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Chapters C.27, C.28, C.29 and C.30 are added: ‘C.27 SEDIMENT-WATER CHIRONOMID TOXICITY TEST USING SPIKED SEDIMENT INTRODUCTION 1. This Test Method is equivalent to OECD Test Guideline (TG) 218 (2004). This Test Method is designed to assess the effects of prolonged exposure of chemicals to the sediment-dwelling larvae of the freshwater dipteran Chironomus sp. It is based on existing toxicity test protocols for Chironomus riparius and Chironomus tentans which have been developed in Europe (1)(2)(3) and North America (4)(5)(6)(7)(8) and ring-tested (1)(6)(9). Other well documented chironomid species may also be used, e.g. Chironomus yoshimatsui (10)(11). 2. The exposure scenario used in this Test Method is spiking of sediment with the test substance. The selection of the appropriate exposure scenario depends on the intended application of the test. The scenario of spiking sediment is intended to simulate accumulated levels of chemicals persisting in the sediment. This exposure system involves spiking sediment of a sediment-water test system. 3. Substances that need to be tested towards sediment-dwelling organisms usually persist in this compartment over long time periods. The sediment-dwelling organisms may be exposed via a number of routes. The relative importance of each exposure route, and the time taken for each to contribute to the overall toxic effects, is dependent on the physical-chemical properties of the chemical concerned. For strongly adsorbing substances (e.g. with log Kow > 5) or for substances covalently binding to sediment, ingestion of contaminated food may be a significant exposure route. In order not to underestimate the toxicity of highly lipophilic substances, the use of food added to the sediment before application of the test substance may be considered. In order to take all potential routes of exposure into account the focus of this Test Method is on long-term exposure. The test duration is in the range of 20-28 days for C. riparius and C. yoshimatsui, and 28-65 days for C. tentans. If short-term data are required for a specific purpose, for example to investigate the effects of an unstable chemical, additional replicates may be removed after a 10-day period. 4. The measured endpoints are the total number of adults emerged and the time to emergence. It is recommended that measurements of larval survival and growth should only be made after a 10-day period if additional short-term data are required, using additional replicates as appropriate. 5. The use of formulated sediment is recommended. Formulated sediment has several advantages over natural sediments: — the experimental variability is reduced because it forms a reproducible “standardised matrix” and the need to find uncontaminated and clean sediment sources is eliminated; — the tests can be initiated at any time without encountering seasonal variability in the test sediment and there is no need to pre-treat the sediment to remove indigenous fauna; the use of formulated sediment also reduces the cost associated with the field collection of sufficient amounts of sediment for routine testing; — the use of formulated sediment allows for comparisons of toxicity and ranking substances accordingly. 6. Definitions used are given in Appendix 1. PRINCIPLE OF THE TEST 7. First instar chironomid larvae are exposed to a concentration range of the test chemical in sediment — water systems. The test substance is spiked into the sediment and first instar larvae are subsequently introduced into test beakers in which the sediment and water concentrations have been stabilised. Chironomid emergence and development rate is measured at the end of the test. Larval survival and weight may also be measured after 10 days if required (using additional replicates as appropriate). These data are analysed either by using a regression model in order to estimate the concentration that would cause × % reduction in emergence or larval survival or growth (e.g. EC15, EC50 etc.), or by using statistical hypothesis testing to determine a NOEC/LOEC. The latter requires comparison of effect values with control values using statistical tests. INFORMATION ON THE TEST SUBSTANCE 8. The water solubility of the test substance, its vapour pressure, measured or calculated partitioning into sediment and stability in water and sediment should be known. A reliable analytical method for the quantification of the test substance in overlying water, pore water and sediment with known and reported accuracy and limit of detection should be available. Useful information includes the structural formula and purity of the test substance. Chemical fate of the test substance (e.g. dissipation, abiotic and biotic degradation, etc.) also is useful information. Further guidance for testing substances with physical-chemical properties that make them difficult to perform the test is provided in (12) REFERENCE CHEMICALS 9. Reference chemicals may be tested periodically as a means of assuring that the test protocol and test conditions are reliable. Examples of reference toxicants used successfully in ring-tests and validation studies are: lindane, trifluralin, pentachlorophenol, cadmium chloride and potassium chloride (1)(2)(5)(6)(13). VALIDITY OF THE TEST 10. For the test to be valid the following conditions apply: — the emergence in the controls must be at least 70 % at the end of the test. (1)(6); — C. riparius and C. yoshimatsui emergence to adults from control vessels should occur between 12 and 23 days after their insertion into the vessels; for C. tentans, a period of 20 to 65 days is necessary. — at the end of the test, pH and the dissolved oxygen concentration should be measured in each vessel. The oxygen concentration should be at least 60 per cent of the air saturation value (ASV) at the temperature used, and the pH of overlying water should be in the 6-9 range in all test vessels; — the water temperature should not differ by more than ± 1,0 °C. The water temperature could be controlled by isothermal room and in that case the room temperature should be confirmed in an appropriate time interval. DESCRIPTION OF THE METHOD Test vessels 11. The study is conducted in glass 600 ml beakers measuring 8 cm in diameter. Other vessels are suitable, but they should guarantee a suitable depth of overlying water and sediment. The sediment surface should be sufficient to provide 2 to 3 cm2 per larvae. The ratio of the depth of the sediment layer to the depth of the overlying water should be 1:4. Test vessels and other apparatus that will come into contact with the test system should be made entirely of glass or other chemically inert material (e.g. Teflon). Selection of species 12. The species to be used in the test is preferably Chironomus riparius. Chironomus tentans is also suitable but more difficult to handle and requires a longer test period. Chironomus yohimatsui may also be used. Details of culture methods are given in Appendix 2 for Chironomus riparius. Information on culture conditions is also available for other species, i.e. Chironomus tentans (4) and Chironomus yoshimatsui (11). Identification of species must be confirmed before testing but is not required prior to every test if organisms come from an in-house culture. Sediment 13. Formulated sediment (also called reconstituted, artificial or synthetic sediment) should preferably be used. However, if natural sediment is used, it should be characterised (at least pH, organic carbon content, determination of other parameters such as C/N ratio and granulometry are also recommended), and it should be free from any contamination and other organisms that might compete with, or consume the chironomids. It is also recommended that, before it is used in a chironomid toxicity test, the natural sediment be conditioned for seven days under the same conditions which prevail in the subsequent test. The following formulated sediment, based on the artificial soil used in Test Method C.8 (14), is recommended for use in this test (1)(15)(16): (a) 4-5 % (dry weight) peat: as close to pH 5,5 to 6,0 as possible; it is important to use peat in powder form, finely ground (particle size ≤ 1 mm) and only air dried. (b) 20 % (dry weight) kaolin clay (kaolinite content preferably above 30 %). (c) 75-76 % (dry weight) quartz sand (fine sand should predominate with more than 50 per cent of the particles between 50 and 200 μm). (d) Deionised water is added to obtain moisture content of the final mixture in a range of 30-50 %. (e) Calcium carbonate of chemically pure quality (CaCO3) is added to adjust the pH of the final mixture of the sediment to 7,0 ± 0,5. Organic carbon content of the final mixture should be 2 % (± 0,5 %) and is to be adjusted by the use of appropriate amounts of peat and sand, according to (a) and (c). 14. The source of peat, kaolin clay and sand should be known. The sediment components should be checked for the absence of chemical contamination (e.g. heavy metals, organochlorine compounds, organophosphorous compounds, etc.). An example for the preparation of the formulated sediment is described in Appendix 3. Mixing of dry constituents is also acceptable if it is demonstrated that after addition of overlying water a separation of sediment constituents (e.g. floating of peat particles) does not occur, and that the peat or the sediment is sufficiently conditioned. Water 15. Any water which conforms to the chemical characteristics of acceptable dilution water as listed in Appendices 2 and 4 is suitable as test water. Any suitable water, natural water (surface or ground water), reconstituted water (see Appendix 2) or dechlorinated tap water are acceptable as culturing water and test water if chironomids will survive in it for the duration of the culturing and testing without showing signs of stress. At the start of the test, the pH of the test water should be between 6 and 9 and the total hardness not higher than 400 mg/l as CaCO3. However, if there is an interaction suspected between hardness ions and the test substance, lower hardness water should be used (and thus, Elendt Medium M4 must not be used in this situation). The same type of water should be used throughout the whole study. The water quality characteristics listed in Appendix 4 should be measured at least twice a year or when it is suspected that these characteristics may have changed significantly. Stock solutions — Spiked sediments 16. Spiked sediments of the chosen concentration are usually prepared by addition of a solution of the test substance directly to the sediment. A stock solution of the test substance dissolved in deionised water is mixed with the formulated sediment by rolling mill, feed mixer or hand mixing. If poorly soluble in water, the test substance can be dissolved in as small a volume as possible of a suitable organic solvent (e.g. hexane, acetone or chloroform). This solution is then mixed with 10 g of fine quartz sand for one test vessel. The solvent is allowed to evaporate and it has to be totally removed from sand; the sand is then mixed with the suitable amount of sediment per test beaker. Only agents which volatilise readily can be used to solubilise, disperse or emulsify the test substance. It should be born in mind that the sand provided by the test substance and sand mixture, has to be taken into account when preparing the sediment (i.e. the sediment should thus be prepared with less sand). Care should be taken to ensure that the test substance added to sediment is thoroughly and evenly distributed within the sediment. If necessary, subsamples can be analysed to determine degree of homogeneity. TEST DESIGN 17. The test design relates to the selection of the number and spacing of the test concentrations, the number of vessels at each concentration and the number of larvae per vessel. Designs for EC point estimation, for estimation of NOEC, and for conducting a limit test are described. Design for analysis by regression 18. The effect concentration (e.g. EC15, EC50) and the concentration range, over which the effect of the test substance is of interest, should be spanned by the concentrations included in the test. Generally, the accuracy and especially validity, with which estimates of effect concentrations (ECx) can be made, is improved when the effect concentration is within the range of concentrations tested. Extrapolating much below the lowest positive concentration or above the highest concentration should be avoided. A preliminary range-finding test is helpful for selecting the range of concentrations to be used (see paragraph 27). 19. If the ECx is to be estimated, at least five concentrations and three replicates for each concentration should be tested. In any case, it is advisable that sufficient test concentrations are used to allow good model estimation. The factor between concentrations should not be greater than two (an exception could be made in cases when the dose response curve has a shallow slope). The number of replicates at each treatment can be reduced if the number of test concentrations with different responses is increased. Increasing the number of replicates or reducing the size of the test concentration intervals tends to lead to narrower confidence intervals for the test. Additional replicates are required if 10-day larval survival and growth are to be estimated. Design for estimation of a NOEC/LOEC 20. If the LOEC or NOEC are to be estimated, five test concentrations with at least four replicates should be used and the factor between concentrations should not be greater than two. The number of replicates should be sufficient to ensure adequate statistical power to detect a 20 % difference from the control at the 5 % level of significance (p = 0,05). With the development rate, an Analysis of Variance (ANOVA) is usually appropriate, such as Dunnett-test and Williams-test (17)(18)(19)(20). In the emergence ratio the Cochran-Armitage, Fisher’s exact (with Bonferroni correction), or Mantel-Haenszel tests may be used. Limit test 21. A limit test may be performed (one test concentration and control) if no effects were seen in the preliminary range-finding test. The purpose of the limit test is to perform a test at a concentration sufficiently high to enable decision makers to exclude possible toxic effects of the test substance, and the limit is set at a concentration which is not expected to appear in any situation. 1 000 mg/kg (dry weight) is recommended. Usually, at least six replicates for both the treatment and control are necessary. Adequate statistical power to detect a 20 % difference from the control at the 5 % level of significance (p = 0,05) should be demonstrated. With metric response (development rate and weight), the t-test is a suitable statistical method if data meet the requirements of this test (normality, homogeneous variances). The unequal-variance t-test or a non parametric test, such as the Wilcoxon-Mann-Whithey test may be used, if these requirements are not fulfilled. With the emergence ratio, the Fisher exact test is appropriate. PROCEDURE Conditions of exposure Preparation of spiked sediment — water system 22. The spiking procedure described in Test Method C.8: Toxicity for Earthworms is recommended for application of the test substance (14). The spiked sediments are placed in the vessels and overlying water is added to produce a sediment-water volume ratio of 1:4 (see paragraphs 11 and 15). The depth of the sediment layer should be in the range of 1,5-3 cm. To avoid separation of sediment ingredients and re-suspension of fine material during addition of test water in the water column, the sediment can be covered with a plastic disc while water is poured onto it, and the disc removed immediately afterwards. Other devices may also be appropriate. 23. The test vessels should be covered (e.g. by glass plates). If necessary, during the study the water levels will be topped to the original volume in order to compensate for water evaporation. This should be performed using distilled or deionised water to prevent build-up of salts. Stabilisation 24. Once the spiked sediment with overlying water has been prepared, it is desirable to allow partitioning of the test substance from the aqueous phase to the sediment (3)(4)(6)(13). This should preferably be done under the conditions of temperature and aeration used in the test. Appropriate equilibration time is sediment and chemical specific, and can be in the order of hours to days and in rare cases up to several weeks (4-5 weeks). As this would leave time for degradation of many chemicals, equilibrium is not awaited but an equilibration period of 48 hours is recommended. At the end of this further equilibration period, the concentration of the test substance should be measured in the overlying water, the pore water and the sediment, at least at the highest concentration and a lower one (see paragraph 38). These analytical determinations of the test substance allow for calculation of mass balance and expression of results based on measured concentrations. Addition of test organisms 25. Four to five days before adding the test organisms to the test vessels, egg masses should be taken from the cultures and placed in small vessels in culture medium. Aged medium from the stock culture or freshly prepared medium may be used. If the latter is used, a small amount of food e.g. green algae and/or a few droplets of filtrate from a finely ground suspension of flaked fish food should be added to the culture medium (see Appendix 2). Only freshly laid egg masses should be used. Normally, the larvae begin to hatch a couple of days after the eggs are laid (2 to 3 days for Chironomus riparius at 20 °C and 1 to 4 days for Chironomus tentans at 23 °C and Chironomus yoshimatui at 25 °C) and larval growth occurs in four instars, each of 4-8 days duration. First instar larvae (2-3 or 1-4 days post hatching) should be used in the test. The instar of midges can possibly be checked using head capsule width (6). 26. Twenty first instar larvae are allocated randomly to each test vessel containing the spiked sediment and water, using a blunt pipette. Aeration of the water has to be stopped while adding the larvae to test vessels and remain so for another 24 hours after addition of larvae (see paragraphs 25 and 32). According to the test design used (see paragraphs 19 and 20), the number of larvae used per concentration is at least 60 for the EC point estimation and 80 for determination of NOEC. Test concentrations 27. A range-finding test may be helpful to determine the range of concentrations for the definitive test. For this purpose a series of widely spaced concentrations of the test substance are used. In order to provide the same density of surface per chironomids, which is to be used for the definitive test, chironomids are exposed to each concentration of the test substance for a period which allows estimation of appropriate test concentrations, and no replicates are required. 28. The test concentrations for the definitive test are decided based on the result of the range-finding test. At least five concentrations should be used and selected as described in paragraphs 18 to 20. Controls 29. Control vessels without any test substance but including sediment should be included in the test with the appropriate number of replicates (see paragraphs 19-20). If a solvent has been used for application of test substance (see paragraph 16), a sediment solvent control should be added. Test system 30. Static systems are used. Semi-static or flow-through systems with intermittent or continuous renewal of overlying water might be used in exceptional cases as for instance if water quality specifications become inappropriate for the test organism or affect chemical equilibrium (e.g. dissolved oxygen levels fall too low, the concentration of excretory products rises too high or minerals leach from sediment and affect pH and/or water hardness). However, other methods for ameliorating the quality of overlying water, such as aeration, will normally suffice and be preferable. Food 31. It is necessary to feed the larvae, preferably daily or at least three times per week. Fish-food (a suspension in water or finely ground food, e.g. TetraMin or TetraPhyll; see details in Appendix 2) in the amount of 0,25-0,5 mg (0,35-0,5 mg for C. yoshimatui) per larvae per day seems adequate for young larvae for the first 10 days. Slightly more food may be necessary for older larvae: 0,5-1 mg per larvae per day should be sufficient for the rest of the test. The food ration should be reduced in all treatments and control if fungal growth is seen or if mortality is observed in controls. If fungal development cannot be stopped the test is to be repeated. When testing strongly adsorbing substances (e.g. with log Kow > 5), or substances covalently binding to sediment, the amount of food necessary to ensure survival and natural growth of the organisms may be added to the formulated sediment before the stabilisation period. For this, plant material must be used instead of fish food, e.g. addition of 0,5 % (dry weight) finely ground leaves of e.g. stinging nettle (Urtica dioica), mulberry (Morus alba), white clover (Trifolium repens), spinach (Spinacia oleracea) or of other plant material (Cerophyl or alpha-cellulose) may be used. Incubation conditions 32. Gentle aeration of the overlying water in test vessels is supplied preferably 24 hours after addition of the larvae and is pursued throughout the test (care should be taken that dissolved oxygen concentration does not fall below 60 per cent of ASV). Aeration is provided through a glass Pasteur pipette fixed 2-3 cm above the sediment layer (i.e. one or few bubbles/sec). When testing volatile chemicals, consideration may be given not to aerate the sediment-water system. 33. The test is conducted at a constant temperature of 20 °C (± 2 °C). For C. tentans and C. yoshimatui recommended temperatures are 23 °C and 25 °C (± 2 °C), respectively. A 16 hours photoperiod is used and the light intensity should be 500 to 1 000 lux. Exposure duration 34. The exposure commences with the addition of larvae to the spiked and control vessels. The maximum exposure duration is 28 days for C. riparius and C. yoshimatsui, and 65 days for C. tentans. If midges emerge earlier, the test can be terminated after a minimum of five days after emergence of the last adult in the control. Observations Emergence 35. The development time and the total number of fully emerged male and female midges are determined. Males are easily identified by their plumose antennae. 36. The test vessels should be observed at least three times per week to make visual assessment of any abnormal behaviour (e.g. leaving sediment, unusual swimming), compared with the control. During the period of expected emergence a daily count of emerged midges is necessary. The sex and number of fully emerged midges are recorded daily. After identification the midges are removed from the vessels. Any egg masses deposited prior to the termination of the test should be recorded and then removed to prevent re-introduction of larvae into the sediment. The number of visible pupae that have failed to emerge is also recorded. Guidance on measurement of emergence is provided in Appendix 5. Growth and survival 37. If data on 10-day larval survival and growth are to be provided, additional test vessels should be included at the start, so that they may be used subsequently. The sediment from these additional vessels is sieved using a 250 μm sieve to retain the larvae. Criteria for death are immobility or lack of reaction to a mechanical stimulus. Larvae not recovered should also be counted as dead (larvae which have died at beginning of the test may have been degraded by microbes). The (ash free) dry weight of the surviving larvae per test vessel is determined and the mean individual dry weight per vessel calculated. It is useful to determine which instar the surviving larvae belong to; for that measurement of the width of the head capsule of each individual can be used. Analytical measurements Concentration of the test substance 38. Prior to test commencement (i.e. addition of larvae), samples of bulk sediment are removed from at least one vessel per treatment for the analytical determination of the test substance concentration in the sediment. It is recommended that, as a minimum, samples of the overlying water, the pore water and the sediment be analysed at the start (see paragraph 24) and at the end of the test, at the highest concentration and a lower one. These determinations of test substance concentration inform about the behaviour/partitioning of the test substance in the water-sediment system. 39. When intermediate measurements are made (e.g. at day 7) and if the analysis needs large samples which cannot be taken from test vessels without influencing the test system, analytical determinations should be performed on samples from additional test vessels treated in the same way (including the presence of test organisms) but not used for biological observations. 40. Centrifugation at e.g. 10 000 g and 4 °C for 30 min. is the recommended procedure to isolate interstitial water. However, if the test substance is demonstrated not to adsorb to filters, filtration may also be acceptable. In some cases it might not be possible to analyse concentrations in the pore water as the sample size is too small. Physical-chemical parameters 41. pH and temperature of the test vessels should be measured in an appropriate manner (see paragraph 10). Hardness and ammonia should be measured in the controls and one test vessel at the highest concentration at the start and the end of the test. DATA AND REPORTING Treatment of results 42. The purpose of this test is to determine the effect of the test substance on the development rate and the total number of fully emerged male and female midges, or in the case of the 10-day test effects on survival and weight of the larvae. If there are no indications of statistically different sensitivities of sexes, male and female results may be pooled for statistical analyses. The sensitivity differences between sexes can be statistically judged by e.g. a χ2-r × 2 table test. Larval survival and mean individual dry weight per vessel must be determined after 10 days where required. 43. Effect concentrations expressed and based on dry weight, are calculated preferably based on measured sediment concentrations at the beginning of the test (see paragraph 38). 44. To compute a point estimate for the EC50 or any other ECx, the per-vessel statistics may be used as true replicates. In calculating a confidence interval for any ECx the variability among vessels should be taken into account, or it should be shown that this variability is so small that it can be ignored. When the model is fitted by Least Squares, a transformation should be applied to the per-vessel statistics in order to improve the homogeneity of variance. However, ECx values should be calculated after the response is transformed back to the original value. 45. When the statistical analysis aims at determining the NOEC/LOEC by hypothesis testing, the variability among vessels needs to be taken into account, e.g. by a nested ANOVA. Alternatively, more robust tests (21) can be appropriate in situations where there are violations of the usual ANOVA assumptions. Emergence ratio 46. Emergence ratios are quantal data, and can be analyzed by the Cochran-Armitage test applied in step-down manner where a monotonic dose-response is expected and these data are consistent with this expectation. If not, a Fisher’s exact or Mantel-Haenszel test with Bonferroni-Holm adjusted p-values can be used. If there is evidence of greater variability between replicates within the same concentration than a binomial distribution would indicate (often referenced as “extra-binomial” variation), then a robust Cochran-Armitage or Fisher exact test such as proposed in (21), should be used. The sum of midges emerged per vessel, ne, is determined and divided by the number of larvae introduced, na: where: ER = emergence ratio ne = number of midges emerged per vessel na = number of larvae introduced per vessel 47. An alternative that is most appropriate for large sample sizes, when there is extra binomial variance, is to treat the emergence ratio as a continuous response and use procedures such as William’s test when a monotonic dose-response is expected and is consistent with these ER data. Dunnett’s test would be appropriate where monotonicity does not hold. A large sample size is defined here as the number emerged and the number not emerging both exceeding five, on a per replicate (vessel) basis. 48. To apply ANOVA methods values of ER should first be transformed by the arcsin-sqrt-transformation or Freeman-Tukey transformation to obtain an approximate normal distribution and to equalise variances. The Cochran-Armitage, Fisher’s exact (Bonferroni), or Mantel-Haenszel tests can be applied when using the absolute frequencies. The arcsin-sqrt transformation is applied by taking the inverse sine (sin-1) of the square root of ER. 49. For emergence ratios, ECx-values are calculated using regression analysis (or e.g. probit (22), logit, Weibull, appropriate commercial software etc.). If regression analysis fails (e.g. when there are less than two partial responses), other non-parametric methods such as moving average or simple interpolation are used. Development rate 50. The mean development time represents the mean time span between the introduction of larvae (day 0 of the test) and the emergence of the experimental cohort of midges. (For the calculation of the true development time, the age of larvae at the time of introduction should be considered). The development rate is the reciprocal of the development time (unit: 1/day) and represents that portion of larval development which takes place per day. The development rate is preferred for the evaluation of these sediment toxicity studies as its variance is lower, and it is more homogeneous and closer to normal distribution as compared to development time. Hence, powerful parametric test procedures may be used with development rate rather than with development time. For development rate as a continuous response, ECx-values can be estimated by using regression analysis (e.g. (23), (24)). 51. For the following statistical tests, the number of midges observed on inspection day × are assumed to be emerged at the mean of the time interval between day x and day x – l (l = length of the inspection interval, usually 1 day). The mean development rate per vessel (x) is calculated according to: where: : mean development rate per vessel i : index of inspection interval m : maximum number of inspection intervals : number of midges emerged in the inspection interval i ne : total number of midges emerged at the end of experiment (= ) xi : development rate of the midges emerged in interval i where: dayi : inspection day (days since application) li : length of inspection interval i (days, usually 1 day) Test report 52. The test report must at least provide the following information: Test substance: — physical nature and, where relevant, physical-chemical properties (water solubility, vapour pressure, partition coefficient in soil (or in sediment if available), stability in water, etc.); — chemical identification data (common name, chemical name, structural formula, CAS number, etc.) including purity and analytical method for quantification of test substance. Test species: — test animals used: species, scientific name, source of organisms and breeding conditions; — information on handling of egg masses and larvae; — age of test animals when inserted into test vessels. Test conditions: — sediment used, i.e. natural or formulated sediment; — for natural sediment, location and description of sediment sampling site, including, if possible, contamination history; characteristics: pH, organic carbon content, C/N ratio and granulometry (if appropriate). — preparation of the formulated sediment: ingredients and characteristics (organic carbon content, pH, moisture, etc. at the start of the test); — preparation of the test water (if reconstituted water is used) and characteristics (oxygen concentration, pH, conductivity, hardness, etc. at the start of the test); — depth of sediment and overlying water; — volume of overlying and pore water; weight of wet sediment with and without pore water; — test vessels (material and size); — method of spiking sediment: test concentrations used, number of replicates and use of solvent if any; — stabilisation equilibrium phase of the spiked sediment-water system: duration and conditions; — incubation conditions: temperature, light cycle and intensity, aeration (frequency and intensity); — detailed information on feeding including type of food, preparation, amount and feeding regime. Results: — the nominal test concentrations, the measured test concentrations and the results of all analyses to determine the concentration of the test substance in the test vessel; — water quality within the test vessels, i.e. pH, temperature, dissolved oxygen, hardness and ammonia; — replacement of evaporated test water, if any; — number of emerged male and female midges per vessel and per day; — number of larvae which failed to emerge as midges per vessel; — mean individual dry weight of larvae per vessel, and per instar, if appropriate; — percent emergence per replicate and test concentration (male and female midges pooled); — mean development rate of fully emerged midges per replicate and treatment rate (male and female midges pooled); — estimates of toxic endpoints e.g. ECx (and associated confidence intervals), NOEC and/or LOEC,, and the statistical methods used for their determination; — discussion of the results, including any influence on the outcome of the test resulting from deviations from this Test Method. 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Test for Growth and Survival in Sediment using Larvae of Freshwater Midges (Chironomus tentans or Chironomus riparius). Biological Test Method. Report SPE 1/RM/32. December 1997. (6) US-EPA (2000). Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates. Second edition. EPA 600/R-99/064. March 2000. Revision to the first edition dated June 1994. (7) US-EPA/OPPTS 850.1735. (1996): Whole Sediment Acute Toxicity Invertebrates. (8) US-EPA/OPPTS 850.1790. (1996): Chironomid Sediment toxicity Test. (9) Milani D, Day KE, McLeay DJ, and Kirby RS (1996). Recent intra- and inter-laboratory studies related to the development and standardisation of Environment Canada’s biological test methods for measuring sediment toxicity using freshwater amphipods (Hyalella azteca) and midge larvae (Chironomus riparius). Technical Report. Environment Canada. National Water Research Institute. Burlington, Ontario, Canada. (10) Sugaya Y (1997). Intra-specific variations of the susceptibility of insecticides in Chironomus yoshimatsui. Jp. J. Sanit. Zool. 48 (4): 345-350. (11) Kawai K (1986). Fundamental studies on Chironomid allergy. I. Culture methods of some Japanese Chironomids (Chironomidae, Diptera). Jp. J. Sanit. Zool. 37(1): 47-57. (12) OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Environment, Health and Safety Publications, Series on Testing and Assessment No 23. (13) Environment Canada (1995). Guidance Document on Measurement of Toxicity Test Precision Using Control Sediments Spiked with a Reference Toxicant. Report EPS 1/RM/30. September 1995. (14) Test Method C.8 of this Annex, Toxicity for Earthworms. (15) Suedel BC and JH Rodgers (1994). Development of formulated reference sediments for freshwater and estuarine sediment testing. Environ. Toxicol. Chem. 13: 1163-1175. (16) Naylor C and C Rodrigues (1995). Development of a test method for Chironomus riparius using a formulated sediment. Chemosphere 31: 3291-3303. (17) Dunnett CW (1964). A multiple comparisons procedure for comparing several treatments with a control. J. Amer. Statis. Assoc., 50: 1096-1121. (18) Dunnett CW (1964). New tables for multiple comparisons with a control. Biometrics, 20: 482-491. (19) Williams DA (1971). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics, 27: 103-117. (20) Williams DA (1972). The comparison of several dose levels with a zero dose control. Biometrics, 28: 510-531. (21) Rao JNK and Scott AJ (1992). A simple method for the analysis of clustered binary data. Biometrics 48: 577-585. (22) Christensen ER (1984). Dose-response functions in aquatic toxicity testing and the Weibull model. Water Research 18: 213-221. (23) Bruce and Versteeg (1992). A statistical procedure for modelling continuous toxicity data. Environmental Toxicology and Chemistry 11: 1485-1494. (24) Slob W (2002). Dose-response modelling of continuous endpoints. Toxicol. Sci. 66: 298-312. Appendix 1 DEFINITIONS For the purpose of this Test Method the following definitions are used: Formulated sediment or reconstituted, artificial or synthetic sediment, is a mixture of materials used to mimic the physical components of a natural sediment. Overlying water is the water placed over sediment in the test vessel. Interstitial water or pore water is the water occupying space between sediment and soil particles. Spiked sediment is sediment to which test substance has been added. Test chemical: Any substance or mixture tested using this Test Method. Appendix 2 Recommendations for culture of Chironomus riparius 1. Chironomus larvae may be reared in crystallising dishes or larger containers. Fine quartz sand is spread in a thin layer of about 5 to 10 mm deep over the bottom of the container. Kieselguhr (e.g. Merck, Art 8117) has also been shown to be a suitable substrate (a thinner layer of up to a very few mm is sufficient). Suitable water is then added to a depth of several cm. Water levels should be topped up as necessary to replace evaporative loss, and prevent desiccation. Water can be replaced if necessary. Gentle aeration should be provided. The larval rearing vessels should be held in a suitable cage which will prevent escape of the emerging adults. The cage should be sufficiently large to allow swarming of emerged adults, otherwise copulation may not occur (minimum is ca. 30 × 30 × 30 cm). 2. Cages should be held at room temperature or in a constant environment room at 20 ± 2 °C with a photo period of 16 hour light (intensity ca. 1 000 lux), 8 hours dark. It has been reported that air humidity of less than 60 % RH can impede reproduction. Dilution water 3. Any suitable natural or synthetic water may be used. Well water, dechlorinated tap water and artificial media (e.g. Elendt “M4” or “M7” medium, see below) are commonly used. The water has to be aerated before use. If necessary, the culture water may be renewed by pouring or siphoning the used water from culture vessels carefully without destroying the tubes of larvae. Feeding larvae 4. Chironomus larvae should be fed with a fish flake food (TetraMin® TetraPhyll® or other similar brand of proprietary fish food), at approximately 250 mg per vessel per day. This can be given as a dry ground powder or as a suspension in water: 1,0 g of flake food is added to 20 ml of dilution water and blended to give a homogenous mix. This preparation may be fed at a rate of about 5 ml per vessel per day (shake before use). Older larvae may receive more. 5. Feeding is adjusted according to the water quality. If the culture medium becomes “cloudy”, the feeding should be reduced. Food additions must be carefully monitored. Too little food will cause emigration of the larvae towards the water column, and too much food will cause increased microbial activity and reduced oxygen concentrations. Both conditions can result in reduced growth rates. 6. Some green algae (e.g. Scenedesmus subspicatus, Chlorella vulgaris) cells may also be added when new culture vessels are set up. Feeding emerged adults 7. Some experimenters have suggested that a cotton wool pad soaked in a saturated sucrose solution may serve as a food for emerged adults. Emergence 8. At 20 ± 2 °C adults will begin to emerge from the larval rearing vessels after approximately 13-15 days. Males are easily distinguished by having plumose antennae. Egg masses 9. Once adults are present within the breeding cage, all larval rearing vessels should be checked three times weekly for deposition of the gelatinous egg masses. If present, the egg masses should be carefully removed. They should be transferred to a small dish containing a sample of the breeding water. Egg masses are used to start a new culture vessel (e.g. 2-4 egg masses/vessel) or are used for toxicity tests. 10. First instar larvae should hatch after 2-3 days. Set-up of new culture vessels 11. Once cultures are established it should be possible to set up a fresh larval culture vessel weekly or less frequently depending on testing requirements, removing the older vessels after adult midges have emerged. Using this system a regular supply of adults will be produced with a minimum of management. Preparation of test solutions “M4” and “M7” 12. Elendt (1990) has described the “M4” medium. The “M7” medium is prepared as the “M4” medium except for the substances indicated in Table 1, for which concentrations are four times lower in “M7” than in “M4”. A publication on the “M7” medium is in preparation (Elendt, personal communication). The test solution should not be prepared according to Elendt and Bias (1990) for the concentrations of NaSiO3 5 H2O, NaNO3, KH2PO4 and K2HPO4 given for the preparation of the stock solutions are not adequate. Preparation of the “M7”-medium 13. Each stock solution (I) is prepared individually and a combined stock solution (II) is prepared from these stock solutions (I) (see Table 1). Fifty ml from the combined stock Solution (II) and the amounts of each macro nutrient stock solution which are given in Table 2 are made up to 1 litre of deionised water to prepare the “M7” medium. A vitamin stock solution is prepared by adding three vitamins to deionised water as indicated in Table 3, and 0,1 ml of the combined vitamin stock solution are added to the final “M7” medium shortly before use. (The vitamin stock solution is stored frozen in small aliquots). The medium is aerated and stabilised. LITERATURE: BBA (1995). Long-term toxicity test with Chironomus riparius: Development and validation of a new test system. Edited by M. Streloke and H. Köpp. Berlin 1995. Table 1 Stock solutions of trace elements for medium M4 and M7 Stock solutions (I) Amount (mg) made up to 1 litre of deionised water To prepare the combined stock solution (II): mix the following amounts (ml) of stock solutions (I) and make up to 1 litre of deionised water Final concentrations in test solutions (mg/l) M4 M7 M4 M7 H3BO3 (15) 57 190 1,0 0,25 2,86 0,715 MnCl2 · 4 H2O (15) 7 210 1,0 0,25 0,361 0,090 LiCl (15) 6 120 1,0 0,25 0,306 0,077 RbCl (15) 1 420 1,0 0,25 0,071 0,018 SrCl2 · 6 H2O (15) 3 040 1,0 0,25 0,152 0,038 NaBr (15) 320 1,0 0,25 0,016 0,004 Na2MoO4 · 2 H2O (15) 1 260 1,0 0,25 0,063 0,016 CuCl2 · 2 H2O (15) 335 1,0 0,25 0,017 0,004 ZnCl2 260 1,0 1,0 0,013 0,013 CaCl2 · 6 H2O 200 1,0 1,0 0,010 0,010 KI 65 1,0 1,0 0,0033 0,0033 Na2SeO3 43,8 1,0 1,0 0,0022 0,0022 NH4VO3 11,5 1,0 1,0 0,00058 0,00058 Na2EDTA · 2 H2O (15) (16) 5 000 20,0 5,0 2,5 0,625 FeSO4 · 7 H2O (15) (16) 1 991 20,0 5,0 1,0 0,249

Table 2 Macro nutrient stock solutions for medium M4 and M7 Amount made up to 1 litre of deionised water (mg) Amount of macro nutrient stock solutions added to prepare medium M4 and M7 (ml/l) Final concentrations in test solutions M4 and M7 (mg/l) CaCl2 · 2 H2O 293 800 1,0 293,8 MgSO4 · 7 H2O 246 600 0,5 123,3 KCl 58 000 0,1 5,8 NaHCO3 64 800 1,0 64,8 NaSiO3 · 9 H2O 50 000 0,2 10,0 NaNO3 2 740 0,1 0,274 KH2PO4 1 430 0,1 0,143 K2HPO4 1 840 0,1 0,184

Table 3 Vitamin stock solution for medium M4 and M7. All three vitamin solutions are combined to make a single vitamin stock solution Amount made up to 1 litre of deionised water (mg) Amount of vitamin stock solution added to prepare medium M4 and M7 (ml/l) Final concentrations in test solutions M4 and M7 (mg/l) Thiamine hydrochloride 750 0,1 0,075 Cyanocobalamin (B12) 10 0,1 0,0010 Biotine 7,5 0,1 0,00075 LITERATURE: Elendt, B.P. (1990). Selenium Deficiency in Crustacean. Protoplasma 154: 25-33. Elendt, B.P. & W.-R. Bias (1990). Trace Nutrient Deficiency in Daphnia magna Cultured in Standard Medium for Toxicity Testing. Effects on the Optimization of Culture Conditions on Life History Parameters of D. magna. Water Research 24 (9): 1157-1167. Appendix 3 PREPARATION OF FORMULATED SEDIMENT Sediment composition The composition of the formulated sediment should be as follows: Constituent Characteristics % of sediment dry weight Peat Sphagnum moss peat, as close to pH 5,5-6,0 as possible, no visible plant remains, finely ground (particle size ≤ 1 mm) and air dried 4-5 Quartz sand Grain size: > 50 % of the particles should be in the range of 50-200 μm 75-76 Kaolinite clay Kaolinite content ≥ 30 % 20 Organic carbon Adjusted by addition of peat and sand 2 (± 0,5) Calcium carbonate CaCO3, pulverised, chemically pure 0,05-0,1 Water Conductivity ≤ 10 μS/cm 30-50 Preparation The peat is air dried and ground to a fine powder. A suspension of the required amount of peat powder in deionised water is prepared using a high-performance homogenising device. The pH of this suspension is adjusted to 5,5 ± 0,5 with CaCO3. The suspension is conditioned for at least two days with gentle stirring at 20 ± 2 °C, to stabilise pH and establish a stable microbial component. pH is measured again and should be 6,0 ± 0,5. Then the peat suspension is mixed with the other constituents (sand and kaolin clay) and deionised water to obtain a homogeneous sediment with a water content in a range of 30-50 per cent of dry weight of the sediment. The pH of the final mixture is measured once again and is adjusted to 6,5 to 7,5 with CaCO3 if necessary. Samples of the sediment are taken to determine the dry weight and the organic carbon content. Then, before it is used in the chironomid toxicity test, it is recommended that the formulated sediment be conditioned for seven days under the same conditions which prevail in the subsequent test. Storage The dry constituents for preparation of the artificial sediment may be stored in a dry and cool place at room temperature. The formulated (wet) sediment should not be stored prior to its use in the test. It should be used immediately after the 7 days conditioning period that ends its preparation. LITERATURE: Chapter C.8 of this Annex. Toxicity for Earthworms. Meller M, Egeler P, Rombke J, Schallnass H, Nagel R, Streit B (1998). Short-term Toxicity of Lindane, Hexachlorobenzene and Copper Sulfate on Tubificid Sludgeworms (Oligochaeta) in Artificial Media. Ecotox. and Environ. Safety 39: 10-20. Appendix 4 Chemical Characteristics of an Acceptable Dilution Water Substance Concentrations Particulate matter < 20 mg/l Total organic carbon < 2 mg/l Unionised ammonia < 1 μg/l Hardness as CaCO3 < 400 mg/l (17) Residual chlorine < 10 μg/l Total organophosphorus pesticides < 50 ng/l Total organochlorine pesticides plus polychlorinated biphenyls < 50 ng/l Total organic chlorine < 25 ng/l Appendix 5 Guidance for monitoring emergence of chironomid larvae Emergence traps are placed on the test beakers. These traps are needed from day 20 to the end of the test. Example of trap used is drawn below: A: the nylon screen B: the inverted plastic cups C: the lipless exposure beaker D: the water exchange screen ports E: water F: sediment C. 28. SEDIMENT-WATER CHIRONOMID TOXICITY TEST USING SPIKED WATER INTRODUCTION 1. This Test Method is equivalent to OECD TG 219 (2004). This Test Method is designed to assess the effects of prolonged exposure of chemicals to the sediment-dwelling larvae of the freshwater dipteran Chironomus sp. It is mainly based on the BBA guideline using a sediment-water test system with artificial soil, and water column exposure scenario (1). It also takes into account existing toxicity test protocols for Chironomus riparius and Chironomus tentans which have been developed in Europe and North America (2)(3)(4)(5)(6)(7)(8) and ring-tested (1)(6)(9). Other well documented chironomid species may also be used, e.g. Chironomus yoshimatsui (10)(11). 2. The exposure scenario used in this Test Method is water spiking. The selection of the appropriate exposure scenario depends on the intended application of the test. The water exposure scenario, involving spiking of the water column, is intended to simulate a pesticide spray drift event and covers the initial peak of concentrations in pore water. It is also useful for other types of exposure (including chemical spills) except accumulation processes lasting longer than the test period. 3. Substances that need to be tested towards sediment-dwelling organisms usually persist in this compartment over long time periods. The sediment-dwelling organisms may be exposed via a number of routes. The relative importance of each exposure route, and the time taken for each to contribute to the overall toxic effects, is dependent on the physical-chemical properties of the chemical concerned. For strongly adsorbing substances (e.g. with log Kow > 5) or for substances covalently binding to sediment, ingestion of contaminated food may be a significant exposure route. In order not to underestimate the toxicity of highly lipophilic substances, the use of food added to the sediment before application of the test substance may be considered. In order to take all potential routes of exposure into account the focus of this Test Method is on long-term exposure. The test duration is in the range of 20-28 days for C. riparius and C. yoshimatsui, and 28-65 days for C. tentans. If short-term data are required for a specific purpose, for example to investigate the effects of unstable chemicals, additional replicates may be removed after a 10-day period. 4. The measured endpoints are the total number of adults emerged and the time to emergence. It is recommended that measurements of larval survival and growth should only be made after a 10-day period if additional short-term data are required, using additional replicates as appropriate. 5. The use of formulated sediment is recommended. Formulated sediment has several advantages over natural sediments: — the experimental variability is reduced because it forms a reproducible “standardised matrix” and the need to find uncontaminated and clean sediment sources is eliminated; — the tests can be initiated at any time without encountering seasonal variability in the test sediment and there is no need to pre-treat the sediment to remove indigenous fauna; the use of formulated sediment also reduces the cost associated with the field collection of sufficient amounts of sediment for routine testing; — the use of formulated sediment allows for comparisons of toxicity and ranking substances accordingly: toxicity data from tests with natural and artificial sediments were comparable for several chemicals (2). 6. Definitions used are given in Appendix 1. PRINCIPLE OF THE TEST 7. First instar chironomid larvae are exposed to a concentration range of the test substance in sediment-water systems. The test starts by placing first instar larvae into the test beakers containing the sediment-water system and subsequently spiking the test substance into the water. Chironomid emergence and development rate is measured at the end of the test. Larval survival and weight may also be measured after 10 days if required (using additional replicates as appropriate). These data are analysed either by using a regression model in order to estimate the concentration that would cause x % reduction in emergence, larvae survival or growth (e.g. EC15, EC50, etc.), or by using statistical hypothesis testing to determine a NOEC/LOEC. The latter requires comparison of effect values with control values using statistical tests. INFORMATION ON THE TEST SUBSTANCE 8. The water solubility of the test substance, its vapour pressure, measured or calculated partitioning into sediment and stability in water and sediment should be known. A reliable analytical method for the quantification of the test substance in overlying water, pore water and sediment with known and reported accuracy and limit of detection should be available. Useful information includes the structural formula and purity of the test substance. Chemical fate of the test substance (e.g. dissipation, abiotic and biotic degradation, etc.) also is useful information. Further guidance for testing substances with physical-chemical properties that make them difficult to perform the test is provided in (12). REFERENCE CHEMICALS 9. Reference chemicals may be tested periodically as a means of assuring that the test protocol and test conditions are reliable. Examples of reference toxicants used successfully in ring-tests and validation studies are: lindane, trifluralin, pentachlorophenol, cadmium chloride and potassium chloride. (1)(2)(5)(6)(13). VALIDITY OF THE TEST 10. For the test to be valid the following conditions apply: — the emergence in the controls must be at least 70 % at the end of the test. (1)(6); — C. riparius and C. yoshimatsui emergence to adults from control vessels should occur between 12 and 23 days after their insertion into the vessels; for C. tentans, a period of 20 to 65 days is necessary. — at the end of the test, pH and the dissolved oxygen concentration should be measured in each vessel. The oxygen concentration should be at least 60 % of the air saturation value (ASV) at the temperature used, and the pH of overlying water should be in the 6-9 range in all test vessels; — the water temperature should not differ by more than ± 1,0 °C. The water temperature could be controlled by isothermal room and in that case the room temperature should be confirmed in an appropriate time intervals. DESCRIPTION OF THE METHOD Test vessels 11. The study is conducted in glass 600 ml beakers measuring 8 cm in diameter. Other vessels are suitable, but they should guarantee a suitable depth of overlying water and sediment. The sediment surface should be sufficient to provide 2 to 3 cm2 per larvae. The ratio of the depth of the sediment layer to the depth of the overlying water should be 1:4. Test vessels and other apparatus that will come into contact with the test system should be made entirely of glass or other chemically inert material (e.g. Teflon). Selection of species 12. The species to be used in the test is preferably Chironomus riparius. Chironomus tentans is also suitable but more difficult to handle and requires a longer test period. Chironomus yohimatsui may also be used. Details of culture methods are given in Appendix 2 for Chironomus riparius. Information on culture conditions is also available for other species, i.e. Chironomus tentans (4) and Chironomus yoshimatsui (11). Identification of species must be confirmed before testing but is not required prior to every test if organisms come from an in-house culture. Sediment 13. Formulated sediment (also called reconstituted, artificial or synthetic sediment) should preferably be used. However, if natural sediment is used, it should be characterised (at least pH, organic carbon content, determination of other parameters such as C/N ratio and granulometry are also recommended), and it should be free from any contamination and other organisms that might compete with, or consume the chironomids. It is also recommended that, before it is used in a chironomid toxicity test, the natural sediment be conditioned for seven days under the same conditions which prevail in the subsequent test. The following formulated sediment, based on the artificial soil used in Test Method C.8 (14), is recommended for use in this test (1)(15)(16): a) 4-5 % (dry weight) peat: as close to pH 5,5 to 6,0 as possible; it is important to use peat in powder form, finely ground (particle size ≤ 1 mm) and only air dried. b) 20 % (dry weight) kaolin clay (kaolinite content preferably above 30 %). c) 75-76 % (dry weight) quartz sand (fine sand should predominate with more than 50 % of the particles between 50 and 200 μm). d) Deionised water is added to obtain moisture of the final mixture in a range of 30-50 %. e) Calcium carbonate of chemically pure quality (CaCO3) is added adjust the pH of the final mixture of the sediment to 7,0 ± 0,5. f) Organic carbon content of the final mixture should be 2 % (± 0,5 %) and is to be adjusted by the use of appropriate amounts of peat and sand, according to (a) and (c). 14. The source of peat, kaolin clay and sand should be known. The sediment components should be checked for the absence of chemical contamination (e.g. heavy metals, organochlorine compounds, organophosphorous compounds, etc.). An example for the preparation of the formulated sediment is described in Appendix 3. Mixing of dry constituents is also acceptable if it is demonstrated that after addition of overlying water a separation of sediment constituents (e.g. floating of peat particles) does not occur, and that the peat or the sediment is sufficiently conditioned. Water 15. Any water which conforms to the chemical characteristics of acceptable dilution water as listed in Appendices 2 and 4 is suitable as test water. Any suitable water, natural water (surface or ground water), reconstituted water (see Appendix 2) or dechlorinated tap water are acceptable as culturing water and test water if chironomids will survive in it for the duration of the culturing and testing without showing signs of stress. At the start of the test, the pH of the test water should be between 6 and 9 and the total hardness not higher than 400 mg/l as CaCO3. However, if there is an interaction suspected between hardness ions and the test substance, lower hardness water should be used (and thus, Elendt Medium M4 must not be used in this situation). The same type of water should be used throughout the whole study. The water quality characteristics listed in Appendix 4 should be measured at least twice a year or when it is suspected that these characteristics may have changed significantly. Stock solutions — Spiked water 16. Test concentrations are calculated on the basis of water column concentrations, i.e. the water overlying the sediment. Test solutions of the chosen concentrations are usually prepared by dilution of a stock solution. Stock solutions should preferably be prepared by dissolving the test substance in test medium. The use of solvents or dispersants may be required in some cases in order to produce a suitably concentrated stock solution. Examples of suitable solvents are acetone, ethanol, methanol, ethylene glycol monoethyl ether, ethylene glycol dimethyl ether, dimethylformamide and triethylene glycol. Dispersants which may be used are Cremophor RH40, Tween 80, methylcellulose 0,01 % and HCO-40. The solubilising agent concentration in the final test medium should be minimal (i.e. ≤ 0,1 ml/l) and should be the same in all treatments. When a solubilising agent is used, it must have no significant effects on survival or no visible adverse effect on the chironomid larvae as revealed by a solvent-only control. However, every effort should be made to avoid the use of such materials. TEST DESIGN 17. The test design relates to the selection of the number and spacing of the test concentrations, the number of vessels at each concentration and the number of larvae per vessel. Designs for EC point estimation, for estimation of NOEC, and for conducting a limit test are described. The analysis by regression is preferred to the hypothesis testing approach. Design for analysis by regression 18. The effect concentration (e.g. EC15, EC50) and the concentration range, over which the effect of the test substance is of interest, should be spanned by the concentrations included in the test. Generally, the accuracy and especially validity, with which estimates of effect concentrations (ECx) can be made, is improved when the effect concentration is within the range of concentrations tested. Extrapolation much below the lowest positive concentration or above the highest concentration should be avoided. A preliminary range-finding test is helpful for selecting the range of concentrations to be used (see paragraph 27). 19. If the ECx is to be estimated, at least five concentrations and three replicates for each concentration should be tested. In any case, it is advisable that sufficient test concentrations are used to allow a good model estimation. The factor between concentrations should not be greater than two (an exception could be made in cases when the dose response curve has a shallow slope). The number of replicates at each treatment can be reduced if the number of test concentrations with different responses is increased. Increasing the number of replicates or reducing the size of the test concentration intervals tends to lead to narrower confidence intervals for the test. Additional replicates are required if 10-day larval survival and growth are to be estimated. Design for estimation of a NOEC/LOEC 20. If the LOEC/NOEC are to be estimated, five test concentrations with at least four replicates should be used and the factor between concentrations should not be greater than two. The number of replicates should be sufficient to ensure adequate statistical power to detect a 20 % difference from the control at the 5 % level of significance (p = 0,05). With the development rate, an Analysis of Variance (ANOVA) is usually appropriate, such as Dunnett-test and Williams-test (17)(18)(19)(20). In the emergence ratio the Cochran-Armitage, Fisher’s exact (with Bonferroni correction), or Mantel-Haenszel tests may be used. Limit test 21. A limit test may be performed (one test concentration and control) if no effects were seen in the preliminary range-finding test. The purpose of the limit test is to indicate that the toxic value of the test substance is greater than the limit concentration tested. No suggestion for a recommended concentration can be made in this Test Method; this is left to the regulators’ judgement. Usually, at least six replicates for both the treatment and control are necessary. Adequate statistical power to detect a 20 % difference from the control at the 5 % level of significance (p = 0,05) should be demonstrated. With metric response (development rate and weight), the t-test is a suitable statistical method if data meet the requirements of this test (normality, homogeneous variances). The unequal-variance t-test or a non parametric test, such as the Wilcoxon-Mann-Whithey test may be used, if these requirements are not fulfilled. With the emergence ratio, the Fisher exact test is appropriate. PROCEDURE Conditions of exposure Preparation of spiked water-sediment system 22. Appropriate amounts of formulated sediment (see paragraphs 13-14 and Appendix 3) are added in the test vessels to form a layer of at least 1,5 cm. Water is added to a depth of 6 cm (see paragraph 15). The ratio of the depth of the sediment layer and the depth of the water should not exceed 1:4 and the sediment layer should not be deeper than 3 cm. The sediment-water system should be left under gentle aeration for seven days prior to addition of test organisms (see paragraph 14 and Appendix 3). To avoid separation of sediment ingredients and re-suspension of fine material during addition of test water in the water column, the sediment can be covered with a plastic disc while water is poured onto it, and the disc is removed immediately afterwards. Other devices may also be appropriate. 23. The test vessels should be covered (e.g. by glass plates). If necessary, during the study the water levels will be topped to the original volume in order to compensate for water evaporation. This should be performed using distilled or deionised water to prevent build-up of salts. Addition of test organisms 24. Four to five days before adding the test organisms to the test vessels, egg masses should be taken from the cultures and placed in small vessels in culture medium. Aged medium from the stock culture or freshly prepared medium may be used. If the latter is used, a small amount of food e.g. green algae and/or a few droplets of filtrate from a finely ground suspension of flaked fish food should be added to the culture medium (see Appendix 2). Only freshly laid egg masses should be used. Normally, the larvae begin to hatch a couple of days after the eggs are laid (2 to 3 days for Chironomus riparius at 20 °C and 1 to 4 days for Chironomus tentans at 23 °C and Chironomus yoshimatui at 25 °C) and larval growth occurs in four instars, each of 4-8 days duration. First instar larvae (2-3 or 1-4 days post hatching) should be used in the test. The instar of midges can possibly be checked using head capsule width (6). 25. Twenty first instar larvae are allocated randomly to each test vessel containing the spiked sediment and water, using a blunt pipette. Aeration of the water has to be stopped while adding the larvae to test vessels and remain so for another 24 hours after addition of larvae (see paragraphs 24 and 32). According to the test design used (see paragraphs 19 and 20), the number of larvae used per concentration is at least 60 for the EC point estimation and 80 for determination of NOEC. 26. Twenty-four hours after adding the larvae, the test substance is spiked into the overlying water column, and slight aeration is again supplied. Small volumes of test substance solutions are applied below the surface of the water using a pipette. The overlying water should then be mixed with care not to disturb the sediment. Test concentrations 27. A range-finding test may be helpful to determine the range of concentrations for the definitive test. For this purpose a series of widely spaced concentrations of the test substance are used. In order to provide the same density of surface per chironomids, which is to be used for the definitive test, chironomids are exposed to each concentration of the test substance for a period which allows estimation of appropriate test concentrations, and no replicates are required. 28. The test concentrations for the definitive test are decided based on the result of the range-finding test. At least five concentrations should be used and selected as described in paragraphs 18 to 20. Controls 29. Control vessels without any test substance but including sediment should be included in the test with the appropriate number of replicates (see paragraphs 19-20). If a solvent has been used for application of test substance (see paragraph 16), a sediment solvent control should be added. Test system 30. Static systems are used. Semi-static or flow-through systems with intermittent or continuous renewal of overlying water might be used in exceptional cases as for instance if water quality specifications become inappropriate for the test organism or affect chemical equilibrium (e.g. dissolved oxygen levels fall too low, the concentration of excretory products rises too high or minerals leach from sediment and affect pH and/or water hardness). However, other methods for ameliorating the quality of overlying water, such as aeration, will normally suffice and be preferable. Food 31. It is necessary to feed the larvae, preferably daily or at least three times per week. Fish-food (a suspension in water or finely ground food, e.g. TetraMin or TetraPhyll; see details in Appendix 2) in the amount of 0,25-0,5 mg (0,35-0,5 mg for C. yoshimatui) per larvae per day seems adequate for young larvae for the first 10 days. Slightly more food may be necessary for older larvae: 0,5-1 mg per larvae per day should be sufficient for the rest of the test. The food ration should be reduced in all treatments and control if fungal growth is seen or if mortality is observed in controls. If fungal development cannot be stopped the test is to be repeated. When testing strongly adsorbing substances (e.g. with log Kow > 5), or substances covalently binding to sediment, the amount of food necessary to ensure survival and natural growth of the organisms may be added to the formulated sediment before the stabilisation period. For this, plant material must be used instead of fish food, e.g. addition of 0,5 % (dry weight) finely ground leaves of e.g. stinging nettle (Urtica dioica), mulberry (Morus alba), white clover (Trifolium repens), spinach (Spinacia oleracea) or of other plant material (Cerophyl or alpha-cellulose) may be used. Incubation conditions 32. Gentle aeration of the overlying water in test vessels is supplied preferably 24 hours after addition of the larvae and is pursued throughout the test (care should be taken that dissolved oxygen concentration does not fall below 60 %of ASV). Aeration is provided through a glass Pasteur pipette fixed 2-3 cm above the sediment layer (i.e. one or few bubbles/sec). When testing volatile chemicals, consideration may be given not to aerate the sediment-water system. 33. The test is conducted at a constant temperature of 20 °C (± 2 °C). For C. tentans and C. yoshimatui, recommended temperatures are of 23 °C and 25 °C (± 2 °C), respectively. A 16 hours photoperiod is used and the light intensity should be 500 to 1 000 lux. Exposure duration 34. The exposure commences with the addition of larvae to the spiked and control vessels. The maximum exposure duration is 28 days for C. riparius and C. yoshimatsui, and 65 days for C. tentans. If midges emerge earlier, the test can be terminated after a minimum of five days after emergence of the last adult in the control. OBSERVATIONS Emergence 35. The development time and the total number of fully emerged male and female midges are determined. Males are easily identified by their plumose antennae. 36. The test vessels should be observed at least three times per week to make visual assessment of any abnormal behaviour (e.g. leaving sediment, unusual swimming), compared with the control. During the period of expected emergence a daily count of emerged midges is necessary. The sex and number of fully emerged midges are recorded daily. After identification the midges are removed from the vessels. Any egg masses deposited prior to the termination of the test should be recorded and then removed to prevent re-introduction of larvae into the sediment. The number of visible pupae that have failed to emerge is also recorded. Guidance on measurement of emergence is provided in Appendix 5. Growth and survival 37. If data on 10-day larval survival and growth are to be provided, additional test vessels should be included at the start, so that they may be used subsequently. The sediment from these additional vessels is sieved using a 250 μm sieve to retain the larvae. Criteria for death are immobility or lack of reaction to a mechanical stimulus. Larvae not recovered should also be counted as dead (larvae which have died at beginning of the test may have been degraded by microbes). The (ash free) dry weight of the surviving larvae per test vessel is determined and the mean individual dry weight per vessel calculated. It is useful to determine which instar the surviving larvae belong to; for that measurement of the width of the head capsule of each individual can be used. Analytical measurements Concentration of the test substance 38. As a minimum, samples of the overlying water, the pore water and the sediment must be analysed at the start (preferably one hour after application of test substance) and at the end of the test, at the highest concentration and a lower one. These determinations of test substance concentration inform on the behaviour/partitioning of the test substance in the water-sediment system. Sampling of sediment at the start of the test may influence the test system (e.g. removing test larvae), thus additional test vessels should be used to perform analytical determinations at the start and during the test if appropriate (see paragraph 39). Measurements in sediment might not be necessary if the partitioning of the test substance between water and sediment has been clearly determined in a water/sediment study under comparable conditions (e.g. sediment to water ratio, type of application, organic carbon content of sediment). 39. When intermediate measurements are made (e.g. at day 7) and if the analysis needs large samples which cannot be taken from test vessels without influencing the test system, analytical determinations should be performed on samples from additional test vessels treated in the same way (including the presence of test organisms) but not used for biological observations. 40. Centrifugation at e.g. 10 000 g and 4 °C for 30 min. is the recommended procedure to isolate interstitial water. However, if the test substance is demonstrated not to adsorb to filters, filtration may also be acceptable. In some cases it might not be possible to analyse concentrations in the pore water as the sample size is too small. Physical-chemical parameters 41. The pH, dissolved oxygen in the test water and temperature of the test vessels should be measured in an appropriate manner (see paragraph 10). Hardness and ammonia should be measured in the controls and one test vessel at the highest concentration at the start and the end of the test. DATA AND REPORTING Treatment of results 42. The purpose of this test is to determine the effect of the test substance on the development rate and the total number of fully emerged male and female midges, or in the case of the 10-day test effects on survival and weight of the larvae. If there are no indications of statistically different sensitivities of sexes, male and female results may be pooled for statistical analyses. The sensitivity differences between sexes can be statistically judged by e.g. a χ2-r × 2 table test. Larval survival and mean individual dry weight per vessel must be determined after 10 days where required. 43. Effect concentrations expressed as concentrations in the overlaying water, are calculated preferably based on measured concentrations at the beginning of the test (see paragraph 38). 44. To compute a point estimate for the EC50 or any other ECx, the per-vessel statistics may be used as true replicates. In calculating a confidence interval for any ECx the variability among vessels should be taken into account, or it should be shown that this variability is so small that it can be ignored. When the model is fitted by Least Squares, a transformation should be applied to the per-vessel statistics in order to improve the homogeneity of variance. However, ECx values should be calculated after the response is transformed back to the original value. 45. When the statistical analysis aims at determining the NOEC/LOEC by hypothesis testing, the variability among vessels needs to be taken into account, e.g. by a nested ANOVA. Alternatively, more robust tests (21) can be appropriate in situations where there are violations of the usual ANOVA assumptions. Emergence ratio 46. Emergence ratios are quantal data, and can be analyzed by the Cochran-Armitage test applied in step-down manner where a monotonic dose-response is expected and these data are consistent with this expectation. If not, a Fisher’s exact or Mantel-Haenszal test with Bonferroni-Holm adjusted p-values can be used. If there is evidence of greater variability between replicates within the same concentration than a binomial distribution would indicate (often referenced as “extra-binomial” variation), then a robust Cochran-Armitage or Fisher exact test such as proposed in (21), should be used. 47. The sum of midges emerged per vessel, ne, is determined and divided by the number of larvae introduced, na: where: ER = emergence ratio ne = number of midges emerged per vessel na = number of larvae introduced per vessel 48. An alternative that is most appropriate for large sample sizes, when there is extra binomial variance, is to treat the emergence ratio as a continuous response and use procedures such as William’s test when a monotonic dose-response is expected and is consistent with these ER data. Dunnett’s test would be appropriate where monotonicity does not hold. A large sample size is defined here as the number emerged and the number not emerging both exceeding five, on a per replicate (vessel) basis. 49. To apply ANOVA methods values of ER should first be transformed by the arcsin square roottransformation or Freeman-Tukey transformation to obtain an approximate normal distribution and to equalise variances. The Cochran-Armitage, Fisher’s exact (Bonferroni), or Mantel-Haenszel tests can be applied when using the absolute frequencies. The arcsin square root transformation is applied by taking the inverse sine (sine–1) of the square root of ER. 50. For emergence ratios, ECx-values are calculated using regression analysis (or e.g. probit (22), logit, Weibull, appropriate commercial software etc.). If regression analysis fails (e.g. when there are less than two partial responses), other non-parametric methods such as moving average or simple interpolation are used. Development rate 51. The mean development time represents the mean time span between the introduction of larvae (day 0 of the test) and the emergence of the experimental cohort of midges. (For the calculation of the true development time, the age of larvae at the time of introduction should be considered). The development rate is the reciprocal of the development time (unit: 1/day) and represents that portion of larval development which takes place per day. The development rate is preferred for the evaluation of these sediment toxicity studies as its variance is lower, and it is more homogeneous and closer to normal distribution as compared to development time. Hence, powerful parametric test procedures may be used with development rate rather than with development time. For development rate as a continuous response, ECx-values can be estimated by using regression analysis (e.g. (23)(24)). 52. For the following statistical tests, the number of midges observed on inspection day x are assumed to be emerged at the mean of the time interval between day x and day x – l (l = length of the inspection interval, usually 1 day). The mean development rate per vessel (x) is calculated according to: where: : mean development rate per vessel i : index of inspection interval m : maximum number of inspection intervals : number of midges emerged in the inspection interval i ne : total number of midges emerged at the end of experiment (= ) xi : development rate of the midges emerged in interval i where: dayi : inspection day (days since application) li : length of inspection interval i (days, usually 1 day) Test report 53. The test report must at least provide the following information: Test substance: — physical nature and, where relevant, physical-chemical properties (water solubility, vapour pressure, partition coefficient in soil (or in sediment if available), stability in water, etc.); — chemical identification data (common name, chemical name, structural formula, CAS number, etc.) including purity and analytical method for quantification of test substance. Test species: — test animals used: species, scientific name, source of organisms and breeding conditions; — information on handling of egg masses and larvae; — age of test animals when inserted into test vessels. Test conditions: — sediment used, i.e. natural or formulated sediment; — for natural sediment, location and description of sediment sampling site, including, if possible, contamination history; characteristics: pH, organic carbon content, C/N ratio and granulometry (if appropriate). — preparation of the formulated sediment: ingredients and characteristics (organic carbon content, pH, moisture, etc. at the start of the test); — preparation of the test water (if reconstituted water is used) and characteristics (oxygen concentration, pH, conductivity, hardness, etc. at the start of the test); — depth of sediment and overlying water; — volume of overlying and pore water; weight of wet sediment with and without pore water; — test vessels (material and size); — method of preparation of stock solutions and test concentrations; — application of test substance: test concentrations used, number of replicates and use of solvent if any; — incubation conditions: temperature, light cycle and intensity, aeration (frequency and intensity); — detailed information on feeding including type of food, preparation, amount and feeding regime. Results: — the nominal test concentrations, the measured test concentrations and the results of all analyses to determine the concentration of the test substance in the test vessel; — water quality within the test vessels, i.e. pH, temperature, dissolved oxygen, hardness and ammonia; — replacement of evaporated test water, if any; — number of emerged male and female midges per vessel and per day; — number of larvae which failed to emerge as midges per vessel; — mean individual dry weight of larvae per vessel, and per instar, if appropriate; — percent emergence per replicate and test concentration (male and female midges pooled); — mean development rate of fully emerged midges per replicate and treatment rate (male and female midges pooled); — estimates of toxic endpoints e.g. ECx (and associated confidence intervals), NOEC and/or LOEC, and the statistical methods used for their determination; — discussion of the results, including any influence on the outcome of the test resulting from deviations from this Test Method. LITERATURE: (1) BBA (1995). Long-term toxicity test with Chironomus riparius: Development and validation of a new test system. Edited by M. Streloke and H. Köpp. Berlin 1995. (2) Fleming R et al. (1994). Sediment Toxicity Tests for Poorly Water-Soluble Substances. Final Report to them European Commission. Report No: EC 3738. August 1994. WRc, UK. (3) SETAC (1993). Guidance Document on Sediment toxicity Tests and Bioassays for Freshwater and Marine Environments. From the WOSTA Workshop held in the Netherlands. (4) ASTM International/E1706-00 (2002). Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates. pp 1125-1241. In ASTM International 2002 Annual Book of Standards. Volume 11.05. Biological Effects and Environmental Fate; Biotechnology; Pesticides. ASTM International, West Conshohocken, PA. (5) Environment Canada (1997). Test for Growth and Survival in Sediment using Larvae of Freshwater Midges (Chironomus tentans or Chironomus riparius). Biological Test Method. Report SPE 1/RM/32. December 1997. (6) US-EPA (2000). Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates. Second edition. EPA 600/R-99/064. March 2000. Revision to the first edition dated June 1994. (7) US-EPA/OPPTS 850.1735. (1996): Whole Sediment Acute Toxicity Invertebrates. (8) US-EPA/OPPTS 850.1790. (1996): Chironomid Sediment toxicity Test. (9) Milani D, Day KE, McLeay DJ, Kirby RS (1996). Recent intra- and inter-laboratory studies related to the development and standardisation of Environment Canada’s biological test methods for measuring sediment toxicity using freshwater amphipods (Hyalella azteca) and midge larvae (Chironomus riparius). Technical Report. Environment Canada. National Water Research Institute. Burlington, Ontario, Canada. (10) Sugaya Y (1997). Intra-specific variations of the susceptibility of insecticides in Chironomus yoshimatsui. Jp. J. Sanit. Zool. 48 (4): 345-350. (11) Kawai K (1986). Fundamental studies on Chironomid allergy. I. Culture methods of some Japanese Chironomids (Chironomidae, Diptera). Jp. J. Sanit. Zool. 37(1): 47-57. (12) OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Environment, Health and Safety Publications, Series on Testing and Assessment No 23. (13) Environment Canada (1995). Guidance Document on Measurement of Toxicity Test Precision Using Control Sediments Spiked with a Reference Toxicant. Report EPS 1/RM/30. September 1995. (14) Chapter C.8 of this Annex, Toxicity for Earthworms, (15) Suedel BC and Rodgers JH (1994). Development of formulated reference sediments for freshwater and estuarine sediment testing. Environ. Toxicol. Chem. 13: 1163-1175. (16) Naylor C and Rodrigues C (1995). Development of a test method for Chironomus riparius using a formulated sediment. Chemosphere 31: 3291-3303. (17) Dunnett CW (1964). A multiple comparisons procedure for comparing several treatments with a control. J. Amer. Statis. Assoc. 50: 1096-1121. (18) Dunnett CW (1964). New tables for multiple comparisons with a control. Biometrics 20: 482-491. (19) Williams DA (1971). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27: 103-117. (20) Williams DA (1972). The comparison of several dose levels with a zero dose control. Biometrics 28: 510-531. (21) Rao JNK and Scott AJ (1992). A simple method for the analysis of clustered binary data. Biometrics 48: 577-585. (22) Christensen ER (1984). Dose-response functions in aquatic toxicity testing and the Weibull model. Water Research 18: 213-221. (23) Bruce and Versteeg (1992). A statistical procedure for modelling continuous toxicity data. Environmental Toxicology and Chemistry 11:1485-1494. (24) Slob W (2002). Dose-response modelling of continuous endpoints. Toxicol. Sci. 66: 298-312. Appendix 1 DEFINITIONS For the purpose of this method the following definitions are used: Formulated sediment or reconstituted, artificial or synthetic sediment, is a mixture of materials used to mimic the physical components of a natural sediment. Overlying water is the water placed over sediment in the test vessel. Interstitial water or pore water is the water occupying space between sediment and soil particles. Spiked water is the test water to which test substance has been added. Test chemical: Any substance or mixture tested using this Test Method. Appendix 2 Recommendations for culture of Chironomus riparius 1. Chironomus larvae may be reared in crystallising dishes or larger containers. Fine quartz sand is spread in a thin layer of about 5 to 10 mm deep over the bottom of the container. Kieselguhr (e.g. Merck, Art 8117) has also been shown to be a suitable substrate (a thinner layer of up to a very few mm is sufficient). Suitable water is then added to a depth of several cm. Water levels should be topped up as necessary to replace evaporative loss, and prevent desiccation. Water can be replaced if necessary. Gentle aeration should be provided. The larval rearing vessels should be held in a suitable cage which will prevent escape of the emerging adults. The cage should be sufficiently large to allow swarming of emerged adults, otherwise copulation may not occur (minimum is ca. 30 × 30 × 30 cm). 2. Cages should be held at room temperature or in a constant environment room at 20 ± 2 °C with a photo period of 16 hour light (intensity ca. 1 000 lux), 8 hours dark. It has been reported that air humidity of less than 60 % RH can impede reproduction. Dilution water 3. Any suitable natural or synthetic water may be used. Well water, dechlorinated tap water and artificial media (e.g. Elendt “M4” or “M7” medium, see below) are commonly used. The water has to be aerated before use. If necessary, the culture water may be renewed by pouring or siphoning the used water from culture vessels carefully without destroying the tubes of larvae. Feeding larvae 4. Chironomus larvae should be fed with a fish flake food (TetraMin®, TetraPhyll® or other similar brand of proprietary fish food), at approximately 250 mg per vessel per day. This can be given as a dry ground powder or as a suspension in water: 1,0 g of flake food is added to 20 ml of dilution water and blended to give a homogenous mix. This preparation may be fed at a rate of about 5 ml per vessel per day (shake before use.) Older larvae may receive more. 5. Feeding is adjusted according to the water quality. If the culture medium becomes “cloudy”, the feeding should be reduced. Food additions must be carefully monitored. Too little food will cause emigration of the larvae towards the water column, and too much food will cause increased microbial activity and reduced oxygen concentrations. Both conditions can result in reduced growth rates. 6. Some green algae (e.g. Scenedesmus subspicatus, Chlorella vulgaris) cells may also be added when new culture vessels are set up. Feeding emerged adults 7. Some experimenters have suggested that a cotton wool pad soaked in a saturated sucrose solution may serve as a food for emerged adults. Emergence 8. At 20 ± 2 °C adults will begin to emerge from the larval rearing vessels after approximately 13-15 days. Males are easily distinguished by having plumose antennae. Egg masses 9. Once adults are present within the breeding cage, all larval rearing vessels should be checked three times weekly for deposition of the gelatinous egg masses. If present, the egg masses should be carefully removed. They should be transferred to a small dish containing a sample of the breeding water. Egg masses are used to start a new culture vessel (e.g. 2-4 egg masses/vessel) or are used for toxicity tests. 10. First instar larvae should hatch after 2-3 days. Set-up of new culture vessels 11. Once cultures are established it should be possible to set up a fresh larval culture vessel weekly or less frequently depending on testing requirements, removing the older vessels after adult midges have emerged. Using this system a regular supply of adults will be produced with a minimum of management. Preparation of test solutions “M4” and “M7” 12. Elendt (1990) has described the “M4” medium. The “M7” medium is prepared as the “M4” medium except for the substances indicated in Table 1, for which concentrations are four times lower in “M7” than in “M4”. A publication on the “M7” medium is in preparation (Elendt, personal communication). The test solution should not be prepared according to Elendt and Bias (1990) for the concentrations of NaSiO3 5 H2O, NaNO3, KH2PO4 and K2HPO4 given for the preparation of the stock solutions are not adequate. Preparation of the “M7”-medium 13. Each stock solution (I) is prepared individually and a combined stock solution (II) is prepared from these stock solutions (I) (see Table 1). 50 ml from the combined stock Solution (II) and the amounts of each macro nutrient stock solution which are given in Table 2 are made up to 1 l of deionised water to prepare the “M7” medium. A vitamin stock solution is prepared by adding three vitamins to deionised water as indicated in Table 3, and 0,1 ml of the combined vitamin stock solution are added to the final “M7” medium shortly before use. (The vitamin stock solution is stored frozen in small aliquots). The medium is aerated and stabilised. Table 1 Stock solutions of trace elements for medium M4 and M7 Stock solutions (I) Amount (mg) made up to 1 litre of deionised water To prepare the combined stock solution (II): mix the following amounts (ml) of stock solutions (I) and make up to 1 litre of deionised water Final concentrations in test solutions (mg/l) M4 M7 M4 M7 H3BO3 (18) 57 190 1,0 0,25 2,86 0,715 MnCl2 · 4 H2O (18) 7 210 1,0 0,25 0,361 0,090 LiCl (18) 6 120 1,0 0,25 0,306 0,077 RbCl (18) 1 420 1,0 0,25 0,071 0,018 SrCl2 · 6 H2O (18) 3 040 1,0 0,25 0,152 0,038 NaBr (18) 320 1,0 0,25 0,016 0,004 Na2MoO4 · 2 H2O (18) 1 260 1,0 0,25 0,063 0,016 CuCl2 · 2 H2O (18) 335 1,0 0,25 0,017 0,004 ZnCl2 260 1,0 1,0 0,013 0,013 CaCl2 · 6 H2O 200 1,0 1,0 0,010 0,010 KI 65 1,0 1,0 0,0033 0,0033 Na2SeO3 43,8 1,0 1,0 0,0022 0,0022 NH4VO3 11,5 1,0 1,0 0,00058 0,00058 Na2EDTA · 2 H2O (18) (19) 5 000 20,0 5,0 2,5 0,625 FeSO4 · 7 H2O (18) (19) 1 991 20,0 5,0 1,0 0,249 Table 2 Macro nutrient stock solutions for medium M4 and M7 Amount made up to 1 litre of deionised water (mg) Amount of macro nutrient stock solutions added to prepare medium M4 and M7 (ml/l) Final concentrations in test solutions M4 and M7 (mg/l) CaCl2 · 2 H2O 293 800 1,0 293,8 MgSO4 · 7 H2O 246 600 0,5 123,3 KCl 58 000 0,1 5,8 NaHCO3 64 800 1,0 64,8 NaSiO3 · 9 H2O 50 000 0,2 10,0 NaNO3 2 740 0,1 0,274 KH2PO4 1 430 0,1 0,143 K2HPO4 1 840 0,1 0,184 Table 3 Vitamin stock solution for medium M4 and M7 All three vitamin solutions are combined to make a single vitamin stock solution.

Amount made up to 1 litre of deionised water (mg) Amount of vitamin stock solution added to prepare medium M4 and M7 (ml/l) Final concentrations in test solutions M4 and M7 (mg/l) Thiamine hydrochloride 750 0,1 0,075 Cyanocobalamin (B12) 10 0,1 0,0010 Biotine 7,5 0,1 0,00075 LITERATURE: BBA (1995). Long-term toxicity test with Chironomus riparius: Development and validation of a new test system. Edited by M. Streloke and H.Köpp. Berlin 1995. Elendt BP (1990). Selenium Deficiency in Crustacean. Protoplasma 154: 25-33. Elendt BP and Bias W-R (1990). Trace Nutrient Deficiency in Daphnia magna Cultured in Standard Medium for Toxicity Testing. Effects on the Optimization of Culture Conditions on Life History Parameters of D. magna. Water Research 24 (9): 1157-1167. Appendix 3 PREPARATION OF FORMULATED SEDIMENT Sediment composition The composition of the formulated sediment should be as follows: Constituent Characteristics % of sediment dry weight Peat Sphagnum moss peat, as close to pH 5,5-6,0 as possible, no visible plant remains, finely ground (particle size ≤ 1 mm) and air dried 4-5 Quartz sand Grain size: > 50 % of the particles should be in the range of 50-200 μm 75-76 Kaolinite clay Kaolinite content ≥ 30 % 20 Organic carbon Adjusted by addition of peat and sand 2 (± 0,5) Calcium carbonate CaCO3, pulverised, chemically pure 0,05-0,1 Water Conductivity ≤ 10 μS/cm 30-50 Preparation The peat is air dried and ground to a fine powder. A suspension of the required amount of peat powder in deionised water is prepared using a high-performance homogenising device. The pH of this suspension is adjusted to 5,5 ± 0,5 with CaCO3. The suspension is conditioned for at least two days with gentle stirring at 20 ± 2 °C, to stabilise pH and establish a stable microbial component. pH is measured again and should be 6,0 ± 0,5. Then the peat suspension is mixed with the other constituents (sand and kaolin clay) and deionised water to obtain a homogeneous sediment with a water content in a range of 30-50 per cent of dry weight of the sediment. The pH of the final mixture is measured once again and is adjusted to 6,5 to 7,5 with CaCO3 if necessary. Samples of the sediment are taken to determine the dry weight and the organic carbon content. Then, before it is used in the chironomid toxicity test, it is recommended that the formulated sediment be conditioned for seven days under the same conditions which prevail in the subsequent test. Storage The dry constituents for preparation of the artificial sediment may be stored in a dry and cool place at room temperature. The formulated (wet) sediment should not be stored prior to its use in the test. It should be used immediately after the 7 days conditioning period that ends its preparation. LITERATURE: Chapter C.8 of this Annex, Toxicity for Earthworms Meller M, Egeler P, Rombke J, Schallnass H, Nagel R and Streit B (1998). Short-term Toxicity of Lindane, Hexachlorobenzene and Copper Sulfate on Tubificid Sludgeworms (Oligochaeta) in Artificial Media. Ecotox. and Environ. Safety 39: 10-20. Appendix 4 Chemical Characteristics of Acceptable Dilution Water Substance Concentrations Particulate matter < 20 mg/l Total organic carbon < 2 mg/l Unionised ammonia < 1 μg/l Hardness as CaCO3 < 400 mg/l (20) Residual chlorine < 10 μg/l Total organophosphorus pesticides < 50 ng/l Total organochlorine pesticides plus polychlorinated biphenyls < 50 ng/l Total organic chlorine < 25 ng/l Appendix 5 Guidance for monitoring emergence of chironomid larvae Emergence traps are placed on the test beakers. These traps are needed from day 20 to the end of the test. Example of trap used is drawn below: A : the nylon screen B : the inverted plastic cups C : the lipless exposure beaker D : the water exchange screen ports E : water F : sediment C.29. READY BIODEGRADABILITY — CO2 IN SEALED VESSELS (HEADSPACE TEST) INTRODUCTION 1. This Test Method is equivalent to OECD Test Guideline (TG) 310 (2006). This Test Method is a screening method for the evaluation of ready biodegradability of chemicals and provides similar information to the six test methods described in chapter C.4 of this Annex A to F. Therefore, a chemical that shows positive results in this Test Method can be considered readily biodegradable and consequently rapidly degradable in the environment. 2. The well established carbon dioxide (CO2) method (1), based on Sturm’s original test (2) for assessing biodegradability of organic chemicals, by the measurement of the carbon dioxide produced by microbial action, has normally been the first choice for testing poorly soluble chemicals and those which strongly adsorb. It is also chosen for soluble (but not volatile) chemicals, since the evolution of carbon dioxide is considered by many to be the only unequivocal proof of microbial activity. Removal of dissolved organic carbon can be effected by physico-chemical processes — adsorption, volatilisation, precipitation, hydrolysis — as well as by microbial action and many non-biological reactions consume oxygen; rarely is CO2 produced from organic chemicals abiotically. In the original and modified Sturm test (1)(2) CO2 is removed from the liquid phase to the absorbing vessels by sparging (i.e. bubbling air treated to remove CO2 through the liquid medium), while in the version of Larson (3)(4) CO2 is transferred from the reaction vessel to the absorbers by passing CO2-free air through the headspace and, additionally, by shaking the test vessel continuously. Only in the Larson modification is the reaction vessel shaken; stirring is specified only for insoluble substances in ISO 9439 (5) and in the original US version (6), both of which specify sparging rather than headspace replacement. In another official US EPA method (7) based on Gledhill’s method (8), the shaken reaction vessel is closed to the atmosphere and CO2 produced is collected in an internal alkaline trap directly from the gaseous phase, as in classical Warburg/Barcroft respirometer flasks. 3. However, inorganic carbon (IC) has been shown to accumulate in the medium during the application of the standard, modified Sturm test