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STUDY TITLE

DETERMINATION OF HYDROLYSIS RATE 

FOLLOWING OECD 111 GUIDELINE

Test Guidelines

OECD Guideline 111 (2004) 

Author(s)

Study Completion Date

26 May 2011 

Sponsor

The Dow Chemical Company

Midland, Michigan 48674 

Performing Laboratory 

Toxicology & Environmental Research and Consulting 

The Dow Chemical Company 

Midland, Michigan 48674 

Laboratory Project Study ID 



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TABLE OF CONTENTS

Page

STUDY TITLE ....................................................................................................................1

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ..................2

QUALITY ASSURANCE STATEMENT ..........................................................................3

SIGNATURE PAGE ...........................................................................................................4

SUMMARY .........................................................................................................................6

INTRODUCTION ...............................................................................................................7

Purpose ............................................................................................................................7

Test Guidelines................................................................................................................7

Quality Assurance ...........................................................................................................7

Archiving ........................................................................................................................7

Safety...............................................................................................................................7

MATERIALS .......................................................................................................................7

Test Material ...................................................................................................................7

Chemical Name ...............................................................................................................7

Synonyms ........................................................................................................................7

Supplier, City, State (Lot, Reference Number) ...............................................................7

Purity/Characterization (Method of Analysis and Reference) ........................................8

Characteristics .................................................................................................................8

Molecular Formula .......................................................................................................8

Molecular Weight .........................................................................................................8

Chemical Structure .......................................................................................................8

Previous Information .......................................................................................................8

EXPERIMENTAL DESIGN ...............................................................................................9

Analytical Procedures .....................................................................................................9

STUDY SPECIFIC PARAMETERS .................................................................................10

Data and Reporting .......................................................................................................10

STATISTICS .....................................................................................................................10

RESULTS AND DISCUSSION ........................................................................................10

CONCLUSIONS................................................................................................................11

REFERENCES ..................................................................................................................12

TABLES ............................................................................................................................13

Table 1. Analytical Testing Conditions and Settings........................................................13

Table 2. Hydrolysis Results .............................................................14

FIGURE .............................................................................................................................16

Figure 1. Ln Concentration vs Time...............................................16
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SUMMARY

A 1st tier hydrolysis study was conducted for in accordance with the 

test procedure described in the OECD “Hydrolysis as a Function of pH” guideline 

(method 111). Precautions were taken to minimize possible biodegradation, 

photodegradation, and oxidation reactions. Buffered solutions containing 3 μg/L of 

at pH 4, 7, and 9 were incubated at 50 ± 0.5°C for five days in the 

dark. Solutions containing 3 μg/L of at pH 1.2 were incubated at 37 

± 0.5°C for 17 hours in the dark. concentrations in the pH 4, 7 and 9 

test solutions were determined on day 0, 1, and 5 by reverse phase high performance 

liquid chromatography with MS detection (HPLC-MS). 

concentrations in the pH 1.2 test solutions were determined for 0, 1, 2 and 17 hours by 

HPLC-MS.

exhibited increasing hydrolysis rates with increasing pH. No 

detectable degradation of the test substance occurred after 17 hours at pH 1.2 at 37oC. 

After five days at 50°C, degradation of the test substance was less than 7% at pH 4, 38% 

at pH 7, and 100% at pH 9. Corresponding half lives of the test substance were >1 year 

at pH 4, 7.5 days at pH 7, and <1 day at pH 9. Since less than 10% degradation occurred 

after 5 days at pH 4, the test substance is considered hydrolytically stable at pH 4. 
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INTRODUCTION

Purpose

The objective of this study was to investigate the hydrolysis of 

following the guidelines of the Organisation for Economic Co-Operation and 

Development (OECD) method 111 “Hydrolysis as a Function of pH” (OECD, 2004).

Test Guidelines

OECD Organisation for Economic Co-operation and Development, (OECD) 

Guidelines for Testing of Chemicals. Guideline Number 111: 

Hydrolysis as a Function of pH. 13 April 2004. 

Quality Assurance 

The study conduct, data, protocol, protocol changes/revisions, and final report were 

inspected by the Quality Assurance Unit, Toxicology & Environmental Research and 

Consulting, The Dow Chemical Company, Midland, Michigan. 

Archiving

The data, protocol, protocol changes/revisions, and final report were archived by the 

Toxicology & Environmental Research and Consulting archivist and stored at The Dow 

Chemical Company, Midland, Michigan. 

Safety

All personnel involved in the study were advised of the safety precautions to follow when 

handling the test material. Chemical safety information was made available.

MATERIALS

Test Material 

 

Chemical Name

Synonyms 

Supplier, City, State (Lot, Reference Number) 

The Dow Chemical Company, Midland, Michigan (Lot# Batch 
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Purity/Characterization (Method of Analysis and Reference)

The purity of the test material was determined to be by gas 

chromatography with identification by Fourier transform infrared spectroscopy and gas 

chromatography mass spectrometry

Characteristics

Molecular Formula

Molecular Weight

Previous Information 

Hydrolysis can be an important abiotic transformation process for chemicals that enter 

the aqueous environment. A tiered testing approach is recommended by the OECD to 

assess the hydrolytic fate of a compound in water. A preliminary 1st tier study is usually 

conducted at 50°C to determine the susceptibility of the test compound to hydrolysis at 

pH 4, 7, and 9. Examination of hydrolysis at pH 1.2 may also be included if a 

physiologically relevant pH is required to support toxicology studies. If the compound is 

shown to be hydrolytically unstable in the preliminary study, a more extensive 
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investigation (2nd tier) may be carried out at different temperatures to better define the 

hydrolysis kinetics for the compound and identify hydrolytic degradation products. 

To better understand the hydrolysis of Tier 1 hydrolysis studies were 

conducted at 50°C over five days in buffered solutions at pH 4, 7, and 9, and at 37°C for 

17 hours in pH 1.2. 

EXPERIMENTAL DESIGN

Hydrolysis studies were conducted following the guideline established by the OECD 

method 111 (OECD, 2004). Buffered solutions (0.05M) were prepared at pH 1.2 (HCl), 

pH 4 (phosphoric acid), pH 7 (monopotassium phosphate), and pH 9 (boric acid). Final 

pH adjustments were made using either 1N sodium hydroxide or 1N hydrochloric acid 

(±0.2 pH units). To reduce the potential for biodegradation on the test compound, the 

buffered solutions were filter-sterilized through a 0.45-μm nylon membrane (Nalge 

Company, Rochester, New York). Glassware was sterilized by autoclaving as 

appropriate. The test compound was added to 10-mL portions of buffered solutions in 

20-mL serum vials at a nominal concentration of 3 μg/L. To minimize possible oxidation 

reactions, test solutions were sparged with argon to reduce the dissolved oxygen 

concentrations in the reaction mixtures and oxygen concentrations in the headspace of the 

serum vials. The serum vials were crimp capped, and labeled with a unique code for 

identification. The test solutions were prepared in duplicate. The pH 1.2 solutions were 

placed in a 37°C incubator and the pH 4, 7 and 9 samples were placed in a 50°C water 

bath (temperature control 0.5±°C). The test solutions were incubated in the dark to 

minimize possible photochemical reactions. 

Analytical Procedures

The concentration of the test compound dissolved in an aqueous solution was determined 

using reverse phase high performance liquid chromatography (HPLC) with mass 

spectrometry (MS) detection. An Agilent 1110 series HPLC was used with the gradient 

program summarized in Table 1. 

The separation of the test substance from possible hydrolysis products was obtained on a 

Agilent XDB C18 column (4.6 x 50 mm 1.8um). eluted from the 

column with an approximate retention time of 7.6 minutes. An analytical stock solution 

of was prepared in acetonitrile and a series of dilutions were 

performed in water to achieve the target concentration of 3 μg/L. 
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Periodic measurements of the water bath temperature were made with a mercury 

thermometer to confirm proper temperature of the water bath. 

STUDY SPECIFIC PARAMETERS

Data and Reporting 

The concentration of the test substance in the pH 4, 7 and 9 test samples was measured at 

each sampling point. Hydrolysis half-lives for the test substance were determined using 

the calculations described in the OECD method 111 guideline. For hydrolysis reactions 

that follow pseudo-first order kinetics, a plot of the natural logarithm of the concentration 

versus time results in a straight line (Figure 1). The slope of the line equals –k, the 

pseudo-first order rate constant. The half-life for the hydrolysis reaction is equal to 

t1/2 = ln 2/k. 

The percent degradation was calculated for the pH 1.2 samples at each sampling point to 

verify that is stable in low pH solutions. 

STATISTICS

Descriptive statistics (mean, standard deviation) were used where applicable in this study.

RESULTS AND DISCUSSION

The results for the OECD 111 hydrolysis study for are summarized 

in Table 2. No hydrolysis of was noted at pH 1.2 but hydrolysis rates increased 

with increasing pH. 

At pH 1.2 there was no loss of the test material from solution over 17 hrs indicating that 

was hydrolytically stable over this time period. At pH 4 minimal (~7%) loss of 

from solution was observed also indicating hydrolytic stability at this pH range). 

Aqueous reaction mixtures poised at pH 7 and pH 9 samples exhibited significant loss of 

over the five days. At pH 7 and 9 the concentration of decreased by 38% 

and 100% resulting in a half lives of 7.5 and less than 1 days respectively. 

The water bath was maintained at 50oC ± 0.0 oC (n=3) during the study based on 

measurements at the time of sampling.
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CONCLUSIONS 

A hydrolysis study was conducted for in accordance with the test 

procedure described in the OECD “Hydrolysis as a Function of pH” guideline (method 

111). The showed increasing hydrolysis rates with increasing pH. 

Percent degradation of the test substance after five days at 50oC was 7% at pH 4, 38% at 

pH 7, and 100% at pH 9. Corresponding half lives of the test substance were >1 year at 

pH 4, 7.5 days at pH 7, and <1 day at pH 9. No detectable degradation of the test 

substance occurred after 17 hours at pH 1.2 at 37oC. 
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REFERENCES

OECD (2004). Organisation for Economic Co-operation and Development, (OECD) 

Guidelines for the Testing of Chemicals. Test Guideline 111: Hydrolysis as a 

Function of pH. Adopted 13 April 2004. 

 
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 DETERMINATION OF HYDROLYSIS RATE FOLLOWING OECD 111 

GUIDELINE

Table 1. Analytical Testing Conditions and Settings

Instrument Components:

Pump: Agilent 1100 Series; G1312A; S/N DE43618194 

Autosampler: Agilent 1200 Series; G1367B; S/N DE60555149 

MS Detector: Agilent 1100 MSD; G1946D; S/N US23412287 

Column: Agilent XBD C18; 4.6mm x 50mm, 1.8μm 

Analytical Conditions: 

HPLC Eluent (A) = Milli-Q water + 0.1% formic acid

HPLC Eluent (B) = Acetonitrile + 0.1% formic acid

Time (minutes) % Eluent A % Eluent B

0 60 40 

3 60 40 

6 5 95 

10 5 95 

12 60 40 

16 60 40 

Flow = 1.0 mL/min 

Detection: MS SIM 359 ion

Retention Time: Approximately 7.6 minutes

Injection Volume: 50 μL
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 DETERMINATION OF HYDROLYSIS RATE FOLLOWING OECD 111 

GUIDELINE

Table 2. Hydrolysis Results

 a

 ± 1 standard deviation 

 b natural logarithm

pH 7

Day Calc Conc (ppb) % Degradation ln (conc)

0 3.23 ± 0.00 - 1.17

1 2.85 ± 0.07 12% 1.05

5 2.01 ± 0.23 38% 0.70 

 pH 7 Half Life = 7.5 Days

pH 1.2

Hour % Degradation

1 0 

2 0 

17 0 

pH 4

Day

Concentration

(μg/L)

% Degradation lnb

(concentration) 

0 3.23 ± 0.00a – 1.17

1 3.41 ± 0.21 None 1.23

5 3.01 ± 0.02 7% 1.10
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 DETERMINATION OF HYDROLYSIS RATE FOLLOWING OECD 111 

GUIDELINE

Table 2. Hydrolysis Results (continued) 

 pH 9 Half Life = < 1 Day

pH 9

Day Calc Conc (ppb) % Degradation ln (conc)

0 3.23 ± 0.00 - 1.17

1 0.00 ± 0.0 100% NA

5 0.00 ± 0.0 100% NA
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 DETERMINATION OF HYDROLYSIS RATE FOLLOWING OECD 111 

GUIDELINE

Figure 1. Ln Concentration vs Time

 

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0 1 2 3 4 5 6

Ln Concentration

Time (Days)

pH 4

pH 7

pH 9

Linear (pH 4)

Linear (pH 7)

Linear (pH 9)