1、BS ISO 10872:2010ICS 13.060.70NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDWater quality Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans(Nematoda)This British Standardwas published
2、 under theauthority of the StandardsPolicy and StrategyCommittee on 31 July 2010 BSI 2010ISBN 978 0 580 64807 6Amendments/corrigenda issued since publicationDate CommentsBS ISO 10872:2010National forewordThis British Standard is the UK implementation of ISO 10872:2010.The UK participation in its pre
3、paration was entrusted to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct appli
4、cation.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS ISO 10872:2010Reference numberISO 10872:2010(E)ISO 2010INTERNATIONAL STANDARD ISO10872First edition2010-06-15Water quality Determination of the toxic effect of sediment and soil samples on growth, fertility and
5、 reproduction of Caenorhabditis elegans (Nematoda) Qualit de leau Dtermination de leffet toxique dchantillons de sdiment et de sol sur la croissance, la fertilit et la reproduction de Caenorhabditis elegans (Nematodes) BS ISO 10872:2010ISO 10872:2010(E) PDF disclaimer This PDF file may contain embed
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10、ster. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2010 All rights reservedBS ISO 10872:2010ISO 10872:2010(E) ISO 2010 All rights reserved iiiContents Page Foreword iv Intro
11、duction.v 1 Scope1 2 Normative references1 3 Terms and definitions .1 4 Principle3 5 Reagents.3 6 Apparatus.5 7 Reference substance 6 8 Organisms7 8.1 Test organism 7 8.2 Food organism.7 9 Stock- and pre-cultures 7 9.1 Stock cultures7 9.2 Pre-culture7 10 Procedure.8 10.1 Preparation of food medium.8
12、 10.2 Preparation of test material and controls .8 10.3 Test .9 10.4 Nematode separation9 10.5 Measurements and calculations 9 10.6 Timetable of the test11 11 Validity criteria.12 12 Expression of results12 13 Test report13 Annex A (informative) Figures of adult worms C. elegans .14 Annex B (informa
13、tive) Precision data .15 Bibliography17 BS ISO 10872:2010ISO 10872:2010(E) iv ISO 2010 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is nor
14、mally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in
15、the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepar
16、e International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that som
17、e of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 10872 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods. BS ISO 10872:2010ISO 10872:2010(E)
18、ISO 2010 All rights reserved vIntroduction 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 organisms that are found at various trophic levels due to the evolution of diff
19、erent 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 occurs in aquatic sediments of polysaprobial fresh-water systems56. C. ele
20、gans 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, elutriates and aqueous extracts. BS ISO 10872:2010BS ISO 10872:2010INTER
21、NATIONAL STANDARD ISO 10872:2010(E) ISO 2010 All rights reserved 1Water quality Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) WARNING Persons using this International Standard should be familiar with normal l
22、aboratory 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. IMPORTA
23、NT It is 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 e
24、legans. The 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 indispen
25、sable for 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 q
26、uality Determination 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
27、 terms and 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 BS ISO 10872:2010ISO 10872:2010(E) 2 ISO 2010 All rights reserved3.3 artificial control sediment defined artificial sediment (5.12
28、) 3.4 bacterial stock culture stock culture of food 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 ma
29、terial is compared (3.2, 3.3, 3.7) 3.7 control soil 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 ar
30、e introduced at the beginning of the test 3.10 food 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 w
31、ith dauer larvae 3.14 test material discrete portion of a contaminated environmental sample (10.2) or solution of the reference substance (Clause 7) BS ISO 10872:2010ISO 10872:2010(E) ISO 2010 All rights reserved 34 Principle Juvenile organisms of the species C. elegans are exposed to the environmen
32、tal sample over a period of 96 h. In the controls, the exposed test organisms are able to complete a whole life cycle within this period. A toxic effect of an environmental sample occurs if the inhibition of growth, fertility or reproduction of C. elegans in comparison to a control (aqueous control,
33、 control sediment or soil) exceeds a certain threshold value. Toxicity can by quantified by the intensity of the effect as percentage inhibition. 5 Reagents Use only reagents of recognized analytical grade. 5.1 Water, distilled or deionized water or water of equivalent purity, conductivity u 10 S/cm
34、. 5.2 LB-medium. Dissolve 0,5 g of casein peptone; 0,25 g of yeast extract; 0,5 g of sodium chloride (NaCl); in 50 ml water in a 250 ml flask and autoclave for 20 min at 121 C. 5.3 Cholesterol stock solution. Dissolve 500 mg of powdered cholesterol in 100 ml of absolute ethanol ( 99 % purity) by sti
35、rring and gentle heating ( 5 % mass fraction can cause deleterious effects on growth, fertility and reproduction of C. elegans. 5.13 Control soil. Standard soil St. 2.2 from LUFA2): soil type: loamy sand; organic carbon: (2,16 0,4) % mass fraction; pH: 5,4 0,1; cation exchange capacity: (10 1) mmolc
36、/100 g; NOTE mmolc/100 g is synonymous with meq/100 g. water holding capacity: (48,2 5) g/100 g; clay content: (6,4 0,9) % mass fraction particles 200 FAU. After centrifugation of the bacterial suspension (20 min, 2 000g), remove the supernatant and resuspend the pellet in M9-medium (5.9). After rep
37、eated centrifugation and removing of the supernatant, resuspend the pellet in approximately x 8 ml of M9-medium and adjust the bacterial density to (12 000 600) FAU for testing sediment, soil and waste; x 100 ml of M9-medium and adjust the bacterial density to (1 000 50) FAU for testing pore water,
38、elutriates, extracts or solutions of reference substance. Finally, the accurate volume of cholesterol stock solution is added (0,2 % of volume of bacterial suspension; e.g. 100 l of cholesterol stock solution in 50 ml bacterial suspension). NOTE Densities of bacteria differ between tests with solid
39、and liquid test material due to different exposure conditions. 10.2 Preparation of test material and controls 10.2.1 Sediment, soil and waste Pass the test material (3.14) through a 2 mm sieve (6.22). Determine the dry mass of the test material and control sediment or control soil in accordance with
40、 ISO 11465 by drying a small portion of the test sample. Determine the pH of the test material and control sediment or control soil in accordance with ISO 10523 (aqueous test material, sediments) and ISO 10390 (soils). Prepare at least four replicates for each test material and the control artificia
41、l control sediment (5.12) or control soil (5.13). Prepare one additional blank replicate (without test organisms) to estimate the number of indigenous nematodes in the samples. For artificial substrates, such as the artificial control sediment, it is not necessary to set up a blank replicate. For te
42、st material with W 40 % water content (based on total mass), transfer (0,500 0,010) g (wet mass) of test material into each test well (6.19). For test material with 40 % water content (based on total mass), artificial control sediment and control soil, transfer m 0,010 g into each test well, add (0,
43、500 m) ml of M9-medium (5.9), and stir with a spatula to achieve a homogenous suspension. Calculate m, expressed in grams, as given in Equation (1): o,t0,5 0,60mm= (1) where mo,tis the measured dry mass of the test material. Store in a refrigerator at (8 2) C to avoid loss of moisture. BS ISO 10872:
44、2010ISO 10872:2010(E) ISO 2010 All rights reserved 9Stir the food medium (3.10, 10.1; 12 000 FAU) to ensure homogeneity and add, immediately before the start of the test, 0,5 ml of homogenized food medium (3.10) to each test well. Mix artificial control sediment or control soil and test material wit
45、h the added food medium thoroughly with a spatula. 10.2.2 Pore water, elutriate, extract Prepare at least four replicates for each test material (3.14) and the control (aqueous control; 3.2). Transfer 0,5 ml of test material and aqueous control into each test well (6.19). Stir the food medium (3.10,
46、 10.1; 1 000 FAU) to ensure homogeneity and add, immediately before the start of the test, 0,5 ml of homogenized food medium to each test well. 10.2.3 Solution of reference substance Prepare at least four replicates for each solution of the reference substance (5.14, Clause 7) and the control (aqueo
47、us control; 3.2). Transfer 0,5 ml of the solution of reference substance and aqueous control into each test well (6.19). Stir the food medium (3.10, 10.1; 1 000 FAU) to ensure homogeneity and add, immediately before the start of the test, 0,5 ml of homogenized food medium to each test well. 10.3 Tes
48、t At the start of the test, transfer ten first-stage juveniles (J1; exposed test organisms) from the filtrate by micropipette (6.8) to each of the test wells containing test material (3.14) and food medium (3.10) (after allowing temperature to equilibrate to room temperature). Non-moving organisms s
49、hould be excluded from the test. After the addition of the test organisms, seal the multidishes (6.19) with an adhesive tape Parafilm4) and incubate at (20 0,5) C in the dark. After 96 h, add approximately 0,5 ml of Rose Bengal stock solution (5.10) to each test well to stain the nematode cuticle for better recovery. Heat the multidishes with the lid on in a drying oven for 10 min at 80 C to terminate the test. This treatment results in s
