The Power of Keratinous Matrices (Head Hair, Body Hair and Nail Clippings) Analysis in a Case of Death Involving Anabolic Agents.

A 29-year-old man with no previous medical history was found dead at home. Anabolic products (tablets and oily solutions) and syringes were found at the scene. The man was known to train regularly at a fitness club and to use anabolic drugs. Following an unremarkable autopsy with normal histology, toxicological analyses were requested by the local prosecutor to provide further information. Blood, head hair (5 cm, black), body hair (axillary and leg) and toe and finger nail clippings were submitted to liquid and gas chromatography coupled to tandem mass spectrometry (LC and GC-MS-MS) methods to test for anabolic steroids. Blood tested positive for testosterone (4 ng/mL), boldenone (26 ng/mL), stanozolol (3 ng/mL) and trenbolone (<1 ng/mL). Segmental head hair tests (2 × 2.5 cm) revealed a repeated consumption of testosterone (65-72 pg/mg), testosterone propionate (930-691 pg/mg), testosterone isocaproate (79 pg/mg to <5 pg/mg), nandrolone decanoate (202-64 pg/mg), boldenone (16 pg/mg), stanozolol (575-670 pg/mg), trenbolone (4 pg/mg-not detected), drostanolone (112-30 pg/mg), drostanolone enanthate (26-5 pg/mg) and drostanolone propionate (15-4 pg/mg). In addition to the substances identified in head hair, testosterone decanoate, testosterone cypionate and nandrolone were identified in both body hair and nails. The experts concluded that the manner of death can be listed as toxic due to massive repetitive use of anabolic steroids during the previous months. For anabolic agents, blood does not seem to be the best matrix to document a fatal intoxication. Indeed, these products are toxics when abused long term and are known to cause cardiac, hepatic and renal diseases. When compared to blood, hair and nails have a much larger window of detection. Therefore, keratinous matrices seem to be the best approach to test for anabolic steroids when a sudden death is observed in the context of possible abuse of steroids.

Anabolic steroids and extreme violence: a case of murder after chronic intake and under acute influence of metandienone and trenbolone.

A 32-year-old male went to the police to claim he just killed his girlfriend by inflicting several stabs with a kitchen knife. He was very nervous and particularly aggressive. About 90 min after the assault, a blood specimen was collected with natrium fluoride as preservative. The blood was free of alcohol, pharmaceuticals and drugs of abuse, but tested positive by LC-MS/MS for metandienone (32 ng/mL) and trenbolone (9 ng/mL). The perpetrator admitted regular consumption of anabolic steroids to enhance his muscular mass, as he was a professional security agent. To document long-term steroid abuse, a hair specimen was collected 3 weeks after the assault, which tested positive for both drugs. Segmental analyses revealed in the proximal 1.5 cm segment, corresponding to the period of the assault, the simultaneous presence of metandienone (11 pg/mg) and trenbolone (14 pg/mg), while only metandienone (3 pg/mg) was identified in the distal 1.5 cm segment. As aggressiveness and violence can be associated with abuse of anabolic steroids, the aetiology of this domestic crime was listed to be due impulsive behaviour in a context of antisocial lifestyle.

Ractopamine and Other Growth-Promoting Compounds in Beef Cattle Operations: Fate and Transport in Feedlot Pens and Adjacent Environments.

The current study represents a comprehensive investigation of the occurrence and fates of trenbolone acetate (TBA) and metabolites 17α-trenbolone (17α-TBOH), 17ß-TBOH, and trendione (TBO); melengesterol acetate (MGA); and the less commonly studied ß-andrenergic agonist ractopamine (RAC) in two 8 month cattle feeding trials and simulated rainfall runoff experiments. Cattle were administered TBA, MGA, or RAC, and their residues were measured in fresh feces, pen floor material, and simulated rainfall runoff from pen floor surfaces and manure-amended pasture. Concentrations of RAC ranged from 3600 ng g-1, dry weight (dw), in pen floor to 58 000 ng g-1 in fresh feces and were, on average, observed at 3-4 orders of magnitude greater than those of TBA and MGA. RAC persisted in pen floors (manure t1/2 = 18-49 days), and contamination of adjacent sites was observed, likely via transport of windblown particulates. Concentrations in runoff water from pen floors extrapolated to larger-scale commercial feedlots revealed that a single rainfall event could result in mobilization of gram quantities of RAC. This is the first report of RAC occurrence and fate in cattle feedlot environments, and will help understand the risks posed by this chemical and inform appropriate manure-management practices.

Photolysis of Trenbolone Acetate Metabolites in the Presence of Nucleophiles: Evidence for Metastable Photoaddition Products and Reversible Associations with Dissolved Organic Matter.

Photolysis of trenbolone acetate (TBA) metabolites in the presence of various nitrogen-, sulfur-, or oxygen-containing nucleophiles (e.g., azide, ammonia, or thiosulfate, respectively) results in rapid (half-lives ∼20-60 min), photochemically induced nucleophile incorporation across the parent steroid's trienone moiety. The formation of such nucleophile adducts limits formation of photohydrates, suggesting competition between the nucleophile and water for photochemical addition into the activated steroid structure. Analogous to previously reported photohydration outcomes, LC/MS analyses suggest that such photonucleophilic addition reactions are reversible, with more rapid elimination rates than thermal dehydration of photohydrates, and regenerate parent steroid structures. Beyond photonucleophilic addition pathways, we also found that hydroxylamine and presumed nucleophilic moieties in model dissolved organic matter (DOM; Fluka humic acid) can react via thermal substitution with TBA metabolite photohydrates, although this reaction with model DOM was only observed for photohydrates of trendione. Most nucleophile addition products [i.e., formed via (photo)reaction with thiosulfate, hydroxylamine, and ammonia] are notably more polar relative to the parent metabolite and photohydration products. Thus, if present, both nucleophilic adducts and bound residues in organic matter will facilitate transport and help mask detection of TBA metabolites in surface waters and treatment systems.

Sorption and transport of trenbolone and altrenogest photoproducts in soil-water systems.

This study evaluated the sorption and transport potential of seven phototransformation products of 17α-trenbolone, 17ß-trenbolone, trendione, and altrenogest, along with the parent trienone steroids in batch and column soil-water systems. In batch systems, the target solutes exhibited linear isotherms, with values for sorption coefficients (log Koc) of parent steroids (2.46-2.76) higher than those for photoproducts (1.92-2.57). In column systems, the estimated retardation factors (Rsol) for parents (2.7-5.1) were ∼2-5 times higher than those for photoproducts (0.84-1.7). The log Koc (R2 = 0.75) and Rsol (R2 = 0.89-0.98) were well correlated with measured log Kow values, indicating that hydrophobic partitioning governed the soil-solute interaction of these biologically potent compounds in soil-water systems. These data indicated that photoproducts exhibited reduced sorption affinity and increased transport potential relative to more hydrophobic parent structures. In agroecosystems, traditional runoff management practices would be expected to exhibit reduced treatment effectiveness for photoproducts relative to the parent compounds of commonly used trienone steroids.

Detection and quantification of metastable photoproducts of trenbolone and altrenogest using liquid chromatography-tandem mass spectrometry.

Here, we developed a novel and sensitive method for the detection and quantification of metastable trenbolone and altrenogest photoproducts in agricultural receiving waters based on solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Primary method analytes were seven cycloaddition or photohydration transformation products of 17α-trenbolone (17α-TBOH), 17ß-trenbolone (17ß-TBOH), trendione (TBO), and altrenogest (ALT), which are key contributors to the fate and environmental risks of these steroidal pharmaceuticals. Because commercial analytical standards are not available, reference standards for photoproducts were generated from trenbolone or ALT with a solar simulator (˜6 h, >10 half-lives). Efficient detection of metastable photoproducts required cold and pH neutral conditions, rapid sample processing, minimal sample storage, and consideration of cationic artifacts. Method detection limits (MDLs) were 0.034-0.40 ng L-1 for parent compounds and 0.16-2.1 ng L-1 for photoproducts, sufficient for their detection in agroecosystems. Matrix suppression was observed and corrected by internal standards, and relative recovery rates were near 100% for all analytes except for 12-OH-17α-TBOH (˜75% recovery). Intra-day variation was <20% and inter-day variation <25% for all the analytes. The developed method is capable of the analysis of trenbolone, altrenogest, and their key bioactive photoproducts in agricultural receiving waters.

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.

Degradation and transformation of 17α-trenbolone in aerobic water-sediment systems.

Synovex® ONE is an extended-release implant containing the active ingredients estradiol benzoate and trenbolone acetate for use in beef steers and heifers. Trenbolone acetate is rapidly hydrolyzed in cattle to form 17ß-trenbolone and its isomer, 17α-trenbolone, which are further transformed to a secondary metabolite, trendione. As part of the environmental assessment for the use of Synovex ONE, data were generated to characterize the fate of 17α-trenbolone, which is the principal metabolite found in cattle excreta, in the environment. A study was conducted to determine the degradation and transformation of [14 C]-17α-trenbolone in 2 representative water-sediment systems under aerobic conditions. The same transformation products, 17ß-trenbolone and trendione, were formed, principally in the sediment phase, in both systems. From the production of these transformation products, the 50% disappearance time (DT50) values of 17ß-trenbolone and trendione were determined, along with the DT50 values of the parent compound and the total drug (17α-trenbolone + 17ß-trenbolone + trendione). The DT50 values for the total system (aqueous and sediment phase) and for the total residues (17α-trenbolone + 17ß-trenbolone + trendione) in the 2 systems were 34.7 d and 53.3 d, respectively. Environ Toxicol Chem 2017;36:630-635. © 2016 SETAC.

Degradation and transformation of 17α-estradiol in water-sediment systems under controlled aerobic and anaerobic conditions.

One of the principal metabolites in cattle excreta following the administration of Synovex® ONE, which contains estradiol benzoate and trenbolone acetate, is 17α-estradiol. As part of the environmental assessment of the use of Synovex ONE, data were generated to characterize the fate of 17α-estradiol in the environment. Studies were conducted to determine the degradation and transformation of 17α-[14 C]-estradiol in 2 representative water-sediment systems each under aerobic and anaerobic conditions. The same transformation products-estriol, 17ß-estradiol, and estrone-were formed, principally in the sediment phase, under both conditions in both systems. From the production of these transformation products, the 50% disappearance time (DT50) values of estrone and 17ß-estradiol were determined, along with the DT50 values of 17α-estradiol and the total drug (17α-estradiol + 17ß-estradiol + estrone). The results indicate that 17 α-[14 C]-estradiol was more persistent under anaerobic conditions than under aerobic conditions and that 17 α-[14 C]-estradiol was less persistent than its transformation products. The DT50 values for the total system (aqueous and sediment phases) and for the total residues (17α-estradiol, 17ß-estradiol, and estrone) were selected for use in modeling the environmental fate of estradiol benzoate. For aerobic degradation in the water-sediment system, the DT50 was 31.1 d, and it was 107.8 d for the anaerobic system. Environ Toxicol Chem 2017;36:621-629. © 2016 SETAC.

Optimisation and validation of a new analytical method for the determination of four natural and synthetic hormones using LC-ESI-MS/MS.

A rapid liquid chromatographic-tandem mass spectrometric method was developed for the simultaneous determination of four natural and synthetic hormone residues (progesterone, testosterone, trenbolone acetate and zeranol) in animal tissue samples. Sample preparation was optimised to minimise time and solvent consumption. Meat samples were mechanically homogenised and digested in a procedure that gave similar recoveries to those enzymatically hydrolysed by Helix pomatia. Efficient extraction was achieved using acidified acetonitrile (1% acetic acid). Chromatographic conditions were optimised to minimise matrix effects. Analytes were separated using a C18 column with gradient elution using ammonium formate solution in methanol (MeOH)/water (1:9) and MeOH mobile phases. Finally, residues were qualitatively and quantitatively determined by electrospray ionisation tandem mass spectrometry in multiple reaction monitoring mode. Different parameters for LC-MS/MS (e.g., declustering potential and collision energy) were optimised using API 6500QT; all analytes were measured using positive-mode electrospray ionisation (ESI+) except zeranol which was measured in negative mode (ESI-). Due to LC-MS/MS signal enhancement/suppression, the determination of hormones was based on matrix-matched standard calculations. The method was validated for the four hormones on meat samples at different fortification levels and showed accepted performance criteria according to European Commission Decision 2002/657/EC. Decision limits and detection capabilities were estimated for all analytes.

Determination of residual levels of naloxone, yohimbine, thiophanate, and altrenogest in porcine muscle using QuEChERS with liquid chromatography and triple quadrupole mass spectrometry.

A quick, easy, cheap, effective, rugged, and safe QuEChERS (method) was used for the simultaneous detection of four veterinary drug residues, namely naloxone, yohimbine, thiophanate, and altrenogest, in porcine muscle, using liquid chromatography with electrospray ionization triple quadrupole tandem mass spectrometry. Because of the unavailability of a suitable internal standard, matrix-matched calibrations were used for quantification, with determination coefficients ≥ 0.9542. The accuracy (expressed as recovery %) ranged from 60.53 to 83.25%, and the intra- and interday precisions (expressed as relative standard deviations) were <12%. The limits of quantification were 5, 0.5, 2, and 5 ng/g for naloxone, yohimbine, thiophanate, and altrenogest, respectively. Samples purchased from local markets in Seoul, Republic of Korea, revealed no traces of the target analytes. The developed method described herein is sensitive and reliable and can be applied to quantify the tested veterinary drugs in animal tissues.

Sorption, uptake, and biotransformation of 17ß-estradiol, 17α-ethinylestradiol, zeranol, and trenbolone acetate by hybrid poplar.

Hormonally active compounds may move with agricultural runoff from fields with applied manure and biosolids into surface waters where they pose a threat to human and environmental health. Riparian zone plants could remove hormonally active compounds from agricultural runoff. Therefore, sorption to roots, uptake, translocation, and transformation of 3 estrogens (17ß-estradiol, 17α-ethinylestradiol, and zeranol) and 1 androgen (trenbolone acetate) commonly found in animal manure or biosolids were assessed by hydroponically grown hybrid poplar, Populus deltoides x nigra, DN-34, widely used in riparian buffer strips. Results clearly showed that these hormones were rapidly removed from 2 mg L(-1) hydroponic solutions by more than 97% after 10 d of exposure to full poplar plants or live excised poplars (cut-stem, no leaves). Removals by sorption to dead poplar roots that had been autoclaved were significantly less, 71% to 84%. Major transformation products (estrone and estriol for estradiol; zearalanone for zeranol; and 17ß-trenbolone from trenbolone acetate) were detected in the root tissues of all 3 poplar treatments. Root concentrations of metabolites peaked after 1 d to 5 d and then decreased in full and live excised poplars by further transformation. Metabolite concentrations were less in dead poplar treatments and only slowly increased without further transformation. Taken together, these findings show that poplars may be effective in controlling the movement of hormonally active compounds from agricultural fields and avoiding runoff to streams.

Sex in troubled waters: Widespread agricultural contaminant disrupts reproductive behaviour in fish.

Chemical pollution is a pervasive and insidious agent of environmental change. One class of chemical pollutant threatening ecosystems globally is the endocrine disrupting chemicals (EDCs). The capacity of EDCs to disrupt development and reproduction is well established, but their effects on behaviour have received far less attention. Here, we investigate the impact of a widespread androgenic EDC on reproductive behaviour in the guppy, Poecilia reticulata. We found that short-term exposure of male guppies to an environmentally relevant concentration of 17ß-trenbolone-a common environmental pollutant associated with livestock production-influenced the amount of male courtship and forced copulatory behaviour (sneaking) performed toward females, as well as the receptivity of females toward exposed males. Exposure to 17ß-trenbolone was also associated with greater male mass. However, no effect of female exposure to 17ß-trenbolone was detected on female reproductive behaviour, indicating sex-specific vulnerability at this dosage. Our study is the first to show altered male reproductive behaviour following exposure to an environmentally realistic concentration of 17ß-trenbolone, demonstrating the possibility of widespread disruption of mating systems of aquatic organisms by common agricultural contaminants.

Rates and product identification for trenbolone acetate metabolite biotransformation under aerobic conditions.

Trenbolone acetate metabolites are endocrine-active contaminants discharged into the aquatic environment in runoff from agricultural fields, rangelands, and concentrated animal feeding operations. To investigate the environmental fate of these compounds and their biotransformation mechanisms, the authors used inocula from a variety of different water sources and dosed biologically active microcosms with approximately 1400 ng/L of trenbolone acetate metabolites, including 17ß-trenbolone, trendione, and 17α-trenbolone. To investigate aerobic biotransformation rates and interconversions between known trenbolone acetate metabolites, gas chromatography-tandem mass spectrometry was used to measure concentrations and assess product distributions as a function of time. High-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to characterize novel transformation products and potential transformation pathways. Kinetic analysis yields observed half-lives of approximately 0.9 d, 1.3 d, and 2.2 d for 17ß-trenbolone, trendione, and 17α-trenbolone, respectively, at 20 °C, although colder conditions increased half-lives to 8.5 d and biphasic transformation was observed. Relative to reported faster attenuation rates in soils, trenbolone acetate metabolites are likely more persistent in aqueous systems. Product distributions indicate an enzymatic preference for biotransformation between trendione and 17ß-trenbolone. The LC-MS/MS characterization indicates dehydrogenation products as the major detectable products and demonstrates that major structural elements responsible for bioactivity in steroids are likely retained during biotransformation.

Development of a rapid method for the analysis of trenbolone, nortestosterone, and zeranol in bovine liver using liquid chromatography tandem mass spectrometry.

A rapid liquid chromatography tandem mass spectrometry method has been developed and validated for the determination of α-trenbolone, ß-trenbolone, α-nortestosterone, ß-nortestosterone, zeranol, and taleranol in bovine liver. The impact of liquid-liquid extraction with methyl tert-butyl ether and optimized solid phase extraction on silica cartridges significantly reduced effort and time of sample preparation. Electrospray ionization gives a significant signal increase compared with atmospheric pressure chemical ionization and atmospheric pressure photoionization. The HPLC gradient was optimized to separate isobaric analytes and matrix constituents from the hormone molecules. The optimized time and temperature of enzymatic hydrolysis of conjugated trenbolone was 4 h at 52 °C. The method validated in the range of 0.5-30 µg kg(-1) for α-trenbolone, ß-trenbolone, zeranol, taleranol, and 2-30 µg kg(-1) for α-nortestosterone, ß-nortestosterone. Combined uncertainty of measurements was in the range of 4%-23%. The matrix effect was negligible (1%-5%) for all analytes except of α-nortestosterone (19%). The developed method with changes concerning sample size and hydrolysis was also applied for the analysis of meat, serum, and urine samples. Graphical Abstract Determination of trenbolone, nortestosterone and zeranol in bovine liver.

Surface and subsurface attenuation of trenbolone acetate metabolites and manure-derived constituents in irrigation runoff on agro-ecosystems.

Although studies have evaluated the ecotoxicity and fate of trenbolone acetate (TBA) metabolites, namely 17α-trenbolone (17α-TBOH), 17ß-trenbolone (17ß-TBOH), and trendione (TBO), their environmental transport processes remain poorly characterized with little information available to guide agricultural runoff management. Therefore, we evaluated TBA metabolite transport in representative agricultural systems with concurrent assessment of other manure-derived constituents. Leachate generated using manure from TBA-implanted cattle was applied to a subsurface infiltration plot (4 m) and surface vegetative filter strips (VFSs; 3, 4, and 5 m). In the subsurface experiment, 17α-TBOH leachate concentrations were 36 ng L(-1) but decreased to 12 ng L(-1) in initial subsurface discharge. Over 75 minutes, concentrations linearly increased to 23 ng L(-1) (C/Co = 0.32-0.64). In surface experiments (n = 4), 17α-TBOH leachate concentrations ranged from 11-150 ng L(-1), remained nearly constant with time, but were attenuated by ∼70-90% after VFS treatment with no statistical dependence on the VFS length. While attenuation clearly occurred, the observations of a highly mobile fraction of all constituents in both surface runoff and subsurface discharge suggest that these treatment strategies may not always be capable of achieving threshold discharge concentrations. To attain no observed adverse effect levels (NOAELs) in receiving waters, concurrent assessment of leachate concentrations and available dilution capacities can be used to guide target treatment performance levels for runoff management. Dilution is usually necessary to achieve NOAELs, and receiving waters with less than 70-100 fold dilution capacity are at the highest risk for steroidal endocrine disruption.

Trenbolone acetate metabolite transport in rangelands and irrigated pasture: observations and conceptual approaches for agro-ecosystems.

To assess the relative ecological risks of trenbolone acetate (TBA) use in agro-ecosystems, we evaluated the spatiotemporal dynamics of TBA metabolite transport during irrigation and rainfall events. Within a pasture, TBA-implanted heifers (40 mg TBA, 8 mg estradiol) were briefly penned (24 h) at high stocking densities (500 animal units (AU)/ha), prior to irrigation. Irrigation runoff concentrations of 17α-trenbolone (17α-TBOH) 0.3 m downslope were 11 ng/L in the wetting front, but quickly decreased to ∼0.5 ng/L, suggesting mass transfer limitations to transport. At 3 and 30 m downslope, efficient attenuation of 17α-TBOH concentrations is best explained by infiltration and surface partitioning. At plot scales, transport through vegetated filter strips resulted in <0.5-7 ng/L 17α-TBOH concentrations in rainfall-induced runoff with partial subsequent attenuation. Thus, even under intense grazing scenarios, TBA-metabolite transport potential is expected to be low in rangelands, with ecological risks primarily arising from uncontrolled animal access to receiving waters. However, 17α-TBOH concentrations in initial runoff were predicted to exceed threshold levels (i.e., no observed adverse effect levels [NOAELs]) for manure concentrations exceeding 2.0 ng/g-dw, which occurs throughout most of the implant life. For comparison, estrone and 17ß-estradiol were modeled and are likely capable of exceeding NOAELs by a factor of ∼2-5 in irrigation runoff, suggesting that both endogenous and exogenous steroids contribute to endocrine disruption potential in agro-ecosystems.

Bioavailability and fate of sediment-associated trenbolone and estradiol in aquatic systems.

Endocrine disrupting effects in aquatic organisms have been observed in systems influenced by steroid hormones. Associating endocrine disruption with aqueous concentrations of steroids alone may overlook the influence of source-sink dynamics in sediments on steroid hormone bioavailability. The objective of this study was to determine the fate of 17ß-estradiol and 17ß-trenbolone in two field sediments and to evaluate the corresponding bioavailability of the compounds to the fathead minnow (Pimephales promelas). Steroid fate was evaluated using analytical chemistry and verified by assessing the biological activity using yeast based in vitro assays. Effective bioavailability of the steroids was inferred from changes in hepatic vitellogenin expression (increased expression in males exposed to 17ß-estradiol, and reduced expression in females exposed to 17ß-trenbolone). In experiments conducted with 17ß-estradiol, no induction of hepatic vitellogenin mRNA expression was observed in male fish exposed to sediment-associated 17ß-estradiol. In contrast, female minnows exposed to sediment-associated 17ß-trenbolone experienced significant reductions in hepatic vitellogenin compared to negative controls. In both systems, the parent compounds were shown to degrade rapidly to the more persistent metabolites, estrone and trendione, both of which were found predominantly associated with the sediments. Results from the yeast estrogen screen indicate a reduction in biological activity as biotransformation of 17ß-estradiol occurs; results from the yeast anti-estrogen screen were inconclusive and unable to substantiate 17ß-trenbolone fate in aquatic systems. Collectively, these data support the contention that steroid hormones associated with the sediment can become bioavailable to fish, and that sediment characteristics influence the observed bioavailability of these compounds.

Analysis of trenbolone acetate metabolites and melengestrol in environmental matrices using gas chromatography-tandem mass spectrometry.

Studies demonstrate that exposure to steroid hormones in receiving waters can adversely impact reproduction of aquatic organisms. In particular, exogenous steroid hormones widely used as growth promoters in animal agriculture are of high concern, yet no gas chromatography-tandem mass spectrometry (GC/MS/MS) analytical methods for the detection of these compounds in complex environmental matrices is described in the literature. This study utilizes analytical methods based upon N-methyl-N-(trimethylsilyl)trifluoro-acetamide-iodine (MSTFA-I(2)) derivatization for the analysis of metabolites of trenbolone acetate (TBA), including 17α-trenbolone, 17ß-trenbolone, and trendione, and melengestrol acetate in receiving waters and surface soils associated with animal agriculture. Results suggest method detection levels of 0.5-1 ng/L for the trenbolone metabolites, while detection of melengestrol is qualitative only. Isotope dilution methods employing d3-17ß-trenbolone were used to improve steroid quantification. Method recoveries in spiked samples collected from a variety of representative receiving waters generally ranged from 80-120% with consistent and low standard deviation (generally<10%) for replicate analysis. Analysis of a storm water runoff sample from a commercial confined animal feeding operation (CAFO) that used TBA implants detected 17ß-trenbolone and trendione at concentrations of 31 and 52 ng/L, respectively. Analysis of surface soils at a commercial CAFO using TBA implants detected 17α-trenbolone at concentrations between 4-6 ng/g dry weight. Method development efforts suggested that the concentration of I(2) in MSTFA, the removal of I(2) from sample extracts after derivatization, and the use of Florisil clean-up to reduce organic matter matrix were vital aspects of steroid hormone quantification at low (<30ng/L) concentrations in complex environmental matrices.

Estrogens and synthetic androgens in manure slurry from trenbolone acetate/estradiol implanted cattle and in waste-receiving lagoons used for irrigation.

The increasing size of concentrated animal feeding operations has led to a concomitant increase in the land-application of manure, which has spawned research on the concentrations and environmental risk assessment of natural and synthetic hormones in animal manures. 17ß-Trenbolone acetate (TBA) is widely used in the United States for improving daily gains in beef cattle and is often administered in combination with 17ß-estradiol (17ß-E2). Trenbolone (TB) and E2 isomers and their metabolites were quantified in manure collection pits and lagoon effluent from beef cattle implanted with the commercial anabolic preparation Ravoler-S (containing 140 mg 17ß-trenbolone acetate and 28 mg 17ß-E2). Manure pit and lagoon effluent samples were collected weekly for 9 weeks post implanting and analyzed using reverse-phase liquid chromatography tandem mass spectrometry. 17α-TB was the most abundant androgen with the highest concentration observed 2 weeks post implant. 17ß-TB and trendione peaked at the end of week 2 and 4, respectively. For the estrogens, the highest concentrations for estrone (E1), estriol (E3), and 17α-E2 were observed after week 4, 6, and 8, respectively. 17ß-E2 concentrations were the lowest of the estrogens and erratic over time. In lagoon water, which is used for irrigation, 17α-TB and E1 had the highest detected hormone concentrations (1.53 and 1.72 µg L(-1), respectively). Assuming a 1-2 order dilution during transport to surface water, these hormone levels could lead to concentrations in receiving waters that exceed some of the lowest observable effect levels (LOELs) reported for hormones (e.g., 0.01-0.03 µg L(-1)).