Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

Young daphnids, aged less than 24 hours at the start of the test, are exposed to the test substance at a range of 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 (see Section 1.2 for definitions). Determination of the EC50 at 24h is optional.

1.4.   INFORMATION ON THE TEST SUBSTANCE

The water solubility and the vapour pressure of the test substance should be known and a reliable analytical method for the quantification of the substance in the test solutions with reported recovery efficiency, and limit of determination should be available. Useful information includes the structural formula, purity of the substance, stability in water or light, Pow and results of a test for ready biodegradability (see method C.4).

Note: guidance for testing substances with physical chemical properties that made them difficult to test is provided in (4).

1.5.   REFERENCE SUBSTANCES

A reference substance may be tested for EC50 as a means of assuring that the test conditions are reliable. Toxicants used in international ring-tests (1)(5) are recommended for this purpose ([^E0059]). Test(s) with a reference substance should be done preferably every month and at least twice a year.

1.6.   QUALITY CRITERIA

For a test to be valid, the following performance criteria apply:

• — in the controls, including the control containing the solubilising agent, not more that 10 % of the daphnids should have been immobilised;

• — the dissolved oxygen concentration at the end of the test should be ≥ 3 mg/l in control and test vessels.

Note: For the first criterion, not more than 10 % of the control daphnids should show immobilisation or other signs of disease or stress, for example, discoloration, unusual behaviour such as trapping at surface of water.

1.7.   DESCRIPTION OF THE TEST METHOD

1.7.1.   Apparatus

Test vessels and other apparatus that will come into contact with the test solutions should be made entirely of glass or other chemically inert material. Test vessels will normally be glass test tubes or beakers; they should be cleaned before each use using standard laboratory procedures. Test vessels should be loosely covered to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions. Volatile substances should be tested in completely filled closed vessels, large enough to prevent oxygen becoming limiting or too low (see Section 1.6 and first paragraph of Section 1.8.3).

In addition some or all of the following equipment will be used: oxygen-meter (with microelectrode or other suitable equipment for measuring dissolved oxygen in low volumes samples); pH-meter; adequate apparatus for temperature control; equipment for the determination of total organic carbon concentration (TOC); equipment for the determination of chemical oxygen demand (COD); equipment for the determination of hardness, etc.

1.7.2.   Test organism

Daphnia magna Straus is the preferred test species although other suitable Daphnia species can be used in this test (e.g. Daphnia pulex). At the start of the test, the animals should be less than 24 hours old and to reduce variability, it is strongly recommended they are not first brood progeny. They should be derived from a healthy stock (i.e. showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discoloured animals, etc.). All organisms used for a particular test should have originated from cultures established from the same stock of daphnids. The stock animals must be maintained in culture conditions (light, temperature, medium) similar to those to be used in the test. If the daphnids culture medium to be used in the test is different from that used for routine daphnids culture, it is good practice to include a pre-test acclimation period. For that, brood daphnids should be maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.

1.7.3.   Holding and dilution water

Natural water (surface or ground water), reconstituted water or dechlorinated tap water are acceptable as holding and dilution water if daphnids will survive in it for the duration of the culturing, acclimation and testing without showing signs of stress. Any water which conforms to the chemical characteristics of an acceptable dilution water as listed in Appendix 1 is suitable as a test water. It should be of constant quality during the period of the test. Reconstituted water can be made up by adding specific amounts of reagents of recognised analytical grade to deionised or distilled water. Examples of reconstituted water are given in (1) (6) and in Appendix 2. Note that media containing known chelating agents, such as M4 and M7 media in Appendix 2, should be avoided for testing substances containing metals. The pH should be in the range of 6 to 9. Hardness between 140 and 250 mg/l (as CaCO3) is recommended for Daphnia magna, while lower hardness may be also appropriate for other Daphnia species. The dilution water may be aerated prior to use for the test so that the dissolved oxygen concentration has reached saturation.

If natural water is used, the quality parameters should be measured at least twice a year or whenever it is suspected that these characteristics may have changed significantly (see previous paragraph and Appendix 1). Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni) should also be made. If dechlorinated tap water is used, daily chlorine analysis is desirable. If the dilution water is from a surface or ground water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) should be measured.

1.7.4.   Test solutions

Test solutions of the chosen concentrations are usually prepared by dilution of a stock solution. Stock solutions should preferably be prepared by dissolving the test substance in the dilution water. As far as possible, the use of solvents, emulsifiers or dispersants should be avoided. However, such compounds may be required in some cases in order to produce a suitably concentrated stock solution. Guidance for suitable solvents, emulsifiers and dispersants is given in (4). In any case, the test substance in the test solutions should not exceed the limit of solubility in the dilution water.

The test should be carried out without the adjustment of pH. If the pH does not remain in the range 6-9, then a second test could be carried out, adjusting the pH of the stock solution to that of the diluition water before addition of the test substance. The pH adjustment should be made in such a way that the stock solution concentration is not changed to any significant extent and that no chemical reaction or precipitation of the test substance is caused. HCl and NaOH are preferred.

1.8.   PROCEDURE

1.8.1.   Conditions of exposure

1.8.1.1.   Test groups and controls

Test vessels are filled with appropriate volumes of dilution water and solutions of test substance. Ratio of air/water volume in the vessel should be identical for test and control group. Daphnids are then placed into test vessels. 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 test may be carried out using semi-static renewal or flow-through system when the concentration of the test substance is not stable.

One dilution-water control series and also, if relevant, one control series containing the solubilising agent must be run in addition to the treatment series.

1.8.1.2.   Test concentrations

A range-finding test may be conducted to determine the range of concentrations for the definitive test unless information on toxicity of the test substance is available. For this purpose, the daphnids are exposed to a series of widely spaced concentrations of the test substance. Five daphnids should be exposed to each test concentration for 48 hours or less, and no replicates are necessary. The exposure period may be shortened (e.g. 24 hours or less) if data suitable for the purpose of the range-finding test can be obtained in less time.

At least five test concentrations should be used. They should be arranged in a geometric series with a separation factor preferably not exceeding 2,2 . Justification should be provided if fewer than five concentrations are used. The highest concentration tested should preferably result in 100 % immobilisation, and the lowest concentration tested should preferably give no observable effect.

1.8.1.3.   Incubation conditions

The temperature should be within the range of 18 ^oC and 22 ^oC, and for each single test it should be constant within ± 1 ^oC. A 16-hour light and eight-hour dark cycle is recommended. Complete darkness is also acceptable, especially for the test substances unstable in light.

The test vessels must not be aerated during the test. The test is carried out without adjustment of pH. The daphnids should not be fed during the test.

1.8.1.4.   Duration

The test duration is 48 hours.

1.8.2.   Observations

Each test vessel should be checked for immobilised daphnids at 24 and 48 hours after the beginning of the test (see Section 1.2 for definitions). In addition to immobility, any abnormal behaviour or appearance should be reported.

1.8.3.   Analytical measurements

The dissolved oxygen and pH are measured at the beginning and end of the test in the control(s) and in the highest test substance concentration. The dissolved oxygen concentration in controls should be in compliance with the validity criterion (see Section 1.6). The pH should normally not vary by more than 1,5 units in any one test. The temperature is usually measured in control vessels or in ambient air and it should be recorded preferably continuously during the test or, as a minimum, at the beginning and end of the test.

The concentration of the test substance should be measured, as a minimum, at the highest and lowest test concentration, at the beginning and end of the test (4). It is recommended that results be based on measured concentrations. However, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within ± 20 % of the nominal or measured initial concentration throughout the test, then the results can be based on nominal or measured initial values.

1.9.   LIMIT TEST

Using the procedures described in this Method, a limit test may be performed at 100 mg/l of test substance or up to its limit of solubility in the test medium (whichever is the lower) in order to demonstrate that the EC50 is greater than this concentration. The limit test should be performed using 20 daphnids (preferably divided into four groups of five), with the same number in the control(s). If any immobilisation occurs, a full study should be conducted. Any observed abnormal behaviour should be recorded.

2. DATA

Data should be summarised in tabular form, showing for each treatment group and control, the number of daphnids used, immobilisation at each observation. The percentages immobilised at 24 hours and 48 hours are plotted against test concentrations. Data are analysed by appropriate statistical methods (e.g. probit analysis, etc.) to calculate the slopes of the curves and the EC50 with 95 % confidence limits (p = 0,05 ) (7) (8).

Where the standard methods of calculating the EC50, are not applicable to the data obtained, the highest concentration causing no immobility and the lowest concentration producing 100 % immobility should be used as an approximation for the EC50 (this being considered the geometric mean of these two concentrations).

3. REPORTING

3.1.   TEST REPORT

The test report must include the following:

Test substance:

• — physical nature and relevant physical-chemical properties,

• — chemical identification data, including purity.

Test species:

• — source and species of Daphnia, supplier of source (if known) and the culture conditions used (including source, kind and amount of food, feeding frequency).

Test conditions:

• — description of test vessels: type of vessels, volume of solution, number of daphnids per test vessel, number of test vessels (replicates) per concentration,

• — methods of preparation of stock and test solutions including the use of any solvent or dispersants, concentrations used,

• — details of dilution water: source and water quality characteristics (pH, hardness, Ca/Mg ratio, Na/K ratio, alkalinity, conductivity, etc.); composition of reconstituted water if used,

• — incubation conditions: temperature, light intensity and periodicity, dissolved oxygen, pH, etc.

Results:

• — the number and percentage of daphnids that were immobilised or showed any adverse effects (including abnormal behaviour) in the controls and in each treatment group, at each observation time and a description of the nature of the effects observed,

• — results and date of test performed with reference substance, if available,

• — the nominal test concentrations and the result of all analyses to determine the concentration of the test substance in the test vessels; the recovery efficiency of the method and the limit of determination should also be reported,

• — all physical-chemical measurements of temperature, pH and dissolved oxygen made during the test,

• — the EC50 at 48 h for immobilisation with confidence intervals and graphs of the fitted model used for their calculation, the slopes of the dose-response curves and their standard error; statistical procedures used for determination of EC50; (these data items for immobilisation at 24 h should also be reported when they were measured),

• — explanation for any deviation from the Testig Method and whether the deviation affected the test results.

4. REFERENCES

• (1)ISO 6341. (1996). Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) — Acute toxicity test. Third edition, 1996.

• (2)EPA OPPTS 850.1010. (1996). Ecological Effects Test Guidelines — Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids.

• (3)Environment Canada. (1996) Biological test method. Acute Lethality Test Using Daphnia spp. EPS 1/RM/11. Environment Canada, Ottawa, Ontario, Canada.

• (4)Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Environmental Health and Safety Publication. Series on Testing and Assessment. No 23. Paris 2000.

• (5)Commission of the European Communities. Study D8369. (1979). Inter-laboratory Test Programme concerning the study of the ecotoxicity of a chemical substance with respect to Daphnia.

• (6)OECD Guidelines for the Testing of Chemicals. Guideline 211: Daphnia magna Reproduction Test, adopted September 1998.

• (7)Stephan C.E. (1977). Methods for calculating an LC50. In Aquatic Toxicology and Hazard Evaluation (edited by F.I. Mayer and J.L. Hamelink). ASTM STP 634 — American Society for Testing and Materials. p. 65-84

• (8)Finney D.J. (1978). Statistical Methods in Biological Assay. 3^rd ed. London. Griffin, Weycombe, UK.

Appendix 1

SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER

Substance	Concentration
Particulate matter	< 20 mg/l
Total organic carbon	< 2 mg/l
Unionised ammonia	< 1 μg/l
Residual chlorine	< 10 μg/l
Total organophosphorus pesticides	< 50 ng/l
Total organochorine pesticides plus polychlorinated biphenyls	< 50 ng/l
Total organic chlorine	< 25 ng/l

Appendix 2

EXAMPLES OF SUITABLE RECONSTITUTED TEST WATER

ISO Test water (1)

Stock solutions (single substance)	Stock solutions (single substance)	To prepare the reconstituted water, add the following volumes of stock solutions to 1 litre water [^E0060]
Substance	Amount added to 1 litre water [^E0060]	To prepare the reconstituted water, add the following volumes of stock solutions to 1 litre water [^E0060]
Calcium chloride CaCl2, 2H2O	11,76 g	25 ml
Magnesium sulfate MgSO4, 7H2O	4,93 g	25 ml
Sodium bicarbonate NaHCO3	2,59 g	25 ml
Potassium chloride KCl	0,23 g	25 ml

Water of suitable purity, e.g. deionised, distilled or reverse osmosis with conductivity preferably not exceeding 10 μS.cm^-1.

Elendt M7 and M4 medium

Acclimation to Elendt M4 and M7 medium

Some laboratories have experienced difficulty in directly transferring Daphnia to M4 and M7 media. However, some success has been achieved with gradual acclimation, i.e. moving from own medium to 30 % Elendt, then to 60 % Elendt and then to 100 % Elendt. The acclimation periods may need to be as long as one month.

Preparation

Trace element

Separate stock solutions (I) of individual trace elements are first prepared in water of suitable purity, e.g. deionised, distilled or reverse osmosis. From these different stock solutions (I) a second single stock solution (II) is prepared, which contains all trace elements (combined solution), i.e.:

Stock solution(s) I (single substance)	Amount added to water (mg/l)	Concentration (related to medium M4)	To prepare the combined stock solution II, add the following amount of stock solution I to water (ml/l)	To prepare the combined stock solution II, add the following amount of stock solution I to water (ml/l)
Stock solution(s) I (single substance)	Amount added to water (mg/l)	Concentration (related to medium M4)	M4	M7
H3 BO3	57 190	20 000 -fold	1,0	0,25
MnCl2.4H2O	7 210	20 000 -fold	1,0	0,25
LiCl	6 120	20 000 -fold	1,0	0,25
RbCl	1 420	20 000 -fold	1,0	0,25
SrCl2.6H2O	3 040	20 000 -fold	1,0	0,25
NaBr	320	20 000 -fold	1,0	0,25
Na2 MoO4.2H2O	1 230	20 000 -fold	1,0	0,25
CuCl2.2H2O	335	20 000 -fold	1,0	0,25
ZnCl2	260	20 000 -fold	1,0	1,0
CoCl2.6H2O	200	20 000 -fold	1,0	1,0
KI	65	20 000 -fold	1,0	1,0
Na2 SeO3	43,8	20 000 -fold	1,0	1,0
NH4 VO3	11,5	20 000 -fold	1,0	1,0
Na2 EDTA.2H2O	5 000	2 000 -fold	—	—
FeSO4.7H2O	1 991	2 000 -fold	—	—
Both Na 2 EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately.	Both Na 2 EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately.	Both Na 2 EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately.	Both Na 2 EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately.	Both Na 2 EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately.
This gives:	This gives:	This gives:	This gives:	This gives:
2 l Fe-EDTA solution		1 000 -fold	20,0	5,0

M4 and M7 media

M4 and M7 media are prepared using stock solution II, the macro-nutrients and vitamin as follows:

	Amount added to water (mg/l)	Concentration (related to medium M4)	Amount of stock solution II added to prepare medium (ml/l)	Amount of stock solution II added to prepare medium (ml/l)
	Amount added to water (mg/l)	Concentration (related to medium M4)	M4	M7
Stock solution II (combined trace elements)		20-fold	50	50
Macro nutrient stock solutions (single substance)
CaCl2 · 2H20	293 800	1 000 -fold	1,0	1,0
MgSO4 · 7H2O	246 600	2 000 -fold	0,5	0,5
KCl	58 000	10 000 -fold	0,1	0,1
NaHCO3	64 800	1 000 -fold	1,0	1,0
Na2 SiO3 · 9H2O	50 000	5 000 -fold	0,2	0,2
NaNO3	2 740	10 000 -fold	0,1	0,1
KH2 PO4	1 430	10 000 -fold	0,1	0,1
K2HPO4	1 840	10 000 -fold	0,1	0,1
Combined Vitamin stock	—	10 000 -fold	0,1	0,1
The combined vitamin stock solution is prepared by adding the 3 vitamin to 1 litre water, as shown below:	The combined vitamin stock solution is prepared by adding the 3 vitamin to 1 litre water, as shown below:	The combined vitamin stock solution is prepared by adding the 3 vitamin to 1 litre water, as shown below:	The combined vitamin stock solution is prepared by adding the 3 vitamin to 1 litre water, as shown below:	The combined vitamin stock solution is prepared by adding the 3 vitamin to 1 litre water, as shown below:
Thiamine hydrochloride	750	10 000 -fold
Cyanocobalamine (B12)	10	10 000 -fold
Biotine	7,5	10 000 -fold

The combined vitamin stock is stored frozen in small aliquots. Vitamins are added to the media shortly before use.

N.B	:	to avoid precipitation of salts when preparing the complete media, add the aliquots of stock solutions to about 500-800 ml deionised water and then fill up to 1 litre.
---	---	---
N.B	:	the first publication of the M4 medium can be found in Elendt, B. P. (1990). Selenium deficiency in crustacea; an ultrastructual approach to antennal damage in Daphnia magna Straus. Protoplasma, 154, 25-33.

C.3.   FRESHWATER ALGA AND CYANOBACTERIA, GROWTH INHIBITION TEST

INTRODUCTION

• 1.This test method is equivalent to OECD test guideline (TG) 201 (2006, annex corrected in 2011). The need to extend the test method to include additional species and update it to meet the requirements for hazard assessment and classification of chemicals has been identified. This 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.

• 2.Definitions used are given in Appendix 1.

PRINCIPLE OF THE TEST

• 3.The purpose of this test is to determine the effects of a chemical on the growth of freshwater microalgae and/or cyanobacteria. Exponentially growing test organisms are exposed to the test chemical 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.

• 4.The system response is the reduction of growth in a series of algal cultures (test units) exposed to various concentrations of a test chemical. 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 nutrient sufficient conditions and continuous light for a sufficient period of time to measure reduction of the specific growth rate.

• 5.Growth and growth inhibition are quantified from 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.

• 6.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 ErCx (e.g. ErC50).

• 7.An additional response variable used in this test method is yield, which may be needed to fulfil specific regulatory requirements in some countries. It is defined as the 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 EyCx (e.g. EyC50).

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

INFORMATION ON THE TEST CHEMICAL

• 9.Information on the test chemical which may be useful in establishing the test conditions includes 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.

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

VALIDITY OF THE TEST

• 11.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 2). This criterion may not be met when species that grow slower than those listed in Appendix 2 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 hours 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 Appendix 1 under ‘coefficient of variation’) must not exceed 35 %. See paragraph 49 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 %.

REFERENCE CHEMICAL

• 12.Reference chemical(s), such as 3,5-dichlorophenol used in the international ring test (1), may be tested as a means of checking the test procedure. Potassium dichromate can also be used as a reference chemical for green algae. It is desirable to test a reference chemical at least twice a year.

APPLICABILITY OF THE TEST

• 13.This test method is most easily applied to water-soluble chemicals which, under the conditions of the test, are likely to remain in the water. For testing of chemicals that are volatile, strongly adsorbing, coloured, having a low solubility in water or chemicals 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).

DESCRIPTION OF THE TEST METHOD

Apparatus

• 14.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.

• 15.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 paragraph 30). Note that the liquid volume must be sufficient for analytical determinations (see paragraph 37).

• 16.In addition some or all of the following equipment may 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), may 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.

Test organisms

• 17.Several species of non-attached microalgae and cyanobacteria may be used. The strains listed in Appendix 2 have been shown to be suitable using the test procedure specified in this test method.

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

Growth medium

• 19.Two alternative growth media, the OECD and the AAP medium, are recommended. The compositions of these media are shown in Appendix 3. 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 chemicals.

• 20.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 should be described in detail and justified (3) (4).

Initial biomass concentration

• 21.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

Concentrations of test chemical

• 22.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 chemicals 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.

Replicates and controls

• 23.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.

• 24.A separate set of test solutions may be prepared for analytical determinations of test chemical concentrations (see paragraphs 36 and 38).

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

Preparation of inoculum culture

• 26.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. Incubate the inoculum culture 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 4. To avoid synchronous cell divisions during the test a second propagation step of the inoculum culture may be required.

Preparation of test solutions

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

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

Incubation

• 29.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 2, 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.

• 30.The pH of the control medium should not increase by more than 1,5 units during the test. For metals and chemicals 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.

• 31.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, select the light intensity at the level of the test solutions 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}). Maintain the light intensity within ±15 % from the average light intensity over the incubation area.

Test duration

• 32.Test duration is normally 72 hours. However, shorter or longer test durations may be used provided that all validity criteria in paragraph 11 can be met.

Measurements and analytical determinations

• 33.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.

• 34.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 (5) (6), or optical density can be used if a satisfactory correlation with biomass can be demonstrated over the range of biomass occurring in the test.

• 35.Measure the pH of the solutions at the beginning and at the end of the test.

• 36.Provided an analytical procedure for determination of the test chemical 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.

• 37.Analysis of the concentration of the test chemical 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 beginning and at the end of the test is recommended where concentrations are unlikely to remain within 80-120 % of the nominal concentration. For volatile, unstable or strongly adsorbing test chemicals, additional samplings for analysis at 24 hour intervals during the exposure period are recommended in order to better define loss of the test chemical. For these chemicals, extra replicates may be needed. In all cases, determination of test chemical concentrations need only be performed on one replicate vessel at each test concentration (or the contents of the vessels pooled by replicate).

• 38.The 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 chemical 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.

• 39.If there is evidence that the concentration of the chemical 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 not within the range of ± 20 %, analysis of the results should be based on geometric mean concentration during exposure or on models describing the decline of the concentration of the test chemical (3) (7).

• 40.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 chemicals tested at low concentrations. In such cases, disappearance of the test chemical 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 chemical 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 the test chemical (7) may be considered. If not, it may be appropriate to base the analysis of the results on the initial (nominal or measured) concentrations.

Other observations

• 41.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 the exposure to the test chemical) at the end of the test.

Limit test

• 42.Under some circumstances, e.g. when a preliminary test indicates that the test chemical has no toxic effects at concentrations up to 100 mg/l 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 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's t-test. If variances of the two groups are unequal, a t-test adjusted for unequal variances should be performed

DATA AND REPORTING

Plotting growth curves

• 43.The biomass in the test vessels may be expressed in units of the surrogate parameter used for measurement (e.g. cell number, fluorescence).

• 44.Tabulate the estimated biomass concentration in test cultures and controls together with the concentrations of test material and the times of measurement, recorded with a resolution of at least whole hours, to produce plots of growth curves. Both logarithmic scales and linear scales can be useful at this first stage, but logarithmic scales are mandatory and generally give a better presentation of variations in growth pattern during the test period. Note that exponential growth produces a straight line when plotted on a logarithmic scale, and inclination of the line (slope) indicates the specific growth rate.

• 45.Using the plots, examine whether control cultures grow exponentially at the expected rate throughout the test. Examine all data points and the appearance of the graphs critically and check raw data and procedures for possible errors. Check in particular any data point that seems to deviate by a systematic error. If it is obvious that procedural mistakes can be identified and/or considered highly likely, the specific data point is marked as an outlier and not included in subsequent statistical analysis. (A zero algal concentration in one out of two or three replicate vessels may indicate the vessel was not inoculated correctly, or was improperly cleaned). State reasons for rejection of a data point as an outlier clearly in the test report. Accepted reasons are only (rare) procedural mistakes and not just bad precision. Statistical procedures for outlier identification are of limited use for this type of problem and cannot replace expert judgement. Outliers (marked as such) should preferably be retained among the data points shown in any subsequent graphical or tabular data presentation.

Response variables

• 46.The purpose of the test is to determine the effects of the test chemical on the growth of algae. This test method describes two response variables, as different jurisdictions have different preferences and regulatory needs. In order for the test results to be acceptable in all jurisdictions, the effects should be evaluated using both response variables (a) and (b) described below.

• (a) Average specific growth rate : this response variable is calculated on the basis of the logarithmic increase of biomass during the test period, expressed per day

• (b) Yield : this response variable is the biomass at the end of the test minus the starting biomass.

• 47.It should be noted that toxicity values calculated by using these two response variables are not comparable and this difference must be recognised when using the results of the test. ECx values based upon average specific growth rate (ErCx) will generally be higher than results based upon yield (EyCx) if the test conditions of this test method are adhered to, due to the mathematical basis of the respective approaches. This should not be interpreted as a difference in sensitivity between the two response variables, simply that the values are different mathematically. The concept of average specific growth rate is based on the general exponential growth pattern of algae in non-limited cultures, where toxicity is estimated on the basis of the effects on the growth rate, without being dependent on the absolute level of the specific growth rate of the control, slope of the concentration-response curve or on test duration. In contrast, results based upon the yield response variable are dependent upon all these other variables. EyCx is dependent on the specific growth rate of the algal species used in each test and on the maximum specific growth rate that can vary between species and even different algal strains. This response variable should not be used for comparing the sensitivity to toxicants among algal species or even different strains. While the use of average specific growth rate for estimating toxicity is scientifically preferred, toxicity estimates based on yield are also included in this test method to satisfy current regulatory requirements in some countries.

Average growth rate

• 48.The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments [1]:

Image omitted; see official source	[1],

• where:

• μi-j is the average specific growth rate from time i to j;

• Xi is the biomass at time i;

• Xj is the biomass at time j

• For each treatment group and control group, calculate a mean value for growth rate along with variance estimates.

• 49.Calculate the average specific growth rate over the entire test duration (normally days 0-3), using the nominally inoculated biomass as the starting value rather than a measured starting value, because in this way greater precision is normally obtained. If the equipment used for biomass measurement allows sufficiently precise determination of the low inoculum biomass (e.g. flow cytometer) then the measured initial biomass concentration can be used. Assess also the section-by-section growth rate, calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and examine whether the control growth rate remains constant (see validity criteria, paragraph 11). A significantly lower specific growth rate on day one than the total average specific growth rate may indicate a lag phase. While a lag phase can be minimised and practically eliminated in control cultures by proper propagation of the pre-culture, a lag phase in exposed cultures may indicate recovery after initial toxic stress or reduced exposure due to loss of test chemical (including sorption onto the algal biomass) after initial exposure. Hence the section-by-section growth rate may be assessed in order to evaluate effects of the test chemical occurring during the exposure period. Substantial differences between the section-by-section growth rate and the average growth rate indicate deviation from constant exponential growth and that close examination of the growth curves is warranted.

• 50.Calculate the percent inhibition of growth rate for each treatment replicate from equation [2]:

Image omitted; see official source	[2],

• where:

%Ir	=	percent inhibition in average specific growth rate;
---	---	---
μC	=	mean value for average specific growth rate (μ) in the control group;
---	---	---
μT	=	average specific growth rate for the treatment replicate.

• 51.When solvents are used to prepare the test solutions, the solvent controls rather than the controls without solvents should be used in calculation of percent inhibition.

Yield

• 52.Yield is calculated as the biomass at the end of the test minus the starting biomass for each single vessel of controls and treatments. For each test concentration and control, calculate a mean value for yield along with variance estimates. The percent inhibition in yield ( %Iy) may be calculated for each treatment replicate as follows:

Image omitted; see official source	[3]

• where:

%Iy	=	percent inhibition of yield;
---	---	---
YC	=	mean value for yield in the control group;
---	---	---
YT	=	value for yield for the treatment replicate.

Plotting concentration response curve

• 53.Plot the percentage of inhibition against the logarithm of the test chemical concentration and examine the plot closely, disregarding any such data point that was singled out as an outlier in the first phase. Fit a smooth line through the data points by eye or by computerised interpolation to get a first impression of the concentration-response relationship, and then proceed with a more detailed method, preferably a computerised statistical method. Depending on the intended usage of data; the quality (precision) and amount of data as well as the availability of data analysis tools, it may be decided (and sometimes well justified) to stop the data analysis at this stage and simply read the key figures EC50 and EC10 (and/or EC20) from the eye fitted curve (see also section below on stimulatory effects). Valid reasons for not using a statistical method may include:

• — Data are not appropriate for computerised methods to produce any more reliable results than can be obtained by expert judgement — in such situations some computer programs may even fail to produce a reliable solution (iterations may not converge etc.)

• — Stimulatory growth responses cannot be handled adequately using available computer programs (see below).

Statistical procedures

• 54.The aim is to obtain a quantitative concentration-response relationship by regression analysis. It is possible to use a weighted linear regression after having performed a linearising transformation of the response data — for instance into probit or logit or Weibull units (8), but non-linear regression procedures are preferred techniques that better handle unavoidable data irregularities and deviations from smooth distributions. Approaching either zero or total inhibition, such irregularities may be magnified by the transformation, interfering with the analysis (8). It should be noted that standard methods of analysis using probit, logit, or Weibull transforms are intended for use on quantal (e.g. mortality or survival) data, and must be modified to accommodate growth or biomass data. Specific procedures for determination of ECx values from continuous data can be found in (9) (10) and (11). The use of non-linear regression analysis is further detailed in Appendix 5.

• 55.For each response variable to be analysed, use the concentration-response relationship to calculate point estimates of ECx values. When possible, the 95 % confidence limits for each estimate should be determined. Goodness of fit of the response data to the regression model should be assessed either graphically or statistically. Regression analysis should be performed using individual replicate responses, not treatment group means. If, however nonlinear curve fitting is difficult or fails because of too great scatter in the data, the problem may be circumvented by performing the regression on group means as a practical way of reducing the influence of suspected outliers. Use of this option should be identified in the test report as a deviation from normal procedure because curve fits with individual replicates did not produce a good result.

• 56.EC50 estimates and confidence limits may also be obtained using linear interpolation with bootstrapping (13), if available regression models/methods are unsuitable for the data.

• 57.For estimation of the LOEC and hence the NOEC, for effects of the test chemical on growth rate, it is necessary to compare treatment means using analysis of variance (ANOVA) techniques. The mean for each concentration must then be compared with the control mean using an appropriate multiple comparison or trend test method. Dunnett's or Williams' test may be useful (12)(14)(15)(16)(17). It is necessary to assess whether the ANOVA assumption of homogeneity of variance holds. This assessment may be performed graphically or by a formal test (17). Suitable tests are Levene's or Bartlett's. Failure to meet the assumption of homogeneity of variances can sometimes be corrected by logarithmic transformation of the data. If heterogeneity of variance is extreme and cannot be corrected by transformation, analysis by methods such as step-down Jonkheere trend tests should be considered. Additional guidance on determining the NOEC can be found in (11).

• 58.Recent scientific developments have led to a recommendation of abandoning the concept of NOEC and replacing it with regression based point estimates ECx. An appropriate value for x has not been established for this algal test. A range of 10 to 20 % appears to be appropriate (depending on the response variable chosen), and preferably both the EC10 and EC20 should be reported.

Growth stimulation

• 59.Growth stimulation (negative inhibition) at low concentrations is sometimes observed. This can result from either hormesis (‘toxic stimulation’) or from addition of stimulating growth factors with the test material to the minimal medium used. Note that the addition of inorganic nutrients should not have any direct effect because the test medium should maintain a surplus of nutrients throughout the test. Low dose stimulation can usually be ignored in EC50 calculations unless it is extreme. However, if it is extreme, or an ECx value for low x is to be calculated, special procedures may be needed. Deletion of stimulatory responses from the data analysis should be avoided if possible, and if available curve fitting software cannot accept minor stimulation, linear interpolation with bootstrapping can be used. If stimulation is extreme, use of a hormesis model may be considered (18).

Non toxic growth inhibition

• 60.Light absorbing test materials may give rise to a growth rate reduction because shading reduces the amount of available light. Such physical types of effects should be separated from toxic effects by modifying the test conditions and the former should be reported separately. Guidance may be found in (2) and (3).

TEST REPORT

• 61.The test report must include the following:

Test chemical:

• — physical nature and relevant physical-chemical properties, including water solubility limit;

• — chemical identification data (e.g., CAS Number), including purity (impurities).

Test species:

• — the strain, supplier or source and the culture conditions used.

Test conditions:

• — date of start of the test and its duration;

• — description of test design: test vessels, culture volumes, biomass density at the beginning of the test;

• — composition of the medium;

• — test concentrations and replicates (e.g., number of replicates, number of test concentrations and geometric progression used);

• — description of the preparation of test solutions, including use of solvents etc.

• — culturing apparatus;

• — light intensity and quality (source, homogeneity);

• — temperature;

• — concentrations tested: the nominal test concentrations and any results of analyses to determine the concentration of the test chemical in the test vessels. The recovery efficiency of the method and the limit of quantification in the test matrix should be reported;

• — all deviations from this test method;

• — method for determination of biomass and evidence of correlation between the measured parameter and dry weight;

Results:

• — pH values at the beginning and at the end of the test at all treatments;

• — biomass for each flask at each measuring point and method for measuring biomass;

• — growth curves (plot of biomass versus time);

• — calculated response variables for each treatment replicate, with mean values and coefficient of variation for replicates;

• — graphical presentation of the concentration/effect relationship;

• — estimates of toxicity for response variables e.g., EC50, EC10, EC20 and associated confidence intervals. If calculated, LOEC and NOEC and the statistical methods used for their determination;

• — if ANOVA has been used, the size of the effect which can be detected (e.g. the least significant difference);

• — any stimulation of growth found in any treatment;

• — any other observed effects, e.g. morphological changes of the algae;

• — discussion of the results, including any influence on the outcome of the test resulting from deviations from this test method.

LITERATURE

• (1)International Organisation for Standardisation (1993). ISO 8692 Water quality — Algal growth inhibition test.

• (2)International Organisation for Standardisation (1998). ISO/DIS 14442. Water quality — Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waster water.

• (3)OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and mixtures. Environmental Health and Safety Publications. Series on Testing and Assessment, no. 23. Organisation for Economic Co-operation and Development, Paris.

• (4)International Organisation for Standardisation (1998). ISO 5667-16 Water quality — Sampling — Part 16: Guidance on Biotesting of Samples.

• (5)Mayer, P., Cuhel, R. and Nyholm, N. (1997). A simple in vitro fluorescence method for biomass measurements in algal growth inhibition tests. Water Research 31: 2525-2531.

• (6)Slovacey, R.E. and Hanna, P.J. (1997). In vivo fluorescence determinations of phytoplancton chlorophyll, Limnology & Oceanography 22: 919-925

• (7)Simpson, S.L., Roland, M.G.E., Stauber, J.L. and Batley, G.E. (2003). Effect of declining toxicant concentrations on algal bioassay endpoints. Environ. Toxicol. Chem. 22: 2073-2079.

• (8)Christensen, E.R., Nyholm, N. (1984). Ecotoxicological Assays with Algae: Weibull Dose-Response Curves. Env. Sci. Technol. 19: 713-718.

• (9)Nyholm, N. Sørensen, P.S., Kusk, K.O. and Christensen, E.R. (1992). Statistical treatment of data from microbial toxicity tests. Environ. Toxicol. Chem. 11: 157-167.

• (10)Bruce, R.D.,and Versteeg, D.J. (1992). A statistical procedure for modelling continuous toxicity data. Environ. Toxicol. Chem. 11: 1485-1494.

• (11)OECD (2006). Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application. Organisation for Economic Co-operation and Development, Paris.

• (12)Dunnett, C.W. (1955). A multiple comparisons procedure for comparing several treatments with a control. J. Amer. Statist. Assoc. 50: 1096-1121

• (13)Norberg-King T.J. (1988). An interpolation estimate for chronic toxicity: The ICp approach. National Effluent Toxicity Assessment Center Technical Report 05-88. US EPA, Duluth, MN.

• (14)Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics 20: 482-491.

• (15)Williams, D.A. (1971). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27: 103-117.

• (16)Williams, D.A. (1972). The comparison of several dose levels with a zero dose control. Biometrics 28: 519-531.

• (17)Draper, N.R. and Smith, H. (1981). Applied Regression Analysis, second edition. Wiley, New York.

• (18)Brain, P. and Cousens, R. (1989). An equation to describe dose-responses where there is stimulation of growth at low doses. Weed Research, 29, 93-96.

Appendix 1

Definitions

The following definitions and abbreviations are used for the purposes of this test 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.

Chemical means a substance or mixture

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 percent value. 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;

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

ECx is the concentration of the test chemical dissolved in 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 growth rate or yield the symbol ‘ErC’ is used for growth rate and ‘EyC’ is used for yield.

Growth medium is the complete synthetic culture medium in which test algae grow when exposed to the test chemical. The test chemical 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 chemical 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 test 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 quotient of the difference of the natural logarithms of a parameter of observation (in this test method, biomass) and the respective time period

Test chemical means any substance or mixture tested using this test method.

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.

Appendix 2

Strains Shown to be Suitable for the Test

Green algae

Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), ATCC 22662, CCAP 278/4, 61.81 SAG

Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus), 86.81 SAG

Diatoms

Navicula pelliculosa, UTEX 664

Cyanobacteria

Anabaena flos-aquae, UTEX 1444, ATCC 29413, CCAP 1403/13A

Synechococcus leopoliensis, UTEX 625, CCAP 1405/1

Sources of Strains

The strains recommended are available in unialgal cultures from the following collections (in alphabetical order):

ATCC: American Type Culture Collection

10801 University Boulevard

Manassas, Virginia 20110-2209

USA

CCAP, Culture Collection of Algae and Protozoa

Institute of Freshwater Ecology,

Windermere Laboratory

Far Sawrey, Amblerside

Cumbria LA22 0LP

UK

SAG: Collection of Algal Cultures

Inst. Plant Physiology

University of Göttingen

Nikolausberger Weg 18

37073 Göttingen

GERMANY

UTEX Culture Collection of Algae

Section of Molecular, Cellular and Developmental Biology

School of Biological Sciences

the University of Texas at Austin

Austin, Texas 78712

USA.

Appearance and characteristics of recommended species

	P. subcapitata	D. subspicatus	N. pelliculosa	A. flos-aquae	S. leopoliensis
Appearance	Curved, twisted single cells	Oval, mostly single cells	Rods	Chains of oval cells	Rods
Size (L × W) μm	8-14 × 2-3	7-15 × 3-12	7,1 × 3,7	4,5 × 3	6 × 1
Cell volume (μm3/cell)	40-60 [^E0061]	60-80 [^E0061]	40-50 [^E0061]	30-40 [^E0061]	2,5 [^E0062]
Cell dry weight (mg/cell)	2-3 × 10- 8	3-4 × 10- 8	3-4 × 10- 8	1-2 × 10- 8	2-3 × 10- 9
Growth rate [^E0063] (day- 1)	1,5 -1,7	1,2-1,5	1,4	1,1-1,4	2,0-2,4

Measured with electronic particle counter

Calculated from size

Most frequently observed growth rate in OECD medium at light intensity approx. 70 μE m^{- 2} s^{- 1} and 21 °C

Specific Recommendations on Culturing and Handling of Recommended Test Species

Pseudokirchneriella subcapitata and Desmodesmus subspicatus

These green algae are generally easy to maintain in various culture media. Information on suitable media is available from the culture collections. The cells are normally solitary, and cell density measurements can easily be performed using an electronic particle counter or microscope.

Anabaena flos-aquae

Various growth media may be used for keeping a stock culture. It is particularly important to avoid allowing the batch culture to go past log phase growth when renewing, recovery is difficult at this point.

Anabaena flos-aquae develops aggregates of nested chains of cells. The size of these aggregates may vary with culturing conditions. It may be necessary to break up these aggregates when microscope counting or an electronic particle counter is used for determination of biomass.

Sonication of sub-samples may be used to break up chains to reduce count variability. Longer sonication than required for breaking up chains into shorter lengths may destroy the cells. Sonication intensity and duration must be identical for each treatment.

Count enough fields on the hemocytometer (at least 400 cells) to help compensate for variability. This will improve reliability of microscopic density determinations.

An electronic particle counter can be used for determination of total cell volume of Anabaena after breaking up the cell chains by careful sonification. The sonification energy has to be adjusted to avoid disruption of the cells.

Use a vortex mixer or similar appropriate method to make sure the algae suspension used to inoculate test vessels is well mixed and homogeneous.

Test vessels should be placed on an orbital or reciprocate shaker table at about 150 revolutions per minute. Alternatively, intermittent agitation may be used to reduce the tendency of Anabaena to form clumps. If clumping occurs, care must be taken to achieve representative samples for biomass measurements. Vigorous agitation before sampling may be necessary to disintegrate algal clumps.

Synechococcus leopoliensis

Various growth media may be used for keeping a stock culture. Information on suitable media is available from the culture collections.

Synechococcus leopoliensis grows as solitary rod-shaped cells. The cells are very small, which complicates the use of microscope counting for biomass measurements. Electronic particle counters equipped for counting particles down to a size of approximately 1 μm are useful. In vitro fluorometric measurements are also applicable.

Navicula pelliculosa

Various growth media may be used for keeping a stock culture. Information on suitable media is available from the culture collections. Note that silicate is required in the medium.

Navicula pelliculosa may form aggregates under certain growth conditions. Due to production of lipids the algal cells sometimes tend to accumulate in the surface film. Under those circumstances special measures have to be taken when sub-samples are taken for biomass determination in order to obtain representative samples. Vigorous shaking, e.g. using a vortex mixer may be required.

Appendix 3

Growth Media

One of the following two growth media may be used:

• — OECD medium: Original medium of OECD TG 201, also according to ISO 8692

• — US. EPA medium AAP also according to ASTM.

When preparing these media, reagent or analytical-grade chemicals should be used and deionised water.