/DT50 (50% decline time) of dicumyl peroxide in aerobic water was 142 days. Thus, biotransformation was slow. As the DT50 is higher than 60 days, dicumyl peroxide should be considered as very persistent, cf. Annex XIII of the REACH regulation.

The major transformation products detected were 2-phenyl-2-propanol, with maximum concentrations of 9.8 % of the applied amount, observed on 37 day of incubation. The corresponding concentration at the end of the study period was 0.4 % of the applied amount. Minor transformation products were not detected. At study termination, evolved ¹⁴CO₂ in solution and in headspace accounted for 13 % of the applied radioactivity. The metabolite 2-phenyl-2-propanol was also formed in the sterile (autoclaved) control replicates. This metabolite was detected in samples analysed at day 64, 70 and at test end with 9.2–9.9 % of applied radioactivity. The concentration of this metabolite remained constant over this time course and its concentration was very similar to the maximum determined concentration in the test item replicates. No formation of ¹⁴CO₂ was observed in the sterile controls. These observations suggest that the test item is first degraded by hydrolysis, followed by microbial degradation of the metabolite 2-phenyl-2-propanol.

Description of key information

The primary degradation half-life of dicumyl peroxide (bis(α,α-dimethylbenzyl) peroxide) in surface water is estimated at DT50 = 142 d, based on (poorly fitting) single first-order kinetics.

Mineralisation reaches a maximum of 12.9 % after 90 days (50 µg/L).

Key value for chemical safety assessment

Half-life in freshwater:

142 d

at the temperature of:

12 °C

Additional information

In pelagic freshwater, dicumyl peroxide (bis(α,α-dimethylbenzyl) peroxide) degrades only slowly. Degradation is probably initiated by a primary hydrolysis step, followed by slow biological degradation. The primary metabolite 2-phenyl-2-propanol reached maximum levels of 9.8 % of AR on days 37 and 48. The degree of mineralisation reached a maximum of 12.9 % on day 90 at the high test concentration (50 µg/L). At the low concentration (0.5 µg/L, specifically designed at measuring mineralisation), CO2 formation reached 60 % on day 97 after a long lag phase. This apparently high mineralisation at test end might be an artefact.