Using emerging science to inform risk characterizations for wildlife within current regulatory frameworks.

Many jurisdictions have regulatory frameworks that seek to reduce the effects of environmental exposures of anthropogenic chemicals on terrestrial wildlife (i.e., mammals, birds, reptiles, and amphibians). The frameworks apply for new and existing chemicals, including pesticides (prospective assessments), and to environmental contamination from releases (retrospective risk assessments). Relatively recently, there have been many scientific advances that could improve risk estimates for wildlife. Here, we briefly describe current regulations from North America (United States and Canada) and from Europe that include risk assessments for wildlife to ascertain whether they are conducive to the use of emerging science and new methods. We also provide examples where new and emerging science may be used to improve wildlife risk characterization and identify areas in need of future research. Integr Environ Assess Manag 2024;20:765-779. © 2024 His Majesty the King in Right of Canada and The Authors. Integrated Environmental Assessment and Management © 2024 Society of Environmental Toxicology & Chemistry (SETAC). Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Toxicological effects assessment for wildlife in the 21st century: Review of current methods and recommendations for a path forward.

Model species (e.g., granivorous gamebirds, waterfowl, passerines, domesticated rodents) have been used for decades in guideline laboratory tests to generate survival, growth, and reproductive data for prospective ecological risk assessments (ERAs) for birds and mammals, while officially adopted risk assessment schemes for amphibians and reptiles do not exist. There are recognized shortcomings of current in vivo methods as well as uncertainty around the extent to which species with different life histories (e.g., terrestrial amphibians, reptiles, bats) than these commonly used models are protected by existing ERA frameworks. Approaches other than validating additional animal models for testing are being developed, but the incorporation of such new approach methodologies (NAMs) into risk assessment frameworks will require robust validations against in vivo responses. This takes time, and the ability to extrapolate findings from nonanimal studies to organism- and population-level effects in terrestrial wildlife remains weak. Failure to adequately anticipate and predict hazards could have economic and potentially even legal consequences for regulators and product registrants. In order to be able to use fewer animals or replace them altogether in the long term, vertebrate use and whole organism data will be needed to provide data for NAM validation in the short term. Therefore, it is worth investing resources for potential updates to existing standard test guidelines used in the laboratory as well as addressing the need for clear guidance on the conduct of field studies. Herein, we review the potential for improving standard in vivo test methods and for advancing the use of field studies in wildlife risk assessment, as these tools will be needed in the foreseeable future. Integr Environ Assess Manag 2024;20:699-724. © 2023 His Majesty the King in Right of Canada and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Wildlife ecological risk assessment in the 21st century: Promising technologies to assess toxicological effects.

Despite advances in toxicity testing and the development of new approach methodologies (NAMs) for hazard assessment, the ecological risk assessment (ERA) framework for terrestrial wildlife (i.e., air-breathing amphibians, reptiles, birds, and mammals) has remained unchanged for decades. While survival, growth, and reproductive endpoints derived from whole-animal toxicity tests are central to hazard assessment, nonstandard measures of biological effects at multiple levels of biological organization (e.g., molecular, cellular, tissue, organ, organism, population, community, ecosystem) have the potential to enhance the relevance of prospective and retrospective wildlife ERAs. Other factors (e.g., indirect effects of contaminants on food supplies and infectious disease processes) are influenced by toxicants at individual, population, and community levels, and need to be factored into chemically based risk assessments to enhance the "eco" component of ERAs. Regulatory and logistical challenges often relegate such nonstandard endpoints and indirect effects to postregistration evaluations of pesticides and industrial chemicals and contaminated site evaluations. While NAMs are being developed, to date, their applications in ERAs focused on wildlife have been limited. No single magic tool or model will address all uncertainties in hazard assessment. Modernizing wildlife ERAs will likely entail combinations of laboratory- and field-derived data at multiple levels of biological organization, knowledge collection solutions (e.g., systematic review, adverse outcome pathway frameworks), and inferential methods that facilitate integrations and risk estimations focused on species, populations, interspecific extrapolations, and ecosystem services modeling, with less dependence on whole-animal data and simple hazard ratios. Integr Environ Assess Manag 2024;20:725-748. © 2023 His Majesty the King in Right of Canada and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Do Pharmaceuticals in the Environment Pose a Risk to Wildlife?

The vast majority of knowledge related to the question "To what extent do pharmaceuticals in the environment pose a risk to wildlife?" stems from the Asian vulture crisis (>99% decline of some species of Old World vultures on the Indian subcontinent related to the veterinary use of the nonsteroidal anti-inflammatory drug [NSAID] diclofenac). The hazard of diclofenac and other NSAIDs (carprofen, flunixin, ketoprofen, nimesulide, phenylbutazone) to vultures and other avian species has since been demonstrated; indeed, only meloxicam and tolfenamic acid have been found to be vulture-safe. Since diclofenac was approved for veterinary use in Spain and Italy in 2013 (home to ~95% of vultures in Europe), the risk of NSAIDs to vultures in these countries has become one of the principal concerns related to pharmaceuticals and wildlife. Many of the other bodies of work on pharmaceutical exposure, hazard and risk to wildlife also relate to adverse effects in birds (e.g., poisoning of scavenging birds in North America and Europe from animal carcasses containing pentobarbital, secondary and even tertiary poisoning of birds exposed to pesticides used in veterinary medicine as cattle dips, migratory birds as a vector for the transfer of antimicrobial and antifungal resistance). Although there is some research related to endocrine disruption in reptiles and potential exposure of aerial insectivores, there remain numerous knowledge gaps for risk posed by pharmaceuticals to amphibians, reptiles, and mammals. Developing noninvasive sampling techniques and new approach methodologies (e.g., genomic, in vitro, in silico, in ovo) is important if we are to bridge the current knowledge gaps without extensive vertebrate testing. Environ Toxicol Chem 2023;00:1-16. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Thyroid disruption and oxidative stress in American kestrels following embryonic exposure to the alternative flame retardants, EHTBB and TBPH.

Brominated flame retardant chemicals, such as 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EHTBB) (CAS #: 183658-27-7) and bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH) (CAS #: 26040-51-7), have been detected in avian tissues and eggs from remote regions. Exposure to EHTBB and TBPH has been shown to cause oxidative stress and altered thyroid function in rodents and fish, yet no controlled studies have examined potential adverse effects of exposure in birds. Because flame retardants have been detected in wild raptors, we used American kestrels (Falco sparverius) as a model raptor to determine whether in ovo exposure to EHTBB or TBPH affected growth, hatching success, oxidative stress, or thyroid function. We exposed kestrel embryos to nominal concentrations (10, 50, or 100 ng g-1 egg weight) of EHTBB and TBPH via egg-injection on embryonic day 5. Embryonic exposure (~23 d) to EHTBB increased thyroid gland mass, reduced glandular colloid and total thyroxine (T4) in hatchling males and females, whereas deiodinase enzyme activity increased in males but decreased in females. Hatchlings exposed to TBPH in ovo exhibited reduced colloid and increased oxidative stress. Although exposure to EHTBB and TBPH caused several physiological effects (e.g., heart and brain mass), only exposure to 50 ng g-1 EHTBB appeared to reduce hatching success. Our results suggest these flame retardants may be hazardous for predatory birds. Future research should evaluate long-term survival and fitness consequences in birds exposed to these chemicals.

Uptake, Metabolism, and Elimination of Fungicides from Coated Wheat Seeds in Japanese Quail (Coturnix japonica).

Pesticides coated to the seed surface potentially pose an ecological risk to granivorous birds that consume incompletely buried or spilled seeds. To assess the toxicokinetics of seeds treated with current-use fungicides, Japanese quail (Coturnix japonica) were orally dosed with commercially coated wheat seeds. Quail were exposed to metalaxyl, tebuconazole, and fludioxonil at either a low dose (0.0655, 0.0308, and 0.0328 mg/kg of body weight, respectively) or a high dose (0.196, 0.0925, and 0.0985 mg/kg of body weight, respectively). Fungicides were rapidly absorbed and distributed to tissues. Tebuconazole was metabolized into tert-butylhydroxy-tebuconazole. All compounds were eliminated to below detection limits within 24 h. The high detection frequencies observed in fecal samples potentially offer a non-invasive matrix to monitor pesticide exposure. With the summation of total body burden across plasma, tissue, and fecal samples, less than 9% of the administered dose was identified as the parent fungicide, demonstrating the importance to monitor both active ingredients and their metabolites in biological samples.

Brodifacoum Toxicity in American Kestrels (Falco sparverius) with Evidence of Increased Hazard on Subsequent Anticoagulant Rodenticide Exposure.

A seminal question in ecotoxicology is the extent to which contaminant exposure evokes prolonged effects on physiological function and fitness. A series of studies were undertaken with American kestrels ingesting environmentally realistic concentrations of the second-generation anticoagulant rodenticide (SGAR) brodifacoum. Kestrels fed brodifacoum at 0.3, 1.0, or 3.0 µg/g diet wet weight for 7 d exhibited dose-dependent hemorrhage, histopathological lesions, and coagulopathy (prolonged prothrombin and Russell's viper venom times). Following termination of a 7-d exposure to 0.5 µg brodifacoum/g diet, prolonged blood clotting time returned to baseline values within 1 wk, but brodifacoum residues in liver and kidney persisted during the 28-d recovery period (terminal half-life estimates >50 d). To examine the hazard of sequential anticoagulant rodenticide (AR) exposure, kestrels were exposed to either the first-generation AR chlorophacinone (1.5 µg/g diet) or the SGAR brodifacoum (0.5 µg/g diet) for 7 d and, following a recovery period, challenged with a low dose of chlorophacinone (0.75 µg/g diet) for 7 d. In brodifacoum-exposed kestrels, the challenge exposure clearly prolonged prothrombin time compared to naive controls and kestrels previously exposed to chlorophacinone. These data provide evidence that the SGAR brodifacoum may have prolonged effects that increase the toxicity of subsequent AR exposure. Because free-ranging predatory and scavenging wildlife are often repeatedly exposed to ARs, such protracted toxicological effects need to be considered in hazard and risk assessments. Environ Toxicol Chem 2020;39:468-481. © 2020 SETAC.

In ovo exposure to brominated flame retardants Part II: Assessment of effects of TBBPA-BDBPE and BTBPE on hatching success, morphometric and physiological endpoints in American kestrels.

Tetrabromobisphenol A bis(2,3-dibromopropyl ether) (TBBPA-BDBPE) and 1,2-bis(2,4,6-tribromophenoxy)ethane (BTPBE) are both brominated flame retardants (BFRs) that have been detected in birds; however, their potential biological effects are largely unknown. We assessed the effects of embryonic exposure to TBBPA-BDBPE and BTBPE in a model avian predator, the American kestrel (Falco sparverius). Fertile eggs from a captive population of kestrels were injected on embryonic day 5 (ED5) with a vehicle control or one of three doses within the range of concentrations that have been detected in biota (nominal concentrations of 0, 10, 50 or 100 ng/g egg; measured concentrations 0, 3.0, 13.7 or 33.5 ng TBBPA-BDBPE/g egg and 0, 5.3, 26.8 or 58.1 ng BTBPE/g egg). Eggs were artificially incubated until hatching (ED28), at which point blood and tissues were collected to measure morphological and physiological endpoints, including organ somatic indices, circulating and glandular thyroid hormone concentrations, thyroid gland histology, hepatic deiodinase activity, and markers of oxidative stress. Neither compound had any effects on embryo survival through 90% of the incubation period or on hatching success, body mass, organ size, or oxidative stress of hatchlings. There was evidence of sex-specific effects in the thyroid system responses to the BTBPE exposures, with type 2 deiodinase (D2) activity decreasing at higher doses in female, but not in male hatchlings, suggesting that females may be more sensitive to BTBPE. However, there were no effects of TBBPA-BDBPE on the thyroid system in kestrels. For the BTPBE study, a subset of high-dose eggs was collected throughout the incubation period to measure changes in BTBPE concentrations. There was no decrease in BTBPE over the incubation period, suggesting that BTBPE is slowly metabolized by kestrel embryos throughout their ∼28-d development. These two compounds, therefore, do not appear to be particularly toxic to embryos of the American kestrel.

Toxicokinetics of Imidacloprid-Coated Wheat Seeds in Japanese Quail ( Coturnix japonica) and an Evaluation of Hazard.

Birds are potentially exposed to neonicotinoid insecticides by ingestion of coated seeds during crop planting. Adult male Japanese quail were orally dosed with wheat seeds coated with an imidacloprid (IMI) formulation at either 0.9 or 2.7 mg/kg body weight (BW) (∼3 and 9% of IMI LD50 for Japanese quail, respectively) for 1 or 10 days. Quail were euthanized between 1 and 24 h postexposure to assess toxicokinetics. Analysis revealed rapid absorption (1 h) into blood and distribution to the brain, muscle, kidney, and liver. Clearance to below detection limits occurred at both dose levels and exposure durations in all tissues within 24 h. Metabolism was extensive, with 5-OH-IMI and IMI-olefin detected at greater concentrations than IMI in tissues and fecal samples. There was no lethality or overt signs of toxicity at either dose level. Furthermore, no evidence of enhanced expression of mRNA genes associated with hepatic xenobiotic metabolism, oxidative DNA damage, or alterations in concentrations of corticosterone and thyroid hormones was observed. Application of the toxicokinetic data was used to predict IMI residue levels in the liver with reasonable results for some field exposure and avian mortality events. It would appear that some affected species of birds are either consuming larger quantities of seeds or exhibit differences in ADME or sensitivity than predicted by read-across from these data.

Examination of contaminant exposure and reproduction of ospreys (Pandion haliaetus) nesting in Delaware Bay and River in 2015.

A study of ospreys (Pandion haliaetus) nesting in the coastal Inland Bays of Delaware, and the Delaware Bay and Delaware River in 2015 examined spatial and temporal trends in contaminant exposure, food web transfer and reproduction. Concentrations of organochlorine pesticides and metabolites, polychlorinated biphenyls (PCBs), coplanar PCB toxic equivalents, polybrominated diphenyl ethers (PBDEs) and other flame retardants in sample eggs were generally greatest in the Delaware River. Concentrations of legacy contaminants in 2015 Delaware Bay eggs were lower than values observed in the 1970s through early 2000s. Several alternative brominated flame retardants were rarely detected, with only TBPH [bis(2-ethylhexyl)-tetrabromophthalate)] present in 5 of 27 samples at <5 ng/g wet weight. No relation was found between p,p'-DDE, total PCBs or total PBDEs in eggs with egg hatching, eggs lost from nests, nestling loss, fledging and nest success. Osprey eggshell thickness recovered to pre-DDT era values, and productivity was adequate to sustain a stable population. Prey fish contaminant concentrations were generally less than those in osprey eggs, with detection frequencies and concentrations greatest in white perch (Morone americana) from Delaware River compared to the Bay. Biomagnification factors from fish to eggs for p,p'-DDE and total PCBs were generally similar to findings from several Chesapeake Bay tributaries. Overall, findings suggest that there have been improvements in Delaware Estuary waterbird habitat compared to the second half of the 20th century. This trend is in part associated with mitigation of some anthropogenic contaminant threats.

Pharmaceuticals in water, fish and osprey nestlings in Delaware River and Bay.

Exposure of wildlife to Active Pharmaceutical Ingredients (APIs) is likely to occur but studies of risk are limited. One exposure pathway that has received attention is trophic transfer of APIs in a water-fish-osprey food chain. Samples of water, fish plasma and osprey plasma were collected from Delaware River and Bay, and analyzed for 21 APIs. Only 2 of 21 analytes exceeded method detection limits in osprey plasma (acetaminophen and diclofenac) with plasma levels typically 2-3 orders of magnitude below human therapeutic concentrations (HTC). We built upon a screening level model used to predict osprey exposure to APIs in Chesapeake Bay and evaluated whether exposure levels could have been predicted in Delaware Bay had we just measured concentrations in water or fish. Use of surface water and BCFs did not predict API concentrations in fish well, likely due to fish movement patterns, and partitioning and bioaccumulation uncertainties associated with these ionizable chemicals. Input of highest measured API concentration in fish plasma combined with pharmacokinetic data accurately predicted that diclofenac and acetaminophen would be the APIs most likely detected in osprey plasma. For the majority of APIs modeled, levels were not predicted to exceed 1 ng/mL or method detection limits in osprey plasma. Based on the target analytes examined, there is little evidence that APIs represent a significant risk to ospreys nesting in Delaware Bay. If an API is present in fish orders of magnitude below HTC, sampling of fish-eating birds is unlikely to be necessary. However, several human pharmaceuticals accumulated in fish plasma within a recommended safety factor for HTC. It is now important to expand the scope of diet-based API exposure modeling to include alternative exposure pathways (e.g., uptake from landfills, dumps and wastewater treatment plants) and geographic locations (developing countries) where API contamination of the environment may represent greater risk.

Predictive framework for estimating exposure of birds to pharmaceuticals.

We present and evaluate a framework for estimating concentrations of pharmaceuticals over time in wildlife feeding at wastewater treatment plants (WWTPs). The framework is composed of a series of predictive steps involving the estimation of pharmaceutical concentration in wastewater, accumulation into wildlife food items, and uptake by wildlife with subsequent distribution into, and elimination from, tissues. Because many pharmacokinetic parameters for wildlife are unavailable for the majority of drugs in use, a read-across approach was employed using either rodent or human data on absorption, distribution, metabolism, and excretion. Comparison of the different steps in the framework against experimental data for the scenario where birds are feeding on a WWTP contaminated with fluoxetine showed that estimated concentrations in wastewater treatment works were lower than measured concentrations; concentrations in food could be reasonably estimated if experimental bioaccumulation data are available; and read-across from rodent data worked better than human to bird read-across. The framework provides adequate predictions of plasma concentrations and of elimination behavior in birds but yields poor predictions of distribution in tissues. The approach holds promise, but it is important that we improve our understanding of the physiological similarities and differences between wild birds and domesticated laboratory mammals used in pharmaceutical efficacy/safety trials, so that the wealth of data available can be applied more effectively in ecological risk assessments. Environ Toxicol Chem 2017;36:2335-2344. © 2017 SETAC.

Evaluation of a Novel Approach for Reducing Emissions of Pharmaceuticals to the Environment.

Increased interest over the levels of pharmaceuticals detected in the environment has led to the need for new approaches to manage their emissions. Inappropriate disposal of unused and waste medicines and release from manufacturing plants are believed to be important pathways for pharmaceuticals entering the environment. In situ treatment technologies, which can be used on-site in pharmacies, hospitals, clinics, and at manufacturing plants, might provide a solution. In this study we explored the use of Pyropure, a microscale combined pyrolysis and gasification in situ treatment system for destroying pharmaceutical wastes. This involved selecting 17 pharmaceuticals, including 14 of the most thermally stable compounds currently in use and three of high environmental concern to determine the technology's success in waste destruction. Treatment simulation studies were done on three different waste types and liquid, solid, and gaseous emissions from the process were analyzed for parent pharmaceutical and known active transformation products. Gaseous emissions were also analyzed for NOx, particulates, dioxins, furans, and metals. Results suggest that Pyropure is an effective treatment process for pharmaceutical wastes: over 99 % of each study pharmaceutical was destroyed by the system without known active transformation products being formed during the treatment process. Emissions of the other gaseous air pollutants were within acceptable levels. Future uptake of the system, or similar in situ treatment approaches, by clinics, pharmacists, and manufacturers could help to reduce the levels of pharmaceuticals in the environment and reduce the economic and environmental costs of current waste management practices.

An in vitro method for determining the bioaccessibility of pharmaceuticals in wildlife.

Wildlife can be exposed to human pharmaceuticals via prey that have accumulated the compounds from wastewater, surface water, sediment, and soil. One factor affecting internal absorption of pharmaceuticals is bioaccessibility, the proportion of the compound that enters solution in the gastrointestinal tract. Currently, the bioaccessibility of most pharmaceuticals in prey remains unknown for most wildlife species. The authors evaluated the potential of a 2-compartment in vitro gastrointestinal tract model to compare the bioaccessibility of the antidepressant fluoxetine from invertebrate prey for birds and mammals. Samples of gizzard (or stomach) and intestinal-phase digestive juices were obtained from the in vitro models along with the residual solid material. High-performance liquid chromatographic analysis revealed that the bioaccessibility of fluoxetine in the avian in vitro models was statistically significantly lower than that in the mammalian models as a percentage of what was recovered; however, there were no statistically or biologically significant interspecies difference in terms of the amount recovered per gram of "food" inserted at the start of the simulation. This in vitro model provides a useful method of comparing the bioaccessibility of pharmaceuticals in different prey for species with different gastrointestinal conditions. There is merit for ecological risk assessments in further developing this in vitro approach to improve estimates of internal exposure for organics. Environ Toxicol Chem 2016;35:2349-2357. © 2016 SETAC.