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Diss Factsheets

Administrative data

Endpoint:
biodegradation in soil: simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA Subdivision N, Section 162-1
GLP compliance:
yes (incl. QA statement)
Test type:
laboratory

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4,6-tri-sec-butylphenol
EC Number:
227-572-6
EC Name:
2,4,6-tri-sec-butylphenol
Cas Number:
5892-47-7
Molecular formula:
C18H30O
IUPAC Name:
2,4,6-tris(butan-2-yl)phenol
Radiolabelling:
yes

Study design

Oxygen conditions:
aerobic
Soil classification:
other: Three representative field soils I (Hoefchen am Hohensee, Germany, HaH, silt loam); II (Wurmwiese, Germany, WuW; sandy loam) and III (Dollendorf II, Germany; Doll, Clay loam) were used in the study.
Year:
2009
Soil propertiesopen allclose all
Soil no.:
#1
Soil type:
silt loam
% Clay:
15
% Silt:
52
% Sand:
33
% Org. C:
2.3
pH:
6.3
CEC:
12.1 meq/100 g soil d.w.
Bulk density (g/cm³):
1.07
Soil no.:
#2
Soil type:
sandy loam
% Clay:
9
% Silt:
30
% Sand:
61
% Org. C:
2
pH:
5.3
CEC:
9.9 meq/100 g soil d.w.
Bulk density (g/cm³):
1.15
Soil no.:
#3
Soil type:
clay loam
% Clay:
35
% Silt:
36
% Sand:
29
% Org. C:
4.4
pH:
7.2
CEC:
19.7 meq/100 g soil d.w.
Bulk density (g/cm³):
0.97
Details on soil characteristics:
Collection of Soils
All soils were sampled freshly from the fields and shipped directly to the laboratory. The sampling depth was about 20 cm. Grass cover and roots were removed for this study before drying.

Properties of Soils
The pH value of the soils suspended in aqueous 0.01 M CaCl2 solution was in the range of pH 5.3 to 7.2, the organic carbon content was in the range of 2.0% to 4.4%, and the clay content was between 9% and 35% (USDA). The data of the individual batches were determined by AGVISE Laboratories (604 Highway 15 West, P.O. Box 510, Northwood, ND 58267, USA) as a separate investigation (under GLP) with the exception of WHCmax. (in-house).
The microbial biomass was performed at test initiation and at the termination of the study (Laboratory Dr. T. Leicher, BCS-D-EnSa/ETX (formerly BCSD- ETX).

Preparation of Soils
The soils were carefully dried in the lab at ambient temperature and sieved to a particle size of ≤ 2 mm about a week before application. Subsequently the soil moisture was determined by drying aliquots of the soils at 105 °C using a balance for humidity determination (Mettler Toledo HB43). Aliquots of the soils, each corresponding to 100-g dry weight, were filled into 300-mL Erlenmeyer flasks. Water (Milli-Q) was added to each soil aliquot in a way that the final moisture (taking into account the water included in the application solution) was 55% of the maximum water holding capacity (WHCmax). The weights of all test vessels were recorded and the samples were pre-equilibrated at 20 °C in the dark for about three days.
Duration of test (contact time)open allclose all
Soil No.:
#1
Duration:
62 d
Soil No.:
#2
Duration:
62 d
Soil No.:
#3
Duration:
62 d
Initial test substance concentrationopen allclose all
Soil No.:
#1
Initial conc.:
0.45 mg/kg soil d.w.
Based on:
act. ingr.
Soil No.:
#2
Initial conc.:
0.45 mg/kg soil d.w.
Based on:
act. ingr.
Soil No.:
#3
Initial conc.:
0.45 mg/kg soil d.w.
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
radiochem. meas.

Results and discussion

Material (mass) balanceopen allclose all
Soil No.:
#1
% Recovery:
100.5
St. dev.:
3.1
Remarks on result:
other: The total mean recoveries in terms of percent of the applied radioactivity ± 3.1%
Soil No.:
#2
% Recovery:
99.3
St. dev.:
1.3
Remarks on result:
other: The total mean recoveries in terms of percent of the applied radioactivity ± 1.3%
Soil No.:
#3
% Recovery:
100
St. dev.:
2.8
Remarks on result:
other: The total mean recoveries in terms of percent of the applied radioactivity ± 2.8%
Half-life / dissipation time of parent compoundopen allclose all
Soil No.:
#1
DT50:
9.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Soil No.:
#1
DT50:
32.9 d
Type:
other: DT90 (d)
Temp.:
20 °C
Soil No.:
#2
DT50:
8.4 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Soil No.:
#2
DT50:
27.9 d
Type:
other: DT90 (d)
Temp.:
20 °C
Soil No.:
#3
DT50:
8.9 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Soil No.:
#3
DT50:
29.6 d
Type:
other: DT90 (d)
Temp.:
20 °C
Transformation products:
no
Evaporation of parent compound:
not measured
Volatile metabolites:
yes
Residues:
yes
Details on results:
Material Balance
In the present study, 388 kBq of test item [ring-UL-14C]2,4,6-tri-sec.-butyl-phenol have been applied to each flask containing 100 g (as dry matter) of soil. The total applied radioactivity was taken as 100 % for the calculation of material balances.
The total recovery of RA for all soils was in the range of 97.0-102.5% (HaH), 97.8- 101.2% (Wurmwiese) and 96.9-103.5% of AR (Doll) for the test systems indicating that no radioactivity was lost during the course of the study (mean values of duplicates).

The amounts of bound residues increased during the course of the study. Volatile metabolites representing carbon dioxide increased steadily with time indicating that bound residues were mineralized.

Distribution and Composition of Residues

Mineralization (Trapped 14CO2) and Other Volatiles
The amount of formed 14CO2 increased steadily during the entire study period. At the end of the study, 60 days after application, evolved 14CO2 was 23.0%, 34.6% and 23.0% of the applied RA in soils HaH, WuW and Doll, respectively
Volatile organic compounds were detected with up to maximum 1.1% of AR.

Extracted Radioactivity
Extractable 14C-residues decreased from 93.9%, 90.7% and 96.1% of the applied amount at DAT-0 to 15.6%, 17.8% and 15.3% of the applied RA at the end of the study in soils Hoefchen a.H., Wurmwiese and Dollendorf, respectively.

During the course of the experiment, a number of zones of RA were detected in the extracts and quantified by HPLC. The biotransformation (sum of organic ambient (cold) and organic hot extracts, ID: CE and HE) expressed as % of AR was calculated.

The parent compound decreased from 91.4%, 85.6% and 93.9% of the applied amount at DAT-0 to 3.1%, 3.3% and 4.1% of the applied radioactivity at the end of the study, respectively.

Carbon dioxide was the main degradation product (up to 34.6% of AR). During the course of the experiment, additionally up to 6 HPLC peaks indicating degradation products were observed and quantified. No degradation product was detected in significant amounts (i.e. > 10% of AR) with the exception of peak HPLC-Ret 14.8 in soil WuW. This zone had reached its maximum with 14.0% of AR at day 31 and decreased then rapidly to 7.0% of AR at day 47.

Non-extracted Radioactivity (Bound Residues)
The remaining RA measured in the soil after cold and hot extraction was determined for each individual test vessel. The formation of bound residues increased with the overall metabolism of test compound. Although the soils were thoroughly extracted, non-extractable residues were formed at DAT-0 (8.6%, 10.5% and 7.5% in soils HaH, WuW and Doll), and increased to 60.6% (DAT-60), 46.8% (DAT-60), and 61.2% (DAT-60), respectively.
The amount of bound residues corresponds with the very high amount of mineralization, which is also observed in other studies and can be explained by irreversible binding and microbial mediated incorporation of transformation products to the organic matter.

Characterization of Bound Residues
Bound residues were separated into fractions of humin, humic acid and fulvic acid.
After extractions, the 20 g of remaining soil (Doll) of the last sampling day (ID: PC44D60Ba) was analyzed.

Identification and Characterization of Transformation Products
Transformation products were not identified.

Kinetic Analysis of Data

Kinetics of Parent Compound Degradation
The data for the parent compound 2,4,6-tri-sec-butyl-phenol were evaluated with the program KINGUI, Version 1.1 based on MatLab®, Version 7.0.4.365. The initial concentration at day 0 was included in the parameter optimization procedure. The degradation rate was fitted with single first order kinetics (SFO), with respect to the number of sampling intervals and the fast degradation.

The half-lives of 2,4,6-tri-sec-butyl-phenol were calculated according to FOCUS as 9.9, 8.4, and 8.9 days for soil HaH, WuW and Doll, respectively. The DT90 were in the range of 27.9 days to 32.9 days.

Decline of Metabolites
One metabolite peak (HPLC-Ret 14.8) was detected in one soil (WuW) at one sampling interval in higher amounts (i.e. > 10% of AR). This zone had reached its maximum with 14.0% of AR at day 31 and then decreased fast to 7.0% of AR at day 47.

Degradation Pathway
The test substance 2,4,6-tri-sec-butyl-phenol is well degraded in soil by formation of bound residues and mineralization to carbon dioxide, finally.

Storage Stability
Extracts were stored in a deep freezer. Extracts were analyzed within a few days after samples. No degradation was observed within that period.

Additional Orientating Investigations with Sterile Soil:
Test vessels with each soil were sterilized before application and analyzed at day 60 (sample ID: -60s) in the same way as the non-sterile samples. After processing in an autoclave, the soils exhibited no microbial activity.
No degradation of 2,4,6-tri-sec-butyl-phenol and none formation of bound residues was observed after incubation for 60 days in case of soils HaH and WuW indicating that the degradation is due to microbial activity. In soil Dollendorf the degradation was slow from 93.9% to 77.6% due to formation of minor amount of bound residues (21.6%) in comparison to non-sterile soil (61.2%). It is considered that in this sterile soil Dollendorf 14C carbon dioxide was formed from non-extractable residue, because 14C-residues were not detected by HPLC.
The non-formation of non-extractable residues in case of sterile soils shows, that in the biological active samples the formation of non-extractable residues is due to microbial activity. Therefore it is considered that the non-extractable residues are irreversibly bound to soil and can be regarded as bound residues.

Applicant's summary and conclusion

Conclusions:
Major Outcome of Study
The data gathered in the current laboratory investigation demonstrated, that the test item 2,4,6-tri-sec-butyl-phenol had well degraded in all 3 soils with a DT50 of 8.4 to 9.9 days (20 °C) according to a single first order kinetics (SFO)
The formation of bound residues is the major degradation step. Metabolites were detected in low amounts only. At the end of incubation, the formation of 14C carbon dioxide was the main degradation path. The amount increased to 23.0% - 34.6% indicating the complete mineralization of the test compound and bound residues.

Significance of Results to Environmental Behavior of Test Compound
The test item 2,4,6-tri-sec-butyl-phenol degraded fast in soil under aerobic conditions. The high amount of 14CO2 indicates the complete mineralization.
The formation of radioactive carbon dioxide was high in all soils, reaching levels of 57.5%, 58.8% and 54.8% of the applied radioactivity in soils I to III, respectively, after 62 days of incubation.
Executive summary:

The biotransformation of [ring-UL-14C]2,4,6-tri-sec-butyl-phenol was studied in three different soils, i.e. silt loam (Hoefchen am Hohenseh, organic carbon 2.3%, pH 6.3, ID: HaH, Burscheid, Germany, sandy loam (Wurmwiese, organic carbon 2.0%, pH 5.3, ID: WuW, Monheim, Germany), and clay loam (Dollendorf II, organic carbon 4.4%, pH 7.2, ID: Doll, Blankenheim, Germany) for 60 days under aerobic conditions in the dark at 20 °C and about 55% WHCmax (max. water holding capacity). [Ring-UL-14C]2,4,6-tri-sec-butyl-phenol was applied at the rate of 407.6 μg/kg soil, which is equivalent to a field rate of about 164 g/ha (calculated for 2.5 cm soil depth).

The experiment was conducted in accordance with the OECD Guideline 307 and US EPA Subdivision N, Section 162-1 and in compliance with the GLP standards of OECD, FIFRA and JMAFF. The test system consisted of biometer-type flasks with traps for the collection of CO2 and volatile organic compounds. Samples were analyzed after 0, 31, 47, and 60 days of incubation. Additional samples were sterilized by pressure steam and analysed after 60 days. The soil samples were extracted three times at ambient temperature and once by hot extraction under reflux conditions with acetonitrile/water (80/20, v/v). The 2,4,6-tri-sec-butyl-phenol residues were analyzed by LSC and HPLC with 14C-detection. Identity of the applied substance was confirmed by GC-MS and NMR techniques.

The test conditions outlined in the study protocol maintained throughout the study. The material balance for the biological active samples was in the range of 97.0%- 102.5% (HaH), 97.8%-101.2% (WuW) and 97.7%-103.5% of AR (Doll) for the test systems indicating that no radioactivity was lost during the course of the study (mean values of duplicates).

The parent compound decreased from 91.4%, 85.6% and 93.9% of the applied amount at DAT-0 to 3.1%, 3.3% and 4.1% of the applied radioactivity at the end of the study, respectively.

The half-lives of 2,4,6-tri-sec-butyl-phenol were calculated according to single first order (SFO) as 9.9, 8.4, and 8.9 days (mean 9.1 days) for soil HaH, WuW and Doll, respectively.

The main step in the degradation was formation of non-extractable residues followed by emerging of carbon dioxide.

Due to the exhaustive cold and hot extraction the portions of non-extracted residues at DAT-0 were low (8.6%, 10.5%, and 7.5% in soils HaH, WuW and Doll), and increased to 60.6% (DAT-60), 46.8% (DAT-60) and 61.2% (DAT-60).

Extractable 14C-residues decreased from 93.9%, 90.7% and 96.1% of the applied amount at DAT-0 to 15.6%, 17.8% and 15.5% of the applied RA at the end of the study, respectively.

The amount of formed 14CO2 increased steadily during the entire study period. At theend of the study, 60 days after application, evolved 14CO2 was 23.0%, 34.6% and 23.0% of the applied RA in soils HaH, WuW and Doll, respectively.

Besides carbon dioxide, no degradation product was detected during the course of the incubation in significant amounts (i.e. > 10% of AR) with the exception of peak HPLC-Ret 14.8 in soil WuW. This zone reached its maximum with 14.0% of AR at day 31 and then decreased fast to 7.0% of AR at day 47.

No degradation was observed in samples with sterile soils HaH and WuW. In sterile soil Dollendorf, the test substance decreased slowly to 77.6% of AR at day 60. Nonextractable residues were formed only in minor amounts (5.7%, 8.2% and 21.6% for

soils HaH, WuW and Doll, respectively) indicating that the formation of bound residues in soils was due to biological activity. Carbon dioxide was not evolved in the sterile soils.

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