The mechanism of action of silver nanoparticles (AgNPs) is unclear due to the particles’ strong tendency to agglomerate. Preventing agglomeration could offer precise control of the physicochemical properties that drive biological response to AgNPs. In an attempt to control agglomeration, we exposed zebrafish embryos to AgNPs of 20 or 110 nm core size, and polypyrrolidone (PVP) or citrate surface coatings in media of varying ionic strength. AgNPs remained unagglomerated in 62.5 μM CaCl2 (CaCl2) and ultrapure water (UP), but not in standard zebrafish embryo medium (EM). Zebrafish embryos developed normally in the low ionic strength environments of CaCl2 and UP. Exposure of embryos to AgNPs suspended in UP and CaCl2 resulted in higher toxicity than suspensions in EM. 20 nm AgNPs were more toxic than 110 nm AgNPs, and the PVP coating was more toxic than the citrate coating at the same particle core size. The silver tissue burden correlated well with observed toxicity but only for those exposures where the AgNPs remained unagglomerated. Our results demonstrate that size- and surface coating-dependent toxicity is a result of AgNPs remaining unagglomerated, and thus a critical-design consideration for experiments to offer meaningful evaluations of AgNP toxicity.

The fish early life-stage (FELS) test (OECD Test Guideline 210) is the primary test used internationally to estimate chronic fish toxicity in support of ecological risk assessments and chemical management programs. As part of an on-going effort to develop efficient and cost-effective alternatives to the FELS test, there is a need to identify and describe potential adverse outcome pathways (AOPs) relevant to FELS toxicity. To support this endeavor, we outline and illustrate an overall strategy for discovery and annotation of FELS AOPs. Key events represented by major developmental landmarks were organized into a preliminary conceptual model of fish development. Using swimbladder inflation as an example, a weight-of-evidence-based approach was used to support linkage of key molecular initiating events to adverse phenotypic outcomes and reduced young-of-year survival. Based on an iterative approach, we explored the feasibility of using key events as the foundation for expanding a network of plausible linkages and AOP knowledge and, in the process, identify important knowledge gaps. Given the scope and scale of the task, prioritization of AOP development was recommended and key research objectives were defined relative to factors such as current animal use restrictions in the European Union and increased demands for fish toxicity data in chemical management programs globally. The example and strategy described are intended to guide collective efforts to define FELS-related AOPs and develop resource-efficient predictive assays that address the toxicological domain of the OECD 210 test.

Synthetic glucocorticoids are pharmaceutical compounds prescribed in human and veterinary medicine as anti-inflammatory agents and have the potential to contaminate natural watersheds via inputs from wastewater treatment facilities and confined animal-feeding operations. Despite this, few studies have examined the effects of this class of chemicals on aquatic vertebrates. To generate data to assess potential risk to the aquatic environment, we used fathead minnow 21-d reproduction and 29-d embryo-larvae assays to determine reproductive toxicity and early-life-stage effects of dexamethasone. Exposure to 500 µg dexamethasone/L in the 21-d test caused reductions in fathead minnow fecundity and female plasma estradiol concentrations and increased the occurrence of abnormally hatched fry. Female fish exposed to 500 µg dexamethasone/L also displayed a significant increase in plasma vitellogenin protein levels, possibly because of decreased spawning. A decrease in vitellogenin messenger ribonucleic acid (mRNA) expression in liver tissue from females exposed to the high dexamethasone concentration lends support to this hypothesis. Histological results indicate that a 29-d embryo-larval exposure to 500 µg dexamethasone/L caused a significant increase in deformed gill opercula. Fry exposed to 500 µg dexamethasone/L for 29 d also exhibited a significant reduction in weight and length compared with control fry. Taken together, these results indicate that nonlethal concentrations of a model glucocorticoid receptor agonist can impair fish reproduction, growth, and development.

Engineered metal oxide nanoparticles (MO NPs) are finding increasing utility in the medical field as anticancer agents. Before validation of in vivo anticancer efficacy can occur, a better understanding of whole-animal toxicity is required. We compared the toxicity of seven widely used semiconductor MO NPs made from zinc oxide (ZnO), titanium dioxide, cerium dioxide and tin dioxide prepared in pure water and in synthetic seawater using a five-day embryonic zebrafish assay. We hypothesized that the toxicity of these engineered MO NPs would depend on physicochemical properties. Significant agglomeration of MO NPs in aqueous solutions is common making it challenging to associate NP characteristics such as size and charge with toxicity. However, data from our agglomerated MO NPs suggests that the elemental composition and dissolution potential are major drivers of toxicity. Only ZnO caused significant adverse effects of all MO particles tested, and only when prepared in pure water (point estimate median lethal concentration = 3.5–9.1 mg/L). This toxicity was life stage dependent. The 24 h toxicity increased greatly (~22.7 fold) when zebrafish exposures started at the larval life stage compared to the 24 hour toxicity following embryonic exposure. Investigation into whether dissolution could account for ZnO toxicity revealed high levels of zinc ion (40–89% of total sample) were generated. Exposure to zinc ion equivalents revealed dissolved Zn2+ may be a major contributor to ZnO toxicity.

The vertebrate hypothalamic-pituitary-gonadal (HPG) axis is controlled through various feedback mechanisms that maintain a dynamic homeostasis in the face of changing environmental conditions, including exposure to chemicals. We assessed the effects of prochloraz on HPG axis function in adult fathead minnows (Pimephales promelas) at multiple sampling times during 8-day exposure and 8-day depuration/recovery phases. Consistent with one mechanism of action of prochloraz, inhibition of cytochrome P450 (CYP) 19 aromatase activity, the fungicide depressed ex vivo ovarian production and plasma concentrations of 17b-estradiol (E2) in female fish. At a prochloraz water concentration of 30 mg/l, inhibitory effects on E2 production were transitory and did not persist during the 8-day exposure phase. At 300 mg/l prochloraz, inhibition of E2 production was evident throughout the 8-day exposure but steroid titers recovered within 1 day of cessation of exposure. Compensation or recovery of steroid production in prochloraz-exposed females was accompanied by upregulation of several ovarian genes associated with steroidogenesis, including cyp19a1a, cyp17 (hydroxylase/lyase), cyp11a (cholesterol side-chain cleavage), and follicle-stimulating hormone receptor. In male fathead minnows, the 8-day prochloraz exposure decreased testosterone (T) production, possibly through inhibition of CYP17. However, as for E2 in females, ex vivo testicular production and plasma concentrations of T recovered within 1 day of stopping exposure. Steroidogenic genes upregulated in testis included cyp17 and cyp11a. These studies demonstrate the adaptability of the HPG axis to chemical stress and highlight the need to consider the dynamic nature of the system when developing approaches to assess potential risks of endocrine-active chemicals.