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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Dec 2017 - 12 Feb 2018
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)
Version / remarks:
March 04, 2016
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
April 13, 2004
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)
Version / remarks:
October 2008
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method and interval:
The concentration of the test item in the test samples was determined immediately after preparation (t=0) and after 5 days. The samples taken at t=5 days were cooled to room temperature using running tap water. Blank buffer solutions containing a similar content of blank spiking solution were treated similarly as the test samples and analyzed at t=0.

Analysis was performed on subsamples of 700 µL. The samples were diluted in a 7:3 (v:v) ratio with acetonitrile and analyzed.
Blank buffer solutions containing a similar content of blank spiking solution were treated similarly as the test samples and analyzed at t=0.

- Sampling intervals/times for pH measurements: The pH of each of the test solutions (except for the blanks) was determined at each sampling time.
Buffers:
BUFFER SOLUTIONS

Acetate buffer pH 4, 0.1:
solution of 16.7% 0.1 M sodium acetate in water and 83.3% 0.1 M acetic acid in water. Buffer contained 0.0009% (w/v) sodium azide.

Phosphate buffer pH 7, 0.1 M:
Solution of 0.1 M potassium di-hydrogen-phosphate in water adjusted to pH 7 using 1N sodium hydroxide. Buffer contained 0.0009% (w/v) sodium azide.

Borate buffer pH 9, 0.1 M:
Solution of 0.1 M boric acid in water and 0.1 M potassium chloride in water adjusted to pH 9 using 10N sodium hydroxide. Buffer contained 0.0009% (w/v) sodium azide.
Details on test conditions:
TEST SYSTEM
- Sterilisation method: The buffer solutions were filter-sterilised through a 0.2 µm FP 30/0.2 CA-S filter (Whatman, Dassel, Germany) and transferred into a sterile vessel.
- Measures taken to avoid photolytic effects: sterile vessels under vacuum were placed in the dark
- Measures to exclude oxygen: To exclude oxygen, nitrogen gas was purged through the solution for 5 minutes.
- Is there any indication of the test material adsorbing to the walls of the test apparatus? No

TEST MEDIUM
- The test item was spiked to the solutions at a target concentration of 20 mg/L using a spiking solution in acetonitrile. For each sampling time, duplicate sterile vessels under vacuum were filled with 6 mL test solution and placed in the dark in a temperature controlled environment at 49.9°C +/- 0.1°C.
- Volume used/treatment: The spiking volume was ≤ 1% of the sample volume. Nominal concentrations were not corrected for the spiking volume.
- Kind and purity of water: Tap water purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA)
- Preparation of test medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H20 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO 41.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L

- Identity and concentration of co-solvent: The samples were diluted in a 7:3 (v:v) ratio with acetonitrile and analyzed.
Duration:
5 d
pH:
4.1
Temp.:
50 °C
Initial conc. measured:
20.1 mg/L
Remarks:
49.9 +/- 0.1 °C
Duration:
5 d
pH:
7.1
Temp.:
50 °C
Initial conc. measured:
>= 20.1 - <= 20.2 mg/L
Remarks:
49.9 +/- 0.1 °C
Duration:
5 d
pH:
9
Temp.:
50 °C
Initial conc. measured:
20.2 mg/L
Remarks:
49.9 +/- 0.1 °C
Number of replicates:
2
Positive controls:
no
Negative controls:
no
Preliminary study:
At pH 4, pH 7 and pH 9, a degree of hydrolysis of < 10% was observed after 5 days. It demonstrated that the half-life time of the test item at 25°C is > 1 year. According to the guideline, no further tests were required. No test item was detected in the blank buffer solutions. The mean recoveries of the of the test item containing buffer solutions at t=0 fell within the criterion range of 90-110%. It demonstrated that the analytical method was adequate to support the hydrolysis study on the test item.
Test performance:
The analytical method was validated for the analysis of N-ethyl-o (or p) toluenesulphonamide in M2-medium for the following parameters:

• Specificity: One major peak was observed in the chromatograms of the QC samples. The peak area of this peak was used as response in the calculationsThe chromatogram of the blank QC sample showed no peak at the retention time of the test item. Since no interferences were detected, the specificity requirements were met and the analytical method was found to be specific for the test item.
• Calibration curve: There was a linear relationship between response and test item concentration in the range of 0.0500 – 20.0 mg/L (in end solution). Since the coefficient of correlation (r) was > 0.99 and the back calculated accuracies of the data points were in the range 85-115% the calibration line was accepted.
• Accuracy and Repeatability: Since the mean accuracy at each concentration level fell in the criterion 70-110% and the coefficient of variation was ≤ 20% the analytical method was accepted for the analysis of the test item in M2-medium in the target concentration range of 0.100 - 100 mg/L.
• Limit of quantification: The limit of quantification (LOQ) was assessed at 0.1 mg/L in M2-medium.
• Stability analytical system and end solutions: Since the coefficient of variation at both concentration levels was ≤ 20% the analytical system and end solutions were stable over at least a 19.32 hour time interval.
• Stability stock solutions: The coefficient of variation on the response factors of the calibration solutions prepared with fresh and stored stock solutions was 1.9%. Since the value was ≤ 10% the stock solutions were stable when stored at room temperature for at least 4 days.
• Storage stability of samples: The mean accuracy of the frozen QC samples fell in the criterion 70-110% the samples were stable when stored in the freezer (≤ -15°C).
Transformation products:
no
Remarks:
no decrease of initial concentration was measured
% Recovery:
100
pH:
4
Temp.:
50 °C
Remarks on result:
other: The mean recoveries of the test item containing buffer solutions were measured at t=0
% Recovery:
ca. 100
pH:
7
Temp.:
50 °C
Remarks on result:
other: The mean recoveries of the test item containing buffer solutions were measured at t=0 and were concluded 101%
% Recovery:
ca. 100
pH:
9
Temp.:
50 °C
Remarks on result:
other: The mean recoveries of the test item containing buffer solutions were measured at t=0 and were concluded 101%
Key result
pH:
4
Temp.:
25 °C
DT50:
> 1 yr
Key result
pH:
7
Temp.:
25 °C
DT50:
> 1 yr
Key result
pH:
9
Temp.:
25 °C
DT50:
> 1 yr

Hydrolysis of the Test Item at pH 4, pH 7 and pH 9

pH code

Sampling time

Analyzed concentration
[mg/L]

Degree of hydrolysis
[%]

pH

Individual

Mean

pH 4

0 hours

20.1

 

 

4.1

 

 

20.1

 

 

4.1

 

5 days

20.7

-3.2

-2.4

4.1

 

 

20.4

-1.6

 

4.1

pH 7

0 hours

20.2

 

 

7.1

 

 

20.1

 

 

7.1

 

5 days

20.4

-1.3

-1.4

7.1

 

 

20.5

-1.6

 

7.1

pH 9

0 hours

20.2

 

 

9.0

 

 

20.2

 

 

9.0

 

5 days

20.6

-2.2

-2.2

9.0

 

 

20.6

-2.3

 

9.0

      
Recoveries

pH code

Nominal concentration
[mg/L]

Analyzed concentration
[mg/L]

Recovery
[%]

Individual

Mean

pH 4

20.0

20.1

100

100

 

20.0

20.1

100

 

pH 7

20.0

20.2

101

101

 

20.0

20.1

101

 

pH 9

20.0

20.2

101

101

 

20.0

20.2

101

 

 


 

Validity criteria fulfilled:
yes
Conclusions:
The preliminary test (Tier 1) was performed for the determination of the rate of hydrolysis of N-ethyl-o (or p) toluenesulphonamide at pH values normally found in the environment (pH 4-9). At each pH value a degree of hydrolysis of < 10% was observed after 5 days. According to the guideline, performance of the main study (Tier 2) was not required. The chemical was considered hydrolytically stable.
Executive summary:

A preliminary test (Tier 1) was performed to determine the rate of hydrolysis of N-ethyl-o (or p) toluenesulphonamide at pH values normally found in the environment (pH 4-9).The study was conducted according to OECD 111 and GLP guidelines. In addition analytical methods were validated prior to the test. Sterile aqueous buffer solutions of  pH values of 4, 7 and 9 were treated with the test substance and incubated in the dark under controlled laboratory conditions at a temperature of 49 ± 1°C. Quantative analysis was performed using an ultra performance liquid chromatographic method with spectrophotometric detection (UPLC-UV) and measured immediately after preparation (t=0) and after 5 days. At each pH value a degree of hydrolysis of < 10% was observed after 5 days. According to the guideline, performance of the main study (Tier 2) was not required. A hydrolysis half-life of > 1 year for all pH at 25°C was determined for the test substance. The chemical is considered hydrolytically stable.

Description of key information

Hydrolysis: the substance is hydrolytically stable as shown from a OECDTG111 guideline study:

Hydrolysis as a function of pH

EC C.7
OECD 111
OPPTS 835.2120

pH 4: t½> 1 year
pH 7: t½> 1 year
pH 9: t½> 1 year

Key value for chemical safety assessment

Half-life for hydrolysis:
1 yr
at the temperature of:
50 °C

Additional information

The half-life is > 1 year for all pH's tested

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