Cyclohexylidenebis[tert-butyl] peroxide

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Please refer to read-across justification attached to IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Reference 2

Endpoint:

biodegradation in water: sediment simulation testing

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

A study on the biodegradation of the test item in sediment requested in REACH Annex IX Section 9.2 is to be performed according to OECD test guideline 308. In section 5 "Applicability of the test" of this guideline states, that:
"The method is generally applicable to chemical substances (unlabelled or labelled) for which an
analytical method with sufficient accuracy and sensitivity is available. It is applicable to slightly volatile, non-volatile, water-soluble or poorly water-soluble compounds. The test should not be applied to chemicals which are highly volatile from water (e.g. fumigants, organic solvents) and thus cannot be kept in water and/or sediment under the experimental conditions of this test."

In a new vapour pressure study (LAUS, 2020), the vapour pressure of the test item at ambient temperature was determined to be 399 Pa at 20°C and 488 Pa at 25 °C, respectively. The old vapour pressure study (Akzo Nobel, 1996) was considered as invalid, as the measurements were conducted at temperature higher than the SADT temperature of the test item and it is highly possible that the measured vapour pressure is not of the test item but its decomposition products. Additionally, the Henry's Law Constant was calculated based on the experimental values for vapour pressure and water solubility. The calculated constant of 60500 Pa.m³/mol clearly showed that the test item is highly volatile.

Based on the newly obtained vapour pressure, it can be concluded that the test item has potential to be moderately to highly volatile. This assumption was confirmed in the newly performed higher tier ecotoxicological studies - FELS test according to OECD 210 and a BCF pre-test according to OECD 305. In both studies evaporation of the test item from the water medium was observed, which led to not being able to reach a steady state concentration in the BCF pte-test. Test item concentration were found in the control vessels in the FELS test, in which the air from the room was used for the aeration of the tanks. For more details, please refer to the respective section in the technical dossier.

An OECD 308 feasibility study with the structural analogue (see supporting study) was performed and it was also marked as technically not feasible due to major deficiencies in obtaining a stable concentrations in the water medium due to the volatile nature of the analogue substance if the application is performed through the water phase. If application through the sediment phase is performed, a half-life of the source substance in sediment only could be derived. For details, please refer to the read-across study records.

In conclusion, the study according to OECD 308 with the test item is considered technically not feasible according REACH Annex XI, Section 2 and OECD 308, Section 5, and therefore, read-across to the structural analogue was performed.

Transformation products:

not measured

Reference 3

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start date: 23 April 2018
Experimental completion date: 26 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Some modifications due to test substance properties.

Reason / purpose for cross-reference:

data waiving: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

Deviations:

yes

Remarks:

The study design was modified from the OECD 308 guideline as the test item was volatilised (Although not inherently volatile). The test item therefore was applied to the sediment layer.

Principles of method if other than guideline:

Test was extended and application of test material was modified to maintain the test chemical concentration in the test system by dosing directly on the sediment. This was investigated during a series of preliminary tests. Sediment application was the only approach that allowwed the test material to remain long enough in the sustem for testing.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical name: 1,1-Bis(tert-butylperoxy)-3,3,5-Trimethylcyclohexane
CAS number: 6731-36-8
Use: Chemical
Molecular formula: C17H34O4
Molecular weight: 302.5
Physical state: Liquid
Solubility in water: 92 µg/L

Radiolabelled Test Item
Source: Sponsor
Batch number: NPE/YH94QT/06 (purified at Envigo from CFQ42912)
Specific activity: 7.17 MBq/mg
Radiochemical purity: ≥95.0%
Storage: At -10ºC to -30ºC


Non-radiolabelled Test Item
Source: Sponsor
Lot number: 0905137109
Purity: 93.4%
Storage: 2ºC to 8ºC
Expiry date: 06 January 2019

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

Calwich Abbey Lake:
Apperanace: Clear, colourless
Temperature (just below surface, °C) 10.2
Temperature (5 cm above sediment, °C): 10.2
pH: 7.81
Oxygen saturation:
Just below surface (%): 86.3
At water/sediment interfance (%): 86.2


Emperor Lake
Apperanace: Slight brown colour, clear
Temperature (just below surface, °C) 10.5
Temperature (5 cm above sediment, °C): 10.3
pH: 5.70
Oxygen saturation:
Just below surface (%): 81.0
At water/sediment interfance (%): 75.1

Details on source and properties of sediment:

Calwich Abbey Lake’ and ‘Emperor Lake’ aquatic sediments were provided by LRA Labsoil, Lockington, Derby, UK. Sediment and water were shipped in separate containers and, upon receipt at the testing facility, stored at approximately 2 - 8 °C for 8 days (recommended maximum is 4 weeks). Prior to use, the sediment was passed through a 2 mm sieve and the water was passed through a 0.2 mm sieve. Characterization of sediment and water was carried out, not to GLP, by NRM Ltd, Bracknell, UK. Microbiological assays of the aquatic sediments were conducted at Envigo as part of this study. Further batches of sediment were used for repeating zero-time samples.

The sediment from Emperor Lake was a slightly acidic sandy clay loam with a low organic carbon content while that from Calwich Abbey Lake was an approximately neutral sandy silt loam with a higher organic carbon content.

Duration of test (contact time):

190 d

Initial conc.:

0.044 other: mg/mL

Based on:

test mat.

Remarks:

Calwich Abbey

Initial conc.:

0.088 other: mg/mL

Based on:

test mat.

Remarks:

Emperor Lake

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

Study Design
The study design was modified from the OECD 308 guideline as the test item was volatilised from water and application of the test item to the water phase was not suitable. This was demonstrated in previous preliminary experiments (Trigonox 29: Preliminary Investigation of Aerobic Transformation in Surface Water and Aquatic Sediment Systems (non-GLP), Envigo study number: VN47RM, 04 April 2018). The test item therefore was applied to the sediment layer. The water layer was decanted off the sediment layer with minimal disturbance. Radiolabelled test item was applied to the sediment layer of samples of two aquatic sediment types at a rate of 0.4 mg/kg, based on the dry weight of the sediment. The water layer was returned to the vessels after treatment. The volume ratio of wet sediment to water in each sample was approximately 1 : 3. Aquatic sediment systems were acclimatised under aerobic conditions (continuously air flow was passing though the systems) prior to dose application until reasonable stability had been established with respect to the pH, oxygen concentration and redox potential in the water and the pH and redox potential in the sediment. The air flow was reduced to periods of two hours on two occasions per week after treatment due to volatility of test item. Samples were arranged in flow-through systems designed to trap volatile radiolabelled compounds including 14CO2. The systems were incubated in darkness at 12  2C for periods of up to 159 days prior to analysis. Sediment and water phases were analysed separately.

Additional samples were established for the determination of the microbiological activity at the start, Day 100 and end of the incubation period and the measurement of the pH and redox potential in both phases and oxygen content in the water phase.

Study Conduct
Preparation and Incubation of the Test Systems
Portions (109.1 g or 220.1 g, dry weight equivalent, of Calwich Abbey Lake or Emperor Lake sediment, respectively) of wet-sieved sediment were added to cylindrical one-litre glass bottles of approximately 9 cm internal diameter. This required 268.6 g of Calwich Abbey Lake sediment and 353.7 g of Emperor Lake sediment. Portions of sieved water (603.1 g from Calwich Abbey Lake and 598.1 g from Emperor Lake) were added to the respective bottles. The volume ratio of wet sediment to water was 1 : 3, where the sediment layer was 3 cm deep and the water layer was 9 cm deep.

For each aquatic sediment, eighteen vessels were set up for treatment with radiolabelled test item at a rate of 0.4 mg/kg, based on the dry weight of the sediment. In addition, five vessels of each aquatic sediment were set up for microbiological activity measurements and two vessels of each sediment type were set up for measurements of pH, oxygen content and redox potential. These vessels were not treated with the test item.

Samples established for treatment with [14C]-test item were incorporated into individual flow-through systems arranged as follows.
1. Humidifying vessel (with sintered stem for uniform gas dispersion) containing water to humidify the air-flow.
2. Test vessel containing the aquatic sediment test system, aerated through a down tube above the surface of the water.
3. Vessel containing ethyl digol (to trap organic volatile compounds) with a polyurethane foam bung in the neck.
4. Vessel containing 1 M aqueous potassium hydroxide solution with phenolphthalein indicator (to trap 14CO2).
5. A non-return valve to prevent accidental backflow through the test apparatus.

The end of the glass stem bringing the air flow into each test vessel was just below the surface of the water during the acclimatization. Air was drawn through each system continuously at a flow rate of approximately 50 mL/minute. Flow rates were checked and adjusted throughout the incubation period. During the acclimatisation period (27 days) all traps except the humidifying water bottle were empty.

After test item application, the end of the glass stem bringing the air flow into each test vessel was adjusted to above the surface of the water and the air flow was reduced to about 30 mL/minute for periods of two hours on two occasions per week.

The samples assigned for measurement of microbiological activity were connected, in series, into a separate flow-through system for each aquatic sediment. Humidified air was passed through these vessels but no traps were included.

The samples assigned for system parameter measurements were connected in series (one for each aquatic sediment) as for treated vessels. Throughout incubation period, these samples were used for measurement of pH and redox potential in both phases and oxygen content in the water phase. Measurements were made twice weekly throughout the acclimatisation period and then, following test item application, at every sampling interval. The data are shown in Appendix 2.

All test systems were maintained in darkness at approximately 12°C in a temperature-controlled room. The temperature in the room was recorded using a temperature probe linked to an electronic monitoring system (REES Scientific).

The new batches of sediment were used to set up the samples for replacement of zero-time samples. Test vessels were acclimatized for 34 days prior to treatment. Samples were treated with 1 mL of the application dose.

Preparation and Application of the Test Item
The [14C]-test item was used without radiodilution for application to Calwich Abbey sediment. An aliquot of the [14C]-test item stock solution (1.1 mg, 7.55 MBq, in hexane) was transferred to a 25 mL volumetric flask and the solvent removed under a gentle steam of nitrogen. The dose was made up to volume with acetonitrile. The application solution was prepared at a concentration of 0.044 mg/mL (18744833 dpm/mL).

The [14C]-test item was used with radiodilution for application to Emperior Lake sediment. An aliquot of the [14C]-test item stock solution (1.14 mg, 8.16 MBq, in hexane) was transferred to a 25 mL volumetric flask and the solvent removed under a gentle steam of nitrogen. An aliquot of a non-radiolabelled test item stock solution (1.05 mg, in acetonitrile) was added to the same flask and made up to volume with acetonitrile. The application solution was prepared at a concentration of 0.088 mg/mL (19724800 dpm/mL).

Prior to application the water layer was decanted off into a beaker with minimal disturbance of the sediment. Due to the nature of sediment it was not possible to remove all of the water from Emperor Lake sediment. An aliquot (1 mL) of the respective application solution was applied to the surface of the sediment phase of each aquatic sediment. The water layer was carefully returned to the test vessel. The vessels (with the exception of those taken for zero-time analysis) were then reincorporated into their respective flow-through system. There was no air flow following treatment.

No test item was added to the samples established for microbiological activity determination or measurement of system parameters. Two of the five microbiology vessels for each sediment type were treated with 1 mL of acetonitrile in the same way as used for main application. For parameter vessels, the water was decanted off the sediment layer causing minimal disturbance then returned to the vessel prior to parameters being measured.

Compartment:

natural water / sediment: freshwater

% Recovery:

67.6

Remarks on result:

other: Calwich abby

Compartment:

natural water / sediment: freshwater

% Recovery:

85.2

Remarks on result:

other:

Remarks:

Emperor lake

Key result

% Degr.:

>= 49.2 - <= 67.6

Parameter:

radiochem. meas.

Sampling time:

159 d

Remarks on result:

other:

Remarks:

Calwich Abbey Lake Sediment. Parent material was rapidly no longer recoverable from the test system. However only low levels of mineralization to Co2 occurred. While this figure is likely representative of Primary degredation/transformation radioactivity indicated remained in the test system and hence should not be confused with removal from the test system.

% Degr.:

>= 77.5 - <= 85.2

Parameter:

radiochem. meas.

Sampling time:

159 d

Remarks on result:

other: Emperor Lake

Remarks:

Emperor Lake Sediment. Parent material was rapidly no longer recoverable from the test system. However only low levels of mineralization to Co2 occurred. While this figure is likely representative of Primary degredation/transformation radioactivity indicated remained in the test system and hence should not be confused with removal from the test system.

Key result

Compartment:

natural water / sediment: freshwater

DT50:

> 159 d

Type:

other: Total radioactivity in test system at the end of the test

Temp.:

12 °C

Remarks on result:

other:

Remarks:

Test material formed bound residue slowly decreasing througout the test and up to 18 low level unidentified degredation products. Totalling approximately 30% in each sediment system. A low level of mineralization

Compartment:

natural sediment: freshwater

DT50:

0.1 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Emperor Lake

Remarks:

This figure is calculated from parent material and is indicative of a rapid change in the parent material. Not to be confused with actual removal / mineralization or biodegradation. High levels of radioactivity remained in the system for both sediment types.

Compartment:

natural sediment: freshwater

DT50:

0.4 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Calwich Abbey Lake

Remarks:

This figure is calculated from parent material and is indicative of a rapid change in the parent material. Not to be confused with actual removal / mineralization or biodegradation. High levels of radioactivity remained in the system for both sediment types.

DT50:

180 d

Temp.:

12 °C

Remarks on result:

not determinable

Transformation products:

yes

Remarks:

Not identifiable multiple radioactive volatile products formed.

No.:

#1

No.:

#2

Details on transformation products:

The test item was shown to degrade to unidentified polar material (Unk 1, up to 15.3% AR) and up to seventeen low level unidentified degradates (≤10.9% AR). Test item was finally incorporated into bound residues and mineralized to carbon dioxide.

Evaporation of parent compound:

yes

Remarks:

Some parent material was removed by the test system by aeration not evaporation. Sediment application allowed the majority to remain in the test system however.

Volatile metabolites:

yes

Remarks:

Unidentified

Residues:

yes

Remarks:

Large percentage of radiolabel remained associated with sediment as unextractable bound residue

Details on results:

Please refer to "Any other information on results incl. tables"

  Incubation Conditions

Results of measurements of water and sediment redox potential, water and sediment pH and water oxygen content are shown inAppendix2. During the incubation period, oxygen levels in the water (approximately 29 to 93% saturation) and redox potentials in the water (greater than +380 mV) and sediment (generally less than +200 mV) were indicative of an aerobic, oxidising water phase and a reducing sediment phase.

The temperature of the room remained generally within the range 12±2°C throughout the incubation period except for several deviations where a maximum temperature of 15.6°C was recorded. The temperature deviations from the range occurred for no longer than 10 to 48 minutes on four occasions. On two other occasions the temperature rose to a maximum of 16.5°C for 6 days and then dropped to a minimum of 9.1°C for 2 days. This was not considered to have affected the outcome or integrity of the study.

 Achieved Application Rates and Radiochemical Purities

The amounts of radioactivity and test item applied to the various samples are shown below.

Aquatic sediment	Radioactivity applied per sample (dpm)	Specific activity of [^14C]–test item (dpm/mg)	Test item applied per sample (mg)	Application rate (mg/kg)
Calwich Abbey Lake	18744833	430000	43.59	0.40
Emperor Lake	19724800	223000	88.45	0.40

 

The radiochemical purity of [¹⁴C]-test item was ≥95.0% (Appendix 1). This value was considered acceptable for use in this study.

 

 Recovery and Distribution of Radioactivity

The total recoveries of radioactivity (‘mass balances’,i.e.the sum of radioactivity in the water layer, extractable and non-extractable sediment radioactivity and volatile radioactivity) were in the range 45.4 to 94.4% applied radioactivity (AR).

Total recoveries of radioactivity (63.3 – 77.2% AR) were low for the zero-time samples. This was considered to be due to losses of the test item during sample processing and therefore new samples were generated to replace original samples. The total recoveries of radioactivity accounted for 91.9 – 94.4 % AR (Calwich Abbey) and 75.1 – 77.7% AR (Emperor Lake) from the repeat zero-time.

In Calwich Abbey Lake aquatic sediment, total radioactivity in the water layer increased to 16.9 – 21.1% AR after 29 days and then slightly declined to 8.1 – 8.8% AR after 159 days. The partitioning of one water sample at Day 7, showed that the recoveries from the organo‑soluble and aqueous phases were the same as in total water sample. In the sediment, total radioactivity decreased from 81.3 – 82.3% AR at zero-time to 32.4 – 50.8% AR after 159 days. Non-extractable radioactivity in the sediment (bound residues) increased from 3.1 – 4.2% AR at zero-time to 24.9 – 42.0% AR after 159 days. Volatile radioactivity recovered from the KOH trap was associated with¹⁴CO₂and accounted for a maximum of 10.2% AR after 159 days. Organic volatile radioactivity was recovered from the ethyl digol trap (up to 3.6% AR) and foam bungs (up to 6.1% AR, all associated with test item). The extraction of vessel head, down tube, rubber seal and tubing recovered up to 18.3% AR. The analysis of tubing extract one showed that all radioactivity was associated with test item. 

Dissipation of radioactivity from the Emperor Lake aquatic sediment system was faster than for Calwich Abbey due to the nature of Emperor Lake sediment (phase separation was poorer and so the sediment contained a watery layer at the time of dosing). Total radioactivity in the water layer accounted for a maximum of 25.5 – 33.2% AR after one day and declined to 13.0 – 14.1% AR after 159 days. The partitioning of one water sample at Day 7, showed that the recoveries in organo-soluble and aqueous phases were similar to the total water sample. In the sediment, total radioactivity decreased from 46.3 – 54.0% AR at zero-time to 14.8 – 22.5% AR after 159 days. Non-extractable radioactivity in the sediment (bound residues) increased from 0.8 – 1.1% AR at zero time to 14.2 – 20.0% AR after 100 days (9.3 – 17.8% AR after 159 days). Volatile radioactivity recovered from the KOH trap was associated with¹⁴CO₂and accounted for a maximum of 5.8% AR after 100 days. Organic volatile radioactivity was recovered from the ethyl digol trap (up to 4.9% AR) and foam bungs (up to 7.1% AR, all associated with test item). The extraction of vessel head, down tube, rubber seal and tubing recovered up to 29.3% AR. The analysis of tubing extract one showed that all radioactivity was associated with test item.

The radioactivity recovered in the tubing extracts suggests that the test item was volatilised from the water and was absorbed by plastic tubing and foam bung. However not all radioactivity reached the traps and there was incomplete trapping of volatile radioactivity resulting in the low total recoveries in most samples. The test item lost from the test system by volatilisation was therefore not available for degradation in the aquatic sediment system.

Characterization of Volatile Radioactivity

A portion of volatile radioactivity was trapped by potassium hydroxide solution. The trapped radioactivity was shown to be associated with¹⁴CO₂by precipitation of the insoluble barium¹⁴C-carbonate.

Chromatographic Analysis

HPLC analysis of the overlying water and extracts of sediment resolved up to 18 components in addition to test item.

There was some movement of radioactivity from sediment into water from zero-time samples. Radioactivity accounted for 9.6 – 13.1% AR (Calwich Abbey, CA26 and CA27,Table 4) and 23.7 – 28.8% AR (Emperor Lake, EL26 and EL27,Table 5), where test item accounted for 3.5 – 4.7% AR (Calwich Abbey) and 11.9 – 20.3% AR (Emperor Lake). This suggests that degradation of test item occurred in the water in the early stages of incubation. There are no data available for the analysis of the water samples from Day 1 to Day 14. The data from Day 29 samples shows that no test item remained in the water samples. One major polar component (Unk 1) was detected (up to 10.9% AR) in Calwich Abbey water samples at 29 days and then declined to 4.4 – 4.6% AR after 159 days. Up to 14 minor unknown components were detected in water samples for both systems (≤7.3% AR).

In the sediment of Calwich Abbey Lake systems, test item declined from 74.1 – 76.4% AR to 26.7 – 28.4% AR after 7 days, little or no test item was detected after 29 days. Up to 8 minor unknown components were detected in sediment samples (≤8.8% AR). In the sediment of Emperor Lake systems, test item declined from 42.2 – 48.6% AR to 22.2 – 22.3% AR after 7 days and then to 0.3% AR after 100 days. Up to 10 minor unknown components were detected in sediment samples (≤4.8% AR).

The polar material (Unk 1) accounted for 12.3 – 15.3% AR (Calwich Abbey) after 62 days and up to 8.1% AR (Emperor Lake) after 159 days. This is assumed to be comprised of multiple low molecule weight components. Up to 17 minor unknown components were detected in both systems (≤10.9% AR). There were no references available to aid identification of unknowns. The polar component would be difficult to identify due to low molecular weight and may consist of multicomponents. The samples were unstable on storage (see Section 6.7), so any further investigation was not technically feasible.

The identity of [¹⁴C]-test item was established by co-chromotographic correspondence with authentic non-radiolabelled test item after co-injection. The identification of the test item is tentative as confirmation in a secondary chromatographic technique was not possible due to instability and volatility of the test item.

Storage Stability

The dose solution was prepared in acetonitrile and kept frozen till the repeat application of zero-time samples. Test item was stable in acetonitrile for approximately 3 months.

Water samples were analyzed on the day of sampling. Re-analysis of water samples was not possible after storage for short times at ambient or frozen conditions due to the volatile nature of the test item from water. This was demonstrated in previous preliminary experiments (Trigonox 29:Preliminary Investigation of Aerobic Transformation in Surface Water and Aquatic Sediment Systems (non-GLP), Envigo study number: VN47RM, 04 April 2018)and the recovery of test item from foam bung and tubing extracts.

Sediment extracts were analyzed after frozen storage as summarized below:

Sampling point	Date sampled	Date analysed by HPLC	Days after sampling
Day 1	24 May 2018	25 July 2018	62
Day 7	30 May 2018	25 July 2018	56
Day 14	06 June 2018	25-26 July 2018	50
Day 29	21 June 2018	08 August 2018	48
Day 62	24 July 2018	21 August 2018	28
Day 100	31 August 2018	04 September 2018	4
Day 159	29 October 2018	Not analysed	N/A

 

The results of the analyses of Day 29 sediment extracts (both systems) were inconsistent in comparison with Day 14 and Day 62. An attempt was made to re-analyze Day 29 extracts after further 1.5 months frozen storage. Re-analysis showed that there was little or no radioactivity remaining in these samples. This suggest that the samples may consist of a high proportion of water and were not stable on storage.

 

The post extraction solids were rinsed with water and combusted wet on the same day. The wet post extraction solids for Day 29 samples were air dried at ambient temperature. Re-analysis of dry solids showed significant loss of radioactivity. This suggest that volatile radioactivity was still present in stored samples.

 

Biotransformation Pathway

Test item was volatilised from water. Test item degraded to a number of low level unidentified degradates and was finally incorporated into bound residues and mineralized to carbon dioxide.

Validity criteria fulfilled:

yes

Conclusions:

Decline of the test item in the overall system corresponded to DT50 values of 0.4 days (Calwich Abbey Lake) and 0.1 days (Emperor Lake). This is indicative of rapid binding of the parent material only. Not Rapid biodegradation.
The test item was volatilised ( a volatility effect caused by purging) from the water. The test item was shown to degrade to polar material (Unk 1, up to 15.3% AR) and up to 17 low level unidentified degradates (≤10.9% AR), incorporated into bound residues and mineralized to carbon dioxide. High levels of radioactivity remained in both test systems indication a DT50 of the total system that corresponds to a Persistance classification (>120d).

Executive summary:

   Summary

The fate of test item has been studied in two natural aquatic sediment systems under laboratory conditions. The sediment from Emperor Lake was a slightly acidic sandy clay loam with a low organic carbon content while that from Calwich Abbey Lake was an approximately neutral sandy silt loam with a higher organic carbon content. Samples of each aquatic sediment system were allowed to acclimatise before being treated with [¹⁴C]-test item at a nominal rate of 0.4 mg/kg based on the dry weight of sediment in the test vessel. Prior to application the water layer was decanted off with minimal disturbance. An aliquot (1 mL) of the application solution was applied to the surface of the sediment phase of each aquatic sediment. The water layer was carefully returned to the test vessel. The samples were incubated under aerobic conditions at about 12 °C in darkness for up to 159 days.

 

The radioactivity in the water layer increased up to a maximum of 21.1 % applied radioactivity (AR, Calwich Abbey) and 33.2 % AR (Emperor) and then declined to 8.1 – 8.8 % AR and 13.0 – 14.1 % AR after 159 days in Calwich Abbey and Emperor Lake, respectively. In sediment, the total radioactivity decreased to 32.4 – 50.8 % AR (Calwich Abbey) and 14.8 – 22.5 % AR (Emperor) after 159 days. The proportion of radioactivity remaining unextracted in the sediment increased to 24.9 – 42.0 % AR and 9.3 – 17.8 % AR after 159 days in Calwich Abbey and Emperor Lake, respectively.

 

Total recoveries of radioactivity (mass balances) were between 45.4 % and 94.4 % AR. Incomplete trapping of volatile radioactivity resulted in low total recoveries for most samples. Radioactivity recovered in trapping solutions and associated with ¹⁴CO₂ accounted for 4.8 – 10.2 % AR after 159 days. Organic volatile radioactivity recovered from ethyl digol traps accounted for up to 4.9 % AR. Up to 7.1 % AR was recovered from polyurethane foam bungs, this was all associated with test item. The extraction of the vessel head, down tube, rubber seal and tubing to trapping vessels (tubing extract) recovered up to 29.3 % AR. The analysis of the tubing extract showed that all radioactivity was associated with test item.

 

The radioactivity recovered in the tubing extracts suggests that test item was volatised from the water and was absorbed by plastic tubing and polyurethane foam bungs. However, not all radioactivity reached the traps and therefore could not be measured to improve the overall recoveries.

 

DT₅₀ and DT₉₀ values for the decline of the test item from the total aquatic sediment system are shown below.

	Calwich Abbey Lake		Emperor Lake
	DT50 (days)	DT90 (days)	DT50 (days)	DT90 (days)
Total system	0.4	15.4	0.1	21.3

DFOP (Double First Order in Parallel) kinetic model is reported.

 

The test item was shown to degrade to unidentified polar material (Unk 1, up to 15.3 % AR) and up to seventeen low level unidentified degradates (≤10.9 % AR). Test item was finally incorporated into bound residues and mineralized to carbon dioxide.

Reference 4

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017

Reliability:

other: Preliminary data to advise best test system

Rationale for reliability incl. deficiencies:

other:

Justification for type of information:

Testing was met with technical difficulties. An Extensive set of feasibility tests were conducted to determine which method the testing laboratory could conduct reliably.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD308&309

Version / remarks:

problems keeping material in system report documents testing and contract labs recommendations for definitive testing

Deviations:

yes

Principles of method if other than guideline:

OECD 308 and 309 methods trialed to determine if test material can be reliably brought into and extracted from the test system.

GLP compliance:

no

Remarks:

Feasibility study to determine best approach for definitive study

Radiolabelling:

yes

Inoculum or test system:

natural water: freshwater

Details on source and properties of surface water:

included in file attached

Details on source and properties of sediment:

included in file attached

Details on inoculum:

included in file attached

Details on study design:

included in file attached

Validity criteria fulfilled:

yes

Conclusions:

The test material did not remain in the treated surface water for long enough to determine if degradation occurred and test material was recovered in the foam bung trapping media (48.6% AR) after 4 hours of incubation. This demonstrates that an OECD 309 study would not be feasible. For aquatic sediment samples treated via the water phase the total recoveries were low (60.1 – 72.0% AR) after 3 days. For aquatic sediment samples treated via the sediment phase the total recovery of radioactivity was in the range of 87.3 – 98.6% AR up to 3 days. Mostly parent was detected in water and sediment extracts. This demonstrates that it should be possible to conduct an OECD 308 study if treatment is via the sediment phase.

Executive summary:

OECD 308 concluded technically feasible, OECD 309 concluded as not technically feasible. These recommendations have been adopted and the OECD 308 has been commissioned and is in progress.

Reference 5

Endpoint:

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

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

The study on ultimate biodegradation in surface water requested in REACH Annex IX Section 9.2 is to be performed according to OECD test guideline 309. In section 7 "Applicability of the test" of this guideline states, that:
"This simulation test is applicable to non-volatile or slightly volatile organic substances tested at low concentrations. Using flasks open to the atmosphere (e.g. cotton wool plugged), substances with Henry’s law constants less than about 1 Pa · m³/mol (approx. 10-5 atm · m³/mol) can be regarded as nonvolatile in practice. Using closed flasks with a headspace, it is possible to test slightly volatile substances (with Henry’s law constants <100 Pa · m³/mol or <10-³ atm · m³/mol) without losses from the test system."

In a new vapour pressure study (LAUS, 2020), the vapour pressure of the test item at ambient temperature was determined to be 399 Pa at 20°C and 488 Pa at 25 °C, respectively. The old vapour pressure study (Akzo Nobel, 1996) was considered as invalid, as the measurements were conducted at temperature higher than the SADT temperature of the test item and it is highly possible that the measured vapour pressure is not of the test item but its decomposition products. Additionally, the Henry's Law Constant was calculated based on the experimental values for vapour pressure and water solubility. The calculated constant of 60500 Pa.m³/mol clearly showed that the test item is highly volatile.

Based on the newly obtained vapour pressure, it can be concluded that the test item has potential to be moderately to highly volatile. This assumption was confirmed in the newly performed higher tier ecotoxicological studies - FELS test according to OECD 210 and a BCF pre-test according to OECD 305. In both studies evaporation of the test item from the water medium was observed, which led to not being able to reach a steady state concentration in the BCF pte-test. Test item concentration were found in the control vessels in the FELS test, in which the air from the room was used for the aeration of the tanks. For more details, please refer to the respective section in the technical dossier.

An OECD 309 feasibility study with the structural analogue (see supporting study) was performed and it was also marked as technically not feasible due to major deficiencies in obtaining a stable concentrations in the water medium due to the volatile nature of the analogue substance.

In conclusion, the study according to OECD 309 with the test item is considered technically not feasible according REACH Annex XI, Section 2 and OECD 309, Section 7, and no study needs to be performed.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Transformation products:

not specified

Reference 6

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Please refer to read-across justification attached to IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Compartment:

natural water / sediment: freshwater

% Recovery:

67.6

Remarks on result:

other: Calwich abby

Compartment:

natural water / sediment: freshwater

% Recovery:

85.2

Remarks on result:

other: Emperor lake

Key result

% Degr.:

>= 49.2 - <= 67.6

Parameter:

radiochem. meas.

Sampling time:

159 d

Remarks on result:

other: Calwich Abbey Lake Sediment.

% Degr.:

>= 77.5 - <= 85.2

Parameter:

radiochem. meas.

Sampling time:

159 d

Remarks on result:

other: Emperor Lake Sediment.

Key result

Compartment:

natural water / sediment: freshwater

DT50:

> 159 d

Type:

other: Total radioactivity in test system at the end of the test

Temp.:

12 °C

Compartment:

natural water / sediment: freshwater

DT50:

0.1 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Emperor Lake

Compartment:

natural water / sediment: freshwater

DT50:

0.4 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Calwich Abbey Lake

DT50:

180 d

Temp.:

12 °C

Remarks on result:

not determinable

Transformation products:

yes

Remarks:

Not identifiable multiple radioactive volatile products formed.

No.:

#1

No.:

#2

Description of key information

A study on ultimate biodegradation in surface water (OECD 309) does not need to be conducted according to REACH Annex XI, Section 2. Due to the volatility of the test item, the study is technically not feasible. A study on sediment biodegradation according to OECD 308 is technically not feasible due to the volatility of the tets item, therefore, read-across to the structural analogue was performed.