Decree No. 389 / 2005 Coll.
Decree amending Decree No 222 / 2004 Coll., laying down basic methods for testing physicochemical properties, explosive properties and environmental hazards for chemicals and chemical preparations
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389
DECLARATION
of 15 September 2005
amending Decree No 222 / 2004 Coll., laying down the basic methods for testing physicochemical properties, explosive and hazardous properties for chemicals and chemical preparations
According to § 8 (5) (c) of Act No. 356 / 2003 Coll., on Chemicals and Chemicals, and on the amendment of certain laws, the Ministry of Environment provides for:
Decree No 222 / 2004 Coll., laying down, for chemicals and chemical preparations, the basic methods for testing physico-chemical properties, explosive and hazardous properties for the environment, are amended as follows:
1. In Article 2 (1), "Part I of Annex 2 'is replaced by" Annex 2';
2. In Article 2, at the end of paragraph 1, the dot is replaced by a comma and the following points (v) to (y) are added:
'(v) the activity of soil micro-organisms in the transformation of nitrogen;
(w) activity of soil micro-organisms in carbon transformation;
x) aerobic and anaerobic transformation in soil,
(y) aerobic and anaerobic transformation in water-sediment systems. ';
3. Paragraph 2 (2) is deleted and paragraph 1 is deleted.
4. The following points XXI to XXIV are added to Annex No 2, Part I:
"XXI. METHOD FOR DETERMINING THE ACTIVITY OF ORIGIN MICROORGANISMS IN THE TRANSFORMATION OF DUSK
(Method C.21 of the Annex to Directive 2004 / 73 / EC of 29 April 2004 adapting to technical progress for the 22nd time Council Directive 67 / 548 / EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances)
XXI.1. METHOD
This test method corresponds to OECD TG 216 (2000).
XXI.1.1 INTRODUCTION
This test method describes the laboratory procedure for investigating the long-term effects of chemicals after a single exposure to soil micro-organism activity in the transformation of nitrogen. It is based on the documentation and recommendations of national and international organisations (see literature 1 to 7). In assessing and evaluating the toxic properties of test substances, it may be necessary to determine effects on the activity of soil micro-organisms, e.g. when data are required on the side effects of plant protection products on soil micro-flora or when exposure of soil micro-organisms to chemicals other than plant protection products is expected. The objective of the nitrogen transformation test is to identify the effects of such chemicals on soil microflora. If agrochemicals (e.g. plant protection products, fertilisers, chemical products for forestry) are tested, both a nitrogen transformation test and a carbon transformation test are performed. If chemicals other than agrochemicals are tested, a nitrogen transformation test is sufficient. However, if these chemicals have EC50 values for nitrogen transformation within the range found for commercially available nitrification inhibitors (e.g. nitrapyrin), a carbon transformation test may be performed to obtain further information.
Soils consist of living and inanimate ingredients that occur in the form of complex and heterogeneous mixtures. Microorganisms play an important role in the degradation and transformation of organic substances in fertile soils, with different species contributing to different aspects of soil fertility. Any long-term breach of these biochemical processes could break the nutrient cycle and this could affect soil fertility. The transformation of carbon and nitrogen occurs on all fertile soils. Although the populations of micro-organisms responsible for these processes differ from soil to soil, they are essentially the same transformation scheme.
The described test method serves to identify the long-term adverse effects of the substance on the nitrogen transformation process in aerobic surface soils. This method also makes it possible to estimate the effects of substances on carbon transformation by soil microflora. Nitrates are formed after the carbon-nitrogen bonds have disintegrated. Therefore, if the same nitrate production rate is found for test and control soils, it is highly likely that the main carbon degradation scheme is intact and functional. The substrate chosen for the test (powder lucerne meal) has a favourable carbon / nitrogen ratio (usually from 12 / 1 to 16 / 1). Thus, the carbon deficiency is reduced during the test and can be restored within 100 days if the micro-organism population is damaged by a chemical.
The test on which this test method is based was initially developed for substances that can be predicted to penetrate the soil. Examples are plant protection products for which the field application dose is known. For agrochemicals it is sufficient to perform a test at two dose levels corresponding to the expected or estimated application dose. For agrochemicals, active ingredients or preparations in commercial treatment may be tested. However, the use of the test is not limited to agrochemicals only. By changing the amount of test substance applied to the soil and the way in which the data are evaluated, the test may also be used for chemicals which do not know how much they penetrate the soil. Therefore, for chemicals other than agrochemicals, the effect of a number of concentrations on nitrogen transformation is determined. The data from these tests shall be used to construct a dose response curve and to calculate ECx values where x is the percentage effect.
XXI.1.2 DEFINITIONS
Nitrogen transformation: is the final degradation of the nitrogen organic substance by micro-organisms through ammonia and nitrification to the relevant final inorganic product - nitrate.
ECx (Effective concentration): is the concentration of the test substance in the soil which results in an x-percent inhibition of nitrogen transformation into nitrate.
EC50 (Median effective concentration): is the concentration of the test substance in soil that results in a 50% inhibition of nitrogen transformation to nitrate.
XXI.1.3 REFERENCE SUBSTANCES
None.
XXI.1.4 CONDITIONS OF THE TEST METHOD
The test substance is then applied to the ground, or the soil is left intact (as control). When testing agrochemicals, it is recommended to perform a test with at least two concentrations chosen to be related to the highest expected concentrations in the field. After 0, 7, 14 and 28 days of incubation, samples of exposed soil and control soil shall be extracted with appropriate solvent and the nitrate content determined. The rate of nitrate formation in the exposed samples shall be compared to the rate in the control samples and the percentage deviation of the experimental samples from the control samples shall be calculated. All tests shall be carried out for at least 28 days. If the 28-day difference between experimental and control soil is 25% or greater, the measurements shall be continued up to 100 days. When testing substances other than agrochemicals, a number of concentrations of the test substance and the amount of nitrates produced in experimental and control samples shall be added to soil samples, measured after 28 days incubation. Multi-concentration test results shall be analysed using a regression model and ECx values shall be calculated (i.e. EC50, EC25 and / or EC10). See definitions.
XXI.1.5 TEST VALIDITY
Assessment of test results for agrochemicals is performed with relatively small differences (i.e. mean ± 25%) between nitrate concentrations in control and exposed samples, so that large variations in control samples may lead to incorrect results. The differences between each control sample should therefore be less than ± 15%.
XXI.1.6 DESCRIPTION OF THE METHOD
XXI.1.6.1 Test equipment
The containers used in the test shall be of chemically inert material. They should have an appropriate volume which should correspond to the procedure used for soil incubation, i.e. either the incubation of the entire soil quantity or the incubation of individual soil samples (see Section 1.7.1.2). During the test, water losses shall be as low as possible and gas exchange shall be allowed (e.g. by covering the containers with perforated polyethylene foil). Closed gas-tight containers shall be used for the testing of volatile substances. They should be large enough to fill approximately one quarter of their volume with a soil sample.
Standard laboratory equipment including:
- shaker: mechanical shaker or equivalent;
- centrifuge (3,000 g) or filtration equipment (using nitrate-free filter paper);
- measuring equipment for the determination of nitrates with appropriate sensitivity and reproducibility.
XXI.1.6.2 Selection and number of soils
One soil shall be used. The soil should have the following characteristics:
- sand content: not less than 50% and not more than 75%;
- pH: 5,5 - 7,5;
- organic carbon content: 0,5 - 1,5%;
- microbial biomass is determined (see literature 8.9) and its carbon should be at least 1% of the total organic carbon in the soil.
In most cases, soil with these characteristics is the most conservative case since adsorption of the test chemical is minimal and its availability for micro-organisms is maximum. As a result, testing with other soils is generally unnecessary. However, under certain circumstances, the use of additional soil may be necessary, for example in the intended main use of the test substance on certain soil types, such as acidic forest land or in the case of electrostatically charged chemicals.
XXI.1.6.3 Soil sampling and storage
XXI.1.6.3.1 Collection
Detailed information on the history of the place from which the land is collected should be available. This information shall include the exact location, vegetation, treatment data of plant protection products, the use of organic and mineral fertilisers, the addition of biological material or accidental contamination. A place should be chosen for land collection that allows long-term use. Permanent pasture, fields with annual crops (except maize) or densely sown green fertilisation area are suitable. The sites selected for sampling should not be treated with plant protection products for at least one year prior to collection. Organic fertilisers should also not be used for at least six months. The use of mineral fertilisers is permitted only if it is necessary for crops and soil samples should not be taken before three months after application. Soils with fertilisers known to have biocidal effects (e.g. calcium cyanide) should not be used.
Samples should not be taken during or after long periods of drought or irrigation (longer than 30 days). The arable soil samples shall be taken from a depth of 0 to 20 cm. In the case of meadows (pastures) or other soils for which no ploughing has been carried out for at least one growing season, the maximum sampling depth may be slightly above 20 cm (e.g. up to 25 cm).
Soil samples should be transported in such containers and at such temperature as to ensure that the original soil characteristics do not change significantly.
XXI.1.6.3.2 Storage
The use of freshly harvested land is preferred. If storage of soil in the laboratory is unavoidable, it may be stored in the dark at (4 ± 2) ° C for a maximum period of three months. During storage, aerobic conditions must be ensured. If the soil is collected from areas where it is frozen for at least three months in the year, storage at -18 ° C to -22 ° C may be considered for six months. Prior to each experiment, microbial biomass stored soils are determined and biomass-derived carbon should be at least 1% of total organic carbon in the soil (see Section 1.6.2).
XXI.1.6.4 Soil treatment and preparation for testing
XXI.1.6.4.1 Preliminary incubation
If the soil has been stored (see Section 1.6.3.2), a preliminary incubation of 2 to 28 days is recommended. The soil temperature and humidity during the preliminary incubation should be similar to the test conditions (see sections 1.6.4.2 and 1.7.1.3).
XXI.1.6.4.2 Physicalchemical characteristics
Large pieces (e.g. stones, parts of plants, etc.) shall be removed by hand from the soil and then spread through particles 2 mm or less without excessive drying. The moisture content of the soil sample shall be adjusted by distilled or deionised water to 40% to 60% of the maximum water capacity.
XXI.1.6.4.3 Enrichment by organic substrate
The soil should be enriched with an appropriate organic substrate such as green grass meal with alfalfa (main ingredient: Medicago sativa) with a ratio of C / N from 12 / 1 to 16 / 1. The recommended dose of lucerne meal is 5 g of lucerne per kilogram of soil (based on dry matter).
XXI.1.6.5. Preparation of the test substance for application to soil
The test substance is usually applied using a carrier. The carrier may be water (for water-soluble substances) or inert solids such as fine silica sand (grain size: 0,1 - 0,5 mm). Liquid carriers other than water (e.g. organic solvents such as acetone, chloroform) should not be used as they can destroy microflora. If used as a sand carrier, it may be coated with a test substance dissolved or dispersed in an appropriate solvent. In this case the solvent is removed by evaporation before mixing with the soil. For optimum distribution of the test substance in soil, 10 g sand per kilogram of soil (expressed on dry matter) is recommended. Only the corresponding amount of water and / or sand shall be applied to the control samples.
When testing volatile chemicals, losses during application should be avoided as far as possible and a homogeneous dispersal of the substance in the soil should be attempted (e.g. spray the substance into the soil in several places).
XXI.1.6.6 Test concentrations
At least two concentrations shall be used for the testing of agrochemicals. The lower concentration should correspond to at least the maximum expected amount of substance that reaches the soil in real conditions, while the higher concentration should be multiple of the lower concentration. Calculation of the concentration of the test substance added to the soil shall be carried out provided that it is evenly adjusted to a depth of 5 cm and for a free soil density of 1,5. For agrochemicals that are applied directly to the soil, or for chemicals for which quantities can be predicted in the soil, the recommended test concentrations are the maximum predicted environmental concentration (PEC) and their five times. For substances that are normally applied to soil several times per season, the test concentrations are derived from the product PEC and the expected maximum number of applications. However, the upper concentration should not exceed 10 times the maximum single dose administered. A geometric series of at least five concentrations shall be used for substances other than agrochemicals. The ECx values should be found in the range of these test concentrations.
XXI.1.7 TEST IMPLEMENTATION
XXI.1.7.1. Exposure conditions
XXI.1.7.1.1 Exposure and control
When testing agrochemicals, the soil is divided into three parts of the same weight. Two parts are mixed with a carrier containing the substance and the third part is only mixed with the carrier (control). It is recommended that the test be carried out with at least three duplicate samples of exposed and unexposed soils. When testing substances other than agrochemicals, the soil is divided into six parts of the same weight. Five samples shall be mixed with the carrier containing the test substance and the sixth sample shall only be mixed with the non-chemical carrier. It is recommended to use three duplicate samples of exposed and unexposed soil. Attention shall be paid to the homogeneous dispersion of the test substance in exposed soil samples. There should be no compaction or accumulation of soil during mixing.
XXI.1.7.1.2 Incubation of soil samples
The incubation of soil samples may be carried out in two ways: with an aggregate sample of exposed soil and an aggregate sample of unexposed soil, or with a series of individual equal subsamples of both exposed and unexposed soil. However, in the case of volatile substances, the test shall only be carried out with a series of individual incremental samples. If aggregate samples are incubated, large quantities of exposed and unexposed soil shall be prepared and sub-samples shall be taken for analysis as necessary during the test. The initial quantity prepared for the samples to be exposed and the control samples shall depend on the size of the incremental samples, the number of duplicate samples used for the analysis and the estimated number of intervals for sampling. Soils incubated as an aggregate sample shall be thoroughly mixed before the incremental samples are taken. If a series of individual soil samples is incubated, the aggregate sample of exposed soil and the aggregate sample of unexposed soil shall be divided into the required number of incremental samples and shall be used as appropriate. For experiments where more than two sampling intervals can be expected, a sufficient number of incremental samples should be prepared for all duplicate samples and all sampling intervals. At least three duplicate samples of test soil should have been incubated under aerobic conditions (see section 1.7.1.1). Suitable containers with sufficient space under the lid should be used for all tests to avoid anaerobic conditions. In the case of volatile substances, the test shall only be carried out with a series of individual incremental samples.
XXI.1.7.1.3 Test conditions and duration
The test shall be carried out in the dark at room temperature (20 ± 2) ° C. The moisture content of the soil sample shall be maintained over the test period between 40% and 60% (± 5%) of the maximum water capacity of the soil (see section 1.6.4.2). Distilled or deionised water is added as needed.
Tests shall last at least 28 days. In the tests of agrochemicals, the rates of nitrate formation in exposed and control samples are compared. If these speeds differ by more than 25% on day 28, the test shall be continued until the difference falls to 25% or less, or 100 days, whichever is the earlier. For substances other than agrochemicals, the test shall be completed after 28 days. On day 28, the amount of nitrate in the treated and control samples shall be determined and ECx values calculated.
XXI.1.7.2 Sampling and soil analysis
XXI.1.7.2.1. Sampling intervals
When testing agrochemicals, the nitrate content of the samples shall be analysed on days 0, 7, 14 and 28. If the test is extended, further measurements shall be made at 14-day intervals after 28 days.
At least five test concentrations shall be used for testing substances other than agrochemicals and the nitrate content shall be analysed in soil samples at the beginning of the exposure period (day 0) and at the end (after 28 days). If deemed necessary, further intermediate measurements may be made, e.g. on Day 7. Data obtained on day 28 shall be used for the determination of ECx of the chemical. If necessary, data of 0 for reference samples may be used in the report as a starting concentration of nitrates in soil.
XXI.1.7.2.2 Analysis of soil samples
For each sampling time, the nitrate content shall be determined in both exposed and control samples. Nitrates are extracted from samples by shaking with suitable extraction solution, e.g. 0,1M potassium chloride solution. It is recommended to use 5 ml KCl solution per gram of dry matter of soil. For optimal extraction, soil vessels and extraction solution should be filled with a maximum of one half. The mixtures are extracted by shaking at 150 rpm for 60 minutes. The mixtures are centrifuged or filtered and the liquid phase is analysed for nitrates. Liquid particulate extract can be stored at (-20 ± 5) ° C for six months before analysis.
XXI.2. DATA
XXI.2.1 PROCESSING OF RESULTS
When testing agrochemicals, the amount of nitrates produced in each duplicate sample shall be recorded and the average values recorded in the table. The rate of nitrogen conversion shall be evaluated by appropriate generally accepted statistical methods (e.g. F-test to 5% of the level of significance). The amount of nitrates produced shall be expressed in mg of nitrates per kg of dry matter per day. The rate of nitrate formation shall be compared for each exposure with that obtained for control samples and a percentage deviation from the control samples shall be calculated.
When testing substances other than agrochemicals, the amount of nitrates in each duplicate sample shall be determined and a dose response curve shall be drawn for the purpose of estimating ECx values. The amount of nitrates (i.e. the quantity of nitrates in mg per kg dry matter of soil) found in exposed samples after 28 days shall be compared to the values found in the control samples. These data shall be calculated for each test concentration as a percentage inhibition. These percentages are drawn into a concentration-versus-concentration chart and then the ECx values are calculated using statistical methods. Standard methods (see literature 10 to 12) also calculate confidence intervals (p = 0,95) for calculated ECx values.
Test substances with high nitrate content may contribute to the amount of nitrate produced during the test. If high concentrations of these substances (e.g. chemicals expected to be re-used) are tested, appropriate control samples (i.e. soil and test substance without plant meal) shall be included. Data from these checks shall be taken into account in the ECx calculations.
XXI.2.2 INTERPRETATION OF RESULTS
If, in assessing the results of the tests with agrochemicals at any sampling time after 28 days, the difference between the rate of nitrate formation at lower exposure (i.e. maximum expected concentration) and the rate of production in controls is 25% or less, it can be concluded that the test substance does not have a long-term effect on the transformation of nitrogen in soils. The EC50, EC25 and / or EC10 values shall be used to evaluate test results of substances other than agrochemicals.
XXI.3. REPORTS
The test report shall include the following information:
Full identification of the land used, including the following:
- geographical location of the site (latitude and longitude);
- information and history of the site (i.e. vegetation cover, treatment of plant protection products, fertiliser use, accidental contamination, etc.);
- use method (e.g. agricultural land, forest, etc.);
- depth of sampling (cm);
- sand / dust / clay content (in% of dry matter);
- pH (in water);
- organic carbon content (in% of dry matter);
- nitrogen content (% of dry matter);
- initial nitrate concentrations (mg nitrates per kg dry matter);
- ion exchange capacity (mmol / kg);
- microbial biomass as a percentage of total organic carbon;
- reference to the methods used to determine each parameter;
- all information concerning the collection and storage of soil samples;
- details of any preliminary land incubation.
Test substance:
- the physical nature and, where appropriate, the physico-chemical characteristics;
- where appropriate, chemical identity data, including structural formula, purity (i.e. percentage of active ingredient for plant protection products), nitrogen content.
Substrate:
- the source of the substrate;
- composition (i.e. lucerne meal, green grass meal with lucerne);
- carbon content, nitrogen (in% of dry matter);
- sieve size (mm).
Test conditions:
- details of soil enrichment by an organic substrate;
- the number of test substance concentrations used and, where appropriate, justification for the choice of concentrations;
- details of application of the test substance to soil;
- incubation temperature;
- soil moisture at the beginning and during the test;
- the soil incubation method used (i.e. incubation of the aggregate sample or incubation of individual soil sub-samples);
- the number of duplicate samples;
- sampling periods;
- the method used to extract nitrate from the soil.
Results:
- the method of analysis and the equipment used for the determination of nitrate;
- individual measured nitrate levels and their average values in tabular form;
- differences between duplicate samples for exposed and soil control samples;
- an explanation of the corrections made to the calculations as necessary;
- percentage deviation at nitrate production rate for each sampling period, or EC50 with 95% confidence interval, other ECx values (i.e. EC25 or EC10) with confidence intervals and a graph of dose response curve;
- statistical processing of results;
- all information and observations useful for interpreting the results.
XXI.4. LITERATURE
(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 roule. September 28, 1987.
(4) SETAC-Europe (1995). Procedures for assessment of the environmental fate and ecotoxicity of pesticides, (ex M. R. Lynch), Pub. SETAC-Europe, Brussels.
(5) ISO 14238 (1997). 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.
(6) OECD (1995). Final Report of the OECD Workshop on Selection of Soils / Sediments, Belgium, 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: Subset-induced respiration method.
(9) ISO 14240-2 (1997). Soil quality - Determination of soil microbial biomass - Part 2: Fumigation-extraction method.
(10) Litchfield, J. T., Wilcoxon F. (1949). A simplified method of evaluation does-effect experiments. Yeah, Pharmacol. Exper. ther., 96, 99 - 113.
(11) Finney, D. J. (1971). Probit Analysis, 3rd edition, Cambridge, London, New York.
(12) Finney, D. J. (1978). Statistical Methods in Biological Assay. Griffin, Weycombe, UK.
XXII. METHOD FOR DETERMINING THE ACTIVITY OF SOCIAL MICROORGANISMS IN THE TRANSFORMATION OF CARBON
(Method C.22 of the Annex to Directive 2004 / 73 / EC of 29 April 2004 adapting to technical progress for the 22nd time Council Directive 67 / 548 / EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances)
XXII.1. METHOD
This method corresponds to OECD TG 217 (2000).
XXII.1.1 INTRODUCTION
This test method describes the laboratory procedure for investigating possible long-term effects of single exposure to plant protection products and, where appropriate, other chemicals on the activity of soil microorganisms in carbon transformation. It is based on the treasures and recommendations of national and international organisations (see literature 1 to 6). In assessing and evaluating the toxic properties of test substances, it may be necessary to determine effects on the activity of soil micro-organisms, e.g. when data are required on the side effects of plant protection products on soil micro-flora or when exposure of soil micro-organisms to chemicals other than plant protection products is expected. The objective of the carbon transformation test is to identify the effects of such chemicals on soil microflora. If agrochemicals (e.g. plant protection products, fertilisers, chemical products for forestry) are tested, both a carbon transformation test and a nitrogen transformation test are performed. If chemicals other than agrochemicals are tested, a nitrogen transformation test is sufficient. However, if these chemicals have EC50 values for nitrogen transformation within the range found for commercially available nitrification inhibitors (e.g. nitrapyrin), a carbon transformation test may be performed to obtain further information.
Soils consist of living and inanimate ingredients that occur in the form of complex and heterogeneous mixtures. Microorganisms play an important role in the degradation and transformation of organic substances in fertile soils, with different species contributing to different aspects of soil fertility. Any long-term breach of these biochemical processes could break the nutrient cycle and this could affect soil fertility. The transformation of carbon and nitrogen occurs on all fertile soils. Although the populations of micro-organisms responsible for these processes differ from soil to soil, they are essentially the same transformation scheme.
The described test method serves to identify the long-term adverse effects of the substance on the carbon transformation process in aerobic surface soils. The test is sensitive to changes in the size and activity of the microbial population responsible for carbon transformation, as these populations will be exposed to both chemical stress and carbon deficiency. Sand soils with low organic content shall be used. These soils are exposed to the test substance and incubated under conditions that allow rapid microbial metabolism. Under these conditions, the sources of readily available carbon are rapidly depleted. This causes carbon shortages, which lead to not only the death of microbial cells, but also induces a resting stage and / or litigation. If the test is performed for more than 28 days, the sum of these reactions can be measured in control samples (unexposed soil) as a gradual loss of metabolic-active microbial biomass (see literature 7). If the biomass in carbon-deficient soil under test conditions is affected by the presence of a chemical, it is unlikely to return to the same state as the control sample. Thus, the failure of the test substance at any time during the test often persists until the end of the test.
The test on which this test method is based was initially developed for substances that can be predicted to penetrate the soil. Examples are plant protection products for which the field application dose is known. For agrochemicals it is sufficient to perform a test at two dose levels corresponding to the expected or estimated application dose. For agrochemicals, active ingredients or preparations in commercial treatment may be tested. However, the test is not limited to chemicals whose concentration in the environment can be predicted. By changing the amount of test substance applied to the soil and the way in which the data are evaluated, the test may also be used for chemicals which do not know how much they penetrate the soil. Therefore, for chemicals other than agrochemicals, the effect of a number of concentrations on carbon transformation is determined. The data from these tests shall be used to construct a dose response curve and to calculate ECx values where x is the percentage effect.
XXII.1.2 DEFINITIONS
Carbon transformation: is the degradation of organic material by the action of micro-organisms resulting in an inorganic end product of carbon dioxide.
ECx (Effective concentration): is the concentration of the test substance in soil that results in an x-percentage inhibition of carbon transformation into carbon dioxide.
EC50 (Median effective concentration): is the concentration of the test substance in soil which results in a 50% inhibition of carbon transformation into carbon dioxide.
XXII.1.3 REFERENCE SUBSTANCES
None.
XXII.1.4 CONDITIONS OF THE TEST METHOD
Stranded soil is either exposed to the test substance or left unexposed (control). When testing agrochemicals, it is recommended to perform a test with at least two concentrations chosen to be related to the highest expected concentration in the field. After 0, 7, 14 and 28 days incubation, samples of exposed and control soil shall be mixed with glucose and the following 12 hours shall be measured at the respiratory rate induced by glucose. The respiratory rate is expressed as released carbon dioxide (mg carbon dioxide per kg dry matter per hour) or as oxygen consumed (mg oxygen per kg of soil per hour). The mean respiratory rate of exposed soil samples shall be compared to the rate of reference samples and the percentage deviation of the exposed samples from the control samples shall be calculated. All tests shall be carried out for at least 28 days. If the difference between experimental and control soil is 25% or greater on day 28, measurements shall be continued at 14-day intervals up to a maximum of 100 days. When testing chemicals other than agrochemicals, a number of concentrations of the test substance shall be added to soil samples and the respiratory rate induced by glucose (i.e. the average amount of carbon dioxide released or consumed oxygen) shall be measured after 28 days. Test results with a number of concentrations shall be analysed using a regression model and ECx values shall be calculated (i.e. EC50, EC25 and / or EC10). See definitions.
XXII.1.5 TEST VALIDITY
Assessment of test results for agrochemicals is performed with relatively small differences (i.e. an average of ± 25%) between carbon dioxide released or oxygen consumed in control and treated samples, so that large variations in control samples may lead to incorrect results. The differences between each control sample should therefore be less than ± 15%.
XXII.1.6 DESCRIPTION OF THE METHOD
XXII.1.6.1 Test equipment
The containers used in the test shall be of chemically inert material. They should have an appropriate volume which should correspond to the procedure used for soil incubation, i.e. either soil incubation or soil incubation (see Section 1.7.1.2). During the test, water losses shall be as low as possible and gas exchange shall be allowed (e.g. by covering the containers with perforated polyethylene foil). Closed gas-tight containers shall be used for the testing of volatile substances. They should be large enough to fill approximately one quarter of their volume with a soil sample.
Incubation systems and instruments for measuring released carbon dioxide or oxygen consumption are necessary to determine glucose-induced respiration. Examples of such systems can be found in literature 8 to 11.
XXII.1.6.2 Selection and number of soils
One soil shall be used. The soil should have the following characteristics:
- sand content: not less than 50% and not more than 75%;
- pH: 5,5 - 7,5;
- organic carbon content: 0,5 - 1,5%;
- microbial biomass is determined (see literature 12 and 13) and its carbon content should be at least 1% of the total organic carbon in the soil.
In most cases, soil with these characteristics is the most conservative case since adsorption of the test chemical is minimal and its availability for micro-organisms is maximum. As a result, testing with other soils is generally unnecessary. Under certain circumstances, additional soil may be necessary, e.g. in the intended main use of the test substance on certain soil types, such as acidic forest land or in the case of electrostatically charged chemicals.
XXII.1.6.3 Collection and storage of soil samples
XXII.1.6.3.1 Collection
Detailed information on the history of the place from which the land is collected should be available. This information shall include the exact location, vegetation, treatment data of plant protection products, the use of organic and mineral fertilisers, the addition of biological material or accidental contamination. A place should be chosen for land collection that allows long-term use. Permanent pasture, fields with annual crops (except maize) or densely sown green fertilisation area are suitable. The sites selected for sampling should not be treated with plant protection products for at least one year prior to collection. Organic fertilisers should also not be used for at least six months. The use of mineral fertilisers is permitted only if it is necessary for crops and soil samples should not be taken before three months after application. Soils with fertilisers known to have biocidal effects (e.g. calcium cyanide) should not be used.
Samples should not be taken during or after long periods of drought or irrigation (longer than 30 days). The arable soil samples shall be taken from a depth of 0 to 20 cm. In the case of meadows (pastures) or other soils for which no ploughing has been performed for at least one growing season, the maximum sampling depth may slightly exceed 20 cm (e.g. up to 25 cm). Soil samples should be transported in such containers and at such temperature as to ensure that the original soil characteristics do not change significantly.
XXII.1.6.3.2 Storage
The use of freshly harvested land is preferred. If storage of soil in the laboratory is unavoidable, it may be stored in the dark at (4 ± 2) ° C for a maximum period of three months. During storage, aerobic conditions must be ensured. If the soil is collected from areas where it is frozen for at least three months in the year, storage at -18 ° C for six months may be considered. Prior to each experiment, microbial biomass stored soils are determined and biomass-derived carbon should be at least 1% of total organic carbon in the soil (see Section 1.6.2).
XXII.1.6.4 Soil treatment and preparation for testing
XXII.1.6.4.1 Preliminary incubation
Where land has been stored (see sections 1.6.3.2 and 1.7.1.3), preliminary incubation of 2 to 28 days is recommended. The soil temperature and humidity during the preliminary incubation should be similar to the test conditions (see sections 1.6.4.2 and 1.7.1.3).
XXII.1.6.4.2 Physical-chemical characteristics
Large pieces (e.g. stones, parts of plants, etc.) shall be removed by hand from the soil and then spread through particles 2 mm or less without excessive drying. The moisture content of the soil sample shall be adjusted by distilled or deionised water to 40% to 60% of the maximum water capacity.
XXII.1.6.5 Preparation of the test substance for application to soil
The test substance is usually applied using a carrier. The carrier may be water (for water-soluble substances) or inert solids such as fine silica sand (grain size: 0,1 - 0,5 mm). Liquid carriers other than water (e.g. organic solvents such as acetone, chloroform) should not be used as they can destroy microflora. If used as a sand carrier, it may be coated with a test substance dissolved or dispersed in an appropriate solvent. In this case the solvent is removed by evaporation before mixing with the soil. For optimum distribution of the test substance in soil, 10 g sand per kilogram of soil (expressed on dry matter) is recommended. Only the corresponding amount of water and / or sand shall be applied to the control samples.
When testing volatile chemicals, losses during application should be avoided and a homogeneous dispersal of the substance in the soil should be attempted (e.g. spray the substance into the soil in several places).
XXII.1.6.6 Test concentrations
At least two concentrations shall be used when testing plant protection products or other chemicals for which concentration can be predicted in the environment. The lower concentration should correspond to at least the maximum expected quantity of the substance that enters the soil under real conditions, while the higher concentration should be a multiple of the lower concentration. Calculation of the concentration of the test substance added to the soil shall be carried out provided that it is evenly adjusted to a depth of 5 cm and for a free soil density of 1,5. For agrochemicals that are applied directly to the soil, or for chemicals for which the quantity can be predicted in the soil, the recommended test concentrations are the predicted environmental concentration (PEC) and their five times. For substances that are normally applied to soil several times per season, the test concentrations are derived from the product PEC and the expected maximum number of applications. However, the upper concentration should not exceed 10 times the maximum single dose administered.
A geometric series of at least five concentrations shall be used for substances other than agrochemicals. The ECx values should be found in the range of these test concentrations.
XXII.1.7 TEST PERFORMANCE
XXII.1.7.1 Exposure conditions
XXII.1.7.1.1 Exposure and control
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Regulation Information
| Citation | Decree No. 389 / 2005 Coll., amending Decree No. 222 / 2004 Coll., laying down, for chemicals and chemical preparations, the basic methods for testing physico-chemical properties, explosive properties and environmental hazards |
|---|---|
| Regulation Type | Order |
| Author | - |
| Collection | Code of Laws |
| Date of Promulgation | 01.10.2005 |
|---|---|
| Effective from | 08.10.2005 |
| Effective until | - |
| Status | Valid |
The regulation text is for informational purposes only.
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