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This Test Guideline describes two methods. These tests are applicable only to water insoluble (solubility < 10-6 g/1) substances.

The first method described (Method A) is designed to provide information on the transportation and sedimentation of insoluble particles in water and air. There are several standard methods available which meet the sensitivity requirements: the sedimentation, the centrifugation or the coulter counter. The sample should be subjected to a simple light microscopic examination to determine the approximate nature of the particles. Data are obtained for a 3 size ranges ›200µm, ‹2 µm and the region 2to 200µm. Method A, which determines the effective hydrodynamic radius, Rs (only in the range 2 μm < Rs < 100 μm), can be used for both fibrous and non-fibrous particulates.

The Method B is used in the special case of materials which can form fibres, it is comparatively specialised, infrequently required and involves microscopic examination. Scanning (SEM) or transmission (TEM) electron microscopy are required. There is no standard procedure at present. The full length (l) and diameter (d) data are needed on fibre of dimensions d≥ 0.1µm and l≥ 5µm. Two histograms should be prepared based on examination of at least 50 fibres each.

This Test Guideline describes a method to determine the fat solubility of solid and liquid substances. The fat solubility of a substance is one of the data for evaluating the storage of lipid soluble materials in biological tissue.

This test method can only be applied to pure substances, non-reactive with triglyceride, which are stable at 50°C for at least 24 hours and which also are not appreciably volatile under the same conditions. The substance is dissolved in a liquid "standard fat" by stirring, and the saturation mass fraction of the substance is achieved by continued addition until a constant value for the mass fraction dependent variable is achieved as determined by a suitable analytical method.The methods use eight samples; each should be twice the quantity necessary for saturation as determined in the preliminary test. After adding a weighed amount of approximately 25 g of liquified and mixed standard fat, four flasks are stirred at 30°C (group I) and the other four at approximately 50°C (group II), each for at least one hour. All the flasks are stirred at 37°C, for liquids during 3 hours for two flasks of each group and for at least 24 hours for the last two flasks. At the end a sample is taken, weighed and the mass fraction is determined with different methods (photometric methods, gas chromatography, and extraction).

This Test Guideline describes a method which is an adaptation of the Soap and Detergent Association semi-continuous activated sludge (SCAS) procedure for assessing the primary biodegradation of alkyl benzene sulphonate. The test does not simulate those conditions experienced in a sewage treatment plant.

Activated sludge from a sewage treatment plant is placed in an aeration (SCAS) unit. The test compound (non-volatile, water soluble, organic, non-inhibitoring to bacteria at the test concentration) and settled domestic sewage are added, and the mixture is aerated for 23 hours. The aeration is then stopped, the sludge allowed to settle and the supernatant liquor is removed. The sludge remaining in the aeration chamber is then mixed with a further aliquot of test compound and sewage and the cycle is repeated. The above fill and draw procedure is repeated daily throughout the test. A high concentration of aerobic micro-organisms is used. The length of the test for compounds showing little or no biodegradation is indeterminate, but experience suggests that this should be at least 12 weeks. Biodegradation is established by determination of the dissolved organic carbon content of the supernatant liquor. This value is compared with that found for the liquor obtained from a control tube dosed with settled sewage only.

This Test Guideline describes methods for determining storage stability of a substance with respect to heat and air. Two methods are applicable to homogeneous solid and liquid substances and to mixtures of these: the accelerated storage test and the thermal analysis methods.

In the thermal analysis methods (TGA and DTA), the sample and the standard reference material are heated up to the final temperature at a constant rate in a defined test atmosphere, either separately in a TGA or DTA apparatus, or in a combined system. The weight change of the sample or the quantities of heat absorbed or given off are measured and recorded. The substance is considered to be stable at room temperature if no decomposition or chemical transformation is found below 150°.

The method described in this Test Guideline is designed for the evaluation of the mineralisation rate of a 14C-labelled compound in soil. The method is applicable to volatile or non-volatile, soluble or insoluble compounds which are not inhibitory to micro-organisms.

The basic test consists on the treatment of a small sample of soil (50g) with the 14C-labelled test chemical (100µl) in a biometer flask apparatus. The soil and the radioactive test solution are mixed. In addition, an equivalent volume of test solution is placed in a 100 ml volumetric flask for direct determination of the added radioactivity. The biometer flask is closed with a stopper through which an Ascarite filter is inserted. The unit is charged by injecting 10 ml of alkali solution into the side tube. Experiment duration times of 1, 2, 4, 8, 16, 32 and - if necessary - 64 days should be chosen for measurement. The test requires parallel determinations. The method can included optional experiments: for chemicals of a vapour pressure higher than 0.0133 Pa and for relatively persistent chemicals. Release of 14CO2 from the test chemical is measured by means of alkali absorption and liquid scintillation counting. The 14CO2 radioactivity recovered is plotted versus time. Incubation time is sufficient when a total of 50 per cent CO2 expressed as 14C originally applied can be measured. Incubation should be stopped after reaching 64 days, whether or not this value is obtained.

This study relates to the analysis, via dermal application, of the health hazard of solid or liquid test substance.

This method is composed of two tests: the main test and the limit test. This Test Guideline is intended for use with the adult rat, rabbit or guinea pig. At least 10 animals (5 female and 5 male) with healthy skin should be used at each dose level (at least three). The highest dose level should result in toxic effects but not produce an incidence of fatalities. The limit test corresponds to one dose level of at least 1000 mg/kg body weight. The method is based on the repeated application of the substance of interest during one limited period (several hours daily during 21/28 days). The test substance should be applied over not less than 10 per cent of the body surface area. The results of this study include: measurements and daily and detailed observations (haematology, clinical biochemistry and urinalysis), as well as gross necropsy and histopathology. A properly conducted 21-day or 28-day study should provide information on the effects of repeated inhalation exposure and can indicate the need for further longer term studies and provide information on the dose levels of the latter.

This Test Guideline describes the polarographic method. The ability of a substance to form complexes with metals can be assessed by means of polarographic techniques which allow the determination of stability constants for some complexes.

The polarographic method can be applied to substances with a water solubility greater than 10-5 M. This method is based on the fact that the reduction potentials of metal ions are shifted, usually to more negative values, as a result of complex formation. A minimum of four known concentrations of the chemical being tested should be investigated with a known concentration of metal ions. The chemical being tested should normally be present in at least a 25-fold excess over the metal ion concentrations. The current should be measured at applied potentials in the range - 0.2 V to -1.0 V. In order to detect complexes which form slowly, it is necessary to allow the solutions to stand under a nitrogen atmosphere for a minimum period of 24 hours. If the results are not significant, it is necessary to use methods based on different physicochemical principles, such as spectrophotometry or nuclear magnetic resonance spectrometry.