Rodent Sperm Analysis: A rationale for and path toward acceptance and use in ecological risk assessment.

The Rodent Sperm Analysis method was devised some two decades ago to advance ecological assessment science for mammals, one of two groups of terrestrial receptors routinely evaluated at chemically contaminated sites. In part, this method recognizes that sufficient time has elapsed at sites, such that a need to anticipate or predict impacts to ecological receptors is an obsolete task. The method therefore, recommends evaluating the very receptors that occupy sites for evidence today of their displaying compromised reproduction, a toxicological endpoint of great concern within the regulatory community. Critically, Rodent Sperm Analysis is not a risk assessment method but rather a direct health status assessment method, a distinction that ecological risk assessors may fail to recognize or appreciate. Further, the method is not intended as a replacement for the conventional approach to ecological assessment, but rather a method to be run in tandem with it, where it may likely reveal that site mammals are commonly free of impacts and that conventional assessments are fully unnecessary. This Discussion paper is a cautious analytical review of ecological assessor perceptions about the vetted and recently ASTM International certified Rodent Sperm Analysis method. The review identifies potential impediments to the method gaining broader acceptance by professionals in the field, prominently among these, resistance to adopting a new ecological assessment paradigm that may be more helpful than the one presently in place.

Commentary: An open appeal to the EPA for Superfund ERA reform.

The ecological risk assessment guidance of virtually all federal and state agencies, private companies, and other interests can be traced to that of the essential design of the U.S. Environmental Protection Agency (EPA). The EPA ecological risk assessment paradigm has remained unchanged for all intents and purposes since its inception 30 years ago, this despite criticism expressed repeatedly by some, for many years. Despite the discipline's name, a core paradigm shortcoming is its inability to express risk, the probability of a receptor-of-concern at a contaminated site developing a toxicological endpoint (e.g., reproductive impairment). Further, common site context and biological realities (e.g., site sizes; home ranges of receptors-of concern) allow for the supported challenges that risk assessments aren't needed altogether, and instances of ecological damage at sites being unknown. This commentary is an open appeal to the EPA to replace the paradigm it has set forth, dispensing with failed processes (e.g., endeavoring to assess risk potential at 75 year-old sites; endeavoring to assess risk potential to wide-ranging species at one-acre properties). The commentary invites the EPA to respond, not with counter-arguments, but rather with explanations for the Agency's resistance to acknowledging problems with its guidance, followed by the Agency commitment to sorely needed ERA reform.

Ingestion of a Single 2.3 mm Lead Pellet by Laying Roller Pigeon Hens Reduces Egg Size and Adversely Affects F1 Generation Hatchlings.

Many aquatic and terrestrial avian species inadvertently ingest lead (Pb) in the form of spent or fragmented ammunition, mistaking it for food or grit. Previous studies in our laboratory have shown that ingestion of even a single 45-mg pellet can significantly increase blood-Pb levels and significantly inhibit the enzyme delta aminolevulinic-acid dehydratase (δ-ALAD) for a period of greater than 4 weeks. In the current study, proven breeder pairs of domestic Roller pigeons were housed in individual cages. The hens were orally gavaged with dH2O vehicle, a single #9 Pb pellet (2.0 mm/45 mg) or a single #7.5 Pb pellet (2.3 mm/95 mg), placed back with the cock bird and allowed to mate for two consecutive clutches. The eggs were monitored for fertilization, shell damage, egg weight, and length during the 16- to 18-day incubation period. Hatchlings remained with the hen and cock through the weaning period (28-35 days post hatch) and were monitored for weight, development, and mortality. Weanling blood was collected for blood-Pb levels, δ-ALAD activity, red blood cell counts, total protein, and packed cell volume. Following euthanasia, weanling liver, spleen, kidney, sciatic nerve, thymus, and brain were collected for histopathology. Egg weight and length were significantly decreased in the #7.5 Pb pellet treatment group for the first clutch, and hatchling weight 7 days post hatch also was significantly less in the #7.5 Pb pellet treatment group during the first clutch. Histopathologic analysis showed increased lesions in liver, kidney, spleen, and thymus of the Pb-treated weanlings, during both the first and second clutch compared with the non-Pb-treated weanlings. These data suggest that maternal consumption of a single 95-mg Pb pellet can adversely impact egg size and hatchling organ development.

Validating mammalian resistance to stressor-mediated reproductive impact using rodent sperm analysis.

Small rodents from chemically and radiologically contaminated areas on the Savannah River Site, SC were evaluated for sensitive reproductive parameters in a dual purpose study. The primary intent was to observe if established reproductive thresholds-for effect could be exceeded in animals that, due to their restricted home ranges, are maximally exposed to local contamination. Secondarily, validation was sought for a principal element of the Rodent Sperm Analysis method that is used in support of ecological risk assessments for contaminated terrestrial properties. The method's fundamental underlying premise is that during decades of elapsed time between contamination release events and ecological assessments being conducted, rodents develop a resilience to potential stressors, evidenced by their continuing presence. During spring 2014 we collected 89 cotton mice (Peromyscus gossypinus) across three contaminated locations and one reference location, and quantified important male and female reproductive parameters (sperm counts and sperm morphology, and ovarian follicle counts, respectively) and organ weights. The outcome of the comprehensive sperm parameter review, in conjunction with the parallel female reproduction review and other corroborative population and tissue-based information gathered, suggests that mammalian assessments at contaminated sites are unnecessary in the common case.

Exposure Duration-Dependent Ovarian Recovery in Methoxychlor-Treated Mice.

The pesticide methoxychlor (MXC) is known to target ovarian antral follicles in the mouse. In previous in vivo studies, MXC administration for 20 days increased atresia, but did not affect female fertility immediately after dosing. Thus, we hypothesized that perhaps not enough time had elapsed between the onset of MXC-induced atresia and actual follicle loss to result in reduced fertility. The current study was undertaken to determine whether MXC treatment for 20 days results in reduced antral follicle numbers and fertility at 30 and 60 days after dosing. To test this hypothesis, adult CD-1 female mice were dosed with vehicle control or MXC (64 mg/kg/day) for 20 days. At 30 and 60 days postdosing, the mice were either subjected to fertility tests or their ovaries were collected and subjected to histological evaluation of follicle numbers and atresia. The results indicate that at 30 days after the completion of dosing, MXC significantly increased atresia and reduced primordial and total follicle numbers, but did not affect fertility compared to controls. At 60 days after completion of dosing, MXC did not significantly affect fertility, follicle numbers, or atresia compared to controls. Collectively, these data indicate that the ovary may be able to recover from MXC treatment for 20 days.

Where deer roam: chronic yet acute site exposures preclude ecological risk assessment.

Global positioning system (GPS) technology has made possible the detailed tracking of the spatial movements of wildlife. Using GPS tracking collars placed on female white-tailed deer (Odocoileus virginianus) over a protracted period, we illustrate that this species is spatially irrelevant for consideration in ecological risk assessments (ERAs) for commonly assessed contaminated sites. Specifically, deer movements do not allow for a sufficiency of chemical exposures to occur such that toxicological endpoints would be triggered. Deer are spatially irrelevant not only because their home ranges and overall utilized areas dwarf prototypical hazardous waste sites. They are also inappropriate for assessment because they only minimally contact reasonably sized preferred locations, this while demonstrating a confounding high degree of site affinity for them. Our spatial movements analysis suggests that deer introduce the ERA novelty of a species displaying elements of both chronic and acute site exposure. We further suggest that other large commonly assessed high-profile mammals could also be found to be spatially irrelevant for ERAs were they to submit to the GPS tracking and subsequent data analysis we performed for the deer. Recognizing that certain receptors need not be considered in ERAs can help to simplify the ERA process.

Methoxychlor-induced ovarian follicle toxicity in mice: dose and exposure duration-dependent effects.

BACKGROUND: Methoxychlor (MXC) is specifically known to target ovarian antral follicles, increasing atresia (death via apoptosis) in them. This is of concern because females are born with a finite pool of ovarian follicles. Only limited studies have explored the phenomenon of a reduced fertility threshold for effect based on the percentage of antral follicle atresia. METHODS: In this article, we report on adult female CD-1 mice exposed intraperitoneally to various doses of MXC for 5, 10, 20, and 30 days. In the 20-day treatment, mice were dosed with either the vehicle or MXC at 64 or 96 mg/kg/day, whereas in the 30-day treatment, mice were dosed with vehicle or MXC at 48, 64, or 96 mg/kg/day. The mice that were dosed with MXC for 30 days were also mated with untreated males for a determination of overall fertility. RESULTS: A significantly increased percentage (50%) of atretic antral follicles was observed only after 20 and 30 days of treatment. Specifically, mice treated with MXC64 for 20 and 30 days had an increased percentage of atretic antral follicles compared with vehicle-treated mice. Interestingly, mice dosed with MXC96 had an increased percentage of atretic antral follicles after 30 days, but not after 20 days of treatment compared with vehicle-treated mice. Overall fertility of the mice was not different compared with controls. CONCLUSIONS: The results indicate that as much as a 50% increase in atretic antral follicles does not affect the immediate fertility of the mice.

Correlational sperm motility analysis: wild rodent progressive sperm motility as a potentially improved reproduction-assessment parameter in mammals.

Progressive sperm motility, describing the directed or linear movement of sperm, is infrequently reported in reproductive studies conducted with rodents. This is a curious circumstance given that progressive motility can be easily reported with current computer-assisted sperm-analysis equipment, and because progressively motile sperm seemingly display a greater likelihood of fertilizing ova than do sperm that move in a random or undefined pattern. This study presents a comparison of the routinely reported total motility (i.e., the percentage of moving sperm cells in a sample) and the progressive motility of sperm from rodents captured at soil-contaminated sites and at habitat-matched noncontaminated reference locations. Progressive motility's trending with total motility at all areas, in terms of direction of shift, magnitude of change, and degree of correlation, was extremely high. Based on these results and in the interest of assisting ecological health assessments at contaminated sites, it would seem prudent to conduct the empirical research to determine progressive sperm motility's threshold-for-effect, which may be lower than that for total motility.

Testing the limits of Rodent Sperm Analysis: azoospermia in an otherwise healthy wild rodent population.

By comparing the sperm parameters of small rodents trapped at contaminated terrestrial sites and nearby habitat-matched noncontaminated locations, the patent-pending Rodent Sperm Analysis (RSA) method provides a direct health status appraisal for the maximally chemical-exposed mammalian ecological receptor in the wild. RSA outcomes have consistently allowed for as definitive determinations of receptor health as are possible at the present time, thereby streamlining the ecological risk assessment (ERA) process. Here, we describe the unanticipated discovery, at a contaminated US EPA Superfund National Priorities List site, of a population of Hispid cotton rats (Sigmodon hispidus), with a high percentage of adult males lacking sperm entirely (azoospermia). In light of the RSA method's role in streamlining ERAs and in bringing contaminated Superfund-type site investigations to closure, we consider the consequences of the discovery. The two matters specifically discussed are (1) the computation of a population's average sperm count where azoospermia is present and (2) the merits of the RSA method and its sperm parameter thresholds-for-effect when azoospermia is masked in an otherwise apparently healthy rodent population.

Demonstrating ecological receptor health at contaminated sites with wild rodent sperm parameters.

Consistently at Superfund and other contaminated terrestrial sites, ecological receptors have been chemically exposed for multiple decades by the time risk assessments are conducted. Given that numerous generations of the receptors have lived through the contaminated site condition by the present day, a paradigm shift from risk assessment, where the potential for health effects are forecasted, to a direct, health status assessment scheme for the site-exposed receptor, would seem to be most appropriate. We applied the only such existing direct health status assessment method, Rodent Sperm Analysis (RSA), with small rodents trapped at contaminated sites and at matched noncontaminated reference locations. Reproductive health, ecological risk assessment's endpoint of greatest concern, is targeted with RSA by comparing the sperm parameters of count, motility, and morphology, for each of which it is known how much of a change from a control condition signifies compromised reproductive capability. Given that sperm parameter thresholds were not exceeded in maximally exposed receptors, the data suggest that in the general case, contaminated terrestrial sites do not need cleanups to afford health protection to ecological species, and particularly the larger, wider-ranging, higher trophic level species. Our findings suggest that RSA has the ability to consistently discriminate between clean and contaminated sites, and that the method can allow for as definitive determinations of terrestrial ecological receptor health as are possible, thereby facilitating early site clean-up decisions.

A critical assessment of the ecological risk assessment process: a review of misapplied concepts.

A frank assessment of present-day ecological risk assessments (ERA) for managed contaminated sites reveals that fundamental concepts regarding the receptors that are considered and the chemical exposures they experience are commonly misapplied. As a consequence, environmental managers are not being supplied with the information needed for proper decision making. The stepwise review of ecological risk issues provided here suggests that the ERA process needs I to be severely revamped. Further, what is likely hindering the development of a refined ecological assessment process that is better suited to environmental problem solving and land management is the unwillingness of stakeholders to agree that much of the current ERA practice and convention is flawed.

Two simple algorithms for refining mammalian receptor selection in ecological risk assessments.

Although guidelines exist for selecting appropriate ecological receptors for risk assessments at contaminated sites, it can be demonstrated that many of the mammals commonly evaluated are spatially irrelevant. Terrestrial risk assessments could be simplified and made more efficient, though, if mammals that are initially considered were screened for their spatial relevance. This article presents 2 simple algorithms that each demonstrate that most mammalian receptors are not spatially relevant for the overwhelming majority of hazardous waste and other contaminated sites. The algorithms use readily available and curiously overlooked spatial distribution (e.g., animal density) information and suggest that contaminated sites need to be 80 to 100 acres in size to justify the inclusion of most mammalian receptors. Given that hazardous waste sites are generally much smaller than this, many ecological risk assessments (ERA) could reasonably dispense with incorporating mammals entirely. An awareness on the part of decision makers and risk managers of the nonsuitability of many mammalian receptors evaluated in terrestrial ERAs could significantly impact the perceived need to monitor or remediate sites. This article also examines the anticipated challenges of regulators and other decision makers when entertaining the notion of a spatial relevance screen for mammals.

Rodent sperm analysis in field-based ecological risk assessment: pilot study at Ravenna army ammunition plant, Ravenna, Ohio.

Ecological risk assessment (ERA) guidance recommends that field-truthing efforts proceed when modeled hazard quotients (HQs) suggest that toxicological effects are occurring to site receptors. To date, no field methods have been proposed by the regulatory community that can lead to definitive determinations of acceptable or unacceptable risk for birds and mammals, the two terrestrial classes of receptors that are commonly assessed using the HQ method. This paper describes rodent sperm analysis (RSA) as a viable method to be applied in the field at sites with historical contamination. RSA is capable of detecting biological differences that bear on reproduction, a highly regarded toxicological endpoint of concern in USEPA Superfund-type ERAs. The results of RSA's first application at a study site are reported and discussed. The paper also provides the rationale for RSA's efficacy in the context of Superfund and other environmental cleanup programs, where limited time and money are available to determine and evaluate the field condition.