Commission Regulation (EC) No 761/2009Show full title

1. METHOD

This method is equivalent to OECD TG 221 (2006) (1). There has been broad agreement by EU authorities that the Lemna test is a suitable alternative to an algal test for strongly coloured substances (2)(3).

1.1. INTRODUCTION

This Testing Method is designed to assess the toxicity of substances to freshwater aquatic plants of the genus Lemna (duckweed). It is based on existing guidelines (4)(5)(6)(7)(8)(9) but includes modifications of those methods to reflect recent research and consultation on a number of key issues. The proposed method has been validated by an international ring-test (10).

This Testing Method describes toxicity testing using Lemna gibba and Lemna minor, both of which have been extensively studied and are the subject of the standards referred to above. The taxonomy of Lemna spp. is difficult, being complicated by the existence of a wide range of phenotypes. Although genetic variability in the response to toxicants can occur with Lemna, there are currently insufficient data on this source of variability to recommend a specific clone for use with this Testing Method. It should be noted that the test is not conducted axenically, but steps are taken at stages during the test procedure to keep contamination by other organisms to a minimum.

Details on testing with renewal (semi-static and flow-through) and without renewal (static) of the test solution are described. Depending on the objectives of the test and on the regulatory requirements, it is recommended to consider the application of semi-static and flow through methods, e.g. for substances that are rapidly lost from solution as a result of volatilisation, photodegradation, precipitation or biodegradation. Further guidance is given in (11).

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. In this test, surrogates for biomass, such as frond counts or frond area, are typically measured and the use of the term ‘biomass’ thus refers to these surrogate measures as well.

Chlorosis: is the yellowing of frond tissue.

Clone: is an organism or cell arisen from a single individual by asexual reproduction. Individuals from the same clone are, therefore, genetically identical.

Colony: means an aggregate of mother and daughter fronds (usually 2 to 4) attached to each other. Sometimes referred to as a plant.

EC_x : is the concentration of the test substance dissolved in test medium that results in an x % (e.g. 50 %) reduction in growth of Lemna 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 growth rate or yield, the symbol ‘E_rC’ is used for growth rate and ‘E_yC’ is used for yield, followed by the measurement variable used, e.g. E_rC (frond number).

Flow-through: is a test in which the test solutions are replaced continuously.

Frond: is an individual/single ‘leaf-like’ structure of a duckweed plant. It is the smallest unit, i.e. individual, capable of reproduction.

Gibbosity: means fronds exhibiting a humped or swollen appearance.

Growth: is an increase in the measurement variable, e.g. frond number, dry weight, wet weight or frond area, over the test period.

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.

Measurement variables: are any type of variables which are measured to express the test endpoint using one ore more different response variables. In this method frond number, frond area, fresh weight and dry weight are measurement variables.

Monoculture: is a culture with one plant species.

Necrosis: is dead (i.e. white or water-soaked) frond tissue.

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

Phenotype: is the observable characteristics of an organism determined by the interaction of its genes with its environment.

Response variables: are variables for the estimation of toxicity derived from any measured variables describing biomass by different methods of calculation. For this method, growth rates and yield are response variables derived from measurement variables like frond number, frond area, fresh weight or dry weight.

Semi-static (renewal) test: is a test in which the test solution is periodically replaced at specific intervals during the test.

Static test: is a test method without renewal of the test solution during the test.

Test endpoint: describes the general factor that will be changed by the test chemical relative to the control as aim of the test. In this method the test endpoint is inhibition of growth, which may be expressed by different response variables which are based on one or more measurement variables.

Test medium: is the complete synthetic growth medium on which test plants grow when exposed to the test substance. The test substance will normally be dissolved in the test medium.

Yield: is the value of a measurement variable to express biomass at the end of the exposure period minus the measurement variable at the start of the exposure period.

1.3. PRINCIPLE OF THE TEST

Exponentially growing plant cultures of the genus Lemna are allowed to grow as monocultures in different concentrations of the test substance over a period of seven days. The objective of the test is to quantify substance-related effects on vegetative growth over this period, based on assessments of selected measurement variables. Frond number is the primary measurement variable. At least one other measurement variable (total frond area, dry weight or fresh weight) is also measured, since some substances may affect other measurement variables much more than frond numbers. To quantify substance-related effects, growth in the test solutions is compared with that of the controls and the concentration bringing about a specified x % inhibition of growth (e.g. 50 %) is determined and expressed as the EC_x (e.g. EC₅₀).

The test endpoint is inhibition of growth, expressed as logarithmic increase in the measurement variable (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).

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 the measurement variables at the end of the exposure period minus the measurement variables 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.

1.4. INFORMATION ON THE TEST SUBSTANCE

An analytical method, with adequate sensitivity for quantification of the substance in the test medium, should be available.

Information on the test substance which may be useful in establishing the test conditions includes the structural formula, purity, water solubility, stability in water and light, pK_a, K_ow, vapour pressure and biodegradability. Water solubility and vapour pressure can be used to calculate Henry’s Law constant, which will indicate if significant losses of the test substance during the test period are likely. This will help indicate whether particular steps to control such losses should be taken. Where information on the solubility and stability of the test substance is uncertain, it is recommended that these be assessed under the conditions of the test, i.e. growth medium, temperature, lighting regime to be used in the test.

When pH control of the test medium is particularly important, e.g. when testing metals or substances which are hydrolytically unstable, the addition of a buffer to the growth medium is recommended (see first paragraph in section 1.7.4). Further guidance for testing substances with physicochemical properties that make them difficult to test is provided in (11).

1.5. REFERENCE SUBSTANCE

Reference substance(s), such as 3,5-dichlorophenol used in the international ring test (10), may be tested as a means of checking the test procedure. It is advisable to test a reference substance at least twice a year or, where testing is carried out at a lower frequency, in parallel to the determination of the toxicity of a test substance.

1.6. VALIDITY OF THE TEST

For the test to be valid, the doubling time of frond number in the control must be less than 2,5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0,275 d^–1. Using the media and test conditions described in this Testing Method, this criterion can be attained using a static test regime (8). It is also anticipated that this criterion will be achievable under semi-static and flow-through test conditions. Calculation of the doubling time is shown in section 2.1.

1.7. DESCRIPTION OF THE METHOD

1.7.1. Apparatus

All equipment in contact with the test media should be made of glass or other chemically inert material. Glassware used for culturing and testing purposes should be cleaned of chemical contaminants that might leach into the test medium and should be sterile. The test vessels should be wide enough for the fronds of different colonies in the control vessels to grow without overlapping at the end of the test. It does not matter if the roots touch the bottoms of the test vessels, but a minimum depth of 20 mm and minimum volume of 100 ml in each test vessel is advised. The choice of test vessels is not critical as long as these requirements are met. Glass beakers, crystallising dishes or glass petri dishes of appropriate dimensions have all proved suitable. Test vessels must be covered to minimise evaporation and accidental contamination, while allowing necessary air exchange. Suitable test vessels, and particularly covers, must avoid shadowing or changes in the spectral characteristics of light.

The cultures and test vessels should not be kept together. This is best achieved using separate environmental growth chambers, incubators, or rooms. Illumination and temperature must be controllable and maintained at a constant level (see section 1.7.8).

1.7.2. Test organism

The organism used for this test is either Lemna gibba or Lemna minor. Short descriptions of duckweed species that have been used for toxicity testing are given in Appendix 1. Plant material may be obtained from a culture collection, another laboratory or from the field. If collected from the field, plants should be maintained in culture in the same medium as used for testing for a minimum of eight weeks prior to use. Field sites used for collecting starting cultures must be free of obvious sources of contamination. If obtained from another laboratory or from a culture collection they should be similarly maintained for a minimum of three weeks. The source of plant material and the species and clone (if known) used for testing should always be reported.

Monocultures, that are visibly free from contamination by other organisms such as algae and protozoa, should be used. Healthy plants of L. minor will consist of colonies comprising between two and five fronds, whilst healthy colonies of L. gibba may contain up to seven fronds.

The quality and uniformity of the plants used for the test will have a significant influence on the outcome of the test and should therefore be selected with care. Young, rapidly growing plants without visible lesions or discoloration (chlorosis) should be used. Good quality cultures are indicated by a high incidence of colonies comprising at least two fronds. A large number of single fronds is indicative of environmental stress, e.g. nutrient limitation, and plant material from such cultures should not be used for testing.

1.7.3. Cultivation

To reduce the frequency of culture maintenance (e.g. when no Lemna tests are planned for a period), cultures can be held under reduced illumination and temperature (4-10 °C). Details of culturing are given in Appendix 2. Obvious signs of contamination by algae or other organisms will require surface sterilisation of a sub-sample of Lemna fronds, followed by transfer to fresh medium (see Appendix 2). In this eventuality, the remaining contaminated culture should be discarded.

At least seven days before testing, sufficient colonies are transferred aseptically into fresh sterile medium and cultured for 7-10 days under the conditions of the test.

1.7.4. Test medium

Different media are recommended for Lemna minor and Lemna gibba, as described below. Careful consideration should be given to the inclusion of a pH buffer in the test medium (MOPS (4-morpholinepropane sulphonic acid, CAS No: 1132-61-2; EINECS No: 214-478-5) in L. minor medium and NaHCO₃ in L. gibba medium) when it is suspected that it might react with the test substance and influence the expression of its toxicity. Steinberg Medium (12) is also acceptable as long as the validity criteria are met.

A modification of the Swedish standard (SIS) Lemna growth medium is recommended for culturing and testing with L. minor. The composition of this medium is given in Appendix 3.

The growth medium, 20X — AAP, as described in Appendix 3, is recommended for culturing and testing with L. gibba.

Steinberg medium, as described in Appendix 3, is also suitable for L. minor, but may also be used for L. gibba as long as the validity criteria are met.

1.7.5. Test solutions

Test solutions are usually prepared by dilution of a stock solution. Stock solutions of the test substance are normally prepared by dissolving the substance in growth medium.

The highest tested concentration of the test substance should not normally exceed the water solubility of the substance under the test conditions. It should be noted however that Lemna spp. float on the surface and may be exposed to substances that collect at the water-air interface (e.g. poorly water-soluble or hydrophobic substances or surface-active substances). Under such circumstances, exposure will result from material other than in solution and test concentrations may, depending on the characteristics of the test substance, exceed water solubility. For test substances of low water solubility it may be necessary to prepare a concentrated stock solution or dispersion of the substance using an organic solvent or dispersant in order to facilitate the addition of accurate quantities of the test substance to the test medium and aid in its dispersion and dissolution. Every effort should be made to avoid the use of such materials. There should be no phytotoxicity resulting from the use of auxiliary solvents or dispersants. For example, commonly used solvents which do not cause phytotoxicity at concentrations up to 100 μl·l^–1 include acetone and dimethylformamide. If a solvent or dispersant is used, its final concentration should be reported and kept to a minimum (≤ 100 μl·l^–1), and all treatments and controls should contain the same concentration of solvent or dispersant. Further guidance on the use of dispersants is given in (11).

1.7.6. Test and control groups

Prior knowledge of the toxicity of the test substance to Lemna, e.g. from a range-finding test, will help in selecting suitable test concentrations. In the definitive toxicity test, there should normally be at least five test concentrations arranged in a geometric series. Preferably the separation factor between test concentrations should not exceed 3,2, but a larger value may be used where the concentration-response curve is flat. Justification should be provided if fewer than five concentrations are used. At least three replicates should be used at each test concentration.

In setting the range of test concentrations (for range-finding and/or for the definitive toxicity test), the following should be considered:

• To determine an EC_x, test concentrations should bracket the EC_x value to ensure an appropriate level of confidence. For example, if estimating the EC₅₀, the highest test concentration should be greater than the EC₅₀ value. If the EC₅₀ value lies outside the range of test concentrations, associated confidence intervals will be large and a proper assessment of the statistical fit of the model may not be possible.

• If the aim is to estimate the LOEC/NOEC, the lowest test concentration should be low enough so that growth is not significantly less than that of the control. In addition, the highest test concentration should be high enough so that growth is significantly lower than that in the control. If this is not the case, the test will have to be repeated using a different concentration range (unless the highest concentration is at the limit of solubility or the maximum required limit concentration, e.g. 100 mg·l^–1).

Every test should include controls consisting of the same nutrient medium, number of fronds and colonies, environmental conditions and procedures as the test vessels but without the test substance. If an auxiliary solvent or dispersant is used, an additional control treatment with the solvent/dispersant present at the same concentration as that in the vessels with the test substance should be included. The number of replicate control vessels (and solvent vessels, if applicable) should be at least equal to, ideally twice, the number of vessels used for each test concentration.

If determination of NOEC is not required, the test design may be altered to increase the number of concentrations and reduce the number of replicates per concentration. However, the number of control replicates must be at least three.

1.7.7. Exposure

Colonies consisting of 2 to 4 visible fronds are transferred from the inoculum culture and randomly assigned to the test vessels under aseptic conditions. Each test vessel should contain a total of 9 to 12 fronds. The number of fronds and colonies should be the same in each test vessel. Experience gained with this method and ring-test data have indicated that using three replicates per treatment, with each replicate containing 9 to 12 fronds initially, is sufficient to detect differences in growth of approximately 4 to 7 % of inhibition calculated by growth rate (10 to 15 % calculated by yield) between treatments (10).

A randomised design for location of the test vessels in the incubator is required to minimise the influence of spatial differences in light intensity or temperature. A blocked design or random repositioning of the vessels when observations are made (or repositioning more frequently) is also required.

If a preliminary stability test shows that the test substance concentration cannot be maintained (i.e. the measured concentration falls below 80 % of the measured initial concentration) over the test duration (7 days), a semi-static test regime is recommended. In this case, the colonies should be exposed to freshly prepared test and control solutions on at least two occasions during the test (e.g. days 3 and 5). The frequency of exposure to fresh medium will depend on the stability of the test substance; a higher frequency may be needed to maintain near-constant concentrations of highly unstable or volatile substances. In some circumstances, a flow-through procedure may be required (11)(13).

The exposure scenario through a foliar application (spray) is not covered in this Testing Method, instead see (14).

1.7.8. Incubation conditions

Continuous warm or cool white fluorescent lighting should be used to provide a light intensity selected from the range of 85-135 μE·m^–2·s^–1 when measured in a photosynthetically active radiation (400-700 nm) at points the same distance from the light source as the Lemna fronds (equivalent to 6 500-10 000 lux). Any differences from the selected light intensity over the test area should not exceed ± 15 %. The method of light detection and measurement, in particular the type of sensor, will affect the measured value. Spherical sensors (which respond to light from all angles above and below the plane of measurement) and ‘cosine’ sensors (which respond to light from all angles above the plane of measurement) are preferred to unidirectional sensors, and will give higher readings for a multi-point light source of the type described here.

The temperature in the test vessels should be 24 ± 2 °C. The pH of the control medium should not increase by more than 1,5 units during the test. However, deviation of more than 1,5 units would not invalidate the test when it can be shown that validity criteria are met. Additional care is needed on pH drift in special cases such as when testing unstable substances or metals. See (11) for further guidance.

1.7.9. Duration

The test is terminated 7 days after the plants are transferred into the test vessels.

1.7.10. Measurements and analytical determinations

At the start of the test, the frond number in the test vessels is counted and recorded, taking care to ensure that protruding, distinctly visible fronds are accounted for. Frond numbers appearing normal or abnormal, need to be determined at the beginning of the test, at least once every 3 days during the exposure period (i.e. on at least 2 occasions during the 7 day period), and at test termination. Changes in plant development, e.g. in frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up or loss of buoyancy, and in root length and appearance, should be noted. Significant features of the test medium (e.g. presence of undissolved material, growth of algae in the test vessel) should also be noted.

In addition to determinations of frond number during the test, effects of the test substance on one (or more) of the following measurement variables are also assessed:

(i)

total frond area;

(ii)

dry weight;

(iii)

fresh weight.

Total frond area has an advantage that it can be determined for each test and control vessel at the start, during, and at the end of the test. Dry or fresh weight should be determined at the start of the test from a sample of the inoculum culture representative of what is used to begin the test, and at the end of the test with the plant material from each test and control vessel. If frond area is not measured, dry weight is preferred over fresh weight.

Total frond area, dry weight and fresh weight may be determined as follows:

(i)

Total frond area: The total frond area of all colonies may be determined by image analysis. A silhouette of the test vessel and plants can be captured using a video camera (i.e. by placing the vessel on a light box) and the resulting image digitised. By calibration with flat shapes of known area, the total frond area in a test vessel may then be determined. Care should be taken to exclude interference caused by the rim of the test vessel. An alternative but more laborious approach is to photocopy test vessels and plants, cut out the resulting silhouette of colonies and determine their area using a leaf area analyser or graph paper. Other techniques (e.g. paper weight ratio between silhouette area of colonies and unit area) may also be appropriate.

(ii)

Dry weight: All colonies are collected from each of the test vessels and rinsed with distilled or deionised water. They are blotted to remove excess water and then dried at 60 °C to a constant weight. Any root fragments should be included. The dry weight should be expressed to an accuracy of at least 0,1 mg.

(iii)

Fresh weight: All colonies are transferred to pre-weighed polystyrene (or other inert material) tubes with small (1 mm) holes in the rounded bottoms. The tubes are then centrifuged at 3 000 rpm for 10 minutes at room temperature. Tubes, containing the now dried colonies, are re-weighed and the fresh weight is calculated by subtracting the weight of the empty tube.

1.7.10.1. Frequency of measurements and analytical determinations

If a static test design is used, the pH of each treatment should be measured at the beginning and end of the test. If a semi-static test design is used, the pH should be measured in each batch of ‘fresh’ test solution prior to each renewal and also in the corresponding ‘spent’ solutions.

Light intensity should be measured in the growth chamber, incubator or room at points the same distance from the light source as the Lemna fronds. Measurements should be made at least once during the test. The temperature of the medium in a surrogate vessel held under the same conditions in the growth chamber, incubator or room should be recorded at least daily.

During the test, the concentrations of the test substance are determined at appropriate intervals. In static tests, the minimum requirement is to determine the concentrations at the beginning and the end of the test.

In semi-static tests where the concentration of the test substance is not expected to remain within ± 20 % of the nominal concentration, it is necessary to analyse all freshly prepared test solutions and the same solutions at each renewal (see third paragraph in section 1.7.7). However, for those tests where the measured initial concentration of the test substance is not within ± 20 % of nominal, but where sufficient evidence can be provided to show that the initial concentrations are repeatable and stable (i.e. within the range 80-120 % of the initial concentration), chemical determinations may be carried out on only the highest and lowest test concentrations. In all cases, determination of test substance concentrations prior to renewal need only be performed on one replicate vessel at each test concentration (or the contents of the vessels pooled by replicate).

If a flow-through test is used, a similar sampling regime to that described for semi-static tests, including analysis at the start, mid-way through and at the end of the test, is appropriate, but measurement of ‘spent’ solutions is not appropriate in this case. In this type of test, the flow-rate of diluent and test substance or test substance stock solution should be checked daily.

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 the geometric mean concentration during exposure or models describing the decline of the concentration of the test substance (11).

1.7.11. 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 doubled. Growth in the control and treatment group may be analysed using a statistical test to compare means, e.g. a Student's t-test.