[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR796.3500]
[Page 96-101]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 796_CHEMICAL FATE TESTING GUIDELINES--Table of Contents
Subpart D_Transformation Processes
Sec. 796.3500 Hydrolysis as a function of pH at 25 [deg]C.
(a) Introduction--(1) Background and purpose. (i) Water is one of
the most widely distributed substances in the environment. It covers a
large portion of the earth's surface as oceans, rivers, and lakes. The
soil also contains water, as does the atmosphere in the form of water
vapor. As a result of this ubiquitousness, chemicals introduced into the
environment almost always come into contact with aqueous media. Certain
classes of these chemicals, upon such contact, can undergo hydrolysis,
[[Page 97]]
which is one of the most common reactions controlling chemical stability
and is, therefore, one of the main chemical degradation paths of these
substances in the environment.
(ii) Since hydrolysis can be such an important degradation path for
certain classes of chemicals, it is necessary, in assessing the fate of
these chemicals in the environment, to know whether, at what rate, and
under what conditions a substance will hydrolyze. Some of these
reactions can occur so rapidly that there may be greater concern about
the products of the transformation than about the parent compounds. In
other cases, a substance will be resistant to hydrolysis under typical
environmental conditions, while, in still other instances, the substance
may have an intermediate stability that can result in the necessity for
an assessment of both the original compound and its transformation
products. The importance of transformation of chemicals via hydrolysis
in aqueous media in the environment can be determined quantitatively
from data on hydrolysis rate constants. This hydrolysis Test Guideline
represents a test to allow one to determine rates of hydrolysis at any
pH of environmental concern at 25[deg]C.
(2) Definitions and units. (i) ``Hydrolysis'' is defined as the
reaction of an organic chemical with water, such that one or more bonds
are broken and the reaction products of the transformation incorporate
the elements of water (H2O).
(ii) ``Elimination'' is defined in this Test Guideline to be a
reaction of an organic chemical (RX) in water in which the X group is
lost. These reactions generally follow the same type of rate laws that
hydrolysis reactions follow and, thus, are also covered in this Test
Guideline.
(iii) A ``first-order reaction'' is defined as a reaction in which
the rate of disappearance of the chemical substance being tested is
directly proportional to the concentration of the chemical substance and
is not a function of the concentrations of any other substances present
in the reaction mixture.
(iv) The ``half-life'' of a chemical is defined as the time required
for the concentration of the chemical substance being tested to be
reduced to one-half its initial value.
(v) ``Hydrolysis'' refers to a reaction of an organic chemical with
water such that one or more bonds are broken and the reaction products
incorporate the elements of water (H2O). This type of
transformation often results in the net exchange of a group X, on an
organic chemical RX, for the OH group from water. This can be written
as:
RX+HOH[rarr]ROH+HX.
(A) Another result of hydrolysis can be the incorporation of both H
and OH in a single product. An example of this is the hydrolysis of
epoxides, which can be represented by
(B) The hydrolysis reaction can be catalyzed by acidic or basic
species, including OH- and H3O=
(H=). The promotion of the reaction by
H3O- or OH- is called specific acid or
specific base catalysis, respectively, as contrasted with general acid
or base catalysis encountered with other cationic or anionic species.
Usually, the rate law for chemical RX can be written as:
Equation 1
-d[RX]/d= = kh[RX]=kA[H=]
[RX]
+kB[OH-] [RX]+k'N [H2O]
[RX],
where KA, kB and k'N are the second-
order rate constants for acid and base catalyzed and neutral water
processes, respectively. In dilute solutions, such as are encountered in
following this Test Guideline, water is present in great excess and its
concentration is, thus, essentially constant during the course of the
hydrolysis reaction. At fixed pH, the reaction, therefore, becomes
pseudo first-order, and the rate constant (kh) can be written
as:
Equation 2
kh=kA [H=]+kB
[OH-]+kN,
where kN is the first-order neutral water rate constant.
Since this is a
[[Page 98]]
pseudo first-order process, the half-life is independent of the
concentration and can be written as:
Equation 3
t1/2=0.693/kh.
At constant pH, Equation 1 can be integrated to yield the first order
rate expression
Equation 4
log10C=- (kh t/
2.303)+log10Co,
where C is the concentration of the test chemical at time t and
Co is the initial chemical concentration (t=0).
(C) At a given pH, Equation 2 under paragraph (a)(2)(v)(B) of this
section contains three unknowns, kA, kB, and
kN. Therefore, three equations (i.e., measurements at three
different pH's at a fixed temperature) are required if one wishes to
solve for these quantities. Making suitable approximations for
quantities that are negligible, the expressions for kA,
kB, and kN using values of kh measured
at pH 3, 7, and 11 are:
Equation 5
kA=10\3\ [kh (3)-kh (7)+10-4
kh (11)]
kB=10\3\ [kh (11)-kh
(7)+10-4 kh (3)]
kN=kh (7)-10-4 [kh
(3)+kh (11)]
The calculated rate constants from equation 5 under this paragraph can
be employed in equation 2 under paragraph (a)(2)(v)(B) of this section
to calculate the hydrolysis rate of a chemical at any pH of
environmental concern.
(D) The equations under paragraph (a)(2) of this section apply
whether the test chemical has one or more hydrolyzable groups. In the
latter case, the rate may be written as:
Equation 6
-d[RX]/dt=[RX]=k2 [RX]+ . . . . +kn
[RX]=(k1+k2+ . . . . . kn)
[RX]=kh [RX].
Equation 6 applies to the hydrolysis rate of a molecule having n
hydrolyzable groups, each of which follows first-order reaction
kinetics. The measured kh is now the sum of the individual
reaction rates and is the only rate constant required in this section.
(3) Principle of the test method. Procedures described in this
section enable sponsors to obtain quantitative information on hydrolysis
rates through a determination of hydrolysis rate constants and half-
lives of chemicals at pH 3.00, 7.00, and 11.00 at 25 [deg]C. The three
measured rate constants are used to determine the acidic, basic, and
neutral rate constants associated with a hydrolytic reaction. The latter
constants can then be employed in determining the hydrolysis rates of
chemicals at any pH of environmental concern at 25 [deg]C.
(4) Applicability and specificity. There are several different
common classes of organic chemicals that are subject to hydrolysis
transformation, including esters, amides, lactones, carbamates,
organophosphates, and alkyl halides. Processes other than nucleophilic
displacement by water can also take place. Among these are elimination
reactions that exhibit behavior similar to hydrolysis and, therefore,
are also covered in this section.
(b) Test procedures--(1) Test conditions--(i) Special laboratory
equipment. (A) A thermostatic bath that can be maintained at a
temperature of 25 1 [deg]C.
(B) A pH meter that can resolve differences of 0.05 pH units or
less.
(C) Stoppered volumetric flasks (no grease) or glass ampoules that
can be sealed.
(ii) Purity of water. Reagent-grade water (e.g., water meeting ASTM
Type IIA standards or an equivalent grade) shall be used to minimize
biodegradation. ASTM Type IIA water is described in ASTM D 1193-77
(Reapproved 1983), ``Standard Specification for Reagent Water.'' ASTM D
1193-77 (Reapproved 1983) is available for inspection at the National
Archives and Records Administration (NARA). For information on the
availability of this material at NARA, call 202-741-6030, or go to:
http://www.archives.gov/federal--register/code--of--federal--
regulations/ibr--locations.html. This incorporation by reference was
approved by the Director of the Office of the Federal Register. This
material is incorporated as it exists on the date of approval and a
notice of any change in this material
[[Page 99]]
will be published in the Federal Register. Copies of the incorporated
material may be obtained from the Non-Confidential Information Center
(NCIC) (7407), Office of Pollution Prevention and Toxics, U.S.
Environmental Protection Agency, Room B-607 NEM, 401 M St., SW.,
Washington, DC 20460, between the hours of 12 p.m. and 4 p.m. weekdays
excluding legal holidays, or from the American Society for Testing and
Materials (ASTM), 1916 Race Street, Philadelphia, PA 19103.
(iii) Sterilization. All glassware shall be sterilized. Aseptic
conditions shall be used in the preparation of all solutions and in
carrying out all hydrolysis experiments to eliminate or minimize
biodegradation. Glassware can be sterilized in an autoclave or by any
other suitable method.
(iv) Precautions for volatility. If the chemical is volatile the
reaction vessels shall be almost completely filled and sealed.
(v) Temperature controls. All hydrolysis reactions shall be carried
out at 25 [deg]C (1 [deg]C) and with the
temperature controlled to 0.1 [deg]C.
(vi) pH conditions. It is recommended that all hydrolysis
experiments be performed at pH 3.00, 7.00, and 11.00 0.05 using the appropriate buffers described in
paragraph (b)(2)(i)(A) of this section.
(vii) Concentration of solutions of chemical substances. The
concentration of the test chemical shall be less than one-half the
chemical's solubility in water but not greater than 10-3 M.
(viii) Effect of acidic and basic groups. Complications can arise
upon measuring the rate of hydrolysis of chemicals that reversibly
ionize or are protonated in the pH range 3.00 to 11.00. Therefore, for
these chemicals, it is recommended that these hydrolysis tests be
performed at pH 5.00, 7.00, and 900 0.05 using the
appropriate buffers described in paragraphs (b)(2)(i) (A) and (B) of
this section. If a test chemical reversibly ionizes or protonates in the
pH range 5.00 to 9.00, then it is recommended that additional hydrolysis
tests should be carried out at pH 6.00 and 8.00 0.05 using the buffers described in paragraph
(b)(2)(i)(B) of this section.
(ix) Buffer catalysis. For certain chemicals, buffers may catalyze
the hydrolysis reaction. If this is suspected, hydrolysis rate
determination shall be carried out with the appropriate buffers and the
same experiments repeated at buffer concentrations lowered by at least a
factor of five. If the hydrolysis reaction produces a change of greater
than 0.05 pH units in the lower concentration buffers at the end of the
measurement time, the test chemical concentrations also shall be lowered
by at least a factor of five. Alternatively, test chemical
concentrations and buffer concentrations may both be lowered
simultaneously by a factor of five. A sufficient criterion for
minimization of buffer catalysis is an observed equality in the
hydrolysis rate constant for two different solutions differing in buffer
or test chemical concentration by a factor of five.
(x) Photosensitive chemicals. The solution absorption spectrum can
be employed to determine whether a particular chemical is potentially
subject to photolytic transformation upon exposure to light. For
chemicals that absorb light of wavelengths greater than 290 nm, the
hydrolysis experiment shall be carried out in the dark, under amber or
red safelights, in amber or red glassware, or employing other suitable
methods for preventing photolysis. The absorption spectrum of the
chemical in aqueous solution can be measured under Sec. 796.1050.
(xi) Chemical analysis of solutions. In determining the
concentrations of the test chemicals in solution, any suitable
analytical method may be employed, although methods which are specific
for the compound to be tested are preferred. Chromatographic methods are
recommended because of their compound specificity in analyzing the
parent chemical without interferences from impurities. Whenever
practicable, the chosen analytical method should have a precision within
5 percent.
(2) Preparation--(i) Reagents and solutions--(A) Buffer solutions.
Prepare buffer solutions using reagent-grade chemicals and reagent-grade
water as follows:
(1) pH 3.00: use 250 mL of 0.100M potassium hydrogen phthalate; 111
mL of 0.100M hydrochloric acid; and adjust
[[Page 100]]
volume to 500 mL with reagent-grade water.
(2) pH 7.00: use 250 mL of 0.100M potassium dihydrogen phosphate;
145 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with
reagent-grade water.
(3) pH 11.00: use 250 mL of 0.0500M sodium bicarbonate; 113 mL of
0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-grade
water.
(B) Additional buffer solutions. For chemicals that ionize or are
protonated as discussed in paragraph (b)(1)(viii) of this section,
prepare buffers using reagent-grade water and reagent-grade chemicals as
follows:
(1) pH 5.00: use 250 mL of 0.100M potassium hydrogen phthalate; 113
mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-
grade water.
(2) pH 6.00: use 250 mL of 0.100M potassium dihydrogen phosphate; 28
mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with reagent-
grade water.
(3) pH 8.00: use 250 mL of 0.100M potassium dihydrogen phosphate;
234 mL of 0.100M sodium hydroxide; and adjust volume to 500 mL with
reagent-grade water.
(4) pH 9.00: use 250 mL of 0.0250M borax (Na2
B4O7); 23 mL of 0.100M hydrochloric aid; and
adjust volume to 500 mL with reagent-grade water.
(C) Adjustment of buffer concentrations. (1) The concentrations of
all the above buffer solutions are the maximum concentration to be
employed in carrying out hydrolysis measurements. If the initial
concentration of the test chemical is less than 10-3 M, the
buffer concentration shall be lowered by a corresponding amount; e.g.,
if the initial test chemical concentration is 10-4 M, the
concentration of the above buffers shall be reduced by a factor of 10.
In addition, for those reactions in which an acid or base is not a
reaction product, the minimum buffer concentration necessary for
maintaining the pH within +0.05 units shall be employed.
(2) Check the pH of all buffer solutions with a pH meter at 25
[deg]C and adjust the pH to the proper value, if necessary.
(D) Preparation of test solution. (1) If the test chemical is
readily soluble in water, prepare an aqueous solution of the chemical in
the appropriate buffer and determine the concentration of the chemical.
Alternatively, a solution of the chemical in water may be prepared and
added to an appropriate buffer solution and the concentration of the
chemical then determined. In the latter case, the aliquot shall be small
enough so that the concentration of the buffer in the final solution and
the pH of the solution remain essentially unchanged. Do not employ heat
in dissolving the chemical. The final concentration shall not be greater
than one-half the chemical's solubility in water and not greater than
10-3 M.
(2) If the test chemical is too insoluble in pure water to permit
reasonable handling and analytical procedures, it is recommended that
the chemical be dissolved in reagent-grade acetonitrile and buffer
solution and then added to an aliquot of the acetonitrile solution. Do
not employ heat to dissolve the chemical in acetonitrile. The final
concentration of the test chemical shall not be greater than one-half
the chemical's solubility in water and not greater than 10-3
M. In addition, the final concentration of the acetonitrile shall be one
volume percent or less.
(3) Performance of the test. Carry out all hydrolysis experiments by
employing one of the procedures described in this paragraph. Prepare the
test solutions as described in paragraph (b)(2)(i) of this section at pH
3.00, 7.00, and 11.00 0.05, and determine the
initial test chemical concentration (Co) in triplicate.
Analyze each reaction mixture in triplicate at regular intervals,
employing one of the following procedures:
(i) Procedure 1. Analyze each test solution at regular intervals to
provide a minimum of six measurements with the extent of hydrolysis
between 20 to 70 percent. Rates should be rapid enough so that 60 to 70
percent of the chemical is hydrolyzed in 672 hours.
(ii) Procedure 2. If the reaction is too slow to conveniently follow
hydrolysis to high conversion in 672 hours but still rapid enough to
attain at least 20 percent conversion, take 15 to 20 time points at
regular intervals after 10 percent conversion is attained.
[[Page 101]]
(iii) Procedure 3. (A) If chemical hydrolysis is less than 20
percent after 672 hours, determine the concentration (C) after this time
period.
(B) If the pH at the end of concentration measurements employing any
of the above three procedures has changed by more than 0.05 units from
the initial pH, repeat the experiment using a solution having a test
chemical concentration lowered sufficiently to keep the pH variation
within 0.05 pH units.
(iv) Analytical methodology. Select an analytical method that is
most applicable to the analysis of the specific chemical being tested
under paragraph (b)(1)(xi) of this section.
(c) Data and reporting--(1) Treatment of results. (i) If Procedure 1
or 2 were employed in making concentration measurements, use a linear
regression analysis with Equation 4 under paragraph (a)(2)(v)(B) of this
section to calculate kh at 25 [deg]C for each pH employed in
the hydrolysis experiments. Calculate the coefficient of determination
(R\2\) for each rate constant. Use Equation 3 under paragraph
(a)(2)(v)(B) of this section to calculate the hydrolysis half-life using
kh.
(ii) If Procedure 3 was employed in making rate measurements, use
the mean initial concentration (Co) and the mean
concentration of chemical (C) in Equation 4 under paragraph (a)(2)(v)(B)
of this section to calculate kh for each pH used in the
experiments. Calculate the hydrolysis half-life using kh in
Equation 3 under paragraph (a)(2)(v)(B) of this section.
(iii) For each set of three concentration replicates, calculate the
mean value of C and the standard deviation.
(iv) For test chemicals that are not ionized or protonated between
pH 3 and 11, calculate kA, kB, and kN
using Equation 5.
(2) Specific analytical and recovery procedures. (i) Provide a
detailed description or reference for the analytical procedure used,
including the calibration data and precision.
(ii) If extraction methods were used to separate the solute from the
aqueous solution, provide a description of the extraction method as well
as the recovery data.
(3) Test data report. (i) For Procedures 1 and 2, report
kh, the hydrolysis half-life (t1/2), and the
coefficient of determination (R\2\) for each pH employed in the rate
measurements. In addition, report the individual values, the mean value,
and the standard deviation for each set of replicate concentration
measurements. Finally, report kA, kB, and
kN.
(ii) For Procedure 3, report kh and the half-life for
each pH employed in the rate measurements. In addition, report the
individual values, the mean value, and the standard deviation for each
set of replicate concentration measurements. Finally, report
kA, kB, and kN.
(iii) If, after 672 hours, the concentration (C) is the same as the
initial concentration (Co) within experimental error, then
kh cannot be calculated and the chemical can be reported as
being persistent with respect to hydrolysis.
[50 FR 39252, Sept. 27, 1985, as amended at 53 FR 10391, Mar. 31, 1988;
53 FR 12526, Apr. 15, 1988; 53 FR 22323, June 15, 1988; 60 FR 34467,
July 3, 1995; 69 FR 18803, Apr. 9, 2004]