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

Of course, for the Daphnia reproduction test the table should be extended to cover 21 days.

It is clear that when observations are taken only at the start and end of each renewal period, it is not possible to confirm that the decay process in, in fact, exponential. A different curve would result in a different calculation for Area. However, an exponential decay process is not implausible and is probably the best curve to use in the absence of other information.

However, a word of caution is required if the chemical analysis fails to find any chemical at the end of the renewal period. Unless it is possible to estimate how quickly the chemical disappeared from the solution, it is impossible to obtain a realistic area under the curve, and hence it is impossible to obtain a reasonable time-weighted mean.

Appendix 7

GUIDANCE FOR THE IDENTIFICATION OF NEONATE SEX

Production of male neonates can occur under changing environmental conditions, such as shortening photoperiod, temperature, decreasing food concentration, and increasing population density (Hobaek and Larson, 1990; Kleiven et al., 1992). Male production is also a known response to certain insect growth regulators (Oda et al., 2005). Under conditions where chemical stressors are inducing a decrease in reproductive offspring from the parthenogenic females, an increased number of males would be expected (OECD, 2008). On the basis of available information, it is not possible to predict which of the sex ratio or of the reproduction endpoint will be more sensitive; however, there are indications (reference ‘validation report’, part 1) this increase in the number of males might be less sensitive than the decrease in offspring. Since the primary purpose of the test method is to assess the number of offspring produced, the appearance of males is an optional observation. If this optional endpoint is evaluated in a study, then an additional test validity criterion of no more than 5 % males in the controls should be employed.

The most practical and easy way to differentiate sex of Daphnia is to use their phenotypic characteristics, as males and females are genetically identical and their sex is environmentally determined. Males and females are different in the length and morphology of the first antennae, which are longer in males than females (Fig. 1). This difference is recognizable right after birth, although other secondary sex characteristics develop as they grow up (e.g. see Fig. 2 in Olmstead and LeBlanc, 2000).

To observe the morphological sex, neonates produced by each test animal should be transferred by pipet and placed into a petri dish with test medium. The medium is kept to a minimum to restrain movement of the animals. Observation of the first antennae can be conducted under a stereomicroscope (× 10-60).

Figure 1

24-hour-old male (left) and female (right) of D. magna. Males can be distinguished from females by the length and morphology of the first antennae as shown in the circles (Tatarazako et al., 2004)

Image omitted; see official source

REFERENCES:

Hobaek A and Larson P. 1990. Sex determination in Daphnia magna. Ecology 71: 2255-2268.

Kleiven O.T., Larsson P., Hobaek A. 1992. Sexual reproduction in Daphnia magna requires three stimuli. Oikos 65, 197-206.

Oda S., Tatarazako N, Watanabe H., Morita M., and Iguchi T. 2005. Production of male neonates in Daphnia magna (Cladocera, Crustacea) exposed to juvenile hormones and their analogs. Chemosphere 61:1168-1174.

OECD, 2008. Validation report for an enhancement of OECD TG 211 Daphnia magna reproduction test. OECD Series on Testing and Assessment, Number 88. Organisation for Economic Co-operation and Development, Paris.

Olmstead, A.W., LeBlanc, G.A., 2000. Effects of endocrine-active chemicals on the development characteristics of Daphnia magna. Environmental Toxicology and Chemistry 19:2107-2113.

Tatarazako, N., Oda, S., Abe, R., Morita M. and Iguchi T., 2004. Development of a screening method for endocrine disruptors in crustaceans using Daphnia magna (Cladocera, Crustacea). Environmental Science 17, 439-449.

C.21.   SOIL MICROORGANISMS: NITROGEN TRANSFORMATION TEST

1. METHOD

This test method is a replicate of OECD TG 216 (2000).

1.1.   INTRODUCTION

This testing method describes a laboratory method designed to investigate the long-term effects of chemicals, after a single exposure, on nitrogen transformation activity of soil microorganisms. The test is principally based on the recommendations of the European and Mediterranean Plant Protection Organization (1). However, other guideline, including those of the German Biologische Bundesanstalt (2), the US Environmental Protection Agency (3) SETAC (4) and the International Organization for Standardization (5), were also taken into account. An OECD workshop on soil/sediment selection held at Belgirate, Italy, in 1995 (6) agreed on the number and type of soils for use in this test. Recommendations for collection, handling and storage of soil sample are based on an ISO Guidance Document (7) and recommendations from the Belgirate workshop. In the assessment and evaluation of toxic characteristics of test substances, determination of effects on soil microbial activity may be required, e.g. when data on the potential side effects of crop protection products on soil microflora are required or when exposure of soil microorganisms to chemicals other than crop protection products is expected. The nitrogen transformation test is carried out to determine the effects of such chemicals on soil microflora. If agrochemicals (e.g. crop protection products, fertilisers, forestry chemicals) are tested, both nitrogen transformation and carbon transformation tests are conducted. If non-agrochemicals are tested, the nitrogen transformation test is sufficient. However, if EC50 values of the nitrogen transformation test for such chemicals fall within the range found for commercially available nitrification inhibitors (e.g. nitrapyrin), a carbon transformation test can be conducted to gain further information.

Soils consist of living and non-living components which exist in complex and heterogeneous mixtures. Microorganisms play an important role in break-down and transformation of organic matter in fertile soils with many species contributing to different aspects of soil fertility. Any long-term interference with these biochemical processes could potentially interfere with nutrient cycling and this could alter soil fertility. Transformation of carbon and nitrogen occurs in all fertile soils. Although the microbial communities responsible for these processes differ from soil to soil, the pathways of transformation are essentially the same.

This testing method described is designed to detect long-term adverse effects of a substance on the process of nitrogen transformation in aerobic surface soils. The test method also allows estimation of the effects of substances on carbon transformation by the soil microflora. Nitrate formation takes place subsequent to the degradation of carbon-nitrogen bonds. Therefore, if equal rates of nitrate production are found in treated and control soils, it is highly probable that the major carbon degradation pathways are intact and functional. The substrate chosen for the test (powdered lucerne meal) has a favourable carbon to nitrogen ratio (usually between 12/1 and 16/1). Because of this, carbon starvation is reduced during the test and if microbial communities are damaged by a chemical, they might recover within 100 days.

The tests from which this testing method was developed were primarily designed for substances for which the amount reaching the soil can be anticipated. This is the case, for example, for crop protection products for which the application rate in the field is known. For agrochemicals, testing of two doses relevant to the anticipated or predicted application rate is sufficient. Agrochemicals can be tested as active ingredients (a.i.) or as formulated products. However, the test is not limited to agrochemicals. By changing both the amounts of test substance applied to the soil, and the way in which the data are evaluated, the test can also be used for chemicals for which the amount expected to reach the soil is not known. Thus, with chemicals other than agrochemicals, the effects of a series of concentrations on nitrogen transformation are determined. The data from these tests are used to prepare a dose-response curve and calculate ECx values, where x is defined % effect.

1.2.   DEFINITIONS

Nitrogen transformation: is the ultimate degradation by microorganisms of nitrogen-containing organic matter, via the process of ammonification and nitrification, to the respective inorganic end-product nitrate.

EC x (effective concentration): is the concentration of the test substance in soil that results in a x percent inhibition of nitrogen transformation to nitrate.

EC 50 (median effective concentration): is the concentration of the test substance in soil that results in a 50 percent (50 %) inhibition of nitrogen transformation to nitrate.

1.3.   REFERENCE SUBSTANCES

None.

1.4.   PRINCIPLE OF THE TEST METHOD

Sieved soil is amended with powdered plant meal and either treated with the test substance or left untreated (control). If agrochemicals are tested, a minimum of two test concentrations are recommended and these should be chosen in relation to the highest concentration anticipated in the field. After 0, 7, 14 days and 28 days of incubation, samples of treated and control soils are extracted with an appropriate solvent, and the quantities of nitrate in the extracts are determined. The rate of nitrate formation in treated samples is compared with the rate in the controls, and the percent deviation of the treated from the control is calculated. All tests run for at least 28 days. If, on the 28th day, differences between treated and untreated soils are equal to or greater than 25 %, measurements are continued to a maximum of 100 days. If non-agrochemicals are tested, a series of concentrations of the test substance are added to samples of the soil, and the quantities of nitrate formed in treated and control samples are measured after 28 days of incubation. Results from tests with multiple concentrations are analysed using a regression model, and the ECx values are calculated (i.e. EC50, EC25 and/or EC10). See definitions.

1.5.   VALIDITY OF THE TEST

Evaluations of test results with agrochemicals are based on relatively small differences (i.e. average value ±25 %) between nitrate concentrations in control and treated soil samples, so large variations in the controls can lead to false results. Therefore, the variation between replicate control samples should be less than ±15 %.

1.6.   DESCRIPTION OF THE TEST METHOD

1.6.1.   Apparatus

Test containers made of chemically inert material are used. They should be of a suitable capacity in compliance with the procedure used for incubation of soils, i.e. incubation in bulk or as a series of individual soil samples (see Section 1.7.1.2). Care should be taken both to minimise water loss and to allow gas exchange during the test (e.g. the test containers may be covered with perforated polyethylene foil). When volatile substances are tested, sealable and gas-tight containers should be used. These should be of a size such that approximately one quarter of their volume is filled with the soil sample.

Standard laboratory equipment including the following is used:

• — agitation device: mechanical shaker or equivalent equipment;

• — centrifuge (3 000 g) or filtration device (using nitrate-free filter paper);

• — instrument of adequate sensitivity and reproducibility for nitrate analysis.

1.6.2.   Selection and number of soils

One single soil is used. The recommended soil characteristics are as follows:

• — sand content: not less than 50 % and not greater than 75 %,

• — pH: 5,5 -7,5 ,

• — organic carbon content: 0,5 -1,5 %,

• — the microbial biomass should be measured (8)(9) and its carbon content should be at least 1 % of the total soil organic carbon.

In most cases, a soil with these characteristics represents a worst case situation, since adsorption of the test chemical is minimum and its availability to the microflora is maximum. Consequently, tests with other soils are generally unnecessary. However, in certain circumstances, e.g. where the anticipated major use of the test substance is in particular soils such as acidic forest soils, or for electrostatically charged chemicals, it may be necessary to use an additional soil.

1.6.3.   Collection and storage of soil samples

1.6.3.1.   Collection

Detailed information on the history of the field site from where the test soil is collected should be available. Details include exact location, vegetation cover, dates of treatments with crop protection products, treatments with organic and inorganic fertilisers, additions of biological materials or accidental contaminations. The site chosen for soil collection should be one which allows long-term use. Permanent pastures, fields with annual cereal crops (except maize) or densely sown green manures are suitable. The selected sampling site should not have been treated with crop protection products for a minimum of one year before sampling. Also, no organic fertiliser should have been applied for at least six months. The use of mineral fertiliser is only acceptable when in accordance with the requirements of the crop and soil samples should not be taken until at least three months after fertiliser application. The use of soil treated with fertilisers with known biocidal effects (e.g. calcium cyanamide) should be avoided.

Sampling should be avoided during or immediately following long periods (greater than 30 days) of drought or water logging. For ploughed soils, samples should be taken from a depth of 0 down to 20 cm. For grassland (pasture) or other soils where ploughing does not occur over longer periods (at least one growing season), the maximum depth of sampling may be slightly more than 20 cm (e.g. to 25 cm).

Soil samples should be transported using containers and under temperature conditions which guarantee that the initial soil properties are not significantly altered.

1.6.3.2.   Storage

The use of soils freshly collected from the field is preferred. If storage in the laboratory cannot be avoided, soils may be stored in the dark at 4 ± 2 ^oC for a maximum of three months. During the storage of soils, aerobic conditions must be ensured. If soils are collected from areas where they are frozen for at least three months per year, storage for six months at minus 18 ^oC to minus 22 ^oC can be considered. The microbial biomass of stored soils is measured prior to each experiment and the carbon in the biomass should be at least 1 % of the total soil organic carbon content (see Section 1.6.2).

1.6.4.   Handling and preparation of soil for the test

1.6.4.1.   Pre-incubation

If the soil was stored (see Section 1.6.3.2), pre-incubation is recommended for a period between two and 28 days. The temperature and moisture content of the soil during pre-incubation should be similar to that used in the test (see Sections 1.6.4.2 and 1.7.1.3).

1.6.4.2.   Physical-chemical characteristics

The soil is manually cleared of large objects (e.g. stones, parts of plants, etc.) and then moist sieved without excess drying to a particle size less than or equal to 2 mm. The moisture content of the soil sample should be adjusted with distilled or deionised water to a value between 40 % and 60 % of the maximum water holding capacity.

1.6.4.3.   Amendment with organic substrate

The soil should be amended with a suitable organic substrate, e.g. powdered lucerne-grass-green meal (main component: Medicago sativa) with a C/N ratio between 12/1 and 16/1. The recommended lucerne-soil ratio is 5 g of lucerne per kilogram of soil (dry weight).

1.6.5.   Preparation of the test substance for the application to soil

The test substance is normally applied using a carrier. The carrier can be water (for water soluble substances) or an inert solid such as fine quartz sand (particle size: 0,1 -0,5 mm). Liquid carriers other than water (e.g. organic solvents such as acetone, chloroform) should be avoided since they can damage the microflora. If sand is used as a carrier, it can be coated with the test substance dissolved or suspended in an appropriate solvent. In such cases, the solvent should be removed by evaporation before mixing with the soil. For an optimum distribution of the test substance in soil, a ratio of 10 g of sand per kilogram of soil (dry weight) is recommended. Control samples are treated with an equivalent amount of water and/or quartz sand only.

When testing volatile chemicals, losses during treatment should be avoided as far as possible and an attempt should be made to ensure homogeneous distribution in the soil (e.g. the test substance should be injected into the soil at several places).

1.6.6.   Test concentrations

If agrochemicals are tested, at least two concentrations should be used. The lower concentration should reflect at least the maximum amount expected to reach the soil under practical conditions whereas the higher concentration should be a multiple of the lower concentration. The concentrations of test substance added to soil are calculated assuming uniform incorporation to a depth of 5 cm and a soil bulk density of 1,5 . For agrochemicals that are applied directly to soil, or for chemicals for which the quantity reaching the soil can be predicted, the test concentrations recommended are the maximum Predicted Environmental Concentration (PEC) and five times that concentration. Substances that are expected to be applied to soils several times in one season should be tested at concentrations derived from multiplying the PEC by the maximum anticipated number of applications. The upper concentration tested, however, should not exceed 10 times the maximum single application rate. If non-agrochemicals are tested, a geometric series of at least five concentrations is used. The concentrations tested should cover the range needed to determine the ECx values.

1.7.   PERFORMANCE OF THE TEST

1.7.1.   Conditions of exposure

1.7.1.1.   Treatment and control

If agrochemicals are tested, the soil is divided into three portions of equal weight. Two portions are mixed with the carrier containing the product, and the other is mixed with the carrier without the product (control). A minimum of three replicates for both treated and untreated soils is recommended. If non-agrochemicals are tested, the soil is divided into six portions of equal weight. Five of the samples are mixed with the carrier containing the test substance, and the sixth sample is mixed with the carrier without the chemical. Three replicates for both treatments and control are recommended. Care should be taken to ensure homogeneous distribution of the test substance in the treated soil samples. During mixing, compacting or balling of the soil should be avoided.

1.7.1.2.   Incubation of soil samples

Incubation of soil samples can be performed in two ways: as bulk samples of each treated and untreated soil or as a series of individual and equally sized subsamples of each treated and untreated soil. However, when volatile substances are tested, the test should only be performed with a series of individual subsamples. When soils are incubated in bulk, large quantities of each treated and untreated soils are prepared and subsamples to be analysed are taken as needed during the test. The amount initially prepared for each treatment and control depends on the size of the subsamples, the number of replicates used for analysis and the anticipated maximum number of sampling times. Soils incubated in bulk should be thoroughly mixed before subsampling. When soils are incubated as a series of individual soil samples, each treated and untreated bulk soil is divided into the required number of subsamples, and these are utilised as needed. In the experiments where more than two sampling times can be anticipated, enough subsamples should be prepared to account for all replicates and all sampling times. At least three replicate samples of the test soil should be incubated under aerobic conditions (see Section 1.7.1.1). During all tests, appropriate containers with sufficient headspace should be used to avoid development of anaerobic conditions. When volatile substances are tested, the test should only be performed with a series of individual subsamples.

1.7.1.3.   Test conditions and duration

The test is carried out in the dark at room temperature of 20 ± 2 ^oC. The moisture content of soil samples should be maintained during the test between 40 % and 60 % of the maximum water holding capacity of the soil (see Section 1.6.4.2) with a range of ±5 %. Distilled, deionised water can be added as needed.

The minimum duration of tests is 28 days. If agrochemicals are tested, the rates of nitrate formation in treated and control samples are compared. If these differ by more than 25 % on day 28, the test is continued until a difference equal to or less than 25 % is obtained, or for a maximum of 100 days, whichever is shorter. For non-agrochemicals, the test is terminated after 28 days. On day 28, the quantities of nitrate in treated and control soil samples are determined and the ECx values are calculated.

1.7.2.   Sampling and analysis of soils

1.7.2.1.   Soil sampling schedule

If agrochemicals are tested, soil samples are analysed for nitrate on days 0, 7, 14 and 28. If a prolonged test is required, further measurements should be made at 14 days intervals after day 28.

If non-agrochemicals are tested, at least five test concentrations are used and soil samples are analysed for nitrate at the beginning (day 0) and at the end of the exposure period (28 days). An intermediate measurement, e.g. at day 7, may be added if deemed necessary. The data obtained on day 28 are used to determine ECx value for the chemical. If desired, data from day 0 control samples can be used to report the initial quantity of nitrate in the soil.

1.7.2.2.   Analysis of soil samples

The amount of nitrate formed in each treated and control replicate is determined at each sampling time. Nitrate is extracted from soil by shaking samples with a suitable extraction solvent, e.g. a 0,1 M potassium chloride solution. A ratio of 5 ml of KCl solution per gram dry weight equivalent of soil is recommended. To optimise extraction, containers holding soil and extraction solution should not be more than half full. The mixtures are shaken at 150 rpm for 60 minutes. The mixtures are centrifuged or filtered and the liquid phases are analysed for nitrate. Particle-free liquid extracts can be stored prior to analysis at minus 20 ± 5 ^oC for up to six months.

2. DATA

2.1.   TREATMENT OF RESULTS

If tests are conducted with agrochemicals, the quantity of nitrate formed in each replicate soil sample should be recorded, and the mean values of all replicates should be provided in tabular form. Nitrogen transformation rates should be evaluated by appropriate and generally acceptable statistical methods (e.g. F-test, 5 % significance level). The quantities of nitrate formed are expressed in mg nitrate/kg dry weight soil/day. The nitrate formation rate in each treatment is compared with that in the control, and the percent deviation from the control is calculated.

If tests are conducted with non-agrochemicals, the quantity of nitrate formed in each replicate is determined, and a dose-response curve is prepared for estimation of the ECx values. The quantities of nitrate (i.e. mg nitrate/kg dry weight soil) found in the treated samples after 28 days are compared to that found in the control. From these data, the % inhibition values for each test concentration are calculated. These percentages are plotted against concentration, and statistical procedures are then used to calculate the ECx values. Confidence limits (p = 0,95 ) for the calculated ECx are also determined using standard procedures (10)(11)(12).

Test substances that contain high quantities of nitrogen may contribute to the quantities of nitrate formed during the test. If these substances are tested at a high concentration (e.g. chemicals which are expected to be used in repeated applications) appropriate controls must be included in the test (i.e. soil plus test substance but without plant meal). Data from these controls must be accounted for in the ECx calculations.

2.2.   INTERPRETATION OF RESULTS

When results from tests with agrochemicals are evaluated, and the difference in the rates of nitrate formation between the lower treatment (i.e. the maximum predicted concentration) and control is equal to or less than 25 % at any sampling time after day 28, the product can be evaluated as having no long-term influence on nitrogen transformation in soils. When results from tests with chemicals other than agrochemicals are evaluated, the EC50, EC25 and/or EC10 values are used.

3. REPORTING

The test report must include the following information:

Complete identification of the soil used including:

• — geographical reference of the site (latitude, longitude),

• — information on the history of the site (i.e. vegetation cover, treatments with crop protection products, treatments with fertilisers, accidental contamination, etc.),

• — use pattern (e.g. agricultural soil, forest, etc.),

• — depth of sampling (cm),

• — sand/silt/clay content (% dry weight),

• — pH (in water),

• — organic carbon content (% dry weight),

• — nitrogen content (% dry weight),

• — initial nitrate concentration (mg nitrate/kg dry weight),

• — cation exchange capacity (mmol/kg),

• — microbial biomass in terms of percentage of the total organic carbon,

• — reference of the methods used for the determination of each parameter,

• — all information relating to the collection and storage of soil samples,

• — details of pre-incubation of soil if any.

Test substance:

• — physical nature and, where relevant, physical-chemical properties,

• — chemical identification data, where relevant, including structural formula, purity (i.e. for crop protection products the percentage of active ingredient), nitrogen content.

Substrate:

• — source of substrate,

• — composition (i.e. lucerne meal, lucerne-grass-green meal),

• — carbon, nitrogen content (% dry weight),

• — sieve size (mm).

Test conditions:

• — details of the amendment of soil with organic substrate,

• — number of concentrations of test chemical used and, where appropriate, justification of the selected concentrations,

• — details of the application of test substance to soil,

• — incubation temperature,

• — soil moisture content at the beginning and during the test,

• — method of soil incubation used (i.e. as bulk or as a series of individual subsamples),

• — number of replicates,

• — sampling times,

• — method used for extraction of nitrate from soil,

Results:

• — analytical procedure and equipment used to analyse nitrate,

• — tabulated data including individual and mean values for nitrate measurements,

• — variation between the replicates in treated and control samples,

• — explanations of corrections made in the calculations, if relevant,

• — the percent variation in nitrate formation rates at each sampling time or, if appropriate, the EC50 value with 95 % confidence limit, other ECx (i.e. EC25 or EC10) with confidence intervals, and a graph of the dose-response curve,

• — statistical treatment of results,

• — all information and observations helpful for the interpretation of the results.

4. REFERENCES

• (1)EPPO, (1994) Decision-Making Scheme for the Environmental Risk Assessment of Plant Protection Chemicals. Chapter 7: Soil Microflora. EPPO Bulletin 24: 1-16, 1994.

• (2)BBA, (1990) Effects on the Activity of the Soil Microflora. BBA Guidelines for the Official Testing of Plant Protection Products, VI, 1-1 (2nd eds., 1990).

• (3)EPA (1987) Soil Microbial Community Toxicity Test. EPA 40 CFR Part 797.3700. Toxic Substances Control Act Test Guidelines; Proposed rule. September 28, 1987.

• (4)SETAC-Europe, (1995) Procedures for assessing the environmental fate and ecotoxicity of pesticides, Ed. M.R. Lynch, Pub. SETAC-Europe, Brussels.

• (5)ISO/DIS 14238 (1995) Soil Quality — Determination of Nitrogen Mineralisation and Nitrification in Soils and the Influence of Chemicals on these Processes. Technical Committee ISO/TC 190/SC 4: Soil Quality — Biological Methods.

• (6)OECD, (1995) Final Report of the OECD Workshop on Selection of Soils/Sediments, Belgirate, Italy, 18-20 January 1995.

• (7)ISO 10381-6 (1993) Soil quality — Sampling. Guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory.

• (8)ISO 14240-1, (1997) Soil quality — Determination of soil microbial biomass — Part 1: Substrate-induced respiration method.

• (9)ISO 14240-2, (1997) Soil quality — Determination of soil microbial biomass — Part 2: Fumigation-extraction method.

• (10)Litchfield, J.T. and Wilcoxon F., (1949) A simplified method of evaluating dose-effect experiments. Jour. Pharmacol. and Exper. Ther., 96, p. 99-113.

• (11)Finney, D.J., (1971) Probit Analysis. 3rd ed., Cambridge, London and New-York.

• (12)Finney, D.J., (1978) Statistical Methods in biological Assay. Griffin, Weycombe, UK.

C.22.   SOIL MICROORGANISMS: CARBON TRANSFORMATION TEST

1. METHOD

This method is a replicate of OECD TG 217 (2000).

1.1.   INTRODUCTION

This testing method describes a laboratory method designed to investigate long term potential effects of a single exposure of crop protection products and possibly other chemicals on carbon transformation activity of soil microorganisms. The test is principally based on the recommendations of the European and Mediterranean Plant Protection Organization (1). However, other guideline, including those of the German Biologische Bundesanstalt (2), the US Environmental Protection Agency (3) and SETAC (4), were also taken into account. An OECD Workshop on Soil/Sediment Selection held at Belgirate, Italy, in 1995 (5) agreed on the number and type of soils for use in this test. Recommendations for collection, handling and storage of soil sample are based on an ISO Guidance Document (6) and recommendations from the Belgirate Workshop.

In the assessment and evaluation of toxic characteristics of test substances, determination of effects on soil microbial activity may be required, e.g. when data on the potential side effects of crop protection products on soil microflora are required or when exposure of soil microorganisms to chemicals other than crop protection products is expected. The carbon transformation test is carried out to determine the effects of such chemicals on soil microflora. If agrochemicals (e.g. crop protection products, fertilisers, forestry chemicals) are tested, both carbon transformation and nitrogen transformation tests are conducted. If non-agrochemicals are tested, the nitrogen transformation test is sufficient. However, if EC50 values of the nitrogen transformation test for such chemicals fall within the range found for commercially available nitrification inhibitors (e.g. nitrapyrin), a carbon transformation test can be conducted to gain further information.

Soils consist of living and non-living components which exist in complex and heterogeneous mixtures. Microorganisms play an important role in breakdown and transformation of organic matter in fertile soils with many species contributing to different aspects of soil fertility. Any long-term interference with these biochemical processes could potentially interfere with nutrient cycling and this could alter the soil fertility. Transformation of carbon and nitrogen occurs in all fertile soils. Although the microbial communities responsible for these processes differ from soil to soil, the pathways of transformation are essentially the same.

This testing method is designed to detect long-term adverse effects of a substance on the process of carbon transformation in aerobic surface soils. The test is sensitive to changes in size and activity of microbial communities responsible for carbon transformation since it subjects these communities to both chemical stress and carbon starvation. A sandy soil low in organic matter is used. This soil is treated with the test substance and incubated under conditions that allow rapid microbial metabolism. Under these conditions, sources of readily available carbon in the soil are rapidly depleted. This causes carbon starvation which both kills microbial cells and induces dormancy and/or sporulation. If the test runs for more than 28 days, the sum of these reactions can be measured in (untreated soil) controls as a progressive loss of metabolically active microbial biomass (7). If the biomass in carbon-stressed soil, under the conditions of the test, is affected by the presence of a chemical, it may not return to the same level as the control. Hence, disturbances caused by the test substance at any time during the test will often last until the end of the test.

The tests from which this testing method was developed were primarily designed for substances for which the amount reaching the soil can be anticipated. This is the case, for example, for crop protection products for which the application rate in the field is known. For agrochemicals, testing of two doses relevant to the anticipated or predicted application rate is sufficient. Agrochemicals can be tested as active ingredients (a.i.) or as formulated products. However, the test is not limited to chemicals with predictable environmental concentrations. By changing both the amounts of test substance applied to the soil, and the way in which the data are evaluated, the test can also be used for chemicals for which the amount expected to reach the soil is not known. Thus, with non-agrochemicals, the effects of a series of concentrations on carbon transformation are determined. The data from these tests are used to prepare a dose-response curve and calculate ECx values, where x is defined % effect.

1.2.   DEFINITIONS

Carbon transformation: is the degradation by microorganisms of organic matter to form inorganic end-product carbon dioxide.

EC x (Effective Concentration): is the concentration of the test substance in soil that results in a x % inhibition of carbon transformation in carbon dioxide.

EC 50 (Median Effective Concentration): is the concentration of test substance in soil that results in a 50 % inhibition of carbon transformation in carbon dioxide.

1.3.   REFERENCE SUBSTANCES

None.

1.4.   PRINCIPLE OF THE TEST METHOD

Sieved soil is either treated with the test substance or left untreated (control). If agrochemicals are tested, a minimum of two test concentrations are recommended and these should be chosen in relation to the highest concentration anticipated in the field. After 0, 7, 14 and 28 days incubation, samples of treated and control soils are mixed with glucose, and glucose-induced respiration rates are measured for 12 consecutive hours. Respiration rates are expressed as carbon dioxide released (mg carbon dioxide/kg dry soil/h) or oxygen consumed (mg oxygen/kg soil/h). The mean respiration rate in the treated soil samples is compared with that in control and the percent deviation of the treated from the control is calculated. All tests run for at least 28 days. If, on the 28th day, differences between treated and untreated soils are equal to or greater than 25 % measurements are continued in 14 day intervals for a maximum of 100 days. If chemicals other than agrochemicals are tested, a series of concentrations of the test substance are added to samples of the soil, and glucose induced respiration rates (i.e. the mean of the quantities of carbon dioxide formed or oxygen consumed) are measured after 28 days. Results from tests with a series of concentrations are analysed using a regression model, and the ECx values are calculated (i.e. EC50, EC25 and/or EC10). See definitions.

1.5.   VALIDITY OF THE TEST

Evaluations of test results with agrochemicals are based on relatively small differences (i.e. average value ±25 %) between the carbon dioxide released or the oxygen consumed in (or by) control and treated soil samples, so large variations in the controls can lead to false results. Therefore, the variation between replicate control samples should be less than ±15 %.

1.6.   DESCRIPTION OF THE TEST METHOD

1.6.1.   Apparatus

Test containers made of chemically inert material are used. They should be of a suitable capacity in compliance with the procedure used for incubation of soils, i.e. incubation in bulk or as a series of individual soil samples (see Section 1.7.1.2). Care should be taken both to minimise water loss and to allow gas exchange during the test (e.g. the test containers may be covered with perforated polyethylene foil). When volatile substances are tested, sealable and gas-tight containers should be used. These should be of a size such that approximately one quarter of their volume is filled with the soil sample.

For determination of glucose-induced respiration, incubation systems and instruments for measurement of carbon dioxide production or oxygen consumption are required. Examples of such systems and instruments are found in the literature (8) (9) (10) (11).

1.6.2.   Selection and number of soils

One single soil is used. The recommended soil characteristics are as follows:

• — sand content: not less than 50 % and not greater than 75 %,

• — pH: 5,5 -7,5 ,

• — organic carbon content: 0,5 -1,5 %,

• — the microbial biomass should be measured (12)(13) and its carbon content should be at least 1 % of the total soil organic carbon.

In most cases, a soil with these characteristics represents a worst case situation, since adsorption of the test chemical is minimised and its availability to the microflora is maximum. Consequently, tests with other soils are generally unnecessary. However, in certain circumstances, e.g. where the anticipated major use of the test substance is in particular soils such as acidic forest soils, or for electrostatically charged chemicals, it may be necessary to substitute an additional soil.

1.6.3.   Collection and storage of soil samples

1.6.3.1.   Collection

Detailed information on the history of the field site from where the test soil is collected should be available. Details include exact location, vegetation cover, dates of treatments with crop protection products, treatments with organic and inorganic fertilisers, additions of biological materials or accidental contaminations. The site chosen for soil collection should be one which allows long-term use. Permanent pastures, fields with annual cereal crops (except maize) or densely sown green manures are suitable. The selected sampling site should not have been treated with crop protection products for a minimum of one year before sampling. Also, no organic fertiliser should have been applied for at least six months. The use of mineral fertiliser is only acceptable when in accordance with the requirements of the crop and soil samples should not be taken until at least three months after fertiliser application. The use of soil treated with fertilisers with known biocidal effects (e.g. calcium cyanamide) should be avoided.

Sampling should be avoided during or immediately following long periods (greater than 30 days) of drought or water logging. For ploughed soils, samples should be taken from a depth of 0 down to 20 cm. For grassland (pasture) or other soils where ploughing does not occur over longer periods (at least one growing season), the maximum depth of sampling may be slightly more than 20 cm (e.g. to 25 cm). Soil samples should be transported using containers and under temperature conditions which guarantee that the initial soil properties are not significantly altered.

1.6.3.2.   Storage

The use of soils freshly collected from the field is preferred. If storage in the laboratory cannot be avoided, soils may be stored in the dark at 4 ± 2 ^oC for a maximum of three months. During the storage of soils, aerobic conditions must be ensured. If soils are collected from areas where they are frozen for at least three months per year, storage for six months at minus 18 ^oC can be considered. The microbial biomass of stored soils is measured prior to each experiment and the carbon in the biomass should be at least 1 % of the total soil organic carbon content (see Section 1.6.2).

1.6.4.   Handling and preparation of soil for the test

1.6.4.1.   Pre-incubation

If the soil was stored (see Sections 1.6.4.2 and 1.7.1.3), pre-incubation is recommended for a period between two and 28 days. The temperature and moisture content of the soil during pre-incubation should be similar to that used in the test (see Sections 1.6.4.2 and 1.7.1.3).

1.6.4.2.   Physical-chemical characteristics

The soil is manually cleared of large objects (e.g. stones, parts of plants, etc.) and then moist sieved without excess drying to a particle size less than or equal to 2 mm. The moisture content of the soil sample should be adjusted with distilled or deionised water to a value between 40 % and 60 % of the maximum water holding capacity.

1.6.5.   Preparation of the test substance for the application to soil

The test substance is normally applied using a carrier. The carrier can be water (for water soluble substances) or an inert solid such as fine quartz sand (particle size: 0,1 -0,5 mm). Liquid carriers other than water (e.g. organic solvents such as acetone, chloroform) should be avoided since they can damage the microflora. If sand is used as a carrier, it can be coated with the test substance dissolved or suspended in an appropriate solvent. In such cases, the solvent should be removed by evaporation before mixing with the soil. For an optimum distribution of the test substance in soil, a ratio of 10 g of sand per kilogram of soil (dry weight) is recommended. Control samples are treated with the equivalent amount of water and/or quartz sand only.

When testing volatile chemicals, losses during treatment should be avoided and an attempt should be made to ensure homogeneous distribution in the soil (e.g. the test substance should be injected into the soil at several places).

1.6.6.   Test concentrations

If crop protection products or other chemicals with predictable environmental concentrations are tested, at least two concentrations should be used. The lower concentration should reflect at least the maximum amount expected to reach the soil under practical conditions whereas the higher concentration should be a multiple of the lower concentration. The concentrations of test substance added to soil are calculated assuming uniform incorporation to a depth of 5 cm and a soil bulk density of 1,5 . For agrochemicals that are applied directly to soil, or for chemicals for which the quantity reaching the soil can be predicted, the test concentrations recommended are the Predictable Environmental Concentration (PEC) and five times that concentration. Substances that are expected to be applied to soils several times in one season should be tested at concentrations derived from multiplying the PEC by the maximum anticipated number of applications. The upper concentration tested, however, should not exceed 10 times the maximum single application rate.

If non-agrochemicals are tested, a geometric series of at least five concentrations is used. The concentrations tested should cover the range needed to determine the ECx values.

1.7.   PERFORMANCE OF THE TEST

1.7.1.   Conditions of exposure

1.7.1.1.   Treatment and control

If agrochemicals are tested, the soil is divided into three portions of equal weight. Two portions are mixed with the carrier containing the product, and the other is mixed with the carrier without the product (control). A minimum of three replicates for both treated and untreated soils is recommended. If non-agrochemicals are tested, the soil is divided into six portions of equal weight. Five of the samples are mixed with the carrier containing the test substance, and the sixth sample is mixed with the carrier without the chemical. Three replicates for both treatments and control are recommended. Care should be taken to ensure homogeneous distribution of the test substance in the treated soil samples. During mixing, compacting or balling of the soil should be avoided.

1.7.1.2.   Incubation of soil samples

Incubation of soil samples can be performed in two ways: as bulk samples of each treated and untreated soil or as a series of individual and equally sized subsamples of each treated and untreated soil. However, when volatile substances are tested, the test should only be performed with a series of individual subsamples. When soils are incubated in bulk, large quantities of each treated and untreated soils are prepared and subsamples to be analysed are taken as needed during the test. The amount initially prepared for each treatment and control depends on the size of the subsamples, the number of replicates used for analysis and the anticipated maximum number of sampling times. Soils incubated in bulk should be thoroughly mixed before subsampling. When soils are incubated as a series of individual soil samples, each treated and untreated bulk soil is divided into the required number of subsamples, and these are utilised as needed. In the experiments where more than two sampling times can be anticipated, enough subsamples should be prepared to account for all replicates and all sampling times. At least three replicate samples of the test soil should be incubated under aerobic conditions (see Section 1.7.1.1). During all tests, appropriate containers with sufficient headspace should be used to avoid development of anaerobic conditions. When volatile substances are tested, the test should only be performed with a series of individual subsamples.

1.7.1.3.   Test conditions and duration

The test is carried out in the dark at room temperature of 20 ± 2 ^oC. The moisture content of soil samples should be maintained during the test between 40 % and 60 % of the maximum water holding capacity of the soil (see Section 1.6.4.2) with a range of ±5 %. Distilled, deionised water can be added as needed.

The minimum duration of tests is 28 days. If agrochemicals are tested, the quantities of carbon dioxide released or oxygen consumed in treated and control samples are compared. If these differ by more than 25 % on day 28, the test is continued until a difference equal to or less than 25 % is obtained, or for a maximum of 100 days, whichever is shorter. If non-agrochemicals are tested, the test is terminated after 28 days. On day 28, the quantities of carbon dioxide released or oxygen consumed in treated and control soil samples are determined and the ECx values are calculated.

1.7.2.   Sampling and analysis of soils

1.7.2.1.   Soil sampling schedule

If agrochemicals are tested, soil samples are analysed for glucose-induced respiration rates on days 0, 7, 14 and 28. If a prolonged test is required, further measurements should be made at 14 days intervals after day 28.

If non-agrochemicals are tested, at least five test concentrations are used and soil samples are analysed for glucose-induced respiration at the beginning (day 0) and at the end of the exposure period (28 days). An intermediate measurement, e.g. at day 7, may be added if deemed necessary. The data obtained on day 28 are used to determine ECx value for the chemical. If desired, data from day 0 control samples can be used to estimate the initial quantities of metabolically active microbial biomass in the soil (12).

1.7.2.2.   Measurement of glucose-induced respiration rates

The glucose-induced respiration rate in each treated and control replicate is determined at each sampling time. The soil samples are mixed with a sufficient amount of glucose to elicit an immediate maximum respiratory response. The amount of glucose needed to elicit a maximum respiratory response from a given soil can be determined in a preliminary test using a series of concentrations of glucose (14). However, for sandy soils with 0,5 -1,5 % organic carbon, 2 000 mg to 4 000 mg glucose per kg dry weight soil is usually sufficient. The glucose can be ground to a powder with clean quartz sand (10 g sand/kg dry weight soil) and homogeneously mixed with the soil.

The glucose amended soil samples are incubated in a suitable apparatus for measurement of respiration rates either continuously, every hour, or every two hours (see Section 1.6.1) at 20 ± 2 ^oC. The carbon dioxide released or the oxygen consumed is measured for 12 consecutive hours and measurements should start as soon as possible, i.e. within one to two hours after glucose supplement. The total quantities of carbon dioxide released or oxygen consumed during the 12 hours are measured and mean respiration rates are determined.

2. DATA

2.1.   TREATMENT OF RESULTS

If agrochemicals are tested, the carbon dioxide released from, or oxygen consumed by each replicate soil sample should be recorded, and the mean values of all replicates should be provided in tabular form. Results should be evaluated by appropriate and generally acceptable statistical methods (e.g. F-test, 5 % significance level). Glucose-induced respiration rates are expressed in mg carbon dioxide/kg dry weight soil/h or mg oxygen/dry weight soil/h. The mean carbon dioxide formation rate or mean oxygen consumption rate in each treatment is compared with that in control, and the percent deviation from the control is calculated.

If tests are conducted with non-agrochemicals, the quantities of carbon dioxide released or oxygen consumed by each replicate is determined, and a dose-response curve is prepared for estimation of the ECx values. The glucose-induced respiration rates (i.e. mg carbon dioxide/kg dry weight soil/h or mg oxygen/dry weight soil/h) found in the treated samples after 28 days are compared to that found in control. From these data, the % inhibition values for each test concentration are calculated. These percentages are plotted against concentration, and statistical procedures are used to calculate the ECx values. Confidence limits (p = 0,95 ) for the calculated ECx are also determined using standard procedures (15)(16)(17).

2.2.   INTERPRETATION OF RESULTS

When results from tests with agrochemicals are evaluated, and the difference in respiration rates between the lower treatment (i.e. the maximum predicted concentration) and control is equal to or less than 25 % at any sampling time after day 28, the product can be evaluated as having no long-term influence on carbon transformation in soils. When results from tests with chemicals other than agrochemicals are evaluated, the EC50, EC25 and/or EC10 values are used.

3. REPORTING

TEST REPORT

The test report must include the following information:

Complete identification of the soil used including:

• — geographical reference of the site (latitude, longitude),

• — information on the history of the site (i.e. vegetation cover, treatments with crop protection products, treatments with fertilisers, accidental contamination, etc.),

• — use pattern (e.g. agricultural soil, forest, etc.),

• — depth of sampling (cm),

• — sand/silt/clay content (% dry weight),

• — pH (in water),

• — organic carbon content (% dry weight),

• — nitrogen content (% dry weight);

• — cation exchange capacity (mmol/kg),

• — initial microbial biomass in terms of percentage of the total organic carbon,

• — reference of the methods used for the determination of each parameter,

• — all information relating to the collection and storage of soil samples,

• — details of pre-incubation of soil if any.

Test substance:

• — physical nature and, where relevant, physical-chemical properties,

• — chemical identification data, where relevant, including structural formula, purity (i.e. for crop protection products the percentage of active ingredient), nitrogen content.

Test conditions:

• — details of the amendment of soil with organic substrate,

• — number of concentrations of test chemical used and, where appropriate, justification of the selected concentrations,

• — details of the application of test substance to soil,

• — incubation temperature,

• — soil moisture content at the beginning and during the test,

• — method of soil incubation used (i.e. as bulk or as a series of individual subsamples),

• — number of replicates,

• — sampling times.

Results:

• — method and equipment used for measurement of respiration rates,

• — tabulated data including individual and mean values for quantities of carbon dioxide or oxygen,

• — variation between the replicates in treated and control samples,

• — explanations of corrections made in the calculations, if relevant,

• — the percent variation of glucose-induced respiration rates at each sampling time or, if appropriate, the EC50 with 95 % confidence limit, other ECx (i.e. EC25 or EC10) with confidence intervals, and a graph of the dose-response curve,

• — statistical treatment of results, where appropriate,

• — all information and observations helpful for the interpretation of the results.

4. REFERENCES

• (1)EPPO, (1994) Decision-Making Scheme for the Environmental Risk Assessment of Plant Protection Chemicals. Chapter 7: Soil Microflora. EPPO Bulletin 24: 1-16, 1994.

• (2)BBA, (1990) Effects on the Activity of the Soil Microflora. BBA Guidelines for the Official Testing of Plant Protection Products, VI, 1-1 (2nd eds., 1990).

• (3)EPA, (1987) Soil Microbial Community Toxicity Test. EPA 40 CFR Part 797.3700. Toxic Substances Control Act Test Guidelines; Proposed rule. September 28, 1987.

• (4)SETAC-Europe, (1995) Procedures for assessing the environmental fate and ecotoxicity of pesticides, Ed. M.R. Lynch, Pub. SETAC-Europe, Brussels.

• (5)OECD, (1995) Final Report of the OECD Workshop on Selection of Soils/Sediments, Belgirate, Italy, 18-20 January 1995.

• (6)ISO 10381-6, (1993) Soil quality — Sampling. Guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory.

• (7)Anderson, J.P.E., (1987) Handling and Storage of Soils for Pesticide Experiments, in ‘Pesticide Effects on Soil Microflora’. Eds. L. Somerville and M.P. Greaves, Chap. 3: 45-60.

• (8)Anderson, J.P.E., (1982) Soil Respiration, in ‘Methods of Soil Analysis — Part 2: Chemical and Microbiological Properties’. Agronomy Monograph No 9. Eds. A.L. Page, R.H. Miller and D.R. Keeney. 41: 831- 871.

• (9)ISO 11266-1, (1993) Soil Quality — Guidance on Laboratory Tests for Biodegradation in Soil: Part 1. Aerobic Conditions.

• (10)ISO 14239, (1997E) Soil Quality — Laboratory incubation systems for measuring the mineralisation of organic chemicals in soil under aerobic conditions.

• (11)Heinemeye r O., Insam, H., Kaiser, E.A, and Walenzik, G., (1989) Soil microbial biomass and respiration measurements; an automated technique based on infrared gas analyses. Plant and Soil, 116: 77-81.

• (12)ISO 14240-1, (1997) Soil quality — Determination of soil microbial biomass — Part 1: Substrate-induced respiration method.

• (13)ISO 14240-2, (1997) Soil quality — Determination of soil microbial biomass — Part 2: Fumigation-extraction method.

• (14)Malkomes, H.-P., (1986) Einfluß von Glukosemenge auf die Reaktion der Kurzzeit-Atmung im Boden Gegenüber Pflanzenschutzmitteln, Dargestellt am Beispiel eines Herbizide. (Influence of the Amount of Glucose Added to the Soil on the Effect of Pesticides in Short-Term Respiration, using a Herbicide as an Example). Nachrichtenbl. Deut. Pflanzenschutzd., Braunschweig, 38: 113-120.

• (15)Litchfield, J.T. and Wilcoxon, F., (1949) A simplified method of evaluating dose-effect experiments. Jour. Pharmacol. and Exper. Ther., 96, 99-113.

• (16)Finney, D.J., (1971) Probit Analysis. 3rd ed., Cambridge, London and New-York.

• (17)Finney D.J., (1978) Statistical Methods in biological Assay. Griffin, Weycombe, UK.

C.23.   AEROBIC AND ANAEROBIC TRANSFORMATION IN SOIL

1. METHOD

This test method is a replicate of the OECD TG 307 (2002)

1.1.   INTRODUCTION

This test method is based on existing guidelines (1)(2)(3)(4)(5)(6)(7)(8)(9). The method described in this test Method is designed for evaluating aerobic and anaerobic transformation of chemicals in soil. The experiments are performed to determine (i) the rate of transformation of the test substance, and (ii) the nature and rates of formation and decline of transformation products to which plants and soil organisms may be exposed. Such studies are required for chemicals which are directly applied to soil or which are likely to reach the soil environment. The results of such laboratory studies can also be used to develop sampling and analysis protocols for related field studies.

Aerobic and anaerobic studies with one soil type are generally sufficient for the evaluation of transformation pathways (8)(10)(11). Rates of transformation should be determined in at least three additional soils (8)(10).

An OECD Workshop on soil and sediment selection, held at Belgirate, Italy in 1995 (10) agreed, in particular, on the number and types of soils for use in this test. The types of soils tested should be representative of the environmental conditions where use or release will occur. For example, chemicals that may be released in subtropical to tropical climates should be tested with Ferrasols or Nitosols (FAO system). The Workshop also made recommendations relating to collection, handling and storage of soil samples, based on the ISO Guidance (15). The use of paddy (rice) soils is also considered in this method.

1.2.   DEFINITIONS

Test substance: any substance, whether the parent compound or relevant transformation products.

Transformation products: all substances resulting from biotic or abiotic transformation reactions of the test substance including CO2 and products that are in bound residues.

Bound residues: ‘Bound residues’ represent compounds in soil, plant or animal, which persist in the matrix in the form of the parent substance or its metabolite(s)/transformation products after extraction. The extraction method must not substantially change the compounds themselves or the structure of the matrix. The nature of the bond can be clarified in part by matrix-altering extraction methods and sophisticated analytical techniques. To date, for example, covalent ionic and sorptive bonds, as well as entrapments, have been identified in this way. In general, the formation of bound residues reduces the bioaccessibility and the bioavailability significantly (12) [modified from IUPAC 1984 (13)].

Aerobic transformation: reactions occurring in the presence of molecular oxygen (14).

Anaerobic transformation: reactions occurring under exclusion of molecular oxygen (14).

Soil: is a mixture of mineral and organic chemical constituents, the latter containing compounds of high carbon and nitrogen content and of high molecular weights, animated by small (mostly micro-) organisms. Soil may be handled in two states:

• (a) undisturbed, as it has developed with time, in characteristic layers of a variety of soil types;

• (b) disturbed, as it is usually found in arable fields or as occurs when samples are taken by digging and used in this test method (14).

Mineralisation: is the complete degradation of an organic compound to CO2 and H2O under aerobic conditions, and CH4, CO2 and H2O under anaerobic conditions. In the context of this test method, when ¹⁴C-labelled compound is used, mineralisation means extensive degradation during which a labelled carbon atom is oxidised with release of the appropriate amount of ¹⁴CO2 (14).

Half-life: t0,5 , is the time taken for 50 % transformation of a test substance when the transformation can be described by first-order kinetics; it is independent of the concentration.

DT 50 (Disappearance Time 50): is the time within which the concentration of the test substance is reduced by 50 %; it is different from the half-life t0,5 when transformation does not follow first order kinetics.

DT 75 (Disappearance Time 75): is the time within which the concentration of the test substance is reduced by 75 %.

DT 90 (Disappearance Time 90): is the time within which the concentration of the test substance is reduced by 90 %.

1.3.   REFERENCE SUBSTANCES

Reference substances should be used for the characterisation and/or identification of transformation products by spectroscopic and chromatographic methods.

1.4.   APPLICABILITY OF THE TEST

The method is applicable to all chemical substances (non-labelled or radiolabelled) for which an analytical method with sufficient accuracy and sensitivity is available. It is applicable to slightly volatile, non-volatile, water-soluble or water-insoluble compounds. The test should not be applied to chemicals which are highly volatile from soil (e.g. fumigants, organic solvents) and thus cannot be kept in soil under the experimental conditions of this test.

1.5.   INFORMATION ON THE SUBSTANCE

Non-labelled or labelled test substance can be used to measure the rate of transformation. Labelled material is required for studying the pathway of transformation and for establishing a mass balance. ¹⁴C-labelling is recommended but the use of other isotopes, such as ¹³C, ¹⁵N, ³H, ³²P, may also be useful. As far as possible, the label should be positioned in the most stable part(s) of the molecule ([^E0158]). The purity of the test substance should be at least 95 %.

Before carrying out a test on aerobic and anaerobic transformation in soil, the following information on the test substance should be available:

• (a) solubility in water (Method A.6)

• (b) solubility in organic solvents;

• (c) vapour pressure (Method A.4) and Henry's law constant;

• (d) n-octanol/water partition coefficient (Method A.8);

• (e) chemical stability in dark (hydrolysis) (Method C.7);

• (f) pKa if a molecule is liable to protonation or deprotonation [OECD Guideline 112] (16).

Other useful information may include data on toxicity of the test substance to soil micro-organisms [testing methods C.21 and C.22] (16).

Analytical methods (including extraction and clean-up methods) for quantification and identification of the test substance and its transformation products should be available.

1.6.   PRINCIPLE OF THE TEST METHOD

Soil samples are treated with the test substance and incubated in the dark in biometer-type flasks or in flow-through systems under controlled laboratory conditions (at constant temperature and soil moisture). After appropriate time intervals, soil samples are extracted and analysed for the parent substance and for transformation products. Volatile products are also collected for analysis using appropriate absorption devices. Using ¹⁴C-labelled material, the various mineralisation rates of the test substance can be measured by trapping evolved ¹⁴CO2 and a mass balance, including the formation of soil bound residues, can be established.

1.7.   QUALITY CRITERIA

1.7.1.   Recovery

Extraction and analysis of, at least, duplicate soil samples immediately after the addition of the test substance gives a first indication of the repeatability of the analytical method and of the uniformity of the application procedure for the test substance. Recoveries for later stages of the experiments are given by the respective mass balances. Recoveries should range from 90 % to 110 % for labelled chemicals (8) and from 70 % to 110 % for non-labelled chemicals (3).

1.7.2.   Repeatability and sensitivity of analytical method

Repeatability of the analytical method (excluding the initial extraction efficiency) to quantify test substance and transformation products can be checked by duplicate analysis of the same extract of the soil, incubated long enough for formation of transformation products.

The limit of detection (LOD) of the analytical method for the test substance and for the transformation products should be at least 0,01 mg· kg^-1 soil (as test substance) or 1 % of applied dose whichever is lower. The limit of quantification (LOQ) should also be specified.

1.7.3.   Accuracy of transformation data

Regression analysis of the concentrations of the test substance as a function of time gives the appropriate information on the reliability of the transformation curve and allows the calculation of the confidence limits for half-lives (in the case of pseudo first order kinetics) or DT50 values and, if appropriate, DT75 and DT90 values.

1.8.   DESCRIPTION OF THE METHOD

1.8.1.   Equipment and chemical reagents

Incubation systems consist of static closed systems or suitable flow-through systems (7)(17). Examples of suitable flow-through soil incubation apparatus and biometer-type flask are shown in Figures 1 and 2, respectively. Both types of incubation systems have advantages and limitations (7)(17).

Standard laboratory equipment is required and especially the following:

• — analytical instruments such as GLC, HPLC, TLC-equipment, including the appropriate detection systems for analysing radiolabelled or non-labelled substances or inverse isotopes dilution method,

• — instruments for identification purposes (e.g. MS, GC-MS, HPLC-MS, NMR, etc.),

• — liquid scintillation counter,

• — oxidiser for combustion of radioactive material,

• — centrifuge,

• — extraction apparatus (for example, centrifuge tubes for cold extraction and Soxhlet apparatus for continuous extraction under reflux),

• — instrumentation for concentrating solutions and extracts (e.g. rotating evaporator),

• — water bath,

• — mechanical mixing device (e.g. kneading machine, rotating mixer).

Chemical reagents used include, for example:

• — NaOH, analytical grade, 2 mol· dm^-3, or other appropriate base (e.g. KOH, ethanolamine),

• — H2SO4, analytical grade, 0,05 mol· dm^-3,

• — ethylene glycol, analytical grade,

• — solid absorption materials such as soda lime and polyurethane plugs,

• — organic solvents, analytical grade, such as acetone, methanol, etc.,

• — scintillation liquid.

1.8.2.   Test substance application

For addition to and distribution in soil, the test substance can be dissolved in water (deionised or distilled) or, when necessary, in minimum amounts of acetone or other organic solvents (6) in which the test substance is sufficiently soluble and stable. However, the amount of solvent selected should not have a significant influence on soil microbial activity (see Sections 1.5 and 1.9.2-1.9.3). The use of solvents which inhibit microbial activity, such as chloroform, dichloromethane and other halogenated solvents, should be avoided.

The test substance can also be added as a solid, e.g. mixed in quartz sand (6) or in a small sub-sample of the test soil which has been air-dried and sterilised. If the test substance is added using a solvent the solvent should be allowed to evaporate before the spiked sub-sample is added to the original non-sterile soil sample.

For general chemicals, whose major route of entry into soil is through sewage sludge/farming application, the test substance should be first added to sludge which is then introduced into the soil sample. (see Sections 1.9.2 and 1.9.3)

The use of formulated products is not routinely recommended. However, e.g. for poorly soluble test substances, the use of formulated material may be an appropriate alternative.

1.8.3.   Soils

1.8.3.1.   Soil selection

To determine the transformation pathway, a representative soil can be used; a sandy loam or silty loam or loam or loamy sand (according to FAO and USDA classification (18)), with a pH of 5,5 -8,0 , an organic carbon content of 0,5 -2,5 % and a microbial biomass of at least 1 % of total organic carbon is recommended (10).

For transformation rate studies at least three additional soils should be used representing a range of relevant soils. The soils should vary in their organic carbon content, pH, clay content and microbial biomass (10).

All soils should be characterised, at least, for texture (% sand, % silt, % clay) [according to FAO and USDA classification (18)], pH, cation exchange capacity, organic carbon, bulk density, water retention characteristic ([^E0159]) and microbial biomass (for aerobic studies only). Additional information on soil properties may be useful in interpreting the results. For determination of the soil characteristics the methods recommended in references (19)(20)(21)(22)(23) can be used. Microbial biomass should be determined by using the substrate-induced respiration (SIR) method (25)(26) or alternative methods (20).

1.8.3.2.   Collection, handling, and storage of soils