Reaction mass of 2,6-bis(1-phenylethyl)phenol and 2,4,6-tris(1-phenylethyl)phenol

Biodegradation in soil

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29.11.2016 - 06.06.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4100 (Aerobic Soil Metabolism)

GLP compliance:

yes

Test type:

laboratory

Specific details on test material used for the study:

Radiolabeled Test Substance:
- Name: tristyrenated [U-14C]phenol
- Lot No.: CFQ42881
- Radiochemical Purity: 99.7%
- Molecular Weight: 408.3 g/mol
- Specific Activity: 53 mCi/mmol, 288,170 dpm/μg
- Positiion of the radiolabel: in the central phenol ring
- Storage conditions: in the freezer in the original container

Non-Radiolabeled Reference Substance:
- Name: SOPROPHOR TSP
- Batch No.: 3688102
- CAS No.: 61788-44-1
- Purity: 70 to 74%
- Storage conditions: room temperature, in the dark in a ventilated cabinet in the original container

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

other: sandy loam, silt loam, loam

Year:

2016

Soil no.:

#1

Soil type:

sandy loam

% Clay:

16

% Silt:

24

% Sand:

60

% Org. C:

2.2

pH:

5.7

CEC:

14.9 meq/100 g soil d.w.

Bulk density (g/cm³):

1.14

Soil no.:

#2

Soil type:

silt loam

% Clay:

8

% Silt:

52

% Sand:

40

% Org. C:

2.3

pH:

7.1

CEC:

8.9 meq/100 g soil d.w.

Bulk density (g/cm³):

0.93

Soil no.:

#3

Soil type:

silt loam

% Clay:

20

% Silt:

60

% Sand:

20

% Org. C:

1.3

pH:

6.6

CEC:

12.7 meq/100 g soil d.w.

Bulk density (g/cm³):

1.02

Soil no.:

#4

Soil type:

loam

% Clay:

22

% Silt:

40

% Sand:

38

% Org. C:

2.1

pH:

7.7

CEC:

10.5 meq/100 g soil d.w.

Bulk density (g/cm³):

0.98

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location: sample 1: 18 Acres; Bracknell, United Kingdom, sample 2: Gartenacker; Gartenacker SW1, Switzerland, sample 3: Krone; Möhlin, United Kingdom, sample 4: Vetroz; Vetroz, Switzerland
- Provided history indicates no treatment with the test substance or its structural analogs during the last four years for all soils.
- Pesticide use history at the collection site: No pesticide history was available for the 18 Acres Soil. No pesticides were used on the Gartenacker, Krone, or Vetroz Soils
- Collection procedures: The test soils were collected by Study Sponsor designees and were received by Smithers Viscient on 28 September 2016
- Sampling depth (cm): not specified
- Storage conditions: not specified
- Storage length: approx. 2 month till start of the experimental phase
- Soil preparation (e.g., 2 mm sieved; air dried etc.): The soils were passed through a 2-mm sieve to assure uniformity and to remove small stones, fauna, and plant debris. The microbial biomass of the soils was determined by fumigation/extraction near the start, middle, and end of the incubation period

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture (%): adjusted to 20.1 (18 Acres), 32.4 (Gartenacker), 29.3 (Krone), 31.1 (Vetroz)
- Bulk density (g/cm3): 1.0 g/cm3 is used in the calculation of the application dosing, based on assuming a typical value for a typical soil

Soil No.:

#1

Duration:

180 d

Soil No.:

#2

Duration:

120 d

Soil No.:

#3

Duration:

120 d

Soil No.:

#4

Duration:

121 d

Soil No.:

#1

Initial conc.:

0.6 other: µg/g d.w.

Based on:

test mat.

Soil No.:

#2

Initial conc.:

0.6 other: µg/g d.w.

Based on:

test mat.

Soil No.:

#3

Initial conc.:

0.6 other: µg/g d.w.

Based on:

test mat.

Soil No.:

#4

Initial conc.:

0.6 other: µg/g d.w.

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

other: rate and route of transformation of the test item

Soil No.:

#1

Temp.:

12 ± 2 °C

Humidity:

20.1 %

Soil No.:

#1

Temp.:

20 ± 2 °C

Humidity:

20.1%

Soil No.:

#2

Temp.:

12 ± 2 °C

Humidity:

32.4%

Soil No.:

#2

Temp.:

20 ± 2 °C

Humidity:

32.4%

Soil No.:

#3

Temp.:

12 ± 2 °C

Humidity:

29.3%

Soil No.:

#3

Temp.:

20 ± 2 °C

Humidity:

29.3%

Soil No.:

#4

Temp.:

12 ± 2 °C

Humidity:

31.1%

Soil No.:

#4

Temp.:

20 ± 2 °C

Humidity:

31.1%

Details on experimental conditions:

1. PRELIMINARY EXPERIMENTS:
A 14-day preliminary study was conducted to determine the analytical methods for this study.


2. EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): not applicable
- Soil condition: fresh
- Soil (g/replicate): 100 g soil (dry weight equivalent)
- No. of replication treatments: Two test vessels for each soil at each incubation temperature
- Test condition: aerobic conditions in the dark at 12 ± 2 °C and 20 ± 2 °C
- Test apparatus (Type/material/volume): not specified
- Details of traps for CO2 and organic volatile, if any: volatile trap: solutions of ethylene glycol; CO2 trap: 1.0 M potassium hydroxide. Volatile traps were analyzed beginning at day 3 and at frequent intervals thereafter for total radioactivity. Ethylene glycol and KOH traps were removed from each test system, volumes recorded and analyzed by LSC for quantification of volatile radioactivity. The presence of 14CO2 in the KOH traps was confirmed by BaCl2 precipitation for representative volatile traps.
- Identity and concentration of co-solvent: not applicable

Test material application
- Volume of test solution used/treatment: [14C]Tristyrenated phenol at a nominal concentration of 0.6 μg/g
- Application method (e.g. applied on surface, homogeneous mixing etc.): not specified
- Is the co-solvent evaporated: not applicable

Any indication of the test material adsorbing to the walls of the test apparatus: not specified

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: periodically weighing the test systems. Any moisture loss was adjusted by the addition of purified reagent water
- Moisture: pF 2.5.
- Continuous darkness: Yes


3. OXYGEN CONDITIONS
- Methods used to create the aerobic conditions: each test vessel chain is connected to a vacuum pump and flushed continuously with hydrated air.
- Evidence that an/aerobic conditions were maintained during the experiment (e.g. redox potential): not specified


4. SUPPLEMENTARY EXPERIMENTS:
4.1 Non-Extractable Residues (NER) Analysis
Aliquots of the post-extraction solids (PES) were analyzed by combustion to determine the amount of non-extractable residues. The solids remaining after extraction were dried, prior to combustion. The weight of each dried PES sample was recorded and the samples were homogenized using a mortar and pestle. Triplicate subsamples (approximately 0.2 g each) were then combusted on a Harvey Oxidizer Model OX-500 or OX-501 for three minutes. The released CO2 was collected in Harvey cocktail and quantified by LSC analysis to determine total residual radioactivity in the subsamples. The residual radioactivity in each sample was calculated by determining the dpm per dry weight equivalent of each solid subsample combusted.

4.2 Characterisation of Non Extractable Residues
Following combustion analysis, representative PES samples from each soil (day 120 and 121 from the 12 °C and 20 °C test systems, respectively) were further extracted using two additional solvents, tetrahydrofuran and hexane. Tetrahydrofuran was selected as an extraction solvent to remove any radioactivity remaining as polar compound residues and hexane was used to remove any non-polar compounds. The solvents used for these additional extractions were chosen to cover all possible polarity classifications mentioned in the OCSPP 835.4100 Guideline (U.S. EPA, 2008).
Representative PES samples were extracted with approximately 150 mL of tetrahydrofuran by sonicating for 10 minutes and then placing on a shaker table at approximately 200 rpm for 30 minutes. Samples were then centrifuged at 3000 rpm for 10 minutes and transferred to a graduated cylinder. The volume was recorded and duplicate 1.0-mL aliquots were analyzed by LSC. This extraction procedure was repeated once as described above using hexane.

4.3 Soxhlet Extraction
After the polar and non-polar extractions, further extractions of these samples (day 120 and 121 from 12 °C and 20 °C test systems, respectively) were performed using the harsh methods of Soxhlet extraction and acidic conditions. Approximately 20 g of the remaining solids from each sample was weighed into porous cellulose thimbles and further extracted by using a Soxhlet apparatus. An aliquot (250 mL) of 85/11/4.0 acetone/purified reagent water/phosphoric acid was added to each labeled round bottom flask. The cellulose thimble containing the sample was then placed in a Soxhlet extractor for approximately 16 hours. The total volume of extract was recorded and duplicate 0.50 mL aliquots were analyzed by LSC. Select Soxhlet extracts were analyzed by HPLC-RAM, using co-chromatography with one of the mixtures of reference standards within one day after completion of the extraction procedure. The remaining Soxhlet extracts were stored frozen for future analysis, if needed.

4.4 Organic Matter Fractionation (OMF)
After the polar and non-polar extractions, a portion of the remaining solids from each sample was subjected to organic-matter fractionation (OMF) into humic acid, fulvic acid, and humin. These fractions are operationally defined based on solubility as follows:
• fulvic acid is the fraction of soil organic matter (SOM) that is soluble in aqueous solution regardless of the pH of the solution.
• humic acid is the fraction of SOM that is soluble in aqueous alkaline conditions but insoluble in acidic conditions.
• humin is the fraction of the SOM that is insoluble in aqueous solution at all pH values; it is the insoluble fraction left after fulvic acid and humic acid fractions have been extracted.
Approximately 10 g dry weight, (10% by weight) was transferred to a 40-mL plastic centrifuge tube to which approximately 25 mL of 2M sodium hydroxide was added. The containers were sonicated for two minutes and shaken at room temperature at approximately 200 rpm for
30 minutes. Samples were then centrifuged at approximately 3000 rpm for 10 minutes to pellet the solids and the supernatants were decanted. The procedure was repeated twice, the respective supernatants were pooled, and the total volume was recorded. Duplicate 0.05-mL aliquots were analyzed by LSC.
The supernatants were transferred to containers and the pH was adjusted to < 1 with concentrated hydrochloric acid to precipitate the humic acid fraction. The sample was centrifuged at approximately 3000 rpm for 10 minutes to pellet any precipitate. The supernatant containing the fulvic acid fraction was decanted and the volume was recorded. Duplicate 0.05-mL aliquots were analyzed by LSC. The remaining precipitate containing the humic acid fraction was re-suspended in 2M sodium hydroxide and the volume was recorded. Duplicate 0.05-mL aliquots were analyzed by LSC to quantify radioactivity associated with the humic acid fraction. The radioactivity in the humin fraction was determined by subtraction of fulvic acid and humic acid fraction residues from the available radioactivity in the non-extractable residue of each subsample after polar and non-polar extractions.



5. SAMPLING DETAILS
- Sampling intervals:
-- Soil samples incubated at 12 ± 2 °C were analyzed at 0, 3, 7, 14, 21, 29, 60, and 120 days of incubation
-- Soil samples incubated at 20 ± 2 °C were analyzed at 0, 3, 7, 14, 21, 29, 59, and 121 days of incubation.
-- The 18 Acres soil, incubated at 12 ± 2 °C and 20 ± 2 °C was also sampled at 180 and 178 days, respectively
- Sampling method for soil samples: For each sampling interval, duplicate samples for each soil type were removed from each test system.
- Method of collection of CO2 and volatile organic compounds: via volatile and CO2 traps, see above.
- Sampling intervals/times for:
> Moisture content: periodically weighing
> Sample storage before analysis: soil extracts were processed and analyzed immediately after sampling to minimize potential degradation under storage conditions
- Other observations, if any: not specified


6. CALCULATIONS
6.1 Material Balance
The radioactivity measured in the soil extracts, the volatile trapping solutions and the non-extractable residues was summed to determine the total radioactivity measured in each test vessel at each sampling interval. Volatile radioactivity was calculated as a cumulative value for each sampling interval. The percent total recovery was calculated by dividing the total measured radioactivity by the total radioactivity applied to the test system, as measured by LSC of the aliquot of the dosing solution.

6.2 Transformation / Dissipation Rate
The transformation / dissipation rate of the test item and major transformation products was evaluated in soil following FOCUS (2006) Guidance recommendations.
All calculation were performed in MS Excel and CAKE v3.3 (TESSELA) using unrounded values. Reported values were rounded.
CAKE was used to evaluate the study data. The suitability of the fit of the models was evaluated both visually and statistically by calculating the minimum percent error required to pass the Chi-square test at a probability of 0.05 (acceptability criteria Chi-square <= 15%).

Soil No.:

#1

% Recovery:

100.4

Remarks on result:

other: 12 ± 2 °C (overall average)

Soil No.:

#1

% Recovery:

103.7

Remarks on result:

other: 20 ± 2 °C (overall average)

Soil No.:

#2

% Recovery:

100.4

Remarks on result:

other: 12 ± 2 °C (overall average)

Soil No.:

#2

% Recovery:

103.3

Remarks on result:

other: 20 ± 2 °C (overall average)

Soil No.:

#3

% Recovery:

101.1

Remarks on result:

other: 12 ± 2 °C (overall average)

Soil No.:

#3

% Recovery:

103.2

Remarks on result:

other: 20 ± 2 °C (overall average)

Soil No.:

#4

% Recovery:

100.9

Remarks on result:

other: 12 ± 2 °C (overall average)

Soil No.:

#4

% Recovery:

103

Remarks on result:

other: 20 ± 2 °C (overall average)

Key result

Soil No.:

#1

DT50:

5.95 d

Type:

other: non-linear kinetics

Temp.:

12 °C

Key result

Soil No.:

#2

DT50:

7.66 d

Type:

other: non-linear kinetics

Temp.:

12 °C

Key result

Soil No.:

#3

DT50:

3.13 d

Type:

other: non-linear kinetics

Temp.:

12 °C

Key result

Soil No.:

#4

DT50:

12.5 d

Type:

other: non-linear kinetics

Temp.:

12 °C

Soil No.:

#1

DT50:

3.65 d

Type:

other: non-linear kinetics

Temp.:

20 °C

Soil No.:

#2

DT50:

8.46 d

Type:

other: non-linear kinetics

Temp.:

20 °C

Soil No.:

#3

DT50:

2.88 d

Type:

other: non-linear kinetics

Temp.:

20 °C

Soil No.:

#4

DT50:

3.73 d

Type:

other: non-linear kinetics

Temp.:

20 °C

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

No.:

#5

Details on transformation products:

- Met 0 is proposed to be DSP monooxide (i.e. 2,6-di(1-phenylethyl)phenol or 2,4-di(1-phenylethyl)lphenol with one additional hydroxyl group in an unspecified location)
- Met 3 and Met 4 are proposed to be demethylated oxo-TSP (i.e. TSP from which one methyl group is lost, and the benzyl-CH being oxidized to the keto form)
- Met 5 is proposed to be TSP trioxide methyl ester
- Met 7 and Met 8 are proposed to TSP monoxides (i.e. 2,4,6-tri(1-phenylethyl)henol with one additional hydroxyl group in an unspecified location)
- Met 9 is proposed to be TSP dioxide (i.e. 2,4,6-tri(1-phenylethyl)phenol with two additional hydroxyl groups in unspecified locations)

Samples representing the highest concentration of major transformation products (> 10% AR or >5% AR at two consecutive time points; DSP monoxide, demethylated TSP, TSP trioxide methyl ester, TSP monoxides, and TSP dioxide), were identified using LC-MS/MS

Evaporation of parent compound:

no

Remarks:

no significant loss of the test item was observed during incubation

Volatile metabolites:

yes

Remarks:

Volatile organic compounds generally remained below the limit of detection and 14CO2 increased in all soil test systems over the course of the incubation period, up to a maximum of 22% AR

Residues:

yes

Remarks:

Non-extractable residues increased over the course of the incubation period

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): no
Two sets of test system samples were incubated and maintained aerobically in the dark at either 12 ± 2 °C (actual range 10.7 to 13.1 °C) or 20 ± 2 °C (actual range 19.7 to 22.0 °C with two excursions to 26.3 and 28.6 °C) for the duration of the study

MICROBIAL BIOMASS
The microbial biomass was measured in each soil test system (12 °C and 20 °C) at the start of incubation, the approximate mid-point, and at the end of incubation.
The microbial biomass measured at the start of incubation at 12 °C, as percent of organic carbon, was 3.05, 3.85, 5.82, and 1.74% OC, for the 18 Acres, Gartenacker, Krone, and Vetroz soil test systems, respectively. The microbial biomass measured at the approximate mid-point and at the end of incubation remained fairly consistent. Microbial biomass, as percent of organic carbon, was measured at end of incubation as 3.47, 2.27, 4.96, and 2.09% OC for the 18 Acres, Gartenacker, Krone, and Vetroz soil test systems, respectively.
A microbial biomass of approximately 1% OC is the recommended guideline for soils at the start of incubation. There is generally no recommendation for biomass levels after this; however, it is typical for microbial populations to decline throughout incubation. Therefore, these results indicate that the microbial biomass was quite healthy and viable for the duration of incubation. The microbial biomass observed in each test system indicated that there was little difference between microbial populations at the different temperatures and the microbial populations were unaffected by the incubation at lower temperature (12 ± 2 °C).

TEST CONCENTRATION
The nominal test substance concentration for this study was 0.6 μg/g (dry weight). The actual concentration of [14C]tristyrenated phenol in each test vessel incubated at 12 ± 2 °C and 20 ± 2 °C was determined to be 0.617 and 0.598 μg/g (dry weight), respectively, based on LSC analysis of the pre-dose and post-dose solutions.

MATERIAL BALANCE
Material balance over the course of the incubation period was within the acceptable range of 90 to 110% applied radioactivity. Therefore, it can be concluded that no significant loss of the test item was observed during incubation. Cfr. table further on in this RSS.

SOIL EXTRACTED RESIDUES
Soil extracted residue decreased over the course of the incubation period. Cfr. table further on in this RSS.
The amounts of extracted radioactivity between the soils incubated at 12 °C and 20 °C were similar at the start of the test. The extraction scheme was developed to provide optimal extraction efficiency, based on the characteristics of the soil types (clay content). The extractability at the start of the study is similar and within expected ranges.
Throughout incubation, the extractable radioactivity decreased in both test systems (12 °C and 20 °C), with only slightly less residue extracted in test systems incubated at 20 °C. This slight difference is accounted for by the higher extent of mineralization in the test systems incubated at 20 °C, in which up to 22% of 14CO2 was observed, compared to a maximum of 14.3% of 14CO2 in the test systems incubated at 12 °C, and does not appear to be related to soil type.

NON-EXTRACTABLE RESIDUES
Radioactivity not removed during extraction was considered non-extractable residue.
Non-extractable residues increased over the course of the incubation period. Cfr. table further on in this RSS. There was no significant difference in amounts of non-extracted radioactivity between the soils incubated at 12 and 20 °C at the start and the end of the test.

CHARACTERISATION OF NON-EXTRACTABLE RESIDUES
Since non-extractable residues exceeded 10% AR, they were additionally characterized in representative samples using a polar solvent (tetrahydrofuran, THF) to remove and quantify polar compounds and a non-polar solvent (hexane) to remove and quantify non-polar compounds.
No analysis by HPLC-RAM was performed, as the radioactive recovery in each additional extract was < 1.5% AR. The amount of additional extracts is very negligible, indicating that any compounds in the non-extractable residues are sequestered and non-bioavailable.

SOXHLET EXTRACTION
Radioactivity remaining in the non-extractable residue samples after the polar and non-polar extractions was further characterized using Soxhlet extraction methods, which represents a more harsh extraction method (acidic conditions).
Analysis by HPLC-RAM was performed on representative Soxhlet extracts. Cfr. table further on in this RSS. A single polar transformation product was observed in the chromatograms of each soil, indicating that there was most likely no more parent material remaining in the non-extractable residue. However, since Soxhlet extraction is considered to be a harsh method (acidic conditions and elevated temperature), there is potential for conversion of parent to have occurred during this continuous extraction procedure under reflux.

ORGANIC MATTER FRACTIONATION
Radioactivity remaining in the non-extractable residue samples after the polar and non-polar extractions was characterized by fractionation into fulvic acid, humic acid, and humin fractions. Cfr. table further on in this RSS.
The results indicate that most of the radioactive residue (average of 25.3% AR in 12 °C systems and 28.6% AR in 20 °C systems) is associated with the insoluble humin fraction of the SOM. The majority of residue being in the insoluble fraction indicates that it is not bioavailable, which is also consistent with the results of the polar and non-polar extractions. The average amount of radioactive residue in the fulvic acid fraction is 10.4% AR and 11.5% AR in the 12 and 20 °C systems, respectively. This is the extent of the non-extractable residues that is potentially bioavailable in the environment since fulvic acid is soluble at all pH levels. The average amount of radioactive residue over both test temperatures in the humic acid fraction of 18 Acres and Krone soil is 9.7% AR and 8.7% AR, respectively. Since these two soils have acidic pH values, this fraction will be insoluble, thereby preventing the radioactive residues from going into solution. Therefore, this amount of residue associated with the humic acid fraction is not bioavailable. Gartenacker and Vetroz soils have slightly basic pH values (7.1 and 7.7, respectively), and the radioactive residue in their humic acid fractions will be correspondingly slightly potentially bioavailable in the environment.

VOLATILE RADIOACTIVITY AND CONFIRMATION OF 14CO2
Radioactivity trapped in ethylene glycol trapping solutions was considered volatile organic compounds (VOC), and radioactivity trapped in 1.0 M potassium hydroxide trapping solutions was considered 14CO2. Volatile organic compounds generally remained below the limit of detection and 14CO2 increased in all soil test systems over the course of the incubation period, up to a maximum of 22% AR. Cfr. table further on in this RSS. This is an indication of significant mineralization and non-persistent nature of the parent compound.
Select potassium hydroxide trapping solutions were chosen to further characterize the nature of the radioactive residues in these trapping solutions, using a barium chloride precipitation method. Representative potassium hydroxide traps, containing >10% AR, from selected sampling intervals were processed according to this procedure. The barium carbonate formed during processing is pelletized by centrifuging and the supernatant analyzed by LSC. The absence of radioactivity in the supernatants confirmed the nature of the precipitate as Ba14CO3.
These results confirmed that radioactivity in the potassium hydroxide trapping solutions was 14CO2 for each test system and incubation temperature.

DISTRIBUTION OF [14C]TRISTYRENATED PHENOL AND TRANSFORMATION PRODUCTS
The amount of test substance recovered at the end of incubation is slightly lower in the system incubated at 20 °C. This effect may be attributed to the higher mineralization rate observed and the increase in non-extractable residues in these systems, as previously cited. These differences are not necessarily considered significant nor based on soil type. Cfr. table further on in this RSS.
Several major regions of radioactivity (> 10% AR or > 5% AR in at least two consecutive intervals) were observed in some of the soil extract chromatograms for all test systems.
Several minor radioactive peaks were observed at random intervals during the HPLC-RAM analysis of the soil extracts. In all cases these individual peaks represented less than 10% AR, and were not investigated further.

IDENTIFICATION OF UNKNOWN TRANSFORMATION PRODUCTS
Several major transformation products were observed during the study. These transformation products were identified using LC-MS/MS analysis.

KINETICS
Selection for the best fit model that best describes each set of the experimental data was based on recommendation provided in the FOCUS Guidance (FOCUS, 2006). In general, a visual assessment of fitted and observed data versus time is considered. In addition to visual assessment, the error at which the χ2 test is passed at the 5% significance level with an error percent of < 15% is also considered.
Most of the results presented here pass the χ2 test with an error much smaller than 15%. Another criterion considered is the determination coefficient (r2) which should fall between 0.85 and 1.0. For most of these models, the r2 values fall in this range. For transformation product kinetics, an r2 ≥ 0.7 can also be acceptable.

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: 2.4% - 7.3% at 12 ± 2 °C; 1.4% - 7.3% at 20 ± 2 °C
- % of applied amount at end of study period: 43.5% - 51.6% at 12 ± 2 °C; 45.0% - 50.4% at 20 ± 2 °C

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 10.4% - 14.3% at 12 ± 2 °C; 17.5% - 22.0% at 20 ± 2 °C

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: 0.1% in only one case (Vetroz) at 20 ± 2 °C

STERILE TREATMENTS (if used): not applicable

Results with reference substance:

not applicable

The rate of transformation of [¹⁴C]tristyrenated phenol

Incubated at 12 ± 2°C

Soil Type	Parent DT50 (days)	Parent DT90 (days)	Max. NER (%AR)	Max. VOC (%AR)	Max.^14CO2 (%AR)
18 Acres	5.95	463	49.5	-	10.4
Gartenacker	7.66	104	46.2	-	13.6
Krone	3.13	111	51.6	-	14.3
Vetroz	12.5	407	43.5	-	11.8

“-” = < Limit of Detection (LOD); < 0.000671% AR

18 Acres Soil Test System
Transformation Product ID	Initial Observation		Maximum Observation		End of Incubation (% AR)
	Day	% AR	Day	% AR
Met 0	3	4.3	7	11.1	-
Met 5	7	4.2	29	5.3	2.6
Met 8	3	3.9	14	7.0	4.5

“-” = < Limit of Detection (LOD); < 1.82% AR

Gartenacker Soil Test System
Transformation Product ID	Initial Observation		Maximum Observation		End of Incubation (% AR)
	Day	% AR	Day	% AR
Met 0	3	4.4	21	7.3	-
Met 2	3	5.7	14	6.7	1.4
Met 4	7	2.6	60	6.3	3.3
Met 8	3	5.9	7	6.2	2.2

“-” = < Limit of Detection (LOD); < 1.82% AR

Krone Soil Test System
Transformation Product ID	Initial Observation		Maximum Observation		End of Incubation (% AR)
	Day	% AR	Day	% AR
Met 0	3	8.1	7	12.2	-
Met 2	3	5.3	3	5.3	3.2
Met 4	3	3.3	60	5.3	2.1
Met 8	3	5.6	7	6.4	3.9
Met 9	7	4.7	21	6.0	-

“-” = < Limit of Detection (LOD); < 1.82% AR

Vetroz Soil Test System
Transformation Product ID	Initial Observation		Maximum Observation		End of Incubation (% AR)
	Day	% AR	Day	% AR
Met 0	3	3.1	7	6.4	-
Met 8	3	3.9	14	6.8	3.9

“-” = < Limit of Detection (LOD); < 1.82% AR

Kinetic Results for Transformation Products at 12 °C
Transformation Product	Soil	Kinetic Model	Parameter Estimates	chi²	r^2	DT50 (days)	DT90 (days)
Met 0	18 Acres	DFOP	M0 = 101.2 K1 = 0.03483	30.1	0.7267	19.9	66.1
	Gartenacker	DFOP	M0 = 96.36 K1 = 0.03407	25.1	0.7296	20.3	67.6
	Krone	SFO	M0= 101.8 K = 0.0224	19	0.8542	30.9	103
	Vetroz	FOMC	M0 = 98.29 K = 0.0314	18.4	0.8358	22.1	73.3
Met 2	Gartenacker	DFOP	M0 = 96.36 K1= 0.04455	11.1	0.8767	15.6	51.7
	Krone	FOMC	M0 = 94.79 K = 0.04165	26.2	0.6255	16.6	55.3
Met 4	Gartenacker	SFO	M0 = 78.65 K = 0.01086	11.3	0.9582	63.8	212
	Krone	DFOP	M0 = 95.43 K1= 0.007372	11.7	0.8543	94	312
Met 5	18 Acres	DFOP	M0 = 101.2 K1= 0.02673	28.5	0.6967	25.9	86.2
Met 8	18 Acres	DFOP	M0 = 101.2 K1= 0.04398	40.3	0.3887	15.8	52.4
	Gartenacker	FOMC	M0 = 96.74 K = 0.02667	13.4	0.8708	26	86.3
	Krone	SFO	M0 = 101.8 K = 0.006292	17.1	0.6805	110	366
	Vetroz	DFOP	M0 = 98.67 K1 = 0.01399	10.6	0.8722	49.5	165
Met 9	Krone	DFOP	M0 = 95.02 K1 = 0.04712	17.1	0.896	14.7	48.9

Incubated at 20 ±

Soil Type	Parent DT50 (days)	Parent DT90 (days)	Max. NER (%AR)	Max. VOC (%AR)	Max.^14CO2 (%AR)
18 Acres	3.65	200	50.4	-	17.5
Gartenacker	8.56	45.5	48.5	-	20.1
Krone	2.88	55.2	48.7	-	22.0
Vetroz	3.73	51.5	50.1	0.1	20.1

“-” = < Limit of Detection (LOD); < 0.000671% AR

18 Acres Soil Test System
Transformation Product ID	Initial Observation		Maximum Observation		End of Incubation (% AR)
	Day	% AR	Day	% AR
Met 8	3	6.8	3	6.8	3.2

 

Gartenacker Soil Test System
Transformation Product ID	Initial Observation		Maximum Observation		End of Incubation (% AR)
	Day	% AR	Day	% AR
Met 2	3	4.9	21	6.3	2.5
Met 3	3	1.8	29	5.5	2.0
Met 4	3	2.8	21	7.4	3.4
Met 8	3	4.7	14	5.6	1.8

 

Krone Soil Test System
Transformation Product ID	Initial Observation		Maximum Observation		End of Incubation (% AR)
	Day	% AR	Day	% AR
Met 0	3	4.4	14	8.9	1.2
Met 1	3	3.8	7	5.5	1.0
Met 2	3	5.3	7	6.1	1.0
Met 4	3	3.5	29	6.9	1.4
Met 8	3	5.4	7	5.8	1.3
Met 9	3	2.1	14	7.2	-

“-” = < Limit of Detection (LOD); < 1.82% AR

Vetroz Soil Test System
Transformation Product ID	Initial Observation		Maximum Observation		End of Incubation (% AR)
	Day	% AR	Day	% AR
Met 2	3	5.6	7	6.1	1.6
Met 4	3	4.3	14	7.3	2.8
Met 8	3	6.1	7	6.3	1.4

Kinetic Results for Transformation Products at 20 °C
Transformation Product	Soil	Kinetic Model	Parameter Estimates	chi²	r^2	DT50 (days)	DT90 (days)
Met 0	Krone	SFO	M0 = 92.39 K = 0.04491	14.7	0.8188	15.4	51.3
Met 1	Krone	DFOP	M0 = 99.05 K1= 0.03913	16.7	0.7991	17.7	58.9
Met 2	Gartenacker	SFO	M0 = 94.66 K = 0.05699	19.4	0.8234	12.2	40.4
	Krone	DFOP	M0 = 99.05 K1= 0.01966	16.6	0.7427	35.3	117
	Vetroz	DFOP	M0 = 102.7 K1 = 0.0907	19.6	0.7759	7.64	25.4
Met 3	Gartenacker	SFO	M0 = 94.1 K = 0.01947	9.82	0.9439	35.6	118
Met 4	Krone	FOMC	M0 = 94.6 K = 0.01483	9.6	0.9280	46.7	155
	Gartenacker	SFO	M0 =94.66 K = 0.01737	11.5	0.9275	39.9	133
	Vetroz	FOMC	M0 = 103.3 K = 0.01465	7.12	0.9235	47.3	157
Met 8	18 Acres	DFOP	M0 = 106.1 K1= 0.008348	19.5	0.6485	83.0	276
	Gartenacker	SFO	M0 = 94.1 K = 0.09508	18.2	0.8616	7.29	24.2
	Krone	FOMC	M0 = 94.6 K = 0.1449	32.6	0.7735	4.78	15.9
	Vetroz	DFOP	M0 = 102.7 K1= 0.03951	6.48	0.9311	17.5	58.3
Met 9	Krone	SFO	M0 = 92.39 K = 0.09734	12.2	0.8638	7.12	23.7

 Material Balance

Incubation Temperature	Soil	Min. Average (%AR)	Max. Average (%AR)	Overall Average (%AR)
12 ±2°C	18 Acres	95.8	102.7	100.4
	Gartenacker	94.7	103.1	100.4
	Krone	97.7	102.8	101.1
	Vetroz	97.2	102.4	100.9
20 ±2°C	18 Acres	101.0	107.2	103.7
	Gartenacker	96.0	106.6	103.3
	Krone	95.9	105.9	103.2
	Vetroz	97.2	107.0	103.0

Soil Extracted Residues

Incubation Temperature	Soil	Incubation Start Average (%AR)	Incubation End Average (%AR)
12 ±2°C	18 Acres	100.4	40.9
	Gartenacker	96.7	38.3
	Krone	94.8	33.1
	Vetroz	97.3	43.8
20 ±2°C	18 Acres	105.8	33.4
	Gartenacker	101.7	28.2
	Krone	98.6	28.9
	Vetroz	103.4	27.5

Non-Extractable Residues

Incubation Temperature	Soil	Incubation Start Average (%AR)	Incubation End Average (%AR)
12 ± 2 °C	18 Acres	2.4	49.5
	Gartenacker	6.4	46.2
	Krone	7.3	51.6
	Vetroz	4.8	43.5
20 ± 2 °C	18 Acres	1.4	50.4
	Gartenacker	4.1	47.7
	Krone	7.3	45.0
	Vetroz	3.6	50.1

Soxhlet Extraction

Incubation Temperature	Test Day	Sample ID	Available NER (% AR)	Soxhlet (% AR)
12 ± 2 °C	120	18 Acres	38.5	8.8
	120	Gartenacker	45.8	9.8
	120	Krone	51.2	9.3
	120	Vetroz	42.4	6.0
20 ± 2 °C	59	18 Acres	48.3	11.1
	59	Gartenacker	48.8	11.5
	121	Krone	48.4	10.2
	121	Vetroz	50.6	5.8

Organic Matter Factionation

Incubation Temperature	Day	Sample ID	Soil pH	Available NER (% AR)	Fulvic Acid (% AR)	Humic Acid (% AR)	Humin (% AR)
12 ± 2 °C	120	18 Acres Soil	5.7	38.5	9.1	9.5	19.9
	120	Gartenacker Soil	7.1	45.8	9.9	8.4	27.6
	120	Krone Soil	6.6	51.2	13.3	9.2	28.7
	120	Vetroz Soil	7.7	42.4	9.3	8.2	24.9
20 ± 2 °C	59	18 Acres Soil	5.7	48.3	10.3	9.9	28.1
	59	Gartenacker Soil	7.1	48.8	11.9	8.9	28.0
	121	Krone Soil	6.6	48.4	13.1	8.1	27.2
	121	Vetroz Soil	7.7	50.6	10.6	9.0	31.1

Distribution of [14C]Tristyrenated phenol and transformation products

Incubation Temperature	Soil	Average [14C]Tristyrenated Phenol at Incubation Start (%AR)	Average [14C]Tristyrenated Phenol at Incubation End (%AR)
12 ± 2 °C	18 Acres	100.4	20.6
	Gartenacker	96.7	12.5
	Krone	94.8	11.3
	Vetroz	97.3	18.2
20 ± 2 °C	18 Acres	105.8	15.6
	Gartenacker	101.7	4.7
	Krone	98.6	4.8
	Vetroz	103.4	3.9

Conclusions:

[14C]Tristyrenated phenol transformed rapidly over the incubation period under the conditions of this aerobic soil study, with DT50 ranging from 3.13 to 12.5 days at 12 °C and 2.88 to 8.46 days at 20 °C.
The rapid degradation of [14C]Tristyrenated phenol combined with a significant amount (≤ 22% AR) of 14CO2 generated over the course of the incubation period, is an indication of significant mineralization and non-persistent nature of the parent compound.

Executive summary:

In this study the rate and route of transformation of [14C]tristyrenated phenol was studied in four European soils, a sandy loam (18 Acres, from the United Kingdom), a silt loam (Gartenacker, from Switzerland), a silt loam (Krone, from the United Kingdom), and a loam (Vetroz, from Switzerland). The study was performed according to OECD TG 307 and in compliance to GLP.

Test systems consisted of 100 g soil (dry weight equivalent) aliquots, which were adjusted to a moisture level of approximately pF 2.5, connected to volatile traps, and incubated under aerobic conditions in the dark at 12 ± 2 °C and 20 ± 2 °C. [14C]Tristyrenated phenol was applied at a nominal concentration of 0.6 μg/g.

Soil samples incubated at 12 ± 2 °C were analyzed at 0, 3, 7, 14, 21, 29, 60, and 120 days of incubation. Soil samples incubated at 20 ± 2 °C were analyzed at 0, 3, 7, 14, 21, 29, 59, and 121 days of incubation. One soil, 18 Acres, incubated at 12 ± 2 °C and 20 ± 2 °C was sampled at an additional interval of 180 and 178 days, respectively, in an attempt to better characterize the decline of the test substance.

Soil samples were extracted according to the extraction method and analyzed by LSC and HPLC-RAM for determination and profiling of extractable residues. The post-extraction soils were combusted and analyzed by LSC for determination of non-extractable residues. The volatile traps were analyzed by LSC for determination of 14CO2 and volatile organics. Representative post-extraction soil samples were additionally characterized by harsh extraction and organic matter fractionation. Representative volatile traps were additionally characterized by barium chloride precipitation.

Average material balance ranged from 94.7 to 103.1% AR and 95.9 to 107.2% AR over the course of the study for the 12 ± 2 °C and 20 ± 2 °C test systems, respectively. All validity criteria were fulfilled, all amendments do not affect the final results, and in the end, the study results are considered valid.

[14C]Tristyrenated phenol transformed rapidly over the incubation period under the conditions of this aerobic soil study, with DT50 ranging from 3.13 to 12.5 days at 12 °C and 2.88 to 8.46 days at 20 °C. The range of DT50 values calculated for the soil incubated at 12 °C did not differ significantly from the values obtained for the soil incubated at 20 °C. The expectation was that [14C]Tristyrenated phenol would degrade faster at a higher temperature, but this was not observed. However, the decline of formed degradation products was generally more rapid at 20 °C, especially for Metabolite 8. The rapid degradation of [14C]Tristyrenated phenol combined with a significant amount (≤ 22% AR) of 14CO2 generated over the course of the incubation period, is an indication of significant mineralization and non-persistent nature of the parent compound.

The amount of non-extractable residues increased to a maximum of 51.6% AR. The additional extractions with THF and hexane did not remove appreciable amounts of residues. Characterization by organic matter fractionation demonstrated that most of the non-extractable residue is associated with the insoluble humin fraction of the SOM; therefore, it is not bioavailable.

Description of key information

The rate and route of transformation of [14C]tristyrenated phenol (TSP) was studied in four European soils: a sandy loam (18 Acres, from the United Kingdom), a silt loam (Gartenacker, from Switzerland), a silt loam (Krone, from the United Kingdom), and a loam (Vetroz, from Switzerland). The radiolabeled carbon atom in [14C]TSP is located randomly at one of the 6 possible locations in the central phenol ring. The study was performed according to OECD TG 307 and in compliance to GLP.

The test was run in the dark, under aerobic conditions, at two test temperatures (12 ± 2 °C and 20 ± 2 °C) using a nominal concentration of 0.6 μg/g applied to 100 g of soil. The test duration was ca. 120 days for all soils except 18 Acres, which was continued up to ca. 180d. Tables compiling the results for the different soils are available in the RSS. The main findings are described below.

Ultimate degradation of the test item was monitored by means of 14CO2 evolution, which increased in all soil test systems over the course of the incubation period, up to a maximum of 14.3% of applied radioactivity in the test at 12°C and up to 22% of applied radioactivity in the test at 20°C. Small but nevertheless significant mineralization of the test item thus is demonstrated.

Decrease of TSP concentration was monitored by means of mild extraction of the soil samples. At incubation start, the radioactivity was (almost) completely extractable from the soil samples (ranging from 94.8% to 105.8%). The radioactive material was confirmed to be the parent chemical, TSP, thus demonstrating that the (mild) extraction protocol does not lead to conversion / transformation of the parent substance. At the end of the incubation period, 11.3 -20.6% and 3.9-­15.6% of applied radioactivity was still present as parent TSP in the test system at 12°C and 20°C, respectively.

Primary degradation of the test item was also monitored by means of examination of the mild extraction fractions. Metabolites that were formed at concentrations >10% of applied radioactivity, or >5% in at least 2 consecutive intervals, were identified. The identified primary metabolites are in general oxidation products, i.e. containing additional hydroxyl groups. At day 3 after incubation, up to 22.3% and 22.0% of applied radioactivity was identified as primary metabolites in the 12°C and 20°C experiment, respectively. This indicates that primary degradation of TSP starts quickly after application of the test item to the soil. In total, 9 metabolites were investigated and potential chemical structures were proposed where possible.

Any radioactivity not removed during the (mild) extraction process was considered ‘Non-Extractable Residue’ (NER). The extracted soil samples were divided in two parts and submitted either to Soxhlet extraction or to Organic Matter Fractionation (OMF).

Soxhleting allowed to further extract 5.8-­11.5% of applied radioactivity from the soil samples. Characterisation of this fraction identified it as polar transformation product. However, as Soxhlet is a harsh technique, it cannot be excluded that conversion of parent chemical occurred during the process.

The organic matter fractionation separates the fulvic acid, humic acid and humin fractions of the soil organic matter. For the TSP soil samples, the radioactivity was found to be mainly (present in the insoluble humin fraction: 19.9-­31.1% of applied radioactivity. This indicates that it is not bioavailable.

Finally, kinetic modelling was performed in order to obtain the transformation/dissipation rate of [14C]TSP and its major transformation products. All calculations were carried out by means of the TESSELLA model CAKE v3.3 (Computational Assisted Kinetic Evaluation). Three kinetic models were used: i) Single first-­order (SFO), ii) Biphasic double first-­order (DFOP) and iii) First-order multi-­compartment (FOMC).

[14C]Tristyrenated phenol was found to transform rapidly over the incubation period under the conditions of this aerobic soil study, with DT50 ranging from 3.13 to 12.5 days at 12 °C and 2.88 to 8.46 days at 20 °C.

The DT50 values of the identified transformation products were also determined by means of the same kinetic model, and ranged from 14.7 to 110 days at 12°C and from 4.78 to 83 days at 20°C.

Conclusion:

The rapid degradation of [14C]Tristyrenated phenol, with the formation of several metabolites, combined with a significant amount (≤ 22% of applied radioactivity) of 14CO2 generated over the course of the incubation period, is an indication of significant mineralization and non-persistent nature of the parent compound.

The amount of non-­extractable residues increased to a maximum of 51.6% AR. Characterization by organic matter fractionation demonstrated that most of the non-extractable residue is associated with the insoluble humin fraction of the SOM; therefore, it is not bioavailable.

The DT50 of TSP is determined to be ranging from 3.13 to 12.5 days at 12 °C and 2.88 to 8.46 days at 20 °C. The DT50 values of the identified transformation products ranged from 14.7 to 110 days at 12°C and from 4.78 to 83 days at 20°C.

Additional information

For 2,4,6­tristyryl phenol (TSP), an aerobic mineralization test in surface water (OECD 309) was requested, if technically feasible. If the OECD 309 test is found to be not feasible, ECHA’s Final Decision Letter provides a soil simulation test (OECD 307) or a sediment simulation test (OECD

308) as alternative options.

The registrants have assessed the technical feasibility of the OECD 309 test, and concluded on the grounds described below, that such a test is not technically feasible, and that, as a result of the anticipated technical difficulties, the outcome of such an OECD 309 test would not contribute to a better understanding of the persistence properties of TSP.

According to OECD Guideline 309, “the principal objective of the simulation test is to determine the mineralisation of the test substance in surface water, and mineralisation constitutes the basis for expressing degradation kinetics. However, an optional secondary objective of the test is to obtain information on the primary degradation and the formation of major transformation products. Identification of transformation products, and if possible quantification of their concentrations, are especially important for substances that are very slowly mineralised (e.g. with half-­lives for total residual 14C exceeding 60 days). Higher concentrations of the test substance (e.g., >100 μg/l) should normally be used for identification and quantification of major transformation products due to analytical limitations.”

In the registration dossier, a ready biodegradation test in accordance with OECD Guideline 301B (modified Sturm test) is available. The test item used for this ready biodegradation test consists of ca. 80% TSP and 20% DSP. In this test, less than 1% mineralization (CO2 evoluation) was noted after 28 days. These results clearly indicate the slow mineralization rate for TSP. As quoted above, the OECD 309 Guideline in this case advises that it is important to assess primary degradation, by means of identification and – where possible – quantification of major transformation products.

For the assessment of (ultimate) degradation kinetics, the OECD 309 guideline prescribes a test concentration of < 1 μg/L in order to ensure that the biodegradation follows first order kinetics. However, when intending for the identification of major transformation products in an OECD 309 test, the test should be run at a higher test concentration. According to the guideline, a concentration > 100 μg/L or sometimes even > 1000 μg/L should be used, due to the analytical limitations related to chemical structure identification techniques. However, 2,4,6-­Tristyrenated phenol has an experimentally determined water solubility of 7.07 μg/L. Thus, it is not feasible to obtain sufficiently high test item concentrations in order to allow for the evaluation of primary degradation by means of an OECD 309 test.

The PBT criteria as described in Annex XIII refer to the degradation half-­life of a substance in the different compartments. As further clarified in ECHA Guidance R11: “Degradation may be biotic and/or abiotic (e.g. hydrolysis) and result in complete mineralisation, or simply in the transformation of the parent substance (primary degradation). Where only primary degradation is observed, it is necessary to identify the degradation products and to assess whether they possess PBT/vPvB properties.”

Hence, it is clear that the goal of the requested persistence testing should be to identify a degradation half-­life for the parent substance (TSP), and – in case primary degradation is observed, but not complete mineralization – to identify the degradation products and examine their persistence.

Based on the above, the registrants concluded that:

i) It is technically impossible to assess the primary degradation potential of the parent substance TSP by means of an aqueous OECD 309 test due to the insufficient solubility of the test item in water.

ii) Assessment of the ultimate degradation potential of TSP by means of an OECD 309 test at low test concentration – although maybe technically possible – will not be of added value to the PBT assessment. The slow mineralization of the test item has already been confirmed in the available ready biodegradation test (OECD 301B).