United States Environmental Protection Agency Environmental Research Laboratory Athens GA 30613 Research and Development EPA/600/S3-88/014 Sept. 1988 v°/EPA Project Summary Interim Protocol for Measuring Hydrolysis Rate Constants in Aqueous Solutions J. Jackson Ellington, Frank E. Stancil, Jr., William D. Payne, and Cheryl D. Trusty A detailed protocol was developed to measure first- and second-order hydrolysis rate constants for organic chemicals for use in predicting persis- tence In aquatic systems. The protocol delineates theoretical considerations, laboratory experiments, and calculation procedures. Repetitive application of the protocol to measure hydrolysis rate con- stants for four standard reference com- pounds over a period of 2 years yielded coefficients of variation of less than 12% in the measurements. This Project Summary was developed by EPA's Environmental Research Laboratory, Athens, GA, to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report order- ing information at back). Introduction Under the Toxic Substances Control Act (PL 94-469) of 1976 Office of Toxic Substances (OTS) screens new chemicals proposed for manufacture and reviews the safety of existing chemicals already on the market. To assess potential risk to human health and the environment, OTS must evaluate both effects and exposure poten- tial. Transport and transformation characteristics in ambient environments are major considerations in assessing poten- tial exposure. Essential to transport and transformation assessments are physical and chemical data that permit estimation of chemical fate either by use of mathematical models or other techniques. To obtain necessary data, OTS either re- quests information from manufacturers or estimates values by comparing the subject chemical to analogous chemicals whose properties are known. In either case, reliable data are necessary. A major transformation process for many chemicals is chemical hydrolysis; therefore, measurements of hydrolysis rate constants are often required. In the measurement of hydrolysis rate constants, some means are needed to en- sure that the measurements are reliable and reproducible. Suggested laboratory protocols for measuring hydrolysis as a function of pH and temperature have been published; however, these previously published protocols fail to specify some of the step-wise procedures in sufficient detail to reproducibility of measurements made by different investigators. This report, on the other hand, provides specific guidance in a protocol for deriving hydrolysis rate con- stants for use in mathematical models to predict the fate of chemicals in aquatic systems. The protocol has evolved over the past several years at the Environmental Research Laboratory, Athens, GA, and has been found to provide reproducible rate constants as a function of pH and temperature. Kinetics of Hydrolysis Hydrolysis Kinetics The importance of hydrolysis as a transformation process for chemicals in water can be determined from data on rate constants and half-lives coupled with data describing environmental conditions. Hydrolysis of organic compounds refers to reaction of the compound with water in which bonds are broken and new bonds with HO- and H- are formed. A typical ex- ------- ample is the reaction of an alkyl halide with water resulting in the formation of halide ion (X-): RX + HOH-*-ROH + HX (or H + , X") (1) The rate of hydrolysis may be promoted by the hydronium ion (H + , H3O+) or the hydroxyl ion (OH"). The former is referred to as specific acid catalysis and the latter as base mediated hydrolysis. These two processes together with the pH indepen- dent reaction with water are the only mechanisms considered in this protocol. The H3O+ activity is measured directly and the OH" activity is calculated from ac- curate determination (calibration between secondary buffers) of solution pH. Some chemicals undergo an elimination reaction: (2) In this protocol, only the disappearance of substrate is monitored with no attempts to identify mechanisms or reaction products. Reactions represented by equations 1 and 2 are included in a broad definition of hydrolysis. Rate Laws In both processes referred to in the discussion of hydrolysis mechanisms, the rate of disappearance of the organic com- pound is given by the equation, (3) kB[OH1[C] + kN'[H20][C] where [C] is the concentration of organic and kh is the observed pseudo-first-order rate constant at a specific pH and temperature; kA and kB are second-order rate constants; and kN' is the neutral hydrolysis rate constant for the acid, base and neutral promoted processes, respec- tively. The water concentration, because of the large excess, does not change during the reaction, thus kN'[H2O] is a constant (kN). Equation 3 assumes each individual rate process is first-order in substrate, thus kh can be defined as: kh = kA[H + ] + kB[OHl + kN (4) Using the autoprotolysis equilibrium expression Kw = [H+][OHT (5) equation 4 may be rewritten as kh =» kA[H+] +*&"- + kN Equation 6 shows the dependence of kh on hydronium ion concentration (pH) and on the relative values of kA kB, and kN. When the disappearance rate constants are determined at pHs 3, 7, and 11, the second-order rate constants for acid hydrolysis and for base hydrolysis can be calculated by dividing the pseudo-first-order rate constant obtained at the appropriate pH by the hydronium ion or hydroxyl ion ac- tivity, respectively. The neutral contribution is determined by solving equation 4 for kN and substituting the observed rate (kh) at pH 7 together with values for KA and kg. The half-life of a compound at a given pH and temperature can be calculated from equation 7, where kh is the observed rate at the given pH and temperature and 0.693 where [C0] equals concen- tration at time zero and [Ct] equals con- centration at 50% reaction. tl/2 0.693 kh (7) Standard Reference Compounds Chemical standards of known concen- tration have long been used for assuring reliability of quantitative chemical analyses, calibrating instruments, and measuring recoveries of analytes from various matrices. Analogous to using chemicals of known concentration as standards for con- centration measurement, chemicals whose hydrolysis constants have been measured with established precision by one experi- menter or group can be used as standard reference compounds (SRCs) by other ex- perimenters in establishing and maintain- ing quality control in rate measurements. Precise measurement of established hydrolysis rate constants for SRCs in- terspersed with other rate constant measurements will help assure reliability and comparability of the measured constants. Standard reference compounds are used as quality assurance standards and as references in inter-laboratory generation of hydrolysis data. Repetition of rate constant measurements in our laboratory for these compounds over the course of 2 years has established baseline information for evaluating experimental techniques and for all aspects of quality assurance. Four com- pounds were selected, one each for acid and neutral hydrolysis, and two for basic hydrolysis (Table 1). Reproduction of the hydrolysis rate con- stants of the SRCs at the established con- centrations, pHs, and temperatures en- sured that the experimental conditions were reproducible and helped evaluate the ac- curacy and precision of measurements for other compounds. Pseudo-first-order hydrolysis rate constants for all SRCs at various temperatures and pHs and second- order rate constants for the acidic and basic reference compound were established from these determinations. Hydrolysis Experiments Screening Test (Level I) Laboratory experiments should be divided into three levels. In Level I experiments, the hydrolysis protocol is tested in the laboratory by selecting and determining the hydrolysis rate con- stants established for the SRCs. The complete data generated in hydrolysis experiments on the SRCs are converted to rate constants using the computation techniques discussed in the "Data and Reporting" section and the appendices of the complete text. The SRC rate constant measurements are repeated until the desired precision is attained in all phases of rate constant measurement. Screening Test (Level II) Level II experiments are screening tests to determine the approximate half-life and dependence of hydrolysis on pH of chemicals of interest at pHs 3, 7, and 11 at a selected temperature. Results from Level II experiments are then used to set pH and temperature for Level III experiments. Level II experiments are intended to quantify the effects of temperature and pH on the hydrolysis rate of the chemical of interest. Buffer solutions at pHs 3, 7, and 11 are prepared by following instructions in the section entitled "Buffers." For each chemical, reaction mixtures should be prepared in each of the three buffer solu- tions without the use of heat. The chemical concentration should be less than one-half its water solubility and at less than 1E-5M (see section on "Test Chemical Solution"). The test chemical solution is transferred to sealed ampules or test tubes with Teflon- lined screw caps (15-ml tubes, 10-ml solu- tion). A minimum of six tubes are prepared at each pH. Then based on a "best guess" half-life, the tubes are placed in either a 25, 45, 65, or 85°C constant temperature bath. After sufficient time to equilibrate (30 to 60 ------- Tabte 1. Standard Reference Compounds Name DL-trans-4-chlorostilbene oxide (CSO) Benzyl chloride Methyl-2, 4-dichlorophenoxy acetate (2,4-DME) LJndane pH Range Ea(kJ/mol) In A ,. — j 2-5 84.7 ± 13.2 37.1 ± 5.30 Neutral 84.1 ±5.8 45.3 ± 2.21 8-9.5 40.1 ± 4.9 22.7 ± 1.91 9.5- 11 65.3 ± 1.9 27.5 ± 0.75 mm), one sample is taken to determine the time zero concentration. The screening test decision tree (Figure 1) is then used to determine sampling times for subsequent tubes. Data are calculated according to pro- cedures given in the "Data and Reporting" section of the complete text. Detailed Tests (Level III) The objective of this set of experiments is to determine kA, kN, and kB (if all three processes are operative) at two or more temperatures (separated by 20°C or more) such that activation energies for each pro- cess can be calculated and used to predict hydrolysis rate constants at other tempera- tures and pHs. The preliminary values of kA, kN, and KB calculated according to in- struction in the "Treatment of Results" sec- tion and the screening test are used to design the Level III detailed tests. Assum- ing the kh values at pH 3 and 11 are due solely to acid- and base-catalyzed reactions (pseudo-first-order kinetics), the value of kh Conditions: pH3, 7, 11 85 Degrees C <40% ^- \^ 40-90% Percent Remaining Repeat Run Using Lower Temperature Pull T(2J at +2 hrs Pull T(3) - T(5) at hr Intervals i [ Analyze T(0) - T(5) \ Pull T(3) - T<5) at 1 Day Intervals Analyze T(0) - T/5) Analyze T(0) - T(5) Figure 1. Screening test decision tree. will decrease by a factor of 10 for every change of pH unit toward neutrality. If the kh values at pH 3 and 11 are the same as the pH 7 value, then hydrolysis is indepen- dent of pH and controlled only by temperature. Level III experiments for pro- cesses independent of pH are conducted at pH 7 and at temperatures that result in 50-80% hydrolysis between one day and two weeks. If either or both acid catalysis or hydrozide ion mediated hydrolysis is in- dicated in the screening test, then Level III experiments are conducted at pHs and temperatures (including pH 7) such that rate constants (first- and second-order) and activation energies can be calculated for the processes (kA, kN, KB). Ideally, rate constants would be determined at a cons- tant temperature and at two or more pHs on each side of neutrality. Establishment of a pH versus first-order disappearance rate constant curve would allow prediction of rates at other pHs. To quantify the effect of temperature and pH on the disappearance rate constant for hydrolysis, two objectives must be attain- ed: (1) expertise must be established in rate constant measurements by reproducibly measuring values for the SRCs and (2) rate constants must be replicated (minimum of three) for the compounds of interest at two or more temperatures separated by 20°C. Level III experiments are set up and con- ducted similar to Level II measurements. Water, buffers, and test solutions are prepared in the same manner as for the screening tests (6 to 8 tubes or ampules). The temperatures and pHs are adjusted after consideration of the screening test results to yield experimental conditions that allow 50-80% reduction in concentration of the chemical in two weeks or less. The tubes are removed at regular intervals and the percent remaining of the test chemical is determined by a method of established accuracy and precision (±5% acceptable). Two concentrations of chemical, differing by a factor of ten, can be used as a test to support the first-order kinetics hydrolytic mechanism. If plots of In % chemical re- maining versus time are linear and have the same slope within experimental error, then first-order kinetics are assumed. Rate constants and activation energies are calculated by methods outlined in the complete text. Treatment of Results An observed rate constant (kh) for the pH 3,7, and 11 runs is calculated by the ap- propriate method described in the Appen- dices (G-1, G-2, H, or I). At constant temperature, the kh values for pHs 3 and 11 are compared to the value calculated at ------- pH 7 for evidence of H + or OH" enhance- ment of rates. If the observed rates are within experimental error, hydrolysis is con- sidered to be independent of pH in the range 3 to 11 and Level III experiments are conducted at pH 7. The rate studies at pH 7 should be conducted at two or more temperatures to allow calculation of the ac- tivation energy (Ea) and collision frequen- cy (A) by the method outlined in Appendix J. If kh at pHs 3 and 11 are greater than ex- perimental error from the pH 7 rate, then second-order rate constants (kA and KB) can be calculated from each first-order rate constant at each temperature by dividing the measured kh at the particular pH by the hydronium ion activity (kA, pH 3) or hydroxide ion activity (ke, pH 11). this calculation assumes hydrolysis at pHs 3 and 11 are second-order reactions (first- order in compound and first-order in hydronium or hydroxide activity). The effect of temperature on Kw is taken into account when using equation 5 to calculate hydrox- ide ion activity. The results of the screening tests are us- ed to estimate the hydroysis rate at other pH values and are used to determine the pHs and temperatures for the Level III hydrolysis rate determinations. Assuming that the kh values at pH 3 and 11 are due solely to acid catalyzed and base mediated reactions, the value of kh will decrease by a factor of 10 for every change of pH unit toward neutrality, and vary approximately by a factor of 10 for each 20°C change in temperature (Ea = 84 kJ/mol). Under ideal conditions the Level III hydrolysis studies are conducted at three temperatures (separated by 20°C) and three pHs 3, 7, and 11. Values of kA, kN, and ke are calculated at each temperature. Regression analysis on the three sets of three constants at three temperatures yields values for EA, EN, EB, log AA, log AN, and log AB. These values of E and A can be used to calculate values of kx (X = A, N, B) at temperatures of interest. The calculated kx values at a particular temperature are used to calculate kh at a chosen pH (Kw at the particular temperature used when calculating [OH']. Values of A and E for each process can be estimated by plotting log kx versus 1 /T (Ar- rhenius plot) and taking the best straight line through the data points. Slope will equal -E/2.303R with intercept of log A. A two-point Arrhenius plot can be used when rate data are available at two or more • temperatures. The EPA authors J. Jackson Ellington (also the EPA Project Officer, see below). and Frank E. Stancil. Jr., are with the Environmental Research Laboratory, Athens, GA 30613; William D. Payne and Cheryl Trusty are with Technology Applications. Inc., Athens GA 30613. The complete report, entitled "Interim Protocol for Measuring Hydrolysis Rate Constants in Aqueous Solutions," (Order No. PB 88-225 081/AS; Cost: $14.95, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Environmental Research Laboratory U.S. Environmental Protection Agency College Station Road Athens, GA 30613 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Official Business Penalty for Private Use S300 EPA/600/S3-88/014 0000329 PS u s Eims,?lgHSTI011 *GENCY ° ------- |