Declines in Reproductive Condition of Male Largemouth Bass (Micropterus salmoides) Following Seasonal Exposure to Estrogenic Endocrine-Disrupting Compounds.

Reproductive abnormalities, that could lead to possible effects at the population level, have been observed in wild fish throughout the United States, with high prevalence in largemouth bass (LMB; Micropterus salmoides) and smallmouth bass (Micropterus dolomieu). Estrone (E1) and atrazine (ATR) are common environmental contaminants often associated with agricultural land use. 17alpha-ethinylestradiol (EE2) is a contaminant associated with wastewater treatment effluent, and a representative, well-studied estrogen commonly used for fish toxicity testing. Our objective was to assess whether early gonad recrudescence in adult fish was a period of sensitivity for alterations in reproductive condition and function. Adult male LMB were exposed from post-spawning to early gonad recrudescence to either a mixture of E1 (47.9 ng/L) + ATR (5.4 µg/L), or EE2 (2.4 ng/L) in outdoor experimental ponds. Gonad samples were collected from fish just prior to the start of exposure (July), at the end of the exposure period (December), the following spring just prior to spawning (April), and post spawning (May). Gonadosomatic index (GSI) was significantly reduced in E1 + ATR-exposed and EE2-exposed males compared to control at every post-exposure time point. Reduced sperm count and sperm motility were observed in the mixture treatment (E1 + ATR) compared to the control. Sperm motility was also reduced in the EE2 treatment. These data together indicate that estrogenic endocrine-disrupting compounds can lessen the reproductive condition of adult male LMB, and that effects of exposure during early gonad recrudescence can persist at least through the subsequent spawning cycle.

Exposure to 17α-Ethinylestradiol Results in Differential Susceptibility of Largemouth Bass (Micropterus salmoides) to Bacterial Infection.

Disease outbreaks, skin lesions, mortality events, and reproductive abnormalities have been observed in wild populations of centrarchids. The presence of estrogenic endocrine disrupting compounds (EEDCs) has been implicated as a potential causal factor for these effects. The effects of prior EEDC exposure on immune response were examined in juvenile largemouth bass (Micropterus salmoides) exposed to a potent synthetic estrogen (17α-ethinylestradiol, EE2) at a low (EE2Low, 0.87 ng/L) or high (EE2High, 9.08 ng/L) dose for 4 weeks, followed by transfer to clean water and injection with an LD40 dose of the Gram-negative bacteria Edwardsiella piscicida. Unexpectedly, this prior exposure to EE2High significantly increased survivorship at 10 d post-infection compared to solvent control or EE2Low-exposed, infected fish. Both prior exposure and infection with E. piscicida led to significantly reduced hepatic glycogen levels, indicating a stress response resulting in depletion of energy stores. Additionally, pathway analysis for liver and spleen indicated differentially expressed genes associated with immunometabolic processes in the mock-injected EE2High treatment that could underlie the observed protective effect and metabolic shift in EE2High-infected fish. Our results demonstrate that exposure to a model EEDC alters metabolism and immune function in a fish species that is ecologically and economically important in North America.

Current ecotoxicity testing needs among selected U.S. federal agencies.

U.S. regulatory and research agencies use ecotoxicity test data to assess the hazards associated with substances that may be released into the environment, including but not limited to industrial chemicals, pharmaceuticals, pesticides, food additives, and color additives. These data are used to conduct hazard assessments and evaluate potential risks to aquatic life (e.g., invertebrates, fish), birds, wildlife species, or the environment. To identify opportunities for regulatory uses of non-animal replacements for ecotoxicity tests, the needs and uses for data from tests utilizing animals must first be clarified. Accordingly, the objective of this review was to identify the ecotoxicity test data relied upon by U.S. federal agencies. The standards, test guidelines, guidance documents, and/or endpoints that are used to address each of the agencies' regulatory and research needs regarding ecotoxicity testing are described in the context of their application to decision-making. Testing and information use, needs, and/or requirements relevant to the regulatory or programmatic mandates of the agencies taking part in the Interagency Coordinating Committee on the Validation of Alternative Methods Ecotoxicology Workgroup are captured. This information will be useful for coordinating efforts to develop and implement alternative test methods to reduce, refine, or replace animal use in chemical safety evaluations.

Effects of early life stage exposure of largemouth bass to atrazine or a model estrogen (17α-ethinylestradiol).

Endocrine disrupting contaminants are of continuing concern for potentially contributing to reproductive dysfunction in largemouth and smallmouth bass in the Chesapeake Bay watershed (CBW) and elsewhere. Exposures to atrazine (ATR) have been hypothesized to have estrogenic effects on vertebrate endocrine systems. The incidence of intersex in male smallmouth bass from some regions of CBW has been correlated with ATR concentrations in water. Fish early life stages may be particularly vulnerable to ATR exposure in agricultural areas, as a spring influx of pesticides coincides with spawning and early development. Our objectives were to investigate the effects of early life stage exposure to ATR or the model estrogen 17α-ethinylestradiol (EE2) on sexual differentiation and gene expression in gonad tissue. We exposed newly hatched largemouth bass (LMB, Micropterus salmoides) from 7 to 80 days post-spawn to nominal concentrations of 1, 10, or 100 µg ATR/L or 1 or 10 ng EE2/L and monitored histological development and transcriptomic changes in gonad tissue. We observed a nearly 100% female sex ratio in LMB exposed to EE2 at 10 ng/L, presumably due to sex reversal of males. Many gonad genes were differentially expressed between sexes. Multidimensional scaling revealed clustering by gene expression of the 1 ng EE2/L and 100 µg ATR/L-treated male fish. Some pathways responsive to EE2 exposure were not sex-specific. We observed differential expression in male gonad in LMB exposed to EE2 at 1 ng/L of several genes involved in reproductive development and function, including star, cyp11a2, ddx4 (previously vasa), wnt5b, cyp1a and samhd1. Expression of star, cyp11a2 and cyp1a in males was also responsive to ATR exposure. Overall, our results confirm that early development is a sensitive window for estrogenic endocrine disruption in LMB and are consistent with the hypothesis that ATR exposure induces some estrogenic responses in the developing gonad. However, ATR-specific and EE2-specific responses were also observed.

Factors Affecting Sampling Strategies for Design of an Effects-Directed Analysis for Endocrine-Active Chemicals.

Effects-directed analysis (EDA) is an important tool for identifying unknown bioactive components in a complex mixture. Such an analysis of endocrine-active chemicals (EACs) from water sources has promising regulatory implications but also unique logistical challenges. We propose a conceptual EDA (framework) based on a critical review of EDA literature and concentrations of common EACs in waste and surface waters. Required water volumes for identification of EACs under this EDA framework were estimated based on bioassay performance (in vitro and in vivo bioassays), limits of quantification by mass spectrometry (MS), and EAC water concentrations. Sample volumes for EDA across the EACs showed high variation in the bioassay detectors, with genistein, bisphenol A, and androstenedione requiring very high sample volumes and ethinylestradiol and 17ß-trenbolone requiring low sample volumes. Sample volume based on the MS detector was far less variable across the EACs. The EDA framework equation was rearranged to calculate detector "thresholds," and these thresholds were compared with the literature EAC water concentrations to evaluate the feasibility of the EDA framework. In the majority of instances, feasibility of the EDA was limited by the bioassay, not MS detection. Mixed model analysis showed that the volumes required for a successful EDA were affected by the potentially responsible EAC, detection methods, and the water source type, with detection method having the greatest effect on the EDA of estrogens and androgens. The EDA framework, equation, and model we present provide a valuable tool for designing a successful EDA. Environ Toxicol Chem 2020;39:1309-1324. © 2020 SETAC.

Tris(1,3-dichloro-2-propyl) phosphate disrupts dorsoventral patterning in zebrafish embryos.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a high-production volume organophosphate flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) results in genome-wide alterations in methylation during cleavage (2 hpf) as well as epiboly delay or arrest (at higher concentrations) during late-blastula and early-gastrula (4-6 hpf). To determine whether these TDCIPP-induced effects were associated with impacts on the transcriptome, embryos were exposed to vehicle (0.1% DMSO) or 2 µM TDCIPP from 0.75 hpf to 6 hpf, and total RNA was extracted from triplicate embryo pools per treatment and hybridized onto duplicate Affymetrix Zebrafish Gene 1.0 ST Arrays per RNA sample. Based on transcriptome-wide profiling, TDCIPP resulted in a significant impact on biological processes involved in dorsoventral patterning and bone morphogenetic protein (BMP) signaling. Consistent with these responses, TDCIPP exposure also resulted in strongly dorsalized embryos by 24 hpf-a phenotype that mimicked the effects of dorsomorphin, a potent and selective BMP inhibitor. Moreover, the majority of dorsalized embryos were preceded by epiboly arrest at 6 hpf. Our microarray data also revealed that the expression of sizzled (szl)-a gene encoding a secreted Frizzled-related protein that limits BMP signaling-was significantly decreased by nearly 4-fold at 6 hpf. Therefore, we used a splice-blocking morpholino to test the hypothesis that knockdown of szl phenocopies TDCIPP-induced delays in epiboly progression. Interestingly, contrary to our hypothesis, injection of szl MOs did not affect epiboly progression but, similar to chordin (chd) morphants, resulted in mildly ventralized embryos by 24 hpf. Overall, our findings suggest that TDCIPP-induced epiboly delay may not be driven by decreased szl expression, and that TDCIPP-induced dorsalization may-similar to dorsomorphin-be due to interference with BMP signaling during early zebrafish development.

Tris(1,3-dichloro-2-propyl)phosphate Induces Genome-Wide Hypomethylation within Early Zebrafish Embryos.

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is a high-production volume organophosphate-based plasticizer and flame retardant widely used within the United States. Using zebrafish as a model, the objectives of this study were to determine whether (1) TDCIPP inhibits DNA methyltransferase (DNMT) within embryonic nuclear extracts; (2) uptake of TDCIPP from 0.75 h postfertilization (hpf, 2-cell) to 2 hpf (64-cell) or 6 hpf (shield stage) leads to impacts on the early embryonic DNA methylome; and (3) TDCIPP-induced impacts on cytosine methylation are localized to CpG islands within intergenic regions. Within this study, 5-azacytidine (5-azaC, a DNMT inhibitor) was used as a positive control. Although 5-azaC significantly inhibited zebrafish DNMT, TDCIPP did not affect DNMT activity in vitro at concentrations as high as 500 µM. However, rapid embryonic uptake of 5-azaC and TDCIPP from 0.75 to 2 hpf resulted in chemical- and chromosome-specific alterations in cytosine methylation at 2 hpf. Moreover, TDCIPP exposure predominantly resulted in hypomethylation of positions outside of CpG islands and within intragenic (exon) regions of the zebrafish genome. Overall, these findings provide the foundation for monitoring DNA methylation dynamics within zebrafish as well as identifying potential associations among TDCIPP exposure, adverse health outcomes, and DNA methylation status within human populations.

Environmental hormones and their impacts on sex differentiation in fathead minnows.

Runoff from lands fertilized with animal manure from concentrated animal feeding operations (CAFOs) is a source of hormones to surface water. In this study we tested the hypothesis that larval fathead minnows exposed to sex steroids singly or in a "typical" CAFO mixture during sex differentiation would respond with changes in the expression of a set of target genes, leading to gonadal abnormalities later in life. In the first experiment, a static daily-renewal system was used to expose larvae during the period of 10-20 days post-hatch (dph) to either 5 ng/L 17ß-trenbolone (17ß-TRB) or 5 ng/L 17α-ethinylestradiol (EE2). In a second experiment, fish were exposed from 0 to 45 dph in a flow-through system to a CAFO mixture composed of steroids and degradates (2-16 ng/L), atrazine and degradates (15-250 ng/L), and nitrate (3-11 mg/L). In the single hormone experiment, expression of genes involved in steroidogenesis (cyp19a, cyp17, and star) was decreased in females. In contrast, no differences in gene expression were observed in fish exposed to the CAFO mixture. However, the majority (84%) of treated males had testes containing an ovarian cavity, indicative of feminization, compared to 0% in the control males. Overall, our results show that: (1) changes in gene expression after single hormone exposures are sex-specific, with females more responsive than males; and (2) phenotypic alterations in testicular development can be elicited by a simulated "CAFO" mixture when fathead minnows are exposed during the first 45 days of development. More research is needed to further discern the complex response of fish to steroid mixtures, especially those associated with runoff from land-applied CAFO waste.

Presence and effects of pharmaceutical and personal care products on the Baca National Wildlife Refuge, Colorado.

Pharmaceuticals and personal care products (PPCPs) have raised concerns due to their potential effects to aquatic organisms. These chemicals appear in mixtures at very low concentrations thus making their detection and quantification difficult. Polar organic chemical integrative samplers (POCIS) concentrate trace levels of chemicals over time increasing method sensitivity and thus represent a cost-effective screening tool for biomonitoring studies. The Baca National Wildlife Refuge (BNWR), Colorado, is home for several endemic fish species, including Rio Grande chub (Gila pandora). The objectives of this study were to (1) determine the types and concentrations of PPCPs in the Refuge, (2) compare and contrast two methods (grab and POCIS) for the quantification of PPCPs from surface water, and (3) determine effects due to PPCP exposure in fish. Between 2011 and 2013, 141 PPCPs were quantified using a combination of grab samples and POCIS. Although no PPCPs were detected from the grab samples, high concentrations of N,N-Diethyl-meta-toluamide (DEET) and triclosan were detected in all fish sampling sites with POCIS. Fathead minnows (Pimephales promelas) and Rio Grande chubs of both sexes were collected in 2011 and 2012. Several biological responses were observed in both species from creeks contaminated with PPCPs; however the presence of PPCPs in the reference site did not allow for valid data comparison and interpretation. We conclude that POCIS is a sensitive method for the detection and quantification of PPCPs and for identification of reference sites and that appropriate "reference" sites need to be identified at the BNWR for follow-up studies with native fish.

Butafenacil: A positive control for identifying anemia- and variegate porphyria-inducing chemicals.

Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPOX), an enzyme that catalyzes oxidation of protoporphyrinogen IX to protoporphyrin IX during chlorophyll and heme biosynthesis. Based on a high-content screen, we previously identified butafenacil as a potent inducer of anemia in zebrafish embryos. Therefore, the objective of this study was to begin investigating the utility of butafenacil as a positive control for identifying anemia- and variegate porphyria-inducing chemicals. Static exposure to butafenacil from 5 to 72 h post-fertilization (hpf) in glass beakers resulted in a concentration-dependent decrease in arterial circulation at low micromolar concentrations. At 72 hpf, the magnitude of butafenacil-induced anemia was similar when embryos were exposed in the presence or absence of light, whereas protoporphyrin accumulation and acute toxicity were significantly lower or absent when embryos were exposed under dark conditions. To identify sensitive developmental windows, we treated embryos to butafenacil from 5, 10, 24, or 48 hpf to 72 hpf in the presence of light, and found that anemia and protoporphyrin accumulation were present at 72 hpf following initiation of exposure at 5 and 10 hpf. On the contrary, protoporphyrin accumulation - but not anemia - was present following initiation of exposure at 24 hpf. Lastly, protoporphyrin accumulation at 72 hpf after exposure from 24 to 48 hpf suggests that protoporphyrin was not eliminated over a 24-h recovery period. Collectively, our data suggests that butafenacil may be a reliable positive control for identifying anemia- and variegate porphyria-inducing chemicals.

High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia.

Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO)--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism-focused investigations within zebrafish and mammalian models.

Reproductive physiology in eastern snapping turtles (Chelydra serpentina) exposed to runoff from a concentrated animal feeding operation.

The eastern snapping turtle (Chelydra serpentina) is widely distributed throughout the eastern and central US and is a useful model organism to study land-use impacts on water quality. We compared the reproductive condition of turtles from a pond impacted by runoff from land applied with animal manure from a concentrated animal feeding operation (CAFO) relative to animals from a control pond. Turtles from the CAFO site were heavier and had higher plasma concentrations of vitellogenin (VTG, mean ± SE; females; 859 ± 115 vs. 401 ± 127 ng/mL from controls) and testosterone (T, males; 39 ± 7.0 vs. 3.8 ± 6.9 ng/mL from controls). No VTG was detected in males. Body mass was positively correlated with VTG and T. Our results suggest that nutrient pollution of the CAFO pond indirectly resulted in higher plasma VTG in females and T in males because of an increase in body mass. The population-level consequences of these effects are not clear, but could result in females producing larger clutches.

Assessing impacts of land-applied manure from concentrated animal feeding operations on fish populations and communities.

Concentrated animal feeding operation (CAFO) manure is a cost-effective fertilizer. In the Midwest, networks of subsurface tile-drains expedite transport of animal hormones and nutrients from land-applied CAFO manure to adjacent waterways. The objective of this study was to evaluate impacts of land-applied CAFO manure on fish populations and communities. Water chemistry including hormone, pesticide, and nutrient concentrations was characterized from study sites along with fish assemblage structure, growth, and endocrine disruption assessed in selected fish species. Although most CAFO water samples had hormone concentrations <1 ng/L, equivalent concentrations for 17ß-E2 and 17α-TB peaked at >30 ng/L each during the period of spawning, hatching, and development for resident fishes. CAFO sites had lower fish species richness, and fishes exhibited faster somatic growth and lower reproductive condition compared to individuals from the reference site. Fathead minnows (Pimephales promelas) exposed to CAFO ditchwater during early developmental stages exhibited significantly skewed sex ratios toward males. Maximum observed hormone concentrations were well above the lowest observable effect concentrations for these hormones; however, complexities at the field scale make it difficult to directly relate hormone concentration and impacts on fish. Complicating factors include the consistent presence of pesticides and nutrients, and the difference in temperature and stream architecture of the CAFO-impacted ditches compared to the reference site (e.g., channelization, bottom substrate, shallow pools, and riparian cover).

A review of studies on androgen and estrogen exposure in fish early life stages: effects on gene and hormonal control of sexual differentiation.

Teleost fish are unique among vertebrates in that phenotypic sex or onset of sex inversion can be easily manipulated by hormonal treatments. In recent years, researchers have begun reporting concentrations of synthetic and natural hormones in the environment. Although concentrations are very low (in the parts per trillion to low parts per billion), they are still of concern because of the high potency of synthetic hormones and the enhanced susceptibility of teleost fishes, especially early life stages, to hormonal exposures. In this review, we will focus on sex differentiation in teleost fishes and how these processes in fish early life stages may be impacted by environmental hormones which are known to contaminate aquatic environments. We will start by reviewing information on sources and concentrations of hormones in the environment and continue by summarizing the state of knowledge of sex differentiation in teleost gonochoristic fishes, including information on genes involved (e.g. cyp19, dmrt1, sox9 and foxl2). We will end our review with a summary of studies that have examined the effects of androgens and estrogens on fish sex differentiation after exposure of fish embryos and larvae and with ideas for future research.