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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Analytical monitoring:
yes
Details on test conditions:
Preparation of the test solution:
Stock solutions of the test item were prepared by weighting of approx. 20 mg of the test item in 100 ml acetonitrile. Separate hydrolysis test solutions for each pH were prepared by 1:500 dilution of the stock solution with the corresponding buffer systems. Aliquots of these hydrolysis test solutions were taken without further treatment to obtain individual vials for every test point. Preparation was carried out under nitrogen as flushing gas, to avoid oxygen. The vials were closed and incubated at 50 °C in a heat regulator under dark to avoid any photolytic effects.
The pH of the blank buffer solution was checked at the beginning of the test. Additionally the pH of the hydrolysis solution was measured at each test point. The temperature during the experiment was also checked at each test point.
Calibration was done at each point in time at the defined time intervals for the analysis of the hydrolysis test solution. The calibration was verified at each test point at the level of 200 µg/l by using a control calibration solution.
At each test point the hydrolysis solution was directly led to the chromatographic measurement.

Pre-tests for hydrolysis:
According to OECD TG 111 the concentration of the test item should not exceed 0.01 M or half of the saturation concentration of the water solubility.
The test item was applied as an aqueous solution (including 1 % acetonitrile) with a concentration of approx. 0.4 mg/l which fulfills the requirements of OECD TG 111.
In compliance with OECD 111 Guideline no further solubility test with organic solvent additives > 1% v/v was performed.
Transformation products:
yes
No.:
#1
Details on hydrolysis and appearance of transformation product(s):
Identification of hydrolysis product by HPLC-MS.
No hydrolysis product could be determined during the hydrolysis tests. Therefore 10.1 mg of the test substance were weighed to the nearest 0.1 mg into a 100 ml measuring flask. Buffer solution pH 4 / acetonitrile 9:1 (v/v) were added and the mixture was dissolved by manual shaking. The flask was then filled up to the mark with buffer solution pH 4 / acetonitrile 9:1 (v/v). Exactly 10 ml of this solution was diluted with buffer solution pH 4 to 100 ml. This solution was agitated in a thermostated water bath at 60 °C for 48 hours.
Preparation of the calibration solution of Tetrabromophthalic acid:
14.0 mg of the calibration substance Tetrabromophthalic anhydride were weighed to the nearest 0.1 mg into a 100 ml measuring flask. Buffer solution pH 4 / acetonitrile 9:1 (v/v) were added and the mixture was dissolved by manual shaking (Tetrabromophthalic anhydride reacts with water directly to Tetrabromophthalic acid). The flask was then filled up to the mark with buffer solution pH 4 / acetonitrile 9:1 (v/v). Exactly 10 ml of this solution was diluted with buffer solution pH 4 to 100 ml.

Description of the analytical method and method parameter
Test: HPLC-MS / HPLC-UV/VIS
Procedure: High Performance Liquid Chromatography (HPLC-MS-MS and HPLC-UV/VIS detection)
Apparatus: Agilent 1200/1100 + Applied Biosystems 4000 Q TRAP
Column type: Length: 50 mm; Inner diameter: 2.1 mm
Stationary phase: Perfectsil 300 C4; Particle diameter: 5 μm
Mobile phase: A: 1000 ml demineralized water + 0.5 ml formic acid; B: 1000 ml acetonitrile + 0.5 ml formic acid
Solvent Program:
0 min: 100% A; 0% B
0.5 min: 0% A; 100% B
3.51 min: 100% A; 0% B
5.51 min: stop

Injection volume: 10 μl
Flow rate: 400 μl / minute
Column temperature: 80 °C
UV/VIS-detection: 200 nm - 700 nm
MS-detection: 4000 Q TRAP; Multiple Ions, negative; Q1 Mass 480.8 Da
Dwell: 100 msec

Result:
The HPLC-peak at RT = 1.66 minutes of the HPLC chromatograms and its corresponding MS-spectra were observed. Therefore the unknown hydrolysis product could be identified as Tetrabromophthalic acid.
% Recovery:
2
pH:
4
Temp.:
50 °C
Duration:
6 d
% Recovery:
1
pH:
7
Temp.:
50 °C
Duration:
12 d
% Recovery:
8
pH:
9
Temp.:
50 °C
Duration:
12 d
pH:
4
Temp.:
50 °C
Hydrolysis rate constant:
0 s-1
DT50:
30.39 h
Remarks on result:
other: r= 0.97964
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0 s-1
DT50:
44.08 h
Remarks on result:
other: r= 0.98043
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
0 s-1
DT50:
77.52 h
Remarks on result:
other: r= 0.97892
Details on results:
The overall degradation rates of the test item observed with the determination of the content of the substance at 50 °C and pH 4, 7 and 9 are greater than 10 % after 5 days. The degradation of the test item was > 91% after the study period at each pH. As a conclusion the substance is hydrolytically unstable at environmental relevant pHs.
Results of the solutions for verification of the calibration show stability of the chromatographic system. Recoveries of the test item are in the range from 95.5 % to 104.0 % indicating a satisfying repeatability of the method applied to quantify the test item concentrations.
Regarding the chromatogram of the lowest concentration of 2 µg/l used for calibration, the analytical method is sufficiently sensitive to quantify test item concentrations down to 10 % or less of the initial concentration used in the hydrolysis experiment.

According to OECD 111 Guideline a sterility test was conducted at the end of the hydrolysis test of samples from all pH values used (pH 4, 7 and 9). No microbes (colonies) were found. Therefore biotic degradation can be excluded.
Conclusions:
The substance is hydrolytically unstable showing half-life-times at 50°C of 30.39 h at pH 4, 44.08 h at pH 7 and 77.52 h at pH 9. One transformation product was identified as tetrabromophthalic acid.
Executive summary:

The hydrolytical behavior of the substance was investigated with the OECD guideline 111. The substance is hydrolytically unstable showing half-life-times at 50°C of 30.39 h at pH 4, 44.08 h at pH 7 and 77.52 h at pH 9. One transformation product was identified as tetrabromophthalic acid.

Description of key information

The substance is hydrolytically unstable showing half-life-times at 50°C of 30.39 h at pH 4, 44.08 h at pH 7 and 77.52 h at pH 9. One transformation product was identified as tetrabromophthalic acid.

Key value for chemical safety assessment

Half-life for hydrolysis:
77.52 h
at the temperature of:
50 °C

Additional information

Using the van´t Hoff equation the half-life-times can be extrapolated to a temperature of 20°C:

pH4: t1/2= 30.39h at 50°C is corresponding to t1/2= 10.1 days at 20°C

pH7: t1/2= 44.08h at 50°C is corresponding to t1/2= 14.7 days at 20°C

pH9: t1/2= 77.52h at 50°C is corresponding to t1/2= 25.8 days at 20°C