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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 laboratory test method to assess abiotic hydrolytic transformations of chemicals in aquatic systems at pH values normally found in the environment (pH 4 – 9). This Guideline is designed as a tiered approach; each tier is triggered by the results of the previous tier.

Sterile aqueous buffer solutions of different pH values (pH 4, 7 and 9) are treated with the non-labelled or labelled test substance (only one concentration, which should not exceed 0.01 M or half of the saturation concentration). They are incubated in the dark under controlled laboratory conditions (at constant temperatures). After appropriate time intervals, buffer solutions are analysed for the test substance and for hydrolysis products. The preliminary test should be carried out for 5 days at 50 ± 0.5°C and pH 4.0, 7.0 and 9.0. The second tier consists of the hydrolysis of unstable substances, and the third tier is the identification of hydrolysis products. The higher Tier tests should be conducted until 90 % hydrolysis of the test substance is observed or for 30 days whichever comes first.

This Test Guideline describes the methods allowing the determination of the dissociation constants in water. The dissociation is the reversible splitting into two or more chemical species which may be ionic.

The determination of the dissociation constant requires a measure of the concentrations of the dissociated and undissociated forms of the chemical substance. From a knowledge of the stoichiometry of the dissociation reaction the appropriate constant can be determined. In the particular case described in this Guideline the substance is behaving as an acid or a base, and the determination is most conveniently done by determining the relative concentrations of ionised and unionised forms of the substance and the pH of the solution. The relationship between these terms is given in the equation for pKa. Some compounds exhibit more than one dissociation constant. Some of the methods described are also suitable for non-acid/base dissociation. There are two basic approaches to the determination of pKa. One involves titrating a known amount of substance with standard acid or base, as appropriate; the other involves determining the relative concentrations of the ionised and unionised forms and their pH dependence. Methods based on those principles may be classified as titration, spectrophotometric and conductometric procedures.

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 accelerated storage test (CIPAC), a long duration storage instability can be simulated by applying a higher temperature during a short test. This test calls for the controlled storage at 54°C to 55°C for 14 days and subsequent analysis. In simple cases, it will be enough to determine a characteristic property before and after storage. The substance is considered to be stable at room temperature if the melting point has remained constant or the content of original substance as determined by analysis has decreased by not more than 5 per cent.

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°.

This Test Guideline describes methods to measure the viscosity of liquids.

The methods listed are appropriate in principle for the investigation of Newtonian liquids. The measurement of non-Newtonian liquids is only possible with the rotational viscometer. Viscosity measurements are carried out predominantly according to five methods: the capillary viscometer, the flow cup, the rotational viscometer, the rolling ball viscometer and the drawing ball viscometer. Each determination of viscosity should preferably be made at a temperature of 20°C and at one other temperature approximately 20°C higher. At least two determinations should be made at each temperature. The evaluation of the viscosity measurement is to be carried out according to the standards in the case of capillary and forced ball viscometers. In the case of rotational viscometers, the specification of a viscosity is appropriate only for Newtonian fluids. For non- Newtonian fluids the results obtained are preferred in the form of flow curves which must be interpreted, assuming the validity of various laws of flow.

This Test Guideline describes methods to determine the surface tension (in N/m) of aqueous solutions. The methods are based on the measurement of the force which it is necessary to exert vertically on a stirrup or ring, in contact with the surface of the liquid, in order to separate it from the surface, or on a plate, with an edge in contact with the surface, in order to draw up the film that has formed.

There are four different methods: the plate method, the stirrup method, the ring method and the OECD harmonized ring method. They are described in detail in the ISO Standard 304-1985. The methods described are applicable to aqueous solutions of most substances regardless of their degree of purity. The concentration should be 90% of the saturation solubility, but must below 1g/l. This shall therefore be carried out under a protective cover to avoid interference at 20°C approximately. The ring is immersed below the surface of the solution. Then the table top, where the measurement vessel is placed, is lowered gradually and evenly at a rate of approximately 0.5 cm/min to detach the ring from the surface until the maximum force is reached. The force is read on the tensiometer. After completing the first measurement, measurements are repeated until a constant surface tension value is reached.

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 the HPLC method, which permits to determine the partition coefficient (n-octanol/water) (Pow).

Reverse phase HPLC is performed on analytical columns packed with a solid phase containing long hydrocarbon chains chemically bound onto silica. The chemicals are retained in the column in proportion to their hydrocarbon-water partition coefficient, with hydrophilic chemicals eluted first and lipophilic chemicals last. The HPLC method covers log Pow in the range of 0 to 6, but it can be expanded to cover the log Pow range between 6 and 10 in exceptional cases. The HPLC operation mode is isocratic. The test substance is injected in the smallest detectable quantities in the column. The retention time is determined in duplicate. The partition coefficient of the test substance is obtained by interpolation of the calculated capacity factor on the calibration graph. For very low and very high partition coefficients extrapolation is necessary.

This Test guideline describes the Gel Permeation Chromatography (GPC). This method permits to determine the molecular weight distribution and the average molecular weights (Mn, Mw). GPC is a special type of liquid chromatography in which the sample is separated according to the hydrodynamic volumes of the individual constituents.

According to their size, the eluted molecules can or not penetrate in the porous material (typically an organic gel) of which the columns are filled. Thus, the smallest molecules are retained whereas largest elute more quickly. At exit of column, detectors (generally by differential refractometry) provide the refractive index or UV-absorption and yield a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate the column by passing down polymers of known molecular weight and, ideally, of broadly similar structure, e.g. various polystyrene standards. For each sample analyzed, two independent experiments must be undertaken. They have to be analysed individually. Mn, Mw, Mw/Mn must be provided for every measurement.

This Test guideline describes the Gel Permeation Chromatography (GPC). This method permits to determine the molecular weight distribution and the average molecular weights (Mn, Mw). GPC is a special type of liquid chromatography in which the sample is separated according to the hydrodynamic volumes of the individual constituents. Low molecular weight is arbitrarily defined as a molecular weight below 1000 dalton.

According to their size, the eluted molecules can or not penetrate in the porous material (typically an organic gel) of which the columns are filled. Thus, the smallest molecules are retained whereas largest elute more quickly. At exit of column, detectors (generally by differential refractometry) provide the refractive index or UV-absorption and yield a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate the column by passing down polymers of known molecular weight and, ideally, of broadly similar structure, e.g. various polystyrene standards. For each sample analyzed, two independent experiments must be undertaken.

This Test Guideline describes a method to analyse the solution/extraction behaviour of polymers in water, which uses the flask method with modifications.

Three samples of 10 g of particles of the test substance are weighed into each of three vessels fitted with glass stoppers. 1000 ml of water is added to each vessel. The vessels are tightly stoppered and then agitated at 20 °C normally during a period of 24 hours. Then, the content of each vessel is centrifuged or filtered and the concentration of polymer in the clear aqueous phase is determined by a suitable analytical method. For a direct analysis of the aquous phase one sample size is sufficent. Analysis of the aqueous phase for the total polymeric species is possible either by a sufficiently sensitive method or by vacuum evaporation to dryness of the aqueous extract and spectroscopic or AAS/ICP analysis of the residue. When determination of the solution/extraction behaviour of the polymer consists in determining only the total organic carbon content (TOC) of the aqueous extract, an additional test should be conducted, with two different sample sizes in triplicate, using ten times smaller polymer samples and the same amounts of water as those used in the first test. A gravimetric determination is performed on the undissolved/not-extracted part of the sample.

This Test Guideline describes an experimental method using HPLC for the estimation of the adsorption coefficient Koc in soil and in sewage sludge. The adsorption coefficient is defined as the ratio between the concentration of the substance in the soil/sludge and the concentration of the substance in the aqueous phase at adsorption equilibrium.

The test substance is injected in the column packed with cyanopropyl solid phase containing lipophilic and polar moieties. As a result of partitioning between mobile and stationary phases the test substance is retarded. The retention time tR is determined in duplicate. The HPLC dead time t0 may be determined by two methods: the determination by means of a homologous series and the determination by inert substances which are not retained by the column. If the actual results show that the log Koc of the test substance is outside the calibration range the test should be repeated using different, more appropriate reference substances.

This Test Guideline describes the procedure for the electronic determination of pH of an undiluted aqueous solution or dispersion, the pH of a dilution of a solution or dispersion in water, or the pH of a chemical diluted to end-use concentration.  It also describes procedures to determine acid reserve or alkali reserve for a chemical that is acidic or alkaline with either strong or weak acid or alkali.

The pH of an aqueous solution or dispersion in water is determined with a pH-meter equipped with an appropriate electrode system. The acidity or alkalinity of a solution or dispersion in water is determined by titration with standard acid or alkali using electrometric endpoint detection.

This Test Guideline describes the slow-stirring method, which permits the determination of the 1-octanol/water partition coefficient (POW) values up to a log POW of 8.2. The partition coefficient between water and 1-octanol (POW) is defined as the ratio of the equilibrium concentrations of the pure test substance in 1-octanol saturated with water (CO) and water saturated with 1-octanol (CW).

In order to determine the partitioning coefficient, water, 1-octanol, and the test substance are equilibrated with each other at constant temperature in a thermostated stirred reactor at 25 °C and protected from daylight. Exchange between the phases is accelerated by stirring. The concentrations of the test substance in the two phases are determined. Each POW determination has to be performed employing at least three independent slow-stirring experiments with identical conditions. The regression used to demonstrate attainment of equilibrium should be based on the results of at least four determinations of CO / CW at consecutive time points.

The purpose of this test is to determine the effects of a substance on the growth of freshwater microalgae and/or cyanobacteria. Exponentially growing test organisms are exposed to the test substance in batch cultures over a period of normally 72 hours.

The system response is the reduction of growth in a series of algal cultures exposed to, at least, five concentrations of a test substance. Three replicates at each test concentration should be used. The response is evaluated as a function of the exposure concentration in comparison with the average growth of control cultures. The cultures are allowed unrestricted exponential growth under nutrient sufficient conditions (two alternative growth media: the OECD and the AAP) and continuous fluorescent illumination. Growth and growth inhibition are quantified from measurements of the algal biomass as a function of time. The limit test corresponds to one dose level of 100 mg/L. This study includes: the determination, at least daily, of the algal biomass; the measure of the pH (at the beginning and at the end); microscopic observation. This Test Guideline describes two response variables: average specific growth rate, and yield.

 

This Test Guideline describes an acute toxicity test to assess effects of chemicals towards daphnids (usually Daphnia magna Staus).

Young daphnids, aged less than 24 hours at the start of the test, are exposed to the test substance at a range of concentrations (at least five concentrations) for a period of 48 hours. Immobilisation is recorded at 24 hours and 48 hours and compared with control values. The results are analysed in order to calculate the EC50 at 48h. Determination of the EC50 at 24h is optional. At least 20 animals, preferably divided into four groups of five animals each, should be used at each test concentration and for the controls. At least 2 ml of test solution should be provided for each animal (i.e. a volume of 10 ml for five daphnids per test vessel). The limit test corresponds to one dose level of 100 mg/L. The study report should include the observation for immobilized daphnids at 24 and 48 hours after the beginning of the test and the measures of dissolved oxygen, pH, concentration of the test substance, at the beginning and end of the test.