Fatty acids, C18-unsatd., phosphates

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January-July 2018

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:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0101891886
- Expiration date of the lot/batch: 14 September 2018 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

Radiolabelling:

not specified

Analytical monitoring:

yes

Buffers:

Buffer Solutions:
Acetate buffer pH 4, 0.01 M: Solution of 16.7% 0.01 M sodium acetate in water and 83.3% 0.01 M acetic acid in water. Buffer contained 0.0009% (w/v) sodium azide.
Phosphate buffer pH 7, 0.01 M: Solution of 0.01 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, 0.01 M: Solution of 0.01 M boric acid in water and 0.01 M potassium chloride in water adjusted to pH 9 using 1N sodium hydroxide. Buffer contained 0.0009% (w/v) sodium azide.

Details on test conditions:

The rate of hydrolysis of the test item as a function of pH was determined at pH values normally found in the environment (pH 4-9).

Preliminary test:
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. To exclude oxygen, nitrogen gas was purged through the solution for 5 minutes. The test item was spiked to the solutions at a target concentration of 10 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 50.1°C  0.1°C.
The spiking volume was ≤ 1% of the sample volume. Nominal concentrations were not corrected for the spiking volume.
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.
Analysis was performed on subsamples of 900 µL. The samples were diluted in a 9:1 (v:v) ratio with acetonitrile. The samples were 10-fold further diluted with 10/90 (v/v) acetonitrile / buffer pH4, 7 or 9 to obtain concentration levels within the calibrated range. Blank buffer solutions containing a similar content of blank spiking solution were diluted in a 9:1 (v:v) ratio with acetonitrile and analyzed.
The pH of each of the test solutions (except for the blanks) was determined at each sampling time.

Main study:
Test samples were prepared and treated similarly as during the preliminary test. The concentrations of the test item were determined immediately after preparation (t=0) and at several sampling points after t=0.
Blank buffer solutions were treated similarly as the test samples and analyzed at t=0.
The pH of each of the test solutions (except for the blanks) was determined at least at the beginning and at the end of the test.

The study was performed at the following temperatures.

pH Temperature I Temperature II Temperature III
pH 4 19.9°C  0.2°C 49.7°C  0.9°C Study stopped, test item is stable
pH 7 19.9°C  0.2°C 50.1°C  0.5°C 60.6°C  0.2°C
pH 9 19.6°C  0.5°C 50.0°C  0.1°C 60.5°C  0.1°C



Chemicals and Reagents used in the test:

Chemical / Reagent: Supplier
Water: Tap water purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA)
Acetonitrile: Biosolve, Valkenswaard, The Netherlands
Formic acid: Biosolve.
Sodium acetate: Merck, Darmstadt, Germany
Acetic acid, 100%: Merck
Potassium di-hydrogenphosphate: Merck
Boric acid: Merck
Potassium chloride: Merck
Sodium hydroxide, 1N: Merck
Sodium azide: Merck

All chemicals and reagents were of analytical grade, unless specified otherwise.

Duration:

14 d

pH:

4

Temp.:

20 °C

Initial conc. measured:

10.6 mg/L

Duration:

12 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

10.2 mg/L

Duration:

18 d

pH:

7

Temp.:

20 °C

Initial conc. measured:

10.4 mg/L

Duration:

16 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

10 mg/L

Duration:

18 d

pH:

9

Temp.:

20 °C

Initial conc. measured:

10 mg/L

Duration:

10 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

10 mg/L

Duration:

17 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

10.4 mg/L

Duration:

6 h

pH:

0

Temp.:

60 °C

Initial conc. measured:

10 mg/L

Positive controls:

not specified

Negative controls:

not specified

Preliminary study:

At each pH, calibration curves at m/z 151.0 and at m/z 391.3 were constructed using six concentration levels. For each level, duplicate responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration2 weighting factor. If necessary, one or two data points were excluded from the curve since the back calculated accuracy was > 15% from the nominal concentration or a deviation was observed in the response factor. The coefficient of correlation (r) was > 0.99 for each curve.
Details of the results are found in tables 20-23 in the section "other inforamtion includign tables"

Test performance:

At each pH, calibration curves at m/z 391.3 were constructed using six concentration levels. For each level, duplicate responses were used. Linear or quadratic regression analysis was performed using the least squares method with a 1/concentration2 weighting factor. If necessary, one, two or three data points were excluded from the curve since the back calculated accuracy was > 15% from the nominal concentration or a deviation was observed in the response factor. The coefficient of correlation (r) was > 0.99 for each curve.
Details of the results are found in tables 24-35 in the section "other inforamtion includign tables"

Transformation products:

not measured

Key result

pH:

4

Temp.:

25 °C

DT50:

> 1 yr

Type:

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

Key result

pH:

7

Temp.:

25 °C

DT50:

> 1 yr

Type:

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

Key result

pH:

9

Temp.:

20 °C

DT50:

18 d

Type:

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

Key result

pH:

9

Temp.:

25 °C

DT50:

10 d

Type:

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

Key result

pH:

9

Temp.:

50 °C

DT50:

17 h

Type:

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

Key result

pH:

9

Temp.:

60 °C

DT50:

5.7 h

Type:

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

Details on results:

Details of the results are found in tables 20-34 in the section "other inforamtion includign tables". As follows a description of the behaviour of the substance at the measured pHs:
pH 4
The analytical results of the main study at pH 4 are given in Table 24 to Table 26.
A small response at the retention time of the test item was detected in the chromatograms of the blank buffer solutions. This was probably caused by carry-over since a similar or higher response was observed in the analytical blank.
The mean recoveries of the test item containing buffer solutions at t=0 fell not within the criterion range of 90-110. Since hydrolysis is determined as relative to initial, this had no impact in the study
All logarithms of the relative concentrations were correlated with time using linear regression analysis. Figure 13 illustrates the regression curves. A degree of hydrolysis of < 10% was observed after 337 hours at pH 4, 20°C and after 288 hours at pH4, 50°C. 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.
pH 7
The analytical results of the main study are given in Table 27 to Table 29.
A small response at the retention time of the test item was detected in one of the chromatograms of the blank buffer solutions. This was probably caused by carry-over since a similar or higher response was observed in the analytical blank. In all other chromatograms, no test item was detected.
The mean recoveries 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.
All logarithms of the relative concentrations were correlated with time using linear regression analysis. Figure 14 illustrates the regression curves. A degree of hydrolysis of < 10% was observed after 432 hours at pH 7, 20°C and after 384 hours at pH7, 50°C. 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.
pH 9
The analytical results of the main study are given in Table 30 to Table 33.
A small response at the retention time of the test item was detected in one of the chromatograms of the blank buffer solutions. This was probably caused by carry-over since a similar or higher response was observed in the analytical blank. In all other chromatograms, no test item was detected.
The mean recoveries of the test item containing buffer solutions at t=0 were in the range of 102 -111%. It demonstrated that the analytical method was adequate to support the hydrolysis study on the test item.
For testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 10% and 90% were plotted against time. At all temperatures linear relationships were obtained.
The half-life times of the test item were determined according to the model for pseudo-first order reactions. All logarithms of the relative concentrations were correlated with time using linear regression analysis. Figure 15 illustrates the regression curves and Table 34 shows the statistical parameters.

Table 20      
Preliminary Test: Hydrolysis of the Test Item at pH 4, pH 7 and pH 9m/z151.0

pH code	Sampling time	Analyzed concentration [mg/L]	Degree of hydrolysis [%]		pH
			Individual	Mean
pH 4	0 hours	10.6			4.1
		10.3			4.1
	5 days	10.4	0.73	-0.53	4.2
		10.6	-1.8		4.2
pH 7	0 hours	10.2			7.0
		10.3			7.0
	5 days	10.2	0.32	-1.1	7.1
		10.5	-2.6		7.1
pH 9	0 hours	10.4			9.1
		10.5			9.1
	5 days	10.2	2.2	1.7	9.0
		10.3	1.2		9.1

   

Table 21      
Preliminary Test: Hydrolysis of the Test Item at pH 4, pH 7 and pH 9m/z391.3

pH code	Sampling time	Analyzed concentration [mg/L]	Degree of hydrolysis [%]		pH
			Individual	Mean
pH 4	0 hours	8.70			4.1
		8.98			4.1
	5 days	5.73	35	38	4.2
		5.20	41		4.2
pH 7	0 hours	10.1			7.0
		10.8			7.0
	5 days	8.93	14	12	7.1
		9.47	9.3		7.1
pH 9	0 hours	12.7			9.1
		10.7			9.1
	5 days	0.077^1	99	99	9.0
		0.077^1	99		9.1

¹Estimated value, calculated by the extrapolation of the calibration curve.

Table 22      
Preliminary Test: Recoveriesm/z151.0

pH code	Nominal concentration [mg/L]	Analyzed concentration [mg/L]	Recovery [%]
			Individual	Mean
pH 4	10.0	10.6	106	104
	10.0	10.3	103
pH 7	10.0	10.2	102	103
	10.0	10.3	103
pH 9	10.0	10.4	104	105
	10.0	10.5	105

 

Table 23      
Preliminary Test: Recoveriesm/z391.3

pH code	Nominal concentration [mg/L]	Analyzed concentration [mg/L]	Recovery [%]
			Individual	Mean
pH 4	10.0	8.70	87	88
	10.0	8.98	90
pH 7	10.0	10.1	101	104
	10.0	10.8	108
pH 9	10.0	12.7	127	117
	10.0	10.7	107

Table 24      
Main Test pH 4: Hydrolysis of the Test Item at 20°Cm/z391.3

Sampling time [hours]	Analyzed concentration [mg/L]	Relative concentration [%]	Logarithm relative concentration	pH
0.00	9.19	96	1.98	4.0
0.00	9.86	104	2.02	4.0
48.73	7.69	81	1.91	4.0
48.73	6.72	71	1.85	4.0
93.22	7.88	83	1.92	4.0
93.22	6.95	73	1.86	4.0
168.62	10.6	111	2.05	-
168.62	8.84	93	1.97	-
240.78	13.4	141	2.15	4.0
240.78	10.1	106	2.03	4.0
336.60	11.3	119	2.07	4.0
336.60	10.8	114	2.06	4.0

 

 

 

Table 25      
Main Test at pH 4: Hydrolysis of the Test Item at 50°Cm/z391.3

Sampling time [hours]	Analyzed concentration [mg/L]	Relative concentration [%]	Logarithm relative concentration	pH
0.00	5.66	89	1.95	4.0
0.00	7.01	111	2.04	4.0
45.70	7.86	124	2.09	4.0
45.70	8.22	130	2.11	4.0
120.62	7.91	125	2.10	-
120.62	8.54	135	2.13	-
192.72	9.59	151	2.18	4.0
192.72	11.1	175	2.24	4.0
288.12	8.14	129	2.11	4.0
288.12	9.33	147	2.17	4.0

 

Table 26      
Main Test pH 4: Recoveries

Temperature (°C)	Nominal concentration [mg/L]	Analyzed concentration [mg/L]	Recovery [%]
			Individual	Mean
20	10.0	9.19	92	95
	10.0	9.86	99
50	10.0	5.66	57	63
	10.0	7.01	70

Table 27      
Main Test pH 7: Hydrolysis of the Test Item at 20°Cm/z391.3

Sampling time [hours]	Analyzed concentration [mg/L]	Relative concentration [%]	Logarithm relative concentration	pH
0.00	10.3	99	1.99	7.0
0.00	10.6	101	2.01	7.0
48.40	9.65	92	1.97	7.0
48.40	11.1	106	2.03	7.0
92.88	5.25	50	1.70	7.0
92.88	10.6	101	2.01	7.0
240.45	12.6	120	2.08	7.0
240.45	12.3	118	2.07	7.0
432.83	11.8	113	2.05	7.0
432.83	13.7	131	2.12	7.0

 

Table 28      
Main Test pH 7: Hydrolysis of the Test Item at 50°Cm/z391.3

Sampling time [hours]	Analyzed concentration [mg/L]	Relative concentration [%]	Logarithm relative concentration	pH
0.00	9.65	95	1.98	7.0
0.00	10.7	105	2.02	7.0
45.33	7.10	70	1.84	7.0
45.33	9.59	94	1.97	7.0
192.35	9.57	94	1.97	7.0
192.35	9.59	94	1.97	7.0
384.63	9.11	90	1.95	7.0
384.63	9.51	93	1.97	7.0

 

Table 29      
Main Test pH 7: Recoveries

Temperature (°C)	Nominal concentration [mg/L]	Analyzed concentration [mg/L]	Recovery [%]
			Individual	Mean
20	10.0	10.3	103	105
	10.0	10.6	106
50	10.0	9.65	97	102
	10.0	10.7	107

Table 30      
Main Test pH 9: Hydrolysis of the Test Item at 20°Cm/z391.3

Sampling time [hours]	Analyzed concentration [mg/L]	Relative concentration [%]	Logarithm relative concentration	pH
0.00	10.3	99	1.99	9.0
0.00	10.6	101	2.01	9.0
48.00	8.75	84	1.92	9.0
48.00	9.61	92	1.96	9.0
92.48	8.93	86	1.93	9.0
92.48	9.21	88	1.95	9.0
240.05	7.90	76	1.88	9.0
240.05	8.71	83	1.92	9.0
335.87	6.60	63	1.80	9.0
335.87	7.16	69	1.84	9.0
432.43	6.20	59	1.77	9.0
432.43	6.82	65	1.81	9.0
503.42	5.18	50	1.70	9.0
503.42	5.18	50	1.70	9.0
595.18	4.53	43	1.64	9.0
595.18	4.69	45	1.65	9.0
693.88	2.86	27	1.44	9.0
693.88	3.25	31	1.49	9.0
741.38	2.99	29	1.46	9.0
741.38	3.41	33	1.51	9.0

 

Table 31      
Main Test at pH 9: Hydrolysis of the Test Item at 50°Cm/z391.3

Sampling time [hours]	Analyzed concentration [mg/L]	Relative concentration [%]	Logarithm relative concentration	pH
0.00	9.93	97	1.99	9.0
0.00	10.6	103	2.01	9.0
2.05	8.95	87	1.94	9.0
2.05	9.88	96	1.98	9.0
3.08	9.25	90	1.96	9.0
3.08	9.88	96	1.98	9.0
20.77	4.14	40	1.61	9.0
20.77	4.55	44	1.65	9.0
23.10	3.71	36	1.56	9.0
23.10	3.75	37	1.56	9.0
25.25	2.92	28	1.45	9.0
25.25	3.49	34	1.53	9.0
26.22	3.27	32	1.50	9.0
26.22	3.46	34	1.53	9.0
26.88	3.32	32	1.51	9.0
26.88	3.46	34	1.53	9.0
44.98	1.60	16	1.19	9.0
44.98	1.45	14	1.15	9.0
52.47	1.00	10	0.99	9.0
52.47	1.03	10	1.00	9.0
93.02	0.23^1	2.2	0.35	9.0
93.02	0.22^1	2.2	0.33	9.0

¹Estimated value, calculated by the extrapolation of the calibration curve.

 

Table 32      
Main Test at pH 7: Hydrolysis of the Test Item at 60°Cm/z391.3

Sampling time [hours]	Analyzed concentration [mg/L]	Relative concentration [%]	Logarithm relative concentration	pH
0.00	11.2	101	2.00	9.0
0.00	11.0	99	2.00	9.0
1.87	7.98	72	1.86	9.0
1.87	8.51	77	1.88	9.0
2.33	7.61	69	1.84	9.0
2.33	8.18	74	1.87	9.0
2.95	7.59	68	1.83	9.0
2.95	8.05	73	1.86	9.0
3.90	6.55	59	1.77	9.0
3.90	6.84	62	1.79	9.0
4.67	5.90	53	1.73	9.0
4.67	6.25	56	1.75	9.0
5.22	5.68	51	1.71	9.0
5.22	5.67	51	1.71	9.0
5.65	5.42	49	1.69	9.0
5.65	5.80	52	1.72	9.0
23.75	0.567	5.1	0.71	9.0
23.75	0.610	5.5	0.74	9.0
31.23	0.25^1	2.3	0.36	9.0
31.23	0.26^1	2.3	0.37	9.0

¹Estimated value, calculated by the extrapolation of the calibration curve.

Table 33      
Main Test pH 9: Recoveries

Temperature (°C)	Nominal concentration [mg/L]	Analyzed concentration [mg/L]	Recovery [%]
			Individual	Mean
20	10.0	10.3	103	104
	10.0	10.6	106
50	10.0	9.93	99	102
	10.0	10.6	106
60	10.0	11.2	112	111
	10.0	11.0	110

Table 34      
Main Test pH 9: Statistical Parameters Regression Curves

Temperature (°C)	Slope [hours^-1]	Intercept	Coefficient of correlation
20	-6.87´10^-4	2.02	0.96
50	-1.81´10^-2	1.99	0.998
60	-5.24´10^-2	1.99	0.9995

Table 35      
Main Test pH 9: Rate Constants (k_obs) and Half-life Time (t_½)

Temperature [°C]	kobs [hours^-1]	t½
20	1.58´10^-3	18 days
25	2.86´10^-3	10 days
50	4.17´10^-2	17 hours
60	1.21´10^-1	5.7 hours

Validity criteria fulfilled:

yes

Conclusions:

The preliminary test (Tier 1) and main study (Tier 2) were performed for the determination of the rate of hydrolysis of Fatty acids, C18-unsatd., phosphates at pH values normally found in the environment (pH 4-9).
At pH 4 and pH 7 a degree of hydrolysis of < 10% was observed after 5 days at 50°C. According to the guideline, performance of the main study (Tier 2) was not required.
At pH 9, a degree of hydrolysis of ≥ 10% was observed after 5 days at 50°C and the main study was performed to determine the half-life time of the test item.
The half-life times (t(1/2)) of the test item were as follows:
pH 4 at 25 degrees celsisus: t(1/2) > 1 year
pH 25 at 25 degrees celsius: t(1/2) > 1 year
pH 9 at the following temperatures:
20 degrees celsius: t(1/2) 18 days
25 degrees celsius: t(1/2) 10 days
50 degrees celsius: t(1/2) 17 hours
60 degrees celsius: t(1/2) 5.7 hours

Executive summary:

The stability of Fatty acirds, C18 -unsatd., phosphates to hydrolysis at pH 4, 7 and 9 was tested on a preliminary study at 20, 25,50 and 60ºC (Ciric, J., 2018). The study was conducted according to OECD 111 andt the EEC C.7 guideline, "abiotic degradation: hydrolysis as a function of pH".

The preliminary test (Tier 1) and main study (Tier 2) were performed for the determination of the rate of hydrolysis of Fatty acids, C18-unsatd., phosphates at pH values normally found in the environment (pH 4-9).

At pH 4 and pH 7 a degree of hydrolysis of < 10% was observed after 5 days at 50°C.  According to the guideline, performance of the main study (Tier 2) was not required.

At pH 9, a degree of hydrolysis of ≥ 10% was observed after 5 days at 50°C and the main study was performed to determine the half-life time of the test item.

The half-life times (t(1/2)) of the test item were as follows:

pH 4 at 25 degrees celsisus:  t(1/2) > 1 year

pH 7 at 25 degrees celsius:  t(1/2) > 1 year

pH 9 at the following temperatures:

20 degrees celsius: t(1/2) 18 days

25 degrees celsius: t(1/2) 10 days

50 degrees celsius: t(1/2) 17 hours

60 degrees celsius: t(1/2) 5.7 hours

Description of key information

The stability of Fatty acirds, C18 -unsatd., phosphates to hydrolysis at pH 4, 7 and 9 was tested on a preliminary study at 20, 25,50 and 60ºC (Ciric, J., 2018). The study was conducted according to OECD 111 andt the EEC C.7 guideline, "abiotic degradation: hydrolysis as a function of pH".

The preliminary test (Tier 1) and main study (Tier 2) were performed for the determination of the rate of hydrolysis of Fatty acids, C18-unsatd., phosphates at pH values normally found in the environment (pH 4-9).

At pH 4 and pH 7 a degree of hydrolysis of < 10% was observed after 5 days at 50°C.  According to the guideline, performance of the main study (Tier 2) was not required.

At pH 9, a degree of hydrolysis of ≥ 10% was observed after 5 days at 50°C and the main study was performed to determine the half-life time of the test item.

The half-life times (t(1/2)) of the test item were as follows:

pH 4 at 25 degrees celsisus:  t(1/2) > 1 year

pH 7 at 25 degrees celsius:  t(1/2) > 1 year

pH 9 at the following temperatures:

20 degrees celsius: t(1/2) 18 days

25 degrees celsius: t(1/2) 10 days

50 degrees celsius: t(1/2) 17 hours

60 degrees celsius: t(1/2) 5.7 hours

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

20 °C