United States Prevention, Pesticides EPA712-C-96-042
Environmental Protection and Toxic Substances August 1996
Agency (7101)
&EPA Product Properties
Test Guidelines
OPPTS 830.7860
Water Solubility
(Generator Column
Method)
-------
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 Public Access Gopher (gopher.epa.gov) under the heading
"Environmental Test Methods and Guidelines."
-------
OPPTS 830.7860 Water solubility (generator column 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
796.1860 Water solubility (generator column method), and OPP guideline
63-8 Solubility (Pesticide Assessment Guidelines, Subdivision D: Product
Chemistry, EPA Report 540/9-82-018, October 1982).
(b) Introduction—(1) Purpose, (i) The water solubility of a chemical
is defined as the equilibrium concentration of the chemical in a saturated
aqueous solution at a given temperature and pressure. The aqueous phase
solubility is an important factor in governing the movement, distribution,
and rate of degradation of chemicals in the environment. Substances that
are relatively water soluble are more likely to be widely distributed by
the hydrologic cycle than those which are relatively insoluble. Further-
more, substances with higher water solubility are more likely to undergo
microbial or chemical degradation in the environment because dissolution
makes them "available" to interact and, therefore, react with other chemi-
cals and microorganisms. Both the extent and rate of degradation via hy-
drolysis, photolysis, oxidation, reduction, and biodegradation depend on
a chemical being soluble in water (i.e., homogeneous kinetics).
(ii) Water provides the medium in which many organisms live, and
water is a major component of the internal environment of all living orga-
nisms (except for dormant stages of certain life forms). Even organisms
which are adapted to life in a gaseous environment require water for nor-
mal functioning. Water is thus the medium through which most other
chemicals are transported to and into living cells. As a result, the extent
to which chemicals dissolve in water will be a major determinant for
movement through the environment and entry into living systems.
(iii) The water solubility of a chemical also has an effect on its sorp-
tion into and desorption from soils and sediments, and on volatilization
from aqueous media. The more soluble a chemical substance is, the less
likely it is to sorb to soils and sediments and the less likely it is to volatil-
ize from water. Finally, the design of most chemical tests and many eco-
logical and health tests requires precise knowledge of the water solubility
of the chemical to be tested.
(2) Definitions and units, (i) Concentration of a solution is the
amount of solute in a given amount of solvent or solution and can be
expressed as a weight/weight or weight/volume relationship. The conver-
sion from a weight relationship to one of volume incorporates density as
a factor. For dilute aqueous solutions, the density of the solvent is approxi-
-------
mately equal to the density of the solution; thus, concentrations expressed
in milligrams per liter (mg/L) are approximately equal to 10'3 g/103 g
or parts per million (ppm); those expressed in micrograms per liter
(|-ig/L) are approximately equal to 10-6 g/103 g or parts per billion (ppb).
In addition, concentration can be expressed in terms of molarity, normality,
molality, and mole fraction. For example, to convert from weight/volume
to molarity molecular mass is incorporated as a factor.
(ii) Density is the mass of a unit volume of a material. It is a function
of temperature, hence the temperature at which it is measured should be
specified. For a solid, it is the density of the impermeable portion rather
than the bulk density. For solids and liquids, suitable units of measurement
are grams per cubic centimeter (g/cm3). The density of a solution is the
mass of a unit volume of the solution and suitable units of measurement
are grams per cubic centimeter.
(iii) A saturated solution is a solution in which the dissolved solute
is in equilibrium with an excess of undissolved solute; or a solution in
equilibrium such that at a fixed temperature and pressure, the concentration
of the solute in the solution is at its maximum value and will not change
even in the presence of an excess of solute.
(iv) A solution is a homogeneous mixture of two or more substances
constituting a single phase.
(v) A generator column is used to produce or generate saturated solu-
tions of a solute in a solvent. The column (see figure 1 under paragraph
(c)(l)(i)(A) of this guideline) is packed with a solid support coated with
the solute, i.e., the organic compound whose solubility is to be determined.
When water (the solvent) is pumped through the column, saturated solu-
tions of the solute are generated. Preparation of the generator column is
described under paragraph (c)(l)(i)(A) of this guideline.
(vi) An extractor column is used to extract the solute from the satu-
rated solutions produced by the generator column. After extraction onto
a chromatographic support, the solute is eluted with a solvent/water mix-
ture and subsequently analyzed by high pressure liquid chromatography
(HPLC), gas chromatography (GC), or any other suitable analytical proce-
dure. A detailed description of the preparation of the extractor column
is given in paragraph (c)(l)(i)(D) of this guideline.
(vii) The sample loop is a Vie in O.D. (1.6 mm) stainless steel tube
with an internal volume between 20 and 50 (iL. The loop is attached to
the sample injection valve of the HPLC and is used to inject standard
solutions into the mobile phase of the HPLC when determining the re-
sponse factor for the recording integrator. The exact volume of the loop
must be determined as described in paragraph (c)(3)(ii)(B)(7) of this guide-
line when the HPLC method is used.
-------
(viii) The response factor (RF) is the solute concentration required
to give a 1 unit area chromatographic peak or 1 unit output from the HPLC
recording integrator at a particular recorder attenuation. The factor is re-
quired to convert from units of area to units of concentration. The deter-
mination of the response factor is given in paragraph (c)(3)(ii)(B)(2) of
this guideline.
(3) Principle of the test method, (i) This test method is based on
the dynamic coupled column liquid chromatographic (DCCLC) technique
for determining the aqueous solubility of organic compounds that was ini-
tially developed by May et al. (see paragraphs (e)(5) and (e)(6) of this
guideline), modified by DeVoe et al. (see paragraph (e)(l) of this guide-
line), and finalized by Wasik et al. (see paragraph (e)(ll) of this guide-
line). The DCCLC technique utilizes a generator column, extractor column
and HPLC coupled or interconnected to provide a continuous closed flow
system. Saturated aqueous solutions of the test compound are produced
by pumping water through the generator column that is packed with a
solid support coated with the compound. The compound is extracted from
the saturated solution onto an extractor column, then eluted from the ex-
tractor column with a solvent/water mixture and subsequently analyzed
by HPLC using a variable wavelength UV detector operating at a suitable
wavelength. Chromatogram peaks are recorded and integrated using a re-
cording integrator. The concentration of the compound in the effluent from
the generator column, i.e., the water solubility of the compound, is deter-
mined from the mass of the compound (solute) extracted from a measured
volume of water (solvent).
(ii) Since the HPLC method is only applicable to compounds that
absorb in the UV, an alternate gas chromatographic (GC) method, or any
other reliable procedure (which must be approved by OPPTS), can be used
for those compounds that do not absorb in the UV. In the GC method
the saturated solutions produced in the generator column are extracted
using an appropriate organic solvent that is subsequently injected into the
GC, or any other suitable analytical device, for analysis of the test
compound.
(4) Reference chemicals. The following table 1 lists the water
solubilities at 25 °C for a number of reference chemicals as obtained from
the scientific literature. The data from Wasik et al. (see paragraph (e)(ll)
of this guideline), Miller et al. (see paragraph (e)(7) of this guideline),
and Tewari et al. (see paragraph (e)(10) of this guideline) were obtained
from the generator column method. The water solubilities were also ob-
tained from Yalkowski et al. (see paragraph (e)(13) of this guideline),
Mackay et al. (see paragraph (e)(4) of this guideline) and other scientists
by the conventional shake-flask method. These data have been provided
primarily so that the generator column method can be calibrated from time
to time and to allow the chemical testing laboratory an opportunity to com-
pare its results with those listed in the following table 1. The water solu-
-------
bility values at 25 °C reported by Yalkowski et al. (see paragraph (e)(13)
of this guideline) are their preferred values and, in general, represent the
best available water solubility data at 25 °C. The testing laboratory has
the option of choosing its own reference chemicals, but references must
be given to establish the validity of the measured values of the water solu-
bility and these chemicals must be approved by OPPTS.
Table 1.—Water Solubilities at 25 tC of Some Reference Chemicals
Reference chemical
2-Heptanone
1-Chlorobutane
Ethylbenzene
1 ,2,3-Trimethylbenzene
Biphenyl
Phenanthrene
2 4 6-Trichlorobiphenyl
2,3,4,5-Tetrachlorobiphenyl
Hexachlorobenzene
2.3.4.5.6-PentachlorobiDhenvl
Water solubility (ppm at 25 °C)
Wasik (gen-
erator col-
umn meth-
od)
40802
8732
1872
65. 52
6.71310
1 .0024
0.2263 10
0.02093 10
0.005483 10
Yalkowski1
5
4300
872.9
208
75.2
7.48
1.212
0.225
0.01396
0.004669
0.004016
Other lit-
erature ref-
erences
43305
6667
1627
48.27
6.628
0.1198
0.01 928
0.009969
0.00688
1 Preferred water solubility at 25 °C by Yalkowski et al. (1990) under paragraph (e)(13) of this guide-
line based on a critical review of all the experimental water solubility data published.
2 Tewari et al. (1982) under paragraph (e)(10) of this guideline.
3 Leifer et al. (1983) under paragraph (e)(3) of this guideline.
4 May, Wasik, and Freeman (1978, 1978a) under paragraphs (e)(5) and (6) of this guideline.
5 Yalkowski et al. (1990) under paragraph (e)(13) of this guideline.
6 Hansch et al. (1968) under paragraph (e)(2) of this guideline.
7 Button and Calder (1975) under paragraph (e)(9) of this guideline.
8 Mackay et al. (1980) under paragraph (e)(4) of this guideline.
9 The elution chromatographic method from OECD (1981) under paragraph (e)(8) of this guideline.
10 Miller et al. (1984) under paragraph (e)(7) of this guideline.
(5) Applicability and specificity, (i) Procedures are described in this
test guideline to determine the water solubility for liquid or solid com-
pounds. The water solubility can be determined in very pure water, buffer
solution for compounds that reversibly ionize or protonate, or in artificial
seawater as a function of temperature (i.e., in the range of temperatures
of environmental concern). This guideline is not applicable to the water
solubility of gases.
(ii) This test guideline is designed to determine the water solubility
of a solid or liquid test chemical in the range of 1 ppb to 5000 ppm.
For chemicals whose solubility is below 1 ppb, the water solubility should
be characterized as "less than 1 ppb" with no further quantification. For
solubilities greater than 5000 ppm, the shake flask method under OPPTS
830.7840 should be used.
(c) Test procedure—(1) Test conditions—(i) Special laboratory
equipment. (A) Generator column. Either of two different designs shall
-------
be used depending on whether the eluted aqueous phase is analyzed by
HPLC under paragraph (c)(3)(ii) of this guideline or by solvent extraction
followed by GC (or any other reliable quantitative) analysis of solvent
extract under paragraph (c)(3)(iv) of this guideline. The design of the gen-
erator column is shown in the following figure 1:
FIGURE 1—GENERATOR COLUMN
- GLASS WOOL
-SUPPORT (100-120 MESH
CHROMOSORB W)
-6mm
-GLASS WOOL
The column consists of a 6 mm (V4-inch) O.D. Pyrex tube joined to a
short enlarged section of 9 mm Pyrex tubing which in turn is connected
to another section of 6 mm (V4-inch) O.D. Pyrex tubing. Connections to
the inlet Teflon tubing (Vs-inch O.D.) and to the outlet stainless steel tub-
ing (Vie-inch O.D.) shall be made by means of stainless steel fittings with
Teflon ferrules. The column is enclosed in a water jacket for temperature
control as shown in the following figure 2:
-------
FIGURE 2—SETUP SHOWING GENERATOR COLUMN ENCLOSED IN A
WATER JACKET AND OVERALL ARRANGEMENT OF THE APPARATUS USED
IN THE GC METHOD
I-—GENERATOR
01 COLUMN
I I INLET
BATH —
RETURN
COLLECTING-
VESSEL
-GENERATOR
COLUMN
-TO CONSTANT
TEMPERATURE
BATH
-EXTRACTING
SOLVENT
-WATER CONTAINING
SOLUTE
(B) Constant temperature bath with circulation pump-bath and capable
of controlling temperature to + 0.05 °C. (See paragraph (c)(3) of this
guideline.)
(C) High pressure liquid chromatograph equipped with a variable
wavelenth UV absorption detector operating at a suitable wavelength and
a recording integrator under paragraph (c)(3)(ii) of this guideline.
(D) Extractor column. 6.6 x 0.6 cm stainless steel tube with end fit-
tings containing 5 (im frits filled with a superficially porous phase packing
(Bondapack C7S/Corasil: Waters Associates) under paragraph (c)(3)(ii) of
this guideline.
(E) Two 6-port high pressure rotary switching valves under paragraph
(c)(3)(ii) of this guideline.
-------
(F) Collection vessel. An 8 x 3/4 in section of Pyrex tubing with a
flat bottom connected to a short section of 3/s-in O.D. borosilicate glass
tubing in figure 2 under paragraph (c)(l)(i)(A) of this guideline. The col-
lecting vessel is sealed with a 3/s-in Teflon cap fitting under paragraph
(c)(3)(iii) of this guideline.
(G) Gas chromatograph, or any other reliable analytical equipment,
which has a detector sensitive to the solute of interest under paragraph
(c)(3)(iii) of this guideline.
(ii) Purity of water. Water meeting ASTM Type II standards, or an
equivalent grade, shall be used to minimize the effects of dissolved salts
and other impurities on water solubility. ASTM Type II water is described
in ASTM D 1193-77, "Standard Specification for Reagent Water,"
ASTM D 1193-77. Copies of this material may be obtained from the
American Society for Testing and Materials (ASTM), 1916 Race Street,
Philadelphia, PA 19103.
(iii) Purity of solvents. All solvents used in this method shall be
reagent or HPLC grade. Solvents shall contain no impurities which could
interfere with the determination of the test compound.
(iv) Seawater. When the water solubility in seawater is desired, the
artificial seawater described in paragraph (c)(2)(ii) of this guideline shall
be used.
(v) Effect of pH on solubility. For chemicals that reversibly ionize
or protonate with a pKa or pKb between 3 and 11, experiments shall be
performed at pH's 5.0, 7.0, and 9.0 (or any other pH's specified by
OPPTS) using appropriate buffers.
(2) Preparation of reagents and solutions—(i) Buffer solutions.
Prepare buffer solutions as follows:
(A) pH 3.0—To 250 mL of 0.10M potassium hydrogen phosphate
add 111 mL of 0.10 M hydrochloric acid; adjust the final volume to
500 mL with reagent grade water.
(B) pH 5.0—To 250 mL of 0.1M potassium hydrogen phthalate add
113 mL of 0.1M sodium hydroxide; adjust the final volume to 500 mL
with reagent grade water.
(C) pH 7.0—To 250 mL of 0.1M potassium dihydrogen phosphate
add 145 mL of 0.1M sodium hydroxide; adjust the final volume to
500 mL with reagent grade water.
(D) pH 9.0—To 250 mL of 0.075M borax add 69 mL of 0.1M HC1;
adjust the final volume to 500 mL with reagent grade water.
-------
(E) pH 11.0—To 250 mL of 0.05 M sodium bicarbonate add
113 mL of 0.10 M sodium hydroxide; adjust the final volume to
500 mL with reagent grade water.
Check the pH of each buffer solution with a pH meter at 25 °C and adjust
to pH 5.0, 7.0, or 9.0, if necessary. If the pH of the solution has changed
by ±0.2 pH units or more after the addition of the test compound, then
a more concentrated buffer is required for that pH determination. The
sponsor should then choose a more suitable buffer.
(ii) Artificial seawater. Add the reagent-grade chemicals listed in
the following table 2 in the specified amounts and order to 890 mL of
reagent-grade water. Each chemical shall be dissolved before another one
is added.
Table 2.—Constituents of Artificial Seawater1
Chemical
NaF
SrCI26H2O
H3BO3
KBr
KCI
CaCI22H2O
Na2SO4
MgCI26H2O
NaCI
Na2SiO39H2O
NaHCO,
Amount
3 mg
20 mg
30 mg
100 mg
700 mg
1 47 q
400 q
1078 g
23.50 g
20 mg
200 ma
1 If the resulting solution is diluted to 1 cubic decimeter (1 L), the salinity should be 34+0.5 g/kg and
the pH 8.0+0.2. The desired test salinity is attained by dilution at time of use.
(3) Performance of the test. Using either the procedures under para-
graph (c)(3)(ii) or (c)(3)(iii) of this guideline, determine the water solu-
bility of the test compound at 25 °C in reagent grade water or buffer solu-
tion, as appropriate. Under certain circumstances, it may be necessary to
determine the water solubility of a test compound at 25 °C in artificial
seawater. The water solubility can also be determined at other temperatures
of environmental concern by adjusting the temperature of the water bath
to the appropriate temperature.
(i) Prior to the determination of the water solubility of the test chemi-
cal, two procedures shall be followed.
(A) The saturated aqueous solution leaving the generator column must
be tested for the presence of an emulsion, using a Tyndall procedure. If
colloids are present, they must be eliminated prior to the injection into
the extractor column. This may be achieved by lowering the flow rate
of the water.
8
-------
(B) The efficiency of the removal of the solute (i.e. test chemical)
by the solvent extraction from the extraction column must be determined
and used in the determination of the water solubility of the test chemical.
(ii) Procedure A—HPLC Method—(A) Scope. (7) Procedure A
covers the determination of the aqueous solubility of compounds which
absorb in the UV. The HPLC analytical system is shown schematically
in the following figure 3:
FIGURE 3—SCHEMATIC OF HPLC—GENERATOR COLUMN FLOW SYSTEM
GENERATOR COLUMN
SAMPLE INJECTION VALVE
SAMPLE LOOP
EXTRACTOR COLUMN
WASTE
Two reciprocating piston pumps deliver the mobile phase (water or sol-
vent/water mixture) through two 6-port high pressure rotary valves and
a 30 x 0.6 cm C7S/Corasil analytical column to a variable wavelength UV
absorption detector operating at a suitable wavelength; chromatogram
peaks are recorded and integrated with a recording integrator. One of the
6-port valves is the sample injection valve used for injecting samples of
standard solutions of the solute in an appropriate concentration for deter-
mining response factors of standard solutions of basic chromate for deter-
mining the sample loop volume. The other 6-port valve in the system
serves as a switching valve for the extractor column which is used to re-
move solute from the aqueous solutions.
(2) The general procedure for analyzing the aqueous phase is as fol-
lows (a detailed procedure is given in paragraph (c)(3)(ii)(B)(/) of this
guideline).
(/) Direct the aqueous solution to "Waste" (see figure 3 under para-
graph (c)(3)(ii)(A)(7) of this guideline) with the switching valve in the
inject position in order to equilibrate internal surfaces with the solution,
thus ensuring that the analyzed sample would not be depleted by solute
adsorption on surfaces upstream from the valve.
-------
(//) At the same time, water is pumped from the HPLC pumps in
order to displace the solvent from the extractor column.
(///) The switching valve is next changed to the load position to divert
a sample of the solution through the extractor column, and the liquid leav-
ing this column is collected in a weighing bottle. During this extraction
step, the mobile phase is changed to a solvent/water mixture to condition
the analytical column.
(iv) After the desired volume of sample is extracted, the switching
valve is returned to the inject position for elution and analysis. Assuming
that there is no breakthrough of solute from the extractor column during
the extraction step, the chromatographic peak represents all of the solute
in the sample, provided that the extraction efficiency is 100 percent. If
the extraction efficiency is less than 100 percent, then the extraction effi-
ciency shall be used to determine the actual weight of the solute extracted.
(v) The solute concentration in the aqueous phase is calculated from
the peak area and the weight of the extracted liquid collected in the weigh-
ing bottle.
(B) Determinations—(7) Sample Loop Volume. Accurate measure-
ment of the sample loop may be accomplished by using the
spectrophotometric method of Devoe et al. (see paragraph (e)(l) of this
guideline). For this method measure absorbance, Aioop, at 373 nm of at
least three solutions, each of which is prepared by collecting from the
sample valve an appropriate number, n, of loopfuls of an aqueous stock
solution of K.2CrO4 (1.3 percent by weight) and diluting to 50 mL with
0.2 percent KOH. (For a 20 (iL loop, use n = 5; for a 50 |iL loop, use
n = 2.) Also measure the absorbance, Ast0ck, of the same stock solution
after diluting 1:500 with 0.2 percent KOH. Calculate the loop volume to
the nearest 0.1 [iL using the equation:
Vioop =(Aioop/Astock)(10-4/n)
(2) Response Factor (RF). (/) For all determinations adjust the mo-
bile phase solvent/water ratio and flow rate to obtain a reasonable retention
time on the HPLC column. For example, typical concentrations of solvent
in the mobile phase range from 50 to 100 percent while flow rates range
from 1 to 3 mL/min; these conditions give a 3 to 5 min retention time.
(//) Prepare standard solutions of known concentrations of the solute
in a suitable solvent. Concentrations must give a recorder response within
the maximum response of the detector. Inject samples of each standard
solution into the HPLC system using the calibrated sample loop. Obtain
an average peak area from at least three injections of each standard sample
at a set absorbance unit full scale (AUFS), i.e., at the same absorbance
scale attenuation setting.
10
-------
(///) Calculate the response factor from the following equation:
Concentration (M)
Response Factor (RF) =
(Average Area) (AUFS)
Loading of the Generator Column. (/) The design of the genera-
tor column was described in paragraph (c)(l)(i) of this guideline and is
shown in figure 1 under paragraph (c)(l)(i)(A) of this guideline. To pack
the column, a plug of silanized glass wool is inserted into one end of
the 6 mm Pyrex tubing. Silanized diatomaceous silica support (about 0.5g
100-120 mesh Chromosorb W chromatographic support material) is
poured into the tube with tapping and retained with a second plug of
silanized glass wool.
(//) If the solute is a liquid, the column is loaded by pulling the liquid
solute through the dry support with gentle suction. If the solute is a solid,
a 1 percent solution of the solid in a volatile solvent is added to the dry
packing. The solvent is then distilled off the column under reduced pres-
sure. After loading the column draw water up through the column to re-
move entrapped air.
(4) Analysis of the solute. Use the following procedure to collect
and analyze the solute.
(/) Pump water to the generator column by means of a minipump
or pressurized water reservoir as shown in the following figure 4:
11
-------
FIGURE 4—WATER RESERVOIR FOR GC METHOD
'TO
COMPRESSED
GAS CYLINDER
TO GENERATOR
COLUMN INLET
With the switching valve (figure 3 under paragraph (c)(3)(ii)(A)(7) of this
guideline) in the inject position (i.e., water to waste), pump water through
the generator column at a flow rate of approximately 1 mL/min for ap-
proximately 5 minutes to bring the system into equilibrium.
(//) Flush out the solvent that remains in the system from previous
runs by changing the mobile phase to 100 percent H2O and allowing the
water to reach the HPLC detector, as indicated by a negative reading. As
soon as this occurs, place a 25 mL weighing bottle (weighed to the nearest
milligram) at the waste position and immediately turn the switching valve
to the load position.
(///) Collect an amount of water (as determined by trial and error)
in the weighing bottle, corresponding to the amount of solute adsorbed
by the extractor column that gives a large on-scale detector response. Dur-
ing this extraction step, switch back to the original HPLC mobile phase
composition, i.e., solvent/water mixture, to condition the HPLC analytical
column.
(iv) After the desired volume of sample has been extracted, turn the
switching valve back to the inject position (figure 3 under paragraph
12
-------
(c)(3)(ii)(A)(7) of this guideline); at the same time turn on the recording
integrator. The solvent/water mobile phase will elute the solute from the
extractor column and transfer the solute to the HPLC analytical column.
(v) Remove the weighing bottle, cap it and replace it with the waste
container. Determine the weight of water collected to the nearest milligram
and record the corresponding peak area. Using the same AUFS setting
repeat the analysis of the solute at least 2 more times and determine the
average ratio of peak area to grams of water collected. Calculate the solute
solubility in water using the following equation:
s = (997 g/L)(RF)(Vioop)(AUFS)(R)
where
s = solubility (M)
RF = response factor
Vioop = sample loop volume (L)
R = ratio of area to grams of water.
(iii) Procedure B—GC Method. (A) Scope. In the GC method, or
any other analytical method, aqueous solutions from the generator column
enter a collecting vessel (figure 2 under paragraph (c)(l)(i)(A) of this
guideline) containing a known weight of extracting solvent which is im-
miscible in water. The outlet of the generator column is positioned such
that the aqueous phase always enters below the extracting solvent. After
the aqueous phase is collected, the collecting vessel is stoppered and the
quantity of aqueous phase is determined by weighing. The solvent and
the aqueous phase are equilibrated by slowly rotating the collecting vessel.
The extraction efficiency of the solvent must be determined at this time.
A small amount of the extracting solvent is removed and injected into
a gas chromatograph equipped with an appropriate detector. The solute
concentration in the aqueous phase is determined from a calibration curve
constructed using known concentrations of the solute.
(B) Alternative Method. If another (approved) analytical method is
used instead of the GC, that method shall be used to determine quan-
titatively the amount of solute present in the extraction solvent.
(C) Determinations—(7) Calibration curve. (/) Prepare solute stand-
ard solutions of concentrations covering the range of the solute solubility.
Select a column and optimum GC operating conditions for resolution be-
tween the solute and solvent and the solute and extracting solvent. Inject
a known volume of each standard solution into the injection port of the
GC. For each standard solution determine the average of the ratio R of
peak area to volume (in microliters) for three chromatographic peaks from
three injections.
13
-------
(//) After running all the standard solutions, determine the coeffi-
cients, a and b, using a linear regression equation of concentration
(C) vs. R in the following form
C = aR + b
(///) If another analytical method is used, the procedures described
in paragraph (c)(3)(iii)(C)(7) of this guideline shall be used to determine
quantitatively the amount of solute in the extraction solvent.
(2) Loading of the generator column. The generator column is
packed and loaded with solute in the same manner as for the HPLC meth-
od (see paragraph (c)(3)(ii)(C)(3) of this guideline). As shown in figure
2 under paragraph (c)(l)(i)(A) of this guideline, attach approximately 20
cm of straight stainless steel tubing to the bottom of the generator column.
Connect the top of the generator column to a water reservoir (figure 4
under paragraph (c)(3)(ii)(C)(/)(/) of this guideline) using Teflon tubing.
Use air or nitrogen pressure (5 PSI) from an air or nitrogen cylinder to
force water from the reservoir through the column. Collect water in an
Erlenmeyer flask for approximately 15 min while the solute concentration
in water equilibrates; longer time may be required for less soluble com-
pounds.
Collection and extraction of the solute. During the equilibration
time, add a known weight of extracting solvent to a collection vessel which
can be capped. The extracting solvent should cover the bottom of the col-
lection vessel to a depth sufficient to submerge the collecting tube but
still maintain 100:1 water/solvent ratio. Record the weight (to the nearest
milligram) of a collection vessel with cap and extracting solvent. Place
the collection vessel under the generator column so that water from the
collecting tube enters below the level of the extracting solvent (figure 2
under paragraph (c)(l)(i)(A) of this guideline). When the collection vessel
is filled, remove it from under the generator column, replace cap, and
weigh the filled vessel. Determine the weight of water collected. Before
analyzing for the solute, gently shake the collection vessel contents for
approximately 30 min, controlling the rate of shaking so as not to form
an emulsion; rotating the flask end over end 5 times per minute is suffi-
cient.
(4) Analysis of the solute. (/) After shaking, allow the collection ves-
sel to stand for approximately 30 min; then remove a known volume of
the extracting solvent from the vessel using a microliter syringe and inject
it into the GC. Record the ratio of peak area to volume injected and, from
the regression equation of the calibration line, determine the concentration
of solute in the extracting solvent. The concentration of solute in water
C(M) is determined from the following equation:
C(M) = (Ces) [(dH20/des)][(ges/gH2o)]
14
-------
where
Ces is the concentration of solute in extracting solvent (M),
dmo and des are the densities of water and extracting solvent, respec-
tively, and
ges and gH2o are the grams of extracting solvent and water, respec-
tively, contained in the collection vessel.
(//) Make replicate injections from each collecting vessel to determine
the average solute concentration in water for each vessel. To make sure
the generator column has reached equilibrium, run at least two additional
(for a total of three) collection vessels and analyze the extracted solute
as described above. Calculate the water solubility of the solute from the
average solute concentration in the three vessels.
(iv) Modification of procedures for potential problems. If the test
compound decomposes in one or more of the aqueous solvents required
during the period of the test at a rate such that an accurate value for water
solubility cannot be obtained, then it will be necessary to carry out detailed
transformation studies; e.g., hydrolysis under OPPTS 830.2110. If decom-
position is due to aqueous photolysis, then it will be necessary to carry
out water solubility studies in the dark, under red or yellow lights, or by
any other suitable method to eliminate this transformation process.
(d) Data and reporting—(1) Test report, (i) For each set of condi-
tions, (e.g., temperature, pure water, buffer solution, artificial seawater)
required for the study, provide the water solubility value for each of three
determinations, the mean value, and the standard deviation.
(ii) For compounds that decompose at a rate such that a precise value
for the water solubility cannot be obtained, provide a statement to that
effect.
(iii) For compounds with water solubility below 1 ppb, report the
value as "less than 1 ppb".
(2) Specific analytical, calibration and recovery procedures, (i) For
the HPLC method describe and/or report:
(A) The method used to determine the sample loop volume and the
average and standard deviation of that volume.
(B) The average and standard deviation of the response factor.
(C) Any changes made or problems encountered in the test procedure.
(ii) For the GC, or any other analytical, method report:
15
-------
(A) The column and GC operating conditions of temperature and flow
rate, or the operating conditions of any other analytical method used.
(B) The average and standard deviation of the average area per
microliter obtained for each of the standard solutions.
(C) The form of the regression equation obtained in the calibration
procedure.
(D) The extracting solvent used, and its extraction efficiency.
(E) The average and standard deviation of solute concentration in
each collection vessel.
(F) Any changes made or problems encountered in the test procedure.
(G) If applicable, a complete description of the analytical method
which was used instead of the GC method.
(e) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) DeVoe, H. et al., Generator columns and high pressure liquid
chromatography for determining aqueous solubilities and octanol-water
partition coefficients of hydrophobic substances. Journal of Research, Na-
tional Bureau of Standards, 86:361-366 (1981).
(2) Hansch, C. et al., The linear free-energy relationship between par-
tition coefficients, and the aqueous solubility of organic liquids. Journal
ofOranic Chemistry 33:347-350 (1968).
(3) Leifer, A. et al., Environmental transport and transformation of
polychlorinated biphenyls. Chapter 1. U.S. Environmental Protection
Agency Report: EPA-560/5-83-005 (1983).
(4) Mackay, D. et al., Relationship between aqueous solubility and
octanol-water partition coefficient. Chemosphere 9:701-711 (1980).
(5) May, W.E. et al., Determination of the aqueous solubility of
polynuclear aromatic hydrocarbons by a coupled column liquid
chromatographic technique. Analytical Chemistry 50:175-179 (1978).
(6) May, W.E. et al. Determination of the solubility behavior of some
polycyclic aromatic hydrocarbons in the water. Analytical Chemistry,
50:997-1000 (1978a).
(7) Miller, N.M. et al., Aqueous solubilities, octanol/water partition
coefficients, and entropy of melting of chlorinated benzenes and biphenyls.
Journal of Chemical and Engineering Data 29:184-190 (1984).
16
-------
(8) OECD/Organization for Economic Cooperation and Development.
Test Guideline No. 105. Water solubility column elution-flask method
(1981).
(9) Sutton, C. and Calder, J.A., Solubility of alkylbenzenes in distilled
water and seawater at 25 °C. Journal of Chemical and Engineering Data
20:320-322 (1975).
(10) Tewari , Y.B. et al., Aqueous solubility and octanol/water parti-
tion coefficient of organic compounds at 25 °C. Journal of Chemical and
Engineering Data 27:451-454 (1982).
(11) Wasik, S.P. et al., Octanol/Water Partition Coefficient and Aque-
ous Solubilities of Organic Compounds. NBS Report NBSIR 81-2406.
Washington, DC: National Bureau of Standards, U.S. Department of Com-
merce (1981).
(12) Wasik. S.P. et al., Water solubility and octanol/water partition
coefficient of polychlorinated biphenyls and other selected substances.
Task 1C. Interagency Agreement EPA-80-D-X0958 between the U.S. En-
vironmental Protection Agency and the National Bureau of Standards
(1982).
(13) Yalkowski, S.H. et al., "Arizona database of aqueous solubilities
of organic compounds"; Fifth Edition. University of Arizona, College of
Pharmacy, Tucson, AZ 85721 (1990).
17
------- |