Commission Directive 92/69/EEC of 31 July 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances
Date: 1992-07-31
Institutions: European Commission
Subjects: approximation of laws, classification, dangerous substance, labelling, packaging













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31992L0069

Commission Directive 92/69/EEC of 31 July 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances  


Official Journal L 383 , 29/12/1992 P. 0113 - 0115 Finnish special edition: Chapter 6 Volume 6 P. 0003  Swedish special edition: Chapter 6 Volume 6 P. 0003  L 383A 29/12/1992 P. 0001 - 0235 





COMMISSION DIRECTIVE 92/69/EEC of 31 July 1992  adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substancesTHE COMMISSION OF THE EUROPEAN COMMUNITIES,Having regard to the Treaty establishing the European Economic Community,Having regard to Council Directive 67/548/EEC of 27 June 1967 on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (1), as last amended by Directive 92/32/EEC (2), and in particular Articles 28 and 29 thereof,(1) OJ No 196, 16. 8. 1967, p. 1.(2) OJ No L 154, 5. 6. 1992, p. 1.Whereas Article 3 (1) of Directive 67/548/EEC and Article 3 of Council Directive 88/379/EEC of 7 June 1988 on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous preparations (3), as last amended by Commission Directive 90/492/EEC (4), provide that the physico-chemical properties, toxicity and ecotoxicity of substances and preparations shall be determined according to the methods specified in Annex V of Directive 67/548/EEC;(1) OJ No 196, 16. 8. 1967, p. 1.(3) OJ No L 187, 16. 7. 1988, p. 14.(4) OJ No L 275, 5. 10. 1990, p. 35.Whereas the text of Annex V to Directive 67/548/EEC is currently published in two parts, these respectively being the Annex to Commission Directive 84/449/EEC (5), and the Annex to Commission Directive 88/302/EEC (6);(5) OJ No L 251, 19. 9. 1984, p. 1.(6) OJ No L 133, 30. 5. 1988, p. 1 and OJ No L 136, 2. 6. 1988, p. 20.Whereas, in order to take account of technical developments it is necessary to revise the test methods to be found in the Annex to Commission Directive 84/449/EEC;Whereas, in order to take account of technical developments it is also necessary to revise the test method for the algal inhibition test currently to be found in the Annex to Commission Directive 88/302/EEC and on this occasion to transfer this test method to the Annex to Directive 84/449/EEC;Whereas it is appropriate to reduce to a minimum the number of animals used for experimental purposes, in accordance with Council Directive 86/609/EEC on the approximation of the laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental purposes (7);(7) OJ No L 358, 18. 12. 1986, p. 1.Whereas the provisions of this Directive are in accordance with the opinion of the Committee for the Adaptation to Technical Progress of the Directives on the Removal of Technical Barriers to Trade in Dangerous Substances and Preparations,HAS ADOPTED THIS DIRECTIVE: Article 1The Annex to Directive 84/449/EEC is hereby replaced by the Annex to the present Directive. Article 2The algal inhibition test method described to the Annex to Directive 88/302/EEC is hereby deleted. Article 3The Member States shall bring into force the laws, regulations and administrative provisions necessary to comply with this Directive not later than 30 October 1993. Member States shall immediately inform the Commission thereof.When Member States adopt these provisions these shall contain a reference to this Directive or shall be accompanied by such reference at the time of their official publication.The procedure for such reference shall be adopted by the Member States.Article 4This Directive is addressed to the Member States.Done at Brussels, 31 July 1992. For the CommissionKarel VAN MIERTMember of the CommissionANNEXThis Annex will be published in Official Journal of the European Communities No L 383 A.(See the notice published on the inside back cover of this Official Journal.)Annex to Commission Directive 92/69/EEC of 31 July 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (1)CONTENTSINTRODUCTIONPART A: METHODS FOR THE DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES //5A.1. Melting / freezing temperature //5A.2. Boiling temperature //15A.3. Relative density //21A.4. Vapour pressure //26A.5. Surface tension //47A.6. Water solubility //54A.8. Partition coefficient //63A.9. Flash-point //74A.10. Flammability (solids) //76A.11. Flammability (gases) //79A.12. Flammability (contact with water) //81A.13. Pyrophoric properties of solids and liquids //85A.14. Explosive properties //87A.15. Auto-ignition temperature (liquids and gases)//98A.16. Relative self-ignition temperature for solids//99A.17. Oxidizing properties (solids) //102PART B: METHODS FOR THE DETERMINATION OF TOXICITY //107General introduction//107B.1. Acute toxicity (oral) //110B.1 bis Acute toxicity (oral) Fixed Dose Method //113B.2. Acute toxicity (inhalation) //117B.3. Acute toxicity (dermal) //121B.4. Acute toxicity (skin irritation) //124B.5. Acute toxicity (eye irritation) //127B.6. Skin sensitization //131B.7. Repeated dose (28 days) toxicity (oral) //136B.8. Repeated dose (28 days) toxicity (inhalation) //140B.9. Repeated dose (28 days) toxicity (dermal) //144B.10. Mutagenicity (in vitro mammalian cytogenetic test) //148B.11. Mutagenicity (in vivo mammalian bone-marrow cytogenetic test, chromosomal analysis) //151B.12. Mutagenicity (micronucleus test) //154B.13. Mutagenicity (Escherichia coli - reverse mutation assay) //157B.14. Mutagenicity (Salmonella typhimurium - reverse mutation assay) //160PART C: METHOD FOR THE DETERMINATION OF ECOTOXICITY //163C.1. Acute toxicity for fish //163C.2. Acute toxicity for Daphnia //172C.3. Algal inhibition test //179C.4. Biodegradation: determination of the ready biodegradability //187C.4-A: Dissolved organic carbon (DOC) die-away //194C.4-B: Modified OECD screening test //197C.4-C: Carbon dioxide (CO2) evolution //202C.4-D: Manometric respirometry //207C.4-E: Closed bottle //211C.4-F: MITI (Ministry of International Trade and Industry - Japan) //216Annexes //221C.5. Degradation: biochemical oxygen demand //226C.6. Degradation: chemical oxygen demand //227C.7. Degradation: abiotic degradation: hydrolysis as a function of pH //229INTRODUCTION The Annex sets out test methods for the determination of physicochemical, toxicological and ecotoxicological properties listed in Annexes VII and VIII to Directive 79/831/EEC. The methods are based on those recognized and recommended by competent international bodies (in particular OECD).When such methods were not available, national standards or scientific consensus methods have been adopted. Generally, tests should be performed with the substance as defined by the Directive. Attention should be given to the possible influence of impurities on the test results.When the methods of this Annex are inappropriate for the investigation of a certain property, the notifier must justify the alternate method used.Animal tests and studies shall be conducted in accordance with national regulations and shall take into account humane principles and international developments in the field of animal welfare.Among equivalent testing methods, the method using the minimum number of animals is chosen.PART A: METHODS FOR THE DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES A.1. MELTING/FREEZING TEMPERATURE1. METHODThe majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).1.1. INTRODUCTIONThe methods and devices described are to be applied for the determination of the melting temperature of substances, without any restriction in respect to their degree of purity.The selection of the method is dependent on the nature of the substance to be tested. In consequence the limiting factor will be according to whether or not the substance can be pulverized easily, with difficulty, or not at all.For some substances, the determination of the freezing or solidification temperature is more appropriate and the standards for these determinations have also been included in this method.Where, due to the particular properties of the substance, none of the above parameters can be conveniently measured, a pour point may be appropriate.1.2. DEFINITIONS AND UNITSThe melting temperature is defined as the temperature at which the phase transition from solid to liquid state occurs at atmospheric pressure and this temperature ideally corresponds to the freezing temperature.As the phase transition of many substances takes place over a temperature range, it is often described as the melting range.Conversion of units (K to  C)t = T   273,15t: Celsius temperature, degree Celsius ( C)T: thermodynamic temperature, kelvin (K)1.3. REFERENCE SUBSTANCESReference substances do not need to be employed in all cases when investigating a new substance.They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.Some calibration substances are listed in the references (4).1.4. PRINCIPLE OF THE TEST METHODThe temperature (temperature range) of the phase transition from the solid to the liquid state or from the liquid to the solid state is determined. In practice while heating/cooling a sample of the test substance at atmospheric pressure the temperatures of the initial melting/freezing and the final stage of melting/freezing are determined. Five types of methods are described, namely capillary method, hot stage methods, freezing temperature determinations, methods of thermal analysis, and determination of the pour point (as developed for petroleum oils). In certain cases, it may be convenient to measure the freezing temperature in place of the melting temperature.1.4.1. Capillary method1.4.1.1. Melting temperature devices with liquid bathA small amount of the finely ground substance is placed in a capillary tube and packed tightly. The tube is heated, together with a thermometer, and the temperature rise is adjusted to less than about 1 K/min during the actual melting. The initial and final melting temperatures are determined.1.4.1.2. Melting temperature devices with metal blockAs described under 1.4.1.1., except that the capillary tube and the thermometer are situated in a heated metal block, and can be observed through holes in the block.1.4.1.3. Photocell detectionThe sample in the capillary tube is heated automatically in a metal cylinder. A beam of light is directed through the substance, by way of a hole in the cylinder, to a precisely calibrated photocell. The optical properties of most substances change from opaque to transparent when they are melting. The intensity of light reaching the photocell increases and sends a stop signal to the digital indicator reading out the temperature of a platinum resistance thermometer located in the heating chamber. This method is not suitable for some highly coloured substances.1.4.2. Hot Stages1.4.2.1. Kofler hot barThe Kofler hot bar uses two pieces of metal of different thermal conductivity, heated electrically, with the bar designed so that the temperature gradient is almost linear along its length. The temperature of the hot bar can range from 283 to 573 K with a special temperature-reading device including a runner with a pointer and tab designed for the specific bar. In order to determine a melting temperature, the substance is laid, in a thin layer, directly on the surface of the hot bar. In a few seconds a sharp dividing line between the fluid and solid phase develops. The temperature at the dividing line is read by adjusting the pointer to rest at the line.1.4.2.2. Melt microscopeSeveral microscope hot stages are in use for the determination of melting temperatures with very small quantities of material. In most of the hot stages the temperature is measured with a sensitive thermocouple but sometimes mercury thermometers are used. A typical microscope hot stage melting temperature apparatus has a heating chamber which contains a metal plate upon which the sample is placed on a slide. The centre of the metal plate contains a hole permitting the entrance of light from the illuminating mirror of the microscope. When in use, the chamber is closed by a glass plate to exclude air from the sample area.The heating of the sample is regulated by a rheostat. For very precise measurements on optically anisotropic substances, polarized light may be used.1.4.2.3. Meniscus methodThis method is specifically used for polyamides.The temperature at which the displacement of a meniscus of silicone oil, enclosed between a hot stage and a cover-glass supported by the polyamide test specimen, is determined visually.1.4.3. Method to determine the freezing temperatureThe sample is placed in a special test tube and placed in an apparatus for the determination of the freezing temperature. The sample is stirred gently and continuously during cooling and the temperature is measured at suitable intervals. As soon as the temperature remains constant for a few readings this temperature (corrected for thermometer error) is recorded as the freezing temperature.Supercooling must be avoided by maintaining equilibrium between the solid and the liquid phases.1.4.4. Thermal analysis1.4.4.1 Differential thermal analysis (DTA)This technique records the difference in temperatures between the substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic (melting) or exothermic (freezing) departure from the base line of the temperature record.1.4.4.2 Differential scanning calorimetry (DSC)This technique records the difference in energy inputs into a substance and a reference material, as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic (melting) or exothermic (freezing) departure from the base line of the heat flow record.1.4.5. Pour pointThis method was developed for use with petroleum oils and is suitable for use with oily substances with low melting temperatures. After preliminary heating, the sample is cooled at a specific rate and examined at intervals of 3 K for flow characteristics. The lowest temperature at which movement of the substance is observed is recorded as the pour point.1.5. QUALITY CRITERIAThe applicability and accuracy of the different methods used for the determination of the melting temperature/melting range are listed in the following table:TABLE: APPLICABILITY OF THE METHODS>TABLE>>TABLE>>TABLE>>TABLE>1.6. DESCRIPTION OF THE METHODSThe procedures of nearly all the test methods have been described in international and national standards (see Appendix 1).1.6.1. Methods with capillary tubeWhen subjected to a slow temperature rise, finely pulverised substances usually show the stages of melting shown in figure 1.Figure 1>START OF GRAPHIC>>END OF GRAPHIC>Stage A (Beginning of melting): fine droplets adhere uniformly to the inside wall of the capillary tube.Stage B a clearance appears between the sample and the inside wall due to shrinkage of the melt.Stage C the shrunken sample begins to collapse downwards and liquefies.Stage D a complete meniscus is formed at the surface but an appreciable amount of the sample remains solid.Stage E (Final stage of melting): there are no solid particles.During the determination of the melting temperature, the temperatures are recorded at the beginning of melting and at the final stage.1.6.1.1. Melting temperature devices with liquid bath apparatusFigure 2 shows a type of standardized melting-temperature apparatus made of glass (JIS K 0064); all specifications are in millimetres.Figure 2>START OF GRAPHIC>>END OF GRAPHIC>A: Measurement vessel B: Stopper C: Vent D: Thermometer E: Auxiliary thermometer F: Bath liquid G: Capillary tube made of glass, 80 to 100 mm in length, 1,0 ± 0,2 mm inner diameter, 0,2 to 0,3 mm wall thickness H: Side tubeBath liquid: A suitable liquid should be chosen. The choice of the liquid depends upon the melting temperature to be determined, e.g. liquid paraffin for melting temperatures no higher than 473 K, silicone oil for melting temperatures no higher than 573 K.For melting temperatures above 523 K, a mixture consisting of three parts sulphuric acid and two parts potassium sulphate (in mass ratio) can be used. Suitable precautions should be taken if a mixture such as this is used.Thermometer: Only those thermometers should be used which fulfill the requirements of the following or equivalent standards:ASTM E 1-71, DIN 12770, JIS K 8001.Procedure: The dry substance is finely pulverized in a mortar and is put into the capillary tube, fused at one end, so that the filling level is approximately 3 mm after being tightly packed. To obtain a uniform packed sample, the capillary tube should be dropped from a height of approximately 700 mm through a glass tube vertically onto a watch glass.The filled capillary tube is placed in the bath so that the middle part of the mercury bulb of the thermometer touches the capillary tube at the part where the sample is located. Usually the capillary tube is introduced into the apparatus about 10 K below the melting temperature.The bath liquid is heated so that the temperature rise is approximately 3 K/min. The liquid should be stirred. At about 10 K below the expected melting temperature the rate of temperature rise is adjusted to a maximum of 1 K/min .Calculation: The calculatin of the melting temperature is as follows:T =  TD + 0,00016 (TD   TE)nwhere:T = corrected melting temperature in KTD = temperature reading of thermometer D in KTE = temperature reading of thermometer E in Kn = number of graduations of mercury thread on thermometer D at emergent stem.1.6.1.2. Melting temperature devices with metal blockApparatus:This consists of:-  a cylindrical metal block, the upper part of which is hollow and forms a chamber (see figure 3),-  a metal plug, with two or more holes, allowing tubes to be mounted into the metal block,-  a heating system, for the metal block, provided for example by an electrical resistance enclosed in the block,-  a rheostat for regulation of power input, if electric heating is used,-  four windows of heat-resistant glass on the lateral walls of the chamber, diametrically disposed at right-angles to each other. In front of one of these windows is mounted an eye-piece for observing the capillary tube. The other three windows are used for illuminating the inside of the enclosure by means of lamps,-  a capillary tube of heat-resistant glass closed at one end (see 1.6.1.1).Thermometer:See standards mentioned in 1.6.1.1. Thermoelectrical measuring devices with comparable accuracy are also applicable.Figure 3>START OF GRAPHIC>>END OF GRAPHIC>1.6.1.3. Photocell detectionApparatus and procedure:The apparatus consists of a metal chamber with automated heating system. Three capillary tubes are filled according to 1.6.1.1 and placed in the oven.Several linear increases of temperature are available for calibrating the apparatus and the suitable temperature rise is electrically adjusted at a pre-selected constant and linear rate. Recorders show the actual oven temperature and the temperature of the substance in the capillary tubes.1.6.2. Hot stages1.6.2.1. Kofler hot bar See Appendix.1.6.2.2. Melt microscope See Appendix.1.6.2.3. Meniscus method (polyamides)See Appendix.The heating rate through the melting temperature should be less than 1 K/min.1.6.3. Methods for the determination of the freezing temperatureSee Appendix.1.6.4. Thermal analysis1.6.4.1. Differential thermal analysisSee Appendix.1.6.4.2. Differential scanning calorimetrySee Appendix.1.6.5. Determination of the pour pointSee Appendix.2. DATAA thermometer correction is necessary in some cases.3. REPORTINGThe test report shall, if possible, include the following information:- method used,- precise specification of the substance (identity and impurities) and preliminary purification step, if any,- an estimate of the accuracy.The mean of at least two measurements which are in the range of the estimated accuracy (see tables) is reported as the melting temperature.If the difference between the temperature at the beginning and at the final stage of melting is within the limits of the accuracy of the method, the temperature at the final stage of melting is taken as the melting temperature; otherwise the two temperatures are reported.If the substance decomposes or sublimes before the melting temperature is reached, the temperature at which the effect is observed shall be reported.All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.4. REFERENCES(1) OECD, Paris, 1981, Test Guideline 102, Decision of the Council C(81) 30 final.(2) IUPAC, B. Le Neindre, B. Vodar, eds. Experimental thermodynamics, Butterworths, London 1975, vol. II, 803-834.(3) R. Weissberger ed.: Technique of organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, vol. I, Part I, Chapter VII.(4) IUPAC, Physicochemical measurements: Catalogue of reference materials from national laboratories, Pure and applied chemistry, 1976, vol. 48, 505-515.AppendixFor additional technical details, the following standards may be consulted for example.1. Capillary methods1.1. Melting temperature devices with liquid bathASTM E 324-69 Standard test method for relative initial and final melting points and the melting range of organic chemicalsBS 4634 Method for the determination of melting point and/or melting rangeDIN 53181 Bestimmung des Schmelzintervalles von Harzen nach Kapillarverfahren.JIS K 00-64 Testing methods for melting point of chemical products.1.2. Melting temperature devices with metal blockDIN 53736 Visuelle Bestimmung der Schmelztemperatur von teilkristallinen KunststoffenISO 1218 (E) Plastics - polyamides - determination of 'melting point`2. Hot stages2.1. Kofler hot barANSI/ASTM D 3451-76 Standard recommended practices for testing polymeric powder coatings2.2. Melt microscopeDIN 53736 Visuelle Bestimmung der Schmelztemperatur von teilkristallinen Kunststoffen.2.3. Meniscus method (polyamides)ISO 1218 (E) Plastics - polyamides - determination of 'melting point`ANSI/ASTM D 2133-66 Standard specification for acetal resin injection moulding and extrusion materialsNF T 51-050 Résines de polyamides. Détermination du 'point de fusion` Méthode du ménisque3. Methods for the determination of the freezing temperatureBS 4633 Method for the determination of crystallizing pointBS 4695 Method for Determination of Melting Point of petroleum wax (Cooling Curve)DIN 51421 Bestimmung des Gefrierpunktes von Flugkraftstoffen, Ottokraftstoffen und MotorenbenzolenISO 2207 Cires de pétrole: détermination de la température de figeageDIN 53175 Bestimmung des Erstarrungspunktes von FettsäurenNF T 60-114 Point de fusion des paraffinesNF T 20-051 Méthode de détermination du point de cristallisation (point de congélation)ISO 1392 Method for the determination of the freezing point4. Thermal analysis4.1. Differential thermal analysisASTM E 537-76 Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysisASTM E 473-85 Standard definitions of terms relating to thermal analysisASTM E 472-86 Standard practice for reporting thermoanalytical dataDIN 51005 Thermische Analyse, Begriffe4.2. Differential scanning calorimetryASTM E 537-76 Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysisASTM E 473-85 Standard definitions of terms relating to thermal analysisASTM E 472-86 Standard practice for reporting thermoanalytical dataDIN 51005 Thermische Analyse, Begriffe5. Determination of the pour pointNBN 52014 Echantillonnage et analyse des produits du pétrole: Point de trouble et point d'écoulement limite - Monsterneming en ontleding van aardolieproducten: Troebelingspunt en vloeipuntASTM D 97-66 Standard test method for pour point of petroleum oilsISO 3016 Petroleum oils - Determination of pour point.A.2. BOILING TEMPERATURE1. METHODThe majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).1.1. INTRODUCTIONThe methods and devices described here can be applied to liquid and low melting substances, provided that these do not undergo chemical reaction below the boiling temperature (for example: auto-oxidation, rearrangement, degradation, etc.). The methods can be applied to pure and to impure liquid substances.Emphasis is put on the methods using photocell detection and thermal analysis, because these methods allow the determination of melting as well as boiling temperatures. Moreover, measurements can be performed automatically.The 'dynamic method` has the advantage that it can also be applied to the determination of the vapour pressure and it is not necessary to correct the boiling temperature to the normal pressure (101,325 kPa) because the normal pressure can be adjusted during the measurement by a manostat.Remarks:The influence of impurities on the determination of the boiling temperature depends greatly upon the nature of the impurity. When there are volatile impurities in the sample, which could affect the results, the substance may be purified.1.2 DEFINITIONS AND UNITSThe normal boiling temperature is defined as the temperature at which the vapour pressure of a liquid is 101,325 kPa.If the boiling temperature is not measured at normal atmospheric pressure, the temperature dependence of the vapour pressure can be described by the Clausius-Clapeyron equation:log p = 2,3 RTD Hv + const.where:p = the vapour pressure of the substance in pascalsÄ Hv = its heat of vaporization in J mol 1R = the universal molar gas constant = 8,314 J mol 1 K 1T = thermodynamic temperature in KThe boiling temperature is stated with regard to the ambient pressure during the measurement.ConversionsPressure (units: kPa)100 kPa = 1 bar = 0,1 MPa('bar` is still permissible but not recommended)133 Pa = 1 mm Hg = 1 Torr(the units 'mm Hg` and 'Torr` are not permitted).1 atm = standard atmosphere = 101 325 Pa(the unit 'atm` is not permitted).Temperature (units: K)t = T   273,15t: Celsius temperature, degree Celsius ( C)T: thermodynamic temperature, kelvin (K)1.3. REFERENCE SUBSTANCESReference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.Some calibration substances can be found in the methods listed in the Appendix.1.4. PRINCIPLE OF THE TEST METHODFive methods for the determination of the boiling temperature (boiling range) are based on the measurement of the boiling temperature, two others are based on thermal analysis.1.4.1. Determination by use of the ebulliometerEbulliometers were originally developed for the determination of the molecular weight by boiling temperature elevation, but they are also suited for exact boiling temperature measurements. A very simple apparatus is described in ASTM D 1120-72 (see Appendix). The liquid is heated in this apparatus under equilibrium conditions at atmospheric pressure until it is boiling.1.4.2. Dynamic methodThis method involves the measurement of the vapour recondensation temperature by means of an appropriate thermometer in the reflux while boiling. The pressure can be varied in this method.1.4.3. Distillation method for boiling temperatureThis method involves distillation of the liquid and measurement of the vapour recondensation temperature and determination of the amount of distillate.1.4.4. Method according to SiwoloboffA sample is heated in a sample tube, which is immersed in a liquid in a heat-bath. A fused capillary, containing an air bubble in the lower part, is dipped in the sample tube.1.4.5. Photocell detectionFollowing the principle according to Siwoloboff, automatic photo-electrical measurement is made using rising bubbles.1.4.6. Differential thermal analysisThis technique records the difference in temperatures between the substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the temperature record.1.4.7. Differential scanning calorimetryThis technique records the difference in energy inputs into a substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the heat flow record.1.5. QUALITY CRITERIAThe applicability and accuracy of the different methods used for the determination of the boiling temperature/boiling range are listed in table 1.>TABLE>1.6. DESCRIPTION OF THE METHODSThe procedures of some test methods have been described in international and national standards (see Appendix).1.6.1. EbulliometerSee Appendix.1.6.2. Dynamic methodSee test method A.4. for the determination of the vapour pressure.The boiling temperature observed with an applied pressure of 101,325 kPa is recorded.1.6.3. Distillation process (boiling range)See Appendix.1.6.4. Method according to SiwoloboffThe sample is heated in a melting temperature apparatus in a sample tube, with a diameter of approximately 5 mm (figure 1).Figure 1 shows a type of standardized melting and boiling temperature apparatus (JIS K 0064) (made of glass, all specifications in millimetres).Figure 1>START OF GRAPHIC>>END OF GRAPHIC>A: Measuring vesselB: StopperC: VentD: ThermometerE: Auxiliary thermometerF: Bath liquidG: Sample tube, maximum 5 mm outer diameter; containing a capillary tube, approximately 100 mm long, approximately 1 mm inner diameter and approximately 0,2 to 0,3 mm wall-thicknessH: Side tubeA capillary tube (boiling capillary) which is fused about 1 cm above the lower end is placed in the sample tube. The level to which the test substance is added is such that the fused section of the capillary is below the surface of the liquid. The sample tube containing the boiling capillary is fastened either to the thermometer with a rubber band or is fixed with a support from the side (see figure 2).Figure 2>START OF GRAPHIC>>END OF GRAPHIC>Principle according to SiwoloboffFigure 3>START OF GRAPHIC>>END OF GRAPHIC>Modified principleThe bath liquid is chosen according to boiling temperature. At temperatures up to 573 K, silicone oil can be used. Liquid paraffin may only be used up to 473 K. The heating of the bath liquid should be adjusted to a temperature rise of 3 K/min at first. The bath liquid must be stirred. At about 10 K below the expected boiling temperature, the heating is reduced so that the rate of temperature rise is less than 1 K/min. Upon approach of the boiling temperature, bubbles begin to emerge rapidly from the boiling capillary.The boiling temperature is that temperature when, on momentary cooling, the string of bubbles stops and fluid suddenly starts rising in the capillary. The corresponding thermometer reading is the boiling temperature of the substance.In the modified principle (figure 3) the boiling temperature is determined in a melting temperature capillary. It is stretched to a fine point about 2 cm in length (a) and a small amount of the sample is sucked up. The open end of the fine capillary is closed by melting, so that a small air bubble is located at the end. While heating in the melting temperature apparatus (b), the air bubble expands. The boiling temperature corresponds to the temperature at which the substance plug reaches the level of the surface of the bath liquid (c).1.6.5. Photocell detectionThe sample is heated in a capillary tube inside a heated metal block.A light beam is directed, via suitable holes in the block, through the substance onto a precisely calibrated photocell.During the increase of the sample temperature, single air bubbles emerge from the boiling capillary. When the boiling temperature is reached the number of bubbles increases greatly. This causes a change in the intensity of light, recorded by a photocell, and gives a stop signal to the indicator reading out the temperature of a platinum resistance thermometer located in the block.This method is especially useful because it allows determinations below room temperature down to 253,15 K ( 20  C) without any changes in the apparatus. The instrument merely has to be placed in a cooling bath.1.6.6. Thermal analysis1.6.6.1. Differential thermal analysisSee Appendix.1.6.6.2. Differential scanning calorimetrySee Appendix.2. DATAAt small deviations from the normal pressure (max. ± 5 kPa) the boiling temperatures are normalized to Tn by means of the following number-value equation by Sidney Young:Tn = T + (fT × D p)where:Ä p = (101,325   p) [note sign]p = pressure measurement in kPafT = rate of change of boiling temperature with pressure in K/kPaT = measured boiling temperature in KTn = boiling temperature corrected to normal pressure in KThe temperature-correction factors, fT, and equations for their approximation are included in the international and national standards mentioned above for many substances.For example, the DIN 53171 method mentions the following rough corrections for solvents included in paints:>TABLE>3. REPORTINGThe test report shall, if possible, include the following information:- method used,- precise specification of the substance (identity and impurities) and preliminary purification step, if any,- an estimate of the accuracy.The mean of at least two measurements which are in the range of the estimated accuracy (see table 1) is reported as the boiling temperature.The measured boiling temperatures and their mean shall be stated and the pressure(s) at which the measurements were made shall be reported in kPa. The pressure should preferably be close to normal atmospheric pressure.All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.4. REFERENCES(1) OECD, Paris, 1981, Test Guideline 103, Decision of the Council C (81) 30 final.(2) IUPAC, B. Le Neindre, B. Vodar, editions. Experimental thermodynamics, Butterworths, London 1975, volume II.(3) R. Weissberger edition: Technique of organic chemistry, Physical methods of organic chemistry, Third Edition, Interscience Publications, New York, 1959, volume I, Part I, Chapter VIII.AppendixFor additional technical details, the following standards may be consulted for example:1. EbulliometerASTM D 1120-72 Standard test method for boiling point of engine anti-freezes2. Distillation process (boiling range)ISO/R 918 Test Method for Distillation (Distillation Yield and Distillation Range)BS 4349/68 Method for determination of distillation of petroleum productsBS 4591/71 Method for the determination of distillation characteristicsDIN 53171 Lösungsmittel für Anstrichstoffe, Bestimmung des SiedeverlaufesNF T 20-608 Distillation:détermination du rendement et de l'intervalle de distillation3. Differential thermal analysis and differential scanning calorimetryASTM E 537-76 Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysisASTM E 473-85 Standard definitions of terms relating to thermal analysisASTM E 472-86 Standard practice for reporting thermoanalytical dataDIN 51005 Thermische Analyse: BegriffeA.3 RELATIVE DENSITY1. METHODThe methods described are based on the OECD Test Guideline (1). The fundamental principles are given in reference (2).1.1. INTRODUCTIONThe methods for determining relative density described are applicable to solid and to liquid substances, without any restriction in respect to their degree of purity. The various methods to be used are listed in table 1.1.2. DEFINITIONS AND UNITSThe relative density, D420, of solids or liquids is the ratio between the mass of a volume of substance to be examined, determined at 20  C, and the mass of the same volume of water, determined at 4  C. The relative density has no dimension.The density, ñ, of a substance is the quotient of the mass, m, and its volume, v.The density, ñ, is given, in SI units, in kg/m3.1.3. REFERENCE SUBSTANCES Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.1.4. PRINCIPLE OF THE METHODSFour classes of methods are used.1.4.1. Buoyancy methods1.4.1.1. Hydrometer (for liquid substances)Sufficiently accurate and quick determinations of density may be obtained by the floating hydrometers, which allow the density of a liquid to be deduced from the depth of immersion by reading a graduated scale.1.4.1.2. Hydrostatic balance (for liquid and solid substances)The difference between the weight of a test sample measured in air and in a suitable liquid (e.g. water) can be employed to determine its density.For solids, the measured density is only representative of the particular sample employed. For the determination of density of liquids, a body of known volume, v, is weighed first in air and then in the liquid.1.4.1.3. Immersed body method (for liquid substances) In this method, the density of a liquid is determined from the difference between the results of weighing the liquid before and after immersing a body of known volume in the test liquid.1.4.2. Pycnometer methodsFor solids or liquids, pycnometers of various shapes and with known volumes may be employed. The density is calculated from the difference in weight between the full and empty pycnometer and its known volume.1.4.3. Air comparison pycnometer (for solids)The density of a solid in any form can be measured at room temperature with the gas comparison pycnometer. The volume of a substance is measured in air or in an inert gas in a cylinder of variable calibrated volume. For the calculation of density one mass measurement is taken after concluding the volume measurement.1.4.4. Oscillating densitimeter (5) (6) (7)The density of a liquid can be measured by an oscillating densitimeter. A mechanical oscillator constructed in the form of a U-tube is vibrated at the resonance frequency of the oscillator which depends on its mass. Introducing a sample changes the resonance frequency of the oscillator. The apparatus has to be calibrated by two liquid substances of known densities. These substances should preferably be chosen such that their densities span the range to be measured.1.5. Applicability of the different methods used for the determination of the relative density is listed in the table.1.6. DESCRIPTION OF THE METHODSThe standards given as examples, which are to be consulted for additional technical details, are attached in the Appendix.The tests have to be run at 20  C, and at least two measurements performed.2. DATASee standards.3. REPORTINGThe test report shall, if possible, include the following information:-  method used,-  precise specification of the substance (identity and impurities) and preliminary purification step, if any.The relative density, D420, shall be reported as defined in 1.2, along with the physical state of the measured substance.All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.>TABLE>4. REFERENCES(1) OECD, Paris, 1981, Test Guideline 109, Decision of the Council C(81) 30 final.(2) R. Weissberger ed., Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Chapter IV, Interscience Publ., New York, 1959, vol. I, Part 1.(3) IUPAC, Recommended reference materials for realization of physico-chemical properties, Pure and applied chemistry, 1976, vol. 48, 508.(4) Wagenbreth, H., Die Tauchkugel zur Bestimmung der Dichte von Flüssigkeiten, Technisches Messen tm, 1979, vol.11, 427-430.(5) Leopold, H., Die digitale Messung von Flüssigkeiten, Elektronik, 1970, vol. 19, 297-302.(6) Baumgarten, D., Füllmengenkontrolle bei vorgepackten Erzeugnissen - Verfahren zur Dichtebestimmung bei flüssigen Produkten und ihre praktische Anwendung, Die Pharmazeutische Industrie, 1975, vol. 37, 717 - 726.(7) Riemann, J., Der Einsatz der digitalen Dichtemessung im Brauereilaboratorium, Brauwissenschaft, 1976, vol. 9, 253-255.AppendixFor additional technical details, the following standards may be consulted for example:1. BUOYANCY METHODS1.1. HydrometerDIN 12790, ISO 387 Hydrometer; general instructionsDIN 12791 Part I: Density hydrometers; construction, adjustment and usePart II: Density hydrometers; standardized sizes, designationPart III: Use and testISO 649-2 Laboratory glassware: Density hydrometers for general purposeNF T 20-050 Chemical products for industrial use - Determination of density of liquids - Areometric methodDIN 12793 Laboratory glassware: range find hydrometers1.2. Hydrostatic balanceFor solid substancesISO 1183 Method A: Methods for determining the density and relative density of plastics excluding cellular plasticsNF T 20-049 Chemical products for industrial use - Determination of the density of solids other than powders and cellular products - Hydrostatic balance methodASTM-D-792 Specific gravity and density of plastics by displacementDIN 53479 Testing of plastics and elastomers; determination of densityFor liquid substancesISO 901 ISO 758DIN 51757 Testing of mineral oils and related materials; determination of densityASTM D 941-55, ASTM D 1296-67 and ASTM D 1481-62ASTM D 1298 Density, specific gravity or API gravity of crude petroleum and liquid petroleum products by hydrometer methodBS 4714 Density, specific gravity or API gravity of crude petroleum and liquid petroleum products by hydrometer method1.3. Immersed body methodDIN 53217 Testing of paints, varnishes and similar coating materials; determination of density; immersed body method2. PYCNOMETER METHODS2.1. For liquid substancesISO 3507 PycnometersISO 758 Liquid chemical products; determination of density at 20  CDIN 12797 Gay-Lussac pycnometer (for non-volatile liquids which are not too viscous)DIN 12798 Lipkin pycnometer (for liquids with a kinematic viscosity of less than 100  7 10 6 m2 s 1 at 15  C)DIN 12800 Sprengel pycnometer (for liquids as DIN 12798)DIN 12801 Reischauer pycnometer (for liquids with a kinematic viscosity of less than 100  7 10 6 m2 s 1 at 20  C, applicable in particular also to hydrocarbons and aqueous solutions as well as to liquids with higher vapour pressure, approximately 1 bar at 90  C)DIN 12806 Hubbard pycnometer (for viscous liquids of all types which do not have too high a vapour pressure, in particular also for paints, varnishes and bitumen)DIN 12807 Bingham pycnometer (for liquids, as in DIN 12801)DIN 12808 Jaulmes pycnometer (in particular for ethanol- water mixture)DIN 12809 Pycnometer with ground-in thermometer and capillary side tube (for liquids which are not too viscous)DIN 53217 Testing of paints, varnishes and similar products; determination of density by pycnometerDIN 51757 Point 7: Testing of mineral oils and related materials; determination of densityASTM D 297 Section 15: Rubber products - chemical analysisASTM D 2111 Method C: Halogenated organic compoundsBS 4699 Method for determination of specific gravity and density of petroleum products (graduated bicapillary pycnometer method)BS 5903 Method for determination of relative density and density of petroleum products by the capillary- stoppered pycnometer methodNF T 20-053 Chemical products for industrial use - Determination of density of solids in powder and liquids - Pyknometric method2.2. For solid substancesISO 1183 Method B: Methods for determining the density and relative density of plastics excluding cellular plastics.NF T 20-053 Chemical products for industrial use - Determination of density of solids in powder and liquids - Pyknometric methodDIN 19683 Determination of the density of soils3. AIR COMPARISON PYCNOMETERDIN 55990 Part 3: Prüfung von Anstrichstoffen und ähnlichen Beschichtungsstoffen; Pulverlack; Bestimmung der DichteDIN 53243 Anstrichstoffe; Chlorhaltige Polymere; PrüfungA.4. VAPOUR PRESSURE1. METHODThe majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).1.1. INTRODUCTIONIt is useful to have preliminary information on the structure, the melting temperature and the boiling temperature of the substance to perform this test.There is no single measurement procedure applicable to the entire range of vapour pressures. Therefore, several methods are recommended to be used for the measurement of vapour pressure from TABLE POSITION>1.6. DESCRIPTION OF THE METHODS1.6.1. Dynamic measurement1.6.1.1. ApparatusThe measuring apparatus typically consists of a boiling vessel with attached cooler made of glass or metal (figure 1), equipment for measuring the temperature, and equipment for regulating and measuring the pressure. A typical measuring apparatus shown in the drawing is made from heat-resistant glass and is composed of five parts:The large, partially double-walled tube consists of a ground jacket joint, a cooler, a cooling vessel and an inlet.The glass cylinder, with a Cottrell 'pump`, is mounted in the boiling section of the tube and has a rough surface of crushed glass to avoid 'bumping' in the boiling process.The temperature is measured with a suitable temperature sensor (e.g. resistance thermometer, jacket thermocouple) immersed in the apparatus to the point of measurement (No. 5, figure 1) through a suitable inlet (e.g. male ground joint).The necessary connections are made to the pressure regulation and measuring equipment.The bulb, which acts as a buffer volume, is connected with the measuring apparatus by means of a capillary tube.The boiling vessel is heated by a heating element (e.g. cartridge heater) inserted into the glass apparatus from below. The heating current required is set and regulated via a thermocouple.The necessary vacuum of between 102 Pa and approximately 105 Pa is produced with a vacuum pump.A suitable valve is used to meter air or nitrogen for pressure regulation (measuring range approximately 102 to 105 Pa) and ventilation.Pressure is measured with a manometer.1.6.1.2. Measurement procedureThe vapour pressure is measured by determining the boiling temperature of the sample at various specified pressures between roughly 103 and 105 Pa. A steady temperature under constant pressure indicates that the boiling temperature has been reached. Frothing substances cannot be measured using this method.The substance is placed in the clean, dry sample vessel. Problems may be encountered with non-powder solids but these can sometimes be solved by heating the cooling jacket. Once the vessel has been filled the apparatus is sealed at the flange and the substance degassed. The lowest desired pressure is then set and the heating is switched on. At the same time, the temperature sensor is connected to a recorder.Equilibrium is reached when a constant boiling temperature is recorded at constant pressure. Particular care must be taken to avoid bumping during boiling. In addition, complete condensation must occur on the cooler. When determining the vapour pressure of low melting solids, care should be taken to avoid the condenser blocking.After recording this equilibrium point, a higher pressure is set. The process is continued in this manner until 105 Pa has been reached (approximately 5 to 10 measuring points in all). As a check, equilibrium points must be repeated at decreasing pressures.1.6.2. Static measurement1.6.2.1. ApparatusThe apparatus comprises a container for the sample, a heating and cooling system to regulate the temperature of the sample and measure the temperature. The apparatus also includes instruments to set and measure the pressure. Figures 2a and 2b illustrate the basic principles involved.The sample chamber (figure 2a) is bounded on one side by a suitable high-vacuum valve. A U-tube containing a suitable manometer fluid is attached to the other side. One end of the U-tube branches off to the vacuum pump, the nitrogen cylinder or ventilation valve, and a manometer.A pressure gauge with a pressure indicator can be used instead of a U-tube (figure 2b).In order to regulate the temperature of the sample, the sample vessel together with valve and U-tube or pressure gauge is placed in a bath which is kept at a constant temperature of ± 0,2 K. The temperature measurements are taken on the outside wall of the vessel containing the sample or in the vessel itself.A vacuum pump with an upstream cooling trap is used to evacuate the apparatus.In method 2a the vapour pressure of the substance is measured indirectly using a zero indicator. This takes into account the fact that the density of the fluid in the U-tube alters if the temperature changes greatly.The following fluids are suitable for use as zero indicators for the U-tube, depending on the pressure range and the chemical behaviour of the test substance: silicone fluids, phthalates. The test substance must not dissolve noticeably in or react with the U-tube fluid.For the manometer, mercury can be used in the range of normal air pressure to 102 Pa, while silicone fluids and phthalates are suitable for use below 102 Pa down to 10 Pa. Heatable membrane capacity manometers can even be used at below 10 1 Pa. There are also other pressure gauges which can be used below 102 Pa.1.6.2.2. Measurement procedureBefore measuring, all components of the apparatus shown in figure 2 must be cleaned and dried thoroughly.For method 2a, fill the U-tube with the chosen liquid, which must be degassed at an elevated temperature before readings are taken.The test substance is placed in the apparatus, which is then closed and the temperature is reduced sufficiently for degassing. The temperature must be low enough to ensure that the air is sucked out, but - in the case of multiple component system - it must not alter the composition of the material. If required, equilibrium can be established more quickly by stirring.The sample can be supercooled with e.g. liquid nitrogen (taking care to avoid condensation of air or pump fluid) or a mixture of ethanol and dry ice. For low-temperature measurements use a temperature-regulated bath connected to an ultra-cryomat.With the valve over the sample vessel open, suction is applied for several minutes to remove the air. The valve is then closed and the temperature of the sample reduced to the lowest level desired. If necessary, the degassing operation must be repeated several times.When the sample is heated the vapour pressure increases. This alters the equilibrium of the fluid in the U-tube. To compensate for this, nitrogen or air is admitted to the apparatus via a valve until the pressure indicator fluid is at zero again. The pressure required for this can be read off a precision manometer at room temperature. This pressure corresponds to the vapour pressure of the substance at that particular measuring temperature.Method 2b is similar but the vapour pressure is read off directly.The temperature-dependence of vapour pressure is determined at suitably small intervals (approximately 5 to 10 measuring points in all) up to the desired maximum. Low-temperature readings must be repeated as a check.If the values obtained from the repeated readings do not coincide with the curve obtained for increasing temperature, this may be due to one of the following :1. The sample still contains air (e.g. high-viscosity materials) or low-boiling substances, which is/are released during heating and can be removed by suction following further supercooling.2. The cooling temperature is not low enough. In this case liquid nitrogen is used as the cooling agent.If either 1 or 2 is the case, the measurements must be repeated.3. The substance undergoes a chemical reaction in the temperature range investigated (e.g. decomposition, polymerization).1.6.3. IsoteniscopeA complete description of this method can be found in reference 7. The principle of the measuring device is shown in figure 3. Similarly to the static method described in 1.6.2, the isoteniscope is appropriate for the investigation of solids or liquids.In the case of liquids, the substance itself serves as the fluid in the auxiliary manometer. A quantity of the liquid, sufficient to fill the bulb and the short leg of the manometer section, is put in the isoteniscope. The isoteniscope is attached to a vacuum system and evacuated, then filled by nitrogen. The evacuation and purge of the system is repeated twice to remove residual oxygen. The filled isoteniscope is placed in an horizontal position so that the sample spreads out into a thin layer in the sample bulb and manometer section (U-part). The pressure of the system is reduced to 133 Pa and the sample gently warmed until it just boils (removal of dissolved fixed gases). The isoteniscope is then placed so that the sample returns to the bulb and short leg of the manometer, so that both are entirely filled with liquid. The pressure is maintained as for degassing; the drawn-out tip of the sample bulb is heated with a small flame until sample vapour released expands sufficiently to displace part of the sample from the upper part of the bulb and manometer arm into the manometer section of the isoteniscope, creating a vapour-filled, nitrogen-free space.The isoteniscope is then placed in a constant temperature bath, and the pressure of nitrogen is adjusted until its pressure equals that of the sample. Pressure balance is indicated by the manometer section of the isoteniscope. At the equilibrium, the vapour pressure of nitrogen equals the vapour pressure of the substance.In the case of solids, depending on the pressure and temperature range, the manometer liquids listed in 1.6.2.1 are used. The degassed manometer liquid is filled into a bulge on the long arm of the isoteniscope. Then the solid to be investigated is placed in the bulb and is degassed at elevated temperature. After that the isoteniscope is inclined so that the manometer liquid can flow into the U-tube. The measurement of vapour pressure as a function of temperature is done according to 1.6.2.1.6.4. Effusion method: Vapour pressure balance1.6.4.1. ApparatusVarious versions of the apparatus are described in the literature (1). The apparatus described here illustrates the general principle involved (figure 4). Figure 4 shows the main components of the apparatus, comprising a high-vacuum stainless steel or glass container, equipment to produce and measure a vacuum and built-in components to measure the vapour pressure on a balance. The following built-in components are included in the apparatus :-  an evaporator furnace with flange and rotary inlet. The evaporator furnace is a cylindrical vessel, made of e.g. copper or a chemically resistant alloy with good thermal conductivity. A glass vessel with a copper wall can also be used. The furnace has a diameter of approximately 3 to 5 cm and is 2 to 5 cm high. There are between one and three openings of different sizes for the vapour stream. The furnace is heated either by a heating plate underneath or a heating spiral around the outside. To prevent heat being dissipated to the base plate, the heater is attached to the base plate by a metal with low thermal conductivity (nickel-silver or chromium-nickel steel), e.g. a nickel-silver pipe attached to a rotary inlet if using a furnace with several openings. This arrangement has the advantage of allowing the introduction of a copper bar. This allows cooling from the outside using a cooling bath,-if the copper furnace lid has three openings of different diameters at 90  to ea ch other, various vapour pressure ranges within the overall measuring range can be covered (openings between approximately 0,30 and 4,50 mm diameter). Large openings are used for low vapour pressure and vice versa. By rotating the furnace the desired opening or an intermediate position in the vapour stream (furnace opening - shield - balance pan) can be set and the stream of molecules is released or deflected through the furnace opening onto the scale pan. In order to measure the temperature of the substance, a thermocouple or resistance thermometer is placed at a suitable point,-  above the shield is a balance pan belonging to a highly sensitive microbalance (see below). The balance pan is approximately 30 mm in diameter. Gold-plated aluminium is a suitable material,-  the balance pan is surrounded by a cylindrical brass or copper refrigeration box. Depending on the type of balance, it has openings for the balance beam and a shield opening for the stream of molecules and should guarantee complete condensation of the vapour on the balance pan. Heat dissipation to the outside is ensured e.g. by a copper bar connected to the refrigeration box. The bar is routed through the base plate and thermally insulated from it, e.g. with a chromium-nickel steel tube. The bar is immersed in a Dewar flask containing liquid nitrogen under the base plate or liquid nitrogen is circulated through the bar. The refrigeration box is thus kept at approximately 120  C. The balance pan is cooled exclusively by radiation and is satisfactory for the pressure range under investigation (cooling approximately 1 hour before the start of measurement),- the balance is positioned above the refrigeration box. Suitable balances are e.g. a highly sensitive 2-arm electronic microbalance (8) or a highly sensitive moving coil instrument (see OECD Test Guideline 104, Edition 12.05.81),-  the base plate also incorporates electrical connections for thermocouples (or resistance thermometers) and heating coils,-  a vacuum is produced in the vessel using a partial vacuum pump or high-vacuum pump (required vacuum approximately 1 to 2  7 10 3 Pa, obtained after 2 h pumping). The pressure is regulated with a suitable ionisation manometer.1.6.4.2. Measurement procedureThe vessel is filled with the test substance and the lid is closed. The shield and refrigeration box are slid across the furnace. The apparatus is closed and the vacuum pumps are switched on. The final pressure before starting to take measurements should be approximately 10 4 Pa. Cooling of the refrigeration box starts at 10 2 Pa.Once the required vacuum has been obtained, start the calibration series at the lowest temperature required. The corresponding opening in the lid is set, the vapour stream passes through the shield directly above the opening and strikes the cooled balance pan. The balance pan must be big enough to ensure that the entire stream guided through the shield strikes it. The momentum of the vapour stream acts as a force against the balance pan and the molecules condense on its cool surface.The momentum and simultaneous condensation produce a signal on the recorder. Evaluation of the signals provides two pieces of information:1. In the apparatus described here the vapour pressure is determined directly from the momentum on the balance pan (it is not necessary to know the molecular weight for this (2)). Geometrical factors such as the furnace opening and the angle of the molecular stream must be taken into account when evaluating the readings.2. The mass of the condensate can be measured at the same time and the rate of evaporation can be calculated from this. The vapour pressure can also be calculated from the rate of evaporation and molecular weight using the Hertz equation (2).p = G2 p RT × 103MwhereG = evaporation rate (kg s 1 m 2)M = molar mass (g mol 1)T = temperature (K)R = universal molar gas constant (J mol 1 K 1)p = vapour pressure (Pa)After the necessary vacuum is reached, the series of measurements is commenced at the lowest desired measuring temperature. For further measurements, the temperature is increased by small intervals until the maximum desired temperature value is reached. The sample is then cooled again and a second curve of the vapour pressure may be recorded. If the second run fails to confirm the results of the first run, then it is possible that the substance may be decomposing in the temperature range being measured.1.6.5. Effusion method - by loss of weight1.6.5.1. ApparatusThe effusion apparatus consists of the following basic parts:- a tank that can be thermostated and evacuated and in which the effusion cells are located,- a high vacuum pump (e.g. diffusion pump or turbomolecular pump) with vacuum gauge,- a trap, using liquefied nitrogen or dry ice.An electrically heated, aluminium vacuum tank with 4 stainless steel effusion cells is shown in figure 5 for example. The stainless steel foil of about 0,3 mm thickness has an effusion orifice of 0,2 to 1,0 mm diameter and is attached to the effusion cell by a threaded lid.1.6.5.2. Measurement procedureThe reference and test substances are filled into each effusion cell, the metal diaphragm with the orifice is secured by the threaded lid, and each cell is weighed to within an accuracy of 0,1 mg. The cell is placed in the thermostated apparatus, which is then evacuated to below one tenth of the expected pressure. At defined intervals of time ranging from 5 to 30 hours, air is admitted into the apparatus, and the loss in mass of the effusion cell is determined by reweighing.In order to ensure that the results are not influenced by volatile impurities, the cell is reweighed at defined time intervals to check that the evaporation rate is constant over at least two such intervals of time.The vapour pressure p in the effusion cell is given by: p =mKAtE2pRTMwherep = vapour pressure (Pa)m = mass of the substance leaving the cell during time t (kg)t = time (s)A = area of the hole (m2)K = correction factorR = universal gas constant (J mol 1 K 1)T = temperature (K)M = molecular mass (kg mol 1)The correction factor K depends on the ratio of length to radius of the cylindrical orifice:ratio:0,10,20,61,02,0K:0,9520,9090,7710,6720,514The above equation may be written:p = EmtTMwhere = E1KA2pR and is the effusion cell constant.This effusion cell constant E may be determined with reference substances (2,9), using the following equation:E =p(r)tmM(r)Twherep(r) = vapour pressure of the reference substance (Pa)M(r) = molecular mass of the reference substance (kg.mol 1)1.6.6. Gas saturation method1.6.6.1. ApparatusA typical apparatus used to perform this test comprises a number of components given in figure 6a and described below (1).Inert gas:The carrier gas must not react chemically with the test substance. Nitrogen is usually sufficient for this purpose but occasionally other gases may be required (10). The gas employed must be dry (see figure 6a, key 4: relative humidity sensor)Flow control:A suitable gas-control system is required to ensure a constant and selected flow through the saturator column.Traps to collect vapour:These are dependent on the particular sample characteristics and the chosen method of analysis. The vapour should be trapped quantitatively and in a form which permits subsequent analysis. For some test substances, traps containing liquids such as hexane or ethylene glycol will be suitable. For others, solid absorbents may be applicable.As an alternative to vapour trapping and subsequent analysis, in-train analytical techniques, like chromatography, may be used to determine quantitatively the amount of material transported by a known amount of carrier gas. Furthermore, the loss of mass of the sample can be measured.Heat exchanger:For measurements at different temperatures it may be necessary to include a heat-exchanger in the assembly.Saturator column:The test substance is deposited from a solution onto a suitable inert support. The coated support is packed into the saturator column, the dimensions of which and the flow rate should be such that complete saturation of the carrier gas is ensured. The saturator column must be thermostated. For measurements above room temperature, the region between the saturator column and the traps should be heated to prevent condensation of the test substance.In order to lower the mass transport occurring by diffusion, a capillary may be placed after the saturator column (figure 6b).1.6.6.2. Measurement procedurePreparation of the saturator column:A solution of the test substance in a highly volatile solvent is added to a suitable amount of support. Sufficient test substance should be added to maintain saturation for the duration of the test. The solvent is totally evaporated in air or in a rotary evaporator, and the thoroughly mixed material is added to the saturator column. After thermostating the sample, dry nitrogen is passed through the apparatus.Measurement:The traps or in-train detector are connected to the column effluent line and the time recorded. The flow rate is checked at the beginning and at regular intervals during the experiment, using a bubble meter (or continuously with a mass flow-meter).The pressure at the outlet to the saturator must be measured. This may be done either:(a) by including a pressure gauge between the saturator and traps (this may not be satisfactory because this increases the dead space and the adsorptive surface); or(b) by determining the pressure drops across the particular trapping system used as a function of flow rate in a separate experiment (this may be not very satisfactory for liquid traps).The time required for collecting the quantity of test substance that is necessary for the different methods of analysis is determined in preliminary runs or by estimates. As an alternative to collecting the substance for further analysis, in-train quantitative analytical technique may be used (e.g. chromatography). Before calculating the vapour pressure at a given temperature, preliminary runs are to be carried out to determine the maximum flow rate that will completely saturate the carrier gas with substance vapour. This is guaranteed if the carrier gas is passed through the saturator sufficiently slowly so that a lower rate gives no greater calculated vapour pressure.The specific analytical method will be determined by the nature of the substance being tested (e.g. gas chromatography or gravimetry).The quantity of substance transported by a known volume of carrier gas is determined.1.6.6.3. Calculation of vapour pressureVapour pressure is calculated from the vapour density, W/V, through the equation:p =WV×RTMwhere:p = vapour pressure (Pa)W = mass of evaporated test substance (g)V = volume of saturated gas (m3)R = universal molar gas constant (J mol 1 K 1)T = temperature (K)M = molar mass of test substance (g mol 1)Measured volumes must be corrected for pressure and temperature differences between the flow meter and the thermostated saturator. If the flow meter is located downstream from the vapour trap, corrections may be necessary to account for any vaporized trap ingredients (1).1.6.7. Spinning rotor (8, 11, 13)1.6.7.1. ApparatusThe spinning rotor technique can be carried out using a spinning rotor viscosity gauge as shown in figure 8. A schematic drawing of the experimental set-up is shown in figure 7.The measuring apparatus typically consists of a spinning rotor measuring head, placed in a thermostated enclosure (regulated within 0,1  C). The sample container is placed in a thermostatted enclosure (regulated within 0,01  C), and all other parts of the set-up are kept at a higher temperature to prevent condensation. A high-vacuum pump device is connected to the system by means of high-vacuum valves.The spinning rotor measuring head consists of a steel ball (4 to 5 mm diameter) in a tube. The ball is suspended and stabilized in a magnetic field, generally using a combination of permanent magnets and control coils.The ball is made to spin by rotating fields produced by coils. Pick-up coils, measuring the always present low lateral magnetization of the ball, allow its spinning rate to be measured.1.6.7.2. Measurement procedureWhen the ball has reached a given rotational speed v(o) (usually about 400 revolutions per second), further energizing is stopped and deceleration takes place, due to gas friction.The drop of rotational speed is measured as a function of time. As the friction caused by the magnetic suspension is negligible as compared with the gas friction, the gas pressure p is given by :p =pcrrs10t×lnv(t)v(o)wherec =  average speed of the gas moleculesr =  radius of the ballñ =  mass density of the balló =  coefficient of tangential momentum transfer (å =1 for an ideal spherical surface of the ball)t =  timev(t) =  rotational speed after time tv(o) =  initial rotational speedThis equation may also be written:p =pcrr10s×tn tn-1 tn × tn-1where tn, tn 1 are the times required for a given number N of revolutions. These time intervals tn and tn 1 succeed one another, and tn > tn 1.The average speed of the gas molecule c is given by:c = (8 RTp M)12where:T = temperatureR = universal molar gas constantM = molar mass2. DATAThe vapour pressure from any of the preceding methods should be determined for at least two temperatures. Three or more are preferred in the range 0 to 50  C, in order to check the linearity of the vapour pressure curve.3. REPORTINGThe test report shall, if possible, include the following information :- method used,- precise specification of the substance (identity and impurities) and preliminary purification step, if any,- at least two vapour pressure and temperature values, preferably in the range 0 to 50  C,- all of the raw data,-  a log p versus 1/T curve,-  an estimate of the vapour pressure at 20 or 25  C.If a transition (change of state, decomposition) is observed, the following information should be noted:-  nature of the change,-  temperature at which the change occurs at atmospheric pressure,-  vapour pressure at 10 and 20  C below the transition temperature and 10 and 20  C above this temperature (unless the transition is from solid to gas).All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.4. REFERENCES(1)  OECD, Paris, 1981, Test Guideline 104, Decision of the Council C(81) 30 final.(2)  Ambrose, D. in B. Le Neindre, B. Vodar, (Eds.): Experimental Thermodynamics, Butterworths, London, 1975, Vol. II.(3)  R. Weissberger ed.: Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed. Chapter IX, Interscience Publ., New York, 1959, Vol. I, Part I.(4)  Knudsen, M. Ann. Phys. Lpz., 1909, vol. 29, 1979; 1911, vol. 34, 593.(5)  NF T 20-048 AFNOR (Sept. 85). Chemical products for industrial use - Determination of vapour pressure of solids and liquids within range from 10 1 to 105 Pa - Static method.(6)  NF T 20-047 AFNOR (Sept. 85). Chemical products for industrial use - Determination of vapour pressure of solids and liquids within range from 10 3 to 1 Pa - Vapour pressure balance method.(7) ASTM D 2879-86, Standard test method for vapour pressure- temperature relationship and initial decomposition temperature of liquids by isoteniscope.(8) G. Messer, P. Röhl, G. Grosse and W. Jitschin. J. Vac. Sci. Technol.(A), 1987, vol. 5 (4), 2440.(9) Ambrose, D.; Lawrenson, I.J.; Sprake, C.H.S. J. Chem. Thermodynamics 1975, vol. 7, 1173.(10) B.F. Rordorf. Thermochimica Acta, 1985, vol. 85, 435.(11) G. Comsa, J.K. Fremerey and B. Lindenau. J. Vac. Sci. Technol., 1980, vol. 17 (2), 642.(12) G. Reich. J. Vac. Sci. Technol., 1982, vol. 20 (4), 1148.(13) J.K. Fremerey. J. Vac. Sci. Technol.(A), 1985, vol. 3 (3), 1715.Appendix 1Estimation MethodINTRODUCTIONCalculated values of the vapour pressure can be used:- for deciding which of the experimental methods is appropriate,- for providing an estimate or limit value in cases where the experimental method cannot be applied due to technical reasons (including where the vapour pressure is very low),- to help identify those cases where omitting experimental measurement is justified because the vapour pressure is likely to be START OF GRAPHIC>>END OF GRAPHIC>Apparatus for determining the vapour pressure curve according to the dynamic method1 = Thermocouple2 = Vacuum buffer volume3 = Pressure gauge4 = Vacuum5 = Measuring point6 = Heating element circa 150 WFigure 2a>START OF GRAPHIC>>END OF GRAPHIC>Apparatus for determining the vapour pressure curve according to the static ethod (using a U-tube manometer)1. Test substance6. Temperature bath2. Vapour phase7. Temperature measuring device3. High vacuum valve8. To vacuum pump4. U-tube (auxiliary manometer)9. Ventilation5. ManometerFigure 2b>START OF GRAPHIC>>END OF GRAPHIC>Apparatus for determining the vapour pressure curve according to the static method (using a pressure indicator)1. Test substance5. Pressure indicator2. Vapour phase6. Temperature bath3. High vacuum valve7. Temperature measuring device4. Pressure gaugeFigure 3>END OF GRAPHIC>>START OF GRAPHIC>Isoteniscope (see reference 7)1. To pressure control and measurement system2. 8 mm OD tube3. Dry nitrogen in pressure system4. Sample vapour5. Small tip6. Liquid sampleFigure 4>END OF GRAPHIC>>START OF GRAPHIC>Apparatus for determining the vapour pressure curve according to the vapour pressure balance method1. Test substance7. Shield2. Vapour phase with vapour stream8. Refrigeration bar for3. Evaporation furnace with rotary inlet refrigeration box3a. Furnace lid with opening9. Balance pan4. Furnace heating (refrigeration)10. Microbalance5. Measurement of temperature of sample11. To recorder          6. Refrigeration box12. To high-vacuum pump  Figure 5>START OF GRAPHIC>>END OF GRAPHIC>Example of apparatus for evaporation at low pressure by effusion methode, with an effusion cell volume of 8 cm1 Connection to vacuum2 Wells for platinum resistance thermometer or temperature measurement and control (2)3 Lid for vacuum tank4 O-ring5 Aluminium vacuum tank6 Device for installing and removing the effusion cells7 Threaded lid8 Butterfly nuts (6)9 Bolts (6)10 Stainless steel effusion cells11 Heater cartridges (6)Figure 6a>END OF GRAPHIC>>START OF GRAPHIC>An example of a flow system for the determination of vapour pressure by the gas saturation method1 = Flow regulator2 = Heat exchanger3 = Needle valves4 = Relative humidity sensor5 = Saturation columns6 = PTFE joints7 = Flow meter8 = Trap (absorber)9 = Oil trap10 = Fritted bubblerFigure 6b>START OF GRAPHIC>>END OF GRAPHIC>An example of a system for the determination of vapour pressure by the gas saturation method, with a capillary placed after the saturation chamber1. Thermal mass flowmeter6. Gas saturation chamber2. Manometer7. Capillary3. Temperature-controlled chamber8. Absorption vessels4. Thermostating coil for carrier gas9. Gas meter5. Thermometer (Pt 100)10. Cold trapFigure 7>START OF GRAPHIC>>END OF GRAPHIC>Example of the experimental set-up for spinning rotor method Vapour pressure apparatusA. spinning rotor sensor head;B. sample cell;C. thermostatD. vacuum line (turbo pump);E. air thermostat.Figure 8>START OF GRAPHIC>>END OF GRAPHIC>Example of spinning rotor measuring head1. Ball;2. Evacuated tubular extension of 63. Permanent magnets (2);4. Coils (2) for vertical stabilization;5. Driving coils (4)6. Connection flnge.A. 5. SURFACE TENSION1. METHODThe methods described are based on the OECD Test Guideline (1). The fundamental principles are given in reference (2).1.1. INTRODUCTIONThe described methods are to be applied to the measurement of the surface tension of aqueous solutions.It is useful to have preliminary information on the water solubility, the structure, the hydrolysis properties and the critical concentration for micelles formation of the substance before performing these tests.The following methods are applicable to most chemical substances, without any restriction in respect to their degree of purity.The measurement of the surface tension by the ring tensiometer method is restricted to aqueous solutions with a dynamic viscosity of less than approximately 200 mPa s.1.2. DEFINITIONS AND UNITSThe free surface enthalpy per unit of surface area is referred to as surface tension.The surface tension is given as:N/m (SI unit) ormN/m (SI sub-unit)1 N/m = 103 dynes/cm1 mN/m = 1 dyne/cm in the obsolete cgs system1.3. REFERENCE SUBSTANCESReference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.Reference substances which cover a wide range of surface tensions are given in references 1 and 3.1.4. PRINCIPLE OF THE METHODSThe methods are based on the measurement of the maximum force which it is necessary to exert vertically, on a stirrup or a ring in contact with the surface of the liquid being examined placed in a measuring cup, in order to separate it from this surface, or on a plate, with an edge in contact with the surface, in order to draw up the film that has formed.Substances which are soluble in water at least at a concentration of 1 mg/l are tested in aqueous solution at a single concentration.1.5. QUALITY CRITERIAThese methods are capable of greater precision than is likely to be required for environmental assessment.1.6. DESCRIPTION OF THE METHODSA solution of the substance is prepared in distilled water. The concentration of this solution should be 90 % of the saturation solubility of the substance in water; when this concentration exceeds 1 g/l, a concentration of 1 g/l is used for testing. Substances with a water solubility lower than 1 mg/l need not be tested.1.6.1. Plate methodSee ISO 304 and NF T 73-060 (Surface active agents - determination of surface tension by drawing up liquid films).1.6.2. Stirrup methodSee ISO 304 and NF T 73-060 (Surface active agents - determination of surface tension by drawing up liquid films).1.6.3. Ring methodSee ISO 304 and NF T 73-060 (Surface active agents - determination of surface tension by drawing up liquid films).1.6.4. OECD harmonized ring method1.6.4.1. ApparatusCommercially available tensiometers are adequate for this measurement. They consist of the following elements:- mobile sample table,- force measuring system,- measuring body (ring),- measurement vessel.1.6.4.1.1. Mobile sample tableThe mobile sample table is used as a support for the temperature-controlled measurement vessel holding the liquid to be tested. Together with the force measuring system, it is mounted on a stand.1.6.4.1.2. Force measuring systemThe force measuring system (see figure) is located above the sample table. The error of the force measurement shall not exceed ± 10 6 N, corresponding to an error limit of ± 0,1 mg in a mass measurement. In most cases, the measuring scale of commercially available tensiometers is calibrated in mN/m so that the surface tension can be read directly in mN/m with an accuracy of 0,1 mN/m.1.6.4.1.3. Measuring body (ring)The ring is usually made of a platinum-iridium wire of about 0,4 mm thickness and a mean circumference of 60 mm. The wire ring is suspended horizontally from a metal pin and a wire mounting bracket to establish the connection to the force measuring system (see figure).Figure>START OF GRAPHIC>>END OF GRAPHIC>Measuring body (All dimensions expressed in millimetres)1.6.4.1.4. Measurement vesselThe measurement vessel holding the test solution to be measured shall be a temperature-controlled glass vessel. It shall be designed so that during the measurement the temperature of the test solution liquid and the gas phase above its surface remains constant and that the sample cannot evaporate. Cylindrical glass vessels having an inside diameter of not less than 45 mm are acceptable.1.6.4.2. Preparation of the apparatus1.6.4.2.1. CleaningGlass vessels shall be cleaned carefully. If necessary they shall be washed with hot chromo-sulphuric acid and subsequently with syrupy phosphoric acid (83 to 98 % by weight of H3PO4), thoroughly rinsed in tap water and finally washed with double-distilled water until a neutral reaction is obtained and subsequently dried or rinsed with part of the sample liquid to be measured.The ring shall first be rinsed thoroughly in water to remove any substances which are soluble in water, briefly immersed in chromo-sulphuric acid, washed in double-distilled water until a neutral reaction is obtained and finally heated briefly above a methanol flame.Note:Contamination by substances which are not dissolved or destroyed by chromo-sulphuric acid or phosphoric acid, such as silicones, shall be removed by means of a suitable organic solvent.1.6.4.2.2. Calibration of the apparatusThe validation of the apparatus consists of verifying the zero point and adjusting it so that the indication of the instrument allows reliable determination in mN/m.Mounting:The apparatus shall be levelled, for instance by means of a spirit level on the tensiometer base, by adjusting the levelling screws in the base.Zero point adjustment:After mounting the ring on the apparatus and prior to immersion in the liquid, the tensiometer indication shall be adjusted to zero and the ring checked for parallelism to the liquid surface. For this purpose, the liquid surface can be used as a mirror.Calibrations:The actual test calibration can be accomplished by means of either of two procedures:(a) Using a mass: procedure using riders of known mass between 0,1 and 1,0 g placed on the ring. The calibration factor, Öa by which all the instrument readings must be multiplied, shall be detemined according to equation (1):fa =  srsa(1)where:sr = mg2b (mN/m)m  = mass of the rider (g)g  = gravity acceleration (981 cm s 2 at sea level)b  = mean circumference of the ring (cm)óa   = reading of the tensiometer after placing the rider on the ring (mN/m).(b) Using water: procedure using pure water whose surface tension at, for instance, 23  C is equal to 72,3 mN/m. This procedure is accomplished faster than the weight calibration but there is always the danger that the surface tension of the water is falsified by traces of contamination by surfactants.The calibration factor, Öb, by which all the instrument readings shall be multiplied, shall be determined in accordance with the equation (2):fb =  sosg(2)where:óo  =  value cited in the literature for the surface tension of water (mN/m)óg  =  measured value of the surface tension of the water (mN/m) both at the same temperature.1.6.4.3. Preparation of samplesAqueous solutions shall be prepared of the substances to be tested, using the required concentrations in water, and shall not contain any non-dissolved substances.The solution must be maintained at a constant temperature (± 0,5  C). Since the surface tension of a solution in the measurement vessel alters over a period of time, several measurements shall be made at various times and a curve plotted showing surface tension as a function of time. When no further change occurs, a state of equilibrium has been reached.Dust and gaseous contamination by other substances interfere with the measurement. The work shall therefore be carried out under a protective cover.1.6.5. Test conditionsThe measurement shall be made at approximately 20  C and shall be controlled to within ± 0,5  C.1.6.6. Performance of testThe solutions to be measured shall be transferred to the carefully cleaned measurement vessel, taking care to avoid foaming, and subsequently the measurement vessel shall be placed onto the table of the test apparatus. The table-top with measurement vessel shall be raised until the ring is immersed below the surface of the solution to be measured. Subsequently, the table-top shall be lowered gradually and evenly (at a rate of approximately 0,5 cm/min) to detach the ring from the surface until the maximum force has been reached. The liquid layer attached to the ring must not separate from the ring. After completing the measurements, the ring shall be immersed below the surface again and the measurements repeated until a constant surface tension value is reached. The time from transferring the solution to the measurement vessel shall be recorded for each determination. Readings shall be taken at the maximum force required to detach the ring from the liquid surface.2. DATAIn order to calculate the surface tension, the value read in mN/m on the apparatus shall be first multiplied by the calibration factor Öa or Öb (depending on the calibration procedure used). This will yield a value which applies only approximately and therefore requires correction.Harkins and Jordan (4) have empirically determined correction factors for surface-tension values measured by the ring method which are dependent on ring dimensions, the density of the liquid and its surface tension.Since it is laborious to determine the correction factor for each individual measurement from the Harkins and Jordan tables, in order to calculate the surface tension for aqueous solutions the simplified procedure of reading the corrected surface-tension values directly from the table may be used. (Interpolation shall be used for readings ranging between the tabular values.)>TABLE>This table has been compiled on the basis of the Harkins-Jordan correction. It is similar to that in the DIN Standard (DIN 53914) for water and aqueous solutions (density ñ = 1 g/cm3) and is for a commercially available ring having the dimensions R = 9,55 mm (mean ring radius) and r = 0,185 mm (ring wire radius). The table provides corrected values for surface-tension measurements taken after calibration with weights or calibration with water.Alternatively, without the preceding calibration, the surface tension can be calculated according to the following formula:s = 4 p Rf × Fwhere:F =  the force measured on the dynamometer at the breakpoint of the filmR =  the radius of the ringf =  the correction factor (1)3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information: -  method used,-  type of water or solution used,-  precise specification of the substance (identity and impurities),-  measurement results: surface tension (reading) stating both the individual readings and their arithmetic mean as well as the corrected mean (taking into consideration the equipment factor and the correction table),-  concentration of the solution,-  test temperature,-  age of solution used; in particular the time between preparation and measurement of the solution,-  description of time dependence of surface tension after transferring the solution to the measurement vessel,-  all information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.3.2. INTERPRETATION OF RESULTSConsidering that distilled water has a surface tension of 72,75 mN/m at 20  C, substances showing a surface tension lower than 60 mN/m under the conditions of this method should be regarded as being surface-active materials.4. REFERENCES(1) OECD, Paris, 1981, Test Guideline 115, Decision of the Council C(81) 30 final.(2) R. Weissberger ed.: Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, Vol. I, Part I, Chapter XIV(3) Pure Appl. Chem., 1976, vol. 48, 511.(4) Harkins, W.D., Jordan, H.F., J. Amer. Chem. Soc., 1930, vol. 52, 1751.A.6 WATER SOLUBILITY1. METHODThe methods described are based on the OECD Test Guideline (1).1.1. INTRODUCTIONIt is useful to have preliminary information on the structural formula, the vapour pressure, the dissociation constant and the hydrolysis (as a function of pH) of the substance to perform this test.N  single method is available to cover the whole range of solubilities in water.The two test methods described below cover the whole range of solubilities but are not applicable to volatile substances :- one which applies to essentially pure substances with low solubilities, ( 10 2 grams per litre), and which are stable in water, referred to as the 'flask method`.The water solubility of the test substance can be considerably affected by the presence of impurities.1.2. DEFINITION AND UNITSThe solubility in water of a substance is specified by the saturation mass concentration of the substance in water at a given temperature. The solubility in water is specified in units of mass per volume of solution. The SI unit is kg/m3 (grams per litre may also be used).1.3. REFERENCE SUBSTANCESReference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.1.4. PRINCIPLE OF THE TEST METHODThe approximate amount of the sample and the time necessary to achieve the saturation mass concentration should be determined in a simple preliminary test.1.4.1. Column elution methodThis method is based on the elution of a test substance with water from a micro-column which is charged with an inert support material, such as glass beads or sand, coated with an excess of test substance. The water solubility is determined when the mass concentration of the eluate is constant. This is shown by a concentration plateau as a function of time.1.4.2. Flask methodIn this method, the substance (solids must be pulverized) is dissolved in water at a temperature somewhat above the test temperature. When saturation is achieved the mixture is cooled and kept at the test temperature, stirring as long as necessary to reach equilibrium. Alternatively, the measurement can be performed directly at the test temperature, if it is assured by appropriate sampling that the saturation equilibrium is reached. Subsequently, the mass concentration of the substance in the aqueous solution, which must not contain any undissolved particles, is determined by a suitable analytical method.1.5. QUALITY CRITERIA1.5.1. RepeatabilityFor the column elution method,  3 % per  C), two other temperatures at least 10  C above and below the initially chosen temperature should also be used. In this case, the temperature control should be ± 0,1  C. The chosen temperature should be kept constant in all relevant parts of the equipment.1.6.2. Preliminary testTo approximately 0,1 g of the sample (solid substances must be pulverized) in a glass-stoppered 10 ml graduated cylinder, increasing volumes of distilled water at room temperature are added according to the steps shown in the table below:>TABLE>After each addition of the indicated amount of water, the mixture is shaken vigorously for 10 minutes and is visually checked for any undissolved parts of the sample. If, after addition of 10 ml of water, the sample or parts of it remain undissolved, the experiment has to be repeated in a 100 ml measuring cylinder with larger volumes of water. At lower solubilities the time required to dissolve a substance can be considerably longer (at least 24 h should be allowed). The approximate solubility is given in the table under that volume of added water in which complete dissolution of the sample occurs. If the substance is still apparently insoluble, more than 24 h should be allowed (96 h maximum), or further dilution should be undertaken to ascertain whether the column elution or flask solubility method should be used.1.6.3. Column elution method1.6.3.1. Support material, solvent and eluentThe support material for the column elution method should be inert. Possible materials which can be employed are glass beads and sand. A suitable volatile solvent of analytical reagent quality should be used to apply the test substance to the support material. Water which has been double distilled in glass or quartz apparatus should be employed as the eluent.Note:Water directly from an organic ion exchanger must not be used.1.6.3.2. Loading of the supportApproximately 600 mg of support material is weighed and transferred to a 50 ml round-bottom flask.A suitable, weighed amount of test substance is dissolved in the chosen solvent. An appropriate amount of this solution is added to the support material. The solvent must be completely evaporated, e.g. in a rotary evaporator; otherwise water saturation of the support is not achieved due to partition effects on the surface of the support material.The loading of support material may cause problems (erroneous results) if the test substance is deposited as an oil or a different crystal phase. The problem should be examined experimentally and the details reported.The loaded support material is allowed to soak for about two hours in approximately 5 ml of water, and then the suspension is added to the microcolumn. Alternatively, dry loaded support material may be poured into the microcolumn, which has been filled with water, and then equilibrated for approximately two hours.Test procedure:The elution of the substance from the support material can be carried out in one of two different ways:- recirculating pump (see figure 1),- levelling vessel (see figure 4).1.6.3.3. Column elution method with recirculating pumpApparatusA schematic arrangement of a typical system is presented in figure 1. A suitable microcolumn is shown in figure 2, although any size is acceptable, provided it meets the criteria for reproducibility and sensitivity. The column should provide for a headspace of at least five bed volumes of water and be able to hold a minimum of five samples. Alternatively, the size can be reduced if make-up solvent is employed to replace the initial five bed volumes removed with impurities.The column should be connected to a recirculating pump capable of controlling flows of approximately 25 ml/h. The pump is connected with polytetrafluoroethylene (P.T.F.E.) and/or glass connections. The column and pump, when assembled, should have provision for sampling the effluent and equilibrating the headspace at atmospheric pressure. The column material is supported with a small (5 mm) plug of glass wool, which also serves to filter out particles. The recirculating pump can be, for example, a peristaltic pump or a membrane pump (care must be taken that no contamination and/or absorption occurs with the tube material).Measurement procedureThe flow through the column is started. It is recommended that a flow rate of approximately 25 ml/hr be used (this corresponds to 10 bed volumes/hr for the column described). The first five bed volumes (minimum) are discarded to remove water-soluble impurities. Following this, the recirculating pump is allowed to run until equilibration is established, as defined by five successive samples whose concentrations do not differ by more than ± 30 % in a random fashion. These samples should be separated from each other by time intervals corresponding to the passage of at least 10 bed volumes of the eluent.1.6.3.4. Column elution method with levelling vesselApparatus (see figures 4 and 3)Levelling vessel: The connection to the levelling vessel is made by using a ground glass joint which is connected by PTFE tubing. It is recommended that a flow rate of approximately 25 ml/hr be used. Successive eluate fractions should be collected and analyzed by the chosen method.Measurement procedureThose fractions from the middle eluate range where the concentrations are constant (± 30 %) in at least five consecutive fractions are used to determine the solubility in water.In both cases (using a recirculating pump or a levelling vessel), a second run is to be performed at half the flow rate of the first. If the results of the two runs are in agreement, the test is satisfactory; if there is a higher apparent solubility with the lower flow rate, then the halving of the flow rate must continue until two successive runs give the same solubility.In both cases (using a recirculating pump or a levelling vessel) the fractions should be checked for the presence of colloidal matter by examination for the Tyndall effect (light scattering). Presence of such particles invalidates the results, and the test should be repeated with improvements in the filtering action of the column.The pH of each sample should be recorded. A second run should be performed at the same temperature.1.6.4. Flask method1.6.4.1. ApparatusFor the flask method the following material is needed:- normal laboratory glassware and instrumentation,- a device suitable for the agitation of solutions under controlled constant temperatures,- a centrifuge (preferably thermostated), if required with emulsions, and- equipment for analytical determination.1.6.4.2. Measurement procedureThe quantity of material necessary to saturate the desired volume of water is estimated from the preliminary test. The volume of water required will depend on the analytical method and the solubility range. About five times the quantity of material determined above is weighed into each of three glass vessels fitted with glass stoppers (e.g. centrifuge tubes, flasks). The chosen volume of water is added to each vessel, and the vessels are tightly stoppered. The closed vessels are then agitated at 30  C. (A shaking or stirring device capable of operating at constant temperature should be used, e.g. magnetic stirring in a thermostatically controlled water bath). After one day, one of the vessels is removed and re-equilibrated for 24 hours at the test temperature with occasional shaking. The contents of the vessel are then centrifuged at the test temperature, and the concentration of test substance in the clear aqueous phase is determined by a suitable analytical method. The other two flasks are treated similarly after initial equilibration at 30  C for two and three days, respectively. If the concentration results from at least the last two vessels agree with the required reproducibility, the test is satisfactory. The whole test should be repeated, using longer equilibration times, if the results from vessels 1, 2 and 3 show a tendency to increasing values.The measurement procedure can also be performed without preincubation at 30  C. In order to estimate the rate of establishment of the saturation equilibrium, samples are taken until the stirring time no longer influences the concentration of the test solution.The pH of each sample should be recorded.1.6.5. AnalysisA substance-specific analytical method is preferred for these determinations, since small amounts of soluble impurities can cause large errors in the measured solubility. Examples of such methods are: gas or liquid chromatography, titration methods, photometric methods, voltammetric methods.2. DATA2.1. COLUMN ELUTION METHODThe mean value from at least five consecutive samples taken from the saturation plateau should be calculated for each run, as should the standard deviation. The results should be given in units of mass per volume of solution.The means calculated on two tests using different flows are compared and should have a repeatability of less than 30 %.2.2. FLASK METHODThe individual results should be given for each of the three flasks and those results deemed to be constant (repeatability of less than 15 %) should be averaged and given in units of mass per volume of solution. This may require the reconversion of mass units to volume units, using the density when the solubility is very high (> 100 grams per litre).3. REPORTING3.1. COLUMN ELUTION METHODThe test report shall, if possible, include the following information:-  the results of the preliminary test,-  precise specification of the substance (identity and impurities),-  the individual concentrations, flow rates and pH of each sample,-  the means and standard deviations from at least five samples from the saturation plateau of each run,-  the average of the two successive, acceptable runs,-  the temperature of the water during the saturation process,-  the method of analysis employed,-  the nature of the support material employed,-  loading of support material,-  solvent used,-  evidence of any chemical instability of the substance during the test and the method used,-  all information relevant for the interpretation of the results, especially with regard to impurities and physical state of the substance.3.2. FLASK METHODThe test report shall, if possible, include the following information:-  the results of the preliminary test,-  precise specification of the substance (identity and impurities),-  the individual analytical determinations and the average where more than one value was determined for each flask,-  the pH of each sample,-  the average of the value for the different flasks which were in agreement,-  the test temperature,-  the analytical method employed,-  evidence of any chemical instability of the substance during the test and the method used,-  all information relevant for the interpretation of the results, especially with regard to impurities and physical state of the substance.4. REFERENCES(1) OECD, Paris, 1981, Test Guideline 105, Decision of the Council C(81) 30 final.(2) NF T 20-045 (AFNOR) (Sept. 85). Chemical products for industrial use - Determination of water solubility of solids and liquids with low solubility - Column elution method(3)  NF T 20-046 (AFNOR) (Sept. 85). Chemical products for industrial use - Determination of water solubility of solids and liquids with high solubility - Flask methodAppendixFigure 1>START OF GRAPHIC>>END OF GRAPHIC>Column elution method with recirculating pumpFigure 2>START OF GRAPHIC>>END OF GRAPHIC>A typical microcolumn(All dimensions in millimetres)Figure 3>START OF GRAPHIC>>END OF GRAPHIC>A typical microcolumn(All dimensions in millimetres)Figure 4 >START OF GRAPHIC>>END OF GRAPHIC>Column elution method with levelling vesselp = Levelling vessel (e.g. 2,5 litre flask)2 = Column (see figure 3)3 = Fraction collector4 = Thermostat5 = Teflon tubing6 = Ground glass joint7 = Water line (between thermostat and column, inner diameter: approximately 8 mm)A.8. PARTITION COEFFICIENT1. METHODThe 'shake flask` method described is based on the OECD Test Guideline (1).1.1. INTRODUCTIONIt is useful to have preliminary information on structural formula, dissociation constant, water solubility, hydrolysis, n-octanol solubility and surface tension of the substance to perform this test.Measurements should be made on ionizable substances only in their non-ionized form (free acid or free base) produced by the use of an appropriate buffer with a pH of at least one pH unit below (free acid) or above (free base) the pK.This test method includes two separate procedures: the shake flask method and high performance liquid chromatography (HPLC). The former is applicable when the log Pow value (see below for definitions) falls within the range  2 to 4 and the latter within the range 0 to 6. Before carrying out either of the experimental procedures a preliminary estimate of the partition coefficient should first be obtained.The shake-flask method applies only to essentially pure substances soluble in water and n-octanol. It is not applicable to surface active materials (for which a calculated value or an estimate based on the individual n-octanol and water solubilities should be provided).The HPLC method is not applicable to strong acids and bases, metal complexes, surface-active materials or substances which react with the eluent. For these materials, a calculated value or an estimate based on individual n-octanol and water solubilities should be provided.The HPLC method is less sensitive to the presence of impurities in the test compound than is the shake-flask method. Nevertheless, in some cases impurities can make the interpretation of the results difficult because peak assignment becomes uncertain. For mixtures which give an unresolved band, upper and lower limits of log P should be stated.1.2. DEFINITION AND UNITSThe partition coefficient (P) is defined as the ratio of the equilibrium concentrations (ci) of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. In the case n-octanol and water:Pow = cn-octanolcwaterThe partition coefficient (P) therefore is the quotient of two concentrations and is usually given in the form of its logarithm to base 10 (log P).1.3. REFERENCE SUBSTANCESShake-flask methodReference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.HPLC methodIn order to correlate the measured HPLC data of a compound with its P value, a calibration graph of log P vs. chromatographic data using at least 6 reference points has to be established. It is for the user to select the appropriate reference substances. Whenever possible, at least one reference compound should have a Pow above that of the test substance, and another a Pow below that of the test substance. For log P values less than 4, the calibration can be based on data obtained by the shake-flask method. For log P values greater than 4, the calibration can be based on validated literature values if these are in agreement with calculated values. For better accuracy, it is preferable to choose reference compounds which are structurally related to the test substance.Extensive lists of values of log Pow for many groups of chemicals are available (2)(3). If data on the partition coefficients of structurally related compounds are not available, then a more general calibration, established with other reference compounds, may be used.A list of recommended reference substances and their Pow values is given in Appendix 2.1.4. PRINCIPLE OF THE METHOD1.4.1. Shake-flask methodIn order to determine a partition coefficient, equilibrium between all interacting components of the system must be achieved, and the concentrations of the substances dissolved in the two phases must be determined. A study of the literature on this subject indicates that several different techniques can be used to solve this problem, i.e. the thorough mixing of the two phases followed by their separation in order to determine the equilibrium concentration for the substance being examined.1.4.2. HPLC methodHPLC is performed on analytical columns packed with a commercially available solid phase containing long hydrocarbon chains (e.g. C8, C18) chemically bound onto silica. Chemicals injected onto such a column move along it at different rates because of the different degrees of partitioning between the mobile phase and the hydrocarbon stationary phase. Mixtures of chemicals are eluted in order of their hydrophobicity, with water-soluble chemicals eluted first and oil-soluble chemicals last, in proportion to their hydrocarbon-water partition coefficient. This enables the relationship between the retention time on such a (reverse phase) column and the n-octanol/water partition coefficient to be established. The partition coefficient is deduced from the capacity factor k, given by the expression:k = tR   t0t0in which, tR = retention time of the test substance, and t0 = average time a solvent molecule needs to pass through the column (dead-time).Quantitative analytical methods are not required and only the determination of elution times is necessary.1.5. QUALITY CRITERIA1.5.1. RepeatabilityShake-flask methodIn order to assure the accuracy of the partition coefficient, duplicate determinations are to be made under three different test conditions, whereby the quantity of substance specified as well as the ratio of the solvent volumes may be varied. The determined values of the partition coefficient expressed as their common logarithms should fall within a range of ± 0,3 log units.HPLC methodIn order to increase the confidence in the measurement, duplicate determinations must be made. The values of log P derived from individual measurements should fall within a range of ± 0,1 log units.1.5.2. SensitivityShake-flask methodThe measuring range of the method is determined by the limit of detection of the analytical procedure. This should permit the assessment of values of log Pow in the range of  2 to 4 (occasionally when conditions apply, this range may be extended to log Pow up to 5) when the concentration of the solute in either phase is not more than 0,01 mol per litre.HPLC methodThe HPLC method enables partition coefficients to be estimated in the log Pow range 0 to 6.Normally, the partition coefficient of a compound can be estimated to within ± 1 log unit of the shake-flask value. Typical correlations can be found in the literature (4)(5)(6)(7)(8). Higher accuracy can usually be achieved when correlation plots are based on structurally-related reference compounds (9).1.5.3. SpecificityShake-flask methodThe Nernst Partition Law applies only at constant temperature, pressure and pH for dilute solutions. It strictly applies to a pure substance dispersed between two pure solvents. If several different solutes occur in one or both phases at the same time, this may affect the results.Dissociation or association of the dissolved molecules result in deviations from the Nernst Partition Law. Such deviations are indicated by the fact that the partition coefficient becomes dependent upon the concentration of the solution.Because of the multiple equilibria involved, this test method should not be applied to ionizable compounds without applying a correction. The use of buffer solutions in place of water should be considered for such compounds; the pH of the buffer should be at least 1 pH unit from the pKa of the substance and bearing in mind the relevance of this pH for the environment.1.6. DESCRIPTION OF THE METHOD1.6.1. Preliminary estimate of the partition coefficientThe partition coefficient is estimated preferably by using a calculation method (see Appendix 1), or where appropriate, from the ratio of the solubilities of the test substance in the pure solvents (10).1.6.2. Shake-flask method1.6.2.1. Preparationn-Octanol: The determination of the partition coefficient should be carried out with high purity analytical grade reagent.Water: water distilled or double distilled in glass or quartz apparatus should be employed. For ionizable compounds, buffer solutions in place of water should be used if justified.Note: Water taken directly from an ion exchanger should not be used.1.6.2.1.1. Pre-saturation of the solventsBefore a partition coefficient is determined, the phases of the solvent system are mutually saturated by shaking at the temperature of the experiment. To do this, it is practical to shake two large stock bottles of high purity analytical grade n-octanol or water each with a sufficient quantity of the other solvent for 24 hours on a mechanical shaker and then to let them stand long enough to allow the phases to separate and to achieve a saturation state.1.6.2.1.2. Preparation for the testThe entire volume of the two-phase system should nearly fill the test vessel. This will help prevent loss of material due to volatilization. The volume ratio and quantities of substance to be used are fixed by the following:- the preliminary assessment of the partition coefficient (see above),- the minimum quantity of test substance required for the analytical procedure, and - the limitation of a maximum concentration in either phase of 0,01 mol per litre.Three tests are carried out. In the first, the calculated volume ratio of n-octanol to water is used; in the second, this ratio is divided by two; and in the third, this ratio is multiplied by two (e.g. 1:1, 1:2, 2:1).1.6.2.1.3. Test substanceA stock solution is prepared in n-octanol pre-saturated with water. The concentration of this stock solution should be precisely determined before it is employed in the determination of the partition coefficient. This solution should be stored under conditions which ensure its stability.1.6.2.2. Test conditionsThe test temperature should be kept constant (± 1  C) and lie in the range of 20 to 25  C.1.6.2.3. Measurement procedure1.6.2.3.1. Establishment of the partition equilibrium Duplicate test vessels containing the required, accurately measured amounts of the two solvents together with the necessary quantity of the stock solution should be prepared for each of the test conditions.The n-octanol phases should be measured by volume. The test vessels should either be placed in a suitable shaker or shaken by hand. When using a centrifuge tube, a recommended method is to rotate the tube quickly through 180  about its transverse axis so that any trapped air rises through the two phases. Experience has shown that 50 such rotations are usually sufficient for the establishment of the partition equilibrium. To be certain, 100 rotations in five minutes are recommended.1.6.2.3.2. Phase separationWhen necessary, in order to separate the phases, centrifugation of the mixture should be carried out. This should be done in a laboratory centrifuge maintained at room temperature, or, if a non-temperature controlled centrifuge is used, the centrifuge tubes should be kept for equilibration at the test temperature for at least one hour before analysis.1.6.2.4. AnalysisFor the determination of the partition coefficient, it is necessary to determine the concentrations of the test substance in both phases. This may be done by taking an aliquot of each of the two phases from each tube for each test condition and analyzing them by the chosen procedure. The total quantity of substance present in both phases should be calculated and compared with the quantity of the substance originally introduced.The aqueous phase should be sampled by a procedure that minimizes the risk of including traces of n-octanol: a glass syringe with a removable needle can be used to sample the water phase. The syringe should initially be partially filled with air. Air should be gently expelled while inserting the needle through the n-octanol layer. An adequate volume of aqueous phase is withdrawn into the syringe. The syringe is quickly removed from the solution and the needle detached. The contents of the syringe may then be used as the aqueous sample. The concentration in the two separated phases should preferably be determined by a substance-specific method. Examples of analytical methods which may be appropriate are:- photometric methods,- gas chromatography,- high-performance liquid chromatography.1.6.3. HPLC method1.6.3.1. PreparationApparatusA liquid chromatograph, fitted with a pulse-free pump and a suitable detection device, is required. The use of an injection valve with injection loops is recommended. The presence of polar groups in the stationary phase may seriously impair the performance of the HPLC column. Therefore, stationary phases should have the minimal percentage of polar groups (11). Commercial microparticulate reverse-phase packings or ready-packed columns can be used. A guard column may be positioned between the injection system and the analytical column.Mobile phaseHPLC grade methanol and HPLC grade water are used to prepare the eluting solvent, which is degassed before use. Isocratic elution should be employed. Methanol/water ratios with a minimum water content of 25 % should be used. Typically a 3:1 (v/v) methanol-water mixture is satisfactory for eluting compounds of log P 6 within an hour, at a flow rate of 1 ml/min. For compounds of high log P it may be necessary to shorten the elution time (and those of the reference compounds) by decreasing the polarity of the mobile phase or the column length.Substances with very low solubility in n-octanol tend to give abnormally low log Pow values with the HPLC method; the peaks of such compounds sometimes accompany the solvent front. This is probably due to the fact that the partitioning process is too slow to reach the equilibrium in the time normally taken by an HPLC separation. Decreasing the flow rate and/or lowering the methanol/water ratio may then be effective to arrive at a reliable value.Test and reference compounds should be soluble in the mobile phase in sufficient concentrations to allow their detection. Only in exceptional cases may additives be used with the methanol-water mixture, since additives will change the properties of the column. For chromatograms with additives it is mandatory to use a separate column of the same type. If methanol-water is not appropriate, other organic solvent-water mixtures can be used, e.g. ethanol-water or acetonitrile-water.The pH of the eluent is critical for ionizable compounds. It should be within the operating pH range of the column, which is usually between 2 and 8. Buffering is recommended. Care must be taken to avoid salt precipitation and column deterioration which occur with some organic phase/buffer mixtures. HPLC measurements with silica-based stationary phases above pH 8 are not advisable since the use of an alkaline mobile phase may cause rapid deterioration in the performance of the column.SolutesThe reference compounds should be the purest available. Compounds to be used for test or calibration purposes are dissolved in the mobile phase if possible.Test conditionsThe temperature during the measurements should not vary by more than ± 2 K.1.6.3.2. MeasurementCalculation of dead time toThe dead time to can be determined by using either a homologous series (e.g. n-alkyl methyl ketones) or unretained organic compounds (e.g. thiourea or formamide). For calculating the dead time to by using a homologous series, a set of at least seven members of a homologous series is injected and the respective retention times are determined. The raw retention times tr(nc + 1) are plotted as a function of tr(nc), and the intercept a and slope b of the regression equation:tr(nc + 1) = a + b tr(nc) are determined (nc = number of carbon atoms). The dead time to is then given by:t0  =  a / (1b)Calibration graphThe next step is to construct a correlation plot of log k values versus log P for appropriate reference compounds. In practice, a set of between 5 and 10 standard reference compounds whose log P is around the expected range are injected simultaneously and the retention times are determined, preferably on a recording integrator linked to the detection system. The corresponding logarithms of the capacity factors, log k, are calculated and plotted as a function of the log P determined by the shake-flask method. The calibration is performed at regular intervals, at least once daily, so that possible changes in column performance can be allowed for.Determination of the capacity factor of the test substance The test substance is injected in as small a quantity of mobile phase as possible. The retention time is determined (in duplicate), permitting the calculation of the capacity factor k. From the correlation graph of the reference compounds, the partition coefficient of the test substance can be interpolated. For very low and very high partition coefficients, extrapolation is necessary. In those cases particular care has to be taken of the confidence limits of the regression line.2. DATAShake-flask methodThe reliability of the determined values of P can be tested by comparison of the means of the duplicate determinations with the overall mean.3. REPORTINGThe test report shall, if possible, include the following information :- precise specification of the substance (identity and impurities),- when the methods are not applicable (e.g. surface active material), a calculated value or an estimate based on the individual n-octanol and water solubilities should be provided,- all information and remarks relevant for the interpretation of results, especially with regard to impurities and physical state of the substance.For shake-flask method:- the result of the preliminary estimation, if any,- temperature of the determination,- data on the analytical procedures used in determining concentrations,- time and speed of centrifugation, if used,- the measured concentrations in both phases for each determination (this means that a total of 12 concentrations will be reported),- the weight of the test substance, the volume of each phase employed in each test vessel and the total calculated amount of test substance present in each phase after equilibration,- the calculated values of the partition coefficient (P) and the mean should be reported for each set of test conditions as should the mean for all determinations. If there is a suggestion of concentration dependency of the partition coefficient, this should be noted in the report,- the standard deviation of individual P values about their mean should be reported,- the mean P from all determinations should also be expressed as its logarithm (base 10),- the calculated theoretical Pow when this value has been determined or when the measured value is > 104,- pH of water used and of the aqueous phase during the experiment,- if buffers are used, justification for the use of buffers in place of water, composition, concentration and pH of the buffers, pH of the aqueous phase before and after the experiment.For HPLC method:- the result of the preliminary estimation, if any,- test and reference substances, and their purity,- temperature range of the determinations,- pH at which the determinations are made,- details of the analytical and guard column, mobile phase and means of detection,- retention data and literature log P values for reference compounds used in calibration,- details of fitted regression line (log k versus log P),- average retention data and interpolated log P value for the test compound,- description of equipment and operating conditions,- elution profiles,- quantities of test and references substances introduced in the column,- dead-time and how it was measured.4. REFERENCES(1) OECD, Paris, 1981, Test Guideline 107, Decision of the Council C(81) 30 final.(2) C. Hansch and A.J. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York 1979.(3) Log P and Parameter Database, A tool for the quantitative prediction of bioactivity (C. Hansch, chairman, A.J. Leo, dir.) - Available from Pomona College Medical Chemistry Project 1982, Pomona College, Claremont, California 91711.(4) L. Renberg, G. Sundström and K. Sundh-Nygärd, Chemosphere, 1980, vol. 80, 683.(5) H. Ellgehausen, C. D'Hondt and R. Fuerer, Pestic. Sci., 1981, vol. 12, 219 (1981).(6) B. McDuffie, Chemosphere, 1981, vol. 10, 73.(7) W.E. Hammers et al., J. Chromatogr., 1982, vol. 247, 1.(8) J.E. Haky and A.M. Young, J. Liq. Chromat., 1984, vol. 7, 675 (9) S. Fujisawa and E. Masuhara, J. Biomed. Mat. Res., 1981, vol. 15, 787 (10) O. Jubermann, Verteilen und Extrahieren, in Methoden der Organischen Chemie (Houben Weyl), Allgemeine Laboratoriumpraxis (edited by E.Muller), Georg Thieme Verlag, Stuttgart, 1958, Band I/1, 223-339.(11) R.F. Rekker and H.M. de Kort, Euro. J. Med. Chem., 1979, vol. 14, 479 (12) A. Leo, C. Hansch and D. Elkins, Partition coefficients and their uses. Chem. Rev., 1971, vol. 71, 525.(13) R.F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977.(14) NF T 20-043 AFNOR (1985). Chemical products for industrial use - Determination of partition coefficient - Flask shaking method.(15) C.V. Eadsforth and P. Moser, Chemosphere, 1983, vol. 12, 1459 (16) A. Leo, C. Hansch and D. Elkins, Chem. Rev., 1971, vol. 71, 525 (17) C. Hansch, A. Leo, S.H. Unger, K.H. Kim, D. Nikaitani and E.J. Lien, J. Med. Chem., 1973, vol. 16, 1207.(18) W.B. Neely, D.R. Branson and G.E. Blau, Environ. Sci. Technol., 1974, vol. 8, 1113.(19) D.S. Brown and E.W. Flagg, J. Environ. Qual., 1981, vol. 10, 382(20) J.K. Seydel and K.J. Schaper, Chemische Struktur und biologische Aktivität von Wirkstoffen, Verlag Chemie, Weinheim, New York 1979.(21) R. Franke, Theoretical Drug Design Methods, Elsevier, Amsterdam 1984,(22) Y.C. Martin, Quantitative Drug Design, Marcel Dekker, New York, Basel 1978.(23)  N.S. Nirrlees, S.J. Noulton, C.T. Murphy, P.J. Taylor; J. Med. Chem., 1976, vol. 19, 615.Appendix 1Calculation/estimation MethodsINTRODUCTIONA general introduction to calculation methods, data and examples are provided in the Handbook of Chemical Property Estimation Methods (a).Calculated values of Pow can be used:- for deciding which of the experimental methods is appropriate (shake-flask range: log Pow:  2 to 4, HPLC range: log Pow : 0 to 6),- for selecting the appropriate test conditions (e.g. reference substances for HPLC procedures, volume ratio n-octanol/water for shake flask method),- as a laboratory internal check on possible experimental errors,- for providing a Pow-estimate in cases where the experimental methods cannot be applied for technical reasons.ESTIMATION METHODPreliminary estimate of the partition coefficientThe value of the partition coefficient can be estimated by the use of the solubilities of the test substance in the pure solvents:For this:Pestimate = saturation cn-octanolsaturation cwaterCALCULATION METHODSPrinciple of the Calculation MethodsAll calculation methods are based on the formal fragmentation of the molecule into suitable substructures for which reliable log Pow-increments are known. The log Pow of the whole molecule is then calculated as the sum of its corresponding fragment values plus the sum of correction terms for intramolecular interactions.Lists of fragment constants and correction terms are available (b)(c)(d)(e). Some are regularly updated (b).Quality CriteriaIn general, the reliability of the calculation method decreases with increasing complexity of the compound under study. In the case of simple molecules with low molecular weight and one or two functional groups, a deviation of 0,1 to 0,3 log Pow units between the results of the different fragmentation methods and the measured value can be expected. In the case of more complex molecules the margin of error can be greater. This will depend on the reliability and availability of fragment constants, as well as on the ability to recognize intramolecular interactions (e.g. hydrogen bonds) and the correct use of the correction terms (less of a problem with the computer software CLOGP-3) (b). In the case of ionizing compounds the correct consideration of the charge or degree of ionization is important.Calculation ProceduresHansch ð-MethodThe original hydrophobic substituent constant, ð, introduced by Fujita et al. (f) is defined as:ðx = log Pow (PhX)   log Pow (PhH) where Pow (PhX) is the partition coefficient of an aromatic derivative and Pow (PhH) that of the parent compoundPestimate = saturation cn-octanolsaturation cwaterAccording to its definition the ð-method is applicable predominantly for aromatic substitution. ð-values for a large number of substituents have been tabulated (b)(c)(d). They are used for the calculation of log Pow for aromatic molecules or substructures.Rekker MethodAccording to Rekker (g) the log Pow value is calculated as follows:log Pow = Siai fi + Sj (interaction terms) where fi represents the different molecular fragment constants and ai the frequency of their occurrence in the molecule under investigation. The correction terms can be expressed as an integral multiple of one single constant Cm (so-called 'magic constant`). The fragment constants fi and Cm were determined from a list of 1 054 experimental Pow values (825 compounds) using multiple regression analysis (c)(h). The determination of the interaction terms is carried out according to set rules described in the literature (e)(h)(i).Hansch-Leo MethodAccording to Hansch and Leo (c), the log Pow value is calculated from:log Pow = Siai fi + Sj bj Fjwhere fi represents the different molecular fragment constants, Fj the correction terms and ai, bj the corresponding frequencies of occurrence. Derived from experimental Pow values, a list of atomic and group fragmental values and a list of correction terms Fj (so-called 'factors`) were determined by trial and error. The correction terms have been ordered into several different classes (a)(c). It is relatively complicated and time consuming to take into account all the rules and correction terms. Software packages have been developed (b).Combined MethodThe calculation of log Pow of complex molecules can be considerably improved, if the molecule is dissected into larger substructures for which reliable log Pow values are available, either from tables (b)(c) or from one's own measurements. Such fragments (e.g. heterocycles, anthraquinone, azobenzene) can then be combined with the Hansch ð-values or with Rekker or Leo fragment constants.Remarksi) The calculation methods can only be applied to partly- or fully-ionized compounds when it is possible to take the necessary correction factors into account.ii) If intramolecular hydrogen bonds can be assumed, the corresponding correction terms (approx. + 0,6 to +1,0 log Pow units) have to be added (a). Indications for the presence of such bonds can be obtained from stereo models or spectroscopic data of the molecule.iii) If several tautomeric forms are possible, the most likely form should be used as the basis of the calculation.iv) The revisions of lists of fragment constants should be followed carefully.ReportWhen using calculation/estimation methods, the test report shall, if possible, include the following information:- description of the substance (mixture, impurities, etc.),- indication of any possible intramolecular hydrogen bonding, dissociation, charge and any other unusual effects (e.g. tautomerism),- description of the calculation method,- identification or supply of database,- peculiarities in the choice of fragments,- comprehensive documentation of the calculation.LITERATURE(a) W.J. Lyman, W.F. Reehl and D.H. Rosenblatt (ed.), Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York, 1983.(b) Pomona College, Medicinal Chemistry Project, Claremont, California 91711, USA, Log P Database and Med. Chem. Software (Program CLOGP-3).(c) C. Hansch, A.J. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York, 1979.(d) A. Leo, C. Hansch, D. Elkins, Chem. Rev., 1971, vol. 71, 525.(e) R.F. Rekker, H.M. de Kort, Eur. J. Med. Chem. - Chim. Ther. 1979, vol. 14, 479.(f) T. Fujita, J. Iwasa and C. Hansch, J. Amer. Chem. Soc., 1964, vol. 86, 5175.(g) R.F. Rekker, The Hydrophobic Fragmental Constant, Pharmacochemistry Library, Elsevier, New York, 1977, vol. 1.(h) C.V. Eadsforth, P. Moser, Chemosphere, 1983, vol. 12, 1459.(i) R.A. Scherrer, ACS, American Chemical Society, Washington D.C., 1984, Symposium Series 255, p. 225.Appendix 2>TABLE>A.9. FLASH-POINT1. METHOD1.1. INTRODUCTIONIt is useful to have preliminary information on the flammability of the substance before performing this test. The test procedure is applicable to liquid substances whose vapours can be ignited by ignition sources. The test methods listed in this text are only reliable for flash-point ranges which are specified in the individual methods.The possibility of chemical reactions between the substance and the sample holder should be considered when selecting the method to be used.1.2. DEFINITIONS AND UNITSThe flash-point is the lowest temperature, corrected to a pressure of 101,325 kPa, at which a liquid evolves vapours, under the conditions defined in the test method, in such an amount that a flammable vapour/air mixture is produced in the test vessel.Units:  Ct = T273,15(t in  C and T in K)1.3. REFERENCE SUBSTANCESReference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.1.4. PRINCIPLE OF THE METHODThe substance is placed in a test vessel and heated or cooled to the test temperature according to the procedure described in the individual test method. Ignition trials are carried out in order to ascertain whether or not the sample flashed at the test temperature.1.5. QUALITY CRITERIA1.5.1. RepeatabilityThe repeatability varies according to flash-point range and the test method used; maximum 2  C.1.5.2. SensitivityThe sensitivity depends on the test method used.1.5.3. SpecificityThe specificity of some test methods is limited to certain flash-point ranges and subject to substance-related data (e.g. high viscosity).1.6. DESCRIPTION OF THE METHOD1.6.1. PreparationsA sample of the test substance is placed in a test apparatus according to 1.6.3.1 and/or 1.6.3.2.For safety, it is recommended that a method utilizing a small sample size, circa 2 cm3, be used for energetic or toxic substances.1.6.2. Test conditionsThe apparatus should, as far as is consistent with safety, be placed in a draught-free position.1.6.3. Performance of the test1.6.3.1. Equilibrium methodSee ISO 1516, ISO 3680, ISO 1523, ISO 3679.1.6.3.2. Non-equilibrium methodAbel apparatus:See BS 2000 part 170, NF M07-011, NF T66-009.Abel-Pensky apparatus:See EN 57, DIN 51755 part 1 (for temperatures from 5 to 65  C), DIN 51755 part 2 (for temperatures below 5  C), NF M07-036.Tag apparatus:See ASTM D 56.Pensky-Martens apparatus:See ISO 2719, EN 11, DIN 51758, ASTM D 93, BS 2000-34, NF M07-019.Remarks:When the flash-point, determined by a non-equilibrium method in 1.6.3.2., is found to be 0 ± 2  C, 21 ± 2  C or 55 ± 2  C, it should be confirmed by an equilibrium method using the same apparatus.Only the methods which can give the temperature of the flash-point may be used for a notification.To determine the flash-point of viscous liquids (paints, gums and similar) containing solvents, only apparatus and test methods suitable for determining the flash-point of viscous liquids may be used.See ISO 3679, ISO 368O, ISO 1523, DIN 53213 part 1.2. DATA3. REPORTINGThe test report shall, if possible, include the following information:- the precise specification of the substance (identification and impurities),- the method used should be stated as well as any possible deviations, - the results and any additional remarks relevant for the interpretation of results.4. REFERENCESNone.A.10. FLAMMABILITY (SOLIDS)1. METHOD1.1. INTRODUCTIONIt is useful to have preliminary information on potentially explosive properties of the substance before performing this test.This test should only be applied to powdery, granular or paste-like substances.In order not to include all substances which can be ignited but only those which burn rapidly or those whose burning behaviour is in any way especially dangerous, only substances whose burning velocity exceeds a certain limiting value are considered to be highly flammable.It can be especially dangerous if incandescence propagates through a metal powder because of the difficulties in extinguishing a fire. Metal powders should be considered highly flammable if they support spread of incandescence throughout the mass within a specified time.1.2. DEFINITION AND UNITSBurning time expressed in seconds.1.3. REFERENCE SUBSTANCESNot specified.1.4. PRINCIPLE OF THE METHODThe substance is formed into an unbroken strip or powder train about 250 mm long and a preliminary screening test performed to determine if, on ignition by a gas flame, propagation by burning with flame or smouldering occurs. If propagation over 200 mm of the train occurs within a specified time then a full test programme to determine the burning rate is carried out.1.5. QUALITY CRITERIANot stated.1.6. DESCRIPTION OF METHOD1.6.1. Preliminary screening testThe substance is formed into an unbroken strip or powder train about 250 mm long by 20 mm wide by 10 mm high on a non-combustible, non-porous and low heat-conducting base plate.A hot flame from a gas burner (minimum diameter 5 mm) is applied to one end of the powder train until the powder ignites or for a maximum of 2 minutes (5 minutes for powders of metals or metal-alloys). It should be noted whether combustion propagates along 200 mm of the train within the 4 minutes test period (or 40 minutes for metal powders). If the substance does not ignite and propagate combustion either by burning with flame or smouldering along 200 mm of the powder train within the 4 minutes (or 40 minutes) test period, then the substance should not be considered as highly flammable and no further testing is required. If the substance propagates burning of a 200 mm length of the powder train in less than 4 minutes, or less than 40 minutes for metal powders, the procedure described below (point 1.6.2. and following) should be carried out.1.6.2. Burning rate test1.6.2.1. PreparationPowdery or granular substances are loosely filled into a mould 250 mm long with a triangular cross-section of inner height 10 mm and width 20 mm. On both sides of the mould in a longitudinal direction two metal plates are mounted as lateral limitations which project 2 mm beyond the upper edge of the triangular cross section (figure). The mould is then dropped three times from a height of 2 cm onto a solid surface. If necessary the mould is then filled up again. The lateral limitations are then removed and the excess substance scraped off. A non-combustible, non-porous and low heat-conducting base plate is placed on top of the mould, the apparatus inverted and the mould removed.Paste-like substances are spread on a non-combustible, non-porous and low heat-conducting base plate in the form of a rope 250 mm in length with a cross section of about 1 cm2.1.6.2.2. Test conditionsIn the case of a moisture-sensitive substance, the test should be carried out as quickly as possible after its removal from the container.1.6.2.3. Performance of the testArrange the pile across the draught in a fume cupboard.The air-speed should be sufficient to prevent fumes escaping into the laboratory and should not be varied during the test. A draught screen should be erected around the apparatus.A hot flame from a gas burner (minimum diameter of 5 mm) is used to ignite the pile at one end. When the pile has burned a distance of 80 mm, the rate of burning over the next 100 mm is measured. The test is performed six times, using a clean cool plate each time, unless a positive result is observed earlier.2. DATAThe burning time from the preliminary screening test (1.6.1.) and the shortest burning time in up to six tests (1.6.2.3.) are relevant for evaluation.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:-  the precise specification of the substance (identification and impurities),-  a description of the substance to be tested, its physical state including moisture content,-  results from the preliminary screening test and from the burning rate test if performed,-  all additional remarks relevant to the interpretation of results.3.2. INTERPRETATION OF THE RESULTPowdery, granular or paste-like substances are to be considered as highly flammable when the time of burning in any tests carried out according to the test procedure described in 1.6.2 is less than 45 seconds. Powders of metals or metal-alloys are considered to be highly flammable when they can be ignited and the flame or the zone of reaction spreads over the whole sample in 10 minutes or less.4. REFERENCES(1)  NF T 20-042 (SEPT 85). Chemical products for industrial use. Determination of the flammability of solids.AppendixFigure>START OF GRAPHIC>>END OF GRAPHIC>Mould and accessories for the preparation of the pile (All dimensions in millimetres)A.11. FLAMMABILITY (GASES)1. METHOD1.1. INTRODUCTIONThis method allows a determination of whether gases mixed with air at room temperature (circa 20  C) and atmospheric pressure are flammable and, if so, over what range of concentrations. Mixtures of increasing concentrations of the test gas with air are exposed to an electrical spark and it is observed whether ignition occurs.1.2. DEFINITION AND UNITSThe range of flammability is the range of concentration between the lower and the upper explosion limits. The lower and the upper explosion limits are those limits of concentration of the flammable gas in admixture with air at which propagation of a flame does not occur.1.3. REFERENCE SUBSTANCESNot specified.1.4. PRINCIPLE OF THE METHODThe concentration of gas in air is increased step by step and the mixture is exposed at each stage to an electrical spark.1.5. QUALITY CRITERIANot stated.1.6. DESCRIPTION OF THE METHOD1.6.1. ApparatusThe test vessel is an upright glass cylinder having a minimum inner diameter of 50 mm and a minimum height of 300 mm. The ignition electrodes are separated by a distance of 3 to 5 mm and are placed 60 mm above the bottom of the cylinder. The cylinder is fitted with a pressure-release opening. The apparatus has to be shielded to restrict any explosion damage.A standing induction spark of 0,5 sec. duration, which is generated from a high voltage transformer with an output voltage of 10 to 15 kV (maximum of power input 300 W), is used as the ignition source. An example of a suitable apparatus is described in reference (2).1.6.2. Test conditionsThe test must be performed at room temperature (circa 20  C).1.6.3. Performance of the testUsing proportioning pumps, a known concentration of gas in air is introduced into the glass cylinder. A spark is passed through the mixture and it is observed whether or not a flame detaches itself from the ignition source and propagates independently. The gas concentration is varied in steps of 1 % vol. until ignition occurs as described above.If the chemical structure of the gas indicates that it would be non-flammable and the composition of the stoichiometric mixture with air can be calculated, then only mixtures in the range from 10 % less than the stoichiometric composition to 10 % greater than this composition need be tested in 1 % steps.2. DATAThe occurrence of flame propagation is the only relevant information data for the determination of this property.3. REPORTINGThe test report shall, if possible, include the following information:- the precise specification of the substance (identification and impurities),- a description, with dimensions, of the apparatus used- the temperature at which the test was performed,- the tested concentrations and the results obtained,- the result of the test: non-flammable gas or highly flammable gas,- if it is concluded that the gas is non-flammable then the concentration range over which it was tested in 1 % steps should be stated,-  all information and remarks relevant to the interpretation of results have to be reported.4. REFERENCES(1) NF T 20-041 (SEPT 85). Chemical products for industrial use. Determination of the flammability of gases.(2) W.Berthold, D.Conrad, T.Grewer, H.Grosse-Wortmann, T.Redeker und H.Schacke. 'Entwicklung einer Standard-Apparatur zur Messung von Explosionsgrenzen`. Chem.-Ing.-Tech. 1984, vol 56, 2, 126-127.A.12. FLAMMABILITY (CONTACT WITH WATER)1. METHOD1.1. INTRODUCTIONThis test method can be used to determine whether the reaction of a substance with water or damp air leads to the development of dangerous amounts of gas or gases which may be highly flammable.The test method can be applied to both solid and liquid substances. This method is not applicable to substances which spontaneously ignite when in contact with air.1.2. DEFINITIONS AND UNITSHighly flammable: substances which, in contact with water or damp air, evolve highly flammable gases in dangerous quantities at a minimum rate of 1 litre/kg per hour.1.3. PRINCIPLE OF THE METHODThe substance is tested according to the step by step sequence described below; if ignition occurs at any step, no further testing is necessary. If it is known that the substance does not react violently with water then proceed to step 4 (1.3.4).1.3.1. Step 1The test substance is placed in a trough containing distilled water at 20  C and it is noted whether or not the evolved gas ignites.1.3.2. Step 2The test substance is placed on a filter paper floating on the surface of a dish containing distilled water at 20  C and it is noted whether or not the evolved gas ignites. The filter paper is merely to keep the substance in one place to increase the chances of ignition.1.3.3. Step 3The test substance is made into a pile approximately 2 cm high and 3 cm diameter. A few drops of water are added to the pile and it is noted whether or not the evolved gas ignites.1.3.4. Step 4The test substance is mixed with distilled water at 20  C and the rate of evolution of gas is measured over a period of seven hours, at one-hour intervals. If the rate of evolution is erratic, or is increasing, after seven hours, the measuring time should be extended to a maximum time of five days. The test may be stopped if the rate at any time exceeds 1 litre/kg per hour.1.4. REFERENCE SUBSTANCESNot specified.1.5. QUALITY CRITERIANot stated.1.6 DESCRIPTION OF METHODS1.6.1. Step 11.6.1.1. Test conditionsThe test is performed at room temperature (circa 20  C).1.6.1.2. Performance of the testA small quantity (approximately 2 mm diameter) of the test substance should be placed in a trough containing distilled water. A note should be made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.1.6.2. Step 21.6.2.1. ApparatusA filter-paper is floated flat on the surface of distilled water in any suitable vessel, e.g. a 100 mm diameter evaporating dish.1.6.2.2. Test conditionsThe test is performed at room temperature (circa 20  C).1.6.2.3. Performance of the testA small quantity of the test substance (approximately 2 mm diameter) is placed onto the centre of the filter-paper. A note should be made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.1.6.3. Step 31.6.3.1. Test conditionsThe test is performed at room temperature (circa 20  C).1.6.3.2. Performance of the testThe test substance is made into a pile approximately 2 cm high and 3 cm diameter with an indentation in the top. A few drops of water are added to the hollow and a note is made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.1.6.4. Step 41.6.4.1. ApparatusThe apparatus is set up as shown in the figure.1.6.4.2. Test conditionsInspect the container of the test substance for any powder START OF GRAPHIC>>END OF GRAPHIC>ApparatusA.13. PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS1. METHOD1.1. INTRODUCTIONThe test procedure is applicable to solid or liquid substances, which, in small amounts, will ignite spontaneously a short time after coming into contact with air at room temperature (circa 20  C).Substances which need to be exposed to air for hours or days at room temperature or at elevated temperatures before ignition occurs are not covered by this test method.1.2 DEFINITIONS AND UNITSSubstances are considered to have pyrophoric properties if they ignite or cause charring under the conditions described in 1.6.The auto-flammability of liquids may also need to be tested using method A.15 Auto-ignition temperature (liquids and gases).1.3. REFERENCE SUBSTANCESNot specified.1.4. PRINCIPLE OF THE METHODThe substance, whether solid or liquid, is added to an inert carrier and brought into contact with air at ambient temperature for a period of five minutes. If liquid substances do not ignite then they are absorbed onto filter paper and exposed to air at ambient temperature (circa 20  C) for five minutes. If a solid or liquid ignites, or a liquid ignites or chars a filter paper, then the substance is considered to be pyrophoric.1.5. QUALITY CRITERIARepeatability: because of the importance in relation to safety, a single positive result is sufficient for the substance to be considered pyrophoric.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. ApparatusA porcelain cup of circa 10 cm diameter is filled with diatomaceous earth to a height of about 5 mm at room temperature (circa 20  C).Note:Diatomaceous earth or any other comparable inert substance which is generally obtainable shall be taken as representative of soil onto which the test substance might be spilt in the event of an accident.Dry filter paper is required for testing liquids which do not ignite on contact with air when in contact with an inert carrier.1.6.2. Performance of the Testa) Powdery Solids1 to 2 cm3 of the powdery substance to be tested is poured from circa 1 m height onto a non-combustible surface and it is observed whether the substance ignites during dropping or within five minutes of settling.The test is performed six times unless ignition occurs.b) LiquidsCirca 5 cm3 of the liquid to be tested is poured into the prepared porcelain cup and it is observed whether the substance ignites within five minutes.If no ignition occurs in the six tests, perform the following tests:A 0,5 ml test sample is delivered from a syringe to an indented filter paper and it is observed whether ignition or charring of the filter paper occurs within five minutes of the liquid being added. The test is performed three times unless ignition or charring occurs.2. DATA2.1. TREATMENT OF RESULTSTesting can be discontinued as soon as a positive result occurs in any of the tests.2.2. EVALUATIONIf the substance ignites within five minutes when added to an inert carrier and exposed to air, or a liquid substance chars or ignites a filter paper within five minutes when added and exposed to air, it is considered to be pyrophoric.3. REPORTINGThe test report shall, if possible, include the following information :- the precise specification of the substance (identification and impurities),- the results of the tests,- any additional remark relevant to the interpretation of the results.4. REFERENCES(1) NF T 20-039 (SEPT 85). Chemical products for industrial use. Determination of the spontaneous flammability of solids and liquids.(2) Recommendations on the Transport of Dangerous Goods, Test and criteria, 1990, United Nations, New York.A.14. EXPLOSIVE PROPERTIES1. METHOD1.1. INTRODUCTIONThe method provides a scheme of testing to determine whether a solid or a pasty substance presents a danger of explosion when submitted to the effect of a flame (thermal sensitivity), or to shock or friction (sensitivity to mechanical stimuli), and whether a liquid substance presents a danger of explosion when submitted to the effect of a flame or shock.The method comprises three parts:(a) a test of thermal sensitivity (1);(b) a test of mechanical sensitivity with respect to shock (1);(c) a test of mechanical sensitivity with respect to friction (1).The method yields data to assess the likelihood of initiating an explosion by means of certain common stimuli. The method is not intended to ascertain whether a substance is capable of exploding under any conditions.The method is appropriate for determining whether a substance will present a danger of explosion (thermal and mechanical sensitivity) under the particular conditions specified in the directive. It is based on a number of types of apparatus which are widely used internationally (1) and which usually give meaningful results. It is recognised that the method is not definitive. Alternative apparatus to that specified may be used provided that it is internationally recognised and the results can be adequately correlated with those from the specified apparatus.The tests need not be performed when available thermodynamic information (e.g. heat of formation, heat of decomposition) and/or absence of certain reactive groups (2) in the structural formula establishes beyond reasonable doubt that the substance is incapable of rapid decomposition with evolution of gases or release of heat (i.e. the material does not present any risk of explosion). A test of mechanical sensitivity with respect to friction is not required for liquids.1.2. DEFINITIONS AND UNITSExplosive:Substances which may explode under the effect of flame or which are sensitive to shock or friction in the specified apparatus (or are more mechanically sensitive than 1,3-dinitrobenzene in alternative apparatus).1.3. REFERENCE SUBSTANCES1,3-dinitrobenzene, technical crystalline product sieved to pass 0,5 mm, for the friction and shock methods.Perhydro-1,3,5-trinitro-1,3,5-triazine (RDX, hexogen, cyclonite - CAS 121-82-4), recrystallised from aqueous cyclohexanone, wet-sieved through a 250 ìm and retained on a 150 ìm sieve and dried at 103 ± 2  C (for 4 hours) for the second series of friction and shock tests.1.4. PRINCIPLE OF THE METHODPreliminary tests are necessary to establish safe conditions for the performance of the three tests of sensitivity.1.4.1. Safety-in-handling tests (3)For safety reasons, before performing the main tests, very small samples (circa 10 mg) of the substance are subjected to heating without confinement in a gas flame, to shock in any convenient form of apparatus and to friction by the use of a mallet against an anvil or any form of friction machine. The objective is to ascertain if the substance is so sensitive and explosive that the prescribed sensitivity tests, particularly that of thermal sensitivity, should be performed with special precautions so as to avoid injury to the operator.1.4.2. Thermal sensitivityThe method involves heating the substance in a steel tube, closed by orifice plates with differing diameters of hole, to determine whether the substance is liable to explode under conditions of intense heat and defined confinement.1.4.3. Mechanical sensitivity (shock)The method involves subjecting the substance to the shock from a specified mass dropped from a specified height.1.4.4. Mechanical sensitivity (friction)The method involves subjecting solid or pasty substances to friction between standard surfaces under specified conditions of load and relative motion.1.5. QUALITY CRITERIANot stated.1.6. DESCRIPTION OF METHOD1.6.1. Thermal sensitivity (effect of a flame)1.6.1.1. ApparatusThe apparatus consists of a non-reusable steel tube with its re-usable closing device (figure 1), installed in a heating and protective device. Each tube is deep-drawn from sheet steel (see Appendix) and has an internal diameter of 24 mm, a length of 75 mm and wall thickness of 0,5 mm. The tubes are flanged at the open end to enable them to be closed by the orifice plate assembly. This consists of a pressure-resistant orifice plate, with a central hole, secured firmly to a tube using a two-part screw joint (nut and threaded collar). The nut and threaded collar are made from chromium-manganese steel (see Appendix) which is spark-free up to 800  C. The orifice plates are 6 mm thick, made from heat-resistant steel (see Appendix), and are available with a range of diameters of opening.1.6.1.2. Test conditionsNormally the substance is tested as received although in certain cases, e.g. if pressed, cast or otherwise condensed, it may be necessary to test the substance after crushing.For solids, the mass of material to be used in each test is determined using a two-stage dry run procedure. A tared tube is filled with 9 cm3 of substance and the substance tamped with 80 N force applied to the total cross-section of the tube. For reasons of safety or in cases where the physical form of the sample can be changed by compression, other filling procedures may be used; e.g. if the substance is very friction sensitive then tamping is not appropriate. If the material is compressible then more is added and tamped until the tube is filled to 55 mm from the top. The total mass used to fill the tube to the 55 mm level is determined and two further increments, each tamped with 80 N force, are added. Material is then either added with tamping, or taken out, as required, to leave the tube filled to a level 15 mm from the top. A second dry run is performed, starting with a tamped quantity of a third of the total mass found in the first dry run. Two more of these increments are added with 80 N tamping and the level of the substance in the tube adjusted to 15 mm from the top by addition or subtraction of material as required. The amount of solid determined in the second dry run is used for each trial; filling being performed in three equal amounts, each compressed to 9 cm3 by whatever force is necessary. (This may be facilitated by the use of spacing rings.)Liquids and gels are loaded into the tube to a height of 60 mm taking particular care with gels to prevent the formation of voids. The threaded collar is slipped onto the tube from below, the appropriate orifice plate is inserted and the nut tightened after applying some molybdenum disulphide based lubricant. It is essential to check that none of the substance is trapped between the flange and the plate, or in the threads.Heating is provided by propane taken from an industrial cylinder, fitted with a pressure regulator (60 to 70 mbar), through a meter and evenly distributed (as indicated by visual observation of the flames from the burners) by a manifold to four burners. The burners are located around the test chamber as shown in figure 1. The four burners have a combined consumption of about 3.2 litres of propane per minute. Alternative fuel gases and burners may be used but the heating rate must be as specified in figure 3. For all apparatus, the heating rate must be checked periodically using tubes filled with dibutyl phthalate as indicated in figure 3.1.6.1.3. Performance of the testsEach test is performed until either the tube is fragmented or the tube has been heated for five minutes. A test resulting in the fragmentation of the tube into three or more pieces, which in some cases may be connected to each other by narrow strips of metal as illustrated in figure 2, is evaluated as giving an explosion. A test resulting in fewer fragments or no fragmentation is regarded as not giving an explosion.A series of three tests with a 6,0 mm diameter orifice plate is first performed and, if no explosions are obtained, a second series of three tests is performed with a 2,0 mm diameter orifice plate. If an explosion occurs during either test series then no further tests are required.1.6.1.4. EvaluationThe test result is considered positive if an explosion occurs in either of the above series of tests.1.6.2. Mechanical sensitivity (shock)1.6.2.1. Apparatus (figure 4)The essential parts of a typical fall hammer apparatus are a cast steel block with base, anvil, column, guides, drop weights, release device and a sample holder. The steel anvil 100 mm (diameter) × 70 mm (height) is screwed to the top of a steel block 230 mm (length) × 250 mm (width) × 200 mm (height) with a cast base 450 mm (length) × 450 mm (width) × 60 mm (height). A column, made from seamless drawn steel tube, is secured in a holder screwed on to the back of the steel block. Four screws anchor the apparatus to a solid concrete block 60 × 60 × 60 cm such that the guide rails are absolutely vertical and the drop weight falls freely. 5 and 10 kg weights, made from solid steel, are available for use. The striking head of each weight is of hardened steel, HRC 60 to 63, and has a minimum diameter of 25 mm.The sample under test is enclosed in a shock device consisting of two coaxial solid steel cylinders, one above the other, in a hollow cylindrical steel guide ring. The solid steel cylinders should be of 10 ( 0,003,  0,005) mm diameter and 10 mm height and have polished surfaces, rounded edges (radius of curvature 0,5 mm) and a hardness of HRC 58 to 65. The hollow cylinder must have an external diameter of 16 mm, a polished bore of 10 (+0,005, +0,010) mm and a height of 13 mm. The shock device is assembled on an intermediate anvil (26 mm diameter and 26 mm height) made of steel and centred by a ring with perforations to allow escape of fumes.1.6.2.2. Test conditionsThe sample volume should be 40 mm3, or a volume to suit any alternative apparatus. Solid substances should be tested in the dry state and prepared as follows:(a) powdered substances are sieved (sieve size 0,5 mm); all that has passed through the sieve is used for testing;(b) pressed, cast or otherwise condensed substances are broken into small pieces and sieved; the sieve fraction from 0,5 to 1 mm diameter is used for testing and should be representative of the original substance.Substances normally supplied as pastes should be tested in the dry state where possible or, in any case, following removal of the maximum possible amount of diluent. Liquid substances are tested with a 1 mm gap between the upper and lower steel cylinders.1.6.2.3. Performance of the testsA series of six tests are performed dropping the 10 kg mass from 0,40 m (40 J). If an explosion is obtained during the six tests at 40 J, a further series of 6 tests, dropping a 5 kg mass from 0,15 m (7,5 J), must be performed. In other apparatus, the sample is compared with the chosen reference substance using an established procedure (e.g. up-and-down technique etc.).1.6.2.4. EvaluationThe test result is considered positive if an explosion (bursting into flame and/or a report is equivalent to explosion) occurs at least once in any of the tests with the specified shock apparatus or the sample is more sensitive than 1,3-dinitrobenzene or RDX in an alternative shock test.1.6.3. Mechanical sensitivity (friction)1.6.3.1. Apparatus (figure 5)The friction apparatus consists of a cast steel base plate on which is mounted the friction device. This consists of a fixed porcelain peg and moving porcelain plate. The porcelain plate is held in a carriage which runs in two guides. The carriage is connected to an electric motor via a connecting rod, an eccentric cam and suitable gearing such that the porcelain plate is moved, once only, back and forth beneath the porcelain peg for a distance of 10 mm. The porcelain peg may be loaded with, for example, 120 or 360 newtons.The flat porcelain plates are made from white technical porcelain (roughness 9 to 32 ìm) and have the dimensions 25 mm (length) × 25 mm (width) × 5 mm (height). The cylindrical porcelain peg is also made of white technical porcelain and is 15 mm long, has a diameter of 10 mm and roughened spherical end surfaces with a radius of curvature of 10 mm.1.6.3.2. Test conditionsThe sample volume should be 10 mm3 or a volume to suit any alternative apparatus.Solid substances are tested in the dry state and prepared as follows:(a) powdered substances are sieved (sieve size 0,5 mm); all that has passed through the sieve is used for testing;(b) pressed, cast or otherwise condensed substances are broken into small pieces and sieved; the sieve fraction START OF GRAPHIC>>END OF GRAPHIC>>START OF GRAPHIC>>END OF GRAPHIC>Figure 2>START OF GRAPHIC>>END OF GRAPHIC>Thermal sensitivity testExamples of fragmentationFigure 3>START OF GRAPHIC>>END OF GRAPHIC>Heating rate calibration for thermal sensitivity testTemperature/time curve obtained on heating dibutyl phthalate (27 cm3) in a closed (1,5 mm orifice plate) tube using a propane flow rate of 3,2 litre/minute. The temperature is measured with a 1 mm diameter stainless steel sheathed chromel/alumel thermocouple, placed centrally 43 mm below the rim of the tube. The heating rate between 135  C and 285  C should be between 185 and 215 K/minute.Figure 4>START OF GRAPHIC>>END OF GRAPHIC>>START OF GRAPHIC>>END OF GRAPHIC>Figure 4 ContinuedFig. 4c Shock device for substances in powdered or paste-like formFig. 4d Shock device for liquid substances (1) steel cylinders(2) guide ring for steel cylinders(3) locating ring with orifices(a) vertical section(b) plan(4) rubber ring(5) liquid substance (40 mm3)(6) space free from liquid>START OF GRAPHIC>>END OF GRAPHIC>Fig. 4e Hammer (drop mass of 5 kg)(1) suspension spigot(2) height marker(3) positioning groove(4) cylindrical striking head(5) rebound catchFigure 5>START OF GRAPHIC>>END OF GRAPHIC>Friction sensitivity apparatusFig. 5a Friction apparatus; elevation and plan view>START OF GRAPHIC>>END OF GRAPHIC>Fig. 5b Starting position of peg on sample>START OF GRAPHIC>>END OF GRAPHIC>A.15. AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES)1. METHOD1.1. INTRODUCTIONExplosive substances and substances which ignite spontaneously in contact with air at ambient temperature should not be submitted to this test. The test procedure is applicable to gases, liquids and vapours which, in the presence of air, can be ignited by a hot surface.The auto-ignition temperature can be considerably reduced by the presence of catalytic impurities, by the surface material or by a higher volume of the test vessel.1.2. DEFINITIONS AND UNITSThe degree of auto-ignitability is expressed in terms of the auto-ignition temperature. The auto-ignition temperature is the lowest temperature at which the test substance will ignite when mixed with air under the conditions defined in the test method.1.3. REFERENCE SUBSTANCESReference substances are cited in the standards (see 1.6.3). They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.1.4. PRINCIPLE OF THE METHODThe method determines the minimum temperature of the inner surface of an enclosure that will result in ignition of a gas, vapour or liquid injected into the enclosure.1.5. QUALITY CRITERIAThe repeatability varies according to the range of auto-ignition temperatures and the test method used.The sensitivity and specificity depend on the test method used.1.6. DESCRIPTION OF THE METHOD1.6.1. ApparatusThe apparatus is described in the method referred to in 1.6.3.1.6.2. Test conditionsA sample of the test substance is tested according to the method referred to in 1.6.3.1.6.3. Performance of the testSee IEC 79-4, DIN 51794, ASTM-E 659-78, BS 4056, NF T 20-037.2. DATARecord the test-temperature, atmospheric pressure, quantity of sample used and time-lag until ignition occurs.3. REPORTINGThe test report shall, if possible, include the following information:- the precise specification of the substance (identification and impurities),- the quantity of sample used, atmospheric pressure,- the apparatus used,- the results of measurements (test temperatures, results concerning ignition, corresponding time-lags), - all additional remarks relevant to the interpretation of results.4. REFERENCESNone.A.16. RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS1. METHOD1.1. INTRODUCTIONExplosive substances and substances which ignite spontaneously in contact with air at ambient temperature should not be submitted to this test.The purpose of this test is to provide preliminary information on the auto-flammability of solid substances at elevated temperatures.If the heat developed either by a reaction of the substance with oxygen or by exothermic decomposition is not lost rapidly enough to the surroundings, self-heating leading to self-ignition occurs. Self-ignition therefore occurs when the rate of heat-production exceeds the rate of heat loss.The test procedure is useful as a preliminary screening test for solid substances. In view of the complex nature of the ignition and combustion of solids, the self-ignition temperature determined according to this test method should be used for comparison purposes only.1.2. DEFINITIONS AND UNITSThe self-ignition temperature as obtained by this method is the minimum ambient temperature expressed in  C at which a certain volume of a substance will ignite under defined conditions.1.3. REFERENCE SUBSTANCENone.1.4. PRINCIPLE OF THE METHODA certain volume of the substance under test is placed in an oven at room temperature; the temperature/time curve relating to conditions in the centre of the sample is recorded while the temperature of the oven is increased to 400  C, or to the melting point if lower, at a rate of 0,5  C/min. For the purpose of this test, the temperature of the oven at which the sample temperature reaches 400  C by self-heating is called the self-ignition temperature.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE METHOD1.6.1. Apparatus1.6.1.1. OvenA temperature-programmed laboratory oven (volume about 2 litres) fitted with natural air circulation and explosion relief. In order to avoid a potential explosion risk, any decomposition gases must not be allowed to come into contact with the electric heating elements.1.6.1.2. Wire mesh cubeA piece of stainless steel wire mesh with 0,045 mm openings should be cut according to the pattern in figure 1. The mesh should be folded and secured with wire into an open-topped cube.1.6.1.3. ThermocouplesSuitable thermocouples.1.6.1.4. RecorderAny two-channel recorder calibrated from 0 to 600  C or corresponding voltage.1.6.2. Test conditionsSubstances are tested as received.1.6.3. Performance of the testThe cube is filled with the substance to be tested and is tapped gently, adding more of the substance until the cube is completely full. The cube is then suspended in the centre of the oven at room temperature. One thermocouple is placed at the centre of the cube and the other between the cube and the oven wall to record the oven temperature.The temperatures of the oven and sample are continuously recorded while the temperature of the oven is increased to 400  C, or to the melting point if lower, at a rate of 0,5  C/min.When the substance ignites the sample thermocouple will show a very sharp temperature rise above the oven temperature.2. DATAThe temperature of the oven at which the sample temperature reaches 400  C by self-heating is relevant for evaluation (see figure 2).3. REPORTINGThe test report shall, if possible, include the following information:- a description of the substance to be tested,- the results of measurement including the temperature/time curve,- all additional remarks relevant for the interpretation of the results.4. REFERENCES(1)  NF T 20-036 (September 85). Chemical products for industrial use. Determination of the relative temperature of the spontaneous flammability of solids.Figure 1>START OF GRAPHIC>>END OF GRAPHIC>Pattern of 20 mm test cubeFigure 2>START OF GRAPHIC>>END OF GRAPHIC>Typical temperature/time curveA.17. OXIDIZING PROPERTIES (SOLIDS)1. METHOD1.1. INTRODUCTIONIt is useful to have preliminary information on any potentially explosive properties of the substance before performing this test.This test is not applicable to liquids, gases, explosive or highly flammable substances, or organic peroxides.This test need not be performed when examination of the structural formula establishes beyond reasonable doubt that the substance is incapable of reacting exothermically with a combustible material.In order to ascertain if the test should be performed with special precautions, a preliminary test should be performed.1.2. DEFINITION AND UNITSBurning time: reaction time, in seconds, taken for the reaction zone to travel along a pile, following the procedure described in 1.6.Burning rate: expressed in millimetres per second.Maximum burning rate: the highest value of the burning rates obtained with mixtures containing 10 to 90 % by weight of oxidizer.1.3. REFERENCE SUBSTANCEBarium nitrate (analytical grade) is used as reference substance for the test and the preliminary test.The reference mixture is that mixture of barium nitrate with powdered cellulose, prepared according to 1.6, which has the maximum burning rate (usually a mixture with 60 % barium nitrate by weight).1.4. PRINCIPLE OF THE METHODA preliminary test is carried out in the interests of safety. N  further testing is required when the preliminary test clearly indicates that the test substance has oxidizing properties. When this is not the case, the substance should then be subject to the full test.In the full test, the substance to be tested and a defined combustible substance will be mixed in various ratios. Each mixture is then formed into a pile and the pile is ignited at one end. The maximum burning rate determined is compared with the maximum burning rate of the reference mixture.1.5. QUALITY CRITERIAIf required, any method of grinding and mixing is valid provided that the difference in the maximum rate of burning in the six separate tests differs from the arithmetic mean value by no more than 10 %.1.6. DESCRIPTION OF THE METHOD1.6.1. Preparation1.6.1.1. Test substanceReduce the test sample to a particle size START OF GRAPHIC>>END OF GRAPHIC>Mould and accessories for the preparation of the pile (All dimensions in millimetres)PART B: METHODS FOR THE DETERMINATION OF TOXICITY GENERAL INTRODUCTION: PART B A. INTRODUCTIONSee General Introduction.B. DEFINITIONS(i) Acute toxicity comprises the adverse effects occurring within a given time (usually 14 days), after administration of a single dose of a substance(ii) LD50 (median lethal dose) is a statistically derived single dose of a substance that can be expected to cause death in 50 % of dosed animals. The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (milligrams per kilogram)(iii) LC50 (median lethal concentration) is a statistically derived concentration of a substance that can be expected to cause death during exposure or within a fixed time after exposure in 50 % of animals exposed for a specified time. The LC50 value is expressed as weight of test substance per standard volume of air (milligrams per litre)(iv) N  adverse effect level is the maximum dose or exposure level used in a test which produces no detectable signs of toxicity(v) Sub-acute/Sub-chronic toxicity comprises the adverse effects occurring in experimental animals as a result of repeated daily dosing with, or exposure to, a chemical for a short part of their expected lifespan(vi) Maximum Tolerated Dose (MTD) is the highest dose level eliciting signs of toxicity in animals without having major effects on survival relative to the test in which it is used (vii) Skin irritation is the production of reversible inflammatory changes in the skin following the application of a test substance(viii) Eye irritation is the production of reversible changes in the eye following the application of a test substance to the anterior surface of the eye(ix) Skin sensitization (allergic contact dermatitis) is an immunologically mediated cutaneous reaction to a substanceSpecific definitions for inhalation toxicity- an aerosol is defined as particles (solid and/or liquid) homogeneously dispersed in air- the aerodynamic diameter is the diameter of a sphere of unit density (1 g cm 3) having the same terminal settling velocity as the particle in question.- the mass median aerodynamic diameter (MMAD) is the calculated aerodynamic diameter which divides the size distribution of the aerosol in half when measured by mass.- the geometric standard deviation (GSD) is the ratio of the estimated 84 percentile to the 50 percentile and indicates the slope of the cumulative particle size distribution curve, assuming the size distribution to be log normal.Specific definitions for the fixed dose procedure in the determination of acute oral toxicity- Evident toxicity refers to toxic effects seen following administration of a test substance, which are of a severity such that administration at the next higher dose level could result in mortality.- Discriminating dose is the highest out of the four fixed dose levels which can be administered without causing compound-related mortality (including humane kills).C. MUTAGENICITY (including carcinogenicity pre-screening test)For the preliminary assessment of mutagenic potential of a substance, it is necessary to obtain information on two categories of end point, namely, gene mutation and chromosomal abberrations.These two end points are evaluated by the following tests:(i) Tests on the production of gene (point) mutations in procaryotic cells such as Salmonella typhimurium; tests using Escherichia coli are also acceptable. The choice between these two test organisms may be determined by the nature of the chemical being tested.(ii) Tests on the production of chromosomal aberrations in mammalian cells grown in vitro; an in vivo procedure (the micronucleus test or the metaphase analysis of bone marrow cells) is also acceptable. However, in the absence of any contraindications the in vitro methods are strongly to be preferred.D. EVALUATION AND INTERPRETATIONThere are limitations in the extent to which the results of animal and in vitro tests can be extrapolated directly to man and this must be borne in mind when tests are evaluated and interpreted.Where available, evidence of adverse effects in humans may be of relevance in determining the potential effects of chemical substances on the human population.E. LITERATURE REFERENCESToxicology is a developing experimental science and there is abundant literature for each topic. Relevant information can be found in the OECD Test Guidelines.Additional remarksAnimal CareStringent control of environmental conditions and proper animal care techniques are essential in toxicity testing.(i) Housing conditionsThe environmental conditions in the experimental animal rooms or enclosures should be appropriate to the test species. For rats, mice and guinea pigs, suitable conditions are a room temperature of 22 ± 3  C with a relative humidity of 30 to 70 %; for rabbits the temperature should be 20 ± 3  C with a relative humidity of 30 to 70 %.Some experimental techniques are particularly sensitive to temperature effects and, in these cases, details of appropriate conditions are included in the description of the test method. In all investigations of toxic effects, the temperature and humidity should be monitored, recorded, and included in the final report of the study.When lighting is artificial, the sequence should normally be 12 hours light, 12 hours dark. Details of the lighting pattern should be recorded and included in the final report of the study.In reports of animal experiments, it is important to indicate the type of caging used and the number of animals housed in each cage both during exposure to the chemical and any subsequent observation period.(ii) Feeding conditionsDiets should meet all the nutritional requirements of the species under test. Where test substances are administered to animals in their diet the nutritional value may be reduced by interaction between the substance and a dietary constituent.The possibility of such a reaction should be considered when interpreting the results of tests.Dietary contaminants which are known to influence the toxicity should not be present in interfering concentrations.Animal WelfareWhen elaborating the test methods due consideration was given to animal welfare. Some examples are briefly given hereunder, this list is not exhaustive. The exact wording and/or conditions should be read in the text of the methods :- For the determination of acute oral toxicity, an alternative method, the 'Fixed Dose Procedure` is introduced. This procedure does not utilize death as specific endpoint. It uses fewer animals and results in less pain and distress than the classical determination of acute oral toxicity.- The number of animals used is reduced to the scientifically acceptable minimum : only 5 animals of the same sex are tested per dose level for methods B.1 and B.3; only 10 animals (and only 5 for the negative control group) are used for the determination of the skin sensitisation by the guinea pig maximization test (method B.6); the number of animals needed for the positive control when testing mutagenicity in vivo is also lowered (methods B.11 and B.12)- Pain and distress of animals during the tests are minimised : animals showing severe and enduring signs of distress and pain may need to be humanely killed; dosing test substances in a way known to cause marked pain and distress due to corrosive or irritating properties need not to be carried out (methods B.1, B.2 and B.3).- Testing with irrelevantly high doses is avoided by the introduction of limit tests, not only in the acute toxicity tests (methods B.1, B.2 and B.3) but also in the in vivo tests for mutagenicity (methods B.11 and B.12).- A strategy of testing for irritancy now allows the non-performance of a test, or its reduction to a single animal study, when sufficient scientific evidence can be provided.Such scientific evidence can be based on the physico-chemical properties of the substance, the results of other tests already performed, or the results of well validated in vitro tests. For example, if an acute toxicity study by the dermal route has been conducted at the limit test dose with the substance (method B.3), and no skin irritation was observed, further testing for skin irritation (method B.4) may be unnecessary; materials which have demonstrated definite corrosion or severe skin irritancy in a dermal irritation tudy (method B.4) should not be further tested for eye irritancy (method B.5).B.1 ACUTE TOXICITY (ORAL)1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSSee General Introduction Part B (B).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODThe test substance is administered orally by gavage in graduated doses to several groups of experimental animals, one dose being used per group. The doses chosen may be based on the results of a range finding test. Subsequently, observations of effects and deaths are made. Animals which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied. This method is directed primarily to studies in rodent species.Animals showing severe and enduring signs of distress and pain may need to be humanely 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.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. PreparationsThe animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. Before the test, healthy young adult animals are randomized and assigned to the treatment groups. Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that wherever possible the use of an aqueous solution is considered first, followed by consideration of a solution in vegetable oil, then by possible solution in other vehicles, or in suspension. For non-aqueous vehicles the relevant toxic characteristics of the vehicle should be known or should be determined before or during the test. In rodents, normally the volume should not exceed 10 ml/kg body weight except in the case of aqueous solutions where 20 ml/kg may be used. Variability in test volume should be minimized by adjusting the concentration to ensure a constant volume at all dose levels.1.6.2. Test Conditions1.6.2.1. Experimental AnimalsUnless there are contra-indications the rat is the preferred species.Commonly used laboratory strains should be employed. For each sex, at the start of the test the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.1.6.2.2. Number and SexAt least five rodents 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. Where information is available demonstrating that a sex is markedly more sensitive, animals of this sex should be dosed.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.1.6.2.3. Dose 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 determination of the LD50.1.6.2.4. Limit TestWhen rodents are used, a limit test at one dose level of at least 2 000 mg/kg bodyweight may be carried out in a group of five males and five females using the procedures described above. If compound-related mortality is produced, a full study may need to be considered.1.6.2.5. Observation PeriodThe observation period should be at least 14 days. However, the duration of observation should not be rigidly fixed. It should be determined by the toxic reactions, their rate of onset and the length of the recovery period; it may thus be extended when considered necessary. The time at which signs of toxicity appear and disappear and the time of death are important, especially if there is a tendency for deaths to be delayed.1.6.3 ProcedureAnimals should be fasted prior to substance administration. For the rat, food should be withheld overnight; for animals with higher metabolic rates a shorter period of fasting is appropriate; water is not restricted. The following day, the animals should be weighed and then the test substance administered by gavage in a single dose. If a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours. After the substance has been administered, food may be withheld for a further three to four hours.Where a dose is administered in fractions over a period it may be necessary to provide the animals with food and water depending on the length of the period.Following administration, observations are made and recorded systematically, individual records should be maintained for each animal. Observations should be made frequently during the first day.A careful clinical examination should be made at least once each working day, other observations should be made daily with appropriate actions taken to minimize loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals. Observations should include changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Particular attention should be directed to obervation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The time of death should be recorded as precisely as possible.Animals that die during the test and those surviving at the termination of the test are subjected to necropsy. All gross pathological changes should be recorded. Where indicated, tissues should be taken for histopathological examination.Assessment of toxicity in the other sexAfter completion of the study in one sex, at least one group of five 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 markedly more sensitive, testing in animals of the other sex may be dispensed with.2. DATAData should be summarized in tabular form, showing for each test group the number of animals at the start of the test, time of death of individual animals, number of animals displaying other signs of toxicity, description of toxic effects and necropsy findings. Individual weights of animals should be determined and recorded shortly before the test substance is administered, weekly thereafter and at death. Changes in weight should be calculated and recorded when survival exceeds one day. Animals which are humanely killed due to compound-related distress and pain are recorded as compound-related deaths. The LD50 may be determined by a recognized method. Data evaluation should include the relationship, if any, between the animals' exposure to the test substance and the incidence and severity of all abnormalities, including behavioural and clinical abnormalities, gross lesions, body weight changes, mortality, and any other toxicological effects.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- species, strain, source, environmental conditions, diet, etc.,- test conditions,-  dose levels (with vehicle, if used, and concentration),- sex of animals dosed,- tabulation of response data by sex and 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 exposed),- time of death after dosing, reasons and criteria used for humane killing of animals,- all observations,- LD50 value for the sex subjected to a full study determined at 14 days (with the method of determination specified),- 95 % confidence interval for the LD50 (where this can be provided),- dose/mortality curve and slope (where permitted by the method of determination),- necropsy findings,- any histopathological findings,- results of any test on the other sex,- discussion of the results (particular attention should be given to the effect that humane killing of animals during the test may have on the calculated LD50 value),- interpretation of results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.1 bis ACUTE TOXICITY (ORAL) - FIXED DOSE METHOD1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSSee General Introduction Part B (B).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODThe acute oral toxicity test provides information on the adverse effects which can follow, within a short period of time, the ingestion of a single dose of the test substance.The fixed dose method is conducted in two stages.In a preliminary sighting study, the effects of various doses administered orally by gavage to single animals of one sex are investigated in a sequential manner. The sighting study yields information on the dose-toxicity relationship, including an estimate of the minimum lethal dose. Normally, no more than five animals are used in this first stage.In the main study, the substance is administered orally by gavage to groups of five male and five female animals at one of the pre-set dose levels (5, 50, 500 or 2 000 mg/kg). The dose used is derived from the sighting study and is that which is likely to produce 'evident toxicity` (see 1.2. Definitions) but no deaths.Following administration, observations for effects are made.When the initial dose level chosen produces evident toxicity but no compound-related mortality, no further testing is needed.Where evident toxicity is not seen at the chosen dose level, the substance should be re-tested at the next higher dose level. Where animals die, or where a severe toxic reaction requires humane killing of animals, the substance should be re-tested at the next lower dose level.This procedure permits the identification of the 'discriminating dose` (see 1.2. Definitions), that is the highest of the pre-set dose levels which can be administered without causing mortality (including humane kills).Animals showing severe and enduring signs of distress and pain may need to be humanely 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.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. Preparations1.6.1.1. Experimental AnimalsUnless there are contra-indications the rat is the preferred species.Commonly used laboratory strains should be employed. For each sex, at the start of the test, the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.The animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. Before the test, healthy young adult animals are randomized and assigned to the sighting study and main study treatment groups. In practice, only one group of each sex may be needed in the main study.1.6.1.2. Dose preparation and administrationWhere necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that wherever possible the use of an aqueous solution is considered first, followed by consideration of a solution in vegetable oil, then by possible solution in other vehicles, or in suspension. For non-aqueous vehicles the relevant toxic characteristics of the vehicle should be known or should be determined before or during the test. In rodents, normally the volume should not exceed 10 ml/kg body weight except in the case of aqueous solutions where 20 ml/kg may be used. Variability in test volume should be minimized by adjusting the concentration to ensure a constant volume at all dose levels.Animals should be fasted prior to substance administration. For the rat, food should be withheld overnight; water is not restricted. The following day, the animals should be weighed and then the test substance administered by gavage in a single dose. If a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours. After the substance has been administered, food may be withheld for a further three to four hours. Where a dose is administered in fractions over a period it may be necessary to provide the animals with food and water depending on the length of the period.1.6.2. Procedure1.6.2.1. Sighting studyThe effects of various doses are investigated in single animals. Normally female animals will be used in the absence of information indicating that males will be the more sensitive sex. Dosing is sequential, allowing at least 24 hours before dosing the next animal. All animals are carefully observed for signs of toxicity for at least seven days; if signs of moderate toxicity persist at seven days, the animal should be observed for up to an additional seven days. The following initial dose levels are considered : 5, 50, 500 and 2 000 mg/kg. If the initial dose chosen does not produce severe toxicity, and the next higher level produces mortality, then it will be necessary to investigate one or more intermediate dose levels as appropriate. In this way it should be possible to build up information on the dose level(s) that produce(s) some signs of toxicity and the minimum dose level that produces mortality.An effort should be made to select the initial dose using evidence from related chemicals. In the absence of such information, it is suggested that the 500 mg/kg dose is used in the first instance. If no signs of toxicity are seen at the initial dose, then the next higher dose level is investigated. If no mortality occurs at 2 000 mg/kg, the sighting test is complete and the main study should be conducted at this dose level. If severe effects, necessitating humane killing are seen at the initial dose (e.g. 500 mg/kg), the next lower dose (e.g. 50 mg/kg) is given to another animal. If this animal survives, further animals may then be dosed with the appropriate intermediate dose levels between the fixed doses. Normally, one would not expect to use more than five animals in this procedure.1.6.2.2. Main studyAt least 10 animals (five female and five male) should be used for each dose level which is investigated. The females should be nulliparous and non-pregnant.It is a principle of the fixed dose method that only moderately toxic doses are used in the main study. Administration of lethal doses of the test substance should be avoided.The dose level to be used in the test should be selected from one of the four fixed dose levels, namely 5, 50, 500 or 2 000 mg/kg body weight. The initial dose level chosen should be that which is likely to produce evident toxicity but no compound-related mortality (including humane kills; accidental deaths are not included but should be recorded). N  further testing is necessary when this dose level produces evident toxicity but no compound-related mortality.Where evident toxicity does not result from administration of the chosen dose level, the substance should be re-tested at the next higher dose level. The animals, however, should continue to be kept under observation until the observation period is complete. Where a severe toxic reaction requires animals to be humanely killed or there is compound-related mortality, the substance should be retested at the next lower dose level. Again, animals that do not need to be humanely killed should be kept under observation for the full observation period.Following administration, observations are made and recorded systematically. Individual records should be maintained for each animal.The observation period should be at least 14 days. However, the duration of observation should not be rigidly fixed. It should be determined by the toxic reactions, their rate of onset and the length of the recovery period; it may thus be extended when considered necessary. The time at which signs of toxicity appear and disappear and the time of death are important, especially if there is a tendency for toxic signs to be delayed.A careful clinical examination should be made at least twice on the day of dosing and at least once each day thereafter. Animals obviously in pain or showing severe signs of distress should be humanely killed. Additional observations will be necessary during the first few days after dosing if the animals continue to display signs of toxicity. The test may be terminated if it becomes apparent that the initial dose level chosen was too high.Observations should include changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Particular attention should be directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.Individual weights of animals should be determined shortly before the test substance is administered, daily for the next three days, and weekly thereafter. Animals that die during the test, and those surviving to termination of the test, are weighed and subjected to necropsy. All gross pathological changes should be recorded. Where indicated, tissues should be taken for histopathological examination.The investigation of a second or, in exceptional circumstances, a third dose level may be required, dependent upon the results of the preceding dose level.In the case in which a substance produces mortality at 5 mg/kg body weight (or where the sighting study indicates that mortality will result at that dose level) the acute toxicity of the substance may need to be further investigated.2. DATAData from both the sighting study and the main study should be summarized in tabular form showing for each dose level tested the number of animals at the start of the test; the number of animals displaying signs of toxicity; the number of animals found dead during the test or killed for humane reasons; a description of the toxic effects and, for the main study, whether compound-related evident toxicity was observed; the time course of any toxic effects; and the necropsy findings. Changes in weight should be calculated and recorded when survival exceeds one day.Animals which are humanely killed due to compound-related distress and pain are recorded as compound-related deaths.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information, for both the sighting study and the main study, as appropriate :- species, strain, source, environmental conditions, diet, etc.- test conditions- dose levels (with vehicle, if used, and concentration)- full results of all dose levels investigated- tabulation of response data by sex and dose level (i.e. number of animals used; changes in body weight; when applicable, number of animals that died or were killed during the test; number of animals showing signs of toxicity; nature, severity and duration of effects)- time course of onset of signs of toxicity and whether these were reversible- when animals died or were killed, time of death after dosing, reasons and criteria used for humane killing of animals- necropsy findings- any histopathological findings- discussion of the results- interpretation of results, including the signs of evident toxicity and the discriminating dose level identified in the test.3.2. EVALUATION AND INTERPRETATION>TABLE>See also General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.2. ACUTE TOXICITY (INHALATION)1. METHOD1.1. INTRODUCTIONIt is useful to have preliminary information on the particle size distribution, the vapour pressure, the melting point, the boiling point, the flash point and explosivity (if applicable) of the substance.See also General Introduction Part B (A).1.2. DEFINITIONSSee General Introduction Part B (B).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODSeveral groups of experimental animals are exposed for a defined period to the test substance in graduated concentrations, one concentration being used per group. Subsequently observations of effects and deaths are made. Animals which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied.Animals showing severe and enduring signs of distress and pain may need to be humanely killed. Dosing test substances in a way known to cause marked pain and distress due to corrosive or severe irritating properties need not be carried out.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. PreparationsThe animals are kept under the experimental housing and feeding conditions for at least five days prior to the experiment. Before the test healthy young animals are randomized and assigned to the required number of groups. They need not be subjected to simulated exposure unless this is indicated by the type of exposure apparatus being used.Solid test substances may need to be micronised in order to achieve particles of an appropriate size.Where necessary a suitable vehicle may be added to the test substance to help generate an appropriate concentration of the test substance in the atmosphere and a vehicle control group should then be used. If a vehicle or other additives are used to facilitate dosing, they should be known not to produce toxic effects. Historical data can be used if appropriate.1.6.2. Test Conditions1.6.2.1. Experimental AnimalsUnless there are contra-indications the rat is the preferred species. Commonly used laboratory strains should be employed. For each sex, at the start of the test the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.1.6.2.2. Number and SexAt least 10 rodents (five female and five male) are used at each concentration level. The females should be nulliparous and non-pregnant.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.1.6.2.3. Exposure ConcentrationsThese 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 concentration mortality curve and, where possible, permit an acceptable determination of an LC50.1.6.2.4. Limit TestIf an exposure of five male and five female test animals to 5 mg per litre of a gas, or aerosol of liquid or solid substance (or, where this 2s not possible due to the physical or chemical, including explosive, properties of the test substance, the maximum attainable concentration) produces, after a four-hour exposure, no compound-related mortality within 14 days, further testing may not be considered necessary.1.6.2.5. Exposure TimeThe period of exposure should be four hours.1.6.2.6. EquipmentThe animals should be tested with inhalation equipment designed to sustain a dynamic airflow of at least 12 air changes per hour, to ensure an adequate oxygen content and an evenly distributed exposure atmosphere. Where a chamber is used its design should minimize crowding of the test animals and maximize their exposure by inhalation to the test substance. As a general rule to ensure stability of a chamber atmosphere the total 'volume` of the test animals should not exceed 5 % of the volume of the test chamber. Oro-nasal, head only, or whole body individual chamber exposure may be used; the first two will help to minimize the uptake of the test substance by other routes.1.6.2.7. Observation PeriodThe observation period should be at least 14 days. However, the duration of observations should not be rigidly fixed. It should be determined by the toxic reactions, their rate of onset and the length of the recovery period; it may thus be extended when considered necessary. The time at which signs of toxicity appear and disappear and the time of death are important, especially if there is a tendency for deaths to be delayed.1.6.3. ProcedureShortly before exposure, the animals are weighed, and then exposed to the test concentration in the designated apparatus for a period of four hours, after equilibration of the chamber concentration. Time for equilibration should be short. The temperature at which the test is performed should be maintained at 22 ± 3  C. Ideally the relative humidity should be maintained between 30 % and 70 %, but in certain instances (e.g. tests of some aerosols) this may not be practicable. Maintenance of a slight negative pressure inside the chamber ( & ge; 5 mm of water) will prevent leakage of the test substance into the surrounding area. Food and water should be withheld during exposure. Suitable systems for the generation and monitoring of the test atmosphere should be used. The system should ensure that stable exposure conditions are achieved as rapidly as possible. The chamber should be designed and operated in such a way that a homogeneous distribution of the test atmosphere within the chamber is maintained.Measurements or monitoring should be made:(a) of the rate of air flow (continuously).(b) of the actual concentration of the test substance measured in the breathing zone at least three times during exposure (some atmospheres, e.g. aerosols at high concentrations, may need more frequent monitoring). During the exposure period the concentration should not vary by more than ± 15 % of the mean value. However in the case of some aerosols, this level of control may not be achievable and a wider range would then be acceptable. For aerosols, particle size analysis should be performed as often as necessary (at least once per test group).(c) of temperature and humidity, continuously if possible.During and following exposure, observations are made and recorded systematically; individual records should be maintained for each animal. Observations should be made frequently during the first day. A careful clinical examination should be made at least once each working day, other observations should be made daily with appropriate actions taken to minimize loss of animals from the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals.Observations should include changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Particular attention should be directed to observation of respiratory behaviour, tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The time of death should be recorded as precisely as possible. Individual weights of animals should be determined weekly after exposure, and at death.Animals that die during the test and those surviving at the termination of the test are subjected to necropsy with particular reference to any changes in the upper and lower respiratory tract. All gross pathological changes should be recorded. Where indicated, tissues should be taken for histopathological examination.2. DATAData should be summarized in tabular form showing for each test group the number of animals at the start of the test, time of death of individual animals, number of animals displaying other signs of toxicity, description of toxic effects and necropsy findings. Changes in weight must be calculated and recorded when survival exceeds one day. Animals which are humanely killed due to compound-related distress and pain are recorded as compound-related deaths. The LC50 should be determined by a recognized method. Data evaluation should include the relationship, if any, between the animal's exposure to the test substance and the incidence and severity of all abnormalities, including behavioural and clinical abnormalities, gross lesions, body weight changes, mortality and any other toxic effects.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- species, strain, source, environmental conditions, diet etc.;- test conditions: description of exposure apparatus including design, type, dimensions, source of air, system for generating aerosols, method of conditioning air and the method of housing animals in a test chamber when this is used. The equipment for measuring temperature, humidity, and aerosol concentrations and particle size distribution should be described.Exposure dataThese should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and shall, if possible, include:(a) airflow rates through the inhalation equipment;(b) temperature and humidity of the air;(c) nominal concentrations (total amount of test substance fed into the inhalation equipment divided by volume of air);(d) nature of vehicle, if used;(e) actual concentrations in test breathing zone;(f) The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD);(g) equilibration period;(h) exposure period;- tabulation of response data by sex and exposure level (i.e. number of animals that died or were killed during the test; number of animals showing signs of toxicity; number of animals exposed);- time of death during or following exposure, reasons and criteria used for humane killing of animals;- all observations;- LC50 value for each sex determined at the end of the observation period (with method of calculation specified);- 95 % confidence interval for the LC50 (where this can be provided);- dose/mortality curve and slope (where permitted by the method of determination);- necropsy findings;- any histopathological findings;- discussions of the results (particular attention should be given to the effect that humane killing of animals during the test may have on the calculated LC50 value);- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.3. ACUTE TOXICITY (DERMAL)1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSee General Introduction Part B (B).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODThe test substance is applied to the skin in graduated doses to several groups of experimental animals, one dose being used per group. Subsequently, observations of effects and deaths are made. Animals which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied.Animals showing severe and enduring signs of distress and pain may need to be humanely 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.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. PreparationsThe animals are kept in their experimental cages under the experimental housing and feeding conditions for at least five days prior to the experiment. Before the test, healthy young adult animals are randomized and assigned to the treatment groups. Approximately 24 hours before the test, fur should be removed by clipping or shaving from the dorsal area of the trunk of the animals. When clipping or shaving the fur, care must be taken to avoid abrading the skin which could alter its permeability. Not less than 10 % of the body surface should be clear for the application of the test substance. When testing solids, which may be pulverized 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.1.6.2. Test Conditions1.6.2.1. Experimental AnimalsThe adult rat or rabbit may be used. Other species may be used but their use would require justification. Commonly used laboratory strains should be employed. For each sex, at the start of the test the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.1.6.2.2. 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. Where information is available demonstrating that a sex is markedly more sensitive, animals of this sex should be dosed.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.1.6.2.3. Dose 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. Any irritant or corrosive effects should be taken into account when deciding on dose levels. The data should be sufficient to produce a dose/response curve and, where possible, permit an acceptable determination of the LD50.1.6.2.4. Limit TestA limit test at one dose level of at least 2 000 mg/kg bodyweight may be carried out in a group of 5 male and 5 female animals, using the procedures described above. If compound-related mortality is produced, a full study may need to be considered.1.6.2.5. Observation PeriodThe observation period should be at least 14 days. However, the duration of observation should not be rigidly fixed. It should be determined by the toxic reactions, their rate of onset and the length of the recovery period; it may thus be extended when considered 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.1.6.3. ProcedureAnimals should be caged individually. The test substance should be applied uniformly over an area which is approximately 10 % of the total body surface area. With highly toxic substances the surface area covered may be less but as much of the area should be covered with a layer as thin and uniform 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 some other appropriate method of cleansing the skin.Observations should be recorded systematically as they are made. Individual records should be maintained for each animal. Observations should be made frequently during the first day. A careful clinical examination should be made at least once each working day, other observations should be made daily with appropriate actions taken to minimize loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals.Observations should include changes in fur, treated skin, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour 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. Animals that die during the test and those surviving at the termination of the test are subjected to necropsy. All gross pathological changes should be recorded. Where indicated, tissues should be taken for histopathological examination.Assessment of toxicity in the other sexAfter 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 markedly more sensitive, testing in animals of the other sex may be dispensed with.2. DATAData should be summarized in tabular form, showing for each test group the number of animals at the start of the test, time of death of individual animals, number of animals displaying other signs of toxicity, description of toxic effects and necropsy findings. Individual weights of animals should be determined and recorded 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. Animals which are humanely killed due to compound-related distress and pain are recorded as compound-related deaths. The LD50 should be determined by a recognized method.Data evaluation should include an evaluation of relationships, if any, between the animal's exposure to the test substance and the incidence and severity of all abnormalities, including behavioural and clinical abnormalities, gross lesions, body weight changes, mortality, and any other toxicological effects.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- species, strain, source, environmental conditions, diet, etc.;- test conditions (including method of skin cleansing and type of dressing: occlusive or not occlusive);- dose levels (with vehicle, if used, and concentrations),- sex of animals dosed;- tabulation of response data by sex and 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 exposed);- time of death after dosing, reasons and criteria used for humane killing of animals;- all observations;- LD50 value for the sex subjected to a full study, determined at 14 days with the method of determination specified;- 95 % confidence interval for the LD50 (where this can be provided);- dose/mortality curve and slope where permitted by the method of determination;- necropsy findings;- any histopathological findings;- results of any test on the other sex;- discussion of results (particular attention should be given to the effect that humane killing of animals during the test may have on the calculated LD50 value);- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.4. ACUTE TOXICITY (SKIN IRRITATION)1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSee General Introduction Part B (B).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODInitial considerationsCareful consideration needs to be given to all the available information on a substance to minimize the testing of substances under conditions that are likely to produce severe reactions. The following information may be useful when considering whether a complete test, a single-animal study, or no further testing is appropriate.i) Physicochemical properties and chemical reactivity. Strongly acidic or alkaline substances (demonstrated pH of 2 or less or 11,5 or greater, for example) may not require testing for primary dermal irritation if corrosive properties can be expected. Alkaline or acidic reserve should also be taken into account.ii) If convincing evidence of severe effects in well validated in vitro tests is available, a complete test may not be required.iii) Results from acute toxicity studies. If an acute toxicity test by the dermal route has been conducted with the substance at the limit test dose level (2 000 mg/kg body weight), and no skin irritation was observed, further testing for skin irritation may be unnecessary. In addition, testing of materials which have been shown to be highly toxic by the dermal route is unnecessary.The substance to be tested is applied in a single dose to the skin of several experimental animals, each animal serving as its own control. The degree of irritation is read and graded after a specific interval, and is further described to provide a complete evaluation of the effects. The duration of the observations should be sufficient to evaluate fully the reversibility of the effects observed.Animals showing severe and enduring signs of distress and pain may need to be humanely killed.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. PreparationsApproximately 24 hours before testing, fur should be removed, by clipping or shaving, from the dorsal area of the trunk of the animal.When clipping or shaving the fur, care should be taken to avoid abrading the skin. Only animals with healthy intact skin should be used.Some strains of rabbit have dense islets of hair which are more prominent at certain times of the year. Test substances should not be applied to these zones of dense hair growth.When testing solids (which may be pulverized if considered necessary) the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle, to ensure good contact with the skin. When vehicles are used, the influence of the vehicle on irritation of skin by the test substance should be taken into account. Liquid test substances are generally used undiluted.1.6.2. Test Conditions1.6.2.1. Experimental AnimalsAlthough several mammalian species may be used, the albino rabbit is the preferred species.1.6.2.2. Number of AnimalsIf it is suspected from in vitro screening results or other considerations that the substance might produce necrosis (i.e. be corrosive) a single-animal test should be considered. If the results of this test do not indicate corrosivity, the test should be completed using at least two additional animals.For the complete test, at least three healthy adult animals are used. Separate animals are not required for an untreated control group. Additional animals may be required to clarify equivocal responses.1.6.2.3. Dose LevelUnless there are contra-indications 0,5 ml of liquid or 0,5 g of solid or semi-solid is applied to the test site. Adjacent areas of untreated skin of each animal serve as controls for the test.1.6.2.4. Observation PeriodThe duration of the observation period should not be fixed rigidly. It should be sufficient to evaluate fully the reversibility or irreversibility of the effects observed, but need not normally exceed 14 days after application.1.6.3. ProcedureAnimals should be caged individually. The test substance should be applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which is held in place with non-irritating tape. In the case of liquids or some pastes it may be necessary to apply the test substance to the gauze patch and then apply that to the skin. The patch should be loosely held in contact with the skin by means of a suitable occlusive or semi-occlusive dressing for the duration of the exposure period. Access by the animal to the patch and resultant ingestion/inhalation of the test substance should be prevented.At the end of the exposure period, residual test substance should be removed, where practicable, using water or an appropriate solvent, without altering the existing response or the integrity of the epidermis.Exposure duration normally is four hours.If it is suspected that the substance might produce necrosis (i.e. be corrosive), the duration of exposure should be reduced (e.g. to one hour or three minutes). Such testing may also employ a single animal in the first instance and, if not precluded by the acute dermal toxicity of the test compound, three patches may be applied simultaneously to this animal. The first patch is removed after three minutes. If no serious skin reaction is observed, the second patch is removed after one hour. If the observations at this stage indicate that a four-hour exposure is necessary and can be humanely conducted, the third patch is removed after four hours and the responses are graded. In this case (i.e. when a four-hour exposure has been possible), the test should then be completed using at least two additional animals, unless it is not considered humane to do so (e.g. if necrosis is observed following the four hour exposure).If a serious skin reaction (e.g. necrosis) is observed at either three minutes or one hour, the test is immediately terminated.Longer exposures may be indicated under certain conditions, e.g. expected pattern of human use and exposure.1.6.3.1. Observation and GradingAnimals should be observed for signs of erythema and oedema and the response graded at 60 minutes, and then at 24, 48 and 72 hours after patch removal. Dermal irritation is graded and recorded according to the system in table 1. Further observations may be needed if reversibility has not been fully established within 72 hours. In addition to the observation of irritation, any serious lesions such as corrosion (irreversible destruction of skin tissue) and other toxic effects should be fully described.Techniques such as histopathological examination or measurement of skin-fold thickness may be used to clarify doubtful reactions or responses masked by staining of the skin by test substance.2. DATAData should be summarized in tabular form, showing for each individual animal the irritation gradings for erythema and oedema throughout the observation period. Any serious lesions, a description of the degree and nature of irritation, reversibility or corrosion and any other toxic effect observed should be recorded.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- species, strain, source, environmental conditions, diet, etc.;- test conditions (including the relevant physicochemical properties of the chemical, the technique of skin preparation and cleansing, and the type of dressing: occlusive or semi-occlusive);- tabulation of irritation response data for each individual animal for each observation time period (e.g. 1, 24, 48 and 72 hours, etc., after patch removal);- description of any serious lesions observed, including corrosivity;- description of the degree and nature of irritation observed and any histopathological findings;- description of any toxic effects other than dermal irritation;- discussion of the results;- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).AppendixB.5. ACUTE TOXICITY (EYE IRRITATION)1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSee General Introduction Part B (B).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODInitial ConsiderationsCareful consideration needs to be given to all the available information on a substance to minimize the testing of substances under conditions that are likely to produce severe reactions. The following information may be useful in this regard.i) Physicochemical properties and chemical reactivity. Strongly acidic or alkaline substances which, for example, can be expected to result in a pH in the eye of 2 or less, or 11,5 or greater, may not require testing if severe lesions can be expected. Alkaline or acidic reserve should also be taken into account.ii) Results from well-validated alternative studies; materials which have been shown to have potential corrosive or severe irritant properties should not be further tested for eye irritation, it being presumed that such substances will produce severe effects on the eyes in a test using this method.iii) Results from skin irritation studies. Materials which have demonstrated definite corrosive or severe skin irritancy in a dermal irritation study should not be further tested for eye irritancy, it being presumed that such substances might produce severe effects on the eyes.The substance to be tested is applied in a single dose to one of the eyes in each of several experimental animals; the untreated eye is used to provide control information. The degree of irritation is evaluated and graded at specific intervals and is further described to provide a complete evaluation of the effects. The duration of the observations should be sufficient to evaluate fully reversibility or irreversibility of the effects observed.Animals showing severe and enduring signs of distress and pain may need to be humanely killed.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. PreparationsBoth eyes of each experimental animal provisionally selected for testing should be examined within 24 hours before testing starts. Animals showing eye irritation, ocular defects or pre-existing corneal injury should be not used.1.6.2. Test Conditions1.6.2.1. Experimental AnimalsAlthough a variety of experimental animals have been used it is recommended that testing be performed using healthy adult albino rabbits.1.6.2.2. Number of AnimalsA single-animal test should be considered if marked effects are anticipated. If the results of this test in one rabbit suggest the substance to be severely irritant (reversible effect) or corrosive (irreversible effect) to the eye using the procedure described, further testing for ocular irritancy in subsequent animals may not need to be carried out. Occasionally, further testing in additional animals may be appropriate to investigate specific aspects.In cases other than a single-animal test at least 3 animals should be used. Additional animals may be required to clarify equivocal responses.1.6.2.3. Dose LevelFor testing liquids, a dose of 0,1 ml is used. In testing solids, pastes, and particulate substances, the amount used should have a volume of 0,1 ml, or weigh approximately 0,1 g (the weight must always be recorded). If the test material is solid or granular it should be ground to a fine dust. The volume of particulates should be measured after gently compacting them, e.g. by tapping the measuring container.For substances contained in pump sprays or pressurized aerosol containers the liquid should be expelled and 0,1 ml collected and instilled into the eye as described for liquids.1.6.2.4. Observation PeriodThe duration of the observation period should not be rigidly fixed. It should be sufficient to evaluate the reversibility or irreversibility of the effects observed, but normally need not exceed 21 days after instillation.1.6.3. ProcedureAnimals should be caged individually. The test substance should be placed in the conjunctival sac of one eye of each animal after gently pulling the lower lid away from the eyeball. The lids are then gently held together for about one second to prevent loss of the material. The other eye, which remains untreated, serves as a control.If it is thought that the substance could cause unreasonable pain, a local anaesthetic may be used prior to instillation of the test substance. The type, concentration, and the time of application of the local anaesthetic should be carefully selected to ensure that no significant differences in reaction to the test substance will result from its use. The control eye should be similarly anaesthetized.The eyes of the test animals should not be washed out for 24 hours following instillation of the test substance. At 24 hours a washout may be used if considered appropriate.For some substances shown to be irritating by this test, additional tests using rabbits with eyes washed soon after instillation of the substance may be indicated. In these cases it is recommended that three rabbits be used. Half a minute after instillation the eyes of the rabbits are washed for half a minute using a volume and velocity of flow which will not cause injury.1.6.3.1. Observation and GradingThe eyes should be examined at 1, 24, 48 and 72 hours. If there is no evidence of ocular lesions at 72 hours the study may be ended.Extended observation may be necessary if there is persistent corneal involvement or other ocular irritation in order to determine the progress of the lesions and their reversibility or irreversibility. In addition to the observations of the cornea, iris and conjunctiva, any other lesions which are noted should be recorded and reported. The grades of ocular reaction (table) should be recorded at each examination. (The grading of ocular responses is subject to various interpretations. To assist testing laboratories and those involved in making and interpreting the observations an illustrated guide to eye irritation may be used.)Examination of reactions can be facilitated by use of a binocular loupe, hand slit-lamp, biomicroscope, or other suitable device. After recording the observations at 24 hours, the eyes of any or all rabbits may be further examined with the aid of fluorescein.2. DATAData should be summarized in tabular form, showing for each individual animal the irritation grades at the designated observation time. A description of the degree and nature of irritation, the presence of serious lesions and any effects other than ocular which were observed, shall be reported.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- animal data (species, strain, source, environmental conditions, diet, etc.);- test conditions (including relevant physicochemical properties of the test substance);- tabulation of irritant/corrosive response data for each individual animal at each observation time point (e.g. 1, 24, 48 and 72 hours);- description of any serious lesions observed;- narrative description of the degree and nature of irritation or corrosion observed, including the area of the cornea involved, and the reversibility;- description of the method used to grade the irritation at 1, 24, 48 and 72 hours (e.g. hand slit-lamp, biomicroscope, fluorescein);- description of any non-ocular topical effects noted;- discussion of the results;- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).AppendixB.6. SKIN SENSITIZATION1. METHOD1.1. INTRODUCTIONRemarks:The sensitivity and ability of tests to detect potential human skin sensitizers are considered important in a classification system for toxicity relevant to public health.There is no single test method which will adequately identify all substances with a potential for sensitizing human skin and which is relevant for all substances.Factors such as the physical characteristics of a substance, including its ability to penetrate the skin, must be considered in the selection of a test.Tests using guinea-pigs can be subdivided into the adjuvant-type tests, in which an allergic state is potentiated by dissolving or suspending the test substance in Freunds Complete Adjuvant (FCA), and the non-adjuvant tests.Adjuvant-type tests are likely to be more accurate in predicting a probable skin sensitizing effect of a substance in humans than those methods not employing Freunds Complete Adjuvant and are thus the preferred methods.The Guinea-Pig Maximization Test (GPMT) is a widely used adjuvant-type test. Although several other methods can be used to detect the potential of a substance to provoke skin sensitization reaction, the GPMT is considered to be the preferred adjuvant technique.With many chemical classes, non-adjuvant tests (the preferred one being the Buehler test) are considered to be less sensitive.In certain cases there may be good reasons for choosing the Buehler test involving topical application rather than the intradermal injection used in the Guinea-Pig Maximization Test. Scientific justification should be given when the Buehler test is used.The Guinea-Pig Maximization Test (GPMT) and the Buehler test are described in this method. Other methods may be used provided that they are well-validated and scientific justification is given.Regardless of the methods used, the sensitivity of the strain of guinea-pig being used for skin sensitization testing must be checked at regular intervals (six months) using a known mild to moderate sensitizer and a satisfactory number of positive responses obtained.See also General Introduction Part B (A).1.2. DEFINITIONSee General Introduction Part B (B).1.3. REFERENCE SUBSTANCESThe following substances, diluted as necessary, are recommended, as well as any other sensitizing substance known either from the literature or which belongs to the group of the substance being tested.- p-phenylenediamineCAS N  106-50-3- 2,4-dinitrochlorobenzeneCAS N  97-00-7- potassium dichromateCAS N  7778-50-9- neomycin sulphateCAS N  1405-10-3- nickel sulphateCAS N  7786-81-41.4. PRINCIPLE OF THE TEST METHODSFollowing initial exposure to a test substance (the 'induction` period) the animals are subjected approximately two weeks after the last induction exposure to a 'challenge` exposure to the test substance in order to establish if a hypersensitive state has been induced. Sensitization is determined by examining the skin reaction to the challenge exposure.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHODS1.6.1. Guinea-Pig Maximization Test (GPMT)1.6.1.1. PreparationsHealthy young albino guinea-pigs are randomized and assigned to the treatment and control groups. Prior to dosing, the hair is removed, by clipping or shaving, from the shoulder region. Care should be taken to avoid damaging the skin.1.6.1.2. Test conditions1.6.1.2.1. Experimental AnimalsCommonly used laboratory strains of albino guinea-pigs are used, weighing less than 500 g.1.6.1.2.2. Number and SexMale and/or female animals can be used. If females are used, they should be nulliparous and non-pregnant. A minimum of 10 animals is used in the treated group and at least 5 in the control group. The use of fewer animals must be justified. In the case of equivocal results, histopathological examination may help to decide if the test should be repeated using another set of animals.When it is not possible to conclude definitively that the test substance is or is not a sensitizer, testing in additional animals to give a total of at least 20 test and 10 control animals is recommended.1.6.1.2.3. Dose levelsThe concentration of the test substance is adjusted to a level that produces some evidence of skin irritation, but that is well tolerated by the animals in each induction stage.The challenge concentration should be the maximum which produces no evidence of skin irritation in non-sensitized animals.These concentrations can be determined by a small scale (two to three animals) pilot study.1.6.1.2.4. Observation periodDuring the induction period observations are carried out to check for possible irritant effects. After the challenge exposure, skin reactions are recorded 24 and 48 hours after the removal of the patch.1.6.1.3. ProcedureThe animals are weighed before the test commences and at the end of the test. The shoulder region is cleared of hair. There are two stages in the procedure:1.6.1.3.1. InductionDay 0 - treated groupThe following pairs of intradermal injections, each of 0,1 ml, are given in the shoulder region so that one of each pair lies on each side of the midline:Injection 1:0,1 ml of Freunds Complete Adjuvant (FCA) mixed with water or physiological saline 1:1,Injection 2:0,1 ml of test substance, when necessary in an appropriate vehicle,Injection 3:0,1 ml of test substance in FCA.In injection 3, water soluble substances are dissolved in 0,05 ml water and 0,05 ml undiluted FCA. If liposoluble or insoluble substances are to be tested, they are mixed with undiluted FCA.In injection 3, the final concentration of test substance shall be equal to that in injection 2.Injections 1 and 2 are given close to each other and nearest the head, while 3 is towards the caudal part of the test area.Day 0 - control groupThe following pairs of intradermal injections are given in the same sites as above:Injection 1:0,1 ml of Freunds Complete Adjuvant (FCA) mixed with water or physiological saline 1:1,Injection 2:0,1 ml of vehicle alone,Injection 3:0,1 ml of vehicle in FCA.Day 6 - Control and treated groupsIf the substance is not a skin irritant, the test area, after clipping and/or shaving, is painted with 0,5 ml of 10 % sodium lauryl sulphate in vaseline, in order to create a local irritation.Day 7 - treated groupThe test area is again cleared of hair. The test substance in a suitable vehicle (the choice of the vehicle should be justified; solids are finely pulverised and incorporated in a suitable vehicle; liquids if appropriate can be applied directly) is spread over a filter paper (2 × 4 cm) and applied to the test area and held in contact by an occlusive dressing for 48 hours.Day 7 - control groupThe test area is again cleared of hair. The vehicle only is applied in a similar manner to the test area and held in contact by an occlusive dressing for 48 hours.1.6.1.3.2. ChallengeDay 21The flanks of treated and control animals are cleared of hair. A patch or chamber containing the test substance is applied to one flank of treated animals and a patch or chamber with vehicle only to the other flank.The patches are held in contact by an occlusive dressing for 24 hours.The control group is exposed in an identical manner.Days 23 and 24- 21 hours after removing the patch the challenge area is cleaned and cleared of hair if necessary,- three hours later (at 48 hours from the start of challenge application) the skin reaction is observed and recorded,- 24 hours after this observation a second observation (72 hours) is made and recorded.To clarify the results obtained in the first challenge, a second challenge, if necessary with a new vehicle control group, should be considered approximately one week after the first one.1.6.1.3.3. Observation and GradingAll skin reactions and any unusual findings resulting from induction and challenge procedures should be recorded and reported.Techniques such as histopathological examination or measurement of skin-fold thickness may be used to clarify doubtful reactions or responses masked by staining of the skin by test substance.1.6.2. Buehler test1.6.2.1. PreparationsHealthy young albino guinea-pigs are randomized and assigned to the treatment and control groups. Prior to dosing, the hair is removed, by clipping and/or shaving, from one flank. Care should be taken to avoid damaging the skin.1.6.2.2. Test conditions1.6.2.2.1. Experimental animalsCommonly used laboratory strains of albino guinea-pigs are used, weighing less than 500 g.1.6.2.2.2. Number and sexMale and/or female animals can be used. If females are used, they should be nulliparous and non-pregnant. At least 20 animals are used in the treated group and at least 10 in the control group. The use of fewer animals must be justified. In the case of equivocal results, histopathological examination may help to decide if the test should be repeated using another set of animals.1.6.2.2.3. Dose levelsFor each induction stage, the concentration of test substance is adjusted to the highest level that can be well tolerated systemically and which, for irritant substances, produces mild to moderate irritation in the majority of test animals. The challenge concentration should be the maximum which produces no evidence of skin irritation in non-sensitised animals. These concentrations can be determined by a small scale (two to three animals) study.1.6.2.2.4. Observation periodDuring the induction period skin observations are carried out to check for irritant effects. After the challenge exposure, skin reactions are recorded 24 and 48 hours after the removal of the patch, i.e. 30 and 54 hours after the beginning of application.1.6.2.3. ProcedureThe animals are weighed before the test commences and at the end of the test.There are two stages in the procedure:1.6.2.3.1. InductionDay 0 - treated groupOne flank is cleared of hair. 0,5 ml of the test substance in a suitable vehicle (the choice of the vehicle should be justified; liquids if appropriate can be applied directly) is spread over a cotton pad. It is applied to the test area and held in contact with the skin by an occlusive patch or chamber and a suitable dressing for 6 hours.Day 0 - control groupOne flank is cleared of hair. The vehicle only is applied in a similar manner to the test area. It is held in contact with the skin by an occlusive patch or chamber and a suitable dressing for 6 hours.Days 7 and 14The same application as on Day 0 is carried out on the same test area (cleared of hair if necessary) on Day 7, and on Day 14.1.6.2.3.2. ChallengeDay 28The other flank of treated and control animals is cleared of hair. An occlusive patch or chamber containing 0,5 ml of the test substance is applied, at the maximum non-irritant concentration, to the posterior of the flank of treated animals. An occlusive patch or chamber with vehicle only is also applied to the anterior of the flank.The occlusive patches are held in contact by a suitable dressing for 6 hours.The control group is exposed in an identical manner.Days 29 and 30- 21 hours after removing the patch the challenge area is cleaned and cleared of hair if necessary,- three hours later (at 30 hours from the start of challenge application) the skin reaction is observed and recorded,- 24 hours after this observation a second observation (54 hours) is made and recorded.1.6.2.3.3. Observation and gradingAll skin reactions and any unusual findings resulting from induction and challenge procedures should be recorded and reported.Techniques such as histopathological examination or measurement of skin-fold thickness may be used to clarify doubtful reactions or responses masked by staining of the skin by test substance.2. DATA (GPMT and Buehler test)Data should be summarized in tabular form, showing for each animal the skin reactions at each observation.3. REPORTING (GPMT and Buehler test)3.1. TEST REPORT (GPMT and Buehler test)The test report shall, if possible, include the following information:- strain of guinea-pig used;- test conditions, vehicle and test substance concentrations used for inductions and challenges;- number, age and sex of animals;- individual weights of animals at the start and at the conclusion of the test;- each observation made on each animal including grading system if one is used;- discussion of the results;- interpretation of the results.3.2. EVALUATION AND INTERPRETATION (GPMT and Buehler test)See General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.7. REPEATED DOSE (28 DAYS) TOXICITY (ORAL)1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSSee General Introduction Part B (B).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODThe test substance is administered orally, daily, in graduated doses to several groups of experimental animals, one dose per group for a period of 28 days. During the period of administration the animals are observed daily to detect signs of toxicity. Animals which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. PreparationsThe animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. Before the test, healthy young animals are randomized and assigned to the treatment groups. The test substance may be administered in the diet, by gavage, in capsules, or in the drinking water. All animals should be dosed by the same method during the entire experimental period. If a vehicle or other additives are used to facilitate dosing, they should be known not to produce toxic effects. Historical data can be used if appropriate.1.6.2. Test conditions1.6.2.1. Experimental animalsUnless there are contra-indications, the preferred species is the rat. Commonly used laboratory strains of young healthy animals should be employed and dosing should begin ideally before the rats are six weeks old, and in any case not more than eight weeks old.At the commencement of the study, the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.1.6.2.2. Number and sexAt least 10 animals (five female and five male) should be used at each dose level. The females should be nulliparous and non-pregnant. If interim sacrifices are planned, the number of animals should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 10 animals (five animals per sex) may be treated with the high dose level for 28 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 14 days post-treatment. A satellite group of 10 control animals (five animals per sex) is also used.1.6.2.3. Dose LevelsAt least three dose levels and a control should be used. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test-group subjects. Where a vehicle is used to facilitate dosing, the controls should be dosed with the vehicle in the same way as the treated groups, and receive the same amount of vehicle as that received by the highest dose level group. The highest dose level should result in toxic effects but produce no, or few, fatalities. The lowest dose level should not produce any evidence of toxicity. Where there is a usable estimation of human exposure, the lowest level should exceed this. Ideally, the middle dose level should produce minimal observable toxic effects. If more than one intermediate dose is used the dose levels should be spaced to produce a gradation of toxic effects. In the low and intermediate groups and in the controls, the incidence of any fatalities should be low in order to permit a meaningful evaluation of results.When the test substance is administered in the diet, either a constant dietary concentration (ppm or mg/kg of food) or a constant dose level in terms of the animal's body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day and dose levels should be adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body weight.1.6.2.4. Limit TestIf a 28-day study conducted in accordance with the method detailed below, at one dose level of 1 000 mg/kg body weight/day or a higher dose level related to possible human exposure where this is known, produces no evidence of toxic effects, further testing may not be considered necessary. For substances of low toxicity it is important to ensure that when administered in the diet the quantities and other properties of the test substance involved do not interfere with normal nutritional requirements.1.6.2.5 Observation PeriodAll the animals should be observed daily and signs of toxicity recorded including their time of onset, degree and duration. The time of death and the time at which signs of toxicity appear and disappear should be recorded.1.6.3. ProcedureThe animals are dosed with the test substance ideally on seven days per week for a period of 28 days. Animals in any satellite group scheduled for follow-up observations should be kept for a further 14 days without treatment to detect recovery from, or persistence of, toxic effects.Observations should include changes in skin and fur, eyes and mucous membranes as well as respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Weekly measurements should be made of food consumption (and water consumption when the test substance is administered in the drinking water) and the animals should be weighed weekly.Regular observation of the animals is necessary to ensure that animals are as far as possible not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the study period, all survivors in the non-satellite treatment groups are necropsied. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.The following examination shall be made at the end of the test period for all animals including controls:1. haematology including at least haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count, and a measure of clotting potential;2. clinical blood biochemistry including at least one parameter of liver and kidney function: alanine aminotransferase (formerly known as glutamic pyruvic transaminase), serum aspartate aminotransferase (formerly known as glutamic oxaloacetic transaminase), urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein.Other determinations which may be necessary for an adequate toxicological evaluation include calcium, phosphorus, chloride, sodium, potassium, fasting glucose, analysis of lipids, hormones, acid/base balance, methaemoglobin, cholinesterase activity.Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.1.6.3.1. Gross NecropsyAll animals in the study should be subjected to a full gross necropsy. At least the liver, kidney, adrenals, and testes should be weighed wet as soon as possible after dissection to avoid drying. Organs and tissues (liver, kidney, spleen, testes, adrenals, heart, and any organs showing gross lesions or changes in size) should be preserved in a suitable medium for possible future histopathological examination.1.6.3.2. Histopathological ExaminationIn the high-dose group and in the control group, histological examination should be performed on preserved organs and tissues. Organs and tissues showing defects attributable to the test substance at the highest dosage level should be examined in all lower-dosage groups. Animals in any satellite group should be examined histologically with particular emphasis on those organs and tissues identified as showing effects in the other treated groups.2. DATAData should be summarized in tabular form, showing for each test group the number of animals at the start of the test and the number of animals displaying each type of lesion.All observed results should be evaluated by an appropriate statistical method. Any recognized statistical method may be used.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- species, strain, source, environmental conditions, diet, etc.;- test conditions;- dose levels (including vehicle, if used) and concentrations;- toxic response data by sex and dose;- no-effect level, when possible;- time of death during the study or whether animals survived to termination;- toxic or other effects;- the time of observation of each abnormal sign and its subsequent course;- food and body-weight data;- haematological tests employed and all results;- clinical biochemistry tests employed and all results;- necropsy findings;- a detailed description of all histopathological findings;- statistical treatment of results where appropriate;- discussion of the results;- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.8. REPEATED DOSE (28 DAYS) TOXICITY (INHALATION)1. METHOD1.1. INTRODUCTIONIt is useful to have preliminary information on the particle size distribution, the vapour pressure, the melting point, the boiling point, the flash point and explosivity (if applicable) of the substance.See also General Introduction Part B (A).1.2. DEFINITIONSee General Introduction Part B (B).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODSeveral groups of experimental animals are exposed daily for a defined period to the test substance in graduated concentrations, one concentration being used per group, for a period of 28 days. Where a vehicle is used to help generate an appropriate concentration of the test substance in the atmosphere, a vehicle control group should be used. During the period of administration the animals are observed daily to detect signs of toxicity. Animals which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. PreparationsThe animals are kept under the experimental housing and feeding conditions for at least five days prior to the experiment. Before the test, healthy young animals are randomized and assigned to the required number of groups. Where necessary, a suitable vehicle may be added to the test substance to help generate an appropriate concentration of the substance in the atmosphere. If a vehicle or other additive is used to facilitate dosing, it should be known not to produce toxic effects. Historical data can be used if appropriate.1.6.2. Test Conditions1.6.2.1. Experimental AnimalsUnless there are contra-indications, the rat is the preferred species. Commonly used laboratory strains of young healthy animals should be employed.At the commencement of the study the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.1.6.2.2. Number and SexAt least 10 animals (five female and five male) should be used for each test group. The females should be nulliparous and non-pregnant. If interim sacrifices are planned, the numbers should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 10 animals (five animals per sex) may be treated with the high concentration level for 28 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 14 days post-treatment. A satellite group of 10 control animals (five animals per sex) is also used.1.6.2.3. Exposure ConcentrationAt least three concentrations are required, with a control or a vehicle control (corresponding to the concentration of vehicle at the highest level) if a vehicle is used. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test-group animals. The highest concentration should result in toxic effects but no, or few, fatalities. The lowest concentration should not produce any evidence of toxicity. Where there is a usable estimation of human exposure, the lowest concentration should exceed this. Ideally, the intermediate concentration should produce minimal observable toxic effects. If more than one intermediate concentration is used the concentrations should be spaced to produce a gradation of toxic effects. In the low and intermediate groups and in the controls, the incidence of fatalities should be low to permit a meaningful evaluation of the results.1.6.2.4. Exposure TimeThe duration of daily exposure should be six hours but other periods may be needed to meet specific requirements.1.6.2.5. EquipmentThe animals should be tested in inhalation equipment designed to sustain a dynamic airflow of at least 12 air changes per hour to ensure an adequate oxygen content and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure by inhalation of the test substance. As a general rule to ensure stability of a chamber atmosphere the total 'volume` of the test animals should not exceed 5 % of the volume of the test chamber. Oro-nasal, head only, or individual whole body chamber exposure may be used; the first two will minimize uptake by other routes.1.6.2.6. Observation PeriodThe experimental animals should be observed daily for signs of toxicity during the entire treatment and recovery period. The time of death and the time at which signs of toxicity appear and disappear should be recorded.1.6.3. ProcedureThe animals are exposed to the test substance daily, five to seven days per week, for a period of 28 days. Animals in any satellite groups scheduled for follow-up observations should be kept for a further 14 days without treatment to detect recovery from, or persistence of, toxic effects. The temperature at which the test is performed should be maintained at 22 ± 3  C.Ideally, the relative humidity should be maintained between 30 and 70 %, but in certain instances (e.g. tests of some aerosols) this may not be practicable. Maintenance of a slight negative pressure inside the chamber (&le; 5 mm of water) will prevent leakage of the test substance into the surrounding area. Food and water should be withheld during exposure.A dynamic inhalation system with a suitable analytical concentration control system should be used. To establish suitable exposure concentrations a trial test is recommended. The airflow should be adjusted to ensure that conditions throughout the exposure chamber are homogeneous. The system should ensure that stable exposure conditions are achieved as rapidly as possible.Measurements or monitoring should be made:(a) of the rate of airflow (continuously);(b) of the actual concentration of the test substance measured in the breathing zone. During the daily exposure period the concentration should not vary by more than ± 15 % of the mean value. However, in the case of some aerosols, this level of control may not be achievable and a wider range would then be acceptable. During the total duration of the study, the day-to-day concentrations should be held as constant as practicable. For aerosols, at least one particle size analysis should be performed per test group weekly;(c) of temperature and humidity, continuously if possible.During and following exposure observations are made and recorded systematically; individual records should be maintained for each animal. All the animals should be observed daily and signs of toxicity recorded including the time of onset, their degree and duration. Observations should include changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Measurements should be made weekly of the animals' weight. It is also recommended that food consumption is measured weekly. Regular observation of the animals is necessary to ensure that animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the study period, all survivors in the non-satellite treatment groups are necropsied. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.The following examinations shall be made at the end of the test on all animals including the controls:(i) haematology, including at least haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count and a measure of clotting potential;(ii) clinical blood biochemistry including at least one parameter of liver and kidney function: serum alanine aminotransferase (formerly known as glutamic pyruvic transaminase), serum aspartate aminotransferase (formerly known as glutamic oxaloacetic transaminase), urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measurements;Other determinations which may be necessary for an adequate toxicological evaluation include calcium, phosphorus, chloride, sodium, potassium, fasting glucose analysis of lipids, hormones, acid/base balance, methaemoglobin and cholinesterase activity.Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed toxic effects.1.6.3.1. Gross NecropsyAll animals in the study should be subjected to a full gross necropsy. At least the liver, kidneys, adrenals, lungs, and testes should be weighed wet as soon as possible after dissection to avoid drying. Organs and tissues (the respiratory tract, liver, kidneys, spleen, testes, adrenals, heart, and any organs showing gross lesions or changes in size) should be preserved in a suitable medium for possible future histopathological examination. The lungs should be removed intact, weighed and treated with a suitable fixative to ensure that lung structure is maintained.1.6.3.2. Histopathological ExaminationIn the high-concentration group and in the control(s), histological examination should be performed on preserved organs and tissues. Organs and tissues showing defects attributable to the test substance at the highest dosage level should be examined in all lower-dosage groups. Animals in any satellite groups should be examined histologically with particular emphasis on those organs and tissues identified as showing effects in the other treated groups.2. DATAData should be summarized in tabular form, showing for each test group the number of animals at the start of the test and the number of animals displaying each type of lesion.All observed results should be evaluated by an appropriate statistical method. Any recognized statistical method may be used.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- species, strain, source, environmental conditions, diet, etc.;- test conditions:Description of exposure apparatus including design, type, dimensions, source of air, system for generating aerosols, method of conditioning air, treatment of exhaust air and the method of housing animals in a test chamber when this is used. The equipment for measuring temperature, humidity and, where appropriate, stability of aerosol concentrations or particle size distribution, should be described.Exposure data:These should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and shall, if possible, include:a) airflow rates through the inhalation equipment;b) temperature and humidity of air;c) nominal concentrations (total amount of test substance fed into the inhalation equipment divided by the volume of air);d) nature of vehicle, if used;e) actual concentrations in test breathing zone;f) the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD);- toxic response data by sex and concentration;- time of death during the study or whether animals survived to termination;- description of toxic or other effects; no-effect level;- the time of observation of each abnormal sign and its subsequent course;- food and body-weight data;- haematological tests employed and results;- clinical biochemistry tests employed and results;- necropsy findings;- a detailed description of all histopathological findings;- a statistical treatment of results where possible;- discussion of the results;- interpretation of results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.9. REPEATED DOSE (28 DAYS) TOXICITY (DERMAL)1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSSee General Introduction Part B (B).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODThe test substance is applied daily to the skin in graduated doses to several groups of experimental animals, one dose per group, for a period of 28 days. During the period of application, the animals are observed daily to detect signs of toxicity. Animals which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. PreparationsThe animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. Before the test, healthy young animals are randomized and assigned to the treatment and control groups. Shortly before testing, fur is clipped from the dorsal area of the trunk of the test animals. Shaving may be employed but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care must be taken to avoid abrading the skin. Not less than 10 % 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 covering. When testing solids, which may be pulverized if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. Liquid test substances are generally used undiluted. Daily application on a five to seven-day per week basis is used.1.6.2. Test Conditions1.6.2.1. Experimental AnimalsThe adult rat, rabbit or guinea-pig may be used. Other species may be used but their use would require justification.At the commencement of the study, the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.1.6.2.2. Number and SexAt least 10 animals (five female and five male) with healthy skin should be used at each dose level. The females should be nulliparous and non-pregnant. If interim sacrifices are planned, the numbers should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 10 animals (five animals per sex) may be treated with the high dose level for 28 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 14 days post-treatment. A satellite group of 10 control animals (five animals per sex) is also used.1.6.2.3. Dose levelsAt least three dose levels are required with a control or a vehicle control if a vehicle is used. The exposure period should be at least six hours per day. The application of the test substance should be made at similar times each day, and adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body-weight. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. Where a vehicle is used to facilitate dosing, the vehicle control group should be dosed in the same way as the treated groups, and receive the same amount as that received by the highest dose level group. The highest dose level should result in toxic effects but produce no, or few, fatalities. The lowest dose level should not produce any evidence or toxicity. Where there is a usable estimation of human exposure, the lowest level should exceed this. Ideally, the intermediate dose level should produce minimal observable toxic effects. If more than one intermediate dose is used the dose levels should be spaced to produce a gradation of toxic effects. In the low and intermediate groups and in the controls, the incidence of fatalities should be low in order to permit a meaningful evaluation of the results.If application of the test substance produces severe skin irritation, the concentrations should be reduced and this may result in a reduction in, or absence of, other toxic effects at the high dose level. Moreover if the skin has been badly damaged it may be necessary to terminate the study and undertake a new study at lower concentrations.1.6.2.4. Limit TestIf a preliminary study at a dose level of 1 000 mg/kg, or a higher dose level related to possible human exposure where this is known, produces no toxic effects, further testing may not be considered necessary.1.6.2.5. Observation PeriodThe experimental animals should be observed daily for signs of toxicity. The time of death and the time at which signs of toxicity appear and disappear should be recorded.1.6.3. ProcedureAnimals should be caged individually. The animals are treated with the test substance, ideally on seven days per week, for a period of 28 days. Animals in any satellite groups scheduled for follow-up observations should be kept for a further 14 days without treatment to detect recovery from or persistence of toxic effects. Exposure time should be at least six hours per day.The test substance should be applied uniformly over an area which is approximately 10 % of the total body surface area. With highly toxic substances, the surface area covered may be less but as much of the area as possible should be covered with as thin and uniform a layer as possible.During exposure the test substance is held in contact with the skin with porous gauze dressing and non-irritating tape. 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 immobilization is not a recommended method. As an alternative a 'collar protective device` may be used.At the end of the exposure period, residual test substance should be removed, where practicable, using water or some other appropriate method of cleansing the skin.All the animals should be observed daily and signs of toxicity recorded including the time of onset, their degree and duration. Observations should include changes in skin and fur, eyes and mucous membranes as well as respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Measurements should be made weekly of the animals' weight. It is also recommended that food consumption is measured weekly. Regular observation of the animals is necessary to ensure that animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the study period, all survivors in the non-satellite treatment groups are necropsied. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.The following examinations shall be made at the end of the test on all animals including the controls:1) haematology, including at least haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count, and a measure of clotting potential;2) clinical blood biochemistry including at least one parameter of liver and kidney function: serum alanine aminotransferase (formerly known as glutamic pyruvic transaminase), serum aspartate aminotransferase (formerly known as glutamic oxaloacetic transaminase), urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein;Other determinations which may be necessary for an adequate toxicological evaluation include calcium, phosphorus, chloride, sodium, potassium, fasting glucose, analysis of lipids, hormones, acid/base balance, methaemoglobin and cholinesterase activity.Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.1.6.4. Gross NecropsyAll animals in the study should be subjected to a full gross necropsy. At least the liver, kidneys, adrenals, and testes should be weighed wet as soon as possible after dissection, to avoid drying. Organs and tissues, i.e. normal and treated skin, liver, kidney, spleen, testes, adrenals, heart, and target organs (that is those organs showing gross lesions or changes in size), should be preserved in a suitable medium for possible future histopathological examination.1.6.5. Histopathological ExaminationIn the high dose group and in the control group, histological examination should be performed on the preserved organs and tissues. Organs and tissues showing defects attributable to the test substance at the highest dosage level should be examined in all lower-dosage groups. Animals in the satellite group should be examined histologically with particular emphasis on those organs and tissues identified as showing effects in the other treated groups.2. DATAData should be summarized in tabular form, showing for each test group the number of animals at the start of the test and the number of animals displaying each type of lesion.All observed results should be evaluated by an appropriate statistical method. Any recognized statistical method may be used.3. REPORTING 3.1. TEST REPORTThe test report shall, if possible, include the following information:- animal data (species, strain, source, environmental conditions, diet, etc.);- test conditions (including the type of dressing: occlusive or not-occlusive);- dose levels (including vehicle, if used) and concentrations;- no-effect level, where possible;- toxic response data by sex and dose;- time of death during the study or whether animals survived to termination;- toxic or other effects;- the time of observation of each abnormal sign and its subsequent course;- food and body-weight data;- haematological tests employed and results;- clinical biochemistry tests employed and results;- necropsy findings;- a detailed description of all histopathological findings;- statistical treatment of results where possible;- discussion of the results;- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.10. MUTAGENICITY ('IN VITRO` MAMMALIAN CYTOGENETIC TEST)1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSee General Introduction Part B (C).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODThe in vitro cytogenetic test is a short-term mutagenicity test for the detection of structural chromosomal aberrations in cultured mammalian cells. Cultures of established cell lines as well as primary cell cultures may be used. After exposure to test chemicals with and without an appropriate metabolic activation system, cell cultures are treated with spindle inhibitors such as colchicine to accumulate cells in a metaphase-like stage of mitosis (c-metaphase). Cells are harvested at appropriate times and chromosome preparations are made. Preparations are stained and metaphase cells are analyzed for chromosomal abnormalities.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. Preparations1.6.1.1. CellsEstablished cell lines or cultures of primary cells are used, e.g. Chinese hamster cells and human lymphocytes. Test chemicals are prepared in culture medium or dissolved in appropriate vehicles prior to treatment of the cells.1.6.1.2. Metabolic activation systemCells should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction prepared from the livers of rodents treated with enzyme-inducing agents.1.6.2. Test conditionsNumber of cultures:At least duplicate cultures are used for each experimental point.Use of negative and positive control:Solvent (when the solvent is not the culture medium or water), liver enzyme activation mixture, liver enzyme activation mixture and solvent, and untreated controls are used as negative controls.In each experiment a positive control is included; when liver enzyme activation mixture is used to activate the test chemical, a compound known to require metabolic activation must be used as a positive control.Dose level:At least three doses of the test compound over at least a one-log dose range are employed. The highest dose should inhibit mitotic activity by approximately 50 % or exhibit some other indication of cytotoxicity. If not toxic, the test substance should be tested up to the solubility limit, or up to a maximum concentration of 5 mg/ml.Culture conditions:Appropriate culture medium, incubation conditions (e.g. temperature, culture vessels used, CO2 concentrations and humidity) are used.1.6.3. Procedure1.6.3.1. Preparation of culturesEstablished cell lines: Cells are generated from stock cultures (e.g. by trypsinization or by shaking off), seeded in culture vessels at appropriate density, and incubated at 37  C.Human lymphocytes: Heparinized whole blood is added to culture medium containing phytohaemagglutinin, fetal calf serum and antibiotics and incubated at 37  C.1.6.3.2. Treatment of the cultures with the test compound(i) Treatment without liver enzyme activation mixtureAll treatments shall when possible cover at least the period of one whole cell cycle and fixation schemes shall ensure the analysis of first post-treatment mitoses of cells treated at different stages in the cycle.When the treatment does not cover the length of one whole cell cycle, fixation times are chosen to sample cells that are in different stages of the cell cycle during the treatment i.e. G1, S and G2.The test chemical is added to cultures of established cell lines when they are in the exponential stage of growth. Human lymphocyte cultures are treated while they are in a semi-synchronous condition.(ii) Treatment with liver enzyme activation mixture For the treatment, the test compound in combination with the activation system should be present for as long as possible without exerting a toxic effect on the cells. If for toxicity reasons this treatment does not cover the length of a whole cell cycle, fixation times are chosen to sample cells that are in different stages of the cell cycle during the treatment, i.e. G1, S and G2.Harvesting cells:Cell cultures are treated with a spindle inhibitor for an appropriate time prior to harvesting. Each culture is harvested and processed separately for the preparation of chromosomes.At least two harvest times are needed. It is recommended that one is at approximately one cell cycle, and another later. This is to ensure that all stages of the cell cycle are covered and to allow for cell cycle delay.1.6.3.3. Chromosome preparationChromosome preparations involve hypotonic treatment of the cells, fixation, spreading on slides, and staining.Analysis:At least 100 well-spread metaphases per culture are analyzed for chromosomal aberrations. Slides are coded before analysis. In human lymphocytes only metaphases containing 46 centromeres are analyzed.In established cell lines only metaphases containing ± 2 centromeres of the modal number are analyzed.Additionally, the mitotic index, or some other indication of cytotoxicity when appropriate, should be assessed during the test for each dose level.2. DATAData are presented in a tabular form. Chromatid-type aberrations (gaps, breaks, interchanges), chromosome-type aberrations (e.g. gaps, breaks, minutes, rings, dicentrics, polycentrics) and the number of aberrant metaphases (including and excluding gaps) are listed separately for all treated and control cultures.The data are evaluated by appropriate statistical methods.Test results must be compared with concurrent negative controls.At least two independent experiments are conducted. However, if it can be scientifically justified, a single experiment may be sufficient. It is not necessary to perform the second one in an identical way to the initial experiment. Indeed, it may be preferable to alter certain test conditions in order to obtain more useful data.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- cells used;- test conditions: composition of medium, CO2 concentration, incubation temperature, incubation time, dose levels, treatment time, duration of treatment with and concentration of the spindle inhibitor used, type of liver enzyme activation mixture used, positive and negative controls;- number of cell cultures;- number of metaphases analyzed (data given separately for each culture);- mitotic index or other indication of cytotoxicity;- type and number of aberrations given separately for each treated and control culture, modal number of chromosomes in established cell lines used;- statistical evaluation;- discussion of the results;- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.11. MUTAGENICITY ('IN VIVO` MAMMALIAN BONE-MARROW CYTOGENETIC TEST, CHROMOSOMAL ANALYSIS)1. METHOD 1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSee General Introduction Part B (C).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODThis in vivo cytogenetic test is a short-term mutagenicity test for the detection of structural chromosomal aberrations. Chromosomal aberrations are generally evaluated in first post-treatment mitoses. With chemical mutagens, the majority of the induced aberrations are of the chromatid type.The method employs bone-marrow cells of mammals which are exposed to test chemicals by appropriate routes and are sacrificed at sequential intervals. Animals are further treated, prior to sacrifice, with a spindle inhibitor such as colchicine to accumulate cells in a metaphase-like stage of mitosis (c-metaphase). Air-dried chromosome preparations from the cells are made and stained and metaphases are analyzed microscopically for chromosomal aberrations.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. PreparationsTest chemicals are dissolved in normal saline. If insoluble, they are dissolved or suspended in appropriate vehicles.Freshly prepared solutions of the test compound are employed. If a vehicle is used to facilitate dosing, it must not interfere with the test compound or produce toxic effects.1.6.2. Test Conditions1.6.2.1. Experimental animalsRodent species, such as rats, mice or Chinese hamsters, are used. Healthy young adult animals are randomized and assigned to treatment and control groups.1.6.2.2. Number and SexAt least five female and five male animals per experimental and control group are employed. Thus, 10 animals would be sacrificed per time period per group if several test times after treatment are included in the experimental schedule.For the positive control group, a single sampling time is sufficient.1.6.2.3. Route of administrationTest compounds should generally be administered only once. Based on toxicological information a repeated treatment schedule can be employed. However, the repeated treatment schedule can only be applied if the test compound does not exhibit cytotoxic effects in bone-marrow. The usual routes of administration are oral and intraperitoneal injection. Other routes of administration may be appropriate.1.6.2.4. Use of negative and positive controlsA compound known to produce chromosomal aberrations in vivo is employed as a positive control and a negative (solvent) control group is also included in the design of each experiment.1.6.2.5. Dose LevelFor the base set, one dose of the test compound is used, the dose being the maximum tolerated dose or that producing some indication of cytotoxicity (e.g. partial inhibition of mitosis).For 'non-toxic` compounds, the maximum (limit) dose that needs to be investigated following single dose administration is 2 000 mg/kg body weight.If a repeated dose schedule is employed, the limit dose is 1 000 mg/kg body weight per day.Additional dose levels may be used where these are indicated by scientific reasons.If the test is being used as a method for verification at least two additional dose levels should be used.1.6.3. ProcedureThe test may be performed in two ways:(i) Animals are treated with the test compound once, at the highest tolerated dose. In the first instance, samples are taken at 24 hours after treatment. If the results are clearly positive at this stage, further sampling may not be necessary. However, if the results are negative or equivocal, since cell cycle kinetics can be influenced by the test chemical, one earlier and one later sampling interval, adequately spaced within the range of six to 48 hours, are applied.When additional dose levels are used, samples should be taken at the particularly sensitive intervals or, if that is not known, 24 hours after treatment.(ii) If pharmacokinetic and metabolic information indicate a repeated treatment schedule, repeated dosage can be employed and samples should be taken six and 24 hours after the last treatment.Bone-marrow preparation:Prior to sacrifice, animals are injected intraperitoneally with an appropriate dose of the spindle inhibitor to obtain an adequate number of cells in c-metaphase. Bone-marrow is obtained from both femora of freshly killed animals by rinsing with an isotonic solution. After appropriate hypotonic treatment the cells are fixed and then spread on slides. After air-drying the slides are stained.Analysis:Slides are coded before microscopic analysis. At least 50 well-spread metaphases with the complete number of centromeres are analyzed per animal for structural chromosomal aberrations. Additionally, the mitotic indexes may be established for each animal.2. DATAData are presented in a tabular form. Chromatid- and isochromatid-type aberrations (gaps, breaks, interchanges), and the mitotic indexes, where established, are listed separately for all treated and control animals. Mean numbers and standard deviations for each experimental and control group are also listed. The data are evaluated by appropriate statistical methods.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- species, strain and age of animals used;- number of animals for each sex in experimental and control groups;- test conditions: detailed description of treatment and sampling schedule, dose levels, duration of treatment with and concentration of the spindle inhibitor used;- number of metaphases analyzed per animal;- mitotic indexes, where established;- type and number of aberrations given separately for each treated and control animal;- signs of toxicity during the course of the study;- statistical evaluation;- discussion of the results;- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESee General Introduction Part B (E).B.12. MUTAGENICITY (MICRONUCLEUS TEST)1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSee General Introduction Part B (C).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODThe micronucleus test is a mammalian short-term in vivo test for the detection of chromosomal damage or damage of the mitotic apparatus by chemicals. The basis of this assay is an increase in micronuclei in the polychromatic erythrocytes of treated animals versus the controls.Micronuclei are formed from chromosomal fragments or whole chromosomes lagging in mitosis. When erythroblasts develop into erythrocytes, the main nucleus is expelled while the micronucleus may be maintained in the cytoplasm. Young polychromatic erythrocytes in the bone-marrow of laboratory mammals which were exposed to test substances by appropriate routes are used in this test. The bone-marrow is extracted and smear preparations are made and stained. Polychromatic erythrocytes are scored for micronuclei under the microscope and the ratio of polychromatic to normochromatic erythrocytes is established.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. PreparationsTest chemicals are dissolved in isotonic solution. If insoluble, they are dissolved or suspended in appropriate vehicles. If a vehicle is used, it must not interfere with the test compound or produce toxic effects. Normally, freshly prepared solutions of the test compound are employed.1.6.2. Test Conditions1.6.2.1. Experimental animalsMice are recommended, but other mammals may be used. Healthy young adult animals are randomized and assigned to treatment and control groups.1.6.2.2. Number and SexAt least five female and five male animals per experimental and control group are employed. Thus, 10 animals would be sacrificed per time period per group if several test times after treatment are included in the experimental schedule. For the positive control group, a single sampling time is sufficient.1.6.2.3. Route of administrationTest compounds should generally be administered only once. Based on toxicological information, a repeated treatment schedule can be employed. However, the repeated treatment schedule can only be applied if the test compound does not exhibit cytotoxic effects in bone-marrow. The usual routes of administration are oral and intraperitoneal injection. Other routes of administration may be appropriate.1.6.2.4. Use of negative and positive controlsBoth positive and negative (solvent) controls are to be used in each experiment.1.6.2.5. Dose LevelFor the base set one dose of the test compound is used, the dose being the maximum tolerated dose or that producing some indication of cytotoxicity, e.g. by a change in the ratio of polychromatic to normochromatic erythrocytes.For 'non-toxic` compounds, the maximum (limit) dose that needs to be investigated following single dose administration is 2 000 mg/kg body weight.If a repeated dose schedule is employed, the limit dose is 1 000 mg/kg body weight per day.Additional dose levels may be used where these are indicated by scientific reasons.If the test is being used as a method for verification at least two additional dose levels should be used.1.6.3. ProcedureThe test may be performed in two ways:(i) Animals are treated with the test compound once. Sampling times should coincide with the maximum response of the assay, which varies with the test compound. Therefore, samples of bone-marrow are taken at least twice starting not earlier than 12 hours after treatment, and not extending beyond 48 hours.When additional dose levels are used, samples should be taken at the maximum sensitive period, or, if that is not known, 24 hours after treatment.(ii) If pharmacokinetic and metabolic information indicate a repeated treatment schedule, repeated dosage can be employed and samples should be taken once, not earlier than 12 hours after the last treatment.Bone-marrow preparationBone-marrow is obtained from both femora of freshly killed animals by rinsing with fetal calf serum. The cells are sedimented by centrifugation and the supernatant is discarded. Drops of the homogeneous cell suspension are put on slides and spread as a smear. After air-drying the slides are stained.Analysis:Slides are coded before microscopic analysis. At least 1 000 polychromatic erythrocytes per animal are scored for the incidence of micronuclei.The ratio of normochromatic to polychromatic erythrocytes is determined for each animal by counting a total of 1 000 erythrocytes.2. DATAData are presented in a tabular form. Thus the number of polychromatic erythrocytes scored, the number of polychromatic erythrocytes with micronuclei, and the percent micronucleated cells are listed separately for each experimental and control animal, as well as the ratio of normochromatic to polychromatic erythrocytes. Mean numbers and standard deviations for each experimental and control group are also listed. The listed data are evaluated by appropriate statistical methods.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- species, strain and age of animals used;- number of animals of each sex in experimental and control groups;- test conditions: detailed description of treatment and sampling schedule, dose levels, toxicity data, negative and positive controls;- criteria for scoring micronuclei;- dose/effect relationship when possible;- signs of toxicity during the course of the study;- statistical evaluation;- discussion of the results;- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.13. MUTAGENICITY (ESCHERICHIA COLI - REVERSE MUTATION ASSAY)1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSee General Introduction Part B (C).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLES OF THE TEST METHODThe Escherichia coli tryptophan (trp) reversion system is a microbial assay which measures trp   . trp+ reversion by chemicals which cause base changes in the genome of the organism.Bacteria are exposed to test chemicals with and without metabolic activation. After a suitable period of incubation on minimal medium, revertant colonies are counted and compared to the number of spontaneous revertants in an untreated and/or solvent control culture.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHODThe following methods may be used to perform the assay: (1) the preincubation method; and (2) the direct incorporation method in which bacteria and test agent are mixed in overlay agar and poured over the surface of a selective agar plate.1.6.1. Preparation1.6.1.1. BacteriaBacteria are grown at 37  C up to late exponential or early stationary phase of growth. Approximate cell density should be 108-109 cells per millilitre.1.6.1.2. Metabolic activationBacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction prepared from the liver of rodents treated with enzyme-inducing agents.1.6.2. Test Condition1.6.2.1. Tester strainsThree strains, WP2, WP2 uvr A and WP2 uvr A pKM 101 should be used. Recognized methods of stock culture preparations and storage are to be used. The growth requirements and the genetic identity of the strains, their sensitivity to UV radiation or mitomycin C and the resistance to ampicillin in strain WP2 uvr A pKM 101 has to be checked. The strains should also yield spontaneous revertants within the frequency ranges expected.1.6.2.2. MediaAn appropriate medium for the expression and selection of mutants is used with an adequate overlay agar.1.6.2.3. Use of negative and positive controlsConcurrent untreated and solvent controls have to be performed. Positive controls have to be conducted also for two purposes:(i) To confirm the sensitivity of bacterial strains.Methyl methane sulphonate, 4-nitroquinoline oxide or ethylnitrosourea may be used as positive controls for tests without metabolic activation.(ii) To ensure the activity of the appropriate metabolizing systems.A positive control for the activity of one metabolizing system for all strains is 2-aminoanthracene. When available, a positive control of the same chemical class as the chemical under test should be used.1.6.2.4. Amount of test substance per plateAt least five different amounts of test chemical are tested, with half-log intervals between plates. Substances are tested up to the limit of solubility or toxicity. Toxicity is evidenced by a reduction in the number of spontaneous revertants, a clearing of the background lawn, or by degree of survival of treated cultures. Non-toxic chemicals should be tested to 5 mg per plate before considering the test substance negative.1.6.2.5. Incubation conditionsPlates are incubated for 48 up to 72 hours at 37  C.1.6.3. ProcedureFor the direct plate incorporation method without enzyme activation, the chemical and 0,1 ml of a fresh bacterial culture are added to 2 ml of overlay agar. For tests with metabolic activation, 0,5 ml of liver enzyme activation mixture containing an adequate amount of post-mitochondrial fraction is added to the agar overlay after the addition of test chemical and bacteria. The contents of each tube are mixed and poured over the surface of a selective agar plate. Overlay agar is allowed to solidify and plates are incubated at 37  C for 48 up to 72 hours. At the end of the incubation period, revertant colonies per plate are counted.For the preincubation method, a mixture of test chemical, 0,1 ml of a fresh bacterial culture and an adequate amount of liver enzyme activation mixture or the same amount of buffer is preincubated before adding 2 ml of overlay agar. All other procedures are the same as for the incorporation method.All plating for both methods is done at least in triplicate.2. DATAThe numbers of revertant colonies per plate are reported for both control and treated series. Individual plate counts, the mean number of revertant colonies per plate and standard deviations should be presented for the tested chemical and the controls.Data should be evaluated using appropriate statistical methods.At least two independent experiments are conducted. It is not necessary to perform the second one in an identical way to the initial experiment. Indeed, it may be preferable to alter certain test conditions in order to obtain more useful data.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- bacteria, strain used;- test conditions: dose levels, toxicity, composition of media; treatment procedures (preincubation incubation); metabolic activation system; reference substances, negative controls;- individual plate count, the mean number of revertant colonies per plate, standard deviation, dose/effect relationship when possible;- discussion of the results;- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).B.14. MUTAGENICITY (SALMONELLA TYPHIMURIUM - REVERSE MUTATION ASSAY)1. METHOD1.1. INTRODUCTIONSee General Introduction Part B (A).1.2. DEFINITIONSee General Introduction Part B (C).1.3. REFERENCE SUBSTANCESNone.1.4. PRINCIPLE OF THE TEST METHODThe Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures his   . his+ reversion by chemicals which cause base substitutions or frameshift mutations in the genome of this organism.Bacteria are exposed to test chemicals with and without metabolic activation and plated on minimal medium. After a suitable period of incubation, revertant colonies are counted and compared to the number of spontaneous revertants in an untreated and/or solvent control culture.1.5. QUALITY CRITERIANone.1.6. DESCRIPTION OF THE TEST METHOD1.6.1. Preparations1.6.1.1. BacteriaFresh cultures of bacteria are grown at 37  C until late exponential or early stationary phase of growth. Approximate cell density should be 108 to 109 cells per millilitre.1.6.1.2. Metabolic activationBacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction prepared from the liver of rodents treated with enzyme-inducing agents.1.6.2. Test conditions1.6.2.1. Tester strainsAt least four strains TA 1535, TA 1537 or TA 97, TA 98 and TA 100 are to be used; other strains, such as TA 1538 and TA 102 may be used in addition. Recognized methods of stock culture preparation and storage are to be used. The growth requirements and the genetic identity of the strains, their sensitivity to UV radiation and crystal violet, and their resistance to ampicillin must be checked. The strains should also yield spontaneous revertants within the frequency ranges expected.1.6.2.2. MediaAn appropriate selective medium is used with an adequate overlay agar.1.6.2.3. Use of negative and positive controlsConcurrent untreated and solvent controls have to be performed. Positive controls have to be conducted also for two purposes:(i) To confirm the sensitivity of the bacterial strains.The following compounds may be used for tests without metabolic activation:StrainsReverts withTA 1535, TA 100Sodium azideTA 1538, TA 98, TA 972-nitrofluoreneTA 15379-aminoacridineTA 102cumene hydroperoxide(ii) To ensure the activity of the appropriate metabolizing system.A positive control for the activity of one metabolizing system for all strains is 2-aminoanthracene. When available a positive control of the same chemical class as the chemical under test should be used.1.6.2.4. Amount of test substance per plate At least five different amounts of test chemical are tested, with half-log intervals between plates. Substances are tested up to the limit of solubility or toxicity. Toxicity is evidenced by a reduction in the number of spontaneous revertants, a clearing of the background lawn, or by degree of survival of treated cultures. Non-toxic chemicals should be tested to 5 mg per plate before considering the test substance negative.1.6.2.5. Incubation conditionsPlates are incubated for 48 to 72 hours at 37  C.1.6.3. ProcedureFor the direct plate incorporation method without enzyme activation, the test chemical and 0,1 ml of fresh bacterial culture are added to 2 ml of overlay agar. For tests with metabolic activation, 0,5 ml of liver enzyme activation mixture containing an adequate amount of post-mitochondrial fraction is added to the agar overlay after the addition of the test chemical and bacteria. The contents of each tube are mixed and poured over the surface of a selective agar plate. Overlay agar is allowed to solidify and plates are incubated at 37  C for 48 to 72 hours. At the end of the incubation period, revertant colonies per plate are counted. For the preincubation method, a mixture of the test chemical, 0,1 ml of fresh bacterial culture and an adequate amount of liver enzyme activation mixture or the same amount of buffer is preincubated before adding 2 ml of overlay agar. All other procedures are the same as for the direct plate incorporation method.All plating for both methods is done at least in triplicate.2. DATAThe number of revertant colonies per plate are reported for both control and treated series.Individual plate counts, the mean number of revertant colonies per plate and standard deviation should be presented for the tested chemical and the controls.Data should be evaluated using appropriate statistical methods.At least two independent experiments are conducted. It is not necessary to perform the second one in an identical way to the initial experiment. Indeed, it may be preferable to alter certain test conditions in order to obtain more useful data.3. REPORTING3.1. TEST REPORTThe test report shall, if possible, include the following information:- bacteria, strain used;- test conditions: dose levels, toxicity, composition of media, treatment procedures (preincubation, incubation) metabolic activation system, reference substances, negative controls;- individual plate count, the mean number of revertant colonies per plate, standard deviation, dose/effect relationship when possible;- discussion of the results;- interpretation of the results.3.2. EVALUATION AND INTERPRETATIONSee General Introduction Part B (D).4. REFERENCESSee General Introduction Part B (E).PART C: METHODS FOR THE DETERMINATION OF ECOTOXICITY C.1. ACUTE TOXICITY FOR FISH1. METHOD1.1. INTRODUCTIONThe purpose of this test is to determine the acute lethal toxicity of a substance to fish in fresh water. It is desirable to have, as far as possible, information on the water solubility, vapour pressure, chemical stability, dissociation constants and biodegradability of the substance to help in the selection of the most appropriate test method (static, semi-static or flow-through) for ensuring satisfactorily constant concentrations of the test substance over the period of the test.Additional information (for instance structural formula, degree of purity, nature and percentage of significant impurities, presence and amounts of additives, and n-octanol/water partition coefficient) should be taken into consideration in both the planning of the test and interpretation of the results.1.2. DEFINITIONS AND UNITSAcute toxicity is the discernible adverse effect induced in an organism within a short time (days) of exposure to a substance. In the present test, acute toxicity is expressed as the median lethal concentration (LC50), that is the concentration in water which kills 50 % of a test batch of fish within a continuous period of exposure which must be stated.All concentrations of the test substance are given in weight by volume (milligrams per litre). They may also be expressed as weight by weight (mg.kg 1).1.3. REFERENCE SUBSTANCESA reference substance may be tested as a means of demonstrating that under the laboratory test conditions the response of tested species have not changed significantly.N  reference substances are specified for this test.1.4. PRINCIPLE OF THE TEST METHODA limit test may be performed at 100 mg per litre in order to demonstrate that the LC50 is greater than this concentration.The fish are exposed to the test substance added to water at a range of concentrations for a period of 96 hours. Mortalities are recorded at least at 24-hour intervals, and the concentrations killing 50 % of the fish (LC50) at each observation time are calculated where possible.1.5. QUALITY CRITERIAThe quality criteria shall apply to the limit test as well as the full test method.The mortality in the controls must not exceed 10 % (or one fish if less than ten are used) by the end of the test.The dissolved oxygen concentration must have been more than 60 % of the air-saturation value throughout.The concentrations of the test substance shall be maintained to within 80 % of the initial concentrations throughout the duration of the test.For substances which dissolve easily in the test medium, yielding stable solutions i.e. those which will not to any significant extent volatilize, degrade, hydrolyze or adsorb, the initial concentration can be taken as being equivalent to the nominal concentration. Evidence shall be presented that the concentrations have been maintained throughout the test and that the quality criteria have been satisfied.For substances that are:(i) poorly soluble in the test medium,or(ii) capable of forming stable emulsions or dispersions,or(iii) not stable in aqueous solutions, the initial concentration shall be taken as the concentration measured in solution (or, if  technically not possible, measured in the water column) at the start of the test. The concentration shall be determined after a period of equilibration but before the introduction of the test fish.In any of these cases, further measurements must be made during the test to confirm the actual exposure concentrations or that the quality criteria have been met.The pH should not vary by more than 1 unit.1.6. DESCRIPTION OF THE TEST METHODThree types of procedure can be used:Static test:Toxicity test in which no flow of test solution occurs. (Solutions remain unchanged throughout the duration of the test.)Semi-static test:Test without flow of test solution, but with regular batchwise renewal of test solutions after prolonged periods (e.g. 24 hours).Flow-through test:Toxicity test in which the water is renewed constantly in the test chambers, the chemical under test being transported with the water used to renew the test medium.1.6.1. Reagents1.6.1.1. Solutions of test substancesStock solutions of the required strength are prepared by dissolving the substance in deionized water or water according to 1.6.1.2.The chosen test concentrations are prepared by dilution of the stock solution. If high concentrations are tested, the substance may be dissolved in the dilution water directly.The substances should normally only be tested up to the limit of solubility. For some substances (e.g. substances having low solubility in water, or high Pow, or those forming stable dispersion rather than true solution in water), it is acceptable to run a test concentration above the solubility limit of the substance to ensure that the maximum soluble/stable concentration has been obtained. It is important, however, that this concentration will not otherwise disturb the test system (e.g. film of the substance on the water surface preventing the oxygenation of the water, etc.).Ultrasonic dispersion, organic solvents, emulsifiers or dispersants may be used as an aid to prepare stock solutions of substances with low aqueous solubility or to help to disperse these substances in the test medium. When such auxiliary substances are used, all test concentrations should contain the same amount of auxiliary substance, and additional control fish should be exposed to the same concentration of the auxiliary substance as that used in the test series. The concentration of such auxiliaries should be minimized, but in no case should exceed 100 mg per litre in the test medium.The test should be carried out without adjustment of the pH. If there is evidence of marked change in the pH, it is advised that the test should be repeated with pH adjustment and the results reported. In that case, the pH value of the stock solution should be adjusted to the pH value of the dilution water unless there are specific reasons not to do so. HCl and NaOH are preferred for this purpose. This pH adjustment should be made in such a way that the concentration of test substance in the stock solution is not changed to any significant extent. Should any chemical reaction or physical precipitation of the test compound be caused by the adjustment, this should be reported.1.6.1.2. Holding and dilution waterDrinking-water supply (uncontaminated by potentially harmful concentrations of chlorine, heavy metals or other substances), good-quality natural water or reconstituted water (See Appendix I) may be used. Waters with a total hardness of between 10 and 250 mg per litre (as CaCO3) and with a pH from 6,0 to 8,5 are preferred.1.6.2. ApparatusAll apparatus must be made of chemically inert material.- automatic dilution system (for flow-through test),- oxygen meter,- equipment for determination of hardness of water,- adequate apparatus for temperature control,- pH meter.1.6.3. Test fishThe fish should be in good health and free from any apparent malformation.The species used should be selected on the basis of practical criteria, such as their ready availability throughout the year, ease of maintenance, convenience for testing, relative sensitivity to chemicals, and any economic, biological or ecological factors which have any bearing. The need for comparability of the data obtained and existing international harmonization (reference 1) should also be borne in mind when selecting the fish species.A list of fish species which are recommended for the performance of this test is given in Appendix 2; Zebra fish and rainbow trout are the preferred species.1.6.3.1. HoldingTest fish should preferably come from a single stock of similar length and age. The fish must be held for at least 12 days, in the following conditions:loading: appropriate to the system (recirculation or flow-through) and the fish species, water: see 1.6.1.2,light: 12 to 16 hours illumination daily,dissolved oxygen concentration:at least 80 % of air-saturation value,feeding: three times per week or daily, ceasing 24 hours before the startof the test.1.6.3.2. MortalityFollowing a 48-hour settling-in period, mortalities are recorded and the following criteria applied:- greater than 10 % of population in seven days: rejection of entire batch,- between 5 and 10 % of population:holding period continued for seven additional days. If no further mortalities occur, the batch is acceptable, otherwise it must be rejected,- less than 5 % of population:acceptance of the batch.1.6.4. AdaptationAll fish must be exposed to water of the quality and the temperature to be used in the test for at least seven days before they are used.1.6.5. Test ProcedureA range-finding test can precede a definitive test, in order to obtain information about the range of concentrations to be used in the main test.One control without the test substance is run and, if relevant, one control containing the auxiliary substance is also run, in addition to the test series.Depending on the physical and chemical properties of the test compound, a static, semi-static, or a flow-through test should be selected as appropriate, to fulfil the quality criteria.Fish are exposed to the substance as described below:- duration: 96 hours- number of animals: at least 7 per concentration,- tanks: of suitable capacity in relation to the recommended loading,- loading: maximum loading of 1 g per litre for static and semi-static tests is recommended; for flow-through systems, higher loading is acceptable,- test concentration: At least five concentrations differing by a constant factor not exceeding 2,2 and as far as possible spanning the range of 0 to 100 % mortality,- water: see 1.6.1.2,- light: 12 to 16 hours illumination daily,- temperature: appropriate to the species (Appendix 2) but within ± 1  C within any particular test,- dissolved oxygen concentration: not less than 60 % of the air-saturation value at the selected temperature,- feeding: none.The fish are inspected after the first 2 to 4 hours and at least at 24-hour intervals. Fish are considered dead if touching of the caudal peduncle produces no reaction, and no breathing movements are visible. Dead fish are removed when observed and mortalities are recorded.Records are kept of visible abnormalities (e.g. loss of equilibrium, changes in swimming behaviour, respiratory function, pigmentation, etc.).Measurements of pH, dissolved oxygen and temperature must be carried out daily.Limit testUsing the procedures described in this test method, a limit test may be performed at 100 mg per litre in order to demonstrate that the LC50 is greater than this concentration.If the nature of the substance is such that a concentration of 100 mg per litre in the test water cannot be attained, the limit test should be performed at a concentration equal to the solubility of the substance (or the maximum concentration forming a stable dispersion) in the medium used (see also point 1.6.1.1).The limit test should be performed using 7 to 10 fish, with the same number in the control(s). (Binomial theory dictates that when 10 fish are used with zero mortality, there is a 99,9 % confidence that the LC50 is greater than the concentration used in the limit test. With 7, 8 or 9 fish, the absence of mortality provides at least 99 % confidence that the LC50 is greater than the concentration used.)If mortalities occur, a full study must be carried out. If sublethal effects are observed, these should be recorded.2. DATA AND EVALUATIONFor each period where observations were recorded (24, 48, 72 and 96 hours), plot percentage mortality for each recommended exposure period against concentration on logarithmic-probability paper.When possible and for each observation time, the LC50 and the confidence limits (p = 0,05) should be estimated using standard procedures; these values should be rounded off to one, or at most two significant figures (examples of rounding off to two figures: 170 for 173,5; 0,13 for 0,127; 1,2 for 1,21).In those cases where the slope of the concentration/percentage response curve is too steep to permit calculation of the LC50, a graphical estimate of this value is sufficient.When two consecutive concentrations, at a ratio of 2,2 give only 0 and 100 % mortality, these two values are sufficient to indicate the range within which the LC50 falls.If it is observed that the stability or homogeneity of the test substance cannot be maintained, this should be reported and care should be taken in the interpretation of the results.3. REPORTINGThe test report shall, if possible, include the following information:- information about test fish (scientific name, strain, supplier, any pretreatment, size and number used in each test concentration);- dilution-water source and major chemical characteristics (pH, hardness, temperature);- in the case of a substance of low aqueous solubility, the method of preparation of stock and test solutions;- concentration of any auxiliary substances;- list of the concentrations used and any available information on the stability at the concentrations of the tested chemical in the test solution;- if chemical analyses are performed, methods used and results obtained;- results of the limit test if conducted;- reasons for the choice and details of the test procedure used (e.g. static, semi-static, dosing rate, flow-through rate, whether aerated, fish loading, etc.);- description of test equipment;- lighting regime;- dissolved oxygen concentrations, pH values and temperatures of the test solutions every 24 hours;- evidence that the quality criteria have been fulfilled;- a table showing the cumulative mortality at each concentration and the control (and control with the auxiliary substance if required) at each of the recommended observation times;- graph of the concentration/percentage response curve at the end of the test;- if possible, the LC50 values at each of the recommended observation times (with 95 % confidence limits);- statistical procedures used for determining the LC50 values;- if a reference substance is used, the results obtained,- highest test concentration causing no mortality within the period of the test;- lowest test concentration causing 100 % mortality within the period of the test.4. REFERENCES(1) OECD, Paris, 1981, Test Guideline 203, Decision of the Council C(81) 30 final and updates.(2) AFNOR - Determination of the acute toxicity of a substance to Brachydanio rerio - Static and Flow Through methods - NFT 90-303 June 1985.(3) AFNOR - Determination of the acute toxicity of a substance to Salmo gairdneri - Static and Flow - Through methods - NFT 90-305 June 1985.(4) ISO 7346/1, /2 and /3 - Water Quality - Determination of the acute lethal toxicity of substances to a fresh water fish (Brachydanio rerio Hamilton-Buchanan - Teleostei, Cyprinidae). Part 1: Static method. Part 2: Semi-static method. Part 3: Flow-through method.(5) Eidgenössisches Department des Innern, Schweiz: Richtlinien fur Probenahme und Normung von Wasseruntersuchungsmethoden - Part II 1974.(6) DIN Testverfahren mit Wasserorganismen, 38 412 (L1) und L (15).(7) JIS K 0102, Acute toxicity test for fish.(8) NEN 6506 - Water - Bepaling van de akute toxiciteit met behulp van Poecilia reticulata, 1980.(9) Environmental Protection Agency, Methods for the acute toxicity tests with fish, macroinvertebrates and amphibians. The Committee on Methods for Toxicity Tests with Aquatic Organisms, Ecological Research Series EPA-660-75-009, 1975.(10) Environmental Protection Agency, Environmental monitoring and support laboratory, Office of Research and Development, EPA-600/4-78-012, January 1978.(11) Environmental Protection Agency, Toxic Substance Control, Part IV, 16 March 1979.(12) Standard methods for the examination of water and wastewater, fourteen edition, APHA-AWWA-WPCF, 1975.(13) Commission of the European Communities,Inter-laboratory test programme concerning the study of the ecotoxicity of a chemical substance with respect to the fish. EEC Study D.8368, 22 March 1979.(14) Verfahrensvorschlag des Umweltbundesamtes zum akuten Fisch-Test. Rudolph, P. und Boje, R. Okotoxikologie, Grundlagen für die okotoxikologische Bewertung von Umweltchemikalien nach dem Chemikaliengesetz, ecomed 1986.(15) Litchfield, J.T. and Wilcoxon, F., A simplified method for evaluating dose effects experiments, J. Pharm, Exp. Therap., 1949, vol. 96, 99.(16) Finney, D.J. Statistical Methods in Biological Assay. Griffin, Weycombe, U.K., 1978.(17) Sprague, J.B. Measurement of pollutant toxicity to fish. Bioassay methods for acute toxicity. Water Res., 1969, vol. 3, 793-821.(18) Sprague, J.B. Measurement of pollutant toxicity to fish. II Utilising and applying bioassay results. Water Res. 1970, vol. 4, 3-32.(19) Stephan, C.E. Methods for calculating an LC50. In Aquatic Toxicology and Hazard Evaluation (edited by F.I. Mayer and J.L. Hamelink). American Society for Testing and Materials, ASTM STP 634, 1977, 65-84.(20) Stephan, C.E., Busch, K.A., Smith, R., Burke, J. and Andrews, R.W. A computer program for calculating an LC50. US EPA.Appendix 1Reconstituted waterExample of a suitable dilution waterAll chemicals must be of analytical grade.The water should be good-quality distilled water, or deionized water with a conductivity less than 5 ìScm 1.Apparatus for distillation of water must not contain any parts made of copper.Stock solutionsCaCl2. 2H2O (calcium chloride dihydrate): 11,76 g Dissolve in, and make up to 1 litre with water.MgSO4. 7H2O (magnesium sulphate heptahydrate): 4,93 g Dissolve in, and make up to 1 litre with water.NaHCO3 (sodium hydrogen carbonate): 2,59 g Dissolve in, and make up to 1 litre with water.KCl (potassium chloride): 0,23 g Dissolve in, and make up to 1 litre with water.Reconstituted dilution waterMix 25 ml of each of the four stock solutions and make up to 1 litre with water.Aerate until the dissolved oxygen concentration equals the air-saturation value.The pH should be 7,8 ± 0,2.If necessary adjust the pH with NaOH (sodium hydroxide) or HCl (hydrochloric acid).The dilution water so prepared is set aside for about 12 hours and must not be further aerated.The sum of the Ca and Mg ions in this solution is 2,5 mmol per litre. The ratio of Ca:Mg ions is 4:1 and of Na:K ions is 10:1. The total alkalinity of this solution is 0,8 mmol per litre.Any deviation in the preparation of the dilution water must not change the composition or properties of the water.Appendix 2CollectionThe fish listed above are easy to rear and/or are widely available throughout the year. They are capable of being bred and cultivated either in fish farms or in the laboratory, under disease- and parasite-controlled conditions, so that the test animal will be healthy and of known parentage. These fish are available in many parts of the world.Appendix 3Example of concentration: percentage mortality Example of >START OF GRAPHIC>>END OF GRAPHIC>determination of LC50 using log-probit paperC.2. ACUTE TOXICITY FOR DAPHNIA1. METHOD1.1. INTRODUCTIONThe purpose of this test is to determine the median effective concentration for immobilization (EC50) of a substance to Daphnia in fresh water. It is desirable to have, as far as possible, information on the water solubility, vapour pressure, chemical stability, dissociation constants and biodegradability of the substance before starting the test.Additional information (for instance structural formula, degree of purity, nature and percentage of significant impurities, presence and amount of additives, and n-octanol/water partition coefficient) should be taken into consideration in both the planning of the test and interpretation of the results.1.2. DEFINITIONS AND UNITSThe Directive requirement for the LC50 for Daphnia is considered to be fulfilled by the determination of the EC50 as described in this test method.Acute toxicity is expressed in this test as the median effective concentration (EC50) for immobilization. This is the concentration, in terms of initial values, which immobilizes 50 % of the Daphnia in a test batch within a continuous period of exposure which must be stated.Immobilization:Those animals which are not able to swim within 15 seconds after gentle agitation of the test container are considered to be immobile.All concentrations of the test substance are given in weight by volume (milligrams per litre). They may also be expressed as weight by weight (mg.kg 1).1.3. REFERENCE SUBSTANCESA reference substance may be tested as a means of demonstrating that under the laboratory test conditions the sensitivity of the test species has not changed significantly.The summary of the results of an EEC ring-test, using four different substances, is given in Appendix 2.1.4. PRINCIPLE OF THE TEST METHODA limit test may be performed at 100 mg per litre in order to demonstrate that the EC50 is greater than this concentration.The Daphnia are exposed to the test substance added to water at a range of concentrations for 48 hours. If a shorter test is used, justification should be given in the test report.Under otherwise identical test conditions, and an adequate range of test substance concentrations, different concentrations of a test substance exert different average degrees of effect on the swimming ability of Daphnia. Different concentrations result in different percentages of Daphnia being no longer capable of swimming at the end of the test. The concentrations causing zero or 100 % immobilization are derived directly from the test observations whereas the 48-hour EC50 is determined by calculation if possible.A static system is used for this method, hence test solutions are not renewed during the exposure period.1.5. QUALITY CRITERIAThe quality criteria shall apply to the limit test as well as the full test method.Immobilization in the controls must not exceed 10 % at the end of the test.Test Daphnia in the control groups must not have been trapped at the surface of the water.It is desirable that the concentration of dissolved oxygen in the test vessels should remain above 3 mg l 1 throughout the course of the test. However, in no circumstances should the dissolved oxygen concentration fall below 2 mg l 1.The concentration of the test substance shall be maintained to within 80 % of the initial concentration throughout the duration of the test.For substances which dissolve easily in the test medium, yielding stable solutions i.e. those which will not to any significant extent volatilize, degrade, hydrolyze or adsorb, the initial concentration can be taken as being equivalent to the nominal concentration. Evidence shall be presented that the concentrations have been maintained throughout the test and that the quality criteria have been satisfied.For substances that are:(i) poorly soluble in the test medium,or(ii) capable of forming stable emulsions or dispersions,or(iii) not stable in aqueous solutions,the initial concentration shall be taken as the concentration measured in solution (or, if technically not possible, measured in the water column) at the start of the test. The concentration shall be determined after a period of equilibration but before the introduction of the test organisms.In any of these cases, further measurements must be made during the test to confirm the actual exposure concentrations or that the quality criteria have been met.The pH should not vary by more than 1 unit.1.6. DESCRIPTION OF TEST METHOD1.6.1. Reagents1.6.1.1. Solutions of test substancesStock solutions of the required strength are prepared by dissolving the substance in deionized water or water according to 1.6.1.2.The chosen test concentrations are prepared by dilution of the stock solution. If high concentrations are tested, the substance may be dissolved in the dilution water directly.The substances should normally only be tested up to the limit of solubility. For some substances (e. g. substances having low solubility in water, or high Pow, or those forming stable dispersion rather than true solution in water), it is acceptable to run a test concentration above the solubility limit of the substance to ensure that the maximum soluble/stable concentration has been obtained. It is important, however, that this concentration will not otherwise disturb the test system (e. g. film of the substance on the water surface preventing the oxygenation of the water, etc.).Ultrasonic dispersion, organic solvents, emulsifiers or dispersants may be used as an aid to prepare stock solutions of substances with low aqueous solubility or to help to disperse these substances in the test medium. When such auxiliary substances are used, all test concentrations should contain the same amount of auxiliary substances, and additional control Daphnia should be exposed to the same concentration of the auxiliary substance as that used in the test series. The concentration of such auxiliaries should be minimized, but in no case should exceed 100 mg per litre in the test medium.The test should be carried out without adjustment of the pH. If there is evidence of marked change in the pH, it is advised that the test should be repeated with pH adjustment and the results reported. In that case, the pH value of the stock solution should be adjusted to the pH value of the solution water unless there are specific reasons not to do so. HCl and NaOH are preferred for this purpose. This pH adjustment should be made in such a way that the concentration of test substance in the stock solution is not changed to any significant extent. Should any chemical reaction or physical precipitation of the test compound be caused by the adjustment, this should be reported.1.6.1.2. Test waterReconstituted water is used in this test (see Appendix 1 and reference (2) : ISO 6341). To avoid the necessity for acclimation prior to the test, it is recommended that the culture water should be of similar quality (pH, hardness) as the water used for the test.1.6.2. ApparatusNormal laboratory apparatus and equipment should be used. Equipment which will come into contact with the test solutions should preferably be made entirely of glass:- Oxygen meter (with microelectrode or other suitable equipment for measuring dissolved oxygen in low-volume samples),- adequate apparatus for temperature control,- pH meter,- equipment for the determination of hardness of water.1.6.3. Test organismDaphnia magna is the preferred test species although Daphnia pulex is also permitted. The test animals shall be less than 24 hours old, at the beginning of the test, laboratory bred, free from overt disease and with a known history (e.g. breeding - any pretreatments, etc.).1.6.4. Test procedureA range-finding test can precede the definitive test, in order to obtain information about the range of concentrations to be used in the main test.One control without the test substance is run and, if relevant, one control containing the auxiliary substance is also run in addition to the test series.Daphnia are exposed to the substance as described below:- duration: preferably 48 hours,- number of animals: at least 20 animals at each test concentration preferably divided into four batches of five animals each or two batches of 10,- loading: at least 2 ml of test solutions should be provided for each animal,- test concentration: the test solution should be prepared immediately before introduction of the Daphnia, preferably without using any solvent other than water. The concentrations are made up in a geometric series, at a concentration ratio not exceeding 2.2. Concentrations sufficient to give 0 and 100 % immobilization after 48 hours and a range of intermediate degrees of immobilizations permitting calculation of the 48 hour EC50 should be tested together with controls,- water: see 1.6.1.2,- light: a light-dark cycle is optional,- temperature: the test temperature should be between 18 and 22  C, but for each single test it should be constant within ± 1  C,- aeration: the test solutions must not be bubble-aerated,- feeding: none.The pH and the oxygen concentration of the controls and of all the test concentrations should be measured at the end of the test; the pH of the test solutions should not be modified.Volatile compounds should be tested in completely filled closed containers, large enough to prevent lack of oxygen.Daphnia are inspected at least after 24 hours exposure and again after 48 hours.Limit testUsing the procedures described in this method, a limit test may be performed at 100 mg per litre in order to demonstrate that the EC50 is greater than this concentration.If the nature of the substance is such that a concentration of 100 mg per litre in the test water cannot be attained, the limit test should be performed at a concentration equal to the solubility of the substance (or the maximum concentration forming a stable dispersion) in the medium used (see also point 1.6.1.1).The limit test should be performed using 20 Daphnia, divided in two or four batches, with the same number in the control(s). If immobilisations occur, a full study must be carried out.2. DATA AND EVALUATIONFor each period where observations were recorded (24 and 48 h), the percentage mortality is plotted against concentration on logarithmic-probability paper.When possible and for each observation time, the EC50 and the confidence limits (p = 0,05) should be estimated using standard procedures; these values should be rounded off to one, or at most two significant figures (examples of rounding off to two figures : 170 for 173,5; 0,13 for 0,127; 1,2 for 1,21).In those cases where the slope of the concentration/percentage response curve is too steep to permit calculation of the EC50, a graphical estimate of this value is sufficient.When two immediately consecutive concentrations at a ratio of 2,2 give only 0 and 100 % immobilization these two values are sufficient to indicate the range within which the EC50 falls.If it is observed that the stability or homogeneity of the test substance cannot be maintained, this should be reported and care taken in the interpretation of the results.3. REPORTINGThe test report shall, if possible, include the following information:- information about the test organism (scientific name, strain, supplier or source, any pretreatment, breeding method - including source, kind and amount of food, feeding frequency);- dilution water source and major chemical characteristics (i. e. pH, temperature, hardness);- in the case of substance of low aqueous solubility, the method of preparation of stock and test solution;- concentration of any auxiliary substances;- list of the concentrations used and any available information on the stability at the concentrations of the tested chemical in the test solutions;- if chemical analyses are performed, methods used and results obtained;- results of the limit test, if conducted;- description of test equipment;- lighting regime;- dissolved oxygen concentrations, pH values and temperatures of the test solutions;- evidence that the quality criteria have been fulfilled;- a table showing the cumulative immobilisation at each concentration and the control (and control with the auxiliary substance if required) at each of the recommended observation times (24 and 48 h);- graph of the concentration/percentage response curve at the end of the test;- if possible, the EC50 values at each of the recommended observation times (with 95 % confidence limits);- statistical procedures used for determining the EC50 values;- if a reference substance is used, the results obtained;- highest tested concentration causing no immobilization within the period of the test;- lowest tested concentration causing 100 % immobilization within the period of the test.4. REFERENCES(1) OECD, Paris, 1981, Test Guidelines 202, Decision of the Council C(81) 30 final and updates.(2) International Standard ISO, Water Quality - Determination of inhibition of mobility of Daphnia magna Straus, ISO 6341-1989(3) AFNOR Inhibition of mobility of Daphnia magna Straus (Cladocera - crustacea) NFT 90 301 (January 1983).(4) Verfahrensvorschlag des Umweltbundesamtes zum akuten Daphnien-Test. Rudolph, P. und Boje, R. Ökotoxikologie, Grundlagen für die ökotoxikologische Bewertung von Umweltchemikalien nach dem Chemikaliengesetz, ecomed 1986.(5) DIN Testverfahren mit Wasserorganismen 38412 (L1) und (L11).(6) Finney, D.J. Statistical Methods in Biological Assay. Griffin, Weycombe, U.K., 1978.(7) Litchfield, J.T. and Wilcoxon, F. A simplified method of evaluating dose-effect experiments. J. Pharmacol. and Exper. Ther., 1949, vol. 96, 99-113.(8) Sprague, J.B. Measurement of pollutant toxicity to fish. I Bioassay methods for acute toxicity. Water Res., 1969, vol. 3, 793-821.(9) Sprague, J.B. Measurement of pollutant toxicity to fish. II Utilising and applying bioassay results. Water Res. 1970, vol. 4, 3-32.(10) Stephan, C.E. Methods for calculating an LC50. In Aquatic Toxicology and Hazard Evaluation (edited by F.I. Mayer and J.L. Hamelink). American Society for Testing and Materials. ASTM, 1977, STP 634, 65-84.(11) Stephan, C.E., Busch, K.A., Smith, R., Burke, J. and Andrews, R.W. A computer program for calculating an LC50. US EPA.Appendix 1Reconstituted waterExample of a suitable dilution water (according to ISO 6341)All chemicals must be of analytical grade.The water should be good-quality distilled water, or deionized water with a conductivity less than 5 ìScm 1.The apparatus for distillation of water must not contain any parts made of copper.Stock solutionsCaCl2.2 H2O (calcium chloride dihydrate): 11,76 g dissolve in, and make up to 1 litre with waterMgSO4.7H2O (magnesium sulphate heptahydrate): 4,93 g dissolve in, and make up to 1 litre with waterNaHCO3 (sodium hydrogen carbonate): 2,59 g dissolve in, and make up to 1 litre with waterKCl (potassium chloride): 0,23 g dissolve in, and make up to 1 litre with waterReconstituted dilution waterMix 25 ml of each of the four stock solutions and make up to 1 litre with water.Aerate until the dissolved oxygen concentration equals the air-saturation value.The pH should be 7,8 ± 0,2.If necessary adjust the pH with NaOH (sodium hydroxide) or HCl (hydrochloric acid).The dilution water so prepared is set aside for about 12 hours and need not be further aerated.The sum of the Ca and Mg ions in this solution is 2,5 mmol per litre. The ratio of Ca:Mg ions is 4:1 and of Na:K ions is 10:1. The total alkalinity of this solution is 0,8 mmol per litre.Any deviation in the preparation of the dilution water must not change the composition or properties of the water.Appendix 2Summary of the results of an EEC ring-test performed in 1978 (also cited in reference 2)Caution: the purpose of this ring-test was the determination of the EC50 24 hours.Substances used:1) Potassium dichromate2) Tetrapropylbenzenesulphonic acid3) Tetrapropylbenzenesulphonic acid, sodium salt4) Trichloro-2,4,5-phenoxyacetic acid, potassium salt>TABLE>Appendix 3Example of concentration: percentage immobilisation >START OF GRAPHIC>>END OF GRAPHIC>Example of determination of EC50 using log-probit paper Immobilization 10 %C.3. ALGAL INHIBITION TEST1. METHOD1.1. INTRODUCTIONThe purpose of this test is to determine the effects of a substance on the growth of a unicellular green algal species. Relatively brief (72 hours) tests can assess effects over several generations. This method can be adapted for use with several unicellular algal species, in which case a description of the method used must be provided with the test report.This method is most easily applied to water-soluble substances which, under the conditions of the test, are likely to remain in the water.The method can be used for substances that do not interfere directly with the measurement of algal growth.It is desirable to have, as far as possible, information on the water solubility, vapour pressure, chemical stability, dissociation constants and biodegradability of the substance before starting the test.Additional information (for instance structural formula, degree of purity, nature and percentage of significant impurities, presence and amount of additives, and n-octanol/water partition coefficient) should be taken into consideration in both the planning of the test and interpretation of the results.1.2. DEFINITIONS AND UNITSCell density: the number of cells per millilitre;Growth: the increase in cell density over the test period;Growth rate: the increase in cell density per unit time;EC50: in this method, that concentration of test substance which results in a 50 % reduction in either growth (EbC50) or growth rate (ErC50) relative to the control;NOEC (no observed effect concentration): in this method, the highest tested concentration at which no significant inhibition of growth is observed relative to the control.All concentrations of the test substance are given in weight by volume (milligrams per litre). They may also be expressed as weight by weight (mg.kg 1).1.3. REFERENCE SUBSTANCESA reference substance may be tested as a means of demonstrating that under the laboratory test conditions the sensitivity of the test species has not changed significantly.If a reference substance is used, the results should be given in the test report. Potassium dichromate can be used as a reference substance, but its colour may affect the light quality and intensity available to the cells and also the spectrophotometric determinations if used. Potassium dichromate has been used in an international inter-laboratory test (see ref. (3) and Appendix 2).1.4. PRINCIPLE OF THE TEST METHODA limit test may be performed at 100 mg per litre in order to demonstrate that the EC50 is greater than this concentration.Exponentially-growing cultures of selected green algae are exposed to various concentrations of the test substance over several generations under defined conditions.The test solutions are incubated for a period of 72 hours, during which the cell density in each solution is measured at least every 24 hours. The inhibition of growth in relation to a control culture is determined.1.5. QUALITY CRITERIAThe quality criteria shall apply to the limit test as well as the full test method.The cell density in the control cultures should have increased by a factor of at least 16 within three days.The concentrations of the test substance shall be maintained to within 80 % of the initial concentrations throughout a time corresponding to the duration of the test.For substances which dissolve easily in the test medium, yielding stable solutions i.e. those which will not to any significant extent volatilize, degrade, hydrolyze or adsorb, the initial concentration can be taken as being equivalent to the nominal concentration. Evidence shall be presented that the concentrations have been maintained throughout the test and that the quality criteria have been satisfied.For substances that are:(i) poorly soluble in the test medium,or(ii) capable of forming stable emulsions or dispersions,or(iii) not stable in aqueous solutions, the initial concentration shall be taken as the concentration measured at the start of the test. The concentration shall be determined after a period of equilibration.In any of these cases, further measurements must be made during the test to confirm the actual exposure concentrations or that the quality criteria have been met.It is recognized that significant amounts of the test substance may be incorporated into the algal biomass during the period of the test. Therefore, for the purpose of demonstrating compliance with the above quality criteria, both the amount of the substance incorporated into the algal biomass and the substance in solution (or, if not technically possible measured in the water column) should be taken into account. However, as determination of the substance concentration in the algal biomass may pose significant technical problems, compliance with the quality criteria may be demonstrated by running a test vessel at the highest substance concentration but without algae and measuring concentrations in solution (or, if not technically possible in the water column) at the beginning and at the end of the test period.1.6. DESCRIPTION OF THE TEST PROCEDURE1.6.1. Reagents1.6.1.1. Solutions of test substancesStock solutions of the required strength are prepared by dissolving the substance in deionized water or water according to 1.6.1.2.The chosen test concentrations are prepared by adding suitable aliquots to algal pre-cultures (see Appendix 1). Substances should normally only be tested up to the limit of solubility. For some substances (e.g. substances having low solubility in water, or high Pow, or those forming stable dispersion rather than true solution in water), it is acceptable to run a test concentration above the solubility limit of the substance to ensure that the maximum soluble/stable concentration has been obtained. It is important, however, that this concentration will not otherwise disturb the test system (e.g. film of the substance on the water surface preventing the oxygenation of the water, etc.).Ultrasonic dispersion, organic solvents, emulsifiers or dispersants may be used as an aid to prepare stock solutions of substances with low aqueous solubility or to help to disperse these substances in the test medium. When such auxiliary substances are used, all test concentrations should contain the same amount of auxiliary substances, and additional controls should be exposed to the same concentration of the auxiliary substance as that used in the test series. The concentration of such auxiliaries should be minimized, but in no case should exceed 100 mg per litre in the test medium.The test should be carried out without adjustment of the pH. If there is evidence of marked change in the pH, it is advised that the test should be repeated with pH adjustment and the results reported. In that case, the pH value of the stock solution should be adjusted to the pH value of the solution water unless there are specific reasons not to do so. HCl and NaOH are preferred for this purpose. This pH adjustment should be made in such a way that the concentration of test substance in the stock solution is not changed to any significant extent. Should any chemical reaction or physical precipitation of the test compound be caused by the adjustment, this should be reported.1.6.1.2. Test mediumThe water should be good-quality distilled water, or deionized water with a conductivity less than 5 ìS.cm 1. The apparatus for distillation of water must not contain any part made of copper.The following medium is recommended.Four stock solutions are prepared, according to the following table. The stock solutions are sterilised by membrane filtration or by autoclaving, and stored in the dark at 4  C. Stock solution no. 4 should be sterilised only by membrane filtration. These stock solutions are diluted to achieve the final nutrient concentrations in the test solutions.>TABLE>The pH of the medium after equilibration with air is approximately 8.1.6.2. Apparatus- Normal laboratory equipment,- Test flasks of suitable volume (e.g. 250 ml conical flasks are suitable when the volume of the test solution is 100 ml). All test flasks should be identical as regards to material and dimensions.- Culturing apparatus: cabinet or chamber in which a temperature in the range 21  C to 25  C can be maintained at ± 2  C, and continuous uniform illumination provided in the spectral range 400 to 700 nm. If algae in control cultures have achieved the recommended growth rates, it can be assumed that the conditions for growth, including light intensity, have been adequate.It is recommended to use, at the average level of the test solutions, a light intensity in the range 60 to 120 ìE.m 2.s 1 (35 to 70 × 1018 photons.m 2.s 1) when measured in the range 400 to 700 nm using an appropriate receptor. For light measuring instruments calibrated in lux, an equivalent range of 6 000 to 10 000 lx is acceptable.The light intensity could be obtained using four to seven 30 W fluorescent lamps of the universal white type (colour temperature of approximately 4 300 K), at a distance of 0,35 m from the algal culture.- Cell density measurements should be made using a direct counting method of living cells, e.g. a microscope with counting chambers. However, other procedures (photometry, turbidimetry,...) may be used if sufficiently sensitive and if shown to be sufficiently well correlated with cell density.1.6.3. Test organismsIt is suggested that the species of green algae used be a fast-growing species that is convenient for culturing and testing. The following species are preferred:- Selenastrum capricornutum, e.g. ATCC 22662 or CCAP 278/4,- Scenedesmus subspicatus, e.g. 86.81 SAG,Note:ATCC = American Type Culture Collection (U.S.A.)CCAP = Culture Centre of Algae and Protozoa (U.K.)SAG = Collection of algal culture (Göttingen, F.R.G.)If other species are used, the strain should be reported.1.6.4. Test procedureThe concentration range in which effects are likely to occur is determined on the basis of results from range-finding tests.The two measures of growth (biomass and growth rate) may result in widely disparate measures of growth inhibition; both should be used in the range finding test to ensure that the geometric progression of concentrations will allow estimation of both the EbC50 and the ErC50.Initial cell densityIt is recommended that the initial cell density in the test cultures be approximately 104 cells/ml for Selenastrum capricornutum and Scenedesmus subspicatus. When other species are used the biomass should be comparable.Concentrations of test substanceFor the test, at least five concentrations are made up in a geometric series at a concentration ratio not exceeding 2,2. The lowest concentration tested should have no observed effect on the growth of the algae. The highest concentration tested should inhibit growth by at least 50 % relative to the control and, preferably, stop growth completely.Replicates and controlsThe test design should include three replicates at each test concentration. Three controls without test substance are run and, if relevant, three controls containing the auxiliary substance are also run. If justified, the test design may be altered to increase the number of concentrations and reduce the number of replicates per concentration.Performance of the testTest cultures containing the desired concentrations of test substance and the desired quantity of algal inoculum are prepared by adding aliquots of stock solutions of the test substance to suitable amounts of algal pre-cultures (see Appendix 1).The culture flasks are shaken and placed in the culturing apparatus. The algal cells are kept in suspension by shaking, stirring or bubbling with air, in order to improve gas exchange and reduce pH variation in the test solutions. The cultures should be maintained at a temperature in the range of 21 to 25  C, controlled at ± 2  C.The cell density in each flask is determined at least at 24, 48 and 72 hours after the start of the test. Filtered algal medium containing the appropriate concentration of the test chemical is used to determine the background when using cell density measurements other than direct counting methods.The pH is measured at the beginning of the test and at 72 hours.The pH of the controls should not normally deviate by more than 1,5 units during the test.Testing volatile substancesThere is to date no generally accepted way to test volatile substances. When a substance is known to have a tendency to vaporize, closed test flasks with increased head-space may be used. The possibility of shortage of CO2 should be considered when calculating the head-space of the closed flasks. Variations to this method have been proposed (see reference (4)).Attempts should be made to determine the amount of the substance which remains in solution, and extreme caution is advised when interpreting results of tests with volatile chemicals using closed systems.Limit testUsing the procedures described in this method, a limit test may be performed at 100 mg per litre in order to demonstrate that the EC50 is greater than this concentration.If the nature of the substance is such that a concentration of 100 mg per litre in the test water cannot be attained, the limit test should be performed at a concentration equal to the solubility of the substance (or the maximum concentration forming a stable dispersion) in the medium used (see also point 1.6.1.1).The limit test should be performed at least in triplicate, with the same number of controls. The two measures of growth (biomass and growth rate) should be used for the limit test.If, in a limit test, a mean decrease of 25 % or more is found in either biomass or growth rate between the limit test and the control, a full test should be carried out.2. DATA AND EVALUATIONThe measured cell density in the test cultures and controls are tabulated together with the concentrations of the test substance and the times of measurements. The mean value of the cell density for each test substance concentration and for the controls is plotted against time (0-72 h) to produce growth curves.To determine the concentration/effect relationship, the two following approaches should be used. Some substances can stimulate the growth at low concentrations. Only data points indicating inhibition between 0 and 100 % should be considered.2.1. COMPARISON OF AREAS UNDER THE GROWTH CURVESThe area between the growth curves and the horizontal line N = N0 may be calculated according to the formula:A =N1N02 × t1 +N1 + N22N02 × (t2t1) +  +Nn 1 + Nn 2N02× (tn  tn 1)whereA = area,N0 = number of cells/ml at time t0 (beginning of the test),0,N1 = measured number of cells/ml at t1,Nn = measured number of cells/ml at time tn,t1 = time of first measurement after beginning of test,tn = time of nth measurement after beginning of test.n = number of measurements taken after the beginning of the test.The percentage inhibition of the cell growth at each test substance concentration (IA) is calculated according to the formula:IA = Ac   AtAc × 100whereAc = area between the control growth curve and the horizontal line N = N0.At = area between the growth curve at the concentration t and the horizontal line N = N0.IA values are plotted on semilogarithmic paper or on semilogarithmic probit paper against the corresponding concentrations. If plotted on probit paper, the points are fitted by a straight line, either by eye or by a computed regression.The EC50 is estimated from the regression line by reading off the concentration that is equivalent to a 50 % inhibition (IA = 50 %). To denote this value unambiguously in relation to this method of calculation, it is proposed to use the symbol EbC50. It is essential that the EbC50 is quoted with the appropriate exposure period, e.g. EbC50(0-72h).2.2. COMPARISON OF GROWTH RATESThe average specific growth rate (ì) for exponentially growing cultures can be calculated asm = ln Nn ln N0tn t0where t0 is the time at the beginning of the test.Alternatively, the average specific growth rate may be derived from the slope of the regression line in a plot of ln N versus time.The percentage inhibition of specific growth rate at each test substance concentration (Iìt) is calculated according to the formula:Imt = mc mtmc × 100whereìc = mean control specific growth rateìt = mean specific growth rate for the test concentration tThe percentage reduction in average specific growth rate at each test substance concentration compared to the control value is plotted against the logarithm of the concentration. The EC50 may be read from the resulting graph. To denote unambiguously the EC50 derived by this method it is proposed to use the symbol ErC50. The times of measurement must be indicated, e.g. if the value relates to times 0 and 72 hours, the symbol becomes ErC50 (0-72h).Note: specific growth rate is a logarithmic term, and small changes in growth rate may lead to great changes in biomass. EbC and ErC values are therefore not numerically comparable.2.3. CALCULATION OF THE NOECThe N  Observed Effect Concentration is determined by a suitable statistical procedure for multisample comparison (e.g. analysis of variance and Dunnett's test), using the individual replicates values of the areas under the growth curves A (see point 2.1) or the specific growth rates ì (see point 2.2).3. REPORTINGThe test report shall, if possible, include the following information:- test substance: chemical identification data;- test organisms: origin, laboratory culture, strain number, method of cultivation;- test conditions:- date of the start and the end of the test and its duration,- temperature,- composition of medium,- culturing apparatus,- pH of solutions at the start and end of the test (an explanation should be provided if pH deviations of more than 1,5 unit are observed),- vehicle and method used for solubilizing the test substance and concentration of the vehicle in the test solutions,- light intensity and quality,- concentrations tested (measured or nominal).- results:- cell density for each flask at each measuring point and method for measuring cell density,- mean values of cell density,- growth curves,- graphical presentation of the concentration effect relationship,- EC values and method of calculation,- NOEC,- other observed effects.4. REFERENCES(1) OECD, Paris, 1981, Test Guideline 201, Decision of the Council C(81) 30 Final.(2) Umweltbundesamt, Berlin, 1984, Verfahrensvorschlag 'Hemmung der Zellvermehrung bei der Grünalge Scenedes¹us subspicatus`, in: Rudolph/Boje: Ökotoxikologie, ecomed, Landsberg, 1986.(3) ISO 8692 - Water quality - Fresh water algal growth inhibition test with Scenedesmus subspicatus and Selenastrum capricornutum.(4) S.Galassi and M.Vighi - Chemosphere, 1981, vol.10, 1123-1126.Appendix 1Example of a procedure for the culturing of algaeGeneral observationsThe purpose of culturing on the basis of the following procedure is to obtain algal cultures for toxicity tests.Suitable methods should be used to ensure that the algal cultures are not infected with bacteria (ISO 4833). Axenic cultures may be desirable but unialgal cultures are essential.All operations should be carried out under sterile conditions in order to avoid contamination with bacteria and other algae. Contaminated cultures should be rejected.Procedures for obtaining algal culturesPreparation of nutrient solutions (media):The medium can be prepared by diluting concentrated stock solutions of nutrients. For solid medium, 0,8 % of agar is added. The medium used should be sterile. Sterilisation by autoclaving may lead to a loss of NH3.Stock culture:The stock cultures are small algal cultures that are regularly transferred to fresh medium to act as initial test material. If the cultures are not used regularly they are streaked out on sloped agar tubes. These are transferred to fresh medium at least once every two months.The stock cultures are grown in conical flasks containing the appropriate medium (volume about 100 ml). When the algae are incubated at 20  C with continuous illumination, a weekly transfer is required.During transfer an amount of 'old' culture is transferred with sterile pipettes into a flask of fresh medium, so that with the fast-growing species the initial concentration is about 100 times smaller than in the old culture.The growth rate of a species can be determined from the growth curve. If this is known, it is possible to estimate the density at which the culture should be transferred to new medium. This must be done before the culture reaches the death phase.Pre-culture:The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture is incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they must be discarded.Appendix 2The ISO 8692 - Water quality - Fresh water algal growth inhibition test with Scenedesmus subspicatus andSelenastrum capricornutum reports the following results for an inter-laboratory test among 16 laboratories, testing potassium dichromate :>TABLE>C.4. DETERMINATION OF 'READY` BIODEGRADABILITYPART I. GENERAL ONSIDERATIONSI.1. INTRODUCTIONSix test methods are described that permit the screening of chemicals for ready biodegradability in an aerobic aqueous medium:(a) Dissolved Organic Carbon (DOC) Die-Away (Method C.4-A)(b) Modified OECD Screening - DOC Die-Away (Method C.4-B)(c) Carbon dioxide (CO2) Evolution (Modified Sturm Test) (Method C.4-C)(d) Manometric Respirometry (Method C.4-D)(e) Closed Bottle (Method C.4-E)(f) MITI (Ministry of International Trade and Industry - Japan) (Method C.4-F)General and common considerations to all six tests are given in Part I of the method. Items specific for individual methods are given in Parts II to VII. The annexes contain definitions, formulas and guidance material.An OECD inter-laboratory comparison exercise, done in 1988, has shown that the methods give consistent results. However, depending on the physical characteristics of the substance to be tested, one or other of the methods may be preferred.I.2. SELECTION OF THE APPROPRIATE METHODIn 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, COD (see Annexes I and II).Test chemicals which are soluble in water to at least 100 mg/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 Table 1. The manner in which poorly water-soluble chemicals and volatile chemicals can be dealt with is described in Annex III. 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.>TABLE>Information on the purity or the relative proportions of major components of the test material is required to interpret the results obtained, especially when the results are low or marginal.Information on the toxicity of the test chemical to bacteria (Annex IV) may be very useful for selecting appropriate test concentrations and may be essential for the correct interpretation of low biodegradation values.I.3. REFERENCE SUBSTANCESIn order to check the procedure, reference chemicals which meet the criteria for ready biodegradability are tested by setting up an appropriate flask in parallel to the normal test runs.Suitable chemicals are aniline (freshly distilled), sodium acetate and sodium benzoate. These reference chemicals all degrade in these methods even when no inoculum is deliberately added.It was suggested that a reference chemical 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 substance before it can be accepted as a reference substance.In the respirometric tests, nitrogen-containing compounds may affect the oxygen uptake because of nitrification (see Annexes II and V).I.4. PRINCIPLE OF THE TEST METHODSA 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 to the amount of DOC due to the test substance. Allowance is made for the endogenous activity of the inoculum by running parallel blank tests with inoculum but without test substance, although the endogenous activity of cells in the presence of the substance will not exactly match that in the endogenous control. A reference substance is run in parallel to check the operation of the procedures.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 the 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 (obligatory in the MITI test).Normally, the test lasts for 28 days. Tests however may be ended before 28 days, i.e. as soon as the biodegradation curve has reached a plateau for at least 3 determinations. Tests may also be prolonged beyond 28 days when the curve shows that biodegradation has started but that the plateau has not been reached day 28.I.5. QUALITY CRITERIAI.5.1. ReproducibilityBecause 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 common experience 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.I.5.2. Validity of the testA test is considered valid if the difference of extremes of replicate values of the removal of test chemical at the plateau, at the end of the test or at the end of the 10-day window, as appropriate, is less than 20 % and if the percentage degradation of the reference substance has reached the level for ready biodegradability by 14 days. 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.If in a toxicity test, containing both the test substance and a reference chemical, less than 35 % degradation (based on DOC) or less than 25 % (based on ThOD or ThCO2) occurred in 14 days, the test chemicals can be assumed to be inhibitory (see also Annex IV). The test series should be repeated, if possible using a lower concentration of test chemical and/or a higher concentration of inoculum, but not greater than 30 mg solids/litre.I.6. GENERAL PROCEDURES AND PREPARATIONSGeneral conditions applying to the tests are summarised in Table 2. Apparatus and other experimental conditions pertaining specifically to an individual test are described later under the heading for that test.>TABLE>I.6.1. Dilution waterDeionized or distilled water, free from inhibitory concentrations of toxic substances (e.g. Cu++ ions) is used. It must contain no more than 10 % of the organic carbon content introduced by the test material. The high purity of the test water is necessary to eliminate high blank values. Contamination may result from inherent impurities and also from the ion-exchange resins and lysed material from bacterial and algae. For each series of tests use only one batch of water, checked beforehand by DOC analysis. Such a check is not necessary for the closed bottle test, but the oxygen consumption of the water must be low.I.6.2. Stock solutions of mineral componentsTo make up the test solutions, stock solutions of appropriate concentrations of mineral components are made up. The following stock solutions may be used (with different dilution factors) for the methods DOC Die-Away, Modified OECD Screening, CO2 Evolution, Manometric Respirometry, Closed Bottle test.The dilution factors and, for the MITI test, the specific preparation of the mineral medium are given under the headings of the specific tests.Stock solutions:Prepare the following stock solutions, using analytical grade reagents.(a) Monopotassium dihydrogen orthophosphate, KH2PO4 8,50 gDipotassium monohydrogen orthophosphate, K2HPO4 21,75 gDisodium monohydrogen orthophosphate dihydrate Na2HPO4. 2 H2O 33,40 gAmmonium chloride, NH4Cl 0,50 gDissolve in water and make up to 1 litre The pH of the solution should be 7,4.(b) Calcium chloride, anhydrous, CaCl2 27,50 gor Calcium chloride dihydrate, CaCl2. 2 H2O 36,40 gDissolve in water and make up to 1 litre(c) Magnesium sulphate heptahydrate, MgSO4. 7 H2O 22,50 gDissolve in water and make up to 1 litre(d) Iron (III) chloride hexahydrate, FeCl3. 6 H2O 0,25 gDissolve in water and make up to 1 litre.Note: in order to avoid having to prepare this solution immediately before use add one drop of conc. HCL or 0,4 g ethylenediaminetetra-acetic acid disodium salt (EDTA) par litre.I.6.3. Stock solutions of chemicalsFor example, dissolve 1-10 g, as appropriate, of test or reference chemical in deionized water and make up to 1 litre when the solubility exceeds 1 g/l. Otherwise, prepare stock solutions in the mineral medium or add the chemical direct to the mineral medium. For the handling of less soluble chemicals, see Annex III, but in the MITI test (Method C.4-F), neither solvents nor emulsifying agents are to be used.I.6.4. InoculaThe 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, CO2 Evolution and Manometric Respirometry tests, 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 have been found to give higher scattering of results. For the Modified OECD Screening and the Closed Bottle tests 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 test the inoculum is derived from a mixture of sources and is described under the heading of this specific test.I.6.4.1. Inoculum from activated sludgesCollect a sample of activated sludge freshly 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.Alternatively, settle or centrifuge (e.g. at 1 100 g for 10 min.) after removal of any coarse particles. Discard the supernatant. The sludge may be washed in the mineral medium. Suspend the concentrated sludge in mineral medium to yield a concentration of 3-5 g suspended solids/l and aerate until required.Sludge should be taken from a properly working conventional 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.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.A further alternative is to homogenise activated sludge (3-5 g suspended solids/l). Treat the sludge in a mechanical blender for 2 min. at medium speed. Settle the blended sludge for 30 min. or longer if required and decant liquid for use as inoculum at the rate of 10 ml/l of mineral medium.I.6.4.2. Other sources of inoculumIt 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 for 1 h. or filter through a coarse filter paper and keep the decanted effluent or filtrate aerobic until required. Up to 100 ml of this type of inoculum may be used per litre of medium.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.I.6.5. Pre-conditioning of inoculaInocula may be pre-conditioned to the experimental conditions, but not pre-adapted to the test chemical. Pre-conditioning consists of aerating activated sludge in mineral medium or secondary effluent for 5-7 days at the test temperature. Pre-conditioning sometimes improves the precision of the test methods by reducing blank values. It is considered unnecessary to pre-condition MITI inoculum.I.6.6. Abiotic controlsWhen required, check for the possible abiotic degradation of the test substance by determining the removal of DOC, oxygen uptake or carbon dioxide evolution in sterile controls containing no inoculum. Sterilize by filtration through a membrane (0,2-0,45 micrometre) or by the addition of a suitable toxic substance at an appropriate concentration. If membrane filtration is used, take samples aseptically to maintain sterility. Unless adsorption of the test chemical 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.I.6.7. Number of flasksThe number of flasks in a typical run is described under the headings of each tests.The following type of flask may be used:Test suspension: containing test substance and inoculumInoculum blank: containing only inoculumProcedure control: containing reference substance and inoculumAbiotic sterile control: sterile, containing test substance (see I.6.6)Adsorption control: containing test substance, inoculum and sterilising agentToxicity control: containing test substance, reference substance and inoculumIt is mandatory that determination in test suspension and inoculum blank is made in parallel. It is advisable to make the determinations in the other flasks in parallel as well.This may, however, not always be possible. Ensure that sufficient samples or readings are taken to allow the percentage removal in the 10-day window to be assessed.I.7. DATA AND EVALUATIONIn the calculation of Dt, percentage degradation, the mean values of the duplicate measurement of the parameter in both test vessels and inoculum blank are used. The formulas are set out in the sections below on specific tests. The course of degradation is displayed graphically and the 10-day window is indicated. Calculate and report the percentage removal achieved at the end of the 10-day window and the value at the plateau or at the end of the test, whichever is appropriate.In respirometric tests nitrogen-containing compounds may affect the oxygen uptake because of nitrification (see Annexes II and V).I.7.1. Degradation measured by means of DOC determinationThe percentage degradation Dt at each time a sample was taken should be calculated separately for the flasks containing test substance using mean values of duplicate DOC measurements in order that the validity of the test can be assessed (see I.5.2.). It is calculated using the following equation:Dt = (1CtCbtCoCbo) × 100where: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),Cbo = mean starting concentration of DOC in blank inoculated mineral medium (mg DOC/l),Cbt = mean concentration of DOC blank inoculated mineral medium at time t (mg DOC/l).All concentrations are measured experimentally.I.7.2. Degradation measured by means of specific analysisWhen specific analytical data are available, calculate primary biodegradation from:Dt = Sb SaSb × 100where:Dt = % degradation at time t, normally 28 days,Sa = residual amount of test substance in inoculated medium at end of test (mg),Sb = residual amount of test substance in the blank test with water/medium to which only the test substance was added (mg).I.7.3. Abiotic degradationWhen an abiotic sterile control is used, calculate the percentage abiotic degradation using% abiotic degradation = Cs(o)   Cs(t)Cs(o) × 100whereCs(o) = DOC Concentration in sterile control at day 0Cs(t) = DOC Concentration in sterile control at day tI.8. REPORTINGThe test report shall, if possible, contain the following:- test and reference chemicals, and their purity;- test conditions;- inoculum: nature and sampling site(s), concentration and any pre-conditioning treatment;- proportion and nature of industrial waste present in sewage if known;- test duration and temperature;- in the case of poorly soluble test chemicals, treatment given;- test method applied; scientific reasons and explanation should be given for any change of procedure;- data sheet;- 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 test and reference substances; the lag phase, degradation phase, 10-day window and slope should be clearly indicated (Annex I). If the test has complied with the validity criteria, the mean of the degradation percentages of the flasks containing test substance may be used for the graph.- percentage removal after 10-day window, and at plateau or at end of the test.PART II. DOC DIE-AWAY TEST (Method C.4-A)II.1. PRINCIPLE OF THE METHODA measured volume of inoculated mineral medium containing a known concentration of the test substance (10-40 mg DOC/l) as the nominal sole source of organic carbon is aerated in the dark or diffused light at 22 ± 2  C.Degradation is followed by DOC analysis at frequent intervals over a 28-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. The degree of primary biodegradation may also be calculated from supplemental chemical analysis made at the beginning and end of incubation.II.2. DESCRIPTION OF THE METHODII.2.1. Apparatus(a) Conical flasks, e. g. 250 ml to 2 l, depending on the volume needed for DOC analysis;(b) Shaking machine to accommodate the conical flasks, either with automatic temperature control or used in a constant temperature room, and of sufficient power to maintain aerobic conditions in all flasks;(c) Filtration apparatus, with suitable membranes;(d) DOC analyser;(e) Apparatus for determining dissolved oxygen;(f) Centrifuge.II.2.2. Preparation of mineral mediumFor the preparation of the stock solutions, see I.6.2.Mix 10  ml of solution (a) with 800 ml dilution water, add 1  ml of solutions (b) to (d) and make up to 1 l with dilution water.II.2.3. Preparation and pre-conditioning of inoculumThe inoculum may be derived from a variety of sources: activated sludge; sewage effluents; surface waters; soils or from a mixture of these.See I.6.4., I.6.4.1., I.6.4.2. and I.6.5.II.2.4. Preparation of flasksAs an example, introduce 800 ml portions of mineral medium into 2 l 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 to 10-40 mg DOC/l. Check the pH values and adjust, if necessary, to 7,4. Inoculate the flasks with activated sludge or other source of inocula (see I.6.4.), to give a final concentration not greater than 30 mg suspended solids/l. Also prepare inoculum controls in the mineral medium but without test or reference chemical.If needed, use one vessel to check the possible inhibitory effect of the test chemical by inoculating a solution containing, in the mineral medium, comparable concentrations of both the test and a reference chemical.Also, if required, set up a further, sterile flask to check whether the test chemical is degraded abiotically by using an uninoculated solution of the chemical (see I.6,6.).Additionally, if the test chemical is suspected of being significantly adsorbed on to 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 Table 1). Set up a flask containing the test substance, inoculum and sterilizing agent.Make up the volumes in all flasks to 1 l with mineral medium and, after mixing, take a sample from each flask to determine the initial concentration of DOC (see Annex II.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. Then insert the vessels into the shaking machine for starting the test.II.2.5. Number of flasks in typical runFlasks 1 and 2: Test suspension Flasks 3 and 4: Inoculum blank Flask 5: Procedure control preferably and when necessary: Flask 6: Abiotic sterile control Flask 7: Adsorption control Flask 8: Toxicity controlSee also I.6.7.II.2.6. Performance of the testThroughout the test, determine the concentrations of DOC in each flask in duplicate at known time intervals, sufficiently frequently to be able to determine the start of the 10-day window and the percentage removal at the end of the 10-day window. Take only the minimal volume of test suspension necessary for each determination.Before sampling make good evaporation losses from the flasks by adding dilution water (I.6.1) in the required amount if necessary. Mix the culture medium thoroughly before withdrawing a sample and ensure that material adhering to the walls of the vessels is dissolved or suspended before sampling. Membrane-filter or centrifuge (see Annex II.4) immediately after the sample has been taken. Analyse the filtered or centrifuged samples on the same day, otherwise store at 2-4  C for a maximum of 48 h, or below  18  C for a longer period.II.3. DATA AND REPORTINGII.3.1. Treatment of resultsCalculate the percentage degradation at time t as given under I.7.1. (DOC determination) and, optionally, under 1.7.2. (specific analysis).Record all results on the data sheets provided.II.3.2. Validity of resultsSee I.5.2.II.3.3. ReportingSee I.8.II.4. DATA SHEETAn example of a data sheet is given hereafter.DOC DIE-AWAY TEST1. LABORATORY2. DATE AT START OF TEST3. TEST SUBSTANCEName:Stock solution concentration: mg/l as chemicalInitial concentration in medium, to: mg/l as chemical4. INOCULUMSource:Treatment given:Pre-conditioning, if any:Concentration of suspended solids in reaction mixture: mg/l5. CARBON DETERMINATIONSCarbon analyser:>TABLE>6. EVALUATION OF RAW DATA>TABLE>7. ABIOTIC CONTROL (optional)>TABLE>% abiotic degradation = Cs(o) Cs(t)Cs(o) × 1008. SPECIFIC CHEMICAL ANALYSIS (optional)>TABLE>PART III. MODIFIED OECD SCREENING TEST (Method C.4-B)III.1. PRINCIPLE OF THE METHODA measured volume of mineral medium containing a known concentration of the test substance (10-40 mg DOC/litre) as the nominal sole source of organic carbon is inoculated with 0,5 ml effluent per litre of medium. The mixture is aerated in the dark or diffused light at 22 ± 2  C.Degradation is followed by DOC analysis at frequent intervals over a 28-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. The degree of primary biodegradation may also be calculated from supplemental chemical analysis made at the beginning and end of incubation.III.2. DESCRIPTION OF THE METHODIII.2.1. Apparatus(a) Conical flasks, e.g. 250 ml to 2 litres, depending on the volume needed for DOC analysis;(b) Shaking machine - to accommodate the conical flasks, either with automatic temperature control or used in a constant temperature room, and of sufficient power to maintain aerobic conditions in all flasks;(c) Filtration apparatus, with suitable membranes;(d) DOC analyser;(e) Apparatus for determining dissolved oxygen;(f) Centrifuge.III.2.2. Preparation of mineral mediumFor the preparation of the stock solutions, see I.6.2.Mix 10 ml of solution (a) with 80 ml dilution water, add 1 ml of solutions (b) to (d) and make up to 1 litre with dilution water.This method uses only 0,5 ml effluent/litre as inoculum and therefore the medium may need to be fortified with trace elements and growth factors. This is done by adding 1 ml each of the following solutions per litre of final medium:Trace element solution:Manganese sulfate tetrahydrate, MnSO4. 4H2O 39,9 mgBoric acid, H3BO3 57,2 mgZinc sulfate heptahydrate, ZnSO4. 7H2O 42,8 mgAmmonium heptamolybdate (NH4)6 Mo7O24 34,7 mgFe-chelate (FeCl3 ethylenediamine-tetra-acetic acid) 100,0 mgDissolve in, and make up to 1 000 ml with dilution waterVitamin solution:Yeast extract 15,0 mgDissolve the yeast extract in 100 ml water. Sterilise by passage through a 0,2 micron membrane, or make up freshly.III.2.3. Preparation and pre-conditioning of inoculumThe inoculum is derived from the secondary effluent of a treatment plant or laboratory scale unit receiving predominantly domestic sewage. See I.6.4.2. and I.6.5.0,5 ml per litre of mineral medium is used.III.2.4. Preparation of flasksAs an example, introduce 800 ml portions of mineral medium into 2-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 to 10-40 mg DOC/litre. Check the pH value and adjust, if necessary, to 7,4. Inoculate the flasks with sewage effluent at 0,5 ml/litre (see I.6.4.2.). Also prepare inoculum controls in the mineral medium but without test or reference chemical.If needed, use one vessel to check the possible inhibitory effect of the test chemical by inoculating a solution containing, in the mineral medium, comparable concentrations of both the test and a reference chemical.Also, if required, set up a further, sterile flask to check whether the test chemical is degraded abiotically by using an uninoculated solution of the chemical (see I.6.6.).Additionally, if the test chemical is suspected of being significantly adsorbed on to 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 Table 1). Set up a flask containing the test substance, inoculum and sterilizing agent.Make up the volumes in all flasks to 1 litre with mineral medium and, after mixing, take a sample from each flask to determine the initial concentration of DOC (see Annex II.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. Then insert the vessels into the shaking machine for starting the test.III.2.5. Number of flasks in typical runFlasks 1 and 2: Test suspensionFlasks 3 and 4: Inoculum blankFlask 5: Procedure control and preferably and when necessary:Flask 6: Abiotic sterile controlFlask 7: Adsorption controlFlask 8: Toxicity controlSee also I.6.7.III.2.6. Performance of the testThroughout the test, determine the concentrations of DOC in each flask in duplicate at known time intervals, sufficiently frequently to be able to determine the start of the 10-day window and the percentage removal at the end of the 10-day window. Take only the minimal volume of test suspension necessary for each determination.Before sampling make good evaporation losses from the flasks by adding dilution water (I.6.1) in the required amount if necessary. Mix the culture medium thoroughly before withdrawing a sample and ensure that material adhering to the walls of the vessels is dissolved or suspended before sampling. Membrane-filter or centrifuge (see Annex II.4) immediately after the sample has been taken. Analyse the filtered or centrifuged samples on the same day, otherwise store at 2-4  C for a maximum of 48 h, or below  18  C for a longer period.III.3. DATA AND REPORTINGIII.3.1. Treatment of resultsCalculate the percentage degradation at time t as given under I.7.1. (DOC determination) and, optionally, under I.7.2. (specific analysis).Record all results on the data sheets provided.III.3.2. Validity of resultsSee I.5.2.III.3.3. ReportingSee I.8.III.4. DATA SHEETAn example of a data sheet is given hereafter.MODIFIED OECD SCREENING TEST1. LABORATORY2. DATE AT START OF TEST3. TEST SUBSTANCEName:Stock solution concentration: mg/litre as chemicalInitial concentration in medium, to: mg/litre as chemical4. INOCULUMSource:Treatment given:Pre-conditioning, if any:Concentration of suspended solids in reaction mixture: mg/l5. CARBON DETERMINATIONSCarbon analyser:>TABLE>6. EVALUATION OF RAW DATA>TABLE>7. ABIOTIC CONTROL (optional)>TABLE>% abiotic degradation = Cs(o)   Cs(t)Cs(o) × 1008. SPECIFIC CHEMICAL ANALYSIS (optional)>TABLE>PART IV. CO2 EVOLUTION TEST (Method C.4-C)IV.1. PRINCIPLE OF THE METHODA measured volume of inoculated mineral medium containing a known concentration of the test chemical (10-20 mg DOC or TOC/l) as the nominal sole source of organic carbon is aerated by the passage of carbon dioxide-free air at a controlled rate in the dark or in diffuse light. Degradation is followed over 28 days by determining the carbon dioxide produced, which is trapped in barium or sodium hydroxide and which is measured by titration of the residual hydroxide or as inorganic carbon. The amount of carbon dioxide produced from the test chemical (corrected for that derived from the blank inoculum) is expressed as a percentage of ThCO2. The degree of biodegradation may also be calculated from supplemental DOC analysis made at the beginning and end of incubation.IV.2. DESCRIPTION OF THE METHODIV.2.1. Apparatus(a) Flasks, 2-5 litres, each fitted with an aeration tube reaching nearly the bottom of the vessel and an outlet;(b) Magnetic stirrers, when assessing poorly soluble chemicals;(c) Gas-absorption bottles;(d) Device for controlling and measuring airflow;(e) Apparatus for carbon dioxide scrubbing, for preparation of air which is free from carbon dioxide; alternatively, a mixture of CO2-free oxygen and CO2-free nitrogen, from gas cylinders, in the correct proportions (20 % O2:80 % N2) may be used;(f) Device for determination of carbon dioxide, either titrimetrically or by some form of inorganic carbon analyser;(g)  Membrane filtration device (optional);(h) DOC analyser (optional).IV.2.2. Preparation of mineral mediumFor the preparation of the stock solutions, see I.6.2.Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of solutions (b) to (d) and make up to 1 l with dilution water.IV.2.3. Preparation and pre-conditioning of inoculumThe inoculum may be derived from a variety of sources: activated sludge; sewage effluents; surface waters; soils or from a mixture of these.See I.6.4., I.6.4.1., I.6.4.2. and I.6.5.IV.2.4. Preparation of flasksAs an example the following volumes and weights indicate the values for 5-litre flasks containing 3 l of suspension. If smaller volumes are used modify the values accordingly, but ensure that the carbon dioxide formed can be measured accurately.To each 5-litre flask add 2 400 ml mineral medium. Add an appropriate volume of the prepared activated sludge (see I.6.4.1. and I.6.5.) to give a concentration of suspended solids of not more than 30 mg/l in the final 3 l of inoculated mixture. Alternatively first dilute the prepared sludge to give a suspension of 500-1 000 mg/l in the mineral medium before adding an aliquot to the contents of the 5-litre flask to attain a concentration of 30 mg/l; this ensures greater precision. Other sources of inoculum may be used (see I.6.4.2.).Aerate these inoculated mixtures with CO2-free air overnight to purge the system of carbon dioxide.Add the test material and reference substance, separately, as known volume of stock solutions, to replicate flasks to yield concentrations, contributed by the added chemicals, of 10 to 20 mg DOC or TOC/l; leave some flasks without addition of chemicals as inoculum controls. Add poorly soluble test substances directly to the flasks on a weight or volume basis or handle as described in Annex III.If required, use one flask to check the possible inhibitory effect of the test chemical by adding both the test and reference chemicals at the same concentrations as present in the other flasks.Also, if required, use a sterile flask to check whether the test chemical is degraded abiotically by using an uninoculated solution of the chemical (see I.6.6.). Sterilise by the addition of a toxic substance at an appropriate concentration.Make up the volumes of suspensions in all flasks to 3 l by the addition of mineral medium previously aerated with CO2-free air. Optionally, samples may be withdrawn for analysis of DOC (see Annex II.4.) and/or specific analysis. Connect the absorption bottles to the air outlets of the flasks.If barium hydroxide is used, connect three absorption bottles, each containing 100 ml of 0,0125 M barium hydroxide solution, in series to each 5-litre flask. The solution must be free of precipitated sulphate and carbonate and its strength must be determined immediately before use. If sodium hydroxide is used, connect two traps, the second acting as a control to demonstrate that all the carbon dioxide was absorbed in the first. Absorption bottles fitted with serum bottle closures are suitable. Add 200 ml 0,05 M sodium hydroxide to each bottle, which is sufficient to absorb the total quantity of carbon dioxide evolved when the test chemical is completely degraded. The sodium hydroxide solution, even when freshly prepared, will contain traces of carbonates; this is corrected by deduction of the carbonate in the blank.IV.2.5. Number of flasks in a typical runFlasks 1 and 2: Test suspensionFlasks 3 and 4: Inoculum blankFlask 5: Procedure control and, preferably and when necessary:Flask 6: Abiotic sterile controlFlask 7: Toxicity controlSee also I.6.7.IV.2.6. Performance of the testStart the test by bubbling CO2-free air through the suspensions at a rate of 30-100 ml/min. Take samples of the carbon dioxide absorbent periodically for analysis of the CO2-content. During the first ten days it is recommended that analyses should be made every second or third day and then every fifth day until the 28th day so that the 10-day window period can be identified.On the 28th day, withdraw samples (optionally) for DOC and/or specific analysis, measure the pH of the suspensions and add 1 ml of concentrated hydrochloric acid to each flask; aerate the flasks overnight to drive off the carbon dioxide present in the test suspensions. On day 29 make the last analysis of evolved carbon dioxide.On the days of measurement of CO2, disconnect the barium hydroxide absorber closest to the flask and titrate the hydroxide solution with HCl 0,05 M using phenolphthalein as the indicator. Move the remaining absorbers one place closer to the flask and place a new absorber containing 100 ml fresh 0,0125 M barium hydroxide at the far end of the series. Make titrations as needed, for example, when substantial precipitation is seen in the first trap and before any is evident in the second, or at least weekly. Alternatively, with NaOH as absorbent, withdraw with a syringe a small sample (depending on the characteristics of the carbon analyser used) of the sodium hydroxide solution in the absorber nearer to the flask. Inject the sample into the IC part of the carbon analyser for analysis of evolved carbon dioxide directly.Analyse the contents of the second trap only at the end of the test to correct for any carry over of carbon dioxide.IV.3. DATA AND REPORTINGIV.3.1. Treatment of resultsThe amount of CO2 trapped in an absorber when titrated is given by:mgCO2 = (100 × CB 0,5 × V × CA) × 44where:V = volume of HCl used for titration of the 100 ml in the absorber (ml),CB = concentration of the barium hydroxide solution (M),CA = concentration of the hydrochloric acid solution (M),if CB is 0,0125 M and CA is 0,05 M, the titration for 100 ml barium hydroxide is 50 ml and the weight of CO2 is given by:0.052 × 44 × ml HCl titrated = 1.1 × ml HClThus, in this case, to convert volume of HCl titrated to mg CO2produced the factor is 1,1.Calculate the weights of CO2 produced from the inoculum alone and from the inoculum plus test chemical using the respective titration values and the difference is the weight of CO2 produced from the test chemical alone.For example, if the inoculum alone gives a titration of 48 ml and inoculum plus test chemical gives 45 ml,CO2 from inoculum = 1,1 × (50-48) = 2,2 mgCO2 from inoculum plus test chemical = 1,1 × (50-45) = 5,5 mg and thus the weight of CO2 produced from the test chemical is 3,3 mg.The percentage biodegradation is calculated from:% degradation = ThCO2 × mg test chemical addedmg CO2 produced × 100or,% degradation = mg TOC added in test × 3.67mg CO2 produced × 1003,67 being the conversion factor (44/12) for carbon to carbon dioxide.Obtain the percentage degradation after any time interval by adding the percentage of ThCO2 values calculated for each of the days, up to that time, on which it was measured.For sodium hydroxide absorbers, calculate the amount of carbon dioxide produced, expressed as IC (mg), by multiplying the concentration of IC in the absorbent by the volume of the absorbent.Calculate the percentage degradation from:% of ThCO2 = mg IC from test flask   mg IC from blankmg TOC added as test chemical × 100Calculate DOC removals (optional) as described under I.7. Record these and all other results on the data sheets provided.IV.3.2. Validity of resultsThe IC content of the test chemical suspension in the mineral medium at the beginning of the test must be less than 5 % of the TC, and the total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/l medium. If values greater than 70 mg CO2/litre are obtained, the data and experimental technique should be examined critically.See also I.5.2.IV.3.3. ReportingSee I.8.IV.4. DATA SHEETAn example of a data sheet is given hereafter.CARBON DIOXIDE EVOLUTION TEST1. LABORATORY2. DATE AT START OF TEST3. TEST SUBSTANCEName:Stock solution concentration: mg/litre as chemicalInitial conc. in medium: mg/litre as chemicalTotal C added to flask: mg CThCO2: mg CO24. INOCULUMSource:Treatment given:Pre-conditioning if any:Concentration of suspended solids in reaction mixture: mg/litre5. CARBON DIOXIDE PRODUCTION AND DEGRADABILITYMethod: Ba(OH)2/NaOH/other>TABLE>6. CARBON ANALYSIS (optional)Carbon analyser:>TABLE>% DOC removed = 1 Ct Cb(t) CoCb(o) × 1007. ABIOTIC DEGRADATION (optional)% abiotic degradation = CO2 formation in sterile flask after 28 day (mg)ThCO2 (mg) × 100PART V. MANOMETRIC RESPIROMETRY TEST (Method C.4-D)V.1. PRINCIPLE OF THE METHODA measured volume of inoculated mineral medium, containing a known concentration of test chemical (100 mg/litre of the test substance, to give at least 50-100 mg ThOD/litre) as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (± 1  C or closer) for up to 28 days. The consumption of oxygen is determined either by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas volume in the respirometer flask, or from the change in volume or pressure (or a combination of the two) in the apparatus. Evolved carbon dioxide is absorbed in a solution of potassium hydroxide or another suitable absorbent. The amount of oxygen taken up by the test chemical (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD or COD. Optionally, primary biodegradation may also be calculated from supplemental specific analysis made at the beginning and end of incubation, and ultimate biodegradation by DOC analysis.V.2. DESCRIPTION OF THE METHODV.2.1. Apparatus(a) suitable respirometer;(b) temperature control, maintaining ± 1  C or better;(c) membrane-filtration assembly (optional);(d) carbon analyser (optional).V.2.2. Preparation of mineral mediumFor the preparation of the stock solutions, see I.6.2.Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of solutions (b) to (d) and make up to 1 litre with dilution water.V.2.3. Preparation and pre-conditioning of inoculumThe inoculum may be derived from a variety of sources: activated sludge; sewage effluents; surface waters and soils or from a mixture of these.See I.6.4., I.6.4.1., I.6.4.2. and I.6.5.V.2.4. Preparation of flasksPrepare solutions of the test and reference chemicals, in separate batches, in mineral medium equivalent to a concentration, normally, of 100 mg chemical/litre (giving at least 50-100 mg ThOD/litre), using stock solutions.Calculate the ThOD on the basis of formation of ammonium salts unless nitrification is anticipated, when the calculation should be based on nitrate formation (see Annex II.2.)Determine the pH values and if necessary adjust to 7,4 ± 0,2.Poorly soluble substances should be added at a later stage (see below).If the toxicity of the test chemical is to be determined, prepare a further solution in mineral medium containing both test and reference chemicals at the same concentrations as in the individual solutions.If measurement of the physico-chemical uptake of oxygen is required, prepare a solution of the test chemical at, normally, 100 mg ThOD/litre which has been sterilised by the addition of a suitable toxic substance (see I.6,6.).Introduce the requisite volume of solutions of test and reference chemicals, respectively, into at least duplicate flasks. Add to further flasks mineral medium only (for inoculum controls) and, if required, the mixed test/reference chemical solution and the sterile solution.If the test chemical is poorly soluble, add it directly at this stage on a weight or volume basis or handle it as described in Annex III. Add potassium hydroxide, soda lime pellets or other absorbent to the CO2-absorber compartments.V.2.5. Number of flasks in a typical runFlasks 1 and 2: Test suspensionFlasks 3 and 4: Inoculum blankFlask 5: Procedure controlpreferably, and when necessary:Flask 6: Sterile controlFlask 7: Toxicity controlSee also I.6.7.V.2.6. Performance of the testAllow the vessels to reach the desired temperature and inoculate appropriate vessels with prepared activated sludge or other source of inoculum to give a concentration of suspended solids not greater than 30 mg/litre. Assemble the equipment, start the stirrer and check for air-tightness, and start the measurement of oxygen uptake. Usually no further attention is required other than taking the necessary readings and making daily checks to see that the correct temperature and adequate stirring are maintained.Calculate the oxygen uptake from the readings taken at regular and frequent intervals, using the methods given by the manufacturer of the equipment. At the end of incubation, normally 28 days, measure the pH of the contents of the flasks, especially if oxygen uptakes are low or greater than ThODNH4 (for nitrogen-containing compounds).If required, withdraw samples from the respirometer flasks, initially and finally, for analysis of DOC or specific chemical (see Annex II.4). At the initial withdrawal, ensure that the volume of test suspension remaining in the flask is known. When oxygen is taken up by N-containing test substance, determine the increase in concentration of nitrite and nitrate over 28 days and calculate the correction for the oxygen consumed by nitrification (Annex V).V.3. DATA AND REPORTINGV.3.1. Treatment of resultsDivide the oxygen uptake (mg) of the test chemical after a given time (corrected for that by the blank inoculum control after the same time) by the weight of the test chemical used. This yields the BOD expressed as mg oxygen/mg test chemical, that isBOD = mg O2 uptake by test chemical   mg O2 uptake by blankmg test chemical in flask= mg O2 per mg test chemical.calculate the percentage biodegradation either from:% biodegradation = % ThOD =  BOD (mg O2/mg chemical)ThOD (mgO2 chemical) × 100,or form% COD =  BOD (mg O2/mg chemical) COD (mg O2/mg chemical) × 100It should be noted that these two methods do not necessarily give the same value; it is preferable to use the former method.For test substances containing nitrogen, use the appropriate ThOD (NH4 or NO3) according to what is known or expected about the occurrence of nitrification (Annex II.2). If nitrification occurs but is not complete, calculate a correction for the oxygen consumed by nitrification from the changes in concentration of nitrite and nitrate (Annex V).When optional determinations of organic carbon and/or specific chemical are made, calculate the percentage degradation, as described under I.7.Record all results on the data sheets attached.V.3.2. Validity of resultsThe oxygen uptake of the inoculum blank is normally 20-30 mg O2/litre and should not be greater than 60 mg/litre in 28 days. Values higher than 60 mg/litre require critical examination of the data and experimental techniques. If the pH value is outside the range 6-8,5 and the oxygen consumption by the test chemical is less than 60 %, the test should be repeated with a lower concentration of test chemical.See also I.5.2.V.3.3. ReportingSee I.8.V.4. DATA SHEETAn example of a data sheet is given hereafter.MANOMETRIC RESPIROMETRY TEST1. LABORATORY2. DATE AT START OF TEST3. TEST SUBSTANCEName:Stock solution concentration: mg/litreInitial concentration in medium, Co: mg/litreVolume in test flask (V): mlThOD or COD: mg O2/mg test substance (NH4, NO3)4. INOCULUMSource:Treatment given:Pre-conditioning, if any:Concentration of suspended solids in reaction mixture: mg/l5. OXYGEN UPTAKE: BIODEGRADABILITY>TABLE>6. CORRECTION FOR NITRIFICATION (see Annex V)>TABLE>7. CARBON ANALYSIS (optional)Carbon analyser:>TABLE>% DOC removed = 1    Ct   Cblt Co   Cblo × 1008. SPECIFIC CHEMICAL (optional)Sb = concentration in physico-chemical (sterile) control at 28 daysSa = concentration in inoculated flask at 28 days.% biodegradation = Sb   SaSb × 1009. ABIOTIC DEGRADATION (optional)a = oxygen consumption in sterile flasks after 28 days, (mg)oxygen consumption per mg test chemical = CoVa (see sections 1 and 3)% abiotic degradation = CoV × ThODa × 100PART VI. CLOSED BOTTLE TEST (Method C.4-E)VI.1. PRINCIPLE OF THE TEST METHODThe solution of the test chemical in mineral medium, usually at 2-5 mg/litre, is inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely full, closed bottles in the dark at constant temperature. Degradation is followed by analysis of dissolved oxygen over a 28-day period. The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of ThOD or COD.VI.2. DESCRIPTION OF THE METHODVI.2.1. Apparatusa) BOD bottles, with glass stoppers, e.g. 250-300 ml;b) Water bath or incubator, for keeping bottles at constant temperature (± 1  C or better) with the exclusion of light;c) Large glass bottles (2-5 litres) for the preparation of media and for filling the BOD bottles;d) Oxygen electrode and meter, or equipment and reagents for Winkler titration.VI.2.2. Preparation of mineral mediumFor the preparation of the stock solutions, see I.6.2.Mix 1 (one) ml of solution (a) to (d) and make up to 1 litre with dilution water.VI.2.3. Preparation of the inoculumThe inoculum is normally derived from the secondary effluent of a treatment plant or laboratory-scale unit receiving predominantly domestic sewage. An alternative source for the inoculum is surface water. Normally use from one drop (0,05 ml) to 5 ml of filtrate per litre of medium; trials may be needed to discover the optimum volume for a given effluent (See I.6.4.2. and I.6.5.).VI.2.4. Preparation of flasksStrongly aerate mineral medium for at least 20 min. Carry out each test series with mineral medium derived from the same batch. Generally, the medium is ready for use after standing for 20 h, at the test temperature. Determine the concentration of dissolved oxygen for control purposes; the value should be about 9 mg/litre at 20  C. Conduct all transfer and filling operations of the air-saturated medium bubble-free, for example, by the use of siphons.Prepare parallel groups of BOD bottles for the determination of the test and reference chemicals in simultaneous experimental series. Assemble a sufficient number of BOD bottles, including inoculum blanks, to allow at least duplicate measurements of oxygen consumption to be made at the desired test intervals, for example, after 0, 7, 14, 21 and 28 days. To ensure being able to identify the 10-day window, more bottles may be required.Add fully aerated mineral medium to large bottles so that they are about one-third full. Then add sufficient of the stock solutions of the test chemical and reference chemical to separate large bottles so that the final concentration of the chemicals is normally not greater than 10 mg/litre. Add no chemicals to the blank control medium contained in a further large bottle.In order to ensure that the inoculum activity is not limited, the concentration of dissolved oxygen must not fall below 0,5 mg/litre in the BOD bottles. This limits the concentration of test chemical to about 2 mg/litre. However, for poorly degradable compounds and those with a low ThOD, 5-10 mg/litre can be used. In some cases, it would be advisable to run parallel series of test chemical at two different concentrations, for example, 2 and 5 mg/litre. Normally, calculate the ThOD on the basis of formation of ammonium salts but, if nitrification is expected or known to occur, calculate on the basis of the formation of nitrate (ThODNO3: see Annex II.2). However, if nitrification is not complete but does occur, correct for the changes in concentration of nitrite and nitrate, determined by analysis, (see Annex V).If the toxicity of the test chemical is to be investigated (in the case, for example, of a previous low biodegradability value having been found), another series of bottles is necessary.Prepare another large bottle to contain aerated mineral medium (to about one-third of its volume) plus test chemical and reference chemical at final concentrations normally the same as those in the other large bottles.Inoculate the solutions in the large bottles with secondary effluent (one drop or about 0,05 ml, to 5 ml/litre) or with another source such as river water (see I.6.4.2.). Finally, make up the solutions to volume with aerated mineral medium using a hose which reaches down to the bottom of the bottle to achieve adequate mixing.VI.2.5. Number of flasks in a typical runIn a typical run the following bottles are used:at least 10 containing test chemical and inoculum (test suspension),at least 10 containing only inoculum (inoculum blank),at least 10 containing reference chemical and inoculum (procedure control),and, when necessary, 6 bottles containing test chemical, reference chemical and inoculum (toxicity control). However, to ensure being able to identify the 10-day window, about twice as many bottles would be necessary.VI.2.6. Performance of the testDispense each prepared solution immediately into the respective group of BOD bottles by hose from the lower quarter (not the bottom) of the appropriate large bottle, so that all the BOD bottles are completely filled. Tap gently to remove any air bubbles. Analyse the zero-time bottles immediately for dissolved oxygen by the Winkler or electrode methods. The contents of the bottles can be preserved for later analysis by the Winkler method by adding manganese (II) sulfate and sodium hydroxide (the first Winkler reagent). Store the carefully stoppered bottles, containing the oxygen fixed as brown manganese (III) hydrated oxide, in the dark at 10-20  C for no longer than 24 hours before proceeding with the remaining steps of the Winkler method. Stopper the remaining replicate bottles ensuring that no air bubbles are enclosed, and incubate at 20  C in the dark. Each series must be accompanied by a complete parallel series for the determination of the inoculated blank medium. Withdraw at least duplicate bottles of all series for dissolved oxygen analysis at time intervals (at least weekly) over the 28 days incubation.Weekly samples should allow the assessment of percentage removal in a 14-day window, whereas sampling every 3-4 days should allow the 10-day window to be identified, which would require about twice as many bottles.For N-containing test substances, corrections for uptake of oxygen by any nitrification occurring should be made. To do this, use the O2-electrode method for determining the concentration of dissolved oxygen and then withdraw a sample from the BOD bottle for analysis for nitrite and nitrate. From the increase in concentration of nitrite and nitrate, calculate the oxygen used (see Annex V).VI.3. DATA AND REPORTINGVI.3.1. Treatment of resultsFirst calculate the BOD exerted after each time period by subtracting the oxygen depletion (mg O2/litre) of the inoculum blank from that exhibited by the test chemical. Divide this corrected depletion by the concentration (mg/litre) of the test chemical, to obtain the specific BOD as mg oxygen per mg test chemical. Calculate the percentage biodegradability by dividing the specific BOD by the specific ThOD (calculated according to Annex II.2) or COD (determined by analysis, see Annex II.3),thus:BOD = mg O2 uptake by test chemical   mg O2 uptake by blankmg test chemical in flask= mg O2 per mg test chemical% degradation =  BOD (mg O2/mg test chemical) ThOD (mg O2/mg test chemical) × 100or% degradation=  BOD (mg O2/mg test chemical) COD (mg O2/mg test chemical) × 100It should be noted that these two methods do not necessarily give same value; it is preferable to use the former method.For test substances containing nitrogen, use the appropriate ThOD (NH4 or NO3) according to what is known or expected about the occurrence of nitrification (Annex II.2). If nitrification occurs but is not complete, calculate a correction for the oxygen consumed by nitrification from the changes in concentration of nitrite and nitrate (Annex V).VI.3.2. Validity of resultsOxygen depletion in the inoculum blank should not exceed 1,5 mg dissolved oxygen/litre after 28 days. Values higher than this require investigation of the experimental techniques. The residual concentration of oxygen in the test bottles should not fall below 0,5 mg/litre at any time. Such low oxygen levels are valid only if the method of determining dissolved oxygen used is capable of measuring such levels accurately.See also I.5.2.VI.3.3. ReportingSee I.8.VI.4. DATA SHEETAn example of a data sheet is given hereafter.CLOSED BOTTLE TEST1. LABORATORY2. DATE AT START OF TEST3. TEST SUBSTANCEName:Stock solution concentration: mg/litreInitial concentration in bottle: mg/litreThOD or COD: mgO2/mg test substance4. INOCULUMSource:Treatment given:Pre-conditioning if any:Concentration in the reaction mixture: mg/litre5. DO DETERMINATIONMethod: Winkler/electrode>TABLE>6. CORRECTION FOR NITRIFICATION (see Annex V)>TABLE>7. DO DEPLETION: % DEGRADATION>TABLE>mto = value in the test flask at time 0mtx = value in the test flask at time xmbo = mean blank value at time 0mbx = mean blank value at time xApply also correction for nitrification from iii+vi in section 6.8. BLANK DO DEPLETIONSOxygen consumption by blank: (mbo   mb28) mg/litre. This consumption is important for the validity of the test. It should be less than 1,5 mg/litre.PART VII. M.I.T.I. TEST (Method C.4-F)VII.1. PRINCIPLE OF THE METHODThe oxygen uptake by a stirred solution, or suspension, of the test chemical in a mineral medium, inoculated with specially grown, unadapted micro-organisms, is measured automatically over a period of 28 days in a darkened, enclosed respirometer at 25 ± 1  C. Evolved carbon dioxide is absorbed by soda lime. Biodegradability is expressed as the percentage oxygen uptake (corrected for blank uptake) of the theoretical uptake (ThOD). The percentage of primary biodegradability is also calculated from supplemental specific chemical analysis made at the beginning and end of incubation and, optionally, by DOC analysis.VII.2. DESCRIPTION OF THE METHODVII.2.1. Apparatus(a) Automatic electrolytic BOD meter or respirometer normally equipped with 6 bottles, 300 ml each and equipped with cups to contain CO2 absorbent;(b) Constant temperature room and/or water-bath at 25  C ± 1  C or better;(c) Membrane-filtration assembly (optional);(d) Carbon analyser (optional).VII.2.2. Preparation of mineral mediumPrepare the following stock solutions, using analytical grade reagents and water (I.6.1.):(a) Monopotassium dihydrogen ortho phosphate, KH2PO4 8,50 gDipotassium monohydrogen ortho phosphate, K2HPO4 21,75 gDisodium monohydrogen ortho phosphate dodecahydrate Na2HPO4 12 H2O 44,60 gAmmonium chloride, NH4Cl 1,70 gDissolve in water and make up to 1 litreThe pH value of the solution should be 7,2(b) Magnesium sulphate heptahydrate, MgSO4 7 H2O 22,50 gDissolve in water and make up to 1 litre(c) Calcium chloride anhydrous, CaCl2 27,50 gDissolve in water and make up to 1 litre(d) Iron (III) chloride hexahydrate, FeCl3 6 H2O 0,25 gDissolve in water and make up to 1 litreTake 3 ml of each solution (a), (b), (c) and (d) and make up to 1 litre.VII.2.3. Preparation of inoculumCollect fresh samples from no fewer than ten sites, mainly in areas where a variety of chemicals are used and discharged. From sites such as sewage treatment works, industrial waste-water treatment, rivers, lakes, seas, collect 1 l samples of sludge, surface soil, water, etc. and mix thoroughly together. After removing floating matter and allowing to stand, adjust the supernatant to pH 7 ± 1 with sodium hydroxide or phosphoric acid.Use an appropriate volume of the filtered supernatant to fill a fill-and-draw activated sludge vessel and aerate the liquid for about 23 1/2 h. Thirty minutes after stopping aeration, discard about one third of the whole volume of supernatant and add an equal volume of a solution (pH 7) containing 0,1 % each of glucose, peptone and monopotassium ortho phosphate, to the settled material and recommence aeration. Repeat this procedure once per day. The sludge unit must be operated according to good practice: effluents should be clear, temperature should be kept at 25 ± 2  C, pH should be 7 ± 1, sludge should settle well, sufficient aeration to keep the mixture aerobic at all times, protozoa should be present and the activity of the sludge should be tested against a reference substance at least every three months. Do not use sludge as inoculum until after at least one month's operation, but not after more than four months. Thereafter, sample from at least 10 sites at regular intervals, once every three months.In order to maintain fresh and old sludge at the same activity, mix the filtered supernatant of an activated sludge in use with an equal volume of the filtered supernatant of a freshly collected ten-source mixture and culture the combined liquor as above. Take sludge for use as inoculum 18-24 h after the unit has been fed.VII.2.4. Preparation of flasksPrepare the following six flasks:Nr. 1: test chemical in dilution water at 100 mg/lNr. 2, 3 and 4: test chemical in mineral medium at 100 mg/lNr. 5: reference chemical (e.g. aniline) in mineral medium at 100 mg/lNr. 6: mineral medium onlyAdd poorly soluble test chemicals directly on a weight or volume basis or handle as described in Annex III, except that neither solvents nor emulsifying agents should be used. Add the CO2 absorbent to all flasks in the special cups provided. Adjust the pH in flasks nr. 2, 3 and 4 to 7,0.VII.2.5. Performance of the testInoculate flasks nr. 2, 3 and 4 (test suspensions), nr. 5 (activity control) and nr. 6 (inoculum blank) with a small volume of the inoculum to give a concentration of 30 mg/l suspended solids. N  inoculum is added to flask nr. 1 which serves as an abiotic control. Assemble the equipment, check for air-tightness, start the stirrers, and start the measurement of oxygen uptake under conditions of darkness. Daily check the temperature, stirrer and coulometric oxygen uptake recorder, and note any changes in colour of the contents of the flasks. Read the oxygen uptakes for the six flasks directly by an appropriate method, for example, from the six-point chart recorder, which produces a BOD curve. At the end of incubation, normally 28 days, measure the pH of the contents of the flasks and determine the concentration of the residual test chemical and any intermediate and, in the case of water soluble substance, the concentration of DOC (Annex II.4). Take special care in the case of volatile chemicals. If nitrification is anticipated, determine nitrate and nitrite concentration, if possible.VII.3. DATA AND REPORTINGVII.3.1. Treatment of resultsDivide the oxygen uptake (mg) by the test chemical after a given time, corrected for that taken up by the blank inoculum control after the same time, by the weight of the test chemical used. This yields the BOD expressed as mg oxygen/mg test chemical, that is:BOD = mg O2 uptake by test chemical   mg O2 uptake by blankmg test chemical in flask = mg O2/mg test chemical.The percentage biodegradation is then obtained from:% biodegradation = % ThOD =   BOD (mg O2/mg chemical)  ThOD (mg O2/mg chemical) × 100For mixtures, calculate the ThOD from the elemental analysis, as for simple compound. Use the appropriate ThOD (ThODNH4 or ThODNO3) according to whether nitrification is absent or complete (Annex II.2). If however, nitrification occurs but is incomplete, make a correction for the oxygen consumed by nitrification calculated from the changes in concentrations of nitrite and nitrate (Annex V).Calculate the percentage primary biodegradation from loss of specific (parent) chemical (see I.7,2).Dt = Sb   SaSb × 100 %If there has been a loss of test chemical in the flask nr. 1 measuring physico-chemical removal, report this and use the concentration of test chemical (Sb) after 28 days in this flask to calculate the percentage biodegradation.When determinations of DOC are made (optional), calculate the percentage ultimate biodegradation from:Dt = 1    Ct    Cbt Co   Cbo × 100%as described under point I.7.1. If there has been a loss of DOC in the flask nr. 1, measuring physico-chemical removal, use the DOC concentration in this flask to calculate the percentage biodegradation.Record all results on the data sheets attached.VII.3.2. Validity of resultsThe oxygen uptake of the inoculum blank is normally 20-30 mg O2/l and should not be greater than 60 mg/l in 28 days. Values higher than 60 mg/l require critical examination of the data and experimental techniques. If the pH value is outside the range 6-8,5 and the oxygen consumption by the test chemical is less than 60 %, the test should be repeated with a lower concentration of test chemical.See also I.5.2.If the percentage degradation of aniline calculated from the oxygen consumption does not exceed 40 % after 7 days and 65 % after 14 days, the test is regarded as invalid.VII.3.3. ReportingSee I.8.VII.4. DATA SHEETAn example of a data sheet is given below.MITI (I) TEST1. LABORATORY2. DATE AT START OF TEST3. TEST SUBSTANCEName:Stock solution concentration: mg/l as chemicalInitial concentration in medium, Co: mg/l as chemicalVolume of reaction mixture, V: mlThOD: mg O2/l4. INOCULUMSludge sampling sites:1) ...2) ...3) ...4) ...5) ...6) ...7) ...8) ...9) ...10) ...Concentration of suspended solids in activated sludge after acclimatization with synthetic sewage = ... mg/lVolume of activated sludge per litre of final medium = ... mlConcentration of sludge in final medium = ... mg/l5. OXYGEN UPTAKE: BIODEGRADABILITYType of respirometer used:>TABLE>6. CARBON ANALYSIS (optional):Carbon analyser:>TABLE>% DOC removed: a   (b   c)a × 1007. SPECIFIC CHEMICAL ANALYTICAL DATA>TABLE>% degradation = Sb   SaSb × 100Calculate % degradation for flasks a1, a2 and a3 respectively8. REMARKSBOD curve against time, if available, should be attached.ANNEX I ABBREVIATIONS AND DEFINITIONSDO: Dissolved oxygen (mg/l) is the concentration of oxygen dissolved in an aqueous sample.BOD: Biochemical oxygen demand (g) is the amount of oxygen consumed by micro-organisms when metabolizing a test compound; also expressed as g oxygen uptake per g test compound. (See method C.5).COD: Chemical oxygen demand (g) is the amount of oxygen consumed during oxidation of a test compound with hot, acidic dichromate; it provides a measure of the amount of oxidisable matter present; also expressed as g oxygen consumed per g test compound. (See method C.6).DOC: Dissolved organic carbon is the organic carbon present in solution or that which passes through a 0,45 micrometre filter or remains in the supernatant after centrifuging at 40 000 m.s 2 (± 4 000 g) for 15 min.ThOD: Theoretical oxygen demand (mg) is the total amount of oxygen required to oxidise a chemical completely; it is calculated from the molecular formula (see Annex II.2) and is also expressed as mg oxygen required per mg test compound.ThCO2: Theoretical carbon dioxide (mg) is the quantity of carbon dioxide calculated to be produced from the known or measured carbon content of the test compound when fully mineralized; also expressed as mg carbon dioxide evolved per mg test compound.TOC: Total organic carbon of a sample is the sum of the organic carbon in solution and in suspension.IC: Inorganic carbonTC: Total carbon, is the sum of the organic and inorganic carbon present in a sample.Primary Biodegradation:is the alteration in the chemical structure of a substance, brought about by biological action, resulting in the loss of specific property of that substance.Ultimate Biodegradation (aerobic):is the level of degradation achieved when the test compound is totally utilised by micro-organisms resulting in the production of carbon dioxide, water, mineral salts and new microbial cellular constituents (biomass).Readily Biodegradable:an arbitrary classification of chemicals which have passed certain specified screening tests for ultimate biodegradability; these tests are so stringent that it is assumed that such compounds will rapidly and completely biodegrade in aquatic environments under aerobic conditions.Inherently Biodegradable:a classification of chemicals for which there is unequivocal evidence of biodegradation (primary or ultimate) in any recognized test of biodegradability.Treatability:is the amenability of compounds to removal during biological wastewater treatment without adversely affecting the normal operation of the treatment processes. Generally, readily biodegradable compounds are treatable but not all inherently biodegradable compounds are. Abiotic processes may also operate.Lag time is the time from inoculation, in a die-away test, until the degradation percentage has increased to at least 10 %. The lag time is often highly variable and poorly reproducible.Degradation time is the time from the end of the lag time till the time that 90 % of maximum level of degradation has been reached.10-day window is the 10 days immediately following the attainment of 10 % degradation.ANNEX IICALCULATION AND DETERMINATION OF SUITABLE SUMMARY PARAMETERSDepending on the method chosen, certain summary parameters will be required. The following section describes the derivation of these values. The use of these parameters is described in the individual methods.1. Carbon ContentThe carbon content is calculated from the known elemental composition or determined by elemental analysis of the test substance.2. Theoretical oxygen demand (ThOD)The theoretical oxygen demand (ThOD) may be calculated if the elemental composition is known or determined by elemental analysis. It is for the compound:CcHhClclNnNanaOoPpSs without nitrification,ThODNH4 = 16 [2 c + 1/2 (h   cl   3 n) + 3 s + 5/2 p + 1/2 na   o]MW mg/mgor with nitrification,ThODNO3 = 16 [2 c + 1/2 (h   cl) + 5/2 n + 3 s + 5/2 p + 1/2 na   o]MW mg/mg3. Chemical Oxygen Demand (COD)The Chemical oxygen demand (COD) is determined according to method C.6.4. Dissolved Organic Carbon (DOC)Dissolved organic carbon (DOC) is by definition the organic carbon of any chemical or mixture in water passing through a 0,45 micrometre filter.Samples from the test vessels are withdrawn and filtered immediately in the filtration apparatus using an appropriate membrane filter. The first 20 ml (amount can be reduced when using small filters) of the filtrate are discarded. Volumes of 10-20 ml or lower, if injected (volume depending on the amount required for carbon analyser) are retained for carbon analysis. The DOC-concentration is determined by means of an organic carbon analyser which is capable of accurately measuring a carbon concentration equivalent or lower than 10 % of the initial DOC-concentration used in the test.Filtered samples which cannot be analysed on the same working day can be preserved by storage in a refrigerator at 2-4  C for 48 h, or below  18  C for longer periods.Remarks:Membrane filters are often impregnated with surfactants for hydrophilisation. Thus the filter may contain up to several mg of soluble organic carbon which would interfere in the biodegradability determinations. Surfactants and other soluble organic compounds are removed from the filters by boiling them in deionised water for three periods each of one hour. The filters may then be stored in water for one week. If disposable filter cartridges are used each lot must be checked to confirm that it does not release soluble organic carbon.Depending on the type of membrane filter the test chemical may be retained by adsorption. It may therefore be advisable to ensure that the test chemical is not retained by the filter.Centrifugation at 40 000 m.sec 2 (4 000 g) for 15 min may be used for differentiation of TOC versus DOC instead of filtration. The method is not reliable at initial concentration of TABLE POSITION>0,05 M borax + 0,1 N NaOH>TABLE>