1、June 2010 Translation by DIN-Sprachendienst.English price group 14No 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).ICS 1
2、3.080.10!$i/Q“1701246www.din.deDDIN ISO 17512-1Soil quality Avoidance test for determining the quality of soils and effects ofchemicals on behaviour Part 1: Test with earthworms (ISO 17512-1:2008)English translation of DIN ISO 17512-1:2010-06Bodenbeschaffenheit Vermeidungsprfung zur Bestimmung der B
3、odenbeschaffenheit und der Auswirkungenvon Chemikalien auf das Verhalten Teil 1: Prfung von Regenwrmern (ISO 17512-1:2008)Englische bersetzung von DIN ISO 17512-1:2010-06Qualit du sol Essai dvitement pour contrler la qualit des sols et les effets des produits chimiquessur le comportement Partie 1: E
4、ssai avec des vers de terre (ISO 17512-1:2008)Traduction anglaise de DIN ISO 17512-1:2010-06www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 29 pages(Eisenia fetida and Eisenia andrei) (Eisenia fetida und Eisenia andrei) (Eisenia fetida
5、et Eisenia andrei) DIN ISO 17512-1:2010-06 A comma is used as the decimal marker. Contents Page National foreword .3 National Annex NA (informative) Bibliography 3 Introduction.4 1 Scope 5 2 Normative references 5 3 Terms and definitions .6 4 Principle6 5 Reagents and materials 7 6 Apparatus .8 7 Pr
6、ocedure .9 7.1 Appropriate concentration range.9 7.2 Testing of soils.9 7.3 Testing of chemical 10 7.4 Reference substance 10 7.5 Validity criteria 10 8 Calculation and expression of results 10 9 Test report . 11 Annex A (informative) Test chambers 12 Annex B (informative) Example of a breeding tech
7、nique for Eisenia fetida and Eisenia andrei . 15 Annex C (informative) Further test organisms 16 Annex D (informative) Contaminants that earthworms can detect and avoid in the avoidance test 17 Annex E (normative) Testing of chemicals in the avoidance test. 18 Annex F (normative) Determination of wa
8、ter-holding capacity 20 Annex G (informative) Comparison of the results obtained in the two section chamber and six section chamber system 21 Annex H (informative) Influence of soil properties on avoidance behaviour Basis for the threshold value of 20 % 24 Annex I (informative) Data gained in “dual”
9、 tests with the same (untreated) control soil on both sides of the test vessels 26 Bibliography. 28 2 DIN ISO 17512-1:2010-06 3 National foreword This standard has been prepared by Technical Committee ISO/TC 190 “Soil quality”, Subcommittee SC 4 “Biological methods”. The responsible German body invo
10、lved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Working Committee NA 119-01-02-04 UA Biologische Verfahren. Expert assistance and specialized laboratories will be recommended to perform the procedures described in this standard. It should be noted th
11、at, in Germany, use of the two section test vessel is recommended. 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 17512 consists of the foll
12、owing parts, under the general title Soil quality Avoidance test for determining the quality of soils and effects of chemicals on behaviour: Part 1: Test with earthworms (Eisenia fetida and Eisenia andrei) Part 2: Test with collembolans (Folsomia candida) The DIN Standards corresponding to the Inter
13、national Standards referred to in this document are as follows: ISO 10390 DIN ISO 10390 ISO 11268-1:1993 DIN ISO 11268-1:1997 ISO 11268-2:1998 DIN ISO 11268-2:2000 ISO 11269-2 DIN ISO 11269-2 ISO 11465 DIN ISO 11465 ISO 15799 DIN ISO 15799 National Annex NA (informative) Bibliography DIN ISO 10390,
14、Soil quality Determination of pH DIN ISO 11268-1:1997, Soil quality Effects of pollutants on earthworms (Eisenia fetida) Part 1: Determination of acute toxicity using artificial soil substrate DIN ISO 11268-2:2000, Soil quality Effects of pollutants on earthworms (Eisenia fetida) Part 2: Determinati
15、on of effects on reproduction (ISO 11268-2:1998) DIN ISO 11269-2, Soil quality Determination of the effects of pollutants on soil flora Part 2: Effects of chemicals on the emergence and growth of higher plants DIN ISO 11465, Soil quality Determination of dry matter and water content on a mass basis
16、Gravimetric method DIN ISO 15799, Soil quality Guidance on the ecotoxicological characterization of soils and soil materials Introduction Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil and are proposed to complement conventional chemical ana
17、lysis (see ISO 15799). ISO 15799 includes a list and short characterisation of recommended and standardised test systems. Aquatic test systems with soil eluate are applied to obtain information about the fraction of contaminants potentially reaching the groundwater by the water path (retention funct
18、ion of soils), whereas terrestrial test systems are used to assess the habitat function of soils. As standardised test systems, a mortality test (ISO 11268-1) and a reproduction test (ISO 11268-2) exist to investigate the habitat function of a soil with respect to earthworms as representatives of th
19、e soil biocenosis. The reproduction test with earthworms (ISO 11268-2) is applied to detect effects resulting from sublethal concentrations. Such endpoints are preferably applied to obtain information on environmental effects. However, the reproduction test is very labour-intensive and time-consumin
20、g, needing long incubation periods with results obtained only after 56 days. As the test period and the work expense dictate the costs of a given test, it is preferable to obtain the results within a short test period and at a high level of sensitivity. That is especially the case for the assessment
21、 of remediated soils. This feature is offered by the avoidance test with Eisenia fetida and Eisenia andrei. Experiences gained in a laboratory comparison test with eight contaminated soils in three laboratories point out that the avoidance test is as sensitive as the reproduction test (Reference 5).
22、 However, it is not intended to use this test to replace the earthworm reproduction test. NOTE The results were compared with those of the earthworm acute and reproduction tests carried out with the same soils. The results showed that with a criterion of 80 % avoidance response, a 72 % agreement of
23、the results was achieved. DIN ISO 17512-1:2010-06 Soil quality Avoidance test for determining the quality of soils and effects of chemicals on behaviour Part 1: Test with earthworms (Eisenia fetida and Eisenia andrei) 4 1 Scope This part of ISO 17512 specifies a rapid screening method for evaluating
24、 the habitat function of soils and the influence of contaminants and chemicals on earthworm behaviour. The sublethal test is a rapid method that reflects the bioavailability of contaminant mixtures in natural soils and substances spiked into soils to Eisenia fetida and Eisenia andrei. The avoidance
25、behaviour of the worms is the measurement endpoint of the test. This test is not intended to replace the earthworm reproduction test. Two different designs (a two section unit and a six section unit) have been developed and successfully applied. Both designs are applicable to either single-concentra
26、tion (e.g. for assessing the quality of a field soil) or multi-concentration (e.g. for assessing the toxicity of a spiked chemical) tests. In both cases, the earthworms are allowed to make the initial choice on which compartment, control and a treatment in the two section test vessel between right a
27、nd left side; in the six section test vessel between the (3 + 3) alternating compartments, to enter. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the lat
28、est edition of the referenced document (including any amendments) applies. ISO 10390, Soil quality Determination of pH 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 e
29、ffects of pollutants on soil flora Part 2: Effects of chemicals on the emergence and 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 soil and
30、soil materials DIN ISO 17512-1:2010-06 5 3 Terms and definitions 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 3.2 habitat function ability of soils/soil materia
31、ls to serve as habitat for micro-organisms, plants and soil-living animals and their interactions ISO 15799:2003 NOTE Ecotoxicological tests as indicators for the habitat function provide information concerning the respective test parameter, e.g. acute test for survival, or chronic tests for reprodu
32、ction. 3.3 limited habitat function habitat function (3.2) is limited if on average 80 % of worms are found in the control soil (indication as an impact on behaviour) 3.4 effective concentration ECxconcentration at which a specific effect is detected where x is a percentage (10, 25, 50) of this effe
33、ct; e.g. avoidance EXAMPLE In this part of ISO 17512, an EC50means the concentration of a substance or mixture of substances in soil that is estimated to cause a behavioural response in 50 % of the test earthworms. 4 Principle Ten adult earthworms (species Eisenia fetida or Eisenia andrei) are expos
34、ed at the same time to a control soil and a contaminated soil or a soil containing test substances. Test soil and control soil are placed into each test vessel and the earthworms are thus presented with a choice between the test soil and the control soil. Two test-vessel designs are available: a) a
35、two section test vessel; and b) a six section test vessel. After an incubation period of two days, the number of worms is determined in all sections of the vessels. Individual studies (e.g. testing boric acid in one of the two designs in different laboratories) or comparative investigations (testing
36、 the same chemical or soil in the same laboratory, e.g. Reference 8) have in some cases shown different results. Recently, both designs were validated in interlaboratory tests in Canada (Reference 2) and France; however, no international ring test using both designs in parallel has been performed so
37、 far. Therefore, for the time being, the choice of the design is up to the experimenter. When doing so, practical considerations like costs of the units as well as the amount of waste produced should also be taken into consideration. DIN ISO 17512-1:2010-06 6 5 Reagents and materials 5.1 Boric acid
38、reference toxicant, recommended. H3BO3has been used historically as a soil chemosterilant and is an effective non-selective biocide (relative molecular mass: 61,81). Earthworms can detect and avoid sublethal concentrations that adversely affect reproduction. Boric acid satisfies the following criter
39、ia that attest to its suitability as a reference toxicant: a) it is effective at relatively low concentrations that are not strongly influenced by the nature of the substrate; b) it is relatively stable and persistent so that concentrations do not change rapidly over the duration of the test; c) it
40、is reasonably water soluble or miscible in water, does not volatilise readily, and can be readily mixed with soils; d) there is a standard method for measuring boric acid concentrations in soil; e) it represents a minimal hazard to technicians and it is free of disposal problems. 5.2 Biological mate
41、rial, consisting of adult earthworms of the species Eisenia fetida or Eisenia andrei (individual mass: between 300 mg and 600 mg). Synchronisation of breeding of the organisms for this test is not necessary. An example of how to breed compost worms is given in Annex B. Condition the selected worms f
42、or at least one day in the selected control soil (5.4). NOTE Eisenia fetida and Eisenia andrei are compost worms. Ecologically, these species are not the most important in soils (Reference 7). On the other hand, from a practical point of view, compost worms are much more suitable than any other lumb
43、ricid species due to the fact that they reproduce very quickly and easily in the laboratory (i.e. mass cultures can be obtained). In addition, the sensitivity of these species is more or less of the same order of magnitude in comparison to other earthworm species. In most cases, the differences betw
44、een species are depending on the chemical or contaminant mixture tested not larger than a factor of 10 in acute or chronic tests (References 6, 7). Despite the fact that other earthworm species have already successfully been used in avoidance tests (see Annex C), a factor describing their range of a
45、voidance response is not yet known. 5.3 Test substrate. The soil to be tested should be sieved (size of openings, 2 mm) adjusted to about 60 % of the maximum water holding capacity. The optimum water content is achieved, if there is no standing water or free water appearing when the soil is compress
46、ed. NOTE For highly silty and loamy soils, it can be difficult to get the necessary amount of soil sieved to u 2 mm with an acceptable expenditure of work. The holes of the sieves may plug up within several minutes. Frequent cleaning is necessary. In this case, it is acceptable to sieve the amount o
47、f soil needed for the test to u 4 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 capacity shall be determined according to Annex F. 5
48、.4 Control soil: three choices are possible (see also ISO 15799). Option a) is preferred, but since such a soil is often not available either a standard soil, b), or an artificial soil, c), is possible (potential influences of these soils are covered by the 80 % assessment criterion, see Clause 8).
49、a) A control soil as similar as the test soil in all characteristics other than the presence of contaminants. b) A soil with the characteristics according to ISO 11269-2 Corgu 1,5 %, sand (0,063 mm to 2 mm) content of 50 % to 75 %, 10 % per treatment. To validate the test set, check the homogeneity of distribution of the worms. For this purpose, fill the whole test vessel with the same soil and ensure that the orientation of the test vessels in the room is the same. On average, the ratio of worms should be
