1.1 本指南描述了获取和解释与受化学污染的陆地场所哺乳动物受体直接健康状况评估相关的数据的程序，在这些场所，ERA工作已安排或正在进行，且与可能存在的化学品的数量和类型无关。通过回顾精子特征，RSA方法报告了雄性啮齿动物在其自然环境中的生殖健康，这些动物作为其他（和更大）部位哺乳动物的替代物 ( 4. ) . 1.2 这些程序适用于支持小型哺乳动物（例如，老鼠、田鼠、老鼠、松鼠）且土壤受到污染的任何陆地财产。重要的是，场地土壤中引起关注的化学物质不一定是精蛋白。 此外，RSA方法认为，化学品或其他部位应激源的任何组合可能共同影响生殖，被认为是哺乳动物的敏感毒理学终点。该方法的预期主要应用将在历史上受污染的场所（如超级基金场所）。这些程序描述了在现场和实验室中执行的任务。对于后者，可以在现场移动实验室或更传统的实验室环境中执行任务。对于某些任务，临时工作空间也可能合适（参见 7.3 ). 1.3 通过仔细比较精子参数，初步确定居住雄性小型啮齿类动物的受损或非受损生殖。 简而言之，对于在受污染地点和栖息地匹配（未受污染）参考位置收集的给定物种的啮齿动物，首先计算三个精子参数（计数、活力和形态）的算术平均值。如果受污染场所啮齿动物的一个或多个参数均值反映出不利的变化（即，计数或运动能力低于参考位置动物；异常形状精子的百分比相对于参考位置动物更大），则将每个均值的减少或增加百分比与相关既定精子参数基准进行比较，每一种都以这种程度的向不利方向转移的形式出现，这意味着生殖成功率较低 ( 2. ) （参见 9.3 ). 1.4 RSA方法和本指南的真正重点是通过应用基于空间运动的外推方案，对接触较大部位的哺乳动物中的妥协或非妥协繁殖进行高级确定。如果在受污染的场所啮齿动物中未观察到既定的精子参数基准超标，则也假设接触该场所的其他哺乳动物具有非危害性繁殖。这源于后者，由于家庭活动范围远大于啮齿动物，因此所有人的现场暴露程度都明显较低。举例来说，红狐和白尾鹿各自的活动范围分别为400多英亩和640英亩 ( 10- 14 ) ，这些物种将花费最少的时间（例如，5 %) 在占地25英亩或更少的典型污染场所 ( 15 , 16 ) . 如果在受污染的场地啮齿动物中超过一个或多个精子参数基准（当然表明啮齿动物的生殖能力受到损害），其他场地哺乳动物也可能受到生殖能力的损害。目标物种（即现场啮齿动物）的家域与已知接触受污染现场的任何其他哺乳动物之间的差距越大，后者生殖能力受损的可能性就越小。RSA方法采用的毒理学外推原理与传统桌面电子逆向拍卖中用于哺乳动物的毒理学外推原理相同。在这些时代，啮齿动物的应激反应（基于实验室的研究）有助于解释无法方便地直接评估健康状况的哺乳动物（例如狐狸、臭鼬、浣熊、鹿、郊狼等）的扩展列表的健康影响。 ). 1.5 本指南安排如下: 部分 范围 1. 参考文件 2. 术语 3. 指南摘要 4. 意义和用途 5. 安全注意事项 6. 仪器 7. 程序 8. 报告 9 关键词 10 1.6 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。 1.7 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 RSA方法使风险和资源管理者能够更好地了解他们所监管地点的生态健康问题，因为与传统陆地ERA不同，实际地点的哺乳动物是被评估的。此外，桌面工作的总部仅报告摄入的污染物暴露途径，只能单独评估化学品，而RSA的发现反映了所有三种暴露途径以及多种化学品对高价值终点的综合影响。关键的是，RSA方法结合了必然影响生物反应现象的现场历史因素。如果曾经在受污染的场所引发生殖影响，这在今天将是显而易见的，因为被评估的场所至少已经连续几十年将其生态受体暴露于化学品中，在这几十年中，已经过了几十代，而且往往超过了一百代 ( 5. ) . 5.2 主题指南的应用使补救决策者和风险管理者熟悉两个概念。首先，与其试图预测位点受体产生的健康影响，不如记录已证实的健康影响，如果在实际位点暴露的哺乳动物受体中存在这种影响的话。其次，存在这样一种可能性，即自场地首次受到污染以来，多年来场地受体从未经历过压力或影响。 5.3 应用主题指南可以节省大量成本。通常，基于总部的评估结果被概括地用于进行正在进行的研究、监测现场或实施现场清理，所有这些可能都是不必要的。RSA应用程序应最大限度地证明- 暴露的哺乳动物受体（定义见第 4.1 )在生殖成功的敏感终点方面没有遇到妥协，很明显，不需要代表哺乳动物进行土壤修复工作。 5.4 在ERA过程中，一种或多种哺乳动物物种的HQ值大于1.0时，所述RSA方法通常可以应用于ERA过程中的该点，如该过程的步骤2（筛查水平暴露估计和风险计算；其中生态威胁是以一般方式而不是特定方式评估的）。或者，特别是在没有像《综合环境反应、赔偿和责任法案》（CERCLA）那样严格管理的现场； 又名超级基金类型）场地，一旦确定某个场地有感兴趣的化学污染足迹（即土壤浓度高到足以对哺乳动物场地受体有害），即可应用该指南。鉴于初步和完善的HQ倾向于表明哺乳动物摄入了不健康剂量的现场污染物，这通常会导致进入现场进行验证工作，将RSA作为第一次评估工作的应用旨在节省时间和成本。 5.5 本指南的意义在于，方法设计反映了对某些不可避免的ERA过程约束的理解，特别是在野外哺乳动物采集和后续组织分析领域。 首先，RSA方法认识到，小型啮齿动物是唯一可以定期从野外扑杀的哺乳动物（即，被清除而不被退回），此外，这一现实不太可能改变。当地管理机构和动物护理机构可能会挑战定期捕捞大型哺乳动物（例如狐狸）的努力。此外，由于动物密度相对较小，获取足够的大型哺乳动物是耗时和劳动密集型的。此外，并非所有栖息地或所有州都有许多大型哺乳动物（例如长尾鼬、獾）。相反，小型啮齿动物几乎出现在每个栖息地，相对容易收集，数量足够多，可以在地点之间进行防御性比较。 在选择接触量最大的小型啮齿类动物（即，由于家畜面积几乎总是在一英亩或更少，因此在其一生中被限制在受污染的环境中的动物）时，RSA方法具有一个共同的比较基础（当然，在美国应该应用它的任何地方）。 5.6 RSA理论理解，通常在受污染的陆地场所，人们担心所关注的受体可能在繁殖方面受到损害。将生殖作为主要毒理学关注点 ( 6. , 7. ) 认识到已经开发了许多方法来研究啮齿动物的生殖效应（支持人类健康） ( 9 , 17 ) . 在生态风险评估师为支持HQ计算而经常选择的首选毒性参考值（TRV）层次结构中，生殖具有这种状态是显而易见的。 人们还认识到，不存在有效评估现场采集生物体中其他感兴趣端点的标准化方法，例如神经毒性或行为。如果在受污染的场所啮齿动物中未观察到既定的精子参数基准超标，则这可以构成一条重要的证据线，支持确定生殖正在充分进行。RSA方法认识到，尽管繁殖过程正常，但受污染地区啮齿动物的其他生物功能（例如，行为、神经冲动传递）可能会受到损害 ( 2. , 3. ) . 在这种情况下，该方法的支持理论理解到，正在达到的其他终点不一定会引起关注，因为它们没有阻碍最大程度暴露的啮齿动物存活到繁殖年龄、寻找配偶和产生有活力的幼崽的能力 ( 2. , 18 ) . 5.7 本指南承认，目前还没有针对雌性啮齿动物的肛门生殖评估方法。重要的是，缺乏针对女性的生殖评估方法并不构成主题指南的缺陷。例如，美国环境保护局的相关指南支持评估涉及药物和化学品监管的物种的性别，并根据这些信息得出结论 ( 19 ) . 在这方面，有几点值得注意。首先，98岁以上 % 在制定美国环境保护局ERA土壤筛选水平（SSL）时考虑的所有哺乳动物毒性研究中（约17种无机和4种有机化学物种），有35种为单性类型 % 研究对象为男性- 只有 ( 20 ) . 此外，对于37 % 在具有SSL的化学品中，纯男性毒性研究的数量超过了纯女性毒性研究的数量。最后，最常用的野生动物毒理学基准中有很大一部分 ( 21 ) 来自单性别研究。关键的是，由于RSA专注于直接评估雄性啮齿动物的繁殖，其破坏性明显低于从野外扑杀雌性啮齿动物的方法，因为后者是生育后代的啮齿动物。 5.8 本指南认识到在基于现场的哺乳动物受体评估中使用野生啮齿动物的价值。除了啮齿动物可能是唯一可以定期从现场扑杀的哺乳动物（如上所述）这一事实外，与这些动物合作还有一些关键优势。 几乎所有陆地栖息地都有小型啮齿动物，因此本指南在地理意义上具有广泛的适用性。第二个优点是，小型啮齿动物可能无一例外地是陆地环境中接触最多的哺乳动物，这也是直接接触受污染土壤的一个方面。这是因为啮齿动物本质上是非迁徙性的，在受污染的场所有效控制它们的活动范围极其有限，它们几乎所有的时间都直接接触地面（即受污染的土壤； 2. , 4. , 18 ). 5.9 在提供支持哺乳动物电子逆向拍卖的有用证据时，本指南采用了一种直接的外推方法 ( 2. , 18 ) ，这与传统的基于总部的评估中应用的评估是同构的。如果与任何其他场地哺乳动物相比，与受影响场地土壤有更频繁和更密切接触的场地啮齿动物未被发现存在生殖危害，那么接触场地（即受污染土壤）程度相当低的更大、范围更广的哺乳动物也应不存在生殖危害。对这种外推方案的赞赏来自对传统应用的基于桌面的电子逆向拍卖的主要外推方案的审查。在那里，基于实验室的小鼠或大鼠研究通常用于确定另一种哺乳动物（例如，鹿、狐狸、兔子）是否摄入了不健康数量的给定化学物质。 与传统的ERA方案相比，有许多差异需要承认，甚至在啮齿动物的水平上。因此，试验动物和居住在待评估地点的野生动物在物种、饲养、环境/栖息地或饲养设计方面不匹配，这些差异削弱了可以得出的结论。相比之下，受试者标准在其初始推断中比较了精子测量值，每一个测量值都是生殖成功的晴雨表 ( 22- 25 ) 在相距不到一公里的同种种群中，一个种群居住在土壤污染区，另一个种群是无污染区。RSA方法认识到，受污染场所的小型啮齿动物是潜在施加环境压力的集成商，这些压力超出了土壤和饮食项目中可能存在的化学物质，包括物理栖息地干扰（例如，噪音或土地振动）。 RSA理解，传统的生态评估必然努力了解小型啮齿动物的繁殖能力，因为这种分组被认为是关键的生态系统要素。在RSA结果中未观察到生殖损害的情况下，有证据表明，一个部位的详尽的部位应激源列表，在其发生的实际阵列中，不会影响通常被认为是最重要的毒理学终点。 5.10 本指南的一个局限性是，所应用的（生殖）效应的生物学重要阈值是由实验室得出的。本指南的第二个局限性是，鼩通常不能接受RSA方法，因为它们的新陈代谢非常高，干扰了它们的生存- 被困在野外。在罕见的情况下，在受污染的关注场所，只有鼩鼠存在，RSA方法可能无法成功应用。如果由于任何原因，给定的受污染场地没有提供一个完整的小啮齿动物种群，或者如果在感兴趣的场地和合适的栖息地匹配的参考位置都没有至少一种常见的小啮齿动物物种，或者找不到合适的参考位置（参见 8.1 )，该方法不适用。RSA仅用于确定现场哺乳动物是否生殖受损。该方法本身并不涉及确定导致观察到的精子参数阈值的化学或物理部位应激源- 对于效果超标，或清理水平的确定，这些都不是方法限制。这种情况类似于对各种水生试验物种（例如， 眼底sp。 ). 在那里，目标只是确定废水处理程度是否足以支持居住在受纳水体混合区的水生生物。（主要不是设计标准的整体废水毒性测试，以确定废水中可能导致不可接受测试结果的一种或多种成分。） 5.11 本指南与ERA指南和指南一致 ( 26 , 27 ) ，在这一领域中，对现场受体（so）的健康状况进行了与环境相关的评估- 称为“现场验证”）是公认的正式步骤。在了解到在受污染的场所已经过了足够的时间来证明生殖危害是明显的（如果该终点曾被触发），本指南旨在记录此类已证明的危害。关键是，RSA不是一种旨在预测或预测持续接触污染物对哺乳动物健康影响的风险评估方法。因此，本指南与其他与ERA毒理学效应预测方面有关的ASTM标准相关，但明显不同（指南 E1527 -13, E1689 , E1848 -96, E2081年 , E2205 -02, E2616 和 E2790 ). 该指南还符合美国《生殖毒性风险评估指南》。 S、 美国环保局 ( 19 ) . 具体来说，仅评估一个物种的一种性别的生殖健康被认为足以进行整体物种评估 ( 17 ) . 然而，在一个关键领域，本指南与传统电子逆向拍卖截然不同，传统电子逆向拍卖在很大程度上局限于桌面分析水平。而传统的评估要么依赖于结果的统计显著差异，要么依赖于通常协商的生物反应差异（例如，20） %) 在得出结论时，本指南主要利用了先前提到的一系列已确定的生物学重要阈值 ( 22- 25 ) . 此外，统计比较只需应用于两种可能的RSA结果之一（参见 9.3.1 和 9.4 ).

1.1 This guide describes the procedures for obtaining and interpreting data associated with a direct health status assessment for mammalian receptors at chemically contaminated terrestrial sites where ERA work is either scheduled or ongoing, and irrespective of the number and type of chemicals that may be present. Through reviewing sperm features, the RSA method reports on the reproductive health of male rodents in their natural environmental settings, with these animals serving as surrogates for other (and larger) site mammals ( 4 ) . 1.2 These procedures are applicable at any terrestrial property that supports small mammals (for example, mice, voles, rats, squirrels) and has contaminated soil. Importantly, chemicals of concern in site soils need not be spermatoxins. Additionally, the RSA method considers that any combination of chemicals or other site stressors might collectively act to compromise reproduction, held to be a sensitive toxicological endpoint for mammals. The anticipated primary application of the method will be at historically contaminated sites (such as Superfund sites). The procedures describe tasks conducted in the field and in a laboratory. For the latter, tasks may be conducted either in an on-site mobile laboratory, or in a more conventional laboratory setting. For certain tasks, a make-shift work space may be suitable as well (see 7.3 ). 1.3 Initial determinations of compromised or non-compromised reproduction in resident male small rodents are made through a cautious comparative review of sperm parameters. Briefly, for the rodents of a given species collected at both a contaminated site and a habitat-matched (non-contaminated) reference location, arithmetic means are first computed for each of the three sperm parameters of count, motility, and morphology. If one or more of the parameter means of the contaminated site rodents reflect an unfavorable shift (that is, count or motility is less than that of reference location animals; the percentage of abnormally-shaped sperm is greater relative to reference location animals), the percent decrease or increase in each mean is compared to the relevant established sperm parameter benchmark, each in the form of that degree of shift in an unfavorable direction that signifies lesser reproductive success ( 2 ) (see 9.3 ). 1.4 Advanced determinations of compromised or non-compromised reproduction in larger site-contacting mammals, the true focus of the RSA method and this guide, are made through an applied spatial movements-based extrapolation scheme. Where established sperm parameter benchmark exceedances are not observed in contaminated-site rodents, other mammals contacting a site are also assumed to have non-compromised reproduction. This follows from the latter all having notably lesser degrees of site exposure due to home ranges that are vastly larger than those of rodents. By way of example, with respective home ranges of 400+ and 640 acres for the red fox and white-tailed deer ( 10- 14 ) , these species would spend minimal amounts of their time (for example, 5 %) at prototypical contaminated sites that cover areas of 25 acres or less ( 15 , 16 ) . Where one or more sperm parameter benchmarks are exceeded in contaminated-site rodents (certainly indicating that the rodents are reproductively compromised), other site mammals may also be reproductively compromised. The greater the disparity between the home ranges of the target species (that is, the site rodent) and any of the other mammals known to contact the contaminated site in question, the less likely it will be that the latter are reproductively compromised. The RSA method employs the same toxicological extrapolation principles as that used for mammals in conventional desktop-based ERAs. In those ERAs, stressor-mediated responses of rodents (of a laboratory-based study) assist with the interpretation of health effects for an expanded list of mammals that cannot conveniently be evaluated directly for health status (for example, fox, skunk, raccoon, deer, coyote, etc.). 1.5 This guide is arranged as follows: Section Scope 1 Referenced Documents 2 Terminology 3 Summary of Guide 4 Significance and Use 5 Safety Precautions 6 Apparatus 7 Procedure 8 Reporting 9 Keywords 10 1.6 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. 1.7 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 The RSA method provides risk and resource managers with an enhanced understanding of the ecological health concerns at the sites they oversee because unlike conventional terrestrial ERAs, actual site mammals are the ones evaluated. Additionally, the HQs of desktop efforts report only on the contaminant exposure route of ingestion, and can only evaluate chemicals singly, whereas RSA findings reflect all three exposure routes as well as the combined effects of multiple chemicals on a highly valued endpoint. Critically, the RSA method incorporates site history considerations that necessarily influence the phenomenon of biological response. If reproductive impacts at contaminated sites were ever to be elicited, such would be apparent today because evaluated sites have, at a minimum, continuously exposed their ecological receptors to chemicals for multiple decades during which time tens and often more than one hundred generations have passed ( 5 ) . 5.2 Application of the subject guide familiarizes remedial decision-makers and risk managers with two concepts. First, rather than attempting to predict health effects arising in site receptors, there may be more value in documenting demonstrated health effects, should such exist in actual site-exposed mammalian receptors. Second, the possibility exists that site receptors never experienced stress or impact over the years since a site first became contaminated. 5.3 Application of the subject guide can allow for substantial cost savings. Often, the outcomes of HQ-based assessments are summarily relied upon to conduct ongoing studies, monitor sites, or implement site cleanups, all of which may be unnecessary. Where RSA applications should demonstrate that maximally site-exposed mammalian receptors (as defined in section 4.1 ) are not experiencing compromise with regard to the sensitive endpoint of reproductive success, it can become apparent that soil remediation efforts on behalf of mammals are not needed. 5.4 The described RSA method can typically be applied at that point in the ERA process where HQs for one or more mammalian species are found to be greater than 1.0, as in the process’s Step 2 (Screening-Level Exposure Estimate and Risk Calculation; where ecological threats are evaluated in a general, as opposed to a specific fashion). Alternatively and particularly at sites that are not governed as rigidly as, for example, Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA; aka Superfund-type) sites, the guide can be applied once it is established that a site has a chemical contamination footprint of interest (that is, that soil concentrations are high enough to potentially be harmful to mammalian site receptors). In light of the propensity for preliminary and refined HQs to suggest mammals are ingesting unhealthful doses of site contaminants, in turn commonly leading to advancing to the field for a verification effort, the application of RSA as a first evaluative effort is intended to be a time- and cost-saving effort. 5.5 The significance of this guide is the method design that reflects an understanding of certain unavoidable ERA process constraints, specifically in the areas of field mammal collection and subsequent tissue analysis. First, the RSA method recognizes that small rodents are the only mammals that can be routinely culled from the field (that is, to be removed and not returned), and further, that this reality is unlikely to ever change. Efforts to regularly harvest larger mammals (for example, fox) may be challenged by local governing agencies and animal care institutions. Additionally, acquiring a sufficiency of larger mammals is time-consuming and labor-intensive, owing to relatively miniscule animal densities. Further, many larger mammals (for example, long-tailed weasel, badger) are not found in all habitats or in all states. In contrast, small rodents occur in virtually every habitat, are relatively easy to collect, and are numerous enough to allow for defensible comparisons between or among sites. In selecting the maximally exposed small rodent to work with (that is, an animal confined to contaminated surroundings throughout its life due to a home range that is almost always of one acre or less), the RSA method features a common basis of comparison (and certainly wherever it should be applied in the United States). 5.6 RSA theory understands that, generally at contaminated terrestrial sites, there is worry that receptors-of-concern might be reproductively compromised. The focus on reproduction as the dominant toxicological endpoint of concern ( 6 , 7 ) , recognizes that much method development for reproductive effects in rodents (in support of human health) has occurred ( 9 , 17 ) . That reproduction bears this status is evident in the hierarchy of preferred toxicity reference values (TRVs) that ecological risk assessors often select in support of HQ computation. Additional recognition is given to the reality that standardized means for effectively assessing other endpoints of interest in field-collected organisms, such as neurotoxicity or behavior, do not exist. Where established sperm parameter benchmark exceedances are not observed in contaminated site rodents, such can constitute a significant line of evidence in support of a determination that reproduction is proceeding adequately. The RSA method recognizes that impairments to other biological functions (for example, behavior, nerve impulse transmission) of contaminated-site rodents may be occurring despite reproduction proceeding normally ( 2 , 3 ) . Where such is the case, the method’s supporting theory understands that other endpoints being reached do not necessarily pose a concern for they have not impeded the ability of maximally exposed rodents to survive to the age of reproduction, find mates, and produce viable young ( 2 , 18 ) . 5.7 This guide recognizes that an analagous reproductive assessment approach for female rodents, is not available at the present time. Importantly, an absent reproductive assessment approach for females does not constitute a shortcoming of the subject guide. Relevant U.S. EPA guidance, for example, supports evaluating one sex of a species where drug and chemical regulation is concerned, and drawing conclusions based on such information ( 19 ) . In this context several noteworthy points follow. First, over 98 % of all mammalian toxicity studies considered in crafting the U.S. EPA’s Soil Screening Levels (SSLs) for ERA (for some 17 inorganic and 4 organic chemical species) are of the single-sex type, with 35 % of the studies being male-only ( 20 ) . Additionally, for 37 % of the universe of chemicals with SSLs, the number of male-only toxicity studies exceeds the number of female-only toxicity studies. Finally, a significant percentage of the most commonly applied toxicological benchmarks for wildlife ( 21 ) derive from single-sex studies. Critically, with its focus on directly assessing reproduction in male rodents, RSA is notably far less destructive than would be a method involving the culling of female rodents from the field, given that the latter are the ones that bear the young. 5.8 This guide recognizes the value in employing the wild rodent in field-based mammalian receptor assessment. Aside from the reality that rodents may constitute the only mammals that can regularly be culled from sites (discussed above), there are key advantages that accrue to working with these animals. Small rodents occur in nearly all terrestrial habitats, allowing the guide to be broadly applicable in a geographical sense. A second advantage is that the small rodent with perhaps no exception, will likely be the maximally-exposed mammal in terrestrial settings, this again, in terms of having direct contact with contaminated soils. This follows from rodents being non-migratory in nature, having extremely limited home ranges that effectively contain them at contaminated sites, and their spending nearly all of their time directly contacting the ground (that is, contaminated soils; 2 , 4 , 18 ). 5.9 In providing a useful line of evidence in support of ERAs for mammals, this guide employs a straightforward extrapolation approach ( 2 , 18 ) , one that is isomorphic to that applied in conventional HQ-based assessments. If site rodents, that have more constant and intimate contact with affected site soils than that of any other site mammal, are not found to have compromised reproduction, larger and wider-ranging mammals, with their considerably lesser degrees of site (that is, contaminated soil) contact, should also be free of reproductive compromise. An appreciation for this extrapolation scheme derives from a review of the principal extrapolation scheme of conventionally-applied desktop-based ERAs. There, a laboratory-based mouse or rat study is routinely used to determine if another mammal (for example, deer, fox, rabbit) is ingesting an unhealthful quantity of a given chemical. With the conventional ERA scheme, there are numerous differences to acknowledge, and even at the level of the rodent. Thus the test animal and the wild form inhabiting the site of interest that is to be assessed, do not match in terms of species, rearing, environment/habitat, or feeding design, and these differences weaken conclusions that can be drawn. In contrast, the subject standard in its initial extrapolation, compares sperm measures, each a proven barometer of reproductive success ( 22- 25 ) , in populations of conspecifics living less than a kilometer apart, with one population inhabiting a soil-contaminated area, and the other a contaminant-free one. The RSA method recognizes that small rodents of contaminated sites are integrators of potentially imposing environmental stressors that extend beyond chemicals that may be present in soil and diet items, to include such things as physical habitat disturbances (for example, noise or land vibration). RSA understands that conventional ecological assessments necessarily strive to know of small rodent reproductive capability, as this grouping is held to be a keystone ecosystem element. Where reproductive compromise is not observed in an RSA outcome, there is demonstration that a site’s exhaustive list of site stressors, in the actual arrays in which they occur, are not impinging on what is generally held to be the most important toxicological endpoint. 5.10 One limitation of this guide is that the biologically-significant thresholds-for- (reproductive)-effect that are applied, are laboratory-derived. A second limitation of this guide is that shrews generally cannot submit to the RSA method, owing to their exceedingly high metabolism that interferes with their being live-trapped in the field. In the rare case where the only rodents present at a contaminated site of concern should be shrews, the RSA method can probably not be successfully applied. If for any reason a given contaminated site does not offer a small rodent population altogether, or if there is not at least one common small rodent species occurring at both the site of interest and a suitable habitat-matched reference location, or an appropriate reference location cannot be found (see 8.1 ), the method is not applicable. RSA is intended only to identify if site mammals are reproductively compromised. The method does not concern itself with identifying the chemical(s) or physical site stressors responsible for observed sperm parameter threshold-for-effect exceedances, or the determination of cleanup levels, and such are not method limitations. The situation is analogous to standardized whole effluent toxicity tests conducted with various aquatic test species (for example, Fundulus sp. ). There, the objective is only to ascertain if the degree of wastewater treatment is adequate to support the aquatic life inhabiting a receiving waterbody’s mixing zone. (Standard whole effluent toxicity testing is not designed in the main, to identify the constituent or constituents in effluent that may be responsible for unacceptable test outcomes.) 5.11 This guide is consistent with ERA guidance and guidelines ( 26 , 27 ) , where advancing to the field for an environmentally relevant assessment of the health of site receptors (so-called ‘field verification’) is a recognized formal step. In understanding that sufficient time has elapsed at contaminated sites for reproductive compromise to be evident (if that endpoint was ever to be triggered), this guide is designed to document such demonstrated compromise. Critically, RSA is not a risk assessment method that aims to forecast or predict health effects arising in mammals with ongoing contaminant exposures. The guide then is related to, but distinctly different from other ASTM standards that bear on the toxicological effects prediction aspect of ERA (Guides E1527 -13, E1689 , E1848 -96, E2081 , E2205 -02, E2616 , and E2790 ). The guide is also consistent with guidelines for reproductive toxicity risk assessment as per the U.S. EPA ( 19 ) . Specifically, assessing the reproductive health of only one sex of a species is deemed adequate for an overall species assessment ( 17 ) . In one key area however, this guide is quite unlike conventional ERAs that are largely restricted to the level of desktop analysis. Whereas conventional assessments rely on either statistically-significant differences in outcome, or on a commonly negotiated difference in biological response (for example, 20 %) when drawing conclusions, this guide primarily avails itself to the utility of a series of established biologically-significant thresholds alluded to previously ( 22- 25 ) . Further, a statistical comparison need only be applied for one of two possible RSA outcomes (see 9.3.1 and 9.4 ).