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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable GLP-study following current OECD guideline.
Qualifier:
according to
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report):[14C]-2,4-di-tert.-butylphenol
- Substance type: Alkylphenol
- Physical state: solid
- Radiochemical purity (if radiolabelling): 98.8 %
- Specific activity (if radiolabelling):63.7 mCi/mmol
- Locations of the label (if radiolabelling): Phenyl ring
- Expiration date of radiochemical substance (if radiolabelling): 12 August 2016
- Storage condition of test material: Frozen
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water / sediment
Details on source and properties of surface water:
Water-sediment system #1:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Swiss Lake, Chatsworth, Derbyshire, England, sampled at 5-10 cm, scooped from lake in bucket and sieved, no pesticide use for 5 years
- Storage conditions: approximately 4°C, water logged with free access to air
- Storage length: 18 days
- Temperature (°C) at time of collection: 19°C
- pH at time of collection: 7.4
- Electrical conductivity: 117 µS/cm
- Redox potential (mv) initial/final: 168.2
- Oxygen concentration (mg/l) initial/final: 8.73
- Hardness (CaCO3): 273
- Dissolved organic carbon (%): 0.47%
- Water filtered: no

Water-sediment system #2:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Calwich Abbey Lake, Calwich, Staffordshire, England, sampled at 30-40 cm, scooped from lake in bucket and sieved, no pesticide use for 5 years
- Storage conditions: approximately 4°C, water logged with free access to air
- Storage length: 18 days
- Temperature (°C) at time of collection: 17°C
- pH at time of collection: 8.41
- Electrical conductivity: 568 µS/cm
- Redox potential (mv) initial/final: 151.2
- Oxygen concentration (mg/l) initial/final: 8.05
- Hardness (CaCO3): 274
- Dissolved organic carbon (%): 4.8%
- Water filtered: no
Details on source and properties of sediment:
Water-sediment system #1:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Swiss Lake, Chatsworth, Derbyshire, England, sampled at 0-5 cm, scooped from top layer of sediment and sieved, no pesticide use for 5 years
- Storage conditions: approximately 4 °C, water logged with free access to air
- Storage length: 18 days
- Textural classification (i.e. %sand/silt/clay): sand
- pH at time of collection: 6.734
- Organic carbon (%): 0.47
- Redox potential (mv) initial/final: -36.6
- CEC (meq/100 g): 2.8
- Bulk density (g/cm³): 1.20
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): 579.6 µg/g
- Sediment samples sieved: yes

Water-sediment system #2:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Calwich Abbey Lake, Calwich, Staffordshire, England, sampled at 1 m, scooped from top layer of sediment and sieved, no pesticide use for 5 years
- Storage conditions: approximately 4 °C, water logged with free access to air
- Storage length: 18 days
- Textural classification (i.e. %sand/silt/clay): loam
- pH at time of collection: 7.7
- Organic carbon (%): 4.8
- Redox potential (mv) initial/final: -164.6
- CEC (meq/100 g): 10.6
- Bulk density (g/cm³): 0.69
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): 754.0 µg/g
- Sediment samples sieved: yes
Details on inoculum:
n/a
Duration of test (contact time):
105 d
Initial conc.:
1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 270 µL
- Test temperature: 20 ± 2°C
- pH: Swiss Lake: 8.3 (water) and 5.6 (sediment), Calwich Abbey Lake: 8.3 (water) and 7.5 (sediment)
- pH adjusted: no
- CEC (meq/100 g): Swiss Lake: 2.8, Calwich Abbey Lake: 10.6
- Aeration of dilution water: yes
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: slight indication

TEST SYSTEM
- Culturing apparatus: Air was pulled through a water hydrator, then into the series of test bottles. Air exiting the series of test bottles was passed through a series of volatile traps.
- Number of culture flasks/concentration: 18 flasks per system/ 1 µg/mL
- Method used to create aerobic conditions: vacuum
- Measuring equipment: redox pH meter

- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: Supelco ORBO™-32 adsorbent tube, then through two traps containing 30 mL 1 N KOH

SAMPLING
- Sampling frequency: 0, 7, 14, 28, 45, 59, 80, and 105 days
- Sampling method: Water and sediment layers were separated by decanting an initial 100 mL of water into a 500-mL graduated cylinder. The remaining water layer was carefully transferred to a 250-mL Nalgene bottle for centrifugation at approximately 3,700 x g for 10 minutes. The centrifuged water layer was then decanted into 500 mL mixing cylinders container initial 100 mL. The volumes of water collected in the mixing cylinders were measured and adjusted, as necessary, to a consistent volume with reagent water. Triplicate aliquots of this sample were analyzed by LSC. An aliquot was vialed for HPLC analysis. Following decanting of the water phase, a 100-mL aliquot of 4:1 acetone:0.1 M ammonium carbonate was added to the Chemglass bottles, the sediment layers were quantitatively transferred from the glass test vessels to the correspondingly labeled Nalgene bottles, and the samples were shaken on a platform shaker for 60 minutes. The sediment pellets were manually broken up. The bottles were then centrifuged at approximately 3,700 x g for 20 minutes. Following centrifugation, successive supernatants were decanted into a 250-mL mixing cylinder. This process was repeated twice more, with each extraction kept separate, and the volume of each extraction was adjusted to a consistent volume with extraction solvent. Triplicate aliquots of this sample were analyzed by LSC. The sediment extract samples were combined for analysis using the following process. A 50 mL aliquot of each extract (one through three) was combined and adjusted to 150 mL with extraction solvent. A 500-µL aliquot of combined sample was mixed with a 500-µL of water. Aliquots of the diluted sample were analyzed by HPLC with fraction collection.
- Sterility check if applicable: no
- Sample storage before analysis: yes
Test performance:
Water-sediment system #1:
The mean material balance for the Swiss Lake sediment system was 42.0 ± 26.2% AR for the 14C-2,4-DTBP treated samples (Day 0 was 97.6 to 99.4%, range of 20.3% to 99.4% AR).
Water-sediment system #2:
The mean material balance for the Calwich Abbey Lake sediment system was 43.4 ± 27.0% AR for the 14C-2,4-DTBP treated samples (Day 0 was 97.5 to 98.1%, range of 17.4% to 98.1% AR).
Compartment:
other: water / sediment, material (mass) balance
Remarks on result:
other: see above
Key result
Compartment:
natural water: freshwater
DT50:
0.979 d
Type:
other: SFO (single first order)
Temp.:
20 °C
Remarks on result:
other: [system #1] M0 = 80.3 ± 1.912 k (d-1) = 0.7082 ± 0.4836 r2 = 0.9937 DT90 = 3.25
Key result
Compartment:
entire system
DT50:
4.02 d
Type:
other: SFO
Temp.:
20 °C
Remarks on result:
other: [sytem #1] M0 = 97.15 ± 13.85 k (d-1) = 0.1724 ± 0.06094 r2 = 0.7941 DT90 = 13.4
Key result
Compartment:
natural water: freshwater
DT50:
2.53 d
Type:
other: SFO
Temp.:
20 °C
Remarks on result:
other: [system #2] M0 = 83.7 ± 0.2306 k (d-1) = 0.274 ± 0.002598 r2 = 0.9999 DT90 = 8.41
Key result
Compartment:
entire system
DT50:
7.34 d
Type:
other: DFOP
Temp.:
20 °C
Remarks on result:
other: [system #2] M0 = 100.6 ± 5.479 k1 (d-1) = 0.1109 ± 0.02413 k2 (d-1) = 1.20E-012 ± 0.01114 g = 0.8976 ± 0.08178 r2 = 0.9568 DT90 = 161
Other kinetic parameters:
other: see above
Transformation products:
no
Evaporation of parent compound:
yes
Volatile metabolites:
yes
Residues:
yes
Details on results:
TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): Loss of substance/Mass balance

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:

EXTRACTABLE RESIDUES
- % of applied amount at day 0:
[System #1] Swiss Lake water: 82.8
[System #1] Swiss Lake sediment: 18.8
[System #1] Swiss Lake total system: 98.6
[System #2] Calwich Abbey Lake water: 83.8
[System #2] Calwich Abbey Lake sediment: 13.2
[System #2] Calwich Abbey Lake total system: 97.0
- % of applied amount at end of study period:
[System #1] Swiss Lake water: 1.8
[System #1] Swiss Lake sediment: 11.1
[System #1] Swiss Lake total system: 13.3
[System #2] Calwich Abbey Lake water: 1.4
[System #2] Calwich Abbey Lake sediment: 17.5
[System #2] Calwich Abbey Lake total system: 18.9

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0:
[System #1]Swiss Lake sediment: 0.7
[System #2] Calwich Abbey Lake sediment: 1.0
- % of applied amount at end of study period:
[System #1]Swiss Lake sediment: 8.7
[System #2] Calwich Abbey Lake sediment: 14.8
Results with reference substance:
not applicable

The mean material balance for the Swiss Lake sediment system was 42.1 ± 26.2% AR for the 14C-2,4-DTBP treated samples (Day 0 was 97.6 to 99.4%, range of 20.5% to 99.4% AR over the 105 day study duration). The amount of radioactivity in the water layer generally decreased over the course of the study from 82.8% AR at initiation to 1.8% AR at 105 days. At initiation, the observed amount of extractable radioactivity in the sediment layer was 18.8% AR, which decreased to approximately 14.3% AR at Day 14, yet increased to 49.1% AR at Day 28 before decreasing to 11.1% AR by termination. Non extractable residues reached a maximum of 13.5% AR at Day 45. Captured volatile radioactivity did not exceed 0.3% AR in any of the 14C volatile or KOH traps.

The mean material balance for the Calwich Abbey Lake sediment system was 43.5 ± 26.9% AR for the 14C-2,4-DTBP treated samples (Day 0 was 97.5 to 98.1%, range of 17.7% to 98.1% AR over the 105 day study duration). The amount of radioactivity in the water layer generally decreased over the course of the study from 83.8% AR at initiation to 1.4% AR at 105 days (termination). At initiation, the observed amount of extractable radioactivity in the sediment layer was 13.2% AR, and then increased to maximum level of 57.7% AR by Day 7 before decreasing to 17.5% AR at termination. Non-extractable residues reached a maximum of 16.4% AR at 80 days. Captured volatile radioactivity did not exceed 0.3% AR in any of the 14C volatile or KOH traps.

Evidence that loss of mass balance in this study is associated with volatility of 2,4-DTBP and/or its metabolites is supported by similar results from other studies that were conducted in parallel. Further testing within this study was performed by setting up an additional sample of pond water. This sample was connected by Teflon lined tubing to multiple trapping solutions consisting of an ORBO™ 47 sampling tube, ethylene glycol, potassium hydroxide, a combustion tube furnace, and addition potassium hydroxide traps for converted organics to CO2. When testing was complete, there was 18.9%, 5.4%, 58%, and 2.1% AR remaining in the dosed sample, the glassware that held the sample, the ORBO™-47 sampling tube, and remaining traps and Teflon tubing, respectively. Testing showed that 95% of the material analyzed within the ORBO™ tube remained as 2,4-DTBP.

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, 14C-2,4-DTBP rapidly dissipates from the water phase to the sediment and/or is ultimately volatilized from the system as parent. No significant metabolites were observed in this study, which would indicate that the pathway of degradation using this study set-up would be loss of parent from an aerobic aquatic environment to the atmosphere. Similar results were observed for contrasting sediment systems, so there is no expected dependency on the nature of the residues that could be found in aerobic aquatic environments.
Executive summary:

The transformation of 14C-2,4-di-tert-butylphenol (2,4-DTBP) was studied in two different water/sediment systems under aerobic conditions. The water/sediment systems were from Swiss Lake, Chatsworth, Derbyshire, England (Swiss Lake) and Calwich Abbey Lake, Calwich, Staffordshire, England (Calwich Abbey Lake). The Swiss Lake water had a pH of 8.3, while the sediment was characterized as a sand (International textural class) with a pH of 6.1 (1:2 soil:0.01M CaCl2) and organic carbon content of 0.47% (Walkley Black method). The Calwich Abbey Lake water had a pH of 8.3, while the sediment was characterized as loam (International textural class) with a pH of 7.5 (1:2 soil:0.01M CaCl2) and organic carbon content of 4.8% (Walkley Black method).

The water phase of the test system was treated with 14C-2,4-DTBP at a rate of 1 µg/mL. The test systems were incubated in darkness at approximately 20 ± 2°C for up to 105 days. Aerobic conditions were maintained by passing a steady stream of humidified air through the test apparatus. The flow-through systems were designed to trap evolved carbon dioxide (CO2) and volatile organic compounds. Duplicate samples from each radiolabeled treatment were taken at 0, 7, 14, 28, 45, 59, 80, and 105 days after application. The water and sediment layers were separated by centrifugation and decanting. The water layer was analyzed by Liquid Scintillation Counting (LSC) for radioactivity content and then analyzed by direct injection using reversed phase High Performance Liquid Chromatography (HPLC) in order to determine the biotransformation products. The sediment layer was extracted with appropriate organic solvent. Extracts were analyzed by LSC and analyzed by reversed-phase HPLC. The post-extracted sediment pellet was homogenized, combusted, and the radioactive residue quantified by LSC. Volatiles traps (1N KOH) were also analyzed by LSC.

The mean material balance was 42.1 ± 26.2% of the applied radioactivity (AR) (Day 0 was 97.6 to 99.4%, range of 20.5% to 99.4% AR over the 105 day study duration) and 43.5 ± 26.9% AR (Day 0 was 97.5 to 98.1%, range of 17.7% to 98.1% AR over the 105 day study duration) for the 14C-2,4-DTBP treated Swiss Lake and Calwich Abbey Lake samples, respectively. The partitioning of the radioactivity over the course of the study was as follows. The amount of radioactivity in the water layer generally decreased over the course of the study from 82.8% and 83.8% AR at Day 0 to 1.8% and 1.4% AR at Day 105 for the Swiss Lake and Calwich Abbey Lake samples, respectively. At Day 0, the observed amount of radioactivity in the sediment extract was 18.8% and 13.2% AR, and then decreased to levels of 11.1%AR at Day 105 for the Swiss Lake samples and increased to a minimum of 17.5% AR at Day 105 for the Calwich Abbey Lake samples. Non extractable residues remained relatively low, reaching a maximum of 13.5% and 16.4% AR at Day 45 and Day 80 for the Swiss Lake and Calwich Abbey Lake samples, respectively. Levels of CO2 were ≤ 0.3% AR and ≤ 0.2% AR for for the Swiss Lake and Calwich Abbey Lake samples, respectively. Further testing showed that significant loss of mass balance is likely due to the volatility of primarily 2,4-DTBP. Since the loss of mass balance could be accounted for in these additional tests, the kinetic modeling results would accurately reflect the fate of the parent in an aerobic aquatic environment.

Metabolite Profile in the Swiss Lake Sediment System: Water Layer

In the water layer of the Swiss Lake system, levels of 2,4-DTBP decreased from 82.8% AR at Day 0 to < Limit of Detection (LOD) by Day 80 in the treated system. The total unassigned radioactivity in the water phase was ≤ 6.3% AR throughout the study. The largest unassigned peak was a maximum of 1.6% AR (Day 45), meaning there was no major transformation products (≥ 5% AR) observed in the water phase.

Metabolite Profile in the Swiss Lake Sediment System: Sediment Layer

In the sediment layer of the Swiss Lake system, levels of 2,4-DTBP increased from 14.8% AR at Day 0 to 38.1% AR by Day 28 and then decreased to 4.4% AR by termination in the treated system. The total unassigned radioactivity in the sediment phase was ≤ 11.2% AR throughout the study. The largest unassigned peak was a maximum of 6.7% AR (Day 28) which was < 5% AR by the next sampling (Day 45), meaning there was no major transformation products (≥ 5% AR) observed at consecutive samplings observed in the sediment phase.

Metabolite Profile in the Swiss Lake Sediment System: Total System

In the total system of the Swiss Lake system, levels of 2,4-DTBP decreased from 97.6% AR at Day 0 to 4.4% AR by Day 105 in the treated system. The total unassigned radioactivity in the total system was ≤ 15.5% AR throughout the study. The largest unassigned peak was a maximum of 7.5% AR (Day 28), which was < 5% AR by the next sampling (Day 45), meaning there was no major transformation products (≥ 5% AR) observed at consecutive samplings observed in the total system.

Metabolite Profile in the Calwich Abbey Lake Sediment System: Water Layer

In the water layer of the Calwich Abbey Lake system, levels of 2,4-DTBP decreased from 83.8% AR at Day 0 to < LOD by Day 80 in the treated system. The total unassigned radioactivity in the water phase was ≤ 1.9% AR throughout the study. The largest unassigned peak was a maximum of 1.6% AR (Day 59), meaning there was no major transformation products (≥ 5% AR) observed in the water phase.

Metabolite Profile in the Calwich Abbey Lake Sediment System: Sediment Layer

In the sediment layer of the Calwich Abbey Lake system, levels of 2,4-DTBP increased from 11.9% AR at Day 0 to 52.7% AR at Day 7 before decreasing to 11.0% AR by Day 105 in the treated system. The total unassigned radioactivity in the sediment phase was ≤ 6.6% AR throughout the study. The largest unassigned peak was a maximum of 3.7% AR (Day 28), meaning there was no major transformation products (≥ 5% AR) observed in the sediment phase.

Metabolite Profile in the Calwich Abbey Lake Sediment System: Total System

In the total system of the Calwich Abbey Lake system, levels of 2,4-DTBP decreased from 95.7% AR at Day 0 to 11.0% AR by Day 105 in the treated system. The total unassigned radioactivity in the total system was ≤ 8.4% AR throughout the study. The largest unassigned peak was a maximum of 4.3% AR (Day 28), meaning there was no major transformation products (≥ 5% AR) observed in the total system.

Non-Extractable Residue (NER) Analysis

Levels of NER increased slightly above 10% AR after the primary extraction method, but then decreased over the remainder of the study. Thus, further NER analysis was not necessary.

Kinetics

The DT50 and DT90 values for 14C-2,4-DTBP were evaluated by single first order (SFO) and double first order parallel (DFOP) kinetic models using Computer Assisted Kinetic Evaluation (CAKE) ver. 2.0.

The SFO model provided an adequate fit (visually and statistically) for the water and total system compartments for each of the two sediment systems.

In conclusion, 14C-2,4-DTBP rapidly dissipates from the water phase to the sediment and/or is ultimately volatilized from the system as parent. No significant metabolites were observed in this study, which would indicate that the pathway of degradation using this study set-up would be loss of parent from an aerobic aquatic environment to the atmosphere. Similar results were observed for contrasting sediment systems, so there is no expected dependency on the nature of the residues that could be found in aerobic aquatic environments.

Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable GLP-study following current OECD guideline.
Qualifier:
according to
Guideline:
OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report):[14C]-2,4-di-tert.-butylphenol
- Substance type: Alkylphenol
- Physical state: solid
- Radiochemical purity (if radiolabelling): 98.8 %
- Specific activity (if radiolabelling):63.7 mCi/mmol
- Locations of the label (if radiolabelling): Phenyl ring
- Expiration date of radiochemical substance (if radiolabelling): 12 August 2016
- Storage condition of test material: Frozen
Radiolabelling:
yes
Oxygen conditions:
anaerobic
Inoculum or test system:
natural water / sediment
Details on source and properties of surface water:
Water-sediment system #1:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Swiss Lake, Chatsworth, Derbyshire, England, sampled at 5-10 cm, scooped from lake in bucket and sieved, no pesticide use for 5 years
- Storage conditions: approximately 4°C, water logged with free access to air
- Storage length: 18 days
- Temperature (°C) at time of collection: 19°C
- pH at time of collection: 7.4
- Electrical conductivity: 117 µS/cm
- Redox potential (mv) initial/final: 168.2
- Oxygen concentration (mg/l) initial/final: 8.73
- Hardness (CaCO3): 273
- Dissolved organic carbon (%): 0.47%
- Water filtered: no

Water-sediment system #2:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Calwich Abbey Lake, Calwich, Staffordshire, England, sampled at 30-40 cm, scooped from lake in bucket and sieved, no pesticide use for 5 years
- Storage conditions: approximately 4°C, water logged with free access to air
- Storage length: 18 days
- Temperature (°C) at time of collection: 17°C
- pH at time of collection: 8.41
- Electrical conductivity: 568 µS/cm
- Redox potential (mv) initial/final: 151.2
- Oxygen concentration (mg/l) initial/final: 8.05
- Hardness (CaCO3): 274
- Dissolved organic carbon (%): 4.8%
- Water filtered: no
Details on source and properties of sediment:
Water-sediment system #1:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Swiss Lake, Chatsworth, Derbyshire, England, sampled at 0-5 cm, scooped from top layer of sediment and sieved, no pesticide use for 5 years
- Storage conditions: approximately 4 °C, water logged with free access to air
- Storage length: 18 days
- Textural classification (i.e. %sand/silt/clay): sand
- pH at time of collection: 6.734
- Organic carbon (%): 0.47
- Redox potential (mv) initial/final: -36.6
- CEC (meq/100 g): 2.8
- Bulk density (g/cm³): 1.20
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): 579.6 µg/g
- Sediment samples sieved: yes

Water-sediment system #2:
- Details on collection (e.g. location, sampling depth, contamination history, procedure): Calwich Abbey Lake, Calwich, Staffordshire, England, sampled at 1 m, scooped from top layer of sediment and sieved, no pesticide use for 5 years
- Storage conditions: approximately 4 °C, water logged with free access to air
- Storage length: 18 days
- Textural classification (i.e. %sand/silt/clay): loam
- pH at time of collection: 7.7
- Organic carbon (%): 4.8
- Redox potential (mv) initial/final: -164.6
- CEC (meq/100 g): 10.6
- Bulk density (g/cm³): 0.69
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): 754.0 µg/g
- Sediment samples sieved: yes
Details on inoculum:
n/a
Duration of test (contact time):
105 d
Initial conc.:
1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 270 µL
- Test temperature: 20 ± 2°C
- pH: Swiss Lake: 8.3 (water) and 5.6 (sediment), Calwich Abbey Lake: 8.3 (water) and 7.5 (sediment)
- pH adjusted: no
- CEC (meq/100 g): Swiss Lake: 2.8, Calwich Abbey Lake: 10.6
- Aeration of dilution water: yes
- Continuous darkness: yes
- Any indication of the test material adsorbing to the walls of the test apparatus: slight indication

TEST SYSTEM
- Culturing apparatus: Air was pulled through a water hydrator, then into the series of test bottles. Air exiting the series of test bottles was passed through a series of volatile traps.
- Number of culture flasks/concentration: 18 flasks per system/ 1 µg/mL
- Method used to create aerobic conditions: vacuum
- Measuring equipment: redox pH meter

- Test performed in closed vessels due to significant volatility of test substance: yes
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: Supelco ORBO™-32 adsorbent tube, then through two traps containing 30 mL 1 N KOH

SAMPLING
- Sampling frequency: 0, 7, 14, 28, 45, 59, 80, and 105 days
- Sampling method: Water and sediment layers were separated by decanting an initial 100 mL of water into a 500-mL graduated cylinder. The remaining water layer was carefully transferred to a 250-mL Nalgene bottle for centrifugation at approximately 3,700 x g for 10 minutes. The centrifuged water layer was then decanted into 500 mL mixing cylinders container initial 100 mL. The volumes of water collected in the mixing cylinders were measured and adjusted, as necessary, to a consistent volume with reagent water. Triplicate aliquots of this sample were analyzed by LSC. An aliquot was vialed for HPLC analysis. Following decanting of the water phase, a 100-mL aliquot of 4:1 acetone:0.1 M ammonium carbonate was added to the Chemglass bottles, the sediment layers were quantitatively transferred from the glass test vessels to the correspondingly labeled Nalgene bottles, and the samples were shaken on a platform shaker for 60 minutes. The sediment pellets were manually broken up. The bottles were then centrifuged at approximately 3,700 x g for 20 minutes. Following centrifugation, successive supernatants were decanted into a 250-mL mixing cylinder. This process was repeated twice more, with each extraction kept separate, and the volume of each extraction was adjusted to a consistent volume with extraction solvent. Triplicate aliquots of this sample were analyzed by LSC. The sediment extract samples were combined for analysis using the following process. A 50 mL aliquot of each extract (one through three) was combined and adjusted to 150 mL with extraction solvent. A 500-µL aliquot of combined sample was mixed with a 500-µL of water. Aliquots of the diluted sample were analyzed by HPLC with fraction collection.
- Sterility check if applicable: no
- Sample storage before analysis: yes
Test performance:
Water-sediment system #1:
The mean material balance for the Swiss Lake sediment system was 35.5 ± 27.9% AR for the [14C]-2,4-DTBP treated samples (Day 0 was 97.3 to 100.1%, range of 11.6% to 100.1% AR).
Water-sediment system #2:
The mean material balance for the Calwich Abbey Lake sediment system was 46.0 ± 31.7% AR for the [14C]-2,4-DTBP treated samples (Day 0 was 97.7 to 99.5%, range of 12.1% to 99.5% AR).
Compartment:
other: water / sediment, material (mass) balance
Remarks on result:
other: see above
Key result
Compartment:
natural water: freshwater
DT50:
3.7 d
Type:
other: DFOP
Temp.:
20 °C
Remarks on result:
other: [system #1] M0 = 76.49 ± 1.353 k1 (d-1) = 0.202 ± 0.01655 k2 (d-1) = 0.01278 ± 0.01328 g = 0.9451 ± 0.03464 r2 = 0.9957 DT90 = 14.2
Key result
Compartment:
entire system
DT50:
3.81 d
Type:
other: DFOP
Temp.:
20 °C
Remarks on result:
other: [sytem #1] M0 = 97.52 ± 2.121 k1 (d-1) = 0.2368 ± 0.02215 k2 (d-1) = 0.001339 ± 0.002281 g = 0.8395 ± 0.02349 r2 = 0.9915 DT90 = 354
Key result
Compartment:
natural water: freshwater
DT50:
4.33 d
Type:
other: DFOP
Temp.:
20 °C
Remarks on result:
other: [system #2] M0 = 82.09 ± 2.616 k1 (d-1) = 0.1664 ± 0.02266 k2 (d-1) = 0.006465 ± 0.03256 g = 0.9718 ± 0.0555 r2 = 0.9858 DT90 = 15.4
Key result
Compartment:
entire system
DT50:
6.22 d
Type:
other: DFOP
Temp.:
20 °C
Remarks on result:
other: [system #2] M M0 = 102.2 ± 7.565 k1 (d-1) = 0.1555 ± 0.05526 k2 (d-1) = 8.17E-04 ± 0.007621 g = 0.8051 ± 0.1039 r2 = 0.8978 DT90 = 871
Other kinetic parameters:
other: see above
Transformation products:
no
Details on transformation products:
not applicable
Evaporation of parent compound:
yes
Volatile metabolites:
yes
Residues:
yes
Details on results:
TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): Loss of substance/Mass balance

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:

EXTRACTABLE RESIDUES
- % of applied amount at day 0:
[System #1] Swiss Lake water: 79.2
[System #1] Swiss Lake sediment: 20.0
[System #1] Swiss Lake total system: 99.2
[System #2] Calwich Abbey Lake water: 80.0
[System #2] Calwich Abbey Lake sediment: 16.8
[System #2] Calwich Abbey Lake total system: 97.5
- % of applied amount at end of study period:
[System #1] Swiss Lake water: 1.2
[System #1] Swiss Lake sediment: 12.4
[System #1] Swiss Lake total system: 13.5
[System #2] Calwich Abbey Lake water: 0.6
[System #2] Calwich Abbey Lake sediment: 10.9
[System #2] Calwich Abbey Lake total system: 11.4

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0:
[System #1] Swiss Lake sediment: 0.9
[System #2] Calwich Abbey Lake sediment: 2.0
- % of applied amount at end of study period:
[System #1] Swiss Lake sediment: 1.1
[System #2] Calwich Abbey Lake sediment: 1.8

Results with reference substance:
not applicable

The mean material balance for the Swiss Lake sediment system was 35.5 ± 27.9% AR for the [14C]-2,4-DTBP treated samples (Day 0 was 97.3 to 100.1%, range of 11.6% to 100.1% AR). The amount of radioactivity in the water layer generally decreased over the course of the study from 79.2% AR at initiation to 1.2% AR at 105 days (termination). At initiation, the observed amount of extractable radioactivity in the sediment layer was 20.0% AR, which decreased to approximately 12.4% AR at Day 105. Non extractable residues reached a maximum of 1.6% AR at 59 days. Captured volatile radioactivity did not exceed 1.4% AR in any of the 14C-volatile or KOH traps.

The mean material balance for the Calwich Abbey Lake sediment system was 46.0 ± 31.7% AR for the [14C]-2,4-DTBP treated samples (Day 0 was 97.7 to 99.5%, range of 12.1% to 99.5% AR). The amount of radioactivity in the water layer generally decreased over the course of the study from 80.8% AR at initiation to 0.6% AR at 105 days (termination). At initiation, the observed amount of extractable radioactivity in the sediment layer was 16.8% AR, and then increased to maximum level of 30.5% AR by Day 7, decreasing to 10.9% AR at termination. Non-extractable residues reached a maximum of 3.2% AR at 2 days. Captured volatile radioactivity did not exceed 1.5% AR in any of the 14C-volatile or KOH traps.

Evidence that loss of mass balance in this study is associated with volatility of 2,4-DTBP and/or its metabolites is supported by similar results from other studies that were conducted in parallel. Further testing within the 2,4-DTBP aerobic aquatic metabolism study was performed by setting up an additional sample of pond water. This sample was connected by Teflon lined tubing to multiple trapping solutions consisting of an ORBO™ 47 sampling tube, ethylene glycol, potassium hydroxide, a combustion tube furnace, and addition potassium hydroxide traps for converted organics to CO2. When testing was complete, there was 18.9%, 5.4%, 58%, and 2.1% AR remaining in the dosed sample, the glassware that held the sample, the ORBO™-47 sampling tube, and remaining traps and Teflon tubing, respectively. Testing showed that 95% of the material analyzed within the ORBO™ tube remained as 2,4-DTBP.

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, 14C-2,4-DTBP rapidly dissipates from the water phase to the sediment and/or is ultimately volatilized from the system as parent. No significant metabolites were observed in this study, which would indicate that the pathway of degradation using this study set-up would be loss of parent from an anaerobic aquatic environment to the atmosphere. Similar results were observed for contrasting sediment systems, so there is no expected dependency on the nature of the residues that could be found in anaerobic aquatic environments.
Executive summary:

The transformation of 2,4-di-tert-butylphenol (2,4-DTBP) was studied in two different water/sediment systems under anaerobic conditions. The water/sediment systems were from Swiss Lake, Chatsworth, Derbyshire, England (Swiss Lake) and Calwich Abbey Lake, Calwich, Staffordshire, England (Calwich Abbey Lake). The Swiss Lake water had a pH of 8.3, while the sediment was characterized as a sand (International textural class) with a pH of 5.6 (1:2 soil:0.01M CaCl2) and organic carbon content of 0.47% (Walkley Black method). The Calwich Abbey Lake water had a pH of 8.3, while the sediment was characterized as loam (International textural class) with a pH of 7.5 (1:2 soil:0.01M CaCl2) and organic carbon content of 4.8% (Walkley Black method).

The water phase of the test system was treated with 14C-2,4-DTBP at a rate of 1 µg/mL. The test systems were incubated in darkness at approximately 20 ± 2°C for up to 105 days. Anaerobic conditions were maintained by passing a steady stream of humidified nitrogen through the test apparatus. The flow-through systems were designed to trap evolved carbon dioxide (CO2) and volatile organic compounds. Duplicate samples from each radiolabeled treatment were taken at 0, 2, 7, 14, 28, 45, 59, 80, and 105 days after application. The water and sediment layers were separated by centrifugation and decanting. The water layer was analyzed by Liquid Scintillation Counting (LSC) for radioactivity content and then analyzed by direct injection using reversed phase High Performance Liquid Chromatography (HPLC) in order to determine the biotransformation products. The sediment layer was extracted with appropriate organic solvent. Extracts were analyzed by LSC and analyzed by reversed-phase HPLC. The post-extracted sediment pellet was homogenized, combusted, and the radioactive residue quantified by LSC. Volatiles traps (1N KOH) were also analyzed by LSC.

The mean material balance was 35.5 ± 27.9% of the applied radioactivity (AR) (Day 0 was 97.3 to 100.1%, range of 11.6% to 100.1% AR over the 105 day study duration) and 42.5 ± 31.4% AR (Day 0 was 97.7 to 99.5%, range of 12.1% to 99.5% AR over the 105 day study duration) for the 14C-2,4 DTBP treated Swiss Lake and Calwich Abbey Lake samples, respectively. The partitioning of the radioactivity over the course of the study was as follows. The amount of radioactivity in the water layer generally decreased over the course of the study from 79.2% and 80.8% AR at Day 0 to 1.2% and 0.6% AR at Day 105 for the Swiss Lake and Calwich Abbey Lake samples, respectively. At Day 0, the observed amount of radioactivity in the sediment extract was 20.0% and 16.8% AR, and then decreased to levels of 12.4% and 10.9% AR at Day 105 for the Swiss Lake and Calwich Abbey Lake samples, respectively. Non extractable residues remained relatively low, reaching a maximum of 1.6% and 3.3% AR at Day 59 and Day 7 for the Swiss Lake and Calwich Abbey Lake samples, respectively. Levels of 14C volatiles were less than or equal to 1.5% AR for both sediment systems. Further testing showed that significant loss of mass balance is likely due to the volatility of primarily 2,4-DTBP. Since the loss of mass balance could be accounted for in these additional tests, the kinetic modeling results would accurately reflect the fate of the parent in an anaerobic aquatic environment.

Metabolite Profile in the Swiss Lake Sediment System: Water Layer

In the water layer of the Swiss Lake system, levels of 2,4-DTBP decreased from 79.2% AR at Day 0 to 1.2% AR by Day 105 in the treated system. The total unassigned radioactivity in the water phase was ≤ 0.8% AR throughout the study. The largest mean unassigned peak was a maximum of 0.7% AR (Day 45), meaning there was no major transformation products (≥ 5% AR) observed in the water phase.

Metabolite Profile in the Swiss Lake Sediment System: Sediment Layer

In the sediment layer of the Swiss Lake system, levels of 2,4-DTBP decreased from 19.8% AR at Day 0 to 12.1% AR by Day 105 in the treated system. The total mean unassigned radioactivity in the sediment phase was ≤ 3.4% AR throughout the study. The largest mean unassigned peak was a maximum of 0.5% AR (Day 14), meaning there was no major transformation products (≥ 5% AR) observed in the sediment phase.

Metabolite Profile in the Swiss Lake Sediment System: Total System

In the total system of the Swiss Lake system, levels of 2,4-DTBP decreased from 99.0% AR at Day 0 to 13.2% AR by Day 105 in the treated system. The total mean unassigned radioactivity in the total system was ≤ 3.9% AR throughout the study. The largest mean unassigned peak was a maximum of 0.7% AR (Days 14 and 45), meaning there were no major transformation products observed (≥ 5% AR) in the total system.

Metabolite Profile in the Calwich Abbey Lake Sediment System: Water Layer

In the water layer of the Calwich Abbey Lake system, levels of 2,4-DTBP decreased from 80.8% AR at Day 0 to 0.6% AR by Day 105 in the treated system. The total mean unassigned radioactivity in the water phase was ≤ 0.5% AR throughout the study. The largest mean unassigned peak was a maximum of 0.4% AR (Day 7), meaning there was no major transformation products (≥ 5% AR) observed in the water phase.

Metabolite Profile in the Calwich Abbey Lake Sediment System: Sediment Layer

In the sediment layer of the Calwich Abbey Lake system, levels of 2,4-DTBP decreased from 16.5% AR at Day 0 to 10.6% AR by Day 105 in the treated system. The total mean unassigned radioactivity in the sediment phase was ≤ 2.1% AR throughout the study. The largest mean unassigned peak was a maximum of 0.3% AR (Days 2 and 28), meaning there was no major transformation products (≥ 5% AR) observed in the sediment phase.

Metabolite Profile in the Calwich Abbey Lake Sediment System: Total System

In the total system of the Calwich Abbey Lake system, levels of 2,4-DTBP decreased from 97.3% AR at Day 0 to 11.2% AR by Day 105 in the treated system. The total mean unassigned radioactivity in the total system was ≤ 2.3% AR throughout the study. The largest mean unassigned peak was a maximum of 0.6% AR (Day 7), meaning there were no major transformation products observed (≥ 5% AR) in the total system.

Non-Extractable Residue (NER) Analysis

Levels of NER never exceeded 10% AR after the primary extraction method so further NER analysis was not necessary.

Kinetics

The DT50 and DT90 values for 14C-2,4-DTBP were evaluated by single first order (SFO) and double first-order in parallel (DFOP) kinetic models using Computer Assisted Kinetic Evaluation (CAKE) ver. 2.0.

The SFO model provided an adequate fit (visually and statistically) for the water compartments for each of the two sediment systems. The DFOP model provided an adequate fit (visually and statistically) for the total system compartments for each of the two sediment systems.

In conclusion, 14C-2,4-DTBP rapidly dissipates from the water phase to the sediment and/or is ultimately volatilized from the system as parent. No significant metabolites were observed in this study, which would indicate that the pathway of degradation using this study set-up would be loss of parent from an anaerobic aquatic environment to the atmosphere. Similar results were observed for contrasting sediment systems, so there is no expected dependency on the nature of the residues that could be found in anaerobic aquatic environments.

Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable GLP-study following current OECD guideline.
Qualifier:
according to
Guideline:
OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report):[14C]-2,4-di-tert.-butylphenol
- Substance type: Alkylphenol
- Physical state: solid
- Radiochemical purity (if radiolabelling): 98.8 %
- Specific activity (if radiolabelling):63.7 mCi/mmol
- Locations of the label (if radiolabelling): Phenyl ring
- Expiration date of radiochemical substance (if radiolabelling): 12 August 2016
- Storage condition of test material: Frozen
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Details on source and properties of surface water:
Definitive test
- pH at collection: 7.116
- Electrical conductivity:0.11 mmhos/cm
- Redox potential (mv): 178.3
- Oxygen concentration (mg/L) initial/final: 5.39
- Hardness (CaCO3/L): 44
- Total suspended solids (ppm): 36
- Turbidity (NTU): 31.3
- Dissolved organic carbon (ppm): 1.0409
- Biological Oxygen Demand (mg-O2/L):3.2
- Total nitrogen (ppm): 2.1
- Ammonium ion (ppm): 0.5
- Total phosphorous (ppm): 1.8
- Water filtered: yes
- Type and size of filter used, if any: glass wool for large debris

Repeated Mass Balance Test
- pH at collection: 6.858
- Electrical conductivity: 0.10 mmhos/cm
- Redox potential (mv): 260.3
- Oxygen concentration (mg/L) initial/final: 9.16
- Total suspended solids (ppm): 8
- Turbidity (NTU): 8.62
- Hardness (CaCO3/L): 39
- Dissolved organic carbon (%): not determined
- Biological Oxygen Demand (mg-O2/L): Not determined
- Ammonium ion: not determined
- Total phosphorous (ppm): 1.5
- Water filtered: no

SURFACE WATER COLLECTION AND STORAGE (Defintive Test)
- Geographic location: Tift County, Georgia, USA
- Pesticide use history at the collection site: None applied
- Collection procedures: Bucket
- Sampling depth (cm): 20 cm depth of the water surface
- Storage conditions: ambient
- Storage length: two days
-Surface water preparation: Large debris filtered through glass wool

SURFACE WATER COLLECTION AND STORAGE (Repeated Mass Balance Test)
- Geographic location: Tift County, Georgia, USA
- Pesticide use history at the collection site: None applied
- Collection procedures: Bucket
- Sampling depth (cm): 20 cm depth of the water surface
- Storage conditions: refrigerated (4°C)
- Storage length:30 days
-Surface water preparation: Large debris filtered through glass wool


Details on source and properties of sediment:
Not applicable
Details on inoculum:
Not applicable
Duration of test (contact time):
62 d
Initial conc.:
10 µg/L
Based on:
test mat.
Initial conc.:
100 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
1. PRELIMINARY EXPERIMENTS: None

2. EXPERIMENTAL DESIGN (definitive)
- Bioreactors with bulk sample 62 days, 20°C in dark
- Surface water condition: fresh
- Surface water (mL): 700
- Control conditions, if used (present differences from other treatments, i.e., sterile/non-sterile, experimental conditions): sterile samples in parallel
- No. of replication controls, if used: 2
- No. of replication treatments: 2
Test apparatus: 1-Liter glass bottles (bioreactors) with plastic cap to seal. Constant agitation via magnetic stir bar and stirrer.

- EXPERIMENTAL DESIGN (repeated mass balance samples)
- Bioreactors with bulk sample 60 days, 20°C in dark
- Surface water condition: refrigerated storage for 30 days prior to use
- Surface water (mL): 200
- Control conditions: none
- No. of replication controls, if used: 2
- No. of replication treatments: 2
- Test apparatus (Repeat mass balance samples): Air pulled through reagent water pre trap humidifier, blank trap, then into 250-mL Nalgene bottles. Air exiting the bottles was then passed through blank trap for overflow, Supelco ORBO™-47 tubes, and 1 N KOH traps (~45 mL) to collect volatiles, all connected with Tygon tubing (day 30) or Teflon Tubing (Day 60).
- Test apparatus (Repeat mass balance samples): Air pulled through reagent water pre trap humidifier, blank trap, then into 250-mL Nalgene bottles. Air exiting the bottles was then passed through blank trap for overflow, Supelco ORBO™-32 tubes, and 1 N KOH traps (~45 mL) to collect volatiles, all connected with Tygon tubing
- Details of traps for CO2 and organic volatile, if any: Supelco ORBO™-32 Tube and two 1 N KOH traps (45 mL)
- If no traps were used, is the system closed/open: n/a
- Identity and concentration of co-solvent: Acetonitrile

Test material application
- Volume of test solution used/treatment: 65µL
- Application method (e.g. applied on surface, homogeneous mixing etc.): applied on surface
- Is the co-solvent evaporated: no

Any indication of the test material adsorbing to the walls of the test apparatus: yes (up to 14%AR)

Experimental conditions (in addition to defined fields)
- Aerobic conditions: Constant stirring
- Continuous darkness: Yes


3. OXYGEN CONDITIONS (delete elements as appropriate)
- Methods used to create the an/aerobic conditions: Constant stirring via a stir plate and magnetic stirrer
- Evidence that an/aerobic conditions were maintained during the experiment (e.g. redox potential): Overall dissolved oxygen levels maintained at 8.47 ±0.18 mg/L

4. SUPPLEMENTARY EXPERIMENTS:
Water sample dosed with parent 2,4-DTBP with direct aeration via bubbling below the water surface resulted in up to 80% of the parent being volatilized. Supplemental data showed losses to plastic tubing that was not Teflon-lined.

5. SAMPLING DETAILS
Definitive (non-Sterile): sub-samples removed from bulk samples at : 0, 3, 7, 10, 14, 21, 31, 45, 62 days after dose
Definitive (Sterile): sub-samples removed from bulk samples at : 7, 14, 21, 31, 45, 62 days after dose
Definitive test (Reference article): sub-samples removed from bulk samples at : 0, 3, 7, 10, 14, 21, 31, 45, 62 days after dose
Mass Balance (repeated test): 30 and 60 days after dose.
Test performance:
Material balances at the beginning and the end of the experiment were below the guideline range of acceptance 90-110%. The mean material balances at Day 30 were 88.4% and 86.5% AR for the low and high dose, respectively. Mass balances at termination (Day 65) were 86.6% and 87.1% AR for the low and high dose, respectively. A summary of individual results of the material balance are presented in, which included significant amounts detected in the solvent rinse of the glassware (range 7.3 % to 13.5 % AR). Additionally low mass balance was attributed to volatile losses of the parent 2,4-DTBP and its metabolites.
Compartment:
other: water / sediment, material (mass) balance
Remarks on result:
other: see above
Key result
Compartment:
natural water
DT50:
5 d
Type:
other: SFO
Temp.:
20 °C
Remarks on result:
other: [Non-Sterile system] M0 = 97.76 ± 6.289; k(d-1) = 0.1332 ± 0.01671; r²=0.9234; DT90=17.3
Key result
Compartment:
other: Natural water (sterile)
DT50:
186 d
Type:
other: SFO
Temp.:
20 °C
Remarks on result:
other: [Sterile system] M0 = 99.88 ± 2.526; k(d-1) = 0.003732 ± 8.45E-4; r2 = 0.6385; DT90 = 617
Transformation products:
yes
No.:
#1
No.:
#2
No.:
#3
No.:
#4
Details on transformation products:
In the surface water of the non-sterile samples, four major biotransformation products (≥ 5% AR) were observed. These products were identified (see Section 4.7.2) as 2-tert-butyl-4-nitrophenol (2-TB-4-NP), 4-tert-butyl-2,6-dinitrophenol (4-TB-2,6-DNP), 2-tert-butyl-4,6-dinitrophenol (2-TB-4,6-DNP), and 2,4-di-tert-butyl-6-nitrophenol (2,4-DTB-6-NP). Mean levels of 2-TB-4-NP reached a maximum of 14.4% AR by Day 45 and then decreased to 13.2 AR at termination (Day 62). Mean levels of 4-TB-2,6-DNP reached a maximum of 12.2% AR by Day 45 and then decreased to 10.2 AR at termination. Mean levels of 2-TB-4,6-DNP reached a maximum of 15.1% AR by Day 45 and then decreased to 14.7 AR at termination. Mean levels of 2,-DTB-6-NP reached 7.4% AR by Day 7 then gradually decreased to 3.3% AR at termination (Day 62). Multiple minor products were observed that were less than 5% AR. No peak greater than 4.2% AR (Day 62) was detected over course of the study.
In the surface water of the sterile samples, levels of 2,4-DTBP decreased from a mean of 93.4% AR at Day 0 to 74.8% AR at termination. Multiple minor products were observed that were less than 5% AR. No peak greater than 3.3% AR (Day 62) was detected over course of the study.
When comparing the results of the test and sterile systems, it can be concluded that the degradation of 2,4-DTBP increases in microbially active surface water.
Evaporation of parent compound:
yes
Volatile metabolites:
yes
Residues:
no
Details on results:
TEST CONDITIONS
- Aerobicity, constant stiring, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): Volatilization of parent and metabolites

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
2-tert-butyl-4-nitrophenol: 15.9% (Day 45)
4-tert-butyl-2,6-dinitrophenol: 15.0% (Day 10)
2-tert-butyl-4,6-dinitrophenol: 19.2% (Day 7)
2,4-di-tert-butyl-6-nitrophenol: 9.3% (Day 7)

MINOR TRANSFORMATION PRODUCTS: Largest single unassigned
Non-Sterile: < 3.3%AR
Sterile: < 4.1%AR

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:
Non-Sterile: mean of 20.7% AR at termination (Day 62)
Sterile: mean of 18.8% AR at termination (Day 62)

MINERALISATION
None observed

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: could not be determined from the indirect method

STERILE TREATMENTS (if used)
- Transformation of the parent compound: Mean of 73.5% AR of parent at termination (Day 62)
- Formation of transformation products: none assigned
- Formation of extractable and non-extractable residues: not applicable
- Volatilization: could not be determined from the indirect method
Material Balance (repeated test) Day 30
Concentration Level / Replicate Water Phase KOH Trap ORBO Tube Rinse KOH Trap (After Acid) ORBO Tube Rinse Solvent Rinse of Glassware Total
(Before Acid) (Before Acid) (Before Acid) (After Acid) Recovered
(%AR) (%AR) (%AR) (%AR) (%AR) (%AR) (%AR)
10 µg/L / R1 69.7 0.4 3.1 0.5 1.2 10.8 85.8
10 µg/L / R2 73.5 0.3 1.9 0.3 2.2 13.5 91.8
Mean 71.6 0.4 2.5 0.4 1.7 12.1 88.8
100 µg/L / R1 70.0 0.05 3.5 0.1 0.5 10.6 84.7
100 µg/L / R2 73.3 0.1 1.6 0.1 1.4 12.2 89.1
Mean 71.8 0.1 2.5 0.1 0.9 11.4 86.9
Material Balance (repeated test) Day 60
Concentration Level / Replicate Water Phase KOH Trap1 KOH Trap2 Solvent Rinse of Glassware (After Acid) ORBO Tube Rinse Solvent Rinse of Connective Tubing (After Acid) Total
(Before Acid) (After Acid) (After Acid) (After Acid) Recovered
(%AR) (%AR) (%AR) (%AR) (%AR) (%AR) (%AR)
10 µg/L / R1 78.2 0.6 0.0 7.3 0.4 0.1 86.6
10 µg/L / R2 77.9 1.1 0.0 10.9 0.1 0.2 90.4
Mean 78.0 0.9 0.0 9.1 0.3 0.2 88.5
100 µg/L / R1 74.1 0.7 0.0 11.2 0.9 0.6 87.5
100 µg/L / R2 68.2 3.4 0.1 11.3 2.5 1.9 87.4
Mean 71.2 2.1 0.0 11.3 1.7 1.2 87.5

Material balances at the beginning and the end of the experiment were below the guideline range of acceptance 90-110%. The mean material balances at Day 30 were 88.8% and 86.9% AR for the low and high dose, respectively. Mass balances at termination (Day 60) were 88.5% and 87.5% AR for the low and high dose, respectively. A summary of individual results included significant amounts detected in the solvent rinse of the glassware (range 7.3 % to 13.5 % AR). Additionally low mass balance was attributed to volatile losses of the parent 2,4-DTBP and its metabolites.

Validity criteria fulfilled:
not applicable
Conclusions:
The aerobic mineralization of radiolabeled 2,4-di-tert-butylphenol was studied in one surface water under aerobic conditions incubated in darkness at approximately 20 ± 2°C. 2,4-di-tert-butylphenol did not mineralize significantly during the 60-day study.
The overall mean mass balance of the closed bioreactors was 87.5% and 88.5% AR for the [14C] 2,4-di-tert-butylphenol radiolabeled treatments. Losses from the definitive samples during sampling, processing, and HPLC analyses as well as method development testing demonstrated that 2,4-DTBP and its metabolites are volatile.
DT50 and DT90 values for 2,4-DTBP in surface water were 5.2 and 17.3 days, respectively.
Four major transformation products (>5% AR) were observed and identified by GC-MS as 2-tert-butyl-4-nitrophenol, 4-tert-butyl-2,6-dinitrophenol, 2-tert-butyl-4,6-dinitrophenol, and 2,4-di-tert-butylphenol All other individual components were minor (< 5% AR). Based on the results of this study 2,4-di-tert-butylphenol would not be expected to hydrolyze in surface water or further mineralize to CO2 within 60 days, but would expect to dissipate from a surface water in the environment or degrade to multiple components. The four major transformation products would not be expected to persist in surface water based on the volatile losses of material from the test system in this study.
Executive summary:

The aerobic mineralization of [14C]2,4-di-tert-butylphenol (2,4-DTBP) was studied in one surface water system from North America under pelagic conditions at 20 ± 2ºC in the dark. The pH, biological oxygen demand, and total suspended solids were 7.1, 10.3 mg-O2/L, and 36 ppb, respectively.

A total of twelve bioreactors (1-liter capacity) containing 700 mL of surface water were used. To the bioreactors, 14C]2,4-DTBPwas applied at nominal rates of 10 µg a.i./L and 100 µg a.i./L. Four bioreactors, one for each radiolabel and concentration, were used for mineralization testing at zero time, 3, 7, 10, 14, 21, 30, 45, and 62 days after dosing. To compare biotic and abiotic processes of the surface water with respect to mineralization, two additional bioreactors were sterilized and prepared at a nominal rate of 100 µg/L. These samples were analyzed at 7,14,21,30, 45, and 62 days after dosing interval for mineralization. To confirm microbial viability of the surface water, two additional bioreactors were prepared with a reference item (sodium benzoate) at a nominal rate of 100 µg/L. These samples were analyzed in parallel at each interval for mineralization.

For mineralization testing, a sub-sample of the surface water was removed and assayed for radioactivity. At time zero, < 2% AR remained in the water phase following by acidification (pH 2–3 using concentrated hydrochloric acid) and purging with air. Therefore indirect determination of CO2 formation by acidification and purging was not possible. The potential for mineralization was assessed via HPLC analysis of the water phase without acidification and monitoring any diffuse polar radioactivity (dissolved carbonates) observed immediately after injection. The potential for biotransformation of 2,4-DTBP was also determined by HPLC analysis.

The results indicated that 2,4-DTBP did not significantly mineralize (<5% applied radioactivity (AR) in phase) over the study duration. Due to the lack of mineralization, no mineralization rate constants were derived for this study. The reference item (sodium benzoate) was mineralized up to a mean of 95% AR after 21 days, confirming the viability of the system.

The amount of [14C]-2,4-DTBP in the surface water showed a mean (non-sterile and sterile samples) of 91.7% AR at time zero that decreased to < 1% AR (non-sterile samples) and 73.5% AR (sterile samples) by Day 60. Four major transformation products (> 5% AR) were observed as 2-tert-butyl-4-nitrophenol, 4-tert-butyl-2,6-dinitrophenol, 2-tert-butyl-4,6-dinitrophenol, and 2,4-di-tert-butyl-6-nitrophenol. Material balance losses from the non-sterile samples (ca. 40% AR) as compared to the sterile samples ( ca. 8% AR)suggested that that these metabolites are volatile in nature.

DT50 and DT90 values for 2,4-DTBP in surface water were 5.2 and 17.3 days, respectively.

Based on the results of this study 2,4-di-tert-butylphenol would not be expected to hydrolyze in surface water under laboratory conditions or further mineralize to CO2 within 60 days, but would be expected to dissipate from a surface water in the environment via volatilization or degrade to multiple components.

Description of key information

In conclusion, 14C-2,4-DTBP rapidly dissipates in sediment-water systems from the water phase to the sediment and/or is ultimately volatilized from the system as parent.

No significant metabolites were observed in these studies, which would indicate that the pathway of degradation using this study set-up would be loss of parent from an aerobic and anaerobic aquatic environment to the atmosphere.

Similar results were observed for contrasting sediment systems, so there is no expected dependency on the nature of the residues that could be found in aerobic and anaerobic aquatic environments.

Key value for chemical safety assessment

Half-life in freshwater:
2.53 d
at the temperature of:
20 °C
Half-life in freshwater sediment:
7.34 d
at the temperature of:
20 °C

Additional information

The maximum aerobic half-life in water and in the total system were selected as key values for the chemical safety assessment.

OECD 308

The degradation of 14C-2,4-DTBP was studied in two different sediment-water-systems, both under aerobic and anaerobic conditions.

For the aerobic studies, the DT50 and DT90 values for 14C-2,4-DTBP were evaluated by single first order (SFO) and double first order parallel (DFOP) kinetic models using Computer Assisted Kinetic Evaluation (CAKE) ver. 2.0. The SFO model provided an adequate fit (visually and statistically) for the water for each of the two sediment systems and for one system’s total kinetics under aerobic conditions.

The SFO model provided an adequate fit (visually and statistically) for the water compartments for each of the two sediment systems under anaerobic conditions. The DFOP model provided an adequate fit (visually and statistically) for the total system compartments for each of the two sediment systems.

The mean material mass balances in all studies were only around 40 % of applied radioactivity in all test systems, independent of the applied conditions (aerobic/anaerobic). Further testing showed that significant loss of mass balance is likely due to the volatility of primarily 2,4-DTBP. Since the loss of mass balance could be accounted for in these additional tests, the kinetic modeling results would accurately reflect the fate of the parent in an aerobic aquatic environment.

In conclusion, 14C-2,4-DTBP rapidly dissipates in sediment-water systems from the water phase to the sediment and/or is ultimately volatilized from the system as parent. No significant metabolites were observed in these studies, which would indicate that the pathway of degradation using this study set-up would be loss of parent from an aerobic and anaerobic aquatic environment to the atmosphere. Similar results were observed for contrasting sediment systems, so there is no expected dependency on the nature of the residues that could be found in aerobic and anaerobic aquatic environments.

OECD 309

The aerobic mineralization of radiolabeled 2,4-di-tert-butylphenol was studied in one surface water under aerobic conditions. 2,4-di-tert-butylphenol did not mineralize significantly during the 60-day study. The overall mean mass balance of the closed bioreactors was 87% AR for the 14C-2,4-di-tert-butylphenol radiolabeled treatments. Losses from the definitive samples during sampling, processing, and HPLC analyses as well as method development testing demonstrated that 2,4-DTBP and its metabolites are volatile. DT50 and DT90 values for 2,4-DTBP in surface water were 5.0 and 16.6 days, respectively. Four major transformation products (>5% AR) were observed and identified by GC-MS as 2-tert-butyl-4-nitrophenol, 4-tert-butyl-2,6-dinitrophenol, 2-tert-butyl-4,6-dinitrophenol, and 2,4-di-tert-butylphenol. Based on the results of this study 2,4-di-tert-butylphenol would not be expected to hydrolyze in surface water or further mineralize to CO2 within 60 days, but would expect to dissipate from a surface water in the environment or degrade to multiple components.