1、October 2012 Translation by DIN-Sprachendienst.English price group 13No part of this translation 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).IC
2、S 13.060.70!$wu“1918482www.din.deDDIN ISO 10872Water quality Determination of the toxic effect of sediment and soil samples ongrowth, fertility and reproduction of Caenorhabditis elegans (Nematoda)(ISO 10872:2010),English translation of DIN ISO 10872:2012-10Wasserbeschaffenheit Bestimmung der toxisc
3、hen Wirkung von Sediment- und Bodenproben auf Wachstum,Fertilitt und Reproduktion von Caenorhabditis elegans (Nematoda) (ISO 10872:2010),Englische bersetzung von DIN ISO 10872:2012-10Qualit de leau Dtermination de leffet toxique dchantillons de sdiment et de sol sur la croissance, lafertilit et la r
4、eproduction de Caenorhabditis elegans (Nematodes) (ISO 10872:2010),Traduction anglaise de DIN ISO 10872:2012-10www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.2301.13A comma is used as the decimal marker. Contents Page National for
5、eword .3 National Annex NA (informative) Bibliography 5 Introduction .6 1 Scope 7 2 Normative references 7 3 Terms and definitions .7 4 Principle 9 5 Reagents .9 6 Apparatus 11 7 Reference substance 12 8 Organisms . 13 8.1 Test organism . 13 8.2 Food organism 13 9 Stock- and pre-cultures 13 9.1 Stoc
6、k cultures . 13 9.2 Pre-culture . 13 10 Procedure 14 10.1 Preparation of food medium 14 10.2 Preparation of test material and controls 14 10.3 Test . 15 10.4 Nematode separation . 15 10.5 Measurements and calculations . 15 10.6 Timetable of the test . 17 11 Validity criteria 18 12 Expression of resu
7、lts . 18 13 Test report . 19 Annex A (informative) Figures of adult worms C. elegans 20 Annex B (informative) Precision data 21 Bibliography . 23 2 DIN ISO 10872:2012-10 National foreword This standard has been prepared by Technical Committee ISO/TC 147 “Water quality” (Secretariat: DIN, Germany). T
8、he responsible German body involved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Working Committee NA 119-01-03 AA Wasseruntersuchung, Working Group NA 119-01-03-05-01 AK Biotests. This standard has been published to implement the Water Framework Direc
9、tive (WFD), Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy*). Considering the environmental significance of free-living non-parasitic nematodes for preserving soil function, this method
10、for testing Caenorhabditis elegans (nematoda) fills an important gap in the “battery” of standard chronic test methods for the biological characterization of soils or soil substrates, even though this method is essentially a water quality testing component. Because the habitats in which these nemato
11、des are found include fresh-water sediments and the water-saturated pore space of soils, it could be shown that this method is suitable for assessing the toxicity of contaminated soils for a wide range of soil contamination levels (Hss et al. 2009). Potentially toxic soils are generally tested by me
12、ans of comparison with a control soil that does not have an effect on the test organism. However, it is difficult to define a suitable control soil for long-term sublethal toxicity tests because test parameters such as growth and reproduction can be influenced not only by materials of anthropogenic
13、origin, but also by the soil characteristics that are typical of the respective habitat. Consequently, the high variability of the test parameters make it difficult to clearly identify the causes of the various effects. For example, in the test specified here it must be borne in mind that the clay c
14、ontent and humus content of soils can have either a direct (Hss et al., 1999, 2001; Steinberg et al., 2002) or an indirect influence on the bioavailability of organic contaminants (Haitzer et al., 1998). Often it is expedient to use standard soils (e.g. LUFA 2.2)*)with known effects on the test para
15、meters in the investigation. For LUFA 2.2, as one of 9 control soils examined, Hss et al. 2009 identified acceptable inhibition values for growth and reproduction (2 % and 34 %, respectively) due to the observed inherent test variability. For all 9 uncontaminated control soils examined, acceptable i
16、nhibition values for growth and reproduction were obtained, namely 10 % and 40 %, respectively. In analogy to the validity criterion, a tolerable deviation of 20 % was attributed to the test parameter “fertility”, which was not much influenced. Nevertheless, such test-specific uncertainty ranges are
17、 not unusual for biological testing and do not reduce the suitability of the test method. Furthermore, the investigations described by Hss et al. 2009 show that the observed toxic effects on C. elegans do not correlate with those observed in other soil quality tests (earthworms, collembola, higher p
18、lants). Regardless of the factors attributed to these observations, it can be concluded that this test method effectively supplements the currently available test methods. As with the other test methods, it may be necessary to supplement the method with additional information regarding the performan
19、ce of the soil tests and the analysis of results once further experience with this test method has been gained. This standard is part of the series Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlamm-untersuchung Testverfahren mit Wasserorganismen (Gruppe L) (German standard methods for th
20、e examination of water, waste water and sludge Test methods using water organisms (group L) and describes method L 55. *) Registered in the DITR database of DIN Software GmbH, obtainable from: Beuth Verlag GmbH, 10772 Berlin. *) Obtainable from: Landwirtschaftliche Untersuchungs- und Forschungsansta
21、lt Speyer (Agricultural Investigation and Research Institute), stdbodenlufa-speyer.de 3 DIN ISO 10872:2012-10 Designation of the method: Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) (L 55): Method DIN ISO 10
22、872 L 55 The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 5667-16 DIN EN ISO 5667-16 ISO 7027 DIN EN ISO 7027 ISO 10390 DIN ISO 10390 ISO 10523 DIN EN ISO 10523 ISO 11465 DIN ISO 11465 Expert assistance and specialized laboratories will
23、be required to perform the analysis described in this standard. Existing safety regulations are to be taken into account. Depending on the objective of the analysis, a check shall be made on a case-by-case basis as to whether and to what extent additional conditions will have to be specified. German
24、 standard methods for the examination of water, waste water and sludge Standard methods published as DIN Standards are obtainable from Beuth Verlag GmbH, either individually or grouped in volumes. The standard methods included in the loose-leaf publication entitled Deutsche Einheitsverfahren zur Was
25、ser-, Abwasser- und Schlammuntersuchung will continue to be published jointly by Wiley-VCH Verlag and Beuth Verlag GmbH. All standard methods relevant to the Abwasserverordnung (Waste Water Regulation) (AbwV*) included in the new Regulation on Section 57, paragraph 1, number 1, of the Gesetz zur Ord
26、nung des Wasserhaushaltes (German Water Management Act*)concerning Anforderungen an das Einleiten von Abwasser in Gewsser together with the Abwasserverordnung and the Gesetz zur Ordnung des Wasserhaushalts have been published by Beuth Verlag GmbH as a loose-leaf collection Analysenverfahren in der A
27、bwasserverordnung Rechtsvorschriften und Normen (Supplements). Standards or draft standards bearing the group title “German standard methods for the examination of water, waste water and sludge” are classified under the following categories (main titles): General information (group A) (DIN 38402) Se
28、nsory analysis (group B) (DIN 38403) Physical and physicochemical parameters (group C) (DIN 38404) Anions (group D) (DIN 38405) Cations (group E) (DIN 38406) Substance group analysis (group F) (DIN 38407) Gaseous constituents (group G) (DIN 38408) Parameters characterizing effects and substances (gr
29、oup H) (DIN 38409) Biological-ecological methods of analysis (group M) (DIN 38410) Microbiological methods (group K) (DIN 38411) Test methods using water organisms (group L) (DIN 38412) Individual constituents (group P) (DIN 38413) Sludge and sediments (group S) (DIN 38414) Bio-assays with microorga
30、nisms (group T) (DIN 38415) *) Registered in the DITR database of DIN Software GmbH, obtainable from: Beuth Verlag GmbH, 10772 Berlin. 4 DIN ISO 10872:2012-10 DIN ISO 10872:2012-10 5 In addition to the methods described in the DIN 38402 to DIN 38415 series of standards, there are a number of Europea
31、n and International Standards available as DIN EN, DIN EN ISO and DIN ISO Standards, which also form part of the collection of German standard methods. Information on Parts of these series of standards that have already been published can be obtained from the offices of the Normenausschuss Wasserwes
32、en, telephone +49 30 2601-2448, or from Beuth Verlag GmbH, Burggrafenstr. 6, 10787 Berlin. National Annex NA (informative) Bibliography DIN EN ISO 5667-16, Water quality Sampling Part 16: Guidance on biotesting of samples DIN EN ISO 7027, Water quality Determination of turbidity DIN EN ISO 10523, Wa
33、ter quality Determination of pH DIN ISO 10390, Soil quality Determination of pH DIN ISO 11465, Soil quality Determination of dry matter and water content on a mass basis Gravimetric method Haitzer, M., Hss, S., Traunspurger, W., Steinberg, C.E.W., 1998. Effects of dissolved organic matter (DOM) on t
34、he bioconcentration of organic chemicals in aquatic organisms a review. Chemosphere 37, pp. 13351362 Hss, S., Haitzer, M., Traunspurger, W., Steinberg, C.E.W., 1999. Growth and fertility of Caenorhabditis elegans (Nematoda) in unpolluted freshwater sediments response to particlesize distribution and
35、 organic content. Environ. Toxicol. Chem. 18, pp. 29212925 Hss, S., Bergtold, M., Haitzer, M., Traunspurger, W., Steinberg, C.E.W., 2001. Refractory dissolved organic matter can influence the reproduction of Caenorhabditis elegans (Nematoda). Freshw. Biol. 46, pp. 110 Hss, S., Jnsch, S., Moser, T.,
36、Junker, T., Rmbke, J., 2009. Assessing the toxicity of contaminated soils using the nematode Caenorhabditis elegans as test organism. Ecotoxicology and Environmental Safety 72, pp. 18111818 Steinberg, C.E.W., Hss, S., Brggemann, R., 2002. Further evidence that humic substances have the potential to
37、modulate there production of the nematode Caenorhabditis elegans. Int. Rev. Hydrobiol. 87, pp. 121133 Introduction Nematodes are the most abundant and species-rich group of metazoans in sediments and soils12and play an important role in benthic and soil food webs34. Nematodes are endobenthic organis
38、ms that are found at various trophic levels due to the evolution of different feeding types (bacterivorous, algal feeder, omnivorous, predators). The test organism Caenorhabditis elegans (Maupas, N2 var. Bristol) is a bacterivorous nematode that is found primarily in terrestrial soils but it also oc
39、curs in aquatic sediments of polysaprobial fresh-water systems56. C. elegans is a well-studied organism and very easy to cultivate7. The test is designed for measurement of the response to dissolved and particle-bound substances8910. It applies to the testing of sediments, soils, waste, pore water,
40、elutriates and aqueous extracts. Water quality Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) 6 DIN ISO 10872:2012-10 WARNING Persons using this International Standard should be familiar with normal laboratory
41、 practice. This International Standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions. IMPORTANT It is
42、absolutely essential that tests conducted according to this International Standard be carried out by suitably trained staff. 1 Scope This International Standard specifies a method for determining the toxicity of environmental samples on growth, fertility and reproduction of Caenorhabditis elegans. T
43、he method applies to contaminated whole fresh-water sediment (maximum salinity 5 ), soil and waste, as well as to pore water, elutriates and aqueous extracts that were obtained from contaminated sediment, soil and waste. 2 Normative references The following referenced documents are indispensable for
44、 the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 5667-16, Water quality Sampling Part 16: Guidance on biotesting of samples ISO 7027, Water quality De
45、termination of turbidity ISO 10390, Soil quality Determination of pH ISO 10523, Water quality Determination of pH ISO 11465, Soil quality Determination of dry matter and water content on a mass basis Gravimetric method 3 Terms and definitions For the purposes of this document, the following terms an
46、d definitions apply. 3.1 agar plate Petri dish filled with NGM agar (5.8) 3.2 aqueous control water that serves as negative control for tests in aqueous samples 7 DIN ISO 10872:2012-10 3.3 artificial control sediment defined artificial sediment (5.12) 3.4 bacterial stock culture stock culture of foo
47、d bacteria 3.5 blank replicate additional replicate that contains no test organism, but is treated in the same way as the other replicates of a sample 3.6 control treatment that serves as negative control to which the effect in the respective test material is compared (3.2, 3.3, 3.7) 3.7 control soi
48、l defined standard soil (5.13) 3.8 dauer larva developmental stage adopted by C. elegans to endure periods of lack of food NOTE Dauer larvae continue normal development if food is supplied. 3.9 exposed test organisms individuals of C. elegans that are introduced at the beginning of the test 3.10 foo
49、d medium defined aqueous bacterial suspension (10.1) 3.11 J1stage first of four juvenile stages (J1to J4) in the development of C. elegans 3.12 overnight culture defined culture of Escherichia coli in LB-medium (9.1.2) 3.13 starved plate agar plate with dauer larvae 3.14 test material discrete portion of a contaminated environmental sample (10.2) or solution of the reference substance (Clause 7) 8 DIN ISO 10872:2012-10 4 Principle Juvenile organisms of the species C. elegans are
