799.6755 TSCA partition coefficient (n-octanol/water), shake flask method.§ 799.6755 TSCA partition coefficient (n-octanol/water), shake flask method.
(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:
(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.
Table 1 - Data for Reference Substances
|Tested substance 1||Pow 2|
|Di(2-ethylhexyl)phthalate (OECD)||1.3 × 10 5 (4.6 × 10 4 − 2.8 × 10 5)|
|Hexachlorobenzene (OECD)||3.6 × 10 5 (1.1 × 10 5 − 8.3 × 10 5)|
|o-Dichlorobenzene European Economic Community (EEC)||5.1 × 10 3 (1.5 × 10 3 − 2.3 × 10 4)|
|Dibutyl phthalate (EEC)||1.3 × 10 4 (1.7 × 10 3 − 2.8 × 10 4)|
|Trichloroethylene (OECD)||2.0 × 10 3 (5.2 × 10 2−3.7 × 10 3)|
|Urea (OECD)||6.2 × 10−2 (2.0 × 10−2 - 2.4 × 10−1)|
1 Substances not tested: Ethyl acetate, 4-methyl-2,4-pentanediol.
2 Total, mean, and range of mean values (in parentheses) submitted by the participants of the OECD or EEC Laboratory Intercomparison Testing.
(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:
(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.
(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]