2,6-di-tert-butyl-p-cresol

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

Study period:

02.06.2017 - 16.12.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Due to the tendency of the test substance to be lost by evaporation in typically used flow-through test systems, the guideline had to be adapted and a closed system has been used.

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Principles of method if other than guideline:

Due to the tendency of the test substance to be lost by evaporation in typically used flow-through test systems, the guideline had to be adapted and a closed system has been used.

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Specific details on test material used for the study:

14C-labelled test item:
Chemical Name: 2,6-di-tert-butyl-4-methyl[U-14C]phenol
Specific activity: 2.22 GBq/mmol corresponding to 9.9865 MBq/mg
Molecular weight: 222.3 g/mol (at this specific activity)
Radiochemical purity: 98.5 % (CoA)
Label position: benzene ring
Impurities: not determined

Radiolabelling:

yes

Remarks:

14C-labelling in the phenyl ring

Oxygen conditions:

aerobic

Soil classification:

not specified

Year:

2017

Soil no.:

#1

Soil type:

loamy sand

% Clay:

6

% Silt:

20

% Sand:

74

% Org. C:

0.93

pH:

5.7

CEC:

16 other: mmol/kg

Soil no.:

#2

Soil type:

silt loam

% Clay:

16

% Silt:

78

% Sand:

6

% Org. C:

0.95

pH:

6.6

CEC:

47 other: mmol/kg

Soil no.:

#3

Soil type:

loam

% Clay:

27

% Silt:

41

% Sand:

32

% Org. C:

2.04

pH:

7.4

CEC:

265 other: mml/kg

Soil no.:

#4

Soil type:

clay

% Clay:

41

% Silt:

35

% Sand:

24

% Org. C:

1.78

pH:

7.2

CEC:

257 other: mmol/kg

Details on soil characteristics:

The soils were sieved < 2 mm before the start of the experiments and analysed.
Before start of the experiments the moisture content was adjusted to about 45 % of its maximum water holding capacity (= WHCmax). Before application the soils were preincubated at 12 +- 2°C in the dark. Immediately before the removal of soil for sample preparation and during the pre-incubation the water content was checked and adjusted if necessary.

Further soil properties: maximum water holding capacity (WHC(max))
Soil no. #1 (refeSol 01-A): 293 g/kg
Soil no. #2 (refesol 02-A): 416 g/kg
Soil no. #3 (LUFA 2.4): 446 g/kg
Soil no. #4 (LUFA 6S): 416 g/kg

Characterisation of the microbial status of the soils
Biomass measurements of the four soils were performed by means of the substrate induced respiration method described in section 4.5.9. Microbial biomass measurement before incubation was determined in untreated samples only. Microbiological status expressed as biomass in mg microbial carbon per kg soil was measured to be 214 mg Cmic/kg dry mass (RefeSol 01-A), 237.8 mg Cmic/kg (RefeSol 02-A), 550.8 mg Cmic/kg (LUFA 2.4) and 1005.6 mg Cmic/kg dry mass (LUFA 6S). Correlated to the organic carbon (Corg) content of the soils (see Table 1) this was corresponding to a Cmic/Corg rate of 2.3% (RefeSol 01-A, RefeSol 01-A), 2.7 % (LUFA 2.4) and 5.6 % (LUFA 6S). The value indicated a normal microbial activity of the soil.
The microbial biomass status during the incubation was carried out in the beginning, in the mid and in the end of the aerobic incubation. Table 8 presents the results of biomass measurement of all soils during the incubation period. Microbial biomass was determined in untreated samples, in samples treated with the organic solvent acetonitrile and in samples treated with 2,6-di-tert-butyl-p-cresol (nominal 25 µg/50 g soil dry mass). Microbiological status is expressed as biomass in mg microbial carbon per kg soil in the following table as mean of three replicates.
The microbial biomass in the soil samples remained relatively stable throughout the incubation period except for RefeSol 02-A where a decreased biomass was determined after 120 days of incubation especially for the treated samples. A decreasing microbial activity at the end of the 120 d incubation time is commonly seen in soil batch tests.
The results of microbial biomass show the existence of an active microbial population throughout the incubation period. No significant adverse effect on microorganism activity by application of the solvent or 2,6-di-tert-butyl-p-cresol was observed

Table 8: Microbial biomass determined by means of substrate induced respiration method during the study.
Mean values of three replicates.
Soil type Soil sample Biomass [mg Cmic/kg dry mass]
2d 62d 120d
RefeSol 01-A Non-treated 317 163 114
Treated with solvent 674 212 81
Treated with test item 426 187 106
RefeSol 02-A Non-treated 201 168 161
Treated with solvent 701 812 45
Treated with test item 426 1084 29
LUFA 2.4 Non-treated 371 503 383
Treated with solvent 651 497 519
Treated with test item 618 549 481
LUFA 6S Non-treated 9 81 706 692
Treated with solvent 1623 1461 1084
Treated with test item 1244 1483 1149

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.:

0.5 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#2

Initial conc.:

0.5 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#3

Initial conc.:

0.5 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#4

Initial conc.:

0.5 mg/kg soil d.w.

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

test mat. analysis

Soil No.:

#1

Temp.:

12+-2°C

Humidity:

45 % WHCmax

Microbial biomass:

214 mg Cmic/kg soil dw

Soil No.:

#2

Temp.:

12+-2°C

Humidity:

45 % WHCmax

Microbial biomass:

265.7 mg Cmic/kg soil dw

Soil No.:

#3

Temp.:

12+-2°C

Humidity:

45 % WHCmax

Microbial biomass:

531.8 mg Cmic/kg soil dw

Soil No.:

#4

Temp.:

12+-2°C

Humidity:

45 % WHCmax

Microbial biomass:

938.7 mg Cmic/kg soil dw

Details on experimental conditions:

- Sterile control samples
For sterile controls, 50 g soil, based on dry weight, were weighed into glass vessels. Vessels were sterilized twice at an interval of 3 days by autoclaving for 20 minutes at 121 °C and 2 bar. The water content of the sterilized soil samples was adjusted after sterilization by means of sterilized water under a clean bench using sterilized equipment.

- Incubation conditions
The incubation of the applied soil samples was carried out in a temperature controlled room at a test temperature of 12 ± 2 °C.
Incubation was performed in glass vessels which were closed gas tight. Trapping of the exhaust gases was carried out by NaOH absorption traps integrated in the vessels and Tenax tubes. Oxygen sensors were placed within the gas phase of the vessels of three representative samples per soil. Depending on the measured oxygen concentration, the vessels were aerated manually to maintain optimal conditions for degradation by the soil microorganisms. No areation of the sterilized subsamples was performed.
Additional subsamples were incubated for the determination of microbial biomass during the incubation period. Incubation of soil subsamples for biomass determination was carried out in glass vessels which were closed gas tight but no trapping of gases was performed. Oxygen sensors were placed within the gas phase of the vessels of soil samples treated with 2,6-di-tert-butyl-p-cresol and of non-treated vessels of soils RefeSol 02-A, LUFA 2.4 and LUFA 6S.
Depending on the measured oxygen concentration, the vessels were aerated manually to maintain optimal conditions for degradation by the soil microorganisms.

- Absorption traps
Trapping of the exhaust gases was carried out by solutions of sodium hydroxide and Tenax tubes.
The sodium hydroxide absorption solution consisted of 5 mL 2 M NaOH with 5-15 drops of phenolphthalein indicator solution in glass vessels which were located inside the test vessels. When the phenolphthalein indicated a decrease of the pH-value of the absorption solution, the NaOH absorption traps were sampled and renewed by 5 mL of fresh solution. In addition, the absorption solutions were sampled at each sampling time. The total radioactivity in each solution was determined by LSC. The pH-value of the NaOH-absorption traps was determined by pH-indicator strips.
Tenax tubes were attached to the test vessels to trap organic volatile compounds. Tenax tubes were sampled at the respective sampling time and were extracted using in total 2 mL acetonitrile with 1% formic acid. The radioactivity in the acetonitrile with 1% formic acid eluate was determined by LSC.

- Characterization of the microbiological status of the soil
The soil samples were analysed for their actual microbial biomass during soil preparation as well as at the beginning, during and at the end of the incubation phase.
The actual microbial biomass was determined by the substrate induced respiration method according to the DIN procedure DIN ISO 14240-1 [6]. Three replicate samples of soil (50 g dry mass) were spiked with glucose and the respiration activity was determined by means of a respiration monitor


- Sampling
Sampling was performed after the following incubation times: 0 d (immediately after application), 3 d, 7 d, 14 d, 30 d, 62 d, 90 d and 120 d after application. Sterilised subsamples were taken after 62 d and 120 d. After sampling, the soil samples were extracted and worked-up immediately. In addition, the corresponding NaOH absorption traps were removed and the trapping solutions were analysed. The Tenax traps were extracted and analysed.
For biomass measurement untreated samples were taken during soil preparation and before application and non-treated samples , samples treated with acetonitrile and samples treated with 2,6-di-tert-butyl-p-cresol were taken at the beginning (2d), in the mid (62 d) and at the end of the incubation period (120 d).

- Sample processing
Soil extraction
The soil subsamples were transferred into glass centrifuge tubes. After that, 50 mL acetonitrile with 1% formic acid was used to rinse the test vessel and was then added to the glass centrifuge tube. The soil samples were extracted three times for 30 minutes on a horizontal shaker. Soil and solvent phase were separated by centrifugation for 10 minutes at 2000 rpm, and afterwards, the extracts were combined. The total radioactivity in the combined extracts was determined by LSC. Thereafter, aliquots were submitted to radio-HPLC and radio-TLC.
Soil samples of LUFA 6S were subsequently extracted using accelerated solvent extraction (ASE™) with acetonitrile with 1% formic acid. Extraction conditions were as follows: 100 bar, 100°C, 2 cycles, heat: 5 minutes, static time: 15 minutes. The total radioactivity in the combined extracts was determined by LSC. Thereafter, aliquots were submitted to radio-HPLC and radio-TLC.

Quantification of non-extractable radioactivity after the first extraction step (shaking)
The extracted soil samples were air dried in a fume hood and ground to an uniform consistency and then five replicates (100 - 300 mg each) of each soil sample were combusted using an Oxidizer. The resulting 14CO2 was trapped in Oxysolve C-400 and afterwards quantified by LSC. For that purpose, each vial (volume: 20 ml) was measured for 5 minutes by means of a Packard Tri-Carb scintillation analyser. The efficiency of oxidation was determined by combustion of quality control standards. Using the mass data and the LSC data the radioactivity remaining in the soil after the extraction procedure described above was calculated for each sample.

Additional extraction by ASE™
Aliquots of approximately 6 g (LUFA 6S) or 10 g (RefeSol 01-A, RefeSol 02-A, LUFA 2.4) of the soil residue was transferred into ASE™ extraction tubes of stainless steel and subjected to an ASE™ after addition of diatomaceous earth. The samples were extracted with acetonitrile with 1% formic acid (all soils except LUFA 6S; 100°C, 100 bar, 2 cycles, heat: 5 minutes, static time: 15 minutes) and subsequently with acetone (all soils; 100°C, 100 bar, 2 cycles, heat: 5 minutes, static time: 15 minutes) and acetonitrile : water 50:50 (v:v) (all soils; 40°C, 100 bar, 100°C, 2 cycles, heat: 5 minutes, static time: 15 minutes). In order to check for potential higher extraction portions representative samples of all soils (replicates 3d-1, 62d-2, 120d-1 and 120d-2 sterile) were additionally extracted with methanol : acetone : water 2:1:1 (v:v:v) (40°C, 100 bar, 100°C, 2 cycles, heat: 5 minutes, static time: 15 minutes) and acetonitrile : ammonium hydroxide pH 9 50:50 (v:v) (40°C, 100 bar, 100°C, 2 cycles, heat: 5 minutes, static time: 15 minutes).
The extracts were separately analysed by LSC. ASE™-extracts 1 and 2 (acetonitrile with 1% formic acid extract and acetone extract) were combined and were subjected to radio-HPLC analysis.
The remaining soil samples were air dried in a fume hood and ground to a uniform consistency and then five replicates (100 - 200 mg each) of each soil sample were combusted using an Oxidizer. The resulting 14CO2 was trapped in Oxysolve C-400 and afterwards quantified by LSC. For that purpose, each vial (volume: 20 ml) was measured for 5 minutes by means of a Packard Tri-Carb scintillation analyser. The efficiency of oxidation was determined by combustion of quality control standards.

Sample preparation for radio-HPLC analysis
Appropriate aliquots of the acetonitrile with 1% formic acid extracts (obtained by shaking during the first extraction step) were concentrated before radio-HPLC analysis. Aliquots of acetonitrile with 1% formic acid extracts were evaporated to a residual volume of ca. 100 µL – 750 µL under a gentle stream of nitrogen. The volume of the residual solution was determined and the concentrated sample was filled up to 1 mL using acetonitrile with 1% formic acid. The resulting solution was homogenised thoroughly and the total radioactivity was analysed by LSC before radio-HPLC analysis.
Radioactivity recovered after this concentration step was always > 90 % except for six replicates (procedural recovery in the range of 84.1 % - 89.8 %). HPLC column recovery was carried out for representative samples (in total 28 replicates). The column recoveries were all in the range of 89.7 % - 105.7 %.
Appropriate aliquots of the ASE™ extracts 1 of soil LUFA 6S (acetonitrile with 1% formic acid extracts) were also concentrated before radio-HPLC analysis. Aliquots of ASE™ extracts 1 of soil LUFA 6S were evaporated to a residual volume of ca. 150 µL – 200 µL under a gentle stream of nitrogen. The volume of the residual solution was determined and the concentrated sample was filled up to 1 mL using acetonitrile with 1% formic acid. The resulting solution was homogenised thoroughly and the total radioactivity was analysed by LSC before radio-HPLC analysis.
Radioactivity recovered after this concentration step was in the range of 70.1 % - 92.9 %. HPLC column recovery was carried out for representative samples of the sampling times 7 days, 30 days, 62 days, 90 days and 120 days (in total 7 replicates). The column recoveries were in the range of 91.0 % - 99.4 % except for the sterile samples which were 86.4 % and 88.1 %, respectively.

- Additional ASE™ extraction of non-extractable residues
Samples taken at incubation day 3 and thereafter were submitted to additional extraction steps using accelerated solvent extraction (ASE™). In addition, sterilised soil samples of 120 days of incubation were subjected to ASE™ extraction. Three subsequent extraction steps were performed using acetonitrile with 1 % formic acid (for soils RefeSol 01- A, RefeSol 02-A and LUFA 2.4), acetone (all soils) and acetonitrile:water (50:50, v:v). Representative replicates were further subjected to ASE™ extraction using subsequently methanol:acetone:water (2:1:1, v:v:v) and acetonitrile:ammonium hydroxide pH 9 (50:50, v:v).

Appropriate aliquots of the ASE™-extracts 1 and 2 (acetonitrile with 1% formic acid extract and acetone extract) were concentrated before radio-HPLC analysis for all soils. Aliquots of the acetone ASE™-extracts were evaporated to a residual volume of ca. 1 mL under a gentle stream of nitrogen. Afterwards, aliquots of the acetonitrile with 1% formic acid ASE™-extracts and 250 µL of water were added and evaporated under a gentle stream of nitrogen down to a final volume of 1mL. The resulting solution was homogenised thoroughly and the total radioactivity was analysed by LSC before radio-HPLC analysis.
Radioactivity recovered after this concentration step was in the range of 62.2 % and 91.3 % except for one replicate which was 47.7 % (sterile sample 120 d-2 of soil RefeSol 02-A). However, losses of radioactivity during this concentration step represents only ≤ 4.3 % AR.
HPLC column recovery was carried out for a representative sample (RefeSol 01-A, 3 d-1). The column recovery was 97.3 %.

Sample preparation for radio-TLC analysis
Aliquots of extracts were subjected to radio-TLC analysis without any further preparation step.

Characterisation of NER type I (by silylation)
The procedure was based on a draft guidance document (ECHA 2018, “Proposal for amendment to ECHA guidance (Chapter R.11: PBT/vPvB assessment) Appendix R.11-X: Extraction steps and approach for characterizing NER types (I, II and III)”, 10 October 2018 [7]) and Kästner et al. 2018 [8].
The characterisation was carried out on representative samples of sampling date 120 days of all soils. For this reason, 2 x 0.5 g of the remaining soil after ASE extraction (replicate 120 d-2 of each soil) were weighed into schlenk flasks. The samples were then dried for 15 minutes at 105 °C in a drying oven. Afterwards, 1.5 g granulated sodium hydroxide, a stirring bar, 30 mL chloroform (dried over a molecular sieve) and 5 mL trimethylchlorosilane (TMCS) were added to each flask. Samples were then stirred at 500-600 rpm under an argon atmosphere for 3 hours. After this time period, 1.5 g sodium hydroxide and 5 mL TMCS were added once more. The samples were then stirred overnight under an argon atmosphere.
On the next day, the stirrer was switched off so that the supernatant could be removed by a Pasteur pipette. The supernatants were transferred into centrifuge tubes and were centrifuged for 10 minutes at 5 °C and 4000 rpm. Acetone (10 mL, dried over a molecular sieve) was added to the remaining soil in the schlenk flasks and the samples were stirred for 2 minutes. The supernatant was separated by centrifugation for 10 minutes at 5 °C and 4000 rpm. The acetone washing step was repeated twice. Afterwards, the remaining soil in the schlenk flask was washed with 30 mL chloroform by stirring for 2 minutes. The supernatant was separated by centrifugation for 10 minutes at 5 °C and 4000 rpm. All supernatants of one sample were combined and aliquots were analysed by LSC.
The combined supernatants were transferred into 100 mL pear shaped flasks and the solvent was evaporated by a rotary evaporator (40 °C, down to 40 mbar) until an oily remainder. The residue was redissolved in 8 mL chloroform. Aliquots of this solution were analysed by LSC and TLC.

- Mass balance
For each sample a mass balance was performed by summing the radioactivity detected in the sodium hydroxide trap, in the Tenax trap, in the organic soil extracts plus the radioactivity detected as non-extractable radioactivity. In addition, this sum was compared with the total radioactivity which had initially been applied to the samples determined by means of application controls (refer to section 4.5.5). According to this, soil samples were applied with 253.8 kBq/sample of [14C]-labelled 2,6-di-tert-butyl-p-cresol except for 0d-samples and sterile samples which were applied with 256.4 kBq/sample. The applied radioactivity corresponds to 101.6 % and 102.7 % of the target application rate, respectively.


- Distribution of radioactivity to compartments
The amounts of radioactivity (radiolabelled test item and transformation products) and its distribution in soil phase, volatile substances and non-extractable residues were calculated as % of initially applied radioactivity (AR).

- Distribution of radioactivity in non-volatile extractables of soil and organic volatiles to parent and metabolites
The amount of test item and metabolites at each sampling time was calculated from determined radioactivity in the extract (LSC) in combination with the relative distribution of parent compound and metabolites in the extract analysed by means of HPLC. The sum of each individual (parent of metabolite) gives the total amount of test item and metabolite at the respective sampling date as % of the initially applied radioactivity (AR) in each compartment.

- Calculation of the DT50/DT90 values
Based on the achieved data set, the calculation of rate constants and DT50/DT90 values of 2,6-di-tert-butyl-p-cresol and of its main metabolites in aerobic soil was calculated by means of the computer software “CAKE” version 1.4 (Release) running on R version 2.15.1 (2012-06-22).
The kinetics considered for all data were “single first order” (SFO), “First order multi compartment” (FOMC), "hockey stick (HS), and "Double first order in parallel" (DFOP) for the parent compound. In addition to the parent compound, the residues of five transformation products were calculated which reached at least in one soil 10% AR or two times 5 % AR. Since the optimisation tool is not able to handle five transformation products in parallel, two separate optimisations were performed.
Model 1 (see Figure 1) considers BHT-CH2OH (=A1) , BHT-CHO (=B1), BHT-COOH (=B2)
Model 2 (see Figure 2) considers BHT-OH (=A1), BHT-quinone (=B1)
The analysis for the metabolites were based on the best fit kinetics for the parent compound. For the metabolites always SFO (Single First Order) was considered. The structures of the models used are
attached to this IUCLID file under 'attached background material'.

Soil No.:

#1

% Recovery:

101.6

Remarks on result:

other: applied radioactivity after 0 d / overall recovery

Soil No.:

#1

% Recovery:

104.4

Remarks on result:

other: applied radioactivity after 120 d / overall recovery

Soil No.:

#2

% Recovery:

99.9

Remarks on result:

other: applied radioactivity after 0 d / overall recovery

Soil No.:

#2

% Recovery:

98.4

Remarks on result:

other: applied radioactivity after 120 d / overall recovery

Soil No.:

#3

% Recovery:

105.2

Remarks on result:

other: applied radioactivity after 0 d / overall recovery

Soil No.:

#3

% Recovery:

97.3

Remarks on result:

other: applied radioactivity after 120 d / overall recovery

Soil No.:

#4

% Recovery:

103.6

Remarks on result:

other: applied radioactivity after 0 d / overall recovery

Soil No.:

#4

% Recovery:

99.9

Remarks on result:

other: applied radioactivity after 120 d / overall recovery

Key result

Soil No.:

#1

DT50:

0.14 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Double First Order Parallel (DFOP)

Remarks:

results mathematically rounded

Key result

Soil No.:

#2

DT50:

0.523 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Double First Order Parallel (DFOP)

Remarks:

results mathematically rounded

Key result

Soil No.:

#3

DT50:

0.001 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Double First Order Parallel (DFOP)

Remarks:

results mathematically rounded

Key result

Soil No.:

#4

DT50:

0.595 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: Double First Order Parallel (DFOP)

Remarks:

results mathematically roundedn

Soil No.:

#1

DT50:

1.3 d

Type:

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

Temp.:

12 °C

Remarks on result:

other:

Remarks:

result mathematically rounded

Soil No.:

#2

DT50:

0.7 d

Type:

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

Temp.:

12 °C

Remarks on result:

other:

Remarks:

result mathematically rounded

Soil No.:

#3

DT50:

0.6 d

Type:

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

Temp.:

12 °C

Remarks on result:

other:

Remarks:

result mathematically rounded

Soil No.:

#4

DT50:

0.8 d

Type:

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

Temp.:

12 °C

Remarks on result:

other:

Remarks:

result mathematically rounded

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

No.:

#5

No.:

#6

Details on transformation products:

The metabolites were determined in the acetonitrile with 1% formic acid extracts of the soils and in the ASE extracts 1 and 2 (solvents acetonitrile with 1% formic acid and acetone). Determinations are based on radio-HPLC analysis of the extracts. Radio-HPLC was chosen as primary analytical method due to the sensitivity of the parent compound towards oxygen. HPLC ensured chromatographic conditions avoiding oxidation or transformation of the test item due to the extensive contact with atmospheric oxygen compared to TLC.

Evaporation of parent compound:

no

Remarks:

Test item and the metabolites: high vapour pressure. In pre-tests: in flow-through system a high portion of radioactivity was lost by evaporation (no subject to biodegradation in soil); thus, incubation in closed gas tight glass vessels ,

Volatile metabolites:

yes

Remarks:

Thus, the incubation was performed in glass vessels which were closed gas tight

Residues:

yes

Remarks:

By applying ASE extraction, relevant parts of the AR could be extracted using the first two ASE steps. Silylation of representative samples of NER remaining after ASE extraction was carried out to further characterise the NER.

Details on results:

Mass balance
For each sample a mass balance was performed by summing the radioactivity detected in the in the sodium hydroxide trap, in the Tenax trap, in the organic soil extracts plus the radioactivity detected as non-extractable radioactivity. In addition, this sum was compared with the total radioactivity which had initially been applied to the samples determined by means of application controls. According to this, soil samples were applied with 253.8 kBq/sample of [14C]-labelled 2,6-di-tert-butyl-p-cresol except for 0d-samples and sterile samples which were applied with 256.4 kBq/sample. The applied radioactivity corresponds to 101.6 % and 102.7 % of the target application rate, respectively.

Distribution of radioactivity to compartments
The amounts of radioactivity (radiolabelled test item and transformation products) and its distribution in soil phase, volatile substances and non-extractable residues were calculated as % of initially applied radioactivity (AR).

Distribution of radioactivity in non-volatile extractables of soil and organic volatiles to parent and metabolites
The amount of test item and metabolites at each sampling time was calculated from determined radioactivity in the extract (LSC) in combination with the relative distribution of parent compound and metabolites in the extract analysed by means of HPLC. The sum of each individual (parent of metabolite) gives the total amount of test item and metabolite at the respective sampling date as % of the initially applied radioactivity (AR) in each compartment.

Volatiles:
In the Tenax® traps of the samples, the radioactivity was always ≤ 0.5 % AR. In sterile samples slightly increased amounts of radioactivity were found in the range of 0.5 % AR and 1.3 % AR. Thus, only small amounts of the test substance or volatile metabolites with low molecular weight or high Henry constants volatilize during the aerobic degradation in the closed test system.
In the sodium hydroxide traps, radioactivity increased from 1.4 % AR - 2.1 % AR at 3 days up to a maximum of 17.8 % AR (LUFA 2.4) at the end of incubation of 28 days. In the other soils values of 5.4 % AR (RefeSol 01-A), 10.5 % AR (RefeSol 02-A) and 11.4 % (LUFA 6S) were detected in the sodium hydroxide traps until the end of incubation (mean values of two replicates). In the sodium hydroxide traps of the sterile samples radioactivity in amounts of 2.5 % AR and 4.2 % AR were detected. The results show that mineralization of the test item occurred during the aerobic incubation of 2,6-di-tert-butyl-p-cresol demonstrating complete degradation of the test item. However, also under abiotic conditions 14CO2 formation was detected in minor amounts indicating that also abiotic transformation led to a complete destruction of the parent molecule.

Extractable radioactivity:
The amount of radioactivity extracted from soil by acetonitrile with 1 % formic acid decreased severely in all soils from 94.9 % – 100.4 % AR at the beginning of incubation to 33.2 % – 44.5 % AR at day 3. Afterwards, the amount of extractable radioactivity remained relatively stable (soil RefeSol 02-A and LUFA 6S) or decreased slightly (soil RefeSol 01-A and LUFA 2.4) until the end of aerobic degradation. After 120 days of incubation the radioactivity in the soil extracts amounted to 23.7 % – 42.7 % AR.
In sterilised soil samples the extractable radioactivity remained relatively stable at levels comparable or slightly higher than the microbial active soil samples throughout the experiments: The radioactivity extractable with acetonitrile with 1 % formic acid was always in the range of 37.4 % - 43.3 % AR for both sampling time points (61 days and 120 days).

Non-extractable radioactivity (NER):
Generally, the amount of non-extractable radioactivity (NER) increased from 3.4 % - 8.7 % AR at day 0 to maximum values of 50.5 % (LUFA 6S) – 71.4 % AR (RefeSol 02-A) after the first 3 days of incubation. During the incubation the non-extractable radioactivity decreased continuously until the end of incubation time to values between 44.1 % (LUFA 6S) and 61.0 % AR (RefeSol 01-A).
In sterile soil samples radioactive amounts of radioactivity in the range of 53.4 % – 58.7 % AR were found as non-extractable radioactivity which are on levels higher (LUFA 6S), lower (RefeSol 01-A) or comparable to the microbial active samples (RefeSol 02-A, LUFA 2.4).

Additional ASE™ extraction of non-extractable residues
By applying ASE™ extraction amounts of non-extractable residues can be extracted additionally with each ASE™ step. It was found that most of the AR could be extracted using the first two ASE steps (using solvents acetonitrile with 1% formic acid and acetone) whereas additional steps yielded only in minor amounts of radioactivity.
The radioactivity found in total in the ASE™ extracts was always in the range of 9.2 % - 17.4 % ITR for the soils RefeSol 01-A, RefeSol 02-A and LUFA 2.4. Maximum amounts of radioactivity were liberated after 3 days of incubation (16.7 % - 17.4 % AR). Thereafter, decreasing amounts of radioactivity were found in the ASE™ extracts. After 120 days of incubation radioactivity levels in the range of 9.2 % - 13.1 % AR were quantified in the ASE™ extracts of the soils RefeSol 01-A, RefeSol 02-A and LUFA 2.4. In the case of soil LUFA 6S a ASE™ extraction using acetonitrile with 1 % formic acid was included in the validated sample extraction procedure due to the high sorption and binding capacity of this soil. Therefore, additional ASE™ extraction steps liberated lower levels of radioactivity in the range of 4.2 % to 6.7 % AR. At the end of incubation slightly decreased levels of radioactivity were detected in the ASE™ extracts.
The radioactivity found in the ASE™ extracts of the sterile sample was in a comparable range of 9.7 % - 11.9 % AR for soils RefeSol 01-A, RefeSol 02-A and LUFA 2.4 and of 3.4 % to 4.4 % AR for LUFA 6S.
Additional ASE™ extraction steps using methanol:acetone:water (2:1:1, v:v:v) and acetonitrile:ammonium hydroxide pH 9 (50:50, v:v) as solvents were carried out for representative replicates. These extraction steps liberated only minor amounts of radioactivity in the range of 0.7 % to 1.7 % (methanol:acetone:water extract) and 0.3 % - 0.6 % AR (acetonitrile:ammonium hydroxide pH 9), respectively.

Identification of extractable radioactivity
Determinations are based on radio-HPLC analysis of the extracts. The values are expressed in percent of the total initially applied radioactivity (AR). Radio-HPLC was chosen as primary analytical method due to the sensitivity of the parent compound towards
oxygen. HPLC ensured chromatographic conditions avoiding oxidation or transformation of the test item due to the extensive contact with atmospheric oxygen compared to TLC. Formation of metabolites showed the degradation of 2,6-di-tert-butyl-p-cresol in four soils.
During 120 days of incubation, the parent substance was degraded to several transformation products which were characterised by co-chromatography with reference standards of known transformation products or, if no reference compounds were available - by their retention time during HPLC analysis. By this way, the known transformation products BHT-CHO, BHT-OH, BHT-COOH, BHT-CH2OH and BHT-quinone were detected in the soil extracts as well as 1-3 unknown transformation products exceeding 10 % AR or two times 5 % AR which were specified as unassigned metabolites with the retention times of 2.5 min, 6.5 min and 22.0 min.
After 3 days of incubation the metabolites BHT-CHO and BHT-quinone were detected in maximum amounts of 5.4 % - 12.4 % AR. During the further incubation decreasing amounts of both metabolites were found in the soil extracts except for BHT-quinone in soil LUFA 6S which remained on a relatively constant level in the range of 7.3 % - 12.4 % AR. The known metabolite BHT-OH was detected in all soils at relatively constant levels between 4.9 % AR and 10.2 % AR except for soil LUFA 2.4 where decreasing amounts were observed after the maximum level of 8.9 % AR after 3 days of incubation until the end of incubation (2.8 % AR).
Maximum levels of BHT-COOH were found at incubation times of 7 to 30 days for soils RefeSol 02-A and LUFA 2.4. Thereafter, continuously decreasing levels of BHT-COOH were detected in these soils. In soil RefeSol 01-A maximum levels were detected between 62 days and 120 days.
During the continuing incubation the known metabolite BHT-CH2OH reached maximum amounts of 6.6 % to 8.8 % AR at later time points of 14 days to 30 days (soils RefeSol 02-A and LUFA 2.4) or 90 days in soil RefeSol 01-A. Afterwards amounts decreased continuously until the end of incubation.
1-3 unknown transformation products exceeding 10 % AR or two times 5 % AR were detected in soil extracts of all soils and were specified as unassigned metabolites with the retention times of 2.5 min, 6.5 min and 22.0 min. The retention times of the metabolites with the retention times 2.5 min and 6.5 min indicate the more polar characteristics of these transformation products compared to the parent compound and known metabolites. The unassigned metabolite with the retention time of 22.0 min was found exclusively in soil RefeSol 02-A in relevant amounts.

Determination of 2,6-di-tert-butyl-p-cresol

To determine the amount of unchanged parent compound, the acetonitrile with 1 % formic acid extracts as well as the ASE™ extracts 1 and 2 were analysed by means of radio-HPLC. The table shows the sum of these 4 extracts representing the extractable part (NER type 1) according to the draft guidance document (ECHA 2018, "Proposal for amendment to ECHA guidance (Chapter R.11: PBT/vPvB assessment) Appendix R.11-X: Extraction steps and approach for characterizing NER types (I, II and III)", 10 October 2018). The total amounts found were calculated in percent of the initially applied radioactivity (% AR).

 

 

Recovery of 2,6-di-tert-butyl-p-cresol in soil extracts of all four soils used in the study in % AR

Sampling time	RefeSol 01 -A	RefeSol 02 -A	LUFA 2.4	LUFA 6S
0d	99.2	95.7	101.4	95.8
3d	15.5	5.0	2.4	5.8
7d	15 .6	1.7	1.9	2.3
14d	18.4	4.6	1.8	3.6
30d	1.5	4.6	2.2	2.1
62d	12.8	0.7	0.7	n.d.
90d	6.2	2.2	1.6	n.d.
120d	8.0	1.6	0.9	n.d.
62d sterile	12.3	10.9	n.d.	n.d.
120d sterile	4.8	4.0	n.d.	n.d.

n.d.: not determinable

 

As can be seen in the table, the amount of parent compound in the soil extracts decreased rapidly from maximum levels between 95.7 % - 101.4 % AR immediately after application to amounts in the range of 2.4 % - 15.5 % AR during the first 3 days of incubation. Afterwards, decreased further to amounts between 8.0 % AR (RefeSol 01-A) to non-detectable levels (LUFA 6S) after 120 days of incubation.

In extracts of sterile soil samples 2,6-di-tert-butylp-p-cresol was found in amounts in the range of non-detectable levels (LUFA 2.4 and LUFA 6S) to 4.8 % AR at the end of the incubation period.

In the analysed ASE extracts 1 and 2 unchanged 2,6-di-tert-butyl-p-cresol could not be found.

 

Details on analytical results (individual measurements) used for CAKE input

Results for t-butyl-p-cresol (BHT)

Sampling time	Replicate	RefeSol 01-A	RefeSol 02-A	LUFA 2.4	LUFA 6S
		[% AR]
0d	1	93.5	101.0	100.1	94.0
	2	103.0	90.3	100.7	95.8
3d	1	15.6	4.9	2.7	5.8
	2	15.4	5.0	2.1	5.8
7d	1	16.4	0.0	1.9	2.3
	2	14.8	3.3	1.9	2.3
14d	1	17.9	5.3	2.4	3.2
	2	18.8	4.0	1.3	4.0
30d	1^1		5.0		2.4
	2	15.5	4.1	2.2	1.8
62d	1	9.8	1.4	1.4	0.0
	2	11.5	0.0	0.0	0.0
90d	1	9.0	2.8	1.4	0.0
	2	3.5	1.5	1.7	0.0
120d	1	7.8	2.0	0.8	0.0
	2	8.3	1.2	1.1	0.0

¹Sample of RefeSol 01-A and LUFA 2.4 were lost during the sample preparation procedure.

Metabolite BHT-CHO in soil treated with 2,6-di-tert-butyl-p-cresol determined in the acetonitrile with 1% formic acid soil extracts and ASE extracts 1 and 2.

Values are given in percent of the applied radioactivity (% AR).

Sampling time	Replicate	RefeSol 01-A	RefeSol 02-A	LUFA 2.4	LUFA 6S
		[% AR]
0d	1	n.d.	n.d.	n.d.	n.d.
	2	n.d.	n.d.	n.d.	n.d.
3d	1	8.5	6.2	7.2	5.6
	2	8.6	5.2	6.7	5.2
7d	1	4.1	2.8	4.2	2.9
	2	5.9	4.2	3.9	4.8
14d	1	4.0	3.5	3.8	4.0
	2	4.4	3.7	3.7	1.4
30d	1^1		1.8		2.3
	2	2.9	2.4	3.3	2.8
62d	1	1.6	0.6	1.2	1.7
	2	1.3	0.8	2.5	1.5
90d	1	5.1	n.d.	1.3	n.d.
	2	2.5	n.d.	0.9	n.d.
120d	1	5.1	2.0	0.8	0.8
	2	3.6	1.6	1.2	0.5

n.d. not detected

1 Sample of RefeSol 01-A and LUFA 2.4 were lost during the sample preparation procedure.

Metabolite BHT-OH in soil treated with 2,6-di-tert-butyl-p-cresol determined in the acetonitrile with 1% formic acid soil extracts and ASE extracts 1 and 2.

Values are given in percent of the applied radioactivity (% AR).

Sampling time	Replicate	RefeSol 01-A	RefeSol 02-A	LUFA 2.4	LUFA 6S
		[% AR]
0d	1	n.d.	n.d.	n.d.	n.d.
	2	n.d.	n.d.	n.d.	n.d.
3d	1	7.1	7.4	9.1	5.5
	2	8.7	7.6	8.6	6.3
7d	1	8.5	10.6	6.6	7.1
	2	7.2	7.2	6.9	6.5
14d	1	8.0	7.7	6.2	6.0
	2	6.7	8.1	5.7	7.3
30d	1^1		6.6		6.5
	2	8.5	6.7	4.3	6.5
62d	1	5.4	7.7	4.3	7.6
	2	6.0	8.3	2.6	8.6
90d	1	4.0	7.4	4.5	10.8
	2	5.9	7.4	5.4	9.5
120d	1	8.4	7.2	2.5	6.2
	2	9.9	6.4	3.2	7.7

n.d. not detected

1 Sample of RefeSol 01-A and LUFA 2.4 were lost during the sample preparation procedure.

Metabolite BHT-COOH in soil treated with 2,6-di-tert-butyl-p-cresol determined in the acetonitrile with 1% formic acid soil extracts and ASE extracts 1 and 2.

Values are given in percent of the applied radioactivity (% AR).

Sampling time	Replicate	RefeSol 01-A	RefeSol 02-A	LUFA 2.4	LUFA 6S
		[% AR]
0d	1	n.d.	n.d.	n.d.	n.d.
	2	n.d.	n.d.	n.d.	n.d.
3d	1	n.d.	6.4	5.8	4.3
	2	3.2	7.1	5.6	5.3
7d	1	5.9	8.1	8.4	8.9
	2	6.3	9.8	12.6	9.1
14d	1	4.5	14.2	4.4	9.5
	2	9.0	11.9	5.0	7.4
30d	1^1		11.8		10.8
	2	7.4	10.8	4.6	7.5
62d	1	5.7	8.3	3.2	n.d.
	2	9.8	8.5	1.9	8.5
90d	1	8.0	9.2	3.2	3.9
	2	n.d.	8.9	2.9	3.3
120d	1	9.7	7.7	2.4	2.1
	2	9.5	6.9	n.d.	2.7

n.d. not detected

1 Sample of RefeSol 01-A and LUFA 2.4 were lost during the sample preparation procedure.

Metabolite BHT-CH₂OH in soil treated with 2,6-di-tert-butyl-p-cresol determined in the acetonitrile with 1% formic acid soil extracts and ASE extracts 1 and 2.

Values are given in percent of the applied radioactivity (% AR).

Sampling time	Replicate	RefeSol 01-A	RefeSol 02-A	LUFA 2.4	LUFA 6S
		[% AR]
0d	1	n.d.	n.d.	n.d.	n.d.
	2	n.d.	n.d.	n.d.	n.d.
3d	1	2.1	3.1	8.1	4.3
	2	1.7	n.d.	4.7	3.6
7d	1	3.5	n.d.	5.4	3.7
	2	5.0	2.5	5.9	2.0
14d	1	0.7	3.3	7.0	7.9
	2	4.3	6.3	6.3	8.8
30d	1^1		7.8		5.9
	2	n.d.	9.8	1.3	5.7
62d	1	5.0	4.8	1.2	10.0
	2	4.1	4.2	3.4	n.d.
90d	1	6.0	4.1	n.d.	2.8
	2	7.2	3.3	n.d.	2.1
120d	1	3.9	2.9	n.d.	1.3
	2	4.3	2.9	2.4	1.7

n.d. not detected

1 Sample of RefeSol 01-A and LUFA 2.4 were lost during the sample preparation procedure.

Calculated DT50 and DT90 for the parent compound based on SFO kinetics.

      

Soil                            chi2                     r2        Prob. > t         DT50       DT90

                                    (%)                      (-)        k_deg              (d)        (d)

RefeSol 01-A              37.63              0.9563       0.001042        1.282       4.258

RefeSol 02-A              14.08              0.9963       1.662 ∙ 10-5       0.7056       2.344

LUFA 2.4               7.956              0.9995       4.692 ∙ 10-7       0.5588       1.856

LUFA 6S              9.734              0.9981       2.043 ∙ 10-9       0.7497       2.491

Calculated DT50 and DT90 for the parent compound based on DFOP kinetics.

      

Soil                            chi2       r2       Prob. > t k_deg       DT50       DT90

                                  (%)       (-)       fast rate                     (d)       (d)

RefeSol 01-A       6.273       0.9922       not given              0.1396       72.67

RefeSol 02-A       7.255       0.9943       0.0161                     0.5225       1.949

LUFA 2.4              2.284       0.9998       8.768 ∙ 10-24       0.001078       0.001078

LUFA 6S              3.809       0.9995       1.263 ∙ 10-7       0.5949       2.22

Geometric mean                            0.07      

Calculated DT50 and DT90 for the parent compound based on FOMC kinetics.

      

Soil                            chi2       r2              Prob. > t       DT50              DT90

                                  (%)       (-)              beta              (d)                     (d)

RefeSol 01-A       9.506       0.9876       0.3939       0.0112              86.32

RefeSol 02-A       7.409       0.994       0.4671       0.0001051              0.1174

LUFA 2.4              2.238       0.9998       0.4383       1.758 ∙ 10-6       0.003614

LUFA 6S              5.05       0.9992       0.1658       0.07615              1.349

According to the results there is evidence that the degradation follows biphasic kinetics. The results in bold characters (DFOP) should therefore be considered in further risk assessment. FOMC kinetics should not be used since the Prob.>t for the degradation parameter beta was above 0.1 which means that the optimised value for beta did not significantly differ from zero. The geometric mean DT50 of the recommended optimisation was found to be 0.07 days for the parent compound.

 

     

Metabolite BHT-quinone in soil treated with 2,6-di-tert-butyl-p-cresol determined in the acetonitrile with 1% formic acid soil extracts and ASE extracts 1 and 2.

Values are given in percent of the applied radioactivity (% AR).

Sampling time	Replicate	RefeSol 01-A	RefeSol 02-A	LUFA 2.4	LUFA 6S
		[% AR]
0d	1	n.d.	n.d.	n.d.	n.d.
	2	n.d.	n.d.	n.d.	n.d.
3d	1	9.0	8.7	7.4	6.9
	2	9.3	10.3	11.1	17.9
7d	1	4.8	5.3	7.2	8.5
	2	8.0	5.0	7.8	6.1
14d	1	2.9	4.7	5.6	8.8
	2	5.0	6.4	7.0	13.5
30d	1^1		3.9		7.9
	2	1.4	3.1	4.1	8.6
62d	1	2.4	3.2	3.0	9.5
	2	2.4	5.3	2.9	7.4
90d	1	1.8	4.5	2.4	11.7
	2	5.3	4.4	3.1	8.3
120d	1	2.1	5.9	4.7	12.6
	2	1.9	7.1	3.6	9.8

n.d. not detected

¹ Sample of RefeSol 01-A and LUFA 2.4 were lost during the sample preparation procedure.

Details on ASE Extraction

During the aerobic incubation of 2,6-di-tert-butyl-p-cresol the amounts of NER determined by combustion were between 41.4　% and 71.4　% AR throughout the incubation period of 3　days to 120　days.

Therefore, samples taken at incubation day 3 and thereafter were submitted to additional extraction steps using accelerated solvent extraction (ASE™). In addition, sterilised soil samples of 120 days of incubation were subjected to ASE™ extraction. Three subsequent extraction steps were performed using acetonitrile with 1　% formic acid (for soils RefeSol 01-A, RefeSol 02-A and LUFA 2.4), acetone (all soils) and acetonitrile:water (50:50, v:v). Further analysis (HPLC and silylation) was performed on the basis of soil samples which had been extracted by the shaking procedure and additionally by two ASE extractions.

Representative replicates were further subjected to ASE™ extraction using subsequently methanol:acetone:water (2:1:1, v:v:v) and acetonitrile:ammonium hydroxide pH　9 (50:50, v:v).

The results are summarised in the following tables:

Detailed results of additional ASE^™ extraction of remaining soil after the first extraction step of RefeSol 01-A samples treated with 2,6-di-tert-butyl-p-cresol. Values in % of applied radioactivity (AR) as non-extractable radioactivity (NER) before additional extraction and in the resulting ASE^™ extracts.
Sampling time	Replicate	Soil NER^1	Additional ASE™ extraction
		(before ASE extraction)	Extract 1 acetonitrile with 1% formic acid	Extract 2 acetone	Extract 3 acetonitrile: water (50:50)	Extract 4 methanol: acetone: water (50:50)	Extract 5 acetonitrile: ammonium hydroxide pH　9 (50:50)	Sum ASE extracts	Soil NER (after ASE extraction)
		[% AR]
0d	1	3.4	-	-	-	-	-	-	-
	2	3.4	-	-	-	-	-	-	-
3d	1	63.5	8.6	5.4	1.6	1.7	0.5	17.8	37.2
	2	62.3	9.3	4.7	1.6			15.5	39.6
7d	1	64.1	6.6	2.0	1.4			9.7	44.1
	2	62.7	6.7	4.4	1.5			12.7	44.0
14d	1	63.9	7.0	3.9	1.6			12.6	43.9
	2	60.9	6.0	4.3	1.8			12.1	44.4
30d	1^2
	E2	63.2	6.3	3.9	1.8			11.9	44.4
62d	1	61.7	5.6	3.4	1.4			10.4	45.7
	2	61.5	4.8	3.8	1.6	1.5	0.4	12.1	45.5
90d	1	62.1	6.0	1.9	2.0			9.9	44.4
	2	58.8	5.9	2.3	1.8			10.0	46.1
120d	1	61.7	4.6	3.1	1.7	1.4	0.4	11.1	46.7
	2	60.3	4.9	2.7	1.7			9.3	50.5
62d sterile	1	58.0	6.8	3.5	1.9			12.2	38.1
	2	59.5	7.2	2.5	1.6			11.4	40.8
120d sterile	1	57.8	5.8	4.1	1.9			11.8	41.8
	2	56.1	6.7	1.1	2.6	1.1	0.3	11.9	40.0

¹ Non-extractable residues (NER) before ASE extraction were calculated from the radioactivity remaining in the soil after soil extraction with acetonitrile with 1 % formic acid by shaking on a horizontal shaker (first extraction step, refer to section).

² Sample was lost during the sample preparation procedure.

Detailed results of additional ASE^™ extraction of remaining soil after the first extraction step of RefeSol 02-A samples treated with 2,6-di-tert-butyl-p-cresol. Values in % of applied radioactivity (AR) as non-extractable radioactivity (NER) before additional extraction and in the resulting ASE^™ extracts.
Sampling time	Replicate	Soil NER^1	Additional ASE™ extraction
		(before ASE extraction)	Extract 1 acetonitrile with 1% formic acid	Extract 2 acetone	Extract 3 acetonitrile: water (50:50)	Extract 4 methanol: acetone: water (50:50)	Extract 5 acetonitrile: ammonium hydroxide pH　9 (50:50)	Sum ASE extracts	Soil NER (after ASE extraction)
		[% AR]
0d	1	4.2	-	-	-	-	-	-	-
	2	4.2	-	-	-	-	-	-	-
3d	1	71.5	10.9	3.9	2.0	1.5	0.4	18.7	52.7
	2	71.3	8.7	4.7	2.8			16.1	53.6
7d	1	62.8	6.9	4.0	2.0			13.0	50.9
	2	64.2	6.6	1.6	1.8			10.1	52.6
14d	1	61.9	5.4	3.9	2.2			11.5	50.3
	2	64.0	6.4	3.6	2.2			12.2	50.6
30d	1	61.6	6.0	2.8	1.8			10.6	50.9
	2	60.8	5.9	2.9	2.9			11.6	50.5
62d	1	57.9	4.1	2.1	1.5			7.7	50.0
	2	57.8	5.5	2.5	1.7	1.0	0.3	10.9	47.3
90d	1	52.1	6.8	3.4	2.4			12.6	44.1
	2	51.5	7.2	3.5	2.8			13.5	44.1
120d	1	53.8	6.6	2.9	2.4	1.3	0.5	13.7	40.4
	2	55.6	6.0	3.8	2.6			12.5	42.4
62d sterile	1	59.5	4.4	3.3	1.9			9.5	50.5
	2	59.2	5.3	2.8	1.7			9.9	47.9
120d sterile	1	53.3	6.2	1.7	1.4			8.4	44.8
	2	53.5	6.6	1.7	1.5	0.7	0.3	10.8	43.3

¹ Non-extractable residues (NER) before ASE extraction were calculated from the radioactivity remaining in the soil after soil extraction with acetonitrile with 1 % formic acid by shaking on a horizontal shaker (first extraction step, refer to section).

Detailed results of additional ASE^™ extraction of remaining soil after the first extraction step of LUFA 2.4 samples treated with 2,6-di-tert-butyl-p-cresol. Values in % of applied radioactivity (AR) as non-extractable radioactivity (NER) before additional extraction and in the resulting ASE^™ extracts.
Sampling time	Replicate	Soil NER^1	Additional ASE™ extraction
		(before ASE extraction)	Extract 1 acetonitrile with 1% formic acid	Extract 2 acetone	Extract 3 acetonitrile: water (50:50)	Extract 4 methanol: acetone: water (50:50)	Extract 5 acetonitrile: ammonium hydroxide pH　9 (50:50)	Sum ASE extracts	Soil NER (after ASE extraction)
		[% AR]
0d	1	4.8	-	-	-	-	-	-	-
	2	4.8	-	-	-	-	-	-	-
3d	1	69.9	11.5	2.9	2.0	1.4	0.5	18.2	48.7
	2	67.9	10.8	3.6	1.8			16.2	51.4
7d	1	67.4	9.9	2.5	1.8			14.3	52.8
	2	67.1	9.8	2.7	2.1			14.7	51.0
14d	1	64.4	7.9	2.6	1.7			12.2	54.4
	2	63.1	8.0	2.8	1.8			12.5	52.1
30d	1^2
	2	62.1	7.9	1.7	1.9			11.6	50.2
62d	1	58.5	5.9	2.0	2.1			9.9	49.9
	2	58.9	6.4	1.5	1.8	0.9	0.4	11.0	49.3
90d	1	52.9	6.1	1.3	1.8			9.2	50.4
	2	52.5	5.3	2.2	1.8			9.3	49.7
120d	1	54.2	4.4	2.1	1.9	1.0	0.4	9.8	45.7
	2	57.9	5.2	1.6	1.9			8.7	50.1
62d sterile	1	55.4	7.7	2.1	1.5			11.4	44.2
	2	56.9	8.1	2.4	1.4			11.9	45.4
120d sterile	1	54.6	7.6	2.3	1.6			11.5	42.7
	2	53.6	5.7	3.1	1.6	1.1	0.4	11.8	43.1

¹ Non-extractable residues (NER) before ASE extraction were calculated from the radioactivity remaining in the soil after soil extraction with acetonitrile with 1 % formic acid by shaking on a horizontal shaker (first extraction step, refer to section).

² Sample was lost during the sample preparation procedure.

Detailed results of additional ASE^™ extraction of remaining soil after the first extraction step of LUFA 6S samples treated with 2,6-di-tert-butyl-p-cresol. Values in % of applied radioactivity (AR) as non-extractable radioactivity (NER) before additional extraction and in the resulting ASE^™ extracts.
Sampling time	Replicate	Soil NER^1	Additional ASE™ extraction
		(before ASE extraction)	Extract 1 acetonitrile with 1% formic acid	Extract 2 acetone	Extract 3 acetonitrile: water (50:50)	Extract 4 methanol: acetone: water (50:50)	Extract 5 acetonitrile: ammonium hydroxide pH　9 (50:50)	Sum ASE extracts	Soil NER (after ASE extraction)
		[% AR]
0d	1	8.7	*	-	-	-	-	-	-
	2	8.6	*	-	-	-	-	-	-
3d	1	50.8	*	2.8	2.4	1.3	0.5	7.0	44.2
	2	50.2	*	3.3	3.0			6.3	42.0
7d	1	47.9	*	3.5	2.7			6.2	42.0
	2	48.4	*	2.9	2.6			5.5	41.8
14d	1	48.4	*	2.8	2.4			5.1	42.9
	2	50.0	*	3.1	2.5			5.6	42.1
30d	1^1	46.8	*	2.8	2.5			5.3	42.5
	2	47.1	*	2.4	2.4			4.8	42.6
62d	1	49.5	*	2.4	2.6			4.9	51.5
	2	47.3	*	1.8	2.8	1.4	0.6	6.7	39.7
90d	1	41.2	*	1.5	2.7			4.3	38.9
	2	41.6	*	1.6	2.5			4.1	39.8
120d	1	44.3	*	1.6	3.3	1.3	0.6	6.8	39.1
	2	43.8	*	1.4	3.0			4.4	40.1
62d sterile	1	56.5	*	2.5	1.9			4.3	48.7
	2	57.8	*	2.5	1.9			4.4	49.9
120d sterile	1	54.4	*	2.3	2.0			4.2	49.9
	2	52.7	*	1.3	0.3	0.9	0.2	2.6	45.8

¹ Non-extractable residues (NER) before ASE extraction were calculated from the radioactivity remaining in the soil after soil extraction with acetonitrile with 1　% formic acid by shaking on a horizontal shaker (first extraction step). ^*ASE™ extraction suing acetonitrile with 1% formic acid (extract 1) was carried out during the validated extraction procedure for soil LUFA 6S, i.e. the radioactivity found in the ASE™ extract 1 is already included in the general acetonitrile with 1 % formic acid soil extract.

Differentiation of the NER

In the study, different types of NER were differentiated. NER Type I consists of parent compound, transformation products or both adsorbed or physically entrapped into the soil matrix (results see Table above). NER type II are residues which are strongly bound to the matrix and NER type III consists of radioactivity incorporated into the biomass. Since NER type I is regarded to be remobilisable and, therefore, still of potential concern, silylation of representative samples was carried out during this study according to the procedures described in the ECHA draft Guidance documents [7, 8] to characterise the NER formed during the degradation of 2,6-di-tert-butyl-p-cresol further.

A representative sample of the 120d sampling time of each soil (replicate 2) was subjected to the silylation procedure described in section 4.5.10. Results presented in Table 22 show the amounts of radioactivity and of 2,6-di-tert-butyl-p-cresol which can be liberated by this procedure.

 

Silylation  of representative samples of NER remaining after ASE extraction was carried out during this study to further characterise the NER formed during the degradation of 2,6-di-tert-butyl-p-cresol. For this purpose, representative soil samples after ASE extraction samples of the 120 d sampling of all four soils were subjected to silylation procedure according to ECHA Guidance documents “Proposal for amendment to ECHA guidance (Chapter R.11: PBT/vPvB assessment) Appendix R.11-X: Extraction steps and approach for characterizing NER types (I, II and III)”, 10 October 2018. The NER after 120 days of incubation amounted between 40.1 % AR and 50.5 % AR in the respective samples. By silylation radioactivity in the range of 10.3 % AR and 16.3 % AR could be liberated from these soil samples. Following sample preparation radio-TLC analysis was carried out. TLC analysis showed that only minor amounts of 2,6-di-tert-butyl-p-cresol up to 1.2 % AR could be released out of soil sampled 120 days after treatment with 2,6-di-tert-butyl-p-cresol indicating that the NER does not contain relevant amounts of parent substance.

Table below shows the radioactivity and amount of 2,6-di-tert-butyl-p-cresol released by the silylation procedure out of NER after ASE extraction of representative samples of the 120 d sampling. Two replicates per soil sample were subjected to silylation procedure; values are given in percent of the applied radioactivity (% AR).

Sample	Replicate	NER present after ASE extractions	Radioactivity released by silylation
			Radioactivity in supernatant (measured without any sample preparation)	Radioactivity after concentration step (measured after evaporation and centrifugation of the supernatant)	Released BHT (determined by radio-TLC)
RefeSol 01-A 120d-2	1	50.5	10.9	9.6	1.2
	2		10.3	9.2	1.1
RefeSol 02-A 120d-2	1	42.4	13.3	9.4	n.d.
	2		16.3	10.4	0.5
LUFA 2.4 120d-2	1	50.1	14.3	8.0	0.8
	2		13.9	9.2	1.1
LUFA 6S 120d-2	1	40.1	14.2	10.9	n.d.
	2		4.4	7.6	0.4

n.d. not detected

As can be seen in the Table above, NER after 120 days of incubation amounted between 40.1% AR and 50.5% AR in the respective samples subjected to silylation procedure. By silylation radioactivity in the range of 10.3% AR and 16.3% AR could be liberated from these soil samples (except one replicate where only 4.4% AR was released). To analyse the released radioactivity in the supernatants by radio-TLC, the supernatants were concentrated by evaporation and centrifuged. The radioactivity after this concentration step was between 8.0% AR and 10.9% AR for all soils tested (except one replicate with 7.6　% AR). TLC analysis showed that only minor amounts of 2,6-di-tert-butyl-p-cresol up to 1.2　% AR could be released out of soil sampled 120　days after treatment with 2,6-di-tert-butyl-p-cresol.

As only very small part of the bound radioactivity could be attributed to BHT at 120d, no further attempts were made to other samples for two reasons:

• Relevant impact of liberated BHT using the silylation procedure would not have a significant impact on the half-life which is far below any trigger values for the P/vP evaluation.

• The silylation procedure is highly complex and costly and goes beyond the current analytical methodology

Detailed reults on the degradation products

 

Determination of the DT50 values

In addition to the parent compound, five transformation products were calculated which reached 10% AR or two times 5% AR in at least one soil. Due to the complexity of the metabolism scheme, the optimisation tool isn’t able to handle all five transformation products in parallel. Therefore, two separate optimisation were performed to cover all substances.

Model 1 considers BHT-CH₂OH (=A1), BHT-CHO (=B1), BHT-COOH (=B2)

Model 2 considers BHT-OH (=A1), BHT-quinone (=B1)

analyses for the metabolites were based on the best fit kinetics for the parent compound. For the metabolites always SFO (single first order) was considered. The results of the optimisations are presented in the following five tables:

 

Calculated DT50 and DT90 for BHT-CH2OH (= Model 1, A1) based on SFO kinetics.
Soil	chi^2	f	DT50	DT90
	(%)	P → A1	(d)	(d)
RefeSol 01-A*	42.95	0.043	-**	-**
RefeSol 02-A	59.04	0.045	518.9	1724
LUFA 2.4*	26.24	0.07326	28.88	95.94
LUFA 6S	42.5	0.06618	75.25	250
Geomean DT50 (d)			104 (n=3)

* Final step failed, results from next to last step considered

** no degradation found in optimisation

 

Calculated DT50 and DT90 for BHT-CHO (= Model 1, B1) based on SFO kinetics.
Soil	chi^2	f	f	DT50	DT90
	(%)	P → B1	A1 → B1	(d)	(d)
RefeSol 01-A*	31.75	0.074	1	77.02	255.8
RefeSol 02-A	62.95	0.144	1	3.285	10.91
LUFA 2.4*	18.7	0.06386	1	12.67	42.08
LUFA 6S	56.86	0.123	1	3.155	10.48
Geomean DT50 (d)				10 (n=4)

* Final step failed, results from next to last step considered

 

Calculated DT50 and DT90 for BHT-COOH (= Model 1, B2) based on SFO kinetics.
Soil	chi^2	f	DT50	DT90
	(%)	B1 → B2	(d)	(d)
RefeSol 01-A*	62.6	1	-**	-**
RefeSol 02-A	9.035	1	111.6	370.6
LUFA 2.4	22.78	1	22.78	75.66
LUFA 6S	13.59	1	33.63	111.7
Geomean DT50 (d)			44 (n=3)

* Final step failed, results from next to last step considered

** no degradation found in optimisation

 

Calculated DT50 and DT90 for BHT-OH (= Model 2, A1) based on SFO kinetics.
Soil	chi^2	f	DT50	DT90
	(%)	P → A1	(d)	(d)
RefeSol 01-A	18.74	0.092	381.4	1267
RefeSol 02-A	7.873	0.08704	506.7	1683
LUFA 2.4	20.3	0.076	81.72	271.5
LUFA 6S	16.51	0.0787	-**	-**
Geomean DT50 (d)			251 (n=3)

** no degradation found in optimisation

 

Calculated DT50 and DT90 for BHT-quinone (= Model 2, B1) based on SFO kinetics.
Soil	chi^2	f	DT50	DT90
	(%)	P → B1	(d)	(d)
RefeSol 01-A	41.89	0.091	25	123.1
RefeSol 02-A	31.99	0.06657	280	930.1
LUFA 2.4	22.91	0.083	60.19	199.9
LUFA 6S	18.04	0.1056	-**	-**
Geomean DT50 (d)			85 (n=3)

** no degradation found in optimisation

It can be seen, that the chi² values are relatively high with values in the range of 7.9　% to 63.0　% indicating a relative high uncertainty of the calculations. Ideally, the error value at which the chi²-test is passed for the metabolite should be below 15%, like for parent substance, and the fit must be visually acceptable.

However, this value should only be considered as guidance and not absolute cut-off criterion. There will be cases where the error value to pass the chi²-test for a metabolite is higher, but the fit still represents a reasonable description of its formation and degradation behaviour. This is especially the case when metabolite residues are low.

According to the results of the fitting following geometric mean DT50 values were found:

BHT-CH₂OH 104 d (n=3)

BHT-CHO 10 d (n=4)

BHT-COOH 44 d (n=3)

BHT-OH 251 d (n=3)

BHT-quinone 85 d (n=3).

 

 

Conclusions:

Formation of metabolites showed the degradation of 2,6-di-tert-butyl-p-cresol in four soils. During 120 days of incubation, the parent substance was degraded to several transformation products which were characterised by co-chromatography with reference standards of known transformation products or by their retention time during HPLC analysis. By this way, the known transformation products BHT-CHO, BHT-OH, BHT-COOH, BHT-CH2OH and BHT-quinone were detected in the soil extracts as well as two unknown transformation products exceeding 5 % or 10 % AR.
The obtained data sets were analysed using the program CAKE version 1.4. For the determination of the disappearance time DT50 values for the parent compound there is evidence that the degradation follows biphasic kinetics. The results are in the range of 0.001 to 0.6 days.
The geometric mean DT50 of the recommended optimisation was found to be 0.07 days for the parent compound.

Executive summary:

In the present study the transformation of 2,6-di-tert-butyl-p-cresol (BHT) was investigated under aerobic conditions according to the OECD-Guideline 307 "Aerobic and anaerobic Transformation in Soil” in four biologically active soils. The incubation was performed using 14C-labelled test item at an application rate of 0.5 mg/kg soil dry weight.

Soil subsamples were prepared by placing 50 g soil samples (dry weight basis) into glassvessels which were then incubated at 12 ± 2 °C in the dark. The test was carried out using a closed system without continuous aeration of the soil subsamples.

The test substance and their metabolites have a rather high vapour pressure. Several pretests have shown that in the commonly used flow-through system a high portion of radioactivity was lost by evaporation and was therefore not subject to biodegradation in the soil.

For this reason, the incubation was performed in glass vessels which were closed gas tight. Trapping of the exhaust gases was carried out by a NaOH absorption trap integrated in the vessel and a Tenax tube. Oxygen sensors were placed within the vessel of three representative samples per soil. Depending on the measured oxygen concentration, the vessels were aerated manually to maintain natural conditions over the test period.

Microbiological activity of the test soil was monitored at the beginning of incubation, during the incubation and at the end of the incubation. Biomass determination was performed by means of the substrate induced respiration method. The results of microbial biomass show the existence of an active microbial population throughout the incubation period.

Replicate soil samples were taken for analyses at 0, 3, 7, 14, 62, 90 and 120 days after application. Soil samples were extracted by several extraction techniques and solvent systems. Selected extracts were analysed for the test substance and possible degradation products by HPLC and TLC.

A total radioactivity balance and the distribution of radioactivity in every subsample were established at each sampling day. The total recoveries ranged between 90 and 110% of applied radioactivity.

Only minor amounts of the test substance or volatile metabolites with low molecular weight or high Henry constants volatilize during the aerobic degradation in the closed test system: In the Tenax® traps of the samples, the radioactivity was always ≤ 0.5 % AR.

Mineralisation of 2,6-di-ter-butyl-p-cresol was detected in amounts of 5.4 % AR (RefeSol 01-A), 10.5 % AR (RefeSol 02-A), 17.8 % AR (LUFA 2.4) and 11.4 % (LUFA 6S) at the end of incubation.

The amount of radioactivity extracted from soil by acetonitrile with 1 % formic acid decreased severely in all soils from 94.9 % – 100.4 % AR at the beginning of incubation to (0 days) to 23.7 % – 42.7 % AR after 120 days of incubation. By applying ASE™ extraction of non-extractable residues, amounts of radioactivity can be extracted by up to 5 additional ASE™ steps. The radioactivity found in total in the ASE™ extracts was always in the range of 4.2 % - 17.4 %.

 

 

After the different extraction procedures including the harsher accelerated solvent extraction (ASE™) have been intensively applied, the non-extractable radioactivity (NER) after ASE extraction was still in the range of 40% AR to 60% AR. The amount of NER remained relatively stable throughout the incubation time.

The amount of parent compound in the soil extracts (including the first extraction step by shaking and after 2 ASE extractions) decreased rapidly from maximum levels between 95.8 % AR (LUFA 6S) - 101.4 % AR (LUFA 2.4) immediately after application to amounts in the range of 2.4 % AR (LUFA 2.4) - 15.5 % AR (RefeSol 01-A) during the first 3 days of incubation. Afterwards, it decreased further to amounts between non-detectable levels (LUFA 6S) and 8.0 % AR (RefeSol 01-A) after 120 days of incubation.

 

Formation of metabolites showed the degradation of 2,6-di-tert-butyl-p-cresol in four soils. During 120 days of incubation, the parent substance was degraded to several transformation products which were characterised by co-chromatography with reference standards of known transformation products or by their retention time during HPLC analysis. By this way, the known transformation products BHT-CHO, BHT-OH, BHT-COOH, BHT-CH2OH and BHTquinone were detected in the soil extracts as well as 1 -2 unknown transformation products exceeding 5 % or 10 % AR, which suppose to be unknown metabolites. The pattern of 2,6-di-tert-butyl-p-cresol and its metabolites in the different soils are summarized in the following tables:

 

 

Pattern of 2,6-di-tert-butyl-p-cresol and its metabolites in RefeSol 01 -A determined in the acetonitrile with 1% formic acid soil extracts and ASE extracts 1 and 2.

      Mean values of two replicates except for the sampling time 30d consisting of one replicate; values are given in percent of the applied radioactivity (% AR).

	Incubation time [d]
Radioactive fraction	0	3	7	14	30	62	90	120
BHT	99.2	15.5	15.6	18.4	15.5	10.7	6.2	8.0
BHT-CHO	*	8.5	5.0	4.2	2.9	1.5	3.8	4.3
BHT-OH	*	7.9	7.8	7.3	8.5	5.7	4.9	9.1
BHT-COOH	*	1.6	6.1	6.7	7.4	7.7	4.0	9.6
BHT-CH2OH	*	1.9	4.3	2.5	*	4.6	6.6	4.1
BHT-quinone	*	9.1	6.4	3.9	1.4	2.4	3.5	2.0
Unassigned Ret. time 16.0 min	*	0.7	*	3.6	7.9	1.0	3.8	0.8
Unassigned Ret. time 2.5 min	*	*	*	0.9	1.1	7.4	4 .2	5.3

* = not detected

 

 

Pattern of 2,6-di-tert-butyl-p-cresol and its metabolites in RefeSol 02 -A determined in the acetonitrile with 1% formic acid soil extracts and ASE extracts 1 and 2.

      Mean values of two replicates; values are given in percent of the applied radioactivity (% AR).

	Incubation time [d]
Radioactive fraction	0	3	7	14	30	62	90	120
BHT	95.7	5.0	1.7	4.6	4.6	0.7	2.2	1.6
BHT-CHO	*	5.7	3.5	3.6	2.1	0.7	*	1.8
BHT-OH	*	7.5	8.9	7.9	6.7	8.0	7.4	6.8
BHT-COOH	*	6.7	9.0	13.0	11.3	8.4	9.0	7.3
BHT-CH2OH	*	1.6	1.2	4.8	8.8	4.5	3.7	2.9
BHT-quinone	*	9.5	5.1	5.6	3.5	4.3	4.4	6.5
Unassigned Ret. time 22.0 min	*	5.0	7.3	4.4	1.9	2.0	1.5	0.8
Unassigned Ret. time 6.4 min	*	*	*	*	*	8.4	10.1	8.4
Unassigned Ret. time 2.5 min	*	*	*	0.6	2.9	3.6	5.9	6.6

* = not detected

 

Pattern of 2,6-di-tert-butyl-p-cresol and its metabolites in LUFA 2.4 determined in the acetonitrile with 1% formic acid soil extracts and ASE extract 1 and 2.

      Mean values of two replicates except for the sampling time 30d consisting of one replicate; values are given in percent of the applied radioactivity (% AR).

	Incubation time [d]
Radioactive fraction	0	3	7	14	30	62	90	120
BHT	100.4	2.4	1.9	1.8	2.2	0.7	1.6	0.9
BHT-CHO	*	7.0	4.0	3.7	3.3	1.9	1.1	1.0
BHT-OH	*	8.9	6.8	5.9	4.3	3.4	5.0	2.8
BHT-COOH	*	5.7	10.5	4.7	4.6	2.6	3.0	1.2
BHT-CH2OH	*	6.4	5.7	6.6	1.3	2.3	*	1.2
BHT-quinone	*	9.3	7.5	6.3	4.1	3.0	2.8	4.2
Unassigned Ret. time 22.0 min	*	4.3	3.3	2.0	1.7	0.9	1.0	*
Unassigned Ret. time 6.7 min	*	*	*	4.3	5.9	6.6	3.4	7.1
Unassigned Ret. time 2.5 min	*	*	1.4	6.7	4.9	12.4	5.9	5.7

* = not detected

 

Pattern of 2,6-di-tert-butyl-p-cresol and its metabolites in LUFA 6S determined in the acetonitrile with 1% formic acid soil extracts and ASE extracts 1 and 2.

      Mean values of two replicates; values are given in percent of the applied radioactivity (% AR).

 

	Incubation time [d]
Radioactive fraction	0	3	7	14	30	62	90	120
BHT	94.9	5.8	2.3	3.6	2.1	*	*	*
BHT-CHO	*	5.4	3.8	2.7	2.6	1.6	*	0.6
BHT-OH	*	5.9	6.8	6.7	6.5	8.1	10.2	6.9
BHT-COOH	*	4.8	9.0	8.5	9.2	4.3	3.6	2.4
BHT-CH2OH	*	4.0	2.8	8.3	5.8	5.0	2.5	1.5
BHT-quinone	*	12.4	7.3	11.1	8.3	8.4	10.0	11.2
Unassignet Ret. time 22.0 min	*	3.2	5.4	4.0	2.2	1.3	*	*
Unassigned Ret. time 6.5 min	*	*	0.8	*	8.5	11.9	8.1	7.9
Unassigned Ret. time 2.5 min	*	*	0.8	*	2.4	6.1	7.7	8.2

* = not deteceted

In the studydifferent types of NER were differentiated. NER Type I consists of parent compound, transformation products or both adsorbed or physically entrapped into the soil matrix.  NER type II are residues which are strongly bound to the matrix and NER type III consists of radioactivity incorporated into the biomass. Since NER type I is regarded to be remobilisable and, therefore, still of potential concern, silylation of representative samples was carried out during this study according to the procedures described in the ECHA draft Guidance documents [7, 8] to characterise the NER formed during the degradation of 2,6-di-tert-butyl-p-cresol further.

In order to characterise the unknown metabolites further, LC-HRMS analysis of representative extracts (LUFA 2.4 120 d, replicate 2 and RefeSol 02-A 7 d, replicate 2) was carried out. However, radiolabelled 2,6-di-tert-butyl-p-cresol could not be ionised using both LC-HRMS devices and both ionisation methods ES (electrospray ionisation) and APCI (atmospheric pressure chemical ionisation).

 

Silylation  of representative samples of NER remaining after ASE extraction was carried out during this study to further characterise the NER formed during the degradation of 2,6-di-tert-butyl-p-cresol. For this purpose, representative soil samples after ASE extraction samples of the 120 d sampling of all four soils were subjected to silylation procedure according to ECHA Guidance documents “Proposal for amendment to ECHA guidance (Chapter R.11: PBT/vPvB assessment) Appendix R.11-X: Extraction steps and approach for characterizing NER types (I, II and III)”, 10 October 2018. The NER after 120 days of incubation amounted between 40.1 % AR and 50.5 % AR in the respective samples. By silylation radioactivity in the range of 10.3 % AR and 16.3 % AR could be liberated from these soil samples. Following sample preparation radio-TLC analysis was carried out. TLC analysis showed that only minor amounts of 2,6-di-tert-butyl-p-cresol up to 1.2 % AR could be released out of soil sampled 120 days after treatment with 2,6-di-tert-butyl-p-cresol indicating that the NER does not contain relevant amounts of parent substance.

 

Results are summarized in the following table:

Radioactivity and amount of 2,6-di-tert-butyl-p-cresol released by the silylation procedure out of NER after ASE extraction of representative samples of the 120 d sampling.

      Two replicates per soil sample were subjected to silylation procedure; values are given in percent of the applied radioactivity (% AR).

Sample	Replicate	NER present after ASE extractions	Radioactivity released by silylation
			Radioactivity in supernatant (measured with any sample preparation)	Radioactivity after concentration step (measured after evaporation and centrifugation of the supernatant)	Released BHT (determined by radio-TLC)
RefeSol 01-A 120d-2	1	50.5	10.9	9.6	1.2
	2		10.3	9.2	1.1
RefeSol 02-A 120d-2	1	42.4	13.3	9.4	n.d. *
	2		16.3	10.4	0.5
LUFA 2.4 120d-2	1	50.1	14.3	8.0	0.8
	2		13.9	9.2	1.1
LUFA 6S 120d-2	1	40.1	14.2	10.9	n.d.
	2		4.4	7.6	0.4

*n.d. not detected

As only very small part of the bound radioactivity could be attributed to BHT at 120d, no further attempts were made to other samples for two reasons:

• Relevant impact of liberated BHT using the silylation procedure would not have a significant impact on the half-life which is far below any trigger values for the P/vP evaluation.

• The silylation procedure is highly complex and costly and goes beyond the current analytical methodology

 

The obtained data sets (sum of Soxhlet and ASE extracts) were analysed using the program CAKE version 1.4. For the determination of the disappearance time DT50 values there is evidence that the degradation follows biphasic kinetics. The results are in the range of 0.001 to 0.6 days. The geometric mean DT50 of the recommended optimisation was found to be 0.07 days for the parent compound.

Reference 2

Endpoint:

biodegradation in soil, other

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 304 A (Inherent Biodegradability in Soil)

Principles of method if other than guideline:

Study design comparable to later OECD Guideline 304 A "Inherent biodegradability in soil", 1981

GLP compliance:

no

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

other: Japanese classification

Soil no.:

#1

Soil type:

other: Light clay

% Clay:

29

% Silt:

40

% Sand:

31

pH:

5.5

CEC:

53.7 meq/100 g soil d.w.

Soil no.:

#2

Soil type:

sandy clay loam

% Clay:

17

% Silt:

18

% Sand:

65

pH:

6.3

CEC:

13.5 meq/100 g soil d.w.

Soil no.:

#3

Soil type:

sandy loam

% Clay:

2

% Silt:

3

% Sand:

95

pH:

7

CEC:

9.6 meq/100 g soil d.w.

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location: #1: Kodaira, Tokyo metropolis, Japan; #2: Azuchi, Shiga pref., Japan; #3: Takarazuka, Hyogo pref., Japan
- Storage conditions: after sieving, stored undried at 5 °C prior to use
- Soil preparation (e.g., 2 mm sieved; air dried etc.): 2 mm sieved,

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at 1/3 atm (%):
- Bulk density (g/cm3):
- Organic matter content (%): #1: 15.3; #2: 2.5; #3: 2.7

Soil No.:

#1

Duration:

24 d

Soil No.:

#2

Duration:

24 d

Soil No.:

#3

Duration:

24 d

Soil No.:

#1

Initial conc.:

1 mg/kg soil d.w.

Based on:

act. ingr.

Soil No.:

#2

Initial conc.:

1 mg/kg soil d.w.

Based on:

act. ingr.

Soil No.:

#3

Initial conc.:

1 mg/kg soil d.w.

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

radiochem. meas.

Soil No.:

#1

Temp.:

25°C

Humidity:

40% of maximum water-holding capacity

Soil No.:

#2

Temp.:

25°C

Humidity:

40% of maximum water-holding capacity

Soil No.:

#3

Temp.:

25°C

Humidity:

40% of maximum water-holding capacity

Details on experimental conditions:

1. PRELIMINARY EXPERIMENTS: no data

2. EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): 1 week, 25 °C, 100-mL beaker, 40% maximum water-holding capacity
- Soil condition: fresh
- Soil (g/replicate):
- Control conditions, if used (present differences from other treatments, i.e., sterile/non-sterile, experimental conditions):
- No. of replication controls, if used:
- No. of replication treatments:
- Test apparatus (Type/material/volume):
- Details of traps for CO2 and organic volatile, if any: Each jar was continuously purged with C02~free air at 100 ml/hr and the effluent air was passed serially through a polyurethane foam plug and gas-washing bottles containing 400 ml of 0.5 N NaOH solution to trap volatile 14C including 14C02 .
- If no traps were used, is the system closed/open:
- Identity and concentration of co-solvent: ethanol

Test material application
- Volume of test solution used/treatment: methanol solution of 14C-BHT (50 µl; final concentration 1 ppm)
- Application method (e.g. applied on surface, homogeneous mixing etc.): homogeneous mixing
- Is the co-solvent evaporated:

Any indication of the test material adsorbing to the walls of the test apparatus:

Experimental conditions (in addition to defined fields)
- Moisture maintenance method:
- Continuous darkness: Yes

Other details, if any: For sterilized condition, moist soil samples were auto-claved at 20 psi and 120 °C and then treated with 1 ppm of 14C-BHT. All procedures up to extraction were carried out aseptically.

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

4. SUPPLEMENTARY EXPERIMENTS:

5. SAMPLING DETAILS
- Sampling intervals:
- Sampling method for soil samples:
- Method of collection of CO2 and volatile organic compounds: Each jar (3-L) was continuously purged with C02~free air at 100 ml/hr and the effluent air was passed serially through a polyurethane foam plug and gas-washing bottles containing 400 ml of 0.5 N NaOH solution to trap volatile 14C including 14C02 .
- Sampling intervals/times for:
- Other observations, if any:

Soil No.:

#1

% Degr.:

> 85

Parameter:

radiochem. meas.

Sampling time:

1 d

Soil No.:

#2

% Degr.:

> 80

Parameter:

radiochem. meas.

Sampling time:

1 d

Soil No.:

#3

% Degr.:

> 75

Parameter:

radiochem. meas.

Sampling time:

1 d

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

No.:

#5

Details on transformation products:

- Formation and decline of each transformation product during test: see table under "Any other information on results incl. tables"
- Description of biotransformation pathway: see attached figure
- Figure attached: Yes

Evaporation of parent compound:

not specified

Volatile metabolites:

yes

Residues:

yes

Details on results:

Nonsterilized conditions: After one day 63-82% of BHT were decomposed (about 1-2% mineralized to CO2). After 24 days of incubation 77-92% were decomposed (21-29% mineralized to CO2). Sterilized conditions: After one day 25-35% of BHT were decomposed. After 24 days of incubation 27-41% were decomposed. In both cases mineralization was negligible (<2%). After one day 57-68% of BHT and after 24 days 50-61% remained unchanged. Under sterilized and nonsterilized conditions BHT-OOH, BHT-OH, BHT-CH2OH, BHT-CHO, BHT-COOH were identified as degradation products of BHT.

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): no

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
- Range of maximum concentrations in % of the applied amount at end of study period:
on the - the and -th day of incubation, respectively. At the end of the study period, the corresponding concentrations were - and -- % of the applied amount, respectively.

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
- Range of maximum concentrations in % of the applied amount at end of study period:

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:

EXTRACTABLE RESIDUES
- % of applied amount at day 0: see table below
- % of applied amount at end of study period: see table below

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: 27% to 39%
- % of applied amount at end of study period: 26% to 36%

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 21% to 29%

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: 21% to 43%

STERILE TREATMENTS (if used)
- Transformation of the parent compound: max. 0.6% CO2
- Formation of transformation products: yes (see table below)
- Formation of extractable and non-extractable residues: bound residues: 11% to 22%
- Volatilization: 47% to 56%

Table: Relative amounts of degradation products 1 or 24 days after application of 14C-BHT to three types of soil under nonsterilized and sterilized upland conditions

	% of applied^14C
	nonsterilized						sterilized
	Kodaira		Azuchi		Takarazuka		Kodaira		Azuchi		Takarazuka
	1	24	1	24	1	24	1	24	1	24	1	24
Volatile^14C	2.5	31.7	3.9	26.1	12.8	42.8	12.4	43.6	9.7	49. 1	19.4	55.9
Polyurethane plug	1.8	5.8	3.2	4.9	10.8	14.0	12.4	43.5	9.7	47.2	19.4	55.3
BHT	1.6	3.7	2.9	3.8	10.3	12.6	12.1	40. 3	9.5	44.2	19.1	51.6
BHT-OH	0.1	0.8	0.1	0.2	0.2	0.3	<0.1	0.4	<0.1	0.2	<0.1	0.5
Others	0.1	1.3	0.2	0.9	0.3	1.1	0.3	2.8	0.2	2.8	0.3	3.2
NaOH solution	0.7	25.9	0.7	21.2	2.0	28.8	<0.1	<0.1	0.1	1.9	<0.1	0.6
14 Extract C	54.1	32.8	50.1	28.3	46.4	25.7	79.0	25.3	78.7	28.4	70.4	21.7
BHT	11.7	2.6	15.9	5.1	12.7	5.0	50.6	9.9	47.4	7.6	49.0	9.6
BHT-OOH	2.5	0.5	4.0	0.8	3.6	0.9	7.7	0.6	2.2	0.3	1.2	0.2
BHT-OH	17.1	15.2	8.9	5.3	8.0	4.0	7.2	1.4	7.8	2.7	6.5	1.2
BHT-CH2OH	1.1	0.7	1.4	0.5	1.1	0.7	0.5	0.6	1.5	0.5	0.8	0.3
BHT-CHO	0.9	0.8	1.4	1.0	1.0	0.7	3.1	1.3	5.3	2.1	3.0	1.5
BHT-COOH	1.4	0.4	0.6	0.4	0.5	0.4	0.5	0.2	1.2	0.2	0.5	0.8
Others	19.1	12.6	17.9	15.2	19. 5	13.0	9.9	11.3	13.3	11.0	9.4	8.1
Bound^14C	38.8	33.7	34.7	35.8	27.1	26.0	5.8	21.6	1.6	19.7	2.9	10.8
Total	95.4	98.2	88. 7	90. 2	86.3	94.5	96.9	90.5	90.0	89. 2	92.7	88.4

Conclusions:

BHT was quite unstable in soils and approximately 20 % of the applied BHT was degraded immediately after treatment while recovery of 14C ranged from 90 to 100 %. BHT as well as its degradation products is quite biodegradable and hardly persists in the soil.

Executive summary:

To characterize biodegradation of 2,6 -di-tert-butyl-p-cresol (BHT) in soil, an experiment was performed by Mikami et al. (1979) following a test design in accordance to later OECD Guideline 304 A (Inherent biodegradability in soil). Light clay, sandy clay loam, and sandy loam were used for this study which was carried out under aerobic conditions and for a time duration of 24 days. The test temperature was 25 °C, all soils were humid to 40% of their maximum water-holding capacity. Under nonsterilized conditions 63-82% of BHT were decomposed (about 1-2% mineralized to CO2) after one day. After 24 days of incubation 77-92% were decomposed (21-29% mineralized to CO2).

Under sterilized conditions 25 -35% of BHT were decomposed after 24 hours. After 24 days of incubation 27-41% were decomposed. In both cases mineralization was negligible (< 2%). After one day 57-68% of BHT and after 24 days 50-61% remained unchanged. Under sterilized and nonsterilized conditions BHT-OOH, BHT-OH, BHT-CH2OH, BHT-CHO, BHT-COOH were identified as degradation products of BHT. All degradation products did not build up over the study period but declined over time.

As overall result the half-life (DT50) of BHT in soil is less than 24 hours at a temperature of 25 °C.

Description of key information

In a study performed according to OECD 307 under aerobic conditions at 12°C (Derz 2019), 2,6 -di-tert.-butyl-p-cresol was found to degrade rapidly. For the determination of the disappearance time DT50 values there is evidence that the degradation follows biphasic kinetics. The results are in the range of 0.001 to 0.6 days. The geometric mean DT50 of the recommended optimisation was found to be 0.07 days for the parent compound. The highest value (0.6 days) was used as key value as it represents the worst case. Five metabolites were identified ("BHT-CHO, BHT-OH, BHT-CH2OH, BHT-COOH, BHT-quinone") and quantified.

Even after exhaustive extractions including 2 ASE steps.about f 40 -50% of the total radioactivity remained in the soils. Following a draft ECHA guidance document, silylation was performed in order to differentiate between NER type 1 and 2. While additional 10.3 -16.3% AR could be resolved by silylation, only minor traces (up to 1.2%) consisted of the unchanged parent substance.

Key value for chemical safety assessment

Half-life in soil:

0.6 d

at the temperature of:

12 °C

Additional information

In the study (Derz 2019) the transformation of 2,6-di-tert-butyl-p-cresol (BHT) was investigated under aerobic conditions according to the OECD-Guideline 307 "Aerobic and anaerobic Transformation in Soil” in four biologically active soils. The incubation was performed using 14C-labelled test item at an application rate of 0.5 mg/kg soil dry weight.

Soil subsamples were prepared by placing 50 g soil samples (dry weight basis) into glassvessels which were then incubated at 12 ± 2 °C in the dark. The test was carried out using a closed system without continuous aeration of the soil subsamples.

The test substance and their metabolites have a rather high vapour pressure. Several pretests have shown that in the commonly used flow-through system a high portion of radioactivity was lost by evaporation and was therefore not subject to biodegradation in the soil.

For this reason, the incubation was performed in glass vessels which were closed gas tight. Trapping of the exhaust gases was carried out by a NaOH absorption trap integrated in the vessel and a Tenax tube. Oxygen sensors were placed within the vessel of three representative samples per soil. Depending on the measured oxygen concentration, the vessels were aerated manually to maintain natural conditions over the test period.

Microbiological activity of the test soil was monitored at the beginning of incubation, during the incubation and at the end of the incubation. Biomass determination was performed by means of the substrate induced respiration method. The results of microbial biomass show the existence of an active microbial population throughout the incubation period.

Replicate soil samples were taken for analyses at 0, 3, 7, 14, 62, 90 and 120 days after application. Soil samples were extracted by several extraction techniques and solvent systems. Selected extracts were analysed for the test substance and possible degradation products by HPLC and TLC.

A total radioactivity balance and the distribution of radioactivity in every subsample were established at each sampling day. The total recoveries ranged between 90 and 110% of applied radioactivity.

Only minor amounts of the test substance or volatile metabolites with low molecular weight or high Henry constants volatilize during the aerobic degradation in the closed test system: In the Tenax® traps of the samples, the radioactivity was always ≤ 0.5 % AR.

Mineralisation of 2,6-di-ter-butyl-p-cresol was detected in amounts of 5.4 % AR (RefeSol 01-A), 10.5 % AR (RefeSol 02-A), 17.8 % AR (LUFA 2.4) and 11.4 % (LUFA 6S) at the end of incubation.

The amount of radioactivity extracted from soil by acetonitrile with 1 % formic acid decreased severely in all soils from 94.9 % – 100.4 % AR at the beginning of incubation to (0 days) to 23.7 % – 42.7 % AR after 120 days of incubation. By applying ASE™ extraction of non-extractable residues, amounts of radioactivity can be extracted by up to 5 additional ASE™ steps. The radioactivity found in total in the ASE™ extracts was always in the range of 4.2 % - 17.4 %. The sum of these data refect the NER type 1 as defined in "Proposal for amendment to ECHA guidance (Chapter R.11: PBT/vPvB assessment) Appendix R.11-X: Extraction steps and approach for characterizing NER types (I, II and III)", 10 October 2018

 

After the different extraction procedures including the harsher accelerated solvent extraction (ASE have been intensively applied, the non-extractable radioactivity (NER) after ASE extraction was still in the range of 40% AR to 60% AR. The amount of NER remained relatively stable throughout the incubation time.

The amount of parent compound in the soil extracts (including the first extraction step by shaking and after 2 ASE extractions) decreased rapidly from maximum levels between 95.8 % AR (LUFA 6S) - 101.4 % AR (LUFA 2.4) immediately after application to amounts in the range of 2.4 % AR (LUFA 2.4) - 15.5 % AR (RefeSol 01-A) during the first 3 days of incubation. Afterwards, it decreased further to amounts between non-detectable levels (LUFA 6S) and 8.0 % AR (RefeSol 01-A) after 120 days of incubation.

 

Formation of metabolites showed the degradation of 2,6-di-tert-butyl-p-cresol in four soils. During 120 days of incubation, the parent substance was degraded to several transformation products which were characterised by co-chromatography with reference standards of known transformation products or by their retention time during HPLC analysis. By this way, the known transformation products BHT-CHO, BHT-OH, BHT-COOH, BHT-CH2OH and BHTquinone were detected in the soil extracts as well as 1 -2 unknown transformation products exceeding 5 % or 10 % AR.

According to ECHA (2018), NER type II are residues which are strongly bound to the matrix. Therefore, silylation  of representative samples of NER remaining after ASE extraction was carried out during this study to further characterise the NER formed during the degradation of 2,6-di-tert-butyl-p-cresol. For this purpose, representative soil samples after ASE extraction samples of the 120 d sampling of all four soils were subjected to silylation procedure according to ECHA Guidance documents “Proposal for amendment to ECHA guidance (Chapter R.11: PBT/vPvB assessment) Appendix R.11-X: Extraction steps and approach for characterizing NER types (I, II and III)”, 10 October 2018. The NER after 120 days of incubation amounted between 40.1 % AR and 50.5 % AR in the respective samples. By silylation radioactivity in the range of 10.3 % AR and 16.3 % AR could be liberated from these soil samples. Following sample preparation radio-TLC analysis was carried out. TLC analysis showed that only minor amounts of 2,6-di-tert-butyl-p-cresol up to 1.2 % AR could be released out of soil sampled 120 days after treatment with 2,6-di-tert-butyl-p-cresol (=NER type 2 indicating that the NER does not contain relevant amounts of parent substance.

To characterize biodegradation of 2,6 -di-tert-butyl-p-cresol (BHT) in soil, an experiment was performed by Mikami et al. (1979) following a test design in accordance to later OECD Guideline 304 A (Inherent biodegradability in soil). Light clay, sandy clay loam, and sandy loam were used for this study which was carried out under aerobic conditions and for a time duration of 24 days. The test temperature was 25 °C, all soils were humid to 40% of their maximum water-holding capacity. Under nonsterilized conditions 63-82% of BHT were decomposed (about 1-2% mineralized to CO2) after one day. After 24 days of incubation 77-92% were decomposed (21-29% mineralized to CO2).

Under sterilized conditions 25 -35% of BHT were decomposed after 24 hours. After 24 days of incubation 27-41% were decomposed. In both cases mineralization was negligible (< 2%). After one day 57-68% of BHT and after 24 days 50-61% remained unchanged. Under sterilized and nonsterilized conditions BHT-OOH, BHT-OH, BHT-CH2OH, BHT-CHO, BHT-COOH were identified as degradation products of BHT. All degradation products did not build up over the study period but declined over time.

As overall result the half-life (DT50) of BHT in soil is less than 24 hours at a temperature of 25 °C.

BHT was quite unstable in soils and approximately 20 % of the applied BHT was degraded immediately after treatment while recovery of 14C ranged from 90 to 100 %. BHT as well as its degradation products are quite biodegradable and do not accumulate in soil.