Commission Regulation (EC) No 761/2009Dangos y teitl llawn

1. METHOD

This method is equivalent to OECD TG 201 (2006) (1).

1.1. INTRODUCTION

Testing Methods are periodically reviewed and updated in the light of scientific progress. Testing Method C.3 needed to be revised to include additional species and to meet the requirements for hazard assessment and classification of chemicals. The revision has been completed on the basis of extensive practical experience, scientific progress in the field of algal toxicity studies, and extensive regulatory use, which has occurred since the original adoption.

1.2. DEFINITIONS

The following definitions and abbreviations are used for the purposes of this Testing Method:

Biomass: is the dry weight of living matter present in a population expressed in terms of a given volume; e.g. mg algae/litre test solution. Usually ‘biomass’ is defined as a mass, but in this test this word is used to refer to mass per volume. Also in this test, surrogates for biomass, such as cell counts, fluorescence, etc. are typically measured and the use of the term ‘biomass’ thus refers to these surrogate measures as well.

Coefficient of variation: is a dimensionless measure of the variability of a parameter, defined as the ratio of the standard deviation to the mean. This can also be expressed as a percentage value. The mean coefficient of variation of average specific growth rate in replicate control cultures should be calculated as follows:

1.

Calculate % CV of average specific growth rate out of the daily/section-by-section growth rates for the respective replicate.

2.

Calculate the mean value of all the values calculated in point 1 to get the mean coefficient of variation of the daily/section-by-section specific growth rate in replicate control cultures.

EC_x : is the concentration of the test substance dissolved in the test medium that results in an x % (e.g. 50 %) reduction in growth of the test organism within a stated exposure period (to be mentioned explicitly if deviating from full or normal test duration). To unambiguously denote an EC value deriving from the growth rate or from the yield, the symbols ‘E_rC’ and ‘E_yC’ are used respectively.

Growth medium: is the complete synthetic culture medium in which test algae grow when exposed to the test substance. The test substance will normally be dissolved in the test medium.

Growth rate (average specific growth rate): is the logarithmic increase in biomass during the exposure period.

Lowest Observed Effect Concentration (LOEC): is the lowest tested concentration at which the substance is observed to have a statistically significant reducing effect on growth (at p < 0,05) when compared with the control, within a given exposure time. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. When these two conditions cannot be satisfied, a full explanation must be given for how the LOEC (and hence the NOEC) has been selected.

No Observed Effect Concentration (NOEC): is the test concentration immediately below the LOEC.

Response variable: is a variable for the estimation of toxicity derived from any measured parameters describing biomass by different methods of calculation. For this method, growth rates and yield are response variables derived from measuring biomass directly or any of the surrogates mentioned.

Specific growth rate: is a response variable defined as the quotient of the difference of the natural logarithms of a parameter of observation (in this Testing Method, biomass) and the respective time period.

Yield: is the value of a measurement variable at the end of the exposure period minus the measurement variable’s value at the start of the exposure period to express biomass increase during the test.

1.3. APPLICABILITY OF THE TEST

This Testing Method is most easily applied to water-soluble substances which, under the conditions of the test, are likely to remain in the water. For testing of substances that are volatile, strongly adsorbing, coloured, having a low solubility in water or substances that may affect the availability of nutrients or minerals in the test medium, certain modifications of the described procedure may be required (e.g. closed system, conditioning of the test vessels). Guidance on some appropriate modifications is given in (2), (3) and (4).

1.4. PRINCIPLE OF THE TEST

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. In spite of the relatively brief test duration, effects over several generations can be assessed.

The system response is the reduction of growth in a series of algal cultures (test units) exposed to various concentrations of a test substance. The response is evaluated as a function of the exposure concentration in comparison with the average growth of replicate, unexposed control cultures. For full expression of the system response to toxic effects (optimal sensitivity), the cultures are allowed unrestricted exponential growth under sufficient nutrient conditions and continuous light for a sufficient period of time to measure reduction of the specific growth rate.

Growth and growth inhibition are quantified by measurements of the algal biomass as a function of time. Algal biomass is defined as the dry weight per volume, e.g. mg algae/litre test solution. However, dry weight is difficult to measure and therefore surrogate parameters are used. Of these surrogates, cell counts are most often used. Other surrogate parameters include cell volume, fluorescence, optical density, etc. A conversion factor between the measured surrogate parameter and biomass should be known.

The test endpoint is inhibition of growth, expressed as the logarithmic increase in biomass (average specific growth rate) during the exposure period. From the average specific growth rates recorded in a series of test solutions, the concentration bringing about a specified x % inhibition of growth rate (e.g. 50 %) is determined and expressed as the E_rC_x (e.g. E_rC₅₀).

For the application of this method within the EU regulatory framework, calculation of results should be based on an average specific growth rate for the reasons described in section 2.2 below. An additional response variable used in this Testing Method is yield, which may be needed to fulfil specific regulatory requirements in some countries. It is defined as biomass at the end of the exposure period minus the biomass at the start of the exposure period. From the yield recorded in a series of test solutions, the concentration bringing about a specified x % inhibition of yield (e.g. 50 %) is calculated and expressed as the E_yC_x (e.g. E_yC₅₀).

In addition, the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) may be statistically determined.

1.5. INFORMATION ON THE TEST SUBSTANCE

Information on the test substance, which may be useful in establishing the test conditions, includes the structural formula, purity, stability in light, stability under the conditions of the test, light absorption properties, pKa, and results of studies of transformation including biodegradability in water.

The water solubility, octanol water partition coefficient (P_ow) and the vapour pressure of the test substance should be known, and a validated method for the quantification of the substance in the test solutions with reported recovery efficiency and limit of detection should be available.

1.6. REFERENCE SUBSTANCE

Reference substance(s), such as 3,5-dichlorophenol used in the international ring test (4), may be tested as a means of checking the test procedure. Potassium dichromate can also be used as a reference substance for green algae. It is desirable to test a reference substance at least twice a year.

1.7. VALIDITY OF THE TEST

For the test to be valid, the following performance criteria should be met:

• The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period. This corresponds to a specific growth rate of 0,92 day^–1. For the most frequently used species, the growth rate is usually substantially higher (see Appendix 1). This criterion may not be met when species that grow slower than those listed in Appendix 1 are used. In this case, the test period should be extended to obtain at least a 16-fold growth in control cultures, while the growth has to be exponential throughout the test period. The test period may be shortened to at least 48 h to maintain unlimited exponential growth during the test, as long as the minimum multiplication factor of 16 is reached.

• The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures (see section 1.2 under ‘coefficient of variation’) must not exceed 35 %. See the second paragraph in section 2.2.1 for the calculation of section-by-section specific growth rate. This criterion applies to the mean value of coefficients of variation calculated for replicate control cultures.

• The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7 % in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10 %.

1.8. DESCRIPTION OF THE METHOD

1.8.1. Apparatus

Test vessels and other apparatus, which will come into contact with the test solutions should be made entirely of glass or other chemically inert material. The items should be thoroughly washed to ensure that no organic or inorganic contaminants may interfere with the algal growth or composition of the test solutions.

The test vessels will normally be glass flasks of dimensions that allow a sufficient volume of culture for measurements during the test and a sufficient mass transfer of CO₂ from the atmosphere (see the second paragraph in section 1.8.9). Note that the liquid volume must be sufficient for analytical determinations (see the fifth paragraph in section 1.8.11).

In addition, some or all of the following equipment will be required:

• Culturing apparatus: a cabinet or chamber is recommended, in which the chosen incubation temperature can be maintained at ±2 °C.

• Light measurement instruments: it is important to note that the method of measurement of light intensity, and in particular the type of receptor (collector), will affect the measured value. Measurements should preferably be made using a spherical (4 π) receptor (which responds to direct and reflected light from all angles above and below the plane of measurement), or a 2 π receptor (which responds to light from all angles above the measurement plane).

• Apparatus to determine algal biomass. Cell count, which is the most frequently used surrogate parameter for algal biomass, may be made using an electronic particle counter, a microscope with counting chamber, or a flow cytometer. Other biomass surrogates can be measured using a flow cytometer, fluorimeter, spectrophotometer or colorimeter. A conversion factor relating cell count to dry weight is useful to calculate. In order to provide useful measurements at low biomass concentrations when using a spectrophotometer, it may be necessary to use cuvettes with a light path of at least 4 cm.

1.8.2. Test organisms

Several species of non-attached microalgae and cyanobacteria may be used. The strains listed in Appendix 1 have been shown to be suitable using the test procedure specified in this Testing Method.

If other species are used, the strain and/or origin should be reported. It has to be confirmed that exponential growth of the selected test alga can be maintained throughout the test period under the prevailing conditions.

1.8.3. Growth medium

Two alternative growth media, the OECD and the AAP medium, are recommended. The compositions of these media are shown in Appendix 2. Note that the initial pH value and the buffering capacity (regulating pH increase) of the two media are different. Therefore the results of the tests may be different depending on the medium used, particularly when testing ionising substances.

Modification of the growth media may be necessary for certain purposes, e.g. when testing metals and chelating agents or testing at different pH values. Use of a modified medium must be described in detail and justified (3)(4).

1.8.4. Initial biomass concentration

The initial biomass in the test cultures must be the same in all test cultures and sufficiently low to allow exponential growth throughout the incubation period without risk of nutrient depletion. The initial biomass should not exceed 0,5 mg/l as dry weight. The following initial cell concentrations are recommended:

Pseudokirchneriella subcapitata	5 × 103-104	cells/ml
Desmodesmus subspicatus	2-5 × 103	cells/ml
Navicula pelliculosa	104	cells/ml
Anabaena flos-aquae	104	cells/ml
Synechococcus leopoliensis	5 × 104-105	cells/ml

1.8.5. Concentrations of test substance

The concentration range in which effects are likely to occur may be determined on the basis of results from range-finding tests. For the final definitive test, at least five concentrations arranged in a geometric series with a factor not exceeding 3,2 should be selected. For test substances showing a flat concentration response curve, a higher factor may be justified. The concentration series should preferably cover the range causing 5-75 % inhibition of algal growth rate.

1.8.6. Replicates and controls

The test design should include three replicates at each test concentration. If determination of the NOEC is not required, the test design may be altered to increase the number of concentrations and reduce the number of replicates per concentration. The number of control replicates must be at least three, and ideally should be twice the number of replicates used for each test concentration.

A separate set of test solutions may be prepared for analytical determinations of test substance concentrations (see the fourth and sixth paragraphs in section 1.8.11).

When a solvent is used to solubilise the test substance, additional controls containing the solvent at the same concentration as used in the test cultures must be included in the test design.

1.8.7. Preparation of inoculum culture

In order to adapt the test alga to the test conditions and ensure that the algae are in the exponential growth phase when used to inoculate the test solutions, an inoculum culture in the test medium is prepared 2-4 days before start of the test. The algal biomass should be adjusted in order to allow exponential growth to prevail in the inoculum culture until the test starts. The inoculum culture shall be incubated under the same conditions as the test cultures. Measure the increase in biomass in the inoculum culture to ensure that growth is within the normal range for the test strain under the culturing conditions. An example of the procedure for algal culturing is described in Appendix 3. To avoid synchronous cell divisions during the test, a second propagation step of the inoculum culture may be required.

1.8.8. Preparation of test solutions

All test solutions must contain the same concentrations of growth medium and initial biomass of test algae. Test solutions of the chosen concentrations are usually prepared by mixing a stock solution of the test substance with growth medium and inoculum culture. Stock solutions are normally prepared by dissolving the substance in test medium.

Solvents, e.g. acetone, t-butyl alcohol and dimethyl formamide, may be used as carriers to add substances of low water solubility to the test medium (2)(3). The solvent concentration should not exceed 100 µl/l, and the same concentration of solvent should be added to all cultures (including controls) in the test series.

1.8.9. Incubation

Cap the test vessels with air-permeable stoppers. The vessels are shaken and placed in the culturing apparatus. During the test it is necessary to keep the algae in suspension and to facilitate transfer of CO₂. To this end, constant shaking or stirring should be used. The cultures should be maintained at a temperature in the range of 21 to 24 °C, controlled at ±2 °C. For species other than those listed in Appendix 1, e.g. tropical species, higher temperatures may be appropriate, providing that the validity criteria can be fulfilled. It is recommended to place the flasks randomly and to reposition them daily in the incubator.

The pH of the control medium should not increase by more than 1,5 units during the test. For metals and compounds that partly ionise at a pH around the test pH, it may be necessary to limit the pH drift to obtain reproducible and well defined results. A drift of < 0,5 pH units is technically feasible and can be achieved by ensuring an adequate CO₂ mass transfer rate from the surrounding air to the test solution, e.g. by increasing the shaking rate. Another possibility is to reduce the demand for CO₂ by reducing the initial biomass or the test duration.

The surface where the cultures are incubated should receive continuous, uniform fluorescent illumination e.g. of ‘cool-white’ or ‘daylight’ type. Strains of algae and cyanobacteria vary in their light requirements. The light intensity should be selected to suit the test organism used. For the recommended species of green algae, the light intensity at the level of the test solutions shall be selected from the range of 60-120 µE·m^–2·s^–1 when measured in the photosynthetically effective wavelength range of 400-700 nm using an appropriate receptor. Some species, in particular Anabaena flos-aquae, grow well at lower light intensities and may be damaged at high intensities. For such species an average light intensity in the range 40-60 µE·m^–2·s^–1 should be selected. (For light-measuring instruments calibrated in lux, an equivalent range of 4 440-8 880 lux for cool white light corresponds approximately to the recommended light intensity 60-120 µE·m^–2·s^–1). The light intensity shall not vary more than ±15 % from the average light intensity over the incubation area.

1.8.10. Test duration

The test duration is normally 72 hours. However, shorter or longer test durations may be used, provided that all validity criteria in section 1.7 can be met.

1.8.11. Measurements and analytical determinations

The algal biomass in each flask is determined at least daily during the test period. If measurements are made on small volumes removed from the test solution by pipette, these should not be replaced.

Measurement of biomass is done by manual cell counting by microscope or an electronic particle counter (by cell counts and/or biovolume). Alternative techniques, e.g. flow cytometry, in vitro or in vivo chlorophyll fluorescence (6)(7), or optical density can be used providing a satisfactory correlation with biomass can be demonstrated over the range of biomass occurring in the test.

The pH of the solutions shall be measured at the beginning and at the end of the test.

Provided an analytical procedure for determination of the test substance in the concentration range used is available, the test solutions should be analysed to verify the initial concentrations and maintenance of the exposure concentrations during the test.

Analysis of the concentration of the test substance at the start and end of the test of a low and high test concentration, and a concentration around the expected EC₅₀ may be sufficient where it is likely that exposure concentrations will vary less than 20 % from nominal values during the test. Analysis of all test concentrations at the start and end of the test is recommended where concentrations are unlikely to remain within 80-120 % of nominal. For volatile, unstable or strongly adsorbing test substances, additional sampling for analysis at 24 hour intervals during the exposure period is recommended in order to better define loss of the test substance. For these substances, extra replicates will be needed. In all cases, determination of test substance concentrations need only be performed on one replicate vessel at each test concentration (or on the contents of the vessels pooled by replicate).

Test media prepared specifically for analysis of exposure concentrations during the test should be treated identically to those used for testing, i.e. they should be inoculated with algae and incubated under identical conditions. If analysis of the dissolved test substance concentration is required, it may be necessary to separate algae from the medium. Separation should preferably be made by centrifugation at a low g-force, sufficient to settle the algae.

If there is evidence that the concentration of the substance being tested has been satisfactorily maintained within ±20 % of the nominal or measured initial concentration throughout the test, analysis of the results can be based on nominal or measured initial values. If the deviation from the nominal or measured initial concentration is greater than ±20 %, analysis of the results should be based on geometric mean concentration during exposure or on models describing the decline of the concentration of test substance (3)(8).

The alga growth inhibition test is a more dynamic test system than most other short-term aquatic toxicity tests. As a consequence, the actual exposure concentrations may be difficult to define, especially for adsorbing substances tested at low concentrations. In such cases, disappearance of the substance from solution by adsorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test is analysed, it should be checked whether a decrease in concentration of the test substance in the course of the test is accompanied by a decrease in growth inhibition. If this is the case, application of a suitable model describing the decline of the concentration of test substance (8) may be considered. If not, it may be appropriate to base the analysis of the results on the initial (nominal or measured) concentrations.

1.8.12. Other observations

Microscopic observation should be performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae (as may be caused by exposure to the test substance) at the end of the test.

1.8.13. Limit test

Under some circumstances, e.g. when a preliminary test indicates that the test substance has no toxic effects at concentrations up to 100 mg·l^–1 or up to its limit of solubility in the test medium (whichever is the lower), a limit test involving a comparison of responses in a control group and one treatment group (100 mg·l^–1 or a concentration equal to the limit of solubility), may be undertaken. It is strongly recommended that this be supported by analysis of the exposure concentration. All previously described test conditions and validity criteria apply to a limit test, with the exception that the number of treatment replicates should be at least six. The response variables in the control and treatment group may be analysed using a statistical test to compare means, e.g. a Student'’t's t-test. If variances of the two groups are unequal, a t-test adjusted for unequal variances should be performed.

1.8.14. Modification for strongly coloured substances

The irradiation (light intensity) should be in the highest end of the range prescribed in this Testing Method: 120µE m-2 s-1 or higher.

The light path should be shortened by reduction of the volume of the test solutions (in the range of 5-25 ml).

Sufficient agitation (for example by moderate shaking) should be performed in order to obtain a high frequency of exposure of the algae to high irradiation at the surface of the culture.