Environmental Toxicology and Chemistry, Vol. 9999, No. 9999, pp. 1–6, 2016 # 2016 SETAC Printed in the USA
Trenbolone DEGRADATION AND TRANSFORMATION OF 17a-TRENBOLONE IN AEROBIC WATER–SEDIMENT SYSTEMS JOSEPH A. ROBINSON,y** QINGLI MA,z JANE P. STAVELEY,*x WALTER J. SMOLENSKI,k and JON ERICSON# yZoetis, Kalamazoo, Michigan, USA zExponent, Washington, DC, USA xExponent, Cary, North Carolina, USA kPhase IIþ, Kalamazoo, Michigan, USA #Pfizer, Worldwide R&D, PDM-Environmental Sciences, Groton, Connecticut, USA
(Submitted 26 August 2015; Returned for Revision 26 October 2015; Accepted 21 January 2016) Abstract: Synovex1 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 17b-trenbolone and its isomer, 17a-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 17a-trenbolone, which is the principal metabolite found in cattle excreta, in the environment. A study was conducted to determine the degradation and transformation of [14C]-17a-trenbolone in 2 representative water–sediment systems under aerobic conditions. The same transformation products, 17b-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 17b-trenbolone and trendione were determined, along with the DT50 values of the parent compound and the total drug (17a-trenbolone þ 17b-trenbolone þ trendione). The DT50 values for the total system (aqueous and sediment phase) and for the total residues (17a-trenbolone þ 17b-trenbolone þ trendione) in the 2 systems were 34.7 d and 53.3 d, respectively. Environ Toxicol Chem 2016;9999:1–6. # 2016 SETAC Keywords: Androgenic compound
Steroid transformation
Trenbolone
Veterinary medicine
Synovex ONE
trenbolone acetate metabolites in water bodies as a result of runoff from agricultural production systems [4,5], animal grazing areas [6], and animal feeding operations [7–10] and effluent discharged from feedlots [11], as well as in surface water and sediments in proximity to animal production facilities [5,8,10,12]. Recent research suggests that steroid hormones and their metabolites may cause endocrine disruption in aquatic organisms such as fish, implying the potential for adverse environmental effects of these compounds in receiving waters. Altered reproductive function in fish [13–15] and effects on amphibian development, sexual differentiation, and survival [16,17] have been observed with exposures to steroid hormones. These findings have drawn increasing attention to exposure of environmental receptors to these synthetic androgenic steroid hormones in animal waste and biosolids. Recent studies have focused on the occurrence and levels of steroid hormones in fresh manure, manure holding systems, feedlot surfaces, feedlot runoff, and the surrounding terrestrial and aquatic environments [6–8,10,11,18–20]. Both endogenous (natural) and synthetic hormones may exist in cattle excreta. However, although extensive studies have focused on estrogenic steroids [20–24], few data are available to characterize the fate of androgenic steroids and their transformation products in aquatic systems. Sangster et al. [25] determined the bioavailability and fate of sediment-associated trenbolone (and estradiol) in 2 aquatic systems and reported that trenbolone degraded rapidly to more persistent trendione in both test systems. They also reported that trendione was found predominantly associated with the sediments. The objective of the present study was to determine the transformation and degradation characteristics of 17a-trenbolone in 2 typical water–sediment systems under aerobic conditions, to
INTRODUCTION 1
Synovex ONE is an extended-release implant containing the active ingredients estradiol benzoate and trenbolone acetate for use in beef steers and heifers [1]. The indications are as follows: Synovex ONE-F for increased rate of weight gain and improved feed efficiency for up to 200 d in steers and heifers fed in confinement for slaughter and Synovex ONE-G for increased rate of weight gain for up to 200 d in pasture-cattle steers and heifers (stocker, feeder, and slaughter). Trenbolone acetate is a synthetic androgenic steroid hormone. It is not excreted from cattle in the form administered; rather, the acetate group is hydrolyzed rapidly in the animal to form 17b-trenbolone [2], which is further transformed into 17a-trenbolone and trendione, with a minor amount of interconversion back to 17b-trenbolone. Based on excretion data and field monitoring data from manure storage structures on feedlots, the principal metabolite identified in cattle excreta following administration of trenbolone acetate is 17a-trenbolone, with minor amounts of 17b-trenbolone and trendione [1]. Following excretion by livestock, the environmental fate of these hormones is determined by sorption, transformation, and degradation during waste storage and disposal. The majority of commercial beef cattle in the United States receive growth implants that contain steroid hormones [3]. Increased use of trenbolone acetate has led to detection of This article includes online-only Supplemental Data. * Address correspondence to [email protected] **Deceased (Contact at Zoetis, Dawn A. Merritt: dawn.a.merritt@zoetis. com). Published online 23 January 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.3381 1
2
Environ Toxicol Chem 9999, 2016
better characterize the fate and transport of the compound in aquatic environments for use in the environmental assessment of Synovex ONE [1]. MATERIALS AND METHODS
Test substance, reference substances, and reagents
The radiolabeled [14C]-17a-trenbolone (Figure 1) was obtained from Pfizer Global Research and Development. The radiochemical purity of the compound was 98.2% by highperformance liquid chromatographic (HPLC) analysis, and the specific activity was 57.9 mCi/mmol. The nonradiolabeled references—17a-trenbolone (Chemical Abstracts Service [CAS] no. 80657-17-6), 17b-trenbolone (CAS no. 10161-33-8), and trendione (CAS no. 4642-95-9)—were from Pfizer, with purities being 98.5%, 95.0%, and 99.4%, respectively. All solutions were prepared using purified reagent water that meets ASTM International type II requirements, purified with 1a 1 Barnstead E-pure II system or Millipore Milli-Q Direct 8 purification system. The filter-sterilized water typically shows >16.7 Mohm-cm resistivity and
Trenbolone DEGRADATION AND TRANSFORMATION OF 17a-TRENBOLONE IN AEROBIC WATER–SEDIMENT SYSTEMS JOSEPH A. ROBINSON,y** QINGLI MA,z JANE P. STAVELEY,*x WALTER J. SMOLENSKI,k and JON ERICSON# yZoetis, Kalamazoo, Michigan, USA zExponent, Washington, DC, USA xExponent, Cary, North Carolina, USA kPhase IIþ, Kalamazoo, Michigan, USA #Pfizer, Worldwide R&D, PDM-Environmental Sciences, Groton, Connecticut, USA
(Submitted 26 August 2015; Returned for Revision 26 October 2015; Accepted 21 January 2016) Abstract: Synovex1 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 17b-trenbolone and its isomer, 17a-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 17a-trenbolone, which is the principal metabolite found in cattle excreta, in the environment. A study was conducted to determine the degradation and transformation of [14C]-17a-trenbolone in 2 representative water–sediment systems under aerobic conditions. The same transformation products, 17b-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 17b-trenbolone and trendione were determined, along with the DT50 values of the parent compound and the total drug (17a-trenbolone þ 17b-trenbolone þ trendione). The DT50 values for the total system (aqueous and sediment phase) and for the total residues (17a-trenbolone þ 17b-trenbolone þ trendione) in the 2 systems were 34.7 d and 53.3 d, respectively. Environ Toxicol Chem 2016;9999:1–6. # 2016 SETAC Keywords: Androgenic compound
Steroid transformation
Trenbolone
Veterinary medicine
Synovex ONE
trenbolone acetate metabolites in water bodies as a result of runoff from agricultural production systems [4,5], animal grazing areas [6], and animal feeding operations [7–10] and effluent discharged from feedlots [11], as well as in surface water and sediments in proximity to animal production facilities [5,8,10,12]. Recent research suggests that steroid hormones and their metabolites may cause endocrine disruption in aquatic organisms such as fish, implying the potential for adverse environmental effects of these compounds in receiving waters. Altered reproductive function in fish [13–15] and effects on amphibian development, sexual differentiation, and survival [16,17] have been observed with exposures to steroid hormones. These findings have drawn increasing attention to exposure of environmental receptors to these synthetic androgenic steroid hormones in animal waste and biosolids. Recent studies have focused on the occurrence and levels of steroid hormones in fresh manure, manure holding systems, feedlot surfaces, feedlot runoff, and the surrounding terrestrial and aquatic environments [6–8,10,11,18–20]. Both endogenous (natural) and synthetic hormones may exist in cattle excreta. However, although extensive studies have focused on estrogenic steroids [20–24], few data are available to characterize the fate of androgenic steroids and their transformation products in aquatic systems. Sangster et al. [25] determined the bioavailability and fate of sediment-associated trenbolone (and estradiol) in 2 aquatic systems and reported that trenbolone degraded rapidly to more persistent trendione in both test systems. They also reported that trendione was found predominantly associated with the sediments. The objective of the present study was to determine the transformation and degradation characteristics of 17a-trenbolone in 2 typical water–sediment systems under aerobic conditions, to
INTRODUCTION 1
Synovex ONE is an extended-release implant containing the active ingredients estradiol benzoate and trenbolone acetate for use in beef steers and heifers [1]. The indications are as follows: Synovex ONE-F for increased rate of weight gain and improved feed efficiency for up to 200 d in steers and heifers fed in confinement for slaughter and Synovex ONE-G for increased rate of weight gain for up to 200 d in pasture-cattle steers and heifers (stocker, feeder, and slaughter). Trenbolone acetate is a synthetic androgenic steroid hormone. It is not excreted from cattle in the form administered; rather, the acetate group is hydrolyzed rapidly in the animal to form 17b-trenbolone [2], which is further transformed into 17a-trenbolone and trendione, with a minor amount of interconversion back to 17b-trenbolone. Based on excretion data and field monitoring data from manure storage structures on feedlots, the principal metabolite identified in cattle excreta following administration of trenbolone acetate is 17a-trenbolone, with minor amounts of 17b-trenbolone and trendione [1]. Following excretion by livestock, the environmental fate of these hormones is determined by sorption, transformation, and degradation during waste storage and disposal. The majority of commercial beef cattle in the United States receive growth implants that contain steroid hormones [3]. Increased use of trenbolone acetate has led to detection of This article includes online-only Supplemental Data. * Address correspondence to [email protected] **Deceased (Contact at Zoetis, Dawn A. Merritt: dawn.a.merritt@zoetis. com). Published online 23 January 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.3381 1
2
Environ Toxicol Chem 9999, 2016
better characterize the fate and transport of the compound in aquatic environments for use in the environmental assessment of Synovex ONE [1]. MATERIALS AND METHODS
Test substance, reference substances, and reagents
The radiolabeled [14C]-17a-trenbolone (Figure 1) was obtained from Pfizer Global Research and Development. The radiochemical purity of the compound was 98.2% by highperformance liquid chromatographic (HPLC) analysis, and the specific activity was 57.9 mCi/mmol. The nonradiolabeled references—17a-trenbolone (Chemical Abstracts Service [CAS] no. 80657-17-6), 17b-trenbolone (CAS no. 10161-33-8), and trendione (CAS no. 4642-95-9)—were from Pfizer, with purities being 98.5%, 95.0%, and 99.4%, respectively. All solutions were prepared using purified reagent water that meets ASTM International type II requirements, purified with 1a 1 Barnstead E-pure II system or Millipore Milli-Q Direct 8 purification system. The filter-sterilized water typically shows >16.7 Mohm-cm resistivity and
