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Environmental fate & pathways

Biodegradation in soil

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Reference
Endpoint:
biodegradation in soil
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-Oct-2017 to 25-09-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)
GLP compliance:
yes (incl. certificate)
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
USDA (US Department of Agriculture)
Year:
2017
Soil no.:
#1
Soil type:
sandy loam
% Clay:
11
% Silt:
22
% Sand:
67
% Org. C:
2
pH:
6.9
CEC:
12.9 meq/100 g soil d.w.
Bulk density (g/cm³):
1.09
Details on soil characteristics:
SOIL COLLECTION AND STORAGE
- Geographic location: N 47°41.961ʹ × W 097°31.020ʹ
- Pesticide use history at the collection site: The collection area is pesticide free. Provided history indicates no treatment with the test substance or its structural analogs during the last three years
- Collection procedures: The test soil ('RMN soil') was collected using a shovel by Agvise Laboratories from Grand Forks, North Dakota on 13 October 2017 and was received by Smithers Viscient on 20 October 2017.
- Sampling depth (cm): 0-6"
- Storage conditions: Upon receipt, the soil was stored refrigerated (at approximately 2 to 8 °C) when not in use
- Storage length: 7 days before test initiation
- Soil preparation: The soil was passed through a 2-mm sieve to assure uniformity.

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture at 1/3 atm (%): 17.2
- Microbial biomass (as percentage of organic carbon): 1.31% OC at the start of the incubation
Soil No.:
#1
Duration:
121 d
Soil No.:
#1
Initial conc.:
1 other: µg/g
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
radiochem. meas.
test mat. analysis
Soil No.:
#1
Temp.:
20°C
Microbial biomass:
1.31
Details on experimental conditions:
1. PRELIMINARY EXPERIMENTS: A 14-day preliminary study was conducted to (1) develop a processing method for extraction of [14C]hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate from a soil test system, (2) to develop an analytical method for quantification of [14C]hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate and its transformation products, and (3) to determine a sampling schedule for the corresponding definitive study.

2. EXPERIMENTAL DESIGN (see representative test system in attached background material)
- Soil preincubation conditions (duration, temperature if applicable): The vessels were aerated continuously with hydrated air, in the dark, in an environmental chamber set to maintain a temperature of 20 ± 2 °C for 12 days until dosing.
- Soil condition: fresh
- Soil: 50 g/replicate
- Control conditions: control vessels, dosed with an equivalent volume of methanol (80 µl) were incubated under similar conditions for determination of biomass at the start of the study, at approx. day 63 and at the end of incubation.
- No. of replication controls, if used: 2 (only used for determination of biomass)
- No. of replication treatments: 2
- Test apparatus (Type/material/volume): Test vessels consisted of 250-ml glass bottles with screw caps and septa.
- Details of traps for CO2 and organic volatile, if any: the test system was connected to volatile trapping solutions of ethylene glycol and two 1.0 M KOH traps.
- Identity and concentration of co-solvent: methanol

Test material application
- Volume of test solution used/treatment: 80 µl
- Application method: The application solution was added dropwise onto the soil surface and the soil phase was gently mixed.
- Is the co-solvent evaporated: No

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: Soil moisture was monitored throughout the incubation period and maintained at pF 2.0. Soil samples were weighed approx. once every two weeks and any loss in weight was compensated for by addition of purified regagent water.
- Continuous darkness: Yes

3. OXYGEN CONDITIONS
- Methods used to create the aerobic conditions: During acclimation and incubation, the test systems were maintained under aerobic conditions by drawing humidified air over the soil samples.

4. SAMPLING DETAILS
- Sampling intervals: 0, 3, 14, 30, 59 and 121 days of incubation
- Sampling method for soil samples: At each sampling interval, the entire soil sample in each vessel was transferred to individual 500-mL Nalgene round bottles, using the first extraction solvent.
- Method of collection of CO2 and volatile organic compounds:Volatile trapping solutions for the soil test systems were analyzed beginning at day 3 and at frequent intervals thereafter for total radioactivity. Ethylene glycol and potassium hydroxide volatile trapping solutions were removed from each test system, volumes recorded, and analyzed by LSC for quantification of volatile radioactivity. Potassium hydroxide (KOH) traps were brought back up to original volume, if necessary, with purified reagent water prior to LSC analysis. The presence of 14CO2 in the potassium hydroxide traps was confirmed by barium chloride (BaCl2) precipitation for representative volatile traps.
- Sampling intervals/times for:
> Moisture content: once every 2 weeks
> Sample storage before analysis: Not applicable, the soil samples were processed and analyzed immediately after sampling. Soil samples not directly needed for analysis were stored in a freezer. Post-extraction solids and volatile trapping solutions were stored at room temperature.
Soil No.:
#1
% Recovery:
93.7
St. dev.:
2.4
Soil No.:
#1
DT50:
117 d
Type:
(pseudo-)first order (= half-life)
Temp.:
20 °C
Transformation products:
no
Remarks:
No transformation products, except for 14CO2, have been identified during the study.
Details on transformation products:
No transformation products, except for 14CO2 were identified during the study since no other regions of radioactivity were observed in the soil extract chromatograms. Therefore it is concluded that mineralization is the major route of the degradation. Since identification was not performed to assist in a proposed pathway, the speculated pathway is that the benzene ring was opened through ortho-catechol cleavage by the microbes and released the CO2 as the transformation product.
Evaporation of parent compound:
no
Volatile metabolites:
yes
Details on results:
TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR AND MINOR TRANSFORMATION PRODUCTS
- The distribution of [14C]Hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate and transformation products in test soil as percent of applied radioactivity (% AR) is presented in Table 2 (see section 'Any other information on results' below). No transformation products were identified during the study since no other regions of radioactivity were observed in the soil extract chromatograms.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: The material balance was calculated for each sample by summing calculated radioactivity in soil extracts, non-extractable residues, and volatile trapping solutions. Volatile radioactivity was calculated as cumulative for each sampling interval. The material balance in the test soil as percent of applied radioactivity (% AR) is presented in Table 1 (see section 'Any other information on results' below).

EXTRACTABLE RESIDUES
- % of applied amount at day 0: 91.7
- % of applied amount at end of study period: 45.4

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0: 0.669
- % of applied amount at end of study period: 14.3

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 35.9% The presence of 14CO2 was confirmed in representative potassium hydroxide traps by precipitation of 14CO2 with BaCl2 in the form of barium carbonate (Ba14CO3).

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: 0.0585%

RESULTS OF SUPPLEMENTARY EXPERIMENT:
Since the percentage of non-extractable residues was >10% at sampling on day 121, additional characterization of the residue was performed. Following combustion analysis, day 21 post-extraction solid samples were further extracted with polar solvent (tetrahydrofuran) and a non-polar solvent (hexane). The results show that 0.13%AR was extracted with tetrahydrofuran, nothing was detected after extraction with hexane.
Radioactivity remaining in the non-extractable residue samples after the polar and non-polar extractions was characterized by fractionation into fulvic acid, humic acid, and humin fractions. These data show that 12.4% of the NER (14.3 % AR) at the end of the study was associated with the humin fraction while 1.21 %AR and 0.61%AR are associated to fulvic and humic fraction, respectively.

Table 1: Material Balance in test Soil as Percent of Applied Radioactivity (% AR)

Sampling
Day

%
Soil Extracts

%
Non-Extractable
Residues

%
14CO2

%
Volatile Organic
Compounds

%
Total Material
Balance

0

93.1

0.599

NA

NA

93.7

0

90.3

0.739

NA

NA

91.1

3

88.6

1.98

1.68

-

92.3

3

91.8

2.07

1.68

-

95.6

14

83.5

2.60

7.88

-

94.0

14

83.6

3.05

7.88

-

94.5

30

79.5

3.90

12.6

0.0158

96.1

30

72.3

6.08

12.6

0.0158

91.0

59

60.8

5.26

23.9

0.0459

90.1

59

66.2

4.28

23.9

0.0459

94.4

121

43.2

13.7

35.9

0.0585

92.8

121

47.5

14.8

35.9

0.0585

98.3

 

Table 2: Distribution of [14C]Hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate and Transformation Products in Test Soil as Percent of Applied Radioactivity (% AR)

Sampling
Day

%

[14C]hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate

%
Others

0

93.1

-

0

90.3

-

3

88.6

-

3

91.8

-

14

83.5

-

14

83.6

-

30

79.5

-

30

72.3

-

59

60.8

-

59

66.2

-

121

43.2

-

121

47.5

-

 

Conclusions:
[14C]hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate degraded at a moderate rate over the incubation period under the conditions of this aerobic soil study, with a DT50 value of 117 days. No transformation products, except for 14CO2, have been identified during the study.
Executive summary:

The aerobic transformation of [14C]hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate in soil was studied in a GLP compliant study according to OECD 307. The rate and route of the transformation was studied in one representative soil, a sandy loam. The test system consisted of 50-g dry weight soil aliquots, which were adjusted to a appropriate moisture levels (approximately pF 2.0), connected to volatile trapping solutions, and incubated under aerobic conditions in the dark at 20 ± 2°C. [14C]hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate was applied at a nominal concentration of 1.0 μg/g.

Soil samples were analyzed at 0, 3, 14, 30, 59, and 121 days of incubation. Soil samples were extracted and analyzed by liquid scintillation counting (LSC) and high performance liquid chromatography with radiometric detection (HPLC-RAM) for determination and profiling of extractable residues. The post-extraction solids (PES) were combusted and analyzed by LSC for determination of non-extractable residues. The volatile traps were analyzed by LSC for determination of 14CO2 and volatile organics. Representative PES samples were additionally characterized by harsh extraction and organic matter fractionation. Representative volatile traps were additionally characterized by barium chloride precipitation.

The average material balance ranged from 92.3 to 95.5% applied radioactivity (% AR) over the course of the study. [14C]hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate degraded at a moderate rate over the incubation period under the conditions of this aerobic soil study, with a DT50 value of 117 days. No transformation products, except for 14CO2 which was present at 35.9% of the applied radioactivity at the end of the test period, have been identified during the study. Therefore it is concluded that mineralization is the major route of the degradation of [14C]hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate.

Description of key information

The aerobic transformation of [14C]hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate in soil was studied in a GLP compliant study according to OECD 307. The rate and route of the transformation was studied in one representative soil, a sandy loam.

[14C]hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate degraded at a moderate rate over the incubation period under the conditions of this aerobic soil study, with a DT50 value of 117 days. No transformation products, except for 14CO2, have been identified during the study.

Key value for chemical safety assessment

Half-life in soil:
117 d
at the temperature of:
20 °C

Additional information