4,4'-methylene bis(dibutyldithiocarbamate)

Biodegradation in soil

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 September 2019 to 22 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

GLP compliance:

yes

Remarks:

Exception: No claim of GLP Compliance is made for the soil collection data or the kinetic evaluation.

Test type:

laboratory

Specific details on test material used for the study:

Radiolabeled test material: 4,4’-methylene bis(dibutyldithiocarbamate), [CH2-14C]
Non-Radiolabeled test material (for co-chromatography): 4,4’-methylene bis(dibutyldithiocarbamate)

Radiolabelling:

yes

Remarks:

The test material, [14C]-4,4’-methylene bis(dibutyldithiocarbamate), was applied to the test system without radiodilution.

Oxygen conditions:

aerobic/anaerobic

Soil classification:

other: UK and USDA classification

Year:

2019

Soil no.:

#1

Soil type:

silt loam

% Clay:

19

% Silt:

64

% Sand:

17

% Org. C:

4.96

pH:

5.8

CEC:

20.4 meq/100 g soil d.w.

Bulk density (g/cm³):

0.94

Soil no.:

#2

Soil type:

sandy loam

% Clay:

15

% Silt:

20

% Sand:

65

% Org. C:

2.59

pH:

6.3

CEC:

16.8 meq/100 g soil d.w.

Bulk density (g/cm³):

1.04

Soil no.:

#3

Soil type:

loamy sand

% Clay:

5

% Silt:

10

% Sand:

85

% Org. C:

0.64

pH:

7.6

CEC:

5.1 meq/100 g soil d.w.

Bulk density (g/cm³):

1.37

Soil no.:

#4

Soil type:

clay

% Clay:

39

% Silt:

29

% Sand:

32

% Org. C:

3.27

pH:

7.8

CEC:

25.8 meq/100 g soil d.w.

Bulk density (g/cm³):

1.13

Details on soil characteristics:

- Geographic location:
Soil #1: Brierlow soil (Brierlow, Derbyshire, UK);
Soil #2: Calke soil (Calke, Derbyshire, UK);
Soil #3: Kennett soil (Kennett, Cambridgeshire, UK); and
Soil #4: South Witham soil (South Witham, Linolnshire, UK)
- Pesticide use history at the collection site: None within previous 5 years
- Collection procedures: Soils were obtained by LRA Labsoil (non-GLP)
- Handling and storage conditions: in accordance with the International Standard ISO 18400-206:2018 (Soil Quality - Sampling - Part 206)
- Soil preparation: Prior to use the soil was passed through a 2 mm sieve; air-drying of the soil was kept to a minimum.

Duration:

ca. 120 d

Initial conc.:

1 mg/kg soil d.w.

Based on:

other:

Remarks:

Application rate calculated from radioactivity and volume applied, and specific activity of the test material, and confirmed by liquid scintillation counting (LSC).

Parameter followed for biodegradation estimation:

radiochem. meas.

test mat. analysis

Soil No.:

#1

Temp.:

12 +/- 2 deg C

Humidity:

pF 2

Microbial biomass:

Aerobic: 814 mg C/kg soil (untreated, Day 0); 492 mg C/kg soil (untreated, Day 120); 312 mg C/kg soil (solvent-treated, Day 120)

Soil No.:

#2

Temp.:

12 +/- 2 deg C

Humidity:

pF 2

Microbial biomass:

Aerobic: 1260 mg C/kg soil (untreated, Day 0); 659 mg C/kg soil (untreated, Day 120); 552 mg C/kg soil (solvent-treated, Day 120)

Soil No.:

#3

Temp.:

12 +/- 2 deg C

Humidity:

pF 2

Microbial biomass:

Aerobic: 691 mg C/kg soil (untreated, Day 0); 558 mg C/kg soil (untreated, Day 120); 471 mg C/kg soil (solvent-treated, Day 120)

Soil No.:

#4

Temp.:

12 +/- 2 deg C

Humidity:

pF 2

Microbial biomass:

Aerobic: 1360 mg C/kg soil (untreated, Day 0); 1610 mg C/kg soil (untreated, Day 120); 2820 mg C/kg soil (solvent-treated, Day 120)

Soil No.:

#3

Temp.:

12 +/- 2 deg C

Humidity:

pF 2

Microbial biomass:

Untreated (Day 119): Anaerobic bacteria: 7.15x10^4 cfu/g; Anaerobic bacterial spores: 5.70x10^4 cfu/g / Solvent-treated (Day 119): Anaerobic bacteria: 6.80x10^4 cfu/g; Anaerobic bacterial spores: 6.95x10^4 cfu/g

Details on experimental conditions:

The test vessels were incubated under the study conditions for up to four weeks prior to test item application. Aliquots of a stock solution of radiolabeled test material were applied to the surface of individual soil samples (approximately 100 g soil on a dry weight basis) contained in glass dishes and housed in glass chambers, one for each soil type. For the rate of transformation evaluation, individual soil samples will be contained in glass dishes each containing approximately 50 g soil on a dry weight basis. The solvent was allowed to evaporate and the soil was mixed using a spatula. Each dish was weighed and distilled water added as necessary to maintain the moisture content at that equivalent to pF2.

Each chamber was incorporated into a separate gas flow-through system arranged in a series as follows: a humidifying vessel (with sintered stem for uniform gas dispersion) containing water to humidify the air-flow; a chamber containing the dishes of soil; a vessel containing ethyl digol (to trap organic volatile compounds); a vessel containing 1M aqueous potassium hydroxide solution with phenolphthalein indicator to trap 14CO2; and a non-return valve to prevent accidental backflow through the test apparatus. For the anaerobic phase, the non-return valve was placed prior to the humidifying vessel in the series, as nitrogen was pushed through the system (rather than air being drawn through the system). Each chamber only contained dishes of one soil type. During the acclimatization phase all traps except the humidifying water bottle were empty.

Samples for the route of transformation under anaerobic conditions will be set up and acclimatized once the DT50 under aerobic conditions were established in the same soil (or 30 days have elapsed, whichever was sooner).

Vessels (1000 mL) for microbiological activity measurement, each containing approximately 500 g soil on a dry weight basis, were connected in one or more series to separate gas flow systems. Vessels used for microbial activity measurements were not treated with the test item; however, two of the five vessels for each soil type were used as a solvent-control and were treated with an equivalent volume of organic solvent as used for test item application. Humidified air was passed through these vessels, but no traps were included.

The air flow was maintained at approximately 60 mL/minute. The test systems were incubated at 12 +/- 2 deg C in the dark in a temperature-controlled room.

The period of the aerobic acclimatization (post application, but prior to flooding) for the anaerobic incubation was 30 days. Information from the aerobic route of transformation soil was used to determine the duration of the aerobic phase. At the end of this acclimatization period, the soil in each bottle was flooded with freshly drawn high purity degassed water (purged with nitrogen) to a depth of about 5 cm. The same volume of water was used in all bottles including those for microbial activity and other parameters (redox potential, pH and oxygen). The bottles was returned to the flow-through systems; the gas flow was changed from air to nitrogen and was maintained at about 50 mL/minute.

Redox potential in water and soil and pH and dissolved oxygen in water was measured at approximately monthly intervals in the bottles set up for this purpose. More frequent measurements were made during the initial period of the anaerobic phase to confirm that anaerobic conditions were established.

Duplicate dishes of each soil type were taken for analysis immediately after application and after 7, 13, 30, 62, 90 and 120 days of aerobic incubation. Duplicate dishes of each soil type were taken for analysis immediately after application, at the end of the acclimatization period, and after 7, 14, 28, 60, 90 and 119 days of anaerobic incubation. Trapping solutions were taken for analysis with associated vessels at each sampling interval. Additionally, trapping media associated with all remaining vessels were taken for analysis (and replaced with fresh media as appropriate) after 14 and 30 days of acclimatization period (anaerobic systems only), and after 14 days of aerobic and anaerobic incubation, and subsequently at approximately two-weekly intervals during the aerobic and anaerobic incubations (to coincide with vessel sampling intervals).

One untreated vessel of each soil type, established for the determination of microbial biomass using the substrate-induced respiration method, was taken for analysis on the day of application. One untreated and one solvent-treated vessel of each soil type were taken for analysis after 120 days of aerobic incubation. Soil microbiological activity, as total viable counts, was measured after 119 days of anaerobic incubation; one untreated and one solvent-treated vessel were taken for analysis.

At the final sampling interval for each soil and incubation condition, all remaining samples were transferred to storage at -10 to -30 deg C.

Soil No.:

#1

% Recovery:

99.8

Remarks on result:

other: % applied radioactivity (Day 120)

Remarks:

aerobic

Soil No.:

#2

% Recovery:

96.9

Remarks on result:

other: % applied radioactivity (Day 120)

Remarks:

aerobic

Soil No.:

#3

% Recovery:

100

Remarks on result:

other: (actual) 101.4% applied radioactivity (Day 120)

Remarks:

aerobic

Soil No.:

#4

% Recovery:

98.4

Remarks on result:

other: % applied radioactivity (Day 120)

Remarks:

aerobic

Soil No.:

#3

% Recovery:

97.9

Remarks on result:

other: % applied radioactivity (Day 119)

Remarks:

anaerobic

Key result

Soil No.:

#1

DT50:

285 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: first order multi compartment (FOMC) model

Remarks:

aerobic; parent material

Key result

Soil No.:

#2

DT50:

119 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: single first order (SFO) model

Remarks:

aerobic; parent material

Key result

Soil No.:

#3

DT50:

286 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: single first order (SFO) model

Remarks:

aerobic; parent material

Key result

Soil No.:

#4

DT50:

200 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: single first order (SFO) model

Remarks:

aerobic; parent material

Key result

Soil No.:

#1

DT50:

78.5 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: single first order (SFO) model; major degradate (S-(((dibutylcarbamothioyl)thio)methyl) dibutylcarbamothioate)

Key result

Soil No.:

#3

DT50:

516 d

Type:

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

Temp.:

12 °C

Remarks on result:

other: single first order (SFO) model

Remarks:

anaerobic; parent material

Transformation products:

yes

Remarks:

The parent material degraded to one major degradate (initially designated as ‘Unknown 1’) and five low level degradates, was incorporated into bound (non-extractable) radioactivity, or was mineralized to carbon dioxide.

Details on transformation products:

The parent material degraded to one major transformation product (degradate), initially designated as ‘Unknown 1’ (up to a mean of 19.0% applied radioactivity), and five low level unidentified transformation products (mean recovery did not exceed 6.6% applied radioactivity for any one minor transformation product on any sampling occasion), was incorporated into bound (non-extractable) radioactivity, or was mineralized to carbon dioxide. A major degradate selected for identification was defined in this study, based on Regulation (EC) No 1107/2009, as a degradate which account for: (i) more than 10% applied radioactivity at any time; (ii) more than 5% applied radioactivity at two or more sequential sampling intervals; or (iii) more than 5% applied radioactivity which have not reached maximum formation at the end of the incubation period.

Identification of the major transformation product designated as ‘Unknown 1’ was attempted
by liquid chromatograph with mass spectrometry (LC-MS/MS). Analysis tentatively identified Unknown 1 as S-(((dibutylcarbamothioyl)thio)methyl) dibutylcarbamothioate, an oxidative transformation product of 4,4’-methylene bis(dibutyldithiocarbamate).

The proposed pathway for the transformation of 4,4’-methylene bis(dibutyldithiocarbamate)
in aerobic and anaerobic soils is provide in the attached figure.

Details on results:

RECOVERY OF RADIOACTIVITY
Total recoveries of radioactivity (mass balances) ranged from 96.1 to 104.1% of applied radioactivity (AR) in the four soils incubated under aerobic conditions for 120 days and 96.9 to 99.6% AR in the soil (#3, Kennett) incubated under anaerobic conditions for 119 days.

Extractable radioactivity showed a small decline during the aerobic incubation of four soils
treated with the radiolabeled test material. In Brierlow soil (#1), mean extractable radioactivity ranged from 95.7% AR at time zero to 82.4% AR after 120 days. In Calke soil (#2), mean extractable radioactivity ranged from 95.4% AR at time zero to 73.4% after 120 days. In Kennett (#3) soil, mean extractable radioactivity ranged from 98.6% AR at time zero to 83.1% after 120 days. In South Witham (#4) soil, mean extractable radioactivity ranged from 95.6% AR at time zero to 77.3% after 120 days.

Non-extractable radioactivity increased slightly during the aerobic incubation of four aerobic soils treated with the radiolabeled test material, from 3.3-8.4% AR at time zero to 9.5-11.6% AR after 120 days. No organic volatiles were detected in the aerobic soils, but mean radioactivity measured in the trapping solutions (attributed to 14CO2) reached 6.9-12.2% AR in the four aerobic soils after 120 days. The radioactivity of the non-extractable residues in the four aerobic soils on Day 120 was characterized as the radioactivity of fulvic and humic acids and, by difference, humin, which accounted for 2.0-3.1%AR, 1.0-3.1%AR and 4.6-9.3%AR, respectively.

Mean extractable radioactivity also showed a small decline during the incubation of the anaerobic Kennett soil (#3) treated with the radiolabeled test material, ranging from from 95.5% AR at time zero to 85.3% AR 119 days under anaerobic conditions. Non-extractable radioactivity increased slightly during the aerobic incubation of the anaerobic soil, reaching a mean maximum of 7.6% AR on Day 90. No organic volatiles were detected in the anaerobic soil, but the mean radioactivity measured in the traps (attributed to 14CO2) ranged from 2.0 to 3.5% AR over 119 days under anaerobic conditions (post-flooding).

CHROMATOGRAPHIC ANALYSIS
Proportions of parent test material and its radioactive degradates were determined by HPLC analysis for soil extracts in the aerobic and anaerobic aquatic systems.

In the aerobic Brierlow soil (#1), parent test material declined from a mean of 89.4% AR at time zero to 58.1% AR after 120 days of incubation. In the aerobic Calke soil (#2), parent test material declined from a mean of 88.6% AR at time zero to 46.5% AR after 120 days of incubation. In the aerobic Kennett (#3) soil, parent test material declined from a mean of 93.9% AR at time zero to 70.1% AR after 120 days of incubation. In the aerobic South Witham (#4) soil, parent test material declined from a mean of 87.9% AR at time zero to 59.3% AR after 120 days of incubation.

In the anaerobic Kennett (#3) soil, parent test material declined from a mean of 93.2% AR to 79.4% AR during the 30-day acclimatization period, and continued to decline through the anaerobic incubation to 67.7% AR after 119 days of incubation.

One major degradate, preliminarily designated as Unknown 1 (RT 27.2 min), detected in the four aerobic soils and one anaerobic soil reached a maximum mean value of 19.0% AR (aerobic Brierlow soil #1, Day 62). See further details above regarding the identity of this transformation product. The remaining extractable radioactivity was attributed to up to five low level degradation products (aerobic soils) or four low level degradation products (anaerobic soil) and other components that did not constitute any discrete regions of radioactivity in the chromatograms.

KINETIC ANALYSIS
Kinetic analysis of the data was used to estimate the DT50 and DT90 values based on single first order and first order multi compartment models, where possible.

Conclusions:

The mean total recoveries of radioactivity ranged from 96.1 to 104.1% of applied radioactivity (AR) in the four soils incubated under aerobic conditions for 120 days, and 96.9 to 99.6% AR in the soil (#3, Kennett) incubated under anaerobic conditions for 119 days.

Extractable radioactivity declined during the aerobic incubation for all four soils treated with the radiolabeled test material, with mean values ranging from 95.2% applied radioactivity at time zero to 72.1% after 120 days. Extractable radioactivity also showed a small decline during the incubation of the anaerobic Kennett soil (#3) treated with the radiolabeled test material, ranging from 98.5% AR to 89.5% AR over the acclimation period, declining to 85.3% AR 119 days under anaerobic conditions.

One major degradate, preliminarily designated as Unknown 1 (RT 27.2 min), was detected in the four aerobic soils and one anaerobic soil (maximum mean value of 19.0% AR was observed in the aerobic Kennett #3 on Day 62). Analysis by LC-MS/MS suggests that this is an oxidative metabolite of the test material i.e., S-(((dibutylcarbamothioyl)thio)methyl) dibutylcarbamothioate. An authentic reference would be required to definitively confirm this proposed identification. The remaining extractable radioactivity was attributed to up to five low level degradation products (aerobic soils) or four low level degradation products (anaerobic soil) and other components that did not constitute any discrete regions of radioactivity in the chromatograms.

Based on first order kinetic models (single or multi-compartment), DT50 values in the aerobic soils were determined to be 285 days (Brierlow, #1), 119 days (Calke, #2), 286 days (Kennett, #3) and 200 days (South Witham, #4) for the parent material and 285 days for the major degradate (RT 27.2 min). Based on the single first order kinetic model, the DT50 value for the parent material in the anaerobic soil (Kennett, #3) was determined to be 516 days.

Executive summary:

The fate of the test material was studied in the laboratory under aerobic (four soils) and anaerobic (one soil) conditions in accordance with OECD Guideline 307. Samples of each soil type were acclimatized for treatment with radiolabeled test material at a rate of 1.0 mg/kg. The test systems were incubated in the dark at approximately 12 deg C and maintained at a moisture content equivalent to pF2 for up to 120 days.

The overall recoveries of radioactivity ranged from 96.1 to 104.1% of applied radioactivity in the four soils incubated under aerobic conditions for 120 days, and 96.9 to 99.6% AR in the soil (#3, Kennett) incubated under anaerobic conditions for 119 days.

Extractable radioactivity declined during the aerobic incubation for all four soils treated with the radiolabeled test material, with mean values ranging from 95.2% applied radioactivity at time zero to 72.1% after 120 days. Extractable radioactivity also showed a small decline during the incubation of the anaerobic Kennett soil (#3) treated with the radiolabeled test material, ranging from 98.5% AR to 89.5% AR over the acclimation period, declining to 85.3% AR 119 days under anaerobic conditions. 

Non-extractable radioactivity increased slightly during the aerobic incubation of four soils treated with the radiolabeled test material, from 3.1-4.8% AR at time zero to 10.5-12.3% AR after 120 days. Non-extractable radioactivity increased during the aerobic incubation of the anaerobic soil to a maximum of 8.0% AR on Day 90. The radioactivity of the non-extractable residues in the four aerobic soils on Day 120 was characterized as the radioactivity of fulvic and humic acids and, by difference, humin, which accounted for 2.0-3.1%AR, 1.0-3.1%AR and 4.6-9.3%AR, respectively.

No organic volatiles were detected in the aerobic or anaerobic soils, but mean radioactivity measured in the trapping solutions (attributed to 14CO2) reached 6.9-12.2% AR in the four aerobic soils after 120 days and 3.5% AR after 119 days under anaerobic conditions (post-flooding). 

One major degradate, preliminarily designated as Unknown 1 (RT 27.2 min), was detected in the four aerobic soils and one anaerobic soil (maximum mean value of 19.0% AR was observed in the aerobic Kennett #3 on Day 62). Analysis by LC-MS/MS suggests that this is an oxidative metabolite of the test material i.e., S-(((dibutylcarbamothioyl)thio)methyl) dibutylcarbamothioate. An authentic reference would be required to definitively confirm this proposed identification. The remaining extractable radioactivity was attributed to up to five low level degradation products (aerobic soils) or four low level degradation products (anaerobic soil) and other components that did not constitute any discrete regions of radioactivity in the chromatograms.

Based on first order kinetic models (single or multi-compartment), DT50 values in the aerobic soils were determined to be 285 days (Brierlow, #1), 119 days (Calke, #2), 286 days (Kennett, #3) and 200 days (South Witham, #4) for the parent material and 285 days for the major degradate (RT 27.2 min). Based on the single first order kinetic model, the DT50 value for the parent material in the anaerobic soil (Kennett, #3) was determined to be 516 days.

Key value for chemical safety assessment

Half-life in soil:

516 d

at the temperature of:

12 °C

Additional information

The mean total recoveries of radioactivity ranged from 96.1 to 104.1% of applied radioactivity (AR) in the four soils incubated under aerobic conditions for 120 days, and 96.9 to 99.6% AR in the soil (#3, Kennett) incubated under anaerobic conditions for 119 days.

Extractable radioactivity declined during the aerobic incubation for all four soils treated with the radiolabeled test material, with mean values ranging from 95.2% applied radioactivity at time zero to 72.1% after 120 days. Extractable radioactivity also showed a small decline during the incubation of the anaerobic Kennett soil (#3) treated with the radiolabeled test material, ranging from 98.5% AR to 89.5% AR over the acclimation period, declining to 85.3% AR 119 days under anaerobic conditions.

One major degradate, preliminarily designated as Unknown 1 (RT 27.2 min), was detected in the four aerobic soils and one anaerobic soil (maximum mean value of 19.0% AR was observed in the aerobic Kennett #3 on Day 62). Analysis by LC-MS/MS suggests that this is an oxidative metabolite of the test material i.e., S-(((dibutylcarbamothioyl)thio)methyl) dibutylcarbamothioate. An authentic reference would be required to definitively confirm this proposed identification. The remaining extractable radioactivity was attributed to up to five low level degradation products (aerobic soils) or four low level degradation products (anaerobic soil) and other components that did not constitute any discrete regions of radioactivity in the chromatograms.

Based on first order kinetic models (single or multi-compartment), DT50 values in the aerobic soils were determined to be 285 days (Brierlow, #1), 119 days (Calke, #2), 286 days (Kennett, #3) and 200 days (South Witham, #4) for the parent material and 285 days for the major degradate (RT 27.2 min). Based on the single first order kinetic model, the DT50 value for the parent material in the anaerobic soil (Kennett, #3) was determined to be 516 days.