3,5-bis(difluoromethyl)-1H-pyrazole

Biodegradation in soil

Administrative data

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07. June 2018 -11. March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Version / remarks:

2002

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.4100 (Aerobic Soil Metabolism)

Version / remarks:

2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF Test Guidelines 12 Nousan 8147, No. 2-5-2

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Test material

Test material information

Constituent 1

Reference substance name:

3,5-bis(difluoromethyl)-1H-pyrazole

EC Number:

814-072-3

Cas Number:

77614-79-0

Molecular formula:

C5H4F4N2

IUPAC Name:

3,5-bis(difluoromethyl)-1H-pyrazole

Radiolabelling:

yes

Study design

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

2018

Soil properties

Soil properties 1

Soil no.:

#1

Soil type:

silt loam

% Clay:

21

% Silt:

60

% Sand:

19

% Org. C:

2.9

pH:

5.2

CEC:

10 meq/100 g soil d.w.

Bulk density (g/cm³):

1

Soil properties 2

Soil no.:

#2

Soil type:

sandy loam

% Clay:

11

% Silt:

18

% Sand:

71

% Org. C:

1.7

pH:

6.9

CEC:

8.6 meq/100 g soil d.w.

Bulk density (g/cm³):

1.2

Soil properties 3

Soil no.:

#3

Soil type:

silt loam

% Clay:

19

% Silt:

66

% Sand:

15

% Org. C:

2.3

pH:

5.7

CEC:

11.2 meq/100 g soil d.w.

Bulk density (g/cm³):

1.1

Soil properties 4

Soil no.:

#4

Soil type:

loam

% Clay:

27

% Silt:

44

% Sand:

29

% Org. C:

5.4

pH:

7.2

CEC:

19.8 meq/100 g soil d.w.

Bulk density (g/cm³):

0.9

Details on soil characteristics:

SOIL COLLECTION AND STORAGE (valid for all four soils)
- Geographic location: North Rhine Westphalia / Germany
- Pesticide use history at the collection site: No plant protection products used for previous 5 years
- Collection procedures: Sample taken with shovel and placed in plastic bag
- Sampling depth (cm): 0 -20
- Storage conditions: Stored at ambient temperature prior to use
- Storage length: 9 - 10 days prior to equilibration
- Soil preparation (e.g., 2 mm sieved; air dried etc.): Soils were passed through a 2 mm sieve

PROPERTIES OF THE SOILS (in addition to defined fields)
- Moisture of sieved soil): #1 26.3 g/100 g soil dry weight
#2 12.8 g/100 g soil dry weight
#3 21.9 g/100 g soil dry weight
#4 34.5 g/100 g soil dry weight

Duration of test (contact time)

Duration of test (contact time) 1

Soil No.:

#1

Duration:

120 d

Duration of test (contact time) 2

Soil No.:

#2

Duration:

28 d

Duration of test (contact time) 3

Soil No.:

#3

Duration:

120 d

Duration of test (contact time) 4

Soil No.:

#4

Duration:

91 d

Initial test substance concentration

Initial test substance concentration 1

Soil No.:

#1

Initial conc.:

0.07 mg/kg soil d.w.

Initial test substance concentration 2

Soil No.:

#2

Initial conc.:

0.07 mg/kg soil d.w.

Initial test substance concentration 3

Soil No.:

#3

Initial conc.:

0.07 mg/kg soil d.w.

Initial test substance concentration 4

Soil No.:

#4

Initial conc.:

0.07 mg/kg soil d.w.

Parameter followed for biodegradation estimation:

radiochem. meas.

test mat. analysis

Experimental conditions

Experimental conditions 1

Soil No.:

#1

Temp.:

19.5

Microbial biomass:

710 mg microbial carbon per kg soil dry weight at experimental start

Experimental conditions 2

Soil No.:

#2

Temp.:

19.5

Microbial biomass:

778 mg microbial carbon per kg soil dry weight at experimental start

Experimental conditions 3

Soil No.:

#3

Temp.:

19.5

Microbial biomass:

950 mg microbial carbon per kg soil dry weight at experimental start

Experimental conditions 4

Soil No.:

#4

Temp.:

19.5

Microbial biomass:

2424 mg microbial carbon per kg soil dry weight at experimental start

Details on experimental conditions:

EXPERIMENTAL DESIGN
- Soil preincubation conditions (duration, temperature if applicable): Equilibration to study conditions for 4 days
- Soil condition: fresh
- Soil (g/replicate): 100 g dry weight equivalents per replicate
- Control conditions, if used (present differences from other treatments, i.e., sterile/non-sterile, experimental conditions): Samples for determination of soil microbial biomass:native soil with and without application solvent
- No. of replication controls, if used: not used
- No. of replication treatments: Duplicate samples for each sampling interval
- Test apparatus (Type/material/volume): 300 mL glass Erlenmeyer flasks
- Details of traps for CO2 and organic volatile, if any: Each test vessel was fitted with a trap attachment (permeable for oxygen) containing soda lime for absorption of carbon dioxide and a polyurethane (PU) foam plug for adsorption of volatile organic compounds (VOC).
- If no traps were used, is the system closed/open: closed system
- Identity and concentration of co-solvent: Acetonitrile 99.5 %

Test material application
- Volume of test solution used/treatment: The total amount of test item was dissolved in 1.75 mL acetonitrile/water (1/1, v/v), resulting in a stock solution with a nominal concentration of 1 mg/mL (3.99 MBq/mL). 952 µL of the stock solution were pipetted into a flask, diluted with 119 mL of acetate buffer pH 4 and sonicated for 5 minutes, resulting in an application solution with a nominal concentration of 7.8 µg/mL (31.2 kBq/mL).

- Application method (e.g. applied on surface, homogeneous mixing etc.): 1000 µL of application solution were applied dropwise evenly distributed using a pipette onto the soil surface of the respective equilibrated test systems.
- Is the co-solvent evaporated: No.

Any indication of the test material adsorbing to the walls of the test apparatus: No.

Experimental conditions (in addition to defined fields)
- Moisture maintenance method: Re-weighing and addition of lost water
- Continuous darkness: Yes

Other details, if any:

OXYGEN CONDITIONS (delete elements as appropriate)
- Methods used to create the aerobic conditions: Trap attachment (permeable for oxygen)

SAMPLING DETAILS
- Sampling intervals: Seven to eleven sampling intervals were distributed over the entire incubation period of 28 days (soil #2), 91 days (soil #4) and 120 days (soils #1 and #4). Duplicate samples were processed and analyzed 0, 1, 3, 7, 10, 14 and 28 days after treatment (DAT) for all soils. Samples of soil #4 were additionally processed 45, 63 and 91 days after treatment while samples of soils #1 and #3 were additionally processed 45, 63, 91 and 120 days after treatment. Microbial soil biomass was determined at start, middle and end of the study (DAT-2 (all soils), DAT-29 (only soil #2), DAT-63 (soils #1, #3 and #4) and DAT-120 (soils #1 and #3)).

- Sampling method for soil samples: Processing of whole replicates.
- Method of collection of CO2 and volatile organic compounds: Collection of CO2 by liberation of CO2 trapped in soda lime using hydrochloric acid
Collection of volatile organic compounds via extraction of the PU plug using 50 mL ethyl acetate
- Sampling intervals/times for:
> Sterility check, if sterile controls are used
> Moisture content: each sampling day

- Sample storage before analysis: The trap attachments containing soda lime and PU foam were stored before processing at ambient temperature in the laboratory for a maximum period of 8 days
The first analyses of soil extracts by LSC and the primary chromatographic method were usually done within three days after sampling. After analysis, soil extracts were stored at < -18 °C in the dark.
The exhaustively extracted soils were dried, homogenized and stored at ambient temperature in the laboratory. For determination of non-extractable residues (NER) by combustion/LSC soils were stored for a maximum period of 19 days. For characterization of soils were stored for a maximum period of 98 days.

- Other observations, if any: No.

Results and discussion

Material (mass) balance

Material (mass) balance 1

Soil No.:

#1

% Recovery:

98.6

Material (mass) balance 2

Soil No.:

#2

% Recovery:

97.4

Material (mass) balance 3

Soil No.:

#3

% Recovery:

98.6

Material (mass) balance 4

Soil No.:

#4

% Recovery:

98.2

Half-life / dissipation time of parent compound

Half-life / dissipation time of parent compound 1

Soil No.:

#1

DT50:

15.8 d

Type:

other: double first order in parallel

Temp.:

19.5 °C

Half-life / dissipation time of parent compound 2

Soil No.:

#2

DT50:

3.3 d

Type:

other: double first order in parallel

Temp.:

19.5 °C

Half-life / dissipation time of parent compound 3

Soil No.:

#3

DT50:

18.6 d

Type:

other: double first order in parallel

Temp.:

19.5 °C

Half-life / dissipation time of parent compound 4

Soil No.:

#4

DT50:

6.9 d

Type:

other: double first order in parallel

Temp.:

19.5 °C

Transformation products:

yes

Remarks:

5-(difluoromethyl)-1H-pyrazole-3-carboxylic acid

Identity of transformation products

No.:

#1

Reference

Reference substance name:

Unnamed

IUPAC name:

5-(difluoromethyl)-1H-pyrazole-3-carboxylic acid

CAS number:

681034-51-5

Molecular formula:

C5 H4 F2 N2 O2

Molecular weight:

ca. 162.1

SMILES notation:

OC(=O)c1cc(nn1)C(F)F

Details on transformation products:

The following degradation product was identified by HPLC-MS(/MS) including accurate mass determination as well as by HPTLC co-chromatography after isolation from soil extracts: 5-(difluoromethyl)-1H-pyrazole-3-carboxylic acid

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes
- Anomalies or problems encountered (if yes): No

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount at end of study period: 42.5 % AR


EXTRACTABLE RESIDUES
Extractable residues decreased from DAT-0 to the last sampling interval from 104 to 12.5% AR at DAT-120 in soil #1, from 103 to 26.3% AR at DAT-28 in soil #2, from 104 to 15.6% AR at DAT-120 in soil #3 and from 103 to 11.8% AR at DAT-91 in soil #4.

NON-EXTRACTABLE RESIDUES
Non-extractable residues (NER) increased from < 0.3% AR at DAT-0 to 35.5% AR at DAT-28 in soil #2 and from 0.5% AR at DAT-0 to 37.6% AR at DAT-91 in soil #4. NER increased in soil #1 from DAT-0 to DAT-91 from 0.3 to 32.4% AR and then further decreased slightly to 30.2% AR at DAT-120. In soil #3, NER increased from DAT-0 to DAT-91 from < 0.3 to 32.0% AR and then further decreased slightly to 30.3% AR at DAT-120

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: The maximum amount of carbon dioxide was 49.7, 29.6, 44.3 and 43.6% AR at the last sampling interval in soil #1, #2, #3 and #4, respectively.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: <= 1.8 % AR for all soils at all sampling intervals

Applicant's summary and conclusion

Conclusions:

The test item undergoes degradation with mineralization in soil under aerobic conditions in the laboratory in the dark. Besides carbon dioxide, one further degradation product was identified.