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)

A liquid chromatograph, fitted with a pulse-free pump and a suitable detection device is required. The use of an injection valve with an injection loop is recommended. Commercial cyanopropyl chemically bound resins on a silica base shall be used (e.g. Hypersil and Zorbax CN). A guard column of the same material may be positioned between the injection system and the analytical column. Columns from different suppliers may vary considerably in their separation efficiency. As a guidance, the following capacity factors k' should be reached: log k'> 0,0 for log Koc = 3,0 and log k' > — 0,4 for log Koc = 2,0 when using methanol/water 55/45 % as mobile phase.

1.7.3.   Mobile phases

Several mobile phases have been tested and the following two are recommended:

• — methanol/water (55/45 % v/v)

• — methanol/0,01 M citrate-buffer pH 6,0 (55/45 % v/v)

HPLC grade methanol and distilled water or citrate-buffer are used to prepare the eluting solvent. The mixture is degassed before use. Isocratic elution should be employed. If methanol/water mixtures are not appropriate, other organic solvent/water mixtures may be tried, e.g. ethanol/water or acetonitrile/water mixtures. For ionisable compounds the use of buffer solution is recommended to stabilise pH. Care must be taken to avoid salt precipitation and column deterioration, which may occur with some organic phase/buffer mixtures.

No additives such as ion pair reagents may be used because they can affect the sorption properties of the stationary phase. Such changes of the stationary phase may be irreversible. For this reason, it is mandatory that experiments using additives are carried out on separate columns.

1.7.4.   Solutes

Test and reference substances should be dissolved in the mobile phase.

1.8.   PERFORMANCE OF THE TEST

1.8.1.   Test condition

The temperature during the measurements should be recorded. The use of a temperature controlled column compartment is highly recommended to guarantee constant conditions during calibration and estimation runs and measurement of the test substance.

1.8.2.   Determination of dead time to

For the determination of the dead time to two different methods may be used (see also Section 1.2).

1.8.2.1.   Determination of the dead time to by means of a homologous series

This procedure has proven to yield reliable and standardised to values. For details see Testing Method A.8: Partition coefficient (n-octanol/water), HPLC method.

1.8.2.2.   Determination of the dead time to by inert substances which are not retained by the column

This technique is based on the injection of solutions of formamide, urea or sodium nitrate. Measurements should be performed at least in duplicate.

1.8.3.   Determination of the retention times tR

Reference substances should be selected as described in Section 1.3. They may be injected as a mixed standard to determine their retention times, provided it has been confirmed that the retention time of each reference standard is unaffected by the presence of the other reference standards. The calibration should be performed at regular intervals at least twice daily in order to account for unexpected changes in column performance. For best practice the calibration injections should be carried out before and after injections of the test substance to confirm retention times have not drifted. The test substances are injected separately in quantities as small as possible (to avoid column overload) and their retention times are determined.

In order to increase the confidence in the measurement, at least duplicate determinations should be made. The values of log Koc derived from individual measurements should fall within a range of 0,25 log unit.

1.8.4.   Evaluation

The capacity factors k' are calculated from the dead time to and retention times tR of the selected reference substances according to equation 4 (see Section 1.2). The log k' data of the reference substances are then plotted against their log Koc values from batch equilibrium experiments given in Tables 1 and 3 of the Appendix. Using this plot, the log k' value of a test substance is then used to determine its log Koc value. If the actual results show that the log Koc of the test substance is outside the calibration range the test should be repeated using different, more appropriate reference substances.

2. DATA AND REPORTING

The report must include the following information:

• — identity of test and reference substances and their purity, and pKa values if relevant,

• — description of equipment and operating conditions, e.g. type and dimension of analytical (and guard) column, means of detection, mobile phase (ratio of components and pH), temperature range during measurements,

• — dead time and the method used for its determination,

• — quantities of test and reference substances introduced in the column,

• — retention times of reference compounds used for calibration,

• — details of fitted regression line (log k' vs log Koc) and a graph of the regression line,

• — average retention data and estimated d log Koc value for the test compound,

• — chromatograms.

3. REFERENCES

• (1)W.J. Lyman, W.F. Reehl, D.H. Rosenblatt (ed), (1990) Handbook of chemical property estimation methods, chapt. 4, McGraw-Hill, New York.

• (2)J. Hodson, N.A. Williams, (1988) The estimation of the adsorption coefficient (Koc) for soils by HPLC. Chemosphere, 17, p. 167.

• (3)G.G. Briggs, (1981) Theoretical and experimental relationships between soil adsorption, octanol-water partition coefficients, water solubilities, bioconcentration factors, and the parachor. J. Agric. Food Chem., 29, p. 1050-1059.

• (4)C.T. Chiou, P.E. Porter, D.W. Schmedding, (1983) Partition equilibria of nonionic organic compounds between soil organic matter and water. Environ. Sci. Technol., 17, p. 227-231.

• (5)Z. Gerstl, U. Mingelgrin (1984) Sorption of organic substances by soils and sediment. J. Environm. Sci. Health, B19, p. 297-312.

• (6)C.T. Chiou, L.J. Peters, V. H. Freed, (1979) A physical concept of soil water equilibria for nonionic organic compounds, Science, 106, p. 831-832.

• (7)S.W. Karickhoff, (1981) Semi-empirical estimation of sorption of hydrophobic pollutants on natural sediments and soils. Chemosphere, 10, p. 833-846.

• (8)W. Kördel, D. Hennecke, M. Herrmann, (1997) Application of the HPLC-screening method for the determination of the adsorption coefficient on sewage sludges. Chemosphere, 35(1/2), p. 121-128.

• (9)M. Mueller, W. Kördel (1996) Comparison of screening methods for the estimation of adsorption coefficients on soil. Chemosphere, 32(12), p. 2493-2504.

• (10)W. Kördel, J. Stutte, G. Kotthoff (1993) HPLC-screening method for the determination of the adsorption coefficient in soil-comparison of different stationary phases, Chemosphere, 27(12), p. 2341-2352.

• (11)B. von Oepen, W. Kördel, W. Klein (1991) Sorption of nonpolar and polar compounds to soils: Processes, measurements and experience with the applicability of the modified OECD Guideline 106, Chemosphere, 22, p. 285-304.

• (12)W. Kördel, G. Kotthoff, J. Müller (1995) HPLC-screening method for the determination of the adsorption coefficient on soil-results of a ring test. Chemosphere, 30(7), p. 1373-1384.

Appendix

Table 1

Comparison of Koc values for soils and sewage sludges, and calculated values by the HPLC screening method [^E0146], [^E0147]

substance	CAS-No	log Koc sewage sludges	log Koc HPLC	Δ	log Koc soils	log Koc HPLC	Δ
Atrazine	1912-24-9	1,66	2,14	0,48	1,81	2,20	0,39
Linuron	330-55-2	2,43	2,96	0,53	2,59	2,89	0,30
Fenthion	55-38-9	3,75	3,58	0,17	3,31	3,40	0,09
Monuron	150-68-5	1,46	2,21	0,75	1,99	2,26	0,27
Phenanthrene	85-01-8	4,35	3,72	0,63	4,09	3,52	0,57
Benzoic acid phenylester	93-99-2	3,26	3,03	0,23	2,87	2,94	0,07
Benzamide	55-21-0	1,60	1,00	0,60	1,26	1,25	0,01
4-Nitrobenzamide	619-80-7	1,52	1,49	0,03	1,93	1,66	0,27
Acetanilide	103-84-4	1,52	1,53	0,01	1,26	1,69	0,08
Aniline	62-53-3	1,74	1,47	0,27	2,07	1,64	0,43
2,5-Dichloroaniline	95-82-9	2,45	2,59	0,14	2,55	2,58	0,03

W. Kördel, D. Hennecke, M. Herrmann, (1997) Application of the HPLC-screening method for the determination of the adsorption coefficient on sewage sludges. Chemosphere, 35(1/2), p. 121-128.

W. Kördel, D. Hennecke, C. Franke, (1997) Determination of the adsorption-coefficients of organic substances on sewage sludges. Chemosphere, 35 (1/2), p. 107-119.

Table 2

Results of a laboratory inter-comparison test (11 participating laboratories) performed to improve and validate the HPLC-method [^E0148]

substance	CAS-No	log Koc	Koc	log Koc
substance	CAS-No	[OECD 106]	[HPLC-method]	[HPLC-method]
Atrazine	1912-24-9	1,81	78 ± 16	1,89
Monuron	150-68-5	1,99	100 ± 8	2,00
Triapenthenol	77608-88-3	2,37	292 ± 58	2,47
Linuron	330-55-2	2,59	465 ± 62	2,67
Fenthion	55-38-9	3,31	2062 ± 648	3,31

W. Kördel, G. Kotthoff, J. Müller (1995). HPLC-screening method for the determination of the adsorption coefficient on soil-results of a ring test. Chemosphere, 30(7), 1373-1384.

Table 3

Recommended reference substances for the HPLC screening method based on soil adsorption data.

Reference substance	CAS-No	log Koc mean values from batch equilibrium	number of Koc data	log S.D.	source
Acetanilide	103-84-4	1,25	4	0,48	[^E0149]
Phenol	108-95-2	1,32	4	0,70	[^E0149]
2-Nitrobenzamide	610-15-1	1,45	3	0,90	[^E0150]
N.N-dimethylbenzamide	611-74-5	1,52	2	0,45	[^E0149]
4-Methylbenzamide	619-55-6	1,78	3	1,76	[^E0149]
Methylbenzoate	93-58-3	1,80	4	1,08	[^E0149]
Atrazine	1912-24-9	1,81	3	1,08	[^E0151]
Isoproturon	34123-59-6	1,86	5	1,53	[^E0151]
3-Nitrobenzamide	645-09-0	1,95	3	1,31	[^E0150]
Aniline	62-53-3	2,07	4	1,73	[^E0149]
3,5-Dinitrobenzamide	121-81-3	2,31	3	1,27	[^E0150]
Carbendazim	10605-21-7	2,35	3	1,37	[^E0151]
Triadimenol	55219-65-3	2,40	3	1,85	[^E0151]
Triazoxide	72459-58-6	2,44	3	1,66	[^E0151]
Triazophos	24017-47-8	2,55	3	1,78	[^E0151]
Linuron	330-55-2	2,59	3	1,97	[^E0151]
Naphthalene	91-20-3	2,75	4	2,20	[^E0149]
Endosulfan-diol	2157-19-9	3,02	5	2,29	[^E0151]
Methiocarb	2032-65-7	3,10	4	2,39	[^E0151]
Acid Yellow 219	63405-85-6	3,16	4	2,83	[^E0149]
1,2,3-Trichlorobenzene	87-61-6	3,16	4	1,40	[^E0149]
γ-HCH	58-89-9	3,23	5	2,94	[^E0149]
Fenthion	55-38-9	3,31	3	2,49	[^E0151]
Direct Red 81	2610-11-9	3,43	4	2,68	[^E0149]
Pyrazophos	13457-18-6	3,65	3	2,70	[^E0151]
α-Endosulfan	959-98-8	4,09	5	3,74	[^E0151]
Diclofop-methyl	51338-27-3	4,20	3	3,77	[^E0151]
Phenanthrene	85-01-8	4,09	4	3,83	[^E0149]
Basic Blue 41 (mix)	26850-47-5 12270-13-2	4,89	4	4,46	[^E0149]
DDT	50-29-3	5,63	1	—	[^E0150]

W. Kördel, J. Müller (1994). Bestimmung des Adsorptionskoeffizienten organischer Chemikalien mit der HPLC. UBA R & D Report No. 106 01 044 (1994).

B.V. Oepen, W. Kördel, W. Klein., (1991) Chemosphere, 22, p. 285-304.

Data provided by industry.

C.20.   DAPHNIA MAGNA REPRODUCTION TEST

INTRODUCTION

This test method is equivalent to OECD test guideline (TG) 211 (2012). OECD test guidelines are periodically reviewed in the light of scientific progress. The reproduction test guideline 211 originates from test guideline 202, Part II, Daphnia sp. reproduction test (1984). It had generally been acknowledged that data from tests performed according to that TG 202 could be variable. This led to considerable effort being devoted to the identification of the reasons for this variability with the aim of producing a better test method. Test guideline 211 is based on the outcome of these research activities, ring-tests and validation studies performed in 1992 (1), 1994 (2) and 2008 (3).

The main differences between the initial version (TG 202, 1984), and second version (TG 211, 1998) of the reproduction test guideline are:

• — the recommended species to be used is Daphnia magna;

• — the test duration is 21 days;

• — for semi-static tests, the number of animals to be used at each test concentration has been reduced from at least 40, preferably divided into four groups of 10 animals, to at least 10 animals held individually (although different designs can be used for flow-through tests);

• — more specific recommendations have been made with regard to test medium and feeding conditions.

• — the main differences between the second version of the reproduction test guideline (TG 211, 1998) and this version are:

• — appendix 7 has been added to describe procedures for the identification of neonate sex if required. In line with previous versions of this test method sex ratio is an optional endpoint;

• — the response variable number of living offspring produced per surviving parental animal has been supplemented with an additional response variable for Daphnia reproduction, i.e. the total number of living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality. The purpose of the added response variable is to align this response variable with other reproduction test methods on invertebrates. Furthermore, in relation to this response variable, it is possible, in this test method, to remove a source of error, namely the effect of inadvertent and/or accidental parental mortality, should that occur during the exposure period.

• — additional statistical guidance for test design and for treatment of results has been included both for ECx (e.g. EC10 or EC50) and for NOEC/LOEC approach.

• — a limit test is introduced.

Definitions used are given in Appendix 1.

PRINCIPLE OF THE TEST

The primary objective of the test is to assess the effect of chemicals on the reproductive output of Daphnia magna. To this end, young female Daphnia (the parent animals), aged less than 24 hours at the start of the test, are exposed to the test chemical added to water at a range of concentrations. The test duration is 21 days. At the end of the test, the total number of living offspring produced is assessed. Reproductive output of the parent animals can be expressed in other ways (e.g. number of living offspring produced per animal per day from the first day offspring were observed) but these should be reported in addition to the total number of living offspring produced at the end of the test. Because of the particular design of the semi-static test compared to other invertebrate reproduction test methods, it is also possible to count the number of living offspring produced by each individual parent animal. This enables that, contrary to other invertebrate reproduction test methods, if the parent animal dies accidentally and/or inadvertently during the test period, its offspring production can be excluded from data assessment. Hence, if parental mortality occurs in exposed replicates, it should be considered whether or not the mortality follows a concentration-response pattern, e.g. if there is a significant regression of the response versus concentration of the test chemical with a positive slope (a statistical test like the Cochran-Armitage trend test may be used for this). If the mortality does not follow a concentration-response pattern, then those replicates with parental mortality should be excluded from the analysis of the test result. If the mortality follows a concentration-response pattern, the parental mortality should be assigned as an effect of the test chemical and the replicates should not be excluded from the analysis. If the parent animal dies during the test i.e. accidentally from mishandling or accident, or inadvertently due to unexplained incident not related to the effect of the test chemical or turns out to be male, then the replicate is excluded from the analysis (see more in paragraph 51). The toxic effect of the test chemical on reproductive output is expressed as ECx by fitting the data to an appropriate model by non-linear regression to estimate the concentration that would cause x % reduction in reproductive output, respectively, or alternatively as the NOEC/LOEC value (4). The test concentrations should preferably bracket the lowest of the used effect concentrations (e.g. EC10) which means that this value is calculated by interpolation and not extrapolation.

The survival of the parent animals and time to production of first brood should also be reported. Other chemical-related effects on parameters such as growth (e.g. length), and possibly intrinsic rate of population increase, can also be examined (see paragraph 44).

INFORMATION ON THE TEST CHEMICAL

Results of an acute toxicity test (see chapter C.2 of this Annex: Daphnia sp. acute immobilisation test) performed with Daphnia magna may be useful in selecting an appropriate range of test concentrations in the reproduction tests. The water solubility and the vapour pressure of the test chemical should be known and a reliable analytical method for the quantification of the chemical in the test solutions with reported recovery efficiency and limit of determination should be available.

Information on the test chemical which may be useful in establishing the test conditions includes the structural formula, purity of the chemical, stability in light, stability under the conditions of the test, pKa, Pow and results of a test for ready biodegradability (see chapters C.4 (determination of ‘ready’ biodegradability), and C.29 (ready biodegradability — CO2 in sealed vessels) of this Annex).

VALIDITY OF THE TEST

For a test to be valid, the following performance criteria should be met in the control(s):

• — the mortality of the parent animals (female Daphnia) does not exceed 20 % at the end of the test;

• — the mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60.

Note: The same validity criterion (20 %) can be used for accidental and inadvertent parental mortality for the controls as well as for each of the test concentrations.

DESCRIPTION OF THE METHOD

Apparatus

Test vessels and other apparatus, which will come into contact with the test solutions, should be made entirely of glass or other chemically inert material. The test vessels will normally be glass beakers.

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

• — oxygen meter (with microelectrode or other suitable equipment for measuring dissolved oxygen in low volume samples);

• — adequate apparatus for temperature control;

• — pH-meter;

• — equipment for the determination of the hardness of water;

• — equipment for the determination of the total organic carbon concentration (TOC) of water or equipment for the determination of the chemical oxygen demand (COD);

• — adequate apparatus for the control of the lighting regime and measurement of light intensity.

Test Organism

The species to be used in the test is Daphnia magna Straus ([^E0152]).

Preferably, the clone should have been identified by genotyping. Research (1) has shown that the reproductive performance of Clone A (which originated from IRCHA in France) (5) consistently meets the validity criterion of a mean of ≥ 60 living offspring per parent animal surviving when cultured under the conditions described in this test method. However, other clones are acceptable provided that the Daphnia culture is shown to meet the validity criteria for the test.

At the start of the test, the animals should be less than 24 hours old and should not be 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.). The stock animals should be maintained in culture conditions (light, temperature, medium, feeding and animals per unit volume) similar to those to be used in the test. If the Daphnia culture medium to be used in the test is different from that used for routine Daphnia culture, it is good practice to include a pre-test acclimation period of normally about 3 weeks (i.e. one generation) to avoid stressing the parent animals.

Test medium

It is recommended that a fully defined medium be used in this test. This can avoid the use of additives (e.g. seaweed, soil extract), which are difficult to characterise, and therefore improves the opportunities for standardisation between laboratories. Elendt M4 (6) and M7 media (see Appendix 2) have been found to be suitable for this purpose. However, other media (e.g. (7) (8)) are acceptable provided the performance of the Daphnia culture is shown to meet the validity criteria for the test.

If media are used which include undefined additives, these additives should be specified clearly and information should be provided in the test report on composition, particularly with regard to carbon content as this may contribute to the diet provided. It is recommended that the total organic carbon (TOC) and/or chemical oxygen demand (COD) of the stock preparation of the organic additive be determined and an estimate of the resulting contribution to the TOC/COD in the test medium made. It is further recommended that TOC levels in the medium (i.e. before addition of the algae) be below 2 mg/l (9).

When testing chemicals containing metals, it is important to recognise that the properties of the test medium (e.g. hardness, chelating capacity) may have a bearing on the toxicity of the test chemical. For this reason, a fully defined medium is desirable. However, at present, the only fully defined media which are known to be suitable for long-term culture of Daphnia magna are Elendt M4 and M7. Both media contain the chelating agent EDTA. Work has shown (2) that the ‘apparent toxicity’ of cadmium is generally lower when the reproduction test is performed in M4 and M7 media than in media containing no EDTA. M4 and M7 are not, therefore, recommended for testing chemicals containing metals, and other media containing known chelating agents should also be avoided. For metal-containing chemicals it may be advisable to use an alternative medium such as, for example, ASTM reconstituted hard fresh water (9), which contains no EDTA. This combination of ASTM reconstituted hard fresh water and seaweed extract (10) is suitable for long-term culturing of Daphnia magna (2).

The dissolved oxygen concentration should be above 3 mg/l at the beginning and during the test. The pH should be within the range 6 - 9, and normally it should not vary by more than 1,5 units in any one test. Hardness above 140 mg/l (as CaCO3) is recommended. Tests at this level and above have demonstrated reproductive performance in compliance with the validity criteria (11) (12).

Test solutions

Test solutions of the chosen concentrations are usually prepared by dilution of a stock solution. Stock solutions should preferably be prepared, without using any solvents or dispersants if possible, by mixing or agitating the test chemical in test medium using mechanical means such as agitating, stirring or ultrasonication, or other appropriate methods. It is preferable to expose test systems to concentrations of the test chemical to be used in the study for as long as is required to demonstrate the maintenance of stable exposure concentrations prior to the introduction of test organisms. If the test chemical is difficult to dissolve in water, procedures described in the OECD Guidance for handling difficult substances should be followed (13). The use of solvents or dispersants should be avoided, but may be necessary in some cases in order to produce a suitably concentrated stock solution for dosing.

A dilution water control with adequate replicates and, if unavoidable, a solvent control with adequate replicates should be run in addition to the test concentrations. Only solvents or dispersants that have been investigated to have no significant or only minimal effects on the response variable should be used in the test. Examples of suitable solvents (e.g. acetone, ethanol, methanol, dimethylformamide and triethylene glycol) and dispersants (e.g. Cremophor RH40, methylcellulose 0,01 % and HCO-40) are given in (13). Where a solvent or dispersant is used, its final concentration should not be greater than 0,1 ml/l (13) and it should be the same concentration in all test vessels, except the dilution water control. However, every effort should be made to keep the solvent concentration to a minimum.

PROCEDURE

Conditions of Exposure

Duration

The test duration is 21 days.

Loading

Parent animals are maintained individually, one per test vessel, usually with 50 - 100 ml (for Daphnia magna, smaller volumes may be possible especially for smaller daphnids e.g. Ceriodaphnia dubia) of medium in each vessel, unless a flow-through test design is necessary for testing.

Larger volumes may sometimes be necessary to meet requirements of the analytical procedure used for determination of the test chemical concentration, although pooling of replicates for chemical analysis is also allowable. If volumes greater than 100 ml are used, the ration given to the Daphnia may need to be increased to ensure adequate food availability and compliance with the validity criteria.

Test animals

For semi-static tests, at least 10 animals individually held at each test concentration and at least 10 animals individually held in the control series.

For flow-through tests, 40 animals divided into four groups of 10 animals at each test concentration has been shown to be suitable (1). A smaller number of test organisms may be used and a minimum of 20 animals per concentration divided into two or more replicates with an equal number of animals (e.g. four replicates each with five daphnids) is recommended. Note that for tests where animals are held in groups, it will not be possible to exclude any offspring from the statistical analysis if inadvertent/ accidental parental mortality occurs when the reproduction has begun, and hence in these cases the reproductive output should be expressed as total number of living offspring produced per parent present at the beginning of the test.

Treatments should be allocated to the test vessels and all subsequent handling of the test vessels should be done in a random fashion. Failure to do this may result in bias that could be construed as being a concentration effect. In particular, if experimental units are handled in treatment or concentration order, then some time-related effect, such as operator fatigue or other error, could lead to greater effects at the higher concentrations. Furthermore, if the test results are likely to be affected by an initial or environmental condition of the test, such as position in the laboratory, then consideration should be given to blocking the test.

Feeding

For semi-static tests, feeding should preferably be done daily, but at least three times per week (i.e. corresponding to media changes). The possible dilution of the exposure concentrations by food addition should be taken into account and avoided as much as possible with well concentrated algae suspensions. Deviations from this (e.g. for flow-through tests) should be reported.

During the test, the diet of the parent animals should preferably be living algal cells of one or more of the following: Chlorella sp., Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) and Desmodesmus subspicatus (formerly Scenedesmus subspicatus). The supplied diet should be based on the amount of organic carbon (C) provided to each parent animal. Research (14) has shown that, for Daphnia magna, ration levels of between 0,1 and 0,2 mg C/Daphnia/day are sufficient for achieving the required number of living offspring to meet the test validity criteria. The ration can be supplied either at a constant rate throughout the period of the test, or, if desired, a lower rate can be used at the beginning and then increased during the test to take account of growth of the parent animals. In this case, the ration should still remain within the recommended range of 0,1 - 0,2 mg C/Daphnia/day at all times.

If surrogate measures, such as algal cell number or light absorbance, are to be used to feed the required ration level (i.e. for convenience since measurement of carbon content is time consuming), each laboratory should produce its own nomograph relating the surrogate measure to carbon content of the algal culture (see Appendix 3 for advice on nomograph production). Nomographs should be checked at least annually and more frequently if algal culture conditions have changed. Light absorbance has been found to be a better surrogate for carbon content than cell number (15).

A concentrated algal suspension should be fed to the Daphnia to minimise the volume of algal culture medium transferred to the test vessels. Concentration of the algae can be achieved by centrifugation followed by re-suspension in Daphnia culture medium.

Light

16 hours light at an intensity not exceeding 15-20 μE · m^{– 2} · s^{– 1} measured at the water surface of the vessel. For light-measuring instruments calibrated in lux, an equivalent range of 1 000 -1 500 lux for cool white light corresponds close to the recommended light intensity 15-20 μE · m-2 · s-1.

Temperature

The temperature of the test media should be within the range 18-22 °C. However, for any one test, the temperature should not, if possible, vary by more than 2 °C within these limits (e.g. 18-20, 19-21 or 20-22 °C) as daily range. It may be appropriate to use an additional test vessel for the purposes of temperature monitoring.

Aeration

The test vessels should not be aerated during the test.

Test design

Range finding test

When necessary, a range-finding test is conducted with, for example five test chemical concentrations and two replicates for each treatment and control. Additional information, from tests with similar chemicals or from literature, on acute toxicity to Daphnia and/or other aquatic organisms may also be useful in deciding on the range of concentrations to be used in the range-finding test.

The duration of the range-finding test is 21 days or of a sufficient duration to reliably predict effect levels. At the end of the test, reproduction of the Daphnia is assessed. The number of parents and the occurrence of offspring should be recorded.

Definitive test

Normally there should be at least five test concentrations, bracketing effective concentration (e.g. ECx), and arranged in a geometric series with a separation factor preferably not exceeding 3,2 An appropriate number of replicates for each test concentration should be used (see paragraphs 24-25). Justification should be provided if fewer than five concentrations are used. Chemicals should not be tested above their solubility limit in test medium. Before conducting the experiment it is advisable to consider the statistical power of the tests design and using appropriate statistical methods (4). In setting the range of concentrations, the following should be borne in mind:

• (i) When ECx for effects on reproduction is estimated, it is advisable that sufficient concentrations are used to define the ECx with an appropriate level of confidence. Test concentrations used should preferably bracket the estimated ECx such that ECx is found by interpolation rather than extrapolation. It is an advantage for the following statistical analysis to have more test concentrations (e.g. 10) and fewer replicates of each concentration (e.g. 5 thus holding the total number of vessels constant) and with 10 controls.

• (ii) When estimating the LOEC and/or NOEC, the lowest test concentration should be low enough so that the reproductive output at that concentration is not significantly lower than that in the control. If this is not the case, the test should be repeated with a reduced lowest concentration.

• (iii) When estimating the LOEC and/or NOEC, the highest test concentration should be high enough so that the reproductive output at that concentration is significantly lower than that in the control. If this is not the case, the test should be repeated with an increased highest concentration unless the maximum required test concentration for chronic effects testing (i.e., 10 mg/l) was used as the highest test concentration in the initial test.

If no effects are observed at the highest concentration in the range-finding test (e.g. at 10 mg/l), or when the test chemical is highly likely to be of low/ no toxicity based on lack of toxicity to other organisms and/or low/no uptake, the reproduction test may be performed as a limit test, using a test concentration of e.g.10 mg/l and the control. Ten replicates should be used for both the treatment and the control groups. When a limit test might need to be done in a flow-through system less replicates would be adequate. A limit test will provide the opportunity to demonstrate that there is no statistically significant effect at the limit concentration, but if effects are recorded a full test will normally be required.

Controls

One test-medium control series and also, if relevant, one control series containing the solvent or dispersant should be run in addition to the test series. When used, the solvent or dispersant concentration should be the same as that used in the vessels containing the test chemical. The appropriate number of replicates should be used (see paragraphs 23-24).

Generally in a well-run test, the coefficient of variation around the mean number of living offspring produced per parent animal in the control(s) should be ≤ 25 %, and this should be reported for test designs using individually held animals.

Test medium renewal

The frequency of medium renewal will depend on the stability of the test chemical, but should be at least three times per week. If, from preliminary stability tests (see paragraph 7), the test chemical concentration is not stable (i.e. outside the range 80 - 120 % of nominal or falling below 80 % of the measured initial concentration) over the maximum renewal period (i.e. 3 days), consideration should be given to more frequent medium renewal, or to the use of a flow-through test.

When the medium is renewed in semi-static tests, a second series of test vessels are prepared and the parent animals transferred to them by, for example, a glass pipette of suitable diameter. The volume of medium transferred with the Daphnia should be minimised.

Observations

The results of the observations made during the test should be recorded on data sheets (see examples in Appendixes 4 and 5). If other measurements are required (see paragraph 44), additional observations may be required.

Offspring

The offspring produced by each parent animal should preferably be removed and counted daily from the appearance of the first brood to prevent them consuming food intended for the parent. For the purpose of this test method it is only the number of living offspring that needs to be counted, but the presence of aborted eggs or dead offspring should be recorded.

Mortality

Mortality among the parent animals should be recorded preferably daily, or at least as frequently as offspring are counted.

Other parameters

Although this test method is designed principally to assess effects on reproductive output, it is possible that other effects may also be sufficiently quantified to allow statistical analysis. Reproductive output per surviving parent animal, i.e. number of living offspring produced during the test per surviving parent, may be recorded. This may be compared with the main response variable (reproductive output per parent animal in the start of the test which did not inadvertently or accidentally die during the test). If parental mortality occurs in exposed replicates it should be considered whether or not the mortality follows a concentration-response pattern, e.g. if there is a significant regression of the response versus concentration of the test chemical with a positive slope (a statistical test like the Cochran-Armitage trend test may be used for this). If the mortality does not follow a concentration-response pattern, then those replicates with parental mortality should be excluded from the analysis of the test result. If the mortality follows a concentration-response pattern, the parental mortality should be assigned as an effect of the test chemical and the replicates should not be excluded from the analysis of the test result. Growth measurements are highly desirable since they provide information on possible sublethal effects which may be useful in addition to reproduction measures alone; the measurement of the length of the parent animals (i.e. body length excluding the anal spine) at the end of the test is recommended. Other parameters that can be measured or calculated include time to production of first brood (and subsequent broods), number and size of broods per animal, number of aborted broods, presence of male neonates (OECD, 2008) or ephippia and possibly the intrinsic rate of population increase (see Appendix 1 for definition and Appendix 7 for the identification of the sex of neonates).

Frequency of analytical determinations and measurements

Oxygen concentration, temperature, hardness and pH values should be measured at least once a week, in fresh and old media, in the control(s) and in the highest test chemical concentration.

During the test, the concentrations of test chemical are determined at regular intervals.

In semi-static tests where the concentration of the test chemical is expected to remain within ± 20 per cent of the nominal (i.e. within the range 80 - 120 per cent- see paragraphs 6, 7 and 39), it is recommended that, as a minimum, the highest and lowest test concentrations be analysed when freshly prepared and at the time of renewal on one occasion during the first week of the test (i.e. analyses should be made on a sample from the same solution — when freshly prepared and at renewal). These determinations should be repeated at least at weekly intervals thereafter.

For tests where the concentration of the test chemical is not expected to remain within ± 20 per cent of the nominal, it is necessary to analyse all test concentrations, when freshly prepared and at renewal. However, for those tests where the measured initial concentration of the test chemical is not within ± 20 per cent 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 per cent of initial concentrations), chemical determinations could be reduced in weeks 2 and 3 of the test to the highest and lowest test concentrations. In all cases, determination of test chemical concentrations prior to renewal need only be performed on one replicate vessel at each test concentration.

If a flow-through test is used, a similar sampling regime to that described for semi-static tests is appropriate (but measurement of ‘old’ solutions is not applicable in this case). However, it may be advisable to increase the number of sampling occasions during the first week (e.g. three sets of measurements) to ensure that the test concentrations are remaining stable. In these types of test, the flow-rate of diluent and test chemical should be checked daily.

If there is evidence that the concentration of the chemical being tested has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the test, then results can be based on nominal or measured initial values. If the deviation from the nominal or measured initial concentration is greater than ± 20 per cent, results should be expressed in terms of the time-weighted mean (see guidance for calculation in Appendix 6).

DATA AND REPORTING

Treatment of results

The purpose of this test is to determine the effect of the test chemical on the reproductive output. The total number of living offspring per parent animal should be calculated for each test vessel (i.e. replicate). In addition, the reproduction can be calculated based on the production of living offspring by the surviving parent organism. However, the ecologically most relevant response variable is the total number of living offspring produced per parent animal which does not die accidentally ([^E0153]) or inadvertently ([^E0154]) during the test. If the parent animal dies accidentally or inadvertently during the test, or turns out to be male, then the replicate is excluded from the analysis. The analysis will then be based on a reduced number of replicates. If parental mortality occurs in exposed replicates it should be considered whether or not the mortality follows a concentration-response pattern, e.g. if there is a significant regression of the response versus concentration of the test chemical with a positive slope (a statistical test like the Cochran-Armitage trend test may be used for this). If the mortality does not follow a concentration-response pattern, then those replicates with parental mortality should be excluded from the analysis of the test result. If the mortality follows a concentration-response pattern, the parental mortality should be assigned as an effect of the test chemical and the replicates should not be excluded from the analysis of the test result.

In summary, when LOEC and NOEC or ECx are being used to express the effects, it is recommended to calculate the effect on reproduction by the use of both response variables mentioned above i.e.

• — as the total number of living offspring produced per parent animal which does not die accidentally or inadvertently during the test and;

• — as the number of living offspring produced per surviving parental animal;

and then to use as the final result the lowest NOEC and LOEC or ECx value calculated by using either of these two response variables.

Before employing the statistical analysis, e.g. ANOVA procedures, comparison of treatments to the control by Student t-test, Dunnett's test, Williams' test, or stepdown Jonckheere-Terpstra test, it is recommended to consider transformation of data if needed for meeting the requirements of the particular statistical test. As non-parametric alternatives one can consider Dunn's or Mann-Whitney's tests. 95 % confidence intervals are calculated for individual treatment means.

The number of surviving parents in the untreated controls is a validity criterion, and should be documented and reported. Also all other detrimental effects, e.g. abnormal behavior and toxicological significant findings, should be reported in the final report as well.

ECx

ECx-values, including their associated lower and upper confidence limits, are calculated using appropriate statistical methods (e.g. logistic or Weibull function, trimmed Spearman-Karber method, or simple interpolation). To compute the EC10, EC50 or any other ECx, the complete data set should be subjected to regression analysis.

NOEC/LOEC

If a statistical analysis is intended to determine the NOEC/LOEC appropriate statistical methods should be used according to OECD Document 54 on the Current Approaches in the Statistical Analysis of Ecotoxicity Data: a Guidance to Application (4). In general, adverse effects of the test chemical compared to the control are investigated using one-tailed hypothesis testing at p ≤ 0,05.

Normal distribution and variance homogeneity can be tested using an appropriate statistical test, e.g. the Shapiro-Wilk test and Levene test, respectively (p≤ 0,05). One-way ANOVA and subsequent multi-comparison tests can be performed. Multiple comparisons (e.g. Dunnett's test) or step-down trend tests (e.g. Williams' test, or stepdown Jonckheere-Terpstra test) can be used to calculate whether there are significant differences (p ≤ 0,05) between the controls and the various test chemical concentrations (selection of the recommended test according to OECD Guidance Document 54 (4)). Otherwise, non-parametric methods (e.g. Bonferroni-U-test according to Holm or Jonckheere-Terpstra trend test) could be used to determine the NOEC and the LOEC.

Limit test

If a limit test (comparison of control and one treatment only) has been performed and the prerequisites of parametric test procedures (normality, homogeneity) are fulfilled, metric responses can be evaluated by the Student test (t-test). An unequal-variance t-test (such as Welch test) or a non-parametric test such as the Mann-Whitney-U-test may be used, if these requirements are not fulfilled.

To determine significant differences between the controls (control and solvent or dispersant control), the replicates of each control can be tested as described for the limit test. If these tests do not detect significant differences, all control and solvent control replicates may be pooled. Otherwise all treatments should be compared with the solvent control.

Test report

The test report includes the following:

Test chemical:

• — physical nature and relevant physicochemical properties;

• — chemical identification data, including purity.

Test species:

• — the clone (whether it has been genetically typed), supplier or source (if known) and the culture conditions used. If a different species to Daphnia magna is used, this should be reported and justified.

Test conditions:

• — test procedure used (e.g. semi-static or flow-through, volume, loading in number of Daphnia per litre);

• — photoperiod and light intensity;

• — test design (e.g. number of replicates, number of parents per replicate);

• — details of culture medium used;

• — if used, additions of organic material including the composition, source, method of preparation, TOC/COD of stock preparations, estimation of resulting TOC/COD in test medium;

• — detailed information on feeding, including amount (in mg C/daphnia/day) and schedule (e.g. type of food(s), including, for algae the specific name (species) and, if known, the strain, the culture conditions);

• — method of preparation of stock solutions and frequency of renewal (the solvent or dispersant and its concentration should be given, when used).

Results:

• — results from any preliminary studies on the stability of the test chemical;

• — the nominal test concentrations and the results of all analyses to determine the concentration of the test chemical in the test vessels (see example data sheets in Appendix 5); the recovery efficiency of the method and the limit of determination should also be reported;

• — water quality within the test vessels (i.e. pH, temperature and dissolved oxygen concentration, and TOC and/or COD and hardness where applicable) (see example data sheet in Appendix 4);

• — the full record of the production of living offspring during the test by each parent animal (see example data sheet in Appendix 4);

• — the number of deaths among the parent animals and the day on which they occurred (see example data sheet in Appendix 4);

• — the coefficient of variation for control reproductive output (based on total number of living offspring per parent animal alive at the end of the test);

• — plot of total number of living offspring produced per parent animal in each replicate excluding any parent animal which may have accidentally or inadvertently died during the test vs. concentration of the test chemical;

• — as appropriate plot of total number of living offspring produced per surviving parent animal in each replicate vs. concentration of the test chemical

• — where appropriate the Lowest Observed Effect Concentration (LOEC) for reproduction, including a description of the statistical procedures used and an indication of what size of effect could be expected to be detected (a power analysis can be performed before the start of the experiment to provide this) and the No Observed Effect Concentration (NOEC) for reproduction; information on which response variable that has been used for calculating the LOEC and NOEC value (either as total living offspring per maternal organism which did not die accidentally or inadvertently during the test or as total number of living offspring per surviving maternal organism), where appropriate, the LOEC or NOEC for mortality of the parent animals should also be reported;

• — where appropriate, the ECx for reproduction and confidence intervals (e.g. 90 % or 95 %) and a graph of the fitted model used for its calculation, the slope of the concentration-response curve and its standard error;

• — other observed biological effects or measurements: report any other biological effects which were observed or measured (e.g. growth of parent animals) including any appropriate justification;

• — an explanation for any deviation from the test method.

LITERATURE:

• (1) OECD Test Guidelines Programme. Report of the Workshop on the Daphnia magna Pilot Ring Test, Sheffield University, U.K., 20-21 March 1993.

• (2) OECD (1997). Report of the Final Ring Test of the Daphnia magna Reproduction Test. Environmental Health and Safety Publications, Series on Testing and Assessment No.6. OECD, Paris.

• (3) OECD (2008). Validation report for an enhancement of OECD TG 211 Daphnia magna reproduction test. Environmental Health and Safety Publications, Series on Testing and Assessment, No.88. OECD, Paris.

• (4) OECD (2006). Current approaches in the statistical analysis of ecotoxicity data: a guidance to application. Environmental Health and Safety Publications, Series on Testing and Assessment Number 54. OECD, Paris.

• (5) Baird, D.J., et al. (1991). A comparative study of genotype sensitivity to acute toxic stress using clones of Daphnia magna Straus. Ecotox. and Environ. Safety, 21, 257-265.

• (6) Elendt, B.-P. (1990). Selenium deficiency in Crustacea; An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma, 154, 25-33.

• (7) EPA (2002). Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition. EPA/821/R-02/012. U.S. Environmental Protection Agency, Office of Water, Washington, DC. www.epa.gov/waterscience/methods

• (8) Vigano, L. (1991). Suitability of commercially available spring waters as standard medium for culturing Daphnia magna. Bull. Environ. Contam. Toxicol., 47, 775-782.

• (9) ASTM. (2008) Standard Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. In: Annual Book of ASTM Standards; Water and Environmental Technology, vol. 11.04; ASTM E729 — 96 (2007) American Society for Testing and Materials, Philadelphia, PA

• (10) Baird, D.J., et al. (1989). The long term maintenance of Daphnia magna Straus for use in ecotoxicological tests; problems and prospects. In: Proceedings of the 1st European Conference on Ecotoxicology. Copenhagen 1988. (H. Løkke, H. Tyle and F. Bro-Rasmussen. Eds.) pp 144-148.

• (11) Parkhurst, B.R., J.L Forte. And G.P. and Wright (1981) Reproducibility of a life-cycle toxicity test with Daphnia magna. Bull. Environ. Contam. and Toxicol., 26: 1-8.

• (12) Cowgill, U.M. and Milazzo, D.P. (1990). The sensitivity of two cladocerans to water quality variables: salinity and hardness. Arch. Hydrobiol., 120(2): 185-196.

• (13) 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. OECD, Paris.

• (14) Sims, I.R., S. Watson. and D. Holmes (1993) Toward a standard Daphnia juvenile production test. Environ. Toxicol. and Chem., 12, 2053-2058.

• (15) Sims, I. (1993). Measuring the growth of phytoplankton: the relationship between total organic carbon with three commonly used parameters of algal growth. Arch. Hydrobiol., 128, 459-466.

Appendix 1

DEFINITIONS:

For the purposes of this test method the following definitions are used:

Accidental mortality : non chemical related mortality caused by an accidental incidence (i.e. known cause).

Chemical : a substance or mixture.

ECx : the concentration of the test chemical dissolved in water that results in a x per cent reduction in reproduction of Daphnia within a stated exposure period.

Inadvertent mortality : non chemical related mortality with no known cause.

Intrinsic rate of population increase : a measure of population growth which integrates reproductive output and age-specific mortality (1) (2) (3). In steady state populations it will be zero. For growing populations it will be positive and for shrinking populations it will be negative. Clearly the latter is not sustainable and ultimately will lead to extinction.

Limit of detection : the lowest concentration that can be detected but not quantified.

Limit of determination : the lowest concentration that can be measured quantitatively.

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

Mortality : an animal is recorded as dead when it is immobile, i.e. when it is not able to swim, or if there is no observed movement of appendages or postabdomen, within 15 seconds after gentle agitation of the test container. (If another definition is used, this should be reported together with its reference).

No Observed Effect Concentration (NOEC) : the test concentration immediately below the LOEC, which when compared with the control, has no statistically significant effect (p < 0,05), within a stated exposure period.

Offspring : the young Daphnia produced by the parent animals in the course of the test.

Parent Animals : those female Daphnia present at the start of the test and of which the reproductive output is the object of study.

Reproductive output : the number of living offspring produced by parental animals within the test period

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

LITERATURE

• (1) Wilson, E.O. and Bossert, W.H. (1971). A Primer of Population Biology. Sinauer Associates Inc. Publishers.

• (2) Poole, R.W. (1974). An Introduction to quantitative Ecology. Mc Graw Hill Series in Population Biology, New York, p 532.

• (3) Meyer, J. S., Ingersoll, C. G., McDonald, L.L. and Boyce, M.S. (1986). Estimating uncertainty in population growth rates: Jackknife vs bootstrap techniques. Ecology, 67, 1156-1166.

Appendix 2

PREPARATION OF FULLY DEFINED ELENDT M7 AND M4 MEDIA

Acclimation to Elendt M7 and M4 media

Some laboratories have experienced difficulty in directly transferring Daphnia to M4 (1) 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 elements

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	Concentration (related to medium M4)	To prepare the combined stock-solution II add the following amount of stock solution I to water	To prepare the combined stock-solution II add the following amount of stock solution I to water
	mg/l		ml/l	ml/l
			M 4	M 7
H3BO3	57 190	20 000 -fold	1,0	0,25
MnCl2 · 4 H2O	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 · 6 H2O	3 040	20 000 -fold	1,0	0,25
NaBr	320	20 000 -fold	1,0	0,25
Mo Na2O4 · 2 H2O	1 260	20 000 -fold	1,0	0,25
CuCl2 · 2 H2O	335	20 000 -fold	1,0	0,25
ZnCl2	260	20 000 -fold	1,0	1,0
CoCl2 · 6 H2O	200	20 000 -fold	1,0	1,0
KI	65	20 000 -fold	1,0	1,0
Na2SeO3	43,8	20 000 -fold	1,0	1,0
NH4VO3	11,5	20 000 -fold	1,0	1,0
Na2EDTA · 2 H2O	5 000	2 000 -fold	—	—
FeSO4 · 7 H2O	1 991	2 000 -fold	—	—
Both Na2EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately. This gives:	Both Na2EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately. This gives:	Both Na2EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately. This gives:	Both Na2EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately. This gives:	Both Na2EDTA and FeSO4 solutions are prepared singly, poured together and autoclaved immediately. This gives:
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 vitamins as follows:

	Amount added to water	Concentration (related to medium M4)	Amount of stock solution added to prepare medium	Amount of stock solution added to prepare medium
	mg/l		ml/l	ml/l
			M 4	M 7
Stock solution II (combined trace elements)		20-fold	50	50
Macro nutrient stock solutions (single substance)
CaCl2 · 2 H2O	293 800	1 000 -fold	1,0	1,0
MgSO4 · 7 H2O	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
Na2SiO3 · 9 H2O	50 000	5 000 -fold	0,2	0,2
NaNO3	2 740	10 000 -fold	0,1	0,1
KH2PO4	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 vitamins to 1 litre water, as shown below:	The combined vitamin stock solution is prepared by adding the 3 vitamins to 1 litre water, as shown below:	The combined vitamin stock solution is prepared by adding the 3 vitamins to 1 litre water, as shown below:	The combined vitamin stock solution is prepared by adding the 3 vitamins to 1 litre water, as shown below:	The combined vitamin stock solution is prepared by adding the 3 vitamins to 1 litre water, as shown below:
	mg/l
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 deionized water and then fill it up to 1 litre.

• N.N.B. The first publication of the M4 medium can be found in Elendt, B.P. (1990). Selenium deficiency in crustacea; an ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma, 154, 25-33.

Appendix 3

TOTAL ORGANIC CARBON (TOC) ANALYSIS AND PRODUCTION OF A NOMOGRAPH FOR TOC CONTENT OF ALGAL FEED

It is recognised that the carbon content of the algal feed will not normally be measured directly but from correlations (i.e. nomographs) with surrogate measures such as algal cell number or light absorbance).

TOC should be measured by high temperature oxidation rather than by UV or persulphate methods. (For advice see: The Instrumental Determination of Total Organic Carbon, Total Oxygen Demand and Related Determinands 1979, HMSO 1980; 49 High Holborn, London WC1V 6HB).

For nomograph production, algae should be separated from the growth medium by centrifugation followed by resuspension in distilled water. Measure the surrogate parameter and TOC concentration in each sample in triplicate. Distilled water blanks should be analysed and the TOC concentration deducted from that of the algal sample TOC concentration.

Nomographs should be linear over the required range of carbon concentrations. Examples are shown below.

• N.B. These should not be used for conversions; it is essential that laboratories prepare their own nomographs.

Image omitted; see official source

Chlorella vulgaris var. viridis (CCAP 211/12).

Regression of mg/l dry weight on mg C/1. Data from concentrated suspensions of semi continuous batch cultured cells, re-suspended in destilled water.

x-axis: mg C/1of concentrated algal feed

y-axis: mg/1 dry weight of concentrated algal feed

Correction coefficient – 0,980

Image omitted; see official source

Chlorella vulgaris var. viridis (CCAP 211/12).

Regression of cell number on mg C/1. Data from concentrated suspensions of semi continuous batch cultured cells, re-suspended in destilled water.

x-axis: mg C/1of concentrated algal feed

y-axis: No. cells/1 of concentrated algal feed

Correction coefficient – 0,926

Image omitted; see official source

Chlorella vulgaris var. viridis (CCAP 211/12).

Regression of absorbance on mg C/1 (1 cm path length). Data from concentrated suspensions of semi continuous batch cultured cells, re-suspended in destilled water.

x-axis: mg C/1of concentrated algal feed

y-axis: Absorbance at 440 nm of a 1/10 dilution of concentrated algal feed

Correction coefficient – 0,998

Appendix 4

EXAMPLE DATA SHEET FOR RECORDING MEDIUM RENEWAL, PHYSICAL/CHEMICAL MONITORING DATA, FEEDING, DAPHNIA REPRODUCTION AND PARENT MORTALITY

Experiment No:	Date started:	Date started:	Date started:	Date started:	Date started:		Clone:	Clone:		Medium:	Medium:	Medium:		Type of food:	Type of food:	Type of food:		Test Chemical:	Test Chemical:	Test Chemical:			Nominal conc:	Nominal conc:
Day	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Medium renewal (tick)
pH [^E0155]																							new
																							old
O2 (mg/l) [^E0155]																							new
																							old
Temp (°C) [^E0155]																							new
																							old
Food provided (tick)
No. live offspring [^E0156]																								Total
Vessel 1
2
3
4
5
6
7
8
9
10
																							Total
Cumulative parent mortality [^E0157]	Cumulative parent mortality [^E0157]

Indicate which vessel was used for the experiment

Record aborted broods as ‘AB’ in relevant box

Record mortality of any parentalanimals as ‘M’ in relevant box

Appendix 5

EXAMPLE DATA SHEET FOR RECORDING RESULTS OF CHEMICAL ANALYSIS

(a) Measured concentrations

Nominal conc.	Week 1 sample	Week 1 sample	Week 2 sample	Week 2 sample	Week 3 sample	Week 3 sample
	Fresh	Old	Fresh	Old	Fresh	Old

(b) Measured concentrations as a percentage of nominal

Nominal conc.	Week 1 sample	Week 1 sample	Week 2 sample	Week 2 sample	Week 3 sample	Week 3 sample
	Fresh	Old	Fresh	Old	Fresh	Old

Appendix 6

CALCULATION OF A TIME-WEIGHTED MEAN

Time-weighted mean

Given that the concentration of the test chemical can decline over the period between medium renewals, it is necessary to consider what concentration should be chosen as representative of the range of concentrations experienced by the parent Daphnia. The selection should be based on biological considerations as well as statistical ones. For example, if reproduction is thought to be affected mostly by the peak concentration experienced, then the maximum concentration should be used. However, if the accumulated or longer term effect of the toxic chemical is considered to be more important, then an average concentration is more relevant. In this case, an appropriate average to use is the time-weighted mean concentration, since this takes account of the variation in instantaneous concentration over time.

Figure 1

Example of time-weighted mean

Image omitted; see official source

Text of image

Days

Figure 1 shows an example of a (simplified) test lasting seven days with medium renewal at Days 0, 2 and 4.

• — The thin zig-zag line represents the concentration at any point in time. The fall in concentration is assumed to follow an exponential decay process.

• — The 6 plotted points represent the observed concentrations measured at the start and end of each renewal period.

• — The thick solid line indicates the position of the time-weighted mean.

The time-weighted mean is calculated so that the area under the time-weighted mean is equal to the area under the concentration curve. The calculation for the above example is illustrated in Table 1.

Table 1

Calculation of Time-weighted mean

Renewal No.	Days	Conc 0	Conc 1	Ln(Conc 0)	Ln(Conc 1)	Area
1	2	10,000	4,493	2,303	1,503	13,767
2	2	11,000	6,037	2,398	1,798	16,544
3	3	10,000	4,066	2,303	1,403	19,781
Total Days:	7				Total Area:	50,092
					TW Mean:	7,156

Days is the number of days in the renewal period

Conc 0 is the measured concentration at the start of each renewal period

Conc 1 is the measured concentration at the end of each renewal period

Ln(Conc 0) is the natural logarithm of Conc 0

Ln(Conc 1) is the natural logarithm of Conc 1

Area is the area under the exponential curve for each renewal period. It is calculated by:

Image omitted; see official source

The time-weighted mean (TW Mean) is the Total Area divided by the Total Days.