GB/T 27854-2011 Test for soil microbial nitrogen transformation
of chemicals GB/T27854-2011 Standard compression package decompression password: www.bzxz.net This standard specifies the method overview, instrumentation, test system, test procedure, quality assurance and quality control, data and report of the test for soil microbial nitrogen transformation of chemicals. This standard is applicable to the assessment of the long-term negative impact of a single exposure to chemicals on the nitrogen transformation activity of soil microorganisms.
class="f14" style="padding-top:10px; padding-left:12px; padding-bottom:10px;"> This standard was drafted in accordance with the rules given in GB/T1.1-2009.
This standard has the same technical content as the OECD Chemical Testing Guidelines 216 "Soil Microbial Nitrogen Transformation Test" (English version) of the Organization for Economic Cooperation and Development
.
This standard has been modified in the following structural and editorial aspects:
———The preface of the original text has been adjusted to be the introduction;
———The terms and definitions have been adjusted from the appendix of the original text to the main text;
———The measurement units have been changed to the legal measurement units of China.
This standard was proposed and managed by the National Technical Committee for Standardization of Dangerous Chemicals Management (SAC/TC251). The
drafting units of this standard are: Guangdong Microbiological Analysis and Testing Center, Hubei Entry-Exit Inspection and Quarantine Bureau, Chemical Registration Center of the Ministry of Environmental Protection, China Inspection and Quarantine Scientific Research Institute, Guangdong Demei Fine Chemical Co., Ltd., Dongguan Shengyuan Environmental Protection Technology Co., Ltd.
The main drafters of this standard are: Mei Chengfang, Zeng Guoqu, Xu Meiying, Guo Jian, Zhou Hong, Chen Huiming, Xiao Yijin, Guo Yuliang.

ICS13.300,13.020
National Standard of the People's Republic of China
GB/T27854——2011
Chemicals
Soil microorganisms
Nitrogen transformation test
Chemicals-Soil mnicroorganisms-Nitrogen transformation test2011-12-30Issued
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaStandardization Administration of the People's Republic of China
2012-08-01Implementation
This standard was drafted in accordance with the rules given in GB/T1.1-2009. GB/T27854—2011
This standard has the same technical content as the OECD Chemical Testing Guidelines 216 Soil Microbial Count Transformation Test (English version).
This standard has been modified in the following ways: the preface of the original text is adjusted as the introduction; the terms and definitions are adjusted from the appendix of the original text to the main text; the measurement units are changed to the legal measurement units of my country. This standard is proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals (SAC/TC251). The drafting units of this standard are: Guangdong Microbiological Analysis and Testing Center, Chaobei Entry-Exit Inspection and Quarantine Bureau, Chemical Registration Center of the Ministry of Environmental Protection, China Inspection and Quarantine Science Research Institute, Guangdong Demei Fine Chemical Co., Ltd., Dongguan Shengyuan Environmental Protection Technology Co., Ltd. The main drafters of this standard are: Mei Chengfang, Zeng Guoqu, Xu Meiying, Guo Jian, Zhou Hong, Chen Huiming, Xiao Yijin, Guo Yuliang. I
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GB/T 27854—201t
This test method is used to evaluate the long-term effects of a single exposure to chemicals on the bioammonia conversion activity of soil. This test is mainly based on the test methods recommended by the European and Mediterranean Plant Protection Organization1, and also refers to the test methods from the German Federal Institute for Biological Research-3], the US Environmental Protection Agency, the Institute of Environmental Toxicology and Chemistry and the International Organization for Standardization. In 1995, the OECD Soil Fill/Sediment Working Group reached a consensus in Belgirate, Italy on the type and quantity of soil to be used in this test. The collection, processing and storage of soil samples mainly refer to the ISO guidance documents [and the recommendations of the Belgirate Working Group. When evaluating the toxicity of the test substance, for example, when it is necessary to provide potential negative effects of crop protection products on soil microbial flora, or when soil microorganisms may be exposed to substances other than crop protection products. When other chemicals are used in the test, it is necessary to determine the effect of the test substance on the activity of soil microorganisms. Nitrogen conversion tests are used to test the effects of these chemicals on soil microbial flora. If the test substance is an agrochemical (such as crop protection products, fertilizers, forest chemicals), nitrogen conversion tests and carbon conversion tests are required. If the test substance is a non-agricultural chemical, only nitrogen conversion tests are required. However, if the EC value of the nitrogen conversion test of the chemical falls within the ECs value range of commercial nitrification inhibitors (such as 2-chloro-6-triaminomethylpyridine), a carbon conversion test is required to obtain further information.
Soil is composed of a complex, heterogeneous mixture of biological and non-biological organisms. Microorganisms play an important role in the degradation and transformation of organic matter in fertile soil, and different species Microorganisms of this type contribute to all aspects of soil fertility. Any long-term intervention in these biochemical processes will disrupt nutrient cycles and thus change soil fertility. Carbon and nitrogen conversion occurs in all fertile soils. Although the microbial communities involved in these conversion processes vary in different soils, the conversion pathways are essentially the same. This test is used to investigate the long-term negative effects of the test substance on the nitrogen conversion process in aerobic topsoil. This test method can also be used to evaluate the effects of the test substance on the carbon conversion process of soil microbial flora. The formation of nitrate usually occurs after the carbon-hydrogen bond is broken. Therefore, when the nitrate production rates of the control group and the treatment group are the same, it can be inferred that the main carbon degradation pathway is complete and effective. The culture medium selected for the test (such as purple flower head hunting powder) should have The appropriate carbon-nitrogen ratio is usually in the range of 12/1 to 16/1. In this way, the phenomenon of "carbon starvation" of microbial cells during the test will be reduced, and even if the microbial community is damaged by chemicals, it may recover within 100 days. This test is mainly for substances whose actual uptake in the soil can be expected, for example, the application rate of crop protection products in the field is known. For agricultural chemicals, two doses can be used in the test according to their expected application rate. Agricultural chemicals can be tested in the form of their active ingredients (AI) or as products. However, this test is not only applicable to agricultural chemicals. By simultaneously changing the amount of the test substance in the soil during the test and the way of data evaluation, this method is also applicable to chemicals whose uptake in the soil is still unknown. Therefore, for non-agricultural chemicals, it is necessary to determine the effect of a series of different concentrations on the conversion of nitrogen. Dose-effect curves are drawn based on the obtained test data, and EC values ​​are calculated, which are defined as the percentage inhibition of nitrogen conversion. TTKANYKAcA
1 Scope
ChemicalsWww.bzxZ.net
Test for ammonia transformation of soil microorganisms
GB/T 27854—2011
This standard specifies the method description, instrumentation, test system, test procedure, quality assurance and quality control, data and report of the test for nitrogen transformation of soil microorganisms of chemicals.
This standard is applicable to the evaluation of the long-term negative impact of a single exposure of chemicals on the nitrogen transformation activity of host microorganisms. 2 Terms and definitions
The following terms and definitions apply to this document. 2.1
Nitrogen transformation The process by which microorganisms degrade nitrogen-containing organic matter into inorganic end products, nitrates, through ammonification and nitrification. 2.2
Effect concentration, EC, is the concentration of the test substance in the soil at 1 pm that causes the percentage inhibition of nitrogen transformation. 2.3
Median effective concentration, ECsp is the concentration of the test substance in the soil when the inhibition percentage of nitrogen transformation is 50%. 3 Method Overview
Sieve soil is mixed with plant debris and then treated with the test substance or not treated (control group). If the test substance is an agricultural chemical, at least two test concentrations are selected. The concentration setting can refer to the highest concentration of the test substance expected to be applied during the year. After 0?d, 14d and 28d of incubation, a certain amount of soil samples are taken from the treatment group and the control group, extracted with a suitable solvent and the content of nitraldehyde salt in the extract is determined. The nitrate formation rate of the treatment group is compared with that of the control group, and the percentage difference of the treatment group relative to the control group is calculated. All experiments last for at least 28d. If the difference between the treatment group and the control group on the 28th day is not less than 25%, the experiment can be extended to a maximum of 100 days. If the test substance is a non-clothing chemical, a series of different concentrations of the test substance are added to the soil samples and the production of nitrate in the treated and control groups is determined on the 28th day. The test results of the series of concentrations are analyzed using a regression model and the EC values ​​(i.e. ECs, ECs and/or EC1) are calculated. 4.1 The test container should be made of chemical materials and should have a suitable volume to match the soil culture method, that is, the soil samples are cultured in large batches or in a series of separate packages (see 6.1.2). During the test, water loss should be minimized and gas exchange should be maintained (for example: the test container can be covered with a ethylene foil with small holes). If the test substance is volatile, a closed or airtight test container should be used. The size of the test container should be such that the soil sample filled accounts for about 1/4 of its volume. 4.2 Laboratory equipment with the following standards: A stirring device (mechanical vibrator or similar device), a centrifuge (3000 g) or a filter (using nitrate-free filter paper) 1
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GB/T 27854—2011
An instrument for nitrate analysis with sufficient sensitivity and reproducibility. 5 Test system
5. 1 Soil
5.1.1 Selection of soil
Use a single soil. The recommended soil characteristics are as follows: sand content: 50%~75%;
pH+5. 5~~7. 5;
Organic carbon content: 0. 5%~1. 5%;
The biomass of microorganisms [B-,Its carbon content should be not less than 1% of the total organic carbon content of the soil. In most cases, soils with the above characteristics represent the worst case, because they have the smallest adsorption disk for the test substance and are most suitable for the growth of microorganisms. Therefore, it is usually not necessary to use other soils for testing. However, in some cases, such as when the test substance is expected to be mainly used in certain special soil spots (such as acidic forest soils) or the test substance is electrostatically charged, another type of soil should be used. 5.1.2 Collection and storage of soil samples
5.1.2.1 Collection
Detailed background information on the soil collection point used for the test should be collected, including: exact location, mold cover, date of application of crop protection products, application of organic and inorganic feeds, added biological materials or accidental contaminants. The soil collection point selected should be able to be used for a long time, such as permanent grassland pasture, cultivated land with annual cereal crops (except corn), or densely planted green manure fields. The sampling site should not have been used with crop protection products for at least one year and organic fertilizers for at least six months before sampling. Unless inorganic fertilizers are applied to meet crop needs, sampling should be done at least three months after the soil has been fertilized. Soils that have been treated with biocidal fertilizers (such as calcium cyanamide) should be avoided. Sampling should be avoided during long dry or flooded periods (more than 30 days) or immediately after such periods. The sampling depth in cultivated land is 0 cm to 20 cm. When sampling in grassland or other types of soil that have not been cultivated for a long time (at least one growing season), the maximum depth can be slightly more than 20 cm (for example, 25 cm). Soil samples should be transported in containers and maintained at an appropriate humidity to ensure that the properties of the soil do not change significantly. 5.1.2.2 Storage
It is best to use soil freshly collected from the field for the test. If storage in the laboratory cannot be avoided, it can be placed in a dark place with 420 degrees Celsius for up to 3 months. The soil should be kept in aerobic conditions during storage. If the sampling area has at least 3 months of ice aging each year, it can also be stored at -18℃~-22℃ for 6 months. The biomass of soil microorganisms should be determined before each test, and the carbon content of the biomass should account for at least 1% of the total organic carbon content of the soil. 5.1.3 Treatment and preparation of test soil
5.1.3.1 Pre-culture
If the soil has been stored, it is recommended to add a pre-culture process for 2d to 28d. The temperature and humidity of the soil during the pre-culture period should be consistent with the test conditions.
5.1.3.2 Physical and chemical properties
Artificially remove coarse objects (such as stones, plant residues, etc.) from the soil, and then sieve it in a wet state (avoid excessive drying) to make the particle size no larger than 2mn. Soil moisture can be adjusted with distilled or deionized water to a moisture content equivalent to 40% to 60% of the maximum water holding capacity. 5.1. 3.3 Supplementation of organic substrate
The soil needs to be supplemented with appropriate organic substrate for adjustment, for example, the main component of which is Medicago sativa grass-green grain meal, with a carbon-nitrogen ratio ofAnalyze the soil sample for nitrate at the same time. If necessary, an additional period may be added, for example on day 7. The data obtained on day 28 can be used to calculate the EC value of the chemical. If necessary, the data from day 0 for the control group can be used to report the initial nitrate content of the soil. 6.2.2 Analysis of soil fill samples
Nitrate content of each treatment and control sample should be determined at each sampling. Extract nitrate by mixing and shaking the soil sample with a suitable extractant (e.g. 0.1 mol/L potassium chloride solution). It is recommended to add 5 mL of potassium chloride solution per gram dry weight of soil. For optimal extraction, the container should not contain more than half the volume of the container. The mixture is shaken at 15.7 rad/s for 60 minutes. Centrifuge or filter the mixture and analyze the liquid phase for nitrate content. The liquid phase, free of solid particles, can be stored for up to 6 months at 20℃±5℃. 7 Quality Assurance and Quality Control
Evaluation of the results of the test of agricultural chemicals is based on small differences in nitrate concentrations between the treatment and control groups (e.g., an average of 25% per soil). Large differences between replicates of the control group will lead to erroneous results. Therefore, the difference between replicates of the control group should be less than ±15%.
8 Data and Reporting
8.1 Data
If agricultural chemicals are tested, the amount of nitrate formed in each replicate soil sample should be recorded and the average of all replicates should be tabulated. Ammonia conversion should be evaluated using appropriate and widely accepted statistical methods (e.g., F test, 5% significance level). The amount of nitrate formed is expressed in milligrams of nitrate per kilogram of soil per day, in units of g/(kd). Compare the nitrate formation rate of 4 soil samples in each treatment group and the control group, and calculate the percentage deviation of the treatment group from the control group. GB/T 27854--2011
If non-agricultural chemicals are tested, the nitrate content of each replicate needs to be determined and a concentration-effect curve needs to be drawn to calculate the EC value. The amount of nitrate formed in the samples of the treated group after 28 days (i.e., milligrams of nitrate produced per gram of dry weight of soil, mg/kg) is compared with the control group. Based on these data, the percentage inhibition at each test concentration is calculated, and these percentages are plotted against the test concentrations. The EC value is calculated using statistical methods, and the confidence limits of the EC value (force = 0.95) [10-121] are determined according to standard procedures. Variables with higher nitrogen content may result in increased amounts of nitrate formed in the test. If these test substances are tested at higher concentrations (e.g., chemicals that may be reused), the test should include corresponding controls (i.e., soil with the test substance added but without plant powder). The data of these control groups need to be referenced when calculating the EC. 8.2 Interpretation of results
When evaluating the test results of agricultural chemicals, if the difference in the rate of salt formation between the low concentration treatment group (i.e. the expected maximum actual use concentration) and the control group is not more than 5, it can be considered that the chemical has no long-term effect on the conversion of ammonia in the soil. When evaluating the test results of chemicals other than agricultural chemicals, ECs, EC and (or) ECo values ​​can be used.
8.3 Test report
The test report should include the following:
a) Complete test soil identification information
Geographic location of sampling point (latitude, longitude); Background information of sampling point (vegetation cover, use of crop protection products and fertilizers, accidental pollution, etc.); Utilization method (agricultural soil, forest, etc.); Sampling depth (cm):
Sand/silt/clay content in soil (thousand weight) (%); pH value (in water);
Organic carbon content in soil (dry weight) (%), ...·Nitrogen content in soil (thousand weight) (%); Initial acetate concentration (nitrate per kg dry weight of soil, mg/kg); Ions Exchange capacity (mmol/kg);
microbial biomass (expressed as a percentage of total organic carbon); references to the methods used to determine each parameter; complete information on soil collection and preservation: details of soil pre-culture (if any);
b) physical properties of the test substance, and relevant physicochemical properties: - information on the test substance, including: structural formula, purity (for crop protection products, the percentage of active ingredients), ammonia content,
e) substrate
source,
composition (i.e. first powder, first grass - green cereal powder); - carbon and ammonia content (%) in the substrate (dry weight) - sieve aperture (mm).
d) Test conditions
Details of soil improvement using organic substrate: 5
GB/T 27854--2011
Set the number of test concentration groups for the test substance and explain the rationality of the selected concentration appropriately; Detailed steps for applying the test substance to the soil; - Incubation temperature:
Soil humidity at the beginning of the test and during the test, - Soil incubation method (whole method, or method of a series of separate sub-samples): - Number of replicates for a test group;
- Number of joint samples,
Set up a method for extracting nitrate from the soil.
Procedures and instruments used for analytical determination of nitrate content; -Tabulation of data, including individual data for nitrate determination and mean + differences between replicates in treatment and control groups; if corrections are involved in the calculation, these should be stated; percentage change in nitrate formation rate for each sampling, if necessary, as well as ECs values ​​and 95% confidence limits, other ECs (such as EC25 or ECm) and their confidence intervals, and concentration-effect curves; -Statistical treatment;
All information and observations that will aid in the interpretation of the results. References
GB/T 27854—2Dt1
[1] EPPO (1994). Decision-Making Schemc for the Environmental Risk Assessment of PlantProtectian Chemicals, Chapter 7:Soil Microflara, EPPO Bulletin.1994,24; 1-16[2] BBA (1990). Effccts on thc Activity of thc Soil Microflora. Guidelincs for the Official Testing of Plant Protectian Products, VI,1-l(2nd cds, >1990[31 EPA (1987). Soil Microbial Community Toxicity Test, EPA 40 CFR Part 797. 3700. TaxicSubstances Control Aet Test Guidelines; Proposed rule.September28,1987[4] SETAC-Europe (1995).Procedures for assessing the environmental fate and ecotoxicity ofpesticides,Ed.MRLynch,Pub.SETAC-Europc,Brussels[5] ISO/DIS14238<1995).Soil Quality-Determination of Nitrogen Mineralisation andNitrification in Soils and the Influence af Chemicals on these Processes. Technical Committee ISO/Tu190/SC 4 Sotl Quality -Biological Method s[6] OECD (1995). Final Report of the OECD Workshop on Selection of Soils/Sediments,Belgirate,Italy,1995:18-20
[7] ISQ 10381-6 (1993). Soil quality-Sampling. Guidance on the collcetion,handling and storagcof soil for the assessment of aerobic microbial processes in the laboratory[8] ISO 14240-1 (1997), Soil quality-Determination of soil microbial biotnass-Part l. Substrate-induced respiration tmethad
[9 ] ISO 14240-2 (1997). Soil quality-Deterrmination of soil microbial biomass-Part 2:Fumigation-extractionmethod
E10] Litehfield,JT and Wilcoxon F. (1949). A simplified method of evaluating dosc-effectexperimcnts .Jour,Pharmacol.andExper.Ther.,96,99-113[11J Finney,DJ(1971).Probit Analysis.3rd ed.,Cambridge,London and New-York[12]Finney,DJ(197).Statistical Methods in biological Assay.Griffin,Weycombe,UK
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