How is soil fertility tested

(valid until June 1st, 2008 according to RL 2006/121 / EG)
Determination of ecotoxicity
C.22. Soil microorganisms: carbon transformation test

Status RL 2004/73 / EG
Appendix V
to RL 67/548 / EEC

to the current version

C.22. 1st method

This method corresponds to the test guideline OECD TG 217 (2000).

C.22. 1.1 Introduction

This test method describes a laboratory method for investigating the potential long-term effects of a single exposure to pesticides and any other chemicals on the carbon transformation activity of soil microorganisms. The test is essentially based on the recommendations of the plant protection organization for Europe and the Mediterranean region (1), but other guidelines were also taken into account, such as those of the German Federal Biological Institute (2), the US Environmental Protection Agency (3), and SETAC ( 4). At an OECD workshop on soil / sediment selection held in Belgirate, Italy in 1995 (5), the number and type of soils to be used in this test were agreed. The recommendations for the collection, treatment and storage of soil samples are based on ISO instructions (6) and recommendations from the Belgirate workshop.

When assessing and evaluating toxic properties of test substances, it may be necessary to determine the effects on the microbial activity of the soil, e.g. if data on possible side effects of pesticides on the soil microflora are required or if soil microorganisms are exposed to chemicals other than pesticides is expected. The carbon transformation test is carried out to determine the influence of such chemicals on the soil microflora. When testing agrochemicals (e.g. pesticides, fertilizers, forest chemicals), both carbon and nitrogen transformation tests are carried out. The nitrogen transformation test is sufficient for substances other than agrochemicals. However, if the EC50- Values ​​of the nitrogen transformation test for these chemicals in the range determined for commercially available nitrification inhibitors (e.g. nitrapyrin), a carbon transformation test can be carried out to gain further information.

Soil consists of both living and non-living components that occur in complex and heterogeneous mixtures. Microorganisms play an important role in the breakdown of organic material and its transformation in fertile soils, with many species responsible for different aspects of soil fertility. Any long-term disruption of these biochemical processes can potentially affect the nutrient cycle and thereby in turn affect soil fertility. The transformation of carbon and nitrogen occurs in all fertile soils. The transformation paths are essentially the same, even if different microbial populations are responsible for these processes depending on the soil.

The test method described here can be used to determine long-term adverse effects caused by a substance on the process of carbon transformation in aerobic topsoils. The test is sensitive to changes in the size and activity of the microbial populations responsible for carbon transformation, as these populations are exposed to both chemical stress and carbon deficiency. A sandy soil poor in organic material 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 a carbon deficiency that not only kills microbial cells but also induces dormancy and / or spore formation. If the test runs for more than 28 days, the sum of these reactions in the control samples (untreated soil) can be measured as a progressive loss of metabolically active microbial biomass (7). If the biomass in carbon - stressed soil is affected by the presence of a chemical under the test conditions, it may not return to the level in the control samples. Consequently, disturbances caused by the test substance at any point during the experiment often persist until the end of the test.

The tests on the basis of which this test method was developed were primarily designed for substances for which the amount of the soil can be determined in advance. This is the case, for example, with pesticides for which the amount applied in the field is known. In the case of agrochemicals, it is sufficient to test two concentrations that are relevant to the expected or predicted application rate. Agrochemicals can be tested as active ingredients (a.i.) or as formulated commercial 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 interpreted, the test can also be used for chemicals for which it is not known how much they will end up in the soil. Thus, for substances other than agrochemicals, the effects of a range of concentrations on carbon transformation are determined. The data from these tests are used to generate a dose-response curve and ECxValues, where x is defined as the effect in%.

C.22. 1.2 Definitions

Carbon transformation: the breakdown of organic material by microorganisms into the inorganic end product carbon dioxide.

ECx (Effect concentration): that concentration of the test substance in the soil that inhibits the transformation of carbon into carbon dioxide by x%.

EC50) (Median value of the effect concentration): that concentration of the test substance in the soil that inhibits the transformation of carbon into carbon dioxide by 50 percent.

C.22. 1.3 Reference substances


C.22. 1.4 Principle of the test method

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

C.22. 1.5 Test validity

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

C.22. 1.6 Description of the test method

C.22. 1.6.1 Devices

Test vessels made of chemically inert material are used. Their capacity should depend on the chosen incubation method of the soil, i. H. for the incubation of bulk samples or a series of individual soil samples (see Section During the test, it must be ensured that the loss of water is kept as low as possible and that gas exchange can take place (for example, the test containers could be covered with perforated polyethylene film). When testing volatile substances, sealable and gas-tight containers must be used. The size of these should be such that they are filled with the soil sample to about a quarter of their volume.

To determine the glucose-induced respiration, incubation systems and devices for measuring carbon dioxide formation and oxygen consumption are required. Examples of such systems can be found in the literature (8) (9) (10) (11).

C.22. 1.6.2 Selection and number of floors

A single floor is used. We recommend floors with the following properties:

Sand content: at least 50% and at most 75%:

pH: 5.5-7.5;

  • organic carbon content: 0.5 - 1.5%;
  • the microbial biomass must be determined (12) (13) and its carbon content should be at least 1% of the total organic carbon in the soil.

Usually a soil with these properties represents the "worst case", since its adsorption is minimal and the availability of the test chemical for the microflora is maximal. Accordingly, tests with other soils are generally not necessary. However, under certain circumstances, e.g. if the expected main use of the test substance takes place on certain soils such as acidic forest soils, or in the case of electrostatically charged chemicals, it may be necessary to use an additional soil.

C.22. 1.6.3 Collection and storage of soil samples

C.22. Removal

Detailed information is required about the history of the field location from which the test soil is being taken. This information includes, among other things, the exact location, vegetation, treatment with pesticides and organic and inorganic fertilizers, additions of biological materials or unintentional contamination. The location chosen for the soil extraction must be usable over a longer period of time. Permanent pastures, fields with annual grain crops (except maize) or densely sown green manure plants are suitable. The selected sampling location should not have been treated with pesticides for at least one year prior to sampling. Furthermore, no organic fertilizers should have been applied for at least six months beforehand. The use of mineral fertilizers is only permitted if this is necessary for the cultivation, and the soil samples should be taken no earlier than three months after the fertilizer has been applied. Avoid using soil that has been treated with fertilizers with a known biocidal effect (e.g. calcium cyanamide).

Sampling during or after longer (more than 30 days) periods of drought or flooding should be avoided. In the case of plowed soils, the samples should be taken from a depth of 0 to 20 cm. In the case of grassland (pastures) or other soils that are not plowed for longer periods of time (at least one vegetation period), the maximum depth of the sampling can be slightly more than 20 cm (e.g. up to 25 cm). The soil samples should be transported in containers and under temperature conditions that ensure that the original soil properties are not significantly altered.

C.22. Storage

The use of soil fresh from the field is preferred. If storage in the laboratory cannot be avoided, the floors should be stored in the dark at 4 ± 2 ° C for a maximum of three months. Aerobic conditions must be ensured during the storage of the soils. If soils are removed from areas that have been frozen for at least three months a year, storage for six months at -18 ° C can be considered. Before each experiment, the microbial biomass of the stored soils must be determined and the carbon content in the biomass should be at least 1% of the total organic carbon in the soil (see Section 1.6.2).

C.22. 1.6.4 Handling and preparing the soil for the test

C.22. Pre-incubation

If the soil has been stored (see Sections and, pre-incubation for a period of 2 to 28 days is recommended. The temperature and moisture content of the soil during the pre-incubation should correspond to the test conditions as far as possible (see Sections and

C.22. Physico-chemical properties

Large objects (e.g. stones, parts of plants, etc.) are manually removed from the soil and, when moist, they are sieved to a particle size of less than or equal to 2 mm without drying out excessively. The moisture content of the soil sample should be adjusted with distilled or deionized water to a value between 40% and 60% of the maximum water holding capacity.

C.22. 1.6.5 Preparation of the test substance for application to the soil

Usually the test substance is added using a carrier. This carrier can be water (in the case of 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 or chloroform) should be avoided as they can damage the microflora. If sand is used as a carrier, it can be coated with the test substance dissolved or suspended in a suitable solvent. In these cases, the solvent should be removed by evaporation before mixing with the soil. A ratio of 10 g sand per kg soil (dry weight) is recommended for optimal distribution of the test substance in the soil. The control samples are only treated with an equivalent amount of water and / or quartz sand.

When testing volatile chemicals, losses during treatment should be avoided as far as possible and homogeneous distribution in the soil should be ensured wherever possible (e.g. by injecting the test substance in different places in the soil).

C.22. 1.6.6 Test Concentrations

If pesticides or other chemicals with predictable environmental concentrations are tested, at least two concentrations should be used. The lower concentration should at least correspond to the maximum amount that is likely to get into the soil under normal practical conditions, while the higher concentration should be a multiple of the lower concentration. The concentrations of the test substance added to the soil are calculated assuming a uniform incorporation to a depth of 5 cm and a soil density of 1.5. For agrochemicals that are directly applied to the soil, or for chemicals where the amount of the amount reaching the soil is predictable, the recommended test concentrations are the highest predicted environmental concentration (PEC) and five times that concentration. Substances that are likely to be applied to the soil several times in a culture period should be tested at concentrations obtained by multiplying the PEC by the highest expected number of applications. However, the upper concentration tested should not exceed ten times the highest single application rate.

When testing substances other than agrochemicals, a geometric series of at least five concentrations is used. The concentrations tested should be those used to determine the ECx-Values ​​cover the necessary range.

C.22. 1.7 Performing the test

C.22. 1.7.1 Conditions of exposure

C.22. Treatment and control

When testing agrochemicals, the soil is divided into three parts of equal weight. Two parts are mixed with the carrier containing the product and the third is mixed with the carrier without the product (control sample). A minimum of three replicates is recommended for both treated and untreated soils. If substances other than agrochemicals are tested, the soil is divided into six parts of equal weight. Five of these samples are mixed with the carrier containing the test substance, and the sixth is mixed with the carrier without the chemical. Three replicates are recommended for both the treated samples and the control samples. Care must be taken to ensure that the test substance is homogeneously distributed in the treated soil samples. Avoid compacting or clumping the soil during mixing.

C.22. Incubation of soil samples

The incubation of the soil samples can be carried out in two ways: as bulk samples from each treated and untreated soil or as a series of individual, equal-sized subsamples from each treated and untreated soil. In the case of volatile substances, however, the test should only be carried out on a number of individual sub-samples. When soils are incubated as bulk samples, large amounts of each treated and untreated soil are prepared and subsamples are taken during the test to be analyzed as necessary. The amount prepared at the beginning for each treatment and control depends on the size of the sub-samples, the number of replicates used for the analysis and the highest expected number of samples.Before partial samples are taken, the incubated bulk samples must be mixed thoroughly. In the case of soil incubation as a series of individual soil samples, each treated and untreated collecting soil is divided into the required number of subsamples, and these subsamples are used as necessary. For investigations with more than two sampling times, sufficient partial samples must be prepared to take into account all replicates and sampling times. At least three replicate samples of the test soil should be incubated under aerobic conditions (see Section Suitable containers with sufficient headspace should be used for all tests to avoid the development of anaerobic conditions. If volatile substances are tested, the test should only be carried out with a number of individual sub-samples.

C.22. Test conditions and duration

The test is carried out in the dark at room temperature (20 ± 2 ° C). The moisture content of the soil samples must be kept at 40% - 60% (± 5%) of the maximum water holding capacity of the soil during the course of the test (see Section Distilled or deionized water can be added as required.

The minimum duration of the tests is 28 days. In the case of agrochemicals, the amounts of carbon released or oxygen consumed in the treated samples are compared with those in the control samples. If these differ by more than 25% on the 28th day, the test is continued until a difference of equal to or less than 25% is reached or for a maximum of 100 days, whichever is shorter. If substances other than agrochemicals are tested, the test is terminated after 28 days. On the 28th day, the amounts of released carbon dioxide or consumed oxygen in the treated samples and in the control samples are determined and the ECxValues ​​calculated.

C.22. 1.7.2 Soil sampling and analysis

C.22. Sampling intervals

When testing agrochemicals, soil samples are analyzed on days 0, 7, 14 and 28 for glucose-induced respiration rates. If a test extension is necessary, further measurements are to be carried out from the 28th day at intervals of 14 days.

When testing substances other than agrochemicals, at least five test concentrations are used and soil samples are analyzed for glucose-induced respiration at the beginning (day 0) and at the end of the exposure period (28 days). If necessary, an intermediate measurement, e.g. on the 7th day, can be inserted. The data obtained on the 28th day are used to determine the ECx value of the chemical. If desired, the data from the control samples from day 0 can be used to estimate the initial quantities of metabolically active microbial biomass in the soil (12).

C.22. Measurement of glucose-induced respiration rates

The glucose-induced respiration rate in each treated sample and in each control replica is determined at each sampling time. The soil samples are mixed with an amount of glucose large enough to immediately reach a maximum respiratory value. The amount of glucose necessary to achieve a maximum respiratory value in a certain soil can be determined in a preliminary test with a series of glucose concentrations (14). In sandy soils with an organic carbon content of 0.5 - 1.5%, however, 2,000 mg to 4,000 mg glucose per kg soil (dry weight) are usually sufficient. The glucose can be pulverized with clean quartz sand (10 g sand / kg dry weight soil) and mixed homogeneously with the soil.

The soil samples enriched with glucose are incubated in a suitable device with which the respiration rates at 20 ± 2 ° C can be measured continuously, hourly or at 2-hour intervals (see Section 1.6.1). The carbon dioxide released or oxygen consumed is measured for 12 hours and the measurements should start as early as possible; H. within one to two hours of the addition of glucose. The total amounts of carbon dioxide released or oxygen consumed in the 12 hours are measured and the mean respiration rates are determined.

C.22. 2 dates

C.22. 2.1 Preparation of the results

If agrochemicals are tested, the amount of carbon dioxide released or oxygen consumed in each case must be recorded for each replica, and the mean values ​​of all replicas are to be presented in tabular form. The results are to be assessed using suitable and generally recognized statistical methods (e.g. F-test, 5% level of significance). The glucose-induced respiration rates are expressed in mg carbon dioxide / kg dry weight soil / h or mg oxygen / dry weight soil / h. The mean rate of carbon dioxide formation or the mean rate of oxygen consumption of each treatment is compared with that in the control sample, and the percentage deviation from the control sample is calculated.

If substances other than agrochemicals are tested, the amount of carbon dioxide released or oxygen consumed is determined for each replicate, and a dose-effect curve is drawn up to estimate the ECK values. The glucose-induced respiration rates found in the treated samples after 28 days (i.e. mg carbon dioxide / kg dry weight soil / h or mg oxygen / dry weight soil / h) are compared with those found in the control sample. On the basis of this data, the inhibition values, expressed in%, are calculated for each test concentration. These percentages are plotted against the concentration, and then the ECx values ​​are calculated using statistical methods. Using standard procedures, confidence intervals (p = 0.95) are also determined for the calculated ECx values ​​(15) (16) (17).

C.22. 2.2 Interpretation of the results

If, when evaluating the test results for agrochemicals, the difference in respiration rates between the low treatment (i.e. the highest expected concentration) and the control samples at each sampling time after the 28th day is equal to or less than 25%, then the product shall be evaluated such that it has no long-term impact on carbon transformation in soils. The EC50-, EC25- and / or EC10Values ​​are used.

C.22. 3 final report


The test report must contain the following information:

Full details of the floors used, including:

geographical information on the location (latitude, longitude);

  • Information about the history of the field location (i.e. vegetation, treatments with pesticides and fertilizers, accidental contamination, etc.)
    Use structure (e.g. agricultural land, forest, etc.);
  • Sampling depth (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 value of the microbial biomass as a share (in%) of the total organic carbon;
  • Information on the methods used to determine the individual parameters;
  • all information on the taking and storage of the soil samples;
  • if necessary, details on pre-incubation of the soil.

Test substance:

  • physical state and, if relevant, physico-chemical properties;
  • Chemical characteristics, if relevant with structural formula, purity (i.e. in the case of pesticides, the active ingredient content in%), nitrogen content.

Test conditions: characteristics, if relevant with structural formula, purity (i.e. in the case of pesticides, the active ingredient content

  • precise information on the enrichment of the soil with organic substrate;
  • Number of concentrations of the test chemical used and, if applicable, justification for the selected concentrations:
  • precise information on the application of the test substance to the soil;
  • Incubation temperature;
  • Moisture content of the soil at the beginning and during the test;
  • the method used for soil incubation (i.e. as bulk samples or as a series of individual subsamples);
  • Number of replicas;
  • Number of sampling times.


  • methods and equipment used to measure respiratory rates;
  • Data in tabular form, including individual and mean values ​​of the amounts of carbon dioxide and oxygen;
  • Deviation between replicates of treated samples and control samples; Explanations of the corrections to the calculations, if relevant;
  • the deviation (in%) of the glucose-induced respiration rates at each sampling time or, if applicable, the EC50Value with 95% confidence interval, further ECx (i.e. EC25 or EC10) with confidence intervals and a graphical representation of the dose-response curve;
  • if necessary, statistical processing of the results;
  • any information and observations that are helpful in interpreting the test results.

C.22. 4 Bibliography

(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 edition, 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 fit 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 - Part 6: Instructions for sampling, treatment and storage of soil for the determination of aerobic microbial processes under laboratory conditions.

(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 ° 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 to measure the mineralization of organic chemicals in soil under aerobic conditions.

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

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

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

(14) Malkomes, H.-P. (1986). Influence of the amount of glucose on the reaction of short-term respiration in the soil to pesticides, illustrated using the example of a herbicide. (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). News sheet Deut. Plant protection d., 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.