1、Soil quality Avoidance test for determining the quality of soils and effects of chemicals on behaviour Part 2: Test with collembolans (Folsomia candida) Qualit du sol Essai dvitement pour contrler la qualit des sols et les effets des produits chimiques sur le comportement Partie 2: Essai avec des co
2、llemboles (Folsomia candida) ISO 2011 Reference number ISO 17512-2:2011(E) First edition 2011-10-01 ISO 17512-2 INTERNATIONAL STANDARDISO 17512-2:2011(E)COPYRIGHT PROTECTED DOCUMENT ISO 2011 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in
3、 any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22
4、 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2011 All rights reservedISO 17512-2:2011(E)Contents Page Foreword iv Introduction v 1 Scope 1 2 Normative references . 1 3 Terms and definitions . 1 4 Principle . 2 5 Reagents and materials . 2 6 Apparatus 3 7 Procedu
5、re 4 7.1 Appropriate concentration/dilution range 4 7.2 Testing of soils . 4 7.3 Reference substance 4 7.4 Validity criteria . 5 8 Calculation and expression of results . 5 8.1 General . 5 8.2 Calculation of the percentage of avoidance 5 8.3 NOEC/LOEC calculation 5 8.4 EC xcalculation . 6 9 Test rep
6、ort . 6 Annex A (informative) Techniques for rearing and breeding of Collembola 7 Annex B (informative) Test chambers . 9 Annex C (normative) Testing of chemicals in the avoidance test .10 Annex D (normative) Determination of the water-holding capacity 12 Bibliography .13 ISO 2011 All rights reserve
7、d iiiForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for whic
8、h 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 the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all ma
9、tters 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 prepare International Standards. Draft International Standards adopted by the technical committees are circ
10、ulated 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 some of the elements of this document may be the subject of patent rights. ISO shall not be held respons
11、ible for identifying any or all such patent rights. ISO 17512-2 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods. ISO 17512 consists of the following parts, under the general title Soil quality Avoidance test for determining the quality of soils and
12、 effects of chemicals on behaviour: Part 1: Test with earthworms (Eisenia fetida and Eisenia andrei) Part 2: Test with collembolans (Folsomia candida) ISO 17512-2:2011(E)iv ISO 2011 All rights reservedIntroduction The use of the avoidance behaviour of soil invertebrates as an indicator of unfavourab
13、le conditions allows a preliminary assessment of contaminated soils in a short period of time, with a high degree of sensitivity. Being rapid, cost-effective and ecologically relevant, the avoidance tests with earthworms were proposed to complement conventional chemical analysis. Supporting the resu
14、lts obtained in the chronic tests, the avoidance bioassays can be used as a first screening tool in the assessment of the habitat function of soils. Considering the fact that the avoidance response of soil invertebrates differs between species due to their distinct sensitivity to contaminants and mo
15、des of exposure, it is recommended to standardize a second rapid cost-effective and ecologically relevant avoidance bioassay. Springtails have shown a distinct sensitivity towards several contaminants when compared with earthworms, complementing the information obtained in the avoidance tests with e
16、arthworms12 . Until now, the species Folsomia candida has been the most commonly used collembolan test species due to a great facility to keep laboratory cultures and due to their high locomotor ability3 . Folsomia candida is considered to be a hemi- edaphic species, meaning that it lives mainly in
17、the soil. Furthermore, this species is already used in ISO 11267. ISO 17512-2:2011(E) ISO 2011 All rights reserved vINTERNATIONAL STANDARD ISO 17512-2:2011(E) Soil quality Avoidance test for determining the quality of soils and effects of chemicals on behaviour Part 2: Test with collembolans (Folsom
18、ia candida) 1 Scope This part of ISO 17512 specifies a rapid screening method for evaluating the habitat function of soils based on the avoidance behaviour of springtails. The test is a rapid method that reflects the bioavailability of contaminants in natural soils and substances spiked into soils t
19、o Folsomia candida. In both cases, it is possible to establish a dose-response-relationship. The avoidance behaviour of the springtails is the measurement endpoint of the test. This test is not intended to replace the Collembola reproduction test. 2 Normative references The following referenced docu
20、ments are indispensable 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 10390, Soil quality Determination of pH ISO 11267:1999, Soil quality Inhib
21、ition of reproduction of Collembola (Folsomia candida) by soil pollutants ISO 11268-2:1998, Soil quality Effects of pollutants on earthworms (Eisenia fetida) Part 2: Determination of effects on reproduction ISO 11269-2, Soil quality Determination of the effects of pollutants on soil flora Part 2: Ef
22、fects of contaminated soils on the emergence and early growth of higher plants ISO 11465, Soil quality Determination of dry matter and water content on a mass basis Gravimetric method ISO 15799, Soil quality Guidance on the ecotoxicological characterization of soils and soil materials 3 Terms and de
23、finitions For the purposes of this document, the following terms and definitions apply. 3.1 avoidance behaviour tendency (of an organism) to avoid the test soil while preferring the control soil ISO 17512-1:2008 3.2 test soil either a natural or an artificial clean soil that is spiked with the test
24、substance or a contaminated natural soil (a site soil) 3.3 control soil natural or artificial uncontaminated soil ISO 2011 All rights reserved 1See 5.3. 3.4 limited habitat function habitat function is limited if, on average, 70 % of springtails are found in the control soil (indication of an impact
25、 on behaviour) after they were allowed to choose between the control soil and tested soil 3.5 effective concentration EC x concentration at which a specific effect is detected where x is a percentage (10, 25, 50) of this effect; e.g. avoidance NOTE In this part of ISO 17512-2, an EC 50means the conc
26、entration of a test substance, or dilution of a contaminated test soil, that is estimated to cause an avoidance response of 50 %. EXAMPLE An avoidance of 50 % occurs when the number of springtails in the test soil is 50 % of the number that should be there in the case of no avoidance: no avoidance (
27、control soil = 10 individuals; test soil = 10 individuals); 50 % avoidance (control soil = 15 individuals; test soil = 5 individuals). 3.6 lowest observed effect concentration LOEC lowest tested concentration of a test substance, or dilution of a contaminated test soil, that is observed to cause a s
28、tatistically significant avoidance response ( p 0,05) NOTE 1 In the case of a test substance, the concentration is expressed as mass of the test substance per dry mass of test substrate; in the case of a contaminated test soil, the concentration is expressed as the percentage dilution of the test so
29、il. NOTE 2 All tested concentrations/dilutions above the LOEC should have a harmful effect equal to or greater than those observed at the LOEC. When this condition is not observed, a full explanation should be given for how the LOEC (and hence the NOEC) has been selected. 3.7 no observed effect conc
30、entration NOEC test concentration/dilution tested immediately below the LOEC, which causes a not statistically significant avoidance response ( p 0,05) 4 Principle Springtails (Folsomia candida) are exposed at the same time to the control soil and the test soil. These soils are filled into the two s
31、ections of the same vessel. After an incubation period of two days, the number of springtails is determined in each section of the vessels. 5 Reagents and materials 5.1 Biological material, only springtails of the species Folsomia candida (Willem) (see A.2.6) coming from synchronized cultures should
32、 be used. NOTE Typically, in this test springtails can be used when they are 10 d to 12 d old (or alternatively adult, e.g. 20 d to 22 d old). 5.2 Test substrate, in the case of a natural soil, the substrate to be tested should be sieved (2 mm) and moisture-adjusted to about 40 % to 60 % of the maxi
33、mum water-holding capacity. If standing water or free water appears when the soil is compressed before achieving the desired percentage of maximum WHC, a lower percentage might be used. The optimum water content is achieved if there is no standing water or free water appearing when the soil is compr
34、essed. ISO 17512-2:2011(E)2 ISO 2011 All rights reservedNOTE For highly silty and loamy soils, it can be difficult to get the necessary amount of soil sieved to 2 mm with an acceptable expenditure of work. The holes of the sieves might plug up within several minutes. Frequent cleaning is necessary.
35、In this case, it is acceptable to sieve the amount of soil needed for the test to 5 mm. Determine the water content and the pH in the presence of 1 mol/l KCl, in accordance with ISO 11465 and ISO 10390, respectively, immediately before the start of the test. In addition, the maximum water-holding ca
36、pacity shall be determined according to Annex D. If testing a substance, a different procedure should be followed (see Annex C). 5.3 Control soil, two choices are possible (see also ISO 15799). Either a) a reference soil or b) a standard soil that allows the presence of springtails. a) If reference
37、soils from uncontaminated areas near a contaminated site are available, they should be treated and characterized like the test soils. If a toxic contamination or unusual soil properties cannot be ruled out, standard control soils should be preferred. b) For testing the effects of substances mixed in
38、to soil or making dilutions of the test soil, standard soils (e.g. LU FA 2 . 2) shall be used as the test substrate. The properties of the field-collected standard soil shall be reported. The substrate called artificial soil can be used as a standard soil and has the following composition:Percentage
39、 expressed on dry-mass basis Sphagnum peat finely ground and with no visible plant remains 10 % Kaolinite clay containing not less than 30 % Kaolinite 20 % Industrial quartz sand (dominant fine sand with more than 50 % of particle size 0,05 mm to 0,2 mm) 69 % Approximately 0,3 % to 1,0 % calcium car
40、bonate (CaCO 3 , pulverized, analytical grade) are necessary to get a pH of 6,0 0,5. Natural soil should be sieved and the water content should be adjusted according to 5.2. 5.4 Reference substance, having Phenmedipham as the only active ingredient. 6 Apparatus Usual laboratory equipment and the fol
41、lowing. 6.1 Containers (see Annex B). Cylindrical containers of capacity 200 ml to 300 ml with a cross-sectional area of about 50 cm 2 , such that a depth of 3 cm to 4 cm of soil is achieved. Test containers shall permit gaseous exchange between the medium and the atmosphere and access of light (e.g
42、. by means of a perforated transparent cover), and shall have provisions to prevent springtails from escaping (e.g. by using a tape to fix the cover). To avoid lateral effects of light, test vessels should be made of opaque material, otherwise they should be wrapped with aluminium foil. NOTE Due to
43、the short test period and the proportionally large volume of soil in the vessels (considering the small amount of soil needed by the springtails), a reduction of the chemical concentration in the soil resulting from sorption to the vessel walls is negligible. Therefore, plastic vessels can be used,
44、although, when available, the use of inert material (e.g. glass or stainless steel) is preferred. 6.2 Divider, made of plastic or thin sheets of metal. It shall divide the test containers vertically into two identical sections. ISO 17512-2:2011(E) ISO 2011 All rights reserved 36.3 Equipment for meas
45、uring the water content of a substrate (in accordance with ISO 11465). 6.4 Apparatus, for measuring the pH of the substrate. 6.5 Exhaustor, to transfer the springtails (see A.3). 6.6 Test environment. 6.6.1 Enclosure or environmental chamber, capable of being maintained at (20 2) C. 6.6.2 Light sour
46、ce, capable of delivering a constant light intensity of 400 lx to 800 lx on the containers at a controlled light/dark cycle of between 12 h:12 h and 16 h:8 h. 7 Procedure 7.1 Appropriate concentration/dilution range The avoidance test is designed to detect sublethal effects. Therefore, the test is i
47、nvalid if more than 20 % of the total number of springtails are dead or missing at the end of the test (see 7.4). In order to avoid mortality, a range-finding test should be performed in those cases when testing a substance or a dilution gradient of contaminated natural soil. 7.2 Testing of soils At
48、 the beginning of the test, the containers (6.1) are divided into two equal sections by means of a vertically introduced divider (6.2). Vessels are filled with sieved soil up to a height of 3 cm to 4 cm ( 30 g wet mass per side; the soil is freshly moistened and should not be pressed). One half of t
49、he vessel is filled with test soil (section A), the other half is filled with control soil (section B). Then the divider is removed and 20 springtails are placed on top of the line separating the two soils on each test vessel. The containers are covered according to 6.1 and placed into the environmental chamber or in the enclosure (6.6.1). No feeding of the animals is required during the test. The test runs with five replicates. To facilitate checking of the pH and humidity of the test substr
