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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
Adequacy of study:
key study
Study period:
from 2008-03-04 to 2008-05-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study according to GLP and EPA guidelines with some deviations from the followed guideline and the equivalent European guideline. Only one water/sediment system tested instead of two; study was conducted at a temperature not relevant to the environment and would require recalculation to a more conservative 12 ºC. Anaerobic conditions only were tested making the aerobic sediment degradation impossible to assess. Extraction efficiency of degradation products was not optimal and a mass balance was not achieved.Nevertheless the study is well documented and the results are considered scientifically acceptable, and demonstrate a strong partitioning to the sediment and lack of persistence of the parent substance in the sediment under anaerobic conditions.
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
EPA Subdivision N Pesticide Guideline 162-3 (Anaerobic Aquatic Metabolism)
Deviations:
yes
Remarks:
A single soil type only was tested,temperature of study to high requires recalculation to 12 ºC,anaerobic conditions only were tested, information on degradation products was not optimal
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
Deviations:
yes
Remarks:
A single soil type only was tested,temperature of study to high requires recalculation to 12 ºC,anaerobic conditions only were tested, information on degradation products was not optimal
Principles of method if other than guideline:
OECD 308 recommends a minimum of 6 sample points in a test that should not exceed 100 days. The short duration of this study was such due to
the rapid removal of the test substance and hence less samples were taken. Sediment was stored for 7 months that could have resulted in reduction
of the microbial component of the sediment.However a long pre incubation for 5 weeks under test conditions was used to allow re-stabilization of the
microbial component. Adsorbtion and behaviour of the metabolites due to the poor extraction efficiency and limited sampling is inconclusive.
GLP compliance:
yes (incl. certificate)
Remarks:
2008-07-29
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data
Radiolabelling:
no
Oxygen conditions:
anaerobic
Inoculum or test system:
natural water / sediment
Details on source and properties of surface water:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Sediment and pond water were collected on august 2, 2007 from Rfugio Park Pond in Hercules, California at 0-6cm depth (submerged sediment) and 3-4 feet from shore in a municipal park with no herbicide or pesticide use except for copper sulfate. Samples were transported and received at PTRL West, Inc. on the same day.
- Storage conditions: refrigerated in the dark until use.
- Storage length:7 months
- Temperature (°C) at time of collection: no data
- pH at time of collection: 8.4
- Electrical conductivity: 2.3 mmhos/cm
- Redox potential (mv) initial/final: 129-150/ -307/-175
- Oxygen concentration (mg/l) initial/final: 0.29-1.26/-0.6/0.75 ppm
- Hardness (CaCO3): 980 mg eq. CaCO3/l
- Dissolved organic carbon (%): no data
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): no data
- Water filtered: No
- Type and size of filter used, if any: not applicable
Details on source and properties of sediment:
- Details on collection (e.g. location, sampling depth, contamination history, procedure):Sediment and pond water were collected on august 2, 2007 from Rfugio Park Pond in Hercules, California and received at PTRL West, Inc. on the same day.
- Storage conditions:refrigerated in the dark until use.
- Storage length:7 months
- Textural classification (i.e. %sand/silt/clay): Sandy loam soil (59% sand/ 30% silt/ 11% clay)
- pH at time of collection: 8.2
- Organic carbon (%): 2.2
- Redox potential (mv) initial/final: 129-150/ -307/-175
- CEC (meq/100 g): 11.7 meq/100 g
- Bulk density (g/cm³): 0.97
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): no data
- Sediment samples sieved: yes (2mm sieve)
Details on inoculum:
Not applicable
Duration of test (contact time):
14 d
Initial conc.:
0.989 other: µg/g
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
other: degradation product analysis 3.3.5 - trimethylcyclohexanone (CAS 873-94-9) ... (see attached file)
Details on study design:
PREPARATION OF TEST SUBSTANCE
the dose solution (296.8 µg/ml) was prepared by transferring 8 ml of CAS 6731-36-8 stock solution (0.9274 mg/ml) to a 25 ml volumetric flask then filled to the mark with ethyl acetate, mix and stored in an amber vial with Teflon cap. A 100µl aliquot of the dose solution was added to 16 previously prepared sediment/water samples by unscrewing the cap and adding the dosing directly into the water layer, using a 100 µl glass syringe. The remaining 15 samples were not dosed but were incubated alonside the dosed samples for use as fortification and control samples at each time point. One undosed sample was taken for control/fortified at T0.

TEST CONDITIONS
- Volume of test solution/treatment: the test system consisted of the test substance applied to 30g dry weight equivalent of sediment (43.2 g ww) with 83 ml of pond water.
- Composition of medium: not applicable
- Additional substrate: no data
- Solubilising agent (type and concentration if used): none
- Test temperature: 25°C
- pH: 6.63 on average
- pH adjusted: no
- CEC (meq/100 g): 11.7 meq/100g
- Aeration of dilution water: none
- Suspended solids concentration: 2032 ppm
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: none
- Other: none

TEST SYSTEM
- Culturing apparatus: 125 ml amber bottles with Teflon-lined septum caps
- Number of culture flasks/concentration: Sediment/water were in duplicate with a control and a fortified flask (16 flasks in total)
- Method used to create aerobic conditions: not applicable
- Method used to create anaerobic conditions: not specified but use of closed vessels. All samples were pre-incubated at 25°C for 5 weeks under anoxic conditions prior to test substance application and a nitrogen stream was set up above the dosing area to avoid exposure to air.
- Measuring equipment: analysis by gas chromatography with mass selective detector (GC-MSD)
- Test performed in closed vessels due to significant volatility of test substance: not applicable
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: no
- Other: none

SAMPLING
- Sampling frequency: at time zero, and following 7 and 14 days after application.
- Sampling method: at each time interval, duplicate dosed samples were removed from the incubator for sampling and analysis, along with two untreated samples for fortification and control.The dissolved oxygen, pH and redox of the samples were measured at sampling, immediately after opening the cap of the bottles.
- Sterility check if applicable: None
- Sample storage before analysis: samples were processed and extracted on the same day of collection.
- Other: none

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: None
- Toxicity control: None
- Other: none

STATISTICAL METHODS: averages, linear regression, standard deviation, relative standard deviation and relative error were the only statistical methods employed in this study.
Test performance:
Not applicable
Compartment:
other: water, material (mass) balance
% Recovery:
3
Compartment:
other: sediment, material (mass) balance
% Recovery:
97
% Degr.:
89.6
Parameter:
test mat. analysis
Sampling time:
14 d
Remarks on result:
other: 10.4% remaining in the sediments
Compartment:
entire system
DT50:
4.3 d
Type:
(pseudo-)first order (= half-life)
Temp.:
25 °C
Remarks on result:
other: r2=0.9505 Ensure use of correct temp input in Iuclid
Compartment:
entire system
DT50:
12.16 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: Recalculated for environmental halflife
Transformation products:
yes
No.:
#1
No.:
#2
Details on transformation products:
- Formation and decline of each transformation product during test:
_ 3.3.5 -trimethylcyclohexanone (CAS 873 -94 -9), an impurity observed in T0 samples, was monitored at all time points. A day 0, an average of 4.90 ng/ml was detected in the test system extracts. The majority of the mass resided in the water layer with an average concentration in the extract of 4.10 ng/ml. After 14 days of incubation, an average of 10.7 ng/ml was detected in the test system extracts, 63% of recovered mass in extracts the water layer.
_ Tert-butanol (CAS75-65-0) was detected in the day 7 and 14 water layers and sediment layers, but the day 14 water layers were the only samples
that contained enough to quantify, with an average of 1005 ng.

-For both transformation products the limited sample points and poor or the absence of extraction methods resulted in an incomplete picture in
the behaviour of these substances. Much data does however exist in open literature or is possible to calculate for these substances and will be
required in order to make a more complete assessment.

- Pathways for transformation: no data
- Other: none
Evaporation of parent compound:
no
Volatile metabolites:
not measured
Residues:
not measured
Results with reference substance:
Not applicable

None

Validity criteria fulfilled:
not applicable
Conclusions:
The parent substance 6731-36-8 can be concluded to be degraded in anaerobic environmental sediments with a halflife of 12.16 days at 12 ºC.
Conclusions are not possible for aerobic sediments and a complete picture of transformation product behaviour was not achieved. Study was
otherwise well conducted and provides valuable information but with the restrictions mentioned.
Executive summary:

Study according to GLP and EPA guidelines with some deviations from the followed guideline and the equivalent European guideline. Only one water/sediment system tested instead of two. The study was conducted at a temperature not relevant to the environment and has been recalculated for a more conservative 12 ºC average environmental temperature . Anaerobic conditions only were tested making the aerobic water / sediment degradation impossible to assess. Extraction efficiency of degradation products was not optimal and a mass balance was not achieved. Nevertheless the study is well documented and the results are considered scientifically

acceptable, and demonstrate a lack of persistence of the parent substance under anaerobic sediment conditions only. Data on degradation products should be supplemented from available sources or via further testing to allow a complete picture to be made and clarification as the the aerobic sediment / water degradation or equivalent is desirable.

Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
key 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:
data waiving: supporting information
Qualifier:
according to
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. certificate)
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
% 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.
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.
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.
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 and up to 17 low level unidentified degredation products. A low level of biodegredation
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 with14CO2accounted 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.

DT50and DT90values 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.


 

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:
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
Oxygen conditions:
other:
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.

Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Data waiving:
study technically not feasible
Justification for data waiving:
other:

Description of key information

In order to refine the estimations of release to the environment and investigate biodegradation beyond what was found in the closed bottle test a sewage

treatment simulation test has been conducted .  This was considered a relevant test due to the substance being used  in  industrial

settings only therefore having no untreated release to the environment (no dispersive use). Results of the continuous activated sludge simulation study

demonstrated 99.5 % of the test material in the influent was removed. 2% of the test material was lost to the air and 15.4% was adsorbed to the sludge. A total of 82.6 % biodegradation/other form of reactive removal of the parent material was measured after correction. The test material can therfore be expected to be significantly removed from industrial waste water streams during water treatment.

A sediment-water study using anaerobic aquatic sediment was conducted with (3,3,5-trimethylcyclohexylidene)bis[(1,1 dimethylethyl)peroxide] applied to a collected pond water/sediment system. 89.6% of the substance was degraded by day 14. Half-life in sediment and water were not determined individually but half-life in the whole system was determined as 4.3 days at 25ºC or 12.16 days at 12 ºC.  The substance was found to partition almost completely to the sediment

97%. Extraction efficiency of the parent material from the sediment met the required guideline criteria and a halflife in anaerobic sediment can therefore be

reliably determined. Conclusive information on transformation products under anaerobic conditions was lacking although positive identification of the

transformation products was achieved. Study lacks information on degradation of the parent substance and its transformation products under aerobic

sediment / water conditions evidence suggests possible reactive removal and subsequent binding as a mechanism for removal of the parent material.

On the request of the authorities additional simulation testing was requested. The original request of an OECD 309 was concluded as technically unfeasible due to the test material not remaining in the test system. The test substance has the tendancy to assosciate with water as a hydrotrope and due to the significant aeration in an aerobic system (and vigerous purging) is blown out of the system as parent material. The OECD 309 was therefore concluded to be not feasible and an OECD 308 was conducted. This study yielded similar half-life data (for the parent material) as the previous simulation test. In the two tested sediment types the DT 50 values of the parent material were 0.4 and 0.1 days respectively. DT90 values were 15.4 and 21.3 days, respectively. While the parent material rapidly became non quantifiable in the test system the majority of the radioactivity remained. The test substance was not completely mineralised. Up to 10% radioactive CO2 was formed at the end of the study. The remainder of the test material / transformation products were identified as radioactive bound non extractable residue in the sediment and up to 17 low level radioactive polar volatile transformation products in the water phase. The DT50 based on total radioactivity remaining in the system is therefore >120 days.

Key value for chemical safety assessment

Half-life in freshwater sediment:
120 d
at the temperature of:
12 °C

Additional information

Results of the continuous activated sludge simulation study demonstrated 99.5 % of the parent test material in the influent was removed. 2% of the test material was lost to the air and 15.4% was found adsorbed to the sludge. A total of 82.6 % removal was measured after correction. The parent test material can therefore be expected to be significantly removed from industrial waste water streams during water treatment.

A biodegradation study in anaerobic sediment according to GLP and EPA guidelines is available with some deviations from the followed guideline and the equivalent European guideline. Only one water/sediment system tested instead of two. The study was conducted at a temperature not relevant to the environment and has been recalculated for a more conservative 12 ºC average environmental temperature . Anaerobic conditions only were tested making the aerobic water / sediment degradation impossible to assess. Extraction efficiency of degradation products was not optimal and a mass balance was not achieved. Nevertheless the study is well documented and the results are considered scientifically acceptable and extraction efficiency of parent material was acceptable. The parent material was rapidy not quantifiable in this test system.

An OECD 308 with adapted test material application methods did demonstrate that the test material could be kept in the test system. As seen with multiple endpoints previously rapid loss of parent material was observed likely for the majority by reactive not biological processes. The OECD308 has confirmed a rapid half life of the parent material and formation of bound residues and unidentified degredation products. Some biodegadation was observed in the later stages of the study.