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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study planned (based on read-across)
Justification for type of information:
A feasibility study on aq sediment has been conducted for 25155-25-3. A full study is planned and data will be submitted when ready.
Qualifier:
according to guideline
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
Endpoint:
biodegradation in water: sediment simulation testing
Remarks:
Feasibility Study in Aerobic and Anaerobic Aquatic Sediment
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
October 2020 to end of January 2021
Reliability:
other: The test is a fesibility study
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read Across justification is attached in IUCLD section 13.2.
Reason / purpose for cross-reference:
read-across source
Specific details on test material used for the study:
Identification: [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide and [1,4-phenylenebis(1-methylethylidene)] bis[tert-butyl] peroxide, CAS 25155-25-3

[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
Specific activity: 3.08 MBq/mg
Source: Selcia
Batch/Lot number: 11722CEO001-1
Radiochemical purity: ≥97.4%
Storage: Frozen at -10 to -30ºC

[1,4-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
Specific activity: 3.87 MBq/mg
Source: Selcia
Batch/Lot number: 11723CEO001-1
Radiochemical purity: ≥93.5%
Storage: Frozen at -10 to -30ºC
Radiolabelling:
yes
Oxygen conditions:
aerobic/anaerobic
Inoculum or test system:
natural sediment: freshwater
Remarks:
Calwich Abbey Lake provided by LRA Labsoil, Lockington,
Details on source and properties of sediment:
The sediment was passed through a 2 mm sieve and the water was passed through a 0.2 mm sieve. This was carried out under nitrogen for the anaerobic sediment. Sediment and water were shipped in separate containers and, upon receipt at the testing facility, stored at approximately 4°C.

Batchnumber Aerobic: H081020A (water), H081020B (sediment), Aneaerobic: H081020C (water), H081020D (sediment)
Origin: 52° 59’ 6.4” N, 1° 48’ 38.3” W Staffordshire
Date of sampling: 05 October 2020
Water body type: Large perennial lake
Pesticide use: None for 5 years

Sediment:
Appearance and odor Greyish brown loam with a few twigs, leaves and small stones. Slight smell.
Depth of sediment layer (cm) ~100
Depth of sampled layer (cm) 5 (aerobic sample). Anaerobic sample taken from anaerobic layer and transferred to container under water to exclude air.
Particle size distribution (UK) 0.063 mm – 2 mm : 9% 0.002 mm – 0.063 mm: 74 % <0.002 mm: 17 %
Texture class: Silt loam
pH (1:2.5 w/v) in water: 7.8
pH in 0.01 M CaCl2: 7.3
Organic carbon: 2.9%
Total nitrogen: 0.48%
Total phosphorus: 767 mg/kg
Calcium carbonate: 56.8 %
Cation exchange capacity: 12.1meq/100 g

Water:
Appearance Slight brown colour, slightly turbid
Depth over sample area (cm) 20 - 30
Temperature °C: Just below surface 12.8 °C, 5 cm above sediment 12.8 °C
pH 7.28
Oxygen saturation: Just below water surface 74.4%, 5 cm above sediment 74.0%
Total organic carbon: 4.1 mg/l
Total nitrogen: 7.1 mg/l
Total phosphorus: <0.1 mg/l
Total suspended solids: 19 mg/l
Hardness as CaCO3: 264 mg/l


Duration of test (contact time):
14 d
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
Portions of Calwich Abbey Lake sediment (94.88 g aerobic or 104.71 g anaerobic, dry weight equivalent) were added to one-litre glass vessels. Portions of Calwich Abbey Lake water (655 g aerobic or 605 g anaerobic) were added to the vessels to achieve the volume ratio of approximately 1 : 3, where the sediment layer was 3 cm deep. The sediment samples were incubated for 27 days prior to dosing.
Three aerobic vessels and three anaerobic vessels were established for treatment with a mixture of radiolabeled 1,3-bis(tert-butylperoxy isopropyl)benzene and 1,4-bis(tertbutylperoxy isopropyl)benzene were incorporated into individual flow-through systems (through which air or nitrogen was drawn) arranged as follows:
(i) Humidifying vessel (with sintered stem for uniform gas dispersion) containing water to humidify the gas flow;
(ii) Test vessel containing the aquatic sediment test system, bubbled with gas through a dip tube immersed just below the surface of the water;
(iii) Vessel containing a polyurethane bung
(iv) Vessel containing ethyl digol to trap volatile organic compounds (Trap 1);
(v) Vessel containing 1 M aqueous potassium hydroxide solution with phenolphthalein indicator to trap 14CO2 (Trap 2);
(vi) A non-return valve to prevent accidental backflow through the test apparatus.

The end of the glass stem (dip tube) bringing the gas flow into each test vessel was just below the surface of the water. Air or nitrogen was drawn through each system at a flow rate of approximately 50 mL/minute during the acclimatization phase. After treatment the gas flow was changed to about 20 mL/min for 15 minutes each day or 30 minutes prior to sampling.
Samples were incubated at 12°C in the dark. Designated vessels were set up for measurement of oxygen concentration, redox potential and pH, as appropriate, twice a week during acclimatization and at each sampling occasion.

A treatment solution of the two radiolabeled isomers 1,3-bis(tert-butylperoxy isopropyl)benzene and 1,4-bis(tert-butylperoxy isopropyl)benzene in the ratio 55.9% : 34.5% was prepared in acetonitrile at nominal concentration of 0.15 mg/mL. For each vessel an aliquot (1.0 mL) of the treatment solution was thoroughly mixed with silica gel (4 g) to give a dry looking powder. The water layer from each sediment system was removed by syringe and the treated silica gel was added to the surface of the sediment. The water layer was then returned to the vessel causing as little disturbance as possible. For the anaerobic vessels this procedure was carried out under nitrogen. This achieved application rate of 0.15 mg/vessel. The purity of the application solution was ≥95.0% (Appendix 2). Single samples of aquatic sediment, incubated under aerobic or anaerobic conditions, were taken for analysis at time zero, and after 7 and 14 days of incubation.
Trapping media were taken for analysis with the associated samples at sampling. Additionally, trapping solutions associated with the 14 day vessels were taken for analysis (and replaced with fresh media as appropriate) after 7 days.
The zero-time samples were allowed to stand for 15 minutes before removal of the water layer. The water was decanted, the weight recorded and duplicate weighed aliquots (1.0 mL) taken for radioassay. Water samples were analysed by HPLC directly on the day of sampling. The sediment was extracted (3 times) with acetonitrile (200 mL) followed by acetonitrile : water 2 : 1 (v : v) by sonicating for 15 minutes then shaking for 15 minutes. The sediment and solvent were separated by centrifugation (2500 rpm for 15 minutes). The extracts were weighed and duplicate weighed aliquots (1.0 mL) were taken for radioassay.

The sediment debris remaining after extraction was rinsed with water (200 mL) by sonication for 15 minutes and shaking for 15 minutes. The sediment and rinse were separated by centrifugation at 2500 rpm for 15 minutes. The rinse was weighed and duplicate weighed aliquots (1.0 mL) taken for radioassay. The wet post extraction solid was weighed and duplicate weighed aliquots (approximately 0.3 g) were immediately taken for combustion.
Sediment extracts were combined proportionally to their total weights and analysed by HPLC. In addition, the dip tube and tubing connected to trapping vessels were extracted with acetonitrile, the extract weight was recorded and duplicate weighed aliquots (1 mL) taken for radioassay. The dip tube extracts were analysed by HPLC. The volumes of trapping solutions were measured and duplicate (1.0 mL) aliquots taken for radioassay. The polyurethane foam bung was extracted with acetonitrile by sonication for
5 minutes, the extract was weighed and duplicate weighed aliquots (1.0 mL) taken for radioassay.
Compartment:
natural sediment: freshwater
DT50:
< 14 d
Details on results:
Parameter measurements indicate that the aerobic and anaerobic systems complied with the requirements of the OECD Guideline 308. The temperature of the room remained within the
range 12 ° plus minus 2°C throughout the incubation period. The achieved application rate was 0.15 mg/vessel. The purity of the application solution was ≥95.0%.

The total recoveries of radioactivity (i.e., ‘mass balances’, the sum of radioactivity in the water layer, extractable and non-extractable sediment radioactivity and volatile radioactivity)
were in the range 81.1 to 95.6% applied radioactivity (AR).In the aerobic system, the total radioactivity in the water layer increased from 7.5% AR at time zero to 24.0% AR after 14 days. In the sediment, extractable radioactivity decreased
from 80.9% AR at time zero to 54.8% AR at 14 days. Non-extractable radioactivity in the sediment (bound residues) was in the range 2.5% AR to 3.6% AR. The dip tube extract recovered a further 12.1% AR after 14 days and 0.4% AR was detected in the tubing extract.

No volatile radioactivity was detected in the trapping system.

In the anaerobic system a similar pattern was seen with the total radioactivity in the water layer increasing from 23.2% AR at time zero to 36.9% AR after 14 days. In the sediment, extractable radioactivity decreased from 56.3% AR at time zero to 44.6% AR at 14 days. Non-extractable radioactivity in the sediment accounted for 1.3% AR after 14 days. The dip tube extract recovered a further 11.2% AR after 14 days and 0.4% AR was detected in the tubing extract. No volatile radioactivity was detected in the trapping system. In the aerobic system HPLC analyses of the water samples showed quick degradation of the test item, especially the 1, 4 isomer, with levels declining from a total of 5.9% AR at time zero to 1.2% after 14 days. In the sediment extracts, test item declined from a total of 75.6% AR to 28.7% AR after 14 days. Up to 8 unknown components were detected in sediment samples (≤8.8% AR).

In the anaerobic system HPLC analyses of the water samples again showed quick degradation of the test item, with levels declining from a total of 17.6% AR at time zero to 2.2% after 14 days. In the sediment extracts, test item declined from a total of 51.9% AR to 23.4% AR after 14 days. Up to 6 unknown components were detected in sediment samples (≤9.9% AR).

Most of the radioactive material detected in organo-soluble extracts from the dip tubes was comprised of the parent substance.
Executive summary:

1,3-bis(tert-butylperoxy isopropyl)benzene and 1,4-bis(tert-butylperoxy isopropyl)benzene degraded rapidly from the aquatic sediments, aerobic and anaerobic, with an estimated DT50 value of <14 days in the total system.

The test item was shown to degrade to up to 11 unidentified degradates (≤19.3% AR).

Mostly parent substance was detected in the dip tube extracts after 7 and 14 days incubation.

The results of this study demonstrate the feasibility of conducting a full OECD Guideline 308 study to investigate degradation of 1,3-bis(tert-butylperoxy isopropyl)benzene and 1,4- bis(tert-butylperoxy isopropyl)benzene in the aerobic and anaerobic aquatic sediments.

Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
September 2018 to September 2019
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Justification for type of information:
Read across justification is attached to IUCLID section 13.2
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Details on source and properties of surface water:
The test water and sediment was collected from Brandywine Creek, Chadds Ford, PA on 18 January 2019. The collection date, temperature, dissolved oxygen content and pH at collection are presented in Table 2. The water was transported in a cool, sealed container with enough headspace to provide access to air and arrived at Eurofins EAG Agroscience, LLC on 23 January 2019. The water was characterized with respect to total suspended solids, dissolved organic carbon, and total organic carbon. The sediment was characterized with respect to texture (% sand-silt-clay), USDA textural class, bulk density, cation exchange capacity (CEC), % moisture at 1/3 bar, organic matter (%) (Walkley-Black method), pH, calcium, magnesium, sodium, potassium, and hydrogen. Water and sediment characterization was conducted at Agvise Laboratories, Inc. (Northwood, North Dakota).

Upon arrival, the water was filtered through a 0.2 mm sieve and the sediment was filtered through a 2 mm sieve to remove coarse particulate matter and debris. Approximately six aliquots of Brandywine creek sediment (15 mg each) were transferred into six subaliquots of filtered Brandywine creek water (one liter each), creating water amended with sediment. The six bottles of water amended with sediment were continuously mixed using a stir plate and stir bar. Samples were prepared by measuring 100 mL of water amended with sediment via a graduated cylinder and dispensing into 250-mL amber bottles. One bottle of the water amended with sediment was placed in the autoclave for 20 minutes at 250°F (15 psi) to be sterilized for the sterile sample set. The sterilized water amended with sediment was dispensed using sterilized graduated cylinders into 250-mL sterilized amber glass bottles (which had also been autoclaved at 250°F and 15 psi for 20 minutes). After dosing, all samples were placed in a constant temperature room maintained at 12 ± 2°C with access to air. The pH and dissolved oxygen content of the filtered water was measured using two surrogate samples prior to use in the study and prior to sampling at each time interval.

The water sample was analyzed for total organic carbon (TOC) at the beginning of the incubation (experimental start) and at the end of incubation (experimental end) during the study, and is expressed in ppm.
Details on source and properties of sediment:
See section 'Details on source and properties of surface water'
Duration of test (contact time):
61 d
Initial conc.:
39.9 µg/L
Based on:
act. ingr.
Initial conc.:
0.8 µg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
test mat. analysis
Details on study design:
The study design was as follows:
- 18 treated samples to be dosed at the high dose rate, sufficient for 6 sampling intervals in duplicate and six extra samples
- 18 treated samples to be dosed at the low dose rate, sufficient for 6 sampling intervals in duplicate and six extra samples
- 8 treated samples to be dosed with [14C]benzoic acid as a control substance, sufficient for 2 sampling intervals in duplicate and four extra samples
- 10 sterile treated samples dosed at the high dose rate, sufficient for 3 sampling intervals in duplicate and 4 extra samples
- 2 untreated incubated samples for TOC measurement

The high-dose and low-dose viable samples were connected to trapping vessels for continuous trapping throughout the study. A peristaltic pump was used to draw ambient air through the sample headspace and traps. The ambient air was passed first through a vial containing deionized water to moisturize the air, followed by the sample container and then the volatile traps. The samples were connected via Teflon tubing threaded through the septum caps and connected to manifolds. Trap solutions were housed in glass vials (40 mL capacity) fitted with open top caps with Teflon-lined silicon septa through which the Teflon tubing was threaded in the same fashion as the samples. Samples were placed in a constant temperature room maintained at 12 ± 2°C during the incubation period. Sample bottles were placed on an orbital shaker for continuous gentle shaking during the incubation period. The temperature of the constant temperature room was continuously monitored with a REES temperature monitoring system.
The setup of the reference control samples (dosed with [14C]benzoic acid) was similar to that of the [14C](1,3)/(1,4)bis-peroxide high-dose/low-dose viable samples.
For sterile sample set, all glassware was autoclaved at 250°F and 15 psi for 20 minutes prior to use. The sterile samples were prepared with a sterilized foam plug inserted into the opening of the test vessel, and no traps were required or connected for volatile/CO2 collection. Sterile samples were placed on an orbital shaker in a constant temperature room maintained at 12 ± 2°C during the incubation period.
The untreated incubated samples were capped with Teflon lined silicon caps and connected to a humidifier (D.I. water) as well as a peristaltic pump for air flow. Untreated samples were placed on an orbital shaker and incubated in a constant temperature room maintained at 12 ± 2°C during the incubation period. The temperature of the constant temperature room was continuously monitored with a REES temperature monitoring system.
Two preliminary tests were conducted to confirm the microbial viability of the natural water to be used in the study and to determine the dose rates, sampling methods, and sampling intervals for the definitive test. Based on the results of the preliminary test, sampling intervals of 0, 5, 14, 29, 44, and 61 days were used for the definitive test.
Reference substance:
benzoic acid, sodium salt
Compartment:
natural water: freshwater
DT50:
17.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: (1,3) + (1,4)bis-peroxide
Compartment:
natural water: freshwater
DT50:
16.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: (1,3)bis-peroxide
Compartment:
natural water: freshwater
DT50:
19.3 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: (1,4)bis-peroxide
Transformation products:
no
Details on results:
- Radiochemical Purity of Test and Reference Substances:
The radiochemical purity of [14C](1,3)/(1,4)bis-peroxide in the dose solution was determined to be 98.2% (both components of (1,3)/(1,4)bis-peroxide combined) following the application processes, confirming the stability of the test substance during application.

- Application Rate:
The target dose rate was 40.0 (high dose, sterile, reference) and 8.0 (low dose) µg/L. This corresponds to 4.0 and 0.8 µg/100 mL sample. The achieved dose rates were 0.0399 ppm or 3.99 µg/sample (39.9 µg/L) for high dose, sterile, and reference samples, and 0.0080 ppm or 0.80 µg/sample (8.0 µg/L) for low dose. The homogeneity of the dose solution during the dosing procedure was confirmed by radioassay of aliquots of the dose solution taken before and after application. The relative standard deviations for these aliquots were = 1.85% (Table 5).
The ratio of the two test substances achieved as demonstrated by the post dose purity checks are as follows:
Dose % HPLC (1,3)bis-peroxide % HPLC (1,4)bis-peroxide Ratio of (1,3):(1,4) achieved
High Dose 63.3 34.6 1.83:1.00
Low Dose 62.8 34.9 1.80:1.00

- Properties of Test System:
Dissolved oxygen (DO) content measurements ranged from an average of 6.75 – 12.12 ppm, demonstrating that the test systems were maintained under aerobic conditions throughout the study. pH measurements ranged from an average of 7.11 to 7.86.
All samples were incubated at 12 ± 2°C in a temperature-controlled room throughout the study.

- Microbial Activity:
The reference samples were treated with [14C]benzoic acid at a concentration of 3.76 µg/sample. Duplicate samples were sacrificed after 5 and 14 days of incubation. Results showed an average of 44.6% of the applied radiocarbon was converted to 14CO2 (recovered in NaOH traps) after 5 days of incubation, and an average of 75.5% AR of the applied radiocarbon was converted to 14CO2 (recovered in NaOH traps) after 14 days of incubation (Table 6). This demonstrated the viability of the test system and showed that the microbial activity of the water used in the study was sufficient to conduct the test.

- HPLC Column Recovery:
HPLC column recoveries were determined by directly counting an aliquot of the injected sample and comparing to the radioactivity eluted from the column. HPLC recoveries ranged from 90.3 to 112.0% throughout the study (Appendix 6).

- Mass Balance
* High Dose
Acceptable mass balance (90 – 110% AR) was achieved in both day 0 high dose samples (average of 95.5% AR), but recoveries declined in all future sampling intervals (Table 6). Mass balance ranged from 53.3 to 90.6% AR for all samples between day 5 and day 61. Average water layer recoveries decreased from 82.3% AR at time 0 to 6.6% AR at day 61. Average ACN rinse recoveries increased from 13.2% AR at time 0 to a maximum of 36.0% AR at day 5, and then declined to 1.0% AR at day 61. Recoveries in hexane rinses did not exceed 4.0% AR in any single sample, and the average hexane rinse recovery did not exceed 3.7% AR in any sampling interval. Recoveries in DCM rinses did not exceed 0.3% AR in any sampling interval. Recoveries in tubing and septa rinses did not exceed 3.3% AR in any single sample. Recoveries in day 5 foam plugs averaged 1.1% AR (acetonitrile extract) and 0.2% AR (hexane extract). The foam plug recoveries (hexane extract) increased to a maximum average of 59.9% AR at day 61. No significant radiocarbon was detected in either ethylene glycol or sodium hydroxide traps.
* Low Dose
Acceptable mass balance (Table 6) was achieved in both day 0 low dose samples (average of 97.8% AR), but similarly to the high dose rate set, recoveries declined in all future samplings (Table 6). Mass balance ranged 42.8 to 90.5% AR for all samples between day 5 and day 61. Average water layer recoveries decreased from 81.2% at time 0 to 13.3% AR at day 61. Average ACN rinse recoveries increased from 16.6% AR at time 0 to a maximum of 21.6% AR at day 5, and then declined to 2.6% AR at day 61. Recoveries in hexane rinses did not exceed 3.0% AR in any single sample, or more than 2.7% AR (average) in any sampling interval. Recoveries in DCM rinses did not exceed 0.7% AR in any sampling interval. Recoveries in tubing and septa rinses did not exceed 12.2% AR in any single sample, or 6.4% AR (average) in any sampling interval. Recoveries in day 5 foam plugs averaged 5.3% AR (acetonitrile extract) and 0.7% AR (hexane extract). The foam plug recoveries (hexane extract) increased to a maximum average of 41.4% AR at day 61. No significant radiocarbon was detected in either ethylene glycol or sodium hydroxide traps, with the exception of day 29 sodium hydroxide traps, which averaged 5.8% AR. No significant difference was observed in the recoveries in NaOH traps between high-dose and low-dose viable samples, demonstrating that the mineralization of (1,3)/(1,4)bis-peroxide was not concentration dependent.
* Sterile
Acceptable mass balance was achieved in the day 14 and day 61 sterile samples, averaging 94.8 and 99.6% AR, respectively (Table 6). The day 29 samples had variable recoveries, 122.9 and 73.8% AR in rep A and rep B, respectively. Average water layer recoveries decreased from 20.7% at day 14 to 5.3% AR at day 61. Average ACN rinse recoveries decreased from 19.6% AR at day 14 to 1.7% AR at day 61. Recoveries in hexane rinses did not exceed 0.9% AR in any sampling interval. No significant radiocarbon was detected in the DCM rinses. Average recoveries in foam plugs (hexane extract) increased from 53.7% AR at day 14 to 92.6% AR at day 61. No volatile traps were used for the sterile samples.


- Product Balance
* High Dose
The high dose water layer product balance table is a composite of water layer product distribution and ACN rinse product distribution as determined by HPLC analysis of components that exceeded 5% AR (Table 7). The product distribution for all samples analyzed throughout the study indicated that the majority of radiocarbon analyzed was (1,3) and (1,4)bis-peroxide (Appendix 6). At time 0, (1,3) and (1,4)bis-peroxide represented averages of 58.5 and 36.0% AR, respectively, and this ratio was similar to the results obtained from post-dose HPLC analysis of the test substance. Both components declined throughout the study, representing averages of 0.7 and 0.9% AR for (1,3) and (1,4)bis-peroxide, respectively, at day 61. Several uncharacterized peaks were detected, but no individual peak exceeded 4.0% AR in any single sample.
The high dose foam plug was regarded as a separate compartment and was analyzed separately from the water layers. The product distribution for all samples analyzed throughout the study indicated that the majority of radiocarbon analyzed was comprised of (1,3) and (1,4)bis-peroxide (Appendix 6). At day 14 (the first sampling interval in which foam plug recoveries exceeded 5% AR), (1,3) and (1,4)bis-peroxide represented averages of 13.8 and 5.0% AR, respectively. Both components increased to averages of 39.1 and 20.8% AR for (1,3) and (1,4)bis-peroxide, respectively, at day 61. Several uncharacterized peaks were detected, but no individual peak exceeded 0.8% AR in any single sample.
* Low Dose
Low Dose samples were not analyzed in this study, as the high dose samples were considered representative.
* Sterile
The sterile water layer product balance table is a composite of water layer product distribution and ACN rinse product distribution as determined by HPLC analysis of the components that exceeded 5% AR (Table 7). The product distribution for all samples analyzed throughout the study indicated that the majority of radiocarbon analyzed was comprised of (1,3) and (1,4)bis-peroxide (Appendix 6). At day 14, (1,3) and (1,4)bis-peroxide represented averages of 20.6 and 17.0% AR, respectively. Both components declined to averages of 1.3% and 0.8% AR for (1,3) and (1,4)bis-peroxide, respectively, at day 61. Several uncharacterized peaks were detected, but no individual peak exceeded 2.8% AR in any single sample.
The sterile foam plug was regarded as a separate compartment and was analyzed separately from the water layers. The product distribution for all the samples analyzed throughout the study indicated that the majority of radiocarbon analyzed was comprised of (1,3) and (1,4)bis-peroxide (Appendix 6). At day 14, (1,3) and (1,4)bis-peroxide represented averages of 35.2 and 18.5% AR, respectively. Both components increased to averages of 59.6 and 33.1% AR for (1,3) and (1,4)bis-peroxide, respectively, at day 61. This ratio was similar to that observed in the test substance post-dose purity analysis, demonstrating the high volatility and stability of bis-peroxide over 61 days. No uncharacterized peaks were detected.

- Confirmation of [14C] (1,3)/(1,4)bis-peroxide:
The identification of (1,3)/(1,4)bis-peroxide was based on HPLC retention times and co-elution with the corresponding reference standards. Typical retention factor values (Rf) are presented in Table 1. The HPLC/ß-ram assignments of (1,3)/(1,4)bis-peroxide in the surface water amended with sediment were confirmed by TLC analysis of representative samples with reference standards.

- Proposed Degradation Pathway of (1,3)/(1,4)bis-peroxide:
The degradation pathway of (1,3)/(1,4)bis-peroxide in natural water amended with sediment under aerobic conditions cannot be elucidated based on the results of this study.

- Kinetic Analysis:
Since there was no degradation observed and mass balance between 90 – 110% AR was not maintained during the study, it was not possible to provide kinetic analyses of decline of parent and/or products. DT50 and DT90 of the dissipation were calculated using CAKE software version 3.3 (Reference 3) according to FOCUS kinetics (Reference 2). The Single First-Order (SFO) model was used in order to obtain the best fit to the experimental data. Calculations for the best-fit model is presented in Appendix 9. The statistics and DT50/DT90 values from the SFO (single first order) model are presented in the section "Helf-life of parent compound/50% disappearance time (DT50).
Results with reference substance:
- Radiochemical Purity of Test and Reference Substances:
The purity of [14C]benzoic acid in the dose solution was determined to be 96.3% following application.

- Mass Balance
* Reference Benzoic Acid
Mass balance in the control substance (benzoic acid) averaged 77.9 and 92.9% AR at day 5 and day 14, respectively (Table 6). Average water layer recoveries in the reference samples decreased from 31.9% at day 5 to 13.1% AR at day 14. Average NaOH trap recoveries increased from 44.6% AR at day 5 to 75.5% at day 14. The recoveries in the NaOH traps indicate the viability of the test system.

- Product Balance
* Reference
Reference benzoic acid samples were not analyzed.
Validity criteria fulfilled:
no
Remarks:
Acceptable mass balance (90–110% AR) was achieved at D0 for all experimental sets, but ranged from 53.3 to 90.6% AR in the high dose samples and 42.8 to 90.5% AR in the low dose samples for subsequent sampling intervals (D5 through D61)
Conclusions:
An aerobic mineralization study was conducted with [14C] (1,3)/(1,4)bis-peroxide in surface water amended with sediment, both from Brandywine Creek, Pennsylvania, USA for up to 61 days at two concentrations, 39.9 and 8.0 µg/L. Acceptable mass balance (90 – 110% AR) was obtained in all day 0 samples, and recoveries thereafter ranged from 53.3 to 90.6% AR from day 5 through day 61 in the high dose samples, and from 42.8 to 90.5% AR from day 5 through day 61 in the low dose samples. Acceptable mass balance was obtained in the day 14 and day 61 sterile samples, with average recoveries of 94.8 and 99.6% AR, respectively. The day 29 sterile samples yielded variable recoveries, 122.9 and 73.8% AR in rep A and rep B, respectively. As recoveries in the water layers declined, the recoveries in foam plug traps increased. Most notably, the majority of the radiocarbon recovered in day 61 sterile samples was found in the foam plugs (average of 92.6% AR).
The product distribution for the samples analyzed throughout the study indicated that the majority of radiocarbon analyzed was comprised of (1,3) and (1,4)bis-peroxide. In the time 0 high dose water layers, (1,3) and (1,4)bis-peroxide represented averages of 58.5 and 36.0% AR, respectively, which were similar to the ratio of the test substances obtained in post-dose HPLC analysis of the dosing solution. Both components declined to averages of 0.7 and 0.9% AR for (1,3) and (1,4)bis-peroxide, respectively, at day 61. In the high dose foam plugs, starting at day 14 (the first sampling interval for which foam plug recoveries exceeded 5% AR), (1,3) and (1,4)bis-peroxide represented averages of 13.8 and 5.0% AR, respectively. Both components increased to averages of 39.1 and 20.8% AR for (1,3) and (1,4)bis-peroxide, respectively, at day 61.
In the sterile water layers, at day 14 (1,3) and (1,4)bis-peroxide represented averages of 20.6 and 17.0% AR, respectively. Both components declined to averages of 1.3 and 0.8% AR for (1,3) and (1,4)bis-peroxide, respectively, at day 61. In the day 14 sterile foam plugs, (1,3) and (1,4)bis-peroxide represented averages of 35.2 and 18.5% AR, respectively. Both components increased to averages of 59.6 and 33.1% AR for (1,3) and (1,4)bis-peroxide, respectively, at day 61. This ratio of the test substances was also similar to the ratio obtained in the post-dose HPLC analysis of the dosing solution, demonstrating the high volatility and stability of bis-peroxide over 61 days under these conditions.
The rate of dissipation of combined (1,3)/(1,4)bis-peroxide from water over 61 days of incubation at 12 ± 2°C was determined to be 17.7 days (SFO model). The rate of dissipation of (1,3)bis-peroxide from water over 61 days of incubation at 12 ± 2°C was determined to be 16.7 days (SFO model). The rate of dissipation of (1,4)bis-peroxide from water over 61 days of incubation at 12 ± 2°C was determined to be 19.3 days (SFO model).
There was no consistently observed mineralization of test substance to 14CO2, as recoveries in NaOH traps were less than 0.1% AR for all high-dose and low-dose sampling intervals, with one exception (low dose 29 day). Since there was no significant difference in 14CO2 production between high- and low-dose samples, this demonstrates that the mineralization of (1,3)/(1,4)bis-peroxide was not concentration dependent.
Overall, the viable samples did not achieve acceptable mass balance. This was likely due to one or more factors: Volatilization (as demonstrated by the high recoveries of radiocarbon in the foam plug traps), poor water solubility (as demonstrated by high recoveries of radiocarbon in rinses of the glassware and bottles), inefficient trapping of radiocarbon (due to active air flow in the viable samples compared to passive aeration in the sterile samples), irreversible adherence of bis-peroxide to the surfaces, and/or formation of degradation products not trapped by the volatile traps used in the study.
Due to the low mass balance, it is not possible to make any definitive conclusions on whether volatilization or mineralization occurs when the test substance leaves the test system.
Executive summary:

An aerobic mineralization study was conducted with [14C] (1,3)/(1,4)bis-peroxide in surface water amended with sediment (at 15 mg sediment per liter of water) from Brandywine Creek, Pennsylvania, USA for up to 61 days at two concentrations, 39.9 and 8.0 µg/L. The study was conducted in accordance with OECD Guidelines for the Testing of Chemicals - Guideline 309. The samples were incubated in the dark on orbital shakers (to provide constant agitation) under aerobic conditions at 12 ± 2 °C for 61 days. In addition, reference and sterile control samples were incubated under the same conditions to confirm the microbial activity of the test water with amended sediment and examine possible abiotic degradation, respectively. The reference control samples were treated with [14C]benzoic acid at a concentration of 37.6 µg/L, and the sterile control samples were treated with [14C](1,3)/(1,4)bis-peroxide at 39.9 µg/L. The test was performed in flow-through systems which allowed humidified air to pass over the sample headspace and through the traps to collect volatile organic components (foam plugs and ethylene glycol) and 14C-carbon dioxide (aqueous sodium hydroxide). Sterile samples were passively aerated and did not include traps for volatiles. At each sampling interval, the amount of radioactivity in the test system and traps for volatiles (if applicable) was determined by liquid scintillation counting (LSC), and those high-dose and sterile subsamples which represented > 5% of the applied radiocarbon (AR) were analyzed by high-performance liquid chromatography (HPLC) coupled with ß-ram detector.

The samples containing the reference control substance [14C]benzoic acid were sacrificed after 5 and 14 days of incubation. The mineralization of benzoic acid to 14CO2 observed after 14 days of incubation (average of 75.5% AR recovered in NaOH traps) confirmed the microbial activity of the test system. Recovery in NaOH traps was confirmed by addition of barium chloride solution, which caused precipitation of 14CO2 as Ba14CO3.

Acceptable mass balance (90 – 110% AR) was achieved in the time 0 samples for all experimental sets, but ranged from 53.3 to 90.6% AR in the high dose samples for subsequent sampling intervals (day 5 through day 61), and ranged from 42.8 to 90.5% AR in the low dose samples for subsequent sampling intervals (day 5 through day 61). Acceptable mass balance was achieved in the day 14 and day 61 sterile samples with averages of 94.8 and 99.6% AR, respectively. The sterile 29 day samples had variable recoveries, 122.9 and 73.8% AR in rep A and rep B, respectively. As recoveries in the water layers declined, the recoveries in foam plugs increased. Most notably, the majority of the radiocarbon in day 61 sterile samples was recovered in the foam plugs (average of 92.6% AR).

The product distribution for all samples analyzed throughout the study indicated that the majority of all radiocarbon analyzed was (1,3) and (1,4)bis-peroxide. In the time 0 high dose water layers, (1,3) and (1,4)bis-peroxide represented averages of 58.5 and 36.0% AR, respectively, which was similar to the ratio of the test substance from post-dose HPLC analysis. Both components declined throughout the study, reaching averages of 0.7 and 0.9% AR ((1,3) and (1,4)bis-peroxide, respectively) by day 61. In the 14 day high dose foam plugs (the first interval in which foam plug recoveries exceeded 5% AR), (1,3) and (1,4)bis-peroxide represented averages of 13.8 and 5.0% AR, respectively. Both components increased throughout the study to averages of 39.1 and 20.8% AR for (1,3) and (1,4)bis-peroxide, respectively, by day 61.

In the sterile water layers, (1,3) and (1,4)bis-peroxide represented averages of 20.6 and 17.0% AR, respectively, after 14 days of incubation. Both components declined to averages of 1.3 and 0.8% AR for (1,3) and (1,4)bis-peroxide, respectively, by day 61. In the sterile foam plugs, (1,3) and (1,4)bis-peroxide represented averages of 35.2 and 18.5% AR, respectively, after 14 days of incubation. Both components increased to reach averages of 59.6 and 33.1% AR for (1,3) and (1,4)bis-peroxide, respectively, by day 61. The similarity of this ratio to the test substance ratio immediately following dosing demonstrates the high volatility and stability of bis-peroxide over 61 days.

The rate of dissipation of combined (1,3)/(1,4)bis-peroxide from water over 61 days of incubation at 12 ± 2°C was determined to be 17.7 days by single-first order (SFO) kinetics. The rate of dissipation of (1,3)bis-peroxide from water over 61 days of incubation at 12 ± 2°C was determined to be 16.7 days (SFO). The rate of dissipation of (1,4)bis-peroxide from water over 61 days of incubation at 12 ± 2°C was determined to be 19.3 days (SFO).

Test Substance

Experimental Set

Sample Phase

DT50

(days)

DT90

(days)

Chi2

Err%

R2

Kinetic Model

(1,3) + (1,4)bis-peroxide

High Dose

water layer

17.7

58.8

20.0

0.8636

SFO

(1,3)bis-peroxide

High Dose

water layer

16.7

55.4

21.6

0.8523

SFO

(1,4)bis-peroxide

High Dose

water layer

19.3

64.0

18.1

0.8734

SFO

Mineralization of test substance to 14CO2 was observed in only one sampling interval during the study. There was no 14CO2 production in any other high- and low-dose samples, demonstrating that the mineralization of (1,3)/(1,4)bis-peroxide was not concentration dependent.

Overall, viable samples did not achieve acceptable mass balance. Based on the results of the study, this can be attributed to several factors. First, bis-peroxide is highly prone to volatilization, as demonstrated by the high recoveries of radiocarbon in the foam plug traps and the product distribution, and has poor solubility in water, as demonstrated by high recoveries of radiocarbon in rinses of the glassware and bottles. It is likely that the low mass balance observed in the viable samples was due to inefficient trapping of radiocarbon (viable samples utilized active air flow whereas sterile samples were only passively aerated) and/or irreversible adherence of bis-peroxide to surfaces. Furthermore, it was observed that the study design used for the sterile samples was more successful at achieving mass balance than the viable samples. It is possible that the viable samples formed products not trapped by the volatile traps used in the study. Due to lack of mass balance, it is not possible to make any definitive conclusions on whether volatilization or mineralization occurs when the test substance leaves the test system.

COMMENT FROM THE REVIEWER:

The test system was not suitable for the test substance as the test material leaved the test system and hence, the study did not meet the validy critera for the test guideline as viable samples did not achieve acceptable mass balance. Due to the poor mass balance and the inability to allow sufficient contact time for biodegradation to occur due to the volatility, the test is concluded as unsuitable for the substance. Based on the study it is not possible to draw any conclusions with regards to surface water degradation. Hence, a OECD 308 test was initiated.

Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
August 2008 to May 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1a: GLP guideline study
Justification for type of information:
see read across rationale in Section 13.2
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
EPA Subdivision N Pesticide Guideline 162-3 (Anaerobic Aquatic Metabolism)
Deviations:
not specified
GLP compliance:
yes
Radiolabelling:
no
Oxygen conditions:
anaerobic
Inoculum or test system:
natural water / sediment
Details on source and properties of surface water:
Sediment and pond water were collected on september 15, 2008 from Refugio Park Pond in Hercules, California and received at the laboratory on the same day. The test system was refrigerated in the dark until use.

pH: 8.1
Calcium: 263 ppm
Mg: 122 ppm
Na: 287 ppm
Hardness: 1168 mg eq. CaCO3/L
Conductivity: 2.48 mmhas/cm
Sodium Absorption Ratio (SAR): 3.67
Total Dissolved Solids: 2386 ppm
Turbidity: 10.0 NTU
Dissolved Oxygen: 9.7 mg/L

Details on source and properties of sediment:
Sediment and pond water were collected on september 15, 2008 from Refugio Park Pond in Hercules, California and received at the laboratory on the same day. The test system was refrigerated in the dark until use.

Sediment: described as a loam soil (Agvise Laboratories, Inc.), was passed throught a 2 mm sieve then stored in plastic bags.

Sand: 46%
Silt: 38%
Clay: 16%
USDA textural class: loam
bulk density: 0.95
CEC (meq/100g): 15.4
% moisture at 1/3 bar: 20.1
% oragnic matter: 3.1
ph in 1:1 soil:water ratio: 7.8
Duration of test (contact time):
90 d
Initial conc.:
1 other: µg/g (dry soil weight) (1.0 ppm)
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
Preparation of Test System
 
The Test System consisted of the test substance applied to 30 g dry weight equivalent of sediment flooded with 120 mL of pond water. Samples were prepared by combining approximately 53.43 g wet weight of sediment (30 g dry weight equivalent) and pond water to a final volume of 120 mL. The samples were prepared in amber bottles with Teflon-lined septum caps. A total of 31 samples were prepared.
 
The anaerobicity of the samples was checked by measuring the dissolved oxygen and redox potential of random samples. All samples were pre-incubated at 25°C for 4 weeks under anoxic conditions prior to test application.

Preparation of test substance, chemical application
 
Concentration of the dose solution: 290.3 µg/mL.
 The dosing was carried out under a nitrogen atmosphere (nitrogen stream above the dosing area).
 100µL aliquot of the dosing solution was added to 12 previously prepared sediment/water samples by unscrewing the cap and adding the dosing solution directly into the water layer. Nitrogen was bubbled through the water layers of each sample after dosing. Caps were replaced immediately and the bottles were placed in an incubator after dosing and maintained at 25°C in the dark (samples were maintained in the dark at 25 +/- 1°C for up to 90 days).

Sampling:
duplicate dosed samples were removed for sampling and analysis at each time interval (0, 7, 13, 69 and 90 days), processed and axtracted the same day of collection.
Water layer was separated by centrifugation
solutions were filtered (0.45 µm nylon filters) prior to analysis.

Sediment layer was extracted with acetonitrile and filtered (0.45 µm nylon filters) prior to analysis.

Statistics
Averages, linear regression, standard deviation, relative tandard deviation and relative error were the only statistical methods employed (Microsoft Excel)
Reference substance:
not specified
Compartment:
other: water, material (mass) balance
% Recovery:
1
Compartment:
other: sediment, material (mass) balance
% Recovery:
94.7
% Degr.:
ca. 30
Parameter:
test mat. analysis
Sampling time:
7 d
% Degr.:
ca. 30
Parameter:
test mat. analysis
Sampling time:
13 d
% Degr.:
ca. 80
Parameter:
test mat. analysis
Sampling time:
69 d
% Degr.:
> 90
Parameter:
test mat. analysis
Sampling time:
90 d
Compartment:
entire system
DT50:
29 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: r²=0.991
Remarks on result:
other: DT90=96.3 days
Transformation products:
yes
No.:
#1
Details on transformation products:
tert-butanol was detected in the sediment and water layers for all time points after 0, but the amount was below quantifiable limits for all except Day 90 samples. in the day 90 sediment layers, tert-butanol was detected but below the limit of quantification. in water layers, an average of 8.3 µg were recovererd in the water extracts. the mass of tert-butanol in the system at day 90 is equivalent to 61.5%n yield from the initial test substance dose.
Evaporation of parent compound:
not specified
Volatile metabolites:
not specified
Residues:
not specified
Details on results:
pH were comprised between 7.13 and 8.11
O2 concentrations were comprised between 0.08 and 1.48 ppm
Eh values were comprised between -309 and 113
Validity criteria fulfilled:
not specified
Executive summary:

A study was conducted to determine the biodegradation of structurally analogous [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[1,1-dimethylethyl] peroxide in water/sediment simulation test, according to US EPA guideline.

The substance was not found in the water layers at all time points. In the sediment layer, an average of 94.7% of the applied dose was detected at day 0. The substance has decreased to an average of 9.7% of the applied dose at day 90. The half-life in sediment/water sediment under anaerobic conditions was determined as 29 days.

Tert-butanol, the main expected breakdown product, was detected in water and sediment layers at all time point (except day 0) but below a quantifiable level until day 90: it represented an average of 61.5% of the test substance applied dose.

Description of key information

The ready biodegradability on 2212 -81 -9 was evaluated in a study performed in accordance with OECD testing guideline 301 D and GLP requirements.

The test substance is not biodegraded in the closed bottle test, and therefore should not be classified as readily biodegradable. This lack of biodegradation is not due to toxicity of the test compound because the endogenous respiration is not inhibited by bis (tert-butyl peroxy isopropyl) benzene.

Based upon the adsorption potential of the substance of interest, a study was conducted to determine the biodegradation of the read across source [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[1,1-dimethylethyl], peroxide, CAS 25155 -25 - 3 in water/sediment simulation test, according to US EPA guideline. The substance was not found in the water layers at all time points. In the sediment layer, an average of 94.7% of the applied dose was detected at day 0. The substance has decreased to an average of 9.7% of the applied dose at day 90. The half-life in sediment/water compartment under anaerobic conditions was determined as 29 days.Tert-butanol, the main expected breakdown product, was detected in water and sediment layers at all time point (except day 0) but below a quantifiable level until day 90: it represented an average of 61.5% of the test substance applied dose.

A mineralization test in surface water (OECD 309) was conducted on the read across source CAS 25155 -25 -3. The test system was not suitable for the test substance as the test material left the test system and hence, the study did not meet the validy critera for the test guideline as viable samples did not achieve acceptable mass balance. Due to the poor mass balance and the inability to allow sufficient contact time for biodegradation to occur due to the volatility, the test is concluded as unsuitable for the substance. Based on the study it is not possible to draw any conclusions with regards to surface water degradation. Hence, a OECD 308 test was initiated and the performed feasibility study shows that the sediment test is feasible for the substance.

The water/sediment simulation test, according to US EPA guideline was considered to not reflecting environmental conditions. The risk assessment also shows that sediment is the most relevant compartment, therefore a new study was started according to theOECD 308 guideline.

A feasilbility study for aquatic sediment was performed. The results of the study demonstrate the feasibility of conducting a full OECD Guideline 308 study to investigate degradation of 1,3-bis(tert-butylperoxy isopropyl)benzene and 1,4- bis(tert-butylperoxy isopropyl)benzene in the aerobic and anaerobic aquatic sediments.

As the OECD 309 did not meet the validy criteria for the test and the full OECD 308 is yet not conducted the key values for the chemical safety assessment has not yet been established.

Key value for chemical safety assessment

Additional information