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,

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).

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).

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:

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:

where

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):

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.

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.

12.

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

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).

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

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.

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.

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.

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.

22.

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

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.

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.

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.

26.

The following must be included in the report:

(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.

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.

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.

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.

11.

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

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.

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).

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:

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.

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).

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:

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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]:

where:

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]:

where:

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.

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:

where:

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:

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.

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).

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).

61.

The test report must include the following:

(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.

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.

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.

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).

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.

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).

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).

13.

Usual laboratory equipment and the following should be used:

14.

Analytical grade reagents should be used throughout.

15.

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

16.

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

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.

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.

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.

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.

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.

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.

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.

31.

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

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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:

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.

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:

where:

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:

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:

49.

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

where:

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:

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.

52.

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

53.

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

54.

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

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.

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.

59.

The test report should include the following information:

(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.

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.

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.

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).

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.

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.

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).

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.

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.

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.