Commission Directive 2004/73/EC of 29 April 2004 adapting to technical progress for the twenty-ninth time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (Text with EEA relevance)

Status:

This is the original version (as it was originally adopted).

ANNEX 2I

C.21. SOIL MICROORGANISMS: NITROGEN TRANSFORMATION TEST

1. METHOD

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

1.1INTRODUCTION

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) SET AC (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 EC_x values, where x is defined % effect.

1.2DEFINITIONS

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

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

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.

1.3REFERENCE SUBSTANCES

None.

1.4PRINCIPLE 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 EC_X values are calculated (i.e. EC₅₀, EC₂₅ and/or EC₁₀). See definitions.

1.5VALIDITY 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%.

1.6DESCRIPTION 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 (3000 g) or filtration device (using nitrate-free filter paper);
instrument of adequate sensitivity and reproducibility for nitrate analysis.

1.6.2 Selection and number of soils

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

sand content: not less than 50% and not greater than 75%;
pH: 5.5 -7.5;
organic carbon content: 0.5 - 1.5%;
the microbial biomass should be measured (8)(9) and its carbon content should be at least 1 % of the total soil organic carbon.

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

1.6.3 Collection and storage of soil samples

1.6.3.1 Collection

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

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

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

1.6.3.2 Storage

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

1.6.4 Handling and preparation of soil for the test

1.6.4.1 Pre-incubation

If the soil was stored (see section 1.6.3.2), pre-incubation is recommended for a period between 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 EC_x values.

1.7PERFORMANCE OF THE TEST

1.7.1 Conditions of exposure

1.7.1.1 Treatment and control

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

1.7.1.2 Incubation of soil samples

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

1.7.1.3 Test conditions and duration

The test is carried out in the dark at room temperature of 20±2^oC. The moisture content of soil samples should be maintained during the test between 40% and 60% of the maximum water holding capacity of the soil (see section 1.6.4.2) with a range of ±5%. Distilled, 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 EC_x 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 EC_x 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 KC1 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 cenrrifuged or filtered and the liquid phases are analysed for nitrate. Particle-free liquid extracts can be stored prior to analysis at minus 20±5 ^oC for up to six months.

2 DATA

2.1TREATMENT 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 EC_x 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 EC_x values. Confidence limits (p = 0.95) for the calculated EC_x are also determined using standard procedures (10)(l 1)(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 EC_x calculations.

2.2INTERPRETATION 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);
micfobial 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 EC_x (i.e. EC₂₅ or EC₁₀) with confidence intervals, and a graph of the dose-response curve;
statistical treatment of results;
all information and observations helpful for the interpretation of the results.

4 REFERENCES

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

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

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

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

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

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

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

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

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

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

1.1INTRODUCTION

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 SET AC (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 EC_x values, where x is defined % effect.

1.2DEFINITIONS

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

EC_x (Effective Concentration): is the concentration of the test substance in soil that results in a x 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.

1.3REFERENCE SUBSTANCES

None.

1.4PRINCIPLE 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 EC_x values are calculated (i.e. EC₅₀, EC₂₅ and/or EC₁₀). See definitions.

1.5VALIDITY 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%.

1.6DESCRIPTION OF THE TEST METHOD

1.6.1 Apparatus

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

1.6.3.2 Storage

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

1.6.4 Handling and preparation of soil for the test

1.6.4.1 Pre-incubation

If the soil was stored (see sections 1.6.4.2 and 1.7.1.3), pre-incubation is recommended for a period between 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

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 EC_x values.

1.7PERFORMANCE OF THE TEST

1.7.1 Conditions of exposure

1.7.1.1 Treatment and control

1.7.1.2 Incubation of soil samples

1.7.1.3 Test conditions and duration

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 EC_x 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 EC_x 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, 2000 mg to 4000 mg glucose per kg dry weight soil is usually sufficient. The glucose can be ground to a powder with clean quartz sand (10 g sand/kg dry weight soil) and homogeneously mixed with the soil.

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

2.1TREATMENT 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 EC_x 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 EC_x values. Confidence limits (p = 0.95) for the calculated EC_x are also determined using standard procedures (15)(16)(17).

2.2INTERPRETATION OF RESULTS

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

3 REPORTING

TEST REPORT

The test report must include the following information:

Complete identification of the soil used including:

geographical reference of the site (latitude, longitude);
information on the history of the site (i.e. vegetation cover, treatments with crop protection products, treatments with fertilisers, accidental contamination, etc.)
use pattern (e.g. agricultural soil, forest, etc.);
depth of sampling (cm);
sand/silt/clay content (% dry weight);
pH (in water);
organic carbon content (% dry weight);
nitrogen content (% dry weight);
cation exchange capacity (mmol/kg);
initial microbial biomass in terms of percentage of the total organic carbon;
reference of the methods used for the determination of each parameter;
all information relating to the collection and storage of soil samples;
details of pre-incubation of soil if any.

Test substance:

physical nature and, where relevant, physical-chemical properties;
chemical identification data, where relevant, including structural formula, purity (i.e. for crop protection products the percentage of active ingredient), nitrogen content.

Test conditions:

details of the amendment of soil with organic substrate;
number of concentrations of test chemical used and, where appropriate, justification of the selected concentrations;
details of the application of test substance to soil;
incubation temperature;
soil moisture content at the beginning and during the test;
method of soil incubation used (i.e. as bulk or as a series of individual subsamples);
number of replicates;
sampling times.

Results:

method and equipment used for measurement of respiration rates;
tabulated data including individual and mean values for quantities of carbon dioxide or oxygen;
variation between the replicates in treated and control samples;
explanations of corrections made in the calculations, if relevant;
the percent variation of glucose-induced respiration rates at each sampling time or, if appropriate, the EC₅₀ with 95 per cent confidence limit, other EC_x (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 N^o 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)

1.1INTRODUCTION

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)(l1). 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.

1.2DEFINITIONS

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: t_0.5, 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 to.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%.

1.3REFERENCE SUBSTANCES

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

1.4APPLICABILITY 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.

1.5INFORMATION 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(1). 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)

pK_a 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.

1.6PRINCIPLE 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 esstablished.

1.7QUALITY 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.

1.8DESCRIPTION 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(2) 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^oC for a maximum of three months) to maintain microbial activity(3). 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.

1.9PERFORMANCE 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 ^oC 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 ^oC).

1.9.1.2 Moisture content

For transformation tests under aerobic conditions, the soil moisture content(4) 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)(5). 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(6) (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(7) 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

2.1TREATMENT 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 mgkg-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:

k = A·e^-B/T or ,

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.

2.2EVALUATION 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

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 mgkg-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 fur 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 (RJ. 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 16^th 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. PflRrankh 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)(1)

Height of Water Column [cm]	pF^a	bar^b	Remarks
^a pF = log of cm water column.
^b 1 bar = 10⁵Pa.
^c Corresponds to an approximate water content of 10% in sand, 35% in loam and 45% in clay.
^d Field capacity is not constant but varies with soil type between pF 1.5 and 2.5.
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^c
10²	2	0.1	Range of Field capacity^d WHC (approximation) Water saturated soil
60	1.8	0.06
33	1.5	0.033
10	1	0.01
1	0	0.001

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.

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

ANNEX 2 SOIL MOISTURE CONTENTS (g water per 100 g dry soil) OF VARIOUS SOIL TYPES FROM VARIOUS COUNTRIES

Soil Moisture Content at
^a Water Holding Capacity
Soil Moisture Content at			Soil Type	Country
		WHC^a	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)

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).

1.1INTRODUCTION

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).

1.2DEFINITIONS

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, t_0.5, 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%.

1.3REFERENCE SUBSTANCES

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

1.4INFORMATION 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(8). 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 (K_d, K_f or K_oc, where appropriate) (Method C.18);

(f)

hydrolysis (Method C.7);

(g)

dissociation constant (pK_a) [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).

1.5PRINCIPLE 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.

1.6APPLICABILITY 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.

1.7QUALITY 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 mgkg^-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.

1.8DESCRIPTION OF THE METHOD

1.8.1 Test system and apparatus

The study should be performed in glass containers (e.g. bottles, centrifuge tubes), unless preliminary information (such as n-octanol-water partition coefficient, sorption data, etc.) indicates that the test substance may adhere to glass, in which case an alternative material (such as Teflon) may have to be considered. Where the test substance is known to adhere to glass, it may be possible to alleviate this problem using one or more of the following methods:

determine the mass of test substance and transformation products sorbed to glass;
ensure a solvent wash of all glassware at the end of the test;
use of formulated products (see also section 1.9.2);
use an increased amount of co-solvent for addition of test substance to the system; if a co-solvent is used it should be a co-solvent that does not solvolyse the test substance.

Examples of typical test apparatus, i.e. gas flow-through and biometer-type systems, are shown in Annexes 2 and 3, respectively (14). Other useful incubation systems are described in reference 15. The design of the experimental apparatus should permit the exchange of air or nitrogen and the trapping of volatile products. The dimensions of the apparatus must be such that the requirements of the test are complied with (see section 1.9.1). Ventilation may be provided by either gentle bubbling or by passing air or nitrogen over the water surface. In the latter case gentle stirring of the water from above may be advisable for better distribution of the oxygen or nitrogen in the water. CO₂-free air should not be used as this can result in increases in the pH of the water. In either case, disturbance of the sediment is undesirable and should be avoided as far as possible. Slightly volatile chemicals should be tested in a biometer-type system with gentle stirring of the water surface. Closed vessels with a headspace of either atmospheric air or nitrogen and internal vials for the trapping of volatile products can also be used (16). Regular exchange of the headspace gas is required in the aerobic test in order to compensate for the oxygen consumption by the biomass.

Suitable traps for collecting volatile transformation products include but are not restricted to 1 moldm^-3 solutions of potassium hydroxide or sodium hydroxide for carbon dioxide(9) and ethylene glycol, ethanolamine or 2% paraffin in xylene for organic compounds. Volatiles formed under anaerobic conditions, such as methane, can be collected, for example, by molecular sieves. Such volatiles can be combusted, for example, to CO₂ by passing the gas through a quartz tube filled with CuO at a temperature of 900 ^oC and trapping the CO₂ formed in an absorber with alkali (17).

Laboratory instrumentation for chemical analysis of test substance and transformation products is required (e.g. gas liquid chromatography (GLC), high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), mass spectroscopy (MS), gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), etc.), including detection systems for radiolabelled or non-labelled chemicals as appropriate. When radiolabelled material is used a liquid scintillation counter and combustion oxidiser (for the combustion of sediment samples prior to analysis of radioactivity) will also be required.

Other standard laboratory equipment for physical-chemical and biological determinations (see section Table 1, section 1.8.2.2), glassware, chemicals and reagents are required as appropriate.

1.8.2 Selection and number of aquatic sediments

The sampling sites should be selected in accordance with the purpose of the test in any given situation. In selecting sampling sites, the history of possible agricultural, industrial or domestic inputs to the catchment and the waters upstream must be considered. Sediments should not be used if they have been contaminated with the test substance or its structural analogues within the previous 4 years.

1.8.2.1 Sediment selection

Two sediments are normally used for the aerobic studies (7). The two sediments selected should differ with respect to organic carbon content and texture. One sediment should have a high organic carbon content (2.5-7.5%) and a fine texture, the other sediment should have a low organic carbon content (0.5-2.5%) and a coarse texture. The difference between the organic carbon contents should normally be at least 2%. "Fine texture" is defined as a [clay + siltj(10) content of >50% and "coarse texture" is defined as a [clay + silt] content of <50%. The difference in [clay + silt] content for the two sediments should normally be at least 20%. In cases, where a chemical may also reach marine waters, at least one of the water-sediment systems should be of marine origin.

For the strictly anaerobic study, two sediments (including their associated waters) should be sampled from the anaerobic zones of surface water bodies (7). Both the sediment and the water phases should be handled and transported carefully under exclusion of oxygen.

Other parameters may be important in the selection of sediments and should be considered on a case-by-case basis. For example, the pH range of sediments would be important for testing chemicals for which transformation and/or sorption may be pH-dependent. pH-dependency of sorption might be reflected by the pK_a of the test substance.

1.8.2.2 Characterisation of water-sediment samples

Key parameters that must be measured and reported (with reference to the method used) for both water and sediment, and the stage of the test at which those parameters are to be determined are summarised in the Table hereafter. For information, methods for determination of these parameters are given in references (18)(19)(20)(21).

In addition, other parameters may need to be measured and reported on a case by case basis (e.g. for freshwater: particles, alkalinity, hardness, conductivity, NO₃/PO₄ (ratio and individual values); for sediments: cation exchange capacity, water holding capacity, carbonate, total nitrogen and phosphorus; and for marine systems: salinity). Analysis of sediments and water for nitrate, sulfate, bioavailable iron, and possibly other electron acceptors may be also useful in assessing redox conditions, especially in relation to anaerobic transformation.

Measurement of parameters for characterisation of water-sediment samples (7)(22)(23)

Parameter	Stage of test procedure
^a Recent research results have shown that measurements of water oxygen concentrations and of redox potentials have neither a mechanistic nor a predictive value as far as growth and development of microbial populations in surface waters are concerned (24)(25). Determination of the biochemical oxygen demand (BOD, at field sampling, start and end of test) and of concentrations of micro/macro nutrients Ca, Mg and Mn (at start and end of test) in water and the measurement of total N and total P in sediments (at field sampling and end of test) may be better tools to interpret and evaluate aerobic biotransformation rates and routes.
^b Microbial respiration rate method (26), fumigation method (27) or plate count measurements (e.g. bacteria, actinomycetes, fungi and total colonies) for aerobic studies; methanogenesis rate for anaerobic studies.
Parameter	field sampling	post-handling	start of acclimation	start of test	during test	end of test
Water
Origin/source	X
Temperature	X
PH	X		X	X	X	X
TOC			X	X		X
O₂ concentration^a	X		X	X	X	X
Redox Potential^a			X	X	X	X
Sediment
Origin/source	X
Depth of layer	X
PH		X	X	X	X	X
Particle size distribution		X
TOC		X	X	X		X
Microbial biomass^b		X		X		X
Redox potential^a	Observation (colour/smell)		X	X	X	X

1.8.3 Collection, Handling and Storage

1.8.3.1 Collection

The draft ISO guidance on sampling of bottom sediment (8) should be used for sampling of sediment. Sediment samples should be taken from the entire 5 to 10 cm upper layer of the sediment. Associated water should be collected from the same site or location and at the same time as the sediment. For the anaerobic study, sediment and associated water should be sampled and transported under exclusion of oxygen (28)(see section 1.8.2.1). Some sampling devices are described in the literature (8)(23).

1.8.3.2 Handling

The sediment is separated from the water by filtration and the sediment wet-sieved to a 2 mm-sieve using excess location water that is then discarded. Then known amounts of sediments and water are mixed at the desired ratio (see section 1.9.1) in incubation flasks and prepared for the acclimation period (see section 1.8.4). For the anaerobic study, all handling steps have to be done under exclusion of oxygen (29)(30)(31)(32)(33).

1.8.3.3 Storage

Use of freshly sampled sediment and water is strongly recommended, but if storage is necessary, sediment and water should be sieved as described above and stored together, water-logged (6-10 cm water layer), in the dark, at 4 ± 2^oC(11) for a maximum of 4 weeks (7)(8)(23). Samples to be used for aerobic studies should be stored with free access of air (e.g. in open containers), whereas those for anaerobic studies under exclusion of oxygen. Freezing of sediment and water and drying-out of the sediment must not occur during transportation and storage.

1.8.4 Preparation of the sediment/water samples for the test

A period of acclimation should take place prior to adding the test substance, with each sediment/water sample being placed in the incubation vessel to be used in the main test, and the acclimation to be carried out under exactly the same conditions as the test incubation (see section 1.9.1). The acclimation period is the time needed to reach reasonable stability of the system, as reflected by pH, oxygen concentration in water, redox potential of the sediment and water, and macroscopic separation of phases. The period of acclimation should normally last between one week and two weeks and should not exceed four weeks. Results of determinations performed during this period should be reported.

1.9PERFORMANCE OF THE TEST

1.9.1 Test conditions

The test-should be performed in the incubation apparatus (see section 1.8.1) with a water sediment volume ratio between 3:1 and 4:1, and a sediment layer of 2.5 cm (± 0.5 cm).¹ A minimum amount of 50 g of sediment (dry weight basis) per incubation vessel is recommended.

The test should be performed in the dark at a constant temperature in the range of 10 to 30 ^oC. A temperature of (20 ± 2)^oC is appropriate. Where appropriate, an additional lower temperature (e.g. 10^oC) may be considered on a case-by-case basis, depending on the information required from the test. Incubation temperature should be monitored and reported.

1.9.2 Treatment and application of test substance

One test concentration of chemical is used(12). For crop protection chemicals applied directly to water bodies, the maximum dosage on the label should be taken as, the maximum application rate calculated on the basis of the surface area of the water in the test vessel. In all other cases, the concentration to be used should be based on predictions from environmental emissions. Care must be taken to ensure that an adequate concentration of test substance is applied in order to characterise the route of transformation and the formation and decline of transformation products. It may be necessary to apply higher doses (e.g. 10 times) in situations where test substance concentrations are close to limits of detection at the start of the study and/or where major transformation products could not readily be detected when present at 10% of the test substance application rate. However, if higher test concentrations are used they should not have a significant adverse effect on the microbial activity of the water-sediment system. In order to achieve a constant concentration of test substance in vessels of differing dimensions an adjustment to the quantity of the material applied may be considered appropriate, based on the depth of the water column in the vessel in relation to the depth of water in the field (which is assumed to be 100 cm, but other depths can be used). See Annex 4 for an example calculation.

Ideally the test substance should be applied as an aqueous solution into the water phase of the test system. If unavoidable, the use of low amounts of water miscible solvents (such as acetone, ethanol) is permitted for application and distribution of the test substance, but this should not exceed 1% v/v and should not have adverse effects on microbial activity of the test system. Care should be exercised in generating the aqueous solution of the test substance - use of generator columns and pre-mixing may be appropriate to ensure complete homogeneity. Following addition of the aqueous solution to the test system, gentle mixing of the water phase is recommended, disturbing the sediment as little as possible.

The use of formulated products is not routinely recommended as the formulation ingredients may affect the distribution of the test substance and/or transformation products between water and sediment phases. However, for poorly water-soluble test substances, the use of formulated material may be an appropriate alternative.

The number of incubation vessels depends on the number of sampling times (see section 1.9.3). A sufficient number of test systems should be included so that two systems may be sacrificed at each sampling time. Where control units of each aquatic sediment system are employed, they should not be treated with the test substance. The control units can be used to determine the microbial biomass of the sediment and the total organic carbon of the water and sediment at the termination of the study. Two of the control units (i.e. one control unit of each aquatic sediment) can be used to monitor the required parameters in the sediment and water during the acclimation period (see Table in section 1.8.2.2). Two additional control units have to be included in case the test substance is applied by means of a solvent to measure adverse effects on the microbial activity of the test system.

1.9.3 Test duration and sampling

The duration of the experiment should normally not exceed 100 days (6), and should continue until the degradation pathway and water/sediment distribution pattern are established or when 90 % of the test substance has dissipated by transformation and/or volatilisation. The number of sampling times should be at least six (including zero time), with an optional preliminary study (see section 1.9.4) being used to establish an appropriate sampling regime and the duration of the test, unless sufficient data is available on the test substance from previous studies. For hydrophobic test substances, additional sampling points during the initial period of the study may be necessary in order to determine the rate of distribution between water and sediment phases.

At appropriate sampling times, whole incubation vessels (in replicate) are removed for analysis. Sediment and overlying water are analysed separately(13). The surface water should be carefully removed with minimum disturbance of the sediment. The extraction and characterisation of the test substance and transformation products should follow appropriate analytical procedures. Care should be taken to remove material that may have adsorbed to the incubation vessel or to interconnecting tubing used to trap volatiles.

1.9.4 Optional preliminary test

If duration and sampling regime cannot be estimated from other relevant studies on the test substance, an optional preliminary test may be considered appropriate, which should be performed using the same test conditions proposed for the definitive study. Relevant experimental conditions and results from the preliminary test, if performed, should be briefly reported.

1.9.5 Measurements and analysis

Concentration of the test substance and the transformation products at every sampling time in water and sediment should be measured and reported (as a concentration and as percentage of applied). In general, transformation products detected at >10% of the applied radioactivity in the total water-sediment system at any sampling time should be identified unless reasonably justified otherwise. Transformation products for which concentrations are continuously increasing during the study should also be considered for identification, even if their concentrations do not exceed the limits given above, as this may indicate persistence. The latter should be considered on a case by case basis, with justifications being provided in the report.

Results from gases/volatiles trapping systems (CO₂ and others, i.e. volatile organic compounds) should be reported at each sampling time. Mineralisation rates should be reported. Non-extractable (bound) residues in sediment are to be reported at each sampling point.

2 DATA

2.1TREATMENT OF RESULTS

Total mass balance or recovery (see section 1.7.1) of added radioactivity is to be calculated at every sampling time. Results should be reported as a percentage of added radioactivity. Distribution of radioactivity between water and sediment should be reported as concentrations and percentages, at every sampling time.

Half-life, DT₅₀ and, if appropriate, DT₇₅ and DT₉₀ of the test substance should be calculated along with their confidence limits (see section 1.7.3). Information on the rate of dissipation of the test substance in the water and sediment can be obtained through the use of appropriate evaluation tools. These can range from application of pseudo-first order kinetics, empirical curve-fitting techniques which apply graphical or numerical solutions and more complex assessments Using, for example, single- or multi-compartment models. Further details can be obtained from the relevant published literature (35)(36)(37).

All approaches have their strengths and weaknesses and vary considerably in complexity. An assumption of first-order kinetics may be an oversimplification of the degradation and distribution processes, but when possible gives a term (the rate constant or half-life) which is easily understood and of value in simulation modelling and calculations of predicted environmental concentrations. Empirical approaches or linear transformations can result in better fits of curves to data and therefore allow better estimation of half-lives, DT₅₀ and, if appropriate, DT₇₅ and DT₉₀ values., The use of the derived constants, however, is limited. Compartment models can generate a number of useful constants of value in risk assessment that describe the rate of degradation in different compartments and the distribution of the chemical. They should also be used for estimation of rate constants for the formation and degradation of major transformation products. In all cases, the method chosen must be justified and the experimenter should demonstrate graphically and/or statistically the goodness of fit.

3 REPORTING

3.1TEST REPORT

The report must include the following information:

Test substance:

common name, chemical name, CAS number, structural formula (indicating position of the label(s) when radiolabelled material is used) and relevant physical-chemical properties;
purity (impurities) of test substance;
radiochemical purity of labelled chemical and molar activity (where appropriate).

Reference substances:

chemical name and structure of reference substances used for the characterisation and/or identification of transformation products

Test sediments and waters:

location and description of aquatic sediment sampling site(s) including, if possible, contamination history;
all information relating to the collection, storage (if any) and acclimation of water-sediment systems;
characteristics of the water-sediment samples as listed in Table in section 1.8.2.2.

Test conditions:

test system used (e.g. flow-through, biometer, way of ventilation, method of stirring, water volume, mass of sediment, thickness of both water and sediment layer, dimension of test vessels, etc.)
application of test substance to test system: test concentration used, number of replicates and controls mode of application of test substance (e.g. use of solvent if any), etc.
incubation temperature;
sampling times;
extraction methods and efficiencies as well as analytical methods and detection limits;
methods for characterisation/identification of transformation products;
deviations from the test protocol or test conditions during the study.

Results:

raw data figures of representative analyses (all raw data have to be stored in the GLP-archive);
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 the applied dose and in mgkg^-1 in water, sediment and total system (% only) for the test substance and, if appropriate, for transformation products and non-extractable radioactivity;
mass balance during and at the end of the studies;
a graphical representation of the transformation in the water and sediment fractions and in total system (including mineralisation);
mineralisation rates;
half-life, DT₅₀ and, if appropriate, DT₇₅ and DT₉₀ values for the test substance and, where appropriate, for major transformation products including confidence limits in water, sediment and in total system;
an assessment of the transformation kinetics of the test substance and, where appropriate, the major transformation products;
a proposed pathway of transformation, where appropriate;
discussion of results.

4 REFERENCES

(1)BBA-Guidelines for the examination of plant protectors in the registration process. (1990). Part IV, Section 5-1: Degradability and fate of plant protectors in the water/sediment system. Germany.

(2)Commission for registration of pesticides: Application for registration of a pesticide. (1991). Part G. Behaviour of the product and its metabolites in soil, water and air, Section G.2.1 (a). The Netherlands.

(3)MAFF Pesticides Safety Directorate. (1992). Preliminary guideline for the conduct of biodegradability tests on pesticides in natural sediment/water systems. Ref No SC 9046. United-Kingdom.

(4)Agriculture Canada: Environmental chemistry and fate. (1987). Guidelines for registration of pesticides in Canada. Aquatic (Laboratory) - Anaerobic and aerobic. Canada, pp 35-37.

(5)US-EPA: Pesticide assessment guidelines, Subdivision N. Chemistry: Environmental fate (1982). Section 162-3, Anaerobic aquatic metabolism.

(6)SETAC-Europe publication. (1995). Procedures for assessing the environmental fate and ecotoxicity of pesticides. Ed. Dr Mark R. Lynch. SETAC-Europe, Brussels.

(7)OECD Test Guidelines Programme. (1995). Final Report of the OECD Workshop on Selection of Soils/sediments, Belgirate, Italy, 18-20 January 1995.

(8)ISO/DIS 5667-12. (1994). Water quality - Sampling - Part 12: Guidance on sampling of bottom sediments.

(9)US-EPA (1998a). Sediment/water microcosm biodegradarion test. Harmonised Test Guidelines (OPPTS 835.3180). EPA 712-C-98-080.

(10)DFG: Pesticide Bound Residues in Soil. Wiley-VCH (1998).

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

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

(13)OECD (1993): Guidelines for Testing of Chemicals. Paris. OECD (1994-2000): Addenda 6-11 to Guidelines for the Testing of Chemicals.

(14)Scholz, K., Fritz R., Anderson C. and Spiteller M. (1988) Degradation of pesticides in an aquatic model ecosystem. BCPC - Pests and Diseases, 3B-4, 149-158.

(15)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.), Vol. 1, 85-114. J. Wiley & Sons.

(16)Madsen, T., Kristensen, P. (1997). Effects of bacterial inoculation and non-ionic surfactants on degradation of polycyclic aromatic hydrocarbons in. soil. Environ. Toxicol. Chem. 16, 631-637.

(17)Steber, J., Wierich, P. (1987). The anaerobic degradation of detergent range fatty alcohol ethoxylates. Studies with ¹⁴C-labelled model surfactants. Water Research 21, 661-667.

(18)Black, C.A. (1965). Methods of Soil Analysis. Agronomy Monograph No. 9. American Society of Agronomy, Madison.

(19)APHA (1989). Standard Methods for Examination of Water and Wastewater (17^th edition). American Public Health Association, American Water Works Association and Water Pollution Control Federation, Washington D.C.

(20)Rowell, D.L. (1994). Soil Science Methods and Applications. Longman.

(21)Light, T.S. (1972). Standard solution for redox potential measurements. Anal. Chemistry 44, 1038-1039.

(22)SETAC-Europe publication (1991). Guidance document on testing procedures for pesticides in freshwater mesocosms. From the Workshop “A Meeting of Experts on Guidelines for Static Field Mesocosms Tests”, 3-4 July 1991.

(23)SETAC-Europe publication. (1993). Guidance document on sediment toxicity tests and bioassays for freshwater and marine environments. From the Workshop On Sediment Toxicity Assessment (WOSTA), 8-10 November 1993; Eds.: I.R. Hill, P. Matthiessen and F. Heimbach.

(24)Vink, J.P.M., van der Zee, S.E.A.T.M. (1997). Pesticide biotransformation in surface waters: multivariate analyses of environmental factors at field sites. Water Research 31, 2858-2868.

(25)Vink, J.P.M., Schraa, G., van der Zee, S.E.A.T.M. (1999). Nutrient effects on microbial transformation of pesticides in nitrifying waters. Environ. Toxicol, 329-338.

(26)Anderson, T.H., Domsch, K.H. (1985). Maintenance carbon requirements of actively-metabolising microbial populations under in-situ conditions. Soil Biol. Biochem. 17, 197-203.

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

(28)Beelen, P. Van and F. Van Keulen. (1990), The Kinetics of the Degradation of Chloroform and Benzene in Anaerobic Sediment from the River Rhine. Hydrobiol. Bull. 24 (1), 13-21.

(29)Shelton, D.R. and Tiedje, J.M. (1984). General method for determining anaerobic biodegradation potential. App. Environ. Microbiol. 47, 850-857.

(30)Birch, R.R., Biver, C, Campagna, R., Gledhill, W.E., Pagga, U., Steber, J., Reust, H. and Bontinck, W.J. (1989). Screening of chemicals for anaerobic biodegradation. Chemosphere 19, 1527-1550.

(31)Pagga, U. and Beimborn, D.B. (1993). Anaerobic biodegradation tests for organic compounds. Chemoshpere 27, 1499-1509.

(32)Nuck, B.A. and Federle, T.W. (1986). A batch test for assessing the mineralisation of ¹⁴C-radiolabelled compounds under realistic anaerobic conditions. Environ. Sci. Technol. 30, 3597-3603.

(33)US-EPA (1998b). Anaerobic biodegradability of organic chemicals. Harmonised Test Guidelines (OPPTS 835.3400). EPA 712-C-98-090.

(34)Sijm, Haller and Schrap (1997). Influence of storage on sediment characteristics and drying sediment on sorption coefficients of organic contaminants. Bulletin Environ. Contam. Toxicol. 58, 961-968.

(35)Timme, G., Frehse H. and Laska V. (1986) Statistical interpretation and graphic representation of the degradational behaviour of pesticide residues II. Pflanzenschutz - Nachrichten Bayer, 39, 187 - 203.

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

(37)Carlton, R.R. and Allen, R. (1994). The use of a compartment model for evaluating the fate of pesticides in sediment/water systems. Brighton Crop Protection Conference - Pest and Diseases, pp 1349-1354.

ANNEX 1 GUIDANCE ON THE AEROBIC AND THE ANAEROBIC TEST SYSTEMS

Aerobic test system

The aerobic test system described in this test method consists of an aerobic water layer (typical oxygen concentrations range from 7 to 10 mgl^-1 ) and a sediment layer, aerobic at the surface and anaerobic below the surface (typical average redox potentials (E_h) in the anaerobic zone of the sediment range from -80 to -190 mV). Moistened air is passed over the surface of the water in each incubation unit to maintain sufficient oxigen in the head space.

Anaerobic test system

For the anaerobic test system, the test procedure is essentially the same as that outlined for the aerobic system with the exception that moistened nitrogen is passed above the surface of the water in each incubation unit to maintain a head space of nitrogen. The sediment and water are regarded as anaerobic once the redox potential (E_h) is lower than -100 mV.

In the anaerobic test, assessment of mineralisation includes measurement of evolved carbon dioxide and methane.

ANNEX 2 EXAMPLE OF A GAS FLOW-THROUGH APPARATUS

ANNEX 3 EXAMPLE OF A BIOMETER APPARATUS

ANNEX 4 EXAMPLE CALCULATION FOR APPLICATION DOSE TO TEST VESSELS

Cylinder internal diameter:	= 8 cm
Water column depth not including sediment:	= 12 cm
Surface area: 3.142 x 4²	= 50.3 cm²
Application rate: 500 g test substance/ha corresponds to 5 µg/cm²
Total µg: 5 x 50.3	= 251.5 µg
Adjust quantity in relation to a depth of 100 cm: 12 x 251.5 ÷ 100	= 30.18 µg
Volume of water column: 50.3 x 12	= 603 ml
Concentration in water: 30.18 ÷ 603	= 0.050 µg/ml or 50 µg/l

(1)

For example, if the test substance contains one ring, labelling on this ring is required; if the test substance contains two or more rings, separate studies may be needed to evaluate the fate of each labelled ring and to obtain suitable information on formation of transformation products.

(2)

Water retention characteristic of a soil can be measured as field capacity, as water holding capacity or as water suction tension (pF). For explanations see Annex 1. It should be reported in the test report whether water retention characteristics and bulk density of soils were determined in undisturbed field samples or in disturbed (processed) samples.

(3)

Recent research results indicate that soils from temperate zones can also be stored at -20^oC for more than three months (28)(29) without significant losses of microbial activity.

(4)

The soil should neither be too wet nor too dry to maintain adequate aeration and nutrition of soil microflora. Moisture contents recommended for optimal microbial growth range from 40-60% water holding capacity (WHC) and from 0.1-0.33 bar (6). The latter range is equivalent to a pF-range of 2.0 - 2.5. Typical moisture contents of various soil types are given in Annex 2.

(5)

Aerobic conditions are dominant in surface soils and even in sub-surface soils as shown in an EU sponsored research project [K. Takagi et al. (1992). Microbial diversity and activity in subsoils: Methods, field site, seasonal variation in subsoil temperatures and oxygen contents. Proc. Internat. Symp. Environm. Aspects Pesticides Microbiol., 270-277, 17-21 August 1992, Sigtuna, Sweden]. Anaerobic conditions may only occur occasionally during flooding of soils after heavy rainfalls or when paddy conditions are established in rice fields.

(6)

Aerobic studies might be terminated much before 120 days provided that ultimate transformation pathway and ultimate mineralisation are clearly reached at that time. Termination of the test is possible after 120 days, or when at least 90% of the test substance is transformed, but only if at least 5% CO₂ is formed.

(7)

Calculation of the initial concentration on an area basis using the following equation:

C_soil= Initial concentration in soil [mg·kg^-1]

A = Application rate [kg·ha^-1]; l = thickness of field soil layer [m]; d = dry bulk density of soil [kg·m^-3].

As a rule of thumb, an application rate of 1 kg·ha^-1 results in a soil concentration of approximately 1 mg·kg^-1 in a 10 cm layer (assuming a bulk density of 1 g · cm^-3).

(8)

For example, if the substance contains one ring, labelling on this ring is required; if the test substance contains two or more rings, separate studies may be needed to evaluate the fate of each labelled ring and to obtain suitable information on formation of transformation products.

(9)

As these alkaline absorption solutions also absorb the carbon dioxide from the ventilation air and that formed by respiration in aerobic experiments, they have to be exchanged in regular intervals to avoid their saturation and thus loss of their absorption capacity.

(10)

[Clay + silt] is the mineral fraction of the sediment with particle size of < 50 µm

(11)

Recent studies have shown that storage at 4 ^oC can lead to a decrease of the organic carbon content of the sediment which may possibly result in a decrease of microbial activity (34).

(12)

Test with a second concentration can be useful for chemicals that reach surface waters by different entry routes resulting in significantly different concentrations, as long as the lower concentration can be analysed with sufficient accuracy.

(13)

In cases where rapid re-oxidation of anaerobic transformation products may readily occur, anaerobic conditions should be maintained during sampling and analyses.

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ANNEX 2I

C.21. SOIL MICROORGANISMS: NITROGEN TRANSFORMATION TEST

1. METHOD

1.1INTRODUCTION

1.2DEFINITIONS

1.3REFERENCE SUBSTANCES

1.4PRINCIPLE OF THE TEST METHOD

1.5VALIDITY OF THE TEST

1.6DESCRIPTION OF THE TEST METHOD

1.6.1 Apparatus

1.6.2 Selection and number of soils

1.6.3 Collection and storage of soil samples

1.6.3.1 Collection

1.6.3.2 Storage

1.6.4 Handling and preparation of soil for the test

1.6.4.1 Pre-incubation

1.6.4.2 Physical-chemical characteristics

1.6.4.3 Amendment with organic substrate

1.6.5 Preparation of the test substance for the application to soil

1.6.6 Test concentrations

1.7PERFORMANCE OF THE TEST

1.7.1 Conditions of exposure

1.7.1.1 Treatment and control

1.7.1.2 Incubation of soil samples

1.7.1.3 Test conditions and duration

1.7.2 Sampling and analysis of soils

1.7.2.1 Soil sampling schedule

1.7.2.2 Analysis of soil samples

2 DATA

2.1TREATMENT OF RESULTS

2.2INTERPRETATION OF RESULTS

3 REPORTING

4 REFERENCES

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

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

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

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

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

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

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

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

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

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

1.1INTRODUCTION

1.2DEFINITIONS

1.3REFERENCE SUBSTANCES

1.4PRINCIPLE OF THE TEST METHOD

1.5VALIDITY OF THE TEST

1.6DESCRIPTION OF THE TEST METHOD

1.6.1 Apparatus

1.6.2 Selection and number of soils

1.6.3 Collection and storage of soil samples

1.6.3.1 Collection

1.6.3.2 Storage

1.6.4 Handling and preparation of soil for the test

1.6.4.1 Pre-incubation

1.6.4.2 Physical-chemical characteristics

1.6.5 Preparation of the test substance for the application to soil

1.6.6 Test concentrations

1.7PERFORMANCE OF THE TEST

1.7.1 Conditions of exposure

1.7.1.1 Treatment and control

1.7.1.2 Incubation of soil samples

1.7.1.3 Test conditions and duration

1.7.2 Sampling and analysis of soils

1.7.2.1 Soil sampling schedule

(8)Anderson, J.P.E. (1982). Soil Respiration, in "Methods of Soil Analysis - Part 2: Chemical and Microbiological Properties". Agronomy Monograph N^o 9. Eds. A.L. Page, R.H. Miller and D.R. Keeney. 41:831-871.