spirodiclofen (ISO);3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutyrate

Dissociation constant

Administrative data

Endpoint:

dissociation constant

Data waiving:

study technically not feasible

Justification for data waiving:

the study does not need to be conducted because the substance is insoluble

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This experimental study report is reliable for use in the risk assessment as it has been conducted by a trusted laboratory, under GLP and following EPA, EEC and SETAC Guidelines relevant at the time of the study being conducted. There are no stated deviations from the current guidelines.

Qualifier:

according to guideline

Guideline:

other: US EPA, Pesticide Assessment Guidelines, Subdivision N, Chemistry: Environmental Fate §161-1 Hydrolysis Studies

Version / remarks:

Oct. 18, 1982.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: EC Commission Directive 95/36/EC amending Council Directive 91/414/EEC Annexes I and II, Fate and Behavior in the Environment

Version / remarks:

July 14, 1995.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: SETAC Procedures for Assessing the Environmental Fate and Ecotoxicity of Pesticides

Version / remarks:

March 1995

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

[dihydrofuranone-3-14C]Spirodiclofen

Analytical monitoring:

yes

Details on sampling:

Samples (7.5 mL of buffer in a crimp top glass vessel) were analyzed at 0, 2, 4, 7, 14, 21, and 30 days. Additional samples were taken for pH 9 at 1 and 6 hours and 3 days. For processing of the test solutions (7.5 mL), three aliquots (500 µL) were analyzed by LSC, and two aliquots (500 µL) were analyzed by TLC. Acetonitrile (5 mL) was added to the remaining test solution (5 mL), to solubilize adsorbed radioactivity from the glass walls, and two aliquots (500 µL) were analyzed by LSC.

Buffers:

0.01 M acetate buffer (pH 4) - A 0.04 M stock solution of acetate buffer was prepared by dissolving 1.36 g CH3CO2Na x 3 H20 in 250 mL of water. The pH of this solution was measured with a pH-electrode and adjusted to pH 4.0 using acetic acid. The final 0.01 M acetate buffer solution was prepared by diluting the 0.04 M buffer solution to the desired molarity with purified water (1:3 buffer:water; v/v) and sterilized

0.01 M TRlS buffer (pH 7) - A 0.05 M TRlS buffer stock solution was prepared by mixing 50 mL of a 0.1 M tris(hydroxymethyl)aminomethane solution (12.1 g/L H20) with 46 mL of 0.1 N hydrochloric acid (3.65 g HCl/L H20) followed by dilution to 100 mL with purified water. The pH of this solution was measured with a pH electrode and adjusted to pH 7.0 using 0.1 N hydrochloric acid and 0.1 N sodium hydroxide, respectively. The final 0.01 M TRlS buffer solution was prepared by diluting to the desired molarity of 0.01 M with purified water (1:4 buffer:water; v/v) and sterilized.

0.01 M borate buffer (pH 9) - A 0.02 M borate buffer stock solution was prepared by dissolving 0.62 g boric acid (H3BO3) and 0.75 g KCl in water to a final volume of 250 mL. To 125 mL of this solution 53 mL of a 0.04 M sodium hydroxide solution (1.6 g NaOH/L H20) was added, and the mixture was diluted with water to a total volume of 250 mL. The pH of this solution was measured with a pH electrode and adjusted to pH 9.0 using 0.4 M sodium hydroxide or boric acid, respectively. The final 0.01 M borate buffer solution was prepared by diluting the 0.04 M buffer stock solution to the desired molarity of 0.01 M with purified water (1:1 buffer:water; v/v) and sterilized.

Details on test conditions:

Test concentrations (mg a.i./L):
nominal: 0.025 mg a.i./L (50% of maximum water solubility).
measured: 0.0235 mg/L (pH 4); 0.0266 mg/L (pH 7); 0.0250 mg/L (pH 9)

Preparation of test medium:
volume used/treatment: 7.5 mL of the corresponding buffer solution.
method of sterilization: Buffer solutions and all glass ware used for preparation of solutions and sampling procedure were sterilized by steam pressure sterilization before application of the test substance.
co-solvent (type/concentration): Dose was applied in 75 µL of acetonitrile (ACN) to 7.5 mL of the corresponding buffer giving a final concentration of 1% ACN (v/v).

Test apparatus (type/material/volume): 10 mL glass crimp-top vials filled with ca. 7.5 mL test solution and stoppered using crimp caps with Teflon-faced septa. The crimp caps of the samples were marked with different colors to distinguish between the pH values.

Traps for volatiles: no traps. Closed test system with crimp caps to maintain sterility.

Is there any indication of the test material adsorbing to the walls of the test apparatus?: yes. Glassware was silanized prior to sterilization and additional samples were prepared and analyzed to ascertain degree of binding. Each flask was rinsed with ACN, and the rinse was radioassayed. Additional flasks were prepared for the analysis of the glass-bound (i.e., ACN soluble) residues.

Experimental conditions:
Temperature (ºC): 25ºC (pre-test performed at 50ºC).
Lighting: dark, no lights.

Duration:

30 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

0.025 mg/L

Duration:

30 d

pH:

7

Temp.:

25 °C

Initial conc. measured:

0.027 mg/L

Duration:

30 d

pH:

4

Temp.:

25 °C

Initial conc. measured:

0.024 mg/L

Number of replicates:

2 per interval for analysis for quantitation of radioactive residues.

Preliminary study:

In a preliminary test, the hydrolysis
study was conducted at 50ºC using the same procedures and buffers as were used in the test at
25ºC. The half-lives at 50ºC and pH 4, 7, and 9 were 3.1, 2.5, and 0.4 days, respectively. Using
the data from the hydrolysis at 50ºC and 25ºC, the half-lives of Spirodiclofen at 20ºC and pH 4, 7,
and 9 were calculated via the Arrhenius equation at 119.6, 52.1, and 2.5 days, respectively.

Transformation products:

yes

No.:

#1

% Recovery:

97.7 - 106.7

pH:

9

Temp.:

25 °C

Duration:

>= 0 - <= 30 d

Remarks on result:

other: recovered radioactivity

% Recovery:

97.4 - 103.5

pH:

7

Temp.:

25 °C

Duration:

>= 0 - <= 30 d

Remarks on result:

other: recovered radioactivity

% Recovery:

98.8 - 103.1

pH:

4

Temp.:

25 °C

Duration:

>= 0 - <= 30 d

Remarks on result:

other: recovered radioactivity

Key result

pH:

9

Temp.:

25 °C

DT50:

1.9 d

Type:

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

Key result

pH:

7

Temp.:

25 °C

DT50:

30.8 d

Type:

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

Key result

pH:

4

Temp.:

25 °C

DT50:

63.6 d

Type:

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

Details on results:

TEST CONDITIONS: The pH and temperature and other experimental conditions were maintained throughout the study. The initial pH 7 hydrolysis did not maintain sterility, but this was repeated and sterility was maintained on the second attempt. Residues of Spirodiclofen adsorbed to the walls of the glassware during the study. These residues were shown to be comprised of only one component, Spirodiclofen. A minor artifact was formed during the analysis of the major degradate, Spirodiclofen-Enol, and additional experiments performed with a standard of the Enol clearly showed that this product (M2, Spirodiclofen-Lactide) was an artifact produced during chromatographic analyses.

MASS BALANCE: Total radiocarbon recovery ranged from 98.8 to 103.1% of the applied amount at pH 4, 97.4 to 103.5% of the applied amount at pH 7, and 97.7 to 106.7% of the applied amount at pH 9.

TRANSFORMATION OF PARENT COMPOUND: At the end of the study, the concentration of the parent compound decreased from 94.9% at day 0 to 70.3% of the initial at pH 4, 101.3 to 51.0% of the initial at pH 7, and 100.4 to 1.1% of the initial at pH 9.

TRANSFORMATION PRODUCTS: The only transformation product detected at pH 4, 7, and 9 was Spirodiclofen-Enol [3-(2,4-dichlorophenyl)-4-hydroxy-1-oxaspiro[4.5]dec-3-en2-one]. Spirodiclofen-Enol reached a maximum concentration of 28.9, 52.2, and 100.8% of the applied amount observed at test termination at pH 4, 7, and 9, respectively. A minor transformation product, M2, was shown to be an artifact formed during chromatographic analysis of Spirodiclofen-Enol. No volatile compounds were formed during the study, and no unidentified radioactivity was observed at any pH.

PATHWAYS: The hydrolysis pathway for Spirodiclofen involved the hydrolysis of an ester sidechain to form Spirodiclofen-Enol.

HALF-LIFE: The half-lives for the hydrolysis of [dihydrofuranone-3-14C]Spirodiclofen at pH 4, 7, and 9 at 50ºC and 25ºC were determined using first-order kinetics. The half-lives for the hydrolysis of [dihydrofuranone-3-14C]Spirodiclofen at pH 4, 7, and 9 at 20ºC were calculated using the Arrhenius equation.

Table 1: Hydrolysis of [dihydrofuranone-3-14C]Spirodiclofen, expressed as % applied radioactivity, at pH 4

Compound	day 0	day 2	day 4	day 7	day 14	day 21	day 30
Spirodiclofen	94.9	93.6	91.6	91.5	82.7	73.6	70.3
Spirodiclofen-Enol	4.6	7.4	9.8	8.4	16.8	27.5	28.9
Unidentified radioactivity	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Volatiles (CO2 and VOCs)	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Total % recovery	99.5	101.0	101.4	99.9	99.5	101.1	99.2

 

Table 2: Hydrolysis of [dihydrofuranone-3-14C]Spirodiclofen, expressed as % applied radioactivity, at pH 7.

Compound	day 0	day 1	day 3	day 7	day 14	day 21	day 30
Spirodiclofen	101.3	95.8	87.3	84.1	70.9	58.6	51.0
Spirodiclofen-Enol	0.7	1.9	12.5	16.7	27.6	40.4	52.2
Unidentified radioactivity	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Volatiles (CO2 and VOCs)	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Total % recovery	102.0	97.7	99.8	100.8	98.5	99.0	103.2

 

Table 3: Hydrolysis of [dihydrofuranone-3-14C]Spirodiclofen, expressed as % applied radioactivity, at pH 9.

Compound	day 0	day 0.1	day 0.25	day 1	day 2	day 3	day 4	day 7	day 14	day 21	day 30
Spirodiclofen	100.4	94.3	82.5	62.5	43.6	27.6	17.5	7.7	2.4	2.8	1.1
Spirodiclofen-Enol	6.2	11.8	17.0	40.9	60.5	71.0	80.7	92.1	98.1	90.9	100.8
Unidentified radioactivity	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Volatiles (CO2 and VOCs)	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Total % recovery	106.6	106.1	99.5	103.4	104.1	98.6	98.2	99.8	100.5	93.7	101.9

 

Table 4: Half Lives

Test Solution	Half-life [days] at 50ºC	Half-life [days] at 25ºC	Half-life [days] at 20ºC
pH 4 (0.01 M acetate buffer)	3.1	63.6	119.6
pH 7 (0.01 M TRlS buffer)	2.5	30.8	52.1
pH 9 (0.01 M borate buffer)	0.4	1.9	2.5

 

Table 5: First order half-lives

pH	half-life	Regression equation	R^2	DT50	DT90
4	63.6 days	y = 4.5361 - 0.0109(x)	0.9716	63.6 days	211.3 days
7	30.8 days	y = 4.5798 - 0.0225(x)	0.9861	30.8 days	102.4 days
9	44.4 hours (1.9 days)	y = 4.5228 - 0.0156(x)	0.989	44.4 hours (1.9 days)	147.6 hours (6.2 days)

Validity criteria fulfilled:

yes

Conclusions:

The half-life (DT50) of the test substance at 25°C was determined to be 63.6 days at pH 4, 30.8 days at pH 7 and 1.9 days at pH 9.

Executive summary:

Hydrolysis of [dihydrofuranone-3-14C]Spirodiclofen at 0.025 mg a.i/L was studied in the dark at 25°C in sterile aqueous buffered solutions at pH 4 (0.01 M acetate buffer), pH 7 (0.01 M TRlS buffer) and pH 9 (pH 9: 0.01 M borate buffer) for a maximum of 30 days. The experiment was conducted in accordance with the EPA Pesticide Assessment Guideline, Subdivision N, Section §161-1, 1982 as well as EC Commission Directive 95/36/EC, 1995, and in compliance with the OECD–GLP standards. Samples were analyzed at 0, 2, 4, 7, 14, 21, and 30 days (pH 4); 0, 1, 3, 7, 14, 21, and 30 days (pH 7), and 0, 0.1, 0.25, 1, 2, 3, 4, 7, 14, 21, and 30 days (pH 9). Aliquots of the solutions were analyzed directly after addition of acetic acid which was used to stablilize residues of Spirodiclofen. Due to the tendency of Spirodiclofen to adsorb on glass, all glassware were silanized. The radioactive components in the buffer solutions were separated and quantitated using two different reverse phase TLC systems, and the identification/confirmation of radioactive residues was performed either by co-chromatography with reference substances using silica TLC (three systems) or by LC-MS.

The radioactivity balance was 101.0 ± 1.4, 100.2 ± 2.1, and 102.3 ± 2.7% of the applied at pH 4, pH 7 and pH 9, respectively. At test termination, the concentration of the parent compound decreased from 94.9% at day 0, to 70.3% of the initial at pH 4, from 101.3 to 51.0% of the initial at pH 7, and from 100.4 to 1.1% of the initial at pH 9. The only transformation product detected at pH 4, 7, and 9 was Spirodiclofen-Enol [3-(2,4-dichlorophenyl)-4-hydroxy-1-oxaspiro[4.5]dec-3-en-2-one]. Spirodiclofen-Enol reached a maximum concentration of 28.9, 52.2, and 100.8% of the applied amount observed at test termination at pH 4, 7, and 9, respectively. A minor transformation product, M2, was shown to be an artifact formed during chromatographic analysis of Spirodiclofen-Enol. No volatile compounds were formed during the study, and no unidentified radioactivity was observed at any pH. The half-lives for the hydrolysis of [dihydrofuranone-3-14C]Spirodiclofen at pH 4, 7, and 9 at 50ºC and 25ºC were determined using first-order kinetics. The half-lives for the hydrolysis of [dihydrofuranone-3-14C]Spirodiclofen at pH 4, 7, and 9 at 20ºC were calculated using the Arrhenius equation.

In a preliminary test, the hydrolysis study was conducted at 50ºC using the same procedures and buffers as were used in the test at 25ºC. The half-lives at 50ºC and pH 4, 7, and 9 were 3.1, 2.5, and 0.4 days, respectively. Using the data from the hydrolysis at 50ºC and 25ºC, the half-lives of Spirodiclofen at 20ºC and pH 4, 7, and 9 were calculated via the Arrhenius equation at 119.6, 52.1, and 2.5 days, respectively. Data from the additional samples that were analyzed for the identification of glass-adsorbed radioactivity clearly demonstrated that all of the glass-bound (i.e., ACN soluble) residues were Spirodiclofen.

The large scale hydrolyses provided material that was used for LC/MS confirmation of Spirodiclofen-Enol and Spirodiclofen-Lactide. Additionally, data from these samples and from the chromatography of a Spirodiclofen-Enol standard clearly showed that Spirodiclofen-Lactide was an artifact that formed in variable amounts during chromatographic analysis of the Enol. This study is classified acceptable and satisfies the guideline requirement for hydrolysis study.