OECD GUIDELINE FOR THE TESTING OF CHEMICALS——Absorption vitro test

OECD/OCDE202Adopted :13 April 2004H867Á@TB78EBG8Á9HQÁS8RSBG@ÁH9Á6A8FB64ERÁ%BQIOJBÁ Á4h pÁBxx gtwt f t yÁSp ÁÁBGSQH7T6SBHGÁ1. OECD Guidelines for the Testing of chemicals are periodically reviewed in the light of scientific progress. Guideline 202 on “Daphnia sp., Acute Immobilisation Test and Reproduction Test”, adopted in April 1984, included two parts: Part I - the 24h EC50 acute immobilisation test and Part II - the reproduction test (at least 14 days). Revision of the reproduction test has resulted in the adoption and publication of Test Guideline 211 on “Daphnia magna Reproduction Test” in September 1998. Consequently, the new version of Guideline 202 is restricted to the acute immobilisation test.2. This guideline describes an acute toxicity test to assess effects of chemicals towards daphnids. Existing test methods were used to the extent possible (1)(2)(3). The main differences in comparison with the earlier version are the extension of the test duration to 48 hours, the provision for more information on recommended culture and test media, and the introduction of a limit test at 100 mg/l of test substance.IQBG6BIE8ÁH9ÁSA8ÁS8RSÁ3. Young daphnids, aged less than 24 hours at the start of the test, are exposed to the test substance at a range of concentrations for a period of 48 hours. Immobilisation is recorded at 24 hours and 48 hours and compared with control values. The results are analysed in order to calculate the EC50 at 48h (see Annex 1 for definitions). Determination of the EC50 at 24h is optional.BG9HQF4SBHGÁHGÁSA8ÁS8RSÁRT5RS4G68Á4. The water solubility and the vapour pressure of the test substance should be known and a reliable analytical method for the quantification of the substance in the test solutions with reported recovery efficiency, and limit of determination should be available. Useful information includes the structural formula, purity of the substance, stability in water and light, P ow and results of a test for ready biodegradability (see Guideline 301).ÁNote: Guidance for testing substances with physical chemical properties that make them difficult to test is provided in a separate document (4).ÁÁ202 OECD/OCDEQ898Q8G68ÁRT5RS4G68RÁ5. A reference substance may be tested for EC50 as a means of assuring that the test conditions are reliable. Toxicants used in international ring-tests (1)(5) are recommended for this purpose1. Test(s) with a reference substance should be done preferably every month and at least twice a year.ÁÁU4EB7BSXÁH9ÁSA8ÁS8RSÁ6. For a test to be valid, the following performance criteria apply:- In the control, including the control containing the solubilising agent, not more that 10 per cent of the daphnids should have been immobilised;- The dissolved oxygen concentration at the end of the test should be t 3 mg/l in control and test vessels.ÁNote: For the first criterion, not more than 10 percent of the control daphnids should show immobilisation or other signs of disease or stress, for example, discoloration or unusual behaviour such as trapping at surface of water.78R6QBISBHGÁH9ÁSA8ÁF8SAH7Á4 f f Á7. Test vessels and other apparatus that will come into contact with the test solutions should be made entirely of glass or other chemically inert material. Test vessels will normally be glass test tubes or beakers; they should be cleaned before each use using standard laboratory procedures. Test vessels should be loosely covered to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions. Volatile substances should be tested in completely filled closed vessels large enough to prevent oxygenÁbecoming limiting or too low (see paragraphs 6 and 22).8. In addition some or all of the following equipment will be used: oxygen-meter (with microelectrode or other suitable equipment for measuring dissolved oxygen in low volumes samples); pH-meter; adequate apparatus for temperature control; equipment for the determination of total organic carbon concentration (TOC); equipment for the determination of chemical oxygen demand (COD); equipment for the determination of hardness, etc.Sp Á rfyt xÁ9. 7f sytfÁxfryfÁStraus is the preferred test species although other suitable 7f sytf species can be used in this test (e.g. 7f sytfÁ wp ). At the start of the test, the animals should be less than 24 hours old and, to reduce variability, it is strongly recommended they areÁnot first brood progeny. They should be derived from a healthy stock (i.e. showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discoloured animals, etc.). All organisms used for a1The results of these inter laboratory tests and a Technical Corrigendum to ISO 6341 give an EC-24 h of the potassium50dichromate (K2Cr2O7) within the range 0.6 mg/l to 2.1 mg/lOECD/OCDE 202 particular test should have originated from cultures established from the same stock of daphnids. The stock animals must be maintained in culture conditions (light, temperature, medium) similar to those to beused in the test. If the daphnids culture medium to be used in the test is different from that used for routine daphnids culture, it is good practice to include a pre-test acclimation period. For that, brood daphnidsshould be maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.A wityrÁfyiÁitw t yÁ f p ÁÁ10. Natural water (surface or ground water), reconstituted water or dechlorinated tap water are acceptable as holding and dilution water if daphnids will survive in it for the duration of the culturing,acclimation and testing without showing signs of stress. Any water which conforms to the chemical characteristics of an acceptable dilution water as listed in Annex 2 is suitable as a test water. It should beof constant quality during the period of the test. Reconstituted water can be made up by adding specific amounts of reagents of recognised analytical grade to deionised or distilled water. Examples of reconstituted water are given in (1)(6) and in Annex 3. Note that media containing known chelating agents,such as M4 and M7 media in Annex 3, should be avoided for testing substances containing metals. The pH should be in the range of 6 to 9. Hardness between 140 and 250 mg/l (as CaCO3) is recommended for 7f sytfÁxfryf, while lower hardness may be also appropriate for other 7f sytf species. The dilution water may be aerated prior to use for the test so that the dissolved oxygen concentration has reachedsaturation.11. If natural water is used, the quality parameters should be measured at least twice a year orwhenever it is suspected that these characteristics may have changed significantly (see paragraph 10 and Annex 2). Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni) should also be made. If dechlorinated tap water is used, daily chlorine analysis is desirable. If the dilution water is from a surface or ground water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) should be measured.Sp Á w t y Á12. Test solutions of the chosen concentrations are usually prepared by dilution of a stock solution.Stock solutions should preferably be prepared by dissolving the test substance in the dilution water. As far as possible, the use of solvents, emulsifiers or dispersants should be avoided. However, such compounds may be required in some cases in order to produce a suitably concentrated stock solution. Guidance for suitable solvents, emulsifiers and dispersants is given in (4). In any case, the test substance in the test solutions should not exceed the limit of solubility in the dilution water.13. The test should be carried out without the adjustment of pH. If the pH does not remain in the range 6-9, then a second test could be carried out, adjusting the pH of the stock solution to that of the dilution water before addition of the test substance. The pH adjustment should be made inÁsuch a way that the stock solution concentration is not changed to any significant extent and that no chemical reaction or precipitation of the test substance is caused. HCl and NaOH are preferred.Á202 OECD/OCDEIQH687TQ8ÁÁ6 yit t y Á qÁp pÁÁSp Ár ÁfyiÁh y w ÁÁ14. Test vessels are filled with appropriate volumes of dilution water and solutions of test substance. Ratio of air/water volume in the vessel should be identical for test and control groups. Daphnids are then placed into test vessels. At least 20 animals, preferably divided into four groups of five animals each, should be used at each test concentration and for the controls. At least 2 ml of test solution should be provided for each animal (i.e. a volume of 10 ml for five daphnids per test vessel). The test may be carried out using semi-static renewal or flow-through system when the concentration of the test substance is not stable.15. One dilution-water control series and also, if relevant, one control series containing the solubilising agent (solvent control) at the level used in treatments must be run in addition to the treatment series.Sp Áh yhpy f t y Á16. A range-finding test mayÁbe conducted to determine the range of concentrations for the definitive test unless information on toxicity of the test substance is available. For this purpose, the daphnids are exposed to a series of widely spaced concentrations of the test substance. Five daphnids should be exposed to each test concentration for 48 hours or less, and no replicates are necessary. The exposure period may be shortened (e.g. 24 hours or less) if data suitable for the purpose of the range-finding test can be obtained in less time.17. At least five test concentrations should be used. They should be arranged in a geometric series with a separation factor preferably not exceeding 2.2. Justification should be provided if fewer than five concentrations are used ÁÁThe highest concentration tested should preferably result in 100 per cent immobilisation Áand the lowest concentration tested should preferably give no observable effect.Byh gf t yÁh yit t y Á18. The temperature should be within the range of 18q C and 22q C, and for each single test it should be constant within r 1q C. A 16-hour light and 8-hour dark cycle is recommended ÁÁComplete darkness is also acceptable, especially for test substances unstable in light.19. The test vessels must not be aerated during the test. The test is carried out without adjustment of pH. The daphnids should not be fed during the test.7 f t yÁ20. The test duration is 48 hours.Hg p f t y Á21. Each test vessel should be checked for immobilised daphnids at 24 and 48 hours after the beginning of the test. (see Annex 1 for definitions). In addition to immobility, any abnormal behaviour or appearance should be reported.OECD/OCDE 2024yfw thfwÁxpf pxpy Á22. The dissolved oxygen and pH are measured at the beginning and end of the test in the control(s) and in the highest test substance concentration. The dissolved oxygen concentration in controls should be in compliance with the validity criterion (see paragraph 6). The pH should normally not vary by more than 1.5 units in any one test. The temperature is usually measured in control vessels or in ambient air and it should be recorded preferably continuously during the test or, as a minimum, at the beginning and end of the test.23. The concentration of the test substance should be measured, as a minimum, at the highest and lowest test concentration, at the beginning and end of the test (4). It is recommended that results be based on measured concentrations. However, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within r 20 per cent of the nominal or measured initial concentration throughout the test, then the results can be based on nominal or measured initial values. EBFBSÁS8RSÁ24. Using the procedures described in this Guideline, a limit test may be performed at 100 mg/l of test substance or up to its limit of solubility in the test medium (whichever is the lower) in order to demonstrate that the EC50 is greater than this concentration. The limit test should be performed using 20 daphnids (preferably divided into four groups of five), with the same number in the control(s). If the percentage of immobilisation exceeds 10% at the end of the test, a full study should be conducted. Any observed abnormal behaviour should be recorded.74S4Á4G7ÁQ8IHQSBG@ÁÁ7f fÁ25. Data should be summarised in tabular form, showing for each treatment group and control, the number of daphnids used, and immobilisation at each observation. The percentages immobilised at 24 hours and 48 hours are plotted against test concentrations. Data are analysed by appropriate statistical methods (e.g. probit analysis, etc.) to calculate the slopes of the curves and the EC50 with 95% confidence limits (p = 0.95) (7)(8).26. Where the standard methods of calculating the EC50 are not applicable to the data obtained, the highest concentration causing no immobility and the lowest concentration producing 100 per cent immobility should be used as an approximation for the EC50 (this being considered the geometric mean of these two concentrations).Sp Á p Á27. The test report must include the following:Testsubstance:- physical nature and relevant physical-chemical properties;- chemical identification data, including purity.202 OECD/OCDEspecies:Test- source and species of 7f sytf, supplier of source (if known) and the culture conditions used (including source, kind and amount of food, feeding frequency).conditions:Test- description of test vessels: type and volume of vessels, volume of solution, number of daphnids per test vessel, number of test vessels (replicates) per concentration;- methods of preparation of stock and test solutions including the use of any solvent or dispersants, concentrations used;- details of dilution water: source and water quality characteristics (pH, hardness, Ca/Mg ratio, Na/K ratio, alkalinity, conductivity, etc.); composition of reconstituted water if used;- incubation conditions: temperature, light intensity and periodicity, dissolved oxygen, pH, etc.Results:- the number and percentage of daphnids that were immobilised or showed any adverse effects (including abnormal behaviour) in the controls and in each treatment group, at eachobservation time and a description of the nature of the effects observed;- results and date of test performed with reference substance, if available;- the nominal test concentrations and the result of all analyses to determine the concentration of the test substance in the test vessels; the recovery efficiency of the method and the limit ofdetermination should also be reported;- all physical-chemical measurements of temperature, pH and dissolved oxygen made during the test;EC50 at 48h for immobilisation with confidence intervals and graphs of the fitted model the-used for their calculation, the slopes of the dose-response curves and their standard error;statistical procedures used for determination of EC50; (these data items for immobilisation at24h should also be reported when they were measured.)- explanation for any deviation from the Test Guideline and whether the deviation affected the test results.EBS8Q4STQ8Á(1) ISO 6341. (1996). Water quality - Determination of the inhibition of the mobility of 7f sytfÁxfryf Straus (Cladocera, Crustacea) - Acute toxicity test. Third edition, 1996.(2) EPA OPPTS 850.1010. (1996). Ecological Effects Test Guidelines - Aquatic Invertebrate AcuteToxicity Test, Freshwater Daphnids.(3) Environment Canada. (1996) Biological test method. Acute Lethality Test Using 7f sytf spp.EPS 1/RM/11. Environment Canada, Ottawa, Ontario, Canada.(4) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECDEnvironmental Health and Safety Publication. Series on Testing and Assessment. No. 23. Paris 2000.OECD/OCDE 202 (5) Commission of the European Communities. Study D8369. (1979). Inter-laboratory TestProgramme concerning the study of the ecotoxicity of a chemical substance with respect to 7f sytf ÁÁ(6) OECD Guidelines for the Testing of Chemicals. Guideline 211: 7f sytfÁxfryf ReproductionTest, adopted September 1998.(7) Stephan C.E. (1977). Methods for calculating an LC50. In Aquatic Toxicology and HazardEvaluation (edited by F.I. Mayer and J.L. Hamelink). ASTM STP 634 - American Society for Testing and Materials. Pp65-84(8) Finney D.J. (1978). Statistical Methods in Biological Assay. 3rd ed. London. Griffin, Weycombe,UK.202 OECD/OCDE4GG8WÁ789BGBSBHGRÁIn the context of this guideline, the following definitions are used:EC50 is the concentration estimated to immobilise 50 per cent of the daphnids within a stated exposure period. If another definition is used, this must be reported, together with its reference. Immobilisation: Those animals that are not able to swim within 15 seconds, after gentle agitation of the test vesselÁare considered to be immobilised (even if they can still move their antennae).OECD/OCDE 2024GG8WÁ ÁÁRHF8Á6A8FB64EÁ6A4Q46S8QBRSB6RÁH9Á4GÁ4668IS45E8Á7BETSBHGÁV4S8QÁR g fyhpÁ6 yhpy f t yÁParticulate matter <20 mg/lTotal organic carbon < 2 mg/lUnionised ammonia < 1 P g/lResidual chlorine <10 P g/lTotal organophosphorus pesticides <50 ng/lTotal organochorine pesticides plus polychlorinated biphenyls <50 ng/lTotal organic chlorine <25 ng/l202 OECD/OCDE4GG8WÁ8W4FIE8RÁH9ÁRTBS45E8ÁQ86HGRSBSTS87ÁS8RSÁV4S8QÁÁBRHÁSp Á f p ÁÉ ÁR hvÁ w t y ÁÉ tyrwpÁ g fyhp ÁR g fyhpÁ4x y ÁfiipiÁ ÁÁÁwt pÁ f p ÁS Á p f pÁ spÁ ph y t piÁ f p ÁfiiÁ spÁq ww tyrÁ w xp Á qÁ hvÁ w t y Á Á Áwt pÁ f p ÁCalcium chlorideCaCl2, 2H2O11.76 g 25 mlMagnesium sulfateMgSO4, 7H2O4.93 g 25 mlSodium bicarbonateNaHCO32.59 g 25 mlPotassium chlorideKCl0.23 g 25 ml* Water of suitable purity, for example deionised, distilled or reverse osmosis with conductivity preferably not exceeding 10 P S.cm-1.OECD/OCDE 2024GG8WÁ É6 y ÁÁÁ8wpyi ÁF$ÁfyiÁF!Áxpit xÁÁÁ4hhwtxf t yÁ Á8wpyi ÁF!ÁfyiÁF$Áxpit xÁÁSome laboratories have experienced difficulty in directly transferring Daphnia to M4 and M7 media.However, some success has been achieved with gradual acclimation, i.e. moving from own medium to30% Elendt, then to 60% Elendt and then to 100% Elendt. The acclimation periods may need to be as longas one month.I p f f t yÁ S fhpÁpwpxpy ÁÁSeparate stock solutions (I) of individual trace elements are first prepared in water of suitable purity, forexample deionised, distilled or reverse osmosis. From these different stock solutions (I) a second singlestock solution (II) is prepared, which contains all trace elements (combined solution), i.e.:To prepare the combined stock solution II, add the following amount of stock solution I to water (ml/l)Stock solution(s) I(single substance) Amount added to water(mg/l) Concentration (related to medium M4) M4 M7H 3BO 3 57 190 20 000-fold 1.0 0.25MnCl 2x 4H 2O7 210 20 000-fold 1.0 0.25 LiCl 6 120 20 000-fold 1.0 0.25RbCl 1 420 20 000-fold 1.0 0.25SrCl 2x 6H 2O3 040 20 000-fold 1.0 0.25 NaBr 320 20 000-fold 1.0 0.25Na 2MoO 4x 2H 2O1 230 20 000-fold 1.0 0.25 CuCl 2x 2H 2O335 20 000-fold 1.0 0.25 ZnCl 2 260 20 000-fold 1.0 1.0CoCl 2x 6H 2O200 20 000-fold 1.0 1.0 KI 65 20 000-fold 1.0 1.0 Na 2SeO 3 43.8 20 000-fold 1.0 1.0 NH 4VO 3 11.5 20 000-fold 1.0 1.0Na 2EDTA x 2H 2O5 000 2 000-fold - - FeSO 4x 7H 2O1991 2 000-fold - - Both Na 2EDTA and FeSO 4 solutions are prepared singly, poured together and autoclaved immediately.This gives:21 Fe-EDTA solution1 000-fold 20.0 5.0202OECD/OCDEF!ÁfyiÁF$ÁxpitfÁÁM4 and M7 media are prepared using stock solution II, the macro-nutrients and vitamin as follows:Amount of stock solution II added to prepare medium (ml/l) Amount added to water (mg/l) Concentration (related to medium M4) M4 M7Stock solution II (combined trace elements)20-fold 50 50 Macro nutrient stocksolutions (single substance)CaCl 2x 2H 2O293 800 1 000-fold 1.0 1.0 MgSO 4x 7H 2O246 600 2 000-fold 0.5 0.5 KCl 58 000 10 000-fold 0.1 0.1 NaHCO 3 64 800 1 000-fold 1.0 1.0Na 2SiO 3x 9H 2O50 000 5 000-fold 0.2 0.2 NaNO 3 2 740 10 000-fold 0.1 0.1 KH 2PO 4 1 430 10 000-fold 0.1 0.1 K 2HPO 4 1 840 10 000-fold 0.1 0.1 Combined Vitamin stock - 10 000-fold 0.1 0.1 The combined vitamin stock solution is prepared by adding the 3 vitamin to 1 litre water, as shown below: Thiamine hydrochloride 750 10 000-fold Cyanocobalamine (B 12) 10 10 000-fold Biotine 7.5 10 000-foldThe combined vitamin stock is stored frozen in small aliquots. Vitamins are added to the media shortly before use.N.B: To avoid precipitation of salts when preparing the complete media, add the aliquots of stocksolutions to about 500 – 800 ml deionised water and then fill up to 1 litre.N.N.B: The first publication of the M4 medium can be found in Elendt, B. P. (1990). Seleniumdeficiency in crustacea; an ultrastructual approach to antennal damage in 7f sytfÁxfryf Straus. Protoplasma, 154, 25-33.。

OECD/OCDE 122Adopted:26 July 20131© OECD, (2013)You are free to use this material for personal, non-commercial purposes without seeking prior consent from the OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written permission from the OECD.OECD GUIDELINES FOR THE TESTING OF CHEMICALS Determination of pH, Acidity and AlkalinityINTRODUCTION1. This Test Guideline 122 describes the procedure for the electrometric determination of pH of an undiluted aqueous solution or dispersion; the pH of a 1% (w/v) dilution of a solution or dispersion in distilled or deionised water; or the pH of a chemical, diluted to end-use concentration. It also describes procedures to determine acid reserve or alkali reserve for a chemical that is acidic (pH < 4) or alkaline (pH > 10) with either strong or weak acid or alkali.2. CIPAC MT 75.3 (1) and OPPTS 830.7000 (2) describe procedures for the determination of pH of a chemical 1 or a 1% (w/v) aqueous dilution or dispersion of the chemical using a pH meter, electrode and calibration solutions. ASTM D1193 (3) and CIPAC MT 191 (4) provide guidance on the reagent water used for dilution. CIPAC MT 191 and ASTM D1067 (5) describe the procedures to determine acidity or alkalinity of chemicals 1 using titrimetry and electrometric fixed endpoint determination.3. This guideline is based on CIPAC MT 75.3 "Determination of pH Values" and CIPAC MT 191 "Acidity or Alkalinity of Formulations 1". CIPAC MT 191 was adopted from CIPAC MT 31 "Free Acidity or Alkalinity".SIGNIFICANCE4. This Test Guideline provides procedures to obtain data on pH, acidity and alkalinity of aqueous solutions or aqueous dispersions of chemicals (substances and mixtures). The data will be used to assess the effects that the chemical may pose to human health and safety and the potential impact upon the environment.SCOPE5. The method is suitable for determining the pH of an aqueous solution or aqueous dispersion, in the range of 0 ≤ pH ≤ 14. A non-aqueous solution or dispersion should be diluted in water to obtain the pH measurement.1 CIPAC MT publications 75.3 and 191 use the word "formulation"122OECD/OCDE6. If the pH is lower than 4, acidity is determined by titration with standardized strong base. Likewise, if the pH is higher than 10, alkalinity is determined with standardized strong acid. DEFINITION AND UNITS7. Electrometric determination of pH measures the negative log10aqueous hydronium ion concentration [H3O+] of ideal solutions.8. Consistent with CIPAC MT 31 and CIPAC MT 191, alkalinity is calculated as % NaOH (mass/mass) in the solution or dispersion; and, acidity is calculated as % H2SO4 (mass/mass) in the solution or dispersion.Note 1: Whereas OECD governmental agencies stipulate the units of % acid or alkali per mass of chemical as per CIPAC MT 191, there are other units encountered to express acidity and alkalinity. For example, g/kg (g acid or alkali per kg chemical1) is sometimes used. As carbonate and bicarbonate are naturally occurring buffers in the environment, alkalinity and acidity of a test substance may sometimes be reported as the meq/L (milliequivalent) of either carbonate or bicarbonate.OUTLINE OF THE METHOD9. The pH of an aqueous solution or dispersion in water is determined with a pH meter equipped with an appropriate electrode system.10. The acidity or alkalinity of a solution or dispersion in water is determined by titration with standard acid or alkali using electrometric endpoint detection.DESCRIPTION OF THE METHODReagents11. The reagents used are:Buffer Solutions: pH 7, pH 4 and pH 10. These may be commercially available reference solutions or solutions prepared in the laboratory. If the solutions are prepared in the laboratory, document the preparation of the buffer solutions and assign an expiration date.Water: Distilled or deionised water:a.With an electrical resistivity ≥ 1 MΩ.cm.b.Freshly distilled/deionised or stored to prevent accumulation of CO2 from the atmosphere,e.g. CIPAC RE 130 (6).Sodium Hydroxide Standard Solution: NaOH 0.01 to 0.2 mol/L standardized solution. This solution may be a commercially available standard solution or prepared in the laboratory,e.g. CIPAC RE 25 (7).Acid standard solution: H2SO40.01 to 0.2 mol/L standardized solution. This solution may be a commercially available standard solution or prepared in the laboratory, e.g. CIPAC RE 28 (8).Acetone: An appropriate grade of acetone that reports acidity and alkalinity < 0.01%.2© OECD, (2013)OECD/OCDE 122 Apparatus12. The apparatus used are:pH Meter: Capable of at least a two-point calibration.pH Electrode System:e.g.a single or dual glass electrode system conditioned and maintained according to the manufacturer’s instructions.Graduated Mixing Cylinders: 50 mL and 100 mL with stoppers.Burette: 25 mL.Beakers: 100 to 250 mL (or other suitable containers for titration).Magnetic Stirrer: Magnetic stirrer and stir bars suitable for titration.Automatic Titrator: as alternative to the pH meter, pH electrode system, burette and stirrer. Procedure13. Determination of pH value of a chemical:a. Calibration: Operate the pH meter and the pH electrode system according to the manufact urer’soperating instructions. Calibrate the measurement system (i.e.pH meter and pH electrode system) according to the manufacturer’s operating instructions using at least two appropriate buffer solutions.b. pH Measurement of a diluted (1%) solution or dispersion:i. Weigh 1.0 g of sample into a mixing cylinder containing ~50 mL reagent water. Addreagent water to bring the total volume to 100 mL, stopper and shake vigorously until thechemical is completely dissolved or dispersed.ii. Transfer the solution or dispersion to a 200-mL beaker and allow any suspended material to settle for one minute.iii. Ensure that the temperature of the diluted solution or dispersion of the chemical does not differ from the reference solutions used for calibration. Immerse the electrode into thediluted solution or dispersion of the chemical and immediately start the stopwatch. Recordthe pH value after one minute and two minutes, without stirring during the measurement. Ifthe pH values differ by more than 0.1 pH units, record and report the pH value 10 minutesafter immersion of the electrode. (Note: When using an automatic pH meter, where themeasurement stops when the change in measured pH is less than a pre-set drift value of0.1 pH units/min, a measurement period of less than 10 min. is acceptable).Note 2: Fluctuation of the pH reading may be observed. This may be the result of insufficient ion concentration. The ion concentration can be increased, and pH reading stabilized, with the addition of some drops of a concentrated sodium chloride solution.3122OECD/OCDE4© OECD, (2013)c. pH Measurement of an undiluted aqueous solution or dispersion : Transfer sufficient solutionor dispersion to a 100-mL beaker and proceed with 13-b-iii above.14. Determination of acidity or alkalinity of a chemical:a. Calibration : Operate the pH meter and the pH electrode system according to the manufacturer’soperating instructions. Calibrate the pH meter and pH electrode system according to the manufacturer’s operating instructions using at least two appropriate buffer solutions.b. Titration of Acidity or Alkalinity :i. If the pH from 13 above is < 4.0, acidity will be determined using standardized sodiumhydroxide solution. If the pH from 13 above is > 10.0, alkalinity will be determined usingstandardized sulphuric acid solution.ii. Weigh 10.0 g (record mass to the nearest mg) of sample into a 200-mL beaker. Add 100 mLreagent water and stir until the complete dissolution or dispersion (see Note 2). Note 3: If the solution or dispersion cannot be titrated due to plugging of the electrodes, the solution or dispersion may be pre-treated with 10 mL of acetone prior to adding the deionised water. The use of acetone must be reported.iii.Stir and titrate electrometrically with an appropriate concentration of sodium hydroxide solution or sulphuric acid solution, at ambient temperature to an endpoint of pH 7. iv. Calculate acidity or alkalinity using the appropriate equation below:cidity ( alculated as H O ) . t c 1wm m) l alinity ( alculated as aOH ). s c w m m) where:c 1 = c (NaOH), mol/L (normality) of the solutionc 2 = c (H 2SO 4), mol/L (normality) of the solutiont = volume (mL) NaOH solution (endpoint pH 7)s = volume (mL) of H 2SO 4 solution (endpoint pH 7)w = weight (g) of sampleNote 4: The sample weight (w) may be reduced if high acidity or alkalinity (i.e. exceeding 25 mL titrant) is anticipated.OECD/OCDE 122 DATA AND REPORTINGRaw Data15. All raw data related to pH, alkalinity and acidity should be retained. This includes test facility worksheets, original observations, printouts from automated equipment, etc.Test Report16. The test report should include the following information:Chemicali. Name, batch number (if any)ii. Relevant physical-chemical properties or characteristicsTest conditionsi. Dates of the performance of the measurementsii. Temperature during the experimentiii. pH measurement time and observations , if necessaryiv. Weights of samplesv. Volume and titer of titrant usedvi. Dilution of test substancevii. Use of acetone, if necessaryviii. Description or identification of apparatus usedResultsi. pHii. Temperatureiii. Acidity or alkalinity, as appropriateiv. Measurement uncertaintyLITERATURE(1) Collaborative International Pesticide Analytical Council, Ltd. (CIPAC) (2000), HandbookJ“MT 75.3 Determination of pH Values”CIPAC () as amended by erratum /errata.htm: Handbook J. CIPAC Publications available from: Marston Book Services Ltd.: ().5122OECD/OCDE(2) United States Environmental Protection Agency (EPA) (1996), Product Properties TestGuidelines OCSPP 830.7000 “pH” EPA 712-C-96-030.(3) ASTM International (2006), Standard Specification for Reagent Water, Annual Book of ASTMStandards, ASTM D 1193-06, American Society for Testing and Materials, Philadelphia, PA. (4) Collaborative International Pesticide Analytical Council, Ltd. (CIPAC) (2006), Handbook L“MT191 Acidity or Alkalinity of Formulations” CIPAC (). CIPAC Publications available from: Marston Book Services Ltd.: ().(5) ASTM International (2006),Standard Test Methods for Acidit y or Alkalinity of Water AnnualBook of ASTM Standards,ASTM D 1067-06,American Society for Testing and Materials, Philadelphia, PA.(6) CIPAC RE 130 (Water for Laboratory Use) –Collaborative International Pesticide AnalyticalCouncil, Ltd. (CIPAC) (1993), Handbook E, “Reagen ts, Indicators, and Solvents –RE 130 Water for Laboratory Use” IP ). CIPAC Publications available from: Marston Book Services Ltd.: ().(7) CIPAC RE 25 (Sodium Hydroxide) –Collaborative International Pesticide Analytical Council,Ltd. (CIPAC) (1993), Handbook E, “Reagen ts, Indicators, and Solvents –RE 25 Sodium Hydroxide” IP ). CIPAC Publications available from: Marston Book Services Ltd.: ()(8) CIPAC RE 28 (Sulphuric Acid) –Collaborative International Pesticide Analytical Council, Ltd.(CIPAC) (1993), Handbook E, “Reagents, Indicators, and Solvent s –RE 28 Sulphuric Acid”CIPAC (). CIPAC Publications available from: Marston Book Services Ltd.: ().6© OECD, (2013)。

oecd化学品测试准则
OECD（经济合作与发展组织）化学品测试准则是一组广泛使
用的标准，用于评估和分类化学品的安全性和环境影响。

这些准则由OECD成员国制定，旨在确保全球范围内对化学品进
行一致和可比较的测试和评估。

OECD化学品测试准则涵盖了各种不同类型的化学品，包括工业化学品、农药、生物技术产品和化妆品等。

这些准则指导了测试方法、试验方案和数据分析等方面的标准化程序，以确保所得到的数据准确可靠。

准则还包括了对化学品的毒性、生态毒性、代谢动力学、物理化学性质和环境行为进行评估的要求。

通过使用OECD化学品测试准则，全球范围内的化学品测试
结果可以得到标准化和互认，从而可以更好地保护人类健康和环境。

这些准则在国际化学品评估和监管中发挥着重要作用，并被广泛接受和采用。

Users of this Test Guideline should consult the Preface,in particular paragraphs 3, 4, 7 and 8.402Adopted: 24 Feb 1987OECD GUIDELINE FOR TESTING OF CHEMICALS"Acute Dermal Toxicity"1.I N T R O D U C T O R Y I N F O R M A T I O N•P r e r e q u i s i t e s–Solid or liquid test substance–Chemical identification of test substance–Purity (impurities) of test substance–Solubility characteristics–Melting point/boiling point–pH (where appropriate)•S t a n d a r d d o c u m e n t sThere are no relevant international standards.2.M E T H O DA.INTRODUCTION, PURPOSE, SCOPE, RELEVANCE,APPLICATION AND LIMITS OF TESTIn the assessment and evaluation of the toxic characteristics of a substance, determination of acute dermal toxicity is useful where exposure by the dermal route is likely. It provides information on health hazards likely to arise from a short-term exposure by the dermal route. Data from an acute dermal toxicity study may serve as a basis for classification and labelling. It is an initial step in establishing a dosage regimen in subchronic and other studies and may provide information on dermal absorption and the mode of toxic action of a substance by this route.• D e f i n i t i o n sAcute dermal toxicity is the adverse effects occurring within a short time of dermal application of a single dose of a test substance.Dose is the amount of test substance applied. Dose is expressed as weight (g, mg) or as weight of test substance per unit weight of test animal (e.g. mg/kg).The LD50 (median lethal dose), dermal, is a statistically derived single dose of a substance that can be expected to cause death in 50 per cent of treated animals when applied to the skin. The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (mg/kg).402page 2"Acute Dermal Toxicity"Dosage is a general term comprising the dose, its frequ ency and the duration of dosing.Dose-response is the relationship between the dose and the proportion of a population sample showing a defined effect.Dose-effect is the relationship between the dose and the magnitude of a defined biological effect either in an individual or in a popu lation sample.•P r i n c i p l e o f t h e t e s t m e t h o dThe test substance is applied to the skin in graduated doses to several groups of experimental animals, one dose being used per gro up. Subsequently, observations of effects and deaths are made. Animals which die during the test are necropsied, and at the conclusion of the test the surviving animals are sacrificed and necropsied. Animals showing severe and enduring signs of distress and pain may need to be hum anely killed. Dosing test substances in a way known to cause marked pain and distress due to corrosive or irritating properties need not be carried out.B.DESCRIPTION OF THE TEST PROCEDURE•P r e p a r a t i o n sHealthy young adult animals are acclimatised to the laboratory conditions for at least 5 days prior to the test. Before the test, animals are randomised and assigned to the treatment groups. Approximately 24 hours before the test, fur should be removed from the dorsal area of the trunk of the test animals by clipping or shaving. Care must be taken to avoid abrading the skin, which could alter its permeability.Not less than 10 per cent of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of the co vering.When testing solids, which may be pulverised if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on penetration of skin by the test substance should be taken into account. Liquid test substances are generally used undiluted.402page 3 "Acute Dermal Toxicity"• E x p e r i m e n t a l a n i m a l sSelection of speciesThe adult rat, rabbit or guinea pig may be used. Other species may be used but their use would require justification. The following weight ranges are suggested to provide animals of a size which facilitates the conduct of the test: rats, 200 to 300 g; rabbits, 2.0 to 3.0 kg; guinea pigs, 350 to 450 g. Animals with healthy, intact skin are required.Note:In acute toxicity tests with animals of a higher order than rodents, the use of smaller numbers should be considered. Doses should be carefully selected, and every effort should be made not to exceed moderately toxic doses. In such tests, administration of lethal doses of the test substance should be avoided.Number and sexAt least 5 animals are used at each dose level. They should all be of the same sex. If females are used, they should be nulliparous and non-pregnant. The use of a sm aller number of animals may be justified in some cases. Where information is available demonstrating that a sex is markedly more sensitive, animals of this sex should be dosed.Housing and feeding conditionsAnimals should be caged individually. The temperature of the experimental animal room should be 22°C (± 3°) for rodents, 20° (± 3°) for rabbits, and the relative humidity 30-70 per cent. Where the lightings is artificial, the sequence should be 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.•T e s t c o n d i t i o n sDose levelsThese should be sufficient in number, at least three, and spaced appropriately to produce test groups with a range of toxic effects and mortality rates. The data should be sufficient to produce a dose-response curve and, where possible, permit an acceptable determ ination of the LD50.Limit testA limit test at one dose level of at least 2000 mg/kg bodyweight may be carried out in a group of 5 male and 5 female animals, using the procedures described abo ve. If compound-related mortality is produced, a full study may need to be considered.402page 4"Acute Dermal Toxicity"Observation periodThe observation period should be at least 14 days. How ever, the duration of observation should not be fixed rigidly. It should be determined by the toxic reactions, rate of onset and length of recovery period, and may thus be extended when co nsidered necessary. The time at which signs of toxicity appear and disappear, their duration and the time of death are important, especially if there is a tendency for deaths to be delayed.•P e r f o r m a n c e o f t h e t e s tThe test substance should be applied u niformly over an area which is approximately 10 per cent of the total body surface area. With highly toxic substances the surface area co vered may be less, but as much of the area should be covered with as thin and u niform a film as possible.Test substances should be held in contact with the skin with a porous gauze dressing and non-irritating tape throughout a 24-hour exposure period. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance, but complete immobilisation is not a recommended method.At the end of the exposure period, residual test substance should be removed, where practicable using water or an appropriate solvent.• C l i n i c a l e x a m i n a t i o n sObservations should be recorded systematically as they are made. I ndividual records should be maintained for each animal. Following application of the test su bstance, the animals should be observed frequently during the first day and then a careful clinical examination should be made at least once each day. Additional observations should be m ade daily with appropriate actions taken to minimise loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or mo ribund animals. Cageside observations should include changes in fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behaviour402page 5 "Acute Dermal Toxicity"pattern. Particular attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The time of death must be recorded as precisely as possible.Individual weights of animals should be determ ined shortly before the test substance is applied, weekly thereafter, and at death; changes in weight should be calculated and recorded when survival exceeds one day. At the end of the test surviving animals are weighed and then sacrificed.•P a t h o l o g yNecropsy of all animals should be carried out and all gross pathological changes should be recorded. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24 or more hours should also be considered because it may yield useful information.• A s s e s s m e n t o f t o x i c i t y i n t h e o t h e r s e xAfter completion of the study in one sex, at least one group of 5 animals of the other sex is dosed to establish that animals of this sex are not markedly more sensitive to the test substance. The use of fewer animals may be justified in individual circumstances. Where adequate information is available to demonstrate that animals of the sex tested are m arkedly more sensitive, testing in animals of the other sex may be dispensed w ith.3. D A T A A N D R E P O R T I N G•T r e a t m e n t o f r e s u l t sData may be summarised in tabular form, showing for each test group the number of animals at the start of the test, time of death of individual animals at different dose levels, number of animals displaying other signs of toxicity, description of toxic effects and necropsy findings.Animals which are hum anely killed due to compound-related distress and pain are recorded as compound-related deaths.The LD50 may be determined by any accepted method, e.g. Bliss (4), Litchfield and Wilcoxon (3), Finney (5), Weil (6), Thompson (7), Miller and Tainter (8).402page 6"Acute Dermal Toxicity"• E v a l u a t i o n o f r e s u l t sThe dermal LD50 value should always be considered in conjunction with the observed toxic effects and the necropsy findings. The LD50 value is a relatively coarse measurement, useful only as a reference value for classification and labelling purpo ses, and an expression of the lethal potential of the test substance following dermal exposure.Reference should always be made to the experimental animal species in which the LD50 value was obtained. An evaluation should include an evaluation of relationships, if any, between the animals' exposure to the test substance and the incidence and severity of all abnorm alities, including behavioural and clinical abnormalities, gross lesions, body weight changes, effect on mortality, and any o ther toxic effects.•T e s t r e p o r tThe test report should include the following information:–species/strain/source used; environmental conditions;–sex of animals do sed;–tabulation of response data by dose level (i.e. number of animals that died or were killed during the test, number of animals showing signs of toxicity, number of animals expo sed);–time of dosing and time of death after dosing;–LD50 value for the sex dosed (intact skin) determined at 14 days with the method of determination specified;–95 per cent confidence interval for the LD50 (where this can be provided);–dose-mortality curve and slope (where perm itted by the method of determination);–pathology findings; and–results of any test on the other sex.•I n t e r p r e t a t i o n o f t h e r e s u l t sA study of acute toxicity by the dermal (percutaneous) route and determination of a dermal LD50 provides an estimate of the relative toxicity of a substance by the dermal route of exposure.402page 7 "Acute Dermal Toxicity"Extrapolation of the results of acute dermal toxicity studies and dermal LD50 values in animals to man is valid only to a lim ited degree. The results of an acute dermal toxicity study should be considered in conjunction with data from acute toxicity studies by other routes.4.L I T E R A T U R E1.WHO Publication: Environmental Health Criteria 6, P rinciples and Methods for Evaluatingthe T oxicity of Chemicals. Part I, Geneva, 1978.2.National Academy of Sciences, Committee for the Revision of NAS Publication 1138,Principles and Procedures for Evaluating the T oxicity of Household S ubstances, Washington, 1977.3.Litchfield, J.T. and Wilcoxon, F., J. Pharm acol., Exp. T her., 96, 99-113, 1949.4.Bliss, C.I., Quart. J. Pharm. Pharm acol., 11, 192-216, 1938.5.Finney, D.G., Probit A nalysis. (3rd Ed.) London, Cambridge University Press, 1971.6.Weil, C.S., Biometrics, 8, 249-263, 1952.7.Thompson, W., B act. Rev., 11, 115-141, 1947.ler, L.C. and Tainter, M.L., Proc. S oc. Exp. Biol. Med. NY, 57, 261-264, 1944.。

OECD化学品测试准则第501号：在农作物中的代谢第502号：在后茬农作物中的代谢第503号：在家畜中的代谢第504号：在后茬农作物中的残留（大田中残留限量的研究）第505号：在家畜中的残留第506号：农药残留在贮藏日用品中的稳定性第507号：农药残留在加工品中的性质——高温水解第508号：加工商品中农药残留的量第509号：作物的田间试验摘要OECD化学品测试准则收集了大约100种被政府部门、工业上和独立实验室对新创制及已存在的化学物质、化学配制品和化学混合物做出鉴别和确认其潜在危险后使用的，并在国际上获得广泛认可的检测方法。

它们是一套最初用于调整安全检测和随后的化学品及化工产品通告、化学品登记的基本工具。

另外，它们也可用在挑选和将候选化学品分等级来开发新化学品和化工产品以及相应的毒理学研究中。

它们包含了化学品的物理-化学性质的测定，对人类健康的影响，对环境的影响，包括在环境中的降解和富集等方面。

自1981年被采用以来，这套准则已经成为被从事化学品检测和对它们的潜在危险作出评估的专业人员认可的参考工具。

第501号：农作物中的代谢在农作物中的代谢研究常用于阐释活性成分的代谢途径，以及农药以直接或间接的方式应用于作物后，对它的代谢物和/或降解产物的鉴定。

此项研究应该提交代谢物在每一种农作物群组类型中的情况。

五种类别的农作物代谢物研究是：根茎类蔬菜，叶类作物，水果，种子植物和油料作物，以及谷类。

活性成分的管理应该表现在诸如叶片、土壤/种子，或者反映其预期使用模式的收割后期的处理。

应当做标记，允许量化；首选同位素是14C，32P、35S也可以运用。

该研究通过使用一种以放射性同位素检测物质处理过的含有沙和有机质的土壤来完成。

可以在温室内或者户外的田间小区或植物生长大棚内进行实验。

应当达到最大使用率（建议的良好农业实践(GAP)使用率）。

我们收集所有原始未加工的农产品样本来描述和/或鉴定它们的残留情况和总体的放射性残留的判定。

301Adopted:17.07.92OECD GUIDELINE FOR TESTING OF CHEMICALSAdopted by the Council on17th July1992Ready BiodegradabilityINTRODUCTION1.In this Guideline six methods are described that permit the screening of chemicals for ready biodegradability in an aerobic aqueous medium.They are:301A:DOC Die-Away301B:CO2Evolution(Modified Sturm Test)301C:MITI(I)(Ministry of International Trade andIndustry,Japan)301D:Closed Bottle301E:Modified OECD Screening301F:Manometric RespirometryMethod301A is similar to the ISO Standard7827-1984and replaces the Modified AFNOR method;AFNOR has adopted the ISO standard.Methods301B,301D and301E are modified versions of the earlier OECD Guidelines adopted in1981.Method301C is virtually identical with earlier Guideline301C(MITI I).Method301F is new;it is similar to301C differing mainly in the inocula employed.2.Much experience has accumulated with the six methods over the years including an OECD inter-laboratory comparison exercise(ring test)in1988.The accumulated experience,and the ring test,have confirmed that the methods may be used for the assessment of ready biodegradability. However,depending on the physical characteristics of the substance to be tested,a particular method may be preferred.3.General considerations including those common to all six methods are given hereafter.Details of individual methods are given under separate headings(301A to F).Throughout the text the reader is referred to the Annexes which contain definitions(Annex I),formulas and useful guidance material. GENERAL PRINCIPLE OF THE TESTS4.A solution,or suspension,of the test substance in a mineral medium is inoculated and incubated under aerobic conditions in the dark or in diffuse light.The amount of DOC in the test solution due to the inoculum should be kept as low as possible compared with the amount of organic carbon due to the test substance.Allowance is made for the endogenous activity of the inoculum by running parallel blanks with inoculum but without test substance,although the endogenous activity of cells in the presence of a chemical will not exactly match that in the endogenous control.A reference compound is run in parallel to check the operation of the procedures.301OCDE/OECD5.In general,degradation is followed by the determination of parameters such as DOC,CO2 production and oxygen uptake and measurements are taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation.With automatic respirometers the measurement is continuous.DOC is sometimes measured in addition to another parameter but this is usually done only at the beginning and end of the test.Specific chemical analysis can also be used to assess primary degradation of the test substance and to determine the concentration of any intermediate substances formed.It is obligatory in the MITI method(301C).6.Normally,the test lasts for28days.Tests however may be ended before28days,i.e.as soonas the biodegradation curve has reached a plateau for at least three determinations.Tests may also be prolonged beyond28days when the curve shows that biodegradation has started but that the plateau has not been reached by day28,but in such cases the chemical would not be classed as readily biodegradable.INFORMATION ON THE TEST SUBSTANCE7.In order to select the most appropriate method,information on the chemical’s solubility,vapour pressure and adsorption characteristics is essential.The chemical structure or formula should be known in order to calculate theoretical values and/or check measured values of parameters,e.g.ThOD,ThCO2,DOC,TOC,and rmation on the purity or the relative proportions of majorcomponents of the test material is required in order to interpret the results obtained,especially when the result lies close to the pass level.rmation on the toxicity of the test substance to bacteria(Annex II)may be very useful for selecting appropriate test concentrations and may be essential for the correct interpretation of low biodegradation values.APPLICABILITY AND SELECTION OF METHODS9.Test substances which are soluble in water to at least100mg/l may be assessed by all methods,provided they are non-volatile and non-adsorbing.For those chemicals which are poorly soluble in water,volatile or adsorbing,suitable methods are indicated in Table1.The manner in which poorly water-soluble chemicals and volatile chemicals can be dealt with is described in Annex III,but in the MITI method neither solvents nor emulsifying agents are to be used.Moderately volatile chemicals may be tested by the DOC Die-Away method if there is sufficient gas space in the test vessels(which should be suitably stoppered).In this case,an abiotic control must be set up to allow for any physical loss.OCDE/OECD301 TABLE1APPLICABILITY OF TEST METHODSPASS LEVELS10.The pass levels for ready biodegradability are70%removal of DOC and60%of ThOD orThCO2production for respirometric methods.They are lower in the respirometric methods since,assome of the carbon from the test chemical is incorporated into new cells,the percentage of CO2 produced is lower than the percentage of carbon being used.These pass values have to be reached in a10-d window within the28-d period of the test,except where mentioned below.The10-d windowbegins when the degree of biodegradation has reached10%DOC,ThOD or ThCO2and must endbefore day28of the test.Chemicals which reach the pass levels after the28-d period are not deemed to be readily biodegradable.The10-d window concept does not apply to the MITI method.The value obtained in a14-d window would be acceptable in the Closed Bottle method if it is considered that the number of bottles necessary to evaluate the10-d window causes the test to become too unwieldy. REFERENCE COMPOUNDS11.In order to check the procedure,reference compounds which meet the criteria for ready biodegradability are tested by setting up an appropriate vessel in parallel as part of normal test runs. Suitable compounds are aniline(freshly distilled),sodium acetate and sodium benzoate.These reference compounds all degrade in these methods even when no inoculum is deliberately added.It was suggested that a reference compound should be sought which was readily biodegradable but required the addition of an inoculum.Potassium hydrogen phthalate has been proposed but more evidence needs to be obtained with this chemical before it can be accepted as a reference compound.301OCDE/OECDREPRODUCIBILITY OF TESTS12.Because of the nature of biodegradation and of the mixed bacterial populations used as inocula,determinations should be carried out at least in duplicate.It is usually found that the larger the concentration of micro-organisms initially added to the test medium,the smaller will be the variation between replicates.Ring tests have also shown that there can be large variations between results obtained by different laboratories,but good agreement is normally obtained with easily biodegradable compounds.GENERAL PROCEDURES AND PREPARATIONS13.General conditions applying to the methods are summarised in Table2.Apparatus and otherexperimental conditions pertaining specifically to an individual method are described later under the heading for that method.Water14.Deionised or distilled water,free from inhibitory concentrations of toxic substances(e.g.Cu2+ions)is used.It must contain no more than10%of the organic carbon content introduced by the test material.The high purity of the test water is necessary in order to eliminate high blank values.Contamination may result from inherent impurities and also from the ion-exchange resins and lysed material from bacteria and algae.For each series of tests,use only one batch of water,previously checked by DOC analysis.Such a check is not necessary for the Closed Bottle method,but the oxygen consumption of the water must be low(see301D,paragraph25).Mineral media15.Mineral media are prepared from stock solutions of appropriate concentrations of mineralcomponents,namely,potassium and sodium phosphates plus ammonium chloride,calcium chloride, magnesium sulphate and iron(III)chloride.Since only a very small inoculum,containing low concentrations of trace elements and growth factors,is used in the Modified OECD Screening method (301E),the medium for this test may need to be fortified with additional compounds.The details of the stock solutions of mineral salts,trace elements and growth factors and the proportions used are given under the headings for the separate tests.Methods of adding the test and reference substances16.The method used for adding the test and reference substances to the reaction mixture dependsupon the nature of the chemical,especially its water solubility.For substances of adequate solubility, greater than about1g/l,prepare stock solutions at appropriate concentrations and use aliquots to prepare the final test solution.Dissolve less soluble substances in the mineral medium to avoid diluting the buffer solution.Add substances which are even less soluble directly to the final mineral medium.Finally,refer to Annex III for the handling of poorly and insoluble substances,but note that in the MITI method(301C)neither organic solvents nor emulsifying agents are to be used.Inoculum17.The inoculum may be derived from a variety of sources:activated sludge;sewage effluents(unchlorinated);surface waters and soils;or from a mixture of these.For the DOC Die-Away(301A),CO2Evolution(301B)and Manometric Respirometry(301F)methods if activated sludge is used,it should be taken from a treatment plant or laboratory-scale unit receiving predominantly domestic sewage.Inocula from other sources,usually yielding lower cell densities,have been found to give higher scattering of results.For the Modified OECD Screening(301E)and Closed Bottle(301D)OCDE/OECD301 methods,a more dilute inoculum without sludge flocs is needed and the preferred source is a secondary effluent from a domestic waste water treatment plant or laboratory-scale unit.For the MITI(I)method,the inoculum is derived from a mixture of sources.Details of the sources and preparationof inocula are described under the headings of the specific test methods.Pre-conditioning of inoculum18.Inoculum may be pre-conditioned to the experimental conditions,but not pre-adapted to thetest substance.Pre-conditioning consists of aerating activated sludge(in mineral medium)or secondary effluent for5-7days at the test temperature.Pre-conditioning sometimes improves the precision of the test methods by reducing blank values.It is considered unnecessary to pre-conditionMITI(I)inoculum.Abiotic controls19.When required,check for the possible abiotic degradation of the test substance by determiningthe removal of DOC,oxygen uptake or carbon dioxide evolution in sterile controls containing no inoculum.Sterilize by filtration through a membrane(0.2-0.45µm)or by the addition of a suitabletoxic substance at an appropriate concentration.If membrane filtration is used,take samples aseptically to maintain sterility.Unless adsorption of the test substance has been ruled out beforehand,tests which measure biodegradation as the removal of DOC,especially with activated sludge inocula, should include an abiotic control which is inoculated and poisoned.Number of flasks and samples20.At least two flasks or vessels containing the test substance plus inoculum,and at least two containing inoculum only should be used.Single vessels suffice for reference compounds plus inoculum and,when required,for toxicity,abiotic removal and adsorption controls.The Closed Bottleand MITI(I)methods have special requirements for the number of flasks.These are given under the specific headings.It is mandatory to follow DOC and/or the other parameters in the test suspensionand inoculum blanks in parallel.It is advisable to follow DOC in the other flasks in parallel as well.This may,however,not always be possible.21.Although it is necessary to ensure that sufficient samples or readings are taken to allow the percentage removal in the10-d window to be assessed,it is not possible to specify accurately the frequency of sampling because of the wide range of the lag phases and rates of degradation.In theMITI method(301C)and,if an automatic respirometer is used in the Manometric Respirometry method(301F),sampling for oxygen uptake presents no problems.In the latter method,daily readings are adequate when non-automatic respirometers are employed.Specific advice on samplingis given under the headings of the other four tests.DATA AND REPORTINGTreatment of results22.In the calculation of Dt ,percentage degradation,the mean values of the duplicate measurementof the parameter in both test vessels and inoculum blank are used.The formulas are set out in the sections below on specific methods.The course of degradation is displayed graphically and the10-d window is indicated where applicable.Calculate and report the percentage removal achieved and the value at the plateau,or at the end of the test,and/or at the end of the10-d window,whichever is appropriate.In respirometric methods,N-containing chemicals may affect the oxygen uptake because of nitrification(see Annexes IV and V).Also,if the ThOD cannot be calculated because the test material is insufficiently defined,the COD value may be used to calculate the percentage degradation.301OCDE /OECDHowever,it must be borne in mind that the COD is often not as high as the ThOD as some chemicals are very poorly oxidised in the COD test,resulting in falsely high values for percentage biodegradation.23.When specific chemical analytical data are available,calculate primary biodegradation from:where:D t =%primary degradation at time t,normally 28days;S a =residual amount of test chemical in inoculated medium at end of the test (mg);S b =residual amount of test chemical in the abiotic control at the end of the test (mg).Validity of tests24.A test is considered valid if the difference of extremes of replicate values of the removal of the test chemical at the plateau,at the end of the test or at the end of the 10-d window,as appropriate,is less than 20%and if the percentage degradation of the reference compound has reached the pass levels by day 14.If either of these conditions is not met,the test should be repeated.Because of the stringency of the methods,low values do not necessarily mean that the test substance is not biodegradable under environmental conditions,but indicates that more work will be necessary to establish biodegradability.25.If in a toxicity test,containing both the test substance and a reference compound,less than 35%degradation (based on total DOC)or less than 25%(based on total ThOD or ThCO 2)occurred within 14days,the test substance can be assumed to be inhibitory (see Annex II for other toxicity tests).The test series should be repeated,using a lower concentration of test substance (if this can be done without seriously impairing the accuracy of the DOC determination)and/or a higher concentration of inoculum,but not greater than 30mg solids/l.26.Other conditions for the validity of test results specific to individual methods are set out under the headings for those tests.Test report 27.The test report must include the following:Test substance:-physical nature and,where relevant,physicochemical properties;-identification data.Test conditions:-inoculum:nature and sampling site(s),concentration and any pre-conditioning treatment;-proportion and nature of industrial waste water in sewage,if known;-test duration and temperature;-in the case of poorly soluble test substances,methods of preparation of test solutions/suspensions;OCDE/OECD301 -test method applied;scientific reasons and explanation forany change of procedure.Results:-data in tabular form;-any observed inhibition phenomena;-any observed abiotic degradation;-specific chemical analytical data,if available;-analytical data on intermediates,if available;-the graph of percentage degradation against time for the testand reference substances,the lag phase,degradation phase,the10-d window and slope(see Annex I for definitions);-percentage removal at plateau,at end of test,and/or after10-d window.Discussion of results.301OCDE /OECDT A B L E 2:T E S T C O N D I T I O N SD O C =D i s s o l v e d O r g a n i c C a r b o nT h O D =T h e o r e t i c a l O x y g e n D e m a n dS S =S u s p e n d e d S o l i d sOCDE/OECD301301A"DOC DIE-AWAY TEST"INTRODUCTION1.Matters of general interest concerning the assessment of biodegradability are discussed in "General Procedures and Preparations"and it is advisable to read this before proceeding.For this method,the test substance should be non-volatile and have a solubility in water of at least100mg/l.Also the carbon content and,preferably,the purity or relative proportions of major components shouldbe known.This test is virtually the same as the ISO Standard7827-1984.It is similar to the Modified OECD Screening test(301E)but allows the use of much higher microbial cell densities. PRINCIPLE OF THE TEST2.A measured volume of inoculated mineral medium,containing a known concentration of thetest substance(10-40mg DOC/l)as the nominal sole source of organic carbon,is aerated in the darkor diffuse light at22±2o C.Degradation is followed by DOC analysis at frequent intervals over a28-day period.The degree of biodegradation is calculated by expressing the concentration of DOC removed(corrected for that in the blank inoculum control)as a percentage of the concentration initially present.Primary biodegradation may also be calculated from supplemental chemical analysis for parent compound made at the beginning and end of incubation.DESCRIPTION OF THE METHODApparatus3.Normal laboratory apparatus and:(a)Conical flasks,e.g.250ml to2litre,depending on the volume needed for DOCanalysis.The flasks must be carefully cleaned with,for example,alcoholichydrochloric acid,rinsed and dried before each test;(b)Shaking machine-to accommodate the conical flasks,either with automatictemperature control or used in a constant temperature room,and of sufficient powerto maintain aerobic conditions in all flasks;(c)Filtration apparatus,with suitable membranes;(d)DOC analyser;(e)Apparatus for determining dissolved oxygen,to check that the flask contents areaerobic;(f)Centrifuge.Water4.A description of the water to be used is given in the"General Procedures and Preparations"(p.5).301OCDE/OECDStock solutions for mineral medium5.Prepare the following stock solutions using analytical grade reagents:(a)Potassium dihydrogen orthophosphate,KH2PO4.............8.50gDipotassium hydrogen orthophosphate,K2HPO4............21.75gDisodium hydrogen orthophosphate dihydrate,Na2HPO4.2H2O...................................33.40gAmmonium chloride,NH4Cl...........................0.50g Dissolve in water and make up to1litre.The pH of the solution should be7.4.(b)Calcium chloride,anhydrous,CaCl2....................27.50gorCalcium chloride dihydrate,CaCl2.2H20..................36.40gDissolve in water and make up to1litre.(c)Magnesium sulphate heptahydrate,MgS04.7H20............22.50gDissolve in water and make up to1litre.(d)Iron(III)chloride hexahydrate,FeCl3.6H20.................0.25gDissolve in water and make up to1litre.Note:In order to avoid having to prepare this solution immediately before use,add one drop of concentrated HCl or0.4g ethylene-diaminetetra-acetic acid(EDTA disodium salt)per litre.If a precipitate forms in a stock solution replace with a freshly made solution. Preparation of mineral medium6.Mix10ml of solution(a)with800ml water,then add1ml of solutions(b),(c)and(d)and make up to1litre with water.Stock solutions of test substances7.When the solubility of the substance exceeds1g/l,dissolve1-10g,as appropriate,of test or reference substance in water and make up to1litre.Otherwise,prepare stock solutions in mineral medium or add the chemical directly to the mineral medium,making sure it dissolves.Inoculum8.The inoculum may be derived from a variety of sources:activated sludge;sewage effluents; surface waters;soils;or from a mixture of these.Inoculum from activated sludge9.Collect a fresh sample of activated sludge from the aeration tank of a sewage treatment plant or laboratory-scale unit treating predominantly domestic sewage.Remove coarse particles if necessary by filtration through a fine sieve and keep the sludge aerobic thereafter.10.Alternatively,after removal of any coarse particles,settle or centrifuge(e.g.at1100g for10 minutes).Discard the supernatant.The sludge may be washed in the mineral medium.Suspend the concentrated sludge in mineral medium to yield a concentration of3-5g suspended solids/l. Thereafter aerate until required.11.Sludge should be taken from a properly working conventional treatment plant.If sludge has to be taken from a high rate treatment plant,or is thought to contain inhibitors,it should be washed. Settle or centrifuge the re-suspended sludge after thorough mixing,discard the supernatant and again re-suspend the washed sludge in a further volume of mineral medium.Repeat this procedure until the sludge is considered to be free from excess substrate or inhibitor.12.After complete re-suspension is achieved,or with untreated sludge,withdraw a sample just before use for the determination of the dry weight of the suspended solids.13.A further alternative is to homogenise activated sludge(3-5g suspended solids/l).Treat the sludge in a Waring blender for2minutes at medium speed. Settle the blended sludge for30minutes or longer if required and decant liquid for use as inoculum at the rate of about10ml/l of mineral medium.Other sources of inoculum14.Alternatively,the inoculum can be derived from the secondary effluent of a treatment plant or laboratory-scale unit receiving predominantly domestic sewage.Collect a fresh sample and keep it aerobic during transport.Allow to settle for1hour or filter through a coarse filter paper and keep the decanted effluent or filtrate aerobic until required.Up to100ml of this type of inoculum may be used per litre of medium.15.A further source for the inoculum is surface water.In this case,collect a sample of an appropriate surface water,e.g.river,lake,and keep aerobic until required.If necessary,concentrate the inoculum by filtration or centrifugation.Pre-conditioning of inoculum16.Inoculum may be pre-conditioned to the experimental conditions,but not pre-adapted to the test substance.Pre-conditioning consists of aerating activated sludge(in mineral medium)or secondary effluent for5-7days at the test temperature.Pre-conditioning sometimes improves the precision of the test method by reducing blank values.Preparation of flasks17.As an example,introduce800ml portions of mineral medium into2-litre conical flasks and add sufficient volumes of stock solutions of the test and reference substances to separate flasks to give a concentration of chemical equivalent to10-40mg DOC/l.Check the pH values and adjust,if necessary,to7.4.Inoculate the flasks with activated sludge or other source of inoculum to give a final concentration not greater than30mg suspended solids/l.Also prepare inoculum controls in the mineral medium but without test or reference substance.18.If needed,use one vessel to check the possible inhibitory effect of the test substance by inoculating a solution containing comparable concentrations of both the test and a reference substance in the mineral medium.19.Also,if required,check whether the test substance is degraded abiotically by setting up a flask containing a sterilised uninoculated solution of the substance.Sterilise by filtering though a membrane (0.2-0.45µm)or by the addition of a suitable toxic substance at an appropriate concentration.20.Additionally,if the test substance is suspected of being significantly adsorbed onto glass, sludge,etc.,make a preliminary assessment to determine the likely extent of adsorption and thus the suitability of the test for the chemical(see Table1,p.4).Set up a flask containing the test substance, inoculum and sterilising agent.21.Make up the volumes in all flasks to1litre with mineral medium and,after mixing,take a sample from each flask to determine the initial concentration of DOC in duplicate(see Annex IV.4). Cover the openings of the flasks,e.g with aluminium foil,in such a way as to allow free exchange of air between the flask and the surrounding atmosphere.To start the test,insert the vessels into the shaking machine.Number of flasks22.In a typical run,the following flasks are used:Flasks1&2-containing test substance and inoculum(test suspension);Flasks3&4-containing only inoculum(inoculum blank);Flask5-containing reference compound and inoculum(procedure control);and,preferably and when necessary,alsoFlask6-containing test substance and sterilising agent(abiotic sterile control);Flask7-containing test substance,inoculum and sterilising agent(adsorptioncontrol);Flask8-containing test substance,reference compound and inoculum(toxicitycontrol).PROCEDUREDOC determinations23.Throughout the test,determine the concentrations of DOC in samples from each flask in duplicate at known time intervals.It is mandatory to follow DOC in the test suspension and inoculum blanks in parallel.It is advisable to follow DOC in the other flasks in parallel as well.This may, however,not always be possible.Sampling24.Take only the minimal volume of test suspension necessary for each determination.Before sampling make good any evaporation losses from the flasks by adding water in the required amount. Mix the culture medium thoroughly before withdrawing a sample and ensure that material adhering to the walls of the vessels is re-dissolved or re-suspended before sampling.Membrane-filter or centrifuge the sample(see Annex IV.4)immediately after it has been taken.Analyse the filtered or centrifuged samples on the same day,otherwise store at2-4°C for a maximum of48h or below-18°C for a longer period.Frequency of sampling25.Ensure that a sufficient number of samples are taken to allow the percentage removal in the 10-d window to be assessed.No precise sampling pattern can be described.If analyses are performed on the day of sampling,assess the next sampling time by considering the result of the analysis.If the samples are preserved,take samples daily or every two days.Analyse the last samples(28d)first and,by a stepwise"backwards"selection of appropriate samples for analysis,it is possible to obtain a good description of the biodegradation curve with a relatively small number of determinations.Of course,if the last samples(28d)show no degradation,no further samples need be analysed.DATA AND REPORTINGTreatment of results26.Data from the test should be entered onto the attached data sheet.27.The percentage degradation(Dt)at each time a sample was taken should be calculated separately for both flasks containing test substance(i.e.Flasks1and2)using mean values of duplicate DOC measurements(see data sheet)in order that the validity of the test can be assessed(see"Data and Reporting",p.7).It is calculated using the following equation:where:Dt =%degradation at time t,Co =mean starting concentration of DOC in the inoculated culture medium containing the test substance(mg DOC/l),Ct =mean concentration of DOC in the inoculated culture medium containing test substance at time t(mg DOC/l),Cbl(o)=mean starting concentration of DOC in blank inoculated mineral medium(mg DOC/l),Cbl(t)=mean concentration of DOC blank inoculated mineral medium at time t(mg DOC/l).All concentrations are measured experimentally.28.If the test has complied with the validity criteria,display the course of degradation graphically using the mean of both flasks containing test substance.Indicate the10-d window.Calculate and report the percentage removal achieved at the plateau,at the end of the test and/or at the end of the 10-d window,whichever are appropriate.29.When specific chemical analytical data are available,calculate primary biodegradation(see "Data and Reporting",p.7).30.When a abiotic sterile control is used calculate the percentage abiotic degradation using:where,Cs(o)=DOC concentration in sterile control at day0,Cs(t)=DOC concentration in sterile control at day t.Validity of tests31.The validity criteria apply given in"Data and Reporting"(p.7).。

oecd化学品测试准则 4031.引言1.1 概述随着全球化的发展，化学品的使用和生产逐渐增加，化学品与人类和环境的接触也越来越密切。

然而，随之而来的是对于化学品安全性的关切，以及对化学品潜在风险的评估需求。

为了确保人类和环境的安全，国际社会开始制定一系列化学品测试准则。

其中，OECD（经济合作与发展组织）化学品测试准则403是其中之一。

OECD化学品测试准则403旨在提供一种系统的方法来评估化学品对光敏感人群的皮肤和眼睛的刺激性。

这些刺激性反应可能导致严重的健康问题，包括皮肤炎症和角膜损伤。

这个测试准则的目的是确保化学品在使用和生产过程中对人体和环境的安全性。

1.2 目的OECD化学品测试准则403的目的是评估化学品对光敏感人群的皮肤和眼睛的刺激性。

对于光敏感人群来说，暴露在不适当的化学品中可能会引发一系列不良反应，这些反应可能是迅速发生的、可逆的，也可能是慢性的、持久的。

这个测试准则的目标是通过系统的实验方法，评估和分类化学品对皮肤和眼睛的刺激性，以便为化学品的使用和生产提供科学依据和决策参考。

通过严格的实验步骤和标准化的操作流程，OECD化学品测试准则403旨在提供一种可靠、可重复的评估方法。

这个测试准则要求使用动物模型，通过观察化学品在接触皮肤和眼睛后引发的反应，包括红斑、水肿、溃疡等，并据此进行刺激性评分。

评分的结果将帮助决策者判断化学品对人体的危害程度，并采取相应的防护措施和管理措施，以确保人体和环境的安全。

OECD化学品测试准则403的引入是为了评估和分类化学品对光敏感人群的皮肤和眼睛的刺激性，为化学品的使用和生产提供科学依据和决策参考。

通过严格的实验步骤和标准化的操作流程，这个测试准则的目标是确保化学品在使用和生产过程中对人体和环境的安全。

2.正文2.1 全球化背景下的化学品测试准则403的必要性在当今全球化的背景下，化学品的生产和使用已经成为了现代社会的基本需求。

然而，随着化学品的广泛应用，人们对化学品的安全性和环境影响的关注也越来越高涨。

OECD GUIDELINES FOR TESTING OF CHEMICALSFull List of Test GuidelinesMarch 2006SECTION 1 - PHYSICAL-CHEMICAL PROPERTIES (blue pages)Summary of Considerations in the Report from the OECD Expert Group on Physical ChemistryNo. Title Original Adoption No. ofUpdates Most Recently Updated101 UV-VIS Absorption Spectra 12 May 1981 0 ---102 Melting Point/Melting Range 12 May 1981 1 27 July 1995 103 Boiling Point 12 May 1981 1 27 July 1995 104 Vapour Pressure 12 May 1981 1 23 March 2006 105 Water Solubility 12 May 1981 1 27 July 1995 106 Adsorption/Desorption Using a BatchEquilibrium Method12 May 1981 1 21 January 2000 107 PartitionCoefficient(n-octanol/water):Shake Flask Method12 May 1981 1 27 July 1995 108 Complex Formation Ability in Water 12 May 1981 0 ---109 Density of Liquids and Solids 12 May 1981 1 27 July 1995 110 Particle Size Distribution/Fibre Lengthand Diameter Distributions12 May 1981 0 ---111 Hydrolysis as a function of pH 12 May 1981 1 13 April 2004 112 Dissociation Constants in Water 12 May 1981 0 ---113 Screening Test for Thermal Stabilityand Stability in Air12 May 1981 0 ---114 Viscosity of Liquids 12 May 1981 0 ---115 Surface Tension of Aqueous Solutions 12 May 1981 1 27 July 1995 116 Fat Solubility of Solid and LiquidSubstances12 May 1981 0 ---No. Title Original Adoption No. ofUpdates Most Recently Updated117 PartitionCoefficient(n-octanol/water),HPLC method30 March 1989 1 13 April 2004118 Determination of the Number-AverageMolecular Weight and the MolecularWeight Distribution of Polymers usingGel Permeation Chromatography14 June 1996 0 ---119 Determination of the Low MolecularWeight Content of a Polymer UsingGel Permeation Chromatography14 June 1996 0 ---120 Solution/Extraction Behaviours ofPolymers in Water14 June 1996 1 21 January 2000121 Estimation of the AdsorptionCoefficient (K oc) on Soil and onSewage Sludge using HighPerformance Liquid Chromatography(HPLC)22 January 2001 0 ---123 Partition Coefficient (1-Octanol /Water): Slow-Stirring Method23 March 2006 0 ---SECTION 2 - EFFECTS ON BIOTIC SYSTEMS (green pages)Summary of Considerations in the Report from the OECD Expert Group on EcotoxicologyNo. Title Original Adoption No. ofUpdates Most Recently Updated201 Alga, Growth Inhibition Test 12 May 1981 2 23 March 2006202 Daphnia sp. Acute Immobilisation Test12 May 1981(adopted asDaphnia sp.14-dayReproduction Testincluding an AcuteImmobilisationTest)2 13 April 2004203 Fish, Acute Toxicity Test 12 May 1981 2 17 July 1992204 Fish, Prolonged Toxicity Test: 14-DayStudy4 April 1984 0 --- 205 Avian Dietary Toxicity Test 4 April 1984 0 --- 206 Avian Reproduction Test 4 April 1984 0 --- 207 Earthworm, Acute Toxicity Tests 4 April 1984 0 --- 208 Terrestrial Plants, Growth Test 4 April 1984 0 ---209 Activated Sludge, RespirationInhibition Test4 April 1984 0 --- 210 Fish, Early-Life Stage Toxicity Test 17 July 1992 0 --- 211 Daphnia magna Reproduction Test 21 September 1998 0 ---212 Fish, Short- term Toxicity Test onEmbryo and Sac-fry Stages21 September 1998 0 --- 213 Honeybees, Acute Oral Toxicity Test 21 September 1998 0 ---214 Honeybees, Acute Contact ToxicityTest21 September 1998 0 --- 215 Fish, Juvenile Growth Test 21 January 2000 0 --- 216 SoilMicroorganisms:NitrogenTransformation Test21 January 2000 0 ---No. Title Original Adoption No. ofUpdates Most Recently Updated217 SoilMicroorganisms:CarbonTransformation Test21 January 2000 0 ---218 Sediment-Water Chironomid ToxicityUsing Spiked Sediment13 April 2004 0 ---219 Sediment-Water Chironomid ToxicityUsing Spiked Water13 April 2004 0 --- 220 Enchytraeid Reproduction Test 13 April 2004 0 --- 221 Lemna sp. Growth Inhibition Test 23 March 2006 0 ---222 Earthworm Reproduction Test (Eiseniafetida/Eisenia andrei)13 April 2004 0 ---SECTION 3 - DEGRADATION AND ACCUMULATION (yellow pages)Summary of Considerations in the Report from the OECD Expert Group on Degradation/AccumulationNo. Title Original Adoption No. ofUpdates Most Recently Updated301 Ready Biodegradability301A : DOC Die-Away Test301B : Co2 Evolution Test301C : Modified MITI Test (I)301D : Closed Bottle Test301E : Modified OECD ScreeningTest301F : Manometric Respirometry Test12 May 1981 1 17 July 1992302A InherentBiodegradability:ModifiedSCAS Test12 May 1981 0 ---302B InherentBiodegradability:Zahn-Wellens/EMPA Test12 May 1981 1 17 July 1992 302C InherentBiodegradability:ModifiedMITI Test (II)12 May 1981 0 ---303 Simulation Test – Aerobic SewageTreatmentA: Activated Sludge UnitsB: Biofilms12 May 1981 1 22 January 2001304A Inherent Biodegradability in Soil 12 May 1981 0 ---305 Bioconcentration: Flow-Through FishTest12 May 1981 1 14 June1996 306 Biodegradability in Seawater 17 July 1992 0 ---307 Aerobic and AnaerobicTransformation in Soil24 April 2002 0 ---308 Aerobic and AnaerobicTransformation in Aquatic SedimentSystems24 April 2002 0 ---309 Aerobic Mineralisation in SurfaceWater – Simulation BiodegradationTest13 April 2004 0310 Ready Biodegradability - CO2 insealed vessels (Headspace Test)23 March 2006 0 ---No. Title Original Adoption No. ofUpdates Most Recently Updated311 Anaerobic Biodegradability of OrganicCompounds in Digested Sludge: byMeasurement of Gas Production23 March 2006 0 --- 312 Leaching in Soil Columns 13 April 2004 0SECTION 4 - HEALTH EFFECTS (pink pages)Summary of Considerations in the Report from the OECD Expert Groups on Short and Long Term ToxicologyNo. Title Original Adoption No. ofUpdates Most Recently Updated401 Acute Oral Toxicity 12 May 1981 1 Date of Deletion:20 December 2002 402 Acute Dermal Toxicity 12 May 1981 1 24 February 1987 403 Acute Inhalation Toxicity 12 May 1981 0 ---404 Acute Dermal Irritation/Corrosion 12 May 1981 2 24 April 2002 405 Acute Eye Irritation/Corrosion 12 May 1981 2 24 April 2002 406 Skin Sensitisation 12 May 1981 1 17 July 1992 407 Repeated Dose 28-Day Oral ToxicityStudy in Rodents12 May 1981 1 27 July 1995408 Repeated Dose 90-Day Oral ToxicityStudy in Rodents12 May 1981 1 21 September 1998409 Repeated Dose 90-Day Oral ToxicityStudy in Non-Rodents12 May 1981 1 21 September 1998410 Repeated Dose Dermal Toxicity:90-Day12 May 1981 0 ---411 Subchronic Inhalation Toxicity: 90-Day12 May 1981 0 ---412 Repeated Dose Inhalation Toxicity:28/14-Day12 May 1981 0 ---413 Subchronic Inhalation Toxicity: 90-Day12 May 1981 0 ---414 PrenatalDevelopmentalToxicityStudy12 May 1981 1 22 January 2001 415 One-GenerationReproductionToxicity26 May 1983 0 ---416 Two-generation Reproduction ToxicityStudy26 May 1983 1 22 January 2001No. Title Original Adoption No. ofUpdates Most Recently Updated417 Toxicokinetics 4 April 1984 0 ---418 Delayed Neurotoxicity ofOrganophosphorus SubstancesFollowing Acute Exposure4 April 1984 1 27 July 1995419 Delayed Neurotoxicity ofOrganophosphorus Substances: 29-Day Repeated Dose Study4 April 1984 1 27 July 1995420 Acute Oral toxicity – Acute ToxicClass Method17 July 1992 1 17 December 2001421 Reproduction/DevelopmentalToxicityScreening Test27 July 1995 0 ---422 Combined Repeated Dose ToxicityStudy with theReproduction/Developmental ToxicityScreening Test22 March 1996 0 ---423 Acute Oral Toxicity – Acute ToxicClass Method22 March 1996 1 17 December 2001424 Neurotoxicity Study in Rodents 21 July 1997 0 ---425 Acute Oral Toxicity: Up-and-DownProcedure21 September 1998 2 23 March 2006427 SkinAbsorption:In Vivo Method 13 April 2004 ---428 SkinAbsorption:In Vitro Method 13 April 2004 ---429 Skin Sensitisation: Local Lymph NodeAssay24 April 2002 0 ---430 In Vitro Skin Corrosion:Transcutaneous Electrical ResistanceTest (TER)13 April 2004 0 ---431 In Vitro Skin Corrosion: Human SkinModel Test13 April 2004 0 ---432 In Vitro 3T3 NRU Phototoxicity Test 13 April 2004451 Carcinogenicity Studies 12 May 1981 0 ---No. Title Original Adoption No. ofUpdates Most Recently Updated452 Chronic Toxicity Studies 12 May 1981 0 ---453 CombinedChronictoxicity/Carcinogenicity Studies12 May 1981 0 ---Introduction to the OECD Guidelines on genetic toxicology testing and guidance on the selection and application of assaysNo. Title Original Adoption No. ofUpdates Most Recently Updated471 Bacterial Reverse Mutation Test 26 May 1983 1 21 July 1997472 GeneticToxicology:Escherichia coli, Reverse Assay 26 May 1983 0 Date of deletion :21 July 1997(Method mergedwith TG 471)473 In Vitro Mammalian ChromosomeAberration Test26 May 1983 1 21 July 1997474 MammalianErythrocyteMicronucleusTest26 May 1983 1 21 July 1997475 Mammalian Bone MarrowChromosome Aberration Test4 April 1984 1 21 July 1997476 In Vitro Mammalian Cell GeneMutation Test4 April 1984 1 21 July 1997 477 GeneticToxicology:Sex-LinkedRecessive Lethal Test in Drosophiliamelanogaster4 April 1984 0 ---478 Genetic Toxicology: Rodent dominantLethal Test4 April 1984 0 --- 479 GeneticToxicology:In Vitro SisterChromatid Exchange assay inMammalian Cells23 October 1986 0 --- 480 GeneticToxicology:Saccharomycescerevisiae, Gene Mutation Assay23 October 1986 0 --- 481 GeneticToxicology:Saccharomycescerevisiae, Mitotic Recombination23 October 1986 0 ---No. Title Original Adoption No. ofUpdates Most Recently UpdatedAssay482 GeneticToxicology:DNADamageand Repair, Unscheduled DNASynthesis in Mammalian Cells In Vitro23 October 1986 0 --- 483 MammalianSpermatagonialChromosome Aberration Test23 October 1986 1 21 July 1997 484 Genetic Toxicology: Mouse Spot Test 23 October 1986 0 ---485 Genetic Toxicology: Mouse HeritableTranslocation Assay23 October 1986 0 ---486 Unscheduled DNA Synthesis (UDS)Test with Mammalian Liver Cells InVivo21 July 1997 0 ---。

Users of this Test Guideline should consult the Preface,in particular paragraphs 3, 4, 7 and 8."Activated Sludge, RespirationInhibition Test"1.I N T R O D U C T O R Y I N F O R M A T I O N•P r e r e q u i s i t e s– Water solubility– Vapour pressure•G u i d a n c e i n f o r m a t i o n– Structural formula– Purity of the test substance•Q u a l i f y i n g s t a t e m e n t s–This test guideline is most readily applied to substances which, due to their w ater solubility and low volatiliy, are likely to remain in water.–For test substances with limited solubility in the test media, it may not be possible to determine the EC 50.–Results based on oxygen uptake may lead to erroneous conclusions when the test substance has the propensity to uncouple oxidative phosphorylation.•R e c o m m e n d a t i o nActivated sludge may contain potentially pathogenic organisms and should be handled with care.•S t a n d a r d d o c u m e n t sSee Section 4, Literature.209page 2"Activated Sludge, Respiration Inhibition Test"2.M E T H O DA.INTRODUCTION, PURPOSE, SCOPE, RELEVANCE,APPLICATION AND LIMITS OF TESTThe method described in this test guideline assesses the effect of a test substance on micro-organisms by measuring the respiration rate under defined co nditions in the presence of different concentrations of the test substance. The method is based on that described by ETAD (Ecological and Toxicological Association of the Dyestuffs Manufacturing Industry), in which activated sludge o btained from a sewage treatment plant is used as the m icrobial source.The purpose of this test guideline is to provide a rapid screening method whereby substances which may adversely affect aerobic microbial treatment plants can be identified and to indicate suitable non-inhibitory concentrations of test substances to be used in biodegradability tests.A range-finding test may precede a definitive test. It provides information about the range of concentrations to be used in the main test.Two controls without test substance are included in the test design, one at the start and the other at the end of the test series. E ach batch of activated sludge should also be checked using a reference substance.• D e f i n i t i o n sThe respiration rate is the oxygen consumption of aerobic sludge or waste-water micro-organisms expressed generally as mg 02 per litre per hour.EC 50 in this Test Guideline is the concentration of the test substance at w hich the respiration rate is 50 per cent of that shown by the co ntrol under conditions described in this guideline.•R e f e r e n c e s u b s t a n c e sIt is recommended that 3,5-dichlorophenol as a known inhibitor of respiration be used as a reference substance and tested for EC 50 on each batch of activated sludge as a means of checking that the sensitivity of the slu dge is not abnormal.209page 3 "Activated Sludge, Respiration Inhibition Test"•P r i n c i p l e o f t h e t e s t m e t h o dThe respiration rate of an activated sludge fed w ith a standard amount of synthetic sewage feed is measured after a contact time of 30 minu tes or 3 hours, or both. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under otherwise identical conditions is also measured The inhibitory effect of the test substance at a particular concentration is expressed as a percentage of the mean respiration rates of two controls. An EC 50 value is calculated from determinations at different concentrations.• C o n d i t i o n s f o r t h e v a l i d i t y o f t h e t e s tThe test results are valid if–the two control respiration rates are within 15 per cent of each other ;–the EC 50 (3 hours) of 3,5-dichlorophenol is in the accepted range 5 to 30 mg/l. Figure 1 : Measuring Apparatus*209page 4"Activated Sludge, Respiration Inhibition Test"B.DESCRIPTION OF THE TEST PROCEDURE•P r e p a r a t i o n sEquipmentNormal laboratory equipment and especially the following is necessary :– Measuring apparatus (see Figure 1)– Aeration device– pH-electrode and m easuring equipment– O2-electrode.Solutions of the test substanceSolutions of the test substance are freshly prepared at the start of the study using a stock solution. A stock solution concentration of O.5 g/l is appropriate if the procedure recommended below is followed.[Note : A solution of 3,5-dichlorophenol can be conveniently prepared by dissolving 0.5 g 3,5-dichlorophenol in 10 ml of 1N NaOH, diluting to approximately 30 ml with distilled water, adding under stirring 1N H2SO4 to the point of incipient precipitation approximately 8 ml of 1N H2SO4 will be required and finally diluting the mixture to one litre with distilled water. The pH should then be in the range 7 to 8].Test concentrationsAt least five concentrations, spaced by a constant factor preferably not exceeding 3.2, should be used.Synthetic sewage feedA synthedic sewage feed is made by dissolving the following amounts of substances in1 litre of water :– 16 g peptone– 11 g meat extract– 3 g urea– 0.7 g NaCl209page 5 "Activated Sludge, Respiration Inhibition Test"– 0.4. g CaCl2.2H2O– 0.2 g MgSO4.7H2O– 2.8 g K2 HPO4[Note : This synthetic sewage is a 100 fold co ncentrate of that described in the OECD Technical Report "Proposed method for the determination of the biodegradability of surfactants used in synthetic detergents" June 11, 1976, w ith moreover dipotassium hydrogen phosphate added.]•T e s t s y s t e mMicrobial innoculumActivated sludge from a sewage treatment plant is normally used as the microbial innoculum for the test. Where possible, activated sludge should be obtained from a sewage work treating predominantly domestic sewage. If this is not possible, the activated sludge may be obtained from sewage works treating predominantly industrial waste water but used only following de-adaptation. Even so, results obtained with activated sludge from wo rks treating industrial waste waters may be atypical.On return to the laboratory the slu dge is washed, if necessary, w ith tap water or an isotonic solution. After centrifuging the supernatant is decanted. This procedure is repeated three times. A small amount of the washed sludge is weighed and dried. From this result the amount of wet sludge can be calculated which must be suspended in water in order to obtain an activated sludge with a mixed liquor suspended solids level of 4 g/l (± 10 per cent). This level gives a concentration of 1.6 g/l in the test m edium if the procedure recommended below is followed.If the sludge cannot be used on the day of collection, 50 ml synthetic sewage is added to each litre of the activated sludge prepared as described above ; this is then aerated overnight at 20 ± 2°C. It is then kept aerated for use during the day. Before use the pH is checked and buffered, if necessary, to pH 6.0 to 8.0 using sodium bicarbonate solution. The m ixed liquor suspended solids should be determined as described in the preceeding paragraph.209page 6"Activated Sludge, Respiration Inhibition Test"If the same batch of sludge is required to be used on subsequent days (maximum four days), a further 50 ml of synthetic sewage feed is added at the end of each working day.•T e s t c o n d i t i o n sDuration/30 minutes and/or 3 hours, during which aeration takes place contact time :Vessels :Beakers are suitableWater :Drinking water (dechlorinated if necessary)Air supply :Clean, oil-free air. Air flow 0.5 to 1 litre/minuteMeasuring Flat bottom flask such as a BOD-flask (see Figure 1) apparatus :Oxygen meter :Polarographic oxygen electrode, connectable to a potentiometricrecorder (200 mV range)Nutrient solution :Synthetic sewage feed (see above)Test substance :The test solution is freshly prepared at the start of the test Reference substance : e.g. 3,5-dichlorophenol (at least 3 concentrations)Controls :Innoculated sample without test substanceTemperature :20 ± 2°C•P e r f o r m a n c e o f t h e t e s tA suggested experimental procedure which may be followed for both the test and reference substance for the 3-hour contact period is given below :– Several vessels (e.g. 1-litre beakers) are used.209page 7 "Activated Sludge, Respiration Inhibition Test"–At time "0", 16 ml of the synthetic sewage feed are made up to 300 ml with water. 200 ml of microbial innoculum are added and the total m ixture (500 ml) poured into a first vesel (first control C1). Aeration at 0.5 to 1 litre per minute is commenced using a Pasteur-pipette as aeration device.–At time "15 min" (15 minutes is an arbitrary, but convenient, interval) the above is repeated, except that 100 ml of the test substance stock solution are added to the 16 ml of synthetic sewage before adding water to 300 ml and m icrobial innoculum to make a volume of 500 ml. This mixture is then poured into a second vessel and aerated as abo ve. This process is repeated at 15-minute intervals with different volumes of the test substance stock solution to give a series of vessels containing different concentrations of the test substance. F inally,a second control (C2) is prepared.–After three hours the contents of the first vessel are poured into the measuring apparatus and the respiration rate is measured over a period of up to 10 minutes ; the measuring can also be carried out directly in the vessel.–This determination is repeated on the contents of each vessel at 15-minute intervals, in sucha way that the contact time in each vessel is three hours.The reference substance is tested on each batch of microbial innoculum in the same way.A different regime (e.g. more than one oxygen m eter) will be necessary when measurements are to be made after 30 minutes of contact.If measurement of the chemical oxygen consum ption is required, further vessels are prepared containing test substance, synthetic sewage feed and w ater, but no activated sludge.ObservationsOxygen consumption is measured and recorded after an aeration time of 30 minutes and/or 3 hours (contact time).209page 8"Activated Sludge, Respiration Inhibition Test"3. D A T A A N D R E P O R T I N G•T r e a t m e n t o f r e s u l t sThe respiration rate is calculated from the recorder trace as mg O2/l.h between approximately 6.5 mg O2/l and 2.5 mg O2/l, or over a 10 minute period when the respiration rate is low. The portion of the respiration curve over which the respiration rate is measured should be linear.In order to calculate the inhibitory effect of a test substance at a particular concentration, the respiration rate is expressed as a percentage of the mean of the two control respiration rates :whereR s=oxygen-consumption rate at tested concentration of test substanceR c1=oxygen-consumption rate, Control 1R c2=oxygen-consumption rate, Control 2If the respiration rates of the two controls are not within 15 per cent of each other or the EC 50 (3 h) of the reference substance is not in the accepted range (5 to 30 m g/l for 3,5-dichlorophenol), the test is invalid and must be repeated.The per cent inhibition is calculated at each test concentration as above. The per cent inhibition is plotted against concentration on log-normal (or log-probability) p aper and an EC 50 value derived.95 per cent confidence limits for the EC 50 values can be determined using standard procedures. In view of the variability often observed in the results, it is recommended that the results be expressed in orders of magnitude, e.g. less than 1, 1 to 10, 10 to 100, etc. (in mg/l).209page 9 "Activated Sludge, Respiration Inhibition Test"•I n t e r p r e t a t i o n o f r e s u l t sThe EC 50 value should be regarded merely as a guide to the likely toxicity of the test substance either to activated sludge sewage treatment or to waste-water micro-organisms, since the complex interactions occuring in the environment cannot be accurately simulated in a laboratory test.•T e s t r e p o r tThe test report should include the following information :Test substance : chemical identification dataTest system : source, concentration and any pretreatment of the activated sludge Test conditions :– test temperature– test duration– reference substance and its measu red EC 50– abiotic oxygen uptake (if any)Results :–all measured data–inhibition curve and m ethod for calculation of EC 50–EC 50 and, if possible, 95 per cent confidence limits, EC 20 and EC 80–all observations and any deviations from this test guideline which could have influenced the result4.L I T E R A T U R E1.International Standard ISO/TC 147/SC 5/WC 1, N53 No. D (June 1981).209page 10"Activated Sludge, Respiration Inhibition Test"2. B. Broecker and R. Zahn, W ater Research 11, 165 (1977).3. D. Brown, H.R. Hitz and L. Schaefer, Chemosphere 10, 245 (1981).4.ETAD (Ecological and Toxicological Association of Dyestuffs Manufacturing Industries)Recommended Method No. 103, also described by :5. B. Robra, W asser/Abw asser 117, 80 (1976) and6.W. Schefer, Textilveredlung 6, 247 (1977).。