Commission Directive 2004/73/ECShow full title

C.21. SOIL MICROORGANISMS: NITROGEN TRANSFORMATION TEST

1. METHOD

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

1.1 INTRODUCTION

This Testing method describes a laboratory method designed to investigate the long-term effects of chemicals, after a single exposure, on nitrogen transformation activity of soil microorganisms. The test is principally based on the recommendations of the European and Mediterranean Plant Protection Organization (1). However, other guideline, including those of the German Biologische Bundesanstalt (2), the US Environmental Protection Agency (3) 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.

1.2 DEFINITIONS

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

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

EC₅₀ (Median Effective Concentration): is the concentration of the test substance in soil that results in a 50 percent (50%) inhibition of nitrogen transformation to nitrate.

1.3 REFERENCE SUBSTANCES

None.

1.4 PRINCIPLE OF THE TEST METHOD

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

1.5 VALIDITY OF THE TEST

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

1.6 DESCRIPTION OF THE TEST METHOD

1.6.1 Apparatus

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

Standard laboratory equipment including the following is used:

• agitation device: mechanical shaker or equivalent equipment;

• centrifuge (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.

1.7 PERFORMANCE OF THE TEST

1.7.1 Conditions of exposure

1.7.1.1 Treatment and control

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

1.7.1.2 Incubation of soil samples

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

1.7.1.3 Test conditions and duration

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

2.1 TREATMENT OF RESULTS

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

If tests are conducted with non-agrochemicals, the quantity of nitrate formed in each replicate is determined, and a dose-response curve is prepared for estimation of the 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.

2.2 INTERPRETATION OF RESULTS

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

3 REPORTING

The test report must include the following information:

Complete identification of the soil used including:

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

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

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

• depth of sampling (cm);

• sand/silt/clay content (% dry weight);

• pH (in water);

• organic carbon content (% dry weight);

• nitrogen content (% dry weight);

• initial nitrate concentration (mg nitrate/kg dry weight);

• cation exchange capacity (mmol/kg);

• 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.