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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 11, 2003- October 29, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to U.S. EPA OPPTS 835.2110 and OECD 111, and in compliance with GLP standards.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.2110 (Hydrolysis as a Function of pH)
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
GLP compliance:
yes
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Sampling intervals for the parent/transformation products: In the preliminary test samples were taken on days 0 and5. In the definitive test, samples were taken at six sampling intervals over a period of 30 days (samples taken at days 0, 2, 3, 10,17 and 30).
- Sampling method: In the preliminary test, triplicate sub-samples of each fortified pH buffer were prepared for analysis on Days 0 and 5. For Day 5, each fortified pH buffer solution was used to overfill three separate 20-mL scintillation vials. These subsamples were placed in an incubator set at 50 ºC for a period of five days. On Day 0, three additional 5-mL aliquots from each fortified pH buffer solution were taken for immediate DBS analysis. Following five days, the scintillation vials were removed from the incubator, a 5-mL aliquot was removed from each sub-sample and the aliquots subsequently analyzed for each constituent of DBS. Sampling for the definitive test was performed in a similar manner. Larger volumes were prepared to accommodate at least six sampling intervals over a period of approximately 30 days. Triplicate aliquots were taken from each pH buffer solution at each sampling interval to allow for a single replicate to be analyzed at 15°C and two to be analyzed at 25°C. Approximately 20 mL of each buffer solution was transferred to a separate scintillation vial at each sampling interval. A 5-mL aliquot was removed from each sub-sample, and subsequently analyzed for each constituent of DBS. pH measurements were made on all sub-samples on each sampling day.
- Sampling intervals/times for pH measurements: pH measurements were taken on day 0 and 5 in the preliminary test and in the definitive test pH measurements were taken on sampling days in all sub-samples.
Buffers:
The study was conducted using sterile aqueous solutions buffered at pH 4, 7 and 9 (± 0.1 pH units). Phthalate (pH 4), phosphate (pH 7) and borate (pH 9) buffers were used. Aqueous buffer solutions were prepared using appropriate aqueous solutions of salts, HCl and NaOH, prepared with HPLC grade water, as follows (definitive trial):

pH 4 - 10.2 g of potassium hydrogen phthalate, 4.00 mL of 0.1 N NaOH, or 0.1 N HCl and approximately 986 mL of water.
pH 7 - 6.81 g of potassium dihydrogen phosphate, 295 mL of 0.1 N NaOH and approximately 700 mL of water.
pH 9 - 3.73 g of potassium chloride, 3.09 g of boric acid, 214 mL of 0.1 N NaOH and approximately 780 mL of water.

The pH of each buffer solution was measured using a pH meter with an accuracy of 0.01 pH unit prior to use. The resulting pH-adjusted buffer solutions were vacuum filtered through 0.2-μm filters for sterilization. Buffer solutions were additionally degassed with nitrogen for approximately five minutes to reduce dissolved oxygen concentrations.
Details on test conditions:
Hydrolysis of DBS in aqueous buffered media (pH 4, 7 and 9) was investigated at a concentration of 1.00 mg/L. The selected concentration was less than one-half of the water solubility of the dominant (85%) dibromostyrene constituent of the test substance (6.91 ± 0.0871 mg DBS/L)(1). A preliminary test was conducted with fortified samples in pH 4, 7 and 9 buffer solutions over a five-day period at 50 ± 1°C. The definitive test was conducted with fortified samples in pH 4, 7 and 9 buffer solutions maintained at 15 and 25°C. Samples were subjected to seven sampling intervals over a 30-day period. Samples incubated at 15C were collected on Day 0. Thereafter samples were collected on Days 2, 3, 10, 25, and 30. Samples incubated at 25°C were collected on Days 0, 2, 3, 10, 17, and 30. Duplicate samples were analyzed at each interval for the 25ºC test, while single samples were analyzed from the 15ºC test. Each of the constituents of DBS (mono-, di- and tribromostyrene) were analyzed to determine the test substance concentration in the samples.

TEST SYSTEM
- Sterilisation method: The pH-adjusted buffer solutions were vacuum filtered through 0.2-μm filters for sterilization.
TEST MEDIUM
- Volume used/treatment 500 mL
- Kind and purity of water: Water, Burdick and Jackson, HPLC grade [(equivalent to ASTM Type II Designation D1193-91]
- Preparation of test medium: The study was conducted using sterile aqueous solutions buffered at pH 4, 7 and 9 (± 0.1 pH units). Phthalate (pH 4), phosphate (pH 7) and borate (pH 9) buffers were used. Aqueous buffer solutions were prepared using appropriate aqueous solutions of salts, HCl and NaOH, prepared with HPLC grade water, as follows (definitive trial):
pH 4 10.2 g of potassium hydrogen phthalate, 4.00 mL of 0.1 N NaOH, or 0.1 N HCl and approximately 986 mL of water.
pH 7 6.81 g of potassium dihydrogen phosphate, 295 mL of 0.1 N NaOH and approximately 700 mL of water.
pH 9 3.73 g of potassium chloride, 3.09 g of boric acid, 214 mL of 0.1 N NaOH and approximately 780 mL of water.
Preliminary test samples were prepared at a nominal concentration of 1.00 mg DBS/L by fortifying 100 mL of each pH buffer solution with 100 μL of the primary (1.00 mg DBS/mL) DBS stock solution in ACN. Triplicate sub-samples of each fortified pH buffer were prepared for analysis on Days 0 and 5. For Day 5, each fortified pH buffer solution was used to overfill three separate 20-mL scintillation vials. These subsamples were placed in an incubator set at 50 ºC for a period of five days.
Definitive test samples in pH 4, 7, and 9 buffer solutions were prepared in the same manner with the exception of use of a larger fortification volume (500 μL) and test solution volume (500 mL). Larger volumes were prepared to accommodate at least six sampling intervals over a period of approximately 30 days.
- Identity and concentration of co-solvent: Acetonitrile, Burdick and Jackson, HPLC grade. A primary stock solution of the test material was prepared volumetrically in acetonitrile (ACN) by accurately transferring 56.0 µL of the test substance into a 100-mL volumetric flask using a gas-tight syringe. The flask was then brought to volume with ACN. Based on the reported specific gravity of 1.8, the resulting stock concentration was 1.00 mg DBS/mL. This primary stock solution was used to prepare hydrolysis test solutions. Aliquots of the stock solution were subsequently diluted with ACN to prepare a 0.100-mg DBS/mL secondary stock solution. The secondary stock solution was used to fortify buffered reagent grade water that was subsequently analyzed to assess method efficiency.
Buffer solutions were additionally degassed with nitrogen for approximately five minutes to reduce dissolved oxygen concentrations.
OTHER TEST CONDITIONS
- Dissolved oxygen: Buffer solutions were additionally degassed with nitrogen for approximately five minutes to reduce dissolved oxygen concentrations
Duration:
30 d
pH:
4
Temp.:
15 °C
Initial conc. measured:
1 mg/L
Duration:
30 d
pH:
7
Temp.:
15 °C
Initial conc. measured:
1 mg/L
Duration:
30 d
pH:
9
Temp.:
15 °C
Initial conc. measured:
1 mg/L
Duration:
30 d
pH:
4
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:
Single sample at 15 °C, duplicate samples at 25 °C
Positive controls:
yes
Remarks:
matrix fortification samples were prepared and analyzed on each sampling day to assess method efficiency (see additional information below)
Statistical methods:
DEGRADATION RATE:
The rate of hydrolysis was quantitated by measuring the decrease in concentration versus time. The hydrolysis rate constant was calculated assuming pseudo first order kinetics for each system tested when degradation was observed. Linear regression analysis was performed using the following equation:
ln(Ct) = -kt + ln(C0)
Where k = degradation rate constant
Ct = concentration at time t (mean of replicates if applicable) expressed as percent of the applied concentration remaining
t = time
C0 = initial concentration (t = 0)

The half life was calculated as follows:
t1/2 = degradation half-life
k = degradation rate constant

ARRHENIUS EQUATION AND RATE CONSTANT EXTRAPOLATIONS
For the test substance requiring testing at multiple a multiple elevated temperatures greater than 25 °C, the hydrolysis rate at 25 °C will be determined by extrapolation using the Arrhenius equation.
k = A exp(-Ea/RT)
or
ln(k) = ln(A)-Ea/RT
Where: k is the degradation rate constant (L/mole•second) at temperature T
A is a pre-exponential factor assumed to be independent of temperature
Ea is the activation energy (kJ/mole or J/mole)
T is the temperature at which the rate constant was determined (°K)

The rate constant at 25 °C will be determined from the linear regression equation of the natural logarithm of the rate constants determined at elevated temperatures versus the temperatures at which they were determined. The activation energy and pre-exponential factor will also be calculated and reported.
Preliminary study:
All pH measurements for preliminary test sub-samples were within ±0.1 pH unit of the target pH. The incubator temperature was maintained at 50 ± 1 °C over the five-day period.
Triplicate sub-samples prepared in pH 4 buffer yielded mean measured concentrations on Day 0 of 0.980, 0.870 and 0.960 mg DBS/L using the dibromo-, monobromo-, and tribromostyrene as surrogates for the test substance, respectivelyThe measured values corresponded to 98.0, 87.0 and 96.0%, respectively, of the nominal DBS concentration.
Triplicate sub-samples prepared in pH 7 buffer yielded mean measured concentrations on Day 0 of 0.956, 0.845 and 0.923 mg DBS/L using the dibromo-, monobromo-, and tribromostyrene as surrogates for the test substance, respectively. The measured values corresponded to 95.6, 84.5 and 92.3%, respectively, of the nominal DBS concentration. Triplicate sub-samples prepared in pH 9 buffer yielded mean measured concentrations on Day 0 of 0.883, 0.849 and 0.892 mg DBS/L using the dibromo-, monobromo-, and tribromostyrene as surrogates for the test substance, respectively. The measured values corresponded to 88.3, 84.9 and 89.2%, respectively, of the nominal DBS concentration. In summary, triplicate sub-samples prepared from each fortified pH buffer solution were analyzed for DBS concentrations immediately and following five days incubation at 50 ± 1°C. Significant degradation of DBS was observed at all three pHs at the elevated temperature. Day 5 measured concentrations were less than 0.200 mg DBS/L, equivalent to less than 20% of the initial DBS concentration. Since the mean Day 5 concentrations were significantly less than 90% of the Day 0 concentration, the hydrolytic stability of DBS under acidic, neutral, and basic conditions was further evaluated in the definitive test.
Transformation products:
not measured
% Recovery:
91.2
pH:
4
Temp.:
15 °C
Duration:
30 d
% Recovery:
79.8
pH:
7
Temp.:
15 °C
Duration:
30 d
% Recovery:
90.5
pH:
9
Temp.:
15 °C
Duration:
30 d
% Recovery:
73.7
pH:
4
Temp.:
25 °C
Duration:
30 d
% Recovery:
67.1
pH:
7
Temp.:
25 °C
Duration:
30 d
% Recovery:
64.2
pH:
9
Temp.:
25 °C
Duration:
30 d
pH:
4
Temp.:
15 °C
Remarks on result:
other: no hydrolysis at these conditions
pH:
7
Temp.:
15 °C
Remarks on result:
other: no hydrolysis at these conditions
pH:
9
Temp.:
15 °C
Hydrolysis rate constant:
0 d-1
DT50:
177 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Hydrolysis rate constant = 0.00393 /d
pH:
4
Temp.:
25 °C
Hydrolysis rate constant:
0.01 d-1
DT50:
50.2 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Hydrolysis rate constant = 0.0138 /d
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.01 d-1
DT50:
58.5 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Hydrolysis rate constant = 0.0118 /d
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.01 d-1
DT50:
52 d
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: Hydrolysis rate constant = 0.0133 /d
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: No
- Anomalies or problems encountered (if yes): Incubator temperatures were maintained at ± 1 °C of the desired test temperatures for the 30-day test with the exception of a 12-hour period for the 15°C incubator (incubator malfunction).

Definitive Test

The hydrolytic stability of DBS in the definitive test was evaluated at pH 4 (phthalate buffer), pH 7 (phosphate buffer) and pH 9 (borate buffer) at two temperatures, 15 and 25°C. 500 mL of each pH buffer solution were fortified on Day 0 at a nominal concentration of 1.00 mg DBS/L. Two sub-samples from each pH buffer solution were taken for immediate analysis. At each sampling interval, a single subsample from those incubated at 15°C and duplicate samples from those incubated at 25°C were removed and analyzed. For each pH level, at least six sampling intervals were collected for each temperature with the test duration extending 30 days. All pH measurements for definitive test sub-samples were within ±0.1 pH unit of the target pH. Incubator temperatures were maintained at ± 1 °C of the desired test temperatures for the 30- day test with the exception of a 12-hour period for the 15°C incubator (incubator malfunction).

The following criteria were applied to evaluate the hydrolytic activity of DBS in the definitive test. For a given experimental condition and monitored constituent - if the Day 30 measured concentration was equal to or greater than the concentration measured at test initiation (t0), the test substance was considered hydrolytically stable. Linear regression was used to fit the plot of ln% of the concentration at t0 versus the concentration at the sampling interval (Day) to determine the degradation rate constant and degradation half-life. If the statistical P-value for the slope (-k) was <0.05 and the confidence limits of the slope did not exceed zero, the calculated rate constant (and half-life) was considered statistically valid. If either condition was not met, the test substance was considered hydrolytically stable. DBS was determined to be hydrolytically stable at 15°C when monitoring DBS with the monobromostyrene constituent at pH 4, 7 and 9. Based on the dibromostyrene constituent, DBS was determined to be hydrolytically stable at pH 4 and 9, but was marginally hydrolytically unstable at pH 9 (t1/2 = 177 days). Based on the tribromostyrene constituent, DBS was determined to be hydrolytically stable at pH 7, but was unstable at pH 4 (t1/2 = 46 days) and pH 9 (t1/2 = 149 days).

At 25°C, DBS degraded at all pH levels. Dibromo- and tribromostyrene showed slightly greater stability at pH 7 than at pH 4 or pH 9. The stability of monobromostyrene showed little, if any, pH dependence. Halflives based on monobromo-, dibromo- and tribromostyrene ranged from 39-43, 50-59, and 37-49 days, respectively. Half-lives based on pH ranged from 37-50 (pH 4), 39-59 (pH 7) and 40-52 (pH 9) days.

Table 1: Summary ofRate Constants and Half-Lives for DBS as a Function of Temperature, pH, and Monitored Constituent

 

pH

Monitored constituent

Temperature

15 °C

25 °C

k

t1/2

k

t1/2

4

Dibromostyrene

-

-

0.0138

50.2

Monobromostyrene

-

-

0.0163

42.6

Tribromostyrene

0.0152

45.5

0.0186

37.4

7

Dibromostyrene

-

-

0.0118

58.5

Monobromostyrene

-

-

0.0177

39.2

Tribromostyrene

-

-

0.0141

49.2

9

Dibromostyrene

0.00393

177

0.0133

52

Monobromostyrene

-

-

0.0175

39.6

Tribromostyrene

0.00464

149

0.0149

46.6

Validity criteria fulfilled:
not specified
Remarks:
OECD 111 Quality Criteria require that rate constants for one pH and one temp are determined in duplicate with a deviation <2.5%. This was not discussed in the study; appears to have been met for 25 °C (dup. samples) but not at 15 °C (single samples).
Conclusions:
The hydrolytic stability of DBS in pH 4, 7 and 9 buffer solutions was evaluated in a preliminary test at approximately 50 °C over a five-day period. Using the dibromostyrene major and the monobromo- and tribromostyrene minor constituents of the test substance as surrogates, significant degradation of DBS was observed at all three pHs at the elevated temperature. A 30-day trial was therefore conducted in pH 4, 7 and 9 buffer solutions at temperatures of 15 and 25°C. DBS was determined to be hydrolytically stable at 15°C when monitoring DBS with the monobromostyrene constituent at pH 4, 7 and 9. Based on the dibromostyrene constituent, DBS was determined to be hydrolytically stable at pH 4 and 9, but was marginally hydrolytically unstable at pH 9 (t1/2 = 177 days). Based on the tribromostyrene constituent, DBS was determined to be hydrolytically stable at pH 7, but was unstable at pH 4 (t1/2 = 46 days) and pH 9 (t1/2 = 149 days). At 25°C, DBS degraded at all pH levels. Half-lives based on monobromo-, dibromo- and tribromostyrene ranged from 39-43, 50-59, and 37-49 days, respectively. Half-lives based on pH ranged from 37-50 (pH 4), 39-59 (pH 7) and 40-52 (pH 9) days.

Description of key information

After preliminary test, a 30-day trial was conducted in pH 4, 7 and 9 buffer solutions at temperatures of 15 and 25°C. DBS was determined to be hydrolytically stable at 15°C when monitoring DBS with the monobromostyrene constituent at pH 4, 7 and 9. Based on the dibromostyrene constituent, DBS was determined to be hydrolytically stable at pH 4 and 9, but was marginally hydrolytically unstable at pH 9 (t1/2 = 177 days). Based on the tribromostyrene constituent, DBS was determined to be hydrolytically stable at pH 7, but was unstable at pH 4 (t1/2 = 46 days) and pH 9 (t1/2 = 149 days). At 25°C, DBS degraded at all pH levels. Half-lives based on monobromo-, dibromo- and tribromostyrene ranged from 39-43, 50-59, and 37-49 days, respectively. Half-lives based on pH ranged from 37-50 (pH 4), 39-59 (pH 7) and 40-52 (pH 9) days.

Key value for chemical safety assessment

Additional information

The key study, Raymond L. Van Hoven, Willard B. Nixon (2003) was considered adequate and reliable for assessment to fulfil the data requirement. The study was conducted to OECD 211 and in compliance with GLP, accordingly the study was assigned a reliability score of 1.

Further details on results:

 

Dissipation half-life of parent compound

pH

Temp.

Hydrolysis rate constant

Half-life

Type

Remarks (e.g. regression equation, r², DT90)

4

15 °C

no hydrolysis at these conditions

7

15 °C

no hydrolysis at these conditions

9

15 °C

0 d-1

177 d

(pseudo-)first order (= DT50)

Hydrolysis rate constant = 0.00393 /d

4

25 °C

0.01 d-1

50.2 d

(pseudo-)first order (= DT50)

Hydrolysis rate constant = 0.0138 /d

7

25 °C

0.01 d-1

58.5 d

(pseudo-)first order (= DT50)

Hydrolysis rate constant = 0.0118 /d

9

25 °C

0.01 d-1

52 d

(pseudo-)first order (= DT50)

Hydrolysis rate constant = 0.0133 /d

 

Total recovery of test substance (in %)

%Recovery

pH

Temp.

Duration

91.2

4

15 °C

30 d

79.8

7

15 °C

30 d

90.5

9

15 °C

30 d

73.7

4

25 °C

30 d

67.1

7

25 °C

30 d

64.2

9

25 °C

30 d