PART 795—PROVISIONAL TEST GUIDELINES Authority: 15 U.S.C. 2603. Subpart A [Reserved] Subpart B—Provisional Chemical Fate Guidelines § 795.70 Indirect photolysis screening test: Sunlight photolysis in waters containing dissolved humic substances. (a) Introduction. (2) In general, both indirect and direct processes can proceed simultaneously. Under favorable conditions the measurement of a photoreaction rate constant in sunlight (K pE DE IE Equation 1 k pE DE IE This relationship is obtained when the reaction volume is optically thin so that a negligible fraction of the incident light is absorbed and is sufficiently dilute in test chemical; thus the direct and indirect photoreaction processes become first-order. (3) In pure water only, direct photoreaction is possible, although hydrolysis, biotransformation, sorption, and volatilization also can decrease the concentration of a test chemical. By measuring k pE DE IE (4) Two protocols have been written that measure k DE DE DE IE DE (5) This protocol provides a cost effective test method for measuring k IE (6) To correct for variations in solar irradiance during the reaction period, an actinometer is simultaneously insolated. From these data, an indirect photoreaction rate constant is calculated that is applicable to clear-sky, near-surface, conditions in fresh water bodies. (7) In contrast to k DE IE pE IE (8) The value of k pE IE (9) This protocol consists of three separate phases that should be completed in the following order: In Phase 1, SHW is prepared and adjusted; in Phase 2, the test chemical is irradiated in SHW and pure water (PW) to obtain approximate sunlight photoreaction rate constants and to determine whether direct and indirect photoprocesses are important; in Phase 3, the test chemical is again irradiated in PW and SHW. To correct for photobleaching of SHW and also solar irradiance variations, tubes containing SHW and actinometer solutions are exposed simultaneously. From these data k pE IE DE (b) Phase 1—Preparation and standardization of synthetic natural water Approach. 1 2 2 2 − (ii) The indirect photoreactivity of a chemical in a natural water will depend on its response to these reactive intermediates, and possibly others yet unknown, as well as the ability of the water to generate such species. This latter feature will vary from water-to-water in an unpredictable way, judged by the complexity of the situation. (iii) The approach to standardizing a test for indirect photoreactivity is to use a synthetic humic water (SHW) prepared by water-extracting commercial humic material. This material is inexpensive, and available to any laboratory, in contrast to a specific natural water. The SHW can be diluted to a dissolved organic carbon (DOC) content and uv-visible absorbance typical of most surface fresh waters. (iv) In recent studies it has been found that the reactivity of SHW mixtures depends on pH, and also the history of sunlight exposure (Mill et al. (1983) under paragraph (f)(11) of this section). The SHW solutions initially photobleach with a time-dependent rate constant. As such, an SHW test system has been designed that is buffered to maintain pH and is pre-aged in sunlight to produce, subsequently, a predictable bleaching behavior. (v) The purpose of Phase 1 is to prepare, pre-age, and dilute SHW to a standard mixture under defined, reproducible conditions. (2) Procedure. 2 4 (ii) Pre-aging is accomplished by exposing the concentrated solution in the 2-liter flask to direct sunlight for 4 days in early spring or late fall; 3 days in late spring, summer, or early fall. At this time the absorbance of the solution is measured at 370 nm, and a dilution factor is calculated to decrease the absorbance to 0.50 in a 1 cm path length cell. If necessary, the pH is re-adjusted to 7.0. Finally, the mixture is brought to exact dilution with a precalculated volume of reagent-grade water to give a final absorbance of 0.500 in a 1-cm path length cell at 370 nm. It is tightly capped and refrigerated. (iii) This mixture is SHW stock solution. Before use it is diluted 10-fold with 0.010 M phosphate buffer to produce a pH 7.0 mixture with an absorbance of 5.00 × 10 −2 (3) Rationale. (c) Phase 2—Screening test Introduction and purpose. (ii) In Phase 2, sunlight photoreaction rate constants are measured in round tubes containing SHW and then mathematically corrected to a flat water surface geometry. These rate constants are not corrected to clear-sky conditions. (2) Procedure. 1 1 (ii) This solution should be mixed 9.00:1.00 by volume with PW or SHW stock solution to provide working solutions. In the case of SHW, it gives a ten-fold dilution of SHW stock solution. Six mL aliquots of each working solution should then be transferred to separate 12 × 100 mm quartz tubes with screw tops and tightly sealed with Mininert valves. 2 2 (iii) The sample tubes are mounted in a photolysis rack with the tops facing geographically north and inclined 30° from the horizontal. The rack should be placed outdoors over a black background in a location free of shadows and excessive reflection. (iv) Reaction progress should be measured with an analytical technique that provides a precision of at least ±5 percent. High pressure liquid chromatography (HPLC) or gas chromatograph (GC) have proven to be the most general and precise analytical techniques. (v) Sample and control solution concentrations are calculated by averaging analytical measurements for each solution. Control solutions should be analyzed at least twice at zero time and at other times to determine whether any loss of chemical in controls or samples has occurred by some adventitious process during the experiment. (vi) Whenever possible the following procedures should be completed in clear, warm, weather so that solutions will photolyze more quickly and not freeze. (A) Starting at noon on day zero, expose to sunlight 24 sample tubes mounted on the rack described above. Tape 24 foil-wrapped controls to the bottom of the rack. (B) Analyze two sample tubes and two unexposed controls in PW and SHW for chemical at 24 hours. Calculate the round tube photolysis rate constants (k p SHW p W p SHW p W Equation 2 (k p SHW o t SHW −1 Equation 3 (k p W o t W −1 where the subscript identifies a reaction in SHW or PW; t is the photolysis time in calendar days; C o t (C) If less than 20 percent conversion occurs in SHW in 1 day, repeat the procedure for SHW and PW at 2 days, 4 days, 8 days, or 16 days, or until 20 percent conversion is reached. Do not extend the experiment past 16 days. If less than 20 percent photoreaction occurs in SHW at the end of 16 days the chemical is “photoinert”. Phase 3 is not applicable. (D) If more than 80 percent photoreaction occurs at the end of day 1 in SHW, repeat the experiment with eight each of the remaining foil-wrapped PW and SHW controls. Divide these sets into four sample tubes each, leaving four foil-wrapped controls taped to the bottom of the rack. ( 1 ( 2 p SHW ( 3 p SHW p W ( 4 p SHW p W Equation 4 R = (k p SHW p W The coefficient R, defined by Equation 4, is equal to [(k I D D pE IE (vii) Since the rate of photolysis in tubes is faster than the rate in natural water bodies, values of near-surface photolysis rate constants in natural and pure water bodies, k pE DE p SHW p W Equation 5 k pE p SHW Equation 6 k DE p W The factor 0.45 is an approximate geometric correction for scattered light in tubes versus horizontal surfaces. A rough value of k IE pE DE Equation 7 k IE pE DE (3) Criteria for Phase 2. pE DE (ii) Rate constants determined by the Phase 2 protocol depend upon latitude, season, and weather conditions. Note that (k p SHW D pE DE p SHW D p SHW D p SHW p SHW (4) Rationale. (5) Scope and limitations. (ii) The test procedure is simple and inexpensive, but does require that the chemical dissolve in water at sufficient concentrations to be measured by some analytical technique but not have appreciable absorbance in the range 290 to 825 nm. Phase 2 tests should be done during a clear-sky period to obtain the best results. Testing will be less accurate for chemicals with half-lives of less than 1 day because dramatic fluctuations in sunlight intensity can arise from transient weather conditions and the difficulty of assigning equivalent reaction times. Normal diurnal variations also affect the photolysis rate constant. Phase 3 tests should be started as soon as possible after the Phase 2 tests to ensure that the (k p SHW (6) Illustrative Example. −5 −5 (ii) The SHW solution of A was photolyzed in sealed quartz tubes (12 × 100 mm) in the fall season starting on October 1. At the end of 1 and 2 days, respectively, the concentration of A was found to be 1.13 × 10 −5 −5 −5 (iii) The tube photolysis rate constant of chemical A was calculated from Equation 2 under paragraph (c)(2)(vi)(B) of this section. The first time point at day 1 was used because the fraction of A remaining was in the range 20 to 80 percent: (k p SHW −5 −5 p SHW −1 (iv) From this value, k pE 1 k pE −1 −1 (v) From measurements in pure water, k D −1 p SHW D (d) Phase 3—Indirect photoreaction with actinometer: Calculation of IE and pE Introduction and purpose. (i) The purpose of Phase 3 is to measure k Io pE p SHW D (ii) In the case (k p SHW D p SHW (iii) The actinometer used is the p (iv) SHW is subject to photobleaching that decreases its ability to promote indirect photolysis based on its ability to absorb sunlight. This effect will be significant when the test period exceeds a few days. To correct for photobleaching, tubes containing SHW are irradiated in action to the other tubes above. (v) At any time, the loss of test chemical is given by Equation 8 assuming actinometric correction to constant light flux: Equation 8 −(d[C]/dt) = k I D (vi) The indirect photolysis rate constant, k I I Equation 9 k I Io in which k Io I −(d[C]/[C] = k Io D This expression is integrated to give Equation 10: Equation 10 Pn(C o SHW Io D The term (k Io o W D Equation 11 Pn(C o SHW o W Io The photobleaching fraction, [1-exp(-kt)], is equivalent to the expression [1-(A 370 370 370 370 Io o SHW o W 370 370 SHW (vii) To evaluate k Io A Equation 12 Pn(A° 370 370 A o PNAP where the slope is (k/k A A A a a Table 1—Day Averaged Rate Constant (k a 1 2 Latitude Season Spring Summer Fall Winter 20° N 515 551 409 327 30° N 483 551 333 232 40° N 431 532 245 139 50° N 362 496 154 6 1 a ga g −1 2 The value of k Io Equation 13 k Io Io A A (viii) To obtain k D D A o W o PNAP Equation 13a Pn(C o W D A o PNAP The slope is (k D A D A D D A A (ix) Then, (k p SHW D Io Equation 14 (k p SHW Io D (x) Finally, k pE Equation 5a k pE p SHW (2) Procedure. p SHW′ Equation 15 [PYR]/M = 26.9[(k p SHW a This pyridine concentration makes the actinometer rate constant match the test chemical rate constant. (A) The variable k a ga g (B) The variable k a (ii) Once [PYR] is determined, an actinometer solution is prepared by adding 1.00 mL of 1.0 × 10 −2 3 Equation 16 V/mL=[PYR]/0.0124. The PNAP/PYR solutions should be wrapped with aluminum foil and kept out of bright light after preparation. (iii) The following solutions should be prepared and individually added in 6.00 mL aliquots to 12/100 mm quartz sample tubes; 8 tubes should be filled with each solution: (A) PNAP/PYR actinometer solution. (B) Test chemical in pH 7.0, 0.010 M phosphate buffer. (C) Test chemcial in pH 7.0, 0.010 M phosphate buffer/SHW. (D) pH 7.0, 0.010 M phosphate buffer/SHW. Four tubes of each set are wrapped in foil and used as controls. (iv) The tubes are placed in the photolysis rack (Phase 2, Procedure) at 0900 hours on day zero, with the controls taped to the bottom of the rack. One tube of each composition is removed, along with their respective controls, according to a schedule found in Table 2, which categorizes sampling times on the basis of (k p SHW Table 2—Category and Sampling Procedure for Test and Actinometry Solutions Category k p −1 SHW Sampling procedure A 5.5 J K p Sample at 0, 1, 2, 4, and 8h. B 0.69>k p Sample at 0, 1, 2, 4, and 8d. C 0.17>k p Sample at 0, 4, 8, 16, and 32d. (v) The tubes containing PNAP, test chemical, and their controls are analyzed for residual concentrations soon after the end of the experiment. PNAP is conveniently analyzed by HPLC, using a 30 cm C 18 (vi) If controls are well-behaved and show no significant loss of chemical or absorbance change, then k I o t SHW o t SHW 370 o 370 o 370 370 o PNAP Equation 17 Pn(C o SHW o W Io 370 o 370 plot the quantities [Pn(C o t SHW o t W 370 o 370 Io (vii) According to Equation 12 under paragraph (d)(1)(vii) of this section, plot the quantities Pn(A o 370 370 o t PNAP A (viii) Then, using Equation 13a under paragraph (d)(1)(vii) of this section, determine the slope (S3) by least squares linear regression. Under the assumptions of the protocol, S3 is equal to (k D A (ix) From Equation 18 Equation 18 k A a calculate k A a a (x) The indirect photoreaction rate constant, k Io Equation 19 k Io A by incorporating the quantities k A (xi) The rate constant k D Equation 20 k D A using the quantities S3 and k A (xii) Then, (k p SHW D Io Equation 14 (k p SHW Io D (xiii) Finally, k pE p SNW Equation 5a k pE p SHW As determined, k pE (3) Criteria for Phase 3. (4) Rationale. (5) Scope and limitations of Phase 3 protocol. (6) Illustrative example. p SHW −1 a −1 p SHW a −1 (ii) The actinometer solution was made up by adding a volume of pyridine (1.95 mL) calculated from equation 16 under paragraph (d)(2)(ii) of this section to a 1 liter volumetric flask containing 1.00 mL of 1.00 × 10 −2 −5 (A) Chemical A (1.53 × 10 −5 (B) Chemical A (1.53 × 10 −5 (C) SHW standard solution diluted with water 0.90 to 1.00 to match solution A. (D) PNAP/PYR actinometer solution. Ten additional foil-wrapped controls of each mixture were taped to the bottom of the rack. (iii) The test chemical had been placed in category B, Table 2 under the paragraph (d)(2)(iv) of this section, on the basis of its Phase 2 rate constant under paragraph (c) of this section. Accordingly, two tubes of each irradiated solution and two tubes of each blank solution were removed at 0, 1, 2, 4, and 8 days at 1,200 hours. The averaged analytical results obtained at the end of the experiment are shown in the following Table 3. Table 3—Chemical Analytical Results for Illustrative Example, Phase 3 Day 10 5 SHW 10 5 W A SHW 370 10 5 0 1.53 1.53 0.0500 1.00 1 1.03 1.40 0.0470 0.810 2 0.760 1.30 0.0440 0.690 4 0.300 1.01 0.0370 0.380 8 0.130 0.800 0.0320 0.220 Data for solutions A through D are given in column 2 through 5, respectively. No significant chemical loss was found in the dark controls. (A) From these items the functions Pn(C o SNW o W 370 o 370 SNW o 370 370 o PNAP Table 4—Photoreaction Function for Illustrative Examples, Phase 3, Derived From Table 3 Day Pn(C o SHW Pn(C o W 1-(A 370 o 370 Pn(A o 370 370 Pn(C o PNAP 0 0 0 0 0 0 1 0.396 0.0888 0.0600 0.0618 0.211 2 0.700 0.163 0.120 0.128 0.371 4 1.629 0.415 0.260 0.301 0.968 8 2.465 0.648 0.360 0.446 1.514 (B) Slope S1 = (k Io Figure 1—Graphic determination of S1 = (k Io based on Equation 17 under paragraph (d)(2)(vi) of this section. (C) Slope S2 = (k/k a o 370 370 SHW o PNAP Figure 2—Graphic determination of S2 = (k/k A based on Equation 12 under paragraph (d)(1)(vii) of this section. (D) Using the data in columns 3 and 6 in Table 4 under paragraph (d)(6)(iii)(A) of this section, slope S3 was calculated by regression from Equation 13a under paragraph (d)(1)(viii) of this section and was found to be 0.428 with correlation coefficient equal to 0.99997. (E) Using Equation 18 under paragraph (d)(2)(ix) of this section, k A −1 (F) The values of S1, S2, and k A Io Equation 19 k Io −1 (G) The rate constant k D A Equation 20 k D −1 (H) The sum of k D Io pE Equation 21 k pE −1 −1 (I) Since k pE 1/2E Equation 22 t 1/2E pE Substituting the value of k pE Equation 23 t 1/2E −1 (e) Data and reporting Test conditions Specific analytical and recovery procedures. (B) 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. (ii) Other test conditions. (B) Report the dates of photolysis, weather conditions, times of exposure, and the duration of exposure. (C) If acetonitrile was used to solubilize the test chemical, report the volume percent. (D) If a significant loss of test chemical occurred in the control solutions for pure water and SHW, indicate the causes and how they were eliminated or minimized. (2) Test data report o (B) Report the molar concentration of test chemical, C t (C) Report the molar concentration of test chemical for each replicate control sample and the mean value for each time point. (D) Report the values of (k p SHW p W (E) If small losses of test chemical were observed in SHW and pure water, report a first-order rate constant loss, (k p loss p obs Equation 24 k p p obs p loss (F) Report the value of R calculated from Equation 4 under paragraph (c)(2)(vi)(D)( 4 (G) Report the values of k pE DE (ii) Phase 3—Indirect photoreaction with actinometer. (A) Report the initial molar concentration of test chemical, C o (B) Report the initial absorbance A o 370 (C) Report the initial molar concentration of PNAP of each replicate and the mean value in the actinometer. Report the concentration of pyridine used in the actinometer which was obtained from Equation 15 under paragraph (d)(2)(i) of this section. (D) Report the time and date the photolysis experiments were started, the time and date the experiments were completed, and the elapsed photolysis time in days. (E) For each time point t, report the separate values of the absorbance of the SHW solution, and the mean values. (F) For each time point for the controls, report the separate values of the molar concentrations of test chemical in pure water and SHW, and the absorbance of the SHW solution, and the mean values. (G) Tabulate and report the following data: t, [C] SHW W SNW 370 (H) From the data in (G), tabulate and report the following data: t, Pn(C o SNW o W 370 o 370 o 370 370 o PNAP (I) From the linear regression analysis of the appropriate data in step (H) in Equation 17 under paragraph (d)(2)(vi) of this section, report the slope S1 and the correlation coefficient. (J) From the linear regression analysis of the appropriate data in step (H) in Equation 12 under paragraph (d)(1)(vii) of this section, report the slope S2 and the correlation coefficient. (K) From the linear regression analysis of the appropriate data in step (H) in Equation 13a under paragraph (d)(1)(viii) of this section, report the slope S3 and the correlation coefficient. (L) If loss of chemical was observed during photolysis in pure water and SHW, then report the data Pn(C o corr o obs o loss (M) Report the value of the actinometer rate constant obtained from Equation 18 under paragraph (d)(2)(ix) of this section. (N) Report the value of k Io (O) Report the value of k D (P) Report the value of (k pE SHW pE (Q) Report the half-life, t 1/2E (f) References. (1) Cooper W.J., Zika R.G. “Photochemical formation of hydrogen peroxide in surface and ground waters exposed to sunlight.” Science, (2) Draper W.M., Crosby D.G. “The photochemical generation of hydrogen peroxide in natural waters.” Archives of Environmental Contamination and Toxicology, (3) Draper, W.M. and Crosby D.G. “Solar photooxidation of pesticides in dilute hydrogen peroxide.” Journal of Agricultural and Food Chemistry, (4) Draper W.M., Crosby D.G. “Hydrogen peroxide and hydroxyl radical: Intermediates in indirect photolysis reactions in water.” Journal of Agricultural and Food Chemistry, (5) Dulin D., Mill T. “Development and evaluation of sunlight actinometers.” Environmental Science and Technology, (6) Haag H.R., Hoigne J., Gassman E., Braun A.M. “Singlet oxygen in surface waters—Part I; Furfuryl alcohol as a trapping agent.” Chemosphere, (7) Haag W.R., Hoigne J., Gassman E., Braun A.M. “Singlet oxygen in surface waters—Part II: Quantum yields of its production by some natural humic materials as a function of wavelength.” Chemosphere, (8) Mill T., Winterle J.S., Fischer A., Tse D., Mabey W.R., Drossman H., Liu A., Davenport J.E. Toxic substances process data generation and protocol development. Work assignment 12, test standard development. “Section 3. Indirect photolysis.” Draft final report. EPA Contract No. 68-03-2981. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA, and Office of Pollution Prevention and Toxics, EPA, Washington, DC. (1984). (9) Mill T., Mabey W.R., Bomberger D.C., Chou T.W., Hendry D.G., Smith J.H. “Laboratory protocols for evaluating the fate of organic chemicals in air and water. Chapter 3. Photolysis in water. Chapter 4. Oxidation in water.” EPA 600/3-82-022. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA. (1981). (10) Mill T., Mabey W.R., Winterle J.S., Davenport J.E., Barich V.P., Dulin D.E., Tse D.S., Lee G. “Design and validation of screening and detailed methods for environmental processes. Appendix C. Lower-tier direct photolysis protocol.” Draft final report. EPA Contract No. 68-01-6325. Office of Pollution Prevention and Toxics, EPA, Washington, DC. (1982). (11) Mill T., Davenport J.E., Winterle J.S., Mabey W.R., Dossman H., Tse D., Liu A. Toxic substances process data generation and protocol development. Work assignment 12. “Appendix B. Upper-tier protocol for direct photolysis in water.” Draft final report. EPA Contract No. 68-03-2981. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA, and Office of Pollution Prevention and Toxics, EPA, Washington, DC. (July 1983). (12) Winterle J.S., Mill T. Toxic substances process data generation and protocol development. Work assignment 18. “Indirect photoreaction protocol.” Draft EPA special report. EPA Contract No. 68-03-2981. Environmental Research Laboratory, Office of Research and Development, EPA, Athens, GA and Office of Pollution Prevention and Toxics, EPA, Washington, DC. (1985). (13) Mill T., Hendry D.G., Richardson H. “Free radical oxidants in natural waters.” Science, (14) U.S. Environmental Protection Agency (USEPA), Office of Pollution Prevention and Toxics (OPPT). “Chemical fate test guidelines. Test guideline (CG, CS-6000). Photolysis in aqueous solution.” EPA-560/6-84-003. NTIS publication PB-84-233287. (1984). (15) USEPA, OPPT. “Chemical fate test guidelines. Test guildeline (CG, CS-6010). Laboratory determination of the direct photolysis reaction quantum yield in aqueous solution and sunlight photolysis.” EPA-560/6-84-003. NTIS publication PB-84-233287. (1984). (16) Wolff C.J.M., Halmans M.T.H., Van der Heijde H.B. “The formation of singlet oxygen in surface waters.” Chemosphere, (17) Zepp R.G., Baughman G.L., Schlotzhauer P.F. “Comparison of photochemical behavior of various humic substances in water: I. Sunlight induced reactions of aquatic pollutants photosensitized by humic substances.” Chemosphere, (18) Zepp R.G., Baughman G.L., Schlozhauer P.F. “Comparison of photochemical behavior of various humic substances in water: II. Photosensitized oxygenations.” Chemosphere, (19) Zepp R.G., Cline D.M. “Rates of direct photolysis in aquatic environments.” Environmental Science and Technology, (20) Zepp, R.G., Wolfe N.L., Baughman G.L., Hollis R.C. “Singlet oxygen in natural waters.” Nature, (21) Zepp R.G., Schlotzhauer P.F., Merritt S.R. “Photosensitized transformations involving electronic energy transfer in natural waters: role of humic substances.” Environmental Science and Technology, [53 FR 34522, Sept. 7, 1988; 53 FR 37393, Sept. 26, 1988] Subpart C—Provisional Environmental Effects Guidelines § 795.120 Gammarid acute toxicity test. (a) Purpose. et seq. (b) Definitions. Death Flow-through LC50 Loading Solvent Static system (c) Test procedures Summary of the test. (2) [Reserved] (3) Range-finding test. (ii) The gammarids shall be exposed to a wide-range of concentrations of the test substance (e.g., 1, 10, 100 mg/1, etc.), usually under static conditions. (iii) A minimum of five gammarids should be exposed to each concentration of test substance for a period of 96 hours. The exposure period may be shortened if data suitable for determining concentrations in the definitive test can be obtained in less time. Nominal concentrations of the test substance may be acceptable. (4) Definitive test. (ii) A minimum of 20 gammarids per concentration shall be exposed to five or more concentrations of the test substance chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, 64 mg/L). The range and number of concentrations to which the organisms are exposed shall be such that in 96 hours there is at least one concentration resulting in mortality greater than 50 and less than 100 percent, and one concentration causing greater than zero and less than 50 percent mortality. An equal number of gammarids may be placed in two or more replicate test chambers. Solvents should be avoided, if possible. If solvents have to be used, a solvent control, as well as a dilution control, shall be tested at the highest solvent concentration employed in the treatments. The solvent should not be toxic or have an effect on the toxicity of the test substance. The concentration of solvent should not exceed 0.1 ml/L. (iii) Every test shall include a concurrent control using gammarids from the same population or culture container. The control group shall be exposed to the same dilution water, conditions and procedures, except that none of the test substance shall be is added to the chamber. (iv) The dissolved oxygen concentration, temperature and pH of the test solution shall be measured at the beginning of the test and at 24, 48, 72 and 96 hours in at least one replicate each of the control, and the highest, lowest and middle test concentrations. (v) The test duration is 96 hours. The test is unacceptable if more than 10 percent of the control organisms die during the test. (vi) In addition to death, any abnormal behavior or appearance shall also be reported. (vii) Gammarids shall be randomly assigned to the test chambers. Test chambers shall be positioned within the testing area in a random manner or in a way in which appropriate statistical analyses can be used to determine whether there is any variation due to placement. (viii) Gammarids shall be introduced into the test chambers after the test substance has been added. (ix) Observations on compound solubility shall be recorded. The investigator should record the appearance of surface slicks, precipitates, or material adhering to the sides of the test chambers. (5) [Reserved] (6) Analytical measurements Water quality analysis. (ii) Collection of samples for measurement of test substance. (iii) Measurement of test substance. (B) The analytical methods used to measure the amount of test substance in a sample shall be validated before beginning the test. This involves adding a known amount of the test substance to each of three water samples taken from a chamber containing dilution water and the same number of gammarids as are placed in each test chamber. The nominal concentrations of the test substance in these samples should span the concentration range to be used in the test. Validation of the analytical method should be performed on at least two separate days prior to starting the test. (C) An analytical method is not acceptable if likely degradation products of the test substance give positive or negative interferences, unless it is shown that such degradation products are not present in the test chambers during the test. (D) Among replicate test chambers, the measured concentrations shall not vary more than 20 percent. The measured concentration of the test substance in any chamber during the test shall not vary more than plus or minus 30 percent from the measured concentration in that chamber at zero time. (E) The mean measured concentration of dissolved test substance shall be used to calculate all LC50's and to plot all concentration-response curves. (d) Test conditions for definitive test Test species Selection. Gammarus fasciatus, G. pseudolimnaeus, G. lacustris (B) Gammarids can be cultured in the laboratory or collected from natural sources. If collected, they must be held in the laboratory for at least 14 days prior to testing. (C) Gammarids used in a particular test shall be of similar age and/or size and from the same source or culture population. (ii) Acclimation. (iii) Care and handling. (iv) Feeding. (2) Facilities Apparatus ( 1 ( 2 ( 3 ( 4 ( 5 (B) Facilities should be well ventilated and free of fumes and disturbances that may affect the test organism. (C) Test chambers shall be covered loosely to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions. (ii) Construction materials. (iii) Test substance delivery system. (iv) Test chambers. (v) Cleaning of test system. (vi) Dilution water. Substance Maximum concentration Particulate matter 20 mg/L Total organic carbon (TOC) or 2 mg/L chemical oxygen demand (COD) 5 mg/L Boron, fluoride 100 ug/L Un-ionized ammonia 1 ug/L Aluminum, arsenic, chromium, cobalt, copper, iron, lead, nickel, zinc 1 ug/L Residual chlorine 3 ug/L Cadmium, mercury, silver 100 ng/L Total organophosphorus pesticides 50 ng/L Total organochlorine pesticides plus: polychlorinated biphenyls (PCBs) or 50 ng/L organic chlorine 25 ng/L (B) If the dilution water is from a ground or surface water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) shall be measured. Reconstituted water can be made by adding specific amounts of reagent-grade chemicals to deionized or distilled water. Glass-distilled or carbon-filtered deionized water with a conductivity less than 1 micromho/cm is acceptable as the diluent for making reconstituted water. (C) The concentration of dissolved oxygen in the dilution water shall be between 90 and 100 percent saturation. If necessary, the dilution water can be aerated before the addition of the test substance. All reconstituted water should be aerated before use. (3) Test parameters. (i) Water temperature of 18 ±1 °C. (ii) Dissolved oxygen concentration between 60 and 105 percent saturation. (iii) The number of gammarids placed in a test chamber shall not be so great as to affect the results of the test. Ten gammarids per liter is the recommended level of loading for the static test. Loading requirements for the flow-through test will vary depending on the flow rate of dilution water. The loading should not cause the dissolved oxygen concentration to fall below the recommended levels. (iv) Photoperiod of 16 hours light and 8 hours darkness. (e) Reporting. (1) Name and address of the facility performing the study and the dates on which the study was initiated and completed. (2) Objectives and procedures stated in the approved protocol, including any changes in the original protocol. (3) Statistical methods employed for analyzing the data. (4) The test substance identified by name, Chemical Abstracts (CAS) number or code number, source, lot or batch number, strength, purity, and composition, or other appropriate characteristics. (5) Stability of the test substance under the conditions of the test. (6) A description of the methods used, including: (i) The source of the dilution water, its chemical characteristics (e.g., hardness, pH, etc.) and a description of any pretreatment. (ii) A description of the test substance delivery system, test chambers, the depth and volume of solution in the chamber, the way the test was begun (e.g., test substance addition), the loading, the lighting, and the flow rate. (iii) Frequency and methods of measurements and observations. (7) The scientific name, weight, length, source, and history of the organisms used, and the acclimation procedures and food used. (8) The concentrations tested, the number of gammarids and replicates per test concentration. The reported results should include: (i) The results of dissolved oxygen, pH and temperature measurements. (ii) If solvents are used, the name and source of the solvent, the nominal concentration of the test substance in the stock solution, the highest solvent concentration in the test solution and a description of the solubility determination in water and solvents. (iii) The measured concentration of the test substance in each test chamber just before the start of the test and at all subsequent sampling periods. (iv) In each test chamber at each observation period, the number of dead and live test organisms, the percentage of organisms that died, and the number of test organisms that showed any abnormal effects in each test chamber at each observation period. (v) The 48, 72 and 96-hour LC50's and their 95 percent confidence limits. When sufficient data have been generated, the 24-hour LC50 value also. These calculations should be made using the mean measured test substance concentrations. (vi) The observed no-effect concentration (the highest concentration tested at which there were no mortalities or abnormal behavioral or physiological effects), if any. (vii) Methods and data for all chemical analyses of water quality and test substance concentrations, including method validations and reagent blanks. (9) A description of all circumstances that may have affected the quality or integrity of the data. (10) The names of the sponsor, study director, principal investigator, names of other scientists or professionals, and the names of all supervisory personnel involved in the study. (11) A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the data, and a statement of the conclusions drawn from the analysis. Results of the analysis of data should include the calculated LC50 value, 95 percent confidence limits, slope of the transformed concentration-response line, and the results of a goodness-of-fit test (e.g., chi-square test). (12) The signed and dated reports prepared by any individual scientist or other professional involved in the study, including each person who, at the request or direction of the testing facility or sponsor, conducted an analysis or evaluation of data or specimens from the study after data generation was completed. (13) The locations where all specimens, raw data, and the final report are stored. (14) The statement prepared and signed by the quality assurance unit. [52 FR 24462, July 1, 1987] Subpart D—Provisional Health Effects Guidelines § 795.225 Dermal pharmacokinetics of DGBE and DGBA. (a) Purpose. (1) The absorption of diethylene glycol butyl ether (DGBE) after administration by the dermal route. (2) The biotransformation of DGBE administered dermally. (3) The dermal absorption of DGBE and diethylene glycol butyl ether acetate (DGBA). (b) Test procedures Animal selection Species. (ii) Animals. (iii) Animal care. (B) During the acclimatization period, the rats should be housed in cages on hardwood chip bedding. All animals shall be provided with conventional laboratory diets and water ad libitum. (2) Administration of DGBE and DGBA Test substances. 14 14 14 (ii) Dosage and treatment. (B) For dermal treatment, the doses shall be applied in a volume adequate to deliver the prescribed doses. The backs of the rats should be lightly shaved with an electric clipper shortly before treatment. The dose shall be applied with a micropipette on a specific area (for example, 2 cm 2 (iii) Washing efficiency study. (iv) Determination of absorption, biotransformation, and excretion. 14 (B) Eight animals shall be dosed once dermally with the high dose of 14 (C) Eight animals shall be dosed once dermally with the low dose of 14 (D) Eight animals shall be dosed once dermally with the high dose of 14 (E) The high and low doses of 14 14 (3) Observation of animals Urinary and fecal excretion. 14 (ii) Biotransformation after dermal dosing. (c) Data and reporting Treatment of results. (2) Evaluation of results. (3) Test report. (i) Species, strain, and supplier of laboratory animals. (ii) Information on the degree (i.e., specific activity for a radiolabel) and sites of labeling of the test substances. (iii) A full description of the sensitivity and precision of all procedures used to produce the data. (iv) Relative percent absorption by the dermal route for rats administered low and high doses of 14 14 (v) Quantity of isotope, together with percent recovery of the administered dose, in feces and urine. (vi) Biotransformation pathways and quantities of DGBE and metabolites in urine collected after administering single high and low dermal doses to rats. [53 FR 5946, Feb. 26, 1988, as amended at 54 FR 41834, Oct. 12, 1989] § 795.228 Oral/dermal pharmacokinetics. (a) Purpose. (1) Ascertain whether the pharmacokinetics and metabolism of a chemical substance or mixture (“test substance”) are similar after oral and dermal administration. (2) Determine bioavailability of a test substance after oral and dermal administration. (3) Examine the effects of repeated dosing on the pharmacokinetics and metabolism of the test substance. (b) Definitions. Bioavailability (2) Metabolism (3) Percent absorption (4) Pharmacokinetics (c) Test procedures Animal selection Species. (ii) Test animals. (iii) Animal care. (B) During the acclimatization period, the animals shall be housed in suitable cages. All animals shall be provided with certified feed and tap water ad libitum. (2) Administration of test substance Test substance. (ii) Dosage and treatment Intravenous. (B) Oral. (C) Dermal. 1 Dermal treatment. 2 2 ( 2 Washing efficacy study. 1 (iii) Dosing and sampling schedule Rat studies. ( 1 Intravenous study. ( 2 Oral study. i ( ii ( 3 Dermal studies. ( i ( ii ( 4 Repeated dosing study. (B) Mini-Pig studies. ( 1 Intravenous study. ( 2 Dermal studies. 3 ( i ( ii (3) Types of studies Pharmacokinetics studies Rat studies. (B) Mini-Pig studies. (ii) Metabolism studies—Rat studies. (4) Measurements Pharmacokinetics. (A) Rat studies 1 Bioavailability. ( 2 Extent of absorption. ( 3 Excretion. ( 4 Tissue distribution. ( 5 Changes in pharmacokinetics. (B) Mini-Pig studies—Extent of absorption. (ii) Metabolism. (A) Rat studies 1 Biotransformation. ( 2 Changes in biotransformation. (d) Data and reporting. (1) Presentation of results. (2) Evaluation of results. (3) Reporting results. (i) Species and strains of laboratory animals. (ii) Chemical characterization of the test substance, including: (A) For the radioactive test substances, information on the site(s) and degree of radiolabeling, including type of label, specific activity, chemical purity, and radiochemical purity. (B) For the nonradioactive compound, information on chemical purity. (C) Results of chromatography. (iii) A full description of the sensitivity, precision, and accuracy of all procedures used to generate the data. (iv) Percent of absorption of test substance after oral and dermal exposures to rats and dermal exposure to mini-pigs. (v) Quantity and percent recovery of radioactivity in feces, urine, expired air, and blood. In dermal studies on rats and mini-pigs, include recovery data for skin, skin washings, and residual radioactivity in the covering as well as results of the washing efficacy study. (vi) Tissue distribution reported as quantity of radioactivity in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin and in residual carcass of rats. (vii) Materials balance developed from each study involving the assay of body tissues and excreta. (viii) Biotransformation pathways and quantities of test substance and metabolites in excreta collected after administering single high and low doses to rats. (ix) Biotransformation pathways and quantities of the test substance and metabolites in excreta collected after administering repeated low doses to rats. (x) Pharmacokinetics model(s) developed from the experimental data. [54 FR 33411, Aug. 14, 1989; 54 FR 49844, Dec. 1, 1989; 55 FR 25392, June 21, 1990] § 795.231 Pharmacokinetics of isopropanal. (a) Purpose. (1) Ascertain whether the pharmacokinetics and metabolism of the “test substance” are similar after oral and inhalation administration. (2) Determine bioavailability of the test substance after oral and inhalation administration. (3) Examine the effects of repeated dosing on the pharmacokinetics and metabolism of the test substance. (b) Definitions. Bioavailability (2) “ Metabolism (3) “ Pharmacokinetics (c) Test procedures Animal selection Species. (ii) Test animals. (iii) Animal care. (B) The animals should be housed in environmentally controlled rooms with at least 10 air changes per hour. The rooms shall be maintained at a temperature of 22 ±2 °C and humidity of 50 ±20 percent with a 12-hour light/dark cycle per day. The animals shall be kept in a quarantine facility for at least 7 days prior to use and shall be acclimated to the experimental environment for a minimum of 48 hours prior to treatment. (C) During the acclimatization period, the animals should be housed in suitable cages. All animals shall be provided with certified feed and tap water ad libitum. (2) Administration of test substance Test substance. (ii) Dosage and treatment Intravenous. (B) Oral. (C) Inhalation. (iii) Dosing and sampling schedule. (A) Intravenous study. (B) Oral studies. 1 per os ( 2 per os (C) Inhalation studies. ( 1 ( 2 (D) Repeated dosing study. (3) Types of studies Pharmacokinetics studies. (ii) Metabolism studies. (4) Measurements Pharmacokinetics. (A) Bioavailability. (B) Extent of absorption. (C) Excretion. (D) Tissue distribution. (E) Changes in pharmacokinetics. (F) Biotransformation. (G) Changes in biotransformation. (ii) [Reserved] (d) Data and reporting. (1) Presentation of results. (2) Evaluation of results. (3) Reporting results. (i) Species and strains of laboratory animals. (ii) Chemical characterization of the test substance, including: (A) For the radioactive test substance, information on the site(s) and degree of radiolabeling, including type of label, specific activity, chemical purity, and radiochemical purity. (B) For the nonradioactive substance, information on chemical purity. (C) Results of chromatography. (iii) A full description of the sensitivity, precision, and accuracy of all procedures used to generate the data. (iv) Extent of absorption of the test substance as indicated by: percent absorption of the administered oral dose; and total body burden after inhalation exposure. (v) Quantity and percent recovery of radioactivity in feces, urine, expired air, and blood. (vi) Tissue distribution reported as quantity of radioactivity in blood and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin, spleen and in residual carcass of each rat. (vii) Biotransformation pathways and quantities of the test substance and metabolites in excreta collected after administering single high and low doses to rats. (viii) Biotransformation pathways and quantities of the test substance and metabolites in excreta collected after administering repeated low doses to rats. (ix) Pharmacokinetics model(s) developed from the experimental data. [54 FR 43261, Oct. 23, 1989] § 795.232 Inhalation and dermal pharmacokinetics of commercial hexane. (a) Purposes. (1) Determine the bioavailability of the test substances after dermal and inhalation administration. (2) Compare the pharmacokinetics and metabolism of the test substances after intravenous, dermal, and inhalation administration. (3) Examine the effects of repeated doses on the pharmacokinetics and metabolism of the test substances. (b) Definitions. Bioavailability (2) Metabolism (3) Pharmacokinetics (4) Low dose 1/10 (5) High dose (6) Test substance 14 n 14 (c) Test procedures Animal selection Species. (ii) Test animals. (iii) Animal care. Guidelines for the Care and Use of Laboratory Animals. (B) The animals shall be housed in environmentally controlled rooms with at least 10 air changes per hour. The rooms shall be maintained at a temperature of 18 to 26 degrees centigrade and humidity of 40 to 70 percent with a 12-hour light/dark cycle per day. The animal subjects shall be kept in a quarantine facility for at least 7 days prior to use, and shall be acclimated to the experimental environment for a minimum of 48 hours prior to treatment. (C) During the acclimatization period, the rats shall be housed in suitable cages. All animals shall be provided with certified feed and tap water ad libitum. (2) Administration of test substances Test substances. 14 n 14 14 n n n 1986 Annual Book of ASTM Standards: Petroleum Products and Lubricants, http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html. Federal Register. (ii) Dosage and treatment Intravenous. (B) Inhalation. (C) Dermal. In vivo Journal of Applied Toxicology (iii) Dosing and sampling schedule. (A) Intravenous study. (B) Inhalation studies. ( 1 ( 2 (C) Dermal studies. ( 1 ( 2 (D) Repeated dosing study. (3) Types of studies Pharmacokinetics studies. (ii) Metabolism studies. (4) Measurements Pharmacokinetics. (A) Bioavailability. 14 n (B) Extent of absorption. (C) Excretion. (D) Tissue distribution. (E) Change in pharmacokinetics. (ii) Metabolism. (A) Biotransformation. (B) Changes in biotransformation. (d) Data and reporting. (1) Presentation of results. (2) Evaluation of results. (3) Reporting results. (i) Strain of laboratory animals. (ii) Chemical characterization of the test substances, including: (A) For the radiolabeled test substances, information on the sites and degree of radiolabeling, including type of label, specific activity, chemical purity prior to mixing with the unlabeled hexane mixture, and radiochemical purity. (B) For the unlabeled test substance, information on lot number and the percentage of MCP and n (C) Results of chromatography. (iii) A full description of the sensitivity, precision, and accuracy of all procedures used to obtain the data. (iv) Percent and rate of absorption of the test substance after inhalation and dermal exposures. (v) Quantity and percent recovery of radioactivity in feces, urine, expired air, and blood. For dermal studies, include recovery data for skin and residual radioactivity in the covering apparatus. (vi) Tissue distribution reported as quantity of radioactivity in blood, in various tissues including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin, spleen, thymus, and in residual carcass. (vii) Biotransformation pathways, to the extent possible, and quantities of the test substances and metabolites in excreta collected after administering single high and low doses. (viii) Biotransformation pathways, to the extent possible, and quantities of test substances and metabolites in excreta collected after administering repeated low doses. (ix) Pharmacokinetics models to the extent they can be developed from the experimental data. [55 FR 632, Jan. 8, 1990, as amended at 58 FR 34205, June 23, 1993; 60 FR 34466, July 3, 1995; 69 FR 18803, Apr. 9, 2004; 77 FR 46293, Aug. 3, 2012] § 795.250 Developmental neurotoxicity screen. (a) Purpose. (b) Principle of the test method. (c) Test procedures Animal selection Species and strain. (ii) Age. (iii) Sex. (iv) Number of animals. (A) On day 4 after birth, the size of each litter should be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter. Whenever the number of male or female pups prevents having 4 of each sex per litter, partial adjustment (for example, 5 males and 3 females) is permitted. Adjustments are not appropriate for litters of less than 8 pups. Elimination of runts only is not appropriate. Individual pups should be identified uniquely after standardization of litters. A method that may be used can be found in Adams et al. (1985) under paragraph (e)(1) of this section. (B) After standardization of litters, males and females shall be randomly assigned to one of each of three behavioral tasks. Alternatively, more than one of the behavioral tasks may be conducted in the same animal. In the latter case, a minimum of 1 to 2 days should separate the tests when conducted at about the same age. (C) One male and one female shall be randomly selected from each litter for sacrifice at weaning as specified in paragraph (c)(8) of this section. (2) Control group. (3) Dose levels and dose selection. (ii) If the substance has been shown to be developmentally toxic either in a standard developmental toxicity study or a pilot study, the highest dose level shall be the maximum dose which will not induce in utero (iii) In the absence of standard developmental toxicity, unless limited by the physicochemical nature or biologicial properties of the substance, the highest dose level shall induce some overt maternal toxicity but shall not result in a reduction in weight gain exceeding 20 percent during gestation and lactation. (iv) The lowest dose should not produce any grossly observable evidence of either maternal or developmental neurotoxicity. (v) The intermediate dose(s) shall be equally spaced between the highest and lowest dose. (4) Dosing period. (5) Administration of test substance. (6) Observation of dams. (ii) During the treatment and observation periods, cage-side observations shall include: (A) Any responses with respect to body position, activity level, coordination of movement, and gait. (B) Any unusual or bizarre behavior including, but not limited to headflicking, head searching, compulsive biting or licking, self-mutilation, circling, and walking backwards. (C) The presence of: ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 ( 8 ( 9 ( 10 (iii) Signs of toxicity shall be recorded as they are observed, including the time of onset, the degree and duration. (iv) Animals shall be weighed at least weekly. (v) The day of delivery of litters shall be recorded. (7) Study conduct Observation of offspring. (B) All offspring shall be examined outside the cage for gross signs of toxicity whenever they are weighed or removed from their cages for behavioral testing. The offspring shall be observed by trained technicians, who are blind with respect to the animal's treatment using standardized procedures to maximize inter-observer reliability. Where possible, it is advisable that the same observer be used to evaluate the animals in a given study. If this is not possible, some demonstration of inter-observer reliability is required. At a minimum, the end points outlined in paragraph (c)(6)(ii) of this section shall be monitored as appropriate for the developmental stage being observed. (C) Any gross signs of toxicity in the offspring shall be recorded as they are observed, including the time of onset, the degree, and duration. (ii) Developmental landmarks. (A) Vaginal opening. (B) Testes descent. (iii) Motor activity. (B) Each animal shall be tested individually. The test session shall be long enough to demonstrate habituation of motor activity in control animals, i.e., to approach asymptotic levels by the last 20 percent of the session. Animals' activity counts shall be collected in equal time periods of no greater than 10 minutes duration. All sessions shall have the same duration. Treatment groups shall be counter-balanced across test times. (C) Efforts shall be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables which can affect motor activity are sound level, size, and shape of the test cage, temperature, relative humidity, lighting conditions, odors, use of home cage or novel test cage, and environmental distractions. (D) Additional information on the conduct of a motor activity study may be obtained in the TSCA motor activity guideline, in § 798.6200 of this chapter. (iv) Auditory startle test. (v) Active avoidance test. (A) Mean number of shuttles during the adaptation period preceding each daily session. (B) Mean number and latency of avoidances per session, presented in blocks of 10 trials (2 blocks of 10 trials per session across 5 sessions). (C) Mean number and latency of escapes per session, presented in blocks of 10 trials as above. (D) Mean duration of shocks per session, presented in blocks of 10 trials as above. (E) Mean number of shuttles during the inter-trial intervals. (8) Post-mortem evaluation Age of animals. (ii) Neuropathology. in situ (A) General staining. (B) Special stains. (C) Alternative technique. (iii) Brain weight. (d) Data reporting and evaluation. (1) Description of system and test methods. (ii) Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. These data do not have to be from studies using prenatal exposures. However, the laboratory must demonstrate competence in testing neonatal animals perinatally exposed to chemicals and establish test norms for the appropriate age group. (iii) Procedures for calibrating and assuring the equivalence of devices and balancing treatment groups. (iv) A short justification explaining any decisions where professional judgement is involved such as fixation technique and choice of stains. (2) Results. (i) In tabular form, data for each animal shall be provided showing: (A) Its identification number and litter from which it came. (B) Its body weight and score on each developmental landmark at each observation time; total session activity counts and intrasession subtotals on each day measured; auditory startle response magnitude session counts and intrasession subtotals on each day measured; avoidance session counts and intrasession counts on each day measured; time and cause of death (if appropriate); locations, nature or frequency, and severity of the lesions; total brain weight; absolute weight of each of the four sections; and weight of each section as a percentage of total brain weight. A commonly used scale such as 1 + , 2 + , 3 + , and 4 + for degree of severity of lesions ranging from very slight to extensive may be used for morphologic evaluation. Any diagnoses derived from neurologic signs and lesions, including naturally occurring diseases or conditions, shall also be recorded. (ii) Summary data for each group shall include: (A) The number of animals at the start of the test. (B) Body weights of the dams during gestation and lactation. (C) Litter size and mean weight at birth. (D) The number of animals showing each observation score at each observation time. (E) The percentage of animals showing each abnormal sign at each observation time. (F) The mean and standard deviation for each continuous end point at each observation time. These will include body weight, motor activity counts, acoustic startle responses, performance in active avoidance tests, and brain weights (both absolute and relative). (G) The number of animals in which any lesion was found. (H) The number of animals affected by each different type of lesion, the average grade of each type of lesion, and the frequency of each different type and/or location of lesions. (3) Evaluation of data. (e) References. (1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter, L.W., Sobotka, T.J., Tilson, H.A., and Nelson, B.K. “Collaborative behavioral teratology study: Protocol design and testing procedure.” Neurobehavioral Toxicology and Teratology. (2) Brush, F.R. “The effects of inter-trial interval on avoidance learning in the rat.” Journal of Comparative Physiology and Psychology. (3) Brush, F.R. “Retention of aversively motivated behavior.” In: “Adverse Conditioning and Learning.” Brush, F.R., ed., New York: Academic Press. (1971). (4) Brush, F.R. and Knaff, P.R. “A device for detecting and controlling automatic programming of avoidance-conditioning in a shuttle-box.” American Journal of Psychology. (5) Dixon, W.J. and Massey, E.J. “Introduction to Statistical Analysis.” 2nd ed. New York: McGraw-Hill. (1957). (6) Glowinski, J. and Iversen, L.L. “Regional studies of catecholamines in the rat brain-I.” Journal of Neurochemistry. (7) Ison, J.R. “Reflex modification as an objective test for sensory processing following toxicant exposure.” Neurobehavioral Toxicology and Teratology. (8) Jensen, D.R. “Some simultaneous multivariate procedures using Hotelling's T2 Statistics.” Biometrics. (9) McAllister, W.R. and McAllister, D.E. “Behavioral measurement of conditioned fear.” In: “Adverse Conditioning and Learning.” Brush, F.R., ed., New York: Academic Press (1971). (10) Neter, J. and Wasserman, W. “Applied Linear Statistical Models.” Homewood: Richard D. Irwin, Inc. (1974). (11) Sokal, R.P. and Rohlf, E.J. “Biometry.” San Francisco: W.H. Freeman and Co. (1969). (12) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H., “Neuropathological methods for the detection of neurotoxic disease.” In: “Experimental and Clinical Neurotoxicology.” Spencer, P.S. and Schaumburg, H.H., eds., Baltimore, MD: Williams & Wilkins, pp. 743-757. (1980). [53 FR 5957, Feb. 26, 1988]