Pirimiphos-methyl

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Apr 1995 to 29 Mar 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Analytical monitoring:

yes

Details on sampling:

PRELIMINART EXPERIMENT
- Sampling intervals: Replicate samples were taken on Days 0, 1, 2, 3, 4 and 7 for analysis by TLC and HPLC.

DEFINITE EXPERIMENT
- Sampling intervals: Replicate samples were taken at each interval (pH 4 - Days 0, 1 , 2, 3, 7, 14, 21, 24 and 30 ; pH 5 - Days 0, 2, 6, 14, 21, 24 and 30 ; pH 7 and 9 - Days 0, 3, 7, 14, 21, 24 and 30) for immediate analysis by HPLC. It was more reliable and easier than TLC. All vials were radioassayed by LSC, prior to storage in the laboratory freezer.
- pH: The pH of hydrolysis solutions was measured at each sampling interval of the study using a pH meter. The meter was calibrated using pH 4, 7 and 10 buffer solutions prepared from buffer capsules
- Quantification of Radioactivity in Hydrolysis Solutions: Quantification of the amounts of activity in the radiochemical stock solutions and the
hydrolysis buffer solutions was carried out using LSC. At the time of sampling, three 100 μL aliquots were removed for counting, These were then mixed with 10 mL of Optiphase "Safe" scintillation cocktail. Background samples consisted of 10 mL of scintillation cocktail only.

Buffers:

- pH: Buffer solutions of pH 4, 5, 7 and 9 were prepared
- Composition of buffer:
pH 4 Acetate Buffer - 41.0 mL 0.01 M acetic acid + 9.0 mL 0.01 M sodium acetate
pH 5 Acetate Buffer - 14.8 mL 0.01M acetic acid + 35.2 mL 0.01M sodium acetate
pH 7 Acetate Buffer - 399 mL 0.01M sodium acetate + 1.0 mis 0.01M acetic acid
pH 9 Borate Buffer - 0.01M sodium tetraborate.
High purity bottled water was used for the preparation of all buffer solutions.

Details on test conditions:

PRELIMINART EXPERIMNENTS (50°C at pH 4, 7 and 9)
25 mL of each sterilised buffer solution was transferred, using a sterilised 25 mL glass bulb-pipette, into 30 mL amber vials. Sufficient vials, of each pH, were prepared to allow for duplicate samples at each time-point and duplicate samples for sterility checks. All the vials were sealed using septa and capped. 100 μL of the radiochemical was added to the appropriate vials by injecting directly through the septa with a sterilised 100 μL glass syringe. This resulted in a final 14C- labelled test substance concentration of approximately 1 μg/mL.
At the end of application, aliquots of the radiochemical solution were taken for quantification by liquid scintillation counting (LSC) to determine the actual amount of radioactivity applied to the buffer solutions. The vials were placed, in the darkness, in a pre-heated water bath maintained at 50°C ± 0.5°C for 5 days for the pH 7 and 9 solutions and 7 days for the pH 4 solutions. The temperature of the water bath was checked and recorded at each sampling interval using a digital thermometer.

BUFFER CATALYSIS EXPERIMENT (50°C)
In order to demonstrate that hydrolysis observed was not due to chemical reaction with the components of the buffer solutions, the following experiment was performed at the same time as the pH 7 and 9 preliminary experiments. 25 mL of unbuffered water was transferred, using a sterilised 25 mL glass bulbpipette, into 30 mL amber vials. 4 vials were prepared. All the vials were sealed using septa and capped.
100 μL of the radiochemical was added to the vials by injecting directly through the septa with a sterilised 100 μL glass syringe. This resulted in a final 14C-labelled test substance concentration of approximately 1 μg/mL.

DEFINITE EXPERIMENT (25°C at pH 4, 5, 7 and 9)
- Type, material and volume of test flasks: 30 mL amber vials
- Sterilisation method: All glass syringes and pipettes were sterilised by immersion in a solution of 7:3 ethanol:water for at least 1 hour prior to use. All vials, septa, lids, and buffer solutions were autoclaved at 120°C for 20 minutes. The radiochemical stock solution was sterilised immediately prior to use by passing the solution through a sterile microfilter.
- Volume of buffer solution per vial: 25 mL
- Preparation of test stock solution: The radiolabelled stock solution was prepared by dissolving the radiochemical in acetonitrile, to give a concentration of approximately 250 μg/mL . The initial purities of the test solutions were assessed using thin layer chromatography (TLC) in two solvent systems.
- Application of the test solution: 100 μL of the radiochemical was added to the appropriate vials by injecting directly through the septa with a sterilised 100 μI glass syringe. This resulted in a final 14C-labelled test substance concentration of approximately 1 μg/mL.
- Lighting: maintained in darkness

Duration:

30 d

pH:

4

Temp.:

25 °C

Initial conc. measured:

1 mg/L

Duration:

30 d

pH:

5

Temp.:

25 °C

Initial conc. measured:

1 mg/L

Duration:

30 d

pH:

7

Temp.:

25 °C

Initial conc. measured:

1 mg/L

Duration:

30 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

1 mg/L

Number of replicates:

Duplicate samples at each sampling time-point and duplicate samples for sterility checks

Positive controls:

no

Negative controls:

no

Statistical methods:

Not reported

Preliminary study:

- Radiochemical Purity and Initial concentration: The purity of the radiolabelled stock solution was 98.2 - 99.5%. The final concentration of the parent compound in the buffer vials was 0.91 μg/mL for the pH 7, 9 and buffer catalysis experiments and 1.01 μg/mL for the pH 4 experiment.

- pH Stability during the Hydrolysis Period: All of the buffer solution pH values remained constant to within the following limits:
pH 4 experiment - 4.0 ± 0.1 pH
pH 7 experiment - 7.0 ± 0.1 pH
pH 9 experiment - 9.0 ± 0.3 pH

- Assessment of Buffer Solution Sterility: The sterility of the test system was maintained throughout the hydrolysis period. It can therefore be concluded that any breakdown of the test substance observed in this study was not microbially mediated.

- Material Balance: Radioassay of the hydrolysis buffers was performed after chromatographic
analysis at the designated time points, to check for homogeneity and recovery of radioactivity. This indicated that mean recoveries were within the range 91- 108% throughout the hydrolysis periods.

- Hydrolysis of the test substance: The test substance was hydrolysed rapidly in pH 4 solutions at 50°C, with one product being formed. At the end of a 7 day incubation period parent test substance represented < 1 % of the total radioactive components. The remainder (96.7%) was identified as M1. Hydrolysis in pH 7 and 9 solutions was less rapid. After 5 days incubation the percentages of parent substance in pH 7 and 9 solutions were 52.6% and 28.8% respectively. M1 was, again found to be present at percentages of 9.1% and 33.8% in pH 7 and 9 respectively.
The remainder was due to an unknown component (Product A) which represented 37.6% and 33.9% in pH 7 and pH 9 solutions respectively. The purity of the Day 0 samples (> 94.6%) indicated that the application solution was stable during the application period and that the samples were stable during storage prior to analysis.

Transformation products:

not specified

Remarks:

See additional information provided in overall endpoint summary.

Details on hydrolysis and appearance of transformation product(s):

An overview of the results is provided in Table 2 - Table 5 in ‘Any other information on results incl. tables’.

The test substance was hydrolysed rapidly in pH 4 and 5 solutions at 25°C, with one product being formed. At the end of a 30 day incubation period parent substance represented < 1 % and 5.8% of the total radioactive components at pH 4 and 5 respectively. The remainder (99.5 and 91.5% respectively) was identified as M1. Hydrolysis in pH 7 and 9 solutions was less rapid. After 30 days incubation the percentages of the test substance in pH 7 and 9 solutions were 82.6% and 75.1% respectively. M1 was, again found to be present at percentages of 7.7% and 11.7% in pH 7 and 9 respectively. The remainder was due to M2 which represented 9.8% and 12.8% in pH 7 and pH 9 solutions respectively. The identities of the hydrolysis products were confirmed by both HPLC and TLC.

% Recovery:

99.8

pH:

2

Temp.:

25 °C

Duration:

30 d

% Recovery:

> 100

pH:

5

Temp.:

25 °C

Duration:

30 d

% Recovery:

96

pH:

7

Temp.:

25 °C

Duration:

30 d

% Recovery:

98.9

pH:

9

Temp.:

25 °C

Duration:

30 d

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0.006 d-1

DT50:

117 d

Type:

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

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0.35 d-1

DT50:

2 d

Type:

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

pH:

5

Temp.:

25 °C

Hydrolysis rate constant:

0.1 d-1

DT50:

7 d

Type:

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

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.009 d-1

DT50:

75 d

Type:

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

Details on results:

An overview of the results is provided in Table1 in ‘Any other information on results incl. tables’.

- Radiochemical Purity and Initial concentration: The purity of the radiolabelled stock solution for the full experiments was 97.9%. The final concentrations of the parent compound in the buffer vials were as follows:
pH 4: 0.90 μg/mL (0.89 μg/mL for extra Day 1 and 2 time-points)
pH 5: 0.88 μg/mL
pH 7: 0.87 μg/mL
pH 9: 0.85 μg/mL

- pH Stability during the Hydrolysis Period: The pH measurements taken at each sampling time indicate that all of the buffer solution pH values remained constant to within the following limits :
pH 4 experiment - 4.0 ± 0.1 pH
pH 5 experiment - 5.0 ± 0.1 pH
pH 7 experiment - 7 .0 ± 0.1 pH
pH 9 experiment - 9.0 ± 0.3 pH

- Temperature Stability During the Hydrolysis Period: The water bath maintained a mean temperature of 25 ± 0.3°C.

- Assessment of Buffer Solution Sterility: The sterility of the test system was maintained throughout the hydrolysis period. It can therefore be concluded that any breakdown of the test substance observed in this study was not microbially mediated.

- Material Balance: Radioassay of the hydrolysis buffers was performed after chromatographic analysis at the designated time points, to check for homogeneity and recovery of radioactivity. This indicated that mean recoveries were within the range 93 - 103% throughout the hydrolysis period.
The recoveries in both experiments were within acceptable variation limits. The application solution was, therefore, shown to be homogenous throughout the application period.

- Buffer Catalysis: Hydrolysis in the Buffer Catalysis solutions (ie pure water only) was very similar
to the pH 7 experiment with the test substance, M1 and the Product A representing 52.6%, 10.2% and 32.0% respectively. This result showed that the hydrolysis observed in the buffered solutions was not due to chemical breakdown caused by the presence of the buffering agents.

- Identification of the Unknown Component: Mass spectrometric analysis of the unknown component Product A (as isolated from the Day 5 Buffer Catalysis sample) gave parent and daughter ions that were consistent with the loss of a methyl group from the phosphorothioate moiety in the test substance (in both positive and negative ion modes). This was confirmed by the NMR analysis of the equivalent, more concentrated sample, which had been chromatographically compared with the above sample and shown to be identical. This compound had been found in previous work. The unknown component was, therefore, designated M6.

Statistical analysis of the results (i.e. a plot of the natural log of the ratio of the initial % of the test substance to the final % versus time) showed first order kinetics for all pH values (as a straight line plot was observed). The half-lives observed were as follows:
pH 4: 2 days
pH 5: 7 days
pH 7: 117 days
pH 9: 75 days

The purity of the Day 0 samples (> 94.6%) indicated that the application solution was stable during the application period and that the samples were stable during storage prior to analysis.

Table 1. Summary of Results Obtained from the 25°C Hydrolysis of the test substance

Time intervals (days)	pH 4		pH 5		pH 7		pH 9
	% Rec*	Total % test substance	% Rec*	Total % test substance	% Rec*	Total % test substance	% Rec*	Total % test substance
0	98	99.2	93.8	99.3	95.7	97	98.8	99.5
1	98.4	67.6	-	-	-	-	-	-
2	99.7	45.2	97.6	81.2	-	-	-	-
3	100.8	30.6	-	-	96.8	97.8	100.3	96.1
6	-	-	99.8	54.9	-	-	-	-
7	100.9	9.4	-	-	96	94.7	98	91.8
14	100.7	3.4	102.1	24.5	97.4	89.2	99.5	87.3
21	101.5	2.1	102.4	11.8	99.8	85.2	97.8	82.8
24	98.9	1.8	102.6	9.6	98	83.1	98	78.8
30	99.8	0.6	102.1	5.8	96	82.6	98.9	75.1

* Mean Radioactive Recovery
- No sample was taken at this time point

Table 2. The Rates of Breakdown of the test substance (and of Formation of Breakdown Products) in pH 4 Solutions at 25°C

Time Interval (days)	% test substance	% M2	% M1
0	99.2	0	0
1	67.6	0	32.4
2	45.2	0	54.9
3	30.6	0	68
7	9.4	0	88.2
14	3.4	0	95.3
21	2.1	0	95.7
24	1.8	0	98.2
30	0.6	0	99.5

 Table 3. The Rates of Breakdown of the test substance (and of Formation of Breakdown Products) in pH 5 Solutions at 25°C 

Time Interval (days)	% test substance	% M2	% M1
0	99.3	0	0
2	81.2	0	18.9
6	54.9	0	45.2
14	24.5	0	71.9
21	11.8	0	85
24	9.6	0	89.2
30	5.8	0	91.5

Table 4. The Rates of Breakdown of the test substance (and of Formation of Breakdown Products) in pH 7 Solutions at 25°C

Time Interval (days)	% test substance	% M2	% M1
0	97	0	0
3	97.8	0	0
7	94.7	3	2.3
14	89.2	5.3	4.9
21	85.2	8.4	6.5
24	83.1	11.9	5.1
30	82.6	9.8	7.7

Table 5. The Rates of Breakdown of the test substance (and of Formation of Breakdown Products) in pH 9 Solutions at 25°C

Time Interval (days)	% test substance	% M2	% M1
0	99.5	0	0
3	96.1	0	2.5
7	91.8	3.8	4.5
14	87.3	6.1	6.6
21	82.8	7.3	9.7
24	78.8	9.4	11.9
30	75.1	12.8	11.7

Validity criteria fulfilled:

yes

Conclusions:

Hydrolysis of the test substance is highly pH dependent. The substance hydrolyses relatively quickly under acidic conditions. Hydrolysis is comparably slow at neutral or basic pH values.

Executive summary:

This laboratory aqueous hydrolysis study was performed according to EU Method C.7 and EPA, Subdivision N, Section 161-1 and was in compliance with GLP criteria. In this study, the radiolabelled test substance was incubated at 25 °C in the absence of light in sterile buffers at pH 4, 5, 7 and 9 at a nominal concentration of 1 μg/mL for 30 days. The vials were removed from the water bath for analysis on days 0, 1, 2, 3, 7, 14, 21, 24 and 30 for the pH 4 experiment; days 0, 2, 6, 14, 21, 24 and 30 for pH 5 and days 0, 3, 7, 14, 21, 24 and 30 for pH 7 and 9. The samples were analysed by HPLC to quantify the test substance over time.

 

The analytical determinations showed that the test substance was hydrolytically unstable under the experimental conditions used as >99%, 94.2%, 17.4% and 24.9% degradation was observed at pH 4, 5, 7 and 9 respectively after 30 days. At pH 4 and 5 one hydrolysis product was observed (M1) at a mean level of 99.5% and 91.5% respectively after 30 days. At pH 7 and 9 this product represented a mean level of 7.7% and 11.7% respectively after 30 days. In addition another product, M2, was observed at a mean level of 9.8% and 12.8% at pH 7 and 9 respectively after 30 days incubation. Based on the results, the DT50 values for the test substance were determined to be 2, 7, 117 and 75 days at pH 4, 5, 7 and 9 respectively.

Description of key information

Hydrolytic DT50 at pH 7 at 25 °C = 117 days , EPA guidelines Subdivision N, section 161 -1 and EU Method C.7 guideline, Hand 1996

Key value for chemical safety assessment

Half-life for hydrolysis:

117 d

at the temperature of:

25 °C

Additional information

A laboratory aqueous hydrolysis study was performed according to EU Method C.7 and EPA, Subdivision N, Section 161-1 and in compliance with GLP criteria. The radiolabelled test substance was incubated at 25 °C in the absence of light in sterile buffers at pH 4, 5, 7 and 9 at a nominal concentration of 1 μg/mL for 30 days. The vials were removed from the water bath for analysis on days 0, 1, 2, 3, 7, 14, 21, 24 and 30 for the pH 4 experiment; days 0, 2, 6, 14, 21, 24 and 30 for pH 5 and days 0, 3, 7, 14, 21, 24 and 30 for pH 7 and 9. The samples were analysed by HPLC to quantify the test substance over time.

The analytical determinations showed that the test substance was hydrolytically unstable under the experimental conditions used as >99%, 94.2%, 17.4% and 24.9% degradation was observed at pH 4, 5, 7 and 9 respectively after 30 days. At pH 4 and 5 one hydrolysis product was observed (M1) at a mean level of 99.5% and 91.5% respectively after 30 days. At pH 7 and 9 this product represented a mean level of 7.7% and 11.7% respectively after 30 days. In addition another product, M2, was observed at a mean level of 9.8% and 12.8% at pH 7 and 9 respectively after 30 days incubation. Based on the results, the DT50 values for the test substance were determined to be 2, 7, 117 and 75 days at pH 4, 5, 7 and 9 respectively.