Predictive value of the ToxCast/Tox21 high throughput toxicity screening data for approximating in vivo ecotoxicity endpoints and ecotoxicological risk in eco- surveillance applications.

Ecotoxicology has long relied on assessing the hazard potential of chemicals through traditional in vivo testing methods to understand the possible risk exposure could pose to ecological taxa. In the past decade, the development of non-animal new approach methods (NAMs) for assessing chemical hazard and risk has quickly grown. These methods are often cheaper and faster than traditional toxicity testing, and thus are amenable to high-throughput toxicity testing (HTT), resulting in large datasets. The ToxCast/Tox21 HTT programs have produced in vitro data for thousands of chemicals covering a large space of biological activity. The relevance of these data to in vivo mammalian toxicity has been much explored. Interest has also grown in using these data to evaluate the risk of environmental exposures to taxa of ecological importance such as fish, aquatic invertebrates, etc.; particularly for the purpose of estimating the risk of exposure from real-world complex mixtures. Understanding the relationship and relative sensitivity of NAMs versus standardized ecotoxicological whole organism models is a key component of performing reliable read-across from mammalian in vitro data to ecotoxicological in vivo data. In this work, we explore the relationship between in vivo ecotoxicity data from several publicly available databases and the ToxCast/Tox21 data. We also performed several case studies in which we compare how using different ecotoxicity datasets, whether traditional or ToxCast-based, affects risk conclusions based on exposure to complex mixtures derived from existing large-scale chemical monitoring data. Generally, predictive value of ToxCast data for traditional in vivo endpoints (EPs) was poor (r ≤ 0.3). Risk conclusions, including identification of different chemical risk drivers and prioritized monitoring sites, were different when using HTT data vs. traditional in vivo data.

A generic avian physiologically-based kinetic (PBK) model and its application in three bird species.

Physiologically-based kinetic (PBK) models are effective tools for designing toxicological studies and conducting extrapolations to inform hazard characterization in risk assessment by filling data gaps and defining safe levels of chemicals. In the present work, a generic avian PBK model for male and female birds was developed using PK-Sim and MoBi from the Open Systems Pharmacology Suite (OSPS). The PBK model includes an ovulation model (egg development) to predict concentrations of chemicals in eggs from dietary exposure. The model was parametrized for chicken (Gallus gallus), bobwhite quail (Colinus virginianus) and mallard duck (Anas platyrhynchos) and was tested with nine chemicals for which in vivo studies were available. Time-concentration profiles of chemicals reaching tissues and egg compartment were simulated and compared to in vivo data. The overall accuracy of the PBK model predictions across the analyzed chemicals was good. Model simulations were found to be in the range of 22-79% within a 3-fold and 41-89% were within 10- fold deviation of the in vivo observed data. However, for some compounds scarcity of in-vivo data and inconsistencies between published studies allowed only a limited goodness of fit evaluation. The generic avian PBK model was developed following a "best practice" workflow describing how to build a PBK model for novel species. The credibility and reproducibility of the avian PBK models were scored by evaluation according to the available guidance documents from WHO (2010), and OECD (2021), to increase applicability, confidence and acceptance of these in silico models in chemical risk assessment.

Quantitative Morphometric, Physiological, and Metabolic Characteristics of Chickens and Mallards for Physiologically Based Kinetic Model Development.

Physiologically based kinetic (PBK) models are a promising tool for xenobiotic environmental risk assessment that could reduce animal testing by predicting in vivo exposure. PBK models for birds could further our understanding of species-specific sensitivities to xenobiotics, but would require species-specific parameterization. To this end, we summarize multiple major morphometric and physiological characteristics in chickens, particularly laying hens (Gallus gallus) and mallards (Anas platyrhynchos) in a meta-analysis of published data. Where such data did not exist, data are substituted from domesticated ducks (Anas platyrhynchos) and, in their absence, from chickens. The distribution of water between intracellular, extracellular, and plasma is similar in laying hens and mallards. Similarly, the lengths of the components of the small intestine (duodenum, jejunum, and ileum) are similar in chickens and mallards. Moreover, not only are the gastrointestinal absorptive areas similar in mallard and chickens but also they are similar to those in mammals when expressed on a log basis and compared to log body weight. In contrast, the following are much lower in laying hens than mallards: cardiac output (CO), hematocrit (Hct), and blood hemoglobin. There are shifts in ovary weight (increased), oviduct weight (increased), and plasma/serum concentrations of vitellogenin and triglyceride between laying hens and sexually immature females. In contrast, reproductive state does not affect the relative weights of the liver, kidneys, spleen, and gizzard.

Quantitative Comparison of Avian and Mammalian Physiologies for Parameterization of Physiologically Based Kinetic Models.

Physiologically based kinetic (PBK) models facilitate chemical risk assessment by predicting in vivo exposure while reducing the need for animal testing. PBK models for mammals have seen significant progress, which has yet to be achieved for avian systems. Here, we quantitatively compare physiological, metabolic and anatomical characteristics between birds and mammals, with the aim of facilitating bird PBK model development. For some characteristics, there is considerable complementarity between avian and mammalian species with identical values for the following: blood hemoglobin and hemoglobin concentrations per unit erythrocyte volume together with relative weights of the liver, heart, and lungs. There are also systematic differences for some major characteristics between avian and mammalian species including erythrocyte volume, plasma concentrations of albumin, total protein and triglyceride together with liver cell size and relative weights of the kidney, spleen, and ovary. There are also major differences between characteristics between sexually mature and sexually immature female birds. For example, the relative weights of the ovary and oviduct are greater in sexually mature females compared to immature birds as are the plasma concentrations of triglyceride and vitellogenin. Both these sets of differences reflect the genetic "blue print" inherited from ancestral archosaurs such as the production of large eggs with yolk filled oocytes surrounded by egg white proteins, membranes and a calciferous shell together with adaptions for flight in birds or ancestrally in flightless birds.

Utility of the avian sub-acute dietary toxicity test in ecological risk assessment and a path forward to reduce animal use.

The United States Environmental Protection Agency (USEPA) has long required both avian sub-acute dietary and acute oral studies to inform risk assessments for pesticides. Recently, the USEPA collaborated with People for the Ethical Treatment of Animals to determine whether the results of the acute oral avian toxicity test or the sub-acute dietary toxicity test consistently generated the greatest risk predictions in USEPA tier 1 assessments for pesticides first registered between 1998 and 2017. Their study concluded that in 99% of the cases, risk conclusions were driven by the acute oral study (OPPTS 850.2100, OCSPP 850.2100, or similar) because using these data results in higher risk quotients than sub-acute dietary data. Shortly after publishing these results, the USEPA released a formal memorandum providing guidance for waiving the sub-acute dietary study for most pesticides. The USEPA will, however, retain the option to require sub-acute dietary studies for pesticides with certain chemical properties. However, as the avian sub-acute dietary study has an exposure regimen that is often more representative of how birds are exposed to pesticides under actual use conditions than does the acute oral study (i.e., as part of a dietary item eaten over the course of a day and not a bolus dose), this study can provide useful context for risk assessment on a case-by-case basis. Decision criteria are needed to determine a path forward that both minimizes vertebrate animal testing and positions the avian sub-acute dietary data as an option for risk refinement. Decision criteria are proposed here with recommendations for refining the design of avian sub-acute dietary studies to ensure that the data generated are optimized to support a science-based acute avian risk assessment, supported by a case study demonstrating when and how sub-acute dietary studies may be used in a higher-tier risk assessment. Integr Environ Assess Manag 2022;18:1629-1638. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

The Extended Amphibian Metamorphosis Assay: A Thyroid-Specific and Less Animal-Intensive Alternative to the Larval Amphibian Growth and Development Assay.

The amphibian metamorphosis assay (AMA; US Environmental Protection Agency [USEPA] test guideline 890.1100 and Organisation for Economic Co-Operation and Development test guideline 231) has been used for more than a decade to assess the potential thyroid-mediated endocrine activity of chemicals. In 2013, in the context of the Endocrine Disruptor Screening Program of the USEPA, a Scientific Advisory Panel reviewed the results from 18 studies and recommended changes to the AMA test guideline, including a modification to a fixed-stage design rather than a fixed-time (i.e., 21-d) design. We describe an extended test design for the AMA (or EAMA) that includes thyroid histopathology and time to metamorphosis (Nieuwkoop-Faber [NF] stage 62), to address both the issues with the fixed-time design and the specific question of thyroid-mediated adversity in a shorter assay than the larval amphibian growth and development assay (LAGDA; Organisation for Economic Co-Operation and Development test guideline 241), using fewer animals and resources. A demonstration study was conducted with the EAMA (up to NF stage 58) using sodium perchlorate. Data analyses and interpretation of the fixed-stage design of the EAMA are more straightforward than the fixed-time design because the fixed-stage design avoids confounded morphometric measurements and thyroid histopathology caused by varying developmental stages at test termination. It also results in greater statistical power to detect metamorphic delays than the fixed-time design. By preferentially extending the AMA to NF stage 62, suitable data can be produced to evaluate thyroid-mediated adversity and preclude the need to perform a LAGDA for thyroid mode of action analysis. The LAGDA remains of further interest should investigations of longer term effects related to sexual development modulated though the hypothalamus-pituitary-gonadal axis be necessary. However, reproduction assessment or life cycle testing is currently not addressed in the LAGDA study design. This is better addressed by higher tier studies in fish, which should then include specific thyroid-related endpoints. Environ Toxicol Chem 2021;40:2135-2144. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Critical Review of Read-Across Potential in Testing for Endocrine-Related Effects in Vertebrate Ecological Receptors.

Recent regulatory testing programs have been designed to evaluate whether a chemical has the potential to interact with the endocrine system and could cause adverse effects. Some endocrine pathways are highly conserved among vertebrates, providing a potential to extrapolate data generated for one vertebrate taxonomic group to others (i.e., biological read-across). To assess the potential for biological read-across, we reviewed tools and approaches that support species extrapolation for fish, amphibians, birds, and reptiles. For each of the estrogen, androgen, thyroid, and steroidogenesis (EATS) pathways, we considered the pathway conservation across species and the responses of endocrine-sensitive endpoints. The available data show a high degree of confidence in the conservation of the hypothalamus-pituitary-gonadal axis between fish and mammals and the hypothalamus-pituitary-thyroid axis between amphibians and mammals. Comparatively, there is less empirical evidence for the conservation of other EATS pathways between other taxonomic groups, but this may be due to limited data. Although more information on sensitive pathways and endpoints would be useful, current developments in the use of molecular target sequencing similarity tools and thoughtful application of the adverse outcome pathway concept show promise for further advancement of read-across approaches for testing EATS pathways in vertebrate ecological receptors. Environ Toxicol Chem 2020;39:739-753. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Primary Cell Phenotypic Screening Illuminates ADRs and AOPs.

Preclinical, in vitro screening for adverse drug reactions continues to present challenges in the field of drug development. In this issue of Cell Chemical Biology, Shah et al. (2017) employ a phenotypic screening strategy using a panel of human primary cells to define a signature response and an adverse outcome pathway for delayed type IV skin hypersensitivity.

AHR2 morpholino knockdown reduces the toxicity of total particulate matter to zebrafish embryos.

The zebrafish embryo has been proposed as a 'bridge model' to study the effects of cigarette smoke on early development. Previous studies showed that exposure to total particulate matter (TPM) led to adverse effects in developing zebrafish, and suggested that the antioxidant and aryl hydrocarbon receptor (AHR) pathways play important roles. This study investigated the roles of these two pathways in mediating TPM toxicity. The study consisted of four experiments. In experiment I, zebrafish embryos were exposed from 6h post fertilization (hpf) until 96hpf to TPM0.5 and TPM1.0 (corresponding to 0.5 and 1.0µg/mL equi-nicotine units) in the presence or absence of an antioxidant (N-acetyl cysteine/NAC) or a pro-oxidant (buthionine sulfoximine/BSO). In experiment II, TPM exposures were performed in embryos that were microinjected with nuclear factor erythroid 2-related factor 2 (Nrf2), AHR2, cytochrome P450 1A (CYP1A), or CYP1B1 morpholinos, and deformities were assessed. In experiment III, embryos were exposed to TPM, and embryos/larvae were collected at 24, 48, 72, and 96hpf to assess several genes associated with the antioxidant and AHR pathways. Lastly, experiment IV assessed the activity and protein levels of CYP1A and CYP1B1 after exposure to TPM. We demonstrate that the incidence of TPM-induced deformities was generally not affected by NAC/BSO treatments or Nrf2 knockdown. In contrast, AHR2 knockdown reduced, while CYP1A or CYP1B1 knockdowns elevated the incidence of some deformities. Moreover, as shown by gene expression the AHR pathway, but not the antioxidant pathway, was induced in response to TPM exposure, providing further evidence for its importance in mediating TPM toxicity.

Silver toxicity across salinity gradients: the role of dissolved silver chloride species (AgCl x ) in Atlantic killifish (Fundulus heteroclitus) and medaka (Oryzias latipes) early life-stage toxicity.

The influence of salinity on Ag toxicity was investigated in Atlantic killifish (Fundulus heteroclitus) early life-stages. Embryo mortality was significantly reduced as salinity increased and Ag(+) was converted to AgCl(solid). However, as salinity continued to rise (>5 ‰), toxicity increased to a level at least as high as observed for Ag(+) in deionized water. Rather than correlating with Ag(+), Fundulus embryo toxicity was better explained (R(2) = 0.96) by total dissolved Ag (Ag(+), AgCl2 (-), AgCl3 (2-), AgCl4 (3-)). Complementary experiments were conducted with medaka (Oryzias latipes) embryos to determine if this pattern was consistent among evolutionarily divergent euryhaline species. Contrary to Fundulus data, medaka toxicity data were best explained by Ag(+) concentrations (R(2) = 0.94), suggesting that differing ionoregulatory physiology may drive observed differences. Fundulus larvae were also tested, and toxicity did increase at higher salinities, but did not track predicted silver speciation. Alternatively, toxicity began to increase only at salinities above the isosmotic point, suggesting that shifts in osmoregulatory strategy at higher salinities might be an important factor. Na(+) dysregulation was confirmed as the mechanism of toxicity in Ag-exposed Fundulus larvae at both low and high salinities. While Ag uptake was highest at low salinities for both Fundulus embryos and larvae, uptake was not predictive of toxicity.

Silver nanoparticle toxicity to Atlantic killifish (Fundulus heteroclitus) and Caenorhabditis elegans: a comparison of mesocosm, microcosm, and conventional laboratory studies.

The use of silver nanoparticles (AgNPs) in consumer products and industrial applications, as well as their recent detection in waste streams, has created concern about potential impacts on aquatic ecosystems. The effect of complex environmental media on AgNP toxicity was investigated using wetland mesocosms and smaller scale microcosms. Mesocosms were dosed with 2.5 mg Ag/L as gum arabic (GA)-coated AgNPs, polyvinylpyrrolidone (PVP)-coated AgNPs, or AgNO3. Water samples were taken from mesocosms 24 h after dosing for acute toxicity tests with embryos and larvae of Atlantic killifish (Fundulus heteroclitus) and the nematode Caenorhabditis elegans. Acute toxicity tests were also performed on Atlantic killifish with AgNO3, GA AgNPs, and PVP AgNPs prepared in the laboratory with similar water. For killifish embryos, mesocosm samples were much less toxic than laboratory samples for all types of silver. For larvae, in contrast, all 3 silver mesocosm treatments exhibited toxicity. Interestingly, mesocosm samples of AgNO3 were less toxic than laboratory samples; samples containing GA AgNPs were similar in toxicity, and samples containing PVP AgNPs were more toxic. For C. elegans, results were similar to killifish larvae. Results obtained from the mesocosms were not replicated on the smaller scale of the microcosms. These results indicate that environmental factors unique to the mesocosms acted differentially on AgNO3 to reduce its toxicity in a manner that does not translate to AgNPs for larval fish.

Sulfidation of silver nanoparticles: natural antidote to their toxicity.

Nanomaterials are highly dynamic in biological and environmental media. A critical need for advancing environmental health and safety research for nanomaterials is to identify physical and chemical transformations that affect the nanomaterial properties and their toxicity. Silver nanoparticles, one of the most toxic and well-studied nanomaterials, readily react with sulfide to form Ag(0)/Ag2S core-shell particles. Here, we show that sulfidation decreased silver nanoparticle toxicity to four diverse types of aquatic and terrestrial eukaryotic organisms (Danio rerio (zebrafish), Fundulus heteroclitus (killifish), Caenorhabditis elegans (nematode worm), and the aquatic plant Lemna minuta (least duckweed)). Toxicity reduction, which was dramatic in killifish and duckweed even for low extents of sulfidation (about 2 mol % S), is primarily associated with a decrease in Ag(+) concentration after sulfidation due to the lower solubility of Ag2S relative to elemental Ag (Ag(0)). These results suggest that even partial sulfidation of AgNP will decrease the toxicity of AgNPs relative to their pristine counterparts. We also show that, for a given organism, the presence of chloride in the exposure media strongly affects the toxicity results by affecting Ag speciation. These results highlight the need to consider environmental transformations of NPs in assessing their toxicity to accurately portray their potential environmental risks.

Biotic and abiotic interactions in aquatic microcosms determine fate and toxicity of Ag nanoparticles: part 2-toxicity and Ag speciation.

To study the effects of complex environmental media on silver nanoparticle (AgNP) toxicity, AgNPs were added to microcosms with freshwater sediments and two species of aquatic plants (Potamogeton diversifolius and Egeria densa), followed by toxicity testing with microcosm surface water. Microcosms were designed with four environmental matrices in order to determine the contribution of each environmental compartment to changes in toxicity: water only (W), water + sediment (WS), water + plants (WP), and water + plants + sediment (WPS). Silver treatments included AgNPs with two different coatings, gum arabic (GA-AgNPs) or polyvinylpyrollidone (PVP-AgNPs), as well as AgNO(3). Water samples taken from the microcosms at 24 h postdosing were used in acute toxicity tests with two standard model organisms, early life stage zebrafish (Danio rerio) and Daphnia magna. Speciation of Ag in these samples was analyzed using Ag L3-edge X-ray absorption near edge spectroscopy (XANES). Silver speciation patterns for the nanoparticle treatments varied significantly by coating type. While PVP-AgNPs were quite stable and resisted transformation across all matrices (>92.4% Ag(0)), GA-AgNP speciation patterns suggest significantly higher transformation rates, especially in treatments with plants (<69.2% and <58.8% Ag(0) in WP and WPS, respectively) and moderately increased transformation with sediments (<85.6% Ag(0)). Additionally, the presence of plants in the microcosms (with and without sediments) reduced both the concentration of Ag in the water column and toxicity for all Ag treatments. Reductions in toxicity may have been related to decreased water column concentrations as well as changes in the surface chemistry of the particles induced by organic substances released from the plants.

Long-term transformation and fate of manufactured ag nanoparticles in a simulated large scale freshwater emergent wetland.

Transformations and long-term fate of engineered nanomaterials must be measured in realistic complex natural systems to accurately assess the risks that they may pose. Here, we determine the long-term behavior of poly(vinylpyrrolidone)-coated silver nanoparticles (AgNPs) in freshwater mesocosms simulating an emergent wetland environment. AgNPs were either applied to the water column or to the terrestrial soils. The distribution of silver among water, solids, and biota, and Ag speciation in soils and sediment was determined 18 months after dosing. Most (70 wt %) of the added Ag resided in the soils and sediments, and largely remained in the compartment in which they were dosed. However, some movement between soil and sediment was observed. Movement of AgNPs from terrestrial soils to sediments was more facile than from sediments to soils, suggesting that erosion and runoff is a potential pathway for AgNPs to enter waterways. The AgNPs in terrestrial soils were transformed to Ag(2)S (~52%), whereas AgNPs in the subaquatic sediment were present as Ag(2)S (55%) and Ag-sulfhydryl compounds (27%). Despite significant sulfidation of the AgNPs, a fraction of the added Ag resided in the terrestrial plant biomass (~3 wt % for the terrestrially dosed mesocosm), and relatively high body burdens of Ag (0.5-3.3 µg Ag/g wet weight) were found in mosquito fish and chironomids in both mesocosms. Thus, Ag from the NPs remained bioavailable even after partial sulfidation and when water column total Ag concentrations are low (<0.002 mg/L).

Biotic and abiotic interactions in aquatic microcosms determine fate and toxicity of Ag nanoparticles. Part 1. Aggregation and dissolution.

To better understand their fate and toxicity in aquatic environments, we compared the aggregation and dissolution behavior of gum arabic (GA) and polyvinylpyrrolidone (PVP) coated Ag nanoparticles (NPs) in aquatic microcosms. There were four microcosm types: surface water; water and sediment; water and aquatic plants; or water, sediment, and aquatic plants. Dissolution and aggregation behavior of AgNPs were examined using ultracentrifugation, ultrafiltration, and asymmetrical flow field flow fractionation coupled to ultraviolet-visible spectroscopy, dynamic and static laser light scattering, and inductively coupled plasma mass spectrometry. Plants released dissolved organic matter (DOM) into the water column either through active or passive processes in response to Ag exposure. This organic matter fraction readily bound Ag ions. The plant-derived DOM had the effect of stabilizing PVP-AgNPs as primary particles, but caused GA-AgNPs to be removed from the water column, likely by dissolution and binding of released Ag ions on sediment and plant surfaces. The destabilization of the GA-AgNPs also corresponded with X-ray absorption near edge spectroscopy results which suggest that 22-28% of the particulate Ag was associated with thiols and 5-14% was present as oxides. The results highlight the potential complexities of nanomaterial behavior in response to biotic and abiotic modifications in ecosystems, and may help to explain differences in toxicity of Ag observed in realistic exposure media compared to simplified laboratory exposures.