2,4-di-tert-butylphenol

Biodegradation in soil

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in soil: 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

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

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- 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

Soil classification:

USDA (US Department of Agriculture)

Soil no.:

#1

Soil type:

sand

% Clay:

2

% Silt:

7

% Sand:

91

% Org. C:

0.61

pH:

5.2

CEC:

4.4 meq/100 g soil d.w.

Bulk density (g/cm³):

1

Soil no.:

#2

Soil type:

loamy sand

% Clay:

2

% Silt:

13

% Sand:

85

% Org. C:

2

pH:

6

CEC:

6.7 meq/100 g soil d.w.

Bulk density (g/cm³):

1.35

Soil no.:

#3

Soil type:

sandy loam

% Clay:

8

% Silt:

19

% Sand:

73

% Org. C:

0.68

pH:

6

CEC:

6.7 meq/100 g soil d.w.

Bulk density (g/cm³):

1.35

Soil no.:

#4

Soil type:

clay loam

% Clay:

28

% Silt:

37

% Sand:

35

% Org. C:

2.3

pH:

7.2

CEC:

20.5 meq/100 g soil d.w.

Bulk density (g/cm³):

1.11

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location: Rheinland-Pfalz, Germany
- Site description: Uncultivated(#1),Meadow(#2), Uncultivated(#3), Meadow with apple trees(#4)
- Pesticide use history at the collection site: None applied
- Collection procedures: Shovelled
- Sampling depth (cm): 20
- Shipment conditions: Ambient
- Storage conditions: Refrigerated
- Storage length: 9 days
- Soil preparation (e.g., 2 mm sieved; air dried etc.): 2 mm sieved

Soil No.:

#1

Duration:

120 d

Soil No.:

#2

Duration:

120 d

Soil No.:

#3

Duration:

120 d

Soil No.:

#4

Duration:

120 d

Soil No.:

#1

Initial conc.:

1 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#2

Initial conc.:

1 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#3

Initial conc.:

1 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#4

Initial conc.:

1 mg/kg soil d.w.

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Soil No.:

#1

Temp.:

20±2°C

Humidity:

Aerated with humidified air

Microbial biomass:

245.1 µg/g

Soil No.:

#2

Temp.:

20±2°C

Humidity:

Aerated with humidified air

Microbial biomass:

538.9 µg/g

Soil No.:

#3

Temp.:

20±2°C

Humidity:

Aerated with humidified air

Microbial biomass:

403.2 µg/g

Soil No.:

#4

Temp.:

20±2°C

Humidity:

Aerated with humidified air

Microbial biomass:

855.3 µg/g

Details on experimental conditions:

1. PRELIMINARY EXPERIMENTS:
A short 7-day preliminary test was performed exactly as described for the definitive test and showed acceptable mass accountability and chromatography, which indicated that the test system design and methods for extraction and analysis were acceptable for use in the definitive test.

2. EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): The moisture in the samples was adjusted to 100% of 0.1 bar (pF 2.0), and the main study and biomass samples were placed into incubation at 20°C on 14 August 2015, to acclimatize to the test temperature and achieve aerobic conditions prior to study initiation. The samples were incubated for 11 days prior to application of the test substance.
- Soil condition: fresh
- Soil (g/replicate): 50
- Control conditions, if used (present differences from other treatments, i.e., sterile/non-sterile, experimental conditions): same
- No. of replication controls, if used: 3
- No. of replication treatments: 2
- Test apparatus (Type/material/volume): Air pulled through reagent water pre trap humidifier (150 mL), 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)
- 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: Slight indication

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: halogen moisture analyzer maintained at 100% of 0.1 bar moisture (pF2)
- Continuous darkness: Yes

3. OXYGEN CONDITIONS (delete elements as appropriate)
- Aerobic conditions were maintained in all samples by drawing humidified air through the series containing the soil samples.

4. SUPPLEMENTARY EXPERIMENTS: n/a

5. SAMPLING DETAILS
- Sampling intervals: Days 0, 1, 3, 7, 14, 30, 59, 90, and 120
- Sampling method for soil samples: Soil samples were extracted 4 times using 100 mL of 4:1 acetone:0.1M ammonium carbonate. The soil and 4:1 acetone:0.1M ammonium carbonate mixtures were shaken on a platform shaker for 60 minutes. The mixture was then centrifuged for 20 minutes at approximately 3,600 × g. The supernatant was transferred to a graduated cylinder and was adjusted to 100 mL with 4:1 acetone:0.1M ammonium carbonate. Each extraction solution was analyzed by LSC prior to storing.
- Method of collection of CO2 and volatile organic compounds: Triplicate aliquots from the liquid volatile traps were taken at each sampling point to determine trapped volatiles by LSC analyses. The ORBO™ Tubes were analyzed at the Days 59 and 120 samplings.
- Sampling intervals/times for:
> Moisture content: Days 0, 1, 3, 7, 14, 30, 59, 90, and 120

Soil No.:

#1

% Recovery:

83.6

St. dev.:

8.9

Soil No.:

#2

% Recovery:

87.7

St. dev.:

11.1

Soil No.:

#3

% Recovery:

83

St. dev.:

10.1

Soil No.:

#4

% Recovery:

83.6

St. dev.:

11.4

Key result

Soil No.:

#1

DT50:

4.63 d

Type:

other: DFOP

Remarks on result:

other: M0 = 97.26 ± 2.793 k1 (d-1) = 0.1901 ± 0.02427 k2 (d-1) = 2.65E-12 ± 0.003226 g = 0.8543 ± 0.03695 r2 = 0.9823 DT90 = 107

Key result

Soil No.:

#2

DT50:

4.84 d

Type:

other: DFOP

Remarks on result:

other: M0 = 96.26 ± 2.657 k1 (d-1) = 0.1912 ± 0.02501 k2 (d-1) = 0.002118 ± 0.003023 g = 0.8259 ± 0.03875 r2 = 0.9836 DT90 = 262

Key result

Soil No.:

#3

DT50:

3.29 d

Type:

other: DFOP

Remarks on result:

other: M0 = 100.2 ± 2.112 k1 (d-1) = 0.2618 ± 0.02232 k2 (d-1) = 8.91E-15 ± 0.002185 g = 0.8655 ± 0.02208 r2 = 0.9911 DT90 = 77.8

Key result

Soil No.:

#4

DT50:

3.7 d

Type:

other: DFOP

Remarks on result:

other: M0 = 97.78 ± 2.919 k1 (d-1) = 0.2492 ± 0.03174 k2 (d-1) = 1.80E-13 ± 0.002514 g = 0.8305 ± 0.03222 r2 = 0.9806 DT90 = 84.3

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

Details on transformation products:

- Description of biotransformation pathway: Study results and additional testing indicate that 2,4-DTBP either rapidly dissipates through volatilization or degrades in an aerobic system maintained under dark conditions at 20 ± 2°C.

The additional work performed for the metabolites of 2,4-DTBP was successful at identifying MRT30 as a dimer of 2,4-DTBP. The lack of LC/MS response for 2,4-DTBP (standard and isolated material), as well as, the dimer (MRT30), MRT28, and MRT32 clearly
showed that these compounds were relatively volatile and/or steam distill (i.e., compounds that are volatile or steam distill typically do not ionize well by electrospray in an LC/MS).
In contrast, volatilization experiments clearly demonstrated that the parent and metabolites are not extremely volatile in a static system where air is not being blown onto the sample or removed under vacuum. This volatilization experiment also demonstrated the difficulty of dissolving radioactive residues from 2,4-DTBP since water was needed for complete solubilization even though these compounds are all relatively non-polar as shown by HPLC retention.
The GC/MS and LC/MS data was searched extensively using both a targeted approach (searching molecular weights of potential metabolites) and a non-targeted approach (searching every scan in the chromatograms for the distinct molecular ion pattern shown the 14C-2,4-DTBP used for dosing) without success for MRT28 and MRT32.
Hydrolysis data, while not conclusive, suggested that MRT28 is stable to both acid and base and that MRT32 may be unstable in these conditions. The stability of MRT28 suggested that this material is not a sugar conjugate or a sulfate, and may indicate that this is a methylated compound. The MS data was searched for the corresponding anisole and various methylated catechols and alcohols without success.
Base hydrolysis also generated a small amount of color which suggested the presence conjugated material that may have air oxidized to form quinones. However, this color formation could be due to the presence of naturally occurring catechols extracted from the samples.

Evaporation of parent compound:

yes

Volatile metabolites:

yes

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
Soil #1: MRT 28- 8.8% (Day 59), MRT 30- 15.1% (Day 14), MRT32- 9.2% (Day 120)
Soil #2: MRT 28- 8.7% (Day 14), MRT 30- 14.4% (Day 7), MRT32- 6.6% (Day 90)
Soil #3: MRT 28- 8.7% (Day 59), MRT 30- 20.7% (Day 7), MRT32- 7.8% (Day 59)
Soil #4: MRT 28- 11.8% (Day 7), MRT 30- 12.0% (Day 7), MRT32- 9.4% (Day 120)
- Range of maximum concentrations in % of the applied amount at end of study period:
Soil #1: MRT 28- 5.1%, MRT 30- 12.8%, MRT32- 9.2%
Soil #2: MRT 28- 4.2%, MRT 30- 4.7%, MRT32- 5.8%
Soil #3: MRT 28- 8.3%, MRT 30- 3.6%, MRT32- 6.0%
Soil #4: MRT 28- 9.5%, MRT 30- 7.2%, MRT32- 9.4%

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:

EXTRACTABLE RESIDUES
- % of applied amount at day 0: Soil #1 102.2, Soil #2 99.3, Soil #3 102.8, Soil #4 100.9
- % of applied amount at end of study period: Soil #1 50.6, Soil #2 34.9, Soil #3 36.2, Soil #4 44.7

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: Soil #1 - % of applied amount at end of study period: Soil #1 23.9, Soil #2 43.2, Soil #3 30.4, Soil #4 37.4

MINERALISATION
- % of applied radioactivity present as CO2 at end of study:

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:

Results with reference substance:

na

The mass balance for all the replicates included in the degradation modeling was between 67.1% and 102.9% AR. The average overall mass balances for the Speyer 2.1 (sand), Speyer 2.2 (sand), Speyer 2.3 (sandy loam), and Speyer 2.4 (light clay) systems were 84.0  7.3%, 89.1  7.1%, 83.1  10.1%, and 84.8  9.9%, respectively. The low recoveries in the soil systems are believed to be a result of the loss of volatile 2,4-DTBP and its metabolites. The loss of test material through volatility was not available to go through the degradation process, thus was not considered in kinetic calculations.

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.

Conclusions:

[14C]-2,4-DTBP was applied to four microbially active soils (two sands, a sandy loam, and a light clay) at 1 µg a.i./g, and the soil samples were incubated at 20 ± 2°C in the dark under aerobic conditions for 120 days. The mass balance for all the replicates included in degradation modeling was between 67.1% and 102.9% AR. The loss of mass balance is likely due to the volatility of 2,4-DTBP and its metabolites. In conclusion, 2,4-DTBP dissipates from the test system through volatilization, while the degradation of parent remaining follows a bi-phasic model where the DT50 is rapid in soils under aerobic conditions at 20°C and then degradation levels off with the DT90 is slower.

Executive summary:

The degradation of 2,4-di-tert-butylphenol (2,4-DTBP) was studied in the laboratory under aerobic conditions in four soils. [14C]-2,4-di-tert-butylphenol was applied to four different soils, a sand, a loamy sands, a sandy loam, and a light clay, all from Rheinland-Pfalz, Germany; at a dose rate of 1 µg/g. The soil samples were incubated at 20 ± 2°C in the dark for up to 120 days. Humidified air was drawn through the test systems and then through traps designed to retain radiolabeled volatile components to demonstrate a radiochemical balance.

At zero time and 1, 3, 7, 14, 30, 59, 90, and 120 days after application, duplicate samples were removed from incubation. The soil layer was extracted with 4:1 acetone:0.1M ammonium carbonate. The extracts were analyzed for radioactivity by liquid scintillation counting (LSC), and then analyzed by reverse phase high performance liquid chromatography (HPLC) and fraction collection. The post-extracted soil pellet was homogenized, combusted, and the radioactive residue quantified by LSC. Volatiles traps (1N KOH and extractions from ORBO™ 32) were also analyzed by LSC.

The mass balance for all the replicates included in degradation modeling was between 67.1% and 102.9% of the applied radioactivity (% AR). 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 soil environment.

Analysis of soil extracts for metabolites was performed by HPLC, and the identification of the metabolites will be confirmed using GC-MS/MS. Three major metabolites (>5% AR) were observed in the study and were designated as MRT28, MRT30, and MRT32.

Levels of non-extractable residue (NER) exceeded 10% AR following the primary extraction method by the Day 7 interval. Therefore, additional extraction methods were used within the preliminary testing. Extracted soil samples were extracted sequentially using ethyl acetate, hexane, and 10 mM ammonium hydroxide at ambient temperature.

The mass balance for all the replicates included in the degradation modeling was between 67.1% and 102.9% AR. The average overall mass balances for the Speyer 2.1 (sand), Speyer 2.2 (sand), Speyer 2.3 (sandy loam), and Speyer 2.4 (light clay) systems were 84.0  7.3%, 89.1  7.1%, 83.1  10.1%, and 84.8  9.9%, respectively. The low recoveries in the soil systems are believed to be a result of the loss of volatile 2,4-DTBP and its metabolites. The loss of test material through volatility was not available to go through the degradation process, thus was not considered in kinetic calculations.

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.

In conclusion, 2,4-DTBP degradation follows a bi-phasic model where the DT50 is rapid in soils under aerobic conditions at 20°C and then degradation levels off such that the DT90 is slower. Some loss of the test substance as parent from the aerobic soil systems was shown by further testing to be due to volatilization.

Reference 2

Endpoint:

biodegradation in soil: 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

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

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

Soil classification:

USDA (US Department of Agriculture)

Soil no.:

#1

Soil type:

sand

% Clay:

2

% Silt:

7

% Sand:

91

% Org. C:

0.61

pH:

5.2

CEC:

4.4 meq/100 g soil d.w.

Bulk density (g/cm³):

1

Soil no.:

#2

Soil type:

loamy sand

% Clay:

2

% Silt:

13

% Sand:

85

% Org. C:

2

pH:

6

CEC:

6.7 meq/100 g soil d.w.

Bulk density (g/cm³):

1.35

Soil no.:

#3

Soil type:

sandy loam

% Clay:

8

% Silt:

19

% Sand:

73

% Org. C:

0.68

pH:

6

CEC:

6.7 meq/100 g soil d.w.

Bulk density (g/cm³):

1.35

Soil no.:

#4

Soil type:

clay loam

% Clay:

28

% Silt:

37

% Sand:

35

% Org. C:

2.3

pH:

7.2

CEC:

20.5 meq/100 g soil d.w.

Bulk density (g/cm³):

1.11

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location: Rheinland-Pfalz, Germany
- Site description: Uncultivated(#1),Meadow(#2), Uncultivated(#3), Meadow with apple trees(#4)
- Pesticide use history at the collection site: None applied
- Collection procedures: Shovelled
- Sampling depth (cm): 20
- Shipment conditions: Ambient
- Storage conditions: Refrigerated
- Storage length: 9 days
- Soil preparation (e.g., 2 mm sieved; air dried etc.): 2 mm sieved

Soil No.:

#1

Duration:

120 d

Soil No.:

#2

Duration:

120 d

Soil No.:

#3

Duration:

120 d

Soil No.:

#4

Duration:

120 d

Soil No.:

#1

Initial conc.:

1 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#2

Initial conc.:

1 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#3

Initial conc.:

1 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#4

Initial conc.:

1 mg/kg soil d.w.

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Soil No.:

#1

Temp.:

20±2°C

Humidity:

Aerated with humidified air during aerobic phase

Microbial biomass:

245.1 µg/g

Soil No.:

#2

Temp.:

20±2°C

Humidity:

Aerated with humidified air during aerobic phase

Microbial biomass:

538.9 µg/g

Soil No.:

#3

Temp.:

20±2°C

Humidity:

Aerated with humidified air during aerobic phase

Microbial biomass:

403.2 µg/g

Soil No.:

#4

Temp.:

20±2°C

Humidity:

Aerated with humidified air during aerobic phase

Microbial biomass:

855.3 µg/g

Details on experimental conditions:

1. PRELIMINARY EXPERIMENTS:
The approximate aerobic DegT50 had previously been determined from a preliminary test within the aerobic soil degradation study (6) (total duration 7 days) using the same application rate.

2. EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): 16 days, 4°C
- Soil condition: fresh
- Soil (g/replicate): 50
- Control conditions, if used (present differences from other treatments, i.e., sterile/non-sterile, experimental conditions): same
- No. of replication controls, if used: 3
- No. of replication treatments: 2
- Test apparatus (Type/material/volume): Air pushed through reagent water pre trap humidifier (150 mL), 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: Slight indication

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: halogen moisture analyzer maintained at 100% of 0.1 bar moisture (pF2)
- Continuous darkness: Yes

3. OXYGEN CONDITIONS (delete elements as appropriate)
- Methods used to create the an/aerobic conditions: House nitrogen
- Evidence that an/aerobic conditions were maintained during the experiment (e.g. redox potential): dissolved oxygen, redox potential

4. SUPPLEMENTARY EXPERIMENTS: n/a

5. SAMPLING DETAILS
- Sampling intervals: Days (DAT) 0, 6; Days (DAIA) 1, 3, 7, 15, 28, 58, 90, and 114
- Sampling method for soil samples: Soil samples were extracted 4 times using 100 mL of 4:1 acetone:0.1M ammonium carbonate. The soil and 4:1 acetone:0.1M ammonium carbonate mixtures were shaken on a platform shaker for 60 minutes. The mixture was then centrifuged for 20 minutes at approximately 3,600 × g. The supernatant was transferred to a graduated cylinder and was adjusted to 100 mL with 4:1 acetone:0.1M ammonium carbonate. Each extraction solution was analyzed by LSC prior to storing.
- Method of collection of CO2 and volatile organic compounds: Triplicate aliquots from the liquid volatile traps were taken at each sampling point to determine trapped volatiles by LSC analyses. The ORBO™ Tubes were analyzed at the Day 120 samplings.
- Sampling intervals/times for:
> Moisture content: Days (DAT) 0, 6; Days (DAIA) 1, 3, 7, 15, 28, 58, 90, and 114

Soil No.:

#1

% Recovery:

90

St. dev.:

6.6

Soil No.:

#2

% Recovery:

94.8

St. dev.:

4.6

Soil No.:

#3

% Recovery:

91.8

St. dev.:

7.4

Soil No.:

#4

% Recovery:

84.7

St. dev.:

9.7

Key result

Soil No.:

#1

DT50:

334 d

Type:

other: SFO (single first order)

Remarks on result:

other: M0 = 39.72 ± 3.635 k (d-1) = 0.002075 ± 0.001826 r2 = 0.0922 DT90 = 1.11E+03

Key result

Soil No.:

#2

DT50:

17 400 000 000 000 d

Type:

other: SFO

Remarks on result:

other: M0 = 32.42 ± 2.937 k (d-1) = 3.99E-014 ± 0.001604 r2 = 0.01458 DT90 = 5.77E+13

Key result

Soil No.:

#3

DT50:

572 d

Type:

other: SFO

Remarks on result:

other: M0 = 24.54 ± 2.061 k (d-1) = 0.001213 ± 0.001538 r2 = 0.04932 DT90 = 1.9E+03

Key result

Soil No.:

#4

DT50:

7 070 d

Type:

other: SFO

Remarks on result:

other: M0 = 29.88 ± 2.165 k (d-1) = 9.80E-005 ± 0.001273 r2 = 0.000446 DT90 = 2.35E+04

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

Details on transformation products:

- Description of biotransformation pathway: Study results and additional testing indicate that 2,4-DTBP either rapidly dissipates through volatilization or degrades in an aerobic system maintained under dark conditions at 20 ± 2°C.

The additional work performed for the metabolites of 2,4-DTBP was successful at identifying MRT30 as a dimer of 2,4-DTBP. The lack of LC/MS response for 2,4-DTBP (standard and isolated material), as well as, the dimer (MRT30), MRT28, and MRT32 clearly
showed that these compounds were relatively volatile and/or steam distill (i.e., compounds that are volatile or steam distill typically do not ionize well by electrospray in an LC/MS).
In contrast, volatilization experiments clearly demonstrated that the parent and metabolites are not extremely volatile in a static system where air is not being blown onto the sample or removed under vacuum. This volatilization experiment also demonstrated the difficulty of dissolving radioactive residues from 2,4-DTBP since water was needed for complete solubilization even though these compounds are all relatively non-polar as shown by HPLC retention.
The GC/MS and LC/MS data was searched extensively using both a targeted approach (searching molecular weights of potential metabolites) and a non-targeted approach (searching every scan in the chromatograms for the distinct molecular ion pattern shown the 14C-2,4-DTBP used for dosing) without success for MRT28 and MRT32.
Hydrolysis data, while not conclusive, suggested that MRT28 is stable to both acid and base and that MRT32 may be unstable in these conditions. The stability of MRT28 suggested that this material is not a sugar conjugate or a sulfate, and may indicate that this is a methylated compound. The MS data was searched for the corresponding anisole and various methylated catechols and alcohols without success.
Base hydrolysis also generated a small amount of color which suggested the presence conjugated material that may have air oxidized to form quinones. However, this color formation could be due to the presence of naturally occurring catechols extracted from the samples.

Evaporation of parent compound:

yes

Volatile metabolites:

yes

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
Soil #1: MRT 28- 20.0% (Day 9), MRT 30- 12.9% (Day 34), MRT32- 15.4% (Day 120)
Soil #2: MRT 28- 11.9% (Day 9), MRT 30- 11.7% (Day 13), MRT32- 7.8% (Day 90)
Soil #3: MRT 28- 18.9% (Day 9), MRT 30- 18.9% (Day 6), MRT32- 12.7% (Day 120)
Soil #4: MRT 28- 14.1% (Day 9), MRT 30- 8.4% (Day 34), MRT32- 10.2% (Day 90)
- Range of maximum concentrations in % of the applied amount at end of study period:
Soil #1: MRT 28- 10.3%, MRT 30- 5.%, MRT32- 15.4%
Soil #2: MRT 28- 4.8%, MRT 30- 2.6%, MRT32- 5.5%
Soil #3: MRT 28- 13.0%, MRT 30- 9.1%, MRT32- 12.7%
Soil #4: MRT 28- 11.8%, MRT 30- 3.5%, MRT32- 2.9%


TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:

EXTRACTABLE RESIDUES
- % of applied amount at day 0: Soil #1 101.4, Soil #2 101.5, Soil #3 99.6, Soil #4 101.4
- % of applied amount at end of study period: Soil #1 59.2, Soil #2 99.3, Soil #3 52.9, Soil #4 52.7

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: Soil #1 0.2, Soil #2 0.3, Soil #3 0.3, Soil #4 1.2
- % of applied amount at end of study period: Soil #1 20.8, Soil #2 32.4, Soil #3 32.0, Soil #4 20.3

MINERALISATION
- % of applied radioactivity present as CO2 at end of study:

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:

Results with reference substance:

na

The rate of degradation in the soil during the anaerobic phase was estimated using single first order (SFO) kinetics. The r2 was between 0.000446 and 0.09208 for the anaerobic degradation SFO calculation using 0-114 DAIA. After flooding, the test substance appeared to show no visible trend in degradation, this data is supported by the kinetics running single first order (SFO), which is supplied below to further support this discussion.

A comparison of SFO and double first order in parallel (DFOP) models are presented below.

Soil	Soil pH^a	Model
		SFO			DFOP
		^2	r^2	DT50 (DAYS)	^2	r^2	DT50 (DAYS)
Soil 1	5.2	15.7	0.09208	334 ^b	17.5	0.1237	406 ^b
Soil 2	6.0	17.3	0.01458	1.74E+13 ^b	18.4	0.1167	2.02E+06 ^b
Soil 3	6.0	9.52	0.04932	572 ^b	10.9	0.0499	135^b
Soil 4	7.2	13.3	0.000446	7.07E+03 ^b	15.3	0.000446	7.07E+03 ^b

^a pH in 1:2 soil:0.01M CaCl2 (aq)

 ^b Extrapolated beyond the duration of the study

Conclusions:

[14C]-2,4-DTBP was applied to four microbially active soils (a sand, a sand, a sandy loam, and a light clay) at 1 µg a.i./g, and the soil samples were incubated at 20 ± 2°C in the dark under anaerobic conditions for 114 days.
The mass balance for all the replicates included in degradation modeling was between 68.2 and 105.3% AR. The loss of mass balance is likely due to the volatility of 2,4-DTBP and its metabolites.
2,4-DTBP degrades fairly rapidly in soils incubated under aerobic conditions and then after flooding, the test substance appeared to show no visible trend in degradation, this data is supported by the kinetics running single first order (SFO).

Executive summary:

The degradation of 2,4-di-tert-butylphenol (2,4-DTBP) was studied in the laboratory under anaerobic conditions in four soils. [14C]-2,4-di-tert-butylphenol was applied to two sands, a sandy loam, and a light clay, all from Rheinland-Pfalz, Germany; at a dose rate of 1 µg/g. The soil samples were incubated at 20 ± 2°C in the dark for up to 120 days. Humidified air through six days was drawn through the test systems and then through traps designed to retain radiolabeled volatile components to demonstrate a radiochemical balance. After six days humidified nitrogen was pushed through the test system to maintain anaerobic conditions.

At zero time and 6, 7, 9, 13, 21, 34, 64, 96, and 120 days after application, duplicate samples were removed from incubation. The soil layer was extracted with 4:1 acetone:0.1M ammonium carbonate and combined with the water phase (anaerobic portion). The extracts were analyzed for radioactivity by liquid scintillation counting (LSC), and then analyzed by reverse phase high performance liquid chromatography (HPLC) and fraction collection. The post-extracted soil pellet was homogenized, combusted, and the radioactive residue quantified by LSC. Volatiles traps (1N KOH and extractions from ORBO™ 32) were also analyzed by LSC.

The mass balance for all the replicates included in degradation modeling was between 68.2% and 105.3% of the applied radioactivity (% AR). 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 soil environment.

Analysis of soil extracts for metabolites was performed by HPLC, and the identification of the metabolites will be confirmed using GC-MS/MS. Three major metabolites (>5% AR) were observed in the study and were designated as MRT28, MRT30, and MRT32.

The amount of parent ([14C]-2,4-DTBP) degraded rapidly under aerobic conditions with a DegT50 of approximately 6 days. Following flooding, the amount of parent ([14C] 2,4-DTBP) extracted (ambient temperature) from the soil decreased slowly over time and was between 19.1% and 37.5% at 120 DAT (114 DAIA). The rate of degradation in the soil during the anaerobic phase was estimated using single first order (SFO) kinetics. The r2 was between 0.0004 and 0.0921 for the anaerobic degradation SFO calculation using 0-114 DAIA. After flooding, the test substance appeared to show no visible trend in degradation, this data is supported by the kinetics running single first order (SFO).

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.

In conclusion, 2,4-DTBP degradation follows a single phase model (best fit) where the DT50 is rapid in soils under aerobic conditions at 20°C (< 6 days). The degradation of the metabolites appeared to slightly continue. In the aerobic soil, the primary route of degradation appears to be, but then there is minimal degradation in an anaerobic soil system.

Description of key information

In conclusion, 2,4-DTBP dissipated from the test systems, both aerobic and anaerobic, through volatilization, while the degradation of parent remaining follows a bi-phasic model where the DT50 is rapid in soils under aerobic conditions at 20°C and then degradation levels off with the DT90 is slower under aerobic conditions. Under anaerobic conditions, the test substance appeared to show no visible trend in degradation, this data is supported by the kinetics running single first order (SFO).

Key value for chemical safety assessment

Half-life in soil:

4.84 d

at the temperature of:

20 °C

Additional information

The maximum aerobic half-life was selected as key value for the chemical safety assessment.

The biodegradation in soil of 2,4 -DTBP was assessed under aerobic and anaerobic conditions in two separate OECD 307 studies. In the aerobic study, 14C-2,4-DTBP was applied to four microbial active soils (two sands, a sandy loam, and a light clay) at 1 µg a.i./g, and the soil samples were incubated at 20 ± 2°C in the dark under aerobic conditions for 120 days. The mass balance for all the replicates included in degradation modeling was between 67.1% and 102.9% AR. The loss of mass balance is likely due to the volatility of 2,4-DTBP and its metabolites. Under anaerobic conditions, the amount of parent (14C-2,4-DTBP) degraded rapidly under aerobic conditions with a DegT50 of approximately 6 days. The mass balance for all the replicates included in degradation modeling was between 68.2 and 105.3% AR. After flooding, the test substance appeared to show no visible trend in degradation, this data is supported by the kinetics running single first order (SFO). In conclusion, 2,4-DTBP dissipated from the test system through volatilization, while the degradation of parent remaining, only detected under aerobic conditions, follows a bi-phasic model where the DT50 is rapid in aerobic soils at 20°C and then degradation levels off with the DT90 is slower.

Identification of metabolites (MRT 28, MRT 30, MRT 32)

The additional work performed for the metabolites of 2,4-DTBP was successful at identifying MRT30 as a dimer of 2,4-DTBP. The lack of LC/MS response for 2,4-DTBP (standard and isolated material), as well as, the dimer (MRT30), MRT28, and MRT32 clearly showed that these compounds were relatively volatile and/or steam distill (i.e., compounds that are volatile or steam distill typically do not ionize well by electrospray in an LC/MS).

In contrast, volatilization experiments clearly demonstrated that the parent and metabolites are not extremely volatile in a static system where air is not being blown onto the sample or removed under vacuum. This volatilization experiment also demonstrated the difficulty of dissolving radioactive residues from 2,4-DTBP since water was needed for complete solubilization even though these compounds are all relatively non-polar as shown by HPLC retention.

The GC/MS and LC/MS data was searched extensively using both a targeted approach (searching molecular weights of potential metabolites) and a non-targeted approach (searching every scan in the chromatograms for the distinct molecular ion pattern shown the 14C-2,4-DTBP used for dosing) without success for MRT28 and MRT32.

Hydrolysis data, while not conclusive, suggested that MRT28 is stable to both acid and base and that MRT32 may be unstable in these conditions. The stability of MRT28 suggested that this material is not a sugar conjugate or a sulfate, and may indicate that this is a methylated compound. The MS data was searched for the corresponding anisole and various methylated catechols and alcohols without success.

Base hydrolysis also generated a small amount of color which suggested the presence conjugated material that may have air oxidized to form quinones. However, this color formation could be due to the presence of naturally occurring catechols extracted from the samples.