1、September 2009DEUTSCHE NORM English price group 8No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 13.080.05!$YQS“154464
2、8www.din.deDDIN ISO 10381-6Soil quality Sampling Part 6: Guidance on the collection, handling and storage of soil underaerobic conditions for the assessment of microbiological processes,biomass and diversity in the laboratory (ISO 10381-6:2009)English version of DIN ISO 10381-6:2009-09Bodenbeschaffe
3、nheit Probenahme Teil 6: Anleitung zur Entnahme, Behandlung und Lagerung von Boden unter aerobenBedingungen fr die Beurteilung mikrobiologischer Prozesse sowie der Biomasse und derDiversitt unter Laboratoriumsbedingungen (ISO 10381-6:2009)Englische Fassung DIN ISO 10381-6:2009-09SupersedesDIN ISO 10
4、381-6:1997-05www.beuth.deDocument comprises 11 pagesContents Page National foreword 3 National Annex NA (informative) Bibliography 4 Introduction.5 1 Scope 6 2 Terms and definitions .6 3 Procedure .6 3.1 Selection of sampling locations.6 3.2 Description of field site.7 3.3 Sampling conditions .7 3.4
5、 Sampling methods.7 3.5 Sample marking .7 3.6 Transportation conditions 7 3.7 Soil processing 7 3.8 Storage conditions and storage periods.8 3.9 Pre-incubation9 4 Sampling report .9 Bibliography. 11 2 DIN ISO 10381-6:2009-09 National foreword This standard has been prepared by Technical Committee IS
6、O/TC 190 “Soil quality”, Subcommittee SC 4 “Biological methods”. The responsible German body involved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Technical Committee NA 119-01-02-04 UA Biologische Verfahren. Expert assistance and specialized laborator
7、ies will be recommended to perform the sampling procedures described in this standard. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. DIN shall not be held responsible for identifying any or all such patent rights. DIN ISO 10381
8、consists of the following parts, under the general title Soil quality Sampling: Part 1: Guidance on the design of sampling programmes Part 2: Guidance on sampling techniques Part 3: Guidance on safety Part 4: Guidance on the procedure for investigation of natural, near-natural and cultivated sites P
9、art 5: Guidance on the procedure for the investigation of urban and industrial sites with regard to soil contamination Part 6: Guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory Part 7: Guidance on sampling of soil gas Part 8:
10、 Guidance on sampling of stockpiles The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 11465:1993 DIN ISO 11465:1996 ISO 14238 DIN ISO 14238 ISO 14240-1 DIN ISO 14240-1 ISO 14240-2 DIN ISO 14240-2 ISO 15685 DIN ISO 15685 ISO 16072 DIN ISO
11、16072 ISO 17155 DIN ISO 17155 ISO 23753-1 DIN ISO 23753-1 ISO 23753-2 DIN ISO 23753-2 Amendments This standard differs from DIN ISO 10381-6:1997-05 as follows: a) Clause 3.6 “Transportation conditions” has been revised. b) “Storage conditions and storage periods” have been combined in clause 3.8. 3
12、DIN ISO 10381-6:2009-09 c) Further details have been specified regarding storage conditions and storage periods (e.g. freezing temperature and duration). d) Table 1 relating to storage conditions for the assessment of aerobic microbial processes has been added. e) The title of the standard has been
13、changed. Previous editions DIN ISO 10381-6: 1997-05 National Annex NA (informative) Bibliography DIN ISO 11465:1996, Soil quality Determination of dry matter and water content on a mass basis Gravimetric method DIN ISO 14238, Soil quality Biological methods Determination of nitrogen mineralization a
14、nd nitrification in soils and the influence of chemicals on these processes DIN ISO 14240-1, Soil quality Determination of soil microbial biomass Part 1: Substrate-induced respiration method DIN ISO 14240-2, Soil quality Determination of soil microbial biomass Part 2: Fumigation-extraction method DI
15、N ISO 15685, Soil quality Determination of potential nitrification and inhibition of nitrification Rapid test by ammonium oxidation DIN ISO 16072, Soil quality Laboratory methods for determination of microbial soil respiration DIN ISO 17155, Soil quality Determination of abundance and activity of th
16、e soil microflora using respiration curves DIN ISO 23753-1, Soil quality Determination of dehydrogenase activity in soils Part 1: Method using triphenyltetrazolium chloride DIN ISO 23753-2, Soil quality Determination of dehydrogenase activity in soils Part 2: Method using iodotetrazolium chloride (I
17、NT) 4 DIN ISO 10381-6:2009-09 Introduction Soils are both complex and heterogeneous because they consist of both living and non-living components occurring in different combinations. Therefore, the condition of the soil, from collection to completion of an experiment, should be considered in relatio
18、n to effects on the soil microflora. Temperature, water content, availability of oxygen and duration of storage are all known to affect the soil microflora, and thus the processes they mediate. Soils can however be used effectively in laboratory systems to investigate microbially-mediated processes,
19、 provided that the dynamics of the living microflora are appreciated. This part of ISO 10381 provides guidance on the collection, handling and storage of soil for laboratory use where aerobic microbial activity is the main component of the study. It describes how to minimize the effects of differenc
20、es in temperature, water content and availability of oxygen on aerobic processes to facilitate reproducible laboratory determinations 10, 11. Soil quality Sampling Part 6: Guidance on the collection, handling and storage of soil under aerobic conditions for the assessment of microbiological processe
21、s, biomass and diversity in the laboratory 5 DIN ISO 10381-6:2009-09 1 Scope This part of ISO 10381 provides guidance on the collection, handling and storage of soil for subsequent testing under aerobic conditions in the laboratory. The recommendations in this document are not applicable to the hand
22、ling of soil where anaerobic conditions are to be maintained throughout. This part of ISO 10381 is mainly applicable to temperate soils. Soils collected from extreme climates (e.g. permafrost, tropical soils) may require special handling. 2 Terms and definitions For the purposes of this document, th
23、e following terms and definitions apply. 2.1 aerobic descriptive of a condition in which molecular oxygen is freely available 2.2 anaerobic descriptive of a condition in which molecular oxygen is not available 2.3 water content on a dry mass basis mass of water evaporating from the soil when dried t
24、o constant mass at 105 C divided by the dry mass of the soil and multiplied by 100 ISO 11465:1993, 3.2 3 Procedure 3.1 Selection of sampling locations The locations of the sites from which samples are taken should be selected according to the purpose of the study. These locations should be identifie
25、d and recorded, e.g. on a map by reference to easily recognizable static objects or by using a detailed map reference or by GIS. If practicable, the locations should be marked so that they may be used for comparative tests or for obtaining replicate samples. 6 DIN ISO 10381-6:2009-09 3.2 Description
26、 of field site Selection of a soil sampling site depends on the purpose of a particular study, and knowledge of the field site history is always desirable. The site should be accurately described and its history given. Details of vegetation cover, the morphology of the sampling area (e.g. flat area,
27、 slopes, steepness), and of chemical and biological additions or accidental contamination, should be recorded and reported. 3.3 Sampling conditions Soil required for studies conducted under laboratory conditions should, if practicable, be sampled in the field with a soil water content which facilita
28、tes sieving. Sampling should, unless the purpose of the study requires otherwise, be avoided during or immediately following long periods (e.g. 1 month) of drought, freezing or flooding. If laboratory tests are to be used for field monitoring, conditions existing in the field should be accepted. Soi
29、l samples may be frozen before investigations of, for example, ammonium oxidations. 3.4 Sampling methods The sampling technique depends on the purpose of the study. If aerobic agricultural soil is required, sampling is usually conducted to the actual ploughing depth. Any surface vegetation cover, mo
30、ss-covered litter layer, visible roots, large pieces of plant or woody plant litter and visible soil fauna should be removed to minimize the addition of fresh organic carbon to the soil. Organic constituents introduced from roots or other sources can cause unpredictable changes in the activities and
31、 composition of the soil microflora. If natural soils show distinct horizons, samples should be taken from these horizons. 3.5 Sample marking Sample containers should be clearly and unambiguously marked and identified so that each sample can be related to the location from which it was taken. Use of
32、 containers which either absorb water from the soil or release materials, e.g. solvents or plasticizers, into the soil should be avoided. 3.6 Transportation conditions Samples should be transported in a manner which minimizes changes in the soil water content, and should be kept in the dark with fre
33、e access of air; a loosely-tied polyethylene bag is generally adequate for this purpose. Extreme environmental conditions should be avoided: the soil should be kept as cool as possible but it is essential that it is not allowed to dry out or become water-logged. Exposure to light for extended period
34、s should be avoided as this encourages the growth of algae on the surface of the soil. Physical compaction should be avoided as far as is practicable. Samples for DNA or RNA analysis shall be frozen quickly in the field using dry ice. During transportation to the laboratory, dry ice shall be used to
35、 maintain the temperature of those for RNA analysis. Samples for DNA analysis may be transported in a cooling box unless the circumstances are such that dry ice is needed for these as well. 3.7 Soil processing The soil should be processed as soon as possible after sampling. Vegetation, larger soil f
36、auna and stones should be removed prior to passing the soil through a 2 mm sieve. Sieving soil through a 2 mm sieve facilitates gaseous exchange between particles and is therefore recommended for maintaining the aerobic nature of the soil. It also removes small stones, fauna and plant debris. Some o
37、rganic materials such as moor layers or peat do not pass easily through a 2 mm sieve and should be sieved in the moist condition through a 5 mm sieve. This necessitates manual operation and the quality of the material passing the sieve depends on the operator. If the soil is too wet to sieve, it sho
38、uld be spread out, in a gentle air stream where possible, to facilitate uniform drying. The soil should be finger crumbled and turned over frequently to avoid excessive surface drying. Usually, this should be performed at ambient temperature. If drying is required, the soil should not be dried more
39、than necessary to facilitate sieving. Generally, drying of soils is not recommended although air-drying and rewetting is a common physiological stress for the microbial communities in surface soils. It has been shown that drying-rewetting events can induce significant changes in microbial C and N dy
40、namics which 7 DIN ISO 10381-6:2009-09 can last for more than a month after the last stress 12. Rewetting after drying causes bursts of respiration and growth of distinct populations of bacteria 15. If further storage is necessary following processing, consideration should be given to the parameters
41、 discussed in 3.8. 3.8 Storage conditions and storage periods Samples should be stored in the dark at (4 2) C with free access of air. A loosely tied plastic bag or equivalent is generally adequate for this purpose. Care should be taken to ensure that the soil is not stored in a quantity which allow
42、s anaerobic conditions to occur in the bottom of storage containers. The soil should be processed (see 3.7) before storage in order to ensure stable aerobic conditions. It is essential that the soil is not allowed to dry out or become water-logged during storage. Samples should not be stored on top
43、of one another. It is preferable to use soils as soon as possible after sampling. Any delays due to transportation should be minimized. If storage is unavoidable, this should not exceed three months unless there is evidence showing continued microbial activity. Soil samples subject to DNA analyses s
44、hould be frozen at 20 C if not processed immediately. For RNA analyses, samples should be frozen at 80 C. If soil samples need to be stored for longer periods ( three months), freezing of samples at 20 C, 80 C or 180 C may be appropriate although not generally recommended. Examples for appropriate s
45、torage conditions for various test objectives are given in Table 1. It has been shown for a number of soils from temperate climates that storage at 20 C for up to 12 months does not inhibit microbial activity (e.g. ammonium oxidation). Moreover, soil samples for Phospholipid Fatty Acid (PLFA) and DN
46、A analyses can be stored at 20 C for one to two years. Samples for rRNA analyses can be stored at 80 C for the same period. Shock freezing with liquid nitrogen is recommended for freezing samples subjected to DNA, RNA and PLFA/PLEL analyses. Longer storage periods are mainly needed if the influence
47、of added pollutants on soil microbes and microbial processes is tested with the same soil material or if the community structure (PLFA, DNA, RNA) of soils is evaluated at a distinct point of time during the year. In these cases, the time needed for analyses can easily exceed three months (chemical,
48、pollutant testing). For structural analyses of the microflora, storage at 4 C is not suitable. 8 DIN ISO 10381-6:2009-09 Table 1 Storage conditions for the assessment of aerobic microbial processes when analysis cannot be performed immediately Test objective International Standard Wet 4 C months Wet
49、 20 Ca years Wet 80 C or liquid nitrogen (180 C)a years PLFA, PLEL 2 10 DNA RNA 10 Biomass Substrate-induced respiration method Fumigation-extraction method ISO 14240-1 ISO 14240-2 3 1 10 Potential ammonium oxidation ISO 15685 3 1 10 Nitrogen mineralization ISO 14238 3 1 10 Microbial soil respiration ISO 16072 3 1 10 Soil respiration curves ISO 17155 3 1 10 Dehydrogenase activity ISO 23753-1 ISO 23753-2 3 1 symbols: = no storage possible. aThe soils shall be divided into
