N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Biodegradation in soil

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Aug 1990 to 15 Mar 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: German guideline: Richtlinie für die amtliche Prüfung von Panzenschutzmitteln, Teil IV, 4-1: "Verbleib von Pflanzenschutzmitteln im Boden: Abbau, Umbau und Metabolismus”.

Version / remarks:

December, 1986

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

1991

Soil no.:

#1

Soil type:

sandy loam

% Clay:

6.5

% Silt:

30.7

% Sand:

62.8

% Org. C:

1.08

pH:

7.3

CEC:

7.6 meq/100 g soil d.w.

Soil no.:

#2

Soil type:

sand

% Clay:

6.9

% Silt:

8.5

% Sand:

84.6

% Org. C:

1.78

pH:

5

CEC:

11.5 meq/100 g soil d.w.

Details on soil characteristics:

An overview of the characterasation of the soils is provided in Table 1 in 'Any other information on materials and methods incl. tables'.
- Soil preparation (e.g., 2 mm sieved; air dried etc.): Before use, the soils were air-dried at room temperature and passed through a 2 mm sieve. After treatment the moisture content was adjusted to 40% of the maximum water holding capacity (MWC).
- Storage: The soil was stored biologically active in the greenhouse until use.
SOIL 1 (Sandy loam (Mosimann))
- Source: Les Barges/VS, Switzerland
SOIL 2 (Sand (Standard Soil 2; Neuhofen))
- Source: LUFA Speyer/FRG

Soil No.:

#1

Duration:

100 d

Soil No.:

#2

Duration:

149 d

Initial conc.:

0.1 mg/kg soil d.w.

Based on:

test mat.

Remarks:

Applicable for both soil 1 and 2

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Soil No.:

#1

Temp.:

20˚C

Humidity:

40% MWC

Microbial biomass:

55.5 mg microbial carbon per 100 g (day 0) ; 32.6 mg micobial carbon per 100 g (day 91)

Soil No.:

#1

Temp.:

20˚C

Humidity:

40% MWC

Microbial biomass:

25.0 mg microbial carbon per 100 g (day 0); 25.6 mg micobial carbon per 100 g (day 91)

Details on experimental conditions:

EXPERIMENTAL DESIGN
- Soil condition: Air dried
- Soil (g/replicate): 75 g dw
- No. of replication controls: 20 (100g soil dw/replicate)
- No. of replication treatments: 11
- Test apparatus (Type/material/volume): 200 mL Erlenmeyer flasks
- Details of traps for CO2 and organic volatile: Ventilation with moistened air (60 mL/min) in incubation chambers. The effluent air was passed through two absorption bottles each containing 50 mL 2N NaOH and two further bottles one containing 50 mL ethyleneglycol, the other 50 mL H2SO4.

Test material application
- Preparation of the Test Substance
> Stock solution A: Prior to the preparations of the treatment solutions, the total amount of the 14C-labelled test substance was dissolved in accurately 10 mL of acetone. Thereafter, the amount of radioactive material present was determined by LSC to be 1.23 mg.
> Stock Solutions 3 and C for the 0.1 ppm Target Dose Level: For stock solutions B (for Neuhofen soil) and C (for Mosimann soil) 830 µL stock solution A were transferred into a 5 mL volumetric asks and made up with acetone to a final volume of 5 mL.
- Application method (Neuhofen Soil): The complete volume of stock solution B was slowly added to 1000 g soil (based on dry soil weight) under stirring in a kneeding machine. Thereafter, the soil was mixed for additional 10 minutes and the acetone allowed to evaporate. The soil moisture was adjusted to 40% MWC by adding the appropriate amount of pure water. After mixing for further 10 minutes aliquots of the soil corresponding to 75 g dry soil were transferred into Erlenmeyer asks.
- Application method (Mosimann Soil): A batch of soil corresponding to 1000 g dry soil was treated as above by adding the complete volume of stock solution C. Thereafter, the required soil humidity was adjusted to 40% MWC by adding water (Nanopur).

Experimental conditions
- Moisture maintenance method: During incubation the soil moisture was controlled in about weekly intervals for the first month and thereafter in about two weeks interval.
- Continuous darkness: Yes

SAMPLING DETAILS
- Sampling intervals (soil): Neuhofen soil samples were taken for analysis 0, 7, 14, 30, 61, 82, 100 and 149 days after treatment, Mosimann soil samples were taken for analysis 0, 7, 14, 30, 42, 61, 82 and 100 days after treatment.
- Sampling intervals (volatiles): The absorption solutions were exchanged in about weekly intervals during the first month and thereafter in about two weeks intervals.

Soil No.:

#1

% Total extractable:

33.8

% Non extractable:

56.8

% CO2:

5

% Other volatiles:

0

% Recovery:

95.6

Soil No.:

#2

% Total extractable:

73.2

% Non extractable:

24.6

% CO2:

2

% Other volatiles:

0.1

% Recovery:

99.8

Parent/product:

parent

Soil No.:

#1

% Degr.:

90.5

Parameter:

radiochem. meas.

Sampling time:

100 d

Parent/product:

parent

Soil No.:

#2

% Degr.:

67.6

Parameter:

radiochem. meas.

Sampling time:

149 d

Key result

Soil No.:

#1

DT50:

11.8 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: recalculated; corrected for soil moisture

Key result

Soil No.:

#2

DT50:

75.7 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: recalculated; corrected for soil moisture

Soil No.:

#1

DT50:

17.4 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: Based on original data in the reports

Soil No.:

#2

DT50:

83.11 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: Based on original data in the reports

Soil No.:

#1

DT50:

16.1 d

Type:

other: two-compartment model

Temp.:

20 °C

Remarks on result:

other: From original report

Soil No.:

#2

DT50:

68.2 d

Type:

other: two-compartment model

Temp.:

20 °C

Remarks on result:

other: From original report

Transformation products:

not specified

Remarks:

M4 and M6

Details on transformation products:

An overview of the results is provided in Table 4 - Table 5 in 'Any other information on results incl. tables'.
- Test substance and metabolites: The parent substance was first degraded into M4, reaching its highest concentration with 31.8% of the radioactivity applied in the Mosimann soil after 30 days and in Neuhofen soil with 10.1% after 82 days. Thereafter, the urea was further degraded to M6 showing its highest concentration with 28.0% in the Mosimann soil after 61 days and with 32.8% in Neuhofen soil after 149 days. The structures of the two soil metabolites were proven by co-chromatography on HPLC. At the end of the incubation period both metabolites had decreased in Mosimann soil demonstrating the transient character of the two metabolites. Besides these metabolites, small amounts of carbon dioxide were observed (up to 5%) indicating the mineralization of the dichlorophenylring- labelled parent compound with time.

Evaporation of parent compound:

not specified

Volatile metabolites:

yes

Residues:

yes

Details on results:

An overview of the results is provided in Table 2 - Table 5 in 'Any other information on results incl. tables'.
- Recovery of the Test Substance from Soil: The recoveries ranged from 88.8% to 101.8% and from 95.4% to 101.5% in Mosimann and Neuhofen soil, respectively.
- Microbial Biomass: With increasing incubation time the microbial biomass decreased from 55.5 mg microbial carbon per 100 g of Mosimann soil at the first day of incubation to 32.6 mg after 91 days of incubation. During the same time period the microbial biomass in the Neuhofen soil was very low, with 25.0 mg at the first day of incubation and 25.6 mg at day 91. These figures indicate, that sufficient microbial activity was present in the Mosimann soil during the whole incubation period. For the Neuhofen soil, the microbial biomass was extremely low. Therefore, a significant decrease in the degradation rate of the test substance must be assumed.
- Extractability and Volatiles: In both soils, the extractable radioactivity steadily decreased with time reaching 33.8% and 73.2% of the radioactivity applied to Mosimann and Neuhofen soil, respectively. Accordingly, the non-extractable fraction increased in the corresponding samples up to 56.8% and 24.6% of the radioactivity applied. For the incubation of 14C-labelled test substance the evolution of 14C-CO2 was 5% in Mosimann soil and 2% in Neuhofen soil at the end of experiment. Only negligible amounts of volatile radioactivity other than carbon dioxide were found.
- Rates of degradation: Based on the concentration of the parent molecule found after incubation at various time intervals the degradation rate constants were determined by applying a two compartment model. In Neuhofen soil no degradation was observed in the first 14 days. Therefore the model was applied for the concentrations after these lag-phase. The DT50 and DT90 values calculated for the Neuhofen soil were 68.2 and 589.1 days, respectively. The low degradation rate observed in the German standard soil was most probably due to the low microbial biomass present in the soil during the experiment. In Mosimann soil, the test substance was rapidly proven down with mean DT50 and DT90 values of 16.1 and 64.5 days, respectively. These data demonstrate a rapid breakdown of the insecticide in a biologically active soil.

Table 2. Distribution Pattern of 14C-Activity the Degradation of the test substance in Soil (Mosimann) under aerobic conditions at 20˚C (Values given in % of the 14C-Activity applied)

Incubation time (days)	CO2	Volatiles	Extractables	Non-extractable	Recovery
0	0.0	0.0	99.8	0.6	100.4
7	0.1	0.0	95.1	2.8	98.0
14	0.1	0.0	94.2	5.7	100.1
30	0.5	0.0	81.0	7.2	88.8
42	1.2	0.0	67.0	25.3	93.5
61	2.4	0.0	48.2	51.2	101.8
82	3.4	0.0	37.5	48.9	89.9
100	5.0	0.0	33.8	56.8	95.6

Table 3. Distribution Pattern of 14C-Activity the Degradation of the test substance in Soil (Neuhofen) under aerobic conditions at 20˚C (Values given in % of the 14C-Activity applied)

Incubation time (days)	CO2	Volatiles	Extractables	Non-extractable	Recovery
0	0.0	0.0	96.1	0.3	96.3
7	0.0	0.0	98.8	1.9	100.7
14	0.1	0.0	98.2	3.1	101.4
30	0.1	0.0	94.9	0.4	95.4
61	0.4	0.0	89.2	11.9	101.5
82	0.7	0.0	85.4	13.3	99.4
100	1.0	0.0	82.9	17.2	101.2
149	2.0	0.1	73.2	24.6	99.8

 

Table 4. Distribution 14C-labelled test substance and metabolites in Soil (Mosimann) under aerobic conditions at 20˚C (Values given in % of the 14C-Activity applied)

Incubation time (days)	Parent substance	M7	M4	UK	Total
0	99.3	0.0	0.0	0.5	99.8
7	84.6	1.8	7.9	0.7	95.1
14	59.3	8.1	26.0	0.8	94.2
30	23.3	23.8	31.8	2.1	81.0
42	17.6	26.3	20.5	2.5	67.0
61	11.4	28.0	7.0	1.8	48.2
82	8.1	22.2	4.4	2.8	37.5
100	9.5	17.9	6.0	0.3	33.8

NA = Not analysed

UK: Unknown radioactivity

Table 5. Distribution 14C-labelled test substance and metabolites in Soil (Neuhofen) under aerobic conditions at 20˚C (Values given in % of the 14C-Activity applied)

Incubation time (days)	Parent substance	M7	M4	UK	Total
0	95.0	0.0	0.0	1.1	96.1
7	96.5	0.0	0.0	2.2	98.8
14	97.3	0.0	0.0	0.9	98.2
30	78.8	8.0	7.0	1.0	94.9
61	60.3	20.4	8.0	0.5	89.2
82	47.6	24.9	10.1	2.9	85.4
100	41.8	30.1	9.5	1.6	82.9
149	32.4	32.8	7.7	0.3	73.2

NA = Not analysed

UK: Unknown radioactivity

 

Conclusions:

In a biodegradation study in soil, performed in accordance with German guideline BBLF IV 4-1, the DT50 values were calculated to be 16.1 days and 68.2 days in Mosimann soil and Neuhofen soil, respectively, under aerobic condition. The DT50 values were recalculated using single first-order kinetics and corrected for soil moisture. The final DT50 values were 11.8 days (Mosimann soil) and 75.7 days (Neuhofen soil).

Executive summary:

The rate of degradation of dichlorophenyl ring-labelled test substance was investigated in a sandy loam soil (Mosimann) and in a sand soil (Neuhofen) at a rate of 0.1 mg/kg dry soil. The study was conducted in accordance to German guideline BBLF IV 4-1 and in compliance with GLP criteria. The treated soil was incubated in the dark, at a 20±2°C and ventilated with a stream of moist air. The moisture content of the soil was maintained at 40% of the maximum water holding capacity under conditions that allowed 14CO2 and any other volatilised degradates to be trapped. Samples of incubated, treated soils were taken for analysis at 0, 7, 14, 30, 42 (only for Mosimann), 61, 82, 100 and 149 (only for Neuhofen) days. The 14CO2 was collected in aqueous 2 M NaOH traps over the course of the study. Ethylene glycol and 0.25 N sulphuric acid traps were analysed for any other volatile radioactive degradates. Qualitative analysis and metabolite identification was confirmed by co-chromatography using HPLC. The viability of the soil at the beginning and at day 91 of the study was determined by the method of Andersen and Domsch.

Overall recoveries of applied radioactivity (AR) is ranged from 88.8 - 101.8%. As the study progressed the amounts of extractables decreased and the amounts of bound radioactivity increased reaching 24.6% AR (Neuhofen) and 56.8% AR (Mosimann) by the end of the study. Only low amounts of CO2 were formed (2 - 5% AR). Quantities of extractable, unknown metabolites were low (0.3 - 2.9% AR). The primary metabolite was M4, reaching a maximum of 32% AR by day 30 (Neuhofen) and 10% AR by day 82 (Mosimann). This metabolite was subsequently degraded to the M6, which levels increase until the end of the study in the Neuhofen soil, but were decreasing more rapidly in the Mosimann soil. The test substance degraded more rapidly in the soil with the higher microbial biomass (Mosimann, sandy loam) with a DT50 of 16.1 days. In the sand soil (Neuhofen), the degradation was significantly lower, DT50 of 68.2 days was estimated. Those estimations were conducted assuming the parent molecule to be distributed into two compartment. The DT50 values were recalculated using single first-order kinetics and corrected for soil moisture. The final DT50 values were 11.8 days (Mosimann soil) and 75.7 days (Neuhofen soil).

Reference 2

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jan 1991 to 5 Sep 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: Dutch Registration Guideline, Section G.1: Behavior in Soil

Version / remarks:

January 1987

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Danish Law of the Ministry of the Environment

Version / remarks:

September 1, 1987

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Environmental Chemistry and Fate Guidelines for Registration of Pesticides in Canada; Section C: Biotransformation; 1. Soil-Degradation Pathways and Persistence

Version / remarks:

July 15, 1987

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

1991

Soil no.:

#1

Soil type:

silt loam

% Clay:

13.4

% Silt:

59.7

% Sand:

26.9

% Org. C:

1.95

pH:

7.2

CEC:

13.7 other: mmol/Z/100g soil

Details on soil characteristics:

The characterisation of the soil is provided in Table 1 in 'Any other information on materials and method incl. tables'
- Source: Les Evouettes (Les Barges/VS, Switzerland )
- Storage: The soil was stored biologically active in the greenhouse until use.
- Soil preparation: Before use, the soil was air-dried at room temperature and passed through a 2 mm sieve. After treatment the moisture content was adjusted to 30% and 60% of the field capacity (FC), respectively.

Soil No.:

#1

Duration:

180 d

Soil No.:

#1

Initial conc.:

0.1 mg/kg soil d.w.

Soil No.:

#1

Initial conc.:

1 mg/kg soil d.w.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Soil No.:

#1

Temp.:

20 ± 2 ˚C

Humidity:

30% FC

Microbial biomass:

78.0, 79.6 and 70.3 mg microbial carbon per 100 g dw soil on day 0, day 90 days and day 180, respectively

Soil No.:

#1

Temp.:

10 ± 2 ˚C

Humidity:

60% FC

Microbial biomass:

78.0, 79.6 and 70.3 mg microbial carbon per 100 g dw soil on day 0, day 90 days and day 180, respectively

Soil No.:

#1

Temp.:

20 ± 2 ˚C

Humidity:

60% FC

Microbial biomass:

78.0, 79.6 and 70.3 mg microbial carbon per 100 g dw soil on day 0, day 90 days and day 180, respectively

Details on experimental conditions:

EXPERIMENTAL DESIGN
- Soil condition: Air dried
- Soil (g/replicate): 75 g dw
- No. of replication controls: 20 (100g soil dw/replicate)
- No. of replication treatments: 10
- Test apparatus (Type/material/volume): 200 mL Erlenmeyer flasks
- Details of traps for CO2 and organic volatile: Ventilation with moistened air (60 mL/min) in incubation chambers. The effluent air was passed through two absorption bottles each containing 50 mL 2N NaOH.

Test material application
- Preparation of the Test Substance
> Stock solution A: Prior to the preparations of the treatment solutions, the total amount of the 14C-labelled test substance was dissolved in accurately 10 mL of acetone. Thereafter, the amount of radioactive material present was determined by LSC to be 3.99 mg.
> Stock Solutions B and C for the 0.1 ppm Target Dose Level: For stock solutions B, 750 µL stock solution A were transferred into a 10 mL volumetric flasks and made up with acetone to a final volume of 10 mL. For stock solutions C, 7.5 mL stock solution A were transferred into a 10 mL volumetric flasks and made up with acetone to a final volume of 10 mL.
- Application method (0.1 ppm target dose level): An aliquot of 8.6 mL of stock solution B was slowly added to 2700 g soil (based on dry soil weight) under stirring in a kneeding machine . Thereafter, the soil was mixed for additional 10 minutes and the acetone allowed to evaporate. The soil moisture was adjusted to 30% FC by adding the appropriate amount of pure water. After mixing for further 10 minutes 10 aliquots of the soil corresponding to 75 g dry soil were transferred into Erlenmeyer asks (20°C incubation). Now, the soil moisture was adjusted to 60% FC by adding the appropriate amount of pure water, and for further 10 minutes 20 aliquots of the soil corresponding to 75 g dry soil were transferred into Erlenmeyer asks (10°C and 20°C incubation).

- Application method (1 ppm target dose level): A batch of soil corresponding to 2700 g dry soil was treated as above by adding an aliquot of 8.7 mL of stocksolution C. Thereafter the required soil humidities were adjusted as for the 0.1 ppm target dose level.

Experimental conditions
- Moisture maintenance method: The soil moisture was held at 30% and 60 % FC, respectively. It was adjusted in about weekly intervals during the first month and thereafter in about two weeks intervals. Pure water (bidistilled or adequate quality) was added when necessary. During incubation the soil moisture was controlled in about weekly intervals for the first month and thereafter in about two weeks intervals.
- Continuous darkness: Yes

SAMPLING DETAILS
- Sampling intervals (soil): See Table 2 in 'Any other information on materials and methods incl. tables'
- Sampling intervals (Volatiles): The absorption solutions were exchanged in about weekly intervals during the first month and thereafter in about two weeks intervals.

Soil No.:

#1

% Total extractable:

23.3

% Non extractable:

62.8

% CO2:

11.4

% Recovery:

97.4

Remarks on result:

other: 0.1 PPM; 30% FC; 20 ˚C; 180 days

Soil No.:

#1

% Total extractable:

38.1

% Non extractable:

58.1

% CO2:

6

% Recovery:

100

Remarks on result:

other: 0.1 PPM; 60% FC; 10 ˚C; 180 days

Soil No.:

#1

% Total extractable:

25.7

% Non extractable:

63

% CO2:

11.9

% Recovery:

100

Remarks on result:

other: 0.1 PPM; 60% FC; 20 ˚C; 120 days

Soil No.:

#1

% Total extractable:

35.5

% Non extractable:

52.7

% CO2:

3.5

% Recovery:

91.7

Remarks on result:

other: 1 PPM; 30% FC; 20 ˚C; 180 days

Soil No.:

#1

% Total extractable:

59.8

% Non extractable:

30.9

% CO2:

2.3

% Recovery:

93

Remarks on result:

other: 1 PPM; 60% FC; 10 ˚C; 180 days

Soil No.:

#1

% Total extractable:

38.5

% Non extractable:

56.6

% CO2:

5.1

% Recovery:

100

Remarks on result:

other: 1 PPM; 60% FC; 20 ˚C; 120 days

Parent/product:

parent

Soil No.:

#1

% Degr.:

88.7

Parameter:

radiochem. meas.

Sampling time:

180 d

Remarks on result:

other: 0.1 PPM; 30% FC; 20°C

Parent/product:

parent

Soil No.:

#1

% Degr.:

87.1

Parameter:

radiochem. meas.

Sampling time:

180 d

Remarks on result:

other: 0.1 PPM; 60% FC; 10°C

Parent/product:

parent

Soil No.:

#1

% Degr.:

87.9

Parameter:

radiochem. meas.

Sampling time:

120 d

Remarks on result:

other: 0.1 PPM; 60% FC; 20°C

Parent/product:

parent

Soil No.:

#1

% Degr.:

84.5

Parameter:

radiochem. meas.

Sampling time:

180 d

Remarks on result:

other: 1 PPM; 30% FC; 20°C

Parent/product:

parent

Soil No.:

#1

% Degr.:

86.4

Parameter:

radiochem. meas.

Sampling time:

180 d

Remarks on result:

other: 1 PPM; 60% FC; 10°C

Parent/product:

parent

Soil No.:

#1

% Degr.:

90.8

Parameter:

radiochem. meas.

Sampling time:

120 d

Remarks on result:

other: 1 PPM; 60% FC; 20°C

Key result

Soil No.:

#1

DT50:

13 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 60% FC; average of 0.1 and 1 PPM; corrected for soil moisture

Soil No.:

#1

DT50:

14.8 d

Type:

other: two-compartment model

Temp.:

20 °C

Remarks on result:

other: 0.1 PPM; 60% FC; from original report

Soil No.:

#1

DT50:

13.3 d

Type:

other: two-compartment model

Temp.:

20 °C

Remarks on result:

other: 1 PPM; 60% FC; from original report

Soil No.:

#1

DT50:

16.3 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 60% FC; average of 0.1 and 1 PPM

Transformation products:

not specified

Remarks:

M4 and M6

Details on transformation products:

An overview of the results is provided in Table 3 - Table 8 in 'Any other information on results incl. tables'.
- Parent substance and Metabolites: The test substance was first degraded into M4 reaching its highest with 9.8, 23.5 and 21.6% of the radioactivity applied at the 0.1 PPM target dose level after 14, 42 and 21 days at 30% FC and 20°C, 60% FC and 10°C and 60% FC and 20 °C, respectively. At the 1 PPM target dose level the same metabolite reached its highest concentration with 16.4, 37.1 and 38.7% of the radioactivity applied after 42, 60 and 21 days at 30% FC and 20°C, 60% FC and 10°C and 60% FC and 20 °C, respectively. Thereafter, the urea was further degraded to M6 showing its highest concentration with 14.9, 28.1 and 23.1% of the radioactivity applied at the 0.1 PPM target dose level after 28, 60 and 28 days at 30% FC and 20°C, 60% FC and 10°C and 60% FC and 20 °C, respectively. At the 1 PPM target dose level the same metabolite reached its highest concentration with 25.8, 32.9 and 34.1% of the radioactivity applied after 90, 120 and 42 days at 30% FC and 20°C, 60% FC and 10°C and 60% FC and 20 °C, respectively. At the end of the incubation period both metabolites had decreased under almost all incubation conditions, demonstrating the transient character of the two metabolites. Besides these metabolites, amounts up to 11.9% of carbon dioxide were observed indicating the mineralization of the dichlorophenylring moiety with time. The structures of the two soil metabolites were proven by co-chromatography on HPLC.

Evaporation of parent compound:

not specified

Volatile metabolites:

yes

Residues:

yes

Details on results:

An overview of the results is provided in Table 3 - Table 8 in 'Any other information on results incl. tables'.
- Stability: HPLC-analysis of in the application solution after treatment showed a purity of 96.8 %. Thus, the test substance proved to be stable in the vehicle.
- Microbial Biomass of Soils: The microbial biomass was 78.0, 79.6 and 70.3 mg microbial carbon per 100 g dry weight of Les Evouettes soil at the first day, after 90 days and 180 days of incubation, respectively. These figures indicate, that sufficient microbial activity was present during the Whole incubation period.
- Recovery of the test substance from soil: In the 0.1 PPM target dose level, the recoveries ranged from 92.0 to 107.1%, 94.7 to 104.8% and from 93.9 to 107.1% for 30% FC and 20°C, 60% FC and 10°C and 60% FC and 20 °C, respectively. In the 1 PPM target dose level, the recoveries ranged from 91.7 to 100.5%, 93.0 to 101.5% and from 93.8 to 103.0% for 30% FC and 20°C, 60% FC and 10°C and 60% FC and 20 °C, respectively.
- Distribution of 14C-activity:
> Extractability of radioactivity: For incubation of 14C-labelled test substance at 0.1 PPM target dose level at different conditions, extractable radioactivity decreased with time reaching after 180 days 23.3 and 38.1% of the radioactivity applied for 30% FC and 20 °C and 60% FC and 10 °C, respectively. For 60% FC and 20 °C incubation extractable radioactivity decreased to 25.7% after 120 days. Accordingly the non-extractable fraction increased in the corresponding samples up to 62.8, 58.1 and 63.0% of the dose applied. For 14C-labelled test substance at 1 PPM target dose level under conditions extractable radioactivity decreased in the same periods to 35.5, 59.8 and 38.5% for 30% FC and 20°C, 60% FC and 10°C and 60% FC and 20°C, respectively. The non-extractable fraction accounted at the same time for 52.7, 30.9 and 56.6% of the dose applied.
> Volatiles: At 0.1 PPM target dose level the evolution of 14C-CO2 accounted after 180 days for 11.4 and 6.0% for 30% FC and 20°C and 60% FC and 10°C, respectively. For 60% FC and 20°C incubation the evolution of 14C-CO2 accounted after 120 days for 11.9% of the radioactivity applied. Incubation of 14C-labelled test substance at 1 PPM target dose level yielded Within 180 days to 3.5 and 2.3% of 14C-CO2 evolution for 30% FC and 20°C and 60% FC and 10°C, respectively. For 60% FC and 20 °C incubation the evolution of 14C-CO2 accounted after 120 days for 5.1% of the radioactivity applied.
- Rates of degradation: Based on the concentration of the parent molecule found after various time intervals degradation rate constants were determined by applying a two compartment model. The calculated half-lifes at the 0.1 PPM target dose rate were 30.2, 31.5 and 14.8 days, for 30% FC and 20 °C, 60% FC and 10 °C and 60% FC and 20 °C, respectively. The calculated DT90 values for the same samples were 216, 257 and 156 days. For the 1 PPM target dose level the DT50 values were 32.4, 34.1 and 13.3 days, for 30% FC and 20 °C, 60% FC and 10 °C and 60% FC and 20 °C, respectively. The calculated DT90 values for the same samples were 363, 347 and 79 days.

Table 3a. Distribution Pattern of 14C-Activity for the Degradation of the test substance at a concentration of 0.1 PPM in Soil at 30% FC and 20 ˚C (Values given in % of the radioactivity applied)

Incubation Time (Days)	CO2	Extr.	Non-Extr.	Recovery
0	0.0	96.5	1.7	98.2
7	0.3	102.8	4.0	107.1
14	0.8	94.8	8.4	103.9
21	1.5	84.3	13.9	99.7
28	2.2	79.9	20.3	102.4
42	3.5	58.2	30.3	92.0
60	5.4	47.1	42.3	94.9
90	7.1	33.9	52.4	93.4
120	8.8	29.6	60.5	98.9
180	11.4	23.3	62.8	97.4

Table 3b. Distribution Pattern of 14C-labelled test substance and metabolites at a concentration of 0.1 PPM in Soil at 30% FC and 20 ˚C (Values given in % of the radioactivity applied)

Incubation time (days)	Parent substance	M6	M4	UK	Total
0	95.5	0.0	0.0	1.0	96.5
7	87.8	4.8	8.4	1.8	102.8
14	74.0	8.8	9.8	2.2	94.8
21	58.8	12.8	9.7	3.0	84.3
28	52.9	14.9	9.4	2.7	79.9
42	35.7	11.9	8.7	1.9	58.2
60	29.9	11.7	5.4	0.1	47.1
90	22.0	7.8	3.9	0.2	33.9
120	17.8	5.4	3.2	3.1	29.6
180	11.3	7.4	1.8	2.8	23.3

UK = unknown radioactivity (not analysed)

Table 4a. Distribution Pattern of 14C-Activity for the Degradation of the test substance at a concentration of 0.1 PPM in Soil at 60% FC and 10 ˚C (Values given in % of the radioactivity applied)

Incubation Time (Days)	CO2	Extr.	Non-Extr.	Recovery
0	0.0	103.1	1.6	104.8
7	0.2	102.1	2.5	104.7
14	0.2	101.5	2.9	104.7
28	0.5	86.6	7.6	94.7
42	1.0	86.4	11.2	98.5
60	1.8	76.8	22.5	101.1
90	3.1	61.9	33.7	98.7
120	4.3	51.8	48.4	104.4
180	6.0	38.1	58.1	102.1

 

Table 4b. Distribution Pattern of 14C-labelled test substance and metabolites at a concentration of 0.1 PPM in Soil at 60% FC and 10 ˚C (Values given in % of the radioactivity applied)

Incubation time (days)	Parent substance	M6	M4	UK	Total
0	100.5	0.0	0.0	2.6	103.1
7	89.8	3.2	7.5	1.5	102.1
14	75.7	8.5	14.2	3.2	101.5
28	50.7	12.5	20.5	2.9	86.6
42	40.1	20.7	23.5	2.2	86.4
60	30.7	28.1	17.8	0.2	76.8
90	23.0	27.4	11.1	0.3	61.9
120	18.4	23.7	9.3	0.4	51.8
180	12.9	16.5	8.3	0.4	38.1

UK = unknown radioactivity (not analysed)

Table 5a. Distribution Pattern of 14C-Activity for the Degradation of the test substance at a concentration of 0.1 PPM in Soil at 60% FC and 20 ˚C (Values given in % of the radioactivity applied)

Incubation Time (Days)	CO2	Extr.	Non-Extr.	Recovery
0	0.0	99.7	1.7	101.5
7	0.3	102.1	4.7	107.1
14	1.0	89.7	11.6	102.3
21	2.1	78.5	21.9	102.5
28	3.3	65.8	31.5	100.6
42	5.9	43.1	44.8	93.9
60	8.1	33.6	55.4	97.2
90	10.3	32.5	60.5	103.3
120	11.9	25.7	63.0	100.6

 

Table 5b. Distribution Pattern of 14C-labelled test substance and metabolites at a concentration of 0.1 PPM in Soil at 60% FC and 20 ˚C (Values given in % of the radioactivity applied)

Incubation time (days)	Parent substance	M6	M4	UK	Total
0	98.3	0.0	0.0	1.4	99.7
7	73.3	9.6	16.7	2.5	102.1
14	52.6	16.1	20.7	0.2	89.7
21	37.8	18.9	21.6	0.3	78.5
28	29.8	23.1	12.7	0.2	65.8
42	16.4	18.5	5.5	2.7	43.1
60	15.5	12.5	5.3	0.3	33.6
90	15.7	9.6	6.9	0.3	32.5
120	12.1	10.4	3.0	0.2	25.7

UK = unknown radioactivity (not analysed)

 

Table 6a. Distribution Pattern of 14C-Activity for the Degradation of the test substance at a concentration of 1 PPM in Soil at 30% FC and 20 ˚C (Values given in % of the radioactivity applied)

Incubation Time (Days)	CO2	Extr.	Non-Extr.	Recovery
0	0.0	98.5	1.3	99.7
7	0.3	96.1	3.0	99.4
14	0.5	95.1	4.9	100.5
21	0.6	91.7	7.9	100.2
28	0.8	81.8	11.3	93.9
42	1.3	76.8	16.5	94.6
60	1.9	67.7	24.4	94.0
90	2.7	57.2	35.7	95.7
120	3.3	45.3	49.0	97.6
180	3.5	35.5	52.7	91.7

 

Table 6b. Distribution Pattern of 14C-labelled test substance and metabolites at a concentration of 1 PPM in Soil at 30% FC and 20 ˚C (Values given in % of the radioactivity applied)

Incubation time (days)	Parent substance	M6	M4	UK	Total
0	97.5	0.0	0.0	1.0	98.5
7	81.1	3.4	10.3	1.4	96.1
14	72.4	6.7	14.0	2.0	95.1
21	58.8	15.4	14.7	2.8	91.7
28	53.8	10.6	14.9	2.5	81.8
42	39.1	19.1	16.4	2.2	76.8
60	33.7	22.5	11.4	0.1	67.7
90	22.4	25.8	8.8	0.2	57.2
120	18.0	20.2	6.8	0.3	45.3
180	15.5	15.7	3.8	0.5	35.5

UK = unknown radioactivity (not analysed)

Table 7a. Distribution Pattern of 14C-Activity for the Degradation of the test substance at a concentration of 1 PPM in Soil at 60% FC and 10 ˚C (Values given in % of the radioactivity applied)

Incubation Time (Days)	CO2	Extr.	Non-Extr.	Recovery
0	0.0	99.2	1.6	100.8
7	0.2	98.9	2.4	101.5
14	0.3	97.9	2.5	100.7
28	0.4	91.9	5.2	97.5
42	0.5	88.2	6.6	95.2
60	0.7	84.7	10.2	95.6
90	1.1	79.6	16.6	97.4
120	1.6	73.5	24.8	99.9
180	2.3	59.8	30.9	93.0

 

Table 7b. Distribution Pattern of 14C-labelled test substance and metabolites at a concentration of 1 PPM in Soil at 60% FC and 10 ˚C (Values given in % of the radioactivity applied)

Incubation time (days)	Parent substance	M6	M4	UK	Total
0	97.1	0.0	0.0	2.1	99.2
7	88.2	2.4	7.0	1.4	98.9
14	78.0	3.0	14.6	2.3	97.9
28	54.9	5.6	28.8	2.7	91.9
42	44.0	12.8	29.5	1.9	88.2
60	29.6	17.8	37.1	0.1	84.7
90	23.6	24.6	31.2	0.2	79.6
120	18.1	32.9	22.3	0.2	73.5
180	13.6	32.8	12.9	0.3	59.8

UK = unknown radioactivity (not analysed)

Table 8a. Distribution Pattern of 14C-Activity for the Degradation of the test substance at a concentration of 1 PPM in Soil at 60% FC and 20 ˚C (Values given in % of the radioactivity applied)

Incubation Time (Days)	CO2	Extr.	Non-Extr.	Recovery
0	0.0	99.3	1.8	101.1
7	0.3	99.0	3.6	103.0
14	0.6	93.5	7.6	101.6
21	0.9	87.9	11.5	100.4
28	1.2	80.9	15.9	98.0
42	2.0	67.5	24.5	94.0
60	2.9	56.5	35.8	95.2
90	4.2	45.2	44.5	93.8
120	5.1	38.5	56.6	100.3

 

Table 8b. Distribution Pattern of 14C-labelled test substance and metabolites at a concentration of 1 PPM in Soil at 60% FC and 20 ˚C (Values given in % of the radioactivity applied)

Incubation time (days)	Parent substance	M6	M4	UK	Total
0	97.9	0.0	0.0	1.4	99.3
7	73.2	3.0	21.0	1.9	99.0
14	45.4	10.8	37.2	0.1	93.5
21	33.9	15.1	38.7	0.2	87.9
28	29.0	15.8	35.9	0.2	80.9
42	13.5	34.1	19.6	0.2	67.5
60	11.5	34.1	10.7	0.2	56.5
90	11.5	25.7	7.7	0.3	45.2
120	9.2	24.3	4.7	0.3	38.5

UK = unknown radioactivity (not analysed)

 

Conclusions:

In a biodegradation study in soil, performed in accordance with Dutch registration guideline section G1, the mean DT50 values value, calculated from two applications at 0.1 and 1 mg/kg soil dw in the Les Evouettes soil, is 16.3 days using the single first-order kinetic. After correcting for soil moisture, the mean DT50 values is 13.0 days.

Executive summary:

The rate of degradation of dichlorophenyl ring-labelled test substance was investigated in aerobic soil at various conditions. The substance was incubated at two concentrations (0.1 and 1 kg/kg soil dw) in separated studies in a silt loam soil, Les Evouettes, under various test conditions, i.e. 30% and 60% of its field capacity, in the laboratory at 10ºC and 20ºC. The treated soil was incubated in the dark and ventilated with a stream of moist air under conditions that allowed CO2 and volatiles to be trapped. The moisture content of the soil was maintained at the required water content by addition of distilled water at appropriate intervals. Samples of incubated treated soils were taken for analysis at 0, 7, 14, 21, 28, 42, 60, 90, 120 and 180 days. Soil extracts were partitioned with dichloromethane and the aqueous and organic phases quantified by liquid scintillation counting (LSC). Quantitative analysis of the individual compounds was performed by HPLC.

For incubation of 14C-labelled test substance at 0.1 and 1 mg/kg dw target dose level at different conditions, extractable radioactivity decreased with time reaching after 180 days 13.4 and 13.3% of the AR for 30% FC and 20 °C and 60% FC and 10 °C, respectively. For 60% FC and 20 °C incubation extractable radioactivity decreased to 107% after 120 days. The test substance was first degraded into CGA M4 reaching its highest concentration with 12.6%, 24.3% and 27.5% of the radioactivity applied at the target dose level after 42, 28 and 60 days at 30% FC and 60% FC at 20ºC and at 60% FC and 10 ºC, respectively. Thereafter, the urea was further degraded to M6, showing its highest concentration with 17.1%, 26.3% and 28.3% AR at the 0.1 PPM and 1 PPM target dose level after 60, 120 and 42 days at 30% FC and 60% FC at 20ºC and 60% FC at 10 °C, respectively. At the end of the incubation period both metabolites had decreased under almost all incubation conditions, demonstrating the transient character of the two metabolites. At standard test conditions (moisture at 60 % field capacity and 20ºC), the mean half-life was calculated to be 16.3 days using the single first-order kinetic. After correcting for soil moisture, the mean DT50 values is 13.0 days.

Reference 3

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Dec 1988 to 17 Jul 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA 162-1 and 162-2

Version / remarks:

October 18, 1982

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

1991

Soil no.:

#1

Soil type:

loam

% Clay:

7.3

% Silt:

18.1

% Sand:

74.6

% Org. C:

3

pH:

7.2

CEC:

14.1 other: mmol/Z/ 100g Soil

Soil no.:

#2

Soil type:

loamy sand

% Clay:

16.5

% Silt:

35.2

% Sand:

48.3

% Org. C:

3.7

pH:

6.8

CEC:

13.1 other: mmol/Z/ 100g Soil

Details on soil characteristics:

Characteristics of the soil are provided in Table 1 in 'Any other information on materials and methods incl. tables'
- Soil preparation: Before use the soils were slightly air dried at room temperature and sieved through a 2 mm sieve. Thereafter, an aliquot of the soil was taken for determination of microbial biomass.
- Storage: Soil was stored until treatment in the refrigerator at about 4°C.

Soil No.:

#1

Duration:

<= 361 d

Soil No.:

#2

Duration:

<= 361 d

Soil No.:

#1

Initial conc.:

1 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#2

Initial conc.:

1 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#2

Initial conc.:

1.2 mg/kg soil d.w.

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Temp.:

20 ± 2˚C

Humidity:

75% field capacity

Microbial biomass:

Collombey soil: 32 - 129 mg microbial carbon per 100 g of soil; Les Evouettes: 129 - 300 mg microbial carbon per 100 g of soil

Details on experimental conditions:

EXPERIMENTAL DESIGN
- Soil condition: Air dried
- Soil (g/replicate): 200 g (dry weight)
- Control conditions (sterile): Sieved soil (200 g on a dry weight basis) was placed into asks and sterilized by autoclaving. Until treatment soil samples were under sterile conditions at room temperature.
- Control conditions (solvent control): Soil samples of both soils were treated with the vehicle (acetone) only.
- No. of replication controls: 1
- No. of replication treatments: 1
- Test apparatus: 1 L Steilbrust flasks
- Details of traps for CO2 and organic volatile: 50 mL ethylene glycol, sulfuric acid (0.25 N) and sodium hydroxide (2 N) to trap volatiles and radioactive carbon dioxide
- If no traps were used, is the system closed/open: Open (gas-flow system)

Test material application
- Application method: Applied on the surface (The test substance stock solutionswere added in portions to the soil surface by means of a Hamilton Syringe. After addition of each portion the soil was mixed thoroughly.

Experimental conditions
- Moisture maintenance method: After treatment the soil moisture was adjusted to 75% field capacity by adding sterile water.
- Continuous darkness: Yes

OXYGEN CONDITIONS
- Methods used to create the anaerobic conditions: The soils were flooded with a water layer of 2 - 3 cm (about 200 mL) after an aerobic pre-incubation period of 30 days and thereafter ventilated four times per day for about 15 minutes with nitrogen at a flow rate of 60 mL/min.

SAMPLING DETAILS
- Sampling intervals and methods:
> Absorption solutions: Absorption solutions were exchanged and monitored for radioactivity in intervals of one week during the first month and thereafter in two-weeks intervals.
> Soil samples: Soil samples were taken for analysis of parent molecule and number and amount of metabolites up to about one year for the aerobic experiments and up to 90 days for the aerobic/anaerobic and aerobic/sterile experiments.
> Microbial Biomass: Pesticide untreated soil samples were taken for microbial biomass determination prior to the treatment and 91, 181 and 360 days after the treatment.

Soil No.:

#1

% Total extractable:

21.64

% Non extractable:

66.81

% CO2:

9.88

% Other volatiles:

0.76

% Recovery:

99.09

Remarks on result:

other: aerobic, Day 361, 14C-dichlorophenyl-labelled

Soil No.:

#2

% Total extractable:

14.1

% Non extractable:

74.87

% CO2:

15.12

% Other volatiles:

0.74

% Recovery:

100

Remarks on result:

other: aerobic, Day 361, 14C-dichlorophenyl-labelled

Soil No.:

#2

% Total extractable:

2.95

% Non extractable:

28.34

% CO2:

58.58

% Other volatiles:

0.02

% Recovery:

89.89

Remarks on result:

other: aerobic, Day 360, 14C-difluorophenyl-labelled

Parent/product:

parent

Soil No.:

#1

% Degr.:

91.84

Parameter:

radiochem. meas.

Sampling time:

361 d

Remarks on result:

other: aerobic, 14C-dichlorophenyl-labelled

Parent/product:

parent

Soil No.:

#2

% Degr.:

95.76

Parameter:

radiochem. meas.

Sampling time:

361 d

Remarks on result:

other: aerobic, 14C-dichlorophenyl-labelled

Parent/product:

parent

Soil No.:

#2

% Degr.:

98.21

Parameter:

radiochem. meas.

Sampling time:

360 d

Remarks on result:

other: aerobic, 14C-difluorophenyl-labelled

Key result

Soil No.:

#1

DT50:

24.2 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 14C-dichlorophenyl-labelled

Remarks:

recalculated outside of the study

Key result

Soil No.:

#2

DT50:

17.3 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 14C-dichlorophenyl-labelled

Remarks:

recalculated outside of the study

Key result

Soil No.:

#2

DT50:

11.1 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: 14C-difluorophenyl-labelled

Remarks:

recalculated outside of the study

Soil No.:

#1

DT50:

23.7 d

Type:

other: two compartment model

Temp.:

20 °C

Remarks on result:

other: 14C-dichlorophenyl-labelled

Remarks:

original reported

Soil No.:

#2

DT50:

19.4 d

Type:

other: two compartment model

Temp.:

20 °C

Remarks on result:

other: 14C-dichlorophenyl-labelled

Remarks:

original reported

Soil No.:

#2

DT50:

13 d

Type:

other: two compartment model

Temp.:

20 °C

Remarks on result:

other: 14C-difluorophenyl-labelled

Remarks:

original reported

Transformation products:

not specified

Remarks:

M4 and M6

Details on transformation products:

An overview of the results is provided in Table 2 - Table 7 in 'Any other information on results incl. tables'.
- Pathway of Degradation of the test substance: The test substance was mainly metabolized under aerobic conditions. No degradation was observed under sterile and only minor degradation was found under anaerobic conditions. Therefore, the discussion will be limited to the aerobic breakdown of the molecule. The 14C-dichlorophenyl-labelled test substance was first degraded into M4 reaching with 24.3 and 23.05% of the dose applied its highest concentration after 14 days in the Collombey and Les Evouettes soil, respectively. Thereafter, the urea was further degraded to M6 showing its highest concentration with 26.85 and 21.64% after 59 days in the Collombey and Les Evouettes soil, respectively. The structures of the two soil metabolites were proven by co-chromatography on TLC, HPLC and by comparative mass spectrometry with authentic reference compounds. At the end of the incubation period both metabolites had decreased to about 2 - 5% in both soils thus demonstrating the transient character of the two metabolites. Besides these metabolites, considerable amounts of carbon dioxide were observed (up to 10 - 15%) indicating the mineralization of a significant fraction of the dichlorophenylring moiety. The most prominent degradation product observed was the non-extractable radioactivity reaching its highest concentration with 70.7 and 78.6% after 240 days of incubation in the Collombey and Les Evouettes soil, respectively. However, after one year its concentration had slightly dropped to 66.8 and 74.9% of the dose applied in the corresponding soils. Besides the above mentioned degradation products up to at least three minor, unknown metabolites were observed accounting at highest for 3.2%. By far, the most prominent metabolite observed for 14C-difluorophenyl-labelled test substance, i.e. the difluorophenylring labelled molecule was carbon dioxide reaching after 360 days of aerobic incubation 58.6% of the dose applied. Besides the mineralization of the difluorophenylring considerable amounts of non-extractable radioactivity were formed reaching with 36.1% their highest concentration after 60 days. Thereafter, a slight but steady decrease of non-extractable radioactivity was observed reaching after one year 28.3%.These data show that non-extractable radioactivity was susceptible to further microbial breakdown in the soil. Analysis of the extractable radioactivity in the soil showed besides the parent molecule only negligible amounts of unknown, extractable metabolites accounting at highest for 0.66%.

Evaporation of parent compound:

not specified

Volatile metabolites:

yes

Residues:

yes

Details on results:

An overview of the results is provided in Table 2 - Table 7 in 'Any other information on results incl. tables'.
- Recovery: For 14C-dichlorophenyl-labelled test substance, the recoveries ranged from 94.5 to 107.3 (one outlying value with 89.2% at day 361), 99.7 to 109.7%, 103.4 to 105.3% and 102.3 to 108.0% of the dose applied for the aerobic incubation in Collombey loamy sand soil, the aerobic, aerobic-anaerobic and aerobic-sterile incubation in Les Evouettes loam, respectively. For 14C-difluorophenyl-labelled test substance the recoveries ranged from 89.9 to 103.1% and 89.4 to 103.5% (one outlying value with 69.6% at day 60) of the dose applied for aerobic and aerobic-anaerobic incubation in Les Evouettes silt loam, respectively.

- Extractability: Under aerobic conditions extractable radioactivity steadily decreased with time finally reaching after one year 21.6 and 14.1% of 14C-dichlorophenyl-labelled test substance applied to Collombey loamy sand soil and Les Evouettes loam. Accordingly the non-extractable fraction increased in the corresponding samples up to 66.8 and 74.9% of the dose applied. For 14C-difluorophenyl-labelled test substance extractable radioactivity decreased within one year to 3.0%. But, for reasons discussed below the non-extractable fraction accounted only for 28.3% of the dose applied. Under aerobic-anaerobic conditions the extractable fraction decreased within the 30 days aerobic incubation phase in Les Evouettes soil to 72.7 and 25.6% of the dose applied for 14C-dichlorophenyl-labelled and 14C-difluorophenyl-labelled test substance, respectively. The reason for the lower amount of extractable radioactivity found for 14C-difluorophenyl-labelled test substance was due to the formation of volatiles. During anaerobic incubation no drastic changes were observed. Thus, at the end of the study after three months extractables accounted for 69.2 and 20.2% in the corresponding experiments and non-extractable radioactivity was 32.4 and 34.4%. Under aerobic-sterile conditions no significant changes in the amounts of extractable and non-extractable radioactivity were observed during 90 days of incubation.

- Volatiles: For aerobic incubation of 14C-dichlorophenyl-labelled test substance, the evolution of 14C-CO2 was after one year 9.9 and 15.1% in Collombey and Les Evouettes soil, respectively. Only negligible amounts of volatile radioactivity other than carbon dioxide were found. Incubation of 14C-difluorophenyl-labelled test substance yielded under the same conditions within one year 58.6% of 14C-CO2 evolution indicating a rapid mineralization of the difluorophenylring moiety. Here again, no other volatile radioactivity was observed. Under aerobic-anaerobic conditions, 14C-CO2 was mainly observed during the aerobic incubation phase accounting for 14C-dichlorophenyl-labelled and 14C-difluorophenyl-labelled test substance for 2.7 and 29.3 %, respectively. The latter figure is regarded to be too low due to losses of carbon dioxide in the range of about 20% resulting from leaking of the absorption equipment. This becomes obvious for the 60 days samples when recovery and carbon dioxide evolution are compared. The amounts of other volatile radioactivity were again very low. Under aerobic-sterile conditions practically no volatile radioactivity was observed.

- Rate of Degradation of the Test Substance: Based on the concentration of the parent molecule found after aerobic incubation at various time intervals the degradation rate constants were determined by applying a two compartment model. The DT-50 and DT-90 values calculated for the Collombey sandy soil were 23.7 and 257 days, respectively, the latter figure most probably due to the low biomass present in the soil from day 180 on. In Les Evouettes loam the mean DT- 50 and DT-90 values were found to be 16.2 and 74.8 days, respectively. Except for the DT-90 value in the Collombey soil, the data demonstrate a rapid breakdown of the insecticide. Under aerobic-anaerobic conditions a rapid degradation of the test substance was observed during the 30 days aerobic incubation period leaving 25.7 and 25.1% as intact parent molecule for 14C-dichlorophenyl-labelled and 14C-difluorophenyl-labelled test substance, respectively. During the following two months of anaerobic incubation only minor amounts (6.7 - 7.4%) of the parent molecule were additionally broken down. Incubation under aerobic-sterile conditions resulted in no degradation of the parent molecule thus demonstrating that degradation of the parent molecule was mainly due the activity of aerobic microorganisms.

Table 2. Distribution Pattern of 14C-Activity for the Degradation of 14C-dichlorophenyl-labelled test substance in Soil (Collombey) under aerobic conditions (Values given in % of the 14C-activity applied)

Incubation Time (Days)	CO2	Volatiles	Extractable	Non-extractable	Recovery
0	0.00	0.00	103.10	1.10	104.20
7	0.09	0.01	105.76	1.52	107.29
14	0.20	0.08	100.41	4.68	105.17
30	0.92	0.36	86.34	16.85	103.55
59	2.06	0.26	66.31	34.63	101.20
91	3.35	0.60	48.14	48.85	97.59
181	6.74	0.66	26.41	67.47	94.54
240	6.39	0.65	26.65	70.69	97.99
361	9.88	0.76	21.64	66.81	99.09

 

Table 3. Distribution of 14C-dichlorophenyl-labelled test substance and metabolites in soil (collombey) under aerobic conditions (Values given in % of the 14C-activity applied)

Incubation time (days)	parent substance	M6	M4	UK	UK	UK	UK	Total
0	103.6	0	0	0	0	0	0	103.6
7	83.52	4.53	17.71	0	0	0	0	105.76
14	67.38	8.74	24.3	0	0	0	0	100.42
30	38.33	22.43	23.24	0	2.33	0	0	86.33
59	29.28	26.85	10	0.18	0	0	0	66.31
91	19.52	22.39	5.78	0	0	0	0.46	48.15
181	11.31	11.44	3.36	0	0	0	0.3	26.41
240	11.05	9.97	1.55	3.24	0.46	0	0.38	26.65
361	8.16	5.24	1.62	2.84	1.3	1.69	0.6	21.65

Table 4. Distribution Pattern of 14C-Activity for the Degradation of 14C-dichlorophenyl-labelled test substance in Soil (Les Evouettes) under aerobic conditions (Values given in % of the 14C-activity applied)

Incubation Time (Days)	CO2	Volatiles	Extractable	Non-extractable	Recovery
0	0.00	0.00	102.60	2.10	104.70
7	0.13	0.02	104.51	2.72	107.38
14	0.63	0.06	98.11	7.98	106.78
30	2.62	0.35	75.69	25.60	104.26
59	3.46	0.60	48.93	47.50	100.49
91	7.42	0.54	32.40	59.29	99.65
181	12.60	0.63	17.92	70.15	101.30
240	12.94	0.33	17.87	78.55	109.69
361	15.12	0.74	14.10	74.87	104.83

 

Table 5. Distribution of 14C-dichlorophenyl-labelled test substance and metabolites in soil (Les Evouettes) under aerobic conditions (Values given in % of the 14C-activity applied)

Incubation time (days)	parent substance	M6	M4	UK	UK	UK	UK	Total
0	103.15	0.00	0.00	0.00	0.00	0.00	0.00	103.15
7	75.61	7.99	20.91	0.00	0.00	0.00	0.00	104.51
14	60.00	15.06	23.05	0.00	0.00	0.00	0.00	98.11
30	37.08	19.97	15.99	0.00	2.65	0.00	0.00	75.69
59	16.86	21.64	7.47	0.00	2.97	0.00	0.00	48.94
91	9.90	17.09	4.71	0.00	0.00	0.00	0.70	32.40
181	5.07	8.50	2.39	0.00	1.77	0.00	0.19	17.92
240	6.84	6.85	3.22	0.35	0.17	0.13	0.31	17.87
361	4.24	4.13	2.68	0.35	1.10	1.45	0.15	14.10

 

Table 6. Distribution Pattern of 14C-Activity for the Degradation of 14C-difluorophenyl-labelled test substancein Soil (Les Evouettes) under aerobic conditions (Values given in % of the 14C-activity applied

Incubation Time (Days)	CO2	Volatiles	Extractable	Non-extractable	Recovery
0	0	0.00	0.00	2.10	103.12
7	7	13.16	18.44	102.11	102.11
14	14	20.78	47.11	28.00	95.90
30	30	40.77	21.21	35.82	97.80
59	60	46.58	10.51	36.06	93.16
91	90	53.16	6.57	34.42	94.16
181	180	46.10	4.18	30.03	80.32
240	217	56.61	3.88	33.07	93.57
361	360	58.58	2.95	28.34	89.89

 

Table 7. Distribution of 14C-difluorophenyl-labelled test substanceand metabolites in soil (Les Evouettes) under aerobic conditions (Values given in % of the 14C-activity applied)

Incubation time (days)	parent substance	UK	UK	UK	UK	UK	Total
0	101.02	0.00	0.00	0.00	0.00	0.00	101.02
7	70.43	0.00	0.08	0.00	0.00	0.00	70.51
14	46.99	0.00	0.12	0.00	0.00	0.00	47.11
30	20.80	0.14	0.17	0.06	0.05	0.00	21.22
60	10.13	0.06	0.19	0.05	0.07	0.00	10.50
90	5.89	0.11	0.00	0.00	0.57	0.00	6.57
217	2.88	0.09	0.66	0.00	0.09	0.16	3.88
360	1.79	0.43	0.59	0.00	0.00	0.14	2.95
0	101.02	0.00	0.00	0.00	0.00	0.00	101.02

UK= unknown radioactivity

 

 

Conclusions:

In a biodegradation study in soil, performed in accordance with EPA guidelines 162-1 and 162-2, under aerobic condition, the calculated DT50 values are 23.7 days for the 14C-dichlorophenyl-labelled test substance in Collombey Soil, 19.4 and 13.0 days for the 14C-dichlorophenyl-labelled and 14C-difluorophenyl-labelled test substance, respectively, in Les Evouettes soil. These values were re-calculated outside of the original study using single first-order kinetics. The re-calculated DT50 values are 24.2 days (14C-dichlorophenyl-labelled; Collombey Soil), 17.3 days (14C-dichlorophenyl-labelled; Les Evouettes soil) and 11.1 days (14C-difluorophenyl-labelled; Les Evouettes soil).

Executive summary:

The degradation of the test substance was studied with 14C-dichlorophenyl ring-labelled and 14C-difluorophenyl ring-labelled test substance. The study was performed in accordance with EPA guideline 162-1 and 162-2, and in compliance with GLP criteria. The metabolism of the test substance (labelled in the dichlorophenyl ring) was studied in two different soil types, a sandy loam soil (Les Evouettes) and a loam (Collombey) at a rate of 1 mg/kg dry soil. The metabolism of difluorophenyl ring-labelled test substance was investigated in Les Evouettes soil at a rate of 1.2 mg/kg. The treated soil was incubated in the dark, at 20 ± 2 °C and ventilated with a stream of moist air. The moisture content of the soil was maintained at 75% of 1/3 bar under conditions that allowed 14CO2 and any other volatilised degradates to be trapped. A further series of Les Evouettes soil samples, fortified at 1 mg/kg dry soil, were incubated under sterile conditions. Duplicate samples of incubated, treated soils were taken for analysis at 0, 7, 14, 30, 59, 91, 181, 240 and 361 days. 14CO2 was collected in aqueous 2M NaOH traps over the course of the study. Soil extracts were quantified by LSC. Qualitative analysis and metabolite identification was confirmed by co-chromatography using HPLC or TLC followed by mass spectrometry. The viability of the soil at the beginning and at days 91, 181 and 360 was determined.

For dichlorophenyl labelled test substance the recoveries ranged from 99.1 to 107.4% of AR for the aerobic incubation in the Collombey sandy loam soil. Recoveries from the loam soil ranged from 99.7% to 109.7% of AR. Under sterile conditions recoveries ranged from 102.3% to 108.1%. For aerobic incubation of the dichlorophenyl labelled test substance, 14C-CO2 increased to 9.9 and 15.1% in Collombey and Les Evouettes soil, respectively after one year. Only negligible amounts of volatile radioactivity other than CO2 were found. Under aerobic-sterile conditions practically no volatile radioactivity was observed. The test substance was mainly metabolized under aerobic conditions. No degradation was observed under sterile conditions. The test substance was first degraded into M4 reaching the highest concentration of 24.3% and 23.1% of the dose applied after 14 days in the Collombey and Les Evouettes soil, respectively. Thereafter, it was further degraded to M6, which showing its highest concentration with 26.9% and 21.6% after 59 days in the Collombey and Les Evouettes soil, respectively. At the end of the incubation period both metabolites had decreased to about 2 - 5% in both soils thus demonstrating the transient character of the two metabolites. The above mentioned degradation products up to at least three minor, unknown metabolites were observed accounting at highest for 3.2%. Based on the findings, the DT50 values were calculated, using a two compartment model, to be 23.7 days for the 14C-dichlorophenyl-labelled test substance in Collombey Soil, 19.4 and 13.0 days for the 14C-dichlorophenyl-labelled and 14C-difluorophenyl-labelled test substance, respectively, in Les Evouettes soil. These values were re-calculated outside of the original study using single first-order kinetics. The re-calculated DT50 values are 24.2 days (14C-dichlorophenyl-labelled; Collombey Soil), 17.3 days (14C-dichlorophenyl-labelled; Les Evouettes soil) and 11.1 days (14C-difluorophenyl-labelled; Les Evouettes soil).

Reference 4

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Feb 1994 to 30 May 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The degradation rate of the test substance was investigated in silt loam soil under aerobic laboratory conditions using different modes of application. The test substance was incorporated into the soil, or applied on the surface of the soil, or applied on the surface of the soil followed by mixing with the soil. Half-lives of the test substance in these conditions were calculated.

GLP compliance:

not specified

Test type:

laboratory

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

1993

Soil no.:

#1

Soil type:

silt loam

% Clay:

13.9

% Silt:

54.3

% Sand:

31.8

% Org. C:

2.1

pH:

7.3

CEC:

14 meq/100 g soil d.w.

Details on soil characteristics:

An overview of the soil properties is provided in Table 1 in 'Any other information on materials and methods incl. tables'
- Soil preparation: Before use the soil was passed to a 2 mm sieve.

Soil No.:

#1

Duration:

92 d

Soil No.:

#1

Initial conc.:

0.1 mg/kg soil d.w.

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

radiochem. meas.

Soil No.:

#1

Temp.:

20 °C

Humidity:

60% of the field capacity

Microbial biomass:

92.4 mg/100 dry soil (start of the test )

Details on experimental conditions:

EXPERIMENTAL DESIGN
- Soil condition: Aerobic/non-sterile
- Soil (g/replicate): 75
- No. of replication treatments: 24
- Test apparatus: 200 mL Erlenmeyer flasks

Test material application
- Preparation: A stock solution (I) of 14C-labelled test substance in 1.0 mL acetone was prepared and checked for concentration by liquid scintillation counting and analyzed by HPLC for radiochemical purity. The amount determined was 0.2068 mg. Thereafter, the soil batches were prepared and treated with the treatment solutions.
- Volume of test solution used/treatment: See Table 2 in 'Any other information on materials and methods incl. tables'
- Application method: The test substance solution was added to the soil in about three portions. After each portion the soil was carefully mixed in a Hobart kitchen machine for about 1 - 2 minutes. Thereafter, the soil moisture was adjusted by adding water and soil samples corresponding to 75 g dry soil equivalent weighed into the Erlenmeyer flasks. Subsamples of soil up to 1 g were removed from the batches at the beginning, middle and end of the weighing for determination of the accurate amount applied and homogeneity by combustion. After completion of treatment, the Erlenmeyer asks were connected to the other of the metabolism apparatus and incubated in climatization chambers.

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: It was adjusted weekly during the first month and thereafter in about two-weeks intervals
- Continuous darkness: Yes/No

OXYGEN CONDITIONS
- Methods used to create the aerobic conditions: The aerobic experiments were continuously ventilated with air at a low rate of 60 mL/min.

SAMPLING DETAILS
- Sampling intervals (sampling of soil): See Table 3 in 'Any other information on materials and methods incl. tables'
- Sampling intervals (microbial biomass of the soil): Microbial biomass was determined prior to treatment from vehicle only treated soil samples
- Sampling intervals (Absorption solutions): During the first month, the absorption solutions were exchanged in about-weekly intervals, thereafter in about two-weeks intervals preferably at the time of sampling of soil.

Soil No.:

#1

% Total extractable:

8.83

% Non extractable:

37.22

% CO2:

52.04

% Recovery:

98.09

Remarks on result:

other: incorporated into the Soil

Remarks:

day 92

Soil No.:

#1

% Total extractable:

36.75

% Non extractable:

36.4

% CO2:

20.67

% Recovery:

93.82

Remarks on result:

other: applied to soil surface without further Incorporation

Remarks:

day 91

Soil No.:

#1

% Total extractable:

12.81

% Non extractable:

53.26

% CO2:

26.15

% Recovery:

92.22

Remarks on result:

other: applied to the Soil Surface and incorporated 14 days after Treatment

Remarks:

day 91

Parent/product:

parent

Soil No.:

#1

% Degr.:

92.1

Parameter:

radiochem. meas.

Sampling time:

92 d

Remarks on result:

other: incorporated into the Soil

Parent/product:

parent

Soil No.:

#1

% Degr.:

64.78

Parameter:

radiochem. meas.

Sampling time:

92 d

Remarks on result:

other: surface without further incorporation

Parent/product:

parent

Soil No.:

#1

% Degr.:

88.25

Parameter:

radiochem. meas.

Sampling time:

92 d

Remarks on result:

other: surface with incorporation after two weeks of incubation

Key result

Soil No.:

#1

DT50:

9.3 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: correct for soil moisture; average of all types of applications

Soil No.:

#1

DT50:

11.5 d

Type:

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

Temp.:

20 °C

Remarks on result:

other: average of all types of applications

Soil No.:

#1

DT50:

9.1 d

Type:

other: two-compartment model

Temp.:

20 °C

Remarks on result:

other: incorporated into the Soil

Soil No.:

#1

DT50:

32.5 d

Type:

other: two-compartment model

Temp.:

20 °C

Remarks on result:

other: surface without further incorporation

Soil No.:

#1

DT50:

32.3 d

Type:

other: two-compartment model

Temp.:

20 °C

Remarks on result:

other: surface with incorporation after two weeks of incubation; first 14 days

Soil No.:

#1

DT50:

13.8 d

Type:

other: two-compartment model

Temp.:

20 °C

Remarks on result:

other: surface with incorporation after two weeks of incubation; after 14 days

Transformation products:

not measured

Evaporation of parent compound:

not specified

Volatile metabolites:

yes

Residues:

yes

Details on results:

An overview of the results is provided in Table 4 - Table 8 in 'Any other information on reults incl. tables'.
- Microbial Biomass: The microbial biomass of the soil amounted to 92.4 mg microbial carbon per 100 g of dry soil equivalent at the beginning of the study.
- Recovery of Radioactivity: the recoveries ranged for all experimental groups from 92.22 to 100.67% of the radioactivity applied.
- Group1: After incorporation of the test substance, a continuous decline of extractables was observed ending 92 days with 8.8 % of the radioactivity applied. Non-extractables amounted at the start of the experiment to 4.4 % and rapidly increased reaching already two weeks an apparent plateau with 35.3 %. At the end of the study they were 37.2 %. By far the most prominent radioactive fraction observed was CO2 reaching at the end of the study 52.0 %.
- Group 2: For group 2 where the test substance was applied to the soil surface a slow decline of extractables with time was observed reaching steady state conditions already after 34 days of incubation with 44.4 %. At the end of the study extractables were still 36.8%. Non-extractables increased at the same time from 3.6% at the start to 36.4 % at the end of the study but the formation was found to be much slower than observed for Group 1. The most significant difference observed between these two treatment groups was the lower amount of CO2 formed after surface treatment. It was at the end of the study 20.7% only.
- Group 3: Soil samples of group 3 were surface treated with the test compound but after two weeks of incubation the radioactivity was incorporated into the soil. The kinetics of radioactivity, i.e. formation of carbon dioxide, non-extractables and decline of extractables were almost identical with those observed for group 2 but after incorporation a significant increase in formation of carbon dioxide and bound residues was observed. At the end of the study the amounts of extractables, non-extractables and carbon dioxide were 12.8, 53.3 and 26.2 %, respectively. These data clearly state that after incorporation the test compound was further broken down whereas for group 2 steady state conditions were reached after 34 days.
- Rate of Degradation of the Parent Molecule: These data clearly demonstrate that the test substance readily degrades in soil when incorporated into the soil. (Group 1) or when later incorporated into the soil (Group 3). But when the test compound was left on the soil surface a much slower degradation rate was observed reaching after about one month steady state conditions, i.e. in essence no further, significant degradation was observed.

Table 4. Balance of Radioactivity after Incubation of [U-14C] difluorophenyl-ring labelled test substance incorporated into the Soil. (Values in % of the radioactivity applied) 

Time (days)	CO2	Extractable	Non-extractable	Recovery
0	0.00	96.28	4.39	100.67
7	14.52	58.59	27.23	100.34
14	27.50	36.89	35.27	99.66
21	35.01	26.00	37.85	98.87
35	42.43	16.87	37.64	96.94
72	50.23	9.80	35.78	95.81
92	52.04	8.83	37.22	98.09

  

Table 5. Balance of Radioactivity after Incubation of [U-14C] difluorophenyl-ring labelled test substance applied to the Soil Surface without further Incorporation. (Values in % of the radioactivity applied)

Time (days)	CO2	Extractable	Non- extractable	Recovery
0	0.00	94.32	3.64	97.96
7	4.37	81.06	13.72	99.15
14	9.07	73.54	17.60	100.21
21	12.60	59.41	25.24	97.26
34	16.68	44.41	33.21	94.30
71	20.29	24.88*	47.46*	92.64
91	20.67	36.75	36.40	93.82

*Considered to be an outlier.

Table 6. Balance of Radioactivity after Incubation of [U-14C] difluorophenyl-ring labelled test substance applied to the Soil Surface and incorporated 14 days after Treatment. (Values in % of the radioactivity applied)

Time (days)	CO2	Extractable	Non- extractable	Recovery
0	0.00	95.99	2.97	98.96
7	4.71	80.34	14.06	99.10
14	9.76	71.52	18.92	100.19
21	15.21	52.87	28.84	96.92
34	21.16	31.02	44.09	96.26
71	25.62	17.39	55.50	98.51
91	26.15	12.81	53.26	92.22

 

Table 7. Decline of the parent molecule and formation and decline of degradates after incubation of U-14C] difluorophenyl-ring labelled test substance differently applied to soil. (Values in % of the radioactivity applied)

Time (days)	Group I			Group II			Group III
	Parent substance	NA	Recovery	Parent substance	NA	Recovery	Parent substance	NA	Recovery
0	96.28	0.00	96.28	94.32	0.00	94,32	95.99	0.00	95.99
7	57.44	0.00	58.59	81.06	0.00	81.06	80.34	0.00	80.34
14	36.89	0.00	36.89	73.54	0.00	73.54	71.52	0.00	71.52
21	26.00	0.00	26.00	59.41	0.00	59,41	52.87	0.00	52.87
35	15.03	1.83	16.86	44.41	0.00	44.41	31.02	0.00	31.02
72	8.90	0.90	9.80	20.09	1.71	24.88	16.18	1.21	17.39
92	7.90	0.93	8.83	35.22	1.53	36.75	11.75	1.05	12.80

Group I: Treatment by incorporation

Group II: Treatment to the surface without further incorporation

Group III: Treatment to the surface with incorporation after two weeks of incubation

 

Table 8. Rate constants and half-live

Parameter	Group 1	Group 2	Group 3
			Part 1	Part 2
C1	80.73	63.50	95.27906	124.3043
C2	15.46	32.43		10
K1	0.08982	0.03926	0.02145	0.05031
K2	0.00741	0.00012		0.0007
Chi^2	0.258	13.814	4.16022	0.7001
DT-50	9.1	32.5	32.3	13.8
DT-90	62.8	-		103

 

 

Conclusions:

In a biodegradation study in soil, performed without following guideline but well documented and meeting generally accepted scientific principles, the DT50 value of the Les Evouettes soil was calculated to be 11.5 days using the single first-order kinetic. After correcting for soil moisture, the DT50 values is 9.3 days.

Executive summary:

The purpose of this study was to discover why there was a difference in degradation rate between laboratory and field studies. It was postulated that the test substance incorporated into moist, microbial active soil was more readily degraded then the test substance applied to dry soil without a growing crop during summer months in the field. This study was carried out without following guidance, but it is well documented and meets generally accepted scientific principles. The rate of degradation of 14C–difluorophenyl ring-labelled the substance was investigated in a silt loam soil (Les Evouettes) under a range of test conditions. 14C-labelled test substance was applied to soil held at 60% MHC and 20ºC at 0.1 mg/kg. In Group 1 the chemical was incorporated into the soil. In the other two tests, a surface application was made. One of the latter two tests was left unchanged whilst for the other, the test substance was incorporated by mixing after test 1 reached half-life (after 14 days). Samples of incubated, treated soils were taken for analysis at 0, 7, 14, 21, 35, 72 and 92 days. Soil extracts were partitioned with dichloromethane and the aqueous and organic phases quantified by liquid scintillation counting (LSC). Quantitative analysis of the individual compounds was performed by HPLC.

Overall recoveries of applied radioactivity (AR) ranged from 92.2 - 100.7%. For the incorporation of the test substance into the soil group (Group 1), a continuous decline of extractables was observed ending 92 days with 8.8 % of the radioactivity applied. Non-extractables amounted at the start of the experiment to 4.4 % and rapidly increased reaching already two weeks an apparent plateau with 35.3 %. At the end of the study they were 37.2 %. By far the most prominent radioactive fraction observed was CO2 reaching at the end of the study 52.0 %. For the test substance that was applied to the soil surface group (Group 2), a slow decline of extractables with time was observed reaching steady state conditions already after 34 days of incubation with 44.4 %. At the end of the study extractables were still 36.8%. Non-extractables increased at the same time from 3.6% at the start to 36.4 % at the end of the study but the formation was found to be much slower than observed for Group 1. The most significant difference observed between these two treatment groups was the lower amount of CO2 formed after surface treatment. It was 20.7% only at the end of the study. For the surface treated with the test compound with incorporation after two weeks group (Group 3), the kinetics of radioactivity, i.e. formation of carbon dioxide, non-extractables and decline of extractables were almost identical with those observed for group 2 but after incorporation a significant increase in formation of carbon dioxide and bound residues was observed. At the end of the study the amounts of extractables, non-extractables and carbon dioxide were 12.8, 53.3 and 26.2 %, respectively. These data clearly state that after incorporation the test compound was further broken down whereas for group 2 steady state conditions were reached after 34 days. Overall, the DT50 value of the Les Evouettes soil was calculated to be 11.5 days using single first-order kinetics. After correcting for soil moisture, the DT50 values is 9.3 days.

Description of key information

All available data was assessed and the aerobic studies following standard test guidelines are included here in a weight of evidence approach.

Geometric mean DT50 in soil = 18.3 d, 20 °C, EPA 162 -1 & 162 -2; German BBLF IV 4 -1, Dutch guideline G1, or no guideline followed but well documented and scientifically acceptable studies, Ellgehausen 1991, Gonzalez-Valero 1991 a, Gonzalez-Valero 1991b and Ellgehausen 1994.

Key value for chemical safety assessment

Half-life in soil:

18.3 d

at the temperature of:

20 °C

Additional information

Four studies are available for this endpoint. Three of them followed standard test guideline and complied with GLP (Reliability 1). One study (Reliability 2) did not follow standard test guideline but was well documented, met generally accepted scientific principles, and was compliant with GLP criteria. The effect values from all four studies were used in a weight of evidence approach for the CSA. The test conditions and key effect values were summarized in the table below. Calculation of degradation half-lives according to a standardized methodology across all studies is presented in Sapiets (2003).

Table 1. DT50 values for the test substance in soil under aerobic test conditions 

Parent
USDA / Name / Origin	OC [%] / pH (water)	T. [°C] / Moisture	DT50 [d] - Kinetic model in original report (a)	DT50 [d] - Kinetic model recalculated (b)	Author / Year
Sandy loam / Collombey / CH	3.0 / 7.2	20 / 75% FC at 1/3 bar	29.6 (SFO)	24.2* (SFO)	Ellgehausen / 1991 Sapiets/ 2003 (d)
Loam / Les Evouettes / Vouvry-CH	3.7 / 6.8	20 / 75% FC at 1/3 bar	21.2 / 13.6 (c) (SFO)	17.3 / 11.1* (c) (SFO)	Ellgehausen / 1991 Sapiets/ 2003 (d)
Sandy loam / Mosimann / LUFA Speyer- D	1.1 / 7.3	20 / 40% MWHC	17.4 (SFO)	11.8* (SFO)	Gonzalez-Valero / 1991a Sapiets / 2003
Loamy sand / Neuhofen / Les Barges- CH	1.8 / 5.0	20 / 40% MWHC	83.1 (SFO)	75.7* (SFO)	Gonzalez-Valero / 1991a Sapiets / 2003
Silt loam / Les Evouettes / Vouvry- CH	2.0 / 7.2	20 / 60% FC at 1/3 bar	16.3 (SFO) (e)	13.0* (SFO) (e)	Gonzalez-Valero / 1991b Sapiets / 2003
Silt loam / Les Evouettes / Vouvry- CH	2.1 / 7.3	20 / 60% FC at 1/3 bar	11.5 (SFO)	9.3* (SFO)	Ellgehausen / 1994 Sapiets / 2003
			Geomean	18.3
			Median	13.6

* Value used for determination of the geomean

(a) Half-lives calculated (single first order rates) using the original data in the reports.

(b) Half-lives (single first order rates) corrected for soil moisture according to FOCUS kinetic guidelines where necessary (Sapiets, 2003).

(c) Derived from studies with dichlorophenyl and difluorophenyl ring labelled test substance, respectively. Average of both values (14.2 d) used for calculation of the geomean.

(d) Summary report produced to standardise the methodology for all the studies and to provide best-fit degradation rates from each soil

experiment. Half-lives were also calculated in those cases where the best-fit was not simple first-order and, where appropriate, corrected to FOCUS standards.

(e) Mean value calculated from two applications at 0.1 and 1 mg/kg in the same soil.

Field dissipation - additional information

A large number of soil dissipation studies were conducted with the test material. As information on soil dissipation is outside the scope of REACH, the studies are not summarized as an endpoint study entry but briefly discussed here. The test material was tested for different applications in different crops and bare grounds with a rate ranging between 130 - 300 g/ha. The DT50 reported in the original reports as calculated with SFO model were between 34.2 - 344 days. The DT50 as recalculated SFO model were between 98.4 - 315 days. The geometric mean DT50 (n=8) for soil dissipation was calculated to be 213 days (Emburey 2007; Emburey 2008; Stingelin 1992; Hardy 2013; Capps 1988; Simon 2008a and Simon 2008b).