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Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 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)

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1.3.2016   

EN

Official Journal of the European Union

L 54/1


COMMISSION REGULATION (EU) 2016/266

of 7 December 2015

amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 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)

THE EUROPEAN COMMISSION,

Having regard to the Treaty on the Functioning of the European Union,

Having regard to Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC (1), and in particular Article 13(2) thereof,

Whereas:

(1)

Commission Regulation (EC) No 440/2008 (2) contains the test methods for the purposes of the determination of the physicochemical properties, toxicity and ecotoxicity of chemicals to be applied for the purposes of Regulation (EC) No 1907/2006.

(2)

It is necessary to update Regulation (EC) No 440/2008 to include new and updated test methods recently adopted by the OECD in order to take into account technical progress, and to ensure the reduction in the number of animals to be used for experimental purposes, in accordance with Directive 2010/63/EU of the European Parliament and of the Council (3). Stakeholders have been consulted on this draft.

(3)

The adaptation contains twenty test methods: one new method for the determination of a physicochemical property, eleven new test methods and three updated test methods for the assessment of ecotoxicity, and five new test methods to assess the environmental fate and behaviour.

(4)

Regulation (EC) No 440/2008 should therefore be amended accordingly.

(5)

The measures provided for in this Regulation are in accordance with the opinion of the Committee established under Article 133 of Regulation (EC) No 1907/2006,

HAS ADOPTED THIS REGULATION:

Article 1

The Annex to Regulation (EC) No 440/2008 is amended in accordance with the Annex to this Regulation.

Article 2

This Regulation shall enter into force on the third day following that of its publication in the Official Journal of the European Union.

This Regulation shall be binding in its entirety and directly applicable in all Member States.

Done at Brussels, 7 December 2015.

For the Commission

The President

Jean-Claude JUNCKER


(1)  OJ L 396, 30.12.2006, p. 1.

(2)  Commission 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) (OJ L 142, 31.5.2008, p. 1).

(3)  Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (OJ L 276, 20.10.2010, p. 33).


ANNEX

The Annex to Regulation (EC) No 440/2008 is amended as follows:

(1)

A note is inserted at the beginning of the Annex, before Part A:

‘Note:

Before using any of the following test methods to test a multi-constituent substance (MCS), a substance of unknown or variable composition, complex reaction product or biological material (UVCB), or a mixture and where its applicability for the testing of MCS, UVCB, or mixtures is not indicated in the respective test method, it should be considered whether the method is adequate for the intended regulatory purpose.

If the test method is used for the testing of a MCS, UVCB or mixture, sufficient information on its composition should be made available, as far as possible, e.g. by the chemical identity of its constituents, their quantitative occurrence, and relevant properties of the constituents.’

(2)

Chapter A.24 is added:

A.24.   PARTITION COEFFICIENT (N-OCTANOL/WATER), HIGH PERFORMANCELIQUID CHROMATOGRAPHY (HPLC) METHOD

INTRODUCTION

This test method is equivalent to OECD test guideline (TG) 117 (2004)

1.

The partition coefficient (P) is defined as the ratio of the equilibrium concentrations of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. In the case of n-octanol and water,

Formula

The partition coefficient being the quotient of two concentrations, is dimensionless and is usually given in the form of its logarithm to base ten.

2.

Pow is a key parameter in studies of the environmental fate of chemical substances. A highly-significant relationship between the Pow of non-ionised form of substances and their bioaccumulation in fish has been shown. It has also been shown that Pow is a useful parameter in the prediction of adsorption on soil and sediments and for establishing quantitative structure-activity relationships for a wide range of biological effects.

3.

The original proposal for this test method was based on an article by C.V. Eadsforth and P. Moser (1). The development of the test method and an OECD inter-laboratory comparison test were coordinated by the Umweltbundesamt of the Federal Republic of Germany during 1986 (2).

INITIAL CONSIDERATIONS

4.

log Pow values in the range – 2 to 4 (occasionally up to 5 and more) (1) can be experimentally determined by the Shake-Flask method (Chapter A.8 of this Annex, OECD Test Guideline 107). The HPLC method covers log Pow in the range of 0 to 6 (1)(2)(3)(4)(5). This method may require an estimation of Pow to assign suitable reference substances and support any conclusions drawn from the data generated by the test. Calculation methods are briefly discussed in the Appendix to this test method. The HPLC operation mode is isocratic.

5.

The Pow values depend on the environmental conditions such as temperature, pH, ionic strength etc, and these should be defined in the experiment for the correct interpretation of Pow data. For ionisable substances, another method (e.g. draft OECD guideline on pH metric method for ionised substances (6)) may become available and could be used as an alternative method. Although this draft OECD guideline may appropriate be suitable to determine Pow for those ionisable substances, in some cases it is more appropriate to use the HPLC method at an environmentally relevant pH (see paragraph 9).

PRINCIPLE OF THE METHOD

6.

Reverse phase HPLC is performed on analytical columns packed with a commercially available solid phase containing long hydrocarbon chains (e.g. C8, C18) chemically bound onto silica.

7.

A chemical injected on such a column partitions between the mobile solvent phase and the hydrocarbon stationary phase as it is transported along the column by the mobile phase. The substances are retained in proportion to their hydrocarbon-water partition coefficient, with hydrophilic substances eluted first and lipophilic substances last. The retention time is described by the capacity factor k given by the expression:

Formula

where tR is the retention time of the test substance, and t0 is the dead-time, i.e. the average time a solvent molecule needs to pass the column. Quantitative analytical methods are not required and only the determination of retention times is necessary.

8.

The octanol/water partition coefficient of a test substance can be computed by experimentally determining its capacity factor k and then inputting k into the following equation:

Formula

where

a, b

=

linear regression coefficients.

The equation above can be obtained by linearly regressing the log of octanol/water partition coefficients of reference substances against the log of capacity factors of the reference substances.

9.

Reverse phase HPLC method enables partition coefficients to be estimated in the log Pow range between 0 and 6, but can be expanded to cover the log Pow range between 6 and 10 in exceptional cases. This may require that the mobile phase is modified (3). The method is not applicable to strong acids and bases, metal complexes, substances which react with the eluent, or surface-active agents. Measurements can be performed on ionisable substances in their non-ionised form (free acid or free base) only by using an appropriate buffer with a pH below the pKa for a free acid or above the pKa for a free base. Alternatively, the pH-metric method for the testing of ionisable substances (6) may become available and could be used as an alternative method (6). If the log Pow value is determined for the use in environmental hazard classification or in environmental risk assessment, the test should be performed in the pH range relevant for the natural environment, i.e. in the pH range of 5,0 - 9.

10.

In some cases impurities can make the interpretation of the results difficult due to uncertainty in peak assignments. For mixtures which result in an unresolved band, upper and lower limits of log Pow, and the area % of each log Pow peak should be reported. For mixtures which are a group of homologues, the weighted average log Pow should also be stated (7), calculated based on the single Pow values and the corresponding area % values (8). All peaks that contribute an area of 5 % or more to the total peak area should be taken into consideration in the calculation (9):

Formula

The weighed average log Pow is valid only for substances or mixtures (e.g. tall oils) consisting of homologues (e.g. series of alkanes). Mixtures can be measured with meaningful results, provided that the analytical detector used has the same sensitivity towards all the substances in the mixture and that they can be adequately resolved.

INFORMATION ON THE TEST SUBSTANCE

11.

The dissociation constant, structural formula, and solubility in the mobile phase should be known before the method is used. In addition, information on hydrolysis would be helpful.

QUALITY CRITERIA

12.

In order to increase the confidence in the measurement, duplicate determinations must be made.

Repeatability: The value of log Pow derived from repeated measurements made under identical conditions and using the same set of reference substances should fall within a range of ± 0,1 log units.

Reproducibility: If the measurements are repeated with a different set of reference substances, results may differ. Typically, the correlation coefficient R for the relationship between log k and log Pow for a set of test substances is around 0,9, corresponding to an octanol/water partition coefficient of log Pow ± 0,5 log units.

13.

The inter-laboratory comparison test has shown that with the HPLC method log Pow values can be obtained to within ± 0,5 units of the Shake-Flask values (2). Other comparisons can be found in the literature (4)(5)(10)(11)(12). Correlation graphs based on structurally related reference substances give the most accurate results (13).

REFERENCE SUBSTANCES

14.

In order to correlate the measured capacity factor k of a substance with its Pow, a calibration graph using at least 6 points has to be established (see paragraph 24). It is up to the user to select the appropriate reference substances. The reference substances should normally have log Pow values which encompass the log Pow of the test substance, i.e. at least one reference substance should have a Pow above that of the test substance, and another a Pow below that of the test substance. Extrapolation should only be used in exceptional cases. It is preferable that these reference substances should be structurally related to the test substance. log Pow values of the reference substances used for the calibration should be based on reliable experimental data. However, for substances with high log Pow (normally more than 4), calculated values may be used unless reliable experimental data are available. If extrapolated values are used a limit value should be quoted.

15.

Extensive lists of log Pow values for many groups of chemicals are available (14)(15). If data on the partition coefficients of structurally related substances are not available, a more general calibration, established with other reference substances, may be used. Recommended reference substances and their Pow values are listed in Table 1. For ionisable substances the values given apply to the non-ionised form. The values were checked for plausibility and quality during the inter-laboratory comparison test.

Table 1

Recommended reference substances

 

CAS Number

Reference substance

log Pow

pKa

1

78-93-3

2-Butanone

(Methylethylketone)

0,3

 

2

1122-54-9

4-Acetylpyridine

0,5

 

3

62-53-3

Aniline

0,9

 

4

103-84-4

Acetanilide

1,0

 

5

100-51-6

Benzyl alcohol

1,1

 

6

150-76-5

4-Methoxyphenol

1,3

pKa = 10,26

7

122-59-8

Phenoxyacetic acid

1,4

pKa = 3,12

8

108-95-2

Phenol

1,5

pKa = 9,92

9

51-28-5

2,4-Dinitrophenol

1,5

pKa = 3,96

10

100-47-0

Benzonitrile

1,6

 

11

140-29-4

Phenylacetonitrile

1,6

 

12

589-18-4

4-Methylbenzyl alcohol

1,6

 

13

98-86-2

Acetophenone

1,7

 

14

88-75-5

2-Nitrophenol

1,8

pKa = 7,17

15

121-92-6

3-Nitrobenzoic acid

1,8

pKa = 3,47

16

106-47-8

4-Chloroaniline

1,8

pKa = 4,15

17

98-95-3

Nitrobenzene

1,9

 

18

104-54-1

Cinnamyl alcohol

(Cinnamic alcohol)

1,9

 

19

65-85-0

Benzoic acid

1,9

pKa = 4,19

20

106-44-5

p-Cresol

1,9

pKa = 10,17

21

140-10-3

(trans)

Cinnamic acid

2,1

pKa = 3,89 (cis)

4,44 (trans)

22

100-66-3

Anisole

2,1

 

23

93-58-3

Methyl benzoate

2,1

 

24

71-43-2

Benzene

2,1

 

25

99-04-7

3-Methylbenzoic acid

2,4

pKa = 4,27

26

106-48-9

4-Chlorophenol

2,4

pKa = 9,1

27

79-01-6

Trichloroethylene

2,4

 

28

1912-24-9

Atrazine

2,6

 

29

93-89-0

Ethyl benzoate

2,6

 

30

1194-65-6

2,6-Dichlorobenzonitrile

2,6

 

31

535-80-8

3-Chlorobenzoic acid

2,7

pKa = 3,82

32

108-88-3

Toluene

2,7

 

33

90-15-3

1-Naphthol

2,7

pKa = 9,34

34

608-27-5

2,3-Dichloroaniline

2,8

 

35

108-90-7

Chlorobenzene

2,8

 

36

1746-13-0

Allyl phenyl ether

2,9

 

37

108-86-1

Bromobenzene

3,0

 

38

100-41-4

Ethylbenzene

3,2

 

39

119-61-9

Benzophenone

3,2

 

40

92-69-3

4-Phenylphenol

3,2

pKa = 9,54

41

89-83-8

Thymol

3,3

 

42

106-46-7

1,4-Dichlorobenzene

3,4

 

43

122-39-4

Diphenylamine

3,4

pKa = 0,79

44

91-20-3

Naphthalene

3,6

 

45

93-99-2

Phenyl benzoate

3,6

 

46

98-82-8

Isopropylbenzene

3,7

 

47

88-06-2

2,4,6-Trichlorophenol

3,7

pKa = 6

48

92-52-4

Biphenyl

4,0

 

49

120-51-4

Benzyl benzoate

4,0

 

50

88-85-7

2,4-Dinitro-6-sec-butylphenol

4,1

 

51

120-82-1

1,2,4-Trichlorobenzene

4,2

 

52

143-07-7

Dodecanoic acid

4,2

pKa = 5,3

53

101-84-8

Diphenyl ether

4,2

 

54

85-01-8

Phenanthrene

4,5

 

55

104-51-8

n-Butylbenzene

4,6

 

56

103-29-7

Dibenzyl

4,8

 

57

3558-69-8

2,6-Diphenylpyridine

4,9

 

58

206-44-0

Fluoranthene

5,1

 

59

603-34-9

Triphenylamine

5,7

 

60

50-29-3

DDT

6,5

 

DESCRIPTION OF THE METHOD

Preliminary estimate of the partition coefficient

16.

If it is necessary, the partition coefficient of the test substance may be estimated preferably by using a calculation method (see Appendix, or where appropriate, by using the ratio of the solubility of the test substance in the pure solvents.

Apparatus

17.

A liquid-phase chromatograph fitted with a low-pulse pump and a suitable detection system is required. A UV detector, using a wavelength of 210 nm, or an RI detector is applicable to the wide variety of chemical groups. The presence of polar groups in the stationary phase may seriously impair the performance of the HPLC column. Therefore, stationary phases should have a minimal percentage of polar groups (16). Commercial microparticulate reverse-phase packing or ready-packed columns can be used. A guard column may be positioned between the injection system and the analytical column.

Mobile phase

18.

HPLC-grade methanol and distilled or de-ionised water are used to prepare the eluting solvent, which is degassed before use. Isocratic elution should be employed. Methanol/water ratios with minimum water content of 25 % should be used. Typically a 3:1 (v/v) methanol-water mixture is satisfactory for eluting substances with a log P of 6 within an hour, at a flow rate of 1 ml/min. For substances with a log P above 6 it may be necessary to shorten the elution time (and those of the reference substances) by decreasing the polarity of the mobile phase or the column length.

19.

The test substance and the reference substances must be soluble in the mobile phase in sufficient concentration to allow their detection. Additives may be used with the methanol-water mixture in exceptional cases only, since they will change the properties of the column. In these cases it must be confirmed that the retention time of the test and reference substances are not influenced. If methanol-water is not appropriate, other organic solvent-water mixtures can be used, e.g. ethanol-water, acetonitrile-water or isopropyl alcohol (2-propanol)-water.

20.

The pH of the eluent is critical for ionisable substances. It should be within the operating pH range of the column, usually between 2 and 8. Buffering is recommended. Care must be taken to avoid salt precipitation and column deterioration which occur with some organic phase/buffer mixtures. HPLC measurements with silica-based stationary phases above pH 8 are not normally advisable since the use of an alkaline mobile phase may cause rapid deterioration in the performance of the column.

Solutes

21.

The test and reference substances must be sufficiently pure in order to assign the peaks in the chromatograms to the respective substances. Substances to be used for test or calibration purposes are dissolved in the mobile phase if possible. If a solvent other than the mobile phase is used to dissolve the test and reference substances, the mobile phase should be used for the final dilution prior to injection.

Test conditions

22.

The temperature during the measurement should not vary by more than ± 1 °C.

Determination of dead time to

23.

The dead time t0 can be measured by using unretained organic substances (e.g. thiourea or formamide). A more precise dead time can be derived from the retention times measured or a set of approximately seven members of a homologous series (e.g. n-alkyl methyl ketones) (17). The retention times tR (nC + 1) are plotted against tR (nC), where nC is the number of carbon atoms. A straight line, tR (nC + 1) = A tR (nC) + (1 – A)t0, is obtained, where A, representing k(nC + 1)/k(nC), is constant. The dead time t0 is obtained from the intercept (1 – A)t0 and the slope A.

Regression Equation

24.

The next step is to plot a correlation log k versus log P for appropriate reference substances with log P values near the value expected for the test substance. In practice, from 6 to 10 reference substances are injected simultaneously. The retention times are determined, preferably on a recording integrator linked to the detection system. The corresponding logarithms of the capacity factors, log k, are plotted as a function of log P. The regression equation is performed at regular intervals, at least once daily, so that account can be taken of possible changes in column performance.

DETERMINATION OF THE POW OF THE TEST SUBSTANCE

25.

The test substance is injected in the smallest detectable quantities. The retention time is determined in duplicate. The partition coefficient of the test substance is obtained by interpolation of the calculated capacity factor on the calibration graph. For very low and very high partition coefficients extrapolation is necessary. Especially in these cases attention must be given to the confidence limits of the regression line. If the retention time of sample is outside the range of retention times obtained for the standards, a limit value should be quoted.

DATA AND REPORTING

Test report

26.

The following must be included in the report:

if determined the preliminary estimate of the partition coefficient, the estimated values and the method used; and if a calculation method was used, its full description including identification of the data base and detailed information on the choice of fragments;

test and reference substances: purity, structural formula and CAS number,

description of equipment and operating conditions: analytical column, guard column,

mobile phase, means of detection, temperature range, pH;

elution profiles (chromatograms);

deadtime and how it was measured;

retention data and literature log Pow values for reference substances used in calibration;

details on fitted regression line (log k versus log Pow) and the correlation coefficient of the line including confidence intervals;

average retention data and interpolated log Pow value for the test substance;

in case of a mixture: elution profile chromatogram with indicated cut-offs;

log Pow values relative to area % of the log Pow peak;

calculation using a regression line;

calculated weighted average log Pow values, when appropriate.

LITERATURE

(1)

C.V. Eadsforth and P. Moser. (1983). Assessment of Reverse Phase Chromatographic Methods for Determining Partition Coefficients. Chemosphere. 12, 1459.

(2)

W. Klein, W. Kördel, M. Weiss and H.J. Poremski. (1988). Updating of the OECD Test Guideline 107 Partition Coefficient n-Octanol-Water, OECD Laboratory Intercomparison Test on the HPLC Method. Chemosphere. 17, 361.

(3)

C.V. Eadsforth. (1986). Application of Reverse H.P.L.C. for the Determination of Partition Coefficient. Pesticide Science. 17, 311.

(4)

H. Ellgehausen, C. D'Hondt and R. Fuerer (1981). Reversed-phase chromatography as a general method for determining octan-1-ol/water partition coefficients. Pesticide. Science. 12, 219.

(5)

B. McDuffie (1981). Estimation of Octanol Water Partition Coefficients for Organic Pollutants Using Reverse Phase High Pressure Liquid Chromatography. Chemosphere. 10, 73.

(6)

OECD (2000). Guideline for Testing of Chemicals — Partition Coefficient (n-octanol/water): pH-metric Method for Ionisable Substances. Draft Guideline, November 2000.

(7)

OSPAR (1995). “Harmonised Offshore Chemicals Notification Format (HOCFN) 1995”, Oslo and Paris Conventions for the Prevention of Marine Pollution Programmes and Measures Committee (PRAM), Annex 10, Oviedo, 20–24 February 1995.

(8)

M. Thatcher, M. Robinson, L. R. Henriquez and C. C. Karman. (1999). An User Guide for the Evaluation of Chemicals Used and Discharged Offshore, A CIN Revised CHARM III Report 1999. Version 1.0, 3. August.

(9)

E. A. Vik, S. Bakke and K. Bansal. (1998). Partitioning of Chemicals. Important Factors in Exposure Assessment of Offshore Discharges. Environmental Modelling & Software Vol. 13, pp. 529-537.

(10)

L.O. Renberg, S.G. Sundstroem and K. Sundh-Nygård. (1980). Partition coefficients of organic chemicals derived from reversed-phase thin-layer chromatography. Evaluation of methods and application on phosphate esters, polychlorinated paraffins and some PCB-substitutes. Chemosphere. 9, 683.

(11)

W.E. Hammers, G.J.Meurs and C.L. De-Ligny. (1982). Correlations between liquid chromatographic capacity ratio data on Lichrosorb RP-18 and partition coefficients in the octanol-water system. J. Chromatography 247, 1.

(12)

J.E. Haky and A.M. Young. (1984). Evaluation of a simple HPLC correlation method for the estimation of the octanol-water partition coefficients of organic compounds. J. Liq. Chromatography. 7, 675.

(13)

S. Fujisawa and E. Masuhara. (1981). Determination of Partition Coefficients of Acrylates Methacrylates and Vinyl Monomers Using High Performance Liquid Chromatography. Journal of Biomedical Materials Research. 15, 787.

(14)

C. Hansch and A. J. Leo. (1979). Substituent Constants for Correlation Analysis in Chemistry and Biology. John Willey, New York.

(15)

C. Hansch, chairman; A.J. Leo, dir. (1982). Log P and Parameter Database: A tool for the quantitative prediction of bioactivity — Available from Pomona College Medical Chemistry Project, Pomona College, Claremont, California 91711.

(16)

R. F. Rekker, H. M. de Kort. (1979). The hydrophobic fragmental constant: An extension to a 1 000 data point set. Eur. J. Med. Chem. — Chim. Ther. 14, 479.

(17)

G.E. Berendsen, P.J. Schoenmakers, L. de Galan, G. Vigh, Z. Varga-Puchony, and J. Inczédy. (1980). On determination of hold-up time in reversed-phase liquid chromatography. J. Liq. Chromato. 3, 1669.

Appendix

POW calculation methods

INTRODUCTION

1.

This appendix provides a short introduction to the calculation of Pow. For further information the reader is referred to textbooks (1)(2).

2.

Calculated values of Pow are used for:

deciding which experimental method to use: Shake Flask method for log Pow between – 2 and 4 and HPLC method for log Pow between 0 and 6;

selecting conditions to be used in HPLC (reference substances, methanol/water ratio);

checking the plausibility of values obtained through experimental methods;

providing an estimate when experimental methods cannot be applied.

Principle of calculation methods

3.

The calculation methods suggested here are based on the theoretical fragmentation of the molecule into suitable substructures for which reliable log Pow increments are known. The log Pow is obtained by summing the fragment values and the correction terms for intramolecular interactions. Lists of fragment constants and correction terms are available (1)(2)(3)(4)(5)(6). Some are regularly updated (3).

Reliability of calculated values

4.

In general, the reliability of calculation methods decreases as the complexity of the substance under study increases. In the case of simple molecules of low molecular weight and with one or two functional groups, a deviation of 0,1 to 0,3 log Pow units between the results of the different fragmentation methods and the measured values can be expected. The margin of error will depend on the reliability of the fragment constants used, the ability to recognise intramolecular interactions (e.g. hydrogen bonds) and the correct use of correction terms. In the case of ionising substances the charge and degree of ionisation must be taken into consideration (10).

Fujita-Hansch π-method

5.

The hydrophobic substituent constant, π, originally introduced by Fujita et al. (7) is defined as:

πX = log Pow (PhX) – log Pow (PhH)

where PhX is an aromatic derivative and PhH the parent substance.

e.g.

πCl

= log Pow (C6H5Cl) – log Pow (C6H6)

= 2,84 – 2,13

= 0,71

The π-method is primarily of interest for aromatic substances. π-values for a large number of substituents are available (4)(5).

Rekker method

6.

Using the Rekker method (8) the log Pow value is calculated as:

Formula

where ai is the number of times a given fragment occurs in the molecule and fi is the log Pow increment of the fragment. The interaction terms can be expressed as an integral multiple of one single constant Cm (so-called “magic constant”). The fragment constants fi and Cm have been determined from a list of 1 054 experimental Pow values of 825 substances using multiple regression analysis (6)(8). The determination of the interaction terms is carried out according to set rules (6)(8)(9).

Hansch-Leo method

7.

Using the Hansch and Leo method (4), the log Pow value is calculated as:

Formula

where fi is a fragment constant, Fj a correction term (factor), ai and bj the corresponding frequency of occurence. Lists of atomic and group fragmental values and of correction terms Fj were derived by trial and error from experimental Pow values. The correction terms have been divided into several different classes (1)(4). Sofware packages have been developed to take into account all the rules and correction terms (3).

COMBINED METHOD

8.

The calculation of log Pow of complex molecules can be considerably improved, if the molecule is dissected into larger substructures for which reliable log Pow values are available, either from tables (3)(4) or by existing measurements. Such fragments (e.g. heterocycles, anthraquinone, azobenzene) can then be combined with the Hansch- π values or with Rekker or Leo fragment constants.

Remarks:

(i)

The calculation methods are only applicable to partly or fully ionised substances when the necessary correction factors are taken into account.

(ii)

If the existence of intramolecular hydrogen bonds can be assumed, the corresponding correction terms (approx. + 0,6 to + 1,0 log Pow units) must be added (1). Indications on the presence of such bonds can be obtained from stereo models or spectroscopic data.

(iii)

If several tautomeric forms are possible, the most likely form should be used as the basis of the calculation.

(iv)

The revisions of lists of fragment constants should be followed carefully.

LITERATURE ON CALCULATION METHODS

(1)

W.J. Lyman, W.F. Reehl and D.H. Rosenblatt (ed.). Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York (1982).

(2)

W.J. Dunn, J.H. Block and R.S. Pearlman (ed.). Partition Coefficient, Determination and Estimation, Pergamon Press, Elmsford (New York) and Oxford (1986).

(3)

Pomona College, Medicinal Chemistry Project, Claremont, California 91711, USA, Log P Database and Med. Chem. Software (Program CLOGP-3).

(4)

C. Hansch and A.J. Leo. Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York (1979).

(5)

Leo, C. Hansch and D. Elkins. (1971) Partition coefficients and their uses. Chemical. Reviews. 71, 525.

(6)

R. F. Rekker, H. M. de Kort. (1979). The hydrophobic fragmental constant: An extension to a 1 000 data point set. Eur. J. Med. Chem. — Chim. Ther. 14, 479.

(7)

Toshio Fujita, Junkichi Iwasa & Corwin Hansch (1964). A New Substituent Constant, π, Derived from Partition Coefficients. J. Amer. Chem. Soc. 86, 5175.

(8)

R.F. Rekker. The Hydrophobic Fragmental Constant, Pharmacochemistry Library, Vol. 1, Elsevier, New York (1977).

(9)

C.V. Eadsforth and P. Moser. (1983). Assessment of Reverse Phase Chromatographic Methods for Determining Partition Coefficients. Chemosphere. 12, 1459.

(10)

R.A. Scherrer. ACS — Symposium Series 255, p. 225, American Chemical Society, Washington, D.C. (1984).

(3)

Chapter C.3 is replaced by the following:

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]:

Formula

[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]:

Formula

[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:

Formula

[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;

2.

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 (2)

60-80 (2)

40-50 (2)

30-40 (2)

2,5 (3)

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 (4) (day- 1)

1,5 -1,7

1,2-1,5

1,4

1,1-1,4

2,0-2,4

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.

Composition of the AAP-medium (US. EPA) and the OECD TG 201 medium.

Component

AAP

OECD

 

mg/l

mM

mg/l

mM

NaHCO3

15,0

0,179

50,0

0,595

NaNO3

25,5

0,300

 

 

NH4Cl

 

 

15,0

0,280

MgCl2·6(H2O)

12,16

0,0598

12,0

0,0590

CaCl2·2(H2O)

4,41

0,0300

18,0

0,122

MgSO4·7(H2O)

14,6

0,0592

15,0

0,0609

K2HPO4

1,044

0,00599

 

 

KH2PO4

 

 

1,60

0,00919

FeCl3·6(H2O)

0,160

0,000591

0,0640

0,000237

Na2EDTA·2(H2O)

0,300

0,000806

0,100

0,000269*

H3BO3

0,186

0,00300

0,185

0,00299

MnCl2·4(H2O)

0,415

0,00201

0,415

0,00210

ZnCl2

0,00327

0,000024

0,00300

0,0000220

CoCl2·6(H2O)

0,00143

0,000006

0,00150

0,00000630

Na2MoO4·2(H2O)

0,00726

0,000030

0,00700

0,0000289

CuCl2·2(H2O)

0,000012

0,00000007

0,00001

0,00000006

pH

7,5

8,1

The molar ratio of EDTA to iron slightly exceeds unity. This prevents iron precipitation and at the same time, chelation of heavy metal ions is minimised.

In test with the diatom Navicula pelliculosa both media must be supplemented with Na2SiO3 ·9H20 to obtain a concentration of 1,4 mg Si/l.

The pH of the medium is obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air. An approximate relationship between pH at 25 oC and the molar bicarbonate concentration is:

pHeq = 11,30 + log[HCO3]

With 15 mg NaHCO3/l, pHeq = 7,5 (U.S. EPA medium) and with 50 mg NaHCO3/l, pHeq = 8,1 (OECD medium).

Element composition of test media

Element

AAP

OECD

 

mg/l

mg/l

C

2,144

7,148

N

4,202

3,927

P

0,186

0,285

K

0,469

0,459

Na

11,044

13,704

Ca

1,202

4,905

Mg

2,909

2,913

Fe

0,033

0,017

Mn

0,115

0,115

Preparation of OECD medium

Nutrient

Concentration in stock solution

Stock solution 1:

macro nutrients

NH4Cl

1,5 g/l

MgCl2·6H2O

1,2 g/l

CaCl2·2H2O

1,8 g/l

MgSO4·7H2O

1,5 g/l

KH2PO4

0,16 g/l

Stock solution 2:

iron

FeCl3·6H2O

64 mg/l

Na2EDTA·2H2O

100 mg/l

Stock solution 3:

trace elements

H3BO3

185 mg/l

MnCl2·4H2O

415 mg/l

ZnCl2

3 mg/l

CoCl2·6H2O

1,5 mg/l

CuCl2·2H2O

0,01 mg/l

Na2MoO4·2H2O

7 mg/l

Stock solution 4:

bicarbonate

NaHCO3

50 g/l

Na2SiO3·9H20

 

Sterilise the stock solutions by membrane filtration (mean pore diameter 0,2 μm) or by autoclaving (120 °C, 15 min). Store the solutions in the dark at 4 °C.

Do not autoclave stock solutions 2 and 4, but sterilise them by membrane filtration.

Prepare a growth medium by adding an appropriate volume of the stock solutions 1-4 to water:

 

Add to 500 ml of sterilised water:

 

10 ml of stock solution 1

 

1 ml of stock solution 2

 

1 ml of stock solution 3

 

1 ml of stock solution 4

 

Make up to 1 000 ml with sterilised water.

Allow sufficient time for equilibrating the medium with the atmospheric CO2, if necessary by bubbling with sterile, filtered air for some hours.

Preparation of U.S. EPA medium

1.

Add 1 ml of each stock solution in 2.1–2.7 to approximately 900 ml of deionised or distilled water and then dilute to 1 litre.

2.

Macronutrient stock solutions are made by dissolving the following into 500 ml of deionised or distilled water. Reagents 2.1, 2.2, 2.3, and 2.4 can be combined into one stock solution.

2.1

NaNO3

12,750 g.

2.2

MgCl2·6H2O

6,082 g.

2.3

CaCl2·2H2O

2,205 g.

2.4

Micronutrient Stock Solution(see 3).

2.5

MgSO4·7H2O

7,350 g.

2.6

K2HPO4

0,522 g.

2.7

NaHCO3

7,500 g.

2.8

Na2SiO3·9H2O

See Note 1.

Note 1: Use for diatom test species only. May be added directly (202,4 mg) or by way of stock solution to give 20 mg/l Si final concentration in medium.

3.

The micronutrient stock solution is made by dissolving the following into 500 ml of deionised or distilled water:

3.1

H3BO3

92,760 mg.

3.2

MnCl2·4H2O

207,690 mg.

3.3

ZnCl2

1,635 mg.

3.4

FeCl3·6H2O

79,880 mg.

3.5

CoCl2·6H2O

0,714 mg.

3.6

Na2MoO4·2H2O

3,630 mg.

3.7

CuCl2·2H2O

0,006 mg.

3.8

Na2EDTA·2H2O

150,000 mg. [Disodium (Ethylenedinitrilo) tetraacetate].

3.9

Na2SeO4·5H2O

0,005 mg See Note 2.

Note 2: Use only in medium for stock cultures of diatom species.

4.

Adjust pH to 7,5 ± 0,1 with 0,1 N or 1,0 N NaOH or HCl.

5.

Filter the media into a sterile container through either a 0,22 μm membrane filter if a particle counter is to be used or a 0,45 μm filter if a particle counter is not to be used.

6.

Store medium in the dark at approximately 4 °C until use.

Appendix 4

Example of a procedure for the culturing of algae

General observations

The purpose of culturing on the basis of the following procedure is to obtain algal cultures for toxicity tests.

Use suitable methods to ensure that the algal cultures are not infected with bacteria. Axenic cultures may be desirable but unialgal cultures must be established and used.

All operations must be carried out under sterile conditions in order to avoid contamination with bacteria and other algae.

Equipment and materials

See under test method: Apparatus.

Procedures for obtaining algal cultures

Preparation of nutrient solutions (media):

All nutrient salts of the medium are prepared as concentrated stock solutions and stored dark and cold. These solutions are sterilised by filtration or by autoclaving.

The medium is prepared by adding the correct amount of stock solution to sterile distilled water, taking care that no infection occurs. For solid medium 0,8 per cent of agar is added.

Stock culture:

The stock cultures are small algal cultures that are regularly transferred to fresh medium to act as initial test material. If the cultures are not used regularly they are streaked out on sloped agar tubes. These are transferred to fresh medium at least once every two months.

The stock cultures are grown in conical flasks containing the appropriate medium (volume about 100 ml). When the algae are incubated at 20 °C with continuous illumination, a weekly transfer is required.

During transfer an amount of “old” culture is transferred with sterile pipettes into a flask of fresh medium, so that with the fast-growing species the initial concentration is about 100 times smaller than in the old culture.

The growth rate of a species can be determined from the growth curve. If this is known, it is possible to estimate the density at which the culture should be transferred to new medium. This must be done before the culture reaches the death phase.

Pre-culture:

The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture is incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2 to 4 days. When the algal cultures contain deformed or abnormal cells, they must be discarded.

Appendix 5

Data analysis by nonlinear regression

General considerations

The response in algal tests and other microbial growth tests — growth of biomass — is by nature a continuous or metric variable — a process rate if growth rate is used and its integral over time if biomass is selected. Both are referenced to the corresponding mean response of replicate non-exposed controls showing maximum response for the conditions imposed — with light and temperature as primary determining factors in the algal test. The system is distributed or homogenous and the biomass can be viewed as a continuum without consideration of individual cells. The variance distribution of the type of response for a such system relate solely to experimental factors (described typically by the log-normal or normal distributions of error). This is by contrast to typical bioassay responses with quantal data for which the tolerance (typically binomially distributed) of individual organisms are often assumed to be the dominant variance component. Control responses are here zero or background level.

In the uncomplicated situation, the normalised or relative response, r, decreases monotonically from 1 (zero inhibition) to 0 (100 per cent inhibition). Note, that all responses have an error associated and that apparent negative inhibitions can be calculated as a result of random error only.

Regression analysis

Models

A regression analysis aims at quantitatively describing the concentration response curve in the form of a mathematical regression function Y = f (C) or more frequently F(Z) where Z = log C. Used inversely C = f– 1 (Y) allows the calculation of, ECx figures, including the EC50, EC10 and EC20, and their 95 % confidence limits. Several simple mathematical functional forms have proved to successfully describe concentration — response relationships obtained in algal growth inhibition tests. Functions include for instance the logistic equation, the nonsymmetrical Weibul equation and the log normal distribution function, which are all sigmoid curves asymptotically approaching zero for C → 0 and one for C → infinity.

The use of continuous threshold function models (e.g. the Kooijman model “for inhibition of population growth” Kooijman et al. 1996) is a recently proposed or alternative to asymptotic models. This model assumes no effects at concentrations below a certain threshold EC0+ that is estimated by extrapolation of the response concentration relationship to intercept the concentration axis using a simple continuous function that is not differentiable in the starting point.

Note that the analysis can be a simple minimisation of sums of residual squares (assuming constant variance) or weighted squares if variance heterogeneity is compensated.

Procedure

The procedure can be outlined as follows: Select an appropriate functional equation, Y = f(C), and fit it to the data by non-linear regression. Use preferably the measurements from each individual flask rather than means of replicates, in order to extract as much information from the data as possible. If the variance is high, on the other hand, practical experience suggests that means of replicates may provide a more robust mathematical estimation less influenced by systematic errors in the data, than with each individual data point retained.

Plot the fitted curve and the measured data and examine whether the curve fit is appropriate. Analysis of residuals may be a particular helpful tool for this purpose. If the chosen functional relationship to fit the concentration response does not describe well the whole curve or some essential part of it, such as the response at low concentrations, choose another curve fit option — e.g., a non-symmetrical curve like the Weibul function instead of a symmetrical one. Negative inhibitions may be a problem with for instance the log — normal distribution function likewise demanding an alternative regression function. It is not recommended to assign a zero or small positive value to such negative values because this distorts the error distribution. It may be appropriate to make separate curve fits on parts of the curve such as the low inhibition part to estimate EClowx figures. Calculate from the fitted equation (by “inverse estimation”, C = f– 1(Y)), characteristic point estimates ECx's, and report as a minimum the EC50 and one or two EClow x estimates. Experience from practical testing has shown that the precision of the algal test normally allows a reasonably accurate estimation at the 10 % inhibition level if data points are sufficient — unless stimulation occurs at low concentrations as a confounding factor. The precision of an EC20 estimate is often considerably better than that of an EC10, because the EC20 is usually positioned on the approximately linear part of the central concentration response curve. Sometimes EC10 can be difficult to interpret because of growth stimulation. So while the EC10 is normally obtainable with a sufficient accuracy it is recommended to report always also the EC20.

Weighting factors

The experimental variance generally is not constant and typically includes a proportional component, and a weighted regression is therefore advantageously carried out routinely. Weighting factors for a such analysis are normally assumed inversely proportional to the variance:

Wi = 1/Var(ri)

Many regression programs allow the option of weighted regression analysis with weighting factors listed in a table. Conveniently weighting factors should be normalised by multiplying them by n/Σ wi (n is the number of datapoints) so their sum be one.

Normalising responses

Normalising by the mean control response gives some principle problems and gives rise to a rather complicated variance structure. Dividing the responses by the mean control response for obtaining the percentage of inhibition, one introduces an additional error caused by the error on the control mean. Unless this error is negligibly small, weighting factors in the regression and confidence limits must be corrected for the covariance with the control (Draper and Smith, 1981). Note that high precision on the estimated mean control response is important in order to minimise the overall variance for the relative response. This variance is as follows:

(Subscript i refers to concentration level i and subscript 0 to the controls)

Yi = Relative response = ri/r0 = 1 – I = f(Ci)

with a variance Var(Yi) = Var (ri/r0) ≅ (∂ Yi/∂ ri) · Var(ri) + ((∂ Yi/∂ r0)2 · Var(r0)

and since (∂ Yi/∂ ri) = 1/r0 and (∂ Y I/∂ r0) = ri/r0 2

with normally distributed data and mi and m0 replicates: Var(ri) = σ2/mi

the total variance of the relative response Yi thus becomes

Var(Yi) = σ2/(r0 2 · mi) + ri 2 · σ2/r0 4 · m0

The error on the control mean is inversely proportional to the square root of the number of control replicates averaged, and sometimes it can be justified to include historic data and in this way greatly reduce the error. An alternative procedure is not to normalise the data and fit the absolute responses including the control response data but introducing the control response value as an additional parameter to be fitted by non linear regression. With a usual 2 parameter regression equation, this method necessitates the fitting of 3 parameters, and therefore demands more data points than non-linear regression on data that are normalised using a pre-set control response.

Inverse confidence intervals

The calculation of non-linear regression confidence intervals by inverse estimation is rather complex and not an available standard option in ordinary statistical computer program packages. Approximate confidence limits may be obtained with standard non-linear regression programs with re-parameterisation (Bruce and Versteeg, 1992), which involves rewriting the mathematical equation with the desired point estimates, e.g. the EC10 and the EC50 as the parameters to be estimated. (Let the function be I = f (α, β, Concentration) and utilise the definition relationships f (α, β, EC10) = 0,1 and f (α, β, EC50 ) = 0,5 to substitute f (α, β, concentration ) with an equivalent function g( EC10, EC50, concentration).

A more direct calculation (Andersen et al, 1998) is performed by retaining the original equation and using a Taylor expansion around the means of ri and r0.

Recently “boot strap methods” have become popular. Such methods use the measured data and a random number generator directed frequent re-sampling to estimate an empirical variance distribution.

REFERENCES

Kooijman, S.A.L.M.; Hanstveit, A.O.; Nyholm, N. (1996): No-effect concentrations in algal growth inhibition tests. Water Research, 30, 1625-1632.

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

Bruce, R..D. and Versteeg,, D.J. (1992). A Statistical Procedure for Modelling Continuous Ecotoxicity Data. Environ. Toxicol. Chem. 11, 1485-1494.

Andersen, J.S., Holst, H., Spliid, H., Andersen, H., Baun, A. & Nyholm, N. (1998). Continuous ecotoxicological data evaluated relative to a control response. Journal of Agricultural, Biological and Environmental Statistics, 3, 405-420.

(4)

Chapter C.11 is replaced by the following:

C.11.   ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST (CARBON AND AMMONIUM OXIDATION)

INTRODUCTION

1.

This test method is equivalent to OECD test guideline (TG) 209 (2010). This test method describes a method to determine the effects of a chemical on micro-organisms from activated sludge (largely bacteria) by measuring their respiration rate (carbon and/or ammonium oxidation) under defined conditions in the presence of different concentrations of the test chemical. The test method is based on the ETAD (Ecological and Toxicological Association of the Dyestuffs Manufacturing industry) test (1) ( 2), on the previous OECD TG 209 (3) and on the revised ISO Standard 8192 (4). The purpose of the test is to provide a rapid screening method to assess the effects of chemicals on the microorganisms of the activated sludge of the biological (aerobic) stage of waste-water treatment plants. The results of the test may also serve as an indicator of suitable non-inhibitory concentrations of test chemicals to be used in biodegradability tests (for example Chapters C.4 A-F, C.9, C.10, C12 and C.29 of this Annex, OECD TG302C). In this case, the test can be performed as a screening test, similar to a range-finding or limit test (see paragraph 39), considering the overall respiration only. However, this information should be taken with care for ready biodegradability tests (Chapter C.4 A-F and C.29 of this Annex) for which the inoculum concentration is significantly lower than the one used in this test method. Indeed, an absence of inhibition in this respiration test does not automatically result in non-inhibitory conditions in the ready biodegradability test of Chapters C.4 A-F or C.29 of this Annex.

2.

Overall, the respiration inhibition test seems to have been applied successfully since it was first published, but on some occasions spurious results were reported, e.g. (2) (4) (5). Concentration related respiration curves are sometimes bi-phasic, dose-response plots have been distorted and EC50 values have been unexpectedly low (5). Investigations showed that such results are obtained when the activated sludge used in the test nitrifies significantly and the test chemical has a greater effect on the oxidation of ammonium than on general heterotrophic oxidation. Therefore, these spurious results may be overcome by performing additional testing using a specific inhibitor of nitrification. By measuring the oxygen uptake rates in the presence and absence of such an inhibitor, e.g. N-allylthiourea (ATU), the separate total, heterotrophic and nitrification oxygen uptake rates can be calculated (4) (7) (8). Thus, the inhibitory effects of a test chemical on the two processes may be determined and the EC50 values for both organic carbon oxidation (heterotrophic) and ammonium oxidation (nitrification) may be calculated in the usual way. It should be noted that in some rare cases, the inhibitory effect of N-allylthiourea may be partially or completely nullified as a result of complexation with test chemicals or medium supplements, e.g. Cu++ ions (6). Cu++ ions are essential for Nitrosomonas, but are toxic in higher concentration.

3.

The need for nitrification in the aerobic treatment of wastewaters, as a necessary step in the process of removing nitrogen compounds from wastewaters by denitrification to gaseous products, has become urgent particularly in European countries; the EU has now set lower limits for the concentration of nitrogen in treated effluents discharged to receiving waters (5).

4.

For most purposes, the method to assess the effect on organic carbon oxidation processes alone is adequate. However, in some cases an examination of the effect on nitrification alone, or on both nitrification and organic carbon oxidation separately, are needed for the interpretation of the results and understanding the effects.

PRINCIPLE OF THE TEST METHOD

5.

The respiration rates of samples of activated sludge fed with synthetic sewage are measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Under consideration of the realistic exposure scenario, longer contact times could be appropriate. If the test chemical is rapidly degraded e.g. abiotically via hydrolysis, or is volatile and the concentration cannot be adequately maintained, additionally a shorter exposure period e.g. 30 minutes can be used. The sensitivity of each batch of activated sludge should be checked with a suitable reference chemical on the day of exposure. The test is typically used to determine the ECx (e.g. EC50) of the test chemical and/or the no-observed effect concentration (NOEC).

6.

The inhibition of oxygen uptake by micro-organisms oxidising organic carbon may be separately expressed from that by micro-organisms oxidising ammonium by measurement of the rates of uptake of oxygen in the absence and presence of N-allylthiourea, a specific inhibitor of the oxidation of ammonium to nitrite by the first-stage nitrifying bacteria. In this case the percentage inhibition of the rate of oxygen uptake is calculated by comparison of the rate of oxygen uptake in the presence of a test chemical with the mean oxygen uptake rate of the corresponding controls containing no test chemical, both in the presence and absence of the specific inhibitor, N-allylthiourea.

7.

Any oxygen uptake arising from abiotic processes may be detected by determining the rate in mixtures of test chemical, synthetic sewage medium and water, omitting activated sludge.

INFORMATION OF THE TEST CHEMICAL

8.

The identification (preferably CAS number), name (IUPAC), purity, water solubility, vapour pressure, volatility and adsorption characteristics of the test chemical should be known to enable correct interpretation of results to be made. Normally, volatile chemicals cannot be tested adequately unless special precautions are taken (see paragraph 21).

APPLICABILITY OF THE TEST METHOD

9.

The test method may be applied to water-soluble, poorly soluble and volatile chemicals. However, it may not always be possible to obtain EC50 values with chemicals of limited solubility and valid results with volatile chemicals may only be obtained providing that the bulk (say > 80 %) of the test chemical remains in the reaction mixture at the end of the exposure period(s). Additional analytical support data should be submitted to refine the ECx concentration when there is any uncertainty regarding the stability of the test chemical or its volatility.

REFERENCE CHEMICALS

10.

Reference chemicals should be tested periodically in order to assure that the test method and test conditions are reliable, and to check the sensitivity of each batch of activated sludge used as microbial inoculum on the day of exposure. The chemical 3,5-dichlorophenol (3,5-DCP) is recommended as the reference inhibitory chemical, since it is a known inhibitor of respiration and is used in many types of test for inhibition/toxicity (4). Also copper (II) sulphate pentahydrate can be used as a reference chemical for the inhibition of total respiration (9). N-methylaniline can be used as a specific reference inhibitor of nitrification (4).

VALIDITY CRITERIA AND REPRODUCIBILITY

11.

The blank controls (without the test chemical or reference chemical) oxygen uptake rate should not be less than 20 mg oxygen per one gramme of activated sludge (dry weight of suspended solids) in an hour. If the rate is lower, the test should be repeated with washed activated sludge or with the sludge from another source. The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30 % at the end of definitive test.

12.

In a 2004 international ring test organised by ISO (4) using activated sludge derived from domestic sewage, the EC50 of 3,5-DCP was found to lie in the range 2 mg/l to 25 mg/l for total respiration, 5 mg/l to 40 mg/l for heterotrophic respiration and 0,1 mg/l to 10 mg/l for nitrification respiration. If the EC50 of 3,5-DCP does not lie in the expected range, the test should be repeated with activated sludge from another source. The EC50 of copper (II) sulphate pentahydrate should lie in the range of 53-155 mg/l for the total respiration (9).

DESCRIPTION OF THE TEST METHOD

Test vessels and apparatus

13.

Usual laboratory equipment and the following should be used:

(a)

Test vessels — for example, 1 000 ml beakers to contain 500 ml of reaction mixture (see 5 in Fig.1);

(b)

Cell and attachments for measuring concentration of dissolved oxygen; a suitable oxygen electrode; an enclosed cell to contain the sample with no headspace and a recorder (e.g. 7, 8, 9 in Fig.1 of Appendix 2); alternatively, a BOD bottle may be used with a suitable sleeve adaptor for sealing the oxygen electrode against the neck of the bottle (see Fig. 2 of Appendix 3). To avoid loss of displaced liquid on insertion of the oxygen electrode, it is advisable first to insert a funnel or glass tube through the sleeve, or to use vessels with flared-out rims. In both cases a magnetic stirrer or alternative stirrer method, e.g. self-stirring probe, should be used;

(c)

Magnetic stirrers and followers, covered with inert material, for use in measurement chamber and/or in the test vessels;

(d)

Aeration device: if necessary, compressed air should be passed through an appropriate filter to remove dust and oil and through wash bottles containing water to humidify the air. The contents of vessels should be aerated with Pasteur pipettes, or other aeration devices, which do not adsorb chemicals. An orbital shaker operated at orbiting speeds between 150 and 250 rpm with flasks of, for example, 2 000 ml capacity, can be used to satisfy the oxygen demand for the sludge and overcome difficulties with chemicals that produce excessive foam, are volatile and therefore lost, or are difficult to disperse when aerated by air sparging. The test system is typically a number of beakers aerated continuously and sequentially established (e.g. at ca. 10 - 15 minute intervals), then analysed in a sequential manner. Validated instrumentation that allows the simultaneous aeration and measurement of the oxygen consumption rate in the mixtures may also be used;

(e)

pH-meter;

(f)

Centrifuge, general bench-top centrifuge for sludge capable of 10 000 m/s2.

Reagents

14.

Analytical grade reagents should be used throughout.

Water

15.

Distilled or deionised water, containing less than 1 mg/l DOC, should be used except where chlorine free tap water is specified.

Synthetic sewage feed

16.

The medium should be prepared to contain the following constituents at the stated amounts:

peptone

16 g

meat extract (or a comparable vegetable extract)

11 g

urea

3 g

sodium chloride (NaCl)

0,7 g

calcium chloride dihydrate (CaC12, 2H2O)

0,4 g

magnesium sulphate heptahydrate (MgSO4, 7H2O)

0,2 g

anhydrous potassium monohydrogen phosphate (K2HPO4)

2. 8g

distilled or deionised water to 1 litre

 

17.

The pH of this solution should be 7,5 ± 0,5. If the prepared medium is not used immediately, it should be stored in the dark at 0 °C to 4 °C, for no longer than 1 week or under conditions, which do not change its composition. It should be noted that this synthetic sewage is a 100 fold concentrate of that described in the OECD Technical Report “Proposed method for the determination of the biodegradability of surfactants used in synthetic detergents”June 11, 1976, with moreover dipotassium hydrogen phosphate added.

18.

Alternatively, components of the medium can be sterilised individually prior to storage, or the peptone and meat extract can be added shortly before carrying out the test. Prior to use, the medium should be thoroughly mixed and the pH adjusted if necessary to pH 7,5 ± 0,5.

Test chemical

19.

A stock solution should be prepared for readily water soluble test substances up to the maximum water solubility only (precipitations are not acceptable). Poorly water soluble substances, mixtures with components of different water solubility and adsorptive substances should be directly weighed into the test vessels. In these cases, use of stock solutions may be an alternative if dissolved concentrations of the test chemicals are analytically determined in the test vessels (prior to adding activated sludge). If water accommodated fractions (WAFs) are prepared, an analytical determination of the dissolved concentrations of the test chemicals in the test vessels is also essential. Using organic solvents, dispersants/emulsifiers to improve solubility should be avoided. Ultrasonication of stock solutions and pre-stirring suspensions, e.g. overnight, is possible when there is adequate information available concerning the stability of the test chemical under such conditions.

20.

The test chemical may adversely affect pH within the test system. The pH of the test chemical-treated mixtures should be determined prior to the test set up, in a preliminary trial, to ascertain whether pH adjustment will be necessary prior the main test and again on the day of the main test. Solutions/suspensions of test chemical in water should be neutralised prior to inoculum addition, if necessary. However, since neutralisation may change the chemical properties of the chemical, further testing, depending on the purposes of the study, could be performed to assess the effect of the test chemical on the sludge without pH adjustment.

21.

The toxic effects of volatile chemicals, especially in tests in which air is bubbled through the system, can result in variable effect levels occurring owing to losses of the substance during the exposure period. Caution should be exercised with such substances by performing substance specific analysis of control mixtures containing the substance and modifying the aeration regime.

Reference chemical

22.

If 3,5-dichlorophenol is used as reference chemical, a solution of 1,00 g of 3,5-dichlorophenol in 1 000 ml of water should be prepared (15). Warm water and/or ultrasonication should be used to accelerate the dissolution and make the solution up to volume when it has cooled to room temperature. However, it should be ensured that the reference chemical is not structurally changed. The pH of the solution should be checked and adjusted, if necessary, with NaOH or H2SO4 to pH 7 - 8.

23.

If copper(II)sulphate pentahydrate is used as a reference chemical, concentrations of 58 mg/l, 100 mg/l and 180 mg/l (a factor of 1,8) are used. The substance is weighed in directly into the test vessels (29 - 50 - 90 mg for 500 ml total volume). It is then dissolved with 234 ml of autoclaved tap water. Copper(II)sulphate pentahydrate is easily soluble. When the test is started, 16 ml of synthetic sewage and 250 ml of activated sludge are added.

Specific inhibitor of nitrification

24.

A 2,32 g/l stock solution of N-allylthiourea (ATU) should be prepared. The addition of 2,5 ml of this stock solution to an incubation mixture of final volume of 500 ml results in a final concentration of 11,6 mg ATU/l (10– 4 mol/l) which is known to be sufficient (4) to cause 100 % inhibition of nitrification in a nitrifying activated sludge containing 1,5g/l suspended solids.

Abiotic control

25.

Under some rare conditions, a test chemical with strong reducing properties may cause measurable abiotic oxygen consumption. In such cases, abiotic controls are necessary to discriminate between abiotic oxygen uptake by the test chemical and microbial respiration. Abiotic controls may be prepared by omitting the inoculum from the test mixtures. Similarly, abiotic controls without inoculum may be included when supporting analytical measurements are performed to determine the achieved concentration during the exposure phase of the test, e.g. when using stock solutions of poorly water soluble chemicals with components with different water solubility. In specific cases it may be necessary to prepare an abiotic control with sterilised inoculum (e.g. by autoclaving or adding sterilising toxicants). Some chemicals may produce or consume oxygen only if the surface area is big enough for reaction, even if they normally need a much higher temperature or pressure to do so. In this respect special attention should be given to peroxy substances. A sterilised inoculum provides a big surface area.

Inoculum

26.

For general use, activated sludge should be collected from the exit of the aeration tank, or near the exit from the tank, of a well-operated wastewater treatment plant receiving predominantly domestic sewage. Depending on the purpose of the test, other adequate types or sources of activated sludge, e.g. sludge grown in the laboratory, may also be used at suitable suspended solids concentrations of 2 g/l to 4 g/l. However, sludges from different treatment plants are likely to exhibit different characteristics and sensitivities.

27.

The sludge may be used as collected but coarse particles should be removed by settling for a short period, e.g. 5 to 15 minutes, and decanting the upper layer of finer solids or sieving (e.g. 1 mm2 mesh). Alternatively, the sludge may be homogenised in a blender for a ca. 15 seconds or longer, but caution is needed regarding the shear forces and the temperature change which might occur for long periods of blending.

28.

Washing the sludge is often necessary, e.g. if the endogenous respiration rate is low. The sludge should first be centrifuged for a period to produce a clear supernatant and pellet of sewage solids e.g. 10 minutes at ca. 10 000 m/s2. The supernatant liquid should be discarded and the sludge re-suspended in chlorine-free tap water, with shaking, and the wash-water should then be removed by re-centrifuging and discarding again. The washing and centrifuging process should be repeated, if necessary. The dry mass of a known volume of the re-suspended sludge should be determined and the sludge concentrated by removing liquor or diluted further in chlorine-free tap water to obtain the required sludge solids concentration of 3 g/l. The activated sludge should be continuously aerated (e.g. 2 l/minute) at the test temperature and, where possible used on day of collection. If this is not possible, the sludge should be fed daily with the synthetic sewage feed (50 ml synthetic sewage feed/l activated sludge) for two additional days. The sludge is then used for the test and the results are accepted as valid, provided that no significant change in its activity, assessed by its endogenous heterotrophic and nitrification respiration rate, has occurred.

29.

Difficulties can arise if foaming occurs during the incubation to the extent that the foam and the sludge solids carried on it, are expelled from the aeration vessels. Occasionally, foaming may simply result from the presence of the synthetic sewage, but foaming should be anticipated if the test chemical is, or contains, a surfactant. Loss of sludge solids from the test mixtures will result in artificially lowered respiration rates that could mistakenly be interpreted as a result of inhibition. In addition, aeration of surfactant solution concentrates the surfactant in the foam layer; loss of foam from the test system will lower the exposure concentrations. The foaming can be controlled by simple mechanical methods (e.g. occasional manual stirring using a glass rod) or by adding a surfactant-free silicone emulsion antifoam agent and/or use the shake flask aeration method. If the problem is associated with the presence of the synthetic sewage, the sewage composition should be modified by including an antifoam reagent at a rate of e.g. 50 μl/l. If foaming is caused by the test chemical, the quantity needed for abatement should be determined at the maximum test concentration, and then all individual aeration vessels should be identically treated (including those, e.g. blank controls and reference vessels where foam is absent). If antifoam agents are used, there should be no interaction with inoculum and/or test chemical.

TEST PROCEDURE

30.

The inhibition of three different oxygen uptakes may be determined, total, heterotrophic only and that due to nitrification. Normally, the measurement of total oxygen uptake inhibition should be adequate. The effects on heterotrophic oxygen uptake from the oxidation of organic carbon, and due to the oxidation of ammonium are needed when there is a specific requirement for such two separate end-points for a particular chemical or (optionally) to explain atypical dose-response curves from inhibition of total oxygen uptake.

Test conditions

31.

The test should be performed at a temperature within the range 20 ± 2 °C.

Test mixtures

32.

Test mixtures (FT as in Table 1) containing water, synthetic sewage feed and the test chemical should be prepared to obtain different nominal concentrations of the test chemical (See Table 1 for example of volumes of constituents). The pH should be adjusted to 7,5 ± 0,5, if necessary; mixtures should be diluted with water and the inoculum added to obtain equal final volumes in the vessels and to begin the aeration.

Reference mixtures

33.

Mixtures (FR) should be prepared with the reference chemical, e.g. 3,5-dichlorophenol, in place of the test chemical in the same way as the test mixtures.

Blank controls

34.

Blank controls (FB) should be prepared at the beginning and end of the exposure period in tests in which the test beakers are set up sequentially at intervals. In tests performed using equipment which allows simultaneous measurements of oxygen consumption to be made, at least two blank controls should be included in each batch of simultaneous analysis. Blank controls contain an equal volume of activated sludge and synthetic medium but not test or reference chemical. They should be diluted with water to the same volume as the test and reference mixtures.

Abiotic control

35.

If necessary, for example if a test chemical is known or suspected to have strong reducing properties, a mixture FA should be prepared to measure the abiotic oxygen consumption. The mixture should have the same amounts of test chemical, synthetic sewage feed and the same volume as the test mixtures, but no activated sludge.

General procedure and measurements

36.

Test mixtures, reference mixtures and the blank and abiotic controls are incubated at the test temperature under conditions of forced aeration (0,5 to 1 l/min) to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flocs in suspension. Stirring the cultures is also necessary to maintain sludge flocs in suspension. The incubation is considered to begin with the initial contact of the activated sludge inoculum with the other constituents of the final mixture. At the end of incubation, after the specified exposure times of usually 3 hours, samples are withdrawn to measure the rate of decrease of the concentration of dissolved oxygen in the cell designed for the purpose (Fig.2 of Appendix 3) or in a completely filled BOD bottle. The manner in which the incubations begin also depends on the capacity of the equipment used to measure oxygen consumption rates. For example, if it comprises a single oxygen probe, the measurements are made individually. In this case, the various mixtures needed for the test in synthetic sewage should be prepared but the inoculum should be withheld, and the requisite portions of sludge should be added to each vessel of the series. Each incubation should be started in turn, at conveniently timed intervals of e.g. 10 to 15 minutes. Alternatively, the measuring system may comprise multiple probes that facilitate multiple simultaneous measurements; in this case, inoculum may be added at the same time to appropriate groups of vessels.

37.

The activated sludge concentration in all test, reference and blank (but not abiotic control) mixtures is nominally 1,5 g/l of suspended solids. The oxygen consumption should be measured after 3 hours of exposure. Additional 30-minute exposure measurements should be performed as appropriate and previously described in paragraph 5.

Nitrification potential of sludge

38.

In order to decide whether sludge nitrifies and, if so, at what rate, mixtures (FB) as in the blank control and additional “control” mixtures (FN) but which also contain N-allylthiourea at 11,6 mg/l should be prepared. The mixtures should be aerated and incubated at 20 °C ± 2 °C for 3 hours. Then the rates of oxygen uptake should be measured and the rate of oxygen uptake due to nitrification calculated.

Test designs

Range-finding test

39.

A preliminary test is used, when necessary, to estimate the range of concentrations of the test chemical needed in a definitive test for determining the inhibition of oxygen consumption. Alternatively, the absence of inhibition of oxygen consumption by the test chemical in a preliminary test may demonstrate that a definitive test is unnecessary, but triplicates at the highest tested concentration of the preliminary test (typically 1 000 mg/l, but dependent on the data requirement) should be included.

Table 1

Examples of mixtures for the preliminary test

Reagent

Original Concentration

Test chemical stock solution

10 g/l

Synthetic medium stock solution

See paragraph 16

Activated sludge stock suspension

3 g/l of suspended solids

Components of mixtures

Dosing into test vessels (6)

FT1

FT2

FT3-5

FB1-2

FA

Test chemical stock solution (ml)

(paragraphs 19 to 21)

0,5

5

50

0

50

Synthetic sewage feed stock solution (ml)

(paragraph 16)

16

16

16

16

16

Activated sludge suspension (ml)

(paragraphs 26 to 29)

250

250

250

250

0

Water

(paragraph 15)

233,5

229

184

234

434

Total volume of mixtures (ml)

500

500

500

500

500

Concentrations in the mixture

 

 

 

 

 

Test suspension (mg/l)

Activated sludge

10

100

1 000

0

1 000

(suspended solids) (mg/l)

1 500

1 500

1 500

1 500

0

40.

The test should be performed using at least three concentrations of the test chemical, for example, 10 mg/l, 100 mg/l and 1 000 mg/l with a blank control and, if necessary, at least three abiotic controls with the highest concentrations of the test chemical (see as example Table 1). Ideally the lowest concentration should have no effect on oxygen consumption. The rates of oxygen uptake and the rate of nitrification, if relevant, should be calculated; then the percentage inhibition should be calculated. Depending on the purpose of the test, it is also possible to simply determine the toxicity of a limit concentration, e.g. 1 000 mg/l. If no statistically significant toxic effect occurs at this concentration, further testing at higher or lower concentrations is not necessary. It should be noted that poorly water soluble substances, mixtures with components of different water solubility and adsorptive substances should be directly weighed into the test vessels. In this case, the volume reserved for the test substance stock solution should be replaced with dilution water.

Definitive test

Inhibition of total oxygen uptake

41.

The test should be carried out using a range of concentrations deduced from the preliminary test. In order to obtain both a NOEC and an ECx (e.g. EC50), six controls and five treatment concentrations in a geometric series with five replicates are in most cases recommended. The abiotic control does not need to be repeated if there was no oxygen uptake in the preliminary test, but if significant uptake occurs abiotic controls should be included for each concentration of test chemical. The sensitivity of the sludge should be checked using the reference chemical 3,5-dichlorophenol. The sludge sensitivity should be checked for each test series, since the sensitivity is known to fluctuate. In all cases, samples are withdrawn from the test vessels after 3 hours, and additionally 30 minutes if necessary, for measurement of the rate of oxygen uptake in the oxygen electrode cell. From the data collected, the specific respiration rates of the control and test mixtures are calculated; the percentage inhibition is then calculated from equation 7, below.

Differentiation between inhibition of heterotrophic respiration and nitrification

42.

The use of the specific nitrification inhibitor, ATU, enables the direct assessment of the inhibitory effects of test chemicals on heterotrophic oxidation, and by subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate (no ATU present), the effects on the rate of nitrification may be calculated. Two sets of reaction mixtures should be prepared according to the test designs for ECx or NOEC described in paragraph 41, but additionally, ATU should be added to each mixture of one set at a final concentration of 11,6 mg/l, which has been shown to inhibit nitrification completely in sludge with suspended solids concentrations of up to 3 000 mg/l (4). The oxygen uptake rates should be measured after the exposure period; these direct values represent heterotrophic respiration only, and the differences between these and the corresponding total respiration rates represent nitrification. The various degrees of inhibition are then calculated.

Measurements

43.

After the exposure period(s) a sample from the first aeration vessel should be transferred to the oxygen electrode cell (Fig. 1 of Appendix 2) and the concentration of dissolved oxygen should immediately be measured. If a multiple electrode system is available, then the measurements may be made simultaneously. Stirring (by means of a covered magnet) is essential at the same rate as when the electrode is calibrated to ensure that the probe responds with minimal delay to changing oxygen concentrations, and to allow regular and reproducible oxygen measurements in the measuring vessel. Usually, the self-stirring probe system of some oxygen electrodes is adequate. The cell should be rinsed with water between measurements. Alternatively, the sample can be used to fill a BOD bottle (Fig. 2 of Appendix 3) fitted with a magnetic stirrer. An oxygen probe with a sleeve adaptor should then be inserted into the neck of the bottle and the magnetic stirrer should be started. In both cases the concentration of dissolved oxygen should continuously be measured and recorded for a period, usually 5 to 10 minutes or until the oxygen concentration falls below 2 mg/l. The electrode should be removed, the mixture returned to the aeration vessel and aerating and stirring should be continued, if measurement after longer exposure periods is necessary.

Verification of the test chemical concentration

44.

For some purposes, it may be necessary to measure the concentration of the test chemical in the test vessels. It should be noted that if stock solutions of:

poorly water soluble substances,

mixtures with components with different water solubility, or

substances with good water solubility, but where the concentration of the stock solution is near the maximum water solubility,

are used, the dissolved fraction is unknown, and the true concentration of the test chemical that is transferred into the test vessels is not known. In order to characterise the exposure, an analytical estimation of the test chemical concentrations in the test vessels is necessary. To simplify matters, analytical estimation should be performed before the addition of the inoculum. Due to the fact that only dissolved fractions will be transferred into test vessels, measured concentrations may be very low.

45.

To avoid time-consuming and expensive analytics, it is recommended to simply weigh the test chemical directly into the test vessels and to refer to the initial weighed nominal concentration for subsequent calculations. A differentiation between dissolved, undissolved or adsorbed fractions of the test chemical is not necessary because all these fractions appear under real conditions in a waste water treatment plant likewise, and these fractions may vary depending on the composition of the sewage. The aim of the test method is to estimate a non inhibitory concentration realistically and it is not suitable to investigate in detail which fractions make a contribution to the inhibition of the activated sludge organisms. Finally, adsorptive substances should be also weighed directly into the test vessels; and the vessels should be silanised in order to minimise losses through adsorption.

DATA AND REPORTING

Calculation of oxygen uptake rates

46.

The oxygen uptake rates should be calculated from the mean of the measured values, e.g. from the linear part of the graphs of oxygen concentration versus time, limiting the calculations to oxygen concentrations between 2,0 mg/l and 7,0 mg/l, since higher and lower concentrations may themselves influence rates of consumption. Excursion into concentration bands below or above these values is occasionally unavoidable and necessary, for example, when respiration is heavily suppressed and consequently very slow or if a particular activated sludge respires very quickly. This is acceptable provided the extended sections of the uptake graph are straight and their gradients do not change as they pass through the 2,0 mg/l or 7,0 mg/l O2 boundaries. Any curved sections of the graph indicate that the measurement system is stabilising or the uptake rate is changing and should not be used for the calculation of respiration rates. The oxygen uptake rate should be expressed in milligrammes per litre per hour (mg/lh) or milligrammes per gramme dry sludge per hour (mg/gh). The oxygen consumption rate, R, in mg/lh, may be calculated or interpolated from the linear part of the recorded oxygen decrease graph according to Equation 1:

R = (Q1 – Q2)/Δt × 60

(1)

where:

Q1

is the oxygen concentration at the beginning of the selected section of the linear phase (mg/l);

Q2

is the oxygen concentration at the end of the selected section of the linear phase (mg/l);

Δt

is the time interval between these two measurements (min.).

47.

The specific respiration rate (Rs) is expressed as the amount of oxygen consumed per g dry weight of sludge per hour (mg/gh) according to Equation 2:

Rs = R/SS

(2)

where SS is the concentration of suspended solids in the test mixture (g/l).

48.

The different indices of R which may be combined are:

S

specific rate

T

total respiration rate

N

rate due to nitrification respiration

H

rate due to heterotrophic respiration

A

rate due to abiotic processes

B

rate based on blank assays (mean)

Calculation of oxygen uptake rate due to nitrification

49.

The relationship between total respiration (RT), nitrification respiration (RN) and heterotrophic respiration (RH) is given by Equation 3:

RN = RT – RH

(3)

where:

RN

is the rate of oxygen uptake due to nitrification (mg/lh);

RT

is the measured rate of oxygen uptake by the blank control (no ATU; FB) (mg/lh).

RH

is the measured rate of oxygen uptake of the blank control with added ATU (FN) (mg/lh).

50.

This relationship is valid for blank values (RNB, RTB, RHB), abiotic controls (RNA, RTA, RHA) and assays with test chemicals (RNS, RTS, RHS) (mg/gh). Specific respiration rates are calculated from:

RN S = RN/SS

(4)

RTS = RT/SS

(5)

RHS = RH/SS

(6)

51.

If RN is insignificant (e.g. < 5 % of RT in blank controls) in a preliminary test, it may be assumed that the heterotrophic oxygen uptake equals the total uptake and that no nitrification is occurring. An alternative source of activated sludge would be needed if the tests were to consider effects on heterotrophic and nitrifying micro-organisms. A definitive test is performed if there is evidence of suppressed oxygen uptake rates with different test chemical concentrations.

Calculation of percentage of inhibition

52.

The percentage inhibition, IT, of total oxygen consumption at each concentration of test chemical, is given by Equation 7:

IT = [1 – (RT – RTA)/RTB] × 100 %

(7)

53.

Similarly, the percentage inhibition of heterotrophic oxygen uptake, IH, at each concentration of test chemical, is given by Equation 8:

IH = [1 – (RH – RHA)/RHB] × 100 %

(8)

54.

Finally, the inhibition of oxygen uptake due to nitrification, IN, at each concentration, is given by Equation 9:

IN = [1 – (RT – RH)/(RTB – RHB)] × 100 %

(9)

55.

The percentage inhibition of oxygen uptake should be plotted against logarithm of the test chemical concentration (inhibition curve, see Fig.3 of Appendix 4). Inhibition curves are plotted for each aeration period of 3 h or additionally after 30 min. The concentration of test chemical which inhibits the oxygen uptake by 50 % (EC50) should be calculated or interpolated from the graph. If suitable data are available, the 95 % confidence limits of the EC50, the slope of the curve, and suitable values to mark the beginning of inhibition (for example, EC10 or EC20) and the end of the inhibition range (for example, EC80 or EC90) may be calculated or interpolated.

56.

It should be noted that in view of the variability often observed in the results, it may in many cases be sufficient to express the results additionally in order of magnitude, for example:

EC50

< 1 mg/l

EC50

1 mg/l to 10 mg/l

EC50

10 mg/l to 100 mg/l

EC50

> 100mg/l

Interpretation of results

ECx

57.

ECx-values including their associated lower and upper 95 % confidence limits for the parameter are calculated using appropriate statistical methods (e.g. probit analysis, logistic or Weibull function, trimmed Spearman-Karber method or simple interpolation (11)). An ECx is obtained by inserting a value corresponding to x % of the control mean into the equation found. To compute the EC50 or any other ECx, the per-treatment means (x) should be subjected to regression analysis.

NOEC estimation

58.

If a statistical analysis is intended to determine the NOEC, per-vessel statistics (individual vessels are considered as replicates) are necessary. Appropriate statistical methods should be used according to the OECD Document on Current Approaches in the Statistical Analysis of Ecotoxicity Data: a Guidance to Application (11). In general, adverse effects of the test chemical compared to the control are investigated using one-tailed (smaller) hypothesis testing at p ≤ 0,05.

Test report

59.

The test report should include the following information:

 

Test chemical

common name, chemical name, CAS number, purity;

physico-chemical properties of the test chemical (e.g. log Kow, water solubility, vapour pressure, Henry's constant (H) and possible information on the fate of the test chemical e.g. adsorption to activated sludge);

 

Test system

source, conditions of operation of the wastewater treatment plant and influent it receives, concentration, pre-treatment and maintenance of the activated sludge;

 

Test conditions

test temperature, pH during the test and duration of the exposure phase(s);

 

Results

specific oxygen consumption of the controls (mg O2/(g sludge × h);

all measured data, inhibition curve(s) and method for calculation of EC50;

EC50 and, if possible, 95 per cent confidence limits, possibly EC20, EC80; possibly NOEC and the used statistical methods, if the EC50 cannot be determined;

results for total, and if appropriate, heterotrophic and nitrification inhibition;

abiotic oxygen uptake in the physico-chemical control (if used);

name of the reference chemical and results with this chemical;

all observations and deviations from the standard procedure, which could have influenced the result.

LITERATURE

(1)

Brown, D., Hitz, H.R. and Schäfer, L. (1981). The assessment of the possible inhibitory effect of dyestuffs on aerobic waste-water bacteria, Experience with a screening test. Chemosphere 10 (3): 245-261.

(2)

King, E. F. and Painter H. A. (1986). Inhibition of respiration of activated sludge; variability and reproducibility of results. Toxicity Assessment 1(1): 27-39.

(3)

OECD (1984), Activated sludge, Respiration inhibition test, Test Guideline No. 209, Guidelines for the testing of chemicals, OECD, Paris.

(4)

ISO (2007). ISO 8192 Water Quality- Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation, International Organization for Standardization.

(5)

Bealing, D. J. (2003). Document ISO/TC147/WGI/N.183, International Organization for Standardization.

(6)

Painter, H A, Jones K (1963). The use of the wide-bore dropping-mercury electrode for the determination of the rates of oxygen uptake and oxidation of ammonia by micro-orgranisms. Journal of Applied Bacteriology 26 (3): 471-483.

(7)

Painter, H. A. (1986). Testing the toxicity of chemicals by the inhibition of respiration of activated sludge. Toxicity Assessment 1:515-524.

(8)

Robra, B. (1976). Wasser/Abwasser 117, 80.

(9)

Fiebig S. and Noack, U. (2004). The use of copper(II)sulphate pentahydrate as reference substance in the activated sludge respiration inhibition test — acc. to the OECD guideline 209. Fresenius Environmental Bulletin 13 No. 12b: 1556-1557.

(10)

ISO (1995). ISO 10634 Water Quality — Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in aqueous medium, International Organization for Standardization.

(11)

OECD (2006). Current approaches in the statistical analysis of ecotoxicity data: a guidance to application, Series on testing and assessment No. 54, ENV/JM/MONO(2006)18, OECD, Paris.

Appendix 1

Definitions

The following definitions are applicable to this test method.

 

Chemical means a substance or a mixture.

 

ECx (Effect concentration for x % effect) is the concentration that causes an x % of an effect on test organisms within a given exposure period when compared with a control. For example, an EC50 is a concentration estimated to cause an effect on a test end point in 50 % of an exposed population over a defined exposure period.

 

NOEC (no observed effect concentration) is the test chemical concentration at which no effect is observed. In this test, the concentration corresponding to the NOEC, has no statistically significant effect (p < 0,05) within a given exposure period when compared with the control.

 

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

Appendix 2

Fig. 1:   Examples for measuring unit

Image

Key:

1

activated sludge

2

synthetic medium

3

test chemical

4

air

5

mixing vessel

6

magnetic stirrer

7

oxygen measuring cell

8

oxygen electrode

9

oxygen measuring instrument

10

recorder

Appendix 3

Fig. 2:   Example of measuring unit, using a BOD bottle

Image

Key:

1

Test vessel

2

oxygen electrode

3

oxygen measuring instrument

Appendix 4

Fig. 3:   Example of inhibition curves

Image

Key:

X

concentration of 3,5-dichlorophenol (mg/l)

Y

inhibition (%)

Image

inhibition heterotrophic respiration using a nitrifying sludge

Image

inhibition nitrification using a nitrifying sludge

(5)

Chapter C.26 is replaced by the following:

C.26   LEMNA SPECIES GROWTH INHIBITION TEST

INTRODUCTION

1.

This test method is equivalent to OECD Test Guideline (TG) 221 (2006). It is designed to assess the toxicity of chemicals to freshwater aquatic plants of the genus Lemna (duckweed). It is based on existing methods (1)(2)(3)(4)(5)(6) but includes modifications of those methods to reflect recent research and consultation on a number of key issues. This Test Method has been validated by an international ring-test (7).

2.

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

3.

Details of testing with renewal (semi-static and flow-through) and without renewal (static) of the test solution are described. Depending on the objectives of the test and the regulatory requirements, it is recommended to consider the application of semi-static and flow through methods, e.g. for chemicals that are rapidly lost from solution as a result of volatilisation, photodegradation, precipitation or biodegradation. Further guidance is given in (8).

4.

Definitions used are given in Appendix 1.

PRINCIPLE OF THE TEST

5.

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

6.

The test endpoint is inhibition of growth, expressed as logarithmic increase in measurement variable (average specific growth rate) during the exposure period. From the average specific growth rates recorded in a series of test solutions, the concentration bringing about a specified x % inhibition of growth rate (e.g. 50 %) is determined and expressed as the 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 measurement variables at the end of the exposure period minus the measurement variables at the start of the exposure period. From the yield recorded in a series of test solutions, the concentration bringing about a specified x % inhibition of yield (e.g., 50 %) is calculated and expressed as the 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.

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

10.

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

11.

When pH control of the test medium is particularly important, e.g. when testing metals or chemicals which are hydrolytically unstable, the addition of a buffer to the growth medium is recommended (see paragraph 21). Further guidance for testing chemicals with physical-chemical properties that make them difficult to test is provided in (8).

VALIDITY OF THE TEST

12.

For the test to be valid, the doubling time of frond number in the control must be less than 2,5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0,275 d– 1. Using the media and test conditions described in this Test Method, this criterion can be attained using a static test regime (5). It is also anticipated that this criterion will be achievable under semi-static and flow-through test conditions. Calculation of the doubling time is shown in paragraph 49.

REFERENCE CHEMICAL

13.

Reference chemical(s), such as 3,5-dichlorophenol used in the international ring test (7), may be tested as a means of checking the test procedure. It is advisable to test a reference chemical at least twice a year or, where testing is carried out at a lower frequency, in parallel to the determination of the toxicity of a test chemical.

DESCRIPTION OF THE METHOD

Apparatus

14.

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

15.

The cultures and test vessels should not be kept together. This is best achieved using separate environmental growth chambers, incubators, or rooms. Illumination and temperature must be controllable and maintained at a constant level (see paragraphs 35-36).

Test organism

16.

The organism used for this test is either Lemna gibba or Lemna minor. Short descriptions of duckweed species that have been used for toxicity testing are given in Appendix 2. Plant material may be obtained from a culture collection, another laboratory or from the field. If collected from the field, plants should be maintained in culture in the same medium as used for testing for a minimum of eight weeks prior to use. Field sites used for collecting starting cultures must be free of obvious sources of contamination. If obtained from another laboratory or a culture collection they should be similarly maintained for a minimum of three weeks. The source of plant material and the species and clone (if known) used for testing should always be reported.

17.

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

18.

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

Cultivation

19.

To reduce the frequency of culture maintenance (e.g. when no Lemna tests are planned for a period), cultures can be held under reduced illumination and temperature (4 — 10 °C). Details of culturing are given in Appenxix 3. Obvious signs of contamination by algae or other organisms may require surface sterilisation of a sub-sample of Lemna fronds, followed by transfer to fresh medium (see Appendix 3). In this eventuality the remaining contaminated culture should be discarded.

20.

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

Test medium

21.

Different media are recommended for Lemna minor and Lemna gibba, as described below. Careful consideration should be given to the inclusion of a pH buffer in the test medium (MOPS (4-morpholinepropane sulphonic acid, CAS No: 1132-61-2) in L. minor medium and NaHCO3 in L. gibba medium) when it is suspected that it might react with the test chemical and influence the expression of its toxicity. Steinberg Medium (9) is also acceptable as long as the validity criteria are met.

22.

A modification of the Swedish standard (SIS) Lemna growth medium is recommended for culturing and testing with L. minor. The composition of this medium is given in Appendix 4.

23.

The growth medium, 20X — AAP, as described in Appendix 4, is recommended for culturing and testing with L. gibba.

24.

Steinberg medium, as described in Appendix 4, is also suitable for L. minor, but may also be used for L. gibba as long as the validity criteria are met.

Test solutions

25.

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

26.

The highest tested concentration of the test chemical should not normally exceed the water solubility of the chemical under the test conditions. It should be noted however that Lemna spp. float on the surface and may be exposed to chemicals that collects at the water-air interface (e.g. poorly water-soluble or hydrophobic chemicals or surface-active chemicals). Under such circumstances exposure will result from material other than in solution and test concentrations may, depending on the characteristics of the test chemical, exceed water solubility. For test chemicals of low water solubility it may be necessary to prepare a concentrated stock solution or dispersion of the chemical using an organic solvent or dispersant in order to facilitate the addition of accurate quantities of the test chemical to the test medium and aid in its dispersion and dissolution. Every effort should be made to avoid the use of such materials. There should be no phytotoxicity resulting from the use of auxiliary solvents or dispersants. For example, commonly used solvents which do not cause phytotoxicity at concentrations up to 100 μl/l include acetone and dimethylformamide. If a solvent or dispersant is used, its final concentration should be reported and kept to a minimum (≤ 100 μl/l), and all treatments and controls should contain the same concentration of solvent or dispersant. Further guidance on the use of dispersants is given in (8).

Test and control groups

27.

Prior knowledge of the toxicity of the test chemical to Lemna, e.g. from a range-finding test, will help in selecting suitable test concentrations. In the definitive toxicity test, there should normally be at least five test concentrations arranged in a geometric series. Preferably the separation factor between test concentrations should not exceed 3.2, but a larger value may be used where the concentration-response curve is flat. Justification should be provided if fewer than five concentrations are used. At least three replicates should be used at each test concentration.

28.

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

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

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

29.

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

30.

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

Exposure

31.

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

32.

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

33.

If a preliminary stability test shows that the test chemical concentration cannot be maintained (i.e. the measured concentration falls below 80 % of the measured initial concentration) over the test duration (7 days), a semi-static test regime is recommended. In this case, the colonies should be exposed to freshly prepared test and control solutions on at least two occasions during the test (e.g. days 3 and 5). The frequency of exposure to fresh medium will depend on the stability of the test chemical; a higher frequency may be needed to maintain near-constant concentrations of highly unstable or volatile chemicals. In some circumstances, a flow-through procedure may be required (8)(10).

34.

The exposure scenario through a foliar application (spray) is not covered in this test method; instead, see (11).

Incubation conditions

35.

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

36.

The temperature in the test vessels should be 24 ± 2 °C. The pH of the control medium should not increase by more than 1,5 units during the test. However, deviation of more than 1,5 units would not invalidate the test when it can be shown that validity criteria are met. Additional care is needed on pH drift in special cases such as when testing unstable chemicals or metals. See (8) for further guidance.

Duration

37.

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

Measurements and analytical determinations

38.

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

39.

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

(i)

total frond area,

(ii)

dry weight,

(iii)

fresh weight.

40.

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

41.

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

(i)

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

(ii)

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

(iii)

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

Frequency of measurements and analytical determinations

42.

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

43.

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

44.

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

45.

In semi-static tests where the concentration of the test chemical is not expected to remain within ± 20 % of the nominal concentration, it is necessary to analyse all freshly prepared test solutions and the same solutions at each renewal (see paragraph 33). However, for those tests where the measured initial concentration of the test chemical is not within ± 20 % of nominal but where sufficient evidence can be provided to show that the initial concentrations are repeatable and stable (i.e. within the range 80 - 120 % of the initial concentration), chemical determinations may be carried out on only the highest and lowest test concentrations. In all cases, determination of test chemical concentrations prior to renewal need only be performed on one replicate vessel at each test concentration (or the contents of the vessels pooled by replicate).

46.

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

47.

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 ± 20 %, analysis of the results should be based on the geometric mean concentration during exposure or models describing the decline of the concentration of the test chemical (8).

Limit test

48.

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

DATA AND REPORTING

Doubling time

49.

To determine the doubling time (Td ) of frond number and adherence to this validity criterion by the study (paragraph 12), the following formula is used with data obtained from the control vessels:

Td = ln 2/μ

where μ is the average specific growth rate determined as described in paragraphs 54-55.

Response variables

50.

The purpose of the test is to determine the effects of the test chemical on the vegetative growth of Lemna. This Test Method describes two response variables, as different jusidictions 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 changes in the logarithms of frond numbers, and in addition, on the basis of changes in the logarithms of another measurement parameter (total frond area, dry weight or fresh weight) over time (expressed per day) in the controls and each treatment group. It is sometimes referred to as relative growth rate (12).

(b)

Yield: this response variable is calculated on the basis of changes in frond number, and in addition, on the basis of changes in another measurement parameter (total frond area, dry weight or fresh weight) in the controls and in each treatment group until the end of the test.

51.

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 duckweed 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 duckweed species used in each test and on the maximum specific growth rate that can vary between species and even different clones. This response variable should not be used for comparing the sensitivity to toxicants among duckweed species or even different clones. 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 jurisdictions.

52.

Toxicity estimates should be based on frond number and one additional measurement variable (total frond area, dry weight or fresh weight), because some chemicals may affect other measurement variables much more than the frond number. This effect would not be detected by calculating frond number only.

53.

The number of fronds as well as any other recorded measurement variable, i.e. total frond area, dry weight or fresh weight, are tabulated together with the concentrations of the test chemical for each measurement occasion. Subsequent data analysis e.g. to estimate a LOEC, NOEC or ECx should be based on the values for the individual replicates and not calculated means for each treatment group.

Average specific growth rate

54.

The average specific growth rate for a specific period is calculated as the logarithmic increase in the growth variables -frond numbers and one other measurement variable (total frond area, dry weight or fresh weight) — using the formula below for each replicate of control and treatments:

Formula

where:

:

μi-j

:

average specific growth rate from time i to j

:

Ni

:

measurement variable in the test or control vessel at time i

:

Nj

:

measurement variable in the test or control vessel at time j

:

t

:

time period from i to j

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

55.

The average specific growth rate should be calculated for the entire test period (time “i” in the above formula is the beginning of the test and time “j” is the end of the test). For each test concentration and control, calculate a mean value for average specific growth rate along with the variance estimates. In addition, the section-by-section growth rate should be assessed in order to evaluate effects of the test chemical occurring during the exposure period (e.g. by inspecting log-transformed growth curves). 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. In this case, a conservative approach would be to compare specific growth rates from treated cultures during the time period of maximum inhibition to those for controls during the same time period.

56.

Percent inhibition of growth rate (Ir) may then be calculated for each test concentration (treatment group) according to the following formula:

Formula

where:

:

% Ir

:

percent inhibition in average specific growth rate

:

μC

:

mean value for μ in the control

:

μT

:

mean value for μ in the treatment group

Yield

57.

Effects on yield are determined on the basis of two measurement variables, frond number and one other measurement variable (total frond area, dry weight or fresh weight) present in each test vessel at the start and at the end of the test. For dry weight or fresh weight, the starting biomass is determined on the basis of a sample of fronds taken from the same batch used to inoculate the test vessels (see paragraph 20). For each test concentration and control, calculate a mean value for yield along with variance estimates. The mean percent inhibition in yield (% Iy) may be calculated for each treatment group as follows:

Formula

where:

:

% Iy

:

percent reduction in yield

:

bC

:

final biomass minus starting biomass for the control group

:

bT

:

final biomass minus starting biomass in the treatment group

Plotting concentration-response curves

58.

Concentration-response curves relating mean percentage inhibition of the response variable (Ir, or Iy calculated as shown in paragraph 56 or 57) and the log concentration of the test chemical should be plotted.

ECx estimation

59.

Estimates of the ECx (e.g., EC50) should be based upon both average specific growth rate (ErCx) and yield (EyCx), each of which should in turn be based upon frond number and one additional measurement variable (total frond area, dry weight, or fresh weight). This is because there are test chemicals that impact frond number and other measurement variables differently. The desired toxicity parameters are therefore four ECx values for each inhibition level x calculated: ErCx (frond number); ErCx (total frond area, dry weight, or fresh weight); EyCx (frond number); and EyCx (total frond area, dry weight, or fresh weight).

Statistical procedures

60.

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 (13), 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 (13). 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 rate or yield data. Specific procedures for determination of ECx values from continuous data can be found in (14), (15), and (16).

61.

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.

62.

EC50 estimates and confidence limits may also be obtained using linear interpolation with bootstrapping (17), if available regression models/methods are unsuitable for the data.

63.

For estimation of the LOEC and hence the NOEC, 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 (18)(19)(20)(21). 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 (22). 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 (16).

64.

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 Lemna test. However, 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.

Reporting

65.

The test report must include the following:

 

Test chemical:

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

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

 

Test species:

scientific name, clone (if known) and source.

 

Test conditions:

test procedure used (static, semi-static or flow-through);

date of start of the test and its duration;

test medium;

description of the experimental design: test vessels and covers, solution volumes, number of colonies and fronds per test vessel at the beginning of the test;

test concentrations (nominal and measured as appropriate) and number of replicates per concentration;

methods of preparation of stock and test solutions including the use of any solvents or dispersants;

temperature during the test;

light source, light intensity and homogeneity;

pH values of the test and control media;

test chemical concentrations and the method of analysis with appropriate quality assessment data (validation studies, standard deviations or confidence limits of analyses);

methods for determination of frond number and other measurement variables, e.g. dry weight, fresh weight or frond area;

all deviations from this Test Method.

 

Results:

raw data: number of fronds and other measurement variables in each test and control vessel at each observation and occasion of analysis;

means and standard deviations for each measurement variable;

growth curves for each concentration (recommended with log transformed measurement variable, see paragraph 55);

doubling time/growth rate in the control based on the frond number;

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

graphical representation of the concentration/effect relationship;

estimates of toxic endpoints for response variables e.g. EC50, EC10, EC20, and associated confidence intervals. If calculated, LOEC and/or 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 visual signs of phytotoxicity as well as observations of test solutions;

discussion of the results, including any influence on the outcome of the test resulting from deviations from this Test Method.

LITERATURE

(1)

ASTM International. (2003). Standard Guide for Conducting Static Toxicity Test With Lemna gibba G3. E 1415-91 (Reapproved 1998). pp. 733-742. In, Annual Book of ASTM Standards, Vol. 11.05 Biological Effects and Environmental Fate; Biotechnology; Pesticides, ASTM, West Conshohocken, PA.

(2)

US EPA — United States Environmental Protection Agency. (1996). OPPTS 850.4400 Aquatic Plant Toxicity Test Using Lemna spp., “Public draft”. EPA 712-C-96-156. 8pp.

(3)

AFNOR — Association Française de Normalisation. (1996). XP T 90-337: Détermination de l'inhibition de la croissance de Lemna minor. 10pp.

(4)

SSI — Swedish Standards Institute. (1995). Water quality — Determination of growth inhibition (7-d) Lemna minor, duckweed. SS 02 82 13. 15pp. (in Swedish).

(5)

Environment Canada. (1999). Biological Test Method: Test for Measuring the Inhibition of Growth Using the Freshwater Macrophyte, Lemna minor. EPS 1/RM/37 - 120 pp.

(6)

Environment Canada. (1993) Proposed Guidelines for Registration of Chemical Pesticides: Non-Target Plant Testing and Evaluation. Canadian Wildlife Service, Technical Report Series No. 145.

(7)

Sims I., Whitehouse P. and Lacey R. (1999) The OECD Lemna Growth Inhibition Test. Development and Ring-testing of draft OECD Test Guideline. R&D Technical Report EMA 003. WRc plc — Environment Agency.

(8)

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

(9)

International Organisation for Standardisation. ISO DIS 20079. Water Quality — Determination of the Toxic Effect of Water Constituents and Waste Water to Duckweed (Lemna minor) — Duckweed Growth Inhibition Test.

(10)

Walbridge C. T. (1977). A flow-through testing procedure with duckweed (Lemna minor L.). Environmental Research Laboratory — Duluth, Minnesota 55804. US EPA Report No. EPA-600/3-77 108. September 1977.

(11)

Lockhart W. L., Billeck B. N. and Baron C. L. (1989). Bioassays with a floating plant (Lemna minor) for effects of sprayed and dissolved glyphosate. Hydrobiologia, 118/119, 353 — 359.

(12)

Huebert, D.B. and Shay J.M. (1993) Considerations in the assessment of toxicity using duckweeds. Environmental Toxicology and Chemistry, 12, 481-483.

(13)

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

(14)

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.

(15)

Bruce R.D. and Versteeg D.J. (1992) A statistical procedure for modelling continuous toxicity data. Environmental Toxicology and Chemistry, 11, 1485-1494.

(16)

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

(17)

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.

(18)

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

(19)

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

(20)

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.

(21)

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

(22)

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

 

Chemical means a substance or a mixture.

 

Chlorosis is yellowing of frond tissue.

 

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

 

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

 

ECx is the concentration of the test chemical dissolved in test medium that results in a x % (e.g. 50 %) reduction in growth of Lemna within a stated exposure period (to be mentioned explicitly if deviating from full or normal test duration). To unambiguously denote an EC value deriving from growth rate or yield the symbol “ErC” is used for growth rate and “EyC” is used for yield, followed by the measurement variable used, e.g. ErC (frond number).

 

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

 

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

 

Gibbosity means fronds exhibiting a humped or swollen appearance.

 

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

 

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

 

Lowest Observed Effect Concentration (LOEC) is the lowest tested concentration at which the 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.

 

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

 

Monoculture is a culture with one plant species.

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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

Appendix 2

Description of Lemna spp.

The aquatic plant commonly referred to as duckweed, Lemna spp., belongs to the family Lemnaceae which has a number of world-wide species in four genera. Their different appearance and taxonomy have been exhaustively described (1)(2). Lemna gibba and L. minor are species representative of temperate areas and are commonly used for toxicity tests. Both species have a floating or submerged discoid stem (frond) and a very thin root emanates from the centre of the lower surface of each frond. Lemna spp. rarely produce flowers and the plants reproduce by vegetatively producing new fronds (3). In comparison with older plants the younger ones tend to be paler, have shorter roots and consist of two to three fronds of different sizes. The small size of Lemna, its simple structure, asexual reproduction and short generation time makes plants of this genus very suitable for laboratory testing (4)(5).

Because of probable interspecies variation in sensitivity, only comparisons of sensitivity within a species are valid.

Examples of Lemna species which have been used for testing: Species Reference

 

Lemna aequinoctialis : Eklund, B. (1996). The use of the red alga Ceramium strictum and the duckweed Lemna aequinoctialis in aquatic ecotoxicological bioassays. Licentiate in Philosophy Thesis 1996:2. Dep. of Systems Ecology, Stockholm University.

 

Lemna major : Clark, N. A. (1925). The rate of reproduction of Lemna major as a function of intensity and duration of light. J. phys. Chem., 29: 935-941.

 

Lemna minor : United States Environmental Protection Agency (US EPA). (1996). OPPTS 850.4400 Aquatic Plant Toxicity Test Using Lemna spp., “Public draft”. EPA 712-C-96-156. 8pp.

Association Française de Normalisation (AFNOR). (1996). XP T 90-337: Détermination de l'inhibition de la croissance de Lemna minor. 10pp.

Swedish Standards Institute (SIS). (1995). Water quality — Determination of growth inhibition (7-d) Lemna minor, duckweed. SS 02 82 13. 15pp. (in Swedish).

 

Lemna gibba: ASTM International. (2003). Standard Guide for Conducting Static Toxicity Test With Lemna gibba G3. E 1415-91 (Reapproved 1998). pp. 733-742.

United States Environmental Protection Agency (US EPA). (1996). OPPTS 850.4400 Aquatic Plant Toxicity Test Using Lemna spp., “Public draft”. EPA 712-C-96-156. 8pp.

 

Lemna paucicostata : Nasu, Y., Kugimoto, M. (1981). Lemna (duckweed) as an indicator of water pollution. I. The sensitivity of Lemna paucicostata to heavy metals. Arch. Environ. Contam. Toxicol., 10:1959-1969.

 

Lemna perpusilla : Clark, J. R. et al. (1981). Accumulation and depuration of metals by duckweed (Lemna perpusilla). Ecotoxicol. Environ. Saf., 5:87-96.

 

Lemna trisulca : Huebert, D. B., Shay, J. M. (1993). Considerations in the assessment of toxicity using duckweeds. Environ. Toxicol. and Chem., 12:481- 483.

 

Lemna valdiviana : Hutchinson, T.C., Czyrska, H. (1975). Heavy metal toxicity and synergism to floating aquatic weeds. Verh.-Int. Ver. Limnol., 19:2102-2111.

Sources of Lemna species

University of Toronto Culture Collection of Algae and Cyanobacteria

Department of Botany, University of Toronto

Toronto, Ontario, Canada, M5S 3 B2

Tel: +1-416-978-3641

Fax: +1-416-978-5878

e-mail: jacreman@botany.utoronto.ca

North Carolina State University

Forestry Dept

Duckweed Culture Collection

Campus Box 8002

Raleigh, NC 27695-8002

United States

phone 001 (919) 515-7572

astomp@unity.ncsu.edu

Institute of Applied Environmental Research (ITM) Stockholm University

SE-106 91

STOCKHOLM

SWEDEN

Tel: +46 8 674 7240

Fax +46 8 674 7636

Federal Environmental Agency (UBA)

FG III 3.4

Schichauweg 58

12307 Berlin

Germany

e-mail: lemna@uba.de

LITERATURE

(1)

Hillman, W.S. (1961). The Lemnaceae or duckweeds: A review of the descriptive and experimental literature. The Botanical Review, 27:221-287.

(2)

Landolt, E. (1986). Biosystematic investigations in the family of duckweed (Lemnaceae). Vol. 2. Geobotanischen Inst. ETH, Stiftung Rubel, Zürich, Switzerland.

(3)

Björndahl, G. (1982). Growth performance, nutrient uptake and human utilization of duckweeds (Lemnaceae family). ISBN 82-991150-0-0. The Agricultural Research Council of Norway, University of Oslo.

(4)

Wang, W. (1986). Toxicity tests of aquatic pollutants by using common duckweed. Environmental Pollution, Ser B, 11:1-14.

(5)

Wang, W. (1990). Literature review on duckweed toxicity testing. Environmental Research, 52:7-22.

Appendix 3

Maintenance of stock culture

Stock cultures can be maintained under lower temperatures (4-10 °C) for longer times without needing to be re-established. The Lemna growth medium may be the same as that used for testing but other nutrient rich media can be used for stock cultures.

Periodically, a number of young, light-green plants are removed to new culture vessels containing fresh medium using an aseptic technique. Under the cooler conditions suggested here, sub-culturing may be conducted at intervals of up to three months.

Chemically clean (acid-washed) and sterile glass culture vessels should be used and aseptic handling techniques employed. In the event of contamination of the stock culture e.g. by algae or funghi, steps are necessary to eliminate the contaminating organisms. In the case of algae and most other contaminating organisms, this can be achieved by surface sterilisation. A sample of the contaminated plant material is taken and the roots cut off. The material is then shaken vigorously in clean water, followed by immersion in a 0,5 % (v/v) sodium hypochlorite solution for between 30 seconds and 5 minutes. The plant material is then rinsed with sterile water and transferred, as a number of batches, into culture vessels containing fresh growth medium. Many fronds will die as a result of this treatment, especially if longer exposure periods are used, but some of those surviving will usually be free of contamination. These can then be used to re-inoculate new cultures.

Appendix 4

Media

Different growth media are recommended for L. minor and L. gibba. For L. minor, a modified Swedish Standard (SIS) medium is recommended whilst for L. gibba, 20X AAP medium is recommended. Compositions of both media are given below. When preparing these media, reagent or analytical-grade chemicals should be used and deionised water.

Swedish Standard (SIS) Lemna growth medium

Stock solutions I - V are sterilised by autoclaving (120 °C, 15 minutes) or by membrane filtration (approximately 0,2 μm pore size).

Stock VI (and optional VII) are sterilised by membrane filtration only; these should not be autoclaved.

Sterile stock solutions should be stored under cool and dark conditions. Stocks I - V should be discarded after six months whilst stocks VI (and optional VII) have a shelf life of one month.

Stock solution No.

Substance

Concentration in stock solution

(g/l)

Concentration in prepared medium

(mg/ߦl)

Prepared medium

 

 

 

 

Element

Concentration

(mg/ߦl)

I

NaNO3

8,50

85

Na; N

32; 14

KH2PO4

1,34

13,4

K; P

6,0; 2,4

II

MgSO4·7H2O

15

75

Mg; S

7,4; 9,8

III

CaCl2·2H2O

7,2

36

Ca; Cl

9,8; 17,5

IV

Na2CO3

4,0

20

C

2,3

V

H3BO3

1,0

1,00

B

0,17

MnCl2·4H2O

0,20

0,20

Mn

0,056

Na2MoO4·2H2O

0,010

0,010

Mo

0,0040

ZnSO4·7H2O

0,050

0,050

Zn

0,011

CuSO4·5H2O

0,0050

0,0050

Cu

0,0013

Co(NO3)2·6H2O

0,010

0,010

Co

0,0020

VI

FeCl3·6H2O

0,17

0,84

Fe

0,17

Na2-EDTA 2H2O

0,28

1,4

VII

MOPS (buffer)

490

490

To prepare one litre of SIS medium, the following are added to 900 ml of deionised water:

10 ml of stock solution I

5 ml of stock solution II

5 ml of stock solution III

5 ml of stock solution IV

1 ml of stock solution V

5 ml of stock solution VI

1 ml of stock solution VII (optional)

Note: A further stock solution VII (MOPS buffer) may be needed for certain test chemicals (see paragraph 11).

The pH is adjusted to 6,5 ± 0,2 with either 0,1 or 1 mol HCl or NaOH, and the volume adjusted to one litre with deionised water.

20X AAP growth medium

Stock solutions are prepared in sterile distilled or deionised water.

Sterile stock solutions should be stored under cool and dark conditions. Under these conditions the stock solutions will have a shelf life of at least 6 - 8 weeks.

Five nutrient stock solutions (A1, A2, A3, B and C) are prepared for 20X — AAP medium, using reagent-grade chemicals. The 20 ml of each nutrient stock solution is added to approximately 850 ml deionised water to produce the growth medium. The pH is adjusted to 7,5 ± 0,1 with either 0,1 or 1 mol HCl or NaOH, and the volume adjusted to one litre with deionised water. The medium is then filtered through a 0,2 μm (approximate) membrane filter into a sterile container.

Growth medium intended for testing should be prepared 1-2 days before use to allow the pH to stabilise. The pH of the growth medium should be checked prior to use and readjusted if necessary by the addition of 0,1 or 1 mol NaOH or HCl as described above.

Stock solution No.

Sustance

Concentration in stock solution

(g/ߦl) (7)

Concentration in prepared medium

(mg/ߦl) (7)

Prepared medium

 

 

 

 

Element

Concentration

(mg/ߦl) (7)

A1

NaNO3

26

510

Na;N

190;84

MgCl2 · 6H2O

12

240

Mg

58,08

CaCl2 · 2H2O

4,4

90

Ca

24,04

A2

MgSO4 · 7H2O

15

290

S

38,22

A3

K2HPO4 · 3H2 · O

1,4

30

K;P

9.4;3.7

B

H3BO3

0,19

3,7

B

0,65

MnCl2 · 4H2O

0,42

8,3

Mn

2,3

FeCl3 · 6H2O

0,16

3,2

Fe

0,66

Na2EDTA.2H2O

0,30

6,0

ZnCl2

3,3 mg/l

66 μg/l

Zn

31 μg/l

CoCl2 · 6H2O

1,4 mg/l

29 μg/l

Co

7,1 μg/l

Na2MoO4 · 2H2O

7,3 mg/l

145 μg/l

Mo

58 μg/l

CuCl2 · 2H2O

0,012 mg/l

0,24 μg/l

Cu

0,080 μg/l

C

NaHCO3

15

300

Na;C

220; 43

STEINBERG medium (After ISO 20079)

Concentrations and stock solutions

The modified Steinberg medium is used in ISO 20079 for Lemna minor alone (as only Lemna minor is allowed there) but tests showed good results could be reached with Lemna gibba too.

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

Prepare the nutrient medium from stock solutions or the 10 fold concentrated medium which allows maximum concentration of the medium without precipitation.

Table 1

pH-stabilised STEINBERG medium (modified acc. to Altenburger)

Component

Nutrient medium

Macroelements

mol weight

mg/l

mmol/l

KNO3

101,12

350,00

3,46

Ca(NO3)2 · 4H2O

236,15

295,00

1,25

KH2PO4

136,09

90,00

0,66

K2HPO4

174,18

12,60

0,072

MgSO4 · 7H2O

246,37

100,00

0,41

Microelements

mol weight

μg/l

μmol/l

H3BO3

61,83

120,00

1,94

ZnSO4 · 7H2O

287,43

180,00

0,63

Na2MoO4 · 2H2O

241,92

44,00

0,18

MnCl2 · 4H2O

197,84

180,00

0,91

FeCl3 · 6H2O

270,21

760,00

2,81

EDTA Disodium-dihydrate

372,24

1 500,00

4,03


Table 2

Stock solutions (Macroelements)

1.

Macroelements (50-fold concentrated)

g/l

Stock solution 1:

KNO3

17,50

KH2PO4

4,5

K2HPO4

0,63

Stock solution 2:

MgSO4 · 7H2O

5,00

Stock solution 3:

Ca(NO3)2 · 4H2O

14,75


Table 3

Stock solutions (Microelements)

2.

Microelements (1 000-fold concentrated)

mg/l

Stock solution 4:

H3BO3

120,0

Stock solution 5:

ZnSO4 · 7H2O

180,0

Stock solution 6:

Na2MoO4 · 2H2O

44,0

Stock solution 7:

MnCl2 · 4H2O

180,0

Stock solution 8:

FeCl3 · 6H2O

760,00

EDTA Disodium-dihydrate

1 500,00

Stock solutions 2 and 3 and separately 4 to 7 may be pooled (taking into account the required concentrations).

For longer shelf life treat stock solutions in an autoclave at 121 °C for 20 min or alternatively carry out a sterile filtration (0,2 μm). For stock solution 8 sterile filtration (0,2 μm) is strongly recommended.

Preparation of the final concentration of STEINBERG medium (modified)

Add 20 ml of stock solutions 1, 2 and 3 (see table 2) to about 900 ml deionised water to avoid precipitation.

Add 1,0 ml of stock solutions 4, 5, 6, 7 and 8 (see table 3).

The pH should be to 5,5 +/– 0,2 (adjust by addition of a minimised volume of NaOH solution or HCl).

Adjust with water to 1 000 ml.

If stock solutions are sterilised and appropriate water is used no further sterilisation is necessary. If sterilisation is done with the final medium stock solution 8 should be added after autoclaving (at 121 °C for 20 min).

Preparation of 10-fold-concentrated STEINBERG medium (modified) for intermediate storage

Add to 20 ml of stock solutions 1, 2 and 3 (see table 2) to about 30 ml water to avoid precipitation.

Add 1,0 ml of stock solutions 4, 5, 6, 7 and 8 (see table 3). Adjust with water to 100 ml.

If stock solutions are sterilised and appropriate water is used no further sterilisation is necessary. If sterilisation is done with the final medium stock solution 8 should be added after autoclaving (at 121 °C for 20 min).

The pH of the medium (final concentration) should be 5,5 ± 0,2.

(6)

the following Chapters C.31 to C.46 are added:

C.31.   TERRESTRIAL PLANT TEST: SEEDLING EMERGENCE AND SEEDLING GROWTH TEST

INTRODUCTION

1.

This test method is equivalent to OECD Test Guideline (TG) 208 (2006). Test methods are periodically reviewed in the light of scientific progress and applicability to regulatory use. This updated test method is designed to assess potential effects of chemicals on seedling emergence and growth. As such it does not cover chronic effects or effects on reproduction (i.e. seed set, flower formation, fruit maturation). Conditions of exposure and properties of the chemical to be tested must be considered to ensure that appropriate test methods are used (e.g. when testing metals/metal compounds the effects of pH and associated counter ions should be considered) (1). This test method does not address plants exposed to vapours of chemicals. The test method is applicable to the testing of general chemicals, biocides and crop protection products (also known as plant protection products or pesticides). It has been developed on the basis of existing methods (2) (3) (4) (5) (6) (7). Other references pertinent to plant testing were also considered (8) (9) (10). Definitions used are given in Appendix 1.

PRINCIPLE OF THE TEST

2.

The test assesses effects on seedling emergence and early growth of higher plants following exposure to the test chemical in the soil (or other suitable soil matrix). Seeds are placed in contact with soil treated with the test chemical and evaluated for effects following usually 14 to 21 days after 50 % emergence of the seedlings in the control group. Endpoints measured are visual assessment of seedling emergence, dry shoot weight (alternatively fresh shoot weight) and in certain cases shoot height, as well as an assessment of visible detrimental effects on different parts of the plant. These measurements and observations are compared to those of untreated control plants.

3.

Depending on the expected route of exposure, the test chemical is either incorporated into the soil (or possibly into artificial soil matrix) or applied to the soil surface, which properly represents the potential route of exposure to the chemical. Soil incorporation is done by treating bulk soil. After the application the soil is transferred into pots, and then seeds of the given plant species are planted in the soil. Surface applications are made to potted soil in which the seeds have already been planted. The test units (controls and treated soils plus seeds) are then placed under appropriate conditions to support germination/growth of plants.

4.

The test can be conducted in order to determine the dose-response curve, or at a single concentration/rate as a limit test according to the aim of the study. If results from the single concentration/rate test exceed a certain toxicity level (e.g. whether effects greater than x % are observed), a range-finding test is carried out to determine upper and lower limits for toxicity followed by a multiple concentration/rate test to generate a dose-response curve. An appropriate statistical analysis is used to obtain effective concentration ECx or effective application rate ERx (e.g. EC25, ER25, EC50, ER50) for the most sensitive parameter(s) of interest. Also, the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) can be calculated in this test.

INFORMATION ON THE TEST CHEMICAL

5.

The following information is useful for the identification of the expected route of exposure to the chemical and in designing the test: structural formula, purity, water solubility, solubility in organic solvents, 1-octanol/water partition coefficient, soil sorption behaviour, vapour pressure, chemical stability in water and light, and biodegradability.

VALIDITY OF THE TEST

6.

In order for the test to be considered valid, the following performance criteria must be met in the controls:

the seedling emergence is at least 70 %;

the seedlings do not exhibit visible phytotoxic effects (e.g. chlorosis, necrosis, wilting, leaf and stem deformations) and the plants exhibit only normal variation in growth and morphology for that particular species;

the mean survival of emerged control seedlings is at least 90 % for the duration of the study;

environmental conditions for a particular species are identical and growing media contain the same amount of soil matrix, support media, or substrate from the same source.

REFERENCE CHEMICAL

7.

A reference chemical may be tested at regular intervals, to verify that performance of the test and the response of the particular test plants and the test conditions have not changed significantly over time. Alternatively, historical biomass or growth measurement of controls could be used to evaluate the performance of the test system in particular laboratories, and can serve as an intra-laboratory quality control measure.

DESCRIPTION OF THE METHOD

Natural soil — Artificial substrate

8.

Plants may be grown in pots using a sandy loam, loamy sand, or sandy clay loam that contains up to 1,5 percent organic carbon (approx. 3 percent organic matter). Commercial potting soil or synthetic soil mix that contains up to 1,5 percent organic carbon may also be used. Clay soils should not be used if the test chemical is known to have a high affinity for clays. Field soil should be sieved to 2 mm particle size in order to homogenise it and remove coarse particles. The type and texture, % organic carbon, pH and salt content as electronic conductivity of the final prepared soil should be reported. The soil should be classified according to a standard classification scheme (11). The soil could be pasteurised or heat treated in order to reduce the effect of soil pathogens.

9.

Natural soil may complicate interpretation of results and increase variability due to varying physical/chemical properties and microbial populations. These variables in turn alter moisture-holding capacity, chemical-binding capacity, aeration, and nutrient and trace element content. In addition to the variations in these physical factors, there will also be variation in chemical properties such as pH and redox potential, which may affect the bioavailability of the test chemical (12) (13) (14).

10.

Artificial substrates are typically not used for testing of crop protection products, but they may be of use for the testing of general chemicals or where it is desired to minimize the variability of the natural soils and increase the comparability of the test results. Substrates used should be composed of inert materials that minimize interaction with the test chemical, the solvent carrier, or both. Acid washed quartz sand, mineral wool and glass beads (e.g. 0,35 to 0,85 mm in diameter) have been found to be suitable inert materials that minimally absorb the test chemical (15), ensuring that the chemical will be maximally available to the seedling via root uptake. Unsuitable substrates would include vermiculite, perlite or other highly absorptive materials. Nutrients for plant growth should be provided to ensure that plants are not stressed through nutrient deficiencies, and where possible this should be assessed via chemical analysis or by visual assessment of control plants.

Criteria for selection of test species

11.

The species selected should be reasonably broad, e.g., considering their taxonomic diversity in the plant kingdom, their distribution, abundance, species specific life-cycle characteristics and region of natural occurrence, to develop a range of responses (8) (10) (16) (17) (18) (19) (20). The following characteristics of the possible test species should be considered in the selection:

the species have uniform seeds that are readily available from reliable standard seed source(s) and that produce consistent, reliable and even germination, as well as uniform seedling growth;

plant is amenable to testing in the laboratory, and can give reliable and reproducible results within and across testing facilities;

the sensitivity of the species tested should be consistent with the responses of plants found in the environment exposed to the chemical;

they have been used to some extent in previous toxicity tests and their use in, for example, herbicide bioassays, heavy metal screening, salinity or mineral stress tests or allelopathy studies indicates sensitivity to a wide variety of stressors;

they are compatible with the growth conditions of the test method;

they meet the validity criteria of the test.

Some of the historically most used test species are listed in Appendix 2 and potential non-crop species in Appendix 3.

12.