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Environmental fate & pathways

Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 03, 2012 to December 03, 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
adopted 2004-04-13
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Version / remarks:
dated 31 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Analytical monitoring:
yes
Remarks:
The concentration of the test item was determined using LC-MS/MS method. The linearity of the analytical method was proven in the concentration range from 5 μg/L to 250 μg/L. The regression coefficients were determined to be at least 0.9985.
Details on sampling:
Preliminary test:
a) Sampling: Three samples of solutions of each pH value were taken after 0 h for the determination of the applied concentration whereas two samples were taken at each following sampling point.
b) Sampling points: 0, 2.4-2.8, 24, 120-130 h

Main test pH 4 at 18°C:
a) Sampling: Three samples were taken after 0 d for the determination of the applied concentration whereas two samples were taken at each following sampling point.
b) Sampling points: 0, 4, 8, 11, 15, 18, 22, 25, 29 d

Main test pH 4 at 50°C:
a) Sampling: Three samples were taken after 0 d for the determination of the applied concentration whereas two samples were taken at each following sampling point.
b) Sampling points: 0, 1, 4, 6, 8, 11, 13, 15 d

Main test pH 4 at 60°C:
a) Sampling: Three samples were taken after 0 d for the determination of the applied concentration whereas two samples were taken at each following sampling point.
b) Sampling points: 0, 0.08, 1, 2, 4, 7, 9, 11 d
Buffers:
The buffer solutions were prepared as described below. The pH of each buffer solution at the relevant temperatures was checked with a pH meter. Each buffer solution was purged with nitrogen gas and sterilized by heating in an autoclave before using it to prepare the test solution.

pH 4:
A) KH2PO4 buffer (0.01 M): 340-347 mg KH2PO4 were dissolved in 250 mL pure water resulting in a conentration of 0.01 M. The pH of the solution was adjusted by adding dropwise a solution of H3PO4 (85 %) diluted 1:50 v/v with pure water.
B) Citric acid buffer: Commercially available citric acid buffer (Merck, Germany, Certipur, citric acid, sodium hydroxide, hydrogen chloride, pH 4.00 at 20°C)

pH 7:
KH2PO4 buffer (0.001 M): 50 mL potassium dihydrogen phosphate solution (1.36 g KH2PO4 / 100 mL pure water) were mixed with 29.6 mL sodium hydroxide solution (0.1 mol/L NaOH) and filled up to 100 mL with pure water. For preparing the test solution, the buffer solution (0.1 M) was diluted 1:100 v/v with pure water.

pH 9:
Boric acid buffer (0.01 M): 50 mL boric acid solution (618 mg H3BO3 / 100 mL KCl 0.1 M) was mixed with 21.3 mL sodium hydroxide solution (0.1 M NaOH) and filled up to 100 mL with pure water. For preparing the test solution, the buffer solution was diluted 1:10 v/v with pure water.
Details on test conditions:
a) Preliminary test
Test solution:
A stock solution of the test substance was prepared in methanol at a concentration level of approximately 1 g/L. 0.6 mL of the stock solution were transferred into a 200 mL volumetric flask and filled up to the mark with the respective buffer solution resulting in a final concentration of about 3 mg/L. The concentration was below 0.01 M or half of the saturation concentration of the test substance in water (water solubility: 198 mg/L at 20 °C).

Identity and concentration of co-solvent:
The amount of the co-solvent methanol was below 1 % (0.3 %) of the total volume.

Sterility control:
A sterility confirmation test was performed at the end of the incubation period. To do this, separate test vessels were incubated under test conditions as described above. At the end of the maximal incubation period dip slides for fluids (Hycon GK-T/HS, Heipha Dr. Müller GmbH, Eppelheim, Germany) were wetted completely with samples of each pH value. Excess sample was allowed to run off. The dip slides were placed back into their tube and incubated at 20 °C to 22 °C for 4-7 d. No colonies on the agar were observed (expect for the replicate A (with test substance) at pH 7 where one colony was counted after 4 d of incubation).

b) Main test
For the main test two test substance concentrations were used. The lower concentration approx. 3 mg/L was prepared to investigate the degradation behavior and for the verification of the results of the preliminary test. The higher concentration of approx. 30 mg/L was provided for the characterization of possible transformation products. To fulfill the requirements of the OECD guideline (co-solvent in the test solution ≤ 1% v/v) the concentrations of the stock solutions were approximately 1 g/L and 10 g/L. In contrast to the preliminary test the stock solutions were prepared in THF/pure water 70:30 v/v because the test item was not soluble in MeOH at a concentration of 10 g/L.

Test solution A – 3 mg/L
0.6 mL or 0.75 mL of the 1 g/L stock solution were transferred into a 200 mL or 250 mL volumetric flask and filled up to the mark with the buffer solution resulting in a final concentration of about 3 mg/L.

Test solution B – 30 mg/L
0.6 mL or 0.75 mL of the 10 g/L stock solution were transferred into a 200 mL or 250 mL volumetric flask and filled up to the mark with the buffer solution resulting in a final concentration of about 30 mg/L. The concentrations were below 0.01 M or half of the saturation concentration of the test substance in water (water solubility: 198 mg/L at 20°C according to IBACON project 69606185) and the amount of the co-solvent THF was below 1% (0.2%) of the total volume.
Duration:
29 d
pH:
4
Temp.:
18 °C
Initial conc. measured:
ca. 3 mg/L
Duration:
15 d
pH:
4
Temp.:
50 °C
Initial conc. measured:
ca. 30 mg/L
Duration:
15 d
pH:
4
Temp.:
50 °C
Initial conc. measured:
ca. 3 mg/L
Duration:
11 d
pH:
4
Temp.:
60 °C
Initial conc. measured:
ca. 30 mg/L
Duration:
11 d
pH:
4
Temp.:
60 °C
Initial conc. measured:
ca. 3 mg/L
Number of replicates:
3
Positive controls:
no
Negative controls:
no
Preliminary study:
In a preliminary test the test substance was dissolved in aqueous solutions buffered at pH 4, 7 and 9 and incubated at 50°C ± 4.5°C for a maximum of 5 d. No significant reduction of the test substance concentration was observed in the samples incubated at pH 7 and 9 after 5 d of incubation (mean recovery > 90 % of the applied concentration). In the incubated samples at pH 4 a significant reduction of the test substance concentration was observed after 5 d of incubation (mean recovery 63 % of the applied concentration). To assess the impact of the buffer solution on the degradation process the preliminary test at pH 4 was repeated. Two different buffer solutions were used. To verify the results of the first pre-test an equal phosphate buffer was used, whereas the commercially available citric acid buffer was chosen due to the absence of phosphate molecules which were expected to be a degradation product during hydrolytic cleavage of the test substance. A significant reduction of the test substance concentration was observed after 5 d of incubation for both buffer solutions (mean recovery 75 % of the applied concentration). Thus, it is evident that the buffer solution had no impact on the degradation process and the results of the first pre-test are valid. According to the OECD guideline a main test to determine the reaction rate constant and half-life of potassium hexadecyl hydrogen phosphate at pH 4 had to be performed. The test substance was found to be stable at pH 7 and 9 no main test was performed for those pH-values.
Transformation products:
not measured
Details on hydrolysis and appearance of transformation product(s):
In agreement with the sponsor, no identification of degradation products was performed as the test substance partly precipitated during the main test.
% Recovery:
> 90
pH:
7
Temp.:
50 °C
Duration:
ca. 5 d
Remarks on result:
hydrolytically stable based on preliminary test
% Recovery:
> 90
pH:
9
Temp.:
50 °C
Duration:
ca. 5 d
Remarks on result:
hydrolytically stable based on preliminary test
% Recovery:
ca. 63
pH:
4
Temp.:
50 °C
Duration:
ca. 5 d
Remarks on result:
other: significnt reduction in test substance concentration
pH:
4
Remarks on result:
other: could not be determined as the test substance precipitates in course of incubation
pH:
5
Remarks on result:
other: could not be determined as the test substance precipitates in course of incubation
pH:
6
Remarks on result:
other: could not be determined as the test substance precipitates in course of incubation
Details on results:
The test substance is hydrolytically stable at pH 7 and 9. At pH 4-6 a test on the hydrolytic properties of potassium hexadecyl hydrogen phosphate was not feasible as the test item partly precipitated in course of incubation.

Results:

A main test was performed with the commercially available citric acid buffer solution at pH 4. The test substance was dissolved and incubated at 18°C ± 1.3°C, 50°C ± 0.6°C and 60°C ± 1.8°C in the dark. Two test substance concentrations (approx. 3 mg/L and 30 mg/L) were used, both below 0.01 M and half of the saturation concentration of the test substance in water (water solubility: 198 mg/L at 20°C according to IBACON project 69606185). The lower concentration (approx. 3 mg/L) was prepared to investigate the degradation behaviour and for the verification of the results of the preliminary test. The higher concentration (approx. 30 mg/L) was provided for the characterization of possible transformation products. To fulfil the requirements of the OECD guideline (co-solvent in the test solution ≤ 1% v/v) the concentrations of the stock solutions were approx. 1 g/L and 10 g/L. In contrast to the preliminary test the stock solutions were prepared in THF/pure water 70:30 v/v because the test substance was not soluble in MeOH at a concentration of 10 g/L. Further samples preparations (e.g. dilution prior to analysis) were executed equal to the pre-test. When the test solutions were prepared, solutions containing a nominal concentration of approx. 30 mg/L were found to be clear immediately after application but started to become turbid after a short period of time (i.e. minutes) without precipitation of the test substance. Due to the tenfold lower concentration of the 3 mg/L samples, a turbidity of the solution could not been recognized but both concentration levels were far below the water solubility, thus it can be assumed that the behaviour of the test substance at pH 4 was equal for both applied concentrations. As no precipitation could be observed after application the main test was started. However, in course of the test and also in course of sample storage the test substance precipitated by the formation of white solid components. The results in table 1 and 2 indicate that with Amphisol K buffered at pH 4 the mean recoveries did not fit to a common hydrolytic degradation pattern, as the values e.g. increase in course of incubation or reach a steady state after the first few sampling points. In this context it may be possible that Amphisol K interacted on the one hand with counter ions of the buffer solution (e.g. sodium) or on the other hand with itself by the formation of micelles resulting in both cases in the formation of the mentioned precipitate. As it cannot be differentiate between hydrolytic degradation and precipitation, which both leads to a reduction in the measured test substance concentration, an evaluation of the obtained data regarding hydrolysis rate constant, half-life or the calculation of Arrhenius equations was not possible. With all the efforts done including a change of the buffer system (acetate buffer) or without a buffering additive (HCL was used to adjust the pH of the test solution in pure water) to exclude any counter ions it can be concluded that it was not feasible to performed the hydrolysis test with Amphisol K at pH 4. In order to execute the test at a pH between 4 to 7, test solutions were prepared with a concentration of approx. 3 mg/L and 30 mg/L. As buffer systems citric acid and acetate solutions were used, both at pH-values of 5 and 6. The solutions were stored at 20°C ± 1.3°C in the dark over a period of 13 d. In each case, especially within the samples containing the higher test substance concentration, a precipitate could be observed. Thus, in addition to the former results it can be stated that it was not feasible to investigate the hydrolytic properties of Amphisol K at a pH range of 4-6.

Table 1: Results of the main test at pH 4 - Test substance concentration: 3 mg/L

Temperature

Incubation period

Replicate

pH value (measured)

Concentration in samples

Recovery

Found (mg/L)

Dilution factor

Calculated* (mg/L)

% Nominal

Mean (%)

18°C

0

A

4

0.14

20

2.8

101

100

B

4

0.137

20

2.735

98

C

4

0.14

20

2.8

101

4

A

4

0.118

20

2.364

85

85

B

4

0.117

20

2.349

85

8

A

4

0.103

20

2.06

74

74

B

4

0.102

20

2.043

74

11

A

4

0.101

20

2.022

73

72

B

4

0.1

20

1.995

72

15

A

4

0.144

20

2.89

104

87

B

4

0.098

20

1.968

71

18

A

4

0.09

20

1.791

64

68

B

4

0.098

20

1.97

71

22

A

4

0.09

20

1.802

65

68

B

4

0.098

20

1.954

70

25

A

4

0.092

20

1.833

66

67

B

4

0.095

20

1.896

68

29

A

4

0.114

20

2.278

82

85

B

4

0.123

20

2.456

88

50°C

0

A

4

0.137

20

2.735

101

100

B

4

0.138

20

2.751

101

C

4

0.133

20

2.652

98

1

A

4

0.123

20

2.455

90

79

B

4

0.092

20

1.845

68

4

A

4

0.046

20

0.922

34

53

B

4

0.099

20

1.977

73

6

A

4

0.072

20

1.444

53

46

B

4

0.054

20

1.072

39

8

A

4

0.066

20

1.325

49

46

B

4

0.06

20

1.197

44

11

A

4

0.061

20

1.219

45

43

B

4

0.054

20

1.089

40

13

A

4

0.045

20

0.906

33

29

B

4

0.034

20

0.671

25

15

A

4

0.045

20

0.89

33

30

B

4

0.036

20

0.722

27

60°C

0

A

4

0.15

20

2.997

100

100

B

4

0.151

20

3.012

100

C

4

0.149

20

2.982

100

0.08

A

4

0.102

20

2.044

68

68

B

4

0.101

20

2.014

67

1

A

4

0.059

20

1.178

39

31

B

4

0.034

20

0.672

22

2

A

4

0.048

20

0.956

32

34

B

4

0.054

20

1.084

36

4

A

4

0.023

20

454

15

21

B

4

0.04

20

0.801

27

7

A

4

0.022

20

0.444

15

15

B

4

0.023

20

0.45

15

9

A

4

0.028

20

0.553

18

17

B

4

0.023

20

0.468

16

11

A

4

0.035

20

0.699

23

20

B

4

0.024

20

0.479

16

* Values calculated from exact raw data

Nominal concentrations: 20°C = 2.778 mg/L; 50°C = 2.713 mg/L; 60°C = 2.997 mg/L

pH 4: LOD = 2.351 μg/L; LOQ = 7.835 μg/L

Table 2: Results of the main test at pH 4 - Test substance concentration: 30 mg/L

Temperature

Incubation period

Replicate

pH value (measured)

Concentration in samples

Recovery

Found (mg/L)

Dilution factor

Calculated (mg/L)

% Nominal

Mean (%)

18°C

0

A

4

0.144

200

28.77

101

100

B

4

0.142

200

28.36

100

C

4

0.14

200

28.08

99

4

A

4

0.129

200

25.77

91

89

B

4

0.125

200

24.93

88

8

A

4

0.117

200

23.44

83

80

B

4

0.109

200

21.81

77

11

A

4

0.125

200

24.92

88

87

B

4

0.124

200

24.77

87

15

A

4

0.109

200

21.71

76

76

B

4

0.109

200

21.71

76

18

A

4

0.1

200

20.08

71

71

B

4

0.101

200

20.23

71

22

A

4

0.094

200

18.78

66

68

B

4

0.098

200

19.66

69

25

A

4

0.109

200

21.86

77

74

B

4

0.101

200

20.26

71

29

A

4

0.131

200

26.29

93

91

B

4

0.128

200

25.66

90

50°C

0

A

4

0.129

200

25.86

94

100

B

4

0.141

200

28.22

102

C

4

0.144

200

28.77

104

1

A

4

0.118

200

23.65

86

86

B

4

0.12

200

24.06

87

4

A

4

0.1

200

19.92

72

72

B

4

0.1

200

20.09

73

6

A

4

0.087

200

17.36

63

65

B

4

0.093

200

18.69

68

8

A

4

0.077

200

15.43

56

56

B

4

0.077

200

15.43

56

11

A

4

0.108

200

21.51

78

78

B

4

0.107

200

21.36

77

13

A

4

0.087

200

17.48

63

59

B

4

0.074

200

14.89

54

15

A

4

0.071

200

14.27

52

44

B

4

0.051

200

10.14

37

60°C

0

A

4

0.137

200

27.4

103

100

B

4

0.134

200

26.79

101

C

4

0.128

200

25.37

96

0.08

A

4

0.092

200

18.4

69

72

B

4

0.099

200

19.77

74

1

A

4

0.096

200

19.28

73

74

B

4

0.1

200

20.04

75

2

A

4

0.091

200

18.27

69

72

B

4

0.099

200

19.79

74

4

A

4

0.08

200

15.99

60

67

B

4

0.099

200

19.79

74

7

A

4

0.089

200

17.84

67

67

B

4

0.088

200

17.52

66

9

A

4

0.082

200

16.37

62

65

B

4

0.09

200

17.97

68

11

A

4

0.084

200

16.8

63

66

B

4

0.091

200

18.16

68

*Values calculated from exact raw data

Nominal concentrations: 20°C = 28.403 mg/L; 50°C = 27.618 mg/L; 60°C = 26.587 mg/L

pH 4: LOD = 2.351 μg/L; LOQ = 7.835 μg/L

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of the read across study, the test substance, is considered to be hydrolytically stable at pH 7 and 9, whereas at pH 4 to 6 a test is not considered to be feasible as the test substance is expected to partly precipitate in course of incubation.
Executive summary:

A study was conducted to determine the rate of hydrolysis of read across substance, mono- and di- C16 PSE, K+, (purity: ca. 85%), according to OECD Guideline 111 and EU C.7 Method, in compliance with GLP. The study was performed at different environmentally relevant pH-values by quantifying the test substance concentration after different incubation periods and at different temperatures. In a preliminary test the test substance was dissolved in aqueous solutions buffered at pH 4, 7 and 9 and incubated at 50°C ± 4.5°C for a maximum of 5 d. No significant reduction of the test substance concentration was observed in the samples incubated at pH 7 and 9 after 5 d of incubation (mean recovery > 90% of the applied concentration). In the incubated samples at pH 4 a significant reduction of the test substance concentration was observed after 5 d of incubation in phosphate and citric acid buffer, respectively (mean recovery 63-75% of the applied concentration).To assess the impact of the buffer solution on the degradation process the preliminary test at pH 4 was repeated. Two different buffer solutions were used. To verify the results of the first pre-test an equal phosphate buffer was used, whereas the commercially available citric acid buffer was chosen due to the absence of phosphate molecules which were expected to be a degradation product during hydrolytic cleavage of the test substance. A significant reduction of the test substance concentration was observed after 5 days of incubation for both buffer solutions (mean recovery 75 % of the applied concentration). Thus, it was evident that the buffer solution had no impact on the degradation process and the results of the first pre-test are valid. According to the OECD guideline a main test to determine the reaction rate constant and half-life of the test substance at pH 4 was performed. In order to execute the test at a pH 4 to 7, test solutions were prepared with a concentration of approximately 3 mg/L and 30 mg/L. As buffer systems citric acid and acetate solutions were used, both at pH-values of 5 and 6. The solutions were stored at 20°C ± 1.3°C in the dark over a period of 13 d. In each case a precipitate was observed. As it was not possible to differentiate between hydrolytic degradation and precipitation, which both leads to a reduction in the measured test substance concentration, an evaluation of the obtained data regarding hydrolysis rate constant, half-life or the calculation of Arrhenius equations was not possible. Therefore, it was concluded as not feasible to investigate the hydrolytic properties of test substance at a pH range of 4-6. Under the study conditions, the read across substanace, mono- and di- C16 PSE, K+, was determined to be hydrolytically stable at pH 7 and 9, whereas at pH 4 to 6 a test was not feasible as the test substance partly precipitated in course of incubation (Riefer, 2013). Based on the results of the read across study, similar results are expected for the test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE'.

Description of key information

Based on the available weight of evidence from studies on substances representative of the main constituents, the test substancecan be considered to be hydrolytically stable.

Key value for chemical safety assessment

Additional information

In absence of hydrolysis study with the test substance, the endpoint can be assessed based on studies available for substances representative of the main constituents, which can be categorised as phosphate esters (PSE), ethoxylated phosphate ester (AE PSE) and free ethoxylated alcohol (AE). As, representative studies are not available for the constituent, AE PSE, the endpoint assessment has been based on representative studies available on PSE and AE only, under the assumption that AE PSE is likely to hydrolyse to AE and PSE. The results are presented below:

Constituent: PSE - read across study:

A study was conducted to determine the rate of hydrolysis of read across substance, mono- and di- C16 PSE, K+, (purity: ca. 85%), according to OECD Guideline 111 and EU C.7 Method, in compliance with GLP. The study was performed at different environmentally relevant pH-values by quantifying the test substance concentration after different incubation periods and at different temperatures. In a preliminary test the test substance was dissolved in aqueous solutions buffered at pH 4, 7 and 9 and incubated at 50°C ± 4.5°C for a maximum of 5 d. No significant reduction of the test substance concentration was observed in the samples incubated at pH 7 and 9 after 5 d of incubation (mean recovery > 90% of the applied concentration). In the incubated samples at pH 4 a significant reduction of the test substance concentration was observed after 5 d of incubation in phosphate and citric acid buffer, respectively (mean recovery 63-75% of the applied concentration).To assess the impact of the buffer solution on the degradation process the preliminary test at pH 4 was repeated. Two different buffer solutions were used. To verify the results of the first pre-test an equal phosphate buffer was used, whereas the commercially available citric acid buffer was chosen due to the absence of phosphate molecules which were expected to be a degradation product during hydrolytic cleavage of the test substance. A significant reduction of the test substance concentration was observed after 5 days of incubation for both buffer solutions (mean recovery 75 % of the applied concentration). Thus, it was evident that the buffer solution had no impact on the degradation process and the results of the first pre-test are valid. According to the OECD guideline a main test to determine the reaction rate constant and half-life of the test substance at pH 4 was performed. In order to execute the test at a pH 4 to 7, test solutions were prepared with a concentration of approximately 3 mg/L and 30 mg/L. As buffer systems citric acid and acetate solutions were used, both at pH-values of 5 and 6. The solutions were stored at 20°C ± 1.3°C in the dark over a period of 13 d. In each case a precipitate was observed. As it was not possible to differentiate between hydrolytic degradation and precipitation, which both leads to a reduction in the measured test substance concentration, an evaluation of the obtained data regarding hydrolysis rate constant, half-life or the calculation of Arrhenius equations was not possible. Therefore, it was concluded as not feasible to investigate the hydrolytic properties of test substance at a pH range of 4-6. Under the study conditions, the read across substance, mono- and di- C16 PSE, K+, was determined to be hydrolytically stable at pH 7 and 9, whereas at pH 4 to 6 a test was not feasible as the test substance partly precipitated in course of incubation (Riefer, 2013).Based on the results of the read across study, similar results are expected for the test substance, ‘mono- and di- C16-18 PSE and C16-18 AE10 PSE’.

Constituent AE:

Alcohol ethoxylates are not expected to undergo hydrolysis under normal environmental conditions (pH range 4 to 9). Further, hydrolysis has also been discounted for the alcohols (EO=0 homologues) in the OECD SIDS dossier for long chain alcohols (HERA, 2009). Please refer to the attachment in Section 13 for details.

Overall, based on the available weight of evidence from studies on the main constituents, the test substancecan be considered to be hydrolytically stable.