ANNEX 2I

‘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.

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

EC50 (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°C 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°C to minus 22°C 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 2 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: 0.1-0.5mm). 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 ten 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°C. 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, deionized 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 °C 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 per cent 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, Bruxelles.

(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, Italie, 18-20 janvier 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, 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.

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

EC50 (Median Effective Concentration): is the concentration of test substance in soil that results in a 50 per cent 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 °C 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 °C 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 2 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 ten 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°C. 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 2 000 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 °C. 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 1 to 2 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

3.1 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 per cent 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 mineralization 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.

DT50 (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.

DT75 (Disappearance Time 75): is the time within which the concentration of the test substance is reduced by 75 %.

DT90 (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 TEST 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 (19). 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 TEST 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 (20) 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

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, treatments with chemicals, treatments with organic and inorganic fertilisers, additions of biological materials or other contamination. If soils have been treated with the test substance or its structural analogues within the previous four years, these should not be used for transformation studies (10)(15).

The soil should be freshly collected from the field (from the A horizon or top 20 cm layer) with a soil water content which facilitates sieving. For soils other than those from paddy fields, sampling should be avoided during or immediately following long periods (> 30 days) of drought, freezing or flooding (14). Samples should be transported in a manner which minimises changes in soil water content and should be kept in the dark with free access of air, as much as possible. A loosely-tied polyethylene bag is generally adequate for this purpose.

The soil should be processed as soon as possible after sampling. Vegetation, larger soil fauna and stones should be removed prior to passing the soil through a 2 mm sieve which removes small stones, fauna and plant debris. Extensive drying and crushing of the soil before sieving should be avoided (15).

When sampling in the field is difficult in winter (soil frozen or covered by layers of snow), it may be taken from a batch of soil stored in the greenhouse under plant cover (e.g. grass or grass-clover mixtures). Studies with soils freshly collected from the field are strongly preferred, but if the collected and processed soil has to be stored prior to the start of the study storage conditions must be adequate and for a limited time only (4 ± 2 °C for a maximum of three months) to maintain microbial activity (21). Detailed instructions on collection, handling and storage of soils to be used for biotransformation experiments can be found in (8)(10)(15)(26)(27).

Before the processed soil is used for this test, it should be pre-incubated to allow germination and removal of seeds, and to re-establish equilibrium of microbial metabolism following the change from sampling or storage conditions to incubation conditions. A pre-incubation period between 2 and 28 days approximating the temperature and moisture conditions of the actual test is generally adequate (15). Storage and pre-incubation time together should not exceed three months.

1.9 PERFORMANCE OF THE TEST

1.9.1 Test conditions

1.9.1.1 Test temperature

During the whole test period, the soils should be incubated in the dark at a constant temperature representative of the climatic conditions where use or release will occur. A temperature of 20 ± 2 °C is recommended for all test substances which may reach the soil in temperate climates. The temperature should be monitored.

For chemicals applied or released in colder climates (e.g. in northern countries, during autumn/winter periods), additional soil samples should be incubated but at a lower temperature (e.g. 10 ± 2 °C).

1.9.1.2 Moisture content

For transformation tests under aerobic conditions, the soil moisture content (22) should be adjusted to and maintained at a pF between 2.0 and 2.5 (3). The soil moisture content is expressed as mass of water per mass of dry soil and should be regularly controlled (e.g. in 2 week intervals) by weighing of the incubation flasks and water losses compensated by adding water (preferably sterile-filtered tap water). Care should be given to prevent or minimise losses of test substance and/or transformation products by volatilisation and/or photodegradation (if any) during moisture addition.

For transformation tests under anaerobic and paddy conditions, the soil is water-saturated by flooding.

1.9.1.3 Aerobic incubation conditions

In the flow-through systems, aerobic conditions will be maintained by intermittent flushing or by continuously ventilating with humidified air. In the biometer flasks, exchange of air is maintained by diffusion.

1.9.1.4 Sterile aerobic conditions

To obtain information on the relevance of abiotic transformation of a test substance, soil samples may be sterilised (for sterilisation methods see references 16 and 29), treated with sterile test substance (e.g. addition of solution through a sterile filter) and aerated with humidified sterile air as described in section 1.9.1.3. For paddy soils, soil and water should be sterilised and the incubation should be carried out as described in section 1.9.1.6.

1.9.1.5 Anaerobic incubation conditions

To establish and maintain anaerobic conditions, the soil treated with the test substance and incubated under aerobic conditions for 30 days or one half-life or DT50 (whichever is shorter) is then water-logged (1-3 cm water layer) and the incubation system flushed with an inert gas (e.g. nitrogen or argon) (23). The test system must allow for measurements such as pH, oxygen concentration and redox potential and include trapping devices for volatile products. The biometer-type system must be closed to avoid entrance of air by diffusion.

1.9.1.6 Paddy incubation conditions

To study transformation in paddy rice soils, the soil is flooded with a water layer of about 1-5 cm and the test substance applied to the water phase (9). A soil depth of at least 5 cm is recommended. The system is ventilated with air as under aerobic conditions. pH, oxygen concentration and redox potential of the aqueous layer should be monitored and reported. A pre-incubation period of at least two weeks is necessary before commencing transformation studies (see section 1.8.3.2).

1.9.1.7 Test duration

The rate and pathway studies should normally not exceed 120 days (24) (3)(6)(8), because thereafter a decrease of the soil microbial activity with time would be expected in an artificial laboratory system isolated from natural replenishment. Where necessary to characterise the decline of the test substance and the formation and decline of major transformation products, studies can be continued for longer periods (e.g. 6 or 12 months) (8). Longer incubation periods should be justified in the test report and accompanied by biomass measurements during and at the end of these periods.

1.9.2 Performance of the test

About 50 to 200 g of soil (dry weight basis) are placed into each incubation flask (see Figures 1 and 2 in Annex 3) and the soil treated with the test substance by one of the methods described in section 1.8.2. When organic solvents are used for the application of the test substance, they should be removed from soil by evaporation. Then the soil is thoroughly mixed with a spatula and/or by shaking of the flask. If the study is conducted under paddy field conditions, soil and water should be thoroughly mixed after application of the test substance. Small aliquots (e.g. 1 g) of the treated soils should be analysed for the test substance to check for uniform distribution. For alternative method, see below.

The treatment rate should correspond to the highest application rate of a crop protection product recommended in the use instructions and uniform incorporation to an appropriate depth in the field (e.g. top 10 cm layer (25) of soil). For example, for chemicals foliarly or soil applied without incorporation, the appropriate depth for computing how much chemical should be added to each flask is 2.5 cm. For soil incorporated chemicals, the appropriate depth is the incorporation depth specified in the use instructions. For general chemicals, the application rate should be estimated based on the most relevant route of entry; for example, when the major route of entry in soil is through sewage sludge, the chemical should be dosed into the sludge at a concentration that reflects the expected sludge concentration and the amount of sludge added to the soil should reflect normal sludge loading to agricultural soils. If this concentration is not high enough to identify major transformation products, incubation of separate soil samples containing higher rates may be helpful, but excessive rates influencing soil microbial functions should be avoided (see sections 1.5 and 1.8.2).

Alternatively, a larger batch (i.e. 1 to 2 kg) of soil can be treated with the test substance, carefully mixed in an appropriate mixing machine and then transferred in small portions of 50 to 200 g into the incubation flasks (for example with the use of sample splitters). Small aliquots (e.g. 1 g) of the treated soil batch should be analysed for the test substance to check for uniform distribution. Such a procedure is preferred since it allows for more uniform distribution of the test substance into the soil.

Also untreated soil samples are incubated under the same conditions (aerobic) as the samples treated with the test substance. These samples are used for biomass measurements during and at the end of the studies.

When the test substance is applied to the soil dissolved in organic solvent(s), soil samples treated with the same amount of solvent(s) are incubated under the same conditions (aerobic) as the samples treated with the test substance. These samples are used for biomass measurements initially, during and at the end of the studies to check for effects of the solvent(s) on microbial biomass.

The flasks containing the treated soil are either attached to the flow-through system described in Figure 1 or closed with the absorption column shown in Figure 2 (see Annex 3).

1.9.3 Sampling and measurement

Duplicate incubation flasks are removed at appropriate time intervals and the soil samples extracted with appropriate solvents of different polarity and analysed for the test substance and/or transformation products. A well-designed study includes sufficient flasks so that two flasks are sacrificed at each sampling event. Also, absorption solutions or solid absorption materials are removed at various time intervals (7-day intervals during the first month and after one month in 17-day intervals) during and at the end of incubation of each soil sample and analysed for volatile products. Besides a soil sample taken directly after application (0-day sample) at least 5 additional sampling points should be included. Time intervals should be chosen in such a way that pattern of decline of the test substance and patterns of formation and decline of transformation products can be established (e.g. 0, 1, 3, 7 days; 2, 3 weeks; 1, 2, 3 months, etc.).

When using 14C-labelled test substance, non-extractable radioactivity will be quantified by combustion and a mass balance will be calculated for each sampling interval.

In the case of anaerobic and paddy incubation, the soil and water phases are analysed together for test substance and transformation products or separated by filtration or centrifugation before extraction and analysis.

1.9.4 Optional tests

Aerobic, non-sterile studies at additional temperatures and soil moistures may be useful for the estimation of the influence of temperature and soil moisture on the rates of transformation of a test substance and/or its transformation products in soil.

A further characterisation of non-extractable radioactivity can be attempted using, for example, supercritical fluid extraction.

2. DATA

2.1 TREATMENT OF RESULTS

The amounts of test substance, transformation products, volatile substances (in % only), and non-extractable should be given as % of applied initial concentration and, where appropriate, as mg⋅kg-1 soil (based on soil dry weight) for each sampling interval. A mass balance should be given in percentage of the applied initial concentration for each sampling interval. A graphical presentation of the test substance concentrations against time will allow an estimation of its transformation half-life or DT50. Major transformation products should be identified and their concentrations should also be plotted against time to show their rates of formation and decline. A major transformation product is any product representing ≥ 10 % of applied dose at any time during the study.

The volatile products trapped give some indication of the volatility potential of a test substance and its transformation products from soil.

More accurate determinations of half-lives or DT50 values and, if appropriate, DT75 and DT90 values should be obtained by applying appropriate kinetic model calculations. The half-life and DT50 values should be reported together with the description of the model used, the order of kinetics and the determination coefficient (r2). First order kinetics is favoured unless r2 < 0.7. If appropriate, the calculations should also be applied to the major transformation products. Examples of appropriate models are described in references 31 to 35.

In the case of rate studies carried out at various temperatures, the transformation rates should be described as a function of temperature within the experimental temperature range using the Arrhenius relationship of the form:

where ln A and B are regression constants from the intercept and slope, respectively, of a best fit line generated from linearly regressing ln k against 1/T, k is the rate constant at temperature T and T is the temperature in Kelvin. Care should be given to the limited temperature range in which the Arrehenius relationship will be valid in case transformation is governed by microbial action.

2.2 EVALUATION AND INTERPRETATION OF RESULTS

Although the studies are carried out in an artificial laboratory system, the results will allow estimation of the rate of transformation of the test substance and also of rate of formation and decline of transformation products under field conditions (36)(37).

A study of the transformation pathway of a test substance provides information on the way in which the applied substance is structurally changed in the soil by chemical and microbial reactions.

3. REPORTING

3.1 TEST REPORT

The test report must include:

Test substance: — common name, chemical name, CAS number, structural formula (indicating position of label(s) when radiolabelled material is used) and relevant physical-chemical properties (see section 1.5); — purity (impurities) of test substance; — radiochemical purity of labelled chemical and specific activity (where appropriate);

Reference substances: — chemical name and structure of reference substances used for the characterisation and/or identification of transformation product;

Test soils: — details of collection site; — date and procedure of soil sampling; — properties of soils, such as pH, organic carbon content, texture (% sand, % silt, % clay), cation exchange capacity, bulk density, water retention characteristic, and microbial biomass; — length of soil storage and storage conditions (if stored);

Test conditions: — dates of the performance of the studies; — amount of test substance applied; — solvents used and method of application for the test substance; — weight of soil treated initially and sampled at each interval for analysis; — description of the incubation system used; — air flow rates (for flow-through systems only); — temperature of experimental set-up; — soil moisture content during incubation; — microbial biomass initially, during and at the end of the aerobic studies; — pH, oxygen concentration and redox potential initially, during and at the end of the anaerobic and paddy studies; — method(s) of extraction; — methods for quantification and identification of the test substance and major transformation products in soil and absorption materials; — number of replicates and number of controls.

Results: — result of microbial activity determination; — repeatability and sensitivity of the analytical methods used; — rates of recovery (% values for a valid study are given in section 1.7.1); — tables of results expressed as % of applied initial dose and, where appropriate, as mg⋅kg-1 soil (on a dry weight basis); — mass balance during and at the end of the studies; — characterisation of non-extractable (bound) radioactivity or residues in soil; — quantification of released CO2 and other volatile compounds; — plots of soil concentrations versus time for the test substance and, where appropriate, for major transformation products; — half-life or DT50, DT75 and DT90 for the test substance and, where appropriate, for major transformation products including confidence limits; — estimation of abiotic degradation rate under sterile conditions; — an assessment of transformation kinetics for the test substance and, where appropriate, for major transformation products; — proposed pathways of transformation, where appropriate; — discussion and interpretation of results; — raw data (i.e. sample chromatograms, sample calculations of transformation rates and means used to identify transformation products).

4. REFERENCES

(1) US- Environmental Protection Agency (1982). Pesticide Assessment Guidelines, Subdivision N. Chemistry: Environmental Fate.

(2) Agriculture Canada (1987). Environmental Chemistry and Fate. Guidelines for registration of pesticides in Canada.

(3) European Union (EU) (1995). Commission Directive 95/36/EC of 14 July 1995 amending Council Directive 91/414/EEC concerning the placing of plant protection products on the market. Annex II, Part A and Annex III, Part A: Fate and Behaviour in the Environment.

(4) Dutch Commission for Registration of Pesticides (1995). Application for registration of a pesticide. Section G: Behaviour of the product and its metabolites in soil, water and air.

(5) BBA (1986). Richtlinie für die amtliche Prüfung von Pflanzenschutzmitteln, Teil IV, 4-1. Verbleib von Pflanzenschutzmitteln im Boden - Abbau, Umwandlung und Metabolismus.

(6) ISO/DIS 11266-1 (1994). Soil Quality -Guidance on laboratory tests for biodegradation of organic chemicals in soil - Part 1 : Aerobic conditions.

(7) ISO 14239 (1997). Soil Quality – Laboratory incubation systems for measuring the mineralization of organic chemicals in soil under aerobic conditions.

(8) SETAC (1995). Procedures for Assessing the Environmental Fate and Ecotoxicity of Pesticides. Mark R. Lynch, Ed.

(9) MAFF - Japan 2000 - Draft Guidelines for transformation studies of pesticides in soil - Aerobic metabolism study in soil under paddy field conditions (flooded).

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

(11) Guth, J.A. (1980). The study of transformations. In Interactions between Herbicides and the Soil (R.J. Hance, Ed.), Academic Press, 123-157.

(12) DFG: Pesticide Bound Residues in Soil. Wiley – VCH (1998).

(13) T.R. Roberts: Non-extractable pesticide residue in soils and plants. Pure Appl. Chem. 56, 945-956 (IUPAC 1984).

(14) OECD Test Guideline 304 A: Inherent Biodegradability in Soil (adopted 12 May 1981).

(15) ISO 10381-6 (1993). Soil Quality - Sampling - Part 6: Guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory.

(16) Annex V to Dir. 67/548/EEC.

(17) Guth, J.A. (1981). Experimental approaches to studying the fate of pesticides in soil. In Progress in Pesticide Biochemistry. D.H. Hutson, T.R. Roberts, Eds. J. Wiley & Sons. Vol 1, 85-114.

(18) Soil Texture Classification (US and FAO systems): Weed Science, 33, Suppl. 1 (1985) and Soil Sci. Soc. Amer. Proc. 26:305 (1962).

(19) Methods of Soil Analysis (1986). Part 1, Physical and Mineralogical Methods. A. Klute, Ed.) Agronomy Series No 9, 2nd Edition.

(20) Methods of Soil Analysis (1982). Part 2, Chemical and Microbiological Properties. A.L. Page, R.H. Miller and D.R. Kelney, Eds. Agronomy Series No 9, 2nd Edition.

(21) ISO Standard Compendium Environment (1994). Soil Quality - General aspects; chemical and physical methods of analysis; biological methods of analysis. First Edition.

(22) Mückenhausen, E. (1975). Die Bodenkunde und ihre geologischen, geomorphologischen, mineralogischen und petrologischen Grundlagen. DLG-Verlag, Frankfurt, Main.

(23) Scheffer, F., Schachtschabel, P. (1975). Lehrbuch der Bodenkunde. F. Enke Verlag, Stuttgart.

(24) Anderson, J.P.E., Domsch, K.H. (1978) A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biol. Biochem. 10, 215-221.

(25) ISO 14240-1 and 2 (1997). Soil Quality - Determination of soil microbial biomass - Part 1: Substrate-induced respiration method. Part 2: fumigation-extraction method.

(26) Anderson, J.P.E. (1987). Handling and storage of soils for pesticide experiments. In Pesticide Effects on Soil Microflora. L. Somerville, M.P. Greaves, Eds. Taylor & Francis, 45-60.

(27) Kato, Yasuhiro. (1998). Mechanism of pesticide transformation in the environment: Aerobic and bio-transformation of pesticides in aqueous environment. Proceedings of the 16th Symposium on Environmental Science of Pesticide, 105-120.

(28) Keuken O., Anderson J.P.E. (1996). Influence of storage on biochemical processes in soil. In Pesticides, Soil Microbiology and Soil Quality, 59-63 (SETAC-Europe).

(29) Stenberg B., Johansson M., Pell M., Sjödahl-Svensson K., Stenström J., Torstensson L. (1996). Effect of freeze and cold storage of soil on microbial activities and biomass. In Pesticides, Soil Microbiology and Soil Quality, 68-69 (SETAC-Europe).

(30) Gennari, M., Negre, M., Ambrosoli, R. (1987). Effects of ethylene oxide on soil microbial content and some chemical characteristics. Plant and Soil 102, 197-200.

(31) Anderson, J.P.E. (1975). Einfluss von Temperatur und Feuchte auf Verdampfung, Abbau und Festlegung von Diallat im Boden. Z. PflKrankh Pflschutz, Sonderheft VII, 141-146.

(32) Hamaker, J.W. (1976). The application of mathematical modelling to the soil persistence and accumulation of pesticides. Proc. BCPC Symposium: Persistence of Insecticides and Herbicides, 181-199.

(33) Goring, C.A.I., Laskowski, D.A., Hamaker, J.W., Meikle, R.W. (1975). Principles of pesticide degradation in soil. In “Environmental Dynamics of Pesticides”. R. Haque and V.H. Freed, Eds., 135-172.

(34) Timme, G., Frehse, H., Laska, V. (1986). Statistical interpretation and graphic representation of the degradational behaviour of pesticide residues. II. Pflanzenschutz - Nachrichten Bayer 39, 188-204.

(35) Timme, G., Frehse, H. (1980). Statistical interpretation and graphic representation of the degradational behaviour of pesticide residues. I. Pflanzenschutz - Nachrichten Bayer 33, 47-60.

(36) Gustafson D.I., Holden L.R. (1990). Non-linear pesticide dissipation in soil; a new model based on spatial variability. Environm. Sci. Technol. 24, 1032-1041.

(37) Hurle K., Walker A. (1980). Persistence and its prediction. In Interactions between Herbicides and the Soil (R.J. Hance, Ed.), Academic Press, 83-122.

ANNEX 1 WATER TENSION, FIELD CAPACITY (FC) AND WATER HOLDING CAPACITY (WHC) (26 31) Height of Water Column [cm] pF (27) bar (28) Remarks 107 7 104 Dry Soil 1,6 · 104 4,2 16 Wilting point 104 4 10 103 3 1 6 · 102 2,8 0,6 3,3 · 102 2,5 0,33 (29) 102 2 0,1 Range of Field capacity (30) 60 1,8 0,06 33 1,5 0,033 10 1 0,01 WHC (approximation) 1 0 0,001 Water saturated soil Water tension is measured in cm water column or in bar . Due to the large range of suction tension it is expressed simply as pF value which is equivalent to the logarithm of cm water column. Field capacity is defined as the amount of water which can be stored against gravity by a natural soil 2 days after a longer raining period or after sufficient irrigation. It is determined in undisturbed soil in situ in the field. The measurement is thus not applicable to disturbed laboratory soil samples. FC values determined in disturbed soils may show great systematic variances. Water holding capacity (WHC) is determined in the laboratory with undisturbed and disturbed soil by saturating a soil column with water by capillary transport. It is particularly useful for disturbed soils and can be up to 30 % greater than field capacity (1). It is also experimentally easier to determine than reliable FC-values.

ANNEX 2 SOIL MOISTURE CONTENTS (g water per 100 g dry soil) OF VARIOUS SOIL TYPES FROM VARIOUS COUNTRIES Soil Moisture Content at Soil Type Country WHC (26 31) pF = 1,8 pF = 2,5 Sand Germany 28,7 8,8 3,9 Loamy sand Germany 50,4 17,9 12,1 Loamy sand Switzerland 44,0 35,3 9,2 Silt loam Switzerland 72,8 56,6 28,4 Clay loam Brazil 69,7 38,4 27,3 Clay loam Japan 74,4 57,8 31,4 Sandy loam Japan 82,4 59,2 36,0 Silt loam USA 47,2 33,2 18,8 Sandy loam USA 40,4 25,2 13,3

ANNEX 3 Figure 1 Example of a flow-through apparatus to study transformation of chemicals in soil (1)(2) 1: needle valve 2: gas washing bottle containing water 3: ultramembrane (sterile conditions only), pore size 0.2 μm 4: soil metabolism flask (water-logged only for anaerobic and paddy conditions;) 5: ethylene glycol trap for organic volatile compounds 6: sulphuric acid trap for alkaline volatile compounds 7, 8: sodium hydroxide trap for CO2 & other acidic volatiles 9: flow meter. Figure 2 Example of a biometer-type flask for studying the transformation of chemicals in soil (3) (1) Guth, J.A. (1980). The study of transformations. In Interactions between Herbicides and the Soil (R.J. Hance, Ed.), Academic Press, 123-157. (2) Guth, J.A. (1981). Experimental approaches to studying the fate of pesticides in soil. In Progress in Pesticide Biochemistry. D.H. Hutson, T.R. Roberts, Eds. J. Wiley & Sons. Vol 1, 85-114. (3) Anderson, J.P.E. (1975). Einfluss von Temperatur und Feuchte auf Verdampfung, Abbau und Festlegung von Diallat im Boden. Z. PflKrankh Pflschutz, Sonderheft VII, 141-146.

C.24. AEROBIC AND ANAEROBIC TRANSFORMATION IN AQUATIC SEDIMENT SYSTEMS

1. METHOD

This test method is a replicate of the OECD TG 308 (2002).

1.1 INTRODUCTION

Chemicals can enter shallow or deep surface waters by such routes as direct application, spray drift, run-off, drainage, waste disposal, industrial, domestic or agricultural effluent and atmospheric deposition. This Testing Method describes a laboratory method to assess aerobic and anaerobic transformation of organic chemicals in aquatic sediment systems. It is based on existing Guidelines (1)(2)(3)(4)(5)(6). An OECD Workshop on Soil/Sediment Selection, held in Belgirate, Italy in 1995 (7) agreed, in particular, on the number and type of sediments for use in this test. It also made recommendations relating to collection, handling and storage of sediment samples, based on the ISO Guidance (8). Such studies are required for chemicals which are directly applied to water or which are likely to reach the aqueous environment by the routes described above.

The conditions in natural aquatic sediment systems are often aerobic in the upper water phase. The surface layer of sediment can be either aerobic or anaerobic, whereas the deeper sediment is usually anaerobic. To encompass all of these possibilities both aerobic and anaerobic tests are described in this document. The aerobic test simulates an aerobic water column over an aerobic sediment layer that is underlain with an anaerobic gradient. The anaerobic test simulates a completely anaerobic water-sediment system. If circumstances indicate that it is necessary to deviate significantly from these recommendations, for example by using intact sediment cores or sediments that may have been exposed to the test substance, other methods are available for this purpose (9).

1.2 DEFINITIONS

Standard International (SI) units should be used in any case.

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

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

Bound residues:“Bound residues” represent compounds in soil, plant or animal that persist in the matrix in the form of the parent substance or its metabolite(s) after extractions. 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 (10) [modified from IUPAC 1984 (11)].

Aerobic transformation: (oxidising): reactions occurring in the presence of molecular oxygen (12).

Anaerobic transformation: (reducing): reactions occurring under exclusion of molecular oxygen (12).

Natural waters: are surface waters obtained from ponds, rivers, streams, etc.

Sediment: is a mixture of mineral and organic chemical constituents, the latter containing compounds of high carbon and nitrogen content and of high molecular masses. It is deposited by natural water and forms an interface with that water.

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

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 initial concentration.

DT50 (Disappearance Time 50): is the time within which the initial concentration of the test substance is reduced by 50 %.

DT75 (Disappearance Time 75): is the time within which the initial concentration of the test substance is reduced by 75 %.

DT90 (Disappearance Time 90): is the time within which the initial concentration of the test substance is reduced by 90 %.

1.3 REFERENCE SUBSTANCES

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

1.4 INFORMATION ON THE TEST SUBSTANCE

Non-labelled or isotope-labelled test substance can be used to measure the rate of transformation although labelled material is preferred. 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 (32). The chemical and/or radiochemical purity of the test substance should be at least 95 %.

Before carrying out a test, the following information about 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) adsorption coefficient (Kd, Kf or Koc, where appropriate) (Method C.18);

(f) hydrolysis (Method C.7);

(g) dissociation constant (pKa) [OECD Guideline 112] (13);

(h) chemical structure of the test substance and position of the isotope-label(s), if applicable.

Note:

The temperature at which these measurements were made should be reported.

Other useful information may include data on toxicity of the test substance to microorganisms, data on ready and/or inherent biodegradability, and data on aerobic and anaerobic transformation in soil.

Analytical methods (including extraction and clean-up methods) for identification and quantification of the test substance and its transformation products in water and in sediment should be available (see section 1.7.2).

1.5 PRINCIPLE OF THE TEST METHOD

The method described in this test employs an aerobic and an anaerobic aquatic sediment (see Annex 1) system which allows:

(i) the measurement of the transformation rate of the test substance in a water-sediment system,

(ii) the measurement of the transformation rate of the test substance in the sediment,

(iii) the measurement of the mineralisation rate of the test substance and /or its transformation products (when 14C-labelled test substance is used),

(iv) the identification and quantification of transformation products in water and sediment phases including mass balance (when labelled test substance is used),

(v) the measurement of the distribution of the test substance and its transformation products between the two phases during a period of incubation in the dark (to avoid, for example, algal blooms) at constant temperature. Half-lives, DT50, DT75 and DT90 values are determined where the data warrant, but should not be extrapolated far past the experimental period (see section 1.2).

At least two sediments and their associated waters are required for both the aerobic and the anaerobic studies respectively (7). However, there may be cases where more than two aquatic sediments should be used, for example, for a chemical that may be present in freshwater and/or marine environments.

1.6 APPLICABILITY OF THE TEST

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

The method has been applied so far to study the transformation of chemicals in fresh waters and sediments, but in principle can also be applied to estuarine/marine systems. It is not suitable to simulate conditions in flowing water (e.g. rivers) or the open sea.

1.7 QUALITY CRITERIA

1.7.1 Recovery

Extraction and analysis of, at least, duplicate water and sediment 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 (when labelled material is used). Recoveries should range from 90 % to 110 % for labelled chemicals (6) and from 70 % to 110 % for non-labelled chemicals.

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 water or the sediment samples which were incubated sufficiently 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 in water or sediment (as test substance) or 1 % of the initial amount applied to a test system 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 accuracy of the transformation curve and allows the calculation of the confidence limits for half-lives (if pseudo first-order kinetics apply) or DT50 values and, if appropriate, DT75 and DT90 values.

1.8 DESCRIPTI