OECD 111: Hydrolysis as a Function of pH

The purpose of the test is to determine the rate of hydrolysis of a chemical in water at different environmentally relevant pH-values by quantifying the test substance concentration after different incubation periods and at different temperatures. The rate of hydrolysis of the test sustance as a function of pH and the transformation product pattern will be investigated.

Study Design

Test setup

Non-labelled or 14C-labelled test substances can be used to measure the rate of hydrolysis. Incubation in sterilised (buffered) aqueous solution will be accomplished at different pH values (4-9) and at different temperatures in the dark. The test will be conducted using multiple flask design. Stoppered glass flasks will be used for carrying out the tests. All glassware and water will be sterilised before use. A temperature controlled water bath will be used for the incubation of the samples.

Course of the test

One test substance concentration will be used for the test. In a preliminary test the hydrolytic behaviour of the substance will be estimated. The test substance will be incubated at 50°C at three different pH values. If less than 10% hydrolysis will be observed after 5 days of incubation no additional testing is required. At those pH values were the test substance will be found to be unstable, the test will be continued and a main test is carried out. Besides 50°C, at least two further temperatures in the range of <25°C to 70°C will be tested. Each test will be executed until 90% hydrolysis of the test item will be observed or for 30 days whichever will come first. Apart from samples taken directly after application, at least 6 sampling time points will be included. Time intervals will be chosen in such a way that the pattern of decline of the test substance can be established. In case of the use of radiolabelled test substances mass balancing and transformation product pattern will be determined for the test substance.

Analysis of the test substance and transformation products

In case of the 14C-labelled test substance the analytical methods will base on LSC and HPLC coupled with UV and radio detection. LC-MS/MS can be carried out as additional analytical method. Non-labelled test substances will be determined using HPLC coupled with UV detection or LC-MS/MS method. The analytical method and sample preparation will be established to allow a limit of quantification of ≤ 5% of the applied radioactivity.

Special test setup

  • The test can also be performed at pH values other than 4, 7 and 9, if required.
  • The test can also be performed at lower temperatures (e.g. 12°C).


  • Calculation of DT50 values
  • Hydrolysis rate constant as a function of temperature, if possible
  • Transformation product pattern including characterisation/ identification of major products

Guidelines and Literature

  • OECD Guideline for Testing of Chemicals, No. 111, "Hydrolysis as a function of pH", adopted April 13, 2004.