PART 796—CHEMICAL FATE TESTING GUIDELINES Authority: 15 U.S.C. 2603. Subpart A [Reserved] Subpart B—Physical and Chemical Properties § 796.1050 Absorption in aqueous solution: Ultraviolet/visible spectra. (a) Introductory information Guidance information. (ii) Structural formula. (2) Standard documents. (b) Method Introduction, purpose, scope, relevance, application and limits of test. (B) Degradation will depend upon the total energy absorbed in specific wavelength regions. Such energy absorption is characterized by both molar absorption coefficient (molar extinction coefficient) and band width. However, the absence of measurable absorption does not preclude the possibility of photodegradation. (ii) Definitions and units. UV-VIS absorption spectrum 1 i For a resolvable absorbance peak, the band width λ is the wavelength range, expressed in nm = 10 −9 (iii) Reference substances. (B) Reference compounds appropriate for the calibration of the system are: ( 1 2 4 log ε 3.56 3.63 3.16 3.50 λ in nm 235 257 313 350 ( 2 C.R.C. Atlas of Spectral Data, log ε 4.75 4.18 4.73 3.91 3.92 λ in nm 237 236 288 339 357 ( 3 C.R.C. Atlas of Spectral Data, log ε 3.88 4.04 λ in nm 288 311 See also paragraph (d)(1) of this section. (iv) Principle of the test method. (v) Quality criteria—Reproducibility and sensitivity. 2 2 7 (B) In the event that a recording double-beam instrument is not available, it will be necessary to determine the absorbance of the test solution in a single-beam instrument at 5-nm intervals over the entire wavelength range and at 1-nm intervals where there are indicated absorbance maxima. Wavelength and absorbance tests should be done as with the double-beam instrument. (2) Description of the test procedure Preparation Preparation of test solutions. 1 ( 2 ( 3 ( 4 (B) Blank solutions. (C) Cells. (ii) Performance of the test. (c) Data and reporting Treatment of results. where the quantities are as defined above (see Definitions and units). (ii) For each peak which is capable of being resolved, either as recorded or by extrapolated symmetrical peaks, the bandwidth should be recorded. (2) Test report. (ii) For each maximum in each spectrum, the ε value and bandwidth (when applicable) should be calculated and reported, along with the wavelength of the maximum. This should be presented in tabular form. (iii) The various test conditions should be included, such as scan speed, the name and model of the spectrophotom-eter, the slit width (where available), cell type and path length, the concentrations of the test substance, and the nature and acidity of the solvent medium. A recent test spectrum on appropriate reference materials for photometric and wavelength accuracy should also be submitted (see Reproducibility and sensitivity). (d) Literature references. (1) Milazzo, G., Caroli, S., Palumbo-Doretti, M., Violante, N., Analytical Chemistry, (2) Katelaar, J.A.A., Photoelectric Spectrometry Group Bulletin, (3) Chemical Rubber Company, Atlas of Spectral Data, [50 FR 39472, Sept. 27, 1985] § 796.1950 Vapor pressure. (a) Introduction Background and purpose. (ii) Chemicals with relatively low vapor pressures, high adsorptivity onto solids, or high solubility in water are less likely to vaporize and become airborne than chemicals with high vapor pressures or with low water solubility or low adsorptivity to solids and sediments. In addition, chemicals that are likely to be gases at ambient temperatures and which have low water solubility and low adsorptive tendencies are less likely to transport and persist in soils and water. Such chemicals are less likely to biodegrade or hydrolyze and are prime candidates for atmospheric oxidation and photolysis (e.g., smog formation or stratospheric alterations). On the other hand, nonvolatile chemicals are less frequently involved in atmosphere transport, so that concerns regarding them should focus on soils and water. (iii) Vapor pressure data are an important consideration in the design of other chemical fate and effects tests; for example, in preventing or accounting for the loss of violatile chemicals during the course of the test. (2) Definitions and units. (ii) “Pascal” (Pa) is the standard international unit of vapor pressure and is defined as newtons per square meter (N/m 2 (iii) The “torr” is a unit of pressure which equals 133.3 pascals or 1 mm Hg at 0 °C. (iv) “Vapor pressure” is the pressure at which a liquid or solid is in equilibrium with its vapor at a given temperature. (v) “Volatilization” is the loss of a substance to the air from a surface or from solution by evaporation. (3) Principle of the test methods. (ii) Gas saturation (or transpiration) procedures use a current of inert gas passed through or over the test material slowly enough to ensure saturation and subsequent analysis of either the loss of material or the amount (and sometimes kind) of vapor generated. Gas saturation procedures have been described by Spencer and Cliath (1969) under paragraph (d)(2) of this section. Results are easy to obtain and can be quite precise. The same procedures also can be used to study volatilization from laboratory scale environmental simulations. Vapor pressure is computed on the assumption that the total pressure of a mixture of gases is equal to the sum of the pressures of the separate or component gases and that the ideal gas law is obeyed. The partial pressure of the vapor under study can be calculated from the total gas volume and the weight of the material vaporized. If v is the volume which contains w grams of the vaporized material having a molecular weight M, and if p is the pressure of the vapor in equilibrium at temperature T (K), then the vapor pressure, p, of the sample is calculated by p = (w/M)(RT/v), where R is the gas constant (8.31 Pa m 2 −1 −1 (iii) In an effort to improve upon the procedure described by Spencer and Cliath (1969) under paragraph (d)(2) of this section, and to determine the applicability of the gas saturation method to a wide variety of chemical types and structures, EPA has sponsored research and development work at SRI International (EPA 1982) under paragraph (d)(1) of this section. The procedures described in this Test Guideline are those developed under that contract and have been evaluated with a wide variety of chemicals of differing structure and vapor pressures. (4) Applicability and specificity. −5 3 (ii) With respect to the isoteniscope method, if compounds that boil close to or form azeotropes with the test material are present, it is necessary to remove the interfering compounds and use pure test material. Impurities more volatile than the sample will tend to increase the observed vapor pressure above its true value but the purification steps will tend to remove these impurities. Soluble, nonvolatile impurities will decrease the apparent vapor pressure. However, because the isoteniscope procedure is a static, fixed-volume method in which an insignificant fraction of the liquid sample is vaporized, it is subject to only slight error for samples containing nonvolatile impurities. That is, the nonvolatile impurities will not be concentrated due to vaporization of the sample. (iii) The gas saturation method is applicable to solid or liquid chemicals. Since the vapor pressure measurements are made at ambient temperatures, the need to extrapolate data from high temperatures is not necessary and high temperature extrapolation, which can often cause serious errors, is avoided. The method is most reliable for vapor pressures below 10 3 (b) Test procedures Test conditions. (ii) The apparatus used in the gas saturation method is described in paragraph (b)(2)(ii) of this section. (2) Performance of the tests Isoteniscope Procedure. http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html. Federal Register. (ii) Gas saturation procedure. Figure 1—Schematic Diagram of Vapor Saturation Apparatus The insulated box, containing sample holders, may be of any suitable size and shape. The sketch in Figure 1 shows a box containing three solid sample holders and three liquid sample holders, which allows for the triplicate analysis of either a solid or liquid sample. The temperature within the box is controlled to ±0.5° or better. Nitrogen gas, split into six streams and controlled by fine needle valves (approximately 0.79 mm orifice), flows into the box via 3.8 mm (0.125 in.) i.d. copper tubing. After temperature equilibration, the gas flows through the sample and the sorbent trap and exits from the box. The flow rate of the effluent carrier gas is measured at room temperature with a bubble flow meter or other suitable device. The flow rate is checked frequently during the experiment to assure that there is an accurate value for the total volume of carrier gas. The flow rate is used to calculate the total volume (at room temperature) of gas that has passed through the sample and sorbent [(vol/time) × time = volume]. The vapor pressure of the test substance can be calculated from the total gas volume and the mass of sample vaporized. If v is the volume of gas that transported mass w of the vaporized test material having a molecular weight M, and if p is the equilibrium vapor pressure of the sample at temperature T, then p is calculated by the equation p = (w/M)(RT/v). In this equation, R is the gas constant (8.31 Pa m 3 −1 −1 3 (B) Solid samples are loaded into 5 mm i.d. glass tubing between glass wool plugs. The following Figure 2 depicts a drawing of a sample holder and absorber system. Figure 2—Solid Compound Sampling System (C) Liquid samples are contained in a holder as shown in the following Figure 3. Figure 3—Liquid Compound Sampling System The most reproducible method for measuring the vapor pressure of liquids is to coat the liquid on glass beads and to pack the holder in the designated place with these beads. (D) At very low vapor pressures and sorbent loadings, adsorption of the chemical on the glass wool separating the sample and the sorbent and on the glass surfaces may be a serious problem. Therefore, very low loadings should be avoided whenever possible. Incoming nitrogen gas (containing no interfering impurities) passes through a coarse frit and bubbles through a 38 cm column of liquid sample. The stream passes through a glass wool column to trap aerosols and then through a sorbent tube, as described above. The pressure drop across the glass wool column and the sorbent tube are negligible. (E) With both solid and liquid samples, at the end of the sampling time, the front and backup sorbent sections are analyzed separately. The compound on each section is desorbed by adding the sorbent from that section to 1.0 ml of desorption solvent in a small vial and allowing the mixture to stand at a suitable temperature until no more test compound desorbs. It is extremely important that the desorption solvent contain no impurities which would interfere with the analytical method of choice. The resulting solutions are analyzed quantitatively by a suitable analytical method to determine the weight of sample desorbed from each section. The choice of the analytical method, sorbent, and desorption solvent is dictated by the nature of the test material. Commonly used sorbents include charcoal, Tenax GC, and XAD-2. Describe in detail the sorbent, desorption solvent, and analytical methods employed. (F) Measure the desorption efficiency for every combination of sample, sorbent, and solvent used. The desorption efficiency is determined by injecting a known mass of sample onto a sorbent and later desorbing it and analyzing for the mass recovered. For each combination of sample, sorbent, and solvent used, carry out the determination in triplicate at each of three concentrations. Desorption efficiency may vary with the concentration of the actual sample and it is important to measure the efficiency at or near the concentration of sample under gas saturation test procedure conditions. (G) To assure that the gas is indeed saturated with test compound vapor, sample each compound at three differing gas flow rates. Appropriate flow rates will depend on the test compound and test temperature. If the calculated vapor pressure shows no dependence on flow rate, then the gas is assumed to be saturated. (c) Data and reporting. (2) Provide a description of analytical methods used to analyze for the test material and all analytical results. (3) For the isoteniscope procedure, include the plot of p vs. the reciprocal of the temperature in K, developed during the degasing step and showing linearity in the region of 298.15 K (25 °C) and any other required test temperatures. (4) For the gas saturation procedure, include the data on the calculation of vapor pressure at three or more gas flow rates at each test temperature, showing no dependence on flow rate. Include a description of sorbents and solvents employed and the desorption efficiency calculations. (5) Provide a description of any difficulties experienced or any other pertinent information. (d) References. (1) U.S. Environmental Protection Agency. Evaluation of Gas Saturation Methods to Measure Vapor Pressures: Final Report, (2) Spencer, W.F. and Cliath, M.M. “Vapor Density of Dieldrin,” Journal of Agricultural and Food Chemistry, (3) Spencer, W.F. and Cliath, M.M. “Vapor Density and Apparent Vapor Pressure of Lindane,” Journal of Agricultural and Food Chemistry, [50 FR 39252, Sept. 27, 1985, as amended at 53 FR 12525, Apr. 15, 1988; 53 FR 21641, June 9, 1988; 60 FR 34466, July 3, 1995; 69 FR 18803, Apr. 9, 2004; 77 FR 46293, Aug. 3, 2012] Subpart C—Transport Processes § 796.2750 Sediment and soil adsorption isotherm. (a) Introduction Background and purpose. (2) Definitions and units. (ii) “Clay mineral analysis” is the estimation or determination of the kinds of clay-size minerals and the amount present in a sediment or soil. (iii) “Organic matter” is the organic fraction of the sediment or soil; it includes plant and animal residues at various stages of decomposition, cells and tissues of soil organisms, and substances synthesized by the microbial population. (iv) “Particle size analysis” is the determination of the various amounts of the different particle sizes in a sample (i.e., sand, silt, clay), usually by sedimentation, sieving, micrometry, or combinations of these methods. The names and diameter range commonly used in the United States are: Name Diameter range Very coarse sand 2.0 to 1.0 mm Coarse sand 1.0 to 0.5 mm Medium sand 0.5 to 0.25 mm Fine sand 0.25 to 0.125 mm Very fine sand 0.125 to 0.062 mm Silt 0.062 to 0.002 mm Clay <0.002 mm (v) The “pH” of a sediment or soil is the negative logarithm to the base ten of the hydrogen ion activity of the sediment or soil suspension. It is usually measured by a suitable sensing electrode coupled with a suitable reference electrode at a 1/1 solid/solution ratio by weight. (vi) The adsorption ratio, “K d (vii) “Sediment” is the unconsolidated inorganic and organic material that is suspended in and being transported by surface water, or has settled out and has deposited into beds. (viii) “Soil” is the unconsolidated mineral material on the immediate surface of the earth that serves as a natural medium for the growth of land plants. Its formation and properties are determined by various factors such as parent material, climate, macro- and microorganisms, topography, and time. (ix) “Soil aggregate” is the combination or arrangement of soil separates (sand, silt, clay) into secondary units. These units may be arranged in the soil profile in a distinctive characteristic pattern that can be classified according to size, shape, and degree of distinctness into classes, types, and grades. (x) “Soil classification” is the systematic arrangement of soils into groups or categories. Broad groupings are based on general soil characteristics while subdivisions are based on more detailed differences in specific properties. The soil classification system used in this standard and the one used today in the United States is the 7th Approximation-Comprehensive System. The ranking of subdivisions under this system is: Order, Suborder, Great group, family, and series. (xi) A “soil horizon” is a layer of soil approximately parallel to the land surface. Adjacent layers differ in physical, chemical, and biological properties such as color, structure, texture, consistency, kinds and numbers of organisms present, and degree of acidity or alkalinity. (xii) “Soil Order” is the broadest category of soil classification and is based on the general similarities of soil physical/chemical properties. The formation of soil by similar general genetic processes causes these similarities. The Soil Orders found in the United States are: Alfisol, Aridisol, Entisol, Histosol, Inceptisol, Mollisol, Oxisol, Spodosol, Ultisol, and Vertisol. (xiii) “Soil series” is the basic unit of soil classification and is a subdivision of a family. A series consists of soils that were developed under comparable climatic and vegetational conditions. The soils comprising a series are essentially alike in all major profile characteristics except for the texture of the “A” horizon (i.e., the surface layer of soil). (xiv) “Soil texture” is a classification of soils that is based on the relative proportions of the various soil separates present. The soil textural classes are: clay, sandy clay, silty clay, clay loam, silty clay loam, sandy clay loam, loam, silt loam, silt, sandy loam, loamy sand, and sand. (3) Principle of the test method. Equation 1 x/m = C s e l/n where: C e C s K = Freundlich adsorption coefficient m = The mass of the solid in grams l/n x = The mass in micrograms of the chemical adsorbed by m grams of solid. Logarithmetic transformation of the Freundlich equation yields the following linear relationship: Equation 2 log C s e (ii) In order to estimate the environmental movement of the test chemical, the values K and l/n are compared with the values of other chemicals whose behavior in soil and sediment systems is well-documented in scientific literature. (iii) The adsorption isotherm (AI) test has many desirable features. First, adsorption results are highly reproducible. The test provides excellent quantitative data readily amenable to statistical analyses. Also, it has relatively modest requirements for chemicals, soils, laboratory space, and equipment. It allows solution phase organic chemical determinations that are relatively uncomplicated. A chemical extraction-mass balance procedure to elicit information on chemical transformations occurring at colloid interfaces can be incorporated into this test. The ease of performing the isotherm test and mass balance will depend upon the physical/chemical properties of the test chemical and the availability of suitable analytical techniques to measure the chemical. (iv) The papers by Aharonson and Kafkafi (1975) under paragraph (d)(1) of this section, Harvey (1974) under paragraph (d)(3) of this section, Murray (1975) under paragraph (d)(4) of this section, Saltzman (1972) under paragraph (d)(5) of this section, Weber (1971) under paragraph (d)(6) of this section, and Wu (1975) under paragraph (d)(7) of this section served as the basis for this section. The soil and colloid chemistry literature and the analytical chemistry literature substantiate the experimental conditions and procedures specified in this guideline as accepted, standard procedures. (4) Applicability and specificity. (b) Test procedures Test conditions Special laboratory equipment. 2 2 (B) Containers shall be composed of material that ( 1 2 (C) A 150 micron (100 mesh) stainless-steel or brass sieve. (D) Drying oven, with circulating air, that can attain 100 °C. (E) Vortex mixer or a comparable device. (F) Rotary shaker or a comparable device. (G) High speed temperature-controlled centrifuge capable of sedimenting particles greater than 0.5 micron from aqueous solution. (ii) Temperature. (iii) Replications. (iv) Soil pretreatment. (A) Decrease the water content, air or oven-dry soils at or below 50 °C. (B) Reduce aggregate size before and during sieving, crush and grind dried soil very gently. (C) Eliminate microbial growth during the test period using a chemical or physical treatment that does not alter or minimally alters the soil surface properties. (D) Sieve soils with a 100 mesh stainless-steel or brass sieve. (E) Store all solutions and soils at temperatures between 0 and 5 °C. (v) Sediment pretreatment. (A) Decrease the H 2 (B) Eliminate microbial growth during the test period by using a chemical and/or physical treatment that does not alter or minimally alters the colloid surface's properties. (C) Store at temperatures between 0 and 5 °C. (vi) Solid/solution ratio. (vii) Equilibration time. (A) Equilibrate one solution containing a known concentration of the test chemical with the sediment or soil in a solid/solution ratio equal to or greater than 1/10 1/5 1 2 (B) Measure the concentration of the chemical in the solution phase at frequent intervals during the equilibration period. (C) Determine the equilibration time by plotting the measured concentration versus time of sampling; the equilibration time is the minimum period of time needed to establish a rate of change of solution concentration of 5 percent or less per 24 hours. (viii) Centrifugation time. c −5 Equation 3 t c 9 2 1 2 where: t c R 2 R 1 N = number of revolutions of the centrifuge per minute. (ix) Storage of solutions. (x) Solvents for extraction. (2) Test procedure Equilibration. (A) Immediately after the solutions are added to the solids, tightly cap the containers and vigorously agitate them for several minutes with a vortex mixture or similar device. (B) Shake the containers throughout the equilibration period at a rate that suspends all solids in the solution phase. (ii) Centrifugation. c (iii) Chemical extraction. (B) Extract the chemical adsorbed on the sediment or soil colloid surfaces with solvent. (iv) Chemical analysis. (c) Reporting. (1) Temperature at which the test was conducted. (2) Detailed description of the analytical technique(s) used in the chemical extraction, recovery, and quantitative analysis of the parent chemical. (3) Amount of parent test chemical applied, the amount recovered, and the percent recovered. (4) Extent of adsorption by containers and the approach used to correct the data for adsorption by containers. (5) The individual observations, the mean values, and graphical plots of x/m as a function of C e (6) The quantities K, n, and l/n. (7) Soil information: Soil Order, series, texture, sampling location, horizon, general clay fraction mineralogy. (8) Sediment information: sampling location, general clay fraction mineralogy. (9) Sediment and soil physical-chemical properties: percent sand, silt, and clay (particle size analysis); percent organic matter; pH (1/1 solids/H 2 (10) The procedures used to determine the physical/chemical properties listed under paragraphs (c) (7) through (9) of this section. (d) References. (1) Aharonson, N., Kafkafi, U. “Adsorption, mobility and persistence of thiabendazole and methyl 2-benzimidasole carbamate in soils,” Journal of Agricultural and Food Chemistry, (2) Goring, C.A.I., Hamaker, J.W., (eds). Organic Chemicals in the Soil Environment. (3) Harvey, R.G. et al. “Soil adsorption and volatility of dinitroaniline herbicides,” Weed Science, (4) Murray, D.S. et al. “Comparative adsorption, desorption, and mobility of dipropetryn and prometryn in soil,” Journal of Agricultural and Food Chemistry, (5) Saltzman, S.L. et al. “Adsorption, desorption of parathion as affected by soil organic matter,” Journal of Agricultural and Food Chemistry, (6) Weber, J.B. “Model soil system, herbicide leaching, and sorption,” Weed Science, (7) Wu, C.H., et al. “Napropamide adsorption, desorption, and movement in soils,” Weed Science, [50 FR 39252, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987; 54 FR 29715, July 14, 1989] Subpart D—Transformation Processes § 796.3100 Aerobic aquatic biodegradation. (a) Introduction Purpose. (ii) On the contrary, a low biodegradation result may have other causes than poor biodegradability of the test substance. Inhibition of the microbial inoculum by the test substance at the test concentration may be observed. In such cases, further work is needed to assess the aerobic aquatic biodegradability and to determine the concentrations at which toxic effects are evident. An estimate of the expected environmental concentration will help to put toxic effects into perspective. (2) Definitions. (ii) “Ultimate Biodegradability” is the breakdown of an organic compound to CO 2 (iii) “Ready Biodegradability” is an expression used to describe those substances which, in certain biodegradation test procedures, produce positive results that are unequivocal and which lead to the reasonable assumption that the substance will undergo rapid and ultimate biodegradation in aerobic aquatic environments. (3) Principle of the test method. 2 2 2 2 (4) Prerequisites. (5) Guideline information. (ii) Information on the toxicity of the chemical may be useful in the interpretation of low results and in the selection of appropriate test concentrations. (6) Reference substances. 2 (7) Reproducibility. (8) Sensitivity. 2 14 (9) Possibility of standardization. (10) Possibility of automation. (b) Test procedures Preparations Apparatus. 2 Figure 1—Shake-Flask System for Carbon Dioxide Evolution The Ba(OH) 2 2 2 2 2 (ii) Reagents and stock solutions. (B) Yeast extract. (C) Vitamin-free casamino acids. (D) 70 percent O 2 2 (E) 0.2N Ba(OH) 2 (F) 0.1 N HCl. (G) 20 percent H 2 4 (H) Phenolphthalein. (I) Dilution water—distilled, deionized water (DIW). (iii) Soil inoculum. Table 1—Medium Employed for Assay of CO 2 Solution 1 Compound Stock Solution Conc. (g/L) I NH 4 35 KNO 3 15 K 2 4 2 750 NaH 2 4 2 25 II 2 KCl 10 MgSO 4 20 FeSO 4 2 1 III CaCl 2 5 ZnCl 2 0.05 MnCl 2 2 0.5 CuCl 2 0.05 CoCl 2 0.001 H 3 3 0.001 MoO 3 0.0004 1 2 (iv) Acclimation Medium. (2) Procedures. 2 2 2 2 2 2 2 4 2 (ii) For each set of experiments, each test substance shall be tested in triplicate. (iii) For each set of experiments, one or two reference compounds are included to assess the microbial activity of the test medium. Duplicate reference flasks are prepared by adding reference compound equivalent to 10 mg/liter carbon to each of two flasks containing the test medium. Reference compounds which are positive for ultimate biodegradability include: sodium citrate, dextrose, phthalic acid, trimellitic acid, and aniline. (iv) For each test set, triplicate controls receiving inoculated medium and no test compound, plus all test and reference flasks, are analyzed for CO 2 2 (v) A test system containing a growth inhibitor should be established as a control for each substance tested for biodegradation by this method. That inhibited system must contain the same amount of water, mineral nutrients, inoculum, and test substance used in the uninhibited test systems, plus 50 mg/L mercuric chloride (HgCl 2 (vi) Flasks shall be incubated in the dark to minimize both photochemical reactions and algal growth. Appropriate sterile controls or controls containing a metabolic inhibitor, such as 50 mg/1 HgCl 2 2 2 (3) Analytical measurements. 2 2 2 2 2 (c) Data and reporting Treatment of results. 2 −1 2 3 2 2 2 Percent CO 2 where: TF = mL 0.1 N HCl required to titrate Ba(OH) 2 CF = mL 0.1 N HCl required to titrate Ba(OH) 2 (ii) The cumulative percent CO 2 2 (iii) The percent DOC disappearance from the test compound is calculated from the following equation: Percent DOC Removal=[1−(DTF x x o o where: DTF= Dissolved organic carbon from test flask DCF= Dissolved organic carbon from control flask o= Day zero measurements x= Day of measurements during test. (iv) The difference between the amount of 0.1 N HCl used for the Ba(OH) 2 2 (v) CO 2 2 2 2 (vi) Inhibition by the test compound is indicated by lower CO 2 2 (vii) The use of 14 (2) Test report. (ii) Information on the inoculum, including source, collection date, handling, storage and adaptation possibilities (i.e., that the inoculum might have been exposed to the test substance either before or after collection and prior to use in the test). (iii) Results from each test, reference, inhibited (with HgCl 2 (iv) Average cumulative percent theoretical CO 2 (v) Dissolved organic carbon due to test compound at each sampling time (DTF-DCF). (vi) Average percent DOC removal at each sampling time. (vii) Twenty-eight day standard deviation for percent CO 2 (d) References. (1) Gledhill, W.E. “Screening Test for Assessment of Ultimate Biodegradability: Linear Alkyl Benzene Sulfonate,” Applied Microbiology, (2) Pramer, D., Bartha, R. “Preparation and Processing of Soil Samples for Biodegradation Testing,” Environmental Letters, [50 FR 39252, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987] § 796.3500 Hydrolysis as a function of pH at 25 °C. (a) Introduction Background and purpose. (ii) Since hydrolysis can be such an important degradation path for certain classes of chemicals, it is necessary, in assessing the fate of these chemicals in the environment, to know whether, at what rate, and under what conditions a substance will hydrolyze. Some of these reactions can occur so rapidly that there may be greater concern about the products of the transformation than about the parent compounds. In other cases, a substance will be resistant to hydrolysis under typical environmental conditions, while, in still other instances, the substance may have an intermediate stability that can result in the necessity for an assessment of both the original compound and its transformation products. The importance of transformation of chemicals via hydrolysis in aqueous media in the environment can be determined quantitatively from data on hydrolysis rate constants. This hydrolysis Test Guideline represents a test to allow one to determine rates of hydrolysis at any pH of environmental concern at 25 °C. (2) Definitions and units. 2 (ii) “Elimination” is defined in this Test Guideline to be a reaction of an organic chemical (RX) in water in which the X group is lost. These reactions generally follow the same type of rate laws that hydrolysis reactions follow and, thus, are also covered in this Test Guideline. (iii) A “first-order reaction” is defined as a reaction in which the rate of disappearance of the chemical substance being tested is directly proportional to the concentration of the chemical substance and is not a function of the concentrations of any other substances present in the reaction mixture. (iv) The “half-life” of a chemical is defined as the time required for the concentration of the chemical substance being tested to be reduced to one-half its initial value. (v) “Hydrolysis” refers to a reaction of an organic chemical with water such that one or more bonds are broken and the reaction products incorporate the elements of water (H 2 RX + HOH → ROH + HX. (A) Another result of hydrolysis can be the incorporation of both H and OH in a single product. An example of this is the hydrolysis of epoxides, which can be represented by (B) The hydrolysis reaction can be catalyzed by acidic or basic species, including OH − 3 = = 3 − − Equation 1 −d[RX]/ d= h A = + k B − N 2 where K A B N h Equation 2 k h A = B − N where k N Equation 3 t 1/2 h At constant pH, Equation 1 can be integrated to yield the first order rate expression Equation 4 log 10 h 2.303 10 o where C is the concentration of the test chemical at time t and C o (C) At a given pH, Equation 2 under paragraph (a)(2)(v)(B) of this section contains three unknowns, k A B N A B N h Equation 5 k A 3 h h −4 h k B 3 h h −4 h k N h −4 h h The calculated rate constants from equation 5 under this paragraph can be employed in equation 2 under paragraph (a)(2)(v)(B) of this section to calculate the hydrolysis rate of a chemical at any pH of environmental concern. (D) The equations under paragraph (a)(2) of this section apply whether the test chemical has one or more hydrolyzable groups. In the latter case, the rate may be written as: Equation 6 −d[RX]/ dt 2 n [RX] = (k 1 2 n h Equation 6 applies to the hydrolysis rate of a molecule having n hydrolyzable groups, each of which follows first-order reaction kinetics. The measured k h (3) Principle of the test method. (4) Applicability and specificity. (b) Test procedures Test conditions Special laboratory equipment. (B) A pH meter that can resolve differences of 0.05 pH units or less. (C) Stoppered volumetric flasks (no grease) or glass ampoules that can be sealed. (ii) Purity of water. http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html. Federal Register. (iii) Sterilization. (iv) Precautions for volatility. (v) Temperature controls. (vi) pH conditions. (vii) Concentration of solutions of chemical substances. −3 (viii) Effect of acidic and basic groups. (ix) Buffer catalysis. (x) Photosensitive chemicals. (xi) Chemical analysis of solutions. (2) Preparation Reagents and solutions Buffer solutions. ( 1 ( 2 ( 3 (B) Additional buffer solutions. ( 1 ( 2 ( 3 ( 4 2 4 7 (C) Adjustment of buffer concentrations. 1 −3 −4 ( 2 (D) Preparation of test solution. 1 −3 ( 2 −3 (3) Performance of the test. o (i) Procedure 1. (ii) Procedure 2. (iii) Procedure 3. (B) If the pH at the end of concentration measurements employing any of the above three procedures has changed by more than 0.05 units from the initial pH, repeat the experiment using a solution having a test chemical concentration lowered sufficiently to keep the pH variation within 0.05 pH units. (iv) Analytical methodology. (c) Data and reporting Treatment of results. h 2 h (ii) If Procedure 3 was employed in making rate measurements, use the mean initial concentration (C o h h (iii) For each set of three concentration replicates, calculate the mean value of C and the standard deviation. (iv) For test chemicals that are not ionized or protonated between pH 3 and 11, calculate k A B N (2) Specific analytical and recovery procedures. (ii) If extraction methods were used to separate the solute from the aqueous solution, provide a description of the extraction method as well as the recovery data. (3) Test data report. h 1/2 2 A B N (ii) For Procedure 3, report k h A B N (iii) If, after 672 hours, the concentration (C) is the same as the initial concentration (C o h [50 FR 39252, Sept. 27, 1985, as amended at 53 FR 10391, Mar. 31, 1988; 53 FR 12526, Apr. 15, 1988; 53 FR 22323, June 15, 1988; 60 FR 34467, July 3, 1995; 69 FR 18803, Apr. 9, 2004; 77 FR 46293, Aug. 3, 2012]