1.1 本指南涵盖了获取实验室数据的程序，以评估土壤毒性或生物累积试验中与土壤相关的污染物（例如化学品或生物分子）对蚯蚓（蚓科）和马铃薯（嵌合体科）的不利影响。这些方法旨在评估在短期试验（7至28天）中对蚯蚓或污染物生物累积的致命或亚致命毒性影响，或在陆地系统中短期至长期试验（14至42天）中对马铃薯蠕虫的致命或亚致命毒性影响。待测试土壤可能为 (1) 参考土壤或潜在有毒现场土壤； (2) 掺有化合物的人造、参考或现场土壤； (3) 用参考土壤稀释现场土壤；或 (4) 用人工土壤稀释的现场或参考土壤。描述了该物种的测试程序 赤子爱胜蚓 （参见 附件A1 )对于物种 白化囊藻 （参见 附件A4 ). 本指南中描述的方法也可能有助于对其他陆生蚓类和内生蚓类物种进行土壤毒性试验，尽管可能需要修改。 1.2 根据特殊需要，可能需要修改这些程序。使用非典型程序进行的测试结果可能无法与使用本指南的结果进行比较。比较使用这些程序的修改版本和未修改版本获得的结果，可能会提供有关用陆地蠕虫进行土壤毒性和生物累积试验的新概念和程序的有用信息。 1.3 用于毒性试验以确定土壤毒性的空间或时间分布的现场收集土壤的结果可根据对存活或亚致死终点的生物影响进行报告（见第节） 14 ). 当温度、pH值和土壤特性（例如，粒径、有机质含量和粘土含量）等因素引起兴趣时，或当需要测试污水污泥和油类等材料时，可以适当修改这些程序，以进行土壤毒性测试。 这些方法也可能有助于进行生物累积试验。 1.4 毒性试验结果 (1) 通过实验将材料（例如化学品或废物混合物）添加到人造土壤、参考土壤或现场土壤中， (2) 用参考土壤稀释现场土壤，以及 (3) 用人工土壤稀释的场地或参考土壤，以产生一系列浓度，可以报告LC50（半致死浓度），有时报告EC50（中效应浓度）。试验结果可按NOEC（无观察到的效应浓度）、LOEC（最低观察到的效应浓度）或ECx（浓度，其中x % 生物效应降低。生物累积试验结果报告为高于第0天组织基线分析或来自阴性对照或参考土壤的第28天组织（即2x、5x、10x）的污染物浓度大小（见 A3.9 ). 1.5 本指南安排如下: 范围 1. 参考文件 2. 术语 3. 指南摘要 4. 意义和用途 5. 干扰 6. 仪器 7. 安全注意事项 8. 土壤 9 试验生物体 10 程序 11 分析方法学 12 测试的可接受性 13 计算结果 14 汇报 15 附件 附件A1 . 赤子爱胜蚓 附件A2 . 人工土壤成分 附件A3 . 利用 赤子爱胜蚓 附件A4 . 加密恢复测试（ERT） 工具书类 1.6 以国际单位制表示的数值应视为标准值。本标准不包括其他计量单位。 1.7 本标准并非旨在解决与其使用相关的所有安全问题（如有）。本标准的用户有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 虽然本指南中包含了一些安全注意事项，但包含进行土壤毒性试验所需的所有安全要求超出了本标准的范围。 第节给出了具体的预防说明 8. . 1.8 本国际标准是根据世界贸易组织技术性贸易壁垒（TBT）委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认标准化原则制定的。 ====意义和用途====== 5.1 土壤毒性试验提供了与土壤有关的化学品对陆地生物的毒性和生物利用度的信息。作为土壤动物群的重要成员，蚯蚓和家蚕具有许多特征，使其成为评估潜在危险土壤的合适生物。蚯蚓可以摄取大量土壤，与其他土壤生物量（例如无脊椎动物、根、腐殖质、凋落物和微生物）有密切关系，构成高达92 % 是土壤无脊椎动物生物量的重要组成部分，对养分的循环利用很重要 ( 1. , 2. ) . 4. Enchytraeids贡献高达5.2 % 在许多土壤中（在缺乏蚯蚓的酸性土壤中，生物量最高），在很大程度上影响养分循环和群落代谢 ( 3- 5. ) . 蚯蚓和马铃薯蠕虫会积累并受到各种有机和无机化合物的影响 ( 2- 10 , 11- 14 ) . 此外，蚯蚓和马铃薯蠕虫在陆地食物网中很重要，是多种生物的食物来源，包括鸟类、哺乳动物、爬行动物、两栖动物、鱼类、昆虫、线虫和蜈蚣 ( 15 , 16 , 3. ) . 土壤无脊椎动物（如蚓类或内生藻类）的丰度发生重大变化，无论是作为食物来源还是作为在营养能量转移和养分循环中正常运作的生物体，都可能对整个陆地系统产生严重的不利生态影响。 5.2 美国和欧洲的野外和实验室调查中使用了许多种蚓类和内生线虫。 虽然各种蚓类对特定化学物质的敏感性可能不同，但他们对四种蚯蚓（包括 E、 胎儿 )暴露于代表六类化学品的十种有机化合物中，Neuhauser等人 ( 7. ) 表明蚯蚓试验物种的选择不会显著影响化学品毒性的评估。到目前为止，尚未以可比的方式研究各种内生藻物种的敏感性，但生态重要性和实用性原因强烈支持选择属于该属的物种 附着体 . 5.2.1 E、 胎儿 是一种自然栖息地为有机质含量非常高的物种，如堆肥和粪堆。它之所以被选为试验物种，是因为它( 1. )易于在实验室繁殖；( 2. )蚯蚓是实验室实验中最常用的物种吗 ( 17 ) ; ( 3. )已经进行了广泛的研究，产生了关于各种化合物的毒性和生物累积性的数据库 ( 2. , 7. , 8. , 18- 23 ) ; ( 4. )已被欧盟（EU）和经济合作与发展组织（OECD）批准用于毒性测试；和( 5. )已被环境保护局（EPA）用于危险废物场所的毒性筛选 ( 24 ) . 5.2.2 建议的enchytraeid测试物种为 白化囊藻 Henle 1837（白色马铃薯）。 E、 阿尔比杜斯 是一种最大的（高达15毫米）寡毛类的家庭Enchytraeidae，它是分布在世界各地 ( 25 , 26 ) . E、 阿尔比杜斯 在海洋、湖沼和陆地栖息地中发现，主要存在于腐烂的有机物（海藻、堆肥）中，很少存在于草甸中 ( 4. , 26 ) . 这种广泛的生态耐受性和一些形态变异可能表明该物种有不同的种族。 E、 阿尔比杜斯 可在市场上买到，作为鱼类食品出售，可在多种有机废物中轻松繁殖，生命周期短（33至74天； 27 , 28 ). E、 阿尔比杜斯 在各种试验中进行了研究，涵盖了广泛的化合物 ( 28- 30 ) . 此外，欧洲联盟（EU）、经济合作与发展组织（OECD）和国际标准化组织（ISO）目前正在对其用于毒性测试和土壤质量评估进行调查。本属其他物种 附着体 也适用，例如， E、 布霍尔齐 维多夫斯基1879或 E、 隐球菌 Westheide和Graefe 1992（见 附件A4 ). 这些物种是真正的土壤居民，体型较小。其他物种 附着体 可以使用，但应明确标识，并报告其选择的理由。 5.3 在评估材料对陆生生物的危害时，土壤毒性试验的结果可能是一个重要的考虑因素。 5.4 通过分析被监测化学品的动物组织，也可以获得与土壤有关的化学品生物累积的信息。 这些结果有助于研究化学品的生物可用性。 5.5 土壤毒性试验可用于确定土壤毒性的时间或空间分布。试验方法可用于检测毒性的水平和垂直梯度。 5.6 土壤毒性试验结果可用于比较不同物种的敏感性。 5.7 通过改变土壤特性，如pH值、粘土含量和有机物质，可以了解这些参数对毒性和生物累积的影响。 5.8 土壤毒性试验的结果可能有助于预测野外情况下陆地生物可能产生的影响。 5.8.1 现场调查可以设计为对现场内或现场之间的生物效应进行定性或定量评估。 5.8.2 评估生物效应的土壤调查通常是对生物、化学、地质和水文条件进行更全面分析的一部分。 如果从同一地点的同一抓斗中同时采集用于实验室毒性试验、地球化学分析和群落结构的土壤子样本，则可以改善统计相关性并降低成本。 5.9 土壤毒性和生物累积性测试可以作为一个重要工具，用于决定对受污染的陆地场地采取必要补救措施的程度。

1.1 This guide covers procedures for obtaining laboratory data to evaluate the adverse effects of contaminants (for example, chemicals or biomolecules) associated with soil to earthworms (Family Lumbricidae) and potworms (Family Enchytraeidae) from soil toxicity or bioaccumulation tests. The methods are designed to assess lethal or sublethal toxic effects on earthworms or bioaccumulation of contaminants in short-term tests (7 to 28 days) or on potworms in short to long-term tests (14 to 42 days) in terrestrial systems. Soils to be tested may be (1) reference soils or potentially toxic site soils; (2) artificial, reference, or site soils spiked with compounds; (3) site soils diluted with reference soils; or (4) site or reference soils diluted with artificial soil. Test procedures are described for the species Eisenia fetida (see Annex A1 ) and for the species Enchytraeus albidus (see Annex A4 ). Methods described in this guide may also be useful for conducting soil toxicity tests with other lumbricid and enchytraeid terrestrial species, although modifications may be necessary. 1.2 Modification of these procedures might be justified by special needs. The results of tests conducted using atypical procedures may not be comparable to results using this guide. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting soil toxicity and bioaccumulation tests with terrestrial worms. 1.3 The results from field-collected soils used in toxicity tests to determine a spatial or temporal distribution of soil toxicity may be reported in terms of the biological effects on survival or sublethal endpoints (see Section 14 ). These procedures can be used with appropriate modifications to conduct soil toxicity tests when factors such as temperature, pH, and soil characteristics (for example, particle size, organic matter content, and clay content) are of interest or when there is a need to test such materials as sewage sludge and oils. These methods might also be useful for conducting bioaccumulation tests. 1.4 The results of toxicity tests with (1) materials (for example, chemicals or waste mixtures) added experimentally to artificial soil, reference soils, or site soils, (2) site soils diluted with reference soils, and (3) site or reference soils diluted with artificial soil, so as to create a series of concentrations, may be reported in terms of an LC50 (median lethal concentration) and sometimes an EC50 (median effect concentration). Test results may be reported in terms of NOEC (no observed effect concentration), LOEC (lowest observed effect concentration) or as an ECx (concentration where x % reduction of a biological effect occurs. Bioaccumulation test results are reported as the magnitude of contaminant concentration above either the Day 0 tissue baseline analysis or the Day 28 tissues from the negative control or reference soil (that is, 2x, 5x, 10x) (see A3.9 ). 1.5 This guide is arranged as follows: Scope 1 Referenced Documents 2 Terminology 3 Summary of Guide 4 Significance and Use 5 Interferences 6 Apparatus 7 Safety Precautions 8 Soil 9 Test Organism 10 Procedure 11 Analytical Methodology 12 Acceptability of Test 13 Calculation of Results 14 Report 15 Annexes Annex A1 . Eisenia fetida Annex A2 . Artificial Soil Composition Annex A3 . Bioaccumulation Testing Utilizing Eisenia fetida Annex A4 . Enchytraeid Reporduction Test (ERT) References 1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. While some safety considerations are included in this guide, it is beyond the scope of this standard to encompass all safety requirements necessary to conduct soil toxicity tests. Specific precautionary statements are given in Section 8 . 1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee. ====== Significance And Use ====== 5.1 Soil toxicity tests provide information concerning the toxicity and bioavailability of chemicals associated with soils to terrestrial organisms. As important members of the soil fauna, lumbricid earthworms and enchytraeid potworms have a number of characteristics that make them appropriate organisms for use in the assessment of potentially hazardous soils. Earthworms may ingest large quantities of soil, have a close relationship with other soil biomasses (for example, invertebrates, roots, humus, litter, and microorganisms), constitute up to 92 % of the invertebrate biomass of soil, and are important in recycling nutrients ( 1 , 2 ) . 4 Enchytraeids contribute up to 5.2 % of soil respiration, constitute the second-highest biomass in many soils (the highest in acid soils in which earthworms are lacking) and effect considerably nutrient cycling and community metabolism ( 3- 5 ) . Earthworms and potworms accumulate and are affected by a variety of organic and inorganic compounds ( 2- 10 , 11- 14 ) . In addition, earthworms and potworms are important in terrestrial food webs, constituting a food source for a very wide variety of organisms, including birds, mammals, reptiles, amphibians, fish, insects, nematodes, and centipedes ( 15 , 16 , 3 ) . A major change in the abundance of soil invertebrates such as lumbricids or enchytraeids, either as a food source or as organisms functioning properly in trophic energy transfer and nutrient cycling, could have serious adverse ecological effects on the entire terrestrial system. 5.2 A number of species of lumbricids and enchytraeid worms have been used in field and laboratory investigations in the United States and Europe. Although the sensitivity of various lumbricid species to specific chemicals may vary, from their study of four species of earthworms (including E. fetida ) exposed to ten organic compounds representing six classes of chemicals, Neuhauser, et al ( 7 ) suggest that the selection of earthworm test species does not affect the assessment of a chemical's toxicity markedly. The sensitivity of various enchytraeid species has not been investigated in a comparable way so far, but ecological importance and practicability reasons favor strongly the selection of a species belonging to the genus Enchytraeus . 5.2.1 E. fetida is a species whose natural habitats are those of very high organic matter such as composts and manure piles. It was selected as the test species because it ( 1 ) is bred in the laboratory easily; ( 2 ) is the earthworm species used most commonly in laboratory experiments ( 17 ) ; ( 3 ) has been studied extensively, producing a data pool on the toxicity and bioaccumulation of a variety of compounds ( 2 , 7 , 8 , 18- 23 ) ; ( 4 ) has been approved for use in toxicity testing by the European Union (EU) and the Organization for Economic Cooperation and Development (OECD); and ( 5 ) has been used by the Environmental Protection Agency (EPA) for the toxicity screening of hazardous waste sites ( 24 ) . 5.2.2 The recommended enchytraeid test species is Enchytraeus albidus Henle 1837 (white potworm). E. albidus is one of the biggest (up to 15 mm) species of the oligochaete family Enchytraeidae and it is distributed world-wide ( 25 , 26 ) . E. albidus is found in marine, limnic, and terrestrial habitats, mainly in decaying organic matter (seaweed, compost) and rarely in meadows ( 4 , 26 ) . This broad ecological tolerance and some morphological variations might indicate that there are different races for this species. E. albidus is commercially available, sold as food for fish, can be bred easily in a wide range of organic waste materials and has a short life cycle (33 to 74 days; 27 , 28 ). E. albidus was studied in various tests, which covered a wide range of compounds ( 28- 30 ) . In addition, it is currently under investigation for use in toxicity testing and soil quality assessment by the European Union (EU), the Organization for Economic Cooperation and Development (OECD), and the International Organization for Standardization (ISO). Other species of the genus Enchytraeus are also suitable, for example, E. buchholzi Vejdovsky 1879 or E. crypticus Westheide and Graefe 1992 (see Annex A4 ). Those species are true soil inhabitants and are smaller in size. Other species of Enchytraeus may be used, but they should be identified clearly and the rationale for their selection should be reported. 5.3 Results from soil toxicity tests might be an important consideration when assessing the hazards of materials to terrestrial organisms. 5.4 Information might also be obtained on the bioaccumulation of chemicals associated with soil by analysis of animal tissues for the chemicals being monitored. These results are useful for studying the biological availability of chemicals. 5.5 The soil toxicity test might be used to determine the temporal or spatial distribution of soil toxicity. Test methods can be used to detect horizontal and vertical gradients in toxicity. 5.6 Results of soil toxicity tests could be used to compare the sensitivities of different species. 5.7 An understanding of the effect of these parameters on toxicity and bioaccumulation may be gained by varying soil characteristics such as pH, clay content, and organic material. 5.8 Results of soil toxicity tests may be useful in helping to predict the effects likely to occur with terrestrial organisms in field situations. 5.8.1 Field surveys can be designed to provide either a qualitative or quantitative evaluation of biological effects within a site or among sites. 5.8.2 Soil surveys evaluating biological effects are usually part of more comprehensive analyses of biological, chemical, geological, and hydrographic conditions. Statistical correlation can be improved and costs reduced if subsamples of soil for laboratory toxicity tests, geochemical analyses, and community structure are taken simultaneously from the same grab of the same site. 5.9 Soil toxicity and bioaccumulation tests can be an important tool for making decisions regarding the extent of remedial action necessary for contaminated terrestrial sites.