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-

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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

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 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

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   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
                                  °

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