Impacts of Exposure to Ultraviolet Radiation and an Agricultural Pollutant on Morphology and Behavior of Tadpoles (Limnodynastes tasmaniensis).

Amphibians are the most threatened vertebrate class globally. Multiple factors have been implicated in their global decline, and it has been hypothesized that interactions between stressors may be a major cause. Increased ultraviolet (UV) radiation, as a result of ozone depletion, has been identified as one such stressor. Exposure to UV radiation has been shown to have detrimental effects on amphibians and can exacerbate the effects of other stressors, such as chemical pollutants. Chemical pollution has likewise been recognized as a major factor contributing to amphibian declines, particularly, endocrine-disrupting chemicals. In this regard, 17ß-trenbolone is a potent anabolic steroid used in the agricultural industry to increase muscle mass in cattle and has been repeatedly detected in the environment where amphibians live and breed. At high concentrations, 17ß-trenbolone has been shown to impact amphibian survival and gonadal development. In the present study, we investigated the effects of environmentally realistic UV radiation and 17ß-trenbolone exposure, both in isolation and in combination, on the morphology and behavior of tadpoles (Limnodynastes tasmaniensis). We found that neither stressor in isolation affected tadpoles, nor did we find any interactive effects. The results from our 17ß-trenbolone treatment are consistent with recent research suggesting that, at environmentally realistic concentrations, tadpoles may be less vulnerable to this pollutant compared to other vertebrate classes. The absence of UV radiation-induced effects found in the present study could be due to species-specific variation in susceptibility, as well as the dosage utilized. We suggest that future research should incorporate long-term studies with multiple stressors to accurately identify the threats to, and subsequent consequences for, amphibians under natural conditions. Environ Toxicol Chem 2024;43:1615-1626. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Exposure to an androgenic agricultural pollutant does not alter metabolic rate, behaviour, or morphology of tadpoles.

Globally, amphibian species are experiencing dramatic population declines, and many face the risk of imminent extinction. Endocrine-disrupting chemicals (EDCs) have been recognised as an underappreciated factor contributing to global amphibian declines. In this regard, the use of hormonal growth promotants in the livestock industry provides a direct pathway for EDCs to enter the environment-including the potent anabolic steroid 17ß-trenbolone. Emerging evidence suggests that 17ß-trenbolone can impact traits related to metabolism, somatic growth, and behaviour in non-target species. However, far less is known about possible effects of 17ß-trenbolone on anuran species, particularly during early life stages. Accordingly, in the present study we investigated the effects of 28-day exposure to 17ß-trenbolone (mean measured concentrations: 10 and 66 ng/L) on body size, body condition, metabolic rate, and anxiety-related behaviour of tadpoles (Limnodynastes tasmaniensis). Specifically, we measured rates of O2 consumption of individual tadpoles as a proxy for metabolic rate and quantified their swimming activity and their time spent in the upper half of the water column as indicators of anxiety-related behaviour. Counter to our predictions based on effects observed in other taxa, we detected no effect of 17ß-trenbolone on body size, metabolic rate, or behaviour of tadpoles; although, we did detect a subtle, but statistically significant decrease in body condition at the highest 17ß-trenbolone concentration. We hypothesise that 17ß-trenbolone may induce taxa-specific effects on metabolic function, growth, and anxiety-related behaviour, with anurans being less sensitive to disruption than fish, and encourage further cross-taxa investigation to test this hypothesis.

17ß-Trenbolone binds to androgen receptor, decreases number of primordial germ cells, modulates expression of genes related to sexual differentiation, and affects sexual differentiation in zebrafish (Danio rerio).

Exposure to 17ß-trenbolone caused a skewed sex ratio in fish. However, the molecular initiating event and key molecular event(s) remain unknown. In this study, zebrafish were exposed to 17ß-trenbolone at nominal concentrations of 2 ng/L, 20 ng/L, 200 ng/L, and 2000 ng/L from fertilization to 60 days post fertilization (dpf). First, the sex ratio at 60 dpf was calculated to evaluate adverse outcomes on sexual differentiation. 17ß-Trenbolone caused a skewed sex ratio toward males, with intersex individuals observed in the 20 ng/L group and all-male populations found in the 200 ng/L and 2000 ng/L groups. Then, the distribution and number of primordial germ cells, the expression of sex differentiation-related genes, and plasma vitellogenin concentrations were detected in wild-type zebrafish and the EGFP-nanos-3'UTR transgenic line using whole-mount in situ hybridization, real-time PCR, EGFP fluorescence quantification, and enzyme-linked immunosorbent assay. The results indicated that 17ß-trenbolone exposure decreased the number of primordial germ cells at 1 dpf and 3 dpf, decreased expression of ovarian differentiation-related genes foxl2 and cyp19a1a at 60 dpf, increased expression of testis differentiation-related genes dmrt1, sox9a, and amh at 60 dpf, and decreased plasma vitellogenin levels at 60 dpf, revealing the key molecular events at different time points involved in affected sexual differentiation by 17ß-trenbolone. Finally, molecular docking showed that 17ß-trenbolone docked into ligand-binding domain of zebrafish androgen receptor with high binding energy (-3.72 kcal/mol), suggesting that binding to androgen receptor is the molecular initiating event affecting sexual differentiation by 17ß-trenbolone. We found that 17ß-trenbolone can bind to the zebrafish androgen receptor, decrease the number of primordial germ cells during the early embryonic stage, modulate the expression of genes related to sexual differentiation during gonadal differentiation, and eventually cause a skewed sex ratio toward males in adults.

The endocrine disruptor 17ß-trenbolone alters the relationship between pre- and post-copulatory sexual traits in male mosquitofish (Gambusia holbrooki).

It is now well-established that reproduction in wildlife can be disrupted by anthropogenic environmental changes, such as chemical pollution. However, very little is known about how these pollutants might affect the interplay between pre- and post-copulatory mechanisms of sexual selection. Here, we investigated the impacts of 21-day exposure of male eastern mosquitofish (Gambusia holbrooki) to a field-realistic level (average measured concentration: 11 ng/L) of the endocrine-disrupting chemical 17ß-trenbolone (17ß-TB) on pre- and post-copulatory reproductive traits. We examined male reproductive behaviour by testing the time spent near a female behind a partition, as well as the number of copulation attempts made, and the time spent chasing a female in a free-swimming context. Sperm traits were also assayed for all males. We found that exposure of male fish to 17ß-TB altered the relationship between key pre- and post-copulatory reproductive traits. Furthermore, 17ß-TB-exposed males had, on average, a higher percentage of motile sperm, and performed fewer copulation attempts than unexposed males. However, there was no overall effect of 17ß-TB exposure on either the time males spent associating with or chasing females. Taken together, our findings demonstrate the potential for chemical pollutants to affect both pre- and post-copulatory sexual traits, and the interplay between these mechanisms of sexual selection in contaminated wildlife.

Melatonin attenuates 17ß-trenbolone induced insomnia-like phenotype and movement deficiency in zebrafish.

17ß-trenbolone (17ß-TBOH) is one of the dominant metabolites of trenbolone acetate, which is widely applied in beef cattle operations around the globe. The effects of environmental concentrations of 17ß-trenbolone on the early development of zebrafish embryos have received very little attention. Melatonin could regulate sleep-wake cycle and plays a protective role in various adverse conditions. Here, environmentally realistic concentrations of 17ß-trenbolone (1 ng/L, 10 ng/L, 50 ng/L) has been exposure to zebrafish embryos at 2 h postfertilization (hpf). The results showed that 10 ng/L and 50 ng/L 17ß-trenbolone disturbed the distribution of caudal primary motoneurons and downregulated expression of motoneuron development related genes along with locomotion decreasing. While melatonin could recover the detrimental effects caused by 17ß-trenbolone. Interestingly, 17ß-trenbolone exposure increased waking activity and decreased rest even in a low dose (1 ng/L). Moreover, it upregulated hypocretin/orexin (Hcrt) signaling which promotes wakefulness. Melatonin restored the insomnia-like alternation induced by 17ß-trenbolone exposure. Collectively, we conclude that 17ß-trenbolone disturbed motoneuron development and altered sleep/wake behavior, while melatonin could alleviate the deleterious influence on motoneuron development and recover the circadian rhythm.

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. We assessed the utility of a simple AOP network for predicting the effects of mixtures of an aromatase inhibitor (fadrozole) and an androgen receptor agonist (17ß-trenbolone) on aspects of reproductive endocrine function in female fathead minnows. The fish were exposed to multiple concentrations of fadrozole and 17ß-trenbolone individually or in combination for 48 or 96 h. Effects on 2 shared nodes in the AOP network, plasma 17ß-estradiol (E2) concentration and vitellogenin (VTG) production (measured as hepatic vtg transcripts) responded as anticipated to fadrozole alone but were minimally impacted by 17ß-trenbolone alone. Overall, there were indications that 17ß-trenbolone enhanced decreases in E2 and vtg in fadrozole-exposed fish, as anticipated, but the results often were not statistically significant. Failure to consistently observe hypothesized interactions between fadrozole and 17ß-trenbolone could be due to several factors, including lack of impact of 17ß-trenbolone, inherent biological variability in the endpoints assessed, and/or an incomplete understanding of interactions (including feedback) between different pathways within the hypothalamic-pituitary-gonadal axis. Environ Toxicol Chem 2020;39:913-922. © 2020 SETAC.

Genotoxic biomarkers and histological changes in marine medaka (Oryzias melastigma) exposed to 17α-ethynylestradiol and 17ß-trenbolone.

Endocrine-disrupting pollutants in marine environments have aroused great concern for their adverse effects on the reproduction of marine organisms. This study aimed to seek promising biomarkers for estrogenic/androgenic chemicals. First, two possible male-specific genes, SRY-box containing gene 9a2 (sox9a2) and gonadal soma-derived factor (gsdf), were cloned from marine medaka (Oryzias melastigma). Then the responses of sox9a2, gsdf, choriogenin (chgH and chgL), vitellogenin (vtg1 and vtg2), and cytochrome P450 aromatase (cyp19a and cyp19b) were investigated after exposure to 17α-ethynylestradiol (EE2) and 17ß-trenbolone (TB) at 2, 10, and 50 ng/L. The results showed that gsdf was specifically expressed in the testes and easily induced in the ovaries after TB exposure, indicating that gsdf was a potential biomarker of environmental androgens. ChgL was a useful biomarker of weak estrogen pollution for its high sensitivity to low levels of EE2. In addition, both EE2 and TB exposure damaged gonadal structures and inhibited gonadal development.

A comparative study of 3 alternative avian toxicity testing methods: Effects on hepatic gene expression in the chicken embryo.

There is growing interest in developing alternative methods to screen and prioritize chemical hazards, although few studies have compared responses across different methods. The objective of the present study was to compare 3 alternative liver methods derived from white Leghorn chicken (Gallus gallus domesticus): primary hepatocyte culture, liver slices, and liver from in ovo injected embryos. We examined hepatic gene expression changes after exposure to 3 chemicals (17ß-trenbolone [17ßT], 17ß-estradiol [E2], and 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) using a custom quantitative polymerase chain reaction (qPCR) array with 7 genes (vitellogenin [VTG], apolipoprotein [Apo], cytochrome P450 1A4 [CYP1A4], liver basic fatty acid binding protein [LBFABP], 3ß hydroxysteroid dehydrogenase [HSD3ß1], stearoyl coenzyme A desaturase [SCD], and estrogen sulfotransferase [SULT1E1]). Gene expression across the 3 methods was examined using hierarchical clustering. Up-regulation of CYP1A4 in response to TCDD was consistent across all methods, and the magnitude was higher in hepatocytes (>150-fold) compared with slices (>31-fold) and in ovo liver (>27-fold). In hepatocytes, SCD and VTG up-regulation in response to 17ßT and E2 was >4-fold and 16-fold, respectively. The rank order of cases with significant changes in gene expression among the 3 methods was: hepatocytes (22) > in ovo liver (11) > liver slices (6). Hierarchical clustering grouped liver slices and in ovo liver as more similar, whereas hepatocytes were grouped separately from in ovo liver. More introspective comparisons are needed to understand how and why alternative methods differ and to aid in their integration into toxicity testing. Environ Toxicol Chem 2019;38:2546-2555. © 2019 SETAC.

Gonadosomatic index as a confounding variable in fish-based screening assays for the detection of anti-estrogens and nonaromatizable androgens.

The presence of reproductive endocrine-disrupting compounds (REDCs) in the environment poses a potential threat to fish and wildlife, because exposures are capable of altering sexual development, reproductive success, and behavior. Fish-based screening assays are often utilized to screen for the presence of REDCs in surface waters and to assess single chemicals for potential endocrine-disrupting activity. In an effort to improve such screening assays, the goal of the present study was to determine whether the gonadosomatic index (GSI) of female fathead minnows (Pimephales promelas), as assessed via external characteristics, influences their response to REDC exposure. Specifically, we sought to determine whether low-GSI females differed from high-GSI females in their responses to the model anti-estrogen fadrozole and the model androgen 17ß-trenbolone, and whether there was a preferable classification in the context of REDC screening. Low-GSI females were more sensitive to fadrozole at the lower concentration of fadrozole (5 µg/L) and to the higher concentration of trenbolone (50 ng/L), whereas high-GSI females were more sensitive at the lower concentration of trenbolone (5 ng/L). The differential response of low- and high-GSI females to REDCs indicates that GSI influences exposure outcome, and should subsequently be taken into consideration in the implementation of screening assays, as failure to utilize fish of the appropriate reproductive status may skew the test results. Environ Toxicol Chem 2019;38:603-615. © 2019 SETAC.

Acute pancreatitis secondary to the use of the anabolic steroid trenbolone acetate.

BACKGROUND: The use of performance-enhancing drugs has increased dramatically in the last decade with high prevalence reported among the young athlete population. Many of these drugs contain anabolic steroids and may carry potential significant side effects and health risks. We report a case of anabolic steroid-induced acute pancreatitis (AP) that recurred after the reuse of the same drug by the patient, confirming the causative relationship. CASE REPORT: A 24 year-old male presented with severe epigastric pain. His past medical history was significant for two hospitalizations during the last year with AP. During his hospital admissions, extensive workup was performed ruling out the common and uncommon causes of AP. Upon further pressing, the patient admitted to a history of past and current anabolic steroid use for athletic performance enhancement. He began this use four years ago and most recently started using trenbolone acetate (TA). The correlation between the timing of the anabolic steroids administration and the attacks of AP, along with ruling out other causes, confirmed TA as the cause of pancreatitis. DISCUSSION: The side effects associated with the use of these increasingly prevalent drugs are difficult to study in clinical trials due to the unethical nature of their consumption. In addition, these medications are difficult to study due to the varied usage cycles and patterns, unknown origin and source, as well as often high dose ingestion. Physicians and body builders need to be aware of the possible serious consequences of their use.

Fish on steroids: Temperature-dependent effects of 17ß-trenbolone on predator escape, boldness, and exploratory behaviors.

Hormonal growth promoters (HGPs), widely used in beef cattle production globally, make their way into the environment as agricultural effluent-with potential impacts on aquatic ecosystems. One HPG of particular concern is 17ß-trenbolone, which is persistent in freshwater habitats and can affect the development, morphology and reproductive behaviors of aquatic organisms. Despite this, few studies have investigated impacts of 17ß-trenbolone on non-reproductive behaviors linked to growth and survival, like boldness and predator avoidance. None consider the interaction between 17ß-trenbolone and other environmental stressors, such as temperature, although environmental challenges confronting animals in the wild seldom, if ever, occur in isolation. Accordingly, this study aimed to test the interactive effects of trenbolone and temperature on organismal behavior. To do this, eastern mosquitofish (Gambusia holbrooki) were subjected to an environmentally-relevant concentration of 17ß-trenbolone (average measured concentration 3.0 ±â€¯0.2 ng/L) or freshwater (i.e. control) for 21 days under one of two temperatures (20 and 30 °C), after which the predator escape, boldness and exploration behavior of fish were tested. Predator escape behavior was assayed by subjecting fish to a simulated predator strike, while boldness and exploration were assessed in a separate maze experiment. We found that trenbolone exposure increased boldness behavior. Interestingly, some behavioral effects of trenbolone depended on temperature, sex, or both. Specifically, significant effects of trenbolone on male predator escape behavior were only noted at 30 °C, with males becoming less reactive to the simulated threat. Further, in the maze experiment, trenbolone-exposed fish explored the maze faster than control fish, but only at 20 °C. We conclude that field detected concentrations of 17ß-trenbolone can impact ecologically important behaviors of fish, and such effects can be temperature dependent. Such findings underscore the importance of considering the potentially interactive effects of other environmental stressors when investigating behavioral effects of environmental contaminants.

Field-realistic exposure to the androgenic endocrine disruptor 17ß-trenbolone alters ecologically important behaviours in female fish across multiple contexts.

The capacity of pharmaceutical pollution to alter behaviour in wildlife is of increasing environmental concern. A major pathway of these pollutants into the environment is the treatment of livestock with hormonal growth promotants (HGPs), which are highly potent veterinary pharmaceuticals that enter aquatic ecosystems via effluent runoff. Hormonal growth promotants are designed to exert biological effects at low doses, can act on physiological pathways that are evolutionarily conserved across taxa, and have been detected in ecosystems worldwide. However, despite being shown to alter key fitness-related processes (e.g., development, reproduction) in various non-target species, relatively little is known about the potential for HGPs to alter ecologically important behaviours, especially across multiple contexts. Here, we investigated the effects of exposure to a field-realistic level of the androgenic HGP metabolite 17ß-trenbolone-an endocrine-disrupting chemical that has repeatedly been detected in freshwater systems-on a suite of ecologically important behaviours in wild-caught female eastern mosquitofish (Gambusia holbrooki). First, we found that 17ß-trenbolone-exposed fish were more active and exploratory in a novel environment (i.e., maze arena), while boldness (i.e., refuge use) was not significantly affected. Second, when tested for sociability, exposed fish spent less time in close proximity to a shoal of stimulus (i.e., unexposed) conspecific females and were, again, found to be more active. Third, when assayed for foraging behaviour, exposed fish were faster to reach a foraging zone containing prey items (chironomid larvae), quicker to commence feeding, spent more time foraging, and consumed a greater number of prey items, although the effect of exposure on certain foraging behaviours was dependent on fish size. Taken together, these findings highlight the potential for exposure to sub-lethal levels of veterinary pharmaceuticals to alter sensitive behavioural processes in wildlife across multiple contexts, with potential ecological and evolutionary implications for exposed populations.

A critical review of the environmental occurrence and potential effects in aquatic vertebrates of the potent androgen receptor agonist 17ß-trenbolone.

Trenbolone acetate is widely used in some parts of the world for its desirable anabolic effects on livestock. Several metabolites of the acetate, including 17ß-trenbolone, have been detected at low nanograms per liter concentrations in surface waters associated with animal feedlots. The 17ß-trenbolone isomer can affect androgen receptor signaling pathways in various vertebrate species at comparatively low concentrations/doses. The present article provides a comprehensive review and synthesis of the existing literature concerning exposure to and biological effects of 17ß-trenbolone, with an emphasis on potential risks to aquatic animals. In vitro studies indicate that, although 17ß-trenbolone can activate several nuclear hormone receptors, its highest affinity is for the androgen receptor in all vertebrate taxa examined, including fish. Exposure of fish to nanograms per liter water concentrations of 17ß-trenbolone can cause changes in endocrine function in the short term, and adverse apical effects in longer exposures during development and reproduction. Impacts on endocrine function typically are indicative of inappropriate androgen receptor signaling, such as changes in sex steroid metabolism, impacts on gonadal stage, and masculinization of females. Exposure of fish to 17ß-trenbolone during sexual differentiation in early development can greatly skew sex ratios, whereas adult exposures can adversely impact fertility and fecundity. To fully assess ecosystem-level risks, additional research is warranted to address uncertainties as to the degree/breadth of environmental exposures and potential population-level effects of 17ß-trenbolone in sensitive species. Environ Toxicol Chem 2018;37:2064-2078. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

An endocrine-disrupting agricultural contaminant impacts sequential female mate choice in fish.

The environmental impact of endocrine-disrupting chemicals (EDCs)-compounds that interfere with endocrine system function at minute concentrations-is now well established. In recent years, concern has been mounting over a group of endocrine disruptors known as hormonal growth promotants (HGPs), which are natural and synthetic chemicals used to promote growth in livestock by targeting the endocrine system. One of the most potent compounds to enter the environment as a result of HGP use is 17ß-trenbolone, which has repeatedly been detected in aquatic habitats. Although recent research has revealed that 17ß-trenbolone can interfere with mechanisms of sexual selection, its potential to impact sequential female mate choice remains unknown, as is true for all EDCs. To address this, we exposed female guppies (Poecilia reticulata) to 17ß-trenbolone at an environmentally relevant level (average measured concentration: 2 ng/L) for 21 days using a flow-through system. We then compared the response of unexposed and exposed females to sequentially presented stimulus (i.e., unexposed) males that varied in their relative body area of orange pigmentation, as female guppies have a known preference for orange colouration in males. We found that, regardless of male orange pigmentation, both unexposed and exposed females associated with males indiscriminately during their first male encounter. However, during the second male presentation, unexposed females significantly reduced the amount of time they spent associating with low-orange males if they had previously encountered a high-orange male. Conversely, 17ß-trenbolone-exposed females associated with males indiscriminately (i.e., regardless of orange colouration) during both their first and second male encounter, and, overall, associated with males significantly less than did unexposed females during both presentations. This is the first study to demonstrate altered sequential female mate choice resulting from exposure to an endocrine disruptor, highlighting the need for a greater understanding of how EDCs may impact complex mechanisms of sexual selection.

Veterinary drug, 17ß-trenbolone promotes the proliferation of human prostate cancer cell line through the Akt/AR signaling pathway.

Trenbolone acetate (TBA) is a synthetic anabolic steroidal growth factor that is used for rapid muscle development in cattle. The absorbed TBA is hydrolyzed to the active form, 17ß-trenbolone (17 TB; 17ß-hydroxy-estra-4,9,11-trien-3-one) in meat and milk products, which can cause adverse health effects in humans. Similar to 5α-dihydrotestosterone (DHT), 17 TB was reported to exhibit endocrine disrupting effects on animals and humans due to its androgenic effect via binding to the androgen receptor. The purpose of this study is to investigate the molecular mechanism of cell proliferation in prostate cancer (PCa) cells treated with 17 TB. We found that 17 TB induces AR-dependent cell proliferation in the human prostate cancer cell line, 22Rv1 in a concentration dependent manner. Treatment with 17 TB increased the expression of cell cycle regulatory proteins, cyclin D2/CDK-4 and cyclin E/CDK-2, whereas the expression of p27 was down-regulated. Furthermore, phosphorylation of Rb and activation of E2F were also induced, which suggests the activation of cyclin D2/CDK-4 and cyclin E/CDK-2 in the cells. When 22Rv1 cells were exposed to 30 pM of 17 TB, which is the effective concentration (EC50) value required to observe proliferative effects on 22Rv1 cells, the expression levels of the phosphorylated forms of Akt and GSK3ß were increased. This study demonstrates that 17 TB induces AR-dependent proliferation through the modulation of cell cycle-related proteins in the Akt signaling pathway. The present study provides an effective methodology for identifying cell proliferation signaling of veterinary drugs that exert AR agonistic effects.

The agricultural contaminant 17ß-trenbolone disrupts male-male competition in the guppy (Poecilia reticulata).

Despite a growing literature highlighting the potential impact of human-induced environmental change on mechanisms of sexual selection, relatively little is known about the effects of chemical pollutants on male-male competition. One class of environmental pollutant likely to impact male competitive interactions is the endocrine-disrupting chemicals (EDCs), a large and heterogeneous group of chemical contaminants with the potential to influence morphology, physiology and behaviour at minute concentrations. One EDC of increasing concern is the synthetic, androgenic steroid 17ß-trenbolone, which is used globally to promote growth in beef cattle. Although 17ß-trenbolone has been found to cause severe morphological and behavioural abnormalities in fish, its potential impact on male-male competition has yet to be investigated. To address this, we exposed wild male guppies (Poecilia reticulata) to an environmentally realistic concentration of 17ß-trenbolone (average measured concentration: 8 ng/L) for 21 days using a flow-through system. We found that, in the presence of a competitor, 17ß-trenbolone-exposed males carried out more frequent aggressive behaviours towards rival males than did unexposed males, as well as performing less courting behaviour and more sneak (i.e., coercive) mating attempts towards females. Considering that, by influencing mating outcomes, male-male competition has important consequences for population dynamics and broader evolutionary processes, this study highlights the need for greater understanding of the potential impact of EDCs on the mechanisms of sexual selection.

Validation of the OECD reproduction test guideline with the New Zealand mudsnail Potamopyrgus antipodarum using trenbolone and prochloraz.

The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals.

Effects of the antimicrobial contaminant triclocarban, and co-exposure with the androgen 17ß-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 17ß-trenbolone (TRB) on fish reproductive function. Adult Pimephales promelas were continuously exposed to either 1 µg TCC/L or 5 µg TCC/L, to 0.5 µg TRB/L, or to a mixture (MIX) of 5 µg TCC/L and 0.5 µg TRB/L for 22 d, and a variety of reproductive and endocrine-related endpoints were examined. Cumulative fecundity was significantly reduced in fathead minnows exposed to TRB, MIX, or 5 µg TCC/L. Exposure to 1 µg TCC/L had no effect on reproduction. In general, both TRB and MIX treatments caused similar physiological effects, evoking significant reductions in female plasma vitellogenin, estradiol, and testosterone, and significant increases in male plasma estradiol. Based on analysis of the ovarian transcriptome, there were potential pathway impacts that were common to both TRB- and TCC-containing treatment groups. In most cases, however, those pathways were more plausibly linked to differences in reproductive status than to androgen-specific functions. Overall, TCC was reproductively toxic to fish at concentrations at or near those that have been measured in surface water. There was little evidence that TCC elicits reproductive toxicity through a specific mode of endocrine or reproductive action, nor that it could augment the androgenic effects of TRB. Nonetheless, the relatively small margin of safety between some measured environmental concentrations and effect concentrations suggests that concern is warranted. Environ Toxicol Chem 2017;36:231-242. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Reproductive effects on freshwater fish exposed to 17α-trenbolone and 17α-estradiol.

17α-Trenbolone and 17α-estradiol are principal metabolites in cattle excreta following the administration of Synovex® ONE, which contains trenbolone acetate and estradiol benzoate. As part of the environmental assessment of the use of Synovex® ONE, data were generated to characterize the effects of 17α-trenbolone and 17α-estradiol on the reproduction of freshwater fish. These substances are known endocrine disruptors, so the purpose of testing was not to clarify these properties but to identify concentrations representing population-relevant effects for use in risk characterization. The short-term reproduction assay was conducted with 17α-trenbolone using the fathead minnow (Pimephales promelas) and the medaka (Oryzias latipes) and with 17α-estradiol using the fathead minnow. Adverse effects on the population-relevant endpoints of survival and fecundity were used to establish the no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC) for each study. For 17α-trenbolone, adverse effects on fecundity of the fathead minnow occurred at 120 ng/L; this was the LOEC, and the NOEC was 35 ng/L. 17ß-Trenbolone did not adversely affect survival and fecundity of medaka at the concentrations tested, resulting in a NOEC of 110 ng/L and a LOEC of >110 ng/L. 17α-Estradiol did not adversely impact survival and fecundity of the fathead minnow at the concentrations tested, resulting in a NOEC and LOEC of 250 ng/L and >250 ng/L, respectively. Environ Toxicol Chem 2017;36:636-644. © 2016 SETAC.

Wastewater treatment plant effluent alters pituitary gland gonadotropin mRNA levels in juvenile coho salmon (Oncorhynchus kisutch).

It is well known that endocrine disrupting compounds (EDCs) present in wastewater treatment plant (WWTP) effluents interfere with reproduction in fish, including altered gonad development and induction of vitellogenin (Vtg), a female-specific egg yolk protein precursor produced in the liver. As a result, studies have focused on the effects of EDC exposure on the gonad and liver. However, impacts of environmental EDC exposure at higher levels of the hypothalamic-pituitary-gonad axis are less well understood. The pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are involved in all aspects of gonad development and are subject to feedback from gonadal steroids making them a likely target of endocrine disruption. In this study, the effects of WWTP effluent exposure on pituitary gonadotropin mRNA expression were investigated to assess the utility of Lh beta-subunit (lhb) as a biomarker of estrogen exposure in juvenile coho salmon (Oncorhynchus kisutch). First, a controlled 72-h exposure to 17α-ethynylestradiol (EE2) and 17ß-trenbolone (TREN) was performed to evaluate the response of juvenile coho salmon to EDC exposure. Second, juvenile coho salmon were exposed to 0, 20 or 100% effluent from eight WWTPs from the Puget Sound, WA region for 72h. Juvenile coho salmon exposed to 2 and 10ng EE2L(-1) had 17-fold and 215-fold higher lhb mRNA levels relative to control fish. Hepatic vtg mRNA levels were dramatically increased 6670-fold, but only in response to 10ng EE2L(-1) and Fsh beta-subunit (fshb) mRNA levels were not altered by any of the treatments. In the WWTP effluent exposures, lhb mRNA levels were significantly elevated in fish exposed to five of the WWTP effluents. In contrast, transcript levels of vtg were not affected by any of the WWTP effluent exposures. Mean levels of natural and synthetic estrogens in fish bile were consistent with pituitary lhb expression, suggesting that the observed lhb induction may be due to estrogenic activity of the WWTP effluents. These results suggest that lhb gene expression may be a sensitive index of acute exposure to estrogenic chemicals in juvenile coho salmon. Further work is needed to determine the kinetics and specificity of lhb induction to evaluate its utility as a potential indicator of estrogen exposure in immature fish.