# 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), Division 1.1: INTRODUCTION Extent: E+W+S+N.I. 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 ECx values, where x is defined % effect. --- Source: https://www.legislation.gov.uk/eudr/2004/73/annex/2I/division/C.21/division/1/division/1.1 (the official version) Page: https://openlegislation.uk/eu-directive-2004-73/annex-2I/division-C.21/division-1/division-1.1 Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.