United States Prevention, Pesticides EPA712-C-98-041
Environmental Protection and Toxic Substances March 1998
Agency (7101)
4»EPA Product Properties
Test Guidelines
OPPTS 830.7840
Water Solubility: Column
Elution Method; Shake
Flask Method
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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,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This document is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), internet: http://
fedbbs.access.gpo.gov, or call 202-512-0132 for disks or paper copies.
This guideline is available in ASCII and PDF (portable document format)
from the EPA's World Wide Web site (http://www.epa.gov/epahome/re-
search.htm) under the heading "Researchers and Scientists/Test Methods
and Guidelines/OPPTS Harmonized Test Guidelines."
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OPPTS 830.7840 Water solubility: column elution method; shake
flask method.
(a) Scope (1)—Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.} and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source materials used in developing this har-
monized OPPTS test guideline are the OPPT guideline under 40 CFR
796.1840 Water solubility, OPP guideline 63-8 Solubility (Pesticide As-
sessment Guidelines, Subdivision D: Product Chemistry, EPA Report 5407
9-82-018, October 1982) and OECD guideline 105 Water Solubility (Col-
umn Elution Method—Shake Flask Method).
(b) Introductory information—(1) Prerequisites. Suitable analytical
method; structural formula; vapor pressure curve; dissociation constant;
hydrolysis independence of pH (preliminary test).
(2) Coefficient of variation. The coefficient of variation on the mean
values reported by the participants of the OECD Laboratory Intercompari-
son Testing, part I, 1979, appeared to be dependent on the chemicals tested
and the test temperatures; it ranges from 0.05 to 0.34 for the column
elution method, and from 0.03 to 1.12 for the flask method.
(3) Qualifying statements, (i) The method is not applicable to vola-
tile substances. Care should be taken that the substances examined are
as pure as possible and stable in water. It must be ascertained that the
identity of the substance is not changed during the procedure.
(ii) The column elution method is not suitable for volatile substances.
The carrier material used here may not yet be optimal. This method is
intended for material with solubilities below approximately 10~2 g/L.
(iii) The flask method is intended for materials with solubility above
10-2 g/L. It is not applicable to volatile substances; this method may pose
difficulties in the case of surface-active materials.
(iv) For pesticides, in addition to the water solubility data require-
ments, registrants should also submit data on solubility of the pesticide
in organic solvents (see OPPTS 830.1000, paragraph (e)(2)(x)).
(4) Recommendation. Additional research work is necessary for the
column elution method, e.g. to find the best carrier material, to optimize
the construction of the column and the adjustment of the flow rate.
(5) Standard documents. The column elution method is based on
the test principle as it is described in the reference under paragraph (f)(l)
of this guideline. The DAPA (German Council for Pesticides Analysis)
redrafted this method.
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(c) Method—(1) Introduction, purpose, scope, relevance, applica-
tion, and limits of test, (i) A solution is a homogeneous mixture of dif-
ferent substances in a solvent. The particle sizes of the dispersed sub-
stances are of the same magnitude as molecules and ions; therefore, the
smallest volumes which can be obtained from a solution are always of
uniform composition.
(ii) Solubility in water is a significant parameter because:
(A) The spatial and temporal movement (mobility) of a substance is
largely determined by its solubility in water.
(B) Water soluble substances gain ready access to humans and other
living organisms.
(C) The knowledge of the solubility in water is a prerequisite for
testing biological degradation and bioaccumulation in water and for other
tests.
(iii) No single method is available to cover the whole range of
solubilities in water, from relatively soluble to very low soluble chemicals.
A general test guideline for the determination of the solubility in water
must include methods which cover the whole range of water soluble sub-
stances. This guideline therefore includes two methods:
(A) One which applies to substances with low solubilities
(<10-2 g/L), referred to as the "column elution method."
(B) The other which applies to substances with higher solubilities
(>10-2 g/L), referred to as the "flask method."
(2) Definition and units. The solubility in water of a substance is
specified by the saturation mass concentration of the substance in water
and is a function of temperature. The solubility in water is specified in
units of weight per volume of solution. The Si-unit is kg/m3; g/L may
also be used.
(3) Reference substances. The reference substances need not be em-
ployed in all cases when investigating a new substance. They are provided
primarily so that calibration of the method may be performed from time
to time and to offer the chance to compare the results when another meth-
od is applied. The values presented in the following table 1. are not nec-
essarily representative of the results which can be obtained with this test
method as they have been derived from an earlier version of the test meth-
od.
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Table 1.—Data for Reference Substances
Method
Fluoranthene
Elution method
Hexachlorobenzene
Elution method
y-Hexachlorocyclohexane
Elution method
2,4-Dichlorophenoxyacetic
acid
Flask method
Mercury(ll) chloride:
Flask method
4-Nitrophenol:
Flask method
T, °C
15
25
15
25
15
25
15
25
15
25
15
25
Mean (mg/L)
0.275
0.373
921 x 10-3
9.96 x 10-3
650
9.20
0.633
0.812
53.0
66.4
9.95
14.8
Range (mg/L)
0.104 to 0.920
0.1 98 to 1.050
206x ID-3 to 2 16 x 10-2
1.1 9x10-3 to 2.31 x 10-2
4 43 to 1 0 5
6.64 to 14.5
0.380 to 0.764
0.655 to 0.927
47.7 to 56.5
58.3 to 70.4
8.88 to 10.9
13.8 to 15.9
no. of
labs
6
7
6
7
6
7
5
5
4
4
6
6
(4) Principle of the test methods. The approximate amount of the
sample and the time necessary to achieve the saturation mass concentration
should be determined in a simple preliminary test.
(i) Column elution method. This method is based on the elution of
a test substance with water from a micro column which is charged with
an inert carrier material such as glass beads, silica gel or sand, and an
excess of test substance (see paragraph (f)(l)) of this guideline). The water
solubility is determined when the mass concentration of the eluate is con-
stant. This is shown by a concentration plateau as a function of time in
the following figure 1:
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Figure 1.—Concentration versus Time of Substance in the Eluate
1
o
§
o
o
CO
CO
CO
Saturation Concentration
Time (Eluate Fractions)
(ii) Flask method. In this method, the substance (solids must be pul-
verized) is dissolved in water at a temperature somewhat above the test
temperature. When saturation is achieved, the mixture is cooled and kept
at the test temperature, stirring as long as necessary to reach equilibrium
(see paragraph (f)(3) of this guideline). Subsequently, the mass concentra-
tion of the substance in the aqueous solution, which must not contain any
undissolved particles, is determined by a suitable analytical method.
(5) Quality criteria—(i) Repeatability. For the column elution meth-
od <30 percent is acceptable; for the flask method <15 percent should
be observed.
(ii) Sensitivity. This depends upon the method of analysis, but mass
concentration determinations down to at least 10-6 g/L can be determined.
(iii) Specificity. This method should only be applied to:
(A) Pure substance.
(B) Substances that are stable in water.
(C) Slightly soluble substances, i.e. <10-2 g/L for the column elution
method.
ized.
(D) Organic substances for the column elution method.
(iv) Possibility of standardization. These methods can be standard-
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(d) Description of the test procedures—(1) Preparations—(i) Ap-
paratus—(A) Column elution method. (7) The schematic arrangement
of the system is presented in the following figure 2:
Figure 2.—Schematic Test Arrangement
Flow
Recycling
Pump
Micro-
Column
Atmospheric
Equilibration
Thermostatlc
Controlled
Circulating
Pump
Twb-Way-Valve
for Sampling
(2) A suitable microcolumn is shown in the following figure 3:
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Figure 3.—Microcolumn (all dimensions in millimeters)
ro^f
I \ F\ i / rs.
Interior 5
Connection for ground glass joint
Headspace
t^PKjg of glass wool
Stopcock with 2 way action
Although any size is acceptable, provided it meets the criteria for repro-
ducibility and sensitivity. The column should provide for a head space
of at least five bed-volumes of water and a minimum of five samples.
Alternatively, the size can be reduced if make-up solvent is employed to
replace the initial five bed-volumes removed with impurities.
(3) The column should be connected to a recycling pump capable
of controlling flows of approximately 25 mL/h. The pump is connected
with polytetrafluoroethylene and/or glass connections. The column and
pump, when assembled, should have provision for sampling the effluent
and equilibrating the head space at atmospheric pressure. The column ma-
terial is supported with a small (5 mm) plug of glass wool, which must
also serve to filter particles.
(B) Flask method. For the flask method, the following material is
needed:
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(7) Normal laboratory glassware and instrumentation.
(2) A device suitable for the agitation of solutions under controlled
constant temperatures.
(3) A centrifuge (preferably thermostatted), if required with emul-
sions.
(4) Equipment for analytical determinations.
(2) Reagents. The substance to be tested should be as pure as pos-
sible, particularly in the flask method where purification is not provided.
The carrier material for the column elution method should be inert. Pos-
sible materials which can be employed are glass beads and silica. A suit-
able volatile solvent of analytical reaction quality should be used to apply
the test substance to the carrier material. Double distilled water from glass
or quartz apparatus should be employed as the eluent or solvent. (Note:
Water directly from an ion exchanger must not be used.)
(3) Test conditions. The test is preferably run at 20 + 0.5 °C
(293 K). If temperature dependence is suspected in the solubility
(> 3%/°C), two other temperatures should also be used—both differing
from each other and the initially chosen temperature by 10 °C. In this
case the temperature control should be ±0.1 °C. One of these additional
temperatures should be below the initial temperature. The chosen
temperature(s) should be kept constant in all parts of the equipment (in-
cluding the leveling vessel).
(4) Performance of the tests—(i) Preliminary test. (A) To approxi-
mately 0.1 g of the sample (solid substances must be pulverized) in a
glass-stoppered 10 mL graduated cylinder, increasing volumes of distilled
water at room temperature are added according to the steps shown in the
following table 2:
Table 2.—Determination of Solubility
Solubility data
Total volume h^O added (ml)
ADDroximate solubility (a/D
step 1
0 1
>1.000
step 2
05
200
step 3
1
100
step 4
2
50
step 5
10
10
step 6
100
1
step 7
>100
<1
(B) After each addition of water to give the indicated total volume,
the mixture is shaken vigorously for 10 min and is visually checked for
any undissolved parts of the sample. If, after a total of 10 mL of water
has been added (step 5), the sample or parts of it remain undissolved,
the contents of the measuring cylinder is transferred to a 100 mL measur-
ing cylinder which is then filled up with water to 100 mL (step 6) and
shaken. At lower solubilities the time required to dissolve a substance can
be considerably long (24 h should be allowed). The approximate solubility
is given in the table under that volume of added water in which complete
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dissolution of the sample occurs. If the substance is still apparently insolu-
ble, further dilution should be undertaken to ascertain whether the column
elution or flask solubility method should be used.
(ii) Column elution—(A) Apparatus. (7) The equipment is arranged
as shown in figures 2 and 3 (see paragraphs (d)(l)(i)(A)(7) and
(d)(l)(i)(A)(2) of this guideline). Approximately 600 mg of carrier material
is weighed and transferred to a 50-mL round-bottom flask. A suitable,
weighed amount of test substance is dissolved in the chosen solvent, and
an appropriate amount of the test substance solution is added to the carrier
material. The solvent must be completely evaporated, e.g. in a rotary evap-
orator; otherwise water saturation of the carrier is not achieved due to
partition effects on the surface of the carrier.
(2) The loading of carrier material may cause problems (erroneous
results) if the test substance is deposited as an oil or a different crystal
phase. The problem should be examined experimentally.
(3) The loaded carrier material is allowed to soak for about 2 h in
approximately 5 mL of water, and then the suspension is added to the
microcolumn. Alternatively, dry loaded carrier material may be poured in
the microcolumn, which has been filled with water and then equilibrated
for approximately 2 h.
(B) Test procedure. The elution of the substance from the carrier
material can be carried out in two different ways: Leveling vessel or cir-
culating pump. The two principles should be used alternatively.
(7) Leveling vessel (see figure 3 under paragraph (d)(l)(i)(A)(2) and
figure 4 under paragraph (d)(4)(iii) of this guideline). (/) The connection
to the leveling vessel is made by using a ground glass joint which is con-
nected by PTFE tubing. It is recommended that a flow rate of approxi-
mately 25 mL/h be used. Successive eluate fractions should be collected
and analyzed by the chosen method.
(if) Fractions from the middle eluate range where the concentrations
are constant (±30 percent) in at least five consecutive fractions are used
to determine the solubility in water.
(///) A second run is to be performed at half the flow rate of the
first. If the results of the two runs are in agreement, the test is satisfactory;
if there is a higher apparent solubility with the lower flow rate, then the
halving of the flow rate must continue until two successive runs give the
same solubility.
(2) Circulating pump (see figures 2 and 3 under paragraphs
(d)(l)(i)(A)(7) and (d)(l)(i)(A)(2) of this guideline). (/) With this appara-
tus, the microcolumn must be modified. A stopcock with 2-way action
must be used (see figure 3). The circulating pump can be, e.g. a peristaltic
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pump (be careful that no contamination and/or adsorption occurs with the
tube material) or a membrane pump.
(if) The flow through the column is started. It is recommended that
a flow rate of approximately 25 mL/h be used (approximately 10 bed vol-
umes per hour for the described column). The first five bed volumes (mini-
mum) are discarded to remove water soluble impurities.
(///) Following this, the recycling pump is connected and the appara-
tus allowed to run until equilibration is established, as defined by five
successive samples whose concentrations do not differ by more than 30
percent in a random fashion (see paragraph (f)(2) of this guideline). These
samples should be separated from each other by time intervals correspond-
ing to the passage of at least 10 bed-volumes of the eluent.
(3) In both cases (using a circulation pump or a leveling vessel) the
fractions should be checked for the presence of colloidal matter by exam-
ination for the Tyndall effect (light scattering). Presence of such particles
invalidates the results, and the test should be repeated with improvements
in the filtering action of the column. The pH of each sample should be
recorded. A second run should be performed at the same temperature.
(iii) Flask method—Test procedure. The quantity of material nec-
essary to saturate the desired volume of water is estimated from the pre-
liminary test. The volume of water required will depend on the analytical
method and the solubility range. About 5x the quantity of material deter-
mined under paragraph (d)(4)(i)(A) of this guideline is weighed into each
of three glass vessels fitted with glass stoppers (e.g. centrifuge tubes,
flasks). The chosen volume of water is added to each vessel, and the ves-
sels are tightly stoppered. The closed vessels are then agitated at 30 °C.
(A shaking or stirring device capable of operating at constant temperature
should be used, e.g. magnetic stirring in a thermostatically controlled water
bath.) After 1 day, one of the vessels is removed and reequilibrated for
24 h at the test temperature with occasional shaking. The contents of the
vessel are then centrifuged at the test temperature, and the concentration
of compound in the clear aqueous phase is determined by a suitable analyt-
ical method. The other two flasks are treated similarly after initial equili-
bration at 30 °C for 2 and 3 days, respectively. If the concentration results
from at least the last two vessels agree with the required reproducibility,
the test is satisfactory. The whole test should be repeated, using longer
equilibration times if the results from vessels 1, 2, and 3 show a tendency
to increasing values. The arrangement of the apparatus is shown in the
following figure 4:
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Figure 4.—Test Arrangement for the Determination of Solubility in
Water of Slightly Soluble, Low Volatility Organic Substances
5
1
1 = Leveling vessel (e.g. 2.5-L chemical flask)
2 = Column (See figure 3.)
3 = Fraction accumulator
4 = Thermostat
5 = Teflon tubing
6 = Glass stopper
7 = Water line (between thermostat and column, inner diameter: approxi-
mately 8 mm)
(iv) Analysis. A substance-specific analytical method is required for
these determinations, since small amounts of soluble impurities can cause
large errors in the measured solubility. Examples of such methods are gas
or liquid chromatography, tirration methods, photometric methods, and po-
larographic methods.
(e) Data and reporting—(1) Column elution method—(i) Treat-
ment of results. The mean value from at least five consecutive samples
taken from the saturation plateau (figure 1. under paragraph (c)(4)(i) of
10
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this guideline) should be determined for each run, as should the standard
deviation. A comparison should be made between the two means to ensure
that they agree with a repeatability of less than 30 percent.
(ii) Test report. The report should contain an indication of the results
of the preliminary test plus the following information:
(A) The individual concentrations, flow rates and pHs of each sam-
ples.
(B) The means and standard deviations from at least five samples
from the saturation plateau of each run.
(C) The average of the two successive, acceptable runs.
(D) The temperature of the runs.
(E) The method of analysis employed.
(F) The nature of the carrier material employed.
(G) Loading of carrier material.
(H) Solvent used.
(I) Statement that the identity of the substance in the saturated solu-
tion has been proved.
(2) Flask method—(i) Treatment of results. The individual results
should be given for each of the three flasks and those results deemed to
be constant (repeatability <15 percent) should be averaged and given in
units of mass per volume of solution. This may require the conversion
of mass units to volume units, using the density when the solubility is
very high (100 g/L).
(ii) Test report. The report should include the following information:
(A) The individual analytical determinations and the average where
more than one value was determined for each flask.
(B) The average of the value for the different flasks which were in
agreement.
(C) The test temperature.
(D) The analytical method employed.
(f) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Weil, L. et al. Wasserloslichkeit von insektiziden chlorierten
Kohlenwasserstoffen und polychlorierten Biphenylen im Hinblick auf eine
11
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Gewasserbelastung mit diesen Stoffen. Wasser und Abwasser Forschung
7:169-175(1974).
(2) Veith, G.D. and V.M. Comstock. Apparatus for continuously satu-
rating water with hydrophobic organic chemicals. Journal of the Fishing
Research Board of Canada 32:1849-1851 (1975).
(3) Organization for Economic Cooperation and Development, Guide-
lines for The Testing of Chemicals, OECD 105, Water Solubility (Column
Elution Method—Shake Flask Method), OECD, Paris, France.
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