Examining effects of a novel estrogenic perfluoro-alcohol, 1H,1H,8H,8H-Perfluorooctane-1,8-diol (FC8-diol), using the fathead minnow EcoToxChip.

In a previous in vivo study, adult male fathead minnows (Pimephales promelas) were exposed via water for 4 days to 1H,1H,8H,8H-perfluorooctane-1,8-diol (FC8-diol). The present study expands on the evaluation of molecular responses to this perfluoro-alcohol by analyzing 26 male fathead minnow liver RNA samples from that study (five from each test concentration: 0, 0.018, 0.

Survival, Growth, and Reproduction Responses in a Three-Generation Exposure of the Zebrafish (Danio rerio) to Perfluorooctane Sulfonate.

A prior multigenerational perfluorooctane sulfonic acid (PFOS) exposure investigation in zebrafish reported adverse effects at 0.734 µg/L, among the lowest aquatic effect levels for PFOS reported to date. The present three-generation PFOS exposure quantified survival, growth, reproduction, and vitellogenin (VTG; egg yolk protein) responses in zebrafish, incorporating experimental design and procedural improvements relative to the earlier study.

Putative adverse outcome pathway development based on physiological responses of female fathead minnows to model estrogen versus androgen receptor agonists.

Several adverse outcome pathways (AOPs) have linked molecular initiating events like aromatase inhibition, androgen receptor (AR) agonism, and estrogen receptor (ER) antagonism to reproductive impairment in adult fish. Estrogen receptor agonists can also cause adverse reproductive effects, however, the early key events (KEs) in an AOP leading to this are mostly unknown. The primary aim of this study was to develop hypotheses regarding the potential mechanisms through which exposure to ER agonists might lead to reproductive impairment in female fish.

AOP Report: Adverse Outcome Pathways for Aromatase Inhibition or Androgen Receptor Agonism Leading to Male-Biased Sex Ratio and Population Decline in Fish.

Screening and testing of potential endocrine-disrupting chemicals for ecological effects are examples of risk assessment/regulatory activities that can employ adverse outcome pathways (AOPs) to establish linkages between readily measured alterations in endocrine function and whole organism- and population-level responses. Of particular concern are processes controlled by the hypothalamic-pituitary-gonadal/thyroidal (HPG/T) axes. However, the availability of AOPs suitable to meet this need is currently limited in terms of species and life-stage representation relative to the diversity of endpoints influenced by HPG/T function.

Verification of In Vivo Estrogenic Activity for Four Per- and Polyfluoroalkyl Substances (PFAS) Identified as Estrogen Receptor Agonists via New Approach Methodologies.

Given concerns about potential toxicological hazards of the thousands of data-poor per- and polyfluorinated alkyl substances (PFAS) currently in commerce and detected in the environment, tiered testing strategies that employ high-throughput in vitro screening as an initial testing tier have been implemented. The present study evaluated the effectiveness of previous in vitro screening for identifying PFAS capable, or incapable, of inducing estrogenic responses in fish exposed in vivo. Fathead minnows () were exposed for 96 h to five PFAS (perfluorooctanoic acid [PFOA]; 1H,1H,8H,8H-perfluorooctane-1,8-diol [FC8-diol]; 1H,1H,10H,10H-perfluorodecane-1,10-diol [FC10-diol]; 1H,1H,8H,8H-perfluoro-3,6-dioxaoctane-1,8-diol [FC8-DOD]; and perfluoro-2-methyl-3-oxahexanoic acid [HFPO-DA]) that showed varying levels of in vitro estrogenic potency.

Evaluation of Complex Mixture Toxicity in the Milwaukee Estuary (WI, USA) Using Whole-Mixture and Component-Based Evaluation Methods.

Anthropogenic activities introduce complex mixtures into aquatic environments, necessitating mixture toxicity evaluation during risk assessment. There are many alternative approaches that can be used to complement traditional techniques for mixture assessment. Our study aimed to demonstrate how these approaches could be employed for mixture evaluation in a target watershed.

A framework for prioritizing contaminants in retrospective ecological assessments: Application in the Milwaukee Estuary (Milwaukee, WI).

Watersheds are subjected to diverse anthropogenic inputs, exposing aquatic biota to a wide range of chemicals. Detection of multiple, different chemicals can challenge natural resource managers who often have to determine where to allocate potentially limited resources. Here, we describe a weight-of-evidence framework for retrospectively prioritizing aquatic contaminants.

Case Study in 21st-Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Reproductive Outcomes in Fish In Vivo.

To reduce the use of intact animals for chemical safety testing, while ensuring protection of ecosystems and human health, there is a demand for new approach methodologies (NAMs) that provide relevant scientific information at a quality equivalent to or better than traditional approaches. The present case study examined whether bioactivity and associated potency measured in an in vitro screening assay for aromatase inhibition could be used together with an adverse outcome pathway (AOP) and mechanistically based computational models to predict previously uncharacterized in vivo effects. Model simulations were used to inform designs of 60-h and 10-21-day in vivo exposures of adult fathead minnows (Pimephales promelas) to three or four test concentrations of the in vitro aromatase inhibitor imazalil ranging from 0.

Effects of Metformin and its Metabolite Guanylurea on Fathead Minnow (Pimephales promelas) Reproduction.

Metformin, along with its biotransformation product guanylurea, is commonly observed in municipal wastewaters and subsequent surface waters. Previous studies in fish have identified metformin as a potential endocrine-active compound, but there are inconsistencies with regard to its effects. To further investigate the potential reproductive toxicity of metformin and guanylurea to fish, a series of experiments was performed with adult fathead minnows (Pimephales promelas).

Assessing effects of aromatase inhibition on fishes with group-synchronous oocyte development using western mosquitofish (Gambusia affinis) as a model.

Exposure to certain anthropogenic chemicals can inhibit the activity to cytochrome P450 aromatase (CYP19) in fishes leading to decreased plasma 17β-estradiol (E2), plasma vitellogenin (VTG), and egg production. Reproductive dysfunction resulting from exposure to aromatase inhibitors has been extensively investigated in several laboratory model species of fish. These model species have ovaries that undergo asynchronous oocyte development, but many fishes have ovaries with group-synchronous oocyte development.

Case Study in 21st Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Short-Term In Vivo Responses in Adult Female Fish.

The present study evaluated whether in vitro measures of aromatase inhibition as inputs into a quantitative adverse outcome pathway (qAOP) construct could effectively predict in vivo effects on 17β-estradiol (E2) and vitellogenin (VTG) concentrations in female fathead minnows. Five chemicals identified as aromatase inhibitors in mammalian-based ToxCast assays were screened for their ability to inhibit fathead minnow aromatase in vitro. Female fathead minnows were then exposed to 3 of those chemicals: letrozole, epoxiconazole, and imazalil in concentration-response (5 concentrations plus control) for 24 h.

Adverse Outcome Pathway Network-Based Assessment of the Interactive Effects of an Androgen Receptor Agonist and an Aromatase Inhibitor on Fish Endocrine Function.

Predictive approaches to assessing the toxicity of contaminant mixtures have been largely limited to chemicals that exert effects through the same biological molecular initiating event. However, by understanding specific pathways through which chemicals exert effects, it may be possible to identify shared "downstream" nodes as the basis for forecasting interactive effects of chemicals with different molecular initiating events. Adverse outcome pathway (AOP) networks conceptually support this type of analysis.

Quantitative Response-Response Relationships Linking Aromatase Inhibition to Decreased Fecundity are Conserved Across Three Fishes with Asynchronous Oocyte Development.

Quantitative adverse outcome pathways (qAOPs) describe quantitative response-response relationships that can predict the probability or severity of an adverse outcome for a given magnitude of chemical interaction with a molecular initiating event. However, the taxonomic domain of applicability for these predictions is largely untested. The present study began defining this applicability for a previously described qAOP for aromatase inhibition leading to decreased fecundity developed using data from fathead minnow ().

Differential Sensitivity to In Vitro Inhibition of Cytochrome P450 Aromatase (CYP19) Activity Among 18 Freshwater Fishes.

There is significant concern regarding potential impairment of fish reproduction associated with endocrine disrupting chemicals. Aromatase (CYP19) is a steroidogenic enzyme involved in the conversion of androgens to estrogens. Inhibition of aromatase by chemicals can result in reduced concentrations of estrogens leading to adverse reproductive effects.

Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows (Pimephales promelas).

Cytochrome P450 aromatase catalyzes conversion of C19 androgens to C18 estrogens and is critical for normal reproduction in female vertebrates. Fadrozole is a model aromatase inhibitor that has been shown to suppress estrogen production in the ovaries of fish. However, little is known about the early impacts of aromatase inhibition on steroid production and gene expression in fish.

Prioritization of Contaminants of Emerging Concern in Wastewater Treatment Plant Discharges Using Chemical:Gene Interactions in Caged Fish.

We examined whether contaminants present in surface waters could be prioritized for further assessment by linking the presence of specific chemicals to gene expression changes in exposed fish. Fathead minnows were deployed in cages for 2, 4, or 8 days at three locations near two different wastewater treatment plant discharge sites in the Saint Louis Bay, Duluth, MN and one upstream reference site. The biological impact of 51 chemicals detected in the surface water of 133 targeted chemicals was determined using biochemical endpoints, exposure activity ratios for biological and estrogenic responses, known chemical:gene interactions from biological pathways and knowledge bases, and analysis of the covariance of ovary gene expression with surface water chemistry.

Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid.

Inflation of the posterior and/or anterior swim bladder is a process previously demonstrated to be regulated by thyroid hormones. We investigated whether inhibition of deiodinases, which convert thyroxine (T4) to the more biologically active form, 3,5,3'-triiodothyronine (T3), would impact swim bladder inflation. Two experiments were conducted using a model deiodinase inhibitor, iopanoic acid (IOP).

Re-evaluating the Significance of Estrone as an Environmental Estrogen.

Studies worldwide have demonstrated the occurrence of feminized male fish at sites impacted by human and animal wastes. A variety of chemicals could contribute to this phenomenon, but those receiving the greatest attention in terms of research and monitoring have been 17β-estradiol (β-E2) and 17α-ethinylestradiol, due both to their prevalence in the environment and strong estrogenic potency. A third steroid, estrone (E1), also can occur at high concentrations in surface waters but generally has been of lesser concern due to its relatively lower affinity for vertebrate estrogen receptors.

Derivation and Evaluation of Putative Adverse Outcome Pathways for the Effects of Cyclooxygenase Inhibitors on Reproductive Processes in Female Fish.

Cyclooxygenase (COX) inhibitors are ubiquitous in aquatic systems and have been detected in fish tissues. The exposure of fish to these pharmaceuticals is concerning because COX inhibitors disrupt the synthesis of prostaglandins (PGs), which modulate a variety of essential biological functions, including reproduction. In this study, we investigated the effects of well-characterized mammalian COX inhibitors on female fathead minnow reproductive health.

An integrated approach for identifying priority contaminant in the Great Lakes Basin - Investigations in the Lower Green Bay/Fox River and Milwaukee Estuary areas of concern.

Environmental assessment of complex mixtures typically requires integration of chemical and biological measurements. This study demonstrates the use of a combination of instrumental chemical analyses, effects-based monitoring, and bio-effects prediction approaches to help identify potential hazards and priority contaminants in two Great Lakes Areas of Concern (AOCs), the Lower Green Bay/Fox River located near Green Bay, WI, USA and the Milwaukee Estuary, located near Milwaukee, WI, USA. Fathead minnows were caged at four sites within each AOC (eight sites total).

A study of temporal effects of the model anti-androgen flutamide on components of the hypothalamic-pituitary-gonadal axis in adult fathead minnows.

The aim of this study was to investigate temporal changes in the hypothalamic-pituitary-gonadal (HPG) axis of fathead minnows (Pimephales promelas) treated with the model androgen receptor (AR) antagonist flutamide. Reproductively-mature fish were exposed in a flow-through test to analytically-confirmed concentrations of either 50 or 500μg flutamide/L for 8 d, followed by an 8-d recovery period in clean water. Fish were sampled at 1, 2, 4 and 8days during each phase of the experiment.

Effects of the antimicrobial contaminant triclocarban, and co-exposure with the androgen 17β-trenbolone, on reproductive function and ovarian transcriptome of the fathead minnow (Pimephales promelas).

Triclocarban (TCC) is an antimicrobial agent routinely detected in surface waters that has been hypothesized to interact with the vertebrate endocrine system. The present study examined the effects of TCC alone and in combination with the model endocrine disruptor 17β-trenbolone (TRB) on fish reproductive function. Adult Pimephales promelas were continuously exposed to either 1 µg TCC/L or 5 µg TCC/L, to 0.

Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part I: Fathead minnow.

In the present study, a hypothesized adverse outcome pathway linking inhibition of thyroid peroxidase (TPO) activity to impaired swim bladder inflation was investigated in two experiments in which fathead minnows (Pimephales promelas) were exposed to 2-mercaptobenzothiazole (MBT). Continuous exposure to 1mg MBT/L for up to 22 days had no effect on inflation of the posterior chamber of the swim bladder, which typically inflates around 6 days post fertilization (dpf), a period during which maternally-derived thyroid hormone is presumed to be present. In contrast, inflation of the anterior swim bladder, which occurs around 14dpf, was impacted.

Metabolite profiles of repeatedly sampled urine from male fathead minnows (Pimephales promelas) contain unique lipid signatures following exposure to anti-androgens.

The purpose of this study was twofold. First, we sought to identify candidate markers of exposure to anti-androgens by analyzing endogenous metabolite profiles in the urine of male fathead minnows (mFHM, Pimephales promelas). Based on earlier work, we hypothesized that unidentified lipids in the urine of mFHM were selectively responsive to exposure to androgen receptor antagonists, which is otherwise difficult to confirm using established fish toxicity assays.