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.

Contaminant biomonitoring augmented with a qPCR array indicates hepatic mRNA gene expression effects in wild-collected seabird embryos.

Birds can bioaccumulate persistent contaminants, and maternal transfer to eggs may expose embryos to concentrations sufficient to cause adverse effects during sensitive early-life stages. However, using tissue residue concentrations alone to infer whether contaminant effects are occurring suffers from uncertainty, and efficient, sensitive biomarkers remain limited in wildlife. We studied relationships between whole embryo contaminant concentrations (total mercury, organochlorine pesticides, perfluoroalkyl substances, polychlorinated biphenyls, and halogenated flame retardants) together with mRNA expression in embryonic liver tissue from a Pacific Ocean seabird, the rhinoceros auklet (Cerorhinca monocerata). Fresh eggs were collected, incubated under controlled conditions, and from the pre-hatch embryo, hepatic RNA was extracted for qPCR array analysis to measure gene expression (2-∆Cq), while the remaining embryo was analyzed for contaminant residues. Contaminant and gene expression data were assessed with a combination of multivariate approaches and linear models. Results indicated correlations between embryonic total mercury and several genes such as sepp1, which encodes selenoprotein P. Correlation between the biotransformation gene cyp1a4 and the C7 perfluoroalkyl carboxylic acid PFHpA was also evident. This study demonstrates that egg collection from free-living populations for contaminant biomonitoring programs can relate chemical residues to in ovo mRNA gene expression effects in embryo hepatic tissue.

Polycyclic aromatic compound and trace metal element residues in Mytilus mussels at marine wildlife hotspots on the Pacific coast of Canada.

The Pacific coast of Canada has a rich marine fauna and a growing human population with increasing potential for pollution releases, but there is currently little overlap between marine wildlife hotspots and ongoing biomonitoring efforts for less bioaccumulative contaminants such as polycyclic aromatic compounds (PAC) and trace metals (metals). We surveyed PACs and metals at marine bird breeding colonies in coastal British Columbia in 2018 by analyzing chemical residues in the soft tissue of bivalve Mytilus sp. mussels collected from stations (n = 3) at seven sites. The concentration of sum PACs (∑43PAC) and high molecular weight (HMW) PACs were highest at the Second Narrows colony in Vancouver Harbour, a highly urbanized and industrialized port within the Salish Sea. For conservation areas, two Salish Sea and three Pacific Ocean coast colonies, PACs were generally lower. However, ∑43PAC, ∑HMWPAC, and several HMW congeners at the remote site of Triangle Island, a Marine National Wildlife Area, were not significantly different from Second Narrows. The dominant PAC sources at all sites are likely pyrogenic rather than petrogenic, as suggested by PAC profiles, proportion of parent PACs, and source-indicator congeners. For metals, site differences were found for seven out of eight priority metals, but principal component analysis indicated that site differences, such as high mercury and cadmium at offshore sites, are likely related to environmental and biological variables including salinity, condition index, water temperature, and shell length. Our survey across a broad coastal region shows that PAC and metal biomonitoring programs with mussels should include wildlife hotspots where the exposure of protected vertebrate species to pollutants with low bioaccumulation potential would be less obvious, and shows that collection of data on key covariates (e.g. lipid content, salinity) will be critical to tracking long-term trends and detecting pollution release events.

Effects of diluted bitumen exposure on the survival, physiology, and behaviour of zebra finches (Taeniopygia guttata).

Diluted bitumen (dilbit) is an unconventional crude petroleum increasingly being extracted and transported to market by pipeline and tanker. Despite the transport of dilbit through terrestrial, aquatic, and coastal habitat important to diverse bird fauna, toxicity data are currently only available for fish and invertebrates. We used the zebra finch (Taeniopygia guttata) as a tractable, avian model system to investigate exposure effects of lightly weathered Cold Lake blend dilbit on survival, tissue residue, and a range of physiological and behavioural endpoints. Birds were exposed via oral gavage over 14-days with dosages of 0, 2, 4, 6, 8, 10, or 12 mL dilbit/kg bw/day. We identified an LD50 of 9.4 mL/kg/d dilbit, with complete mortality at 12 mL/kg/d. Mortality was associated with mass loss, external oiling, decreased pectoral and heart mass, and increased liver mass. Hepatic ethoxyresorufin-O-deethylase activity (EROD) was elevated in all dilbit-dosed birds compared with controls but there was limited evidence of sublethal effects of dilbit on physiological endpoints at doses < 10 mL/kg/d (hematocrit, hemoglobin, total antioxidants, and reactive oxygen metabolites). Dilbit exposure affected behavior, with more dilbit-treated birds foraging away from the feeder, more birds sleeping or idle at low dilbit doses, and fewer birds huddling together at high dilbit doses. Naphthalene, dibenzothiophene, and their alkylated congeners in particular (e.g. C2-napthalene and C2-dibenzothiophene) accumulated in the liver at greater concentrations in dilbit-treated birds compared to controls. Although directly comparable studies in the zebra finch are limited, our mortality data suggest that dilbit is more toxic than the well-studied MC252 conventional light crude oil with this exposure regime. A lack of overt sublethal effects at lower doses, but effects on body mass and composition, behaviour, high mortality, and elevated PAC residue at doses ≥ 10 mL/kg/d suggest a threshold effect.

Effects of Avian Eggshell Oiling With Diluted Bitumen Show Sublethal Embryonic Polycyclic Aromatic Compound Exposure.

Breeding birds that become oiled may contaminate the shells of their eggs, and studies of conventional crude oil suggest that even small quantities can be absorbed through the eggshell and cause embryotoxicity. Unconventional crude oils remain untested, so we evaluated whether a major Canadian oil sands product, diluted bitumen (dilbit), would be absorbed and cause toxicity when applied to eggshells of two species, domestic chicken (Gallus gallus domesticus) and double-crested cormorant (Nannopterum auritum). We artificially incubated eggs and applied lightly weathered dilbit (Cold Lake blend) to the eggshells (0.015-0.15 mg g-1 egg in chicken; 0.1-0.4 mg g-1 egg in cormorant) at various points during incubation before sampling prehatch embryos. Polycyclic aromatic compound (PAC) residue in cormorant embryos was elevated only at the highest dilbit application (0.4 mg g-1 egg) closest (day 16) to sampling on day 22. In contrast, cormorant liver cytochrome P450 1a4 (Cyp1a4) mRNA expression (quantitative polymerase chain reaction assay) was elevated only in embryos treated with the earliest and lowest dilbit application (0.1 mg g-1 egg on day 4). These results confirm that dilbit can cross through the eggshell and be absorbed by embryos, and they imply rapid biotransformation of PACs and a nonmonotonic Cyp1a4 response. Despite evidence of exposure in cormorant, we found no detectable effects on the frequency of survival, deformity, and gross lesions, nor did we find effects on physiological endpoints indicative of growth and cardiovascular function in either chicken or cormorant. In ovo dilbit exposure may be less toxic than well-studied conventional crude oils. The effects of an oil spill scenario involving dilbit to bird embryos might be subtle, and PACs may be rapidly metabolized. Environ Toxicol Chem 2022;41:159-174. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effects of petroleum exposure on birds: A review.

Birds are vulnerable to petroleum pollution, and exposure has a range of negative effects resulting from plumage fouling, systemic toxicity, and embryotoxicity. Recent research has not been synthesized since Leighton's 1993 review despite the continued discharge of conventional petroleum, including high-volume oil spills and chronic oil pollution, as well as the emergence of understudied unconventional crude oil types. To address this, we reviewed the individual-level effects of crude oil and refined fuel exposure in avifauna with peer-reviewed articles published 1993-2020 to provide a critical synthesis of the state of the science. We also sought to answer how unconventional crude petroleum effects compare with conventional crude oil. Relevant knowledge gaps and research challenges were identified. The resulting review examines avian exposure to petroleum and synthesizes advances regarding the physical effects of oil hydrocarbons on feather structure and function, as well the toxic effects of inhaled or ingested oil, embryotoxicity, and how exposure affects broader scale endpoints related to behavior, reproduction, and survival. Another outcome of the review was the knowledge gaps and challenges identified. The first finding was a paucity of oil ingestion rate estimates in birds. Characterizing environmentally realistic exposure and ingestion rates is a higher research priority than additional conventional oral dosing experiments. Second, there is an absence of toxicity data for unconventional crude petroleum. Although the effects of air and water contamination in the Canadian oil sands region have received attention, toxicity data for direct exposure to unrefined bitumen produced there in high volumes and other such unconventional oil types are needed. Third, we encountered barriers to the interpretation, replication, broad relevance, and comparability of studies. We therefore propose best practices and promising technological advancements for researchers. This review consolidates our understanding of petroleum's effects on birds and points a way forward for researchers and resource managers.

Urea Release by Intermittently Saturated Sediments from a Coastal Agricultural Landscape.

Urea-N is linked to harmful algal blooms in lakes and estuaries, and urea-N-based fertilizers have been implicated as a source. However, the export of urea-N-based fertilizers appears unlikely, as high concentrations of urea-N are most commonly found in surface waters outside periods of fertilization. To evaluate possible autochthonous production of urea-N, we monitored urea-N released from drainage ditch sediments using mesocosms. Sediments from a cleaned (recently dredged) drainage ditch, uncleaned ditch, forested ditch, riparian wetland, and an autoclaved sand control were isolated in mesocosms and flooded for 72 h to quantify urea-N, NH-N, and NO-N in the floodwater. Sediments were flooded with different N-amended solutions (distilled HO, 1.5 mg L NH-N, 3.0 mg L NH-N, 2.6 mg L NO-N, or 5.1 mg L NO-N) and incubated at three water temperatures (16, 21, and 27°C). Urea-N concentrations in mesocosms representing uncleaned and cleaned drainage ditches were significantly greater than nonagricultural sediments and controls. While flooding sediments with N-enriched solution had no clear effect on urea-N, warmer (27°C) temperatures resulted in significantly higher urea-N. Data collected from field ditches that were flooded by a summer rainstorm showed increases in urea-N that mirrored the mesocosm experiment. We postulate that concentrations of urea-N in ditches that greatly exceed environmental thresholds are mediated by biological production in sediments and release to stagnant surface water. Storm-driven urea-N export from ditches could elevate the risk of harmful algal blooms downstream in receiving waters despite the dilution effect.

Leptin expression affects metabolic rate in zebrafish embryos (D. rerio).

We used antisense morpholino oligonucleotide technology to knockdown leptin-(A) gene expression in developing zebrafish embryos and measured its effects on metabolic rate and cardiovascular function. Using two indicators of metabolic rate, oxygen consumption was significantly lower in leptin morphants early in development [<48 hours post-fertilization (hpf)], while acid production was significantly lower in morphants later in development (>48 hpf). Oxygen utilization rates in <48 hpf embryos and acid production in 72 hpf embryos could be rescued to that of wildtype embryos by recombinant leptin coinjected with antisense morpholino. Leptin is established to influence metabolic rate in mammals, and these data suggest leptin signaling also influences metabolic rate in fishes.