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    ASTM E3155 - 19

    Standard Guide for Assessing Mammal Health at Chemically Contaminated Terrestrial Sites Using Rodent Sperm Analysis

    Active Standard ASTM E3155 | Developed by Subcommittee: E50.47

    Book of Standards Volume: 11.05

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    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).

    1. Scope

    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:





    Referenced Documents




    Summary of Guide


    Significance and Use


    Safety Precautions










    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.

    2. Referenced Documents (purchase separately) The documents listed below are referenced within the subject standard but are not provided as part of the standard.

    ASTM Standards

    E1527 Practice for Environmental Site Assessments: Phase I Environmental Site Assessment Process

    E1689 Guide for Developing Conceptual Site Models for Contaminated Sites

    E1848 Guide for Selecting and Using Ecological Endpoints for Contaminated Sites

    E2081 Guide for Risk-Based Corrective Action

    E2205 Guide for Risk-Based Corrective Action for Protection of Ecological Resources

    E2616 Guide for Remedy Selection Integrating Risk-Based Corrective Action and Non-Risk Considerations

    E2790 Guide for Identifying and Complying With Continuing Obligations

    ICS Code

    ICS Number Code 07.080 (Biology. Botany. Zoology); 13.020.40 (Pollution, pollution control and conservation)

    Referencing This Standard
    Link Here
    Link to Active (This link will always route to the current Active version of the standard.)

    DOI: 10.1520/E3155-19

    Citation Format

    ASTM E3155-19, Standard Guide for Assessing Mammal Health at Chemically Contaminated Terrestrial Sites Using Rodent Sperm Analysis, ASTM International, West Conshohocken, PA, 2019, www.astm.org

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