(a) Scope—(1) Applicability. This section is intended to meet the testing requirements of the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601).

(2) Source. The source material used in developing this TSCA test guideline is the Office of Prevention, Pesticides and Toxics (OPPTS) harmonized test guideline 830.7550 (August 1996, final guideline). The source is available at the address in paragraph (f) of this section.

(b) Introductory information—(1) Prerequisites. Suitable analytical method, dissociation constant, water solubility, and hydrolysis (preliminary test).

(2) Coefficient of variation. The coefficient of variation on the mean values reported by the participants of the Organization for Economic Coopertion and Development (OECD) Laboratory Intercomparison Testing, Part I, 1979, appeared to be dependent on the chemicals tested; it ranges from 0.17 to 1.03.

(3) Qualifying statements. This method applies only to pure, water soluble substances which do not dissociate or associate, and which are not surface active. In order to use the partition coefficient (P) as a screening test for bioaccumulation, it should be ascertained that the impurities in the commercial product are of minor importance. Testing of P (n-octanol/water) cannot be used as a screening test in the case of organometallic compounds.

(4) Alternative methods. High-pressure liquid chromatography (HPLC) methods described in the references in paragraphs (f)(3), (f)(4), and (f)(5) of this section may be considered as an alternative test method.

(c) Method—(1) Introduction, purpose, scope, relevance, application, and limits of test. The P of a substance between water and a lipophilic solvent (n-octanol) is one model variable which may be used to describe the transfer of a substance from the aquatic environment into an organism and the potential bioaccumulation of the substance. Studies show a highly significant relationship between the P of different substances in the system water/n-octanol and their bioaccumulation in fish described in paragraph (f)(1) of this section.

(2) Definitions—Partition coefficient (P) is defined as the ratio of the equilibrium concentrations (Ci) of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. The P therefore is the quotient of two concentrations and is usually given in the form of its logarithm to base 10 (log P). In this case n-octanol and water:

$Equation\; 1:$

(3) Reference substances. The reference substances need not be employed 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 method is applied. The values presented in table 1 of this section are not necessarily representative of the results which can be obtained with this test method as they have been derived from an earlier version of the test guideline.

(4) Principle of the test method. In order to determine a P, equilibrium between all interacting components of the system must be achieved, and the concentrations of the substances dissolved in the two phases must be determined. A study of the literature on this subject indicates that there are many different techniques which can be used to solve this problem, i.e. the thorough mixing of the two phases followed by their separation in order to determine the equilibrium concentration for the substance being examined.

(5) Quality criteria—(i) Repeatability. In order to assure the precision of the P, duplicate determinations are to be made under three different test conditions, whereby the quantity of substance specified as well as the ratio of the solvent volumes may be varied. The determined values of the P expressed as their common logarithms should fall within a range of ±0.3 log units.

(ii) Sensitivity. The sensitivity of the method is determined by the sensitivity of the analytical procedure. This should be sufficient to permit the assessment of values of Pow up to 105 when the concentration of the solute in either phase is not more than 0.01 mol/Liter (L). The substance being tested must not be water insoluble (mass concentration ρ ≤10−6 gram (g)/L.

(iii) Specificity. The Nernst Partition Law applies only at constant temperature, pressure, and pH for dilute solutions. It strictly applies to a pure substance dispersed between two pure solvents. If several different solutes occur in one or both phases at the same time, this may affect the results. Dissociation or association of the dissolved molecules result in deviations from the Nernst Partition Law. Such deviations are indicated by the fact that the P becomes dependent upon the concentration of the solution. Because of the multiple equilibria involved, this test guideline should not be applied to ionizable compounds without corrections being made. The use of buffer solutions in place of water should be considered for such compounds.

(iv) Possibility of standardization. This method can be standardized.

(d) Description of the test procedure—(1) Preparations: Preliminary estimate of the P. The size of the P can be estimated either by means of calculation or by use of published solubilities of the test substance in the pure solvents. Alternatively, it may be roughly determined by performing a simplified preliminary test. For this:

$Equation\; 2:$

(2) Preparation of the solvents—(i) n-Octanol. The determination of the P should be carried out with analytical grade n-octanol. Inorganic contaminants can be removed from commercial n-octanol by washing with acid and base, drying, and distilling. More sophisticated methods will be required to separate the n-octanol from organic contaminants with similar vapor pressure if they are present.

(ii) Water. Distilled water or water twice-distilled from glass or quartz apparatus should be employed. Water taken directly from an ion exchanger should not be used.

(iii) Presaturation of the solvents. Before a P is determined, the phases of the solvent system are mutually saturated by shaking at the temperature of the experiment. For doing this, it is practical to shake two large stock bottles of purified n-octanol or distilled water each with a sufficient quantity of the other solvent for 24 hours on a mechanical shaker, and then to let them stand long enough to allow the phases to separate and to achieve a saturation state.

(3) Preparation for the test. The entire volume of the two-phase system should nearly fill the test vessel. This will help prevent loss of material due to volatilization. The volume ratio and quantities of substance to be used are fixed by the following:

(i) The preliminary assessment of the P as discussed in paragraph (d)(1) of this section).

(ii) The minimum quantity of test substance required for the analytical procedure.

(iii) The limitation of a maximum concentration in either phase of 0.01 mol/L.

(iv) Three tests are carried out. In the first, the calculated volume ratio is added; in the second, twice the volume of n-octanol is added; and in the third, half the volume of n-octanol is added.

(4) Test substance. The test substance should be the purest available. For a material balance during the test a stock solution is prepared in n-octanol with a mass concentration between 1 and 100 milligram/milliliter (mg/mL). The actual mass concentration of this stock solution should be precisely determined before it is employed in the determination of the P. This solution should be stored under stable conditions.

(5) Test conditions. The test temperature should be kept constant (±1 °C) and lie in the range of 20-25 °C.

(6) Performance of the test—(i) Establishment of the partition equilibrium. Duplicate test vessels containing the required, accurately measured amounts of the two solvents together with the necessary quantity of the stock solution should be prepared for each of the test conditions. The n-octanol parts should be measured by volume. The test vessels should either be placed in a suitable shaker or shaken by hand. A recommended method is to rotate the centrifuge tube quickly through 180° about its transverse axis so that any trapped air rises through the two phases. Experience has shown that 50 such rotations are usually sufficient for the establishment of the partition equilibrium. To be certain, 100 rotations in 5 minutes are recommended.

(ii) Phase separation. In order to separate the phases, centrifugation of the mixture should be carried out. This should be done in a laboratory centrifuge maintained at room temperature, or, if a non-temperature-controlled centrifuge is used, the centrifuge tubes should be reequilibrated at the test temperature for at least 1 hour before analysis.

(7) Analysis. (i) For the determination of the P, it is necessary to analyze the concentrations of the test substance in both phases. This may be done by taking an aliquot of each of the two phases from each tube for each test condition and analyzing them by the chosen procedure. The total quantity of substances present in both phases should be calculated and compared with the quantity of the substance originally introduced.

(ii) The aqueous phase should be sampled by the following procedure to minimize the risk of including traces of the n-octanol: A glass syringe with a removable needle should be used to sample the water phase. The syringe should initially be partially filled with air. Air should be gently expelled while inserting the needle through the n-octanol layer. An adequate volume of aqueous phase is withdrawn into the syringe. The syringe is quickly removed from the solution and the needle detached. The contents of the syringe may then be used as the aqueous sample.

(iii) The concentration in the two-separated phases should preferably be determined by a substance-specific method. Examples of physical-chemical determinations which may be appropriate are:

(A) Photometric methods.

(B) Gas chromatography.

(C) HPLC.

(D) Back-extraction of the aqueous phase and subsequent gas chromatography.

(e) Data and reporting—(1) Treatment of results. The reliability of the determined values of P can be tested by comparison of the means of the duplicate determinations with the overall mean.

(2) Test report. The following should be included in the report:

(i) Name of the substance, including its purity.

(ii) Temperature of the determination.

(iii) The preliminary estimate of the P and its manner of determination.

(iv) Data on the analytical procedures used in determining concentrations.

(v) The measured concentrations in both phases for each determination. This means that a total of 12 concentrations must be reported.

(vi) The weight of the test substance, the volume of each phase employed in each test vessel, and the total calculated amount of test substance present in each phase after equilibration.

(vii) The calculated values of the P and the mean should be reported for each set of test conditions as should the mean for all determinations. If there is a suggestion of concentration dependency of the P, this should be noted in the report.

(viii) The standard deviation of individual P values about their mean should be reported.

(ix) The mean P from all determinations should also be expressed as its logarithm (base 10).

(f) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.

(1) Neely, W.B. et al. Partition Coefficients to Measure Bioconcentration Potential of Organic Chemicals in Fish. Environmental Science and Technology 8:1113 (1974).

(2) Leo, A. et al. Partition Coefficients and Their Uses. Chemical Reviews 71:525 (1971).

(3) Miyake, K. and H. Terada, Direct measurements of partition coefficients in an octanol-water system. Journal of Chromatography 157:386 (1978).

(4) Veith G.D. and R.T. Morris, A Rapid Method for Estimating Log P for Organic Chemicals, EPA-600/3-78-049 (1978).

(5) Mirrless, M.S. et al., Direct measurement of octanol-water partition coefficient by high pressure liquid chromatography. Journal of Medicinal Chemistry 19:615 (1976).

(6) EPA Draft Guidance of September 8, 1978 (F-16).

(7) Konemann H. et al. Determination of log Poct values of chlorosubstituted benzenes, toluenes, and anilines by high performance liquid chromatography on ODS silica, Journal of Chromatography 178:559 (1979).

(8) Organization for Economic Cooperation and Development, Guidelines for The Testing of Chemicals, OECD 107, Partition Coefficient (n-octanol/water) (Shake Flask Method, Adopted 27 July 1995), OECD, Paris, France.

[65 FR 78751, Dec. 15, 2000, as amended at 77 FR 46293, Aug. 3, 2012]

(a) Scope—(1) Applicability. This section is intended to meet the testing requirements of the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601).

(2) Source. The source material used in developing this TSCA test guideline is the Office of Pollution Prevention, Pesticides and Toxic Substances (OPPTS) harmonized test guideline 830.7560 (August 1996, final guideline). This source is available at the address in paragraph (e) of this section.

(b)(1) Purpose. (i) The measurement and estimation of the n-octanol/water partition coefficient (Kow), has become the cornerstone of a myriad of structure-activity relationships (SAR) property. The coefficient has been used extensively for correlating structural changes in drugs with changes observed in biological, biochemical, or toxic effects. These correlations are then used to predict the effect of a new drug for which a Kow could be measured.

(ii) In the study of the environmental fate of organic chemicals, the Kow has become a key parameter. Kow is correlated to water solubility, soil/sediment sorption coefficient, and bioconcentration and is important to SAR.

(iii) Of the three properties that can be estimated from Kow, water solubility is the most important because it affects both the fate and transport of chemicals. For example, highly soluble chemicals become quickly distributed by the hydrologic cycle, have low-sorption coefficients for soils and sediments, and tend to be more easily degraded by microorganisms. In addition, chemical transformation processes such as hydrolysis, direct photolysis, and indirect photolysis (oxidation) tend to occur more readily if a compound is soluble.

(iv) Direct correlations between Kow and both the soil/sediment sorption coefficient and the bioconcentration factor are to be expected. In these cases, compounds that are more soluble in n-octanol (more hydrophobic and lipophilic) would be expected to partition out of the water and into the organic portion of soils/sediments and into lipophilic tissue. The relationship between Kow and the bioconcentration factor, are the principal means of estimating bioconcentration factors. This relationship is discussed in the reference listed in paragraph (e)(14) of this section. These factors are then used to predict the potential for a chemical to accumulate in living tissue.

(v) This section describes a method for determining the Kow based on the dynamic coupled column liquid chromatographic (DCCLC) technique, a technique commonly referred to as the generator column method. The method described herein can be used in place of the standard shake-flask method specified in § 799.6755 for compounds with a log10Kow greater than 1.0.

(2) Definitions. The following definitions apply to this section.

Extractor column is used to extract the solute from the aqueous solution produced by the generator column. After extraction onto a bonded chromatographic support, the solute is eluted with a solvent/water mixture and subsequently analyzed by high-performance liquid chromatography (HPLC), gas chromatography (GC), or any other analytical procedure. A detailed description of the preparation of the extractor column is given in paragraph (c)(1)(i) of this section.

Generator column is used to partition the test substance between the n-octanol and water phases. The column in figure 1 in paragraph (c)(1)(i)(A)(2) of this section is packed with a solid support and is coated with the test substance at a fixed concentration in n-octanol. The test substance is eluted from the column with water and the aqueous solution leaving the column represents the equilibrium concentration of the test substance that has partitioned from the n-octanol phase into the water phase. Preparation of the generator column is described in paragraph (c)(1)(i) of this section.

n-Octanol/water partition coefficient (Kow) is defined as the ratio of the molar concentrations of a chemical in n-octanol and water, in dilute solution. The coefficient Kow is a constant for a given chemical at a given temperature. Since Kow is the ratio of two molar concentrations, it is a dimensionless quantity. Sometimes Kow is reported as the decadic logarithm (log10Kow). In this equation, Coctanol and Cwater are the molar concentration of the solute in n-octanol and water, respectively, at a given temperature. This test procedure determines Kow at 25 ±0.05 °C. The mathematical statement of Kow is:

$Equation\; 1:$

Response factor (RF) is the solute concentration required to give a one unit area chromatographic peak or one unit output from the HPLC recording integrator at a particular recorder and detector attenuation. The factor is required to convert from units of area to units of concentration. The determination of the RF is given in paragraph (c)(3)(iii)(C)(2) of this section.

Sample loop is a 1/16 inch (in) outside diameter (O.D.) (1.6 millimeter (mm)) stainless steel tube with an internal volume between 20 and 50 µL. 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 RF for the recording integrator. The exact volume of the loop must be determined as described in paragraph (c)(3)(iii)(C)(1) of this section when the HPLC method is used.

(3) Principle of the test method. (i) This test method is based on the DCCLC technique for determining the aqueous solubility of organic compounds. The development of this test method is described in the references listed in paragraphs (e)(6), (e)(12), and (e)(19) of this section. The DCCLC technique utilizes a generator column, extractor column, and HPLC coupled or interconnected to provide a continuous closed-flow system. Aqueous solutions of the test compound are produced by pumping water through the generator column that is packed with a solid support coated with an approximately 1.0% weight/weight (w/w) solution of the compound in n-octanol. The aqueous solution leaving the column represents the equilibrium concentration of the test chemical which has partitioned from the n-octanol phase into the water phase. The compound is extracted from the aqueous solution onto an extractor column, then eluted from the extractor column with a solvent/water mixture and subsequently analyzed by HPLC using a variable wavelength ultraviolet (UV) absorption detector operating at a suitable wavelength. Chromatogram peaks are recorded and integrated using a recording integrator. The concentration of the compound in the effluent from the generator column is determined from the mass of the compound (solute) extracted from a measured volume of water (solvent). The Kow is calculated from the ratio of the molar concentration of the solute in the 1.0% (w/w) n-octanol and molar concentration of the solute in water as determined using the generator column technique.

(ii) Since the HPLC method is only applicable to compounds that absorb in the UV, an alternate GC method, or any other reliable quantitative procedure must 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. (i) Columns 2, 3, 4, and 5 of table 1 in paragraph (b)(4)(ii) of this section list the experimental values of the decadic logarithm of the n-octanol/water partition coefficient (log10Kow) at 25 °C for a number of organic chemicals as obtained from the scientific literature. These values were obtained by any one of the following experimental methods: Shake-flask; generator column; reverse-phase HPLC; or reverse-phase thin-layer chromatography, as indicated in the footnotes following each literature citation. The estimation method of Hawker and Connell as described in paragraph (e)(8) of this section, correlates log10Kow with the total surface area of the molecule and was used to estimate log10Kow for biphenyl and the chlorinated biphenyls. These estimated values are listed in column 7 of table 1 in paragraph (b)(4)(ii) of this section. Recommended values of log10Kow were obtained by critically analyzing the available experimental and estimated values and averaging the best data. These recommended values are listed in column 8 of table 1 in paragraph (b)(4)(ii) of this section.

(ii) The recommended values listed in table 1 of this section have been provided primarily so that the generator column method can be calibrated and to allow the chemical laboratory the opportunity to compare its results with these values. The testing laboratory has the option of choosing its reference chemicals, but references must be given to establish the validity of the measured values of log10Kow.

(5) Applicability and specificity. The test guideline is designed to determine the Kow of solid or liquid organic chemicals in the range log10Kow 1.0 to ≤6.0 (10 to ≤10 6).

(c) Test procedure—(1) Test conditions—(i) Special laboratory equipment—(A)(1) Generator column. Either of two different methods for connecting to the generator column shall be used depending on whether the eluted aqueous phase is analyzed by HPLC (Procedure A, as described in paragraph (c)(3)(iii) of this section) or by solvent extraction followed by GC analysis, or any other reliable method of solvent extract (Procedure B, as described in paragraph (c)(3)(iv) of this section).

(2)(i) The design of the generator column is shown in the following figure 1:

(ii) The column consists of a 6 mm ( 1/4 in) 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 ( 1/4 in) O.D. pyrex tubing. Connections to the inlet teflon tubing ( 1/8 in O.D.) and to the outlet stainless steel tubing ( 1/16 in O.D.) are 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 GC Method

(B) Constant temperature bath with circulation pump-bath and capable of controlling temperature to 25 ±0.05 °C. (Procedures A and B, as described in paragraphs (c)(3)(iii) and (c)(3)(iv) of this section, respectively).

(C) HPLC equipped with a variable wavelength UV absorption detector operating at a suitable wavelength and a recording integrator (Procedure A, as described in paragraph (c)(3)(iii) of this section).

(D) Extractor column—6.6 × 0.6 centimeter (cm) stainless steel tube with end fittings containing 5 micron frits filled with a superficially porous phase packing (such as Bondapack C18 Corasil: Waters Associates) (Procedure A, as described in paragraph (c)(3)(iii) of this section).

(E) Two 6-port high-pressure rotary switching valves (Procedure A, as described in paragraph (c)(3)(iii) of this section).

(F) Collection vessel—8 × 3/4 in section of pyrex tubing with a flat bottom connected to a short section of 3/8 in O.D. borosilicate glass tubing. The collecting vessel is sealed with a 3/8 in teflon cap fitting (Procedure B, as described in paragraph (c)(3)(iv) of this section).

(G) GC, or any other reliable analytic equipment, equipped with a detector sensitive to the solute of interest (Procedure B, as described in paragraph (c)(3)(iv) of this section).

(ii) Purity of n-octanol and water. Purified n-octanol, described in paragraph (c)(2)(i) of this section, and water meeting appropriate American Society for Testing and Materials Type II standards, or an equivalent grade, are recommended to minimize the effects of dissolved salts and other impurities. An ASTM Type II water standard is presented in the reference listed in paragraph (e)(20) of this section).

(iii) Purity of solvents. It is important that all solvents used in this method be reagent or HPLC grade and contain no impurities which could interfere with the determination of the test compound.

(iv) Reference compounds. In order to ensure that the HPLC system is working properly, at least two of the reference compounds listed in table 1 in paragraph (b)(4)(ii) of this section should be run. Reference compounds shall be reagent or HPLC grade to avoid interference by impurities.

(2) Preparation of reagents and solutions—(i) n-Octanol and water. Very pure n-octanol can be obtained as follows: Wash pure n-octanol (minimum 98% pure) sequentially with 0.1N H2SO4, with 0.1N NaOH, then with distilled water until neutral. Dry the n-octanol with magnesium sulfate and distill twice in a good distillation column under reduced pressure [b.p. about 80 °C at 0.27 kPa (2 torr)]. The n-octanol produced should be at least 99.9% pure. Alternatively, a grade equivalent to Fisher Scientific Co. No. A-402 “Certified Octanol-1” can be used. Reagent-grade water shall be used throughout the test procedure, such as ASTM Type II water, or an equivalent grade, as described in paragraph (c)(1)(ii) of this section.

(ii) Presaturated water. Prepare presaturated water with n-octanol to minimize the depletion of n-octanol from the column when measuring the Kowof a test chemical. This is very important when the test chemical is lipophilic and the log10Kow ≤4.

(3) Performance of the test. Initially, an approximately 1.0% (w/w) solution of the test substance in n-octanol is prepared. Precise measurement of the solute concentration in this solution is required for the Kowcalculation. Subsequently, the 1.0% (w/w) solution is coated on the generator column and using either Procedure A or Procedure B as described in paragraphs (c)(3)(iii) and (c)(3)(iv) of this section, the molar concentration of the test substance in reagent-grade water is determined.

(i) Test solution. The test solution consists of an approximately 1.0% (w/w) solution of the test substance in n-octanol. A sufficient quantity (about 10-20 milliliter (mL)) of the test solution should be prepared to coat the generator column. The solution is prepared by accurately weighing out, using a tared bottle, quantities of both the test substance and n-octanol required to make a 1.0% (w/w) solution. When the weights are measured precisely (to the nearest 0.1 milligram (mg)), knowing the density of n-octanol (0.827 gram (g)/mL at 25 °C), then the molar concentration of the test substance in the n-octanol is sufficiently accurate for the purposes of the test procedure. If desired, however, a separate analytical determination (e.g., by GC, or any other reliable analytical method) may be used to check the concentration in the test solution. If storage is required, the test solution should be kept stoppered to prevent volatilization of the test chemical.

(ii) Test procedures. Prior to the determination of the Kow of the test chemical, two procedures shall be followed:

(A) The saturated aqueous solution leaving the generator column shall be tested for the presence of an emulsion, using a Tyndall procedure (i.e. light scattering). If colloids are present, they must be removed prior to injection into the extractor column by lowering the flow rate of water.

(B) The efficiency of removal of the solute (the test chemical) by solvent extraction from the extractor column shall be determined and used in the determination of the Kow of the test chemical.

(iii) Procedure A—HPLC method. (A) Procedure A covers the determination of the aqueous solubility of compounds which absorb in the UV. Two reciprocating piston pumps deliver the mobile phase (water or solvent/water mixture) through two 6-port high-pressure rotary valves and a 30 × 0.6 cm C18 analytical column to a 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 determining RFs or standard solutions of basic chromate for determining 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 remove solute from the aqueous solutions. The HPLC analytical system is shown schematically in the following figure 3:

Figure 3—Schematic of HPLC—Generator Column Flow System

(B) The general procedure for analyzing the aqueous phase after equilibration is as follows; a detailed procedure is given in paragraph (c)(3)(iii)(C)(4) of this section:

(1) Direct the aqueous solution from the generator column to “Waste” in figure 3 in paragraph (c)(3)(iii)(A) of this section with the switching valve in the inject position in order to equilibrate internal surfaces with the solution, thus insuring that the analyzed sample would not be depleted by solute adsorption on surfaces upstream from the valve.

(2) At the same time, water is pumped from the HPLC pumps in order to displace the solvent from the extractor column.

(3) The switching valve is next changed to the load position to divert a sample of the solution from the generator column through the extractor column, and the liquid leaving the extractor column is collected in a tared weighing bottle. During this extraction step, the HPLC mobile phase is changed to a solvent/water mixture to condition the analytical column.

(4) After the desired volume of sample is extracted, the switching valve is returned to the inject position for elution from the extractor column and analysis. Assuming that all of the solute was adsorbed by the extractor column during the extraction step, the chromatographic peak represents all of the solute in the extracted sample, provided that the extraction efficiency is 100%. If the extraction efficiency is less than 100%, then the extraction efficiency shall be measured and used to determine the actual amount of the solute extracted.

(5) The solute concentration in the aqueous phase is calculated from the peak area, the weight of the extracted liquid collected in the weighing bottle, the extraction efficiency, and the RF.

(C)(1) Determination of the sample-loop volume. Accurate measurement of the sample loop may be accomplished by using a spectrophotometric method such as the one described in the reference listed in paragraph (e)(6) of this section. For this method, measure absorbance, Aloop, at 373 nanometers (nm) for 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 K2CrO4 (1.3% by weight) and diluting to 50 mL with 0.2% KOH. (For a 20 µL loop, use n = 5; for a 50 µL loop, use n = 2.) Also measure the absorbance, Astock, of the same stock solution after diluting 1:500 with 0.2% KOH. Calculate the loop volume to the nearest 0.1 µL using the relation:

$Equation\; 2:$

(2) Determination of the RF. (i) For all determinations adjust the mobile phase solvent/water ratio and flow rate to obtain a reasonable retention time on the HPLC column. For example, typical concentrations of organic solvent in the mobile phase range from 50 to 100% while flow rates range from 1 to 3 mL/minutes (min); these conditions often give a 3 to 5 min retention time.

(ii) 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 detector absorbance unit full scale (AUFS), i.e., at the same absorbance scale attenuation setting.

(iii) Calculate the RF from the following equation:

$Equation\; 3:$

(3) Loading of the generator column. (i) The design of the generator column was described in paragraph (c)(1)(i) of this section and is shown in figure 1 in paragraph (c)(1)(i)(A)(2)(i) of this section. 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 of 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.

(ii) The column is loaded by pulling the test solution through the dry support with gentle suction and then allowing the excess solution to drain out. After loading the column, draw water up through the column to remove any entrapped air.

(4) Analysis of the solute. Use the following procedure to collect and analyze the solute:

(i) With the switching valve in figure 3 in paragraph (c)(3)(iii)(A) of this section 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 approximately 15 min to bring the system into equilibrium. Pump water to the generator column by means of a minipump or pressurized water reservoir as shown in the following figure 4:

Figure 4— Water Reservoir for GC Method

(ii) Flush out the organic solvent that remains in the system from previous runs by changing the mobile phase to 100% 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 mg) at the waste position and immediately turn the switching valve to the load position.

(iii) Collect an amount of water from the generator column (as determined by trial and error) in the weighing bottle, corresponding to the amount of solute adsorbed by the extractor column that gives a reasonable detector response. During 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 in figure 3 in paragraph (c)(3)(iii)(A) of this section. As soon as the switching valve is turned to the inject position, remove the weighing bottle, cap it and replace it with the waste container; 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) Determine the weight of water collected to the nearest mg and record the corresponding peak area. Using the same AUFS setting repeat the analysis of the solute at least two more times and determine the average ratio of peak area to grams of water collected. In this equation, S = solubility (M), RF = response factor, Vloop = sample-loop volume (L), and R = ratio of area to grams of water. Calculate the solute solubility in water using the following equation:

$Equation\; 4:$

(iv) Procedure B—GC Method. In the GC method, or any other reliable quantitative method, aqueous solutions from the generator column enter a collecting vessel in figure 2 in paragraph (c)(1)(i)(A)(2)(ii) of this section containing a known weight of extracting solvent which is immiscible 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. A small amount of the extracting solvent is then removed and injected into a GC 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. The extraction efficiency of the solvent shall be determined in a separate set of experiments.

(A) Determination of calibration curve. (1) Prepare solute standard solutions of concentrations covering the expected range of the solute solubility. Select a column and optimum GC operating conditions for resolution between 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 µL) for the chromatographic peak of interest from at least three separate injections.

(2) After running all the standard solutions, determine the coefficients, a and b, using linear regression analysis on the equation of concentration (C) vs. R in the form:

$Equation\; 5:$

(B) Loading of the generator column. The generator column is packed and loaded with solute in the same manner as for the HPLC method in paragraph (c)(3)(iii) of this section. As shown in figure 2 in paragraph (c)(1)(i)(A)(2)(ii) of this section, 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 in figure 4 in paragraph (c)(3)(iii)(C)(4)(i) of this section 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 compounds.

(C) 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 collection vessel to a depth sufficient to submerge the collecting tube but still maintain 100:1 water/solvent ratio. Record the weight (to the nearest mg) 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 in figure 2 in paragraph (c)(1)(i)(A)(2)(ii) of this section. 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 rotate the collection vessel contents for approximately 30 min, controlling the rate of rotation so as not to form an emulsion; rotating the flask end over end five times per minute is sufficient. The extraction efficiency of the solvent shall be determined in a separate set of experiments.

(D) Analysis of the solute. (1) After rotating, allow the collection vessel 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. If the extraction efficiency is not 100%, the measured extraction efficiency shall be used to obtain the correct concentration of solute extracted. In this equation, Ces is the molar concentration of solute in extracting solvent, dH2O and des are the densities in grams per milliliter of water and extracting solvent, respectively, and ges and gH2O are the grams of extracting solvent and water, respectively, contained in the collection vessels. The molar concentration of solute in water C(M) is determined from the following equation:

$Equation\; 6:$

(2) 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 in paragraph (c)(3)(iv)(D)(1) of this section. Calculate C(M) from the average solute concentration in the three vessels.

(3) If another analytical method is used in place of the GC, then Procedure B, as described in paragraph (c)(3)(iv) of this section, shall be modified and the new analytical procedure shall be used to determine quantitatively the amount of solute extracted in the extraction solvent.

(v) Analysis of reference compounds. Prior to analyzing the test solution, make duplicate runs on at least two of the reference compounds listed in table 1 in paragraph (b)(4)(ii) of this section. When using the reference compounds, follow the same procedure previously described for preparing the test solution and running the test. If the average value obtained for each compound is within 0.1 log unit of the reference value, then the test procedure and HPLC system are functioning properly; if not a thorough checking over of the HPLC and careful adherence to the test procedures should be done to correct the discrepancy.

(vi) Modification of procedures for potential problems—Decomposition of the test compound. 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, such as hydrolysis studies. If decomposition is due to aqueous photolysis, then it will be necessary to carry out the 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 the test solution, report the weights to the nearest 0.1 mg of the test substance and n-octanol. Also report the weight percent and molar concentration of the test substance in the n-octanol; the density of n-octanol at 25 °C is 0.827 grams per milliliter (gm)/mL.

(ii) For each run provide the molar concentration of the test substance in water for each of three determinations, the mean value, and the standard deviation.

(iii) For each of the three determinations calculate the Kow as the ratio of the molar concentration of the test substance in n-octanol to the molar concentration in water. Also calculate and report the mean Kow and its standard deviation. Values of Kow shall be reported as their logarithms (log10Kow).

(iv) Report the temperature (±0.05 °C) at which the generator column was controlled during the test.

(v) For each reference compound report the individual values of log10Kow and the average of the two runs.

(vi) For compounds that decompose at a rate such that a precise value for the solubility cannot be obtained, provide a statement to that effect.

(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 RF.

(C) The extraction solvent and the extraction efficiency used.

(D) Any changes made or problems encountered in the test procedures.

(ii) For the GC method report:

(A) The column and GC operating conditions of temperature and flow rate.

(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 and extraction efficiency used.

(E) The average and standard deviation of solute concentration in each collection vessel.

(F) Any changes made or problems encountered in the test procedure.

(iii) If another approved analytical method is used to determine the concentration of the test chemical in water, then all the important test conditions shall be reported.

(iv) If the concentration of the test substance in n-octanol is determined by an independent analytical method such as GC, provide a complete description of the method.

(e) References. For additional background information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.

(1) Banerjee, S. et al., Water solubility and octanol/water partition coefficient of organics. Limitation of the solubility-partition coefficient correlation. Environmental Science and Technology 14:1227-1229 (1980).

(2) Bruggemann W.A. et al., Reversed-phase thin-layer chromatography of polynuclear aromatic hydrocarbons and chlorinated biphenyls. Relationship with hydrophobicity as measured by aqueous solubility and octanol/water partition coefficient. Journal of Chromatography 238: 335-346 (1982).

(3) Chiou, C.T. et al. Partition coefficient and bioaccumulation of selected organic chemicals. Environmental Science and Technology 11:475-478 (1977).

(4) Chiou, C.T. and Schmedding, D.W., Partitioning of organic compounds in octanol/water systems. Environmental Science and Technology 16:4-10 (1982).

(5) Chiou, C.T et al., Partition equilibria of nonionic organic compounds between soil, organic matter, and water. Environmental Science and Technology 17:227-231 (1983).

(6) 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 of the National Bureau of Standards, 86:361-366 (1981).

(7) Fujita, T. et al. “A New Substituent Constant, Derived from Partition Coefficients.” Journal of the American Chemical Society, 86:5175 (1964).

(8) Hansch, C. and Leo, A. 1985 MEDCHEM Project, version 26. Pomona College, Claremont, CA. USA.

(9) Hansch, C. and Leo, A. Medchem Software Manual. CLOGP3 Users Guide. Release 3.32. December 1984. Medicinal Chemistry Project, Pomona College, Claremont, CA.

(10) Hawker, D.W. and Connell, D.W. Octanol-water partition coefficients of polychlorinated biphenyl congeners. Environmental Science and Technology 22:382-387 (1988).

(11) 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).

(12) May, W.E. et al. “Determination of the Solubility Behavior of Some Polycyclic Aromatic Hydrocarbons in Water,” Analytical Chemistry 50:997-1000 (1978).

(13) Miller, M.M. et al. Aqueous solubilities, octanol/water partition coefficients and entropies of melting of chlorinated benzenes and biphenyls. Journal of Chemical and Engineering Data 29:184-190 (1984).

(14) Neely, W.B. et al. Partition Coefficient to Measure Bioconcentration Potential of Organic Chemicals in Fish, Environmental Science Technology, 8:113-115 (1974).

(15) Rappaport, R.A. and Eisenrich, S.J. Chromatographic determination of octanol-water partition coefficients (Kow's) for 58 polychlorinated biphenyl congeners. Environmental Science and Technology 18:163-170 (1984).

(16) Tewari, Y.B. et al. Aqueous solubility and octanol/water partition coefficients of organic compounds at 25 °C. Journal of Chemical and Engineering Data 27:451-454 (1982).

(17) Tulp, M.T.M. and Hutzinger, O. Some thoughts on aqueous solubilities and partition coefficients of PCB, and the mathematical correlation between bioaccumulation and physio-chemical properties. Chemosphere 10:849-860 (1978).

(18) Veith, G.D. et al. A rapid method for estimating log10 P for organic chemicals, Water Research 13:43-47 (1979).

(19) Wasik, S.P. et al. Octanol/water partition coefficient and aqueous solubilities of organic compounds, Report NBSIR 81-2406 (1981). National Bureau of Standards, U.S. Department of Commerce, Washington, DC.

(20) Woodburn, K.B. Measurement and application of the octanol/water partition coefficients for selected polychlorinated biphenyls. Master's Thesis (1982), University of Wisconsin at Madison, Madison, WI.

(21) Woodburn, K.B. et al. Generator column determination of octanol/water partition coefficients for selected polychlorinated biphenyl congeners. Environmental Science and Technology 18:457-459 (1984).

(22) ASTM D 1193-91 (Approved Sep 15, 1991), “Standard Specification for Reagent Water.” American Society for Testing and Materials (ASTM), 1916 Race St., Philadelphia, PA 19103.

[65 FR 78751, Dec. 15, 2000, as amended at 77 FR 46293, Aug. 3, 2012]

(a) Scope—(1) Applicability. This section is intended to meet the testing requirements of the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601).

(2) Source. The source material used in developing this TSCA test guideline is the Office of Pollution Prevention, Pesticides and Toxics (OPPTS) harmonized test guideline 830.7840 (March 1998, revised final guideline). This source is available at the address in paragraph (f) of this section.

(b) Introductory information—(1) Prerequisites. Suitable analytical method, structural formula, vapor pressure curve, dissociation constant, and hydrolysis independence of pH (preliminary test).

(2) Coefficient of variation. The coefficient of variation on the mean values reported by the participants of the Organization for Economic Cooperation and Development (OECD) Laboratory Intercomparison 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 volatile 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 gram/Liter (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.

(c) Method—(1) Introduction, purpose, scope, relevance, application, and limits of test. (i) A solution is a homogeneous mixture of different substances in a solvent. The particle sizes of the dispersed substances 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 substances. Therefore, this section 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. 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 killogram/meter (kg/m) 3; g/L may also be used.

(3) Reference substances. The reference substances need not be employed 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 method is applied. The values presented in table 1 of this section are not necessarily representative of the results which can be obtained with this test method as they have been derived from an earlier version of the test method.

(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 microcolumn which is charged with an inert carrier material such as glass beads, silica gel, or sand, and an excess of test substance. The water solubility is determined when the mass concentration of the eluate is constant. This is shown by a concentration plateau as a function of time in the following figure 1:

Figure 1—Concentration versus Time of Substance in the Eluate

(ii) Flask method. In this method, the substance (solids must be pulverized) 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. Such a procedure is described in the reference listed in paragraph (f)(2) of this section. Subsequently, the mass concentration 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 method <30% is acceptable; for the flask method <15% 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. These methods 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.

(D) Organic substances for the column elution method.

(iv) Possibility of standardization. These methods can be standardized.

(d) Description of the test procedures—(1) Preparations—(i) Apparatus—(A) Column elution method. (1) The schematic arrangement of the system is presented in the following figure 2:

Figure 2—Schematic Test Arrangement

(2) Although any size is acceptable, provided it meets the criteria for reproducibility 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. A suitable microcolumn is shown in the following figure 3:

Figure 3—Microcolumn (all dimensions in millimeters)

(3) The column should be connected to a recycling pump capable of controlling flows of approximately 25 mL/hours (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 material is supported with a small (5 millimeter (mm)) plug of glass wool, which must also serve to filter particles.

(B) Flask method. For the flask method, the following material is needed:

(1) 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 emulsions.

(4) Equipment for analytical determinations.

(2) Reagents. The substance to be tested should be as pure as possible, particularly in the flask method where purification is not provided. The carrier material for the column elution method should be inert. Possible materials which can be employed are glass beads and silica. A suitable 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. 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 (including the leveling vessel).

(4) Performance of the tests—(i) Preliminary test. (A) To approximately 0.1 g of the sample (solid substances must be pulverized) in a glass-stoppered 10 milliliter (mL) graduated cylinder, increasing volumes of distilled water at room temperature are added according to the steps shown in Table 2 of this section:

(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 measuring 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 dissolution of the sample occurs. If the substance is still apparently insoluble, further dilution should be undertaken to ascertain whether the column elution or flask solubility method should be used.

(ii) Column elution—(A) Apparatus. (1) The equipment is arranged as shown in figures 2 and 3 in paragraphs (d)(1)(i)(A)(1) and (d)(1)(i)(A)(2) of this section. Approximately 600 milligrams (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 evaporator; 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 circulating pump. The two principles should be used alternatively.

(1) Leveling vessel, see figure 3 in paragraph (d)(1)(i)(A)(2) and figure 4 in paragraph (d)(4)(iii) of this section.

(i) The connection to the leveling vessel is made by using a ground glass joint which is connected by teflon tubing. It is recommended that a flow rate of approximately 25 mL/h be used. Successive eluate fractions should be collected and analyzed by the chosen method.

(ii) Fractions from the middle eluate range where the concentrations are constant (±30%) in at least five consecutive fractions are used to determine the solubility in water.

(iii) 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 in paragraphs (d)(1)(i)(A)(1) and (d)(1)(i)(A)(2) of this section.

(i) With this apparatus, the microcolumn must be modified. A stopcock with 2-way action must be used, see figure 3 in paragraph (d)(1)(i)(A)(2) of this section). The circulating pump can be, e.g. a peristaltic pump (be careful that no contamination and/or adsorption occurs with the tube material) or a membrane pump.

(ii) The flow through the column is started. It is recommended that a flow rate of approximately 25 mL/h be used (approximately 10 bed volumes per h for the described column). The first five-bed volumes (minimum) are discarded to remove water soluble impurities.

(iii) Following this, the recycling pump is connected and the apparatus allowed to run until equilibration is established, as defined by five successive samples whose concentrations do not differ by more than 30% in a random fashion (see paragraph (f)(2) of this section). These samples should be separated from each other by time intervals corresponding 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 examination 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 necessary to saturate the desired volume of water is estimated from the preliminary test. The volume of water required will depend on the analytical method and the solubility range. About five times the quantity of material determined in paragraph (d)(4)(i)(A) of this section 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 vessels 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 re-equilibrated 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 analytical method. The other two flasks are treated similarly after initial equilibration 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 one, two, and three show a tendency to increasing values. The arrangement of the apparatus is shown in the following figure 4:

Figure 4—Test Arrangement for the Determination of Solubility in Water of Slightly Soluble, Low Volatility Organic Substances 1 = Leveling vessel (e.g. 2.5 L chemical flask) 2 = Column (see figure 3 in paragraph (d)(1)(i)(A)(2) of this section) 3 = Fraction accumulator 4 = Thermostat 5 = Teflon tubing 6 = Glass stopper 7 = Water line (between thermostat and column, inner diameter: approximately 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, titration methods, photometric methods, and polarographic methods.

(e) Data and reporting—(1) Column elution method—(i) Treatment of results. The mean value from at least five consecutive samples taken from the saturation plateau (figure 1 in paragraph (c)(4)(i) of this section) 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%.

(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 samples.

(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 solution 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%) 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. For additional information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.

(1) Veith, G.D. and V.M. Comstock. Apparatus for continuously saturating water with hydrophobic organic chemicals. Journal of the Fishing Research Board of Canada 32:1849-1851 (1975).

(2) Organization for Economic Cooperation and Development, Guidelines for The Testing of Chemicals, OECD 105, Water Solubility (Column Elution Method—Shake Flask Method), OECD, Paris, France (1981).

[65 FR 78751, Dec. 15, 2000, as amended at 77 FR 46293, Aug. 3, 2012]

(a) Scope—(1) Applicability. This section is intended to meet the testing requirements of the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601).

(2) Source. The source material used in developing this TSCA test guideline is the Office of Pollution Prevention, Pesticides and Toxics (OPPTS) harmonized test guideline 830.7860 (March 1998, revised final guideline). The source is available at the address in paragraph (e) of this section.

(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. Furthermore, 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 chemicals and microorganisms. Both the extent and rate of degradation via hydrolysis, 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 organisms (except for dormant stages of certain life forms). Even organisms which are adapted to life in a gaseous environment require water for normal 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 sorption 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 volatilize from water. Finally, the design of most chemical tests and many ecological and health tests requires precise knowledge of the water solubility of the chemical to be tested.

(2) Definitions. The following definitions apply to this section.

Concentration (C) 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 conversion from a weight relationship to one of volume incorporates density as a factor. For dilute aqueous solutions, the density of the solvent is approximately equal to the density of the solution; thus, concentrations expressed in milligrams per liter (mg/L) are approximately equal to 10−3 g/10 3 g or parts per million (ppm); those expressed in micrograms per liter (µg/L) are approximately equal to 10−6 g/10 3 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.

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/cm 3). The density of a solution is the mass of a unit volume of the solution and suitable units of measurement are g/cm 3.

Extractor column is used to extract the solute from the saturated solutions produced by the generator column. After extraction onto a chromatographic support, the solute is eluted with a solvent/water mixture and subsequently analyzed by high-pressure liquid chromatography (HPLC), gas chromatography (GC), or any other suitable analytical procedure. A detailed description of the preparation of the extractor column is given in paragraph (c)(1)(i)(D) of this section.

Generator column is used to produce or generate saturated solutions of a solute in a solvent. The column, see figure 1 in paragraph (c)(1)(i)(A) of this section, 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 solutions of the solute are generated. Preparation of the generator column is described in paragraph (c)(1)(i)(A) of this section.

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 required to convert from units of area to units of concentration. The determination of the RF is given in paragraph (c)(3)(ii)(B)(2) of this section.

Sample loop is a 1/16 inch (in) outer diameter (O.D.) (1.6 millimeter (mm)) stainless steel tube with an internal volume between 20 and 50 µL. 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 RF for the recording integrator. The exact volume of the loop must be determined as described in paragraph (c)(3)(ii)(B)(1) of this section when the HPLC method is used.

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.

Solution is a homogeneous mixture of two or more substances constituting a single phase.

(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 initially developed by May et al. (as described in the references listed in paragraphs (e)(5) and (e)(6) of this section), modified by DeVoe et al. (as described in the reference listed in paragraph (e)(1) of this section), and finalized by Wasik et al. (as described in the reference listed in paragraph (e)(11) of this section). 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 extractor column with a solvent/water mixture and subsequently analyzed by HPLC using a variable wavelength ultraviolet (UV) detector operating at a suitable wavelength. Chromatogram peaks are recorded and integrated using a recording integrator. The concentration of the compound in the effluent from the generator column, i.e., the water solubility of the compound, is determined 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 GC method, or any other reliable procedure (which must be approved by OCSPP), 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. Table 1 of this section 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. (as described in the reference listed in paragraph (e)(11) of this section), Miller et al. and Tewari et al. (as described in the references listed in paragraphs (e)(7) and (e)(10) of this section, respectively) were obtained from the generator column method. The water solubilities data were also obtained from Mackay et al. and Yalkowski et al. (as described in the references listed in paragraphs (e)(4) and (e)(12) of this section, respectively) 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 compare its results with those listed in table 1 of this section. The water solubility values at 25 °C reported by Yalkowski et al. 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 solubility.

(5) Applicability and specificity. (i) Procedures are described in this section to determine the water solubility for liquid or solid compounds. 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 section is not applicable to the water solubility of gases.

(ii) This section is designed to determine the water solubility of a solid or liquid test chemical in the range of 1 ppb to 5,000 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 5,000 ppm, the shake flask method should be used, see paragraph (e)(15) of this section.

(c) Test procedure—(1) Test conditions—(i) Special laboratory equipment—(A) Generator column. (1) Either of two different designs shall be used depending on whether the eluted aqueous phase is analyzed by HPLC in paragraph (c)(3)(ii) of this section or by solvent extraction followed by GC (or any other reliable quantitative) analysis of solvent extract in paragraph (c)(3)(iv) of this section. The design of the generator column is shown in the following figure 1:

Figure 1—Generator Column

(2) The column consists of a 6 mm ( 1/4 in) 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 ( 1/4 in) O.D. pyrex tubing. Connections to the inlet teflon tubing ( 1/8 in O.D.) and to the outlet stainless steel tubing ( 1/16 in 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

(B) Constant temperature bath with circulation pump-bath and capable of controlling temperature to ±0.05 °C, see paragraph (c)(3) of this section.

(C) HPLC equipped with a variable wavelenth UV absorption detector operating at a suitable wavelength and a recording integrator in paragraph (c)(3)(ii) of this section.

(D) Extractor column—6.6 × 0.6 cm stainless steel tube with end fittings containing 5 µm frits filled with a superficially porous phase packing (Bondapack C18/Corasil: Waters Associates) in paragraph (c)(3)(ii) of this section.

(E) Two 6-port high-pressure rotary switching valves in paragraph (c)(3)(ii) of this section.

(F) Collection vessel—8 × 3/4 in section of pyrex tubing with a flat bottom connected to a short section of 3/8 in O.D. borosilicate glass tubing in figure 2 in paragraph (c)(1)(i)(A)(2) of this section. The collecting vessel is sealed with a 3/8 in teflon cap fitting in paragraph (c)(3)(iii) of this section.

(G) GC, or any other reliable analytical equipment, which has a detector sensitive to the solute of interest in paragraph (c)(3)(iii) of this section.

(ii) Purity of water. Water meeting appropriate American Society for Testing and Materials (ASTM) Type II standards, or an equivalent grade, are recommended to minimize the effects of dissolved salts and other impurities on water solubility. ASTM Type II water is presented in the reference listed in paragraph (e)(13) of this section.

(iii) Purity of solvents. All solvents used in this method must be reagent or HPLC grade. Solvents must 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 section must 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 must be performed at pH's 5.0, 7.0, and 9.0 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 HCl; 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 3 mL of 0.10 M sodium hydroxide; adjust the final volume to 500 mL with reagent grade water.

(ii) 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.

(iii) Artificial seawater. Add the reagent-grade chemicals listed in table 2 of this section in the specified amounts and order to 890 mL of reagent-grade water. Each chemical shall be dissolved before another one is added.

(3) Performance of the test. Using either the procedures in paragraph (c)(3)(ii) or (c)(3)(iii) of this section, determine the water solubility of the test compound at 25 °C in reagent-grade water or buffer solution, 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 chemical, 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.

(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. (1) Procedure A covers the determination of the aqueous solubility of compounds which absorb in the UV.

(i) The HPLC analytical system is shown schematically in the following figure 3:

Figure 3—Schematic of HPLC—Generator Column Flow System

(ii) Two reciprocating piston pumps deliver the mobile phase (water or solvent/water mixture) through two 6-port high-pressure rotary valves and a 30 × 0.6 cm C18/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 determining RFs of standard solutions of basic chromate for determining 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 remove solute from the aqueous solutions.

(2) The general procedure for analyzing the aqueous phase is as follows (a detailed procedure is given in paragraph (c)(3)(ii)(B)(4) of this section).

(i) Direct the aqueous solution to “Waste,” see figure 3 in paragraph (c)(3)(ii)(A)(1)(i) of this section, 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.

(ii) At the same time, water is pumped from the HPLC pumps in order to displace the solvent from the extractor column.

(iii) The switching valve is next changed to the load position to divert a sample of the solution through the extractor column, and the liquid leaving 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%. If the extraction efficiency is less than 100%, then the extraction efficiency 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 weighing bottle.

(B) Determinations—(1) Sample-loop volume. Accurate measurement of the sample loop may be accomplished by using the spectrophotometric method of Devoe et al. under paragraph (e)(1) of this section. For this method measure absorbance, Aloop, 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 K2CrO4 (1.3% by weight) and diluting to 50 mL with 0.2% KOH. (For a 20 µL loop, use n = 5; for a 50 µL loop, use n = 2.) Also measure the absorbance, Astock, of the same stock solution after diluting 1:500 with 0.2% KOH. Calculate the loop volume to the nearest 0.1 µL using the equation:

$Equation\; 1:$

(2) RF. (i) For all determinations adjust the mobile 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% while flow rates range from 1 to 3 mL/min; these conditions give a 3 to 5 min retention time.

(ii) 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.

(iii) Calculate the RF from the following equation:

$Equation\; 2:$

(3) Loading of the generator column. (i) The design of the generator column was described in paragraph (c)(1)(i) of this section and is shown in figure 1 in paragraph (c)(1)(i)(A) of this section. 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.

(ii) 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% solution of the solid in a volatile solvent is added to the dry packing. The solvent is then distilled off the column under reduced pressure. After loading the column draw water up through the column to remove entrapped air.

(4) Analysis of the solute. Use the following procedure to collect and analyze the solute.

(i) With the switching valve (figure 3 in paragraph (c)(3)(ii)(A)(1)(i) of this section) 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 approximately 5 minutes (min) to bring the system into equilibrium. Pump water to the generator column by means of a minipump or pressurized water reservoir as shown in the following figure 4:

Figure 4—Water Reservoir for GC Method

(ii) Flush out the solvent that remains in the system from previous runs by changing the mobile phase to 100% 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 mg) at the waste position and immediately turn the switching valve to the load position.

(iii) 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. During 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 in paragraph (c)(3)(ii)(A)(1)(i) of this section); 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 mg and record the corresponding peak area. Using the same AUFS setting repeat the analysis of the solute at least two more times and determine the average ratio of peak area to grams of water collected. In this equation, s = solubility (M), RF = response factor, Vloop = sample-loop volume (L), and R = ratio of area to grams of water. Calculate the solute solubility in water using the following equation:

$Equation\; 3:$

(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 in paragraph (c)(1)(i)(A)(2) of this section) containing a known weight of extracting solvent which is immiscible 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 chromograph 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 quantitatively the amount of solute present in the extraction solvent.

(C) Determinations—(1) Calibration curve. (i) Prepare solute standard solutions of concentrations covering the range of the solute solubility. Select a column and optimum GC operating conditions for resolution between 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.

(ii) After running all the standard solutions, determine the coefficients, a and b, using a linear regression equation of C vs. R in the following form:

$Equation\; 4:$

(iii) If another analytical method is used, the procedures described in paragraph (c)(3)(iii)(C)(1) of this section 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 method described under paragraph (c)(3)(ii)(B)(3) of this section. As shown in figure 2 in paragraph (c)(1)(i)(A)(2) of this section, 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 in paragraph (c)(3)(ii)(B)(4)(i) of this section) 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 compounds.

(3) 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 collection vessel to a depth sufficient to submerge the collecting tube but still maintain 100:1 water/solvent ratio. Record the weight (to the nearest mg) 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 in paragraph (c)(1)(i)(A)(2) of this section). 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 five times per minute is sufficient.

(4) Analysis of the solute. (i) After shaking, allow the collection vessel 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. In this equation, Ces is the concentration of solute in extracting solvent (M), dH2O and des are the densities of water and extracting solvent, respectively, and ges and gH2O are the grams of extracting solvent and water, respectively, contained in the collection vessel. The concentration of solute in water C(M) is determined from the following equation:

$Equation\; 5:$

(ii) 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 in paragraph (e)(16) of this section. If decomposition 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 conditions, (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 RF.

(C) Any changes made or problems encountered in the test procedure.

(ii) For the GC, or any other analytical, method report:

(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. For additional information on this test guideline, the following references should be consulted. These references are available at the addresses in § 700.17(b)(1) and (2) of this chapter.

(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, National Bureau of Standards, 86:361-366 (1981).

(2) Hansch, C. et al., The linear free-energy relationship between partition coefficients, and the aqueous solubility of organic liquids. Journal of Organic 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., Relationships 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).

(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 partition 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 Aqueous Solubilities of Organic Compounds. NBS Report NBSIR 81-2406. Washington, DC: National Bureau of Standards, U.S. Department of Commerce (1981).

(12) Yalkowski, S.H. et al., “Aquasol database of aqueous solubilities of organic compounds”; Fifth Edition. University of Arizona, College of Pharmacy, Tucson, AZ 85721 (1990) (available at http://www.pharm.arizona.edu/aquasol/index.html).

(13) ASTM D 1193-91, Standard Specification for Reagent Water. American Society for Testing and Materials (ASTM). 1916 Race St., Philadelphia, PA 19103.

[65 FR 78751, Dec. 15, 2000, as amended at 77 FR 46293, Aug. 3, 2012]