United States Prevention, Pesticides EPA712-C-08-012
Environmental Protection And Toxic Substances October 2008
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.2120
Hydrolysis
I
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances
(OPPTS), United States Environmental Protection Agency for use in the testing
of pesticides and toxic substances, and the development of test data to meet the
data requirements of the Agency under the Toxic Substances Control Act (TSCA)
(15 U.S.C. 2601), the Federal Insecticide, Fungicide and Rodenticide Act
(FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and
Cosmetic (FFDCA) (21 U.S.C. 346a).
OPPTS developed this guideline through a process of harmonization of
the testing guidance and requirements that existed for the Office of Pollution
Prevention and Toxics (OPPT) in Title 40, Chapter I, Subchapter R of the Code
of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) in
publications of the National Technical Information Service (NTIS) and in the
guidelines published by the Organization for Economic Cooperation and
Development (OECD).
For additional information about OPPTS harmonized guidelines and to
access this and other guidelines, please go to http://www.epa.gov/oppts and
select "Test Methods & Guidelines" on the left side menu.
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OPPTS 835.2120 Hydrolysis
(a) Scope — (1) Applicability. This guideline is intended for use in meeting testing
requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S. C. 136, et
seq.) and for testing pursuant to the Toxic Substances Control Act (TSCA) (15 U.S. C. 2601, etseq.)
It describes procedures that, if followed, would result in data that would generally be of scientific
merit for the purposes described in paragraph (b) of this guideline.
(2) Background. This harmonized OPPTS test guideline is based largely on OECD
Guidelines for the Testing of Chemicals, OECD 111 Hydrolysis as a Function of pH, with
clarifications derived from 40 CFR 796.3500 Hydrolysis as a Function of pH at 25 °C and OPP 161-
1 Hydrolysis studies (Pesticide Assessment Guidelines Subdivision N - Chemistry: Environmental
Fate, EPA report 540/9-82-021), October 1982) for testing under TSCA and FIFRA, respectively.
(b) Purpose. This guideline describes a laboratory test method to assess abiotic hydrolytic
transformations of chemicals in aquatic systems at pH values normally found in the environment (pH
4 - 9). Chemicals can enter surface waters by such routes as direct application, spray drift, run-off,
drainage, waste disposal, industrial, domestic or agricultural effluent and atmospheric deposition and
may be transformed in those waters by chemical (e.g., hydrolysis, oxidation), photochemical and/or
microbial processes. Experiments are performed to determine the rate of hydrolysis of the test
substance as a function of pH and the identity or nature and rates of formation and decline of
hydrolysis products to which organisms may be exposed. Studies apply to chemicals which are
directly applied to water or that are likely to reach the environment by the other routes described in
this paragraph.
(c) Definitions.
(Disappearance time 50) is the time within which the concentration of the test
substance is reduced by 50%; it is different from the half-life (to.s) when the reaction does not follow
first order kinetics.
Half-life (to.s) is the time taken for 50% hydrolysis of a test substance when the reaction can
be described by first order kinetics: it is independent of the concentration.
Hydrolysis is a reaction of a test substance RX with water, with the net exchange of the
group X with OH at the reaction center: RX + HOH -» ROH + HX.
Hydrolysis products are all substances resulting from hydrolytic transformation reactions of
the test substance.
Test substance is any substance, whether the parent compound or relevant transformation
products.
Transformation products are all substances resulting from biotic or abiotic transformation
reactions of the test substance.
1
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(d) Principle of the test. (1) Sterile aqueous buffer solutions of different pH values (pH 4,7
and 9) are treated with the test substance and incubated in the dark under controlled laboratory
conditions (at constant temperatures). After appropriate time intervals, buffer solutions are analyzed
for the test substance and for hydrolysis products. With labeled test substance (e.g., 14C), a mass
balance can be more easily established (see paragraphs (j)l through (j)(5) of this guideline).
(2) A tiered approach to testing is shown in Figure 1:
Yes
"> "' S 't,'. .lt'',»Sl, Plj.^4' .l
ms^ib}' _•, Ji^h^st ill1."
ir .,-vH4 ' -' is r
ini,.i * In '.iv'T • i t, t i; !fiti|irt">n[u
i "!•*;(« de'iit1 li>i'n>h .mm fn " t
L rjv^lt |1" J »• Hi- 'It
H|v], ,^ t ;,r ,, j f
1 ®> iVfy^Kv- f t !Tj* ^ >t rtl
I -T-B V > >
•ni iV\tni' ) i .li.lilitf tj|'s;i"\ 1,1 il-,, \lnJi ,
K M T , 'I x
(e) Special considerations. (1) The method is generally applicable to chemical substances
(unlabeled or labeled) for which an analytical method with sufficient accuracy and sensitivity is
available. It is applicable to slightly volatile and non-volatile compounds of sufficient solubility in
water. The test should not be applied to chemicals that are highly volatile from water (e.g.,
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fumigants, organic solvents) and thus cannot be kept in solution under the experimental conditions
of this test. The test may be difficult to conduct with substances of minimal solubility in water (see
paragraph (j)(6) of this guideline).
(2) Before carrying out a hydrolysis test, the following information on the test substance
should be available: solubility in water; solubility in organic solvents; vapor pressure; n-
octanol/water partition coefficient; dissociation constant (pKa); and phototransformation rate in
water where appropriate.
(3) Analytical methods for quantification of the test substance and, if it is relevant, for
identification and quantification of hydrolysis products in aqueous solutions should be available (see
paragraph (e)(4)(ii) of this guideline). Where possible, reference substances should be used for the
identification and quantification of hydrolysis products by spectroscopic and chromatographic
methods or other suitably sensitive methods.
(4) Quality criteria, (i) Recovery. Analysis of, at least, duplicate buffer solutions or of their
extracts immediately after the addition of the test substance gives a first indication of the
repeatability of the analytical method and of the uniformity of the application procedure for the test
substance. Recoveries for later stages of the experiments are given by the respective mass balances
(when labeled material is used). Recoveries should range from 90% to 110% for labeled and non-
labeled chemicals. In case it is technically difficult to reach this range, a recovery of 70% for non-
labeled chemicals is acceptable, but justification should be given.
(ii) Repeatability and sensitivity of analytical method. (A) Repeatability of the analytical
method(s) used to quantify the test substance and hydrolysis products at later times can be checked
by duplicate analysis of the same buffer solutions (or of their extracts) after sufficient quantities of
hydrolysis products have formed for quantification.
(B) The analytical method should be sufficiently sensitive to quantify test substance
concentrations down to 10% or less of the initial concentration. If relevant, analytical methods
should also be sufficiently sensitive to quantify any hydrolysis product representing 10% or more of
applied (at any time during the study) down to 25% or less of its peak concentration.
(C) For testing pesticides, identification and quantification of products of known
toxicological or ecotoxicological concern, even if below 10% of the amount applied, should also be
identified.
(iii) Confidence intervals for hydrolysis kinetic data. Confidence intervals should be
computed and presented for all regression coefficients, rate constants, half-lives, and any other
kinetic parameters (e.g., DT50).
(f) Test method. (1) Test substance. Non-labeled or labeled test substance can be used to
measure the rate of hydrolysis. Labeled material is generally preferred for studying the pathway of
hydrolysis and for establishing mass balance; however, in special cases, labeling may not be
3
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absolutely necessary. 14C-labeling is recommended but the use of other isotopes, such as 13C, 15N,
3H, may also be useful. As far as possible, the label should be positioned in the most stable part(s) of
the molecule. For example, if the test substance contains one ring, this ring should be labeled; if the
test substance contains two or more rings, separate studies may be called for to evaluate the fate of
each labeled ring and to obtain suitable information on formation of hydrolysis products. The purity
of the test substance should be at least 95%.
(2) Equipment and apparatus, (i) The study should be performed in glass containers (e.g.,
test tubes, small flasks) under dark and sterile conditions, if necessary, unless preliminary
information (such as the n-octanol-water partition coefficient) indicates that the test substance may
adhere to glass. In such cases, alternative materials (such as Teflon) may have to be considered. It
may also be possible to alleviate the problem of adherence to glass by using one or more of the
following methods:
(A) Determine the mass of test substance and hydrolysis products sorbed to the test vessel.
(B) Use of an ultrasonic bath.
(C) Ensure a solvent wash of all glassware at each sampling interval.
(D) Use of formulated products.
(E) Use an increased amount of co-solvent for addition of test substance to the system; if a
co-solvent is used it should be a co-solvent that does not hydrolyze the test substance.
(ii) Temperature-controlled water bath shakers or thermostatically-controlled incubators for
incubation of the various test solutions are normally used.
(iii) Standard laboratory equipment is used, including, in particular, the following:
(A) pH meter.
(B) Analytical instruments such as GC, HPLC, TLC equipment, including the appropriate
detection systems for analyzing radiolabeled and non-labeled substances or inverse isotopes dilution
method.
(C) Instruments for identification purposes (e.g., MS, GC-MS, HPLC-MS, NMR, etc.).
(D) Liquid scintillation counter.
(E) Separating funnels for liquid-liquid extraction.
(F) Instrumentation for concentrating solutions and extracts (e.g., rotating evaporator).
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(G) Temperature control devise (e.g., water bath).
(iv) Chemical reagents include, for example:
(A) Organic solvents, analytical grade, such as hexane, dichloromethane, etc.
(B) Scintillation liquid.
(C) Buffer solutions (for details see paragraph (f)(4)(i) of this guideline).
(v) All glassware, reagent-grade water and buffer solutions used in the hydrolysis tests
should be sterilized.
(3) Application of test substance, (i) The test substance should be applied as aqueous
solution into the different buffer solutions. If it is necessary for adequate dissolution, the use of low
amounts of water miscible solvents (such as acetonitrile, acetone, ethanol) is permitted for
application and distribution of the test substance but this should not normally exceed 1% v/v. In case
a higher concentration of solvents is considered (e.g., in the case of poorly soluble test substances),
this could only be allowed when it can be shown that the solvent has no effect on the hydrolysis of
the test substances.
(ii) The use of formulated product is not routinely recommended, as it cannot be excluded
that the formulation ingredients may influence the hydrolysis process. However, for poorly water-
soluble test substances or for substances that adhere to glass (see paragraph (f)(2)(i) of this
guideline), the use of formulated material may be an appropriate alternative.
(iii) One concentration of the test substance should be used; it should not exceed 0.01 M or
half of the saturation concentration (see Figure 1 in paragraph (d)(2) of this guideline).
(4) Buffer solutions, (i) The hydrolysis test should be performed at pH values of 4, 7 and 9.
For this purpose, buffer solutions should be prepared using reagent grade chemicals and water.
Some useful buffer systems are presented in Tables 1-4. It should be noted that the buffer system
used may influence the rate of hydrolysis and where this is observed an alternate buffer system
should be employed. (Mabey and Mill recommend the use of borate or acetate buffers instead of
phosphate (see paragraph (j)(7) of this guideline).
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Table 1. Buffer mixtures of Clark and Lubs
Composition
0.2 N HC1 AND 0.2 N KC1 AT 20 °C
47.5 ml. HC1 + 25 ml. KC1 dil. to 100 ml
32.25 ml. HC1 + 25 ml. KC1 dil. to 100 ml
20.75 ml. HC1 + 25 ml. KC1 dil. to 100 ml
13.15 ml. HC1 + 25 ml. KC1 dil. to 100 ml
8.3 ml. HC1 + 25 ml. KC1 dil. to 100 ml
5.3 ml. HC1 + 25 ml. KC1 dil. to 100 ml
3.35 ml. HC1 + 25 ml. KC1 dil. to 100 ml
0.1 M potassium biphthalate + 0.1 N HC1 at 20 °C
46.70 ml. 0. N HC1 + 50 ml. biphthalate to 100 ml
39.60 ml. 0. N HC1 + 50 ml. biphthalate to 100 ml
32.95 ml. 0. N HC1 + 50 ml. biphthalate to 100 ml
26.42 ml. 0. N HC1 + 50 ml. biphthalate to 100 ml
20.32 ml. 0. N HC1 + 50 ml. biphthalate to 100 ml
14.70 ml. 0. N HC1 + 50 ml. biphthalate to 100 ml
9.90 ml. 0. 1 N HC1 + 50 ml. biphthalate to 100 ml
5.97 ml. 0. 1 N HC1 + 50 ml. biphthalate to 100 ml
2.63 ml. 0. 1 N HC1 + 50 ml. biphthalate to 100 ml
0.1 M potassium biphthalate + 0.1 N NaOH at 20 °C
0.40 ml. 0. 1 N NaOH +50 ml. biphthalate to 100 ml
3.70 ml. 0. 1 N NaOH +50 ml. biphthalate to 100 ml
7.50 ml. 0. 1 N NaOH +50 ml. biphthalate to 100 ml
12.15 ml. 0. N NaOH +50 ml. biphthalate to 100 ml
17.70 ml. 0. N NaOH +50 ml. biphthalate to 100 ml
23.85 ml. 0. N NaOH +50 ml. biphthalate to 100 ml
29.95 ml. 0. N NaOH +50 ml. biphthalate to 100 ml
Composition
35.45 ml. 0. N NaOH +50 ml. biphthalate to 100 ml
39.85 ml. 0. N NaOH +50 ml. biphthalate to 100 ml
43.00 ml. 0. N NaOH +50 ml. biphthalate to 100 ml
PH
.0
.2
.4
.6
.8
2.0
2.2
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
PH
5.4
5.6
5.8
0.1 M monopotassium phosphate + 0.1
5.70
8.60
12.60
17.80
23.45
29.63
35.00
39.50
42.80
45.20
46.80
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
0.1
0.1
0.
0.
0.
0.
0.
0.
0.
0.
0.
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
N NaOH at 20 °C
phosphate
phosphate
phosphate
phosphate
phosphate
phosphate
phosphate
phosphate
phosphate
phosphate
phosphate
to
to
to
to
to
to
to
to
to
to
to
100
100
100
100
100
100
100
100
100
100
100
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
0.1 M H3B03 in 0.
2.61
3.97
ml
ml
1M
0.1
0.1
KC1 + 0.
NNaOH
NNaOH
6
6
6
6
6
7
7
7
7
7
8
0
2
4
6
8
0
2
4
6
8
0
1 N NaOH at 20 °C
+ 50
+ 50
ml.
ml.
boric acid
boric acid
to
to
100
100
ml
ml
Composition
7
8
8
0
PH
-------
5.90
8.50
12.00
16.30
21.30
26.70
32.00
36.85
40.80
43.90
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
0
0
0
0
0
0
0
0
0
0
1 N NaOH
1 N NaOH
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
NNaOH
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
+ 50
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
boric
boric
boric
boric
boric
boric
boric
boric
boric
boric
acid
acid
acid
acid
acid
acid
acid
acid
acid
acid
to
to
to
to
to
to
to
to
to
to
100
100
100
100
100
100
100
100
100
100
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
8.2
8.4
8.6
8.8
9.0
9.2
9.4
9.6
9.8
10.0
Note: The pH values reported in these tables have been calculated from the potential measurements using Sorensen's
standard equations (1909), (see paragraph (j)(8) of this guideline). The corresponding pH values are 0.04 units higher
than the tabulated values. See paragraph (f)(7) of this guideline.
Table 2. Citrate buffers of Kolthoff and Vleeschhouwer
Composition
0.1 M
49.7
43.4
36.8
30.2
23.6
17.2
10.7
4.2
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
0
0
0
0
0
0
0
0
N
N
N
N
N
N
N
N
PH
monopotassium citrate and 0.1 N HC1 at 18 °C*
HC1 +
HC1 +
HC1 +
HC1 +
HC1 +
HC1 +
HC1 +
HC1 +
50 ml. citrate to 100 ml
50 ml. citrate to 100 ml
50 ml. citrate to 100 ml
50 ml. citrate to 100 ml
50 ml. citrate to 100 ml
50 ml. citrate to 100 ml
50 ml. citrate to 100 ml
50 ml. citrate to 100 ml
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
2.0
9.0
16.3
23.7
31.5
39.2
46.7
54.2
61.0
68.0
74.4
81.2
0
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
ml.
.1
0
0
0
0
0
0
0
0
0
0
0
0
M monopotassium citrate and 0.1 N NaOH at 18 °C*
N
N
N
N
N
N
N
N
N
N
N
N
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
+ 50 ml. citrate to 100 ml
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
5.8
6.0
*Add tiny crystal of thymol or a similar substance to prevent growth of molds
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Table 3. Borate mixtures of Sorensen
Composition
ml. Borax
ml. HCl/NaOH
Sorensen
18 °C
Walbum, pH at
10 °C
40 °C
70 °C
0.05 M borax + 0.1 N HC1
5.25
5.50
5.75
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
4.75
4.50
4.25
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
7.62
7.94
8.14
8.29
8.51
8.08
8.80
8.91
9.01
9.09
9.17
9.24
7.64
7.98
8.17
8.32
8.54
8.72
8.84
8.96
9.06
9.14
9.22
9.30
7.55
7.86
8.06
8.19
8.40
8.56
8.67
8.77
8.86
8.94
9.01
9.08
7.47
7.76
7.95
8.08
8.28
8.40
8.50
8.59
8.67
8.74
8.80
8.86
0.05 M borax + 0.1 N NaOH
10.0
9.0
8.0
7.0
6.0
0.0
1.0
2.0
3.0
4.0
9.24
9.36
9.50
9.68
9.97
9.30
9.42
9.57
9.76
10.06
9.08
9.18
9.30
9.44
9.67
8.86
8.94
9.02
9.12
9.28
Table 4. Posphate mixtures of Sorensen
Composition
0.0667
99.2
98.4
97.3
95.5
92.8
88.9
83.0
75.4
65.3
53.4
41.3
29.6
19.7
12.8
7.4
3.7
pH
M Monopotassium phosphate + 0.0667 M Disodium phosphate at 20 °C
ml. KH2PO4 + 0.8 ml Na2HPO4
ml. KH2PO4 + 1 .6 ml Na2HPO4
ml. KH2PO4 + 2.7 ml Na2HPO4
ml. KH2PO4 + 4.5 ml Na2HPO4
ml. KH2PO4 + 7.2 ml Na2HPO4
ml. KH2PO4 + 1 1 . 1 ml Na2HPO4
ml. KH2PO4 + 17.0 ml Na2HPO4
ml. KH2PO4 + 24.6 ml Na2HPO4
ml. KH2PO4 + 34.7 ml Na2HPO4
ml. KH2PO4 + 46.6 ml Na2HPO4
ml. KH2PO4 + 58.7 ml Na2HPO4
ml. KH2PO4 + 70.4 ml Na2HPO4
ml. KH2PO4 + 80.3 ml Na2HPO4
ml. KH2PO4 + 87.2 ml Na2HPO4
ml. KH2PO4 + 92.6 ml Na2HPO4
ml. KH2PO4 + 96.3 ml Na2HPO4
5.0
5.2
5.4
5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
(ii) The pH of each buffer solution should be checked with a calibrated pH meter to a
precision of at least 0.1 at the required temperature.
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(5) Test conditions, (i) Test temperature. (A) The hydrolysis experiments should be
carried out at constant temperatures. For extrapolation purposes, it is important to maintain the
temperature to at least ± 0.5 °C.
(B) A preliminary test (Tier 1) should be conducted at a temperature of 50 °C if the
hydrolytic behavior of the test substance is unknown. Higher tier kinetic tests should be carried out
with a minimum of three temperatures (including the test at 50 °C) unless the test substance is stable
to hydrolysis as determined by the Tier 1 testing. A suggested temperature range is 10-70 °C
(preferably with at least one temperature below 25 °C utilized), which will encompass the reporting
temperature of 25°C and most of the temperatures encountered in the field.
(C) For testing pesticides, one test temperature at 25 °C is preferred over obtaining the
hydrolytic rate at 25 °C through extrapolation using the Arrhenius equation.
(ii) Light and oxygen. All hydrolysis tests should be conducted using any suitable method
to avoid photolytic effects. All suitable measures should be taken to avoid oxygen (e.g., by bubbling
helium, nitrogen or argon for 5 minutes before preparation of the solution).
(iii) Test duration. The preliminary test should be carried out for 5 days whereas the higher
Tier tests should be conducted until 90% hydrolysis of the test substance or for 30 days whichever
comes first.
(6) Preliminary test (Tier 1). The preliminary test is performed at 50 ± 0.5 °C and pH 4.0,
7.0 and 9.0. If less than 10% of hydrolysis is observed after 5 days (t0.525°c > 1 year), the test
substance is considered hydrolytically stable and, normally, no additional testing is necessary. If the
substance is known to be unstable at environmentally relevant temperatures, the preliminary test is
not required. The analytical method should be sufficiently precise and sensitive to detect a reduction
of 10% in the initial concentration. (Information regarding stability may come from other sources
such as hydrolysis data of structurally similar compounds from the literature or from other
preliminary, semi-quantitative hydrolysis tests with the test substance at an earlier development
stage.)
(7) Hydrolysis of unstable substances (Tier 2). The higher Tier (advanced) test should be
performed at the pH values at which the test substance was found unstable as defined by the
preliminary test above. The buffered solutions of the test substance should be thermostated at the
selected temperatures. To test for first-order behavior, each reaction solution should be analyzed in
time intervals which provide a minimum of six spaced data points normally between 10% and 90%
hydrolysis of the test substance. Individual replicate test samples (a minimum of duplicate samples
contained in separate reaction vessels) should be removed and the contents analyzed at each of at
least six sampling times (for a minimum of twelve replicate data points). The use of a single bulk
sample from which individual aliquots of the test solution are removed at each sampling interval is
considered to be inadequate, as it does not allow for the analysis of data variability and it may lead
to problems with contamination of the test solution. Sterility confirmation tests should be conducted
-------
at the end of the higher Tier test (i.e., at 90% hydrolysis or 30 days). However, if no degradation
(i.e., transformation) is observed, sterility tests are not considered necessary.
(8) Identification of hydrolysis products (Tier 3). (i) Any major hydrolysis products, at
least those representing > 10% of the applied dose, should be identified by appropriate analytical
methods.
(ii) For testing pesticides, identification and quantification of products of known
toxicological or ecotoxicological concern, even if below 10% of the amount applied, should also be
identified.
(9) Optional tests. Additional tests at pH values other than 4, 7 and 9 may be needed for a
hydrolytically unstable test substance. For example, for physiological purposes a test under more
acidic conditions (e.g., pH 1.2) may be called for employing a single physiologically relevant
temperature (37°C).
(g) Treatment of results. (1) The amounts of test substance and of hydrolysis products, if
relevant, should be given as % of applied initial concentration and, where appropriate, as mg/L for
each sampling interval and for each pH and test temperature. In addition, a mass balance should be
given in percentage of the applied initial concentration when labeled test substance has been used.
(2) A graphical presentation of the log-transformed data of the test substance concentrations
against time should be reported. Any major hydrolysis products, at least those representing > 10%
of the applied dose, should be identified and their log-transformed concentrations should also be
plotted in the same manner as the parent substance to show their rates of formation and decline.
(3) More accurate determinations of half-lives or DTso values should be obtained by applying
appropriate kinetic model calculations. The half-life and/or DTso values (including confidence
limits) should be reported for each pH and temperature together with a description of the model used
the order of kinetics and the coefficient of determination (r2). If appropriate, the calculations should
also be applied to the hydrolysis products.
(4) In the case of rate studies carried out at different temperatures, the pseudo first-order
hydrolysis rate constants (k0bs) should be described as a function of temperature. The calculation
should be based on both the separation of k0bs into rate constants for acid-catalyzed, neutral, and
base catalyzed hydrolysis (kn, kneutrai, and kon respectively) and the Arrhenius equation:
kobs =kH[H+] + kneutral +kOH[OH-]= ^A.e B'/T
i=H,neutral,OH
where A; and B; are regression constants from the intercept and slope, respectively, of the
best fit lines generated from linearly regressing In k; against the reciprocal of the absolute
temperature in Kelvin (T). Through the use of the Arrhenius relationships for acid, neutral and base
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catalyzed hydrolysis, pseudo first-order rate constants, and thus half-lives can be calculated for other
temperatures for which the direct experimental determination of a rate constant is not practicable
(see paragraph (j)(9) of this guideline).
(h) Interpretation and evaluation of results. Most hydrolysis reactions follow apparent
first order reaction rates and, therefore, half-lives are independent of the concentration. This usually
permits the application of laboratory results determined at 10"2 to 10"3 M to environmental conditions
(< 10"6 M) (see paragraph (j)(9) of this guideline). Several examples of good agreement between
rates of hydrolysis measured in both pure and natural waters for a variety of chemicals were reported
by Mabey and Mill (see paragraph (j)(7) of this guideline), provided both pH and temperature had
been measured.
(i) Test report. The report should include the following information: (1) Test substance:
(A) Common name, chemical name, CAS number, structural formula (indicating position of
label when radiolabeled material is used) and relevant physical-chemical properties (see paragraph
(e)(2) of this guideline);
(B) Purity (impurities) of test substance.
(C) Label purity of labeled chemical and molar activity (where appropriate).
(2) Buffer solutions: (A) Dates and details of preparation.
(B) Buffers and waters used.
(C) Molarity and pH of buffer solutions.
(3) Test conditions: (A) Dates of the performance of the studies.
(B) Amount of test substance applied.
(C) Method and solvents (type and amount) used for application of the test substance.
(D) Volume of buffered test substance solutions incubated.
(E) Description of the incubation system used.
(F) pH and temperature during the study.
(G) Sampling times.
(H) Method(s) of extraction.
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(I) Methods for quantification and identification of the test substance and its hydrolysis
products in the buffer solutions.
(J) Number of replicates.
(4) Results. (A) Repeatability and sensitivity of the analytical methods used.
(B) Recoveries (% values for a valid study are given in paragraph (e)(4)(i) of this guideline).
(C) Replicate data and means in a tabular form.
(D) Mass balance during and at the end of the studies (when labeled test substance is used).
(E) Results of preliminary test.
(F) Discussion and interpretation of results.
(G) All original data and figures.
(5) Tables and figures. The following information should be included when hydrolysis rate
is determined:
(A) Plots of concentrations versus time for the test substances and, where appropriate, for the
hydrolysis products at each pH value and temperature.
(B) Tables of results of Arrhenius equation for the temperature 20 °C/25 °C, with pH,
rateconstant [h"1 or day"1], half-life or DTso, temperatures [°C] including confidence limits and the
coefficients of correlation (r2) or comparable information.
(C) Proposed pathway of hydrolysis.
(j) References. The following references should be consulted for additional background
information on this guideline:
(1) Agriculture Canada (1987). Environmental Chemistry and Fate Guidelines for
registration of pesticides in Canada.
(2) European Union (EU) (1995). Commission Directive 95/36/EC amending Council
Directive 91/414/EEC concerning the placing of plant protection products on the market. Annex
V: Fate and Behaviour in the Environment.
(3) Dutch Commission for Registration of Pesticides (1991). Application for registration
of a pesticide. Section G: Behaviour of the product and its metabolites in soil, water and air.
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(4) BBA (1980). Merkblatt Nr. 55, Teil I und II: Priifung des Verhaltens von
Pflanzenbehandlungsmitteln im Wasser (October 1980).
(5) SET AC (1995). Procedures for Assessing the Environmental Fate and Ecotoxicity of
Pesticides.Mark R. Lynch, Ed.
(6) OECD (2000). Guidance document on aquatic toxicity testing of difficult substances
and mixtures, OECD Environmental Health and Safety Publications Series on Testing and
Assessment No.23.
(7) Mabey, W. and Mill, T. (1978). Critical review of hydrolysis of organic compounds
in water under environmental conditions. J. Phys. Chem. Ref. Data 7, 383-415.
(8) Sorensen, S.P.L. (1909). Enzymstudien II. Mitteilung. Uber die Messung und die
Bedentung der Wasserstoffionenkonzentration bie enzymatischen Prozessen. Biochem. Z., 21,
131-304.
(9) Nelson, H, Laskowski D, Thermes S, and Hendley P. (1997) Recommended changes
in pesticide fate study guidelines for improving input to computer models. (Text version of oral
presentation at the 14th Annual Meeting of the Society of Environmental Toxicology and
Chemistry, Dallas TX, November 1993).
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