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Biodegradation in soil

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Endpoint:
biodegradation in soil
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to guideline
Guideline:
other: BBA IV, 4-1
Deviations:
no
GLP compliance:
yes
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
DIN 19863 (Deutsche Industrie-Norm)
Soil no.:
#1
Soil type:
other: Slight humic sand
% Clay:
1.9
% Silt:
9.8
% Sand:
88.4
% Org. C:
0.62
pH:
5.9
Soil No.:
#1
Duration:
101 d
Soil No.:
#1
Initial conc.:
0.8 other: mg/100g soil d.w.
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Soil No.:
#1
Temp.:
20 °C
Humidity:
20 other: % water content
Details on experimental conditions:
1. PRELIMINARY EXPERIMENTS:
A slight humic sand soil specified as a LUFA Speyer soil 2.1 was used in this study. The soil was passed through a 2 mm sieve prior to use, dispensed into incubation flasks and pre-incubated at test conditions for 22 days

2. EXPERIMENTAL DESIGN
A total of 26 glass flasks of soil, each containing 100 g soil dry weight were prepared. The flasks were set up in individual open gas flow systems, each with a trapping system for determination of organic volatiles (one trap containing ca. 50 mL 2-methoxyethanol) and CO2 (two traps containing ca. 50 mL 2N NaOH). Each incubation flask was provided with CO2-free moistened air at a flow rate of ca. 30-60 mL/min.

Test material Preparation and application
A solution of 14C-labelled Diuron was prepared in methanol with an activity of 843 KBq/mL. Non-labelled Diuron was also prepared as a solution in methanol at a concentration of 1.004 g/L.
Following pre-incubation of the 100 g soil samples at 20 ± 5 °C and field moisture capacity (20 % water content), the radio-labelled test substance was applied to the soil samples at a rate of 0.8 mg Diuron per 100 g soil (dry weight) corresponding to 8.0 kg a.s./ha. Application was drop by drop onto the soil surface.

Experimental conditions (in addition to defined fields)
- Temperature: 20.0 ± 2 °C
- Continuous darkness: Yes

3. OXYGEN CONDITIONS
-aerobic

4. SUPPLEMENTARY EXPERIMENTS:

5. SAMPLING DETAILS
Duplicate soil samples were taken for analysis at the following days after treatment: 0, 7, 13, 20, 28, 42, 54, 82 and 101. In case samples were not extracted on the sampling day, they were stored at -20 °C until sample pre-treatment.
Soil microbial biomass was determined one day before treatment. Three days after the end of the experiment, the biomass was determined from samples incubated under experimental conditions and treated with non-labelled Diuron at the experimental application rate.
The trapping solutions were analysed weekly during the first 30 days and thereafter at larger intervals by counting 1 mL of each trap in 10 mL cocktail using LSC.
Soil No.:
#1
% Recovery:
93.87
St. dev.:
3.14
Soil No.:
#1
% Degr.:
45.7
Parameter:
radiochem. meas.
Sampling time:
101 d
Soil No.:
#1
DT50:
112 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: linear regression of log transformed data
Soil No.:
#1
DT50:
118 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: linear regression using log scaling
Soil No.:
#1
DT50:
186 d
St. dev.:
111
Type:
other: 1.5 order kinetic
Remarks on result:
other: Timme model
Soil No.:
#1
DT50:
237.8 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: linear regression of log transformed data
Soil No.:
#1
DT50:
250.5 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: linear regression using log scaling
Soil No.:
#1
DT50:
394.9
Type:
other: 1.5 order kinetic
Temp.:
12 °C
Remarks on result:
other: Timme model
Transformation products:
yes
No.:
#1
No.:
#2
Volatile metabolites:
yes
Details on results:
A good recovery was recorded in this study. Recovery from combined duplicate samples ranged from 90.8 – 99.8 % AR
The amount of 14CO2 increased slowly during the first 54 days (2.1 % AR) and then increased rapidly up to 31.8 % AR on day 101. Organic volatiles did not exceed 0.01 % AR throughout the course of the experiment
The parent substance was found to degrade slowly throughout the incubation period from a maximum of 97.2 % AR at day 0 to 54.3 % AR at the end of the incubation period. The amount of radioactivity bound to soil increased from 1.3 % AR at day 0 to 3.8 % at day 7 and further on did not show significant changes. A maximum of 6.0 % AR was reached on day 20
Two metabolites were detected during the study, metabolite A (DCPU) and metabolite B (DCPMU). The highest amounts were found to form between days 28 and 82. Metabolite A was quantified on day 20 and increased to a maximum of 1.6 % AR on day 82, decreasing to 0.5 % AR on day 101. Metabolite B was the only major degradation product, quantified on day 7, increasing up to a maximum of 19.1 % AR at day 54 and declining to 3.7 % at day 101.
The DT50 of Diuron was calculated using two different patterns of decline. With linear regression using logarithmic scaling of the Y-values (log relative amount of parent compound) the extrapolated DT50 value was 118 days. A better curve fit was obtained with the model of Timme following squareroot 1.5 order kinetics. The DT50 extrapolated from this curve was 186 days ± 111 days and considered as the best estimation in the study. No DT90 was determined due to the slow degradation

Table 2

Mean recovery of radioactivity in % AR and distribution of metabolites after application of14C-labelled Diuron and incubation under aerobic conditions

 

Days after appl.

Extracted

Not extracted

Volatiles

(14CO2)

Total

Diuron

DCPMU

DCPU

0

97.2

1.3

0

98.5

97.2

0

0

7

88.7

3.8

0.1

92.6

86.0

2.7

0

13

88.1

4.4

0.4

92.9

81.9

6.2

0

20

85.7

6.0

0.9

92.5

73.6

11.4

0.7

28

84.4

5.1

1.3

90.8

69.3

14.1

1.0

42

88.0

2.8

1.6

92.4

68.1

18.5

1.4

54

85.8

3.5

2.1

91.4

65.2

19.1

1.5

82

86.0

4.1

9.8

99.8

65.9

18.5

1.6

101

58.5

3.6

31.8

93.9

54.3

3.7

0.5

Minor differences in total recovery percentages must be attributed to rounding

 

Executive summary:

A GLP Guideline study is available for this test substance. Following incubation in a slight humic sand LUFA Speyer soil 2.1 at 20 ± 2 °C in the dark under aerobic conditions for up to 101 days, Diuron degraded slowly. Results determined a maximum of 97.2 % AR at day 0 to 54.3 % AR at the end of the incubation period (day 101) and calculated a DT50 of 186 days following squareroot 1.5 order kinetics. One major metabolite (DCPMU) reached a maximum of 19.1 % AR at day 54.

Endpoint:
biodegradation in soil
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to guideline
Guideline:
other: U.S. EPA Pesticides Guidelines Subdivision N 162-1, corresponds to SETAC-Guidelines Part 1-1.1
Deviations:
no
GLP compliance:
yes
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
USDA (US Department of Agriculture)
Soil no.:
#1
Soil type:
silt loam
% Clay:
19
% Silt:
59
% Sand:
22
% Org. C:
3.7
pH:
4.6
CEC:
8.9 meq/100 g soil d.w.
Bulk density (g/cm³):
0.96
Details on soil characteristics:
A silt loam soil from Newark, Delaware U.S.A was used in this study.
The soil was passed through a 2 mm sieve prior to use
Water holding capacity at 0.33 bar: 26.5 %
Soil No.:
#1
Duration:
365 d
Soil No.:
#1
Initial conc.:
> 19.5 - < 20.3 mg/kg soil d.w.
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Soil No.:
#1
Temp.:
24.9 ± 0.2 °C
Humidity:
75 other: of the water content at 0.33 bar potential
Details on experimental conditions:
1. PRELIMINARY EXPERIMENTS:n/a

2. EXPERIMENTAL DESIGN
A total of 61 flasks of soil were prepared, 32 for non-sterile application, 26 for sterile application and 3 control flask for treatment with cellulose.
After application of the test substance, the samples were kept in darkness in an incubator (model R1201, BHD Ltd) at a nominal temperature of 25 ºC (mean measured 24.9 ºC ¿ 0.1 ºC). The non-sterile flask were flushed with air at weekly intervals. The weight of the flasks was checked weekly and water was added if required.

Test material Preparation:
The sample of 14C-Diuron was dissolved in methanol. This solution was transferred to a 25 mL volumetric flask already containing 32.1 mg Diuron and made-up to volume with methanol. Duplicate aliquots (50 µL) were transferred to separate 25 mL flask and made-up to volume with methanol. Triplicate aliquots (100 µL) were radio-assayed. On the basis of this measurements the stock solution contained 63.3 mg of Diuron with an specific activity of 21.7 µCi/mg.
Regarding the 14C cellulose suspension, a stock solution of 4.0 x 106 dpm/mL (equivalent to ca 2.3 mg/mL) was prepared

Test material application:
Non-sterile flasks: to each flask were added 380 µL of stock solution of Diuron, and water was added to bring the soil to 75% of the water content at 0.33 bar potential. Two of the 32 flasks were designated as spares for microbial investigation. Each non-sterile soil received 4.71x 107 dpm (21.2 µCi) of 4C-Diuron at an application rate or 975 µg Diuron/sample (19.5 µg Diuron/g soil).
Sterile flasks: once the soil samples had been dispensed the flasks were autoclaved at 121 ºC for 1 hour 3 successive days. They were fitted with sterile cotton. Application was done in the same way as above, but under sterile conditions. Two of the flasks were designated for microbial investigation.Each sterile soil received 4.90 x 10 7 dpm (22.1 µCi) of 14-C Diuron at an application rate of 1015 µg Diuron/sample (20.3 µg/g soil).
To the side tube of each flask was added 0.5 M aqueous sodium hydroxide solution. The quantity of test substance added to each soil was assessed by dispensing further 380 µL aliquots into 500 mL volumetric flasks which were made up to volume. Triplicate aliquots (1mL) were radio-assayed.

Experimental conditions
- Continuous darkness: Yes

Other details, if any:

3. OXYGEN CONDITIONS
aerobic

4. SUPPLEMENTARY EXPERIMENTS:
Reference Substance: 14C-cellullose (51 µCi/mg)
Initial concentration: 4.0 x 107 dpm (14C-celluose)
Cellulose-treated control flasks: Water was added to each flask so each was 1.25 g below the required weight. Then, 1.25 mL of the stock suspension of cellulose were added. The flask were shaken, the sodium hydroxide was added and the flask weighed.
The quantity of substance added to each soil was assessed by dispensing further 1.25 mL aliquots into 100 mL volumetric flasks which were made up to volume. Triplicate aliquots (1mL) were radio-assayed. Each soil received 4.0 x 107 dpm (14C-celluose) at an application rate or 2-3 mg/sample

5. SAMPLING DETAILS
Triplicate soil flasks were taken for analysis at the following days: 0, 7, 14, 30, 60, 120, 180, 240, 300 and 365. One flask was stored and the other two were analysed. The control flasks were not sampled.
The trapping solutions were taken and replaced if necessary every two weeks (approximately).
Samples were extracted with different solvent until less than 10% of radioactivity remained un-extracted.
Soil No.:
#1
% Recovery:
90.3
Remarks on result:
other: sterile
Soil No.:
#1
% Recovery:
81
Remarks on result:
other: non-sterile
Soil No.:
#1
% Degr.:
8.9
Parameter:
other: Radiochem. meas. sterile soil
Sampling time:
365 d
Soil No.:
#1
% Degr.:
51
Parameter:
other: Radiochem. meas. non-sterile soil
Sampling time:
365 d
Soil No.:
#1
DT50:
1 920 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: sterile soil
Soil No.:
#1
DT50:
372 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: non-sterile soil
Soil No.:
#1
DT50:
6 392.7 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: sterile soil
Soil No.:
#1
DT50:
1 238.6 d
Type:
(pseudo-)first order (= half-life)
Temp.:
12 °C
Remarks on result:
other: non-sterile soil
Transformation products:
yes
No.:
#1
No.:
#2
Details on transformation products:
- Figure attached: Yes

A good recovery was recorded in this study. Recovery from individual samples from the non-sterile soil lay in the range 98.2 – 104.8 % AR and for the sterile soil it ranged among 94.5 – 101.9 % AR. Of this, mean mineralisation to CO2accounted for 3.3 % of applied radioactivity in the non-sterile soil. In the sterile soil no significant radioactivity evolved.

Only the metabolite DCPMU achieved significant levels of AR at any time through the study. In the non-sterile soil this metabolite increased up to a maximum of 21.7% after 365 days. In the sterile soil it was also present but at a 3% AR. In the non-sterile soil the DCPU was also present although it did not exceed the 1% at any time. A component designated as B1 was present in both sterile and non sterile soil but could not be identified. The polar radioactive material accounted for less than 4% in the non-sterile soil and less than 1% in sterile soil.

The TLC analysis did not detect DCA, TCAB or TCAOB. An HPLC of the soil extracts was conducted in order to try to find these products, however these degradation products were not find in any of the soil extracts analysed (< 0.005 ppm).

Table 2.

Mean recovery and extraction of radioactivity from non-sterile soil (as % of applied radioactivity)

 

Days after appl.

Extracted

Not extracted

Volatiles

(14CO2)

Total

Diuron

DCPMU

DCPU

Polars

0

103.1

0.2

-

103.3

97.8

-

-

-

7

96.9

5.0

0.02

101.9

94.0

1.7

0.3

0.1

14

98.9

4.7

0.02

103.6

95.0

2.0

0.4

0.2

30

96.8

5.7

0.14

102.6

86.4

4.7

0.9

0.2

60

94.7

7.8

0.48

103.0

82.9

8.3

0.6

0.1

120

90.8

9.5

1.39

101.7

70.1

13.6

0.6

2.0

180

87.3

11.4

1.96

100.7

63.4

16.9

0.6

2.3

240

86.1

11.6

2.34

100.0

59.5

17.7

0.7

3.1

300

82.0

14.2

2.90

99.1

55.0

17.5

0.7

2.5

365

81.0

14.9

3.36

99.3

49.0

21.7

0.7

2.9

Mean recovery and extraction of radioactivity from sterile soil (as % of applied radioactivity)

 

Days after appl.

Extracted

Not extracted

Volatiles

(14CO2)

Total

Diuron

DCPMU

DCPU1

Polars

7

93.4

3.9

0.002

97.3

91.2

1.2

0.2

 

14

94.6

5.6

<0.004

100.2

91.3

1.5

0.4

see1)

30

97.7

2.7

<0.01

100.4

92.3

1.7

0.4

 

60

96.0

4.4

<0.02

100.4

91.6

2.0

0.3

 

120

94.6

3.6

<0.04

98.2

89.0

2.3

0.3

0.4

180

94.2

4.9

<0.05

99.1

87.9

2.8

0.2

0.4

240

91.2

4.0

<0.05

95.2

83.8

2.9

0.3

0.7

365

90.3

6.1

<0.07

96.4

81.1

3.4

0.2

0.6

1)                 Up to day 60, DCPU together with polars and area of chromatogram between DCPU and DCPMU, from day 120, DCPU together with area of chromatogram between DCPU and DCPMU

 

The amount of radioactivity that was extracted from the soil in the incubation systems declined as a function of incubation time. Following first order kinetics, degradation half-life in the non-sterile was determined to be 372 days (r2= 0.976). In the sterile soil, the half-life was calculated to be 1920 days (r2= 0.950).

Executive summary:

A GLP Guideline study is available for this test substance. Following incubation in a silt loam soil at 25 °C under aerobic conditions for up to 365 days, Diuron degraded slowly. Following first order kinetics, degradation half-life of Diuron in the non-sterile soil was determined to be 372 days (r2 = 0.976). In the sterile soil, the half-life was calculated to be 1920 days (r2 = 0.950). This indicates that degradation is mainly microbial resulting in only one major metabolite (DCPMU) which reached a maximum of 22% AR at study termination.

Description of key information

Diuron degrades slowly in soil under environmental conditions. The half live is 118-372 days (251 - 1239 days recalculated to 12 °C).

Key value for chemical safety assessment

Additional information

Aerobic degreadtion of Diuron was tested in two studies using two different soil types, with similar results. Following incubation in sterile and non-sterile silt loam soil at 25 °C under aerobic conditions for up to 365 days, Diuron degraded slowly. Following first order kinetics, degradation half-life of Diuron in the non-sterile soil was determined to be 372 days (r2 = 0.976) (1239 days recalculated to 12 °C). In the sterile soil, the half-life was calculated to be 1920 days (r2 = 0.950) (6393 days recalculated to 12 °C). This indicates that degradation is mainly microbial resulting in only one major metabolite (DCPMU) which reached a maximum of 22% AR at study termination (Hawkins et al., 1990).

Following incubation in a slight humic sand at 20 ± 2 °C in the dark under aerobic conditions for up to 101 days, Diuron again degraded slowly. Results determined a maximum of 97.2 % AR at day 0 to 54.3 % AR at the end of the incubation period (day 101) and calculated a DT50 of 118 days (251 days recalculated to 12 °C) following first order kinetics with log transformed data. One major metabolite (DCPMU) reached a maximum of 19.1 % AR at day 54 (De Vries, 1996)

On the basis of these results from soil dissipation tests it can be concluded that Diuron degrades slowly in soil under environmental conditions. The DT 50 values range from 118 days (251 days recalculated to 12 °C) (De Vries, 1996) to 372 d (1239 days recalculated to 12 °C) following first order kinetics with log transformed data (Hawkins, 1990) .