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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