ASTM E3155-19

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

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 des...
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 ...

General Information

• Status: Published
• Publication Date: 31-Dec-2018
• ICS: 07.080 - Biology. Botany. Zoology
• ICS: 13.020.40 - Pollution, pollution control and conservation
• Technical Committee: E50 - Environmental Assessment, Risk Management and Corrective Action
• Drafting Committee: E50.47 - Biological Effects and Environmental Fate

Relations

• Refers: ASTM E2790-20 - Standard Guide for Identifying and Complying With Continuing Obligations
• Effective Date: 01-Jan-2020
• Refers: ASTM E2790-11 - Standard Guide for Identifying and Complying With Continuing Obligations
• Effective Date: 15-May-2011
• Refers: ASTM E2081-00(2010)e1 - Standard Guide for Risk-Based Corrective Action
• Effective Date: 01-Sep-2010
• Refers: ASTM E1689-95(2008) - Standard Guide for Developing Conceptual Site Models for Contaminated Sites
• Effective Date: 01-Feb-2008
• Refers: ASTM E1848-96(2008) - Standard Guide for Selecting and Using Ecological Endpoints for Contaminated Sites
• Effective Date: 01-Feb-2008
• Refers: ASTM E1527-05 - Standard Practice for Environmental Site Assessments: Phase I Environmental Site Assessment Process
• Effective Date: 01-Nov-2005
• Refers: ASTM E2081-00(2004)e1 - Standard Guide for Risk-Based Corrective Action
• Effective Date: 01-Oct-2004
• Refers: ASTM E2205-02 - Standard Guide for Risk-Based Corrective Action for Protection of Ecological Resources
• Effective Date: 10-May-2002
• Refers: ASTM E1527-97 - Standard Practice for Environmental Site Assessments: Phase 1 Environmental Site Assessment Process
• Effective Date: 10-May-2000
• Refers: ASTM E1527-00 - Standard Practice for Environmental Site Assessments: Phase 1 Environmental Site Assessment Process
• Effective Date: 10-May-2000
• Refers: ASTM E2081-00 - Standard Guide for Risk-Based Corrective Action
• Effective Date: 10-Apr-2000

Overview

ASTM E3155-19: Standard Guide for Assessing Mammal Health at Chemically Contaminated Terrestrial Sites Using Rodent Sperm Analysis provides a practical, field-based approach for evaluating the health status of resident mammal populations at contaminated terrestrial sites. Unlike traditional ecological risk assessments (ERAs) that rely on laboratory studies or desktop hazard quotients (HQs), this standard leverages direct analysis of sperm quality in wild rodents as a sensitive indicator of reproductive and overall mammal health. By focusing on actual site-exposed animals, the RSA (Rodent Sperm Analysis) method offers risk managers and site stakeholders a realistic, biologically-relevant assessment tool to inform remedial decisions and resource allocation.

Key Topics

• Direct Health Assessment: The standard describes procedures for obtaining and interpreting data from site-exposed small mammals, reporting on reproductive health indicators like sperm count, motility, and morphology.
• Sensitive Reproductive Endpoint: RSA targets reproductive success as a critical toxicological endpoint, valuable for its ecological significance and the availability of well-established sperm parameter benchmarks.
• Comprehensive Exposure Evaluation: Unlike methods limited to ingestion, the RSA method reflects the effects of all relevant exposure routes - ingestion, inhalation, and dermal contact - as well as the combined impact of multiple chemical contaminants.
• Field and Laboratory Methods: The guide details tasks required in both the field (rodent trapping, specimen collection) and laboratory (sperm analysis), suitable for on-site mobile labs, traditional labs, or makeshift field workspaces.
• Practical and Cost-effective: By establishing the presence or absence of demonstrated reproductive impacts in maximally exposed populations, RSA can prevent unnecessary site remediation efforts, offering significant cost savings.

Applications

ASTM E3155-19 is primarily intended for use at:

• Historically Contaminated Sites: Such as Superfund, brownfield, or legacy industrial sites where ecological receptor health is a concern.
• Any Terrestrial Property with Small Mammals and Soil Contamination: The methodology is broadly applicable as long as small rodents (mice, rats, voles, squirrels) are present and can be ethically collected for assessment.
• Sites with Mixed or Unknown Chemical Profiles: RSA is robust for contexts where multiple chemical stressors or a lack of specific spermatoxins are suspected.
• Risk Management and Remedial Decision Support: RSA supplies direct evidence of ecological health, helping regulators, site managers, and risk assessors decide if further action is necessary or if ecosystem health is uncompromised.
• Supplement to Desktop Risk Assessments: By confirming whether predicted risks from modeled exposure pathways correspond to actual effects in site biota.

Related Standards

The RSA guide is not itself a complete ERA method but is intended to supplement and inform broader ecological risk management frameworks. Related ASTM standards that may be referenced alongside ASTM E3155-19 include:

• ASTM E1527: Standard Practice for Environmental Site Assessments: Phase I ESA Process
• ASTM E1689: Guide for Developing Conceptual Site Models for Contaminated Sites
• ASTM E1848: Guide for Selecting and Using Ecological Endpoints for Contaminated Sites
• ASTM E2081: Guide for Risk-Based Corrective Action
• ASTM E2205: Guide for Risk-Based Corrective Action for Protection of Ecological Resources
• ASTM E2616: Guide for Remedy Selection Integrating Risk-Based Corrective Action
• ASTM E2790: Guide for Identifying and Complying With Continuing Obligations

These documents collectively address various aspects of contaminated site assessment, risk evaluation, endpoint selection, and remedial strategy, making the RSA approach part of an integrated environmental management toolkit.

---

Keywords: ASTM E3155-19, rodent sperm analysis, mammal health assessment, chemically contaminated sites, reproductive toxicology, ecological risk assessment, site remediation, small mammal bioindicator, terrestrial contamination, environmental standards.

Standards Content (Sample)

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.
Designation: E3155 − 19
Standard Guide for
Assessing Mammal Health at Chemically Contaminated
Terrestrial Sites Using Rodent Sperm Analysis
This standard is issued under the fixed designation E3155; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Contaminated terrestrial properties provide opportunities for determining whether or not toxico-
logical responses of concern have arisen in ecological receptors contacting them. The guide,
employing direct health status assessment of rodents captured at contaminated sites, can furnish
information to support such determinations, thereby providing a greater degree of realism in health
assessmentsforresidentmammalsthanthatofferedingenericdesktopassessments.Thisguide’sdirect
health status assessment design involves only wild-type animals in their natural state, with this
arrangement circumventing a commonplace species extrapolation element, one that introduces a
considerable degree of uncertainty in ecological risk assessments (ERAs). Of note, the guide is not
itself a terrestrial ERA method, but rather an additional tool to inform such efforts. In the area of
notatingsignificanteffects,theguide’sbiologicalthresholds-for-effectreplacearbitrarilyornegotiated
differences, or both in response that are only assumed to be biologically meaningful (for example, a
20% decrement in a measure), and that typically are not confirmed in field studies (1). Due to the
availability of the thresholds-for-effect, the guide allows for bright-line determinations to be made,
creating an opportunity to supply a high level of conservatism to outcomes (see 9.4). Importantly,
Rodent Sperm Analysis (RSA) is distinctly different from others in concept and practice because it
aims to make yes/no determinations (that is, that reproductive impact has occurred or that it has not)
as opposed to generating estimates of the likelihood of certain toxicological outcomes arising in the
future. RSAprovides a useful line of evidence for ERA, wherein mammal reproduction is a common
endpoint, and it may simplify remedial decision-making for contaminated terrestrial sites.
This guide, notably removed from testing with laboratory-reared, commercially available, and
chemicallynaiveorganismsthataresubjectedtobriefchemicalexposuresincontrolledenvironments,
is predicated on a fundamental underlying premise consistent with the field condition considered (2,
3). It is recognized that sufficient time has elapsed at contaminated sites for toxicological effects of
concern to have been elicited. Given that typical sites are minimally 30 years old by the time they
submit to ERA (2-8), the described standard understands that if critical effects (here, reproductive
impacts) have not appeared over such a time course, they are unlikely to ever occur. Addressing
mammals, one of only two tetrapod classes evaluated in ERA(ClassAves is the other), the guide has
three unique features that set it apart from conventional ecological assessment tasks. First, the
proceduredirectlyassesseshealthbyevaluatingtheactualanimalsthatinhabitcontaminatedsites.(Of
note,commontoRSAandthedesktop-basedriskcharacterizationsofmanyconventionalERAs,isthe
defining of overall mammal health through the key biological function of reproduction.) Second, the
procedure considers the three chemical uptake routes of ingestion, inhalation, and dermal contact,
whereas conventional ERAs only report hazards for the first of these. The procedure’s third unique
feature is its ability to evaluate the effects of chemical mixtures, whereas the conventional hazard
quotient (HQ)-reliant ERAprocess can only review chemicals singly. The procedure is supported by
the existence of established sperm-based barometers of reproductive capability/success for the rodent
grouping. In contradistinction, for most other current biological measures that can be collected in the
laboratoryorfield(forexample,anenzymelevel,sizeofaninternalorgan),itisnotknownhowmuch
of a contaminated site-mediated sublethal change signifies health compromise (3, 9).
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3155 − 19
1. Scope example, with respective home ranges of 400+ and 640 acres
for the red fox and white-tailed deer (10-14), these species
1.1 This guide describes the procedures for obtaining and
wouldspendminimalamountsoftheirtime(forexample,5%)
interpreting data associated with a direct health status assess-
at prototypical contaminated sites that cover areas of 25 acres
ment for mammalian receptors at chemically contaminated
or less (15, 16). Where one or more sperm parameter bench-
terrestrial sites where ERA work is either scheduled or
marks are exceeded in contaminated-site rodents (certainly
ongoing, and irrespective of the number and type of chemicals
indicating that the rodents are reproductively compromised),
that may be present. Through reviewing sperm features, the
other site mammals may also be reproductively compromised.
RSAmethodreportsonthereproductivehealthofmalerodents
Thegreaterthedisparitybetweenthehomerangesofthetarget
in their natural environmental settings, with these animals
species (that is, the site rodent) and any of the other mammals
serving as surrogates for other (and larger) site mammals (4).
known to contact the contaminated site in question, the less
1.2 These procedures are applicable at any terrestrial prop-
likely it will be that the latter are reproductively compromised.
erty that supports small mammals (for example, mice, voles,
TheRSAmethodemploysthesametoxicologicalextrapolation
rats, squirrels) and has contaminated soil. Importantly, chemi-
principles as that used for mammals in conventional desktop-
cals of concern in site soils need not be spermatoxins.
based ERAs. In those ERAs, stressor-mediated responses of
Additionally, the RSAmethod considers that any combination
rodents (of a laboratory-based study) assist with the interpre-
of chemicals or other site stressors might collectively act to
tation of health effects for an expanded list of mammals that
compromise reproduction, held to be a sensitive toxicological
cannot conveniently be evaluated directly for health status (for
endpoint for mammals. The anticipated primary application of
example, fox, skunk, raccoon, deer, coyote, etc.).
the method will be at historically contaminated sites (such as
1.5 This guide is arranged as follows:
Superfund sites). The procedures describe tasks conducted in
Section
the field and in a laboratory. For the latter, tasks may be
Scope 1
conducted either in an on-site mobile laboratory, or in a more
Referenced Documents 2
conventional laboratory setting. For certain tasks, a make-shift
Terminology 3
Summary of Guide 4
work space may be suitable as well (see 7.3).
Significance and Use 5
Safety Precautions 6
1.3 Initial determinations of compromised or non-
Apparatus 7
compromised reproduction in resident male small rodents are
Procedure 8
made through a cautious comparative review of sperm param-
Reporting 9
Keywords 10
eters. Briefly, for the rodents of a given species collected at
both a contaminated site and a habitat-matched (non- 1.6 This standard does not purport to address all of the
contaminated) reference location, arithmetic means are first
safety concerns, if any, associated with its use. It is the
computed for each of the three sperm parameters of count, responsibility of the user of this standard to establish appro-
motility, and morphology. If one or more of the parameter
priate safety, health, and environmental practices and deter-
means of the contaminated site rodents reflect an unfavorable mine the applicability of regulatory limitations prior to use.
shift (that is, count or motility is less than that of reference
1.7 This international standard was developed in accor-
location animals; the percentage of abnormally-shaped sperm
dance with internationally recognized principles on standard-
is greater relative to reference location animals), the percent ization established in the Decision on Principles for the
decrease or increase in each mean is compared to the relevant
Development of International Standards, Guides and Recom-
established sperm parameter benchmark, each in the form of mendations issued by the World Trade Organization Technical
that degree of shift in an unfavorable direction that signifies
Barriers to Trade (TBT) Committee.
lesser reproductive success (2) (see 9.3).
2. Referenced Documents
1.4 Advanced determinations of compromised or non-
2.1 ASTM Standards:
compromised reproduction in larger site-contacting mammals,
E1527PracticeforEnvironmentalSiteAssessments:PhaseI
the true focus of the RSA method and this guide, are made
Environmental Site Assessment Process
through an applied spatial movements-based extrapolation
E1689Guide for Developing Conceptual Site Models for
scheme.Whereestablishedspermparameterbenchmarkexcee-
Contaminated Sites
dances are not observed in contaminated-site rodents, other
E1848Guide for Selecting and Using Ecological Endpoints
mammals contacting a site are also assumed to have non-
for Contaminated Sites
compromised reproduction. This follows from the latter all
E2081Guide for Risk-Based Corrective Action
having notably lesser degrees of site exposure due to home
E2205Guide for Risk-Based Corrective Action for Protec-
ranges that are vastly larger than those of rodents. By way of
tion of Ecological Resources
E2616Guide for Remedy Selection Integrating Risk-Based
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental Corrective Action and Non-Risk Considerations
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Jan. 1, 2019. Published February 2019. DOI: 10.1520/ For referenced ASTM standards, visit the ASTM website, www.astm.org, or
E3155–19 contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
E3155 − 19
E2790Guide for Identifying and Complying With Continu- promised health. In stark contrast to this arrangement, for the
ing Obligations three classic sperm parameters monitored in laboratory animal
trials (just as they are in human fertility clinics), it is rather
3. Terminology
precisely known how much of a change (for example, reduc-
3.1 Definitions of Terms Specific to This Standard:
tion in sperm count) signifies a compromised reproductive
3.1.1 cauda epididymis, n—that portion of the male repro-
state. This guide describes (a) necessary planning steps for
ductive anatomy in mammals where sperm are stored.
employing the RSA method, (b) details for conducting the
method, involving small rodent trapping, recording of field
3.1.2 sperm count, n—the number of sperm cells in a
data, and the recording of sperm parameters, and (c) analysis
standard preparation expressed as millions of sperm per gram
and interpretation of the collected data. RSAparallels conven-
of cauda epididymis.
tional desktop ecological assessments, in that overall health is
3.1.3 sperm morphology, n—for the purposes of assessing
defined through a review of reproductive capability. If repro-
male reproductive health, the measure of broken, bent, and
duction is not compromised, any chemical exposure-posed
other damaged sperm cells in a standard count of 200 cells,
somatic changes that might be occurring (for example, an
expressed as a percent.
enlarged internal organ), are deemed inconsequential, for they
3.1.4 sperm motility, n—the percentage of moving sperm in
are not impeding a species from surviving and perpetuating its
a properly prepared sample, also known as the “total motile
own (2, 3).TheRSAmethodprovidesausefullineofevidence
measure”.
to assist reproductive capability-based health determinations
3.1.5 target animals, n—adult male rodents of a species
for terrestrial mammals evaluated in ERAs for contaminated
found at both a contaminated terrestrial site of interest, and a sites.
nearby, habitat-matched reference location.
5. Significance and Use
4. Summary of Guide
5.1 The RSA method provides risk and resource managers
4.1 RSA is a direct health status assessment method for with an enhanced understanding of the ecological health
select mammals potentially occurring at contaminated terres- concerns at the sites they oversee because unlike conventional
trial sites, that is, it assesses the reproductive health (and terrestrial ERAs, actual site mammals are the ones evaluated.
thereby, overall health) of animals whose habits do not allow Additionally, the HQs of desktop efforts report only on the
for their moving beyond their site boundaries. It evaluates
contaminantexposurerouteofingestion,andcanonlyevaluate
those mammals that are maximally exposed in the sense of chemicals singly, whereas RSAfindings reflect all three expo-
having the greatest degree of direct soil contact. By way of
sure routes as well as the combined effects of multiple
example, many species of small rodents have home ranges of chemicals on a highly valued endpoint. Critically, the RSA
one acre or less (12). While some might consider mammals methodincorporatessitehistoryconsiderationsthatnecessarily
bearing relatively higher chemical body burdens than others to influence the phenomenon of biological response. If reproduc-
be those who are maximally exposed, there are numerous tiveimpactsatcontaminatedsiteswereevertobeelicited,such
reasons to not apply such reasoning with regard to this guide. would be apparent today because evaluated sites have, at a
Briefly, body burden information is almost never collected for minimum, continuously exposed their ecological receptors to
mammals other than rodents, and there are no databases that chemicals for multiple decades during which time tens and
relate body burdens in mammals to health effects they might often more than one hundred generations have passed (5).
experience. Importantly, there is no way to relate body burden
5.2 Application of the subject guide familiarizes remedial
to the sperm parameter thresholds-for-effect that the RSA
decision-makers and risk managers with two concepts. First,
method utilizes, which unlike other somatic measures, allow
rather than attempting to predict health effects arising in site
for assessment of reproductive system health. Finally, due to
receptors, there may be more value in documenting demon-
the notably reduced densities of larger mammals [for example,
strated health effects, should such exist in actual site-exposed
red fox at 0.019 - 0.03 animals/acre; (10-13)], these species
mammalian receptors. Second, the possibility exists that site
maynotbespatiallyrelevantinanERAcontextaltogether(that
receptors never experienced stress or impact over the years
is, at best, only individuals as opposed to populations might
since a site first became contaminated.
bear chemical effects, and ERAs are to assess the latter). RSA
5.3 Application of the subject guide can allow for substan-
also assesses reproduction as a toxicological endpoint of great
tialcostsavings.Often,theoutcomesofHQ-basedassessments
concern in ERA, one which is potentially the most sensitive of
aresummarilyreliedupontoconductongoingstudies,monitor
endpoints for mammals. RSAcapitalizes on the availability of
sites, or implement site cleanups, all of which may be
an information type that hardly exists in the toxicological
unnecessary. Where RSAapplications should demonstrate that
testing realm, namely proven biological thresholds-for-effect,
maximally site-exposed mammalian receptors (as defined in
and specifically sperm parameter-based thresholds that are all
section 4.1) are not experiencing compromise with regard to
barometers of reproductive success (2, 9) (see 9.3.1). For
the sensitive endpoint of reproductive success, it can become
virtually any biological datum that can be collected (for
apparentthatsoilremediationeffortsonbehalfofmammalsare
example, a decrease in an enzyme level), second-order toxi-
not needed.
cology information is lacking (2, 8). Thus, where sublethal
effects are an interest, it is not known how much of a somatic 5.4 The described RSA method can typically be applied at
change in chemically exposed/treated animals signifies com- that point in the ERA process where HQs for one or more
E3155 − 19
mammalian species are found to be greater than 1.0, as in the reproduction proceeding normally (2, 3). Where such is the
process’sStep2(Screening-LevelExposureEstimateandRisk case, the method’s supporting theory understands that other
Calculation; where ecological threats are evaluated in a endpoints being reached do not necessarily pose a concern for
general, as opposed to a specific fashion). Alternatively and they have not impeded the ability of maximally exposed
particularly at sites that are not governed as rigidly as, for rodents to survive to the age of reproduction, find mates, and
example, Comprehensive Environmental Response, produce viable young (2, 18).
Compensation, and Liability Act (CERCLA; aka Superfund-
5.7 This guide recognizes that an analagous reproductive
type) sites, the guide can be applied once it is established that
assessment approach for female rodents, is not available at the
asitehasachemicalcontaminationfootprintofinterest(thatis,
present time. Importantly, an absent reproductive assessment
that soil concentrations are high enough to potentially be
approach for females does not constitute a shortcoming of the
harmful to mammalian site receptors). In light of the propen-
subject guide. Relevant U.S. EPA guidance, for example,
sity for preliminary and refined HQs to suggest mammals are
supports evaluating one sex of a species where drug and
ingesting unhealthful doses of site contaminants, in turn
chemical regulation is concerned, and drawing conclusions
commonly leading to advancing to the field for a verification
based on such information (19). In this context several note-
effort, the application of RSA as a first evaluative effort is
worthy points follow. First, over 98% of all mammalian
intended to be a time- and cost-saving effort.
toxicity studies considered in crafting the U.S. EPA’s Soil
Screening Levels (SSLs) for ERA (for some 17 inorganic and
5.5 The significance of this guide is the method design that
4 organic chemical species) are of the single-sex type, with
reflects an understanding of certain unavoidable ERA process
35% of the studies being male-only (20). Additionally, for
constraints,specificallyintheareasoffieldmammalcollection
37% of the universe of chemicals with SSLs, the number of
and subsequent tissue analysis. First, the RSA method recog-
male-only toxicity studies exceeds the number of female-only
nizes that small rodents are the only mammals that can be
toxicity studies. Finally, a significant percentage of the most
routinely culled from the field (that is, to be removed and not
commonly applied toxicological benchmarks for wildlife (21)
returned), and further, that this reality is unlikely to ever
derive from single-sex studies. Critically, with its focus on
change. Efforts to regularly harvest larger mammals (for
directlyassessingreproductioninmalerodents,RSAisnotably
example, fox) may be challenged by local governing agencies
far less destructive than would be a method involving the
and animal care institutions. Additionally, acquiring a suffi-
cullingoffemalerodentsfromthefield,giventhatthelatterare
ciency of larger mammals is time-consuming and labor-
the ones that bear the young.
intensive, owing to relatively miniscule animal densities.
Further, many larger mammals (for example, long-tailed
5.8 This guide recognizes the value in employing the wild
weasel, badger) are not found in all habitats or in all states. In
rodent in field-based mammalian receptor assessment. Aside
contrast, small rodents occur in virtually every habitat, are
from the reality that rodents may constitute the only mammals
relativelyeasytocollect,andarenumerousenoughtoallowfor
that can regularly be culled from sites (discussed above), there
defensible comparisons between or among sites. In selecting
are key advantages that accrue to working with these animals.
the maximally exposed small rodent to work with (that is, an
Small rodents occur in nearly all terrestrial habitats, allowing
animal confined to contaminated surroundings throughout its
the guide to be broadly applicable in a geographical sense. A
life due to a home range that is almost always of one acre or
second advantage is that the small rodent with perhaps no
less), the RSAmethod features a common basis of comparison
exception, will likely be the maximally-exposed mammal in
(and certainly wherever it should be applied in the United
terrestrial settings, this again, in terms of having direct contact
States).
with contaminated soils. This follows from rodents being
non-migratory in nature, having extremely limited home
5.6 RSAtheory understands that, generally at contaminated
ranges that effectively contain them at contaminated sites, and
terrestrial sites, there is worry that receptors-of-concern might
their spending nearly all of their time directly contacting the
be reproductively compromised. The focus on reproduction as
ground (that is, contaminated soils; 2, 4, 18).
the dominant toxicological endpoint of concern (6, 7), recog-
nizes that much method development for reproductive effects
5.9 In providing a useful line of evidence in support of
in rodents (in support of human health) has occurred (9, 17). ERAs for mammals, this guide employs a straightforward
Thatreproductionbearsthisstatusisevidentinthehierarchyof extrapolation approach (2, 18), one that is isomorphic to that
preferred toxicity reference values (TRVs) that ecological risk applied in conventional HQ-based assessments. If site rodents,
assessors often select in support of HQ computation. Addi- that have more constant and intimate contact with affected site
tional recognition is given to the reality that standardized soils than that of any other site mammal, are not found to have
means for effectively assessing other endpoints of interest in compromised reproduction, larger and wider-ranging
field-collectedorganisms,suchasneurotoxicityorbehavior,do mammals,withtheirconsiderablylesserdegreesofsite(thatis,
not exist. Where established sperm parameter benchmark contaminated soil) contact, should also be free of reproductive
exceedances are not observed in contaminated site rodents, compromise. An appreciation for this extrapolation scheme
such can constitute a significant line of evidence in support of derives from a review of the principal extrapolation scheme of
a determination that reproduction is proceeding adequately. conventionally-applied desktop-based ERAs. There, a
TheRSAmethodrecognizesthatimpairmentstootherbiologi- laboratory-based mouse or rat study is routinely used to
cal functions (for example, behavior, nerve impulse transmis- determineifanothermammal(forexample,deer,fox,rabbit)is
sion) of contaminated-site rodents may be occurring despite ingestinganunhealthfulquantityofagivenchemical.Withthe
E3155 − 19
conventional ERA scheme, there are numerous differences to (if that endpoint was ever to be triggered), this guide is
acknowledge, and even at the level of the rodent.Thus the test designed to document such demonstrated compromise.
animal and the wild form inhabiting the site of interest that is Critically, RSA is not a risk assessment method that aims to
to be assessed, do not match in terms of species, rearing, forecast or predict health effects arising in mammals with
environment/habitat, or feeding design, and these differences ongoing contaminant exposures. The guide then is related to,
weaken conclusions that can be drawn. In contrast, the subject butdistinctlydifferentfromotherASTMstandardsthatbearon
standard in its initial extrapolation, compares sperm measures, the toxicological effects prediction aspect of ERA (Guides
each a proven barometer of reproductive success (22-25),in E1527-13, E1689, E1848-96, E2081, E2205-02, E2616, and
populations of conspecifics living less than a kilometer apart, E2790). The guide is also consistent with guidelines for
with one population inhabiting a soil-contaminated area, and reproductive toxicity risk assessment as per the U.S. EPA (19).
the other a contaminant-free one. The RSAmethod recognizes Specifically, assessing the reproductive health of only one sex
that small rodents of contaminated sites are integrators of of a species is deemed adequate for an overall species
potentially imposing environmental stressors that extend be- assessment (17). In one key area however, this guide is quite
yond chemicals that may be present in soil and diet items, to unlikeconventionalERAsthatarelargelyrestrictedtothelevel
include such things as physical habitat disturbances (for ofdesktopanalysis.Whereasconventionalassessmentsrelyon
example, noise or land vibration). RSA understands that either statistically-significant differences in outcome, or on a
conventionalecologicalassessmentsnecessarilystrivetoknow commonly negotiated difference in biological response (for
ofsmallrodentreproductivecapability,asthisgroupingisheld example, 20%) when drawing conclusions, this guide primar-
to be a keystone ecosystem element. Where reproductive ily avails itself to the utility of a series of established
compromise is not observed in an RSA outcome, there is biologically-significant thresholds alluded to previously (22-
demonstration that a site’s exhaustive list of site stressors, in 25). Further, a statistical comparison need only be applied for
the actual arrays in which they occur, are not impinging on one of two possible RSA outcomes (see 9.3.1 and 9.4).
what is generally held to be the most important toxicological
6. Safety Precautions
endpoint.
6.1 The most serious potential danger to individuals in-
5.10 One limitation of this guide is that the biologically-
volved with RSA duties is that of becoming infected with
significant thresholds-for- (reproductive)-effect that are
rodent-borne disease (for example, Hantavirus or plague).
applied, are laboratory-derived. A second limitation of this
Many rodents in the wild, even at locations far removed from
guide is that shrews generally cannot submit to the RSA
areas where certain rodent-borne diseases are known or ex-
method, owing to their exceedingly high metabolism that
pected to occur, may carry the virus. With multiple opportuni-
interferes with their being live-trapped in the field. In the rare
tiestobecomeinfectedthroughhandlingrodents(forexample,
case where the only rodents present at a contaminated site of
wherefieldandlaboratorypersonnelhavebreaksintheskinon
concern should be shrews, the RSA method can probably not
the hands), certain safety procedures must be implemented.
besuccessfullyapplied.Ifforanyreasonagivencontaminated
Importantly, the specter of the presence of infected rodents in
site does not offer a small rodent population altogether, or if
the field where RSA is being applied, does not make the
thereisnotatleastonecommonsmallrodentspeciesoccurring
implementation of the safety procedures provided below any
at both the site of interest and a suitable habitat-matched
more burdensome. All individuals checking live animal traps
reference location, or an appropriate reference location cannot
for captures must wear gloves, and must hold the traps
be found (see 8.1), the method is not applicable. RSA is
down-wind and at arm’s length. Traps with adult male rodents
intended only to identify if site mammals are reproductively
to be assessed, must be conveyed to the laboratory by truck
compromised. The method does not concern itself with iden-
(andnotbycar),withthetrapsinaseparatecompartmentfrom
tifyingthechemical(s)orphysicalsitestressorsresponsiblefor
the driver and any other passengers. Individuals euthanizing
observed sperm parameter threshold-for-effect exceedances, or
rodents and doing dissections must wear surgical gloves and
the determination of cleanup levels, and such are not method
conduct all such work under a fume hood. Alternatively, a
limitations. The situation is analogous to standardized whole
powered air-purifying respirator (PAPR) unit may be worn
effluent toxicity tests conducted with various aquatic test
when conducting such activities.
species (for example, Fundulus sp.). There, the objective is
only to ascertain if the degree of wastewater treatment is
6.2 Animal carcasses and remains, other than those that are
adequate to support the aquatic life inhabiting a receiving
to be preserved for species identification, must be disposed of
waterbody’s mixing zone. (Standard whole effluent toxicity
in red plastic biomedical waste bags at a facility licensed to
testing is not designed in the main, to identify the constituent
handle such waste. Animals to be identified to species should
or constituents in effluent that may be responsible for unac-
be preserved in accordance with the stipulations of the agency
ceptable test outcomes.)
or institution overseeing the RSA application (for example,
placinginformalin)untiltheyareturnedovertotheinstitution
5.11 This guide is consistent with ERA guidance and
where a mammalogist will examine the specimens.
guidelines (26, 27), where advancing to the field for an
environmentally relevant assessment of the health of site 6.3 Disposable scalpel blades must be thrown away in a
receptors (so-called ‘field verification’) is a recognized formal biomedical waste/sharps container. Surgical tools (forceps,
step. In understanding that sufficient time has elapsed at scissors) must be cleaned with alconox or a similar biocide
contaminated sites for reproductive compromise to be evident after each use.
E3155 − 19
7. Apparatus 8. Procedure
8.1 Site Selection—The RSAmethod necessitates a prelimi-
7.1 Sperm Analyzer Equipment—The essential apparatus
narysitevisittoallowindividualswhowillbeconductingRSA
needed for RSA is a sperm analyzer (22). A fully automated
tasks in the field to gain a familiarity with the site environs.
sperm analyzer, although not absolutely essential, is highly
8.1.1 The site visit should accomplish the following:
recommended given the accuracy in reporting that it can
8.1.1.1 Ascertaintheboundariesoftheaffectedsite;identify
provide(followingforexample,fromitsabilitytodiscriminate
specific areas at which to place animal traps.
between non-moving or dead sperm and other debris that may
8.1.1.2 Identify two or more nearby suitable reference
appear on a prepared slide) and its rapidity of reporting. An
locations, sufficiently distanced from the contaminated site to
automated sperm analyzer is essential however, where sperm
preclude the possibility of a given small rodent occupying/
motility is to be monitored. Such equipment has the capability
contacting both a contaminated site and its corresponding
to accurately record the percentage of moving sperm in a
reference location. A proper reference location will have
microscope slide prepared within 2-3 minutes post-
multipleecologicalaspectsmatchingthoseofthecontaminated
euthanization. A much-recommended but not absolutely re-
site(s)tobeinvestigated.Invariably,referencelocationfeatures
quired sperm analyzer feature where sperm motion is to be
cannot help but match those of contaminated sites, given that
assessed,isanadjustable,heatedmicroscopestage.Ideallythis
they ideally occur as little as 0.40 km away from sites. In that
stage is set to 37-38°C, matching both the temperature at
case, the atmospheric and geologic conditions (for example,
which sperm are maintained in the rodent body, and that of a
temperature, humidity, precipitation, soil type) of site and
Petridish“swim-out”preparationdescribedbelow(seeSection
reference will undoubtedly be the same, and such should be
8, Procedure).
field-verified nevertheless. Other ecological aspects to review
in the field to further establish a high degree of parity of sites,
7.2 SamplePreparation—Atissuehomogenizerisnecessary
and to establish overall reference location suitability include
foranypreparationmadefromthecaudaepididymistobeused
vegetative cover (degree, cover type), elevation, drainage, and
in determining the sperm count, that is, the most important
biota/species lists.
sperm parameter to track with RSA applications (see 8.4.2).
8.1.1.3 Identify a practical and desirable location for de-
One successful method for counting sperm involves placing a
ploying a mobile on-site laboratory (if one is to be used) or
100 µL aliquot of homogenate of the cauda epididymis into a
identify a building room where animal euthanization and
prepared commercially available snap tube that contains a
method work-up can proceed, or both.
fluorescent dye (for staining the DNAin the sperm heads).An
8.1.2 Otherrelatedinformationtoassimilateinreadinessfor
aliquot of the thoroughly reacted sample in the tube is
RSA application include:
transferred to a microscope slide, which is then placed inside
8.1.2.1 Establish the probable small rodent species list for
the automated sperm analyzer for the count determination.
the areas to be animal-trapped.
7.3 Animal Housing—For a multitude of reasons, a mobile
8.1.2.2 Identify the nearest hospital and the quickest travel
laboratory equipped with a fume hood is strongly recom-
route from the study site to the hospital.
mended for euthanizing animals and for harvesting organs for 8.1.2.3 LocateanearbyvendorforaCO tankandregulator
thevariousmeasurementsandpreparationsthatmakeupRSA. gauge if the agency or institution overseeing the RSAapplica-
tion stipulates that euthanization is to be by asphyxiation with
[It is unlikely that an animal care/handling facility would be
conveniently located in close proximity to the contaminated inhaled CO .
8.1.2.4 Locate a nearby vendor of oats or horse feed to be
sites and habitat-matched reference locations where RSA
used when baiting animal traps.
applicationswouldproceed.Evenifafacilitywouldbelocated
8.1.2.5 Locate a nearby certified facility at which animal
relatively nearby the animal trapping locations, it is unlikely
waste (carcasses) and medical waste (sharps) can be disposed
that the facility would be agreeable to sheltering wild rodents,
of.
for these might carry diseases and other vectors that can
8.1.3 It is recommended that the preliminary site visit occur
potentiallyimperilin-housecolonies.]Arelativelyinexpensive
shortly before the actual RSA field work commences, so that
but highly serviceable mobile laboratory can be made by
site familiarity (for example, recognition of landmarks) is
retrofittingautilitytrailersuchthatthecargoareahaselectrical
maintained. Of note, an area in spring or summer could look
power (to supply fluorescent lighting, air conditioning if
vastly different in the fall or winter.
needed,andrunallmethod-relatedequipment),andhasawork
bench installed to support essential equipment (for example,
8.2 Animal Trapping—Although animal trapping at a con-
the sperm analyzer, portable fume hood, analytical scale,
taminated site of interest and a matched (non-contaminated)
vortex).Forventingtheairaboveadissection,anexhausthose
referencelocationcanoccurconcurrently,itisadvisabletotrap
can be directed from the area inside the fume hood to an
first at the contaminated site. This rule recalls that it is the
opening in the trailer’s roof. If a mobile laboratory such as is
rodentspeciesthatreside(s)atthecontaminatedsiteofinterest
described here is not available, a next choice is a room in a
whose reproductive health needs to be assessed, and critically
building that ideally has a fume hood to facilitate safe
this must be established early on. Pre-selected reference
dissections and organ harvesting. In the absence of a fume locations therefore will only be demonstrated to have been
hood, all technicians handling small rodents must wear PAPR
viable ones for RSA purposes when it is evident that they
units. support the same species that occur at the contaminated site of
E3155 − 19
interest.[Inthecaseofverylargesites,RSAapplicationcanbe datasets reveals that with 15 adult males, a 60% reduction in
simplified by intending to trap at the more/most contaminated sperm count relative to the reference location animals can be
detected with statistical power ranging from 80 to 99% (6).A
portions. Additionally, where multiple sites may need to be
trapped, it could be that a singular reference location will review of all RSA datasets also reveals that detecting a 60%
reduction in sperm count with a statistical power of 80% can
suffice.] Live animal traps should be set out in a saturated
be achieved with as few as 3 to 4 animals.
scheme (that is, many traps within a relatively small area), and
to the extent practicable, with the same array used at both the
8.3 Animal Capture Numbers—The desired adult male cap-
site and reference location. A recommended array for either a
ture figure of 15 animals (for one species) is not an absolute
site or reference location has four 100 × 100 m grids set out,
method catch number; comparisons of contaminated site and
with 100 traps aligned in 10 parallel rows of 10 traps within
reference location population means for the three sperm
each grid. Two of the four grids at a location (either contami-
parameters can still be workable with lesser numbers (for
nated site or reference location) should be trapped for three
example, 8-10). Additionally, lesser capture numbers may
consecutive nights (unless requisite capture numbers occur
likely reflect the reality that nearly the entire male population
sooner than this), after which the remaining two grids are
has been trapped at a given locality. Such can be confirmed
trappedforanotherthreenightsoruntiltherequisitenumberof
when reviewing animal capture data over the latter trapping
animals are obtained. Traps should be spaced 10 m from each
daysofanRSAapplication.Specifically,anobservedreduction
other in both the horizontal and vertical rows. Traps should be
intheadultmalecapturerate,andaconcomitantincreaseinthe
set (that is, with spring-loaded doors in the down position) in
number of recaptured animals (that is, fur-clipped juveniles or
the late afternoon, and appropriately baited for the rodents of
females, or both) suggests that most of the adult males in the
the locale where the field work is being conducted. A small
vicinity have been already culled. If after a week’s worth of
cotton ball should be placed inside the trap to provide comfort trapping at a designated area (contaminated site or reference
to captured rodents. Traps should be checked for captures at
location), less than 3 adult males of a given species have been
daybreak to minimize thermal stress to animals (particularly captured, all future captures of adult males of that species
forthoserodentsthatweretrappedshortlyaftertrapswereset). shouldbereleasedtothefieldatthepointofcapture.Withsuch
Individuals checking traps must wear gloves, and traps should low capture success, it is unlikely that a sufficient number of
be held down-wind and at arm’s length for the initial assess- target animals could be collected to allow for a valid compara-
tive sperm parameter analysis.
ment.Trappedanimalsatthepointofcapturearetransferredto
transparent, re-sealable bags (with a thickness ranging from
8.4 Sperm Parameter Measurements:
approximately 1.43 to 1.75 mil) for identification to species,
8.4.1 Sperm Motility—While a vetted reproductive compro-
sex, and age (see 6.1). General health condition should also be
mise threshold-for-effect exists for sperm motility, exhaustive
recorded in a field notebook. For females, a determination of
reviews of RSA outcomes reveal that this threshold is never
pregnant, lactating, or neither of these conditions should be
approached. Further, in almost all cases, contaminated site
made. All captures are weighed in the plastic bag using an
population motilities are observed to be numerically (but not
appropriate scale. All captures other than adult males should
statistically) greater than those observed in corresponding
have a small patch of fur on the back removed with a scissors
reference location populations. It has consequently been
(so as to circumvent the double-counting of animals over what
opined that this regular occurrence might be reflective of a
may be as long as a two-week trapping period), after which
compensatory mechanism at play (4). That is, since a reduced
they are released in the field at the point of capture. Adult
sperm count has the potential to compromise reproduction
males to be assessed are transferred back to their traps and
(although a bright line threshold-for-effect exists to declara-
conveyed by truck to the onsite or indoor laboratory after all
tively identify instances of such impact and typically it is not
grids have been checked for captures. Over the course of the
exceeded), seemingly it can only be beneficial to have a
field effort, the desirable goal is to capture at both the site and
somewhat boosted motility in such instances. While a motility
its matched reference location, 15 adult males (aka target
reviewmayprovidecorroborativeinformationforagivenRSA
animals)ofatleastonecommonlyoccurringspecies.Thereare
application,collectingmotilitymeasuresisoptional;itisnotan
several reasons for this desirable target animal number. First,
indispensable barometer of reproductive success to track. It is
this is the number of animals that are routinely used in U.S.
criticaltonotethatassessingspermmotilitycan,dependingon
EPA laboratory test trials with rodents (17). Second, it is not
circumstance, substantially increase RSA costs, as follows.
the intention to decimate rodent populations while conducting
Tracking sperm motility necessitates an automated sperm
RSA; removing more than 15 adult males at areas at which
analyzer (28), an expensive piece of equipment, and the
grids are placed (described above) could severely impact local services of a trained technician who can rapidly arrange the
reproduction. Third, site-specific conditions (for example,
delicately and rather precisely-timed tissue preparation (de-
locale, species-specific densities) might not allow for as many scribed immediately below), and efficiently operate the sperm
as 15 adult males to be trapped over a gridded area. Finally, a
analyzer. Small rodents are euthanized in the laboratory ac-
sample size of 15 animals provides assurance that for the most cording to the specifications of the overseeing agency or
importantofthethreespermparameterstrackedwithRSA(that
animalcareinstitution(forexample,withcervicaldislocation).
is, sperm count), the method’s threshold-for-effect of a 60% Where CO asphyxiation is to be used, a rodent is first
count reduction relative to the condition for the reference transferredfromitstraptoatransparent,re-sealableplasticbag
location, can be detected. A review of all RSA application (with a thickness ranging from approximately 1.43 to 1.75
E3155 − 19
mil), and euthanized with carbon dioxide addition. After the sperm heads occur), the snap tube (with its combined
recording a final body weight, the right epididymis (by contents of 200 µL) is vortexed anda9µLsample is removed
by pipette to an appropriate microscope slide.Apreferred and
convention) is immediately excised with care to minimize
blood contamination, and is then placed into a pre-warmed superlative counting method involves using an automated
spermanalyzerprogrammedtocountthesperminfivefieldsof
(37°C) Petri dish containing 3 mLof a solution of 1% bovine
the microscope slide and to instantaneously provide summary
serum albumen dissolved in phosphate buffered saline. The
statistics. The error rate (that is, the likelihood of counting
caudal portion is punctured twice with the tip of a #11 scalpel
debris in the slide preparation as spe
...