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Biodegradation in soil

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Endpoint:
biodegradation in soil: simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.06.2017 - 06.12.2019
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:
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. certificate)
Test type:
laboratory
Specific details on test material used for the study:
14C-labelled test item:
Specific activity: 2.22 GBq/mmol corresponding to 9.9865 MBq/mg
Radiolabelling:
yes
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
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.
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:
- 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.

- 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).

- 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).
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 their metabolites: high vapour pressure; 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:
Characterisation of the microbial status of the soils
Biomass measurements of the four soils were performed by means of the substrate induced respiration method. 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), 265.7 mg Cmic/kg (RefeSol 02-A), 531.8 mg Cmic/kg (LUFA 2.4) and 938.7 mg Cmic/kg dry mass (LUFA 6S). Correlated to the organic carbon (Corg) content of the soils 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. 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).
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

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 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.

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.

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 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 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

50.5    

 10.9

 9.6

 1.2

 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

 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

The obtained data sets 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.

Endpoint:
biodegradation in soil
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Soil treatment: The soils were passed through a 2 mm sieve and stored at 5 ºC prior to use. Each undried soil equivalent to 40 g on a dry weight basis was taken into a 100 mL beaker, moistened to 40% of maximum water-holding capacity and incubated for a week at 25 ºC in the dark. After preincubation, methanol solution of 14C-BHT (50 µL) was added to each soil and mixed thoroughly to give a concentration of 1 ppm. Each of these soil samples was placed in a 3-liter glass jar covered with aluminium foil and kept at 25 ± 2 ºC. Each jar was continuously purged with CO2-free air at 100 mL/h and the effluent air was passed serially through a polyurethane foam plug and gas-washing bottles containing 400 mL of 0.5N NaOH solution to trap volatile 14C including 14CO2. For sterilized condition, moist soil samples were autoclaved at 20 psi and 120 ºC and then treated with 1 ppm of 14C-BHT.

At specified intervals, each polyurethane plug was removed and eluted three times with 30 mL of methanol. Each of the soil samples was extracted with 180 mL of ethyl acetate-1N HCl (2/1). The soil residue separated by centrifugation at 3000 rpm for 10 minutes was further extracted with 200 mL of methanol-1N HCl (3/1) by refluxing for 1 hour. An aliquot of each combined extracts was radioassayed by LSC while the remaining portions were evaporated to ca. 2 mL at 30 ºC for TLC analysis.
GLP compliance:
no
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
not specified
Year:
1979
Soil no.:
#1
Soil type:
other: light clay
% Clay:
29
% Silt:
40
% Sand:
31
% Org. C:
15.3
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
% Org. C:
2.5
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
% Org. C:
2.7
pH:
7
CEC:
9.6 meq/100 g soil d.w.
Soil No.:
#1
Duration:
24 d
Soil No.:
#2
Duration:
24 d
Soil No.:
#3
Duration:
24 d
Soil No.:
#1
Initial conc.:
1 ppm
Based on:
not specified
Soil No.:
#2
Initial conc.:
1 ppm
Based on:
not specified
Soil No.:
#3
Initial conc.:
1 ppm
Based on:
not specified
Parameter followed for biodegradation estimation:
CO2 evolution
Details on experimental conditions:
Soil treatment: The soils were passed through a 2 mm sieve and stored at 5 ºC prior to use. Each undried soil equivalent to 40 g on a dry weight basis was taken into a 100 ml beaker, moistened to 40% of maximum water-holding capacity and incubated for a week at 25 ºC in the dark. After preincubation, methanol solution of 14C-BHT (50 µl) was added to each soil and mixed thoroughly to give a concentration of 1 ppm. Each of these soil samples was placed in a 3-liter glass jar covered with aluminium foil and kept at 25 ± 2 ºC. Each jar was continuously purged with CO2-free air at 100 ml/h and the effluent air was passed serially through a polyurethane foam plug and gas-washing bottles containing 400 ml of 0.5N NaOH solution to trap volatile 14C including 14CO2. For sterilized condition, moist soil samples were autoclaved at 20 psi and 120 ºC and then treated with 1 ppm of 14C-BHT.

At specified intervals, each polyurethane plug was removed and eluted three times with 30 ml of methanol. Each of the soil samples was extracted with 180 ml of ethyl acetate-1N HCl (2/1). The soil residue separated by centrifugation at 3000 rpm for 10 minutes was further extracted with 200 ml of methanol-1N HCl (3/1) by refluxing for 1 hour. An aliquot of each combined extracts was radioassayed by LSC while the remaining portions were evaporated to ca. 2 ml at 30 ºC for TLC analysis.
Key result
% Degr.:
63 - 82
Parameter:
radiochem. meas.
Sampling time:
1 d
Key result
% Degr.:
> 77 - 92
Parameter:
radiochem. meas.
Sampling time:
24 d
Transformation products:
yes
No.:
#1
No.:
#2
Details on transformation products:
The findings suggest that BHT was altered to non-volatile products mainly by biological factors in nonsterilised soils soon after treatment and further degraded to 14CO2 via several intermediates. In soils, more than 10 degradation products were present, among which, five products were identified. Of these products, BHT-OOH and BHT-OH were major components under nonsterilised condition and other products oxidised at 4-methyl group such as BHT-CH2OH, BHT-CHO and BHT-COOH were found in minor amounts. These products were also detected in sterilised condition.

- Description of biotransformation pathway: Fig II - Attached document

Evaporation of parent compound:
yes
Volatile metabolites:
yes
Residues:
yes

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%.

In non-sterilized condition, BHT was degraded promptly to leave only 11 to 16% one day after treatment. After 24 days, BHT decreased to 5% or less. No significant difference was observed between the soils tested. Accompanied with the decrease of 14C in soils, the amounts of 14C in polyurethane plugs and alkaline solutions gradually increased and reached to 5-14 and 21-29% in 24 days, respectively.

Even under sterilised condition, degradation of BHT proceeded, although much more slowly. One day after treatment, about half of the applied BHT remained in soil and gradually degraded afterwards; the 14C trapped in polyurethane plugs rapidly increased with time and accounted for 40 to 50% of the applied in 24 days, whereas 14C in alkaline solutions was less than 2% during the same period. A large portion of 14C in polyurethane traps proved to be intact BHT and the 14C in alkaline solutions was considered to be 14CO2 since more than 95% of the radioactivity was precipitated as Ba14CO3.

Conclusions:
BHT, as well as its degradation products, is quite biodegradable and hardly persists in the soil environment.
Executive summary:

According to the results obtained, BHT is relatively unstable in the three soils tested: With non sterilized soils about 63-82 % of BHT were decomposed after one day (about 1-2 % mineralized to CO2) and 77-92 % (21-29 % mineralized) after 24 days of incubation. Under sterilized conditions 25-35 % BHT were decomposed after one day and 27-41 % after 24 days, mineralization was negligible ( 2 %). After one day 57-68 % of BHT and after 24 days 50-61 % remained unchanged. Under non-sterilized conditions the amount of total volatile 14C was 26 to 42 % (21 to 29 % 14CO2).Under sterilized conditions the amount of volatile 14C was 43 to 56 % after 24 days ( 2 % 14CO2). From these results it can be concluded that BHT is altered to nonvolatile products mainly by biological processes. In soil more than 10 degradation products were found. As major decomposition products BHT-OOH and BHT-OH were identified

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 %.

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.

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.

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.